Programmable Designer Therapeutic Fusogenic Secreted Gectosome Vesicles For Macromolecule Delivery And Genome Modification

A method of selectively delivering a target ligand to a recipient cell comprising the steps of:

• transfecting a donor cell to heterologously express a two component delivery system comprising:
• a protein capable of being incorporated into the membrane of an extracellular vesicle (EV) coupled with a first component of a split complement system and optionally configured to be coupled with at least one target ligand;
• a second component of said split complement system configured to be coupled with at least one target ligand;
• optionally a protein, or protein fragment configured to increase delivery of said target ligand;
• anchoring said at least one target ligand to a membrane capable of forming an EV by reconstituting said split complement system;
• and
• encapsulating said target ligand and said reconstituted split complement system in an EV formed from said donor cell.

A method of transiently or stably transfecting a recipient cell through a programmable extracellular vesicle, comprising the steps of:

• transfecting a donor cell to heterologously express a two component delivery system comprising:
• a viral fusion protein G from Vesicular Stomatitis Virus (VSV-G) incorporated into the membrane of an extracellular vesicle (EV) coupled with a first component of a GFP split complement system and optionally configured to be coupled with at least one target ligand;
• a second component of said GFP split complement system configured to be coupled with at least one target ligand;
• a protein or protein fragment having a Gag peptide motif;
• anchoring the at least one target ligand to a membrane capable of forming an EV by reconstituting said split complement system;
• and
• forming one or more EVs from said donor cell encapsulating said at least one target ligand and said reconstituted split complement system.

A method of selectively delivering a target ligand to a recipient cell comprising the steps of:

• transfecting a donor cell to heterologously express a protein capable of being incorporated into the membrane of an extracellular vesicle (EV) and further being configured to be coupled with at least one target ligand;
• and
• optionally a protein, or protein fragment configured to increase delivery of said target ligand;
• forming one or more EVs from said donor cell encapsulating said target ligand.

A composition comprising:

• an extracellular vesicle (EV) having:
• a membrane-bound protein coupled with a first component of a split complement system and further configured to be capable of being coupled with a target molecule;
• a second component of said split complement system configured to be capable of being coupled with at least one target molecule;
• and
• optionally a protein, or protein fragment configured to increase delivery of said target molecule.

A method of amplifying an immune response in a subject comprising the steps of:

• transfecting a donor cell to heterologously express:
• a fusion deficient fusogenic protein coupled with a first component of a split complement system;
• a second component of a split complement system fused with an antibody peptide or a tumor specific antigen peptide;
• antibody peptide or a tumor specific antigen peptide to a membrane an antibody peptide or a tumor specific antigen peptide to a membrane;
• anchoring said antibody peptide or a tumor specific antigen peptide to a membrane capable of forming an EV by reconstituting said split complement system;
• forming one or more EVs from said donor cell wherein the antibody peptide or a tumor specific antigen peptide is presented on the surface of said one or more EVs;
• isolating said one or more EVs;
• and
• administering a therapeutically effective amount of said isolated EVs to a subject in need thereof wherein the antibody peptide or tumor specific antigen peptide presented on the surface of said isolated EVs elicit an immune response in said subject.

A method of amplifying an immune response in a subject comprising the steps of:

• transfecting a donor cell to heterologously express a fusion deficient protein capable of being incorporated into the membrane of an extracellular vesicle (EV) and further being configured to be coupled with at least one antibody peptide, or a tumor specific antigen peptide;
• forming one or more EVs from said donor cell wherein the antibody peptide or a tumor specific antigen peptide is presented on the surface of said one or more EVs;
• isolating said one or more EVs;
• and
• administering a therapeutically effective amount of said isolated EVs to a subject in need thereof wherein the antibody peptide or tumor specific antigen peptide presented on the surface of said isolated EVs elicit an immune response in said subject.