Lentiviral vector-mediated reporter gene expression in avulsed spinal ventral root is short-term, but is prolonged using an immune "stealth" transgene

Article type: Research Article

Authors: Hendriks, William T.J. | Eggers, Ruben | Carlstedt, Thomas P. | Zaldumbide, Arnaud | Tannemaat, Martijn R. | Fallaux, Frits J. | Hoeben, Rob C. | Boer, Gerard J. | Verhaagen, Joost

Affiliations: Department of Neuroregeneration, Netherlands Institute for Neuroscience, an Institute of the Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam Zuidoost, the Netherlands | Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, the Netherlands | PNI-UNIT, the Royal National Orthopaedic Hospital Trust, Brockley Hill, Stanmore, Middlesex HA7 4LP, UK

Note: [] Corresponding author: J. Verhaagen, Department of Neuroregeneration, Netherlands Institute for Neuroscience, an institute of the Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam Zuidoost, the Netherlands. Tel.: +31 20 5665513; Fax: +31 20 5666121; E-mail: j.verhaagen@nin.knaw.nl

Abstract: Purpose: Spinal root avulsions result in paralysis of the upper and/or lower extremities. Implanting a peripheral nerve bridge or reinsertion of the avulsed roots in the spinal cord are surgical strategies that lead to some degree of functional recovery. In the current study lentiviral (LV) vector-mediated gene transfer of a green fluorescent protein (GFP) reporter gene was used to study the feasibility of gene therapy in the reimplanted root to further promote regeneration of motor axons. Methods: A total of 68 female Wistar rats underwent unilateral root avulsion of the L4, L5 and L6 ventral lumbar roots. From 23 rats intercostal nerves were dissected before ventral root avulsion surgery, injected with a lentiviral vector encoding GFP (LV-GFP) and inserted between the spinal cord and avulsed rootlet. In the remaining 45 rats, the avulsed ventral root was injected with either LV-GFP or a lentiviral vector encoding a fusion between a GlyAla repeat and GFP (LV-GArGFP), and reinserted into the spinal cord. Expression of GFP was evaluated at 1, 2, 4 and 10 weeks, and one group at 4 months. Results: LV-GFP transduction of either nerve implants or reimplanted ventral roots revealed high GFP expression during the first 2 post-lesion weeks, but virtually no expression at 4 weeks. Since this reduction coincided with the appearance of mononuclear cells at the repair site, an immune response against GFP may have occurred. In a subsequent experiment reimplanted ventral roots were transduced with a vector encoding GFP fused with the GlyAla repeat of Epstein-Barr virus Nuclear Antigen 1 known to prevent generation of antigenic peptides from transgene products. Expression of this "stealth" gene persisted for at least 4 months in the reimplanted root. Conclusion: Thus persistent transgene expression can be achieved with non-immunogenic transgene products in reimplanted ventral roots. This demonstrates the feasibility of combining neurosurgical repair with LV vector-mediated gene therapy. The current approach will be used in future experiments with LV vectors encoding neurotrophic factors to enhance the regeneration of spinal motor neurons after traumatic avulsion of spinal nerve roots.

Journal: Restorative Neurology and Neuroscience, vol. 25, no. 5-6, pp. 585-599, 2007