WO2024228030A2 - Silençage double - Google Patents

Silençage double Download PDF

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Publication number
WO2024228030A2
WO2024228030A2 PCT/GB2024/051168 GB2024051168W WO2024228030A2 WO 2024228030 A2 WO2024228030 A2 WO 2024228030A2 GB 2024051168 W GB2024051168 W GB 2024051168W WO 2024228030 A2 WO2024228030 A2 WO 2024228030A2
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Prior art keywords
nucleic acid
seq
double stranded
nucleotide sequence
acid molecule
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PCT/GB2024/051168
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WO2024228030A3 (fr
Inventor
Michael Khan
Johnathan MATLOCK
Stella Khan
Daniel Mitchell
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Argonaute RNA Ltd
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Argonaute RNA Ltd
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Priority claimed from GB2306589.9A external-priority patent/GB2629617B/en
Priority claimed from GBGB2404488.5A external-priority patent/GB202404488D0/en
Priority to CN202480030389.6A priority Critical patent/CN121057819A/zh
Priority to EP24726696.8A priority patent/EP4705458A2/fr
Application filed by Argonaute RNA Ltd filed Critical Argonaute RNA Ltd
Priority to AU2024265748A priority patent/AU2024265748A1/en
Priority to KR1020257035331A priority patent/KR20260006569A/ko
Priority to GB2501121.4A priority patent/GB2635479A/en
Priority to GB2406314.1A priority patent/GB2629679B/en
Priority to GB2414903.1A priority patent/GB2631903B/en
Publication of WO2024228030A2 publication Critical patent/WO2024228030A2/fr
Publication of WO2024228030A3 publication Critical patent/WO2024228030A3/fr
Priority to IL323761A priority patent/IL323761A/en
Priority to MX2025012698A priority patent/MX2025012698A/es
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01076—Retinol O-fatty-acyltransferase (2.3.1.76)
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)

Definitions

  • the disclosure relates to isolated nucleic acid molecules comprising at least two double stranded inhibitory ribonucleic acid (RNA) molecules adapted to silence by RNA interference either the same gene or different genes to enhance silencing thereby modulating gene expression.
  • RNA inhibitory ribonucleic acid
  • siRNA double stranded inhibitory RNA
  • siRNA small inhibitory or interfering RNA
  • the siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule.
  • the siRNA molecule is typically, but not exclusively, derived from exons of the gene which is to be ablated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of siRNA.
  • RNA double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides in length) which become part of a ribonucleoprotein complex.
  • the siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.
  • RNA silencing utilises a single siRNA species directed to a single gene to silence expression. It is known to link siRNAs to enable the silencing of two or more genes.
  • WO2016205410 discloses oligonucleotides linked together directly, via functional end-substitutions, or indirectly by way of a linking agent. The oligonucleotide can be bound directly to a linker. Such bonding can be achieved, for example, through use of 3'-thionucleosides.
  • WO2018145086 discloses oligonucleotides in the form of a multimeric oligonucleotide having monomeric subunits of oligonucleotide joined by covalent linkers to decrease clearance due to glomerular filtration.
  • WO2 013040429A1 discloses multi-oligomeric complexes that comprise two or more targeting oligonucleotides linked together by a cleavable linker.
  • WO2015113922 discloses oligo oligonucleotides conjugates where two or more antisense oligonucleotides are covalently linked by physiologically labile linkers, and to a biocleavable functional group such as a conjugate group.
  • W02010141511 discloses bivalent or multivalent nucleic acid molecules or complexes of nucleic acid molecules having two or more target-specific regions, in which the target-specific regions are complementary to a single target gene at more than one distinct nucleotide site, and/or in which the target regions are complementary to more than one target gene or target sequence.
  • WO2017188707 discloses a dicer substrate RNA nanostructure exhibiting enhanced gene silencing effects.
  • the RNA nanostructure includes a plurality of the same or different RNAi sequences in a single RNA nanostructure.
  • the use of oligomeric nanostructures comprising more than one siRNA or antisense molecule is known in the art.
  • Cardiovascular disease associated with hypercholesterolemia is a common condition and results in heart disease and a high incidence of death and morbidity and can be a consequence of poor diet, obesity, or an inherited dysfunctional gene.
  • LDL-receptor Low Density Lipoprotein Receptor
  • ApoB apolipoprotein B
  • Cholesterol is essential for membrane biogenesis in animal cells. The lack of water solubility means that cholesterol is transported around the body in association with lipoproteins. Apolipoproteins form together with phospholipids, cholesterol and lipids which facilitate the transport of lipids such as cholesterol, through the bloodstream to the different parts of the body.
  • Lipoproteins are classified according to size and can form HDL (High-density lipoprotein), LDL (Low-density lipoprotein), IDL (intermediate-density lipoprotein), VLDL (very low- density lipoprotein) and ULDL (ultra-low-density lipoprotein) lipoproteins.
  • HDL High-density lipoprotein
  • LDL Low-density lipoprotein
  • IDL intermediate-density lipoprotein
  • VLDL very low- density lipoprotein
  • ULDL ultra-low-density lipoprotein
  • Familial hypercholesterolemia is an orphan disease and results from elevated levels of LDL cholesterol (LDL-C) in the blood.
