| Since its inception 3 decades ago,the baculovirus expression vector system(BEVS)is an exceptional tool in molecular biology with enhanced expression efficiency in both cell-culture and insect-based expression systems,and its related techniques are still in continuous evolution.Additionally,baculovirus is also widely employed as a gene delivery vector and successfully used to express foreign genes both in vitro and in vivo.Due to the peculiarities of no viral replication and other associated risks in mammalian cells,it has become a principal gene delivery vector in mammals.Here,we attempted to express chicken interferon alpha(chIFN-?),gamma(chIFN-?)and lambda(chIFN-?3)in BEVS and characterized their antiviral potential against NDV(Newcastle disease virus.We exploited primary fibroblast cells,the chicken embryo fibroblasts(CEF)and analyzed the best interferon(chIFN-?)in cohort of IFNs that can be effectively applied to halt the NDV replication.In order to elucidate the possible signaling pathways associated with IFN-?-mediated immune response,we utilized a transcriptomics approach.RNA-Seq data revealed potential interferon-stimulated genes(ISGs)associated with immune-related signaling.Our findings discussed the antiviral role of chIFN-? and designated it as a novel immuno-modulator.Previously,it was established by both published literature and preliminary work of our laboratory that chIFN-? and chIFN-? obtained by BEVS were used to study the interactions between avian type I and type II interferons and their antiviral potential was investigated.In the present study,the combined effect of chIFN-? & chIFN-? and their antiviral role in the broiler chicken was elaborated.Fascinatingly,we also observed and verified a unique feature of chicken IFNs as growth promoter where chIFN-? & chIFN-? co-expression product displayed a synergistic effect on the weight gain of broiler chicken.The results demonstrated that an increase of 4% weight gain was observed in the treatment group compared to mock(control group).In order to elucidate the possible cellular and molecular basis of this phenomenon,we analyzed the RNA-seq data generated from cells(in vitro)and organs of chicken(in vivo).Our results presented that a huge number of genes related to muscle growth and development were significantly up-regulated in the adipose tissue of the treatment group.The activation of immune-related signaling pathways was also deeply undermined.In order to evaluate the efficiency of gene delivery in BEVS,we selected the human blood clotting factor VIII(FVIII)as a gene presentation target.To analyze the feasibility of gene presentation,an effective and successful preliminary study was conducted in a HEK293 T cell line.By utilizing hemophilia A mice,we attempted both intragastric administration(i.g)and intraperitoneal administration(i.p)treatments to explore the feasible route of drug administration.Three different promoters(CMV,CAG,and HLP)were employed to conduct a comparative analysis to determine the efficiency of distinct promoters in presenting(blood clotting factor)FVIII to gene-deficient mice.We have revealed that all the three promoters were capable of eliciting an effective therapeutic dose and astonishingly,oral administration also achieved a corresponding therapeutic effect.These findings provide corresponding experimental evidence for the possible future role of baculovirus as a highly safe and suitable gene delivery vector in the treatment of cancers and genetic disorders.In short,the present study comprehensively designates the antiviral potential and growth-promoting role of chicken interferons.Furthermore,it also describes BEVS as a highly efficient and safer gene delivery system in the treatment of hemophilia A.These findings provide a sound foundation for the practical application of this system for mass-scale production of advanced therapeutics. |
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