	US 12,404,526 B2
	Enhancing agents for improved cell transfection and/or rAAV vector production
Guang Qu, Sicklerville, NJ (US); Lin Lu, Wayne, PA (US); Jesusa Josue-Almqvist, Havertown, PA (US); and John Fraser Wright, Princeton, NJ (US)
Assigned to SPARK THERAPEUTICS, INC., Philadelphia, PA (US)
Appl. No. 16/619,898
Filed by Spark Therapeutics, Inc., Philadelphia, PA (US)
PCT Filed Jun. 6, 2018, PCT No. PCT/US2018/036344 § 371(c)(1), (2) Date Dec. 5, 2019, PCT Pub. No. WO2018/226887, PCT Pub. Date Dec. 13, 2018.
Claims priority of provisional application 62/531,626, filed on Jul. 12, 2017.
Claims priority of provisional application 62/516,432, filed on Jun. 7, 2017.
Prior Publication US 2020/0165632 A1, May 28, 2020
Int. Cl. C12N 15/87 (2006.01); C12N 5/073 (2010.01)

CPC C12N 15/87 (2013.01) [C12N 5/0603 (2013.01); C12N 2750/14143 (2013.01); C12N 2750/14151 (2013.01)]

37 Claims

1. A method for making transfected cells that produce recombinant adeno-associated viral (rAAV) vector, comprising the steps:

(a) providing a polyethylenimine (PEI)/plasmid mixture of components (i), (ii) and (iii):

(i) one or more plasmids comprising nucleic acids encoding AAV packaging proteins, adenovirus E2 proteins, adenovirus E4 proteins and/or nucleic acids encoding virus-associated RNA (VA RNA);

(ii) a plasmid comprising a transgene that encodes a protein or is transcribed into a transcript of interest; and

(iii) a PEI solution,

(b) contacting cells with the plasmid/PEI mixture of step (a) to produce a plasmid/PEI cell culture;

(c) adding valproic acid, a salt or a derivative thereof to the plasmid/PEI cell culture to produce a second mixture, wherein the valproic acid, salt or derivative thereof is added immediately after step (b); and

(d) incubating said second mixture of step (c);

thereby making transfected cells that produce rAAV vector.