ANTAGONIST OF PCSK9

Abstract

This disclosure relates to a nucleic acid comprising a double stranded RNA molecule comprising sense and antisense strands and further comprising a single stranded DNA molecule covalently linked to the 3′ end of either the sense or antisense RNA part of the molecule wherein the double stranded inhibitory RNA targets proprotein convertase subtilisin kexin type 9 (PCSK9); pharmaceutical compositions comprising said nucleic acid molecule and methods for the treatment of diseases associated with increased levels of PCSK9, for example hypercholesterolemia and cardiovascular disease.

Claims

1. A nucleic acid molecule comprising: a first part that comprises a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand and an antisense strand of at least part of the human PCSK9 nucleotide sequence or polymorphic sequence variant thereof; and a second part that comprises a single stranded deoxyribonucleic acid (DNA) molecule, wherein the 5′ end of said single stranded DNA molecule is covalently linked to the 3′ end of the sense strand of the double stranded inhibitory RNA molecule or wherein the 5′ end of the single stranded DNA molecule is covalently linked to the 3′ of the antisense strand of the double stranded inhibitory RNA molecule, wherein said single stranded DNA molecule comprises a nucleotide sequence that is adapted over at least part of its length to anneal by complementary base pairing to a part of said single stranded DNA to form a double stranded DNA structure comprising a double stranded stem domain and a single stranded loop domain.

2. The nucleic acid molecule according to claim 1 wherein: the 5′ end of said single stranded DNA molecule is covalently linked to the 3′ end of the sense strand of the double stranded inhibitory RNA molecule; or the 5′ end of said single stranded DNA molecule is covalently linked to the 3′ end of the antisense strand of the double stranded inhibitory RNA molecule.

3. (canceled)

4. The nucleic acid molecule according to claim 1 wherein said loop portion comprises a region comprising the nucleotide sequence GNA or GNNA, wherein each N independently represents guanine (G), thymidine (T), adenine (A), or cytosine (C).

5. The nucleic acid molecule according to claim 4 wherein said loop domain comprises the nucleotide sequence GCGAAGC.

6. The nucleic acid molecule according to claim 1 wherein said single stranded DNA molecule comprises the nucleotide sequence TCACCTCATCCCGCGAAGC (SEQ ID NO: 133).

7. The nucleic acid molecule according to claim 1 wherein: said double stranded inhibitory RNA molecule is between 18 and 29 nucleotide base pairs in length, more preferably between 19 and 23 nucleotide base pairs in length; said double stranded inhibitory RNA molecule comprises or consists of between 18 and 29 contiguous nucleotides of the sense nucleotide sequence set forth in SEQ ID NO: 134; or said double stranded inhibitory RNA molecule comprises or consists of 21 contiguous nucleotide bases pairs of the sense nucleotide sequence set forth in SEQ ID NO: 134.

8-9. (canceled)

10. The nucleic acid molecule according to claim 1 wherein: said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence selected from the group consisting of: SEQ ID NO: 8, 1, 2, 3, 4, 5, 6, 7, 9 or 10; said double stranded inhibitory RNA molecule comprises an antisense nucleotide sequence selected from the group consisting of: SEQ ID NO: 18, 11, 12, 13, 14, 15, 16, 17, 19 or 20; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence selected from the group consisting of: SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 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, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and 76; or said double stranded inhibitory RNA molecule comprises an antisense nucleotide sequence selected from the group consisting of: SEQ ID NO: 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 and 132.

11-13. (canceled)

14. The nucleic acid molecule according to claim 1 wherein: said double stranded inhibitory RNA molecule comprises a sense strand comprising SEQ ID NO: 8 and an antisense strand comprising SEQ ID NO: 18; said double stranded inhibitory RNA molecule comprises a sense strand comprising SEQ ID NO: 9 and an antisense strand comprising SEQ ID NO: 19; said double stranded inhibitory RNA molecule comprises a sense strand comprising SEQ ID NO: 10 and an antisense strand comprising SEQ ID NO: 20; or said double stranded inhibitory RNA molecule comprises a sense strand comprising SEQ ID NO: 135 and an antisense strand comprising SEQ ID NO: 136.

15. The nucleic acid molecule according to claim 14 wherein: said single stranded DNA molecule is covalently linked to a sense strand comprising SEQ ID NO: 8; or single stranded DNA molecule is covalently linked to an antisense strand comprising SEQ ID NO: 18.

