OLIGONUCLEOTIDE CONJUGATE COMPOSITIONS AND METHODS OF USE
The disclosure relates to GalNAc moieties comprising at least one GalNAc monomer. The disclosure also relates to GalNAc-oligonucleotide conjugates comprising GalNAc moieties and oligonucleotides, e.g., saRNAs or siRNAs useful in regulating the expression of a target gene. Methods of using the GalNAc-oligonucleotide conjugates are also provided.
1 . A N-Acetyl-Galactosamine (GalNAc) monomer comprising a structure selected from the group consisting of
wherein R1, R2, and R3 can be the same or different, and wherein R1, R2, and R3 are independently selected from an alkyl, aryl, and alkenyl group,
wherein R4 is a suitable protecting group or a C1-6 straight or branched alkyl group,
wherein R5 and R6 are each independently a C1-6 straight or branched alkyl group, and
wherein R7 is a suitable protecting group;
wherein R1, R2, and R3 can be the same or different, and wherein R1, R2, and R3 are independently selected from an alkyl, aryl, and alkenyl group;
wherein R4 is a protecting group or a C1-6 straight or branched alkyl group,
wherein R5 and R6 are each independently C1-6 straight or branched alkyl; and
wherein R7 is a suitable protecting group;
wherein R1, R2, and R3 can be the same or different, and wherein R1, R2, and R3 are independently selected from an alkyl, aryl, and alkenyl group,
wherein R7 is a suitable protecting group, and
wherein Linker1 is a cleavable linker; and
wherein R1, R2, and R3 can be the same or different, and wherein R1, R2, and R3 are independently selected from the group consisting of an alkyl, aryl, and alkenyl group,
wherein R7 is a suitable protecting group, and
wherein Linker1 is a cleavable linker.
2 . A GalNAc moiety comprising at least one GalNAc monomer, wherein the GalNAc monomers are selected from the group consisting of
wherein R 8 is —H or a C1-6 straight or branched alkyl group;
wherein R 8 is —H or a C1-6 straight or branched alkyl group, and wherein X is O or S;
wherein X is O or S;
wherein the GalNAc moiety does not comprise more than one M4, M5 or M6 monomer, and
wherein the GalNAc moiety is not a GalNAc moiety consisting of only one M3 monomer,
only one M6 monomer, or one M6 monomer and two M3 monomers.
3 . The GalNAc moiety of claim 2 , wherein the GalNAc moiety comprises a spacer comprising a structure of HEG, C12, ab, TEG, or C3.
4 . The GalNAc moiety of claim 2 , wherein the GalNAc moiety comprises two or three GalNAc monomers.
5 . The GalNAc moiety of claim 2 , wherein the GalNAc moiety comprises three M1, M2 or M3 monomers.
6 . The GalNAc moiety of claim 5 , wherein the GalNAc moiety comprises three M1 monomers.
7 . The GalNAc moiety of claim 2 , wherein the GalNAc cluster comprises a structure of
8 . A GalNAc moiety prepared by a process comprising the steps of:
1 ). Providing at least one GalNAc monomer selected from the group consisting of any monomer of claim 1 ,
and
2). synthesizing a GalNAc moiety from the GalNAc monomers in step 1 ); optionally adding at least one spacer and optionally removing the protecting groups.
9 . A conjugate comprising an oligonucleotide and the GalNAc moiety of claim 2 , wherein the oligonucleotide regulates the expression of a target gene, and wherein the oligonucleotide and the GalNAc moiety are connected by a bond or a cleavable linker.
10 . The conjugate of claim 9 , wherein the oligonucleotide and the GalNAc moiety are connected by a cleavable linker.
11 . The conjugate of claim 10 , wherein the linker is C6ssC6 or dT.
12 . The conjugate of claim 9 , wherein the oligonucleotide and the GalNAc moiety are connected by a bond, wherein the bond is a phosphodiester bond or a phosphorothioate bond.
