Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation.

Monika Savarin,Vesna Todorovic,Maja Cemazar,Gregor Sersa,Katarina Znidar,Urska Kamensek

doi:10.3390/ijms22063069

Abstract

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.

Highlights

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment [1,2]
The dual targeting plasmid was constructed in two phases: first, the expression cassette encoding shRNA against CD105 and CD146 was assembled using artificial gene synthesis (Figure 1A), and an antibiotic free version of the plasmid was prepared using operator repressor titration (ORT) technology
The plasmids were cut with different combinations of restriction enzymes and the identity of the plasmid was confirmed by positive matching of the pattern of bands on the electrophoresis gel to the expected pattern obtained by a simulation experiment using SnapGene software (Figure 1B,C) both plasmids were confirmed by full length plasmid sequencing and annotated plasmids maps were created based on sequencing results (Figure 1D,E)

Summary

Introduction

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment [1,2]. In the normal conditions its expression is low, upon activation of neoangiogenesis that is necessary for the progression of tumors, it becomes overexpressed, leading to changed regulation of migration, proliferation, differentiation and adhesion of endothelial cells [3,4,5,6]. Another tentative target of tumor vasculature is CD146 (melanoma cell adhesion molecule, MCAM), an adhesion molecule present on many tumors and on vascular endothelial and smooth muscle cells. Attempts to downregulate either CD105 or CD146 have been made in several in vitro and in vivo studies using either specific antibodies or RNA interference [10,11,12,13,14,15,16,17]

Objectives

Methods

Results