WO2020180897A1 - Multimeric oligonucleotides with enhanced bioactivity - Google Patents

Multimeric oligonucleotides with enhanced bioactivity Download PDF

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Publication number
WO2020180897A1
WO2020180897A1 PCT/US2020/020845 US2020020845W WO2020180897A1 WO 2020180897 A1 WO2020180897 A1 WO 2020180897A1 US 2020020845 W US2020020845 W US 2020020845W WO 2020180897 A1 WO2020180897 A1 WO 2020180897A1
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Prior art keywords
oligonucleotide
subunits
multimeric
multimeric oligonucleotide
covalent
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PCT/US2020/020845
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English (en)
French (fr)
Inventor
Jonathan Miles Brown
Kristin K. H. Neuman
Hans-Peter Vornlocher
Philipp Hadwiger
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MPEG LA LLC
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MPEG LA LLC
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Priority to CN202080033528.2A priority Critical patent/CN113994004A/zh
Priority to EP20766332.9A priority patent/EP3935168A4/en
Priority to JP2021552130A priority patent/JP2022523226A/ja
Priority to AU2020233375A priority patent/AU2020233375A1/en
Priority to US17/436,254 priority patent/US20230287406A1/en
Priority to CA3132505A priority patent/CA3132505A1/en
Publication of WO2020180897A1 publication Critical patent/WO2020180897A1/en
Priority to IL286070A priority patent/IL286070A/en
Anticipated expiration legal-status Critical
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers

Definitions

  • the present disclosure relates to oligonucleotide-based therapeutics. More specifically, the present disclosure relates to multimeric therapeutic oligonucleotides containing multiple sub-units each with increased bioactivity in a subject relative to the corresponding monomers.
  • Oligonucleotides are now a well-established class of therapeutics with multiple applications (e.g., RNA interference, or RNAi) and ongoing clinical trials.
  • oligonucleotide therapeutics for example, the delivery of the oligonucleotide to a target cell and the subsequent internalization of the oligonucleotide into the target cell in sufficient quantities to achieve a desired therapeutic effect.
  • LNPs lipid nanoparticles
  • lipid spheroids including positively charged lipids to neutralize the negative charge of the oligonucleotide and to facilitate target cell binding and internalization.
  • LNPs can in some cases facilitate delivery and internalization, they suffer from major drawbacks, for example poor targeting and toxicity, resulting in a narrowed therapeutic window.
  • GalNAc A -acetyl gal actosami ne
  • oligonucleotides containing a large proportion of such groups bind to proteins circulating in the blood, thereby increasing the effective molecular size of the oligonucleotide and decreasing the rate of secretion via the kidney.
  • the use of a high number of phosphorothioate groups has many drawbacks.
  • phosphorothioate oligonucleotides of the appropriate length can block the binding of biologically relevant proteins to their natural receptors resulting in toxic side effects (Stein, CA. J Clin Invest. 2001 Sep 1; 108(5): 641-644).
  • the facilitation of protein binding that is an advantage of high levels of thiophosphorylation is simultaneously a major disadvantage.
  • the present disclosure relates to compositions and related methods to increase the biological activity in a subject of an oligonucleotide therapeutic agent.
  • the disclosure is applicable to all types of oligonucleotide therapeutics, including siRNAs and miRNAs as well as antisense oligonucleotides, independent of phosphorothioate content and resulting protein binding characteristics.
  • the present disclosure provides a multimeric oligonucleotide wherein an oligonucleotide therapeutic agent (a“subunit”) is linked via a covalent linker to a number of copies of the same or differing subunits and wherein the biological activity of each of the agents is increased relative to the activity of the agent alone.
  • the increase in bioactivity is independent of the phosphorothioate content.
  • the increase in bioactivity is independent of the total phosphorothioate content of the multimeric oligonucleotide.
  • the increase in bioactivity is independent of the ratio of phosphorothioates to nucleotide residues in the multimeric oligonucleotide.
  • the multimeric oligonucleotide may contain one or more double-stranded subunits, or may contain four or more subunits overall, or may have a molecular weight of at least about 45 kilodaltons (kD).
  • the improved and advantageous properties of the multimers according to the disclosure may be described in terms of increased in vivo activity.
  • the relative increase in in vivo bioactivity of each of the subunits in the multimer as compared to the corresponding monomer may be in the range of 2-10 and higher; for example, the relative increase may be 2, 5, 10, or more times that of the corresponding monomer.
  • the present disclosure also relates to new synthetic intermediates and methods of synthesizing the multi-conjugate oligonucleotides.
  • the present disclosure also relates to methods of using the multi-conjugate oligonucleotides, for example in reducing gene expression, biological research, treating or preventing medical conditions, and/or to produce new or altered phenotypes.
  • the disclosure provides a multimeric oligonucleotide comprising subunits . , wherein: each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits .
  • the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; and the increase in activity of one or more subunits within the multimeric oligonucleotide is independent of phosphorothioate content in the multimeric oligonucleotide.
  • the disclosure provides a multimeric oligonucleotide comprising subunits . , wherein: each of the subunits .
  • the multimeric oligonucleotide is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ; the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; and at least one subunit within the multimeric oligonucleotide is a double-stranded oligonucleotide.
  • the disclosure provides a multimeric oligonucleotide comprising subunits . , wherein: each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ; the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; and the multimeric oligonucleotide comprises 4 or more subunits.
  • the disclosure provides a multimeric
  • oligonucleotide comprising subunits . , wherein: each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits
  • oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; and the molecular weight of the multimeric oligonucleotide is at least about 45 kD.
  • oligonucleotide is a double-stranded oligonucleotide.
  • the multimeric oligonucleotide comprises 4 or more subunits.
  • the molecular weight of the multimeric oligonucleotide is at least about 45 kD.
  • the disclosure provides a multimeric oligonucleotide comprising subunits . , wherein: each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ; the multimeric oligonucleotide has a molecular weight and/or size configured to decrease its clearance due to glomerular filtration; and the molecular weight of the multimeric oligonucleotide is at least about 45 kD, wherein the multimeric oligonucleotide comprises a hetero-multimer of six or more subunits . , wherein at least two subunits . are substantially different.
  • the disclosure provides a multimeric oligonucleotide comprising subunits . , wherein: each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ; the multimeric oligonucleotide comprises five or more subunits . ; and wherein at least one subunit comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • the disclosure provides a multimeric
  • oligonucleotide comprising subunits - forming Structure 119: - ⁇ - ⁇ - ⁇ - , wherein: each subunit is independently a single or double-stranded oligonucleotide; each of the subunits is joined to another subunit by a covalent linker ⁇ ; and wherein at least one subunit comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • the disclosure provides a multimeric oligonucleotide comprising two subunits . , wherein: each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to the other subunit by a covalent linker ⁇ ; the molecular weight of the compound is at least about 45 kD; and at least one subunit comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • At least two subunits . are substantially different.
  • all of the subunits are substantially different.
  • At least two subunits . are substantially the same or are identical.
  • all of the subunits . are substantially the same or are identical.
  • the multimeric oligonucleotide comprises five, six, seven, eight, nine, or ten subunits . .
  • the multimeric oligonucleotide comprises six subunits [0029] In an embodiment, the multimeric oligonucleotide comprises seven, eight, nine, or ten subunits . .
  • each subunit . is independently 10-30, 17-27,
  • one or more subunits are double-stranded. In an embodiment, one or more subunits are single-stranded. In an embodiment, the subunits comprise a combination of single-stranded and double-stranded oligonucleotides.
  • one or more nucleotides within an oligonucleotide is an RNA, a DNA, or an artificial or non-natural nucleic acid analog.
  • At least one of the subunits is a siRNA. In an embodiment, at least one of the subunits is a miRNA. In an embodiment, at least one of the subunits is an antisense oligonucleotide.
  • two or more siRNA subunits are joined by covalent linkers attached to the sense strand of a first siRNA and the antisense strand of a second siRNA.
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of a subunit.
  • S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of a subunit.
  • the covalent linker of Formula (I) is formed from a
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the nucleotide linker is between 2-6 nucleotides in length. In an embodiment, the nucleotide linker is 3, 4, or 5 nucleotides in length.
  • the nucleotide linker is a dinucleotide linker.
  • each covalent linker ⁇ is the same. In an embodiment, the covalent linkers ⁇ comprise two or more different covalent linkers.
  • At least two subunits are joined by covalent linkers ⁇ between the 3’ end of a first subunit and the 3’ end of a second subunit.
  • the multimeric oligonucleotide further comprises one or more targeting ligands.
  • at least one of the subunits is a targeting ligand.
  • the targeting ligand is an aptamer.
  • the targeting ligand comprises N-Acetylgalactosamine (GalNAc). In an embodiment, the targeting ligand comprises an immunostimulant. In an embodiment, the targeting ligand comprises a CpG oligonucleotide. In an embodiment, the CpG oligonucleotide comprises the sequence TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 162). In an embodiment, the CpG oligonucleotide comprises the sequence
  • the multimeric oligonucleotide is at least 75, 80, 85,
  • the oligonucleotide with complementarity to TTR mRNA comprises UUAUAGAGCAAGAACACUGUUUU (SEQ ID NO: 164).
  • the multimeric oligonucleotide is administered in vivo by intravenous injection.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, the method comprising administering an effective amount of the multimeric oligonucleotide to the subject, the multimeric oligonucleotide comprising subunits . , wherein: each of the subunits
  • . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ;
  • the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; and the increase in activity of one or more subunits within the multimeric oligonucleotide is independent of
  • . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ;
  • the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; and at least one subunit within the multimeric oligonucleotide is a double-stranded oligonucleotide.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, the method comprising administering an effective amount of the multimeric oligonucleotide to the subject, the multimeric oligonucleotide comprising subunits . , wherein: each of the subunits
  • the multimeric oligonucleotide comprises 4 or more subunits.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, the method comprising administering an effective amount of the multimeric oligonucleotide to the subject, the multimeric oligonucleotide comprising subunits . , wherein: each of the subunits
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, the method comprising administering an effective amount of the multimeric oligonucleotide to the subject, the multimeric oligonucleotide comprising subunits . , wherein: each of the subunits
  • the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form;
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, the method comprising administering an effective amount of the multimeric oligonucleotide to the subject, the multimeric oligonucleotide comprising subunits . , wherein: each of the subunits
  • . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ;
  • the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; wherein the multimeric oligonucleotide comprises Structure 117: - ⁇ - ⁇ - ⁇ - ; and wherein at least one subunit comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ; the multimeric oligonucleotide has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form; wherein the multimeric oligonucleotide comprises two subunits . ; wherein the molecular weight of the compound is at least about 45 kD; and wherein at least one subunit comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • the administering comprises intravenous injection.
  • the number of subunits contained in the multimeric oligonucleotide is m, m being an integer selected to enable the multimeric
  • oligonucleotide to have the molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered in monomeric form.
  • all of the subunits are substantially different.
  • At least two subunits . are substantially the same or are identical.
  • all of the subunits . are substantially the same or are identical.
  • the multimeric oligonucleotide comprises five, six, seven, eight, nine, or ten subunits . .
  • the multimeric oligonucleotide comprises seven, eight, nine, or ten subunits . .
  • each subunit . is independently 10-30, 17-27,
  • one or more subunits are double-stranded. In an embodiment, one or more subunits are single-stranded. In an embodiment, the subunits comprise a combination of single-stranded and double-stranded oligonucleotides.
  • At least one of the subunits is RNA. In an embodiment, at least one of the subunits is a siRNA, a saRNA, or a miRNA. In an embodiment, at least one of the subunits is a siRNA. In an embodiment, at least one of the subunits is a miRNA. In an embodiment, at least one of the subunits is an antisense oligonucleotide. In an embodiment, at least one of the subunits is a double-stranded siRNA.
  • two or more siRNA subunits are joined by covalent linkers attached to the sense strands of the siRNA. In an embodiment, two or more siRNA subunits are joined by covalent linkers attached to the antisense strands of the siRNA. In an embodiment, two or more siRNA subunits are joined by covalent linkers attached to the sense strand of a first siRNA and the antisense strand of a second siRNA.
  • one or more of the covalent linkers ⁇ comprise a cleavable covalent linker.
  • the cleavable covalent linker contains an acid cleavable bond, a reductant cleavable bond, a bio-cleavable bond, or an enzyme cleavable bond.
  • the cleavable covalent linker is cleavable under intracellular conditions.
  • R2 is a thiopropionate or disulfide
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of a subunit.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of a subunit.
  • S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of a subunit.
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • one or more of the covalent linkers ⁇ comprise a nucleotide linker.
  • the nucleotide linker is between 2-6 nucleotides in length. In an embodiment, the nucleotide linker is 3, 4, or 5 nucleotides in length. In an embodiment, the nucleotide linker is a dinucleotide linker.
  • each covalent linker ⁇ is the same. In an embodiment, the covalent linkers ⁇ comprise two or more different covalent linkers. [00102] In an embodiment, at least two subunits are joined by covalent linkers ⁇ between the 3’ end of a first subunit and the 3’ end of a second subunit. In an
  • At least two subunits are joined by covalent linkers ⁇ between the 3’ end of a first subunit and the 5’ end of a second subunit. In an embodiment, at least two subunits are joined by covalent linkers ⁇ between the 5’ end of a first subunit and the 3’ end of a second subunit. In an embodiment, at least two subunits are joined by covalent linkers ⁇ between the 5’ end of a first subunit and the 5’ end of a second subunit.
  • the multimeric oligonucleotide further comprises one or more targeting ligands.
  • at least one of the subunits is a targeting ligand.
  • the targeting ligand is an aptamer.
  • the targeting ligand comprises N-Acetylgalactosamine (GalNAc).
  • the targeting ligand comprises an immunostimulant.
  • the targeting ligand comprises a CpG oligonucleotide.
  • the CpG oligonucleotide comprises the sequence TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 162).
