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EP-4741504-A1 - DSRNA MOLECULE FOR REGULATING EXPRESSION OF AGT

EP4741504A1EP 4741504 A1EP4741504 A1EP 4741504A1EP-4741504-A1

Abstract

Provided are a double-stranded RNA for inhibiting the expression of angiotensinogen (AGT) in cells; a vector and cell comprising an encoding nucleotide thereof; and a method for using the dsRNA, vector or cell to treat a disease or symptom mediated by or associated with the expression of AGT in a subject.

Inventors

  • HUANG, JINYU
  • YE, Piao

Assignees

  • Rona Bioscience, Limited

Dates

Publication Date
20260513
Application Date
20240705

Claims (20)

  1. A double-stranded nucleotide (dsRNA) for inhibiting the expression of angiotensinogen (AGT) in a cell, the dsRNA comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 21-40.
  2. The dsRNA of claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-20.
  3. The dsRNA of claim 1 or 2, wherein the dsRNA is an siRNA.
  4. The dsRNA of any one of claims 1-3, wherein the double-stranded region is 15-25 nucleotide pairs, preferably 16-23 nucleotide pairs, or more preferably 18-20 nucleotide pairs in length.
  5. The dsRNA of any one of claims 1-4, wherein the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, a nucleotide sequence of at least 18 contiguous nucleotides, a nucleotide sequence of at least 19 contiguous nucleotides, or a nucleotide sequence of at least 20 contiguous nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 21-40, and preferably the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 21-40.
  6. The dsRNA of any one of claims 1-5, wherein the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, or a nucleotide sequence of at least 18 contiguous nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-20, and preferably the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-20.
  7. The dsRNA of any one of claims 1-6, wherein the siRNA comprises any of the following paired of sense strand sequences and antisense strand sequences: (1) sense strand: SEQ ID NO: 1, antisense strand: SEQ ID NO: 21; (2) sense strand: SEQ ID NO: 2, antisense strand: SEQ ID NO: 22; (3) sense strand: SEQ ID NO: 3, antisense strand: SEQ ID NO: 23; (4) sense strand: SEQ ID NO: 4, antisense strand: SEQ ID NO: 24; (5) sense strand: SEQ ID NO: 5, antisense strand: SEQ ID NO: 25; (6) sense strand: SEQ ID NO: 6, antisense strand: SEQ ID NO: 26; (7) sense strand: SEQ ID NO: 7, antisense strand: SEQ ID NO: 27; (8) sense strand: SEQ ID NO: 8, antisense strand: SEQ ID NO: 28; (9) sense strand: SEQ ID NO: 9, antisense strand: SEQ ID NO: 29; (10) sense strand: SEQ ID NO: 10,antisense strand: SEQ ID NO: 30; (11) sense strand: SEQ ID NO: 11,antisense strand: SEQ ID NO: 31; (12) sense strand: SEQ ID NO: 12,antisense strand: SEQ ID NO: 32; (13) sense strand: SEQ ID NO: 13,antisense strand: SEQ ID NO: 33; (14) sense strand: SEQ ID NO: 14,antisense strand: SEQ ID NO: 34; (15) sense strand: SEQ ID NO: 15,antisense strand: SEQ ID NO: 35; (16) sense strand: SEQ ID NO: 16,antisense strand: SEQ ID NO: 36; (17) sense strand: SEQ ID NO: 17,antisense strand: SEQ ID NO: 37; (18) sense strand: SEQ ID NO: 18,antisense strand: SEQ ID NO: 38; (19) sense strand: SEQ ID NO: 19,antisense strand: SEQ ID NO: 39; and (20) sense strand: SEQ ID NO: 20,antisense strand: SEQ ID NO: 40.
  8. The dsRNA of any one of claims 1-7, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.
  9. The dsRNA of any one of claims 1-8, wherein each of the sense strand and the antisense strand independently comprises one or more nucleotide modifications selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, a SCP modification, a glycol modification, and a phosphorothioate internucleotide linkage modification.
  10. The dsRNA of any one of claims 1-9, wherein the antisense strand has a length of 21 nucleotides and has (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (numbered from the 5' end); and/or (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (numbered from the 5' end).
  11. The dsRNA of any one of claims 1-9, wherein the antisense strand has a length of 21 nucleotides and has (i) 2'-O-methyl modified nucleotides at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (numbered from the 5' end); (ii) a 2'-fluoro modified nucleotide at position 14 (numbered from the 5' end); (iii) 2'-deoxy modifications at positions 2, 5, 7, and 12 (numbered from the 5' end); (iv) an SCP modification at position 1 (numbered from the 5' end); and/or (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (numbered from the 5' end).
