Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster Ovary cells

In Giardia lamblia, enhanced translation of luciferase mRNA, flanked between the 5'-untranslated region (UTR) and 3 '-end of giardiavirus transcript, requires the presence of the initial 264-nucleotide (nt) viral capsid-coding region. By introducing the transcripts of dicistronic viral constructs into Giardia, we demonstrated that the 264-nt downstream region alone is insufficient to function as an internal ribosome entry site (IRES) without including a portion of the 5 '-UTR as well. Deletion analysis showed that efficient internal initiation requires the last 253 nts (nts 114-367) of the 5 '-UTR in combination with the downstream 264 nts. Specific mutations that disrupted the predicted secondary structural elements in either the 5 '-UTR or the 264-nt capsid-coding region completely abolished the IRES-mediated translation of downstream cistron, suggesting that the IRES activity requires the presence of these structures in both regions. Mutations that abolished translation of the first cistron did not, however, affect the IRES-mediated translation of the second cistron, indicating that this IRES-mediated translation is independent of the translation of the upstream cistron. This is, to our knowledge, the first reported identification of a viral IRES with an estimated size of 517 nts that extends to both sides of the initiation site.

Internal ribosome entry sites (IRES) are used in eukaryotic vectors for correlated co-expression of genes in eukaryotic cells. To investigate the IRESs? tissue and species specificity we have compared the translational efficiencies in dicistronic expression vectors mediated by the IRES of the bovine viral diarrhea virus (BVDV), the poliovirus type I IRES and by a vector harboring a random intercistronic region in certain primary cells and permanent cell lines derived from the liver, kidney, brain, cervix and endothelium. We found that IRES mediated expression differs considerably between the IRES elements used in one given cell necessitating the careful choice of IRES elements for a given cell type. BVDV IRES was superior to the strong poliovirus IRES in rodent neuronal and liver cells. The polio-IRES is stronger than the BVDV IRES in human neuronal, liver and kidney cells. The BVDV IRES tends to function optimal in the mouse (rodent) context, the poliovirus IRES in the human context. Interestingly, dicistronic expression differs considerably between primary cells and permanent cell lines. This indicates that for gene therapy of primary cells using dicistronic vectors, permanent cell lines cannot be recommended as models for gene expression.

The optimal use of IRES (internal ribosome entry site) in expression vectors

Translation initiation promoted by picornavirus internal ribosome entry site (IRES) elements is dependent on the association of specific IRES sequences to the initiation factor eIF4G. However the RNA determinants interacting with other components of the translational machinery are still unknown. In this study, we have identified novel RNA-protein interactions between the foot-and-mouth disease virus (FMDV) IRES and three translation initiation factors. A doublet of 116/110 kDa that crosslinked to the FMDV IRES is a component of eIF3. We show here that domain 5 holds the preferential binding site for eIF3, although this complex initiation factor can establish multiple contacts with the IRES structure. We have also identified the phylogenetically conserved hairpin of domain 5 as the RNA motif responsible for eIF4B interaction. Mutation of this stem-loop structure abrogated eIF4B, but not eIF3, binding to the IRES. Remarkably, IRES mutants severely affected in their interaction with eIF4B showed a mild reduction in IRES activity when tested in the context of a bicistronic expression vector in transfected cells. Finally, we provide evidence of the interaction of eIF4GII with FMDV IRES, the RNA determinants for this interaction being shared with its functional homolog eIF4GI. The FMDV Lb protease generated a C-terminal fragment of eIF4GII that binds to the IRES as efficiently as the intact protein. Competition experiments showed that titration of eIF4B or p110/116 interaction with the FMDV IRES required a large excess of competitor relative to eIF4G, strongly suggesting that eIF4G-IRES interaction is a limiting factor to titrate the IRES. Comparative analysis of the activity of IRES mutants affected in domains 4 and 5 regarding their pattern of RNA-protein complex formation demonstrates that while binding of eIF4B with the FMDV IRES is dispensable, interaction of eIF4G is a central feature of the activity of this element.

Generation of Mammalian Cells Stably Expressing Multiple Genes at Predetermined Levels

Baculoviruses are one of the most studies insect viruses both in basic virology research and in biotechnology applications. This insect viruses based baculovirus expression vector system (BEVS) is an important tool for molecular biology and widely used in insect cells. Development of a shuttle baculovirus expression vector which can function in both the mammalian cell as well as in insect cell would have a significant advantages. We had cooperated an internal ribosome entry site (IRES) in to the baculovirus genomes and generated a bicistronic baculoviruses expression vector that can express two genes simultaneously. In this project, we attempt to construct a baculovirus vector with an IRES based bicistronic expression vector which using Bac-to-Bac system. The Bac-to-Bac system bring more benefits than the traditional methods using homologous recombination in insect cells. Bac-to-Bac plasmids: pFastBac1-Polh-MCS-Lir-DsRed2 had been successfully generated. Such IRES-based Bac-to-Bac BEVS will facilitate rapid and efficient generation of recombinant baculoviruses: time saving expression bacmid, one-step purification and amplification, rapid and simultaneous isolation of multiple recombinant viruses as well as titer determination.