  • the CpG oligonucleotide comprises the sequence
  • the multimeric oligonucleotide is at least 75, 80, 85,
  • At least one subunit comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • the subunit with complementarity to TTR mRNA comprises increased activity in vivo relative to a monomeric oligonucleotide with complementarity to TTR mRNA.
  • the oligonucleotide with complementarity to TTR mRNA comprises UUAUAGAGCAAGAACACUGUUUU (SEQ ID NO: 164).
  • the multimeric oligonucleotide is administered in vivo by intravenous injection.
  • the multimeric oligonucleotide is administered in vivo by intravenous injection and has a molecular weight and/or size configured to increase in vivo activity of one or more subunits within the multimeric oligonucleotide relative to in vivo activity of the same subunit when administered subcutaneously in monomeric form.
  • the increase in in vivo activity of one or more subunits within the multimeric oligonucleotide is at least a 2-fold increase relative to in vivo activity of the same subunit when administered in monomeric form. In an embodiment, the increase in in vivo activity of one or more subunits within the multimeric
  • oligonucleotide is at least a 5-fold increase relative to in vivo activity of the same subunit when administered in monomeric form.
  • the increase in in vivo activity of one or more subunits within the multimeric oligonucleotide is at least a 10-fold increase relative to in vivo activity of the same subunit when administered in monomeric form.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 92 or Structure 93 :
  • each - is independently a single-stranded oligonucleotide, each is independently a double stranded oligonucleotide, each ⁇ is a covalent linker joining adjacent oligonucleotides, and m is an integer > 0 and n is an integer > 0,
  • the method comprising the steps of: (i) forming - ⁇ - by: (a) annealing a first single- stranded oligonucleotide - and a second single-stranded oligonucleotide
  • R i a third single- stranded oligonucleotide R 2 , wherein R1 and R2 are chemical moieties capable of reacting directly or indirectly to form a covalent linker ⁇ , thereby forming
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 92 or Structure 93 : (Structure 92); or i _
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each ⁇ is a covalent linker joining adjacent oligonucleotides, and m is an integer > 0 and n is an integer > 0, the method comprising (i) annealing a first single-stranded oligonucleotide - and a first single-stranded heterodimer - ⁇ - , thereby forming - ⁇ - ; (ii) optionally annealing - ⁇ - and a second single-stranded dimer - ⁇
  • n is an integer > 0.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising:
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each ⁇ is a covalent linker joining adjacent oligonucleotides, and p is an integer > 0, q is an integer > 0, and r is an integer > 0, the method comprising: (i) annealing Structure 92 and Structure 93:
  • oligonucleotides the method comprising the steps of: (i) forming a first
  • R1 and R2 are chemical moieties capable of reacting directly or indirectly to form a covalent linker ⁇ , thereby forming * , and annealing
  • a terminus of the multimeric oligonucleotide is conjugated to a targeting ligand.
  • each - and is independently 10-30, 17-
  • one or more nucleotides within - and is an RNA, a DNA, or an artificial or non-natural nucleic acid analog.
  • two or more siRNA are joined by covalent linkers attached to the sense strands of the siRNA. In an embodiment, two or more siRNA are joined by covalent linkers attached to the antisense strands of the siRNA. In an embodiment, two or more siRNA are joined by covalent linkers attached to the sense strand of a first siRNA and the antisense strand of a second siRNA.
  • one or more of the covalent linkers ⁇ comprise a cleavable covalent linker.
  • the cleavable covalent linker contains an acid cleavable bond, a reductant cleavable bond, a bio-cleavable bond, or an enzyme cleavable bond.
  • the cleavable covalent linker is cleavable under intracellular conditions.
  • At least one covalent linker comprises a disulfide bond or a compound of Formula (I): wherein: S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of - or ; each Ri is independently a C2-C1 0 alkyl, alkoxy, or aryl group; R2 is a thiopropionate or disulfide
  • each X is independently selected from: or
  • each Ri is independently a C2-C1 0 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • each Ri is independently a C2-
  • Cio alkyl, alkoxy, or aryl group Cio alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the covalent linker of Formula (I) is formed from a
  • covalent linking precursor of Formula wherein: each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • one or more of the covalent linkers ⁇ comprise a nucleotide linker.
  • the nucleotide linker is between 2-6 nucleotides in length.
  • the nucleotide linker is 3, 4, or 5 nucleotides in length.
  • the nucleotide linker is a dinucleotide linker.
  • each covalent linker ⁇ is the same. In an embodiment, the covalent linkers ⁇ comprise two or more different covalent linkers.
  • two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 3’ end of a first subunit and the 3’ end of a second subunit. In an embodiment, two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 3’ end of a first subunit and the 5’ end of a second subunit. In an embodiment, two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 5’ end of a first subunit and the 3’ end of a second subunit. In an embodiment, two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 5’ end of a first subunit and the 5’ end of a second subunit.
  • the multimeric oligonucleotide further comprises one or more targeting ligands.
  • at least one of the oligonucleotide subunits is a targeting ligand.
  • the targeting ligand is an aptamer.
  • a terminus of the multimeric oligonucleotide is conjugated to a targeting ligand.
  • the targeting ligand comprises N-Acetylgalactosamine (GalNAc).
  • the multimeric oligonucleotide is at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% pure.
  • At least one of the oligonucleotide subunits comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • the oligonucleotide with complementarity to TTR mRNA comprises UUAUAGAGCAAGAACACUGUUUU (SEQ ID NO: 164).
  • one or more subunits comprise one or more phosphorothioate modifications. In an embodiment, one or more subunits comprise 1-3 phosphorothioate modifications at the 5’ and/or 3’ end. In an embodiment, each subunit comprises 0-15 phosphorothioate modifications, or 1-12 phosphorothioate modifications, or 2-8 phosphorothioate modifications. [00138] In another aspect, the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 100
  • each is independently a single-stranded oligonucleotide
  • each _ is independently a single or double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent oligonucleotides
  • a, a’, b, b’, c, c’, d and d’ are each independently 0 or 1
  • R1 and R2 are chemical moieties capable of reacting directly or indirectly to form a covalent linker ⁇ , thereby forming Structure 100
  • the sum of a+a'+b+b'+c+c'+d+d' is greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+b+b'+c+c'+d+d' is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 102
  • each is independently a single-stranded oligonucleotide
  • each _ is independently a double-stranded oligonucleotide
  • each _ is independently a single or double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent oligonucleotides
  • Structure 101 wherein: a is l, and a’, a”, b, b’, b”, c, c’, c”, d, d’, and d” are each independently 0 or 1, thereby forming Structure 102
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 103
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ . is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure 100
  • Structure 101 wherein: a’ is 1, and a, a”, b, b’, b”, c, c’, c”, d, d’, and d” are each independently 0 or 1, thereby forming Structure 103
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 104
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each ______ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • the sum of a+a'+a"+a"'+b+b'+b"+b"'+c+c'+c"+c"'+d+d'+d"+d"' is greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+a"+a"'+b+b'+b"+b"'+c+c'+c"+c"'+d+d'+d"+d"' is 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 107
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • a+a'+a"+a"'+b+b'+b"+b"'+c+c'+c"+c"'+d+d'+d"+d"' is 3, 4, 5, 6, 7, 8, 9, or 10.
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • the sum of a+a'+b+b'+c+c'+d+d' is greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, or greater than or equal to 11.
  • the sum of a+a'+b+b'+c+c'+d+d' is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 111
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each ______ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure 108
  • Structure 112 wherein: d is 1, and a, a’, a”, b, b’, b”, c, c’, c”, d’ and d” are each independently 0 or 1, thereby forming Structure 111
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, or greater than or equal to 11.
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 113
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • Structure 112 wherein: d’ is 1, and a, a’, a”, b, b’, b”, c, c’, c”, d and d” are each independently 0 or 1, thereby forming Structure 113
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, or greater than or equal to 11.
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 114
  • each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising reacting Structure 115
  • the sum of a and b is greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of a and b is 5, 6, 7, 8, 9, or 10.
  • the method further comprises annealing one or more single-stranded oligonucleotides - with a complementary single-stranded oligonucleotide - in Structure 98 to Structure 113, thereby forming a double- stranded oligonucleotide .
  • each single-stranded oligonucleotide - and each single or double strand oligonucleotide _ comprises 0-15 phosphorothioate modifications, or 1-12 phosphorothioate modifications, or 2-8 phosphorothioate modifications.
  • At least one _ is a double-stranded
  • the total number of - and _ in the multimeric oligonucleotide is at least 4.
  • the multimeric oligonucleotide is at least about 45 kD.
  • the multimeric oligonucleotide is———— * - ,
  • each _ is substantially the same or different.
  • the multimeric oligonucleotide is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • a terminus of the multimeric oligonucleotide is conjugated to a targeting ligand.
  • each - , and _ is independently 10-
  • _ is an RNA, a DNA, or an artificial or non-natural nucleic acid analog.
  • At least one of - , and _ is a RNA.
  • At least one of - , and _ . is a siRNA, a saRNA, or a miRNA.
  • At least one of - , , and ______ is a siRNA.
  • At least one of - , , and _ is a miRNA.
  • At least one of - and _ is an antisense oligonucleotide.
  • two or more siRNA are joined by covalent linkers attached to the sense strands of the siRNA. In an embodiment, two or more siRNA are joined by covalent linkers attached to the antisense strands of the siRNA.
  • two or more siRNA are joined by covalent linkers attached to the sense strand of a first siRNA and the antisense strand of a second siRNA.
  • one or more of the covalent linkers ⁇ comprise a cleavable covalent linker.
  • the cleavable covalent linker contains an acid cleavable bond, a reductant cleavable bond, a bio-cleavable bond, or an enzyme cleavable bond.
  • the cleavable covalent linker is cleavable under intracellular conditions.
  • At least one covalent linker comprises a disulfide bond or a compound of Formula (I):
  • the compound of Formula (I) is wherein S is attached by a covalent bond or by a linker to the 3 or 5 terminus of - , , or _ ; each Ri is independently a
  • R2 is a thiopropionate or disulfide group.
  • R2 is a thiopropionate or disulfide group.
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the covalent linker of Formula (I) is formed from a covalent linking precursor of Formula (II):
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • one or more of the covalent linkers ⁇ comprise a nucleotide linker.
  • the nucleotide linker is between 2-6 nucleotides in length. In an embodiment, the nucleotide linker is 3, 4, or 5 nucleotides in length. In an embodiment, the nucleotide linker is a dinucleotide linker.
  • each covalent linker ⁇ is the same. In an embodiment, the covalent linkers ⁇ comprise two or more different covalent linkers.
  • two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 3’ end of a first subunit and the 3’ end of a second subunit. In an embodiment, two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 3’ end of a first subunit and the 5’ end of a second subunit. In an embodiment, two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 5’ end of a first subunit and the 3’ end of a second subunit. In an embodiment, two or more adjacent oligonucleotide subunits are joined by covalent linkers ⁇ between the 5’ end of a first subunit and the 5’ end of a second subunit.
  • the multimeric oligonucleotide further comprises one or more targeting ligands. In an embodiment, at least one of the oligonucleotide subunits is a targeting ligand.
  • the targeting ligand is an aptamer.
  • the targeting ligand comprises N-Acetylgalactosamine (GalNAc).
  • a terminus of the multimeric oligonucleotide is conjugated to a targeting ligand.
  • the multimeric oligonucleotide is at least 75, 80, 85,
  • At least one of the oligonucleotide subunits comprises an oligonucleotide with complementarity to transthyretin (TTR) mRNA.
  • TTR transthyretin
  • the oligonucleotide with complementarity to TTR mRNA comprises UUAUAGAGCAAGAACACUGUUUU (SEQ ID NO: 164).
  • FIG. 1 A presents the chemical structure of a tri-antennary N- acetylgalactosamine ligand.
  • FIG. IB presents the chemical structure of a dithio-bis-maleimidoethane.
  • FIG. 2 presents a 5’-GalNAc-FVII canonical control, which is discussed in connection with Example 9.
  • FIG. 3 presents a GalNAc-homodimer conjugate (XD-06330), which is discussed in connection with Example 10.
  • FIG. 4 presents a schematic diagram of a synthesis of a GalNAc- homodimer conjugate (XD-06360), which is discussed in connection with Example 11.
  • FIG. 5 presents a schematic diagram of a synthesis of a GalNAc- homodimer conjugate (XD-06329), which is discussed in connection with Example 12.
  • FIG. 6 presents data showing FVII activity in mouse serum (knockdown by FVII homodimeric GalNAc conjugates), which are discussed in connection with Example 13.
  • FIGS. 7 A and 7B and 7C present data showing FVII activity in mouse serum (knockdown by FVII homodimeric GalNAc conjugates normalized for GalNAc content), which are discussed in connection with Example 13.
  • FIG. 8 presents canonical GalNAc-siRNAs independently targeting FVII, ApoB and TTR, which are discussed in connection with Example 14.
  • FIG. 9 presents a GalNAc-heterotrimer conjugate (XD-06726), which is discussed in connection with Example 15. Key: In this Example,“GeneA” is siFVII; “GeneB” is siApoB; and“GeneC” is siTTR.
  • FIG. 10 presents a schematic diagram for a synthesis strategy for a GalN Ac-conjugated heterotrimer (XD-06726), which is discussed in connection with Example 15. Key: In this Example,“GeneA” is siFVII;“GeneB” is siApoB; and “GeneC” is siTTR.
  • FIG. 11 presents a GalNAc-heterotrimer conjugate (XD-06727), which is discussed in connection with Example 16. Key: In this Example,“GeneA” is siFVII; “GeneB” is siApoB; and“GeneC” is siTTR.
  • FIG. 12 presents a schematic diagram for a synthesis strategy for GalNAc- conjugated heterotrimer (XD-06727), which is discussed in connection with Example 16. Key: In this Example,“GeneA” is siFVII;“GeneB” is siApoB; and“GeneC” is siTTR.
