Highly efficient gene transfer into murine liver achieved by intravenous administration of naked Epstein–Barr virus (EBV)-based plasmid vectors

Abstract

Naked plasmid DNA (pDNA) injection could become an alternative procedure to viral and nonviral gene delivery systems. We have previously shown that Epstein-Barr virus (EBV)-based plasmid vectors containing the EBV nuclear antigen 1 (EBNA1) gene and the oriP sequence enable quite high and long-lasting expression in various in [9][10][11] and to a lesser extent, in the spleen, kidney, lung and heart. 9 The expression levels critically depended on the injection volume and rate, strongly suggesting that the 'hydrodynamic' pressure plays a key role in the genetic transfer. The Epstein-Barr virus (EBV)-based plasmid vector carries two genetic elements from EBV, ie the EBNA1 gene and the oriP element. 12 The EBNA1 binds to oriP in a sequence-specific manner and exerts various effects on the oriP-bearing plasmid. In human cells, the plasmid replicates in synchrony with chromosomal DNA, resulting in long-term retention and expression. 12 The EBNA1 gene and oriP have been employed to engineer artificial chromosomes. 13,14 On the other hand, the EBNA1 also facilitates nuclear localization of the plasmid, 15,16 binding of plasmid to the nuclear matrix, 17 and transcriptional up-regulation. [18][19][20] Collectively, the EBVbased plasmid vector gives not only a prolonged, but also a stronger expression of the transgene. We have applied the EBV-plasmids to various preclinical studies of gene therapy, including those for hereditary, 21 chronic, 22,23 as well as malignant diseases. [24][25][26][27] In some of these studies, the EBV-based plasmid vectors were combined with cationic liposomes, 25,28 cationic polymers, 23,[25][26][27]29 electroporation 21 or gene gun, 30 while in the other studies, naked EBV-based plasmid was injected into the cardiac muscle. 22,29 In the present study, we transferred the naked EBV-based plasmid vectors to the liver through the intravascular route.EBV-based and conventional plasmid vectors encoding the luciferase or ␤-gal genes as markers (Figure 1, upper panels) were constructed and injected intravenously into the tail vein of mice as described. 9 The injection with pG.GL3, a conventional luciferase-expression vector,

Sonoporation using microbubble BR14 promotes pDNA/siRNA transduction to murine heart

“…The plasmids pGEG.GL3 [28], pGE-G.EGFP [29], and pGEG.b [30] carried GL3 firefly luciferase (Luc), EGFP, and Escherichia coli b-galactosidase (b-gal) genes, respectively, under the control of the CAG promoter. Each plasmid also contained Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene and EBV oriP sequence [31].…”

“…At the same time as the initiation of the injection, transthoracic ultrasound insonation (sonication) was performed through a 6-mm diameter probe with an input frequency of 1 MHz, an output intensity of 1.0-2.0 W/cm 2 , a pulse duty ratio (PDR) of 10-50%, and a duration of 60 s. A Sonitron 2000 (Rich-Mar, Inola, OK, USA) was used to generate the ultrasound. Hydrodynamics-based transduction was performed as described previously [28]. Briefly, 40 lg siRNA-GFP was diluted in 1600 ll PBS and injected intravenously within 4s via the tail vein using a 27-gauge needle.…”

“…Organs other than the heart were also treated as described above. Protein concentration in the extract was determined as described previously [28].…”

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

“…We have previously shown that use of an Epstein-Barr virus (EBV)-based plasmid vector that harbors the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence further improved the hydrodynamics-mediated transfection, in terms of the transfer efficiency as well as magnitude and longevity of expression. 5 The herpes simplex viruses cause a variety of clinical illnesses that are painful and often distressing. 6,7 These include oral-facial, genital, ocular, and cutaneous herpes, herpes encephalitis, neonatal herpes, and erythema multiforme.…”

Cytokine genetic adjuvant facilitates prophylactic intravascular DNA vaccine against acute and latent herpes simplex virus infection in mice

“…[10][11][12] As a result, the animals were highly resistant to a subsequent lethal HSV-1 challenge. 13 The surviving mice also escaped latent infection, which was not reported in earlier studies using intramuscular pDNA vaccination.…”