Despite the best efforts of the medical and research communities, cardiovascular disease (CVD) is getting worse, not better. Because of its inherent complexity, CVD research and therapeutics today largely focus around vigilant screening for known risk factors and managing pathogenic processes. But this approach is not stemming the tide; CVD remains one of the leading causes of death, debilitation, and loss of quality of life.

Scientists and observers alike have hailed CRISPR/Cas gene editing technology as revolutionary. It arrived just in time for researchers like Kiran Musunuru from the University of Pennsylvania. Facing a disease that is projected to double its burden on the health care system in the next twenty years (1), Musunuru is dedicated to finding a more permanent therapeutic solution for CVD. “A ‘one-and-done’ therapy would be utterly transformational for a chronic disease,” said Musunuru. “This is the biggest advantage that gene editing brings for cardiovascular disease therapeutics.” 

A “Cure” for Cardiovascular Disease?

High blood plasma cholesterol levels link to increased CVD risk, and the protein PCSK9 is a major regulator of cholesterol production. In 2014, Musunuru’s research team, then based at the Harvard Stem Cell Institute, used CRISPR/Cas to selectively and permanently knockout the Pcsk9 gene in the mouse liver, significantly decreasing plasma cholesterol levels within four days with no observed unintended mutations (2). Two years later, they accomplished the same thing in mice bearing transplanted human hepatocytes (3), demonstrating similar results in terms of efficacy and safety.

“Imagine a scenario where you had a gene editing therapy that could turn off cholesterol producing genes in the liver—basically the same effect as a statin pill every day for the rest of your life, but with a single therapy,” Musunuru mused. He likens the concept to a vaccination, where a single administration would protect an individual for life.

In addition to the immediate physiological benefits, one of the biggest advantages for such a therapeutic strategy, according to Musunuru, is that it would take adherence to a medication regimen entirely out of the equation. “If you look at various studies, only about half of the patients are still taking their pills regularly a year after their heart attack, and that’s a huge problem.”

A Game-Changer for Research

CRISPR/Cas gene editing technology is a game-changer for CVD research. Clinician-scientist Liam Brunham from the University of British Columbia studies how genetic variations affect lipid and cholesterol levels, as well as responses to therapeutic drugs. For him, CRISPR’s ease of use, precision, efficiency, and ability to alter genes in their genomic contexts give it considerable advantages over existing gene editing strategies when it comes to creating disease models. Where it once took years to create a genetically modified rodent model, now it can be done in as little as three weeks. Moreover, the model creation process has been simplified to take place within a single generation, meaning that it can now be used on animals with longer gestational cycles.

This is critical for CVD research. Right now, rodent models are extremely popular owing to their accessibility, rapid reproductive cycle, and genetic malleability. However, rodent physiology is drastically different from that of humans, and rodent responses to CVD are not the same as those of humans. Larger animals, such as rabbits, dogs, and especially pigs, are more similar to humans physiologically and genetically, but were much more difficult to manipulate from a gene editing perspective—until now (4).

“CRISPR works just as well in mouse embryos as it does in embryos from pretty much any other species, so now you can make all of these genetically modified animals—rats, pigs, monkeys—really pretty much the entire gamut fairly easily,” said Musunuru. “This diversification of the animal models that we can use to study diseases has been transformational, and we’ve already gotten more insights into [CVD] using these large animals that we weren’t able to get from rodents.”

Cautious Optimism

CRISPR-based gene editing, much like any other nascent therapeutic approach, is not without risk. For example, permanence, which may be its greatest asset, could be devastating if a genetic modification resulted in unforeseen negative side effects. Brunham believes that this problem can be minimized by focusing on somatic mutations rather than germline, but Musunuru is more bullish. “It’s only been eight years since CRISPR first came on the scene as a gene editor that can work in human cells,” he said. “With newer iterations of the technology, such as base editing, prime editing, and epigenome editing, we may actually develop the ability to reverse what would ordinarily be permanent changes to the genome.”

