Search

CN-122012497-A - RNAi construct for inhibiting ASGR1 gene expression and use thereof

CN122012497ACN 122012497 ACN122012497 ACN 122012497ACN-122012497-A

Abstract

The present invention relates to RNAi constructs that inhibit expression of asialoglycoprotein receptor 1 (ASGR 1) genes, pharmaceutical compositions comprising the same, and uses thereof. The RNAi constructs achieve disease prevention and/or treatment by inhibiting expression of ASGR1 gene in the liver.

Inventors

  • TANG JINGJIE
  • CHEN XIAOQIONG

Assignees

  • 珂阑(上海)医药科技有限公司

Dates

Publication Date
20260512
Application Date
20251110
Priority Date
20241111

Claims (20)

  1. 1. RNAi constructs inhibiting ASGR1 gene expression comprising an antisense strand and a sense strand, wherein The antisense strand comprises 17-23 contiguous nucleotides differing by 0,1, or 2 nucleotides from any of the antisense strand sequences provided in table 1 or table 2; The sense strand comprises a nucleotide sequence that is at least partially complementary to the antisense strand.
  2. 2. The RNAi construct of claim 1, wherein the antisense strand is a contiguous nucleotide differing by 0 or 1 nucleotide in any one of the antisense strand sequences provided in table 1 or table 2.
  3. 3. The RNAi construct of claim 1, wherein the antisense strand comprises, from 5 'to 3', a contiguous nucleotide selected from the group consisting of SEQ ID No. 137 to SEQ ID No. 219, SEQ ID No. 221 to SEQ ID No. 272, SEQ ID No. 432, or SEQ ID No. 504-510, or consists of a contiguous nucleotide selected from the group consisting of SEQ ID No. 137 to SEQ ID No. 219, SEQ ID No. 221 to SEQ ID No. 272, SEQ ID No. 432, or SEQ ID No. 504-510.
  4. 4. The RNAi construct of claim 1, wherein the antisense strand is selected from the group consisting of, from 5 'to 3' comprising, or consisting of, the contiguous nucleotides shown as SEQ ID NO: 137,138,142,145,147,164,165,166,178,210,212,229,239,241,242,244,249,250,252,257,258,267,269,270,271,504-510 or SEQ ID NO: 137,138,142,145,147,164,165,166,178,210,212,229,239,241,242,244,249,250,252,257,258,267,269,270,271,504-510.
  5. 5. The RNAi construct of claim 1, wherein the RNAi construct is selected from any one of the constructs provided in table 1.
  6. 6. The RNAi construct of claim 1, wherein the RNAi construct is selected from the group consisting of the constructs D-1001 (SEQ ID NO:1 and 137), D-1002 (SEQ ID NO:2 and 138), D-1006 (SEQ ID NO:6 and 142), D-1009 (SEQ ID NO:9 and 145), D-1011 (SEQ ID NO:11 and 147), D-1028 (SEQ ID NO:28 and 164), D-1029 (SEQ ID NO:29 and 165), D-1030 (SEQ ID NO:30 and 166), D-1042 (SEQ ID NO:42 and 178), D-1074 (SEQ ID NO:74 and 210), D-1076 (SEQ ID NO:76 and 212), D-1095 (SEQ ID NO:93 and 229), D-1105 (SEQ ID NO:103 and 239), D-1107 (SEQ ID NO:105 and 241), D-1108 (SEQ ID NO:106 and 242), D-1028 (SEQ ID NO:108 and 244), D-1115 (SEQ ID NO:42 and 178), D-1134 (SEQ ID NO:74 and 210), D-1076 (SEQ ID NO:76 and 212), D-1095 (SEQ ID NO:105 and 241), D-1108 (SEQ ID NO:106 and 242), D-1108 (SEQ ID NO:108 and 244), D-1115 (SEQ ID NO:113 and 135), d-1136 (SEQ ID NO:134 and 270), D-1137 (SEQ ID NO:135 and 271), D-1154 (SEQ ID NO:491 and 