  • LDL-C LDL cholesterol
  • the disease is an autosomal dominant disorder with both the heterozygous (350-550mg/dL LDL-C) and homozygous (650-1000m g/dL LDL-C) states resulting in elevated LDL-C.
  • the heterozygous form of familial hypercholesterolemia is around 1:500 of the population.
  • the homozygous state is much rarer and is approximately 1 :1 ,000,000.
  • the normal levels of LDL-C are in the region 130mg/dL.
  • Hypercholesterolemia is particularly acute in paediatric patients which if not diagnosed early can result in accelerated coronary heart disease and premature death.
  • LDL-C low-density lipoprotein
  • statins which block the de novo synthesis of cholesterol by inhibiting the HMG-CoA reductase.
  • the disclosure relates to isolated nucleic acid molecules comprising at least two double stranded inhibitory ribonucleic acid (RNA) molecules adapted to silence by RNA interference either the same gene or different genes to enhance silencing thereby modulating gene expression.
  • RNA inhibitory ribonucleic acid
  • a nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a gene to be silenced and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to either the 5’ or 3’ end of said sense or antisense strand; and ii) a second nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a different or the same gene to be silenced as set forth in i) above and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to either the 5’ or 3’ end of said sense or antisense strand wherein the single stranded deoxyribonucle
  • said a single stranded deoxyribonucleic acid (DNA) is linked to said first or second double stranded inhibitory RNA molecule wherein said linkage is selected from the group: i) 5’ sense strand of said first double stranded inhibitory RNA and 5’ antisense stand of said second double stranded RNA molecule; ii) 5’ sense strand of said first double stranded inhibitory RNA and 3’ sense strand of said second double stranded RNA molecule; iii) 5’ sense strand of said first double stranded inhibitory RNA and 5’ antisense strand of said second inhibitory RNA molecule; iv) 5’ sense strand of said first double stranded inhibitory RNA and the 3’ antisense strand of said second double stranded inhibitory RNA molecule; v) 3’ sense strand of said first double stranded inhibitory RNA and the 5’ sense strand of said second double stranded inhibitory RNA molecule
  • said first and second gene to be silenced is the same gene.
  • said first and second gene to be silenced are different genes.
  • said first and second double stranded inhibitory RNA molecules comprise different nucleotide sequences.
  • said first or second gene to be silenced is the Apo B gene.
  • said Apo B gene comprises a nucleotide sequence set forth in SEQ ID NO: 1 wherein said double stranded inhibitory RNA is 19- 23 nucleotides in length.
  • said Apo B double stranded inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 607 to 906.
  • said Apo B double stranded inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 907 to 1206.
  • said first or second gene to be silenced is DGAT2.
  • said DGAT2 gene comprises a nucleotide sequence set forth in SEQ ID NO: 6 wherein said double stranded inhibitory RNA is 19- 23 nucleotides in length.
  • said double stranded DGAT2 inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 7 to 306.
  • said double stranded DGAT2 inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 307 to 606.
  • said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4830.
  • said double stranded DGAT2 inhibitory RNA comprises or consists of a sense nucleotide sequence set forth in SEQ ID NO: 4829.
  • said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 4840 to 4848.
  • said double stranded DGAT2 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 4831 to 4839.
  • said first or second gene to be silenced is PCSK9.
  • said PCSK9 gene comprises a nucleotide sequence set forth in SEQ ID NO: 2 wherein said double stranded inhibitory RNA is 19- 23 nucleotides in length.
  • said double stranded PCSK9 inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 1207 to 1510.
  • said double stranded PCSK9 inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 1511 to 1814.
  • said double stranded PCSK9 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO:
  • said double stranded PCSK9 inhibitory RNA comprises or consists of a sense nucleotide sequence set forth in SEQ ID NO: 4821.
  • said first or second gene to be silenced is lipoprotein A (Lp(a)).
  • said lipoprotein A gene comprises a nucleotide sequence set forth in SEQ ID NO: 3 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.
  • said lipoprotein A double stranded inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 1815 to 2114.
  • said lipoprotein A double stranded inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 2115 to 2414.
  • said lipoprotein A double stranded inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4824.
  • said first or second gene to be silenced is angiotensinogen.
  • said angiotensinogen gene comprises a nucleotide sequence set forth in SEQ ID NO: 4 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.
  • said double stranded angiotensinogen inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 2415 to 2714.
  • said double stranded angiotensinogen inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 2715 to 3014.
  • said first or second gene to be silenced is Apo CHI .
  • said Apo CHI gene comprises a nucleotide sequence set forth in SEQ ID NO: 5 wherein said double stranded inhibitory RNA is 19- 23 nucleotides in length.
  • said double stranded ApoCi II inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 3015 to 3314.
  • said double stranded ApoCi II inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 3315 to 3614.
  • said double stranded ApoCi II inhibitory RNA comprise or consists of a sense nucleotide sequence set forth in SEQ ID NO 4860.
  • said double stranded ApoCi II inhibitory RNA comprise or consists of an antisense nucleotide sequence set forth in SEQ ID NO 4859.
  • said first or second gene to be silenced is ANGPTL3.
