After successful editing, photoreceptor cells regain their standard shape. Credit: Editas Medicine.
This approach is distinct from those being tested for cancer and blood disorders because it is an in vivo treatment, meaning the genome editing occurs inside the patient’s body. Compared to ex vivo editing, in vivo editing has more challenges and a different set of risks. One of the biggest risks is that viral delivery tools or genome editing components will provoke dangerous immune reactions in a patient. Another big challenge is making sure that CRISPR enzymes don’t stick around for too long, since that would give them a greater chance of cutting in unwanted places in the DNA.
You may feel squeamish thinking about needles near eyeballs, but the eye is actually an ideal organ for in vivo editing. It is small, so it only requires a single-dose, small-volume treatment. The eye has less immune reactivity than most tissues, making a dangerous immune reaction less likely to occur. Because the eye is relatively contained, the CRISPR components aren’t likely to travel to other parts of the body, so there is a lower risk of unwanted genome editing or immune responses in other tissues. In experiments on a mouse model of LCA with the same mutation, researchers found that about 10% of cells showed the desired edit — this is thought to be the percent needed to get vision improvement. The treatment showed few side-effects in animal models, and studies in human retinal cells showed no off-target effects at over 100 sites with similar sequences. See preclinical data here.
CURRENT CRISPR CLINICAL TRIAL
This is the first in vivo CRISPR therapy trial, meaning, the first time CRISPR is being used to edit someone’s genes within their own body. The first patient-volunteer in this study, sponsored by Editas Medicine, was given a low-dose of the treatment in March 2020. Dosing of all patients in the first, low-dose cohort was completed by November 2020. A medium-dose cohort is expected to be treated soon. No results have been released yet.
WHAT TO WATCH FOR
Researchers will need to follow patients closely to see if cells other than the photoreceptor cells they’re targeting are affected, or if patients have immune reactions to the treatment. Other key research questions include: what percentage of cells will get effectively edited? Will it be enough for patients to regain vision?
If the treatment works, it will be the first trial leading to a direct fix for a genetic disease. Traditional gene therapies, like the ones used for severe combined immunodeficiency, commonly known as “bubble boy disease,” work by adding an extra gene — not by fixing the faulty gene. Likewise, the first CRISPR-based treatments for SCD and beta thalassemia don’t fix the original, disease-causing mutation. Success in this trial would be a major step forward in genetic medicine.
Urinary tract infections (UTIs) are a common infection causing over eight million visits to health care providers every year. UTIs occur when bacteria that shouldn’t be there take up residence in the bladder, kidneys, the tubes that connect the bladders to the kidneys, or the tube through which urine exits the body. E. coli, a common fecal bacteria, is usually the culprit. UTIs are much more common in women for anatomical reasons.
UTIs cause a burning sensation during urination and the need to urinate frequently. Beyond discomfort, they can become dangerous if they affect the kidneys or if bacteria enter the bloodstream. Most are easily treated with a short course of antibiotics, but sometimes antibiotics are ineffective or the infections keep recurring. This is referred to as chronic UTI.
This treatment is a cocktail of three bacteriophages combined with CRISPR-Cas3 to attack the genome of the three strains of E. coli responsible for about 95% of UTIs. The destruction of the genome kills the bacteria.
A common bacteriophage structure. Credit: Christine Liu for IGI CC4
Bacteriophages, or phages for short, are viruses that attack bacteria. They usually work by injecting their genetic material into bacteria and using the bacteria as factory to make more bacteriophages. Eventually, the bacteria will burst, dying as it releases more copies of the phage. Phages are being developed for use against bacterial infections, and have gotten more attention recently as antibiotic resistance has become a major public health threat.
In this treatment, phages have been engineered to be an even more powerful tool against E. coli. In addition to the natural action of phages that kills bacteria, these bacteriophages contain CRISPR-Cas3 in their genome. While the more-famous Cas protein Cas9 makes a precise cut at a single location, Cas3 shreds DNA at the gene regions it is targeted to find. In this treatment, the CRISPR-Cas3 system is made to target the genomes of the targeted E. coli strains and damage them by shredding stretches of DNA. In experiments on isolated cells and in animal experiments, the addition of CRISPR-Cas3 makes phages much more effective at killing E. coli. Locus Biosciences plans to deliver the treatment directly to the bladder by injection.
Credit: Innovative Genomics Institute.
CURRENT CRISPR CLINICAL TRIAL
This is the first trial using a CRISPR-based therapy to treat infection. It is also the first trial to use the Cas3 protein, which targets longer stretches of DNA for destruction, rather than Cas9, which makes a precise cut at one location.
Phages have been considered a possible antibacterial therapy since they were first identified about 100 years ago. The discovery of antibiotics like penicillin soon after, as well as the difficulty of patenting phages, has limited the development and testing of phage therapies. In the time since, phages have occasionally been used by doctors for is known as “compassionate treatment.” Compassionate drug use or compassionate treatment is when an unapproved drug or therapy is used to treat a seriously ill patient as a last resort when no other treatments exist. At least 25 case reports of compassionate phage therapy have been published in the last 20 years. Some cases claim success at healing patients, but reports use different phages in different amounts for different conditions — clinical trials will be necessary to systematically evaluate the safety and efficacy of phage treatments.
