Eyeworld

I GENE THERAPY N FOCUS 36 | EYEWORLD | SEPTEMBER 2020 Contact Jun: aljun@jhmi.edu Moore: tara.moore@ulster.ac.uk it produces the healthy wild-type protein, Dr. Moore added. Dr. Jun detailed how he is currently work- ing on treating tissues but not yet actual pa- tients. The challenge, he said, is getting the treat- ment into the cell, and that can be done in a couple of ways. One way is to coat the compo- nents with chemicals, and they work their way into cells. Depending on the cells, that approach could be good or not, as some cells don't let those kinds of chemicals in very easily. The other more efficient way is with a virus, Dr. Jun said, adding that there are differ- ent viruses for delivering CRISPR components. Viruses can get into some cells easily, but they also must be safe to use, he said. "The way we're doing it is packaging CRISPR into viruses and putting it onto tissue and checking how effective it is in modifying the genes," Dr. Jun said. In addition to looking at what percentage of cells are changed, Dr. Jun said it's important to examine "off-target effects." He noted that this could occur if there is a slight sequence mismatch to the one that is being targeted. "It's unlikely there would be that same match somewhere else in the genome," he said, but if a similar sequence exists, the CRISPR system may still work on it in a more limited fashion, so it's possible there could be some unintended effects. Other promising treatments Dr. Moore added that siRNAs are promising. "The work Avellino and Ulster University are doing with drug delivery company SiSaf is the first time I am seeing promise of delivering gene silencing and gene editing tools to an in- tact eye and seeing successful cargo delivery not only to the cornea but also to the retina—a real promise for the future of injection-free delivery of drugs for many different major ophthalmol- ogy diseases," Dr. Moore said. While a cornea transplant is a good option, the idea of having a non-surgical treatment would benefit patients, giving them an addi- tional therapeutic option. "Now we wait for patients to develop the disease, even though we could do a genetic diagnosis of Fuchs, but since there's no medical treatment, we wait until they need surgery." Using a gene editing approach would be a permanent option, so that a patient might not need a transplant at all. The other group of diseases that he's excited about are the TGFBI disorders. "I think this is particularly exciting because there are no good treatments for these conditions," Dr. Jun said. While a laser treatment or corneal trans- plant can be performed, the disease will come back, he said. Dr. Moore said she's particularly excited about this treatment option for a number of disorders, including TGFBI, toward which her team at Ulster University is working. Addition- ally, she noted LCA10, which shows promise of being the first in vivo CRISPR treatment. Dr. Moore also mentioned retinitis pigmentosa, inherited retinal dystrophies, and Huntington's disease and hemophilia disorders as other disor- ders for which this treatment may be particular- ly applicable. How does this treatment work? According to Dr. Moore, CRISPR's allele speci- ficity can be exploited in therapeutic treatments where only the disease-causing allele is targeted and disrupted, and healthy allele remains and minimizes adverse effects. To achieve that, Cas9 nuclease together with a guide RNA is intro- duced into the cornea, she said. "By using this guide, the Cas9 searches for complementary genomic sequence where Cas9 binds and gener- ates double-strand break at the specific loca- tion," Dr. Moore said. "To repair this break, a DNA repair system called non-homologous end joining is activated, which can introduce disrup- tions at the site of the mutation by introducing indels (insertions and deletions)." The gene is then permanently disrupted, as the translation to mRNA contains premature termination that does not create the protein that contains the mutation. The other allele is not affected, and continued from page 35 About the sources Albert Jun, MD, PhD Professor of ophthalmology Johns Hopkins Hospital Baltimore, Maryland Tara Moore, PhD Professor of personalized medicine Ulster University Coleraine, U.K. Reference 1. Stadtmauer EA, et al. CRISPR- engineered T cells in patients with refractory cancer. Science. 2020;367:eaba7365. Relevant disclosures Jun: Hunterian Medicine Moore: Avellino and SiSaf

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