16-17. (canceled)

18. The nucleic acid molecule according to claim 14 wherein: said single stranded DNA molecule is covalently linked to a sense strand comprising SEQ ID NO: 9; or single stranded DNA molecule is covalently linked to an antisense strand comprising SEQ ID NO: 19.

19-20. (canceled)

21. The nucleic acid molecule according to claim 14 wherein: said single stranded DNA molecule is covalently linked to a sense strand comprising SEQ ID NO: 10; or said single stranded DNA molecule is covalently linked to an antisense strand comprising SEQ ID NO: 20.

22-23. (canceled)

24. The nucleic acid molecule according to claim 1 wherein: N-acetylgalactosamine is linked to the DNA part of said nucleic acid molecule via a terminal 3′ end of the DNA part; or N-acetylgalactosamine is linked to the either the antisense part of said inhibitory RNA or the sense part of said inhibitory RNA.

25. (canceled)

26. The nucleic acid molecule according to claim 24 wherein N-acetylgalactosamine comprises the structure: ##STR00031##

27. A pharmaceutical composition comprising at least one nucleic acid molecule according to claim 1 and a pharmaceutical carrier and/or excipient.

28. The pharmaceutical composition according to claim 27 wherein said composition comprises at least one further, different, therapeutic agent.

29. The pharmaceutical composition according to claim 28 wherein said further therapeutic agent is a statin.

30-34. (canceled)

35. A method to treat a subject that has or is predisposed to hypercholesterolemia comprising administering an effective dose of a nucleic acid according to claim 1, or a pharmaceutical composition thereof, thereby treating or preventing hypercholesterolemia or a disease associated with hypercholesterolemia.

36. The method according to claim 35 wherein the hypercholesterolemia is familial hypercholesterolemia and/or said subject is resistant to statin therapy.

37. The method according to claim 36 wherein familial hypercholesterolemia is associated with elevated levels of PCSK9 expression.

38. (canceled)

39. The method according to claim 35 wherein said disease associated with hypercholesterolemia is selected from the group consisting of: stroke prevention, hyperlipidaemia, cardiovascular disease, atherosclerosis, coronary heart disease, cerebrovascular disease, peripheral arterial disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease and non-alcoholic steatohepatitis.

Description

EXAMPLE 1

[0160] In vivo activity of GalNAc-conjugated Crook ApoB siRNA compared to control siRNA constructs. Plasma ApoB levels (micrograms/ml) from 5 mice in each treatment group, were used to calculate a mean ApoB value+/−standard error of the mean (SEM). Plasma ApoB levels after 96 hours following SC administration of GalNAc-conjugated Crook siRNA were compared to levels in mice receiving either control (i) vehicle saline, or (ii) unconjugated siRNA with Crook. Statistical analysis was applied using the two-tailed paired T test algorithm.

[0161] With reference to FIG. 1 (a), plasma ApoB levels (micrograms/ml) of mice 96 hours following treatment with GalNAc-conjugated ApoB Crook siRNA were compared with the control treatment group administered with saline. Statistical analysis was applied using the two-tailed paired T test algorithm. Results show a substantive reduction in mean plasma ApoB levels in mice treated with GalNAc-conjugated Crook siRNA, compared to control. However, it just fails significance (p=0.08), most likely due to small sample size and variation in ApoB levels between control animals.

[0162] With reference to FIG. 1 (b), plasma ApoB levels (micrograms/ml) measured 96 hours following administration of GalNAc-conjugated ApoB Crook siRNA were compared to the control group, treated with siRNA construct unconjugated (without GalNAc) ApoB Crook siRNA. Statistical analysis was applied using the two-tailed paired T test algorithm. Results show a highly significant reduction in plasma ApoB levels in this GalNAc-conjugated Crook siRNA treatment group when compared to control unconjugated siRNA with Crook (P=0.00435832).

EXAMPLE 2

[0163] FIG. 2a-c compares the relative silencing activities of 20 PCSK9 crook siRNAs in vitro. HepG2 cells were reverse transfected with a library of 20 custom crook siRNAs (10 sense siRNAs and 10 antisense siRNAs) alongside the siRNA controls using conditions identified in the assay development phase. A five-point dose range (100 nM, 25 nM, 6.25 nM, 1.56 nM and 0.39 nM) was used with four replicates per siRNA concentration.

