Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity.

Recombinant adeno-associated virus (rAAV)-mediated gene delivery can efficiently target muscle tissues to serve as "biofactories" for secreted proteins in prophylactic and therapeutic scenarios. Nevertheless, efficient rAAV-mediated gene delivery is often limited by host immune responses against the transgene product. The development of strategies to prevent anti-transgene immunity is therefore crucial. The employment of endogenous microRNA (miRNA)-mediated regulation to detarget transgene expression from antigen presenting cells (APCs) has shown promise for reducing immunogenicity. However, the mechanisms underlying miRNA-mediated modulation of anti-transgene immunity by APC detargeting are not fully understood. Using the highly immunogenic ovalbumin (OVA) protein as a proxy for foreign antigens, we show that rAAV vectors containing miR142 binding sites efficiently repress co-stimulatory signals in dendritic cells, significantly blunt the cytotoxic T cell response, allow for sustained transgene expression in skeletal myoblasts, and attenuate clearance of transduced muscle cells in mice. Furthermore, the blunting of humoral immunity against circulating OVA correlates with detargeting of OVA expression from APCs. This demonstrates that incorporating APC-specific miRNA binding sites into rAAV vectors provides an effective strategy for reducing transgene-specific immune response. This approach holds promise for clinical applications where the safe and efficient delivery of a prophylactic or therapeutic protein is desired.

TLR4, TLR7/8 agonist-induced miR-146a promotes macrophage tolerance to MyD88-dependent TLR agonists.

TLRs facilitate the recognition of pathogens by immune cells and the initiation of the immune response, leading to the production of proinflammatory cytokines and chemokines. Production of proinflammatory mediators by innate immune cells, such as macrophages, is tightly regulated to facilitate pathogen clearance while limiting an adverse impact on host tissue. Exposure of innate immune cells to TLR ligands induces a state of temporary refractoriness to a subsequent exposure of a TLR ligand, a phenomenon referred to as "tolerance." This study sought to evaluate the mechanistic regulation of TLR4 and TLR7/8 ligand-induced tolerance to other TLRs by microRNA-146a. With the use of THP-1 macrophages, as well as human classic and alternative macrophages, we demonstrate that priming with a TLR4 agonist (LPS) or a TLR7/8 agonist (R848) induces homologous and heterologous tolerance to various TLR ligands in macrophages, leading to the impaired production of cytokines and chemokines. We also demonstrate that overexpression of microRNA-146a is sufficient to mimic LPS or R848-induced hyporesponsiveness. Conversely, inhibition of microRNA-146a activity leads to LPS- or R848-induced TLR hyper-responsiveness in TLR signaling tolerance. Furthermore, we demonstrate that microRNA-146a dampens cytokine production following a primary stimulus with MyD88-dependent but not MyD88-independent TLR pathways. Collectively, these data provide comprehensive evidence of the central role of microRNA-146a in TLR signaling tolerance to plasma membrane, as well as endosomal TLR ligands in human macrophages.

Development of porcine ficolin-alpha monoclonal and polyclonal antibodies for determining the binding capacity of multiple GlcNAc-binding proteins to bacterial danger components.

Ficolins are a group of oligomeric defense proteins assembled from collagen-like stalks and fibrinogen-like domains that have common biochemical specificity for N-acetyl-d-glucose amine (GlcNAc) and can function as opsonins. In this report, GlcNAc-binding protein (GBP) purified from porcine nonimmune serum was biochemically characterized as ficolin-α. Ficolin-α was used as an immunogen to generate both rabbit polyclonal and murine monoclonal anti-ficolin-α antibodies, which are not yet commercially available. GBPs have been shown to be present in many animals, including humans; however, their functions are largely unknown. GBPs from chicken, dog, horse, bovine, and human sera were isolated using various chromatography methods. Interestingly, anti-ficolin-α antibody showed cross-reaction with those animal sera GBPs. Furthermore, anti-ficolin-α antibody was reactive with the GlcNAc eluate of Escherichia coli O26-bound and Salmonella-bound porcine serum proteins. Functionally, GBPs and bacteria-reactive pig serum proteins were able to bind with pathogen-associated molecular patterns such as lipopolysaccharides and lipoteichoic acids. Our studies demonstrate that ficolin-α specific antibody was reactive with GBPs from many species as well as bacteria-reactive serum proteins. These proteins may play important roles in innate immunity by sensing danger components that can lead to antibacterial activity.

The potential of adeno-associated viral vectors for gene delivery to muscle tissue.

INTRODUCTION: Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8(+) T-cell response against AAV capsid in humans. AREAS COVERED: In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. EXPERT OPINION: Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.