Neurotoxins with enhanced target specificity
Modified neurotoxins that contain protease cleavage sites susceptible uniquely to proteases present in certain tissues are described. The toxins can be selectively activated by proteases in muscle or selectively inactivated by proteases in blood.
1. A nucleic acid molecule which comprises a nucleotide sequence encoding a modified botulinum A neurotoxin comprising:
a) a binding domain comprising a first heavy chain portion able to interact with a surface receptor present in a target cell, wherein said first heavy chain portion comprises a mammalian blood protease cleavage site, said mammalian blood protease cleavage site comprising a modification in one or more regions selected from the group consisting of amino acids 930-935 of SEQ ID NO: 1, amino acids 1060-1063 of SEQ ID NO: 1, amino acids 1136-1144 of SEQ ID NO: 1 and amino acids 1165-1171 of SEQ ID NO: 1;
b) a translocation domain comprising a second heavy chain portion able to mediate the escape of a light chain portion of said neurotoxin from an endosome to the cytoplasm of said target cell; and
c) an enzymatic domain comprising said light chain portion able to cleave a SNARE-protein present in said target cell;
wherein said modified botulinum A neurotoxin is able to interact with a surface receptor of said target cell, able to mediate endosomal escape into the cytoplasm of said target cell and able to cleave a SNARE protein present in said target cell; and
whereupon said modified botulinum A neurotoxin's ability to interact with a surface receptor of said target cell is inactivated upon cleavage of said mammalian blood protease cleavage site.
2. A nucleic acid molecule which comprises a nucleotide sequence encoding a modified botulinum A neurotoxin comprising:
a) a binding domain comprising a first heavy chain portion able to interact with a surface receptor present in a target cell, wherein said first heavy chain portion comprises a mammalian blood protease cleavage site, said mammalian blood protease cleavage site comprising a modification in one or more regions selected from the group consisting of amino acids 930-935 of SEQ ID NO: 1, amino acids 1060-1063 of SEQ ID NO: 1, amino acids 1136-1145 of SEQ ID NO: 1 and amino acids 1165-1171 of SEQ ID NO: 1;
b) a translocation domain comprising a second heavy chain portion able to mediate the escape of a light chain portion of said neurotoxin from an endosome to the cytoplasm of said target cell; and
c) an enzymatic domain comprising said light chain portion able to cleave a SNARE-protein present in said target cell, wherein said light chain portion comprises a mammalian muscle protease cleavage site, said mammalian muscle protease cleavage site comprising a modification in amino acids 430-452 of SEQ ID NO: 1;
wherein said modified botulinum A neurotoxin is able to interact with a surface receptor of said target cell, able to mediate endosomal escape into the cytoplasm of said target cell and able to cleave a SNARE protein present in said target cell; and
whereupon said modified botulinum A neurotoxin's ability to interact with a surface receptor of said target cell is inactivated upon cleavage of said mammalian blood protease cleavage site; and
whereupon said modified botulinum A neurotoxin's ability to cleave a SNARE protein present in a target cell is activated upon cleavage of said mammalian muscle protease cleavage site.
3. The nucleic acid molecule according to either claim 1 or claim 2 , wherein said mammalian blood protease cleavage site is cleaved by a mammalian blood protease selected from the group consisting of Thrombin, Coagulation Factor VIIa, Coagulation Factor IXa, Coagulation Factor Xa, Coagulation Factor XIa, Coagulation Factor XIIa, Kallikrein, Protein C and MBP-associated serine protease.
4. The nucleic acid molecule according to either claim 1 or claim 2 , wherein said modification of amino acids 930-935 of SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
5. The nucleic acid molecule according to either claim 1 or claim 2 , wherein said modification of amino acids 1060-1063 of SEQ ID NO: 1 is SEQ ID NO: 7.
6. The nucleic acid molecule according to either claim 1 or claim 2 , wherein said modification of amino acids 1136-1145 of SEQ ID NO: 1 is SEQ ID NO: 9.
7. The nucleic acid molecule according to either claim 1 or claim 2 , wherein said modification of amino acids 1165-1171 of SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
8. An expression system which comprises the nucleotide sequence of according to claim 1 operably linked to sequences for effecting its expression.
9. A recombinant host cell modified to contain the expression system of claim 8 .
10. A method to produce a modified botulinum A neurotoxin, the method comprising the steps of culturing the host cell of claim 9 under conditions wherein said modified botulinum A neurotoxin is produced and optionally recovering the modified botulinum A neurotoxin from the culture.
11. A nucleic acid molecule which comprises a nucleotide sequence encoding a modified botulinum A neurotoxin comprising a blood protease cleavage site, wherein said blood protease cleavage site comprises a modification in one or more regions selected from the group consisting of amino acids 930-935 of SEQ ID NO: 1, amino acids 1060-1063 of SEQ ID NO: 1, amino acids 1136-1145 of SEQ ID NO: 1 and amino acids 1165-1171 of SEQ ID NO: 1.
12. A nucleic acid molecule which comprises a nucleotide sequence encoding a modified botulinum A neurotoxin comprising a blood protease cleavage site and a muscle protease cleavage site,
wherein said blood protease cleavage site comprises a modification in one or more regions selected from the group consisting of amino acids 930-935 of SEQ ID NO: 1, amino acids 1060-1063 of SEQ ID NO: 1, amino acids 1136-1145 of SEQ ID NO: 1 and amino acids 1165-1171 of SEQ ID NO: 1; and
wherein said muscle protease cleavage site comprises a modification in amino acids 430-452 of SEQ ID NO: 1.
13. The nucleic acid molecule according to either claim 11 or claim 12 , wherein said mammalian blood protease cleavage site is cleaved by a mammalian blood protease selected from the group consisting of Thrombin, Coagulation Factor VIIa, Coagulation Factor IXa, Coagulation Factor Xa, Coagulation Factor XIa, Coagulation Factor XIIa, Kallikrein, Protein C and MBP-associated serine protease.
14. The nucleic acid molecule according to either claim 11 or claim 12 , wherein said modification of amino acids 930-935 of SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
15. The nucleic acid molecule according to either claim 11 or claim 12 , wherein said modification of amino acids 1060-1063 of SEQ ID NO: 1 is SEQ ID NO: 7.
16. The nucleic acid molecule according to either claim 11 or claim 12 , wherein said modification of amino acids 1136-1145 of SEQ ID NO: 1 is SEQ ID NO: 9.
17. The nucleic acid molecule according to either claim 11 or claim 12 , wherein said modification of amino acids 1165-1171 of SEQ ID NO: 1 is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.
18. An expression system which comprises the nucleotide sequence of according to claim 11 operably linked to sequences for effecting its expression.
19. A recombinant host cell modified to contain the expression system of claim 18 .
20. A method to produce a modified botulinum A neurotoxin, the method comprising the steps of culturing the host cell of claim 19 under conditions wherein said modified botulinum A neurotoxin is produced and optionally recovering the modified botulinum A neurotoxin from the culture.