Cell penetrating peptide (cpp)-mediated ev loading

Abstract

The present invention relates to methods for loading extracellular vesicles (EVs) with a pharmacological agent. The invention discloses the use of cell-penetrating peptides as carriers into EVs, using either a non-covalent or covalent loading approach. Furthermore, the present invention pertains to medical uses and compositions comprising such pharmacological agent-loaded EVs.

1 . A population of exosomes, wherein each exosome comprises at least one pharmacological agent and at least one cell-penetrating peptide (CPP), wherein the at least one pharmacological agent is a nucleic acid. 
     
     
         2 . The population of  claim 1 , wherein the at least one pharmacological agent is conjugated to or complexed with at least one CPP. 
     
     
         3 . The population of  claim 2 , wherein the at least one pharmacological agent is released from at least one CPP conjugate or at least one CPP complex inside each exosome. 
     
     
         4 . The population of  claim 1 , further comprising a targeting moiety. 
     
     
         5 . The population of  claim 4 , wherein the targeting moiety comprises a sequence of amino acids expressed as a fusion protein with an exosome polypeptide, or an antibody, attached to the surface of each exosome. 
     
     
         6 . The population of  claim 1 , wherein the nucleic acid and the CPP are present in the form of covalent conjugates, non-covalent complexes and/or a combination thereof. 
     
     
         7 . The population of  claim 1 , wherein the nucleic acid is an oligonucleotide, siRNA, shRNA, antisense oligonucleotide, splice-switching oligonucleotide, mRNA or CRISPR guide strand. 
     
     
         8 . The population of  claim 1 , wherein the nucleic acid is chemically synthesized. 
     
     
         9 . The population of  claim 1 , wherein the nucleic acid comprises chemically modified nucleotides. 
     
     
         10 . The population of  claim 9 , wherein the chemical modifications may be selected from: 2′-0-Me, 2-0-Allyl, 2′-0-MOE, 2′-F, 2′-CE, 2′-EA 2′-FANA, LNA, CLNA, ENA, PNA, phosphorothioates, tricyclo-DNA, or phosphorodiamidate morpholino oligomer (PMO) modification. 
     
     
         11 . The population of  claim 1 , wherein the CPP is highly cationic, rich in proline, arginine rich, lysine rich, amphipathic, having stretches of hydrophobic amino acids, has aliphatic spacers between the amino acids, comprises synthetic peptide derivatives, comprises artificial peptide derivatives, comprises non-natural amino acids, comprises inverso analogues, comprises retro-inverso analogues, comprises modifications from L- to D-amino acids, comprises linear aliphatic chains, comprises branched aliphatic chains, or any combination thereof. 
     
     
         12 . The population of  claim 1 , wherein the CPP is chemically modified by the introduction of lipid tans, cholesterol, cholesterol analogues, quinolones or their fluorinated analogues, chloroquine or its fluorinated analogue, polyhistidine, or C- and/or N-terminal and/or orthogonal modifications thereof. 
     
     
         13 . The population of  claim 1 , wherein the CPP is transportan 10 (TP10), a CADY peptide, transactivator of transcription (Tat) peptide, (Arg)9 peptide, or PepFect peptide. 
     
     
         14 . The population of  claim 1 , produced by exposing a population of exosome source cells to the at least one pharmacological agent and the at least one cell-penetrating peptide (CPP). 
     
     
         15 . A pharmaceutical composition comprising a population of exosomes of  claim 1  and a pharmaceutically acceptable excipient.