Extracellular vesicle linked to a biologically active molecule via an optimized linker and an anchoring moiety

A method of attaching a biologically active molecule (BAM) to an EV, comprising linking an anchoring moiety (AM) to the EV, wherein the anchoring moiety (AM) is attached to the biologically active moiety (BAM) according to the formula:

• [AM]-L1-[SP1]-L2-[SP2]-L3-[BAM]
• wherein:
• [AM] is the anchoring moiety;
• L1 is a cleavable or non-cleavable linkage;
• L2 and L3 are optional cleavable or non-cleavable linkages;
• SP1 is an optional first spacer;
• and,
• SP2 is an optional second spacer.

A method of increasing the load density of a biologically active molecule (BAM) attached to an EV, comprising screening a library of anchoring moieties (AM) attached to the biologically active moiety (BAM) according to the formula:

• [AM]-L1-[SP1]-L2-[SP2]-L3-[BAM]
• wherein:
• [AM] is the anchoring moiety;
• L1 is a cleavable or non-cleavable linkage;
• L2 and L3 are optional cleavable or non-cleavable linkages;
• SP1 is an optional first spacer;
• and,
• SP2 is an optional second spacer.

An extracellular vesicle (EV) comprising an antisense oligonucleotide [ASO] covalently linked to the EV via an anchoring moiety [AM] according to the formula:

• [AM]-L1-[SP1]-L2-[SP2]-L3-[ASO]
• wherein:
• [AM] is the anchoring moiety selected from the group consisting of cholesterol-C6, cholesterol-TEG, tocopherol-C8, tocopherol, and palmitate-C6;
• L1 is a phosphodiesterase cleavable linkage;
• SP1 is an optional first spacer selected from the group consisting of C3, C6, TEG and HEG;
• L2 is optional phosphorothioate non-cleavable linkage;
• SP2 is an optional second spacer selected from the group consisting of C3, C6, TEG, and HEG;
• and,
• L3 is an optional phosphorothioate non-cleavable linkage.

An exosome comprising an antisense oligonucleotide [ASO] covalently linked to the exosome via an anchoring moiety [AM] according to the formula:

• [AM]-L1-[SP1]-L2-[SP2]-L3-[BAM]
• wherein [AM] is cholesterol-TEG;
• L1 is a phosphodiesterase cleavable bond;
• SP1 is C3;
• L2 is a phosphorothioate non-cleavable bond;
• SP2 is TEG;
• and, L3 is a phosphorothioate non-cleavable bond.

An exosome comprising an antisense oligonucleotide [ASO] covalently linked to the exosome via an anchoring moiety [AM] according to the formula:

• [AM]-L1-[SP1]-L2-[BAM]
• wherein [AM] is cholesterol-TEG;
• L1 is a phosphodiesterase cleavable bond;
• SP1 is TEG;
• and L2 is a phosphorothioate non-cleavable bond.