Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

Lina Li,Juan Li,Emilios K Dimitriadis,Yu Yang,Zhenhua Yuan,Luis Garcia,Richard H Smith,Cyriaque Beley,Chunping Qiao,Robert M Kotin

doi:10.1371/journal.pone.0069879

Abstract

Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×109 Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

Highlights

Non-viral gene transfer typically uses bacterial plasmids to introduce foreign DNA into recipient cells
DNA produced in this manner consists solely of a transgene of interest with flanking associated virus type 2 (AAV) inverted terminal repeats (ITRs), and, since it is produced in eukaryotic cells, is devoid of prokaryotic DNA modifications and bacterial endotoxin contamination
To test the hypothesis that Rep protein expression results in rescue and amplification of the integrated green fluorescent protein (GFP) vector genome, extrachromosomal DNA was extracted from stably transfected Sf9/ITR-GFP cells and analyzed using quantitative PCR (Fig. 2C, upper panel) and agarose gel electrophoresis (Fig. 2C, lower panel)

Summary

Introduction

Non-viral gene transfer typically uses bacterial plasmids to introduce foreign DNA into recipient cells. DNA for non-viral gene transfer would contain only the gene of interest, be devoid of prokaryotic modifications that can trigger an innate immune response [1,2,3,4], be in an exonuclease-resistant form, and lack detectable endotoxin contamination. DNA produced in this manner consists solely of a transgene of interest with flanking AAV ITRs, and, since it is produced in eukaryotic cells, is devoid of prokaryotic DNA modifications and bacterial endotoxin contamination. Structural characterization revealed the ITR-flanked transgenes as exonuclease-resistant, linear DNA molecules with covalently closed ends. We have termed these closed-ended, linear duplex molecules ‘‘CELiD’’ DNA

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Results

Conclusion