High Level of Perforin Expression Is Required for Effective Correction of Hemophagocytic Lymphohistiocytosis

Abstract

K.R., M.J., and P.M. share senior authorship.Perforin-1 mutations result in a potentially fatal hemophagocytic lymphohistiocytosis (HLH) with heightened immune activation, hypercytokinemia, pancytopenia, and end-organ damage. At present, hematopoietic stem cell (HSC) transplantation is curative, but limited by donor availability and associated mortality, making gene therapy an attractive alternative approach for HLH. We reported that perforin expression driven by cellular promoters in lentiviral (LV) vectors resulted in significant, albeit partial, correction of the inflammatory features in a murine model of HLH. We hypothesized that the level of perforin expression achieved per cell from ectopic moderate-strength cellular promoters (phosphoglycerate kinase gene/perforin-1 gene) is inadequate and thus engineered an LV vector using a viral promoter (MND; a modified Moloney murine leukemia virus long terminal repeat with myeloproliferative sarcoma virus enhancer) containing microRNA126 target sequences to restrict perforin expression in HSCs. We show here that the MND-LV vector restored perforin expression to normal levels in a perforin-deficient human natural killer cell line and perforin gene-corrected Perforin1-/-transplant recipients, whereas cellular promoters drove only partial correction. On lymphocytic choriomeningitis virus challenge, the clinical scores and survival improved only with the MND-LV vector, but inflammatory markers and cytotoxicity were improved with all LV vectors. Our studies suggest that although moderate levels of expression can result in partial amelioration of the HLH phenotype, high levels of perforin expression per cell are required for complete correction of HLH.

Evolving Gene Therapy in Primary Immunodeficiency

“…103 X-linked lymphoproliferative disease (XLP), a T and NK lymphoproliferative disorder caused by mutations in the signaling adaptor signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), and familial hemophagocytic lymphohistiocytosis (FHLH), a hemophagocytic disease resulting from defective NK and CD8 + T cell cytotoxicity, are potential candidates for both HSC/P and peripheral T cell-based approaches. [104][105][106] SAP is involved in multiple signaling pathways that affect proliferation, apoptosis, and differentiation, whereas perforin and Munc13-4, which are the most commonly deficient proteins in FHLH, are direct effectors of cytotoxicity. Lineage-restricted regulation of these genes might be required to avoid deleterious effects in cells where these proteins are not normally expressed.…”

Gene-corrected human Munc13-4–deficient CD8+ T cells can efficiently restrict EBV-driven lymphoproliferation in immunodeficient mice

“…4 In addition to HSCT, gene therapy of hematopoietic stem cells has been tested as a treatment of FHL2 in a murine model. 8,9 Given that the main defect in FHL disease is cytotoxic dysfunction of mature T cells, the latter constitute a potentially valuable target for gene therapy approaches. The genetic modification of T cells has produced remarkable clinical outcomes in cancer immunotherapy and in cases of adenosine deaminase deficiency.…”

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

“…In diseases like LAD (11), CGD (13,14), and WAS (15,16), where a high level of transgene expression is required to achieve a therapeutic benefit, cellular promoters were insufficient to mediate a therapeutic effect unless high VCN were present and strong enhancers were used. In fact, we have recently reported that expression of perforin from cellular or endogenous promoters in LV only partially corrects the hemophagocytic lymphohistiocytosis (HLH) phenotype, and strong viral enhancers are necessary for complete disease phenotype correction (55).…”