UCLA researchers from the Department of Microbiology, Immunology, and Molecular Genetics and Pediatrics have used a bioinformatics-guided approach to design regulated lentiviral vectors for the treatment of X-linked lymphoproliferative disease type 1.
BACKGROUND: X-linked lymphoproliferative disease type 1 (XLP1) is a rare primary immunodeficiency caused by mutations in the SH2D1A gene, which encodes the SLAM-associated protein (SAP). In over 90% of cases, disease onset is triggered by Epstein-Barr virus infection, leading to impaired function of B, T, and Natural Killer (NK) cells. The current curative treatment is the use of allogenic hematopoietic stem cell (HSC) transplantation, where healthy donor cells are transplanted into a patient to provide them with a functional immune system. However, many patients are unable to receive curative treatment due to poor donor availability or immunologic complications. To overcome this challenge, researchers have sought to develop autologous HSC transplantation, where a patient’s own HSCs are harvested and a lentiviral vector (LV) is introduced to integrate a functional copy of the SH2D1A gene. However, current LVs fail to restrict expression of a functional copy of SH2D1A to just the target cell populations, which can lead to aggressive lymphoid malignancies. Therefore, there is a demand to design new LV to safely cure patients with XLP1 disease.
INNOVATION: UCLA researchers have implemented a bioinformatics-based approach to design LVs that can regulate the expression of SH2D1A (XLP1-SMART LV) in a cell type-specific manner. Researchers at UCLA (i) used genome-wide enhancer databases to identify regulatory DNA elements for cloning into lentiviral vectors designed to drive SH2D1A expression (ii) transduced primary T, NK, and NKT cells with the barcoded constructs, and (iii) quantified enhancer activity by sequencing DNA and RNA barcodes using next-generation sequencing. Researchers found that select enhancer elements increased SH2D1A gene expression up to 4-fold in the target immune cell populations while maintaining minimal off-target expression. UCLA researchers then differentiated T cells using the artificial thymic organoid and found that the enhancer activity and SAP protein expression varied across T cell differentiation as assessed through flow cytometry. Lastly, UCLA researchers generated SH2D1A-/- knock-out cells through CRISPR/Cas9 technology and confirmed that they could restore the expression of the SAP protein. In summary, UCLA researchers have taken a bioinformatics-based approach to design XLP1-SMART LVs that can regulate the expression of SH2D1A in a cell type-specific manner, with a clinically relevant role in XLP1. This finding provides potential for a novel method to cure XLP1 through HSC transplantation therapy.
POTENTIAL APPLICATIONS:
- XLP1-SMART LV therapy to treat XLP1
- XLP1-SMART LV therapy to treat other hemophagocytic lymphohistiocytosis disorders, such as perforin deficiency, which share similar expression profiles and regulation to that of XLP1
ADVANTAGES:
- XLP1-SMART LV can achieve a high level of cell lineage specificity with minimal off-target expression
- XLP1-SMART LV is physiologically relevant to XLP1
DEVELOPMENT-TO-DATE: UCLA researchers have created an XLP-SMART LV to regulate XLP1 expression in a cell type-specific manner in in vitro settings.
Related Papers (from the inventors only): Ayoub, Paul G., et al. “Lentiviral Vectors for Precise Expression to Treat X‑Linked Lymphoproliferative Disease.” Molecular Therapy – Methods & Clinical Development, vol. 32, no. 4, 2024, p. 101323. DOI:10.1016/j.omtm.2024.101323.
KEYWORDS: XLP1, SH2D1A, SLAM, Epstein-Barr virus, allogenic HSC transplantation, autologous HSC transplantation, XLP1-SMART LV, enhancer, barcodes, next-generation sequencing, artificial thymic organoid, flow cytometry, CRISPR/Cas9, immunoblot