Tetragalnac and peptide containing conjugates and methods for delivery of oligonucleotides
Disclosed herein is a modular composition comprising 1) an oligonucleotide; 2) one or more tetraGalNAc ligands of Formula (I), which may be the same or different; optionally, 3) one or more linkers, which may be the same or different; 4) one or more peptides independently selected from Table 3, which may be the same or different; and optionally, 5) one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
1. A method for preparing a modular composition, comprising:
conjugating, to an oligonucleotide, one or more tetraGalNAc ligands of Formula (I), (II) or (III):
to form a tetraGalNAc-conjugated oligonucleotide;
wherein:
X is —O—, —S—, —CR 1 R 2 — or —NR 1 —;
R 1 and R 2 are each independently selected from the group consisting of hydrogen and C 1 —C 6 alkyl; and
n is 1,2,3, or 4.
2. The method of claim 1 , wherein the conjugating step comprises:
reacting one or more tetraGalNAc compounds having a formula of
with N, N-diisopropylethylamine (DIEA) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) to form one or more modified tetraGalNAc compounds; and
reacting the one or more modified tetraGalNAc compounds with the oligonucleotide to form the tetraGalNAc-conjugated oligonucleotide.
3. The method of claim 1 , wherein the conjugating step comprises:
attaching the one or more tetraGalNAc ligands to the oligonucleotide via one or more linkers.
4. The method of claim 3 , wherein each linker is independently selected from the group consisting of:
wherein:
each R is independently H, Boc (tert-butyloxycarbonyl), Cbz (carboxybenzyl), Ac (acetyl), a PEG, a lipid, a targeting ligand, linker(s), or peptide(s); and
each n is 0 to 750.
5. The method of claim 1 , wherein the conjugating step comprises:
attaching the one or more tetraGalNAc ligands to one or more terminal 3′ and/or 5′-positions and/or 2′positions of the ribose rings of the oligonucleotide.
6. The method of claim 1 , further comprising:
conjugating, to the tetraGalNAc-conjugated oligonucleotide, one or more peptides independently selected from SEQ ID NO. 1-474, a D-amino acid variant thereof, a retro-inverso variant thereof, or a cysteine conjugation point variant thereof, to form a tetraGalNAc- and peptide- conjugated oligonucleotide,
wherein the cysteine conjugation point variants thereof refers to variants of the peptides comprising conjugation through existing cysteines or through a cysteine residue added to a N- or C- terminus of the peptides.
7. The method of claim 6 , wherein the conjugation of one or more peptides comprises:
attaching the one or more peptides to the tetraGalNAc-conjugated oligonucleotide via one or more linkers.
8. The method of claim 7 , wherein each linker is independently selected from the group consisting of:
wherein:
each R is independently H, Boc (tert-butyloxycarbonyl), Cbz (carboxybenzyl), Ac (acetyl), a PEG, a lipid, a targeting ligand, linker(s), or peptide(s); and
each n is 0 to 750.
9. The method of claim 6 , wherein the conjugation of one or more peptides comprises:
attaching the one or more peptides to one or more terminal 3′ and/or 5′-positions and/or 2′-positions of the ribose rings of the tetraGalNAc-conjugated oligonucleotide.
10. The method of claim 6 , further comprising:
conjugating, to the tetraGalNAc- and peptide- conjugated oligonucleotide, one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.
11. The method of claim 1 , further comprising
conjugating, to the tetraGalNAc-conjugated oligonucleotide, one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents, to form a tetraGalNAc- and ligand- conjugated oligonucleotide.
12. The method of claim 11 , wherein the conjugation of one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents comprises:
attaching the one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents to the tetraGalNAc-conjugated oligonucleotide via one or more linkers.
13. The method of claim 12 , wherein each linker is independently selected from the group consisting of:
wherein:
each R is independently H, Boc (tert-butyloxycarbonyl), Cbz (carboxybenzyl), Ac (acetyl), a PEG, a lipid, a targeting ligand, linker(s), or peptide(s); and
each n is 0 to 750.
14. The method of claim 11 , wherein the conjugation of one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents comprises:
attaching the one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents to one or more terminal 3′ and/or 5′-positions and/or 2′-positions of the ribose rings of the tetraGalNAc-conjugated oligonucleotide.
15. The method of claim 11 , further comprising:
conjugating, to the tetraGalNAc- and ligand- conjugated oligonucleotide, one or more peptides independently selected from SEQ ID NO. 1-474, a D-amino acid variant thereof, a retro-inverso variant thereof, or a cysteine conjugation point variant thereof, to form a tetraGalNAc- and peptide- conjugated oligonucleotide,
wherein the cysteine conjugation point variants thereof refers to variants of the peptides comprising conjugation through existing cysteines or through a cysteine residue added to a N- or C- terminus of the peptides.
16. A method for preparing a modular composition, comprising:
conjugating, to an oligonucleotide, one or more tetraGalNAc ligands of formula:
to form a tetraGalNAc-conjugated oligonucleotide;
wherein:
X is —O —, —S—, or —CH 2—; and
n, m, and q are each 1, 2, 3, or 4.
17. The method of claim 16 , wherein the conjugating step comprises:
reacting one or more tetraGalNAc compounds having a formula of
with N, N-diisopropylethylamine (DIEA) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) to form one or more modified tetraGalNAc compounds; and
reacting the one or more modified tetraGalNAc compounds with the oligonucleotide to form the tetraGalNAc-conjugated oligonucleotide.
18. The method of claim 17 , wherein the tetraGalNAc compound has the structure of
and wherein m is 2 and q is 1.
19. The method of claim 16 , further comprising
conjugating, to the tetraGalNAc-conjugated oligonucleotide, one or more peptides independently selected from SEQ ID NO. 1-474, a D-amino acid variant thereof, a retro-inverso variant thereof, or a cysteine conjugation point variant thereof, to form a tetraGalNAc- and peptide- conjugated oligonucleotide,
wherein the cysteine conjugation point variants thereof refers to variants of the peptides comprising conjugation through existing cysteines or through a cysteine residue added to a N- or C- terminus of the peptides.
20. The method of claim 16 , further comprising
conjugating, to the tetraGalNAc-conjugated oligonucleotide, one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents, to form a tetraGalNAc- and ligand-conjugated oligonucleotide.