  • said ANGPTL3 gene comprises a nucleotide sequence set forth in SEQ ID NO: 4819 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.
  • said double stranded ANGPTL 3 inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 3615 to 3914.
  • said double stranded ANGPTL 3 inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 3915 to 4214.
  • said double stranded ANGPTL3 inhibitory RNA comprises or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 4826 or 4828.
  • said double stranded ANGPTL3 inhibitory RNA comprises or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 4825 or 4827.
  • said first or second gene to be silenced is ANGPTL4.
  • said ANGPTL4 gene comprises a nucleotide sequence set forth in SEQ ID NO: 4820 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.
  • said ANGPTL4 double stranded inhibitory RNA comprise or consists of a sense nucleotide sequence selected from the group: SEQ ID NO: 4215 to 4514.
  • said ANGPTL4 double stranded inhibitory RNA comprise or consists of an antisense nucleotide sequence selected from the group: SEQ ID NO: 4515 to 4814.
  • said first or second gene to be silenced is selected from the group consisting of: Apo B, DGAT2, PCSK9, Lp(a), APOCHI, angiotensinogen, ANGPTL3 and ANGPTL4
  • said first gene is Apo B and said second gene is DGAT2.
  • said first gene is Apo B and said second gene is PCSK9.
  • said first gene is Apo B and said second gene is angiotensinogen.
  • said first gene is Apo B and said second gene is Apo CHI.
  • said first gene is Apo B and said second gene is Lp(a).
  • said first gene is PCSK9 and said second gene is angiotensinogen.
  • said first gene is PCSK9 and said second gene is Apo Cll I .
  • said first gene is PCSK9 and said second gene is DGAT2.
  • said first gene is PCSK9 and said second gene is Lp(a).
  • said first gene is angiotensinogen and said second gene is Apo CHI.
  • said first gene is angiotensinogen and said second gene is DGAT2.
  • said first gene is angiotensinogen and said second gene is Lp(a).
  • said first gene is Apo CHI and said second gene is DGAT2.
  • said first gene is Apo CHI and said second gene is Lipoproetin (a).
  • said first gene is DGAT2 and said second gene is Lipoprotein (a).
  • said first gene is ANGPLT3 and said second gene is ApoB.
  • said first gene is ANGPLT3 and said second gene is PCSK9.
  • said first gene is ANGPLT3 and said second gene is angiotensinogen.
  • said first gene is ANGPLT3 and said second gene is APOCHI.
  • said first gene is ANGPLT3 and said second gene is Lp(a).
  • said first gene is ANGPLT3 and said second gene is DGAT2.
  • said first gene is ANGPLT3 and said second gene is ANPTLT4.
  • said first gene is ANGPLT4 and said second gene is Apo B.
  • said first gene is ANGPLT4 and said second gene is PCSK9.
  • said first gene is ANGPLT4 and said second gene is angiotensinogen.
  • said first gene is ANGPLT4 and said second gene is Apo CHI.
  • said first gene is ANGPLT4 and said second gene is Lp(a).
  • said first gene is ANGPLT4 and said second gene is DGAT2.
  • said first gene is DGAT2 and comprises a nucleotide sequence set forth in SEQ ID NO: 6 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length and wherein said double stranded DGAT2 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4830 or 4840 to 4848, and/or a sense nucleotide sequence set forth in SEQ ID NO: 4829 or 4831 to 4839.
  • said first gene is PCSK9 and comprises a nucleotide sequence set forth in SEQ ID NO: 2 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length and wherein said double stranded PCSK9 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4822, and/or comprises or consists of a sense nucleotide sequence set forth in SEQ ID NO: 4821.
  • said first gene is lipoprotein A and comprises a nucleotide sequence set forth in SEQ ID NO: 3 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length and wherein said double stranded lipoprotein A inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4824, and /or of a sense nucleotide sequence set forth in SEQ ID NO: 4823.
  • said first gene is ANGPTL 3 and comprises a nucleotide sequence set forth in SEQ ID NO: 4819 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length and wherein said double stranded ANGPTL 3 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4826 or 4828, and/or comprise or consists of a sense nucleotide sequence set forth in SEQ ID NO: 4825 or 4827.
  • said first gene is ApoC3 and comprises a nucleotide sequence set forth in SEQ ID NO: 5 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length and wherein said double stranded ApoC3 inhibitory RNA comprises or consists of an antisense nucleotide sequence set forth in SEQ ID NO: 4859, and /or of a sense nucleotide sequence set forth in SEQ ID NO: 4860.
  • said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815).
  • said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816).
  • said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the sense nucleotide sequence.
  • said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the antisense nucleotide sequence.
  • said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the sense nucleotide sequence.
  • said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the antisense nucleotide sequence.
  • said first and/or said second double stranded inhibitory RNA molecule comprises or consists of natural nucleotides.
  • said first and/or said second double stranded inhibitory RNA molecule comprises modified nucleotides and/or modified sugar(s).
  • said modified nucleotides/sugars are selected from the group: a 3 '-terminal deoxy- thymine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'- O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2' -hydroxyl- modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0- alkyl-modified nucleotide,
  • dT deoxy
  • said first or and/or second double stranded inhibitory RNA molecule comprises at least one modified nucleotide wherein said modification is 2'-deoxy-2'-fluoro.
