title: Safe two-plasmid production for the first clinical lentivirus vector that achieves >99% transduction in primary cells using a one-step protocol.
abstract: We report the design of a unique two-plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV-G pseudotyped vector derived from HIV-1 that expresses antisense to the HIV envelope gene. We found that a two-plasmid approach to production resulted in higher vector production titers when compared with a three-plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication-competent lentivirus (RCL). A native cis-acting ribozyme is used to prevent read through into the envelope gene from the upstream gag-pol genes in the packaging vector, thus preventing RNAs containing gag-pol and env together for comparable safety to a three-plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID-hu mouse transplantation model, which correlates with the presence of an anti-HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one-step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation.
authors.affiliation: VIRxSYS Corporation, Gaithersburg, MA 20877, USA.
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Lu
authors.forename: Laurent
authors.initials: L
authors.lastname: Humeau
authors.forename: Vladimir
authors.initials: V
authors.lastname: Slepushkin
authors.forename: Gwendolyn
authors.initials: G
authors.lastname: Binder
authors.forename: Qiao
authors.initials: Q
authors.lastname: Yu
authors.forename: Tatiana
authors.initials: T
authors.lastname: Slepushkina
authors.forename: Ziping
authors.initials: Z
authors.lastname: Chen
authors.forename: Randall
authors.initials: R
authors.lastname: Merling
authors.forename: Brian
authors.initials: B
authors.lastname: Davis
authors.forename: Yung-Nien
authors.initials: YN
authors.lastname: Chang
authors.forename: Boro
authors.initials: B
authors.lastname: Dropulic
mesh_terms: D000818:Animals; D016375:Antisense Elements (Genetics) / Q000235:genetics; D002478:Cells, Cultured; D015686:Gene Products, env / Q000235:genetics; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000009:adverse effects / Q000096:biosynthesis / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D016513:Mice, SCID; D010957:Plasmids / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics / Q000502:physiology; D012367:RNA, Viral / Q000378:metabolism; D013601:T-Lymphocytes; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000235:genetics
chemical_list: D016375:Antisense Elements (Genetics); D015686:Gene Products, env; D016337:RNA, Catalytic; D012367:RNA, Viral; D014764:Viral Proteins