  • FIG. 13 presents data for an HPLC analysis of the addition of X20336 to X20366, which are discussed in connection with Example 16.
  • FIG. 14 presents data for an HPLC analysis of the further addition of X19580 to the reaction product of X20336 and X20366, which are discussed in connection with Example 16.
  • FIG. 15 presents data for an HPLC analysis of the further addition of X18795 (5’-siFVIIantisense-3’) to the reaction product of X20336, X20366, and X19580 to yield XD-06727, which are discussed in connection with Example 16.
  • FIGS. 16A and 16B present data for TTR protein levels in serum samples (measured by ELISA), which are discussed in connection with Example 18.
  • FIGS. 17A and 17B present data for FVII enzymatic activity in serum samples, which are discussed in connection with Example 18.
  • FIGS. 18A and 18B present data for ApoB protein levels in serum samples (measured by ELISA), which are discussed in connection with Example 18.
  • FIGS. 19A and 19B present target knockdown in liver data, which are discussed in connection with Example 18.
  • FIG. 20 presents a GalNAc-heterotetramer conjugate (XD-07140), which are discussed in connection with Example 19. Key: In this Example,“GeneA” is siFVII; “GeneB” is siApoB; and“GeneC” is siTTR.
  • FIG. 21 presents a schematic diagram for synthesis of a GalNAc- heterotetramer conjugate (XD-07140), which is discussed in connection with Example 19. Key: In this Example,“GeneA” is siFVII;“GeneB” is siApoB; and“GeneC” is siTTR.
  • FIG. 22 presents HPLC results of the GalNAc-siFVII-siApoB-siTTR- siFVII Tetramer (XD-07140), which are discussed in connection with Example 19.
  • FIG. 23 presents a schematic diagram illustrating the steps for
  • FIGS. 24A and 24B present RP-HPLC results showing yield and purity of the ssRNA X30835, which are discussed in connection with Example 24.
  • FIGS. 24C and 24D present RP-HPLC results showing yield and purity of the ssRNA X30837, which are discussed in connection with Example 24.
  • FIG. 24E presents RP-HPLC results for X30838, which are discussed in connection with Example 24.
  • FIG. 24F presents RP-HPLC results for X30838, X18795 and XD-09795, which are discussed in connection with Example 24.
  • FIG. 25 presents data showing serum concentrations of FVII antisense RNA in mice at various times after injection of XD-09795 or XD-09794, which are discussed in connection with Example 25.
  • FIGS. 26A-J present data showing serum levels of various cytokines in mice at various times after injection of XD-09795 or XD-09794, which are discussed in connection with Example 26.
  • FIG. 27A presents a schematic diagram for a synthesis strategy for monomer of FVII siRNA, which is discussed in connection with Example 28.
  • FIG. 27B presents RP-HPLC results for XD-09794, which are discussed in connection with Example 28.
  • FIG. 28A presents a schematic diagram for a synthesis strategy for homo dimer of FVII siRNA, which are discussed in connection with Example 29.
  • FIG. 28B presents RP-HPLC results for XD-10635, which are discussed in connection with Example 29.
  • FIG. 29A presents a schematic diagram for a synthesis strategy for homo- trimer of FVII siRNA, which is discussed in connection with Example 30.
  • FIG. 29B presents RP-HPLC results for XD-10636, which are discussed in connection with Example 30.
  • FIG. 30A presents a schematic diagram for a synthesis strategy for homo- tetramer of FVII siRNA, which is discussed in connection with Example 31.
  • FIG. 30B presents RP-HPLC results for XD-10637, which are discussed in connection with Example 31.
  • FIG. 32A presents a schematic diagram for a synthesis strategy for homo- hexamer of FVII siRNA, which is discussed in connection with Example 33.
  • FIG. 33A presents a schematic diagram for a synthesis strategy for homo- hexamer of FVII siRNA via mono-DTME conjugate, which is discussed in connection with Example 34.
  • FIG. 33B presents RP-HPLC results for XD-09795, which are discussed in connection with Example 34.
  • FIG. 34A presents a schematic diagram for a synthesis strategy for homo- heptamer of FVII siRNA via mono-DTME conjugate, which is discussed in connection with Example 35.
  • FIG. 35B presents RP-HPLC results for XD- 10641, which are discussed in connection with Example 36.
  • FIG. 36A presents a smooth line scatter plot of FVII siRNA levels in serum for various FVII siRNA multimers over time which is discussed in connection with Example 37.
  • FIG. 36B presents a straight marked scatter plot of FVII siRNA levels in serum for various FVII siRNA multimers over time, which are discussed in connection with Example 37.
  • FIG. 38A presents a bar chart of FVII siRNA exposure levels in serum (area under the curve) for FVII multimers, which are discussed in connection with Example 37.
  • FIG. 38B presents a bar chart of total FVII siRNA levels in serum (area under the curve) for FVII multimers normalized to monomer, which are discussed in connection with Example 37.
  • FIG. 40 represents a schematic diagram for a synthesis strategy for homo- tetrameric siRNA, which is discussed in connection with Example 20.
  • FIG. 41 represents a schematic diagram for a synthesis strategy for homo- tetrameric siRNA having linkages on alternating strands, which is discussed in connection with Example 20.
  • FIG. 42 represents a schematic diagram showing a synthesis strategy for hetero-hexameric siRNA in the format of 4: 1 : 1 FVII: ApoB:TTR targeting siRNA.
  • FIG. 43 represents a schematic diagram for the preparation of FVII targeting sense strands.
  • FIG. 44 depicts RP-HPLC and MS data for the FVII targeting sense strand X39850.
  • FIG. 45 depicts RP-HPLC and MS data for the FVII targeting sense strand X39851.
  • FIG. 46 depicts RP-HPLC and MS data for the FVII targeting antisense strand XI 8795.
  • FIG. 47 depicts RP-HPLC and MS data for the FVII targeting antisense strand linked to the ApoB targeting antisense strand via a disulfide linkage and designated X39855.
  • FIG. 48 depicts RP-HPLC data for the annealed duplex of X39850 and X18795 (X39850-X18795).
  • FIG. 49 depicts RP-HPLC data for the product of the conjugation between the FVII duplex X39850-X18795 and the FVII targeting sense strand X39851 (X39850- X18795-X39851).
  • FIG. 50 depicts RP-HPLC data for the product of annealing X39850- X18795-X39851 to the dimeric FVII / ApoB targeting antisense strand X39855 (X39850- X18795-X39851-X39855).
  • FIG. 51 depicts RP-HPLC and MS data for the FVII targeting sense strand linked to the TTR targeting sense strand via a disulfide linkage and designated X39852.
  • FIG. 52 depicts RP-HPLC and MS data for the FVII targeting antisense strand linked to the TTR targeting antisense strand via a disulfide linkage and designated X39854.
  • FIG. 53 depicts RP-HPLC and MS data for the FVII targeting sense strand linked to the ApoB targeting sense strand via a disulfide linkage and designated X39853.
  • FIG. 54 depicts RP-HPLC data for the product of annealing the dimeric sense strand X39852 to the FVII targeting antisense strand XI 8795 (X39852-X18795).
  • FIG. 55 depicts RP-HPLC data for the product of annealing the dimeric antisense strand X39854 to X39852-X18795 (X39852-X18795-X39854).
  • FIG. 56 depicts RP-HPLC data for the product of annealing the dimeric sense strand X39853 to X39852-X18795-X39854 (X39852-X18795-X39854-X39853).
  • FIG. 57A depicts RP-HPLC (FIG. 57A) and MS (FIG. 57B) data for the product of annealing X39852-X18795-X39854-X39853 of FIG. 56 to X39850-X18795- X39851-X39855 of FIG. 50 to form the final hetero-hexameric siRNA (X39850-X18795- X39851-X39855-X39852-X18795-X39854-X39853).
  • FIG. 58 depicts knockdown of TTR protein by a 4: 1 : 1 FVII: ApoB :TTR hetero-hexamer at 6 mg/kg, equivalent to 1 mg/kg siTTR monomer.
  • FIG. 59 illustrates an evaluation of the stability of disulfide linkers.
  • the present disclosure relates to methods of administering to a subject multimeric oligonucleotides having monomeric subunits joined by covalent linkers.
  • the multimeric oligonucleotide can have a molecular weight of at least about 45 kD, such that clearance due to glomerular filtration of the multimeric oligonucleotide is reduced.
  • the present disclosure also relates to the multimeric oligonucleotide and methods of synthesizing the multimeric oligonucleotide.
  • an oligonucleotide multimer according to the disclosure may have a molecular weight of at least about 45 kD and have a relatively higher circulation half-life (e.g., have a lower rate of clearance due to glomerular filtration rate).
  • the improved and advantageous properties of the multimers according to the disclosure can be described in terms of increased in vivo circulation half-life. They may also be described in terms of increased in vivo activity, or increased bioactivity. Increased bioactivity may be represented by decreased levels of a target protein or mRNA after administration of the multimeric oligonucleotide. The increased bioactivity may be observed depending on the
  • administration route for the multimeric oligonucleotide For example, increased bioactivity may be observed when the multimeric oligonucleotide is administered via the intravenous (i.v.) route compared to the subcutaneous (s.c.) route. This increased bioactivity may be observed relative to a monomeric oligonucleotide.
  • a multimeric oligonucleotide administered via the i.v. route may achieve better bioactivity (i.e., increased reduction of the target protein or mRNA) compared to a monomeric oligonucleotide administered via the i.v. route.
  • the multi -conjugate When combined with a targeting ligand, the multi -conjugate can also deliver a higher payload per ligand/receptor binding event than the monomeric equivalent.
  • the present disclosure also relates to new synthetic intermediates and methods of synthesizing the multi-conjugate oligonucleotides.
  • the present disclosure also relates to methods of using the multi-conjugate oligonucleotides, for example in reducing gene expression, biological research, treating or preventing medical conditions, and/or to produce new or altered phenotypes.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, the method comprising administering an effective amount of the multimeric oligonucleotide to the subject, the multimeric oligonucleotide comprising subunits . , wherein:
  • each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ;
  • the multimeric oligonucleotide has a molecular weight and/or size configured to decrease its clearance due to glomerular filtration
  • the molecular weight of the multimeric oligonucleotide is at least about 45 kD.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein the number of subunits contained in the multimeric oligonucleotide is m, m being an integer selected to enable the multimeric oligonucleotide to have the molecular weight and/or size configured to decrease its clearance due to glomerular filtration.
  • m is (i) > 2; (ii) > 3; (iii) 3 4; (iv) > 4 and ⁇ 17; (v) > 4 and ⁇ 8; or (vi) 4, 5, 6, 7, or 8.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the multimeric oligonucleotide comprises Structure 21 : (Structure 21) and n is an integer > 0.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the subunits are single- stranded oligonucleotides. [00269] In one aspect, the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein n is > 1.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the subunits are double- stranded oligonucleotides.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein:
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration results in increased in vivo circulation half-life of the multimeric oligonucleotide.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration is determined by measuring the in vivo circulation half-life of the multimeric oligonucleotide after administering the multimeric oligonucleotide to the subject.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration is determined by measuring the time required for the serum concentration of the multimeric oligonucleotide to decrease to a predetermined value.
  • the predetermined value can be 90%, 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%,
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration is determined by measuring the serum concentration of the multimeric oligonucleotide at a predetermined time after administering the multimeric oligonucleotide to the subject.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration is determined by measuring the area under a curve of a graph representing serum concentration of the multimeric oligonucleotide over time after administering the multimeric oligonucleotide to the subject.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration increases in vivo bioavailability of the multimeric oligonucleotide.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration increases in vivo cellular uptake of the multimeric oligonucleotide.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, in which the decreased clearance due to glomerular filtration increases in vivo therapeutic index/ratio of the multimeric oligonucleotide.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein the measured parameter has a sigmoidal relationship with respect to the number of subunits in a monomeric, dimeric, trimeric and higher number multimeric oligonucleotides, for example, as shown in FIGS. 37A-37D.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein the measured parameter for the multimeric oligonucleotide and each of its subunits starting with a monomeric subunit, when plotted, define a sigmoidal curve, for example, as shown in FIGS. 38A- 38B.
  • the disclosure provides a multimeric oligonucleotide comprising subunits . , wherein:
  • each of the subunits . is independently a single or double-stranded oligonucleotide, and each of the subunits . is joined to another subunit by a covalent linker ⁇ ;
  • the multimeric oligonucleotide has a molecular weight and/or size configured to decrease its clearance due to glomerular filtration
  • the molecular weight of the multimeric oligonucleotide is at least about 45 kD.
  • the disclosure provides a multimeric oligonucleotide wherein the number of subunits contained in the multimeric oligonucleotide is m, m being an integer selected to enable the multimeric oligonucleotide to have the molecular weight and/or size configured to decrease its clearance due to glomerular filtration.
  • m is (i) > 2; (ii) > 3; (iii) > 4; (iv) > 4 and ⁇ 17; (v) > 4 and ⁇ 8; or (vi) 4, 5, 6, 7, or 8.
  • At least one of the subunits comprises a single strand having one of the covalent linkers ⁇ joined to its 3’ terminus and another of the covalent linkers joined to its 5’ terminus, and n is an integer > 0.
  • the disclosure provides a multimeric oligonucleotide wherein n > 1 and n ⁇ 17.
  • the disclosure provides a multimeric oligonucleotide in which n > 1 and n ⁇ 5. [00291] In one aspect, the disclosure provides a multimeric oligonucleotide in which n is 1, 2, 3, 4, or 5.
  • the disclosure provides a multimeric oligonucleotide wherein each subunit is a double-stranded RNA and n > 1.
  • the disclosure provides a multimeric oligonucleotide in which each subunit is a single-stranded oligonucleotide.
