Non-naturally occurring vesicles comprising a chimeric vesicle localization moiety, methods of making and uses thereof

Abstract

Disclosed herein are non-naturally occurring vesicle comprising a chimeric vesicle localization moiety comprising a surface-and-transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety, the method of making said vesicle and uses thereof.

What is claimed is: 
     
         1 . A non-naturally occurring exosome comprising a chimeric vesicle localization moiety comprising
 a. a surface and transmembrane domain of a first vesicle localization moiety and   b. a cytosolic domain of a second vesicle localization moiety, wherein the chimeric vesicle localization moiety incorporated into the exosome has a topology with amino terminal surface domain external to the exosome, a transmembrane domain in lipid bilayer of the exosome, and carboxy terminal cytosolic domain in lumen of the exosome.   
     
     
         2 . The exosome of  claim 1 , wherein the first vesicle localization moiety is a single pass transmembrane protein. 
     
     
         3 . The exosome of  claim 1 , wherein the second vesicle localization moiety is a single pass transmembrane protein. 
     
     
         4 . The exosome of  claim 1 , wherein the chimeric vesicle localization moiety comprises an amino-terminal surface domain and a carboxyl terminal cytosolic domain connected to each other through a single pass transmembrane domain. 
     
     
         5 . The exosome of  claim 2  or  3 , wherein the single pass transmembrane domain comprises an alpha-helical domain. 
     
     
         6 . The exosome of  claim 2  or  3 , wherein the single pass transmembrane protein is a type I transmembrane protein. 
     
     
         7 . The exosome  claim 1 , wherein the chimeric vesicle localization moiety is a mature chimeric vesicle localization moiety. 
     
     
         8 . The exosome of  claim 7 , wherein the mature chimeric vesicle localization moiety lacks a signal peptide, which precedes a surface domain and is cleaved during maturation of a nascent or newly synthesized full length chimeric vesicle localization moiety. 
     
     
         9 . The exosome of  claim 1 , wherein the two vesicle localization moieties are distinct proteins and not isoforms or allelic variants. 
     
     
         10 . The exosome of  claim 1 , wherein the first and second vesicle localization moieties are from non-homologous proteins. 
     
     
         11 . The exosome of  claim 11 , wherein the first or second vesicle localization moiety is selected from the group consisting of a growth factor receptor, Fc receptor, interleukin receptor, immunoglobulin, MHC-I or MHC-II component, CD antigen, and escort protein. 
     
     
         12 . The exosome of  claim 11 , wherein the first or second vesicle localization moiety is selected from the group consisting of ACE, ADAM10, ADAM15, ADAM9, AGRN, ALCAM, ANPEP, ANTXR2, ATP1A1, ATP1B3, BSG, BTN2A1, CALM1, CANX, CD151, CD19, CD1A, CD1B, CD1C, CD2, CD200, CD200R1, CD226, CD247, CD274, CD276, CD33, CD34, CD36, CD37, CD3E, CD40, CD40LG, CD44, CD47, CD53, CD58, CD63, CD81, CD82, CD84, CD86, CD9, CHMP1A, CHMP1B, CHMP2A, CHMP3, CHMP4A, CHMP4B, CHMP5, CHMP6, CLSTN1, COL6A1, CR1, CSF1R, CXCR4, DDOST, DLL1, DLL4, DSG1, EMB, ENG, EVI2B, F11R, FASN, FCER1G, FCGR2C, FLOT1, FLOT2, FLT3, FN1, GAPDH, GLG1, GRIA2, GRIA3, GYPA, HSPG2, ICAM1, ICAM2, ICAM3, IGSF8, IL1RAP, IL3RA, IL5RA, IST1, ITGA2, ITGA2B, ITGA3, ITGA4, ITGA5, ITGA6, ITGAL, ITGAM, ITGAV, ITGAX, ITGB1, ITGB2, ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, JAG1, JAG2, KIT, LAMP2, LGALS3BP, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LMAN2, LRRC25, LY75, M6PR, MFGE8, MMP14, MPL, MRC1, MVB12B, NECTIN1, NOMO1, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NPTN, NRP1, PDCD1, PDCD1LG2, PDCD6IP, PDGFRB, PECAM1, PLXNB2, PLXND1, PROM1, PTGES2, PTGFRN, PTPRA, PTPRC, PTPRJ, PTPRO, RPN1, SDC1, SDC2, SDC3, SDC4, SDCBP, SDCBP2, SELPLG, SIGLEC7, SIGLEC9, SIRPA, SLIT2, SNF8, SPN, STX3, TACSTD2, TFRC, TLR2, TMED10, TNFRSF8, TRAC, TSG101, TSPAN14, TSPAN7, TSPAN8, TYROBP, VPS25, VPS28, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS4A, VPS4B, VTI1A and VTI1 B, or a variant or homologue thereof. 
     
