No abstract

The prevalence of obesity-related metabolic diseases caused by insulin resistance is rapidly increasing worldwide. Adiponectin (ADPN), a hormone derived from adipose tissue, is a potential therapeutic agent for insulin resistance. Chickens are considered efficient bioreactors for recombinant protein production because they secrete large amounts of high-concentration proteins from the oviduct. Additionally, chickens express high levels of high-molecular-weight (HMW) ADPN, which is considered the active form in the body. Therefore, in this study, a gene-targeted chicken model was produced in which the gene encoding human ADPN was inserted into Ovalbumin (OVA) using the CRISPR/Cas9 system, and the characteristics of the resulting recombinant ADPN protein were evaluated. As a result, human ADPN was expressed in G1 hen oviducts and egg whites of OVA ADPN knock-in (KI) chickens. The concentration of ADPN in egg white ranged from 1.47 to 4.59 mg/mL, of which HMW ADPN accounted for ∼29% (0.24–1.49 mg/mL). Importantly, egg white-derived ADPN promoted expression of genes related to fatty acid oxidation and activated the 5′-AMP-activated protein kinase (AMPK) signaling pathway in muscle cells. In summary, the OVA gene-targeted chicken bioreactor proved to be an advantageous model for production of human ADPN, and the resulting protein was of sufficient quantity and efficacy for industrial use.

The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B.Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function 2 of 11 | PARK et Al.

Intravenous immunoglobulin (IVIG) is a plasma-derived polyclonal IgG used for treatment of autoimmune diseases. Studies show that α-2,6 sialylation of the Fc improves anti-inflammatory activity. Also, afucosylation of the Fc efficiently blocks FcγRIIIA by increasing monovalent affinity to this receptor, which can be beneficial for treatment of refractory immune thrombocytopenia (ITP). Here, we generated genome-edited chickens that synthesize human IgG1 Fc in the liver and secrete α-2,6 sialylated and low-fucosylated human IgG1 Fc (rhIgG1 Fc) into serum and egg yolk. Also, rhIgG1 Fc has higher affinity for FcγRIIIA than commercial IVIG. Thus, rhIgG1 Fc efficiently inhibits immune complex-mediated FcγRIIIA crosslinking and subsequent ADCC response. Furthermore, rhIgG1 Fc exerts anti-inflammatory activity in a passive ITP model, demonstrating chicken liver derived rhIgG1 Fc successfully recapitulated efficacy of IVIG. These results show that genome-edited chickens can be used as a production platform for rhIgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activities.