Rapid generation of a well-matched vaccine seed from a modern influenza A virus primary isolate without recourse to eggs.

Most influenza vaccines are produced in chicken eggs but recent human influenza strains often do not grow well in this substrate. The PER.C6 cell line is an alternative platform for vaccine production. Here we demonstrate that PER.C6 cells faithfully propagate recent clinical isolates, without selecting for mutations in the HA gene. PER.C6 cells support the rescue of recombinant influenza viruses from cDNA. We used sequence data from a surveillance programme to generate a PR8-based seed virus with the HA and NA of a contemporary circulating H3N2 human strain, A/England/611/07 (E611) that did not itself grow in eggs. We engineered mutations that affected receptor-binding, G186V or L194P, into the E611 HA gene. Whilst the L194P mutation conferred efficient growth in eggs, G186V did not. The L194P mutation was also spontaneously selected during egg propagation of E611/PR8 7:1 recombinant virus. This suggests generation of a single recombinant vaccine seed might satisfy manufacturers that utilize either eggs or cells for vaccine production.

Fiber-chimeric adenoviruses expressing fibers from serotype 16 and 50 improve gene transfer to human pancreatic adenocarcinoma.

Survival of patients with pancreatic cancer is poor. Adenoviral (Ad) gene therapy employing the commonly used serotype 5 reveals limited transduction efficiency due to the low amount of coxsackie-adenovirus receptor on pancreatic cancer cells. To identify fiber-chimeric adenoviruses with improved gene transfer, a library of Ad vectors based on Ad5 and carrying fiber molecules consisting of 16 other serotypes were transduced to human pancreatic carcinoma cell lines. Adenoviruses containing fibers from serotype 16 and 50 showed increased gene transfer and were further analyzed. In a gene-directed prodrug activation system using cytosine deaminase, these adenoviruses proved to be effective in eradicating primary pancreatic tumor cells. Fiber-chimeric Ad5 containing fiber 16 and wild-type Ad5 were also transduced ex vivo to slices of normal human pancreatic tissue and pancreatic carcinoma tissue obtained during surgery. It was shown that fiber-chimeric Ad5 with fiber 16 revealed an improved gene delivery to primary pancreatic tumor tissue compared to Ad5. In conclusion, fiber-chimeric adenoviruses carrying fiber 16 and 50 reveal a significantly enhanced gene transfer and an increased specificity to human pancreatic adenocarcinoma compared to Ad5, whereas transduction to normal pancreatic tissue was decreased. These findings expand the therapeutic window of Ad gene therapy for pancreatic cancer.

Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

Human adenovirus type 35 vector for gene therapy of brain cancer: improved transduction and bypass of pre-existing anti-vector immunity in cancer patients.

Clinical trials in malignant glioma have demonstrated excellent safety of recombinant adenovirus type 5 (Ad5) but lack of convincing efficacy. The overall low expression levels of the Coxsackie and Adenovirus receptor and the presence of high anti-Ad5-neutralizing antibody (NAb) titers in the human population are considered detrimental for consistency of clinical results. To identify an adenoviral vector better suited to infect primary glioma cells, we tested a library of fiber-chimeric Ad5-based adenoviral vectors on 12 fresh human glioma cell suspensions. Significantly improved marker gene expression was obtained with several Ad5-chimeric vectors, predominantly vectors carrying fiber molecules derived from B-group viruses (Ad11, Ad16, Ad35 and Ad50). We next tested Ad35 sero prevalence in sera derived from 90 Dutch cancer patients including 30 glioma patients and investigated the transduction efficiency of this vector in glioma cell suspensions. Our results demonstrate that the sero prevalence and the titers of NAb against Ad35 are significantly lower than against Ad5. Also, recombinant Ad35 has significantly increased ability to transfer a gene to primary glioma cells compared to Ad5. We thus conclude that Ad35 represents an interesting candidate vector for gene therapy of malignant glioma.

Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector.

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.

Improved gene delivery to intestinal mucosa by adenoviral vectors bearing subgroup B and d fibers.

A major obstacle to successful oral vaccination is the lack of antigen delivery systems that are both safe and highly efficient. Conventional replication-incompetent adenoviral vectors, derived from human adenoviruses of subgroup C, are poorly efficient in delivering genetic material to differentiated intestinal epithelia. To date, 51 human adenovirus serotypes have been identified and shown to recognize different cellular receptors with different tissue distributions. This natural diversity was exploited in the present study to identify suitable adenoviral vectors for efficient gene delivery to the human intestinal epithelium. In particular, we compared the capacities of a library of adenovirus type 5-based vectors pseudotyped with fibers of several human serotypes for transduction, binding, and translocation toward the basolateral pole in human and murine tissue culture models of differentiated intestinal epithelia. In addition, antibody-based inhibition was used to gain insight into the molecular interactions needed for efficient attachment. We found that vectors differing merely in their fiber proteins displayed vastly different capacities for gene transfer to differentiated human intestinal epithelium. Notably, vectors bearing fibers derived from subgroup B and subgroup D serotypes transduced the apical pole of human epithelium with considerably greater efficiency than a subgroup C vector. Such efficiency was correlated with the capacity to use CD46 or sialic acid-containing glycoconjugates as opposed to CAR as attachment receptors. These results suggest that substantial gains could be made in gene transfer to digestive epithelium by exploiting the tropism of existing serotypes of human adenoviruses.

