Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome

Area of Science:

• Immunology
• Gene Therapy
• Molecular Biology

Background:

• X-linked hyper-immunoglobulin M syndrome (X-HIGM) results from CD40 ligand (CD40L) deficiency, causing immune dysfunction and infections.
• Previous gene therapy attempts using retroviral vectors led to lymphoproliferative disease due to uncontrolled CD40L expression.

Purpose of the Study:

• To develop a safe and effective gene editing strategy for X-HIGM by restoring endogenous CD40L regulation.
• To evaluate the efficacy of homology-directed repair (HDR) for correcting the CD40LG locus in human T cells.

Main Methods:

• Utilized transcription activator-like effector nucleases (TALENs) to create a double-strand break at the CD40LG locus.
• Employed recombinant adeno-associated virus (AAV) to deliver a donor template for HDR-mediated insertion of CD40L or GFP.
• Incorporated the CD40LG 3'-untranslated region to maintain posttranscriptional regulation.

Main Results:

• Achieved efficient, on-target HDR editing in primary human T cells, restoring endogenously regulated CD40L expression.
• Transgene expression kinetics mirrored endogenous CD40L levels under basal and stimulated conditions.
• Restored CD40L binding and rescued B cell immunoglobulin class switching in X-HIGM patient-derived T cells in vitro.

Conclusions:

• Engineered nuclease-directed gene repair offers a feasible strategy for restoring endogenously regulated CD40L.
• This approach holds significant potential for T-cell therapy in X-HIGM syndrome, improving safety and efficacy.