X-linked chronic granulomatous disease (X-CGD) is a rare genetic disorder characterized by high susceptibility to infections that results from genetic mutations in the cytochrome b-245 beta chain (CYBB) gene. Patients with X-CGD experience recurrent invasive infections, hyperinflammation, inflammatory bowel disease, and have an increased morbidity and early mortality. Researchers have sought to optimize gene editing approaches to correct X-CGD mutations and develop a safe and effective treatment for the condition.

Base editors (BEs) have been used in gene therapy designs for the treatment of X-CGD using autologous hematopoietic stem and progenitor cells (HSPCs) transduced with lentiviruses that carry the corrected CYBB gene. However, there are limitations. Base editors have limited utility because they use Cas9 enzymes that require a protospacer adjacent motif (PAM).

Now, new work has optimized a PAMless Cas9 enzyme SpRY with an adenine base editor (ABE8e-SpRY) to correct mutations in CYBB in stem cells from patients with X-CGD. The findings suggest that gene editing correction of an X-CGD mutation using the PAMless BEs occurs with high efficiency and specificity.

The findings are published in Science Translational Medicine in the paper, “High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease.”

This work was borne out of a longstanding collaboration between the laboratories of Suk See De Ravin, MD, PhD, a senior research physician and chief of the gene therapy development unit at the NIAID, and Benjamin Kleinstiver, PhD, an investigator at the Center for Genomic Medicine and department of pathology at MGH.

“We’re excited about the use of base editing to directly correct mutations since this approach is distinguished from traditional gene therapies that overexpress a corrective gene,” said Kleinstiver. “Our results demonstrate how the improved capabilities of engineered CRISPR-Cas9 enzymes can be beneficial, and together motivate additional studies using base editors to correct other mutations that cause inborn errors of immunity and other diseases.”

The researchers took hematopoietic stem and progenitor cells from two patients with different X-CGD-causing mutations, and then treated the cells with various adenine base editors to correct either mutation in the CYBB gene. The approach was highly effective, with an efficiency of more than 3.5 times higher than previous approaches and with minimal off-target effects.

More specifically, the authors noted, “For the prototypical X-CGD mutation CYBB c.676C>T, ABE8e-SpRY achieved up to 70% correction, reaching efficiencies greater than three-and-one-half times higher than previous CRISPR nuclease and donor template approaches.”

The adenine base editors also may overcome some of the challenges associated with other gene therapy approaches. The researchers profiled potential off-target DNA edits, transcriptome-wide RNA edits, and chromosomal perturbations in base-edited HSPCs “which together revealed minimal off-target or bystander edits.”

Based on the team’s findings, a first-in-human clinical trial (NCT06325709) is now underway to test the potential benefits of base-edited stem cell treatments in patients with X-CGD.

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