  • said first and/or said second double stranded inhibitory RNA molecule comprises at least one modified nucleotide wherein said modification is 2'-O-methyl.
  • said first and/or said second double stranded inhibitory RNA molecule comprises at least one phosphorothioate linkage.
  • first and/or said second double stranded inhibitory RNA molecule comprises at least one 5'-vinyl phosphate. In an embodiment of the invention said first and/or said second double stranded inhibitory RNA molecule comprises at least one modified sugar.
  • a sugar modification includes a modified version of the ribosyl moiety, such as -O- modified RNA such as 2'-O-alkyl or 2'-O-(substituted)alkyl e.g. 2'-0-methyl, T-0-(2- cyanoethyl), 2'-0-(2-methoxy)ethyl (2'-MOE), 2'-0-(2-thiomethyl)ethyl, 2'-O-butyryl, -O- propargyl, 2'-O-allyl, 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2- amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O-(haloalkoxy)methyl, e.g.
  • -O- modified RNA such as 2'-O-alkyl or
  • nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand.
  • RNA inhibitory ribonucleic acid
  • nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4821) and an antisense strand (SEQ ID NO: 4822) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand.
  • RNA inhibitory ribonucleic acid
  • nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4823) and an antisense strand (SEQ ID NO: 4824) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand.
  • RNA inhibitory ribonucleic acid
  • nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand.
  • RNA inhibitory ribonucleic acid
  • nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4827) and an antisense strand (SEQ ID NO: 4828) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand.
  • RNA inhibitory ribonucleic acid
  • nucleic acid molecule comprises at least one double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4860) and an antisense strand (SEQ ID NO: 4859) wherein said a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand.
  • RNA inhibitory ribonucleic acid
  • a nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4821) and an antisense strand (SEQ ID NO: 4822) wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense strand (SEQ ID NO: 4823) and an antisense strand (SEQ ID NO: 4824) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (RNA) molecule is substantially
  • a nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4825) and an antisense strand (SEQ ID NO: 4826) wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (RNA) molecule is substantially
  • a nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand (SEQ ID NO: 4829) and an antisense strand (SEQ ID NO: 4830) wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense strand (SEQ ID NO: 4860) and an antisense strand (SEQ ID NO: 4859) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (RNA) molecule is substantially
  • said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815).
  • said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816).
  • said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the sense nucleotide sequence.
  • said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT (SEQ ID NO: 4815) and is attached to the 5’ end of the antisense nucleotide sequence.
  • said single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the sense nucleotide sequence.
  • said single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG (SEQ ID NO: 4816) and is attached to the 5’ end of the antisense nucleotide sequence.
  • said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4849 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4822
  • said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4850 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4822.
  • said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4851 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4824.
  • said nucleic acid comprises or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4852 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4824.
  • nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4853 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4826.
  • nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4854 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4826.
  • said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4855 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4828.
  • said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4856 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4828.
  • said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4857 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4830.
  • said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4858 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4830
  • said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4861 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4859
  • said nucleic acid comprise or consists of a sense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4862 and an antisense strand comprising or consisting of the nucleotide sequence set forth in SEQ ID NO 4859.
  • a nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand selected from the group consisting of SEQ ID NO 607 to 906 and an antisense strand selected from the group consisting of SEQ ID NO 907 to 1206 wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense strand selected from the group consisting of SEQ ID NO: 7 to 306, 4829, 4831 to 4839, 4857 and 4858 and an antisense strand selected from the group consisting of SEQ ID NO 307-606, 4830 and 4840 to 4848 and wherein there is provided a single stranded deoxyribonucleic acid (DNA)
  • said second nucleic acid comprises a double stranded inhibitory (RNA) molecule comprising a sense strand set forth in SEQ ID NO 4857 and an antisense strand set forth in SEQ ID NO 4830.
  • RNA double stranded inhibitory
  • said second nucleic acid comprises a double stranded inhibitory (RNA) molecule comprising a sense strand set forth in SEQ ID NO 4858 and an antisense strand set forth in SEQ ID NO 4830.
  • RNA double stranded inhibitory
  • nucleic acid molecule is covalently linked to A/-acetylgaiactosamine.
  • the sugar moiety in N-acety!galactosamine can comprise glycosidic linkages to improve stability.
  • glycosidic bonds are known in the art and formed between the hemiacetal of the sugar moiety and several chemical groups forming O-, N-, S- or C- glycosidic bonds.
  • the N-acetylgalactosamine comprises an O-, N-, S- or C- glycosidic bond.
  • said N-acetylgalactosamine is linked to either the antisense part of said inhibitory RNA or the sense part of said inhibitory RNA.
  • N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said first nucleic acid.
  • N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said second nucleic.
  • N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said first nucleic acid and is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said second nucleic.
  • N-acetylgalactosamine is linked to the 3’ terminus is of said sense RNA.
  • N-acetylgalactosamine is linked to the 5’ terminus of said sense RNA.
  • N-acetylgalactosamine is linked to the 3’ terminus of said antisense RNA.
  • N-acetylgalactosamine is monovalent.
  • N-acetylgalactosamine is divalent.
  • N-acetylgalactosamine is trivalent.
