Preparation of a Solution of Polymer/Nucleic Acid Complexes
AssigneeOxford Biomedica (UK) Limited
InventorsOliver Goodyear, Lee Davies, Mollie Kingwood, Rui Sanches, Ciaran Lamont
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Abstract
The invention relates to the preparation of a solution of polymer/nucleic acid complexes, and the use of such a solution in methods for the transfection of cells.
Claims66
An in vitro method for producing a solution of polymer/nucleic acid complexes comprising the steps of:
- a) mixing a nucleic acid and a cationic polymer in a substantially salt free aqueous solution;
- b) adding a salt to the mixture produced in step a) to form a salt solution;
- c) optionally incubating the salt solution produced in step b);
- and/or
- d) optionally diluting the salt solution produced in step b) or diluting the salt solution following incubation of step c),
- wherein said salt is not a valproic acid salt, isobutyric acid salt or isovaleric acid salt.
An in vitro method for producing a solution of polymer/nucleic acid complexes comprising the steps of:
- a) mixing a nucleic acid and a cationic polymer in a substantially salt free aqueous solution;
- b) adding a salt to the mixture produced in step a) to form a salt solution;
- c) optionally incubating the salt solution produced in step b);
- and/or
- d) optionally diluting the salt solution produced in step b) or diluting the salt solution following incubation of step c),
- wherein said salt is added only prior to contacting the solution of polymer/nucleic acid complexes with a cell.
An in vitro method for producing a solution of polymer/nucleic acid complexes comprising the steps of:
- a) mixing a nucleic acid and a cationic polymer in a substantially salt free aqueous solution;
- b) adding a salt to the mixture produced in step a) to form a salt solution;
- c) optionally incubating the salt solution produced in step b);
- and/or
- d) optionally diluting the salt solution produced in step b) or diluting the salt solution following incubation of step c),
- wherein the final salt concentration in step b) is between about 10 mM to about 100 mM.
An in vitro method for producing a solution of polymer/nucleic acid complexes comprising the steps of:
- a) mixing a nucleic acid and a cationic polymer in a substantially salt free aqueous solution;
- b) adding a salt to the mixture produced in step a) to form a salt solution;
- c) optionally incubating the salt solution produced in step b);
- and/or
- d) optionally diluting the salt solution produced in step b) or diluting the salt solution following incubation of step c),
- wherein said salt has a degree of dissociation of at least 0.95 in the substantially salt free aqueous solution.
An in vitro method for producing a solution of polymer/nucleic acid complexes comprising the steps of:
- a) mixing a nucleic acid and a cationic polymer in a substantially salt free aqueous solution;
- b) adding a salt to the mixture produced in step a) to form a salt solution;
- c) optionally incubating the salt solution produced in step b);
- and/or
- d) diluting the salt solution produced in step b) or diluting the salt solution following incubation of step c).
An in vitro method for producing a solution of polymer/nucleic acid complexes comprising the steps of:
- a) mixing a nucleic acid and a cationic polymer in a substantially salt free aqueous solution;
- b) adding a salt to the mixture produced in step a) to form a salt solution;
- c) incubating the salt solution produced in step b);
- and/or
- d) optionally diluting the salt solution produced in step b) or diluting the salt solution following incubation of step c).
The method according to any one of the preceding claims, wherein the nucleic acid is selected from DNA, RNA, oligonucleotide molecule, and mixtures thereof.
The method according to any one of the preceding claims, wherein the cationic polymer is a polymer-based transfection reagent.
The method according to any one of the preceding claims, wherein the cationic polymer is selected from polyethylenimine (PEI), a dendrimer, DEAE-dextran, polypropyleneimine (PPI), chitosan [poly-(β-¼)-2-amino-2-deoxy-D-glucopyranose], poly-L-lysine (PLL), poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone) (PCL), and a derivative thereof.
The method according to any one of the preceding claims, wherein the cationic polymer is PEI or a derivative thereof, preferably selected from linear PEI, branched PEI, PEGylated PEI, JetPEI, PEIPro®, PEI MAX and PTG1+.
The method according to any one of the preceding claims, wherein the salt is selected from a sodium salt, a magnesium salt, a potassium salt, a calcium salt, a phosphate salt and a mixture thereof.
The method according to any one of the preceding claims, wherein the salt is phosphate buffered saline (PBS).
The method according to any one of the preceding claims, wherein step a) comprises mixing a plurality of nucleic acid molecules and a plurality of cationic polymer molecules in a substantially salt free aqueous solution.
The method according to any one of claims 1 to 5 and 7 to 17, wherein the method comprises the step of incubating the salt solution produced in step b).
The method according to any one of the preceding claims, wherein the nucleic acid encodes a retroviral vector component.
The method according to any one of the preceding claims, wherein the method further comprises the steps of:
- e) optionally culturing a mammalian cell in a perfusion culture;
- f) transfecting a mammalian cell using the solution of polymer/nucleic acid complexes obtained by the method according to any one of the preceding claims;
- g) optionally introducing a nucleic acid that is different to the nucleic acid of the solution of polymer/nucleic acid complexes into the mammalian cell;
- h) optionally selecting for a mammalian cell which has the nucleic acid(s) integrated within its genome;
- i) optionally culturing the mammalian cell under conditions in which the nucleic acid(s) is (are) expressed;
- j) optionally culturing the mammalian cell under conditions in which the retroviral vector is produced;
- and
- k) optionally isolating the retroviral vector.
A solution comprising polymer/nucleic acid complexes and a salt, wherein the salt concentration is between about 10 mM to about 100 mM.
Dependencies are inferred from the claim text.