Josiah Brown Poster Abstract

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Rachel V. Acree
Caroline Y. Kuo
Joseph D. Long, Donald B. Kohn
A Functional Assay to Assess Restoration of Class Switch Recombination by CD40LG-Edited X-Linked Hyper IgM T Cells
STTP

X-linked hyper-IgM syndrome (XHIM) is an inherited primary immunodeficiency arising from mutations in the CD40LG gene, resulting in defective CD40 ligand (CD40L)-mediated B cell immunoglobulin class switch recombination (CSR) and somatic hypermutation. Since all known disease-causing mutations are located on this single gene, XHIM is an ideal target for gene therapy approaches. Our approach involves site-specific insertion of adeno-associated virus (AAV)-delivered CD40L cDNA into primary T cells and HSCs using the CRISPR-Cas9 platform. XHIM T cells corrected this way demonstrate ~30% gene integration and minimal off-target effects. Critically, functional CD40L expression is inducible by stimulation in corrected CD4+ T cells, but induction of B cell CSR has not yet been demonstrated. In this assay, healthy donor naïve B cells are cocultured with stimulated corrected XHIM CD4+ T cells, and subsequent IgG production, indicating CSR, measured by flow cytometry. Although some background levels of CSR are observed, corrected XHIM CD4+ T cells stimulate CSR in an average of 9.1% of B cells (n=2), which is comparable to levels induced by soluble CD40L, and elevated from 3.4% in uncorrected cells. Though the assay has yet to be fully validated, its successful execution may provide further evidence for the feasibility of this gene therapy approach as a potentially curative treatment for XHIM.

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