     
         13 . The exosome of  claim 12 , wherein the variant is an allelic variant or an isoform. 
     
     
         14 . The exosome of  claim 12 , wherein the homologue is an ortholog or paralog. 
     
     
         15 . The exosome of  claim 1 , wherein the chimeric vesicle localization moiety is incorporated into an exosome. 
     
     
         16 . (canceled) 
     
     
         17 . The exosome of  claim 1 , wherein the surface-and-transmembrane domain of the first vesicle localization moiety is a surface-and-transmembrane domain of LAMP2 or a variant or homologue thereof. 
     
     
         18 . The exosome of  claim 1 , wherein the cytosolic domain of the second vesicle localization moiety is the cytosolic domain selected from the group consisting of PTGFRN, ITGA3, IL3RA, SELPLG, ITGB1, CLSTN1, and a homologue thereof. 
     
     
         19 . The extracellular vesicle of  claim 18 , wherein the cytosolic domain of PTGFRN has an amino acid sequence as provided in  FIG.  5    or  FIG.  10    or a homologue or portion thereof, wherein the homologue or portion retains at least about 90% of cytosolic domain activity of PTGFRN in accumulating at an extracellular vesicle, wherein accumulating at an extracellular vesicle is assessed on the basis of the percent of extracellular vesicle positive for the chimeric vesicle localization moiety and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety. 
     
     
         20 . The extracellular vesicle of  claim 18 , wherein the cytosolic domain of ITGA3 has an amino acid sequence as provided in  FIG.  5    or  FIG.  10    or a homologue or portion thereof, wherein the homologue or portion retains at least about 90% of cytosolic domain activity of ITGA3 in accumulating at an extracellular vesicle, wherein accumulating at an extracellular vesicle is assessed on the basis of the percent of extracellular vesicle positive for the chimeric vesicle localization moiety and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety. 
     
     
         21 . The extracellular vesicle of  claim 18 , wherein the cytosolic domain of IL3RA has an amino acid sequence as provided in  FIG.  6    or  FIG.  10    or a homologue or portion thereof, wherein the homologue or portion retains at least about 90% cytosolic domain activity of IL3RA in accumulating at an extracellular vesicle, wherein accumulating at an extracellular vesicle is assessed on the basis of the percent of extracellular vesicle positive for the chimeric vesicle localization moiety and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety. 
     
     
         22 . The extracellular vesicle of  claim 18 , wherein the cytosolic domain of SELPLG has an amino acid sequence as provided in  FIG.  6    or  FIG.  11    or a homologue or portion thereof, wherein the homologue or portion retains at least about 90% of cytosolic domain activity of SELPLG in accumulating at an extracellular vesicle, wherein accumulating at an extracellular vesicle is assessed on the basis of the percent of extracellular vesicle positive for the chimeric vesicle localization moiety and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety. 
     
     
         23 . The extracellular vesicle of  claim 18 , wherein the cytosolic domain of ITGB1 has an amino acid sequence as provided in  FIG.  7    or  FIG.  11    or a homologue or portion thereof, wherein the homologue or portion retains at least about 90% of cytosolic domain activity of ITGB1 in accumulating at an extracellular vesicle, wherein accumulating at an extracellular vesicle is assessed on the basis of the percent of extracellular vesicle positive for the chimeric vesicle localization moiety and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety. 
     
     
         24 . The extracellular vesicle of  claim 18 , wherein the cytosolic domain of CLSTN1 has an amino acid sequence as provided in  FIG.  7    or  FIG.  12    or a homologue or portion thereof, wherein the homologue or portion retains at least about 90% of cytosolic domain activity of CLSTN1 in accumulating at an extracellular vesicle, wherein accumulating at an extracellular vesicle is assessed on the basis of the percent of extracellular vesicle positive for the chimeric vesicle localization moiety and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety. 
     
