Tetragalnac and peptide containing conjugates and methods for delivery of oligonucleotides

1. A method of delivering an oligonucleotide to a cell of a subject in need, comprising:

providing or obtaining a modular composition comprising:

1) A single stranded or double stranded oligonucleotide;

2) One or more tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different:

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 C1-C6 alkyl;

n is 1, 2, 3, or 4; and

the bond with “ ” indicates the point of attachment of the tetraGalNAc ligands to the oligonucleotide;

optionally, 3) one or more linkers, which may be the same or different;

optionally, 4) one or more peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different, wherein the cysteine conjugation point variants thereof refers to variants of the peptides comprising a cysteine conjugation, or a cysteine or other thiol-containing moiety added to the C- or N-terminus of the peptides; and

optionally, 5) one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents; and

contacting the cell of the subject with the modular composition for a period of time sufficient for the cell to internalize the modular composition.

2. The method of claim 1 , wherein the modular composition comprises:

1) A single stranded or double stranded siRNA;

2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different;

3) 1-24 linkers, which may be the same or different;

optionally, 4) 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different; and

optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.

3. The method of claim 2 , wherein in the modular composition, X of Formula (I), (II) or (III) is —O—, —S— or —CH 2 —; and n is 1, 2 or 3.

4. The method of claim 2 , wherein the modular composition comprises 1-4 tetraGalNAc ligands, which may be the same or different.

5. The method of claim 2 , wherein the modular composition comprises 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different.

6. The method of claim 1 , wherein the modular composition comprises:

1) A double stranded siRNA;

2) 1-8 tetraGalNAc ligands of Formula (IV), (V) or (VI):

optionally, 3) 1-16 linkers, which may be the same or different;

optionally, 4) 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different; and

optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.

7. The method of claim 6 , wherein the tetraGalNAc ligands and/or the peptides, if present, are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides, if present, are attached to the siRNA optionally via linkers.

8. The method of claim 6 , wherein the modular composition comprises 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different.

9. The method of claim 8 , wherein the modular composition comprises 1-8 peptides independently selected from SEQ ID No. 2, 3, 5, 7, 11, 13, 19, 22, 27-32, 55, 56, 63, 64, 69, 71-74, 86, 90, 94, 95, 106, 137, 192, 200, 201, 228, 229, 266, 282, 333, 337, 407, 423, 436, 437, 461-463, 467, 468, 470, 473 and 474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different.

10. The method of claim 1 , wherein the subject in need is human, and the method is carried out in vitro, ex vivo, or in vivo.

11. A method for inhibiting the expression of a target gene in a cell of a subject in need, comprising:

contacting the cell with a modular composition, comprising:

1) A single stranded or double stranded oligonucleotide;

2) One or more tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different:

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 C1-C6 alkyl;

n is 1, 2, 3, or 4; and

the bond with “ ” indicates the point of attachment of the tetraGalNAc ligands to the oligonucleotide;

optionally, 3) one or more linkers, which may be the same or different;

optionally, 4) one or more peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different, wherein the cysteine conjugation point variants thereof refers to variants of the peptides comprising a cysteine conjugation, or a cysteine or other thiol-containing moiety added to the C- or N-terminus of the peptides; and

optionally, 5) one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents,

wherein the oligonucleotide is present in an amount sufficient to inhibit expression of the target gene.

12. The method of claim 11 , wherein the modular composition comprises:

1) A single stranded or double stranded siRNA;

2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), which may be the same or different;

3) 1-24 linkers, which may be the same or different;

optionally, 4) 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different; and

optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.

13. The method of claim 12 , wherein in the modular composition, X of Formula (I), (II) or (III) is —O—, —S— or —CH 2 —; and n is 1, 2 or 3.

14. The method of claim 12 , wherein the modular composition comprises 1-4 tetraGalNAc ligands, which may be the same or different.

15. The method of claim 12 , wherein the modular composition comprises 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different.

16. The method of claim 11 , wherein the modular composition comprises:

1) A double stranded siRNA;

2) 1-8 tetraGalNAc ligands of Formula (IV), (V) or (VI):

optionally, 3) 1-16 linkers, which may be the same or different;

optionally, 4) 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different; and

optionally, 5) 1-8 targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.

17. The method of claim 16 , wherein the tetraGalNAc ligands and/or the peptides, if present, are attached to the siRNA at different 2′-positions of the ribose rings and/or at different terminal 3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligands and/or the peptides, if present, are attached to the siRNA optionally via linkers.

18. The method of claim 16 , wherein the modular composition comprises 1-8 peptides independently selected from SEQ ID No. 1-474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different.

19. The method of claim 18 , wherein the modular composition comprises 1-8 peptides independently selected from SEQ ID No. 2, 3, 5, 7, 11, 13, 19, 22, 27-32, 55, 56, 63, 64, 69, 71-74, 86, 90, 94, 95, 106, 137, 192, 200, 201, 228, 229, 266, 282, 333, 337, 407, 423, 436, 437, 461-463, 467, 468, 470, 473 and 474, or the D-amino acid, retro-inverso, and cysteine conjugation point variants thereof, which may be the same or different.

20. The method of claim 11 , wherein the subject in need is human, and the method is carried out in vitro, ex vivo, or in vivo.