  12. The dsRNA of any one of claims 1-9, wherein the antisense strand has a length of 21 nucleotides and has (i) 2'-O-methyl modified nucleotides at positions 3, 5, 9, 11, 13, 15, 17, 19, 20, and 21 (numbered from the 5' end); (ii) 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (numbered from the 5' end); (iii) a GNA modification at position 7 (numbered from the 5' end); (iv) an SCP modification at position 1 (numbered from the 5' end); and/or (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (numbered from the 5' end).
  13. The dsRNA of any one of claims 1-9, wherein the sense strand has a length of 19 nucleotides and has: (i) 2'-O-methyl modified nucleotides at positions 1 to 6, and 10 to 19, and 2'-fluoro modified nucleotides at positions 7-9 (numbered from the 5' end); and/or (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 (numbered from the 5' end).
  14. The dsRNA of any one of claims 8-12, wherein the antisense strand comprises a sequence selected from any one of the sequences set forth in SEQ ID NOs: 81-105.
  15. The dsRNA of any one of claims 8-9 and 13-14, wherein the sense strand comprises a sequence selected from any one of the sequences set forth in SEQ ID NOs: 41-59.
  16. The dsRNA of claim 15, wherein the dsRNA comprises any of the paired of sense strand sequences and antisense strand sequences selected from: sense strand: SEQ ID NO: 41, antisense strand: SEQ ID NO: 81; sense strand: SEQ ID NO: 42, antisense strand: SEQ ID NO: 82; sense strand: SEQ ID NO: 43, antisense strand: SEQ ID NO: 83; sense strand: SEQ ID NO: 44, antisense strand: SEQ ID NO: 84; sense strand: SEQ ID NO: 45, antisense strand: SEQ ID NO: 85; sense strand: SEQ ID NO: 46, antisense strand: SEQ ID NO: 86; sense strand: SEQ ID NO: 47, antisense strand: SEQ ID NO: 87; sense strand: SEQ ID NO: 48, antisense strand: SEQ ID NO: 88; sense strand: SEQ ID NO: 49, antisense strand: SEQ ID NO: 89; sense strand: SEQ ID NO: 50, antisense strand: SEQ ID NO: 90; sense strand: SEQ ID NO: 51, antisense strand: SEQ ID NO: 91; sense strand: SEQ ID NO: 52, antisense strand: SEQ ID NO: 92; sense strand: SEQ ID NO: 53, antisense strand: SEQ ID NO: 93; sense strand: SEQ ID NO: 54, antisense strand: SEQ ID NO: 94; sense strand: SEQ ID NO: 55, antisense strand: SEQ ID NO: 95; sense strand: SEQ ID NO: 56, antisense strand: SEQ ID NO: 96; sense strand: SEQ ID NO: 57, antisense strand: SEQ ID NO: 97; sense strand: SEQ ID NO: 58, antisense strand: SEQ ID NO: 98; and sense strand: SEQ ID NO: 59, antisense strand: SEQ ID NO: 99.
  17. The dsRNA of any one of claims 8-9 and 13-14, wherein the sense strand comprises a sequence selected from any one of the sequences set forth in SEQ ID NOs: 113-131.
  18. The dsRNA of claim 17, wherein the dsRNA comprises any of the paired of sense strand sequences and antisense strand sequences selected from: sense strand: SEQ ID NO: 113, antisense strand: SEQ ID NO: 81; sense strand: SEQ ID NO: 114, antisense strand: SEQ ID NO: 82; sense strand: SEQ ID NO: 115, antisense strand: SEQ ID NO: 83; sense strand: SEQ ID NO: 116, antisense strand: SEQ ID NO: 84; sense strand: SEQ ID NO: 117, antisense strand: SEQ ID NO: 85; sense strand: SEQ ID NO: 118, antisense strand: SEQ ID NO: 86; sense strand: SEQ ID NO: 119, antisense strand: SEQ ID NO: 87; sense strand: SEQ ID NO: 120, antisense strand: SEQ ID NO: 88; sense strand: SEQ ID NO: 121, antisense strand: SEQ ID NO: 89; sense strand: SEQ ID NO: 122, antisense strand: SEQ ID NO: 90; sense strand: SEQ ID NO: 123, antisense strand: SEQ ID NO: 91; sense strand: SEQ ID NO: 124, antisense strand: SEQ ID NO: 92; sense strand: SEQ ID NO: 125, antisense strand: SEQ ID NO: 93; sense strand: SEQ ID NO: 126, antisense strand: SEQ ID NO: 94; sense strand: SEQ ID NO: 127, antisense strand: SEQ ID NO: 95; sense strand: SEQ ID NO: 128, antisense strand: SEQ ID NO: 96; sense strand: SEQ ID NO: 129, antisense strand: SEQ ID NO: 97; sense strand: SEQ ID NO: 130, antisense strand: SEQ ID NO: 98; and sense strand: SEQ ID NO: 131, antisense strand: SEQ ID NO: 99.
  19. The dsRNA of any one of claims 1-18, wherein the dsRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine, and preferably the 3' end of the sense strand is conjugated to the ligand moiety.
  20. The dsRNA of any one of claim 19, wherein the ligand has the following structure: wherein represents the point of attachment to the sense strand of the dsRNA via a phosphate group or a phosphorothioate group.

