New frontiers in gene editingMaking headway against genetic disorders with CRISPR-Cas9
For babies with Duchenne muscular dystrophy, development starts out normally. They might be slow to start walking, but it’s not until the age of 2 or 3 — when they fall frequently, or struggle to run and jump — that their parents realize something’s amiss. The prognosis is grim: Children with Duchenne experience a progressive weakening of all of their muscles, need a wheelchair by around age 12, and face a drastically shortened lifespan due to weakened heart and lung muscles. A lifelong steroid regimen is one of the only available treatments to slow the inevitable progress of this disease.
Duchenne is caused by an absence of dystrophin, a huge protein on the X chromosome that helps the body form healthy muscle tissue. When some of the protein’s genetic sequence is missing, it causes muscular dystrophy, which like other X-linked diseases, primarily affects boys. Duchenne is the most severe form; there’s zero dystrophin protein present in the cell, so the outcomes are the worst.
With the disease built right into a person’s DNA, the treatment focus for Duchenne has been on managing symptoms. CRISPR-Cas9, a gene-editing tool discovered at Berkeley in 2014, has been shaking up therapeutics from cancer to heart disease and has offered the promise of targeting the mutated dystrophin gene itself. But efforts thus far have fallen flat because, while the CRISPR tool excels at deleting defective genes, it is less efficient at correcting mutations. Now, Berkeley bioengineers think they have cracked the stubborn barriers to correcting the Duchenne gene mutation, potentially optimizing both treatment and diagnosis. If they’re successful, their work could have implications for nearly every genetic disease.
CRISPR, short for the genomic pattern “clustered regularly interspaced short palindromic repeats,” and the Cas9 enzyme, DNA “scissors,” function as bacteria’s natural system to expel viruses by cutting an offending sequence from its genome. The CRISPR-Cas9 gene-editing tool — the groundbreaking innovation first identified by Jennifer Doudna, professor of biochemistry and molecular biology, and her colleague Emmanuelle Charpentier — allows scientists to aim Cas9 at any sequence in a person’s genome, modifying genes to remove mutations or fight disease.
“What CRISPR does really well is it cuts DNA. But with most genetic diseases, you don’t actually want to cut DNA — you want to correct the mutation.”
– Bioengineering professor Niren Murthy
When researchers want to use CRISPR to correct, not remove, a mutated gene, they first must send the unmutated sequence, or wild type “blueprints” — called donor DNA — into the cell. And because genetic mutations are present in every cell in the body, CRISPR must repair the DNA of a large number of cells to successfully treat disease.
The challenges of genetic disease also create its greatest promise. Unlike cancer, for example, where the causes are often murky, “genetic diseases are one of the few types of diseases where we exactly know the mechanism,” says bioengineering professor Niren Murthy. “That means if you had a way of correcting that sequence you have a very high chance of actually curing that disease.”
The golden ticket
Murthy, like many biomedical researchers, targets Duchenne because of the large patient population, large unmet medical need and non-invasive access to muscle tissue. “What CRISPR does really well is it cuts DNA,” he explains. “But with most genetic diseases, you don’t actually want to cut DNA — you want to correct the mutation.” Ideally, he says, that means restoring the wild type gene.
Correcting a gene mutation is more akin to a precision-timed transplant than CRISPR’s typical routine surgeries. Scientists must deliver three components into each cell: guide RNA, a map to the target location; the Cas9 enzyme to cut the DNA; and donor DNA — the correct sequence. If these elements can reach a cell at precisely the same time, it triggers a natural process called homology directed repair: Cas9 cuts the DNA then swaps in the donor gene for the mutated one, “tricking” the DNA that the new gene belongs there.
The current standard is for the components to ride into cells on benign viruses. But viruses present problems. It’s a one-shot effort; once a virus enters the body, antibodies form so the body rejects that virus in future encounters. The process is expensive and can lead to off-target DNA damage.
Murthy and bioengineering professor Irina Conboy, whose lab focuses on biomedical stem cell therapies, think getting the homology directed repair components to cells more efficiently will have huge implications for Duchenne muscular dystrophy and other genetic diseases.
“I absolutely think it’s just a delivery problem,” Murthy says.
Gold nanoparticles are a promising delivery vehicle, according to a 2017 study co-authored by Murthy, Conboy, Doudna
“It’s really easy to coat DNA onto gold,” Conboy says. Gold bonds easily with sulfur, and sulfur-attached DNA is a common lab product. Then the Cas9 enzyme and guide RNA naturally bind to each other and onto that core. When these locked-and-loaded gold particles were delivered to mice, the components hit their target cells simultaneously.
Gold nanoparticles are a promising delivery vehicle: “It’s really easy to coat DNA onto gold.”
