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CRISPR Corrects Genetic Immunodeficiency in Blood Stem Cells

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NEW YORK (GenomeWeb) – A new study from scientists at the National Institutes of Health provides a CRISPR-based method for genetic repair of a mutation that causes chronic X-linked granulomatous disease (X-CGD), an immunodeficiency.


Led by first author Suk See De Ravin and senior author Harry Malech, of the National Institute of Allergy and Infectious Diseases, the team used CRISPR/Cas9 along with a single-strand DNA oligonucleotide to correct a mutation in the CYBB gene, a subunit of the nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) gene, important for production of hydrogen peroxide as a first line of defense against foreign microbes.


Not only were the researchers able to reach high ex vivo editing efficiencies using a commercially available electroporation technology, the edited cells were taken up as grafts in immune-compromised mice and produced a relatively high percentage of mature cells after a period of five months.


It's among the high-water marks for ex vivo CRISPR-edited hematopoietic stem and progenitor cells — which have proven exceptionally tricky to edit — and subsequent long-term populating a mouse model.
Malech told GenomeWeb that the repopulation efficiency, between 10 and 15 percent of human cells in the mice, was "in the low but reasonable range" that if achieved in human patients could lead to significant clinical benefit. "We are in no way claiming we've got the cure for CGD, but we're on the path, hopefully," he said.


The researchers published their results today in Science Translational Medicine.

CGD is one of a number of inherited immunodeficiencies that affects approximately one in 200,000 people in the US, Malech said. X-CGD is one of five genetic forms and accounts for about 70 percent of patients and can feature several mutations to the NOX2 gene.
Patients missing that enzyme often get recurrent infections and while researchers have developed treatment plans including improved prophylactic antibiotics and bone marrow transplants, it's not always enough to prevent infections. Transplants can be effective, but graft versus host disease is always a looming outcome.

Malech has been investigating gene therapy approaches to X-CGD for years and while he's investigated zinc finger nuclease-based approaches, he jumped on the CRISPR train several years ago.

"We've been working on this thing for at least two-and-a-half to three years," he said. "It's taken a lot of tinkering."

That's largely because hematopoietic stem cells are difficult to edit and engraft. With no ability to clone and grow edited cells, a meaningful level of edits must be achieved in the correction stage. Recent advances in reagent delivery using adeno-associated virus particles and pre-complexed Cas9-guide RNA ribonucleoproteins are improving ex vivo cell editing efficiencies, but editing seems to reduce engraftability.

"You've got to do it all in one fell swoop," he said. "You can't do it at low efficiency and grow them up and out. This whole business of the inability to clone and grow up HSPCs and re-engraft in animals or humans, has been very vexing. It makes it a special challenge."

There's a strain of serendipity to the team's results. The specific mutation they corrected is a common one, accounting for approximately 7 percent of X-CGD cases. "But after we tried to edit elsewhere, we realized there's something special," Malech said.

The guide RNA targeting this gene doesn't bind well to the wild-type sequence, preventing CRISPR/Cas9 from obliterating the work it just completed. Moreover, it just seems to be more amenable to CRISPR machinery, even when compared to other sites in the same gene.

This high efficiency has promising implications for treating the disease with gene therapy, he said. Hypothetically, modifying 10 to 15 percent of neutrophils could have major phenotypic consequences. Malech has submitted a paper for review that addresses the question of how many normal cells are needed to ameliorate the phenotype.


Because it's mostly X-linked, CGD primarily affects males, but through X-chromosome silencing females carriers can exhibit CGD-like phenotype. "If you're a female carrier and if you have less than 10 percent of normal circulating neturophils, you have a statistically significant risk of getting CGD-like infections," Malech said. However, at levels between 10 and 20 percent, both female-carriers and transplant recipients look almost normal. "They may occaisonally get a CGD-like infection, but are not statistically significantly more at risk," he said. And above 20 percent, the female carriers have no more risk of infection.


It's tantalizingly close to reaching a level of clinical significance and is starting to form a trend line for similar gene correction studies. In October, scientists led by Dana Carroll of the University of Utah and Jacob Corn of the University of California, Berkeley published a study, also in Science Translational Medicine, following a similar approach to correcting mutations responsible for sickle cell trait in HSPCs. They also saw engraftment into immune-compromised mice, but at levels around 6 percent of cells.

It's just one mutation and Malech said the seamless repair technique his team used could be dismissed as a "parlor trick." But he sees a lot of room for improvement and he's excited at the prospects. "The important thing about this is that what we've done is scalable," he said.


"I've got at least 10 or 11 patients with this mutation who might benefit from this if I did a small-scale trial," Malech said.

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