CRISPR/Cas9-Mediated Targeting of the Integrin Beta-2 Gene as a Gene-Editing Strategy for Leukocyte Adhesion Deficiency Type I.

BACKGROUNDLeukocyte adhesion deficiency (LAD) is a genetic immunodeficiency that severely impairs the leukocyte adhesion cascade and migration to the sites of infection and inflammation. As a result, the main consequence is recurrent and severe infections with bacteria and fungi, which can lead to death. The most common variant, LAD-I, is due to mutations in the integrin beta 2 (ITGB2) gene responsible for the β2 integrin (CD18) subunit. Corrective strategies based on gene therapy and genome editing are the most promising methods for treating genetic defects.

METHODSIn the present experimental in vitro study conducted at Tarbiat Modares University, Tehran, Iran, in 2024, we designed a single-guide RNA (sgRNA) for the CRISPR/Cas9 system to target exon 6 of ITGB2 and performed validation experiments. We also investigated peptide nanocarriers as a potential strategy for delivering the CRISPR components. The PX458 plasmid encoding Cas9 and the designed sgRNA was delivered into HEK293T cells, followed by genome-editing analysis.

RESULTSGenome-editing analysis confirmed a measurable level of indel formation at the target locus. Furthermore, the results of MPG nanopeptide production and purification demonstrate the potential of peptide nanocarriers for genome-editing systems.

CONCLUSIONOverall, this work establishes a foundation for subsequent knock-in and gene-correction studies in clinically relevant immune cell models, paving the way for potential LAD-I gene therapy applications.