Intraarticular Delivery of AAV5-Mir-125a-5p Regulates Key Molecular Mediators of Hemophilic Arthropathy

Abstract

Background: Hemophilic arthropathy (HA) is a major comorbidity in patients with hemophilia characterized by chronic inflammation of the joints, stiffness and loss of function. The expression of microRNAs are known to be altered in various joint diseases such as rheumatoid arthritis and osteoarthritis which share clinical attributes with HA. One such microRNA is miR-125a-5p, known to be involved in regulating inflammatory mediators in the arthritic joints. The current study was designed to investigate the role of miR125a-5p in HA and examine the phenotypic effect of its targeted reconstitution in the joints. Methods: We utilized a chronic arthropathy model in hemophilia A mice to study the impact of miR125a-5p in disease progression. First, we performed a targeted screen of miR125a-5p and its target genes (STAT1 and TRAF6) using digital PCR (dPCR). Further, to validate if reconstitution of miR125a-5p aids in modulating the molecular mediators of HA, we performed the Adeno-associated virus vector (AAV) serotype 5 targeted delivery of miR125a (AAV5-miR125a) either alone (2.13x10 11vgs/joint) or in combination with capsid-modified AAV8-human Factor 8 (F8)(AAV8-F8) gene therapy at a low dose (1x10 11vgs/animal) intravenously in F8 tm1kaz/Jmice. Briefly, the animals were administered with vectors (alone or in combination), and seven days post vector administration, the animals were subjected to multiple bleeding episodes in the right knee (Day 0, 14, and 30) to mimic chronic hemarthrosis. On day 45, blood samples were collected from experimental animals for FVIII specific clotting assay, and joint tissues were isolated to perform further molecular and histochemical analysis. Results: Our dPCR analysis revealed a significant reduction (2-fold) of miR125a-5p (3518 copies/µl vs 7242 copies/µl, p≤0.0001) in injured joints compared to control joints. Absolute quantification of target genes, namely STAT1 and TRAF6 showed significantly higher expression in injured joints (7.6 to 10.6 fold, respectively) when compared to control joints. Further immunohistochemical examination of joint sections from the experimental mice confirmed the elevated expression of these immunological mediators (STAT1, TRAF6). Upon administration of AAV vectors containing miR125a and/or F8, we observed a significantly increased expression of miR125a-5p (~12 fold) in only miR125a vector-administered animals while the combination vector treated group had, ~44-fold higher levels of miR125a-5p. The coagulant assay revealed an increased FVIII activity (~17%) in only F8 vector-treated animals and approximately 20% FVIII levels in mice that received both miR125a and F8 vectors at low doses. Surprisingly, the animals that received only F8 vectors also showed significantly upregulated miR125a-5p expression (~20 fold) signifying the role of miR125a-5p in HA pathology. In line with the above findings, immunohistochemical analysis of joint tissues revealed decreased expression of immunological markers STAT1, TRAF6 (direct targets of miR125a-5p), and the chondro-degenerative matrix metalloproteinases 3, 9, 13 in vector-treated groups when compared to injured joints with no treatment. Hematoxylin and Eosin (H&E) staining of joint tissues from experimental animals was performed which revealed that animals that received both miR125a and F8 vectors showed decreased synovial hyperplasia, villi formation, cartilage erosion, and reduced hemosiderin deposition signifying moderate phenotype rescue. Conclusions: Our data confirms that miR125a-5p is a major molecular target that regulates inflammatory response in chronic HA. Targeted delivery of miR125a in tandem with F8 gene augmentation had an impact on several molecular mediators like STAT1, TRAF6, MMP3,9, and 13. The reconstitution of miR125a and a higher dose of F8 vectors might be beneficial to reverse chronic HA.