Targeted delivery of miR125a-5p and human Factor VIII attenuates molecular mediators of hemophilic arthropathy.

Hemophilic arthropathy (HA) due to repeated bleeding into the joint cavity is a major cause of morbidity in patients with hemophilia. The molecular mechanisms contributing to this condition are not well characterized. MicroRNAs (miRs) are known to modulate the phenotype of multiple joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Since miR125a is known to modulate disease progression in OA and RA, we performed a targeted screen of miR125a-5p and its target genes in a murine model of chronic HA. A digital PCR analysis demonstrated significant downregulation of miR125a-5p (2-fold vs control joint). Further molecular evaluation revealed elevated expression of the immunological markers STAT1 (7.6-fold vs control joint) and TRAF6 (10.6 fold vs control joint), which are direct targets of miR125a-5p. We then studied the impact of targeted overexpression of miR125a-5p using an Adeno-associated virus (AAV) vector in modulating the molecular mediators of HA. AAV5-miR125a vectors were administered intra-articularly either alone or in combination with a low dose of AAV8-based human factor 8 (F8) gene in a murine model of HA. We observed significantly increased expression of miR125a-5p in AAV5-miR125a administered mice (~12 fold vs injured joint) or in combination with AAV8-F8 vectors (~44 fold vs injured joint). The activity assay revealed ~17 %-20 % FVIII levels in mice that received low dose liver-directed F8 gene therapy. Further immunohistochemical analysis, demonstrated a decrease in inflammatory markers (STAT1 and TRAF6) and cartilage-degrading matrix metalloproteinases (MMPs) 3, 9, 13 in the joints of treated animals. These data highlight the crucial role of miR125a-5p in the development of HA.

Involvement of oxidative stress, nuclear factor kappa B and the ubiquitin proteasomal pathway in dysferlinopathy.

AIMS: Dysferlinopathies are autosomal recessive neuromuscular disorders arising from mutations of the protein dysferlin that preferentially affect the limbs which waste and weaken. The pathomechanisms of the diseases are not known and effective treatment is not available. Although free radicals and upstream signaling by the redox sensitive transcription factor, NF-κB, in activation of the ubiquitin pathway are shown to occur in several muscle wasting disorders, their involvement in dysferlinopathy is not known. This study analyzed the role of oxidative stress, NF-κB and the ubiquitin pathway in dysferlinopathic muscle and in dysferlin knockdown human myoblasts and myotubes. MAIN METHODS: Fourteen dysferlinopathic muscle biopsies and 8 healthy control muscle biopsies were analyzed for oxidative stress, NF-κB activation and protein ubiquitinylation and human primary myoblasts and myotubes knocked down for dysferlin were studied for their state of oxidative stress. KEY FINDINGS: Dysferlinopathic muscle biopsies showed NF-κB p65 signaling induced protein ubiquitinylation in response to oxidative stress. Dysferlin knock down primary muscle cell cultures confirmed that oxidative stress is induced in the absence of dysferlin in muscle. SIGNIFICANCE: Anti-oxidants that also inhibit nitrosative stress and NF-κB activation, might prove to be of therapeutic benefit in slowing the progression of muscle wasting that occurs with dysferlinopathy.