13 . The conjugate of claim 9 , wherein the conjugate comprises a structure of CJ1, CJ2, CJ3, CJ4, CJ5, CJ6, CJ7, CJ8, CJ9, CJ10, CJ11, CJ12, CJ13, CJ14, CJ15, CJ16, CJ17, CJ18, CJ19, CJ20, CJ21, CJ22, CJ23, CJ24, or CJ25.
14 . The conjugate of claim 9 , wherein the oligonucleotide is a synthetic isolated small activating RNA (saRNA).
15 . The conjugate of claim 14 , wherein the target gene is CEBPA.
16 . The conjugate of claim 14 , wherein saRNA is a double-stranded saRNA.
17 . The conjugate claim 16 , wherein the GalNAc cluster is connected to the 5′ or 3′ end of the sense strand.
18 . The conjugate of claim 16 , wherein the double-stranded saRNA is selected from the group consisting of XD-03302, S1 (XD-06409), S2 (XD-06410), S3 (XD-06411), S4 (XD-06412), S5 (XD-06413), S6 (XD-06414), S7 (XD-06415), S8, XD-07139, XD-03934, and XD-14369K1.
19 . The conjugate of claim 16 , wherein the saRNA is XD-06414 having an antisense strand comprising SEQ ID NO: 15 and a sense strand comprising SEQ ID NO: 14.
20 . The conjugate of claim 19 , wherein the conjugate is selected from the group consising of
21 . The conjugate of claim 19 , wherein the conjugate is L53.
22 . The conjugate of claim 19 , wherein the conjugate is L55.
23 . The conjugate of claim 9 , wherein the oligonucleotide is a synthetic isolated small inhibiting RNA (siRNA).
24 . The conjugate of claim 23 , wherein siRNA is a double-stranded siRNA.
25 . The conjugate claim 24 , wherein the GalNAc cluster is connected to the 5′ or 3′ end of the sense strand.
26 . A conjugate prepared by a process comprising the steps of:
1 ). providing at least one GalNAc monomer selected from the group consisting of M1′, M2′, M3′, M4′, M5′ and M6′; optionally adding at least one spacer;
2 ). providing at least one oligonucleotide; optionally adding at least one linker; and
3). synthesizing the conjugate from the GalNAc monomer(s) in step 1 ) and the oligonucleotide(s) in step 2 ), optionally removing the protecting groups.
27 . The conjugate of claim 26 , wherein the oligonucleotide is an saRNA.
28 . The conjugate of claim 27 , wherein the saRNA is selected from the group consisting of XD-03302, S1 (XD-06409), S2 (XD-06410), S3 (XD-06411), S4 (XD-06412), S5 (XD-06413), S6 (XD-06414), S7 (XD-06415), S8, XD-07139, XD-03934, and XD-14369K1.
29 . The conjugate of claim 26 , wherein the oligonucleotide is an siRNA.
30 . A pharmaceutical composition comprising the conjugate of claim 9 and at least one pharmaceutically acceptable excipient.
31 . The pharmaceutical composition of claim 30 , wherein the conjugate comprises an saRNA.
32 . The pharmaceutical composition of claim 30 , wherein the conjugate comprises an siRNA.
33 . A method of delivering an oligonucleotide to cells comprising administering the conjugate of claim 9 to cells, wherein no transfection agent is used.
34 . The method of claim 33 , wherein the oligonucleotide comprises saRNA.
35 . The method of claim 33 , wherein the oligonucleotide comprises siRNA.
36 . A method of regulating the expression of a target gene in a patient in need thereof, comprising administering the conjugate of claim 9 .
37 . The method of claim 36 , wherein the conjugate comprises an saRNA.
38 . The method of claim 36 , wherein the expression of the target gene is increased.
39 . The method of claim 37 , wherein the target gene is CEBPA.
40 . The method of claim 39 , wherein the expression of albumin is increased in the patient.
41 . The method of claim 36 , wherein the conjugate comprises an siRNA.
42 . The method of claim 36 , wherein the expression of the target gene is reduced.
43 . The method of claim 42 , wherein the target gene is C5.