CRISPR has had a wild year. The gene-editing tool has been the subject of increasing scrutiny as the first clinical trial using it to treat a genetic disease begins. Scientific controversies about the safety and accuracy of CRISPR gene editing have leapt from the pages of academic journals into the news headlines. And in the biggest bombshell of all, last month a Chinese scientist claimed to create the first two CRISPR-edited embryos born as babies.
With all the drama, it’s easy to overlook a detail that could make or break the gene editor’s future as a therapy: the delivery challenge.
Editing a person’s DNA to treat disease requires moving CRISPR’s two components—a large Cas9 enzyme that cuts DNA and a strand of guide RNA that directs the cutting—across cell membranes. Neither component can easily slip into cells like many small-molecule drugs do. Getting CRISPR into cells of a particular organ while avoiding other organs can pose an even bigger challenge.
“Delivery is really the major barrier,” says Vinod Jaskula-Ranga, CEO of Hunterian Medicine. “In my mind, that’s the thing that keeps CRISPR from flourishing.”
That’s why Hunterian and a handful of other start-ups are positioning themselves as CRISPR delivery specialists rather than CRISPR therapy firms.
Hunterian, which is developing a new, virus-based approach to CRISPR delivery, says it has raised $2.7 million in seed financing. It has plans for a bigger series A round next year.
Last month, Feldan Therapeutics announced raising $13.8 million in series A financing to develop a peptide system to get CRISPR and other protein-based therapies into cells. And on Dec. 11, GenEdit announced raising $8.5 million in seed financing for its polymer nanoparticle approach to CRISPR delivery.
Right now, CRISPR therapy firms are repurposing old techniques and delivery vehicles to get the gene editor into cells—and it’s easier for some diseases than others. For instance, Crispr Therapeutics is launching clinical trials for sickle cell disease and β-thalassemia. It removes blood stem cells from the body and sends them to a lab where scientists zap them with an electric shock to loosen their membranes. That technique, called electroporation, allows CRISPR to slip in and do its editing.
To treat diseases affecting parts of the body that can’t be removed and zapped in a dish, scientists must wrap CRISPR in a package to make it palatable to cells. This in vivo gene editing is much trickier. Intellia Therapeutics is using lipid nanoparticles to get CRISPR into the liver, and Editas Medicine is using adeno-associated viruses to get CRISPR into the eye.
But in these approaches, Editas and Intellia aren’t delivering the actual Cas9 protein of CRISPR. The viral method delivers DNA instructions for making Cas9, and the lipid nanoparticle strategy delivers messenger RNA instructions for making Cas9. That creates a potential problem, because cells will use instructions to continue generating CRISPR after the edits have been made—a scenario that increases the chance of CRISPR making a mistake and introducing a mutation in the DNA.
Feldan, GenEdit, and Hunterian are all crafting systems to improve upon these existing approaches to delivery.
Feldan was founded in 2007 to develop molecules called Feldan shuttles that can nonpermanently coat protein therapies to help them cross cell membranes—something that proteins can’t do on their own. Recently, the company garnered attention and money for using the shuttles to get CRISPR into cells without electroporation. Since electroporation can damage some cells, and other cells appear to resist it altogether, the strategy could provide a promising alternative for CRISPR delivery.
The Feldan shuttle is inspired by an old trick: fuse a positively charged, naturally-occurring molecule called a cell-penetrating peptide (CPP) to proteins to help them cross cell membranes. But so far, CPPs haven’t proven very useful for drug delivery because they tend to get trapped in lipid membrane structures called endosomes as they enter cells. If the CPP-protein pair can’t escape the endosome and enter the cytosol—the cell’s main compartment—the scheme is useless.
During Feldan’s long incubation, CEO Francois-Thomas Michaud developed a solution to the problem. Michaud and his team showed that when a peptide known to destabilize endosome membranes was attached to the CPP, the resulting fusion of peptides—the Feldan shuttle—could help proteins bust out of the endosome.
“It’s way more efficient than the two natural peptides alone,” Michaud says. “It’s a completely new and unnatural peptide.”
In a paper published in April, Feldan showed that creating nonpermanent, or noncovalent, mixtures of the Feldan shuttle and CRISPR helped get the gene-editing complex into the cytosol of several kinds of cells (PLOS One 2018, DOI: 10.1371/journal.pone.0195558). Michaud says the technique is also working well as an injection for in vivo gene editing in mice.
So far, the response to Feldan’s work has been mostly positive. “They have provided a massive and convincing set of data,” says Javier Montenegro, a chemist developing CRISPR delivery methods at the University of Santiago de Compostela. Using the Feldan shuttle to escape the endosome “could represent a huge step ahead for protein-based therapies,” he adds.
