Compostions and methods for nucleic acid transfection using cationic polymers and stabilizers
AssigneePfizer Inc.
InventorsSaurabh Kant Bhardwaj, Chia Hung Chu
Life of this patent
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Abstract
Provided are compositions and methods for stabilizing a transfection cocktail containing DNA-cationic polymer complexes for an extended time, while maintaining high transfection efficiency. Such stabilized transfection cocktail can be used to generate transfected cells that can produce, for example, rAAV vectors on a large scale without impacting the key attributes of the virus production, such as, titer, DNA packaged rAAV particle fraction, and rAAV vector purification profile.
Claims57
A method for transfecting cells with one or more nucleic acids, comprising the following steps:
- (i) preparing a transfection cocktail comprising one or more cationic polymers, a stabilizer and one or more nucleic acids,
- (ii) contacting the transfection cocktail prepared in step (i) with cells to be transfected to form a mixture, and
- (iii) incubating the mixture of step (ii) thereby transfecting the cells with the one or more nucleic acids.
A method for making cells that produce recombinant adeno-associated viral (rAAV) vector, comprising the following steps:
- (i) preparing a transfection cocktail comprising one or more cationic polymers, a stabilizer and one or more nucleic acids,
- (ii) contacting the transfection cocktail prepared in step (i) with cells to be transfected to form a mixture, and
- (iii) incubating the mixture of step (ii) thereby making transfected cells that produce rAAV vector.
A method for increasing transfection of cells with one or more nucleic acids, comprising the following steps:
- (i) preparing a transfection cocktail comprising one or more cationic polymers, a stabilizer and one or more nucleic acids,
- (ii) contacting the transfection cocktail prepared in step (i) with cells to be transfected to form a mixture, and
- (iii) incubating the mixture of step (ii), whereby transfection of the cells with the one or more nucleic acids is increased as compared to the transfection of the cells performed under the same conditions but in the absence of the stabilizer.
A method for producing high titer recombinant adeno-associated viral (rAAV) vector, comprising the following steps:
- (i) preparing a transfection cocktail comprising one or more cationic polymers, a stabilizer and one or more nucleic acids,
- (ii) contacting the transfection cocktail prepared in step (i) with cells to be transfected to form a mixture,
- (iii) incubating the mixture of step (ii) to make transfected cells that produce rAAV vector, and
- (iv) isolating and/or purifying rAAV vector from the transfected cells produced in step (iii), wherein the time between initiation of step (i) and completion of step (ii) is greater than 10 seconds, and wherein the rAAV titer is increased by at least 2-fold and/or by at least 5% as compared to the rAAV titer produced under the same conditions but in the absence of the stabilizer.
A composition comprising one or more cationic polymers and a stabilizer.
The composition of claim 49, further comprising one or more nucleic acids and/or cells.
A composition comprising a stabilizer and one or more nucleic acids.
A composition comprising one or more cationic polymers, a stabilizer, one or more nucleic acids, and cells.
A composition comprising about 12×106 cells/mL, about 2.2 μg per million cells of one or more cationic polymers, about 10% of stabilizer relative to the one or more cationic polymers, and about 1 μg per million cells of one or more nucleic acids.
A composition comprising about 24×106 cells/mL, about 2.2 μg per million cells of one or more cationic polymers, about 25% of stabilizer relative to the one or more cationic polymers, and about 1 μg per million cells of one or more nucleic acids.
Dependencies are inferred from the claim text.