Linear DNAs Concatemerize in Vivo and Result in Sustained Transgene Expression in Mouse Liver

Abstract

The short duration of transgene expression remains a major obstacle for the implementation of nonviral DNA vectors in clinical gene therapy trials. Here, we demonstrate stable, long-term transgene expression in vivo by transfecting a linear DNA expression cassette (LDNA) into mouse liver. Interestingly, despite similar quantities and cellular distribution of injected DNAs in their livers, mice receiving LDNA encoding human alpha1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular (cc) DNA for a period of 9 months (length of study). Furthermore, when a linear human factor IX expression cassette was delivered to factor IX-deficient mice, sustained serum concentrations of more than 4 microg/ml (80% of normal) of the human clotting factor and correction of the bleeding diathesis were obtained. Southern blot analyses indicate that, unlike ccDNA, LDNA rapidly formed large, unintegrated concatemers in vivo, suggesting that transgene persistence from plasmid-based vectors was influenced by the structure of the vector in transfected cells. No differences in transgene expression or DNA molecular structures were observed when AAV ITRs were included to flank the hAAT expression cassette in both ccDNA- and LDNA-treated animals. Linear DNA transfection provides an approach for achieving long-term expression of a transgene in vivo.

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor1α promoter

“…However, we expect that the majority of plasmid DNA delivered to the lung will remain episomal in the absence of any selection and will eventually be diluted from the airway cell population as cell division in the lung proceeds. It is also possible that linear concatamers of the plasmid are present in the cell, which have been shown to result in significant persistence of transgene expression in the liver, 14 and these possibilities are being investigated.…”

Intravascular naked DNA vaccine encoding glycoprotein B induces protective humoral and cellular immunity against herpes simplex virus type 1 infection in mice

“…The hydrodynamics-based procedure was applied to transduce mice with a1-antitrypsin 14 and factor IX 14,15 genes, suggesting the potential usefulness of the system in controlling hereditary diseases. Lui et al 16 as well as the authors 17,18 delivered cytokine genes to the mice that had been implanted with syngenic tumor cells, resulting in significant suppression of the solid 16 or metastatic 17,18 As far as we know, the present study is the first report on the application of the intravenous naked pDNA transfer to the genetic vaccination against an infectious disease.…”

Gene Therapy Progress and Prospects: Nonviral vectors

“…Furthermore, a linear DNA expression cassette originating from this plasmid showed 10-to 100-fold higher expression than the closed circular DNA for a period of 9 months. 60 The Epstein-Barr virus (EBV)-based plasmid vector is known to self-replicate in cells. It carries two genetic elements from EBV, the EBV nuclear antigen 1 (EBNA1) gene and the oriP element.…”