Psoralen Treatment of Adenovirus Particles Eliminates Virus Replication and Transcription While Maintaining the Endosomolytic Activity of the Virus Capsid

Abstract

Adenovirus entry into its host cell transiently permeabilizes the cell allowing the coentry of reagents such as DNA. We compare here adenovirus inactivation with beta-propiolactone and several psoralen derivatives, seeking reagents that disrupt the viral genome without impairing the viral entry functions. No virus replication can be detected after 8-methoxypsoralen (8-MOP) modification. Viral transcription is not detectable by Northern analysis, and reverse transcriptase/PCR analysis demonstrates at least a 1000-fold decrease in viral transcription after 8-MOP treatment. Using [3H]8-MOP, the psoralen is found to enter the virus capsid and react throughout the viral genome, with approximately one psoralen modification per 100 bp of viral DNA. This inactivated adenovirus allows us to deliver DNA to target cells without interference from adenovirus gene expression or replication. Furthermore, we can now study the host cell response to adenovirus entry without the complications of adenovirus gene expression.

GALV expression enhances the therapeutic efficacy of an oncolytic adenovirus by inducing cell fusion and enhancing virus distribution

“…35 To obtain the non-replicating particle control, AdwtRGD UV-psoralen, the AdwtRGD was inactivated by UV-psoralen as previously described. 41 ICOVIR15 is a conditionally replicating adenovirus in which expression of the adenovirus E1A-D24 gene is regulated by a modified endogenous E1A promoter, which contains eight E2F-1-binding sites and one Sp1-binding site. To generate ICOVIR16, a shuttle plasmid, pNKFiberGALV, was constructed as follows.…”

Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo

“…We confirmed the potency of magnetofection with calcium phosphate coprecipitation, naked DNA and polylysine-DNA (not shown). Compared with a naked DNA magnetofectin (which hardly yielded any transfection; data not shown), complexation with cationic lipids or PEI (Figure 2a and b), as well as the incorporation of the endosomolytic peptide INF7 11 (not shown) or a chemically inactivated adenovirus 9 (Figure 2a and c) enhanced transfection several thousand-fold. Therefore, the mechanisms of cellular uptake and vector processing are probably similar to those of the parent vectors, namely endocytotic.…”

“…We formulated state-of-the-art nonviral and viral gene vectors (PEI-DNA, 8 AVET, 9 Lipofectamine, GenePorter and DOTAP-Cholesterol, 10 recombinant adenovirus and a retrovirus) as magnetofectins and compared reporter gene expression levels achieved with short-term incubation to the parent standard vector formulations. The data show that the formulation with paramagnetic nanoparticles alone enhanced transfection, while the application of a magnetic field raised reporter gene expression levels up to three orders of magnitude over those achieved with standard vectors under the same conditions ( Figure 2a, nonviral vectors; Figure 3, adenoviral vector; Figure 4, retroviral vector).…”

Enhancement of calcium phosphate-mediated transfection by inclusion of adenovirus in coprecipitates

“…For studies using a neutralizing antibody (Virostat, 1401, Portland, ME, USA), a 1:100 dilution of antibody was incubated with 2 × 10 10 particles of virus for 30 min at room temperature before formation of coprecipitates. For some experiments, virus was inactivated by psoralen/UV treatment as described by Cotten et al; 29 the inactivated virus showed a greater than 10 6 decrease in titer. As a reporter plasmid, we used p␤Gal-NLS which expresses ␤-galactosidase; this plasmid was a gift of Dr Sam Wadsworth (Genzyme, Framingham, MA, USA).…”