rAAV8-PC Gene Therapy for Severe Protein C Deficiency Using a Novel Murine Model

Introduction Protein C (PC) is a vitamin K-dependent protein, synthesized in the liver. The PROC gene is located on chromosome 2 at position 2q13-14 and encodes 9 exons. Severe protein C deficiency (SPCD) is a rare devastating autosomal recessive disease that presents at birth with purpura fulminans (PF) and is associated with high rates of morbidity and mortality. As current management options are limited and burdensome, there is a large unmet need for new treatments including curative therapies. Because small increases in PC may significantly ameliorate the clinical presentation of SPCD and potentially prevent PF, we hypothesize that SPCD could be treated by PC gene therapy (GT). Aim To establish a murine model enabling the survival of SPCD mice and to evaluate PC expression after recombinant adeno-associated viral vector- PC (rAAV8-PC) gene therapy. Methods The study was conducted with C57Bl/6 PC heterozygous mice (a kind gift of Prof. Weiler, [Wisconsin]), severe hemophilia A (HA) mice and wild type C57Bl/6 mice. Genotyping was performed by PCR using appropriate primers for murine PROC and F8 genes. rAAV8-PC was produced by transfecting HEK293 cells with PX-680 as rAAV helper, AAV8 and PC plasmid with an alpha-1 antitrypsin promotor. Extraction and purification were performed using a commercial kit. Viral concentration was determined by a designated commercial qPCR assay. rAAV8 - PC (10 12 /kg Vg of AAV8-PC) was injected via the tail vein to 5-8 weeks old mice. Blood samples were collected from the facial vein into 1.5 ml tubes containing 0.109M sodium citrate at blood: anticoagulant ratio of 1:1 (vol/vol). PC plasma levels were measured prior and following rAAV8-PC infusion using a commercial ELISA assay for PC antigen. Thrombin generation was measured in plasma samples using a modification of the calibrated automated thrombinoscope assay. Results As expected, breeding of heterozygous PC mice yielded only 3/200 homozygous SPCD mice (PC levels= 0), that survived for less than 24 hours. Thus, GT for heterozygous PC mice was considered; however, PC plasma levels in heterozygous PC mice were not substantially different from those in WT mice (median levels in heterozygous PC and WT mice: 216 ng/ml vs. 190 ng/ml, respectively), which precluded this option. Hence, with the aim of increasing the survival rate and duration of homozygous SPCD mice, we decided to rebalance the severe thrombotic disorder of SPCD, by induction of a genetically engineered combinati