504), D-1155 (SEQ ID NO:492 and 505), D-1156 (SEQ ID NO: 493 and 506), D-1157 (SEQ ID NO: 494 and 507), D-1158 (SEQ ID NO: 495 and 508), D-1159 (SEQ ID NO: 496 and 509), D-1160 (SEQ ID NO: 497 and 510), preferably D-1074 (SEQ ID NO:74 and 210), D-1095 (SEQ ID NO:93 and 229), 1107 (SEQ ID NO:105 and 241), D-1108 (SEQ ID NO:106 and 242), D-1123 (SEQ ID NO:121 and 257), D-1115 (SEQ ID NO:113 and 249), D-1116 (SEQ ID NO:114 and 250), D-1118 (SEQ ID NO: and 252), D-1124 (SEQ ID NOS: 122 and 258), D-1154 (SEQ ID NOS: 491 and 504), D-1155 (SEQ ID NOS: 492 and 505), D-1156 (SEQ ID NOS: 493 and 506), D-1157 (SEQ ID NOS: 494 and 507), D-1158 (SEQ ID NOS: 495 and 508), D-1159 (SEQ ID NOS: 496 and 509), D-1160 (SEQ ID NOS: 497 and 510).
  7. 7. The RNAi construct of any one of claims 1-6, wherein at least one nucleotide of the sense strand and/or the antisense strand is or comprises a modified nucleotide, preferably all nucleotides are modified nucleotides.
  8. 8. The RNAi construct of claim 7, wherein the modified nucleotide is a deoxyribonucleotide, an abasic nucleotide, a2 '-modified nucleotide, an inverted nucleotide, a 2', 3 '-ring-opened nucleotide mimetic, a locked nucleotide, a 2' -F-arabinose nucleotide, a 5'-Me, a 2' -fluoro nucleotide, an inosine-containing nucleotide, or a combination thereof.
  9. 9. The RNAi construct of claim 7, wherein the modifying group in the modified nucleotide is selected from the group consisting of 2' -methoxy, 2' -methoxyalkyl, 2' -O-alkyl, 2' -O-allyl, 2' -C-allyl, 2' -fluoro, 2' -deoxy, 2' -O-N-methylacetamido (2-O-NMA), 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE), 2' -O-aminopropyl (2 ' -O-AP), 2' -ara-F, L-nucleoside modification, LNA, ENA, HNA, cET BNA, UNA, and ethylene Glycol Nucleotide (GNA) or a combination thereof, and the modified internucleoside linkage is a phosphorothioate linkage.
  10. 10. The RNAi construct of claim 7, wherein the modified antisense strand comprises from 5 'to 3' a contiguous nucleotide selected from the group consisting of SEQ ID No. 352 to SEQ ID No. 383, SEQ ID No. 385 to SEQ ID No. 416, SEQ ID No. 418 to SEQ ID No. 430 or SEQ ID No. 455 to SEQ ID No. 476, or consists of the contiguous nucleotides shown by SEQ ID No. 352 to SEQ ID No. 383, SEQ ID No. 385 to SEQ ID No. 416, SEQ ID No. 418 to SEQ ID No. 430 or SEQ ID No. 455 to SEQ ID No. 476.
  11. 11. The RNAi construct of claim 10, wherein the RNAi construct is selected from any one of the constructs provided in table 2.
  12. 12. The RNAi construct of claim 11, wherein the RNAi construct is selected from the group consisting of the following constructs in Table 2, D-3001 (SEQ ID NO:273 and 352), D-3002 (SEQ ID NO:274 and 353), D-3003 (SEQ ID NO:275 and 354), D-3005 (SEQ ID NO:277 and 356), D-3006 (SEQ ID NO:278 and 357), D-3007 (SEQ ID NO:279 and 358), D-3012 (SEQ ID NO:284 and 363), D-3015 (SEQ ID NO:287 and 366), D-3016 (SEQ ID NO:288 and 367), D-3017 (SEQ ID NO:289 and 368), D-3021 (SEQ ID NO:293 and 372), D-3027 (SEQ ID NO: 299) D-3032 (SEQ ID NO:304 and 383), D-3046 (SEQ ID NO:312 and 391), D-3047 (SEQ ID NO: 313) D-3068, D-3016 (SEQ ID NO:313 and 3068), D-3016 (SEQ ID NO:289 and 3068), d-3069 (SEQ ID NOS: 335 and 414), D-3070 (SEQ ID NOS: 336 and 415), D-3071 (SEQ ID NOS: 337 and 416), D-3072 (SEQ ID NOS: 433 and 455), D-3073 (SEQ ID NOS: 434 and 456), D-3074 (SEQ ID NOS: 435 and 457), D-3075 (SEQ ID NOS: 436 and 458), D-3076 (SEQ ID NOS: 437 and 459), D-3077 (SEQ ID NOS: 438 and 460), D-3078 (SEQ ID NOS: 439 and 461), D-3079 (SEQ ID NOS: 440 and 462), D-3080 (SEQ ID NOS: 441 and 463), D-3081 (SEQ ID NOS: 442 and 464), D-3082 (SEQ ID NOS: 443 and 465).
