Artificial synapses

Abstract

Described herein are compositions and techniques related to generation and therapeutic application of artificial synapses. Artificial synapses are engineered extracellular vesicles, including exosomes, which incorporate sticky binders on their surface to anchor signaling domains against biological targets, such as receptors. These engineered additives can be organized in genetic vector constructs, expressed in mammalian cells, wherein the sticky binders attach to extracellular vesicles such as exosomes, thereby presenting their joined signaling domains which are rapidly taken up by recipient cells. Artificial synapses adopt the hallmark biophysical and biochemical features of extracellular vesicles, allowing for rapid deployment and scale-up. Importantly, this strategy can allow for kinetically favorable signal generation and signal propagation. This includes, for example, increasing density of agonist presentation to support receptor clustering—an onerous barrier for traditional receptor targeting strategies.

What is claimed is: 
     
         1 . An engineered extracellular vesicle comprising:
 at least one fusion polypeptide comprising:
 (i) at least one protein of interest (POI) domain or a fragment thereof; and 
 (ii) at least one vesicle targeting domain, 
   wherein the POI domain is in an extracellular position relative to a lipid membrane of the extracellular vesicle.   
     
     
         2 . The engineered extracellular vesicle of  claim 1 , wherein the extracellular vesicle is an exosome. 
     
     
         3 . The engineered extracellular vesicle of  claim 1  or  claim 2 , wherein the protein of interest (PO) domain or a fragment thereof is a N-terminal domain of the fusion polypeptide. 
     
     
         4 . The engineered extracellular vesicle of any one of  claims 1 - 3 , wherein the vesicle targeting domain is a C-terminal domain of the fusion polypeptide. 
     
     
         5 . The engineered extracellular vesicle of any one of  claims 1 - 4 , wherein the fusion polypeptide comprises at least two POI domains and/or at least two exosome targeting domains. 
     
     
         6 . The engineered extracellular vesicle of any one of  claims 1 - 5 , wherein the fusion polypeptide further comprises a peptide linker. 
     
     
         7 . The engineered extracellular vesicle of any one of  claims 1 - 6 , wherein the fusion polypeptide further comprises a fragment crystallizable region (Fc) domain. 
     
     
         8 . The engineered extracellular vesicle of any one of  claims 1 - 7 , wherein the vesicle targeting domain is in a luminal position relative to the lipid membrane of the extracellular vesicle. 
     
     
         9 . The engineered extracellular vesicle of any one of  claims 1 - 7 , wherein the vesicle targeting domain in an exterior position relative to the lipid membrane of the extracellular vesicle. 
     
     
         10 . The engineered extracellular vesicle of any one of  claims 1 - 9 , wherein the POI domain is selected from the group consisting of: Table 1. 
     
     
         11 . The engineered extracellular vesicle of any one of  claims 1 - 10 , wherein the POI domain is PD-L1 or a fragment thereof. 
     
     
         12 . The engineered extracellular vesicle of any one of  claims 1 - 11 , wherein the POI domain is PD-L2 or a fragment thereof. 
     
     
         13 . The engineered extracellular vesicle of any one of  claims 1 - 12 , wherein the POI domain is FGL1 or a fragment thereof. 
     
     
         14 . The engineered extracellular vesicle of any one of  claims 1 - 13 , wherein the POI domain is 4-1BBL or a fragment thereof. 
     
     
         15 . The engineered extracellular vesicle of any one of  claims 1 - 14 , wherein the POI domain is CTLA-4 or a fragment thereof. 
     
     
         16 . The engineered extracellular vesicle of any one of  claims 1 - 15 , wherein the POI domain substantially binds to one or more of a target polypeptide. 
     
     
         17 . The engineered extracellular vesicle of  claim 16 , wherein the target polypeptide is selected from the group consisting of: Table 2. 
     
     
         18 . The engineered extracellular vesicle of any one of  claims 1 - 17 , wherein the vesicle targeting domain is selected from the group consisting of: Table 3. 
     
     
         19 . The engineered extracellular vesicle of any one of  claims 1 - 18 , wherein the linker is in an exterior position relative to the lipid membrane of the extracellular vesicle. 
     
     
         20 . The engineered extracellular vesicle of any one of  claims 1 - 18 , wherein the linker is a transmembrane linker. 
     