  • nucleic acid molecule is covalently linked to a molecule comprising the structure: In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure:
  • a medicament comprising a nucleic acid according to the invention.
  • a pharmaceutical composition comprising a nucleic acid molecule according to the invention.
  • composition further includes a pharmaceutical carrier and/or excipient.
  • nucleic acid molecule or a pharmaceutical composition according to the invention for use in the treatment or prevention of a subject that has or is predisposed to hypercholesterolemia.
  • said use is the treatment or prevention of diseases associated with hypercholesterolemia.
  • said disease associated with hypercholesterolemia is selected from the group consisting of: stroke prevention, hyperlipidaemia, cardiovascular disease, atherosclerosis, coronary heart disease, aortic stenosis, cerebrovascular disease, peripheral arterial disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Buerger’s disease, renal artery stenosis, hyper-apobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and optionally other therapeutic agents, such as cholesterol lowering agents, which can be administered separately from the nucleic acid molecule according to the invention or in a combined preparation if a combination is compatible.
  • nucleic acid according to the invention is administered as simultaneous, sequential, or temporally separate dosages.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal or transepithelial.
  • the compositions of the invention are administered in effective amounts.
  • An “effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response.
  • the desired response is inhibiting or reversing the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods.
  • Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
  • compositions used in the foregoing methods preferably are sterile and contain an effective amount of a nucleic acid molecule according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by determining regression of cardiovascular disease and decrease of disease symptoms etc.
  • the doses of the nucleic acid molecule according to the invention administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. If a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. It will be apparent that the method of detection of the nucleic acid according to the invention facilitates the determination of an appropriate dosage for a subject in need of treatment. In general, doses of the nucleic acid molecules herein disclosed of between 1nM - 1pM generally will be formulated and administered according to standard procedures.
  • doses can range from 1nM- 500nM, 5nM-200nM, 10nM-100nM.
  • Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing.
  • the administration of compositions to mammals other than humans, is carried out under substantially the same conditions as described above.
  • a subject, as used herein, is a mammal, preferably a human, and including a nonhuman primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
  • the pharmaceutical preparations of the invention When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents e.g. statins.
  • the salts When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • compositions may be combined, if desired, with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
  • pharmaceutically acceptable carrier in this context denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate, for example, solubility and/or stability.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • the pharmaceutical compositions may contain suitable buffering agents, including acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • suitable buffering agents including acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
  • the pharmaceutical compositions also may contain, optionally, suitable preservatives.
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of nucleic acid, which is preferably isotonic with the blood of the recipient.
  • This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butane diol.
  • acceptable solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono-or di-glycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
  • a method to treat a subject that has or is predisposed to hypercholesterolemia comprising administering an effective dose of a nucleic acid or a pharmaceutical composition according to the invention thereby treating or preventing hypercholesterolemia.
  • the hypercholesterolemia is familial hypercholesterolemia.
  • said disease associated with hypercholesterolemia is selected from the group consisting of: stroke prevention, hyperlipidaemia, cardiovascular disease, atherosclerosis, coronary heart disease, aortic stenosis, cerebrovascular disease, peripheral arterial disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Buerger’s disease, renal artery stenosis, hyper-apobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis.
  • stock siRNAs were incubated in vehicle (nuclease-free water) or in various concentrations (20% - 80%) of human serum (HS) for 2 hours at 37°C.
  • siRNAs were transfected into primary mouse hepatocytes in triplicates in a 384-well plate (Thermo ScientificTM 164688) at various concentrations ranging from 2.5 nM to 100 nM using 0.15 pL of Lipofectamine RNAiMAX (I nvitrogenTM 13778075) per well. Transfected cells were incubated at 37°C and 5% CO2 for 48 hours. Cells receiving no siRNA treatment were used as control.
  • Standard siRNA controls (mTTR and ApoB_C3_01) and GalNAc-conjugated ApoB- mTTR heterodimers (Table 7) were synthesized at Bio-Synthesis (Lewisville, TX). GalNAc-conjugated constructs and standard siRNA controls were resuspended in nuclease-free water (InvitrogenTM AM9932) to generate a stock solution of 10 pM. Stock siRNAs were distributed in triplicates in 384-well plates (Thermo ScientificTM 164688) to a final concentration of 4 nM, 25 nM and 100 nM and primary mouse hepatocytes were added at a concentration of 5,000 cells per well for free-uptake assay. After treatment, cells were incubated at 37°C and 5% CO2 for 48 hours. Cells receiving no siRNA treatment were used as control.
  • Cells were processed for RT-qPCR read-out using the Cells-to-CT 1-step TaqMan Kit (InvitrogenTM A25603). Briefly, cells were washed with 50pL ice-cold PBS and lysed in 20 pl Lysis solution containing DNase I. Lysis was stopped after 5 minutes by addition of 2 pl STOP Solution for 2 min. For the RT-qPCR analysis, 1 pL of lysate was dispensed per well into a 96-well PCR plate in a 10 pL RT-qPCR reaction volume.