     
         25 . (canceled) 
     
     
         26 . The exosome of  claim 1  or  18 , wherein the cytosolic domain increase the accumulation of the surface and transmembrane domain of the chimeric vesicle localization moiety at an exosome, and thereby increasing the concentration of the localization moiety at the exosome. 
     
     
         27 . The exosome of  claim 1 , wherein the chimeric vesicle localization moiety increases accumulation or concentration at an exosome by at least 1,3-fold over its full-length or mature parent vesicle localization moieties. 
     
     
         28 . The exosome of  claim 1 , wherein the chimeric vesicle localization moiety increases accumulation or concentration at an exosome by at least 2,5-fold over its full-length or mature parent vesicle localization moieties. 
     
     
         29 . The exosome of  claim 1 , wherein the increase is synergistic. 
     
     
         30 . The exosome of  claim 1 , where the chimeric vesicle localization moiety is a fusion, protein comprising domain arrangement from amino-to-carboxyl terminus in the order: surface domain of the surface-and-transmembrane domain, followed by transmembrane domain of the surface-and-transmembrane domain, and followed by the cytosolic domain. 
     
     
         31 . The extracellular vesicle of  claim 1 , wherein the chimeric vesicle localization moiety is any of the chimeric protein as provided in  FIGS.  10 - 12    or a homologue or fragment thereof, wherein the homologue has between at least about 98% but less than 100% sequence identity and the fragment is a functional fragment retaining a range of at least about 80-98% of a vesicle localization activity. 
     
     
         32 . The extracellular vesicle of  claim 31 , wherein the vesicle localization activity is the ability of the chimeric vesicle localization moiety to accumulate at a vesicle asessed by the percent of vesicles positive for the localization moiety and/or the mean abundance of localization moiety in an extracellular vesicle positive for the localization moiety. 
     
     
         33 . A method of manufacturing of an exosome of  claim 1 , wherein the method comprises the following steps:
 a. expressing a nucleic acid encoding a chimeric vesicle localization moiety comprising a surface-and-transmembrane domain of a first vesicle localization moiety and a cytosolic domain of a second vesicle localization moiety in a producer cell; and   b. isolating an exosome comprising, the chimeric vesicle localization moiety, wherein the exosome is secreted into a culture medium by the producer cell.   
     
     
         34 . An exosome produced by the method of  claim 33 . 
     
     
         35 . A fusion protein comprising a chimeric vesicle localization moiety comprising
 a. a surface-and-transmembrane domain of a first vesicle localization moiety and   b. a cytosolic domain of a second vesicle localization moiety.   
     
     
         36 . The fusion protein of  claim 35 , wherein the fusion protein is expressed on the surface of an exosome. 
     
     
         37 . The fusion protein of  claim 35 , further comprising a linker. 
     
     
         38 . The fusion protein of  claim 37 , wherein the linker is a peptide linker. 
     
     
         39 . The fusion protein of  claim 35 , wherein the chimeric vesicle localization moiety is any of the moieties as shown in construct  135  at  FIG.  10   , constructs  140 - 141  at  FIG.  10   , constructs  142 - 143  at  FIG.  11   , and construct  144  at  FIG.  12   . 
     
     
         40 . A nucleic acid encoding the fusion protein of  claim 35 . 
     
     
         41 . A vector comprising the nucleic acid sequence of  claim 40 . 
     
     
         42 . The vector of  claim 41 , further comprising a promoter sequence and optionally one or more additional regulatory elements. 
     
     
         43 . A genetically modified producer cell comprising a nucleic acid sequence encoding the vector of  claim 41 . 
     
     
         44 . A pharmaceutical composition comprising the exosome of  claim 1 , and one or more pharmaceutically acceptable excipients. 
     
     
         45 . A pharmaceutical composition comprising the fusion protein of  claim 35 , and one or more pharmaceutically acceptable excipients. 
     
     
         46 . A kit comprising the exosome of  claim 1  and instructions. 
     
     
         47 . A kit comprising the fusion protein of  claim 35  and instructions. 
     
     
         48 . A kit comprising the vector of  claim 41  and instructions. 
     
     
         49 . A kit comprising the genetically modified producer cell of  claim 43  and instructions.