  • the disclosure provides a multimeric oligonucleotide in which each subunit is a double-stranded oligonucleotide.
  • the disclosure provides a multimeric oligonucleotide in which the subunits comprise a combination of single-stranded and double-stranded oligonucleotides.
  • the disclosure provides a multimeric oligonucleotide in which each subunit is an RNA, a DNA, or an artificial or non-natural nucleic acid analog.
  • the disclosure provides a multimeric oligonucleotide in which each subunit is a RNA.
  • the disclosure provides a multimeric oligonucleotide in which each subunit is a siRNA, a saRNA, or a miRNA.
  • the disclosure provides a multimeric oligonucleotide in which each subunit is a double-stranded siRNA and each of the covalent linkers joins sense strands of the siRNA.
  • the disclosure provides a multimeric oligonucleotide in which the multimeric oligonucleotide comprises a homo-multimer of substantially identical subunits . .
  • the disclosure provides a multimeric oligonucleotide in which the multimeric oligonucleotide comprises a hetero-multimer of two or more substantially different subunits . .
  • the disclosure provides a multimeric oligonucleotide in which the multimeric oligonucleotide is at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% pure.
  • the disclosure provides a multimeric oligonucleotide wherein each subunit . is independently a double-stranded oligonucleotide - , and wherein n is an integer > 1.
  • the disclosure provides a multimeric oligonucleotide wherein each subunit . is independently a double-stranded oligonucleotide wherein n is an integer > 1, and wherein each covalent linker ⁇ is on the same strand: (Structure 54), wherein d is an integer > 1.
  • the disclosure provides a multimeric oligonucleotide comprising Structure 22 or 23: (Structure 22) (Structure 23)
  • each - is a double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent double-stranded oligonucleotides
  • f is an integer > 1
  • g is an integer > 0.
  • the disclosure provides a plurality of a multimeric oligonucleotide wherein substantially all of the multimeric oligonucleotides have a predetermined value of n and/or predetermined molecular weight.
  • the disclosure provides a multimeric oligonucleotide in which the multimeric oligonucleotide further comprises one or more targeting ligands.
  • the disclosure provides a multimeric oligonucleotide in which at least one of the subunits is a targeting ligand.
  • the disclosure provides a multimeric oligonucleotide in which the targeting ligand is an aptamer.
  • the disclosure provides a multimeric oligonucleotide in which one or more of the covalent linkers ⁇ comprise a cleavable covalent linker and include nucleotide linkers, for example, as discussed in Examples 20, 22B, 27, and 41.
  • Nucleotide linker is a linker that contains one or more nucleotides and it can be chosen such that it does not carry out any other designated function.
  • the disclosure provides a multimeric oligonucleotide in which the cleavable covalent linker contains an acid cleavable bond, a reductant cleavable bond, a bio-cleavable bond, or an enzyme cleavable bond.
  • the disclosure provides a multimeric oligonucleotide in which the cleavable covalent linker is cleavable under intracellular conditions.
  • the disclosure provides a multimeric oligonucleotide in which each covalent linker ⁇ is the same.
  • the disclosure provides a multimeric oligonucleotide in which the covalent linkers ⁇ comprise two or more different covalent linkers.
  • the disclosure provides a multimeric oligonucleotide in which each covalent linker ⁇ joins two monomeric subunits . .
  • the disclosure provides a multimeric oligonucleotide in which at least one covalent linker ⁇ joins three or more monomeric subunits . .
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 51 : (Structure 51)
  • each is a single-stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent single-stranded oligonucleotides
  • a is an integer > 1
  • a is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, or greater than or equal to 9. In various aspects, a is 2, 3, 4, 5, 6, 7, 8, or 9.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 54: (Structure 54)
  • each - is a single-stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent single-stranded oligonucleotides
  • a > 1 the method comprising the steps of:
  • a is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, or greater than or equal to 9. In various aspects, a is 2, 3, 4, 5, 6, 7, 8, or 9.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 100
  • each - is independently a single-stranded oligonucleotide
  • each _ is independently a single or double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent oligonucleotides
  • the method comprising the steps of: a) reacting Structure 98 E— 3 sE— 3 ⁇ 4f— HE—— « (Structure 98) with Structure (Structure 99), wherein: a, a’, b, b’, c, c’, d and d’ are each independently 0 or 1, and R1 and R2 are chemical moieties capable of reacting directly or indirectly to form a covalent linker ⁇ , thereby forming Structure 100
  • the sum of a+a'+b+b'+c+c'+d+d' is greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+b+b'+c+c'+d+d' is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 102
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure 100
  • Structure 101 wherein: a is 1, and a’, a”, b, b’, b”, c, c’, c”, d, d’, and d” are each independently 0 or 1, thereby forming Structure 102
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 103
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ . is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure 100
  • Structure 101 wherein: a’ is 1, and a, a”, b, b’, b”, c, c’, c”, d, d’, and d” are each independently 0 or 1, thereby forming Structure 103
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 104
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each ______ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • 103 and a’ are each 1, and a”, a’”, b, b’, b”, b”’, c, c’, c”, c”’, d, d’, d”, and d’” are each independently 0 or 1, thereby forming Structure 104
  • the sum of a+a'+a"+a"'+b+b'+b"+b"'+c+c'+c"+c"'+d+d'+d"+d"' is greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+a"+a"'+b+b'+b"+b"'+c+c'+c"+c"'+d+d'+d"+d"' is 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 107
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • the sum of a+a'+a"+a"'+b+b'+b"+b"'+c+c'+c"+c"'+d+d'+d"+d"' is greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of
  • a+a'+a"+a"'+b+b'+b"+b"'+c+c'+c"+c"'+d+d'+d"+d"' is 3, 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 108
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • the sum of a+a'+b+b'+c+c'+d+d' is greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, or greater than or equal to 11.
  • the sum of a+a'+b+b'+c+c'+d+d' is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 111
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure 108
  • Structure 112 wherein: d is 1, and a, a’, a”, b, b’, b”, c, c’, c”, d’ and d” are each independently 0 or 1, thereby forming Structure 111
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, or greater than or equal to 11.
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 113
  • each - is independently a single-stranded oligonucleotide, each is independently a double-stranded oligonucleotide, each _ is independently a single or double-stranded oligonucleotide, and each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising the step of annealing Structure
  • Structure 112 wherein: d’ is 1, and a, a’, a”, b, b’, b”, c, c’, c”, d and d” are each independently 0 or 1, thereby forming Structure 113
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, greater than or equal to 10, or greater than or equal to 11.
  • the sum of a+a'+a"+b+b'+b"+c+c'+c"+d+d'+d" is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • the method further comprises annealing one or more single- stranded oligonucleotides - with a complementary single-stranded oligonucleotide
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising Structure 114
  • each ⁇ is a covalent linker joining adjacent oligonucleotides, the method comprising reacting Structure 115 u u w u u h
  • the sum of a and b is greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, greater than or equal to 8, greater than or equal to 9, or greater than or equal to 10. In other aspects, the sum of a and b is 5, 6, 7, 8, 9, or 10.
  • Structure 115 and/or Structure 116 further comprise one or more targeting ligands.
  • the targeting ligand is a terminal targeting ligand.
  • a is an integer of 4, 5, 6, 7, 8, 9, or 10.
  • b is an integer of 4, 5, 6, 7, 8, 9, or 10.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof.
  • subjects include, but are not limited to, mammals, such as primates, rodents, and agricultural animals.
  • a primate subject includes, but is not limited to, a human, a chimpanzee, and a rhesus monkey.
  • a rodent subject includes, but is not limited to, a mouse and a rat.
  • Examples of an agricultural animal subject includes, but is not limited to, a cow, a sheep, a lamb, a chicken, and a pig.
  • Mouse glomerular filtration rate can be about 0.15-0.25 ml/min. Human GFR can be about 1.8 ml/min/kg (Mahmood I: (1998) Interspecies scaling of renally secreted drugs. Life Sci 63:2365-2371).
  • mice can have about 1.46 ml of blood. Therefore, the time for glomerular filtration of total blood volume in mice can be about 7.3 minutes (1.46/0.2). Humans can have about 5 liters of blood and weigh about 70 kg. Therefore, the time for glomerular filtration of total blood volume in humans can be 39.7 mins [5000/126(1.8*70)].
  • a person of ordinary skill in the art would recognize that different species can have different rates of clearance by glomerular filtration, at least for the above reasons.
  • a person of ordinary skill in the art can infer that a ratio of rate of clearance by glomerular filtration between human and mouse times can be about 1 :5 or 1 :6.
  • the rate of clearance of a certain substance (e.g., a particular oligonucleotide) by humans can be 5-6 times slower than that of a mouse.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein the in vivo circulation half-life is measured between 30 and 120 minutes after administering the multimeric oligonucleotide to the subject.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein the predetermined time is between 30 and 120 minutes after administering the multimeric oligonucleotide to the subject.
  • the disclosure provides a method of administering a multimeric oligonucleotide to a subject in need thereof, wherein the area under the curve is calculated based on serum concentration of the multimeric oligonucleotide between x and y minutes after administering the multimeric oligonucleotide to the subject.
  • x can be 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 75, 90, 120, 180, 240, or 300 minutes
  • y can be 90, 120, 180, 240, 300, 360, 420, 480, 540, 600, 720, 840, 960,
  • the time range can be 30-120 minutes, 1-1600 minutes, or 300-600 minutes.
  • the disclosure provides a multimeric oligonucleotide or a method for increasing in vivo circulation half-life of the multimeric oligonucleotide, wherein the multimeric oligonucleotide is not formulated in a nanoparticle (NP) or a lipid nanoparticle (LNP).
  • NP nanoparticle
  • LNP lipid nanoparticle
  • the present disclosure also relates to multi-conjugate oligonucleotides having improved pharmacodynamics and/or pharmacokinetics.
  • the multi conjugate oligonucleotides e.g., multimeric oligonucleotides including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more siRNA
  • the multi-conjugate can also deliver a higher
  • the present disclosure also relates to new synthetic intermediates and methods of synthesizing the multi-conjugate oligonucleotides.
  • the present disclosure also relates to methods of using the multi-conjugate oligonucleotides, for example in reducing gene expression, biological research, treating or preventing medical conditions, and/or to produce new or altered phenotypes.
  • the nucleic acid or oligonucleotide is RNA, DNA, or comprises an artificial or non-natural nucleic acid analog.
  • the nucleic acid or oligonucleotide is single-stranded. In various embodiments, the nucleic acid or oligonucleotide is double-stranded (e.g., antiparallel double-stranded).
  • the nucleic acid or oligonucleotide is RNA, for example an antisense RNA (aRNA), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), messenger RNA (mRNA), short hairpin RNA (shRNA), small activating (saRNA), or ribozyme.
  • aRNA antisense RNA
  • crRNA CRISPR RNA
  • IncRNA long noncoding RNA
  • miRNA microRNA
  • piwi-interacting RNA piRNA
  • small interfering RNA siRNA
  • messenger RNA messenger RNA
  • shRNA short hairpin RNA
  • small activating saRNA
  • ribozyme for example an antisense RNA (aRNA), CRISPR RNA (crRNA), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (pi
  • the RNA is siRNA.
  • each double- stranded oligonucleotide is an siRNA and/or has a length of 15-30 base pairs.
  • the nucleic acid or oligonucleotide is an aptamer.
  • siRNA small interfering RNA
  • mRNA target messenger RNA
  • miRNAs are non-coding RNAs that play key roles in post-transcriptional gene regulation. miRNA can regulate the expression of 30 % of all mammalian protein encoding genes. Specific and potent gene silencing by double-stranded RNA (RNAi) was discovered, plus additional small noncoding RNA (Canver, M.C. et ah, Nature (2015)). Pre-miRNAs are short stem loops ⁇ 70 nucleotides in length with a 2-nucleotide 3’- overhang that are exported, into the mature 19-25 nucleotide duplexes.
  • RNAi double-stranded RNA
  • the miRNA strand with lower base pairing stability can be loaded onto the RNA- induced silencing complex (RISC).
  • RISC RNA- induced silencing complex
  • the passenger guide strand can be functional but is usually degraded.
  • MiRNAs mimics are described for example, in US Patent No. 8,765,709.
  • the RNA can be short hairpin RNA (shRNA), for example, as described in US Patent Nos. 8,202,846 and 8,383,599.
  • shRNA short hairpin RNA
  • the RNA can be CRISPR RNA (crRNA), for example, CRISPR array of Type V can be processed into short mature crRNAs of 42-44 nucleotides in length, with each mature crRNA beginning with 19 nucleotides of direct repeat followed by 23-25 nucleotides of spacer sequence.
  • mature crRNAs in Type II systems can start with 20-24 nucleotides of spacer sequence followed by about 22 nucleotides of direct repeat.
  • CRISPR systems are described for example, in US Patent No. 8,771,945, Jinek et al., Science, 337(6096): 816-821 (2012), and International Patent Application Publication No. WO 2013/176772.
  • the nucleic acid or oligonucleotide is 15-30, 17- 27, 19-26, 20-25, 40-50, 40-150, 100-300, 1000-2000, or up to 10000 nucleotides in length.
  • the oligonucleotide is double-stranded and complementary.
  • Complementarity can be 100 % complementary, or less than 100 % complementary where the oligonucleotide nevertheless hybridizes and remains double- stranded under relevant conditions (e.g., physiologically relevant conditions).
  • a double-stranded oligonucleotide can be at least about 80, 85, 90, or 95 % complementary.
  • RNA is long noncoding RNA (IncRNA).
  • IncRNAs are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins. IncRNAs are thought to encompass nearly 30,000 different transcripts in humans, hence IncRNA transcripts account for the major part of the non-coding transcriptome (see, e.g., Derrien et al., The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res, 22(9): 1775-89 (2012)).