  • RT-qPCR was performed using the TaqMan® 1-Step qRT-PCR Mix from the Cells-to-CT 1-step TaqMan Kit, with TaqMan probes for GAPDH (VIC_PL, Assay Id Mm99999915_g1), mTTR (FAM, Assay Id Mm00443267_m1), ApoB (FAM, Assay Id Mm01545150_m1) or DGAT2 (FAM, Assay Id Mm00499536_m1).
  • RT-qPCR was performed using a QuantStudio 5 thermocycling instrument (Applied BioSystems). Relative quantification was determined using the AACT method, where GAPDH was used as internal control and expression changes normalized to the reference sample (no siRNA treatment).
  • Freshly prepared or *serum pre-incubated stock siRNAs were distributed in triplicates in collagen-coated 384-well plates (CorningTM 354666) to a final concentration of 0.1 nM, 1 nM, 4 nM and 25 nM and primary mouse hepatocytes were added at a concentration of 6,000 cells per well. After treatment, cells were incubated at 37°C and 5 % CO2 for 48 hours. Cells receiving no siRNA treatment were used as control. (*Stock siRNAs were exposed to 80 % human serum (HS) for 2 hours at 37°C).
  • HS human serum
  • Cells were processed for RT-qPCR read-out using the Cells-to-CT 1-step TaqMan Kit (InvitrogenTM A25603). Briefly, cells were washed with 50 pL ice-cold PBS and lysed in 20 pl Lysis solution containing DNase I. Lysis was stopped after 5 minutes by addition of 2 pl STOP Solution for 2 min. For the RT-qPCR analysis, 1 pL of lysate was dispensed per well into a 96-well PCR plate in a total of 10 pL RT-qPCR reaction volume.
  • RT-qPCR was performed using the TaqMan® 1-Step qRT-PCR Mix from the Cells-to-CT 1-step TaqMan Kit, with TaqMan probes for GAPDH (VIC_PL, Assay Id Mm99999915_g1), mTTR (FAM, Assay Id Mm00443267_m1), ApoB (FAM, Assay Id Mm01545150_m1) or DGAT2 (FAM, Assay Id Mm00499536_m1).
  • RT-qPCR was performed using a QuantStudio 5 thermocycling instrument (Applied BioSystems). Relative quantification was determined using the AACT method, where GAPDH was used as internal control and expression changes normalized to the reference sample (no siRNA treatment).
  • mice Male C57BL/6J mice (20-25 g) were group housed in the Saretius animal unit at the University of Reading, and maintained under a 12 h light/dark cycle, at 23°C with humidity controlled according to Home Office regulations. Mice were given access to standard rodent chow SDS rat expanded diet (RM3-E-FG) for the duration of the study.
  • siRNA compounds CS15 and CS16 were each formulated in RNase free PBS to concentrations of 2 and 5 mg/mL to provide doses of 10 and 20 mg/kg when given subcutaneously (SC) in a 5 mL/kg dosing volume.
  • Single siRNA control compounds CS8, CS13 and PAR-ApoB control were each formulated in RNAase free PBS to concentrations of 1 and 2 mg/mL to provide doses of 5 and 10 mg/kg when given SC in 5 mL/kg dosing volumes.
  • mTTR control siRNA was formulated in RNAase free PBS to a concentration of 2 mg/mL to provide a dose of 10 mg/kg when given SC in 5 mL/kg dosing volume.
  • each treatment group was terminally sampled by cardiac puncture under isoflurane. Liver tissue was excised and snap frozen in liquid N2. Total RNA was extracted from homogenates of snap-frozen whole liver using QIAGEN RNeasy Mini Kit (74104).
  • Duplex RT-qPCR was performed using the ThermoFisher TaqMan Fast 1-Step Master Mix with TaqMan probes for GAPDH (VIC_PL, Assay Id Mm99999915_g1), mTTR (FAM, Assay Id Mm00443267_m1), ApoB (FAM, Assay Id Mm01545150_m1) or DGAT2 (FAM, Assay Id Mm00499536_m1).
  • Relative quantification (RQ) of target mRNA was determined using the AACT method, where GAPDH was used as internal control and the expression changes of the target gene were normalized to the vehicle control.
  • the mTTR portion showed knockdown of mTTR target comparable to the modified single siRNA mTTR (bottom 2 rows in Table 1) while the ApoB portion showed reduced efficacy (KD range: 64.2% - 76.3%) compared to the single siRNA counterpart ApoB_C3_OR (KD range: 90.1% - 92.8%).
  • siRNA constructs were preincubated in human serum for 2 hours and transfected in primary mouse hepatocytes for 48 hours when knockdown of target gene was measured by RT-qPCR.
  • Table 2 the mTTR portion in the ApoB-mTTR heterodimer was stable across different concentrations of human serum showing KD levels between 87% and 94.5% which were comparable to KD levels of the standard mTTR siRNA (KD between 74.3% and 93.1%).
  • DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers pre-incubated in vehicle showed high level of knockdown (KD) for both target genes DGAT2 and ApoB (KD range: 82.7% - 93%) when transfected in primary mouse hepatocytes at concentration of 25 nM.
  • both DGAT2-ApoB-A and DGAT2- ApoB-B heterodimers showed high KD levels fully comparable to pre-incubation in vehicle (KD range after serum pre-incubation at 25 nM: 75.9% - 89.8%) suggesting stability in 50% human serum.
