Highlights
The PM359 trial represents a landmark in clinical genome editing, demonstrating the first-in-human application of prime editing to correct the NCF1 delGT mutation. Both study participants achieved prompt and stable engraftment of neutrophils and platelets following myeloablative conditioning. Most significantly, the therapy restored NADPH oxidase activity in neutrophils, as measured by dihydrorhodamine (DHR) assays, which was maintained throughout the follow-up period. The safety profile remained consistent with standard busulfan conditioning, with no evidence of therapy-related severe adverse events or genotoxicity.
Introduction: The Genetic Challenge of Chronic Granulomatous Disease
Chronic Granulomatous Disease (CGD) is a group of rare, life-threatening primary immunodeficiencies characterized by the inability of phagocytes to produce superoxide and other reactive oxygen species (ROS). This defect stems from mutations in the genes encoding the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Patients with CGD suffer from recurrent, severe bacterial and fungal infections, as well as aberrant inflammatory complications such as granuloma formation and inflammatory bowel disease. While allogeneic hematopoietic stem-cell transplantation (HSCT) offers a cure, it is often limited by donor availability and the risk of graft-versus-host disease. Autosomal recessive p47phox-deficient CGD (p47-CGD) accounts for approximately 25 percent of CGD cases globally. It is primarily caused by a highly specific two-nucleotide deletion (GT) in exon 2 of the NCF1 gene. Correcting this specific mutation has historically been difficult due to the presence of two nearly identical pseudogenes, NCF1B and NCF1C, which share over 98 percent sequence homology with the functional NCF1 gene. Traditional gene-editing techniques, such as CRISPR-Cas9, pose a high risk of off-target effects or large-scale genomic rearrangements when targeting such complex loci.
The Molecular Basis of p47-CGD and the NCF1 Conundrum
The NCF1 gene, located on chromosome 7, encodes the p47phox protein, a critical cytosolic component of the NADPH oxidase complex. The delGT mutation (c.75_76delGT) leads to a frameshift and a premature stop codon, resulting in a complete lack of functional p47phox. The structural complexity of the NCF1 locus is a major hurdle for genetic interventions. Because the functional gene is flanked by pseudogenes that already contain the delGT mutation, standard nuclease-based editing (which relies on double-strand breaks) can trigger inter-chromosomal recombination or unintended deletions. Prime editing, however, offers a “search-and-replace” capability that does not require double-strand breaks (DSBs), significantly reducing the risk of genomic instability. By using a catalytically impaired Cas9 (nickase) fused to a reverse transcriptase and a prime editing guide RNA (pegRNA), researchers can precisely insert the missing GT nucleotides into the functional NCF1 gene without disrupting the surrounding pseudogenes.
Study Design and Methodology: The PM359 Trial
The PM359-101 trial (NCT06559176) is a Phase 1/2, open-label, single-arm study designed to evaluate the safety, tolerability, and efficacy of PM359. PM359 consists of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) that have been edited ex vivo using prime editing technology to correct the delGT mutation. The study enrolled two participants with confirmed p47-CGD and a history of significant infections or inflammatory complications. Both patients underwent mobilization of CD34+ cells using plerixafor and G-CSF. The harvested cells were processed, edited using the PM359 system, and cryopreserved. Before infusion, the participants received myeloablative conditioning with busulfan to clear the bone marrow niche. The primary endpoints included the safety of PM359 infusion and the time to neutrophil and platelet engraftment. Secondary endpoints focused on the percentage of corrected NCF1 alleles in peripheral blood and the restoration of superoxide production in neutrophils.
Clinical Results: Engraftment and Functional Restoration
The results for the first two participants have been highly encouraging. Participant 1 and Participant 2 both demonstrated rapid hematologic reconstitution. Neutrophil engraftment occurred within 14 to 21 days, and platelet engraftment followed shortly thereafter. The safety profile during the immediate post-transplant period was consistent with the known toxicities of busulfan conditioning, including transient mucositis and cytopenias. Following engraftment, molecular analysis of peripheral blood cells revealed stable levels of gene correction. In both patients, a significant proportion of circulating neutrophils carried the corrected NCF1 allele. Functional restoration was assessed using the DHR assay, which measures the oxidative burst in stimulated neutrophils. Within one month of infusion, both patients showed a clear population of DHR-positive neutrophils. Participant 1 maintained this activity at the 6-month follow-up, and Participant 2 showed consistent results at the 4-month mark. The level of superoxide production observed is traditionally considered sufficient to provide clinical protection against the life-threatening infections typically seen in CGD.
Expert Commentary: Why Prime Editing?
The success of PM359 highlights the clinical maturation of prime editing. Unlike earlier iterations of gene therapy that used viral vectors (which carry a risk of insertional mutagenesis) or standard CRISPR-Cas9 (which can cause unintended genomic damage), prime editing provides a level of surgical precision that is uniquely suited for complex loci like NCF1. Clinicians and researchers emphasize that the lack of double-strand breaks is the “holy grail” of gene editing safety. In the context of CGD, where the target gene is surrounded by decoys (pseudogenes), this precision is not just an advantage—it is a requirement. However, experts also note that while the initial results are promising, long-term follow-up is essential to ensure the durability of the edited stem cell population and to monitor for any delayed adverse events. The use of busulfan conditioning remains a significant burden for patients, and future iterations of this therapy might explore non-genotoxic conditioning regimens to improve the overall benefit-risk profile.
Conclusion
The PM359 trial provides the first clinical evidence that prime editing can safely and effectively correct a pathogenic mutation in human hematopoietic stem cells to treat a primary immunodeficiency. By restoring NADPH oxidase activity in p47-CGD patients, this therapy addresses the root cause of the disease with unprecedented precision. As the trial continues to follow these participants and enroll new ones, it paves the way for a new era of genetic medicine where complex mutations, previously deemed “undruggable” or too risky for standard editing, can be corrected with high fidelity.
Funding and ClinicalTrials.gov
This study was funded by Prime Medicine, Inc. The clinical trial is registered at ClinicalTrials.gov under the number NCT06559176.
References
Gori JL, Haddad E, Frangoul H, et al. Prime Editing for p47phox-Deficient Chronic Granulomatous Disease. N Engl J Med. 2025;392(23). doi:10.1056/NEJMoa2509807. Anzalone AV, Randolph PB, Davis JR, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149-157. Seger RA. Modern management of chronic granulomatous disease. Br J Haematol. 2008;140(3):255-266.
