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Look for CRISPR/Cas9 to Treat Eye Diseases First, Scientists Say

This is the fourth article in an in-depth series on the impact of CRISPR/Cas9 genome editing technology on basic and clinical research, the biotechnology industry, and the world at large. The first installment, on the development of the CRISPR toolkit, can be found here. The previous installment, on CRISPR in drug discovery, can be found here.

NEW YORK (GenomeWeb) – When CRISPR/Cas9 genome editing finds its way into a clinical trial, it will be for an eye disease — that's according to Vinny Ranganathan, a scientist at Johns Hopkins University who is working on the delivery of gene therapies into that organ.

"The eye is sort of a perfect target for a lot of reasons," he told GenomeWeb. Among the reasons he listed: it's a contained organ, is relatively accessible, can be monitored externally, and has well-established measures of function. "If something bad is happening [because of the treatment], it would be readily visible," he said.

And of course, restoring eyesight can make a huge difference in a patient's quality of life. "People generally really appreciate vision," he said.

Saying that CRISPR-based therapies will be first tested in the eye is a popular position among eye researchers.

Last week, scientists from Columbia University and the University of Iowa published a study showing that CRISPR/Cas9 could edit a genetic mutation that causes retinitis pigmentosa (RP), at least in induced pluripotent stem cells derived from patients carrying that mutation. Stephen Tsang, a professor at Columbia and co-senior author of the study, uttered nearly the same words Ranganathan did: "We believe that the first therapeutic use of CRISPR will be to treat an eye disease," he said.

But their prognostication is not necessarily self-serving hype. Editas Medicine, the gene-editing company that recently completed a $94.4 million initial public offering, also thinks the eye is a good place to start. It claims that its program to treat one of the myriad forms of Leber Congenital Amaurosis (LCA) is the most advanced among its dozen or so different projects with clinical trials expected to begin in 2017.

These hereditary eye diseases like RP and LCA are rare. Retinitis pigmentosa affects approximately one in every 4,000 people in the US; LCA is 10 times rarer than RP. But treatments for these diseases are low-hanging fruit, tantalizing possibilities of relief for patients for which there are currently limited options for treatment and no outright cures.

LCA is a group of more than a dozen retinal dystrophies that appears in early childhood. LCA10, the particular form of the disease Editas seeks to treat, is very severe and typically results in blindness in the first few months of life. There are currently no FDA-approved treatments for LCA10.

Ranganathan isn't just idly observing that CRISPR stands a good chance to come first to the eye. Trained as a molecular geneticist, he specifically moved into diseases of the eye because he thought that would give him first crack at developing gene therapies.

"The eye has a history of being an organ where a lot of new technologies are translated first," he said. With that in mind, Ranganathan moved his research career in that direction before the advent of CRISPR. In addition to the aforementioned qualities of the eye that make it a good target for research, it has a huge advantage for gene therapy: immune privilege. "The immune system is not going around scanning and looking for foreign proteins in the same way as the rest of the body," he said.

Gene therapies for hereditary eye diseases are already on the cusp of becoming an option for some patients. For example, last October, Philadelphia-based Spark Therapeutics announced positive results from a Phase III clinical trial for a gene therapy targeting a mutation in RPE65, which is implicated in both LCA and RP. The product candidate, SPK-RPE65, consists of genetic material delivered by an adeno-associated viral vector. The trial showed improvement for the participants in the primary end point, functional vision, as well as two secondary endpoints — full-field light sensitivity threshold testing and mobility testing, when compared to controls.

"We saw substantial restoration of vision in patients who were progressing toward complete blindness," Albert Maguire, principal investigator in the trial and professor of ophthalmology at the University of Pennsylvania, said in a statement. "The majority of the subjects given SPK-RPE65 derived the maximum possible benefit that we could measure on the primary visual function test, and this impressive effect was confirmed by a parallel improvement in retinal sensitivity."

Importantly, Spark did not observe serious adverse events or harmful immune responses during the trial. The firm will seek US Food and Drug Administration approval for the treatment this year.

Many potential applications

As with drug discovery, clinical applications of CRISPR/Cas9 can come in three flavors. The first group consists of applications that researchers are already working on, but which could be improved by the numerous advantages of CRISPR/Cas9. Among these are the aforementioned gene therapies for hereditary eye disease, T cell therapies for leukemia, and treatments for sickle cell disease.

