Targeted delivery of nucleic acids
Aspects of the invention provide compositions and methods for delivering nucleic acids to target cells.
1. A composition comprising:
a polypeptide comprising a first homing domain and a second cell-penetrating domain, wherein the second cell-penetrating domain is N-terminal to the first homing domain and the N-terminus of the polypeptide is modified with a lipophilic group, and
a nucleic acid, wherein the nucleic acid is non-covalently associated with the second cell-penetrating domain, and
wherein the second cell-penetrating domain has two or more basic amino acids, and wherein the number of basic amino acids within the second cell penetrating domain is below a threshold level of amino acids above which the polypeptide targets cells non-specifically.
2. The composition of claim 1 , wherein the nucleic acid is an siRNA, and wherein the polypeptide assembles into a light-scattering complex upon complexation with the siRNA molecule.
3. The composition of claim 2 , wherein the complex is not immunostimulatory.
4. The composition of claim 2 , wherein the average diameter of the complex is between 10-1000 nm, 50-800 nm, 100-700 nm, or 200-600 nm.
5. The composition of claim 2 , wherein the complex is renally excreted.
6. The composition of claim 2 , wherein the number of basic amino acids within the second cell penetrating domain is less than 8, less than 7, less than 6, less than 5, or less than 4.
7. The composition of claim 2 , wherein the charge ratio between the nucleic acid and the second cell-penetrating domain is approximately 2-3:1.
8. The composition of claim 1 , wherein the first homing domain is a cyclic homing peptide.
9. The composition of claim 1 , wherein the homing domain has tumor penetration properties.
10. The composition of claim 9 , wherein the homing domain comprises a penetration motif R/KXXR/K, wherein R is arginine, K is lysine and X is any amino acid.
11. The composition of claim 8 , wherein the cyclic homing peptide is Lyp1 or iRGD.
12. The composition of claim 1 , wherein the second cell-penetrating domain is 6R or transportan (TP).
13. The composition of claim 1 , wherein the lipophilic group is myristic acid.
14. A polypeptide comprising:
a first homing domain, and
a second domain comprising a nucleic acid binding motif wherein the second domain is N-terminal to the first homing domain, and wherein the second domain has less than 25% basic amino acids, has protein transduction function, and is non-covalently associated with a nucleic acid and,
wherein a saturated C12-C18 fatty acid is attached to the N-terminus of the second domain.
15. A complex comprising the polypeptide of claim 14 wherein the nucleic acid is associated with the polypeptide with dissociation constant in the range of 1-100 nM, or 1-10 nM.
16. The polypeptide of claim 14 ,
wherein the first homing domain facilitates destabilization of a cell membrane or the membrane of a cell organelle and allows entry into cytosol.
17. The polypeptide of claim 14 , wherein the polypeptide is a non self-assembling polypeptide that assembles with identical polypeptides into light-scattering complexes upon complexation with siRNA molecules.
18. The polypeptide of claim 14 , wherein upon complexation with nucleic acid molecules and other identical polypeptides, the polypeptides display the first homing domains in a multivalent fashion, thereby increasing binding affinity.
19. The polypeptide of claim 14 , wherein the polypeptide is renally excreted.
20. The composition of claim 2 , wherein the second domain comprises at least 10% basic amino acids.
21. The composition of claim 2 , wherein the second domain comprises at least 2-4 basic amino acids.
22. The polypeptide of claim 14 , wherein the nucleic acid binding motif is about 10-50 or about 10, 20, 30 or 40 amino acids long.
23. The polypeptide of claim 14 , wherein the first homing domain binds a cell-type specific cell-surface moiety.
24. The polypeptide of claim 14 , wherein the first homing domain comprises an antibody, antigen, ligand, receptor, cytokine, lymphokine, hormone, growth factor or fragment thereof.
25. The composition of claim 2 , wherein the lipophilic group is a saturated fatty acid.
26. The composition of claim 2 , wherein the first homing domain comprises Lyp-1.
27. The composition of claim 2 , wherein the second domain is selected from the group consisting of: transportan, viral protein- 22 (VP- 22 ) and penetratin or a portion or a modified variant thereof.
28. The composition of claim 2 , wherein the two domains are connected by a synthetic or peptide linker.
29. A method of delivering a nucleic acid, the method comprising:
administering to a subject a nucleic acid in association with a polypeptide of claim 14 .
30. The method of claim 29 , wherein the nucleic acid is administered in conjunction with one or more iRGD peptides.
31. The polypeptide of claim 14 , wherein the nucleic acid binding motif is a siRNA binding motif.