Advanced RNA targeting technology (ARNATAR) for angiotensinogen

Abstract

This specification discloses highly RNA-targeted (ARNATAR) dsRNA compounds that target angiotensinogen (AGT). Such compounds are useful in methods for reducing AGT expression and in methods for therapeutically treating RAAS-related diseases, disorders and / or conditions or symptoms thereof in subjects.

Description

[Technical Field]

[0001] Specific embodiments relate to methods and compounds for regulating angiotensinogen (AGT) gene expression by advanced RNA targeting technology (ARNATAR). Such methods and compounds are useful for treating AGT-related diseases, disorders, and / or conditions in subjects by reducing AGT expression. [Background technology]

[0002] Angiotensinogen (AGT, also known as SERPINA8 or ANHU) is a member of the serpine family and a component of the renin-angiotensin system (RAS) pathway (also known as the renin-angiotensin-aldosterone system (RAAS)). It is primarily produced in the liver and released into circulation, where renin converts it to angiotensin I. Angiotensin-converting enzyme (ACE) then converts angiotensin I to angiotensin II.

[0003] Angiotensin II, a peptide hormone, has diverse effects on the circulatory system that controls blood pressure and may contribute to hypertension. Angiotensin II causes vasoconstriction, which can raise blood pressure. Angiotensin II stimulates the secretion of aldosterone, a hormone from the adrenal cortex. Aldosterone increases sodium reabsorption in the kidneys and modulates the glomerular filtration rate, which can lead to an increase in body fluid volume and raise blood pressure. Excessive angiotensin II can lead to dysregulation of the RAAS pathway and hypertension.

[0004] Chronic high blood pressure is known as hypertension. In hypertensive individuals, high blood pressure requires the heart to work harder to circulate blood through the blood vessels. Hypertension can lead to many harmful conditions in the body, such as increased oxidative stress, increased inflammation, hypertrophy, cardiac fibrosis, renal fibrosis, and arterial fibrosis, and can result in conditions like left ventricular fibrosis, arterial remodeling, and glomerulosclerosis.

[0005] Hypertension is known to be a major risk factor for a variety of diseases, disorders and conditions, such as cardiovascular morbidity, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysm), peripheral artery disease, cardiac injury (e.g., cardiac hypertrophy or enlargement), and other cardiovascular-related diseases, disorders and / or conditions that shorten life expectancy.

[0006] The prevalence of drug-resistant hypertension (RHTN), or hypertension resistant to conventional medications, is steadily increasing due to the aging population and rising rates of obesity, despite the number of commercially available antihypertensive drugs. Furthermore, commercially available antihypertensive therapies targeting various RAAS pathway components have been ineffective in completely inhibiting or blocking the RAAS pathway. While the mechanism of this ineffective inhibition is not fully understood, it may be caused by ACE avoidance and / or aldosterone avoidance pathways. Therefore, new therapies are needed.

[0007] The use of therapeutic oligomeric compounds (e.g., oligonucleotides) was first proposed more than 40 years ago by Stephenson and Zarecnik (Inhibition of Rous Sarcoma Viral RNA Translation by a Specific Oligodeoxyribonucleotide, PNAS, 1978, 75:285-288).

[0008] RNA interference (RNAi), a sequence-specific gene expression silencing technique, was discovered in 1998 by Fire et al. (Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans, Nature, 1998, 391:806-811). RNAi utilizes double-stranded RNA (dsRNA) to inhibit gene expression via the RNA-induced silencing complex (RISC).

[0009] RISCs contain a complex of multiple proteins that interact with oligomeric compounds to inhibit gene expression. These oligomeric compounds act as templates for RISCs to recognize complementary messenger RNA (mRNA) transcripts and cleave specific mRNA transcripts as targets. Cleavage of the target mRNA blocks translation of the target mRNA, silencing the target gene. Oligomer compounds utilized by RISCs include, but are not limited to, microRNAs (miRNAs), certain oligonucleotides, and single-stranded oligomeric compounds such as single-stranded siRNAs (Lima et al., Single-stranded siRNAs activate RNAi in animals. Cell. 2012, 150(5):883-94), as well as double-stranded RNA (dsRNA) compounds such as short hairpin RNA (shRNA) and small interfering RNA (siRNA).

[0010] In 2001, Elbashir et al. demonstrated that 21-nucleotide siRNA double helixs specifically suppress the expression of endogenous and heterologous genes in mammalian cell lines, theorizing that siRNA could ultimately be used as a gene-specific therapeutic agent (Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells, Nature, 2001, 411:494-498).

[0011] The field of oligomeric therapeutic compounds is still mature, and improvements in delivery, stability, specificity, safety, and efficacy are still needed to enhance the therapeutic response of oligomeric compounds.

[0012] Currently, several clinical trials are underway to evaluate nucleic acid-based therapies targeting AGTs for the treatment of hypertension. These trials evaluate both siRNA and antisense oligonucleotide (ASO) therapies (Ranasinghe et al., J Am Heart Assoc, 2022, 11(20); Cruz-Lopez et al., Hypertension, 2022, 79:2115-2126; Morgan et al., Clinical Res, 2021, 6(6):4865-96), but neither type of nucleic acid therapy has received regulatory approval for patient treatment. Therefore, an unmet need for nucleic acid-based therapies targeting AGTs still exists. This specification discloses improved AGT-targeting dsRNA compounds with advanced RNA targeting (ARNATAR) capabilities that enhance gene silencing activity. [Overview of the project]

[0013] Several embodiments provided herein relate to the discovery of specific ARNATAR-designed dsRNA compounds that target AGT transcripts, which can enhance their effectiveness in modulating AGT gene expression. In some embodiments, the dsRNA compound is a modified shRNA or siRNA compound. The double-stranded RNA compound comprises a sense strand and an antisense strand. The antisense strand may be fully or substantially complementary to the target nucleic acid.

[0014] In some embodiments, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand forming a double-stranded portion, the antisense strand comprising any of the antisense strand sequences in Tables 2, 5, 7, 9, 10, or 13.

[0015] In some embodiments, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand forming a double-stranded portion, the sense strand comprising any of the sense strand sequences in Tables 2, 5, 7, 9, 10, or 13.

[0016] In some embodiments, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand forming a double-stranded portion, wherein the sense strand comprises one of the sense strand sequences from Tables 2, 5, 7, 9, 10, or 13, and the antisense strand comprises the corresponding antisense strand sequence from Tables 2, 5, 7, 9, 10, or 13.

[0017] In one embodiment, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand forming a double-stranded portion, wherein the antisense strand comprises one of the antisense strand sequences in Table 13, and the sense strand comprises the corresponding sense strand sequence in Table 13.

[0018] In one embodiment, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand forming a double-stranded portion, the sense strand comprising the nucleotide sequence of SEQ ID NO: 61, and the antisense strand comprising the nucleotide sequence of SEQ ID NO: 62. In certain embodiments, the dsRNA compound comprises a conjugate. In certain embodiments, the conjugate is N-acetylgalactosamine (GalNAc). In certain embodiments, the conjugate is bound to the 3' end of the sense strand of the AGT-targeting dsRNA compound. In certain embodiments, the conjugate is bound to the 3' end of the sense strand of the AGT-targeting siRNA compound.

[0019] Certain embodiments provide a method for inhibiting angiotensinogen (AGT) expression in a subject, comprising the step of administering a dsRNA compound described herein to the subject in an amount sufficient to inhibit AGT expression. In certain embodiments, the dsRNA compound inhibits AGT expression by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the dsRNA compound includes siRNA.

[0020] In certain embodiments, pharmaceutical compositions for inhibiting angiotensinogen (AGT) expression in cells are provided, comprising a double-stranded ribonucleic acid (dsRNA) compound described herein, either alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0021] Certain embodiments provide a method for inhibiting the expression of angiotensinogen (AGT) in cells, which includes inhibiting the expression of AGT in cells by contacting the cells with a dsRNA compound or pharmaceutical composition described herein in an amount sufficient to inhibit the expression of AGT.

[0022] Certain embodiments provide a method of treating a RAAS-related disease, disorder, and / or condition in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a dsRNA compound or pharmaceutical composition described herein to treat the RAAS-related disease, disorder, and / or condition in the subject.

[0023] Certain embodiments provide a method of treating a subject having a disease, disorder, and / or condition that would benefit from a reduction in angiotensinogen (AGT) expression, comprising administering to a subject in need thereof a therapeutically effective amount of a dsRNA compound or pharmaceutical composition described herein to treat the subject having a disorder that would benefit from a reduction in AGT expression.

[0024] Certain embodiments provide a method of preventing at least one symptom in a subject having a disorder that would benefit from a reduction in angiotensinogen (AGT) expression, comprising administering to a subject in need thereof a prophylactically effective amount of a dsRNA compound or pharmaceutical composition described herein to prevent at least one symptom in the subject having a disorder that would benefit from a reduction in AGT expression.

[0025] In certain embodiments, a kit is provided that comprises a dsRNA compound or pharmaceutical composition described herein, and optionally a label.

[0026] In one embodiment, a process for preparing the sense and / or antisense strands of a double-stranded ribonucleic acid (dsRNA) compound is provided, the process comprising the following steps: a) preparing the sense and / or antisense strands by sequentially coupling modified and / or unmodified nucleotides via phosphoramidite oligonucleotide synthesis on a solid support; b) optionally, coupling a GalNAc-containing moiety to the sense and / or antisense strands on the solid support via phosphoramidite oligonucleotide synthesis; c) cleaving the sense and / or antisense strands from the solid support and removing the solid support; and d) optionally, further purifying the sense and / or antisense strands using chromatography, if desired.

[0027] In one embodiment, a process for preparing the sense and / or antisense strands of a double-stranded ribonucleic acid (dsRNA) compound is provided, the process comprising the following steps: a) coupling a GalNAc-containing moiety to a solid support via phosphoramidite oligonucleotide synthesis; b) coupling modified and / or unmodified nucleotides to the GalNAc-containing moiety on the solid support via phosphoramidite oligonucleotide synthesis; c) sequentially coupling additional modified and / or unmodified nucleotides via phosphoramidite oligonucleotide synthesis to prepare the sense and / or antisense strands; d) cleaving the sense and / or antisense strands from the solid support and removing the solid support; and e) optionally, further purifying the sense and / or antisense strands using chromatography, if desired.

[0028] In one embodiment, a process for preparing a double-stranded ribonucleic acid (dsRNA) compound is provided, comprising: a) contacting a sense strand prepared according to any of the processes described herein with an antisense strand prepared according to any of the processes described herein in equimolar concentration in solution; b) optionally, heating the solution to a temperature of about 94°C; and c) optionally, lowering the temperature of the solution to about 25°C.

[0029] Certain embodiments provide double-stranded ribonucleic acid (dsRNA) compounds for use in pharmaceuticals.

[0030] Certain embodiments provide double-stranded ribonucleic acid (dsRNA) compounds for use in treating or preventing RAAS-related diseases, disorders, and / or conditions in a subject. [Brief explanation of the drawing]

[0031] [Figure 1] Figure 1 shows the AGT siRNA inhibition screen in human primary hepatocytes after 48 hours of free uptake. [Figure 2] Figure 2 shows the dose-response curve of AGT siRNA to AGT expression (percentage) after 24-hour transfection of Hep3B with AGT siRNA, comparing third-party modifications and ARNATAR modifications of the same sequence without GalNAc. [Figure 3] Figure 3 shows ARNATAR AGT siRNA ATsi483 exhibiting inhibitory activity equivalent to that of reference AGT siRNA ATXL-G in human primary hepatocytes after 42 hours of free uptake. [Figure 4] Figure 4 shows in vivo AGT protein levels in plasma after siRNA administration, measured by ELISA, demonstrating comparable activity and durability of ARNATAR AGT siRNA compared to reference AGT siRNA ATXL-G over a period of >4 weeks. [Figure 5] Figure 5 shows in vivo plasma AGT protein levels after administration of ARNATAR AGT siRNA with further modification, demonstrating activity and durability comparable to the reference AGT siRNA ATXL-G over a 4-week period. [Figure 6] Figure 6 shows the in vitro inhibitory activity of ARNATAR-modified AGT siRNA and reference ATXL-G by human primary hepatocytes via 42-hour free uptake. [Figure 7]Figure 7 shows the in vivo plasma AGT protein levels of some ARNATAR AGT siRNAs that exhibit low activity in vitro but activity equivalent to or better than reference ATXL-G in vivo. [Figure 8] Figure 8 shows the dose-response curves of AGT siRNAs for AGT expression (percentage) after transfection of Hep3B with ARNATAR AGT siRNAs for 8 or 12 hours, demonstrating better activity than reference ATXL-G for some ARNATAR siRNAs. [Figure 9] Figure 9 shows the percentage of AGT protein levels in mouse plasma at different time points after administration of siRNA at 3 mg / kg, showing similar or better activity at some time points compared to the reference ATXL-G. [Figure 10] Figure 10 shows the percentage of AGT protein levels in mouse plasma of AGT-AAV transgenic mice at different time points after administration of siRNA at 3 mg / kg, with compound ATsi786 showing potent knockdown compared to reference ATXL-G. Detailed explanation

[0032] It should be understood that both the general description above and the detailed description below are illustrative and descriptive only and do not limit the claimed invention. In this specification, the use of the singular includes the plural unless otherwise specified. As used herein, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" include both elements and components containing one unit, and elements and components containing multiple subunits, unless otherwise specified.

[0033] Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. All documents, or parts thereof, including but not limited to patents, patent applications, articles, books, and papers, cited herein are expressly incorporated herein by reference, with respect to the parts and all thereof discussed herein.

[0034] definition Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK accession numbers, and relevant sequence information and other data referred to throughout the disclosure herein, available through databases such as the National Center for Biotechnology Information (NCBI), are incorporated by reference in the parts of the documents discussed herein, as well as in whole.

[0035] Unless otherwise specified, the following terms have the following meanings: "2'-O-methoxyethyl" (also known as 2'-MOE and 2'-O(CH2)2-OCH3) refers to the O-methoxyethyl modification at the 2' position of the furanose ring. 2'-O-methoxyethyl modified sugars are modified sugars.

[0036] "2'-MOE nucleoside" (or 2'-O-methoxyethyl nucleoside) refers to a nucleoside containing a 2'-MOE modified sugar moiety. "2'-MOE nucleotide" (or 2'-O-methoxyethyl nucleotide) refers to a nucleotide containing a 2'-MOE modified sugar moiety.

[0037] "2'-O-methyl" (also 2'-OCH3 and 2'-OMe) refers to the O-methyl modification at the 2' position of the furanose ring. 2'-O-methyl-modified sugars are modified sugars.

[0038] "2'-OMe nucleoside" (or 2'-O-methyl nucleoside) refers to a nucleoside containing a 2'-OMe modified sugar moiety. "2'-OMe nucleotide" (or 2'-O-methyl nucleotide) refers to a nucleotide containing a 2'-OMe modified sugar moiety.

[0039] "2'-substituted nucleoside" means a nucleoside having a substituent at the 2' position of a furanose ring other than H or OH. In certain embodiments, 2'-substituted nucleosides include fluoro(2'-F), O-methyl(2'-OMe), O-methoxyethyl(2'-MOE), or nucleosides having bicyclic sugar modifications.

[0040] "5-methylcytosine" refers to cytosine with a methyl group attached at the 5th position. 5-methylcytosine is a modified nucleic acid base.

[0041] "Approximately" means within ±7% of the value. For example, if it says, "The compound affected at least approximately 70% inhibition of mRNA," it implies that mRNA levels were inhibited within the range of 63% and 77%.

[0042] "Animals" refers to humans or non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates such as monkeys and chimpanzees.

[0043] "Antibody" refers to a complete antibody molecule or any fragment or part thereof, such as the heavy chain, light chain, Fab portion, and Fc portion.

[0044] "Antisense oligonucleotide" or "ASO" means a single-stranded oligonucleotide having a nucleic acid base sequence that enables hybridization to a corresponding region or segment of a target nucleic acid. In certain embodiments, the antisense oligonucleotide comprises one or more ribonucleoside (RNA) residues and / or deoxyribonucleoside (DNA) residues.

[0045] "Base complementarity" refers to the ability of nucleic acid bases of oligonucleotides to form base pairs (i.e., hybridize) with corresponding nucleic acid bases in a target nucleic acid, mediated by Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds between the corresponding nucleic acid bases. Base complementarity also refers to canonical (e.g., A:U, A:T, C:G) or non-canonical base pairing (e.g., A:G, A:U, G:U, I:U, I:A, I:C).

[0046] A "dicyclic sugar" refers to a furanose ring modified by a bridge between two non-geminal carbon atoms. Dicyclic sugars are modified sugars.

[0047] "Cap structure" or "terminal cap portion" refers to a chemical modification incorporated into one of the ends of an oligomeric compound.

[0048] "Chemical modification" refers to the modification of the molecular structure or elements of a naturally occurring molecule. For example, since siRNA compounds are composed of linked ribonucleosides (sometimes referred to as RNA in this specification), the substitution of ribonucleosides with deoxyribonucleosides (sometimes referred to as DNA in this specification) is considered a chemical modification of an siRNA compound.

[0049] A "chemically different portion" refers to a part of an oligomer compound that is chemically different in some way from other parts of the same oligomer compound. For example, the portion containing a 2'-OMe nucleotide is chemically different from the portion containing a nucleotide that does not have the 2'-OMe modification.

[0050] A “chimeric oligomer compound” means an oligomer compound having at least two chemically distinct parts, each part having multiple subunits. For example, as disclosed herein, an siRNA may include a peripheral part and a central part. The peripheral part includes a motif having various modified or unmodified nucleic acid bases to confer increased stability, specificity, safety, and potency, while the central part includes various modified or unmodified nucleic acid bases to function as a substrate for RISC-mediated degradation.

[0051] "Complementarity" refers to the ability of nucleic acid bases of a first nucleic acid and a second nucleic acid to form pairs.

[0052] In the context of therapy, "to comply" means that the patient adheres to the recommended therapy.

[0053] "Comprise," "comprises," and "comprising" are understood to mean the inclusion of the listed members without the exclusion of other members (e.g., not listed). For example, the inclusion of a listed step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements.

[0054] "Consecutive nucleic acid bases" refers to nucleic acid bases that are adjacent to each other.

