Perforin Gene Transfer Into Hematopoietic Stem Cells Improves Immune Dysregulation in Murine Models of Perforin Deficiency

Abstract

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8+ T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8+ lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.

Gene transfer into hematopoietic stem cells reduces HLH manifestations in a murine model of Munc13-4 deficiency

“…17 One important aspect of the present study is that HSC gene correction with a Munc13-4-expressing LV not only corrected biological manifestation of HLH, but above all enabled effective viral clearance and recovery from liver damage. Unlike perforin (the expression of which is restricted to mature T and NK cells), Munc13-4 is ubiquitously expressed in human and mice, 18 and so its vector-derived expression in HSCs may be less likely to induce undesirable side effects.…”

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.…”

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

“…15 The EcoRI end was blunted and PGK.Perforin1 was ligated into the EcoRV/SalIdigested GFP4T. The same strategy was used to clone LV.NGFR.CMV.PRF.Perforin1.WPRE .4TmiR126 (PRF4T), with the fragment PRF.Perforin1 from the LV.PRF.Perforin1.IRES.GFP (PRF), 15 into GFP4T.…”

“…15 The EcoRI/AgeI MND promoter was isolated from the MND-eGFP-WPRE vector and ligated into XhoI/AgeI pBS-PRF.Perforin1 to create the intermediate plasmid pBS-MND-Perforin1, from which the SpeI (blunt)/XhoI fragment of MND .Perforin1 was ligated into the EcoRV/SalI sites of the GFP4T vector. LV vector was produced by transient transfection of 293T cells and viral titers were determined by transduction of mouse erythroleukemia cells after serial dilutions, followed by flow cytometry (all previously described).…”

“…13,14 We reported significant, but partial, correction of cytopenias and cytotoxicity in Prf1 -/-mice, using lentiviral (LV) vectors expressing perforin from cellular promoters of the phosphoglycerate kinase gene (PGK) or the perforin-1 gene (PRF1). 15 We theorized that this incomplete rescue may have been due to an insufficient level of perforin expression driven by these promoters. It has been previously shown that cellular promoters from ubiquitously expressed or tissue-restricted genes were inadequate for correcting the defect in leukocyte adhesion deficiency, where a strong viral promoter was required for phenotypic correction of this defect.…”