eCM (Eur Cell Mater / e Cells & Materials) Not-for-profit Open Access
Created by Scientists, for Scientists
 ISSN:1473-2262         NLM:100973416 (link)         DOI:10.22203/eCM

2016   Volume No 31 – pages 191-204

Title: Improved osteogenic vector for non-viral gene therapy

Authors: ARA Hacobian, K Posa-Markaryan, S Sperger, M Stainer, D Hercher, GA Feichtinger, CMAP Schuh, H Redl

Address: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, A-1200 Vienna, Austria

E-mail: ara.hacobian at trauma.lbg.ac.at

Key Words: Bone morphogenetic protein, gene therapy, osteogenic differentiation, bone regeneration, codon optimisation, intron, non-viral, secretion signal.

Publication date: March 20th 2016

Abstract: Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2) gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp). Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α) promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays  showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.

Article download: Pages 191-204 (PDF file)
DOI: 10.22203/eCM.v031a13