title: An Optimized Protocol for Packaging Pseudotyped Integrase Defective Lentivirus.
abstract: BACKGROUND
  A number of integrase defective lentiviral (IDLV) packaging systems have been developed to produce integration deficient lentiviruses for gene delivery and epichromosomal expression. However, despite their growing demand, a comparative study to systemically evaluate the performance efficiency of different mutants on virus packaging and gene expression has not been done.
  
  
  RESULTS
  Site-directed mutagenesis was used to generate five integrasedeficient mutants for non-integrative lentiviral packaging (NILVP). The five mutants were then individually incorporated to make different integrase defective lentivirus plasmid packaging mix, keeping other packaging factors constant. CD511B-1, a lentivectorexpressing GFP from an EF1 promoter, was packaged with each of the five different lentivirus packaging mix to make pseudotypedviral particles. The performance and packaging efficiency of each of the integrase deficient mutants was evaluated based on GFP expression in HT1080 cells, while the wild type lentivirus packaging mix was used as a control. Of the five integrase mutant candidates, one with the highestGFP transgene expression level was chosen for further characterization. The non-integrative nature of this candidate was confirmed by quantitative polymerase chain reaction and characterized using both dividing and non-dividing cells. Finally, a detailed standard protocol for NILVP using this integrase defective mutant was developed.
  
  
  CONCLUSIONS
  An efficient lentiviral packaging system for producing on-integrative lentivirus was established. This system is compatible with most existing lentivectors and can be used to transduce both dividing and non-dividing cells.
authors.affiliation: System Biosciences (SBI), 265 North Whisman Rd., Mountain View, CA 94043 USA.
authors.forename: Ranjita
authors.initials: R
authors.lastname: Sengupta
authors.affiliation: System Biosciences (SBI), 265 North Whisman Rd., Mountain View, CA 94043 USA.
authors.forename: Chandreyee
authors.initials: C
authors.lastname: Mukherjee
authors.affiliation: Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA USA.
authors.forename: Nandita
authors.initials: N
authors.lastname: Sarkar
authors.affiliation: System Biosciences (SBI), 265 North Whisman Rd., Mountain View, CA 94043 USA.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Sun
authors.affiliation: System Biosciences (SBI), 265 North Whisman Rd., Mountain View, CA 94043 USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Lesnik
authors.affiliation: System Biosciences (SBI), 265 North Whisman Rd., Mountain View, CA 94043 USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Bioengineering, School of Engineering, Santa Clara University, 500 El Camino Real, Santa Clara, CA 95053 USA.
authors.forename: Biao
authors.identifier: 0000-0002-0307-9901
authors.initials: B
authors.lastname: Lu
keywords: Integrase defective lentivirus; lentiviral package; viral transduction