Nucleic acids, peptides and methods
Disclosed herein are novel nucleic acid molecules which encode novel polypeptides for use in treating polyglutamine diseases such as Huntington's disease. The polypeptides of the invention exhibit reduced immunotoxicity in vivo and may have utility in sustained (mid- or long-term) expression in vivo. Also disclosed are methods for the treatment of polyglutamine diseases, such as Huntington's disease and the use of nucleic acids and polypeptides of the invention in medical therapy. Gene therapy methods and compositions, such as AAV vectors for use in gene therapy methods are also disclosed.
1. An isolated nucleic acid encoding a polypeptide, the polypeptide comprising a zinc finger peptide having 8, 9, 10, 11 or 12 zinc finger domains and which binds to a CAG- and/or a CTG-trinucleotide repeat sequence; wherein the zinc finger peptide comprising the sequence:
N′-[(Formula 4)-L 3 ] n0 -{[(Formula 6)-L 2 -(Formula 6)-L 3 ] n1 -[(Formula 6)-L 2 -(Formula 6)-X L ]} n2 -[(Formula 4)-L 2 -(Formula 6)-L 3 ] n3 -[(Formula 6)-L 2 -(Formula 6)]-C′,
wherein n0, n1, n2 and n3 are integers representing the number of times that the corresponding sequence within square brackets is repeated, and wherein n0 is 0 or 1, n1 is 1 or 2, n2 is 1, and n3 is 2, L 2 is -TGEKP-(SEQ ID NO: 6), L 3 is selected from the group consisting of -TGSERP-(SEQ ID NO: 10) and -TGSQKP-(SEQ ID NO: 16), and X L is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 21, 22, 23 and 24;
Formula 4 is a zinc finger domain of the sequence X 2 C X 2 or 4 C X 5 X −1 X +1 X +2 X +3 X +4 X +5 X +6 H X 3 or 4 or 5 H/C and Formula 6 is a zinc finger domain of the sequence X 2 C X 2 C X 5 X −1 X +1 X +2 X +3 X +4 X +5 X +6 H X 3 H, wherein X is any amino acid unless otherwise defined, the numbers in subscript indicate the numbers of residues represented by X at that position, and the number in superscript indicates the position of the amino acid in the recognition sequence of the zinc finger domain;
and wherein:
(i) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including both members of a group consisting of the amino acid sequences of SEQ ID NOs: 2 and 5; or
(ii) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including both members of a group consisting of the amino acid sequences of SEQ ID NOs: 3 and 4; or
(iii) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including all members of a group consisting of the amino acid sequences of SEQ ID NOs: 2, 3 and 4.
2. The nucleic acid according to claim 1 , wherein the zinc finger peptide has 11 zinc finger domains and wherein the recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 include both the amino acid sequences of SEQ ID NOs: 2 and 5; or wherein the recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 include both the amino acid sequences of SEQ ID NOs: 3 and 4.
3. The nucleic acid according to claim 1 , wherein L 3 is -TGSQKP-(SEQ ID NO: 16).
4. The nucleic acid according to claim 1 , wherein all of the zinc finger domains are defined according to Formula 6.
5. The nucleic acid according to claim 1 , wherein the polypeptide comprises the human Kruppel-associated box (KRAB) repressor domain from Kox-1 according to the amino acid sequence of SEQ ID NO: 39, or the mouse KRAB repressor domain from ZF87 according to the amino acid sequence of SEQ ID NO: 40.
6. The nucleic acid according to claim 1 , wherein the polypeptide comprises the nuclear localisation signal sequence according to the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.
7. The nucleic acid according to claim 1 , wherein the zinc finger peptide has a sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 29, 33 and 35.
8. The nucleic acid according to claim 1 , wherein the polypeptide comprises a sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 51, 52, 54, 55 and 56.
9. The nucleic acid according to claim 1 , wherein the polypeptide comprising a zinc finger peptide having 10, 11 or 12 zinc finger domains.
