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Neuromodulation therapies aim to correct imbalanced neural activity, which underlie diseases such as epilepsy, pain, and spasticity. In the case of spasticity, there is excessive excitatory drive to motor neurons that causes exaggerated muscle reflexes and impaired motor function. Gene-based neuromodulation with the inward rectifying potassium channel 2.1 (Kir2.1) has the potential to inhibit excessive neural activation. Kir2.1 is normally found in excitable cells (e.g. neurons, muscle) and acts to stabilize membrane potential below the activation potential of voltage gated sodium channels. In vivo gene transfer was accomplished using an adenoviral (Ad) vector containing an expression cassette with the inducible RheoSwitch™ (Rheo) promoter upstream of the Kir2.1 transgene (i.e. Ad.Rheo.Kir2.1). The Rheo promoter only allowed gene expression in the presence of its orally administered RheoChem™ ligand. To test the ability of the Ad.Rheo.Kir2.1 viral vector to mediate inducible neuromuscular inhibition, we injected it unilaterally into the L1-4 spinal cord of rats. Behavioral assays demonstrated neuromuscular inhibition exclusively in rats that received the ligand. However, histological analysis showed evidence of both Kir2.1 gene expression and cell death in rats that received ligand. Motor neuron loss was consistently noted in spinal cord segments closest to the injection epicenter, which appeared to have the greatest transgene expression. In the adjacent spinal cord sections, which displayed lower levels of transgene expression, no cell loss was noted but apparently healthy motor neurons expressing Kir2.1 were found. Rats that did not receive ligand did not express the transgene, had no functional deficits, and contained normal numbers of motor neurons. Overall, we conclude that: (1) the Rheo system was able to mediated inducible control of our transgene; (2) the Kir2.1 transgene produced a biologically active ion channel; and (3) future experiments in which we administer the virus at a lower concentration will be necessary to determine whether neuromuscular inhibition can be achieved in the absence
Neuromodulation is the specific manipulation of neural activity. Focal inhibition of neural activity has been proven effective in restoring neural function in disorders that lack regular neuronal activity. Current neuromodulation therapies used to treat spasticity include the ablation of dorsal root axons (i.e. dorsal rhizotomy), which is non-adjustable and permanent, as well as neuromuscular blockade with botulinum toxin that requires repeated administration and is subject to tolerance concerns. Gene-based neuromodulation aims to provide a safer, less invasive method of treatment to correct imbalanced neural activity through the selective control of certain genes. The use of viral vector based therapy offers many advantages over current methods of focal inhibition treatment. Viral gene therapy can provide molecular specificity that can manipulate individual proteins in synaptic transmission. Furthermore, it eliminates destruction and permanent alteration of neural pathways.
The gene we used to induce focal inhibition of neuronal activity was the inward rectifying potassium channel 2.1 (Kir2.1). Potassium (K+) channels found in cells work to regulate the electrical excitability and maintenance of the resting membrane potential. Of the four potassium channel families, the family of inward rectifying potassium (Kir) channels has an important role in the control of nervous impulses, or action potentials, in excitable cells, particularly in neurons where currents flow more easily in the inward direction. The Kir2.1 gene partakes in stabilizing the resting potential of the cell at a sufficiently negative level to prevent activation of voltage gated sodium channels and action potential generation.1 A previous study has shown suppressed excitability in neurons without affecting normal electrical activity by using an adenoviral vector to drive expression of Kir2.1 in vitro. 2 Therefore, we hypothesized that direct viral vector delivery of the Kir2.1 gene to the spinal cord could regulate excitability of nervous impulse. This treatment would be significant for patients who suffer from Parkinson’s disease, spasticity, epilepsy, and pain.
A critical aspect of gene-based neuromodulation is the regulation and control of gene expression. The addition of a promoter upstream of the Kir2.1 gene served to control transcription of the Kir2.1 gene. The promoter known as RheoSwitch® is activated in the presence of the RheoChem™ ligand. When this ligand binds to the promoter region, the RheoSwitch® is turned on and then transcription of the Kir2.1 gene can occur. As expected, we found Kir2.1 gene expression only occurred after the viral vector injection combined with delivery of the ligand.
