Exosomal loading using hydrophobically modified oligonucleotides
In one aspect, the invention relates to a method of loading exosomes with oligonucleotide cargo, by incubating an oligonucleotide comprising one or more hydrophobic modifications with a population of exosomes for a period of time sufficient to allow loading of the exosomes with the oligonucleotide. Exosomes loaded with hydrophobic ally modified oligonucleotide cargo, and uses thereof, are also provided.
1. A method of loading exosomes with a therapeutically effective amount of a hydrophobically modified siRNA cargo by self-assembly, comprising:
providing a hydrophobically modified siRNA at a concentration of 1.25 μM to 20 μM, wherein the hydrophobically modified siRNA is conjugated to a hydrophobic moiety selected from the group consisting of a sterol, GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof, and wherein 65% or more of the nucleotides in the hydrophobically modified siRNA comprise a phosphorothioate modification, a 2′O-Methyl modification, a 2′-Fluoro modification, or a combination thereof, and
incubating the hydrophobically modified siRNA with a population of exosomes, wherein the method takes place at about 4° C.−37° C., without ultracentrifugation, in the absence of electroporation and in the absence of a separately added transfection reagent, thereby loading the exosomes with a therapeutically effective amount of the hydrophobically modified siRNA, wherein the therapeutically effective amount is 1000-3000 molecules of siRNA per exosome.
2. The method of claim 1 , consisting essentially of incubating the hydrophobically modified siRNA with a population of exosomes.
3. The method of claim 1 , wherein loading of the exosomes with the hydrophobically modified siRNA occurs in 30 minutes or less.
4. The method of claim 1 , wherein at least 90% of the exosomes are loaded with the hydrophobically modified siRNA.
5. The method of claim 1 , wherein the hydrophobically modified siRNA has an increased hydrophobicity of at least 2 orders of magnitude relative to unmodified siRNA.
6. The method of claim 1 , wherein at least 70% of the nucleotides in the siRNA are hydrophobically modified.
7. The method of claim 1 , wherein 100% of the nucleotides in the siRNA are hydrophobically modified.
8. The method of claim 1 , wherein the hydrophobic moiety is a sterol.
9. The method of claim 1 , wherein the hydrophobic moiety is a vitamin.
10. The method of claim 8 , wherein the hydrophobic moiety is cholesterol.
11. The method of claim 9 , wherein the hydrophobic moiety is Vitamin E.
12. The method of claim 1 , wherein the exosomes are derived from cultured cells.
13. The method of claim 12 , wherein the exosomes are derived from dendritic cells (DC), immature dendritic cells, B cells, T cells, mast cells, epithelial cells, stem cells, induced pluripotent stem cells (iPS cells), neuronal cells, and tumor cells.
14. The method of claim 1 , wherein the exosomes are synthetic exosomes.
15. The method of claim 1 , wherein the exosomes comprise a targeting peptide.
16. The method of claim 15 , wherein the targeting peptide directs the exosomes to neuronal cells.
17. The method of claim 1 , further comprising purifying the exosomes loaded with the hydrophobically modified siRNA from unloaded siRNA.
18. The method of claim 17 , wherein the exosomes are purified after loading using ultracentrifugation.
19. The method of claim 1 , wherein the hydrophobically modified siRNA directs the silencing of a mutant huntingtin gene or a mutant SOD1 gene.
20. The method of claim 1 , wherein the hydrophobically modified oligonucleotide contains a detectable label.
21. The method of claim 20 , wherein the detectable label is a fluorescent label or a radioactive label.
22. The method of claim 1 , wherein the hydrophobically modified siRNA is provided at a concentration of 5 μM.
23. The method of claim 1 , wherein the hydrophobically modified siRNA is provided at a concentration of 10 μM.
24. The method of claim 1 , wherein the hydrophobically modified siRNA is provided at a concentration of 20 μM.
25. The method of claim 1 , wherein the siRNA contains a duplex region of 19 base pairs.
26. The method of claim 1 , wherein the siRNA contains a duplex region of 14-16 base pairs.
27. The method of claim 26 , wherein the siRNA contains a duplex region of 15 base pairs.
28. The method of claim 1 , wherein the method takes place at about 4° C.
29. The method of claim 1 , wherein the method takes place at about 37° C.
30. The method of claim 1 , wherein the method takes place at room temperature.
31. A composition comprising exosomes loaded with hydrophobically modified siRNA, produced by a method according to claim 1 .
32. The method of claim 1 , wherein the hydrophobically modified siRNA is provided at a concentration of 1.25 μM to 5 μM.
33. The method of claim 1 , wherein the hydrophobically modified siRNA is provided at a concentration of 1.25 μM to 10 μM.
34. A method of loading exosomes with a therapeutically effective amount of hydrophobically modified siRNA cargo by self-assembly, comprising:
providing a hydrophobically modified siRNA at a concentration of 1.25 μM to 20 μM, wherein the hydrophobically modified siRNA is conjugated to cholesterol or Vitamin E, and wherein 65% or more of the nucleotides in the hydrophobically modified siRNA comprise a phosphorothioate modification, a 2′-O-Methyl modification, a 2′-Fluoro modification, or a combination thereof, and
incubating the hydrophobically modified siRNA with a population of exosomes, wherein the method takes place at about 4° C. to 37° C. without ultracentrifugation, in the absence of electroporation and in the absence of a separately added transfection reagent, thereby loading the exosomes with a therapeutically effective amount of the hydrophobically modified siRNA.
35. The method of claim 34 , wherein the hydrophobically modified siRNA is provided at a concentration of 5 μM.
36. The method of claim 34 , wherein the hydrophobically modified siRNA is provided at a concentration of 10 μM.
37. The method of claim 34 , wherein the hydrophobically modified siRNA is provided at a concentration of 20 μM.
38. The method of claim 34 , wherein the therapeutically effective amount is 1000-3000 oligonucleotides per exosome.
39. The method of claim 34 , wherein the exosomes are purified after loading.
40. The method of claim 39 , wherein the exosomes are purified after loading using ultracentrifugation.
41. The method of claim 34 , wherein the hydrophobically modified siRNA is provided at a concentration of 1.25 μM to 5 μM.
42. The method of claim 34 , wherein the hydrophobically modified siRNA is provided at a concentration of 1.25 μM to 10 μM.