Drug Delivery Particle and Method for Producing the Same

Abstract

An object of the present invention is to develop and provide a method for conveniently introducing a nucleic acid, a peptide, and/or a low-molecular-weight compound into an empty capsid with viral early infection activities kept. The present invention provides a method for producing a drug delivery particle, comprising the steps of: mixing an empty capsid or an empty particle with a drug including a nucleic acid, a peptide, and/or a low-molecular-weight compound in a solution comprising 0.1 to 20% of a surfactant; and keeping the obtained mixed solution at −5 to 50° C. to introduce the drug into the empty capsid or the empty particle.

1 . A method for producing a drug delivery particle comprising a capsid or an enveloped particle comprising a drug, comprising the steps of:
 (a) mixing an empty capsid or an empty particle with the drug in a solution comprising 0.1 to 20% of a surfactant; and   (b) keeping the mixed solution after the mixing step at −5 to 50° C. to introduce the drug into the empty capsid or the empty particle.   
     
     
         2 . The production method according to  claim 1 , wherein the introduction step comprises keeping the mixed solution for 5 minutes to 120 minutes. 
     
     
         3 . The production method according to  claim 1  or  2 , further comprising the step of (c) removing the surfactant in the solution after the introduction step. 
     
     
         4 . The production method according to any one of  claims 1  to  3 , wherein the surfactant is one or more surfactant(s) selected from the group consisting of Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween-20, Tween-80, octyl-β-glucoside, OTG, SDS, CHAPS, CHAPSO, and a copolymer of PEG and PPG. 
     
     
         5 . The production method according to any one of  claims 1  to  4 , wherein the drug is a nucleic acid, a peptide, and/or a low-molecular-weight compound. 
     
     
         6 . The production method according to any one of  claims 1  to  5 , wherein the nucleic acid is one or more nucleic acid(s) selected from the group consisting of a functional nucleic acid, an mRNA, an mRNA fragment, and a vector comprising an arbitrary gene or a fragment thereof. 
     
     
         7 . The production method according to  claim 6 , wherein the functional nucleic acid is one or more nucleic acid(s) selected from the group consisting of an siRNA, an miRNA, an shRNA, a nucleic acid aptamer, an antisense DNA, a U1 adaptor, a ribozyme, a molecular beacon, and a riboswitch. 
     
     
         8 . The production method according to  claim 6 , wherein the vector is selected from the group consisting of a plasmid, a cosmid, and an artificial chromosome. 
     
     
         9 . The production method according to any one of  claims 1  to  8 , wherein the empty capsid or the empty particle is modified. 
     
     
         10 . The production method according to any one of  claims 1  to  9 , wherein the empty capsid or the empty particle has an early infection activity to a particular cell. 
     
     
         11 . The production method according to any one of  claims 1  to  10 , wherein the empty capsid is derived from adenovirus or adeno-associated virus. 
     
     
         12 . A drug delivery particle comprising a drug in a capsid or an enveloped particle, wherein the drug is an exogenous nucleic acid free from genes of a virus itself from which the capsid or the enveloped particle is derived, a peptide, and/or a low-molecular-weight compound. 
     
     
         13 . The drug delivery particle according to  claim 12 , wherein the exogenous nucleic acid is one or more nucleic acid(s) selected from the group consisting of a functional nucleic acid, an mRNA, an mRNA fragment, and a vector comprising an arbitrary gene or a fragment thereof. 
     
     
         14 . The drug delivery particle according to  claim 13 , wherein the functional nucleic acid is one or more nucleic acid(s) selected from the group consisting of an siRNA, an miRNA, an shRNA, a nucleic acid aptamer, an antisense DNA, a U1 adaptor, a ribozyme, a molecular beacon, and a riboswitch. 
     
     
         15 . The drug delivery particle according to  claim 13 , wherein the vector is selected from the group consisting of a plasmid, a cosmid, and an artificial chromosome. 
     
     
         16 . The drug delivery particle according to any one of  claims 12  to  15 , wherein the capsid or the enveloped particle is modified. 
     
     
         17 . The drug delivery particle according to any one of  claims 11  to  16 , wherein the capsid is derived from adenovirus or adeno-associated virus. 
     
     
         18 . The drug delivery particle according to any one of  claims 11  to  17 , wherein the capsid or the enveloped particle has an early infection activity to a particular cell. 
     
     
         19 . A drug composition comprising at least one drug delivery particle obtained by a production method according to any one of  claims 1  to  11  and/or at least one drug delivery particle according to any one of  claims 12  to  18  as an active ingredient. 
     
     
         20 . The drug composition according to  claim 19 , wherein the drug composition is intended for the prevention or treatment of disease or disease damage. 
     
     
         21 . A method for purifying an empty capsid or an enveloped particle, or a virion, comprising the step of heating a culture solution of a host cell and/or an extract of the host cell that has produced the empty capsid or the enveloped particle, or the virion, wherein
 the heating time is 10 minutes to 90 minutes in the case of a heating temperature of 45° C. or higher and lower than 55° C., or   the heating time is 3 minutes to 30 minutes in the case of a heating temperature of 55° C. or higher and 60° C. or lower.   
     
     
         22 . A method for producing a cell line having the enhanced ability to produce adeno-associated virus, comprising the step of introducing an E1A gene region, an E1B19 gene, an E2A gene region, an E4orf6 gene, and a VA RNA-encoding gene derived from adenovirus to a HEK293 cell line. 
     
     
         23 . The production method according to  claim 22 , further comprising the step of selecting a cell line stably expressing all of the introduced genes. 
     
     
         24 . The production method according to  claim 22  or  23 , further comprising the steps of:
 introducing a human-derived Bcl-x L  gene to the cell line; and 
 selecting a cell line stably expressing the introduced Bcl-x L  gene.