Scientists at the University of California San Francisco (UCSF) have used a modified version of CRISPR technology to reverse genetic forms of severe obesity in two different mouse models, but without carrying out any genome editing. The technique, called CRISPR activation (CRISPRa), uses the CRISPR guidance system to target a particular gene sequence, but instead of using Cas enzymes as molecular scissors to cut out and repair or replace sequences, the technology amplifies existing gene activity to ramp up protein production.

Research lead Nadav Ahituv, Ph.D., and colleagues applied the CRISPRa technology in mouse models that become obese because they lack one copy of a key gene involved in hunger and satiety, and resulting protein levels can’t be maintained at high enough levels by the remaining copy of the gene. In these mice, the CRISPRa therapy effectively boosted the activity of the functional copy of the gene, which produced enough protein to stop the animals from overeating and becoming obese, even many months after the single treatment.

“These results demonstrate that CRISPRa can be used to up the dosage of genes in diseases that result from a missing copy, providing a potential cure for certain forms of obesity as well as hundreds of other diseases,” said Navneet Matharu, Ph.D., lead author of the team’s published paper in Science, which is titled, “CRISPR-mediated activation of promoter or enhancer rescues obesity caused by haploinsufficiency.”

More than 660 genes are estimated to cause human disease due to haploinsufficiency, when one copy of a gene doesn’t function or is lost, and the remaining, functional copy can’t produce enough of the gene’s protein. Haploinsufficiency can lead to conditions including obesity, cancer, neurological diseases, developmental disorders, immunological diseases, limb malformation, and many others, the authors wrote. Gene therapy is one potential approach for replacing the mutant gene copy, and there are numerous clinical trials of gene therapy ongoing, most of which use recombinant adeno-associated virus (rAAV) vectors to deliver the required gene. However, the authors continued, these approaches do have their drawbacks.

As an alternative approach, the UCSF team tested whether the CRISPRa technology could be used in mice to reverse obesity caused by haploinsufficiency of either the SIM1 or MC4R genes, which are crucial for regulating hunger and satiety, and which are the most commonly observed mutations in severely obese people. When one copy of either of the genes doesn’t function the remaining copy can’t produce enough protein to signal satiation, and so affected people can’t control their food intake.

The CRISPRa technology has been developed in the laboratory of UCSF’s Jonathan Weissman, Ph.D., professor of cellular and molecular pharmacology. For their tests in mice, the team generated CRISPRa constructs that targeted promotor or enhancer sequences that are key to regulating the activity of SIM1 or MC4R. They used an rAAV system to deliver the constructs into hunger-controlling areas of the brain in mice that were engineered to have only one functional copy of either SIM1 or MCR4.

These haploinsufficient mouse models would normally become severely obese, but the results confirmed that the CRISPRa-treated animals produced levels of SIM1 and MC4R that were comparable to those of normal mice with two copies of the relevant gene. Critically, the treated animals didn’t become obese, and weighed 30–40% less than untreated animals.

“We were able to rescue a haploinsufficient phenotype in a long-lasting manner (9 months) via CRISPRa-rAAV by targeting either the promoter or enhancer of a gene,” the authors stated. “The results were dramatic,” Dr. Matharu added. “Mice that were missing one copy of the SIM1 gene received the CRISPRa injections at four weeks of age and maintained a healthy body weight like normal mice. Mice that didn’t receive CRISPRa injections couldn’t stop eating. They started gaining weight at six weeks of age, and by the time they were 10-weeks old, they were severely obese on a regular diet.”

Importantly, the one-time CRISPRa administration had long-lasting effects, with treated mice maintaining a healthy weight over the 10 months that they were monitored. The treatment was also highly specific, and there was no evidence of any off-target effects. “These results demonstrate that CRISPRa can be used to up the dosage of genes in diseases that result from a missing copy, providing a potential cure for certain forms of obesity as well as hundreds of other diseases,” Dr. Matharu stated.

The authors suggest that the CRISPRa technology could feasibly be used to prevent diseases that arise from other haploinsufficient genes, or that are caused by microdeletions—when large segments of chromosomes are deleted—that are too big for CRISPR to repair.

“In this study we used an approach to tackle these hurdles and show how a haploinsufficient phenotype could be corrected by increasing the transcriptional output from the existing functional allele via CRISPRa,” the authors wrote. And in cases where a gene is lost it may also be possible to use CRISPRa to boost the activity of a surrogate gene that has a similar function.

“Our results present a potential strategy for treating haploinsufficiency and additional gene dosage related functional abnormalities,” they continued. “There are numerous phenotypes that are caused by lower gene dosage that could potentially be targeted with CRISPRa. In addition, several human diseases could potentially be rescued by the activation of another gene with a similar function.”

“Though this particular study focused on obesity, we believe our system could be applied to any situation in which having only one functional copy of a gene leads to disease,” Dr. Ahituv noted. “Our method demonstrates tremendous therapeutic potential for numerous diseases, and we show that we can achieve these benefits without making any edits to the genome.”

The researches note that CRISPRa may have advantages over existing CRISPR gene editing approaches. “For therapeutic purposes, CRISPRa may be preferable to conventional CRISPR,” suggested co-author Christian Vaisse, M.D., Ph.D., the Vera M. Long endowed chair in diabetes research at UCSF. “It solves many of the problems associated with making permanent modifications to the genome, and it has the potential to treat a variety of genetic diseases for which gene editing isn’t an option.”