Reconstruction and in silico analysis of an Actinoplanes sp. SE50/110 genome-scale metabolic model for acarbose production

Actinoplanes sp. SE50/110 produces the -glucosidase inhibitor acarbose, which is used to treat type 2 diabetes mellitus. To obtain a comprehensive understanding of its cellular metabolism, a genome-scale metabolic model of strain SE50/110, iYLW1028, was reconstructed on the bases of the genome annotation, biochemical databases, and extensive literature mining. Model iYLW1028 comprises 1028 genes, 1128 metabolites and 1219 reactions. 122 and 81 genes were essential for cell growth on acarbose synthesis and sucrose media, respectively, and the acarbose biosynthetic pathway in SE50/110 was expounded completely. Based on model predictions, the addition of arginine and histidine to the media increased acarbose production by 78% and 59%, respectively. Additionally, dissolved oxygen has a great effect on acarbose production based on model predictions. Furthermore, genes to be overexpressed for the overproduction of acarbose were identified, and the deletion of treY eliminated the formation of by-product component C. Model iYLW1028 is a useful platform for optimizing and systems metabolic engineering for acarbose production in Actinoplanes sp. SE50/110

Anti-CD20 as the B cells targeting agent in the combined therapy to modulate anti-factor VIII immune responses in hemophilia A inhibitor mice

Neutralizing antibody formation against transgene products can represent a major complication following gene therapy with treatment of genetic diseases, such as hemophilia A. Although successful approaches have been developed to prevent the formation of anti-factor VIII (FVIII) antibodies, innovative strategies to overcome pre-existing anti-FVIII immune responses in FVIII-primed subjects are still lacking. Anti-FVIII neutralizing antibodies circulate for long periods in part due to persistence of memory B cells. Anti-CD20 targets a variety of B cells (pre-B cells to mature/memory cells); therefore, we investigated the impact of B cell depletion on anti-FVIII immune responses in hemophilia A mice using anti-CD20 combined with regulatory T (Treg) cell expansion using IL-2/IL-2mAb complexes plus rapamycin. We found that anti-CD20 alone can partially modulate anti-FVIII immune responses in both unprimed and FVIII-primed hemophilia A mice. Moreover, in mice treated with anti-CD20 + IL-2/IL-2mAb complexes + rapamycin + FVIII, anti-FVIII antibody titers were significantly reduced in comparison to mice treated with regimens targeting only B or T cells. In addition, titers remained low after a second challenge with FVIII plasmid . Treg cells and activation markers were transiently and significantly increased in the groups treated with IL-2/IL-2mAb complexes ; however,significant B cell depletion was obtained in anti-CD20-treated groups. Importantly, both FVIII-specific antibody-secreting cells and memory B cells were significantly reduced in mice treated with combination therapy. This study demonstrates that a combination regimen is highly promising as a treatment option for modulating anti-FVIII antibodies and facilitating induction of long-term tolerance to FVIII in hemophilia A mice. <br/