Current Issue

Volume 19, Number 3, Oct-Dec (Autumn) 2017, Serial Number: 75 Pages: 361-375

Multiple Sclerosis Gene Therapy Using Recombinant Viral Vectors: Overexpression of IL-4, IL-10 and Leukemia Inhibitory Factor in Wharton’s Jelly Stem Cells in The EAE Mice Model


Ahmad Hosseini, Ph.D, 1, 2, *, Hajar Estiri, Ph.D, 3, 4, Haleh Akhavan Niaki, Ph.D, 5, 6, Akram Alizadeh, Ph.D, 7, Baharak Abdolhossein Zadeh, Ph.D, 3, Sayyed Mohammad Hossein Ghaderian, M.D., Ph.D, 8, Akbar Farjadfar, Ph.D, 9, Ali Fallah, Ph.D, 3, 10,
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Department of Cell Biology and Anatomical Science, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
Iranian Institute of Cell and Gene Therapy, Tehran, Iran
Cellular and Molecular Biology Research Center, Babol University of Medical Sciences, Babol, Iran
Department of Genetics, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran
Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran
Urogenital Stem Cell Research, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Department of Biotechnology, Fasa University of Medical Sciences, Fasa, Iran
BioViva USA Inc, Bainbridge Island WA, USA
*Corresponding Address: P.O. BOX: 193954719 Cellular and Molecular Biology Research Center Shahid Beheshti University of Medical Sciences Tehran Iran Email:Prof_hosseini@yahoo.com

Abstract

Objective

Immunotherapy and gene therapy play important roles in modern medicine. The aim of this study is to evaluate the overexpression of interleukin-4 (IL-4), IL-10 and leukemia inhibitory factor (LIF) in Wharton’s jelly stem cells (WJSCs) in the experimental autoimmune encephalomyelitis (EAE) mice model.

Materials and Methods

In this experimental study, a DNA construction containing IL- 4, IL-10 and LIF was assembled to make a polycistronic vector (as the transfer vector). Transfer and control vectors were co-transfected into Human Embryonic Kidney 293 (HEK-293T) cells with helper plasmids which produced recombinant lentiviral viruses (rLV). WJSCs were transduced with rLV to make recombinant WJSC (rWJSC). In vitro protein and mRNA overexpression of IL-4, LIF, and IL-10 were evaluated using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot (WB) analysis. EAE was induced in mice by MOG-CFA and pertussis toxin. EAE mice were injected twice with 2×105 rWJSCs. The in vivo level of IL-4, LIF, IL-10 cytokines and IL-17 were measured by ELISA. Brain tissues were analyzed histologically for evaluation of EAE lesions.

Results

Isolated WJSCs were performed to characterize by in vitro differentiation and surface markers were analyzed by flow cytometry method. Cloning of a single lentiviral vector with five genes was done successfully. Transfection of transfer and control vectors were processed based on CaPO4 method with >90% efficiency. Recombinant viruses were produced and results of titration showed 2-3×107 infection-unit/ml. WJSCs were transduced using recombinant viruses. IL-4, IL-10 and LIF overexpression were confirmed by ELISA, WB and qPCR. The EAE mice treated with rWJSC showed reduction of Il-17, and brain lesions as well as brain cellular infiltration, in vivo. Weights and physical activity were improved in gene-treated group.

Conclusion

These results showed that gene therapy using anti-inflammatory cytokines can be a promising approach against multiple sclerosis (MS). In addition, considering the immunomodulatory potential of WJSCs, an approach using a combination of WJSCs and gene therapy will enhance the treatment efficacy.