[0055] "Deoxyribonucleotide" refers to a nucleotide having a hydrogen atom at the 2' position of the sugar portion of the nucleotide. The sequence of a deoxyribonucleotide may be referred to herein as "DNA". Deoxyribonucleotides may be referred to herein as "DNA nucleotide", "d nucleotide", or "D". Deoxyribonucleotides may be modified with various substituents.

[0056] In the context of oligomeric compounds, "design" or "engineered" refers to the process of creating / engineering an oligomeric compound that specifically hybridizes with a target nucleic acid molecule. Design generally includes, for example, providing mutations and / or modifications to the native sequence.

[0057] "Efficacy" refers to the ability to produce a desired effect.

[0058] "Expression" generally encompasses all functions in which the encoded information of a gene is translated into structures that exist and function within a cell. Such structures include, but are not limited to, the products of transcription and / or translation. Within this disclosure, expression refers to proteins as products of expression.

[0059] "Completely complementary" or "100% complementary" means that each nucleic acid base of the first nucleic acid molecule has a complementary nucleic acid base in the second nucleic acid molecule. In certain embodiments, the first nucleic acid molecule is an oligomeric compound, and the target nucleic acid molecule is the second nucleic acid molecule.

[0060] A "fully modified motif" refers to an oligomeric compound containing a sequence of consecutive nucleosides, each of which has a chemical modification.

[0061] "GalNAc" refers to the compound N-acetylgalactosamine or a compound containing an N-acetylgalactosamine compound. The GalNAc-containing portion (or "GalNAc" or "GalNAc portion" as used herein) typically describes a compound containing at least one N-acetylgalactosamine compound bonded to one or more spacers and / or linkers for binding to an oligonucleotide compound.

[0062] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, combinations of oligomeric compounds and nucleic acid molecular targets. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, combinations of siRNA and nucleic acid molecular targets, particularly mRNA target molecules.

[0063] Terms such as "induce," "inhibit," "enhance," "increase," "boost," and "decrease" generally describe actions that result in a quantitative difference between two states.

[0064] "Inhibiting expression or activity" refers to reducing or blocking expression or activity, and does not necessarily mean completely eliminating expression or activity.

[0065] "Nucleoside bond" refers to a chemical bond between two adjacent nucleosides. This bond may be a naturally occurring bond, i.e., a phosphate bond, or an artificial bond, such as a phosphorothioate (also known as thiophosphate or PS) bond.

[0066] "Linked nucleosides" refers to adjacent nucleosides linked by nucleoside bonds (e.g., A, G, C, T, U, and I). Examples of linked nucleosides include sequences of deoxyribonucleosides (sometimes referred to herein as DNA) or sequences of ribonucleosides (sometimes referred to herein as RNA).

[0067] A "mismatch" or "non-complementary nucleic acid base" refers to a situation where the nucleic acid bases of the first nucleic acid cannot pair with the corresponding nucleic acid bases of the second or target nucleic acid via Watson-Crick base pairing (e.g., A:T, A:U, C:G).

[0068] "Modified nucleoside bonds" refer to substitutions or modifications to naturally occurring nucleoside bonds (i.e., phosphodiester nucleoside bonds).

[0069] "Modified nucleic acid bases" refer to any nucleic acid base other than adenine, cytosine, guanine, thymidine, or uracil. "Unmodified nucleic acid bases" refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0070] "Modified nucleoside" means a nucleoside having a modified sugar moiety and / or modified nucleic acid base. As used herein, if the oligomer compound is RNA-based, the substitution of a ribonucleoside with a deoxyribonucleoside (sometimes referred to herein as a DNA nucleoside) is considered a modification of the oligomer compound. Similarly, if the oligomer compound is DNA-based, the substitution of a deoxyribonucleoside with a ribonucleoside (sometimes referred to herein as an RNA nucleoside) is considered a modification of the oligomer compound.

[0071] "Modified nucleotide" means a nucleotide having at least one of the following: a modified sugar moiety, a modified nucleoside bond, a deoxyribonucleoside (sometimes referred to as a DNA nucleotide) substitution for a ribonucleoside (sometimes referred to as an RNA nucleotide) as specified, a ribonucleoside (sometimes referred to as an RNA nucleoside) substitution for a deoxyribonucleoside (sometimes referred to as a DNA nucleoside) as specified, and a modified nucleic acid base.

[0072] "Modified oligonucleotide" means an oligonucleotide comprising at least one of the following: modified nucleoside bonds, modified sugars, deoxyribonucleoside (sometimes referred to as DNA nucleoside) substitutions for ribonucleosides (sometimes referred to as RNA nucleosides) in this specification, ribonucleoside (sometimes referred to as RNA nucleosides) substitutions for deoxyribonucleosides (sometimes referred to as DNA nucleosides) in this specification, and / or modified nucleic acid bases.

[0073] "Modified sugar" refers to the substitution and / or any alteration of the natural sugar portion of a nucleotide found in DNA or RNA.

[0074] "Part" refers to one of the parts into which something is divided, that is, a part or component of something. For example, the sugar portion of a nucleotide is the sugar component of the nucleotide.

[0075] A "monomer" refers to a single unit of an oligomer or a single unit for forming an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

[0076] "Motif" refers to a pattern of modification in an oligomeric compound. For example, as disclosed herein, ARNATAR-designed oligomeric compounds include motifs having various modified nucleic acid bases and nucleoside bonds to improve the delivery, stability, specificity, safety, and potency of the compound.

[0077] "Natural sugar (part)" or generally "sugar (part)" refers to the sugar (part) found in DNA(2'-H) or RNA(2'-OH), namely 2-deoxy-beta-D-ribofuranose or beta-D-ribofuranose, respectively.

[0078] "Naturally occurring nucleoside bonds" refers to phosphodiester bonds from 3' to 5'.

[0079] "Non-complementary nucleic acid bases" refer to pairs of nucleic acid bases that do not form hydrogen bonds with each other or do not support hybridization.

[0080] "Nucleic acid (molecule)" refers to a sequence of monomeric nucleotides. Nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), messenger RNA (mRNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering ribonucleic acid (siRNA), hairpin ribonucleic acid (shRNA), and microRNA (miRNA).

[0081] "Nucleic acid base" means any unmodified nucleic acid base as defined above, any modified nucleic acid base, and / or any artificial nucleic acid base which may be any heterocyclic moiety that can form pairs with a base of a nucleic acid molecule.

[0082] "Nucleic acid base complementarity" refers to the ability of a nucleic acid base to form a base pair with another nucleic acid base (also known as being complementary). If a nucleic acid base at a specific position in an oligomeric compound can form a hydrogen bond with a nucleic acid base at a specific position in a target nucleic acid molecule, the hydrogen bond positions between the oligomeric compound and the target nucleic acid molecule are considered complementary in that nucleic acid base pair. For example, in DNA, adenine (A) is complementary to thymine (T), in RNA, adenine (A) is complementary to uracil (U), and guanine (G) is complementary to cytosine (C) in both DNA and RNA. Base pairs, or complementary nucleic acid bases, are usually canonical Watson-Crick base pairs (C:G, A:U, A:T), but also include Hoogsteen base pairs (e.g., A:G, A:U), fluctuation base pairs (e.g., G:U, I:U, I:A, I:C, where I is hypoxanthine), etc. Nucleoside complementarity facilitates the hybridization of the oligomeric compounds described herein to their target nucleic acids.

[0083] "Nucleic acid base sequence" refers to a sequence of nucleic acid bases independent of any sugars, bonds, and / or nucleic acid base modifications.

[0084] "Nucleoside" refers to a nucleic acid base linked to a natural or modified sugar as defined above.

[0085] "Nuclide mimetic" includes structures used to substitute a sugar or a sugar-base at one or more positions in an oligomeric compound, but not necessarily a bond. Examples include morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclo sugar mimetic, such as nucleoside mimetic with a non-furanose sugar unit. Nucleotide mimetic includes structures used to substitute a nucleoside and a bond at one or more positions in an oligomeric compound. Examples include peptide nucleic acids or morpholino (-N(H)-C(=O)-O- or morpholino linked by other non-phosphodiester bonds). Sugar substitutes overlap with the somewhat broader term nucleoside mimetic, but are intended to indicate substitution of only a sugar unit (furanose ring). The tetrahydropyranyl ring provided herein is an example of a sugar substitute in which a furanose sugar group is replaced by a tetrahydropyranyl ring system. A "mimetic" refers to a group that is substituted in place of a sugar, a nucleic acid base, and / or a nucleoside bond. Generally, mimetic groups are used in place of a sugar or a combination of sugar-nucleoside bonds, while the nucleic acid base is retained for hybridization to a selected target.

[0086] A "nucleotide" refers to a nucleoside that has a binding group (e.g., a phosphate (p) or phosphorothioate (PS) group) covalently bonded to the sugar portion of the nucleoside. Nucleotides include ribonucleotides and deoxyribonucleotides that have phosphate and / or phosphorothioate bonds. Linked sequences of nucleotides, such as ribonucleotides, deoxyribonucleotides, and / or mixtures thereof, form oligonucleotides.

[0087] "Off-target effects" refer to undesirable or harmful biological effects related to the regulation of RNA or protein expression of genes other than the intended target nucleic acid.

[0088] "Oligomer activity" refers to any detectable or measurable activity resulting from the hybridization of an oligomeric compound to its target nucleic acid. In certain embodiments, oligomeric activity is measured as a decrease in the amount or expression of the target nucleic acid. Oligomer activity can be modulated by oligomeric compounds such as dsRNA. Oligomer activity can be modulated by oligomeric compounds such as siRNA.

[0089] An "oligomeric compound" means a compound of linked monomer subunits (also known herein as "subunits") that can undergo hybridization to at least a region of a target nucleic acid via hydrogen bonding. Monomer subunits are, in particular, modified or unmodified nucleotides or nucleosides, and oligomeric compounds are, in particular, oligonucleotides. Oligomers serve as templates for RISC to recognize complementary messenger RNA (mRNA) transcripts and cleave specific mRNA transcripts as targets. Cleavage of the target mRNA blocks translation of the target mRNA and silences the target gene. Examples of oligomeric compounds include single-stranded and double-stranded compounds, e.g., antisense oligonucleotides, ssRNA, siRNA, shRNA, and miRNA. Generally, oligomeric compounds are distinguished from polymeric compounds based on the number of monomers, with oligomers often having about 5 to about 100 monomer units.

[0090] "Oligomer inhibition" refers to a decrease in the level of a target nucleic acid (e.g., mRNA) in the presence of an oligomeric compound complementary to the target nucleic acid, compared to the level of the target nucleic acid in the absence of the oligomeric compound.

[0091] The "oligomeric mechanism" includes RISC or RNase H-related mechanisms involving hybridization between oligomeric compounds and target nucleic acids (e.g., mRNA), with the results or effects of hybridization being targeted degradation and inhibition of gene expression.

[0092] As used herein, “olivone molecule” means a sequence of linked nucleosides and / or nucleotides, each of which may be independently modified or unmodified. Olivone molecules may have linking groups other than phosphate groups (e.g., phosphorothioate groups) used as linking portions between nucleosides. In certain embodiments, oligonucleotides comprise one or more ribonucleoside (RNA) residues and / or deoxyribonucleoside (DNA) residues.

[0093] A "phosphorothioate bond" or "PS" refers to a nucleoside bond modified by replacing one of the non-bridged oxygen atoms with a sulfur atom in a phosphodiester bond. A phosphorothioate bond is a modified nucleoside bond.

[0094] A “part” refers to a defined number of consecutive (i.e., linked) nucleic acid bases of a nucleic acid. In certain embodiments, a part is a defined number of consecutive nucleic acid bases of a target nucleic acid. In certain embodiments, a part is a defined number of consecutive nucleic acid bases of an oligomer compound.

[0095] A “region” is defined as a portion of a target nucleic acid having at least one identifiable structure, function, or characteristic.

[0096] RNA, or ribonucleic acid, is composed of ribose nucleotides or ribonucleotides (nitrogen-containing bases linked to ribose sugar) linked by phosphodiester bonds, forming sequences of various lengths. The nitrogen-containing bases of RNA are adenine, guanine, cytosine, and uracil.

[0097] "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of the sugar portion of the nucleotide. Ribonucleotides can be modified with various substituents and may be linked by covalent bonds other than naturally occurring phosphodiesters such as phosphorothioates. Ribonucleotides may be referred to herein as RNA, "R" or "r".

[0098] A "segment" is defined as a smaller or sub-part of a region within a nucleic acid molecule, particularly a target nucleic acid.

[0099] As used herein, “site” is defined as a nucleic acid, and more particularly as a specific nucleic acid base position within a target nucleic acid.

[0100] "Specifically hybridizable" refers to an oligomeric compound that exhibits a sufficient degree of complementarity between the oligomeric compound (e.g., siRNA) and the target nucleic acid (e.g., mRNA) to induce the desired effect under conditions where specific binding is desired, i.e., physiological conditions in in vivo assays and therapeutic treatments, while showing minimal or no effect on non-target nucleic acids.

[0101] "Strict hybridization conditions" or "strict conditions" refer to conditions under which an oligomeric compound hybridizes to its target nucleic acid with minimal hybridization to other nucleic acid molecules.

[0102] "Subject" means human or non-human animal, in particular human or non-human animal selected for treatment or therapy.

[0103] "Target" generally refers to a protein or nucleic acid molecule whose regulation is desired. As used herein, "target" specifically refers to a nucleic acid molecule (e.g., mRNA) whose regulation is desired.

[0104] A "target gene" refers to a gene that codes for a target.

[0105] "Targeting" refers to the process of designing and selecting oligomeric compounds that specifically hybridize with a target nucleic acid and induce the desired effect.

[0106] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" all refer to nucleic acids that can be targeted by oligomeric compounds.

[0107] The "target region" refers to the portion of a target nucleic acid that is targeted by one or more oligomeric compounds.

[0108] The "target segment" refers to the nucleotide sequence of the target nucleic acid to which the oligomer compound is targeted. The "5' target site" refers to the nucleotide at the 5' end of the target segment. The "3' target site" refers to the nucleotide at the 3' end of the target segment. In one embodiment, the target segment is at least 13 nucleic acid bases (i.e., at least 13 consecutive nucleic acid bases) of the target region to which the oligomer compound is targeted.

[0109] "Therapeutic response" or "therapeutic effectiveness" refers to the efficacy of a compound or composition, such as an oligomeric compound, described herein in a therapeutic application. Therapeutic response can be increased by improvements in the delivery, stability, specificity, safety, and / or potency of the therapeutic compound.

[0110] "Unmodified" RNA nucleic acid bases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). "Unmodified" DNA nucleic acid bases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T) and cytosine (C). In certain embodiments, unmodified RNA nucleic acid bases are considered modified if DNA nucleic acid bases are substituted for RNA nucleic acid bases in an oligomeric compound such as an siRNA compound. In certain embodiments, unmodified DNA nucleic acid bases are considered modified if RNA nucleic acid bases are substituted for DNA nucleic acid bases in a DNA sequence.

[0111] In this specification, "unmodified nucleoside" means a nucleoside composed of naturally occurring nucleic acid bases and naturally occurring sugar moieties. In certain embodiments, the unmodified nucleoside is an RNA nucleoside in an oligomeric compound such as an siRNA compound.

[0112] In this specification, "unmodified nucleotide" means a nucleotide consisting of a naturally occurring nucleic acid base, a naturally occurring sugar moiety, and a nucleoside bond, the nucleotide bond may be a naturally occurring bond (i.e., a phosphate bond) or an artificial bond (e.g., a phosphorothioate bond). In certain embodiments, the unmodified nucleotide is an RNA nucleotide in an oligomeric compound such as an siRNA compound.

[0113] This specification discloses improved compounds that target nucleic acids encoding angiotensinogen (AGT) and have an Advanced RNA Targeting (ARNATAR) design that enhances gene silencing activity. In certain embodiments, the compound is a double-stranded ribonucleic acid (dsRNA) compound. In one embodiment, the dsRNA compound is a short hairpin RNA (shRNA) or a small interfering RNA (siRNA) compound.

[0114] In some embodiments, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand, forming a double-stranded portion, where the antisense strand comprises one of any one antisense nucleotide sequences from Tables 2, 5, 7, 9, 10, or 13. In certain embodiments, the dsRNA compound is a small interfering RNA (siRNA) compound, where the antisense strand comprises one of any one antisense nucleotide sequences from Tables 2, 5, 7, 9, 10, or 13. In certain embodiments, the siRNA compound comprises an oligomeric sequence shown in Tables 2, 5, 7, 9, 10, or 13. In certain embodiments, the oligomeric sequence comprises a portion of any sequence shown in Tables 2, 5, 7, 9, 10, or 13 with a length of 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleosides. In some embodiments, the dsRNA (e.g., siRNA) compound comprises an antisense strand of any one of the antisense nucleotide sequences in Table 13. In some embodiments, the dsRNA (e.g., siRNA) compound comprises an antisense strand of any one of the antisense nucleotides of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 28, 30, 32, 35, 37, 39, 42, 44, 46, 47, 50, 52, and 54. In a particular embodiment, the dsRNA (e.g., siRNA) compound comprises an antisense strand of the nucleotide sequence of SEQ ID NO: 62. In one embodiment, the dsRNA (e.g., siRNA) compound comprises a portion of the sequence of SEQ ID NO: 62 with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In a preferred embodiment, the dsRNA (e.g., siRNA) compound comprises an antisense strand of the sequence of SEQ ID NO: 9 or 19, or a portion thereof with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides.

[0115] In one embodiment, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand, forming a double-stranded portion, where the sense strand comprises one of any one sense nucleotide sequence from Tables 2, 5, 7, 9, 10, or 13. In a particular embodiment, the dsRNA compound is an siRNA compound, where the sense strand comprises one of any one sense nucleotide sequence from Tables 2, 5, 7, 9, 10, or 13. In a particular embodiment, the siRNA compound comprises an oligomeric sequence shown in Tables 2, 5, 7, 9, 10, or 13. In a particular embodiment, the oligomeric sequence comprises a portion of any sequence shown in Tables 2, 5, 7, 9, 10, or 13 with a length of 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleosides. In some embodiments, the dsRNA (e.g., siRNA) compound comprises a sense strand nucleotide sequence from Table 13. In some embodiments, the dsRNA (e.g., siRNA) compound comprises a sense strand of any one of the sense nucleotides of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 27, 29, 31, 33, 34, 36, 38, 40, 41, 43, 45, 48, 49, 51, 53, and 55. In a particular embodiment, the dsRNA (e.g., siRNA) compound comprises the sense strand nucleotide sequence of SEQ ID NO: 61. In one embodiment, the dsRNA (e.g., siRNA) compound comprises a portion of the sequence of SEQ ID NO: 61 with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In a preferred embodiment, the siRNA compound comprises a sense strand of the sequence of SEQ ID NOs: 8, 33, or 55, preferably 55, or a portion thereof with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides.