As resistance to traditional antibiotics like penicillin becomes a major public health threat, there is growing interest in developing and testing phage therapies. Phages could in some ways even be preferable to antibiotics that are effective, because each phage usually only kills a specific bacteria. So, instead of being treated with an antibiotic that is destructive to healthy bacteria as well, phage therapy is ideally much more targeted and precise. In addition to innovations using CRISPR technology, this trial is significant because it is one of the first few well-controlled clinical trials for phage therapy, and the first to combine the CRISPR system with phage therapy.
Locus Biosciences began recruiting patient volunteers in the United States at the end of 2019, and completed their Phase 1b trial in February, 2021. Locus Biosciences reported that results of the trial supported the safety and tolerability of the new therapy, with no drug-related adverse effects. You can learn more in this interview with Joseph Nixon of Locus Biosciences.
WHAT TO WATCH FOR
The completed trial was Phase 1, which means it was primarily designed to test whether the treatment is safe and has tolerable side effects, not how effective the treatment is. However, the results from the trial indicate that the therapy can decrease the level of E. coli in the bladders of infected patients. Locus Biosciences plans to move forward with Phase 2 human efficacy trials, but has not yet announced a date.
CRISPR expert Megan Hochstrasser is interested in what the trial might reveal about the Cas3 protein: “Cas3 is like a lawn mower that plows through DNA and cuts it up. Cas9, on the other hand, makes a double-stranded break in the DNA. The bacteria should be killed with either kind of damage, but perhaps Cas3 could better prevent any straggling survivors that somehow managed to repair the Cas9 cut. But I’d be curious to know if there is a difference, because as much as I love Cas3, it’s part of a huge system that could be hard to deliver in other contexts.”
RARE PROTEIN-FOLDING DISEASE
Hereditary transthyretin amyloidosis (hATTR) is a fatal disease caused by mutations in a single DNA letter in the gene TTR.
When the TTR gene is mutated, the protein it makes folds the wrong way. Incorrectly folded proteins stick together and form clumps called amyloid fibrils, in a process called amyloidosis. The protein clumps accumulate in organs and tissues, interfering with their normal functions.
ATTR first affects patients in early or middle adulthood. Symptoms vary, but ATTR usually has severe effects on the nervous system and/or heart. Nerve pain, loss of movement control, digestive problems, vision loss, dementia, and heart failure are common in ATTR. The effects on the nervous system and/or heart eventually kill patients.
Credit: Benton Cheung for IGI.
ATTR usually occurs spontaneously, but for some patients, the mutated gene is passed down from their parents. This leads to hereditary ATTR, or hATTR. hATTR is a rare disease, affecting about 50,000 people worldwide.
Often, hereditary forms of neurological diseases are better understood than spontaneous cases because they are easier to study. ATTR has similarities to other neurological diseases involving protein misfolding and amyloidosis including Alzheimer’s and Parkinson’s diseases.
This treatment uses CRISPR-Cas9 tools to reduce the amount of faulty TTR protein the body makes. Less faulty TTR means less formation and accumulation of protein clumps (amyloidosis). The treatment is delivered in a single dose by IV.
The goal of hATTR treatment isn’t to fix a gene: it’s to break the gene so that patients stop making the faulty protein altogether. The CRISPR components cut the TTR gene, creating a double-stranded break in the DNA. As the cell tries to repair the DNA without a corrected template, the repair attempts mutate the gene even more. And when a gene is too badly damaged, a cell will stop making the protein it codes for.
Getting the CRISPR components into cells is a major hurdle for in vivo genome-editing therapies. Many treatments in development use viruses to deliver the genome-editing components. This will be the first clinical trial for a CRISPR-Cas9 therapy delivered in a lipid nanoparticle. The lipids, or fat molecules, surround the gene-editing components, and are able to get into the cell.
In animal models, lipid nanoparticles tend to accumulate in the liver. TTR is primarily made in the liver, so researchers are taking advantage of this tendency to naturally get the treatment to where it is needed. See preclinical data here.
CURRENT CRISPR CLINICAL TRIAL
This is the first trial using lipid nanoparticles to deliver the genome-editing treatment. It is also the first trial to deliver genome-editing components systemically, meaning to the whole body rather than to one specific type of cell or tissue.
The trial is sponsored by Intellia Therapeutics in conjunction with Regeneron Pharmaceuticals. The first patient was dosed in November 2020 in the United Kingdom. Right now, more patient-volunteers with neurological symptoms are being recruited in the UK, and they hope to expand to other countries. The researchers plan to enroll up to 38 participants with neurological symptoms for the first part of the trial, and then expand to enroll additional patients with heart symptoms.
