Researchers at the University of California San Diego have developed a gene silencing approach providing mice with a higher pain tolerance threshold while lowering their sensitivity to pain for sustained periods of time.
The researchers feel that in the future, this new approach might be able to offer a safer, non-addictive alternative to treating chronic pain compared to opioids.
"What we have right now does not work,” said first author of the study, Ana Moreno, in a statement. Opioids can increase pain sensitivity over time, meaning patients need higher doses as time goes on. “There’s a desperate need for a treatment that’s effective, long-lasting and non-addictive.”
The new findings are reported in Science Translational Medicine.
Moreno, the first author of the new study, first got thinking of how she could use a common gene-editing technology called CRISPR to treat human diseases. One day, she stumbled across a paper that described a genetic mutation in a sodium channel, NaV1.7, that caused some people not to feel any pain – this may sound pleasant, but pain sensing has important evolutionary advantages, and feeling no pain could actually be very risky. NaV1.7 is a protein involved in pain signaling between nerve cells, and mutations in it prevent pain transmission, and so Moreno had an idea.
During her thesis research, she worked with a specific form of CRISPR using “dead” Cas9. This form of the gene-editing tool lacks the ability to cut DNA – however, it can target and "stick" to a certain part of the DNA, which could silence a gene.
“It’s not cutting out any genes, so there are no permanent changes to the genome. You wouldn’t want to permanently lose the ability to feel pain,” she said. “One of the biggest concerns with CRISPR gene editing is off-target effects. Once you cut DNA, that’s it. You can’t go back. With dead Cas9, we’re not doing something irreversible.”
So Moreno put two and two together and thought that if she could use this silencing approach on the gene that codes for NaV1.7, she might be able to silence pain transmission directly.
"By targeting this gene, we could alter the pain phenotype,” Moreno stated. “What’s also cool is that this gene is only involved in pain. There aren’t any severe side effects observed with this mutation.”
To test if this approach would work, Moreno and her team developed the CRISPR/dead Cas9 construct to suppress the gene coding for NaV1.7 in mice. The researchers gave spinal injections of their construct into animals that had been subjected to inflammatory and chemotherapy-induced pain, compared to animals that did not receive the injections.
Assessing the animals over various time points, they found that mice that received the injection had a higher threshold for pain-inducing experiences compared to animals who had not. Overall, the treated animals had a lower sensitivity to pain, with lasting effects seen up to 44 weeks later for animals that been subjected to forms of inflammatory pain.
“In some common diseases, the issue is that a gene is being misexpressed. You don’t want to completely shut it down,” Prashant Mali, co-senior author of the study stated. “But if you could turn down the dose of that gene, you could bring it to a level where it is not pathogenic. That is what we are doing here. We don’t completely take away the pain phenotype, we dampen it.”
Although the length of how long the treatment lasts may require further investigation, the authors note the animals that received the gene-silencing therapy had not experienced any abnormal function, such as changes to sensitivity (which is guided by nerve signaling), or changes in their motor function.
Going a step further, the researchers wanted to validate their initial results. To do this, they set out to test whether they could replicate the findings using a different, slightly older gene-editing tool called zinc finger proteins. The approach uses zinc fingers that similarly bind to a targeted gene, silencing it. Following the same protocol as the first round of experiments, they treated mice that had induced pain with injections of the modified zinc fingers to silence the gene for NaV1.7.
“We were excited that both approaches worked,” Mali said. “The beauty about zinc finger proteins is that they are built on the scaffold of a human protein. The CRISPR system is a foreign protein that comes from bacteria, so it could cause an immune response. That’s why we explored zinc fingers as well, so we have an option that might be more translatable to the clinic.”
The researchers are optimistic that these findings could help treat an array of chronic pain conditions in humans in the future – especially considering the approach had non-permanent effects and seem safe in the animal research conducted. More research needs to be done to take this forward as a therapeutic target that could make it into the clinic, however, as Tony Yaksh, co-author of the study explains:
"Think of the young athlete or wounded war fighter in which the pain may resolve with wound healing,” he said. “We would not want to permanently remove the ability to sense pain in these people, especially if they have a long life expectancy. This CRISPR/dead Cas9 approach offers this population an alternative therapeutic intervention—that’s a major step in the field of pain management.”