[0116] In one embodiment, a double-stranded ribonucleic acid (dsRNA) compound for inhibiting angiotensinogen (AGT) expression in cells comprises a sense strand and an antisense strand, forming a double-stranded portion, wherein the sense strand comprises one of any one sense nucleotide sequence from Tables 2, 5, 7, 9, 10, or 13, and the antisense strand comprises one of any one antisense nucleotide sequence from Tables 2, 5, 7, 9, 10, or 13. In a particular embodiment, the dsRNA compound is an siRNA compound, wherein the sense strand comprises one of any one sense nucleotide sequence from Tables 2, 5, 7, 9, 10, or 13, and the antisense strand comprises one of any one antisense nucleotide sequence from Tables 2, 5, 7, 9, 10, or 13. In a particular embodiment, the sense strand or antisense strand comprises a portion of any sequence shown in Tables 2, 5, 7, 9, 10, or 13 with a length of 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleosides. In certain embodiments, the dsRNA (e.g., siRNA) compound comprises a sense strand of the nucleotide sequence of SEQ ID NO: 61 and an antisense strand of the nucleotide sequence of SEQ ID NO: 62. In one embodiment, the dsRNA (e.g., siRNA) compound comprises a portion of the sequence of SEQ ID NO: 61 and / or 62 with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In preferred embodiments, the dsRNA (e.g., siRNA) compound comprises the antisense sequence of SEQ ID NO: 9 or 19 and the sense sequence of SEQ ID NO: 8, 33, or 55, or a portion thereof with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In a preferred embodiment, the sense strand comprises the sequence of SEQ ID NO: 55, or a portion thereof with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides, and the antisense strand comprises the sequence of SEQ ID NO: 9 or 19, or a portion thereof with a length of 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides.

[0117] In certain embodiments, the dsRNA contains at least one modified nucleotide. In another embodiment, substantially all of the nucleotides in the sense strand are modified; substantially all of the nucleotides in the antisense strand are modified; or substantially all of the nucleotides in both the sense strand and the antisense strand are modified. "Substantially all" means that at least about 70%, 75%, 80%, 85%, 90%, or 95% of the nucleosides are modified. In yet another embodiment, all of the nucleotides in the sense strand are modified; all of the nucleotides in the antisense strand are modified; or all of the nucleotides in both the sense strand and the antisense strand are modified. The modifications are preferably selected from the group consisting of phosphorothioate nucleotide (PS) bonds, furanose ring modifications (e.g., 2'-substituted nucleosides including fluoro(2'-F), O-methyl(2'-OMe), O-methoxyethyl(2'-MOE), H(deoxyribonucleotide), or nucleosides having bicyclic sugar modifications), and combinations thereof.

[0118] In certain embodiments, the dsRNA comprises a chain containing at least one phosphorothioate nucleotide (PS) bond. In certain embodiments, the dsRNA comprises a chain containing a phosphorothioate nucleotide (PS) bond adjacent to a deoxyribonucleoside (D) or ribonucleoside (R). In certain embodiments, the dsRNA comprises a phosphorothioate nucleotide (PS) bond adjacent to a deoxyribonucleoside (D) or ribonucleoside (R) at the 5' end, 3' end, or both sides. In certain embodiments, the dsRNA comprises a chain containing a phosphorothioate nucleotide (PS) bond adjacent to two nucleosides at the 5' end of the chain and / or two nucleosides at the 3' end of the chain.

[0119] In certain embodiments, the dsRNA includes siRNA. In preferred embodiments, the siRNA includes one of the ARNATAR-designed siRNAs listed in any one of Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the dsRNA includes the nucleotide sequence and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55). In preferred embodiments, the siRNA includes the nucleotide sequence and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55).

[0120] dsRNA can be transported into target cells in various ways. In certain embodiments, dsRNA compounds enter cells via viral delivery vectors, lipid-based delivery, polymer-based delivery, and / or conjugate-based delivery.

[0121] In certain embodiments, the dsRNA compounds described herein further include a conjugate moiety. In certain embodiments, the siRNAs described in any one of Tables 2, 5, 7, 9, 10, or 13 include a conjugate moiety. The conjugate can be selected from cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In preferred embodiments, the conjugate moiety is an N-acetylgalactosamine (GalNAc)-containing moiety. The GalNAc-containing moiety is generally understood to include at least one GalNAc compound and a linker for attachment to the dsRNA. In one embodiment, the conjugate can be attached to the 3' end of the sense strand. In preferred embodiments, the dsRNA compound is an siRNA conjugated to a GalNAc-containing moiety.

[0122] Certain embodiments disclosed herein provide compounds comprising siRNA for inhibiting AGT expression in cells, wherein the siRNA comprises a sense strand and an antisense strand forming a double helix, and the sense strand (ATs786 or ATs787, SEQ ID NO: 55, shown in Table 13) comprises the following formula: JPEG2026520610000002.jpg126159

[0123] Certain embodiments disclosed herein provide compounds comprising siRNA for inhibiting AGT expression in cells, wherein the siRNA comprises an antisense strand and an antisense strand forming a double helix, the antisense strand (ATa786, SEQ ID NO: 9, shown in Table 13) comprises the following formula: JPEG2026520610000003.jpg82159

[0124] Specific embodiments disclosed herein provide compounds comprising siRNA for inhibiting AGT expression in cells, wherein the siRNA comprises an antisense strand and an antisense strand forming a double helix, the antisense strand (ATa787, SEQ ID NO: 19, shown in Table 13) comprises the following formula: JPEG2026520610000004.jpg82159

[0125] Certain embodiments disclosed herein provide compounds comprising siRNA for inhibiting AGT expression in cells, wherein the siRNA comprises a sense strand and an antisense strand forming a double helix, and the sense strand (ATs786, Sequence ID: 55, shown in Table 13) comprises the following formula: JPEG2026520610000005.jpg123159 and the antisense chain (ATa786, sequence number: 9, shown in Table 13) contain the following formula: JPEG2026520610000006.jpg82159

[0126] Certain embodiments disclosed herein provide compounds comprising siRNA for inhibiting AGT expression in cells, wherein the siRNA comprises a sense strand and an antisense strand forming a double helix, and the sense strand (ATs787, Sequence ID: 55, shown in Table 13) comprises the following formula: JPEG2026520610000007.jpg118159 and the antisense chain (ATa787, sequence number: 19, shown in Table 13) contain the following formula: JPEG2026520610000008.jpg89159

[0127] In certain embodiments, the compounds or compositions disclosed herein include salts of dsRNA. In certain embodiments, the compounds or compositions disclosed herein include salts of siRNA disclosed in Tables 2, 5, 7, 9, 10, and 13.

[0128] Certain embodiments provide a method for inhibiting the expression of angiotensinogen (AGT) in cells, comprising contacting cells with a dsRNA compound or pharmaceutical composition described herein in an amount sufficient to inhibit the expression of AGT, thereby inhibiting the expression of AGT in cells. In certain embodiments, the compound or composition comprises a dsRNA that inhibits the expression of angiotensinogen (AGT) in cells by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the dsRNA is an siRNA that inhibits the expression of angiotensinogen (AGT) by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, compounds or compositions that inhibit the expression of AGT are provided in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the sense strand of the siRNA comprises the nucleotide sequence and chemical modifications of SEQ ID NO: 8, 33, or 55. In a preferred embodiment, the antisense strand of the siRNA comprises the nucleotide sequence and chemical modifications of SEQ ID NO: 9 or 19. In a more preferred embodiment, the siRNA comprises the nucleotide sequence and chemical modifications of ATsi786 (SEQ ID NO: 9 and 55) or ATsi787 (SEQ ID NO: 19 and 55). The amount of angiotensinogen (AGT) expression inhibition can generally be measured by evaluating the mRNA and / or protein levels in cells after contact with a dsRNA compound or pharmaceutical composition and comparing the mRNA or protein levels with those of cells that were not contacted with the dsRNA.

[0129] In certain embodiments, siRNA compounds that inhibit angiotensinogen (AGT) expression are provided in Tables 2, 5, 7, 9, 10, or 13 and inhibit AGT expression by at least about 85%. In preferred embodiments, the siRNA comprises the nucleotide sequence and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55). The amount of angiotensinogen (AGT) expression inhibition can generally be measured by evaluating mRNA and / or protein levels in cells after contact with a dsRNA compound or pharmaceutical composition and comparing the mRNA or protein levels with those of cells that were not contacted with the dsRNA.

[0130] In certain embodiments, siRNA compounds that inhibit angiotensinogen (AGT) expression are provided in Tables 2, 5, 7, 9, 10, or 13 and inhibit AGT expression by at least about 90%. In preferred embodiments, the siRNA comprises the nucleotide sequence and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55). The amount of angiotensinogen (AGT) expression inhibition can generally be measured by evaluating mRNA and / or protein levels in cells after contact with a dsRNA compound or pharmaceutical composition and comparing the mRNA or protein levels with those of cells that were not contacted with the dsRNA.

[0131] Certain embodiments of the present invention provide assays for determining the level of AGT inhibition in a sample from a subject. In certain embodiments, the AGT assay comprises: a) administering a compound or composition disclosed herein to a subject in an amount sufficient to inhibit AGT expression; b) collecting a sample from the subject; c) determining the amount of AGT protein present in the sample; thereby determining the amount of AGT inhibition by the compound or composition. In certain embodiments, the sample is from blood, serum, urine, and / or liver. In certain embodiments, the amount of AGT protein present in the sample is determined by isolating the AGT protein from the sample, Western blotting the protein, and probing it with an AGT-specific monoclonal antibody to assess the amount of AGT protein present. In certain embodiments, the amount of AGT protein present in the sample is determined using ELISA.

[0132] In certain embodiments, a pharmaceutical composition for inhibiting angiotensinogen (AGT) expression in cells comprises dsRNA alone or in combination with a pharmaceutically acceptable carrier, diluent, and / or excipient. In certain embodiments, the dsRNA is in a buffer. The buffer may include acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer is phosphate-buffered saline (PBS). In certain embodiments, the dsRNA in the pharmaceutical composition is siRNA. In certain embodiments, the siRNA sequence is as provided in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA comprises the nucleotide sequence and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55).

[0133] Specific embodiments of the present invention provide a method for treating renin-angiotensin-aldosterone system (RAAS) related diseases, disorders and / or conditions in a subject, comprising administering to a subject in need a therapeutically effective amount of a compound or composition disclosed herein in an amount sufficient to inhibit AGT expression, thereby inhibiting AGT expression in the subject and treating the RAAS related disease, disorder and / or condition in the subject. In specific embodiments, compounds or compositions that inhibit AGT expression are provided in Tables 2, 5, 7, 9, 10 or 13. In preferred embodiments, the siRNA comprises the nucleotide sequences of SEQ ID NOs: 61 and 62. In more preferred embodiments, the siRNA comprises the nucleotide sequences and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55).

[0134] Specific embodiments of the present invention provide a method for treating RAAS-related diseases, disorders and / or conditions in a subject, comprising administering to a subject in need a therapeutically effective amount of a compound containing an ARNATAR-designed dsRNA listed in Tables 2, 5, 7, 9, 10, or 13, or a pharmaceutical composition containing a dsRNA listed in Tables 2, 5, 7, 9, 10, or 13, in an amount sufficient to inhibit AGT expression, thereby treating the RAAS-related diseases, disorders and / or conditions in the subject. In specific embodiments, the dsRNA for treating RAAS-related diseases, disorders and / or conditions in a subject is an siRNA listed in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA comprises the nucleotide sequences of SEQ ID NOs: 61 and 62. In even more preferred embodiments, the siRNA comprises the nucleotide sequences and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55).

[0135] In certain embodiments, symptoms of RAAS-related diseases, disorders, and / or conditions are treated in a subject requiring treatment, which includes administering to the subject in need a therapeutically effective amount of a compound containing an ARNATAR-designed dsRNA listed in Table 2, 5, 7, 9, 10, or 13, or a pharmaceutical composition containing a dsRNA listed in Table 2, 5, 7, 9, 10, or 13, in an amount sufficient to inhibit AGT expression, thereby treating the symptoms of RAAS-related diseases, disorders, and / or conditions. In certain embodiments, the dsRNA for treating symptoms in a subject is an siRNA listed in Table 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA comprises the nucleotide sequences of SEQ ID NOs: 61 and 62. In even more preferred embodiments, the siRNA comprises the nucleotide sequences and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55).

[0136] In certain embodiments, RAAS-related diseases, disorders and / or conditions include hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertension associated with low plasma renin activity or plasma renin concentration, increased intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, and atherosclerosis. The following conditions are selected from the group consisting of: rheumatoid artery disease, arteriosclerosis, vascular disorders, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), impaired glucose tolerance, type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), and metabolic syndrome. In a preferred embodiment, the RAAS-related disease, disorder, and / or condition is hypertension.

[0137] Specific embodiments of the present invention provide a method for treating hypertension in a subject, comprising administering to a subject in need a therapeutically effective amount of a compound containing an ARNATAR-designed dsRNA as described in Tables 2, 5, 7, 9, 10, or 13, or a pharmaceutical composition containing a dsRNA as described in Tables 2, 5, 7, 9, 10, or 13, in an amount sufficient to inhibit AGT expression, thereby treating hypertension in the subject. In specific embodiments, the dsRNA for treating hypertension in a subject is an siRNA as described in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA comprises the nucleotide sequences of SEQ ID NOs: 61 and 62. In even more preferred embodiments, the siRNA comprises the nucleotide sequences and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55). Types of hypertension include essential, secondary, resistant, malignant, and isolated hypertension.

[0138] In certain embodiments, the subject requiring treatment with the compounds or compositions disclosed herein is a human subject. In certain embodiments, the subject has a systolic blood pressure of at least 130 mmHg and a diastolic blood pressure of at least 80 mmHg; or a systolic blood pressure of at least 140 mmHg and a diastolic blood pressure of at least 80 mmHg. In certain embodiments, the subject has a systolic blood pressure greater than about 120 mmHg, 125 mmHg, 130 mmHg, 135 mmHg, or 140 mmHg. In certain embodiments, subjects have systolic blood pressures of 130-160 mmHg, 130-165 mmHg, 130-170 mmHg, 130-180 mmHg, 135-160 mmHg, 135-165 mmHg, 135-170 mmHg, 140-170 mmHg, 145-180 mmHg, and 155-180 mmHg. In certain embodiments, subjects are members of a group susceptible to salt sensitivity, are overweight, obese, and / or pregnant.

[0139] Specific embodiments of the present invention provide a method for treating subjects having a disease, disorder and / or condition that would benefit from reduced angiotensinogen (AGT) expression, comprising administering to a subject in need a therapeutically effective amount of a compound containing a dsRNA listed in Tables 2, 5, 7, 9, 10, or 13, or a pharmaceutical composition containing any of the dsRNAs listed in Tables 2, 5, 7, 9, 10, or 13, in an amount sufficient to inhibit AGT expression, thereby treating subjects having a disorder that would benefit from reduced AGT expression. In specific embodiments, the dsRNA is an siRNA listed in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA comprises the nucleotide sequences of SEQ ID NOs: 61 and 62. In even more preferred embodiments, the siRNA comprises the nucleotide sequences and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 9 and 55). In specific embodiments, the disease, disorder and / or condition is a RAAS-related disease, disorder and / or condition.

[0140] Specific embodiments of the present invention provide a method for preventing or alleviating at least one symptom in a subject having a disorder that benefits from reduced angiotensinogen (AGT) expression, comprising administering to a subject in need a prophylactic effective amount of a compound containing a dsRNA listed in Tables 2, 5, 7, 9, 10, or 13, or a pharmaceutical composition containing any of the dsRNAs listed in Tables 2, 5, 7, 9, 10, or 13, in an amount sufficient to inhibit AGT expression, thereby preventing or alleviating at least one symptom in a subject having a disorder that benefits from reduced AGT expression. In specific embodiments, the dsRNA is an siRNA listed in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA comprises the nucleotide sequences of SEQ ID NOs: 61 and 62. In even more preferred embodiments, the siRNA comprises the nucleotide sequences and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55). In specific embodiments, the at least one symptom is a RAAS-related symptom.

[0141] Certain embodiments provide a method for inhibiting AGT expression in a subject, comprising the step of administering a compound or composition containing a dsRNA described herein to the subject in an amount sufficient to inhibit AGT expression. The dsRNA compound is administered to the subject subcutaneously or intravenously. In certain embodiments, the dsRNA inhibits angiotensinogen (AGT) expression by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

[0142] Certain embodiments provide double-stranded ribonucleic acid (dsRNA) compounds described herein for use in pharmaceuticals. Certain embodiments provide double-stranded ribonucleic acid (dsRNA) compounds described herein for use in treating or preventing RAAS-related diseases, disorders and / or conditions in a subject. In certain embodiments, RAAS-related diseases, disorders and / or conditions are as described above.

[0143] In certain embodiments, the dsRNA is administered to the subject at a dose of approximately 0.01 mg / kg to approximately 50 mg / kg. In certain embodiments, the dsRNA is an siRNA that inhibits angiotensinogen (AGT) expression by at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the dsRNA is an siRNA administered to the subject at a dose of approximately 0.01 mg / kg to approximately 50 mg / kg. In certain embodiments, preferred doses are selected from 700 mg, 800 mg, and 900 mg. In certain embodiments, the therapeutically effective dose of the compound or composition containing the dsRNA described herein is administered at a dose of approximately 150 mg, 300 mg, or 600 mg once every three months. In certain embodiments, the therapeutically effective dose of the compound or composition containing the dsRNA described herein is administered at a dose of approximately 150 mg, 300 mg, or 600 mg once every six months.

