ZFN: a rival to CRISPR
While CRISPR gene-editing and the battle over its patents continue to dominate headlines, a lesser known contender is making a name for itself, partly thanks to the drive of one company.
“Without a doubt, zinc finger nuclease (ZFN) editing is superior to CRISPR editing,” claims Sandy Macrae, president and CEO of Sangamo Therapeutics based in Richmond, California.
Sangamo’s proprietary technology is based on a naturally-occurring class of proteins called zinc finger DNA-binding proteins (ZFPs), which recognise and bind to specific sequences of DNA. A nuclease, which is a functional domain that cuts DNA, is attached to the ZFP portion to make a ZFN.
According to Macrae, ZFN is better than CRISPR because it balances the key ingredients of gene-editing: precision, efficiency, and specificity.
Macrae says Sangamo is able to engineer ZFNs with precision to target any nucleotide in the genome, with editing efficiencies now routinely greater than 90%, and with very high specificity with off-target cleavage below the level of detection by state of the art assays.
“By contrast, CRISPR is based on a relatively inflexible bacterial system and is limited in targeting precision by guide sequences which are only able to recognise a fraction of nucleotides.”
The development of new linkers for configuring DNA-binding modules has led to a 300-fold increase in design options for targeting any given DNA sequence, while recent innovations by Sangamo mean that its DNA editing efficiency has increased to as high as 99.5%.
Efficiency is critical in multiplexing (achieving multiple edits in a single step) to generate a higher proportion of final cells with all of the desired edits. However, differences in editing efficiency can play a huge role in how these therapies are developed at clinical stage.
On specificity (engaging a target site without any meaningful off-target effects), the company found the cause of off-target cutting and, after removal of non-specific points of contact between the ZFN and the DNA backbone, there was a 1,000-fold reduction in off-target cleavage to levels undetectable by current assays.
New partners
In February this year, Sangamo teamed up with Kite (now a Gilead company) to develop next-generation engineered cell therapies to treat cancer.
Under the partnership, Kite will work with Sangamo to use ZFN technology in the development of next-generation ex vivo cell therapies for autologous and allogeneic use to treat different cancers.
Current CAR T therapies follow the “one patient, one therapy” (autologous) approach, which presents logistical challenges, particularly because there are too few laboratories to make this work for thousands of people at once, which can ramp up costs.
Allogeneic cell therapies from healthy donor cells provide a treatment option that can be accessed directly within the oncology infusion centre and, as a result, the infusion time for patients, and also potentially the cost of manufacturing, can be reduced.
“The development of a universal CAR T therapy, which uses a healthy donor’s immune cells, gene-edits them for immunocompatibility and then multiplies them to treat hundreds of cancer patients at once (allogeneic), is the only practical option to treat more common cancer types,” explains Macrae.
The collaboration is expected to create ten different products, with the first few being targets that people will recognise, although Macrae is tight-lipped about the specifics.
“For so long, people were not interested in ZFNs but now, big companies including Kite-Gilead are coming to the conclusion that this is the way forward,” he says. “We were a single voice advocating the benefits of ZFN and now we’re being heard and understood.”
A different landscape
Founded in 1995, Sangamo borrowed the name from a company owned by the founder’s great grandfather in Sangamon County, Illinois.
Sangamo has had more than two decades to research new technologies for genome-editing and ensure that the discoveries are well protected.
“We’ve made sure that we have filed very broad and very specific patents,” Macrae says.
“We felt that owning our own IP and taking the time to file is vital to the health of company. This means that we’re very comfortable and have good control over our unique IP.”
In practice, this means having an IP team member that sits next to the scientists (literally) throughout the discovery process.
“Sometimes, in bigger companies, IP may be in the corporate headquarters and the discovery group in another country. But the intimacy of our IP group means that we have an IP lawyer in the room when we advance,” Macrae says.
Sangamo is the leading player in the ZFN patent field. By contrast, in the CRISPR arena, there
are many patent owners in what is a complex landscape.
