Biomaterial constructs for delivery of multiple therapeutic genes: a spatiotemporal evaluation of efficacy using molecular beacons.

Jennifer C Alexander,Abhay Pandit,Yury Rochev,Shane Browne

doi:10.1371/journal.pone.0065749

Abstract

Gene therapy is emerging as a potential therapeutic approach for cardiovascular pathogenesis. An appropriate therapy may require multiple genes to enhance therapeutic outcome by modulating inflammatory response and angiogenesis in a controlled and time-dependent manner. Thus, the aim of this research was to assess the spatiotemporal efficacy of a dual-gene therapy model based on 3D collagen scaffolds loaded with the therapeutic genes interleukin 10 (IL-10), a potent anti-inflammatory cytokine, and endothelial nitric oxide synthase (eNOS), a promoter of angiogenesis. A collagen-based scaffold loaded with plasmid IL-10 polyplexes and plasmid eNOS polyplexes encapsulated into microspheres was used to transfect HUVECs and HMSCs cells.The therapeutic efficacy of the system was monitored at 2, 7 and 14 days for eNOS and IL-10 mRNA expression using RT-PCR and live cell imaging molecular beacon technology. The dual gene releasing collagen-based scaffold provided both sustained and delayed release of functional polyplexes in vitro over a 14 day period which was corroborated with variation in expression levels seen using RT-PCR and MB imaging. Maximum fold increases in IL-10 mRNA and eNOS mRNA expression levels occurred at day 7 in HMSCs and HUVECs. However, IL-10 mRNA expression levels seemed dependent on frequency of media changes and/or ease of transfection of the cell type. It was demonstrated that molecular beacons are able to monitor changes in mRNA levels at various time points, in the presence of a 3D scaffolding gene carrier system and the results complemented those of RT-PCR.

Highlights

Cardiovascular diseases are the leading cause of death in the Western world and account for more than 17 million deaths globally (WHO 2012) [1]
This was evident from endothelial nitric oxide synthase (eNOS) Molecular beacons (MB) images which showed low FAM fluorescence signal at day two in both transfected Human umbilical vein endothelial cells (HUVECs) and Human mesenchymal stem cells (HMSCs), confirmed by RT-PCR data which showed the lowest eNOS mRNA expression at day two
Interleukin 10 (IL-10) mRNA expression for HUVECs remained at relatively constant level, while that of HMSCs was increased to a maximum at day seven and decreased to day two levels by day 14

Summary

Introduction

Cardiovascular diseases are the leading cause of death in the Western world and account for more than 17 million deaths globally (WHO 2012) [1]. Gene therapy is emerging as a potential therapeutic approach to address the challenges of cell-based strategies [3], with local gene transfer being a more effective therapy [4]. Combination therapies are becoming increasingly important strategies to improve the efficacy of therapeutics [5,6]. An appropriate gene therapy approach for cardiovascular pathogenesis may require multiple genes to enhance therapeutic outcome by modulating inflammatory response and angiogenesis in a controlled and time-dependent manner. Interleukin 10 (IL-10) is a potent multifunctional cytokine produced by a variety of cells [7]. It plays a crucial role in vivo in the attenuation of immune and inflammatory responses [8]. It is an enzyme that catalyses the conversion of the amino acid L-

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