[0164] 72 h post transfection, PCSK9 mRNA levels were quantified by duplex RT-qPCR, normalising to housekeeping reference gene GAPDH, and then to the average expression of PCSK9 across the five doses of the corresponding negative (NEG) crook siRNA control (Sense or Antisense).

[0165] Most siRNAs induce some PCSK9 mRNA decrease, however with various efficiency; see FIG. 2a-c. PCSK9 mRNA levels tend to increase at high siRNA concentrations (>6.25 nM for sense and >25 nM for antisense). The optimal concentration is 6.25 nM for sense siRNAs and 25 nM for antisense siRNAs.; see FIG. 3.

[0166] In conclusion 4 crook siRNAs have efficiency >80% (sense siRNAs PC8, PC9, PC10 and antisense siRNA PC18) at optimal concentration; see table 4 below.

TABLE-US-00006 TABLE 4 Sense and antisense pairing. The nucleic acid molecules in each row e.g., SEQ ID NO 1 and 11 are complementary and hybridise forming a double stranded RNA. The pair can either comprise a crook sequence on the sense or antisense sequence. Thus, each combination of sense and antisense forms two different nucleic acid molecules e.g., SEQ ID NO 1 and 11 wherein i) the sense sequence comprises the crook or ii) wherein the antisense sequence comprises the crook. NAME Sense antisense SEQ ID SEQ ID crook crook Sense Sequence NO Antisense Sequence NO PC01 PC11 5′- 1 5′- 11 CCUCAUAGGCCUGGAGU AUAAACUCCAGGCCUAUG UUAU-3′ AGG-3′ PC02 PC12 5′- 2 5′- 12 AGGCCUGGAGUUUAUUC UUCCGAAUAAACUCCAGG GGAA-3′ CCU-3′ PC03 PC13 5′- 3 5′- 13 CCCUCAUAGGCCUGGAG UAAACUCCAGGCCUAUGA UUUA-3′ GGG-3′ PC04 PC14 5′- 4 5′- 14 ACCCUCAUAGGCCUGGA AAACUCCAGGCCUAUGAG GUUU-3′ GGU-3′ PC05 PC15 5′- 5 5′- 15 UAGGCCUGGAGUUUAUU UCCGAAUAAACUCCAGGC CGGA-3′ CUA-3′ PC06 PC16 5′- 6 5′- 16 AGGUCUGGAAUGCAAAG UUGACUUUGCAUUCCAGA UCAA-3′ CCU-3′ PC07 PC17 5′- 7 5′- 17 GGCCUGGAGUUUAUUCG UUUCCGAAUAAACUCCAG GAAA-3′ GCC-3′ PC08 PC18 5′- 8 5′- 18 CAGGUCUGGAAUGCAAA UGACUUUGCAUUCCAGAC GUCA-3′* CUG-3′ PC09 PC19 5′- 9 5′- 19 CCUCACCAAGAUCCUGC ACAUGCAGGAUCUUGGUG AUGU-3′ AGG-3′ PC10 PC20 5′- 10 5′- 20 CACCAGCAUACAGAGUG UGGUCACUCUGUAUGCUG ACCA-3′ GUG-3′ PC21 PC77 5′- 21 5′- 77 AGCAAGCAGACAUUUAU AAAGAUAAAUGUCUGCUU CUUU-3′ GCU-3′ PC22 PC78 5′- 22 5′- 78 AGGUCUGGAAUGCAAAG UUGACUUUGCAUUCCAGA UCAA-3′ CCU-3′ PC23 PC79 5′- 23 5′- 79 GGCCUGGAGUUUAUUCG UUUCCGAAUAAACUCCAG GAAA-3′ GCC-3′ PC24 PC80 5′- 24 5′- 80 CAGGUCUGGAAUGCAAA UGACUUUGCAUUCCAGAC GUCA-3′ CUG-3′ PC25 PC81 5′- 25 5′- 81 CCCAAGCAAGCAGACAU AUAAAUGUCUGCUUGCUU UUAU-3′ GGG-3′ PC26 PC82 5′- 26 5′- 82 CCUCACCAAGAUCCUGC ACAUGCAGGAUCUUGGUG AUGU-3′ AGG-3′ PC27 PC83 5′- 27 5′- 83 UUUUCUAGACCUGUUUU AAGCAAAACAGGUCUAGA GCUU-3′ AAA-3′ PC28 PC84 5′- 28 5′- 84 ACCCAAGCAAGCAGACA UAAAUGUCUGCUUGCUUG UUUA-3′ GGU-3′ PC29 PC85 5′- 29 5′- 85 CACCAGCAUACAGAGUG UGGUCACUCUGUAUGCUG ACCA-3′ GUG-3′ PC30 PC86 5′- 30 5′- 86 AUUCUGGGUUUUGUAGC AAAUGCUACAAAACCCAG AUUU-3′ AAU-3′ PC31 PC87 5′- 31 5′- 87 AUCUCCUAGACACCAGC GUAUGCUGGUGUCUAGGA AUAC-3′ GAU-3′ PC32 PC88 5′- 32 5′- 88 UCCUAGACACCAGCAUA UCUGUAUGCUGGUGUCUA CAGA-3′ GGA-3′ PC33 PC89 5′- 33 5′- 89 GACAUUUAUCUUUUGGG CAGACCCAAAAGAUAAAU UCUG-3′ GUC-3′ PC34 PC90 5′- 34 5′- 90 UAUUCUGGGUUUUGUAG AAUGCUACAAAACCCAGA CAUU-3′ AUA-3′ PC35 PC91 5′- 35 5′- 91 CUGGAGUUUAUUCGGAA GCUUUUCCGAAUAAACUC AAGC-3′ CAG-3′ PC36 PC92 5′- 36 5′- 92 GCCUGGAGUUUAUUCGG UUUUCCGAAUAAACUCCA AAAA-3′ GGC-3′ PC37 PC93 5′- 37 5′- 93 GAGGCAGAGACUGAUCC AAGUGGAUCAGUCUCUGC ACUU-3′ CUC-3′ PC38 PC94 5′- 38 5′- 94 AAGCAAGCAGACAUUUA AAGAUAAAUGUCUGCUUG UCUU-3′ CUU-3′ PC39 PC95 5′- 39 5′- 95 UAGACCUGUUUUGCUUU UACAAAAGCAAAACAGGU UGUA-3′ CUA-3′ PC40 PC96 5′- 40 5′- 96 UUUGCUUUUGUAACUUG UCUUCAAGUUACAAAAGC AAGA-3′ AAA-3′ PC41 PC97 5′- 41 5′- 97 CACUUCUCUGCCAAAGA GACAUCUUUGGCAGAGAA UGUC-3′ GUG-3′ PC42 PC98 5′- 42 5′- 98 UUGCUUUUGUAACUUGA AUCUUCAAGUUACAAAAG AGAU-3′ CAA-3′ PC43 PC99 5′- 43 5′- 99 AUGCAAAGUCAAGGAGC CCAUGCUCCUUGACUUUG AUGG-3′ CAU-3′ PC44 PC100 5′- 44 5′- 100 CCCACCCAAGCAAGCAG AUGUCUGCUUGCUUGGGU ACAU-3′ GGG-3′ PC45 PC101 5′- 45 5′- 101 GGGUAACAGUGAGGCUG UUCCCAGCCUCACUGUUA GGAA-3′ CCC-3′ PC46 PC102 5′- 46 5′- 102 GGUCAUGGUCACCGACU UCGAAGUCGGUGACCAUG UCGA-3′ ACC-3′ PC47 PC103 5′- 47 5′- 103 GGCAGCUGUUUUGCAGG CAGUCCUGCAAAACAGCU ACUG-3′ GCC-3′ PC48 PC104 5′- 48 5′- 104 GGGCAGGUUGGCAGCUG AAAACAGCUGCCAACCUG UUUU-3′ CCC-3′ PC49 PC105 5′- 49 5′- 105 UUGAAGAUAUUUAUUCU ACCCAGAAUAAAUAUCUU GGGU-3′ CAA-3′ PC50 PC106 