  • GS-mTTR-A and GS-mTTR-B heterodimers pre-incubation in vehicle showed high levels of KD for both target genes ApoB and mTTR (KD range: 83.2% - 97%) when transfected in primary mouse hepatocytes at concentration of 25 nM.
  • KD range after serum pre-incubation followed by transfection at 25 nM: 87.5% - 96.2%) suggesting high serum stability for these heterodimers.
  • the KD levels of target genes ApoB and mTTR showed by GS-mTTR-A and GS-mTTR-B heterodimers when pre-incubated in vehicle or 50% human serum followed by transfection, were fully comparable to the KD levels of their single siRNA control counterparts mTTR, mTTR-AC, mTTR-ARG, PAR-ApoB, GS-AC and GS-ARG (KD range for siRNA controls: 90.2% - 98.2%).
  • KD range for siRNA controls 90.2% - 98.2%.
  • ApoB-mTTR_AG1 and ApoB-mTTR_AG2 heterodimers showed ApoB KD levels of 51.9% and 57% respectively at 100 nM and KD levels of 48% and 46.2% respectively at 25 nM in free-uptake.
  • ApoB_C3_01 When primary hepatocytes were treated with the single ApoB siRNA counterpart, ApoB_C3_01 , at 100 nM and 25 nM, KD levels were 68.6% and 54.5% respectively suggesting that the heterodimer worked almost as effectively as single siRNA control in silencing target gene ApoB.
  • ApoB-mTTR heterodimer structures showed to be highly effective in silencing mTTR target in all the ApoB-mTTR heterodimers tested here showing KD levels of mTTR ranging from 88.4% to 97.7% at 25 nM and 100 nM treatment in free-uptake assay.
  • mTTR KD values showed by treatment with heterodimers were similar to KD values obtained with single mTTR siRNA control treatment (KD level at 25 nM and 100 nM: 98.1%).
  • a full description of siRNA structures used can be found in Table 7.
  • ApoB-DGAT2 (CS15 and CS16) heterodimer siRNAs showed ApoB KD levels of 64% and 60% respectively at 25 nM and KD levels of 49% and 50% respectively at 4 nM by receptor-mediated uptake in primary mouse hepatocytes.
  • ApoB KD was 72% and 53% respectively at 25nM and 4nM.
  • heterodimer siRNAs showed KD levels of 76% and 65% at 25nM and 55% and 57% at 4nM.
  • Table 8 Target mRNA Knock-down (%) in primary mouse hepatocytes by heterodimer siRNAs or single siRNA controls, with or without preincubation in 80 % human serum for 2 h at 37°C.
  • siRNA constructs were preincubated in 80% human serum (HS) for 2 hours at 37C and receptor-mediated uptake assays subsequently performed in primary mouse hepatocytes. Knockdown of target genes was measured after 48hrs by RT-qPCR.
  • ApoB-DGAT2 heterodimer siRNAs As shown in Table 8, ApoB-DGAT2 heterodimer siRNAs, CS15 and CS16, showed equivalent levels of target KD when compared to single siRNAs of the same sequence, CS13 and CS8.
  • ApoB KD was 71% and 73% respectively for CS15 and CS16 heterodimer siRNAs at 25nm and 52% and 49% at 4nM. This level of silencing compares favourably with that shown by single siRNA counterpart CS13; 71% and 60% KD at 25nM and 4nM, respectively.
  • levels of DGAT2 KD achieved by heterodimer siRNAs was 75% and 73% at 25nM (64% and 58% at 4nM) versus 80% and 67% from single siRNA CS8 at 25nM and 4nM, respectively.
  • mice for each treatment group were injected subcutaneously (SC) with either vehicle (PBS), or GalNAc-conjugated ApoB-DGAT2 (CS15 or CS16) heterodimer, or ApoB (CS13) or DAGT2 (CS8) single siRNA controls (Table 2).
  • Each compound was administered at either 5mg/kg or 10mg/kg (single siRNAs); equivalent to 10mg/kg and 20mg/kg, respectively, for heterodimer siRNAs.
  • levels of liver target mRNA was measured by RT- qPCR and % knockdown (KD) measured relative to vehicle-treated controls.
  • Table 10 shows in vivo silencing of liver target mRNAs (ApoB and DGAT2) by heterodimer siRNA compounds (CS15 and CS16) compared to single ApoB or DGAT2 siRNA controls (CS13 and CS8, respectively).
  • CS15 heterodimer (20mg/kg) led to a 53% KD of both ApoB and DGAT2 mRNA at Day 5, with CS16 heterodimer providing 62% and 54% KD of ApoB and DGAT2, respectively.
  • a positive control siRNA (PAR-ApoB; Alnylam) performed similarly, leading to 63% ApoB KD at Day 5.
  • the silencing of ApoB was reduced at Day 14 for heterodimer CS15 and single siRNA CS13 (13% and 11%, respectively) which was not unexpected, given the positive control of the same sequence (PAR-ApoB; Alnylam) performed similarly (17% KD).
  • the heterodimer CS16 however, provided a KD of 34% at D14.
  • silencing of DGAT2 mRNA was maintained in mice receiving either heterodimer (CS15 67% KD; CS16 57%) or single siRNA CS8 (51%) at Day14.