[0144] In certain embodiments, the level of AGT in a sample from a subject is determined after administering a compound or composition containing dsRNA to the subject. In certain embodiments, the level of AGT in the subject sample is the level of AGT nucleic acid (e.g., mRNA) or protein in a blood, plasma, urine, or liver tissue sample. In certain embodiments, the dsRNA is siRNA.

[0145] In certain embodiments, additional factors are evaluated after administration of a compound or composition containing dsRNA to a subject. In certain embodiments, levels of bradykinin, prekallikrein, or blood pressure in the subject are determined. In certain embodiments, the dsRNA is siRNA.

[0146] In certain embodiments, a compound or composition containing dsRNA is administered to a subject alone or in combination with additional therapeutic agents to treat RAAS-related diseases, disorders and / or conditions or symptoms thereof. In certain embodiments, the dsRNA is an siRNA listed in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA comprises the nucleotide sequences of SEQ ID NOs: 61 and 62. In even more preferred embodiments, the siRNA comprises the nucleotide sequences and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55). In certain embodiments, additional therapeutic agents are selected from the group consisting of diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists (also known as angiotensin II receptor blockers (ARBs)), β-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, α2 agonists, renin inhibitors, α-blockers, peripheral-acting adrenergic agonists, selective DI receptor partial agonists, non-selective α-adrenergic antagonists, synthetic, steroidal antimineralicoid agents, angiotensin receptor-neprilysin inhibitors (ARNi), Entresto(R), sacubitril / valsartan; or endothelin receptor antagonists (ERAs), cytaxentan, ambrisentan, atrasentan, BQ-123, dibotentan, bosentan, macitentan, and tezosentan; any combination of the above; and antihypertensive agents formulated as combinations of these agents. In certain embodiments, the additional therapeutic agent includes an angiotensin II receptor antagonist. In certain embodiments, the angiotensin II receptor antagonist is selected from the group consisting of losartan, valsartan, olmesartan, eprosartan, and azilsartan. In certain embodiments, when used in combination with a compound or composition containing dsRNA described herein, the additional therapeutic agent may provide a synergistic or additive effect in lowering blood pressure in a subject.

[0147] In one embodiment, a process is provided for preparing sense and / or antisense strands of a double-stranded ribonucleic acid (dsRNA) compound, the process comprising the steps of: (a) preparing the sense and / or antisense strands by sequential coupling of modified and / or unmodified nucleotides via phosphoramidite oligonucleotide synthesis on a solid support; (b) optionally coupling a GalNAc-containing moiety to the sense and / or antisense strands on the solid support via phosphoramidite oligonucleotide synthesis; (c) desorbing the sense and / or antisense strands from the solid support and removing the solid support; and (d) optionally further purifying the sense and / or antisense strands using chromatography. Phosphoramidite oligonucleotide synthesis is generally known in the art. This process can prepare sense or antisense strands that may contain a GalNAc-containing moiety as a conjugate at their 5' end. Preferably, this process is used to prepare antisense strands that do not contain any conjugates (e.g., the antisense strands of SEQ ID NO: 9 or 19).

[0148] In one embodiment, a process is provided for preparing sense and / or antisense strands of a double-stranded ribonucleic acid (dsRNA) compound, the process comprising the steps of: (a) coupling a GalNAc-containing moiety to a solid support via phosphoramidite oligonucleotide synthesis; (b) coupling modified and / or unmodified nucleotides to the GalNAc-containing moiety on the solid support via phosphoramidite oligonucleotide synthesis; (c) sequentially coupling additional modified and / or unmodified nucleotides via phosphoramidite oligonucleotide synthesis to prepare a sense and / or antisense strand; (d) desorbing the sense and / or antisense strand from the solid support and removing the solid support; and (e) optionally further purifying the sense and / or antisense strand using chromatography. This process prepares a sense or antisense strand having a GalNAc-containing moiety as a conjugate at its 3' end. Preferably, this process is used to prepare a sense strand having a 3'-terminal GalNAc conjugate (e.g., the sense strand of SEQ ID NO: 55).

[0149] In one embodiment, a process for preparing a double-stranded ribonucleic acid (dsRNA) compound is provided, comprising: (a) contacting a sense prepared according to any one of the above processes with an antisense strand prepared according to any one of the above processes in equimolar concentrations in solution; (b) optionally heating the solution to a temperature of about 94°C; and (c) optionally lowering the temperature of the solution to about 25°C. Preferably, the sense strand is a sense strand containing a 3'-terminal GalNAc conjugate, and the antisense strand is an antisense strand without any conjugates.

[0150] Certain embodiments provide a process for preparing oligonucleotides (sense or antisense chains) for use in forming compounds or compositions described herein, comprising synthesizing the oligonucleotides on a solid support by: (a) attaching modified or unmodified nucleotides to the solid support by phosphoramidite chemistry; (b) removing excess phosphoramidite reaction solution; (c) removing acid-unstable protecting groups from the nucleotides with an acid solution; (d) removing the acid solution; and (e) coupling the modified or unmodified nucleotides, GalNAc-containing moieties, or lipophilic moieties to the unprotected nucleotides attached to the solid support by phosphoramidite chemistry to form extended oligonucleotides attached to the solid support. (f) Remove excess phosphoramidite reaction solution; (g) Capping unreacted amines or alcohols on oligonucleotides attached to the solid support with acetic anhydride solution to reduce branching and shortening; (h) Remove acetic anhydride solution; (i) Introduce phosphodiester bonds with an oxidizing reagent and / or phosphorothioate bonds with a sulfurizing reagent; (j) Remove the oxidizing and / or sulfurizing reagents; (k) Repeat steps (c)-(j) until the desired length of the oligonucleotide is achieved; (l) Remove the oligonucleotide from the solid support with an alkaline solution; (m) Remove the solid support from the oligonucleotide solution; and (n) Optionally, purify the oligonucleotide using chromatography.

[0151] Certain embodiments provide a process for preparing oligonucleotides (of sense or antisense chains) for use in forming compounds or compositions described herein, comprising synthesizing the oligonucleotides on a partially filled solid support by: (a) attaching modified or unmodified nucleotides to a portion of the solid support by phosphoramidite chemistry; (b) removing excess phosphoramidite reaction solution; (c) removing acid-unstable protecting groups from the nucleotides with an acid solution; (d) removing the acid solution; (e) coupling the modified or unmodified nucleotides by phosphoramidite chemistry to the unprotected nucleotides attached to the solid support to form an extended oligonucleotide attached to the solid support; (f (g) removing excess phosphoramidite reaction solution; (h) capping unreacted amines or alcohols on oligonucleotides attached to the solid support with acetic anhydride solution to reduce branching and shortening; (i) introducing phosphodiester bonds with an oxidizing reagent and / or phosphorothioate bonds with a sulfurizing reagent; (j) removing the oxidizing and / or sulfurizing reagents; (k) repeating steps (c)-(j) until the desired length of the oligonucleotide is achieved; (l) removing the oligonucleotide from the solid support with an alkaline solution; (m) removing the solid support from the oligonucleotide solution; and (n) optionally purifying the oligonucleotide using chromatography. In certain embodiments, the portion packed into the solid support is selected from GalNAc-containing portions, cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, dyes, etc. In a preferred embodiment, the portion filled into the solid phase support is a GalNAc-containing portion.

[0152] The present invention also provides a kit comprising any of the compounds disclosed herein or any of the pharmaceutical compositions disclosed herein, and optionally a label (e.g., instructions for use). The present invention provides a vial comprising any of the compounds disclosed herein or any of the pharmaceutical compositions disclosed herein. The present invention provides a syringe comprising any of the compounds disclosed herein or any of the pharmaceutical compositions disclosed herein. In one embodiment, the present invention provides a kit for carrying out a method of inhibiting AGT expression in a subject by administering to a subject requiring it an amount effective to inhibit AGT expression in the subject. The kit comprises a dsRNA compound and instructions for use, and optionally means for administering the dsRNA to the subject. In certain embodiments, the compound or pharmaceutical composition is a dsRNA listed in Tables 2, 5, 7, 9, 10, or 13. In certain embodiments, the dsRNA is an siRNA listed in Tables 2, 5, 7, 9, 10, or 13. In preferred embodiments, the siRNA of the kit comprises the nucleotide sequence and chemical modifications of ATsi786 (SEQ ID NOs: 9 and 55) or ATsi787 (SEQ ID NOs: 19 and 55).

[0153] The following description applies to all of the embodiments described above.

[0154] Oligomer compounds The oligomeric compounds of the present invention include, but are not limited to, short hairpin RNA (shRNA) and double-stranded RNA (dsRNA) compounds such as small interfering RNA (siRNA), which target AGT genes by targeting AGT mRNA. The oligomeric compounds of the present invention contain an "antisense strand" to the target nucleic acid, which means that they can undergo hybridization with the target nucleic acid via hydrogen bonding.

[0155] In certain embodiments, the oligomeric compound has a nucleic acid base sequence that, when described in the 5'-to-3' direction, includes the reverse complementary sequence of the target segment of the target nucleic acid it targets. For example, in certain such embodiments, the siRNA compound includes an antisense strand having a nucleic acid base sequence that, when described in the 5'-to-3' direction, includes the reverse complementary sequence of the target segment of the target nucleic acid it targets.

[0156] In certain embodiments, the oligomer compound has a subunit length of 12 to 30. In certain embodiments, the oligomer compound has a subunit length of 18 to 30. In certain embodiments, the oligomer compound has a subunit length of 12 to 22. In certain embodiments, the oligomer compound has a subunit length of 14 to 30. In certain embodiments, the oligomer compound has a subunit length of 14 to 21. In certain embodiments, the oligomer compound has a subunit length of 15 to 30. In certain embodiments, the oligomer compound has a subunit length of 15 to 21. In certain embodiments, the oligomer compound has a subunit length of 16 to 30. In certain embodiments, the oligomer compound has a subunit length of 16 to 21. In certain embodiments, the oligomer compound has a subunit length of 17 to 30. In certain embodiments, the oligomer compound has a subunit length of 17 to 21. In certain embodiments, the oligomer compound has a subunit length of 18 to 30. In certain embodiments, the oligomer compound has a subunit length of 18 to 21. In certain embodiments, the oligomer compound has a subunit length of 20 to 30. In certain embodiments, the oligomer compound has a subunit length of 15. In certain embodiments, the oligomer compound has a subunit length of 16. In certain embodiments, the oligomer compound has a subunit length of 17. In certain embodiments, the oligomer compound has a subunit length of 18. In certain embodiments, the oligomer compound has a subunit length of 20. In certain embodiments, the oligomer compound has a subunit length of 21. In certain embodiments, the oligomer compound has a subunit length of 22. In certain embodiments, the oligomer compound has a subunit length of 23. In certain embodiments, the oligomer compound has a subunit length of 25. In certain embodiments, the oligomer compound has a subunit length of 25. In other embodiments, the oligomeric compound is a binding subunit of 8-80, 12-50, 13-30, 13-50, 14-30, 14-50, 15-30, 15-50, 16-30, 16-50, 17-30, 17-50, 18-22, 18-24, 18-30, 18-50, 19-22, 19-30, 19-50, or 20-30.In certain such embodiments, the oligomeric compound is a range defined by a bonded subunit length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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, or 80, or any two of the above values. In some embodiments, the oligomeric compound is siRNA.

[0157] It is possible to increase or decrease the length of oligomeric compounds, such as siRNA compounds, and / or introduce base mismatches without eliminating activity (U.S. Patent No. 7,772,203, incorporated herein by reference). For example, it is possible to introduce non-canonical base pairing (e.g., A:G, A:C, G:U, I:U, I:A, I:C) into oligomeric compounds without eliminating activity. In certain embodiments, the activity of the oligomeric compound is improved by designing oligomeric compounds with one or more non-canonical base pairings, i.e., mismatches.

[0158] Oligomer compounds may include mismatches with the target, mismatches between oligomeric chains within the double helix, or combinations thereof. Mismatches can occur throughout the siRNA, such as in overhangs (parts of the sense or antisense chain at the 5' and / or 3' ends of a double-stranded siRNA that lacks a complementary chain) or in the double-strand portion.

[0159] Oligomer compound motif A motif refers to a modification pattern of an oligomeric compound. Various motifs have been described in the Art and are incorporated herein by reference (e.g., U.S. Patent No. 11,203,755; U.S. Patent No. 10,870,849; EP Patent No. 1,532,248; U.S. Patent No. 11,406,716; U.S. Patent No. 10,668,170; U.S. Patent No. 9,796,974; U.S. Patent No. 8,754,201; U.S. Patent No. 10,837,013; U.S. Patent No. 7,732,593; U.S. Patent No. 7,015,315; U.S. Patent No. 7,750,1 (USSN No. 44; USSN No. 8,420,799; USSN No. 8,809,516; USSN No. 8,796,436; USSN No. 8,859,749; USSN No. 9,708,615; USSN No. 10,233,448; USSN No. 10,273,477; USSN No. 10,612,024; USSN No. 10,612,027; USSN No. 10,669,544; USSN No. 11,401,517; USSN2020 / 0031862; USSN2016 / 0272970). However, there is always a demand for new and improved motifs.

[0160] In certain embodiments, the oligomeric compounds disclosed herein, such as siRNA, have chemically modified subunits arranged in a motif to confer beneficial properties to the oligomeric compound, including, but not limited to, enhanced inhibitory activity to increase potency; enhanced binding affinity to increase specificity to target nucleic acids by limiting off-target effects and increasing safety; or increased stability and durability by enhanced resistance to degradation by in vivonucleases. In certain embodiments, the oligomeric compound is a chimera in which the peripheral nucleic acid bases of the oligomeric compound contain a motif having various modified or unmodified nucleic acid bases to confer increased stability, specificity, safety, and potency, while the central portion of the compound contains various modified or unmodified nucleic acid bases to function as a substrate for RISC-mediated degradation. Each distinct portion may include a homogeneous sugar moiety, a modified sugar moiety, or alternating sugar moieties. Each portion may include various patterns of phosphate and phosphorothioate bonds.

[0161] In certain embodiments, the oligomeric compound targeting the AGT nucleic acid includes a sense strand having the sequence and chemical modification motifs shown in Tables 2, 5, 7, 9, 10, or 13. In certain embodiments, the oligomeric compound targeting the AGT nucleic acid includes an antisense strand having the sequence and chemical modification motifs shown in Tables 2, 5, 7, 9, 10, or 13.

[0162] Target nucleic acid, target region, and nucleotide sequence Some embodiments relate to methods for regulating gene expression by inhibiting dsRNA.

[0163] In certain embodiments, a method for inhibiting angiotensinogen (AGT) gene expression in cells includes administering to cells a dsRNA compound (GenBank NM_001382817.3, incorporated herein as SEQ ID NO: 1) that targets the mRNA (or its corresponding cDNA) transcript of AGT.

[0164] Nucleic acid sequences and chemical modification motifs of dsRNAs targeting AGT transcripts are shown in Tables 2, 5, 7, 9, 10, and 13. It is understood that the sequences described in each sequence number in the examples contained herein are independent of any modifications to the sugar moiety, nucleoside bond, or nucleic acid base. Therefore, the siRNA compounds defined by the sequence number may independently contain one or more modifications to the sugar moiety, nucleoside bond, or nucleic acid base. The siRNA compounds indicated by the ARNATAR name represent a combination of sequence and motif.

[0165] Hybridization In some embodiments, hybridization occurs between the oligomeric compounds disclosed herein and mRNA. The most common mechanism of hybridization involves hydrogen bonding between complementary nucleic acid bases of nucleic acid molecules (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds).

[0166] In Watson-Crick canonical base pairing, adenine (A) is complementary to thymine (T) in DNA, adenine (A) is complementary to uracil (U) in RNA, and guanine (G) is complementary to cytosine (C) in both DNA and RNA. While base pairs, or complementary nucleic acid bases, are typically Watson-Crick base pairs (C:G, A:U, A:T), non-canonical base pairs such as Hoogsteen base pairs (e.g., A:G, A:U) and fluctuating base pairs (e.g., G:U, I:U, I:A, I:C, where I is hypoxanthine) are also acceptable during hybridization of oligomeric compounds to target nucleic acids or target regions. Fluctuating base pairs in RNAi agents have been previously described (see, e.g., U.S. Patent No. 7,732,593; U.S. Patent No. 7,750,144).

[0167] Nucleic acid base complementarity facilitates the hybridization of the oligomeric compounds described herein to their target nucleic acids, and the stronger the pairing (e.g., the more base pairs and / or hydrogen bonds), the stronger the hybridization of the oligomeric compound to the target. Hybridization can occur under a variety of conditions. The stringent conditions are sequence-dependent and determined by the properties and composition of the oligomeric compound being hybridized.

[0168] Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art. In certain embodiments, the oligomeric compounds provided herein can specifically hybridize to target mRNA with little or no off-target binding.

[0169] Complementarity Oligomer compounds and target nucleic acids are complementary if a sufficient number of nucleic acid bases in the oligomer compound can hybridize with the corresponding nucleic acid bases of the target nucleic acid, resulting in the desired effect (e.g., inhibition of the target nucleic acid, such as mRNA).

[0170] Non-complementary nucleic acid bases between the oligomeric compound and the mRNA nucleic acid may be acceptable, as long as the oligomeric compound retains its ability to specifically hybridize to the target nucleic acid. Furthermore, the oligomeric compound can hybridize across one or more segments of the mRNA nucleic acid (e.g., loop structures, mismatches, or hairpin structures) in such a way that intervening or adjacent segments do not participate in the hybridization event.

[0171] In certain embodiments, the oligomeric compounds or specific portions thereof provided herein are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to mRNA nucleic acid, target region, target segment, or specific portion thereof. The percentage complementarity of the oligomeric compound having the target nucleic acid can be determined using routine methods.

[0172] For example, an oligomeric compound in which 18 of the 20 nucleic acid bases in the antisense chain of an oligomeric compound are complementary to the target region, and therefore specifically hybridize, represents 90 percent complementarity. In this example, the remaining non-complementary nucleic acid bases may be clustered or scattered with the complementary nucleic acid bases and do not need to be adjacent to each other or to the complementary nucleic acid bases. The percentage complementarity of an oligomeric compound having a region of the target nucleic acid can be routinely determined using the BLAST program (basic local alignment search tools) and the PowerBLAST program (Altschul et al., 1990, J. Mol. Biol., 215:403-410; Zhang and Madden, 1997, Genome Res., 7:649-656), which are known in the art, and via the website of the National Center for Biotechnology Information (NCBI, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Percent homology, sequence identity, or complementarity can be determined, for example, by NCBI Blast (Johnson et al., Nucleic Acids Res. 2008, 36 (Web Server issue): W5-W9).

