Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors

Hiroyuki Nakai, Theresa A. Storm, Mark A. Kay

Research output: Contribution to journalArticle

159 Scopus citations

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 1α (EF1α) gene enhancer/promoter(s) (EF1αEP) with a splice donor. We co-injected a promoter-less nlslacZ vector with a vector containing either a single EF1αEP or a double copy of the EF1αEP 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 EF1αEP/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.

Original languageEnglish (US)
Pages (from-to)527-532
Number of pages6
JournalNature biotechnology
Volume18
Issue number5
DOIs
StatePublished - May 1 2000

Keywords

  • Adeno-associated virus vector
  • Concatemer
  • Gene therapy
  • Hepatocytes
  • Intermolecular recombination

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

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