US 12,264,371 B2
Methods and reagents for assessing the presence or absence of replication competent virus
Ruth Berry, Seattle, WA (US); and Edwin Webb, Seattle, WA (US)
Assigned to Juno Therapeutics, Inc., Seattle, WA (US)
Filed by Juno Therapeutics, Inc., Seattle, WA (US)
Filed on Nov. 22, 2022, as Appl. No. 17/992,888.
Application 17/992,888 is a continuation of application No. 16/966,414, granted, now 11,535,903, previously published as PCT/US2019/016194, filed on Jan. 31, 2019.
Claims priority of provisional application 62/624,801, filed on Jan. 31, 2018.
Prior Publication US 2023/0193411 A1, Jun. 22, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6851 (2018.01); C07K 14/16 (2006.01); C12Q 1/686 (2018.01); C12Q 1/70 (2006.01)
CPC C12Q 1/702 (2013.01) [C07K 14/161 (2013.01); C07K 14/162 (2013.01); C12Q 1/6851 (2013.01); C12Q 1/686 (2013.01); C12N 2740/13011 (2013.01)] 14 Claims
 
1. A method for detecting a presence, absence, or amount of a viral DNA, comprising:
(a) incubating a mixture comprising (i) DNA from at least one cell that contains a heterologous nucleic acid introduced by transduction of the at least one cell with a lentiviral vector comprising the heterologous nucleic acid, (ii) at least one forward oligonucleotide primer and at least one reverse oligonucleotide primer, each specific for one or more sequence of a viral DNA sequence, (iii) an oligonucleotide probe specific for each of said one or more sequence of a viral DNA, and (iv) a DNA polymerase, under conditions sufficient to amplify one or more sequence of a viral DNA by polymerase chain reaction (PCR), if present in a biological sample; and
(b) detecting the presence, absence, amount or concentration of the amplified viral DNA,
wherein the one or more sequence of a viral DNA includes a viral DNA from at least a portion of an env gene, and the at least one forward and reverse oligonucleotide primers comprise:
a forward oligonucleotide primer specific for the VSV-G env gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in SEQ ID NO: 35, 50, 51, or 54 and a reverse oligonucleotide primer specific for the env gene comprising a sequence with at least 90% identity and/or at least 15 contiguous nucleotides of a sequence set forth in SEQ ID NO: 36, 52, 53, or 55.