Improved osteogenic vector for non-viral gene therapy

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 adiposederived 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.

Delivery of the improved BMP-2-Advanced plasmid DNA within a gene-activated scaffold accelerates mesenchymal stem cell osteogenesis and critical size defect repair

“…In a previous study, to streamline transcription while also increasing protein production, an artificial highly truncated intron of 96 bases was inserted into the BMP-2 gene and codon optimisation was performed. This has led to faster transcription of the encoded gene and enhanced protein production and this novel plasmid is referred to as pBMP-2-Advanced [28] ( Figure 1C).…”

“…However, the elongation factor 1 (EF1 ) promoter has also been shown to be an efficient alternative particularly in mesenchymal stem cells (MSCs) [30]. To test which promoter sequence was more efficient in promoting transcription of the BMP-2 gene, two different versions of the pBMP-2-Advanced plasmid were tested, containing either the CMV or the EF1 promoter [28] ( Figure 1D). The objective of this study was to investigate if using a series of modified BMP-2 plasmids could enhance the efficacy of a pBMP-2 gene-activated scaffold.…”

Comparative analysis of bone regeneration behavior using recombinant human BMP‐2 versus plasmid DNA of BMP‐2

“…). Endogenous BMP‐2 expression, evoked by gene vectors, is more physiological in terms of, for example, host‐specific glycosylation, because of de novo synthesis by local host cells …”

“…Nonviral vectors are advantageous in that they do not tend to insert permanently into the host's genome. Furthermore, the host's immune response is not triggered at such high levels as has been observed for other approaches …”

“…On the other hand, GFs have several adverse effects, such as short half‐life, high costs, and potential systemic side‐effects, and an incompletely understood dose dependency . Because of these disadvantages, local gene therapy for bone regeneration in animal defect models has emerged as a possible alternative strategy by expressing GFs and triggering GF expression in target cells for short or long time periods to stimulate osteogenesis . However, even though several proof‐of‐principle experiments have been published, to our knowledge, no systematic studies have been performed to date to compare the two approaches.…”