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

Gene Therapy volume 25, pages 260–268 (2018)Cite this article

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Abstract

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.

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Acknowledgements

This work was supported by a National Institutes of Health grant to J.R.L. [R01AR057076]. The GAL4FF plasmid was kindly provided by Dr. Koichi Kawakami, National Institute of Genetics, Japan. The authors would also like to thank Frank Gonsalves of the Keck Hospital of USC and Judy Yoho of Dr. Yoho’s Cosmetic surgery practice for their valuable assistance in the study.

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Authors and Affiliations

  1. Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Sofia Bougioukli, Osamu Sugiyama, Ram K. Alluri, Daniel A. Oakes & Jay R. Lieberman

  2. Private practice, Visalia, CA, USA

    Robert Yoho

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  1. Sofia Bougioukli
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  2. Osamu Sugiyama
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  3. Ram K. Alluri
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  4. Robert Yoho
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  6. Jay R. Lieberman
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Correspondence to Jay R. Lieberman.

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Conflict of interest

S.B., O.S., R.K.A., R.Y., and D.A.O. have no conflicts to report. J.R.L. has received royalties and has served as a paid consultant for Depuy, is a shareholder in Hip Innovation Technologies, Inc. and has received royalties, financial or material support from Elsevier.

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Bougioukli, S., Sugiyama, O., Alluri, R.K. et al. In vitro evaluation of a lentiviral two-step transcriptional amplification system using GAL4FF transactivator for gene therapy applications in bone repair. Gene Ther 25, 260–268 (2018). https://doi.org/10.1038/s41434-018-0024-9

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