The battle rages on between the Broad Institute of Harvard and MIT and the University of California (UC), Berkeley in the war over CRISPR technology, and there are other players on the CRISPR battleground too.
With so much interest in CRISPR, across the globe, PhD students are using the technology in their projects and people are publishing papers on segments of it.
The IP owned by Sangamo is treated very seriously—“nothing is talked about until the IP is sorted”, adds Macrae. This can be a challenge, given the enthusiasm of scientists when making new discoveries.
“They are so excited about what they’ve discovered and so eager to move things forward that they will want to reveal data before it’s been appropriately filed and protected,” he cautions.
Macrae adds that Sangamo’s scientists may request to present new data at an upcoming conference or meeting before the IP has been filed and protected, “but our IP team reviews all data before it’s presented publicly and prevents this from happening”.
With great power, comes great responsibility
With a wealth of experience in the life sciences industry, including 15 years at GSK, Macrae is no stranger to the importance of IP.
After taking the helm of Sangamo in June 2016, he restructured the organisation, strengthening the company’s leadership team and focusing on the clinical development of gene-based medicines.
In the first 18 months of Macrae’s tenure, Sangamo initiated four clinical trials across multiple indications, expanded its ZFP technology library, and established collaborations with Gilead/Kite and Pfizer.
Sangamo is working with Pfizer on a potential gene therapy using ZFP transcription factors to treat amyotrophic lateral sclerosis and frontotemporal lobar degeneration.
It is also the first company to undertake an in vivo genome-editing trial for MPS II, also known as Hunter syndrome, using ZFNs.
A corrective gene will be added at a precise ‘safe harbour’ location in the genome of liver cells using ZFNs, which will enable a life-saving enzyme to be produced whenever liver cells naturally make the most common component of blood: albumin.
“What’s truly laudable is how the patient community support each other. When one patient went to be treated in the genome-editing trial, he knew it wasn’t for him. He was an adult and the disease had already had an effect on him, but he was doing it for the community,” explains Macrae.
“The gift that patient gave will hopefully allow children in the future to avoid suffering from Hunter syndrome.”
These kinds of treatments have given Macrae a huge sense of responsibility. Whereas with drug trials, patients consent to having the drug in their system for a limited period of time, this modification will be with that patient for the rest of his life.
Macrae also feels that he owes a responsibility to the scientists who have worked their whole careers for something that was so often disbelieved by others.
“Patient safety is at the centre of everything we do; it forms a part of every conversation we have,” adds Macrae.
People with rare diseases are even brought in to the Sangamo offices so that the scientists who are about to perform the treatment understand the rare disease patient experience and know the responsibility they have and its urgency.
Research and development (R&D) can be a risky business—technology risk and biology risk are the two types that are always on Macrae’s mind.
With technology, it’s about “how well we understand our technology and know what a ZFN will do when it meets a piece of DNA”.
Over the years, Sangamo has become increasingly good at understanding what it does, adds Macrae, claiming that the process is now “a very logical and reliable form of engineering”.
Sangamo then applies that technological know-how to biological questions, which present the other form of risk of understanding the role that a certain gene plays in a disease.
That’s why Sangamo’s initial therapeutic targets are monogenic diseases where the gene is deficient or absent in the patient; replacing or mending the gene has a lower risk.
It can cost between $200 million and $300 million from conception to registration for one treatment, with the cost being driven by clinical studies and the manufacturing process, making it vital to ensure that the technology is predictable and the work is scalable.
“We choose diseases where the medical risk is appropriate. Our technology is used for diseases which have significant consequences and where there is an absence of any real alternatives,” concludes Macrae.
“Our philosophy is translating ground-breaking science into therapies for important diseases.”
Sangamo fact file
• Founded: 1995
• Global collaborations with Pfizer: 2
• Global collaboration with Kite-Gilead: 2
• 2017: conducted first ever in vivo human genome editing studies
• 2018: awarded $8 million grant from the California Institute of Regenerative Medicine
