Graham Rector

Introduction: Chronic granulomatous disease (CGD) is a rare congenital disorder affecting 1 in every 250,000 children born in the United States¹. The disorder is caused by a defect in the phagocytic breakdown pathway, most commonly caused by mutations in genes coding for subunits of leukocyte NADPH Oxidase^1-2. The most common mutations are found in the enzymatic subunit gp91-PHOX, encoded by the CYBB gene. This flaw in the respiratory burst process manifests in severe and persistent bacterial and fungal infections starting early in life. Current treatment involves lifelong medical prophylaxis with antibiotics and antifungals, but recent research has shown the potential of gene editing as the possible future CGD treatment³. Zinc Finger Nucleases (ZFN), TALENs, and CRISPR technology have all explored as potential avenues of care⁴. Methods: ZFNs were used to insert a wild-type CYBB gene into the AAVS1 safe harbor locus on chromosome 19 in human Induced Pluripotent Stem Cells (iPSCs) generated from patients with X-linked CGD. Neutrophils derived from the iPSCs were analyzed for gp91-PHOX activity following treatment^5,6. Unlike in the ZFN trial where the CYBB gene was added elsewhere in the genome, another study used TALENs to replace mutated CYBB exons with wild-type DNA in an attempt to restore function. Exons 1, 2, and 5 were all targeted as potential sites of repair. Neutrophils derived from the treated population were analyzed for gp91-PHOX function⁷. Finally, studies have shown the benefit of using CRIPSR/cas-9 technology to replace various exon mutations found in X-CGD. Mutations in exons 1 and 7 of CYBB were repaired with CRISPR with subsequent analysis of derived monocytes for gp91-PHOX function^8,9. Additionally, the CRISPR-corrected Human Stem and Progenitor Cells (HSPCs) were implanted in immunodeficient mice and researchers later tested the function of the derived myeloid cells at several weeks post transplantation⁹. Results: All the gene editing strategies showed improvement in CYBB and subsequent gp91-PHOX function following treatment, with varying degrees of success. ZFN-treated neutrophils showed 15% of normal expression of gp91-PHOX, whereas neutrophils generated with TALENs restored gp91-PHOX to 73-100% of normal in some cases^5-7. The CRISPR method was found to be the most consistent in its effects, successfully inserting genes 24-38% of the time⁷. Myeloid cells collected from transplanted mice expressed functional gp91-PHOX and serum levels stayed constant from 6-20 weeks after transplantation⁹. Conclusions: These recent studies have provided proof of concept for gene editing in the treatment of CGD. The main benefit of using CRISPR or TALEN technology is the ability to replace the gene in its normal place in the genome, which preserves endogenous regulation of the genes and avoids constitutive expression¹⁰. With its low cost and precise DNA cleavage capacity, CRISPR gene editing has emerged as the potential future in CGD treatment.

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