EXOSOMES COMPRISING THERAPEUTIC POLYPEPTIDES
The present invention pertains to an inventive release mechanism for extracellular vesicle (EV)-mediated intracellular and intramembrane delivery of therapeutic polypeptides. More specifically, the invention relates to EVs comprising polypeptide constructs which comprise a therapeutic polypeptide releasably attached to an exosomal polypeptide. Furthermore, the present invention pertains to manufacturing methods, pharmaceutical compositions, medical uses and applications, and various other embodiments related to the inventive EVs.
1 . A method for producing extracellular vesicles (EVs) comprising:
(i) introducing into an EV-producing cell at least one polynucleotide construct encoding at least one polypeptide construct comprising at least one polypeptide of interest (Pol), an endogenously activatable polypeptide-based release system, and at least one exosomal polypeptide;
(ii) expressing in the EV-producing cell at least one polypeptide construct encoded for by the at least one polynucleotide construct; and
(iii) collecting from the EV-producing cell Evs comprising a Pol.
2 . The method of claim 1 , wherein the polypeptide-based release system is a cis-cleaving release system or a nuclear localization signal (NLS)-nuclear localization signal-binding protein (NLSBP) (NLS-NLSBP) release system.
3 . The method of claim 2 , wherein the polypeptide-based release system is a cis-cleaving release system; further wherein the cis-cleaving release system is an intein release system.
4 . The method of claim 1 , wherein the upon activation of the polypeptide-based release system, the at least one Pol is released from the at least one exosomal polypeptide into the lumen of the EV.
5 . The method of claim 1 , wherein the Pol is a therapeutic polypeptide selected from the group consisting of antibodies, intrabodies, single chain variable fragments, affibodies, enzymes, tumor suppressors, viral or bacterial inhibitors, cell component proteins, DNA and/or RNA binding proteins, DNA repair inhibitors, nucleases, proteinases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins, structural proteins, neurotrophic factors, membrane transporters, nucleotide binding proteins, heat shock proteins, CRISPR-associated proteins, and any combination thereof.
6 . The method of claim 5 , wherein the at least one polypeptide of interest is
(i) a CRISPR-associated (Cas) polypeptide with intact nuclease activity or
(ii) a catalytically inactive CRISPR-associated (Cas) polypeptide.
7 . The method of claim 6 , wherein the polypeptide of interest is a catalytically inactive CRISPR-associated (Cas) polypeptide capable of enabling targeted genetic engineering.
8 . The method according to claim 1 , wherein the at least one exosomal polypeptide is a polypeptide capable of transporting a polypeptide construct comprising the Pol to an EV.
9 . The method of claim 1 , wherein the exosomal polypeptide is selected from the group consisting of CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, tetraspanins, Fc receptors, interleukin receptors, immunoglobulins, MHC-I or MHC-II components, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1 CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, and other exosomal polypeptides.
10 . The method of claim 1 , further comprising exposing the EV-producing cell to serum starvation, hypoxia, bafilomycin, or one or more cytokine.
11 . The method of claim 10 , wherein the one or more cytokine is TNF-alpha and/or IFN-gamma.
12 . The method of claim 1 , further comprising step (iv) purifying the EVs.
13 . The method of claim 12 , wherein the purifying comprises a procedure selected from the group consisting of liquid chromatography (LC), bead-elute LC, size-exclusion LC, high-performance liquid chromatography (HPLC), spin filtration, tangential flow filtration, hollow fiber filtration, centrifugation, immunoprecipitation.
14 . An EV produced by the method of claim 1 .