US 11,866,724 B2
Adenoviral vectors
Adrian Francis Stewart, Dresden (DE); Jun Fu, Dresden (DE); Anja Ehrhardt, Witten (DE); Eric Ehrke-Schulz, Witten (DE); and Wenli Zhang, Witten (DE)
Assigned to Gene Bridges GMBH, Heidelberg (DE)
Appl. No. 16/090,941
Filed by Gene Bridges GMBH, Heidelberg (DE)
PCT Filed Apr. 6, 2017, PCT No. PCT/EP2017/058306
§ 371(c)(1), (2) Date Oct. 3, 2018,
PCT Pub. No. WO2017/174753, PCT Pub. Date Oct. 12, 2017.
Claims priority of application No. 1605903 (GB), filed on Apr. 6, 2016.
Prior Publication US 2019/0382793 A1, Dec. 19, 2019
Int. Cl. C12N 15/86 (2006.01); C12N 15/87 (2006.01); C12N 7/00 (2006.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01)
CPC C12N 15/86 (2013.01) [C12N 7/00 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 2310/20 (2017.05); C12N 2710/10021 (2013.01); C12N 2710/10043 (2013.01); C12N 2800/80 (2013.01)] 10 Claims
 
1. A method for cloning an adenoviral sequence, wherein the adenoviral sequence is a full length adenoviral genome sequence, comprising:
a) providing a first linear nucleic acid molecule which comprises the full length adenoviral genome sequence;
b) providing a linearized medium copy plasmid which shares at least two regions of sequence homology with the first linear nucleic acid molecule; and
c) bringing the first linear nucleic acid molecule and the linearized medium copy plasmid into contact in the presence of a 5′ to 3′ exonuclease and an annealing protein such that the first linear nucleic acid molecule and the linearized medium copy plasmid recombine to form a circular plasmid containing the full length adenoviral genome sequence;
wherein the 5′ to 3′ exonuclease is full length RecE of SEQ ID NO:1412, or a protein with at least 95% sequence identity to SEQ ID NO:1412, and the annealing protein is RecT,
and wherein a first region of the at least two regions of sequence homology is a region of sequence homology with the 5′ ITR of the adenoviral sequence in the first linear nucleic acid molecule, and a second region of the at least two regions of sequence homology is a region of sequence homology with the 3′ ITR of the adenoviral sequence in the first linear nucleic acid molecule,
and wherein each of the first region and the second region is 40-80 nucleotides in length.