  13. 13. The RNAi construct of any one of claims 1-12, wherein the RNAi construct is further conjugated to a targeting ligand, preferably the targeting ligand is an N-acetyl-galactosamine derivative (GalNAc derivative).
  14. 14. The RNAi construct of claim 13, wherein the targeting ligand is selected from L96, LICA-1, LICA-2, LICA-3, (NAG 25) s, (NAG 37) or (NAG 37) s; L96 LICA-1 LICA-2 LICA-3 (NAG25) (NAG25)s (NAG37) (NAG37)s。
  15. 15. The RNAi construct of claim 13, wherein the sense strand conjugated to the targeting ligand comprises, from 5 'end to 3' end, a contiguous nucleotide selected from the group consisting of, or consisting of, SEQ ID NOs 339 to 351, 444 to 454.
  16. 16. The RNAi construct of claim 13, wherein the targeting ligand is conjugated to the 5 'end or 3' end of the sense strand, wherein the RNAi construct is selected from the group consisting of the constructs D-5002 (SEQ ID NOs 339 and 418), D-5003 (SEQ ID NOs 340 and 419), D-5004 (SEQ ID NOs 341 and 420), D-5005 (SEQ ID NOs 342 and 421), D-5006 (SEQ ID NOs 343 and 422), D5007 (SEQ ID NOs 344 and 423), D5008 (SEQ ID NOs 345 and 424), D-5009 (SEQ ID NOs 346 and 425), D-5010 (SEQ ID NOs 347 and 426), D-5011 (SEQ ID NOs 348 and 427), D-5012 (SEQ ID NOs 349 and 428), D-5013 (SEQ ID NOs 350 and 429), D-5014 (SEQ ID NOs 351 and 430), D-5015 (SEQ ID NOs 444 and 466), D-5006 (SEQ ID NOs 345 and 422), D-5009 (SEQ ID NOs 346 and 425), D-5010 (SEQ ID NOs 347 and 427), D-5012 (SEQ ID NOs 5013 and 428), D-5013 (SEQ ID NOs 4 and 5025) D-5024 (SEQ ID NOS: 453 and 475) or D-5025 (SEQ ID NOS: 454 and 476).
  17. 17. The RNAi construct of claim 1, selected from the group consisting of (1) D-1074 (SEQ ID NO:74 and 210), D-1095 (SEQ ID NO:93 and 229), D-1115 (SEQ ID NO:113 and 249), D-1116 (SEQ ID NO:114 and 250), D-1107 (SEQ ID NO:105 and 241), D-1108 (SEQ ID NO:106 and 242), D-1118 (SEQ ID NO:116 and 252), D-1123 (SEQ ID NO:121 and 257) or D-1124 (SEQ ID NO:122 and 258), D-1154 (SEQ ID NO:491 and 504), D-1155 (SEQ ID NO:492 and 505), D-1156 (SEQ ID NO: 493 and 506), D-1157 (SEQ ID NO: 494 and 507), D-1158 (SEQ ID NO: 495 and 508), D-1159 (SEQ ID NO: 496 and 509) or D-1160 (SEQ ID NO: 497 and 510); (2) The antisense strand thereof comprises a sequence substantially or entirely complementary to the region at positions 210-230 of ASGR1 mRNA sequence NM-001671.5, preferably D-3012 (SEQ ID NOS: 284 and 363), D-3046 (SEQ ID NOS: 312 and 391), D-3047 (SEQ ID NOS: 313 and 392), D-3049 (SEQ ID NOS: 315 and 394), D-3072 (SEQ ID NOS: 433 and 455), D-3074 (SEQ ID NOS: 437 and 457), D-3075 (SEQ ID NOS: 436 and 458), D-3077 (SEQ ID NOS: 438 and 460), D-5002 (SEQ ID NOS: 339) and 418), D-5007 (SEQ ID NOS: 344 and 423), D-5008 (SEQ ID NOS: 345 