     
         21 . The engineered extracellular vesicle of any one of  claims 1 - 18 , wherein the linker is in a luminal position relative to the lipid membrane of the extracellular vesicle. 
     
     
         22 . The engineered extracellular vesicle of any one of  claims 1 - 21 , wherein the extracellular vesicle does not comprise an endogenous POI polypeptide. 
     
     
         23 . A composition comprising a plurality of the engineered extracellular vesicles of any one of  claims 1 - 22 . 
     
     
         24 . The composition of  claim 23 , further comprising a pharmaceutically acceptable carrier. 
     
     
         25 . An engineered extracellular vesicle comprising:
 (a) a first fusion polypeptide comprising:
 (i) at least one protein of interest (POI) domain or a fragment thereof, and 
 (ii) at least one vesicle targeting domain, 
   wherein the at least one POI domain is in an extracellular position relative to a lipid membrane of the extracellular vesicle,   (b) a second fusion polypeptide comprising:
 (i) at least one protein of interest (POI) domain or a fragment thereof; and 
 (ii) at least one vesicle targeting domain, 
   wherein the POI domain is in an extracellular position relative to a lipid membrane of the extracellular vesicle,   and wherein the at least one vesicle targeting domain is within a lipid membrane of the extracellular vesicle.   
     
     
         26 . A composition comprising two or more of the engineered extracellular vesicles selected from any one of  claims 1 - 25 . 
     
     
         27 . An extracellular vesicle composition comprising:
 a plurality of artificial synapses,   wherein each artificial synapse comprises (i) an extracellular vesicle; (ii) one or more sticky binders; and (iii) one or more signaling domains.   
     
     
         28 . The composition of  claim 27 , wherein the extracellular vesicle comprises an exosome. 
     
     
         29 . The composition of  claim 27 , wherein the one or more sticky binders is selected from the group consisting of: a GPI anchor, a fatty acetylation site, and a prenylation site. 
     
     
         30 . The composition of  claim 27 , wherein the signaling domain comprises one or more of: PD-L1, PD-L2, CTLA-4 (CD152), 4-1BBL (CD137L), HVEM (CD270), FGL1, OX-2 (CD200), Galectin-9, PVR (CD155), Nectin-2 (CD112) isoform alpha, Nectin-2 (CD112) isoform beta, Nectin-2 (CD112) isoform delta, IL-10, TSG-6, B7-H3 (CD276), B7-H4 (VTCN1), B7-H5 (VISTA), B7-H7 (HHLA2), BTNL1, VSIG8, VSIG3 (IGSF11), VSIG4, TIM-3 (HAVCR2), TIM-4 (TIMD4), CEACAM1, BTN3A1, BTN3A2, BTN2A1, BTNL8, BTN2A2, BTN1A1, TIGIT, CD27L (CD70), CD30L (CD153), GITRL, CD40L (CD154), LIGHT (CD258), TL1, CD80, CD86, LFA-3 (CD58), SLAM (CD150), CD40, CD28, CD28H, CD2, LFA-3 (CD58), CD48, CD226, DR3, DcR3, FasL, TIM-1 (CD365), PD-1, or active fragment thereof. 
     
     
         31 . A method of producing the engineered extracellular vesicle or the composition of any one of  claims 1 - 30 , comprising:
 (a) providing a population of cells expressing a vector construct encoding one or more sticky binder and one or more signaling domains; and   (b) isolating a plurality of artificial synapses from the population of cells.   
     
     
         32 . A method of producing the engineered extracellular vesicle or the composition of any one of  claims 1 - 30 , comprising:
 (a) providing a population of cells expressing a vector construct encoding one or more sticky binder and one or more signaling domains; and   (b) isolating a plurality of artificial synapses from the population of cells; and   (c) purifying the plurality of artificial synapses from the population of cells.   
     
     
         33 . The method of  claim 31  or  claim 32 , the isolating is via size exclusion chromatography. 
     
     
         34 . The method of  claim 32 , wherein the purifying is via multimodal chromatography. 
     
     
         35 . The method of any of  claims 31 - 34 , further comprising performing an assay for POI binding to a target polypeptide. 
     
     
         36 . The method of  claim 35 , wherein the vector construct further encodes a promoter. 
     
     
         37 . The method of  claim 36 , wherein the promoter is a tissue-specific promoter or an inducible promotor. 
     
