Methods for delivering nucleic acid molecules into cells and assessment thereof

Abstract

Methods for delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of the nucleic acids are provided. The methods are designed for introduction of large nucleic acid molecules, including artificial chromosomes, into cells, and are practiced in vitro and in vivo.

1 . A method for introducing a large nucleic acid molecule into a cell, comprising: 
 (a) obtaining a cell from a donor subject;    (b) contacting a large nucleic acid molecule with a first delivery agent;    (c) adding a second delivery agent to a composition containing the cell or applying a second delivery agent to the cell, whereby the second delivery agent contacts the cell; and    (d) contacting the cell with the nucleic acid molecule, whereby the nucleic acid molecule is delivered into the cell, wherein:    steps (b)-(d) are performed sequentially in any order, provided that, if a delivery agent is energy it is not applied to the nucleic acid molecule and it is not applied to the cell after contacting the cell with the nucleic acid molecule;    the first and second delivery agents are different;    the first delivery agent increases contact between the nucleic acid molecule and the cell compared to in the absence of the first delivery agent; and    the second delivery agent, when added to the cell composition, enhances permeability of the cell to the nucleic acid molecule compared to in its absence in the composition.    
     
     
         2 . The method of  claim 1 , further comprising, introducing the cells that contain the nucleic acid molecule into a recipient subject.  
     
     
         3 . The method of  claim 2 , wherein the recipient subject is the same as the donor subject.  
     
     
         4 . The method of  claim 1 , wherein the subject is a human.  
     
     
         5 . The method of  claim 1 , wherein the nucleic acid molecule that is introduced encodes a therapeutic protein.  
     
     
         6 . The method of  claim 1 , wherein the nucleic acid molecule is greater than about 0.6 megabase.  
     
     
         7 . The method of  claim 1 , wherein the nucleic acid molecule is a natural chromosome, an artificial chromosome, a fragment of a chromosome that is greater than about 0.6 megabase or naked DNA molecule that is greater than about 0.6 megabase.  
     
     
         8 . The method of  claim 1 , wherein the nucleic acid molecule is an artificial chromosome expression system (ACes).  
     
     
         9 . The method of  claim 1 , wherein: 
 contacting the nucleic acid with a first delivery agent is effected by mixing the nucleic acid with a delivery agent; and    applying an agent that enhances permeability to the cell comprises applying ultrasound or electrical energy to the cell.    
     
     
         10 . The method of  claim 1 , wherein a delivery agent comprises a cationic compound.  
     
     
         11 . The method of  claim 10 , wherein the cationic compound is selected from the group consisting of a cationic lipid, a cationic polymer, a mixture of cationic lipids, a mixture of cationic polymers, a mixture of a cationic lipid and a cationic polymer, a mixture of a cationic lipid and a neutral lipid, polycationic lipids, non-liposomal forming lipids, activated dendrimers, and a pyridinium chloride surfactant.  
     
     
         12 . The method of  claim 9 , wherein prior to applying the ultrasound energy to the cell, the cell is contacted with a cavitation compound.  
     
     
         13 . The method of  claim 10 , wherein the delivery agent is a composition that comprises one or more cationic compounds, wherein the compound is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), C 52 H 106 N 6 O 4 .4CF 3 CO 2 H, C 88 H 178 N 8 O 4 S 2 .4CF 3 CO 2 H, C 40 H 84 NO 3 P.CF 3 CO 2 H, C 50 H 103 N 7 O 3 .4CF 3 CO 2 H, C 55 H 116 N 8 O 2 .6CF 3 CO 2 H, C 49 H 102 N 6 O 3 A.CF 3 CO 2 H, C 49 H 102 N 6 O 3 .4CF 3 CO 2 H, C 44 H 89 N 5 O 3 .2CF 3 CO 2 H, C 100 H 206 N 12 O 4 S 2 .8CF 3 CO 2 H, C 41 H 78 NO 8 P, C 162 H 330 N 22 O 9 .13CF 3 CO 2 H, C 43 H 88 N 4 O 2 .2CF 3 CO 2 H, C 43 H 88 N 4 O 3 .2CF 3 CO 2 H, and (1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride.  
     
     
         14 . A method for introducing a large nucleic acid molecule into a cell, comprising: 
 (a) obtaining a cell from a donor subject    (b) contacting a large nucleic acid molecule with a first delivery agent;    (c) contacting or applying a second delivery agent to the cell; and    (d) contacting the cell with the nucleic acid molecule, whereby the nucleic acid molecule is delivered into the cell, wherein:    steps (b)-(d) are performed sequentially in any order, provided that if a delivery agent is energy it is not applied to the nucleic acid molecule and it is not applied to the cell after contacting the cell with the nucleic acid molecule.    
     
     
         15 . The method of  claim 14 , further comprising, introducing the cells that contain the nucleic acid molecule into a recipient subject.  
     
     
         16 . The method of  claim 15 , wherein the recipient subject is the same as the donor subject.  
     
     
         17 . The method of  claim 14 , wherein the subject is a human.  
     
     
         18 . The method of  claim 14 , wherein the nucleic acid molecule that is introduced encodes a therapeutic protein.  
     
     
         19 . The method of  claim 14 , wherein the cell is treated with energy.  
     
     
         20 . The method of  claim 14 , wherein the energy is ultrasound energy.  
     
     
         21 . The method of  claim 20 , wherein the ultrasound energy is applied to the cell for about 30 seconds to about 5 minutes.  
     
     
         22 . The method of  claim 20 , wherein the ultrasound energy is applied as one continuous pulse.  
     
     
         23 . The method of  claim 20 , wherein the ultrasound energy is applied as two or more intermittent pulses.  
     
