Successful expression of human factor IX following repeat administration of adenoviral vector in mice.

Adenoviral vectors can direct high-level expression of a transgene, but, due to a host immune response to adenoviral antigens, expression is of limited duration, and repetitive administration has generally been unsuccessful. Exposure to foreign proteins beginning in the neonatal period may alter or ablate the immune response. We injected adult and neonatal (immunocompetent) CD-1 mice intravenously with an adenoviral vector expressing human blood coagulation factor IX. In both groups of mice, expression of human factor IX persisted for 12-16 weeks. However, in mice initially injected as adults, repeat administration of the vector resulted in no detectable expression of the transgene, whereas in mice initially injected in the neonatal period, repeat administration resulted in high-level expression of human factor IX. We show that animals that fail to express the transgene on repeat administration have developed high-titer neutralizing antibodies to adenovirus, whereas those that do express factor IX have not. This experimental model suggests that newborn mice can be tolerized to adenoviral vectors and demonstrates that at least one repeat injection of the adenoviral vector is possible; the model will be useful in elucidating the immunologic mechanisms underlying successful repeat administration of adenoviral vectors.

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of enzyme subunits into an active dimer.

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H4B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H4B biosynthesis and the role of H4B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H4B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared from H4B-deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H4B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNOS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-translational mechanism by which intracellular H4B can regulate iNOS expression.

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals.

A recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigen-secreting rBCG system can be used as a tool for development of HIV vaccines.

Gene therapy for long-term expression of erythropoietin in rats.

The injection of recombinant erythropoietin (Epo) is now widely used for long-term treatment of anemia associated with chronic renal failure, cancer, and human immunodeficiency virus infections. The ability to deliver this hormone by gene therapy rather than by repeated injections could provide substantial clinical and economic benefits. As a preliminary approach, we investigated in rats the expression and biological effects of transplanting autologous vascular smooth muscle cells transduced with a retroviral vector encoding rat Epo cDNA. Vector-derived Epo secretion caused increases in reticulocytes, with peak levels of 7.8-9.6% around day 10 after implantation. The initial elevation in reticulocytes was followed by clinically significant increases in hematocrit and hemoglobin for up to 11 weeks. Ten control and treated animals showed mean hematocrits of 44.9 +/- 0.4% and 58.7 +/- 3.1%, respectively (P < 0.001), and hemoglobin values of 15.6 +/- 0.1 g/dl and 19.8 +/- 0.9 g/dl, respectively (P < 0.001). There were no significant differences between control and treated animals in the number of white blood cells and platelets. Kidney and to a lesser extent liver are specific organs that synthesize Epo in response to tissue oxygenation. In the treated animals, endogenous Epo mRNA was largely down regulated in kidney and absent from liver. These results indicate that vascular smooth muscle cells can be genetically modified to provide treatment of anemias due to Epo deficiency and suggest that this cell type may be targeted in the treatment of other diseases requiring systemic therapeutic protein delivery.

Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.

Retrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and sickle cell anemia), however, has been hampered by the inability to generate recombinant viruses able to efficiently and faithfully transmit the necessary sequences for appropriate gene expression. We have addressed this problem by carefully examining the interactions between retroviral and beta-globin gene sequences which affect vector transmission, stability, and expression. First, we examined the transmission properties of a large number of different recombinant proviral genomes which vary both in the precise nature of vector, beta-globin structural gene, and locus control region (LCR) core sequences incorporated and in the placement and orientation of those sequences. Through this analysis, we identified one specific vector, termed M beta 6L, which carries both the human beta-globin gene and core elements HS2, HS3, and HS4 from the LCR and faithfully transmits recombinant proviral sequences to cells with titers greater than 10(6) per ml. Populations of murine erythroleukemia (MEL) cells transduced by this virus expressed levels of human beta-globin transcript which, on a per gene copy basis, were 78% of the levels detected in an MEL-derived cell line, Hu11, which carries human chromosome 11, the site of the beta-globin locus. Analysis of individual transduced MEL cell clones, however, indicated that, while expression was detected in every clone tested (n = 17), the levels of human beta-globin treatment varied between 4% and 146% of the levels in Hu11. This clonal variation in expression levels suggests that small beta-globin LCR sequences may not provide for as strict chromosomal position-independent expression of beta-globin as previously suspected, at least in the context of retrovirus-mediated gene transfer.

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.

Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions.

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector

Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the TTG in the attB1 site (30bp upstream of the ATG in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes. (c) 2006 Elsevier Inc. All rights reserved.

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.