Inhibition of costimulation allows for repeated systemic administration of adenoviral vector in rhesus monkeys.

Immunogenicity of recombinant adenoviral (Ad) vectors severely hampers the clinical development of gene therapy protocols using repeated vector administrations. Inhibition of costimulation by APCs was explored as a strategy to circumvent the immune response against Ad particles. This strategy was tested in rhesus monkeys, treated transiently with chimeric anti-human CD40 and anti-human CD86 antagonist monoclonal antibodies (MAbs) at the time of systemic administration of a recombinant Ad vector. After Ad vector administration in the absence of immunosuppressive treatment, transgene expression in the serum lasted about 3-4 weeks. All control animals developed a strong neutralizing antibody (NAb) response to the Ad particles, which totally prevented efficient administration of a second vector, as shown by the lack of transgene expression. Treatment with anti-CD40 and anti-CD86 chimeric MAbs delayed or blocked the development of a humoral response against Ad and the infiltration of CD8(+) lymphocytes into the liver. This resulted in (i) increased persistence of Ad-transduced cells after injection of a first vector encoding a nonimmunogenic transgene, and (ii) the possibility of readministering a second Ad vector with significant efficacy. In both respects, the combined blockade of CD40 and CD86 was more efficient than treatment with anti-CD40 alone. This study shows for the first time in non-human primates that blocking CD40 and CD86 costimulatory molecules represents a promising strategy to inhibit immune responses against an Ad vector injected systemically.

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.

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism.

Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CAR-binding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu, Tyr491Asp, Ala494Asp and Ala503Asp did not prevent the incorporation of trimeric fibers in the viral capsid but led to loss of CAR binding in vitro. Infectivity of the mutant viruses could be restored in vitro by introducing a ligand at the C-terminal end of the knob, confirming that the reduced infectivity of the fiber-modified virus was due to an impaired interaction of the viral particle with the CAR receptor. However, after systemic delivery, the in vivo biodistribution of impaired CAR-binding viruses without addition of a specific ligand was not altered when compared with wild-type Ad.

The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines.

Influenza viruses for vaccine production are currently grown on embryonated eggs. This manufacturing system conveys many major drawbacks such as inflexibility, cumbersome down stream processing, inability of some strains to replicate on eggs to high enough yields, and selection of receptor-binding variants with reduced antigenicity. These limitations emphasize the need for a cell line-based production system that could replace eggs in the production of influenza virus vaccines in a pandemic proof fashion. Here we present the efficient propagation of influenza A and B viruses on the fully characterized and standardized human cell line PER.C6.

Radioisotopic imaging allows optimization of adenovirus lung deposition for cystic fibrosis gene therapy.

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

A preclinical model of hepatocyte gene transfer: the in vivo, in situ perfused rat liver.

Delivering retroviruses targeted to hepatocytes in vivo involves the injection of retroviruses directly into the portal vein. The aim of this work was to establish a clinically relevant system for retrovirus-mediated gene transfer in a new model of in vivo, in situ perfused rat liver and to study the transgene expression. At 24 h after partial hepatectomy, the liver was completely excluded from the splanchnic circulation using an extracorporeal shunt. Two independent normothermal, oxygenated perfusion systems were used. First, liver perfusion was carried out with a recirculating system (1 h). Culture supernatant containing retroviruses (1.5 x 10(8) ffu/ml, beta-galactosidase gene) was used as perfusate. Then the liver perfusion was maintained for more 30 min in a single liver passage system using culture medium without retroviruses as perfusate. High hepatocyte transduction rates (up to 34.4%) were obtained. PCR analysis showed no provirus in extrahepatic organs. Viral titrations performed simultaneously (inflow and outflow liver lines) showed that after 1 h of perfusion (up to 30 successive liver passages) retroviruses were still detected in the liver outflow perfusate (up to 2.0 x 10(7) ffu/ml). Washing the liver for 30 min dramatically decreased the leakage of retroviruses in the outflow. In order to be of clinical use, the injection of retroviruses targeted to hepatocytes in vivo should be done while the liver is completely excluded from the splanchnic circulation to avoid any extrahepatic retrovirus diffusion.