     
         24 . The method of  claim 23 , wherein the intermittent pulses of the ultrasound energy are applied for substantially the same length of time, at substantially the same energy level.  
     
     
         25 . The method of  claim 23 , wherein the intermittent pulses vary in energy level, the length of time applied, or energy level and the length of time applied.  
     
     
         26 . The method of  claim 20 , wherein prior to applying the ultrasound energy to the cell, the cell is contacted with a cavitation compound.  
     
     
         27 . The method of  claim 1 , wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, and a cell capable of the generation of a specific organ.  
     
     
         28 . The method of  claim 1 , wherein the cell is selected from the group consisting of an embryonic cell and a tumor cell.  
     
     
         29 . A method for delivering a large nucleic acid molecule into a cell, comprising, either: 
 (a) obtaining a cell from a donor subject;    (b) applying ultrasound or electrical energy to the cell; and    (c) upon conclusion of the application of ultrasound or electrical energy, contacting the cell with a mixture of the nucleic acid molecule and a delivery agent, whereby the nucleic acid molecule is delivered into the cell; or    (a) obtaining a cell from a donor subject;    (b) adding a delivery agent to a composition containing the cell in the absence of the nucleic acid molecule, and applying ultrasound energy or electrical energy to the cell, wherein the contacting and applying are performed sequentially or simultaneously; and then    (c) contacting the cell with the large nucleic acid molecule, whereby the nucleic acid molecule is delivered into the cell.    
     
     
         30 . The method of  claim 29 , further comprising, after step (c), introducing the cells that contain the nucleic acid molecule into a recipient subject.  
     
     
         31 . The method of  claim 30 , wherein the recipient subject is the same as the donor subject.  
     
     
         32 . The method of  claim 29 , wherein the subject is a human.  
     
     
         33 . The method of  claim 29 , wherein the nucleic acid molecule that is introduced encodes a therapeutic protein.  
     
     
         34 . The method of  claim 29 , wherein a delivery agent is a cationic compound.  
     
     
         35 . The method of  claim 29 , wherein the energy is ultrasound energy.  
     
     
         36 . The method of  claim 35 , wherein prior to applying the ultrasound energy, the cell is contacted with a cavitation compound.  
     
     
         37 . The method of  claim 29 , wherein the cell is selected from the group consisting of a nuclear transfer donor cell, a stem cell, a primary cell, and a cell capable of the generation of a specific organ.  
     
     
         38 . The method of  claim 29 , wherein the cell is selected from the group consisting of an embryonic cell and a tumor cell.  
     
     
         39 . The method of  claim 34 , wherein the delivery agent is a composition that comprises one or more cationic compounds, wherein the compound is selected from the group consisting of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), C 52 H 106 N 6 O 4 .4CF 3 CO 2 H, C 88 H 178 N 8 O 4 S 2 .4CF 3 CO 2 H, C 40 H 84 NO 3 P.CF 3 CO 2 H, C 50 H 103 N 7 O 3 .4CF 3 CO 2 H, C 55 H 116 N 8 O 2 .6CF 3 CO 2 H, C 49 H 102 N 6 O 3 .4CF 3 CO 2 H, C 44 H 89 N 5 O 3 .2CF 3 CO 2 H, C 100 H 206 N 12 O 4 S 2 .8CF 3 CO 2 H, C 41 H 78 NO 8 P, C 162 H 330 N 22 O 9 .13CF 3 CO 2 H, C 43 H 88 N 4 O 2 .2CF 3 CO 2 H, C 43 H 88 N 4 O 3 .2CF 3 CO 2 , and (1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride.  
     
     
         40 . The method of  claim 29 , wherein a delivery agent is 1-methyl-4-(1-octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride.  
     
     
         41 . The method of  claim 29 , wherein the nucleic acid molecule is greater than about 1 megabase.  
     
     
         42 . The method of  claim 29 , wherein the nucleic acid molecule is selected from the group consisting of an artificial chromosome, an artificial chromosome expression system (ACes) and a natural chromosome or a fragment thereof that is greater than at least about 0.6 megabase.  
     
     
         43 . The method of  claim 34 , wherein the cationic compound is selected from the group consisting of a cationic lipid, a cationic polymer, a mixture of cationic lipids, a mixture of cationic polymers, a mixture of a cationic lipid and a cationic polymer, a mixture of a cationic lipid and a neutral lipid, polycationic lipids, non-liposomal forming lipids, activated dendrimers and a pyridinium chloride surfactant.  
     
     
         44 . A method for delivering a large nucleic acid molecule into a cell, comprising: 
 (a) obtaining a cell from a donor subject;    (b) contacting the nucleic acid molecule with a composition that comprises a cationic lipid, wherein: the cationic lipid composition comprises 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA) and dioleoylphosphatidylethanolamine (DOPE); and the nucleic molecule is at least 5 megabases; and then    (c) contacting the nucleic acid molecule with a cell, wherein steps (b) and (c) are performed simultaneously or sequentially.    
     
     
         45 . The method of  claim 44 , wherein the nucleic acid molecule is a natural chromosome, an artificial chromosome, a fragment of a chromosome, or naked DNA molecule.  
     
     
         46 . The method of  claim 44 , further comprising, after step (c), introducing the cells that contain the nucleic acid molecule into a recipient subject.  
     
     
         47 . The method of  claim 46 , wherein the recipient subject is the same as the donor subject.  
     
     
         48 . The method of  claim 44 , wherein the subject is a human.  
     
     
         49 . The method of  claim 44 , wherein the nucleic acid molecule that is introduced encodes a therapeutic protein  
     
     
         50 . The method of  claim 44 , wherein the cell is selected from the group consisting of a primary cell, an embryonic cell, a stem cell, a transformed cell and a tumor cell.