On-demand gene editing of autologous cells offers a novel therapeutic option for inborn errors of immunity (IEIs) caused by dominant-negative variants, which are often poorly addressed by conventional viral gene addition or hematopoietic stem cell transplantation (HSCT). IRF4 encodes a transcription factor critical for T cell differentiation, Th17 polarization, memory formation, and tissue homing via integrins such as α4β7. Heterozygous neomorphic variants at T95 disrupt IRF4 DNA binding, leading to combined immunodeficiency with susceptibility to severe infections.
We aimed to develop an allele-specific editing strategy targeting the pathogenic IRF4 p.T95A variant and to evaluate genomic precision, safety, and functional rescue in edited T cells.
Base and prime editing (PE) strategies were designed in a K562 reporter line, prioritizing allele-specific PE to minimize bystander and off-target effects. Patient-derived naïve T cells were selected and edited ex vivo via electroporation of PE and PE guide RNAs (pegRNAs). Functional rescue was assessed by flow cytometry and cytokine profiling of effector memory differentiation, Th17 polarization, and α4β7 expression. Genomic precision was evaluated with methods such as variant-aware in silico modeling and amplicon sequencing.
The proband is a 24-year-old man with a de novo IRF4 T95A variant, multidrug-resistant Mycobacterium avium colitis, recurrent candidiasis, and EBV-driven lymphoproliferation, for whom HSCT carries prohibitive risk. His T cells exhibit reduced effector memory differentiation, impaired Th17 responses, and diminished α4β7 expression. In the K562 reporter with the patient’s IRF4 allele, PE achieved efficient double-digit on-target correction with selective mutant-allele knockout while preserving the wild-type allele. Ongoing work focuses on optimizing PE in primary patient naïve T cells and evaluating phenotypic rescue and genome-wide specificity.
This study demonstrates the feasibility of nonviral, allele-specific prime editing to correct a de novo IRF4 variant in autologous T cells, representing a promising personalized therapy for dominant IEIs.