WHAT TO WATCH FOR
This is the first experimental CRISPR therapy to be administered systemically to edit genes inside the human body. Other treatments edit specific cell types outside of the body, and then put them back in after editing (like treatments for blood disorders and cancer) or deliver the genome-editing therapies to self-contained organs (like treatment for genetic blindness and chronic UTI). These strategies help ensure that edits only occur in the cells or tissue of interest.
One long-standing concern about genome-editing therapies is off-target effects: incorrect edits made by the genome-editing components. This is particularly a concern with CRISPR treatments delivered by viruses, since the genome-editing components may persist in the cell for a long time, giving them more opportunity to make editing errors. Avoiding systemic delivery helps reduce the overall risk.
In this trial, delivery is systemic, which could lead to great risk of off-targets because more tissues are exposed. However, exposure risks are reduced because 1) lipid nanoparticles tend to aggregate in the liver, which is the tissue of interest in this disease and 2) no viruses are used to deliver the genome-editing components. In animal models of the hATTR treatment delivered by lipid nanoparticles, the genome-editing components were cleared from the body in less than a week, dramatically reducing the chance of off-target effects. Systemic administration of editing reagents also poses the risk of triggering a potentially dangerous immune reaction. Doctors will monitor patient-volunteers closely for these reactions.
Efficiency — meaning, what percentage of cells are edited — is a big question. In nonhuman primates, only 35 or 40% of liver cells need to be edited to reduce TTR levels enough to have a therapeutic benefit. Researchers will need to follow patients closely to see what percentage of liver cells are effectively edited, and what percentage is necessary to see a benefit.
“It will be a commentary on lipid nanoparticles and how promising they are for editing the liver,” says IGI CRISPR expert Megan Hochstrasser. It might be a situation where we don’t actually need a very high percentage of cells to get edited to start to see a positive benefit for the patient, which would be great.”
THE BIG PICTURE: WHAT WE HOPE TO LEARN
Taken together, these CRISPR clinical trials are helping scientists learn about the types of DNA changes CRISPR enzymes make in different cells, including unwanted off-target changes and problematic on-target changes; the way the immune system reacts to CRISPR-Cas tools; and how well different delivery methods work. These trials are not a major test of what CRISPR can do, but of how well it does what it does.
Over the last year, there has been encouraging news: Victoria Gray and others seem to be functionally cured of sickle cell disease or beta thalassemia, and the edited cells have taken up residence in the bone marrow, indicating the potential for a long-lasting cure. Trials for cancer therapies are at early stages, but the safety and tolerability of the treatments looks promising for moving forward with more current editing technology. New trials initiated this year widen the scope of CRISPR applications and delivery methods.
Editing efficiency varied between trials, with those started more recently having higher efficiency. Differences in technology — newer CRISPR tools are more efficient — and delivery methods account for these differences. Researchers are continuing to develop new CRISPR editors and expanding delivery options with the aim of increased efficiency.
These studies did show unwanted edits to cells, including on- and off-target effects. In the short-term, they don’t seem to cause trouble. But will unwanted DNA changes have meaningful effects on patients down the line or when given to larger cohorts? And how will potential side effects compare to the severity of the diseases being treated? Patient-volunteers will have to be followed over the coming years to get a more complete understanding of the safety profile of these treatments.
We are starting to see more in vivo editing with the addition of the trials for chronic UTI and hATTR, and preclinical research indicates that more will be coming in the years ahead. The hATTR treatment stands out as the first treatment to be administered systemically: other in vivo treatments are delivered directly to specific, contained organs, or cells are edited outside of the body and then returned to the patient. The systemic introduction of genome-editing components is a big advance, and we’ll be watching this one closely.
What the current CRISPR clinical trials have in common are easier delivery options. Getting the treatments where they need to be, and only where they need to be, is one of the biggest challenges of CRISPR-based therapies. The trials initiated so far rely on convenient solutions to delivery, but as delivery improves the menu of options will expand significantly
MORE MEDICAL CRISPR FIRSTS TO LOOK OUT FOR
While the current CRISPR clinical trials are exciting, they rely on making edits to DNA that are easy for CRISPR-Cas enzymes — they are not really major tests of what CRISPR technology itself can do. In terms of understanding the further reaches of CRISPR’s potential, we’ll learn a lot from future milestones:
- A CRISPR treatment that involves inserting DNA to repair or replace a faulty sequence, in essence“pasting” in new material, is still a big challenge.
- A CRISPR therapy that repairs multiple genes at once. Many common conditions, like diabetes and heart disease, are “polygenic,” meaning multiple genes play a role in their development. While researchers have achieved impressive feats in isolated cells and animal models, we’re a long CRISPRi and CRISPRa are strategies to change the level of protein without changing DNA sequence. way from making multiple changes to the genome in real patients.
- A trial where CRISPR tools are used to turn genes on and off without editing the DNA sequence. These strategies, known as CRISPR activation and inhibition, don’t require making breaks in a patient’s DNA, so they might be safer. But, it’s unclear how long their effects would last in humans.
- A treatment that uses base editing. Base editing uses CRISPR components to directly change single DNA letters without making breaks in the DNA. For diseases caused by single-letter changes to DNA, base editors may be a safer editing option than CRISPR.