Recombinant retroviral vector interferes with the detection of amphotropic replication competent retrovirus in standard culture assays.
Many protocols for gene therapy employ recombinant retroviral vectors, which are replication-defective retroviruses engineered to serve as gene delivery vehicles. The use of retroviral vectors for human gene therapy requires careful screening of vector-producing cell lines and culture supernatants to ensure the absence of replication competent retrovirus (RCR) in clinical products. In this study we have examined several different culture assays routinely used to test for the presence of RCR. Results indicate that cocultivation of a vector-producing cell line with a permissive cell line can reproducibly detect a low level of contaminating RCR. RCR was detected less frequently in direct tests of cell-free culture supernatants from a contaminated vector-producing line. Further studies revealed that recombinant retroviral vector can interfere, to varying degrees, with the detection of low-level RCR in culture supernatants when a marker rescue assay, an extended mink S+L- assay or a PG-4 S+L- assay is used. Interference can be partially overcome by culturing the vector preparation with a permissive cell line for several days before testing on the indicator cell line. The interference phenomenon we have observed may also occur in other culture assays routinely used for the detection of RCR.
Printz
M
M
Viagene, Inc., San Diego, CA 92121-1204.
Reynolds
J
J
Mento
S J
SJ
Jolly
D
D
Kowal
K
K
Sajjadi
N
N
D000818:Animals; D016477:Artifacts; D001681:Biological Assay; D002460:Cell Line / Q000821:virology*; D017077:Culture Media, Conditioned / Q000032:analysis*; D046508:Culture Techniques / Q000379:methods; D003673:Defective Viruses / Q000302:isolation & purification*; D004285:Dogs; D005188:False Negative Reactions; D005816:Genetic Complementation Test; D005819:Genetic Markers; D015316:Genetic Therapy / Q000592:standards*; D005822:Genetic Vectors / Q000302:isolation & purification* / Q000592:standards; D008907:Mink; D009115:Muridae; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012190:Retroviridae / Q000302:isolation & purification* / Q000502:physiology*; D012449:Safety; D012680:Sensitivity and Specificity; D010948:Viral Plaque Assay; D014779:Virus Replication
D017077:Culture Media, Conditioned; D005819:Genetic Markers; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase