Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

Guanghua Yang,Cheng Qian,Veronica Fernandez-Ruiz,Xin Huang,M Gabriela Kramer,Zhongmin Liu,Jesus Prieto,Milosz P Kawa

doi:10.1038/srep17166

Abstract

Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

Highlights

For gene therapy-based anti-tumor treatment, therapeutic genes need to be introduced and highly expressed in neoplastic cells, which remains a challenge in the field
The second cassette consisted of the endothelial specific promoter (VEcad) for driving the expression of the Tet-responsive transactivator in the endothelial cells
In the other construction, where the two cassettes were oriented in the sense direction with respect to the vector RNA, the two cassettes shared the same poly (A) signal of the vector in the 3′ long terminal repeat (LTR)

Summary

Introduction

For gene therapy-based anti-tumor treatment, therapeutic genes need to be introduced and highly expressed in neoplastic cells, which remains a challenge in the field. Mass — up to 95% of the tumor vasculature in the peripheral region[4,5] Transduction of these endothelial cells with therapeutic genes holds the potential to retard the tumor growth—even to eradicate it. One mutant derived from rtTA, named rtTA2S-M2, was introduced into the Tet-on/off system This mutant transactivator binds with much lower efficiency to the tetO regions than rtTA in the uninduced state, and its VP16 domain was shortened to avoid cell toxicity[11,12]. This mutant transactivator is highly sensitive to Dox; it can induce the same gene expression levels at 10% of Dox dose required with the original rtTA12. The cassette placed in the opposite orientation has shown a higher transgene expression level[14]

Objectives

Methods

Results

Conclusion