title: Optimal purification method for Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of vector administration.
abstract: Herpes simplex virus (HSV)-1 amplicon vectors could be packaged in the presence of replication-competent helper virus or in a helper virus-free system. In the latter system, cytotoxicity due to the expression of de novo viral gene expression is greatly reduced due to the absence of helper virus. However, the titers produced are relatively low in the range of 10(7) and 10(8)TU/ml after sucrose gradient concentration. This may become a limitation to certain gene transfer applications, such as brain disorder studies since the volume of vectors that could be administered is restricted. In contrast, amplicon viral vectors of high titers can be easily generated in the presence of helper viruses. Despite the potential cytotoxicity caused by the presence of helper virus in the latter method of viral packaging, studies involving vector targeting would still require the complementing function of helper virus for the generation of recombinant HSV-1 amplicon vectors with modified viral envelopes. In view of this, the optimal method of purifying Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of viral vector administration is examined. Parameters such as the ratio of amplicon versus helper viruses, the percentage of viral lost, and the extent of liver cytotoxicity induced by these viral vectors purified using different methods were investigated. In addition, the maximum recombinant HSV-1 viral dosage was also determined in vivo. Taken together, these findings may be of importance to the efficient production of contaminant-free HSV-1 amplicon viral vectors required for animal and human studies.
authors.affiliation: Gene Vector Laboratory, Division of Cellular and Molecular Research, National Cancer Centre, 11, Hospital Drive, Singapore 169610, Singapore.
authors.forename: Kian Chuan
authors.initials: KC
authors.lastname: Sia
authors.forename: Grace Y
authors.initials: GY
authors.lastname: Wang
authors.forename: Ivy A W
authors.initials: IA
authors.lastname: Ho
authors.forename: Hong Yuan
authors.initials: HY
authors.lastname: Khor
authors.forename: Lv
authors.initials: L
authors.lastname: Miao
authors.forename: Kam M
authors.initials: KM
authors.lastname: Hui
authors.forename: Paula Y P
authors.initials: PY
authors.lastname: Lam
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D014709:Vero Cells