In the second category are things people had thought about doing, but had been impractical to even attempt without the aid of CRISPR. One such example is xenotransplantation. Last year, researchers from the lab of Harvard Medical School's George Church used CRISPR/Cas9 to remove 62 retroviruses from the pig genome. This showed that one of the barriers to using organs grown in that animal in humans could be eliminated. "People have thought about that project for a while," Jacob Corn, a professor at the University of California at Berkeley and scientific director of the Innovative Genomics Institute, told GenomeWeb. "The problem was always the retroviruses."

The last category consists of the applications that have yet to be dreamt up. "Of course, that's hard to predict, the sci-fi type stuff," Corn said. "I've learned the hard way, never predict," he said, adding, "We're hearing a lot about people in the genetic disease space coming up with innovative approaches to cures."

There also are a few other important distinctions for how CRISPR may be used in therapies, for example, whether the editing is done in vivo or ex vivo. The difference is so big that Intellia Therapeutics has launched an entirely separate division to conduct its ex vivo work. There's also the question of whether the treatment can work by simply cutting the genome, which uses the more efficient non-homologous end-joining DNA repair pathway, or whether it needs to take advantage of the powerful but less-efficient "find and replace" functionality, which leverages homology-directed repair (HDR) but requires a donor template.

One of the reasons the particular subtype of LCA that Editas is going after is attractive is that it theoretically can be fixed with a cut to the gene CEP290. "What happens is there's a point mutation that creates what looks like an exon in an intron," Ranganathan explained. "The cell splices in something that has a stop codon, so you end up losing gene function. If you can disrupt that cryptic splice site, the gene will be totally fine."

"What [Editas] needs to do is remove a sequence because it lies in a part of a gene that doesn't become a protein, he said. "Remove it, and then the gene will function normally."

"It is unclear how this will work and if it will be a cure or not," Ranganathan said of Editas' LCA program. Some cases of hereditary blindness are so severe that even intervention at birth might not save the photoreceptors, or may just delay the disease.

"A delay is not a bad thing," he said. "If you can extend vision, anything, that's a good thing. But we don't know for sure if these will be lasting cures.

Still, Ranganathan thinks therapies both cutting and replacing genes will find their way to the clinic. Ultimately, to cure most recessive genetic diseases, researchers will need to replace genes, LCA10 being the rare example of a recessive disease that can be fixed with only a cut.

Though cutting is currently more efficient, gene replacement is improving by great leaps. Corn, the Berkeley professor, recently led a team that discovered a short flap on the 3' end of the non-target strand of DNA that can be used to increase the likelihood of HDR. By rationally designing the donor DNA template to be complementary to that flap, Corn's team was able to achieve HDR efficiencies to around 60 percent, for templates shorter than 30 base pairs.

"The real problem with clinical approaches is, fundamentally, delivery," Ranganathan said. His own work is focused on using adeno associated viruses to deliver CRISPR/Cas9 in vivo. In research applications, electroporation, viral delivery, and even straight injection of CRISPR reagents can work, depending on the cell type. "The viral ways will be the approach in the next couple years. All the other methodologies are a few years behind," he said.

The challenge of delivery is why diseases like sickle cell disease, which could theoretically be cured by editing hematopoietic stem cells ex vivo and re-injecting them, are attractive. Bone marrow transplants are a well-established medical practice and cells can be screened for off-target effects prior to injection.

Delivery could be a big challenge for DMD, another disease where scientists have already shown they can edit out the mutation causing it. In December, scientists from Toronto's Hospital for Sick Children published a study showing how to remove an exon duplication affecting a patient with DMD. Then in January, three studies published in Science indicated that it was possible to improve symptoms in a mouse model of the disease.

Getting the CRISPR/Cas9 into muscle cells, or other cells that need to be edited inside the body, could be exceedingly tricky, and delivery has grown to become almost as big of a concern for therapeutic applications of CRISPR as safety. Meanwhile, safety, specifically off-target effects, still looms large due to uncertainties, though recent events have started to alleviate concerns.

"Now we have modified versions of Cas9 that appear to have sort of zero off-targets," Ranganathan said, referencing high-specificity variants of Cas9 developed at the Massachusetts Institute of Technology and Harvard Medical School.