title: Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors.
abstract: A major shortcoming to the use of adeno-associated virus (rAAV) vectors is their limited packaging size. To overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. The vectors contained either a nuclear localizing Escherichia coli lacZ transgene (nlslacZ) with a splice acceptor, or the human elongation factor 1alpha ( EF1alpha) gene enhancer/promoter(s) (EF1alphaEP) with a splice donor. We co-injected a promoter-less nlslacZ vector with a vector containing either a single EF1alphaEP or a double copy of the EF1alphaEP in a head-to-head orientation, into the portal vein of mice. Gene expression, measured by both transduction efficiency and quantitation of the recombinant protein, was as much as 60-70% of that obtained from mice that received a single vector containing a complete EFalphaEP/nlslacZ expression cassette. This two-vector approach may allow development of gene therapy strategies that will carry exogenous DNA sequences with large therapeutic cDNAs and/or regulatory elements.
authors.affiliation: Program in Human Gene Therapy, Department of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: H
authors.initials: H
authors.lastname: Nakai
authors.forename: T A
authors.initials: TA
authors.lastname: Storm
authors.forename: M A
authors.initials: MA
authors.lastname: Kay
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage*; D007275:Injections, Intravenous; D007763:Lac Operon; D008099:Liver / Q000821:virology; D051379:Mice; D020648:Peptide Elongation Factor 1 / Q000096:biosynthesis / Q000235:genetics; D011169:Portal Vein; D011994:Recombinant Proteins / Q000096:biosynthesis*; D011995:Recombination, Genetic; D019076:Transgenes; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
chemical_list: D020648:Peptide Elongation Factor 1; D011994:Recombinant Proteins; D001616:beta-Galactosidase