[0173] In certain embodiments, the oligomeric compounds, or specific portions thereof, provided herein are fully complementary (i.e., 100% complementary) to a target nucleic acid, or specific portion thereof. For example, an oligomeric compound may be fully complementary to an mRNA nucleic acid, or a target region, or a target segment or target sequence. As used herein, “fully complementary” means that each nucleic acid base of the oligomeric compound can form accurate base pairs with the corresponding nucleic acid base of the target nucleic acid. For example, a 20-nucleotide oligomeric compound is fully complementary to a 400-nucleotide target sequence, insofar as there is a corresponding 20-nucleotide portion of the target nucleic acid that is fully complementary to the oligomeric compound.

[0174] The term "perfectly complementary" can also be used in reference to a specific portion of an oligomeric compound or nucleic acid target. For example, a 20-nucleotide portion of a 30-nucleotide oligomeric compound may be "perfectly complementary" to a 400-nucleotide target sequence. The 20-nucleotide portion of a 30-nucleotide oligomer is perfectly complementary to the target sequence if the target sequence has a corresponding 20-nucleotide portion where each nucleic acid base is complementary to the 20-nucleotide portion of the oligomeric compound. At the same time, the entire 30-nucleotide oligomeric compound may or may not be perfectly complementary to the target sequence, depending on whether the remaining 10 nucleic acid bases of the oligomeric compound are also complementary to the target sequence.

[0175] The location of non-complementary nucleic acid bases can be at the 5' or 3' end of the oligomeric compound. Alternatively, non-complementary nucleic acid bases can be located internally within the oligomeric compound. If two or more non-complementary nucleic acid bases are present, they may be adjacent (i.e., bound) or non-adjacent.

[0176] In certain embodiments, the oligomeric compound having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleic acid bases contains 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer non-complementary nucleic acid bases with respect to a target nucleic acid, such as mRNA nucleic acid, or a specific portion thereof.

[0177] In certain embodiments, an oligomeric compound having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleic acid bases contains 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer non-complementary nucleic acid bases with respect to a target nucleic acid, such as mRNA nucleic acid, or a specific portion thereof.

[0178] The oligomeric compounds provided herein also include those complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of adjacent (i.e., bound) nucleic acid bases within a region or segment of the target nucleic acid. “Portion” may also refer to a defined number of adjacent nucleic acid bases of the oligomeric compound. In certain embodiments, the oligomeric compound is complementary to at least eight nucleic acid base portions of the target segment. In certain embodiments, the oligomeric compound is complementary to at least nine nucleic acid base portions of the target segment. In certain embodiments, the oligomeric compound is complementary to at least ten nucleic acid base portions of the target segment. In certain embodiments, the oligomeric compound is complementary to at least eleven nucleic acid base portions of the target segment. In certain embodiments, the oligomeric compound is complementary to at least twelve nucleic acid base portions of the target segment. In certain embodiments, the oligomeric compound is complementary to at least thirteen nucleic acid base portions of the target segment. In certain embodiments, the oligomeric compound is complementary to at least fourteen nucleic acid base portions of the target segment. In certain embodiments, the oligomeric compound is complementary to at least fifteen nucleic acid base portions of the target segment. Furthermore, oligomeric compounds complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acid base portions of the target segment, or to a range defined by any two of these values, are also intended.

[0179] identity The oligomeric compounds provided herein may also have a defined percentage identity with respect to a compound or part thereof represented by a specific nucleotide sequence, sequence number, or specific ARNATAR number. When used herein, an oligomeric compound is identical to a sequence if it has the same nucleic acid base-pairing ability as the sequence disclosed herein. For example, an RNA containing uracil instead of thymidine in a disclosed DNA sequence is considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shortened and extended versions of the oligomeric compounds described herein, as well as compounds having non-identical bases to the oligomeric compounds provided herein, are also contemplated. Non-identical bases may be adjacent to each other or dispersed throughout the oligomeric compound. The percentage identity of an oligomeric compound is calculated according to the number of bases that have identical base-pairing properties with respect to the sequence it is being compared to.

[0180] In certain embodiments, a portion of the oligomeric compound is compared to an equal-length portion of the target nucleic acid. In certain embodiments, the 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleic acid base portions of the oligomeric compound are compared to an equal-length portion of the target nucleic acid.

[0181] chemical modification A nucleoside is a base-sugar combination. The nucleic acid base (also known as the base) portion of a nucleoside is usually a heterocyclic base. A nucleotide is a nucleoside that further contains a covalent bond (e.g., a phosphate group or a chemically modified bond as described below) to the sugar portion of the nucleoside. Oligonucleotides are formed via covalent bonds between adjacent nucleotides, forming a linear sequence of linked nucleotides. Within an oligonucleotide structure, the binding groups are generally referred to as forming the internucleoside bond of the oligonucleotide. Oligomer compounds consist of one (e.g., ssRNA, antisense oligonucleotide, or miRNA) or multiple oligonucleotides (e.g., siRNA or dsRNA such as shRNA).

[0182] Modification of oligomeric compounds includes substitution or alteration of nucleic acid bases, nucleoside bonds, or sugar moieties. Modified oligomeric compounds disclosed herein are often preferred over the natural or unmodified forms for desirable properties such as enhanced delivery (e.g., increased cellular uptake), enhanced specificity or affinity for target nucleic acids, increased stability in the presence of nucleases, enhanced safety (e.g., fewer side effects after administration of the compound to a subject), or increased potency (e.g., inhibitory activity).

[0183] Nucleic acid base modification Nucleic acid bases are heterocyclic moieties that can form base pairs with the nucleic acid bases of other nucleic acids. Modifications to nucleic acid bases can be advantageous for oligomeric compounds for a variety of reasons, including, but are not limited to, increased stability, increased specificity, decreased immunogenicity, increased affinity, increased potency, and other desirable characteristics of the oligomeric compound.

[0184] Nucleic acid base modifications and examples of their benefits are well known in the art (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101). Nucleic acid base modifications can include substituting nucleic acid bases with nucleic acid base analogs or modifying a portion of nucleic acid bases. Examples of nucleic acid base modifications include, but are not limited to, pseudouridine, 2'-thiouridine, N6'-methyladenosine, and 5'-methylcytidine, 5'-fluoro-2'-deoxyuridine, N-ethylpiperidine 5'-triazole-modified adenosine, 5'-nitroindole, 2',4'-difluorotolylribonucleoside, N-ethylpiperidine 7'-EAA triazole-modified adenosine, and 6'-phenylpyrrolocytosine.

[0185] In certain embodiments, the oligomeric compound targeting mRNA nucleic acid comprises one or more modified nucleic acid bases. In certain embodiments, the modified nucleic acid base is, for example, a deoxyribonucleoside (D) substituted in place of a ribonucleoside (R). In certain embodiments, the modified nucleic acid base may be a thymine substitution for uracil. In certain embodiments, multiple nucleic acid bases of the oligomeric compound are modified. In certain embodiments, each nucleic acid base of the oligomeric compound is modified.

[0186] Nucleoside bond modification The naturally occurring nucleoside-to-nucleoside bond in RNA and DNA is a 3'-to-5' phosphodiester bond. In the case of nucleosides containing furanose sugars, the phosphate group can be attached to the 2', 3', or 5' hydroxyl group portion of the sugar. Oligomer compounds with one or more modified, i.e., non-naturally occurring, nucleoside-to-nucleoside bonds are often preferred over oligomer compounds with naturally occurring nucleoside-to-nucleoside bonds for desirable properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, reduced toxicity, increased stability and durability, reduced degradation, and other desirable characteristics of the oligomer compound. The binding of modified nucleosides and their advantages are well known in the art (Friedrich and Aigner, Therapeutic siRNA: State-of-the-Art and Future Perspectives, 2022, BioDrugs, 36(5):549-571; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101).

[0187] Oligomer compounds having modified nucleoside bonds include nucleoside bonds that retain a phosphorus atom, as well as nucleoside bonds that do not contain a phosphorus atom. Typical phosphorus-containing nucleoside bonds include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates (e.g., 5'-methylphosphonate (5'-MP)), phosphoramidates, and phosphorothioates (e.g., phosphorodithioate Rp isomer (PS,Rp), phosphorodithioate Rp isomer (PS,Sp), 5'-phosphorothioate (5'-PS)), methoxypropylphosphonate, (S)-5'-C-methylphosphorate, peptide nucleic acid (PNA), and 5'-(E)-vinylphosphonate.

[0188] In certain embodiments, the oligomeric compound targeting mRNA nucleic acid comprises one or more modified nucleoside bonds. In certain embodiments, the modified nucleoside bonds are phosphorothioate (PS) bonds. In certain embodiments, one or more nucleoside bonds of the oligomeric compound are phosphorothioate nucleoside bonds. In certain embodiments, the PS bonds are adjacent to a deoxyribonucleoside (sometimes referred to herein as DNA or "D") or a ribonucleoside (sometimes referred to herein as RNA, "R" or "r"). In certain embodiments, each nucleoside bond of the oligomeric compound is a phosphorothioate nucleoside bond.

[0189] sugar modification Natural sugars are the sugar moieties found in DNA(2'-H) or RNA(2'-OH), i.e., 2-deoxy-beta-D-ribofuranose or beta-D-ribofuranose, respectively. The oligomeric compounds provided herein may contain one or more nucleosides modified with natural sugar moieties. Such sugar-modified nucleosides may confer to the oligomeric compounds increased stability, increased durability (e.g., increased half-life), increased binding affinity, decreased off-target effects, decreased immunogenicity, decreased toxicity, increased potency, or other beneficial biological properties. The benefits of sugar modification are known in the art (Friedrich and Aigner, 2022, BioDrugs, 36(5):549-571; Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101; Chiu and Rana, 2003, RNA, 9:1034-1048; Choung et al., Biochem Biophys Res Commun, 2006, 342:919-927; Amarzguioui et al., 2003, Nucleic Acids Res, 31(2):589-595; Braasch et al., 2003, Biochemistry, 42(26):7967-7975; Czauderna et al., 2003, Nucleic Acids Res, 31(11):2705-2716; Allerson et al., 2005, J Med Chem, 48:901-904; Layzer et al., 2004, RNA, 10:766-771; Ui-Tei, et al., 2008, Nucleic Acids Res, 36(7):2136-51; Bramsen and Kjems, 2012, Frontiers in Genetics, 3(154):1-22; Bramsen et al., 2010, Nucleic Acids Res, 38(17):5761-5773; Muhonen et al., 2007, Chem & Biodiversity, 4:858-873; these are incorporated herein by reference).

[0190] In certain embodiments, the nucleoside comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings may include, but are not limited to, the addition of substituents (e.g., 5'-sugar modification, 2'-sugar modification); bridging non-dicotyl atoms to form bicyclic nucleic acids (BNAs); substituting the ribosyl ring oxygen atom with S, N(R), or C(R) (R = H, C1-C12 alkyl, or protecting group); nucleoside mimetic compounds; and combinations thereof.

[0191] A 2'-modified sugar refers to a furanose sugar modified at the 2' position of the furanose ring. A 2'-modified nucleoside refers to a nucleoside containing a sugar modified at the 2' position of the furanose ring. In certain embodiments, such modifications include, but are not limited to, substituents selected from: halogens, substituted and unsubstituted alkoxys, substituted and unsubstituted thioalkyls, substituted and unsubstituted aminoalkyls, substituted and unsubstituted alkyls, substituted and unsubstituted allyls, and substituted and unsubstituted alkynyls. In certain embodiments, the 2' modification is selected from substituents including, but are not limited to, O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, OCH2C(=O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2' substituents may also be selected from C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving pharmacokinetic properties, and group for improving the pharmacodynamic properties of oligomeric compounds, as well as other groups having similar properties.

[0192] Further examples of nucleosides having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 2'-F-5'-methyl, 4'-S, 2'-deoxy-2'-fluoro (2'-F), 2'-OCH3 (2'-O-methyl, 2'-OMe), 2'-O(CH2)2OCH3 (2'-O-methoxyethyl, 2'-O-MOE, 2'-MOE), 2'-O-methyl-4-pyridine, phosphorodiamidate morpholino (PMO), tricyclo-DNA (tcDNA), 2'-arabino-fluoro, 2'-O-benzyl, glycol nucleic acid (GNA), and unlocked nucleic acid (UNA) substituents. The substituent at the 2' position may also be selected from allyl, amino, azide, thio, O-allyl, O-C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(Rm)(Rn), and O-CH2-C(=O)-N(Rm)(Rn), where each Rm and Rn is independently H or a substituted or unsubstituted C1-C10 alkyl. 2'-OMe, 2'-OCH3, or 2'-O-methyl each refer to a nucleoside containing a sugar with an -OCH3 group at the 2' position of the sugar ring. 2'-F refers to a sugar with a fluoro group at the 2' position. 2'-O-methoxyethyl, 2'-O-MOE, or 2'-MOE each refer to a nucleoside containing a sugar with an -O(CH2)2OCH3 group at the 2' position of the sugar ring.

[0193] BNA refers to a modified nucleoside containing a bicyclic sugar moiety in which a bridge connecting two carbon atoms of a sugar ring connects the 2' carbon to another carbon atom of the sugar ring. Examples of bicyclic nucleosides include, but are not limited to, nucleosides containing a bridge between 4' and 2' ribosyl ring atoms, such as loc nucleic acid (LNA). In certain embodiments, the oligomeric compounds provided herein contain one or more bicyclic nucleosides in which the bridge connects a bicyclic nucleoside from 4' to 2'. LNA and UNA are described by Campbell and Wengel (Chem Soc Rev, 2011, 40(12):5680-9) and are incorporated herein by reference.

[0194] In certain embodiments, the oligomeric compound comprises one or more nucleotides having a modified sugar moiety. In certain embodiments, the modified sugar moiety has a 2'-OMe modification. In certain embodiments, the modified sugar moiety has a 2'-F modification. In certain embodiments, the 2'-OMe and / or 2'-F modified nucleotides are arranged in a motif. In preferred embodiments, the modifications are arranged in an Arnatar motif disclosed in PCT / US2023 / 084688, which is incorporated herein by reference.

[0195] In certain embodiments, the nucleic acid-targeting oligomeric compound comprises a sense strand having a motif described by one of the following formulas: Equation (I): 5'M-(Y)nZ-(Y)rD-D3', Formula (II): 5'YZ-(Y)q-FFNM-(Y)qM-(Y)vD-D3', Formula (III): 5'M*F*MMN*MN*MFFNMN*MN*MMFM*D*D3', Formula (X): 5'MFMMNMNMFFNMNMNMMNMDD3', or Formula (XI): 5'MFMMNMNMFFMMNMNMFMDD3', Here, each D is a deoxyribonucleoside (D is a modification of R), Each R is a ribonucleoside. Each N is a modified or unmodified nucleoside (e.g., D, R, M, F, UNA modification, or LNA modification). Each M is a 2'-OMe modified nucleoside. Each F is a 2'-F modified nucleoside. Each * is a phosphorothioate (PS) bond. Each Y is either two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF, or FM) or an unmodified nucleoside adjacent to a modified nucleoside (e.g., DR, RD, MR, or RM). Each Z is either two adjacent unmodified nucleosides, or two adjacent nucleosides with the same modification, or two adjacent unmodified nucleosides (e.g., MM, DD, RR, or FF). Each n is 6-8. Each q is 2-3. Each r is 1-2. Each v is 0-1, and The condition is that a single modification type does not modify more than two consecutive nucleotides. In certain embodiments, FFNM is FFRM or FFMM.

[0196] In certain embodiments, the nucleic acid-targeting oligomeric compound comprises an antisense chain having a motif described by one of the following formulas: Formula (IV): 5'-LM-(D)v-(Y)s-(Z)t-(Y)uZN-(Z)r3', Formula (V): 5'L-(Y)p-NM-(FMM)r-(Y)p-(Z)r3', Formula (VI): 5'LM*N*MNMFNMMFMMNMFMFMMN*M*M3', Formula (VII): 5'LM*D*MFMFNMFMMFMFMFMMN*M*M3', Formula (VIII): 5'L-MNMNMFNMMFMMNMFMFMMNMM3', Equation (IX): 5'LM-(Y)pZ-(Y)p-(Z)r3', or Formula (XII): 5'L-MDMFMFNMFMMFMFMFMMNM3', Here, each D is a deoxyribonucleoside (D is a modification of R), Each R is a ribonucleoside, and each N is a modified or unmodified nucleoside (e.g., D, R, M, F, UNA modified, or LNA modified). Each M is a 2'-OMe modified nucleoside. Each F is a 2'-F modified nucleoside. Each L is 5'-phosphate, 5'-vinylphosphonate, or 5'OH. Each * is a phosphorothioate (PS) bond. Each Y is either two adjacent nucleosides with different modifications (e.g., MD, DM, DF, FD, MF, or FM) or an unmodified nucleoside adjacent to a modified nucleoside (e.g., DR, RD, MR, or RM). Each Z is either two adjacent unmodified nucleosides, or two adjacent nucleosides with the same modification, or two adjacent unmodified nucleosides (e.g., MM, DD, RR, or FF). Each (5p) is 5'-phosphate. Each n is 6-8. Each p is 3-5. Each r is 1-2. Each v is between 0 and 1. Each s is 2-7. Each t is 0-2, and The condition is that a single modification type does not modify more than two consecutive nucleotides. In certain embodiments, FNM is FMM.

[0197] Oligomer compound delivery system Oligomer compounds require entry into target cells to be activated. Various modalities have been used to deliver oligomer compounds to target cells, including viral delivery vectors, lipid-based delivery, polymer-based delivery, and conjugate-based delivery (Paunovska et al., Drug Delivery Systems for RNA Therapeutics, 2022, Nature Reviews Genetics, 23(5):265-280; Chen et al., 2022, Molecular Therapy, Nucleic Acids, 29:150-160).

