Since its debut less than a decade ago, CRISPR-Cas9 gene editing has inspired its share of grandiose and cautionary forecasts: that we might soon be able to resurrect beasts from the ancient past, for example, or (that classic genetic manipulation controversy) create designer babies. While such applications may never see the light of day, the technology is already revolutionizing genetics research, allowing scientists to easily manipulate model organisms in the lab. Moreover, many biomedical scientists see the system as a means to fix problematic DNA at play in countless genetic diseases.

Scientists and companies at the front lines of developing CRISPR-based therapies have started with relatively modest goals, targeting rare single-gene disorders and largely aiming to transplant modified cells rather than set a gene-editing delivery system loose in the body. But if green-lighted by regulators, such therapies could serve as trial balloons for a much broader use of CRISPR...

“Up to now, gene therapy has consisted of trying to get [new copies of] genes into cells in people’s bodies, and that’s been tough,” says David Flannery, a medical geneticist at the Cleveland Clinic. CRISPR, instead, offers a way to tweak genes already present in their cells.  

CRISPR ex vivo

With the approval of Novartis’s Kymriah last year, the US Food and Drug Administration (FDA) for the first time green-lighted a treatment—in this case, a cancer immunotherapy—consisting of genetically modified cells. A patient’s own T cells are extracted from the blood and treated with a virus that inserts a gene for a chimeric antigen receptor (CAR). The resulting CAR T cells are then expanded and infused back into the body. Ex vivo CRISPR-based therapies now in development take a similar approach.

I’m optimistic that given even a decade, we’re going to see widespread therapeutic gene editing.

—Jacob Corn, University of California, Berkeley

University of Pennsylvania oncologist Edward Stadtmauer is starting a Phase 1 trial testing a therapy that will filter T cells from the blood of eligible patients with cancer, then use CRISPR to knock out three of the cells’ existing T-cell receptors (TCRα, TCRβ, and PD-1) and a lentiviral vector to insert a receptor for NY-ESO-1, a protein that appears on the surface of some cancer cells. After the modified cells have been expanded for a few weeks and patients have received a brief course of chemotherapy, researchers will infuse the cells, says Stadtmauer, who is currently recruiting patients to the trial.

CRISPR-based cancer immunotherapies are also in the pipeline of the Switzerland-based company CRISPR Therapeutics, which has announced its plans to file an investigational new drug (IND) application with the FDA for one such therapy by the end of this year. As in the UPenn trial, that therapy is based on knocking out T-cell receptors and adding a CAR programmed to seek out a cancer-associated surface protein—in this case, CD19. But in contrast to UPenn’s approach, the company plans to use cells from healthy donors and remove the major histocompatibility complex 1, a modification that researchers at the firm hope will enable the treatment to be used on multiple patients without provoking an immune response.

The patient cells used in autologous CAR-T therapies have been repeatedly exposed to antigen and inflammatory signals. As a result, they “often are pretty beat up, they’re exhausted, and they’re often not able to expand when they see the antigens,” explains Tony Ho, the company’s head of research and development. “With our technology we can make . . . a universal off-the-shelf [treatment]” that uses cells from a healthy person to avoid that shortcoming.

Beyond cancer, CRISPR Therapeutics aims to use ex vivo CRISPR editing to treat blood disorders such as β-thalassemia and sickle cell anemia. The idea is to extract hematopoietic stem cells from patients’ blood and edit them to make fetal hemoglobin as a workaround for the defective adult hemoglobin at fault in both disorders. The company applied for permission from European regulators in December to begin a Phase 1/2 clinical trial for β-thalassemia and plans to treat its first patient there later this year, but its plans to debut the same treatment in US trials to treat sickle cell anemia were put on hold in May when the FDA requested answers to additional questions during its review of the IND.

In a more direct approach to treating sickle cell anemia, Matthew Porteus, a physician-researcher at Stanford University, and his team have been working to fix the causative mutation in hematopoietic stem cells. Because the disease is caused by a single-nucleotide mutation, “on a chalkboard, it would be easy to say, ‘If we could change that back to something that doesn’t cause disease, we could cure the disease,’” he says.

It’s important to balance the medical benefit with the risks that come from the treatment.

—Jussi Taipale, University of Cambridge

In the lab, the team has used CRISPR and homology-directed repair (HDR), which supplies a bit of DNA to cells to use as a template when repairing CRISPR’s double-strand breaks, to correct mutations in 60 percent to 80 percent of patient cells, which Porteus expects will be more than enough for the therapy to alleviate symptoms of the disease. The next step will be to infuse the edited cells back into patients, where they’re expected to naturally lodge themselves in the marrow and begin producing healthy red blood cells. Porteus estimates that a clinical trial on the treatment could begin by the middle of next year. Ultimately, he hopes to treat not just sickle cell, but also other genetic diseases of the blood, including β-thalassemia. “If we’re successful, we believe we will have the platform to apply it to hundreds of other diseases without even having to change very much,” he says.

Genome editing researcher Jacob Corn of the University of California, Berkeley, is also using HDR to tackle sickle cell disease, with an eye on other diseases centered on hematopoietic stem cells. He likens his group’s approach to performing precision “surgery” on a patient’s genome, with edits customized to each person’s mutation. Corn, who cofounded the gene editing–based biotech company Spotlight Therapeutics and receives honoraria from or owns stock in several others, is hoping to launch a clinical trial for sickle cell disease as early as next year.

“There are a lot of really exciting results coming out every day,” Corn says. Although no such treatment has hit the market yet, “I’m optimistic that given even a decade, we’re going to see widespread [therapeutic] gene editing.”

