Abstract
Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.
Highlights
Gene therapy is currently being evaluated for a wide range of genetic disorders including cancer, cystic fibrosis (CF), Parkinson’s disease (PD), sickle cell anemia, amyotrophic lateral sclerosis (ALS), etc
The gene therapy community has recognized that a general vector for all genetic disorders will be difficult to discover, the gene transfer agent has to be carefully chosen depending on a number of aspects such as: genetic condition of the disorder, targeted cell type, required number of treatments, and size and nature of the gene to be delivered
Negative control experiments were performed with a siNeg sequence, and the results indicated that the presence of siRNA in dual-activity vectors did not have any effect on the gene delivery capacity of these vectors (S2 Figure)
Summary
Gene therapy is currently being evaluated for a wide range of genetic disorders including cancer, cystic fibrosis (CF), Parkinson’s disease (PD), sickle cell anemia, amyotrophic lateral sclerosis (ALS), etc. An interesting approach to increase the antitumour activity of oncolytic viruses has been achieved through the use of RNAi. An interesting approach to increase the antitumour activity of oncolytic viruses has been achieved through the use of RNAi This double targeting approach has recently become more popular and involved the combined effect of oncolysis by adenoviruses and siRNA silencing. Another study showed that co-treatment with siRNA increased DNA replication of oncolytic Ad and, enhanced the spread of virus in the tumour environment [2]. This technology has been extended to other genes which are upregulated in cancer [3,4,5]. The results of these studies indicate that the use of oncolytic Ads to deliver tumour-targeted shRNA offers multiple potential benefits
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