Ross Wilson, a CRISPR scientist at the University of California, Berkeley, also applauds Feldan’s alternative to electroporation. But since the Feldan shuttles are not covalently attached to CRISPR, it’s unclear how well the pair will stay together when injected into the body, especially the bloodstream, Wilson says. “It will be interesting to see what new applications can be improved or conceived thanks to this technology.”
Feldan’s first tests in humans won’t involve CRISPR, Michaud says. Instead, the firm plans to test the ability of the shuttle to deliver therapeutic proteins for diseases of the skin and eye. CRISPR programs will come later, but Feldan is already off to a start with a $2 million National Institutes of Health grant it is sharing with the University of Iowa to develop CRISPR therapies that can treat respiratory diseases like cystic fibrosis or asthma. Michaud also says the company is in discussions with potential drug industry partners.
Feldan’s approach has the advantage of delivering the ready-to-edit Cas9 protein without the potential for CRISPR to linger in cells for too long as it does in the viral delivery approach. GenEdit is also working on a different way to directly deliver CRISPR: wrap it in polymer nanoparticles.
GenEdit was cofounded by UC Berkeley professor Niren Murthy and former graduate students Kunwoo Lee and Hyo Min Park. At Berkeley, the trio created a delivery system called CRISPR-Gold that uses a gold nanoparticle core to help get CRISPR into cells.
Murthy’s team collaborated with the lab of Jennifer Doudna, one of CRISPR’s inventors, to test CRISPR-Gold in a mouse model of Duchenne muscular dystrophy, showing that the system could fix genetic mutations in muscle cells (Nat. Biomed. Eng. 2017, DOI: 10.1038/s41551-017-0137-2). In a separate project, Murthy’s group demonstrated that CRISPR-Gold could even be used to treat genetic diseases of the brain in mice (Nat. Biomed. Eng. 2018, DOI: 10.1038/s41551-018-0252-8)
Lee says he was shocked at how well the teams were able to get CRISPR to work in the muscle and brain. But since gold nanoparticles carry the risk of potentially damaging DNA, Lee says GenEdit doesn’t have any plans to test CRISPR-Gold in humans.
“It’s really the first great demonstration that polymer nanoparticles can deliver Cas9, and it works beautifully,” he says. GenEdit is now focused on designing peptidelike polymers to make nanoparticles that can deliver CRISPR without the potentially toxic gold core.
“We’ve made huge progress in building a polymer library,” Lee says. The company’s scientists screen it to find low-toxicity formulas specific for delivery to tissues like the brain, liver, and muscle.
Perhaps the most tried-and-true method for getting CRISPR into the body is to encode instructions for making the Cas9 protein and guide RNA in an adeno-associated virus (AAV). But this approach, borrowed from gene therapy, comes with the complication of getting the large genetic instructions for CRISPR to fit inside the constrained size of the AAV shells, called capsids.
In fact, the genetic code for one of the most commonly used versions of Cas9 is so big that there’s no room left for the guide RNA in the same capsid. It’s the molecular equivalent of packing a suitcase so full that it won’t close.
Some scientists are using two AAVs to get the Cas9 and guide RNA of CRISPR into cells, but this reduces the rate of successful editing. Jaskula-Ranga founded Hunterian based on technology he developed at Johns Hopkins University School of Medicine that allows instructions for Cas9 and guide RNA to fit into the same capsid.
Jaskula-Ranga hopes that putting Cas9 and the guide RNA in the same AAV will make CRISPR therapies more efficient and also less toxic. Viral capsids have a history of eliciting immune reactions when given at high levels, so companies are looking to reducing the number of AAVs needed for a successful treatment whenever possible.
AAVs are the oldest, most proven delivery shuttles, but the drawback of longevity remains. Since AAVs deliver the DNA instructions for making CRISPR, the gene editing system could theoretically remain in cells forever. “The best thing that we have right now are the viral vectors, and it’s just terrible,” UC Berkeley’s Wilson says.
Even though the sums that Feldan, GenEdit, and Hunterian have raised from investors so far aren’t particularly noteworthy by biotech standards, the problems they are trying to solve could be crucial to CRISPR’s success as a therapy. Similar delivery challenges have stymied other promising treatments until scientists discovered how to properly package the therapies for shipping in the body.
Alnylam, a company that develops RNA interference therapies, was founded in 2002 and didn’t earn its first drug approval until this fall. Its initial programs were vexed by ineffective and toxic delivery formulations. It was the drug-delivery designers who carried the company through its dark days and to success by finally crafting lipid nanoparticle formulations that safely and effectively get its RNA drugs into the liver.
“We know the history,” GenEdit’s Lee says. “That is the reason we started this company.”