A Novel Murine Model Rebalancing Coagulation Enables Gene Therapy Studies for Severe Protein C Deficiency, a Lethal Pro-Thrombotic Disorder

Introduction

The success and regulatory approval of recombinant adeno associated viral (rAAV) gene therapy for hemophilia A (HA), hemophilia B, and other rare genetic disorders has been predicated on viable preclinical animal models. While most rare bleeding disorders are compatible with life, investigations of severe pro-thrombotic diseases, such as severe protein C deficiency (SPCD), are drastically hampered by the limited survival of the affected mice. SPCD is a devastating disease, that can currently only be cured by liver transplant. Prophylaxis with PC replacement has limited availability and is extremely burdensome, but provides the rationale for gene therapy.

The PROC gene is located on chromosome 2 (2q13-14) and encodes 9 exons, which can be easily accommodated into an rAAV expression cassette. In order to begin development of PC gene therapy, we first generated a viable novel model of SPCD that would allow for functional evaluation of transgene-PC. Subsequently, we tested the potential rAAV- PC gene therapy for SPCD.

Methods

The study was conducted using C57Bl/6 PC heterozygous mice, severe HA mice, and wild type C57Bl/6 mice. PC ^-/-/F8^- mice were created by breeding PC ^-/+ and HA (F8^-) mice. Genotyping was performed using primers for murine PROC and F8 genes. The model was validated by mRNA extraction from the murine liver samples.

rAAV8-PC was produced by transfecting HEK293 cells with PX-680 as rAAV helper, AAV8 and a plasmid expressing murine PC by an alpha-1 antitrypsin promoter. Murine PC was used to eliminate xenoprotein immune responses and other cross-species differences. Viral concentration was determined by a designated commercial qPCR assay. rAAV vector was dosed by vector genomes (vg) per kg.

PC plasma antigen and activity levels were measured prior and following rAAV8-PC infusion. PC immunostaining in murine liver biopsy was performed using cryosections of each liver incubated with rabbit anti-protein C.

Results

Breeding of heterozygous PC mice yielded only 3/200 homozygous PC mice (PROC^-/-), instead of 50/200 expected by Mendelian inheritance, indicative of near embryonic lethality. These homozygous PROC^-/-mice had undetectable PC levels and survived <24 hours. In order to create a viable model of SPCD for therapeutic development, we crossed homozygous PC mice with severe HA (F8^-) mice to generate PROC^−/−/F8⁻ mice.

The WT mice (n=20) and HA mice (n=20) exhibited comparable PC antigen levels, while PROC^−/−/F8⁻ mice (n=20) had undetectable PC and FVIII antigen levels. mRNA extracted from the liver confirmed the absence of both PROC and F8 expression. Encouragingly, these PROC^−/−/F8⁻ mice exhibited substantially prolonged survival (>90% survived for >6 months (n=20)).

The generation of a viable mice with SPCD allowed for the evaluation of gene therapy approaches for the pro-thrombotic disorder. Ten 6-8 week-old PROC^−/−/F8⁻ mice were treated with 1 x 10¹² vg/kg of rAAV8-PC. Peak plasma PC concentrations were reached at a median of 6 weeks. Plasma PC antigen levels after vector administration ranged from 166 to 2488 ng/ml, with a median of 714 ng/ml; median PC antigen levels in WT mice (n=10) were 594 ng/ml. Peak PC activity levels were 300 ± 60% in rAAV treated animals, compared to 100 ± 20% in the WT control group (p=0.0034). All mice maintained high levels of PC antigen and activity for at least 12 weeks after vector administration. PC expression in the liver following vector administration was further confirmed by immunofluorescence staining, Overall, rAAV8-PC administration to PROC^−/−/F8⁻ mice, resulted in sustained expression of functional PC.

Conclusion

The lethal prothrombotic phenotype of homozygous PC, is rescued by the procoagulant deficiency of severe HA. The generation of a viable SPCD mouse model allowed for preclinical studies of gene transfer for this disorder. Our initial studies demonstrate the translational potential of rAAV-based gene therapy for SPCD with expression of a high-levels of functional PC. Future studies will evaluate the dose response of rAAV-PC and help define the therapeutic window. Further investigation of this viable SPCD mice model also may provide insights into the role of PC in the development of specific tissues, as well as the mechanisms modulating local PC generation and its regulation under inflammatory conditions.