Compositions and methods for the delivery of molecules into live cells
The present disclosure provides methods and compositions related to the cytosolic delivery of proteins and cell-impermeable small molecules into live cells using an endosomolytic dimer of cell-penetrating peptide TAT.
1. A compound having the formula:
wherein
X is a linking moiety,
Y is an amino acid residue covalently coupled to a hydrophobic moiety,
Z is a cell-penetrating peptide (CPP) moiety, wherein the CPP moiety has a net positive charge and has an amino acid sequence with 50% or more residues having a guanidinium group, and
m and n are independently 0 or 1.
2. The compound of claim 1 , wherein the CPP moiety comprises between 3 and 30 amino acids.
3. The compound of claim 2 , wherein the CPP moiety comprises an amino acid sequence with at least 85% identity to the sequence set forth in SEQ ID NO:1.
4. The compound of claim 3 , wherein the CPP moiety comprises the amino acid sequence set forth in SEQ ID NO:1.
5. The compound of claim 1 , wherein at least one of m and n is 1.
6. The compound of claim 5 , wherein the hydrophobic moiety comprises a C 6 -C 30 straight chain, branched, or cyclic group.
7. The compound of claim 6 , wherein the group includes one or more heteroatoms selected from N, O, and S.
8. The compound of claim 6 , wherein the cyclic group is mono-, bi-, or tricyclic.
9. The compound of claim 6 , wherein the hydrophobic moiety comprises a rhodamine group.
10. The compound of claim 9 , wherein the rhodamine group is tetramethylrhodamine (TMR).
11. The compound of claim 5 , wherein the amino acid residue is lysine (K).
12. The compound of claim 1 , wherein m and n are 0, and wherein Z 1 and/or Z 2 is a CPP that comprises an amino acid residue linked to a hydrophobic moiety.
13. The compound of claim 12 , wherein the amino acid residue is the N-terminal most lysine (K) residue in the CPP amino acid sequence.
14. The compound of claim 1 , wherein X is a cysteine (C) residue.
15. The compound of claim 14 , wherein X 1 and X 2 are cysteine (C) residues linked by a disulfide bond.
16. The compound of claim 1 , wherein X, Y, and/or Z are linked by amide bonds.
17. The compound of claim 1 , wherein the CPP of Z 1 and the CPP of Z 2 have amino acid sequences having at least 85% identity to each other.
18. The compound of claim 1 , wherein the combination of X—Y—Z comprises no more than 30 amino acid residues.
19. The compound of claim 1 , wherein the compound is capable of facilitating endosomal lysis.
20. A method of delivering a cell-impermeable molecule to the cytosol of a cell, the method comprising contacting a cell with a compound of claim 1 and a cell-impermeable molecule under conditions sufficient to permit endocytosis.
21. The method of claim 20 , wherein the cell is contacted with a concentration of the compound of at least 1 μM.
22. The method of claim 20 , wherein the cell-impermeable molecule is a peptide, a polymer including polypeptides and nucleic acids, or a small-molecule pharmaceutical.
23. The method of claim 22 , wherein the polypeptide is a transcription factor.
24. The method of claim 20 , wherein the cell-impermeable molecule is not covalently linked to the compound.
25. The method of claim 20 , wherein the cell-impermeable molecule has a net positive charge.
26. The method of claim 20 , wherein the cell is obtained from a living subject and is contacted with the compound and cell-impermeable molecule ex vivo.
27. The method of claim 20 , wherein the cell is in a living subject and is contacted with the compound and cell-impermeable molecule in vivo.
28. The method of claim 27 , wherein the cell is contacted in vivo by administering an amount of the compound to the subject effective to deliver a cell-impermeable molecule to the cytosol of the cell.
29. The method of claim 20 , wherein the cell is in or is obtained from a mammalian subject.
30. A method of inducing pluripotency in a cell ex vivo, the method comprising contacting a cell obtained from a subject with the compound of claim 1 and a transcription factor under conditions sufficient to allow endocytosis, and culturing the cell to obtain pluripotency of the cell.
31. A method of expanding a stem cell ex vivo, the method comprising contacting a stem cell with the compound of claim 1 and a transcription factor under conditions sufficient to allow endocytosis, and culturing the cell to obtain expansion of the cell.