An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction

Terence R. Flotte, Ximena Barraza-Ortiz, Rikki Solow, Sandra A. Afione, Barrie J. Carter, William B Guggino

Research output: Contribution to journalArticle

Abstract

Adeno-associated virus (AAV) vectors are potentially useful for gene therapy of a number of human diseases. However, the use of these vectors has been limited by the lack of stable vector-packaging cell lines. The difficulties in developing packaging cell lines relate to low levels of rep gene expression from the AAV-p5 promoter, and to the propensity of Rep proteins to suppress continued growth of immortalized cell lines. We describe here two new techniques which allow these problems to be circumvented. First, we have demonstrated that expression of rep from the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter results in a 10-fold improvement of packaging efficiency. Second, we have overcome the inefficiency of vector plasmid transfection by generating cell populations containing rescuable AAV recombinant genomes. These improvements yielded a net increase of 50-fold in the packaging efficiency of the AAVp5neo and AAVp5lacZ recombinant vectors. The AAVp5lacZ vector packaged with this method was administered systemically to recently weaned C57BL mice, and mediated efficient expression of the β-galactosidase reporter gene in cells of the airway epithelium and spleen. This indicates the in vivo activity of these vector stocks, and their potential utility for gene therapy.

Original languageEnglish (US)
Pages (from-to)29-37
Number of pages9
JournalGene Therapy
Volume2
Issue number1
StatePublished - 1995

Fingerprint

Dependovirus
Product Packaging
Cell Line
Genetic Therapy
HIV Long Terminal Repeat
Galactosidases
Inbred C57BL Mouse
Reporter Genes
Transfection
Plasmids
Spleen
Epithelium
Genome
Gene Expression
Growth
Population
Proteins

Keywords

  • Adeno-associated virus
  • Gene therapy
  • Packaging methods

ASJC Scopus subject areas

  • Genetics

Cite this

Flotte, T. R., Barraza-Ortiz, X., Solow, R., Afione, S. A., Carter, B. J., & Guggino, W. B. (1995). An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction. Gene Therapy, 2(1), 29-37.

An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction. / Flotte, Terence R.; Barraza-Ortiz, Ximena; Solow, Rikki; Afione, Sandra A.; Carter, Barrie J.; Guggino, William B.

In: Gene Therapy, Vol. 2, No. 1, 1995, p. 29-37.

Research output: Contribution to journalArticle

Flotte, TR, Barraza-Ortiz, X, Solow, R, Afione, SA, Carter, BJ & Guggino, WB 1995, 'An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction', Gene Therapy, vol. 2, no. 1, pp. 29-37.
Flotte, Terence R. ; Barraza-Ortiz, Ximena ; Solow, Rikki ; Afione, Sandra A. ; Carter, Barrie J. ; Guggino, William B. / An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction. In: Gene Therapy. 1995 ; Vol. 2, No. 1. pp. 29-37.
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