Immunogenicity and protection of a recombinant human adenovirus serotype 35-based malaria vaccine against Plasmodium yoelii in mice.

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.

Gene transfer of the urokinase-type plasminogen activator receptor-targeted matrix metalloproteinase inhibitor TIMP-1.ATF suppresses neointima formation more efficiently than tissue inhibitor of metalloproteinase-1.

Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.

Odocoileus hemionus deer adenovirus is related to the members of Atadenovirus genus.

The Odocoileus hemionus deer adenovirus (OdAdV-1) causes systemic and local vasculitis and proves extremely lethal for mule deer. To characterize the virus, part of the genome flanking the fiber gene was cloned and sequenced. The sequence revealed two open-reading frames that mapped to pVIII hexon-associated protein precursor and fiber protein of several other adenoviruses. The highest amino acid homology for pVIII and fiber was found with the members of the proposed Atadenovirus genus: ovine adenovirus isolate 287 (OAdV-287), bovine adenovirus 4 (BAdV-4) and duck adenovirus 1 (DAdV-1). The homology with bovine adenovirus type 3 (BAdV-3) proved low. The E3 region was not found between the gene for pVIII and fiber. These data suggest that OdAdV-1 is a member of the Atadenovirus genus.

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease.

Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

Highly efficient targeted transduction of undifferentiated human hematopoietic cells by adenoviral vectors displaying fiber knobs of subgroup B.

Human hematopoietic stem cells (HSCs) are poorly transduced by vectors based on adenovirus serotype 5 (Ad5). This is primarily due to the paucity of the coxsackievirus-Ad receptor on these cells. In an attempt to change the tropism of Ad5, we constructed a series of chimeric E1-deleted Ad5 vectors in which the shaft and knob of the capsid fibers were exchanged with those of other Ad serotypes. In all these vectors, the Ad E1 region was replaced by an expression cassette containing the cytomegalovirus immediate-early promoter and the gene for enhanced green fluorescent protein (GFP). Experiments performed in vitro showed an efficient transduction of umbilical cord blood (UCB) monocytes, granulocytes, and their precursors as well as the undifferentiated CD34(+) CD33(-) CD38(-) CD71(-) cells by Ad5 vectors carrying Ad subgroup B-specific fiber chimeras (Ad5FBs). In the latter subpopulation, which comprises less than 1% of the CD34(+) cells and is highly enriched with cells repopulating immunodeficient mice, more than 90% of the cells were GFP(+). Transduction by Ad5FBs of the less primitive fraction within UCB CD34(+) cells was significant lower. Actually, the transduction frequency and GFP level declined gradually with increased expression of the CD33, CD38, and CD71 antigens. Flow cytometric analysis of transduced UCB CD34(+) cells that were cultured for 5 days on an allogeneic human bone marrow stroma layer showed maintenance of the phenotypically defined HSCs at levels similar to those of control cultures. The latter finding indicates that neither the transduction procedure nor the high levels of GFP were toxic for these cells.

Strategies for improved antigen delivery into dendritic cells.

Efficacious vaccines against cancers and infectious diseases will, in general, need to elicit comprehensive immune responses, including cytotoxic T lymphocyte activity. Because of their unique T cell stimulatory capacities, dendritic cells (DC) have emerged as the most potent antigen-presenting cell. Vaccination strategies should therefore aim at the acquisition and display of the antigen(s) of choice by DC. Results from vaccination studies, in animal models and in humans, stress the need for optimized antigen delivery systems to DC, to increase vaccination efficacy as well as to improve control on the immunological outcome. Here, we discuss the advantages and limitations of several recently described methodologies for antigen delivery into DC.

Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells.

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.

Infection efficiency of type 5 adenoviral vectors in synovial tissue can be enhanced with a type 16 fiber.