Another potential problem is that the cardiovascular system in general, and the heart in particular, possesses a relatively limited regenerative capacity. As such, any physical damage to cardiovascular system tissues and organs persist chronically, if not permanently. These often prompt additional co-morbidities. Presently, gene editing therapeutics hold greater potential as preventive measures, and neither Brunham nor Musunuru are aware of any studies demonstrating a role for CRISPR in promoting cardiovascular tissue repair and regeneration. “It’s much better to prevent cardiovascular diseases than to try to fix the problem afterwards,” said Musunuru.

However, Musunuru was unwilling to completely close the door on potentially using gene editing to repair damaged tissue. “I can imagine a scenario where if you have a medication that helps with heart failure or the aftereffects of a heart attack, and you could mimic that genetically, that could be advantageous. I think we’d need to have a better understanding of the genes involved.”

Moving Towards the Clinic

Gene editing offers real potential for new approaches and strategies when it comes to treating cardiovascular disease. Clinical trials for gene editing-based approaches already exist in other fields, such as chimeric antigen receptor-T cell immunotherapy, sickle cell disease, rare liver disorders, and HIV. As such, Musunuru believes that CVD research will follow suit sooner rather than later, citing eight promising gene targets: PCSK9, NPC1L1, ApoB, LPa, ApoC3, ANGPTL3, ANGPTL4, and ASGR1.

These eight genes are involved in lipid metabolism, making it easy to determine whether gene editing elicits the desired effect by measuring blood lipid and cholesterol level. Each gene is prominently expressed in the liver, making targeting and delivery more straightforward. Perhaps most importantly, there are documented examples of people who naturally have loss-of-function mutations for one or more of all eight of these genes. These individuals have not shown any serious adverse consequences from lacking these genes, and they possess substantial protection from CVD.

While direct gene editing-based therapeutic approaches are not yet available, gene editing has already found a place in today’s clinic for CVD patients. Genome analysis is starting to play a bigger role in disease screening and diagnosis. “We’re very good at reading the genome, but we’re very bad at interpreting it. You get a lot of information, but you don’t actually necessarily know what to do with it,” said Musunuru, explaining that many mutations detected during genetic screening are classified as variants of uncertain significance (VUSs). “Sometimes, a detected variant falls in a gene that, when mutated, causes diseases in some people. However, your particular variant might be totally benign, or it might be a disease-causing mutation. The problem is, we don’t know, and this uncertainty, this ‘genetic purgatory,’ is very challenging for the patient. It’s challenging for the physician as well—how do I act on this information? How do I manage the patient? And this uncertainty is just going to become an increasingly large problem as more and more people are sequenced.”

In the face of this, Musunuru has developed a way to characterize VUSs as they are discovered. In his lab, his team uses a gene editing platform to introduce identified VUSs into induced pluripotent stem cells from healthy donors, differentiate them into cardiomyocytes or other cardiovascular system cells, and observe whether the cell’s properties have changed. “We are now in a position where a patient will come into our clinic, get genetic testing, and we can find out whether the VUSs they have in CVD-related genes are benign or pathogenic in less than three months, right in time for the patient’s next regularly scheduled visit.”

This capability has tremendous implications not only for patients, but also for their families, explained Musunuru. “There was one patient who needed a heart transplant, but it wasn’t clear what was driving her disease. We were able to take a VUS found during genetic testing and prove that this variant was in fact the driver of this patient’s disease. Now, this patient had been extremely worried about the thought of passing on her disease on to a child, but [this knowledge] allows her to be more open to potentially doing in vitro fertilization and selecting embryos that do not have this mutation, and therefore have children who would not be at risk for her disease.”

References

1. Benjamin, E.J. et al. Heart disease and stroke statistics-2019 update: a report from the American Heart Association. Circulation 139, e56-e528 (2019).
2. Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ Res 115, 488-492 (2014).
3. Wang, X. et al. CRISPR-Cas9 targeting of PCSK9 in human hepatocytes in vivo—brief report. Arterioscler Thromb Vasc Biol 36, 783-786 (2016).
4. Liao, J. et al. Animal models of coronary heart disease. J Biomed Res 31, 3-10 (2017).