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 hydrophobically modified oligonucleotide cargo, and uses thereof, are also provided.
1 . A method of loading exosomes with a therapeutically effective amount of an oligonucleotide cargo, said method comprising:
providing oligonucleotides comprising one or more hydrophobic modifications at a concentration of 1.25 μM to 20 μM, wherein the one or more hydrophobic modifications comprise a hydrophobic moiety conjugated to the oligonucleotide;
wherein the hydrophobic moiety is selected from the group consisting of a sterol, GM1, a lipid, a vitamin, a small molecule, a peptide, or a combination thereof; and
incubating the oligonucleotides comprising one or more hydrophobic modifications with a population of exosomes, wherein the method takes place in the absence of ultracentrifugation, electroporation and a separately added transfection reagent; thereby loading the exosomes with a therapeutically effective amount of an oligonucleotide cargo;
wherein the therapeutically effective amount is an average of 1000-3000 oligonucleotide molecules per exosome.
2 . The method of claim 1 , wherein the oligonucleotide is selected from the group consisting of siRNA, siRNA-GalNAc, antisense RNA, LNA, hairpin siRNA, PMO, miRNA, miRNA inhibitor and combinations thereof.
3 . The method of claim 2 , wherein the oligonucleotide is siRNA or miRNA.
4 . The method of claim 1 , consisting essentially of incubating the oligonucleotides with a population of exosomes.
5 . The method of claim 1 , wherein loading of the exosomes with the therapeutically effective amount of the oligonucleotide cargo occurs in 30 minutes or less.
6 . The method of claim 1 , wherein at least 90% of the exosomes in the population of exosomes are loaded with the oligonucleotide cargo.
7 . The method of claim 1 , wherein the hydrophobic moiety is a sterol.
8 . The method of claim 7 , wherein the hydrophobic moiety is cholesterol.
9 . The method of claim 1 , wherein the hydrophobic moiety is a vitamin.
10 . The method of claim 9 , wherein the hydrophobic moiety is Vitamin E.
11 . The method of claim 1 , wherein the one or more hydrophobic modifications further comprise a backbone modification, a sugar modification, a base modification, or combinations thereof.
12 . The method of claim 11 , wherein the one or more hydrophobic modifications comprise a backbone modification.
13 . The method of claim 12 , wherein the backbone modification is selected from the group consisting of phosphorothioate modifications, phosphorodithioate modifications, p-ethoxy modifications, methylphosphonate modifications, methylphosphorothioate modifications, alkyl- and aryl-phosphate modifications, alkylphosphotriester modifications, peptide nucleic acid (PNA) modifications, and locked nucleic acid (LNA) modifications.
14 . The method of claim 11 , wherein the one or more hydrophobic modifications comprise a sugar modification.
15 . The method of claim 14 , wherein the sugar is ribose, and wherein the sugar modification comprises 2′O-Methyl, 2′Methoxy-ethyl, 2′Fluor, or 2′FANA.
16 . The method of claim 11 , wherein the one or more hydrophobic modifications comprise a base modification.
17 . The method of claim 16 , wherein the base modification is a phenyl modification, a naphthyl modification, or an isobutyl modification.
18 . The method of claim 1 , wherein the exosomes are derived from cultured cells.
19 . The method of claim 18 , 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 or tumor cells.
20 . The method of claim 1 , wherein the exosomes are synthetic exosomes.
21 . The method of claim 1 , wherein the exosomes comprise a targeting peptide.
22 . The method of claim 21 , wherein the targeting peptide directs the exosomes to neuronal cells.
23 . The method of claim 1 , wherein the method takes place at a temperature of about 4° C. to about 37° C.
24 . The method of claim 1 , further comprising purifying the exosomes loaded with the oligonucleotide cargo from unloaded oligonucleotides.