May, Rahel; Frauchiger, Daniela Angelika; Gazdhar, Amiq Ur Rahman; Geiser, Thomas; Benneker, Lorin Michael; Gantenbein, Benjamin (12 February 2016). Non-Viral Gene Therapy to Cells of the bovine Intervertebral Disc (Unpublished). In: YSBM.ch Symposium 2016.
Background. Low back pain is an increasing global health problem, which is associated with
intervertebral disc (IVD) damage and degeneration. Major changes occur in the nucleus pulposus (NP),
with the degradation of the extracellular matrix (ECM).1 Further studies showed that growth factors from
transforming growth factor β (TGFβ) and bone morphogenic proteins (BMP) family may induce
chondrogenic differentiation of mesenchymal stem cells (MSC).2 Focusing on non-viral gene therapies
and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can
induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing
GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition.
Methods. Bovine nucleus pulposus (bNPC) and annulus fibrosus cells (bAFC) were harvested from
bovine coccygeal IVD. Primary cells were then electroporized with plasmid GDF6 (Origene, vector
RG211366) by optimizing parameters using the Neon Transfection system (Life Technologies, Basel).
After transfection, cells were cultured in 2D monolayer or 3D alginate beads for 7, 14 or 21 days.
Transfection efficiency of pGDF6 was analyzed by immunohistochemistry and fluorescent microscopy.
Cell phenotype was quantified by real-time RT-PCR. To test a non-viral gene therapy applied directly to
3D whole organ culture, coccygeal bovine IVDs were harvested as previously described. Bovine IVDs
were transfected by injection of plasmid GDF6 into the center. Electroporation was performed with
ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) using 2-needle array electrode
or tweezertrodes. 72 h after tranfection discs were fixed and cryosectioned and analyzed by
immunofluorescence against GDF6.
Results. RT-PCR and immunohistochemistry confirmed up-regulation of GFP and GDF6 in the primary
bNPC/bAFC culture. The GFP-tagged GDF6 protein, however, was not visible, possibly due to failure of
dimer formation as a result of fusion structure. Organ IVD culture transfection revealed GDF6 positive
staining in the center of the disc using 2-needle array electrode. Results from tweezertrodes did not show
any GDF6 positive cells.
Conclusion. Non-viral transfection is an appealing approach for gene therapy as it fulfills the
translational safety aspects of transiency and lacks the toxic effects of viral transduction. We identified
novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD
explants electroporation parameters need to be further optimized.
Acknowledgements. This project was funded by the Lindenhof Foundation (Funds “Research &
Teaching”) Project no. 13-02-F. The imaging part of this study was performed with the facility of the
Microscopy Imaging Center (MIC), University of Bern.
References.
Roughly PJ (2004): Spine (Phila), 29:2691-2699
Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014), Arthritis Research
& Therapy, 16:R67
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