Less than a decade after being harnessed as a programmable gene-editing tool, CRISPR has marched into the clinic. Advances continue to be made in refining the editing reagents themselves —finessing both the guide RNAs (gRNAs) and Cas endonucleases to improve cutting precision and minimize genotoxic effects—but, for clinical applications, systemic delivery remains a work in progress, relying on the viral vector and lipid nanoparticle technologies (LNPs) used for other nucleic acid therapeutics.
“Those are fine technologies, but they have some serious flaws,” says Jacob Corn of ETH Zürich, one of Spotlight’s co-founders. “When people are trying to make them better, they’re iterating on the core idea. They’re trying to make new AAV capsids or they’re trying to make different types of lipids for the LNPs. They’re not thinking different[ly] about how you could do this.”
Spotlight Therapeutics was born of a vision to re-imagine CRISPR delivery. The company’s three co-founders—Corn; Alex Marson of the University of California, San Francisco; and Patrick Hsu of the University of California, Berkeley—zeroed in on delivery as an area in the CRISPR field that required new thinking.
“We thought there was an opportunity, based on some preliminary work that was happening in our labs and some things we were seeing in the field, to take advantage of the fact that Cas9 is a protein, and that we could use the power of medicinal biochemistry to start addressing that protein to particular cell types,” explains Marson.
The central idea is to target Cas endonuclease and its gRNA to specific cell types in vivo using a “library of parts” consisting of cell-penetrating peptides (CPPs), antibodies, and ligands. These parts can be recombined into a selection of targeted active gene editors (TAGEs). TAGEs combine a cell-targeting antibody with CPPs linked to Cas endonuclease preloaded with a gRNA of interest. They seek out the intended cells, traverse the cell membrane and penetrate the nucleus, where they can edit the locus of interest.
“We have a really rich toolbox of editors available to us, and one of the remaining hurdles is the ability to deliver these tools carefully to different cell types in vivo,” says Ben Kleinstiver of the Center for Genomic Medicine at Massachusetts General Hospital. “They’re addressing a really critical need in the field, and if they can make progress, it will be very impactful.”
To identify targeting molecules that can be used in TAGEs, Spotlight uses a combination of rational design and high-throughput screening. The company says that their discovery pipeline allows them to identify targeting molecules that work in practice, not just in theory. Their screening platform ensures that a given molecule effectively delivers the editors to the nucleus. “If you just make an antibody, sure, it’s going to bind to the cell surface receptor, but how do you know it gets in?” says Corn. “If it hits in our system, it has actually made it into the nucleus.”
So far, most gene editing in humans has been done using cells engineered outside the body, expanded and then reinfused. Chimeric antigen receptor (CAR)-T cells in cancer are one example; another is the engineering of hematopoetic stem cells to treat blood disorders like sickle cell disease or β-thalassemia. These ex vivo therapies allow direct delivery of the gene editing reagents to the desired cells. But because they’re usually autologous, N-of-1 approaches, they’re also expensive, complicated and labor intensive. In some cases, the patient must endure a toxic conditioning regimen to clear the way for the edited cells.
“That’s where Spotlight has an enormous opportunity,” says Marson. “The strongest motivation to move to in vivo editing is that this will democratize the benefits of CRISPR. Editing will go from an extremely complicated process with, in some cases, toxicities to something that could be done directly inside the body, hopefully with a one-time injection that would have a curative effect.”
One benefit of ex vivo therapies, of course, is the chance to perform quality control on the edited cells. Injecting gene-editing reagents directly into the body takes that control out of researchers’ hands, leaving no room for error. Even as editing reagents become better characterized and controllable, questions remain about off-target or other unintended genomic changes. In vivo delivery is also heavily biased towards certain tissues, such as liver sinusoidal endothelial cells, and hyperabundant asialogylcoprotein receptors on hepatocytes have thus far been the main target for successful conjugate-mediated delivery in the clinic.
Although Spotlight’s focus is on optimizing delivery technology and not the editors themselves, any in vivo gene editing therapy will have to contend with the possibility of the “unknown unknowns” of genome editing that takes place in the body rather than outside the body. “Systematic, unbiased profiling of the genomic consequences of genome editing in in vivo models has been a focus in the field,” says Hsu. “But it will need to be tackled individually for each new therapeutic development candidate.”
“We know that CRISPR can trigger some chromosomal rearrangements,” says Philippe Duchateau, CSO of Cellectis. He cautions that careful dosing will be critical to avoiding off-target effects. “You want to use just enough to have the effect that you want, but not too much,” he says. “If you put too much nuclease into the cell, you start having these off-site effects. This technology is going to be difficult to fine tune the dose that is needed.” He adds, “The gene editing field is still in infancy. In vivo application is the future, but for today, we are not there yet.”
Ready or not, however, in vivo gene editing has begun. In March 2020, Editas Medicine (with Allergan) announced treatment of its first patient with an injected CRISPR therapy to correct a mutation in centrosomal protein 290 (CEP290) that causes blindness. The treatment delivers Cas9/gRNAs specific to CEP290 via an adeno-associated virus serotype 5 (AAV-5) vector injected directly into the retina. Last month, Intellia Therapeutics also published results of durable knockout of transthyretin (TTR) in TTR amyloidosis via a LNP-delivered Cas9 endonuclease infused intravenously.
Manufacturing viruses is complicated and costly, however. “At the time that we got started with Spotlight, the noise that was ringing in our ear was that Editas had just failed a clinical manufacturing batch of AAV,” recalls Corn. “Some of these delivery technologies are very difficult to engineer, they’re not modular, and they’re hard to produce.”
“The idea for TAGEs is that you have all the scalability in terms of manufacturing, quality control, of a biotherapeutic, but you do it with a gene-editing reagent,” Corn explains.
Spotlight has their sights on three applications: ocular indications, where the TAGEs would be delivered directly to the subretina; immuno-oncology, where TAGEs would be injected directly into the tumor microenvironment to potentiate T cells and macrophages; and blood disorders, where TAGEs would be injected into the bone marrow to access hematopoietic stem cells, avoiding cumbersome extraction and reinfusion protocols. Because in many of these cases ex vivo CRISPR gene editing has already been validated by other groups, these provide a solid base on which to build TAGE approaches off the ground.
A key feature for selecting indications, Marson says, is that editing only a subset of cells is enough to deliver a therapeutic benefit. “They’re diseases where you don’t have to correct every cell in the body, where getting a targeted gene modification into even a small number of cells would totally transform the way that the body responds and have a major effect on treating that disease,” he says.
So far, the company hasn’t published many data demonstrating their platform’s efficacy. “The data that’s been driving some of this is with transgenic reporter mice,” says Corn. “When you get editing, you get a reporter signal in the mice. In all three of the programs, there’s very clear evidence of in vivo activity following a single administration.” For the first TAGEs, the company has selected CD34 and c-Kit antibodies to target hematopoietic stem cells, and these are being tested in humanized mice.
Marson acknowledges that Spotlight is launching at a very early stage. “It was not something that was fully de-risked in the academic setting,” he says. “It’s been wonderful to have GV [formerly Google Ventures] as a partner that’s been committed to this, recognizing the importance and showing a willingness to be bold.”
Spotlight closed their series A funding round with $30 million from GV and a few other, unnamed, funders. Mary Haak-Frendscho, former president of Takeda San Francisco, joins the team as president and CEO.
Caroline Seydel, Los Angeles, CA, USA