  • RNA is messenger RNA (mRNA). mRNA and its application as a delivery method for in-vivo production of proteins, is described, for example, in International Patent Application Publication No. WO 2013/151736.
  • the nucleic acid or oligonucleotide is DNA, for example an antisense DNA (aDNA) (e.g., antagomir) or antisense gapmer.
  • aDNA antisense DNA
  • antagomir antisense gapmer.
  • Examples of aDNA, including gapmers and multimers, are described for example in Subramanian et al., Nucleic Acids Res, 43(19): 9123-9132 (2015) and International Patent Application Publication No. WO 2013/040429.
  • antagomirs are described for example, in US Patent No. 7,232,806.
  • the oligonucleotide has a specific sequence, for example any one of the sequences disclosed herein.
  • the nucleic acid or oligonucleotide further comprises a chemical modification.
  • the chemical modification can comprise a modified nucleoside, modified backbone, modified sugar, or modified terminus.
  • Modifications include phosphorus-containing linkages, which include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3’alkylene phosphonates and chiral phosphonates, phosphinates,
  • phosphoramidates comprising 3’ -amino phosphoramidate and
  • aminoalkylphosphoramidates aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’ -5’ linkages, T -5’ linked analogs of these, and those having inverted adjacent nucleoside units that are linked 3’-5’ to 5’-3’ or 2’ -5’ to 5’-2’.
  • the oligonucleotides contained in the multi conjugate may comprise one or more phosphorothioate groups.
  • the oligonucleotides may comprise 1-3 phosphorothioate groups at the 5’ end.
  • the oligonucleotides may comprise 1-3 phosphorothioate groups at the 3’ end.
  • the oligonucleotides may comprise 1-3 phosphorothioate groups at the 5’ end and the 3’ end.
  • each oligonucleotide contained in the multi-conjugate may comprise 0-15 total
  • each oligonucleotide may comprise fewer than 10, fewer than 9, fewer than 8, fewer than 7, fewer than 6, fewer than 5, fewer than 4, or fewer than 3 total phosphorothioate groups.
  • the oligonucleotides contained in the multi-conjugate may possess increased in vivo activity with fewer phosphorothioate groups relative to the same oligonucleotides in monomeric form with more phosphorothioate groups.
  • oligonucleotides contained in the multi-conjugates of this disclosure may be modified using various strategies known in the art to produce a variety of effects, including, e.g., improved potency and stability in vitro and in vivo.
  • artificial nucleic acids e.g., T -O-methyl-substituted RNA; 2’-fluro- 2’deoxy RNA, peptide nucleic acid (PNA); morpholinos; locked nucleic acid (LNA); Unlocked nucleic acids (UNA); bridged nucleic acid (BNA); glycol nucleic acid (GNA) ; and threose nucleic acid (TNA); or more generally, nucleic acid analogs, e.g., bicyclic and tricyclic nucleoside analogs, which are structurally similar to naturally occurring RNA and DNA but have alterations in one or more of the phosphate backbone, sugar, or nucleobase portions of the naturally-occurring molecule.
  • nucleic acid analogs e.g., bicyclic and tricyclic nucleoside analogs, which are structurally similar to naturally occurring RNA and DNA but have alterations in one or more of the phosphate backbone, sugar, or nucleobase portions
  • analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canon bases. Examples of phosphate-sugar backbone analogues include PNA. Morpholino-based oligomeric compounds are described in Braasch et ah, Biochemistry, 41(14): 4503-4510 (2002) and US Patent Nos. 5,539,082; 5,714,331; 5,719,262; and 5,034,506.
  • oligonucleotides are modified at a terminal end by substitution with a chemical functional group.
  • the substitution can be performed at the 3’ or 5’ end of the oligonucleotide, and is preferably performed at the 3’ ends of both the sense and antisense strands of the monomer, but is not always limited thereto.
  • a sulfhydryl group e.g., a sulfhydryl group (-SH), a carboxyl group (-COOH), an amine group (-NH2), a hydroxy group (-OH), a formyl group (-CHO), a carbonyl group (-CO-), an ether group (- 0-), an ester group (-COO
  • nucleobases contained in the multi-conjugates of this disclosure may be modified to also include, additionally or alternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions.
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2’ deoxycytosine and often referred to in the art as 5-Me-C), 5- hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosub stituted
  • Modified nucleobases can include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoro
  • Hydroxy group (— OH) at a terminus of the nucleic acid can be substituted with a functional group such as sulfhydryl group (— SH), carboxyl group (— COOH) or amine group (— NH2). The substitution can be performed at the 3’ end or the 5’ end.
  • a functional group such as sulfhydryl group (— SH), carboxyl group (— COOH) or amine group (— NH2).
  • one or more oligonucleotides may be conjugated to one or more additional oligonucleotides or targeting ligands.
  • the oligonucleotides and targeting ligands may be conjugated via any means known in the art, including, but is not limited to, covalent bonds, ionic bonds, hydrogen bonds, and magnetic linkage.
  • oligonucleotides are linked covalently.
  • Linkers may be cleavable (e.g., under intracellular conditions, to facilitate oligonucleotide delivery and/or action) or non-cleavable.
  • linkers including their composition, synthesis, and use are known in the art and may be adapted for use with the disclosure.
  • a covalent linker can comprise the reaction product of nucleophilic and electrophilic groups.
  • a covalent linker can comprise the reaction product of a thiol and maleimide, a thiol and vinylsulfone, a thiol and pyridyldi sulfide, a thiol and iodoacetamide, a thiol and acrylate, an azide and alkyne, or an amine and carboxyl group.
  • one of these groups is connected to an oligonucleotide (e.g., thiol (-SH) functionalization at the 3’ or 5’ end) and the other group is encompassed by a second molecule (e.g., linking agent) that ultimately links two oligonucleotides (e.g., maleimide in DTME).
  • oligonucleotide e.g., thiol (-SH) functionalization at the 3’ or 5’ end
  • a second molecule e.g., linking agent
  • a covalent linker can comprise an unmodified di nucleotide linkage or a reaction product of thiol and maleimide.
  • a covalent linker can comprise a nucleotide linker of 2-6 nucleotides in length. In various embodiments, the nucleotide linker is 3, 4, or 5 nucleotides in length.
  • a covalent linker can comprise a disulfide bond or a compound of Formula (I):
  • each R1 is independently a C2-C10 alkyl, alkoxy, or aryl group;
  • R2 is a thiopropionate or disulfide group; and each X is independently selected from:
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • each Ri is independently a C2-C10 alkyl, alkoxy, or aryl group; and R2 is a thiopropionate or disulfide group.
  • the compound of Formula (I) is N-(2-aminoethyl)-2-aminoethyl
  • S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of a subunit.
  • S is attached by a covalent bond or by a linker to the 3’ or 5’ terminus of a subunit.
  • the covalent linker of Formula (I) is formed from a covalent linking precursor of Formula (II):
  • each R1 is independently a C2-C10 alkyl, alkoxy, or aryl group
  • R2 is a thiopropionate or disulfide group.
  • oligonucleotide can comprise two orthogonal types of bio-cleavable linkages.
  • the two orthogonal bio-cleavable linkages can comprise an unmodified di nucleotide and a reaction product of thiol and maleimide.
  • the nucleic acid or oligonucleotide is connected to the linker via a phosphodiester or thiophosphodiester (e.g., R1 in Structure l is a phosphodiester or thiophosphodiester).
  • the nucleic acid or oligonucleotide is connected to the linker via a Cl -8 alkyl, C2-8 alkenyl, C2-8 alkynyl, heterocyclyl, aryl, and heteroaryl, branched alkyl, aryl, halo-aryl, and/or other carbon- based connectors.
  • the nucleic acid or oligonucleotide is connected to the linker via a C2-C10, C3-C6, or C6 alkyl (e.g., R2 in Structure 1 is a C2- C10, C3-C6, or C6 alkyl).
  • R2 in Structure 1 is a C2- C10, C3-C6, or C6 alkyl.
  • the nucleic acid or oligonucleotide is connected to the linker via a C6 alkyl.
  • these moieties e.g., R1 and/or R2 in Structure 1 are optional and a direct linkage is possible.
  • the nucleic acid or oligonucleotide is connected to the linker via the reaction product of a thiol and maleimide group (e.g., A in Structure 1 is the reaction product of a thiol and maleimide group).
  • Preferred linking agents utilizing such chemistry include DTME (dithiobismaleimidoethane), BM(PEG)2 (1,8- bis(maleimido)diethylene glycol), BM(PEG)3 (1,11-bismaleimido-triethyleneglycol), BMOE (bismaleimidoethane), BMH (bismaleimidohexane), or BMB (1,4- bismaleimidobutane).
  • oligonucleotides can be linked together directly, via functional end- substitutions, or indirectly by way of a linking agent.
  • the oligonucleotide can be bound directly to a linker (e.g., R1 and R2 of Structure 1 are absent).
  • a linker e.g., R1 and R2 of Structure 1 are absent.
  • Such bonding can be achieved, for example, through use of 3’-thionucleosides, which can be prepared according to the ordinary skill in the art. See, e.g., Sun et al. “Synthesis of 3’-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry” RNA.
  • the linking agent may be a non-ionic hydrophilic polymer such as polyethyleneglycol (PEG), polyvinylpyrolidone and polyoxazoline, or a hydrophobic polymer such as PLGA and PLA.
  • a polymer linking agent used as a mediator for a covalent bond may be non-ionic hydrophilic polymers including PEG, Pluronic, polyvinylpyrolidone, polyoxazoline, or copolymers thereof; or one or more biocleavable polyester polymers including poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-glycolic acid, poly-D-lactic-co-glycolic acid, poly-L-lactic-co-glycolic acid, poly-D,L-lactic-co-glycolic acid, poly-D,L-lactic-co- glycolic acid, polycaprolactone, polyvalerolactone, polyhydroxybutyrate,
  • polyhydroxyvalerate or copolymers thereof, but is not always limited thereto.
  • a linking agent having cleavable bonds (such as a reductant bond that is cleaved by the chemical environment of the cytosol) or a linking agent having non- cleavable bonds can be used herein.
  • the linking agent of the foregoing aspects of present disclosure can have non-cleavable bonds such as an amide bond or a urethane bond.
  • the linking agent of the foregoing aspects of the present disclosure can have cleavable bonds such as an acid cleavable bond (e.g., a covalent bond of ester, hydrazone, or acetal), a reductant cleavable bond (e.g., a disulfide bond), a bio- cleavable bond, or an enzyme cleavable bond (e.g., a peptide bond).
  • the cleavable covalent linker is cleavable under intracellular conditions.
  • any linking agent available for drug modification can be used in the foregoing aspects of the disclosure without limitation.
  • combinations of functional groups and linking agents may include: (a) where the functional groups are amino and thiol, the linking agent may be Succinimidyl 3-(2-pyridyldithio)propionate, or Succinimydyl 6-([3(2- pyridyldithio)propioamido]hexanoate; (b) where the functional group is amino, the linking agent may be 3,3’dithiodipropionic acid di-(N-succinimidyl ester), Dithio- bis(ethyl lH-imidazole-l-carboxylate), or Dithio-bis(ethyl lH-imidazole-l-carboxylate); (c) where the functional groups are amino and alkyne, the linking agent may be Sulfo-N- succinimidyl3-[[2-(p-azidosalicylamido)ethyl]-l,3’-d
  • an additional step of activating the functional groups can be included.
  • Compounds that can be used in the activation of the functional groups include but are not limited to 1 -ethyl-3, 3- dimethylaminopropyl carbodiimide, imidazole, N-hydroxysuccinimide,
  • dichlorohexylcarbodiimide N-beta-Maleimidopropionic acid, N-beta-maleimidopropyl succinimide ester or N-Succinimidyl 3-(2-pyridyldithio)propionate.
  • the disclosure provides an oligonucleotide coupled to a covalent linker, which can be used, for example, in the synthesis of defined multi conjugate oligonucleotides having predetermined sizes and compositions.
  • a covalent linker which can be used, for example, in the synthesis of defined multi conjugate oligonucleotides having predetermined sizes and compositions.
  • X is a nucleic acid bonded to R1 through its 3’ or 5’ terminus
  • R1 is a derivative of phosphoric acid, a derivative of thiophosphoric acid, a sulfate, amide, glycol, or is absent;
  • R2 is a C2-C10 alkyl, alkoxy, or aryl group, or is absent;
  • A is the reaction product of a nucleophile and an electrophile
  • R3 is a C2-C10 alkyl, alkoxy, aryl, alkyldithio group, ether, thioether, thiopropionate, or disulfide;
  • B is a nucleophile or electrophile (e.g., a thiol, maleimide, vinylsulfone, pyridyldisulfide, iodoacetamide, acrylate, azide, alkyne, amine, or carboxyl group).
  • a nucleophile or electrophile e.g., a thiol, maleimide, vinylsulfone, pyridyldisulfide, iodoacetamide, acrylate, azide, alkyne, amine, or carboxyl group.
  • the disclosure provides a compound according to Structure
  • X is a nucleic acid bonded to R1 via a phosphate or derivative thereof, or thiophosphate or derivative thereof at its 3’ or 5’ terminus;
  • each R1 is independently a C2-C10 alkyl, alkoxy, or aryl group
  • R2 is a thiopropionate or disulfide group.
  • the disclosure provides a compound according to Structure 3:
  • X is a nucleic acid bonded to R1 through its 3’ or 5’ terminus
  • R1 is a derivative of phosphoric acid such as phosphate, phosphodiester, phosphotriester, phosphonate, phosphoramidate and the like, a derivative of thiophosphoric acid such as thiophosphate, thiophosphodiester, thiophosphotriester, thiophosphoramidate and the like, a sulfate, amide, glycol, or is absent;
  • R2 is a C2-C10 alkyl, alkoxy, or aryl group, or is absent;
  • A is the reaction product of a first and a second reactive moiety
  • R3 is an C2-C10 alkyl, alkoxy, aryl, alkyldithio group, ether, thioether, thiopropionate, or disulfide;
  • B is a third reactive moiety.