[0198] Lipid-based particles can form specific structures such as micelles, liposomes, and lipid nanoparticles (LPNs) to deliver oligomeric compounds into cells. To form these particles, LPNs may contain one or more cationic or ionizable lipids (e.g., DLin-MC3-DMA, SM-102, ALC-0315), cholesterol, helper lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), poly(ethylene glycol) (PEG) modified lipids (e.g., PEG-2000-C-DMG, PEG-2000-DMG, ALC-0159), C12-200, cKK-E12, etc. Different combinations of lipids can be formulated to influence the delivery of oligomeric compounds to different types of cells. As an example, the therapeutic siRNA patisiran was formulated with the cationic ionizable lipid DLin-MC3-DMA, cholesterol, the polar phospholipid DSPC, and PEG-2000-C-DMG for delivery to hepatocytes.

[0199] Polymer-based particles are also used in oligomeric compound delivery systems. Such polymers include poly(lactic acid-coglycolic acid) (PLGA), polyethyleneimine (PEI), poly(L-lysine) (PLL), poly(beta-aminoester) (PBAE), dendrimers (e.g., poly(amidoamine) (PAMAM) or PLL), and other polymers or modified polymers thereof. The polymer composition can be varied depending on the desired properties for oligomeric compound delivery.

[0200] The oligomeric compounds disclosed herein may be covalently bonded to one or more moieties or conjugates that enhance the activity, cell distribution, or cell uptake of the resulting compound. Conjugate groups may include cholesterol, lipids, carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and pigments. Conjugate-based delivery allows for the active delivery of oligomeric compounds to specific cell types.

[0201] In one example, the N-acetylgalactosamine (GalNAc)-containing portion is conjugated into an oligomeric compound, which is then delivered to liver cells. Various GalNAc conjugates can be found in several publications, including, all of which are incorporated herein by reference: Sharma et al., 2018, Bioconjugate Chem, 29:2478-2488; Nair et al., J.Am.Chem.Soc. 2014, 136(49):16958-16961; Keam, 2022, Drugs, 82:1419-1425; US Patent 10, 087, 208; Prakash et al., 2014, Nucleic Acids Res, 42(13):8796-807; Debacker et al., 2020, Molecular Therapy, 28(8):1759-1771; PCT / US2023 / 084692; US Patent 11, 110, 174; US US Patent 9,796,756; US Patent 9,181,549; US Patent 10,344,275; US Patent 10,570,169; US Patent 9,506,030; and US Patent 7,582,744.

[0202] In certain embodiments, AGT dsRNA can be conjugated at the 3' end of the sense strand using the following GalNAc conjugate precursors having a solid support. In preferred embodiments, the dsRNA is an ARNATAR-designed siRNA selected from Tables 2, 5, 7, 9, 10, or 13. JPEG2026520610000009.jpg72159

[0203] In a preferred embodiment, the GalNAc-containing portion conjugated to the oligonucleotide is GalNAc-AN (also known as GA4 or GA-AN) as shown below: JPEG2026520610000010.jpg68159

[0204] In one embodiment, GalNAc-AN is conjugated to the sense strand of a dsRNA compound. In a preferred embodiment, GalNAc-AN is conjugated to a dsRNA compound selected from any of the compounds in Tables 2, 5, 7, 9, 10, or 13.

[0205] Synthesis of oligomer compounds siRNA was designed, synthesized, and prepared using methods known in the art.

[0206] Solid-phase synthesis of oligonucleotides is performed by MerMade TMThe synthesis is performed using a 48x synthesizer (BioAutomation, LGC, Biosearch Technologies, Hoddesdon, UK), which can produce up to 48 1 μMole or 5 μMole scale oligonucleotides per run using standard phosphoramidite chemistry. Phosphoramidite synthesis of oligonucleotides on solid-phase supports is well known in the art (e.g., Beaucage and Caruthers, 1981, Tetrahedron Letters, 22(20):1859-1862; Roy and Caruthers, 2013, Molecules, 18:14268-14284; and Sandahl et al., 2021, Nature Communications, 12:2760). The solid-phase support is either a controlled porous glass (500-1400A) loaded with a universal linker, or one loaded with a 3'-GalNAc conjugate (e.g., AM Chemicals, Vista, CA, USA; Primetech ALC, Minsk, Belarus; Gene Link, Elmsford, NY, USA; or any of the GalNAc conjugates disclosed herein), or a universal solid-phase support (AM Chemicals, Vista, CA, USA). Auxiliary synthesis reagents and standard 2'-cyanoethyl phosphoramidite monomers (2'-fluoro, 2'-O-methyl, RNA, DNA) were obtained from various sources (Hongene Biotech, Shanghai, China; Sigma-Aldrich, St. Louis, MO, USA; Glen Research, Sterling, VA, USA; ThermoFisher Scientific, Waltham, MA, USA; LGC Biosearch Technologies, Hoddesdon, UK). The phosphoramidite mixtures were prepared in anhydrous acetonitrile or 30% DMF:acetonitrile and coupled with 0.25M 4,5-dicyanoimidazole (DCI) (Sigma-Aldrich, St. Louis, MO, USA) with coupling times ranging from 120 to 360 seconds.Standard phosphodiester bonds were achieved using a mixture of tetrahydrofuran (THF), pyridine, and 0.02 M iodine in water. Phosphothioate bonds were generated using 0.05 M sulfurizing reagent II (3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazol-3-thion, DDTT) (40:60, pyridine / acetonitrile) (LGC Biosearch Technologies, Hoddesdon, UK) with an oxidation time of 6 minutes. All sequences were synthesized by removing the dimethoxytrityl (DMT) protecting group.

[0207] Upon completion of solid-phase synthesis, the oligonucleotides were cleaved from the solid support, and deprotection of the base-unstable group was performed by incubation in ammonium hydroxide at 55°C for 6 hours. The ammonium hydroxide was removed at room temperature using a centrifugal vacuum concentrator until dry. For sequences containing tert-butyldimethylsilyl (TBDMS)-protected native ribonucleotides (2'-OH), a second deprotection was performed using triethylamine;trihydrofluoride (TEA:3HF). 100 μL of DMSO and 125 μL of TEA:3HF were added to each TBDMS-protected oligonucleotide and incubated at 65°C for 2.5 hours. After incubation, 25 μL of 3M sodium acetate was added to the solution, followed by precipitation in butanol at -20°C for 30 minutes. The turbid solution was centrifuged to form a cake, and the supernatant was carefully decanted by pipette at this point. The standard precipitation process was then completed using 75% ethanol:water as the supernatant solution, and then 100% ethanol. The oligonucleotide cake was dried in a centrifugal vacuum concentrator for 30 minutes.

[0208] Desalting without HPLC purification was performed by precipitation with 3M sodium acetate followed by elution on a G25 Sephadex(R) column (Sigma-Aldrich, St. Louis, MO, USA). Oligonucleotides were purified by anion exchange chromatography on a Gilson GX271 preparative HPLC system (Middleton, WI, USA) using BioWorks Q40 resin (Uppsala, Sweden). Final desalting was performed on a Sephadex(R) G25 column. All oligonucleotides were analyzed for purity by ion-pairing reverse-phase HPLC on an Agilent 1200 analytical HPLC (Santa Clara, CA, USA), for intact mass by anion mass spectrometry on an Agilent 6130 single quadrupole mass spectrometer (Santa Clara, CA, USA), and for A260 quantification by UV / visible light on a Tecan Infinite(R)M Plex plate reader (Zurich, Switzerland).

[0209] Double-chain oligomer compound double-chain formation Generally, for double-stranded oligomeric compounds such as siRNA compounds, sense and antisense oligonucleotides are annealed to form a double helix. The double helix is ​​formed by contacting a sense strand, prepared according to one of the processes described herein, with an antisense strand, prepared according to one of the processes described herein, in an equimolar concentration in solution. Optionally, the solution is heated to a temperature of about 94°C, and then the temperature is reduced to about 25°C. In one example, 50-300 mM double helix formation can be achieved by heating the sample in 1x phosphate-buffered saline in a block heater at 94°C for 4 minutes, then removing the heating block containing the sample from the block heater and gradually cooling it to room temperature over a period of 1 hour.

[0210] Compositions and methods for formulating pharmaceutical compositions The dsRNA compounds, such as AGT-targeting siRNAs, described herein can be combined with pharmaceutically acceptable active or inactive substances, such as diluents, excipients, or carriers, for the preparation of pharmaceutical compositions or formulations.

[0211] The compositions and methods for formulating pharmaceutical compositions depend on a number of criteria, including but not limited to the route of administration, the severity of the disease, or the dose administered.

[0212] In certain embodiments, the pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspension, or any other pharmacologically inert vehicle for delivering one or more nucleic acid compounds to an animal. The excipient may be liquid or solid and can be selected with the planned method of administration in mind so as to provide a desirable bulk, consistency, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate); disintegrants (e.g., starch, sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

[0213] pharmaceutically acceptable organic or inorganic excipients suitable for parenteral or non-parenteral administration, which do not react adversely with nucleic acid compounds, can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in compositions delivered parenterally. Therefore, in one embodiment, the method described herein uses a pharmaceutical composition comprising a dsRNA compound targeting an AGT nucleic acid and a pharmaceutically acceptable diluent. In a particular embodiment, the pharmaceutically acceptable diluent is PBS. In a particular embodiment, the dsRNA compound is siRNA.

[0214] A pharmaceutical composition containing a dsRNA compound such as siRNA may include any pharmaceutically acceptable salt, ester, or salt of such ester, or any other dsRNA that can provide (directly or indirectly) a biologically active metabolite or residue thereof upon administration to an animal (including a human). Therefore, for example, this disclosure also relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents of dsRNA compounds. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0215] In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In such certain embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution such as water or a physiologically compatible buffer such as Hanks solution, Ringer solution, or physiological saline buffer (e.g., PBS). In certain embodiments, other components are included (e.g., components that aid solubility or function as preservatives). In certain embodiments, the suspension for injection is prepared using a suitable liquid carrier, suspension, etc. The specific pharmaceutical composition for injection is presented in unit dose form, for example, in an ampoule or multi-dose container.

[0216] dose For the purposes of this disclosure, the amount or dose of the activator (the oligomeric compound of the present invention) administered should be sufficient, for example, to inhibit the expression of AGT in an animal. In animals (e.g., humans), the dose is determined by the potency of the particular activator, the condition of the animal, and the body weight of the animal being treated.

[0217] Many assays for determining the dosage to be administered are known in the art.

[0218] The dosage of the activators of this disclosure will also be determined by the presence, nature, and extent of any adverse side effects that may occur with the administration of the particular activator of this disclosure. Typically, the attending physician will determine the dosage of the activator of this disclosure to treat each individual subject by considering various factors such as age, weight, overall health, diet, sex, the activator of this disclosure being administered, the route of administration, and the severity of the condition being treated.

[0219] Dosing In certain embodiments, the pharmaceutical composition is administered according to an administration regimen (e.g., dose, dose frequency, and duration), which can be selected to achieve a desired effect in the subject, i.e., a therapeutic effect in the subject. The desired effect may be, for example, a reduction in AGT, or prevention, reduction, improvement, or delay of progression of a disease, disorder, and / or condition, or symptom thereof, related to AGT or the RAAS pathway in the subject. In certain embodiments, the variables of the administration regimen are adjusted to yield a desired concentration of the pharmaceutical composition in the subject. As used in relation to the dose regimen, “concentration of the pharmaceutical composition” may refer to the dsRNA compound or active ingredient of the pharmaceutical composition. For example, in certain embodiments, the dose and dose frequency are adjusted to provide a tissue or plasma concentration of the pharmaceutical composition in an amount sufficient to achieve the desired effect.

[0220] The administration depends on the severity and responsiveness of the disease condition being treated, and the treatment process lasts from several days to several months until a cure is achieved or a reduction in the disease condition is achieved. The administration also depends on the drug potency and differences in metabolism between subjects. In certain embodiments, the therapeutically effective dose in a subject is within the range of 0.01 μg to 50 mg per kg of body weight, 0.01 μg to 100 mg per kg of body weight, or 0.001 mg to 1000 mg, and can be administered once or more times daily, once or more times weekly, once or more times monthly, once or more times quarterly, or once or more times a year, or even once every two to twenty years. After successful treatment, it may be desirable to provide maintenance therapy to prevent recurrence of the disease state, and dsRNA may be administered at a maintenance dose of 0.01 μg to 100 mg per kg of body weight, at least once daily, at least once weekly, at least once monthly, at least once quarterly, at least once a year, up to once every 20 years, or in doses ranging from 0.001 mg to 1000 mg. In certain embodiments, it may be desirable to administer the dsRNA compound at a frequency of once daily, once weekly, once monthly, once quarterly, once a year, once every two years, once every three years, once every four years, once every five years, once every ten years, or up to once every 20 years.

[0221] In certain embodiments, the therapeutically effective dosage range is any of the following: 1 mg-1500 mg, 100 mg-1400 mg, 100 mg-1300 mg, 100 mg-1200 mg, 100 mg-1100 mg, 100 mg-1000 mg, 100 mg-900 mg, 200 mg-800 mg, 300 mg-700 mg, 400 mg-600 mg, 100 mg-400 mg, 200 mg-500 mg, 300 mg-600 mg, and 400 mg-700 mg. In certain embodiments, therapeutically effective doses are approximately 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, or 1500 mg. In certain embodiments, preferred doses are selected from 700 mg, 800 mg, and 900 mg.

[0222] In certain embodiments, a therapeutically effective dose of dsRNA is administered twice a year in an amount of approximately 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg. In certain embodiments, a therapeutically effective dose of dsRNA is administered quarterly in an amount of approximately 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, or 900 mg. In certain embodiments, a therapeutically effective dose of dsRNA is administered once every three months in an amount of approximately 150 mg, 300 mg, or 600 mg. In certain embodiments, a therapeutically effective dose of dsRNA is administered once every six months in an amount of approximately 150 mg, 300 mg, or 600 mg.

[0223] Administration The dsRNA compounds such as siRNA or pharmaceutical compositions of the present invention can be administered in a number of ways, depending on whether topical or systemic treatment is desired and the area to be treated. Administration may be oral, inhaled, or parenteral.

[0224] In certain embodiments, the compounds and compositions described herein are administered parenterally. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, for example, intrathecal or intraventricular administration. In certain embodiments, parenteral administration is by infusion. Infusion may be chronic or continuous, or short or intermittent. In certain embodiments, the pharmaceutical agent to be infused is delivered by pump.

[0225] In certain embodiments, parenteral administration is by injection. The injection can be delivered by syringe or pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to tissue or organ.

[0226] In certain embodiments, formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions that also contain buffers, diluents, and other suitable additives such as permeabilizers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0227] In certain embodiments, formulations for oral administration of a compound or composition may include, but are not limited to, pharmaceutical carriers, excipients, powders or granules, fine particles, nanoparticles, suspensions or solutions in water or a non-aqueous medium, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing agents, or binders may be desirable. In certain embodiments, the oral formulation is one in which the compound provided herein is administered in combination with one or more permeabilizers, surfactants, and chelating agents.

[0228] In vitro testing of dsRNA This specification describes methods for the treatment of cells with dsRNA, such as siRNA, which can be appropriately modified for treatment with other oligomeric compounds.

[0229] Cells can be treated with siRNA when they reach approximately 60-80% confluence during culture.

[0230] One reagent commonly used to introduce siRNA into cultured cells is the cationic lipid transfection reagent Lipofectamine. TM RNAiMAX (Invitrogen, Waltham, MA) is included. siRNA is contained in OPTI-MEM1 (Thermo Fisher Scientific, Waltham, MA) with Lipofectamine. TM Mix with RNAiMAX to obtain the desired final concentration of siRNA and Lipofectamine. TM RNAiMAX concentrations can be achieved, which can range from 0.001 to 300 nM siRNA. The transfection procedure is carried out according to the manufacturer's recommended protocol.

[0231] Another technique used to introduce siRNA into cultured cells includes electroporation.

[0232] siRNA conjugated with a GalNAc-containing moiety can be introduced into cells by incubation of the siRNA with cells without the use of transfection reagents, a process referred to herein as "free uptake." The siRNA-GalNAc conjugate is transported via endocytosis to asialoglycoprotein receptor (ASGR)-positive cells, such as hepatocytes.

[0233] Cells are treated with siRNA by routine methods. Cells can be harvested 4–144 hours after siRNA treatment, at which point the mRNA level (harvested at 4–144 hours) or protein level (extracted at 24–96 hours) of the target nucleic acid is measured by methods known in the art and by methods described herein. Generally, the treatment is performed in multiple iterations, and the data are presented as the mean plus standard deviation of the iterations.

[0234] The concentration of siRNA used varies depending on the cell line and target. Methods for determining the optimal siRNA concentration for a particular target in a particular cell line are well known in the art. Generally, cells are treated with siRNA in a dose-dependent manner to enable the calculation of the maximum inhibitory concentration (IC50). siRNA is lipofectamine TM When transfected with RNAiMAX, siRNA is typically used at concentrations ranging from 0.001 nM to 300 nM. When transfected using electroporation or free uptake, siRNA is used at higher concentrations ranging from 625 to 20,000 nM.

[0235] RNA isolation RNA analysis at the AGT mRNA level can be performed using whole cell RNA or poly(A)+mRNA. RNA isolation methods are well known in the art. RNA is prepared using methods well known in the art, such as TRIZOL reagent (Thermo Fisher Scientific, Waltham, MA), Qiagen RNeasy kit (Qiagen, Hilden, Germany), or AcroPrep Advance 96-well filter plate (Pall Corporation, Port Washington, New York) using Qiagen RLT, RW1, and RPE buffers. RNA extraction procedures are carried out according to the manufacturer's recommended protocol.

[0236] In vivo testing of dsRNA compounds dsRNA compounds, such as siRNA, are tested in animals to evaluate their ability to inhibit the expression of AGT and / or the RAAS pathway, thereby inducing phenotypic changes such as a reduction in one or more RAAS pathway-related diseases. Testing can be performed in normal animals or experimental disease models. For administration to animals, dsRNA is formulated in pharmaceutically acceptable dilutions, such as phosphate-buffered saline. Administration routes include intraperitoneal, intravenous, and subcutaneous parenteral administration. Calculation of dsRNA dosage and frequency depends on factors such as the administration route and the animal's body weight. In one embodiment, after a period of dsRNA treatment, mRNA encoding AGT is isolated from liver tissue, and changes in AGT expression are measured. Changes in AGT protein levels can also be measured. Changes in AGT expression can also be measured by determining the level of RAAS pathway inhibition in the animal. RAAS pathway-related diseases, disorders, and / or conditions can be used as markers to determine the level of AGT inhibition in the animal.

