The internal ribosome entry site (IRES) has been widely used to coexpress heterologous gene products by a message from a single promoter. However, little is known about the efficiency of IRES-dependent second gene expression in comparison with that of first gene expression. This study was undertaken to characterize the relative expression of IRES-dependent second gene in a bicistronic vector, which was derived from the 5' untranslated regions of the encephalomyocarditis virus (EMCV). IRES-dependent second gene expression was compared with cap-dependent first gene expression in several cultured cell lines and in mouse liver in vivo. The expression of the IRES-dependent second gene ranged from 6 to 100% (in most cases between 20 and 50%) that of the first gene. Second gene expression in a plasmid without the IRES was 0.1-0.8% (with some exceptions) that of the first gene. These findings have important implications for the use of IRES, i.e., care should be taken regarding the decreased capacity of IRES-dependent downstream gene expression as well as in determining which gene should be positioned as the first or second gene in a bicistronic vector.

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcγRI binding region of the Fc domain.

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Objective— An endothelial nicotinic acetylcholine receptor (nAChR) participates in atherogenesis and tumorigenesis by promoting neovascularization. To date, the mechanisms of nAChR-mediated angiogenesis and their relationship to angiogenic factors, eg, VEGF and bFGF, are unknown. Methods and Results— Nicotine induced dose-dependent human microvascular endothelial cell (HMVEC) migration, a key angiogenesis event, to an extent which was equivalent in magnitude to bFGF (10 ng/mL) but less than for VEGF (10 ng/mL). Unexpectedly, nAChR antagonism not only abolished nicotine-induced HMVEC migration but also abolished migration induced by bFGF and attenuated migration induced by VEGF. Transcriptional profiling identified gene expression programs which were concordantly regulated by all 3 angiogens (nicotine, VEGF, and bFGF), a notable feature of which includes corepression of thioredoxin-interacting protein (TXNIP), endogenous inhibitor of the redox regulator thioredoxin. Furthermore, TXNIP repression by all 3 angiogens induced thioredoxin activity. Silencing thioredoxin by small interference RNA abrogated all angiogen-induced migration while silencing TXNIP strongly induced HMVEC migration. Interestingly, nAChR antagonism abrogates growth factor (VEGF and bFGF)–mediated induction of thioredoxin activity. Conclusions— Nicotine promotes angiogenesis via stimulation of nAChR-dependent endothelial cell migration. Furthermore, growth factor–induced HMVEC migration, a key angiogenesis event, requires nAChR activation—an effect mediated in part by nAChR-dependent regulation of thioredoxin activity.

Non-viral vectors for gene therapy, although less efficient for gene delivery, offer several advantages over viral vectors. Non-viral vectors are particularly suitable with respect to simplicity of use and ease of large-scale production, and they are relatively inexpensive to produce, easier to quality control, and generate little or no specific immune response. Nonviral DNA delivery has also become a powerful and popular research tool for elucidating gene structure, regulation, and function. Various types of synthetic vectors such as cationic lipids and polymers have been developed for gene transfer with high transfection efficiency. 1-3)It is necessary to evaluate the efficiency and safety of gene transfer with gene therapy vectors. Many factors may affect the efficacy and safety of non-viral vector-mediated gene transfer, including the structure, charge, and formulation of the vector, the preparation of DNA/vector complexes, 4,5) DNA/vector ratio, charge and size of complexes, [6][7][8] time of exposure, and interaction with serum and blood cells.9,10) As optimal conditions might well differ for different cell types, careful optimization is required to evaluate the efficacy and safety of vectors for the target cells and target organs. [11][12][13][14] In this study, we compared the transfection efficiency and safety of commercially available non-viral vectors with a variety of human cells including primary cells, blood cell lines, and adherent cell lines. We also clarified important factors that affected the evaluation of these vectors. The transfection efficiency and cytotoxicity of all non-viral vectors used was dependent on the type of vector, vector concentration, presence of serum, and type of cell. These results also provided useful information for understanding the characteristics of each vector and selecting the most suitable vector for gene transfer into target cells. (293); 45% MEM and 45% RPMI1640 medium supplemented with 10% FCS (NB-1); RPMI1640 medium supplemented with 10% FCS (SBC-1, K-562, U937, HL60, IM-9 and Jurkat cells); and 199 medium supplemented with 20% FBS containing 5 U/ml of heparin and 15 mg/ml Endothelial Cell Growth Supplement (Technoclone) on collagen type I-coated dishes (HUVEC, HUVSMC and HSFB). In the case of human hepatocytes, frozen cells were thawed and plated on collagen-type-Icoated 96-well plates the day before the experiments in Hepatocyte Culture Medium (Clonetics Co., Walkersville, MD, U.S.A.). MATERIALS AND METHODS CellsNon-viral Vectors Six commercially available transfection reagents were used as non-viral vectors: Lipofectin, LipofectAMINE PLUS, and DMRIE-C (GIBCO-BRL, Gaithersburg, MD), SuperFect and Effectene (QIAGEN, Hilden, Germany), and DOTAP (Boehringer Mannheim, Mannheim, Germany). The composition of each of the vectors was as follows: Lipofectin 15) : 1 : 1 (w/w) liposome formulation of the cationic lipid N- [1-(2,3-dioleyloxy)propyl]-N,N,Ntrimethylammonium chloride (DOTMA) and dioleoyl phosphatidylethanolamine (DOPE) (1 mg/ml); LipofectAMINE PLUS: 3 : ...

Targeted gene delivery to the tissue of interest by recombinant adenovirus (Ad)

The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.