  • the disclosure also provides methods for synthesizing an oligonucleotide coupled to a covalent linker.
  • the disclosure provides a method for synthesizing a compound according to Structure 1 (or adapted for synthesizing a compounds according to Structure 2 or 3), the method comprising:
  • X is a nucleic acid bonded to R1 through its 3’ or 5’ terminus
  • R1 a phosphodiester, thiophosphodiester, sulfate, amide, glycol, or is absent;
  • R2 is a C2-C10 alkyl, alkoxy, or aryl group, or is absent;
  • A is the reaction product of a nucleophile and an electrophile
  • R3 is a C2-C10 alkyl, alkoxy, aryl, alkyldithio group, ether, thioether, thiopropionate, or disulfide;
  • B is a nucleophile or electrophile (e.g., a thiol, maleimide, vinylsulfone, pyridyldisulfide, iodoacetamide, acrylate, azide, alkyne, amine, or carboxyl group).
  • a nucleophile or electrophile e.g., a thiol, maleimide, vinylsulfone, pyridyldisulfide, iodoacetamide, acrylate, azide, alkyne, amine, or carboxyl group.
  • the method can further comprise the step of synthesizing the
  • A' comprises a thiol (-SH) by (i) introducing the thiol during solid phase synthesis of the nucleic acid using
  • the method for synthesizing the compound of Structure 1 further comprises synthesizing the compound of Structure 2.
  • the oligonucleotide coupled to a covalent linker can include any one or more of the features described herein, including in the Examples.
  • the compounds can include any one or more of the nucleic acids (with or without
  • Example 1 provides an example methodology for generating thiol terminated oligonucleotides.
  • Example 2 provides an example methodology for preparing an oligonucleotide coupled to a linker.
  • the method for synthesizing the compound of Structure 1, 2 or 3 is carried out under conditions that substantially favor the formation of Structure 1, 2 or 3 and substantially prevent dimerization of X.
  • the conditions can improve the yield of the reaction (e.g., improve the purity of the product).
  • the method for synthesizing the compound of Structure 1, 2 or 3 the step of reacting the functionalized nucleic acid X - R1 - R2 - A' and the covalent linker A" - R3 - B is carried out at a X - R1 - R2 - A' concentration of below about 1 mM, 500 mM, 250 pM, 100 pM, or 50 pM.
  • the X - R1 - R2 - A' concentration can be about 1 mM, 500 pM, 250 pM, 100 pM, or 50 pM.
  • the method for synthesizing the compound of Structure 1, 2 or 3 the step of reacting the functionalized nucleic acid X - R1 - R2 - A' and the covalent linker A" - R3 - B is carried out with a molar excess of A" - R3 - B of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 100.
  • the molar excess of A" - R3 - B can be about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 100.
  • the method for synthesizing the compound of Structure 1, 2 or 3 the step of reacting the functionalized nucleic acid X - R1 - R2 - A' and the covalent linker A" - R3 - B is carried out at a pH of below about 7, 6, 5, or 4.
  • the pH can be about 7, 6, 5, or 4.
  • the method for synthesizing the compound of Structure 1, 2 or 3 the step of reacting the functionalized nucleic acid X - R1 - R2 - A' and the covalent linker A" - R3 - B is carried out in a solution comprising water and a water miscible organic co-solvent.
  • the water miscible organic co-solvent can comprise DMF (dimethylformamide), NMP (N-methyl-2-pyrrolidone), DMSO (dimethyl sulfoxide), or acetonitrile.
  • the water miscible organic co-solvent can comprise about 10, 15, 20, 25, 30, 40, or 50 %V (v/v) of the solution.
  • the compound is isolated or substantially pure.
  • the compound can be at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 % pure. In one embodiment, the compound is about 85-95 % pure.
  • the methods for synthesizing the compounds and compositions according to the disclosure can result in a product that is at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 % pure. In one embodiment, the product is about 85-95 % pure. Preparations can be greater than or equal to 50 % pure; preferably greater than or equal to 75 % pure; more preferably greater than or equal to 85 % pure; and still more preferably, greater than or equal to 95 % pure.
  • the term about is used in accordance with its plain and ordinary meaning of approximately.
  • “about X” encompasses approximately the value X as stated, including similar amounts that are within the measurement error for the value of X or amounts that are approximately the same as X and have essentially the same properties as X.
  • isolated includes compounds that are separated from other, unwanted substances.
  • the isolated compound can be synthesized in a substantially pure state or separated from the other components of a crude reaction mixture, except that some amount of impurities, including residual amounts of other components of the crude reaction mixture, may remain.
  • pure or substantially pure means sufficiently free from impurities to permit its intended use (e.g., in a pharmaceutical formulation or as a material for a subsequent chemical reaction).
  • X % pure means that the compound is X % of the overall composition by relevant measure, which can be for example by analytical methods such as HPLC.
  • the disclosure provides dimeric defined multi conjugate oligonucleotides. These compounds include homodimers (e.g., two
  • oligonucleotides that are substantially the same, for example targeting the same gene in vivo and heterodimers (e.g., two oligonucleotides that are substantially different, for example different sequences or targeting different genes in vivo )
  • heterodimers e.g., two oligonucleotides that are substantially different, for example different sequences or targeting different genes in vivo
  • the disclosure provides an isolated compound according to Structure 4:
  • each - is a double-stranded oligonucleotide designed to react with the same molecular target in vivo , and • is a covalent linker joining single strands of adjacent single-stranded oligonucleotides at their 3’ or 5’ termini, and having the structure - R1 - R2 - A - R3 - A - R2 - R1 - wherein:
  • each R1 is a derivative of phosphoric acid such as phosphate, phosphodiester, phosphotriester, phosphonate, phosphoramidate and the like, a derivative of
  • thiophosphoric acid such as thiophosphate, thiophosphodiester, thiophosphotriester, thiophosphoramidate and the like
  • each R2 is independently a C2-C10 alkyl, alkoxy, or aryl group, or is absent;
  • each A is independently the reaction product of a nucleophile and an electrophile
  • the disclosure provides an isolated compound according to Structure 5 :
  • - is a first single-stranded oligonucleotide
  • 'LA/n' is a second single-stranded oligonucleotide having a different sequence from the first
  • each R1 is a derivative of phosphoric acid such as phosphate, phosphodiester, phosphotriester, phosphonate, phosphoramidate and the like, a derivative of
  • thiophosphoric acid such as thiophosphate, thiophosphodiester, thiophosphotriester, thiophosphoramidate and the like
  • each R2 is independently a C2-C10 alkyl, alkoxy, or aryl group, or is absent;
  • each A is independently the reaction product of a thiol and maleimide, a thiol and vinylsulfone, a thiol and pyridyldi sulfide, a thiol and iodoacetamide, a thiol and acrylate, an azide and alkyne, or an amine and carboxyl group, and
  • R3 is an C2-C10 alkyl, alkoxy, aryl, alkyldithio group, ether, thioether, thiopropionate, or disulfide.
  • - is a first double-stranded oligonucleotide
  • oligonucleotide is a second double-stranded oligonucleotide having a different sequence from the first
  • each R1 is a derivative of phosphoric acid such as phosphate, phosphodiester, phosphotriester, phosphonate, phosphoramidate and the like, a derivative of
  • thiophosphoric acid such as thiophosphate, thiophosphodiester, thiophosphotriester, thiophosphoramidate and the like, a sulfate, amide, glycol, or is absent;
  • each R2 is independently a C2-C10 alkyl, alkoxy, or aryl group, or is absent;
  • each A is independently the reaction product of a thiol and maleimide, a thiol and vinylsulfone, a thiol and pyridyldi sulfide, a thiol and iodoacetamide, a thiol and acrylate, an azide and alkyne, or an amine and carboxyl group, and
  • R3 is an C2-C10 alkyl, alkoxy, aryl, alkyldithio group, ether, thioether, thiopropionate, or disulfide.
  • the disclosure provides an isolated compound according to Structure 11 :
  • - is a double-stranded oligonucleotide
  • - is a single-stranded oligonucleotide
  • • is a covalent linker joining single strands of adjacent single-stranded oligonucleotides.
  • the disclosure provides methods for synthesizing dimeric defined multi-conjugate oligonucleotides.
  • the disclosure provides a method for synthesizing a compound of Structure 5 :
  • the method can further comprise the step of annealing complementary and Tuv/ o to yield Structure 6:
  • the disclosure provides a method for synthesizing an isolated compound of Structure 4:
  • each is a double-stranded oligonucleotide and ⁇ is a covalent linker joining single strands of adjacent single-stranded oligonucleotides at their 3’ or 5’ termini, the method comprising the steps of:
  • R2 is a chemical group capable of reacting with 0 , thereby forming a single-stranded dimer ⁇ ;
  • the disclosure provides a method for synthesizing an
  • This methodology can be adapted for synthesizing an isolated compound according to - (Structure 11), for example by omitting step (ii).
  • the disclosure provides a method for synthesizing an
  • dimeric compounds and intermediates can include any one or more of the features described herein, including in the Examples.
  • the compounds can include any one or more of the nucleic acids (with or without modifications), targeting ligands, and/or linkers described herein, or any of the specific structures or chemistries shown in the summary, description, or Examples.
  • Example 3 provides an example methodology for preparing dimerized oligonucleotides and Example 4 provides an example methodology for annealing single- stranded oligonucleotides to form double-stranded oligonucleotides.
  • Example 7 provides an example methodology for preparing various oligonucleotide precursors useful in the syntheses above.
  • Example 8 provides an example methodology for preparing various oligonucleotide multimers, which are also useful in the syntheses above.
  • Rl, R2, and the bifunctional linking moiety O can form a covalent linker ⁇ as described and shown herein.
  • Rl and R2 can each independently comprise a reactive moiety, for example an electrophile or nucleophile.
  • Rl and R2 can each independently be selected from the group consisting of a thiol, maleimide, vinylsulfone, pyridyldisulfide, iodoacetamide, acrylate, azide, alkyne, amine, and carboxyl group.
  • the bifunctional linking moiety 0 comprises two reactive moieties that can be sequentially reacted according to steps (i) and (ii) above, for example a second electrophile/nucleophile that can be reacted with an electrophile/nucleophile in Rl and R2.
  • bifunctional linking moieties 0 include, but are not limited to, DTME, BM(PEG)2, BM(PEG)3, BMOE, BMH, or BMB.
  • the disclosure provides multimeric (n>2) defined multi conjugate oligonucleotides, including defined tri-conjugates and defined tetraconjugates.
  • the disclosure provides a compound according to Structure
  • each - is a double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining single strands of adjacent single- stranded
  • n is an integer > 0.
  • the disclosure provides a compound according to Structure
  • the disclosure provides a compound according to Structure
  • each - is a double-stranded oligonucleotide
  • each - is a single-stranded oligonucleotide
  • each ⁇ is a covalent linker joining single strands of adjacent single- stranded
  • oligonucleotides and m is an integer > 1 and n is an integer > 0.
  • the disclosure provides methods for synthesizing multimeric (n>2) defined multi-conjugate oligonucleotides, including defined tri conjugates and defined tetra-conjugates.
  • the disclosure provides a method for synthesizing a compound according to Structure 7 or 8: (Structure 8)
  • each is a double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining single strands of adjacent single-stranded oligonucleotides
  • m is an integer > 1
  • n is an integer > 0, the method comprising the steps of:
  • R with a third single-stranded oligonucleotide ”r 2 wherein Rl and R2 are chemical moieties capable of reacting directly or indirectly to form a covalent linker ⁇ , thereby forming
  • step (iii) annealing a fourth single-stranded oligonucleotide to the product of step (ii), thereby forming structure 7 or 8.
  • the disclosure provides a method for synthesizing a compound according to Structure 7 or 8:
  • each is a double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining single strands of adjacent single-stranded oligonucleotides
  • m is an integer > 1
  • n is an integer > 0,
  • the disclosure provides a method for synthesizing a compound of Structure 9: (Structure 9), wherein each is a double-stranded oligonucleotide, each ⁇ is a covalent linker joining single strands of adjacent single-stranded oligonucleotides, the method comprising the steps of:
  • the disclosure provides a method for synthesizing a compound of Structure 10: - ⁇ - * (Structure 10), wherein each is a double-stranded oligonucleotide, each ⁇ is a covalent linker joining single strands of adjacent single-stranded oligonucleotides, the method comprising the steps of:
  • dimeric compounds and intermediates can include any one or more of the features described herein, including in the Examples.
  • the compounds can include any one or more of the nucleic acids (with or without modifications), targeting ligands, and/or linkers described herein, or any of the specific structures or chemistries shown in the summary, description, or Examples.
  • Example 7 provides an example methodology for preparing various oligonucleotide precursors useful in the syntheses above.
  • Example 8 provides an example methodology for preparing various oligonucleotide multimers, which are also useful in the syntheses above.
  • Rl, R2, and the bifunctional linking moiety O can form a covalent linker ⁇ as described and shown herein.
  • Rl and R2 can each independently comprise a reactive moiety, for example an electrophile or nucleophile.
  • Rl and R2 can each independently be selected from the group consisting of a thiol, maleimide, vinylsulfone, pyridyldisulfide, iodoacetamide, acrylate, azide, alkyne, amine, and carboxyl group.
  • the bifunctional linking moiety 0 comprises two reactive moieties that can be sequentially reacted according to steps (i) and (ii) above, for example a second electrophile/nucleophile that can be reacted with an electrophile/nucleophile in Rl and R2.
  • Examples of bifunctional linking moieties 0 include, but are not limited to, DTME, BM(PEG)2, BM(PEG)3, BMOE, BMH, or BMB.
