Allele-Specific Silencing of Transforming Growth Factor Beta Induced Gene with R124H Mutation Using Short Interfering RNA
The present disclosure relates to a ribonucleic acid (RNA) complex and methods of use thereof for preventing, amelio-rating or treating symptoms associated with granular corneal dystrophy type 2 in a subject.
1 . A ribonucleic acid (RNA) complex comprising a strand that comprises a sequence having at least 80% identity to one of SEQ ID NOs: 1-19.
2 . The RNA complex of claim 1 , wherein the strand comprises a sequence having at least 80% identity to SEQ ID NO: 4, 11 or 16.
3 . The RNA complex of claim 2 , wherein the strand includes at least one of: an overhang at the 5′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16 or an overhang at the 3′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16.
4 . The RNA complex of claim 3 , wherein the strand includes at least one of: cytidine monophosphate (rC) at the 5′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16, uridine monophosphate-cytidine monophosphate (rU-rC) at the 5′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16, double uridine monophosphate (rU-rU) at the 3′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16, double deoxythymidine monophosphates (dT-dT) at the 3′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16, double 2′-O-methylated guanosine monophosphates (oG-oG) at the 3′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16, and double 2′-O-methyl-uridine monophosphates (oU-oU) at the 3′ end of the sequence having at least 80% identity to SEQ ID NO: 4, 11, or 16.
5 . The RNA complex of claim 2 , further comprising a strand that comprises a sequence having at least 80% identity to SEQ ID NO: 23, 30 or 35.
6 . The RNA complex of claim 2 , further comprising a strand that comprises a sequence of SEQ ID NO: 23, 30 or 35.
7 . The RNA complex of claim 1 , wherein the strand comprises a sequence of SEQ ID NO: 4, 11, or 16.
8 . The RNA complex of claim 1 , wherein the RNA complex comprises a first strand and a second strand, each comprising at least one TGFBI R124H mutation site compared to a wild-type 124C TGFBI gene.
9 . The RNA complex of claim 8 , wherein the first strand comprises a sequence that is identical to the sequence of SEQ ID NO: 4, 9 or 11, except for one base that is mismatched to a juxtaposed base in the second strand.
10 . The RNA complex of claim 9 , wherein the mismatched base is between three and seven bases away from the TGFBI R124H mutation site.
11 . The RNA complex of claim 8 , wherein the first strand and the second strand are each between 16 and 23 bases in length.
12 . The RNA complex of claim 8 , wherein the RNA complex has deoxythymidine overhangs.
13 . A ribonucleic acid (RNA) complex comprising a strand that comprises a sequence having at least 80% identity to one of SEQ ID NOs: 20-38.
14 . The RNA complex of claim 13 , wherein the strand comprises a sequence having at least 80% identity to SEQ ID NO: 23, 30 or 35.
15 . The RNA complex of claim 13 , wherein the strand includes at least one of: an overhang at the 5′ end of the sequence having at least 80% identity to SEQ ID NO: 23, 30 or 35 or an overhang at the 3′ end of the sequence having at least 80% identity to SEQ ID NO: 23, 30 or 35.
16 . A ribonucleic acid (RNA) complex comprising a strand having a sequence overlapping with a sequence of a transforming growth factor beta induced (TGFBI) protein messenger RNA (mRNA), the strand containing adenine at a position corresponding to the c. 371G>A single nucleotide polymorphism (SNP) in exon 4 of the TGFBI gene.
17 . A ribonucleic acid (RNA) complex comprising a sense strand and an antisense strand, wherein at least one of the sense strand and the antisense strand comprises a dTdT overhang.
18 . The RNA complex of claim 17 , wherein the sense strand and the antisense strand each comprises a dTdT overhang.
19 . The RNA complex of claim 17 , wherein the sense strand comprises GG nucleotides as an overhang and the antisense strand comprises UC nucleotides as an overhang.
20 . The RNA complex of claim 17 , wherein
(a) the sense strand comprises a series of repeating 2′-OMe; and (b) the antisense strand comprises 2′-OMe;
(a) the sense strand comprises 15 bases and alternating patterns of 2′-OMe and 2′-F; and
(b) the antisense strand comprises alternating patterns of 2′-OMe and 2′-F, wherein the RNA complex comprises additional phosphorothioate links on the 3′ and 5′ ends of both the sense strand and the antisense strand; or
(a) the sense strand comprises two units of 2′-OMe at the 5′ end and at least two 2′-OMe modifications at either U or G residues other than at position 9; and (b) the antisense strand comprises a single 2′-OMe at position 2 from the 5′ end, PS bonds in a dTdT overhand, and all pyrimidines replaced with 2′F-RNA units.
21 . The RNA complex of claim 1 , wherein the RNA complex includes a short interfering RNA duplex; a double stranded RNA complex configured for forming a short interfering RNA duplex; and/or an RNA hairpin.
22 . A method of preventing, ameliorating, or treating granular corneal dystrophy type 2 in a subject, the method comprising:
administering to the subject the RNA complex of claim 1 .
23 . The method of claim 22 , wherein the administering comprises introducing the RNA complex into a cell containing and expressing a deoxyribonucleic acid (DNA) molecule having the c. 371G>A single nucleotide polymorphism (SNP) in exon 4 of a transforming growth factor beta induced (TGFBI) gene.
24 . The method of claim 22 , further comprising:
prior to administering to the subject the RNA complex:
obtaining sequence information of the subject; and
determining that the subject has an allele having the c. 371G>A SNP in exon 4 of the TGFBI gene and an allele that does not have the c. 371G>A SNP in exon 4 of the TGFBI gene.