Retinoid-liposomes for enhancing modulation of HSP47 expression
What is described are pharmaceutical compositions comprising a double-stranded nucleic acid molecule comprising a sense strand and an antisense strand wherein the sense and antisense strands are selected from the oligonucleotides described as SERPINH 1 — 2 (SEQ ID NOS: 60 and 127), SERPINH 1 — 45 a (SEQ ID NOS: 98 and 165), and SERPINH 1 — 51 (SEQ ID NOS: 101 and 168), and drug carrier comprising a mixture of a retinoid and a lipid vesicle, and methods of using these pharmaceutical compositions to treat a disease associated with hsp47 espresssion, including fibrosis.
1. A pharmaceutical composition comprising a drug carrier and a double-stranded nucleic acid molecule, wherein the drug carrier comprises a stellate cell-specific amount of a diVA-PEG-diVA molecule retinoid and wherein the double-stranded nucleic acid molecule comprises the structure:
5′(N) x —Z 3′ (antisense strand)
3′Z′—(N′) y - z″ 5′ (sense strand)
wherein each of N and N′ is a nucleotide which may be unmodified or modified, or an unconventional moiety;
wherein each of (N) x and (N′) y is an oligonucleotide in which each consecutive N or N′ is joined to the next N or N′ by a covalent bond;
wherein each of Z and Z′ is independently present or absent, but if present independently includes 1-5 consecutive nucleotides or non-nucleotide moieties or a combination thereof covalently attached at the 3′-terminus of the strand in which it is present;
wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′-terminus of (N′) y ;
wherein each of x and y is independently an integer between 18 and 40;
wherein the sequence of (N′) y has complementary to the sequence of (N) x ; and
wherein (N) x includes an antisense sequence to the mRNA coding sequence for human hsp47 exemplified by SEQ ID NO:1.
2. The pharmaceutical composition according to claim 1 , wherein the diVA-PEG-diVA is at least partially exposed on the exterior of the drug carrier before the drug carrier reaches the stellate cell.
3. The pharmaceutical composition according to claim 1 , wherein the drug carrier is in a form selected from the group consisting of a polymer micelle, a liposome, an emulsion, a microsphere, and nanosphere.
4. The pharmaceutical composition according to claim 3 , wherein the drug carrier is in a form of a lipid vesicle comprising a bilayer of lipid molecules.
5. The pharmaceutical composition according to claim 4 , wherein the diVA-PEG-diVA is 0.2 wt % to 20 wt % of the lipid molecules.
6. The pharmaceutical composition according to claim 1 , wherein the double-stranded nucleic acid molecule is exposed on the exterior surface of the lipid vesicle.
7. The pharmaceutical composition according to claim 1 , wherein the double-stranded nucleic acid molecule is encapsulated by the lipid vesicle.
8. The pharmaceutical composition according to claim 7 , wherein the double-stranded nucleic acid molecule is resistant to nucleases.
9. The pharmaceutical composition of claim 1 , wherein one strand of the double-stranded nucleic acid molecule comprises SEQ ID NO:127 and the second strand comprises SEQ ID NO:60.
10. The pharmaceutical composition according to claim 9 , wherein the antisense strand comprises SEQ ID NO:127, and the sense strand comprises SEQ ID NO:60.
11. The pharmaceutical composition according to claim 10 , wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides; a 2′-5′-ribonucleotide in at least one of positions 1, 5, 6, or 7; and a non-nucleotide moiety covalently attached to the 3′-terminus; and wherein the sense strand further comprises at least one 2′-5′-ribonucleotide or 2′-O-methyl modified ribonucleotide; a non-nucleotide moiety covalently attached at the 3′-terminus; and a cap moiety covalently attached at the 5′-terminus.
12. The pharmaceutical composition according to claim 10 , wherein the antisense strand further comprises 2-′O-methyl modified ribonucleotides at positions 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises five consecutive 2′-5′-ribonucleotides in the 3′-terminal positions 15, 16, 17, 18, and 19; a non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.
13. The pharmaceutical composition according to claim 10 , wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, and 19; and a C3C3 non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises a C3Pi non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.
14. The pharmaceutical composition according to claim 10 , wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises five consecutive 2′-5′-ribonucleotides in the 3′-terminal positions 15, 16, 17, 18, and 19; a C3Pi non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.
15. The pharmaceutical composition according to claim 10 , wherein the antisense strand further comprises 2-′O-methyl modified ribonucleotides in positions 1, 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 7, 13, 16 and 18; a 2′-5′-ribonucleotide at position 9; a C3 non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.
16. The pharmaceutical composition of claim 1 , wherein one strand of the double-stranded nucleic acid molecule comprises SEQ ID NO:98 and the second strand comprises SEQ ID NO:165.
17. The pharmaceutical composition according to claim 16 , wherein the sense strand comprises SEQ ID NO:98 and the antisense strand comprises SEQ ID NO:165.
18. The pharmaceutical composition according to claim 17 , wherein the sense strand further comprises 2′-5′-ribonucleotides in positions at or near the 3′-terminus; a non-nucleotide moiety covalently attached at the 3′-terminus; and a cap moiety covalently attached at the 5′-terminus; and the antisense strand further comprises a 2′-O-methyl modified ribonucleotide; a 2′-5′-ribonucleotide in at least one of positions 5, 6 or 7; and a non-nucleotide moiety covalently attached at the 3′-terminus.
19. The pharmaceutical composition according to claim 17 , wherein the sense strand further comprises 2′-5′-ribonucleotides in positions 15, 16, 17, 18, and 19; a C3-OH 3′ moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 2, 4, 6, 8, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached at the 3′-terminus.
20. The pharmaceutical composition of claim 1 , wherein one strand of the double-stranded nucleic acid molecule comprises SEQ ID NO:101 and the second strand comprises SEQ ID NO:168.
21. The pharmaceutical composition according to claim 20 , wherein the sense strand comprises SEQ ID NO:101 and the antisense strand comprises SEQ ID NO:168.
22. The pharmaceutical composition according to claim 21 , wherein the sense strand further comprises 2′-O-methyl modified pyrimidine ribonucleotides; an optional 2′-5′-ribonucleotide in one of position 9 or 10; a non-nucleotide moiety covalently attached at the 3′-terminus; and a cap moiety covalently attached at the 5′-terminus; and the wherein antisense strand further comprises a 2′-O-methyl modified ribonucleotide; a 2′-5′-ribonucleotide in at least one of positions 5, 6, or 7; and a non-nucleotide moiety covalently attached at the 3′-terminus.
23. The pharmaceutical composition according to claim 21 , wherein the sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 4, 11, 13, and 17; a 2′-5′-ribonucleotide in position 9; a C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 1, 4, 8, 11 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalently attached at the 3′-terminus.
24. The pharmaceutical composition according to claim 21 , wherein sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 4, 11, 13, and 17; a C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 1, 4, 8, 13 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalently attached at the 3′-terminus.
25. The pharmaceutical composition according to claim 21 , wherein the sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 2, 4, 11, 13, and 17; a C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 1, 4, 8, 11 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′-terminus.
26. The pharmaceutical composition according to claim 1 , wherein the double-stranded nucleic acid molecule reduces hsp47 expression in the stellate cell.