Canine adenovirus type 2 vector generation via I-Sce1-mediated intracellular genome release.
When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.
Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France.
Sandy
S
Ibanes
Eric J
EJ
Kremer
D006516:Adenoviruses, Canine / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004278:DNA, Superhelical; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004285:Dogs; D005822:Genetic Vectors / Q000235:genetics*; D016678:Genome / Q000235:genetics*; D042541:Intracellular Space / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D004278:DNA, Superhelical; D011960:Receptors, Estrogen; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific