In vitro evaluation of a lentiviral two-step transcriptional amplification system using GAL4FF transactivator for gene therapy applications in bone repair

• Published in: Gene therapy Vol. 25; no. 4; pp. 260 - 268
• Main Authors: Bougioukli, Sofia, Sugiyama, Osamu, Alluri, Ram K., Yoho, Robert, Oakes, Daniel A., Lieberman, Jay R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-07-2018; Nature Publishing Group
• Subjects: 13/100; 13/106; 13/31; 13/44; 38; 42; 631/1647/2300/1850; 631/61/201; Adipose tissue; Biochemistry; Biomedical and Life Sciences; Biomedicine; Bone healing; Bone marrow; Bone morphogenetic protein 2; Bone Morphogenetic Protein 2 - biosynthesis; Bone Morphogenetic Protein 2 - genetics; Bone morphogenetic proteins; Bone Regeneration - genetics; Cell Biology; Cell differentiation; Cell Differentiation - genetics; Cells, Cultured; Cytotoxicity; DNA-Binding Proteins - genetics; Enzyme-linked immunosorbent assay; Female; Flow cytometry; Fluorescence; Gene Expression; Gene Therapy; Genes; Genetic research; Genetic Therapy - methods; Human Genetics; Humans; Lentivirus - genetics; Male; Medical schools; Mesenchymal Stem Cells - physiology; Middle Aged; Nanotechnology; Osteoarthritis - pathology; Osteoarthritis - therapy; Saccharomyces cerevisiae Proteins - genetics; Toxicity; Trans-Activators - genetics; Transcription; Transcription (Genetics); Transcription Factors - genetics; Transcriptional Activation; Transduction, Genetic; Transfection
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/s41434-018-0024-9

In this study, we developed a lentiviral two-step transcriptional amplification (TSTA) system expressing bone morphogenetic protein-2 (BMP-2) under the control of a GAL4FF transactivator to enhance gene expression and limit toxicity for bone repair applications. To this end human MSCs, isolated from bone marrow or adipose tissue, were transduced overnight with a LV-TSTA system (GAL4FF or GAL4vp16) expressing BMP-2 or GFP and evaluated in vitro for transduction efficiency, mean fluorescence intensity, cell viability, and BMP-2 production. FACS analysis of GFP-transduced MSCs confirmed successful transduction with the GAL4FF+GFP vector. Moreover, ELISA demonstrated abundant BMP-2 production by GAL4FF+BMP2-transduced human MSCs over a period of 8 weeks, with minimal cytotoxicity at all time points. Compared to GAL4vp16, GAL4FF was superior with respect to BMP production at 1, 2, 4, 6, and 8 weeks in BMSCs. In ASCs, GAL4FF was still associated with higher BMP-2 production at weeks 2–8, but this difference was not as prominent as in BMSCs. To our knowledge, this is the first report of GAL4FF-mediated BMP-2 production by human BMSCs and ASCs. Compared to the standard GAL4vp16TSTA vector, GAL4FF was associated with lower cytotoxicity and higher in vitro gene expression in both BMSCs and ASCs.