The high cost of viral-based cell therapies has prompted some researchers to focus on CRISPR-Cas9 using non-viral delivery methods.Credit: Natali _Mis/Shutterstock
CAR-T-cell therapies are revolutionizing the treatment of blood cancers, and their full potential is still being explored in hundreds of clinical trials. In this therapy, a patient’s own T cells are genetically edited ex vivo so they express a chimeric antigen receptor (CAR) that targets an antigen on the cancer cells. Most currently approved CAR-T therapies use a viral vector delivery system to introduce the CAR to the T cells. However, viral delivery has some drawbacks, such as the inability to regulate dosage or undesired immune response.
Another issue is the steep cost of developing new therapies. “Clinical-grade retroviruses that you can use in early-phase clinical trials at academic research centres are extremely costly,” says Dimitrios Wagner, head of research and development at the Berlin Center for Advanced Therapies at Charité – Universitätsmedizin Berlin.
The quality control procedure for these vectors is extremely stringent because they will be used in therapy. If a viral vector doesn’t achieve the desired results, it can set development back by more than a year. “You will end up paying millions,” says Wagner, “and that does not include the clinical trial you still have to run.”
However, most current CAR-T therapies still rely on viral delivery mechanisms. One of the reasons for that is the high efficiency of viral delivery, which non-viral methods have traditionally struggled to match. But recent work to optimize non-viral editing efficiency is starting to change the picture, highlighting the advantages of non-viral CRISPR editing systems as an alternative delivery vehicle for the next wave of cell and gene therapies.
Non-viral advantages
Given the costs associated with viral-based cell therapies — typically ranging between US$400,000 and US$1 million per patient1 — Wagner and other researchers have focused on CRISPR-Cas9 using non-viral delivery methods. “Our motivation was to make cellular engineering cheaper by going non-viral,” Wagner says.
Besides the lower cost, this also makes it possible to insert the CAR in a targeted location, such as the alpha chain of the T-cell receptor. “This way,” says Wagner, “we found we can generate CAR T cells that are as good as those transduced with lentivirus.”
One way to achieve non-viral transduction is by using electroporation to introduce single-strand (ssDNA) or double-strand DNA (dsDNA) into the cell. CAR T cells produced with such non-viral delivery methods are just as capable of targeting antigens on cancer cells, but they don’t have the same risks and side effects associated with viral delivery2. For example, viral delivery can lead to too much CAR being expressed, which exhausts or even kills the cell over time.
Another benefit of non-viral delivery is that it’s much faster. “It can take a lot of time to procure a plasmid for a viral vector,” says John Zuris, director of editing technologies at Editas Medicine. “Those are not trivial plasmids to make. They often have highly repetitive components and sequences.”
Non-viral HDR templates like ssDNA along with CRISPR system enables efficient and precise gene insertion into target site with minimum toxicity, which is being explored by researchers for CAR-T therapies.Credit: GenScript
Overcoming the efficiency deficit
When lentiviruses or retroviruses are used to insert CAR into T cells, almost all cells take up the viral vector. Non-viral delivery methods have been much less efficient, in the range of 20-40%. Those numbers have made it difficult for non-viral delivery methods to catch on.
“It's so critical to match the viral vector efficiencies we're seeing,” says Zuris. He has been working on a solution to overcome the low efficiency of non-viral delivery. This system, called SLEEK (SeLection by Essential-gene Exon Knock-in), selects cells that have successfully taken up their knock-in cargo, which increases efficiency to around 90%3.
Having a way to enrich only those cells that took up their cargo also minimizes the need to overload the cells with too much template DNA, which reduces the risk of toxic side effects triggered by the presence of foreign DNA fragments in the cell.
Wagner has also used SLEEK in some of his experiments. “It's a very nice system,” he says. “We see a lot of opportunity to adopt it, especially for multiplex engineering.”
But enrichment alone is not a magic bullet to make non-viral delivery work. “One of the things we found experimentally is that you need really high-quality reagents,” says Zuris.
The building blocks of non-viral delivery
Lumeng Ye, senior scientist at GenScript, has been working on non-viral delivery reagents for the past five years. “At the time, there was a growing interest in using this application for CRISPR gene editing,” she says. “We started looking for non-viral DNA payloads and ways to make the editing more precise and safe.”
Non-viral delivery systems can make the cell editing process much simpler, says Ye. “Instead of two or three steps, we can do knock-out or knock-in in one step.”
GenScript now offers several solutions for non-viral delivery, such as single-stranded DNA. Wagner, who has used GenScript’s ssDNA, points out that this type of delivery system is much less toxic to cells than dsDNA, making it possible to add higher doses and increase knock-in rates.
The landscape of non-viral delivery has changed rapidly in just a few years. “We often wondered whether non-viral delivery could be as efficient as the viral approach,” says Ye, “but researchers in different labs have continued to optimize either the process or the design to make it more efficient.”
While a few years ago, the size of a DNA insert was one of the limiting factors for non-viral delivery, optimization strategies such as SLEEK have paved the way to make even the delivery of larger cargo more efficient. Meanwhile, electroporation equipment is also becoming more efficient and less damaging to cells. “It’s like everybody is working together on different efforts to make it happen,” says Ye.
Wagner also thinks the time is right for non-viral delivery. “Once we have a few pivotal trials which show that, for example, a CAR-T-cell product can be very efficient when it’s manufactured in this way, then more people will adopt these technologies,” he says.
And with non-viral delivery bringing advantages in speed and cost-effectiveness, Zuris thinks it will soon catch on more widely: “Just the ability to bypass viral vector manufacturing would be so important.”