and 424), D-5009 (SEQ ID NOS: and 425), D-5015 (SEQ ID NOS: 444 and 466), D-5017 (SEQ ID NOS: 446 and 447), D-5018 (SEQ ID NOS: 463) and 469), D-3074 (SEQ ID NOS: 438 and 463), D-5007 (SEQ ID NOS: 344 and 463), D-5005 (SEQ ID NOS: 444 and 463), D-5005 (SEQ ID NOS: and 463), D-5 (SEQ ID NOS: 5, and 463). (3) The antisense strand thereof comprises a sequence substantially or completely complementary to the region at positions 213-233 of the ASGR1 mRNA sequence NM-001671.5, preferably D-3054 (SEQ ID NOS: 320 and 399), D-3055 (SEQ ID NOS: 321 and 400), D-3080 (SEQ ID NOS: 441 and 463), D-3081 (SEQ ID NOS: 442 and 464), D-5010 (SEQ ID NOS: 347 and 426), D-5011 (SEQ ID NOS: 348 and 427), D-5023 (SEQ ID NOS: 452 and 474), D-5024 (SEQ ID NOS: 453 and 475); (4) The antisense strand of which comprises a sequence substantially or completely complementary to the regions at positions 1222-1242, 1223-1243, 1224-1244, 1225-1245, 1226-1246, 1227-1247 or 1228-1248 of the ASGR1 mRNA sequence NM-001671.5, preferred RNAi constructs are selected from the group consisting of D-1108 (SEQ ID NOS: 106 and 242), D-1154 (SEQ ID NOS: 491 and 504), D-1155 (SEQ ID NOS: 492 and 505), D-1156 (SEQ ID NOS: 493 and 506), D-1157 (SEQ ID NOS: 494 and 507), D-1158 (SEQ ID NOS: 495 and 508), D-1159 (SEQ ID NOS: 496 and 509), D-1160 (SEQ ID NOS: 497 and 510) or the aforementioned constructs of which the sense strand and/or antisense strand is subjected to optional nucleotide modification and/or optional internucleoside bond modification and/or optional conjugation of the targeting ligand, or D-3032 (SEQ ID NOS: 383 and D-341) and D-341 (SEQ ID: 420 and 383); (5) D-5002 (SEQ ID NOS: 339 and 418), D-5004 (SEQ ID NOS: 341 and 420), D-5009 (SEQ ID NOS: 346 and 425), D-5011 (SEQ ID NOS: 348 and 427), D-5015 (SEQ ID NOS: 444 and 466), D-5017 (SEQ ID NOS: 446 and 468), D-5018 (SEQ ID NOS: 447 and 469), or (6) D-5003 (SEQ ID NOS: 340 and 419), D-5008 (SEQ ID NOS: 345 and 424), D-5010 (SEQ ID NOS: 347 and 426), D-5024 (SEQ ID NOS: 453 and 475).
  18. 18. A pharmaceutical composition comprising the RNAi construct of any one of claims 1-17 and a pharmaceutically acceptable excipient.
  19. 19. The pharmaceutical composition of claim 18, further comprising a second ASGR1 RNAi construct or one or more additional therapeutic agents, wherein the RNAi construct of claim 18 is a first ASGR1 RNAi construct, and wherein the second ASGR1 RNAi construct is selected from any one of claims 1-17 that is different from the first ASGR1 RNAi construct.
  20. 20. A method of treating and/or preventing an ASGR 1-related disease or disorder, the method comprising administering to a subject in need thereof an effective amount of an RNAi construct of any of claims 1-17 or a composition of any of claims 18-19, or The RNAi construct of any of claims 1-17 or the composition of any of claims 18-19, for use in the treatment and/or prevention of an ASGR 1-related disease or disorder, or Use of an RNAi construct according to any one of claims 1-17 or a composition according to any one of claims 18-19 in the manufacture of a medicament for the treatment and/or prevention of an ASGR 1-associated disease or disorder.

