title: Optimization of the generation and propagation of gutless adenoviral vectors.
abstract: Adenoviral vectors devoid of all viral coding regions are referred to by many names, including gutless vectors. Gutless vectors display reduced toxicity and immunogenicity, increased duration of transgene expression, and increased coding capacity compared to early generation vectors, which contain the majority of the viral backbone genes. However, the production of gutless vectors at a scale and purity suitable for clinical use has limited the utility of this technology. In this work we describe the optimization of the production of gutless vectors. We constructed an improved helper virus and generated an alternative gutless vector producer cell line, PERC6-Cre. We demonstrated increased gutless vector yields, minimal helper virus contamination, and no replication-competent adenovirus contamination using the optimized system. Furthermore, the PERC6-Cre cells were adapted to serum-free suspension culture and high-titer gutless vector preparations were produced using bioreactor technology, suggesting the feasibility of gutless vector scale-up for clinical use. Finally, we observed that helper virus lacking a packaging signal could be packaged at a low frequency, revealing an inherent limitation to the differential packaging strategy for gutless vector propagation.
authors.affiliation: Genetic Therapy, Inc (A Novartis Company), Gaithersburg, MD 20878, USA.
authors.forename: Kiran
authors.initials: K
authors.lastname: Sakhuja
authors.forename: P Seshidhar
authors.initials: PS
authors.lastname: Reddy
authors.forename: Shanthi
authors.initials: S
authors.lastname: Ganesh
authors.forename: Floro
authors.initials: F
authors.lastname: Cantaniag
authors.forename: Scott
authors.initials: S
authors.lastname: Pattison
authors.forename: Paullin
authors.initials: P
authors.lastname: Limbach
authors.forename: Dawn B
authors.initials: DB
authors.lastname: Kayda
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kadan
authors.forename: Michael
authors.initials: M
authors.lastname: Kaleko
authors.forename: Sheila
authors.initials: S
authors.lastname: Connelly
mesh_terms: D000256:Adenoviridae; D017930:Genes, Reporter; D005822:Genetic Vectors; D006378:Helper Viruses; D016133:Polymerase Chain Reaction