Optimization of bovine coronavirus hemagglutinin-estrase glycoprotein expression in E3 deleted bovine adenovirus-3.

Adenoviral vectors expressing foreign genes have many desirable properties in applications such as vaccination. Recently, we have generated replication-competent (E3 deleted) bovine adenovirus-3 (BAV-3) recombinants expressing significant amounts of glycoprotein D (gD) of bovine herpesvirus-1 (a DNA virus). However, attempts to express the RNA virus genes using the same strategy were not successful. In an effort to optimize the expression, we have constructed several BAV-3 recombinants carrying the hemagglutinin esterase (HE) gene of bovine coronavirus (BCV) in the E3 region with or without exogenous transcription control elements. The expression studies suggest that the introduction of a 137 bp chimeric intron upstream of the HE cDNA is able to increase the level of HE gene expression. The introduction of a SV40 early promoter or human cytomegalovirus (HCMV) immediate early (IE) promoter into the expression cassette changed the kinetics of the HE expression. However, the recombinant BAV-3 containing HE under the HCMV IE promoter replicated less efficiently than the wild-type BAV-3. These studies should prove useful in expression of other RNA viral genes in the E3 region of BAV-3 expression system.

Phase I clinical trial with IL-2-transfected xenogeneic cells administered in subcutaneous metastatic tumours: clinical and immunological findings.

Various studies have emphasized an immunodepression state observed at the tumour site. To reverse this defect and based upon animal studies, we initiated a phase I clinical trial of gene therapy in which various doses of xenogeneic monkey fibroblasts (Vero cells) genetically engineered to produce human IL-2 were administered intratumorally in 8 patients with metastatic solid tumours. No severe adverse effect was observed in the 8 patients analysed during this clinical trial even in the highest dose (5 yen 107 cells) group. This absence of toxicity seems to be associated with rapid elimination of Vero-IL-2 cells from the organism. Indeed, exogenous IL-2 mRNA could no longer be detected in the peripheral whole blood 48 hours after Vero-IL-2 cell administration. In addition, we did not find any expression of exogenous IL-2 mRNA in post-therapeutic lesions removed 29 days after the start of therapy. A major finding of this trial concerns the two histological responses of two treated subcutaneous nodules not associated with an apparent clinical response. The relationship between local treatment and tumour regression was supported by replacement of tumour cells by inflammatory cells in regressing lesions and marked induction of T and natural killer cell derived cytokines (IL-2, IL-4, IFNg ...) in post-therapeutic lesions analysed 28 days after the start of Vero-IL-2 administration. Gene therapy using xenogeneic cells as vehicle may therefore present certain advantages over other vectors, such as its complete absence of toxicity. Furthermore, the in vivo biological effect of immunostimulatory genes, i.e IL-2-, may be potentiated by the xenogeneic rejection reaction.

Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells.

We describe the generation and the characterization of new lentiviral vectors derived from SIVmac251, a simian immunodeficiency virus (SIV). A methodical approach was used to engineer both efficient and safe packaging constructs allowing the production of SIV viral core proteins. SIV-vectors encoding GFP (green fluorescent protein) were generated as VSV-G-pseudotyped particles upon transient expression of the vector construct and helper functions in 293 cells. The SIV vectors were able to transduce efficiently various target cell types at low multiplicity of infection, including monocyte-differentiated human dendritic cells (DCs) which retained their capacity to differentiate into mature DCs after gene transfer. Transduction of the DCs by the SIV vectors was prevented when infections were performed in the presence of AZT, a reverse-transcriptase inhibitor. After gene transfer, expression of the GFP in the target cells remained constant after several weeks, indicating that the vectors had been stably integrated into the genome of the host cells. Preparations of SIV vectors were systematically checked for the absence of replication-competent and recombinant retroviruses but remained negative, suggesting the innocuousness of these novel gene delivery vectors. Side-to-side comparisons with vectors derived from HIV-1 (human immunodeficiency virus) indicated that the SIV vectors were equally potent in transducing proliferating target cells. Finally, we have determined the infectivity of SIV vectors pseudotyped with surface glycoproteins of several membrane-enveloped viruses.

