Novel integrating adenoviral/retroviral hybrid vector for gene therapy

Stephen J. Murphy, Heung Chong, Stephen Bell, Rosa Maria Diaz, Richard Geoffrey Vile

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

A hybrid adenoviral vector system was designed to incorporate an excisable retroviral cassette that can be stably integrated into the host cell genome. The vector contains the terminal sequences of two Moloney murine leukemia virus retroviral long terminal repeats (LTRs), fused to form a junction fragment, and is flanked by two loxP recognition sequences. Cre recombinase-directed excision liberates a circular, double-stranded DNA molecule containing the LTR junction fragment. Despite the natural intermediate for retroviral integrase being a linear DNA molecule, we show that, in the presence of Cre and retroviral Gag and Pol, the excised circle can be integrated into the target cell genome through both specific integrase (Int)-directed mechanisms and by a random integration process. The loxP cassette, carrying in addition a selectable marker gene, was incorporated into the E1-deleted region of an adenoviral vector. Infection of cells expressing Cre, Gag, and Pol generated clones that survived long term in drug selection (>3 months). Int-mediated integration was demonstrated in seven of nine clones by sequencing of the integration sites. In addition, the introduction of the loxP cassette into 293 cells coexpressing Cre and Int alone in the absence of other Gag and Pol proteins was sufficient to catalyze the integration mechanism. These experiments demonstrate that it is possible to generate high-titer adenovirus-mediated delivery of a C-type retroviral provirus that can subsequently undergo retroviral Int-mediated integration into dividing and nondividing cells.

Original languageEnglish (US)
Pages (from-to)745-760
Number of pages16
JournalHuman Gene Therapy
Volume13
Issue number6
DOIs
StatePublished - 2002

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Integrases
Genetic Therapy
Terminal Repeat Sequences
gag-pol Fusion Proteins
Clone Cells
Genome
Moloney murine leukemia virus
Proviruses
DNA
Adenoviridae
Infection
Pharmaceutical Preparations
Genes

ASJC Scopus subject areas

  • Genetics

Cite this

Novel integrating adenoviral/retroviral hybrid vector for gene therapy. / Murphy, Stephen J.; Chong, Heung; Bell, Stephen; Diaz, Rosa Maria; Vile, Richard Geoffrey.

In: Human Gene Therapy, Vol. 13, No. 6, 2002, p. 745-760.

Research output: Contribution to journalArticle

Murphy, Stephen J. ; Chong, Heung ; Bell, Stephen ; Diaz, Rosa Maria ; Vile, Richard Geoffrey. / Novel integrating adenoviral/retroviral hybrid vector for gene therapy. In: Human Gene Therapy. 2002 ; Vol. 13, No. 6. pp. 745-760.
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