The Rheo.Kir2.1 expression cassette included the green fluorescent protein (GFP) reporter gene and was fused into an adenovirus vector (i.e. Ad.Rheo.Kir2.1). Baseline behavioral assays were performed 5 days prior to surgery. Following a T13 dorsal laminectomy, 5 μl of Ad.Rheo.Kir2.1 was stereotaxically injected into the rat spinal cord in 50 nl boluses over 20 minutes. Rats in the ligand group received 50 mg/kg daily starting at day 0 and ending at day 6. At minimum, behavioral and motor function testing was performed 1, 4, and 7 days post-operation. Behavioral assays included quantitative grip strength measurement, rotarod treadmill performance, and locomotor assessment. In brief, muscle strength was measured using an automated grip strength meter designed to record the peak force of hindlimb grip strength in rats. Each rat was tested in 3 sequential trials and the average was calculated. Accelerating rotarod performance was based on the best of three trials. Locomotor function was assayed using the Basso-Beattie-Bresnahan (BBB) scale.3 The BBB scale analyzes the movements of the hip, knee, and ankle joints as well as the balance and position of the trunk and tail. Rats were perfused and frozen spinal cord sections were collected for Nissl staining to quantify motor neurons. GFP expression was detected under fluorescence microscopy. Data analysis compares behavior results pre-operation and post-operation to determine the viral vectors
The behavioral assays indicate neuromuscular inhibition was exclusive to rats that received the ligand. The effects were predominately found in the ipsilateral hindlimb, which supports our ability to target the vector via stereotaxic injection. After the ligand was discontinued, one rat followed for 3 weeks began to show neuromuscular recovery. Since Ad vectors are known to mediate short-term gene expression (i.e. 1-2 weeks), recovery could have occurred because of down-regulation of Kir2.1 and/or compensatory sprouting by the remaining motor neurons. Histological analysis showed motor neuron loss occurred in ligand subjects at epicenter of injection. Diffusion of the vector from the epicenter to adjacent areas likely led to a gradient of lower vector concentrations. In these areas, we found many health motor neurons that were transduced. Rats that did not receive ligand did not express the transgene, had no functional deficits, and contained normal numbers of motor neurons. Our study demonstrated the ability of RheoSwitch® to mediate inducible gene expression as well as the viral vector’s efficiency to produce biologically active ion channels. Future experiments will aim to achieve optimal neuromuscular inhibition in the absence of cell toxicity through a dose response test of the vector at lower concentrations.
Gene-therapy has the potential to provide a regulateable and reversible method of treating disorders of imbalanced neuronal activity.
The surgical approach of intra-spinal cord injection of therapeutics can be performed safely in rats.
In the presence of its induction ligand, Ad.Rheo.Kir2.1 caused neuromuscular inhibition in the rat hindlimb, which corresponded to the injected spinal cord segments.
A loss of motor neurons occurred at epicenter of injection where we found maximal levels of GFP expression in non-neuronal cells. This indicated that the highest levels of Kir2.1 expression caused motor neuron death.
Normal motor neuron density as well as transduced motor neurons were found at the sites adjacent to the epicenter. This suggested that at lower vector concentrations caused by its diffusion to adjacent areas permitted non-lethal expression levels of Kir2.1.
Evaluation of Ad.Rheo.Kir2.1 intra-spinal cord injection is ongoing. We hypothesize that at lower vector concentrations we can achieve neuromuscular inhibition without motor neuron loss.
This material is based upon work supported by the Howard Huges Medical Institute under Grant No. 52005873 and No. 57006193
1. Pruss H, Derst C, Lommel R, Veh RW. Differential Distribution of Individual Subunits of Strongly Inward Rectifying Potassium Channels (Kir2 family) in Rat Brain. Brain Research. Molecular Brain Research, 2005. 139(1): p.63-79.
2. Johns D.C., Marx R, Mains R, O’Rourke B, Marban E. Inducible Genetic Suppression of Neuronal Excitability. The Journal of Neuroscience, 1999. 19 (5): p. 1961-7.
3. Basso DM, Beattie MS, Bresnahan JC. A sensitivie and reliable locomotor rating scale for open field testing n rats. Journal of Neurotrauma, 1995. 12 (1): p.1-21.
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