Researchers at the University of California San Diego (UCSD) have developed a non-permanent form of gene therapy for chronic pain that they suggest could offer a safer, non-addictive alternative to opioid drugs. The team’s epigenetic repression approach used a catalytically inactivated “dead” Cas9 enzyme (dCas9), to temporarily block a gene called NaV1.7, which is expressed in pain-transmitting neurons in the spinal cord. In vivo tests in multiple mouse models found that the gene-repressing strategy increased the animals’ pain tolerance, lowered sensitivity to pain, and provided months of pain relief without causing numbness. Further experiments in mice using an alternative, zinc finger protein tool to repress the gene’s expression yielded the same results.
The researchers, co-led by Ana Moreno, PhD, a bioengineering alumna from the UC San Diego Jacobs School of Engineering, suggest that their technology, which they’ve dubbed pain LATER (long-lasting analgesia via targeted in vivo epigenetic repression of NaV1.7), could work for a large number of chronic pain conditions arising from increased expression of NaV1.7, including diabetic polyneuropathy, erythromelalgia, sciatica and osteoarthritis. It could also provide relief for patients undergoing chemotherapy. “By targeting this gene, we could alter the pain phenotype,” she said. “What’s also cool is that this gene is only involved in pain. There aren’t any severe side effects observed with this mutation.”
The team reports on its studies in Science Translational Medicine, in a paper titled, “Long-lasting analgesia via targeted in situ repression of Nav1.7 in mice.”
Chronic pain affects between 19% and 50% of the world population, but current standard of care for chronic pain commonly relies on opioids, which have adverse side effects and represent a “profound addiction risk,” the authors wrote. Opioids can also make people more sensitive to pain over time, leading them to rely on increasingly higher doses. And, as the authors pointed out, “Despite decades of research, the goal of achieving broadly effective, long-lasting, non-addictive therapeutics for chronic pain has remained elusive.”
“What we have right now does not work,” Moreno stated. “There’s a desperate need for a treatment that’s effective, long-lasting and non addictive.” The idea for such a treatment emerged when Moreno was a PhD student in the lab of UC San Diego bioengineering professor Prashant Mali, PhD, who is co-senior author of the Science Translational Medicine paper. Mali had been investigating the possibility of applying CRISPR-based gene therapy approaches to rare as well as common human diseases. Moreno’s project focused on exploring potential therapeutic avenues. She came across a paper about a genetic mutation that causes humans to feel no pain. This mutation inactivates a sodium channel protein called NaV1.7, which is associated with signaling in pain-transmitting neurons in the spinal cord. For individuals who lack functional NaV1.7, sensations like touching something hot or sharp do not register as pain. Conversely, a gene mutation that leads to overexpression of NaV1.7 causes individuals to feel more pain.
Moreno saw the potential to reversibly target this gene specifically for pain relief indications. “By targeting this gene, we could alter the pain phenotype,” she said. “What’s also cool is that this gene is only involved in pain. There aren’t any severe side effects observed with this mutation.”
In fact, previous efforts to develop synthetic compounds or antibodies that selectively target NaV1.7 had been hampered because of the sequence similarity between different NaV protein subtypes. “Many small-molecule drugs targeting NaV1.7 have accordingly failed because of side effects caused by lack of targeting specificity or their limited bioavailability by the systemic route,” the investigators acknowledged. “In addition, antibodies have faced a similar situation, because there is a trade-off between selectivity and potency due to the binding of a specific (open or close) conformation of the channel, with binding not always translating into successful channel inhibition.”
Moreno had been working on gene repression using CRISPR gene editing technology as part of her dissertation. Specifically, she was working with a version of CRISPR—also known as CRISPRi—which uses a dead Cas9 that lacks the ability to cut DNA, but instead can stick to a gene target via a guide RNA, and block the gene’s expression.
Moreno saw an opportunity to use this approach to repress the gene that codes for NaV1.7. “One of the biggest concerns with CRISPR gene editing is off-target effects,” she said. “Once you cut DNA, that’s it. You can’t go back … With dead Cas9, we’re not doing something irreversible. 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.”
Moreno and colleagues engineered a CRISPR/dead Cas9 system to target and repress the gene that codes for NaV1.7, and tested the system by administering it as spinal injection in mice with inflammatory and chemotherapy-induced pain. Encouragingly, the team found that the treated mice displayed higher pain thresholds than control animals that had not received the injection. The treated mice were slower to withdraw a paw from painful stimuli (heat, cold, or pressure) and they also spent less time licking or shaking the paw after being hurt.
The effects of therapy were also tested at various time-points. It was found to be still effective after 44 weeks in the mice with inflammatory pain, and after 15 weeks in animals with chemotherapy-induced pain. The duration of effectiveness is still being evaluated, the researchers said, and is expected to be long lasting. Importantly, treated mice did not lose sensitivity or display any changes in normal motor function.
To validate their results, the researchers then performed tests using a zinc finger protein gene editing tool. They designed zinc fingers that similarly bind to the same gene target and block expression of NaV1.7. Spinal injections of these zinc fingers in mice produced the same results as the CRISPR-dead Cas9 system.
“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.”
Mali suggests that the use of dead Cas9 opens the door to using gene therapy to target common diseases and chronic ailments. “In some common diseases, the issue is that a gene is being misexpressed. You don’t want to completely shut it down,” he said. “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.”
The authors concluded, “Overall, these in situ epigenetic approaches represent a viable replacement strategy for opioids and serve as a potential therapeutic approach for long-lasting chronic pain.” And due to its non-permanent effects, this therapeutic platform could address a poorly met need for a large population of patients with long-lasting (weeks to months) but reversible pain conditions, added co-senior author Tony Yaksh, PhD, an expert in pain systems and a professor of anesthesiology and pharmacology at UC San Diego School of Medicine. “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.”
Moreno and Mali co-founded the spinoff company, Navega Therapeutics, to work on translating this gene therapy approach, developed at UC San Diego, into the clinic. Yaksh is a scientific advisor to Navega. Researchers at UC San Diego and Navega will next work on optimizing both approaches (CRISPR and zinc fingers) for targeting the human gene that codes for NaV1.7. Trials in non-human primates to test for efficacy and toxicity will follow. Researchers expect to file for an IND and to commence human clinical trials in a couple of years.