CRISPR in situ

Cambridge, Massachusetts–based Intellia Therapeutics has ex vivo therapies in the preclinical pipeline for cancer and sickle cell disease, but its most advanced program is actually an in vivo treatment. Using lipid nanoparticles to deliver the CRISPR machinery to hepatocytes of patients with transthyretin amyloidosis, researchers hope to disable the mutated allele encoding transthyretin. The protein doesn’t play an important role in the body, and an abnormal version of it can form deposits in the peripheral, and sometimes in the autonomic, nervous system, leading to loss of sensation in the extremities and, in the latter case, impairments of physiological functions. “[Transthyretin] has a minor function in the body, ordinarily, and so if you have this amyloidosis which is killing you, it’s just much better to get rid of the protein entirely,” says Tom Barnes, a senior vice president at Intellia. “Our therapy is designed to knock out both copies.” A one-time treatment is showing sustained results in mice, he says, and the company aims to file an IND late next year.

A potential pitfall of injecting CRISPR into the body is the risk of provoking an immune reaction. At Cambridge, Massachusetts–based Editas Medicine, a five-year-old pharma company whose cofounders include the Broad Institute’s Feng Zhang and Harvard University’s George Church, researchers are hoping to avoid such issues by delivering CRISPR for genetic eye diseases directly to the eyes, organs that are immunoprivileged and are less likely to become inflamed in reaction to the therapy.

The treatment furthest along in the pipeline targets one form of Leber congenital amaurosis, a genetic disease that causes vision loss or blindness. In the subtype of the disease that Editas is targeting, a point mutation in an intron causes abnormal splicing of the CEP290 transcript. The aim of the therapy is to snip out the mutation via nonhomologous end joining (NHEJ), a DNA repair process that doesn’t involve a template. At the American Society of Gene & Cell Therapy meeting in May, an Editas researcher presented results from macaques showing the treatment was well tolerated and successfully edited the CEP290 gene, and the firm announced plans to file an IND application very soon. “We believe that if we restore the CEP290 protein, we’ll restore the [function of the eye’s photoreceptors] and thereby significantly improve vision in these patients that are heavily visually impaired, if not blind,” explains Chief Scientific Officer Charlie Albright.

Editas is also applying lessons learned from the development of its Leber congenital amaurosis therapy in programs to treat other inherited eye diseases, such as Usher’s syndrome 2a, as well as blindness-causing herpes simplex virus–1 infection, Albright says.

Treating with CRISPR

Researchers hope to develop treatments for a wide range of genetic disorders, and even cancer, using CRISPR-Cas9 gene editing. These clinical interventions may take the form of ex vivo therapy, in which cells are edited in the lab and transfused into patients, or in vivo therapy, which delivers gene-editing machinery directly to the affected tissues.

EX VIVO (top)IN VIVO (bottom)
Approach: Extract cells from the blood, modify them using CRISPR, expand the edited cells, and infuse them back into the body. Alternatively, modified cells from a healthy donor could be expanded and infused into multiple patients.Approach: Use a vector, such as a nanoparticle or virus, to deliver CRISPR-Cas9 to targeted cells or tissues within the body.
Examples: Several research groups and companies are developing therapies that modify autologous hematopoietic stem cells to treat sickle cell disease and/or β-thalassemia, with trials beginning as early as this year.

Trials are in progress to modify autologous or donor T cells with customized receptors to better fight cancer.
Examples: Editas Medicine is planning to file an IND for a trial in which an adeno-associated virus carrying CRISPR-Cas9 will be injected into the eye as a therapy for Leber congenital amaurosis.

Intellia Therapeutics plans to file an IND next year for a therapy for transthyretin amyloidosis that would use CRISPR-bearing lipid nanoparticles to knock out production of a disease-causing abnormal protein in the liver.
Hurdles: Regulators have little experience with customized, cell-based therapies, and the manufacturing process is considerably more complex than the production of conventional drugs.Hurdles: Delivering CRISPR-Cas9 to a sufficient number of target cells to have a therapeutic impact is a major challenge. Potential safety concerns include the possibility of off-target edits to the genome, and of provoking an immune response in recipients.

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A long road ahead for CRISPR therapies

Both in vivo and ex vivo routes face hurdles on their way into the clinic. When carrying out gene-editing inside the body, delivery is a thorny challenge. “The great thing about CRISPR is when it’s inside the nucleus it works just as advertised,” Barnes tells The Scientist. “The challenge is the platform around it that you need to build.” And a therapeutic made of a patient’s own manipulated cells is very complex, with an intricate manufacturing process and a less clear-cut path to regulatory approval than other drugs, he says.

In addition to these challenges, new studies continue to crop up on potential harmful side effects of both in vivo and ex vivo CRISPR. In June, for example, two papers found that in certain cell types, CRISPR tended to bring about cell death via the p53 pathway, so cells that survived the editing were likely to carry a p53 mutation—a defect that could be oncogenic. And last month, researchers reported that a CRISPR-induced cut could have unintended effects in distant parts of the genome. In both cases, the news led to drops in the stock prices of companies working to develop CRISPR therapies.

Researchers working to develop CRISPR therapies tell The Scientist they’ve long been aware of such dangers and are taking steps to mitigate them. Still, given that Cas9 induces a double-strand DNA break, “there’s always a possibility that something goes wrong,” says the University of Cambridge’s Jussi Taipale, who coauthored one of the June studies. “For that reason it’s important to balance the medical benefit [with] the risks that come from the treatment.”

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