[0237] Specific indications In certain embodiments, the present invention provides a method for treating a subject, comprising administering one or more compounds and / or pharmaceutical compositions of the present invention to the subject. In certain embodiments, the subject has or is at risk of having a RAAS pathway-related disease, disorder and / or condition, or symptoms thereof. In certain embodiments, the present invention provides a method for prophylactically reducing AGT expression in a subject. In certain embodiments, this involves treating a subject in need by administering a therapeutically effective amount of a dsRNA compound, such as siRNA, that targets AGT nucleic acid to the subject.

[0238] In certain embodiments, administration of a therapeutically effective amount of a dsRNA compound targeting AGT nucleic acid to a subject is accompanied by monitoring of AGT levels in the subject's plasma or tissue to determine the subject's response to the administration of the dsRNA compound. The subject's response to the administration of the dsRNA compound is used by the physician to determine the amount and duration of the therapeutic intervention.

[0239] In certain embodiments, administration of a dsRNA compound targeting AGT nucleic acid to a target results in a reduction of AGT expression by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or any two of these values. In certain embodiments, administration of a dsRNA compound targeting AGT nucleic acid to a target results in inhibition of the RAAS pathway by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or any two of these values. In certain embodiments, administration of a dsRNA compound targeting AGT nucleic acid to a subject results in a change in RAAS pathway-related disease, disorder, condition, symptom, or marker (e.g., hypertension or organ damage) in the subject. In certain embodiments, administration of an AGT dsRNA compound to a subject increases or decreases RAAS-related disease, disorder, condition, symptom, or marker in the subject by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, or any two of these values.

[0240] In certain embodiments, a pharmaceutical composition comprising a dsRNA compound targeting AGT is used for the preparation of a pharmacopoeia for the treatment of a subject suffering from or susceptible to RAAS-related disease, disorder, or condition.

[0241] In certain embodiments, the dsRNA compound is an ARNATAR-designed siRNA targeting an AGT as described in Tables 2, 5, 7, 9, 10, or 13.

[0242] Specific combination therapies In certain embodiments, a first agent comprising a dsRNA compound provided herein is co-administered to a subject with one or more second agents. In certain embodiments, the dsRNA compound is an ARNATAR siRNA listed in Tables 2, 5, 7, 9, 10, or 13.

[0243] In certain embodiments, such a second agent is designed to treat the same RAAS pathway-related disease, disorder, or condition as the first agent described herein. In certain embodiments, such a second agent is designed to treat a different disease, disorder, or condition than the first agent described herein. In certain embodiments, such a second agent is designed to treat an undesirable side effect of one or more pharmaceutical compositions described herein. In certain embodiments, such a first agent is designed to treat an undesirable side effect of the second agent. In certain embodiments, the second agent is co-administered with the first agent to treat an undesirable effect of the first agent. In certain embodiments, the second agent is co-administered with the first agent to produce a synergistic or additive effect. In certain embodiments, the second agent is co-administered with the first agent to produce a synergistic effect.

[0244] In certain embodiments, co-administration of the first and second agents allows for the use of lower doses than those required to achieve therapeutic or prophylactic effects when the agents are administered as independent therapies. In certain embodiments, the dose of the co-administered second agent is the same as the dose administered when the second agent is administered alone. In certain embodiments, the dose of the co-administered second agent is greater than the dose administered when the second agent is administered alone.

[0245] In certain embodiments, the first agent and one or more second agents are administered simultaneously. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical formulation. In certain embodiments, the first agent and one or more second agents are prepared separately.

[0246] In certain embodiments, the second agent includes, but is not limited to, specific treatments to reduce hypertension, dietary changes, lifestyle changes, antifibrotic agents, and antihypertensive agents such as RAAS inhibitors, diuretics, calcium channel blockers, adrenergic receptor antagonists, adrenergic agonists, and vasodilators.

[0247] Examples of treatments that can reduce hypertension include, but are not limited to, renal denervation and baroreceptor activation therapy.

[0248] Examples of RAAS inhibitors include, but are not limited to, ACE inhibitors (e.g., captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, trandolapril, and benazepril), angiotensin II receptor antagonists (e.g., candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan), renin inhibitors (e.g., aliskiren), and aldosterone receptor antagonists (e.g., eplerenone and spironolactone).

[0249] Examples of diuretics include loop diuretics (e.g., bumetanide, ethacrine, furosemide, torsemide), thiazide diuretics (e.g., epitizide, hydrochlorothiazide, chlorothiazide, and bendroflumethiazide), thiazide-like diuretics (e.g., indapamide, chlorthalidone, and metrazone), and potassium-sparing diuretics (e.g., amiloride, triamterene, and spironolactone).

[0250] Examples of calcium channel blockers include dihydropyridines (e.g., amlodipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, and nitrendipine) and non-dihydropyridines (e.g., diltiazem and verapamil).

[0251] Examples of adrenergic receptor antagonists include beta-blockers (e.g., atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, and timolol), alpha-blockers (e.g., doxazosin, phentolamine, indramin, phenoxybenzamine, prazosin, terazosin, and trazolin), and mixed alpha-beta blockers (e.g., bucindolol, carvedilol, and labetalol).

[0252] Examples of vasodilators include sodium nitroprusside, hydralazine, and their derivatives.

[0253] Examples of adrenergic agonists include alpha-2 agonists (e.g., clonidine, guanabenz, methyldopa, and moxonidine).

[0254] Examples of additional antihypertensive drugs include guanethidine and reserpine.

[0255] The second agent may be used in combination with the therapeutic compounds described herein to reduce RAAS pathway-related diseases, disorders and / or conditions in the subject, such as hypertension and organ damage.

[0256] The present invention kit According to another aspect of the present invention, a kit is provided. The kit according to the present invention comprises a package containing either the composition of the present invention or an oligomer compound of the present invention. In various embodiments, the kit contains either the composition of the present invention as a unit dose. For the purposes of this specification, “unit dose” refers to a discrete amount dispersed in a suitable carrier.

[0257] The term "package" means any container containing the compositions presented herein. In preferred embodiments, the package may be a box or packaging. Packaging materials for use in packaging pharmaceutical products are well known to those skilled in the art. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes (including pre-filled syringes), bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0258] The kit may also include items that are not included inside the package but are attached to the outside of the package, such as pipettes.

[0259] The kit may, if necessary, include instructions for administering the composition of the present invention to a subject having a condition requiring treatment. The kit may also include instructions for the approved use of the components of the composition herein by a regulatory body, e.g., the U.S. Food and Drug Administration. The kit may, if necessary, include labeling or product inserts for the composition. The packaging and / or any product inserts may themselves be approved by a regulatory body. The kit may include the composition in a solid phase or the composition in a liquid phase (e.g., a buffer provided) provided in the package. The kit may also include a buffer for preparing a solution for carrying out the method, and a pipette for transferring the liquid from one container to another.

[0260] The kit may also include, if necessary, one or more other compositions for use in combination therapies as described herein. In certain embodiments, the package is a container for any means of administration such as intravitreous delivery, intraocular delivery, intratumoral delivery, peritumoral delivery, intraperitoneal delivery, intrathecal delivery, intramuscular injection, subcutaneous injection, intravenous delivery, intraarterial delivery, intraventricular delivery, intrasternal delivery, intracranial delivery, or intradermal injection.

[0261] Method of use and compounds for use The present invention provides a method for inhibiting the expression of AGT in a subject, and a method for treating RAAS-related diseases, disorders and / or conditions or symptoms thereof in a subject, including administering an effective amount of a dsRNA compound of the present invention or a pharmaceutical composition of the present invention, thereby inhibiting the expression of AGT in the subject. The present invention also provides a compound for use in a therapeutic use for treating or preventing RAAS-related diseases, disorders and / or conditions in a subject.

[0262] In some aspects of the present disclosure, the subject is a mammal, including mammalian rodents (such as mice and hamsters), mammalian lagomorphs (such as rabbits), mammalian carnivores including felines (cats) and canines (dogs), mammalian artiodactyls including bovines (cows) and suids (pigs), or mammalian perissodactyls including equines (horses), but not limited thereto. In some aspects, the mammal is of the order Primates, suborder Omaloxylinae, or infraorder Simiiformes (monkeys), or infraorder Catarrhini (humans and apes). In a preferred aspect, the mammal is a human.

[0263] (Advantages of the present invention) Disclosed herein are dsRNA compounds (e.g., siRNA) that target AGT and are improved with Advanced RNA Targeting (ARNATAR) capabilities to enhance gene silencing activity. The dsRNA compounds utilize an ARNATAR motif in combination with an AGT target sequence to generate a stable and persistent therapeutic compound, enabling longer-term benefits for acute and chronic diseases, and / or enabling less frequent administration of the therapeutic compound. In addition to stability and persistence, dsRNA compounds that utilize the ARNATAR motif may have a more rapid mode of action (e.g., by knocking down AGT expression at an earlier time point than a reference compound), which would be beneficial for acute diseases. In some examples, ARNATAR dsRNA has been found to be more potent than a reference compound.

[0264] Another advantage of ARNATAR-designed dsRNAs targeting AGT is their short length. This short length allows for shorter synthesis protocols and shorter synthesis times, reducing the cost of compound production.

[0265] Furthermore, ARNATAR-designed dsRNA compounds are extremely potent inhibitors of AGT. Their high potency allows for AGT reduction in tissues other than the liver.

[0266] Therefore, there is a need for improved dsRNA compounds to treat diseases. ARNATAR dsRNA compounds targeting AGT are designed to improve speed, stability, specificity, safety, and efficacy in order to produce improved therapeutic agents.

[0267] (Examples) While the specific compounds, compositions, and methods described herein are described in a particular manner according to specific embodiments, the following examples are merely illustrative of the compounds described herein and are not intended to limit them. Each of the references cited herein is incorporated herein by reference in its entirety.

[0268] Example 1: Design of AGT siRNAs with various chemical motifs Angiotensinogen (AGT) transcripts (Table 1) were targeted with siRNAs designed with an ARNATAR motif.

[0269] Table 1: AGT target sequences JPEG2026520610000011.jpg1694

[0270] AGT-targeted siRNAs (also known as AGT siRNAs) were designed as shown in Table 2.

[0271] The following applies to all modified sequences disclosed herein: Each nucleotide is preceded by a notation indicating the type of chemical modification (if any) performed on it. If no modification notation is given before the nucleotide, that nucleotide is a deoxyribonucleotide. Chemical modifications to the chain can be found as follows: (5p) = 5'-phosphate r = ribonucleotide d (or no notation before nucleotide) = deoxyribonucleotide replaced by ribonucleotide f = 2' - F m = 2' - OMe * = Phosphothioate (PS) bond replaced by phosphate (PO) bond grona = glycol nucleic acid If multiple sequences are disclosed in a single row of the table, the sequence number applies to the modified sequence ("sequence + chemistry").

[0272] Each ARNATAR sense strand is conjugated with a GalNAc-containing moiety described by Sharma (Sharma et al., 2018, Bioconjugate Chem, 29:2478-2488, incorporated herein by reference; also known herein as GalNAc3 or GA3; Gene Link, Elmsford, NY, USA) or galnac from AM Chemicals (US Patent 10,087,208, incorporated herein by reference; also known herein as GalNAc2 or GA2). For control, a reference siRNA (also known herein as ATXL-G or benchmark sirnas) was synthesized and included in the studies described herein. This reference siRNA ATXL-G (Table 2A) reflects the compound known in the prior art as compound AD85481 (WO2019 / 222166, incorporated herein by reference), which is considered to be Zilebesiran currently in clinical trials.

[0273] Table 2. AGT siRNA with modifications on both strands. JPEG2026520610000012.jpg210159JPEG2026520610000013.jpg108159

[0274] Table 2A. Reference siRNA JPEG2026520610000014.jpg46159

[0275] In vitro screening of siRNA targeting human AGT mRNA in cells siRNA was incubated with human primary hepatocytes (HPH) and taken up by the cells via free uptake. Specifically, siRNA was incubated with cells in the absence of transfection reagents and entered the cells via endocytosis mediated by the interaction of a GalNAc conjugate with the ASGR receptor. Cells were incubated at various final concentrations for 41–48 hours, and total RNA was prepared using AcroPrep Advance 96-well filter plates (Pall Corporation, Port Washington, New York) and Qiagen's RLT, RW1, and RPE buffers. AGT mRNA levels were determined by qRT-PCR using the AGT-specific primer-probe sets listed in Table 3. The qRT-PCR results were analyzed using the AgPath-ID protocol on a QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). TM The procedure was performed using a One-Step RT-PCR reagent. The AGT target RNA levels detected by the qRT-PCR assay were measured using RiboGreen TM The levels were normalized to the total RNA level measured by ThermoFisher Scientific (Waltham, MA, USA). The results are shown in Figure 1. The IC50 was calculated and is shown in Table 4.

[0276] Table 3: Sequence of human AGT primer probe sets JPEG2026520610000015.jpg24133

[0277] Table 4: AGT siRNA inhibition in HPH cells after 48 hours JPEG2026520610000016.jpg8286*ATXL-G was synthesized in two batches: "old" and "DR" #The sequences and chemistries of these siRNAs are listed in Table 7

[0278] The results showed that some ARNATAR-designed siRNAs appeared to be more potent than the reference siRNA ATXL-G targeting AGT.

[0279] Example 2: In vitro comparison of ARNATAR modification and ESC / ESC+ modification for different siRNA sequences The above results suggest that the ARNATAR chemical modification motif produced potent AGT siRNAs. To further determine the effects of different siRNA modification motifs, siRNAs were designed to target five different regions of human AGT. For comparison, siRNAs were designed with the ARNATAR motif or with a third-party structure and modification design (ESC or ESC+ described in Hu et al., Therapeutic siRNA: State of the Art, Signal Transduction and Targeted Therapy, 2020, 5:101; Zilebesaran uses the ESC+ modification motif). Since the ESC / ESC+ motif has a different chain length and motif from the ARNATAR motif, the AGT siRNA antisense sequences were aligned from the 5'-end to maintain a consistent seed sequence. Furthermore, siRNAs were made without GalNac conjugation to compare only the chemical modification motifs. ARNATAR-designed siRNAs have a 5'-phosphate on the antisense strand. The AGT siRNA sequences and chemistries are listed in Table 5. The third-party design is represented by the letters "AL" or "AL1".

[0280] Table 5: Oligomeric compounds targeting different regions of AGT mRNA JPEG2026520610000017.jpg209159JPEG2026520610000018.jpg129159++gnaG was unavailable, so 612-Ala has a different arrangement of gna modification than the usual ESC+ chemistry, however, gna at position 6 is also widely used in drug discovery (see PCT / US2019 / 032150). Since +++gnaG is unavailable, 598a-AL does not contain gna at position 7 and is similar to ESC chemistry.

[0281] In vitro assay: Comparison of ARNATAR motifs and third-party motifs AGT siRNA without a GalNac conjugate was transfected into Hep3B cells at 0–10 nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for a further 24 hours. siRNA activity was determined by qRT-PCR using the primer-probe sets listed in Table 3. The IC50 values ​​of AGT-targeting siRNAs are shown in Table 6, and the dose-response curves of siRNAs to AGT mRNA levels in Hep3B cells are shown in Figure 2.

[0282] Table 6: siRNA activity targeting AGT in Hep3B cells JPEG2026520610000019.jpg6466

[0283] The results show that the optimized ARNATAR design is more potent than the third-party design (P<0.0001) for all these different sequences, indicating that the optimized ARNATAR motif is generalizable and that the enhanced activity applies to different sequences.

[0284] Example 3: ARNATAR AGT siRNA showed better activity and longer duration in animals compared to reference siRNA. To compare the activity and duration of siRNAs in vitro and in vivo with reference to siRNA ATXL-G, selected ARNATAR siRNAs with different chemistries were designed and synthesized using GalNAc3 (Table 7).

[0285] Table 7: AGT oligomeric compounds with modifications on both strands JPEG2026520610000020.jpg97159

[0286] In vitro assay siRNAs were delivered to human primary hepatocytes (HPH) at various final concentrations through free uptake. That is, the siRNAs were incubated with the cells in the absence of transfection reagents and entered the cells through endocytosis mediated by GalNAc conjugate and ASGR receptor interaction. The cells were incubated for 42 hours, and total RNA was prepared using an AcroPrep Advance 96-well filter plate (Pall Corporation, Port Washington, New York) and Qiagen's RLT, RW1, and RPE buffers. The AGT mRNA levels were determined by qRT-PCR using the AGT-specific primer-probe sets listed in Table 3. qRT-PCR was performed using the AgPath-ID TM One-Step RT-PCR reagent in a QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). The AGT RNA levels detected in the qRT-PCR assay were normalized to the total RNA levels measured by RiboGreen TM (ThermoFisher Scientific, Waltham, MA, USA). The results are shown in Figure 3. The IC50 was calculated and shown in Table 8.

[0287] Table 8: IC50 of siRNA inhibition of AGT mRNA in HPH JPEG2026520610000021.jpg18105

[0288] The results showed that ARNATAR siRNA compounds such as ATsi483 exhibited activity equivalent to that of the reference siRNA ATXL-G.

[0289] In vivo study 1 To evaluate human AGT siRNA activity in vivo, a mouse model expressing human AGT was created. Seven-week-old BALB / c mice were intravenously injected with adeno-associated virus (AAV) serotype AAV8 expressing human AGT mRNA (GenBank NM_001382817.3; Sands MS, AAV-Mediated Liver-Directed Gene Therapy, 2014, Methods Mol Biol, 807:141-157). At least three weeks after viral administration, blood was collected from the mice using lithium heparin as an anticoagulant, and plasma was prepared by centrifugation at 2000×g for 10 minutes at 4°C. Plasma AGT protein levels were then determined using a human AGT-specific ELISA kit (IBL, Minneapolis, MN. Catalog No. 27412). The AGT levels in individual animals at this point were used as the baseline for AGT (protein) expression.

[0290] To determine siRNA activity in vivo, siRNA was subcutaneously administered to AGT transgenic mice at 1 mg / kg (ATsi483, ATsi484, and ATXL-G) or 3 mg / kg (ATsi483, ATsi484, ATsi600, and ATXL-G) the day after baseline blood collection (N=4-6 for each siRNA dose cohort). Blood samples were then collected at specified time points after siRNA administration, and plasma AGT protein levels were measured by ELISA. The mean AGT protein levels relative to baseline levels (measured 1 day prior to administration) are shown in Figure 4.

