Therapeutic method for huntington's disease
The present invention relates to anti-gene oligonucleotides adapted to hybridize to DNA in a HTT gene, which are based on locked nucleic acids, phosphorodiamidate morpholino oligomers, (PMO) or equivalent oligonucleotide analogues comprising a (CAG) n sequence, and whose target is a sequence where the majority of the repeats are CAG/CTG, for use in down regulating the expression of the HTT gene, resulting in reduced HTT mRNA and protein levels in afflicted subjects, or in diagnosis, treatment and/or prevention of Huntington's disease, and where the anti-gene oligonucleotides target non-canonical DNA structures, including hairpin and cruciform. The invention also relates to a delivery system comprising said oligonucleotides and said use thereof.
1. One or more anti-gene oligonucleotides adapted to hybridize to DNA in an HTT gene, which are based on locked nucleic acids, phosphorodiamidate morpholino oligomers (PMO) or equivalent oligonucleotide analogues comprising a (CAG) n sequence, and whose target is a sequence where the majority of the repeats are CAG/CTG, and where the first complete repeat is optionally preceded by a G/G or an AG/TG, and/or where the last repeat is optionally followed by a C/C or a CA/CT, wherein n is between 3 and 200, for use in treatment and/or prevention of Huntington's disease, or for use in diagnosis of Huntington's disease and where the anti-gene oligonucleotides target non-canonical DNA structures, including hairpin and cruciform.
2. One or more anti-gene oligonucleotides according to claim 1 , for use in prevention of Huntington's disease, where prevention is related to blocking the CAG/CTG repeat expansion.
3. One or more anti-gene oligonucleotides according to claim 1 , for use in down regulating the expression of an HTT gene.
4. One or more anti-gene oligonucleotides according to claim 1 , for use in down regulating HTT mRNA and protein levels in afflicted subjects.
5. One or more anti-gene oligonucleotides according to claim 1 , in combination with other Huntington's disease therapy, selected from the group comprising siRNAs, splice-switching oligonucleotides, single nucleotide polymorphism (SNP) targeting oligonucleotides, zinc finger nucleases and antisense oligonucleotides working via either RNase-mediated degradation or steric blocking of the HTT mRNA.
6. One or more anti-gene oligonucleotides according to claim 1 , wherein n is 12, 13, 14, 15, 16, 18 or 19.
7. One or more anti-gene oligonucleotides according to claim 1 , wherein the oligonucleotides comprise a phosphodiester (PO) or a phosphorothioate (PS) backbone.
8. One or more anti-gene oligonucleotides according to claim 1 , wherein the oligonucleotide is based on locked nucleic acids, phosphorodiamidate morpholino oligomers, lipid-modified forms of LNA or related oligonucleotide analogues selected from the group comprising 2′,4′-constrained oligonucleotides and oligonucleotides with modifications in their 2′ or 5 positions.
9. One or more anti-gene oligonucleotides according to claim 8 , wherein the oligonucleotides are selected from one or more of ethyl bicyclic nucleic acids, 2′-O-methyl, 2′-aminoglycyl, piperazino-modified 2′-aminonucleotides and 5-propargylamino-LNA, aminated 2′-amino-LNA nucleotides.
10. One or more anti-gene oligonucleotides according to claim 1 , wherein the oligonucleotides are selected from the group comprising 2′-O-methyl RNA/LNA, and 2′-glycylamino 5-methylcytosine LNA.
11. One or more anti-gene oligonucleotides according to claim 10 , wherein the oligonucleotides are selected from the group comprising CAG-14PS-OMe (2′-O-methyl) and CAG-14PS-gly (2′-aminoglycyl).
12. One or more anti-gene oligonucleotides according to claim 1 , wherein the oligonucleotides are based on 2′-aminoglycyl modified nucleotide, such as modified A, G, C or T.
13. One or more anti-gene oligonucleotides according to claim 1 , wherein the oligonucleotides are selected from the group comprising CAG-10PS, CAG-12PS and CAG-12PO, CAG-13PS and CAG-13PO, CAG-14PS and CAG-14PO, CAG-14PS-OMe, CAG-14PS-gly, CAG-15PS, CAG-16PS, CAG-18PS and CAG-19PS.
14. A delivery system for administration of one or more oligonucleotides, comprising
a) one or more of oligonucleotides adapted to hybridize to DNA in an HTT gene, which are based on locked nucleic acids, phosphorodiamidate morpholino oligomers (PMO) or equivalent oligonucleotide analogues comprising a (CAG) n sequence, and whose target is a sequence where the majority of the repeats are CAG/CTG, and where the first complete repeat is optionally preceded by a G/G or an AG/TG, and/or where the last repeat is optionally followed by a C/C or a CA/CT, and wherein n is between 3 and 200,
b) a liquid in which the one or more of oligonucleotides are dissolved, and optionally
c) a cell penetrating peptide/protein, such as PepFect 14 (PF14), or
d) a cationic lipid, or
e) a nuclear localization signal peptide, or
f) an aptamer facilitating targeting and uptake, optionally together with a pharmaceutically acceptable carrier, adjuvant and/or excipient.
15. A delivery system according to claim 14 , comprising
a cell penetrating peptide/protein, such as PepFect 14 (PF14, optionally together with a pharmaceutically acceptable carrier, adjuvant and/or excipient, wherein the one or more of oligonucleotides comprise a phosphodiester (PO) backbone.
16. A combination of any one or more of the oligonucleotides according to claim 1 , together with other Huntington's disease therapies, such as siRNAs, splice-switching oligonucleotides, single nucleotide polymorphism (SNP) targeting oligonucleotides, zinc finger nucleases and antisense oligonucleotides working via either RNase-mediated degradation or steric blocking of the HTT mRNA.
17. The delivery system according to claim 14 for use in down regulating the expression of a HTT gene, or in targeting non-canonical DNA structures at the CAG/CTG repeat sequence, including hairpin and cruciform, or for use in down regulating HTT mRNA and protein levels in afflicted subjects, or in diagnosis treatment and/or prevention of HD in afflicted subjects or subjects at risk of being afflicted.
18. A method for diagnosis of Huntington's disease, whereby the method comprises the steps of
1) isolating genomic DNA from a biological sample, and optionally cleaving the isolated DNA,
2) adding any one or more anti-gene oligonucleotides adapted to hybridize to DNA in an HTT gene, which are based on locked nucleic acids, phosphorodiamidate morpholino oligomers (PMO) or equivalent oligonucleotide analogues comprising a (CAG) n sequence, and whose target is a sequence where the majority of the repeats are CAG/CTG, and where the first complete repeat is optionally preceded by a G/G or an AG/TG, and/or where the last repeat is optionally followed by a C/C or a CA/CT, and wherein n is between 3 and 200, and
3) determining length and sequence and/or number of CAG/CTG repeats in the HTT gene.
19. The method according to claim 18 , whereby cleaving is done using DNA restriction enzymes, CRISPR-Cas9 based DNA cleavage or by fragmentation using ultra sonication.