– Bioengineering professor Irina Conboy
The study’s findings were highly promising: A single saline-based injection of CRISPR-Gold particles doubled strength and agility in Duchenne mice compared to control groups. The wild type gene increased by up to 5 percent, several percentage points higher than any previous results. Five sounds like a small number compared to drug treatments, which generally must inhibit at least 50 percent of their target to be effective, Murthy notes. But because Duchenne patients start with no dystrophin protein, he says, “When you go from nothing to something, you’re going to see very dramatic health benefits.”
Moreover, adds Conboy, unlike viruses’ single-use problem, the body does not form antibodies to gold. That enables continued saline infusions of these nanoparticles, which “can gradually keep repairing the DNA so the muscles stop dying and eventually even repair the defective dystrophin gene back to the wild type.”
Minute as these nanoparticles are, too much gold can be toxic to the body. So the team’s next step is to make more biodegradable, less toxic formulations. Murthy and Conboy have a National Institutes of Health grant supporting that work.
These successes are tempered by dissimilarities between mouse and human biology, which can lead to disappointments when lab research moves to clinical trials. While there are promising indications that gene editing will avoid some of those issues, no one really knows how effective homology directed repair is going to be in humans.
Still, Murthy and Conboy expect to see clinical trials of their nanoparticle therapy as soon as five years from now, especially if they appeal for “compassionate consideration,” an expedited process. “It’s not like you’re competing against some other therapeutic,” Murthy says, “There’s really nothing out there.”
“What we have now is not adequate” says Alex Fay, a pediatric neurologist and neuromuscular specialist at UCSF Benioff Children’s Hospital San Francisco, who treats muscular dystrophy patients. “There’s an urgent need for treatments that are going to dramatically alter the course of the disease.” Even for families willing to take on added risk, therapies often come too late to be meaningful. “The ideal time to treat these patients is when they’re infants or babies,” Fay says — long before symptoms appear.
Kary Mullis won the 1993 Nobel Prize in Chemistry for developing a laboratory process that finds a specific location on the roughly 3 billion-letter human genome. The technique, called polymerase chain reaction, or PCR, has been refined in labs over many years, but it is still prone to errors.
As a postdoc in both the Conboy and Murthy labs, Kiana Aran created a tool that uses CRISPR to perform similar tasks to PCR, but digitally — like Google for genomes. Users place a drop of purified DNA, extracted from a simple swab sample, directly onto a chip containing thousands of tiny CRISPR-programmed graphene transistors. Then, using Duchenne guide RNA as the “search term,” the transistors scan the genome for target sequences and deliver the search results electronically.
The quick, inexpensive and portable technology, called CRISPR-Chip, could be used for diagnosis and for ongoing monitoring of patients’ gene-therapy progress. Aran’s current chip tests for Duchenne, but her team can load guide RNA for other genetic diseases, including sickle cell anemia and Huntington’s, and her lab is developing more sensitive technologies to detect other types of genomic material, like infectious-disease indicators.
A direct comparison to PCR doesn’t apply, Aran says. “We are starting a new process of making genomics digital. We’ve been developing high-tech instruments and gadgets, and we haven’t been using these technologies for healthcare applications.” CRISPR-Chip is one step toward what she sees as the inevitable digitization of all medicine.
Aran, now an assistant professor of medical devices and diagnostics at the Keck Graduate Institute, didn’t start out with this visionary agenda. “I did not enjoy electrical engineering as an undergrad,” she recalls. “How could I connect with real-world problems by just designing transistors and circuits?” When she discovered biomedical engineering, her attitude changed. Now, as the chief science officer at Cardea, which recently merged with Nanosense, a company she co-founded with industry partners, she’s focused on getting her solutions into the world by first commercializing the device for lab-based quality-control while she completes the long regulatory process for medical uses.
“It’s going to be revolutionary”
Better diagnostic tools can’t come soon enough for Fay, who thinks it’s time to add creatine kinase, the muscle-damage test, to neonatal testing protocols. And if technology like Aran’s could provide easy in utero testing, he’s for it.
But as researchers develop these powerful technologies, they must also grapple with the ethical quandaries and unforeseen consequences of their work. For example, while gene therapies currently under development can’t be inherited, CRISPR also enables germline editing, which creates inherited changes — permanently altering human DNA — an ethical rabbit hole so deep that it is formally prohibited in more than 30 countries, including the United States.
Despite these attendant societal concerns, CRISPR gene editing continues its march from labs to implementation. For medical professionals and patients with genetic diseases, this opens a future of possibilities.
“I think it’s going to be revolutionary,” Fay says of the potential for Duchenne. “The whole mentality of the field is going to change from a supportive care model to a treatable disease model, and it’s going to increase the urgency of early genetic testing.”
Aran says the key to successfully implementing CRISPR to treat genetic disease — and other applications from cancer to agriculture — will be addressing the engineering, societal and safety challenges step by step, worldwide. “The U.S. must maintain a position of technical and ethical leadership,” she says. And, she adds, stepping back isn’t an option. “History has shown that you cannot fight advances like this. We cannot stop it from moving forward.”
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