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


CONCLUSIONS
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.
Hendriks
William T J
WT
Department of Neuroregeneration, Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam Zuidoost, the Netherlands.
Eggers
Ruben
R
Carlstedt
Thomas P
TP
Zaldumbide
Arnaud
A
Tannemaat
Martijn R
MR
Fallaux
Frits J
FJ
Hoeben
Rob C
RC
Boer
Gerard J
GJ
Verhaagen
Joost
J
D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D016086:Lentivirus / Q000502:physiology*; D009416:Nerve Regeneration / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013119:Spinal Cord Injuries / Q000473:pathology* / Q000628:therapy; D013126:Spinal Nerve Roots / Q000293:injuries / Q000378:metabolism*; D013997:Time Factors
D049452:Green Fluorescent Proteins