  • each - ⁇ - may independently comprise two sense or two antisense oligonucleotides.
  • a - ⁇ - may comprise two active strands or two passenger strands.
  • each - ⁇ - may independently comprise one sense and one antisense oligonucleotide.
  • a - ⁇ - may comprise one active strand and one passenger strand.
  • the compound or composition comprises a homo- multimer of substantially identical double-stranded oligonucleotides.
  • the substantially identical double-stranded oligonucleotides can each comprise an siRNA targeting the same molecular target in vivo.
  • the compound or composition comprises a hetero-multimer of two or more substantially different double-stranded oligonucleotides.
  • the substantially different double-stranded oligonucleotides can each comprise an siRNA targeting different genes.
  • the compound can further comprise a targeting ligand.
  • the compound can further comprise 2 or 3 substantially different double-stranded oligonucleotides - each comprising an siRNA targeting a different molecular target in vivo.
  • the compound can further comprise a targeting ligand, one - comprising a first siRNA guide strand targeting Factor VII and a first passenger strand hybridized to the guide strand, one - comprising a second siRNA guide strand targeting
  • the targeting ligand can comprise A -A cetyl gal actosami ne (GalNAc).
  • trimers are provided in Examples 17, 18, and 20.
  • the compound can further comprise a targeting ligand.
  • the compound can further comprise 2, 3, or 4 substantially different double-stranded oligonucleotides - each comprising an siRNA targeting a different molecular target in vivo.
  • the compound can further comprise a targeting ligand, one
  • the targeting ligand can comprise A f - A cety 1 gal act o s am i n e (GalNAc).
  • Example 21 Examples of tetramers are provided in Example 21.
  • each double-stranded oligonucleotide e.g., a double-stranded oligonucleotide
  • the compound further comprises a targeting ligand
  • each double-stranded oligonucleotide e.g., -
  • each double-stranded oligonucleotide comprises an siRNA guide strand and a passenger strand hybridized to the guide strand
  • the compound is at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 % pure.
  • oligonucleotides Having Increased Circulation Half-Life and/or Activity in vivo comprises a second siRNA guide strand targeting Apolipoprotein B and a second passenger strand hybridized the second guide strand.
  • Oligonucleotides Having Increased Circulation Half-Life and/or Activity in vivo comprises a second siRNA guide strand targeting Apolipoprotein B and a second passenger strand hybridized the second guide strand.
  • the disclosure provides multimeric oligonucleotides having increased circulation half-life and/or activity in vivo , as well as compositions including the multimeric oligonucleotides and methods for their synthesis and use.
  • the disclosure provides a multimeric oligonucleotide comprising Structure 21 : (Structure 21)
  • each monomeric subunit . is independently a single or double-stranded oligonucleotide
  • m is an integer > 1
  • each ⁇ is a covalent linker joining adjacent monomeric subunits . , and at least one of the monomeric subunits .
  • the disclosure provides a multimeric oligonucleotide comprising Structure 21 : (Structure 21)
  • each monomeric subunit . is independently a single or double-stranded oligonucleotide, each ⁇ is a covalent linker joining adjacent monomeric subunits
  • m is an integer > 0 selected to (a) increase in vivo circulation half-life of the multimeric oligonucleotide relative to that of the individual monomeric subunits
  • the disclosure provides a multimeric oligonucleotide comprising Structure 21 : (Structure 21)
  • each monomeric subunit . is independently a single or double-stranded oligonucleotide, each ⁇ is a covalent linker joining adjacent monomeric subunits .
  • m is an integer > 0, and wherein the multimeric oligonucleotide has molecular size and/or weight configured to (a) increase in vivo circulation half-life of the multimeric oligonucleotide relative to that of the individual monomeric subunits . and/or (b) increase in vivo activity of the multimeric oligonucleotide relative to that of the individual monomeric subunits . .
  • the disclosure provides a method for increasing in vivo circulation half-life and/or in vivo activity of one or more oligonucleotides, the method comprising administering to a subject the one or more oligonucleotides in the form of a multimeric oligonucleotide comprising Structure 21 : (Structure 21)
  • each monomeric subunit . is independently a single or double- stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent monomeric subunits .
  • m is an integer > 0 selected to (a) increase in vivo circulation half- life of the multimeric oligonucleotide relative to that of the individual monomeric subunits . and/or (b) increase in vivo activity of the multimeric oligonucleotide relative to that of the individual monomeric subunits . .
  • the disclosure provides a method for increasing in vivo circulation half-life and/or in vivo activity of one or more oligonucleotides, the method comprising administering to a subject the one or more oligonucleotides in the form of a multimeric oligonucleotide comprising Structure 21 : (Structure 21)
  • each monomeric subunit . is independently a single or double- stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent monomeric subunits .
  • m is an integer > 0, and
  • the multimeric oligonucleotide has molecular size and/or weight configured to (a) increase in vivo circulation half-life of the multimeric oligonucleotide relative to that of the individual monomeric subunits . and/or (b) increase in vivo activity of the multimeric oligonucleotide relative to that of the individual monomeric subunits . .
  • the disclosure provides a multimeric oligonucleotide comprising m monomeric subunits . , wherein each of the monomeric subunits . is independently a single or double-stranded oligonucleotide, each of the monomeric subunits . is joined to another monomeric subunit by a covalent linker
  • ⁇ , and m is an integer > 3 selected to (a) increase in vivo circulation half-life of the multimeric oligonucleotide relative to that of the individual monomeric subunits .
  • the disclosure provides a multimeric oligonucleotide comprising m monomeric subunits . , wherein each of the monomeric subunits
  • . is independently a single or double-stranded oligonucleotide, each of the monomeric subunits . is joined to another monomeric subunit by a covalent linker
  • the multimeric oligonucleotide has molecular size and/or weight configured to (a) increase in vivo circulation half-life of the multimeric
  • oligonucleotide relative to that of the individual monomeric subunits . and/or (b) increase in vivo activity of the multimeric oligonucleotide relative to that of the individual monomeric subunits . .
  • the increase is relative to the circulation half-life and/or activity of a monomeric subunit of the multimeric oligonucleotide. Circulation half-life (and its relationship to other properties such as glomerular filtration) is discussed in further detail in the Oligonucleotide Uptake and Clearance section and in Examples 25 and 37 below.
  • the in vivo circulation half-life increases by a factor of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, or 1,000.
  • the in vivo circulation half-life can increase by a factor of at least 2.
  • the in vivo circulation half-life can increase by a factor of at least 10.
  • the increase in in vivo activity is measured as the ratio of in vivo activity at tmax.
  • the in vivo activity increases by a factor of at least 2, 3, 4, 5, 6, 7,
  • the in vivo activity can increase by a factor of at least 2.
  • the in vivo activity can increase by a factor of at least 10.
  • the increase is in a mouse. In one embodiment, the increase is in a human.
  • m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
  • m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
  • each of the monomeric subunits is a monomeric subunit.
  • each of the covalent linkers joins sense strands of the siRNA.
  • each of the covalent linkers ⁇ joins two monomeric subunits . .
  • At least one of the covalent linkers ⁇ joins three or more monomeric subunits . .
  • each monomeric subunit . is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • each monomeric subunit . is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • each monomeric subunit . is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • each covalent linker ⁇ is on the same strand:
  • each monomeric subunit . is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • oligonucleotide - independently a double-stranded oligonucleotide - , and m is 3, 4, 5, 6, 7, 8, 9, 10,
  • each monomeric subunit . is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • oligonucleotide - independently a double-stranded oligonucleotide - , m is 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and each covalent linker ⁇ is on the same strand.
  • each monomeric subunit . is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • each monomeric subunit . is independently a double-stranded oligonucleotide - , and m is > 13.
  • each covalent linker ⁇ is on the same strand.
  • Structure 21 is Structure 22 or 23: (Structure 22) ⁇ ⁇ - n (Structure 23)
  • each - is a double-stranded oligonucleotide
  • each ⁇ is a covalent linker joining adjacent double-stranded oligonucleotides
  • m is an integer > 1
  • n is an integer > 0.
  • Structure 21 is not a structure disclosed in
  • each oligonucleotide . is a single-stranded oligonucleotide.
  • each oligonucleotide . is a double- stranded oligonucleotide.
  • the oligonucleotides comprise a combination of single and double-stranded oligonucleotides.
  • the multimeric oligonucleotide comprises a linear structure wherein each of the covalent linkers ⁇ joins two monomeric subunits
  • the multimeric oligonucleotide comprises a branched structure wherein at least one of the covalent linkers ⁇ joins three or more monomeric subunits .
  • Structure 21 could be
  • each monomeric subunit . is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • m is 1 - ⁇ - ⁇ - ⁇ - (Structure 34); m is
  • m is 6, 7, 8, 9, 10, 11, or 12. In some such embodiments, m is an integer > 13. In one such embodiment, at least one single-stranded oligonucleotide
  • each single-stranded oligonucleotide - is independently an antisense oligonucleotide.
  • the multimeric oligonucleotide comprises a homo-multimer of substantially identical oligonucleotides.
  • the substantially identical oligonucleotides can be siRNAs targeting the same molecular target in vivo.
  • the substantially identical oligonucleotides can be miRNAs targeting the same molecular target in vivo.
  • the substantially identical oligonucleotides can be antisense
  • oligonucleotides targeting the same molecular target in vivo can be a combination of siRNA, miRNA, and/or antisense RNA targeting the same molecular target in vivo.
  • the multimeric oligonucleotide comprises a hetero-multimer of two or more substantially different oligonucleotides.
  • the substantially different oligonucleotides can be siRNAs targeting different molecular targets in vivo.
  • the substantially different oligonucleotides can be miRNAs targeting different molecular targets in vivo.
  • the substantially different oligonucleotides can be antisense
  • the multimeric oligonucleotide does not comprise PEG.
  • the multimeric oligonucleotide does not comprise a polyether compound.
  • the multimeric oligonucleotide does not comprise a polymer other than the oligonucleotides.
  • Nanoparticles such as lipid nanoparticles (LNP) have been used in attempts to increase the circulation half-life of certain drugs.
  • Such approaches can have drawbacks, including increased toxicity (e.g., from cationic lipids).
  • the present disclosure can be distinguished from such approaches.
  • the multimeric oligonucleotide is not formulated in an NP or LNP.
  • Phosphorothioate groups have been used in attempts to increase the circulation half-life of certain drugs. Such approaches can have the drawbacks, including lower activity (e.g., due to oligonucleotide/plasma protein aggregation).
  • the present disclosure can be distinguished from such approaches.
  • the multimeric oligonucleotide does not comprise a phosphorothioate.
  • the multimeric oligonucleotide further comprises one or more targeting ligands.
  • the multimeric oligonucleotide consists essentially of Structure 21 and an optional targeting ligand.
  • the multimeric oligonucleotide can use any of the targeting ligands discussed herein (see, e.g., the Targeting Ligands section below).
  • a targeting ligand is conjugated to an oligonucleotide subunit, and/or to a linker between adjacent
  • a targeting ligand can be conjugated to an oligonucleotide through its 3’ or 5’ terminus.
  • intracellular cleavage can convert a single multimeric oligonucleotide into multiple biologically active oligonucleotides after cellular targeting and entry (e.g., a single siRNA construct can deliver four or more active siRNA), increasing potency and decreasing undesired side effects.
  • a single siRNA construct can deliver four or more active siRNA
  • the compound is isolated or substantially pure.
  • the compound can be at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 % pure. In one embodiment, the compound is about 85-95 % pure.
  • the methods for synthesizing the compounds and compositions according to the disclosure can result in a product that is at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 % pure. In one embodiment, the product is about 85-95 % pure. Preparations can be greater than or equal to 50 % pure; preferably greater than or equal to 75 % pure; more preferably greater than or equal to 85 % pure; and still more preferably, greater than or equal to 95 % pure.
  • each oligonucleotide is RNA, DNA, or comprises an artificial or non-natural nucleic acid analog.
  • at least one oligonucleotide is an siRNA, miRNA, or antisense oligonucleotide.
  • siRNA siRNA
  • miRNA miRNA
  • antisense oligonucleotide Various other possible oligonucleotides and substitutions are discussed, for example, in the Nucleic Acids section above.
  • each oligonucleotide is 15-30, 17-27, 19-26, or 20-25 nucleotides in length.
  • the nucleic acid or oligonucleotide is 15-30, 17-27, 19-26, 20-25, 40-50, 40-150, 100-300, 1000-2000, or up to 10000 nucleotides in length.
  • the multimeric oligonucleotides comprising structure 21 have a molecular weight of at least about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 kD. In various embodiments, the multimeric oligonucleotides comprising structure 21 have a molecular weight of at least about 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, or 70-75 kD.
  • multimeric oligonucleotides comprising Structure 21 can be synthesized by various methods (e.g., those described herein for making tetrameric or greater multimers), certain results may call for specific methodologies. For example, the following method (as well as those shown in Example 22) is designed to efficiently produce multimers having each covalent linker ⁇ on the same strand.
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising structure 34:
  • each - is a single-stranded oligonucleotide and each ⁇ is a covalent linker joining adjacent single-stranded oligonucleotides, the method comprising the steps of:
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising structure 35:
  • each - is a single-stranded oligonucleotide and each ⁇ is a covalent linker joining adjacent single-stranded oligonucleotides, the method comprising the steps of:
  • the disclosure provides a method of synthesizing a multimeric oligonucleotide comprising structure 37:
  • each - is a single-stranded oligonucleotide and each ⁇ is a covalent linker joining adjacent single-stranded oligonucleotides, the method comprising the steps of:
  • the disclosure also provides methods for synthesizing single-stranded multimeric oligonucleotides, for example wherein m is
  • the multimeric compounds can include any one or more of the features disclosed herein.
  • the compounds can include any one or more of the nucleic acids (with or without modifications), targeting ligands, and/or linkers described herein, or any of the specific structures or chemistries shown in the summary, description, or Examples.
