RNAi Inhibition of Serum Amyloid A For Treatment of Glaucoma

1 . A method of attenuating expression of serum amyloid A mRNA in an eye of a subject, comprising:

administering by ocular injection to the eye of the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising:

a sense nucleotide sequence, an antisense nucleotide sequence, and a region of at least near-perfect contiguous complementarity of at least 19 nucleotides between the sense and antisense sequences;

wherein the antisense sequence hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively,

wherein the expression of serum amyloid A mRNA is thereby attenuated.

2 . The method of claim 1 , wherein the subject has glaucoma.

3 . The method of claim 1 , wherein the subject is at risk of developing glaucoma.

4 . The method of claim 1 , wherein the antisense sequence has a region of at least near-perfect contiguous complementarity of at least 21 to 23 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO: 1 and comprises an additional TT sequence at the 3′ end of each of the sense and the antisense sequence.

5 . The method of claim 1 , wherein the sense nucleotide sequence and the antisense nucleotide sequence are connected by a loop nucleotide sequence.

6 . The method of claim 1 , wherein the composition is administered via a topical, intravitreal, or transcleral route.

7 . The method of claim 1 , wherein the antisense sequence is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:1 beginning at nucleotide 230, 357, 362, 380, 447, 470, 527, 531, 548, or 557.

8 . The method of claim 1 wherein the antisense sequence is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 43, 170, 175, 193, 260, 283, 339, or 370.

9 . The method of claim 1 wherein the antisense sequence is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 252, 271, 276, 325, 343.

10 . The method of claim 1 wherein the antisense sequence is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:3 beginning at nucleotide 153, 166, 222, 227, 251, 268, 297, 335, 356, 384, 390, 396, 406, or 423.

11 . The method of claim 1 wherein the antisense sequence comprises

CUUUGCCACUCCUGCCCCA,

(SEQ ID NO: 37)

UCGGAAGUGAUUGGGGUCU,

(SEQ ID NO: 38)

UUUGUCUGAGCCGAUGUAA,

(SEQ ID NO: 39)

AACCAGGCCCGUGAGAAGC,

(SEQ ID NO: 40)

CUGAGCCGAUGUAAUUGGC,

(SEQ ID NO: 41)

GCCACUCCUGCCCCAUUUA,

(SEQ ID NO: 69)

CCCCCGAGCAUGGAAGUAU,

(SEQ ID NO: 42)

CUCUGGCAUUGCUGAUCAC,

(SEQ ID NO: 43)

GCCUGUGAGUCUCUGGAUA,

(SEQ ID NO: 44)

GCCACUCCUGCCCCAUUUA,

(SEQ ID NO: 45)

GCCAGCAGGUCGGAAGUGA,

(SEQ ID NO: 46)

AGUCUCUGGAUAUUCUCUC,

(SEQ ID NO: 47)

UUUAUUGGCAGCCUGAUCG,

(SEQ ID NO: 48)

UUGCUGAUCACUUCUGCGG,

(SEQ ID NO: 49)

CUGGAUAUUCUCUCUGGCA,

(SEQ ID NO: 50)

UCUGCCACUCCUGCCCCAU,

(SEQ ID NO: 51)

AACCCCUUGGAGAGCCUCC,

(SEQ ID NO: 52)

UGCCCAUGUCCCCAACCCC,

(SEQ ID NO: 53)

AUAGAGAUAUCUGUUUGAA,

(SEQ ID NO: 54)

CGAGCAUAGAGAUAUCUGU,

(SEQ ID NO: 55)

CUUUGGGCAGCAUCAUAGU,

(SEQ ID NO: 56)

AGACACCCCCAGGUCCUCU,

(SEQ ID NO: 57)

CCUGGAACGGCUGAUGAGU,

(SEQ ID NO: 58)

CCAAAUAAAUAGUAGUCUA,

(SEQ ID NO: 59)

UCCAAUACAGUGCUGCUGU,

(SEQ ID NO: 60)

CUCAGCUUUCUCGUUGGAC,

(SEQ ID NO: 61)

CCAUUCCUCAGCUUUCUCG,

(SEQ ID NO: 62)

CCGGCCCCAUUCCUCAGCU,

(SEQ ID NO: 63)

CUUUGCCACUCCGGCCCCA,

(SEQ ID NO: 64)

or

UCUGAAGCGGUCGGGGUCU.

(SEQ ID NO: 65)

12 . The method of claim 1 , wherein the interfering RNA comprises a modification on a base portion, on a sugar portion or on a phosphate portion.

13 . The method of claim 1 , further comprising administering to the eye of the subject a second interfering RNA having a length of 19 to 49 nucleotides, and comprising

a sense nucleotide sequence, an antisense nucleotide sequence, and a region of at least near-perfect complementarity of at least 19 nucleotides between the sense and antisense sequences;

wherein the antisense sequence of the second interfering RNA hybridizes under physiological conditions to a second portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 and the antisense sequence has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the second hybridizing portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively.

14 . The method of claim 1 , wherein the composition comprises an effective amount of a mixture of at least four interfering RNAs, each interfering RNA having a length of 19 to 49 nucleotides, and a pharmaceutically acceptable carrier, each interfering ENA comprising:

a sense nucleotide sequence, an antisense nucleotide sequence, and a region of at least near-perfect contiguous complementarity of at least 19 nucleotides between the sense and antisense sequences of each of the four interfering RNAs;

wherein the antisense sequences of the mixture hybridize under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 175, 252, 276, and 325, respectively, and have a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 175, 252, 276, and 325, respectively.

15 . A method of treating a serum amyloid A-associated glaucoma in a subject in need thereof, comprising:

administering by ocular injection to the eye of the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising:

a nucleotide sequence having a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with a hybridizing portion of mRNA corresponding to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3,

wherein the expression of serum amyloid A mRNA is thereby attenuated.

16 . The method of claim 15 wherein the composition is administered via a topical, intravitreal, or transcleral route.

17 . The method of claim 15 wherein the interfering RNA is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:1 beginning at nucleotide 230, 357, 362, 380, 447, 470, 527, 531, 548, or 557.

18 . The method of claim 15 wherein the interfering RNA is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 43, 170, 175, 193, 260, 283, 339, or 370.

19 . The method of claim 15 wherein the interfering RNA is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 252, 271, 276, 325, 343.

20 . The method of claim 15 wherein the interfering RNA is designed to target a nucleotide sequence of mRNA corresponding to SEQ ID NO:3 beginning at nucleotide 153, 166, 222, 227, 251, 268, 297, 335, 356, 384, 390, 396, 406, or 423.

21 . The method of claim 15 further comprising administering to the eye of the subject a second interfering RNA having a length of 19 to 49 nucleotides, and comprising

a second nucleotide sequence having a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with a second hybridizing portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

22 . The method of claim 15 wherein the composition comprises an effective amount of a mixture of at least four interfering RNAs, each interfering RNA having a length of 19 to 49 nucleotides, and the mixture comprising:

a first, second, third and fourth nucleotide sequence having a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 175, 252, 276, and 325, respectively.