5′- 50 5′- 106 UGGCAGCUGUUUUGCAG AGUCCUGCAAAACAGCUG GACU-3′ CCA-3′ PC51 PC107 5′- 51 5′- 107 CCGGGGAUACCUCACCA AUCUUGGUGAGGUAUCCC AGAU-3′ CGG-3′ PC52 PC108 5′- 52 5′- 108 ACUGAUCCACUUCUCUG UUGGCAGAGAAGUGGAUC CCAA-3′ AGU-3′ PC53 PC109 5′- 53 5′- 109 AUCCACUUCUCUGCCAA AUCUUUGGCAGAGAAGUG AGAU-3′ GAU-3′ PC54 PC110 5′- 54 5′- 110 ACUUCUCUGCCAAAGAU UGACAUCUUUGGCAGAGA GUCA-3′ AGU-3′ PC55 PC111 5′- 55 5′- 111 GUCUGGAAUGCAAAGUC CCUUGACUUUGCAUUCCA AAGG-3′ GAC-3′ PC56 PC112 5′- 56 5′- 112 CUUCUCUGCCAAAGAUG AUGACAUCUUUGGCAGAG UCAU-3′ AAG-3′ PC57 PC113 5′- 57 5′- 113 GAGUUGAGGCAGAGACU GAUCAGUCUCUGCCUCAA GAUC-3′ CUC-3′ PC58 PC114 5′- 58 5′- 114 GACCUGUUUUGCUUUUG GUUACAAAAGCAAAACAG UAAC-3′ GUC-3′ PC59 PC115 5′- 59 5′- 115 CGGGGAUACCUCACCAA GAUCUUGGUGAGGUAUCC GAUC-3′ CCG-3′ PC60 PC116 5′- 60 5′- 116 UUUCUAGACCUGUUUUG AAAGCAAAACAGGUCUAG CUUU-3′ AAA-3′ PC61 PC117 5′- 61 5′- 117 GGUCUGGAAUGCAAAGU CUUGACUUUGCAUUCCAG CAAG-3′ ACC-3′ PC62 PC118 5′- 62 5′- 118 UAUCUCCUAGACACCAG UAUGCUGGUGUCUAGGAG CAUA-3′ AUA-3′ PC63 PC119 5′- 63 5′- 119 AGGUUGGCAGCUGUUUU CUGCAAAACAGCUGCCAA GCAG-3′ CCU-3′ PC64 PC120 5′- 64 5′- 120 AACUUUUCUAGACCUGU CAAAACAGGUCUAGAAAA UUUG-3′ GUU-3′ PC65 PC121 5′- 65 5′- 121 CUUUUCUAGACCUGUUU AGCAAAACAGGUCUAGAA UGCU-3′ AAG-3′ PC66 PC122 5′- 66 5′- 122 UCCACUUCUCUGCCAAA CAUCUUUGGCAGAGAAGU GAUG-3′ GGA-3′ PC67 PC123 5′- 67 5′- 123 UGGAGUUUAUUCGGAAA GGCUUUUCCGAAUAAACU AGCC-3′ CCA-3′ PC68 PC124 5′- 68 5′- 124 GGCAGGUUGGCAGCUGU CAAAACAGCUGCCAACCU UUUG-3′ GCC-3′ PC69 PC125 5′- 69 5′- 125 UGGAGGUGUAUCUCCUA UGUCUAGGAGAUACACCU GACA-3′ CCA-3′ PC70 PC126 5′- 70 5′- 126 GUCAUCAAUGAGGCCUG GAACCAGGCCUCAUUGAU GUUC-3′ GAC-3′ PC71 PC127 5′- 71 5′- 127 UUCUAGACCUGUUUUGC AAAAGCAAAACAGGUCUA UUUU-3′ GAA-3′ PC72 PC128 5′- 72 5′- 128 UUCUGGGUUUUGUAGCA AAAAUGCUACAAAACCCA UUUU-3′ GAA-3′ PC73 PC129 5′- 73 5′- 129 GAGACUGAUCCACUUCU GCAGAGAAGUGGAUCAGU CUGC-3′ CUC-3′ PC74 PC130 5′- 74 5′- 130 AGUCAAGGAGCAUGGAA GGGAUUCCAUGCUCCUUG UCCC-3′ ACU-3′ PC75 PC131 5′- 75 5′- 131 AUCUUUUGGGUCUGUCC GAGAGGACAGACCCAAAA UCUC-3′ GAU-3′ PC76 PC132 5′- 76 5′- 132 CACCCAAGCAAGCAGAC AAAUGUCUGCUUGCUUGG AUUU-3′ GUG-3′

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