237. Genetic Therapy for Perforin Deficiency Associated Hemophagocytic Lymphohistiocytosis Requires High Level Expression of the Perforin Gene for Adequate Correction

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Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal immune regulatory disorder, whereby heightened immune activation following viral infections causes hypercytokinemia, pancytopenia and end-organ damage. The majority of familial HLH is caused by Perforin-1 (prf1) gene mutations. Perforin is expressed in CD8+ T cells and natural killer (NK) cells and is critical for lymphocyte cytotoxicity. Currently, immunosuppressive therapy followed by allogeneic hematopoietic stem cell (HSC) transplant is the only curative option for HLH, but is restricted by availability of matched donors and a high (20-50%) transplant related mortality. We posited that perforin gene transfer into autologous HSC may be curative, have lower mortality, and not be limited by donor availability. We recently reported significant, but partial, correction of HLH symptoms in prf1-/- mice using lentivirus vectors (LV) expressing perforin from a ubiquitous phosphoglycerate kinase (PGK) promoter or a tissue specific perforin gene promoter (PRF). We hypothesized that negative selection of high perforin expressing HSC and the perforin gene dosage delivered by the LV contributes to the partial correction. We redesigned and developed a series of LV and compared prf1 expression from the PGK/PRF promoters to that from the MND promoter/enhancer; additionally we restricted perforin expression from PGK and MND in HSC with 4 repeats of miR126 (4T) target sequence, a miRNA specifically expressed in HSCs but not in lymphocytes, thus, circumventing the negative selection of high perforin expressing HSC. Improved perforin expression and cytotoxicity was observed with the ‘HSC-restricted’ MND4TLV in the KHYG1 human NK cell line, and in prf1-/- P14 mice, transgenic for a T-cell receptor that recognizes a lymphocytic choriomeningitis virus (LCMV) glycoprotein. These LV were compared in prf1-/- mice that were challenged with LCMV following transplant of gene-modified HSC. High gene marked NK chimerism was achieved: 87.6±1.7%, 83.1±2.4% and 58.1±4.6% with the MND4T, PGK4T and PRF0T LV. Following LCMV challenge, γ-IFN levels in MND4T, PRF0T and PGK4T mice were 1.7±0.3, 1.5±0.5 and 5.9±2.1 ng/ml, respectively, compared to 1.7±0.5 ng/ml in prf1-/- mice receiving WT HSC. Similarly, the degree of anemia in WT and MND4T mice was comparable 15 days after LCMV challenge: hemoglobins declined by 4.3±0.3, 3.7±0.3, 5.6±0.3 and 5.8±0.7 g/dL in WT, MND4T, PGK4T and PRF0T mice, respectively. Most importantly, 66.7% of the mice in the MND4T group survived the LCMV challenge, while only 10% and no mice survived in PGK4T and PRF0T groups, suggesting that perforin expression from PGK4T and PRF0T were insufficient to prevent fatal HLH in the majority of animals. Overall, we show that perforin deficient HLH requires a robust perforin expression in cytotoxic T and NK cells for adequate correction of the disease phenotype.