Method for delivering agents into cells using bacterial toxins

1. A method for delivering a bioactive moiety comprising at least one non-natural component into a cell cytosol of an eukaryotic cell comprising contacting the cell with (a) a fusion molecule comprising the bioactive moiety attached to an A component of a bacterial toxin, a functional wild-type or modified fragment thereof, or an A component surrogate or mimetic; and (b) a corresponding B component of the bacterial toxin or a functional fragment thereof, wherein the non-natural component is selected from the group consisting of a peptide or protein comprising one or more D-amino acids.

2. The method of claim 1 , wherein the A component surrogate is selected from N-terminal unstructured, highly charged segments of bacterial toxin A parts that are 10-35 amino acid residues long wherein the amino acids alternate between positively and negatively charged residues, and wherein the negatively charged amino acids are selected from E and D, or D-amino acid isoforms of the same, and the positively charged amino acids are selected from K, R, and H, or D-amino acid isoforms thereof.

3. The method of claim 1 , wherein the A component of a bacterial toxin, a functional wild-type or modified fragment thereof is selected from amino acid sequences of SEQ ID NO: 14; SSv2; SEQ ID NO: 15; and SSv4.

4. The method of claim 3 , wherein the B component is an anthrax protective antigen (PA).

5. The method of claim 1 further comprising a step of attaching the bioactive moiety to the A component of a bacterial toxin or the functional wild-type or modified fragment thereof to form the fusion molecule.

6. The method of claim 5 wherein the fusion molecule further comprises a protease cleavage sequence between the bioactive moiety and the A component of a bacterial toxin or functional wild-type or modified fragment thereof.

7. The method of claim 5 further comprising a step of introducing a protease cleavage sequence into the fusion molecule to allow protease mediated release of the bioactive moiety from the A component after its entry into the cytosol.

8. The method of claim 7 , wherein the protease cleavage sequence is selected from a calpain, a caspase, and a cathepsin cleavage sites.

9. The method of claim 8 , wherein the protease cleavage sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:3.

10. The method of claim 1 , wherein the A and B components are selected from the family of intraceullarly acting toxins.

11. The method of claim 10 , wherein the A and B components are selected from botulinum neurotoxin, anthrax toxin, diphtheria toxin, shiga toxin, shiga like toxin, exotoxin A, tetanus toxin and cholera toxin.

12. The method of claim 1 , wherein the bioactive moiety is attached to the C-terminus or N-terminus of the A component of the bacterial toxin or a functional wild-type or modified fragment thereof.

13. The method of claim 1 , wherein the B component is separate from the A component.

14. The method of claim 1 , wherein the B component is joined to the A component.

15. The method of claim 1 , wherein the attaching is performed using native ligation or sortase mediated protein ligation.

16. The method of claim 1 , wherein the contacting is performed in vitro.

17. The method of claim 1 , wherein the bioactive moiety comprises an isostere, an N-methyl amide, a circular peptide, an ether peptide, a hydrocarbon linker, a conformation locked peptide, a stapled peptide, a cyclic protein, or a peptidomimetic.

18. The method of claim 1 , wherein the bioactive moiety is a D-peptide or a D-protein.

19. The method of claim 1 , wherein the bioactive moiety is not a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), or a natural peptide or protein containing only L-amino acids.

20. A method for delivering a bioactive moiety comprising at least one non-natural component into a cell cytosol of an eukaryotic cell comprising contacting the cell with (a) a fusion molecule comprising the bioactive moiety attached to an A component surrogate of a bacterial toxin; and (b) a corresponding B component of the bacterial toxin or a functional fragment thereof, wherein the A component surrogate is selected from N-terminal unstructured, highly charged segments of bacterial toxin A parts that are 10-35 amino acid residues long wherein the amino acids alternate between positively and negatively charged residues, and wherein the negatively charged amino acids are selected from E and D, or D-amino acid isoforms of the same, and the positively charged amino acids are selected from K, R, and H, or D-amino acid isoforms thereof.

21. The method of claim 20 , wherein the A component surrogate is selected from amino acid sequences of SEQ ID NO: 15 and SSv4.

22. The method of claim 21 , wherein the B component is an anthrax protective antigen (PA).

23. The method of claim 20 further comprising a step of attaching the bioactive moiety to the A component surrogate to form the fusion molecule.

24. The method of claim 23 wherein the fusion molecule further comprises a protease cleavage sequence between the bioactive moiety and the A component surrogate.

25. The method of claim 23 further comprising a step of introducing a protease cleavage sequence into the fusion molecule to allow protease mediated release of the bioactive moiety from the A component surrogate after its entry into the cytosol.

26. The method of claim 25 , wherein the protease cleavage sequence is selected from a calpain, a caspase, and a cathepsin cleavage sites.

27. The method of claim 26 , wherein the protease cleavage sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

28. The method of claim 20 , wherein the bacterial toxin is selected from the family of intraceullarly acting toxins.

29. The method of claim 28 , wherein the bacterial toxin is selected from botulinum neurotoxin, anthrax toxin, diphtheria toxin, shiga toxin, shiga like toxin, exotoxin A, tetanus toxin and cholera toxin.

30. The method of claim 20 , wherein the bioactive moiety is attached to the C-terminus or N-terminus of the A component surrogate.

31. The method of claim 20 , wherein the B component is separate from the A component surrogate.

32. The method of claim 20 , wherein the B component is joined to the A component surrogate.

33. The method of claim 20 , wherein the attaching is performed using native ligation or sortase mediated protein ligation.

34. The method of claim 20 , wherein the contacting is performed in vitro.

35. The method of claim 20 , wherein the non-natural component is selected from the group consisting of a peptide or protein comprising one or more D-amino acids; a peptide or protein comprising one or more N-methyl amino acids; a peptide or protein comprising one or more homo amino acids; a cyclic peptide; a peptide or protein comprising one or more side-chain modified amino acids containing groups composed of fluorine, bromine, iodine, biotin, azide, alkene, alkyne, glycan, lipid, phosphate, polyethylene glycol, thiol, thioester, keto acid, samarium, lanthanum, terbium, and various fluorophores.

36. The method of claim 20 , wherein the bioactive moiety comprises an isostere, an N-methyl amide, a circular peptide, an ether peptide, a hydrocarbon linker, a conformation locked peptide, a stapled peptide, a cyclic protein, or a peptidomimetic.

37. The method of claim 20 , wherein the bioactive moiety is a D-peptide or a D-protein.

38. The method of claim 20 , wherein the bioactive moiety is not a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), or a natural peptide or protein containing only L-amino acids.

39. A method for delivering a bioactive moiety comprising at least one non-natural component into a cell cytosol of an eukaryotic cell comprising contacting the cell with (a) a fusion molecule comprising the bioactive moiety attached to an A component of a bacterial toxin, a functional wild-type or modified fragment thereof, or an A component surrogate or mimetic; and (b) a corresponding B component of the bacterial toxin or a functional fragment thereof, wherein the bioactive moiety does not comprise a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), or a natural peptide or protein containing only L-amino acids.