For years, scientists have entertained the idea that chemogenetics—the ability to manipulate genetically defined cells using engineered receptors—might eventually become more than a research tool. If harnessed correctly by the pain field, the technology could potentially serve as a treatment for chronic pain, perhaps succeeding in cases where standard drug therapies have failed. Now, a preclinical study from David Bennett, University of Oxford, UK, and colleagues shows the promise of a chemogenetic approach to relieve pain, as well as the obstacles it still faces.
The team virally delivered a modified version of an invertebrate, glutamate-gated chloride channel (GluCl) to peripheral neurons in mice with spared nerve injury (SNI), a model of neuropathic pain. The researchers then systemically administered the non-toxic drug ivermectin, which binds to and opens the channel (which had been engineered so it would not respond to glutamate). Doing so not only silenced aberrant activity in peripheral neurons but also reversed the accompanying allodynia in injured mice.
Rebecca Seal, University of Pittsburgh, US, who was not involved in the study, says the chemogenetic strategy has clear therapeutic potential. “It has advantages over some of the efforts people are thinking about, such as optogenetics,” which uses light to either excite or inhibit neurons but so far has usually required implanting a device to deliver the light.
To Seal, it also has the potential to be more powerful than designer receptors exclusively activated by designer drugs (DREADDs), a form of chemogenetics that involves the expression and activation of engineered G protein-coupled receptors. An ion channel “can really power down the cell directly versus relying on ion channels being coupled to the second messengers,” she said.
“Clearly, the possibility of long-term, controllable silencing of the sensory neurons that drive many neuropathic pain conditions offers tremendous hope that a new era in the management of neuropathic pain can be envisioned: an era of effective pain control, repeated dosing without tolerance, limited adverse effects, and less misuse potential,” writes Allan Basbaum, University of California, San Francisco, US, in a commentary published alongside the paper in the October issue of Brain.
A chemogenetic strategy to ease pain
Bennett and his colleagues have been trying to better understand the contribution of the peripheral nervous system to pain. And so, “we were interested in ways of switching off populations of sensory neurons,” said Bennett. “We always wanted a technique where we could avoid killing neurons or having a big developmental effect, and that was reversible.”
Ultimately, they landed on chemogenetics. “It allows you to control activity in neurons, hopefully in a rapid and reversible fashion,” Bennett said. At first, the researchers sought to place GluCl in sensory neurons by creating transgenic animals, which would carry the genes encoding the channel from birth. But midway through the project, they realized they might achieve far more with a slightly different approach.
“We could put this technology in a virus and use it toward a more translational goal” of treating neuropathic pain, said Bennett.
To target sensory neurons in the dorsal root ganglia (DRG), first author Greg Weir and his colleagues intrathecally injected two adeno-associated viruses, each equipped with a gene encoding one of GluCl’s two subunits; each subunit was tagged with a distinct fluorescent protein. Four weeks later, the researchers observed that two-thirds of DRG neurons, of a range of sizes and subpopulations, expressed the channel. And, as they had hoped, the virus did not spread far enough to transduce motor neurons in the ventral horn of the spinal cord, an important consideration if the manipulation was not to affect movement.
From in vitro experiments using dissociated mouse DRG neurons, the researchers had found that GluCl, when expressed via electroporated plasmid DNA and then opened with ivermectin, could block the generation of action potentials. To test if the chemogenetic technology could have the same effect in vivo, they took advantage of the fact that ivermectin’s impact on neuronal activity is long-lasting. The group injected GluCl-bearing mice with the drug, and a day later made electrophysiological recordings from DRG neurons. Upon injecting current, the researchers confirmed that these cells, too, were unable to fire. And, in contrast to the DRG neurons taken from SNI mice, of which about a third were abnormally active, damaged neurons that carried GluCl were silent after ivermectin treatment.
The inhibitory effect of the modified channel was not only seen at DRG cell bodies. By isolating the axons of dissociated DRG neurons using microfluidic compartments and then adding ivermectin only to the axon compartment, the team showed that GluCl activation could stop action potentials that arose along the neural projections from reaching the soma. What’s more, other experiments showed that GluCl activation also prevented firing in response to current injection when the channel was expressed in sensory neurons that had been differentiated from human induced pluripotent stem cells.
But what was responsible for the silencing? “Sensory neurons are somewhat different from neurons in the central nervous system [CNS] in that they have quite high levels of intracellular chloride,” said Bennett. As a result, opening GluCl when a DRG neuron is at its resting membrane potential would cause chloride ions to flow out of rather than into the cell. “Compared to a CNS neuron where you would predict that activation of a chloride channel would give rise to a hyperpolarized state, in a DRG cell it gives rise to a slightly depolarized state,” Bennett added.
"We think that silencing occurs as a result of the drop in membrane resistance caused by GluCl activation, and that this is sufficient to render excitatory stimuli unable to fully depolarize the membrane potential and trigger an action potential in the neurons,” said Weir.
Consistently, activating GluCl in SNI mice attenuated two forms of allodynia. Injured animals that only had one channel subunit in DRG neurons became hypersensitive to touch, and avoided an area associated with mild cold. By comparison, those with the complete version of GluCl showed less sensitivity to both touch and cold after receiving ivermectin, an improvement that lasted for two days. “That time course could have a therapeutic advantage,” said Seal.
The right type of neuron
Though in its early days, the chemogenetic strategy could, in principle, outperform analgesic drugs in alleviating neuropathic pain. “On a global level, we’re still under-resourced in the treatments we have available to us,” said Bennett. “There’s a problem of both efficacy and tolerability.
“The advantage of using a chemogenetic approach is that if you can deliver your gene to the right place, you can very selectively treat a particular population of sensory neurons. And because you’re using a strong means of electrically silencing a neuronal population, the efficacy is likely to be very good compared to other agents that modulate ion channels,” said Bennett. Additionally, “specific [genetic] expression would hopefully allow you to overcome the problem of side effects.”
Still, the approach as presented has yet to achieve such specificity. The GluCl-encoding genes were delivered to a large swath of peripheral neurons, consisting of many different cell types. And while basic motor ability and proprioception remained intact when the researchers activated the channel in normal mice, deficits in touch, pain, and heat sensation did emerge. “Ideally, one would want to target peripheral neurons without dramatically affecting acute sensations, but how to achieve this is still not clear,” said Seal.
To Bennett, the study is a proof of concept and he thinks that much still needs to be done preclinically. In the long run, he would like to find a way to target the engineered channel only to those sensory neurons that are responsible for the neuropathic pain. Since GluCl is an altered invertebrate protein, it would likely be detected by the immune system if expressed in patients. So, “the other way forward would be to look for mammalian chloride channels that would not evoke an immune response.”
Matthew Soleiman is a science writer currently residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.
Image credit: Weir et al., 2017