  • the compounds can be prepared in an of the compositions (e.g., for experimental or medical use) shown in the summary, description, or Examples.
  • the bioavailability of a drug in the blood stream can be characterized as the balance between target cell uptake versus kidney clearance. From a practical perspective, in vivo circulation half-life and/or in vivo activity are good proxies for kidney clearance/glomerular filtration because they can be readily quantified and measured and because their improvement (e.g., increase) can correlate with improved pharmacodynamics and/or pharmacokinetics.
  • Rate of Uptake f ⁇ (ONT Concentration) x (Rate Blood Flow) x (Receptor Copy Number/cell) x (Number of Cells) x (equilibrium dissociation constant KD) X (Internalization Rate) ⁇ .
  • the Copy Number, KD, Number of cells and Internalization Rate will be constant. This can explain why the GalNAc ligand system is so effective for hepatocytes - it targets the ASGP receptor, which is present at high copy number.
  • oligonucleotides leading to a lower rate of clearance, resulting in a higher concentration of ONT in circulation at a given time t (e.g., increased serum half-life, higher overall uptake, and higher activity).
  • glomerular filtration rates can be difficult to measure directly.
  • compounds that pass through the glomerular capillaries are readily absorbed by cells such as tubular epithelial cells, which can retain compounds like siRNA for significant periods of time (see, e.g., Henry, S. P. et al; Toxicology, 301, 13-20 (2012) and van de Water, F.M et al; Drug metabolism and Disposition, 34, No 8, 1393-1397 (2006)).
  • absorbed compounds can be metabolized to breakdown products, which are then secreted in urine.
  • the concentration (e.g., in urine) of a therapeutic agent such as an siRNA at a specific time point may not necessarily be representative of the glomerular filtration rate.
  • serum half-life which is related to glomerular filtration and which is directly measurable, may be considered to be a suitable proxy for glomerular filtration.
  • Table 1 below shows the dramatic effect increasing the circulation half- life (ti/2) of a component can have on the resulting concentration of the component at time t:
  • a typical siRNA (e.g., double-stranded monomer) has a molecular weight of about 15kD.
  • a siRNA tetramer according to the disclosure can have a molecular weight of about 60 kD.
  • multimers can be configured to have a molecular size and/or weight resulting in decreased glomerular filtration in vivo.
  • Such multimers would have an increased circulation half-life.
  • multimers according to the disclosure can be configured to have increased in vivo circulation half-life and/or increased in vivo activity, relative to that of the individual monomeric subunits.
  • the multimer e.g., tetramer
  • the multimer would deliver many (e.g., four) times the payload per ligand/receptor binding event than the monomeric equivalent.
  • the multimeric oligonucleotide has a structure selected to (a) increase in vivo circulation half-life of the multimeric oligonucleotide relative to that of the individual monomeric subunits and/or (b) increase in vivo activity of the multimeric oligonucleotide relative to that of the individual monomeric subunits.
  • the multimeric oligonucleotide can have a molecular size and/or weight configured for this purpose.
  • compositions including any one or more of the compounds or compositions described above.
  • pharmaceutical compositions include compositions of matter, other than foods, that can be used to prevent, diagnose, alleviate, treat, or cure a disease.
  • various compounds or compositions according to the disclosure should be understood as including embodiments for use as a medicament and/or for use in the manufacture of a medicament.
  • a pharmaceutical composition can include a compound or composition according to the disclosure and a pharmaceutically acceptable excipient.
  • an excipient can be a natural or synthetic substance formulated alongside the active ingredient.
  • Excipients can be included for the purpose of long-term stabilization, increasing volume (e.g., bulking agents, fillers, or diluents), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.
  • Excipients can also be useful manufacturing and distribution, for example, to aid in the handling of the active ingredient and/or to aid in vitro stability (e.g., by preventing denaturation or aggregation).
  • appropriate excipient selection can depend upon various factors, including the route of administration, dosage form, and active ingredient(s).
  • Oligonucleotides can be delivered locally or systemically, and the pharmaceutical compositions of the disclosure can vary accordingly.
  • administration is not necessarily limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous,
  • intramedullary, intraarticular, intramuscular, or intraperitoneal injection rectal, topical, transdermal, or oral.
  • Administration to an individual may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a
  • Physiologically acceptable formulations and standard pharmaceutical formulation techniques, dosages, and excipients are well known to persons skilled in the art (see, e.g., Physicians’ Desk Reference (PDR®) 2005, 59th ed., Medical Economics Company, 2004; and Remington: The Science and Practice of Pharmacy, eds. Gennado et al. 21th ed., Lippincott, Williams & Wilkins, 2005).
  • compositions can include an effective amount of the compound or composition according to the disclosure.
  • effective amount can be a concentration or amount that results in achieving a particular stated purpose, or more amount means an amount adequate to cause a change, for example in comparison to a placebo.
  • the effective amount is a therapeutically effective amount, it can be an amount adequate for therapeutic use, for example and amount sufficient to prevent, diagnose, alleviate, treat, or cure a disease.
  • An effective amount can be determined by methods known in the art.
  • An effective amount can be determined empirically, for example by human clinical trials.
  • Effective amounts can also be extrapolated from one animal (e.g., mouse, rat, monkey, pig, dog) for use in another animal (e.g., human), using conversion factors known in the art. See, e.g., Freireich et al., Cancer Chemother Reports 50(4):219-244 (1966).
  • the disclosure provides any one or more of the compounds or compositions described above formulated in a delivery vehicle.
  • the delivery vehicle can be a lipid nanoparticle (LNP), exosome, microvesicle, or viral vector.
  • the disclosure provides any one or more of the compounds or compositions described above and further comprising a targeting ligand.
  • the targeting ligand comprises A f - A cety 1 gal act o s am i n e (GalNAc), cholesterol, tocopherol, folate, 2-[3-(l,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA), anisamide, phospholipid, phosphatidyl choline, lecithin, or an
  • the immunostimulant may be a CpG oligonucleotide, for example, the CpG oligonucleotides of TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 162) or GGT GC ATC GAT GC AGGGGG (SEQ ID NO: 163).
  • the targeting ligand can be bound to the multimeric oligonucleotide construct directly or indirectly to the nucleic acid, for example through its 3’ or 5’ terminus, to an internal nucleic acid residue, or to a linker within the construct.
  • two targeting ligands are conjugated to the multimeric oligonucleotide, where one ligand is conjugated through the 3’ terminus and the other ligand is conjugated through the 5’ terminus of the oligonucleotide.
  • One or more targeting ligands can be conjugated to the sense strand or the antisense strand of the oligonucleotide, or both the sense strand and the antisense strand. Additional examples that may be adapted for use with the disclosure are discussed below.
  • therapeutic oligonucleotides must overcome a series of physiological hurdles to access the target cell in an organism (e.g., animal, such as a human, in need of therapy).
  • a therapeutic oligonucleotide generally must avoid clearance in the bloodstream, enter the target cell type, and then enter the cytoplasm, all without eliciting an undesirable immune response. This process is generally considered inefficient, for example, 95 % or more of siRNA that enters the endosome in vivo may be degraded in lysosomes or pushed out of the cell without affecting any gene silencing.
  • Drug delivery vehicles have been used to deliver therapeutic RNAs in addition to small molecule drugs, protein drugs, and other therapeutic molecules.
  • Drug delivery vehicles have been made from materials as diverse as sugars, lipids, lipid-like materials, proteins, polymers, peptides, metals, hydrogels, conjugates, and peptides. Many drug delivery vehicles incorporate aspects from combinations of these groups, for example, some drug delivery vehicles can combine sugars and lipids.
  • drugs can be directly hidden in‘cell like’ materials that are meant to mimic cells, while in other cases, drugs can be put into, or onto, cells themselves.
  • Drug delivery vehicles can be designed to release drugs in response to stimuli such as pH change, biomolecule concentration, magnetic fields, and heat.
  • oligonucleotides such as siRNA
  • the dose required for effective siRNA delivery to hepatocytes in vivo has decreased by more than 10,000 fold in the last ten years - whereas delivery vehicles reported in 2006 could require more than 10 mg/kg siRNA to target protein production, with new delivery vehicles target protein production can now be reduced after a systemic injection of 0.001 mg/kg siRNA.
  • the increase in oligonucleotide delivery efficiency can be attributed, at least in part, to developments in delivery vehicles.
  • helper components can include chemical structures added to the primary drug delivery system. Often, helper components can improve particle stability or delivery to a specific organ. For example, nanoparticles can be made of lipids, but the delivery mediated by these lipid nanoparticles can be affected by the presence of hydrophilic polymers and/or hydrophobic molecules.
  • hydrophilic polymers One important hydrophilic polymer that influences nanoparticle delivery is polyethylene glycol). Other hydrophilic polymers include non-ionic surfactants.
  • Hydrophobic molecules that affect nanoparticle delivery include cholesterol, l-2-Distearoyl-sn-glyerco-3-phosphocholine (DSPC), l-2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), and others.
  • DSPC l-2-Distearoyl-sn-glyerco-3-phosphocholine
  • DOTMA l-2-di-0-octadecenyl-3-trimethylammonium propane
  • DOTAP 1,2-dioleoyl- 3-trimethylammonium-propane
  • Drug delivery systems have also been designed using targeting ligands or conjugate systems.
  • oligonucleotides can be conjugated to cholesterols, sugars, peptides, and other nucleic acids, to facilitate delivery into hepatocytes and/or other cell types.
  • conjugate systems may facilitate delivery into specific cell types by binding to specific receptors.
  • delivery vehicles and targeting ligands can generally be adapted for use according to the present disclosure.
  • delivery vehicles and targeting ligands can be found in: Sahay, G., et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat Biotechnol, 31 : 653-658 (2013); Wittrup, A., et al. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat Biotechnol (2015); Whitehead, K.A., Langer, R. & Anderson, D.G. Knocking down barriers: advances in siRNA delivery. Nature reviews.
  • Multivalent N- acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi- mediated gene silencing. J Am Chem Soc, 136: 16958-16961 (2014); Ostergaard, M.E., et al. Efficient Synthesis and Biological Evaluation of 5’-GalNAc Conjugated Antisense Oligonucleotides. Bioconjugate chemistry (2015); Sehgal, A., et al. An RNAi therapeutic targeting antithrombin to rebalance the coagulation system and promote hemostasis in hemophilia. Nature Medicine, 21 : 492-497 (2015); Semple, S.C., et al.
  • Nanoparticles Mol Ther Nucleic Acids 2, el39 (2013); Draz, M.S., et al. Nanoparticle- Mediated Systemic Delivery of siRNA for Treatment of Cancers and Viral Infections. Theranostics, 4: 872-892 (2014); Otsuka, H., Nagasaki, Y. & Kataoka, K. PEGylated nanoparticles for biological and pharmaceutical applications. Advanced Drug Delivery Reviews, 55: 403-419 (2003); Kauffman, K.J., et al. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in vivo with Fractional Factorial and Definitive Screening Designs.
  • the compounds and compositions of the disclosure can be conjugated to or delivered with other chemical or biological moieties, including, e.g., biologically active moieties.
  • a biologically active moiety is any molecule or agent that has a biological effect, preferably a measurable biological effect.
  • Chemical or biological moieties include, e.g., proteins, peptides, amino acids, nucleic acids (including, e.g., DNA, RNA of all types, RNA and DNA aptamers, antisense
  • oligonucleotides and antisense miRNA inhibitors
  • targeting ligands carbohydrates, polysaccharides, lipids, organic compounds, and inorganic chemical compounds.
  • targeting ligand can include a moiety that can be made accessible on the surface of a nanoparticle or as part of a delivery conjugate (e.g., multi-conjugate oligonucleotide, or multimeric oligonucleotide) for the purpose of delivering the payload of the nanoparticle or delivery conjugate to a specific target, such as a specific bodily tissue or cell type, for example, by enabling cell receptor attachment of the nanoparticle or delivery conjugate.
  • a delivery conjugate e.g., multi-conjugate oligonucleotide, or multimeric oligonucleotide
  • Suitable targeting ligands include, but are not limited to, cell specific peptides or proteins (e.g., transferrin and monoclonal antibodies), phospholipid, phosphatidyl choline, lecithin, aptamers, cell growth factors, vitamins (e.g., folic acid), monosaccharides (e.g., galactose and mannose),
  • the ligand may be incorporated into the foregoing compounds of the disclosure using a variety of techniques known in the art, such as via a covalent bond such as a disulfide bond, an amide bond, or an ester bond, or via a non-covalent bond such as biotin-streptavidin, or a metal-ligand complex.
  • Additional biologically active moieties within the scope of the disclosure are any of the known gene editing materials, including for example, materials such as oligonucleotides, polypeptides and proteins involved in CRISPR/Cas systems, TALES, TALENs, and zinc finger nucleases (ZFNs).
  • materials such as oligonucleotides, polypeptides and proteins involved in CRISPR/Cas systems, TALES, TALENs, and zinc finger nucleases (ZFNs).
  • the compounds and compositions of the disclosure can be encapsulated in a carrier material to form nanoparticles for intracellular delivery.
  • carrier materials include cationic polymers, lipids or peptides, or chemical analogs thereof. Jeong et al., BIOCONJUGATE CHEM., Vol. 20, No. 1, pp. 5- 14 (2009).

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EP20766332.9A EP3935168A4 (en) 2019-03-04 2020-03-03 MULTIMERIC OLIGONUCLEOTIDES WITH IMPROVED BIOACTIVITY
JP2021552130A JP2022523226A (ja) 2019-03-04 2020-03-03 増強された生理活性を有する多量体オリゴヌクレオチド
AU2020233375A AU2020233375A1 (en) 2019-03-04 2020-03-03 Multimeric oligonucleotides with enhanced bioactivity
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EP3935168A4 (en) 2024-04-10
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