title: Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects.
abstract: Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.
authors.affiliation: Department of Anatomy, John A Burns School of Medicine, Honolulu, HI 96822, USA.
authors.forename: Johann
authors.initials: J
authors.lastname: Urschitz
authors.forename: Miyuri
authors.initials: M
authors.lastname: Kawasumi
authors.forename: Jesse
authors.initials: J
authors.lastname: Owens
authors.forename: Kazuto
authors.initials: K
authors.lastname: Morozumi
authors.forename: Hideaki
authors.initials: H
authors.lastname: Yamashiro
authors.forename: Ilko
authors.initials: I
authors.lastname: Stoytchev
authors.forename: Joel
authors.initials: J
authors.lastname: Marh
authors.forename: James A
authors.initials: JA
authors.lastname: Dee
authors.forename: Kris
authors.initials: K
authors.lastname: Kawamoto
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Coates
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Kaminski
authors.forename: Pawel
authors.initials: P
authors.lastname: Pelczar
authors.forename: Ryuzo
authors.initials: R
authors.lastname: Yanagimachi
authors.forename: Stefan
authors.initials: S
authors.lastname: Moisyadi
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004249:DNA Damage; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D019895:Transposases / Q000235:genetics*
chemical_list: D019895:Transposases
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