10. An adeno-associated virus (AAV) vector comprising a nucleic acid expression construct capable of expressing a polypeptide, the polypeptide comprising a zinc finger peptide having 8, 9, 10, 11, or 12 zinc finger domains and which binds to a CAG- and/or CTG-trinucleotide repeat sequence; wherein the zinc finger peptide comprising the sequence:
N′-[(Formula 4)-L 3 ] n0 -{[(Formula 6)-L 2 -(Formula 6)-L 3 ] n1 -[(Formula 6)-L 2 -(Formula 6)-X L ]} n2 -[(Formula 4)-L 2 -(Formula 6)-L 3 ] n3 -[(Formula 6)-L 2 -(Formula 6)]-C′,
wherein n0, n1, n2 and n3 are integers representing the number of times that the corresponding sequence within square brackets is repeated, and wherein n0 is 0 or 1, n1 is 1 or 2, n2 is 1, and n3 is 2, L 2 is -TGEKP-(SEQ ID NO: 6), L 3 is selected from the group consisting of -TGSERP-(SEQ ID NO: 10) and -TGSQKP-(SEQ ID NO: 16), and X L is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 21, 22, 23 and 24;
Formula 4 is a zinc finger domain of the sequence X 2 C X 2 or 4 C X 5 X −1 X +1 X +2 X +3 X +4 X +5 X +6 H X 3 or 4 or 5 H/C and Formula 6 is a zinc finger domain of the sequence X 2 C X 2 C X 5 X −1 X +1 X +2 X +3 X +4 X +5 X +6 H X 3 H, wherein X is any amino acid unless otherwise defined, the numbers in subscript indicate the numbers of residues represented by X at that position, and the number in superscript indicates the position of the amino acid in the recognition sequence of the zinc finger domain;
and wherein:
(i) the zinc finger domains the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including both members of a group consisting of the amino acid sequences of SEQ ID NOs: 2 and 5;
(ii) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including both members of a group consisting of the amino acid sequences of SEQ ID NOs: 3 and 4; or
(iii) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including all members of a group consisting of the amino acid sequences of SEQ ID NOs: 2, 3 and 4.
11. The adeno-associated virus (AAV) vector according to claim 10 , wherein the zinc finger peptide has 11 zinc finger domains and wherein the recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 include both the amino acid sequences of SEQ ID NOs: 2 and 5; or wherein the recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 include both the amino acid sequences of SEQ ID NOs: 3 and 4; and wherein the polypeptide comprises the human KRAB repressor domain from Kox-1 according to the amino acid sequence of SEQ ID NO: 39, or the mouse KRAB repressor domain from ZF87 according to the amino acid sequence of SEQ ID NO: 40 arranged C-terminal to the zinc finger peptide.
12. The adeno-associated virus (AAV) vector according to claim 10 , wherein the zinc finger peptide has a sequence selected from the group consisting of the amino acid sequences of SEQ ID Nos: 29, 33 and 35.
13. The adeno-associated virus (AAV) according to claim 10 , wherein the polypeptide comprises a sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 51, 52, 54, 55 and 56.
14. The AAV vector according to claim 10 , the polypeptide comprising a zinc finger peptide having 10, 11, or 12 zinc finger domains.
15. A polypeptide comprising a zinc finger peptide, the zinc finger peptide having 8, 9, 10, 11 or 12 zinc finger domains and which binds to CAG- and/or CTG-trinucleotide repeat sequence; wherein the zinc finger peptide comprising the sequence:
N′-[(Formula 4)-L 3 ] n0 -{[(Formula 6)-L 2 -(Formula 6)-L 3 ] n1 -[(Formula 6)-L 2 -(Formula 6)-X L ]} n2 -[(Formula 4)-L 2 -(Formula 6)-L 3 ] n3 -[(Formula 6)-L 2 -(Formula 6)]-C′,
wherein n0, n1, n2 and n3 are integers representing the number of times that the corresponding sequence within square brackets is repeated, and wherein n0 is 0 or 1, n1 is 1 or 2, n2 is 1, n3 is 2, L 2 is -TGEKP-(SEQ ID NO: 6), L 3 is selected from the group consisting of -TGSERP-(SEQ ID NO: 10) and -TGSQKP-(SEQ ID NO: 16), and X L is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 21, 22, 23 and 24;
Formula 4 is a zinc finger domain of the sequence X 2 C X 2 or 4 C X 5 X −1 X +1 X +2 X +3 X +4 X +5 X +6 H X 3 or 4 or 5 H/C and Formula 6 is a zinc finger domain of the sequence X 2 C X 2 C X 5 X −1 X +1 X +2 X +3 X +4 X +5 X +6 H X 3 H, wherein X is any amino acid unless otherwise defined, the numbers in subscript indicate the numbers of residues represented by X at that position, and the number in superscript indicates the position of the amino acid in the recognition sequence of the zinc finger domain;
and wherein:
(i) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including both members of a group consisting of the amino acid sequences of SEQ ID NOs: 2 and 5; or
(ii) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including both members of a group consisting of the amino acid sequences of SEQ ID NOs: 3 and 4; or
(iii) the zinc finger domains of the zinc finger peptide collectively have recognition sequences X −1 X +1 X +2 X +3 X +4 X +5 X +6 selected from and including all members of a group consisting of the amino acid sequences of SEQ ID NOs: 2, 3 and 4.
16. The polypeptide according to claim 15 , wherein the zinc finger peptide has 11 zinc finger domains, wherein L 3 is -TGSQKP-(SEQ ID NO: 16); and wherein X L is the amino acid sequence of SEQ ID NO: 24.
17. The polypeptide according to claim 15 which comprises a peptide sequence having at least 95% identity to a peptide sequence selected from any of the amino acid sequences of SEQ ID NOs: 33, 35, 49, 50, 51, 52, 54, 55 and 56.
18. The polypeptide according to claim 15 , wherein the zinc finger peptide has 10, 11 or 12 zinc finger domains.