     
         38 . A method of modulating inflammation in a subject, the method comprising:
 administering a composition comprising a plurality of engineered extracellular vesicles to a subject in need thereof,   wherein the engineered extracellular vesicles comprise at least one fusion polypeptide comprising:
 (i) at least one protein of interest (POI) domain or a fragment thereof; and 
 (ii) at least one vesicle targeting domain. 
   
     
     
         39 . The method of  claim 38 , wherein the extracellular vesicle comprises an exosome. 
     
     
         40 . The method of any one of  claims 38 - 39 , further comprising selecting a subject that has or is suspected of having an autoimmune disease or an inflammatory disease or condition. 
     
     
         41 . The method of any one of  claims 38 - 40 , wherein the vesicle targeting domain is selected from the group consisting of: a Glycosylphosphatidylinositol (GPI) anchor, a fatty acetylation site, and a prenylation site. 
     
     
         42 . The method of any one of  claims 38 - 41 , wherein the vesicle targeting domain is a GPI anchor. 
     
     
         43 . The method of any one of  claims 38 - 41 , wherein the vesicle targeting domain is C1C2. 
     
     
         44 . The method of any one of  claims 38 - 43 , wherein the protein of interest (POI) domain comprises one or more of: PD-L1, PD-L2, CTLA-4 (CD152), 4-1BBL (CD137L), HVEM (CD270), FGL1, OX-2 (CD200), Galectin-9, PVR (CD155), Nectin-2 (CD112) isoform alpha, Nectin-2 (CD112) isoform beta, Nectin-2 (CD112) isoform delta, IL-10, TSG-6, B7-H3 (CD276), B7-H4 (VTCN1), B7-H5 (VISTA), B7-H7 (HHLA2), BTNL1, VSIG8, VSIG3 (IGSF11), VSIG4, TIM-3 (HAVCR2), TIM-4 (TIMD4), CEACAM1, BTN3A1, BTN3A2, BTN2A1, BTNL8, BTN2A2, BTN1A1, TIGIT, CD27L (CD70), CD30L (CD153), GITRL, CD40L (CD154), LIGHT (CD258), TL1, CD80, CD86, LFA-3 (CD58), SLAM (CD150), CD40, CD28, CD28H, CD2, LFA-3 (CD58), CD48, CD226, DR3, DcR3, FasL, TIM-1 (CD365), PD-1, or active fragment thereof. 
     
     
         45 . The method of any one of  claims 38 - 44 , wherein the protein of interest (POI) domain is PD-L1 or a fragment thereof. 
     
     
         46 . The method of any one of  claims 38 - 44 , wherein the protein of interest (POI) domain is PD-L2 or a fragment thereof. 
     
     
         47 . The method of any one of  claims 38 - 44 , wherein the protein of interest (POI) domain is CTLA-4 or a fragment thereof. 
     
     
         48 . The method of any one of  claims 38 - 44 , wherein the protein of interest (POI) domain is HVEM or a fragment thereof. 
     
     
         49 . The method of  claim 40 , wherein the inflammatory disease and/or condition is acute. 
     
     
         50 . The method of  claim 40 , wherein the inflammatory related disease and/or condition is chronic. 
     
     
         51 . The method of  claim 38 , wherein administering the composition comprises injection, topical administration, or inhalation. 
     
     
         52 . Use of a composition comprising a plurality of engineered extracellular vesicles, the engineered extracellular vesicles each comprising:
 at least one fusion polypeptide comprising:
 (i) at least one protein of interest (POI) domain or a fragment thereof; and 
 (ii) at least one vesicle targeting domain 
   for the treatment of an inflammatory disease or condition.   
     
     
         53 . Use of a composition comprising a plurality of engineered extracellular vesicles, the engineered extracellular vesicles each comprising:
 at least one fusion polypeptide comprising:
 (i) at least one protein of interest (POI) domain or a fragment thereof, and 
 (ii) at least one vesicle targeting domain 
   for the treatment of an autoimmune disease or condition.   
     
     
         54 . Use of a composition comprising a plurality of engineered extracellular vesicles, the engineered extracellular vesicles each comprising:
 at least one fusion polypeptide comprising:
 (i) at least one protein of interest (POI) domain or a fragment thereof; and 
 (ii) at least one vesicle targeting domain 
   for the treatment of cancer.