OBJECTIVE: To obtain an adenoviral vector with increased infection efficiency in the synovial tissue compared with conventional vectors based on adenovirus serotype 5 (Ad5), without compromising the specificity of infection. METHODS: Coxsackie adenovirus receptor (CAR) expression was assessed in cultured synoviocytes. Chimeric adenoviruses based on Ad5 but carrying the DNA encoding the fiber of adenovirus from subgroup B (Adll, 16, 35) or D (Ad24, 28, 33, 45, or 47) were constructed and produced on PER.C6 cells. The gene transfer efficiency of these chimera was tested on cultured synoviocytes and peripheral blood mononuclear cells (PBMC). RESULTS: No surface expression of CAR protein was observed on synoviocytes. CAR messenger RNA expression of synoviocytes was found to be low. Of all fiber chimeric vectors tested, vectors carrying the fiber of Ad16 (Ad5.fib16) were most potent, yielding approximately150 times increased transgene expression in cultured synoviocytes compared with those of Ad5. Flow cytometry showed that the increase in transgene expression was caused by the transduction of higher percentages of synoviocytes and higher gene expression per synoviocyte. Experiments with 500 virus particles/cell of Ad5.GFP or Ad5.fib16.GFP resulted in an infection efficiency of 0.6% and 1% in PBMC and 43% and 76% in synoviocytes, respectively. CONCLUSION: Synoviocytes hardly express CAR, which hampers Ad5-mediated gene transfer. Ad5.fib16 is superior to Ad5 vectors for transducing synoviocytes, without compromising the specificity of infection. Our data suggest that Ad5.fib16-mediated gene transfer to synovial tissue improves the therapeutic window.

Improved adenovirus vectors for infection of cardiovascular tissues.

To identify improved adenovirus vectors for cardiovascular gene therapy, a library of adenovirus vectors based on adenovirus serotype 5 (Ad5) but carrying fiber molecules of other human serotypes, was generated. This library was tested for efficiency of infection of human primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Based on luciferase, LacZ, or green fluorescent protein (GFP) marker gene expression, several fiber chimeric vectors were identified that displayed improved infection of these cell types. One of the viruses that performed particularly well is an Ad5 carrying the fiber of Ad16 (Ad5.Fib16), a subgroup B virus. This virus showed, on average, 8- and 64-fold-increased luciferase activities on umbilical vein ECs and SMCs, respectively, compared to the parent vector. GFP and lacZ markers showed that approximately 3-fold (ECs) and 10-fold (SMCs) more cells were transduced. Experiments performed with both cultured SMCs and organ cultures derived from different vascular origins (saphenous vein, iliac artery, left interior mammary artery, and aorta) and from different species demonstrated that Ad5.Fib16 consistently displays improved infection in primates (humans and rhesus monkeys). SMCs of the same vessels of rodents and pigs were less infectable with Ad5.Fib16 than with Ad5. This suggests that either the receptor for human Ad16 is not conserved between different species or that differences in the expression levels of the putative receptor exist. In conclusion, our results show that an Ad5-based virus carrying the fiber of Ad16 is a potent vector for the transduction of primate cardiovascular cells and tissues.

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer.

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.

The influence of synovial fluid on adenovirus-mediated gene transfer to the synovial tissue.

OBJECTIVE: To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)-mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained. METHODS: SF was derived from 63 randomly selected R4 patients. Ten samples were used to study the effect of SF on Ad5-mediated gene transfer in synoviocytes. IgG and <100-kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild-type Ad5 (wt-Ad5), wt-Ad26, wt-Ad34, wt-Ad35, and wt-Ad48 was tested in the SF from the remaining 53 patients. RESULTS: Seven of 10 SF samples inhibited Ad5-mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt-Ad35 by SF from RA patients was less frequent than neutralization of other wt-Ad tested (4% versus 42-72%; n = 53). CONCLUSION: SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5-mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.

Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset.

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.

Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences.

In this study, we describe the efficiency of second gene translation in bicistronic constructs containing either a short (36bp) synthetic intercistron or known internal ribosomal entry sites (IRES). Experiments were performed using two different gene combinations: Herpes simplex virus-thymidine kinase (HSV-TK) and neomycine (NEO) or human glucocerebrosidase (hGC) and a methotrexate (MTX) resistant mutant dihydrofolate reductase (DHFR). We demonstrate that upon transfection, second gene translation is efficient using either an IRES or a 36-bp intercistron. Infection with retrovirus carrying the TK and NEO genes linked via a 36-bp intercistron resulted in both G418R (NEO expression) and gancyclovir (GCV) sensitivity (TK expression), indicating that both genes were expressed and thus that the genomic DNA and RNA of this bicistronic construct were intact. Likewise, retrovirus carrying the hGC and mutant DHFR gene separated by a short intercistron was harvested from MTXR murine PsiCRE cells. However, infection of PA317 cells with this virus supernatant did not result in the presence of hGC enzyme activity in these murine cells. Proviral DNA and RNA analyses indicated that the hGC coding region was lost from the original construct in the infected PA317 cells. In contrast, retrovirus carrying the hGC and DHFR cDNAs was linked via an IRES functioned as expected. Based on these results, we conclude that the efficiency of second gene translation using short synthetic intercistrons might prove useful in bicistronic constructs, depending on the gene combination used.