AbstratPlant RNA viruses exploit nonorthodox strategies, such as the use of internal ribosomal entry sites (IRES), to express multiple genes from a single RNA species. IRES elements have been reported in tobacco etch virus (TEV), crucifer infecting tobamovirus (crTMV), hibiscus chlorotic ringspot virus (HCRSV), and many other animal and plant RNA viruses. In this chapter, the methodology used to identify and characterize a plant virus IRES element, including construction of a translation reporter vector for testing the IRES activity, testing the IRES activity in coupled in vitro transcription and translation systems and mammalian cells analysis of RNA stability, and sucrose gradient analysis and polysome profiling, is presented.

Efficient and regulated co-expression of multiple genes is an important consideration in the design of gene therapy vectors. While the augmentation of a single therapeutic gene is often sufficient for gene therapy of simple mendelian disorders, strategies for the treatment of complex disorders and infectious diseases necessitate the introduction of multiple genes into the cell. Complex disorders such as cancer often involve mutations in multiple genes and a combination strategy targeting different defective genes simultaneously are often more effective than any single strategy. Likewise, approaches for treating infectious diseases such as HIV-1 (human immunodeficiency virus) often involve the blocking of multiple steps of the viral replication pathway simultaneously to prevent the emergence of resistant strains of the virus. Even for the treatment of single gene defects, the additional incorporation of a selectable marker gene is often necessary to achieve sustained expression of the therapeutic gene in the cells. Among the several different strategies to co-express multiple genes, the incorporation of an IRES (internal ribosome entry site) into gene therapy vector design represents one of the more promising strategies. IRES functions as a ribosome-landing pad for the efficient internal initiation of translation ensuring coordinate expression of several genes and are located at the 5'UTR (5' untranslated regions) of these genes. Currently, the most popular IRES utilized for gene therapy is the IRES from the EMCV (encephalomyocarditis virus). However, the major caveat with present vector systems utilizing this IRES is that the expression of the downstream gene is significantly less efficient than the upstream gene. This review will examine the growing list of naturally occurring and synthetic IRESes and how they can be exploited for human gene therapy.

Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570-1576, 2016.

Control of Splicing Efficiency by the Mouse Histone H2a Element in a Murine Leukemia Virus–based Retroviral Vector

A novel strategy for the expression of foreign genes from plant virus vectors

The antifolate methotrexate (MTX) is an important chemotherapeutic agent for treatment of osteosarcomas. This drug usually converts intracellulary into polyglutamate derivates by the enzyme folylpolyglutamate synthetase (FPGS). MTX polyglutamates show an enhanced and prolonged cytotoxicity in comparison to the monoglutamate. We, therefore, investigated in the present study if transfer of the human fpgs gene to osteosarcoma cells may augment their MTX sensitivity. For this purpose, we employed the human osteocalcin (OC) promoter which had shown marked osteosarcoma specificity in promoter studies using different luciferase assays in osteosarcoma and non-osteosarcoma cell lines. A recombinant lentiviral vector was generated with the OC promoter driving the expression of fpgs and the gene for enhanced green fluorescent protein (egfp) which was linked to fpgs by an internal ribosomal entry site (IRES). Since the vector backbone contained only a self-inactivating viral LTR promoter, any interference of the OC promoter by unspecific promoter elements was excluded. We tested the expression of fpgs and egfp after lentiviral transduction in various osteosarcoma (murine ROS, human MG-63, human TM791) and non osteoblastic cell lines (293T embryonic human kidney cells, HeLa human cervix carcinoma cells) by assessing egfp expression and MTX sensitivity in comparison to non-transduced controls. Whereas the OC promoter failed to enhance MTX sensitivity via fpgs expression in non osteoblastic cell lines, the OC promoter mediated a markedly increased MTX cytotoxicity in all osteosarcoma cell lines after lentiviral transduction. In future animal studies, we will investigate if our lentiviral-mediated chemogene therapy approach also confers osteosarcoma-selective cytotoxicity in vivo.

IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines

Lentiviral Vectors Harboring a Dual-Gene System Allow High and Homogeneous Transgene Expression in Selected Polyclonal Human Embryonic Stem Cells