Description

TECHNICAL FIELD The present disclosure relates to the field of RNA interference. BACKGROUND Angiotensinogen (AGT), an alpha globulin, is located upstream most in the blood pressure regulation system of the renin-angiotensin-aldosterone system (RAAS) and is the only precursor of all angiotensin peptides. Inactive AGT is converted to active angiotensin II (Ang-II) by sequential cleavage with renin and angiotensin converting enzymes. Then, the Ang-II acts through binding to its receptor (AT1-4). Angiotensinogen is mainly synthesized and secreted by the liver and is present in the plasma α-globulin fraction. In addition, angiotensinogen is also present in a variety of tissues that express local RAAS. Elevated angiotensinogen levels are associated with essential hypertension. Transgenic mice expressing rat angiotensinogen gene had hypertension, while mice lacking angiotensinogen gene had hypotension. Regulation of AGT production is multifactorial and occurs at the transcriptional and posttranscriptional levels. AngII itself, glucocorticoids, estrogens, thyroxine, growth hormone, and various cytokines affect the production of AGT. Physiological and pathological conditions such as pregnancy, use of oral contraceptives, increased glucocorticoid secretion (due to hyperadrenal function or chronic stress), and chronic inflammation can lead to increased AGT production. Conversely, deficiency of the pituitary, thyroid, gonadal, or adrenal is associated with low plasma AGT levels. The main source of circulating AGT is hepatocytes, which secrete AGT through constitutive pathways, resulting in relatively stable plasma levels, regardless of acute stress or cardiovascular changes. In addition to the liver, tissues such as adipocytes and epithelial cells of the proximal tubules of the kidney, and the brain also express Agt, and the generation of these tissues contributes to the activation of local ras. In the brain, AGT is mainly expressed in astrocytes (Milsted et al., 1990), while AT1 is present in brain endothelial cells (Wosik et al., 2007). Interestingly, mice lacking AGT showed blood-brain barrier rupture and disordered occlusion at tight junctions (Wosik et al., 2007), which strongly suggests that astrocytes maintain blood-brain barrier properties through the AGT-Ang-II-AT1 pathway. Furthermore, evidence of a positive correlation between BMI and circulating AGT levels suggests that AGT production in adipocytes contributes to circulating AGT levels. AGT is the first gene found to be associated with essential hypertension. Human genetic evidence and animal research evidence show that blocking or inhibiting the expression of AGT can bring significant antihypertensive effects. In this regard, one method of treating hypertension is to reduce AGT expression through small interfering RNA (siRNA) based on RNA interference mechanism. There is a need in the art for compositions and methods for treating diseases, disorders and conditions associated with angiotensinogen. Reducing AGT expression via double-stranded RNA (dsRNA), particularly small interfering RNA (siRNA), based on RNA interference mechanisms, is a novel approach to treat diseases, disorders and conditions associated with angiotensinogen. SUMMARY The present disclosure provides novel double-stranded RNAs (dsRNAs) for inhibiting the expression of angiotensinogen (AGT) in a cell, as well as a vector, a cell, and a kit and a pharmaceutical composition comprising the same, and a method of inhibiting or reducing angiotensinogen (AGT) gene expression or treating diseases or disorders benefiting from reduced angiotensinogen (AGT) expression with the dsRNA, vector, cell, as well as the pharmaceutical composition, and kit. In a first aspect, the present disclosure provides a double stranded nucleotide (dsRNA) for inhibiting the expression of angiotensinogen (AGT) in a cell, comprising a sense strand and an antisense strand that form a double-stranded region, wherein said sense strand and said antisense strand each independently have a length of 15-30 nucleotides, and said antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides from the nucleotide sequence set forth in any one of SEQ ID Nos: 21-40. In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides from the nucleotide sequence set forth in any one of SEQ ID Nos: 1-20. In some embodiments, the dsRNA is an siRNA. In some embodiments, the double-stranded region is 15-25 nucleotide pairs, preferably 16-23 nucleotide pairs, or more preferably 18-20 nucleotide pairs in length. In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, a nucleotide sequence of at least 18 contiguous nucleotides, a nucleotide sequence of at least 19 contiguous nucleotides, or a nucleotide sequence of at least 20 contiguous nucleotides from the nucleoti