Description

RNAi construct for inhibiting ASGR1 gene expression and use thereof Technical Field The present invention relates to RNAi constructs that inhibit expression of asialoglycoprotein receptor 1 (ASGR 1) genes, pharmaceutical compositions comprising the same, and uses thereof. Background Cardiovascular disease (cardiovascular disease, CVD) is a serious hazard to human health. Atherosclerosis (atherosclerosis, as) serves As the primary pathological and physiological basis for CVD, the development of which is the result of a number of risk factor interactions, where dyslipidemia plays an important role. Numerous clinical studies have shown that correction of dyslipidemia has significant benefits in alleviating atherosclerosis. Asialoglycoprotein receptor 1 (ASGR 1, asialoglycoprotein Receptor 1 also known as ASGPR, ASGPR1, HL-1 and CLEC4H 1) is also known as the ash well-Morell receptor. ASGR1 is a transmembrane protein that plays a major physiological role in binding, internalizing and scavenging asialoglycoproteins from the circulation. ASGR1 is mainly expressed in the liver by the asialoglycoprotein receptor 1 gene (ASGR 1 gene). There is evidence that ASGR1 is associated with other risk factors for atherosclerosis, such as inflammatory activation, platelet abnormalities, etc. Thus, therapeutic agents capable of targeting the ASGR1 gene and reducing the level of ASGR1 protein expression represent a novel way of treating cardiovascular diseases, including coronary artery disease. No ASGR1 targeted product is currently marketed in bulk. RNAi has extremely wide application in therapy, since siRNA and miRNA constructs can be synthesized using any nucleotide sequence directed against the target protein. To date, siRNA constructs have been shown to specifically down-regulate target proteins in vitro and in vivo models. Two problems faced by current siRNA or miRNA constructs are, firstly, their susceptibility to nuclease digestion in plasma, and secondly, their limited ability to enter intracellular compartments in which RISC can be bound when administered systemically as free siRNA or miRNA. Such double stranded constructs can be stabilized by incorporating a chemically modified nucleotide linker within the molecule. In addition, the prior art uses GalNac coupled delivery technology to overcome the problem of easy off-targeting of siRNA, becoming the primary technology for delivering oligonucleotides to the liver. The invention aims to provide an RNAi construct for inhibiting the expression of an asialoglycoprotein receptor 1 (ASGR 1) gene, a pharmaceutical composition comprising the RNAi construct and application thereof, wherein the RNAi construct can inhibit the expression of the ASGR1 gene in liver, so that the aim of treating diseases is fulfilled. Disclosure of Invention The invention provides an RNAi construct inhibiting the expression of an asialoglycoprotein receptor 1 (ASGR 1) gene, a pharmaceutical composition comprising the same and uses thereof. In one aspect, the invention provides an RNAi construct that inhibits ASGR1 gene expression. The RNAi constructs of the invention can degrade or inhibit translation of target ASGR1 mRNA transcripts in a sequence-specific manner through RNA interference mechanisms, thereby achieving inhibition of ASGR1 gene expression. The invention screens out a bare sequence RNAi construct with excellent RNA interference effect. These naked sequence RNAi constructs have good RNA interference effects following the use of nucleic acid modifications and/or GalNAc derivative targeting groups for coupling. In some embodiments, the RNAi construct (also known as a Duplex, siRNA or RNAi agent) comprises an antisense strand comprising 17-23 contiguous nucleotides differing by 0,1, 2,3 or 4 nucleotides from any of the antisense strand sequences provided in table 1 or table 2 or in the claims, and a sense strand comprising a nucleotide sequence at least partially complementary to the antisense strand. In some embodiments, the antisense strand in an RNAi construct is 17-23 consecutive nucleotides differing by 0,1, 2, or 3 nucleotides from any of the antisense strand sequences provided in table 1, table 2, or claims. In some embodiments, the sense strand and the antisense strand are each independently 17-21 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each 21-26 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length, and the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length, and the antisense strand is about 23 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length and the antisense strand is 21 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are 21 nucleotides in length each. In some embodiments, the sense strand is 22 nucleotides in length and the antisense