	US 12,215,335 B2
	Non-integrating DNA vectors for the genetic modification of cells
James A. Williams, Lincoln, NE (US); Matthias Bozza, Heidelberg (DE); and Richard Harbottle, Ladenburg (DE)
Assigned to Deutsches Krebsforschungszentrum, Heidelberg (DE)
Appl. No. 16/648,885
Filed by Deutsches Krebsforschungszentrum, Heidelberg (DE)
PCT Filed Sep. 17, 2018, PCT No. PCT/US2018/051381 § 371(c)(1), (2) Date Mar. 19, 2020, PCT Pub. No. WO2019/060253, PCT Pub. Date Mar. 28, 2019.
Claims priority of application No. 17191829 (EP), filed on Sep. 19, 2017.
Prior Publication US 2020/0277624 A1, Sep. 3, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/85 (2006.01); C12N 15/67 (2006.01); C12N 15/79 (2006.01)

CPC C12N 15/85 (2013.01) [C12N 15/79 (2013.01); C12N 15/67 (2013.01); C12N 2800/106 (2013.01); C12N 2800/108 (2013.01); C12N 2820/55 (2013.01); C12N 2830/46 (2013.01)]

27 Claims

1. A method for improving the expression and establishment efficiency of a self-replicating non-integrative episomal S/MAR expression vector in a target vertebrate cell comprising the following steps:

a. providing an episomal S/MAR expression vector comprising:

i. a bacterial replication-selection region comprising a bacterial origin of replication and a selectable marker gene;

ii. a transcription unit for expression of a transgene in a vertebrate cell, comprising a promoter, a 5′ UTR, a transgene, and a 3′ UTR;

iii. an S/MAR insert located within said 3′ UTR; and

b. modifying the episomal S/MAR expression vector such that the S/MAR is flanked by a 5′ splice donor site and a 3′ splice acceptor site within said 3′ UTR, whereby the resultant self-replicating non-integrative episomal S/MAR expression vector has improved expression and establishment efficiency after transfection of a vertebrate cell.