[0291] The results showed that ARNATAR siRNA exhibited comparable (ATsi483 and ATsi484) or better (ATsi600) activity in vivo compared to the reference siRNA ATXL-G, and showed similar durations over the tested timeframe of over 6 weeks. Interestingly, some siRNAs, such as ATsi484 and ATsi600, appeared to be slightly less active than the reference siRNA in cell culture with free uptake, but showed similar or better activity in animals. These results suggest that some differences in activity exist between cell culture and in vivo systems.

[0292] Example 4. In vivo evaluation of different sequences and modifications To further evaluate the effects of sequence and chemical motifs on AGT siRNA activity, novel ARNATAR siRNAs containing different sequences, different modification motifs, or different lengths were synthesized. The siRNAs were conjugated with GalNAc3 (GA3 from Gene Link, Elmsford, NY, USA). The siRNAs are shown in Table 9.

[0293] Table 9. AGT oligomer compounds with modifications on both chains JPEG2026520610000022.jpg131159

[0294] To create a mouse model expressing human AGT, 7-week-old BALB / c mice were intravenously injected with adeno-associated virus (AAV) serotype AAV8 expressing human AGT mRNA (GenBank NM_001382817.3; Sands MS, AAV-Mediated Liver-Directed Gene Therapy, 2014, Methods Mol Biol, 807:141-157). At least 3 weeks after viral administration, blood was collected from the mice, and plasma was prepared by centrifugation at 2000×g for 10 minutes at 4°C. Plasma AGT protein levels were then measured using a human AGT-specific ELISA kit (IBL, Minneapolis, MN. catalog number 27412). The AGT levels in individual animals at this point were used as the baseline for AGT (protein) expression.

[0295] To measure siRNA activity in vivo, siRNA was subcutaneously administered at a dose of 3 mg / kg the day after baseline blood collection (N=3-4). Subsequently, blood samples were collected at a specified time after siRNA administration, and AGT protein levels in the plasma were measured by ELISA. The mean AGT protein levels relative to baseline levels (measured 1 day prior to administration) are shown in Figure 5. In previous experiments (Example 3, Figure 4), the AGT level at the final time point (31 days) for the reference compound ATXL-G was approximately 40%, similar to that of ATsi610 and ATsi643. The results showed that certain siRNA compounds, such as ATsi643 and ATsi610, were comparable to the reference siRNA in terms of both activity and duration.

[0296] Example 5: In vitro and in vivo evaluation of additional sequences and modifications To further evaluate the effects of sequence and chemical motifs on siRNA activity, novel ARNATAR siRNAs were synthesized with different sequences, different modification motifs, different lengths, or containing native or TT overhangs. For example, the sequences of ATsi603 and ATsi612 contain mismatch mutations relative to SEQ ID NO: 1. The siRNAs were conjugated with GalNAc3 (GA3 from Gene Link, Elmsford, NY, USA). The siRNAs are shown in Table 10.

[0297] Table 10: AGT oligomer compounds with modifications on both chains JPEG2026520610000023.jpg141159

[0298] In vitro assay For better comparison, the two siRNAs evaluated above (ATsi600 and ATsi610) were also included in this evaluation of additional ARNATAR siRNAs.

[0299] siRNA was delivered to human primary hepatocytes (HPH) at different final concentrations via free uptake. Specifically, siRNA was incubated with cells in the absence of transfection reagents, and entry into the cells was via endocytosis mediated by the interaction of a GalNAc conjugate with the ASGR receptor. Cells were incubated for 42 hours, and total RNA was prepared using AcroPrep Advance 96-well filter plates (Pall Corporation, Port Washington, New York) and Qiagen's RLT, RW1, and RPE buffers. AGT mRNA levels were measured by qRT-PCR using the AGT-specific primer-probe sets listed in Table 3. qRT-PCR was performed using the AgPath-ID QS3 real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA). TM The procedure was performed using a One-Step RT-PCR reagent. The AGT RNA levels detected by the qRT-PCR assay were measured using RiboGreen TMThe levels were normalized to the total RNA level measured by ThermoFisher Scientific (Waltham, MA, USA). The results are shown in Figure 6. The IC50 was calculated and is shown in Table 11.

[0300] Table 11: IC50 of AGT mRNA siRNA inhibition in human primary hepatocytes JPEG2026520610000024.jpg19147

[0301] In vivo research To create a mouse model expressing human AGT, 7-week-old BALB / c mice were intravenously injected with adeno-associated virus (AAV) serotype AAV8 expressing human AGT mRNA (GenBank NM_001382817.3; Sands MS, AAV-Mediated Liver-Directed Gene Therapy, 2014, Methods Mol Biol, 807:141-157). At least 3 weeks after viral administration, blood was collected from the mice, and plasma was prepared by centrifugation at 2000×g for 10 minutes at 4°C. Plasma AGT protein levels were then measured using a human AGT-specific ELISA kit (IBL, Minneapolis, MN. Catalog No. 27412). The AGT levels in individual animals at this point were used as the baseline for AGT (protein) expression.

[0302] To measure siRNA activity in vivo, selected siRNAs ATsi603, ATsi612, ATsi665, and benchmark siRNA ATXL-G were subcutaneously administered at 3 mg / kg the day after baseline blood collection (N=2-4). Subsequently, blood samples were collected at specified times after siRNA administration, plasma was prepared, and AGT protein levels in the plasma were measured by ELISA. The mean AGT protein levels relative to baseline levels (measured 1 day prior to administration) are shown in Figure 7 and Table 12.

[0303] Table 12. Plasma AGT protein levels as a percentage of baseline AGT protein levels. JPEG2026520610000025.jpg23108

[0304] The results again showed that while certain siRNAs, such as ATsi612, exhibited slightly lower activity in vitro, in vivo this siRNA compound was slightly better than or equivalent to the reference siRNA in terms of both activity and duration.

[0305] Example 6: In vitro and in vivo evaluation of selected oligomer compounds To further evaluate the effect of GalNAc conjugates on siRNA activity, we designed novel ARNATAR siRNAs containing the sequences and chemical motifs of ATsi603 or ATsi612 (see Table 10), but conjugated to different GalNAc-containing moieties (GA-AN or GA4, also known as GalNAc-AN; see above for a description of GA4). The novel siRNAs are shown in Table 13.

[0306] Table 13: AGT oligomer compounds with modifications on both chains JPEG2026520610000026.jpg81159

[0307] In vitro assay: Time course in Hep3B cells AGT-targeted siRNAs ATsi603, ATsi786, ATsi787, and benchmark siRNA ATXL-G were transfected into Hep3B cells at concentrations of 0nM, 0.008nM, 0.04nM, 0.2nM, 1nM, and 5nM using RNAiMAX (Invitrogen, Waltham, MA), and the cells were cultured for a further 8 or 13 hours. siRNA activity was measured by qRT-PCR using the primer-probe sets listed in Table 3.

[0308] AGT RNA levels detected by qRT-PCR assay are measured by RiboGreen TMThe levels were normalized to total RNA levels measured by ThermoFisher Scientific (Waltham, MA, USA). The percentage of AGT mRNA levels is shown in Figure 8. ATsi786 and ATsi603 were more potent than ATXL-G, with P=0.0047 and 0.0033 at 8 hours and P=0.0019 and 0.0385 at 12 hours.

[0309] In vivo assay To create a mouse model expressing human AGT, 7-week-old BALB / c mice were intravenously injected with adeno-associated virus (AAV) serotype AAV8 expressing human AGT mRNA (GenBank NM_001382817.3; Sands MS, AAV-Mediated Liver-Directed Gene Therapy, 2014, Methods Mol Biol, 807:141-157). At least 3 weeks after viral administration, blood was collected from the mice, and plasma was prepared by centrifugation at 2000×g for 10 minutes at 4°C. Plasma AGT protein levels were then measured using a human AGT-specific ELISA kit (IBL, Minneapolis, MN. catalog number 27412). The AGT levels in individual animals at this point were used as the baseline for AGT (protein) expression.

[0310] To measure siRNA activity in vivo, AGT-targeted siRNAs ATsi786, ATsi787, and benchmark siRNA ATXL-G were subcutaneously administered at 3 mg / kg the day after baseline blood collection (N=3-4). Subsequently, blood samples were collected weekly for up to 6 or 7 weeks after siRNA administration, plasma was prepared, and AGT protein levels in the plasma were measured by ELISA. The mean AGT protein levels relative to baseline levels (measured 1 day prior to administration) are shown in Figure 9. As shown in Figure 9, ATsi786 was slightly more potent than benchmark ATXL-G in reducing AGT expression at several time points.

[0311] Example 7: Evaluation of selected oligomer compounds in AGT transgenic mice To evaluate the initial dynamics of human AGT siRNA activity in vivo, a mouse model expressing human AGT was created. 7-8 week old BALB / c mice were injected with adeno-associated virus (AAV) serotype AAV8 expressing human AGT mRNA (GenBank NM_001382817.3; Sands MS, AAV-Mediated Liver-Directed Gene Therapy, 2014, Methods Mol Biol, 807:141-157) at a dose of 2.5 x 10⁻¹ per animal. 11 The viral genome was administered intravenously. Approximately three weeks after viral administration, blood was collected from mice using lithium heparin as an anticoagulant, and plasma was prepared by centrifuging the blood samples at 2000×g for 10 minutes at 4°C. Plasma AGT protein levels were then measured using a human AGT-specific ELISA kit (IBL, Minneapolis, MN. catalog number 27412). The AGT levels in individual animals at this point were used as a baseline for AGT (protein) expression.

[0312] To measure siRNA activity in vivo, human AGT siRNA ATsi786 or benchmark ATXL-G was administered subcutaneously (SC) at a dose of 3 mg / kg on day 0 (N=3 in each group). Approximately 50 μl of blood was collected by buccal bleeding on day -1 (baseline) and on days 3, 5, 7, and 10, placed in Microvette(R) CB300 lithium heparin LH tubes (Sarstedtstr, Numbrecht, Germany), and kept on ice for at least 20 minutes. Plasma was prepared by centrifugating blood samples at 2000 × g for 20 minutes at 4°C using a 5425R centrifuge (Eppendorf, Hamburg, Germany), and collecting the supernatant. Human AGT protein levels in plasma (1:1000 dilution) on days -1 and 10 were measured using the Human Whole Angiotensinogen Assay Kit (product number 27412; IBL-America, Minneapolis, MN, USA) according to the manufacturer's protocol. Relative AGT protein levels at different time points were calculated in Excel as the percentage of AGT protein levels on day -1 in individual animals. The mean percentage for each group was calculated in Excel as shown in Table 14 and plotted as shown in Figure 10.

[0313] Table 14: Mean expression levels of AGT protein at various time points. JPEG2026520610000027.jpg43104

[0314] The results show that levels of human AGT protein in plasma were rapidly reduced by administration of ATsi786 siRNA to animals. ATsi786 reduced human AGT protein faster than the benchmark ATXL-G (P=0.0201).

Claims

1. A double-stranded ribonucleic acid (dsRNA) compound for inhibiting the expression of angiotensinogen (AGT) in cells, wherein the dsRNA compound comprises a sense strand and an antisense strand forming a double-stranded portion, the sense strand comprising the nucleotide sequence of SEQ ID NO: 61, and the antisense strand comprising the nucleotide sequence of SEQ ID NO:

62.

2. The double-stranded ribonucleic acid (dsRNA) compound according to the prior claim, wherein the dsRNA compound is an shRNA or siRNA compound.

3. The double-stranded ribonucleic acid (dsRNA) compound according to any of the prior claims, wherein the dsRNA compound comprises at least one modified nucleotide.

4. A double-stranded ribonucleic acid (dsRNA) compound according to any of the prior claims, wherein substantially all of the nucleotides in the sense strand are modified nucleotides, substantially all of the nucleotides in the antisense strand are modified nucleotides, or substantially all of the nucleotides in both the sense strand and the antisense strand are modified nucleotides.

5. A double-stranded ribonucleic acid (dsRNA) compound according to any of the prior claims, wherein all nucleotides of the sense strand are modified nucleotides, or all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of both the sense strand and the antisense strand are modified nucleotides.

6. A double-stranded ribonucleic acid (dsRNA) compound according to any of the prior claims, wherein the chain comprises at least one phosphorothioate nucleotide (PS) bond.

7. A double-stranded ribonucleic acid (dsRNA) compound according to any of the prior claims, wherein the sense strand comprises one nucleotide sequence of SEQ ID NOs: 8, 33, and 55, and / or the antisense strand comprises one nucleotide sequence of SEQ ID NOs: 9 and 19.

8. The double-stranded ribonucleic acid (dsRNA) according to any of the prior claims, further comprising a conjugate portion.

9. The double-stranded ribonucleic acid (dsRNA) according to claim 8, wherein the conjugate portion is an N-acetylgalactosamine (GalNAc) containing portion.

10. The double-stranded ribonucleic acid (dsRNA) according to claim 8 or 9, wherein the conjugate portion is bound to the 3' end of the sense strand.

11. The double-stranded ribonucleic acid (dsRNA) compound according to any one of the prior claims, wherein the compound inhibits the expression of angiotensinogen (AGT) in cells by at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

12. A pharmaceutical composition for inhibiting the expression of angiotensinogen (AGT) in cells, wherein the pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA) compound described in any of the prior claims, either alone or in combination with a pharmaceutically acceptable carrier or excipient.

13. A method for inhibiting the expression of angiotensinogen (AGT) in cells, comprising contacting the cells with a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12 in an amount sufficient to inhibit the expression of AGT, thereby inhibiting the expression of AGT in the cells.

14. A method for treating RAAS-related diseases, disorders and / or conditions in a subject, comprising administering to the subject in need of such treatment a therapeutically effective amount of a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12, thereby treating the RAAS-related diseases, disorders and / or conditions in the subject.

15. The aforementioned RAAS-related diseases, disorders and / or conditions include hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertension associated with low plasma renin activity or plasma renin concentration, increased intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, and vascular disorders. The method according to claim 14, selected from the group consisting of: diabetes, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), impaired glucose tolerance, type 2 diabetes (non-insulin-dependent diabetes), and metabolic syndrome.

16. The method according to any one of claims 13 to 15, wherein the compound or pharmaceutical composition inhibits AGT expression by at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.

17. A method for treating a subject having a disease, disorder and / or condition that would benefit from reduced angiotensinogen (AGT) expression, comprising administering to the subject in need of such treatment a therapeutically effective amount of a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12, thereby treating the subject having the disorder that would benefit from reduced AGT expression.

18. A method for preventing at least one symptom in a subject having a disorder that would benefit from reduced angiotensinogen (AGT) expression, comprising administering to the subject in need a preventive amount of a compound according to any one of claims 1 to 11 or a pharmaceutical composition according to claim 12, thereby preventing at least one symptom in the subject having the disorder that would benefit from reduced AGT expression.

19. The method according to claim 17 or 18, wherein the disorder is a RAAS-related disease, disorder and / or condition.

20. The aforementioned RAAS-related diseases, disorders and / or conditions include hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertension associated with low plasma renin activity or plasma renin concentration, increased intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, and vascular disorders. The method according to claim 19, selected from the group consisting of: diabetes, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), impaired glucose tolerance, type 2 diabetes (non-insulin-dependent diabetes), and metabolic syndrome.

21. The method according to any one of claims 13 to 20, further comprising administering to the subject an additional therapeutic agent for the treatment of RAAS-related diseases, disorders and / or conditions in the subject.

22. A kit comprising a compound according to any one of claims 1 to 11, or a pharmaceutical composition according to claim 12, and optionally a label.

23. A process for preparing a sense strand and / or antisense strand of a double-stranded ribonucleic acid (dsRNA) compound according to any one of claims 1 to 11, wherein the process is: a. A step of preparing sense strands and / or antisense strands by sequential coupling of modified and / or unmodified nucleotides via phosphoramidite oligonucleotide synthesis on a solid support, b. Optionally, the step of coupling the GalNAc-containing portion to the sense chain and / or antisense chain on the solid support via phosphoramidite oligonucleotide synthesis, c. The step of detaching the sense chain and / or the antisense chain from the solid phase support and removing the solid phase support, d. Optionally, further purifying the sense strand and / or the antisense strand using chromatography, A process that includes this.

24. A process for preparing a sense strand and / or antisense strand of a double-stranded ribonucleic acid (dsRNA) compound according to any one of claims 1 to 11, wherein the process is: a. A step of coupling a GalNAc-containing portion to a solid support via phosphoramidite oligonucleotide synthesis, b. A step of coupling modified and / or unmodified nucleotides to the GalNAc-containing portion on the solid support via phosphoramidite oligonucleotide synthesis, c. The step of preparing the sense strand and / or antisense strand by sequentially coupling additional modified and / or unmodified nucleotides via phosphoramidite oligonucleotide synthesis, d. The step of detaching the sense chain and / or the antisense chain from the solid support and removing the solid support, e. Optionally, further purifying the sense strand and / or the antisense strand using chromatography, A process that includes this.

25. A process for preparing a double-stranded ribonucleic acid (dsRNA) compound according to any one of claims 1 to 11, a) The step of contacting a sense chain prepared according to claim 23 or 24 with an antisense chain prepared according to claim 23 or 24 in an equimolar concentration in a solution, b) Optionally, the step of heating the solution to a temperature of approximately 94°C, c) Optionally, the step of lowering the temperature of the solution to approximately 25°C, A process that includes this.

26. A double-stranded ribonucleic acid (dsRNA) compound defined in any one of claims 1 to 11, for use in pharmaceuticals.

27. A double-stranded ribonucleic acid (dsRNA) compound as defined in any one of claims 1 to 11, for use in treating or preventing RAAS-related diseases, disorders and / or conditions in a subject.

28. RAAS-related diseases, disorders and / or conditions include hypertension, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, hypertension associated with low plasma renin activity or plasma renin concentration, increased intraocular pressure, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disorders, diabetic nephropathy, and diabetes. A double-stranded ribonucleic acid (dsRNA) compound for use according to claim 27, selected from the group consisting of diseased retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), impaired glucose tolerance, type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), and metabolic syndrome.

Images