  • CS15 heterodimer Day 14
  • Table 11 shows Comparative Efficacy (%) of ApoB-DGAT2 heterodimer siRNA compared to each single siRNA (ApoB or DGAT2) on day 5 and day 14. [%KD ApoB or DGAT2 by heterodimer siRNA divided by %KD by mono siRNA (x100)].
  • heterodimer siRNAs compared to single siRNA controls.
  • ApoB KD heterodimers shows efficacy of 134 - 164% (Day 5) and 210 - 280% (Day 14) and at higher dose; 87-102% (Day 5) and 114 - 298% (D14).
  • comparative efficacy is 84 - 124% (Day 5) and 102 - 147% (Day 14); whereas at higher dose, 109 - 111% (Day 5) and 112 - 131% (Day 14).
  • AAGAAGUUCCAGAAAUACAA SEQ UUGUAUUUCUGGAACUUCUU ( SEQ ID NO 4831 ) ID NO 4840 )
  • ApoC3 antisense strand usCfsasCfuGfagaauAfcUfgUfcCfcGfsu (SEQ ID NO:4859) sense strand: (NAG37)s(invAb)sacgggacaGfllfAfuucucaguias(invAb) (SEQ ID NQ:4860)
  • AGTGGAGTAGGGCGCTTCG (NAG37)s(invAb)sacgggacaGfllfAfuucucaguias(invAb) (SEQ ID NO:4862)
  • a, c, g, i, and u represent 2'-O-methyl adenosine, cytidine, guanosine, inosine, and uridine, respectively
  • Af, Cf, Gf, and Ilf represent 2'-fluoro adenosine, cytidine, guanosine, and uridine, respectively
  • s represents a phosphorothioate linkage
  • (invAb) represents an inverted abasic deoxyribose residue
  • (NAG37) is

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Abstract

L'invention concerne des molécules d'acide nucléique isolées comprenant au moins deux molécules d'acide ribonucléique (ARN) inhibiteur double brin conçues pour effectuer le silençage par interférence ARN soit du même gène soit de différents gènes pour améliorer le silençage, modulant ainsi l'expression génique.
PCT/GB2024/051168 2023-05-04 2024-05-03 Silençage double Ceased WO2024228030A2 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
AU2024265748A AU2024265748A1 (en) 2023-05-04 2024-05-03 Dual silencing
EP24726696.8A EP4705458A2 (fr) 2023-05-04 2024-05-03 Silençage double
KR1020257035331A KR20260006569A (ko) 2023-05-04 2024-05-03 이중 발현 억제
CN202480030389.6A CN121057819A (zh) 2023-05-04 2024-05-03 双重沉默
GB2414903.1A GB2631903B (en) 2023-05-04 2024-05-07 Dual silencing
GB2501121.4A GB2635479A (en) 2023-05-04 2024-05-07 Dual silencing
GB2406314.1A GB2629679B (en) 2023-05-04 2024-05-07 Dual Silencing
IL323761A IL323761A (en) 2023-05-04 2025-10-04 Dual silencing
MX2025012698A MX2025012698A (es) 2023-05-04 2025-10-23 Doble silenciamiento

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB2306589.9A GB2629617B (en) 2023-05-04 2023-05-04 Dual Silencing
GB2306589.9 2023-05-04
GB2404488.5 2024-03-28
GBGB2404488.5A GB202404488D0 (en) 2024-03-28 2024-03-28 Dual silencing

Publications (2)

Publication Number Publication Date
WO2024228030A2 true WO2024228030A2 (fr) 2024-11-07
WO2024228030A3 WO2024228030A3 (fr) 2024-12-19

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PCT/GB2024/051168 Ceased WO2024228030A2 (fr) 2023-05-04 2024-05-03 Silençage double

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Cited By (1)

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WO2010141511A2 (fr) 2009-06-01 2010-12-09 Halo-Bio Rnai Therapeutics, Inc. Polynucléotides pour interférence arn multivalente, compositions et procédés pour les utiliser
WO2013040429A1 (fr) 2011-09-14 2013-03-21 Rana Therapeutics Inc. Composés oligonucléotidiques multimères
WO2016205410A2 (fr) 2015-06-15 2016-12-22 Mpeg La, Llc Oligonucléotides multi-conjugués définis
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WO2018145086A1 (fr) 2017-02-06 2018-08-09 Mpeg La, Llc Oligonucléotides multimères ayant une clairance rénale diminuée

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WO2013040429A1 (fr) 2011-09-14 2013-03-21 Rana Therapeutics Inc. Composés oligonucléotidiques multimères
WO2016205410A2 (fr) 2015-06-15 2016-12-22 Mpeg La, Llc Oligonucléotides multi-conjugués définis
WO2017188707A1 (fr) 2016-04-29 2017-11-02 Ewha University - Industry Collaboration Foundation Nanostructures d'arn de substrat dicer ayant un effet de silençage génique amélioré et leur procédé de préparation
WO2018145086A1 (fr) 2017-02-06 2018-08-09 Mpeg La, Llc Oligonucléotides multimères ayant une clairance rénale diminuée

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