Development of minimal lentivirus vectors derived from simian immunodeficiency virus (SIVmac251) and their use for gene transfer into human dendritic cells.

Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5' cytomegalovirus early promoter, the 5' viral sequences up to the 5' end of gag required for reverse transcription and packaging, the Rev-responsive element, a gene-expressing cassette, and the 3' long terminal repeat (LTR). Production of SIV vector particles was achieved by transfecting 293T cells with the vector DNA and helper constructs coding for the viral genes and the vesicular stomatitis virus glycoprotein G envelope. These SIV vectors were found to have transducing titers reaching 10(7) transducing units/ml on HeLa cells and to deliver a gene without transfer of helper functions to target cells. The central polypurine tract can be included in the minimal vector, resulting in a two- to threefold increase in the transduction titers on dividing or growth-arrested cells. Based on this minimal SIV vector, a sin vector was designed by deleting 151 nucleotides in the 3' LTR U3 region, and this SIV sin vector retained high transduction titers. Furthermore, the minimal SIV vector was efficient at transducing terminally differentiated human CD34(+) cell-derived or monocyte-derived dendritic cells (DCs). Results show that up to 40% of human primary DCs can be transduced by the SIV vectors. This opens a new perspective in the field of immunotherapy.

In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.

Immune response to recombinant adenovirus in humans: capsid components from viral input are targets for vector-specific cytotoxic T lymphocytes.

We previously demonstrated that a single injection of 10(9) PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218-2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8(+) CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses.

Modulation of the inflammatory properties and hepatotoxicity of recombinant adenovirus vectors by the viral E4 gene products.

Liver toxicity and inflammation were assessed in C57BL/6, CBA, and BALB/c mice injected intravenously with a series of recombinant adenoviruses deleted simultaneously in E1/E3, in E1/E3/E2A, or in E1/E3/E4. All vectors were either devoid of transgenes or carried in E1 the human CFTR cDNA under the control of the CMV promoter. Injection of the E1/E3-deleted vector induced a significant liver dystrophy and inflammatory responses that were accompanied by an increased serum transaminase concentration. The vector toxicity remained elevated on additional deletion of the E2A gene and was further enhanced when hCFTR was expressed. In contrast, additional deletion of E4 led to a reduction in hepatotoxicity, suggesting an active role of E4 gene products in liver injury. However, deletion of E4 also led to a loss of transgene expression. To identify the individual E4 product(s) involved in liver toxicity and in the regulation of transgene expression, a series of isogenic E1/E3-deleted vectors, with or without the hCFTR transgene, and containing various combinations of functional E4 open reading frames (ORFs), were evaluated in vitro and in vivo. We demonstrate that liver injury was markedly reduced with vectors containing either ORF3 alone or ORF3,4 while vectors containing ORF4, ORF6,7 or ORF3,6,7 still displayed elevated hepatotoxicity and inflammatory responses. Moreover, transgene expression was restored when ORF3,4 or ORF3,6,7 was retained in the vector. These results highlight the importance of the E4 gene products in the design of improved in vivo gene transfer vectors.

Gene transfer of anti-gp41 antibody and CD4 immunoadhesin strongly reduces the HIV-1 load in humanized severe combined immunodeficient mice.

OBJECTIVE: To study the anti-HIV-1 effects of the delivery of anti-gp41 monoclonal antibody (mAb) and soluble CD4 (sCD4) immunoadhesin by genetically modified cells in HIV-1-infected, humanized severe combined immunodeficient (SCID) mice. DESIGN: The complementary DNA of mAb 2F5, an anti-HIV-1 gp41 antibody, and of sCD4-IgG chimeric immunoadhesin were transferred into 3T3 cells using Moloney murine leukaemia virus vectors. The cells were then incorporated into a collagen structure called the neo-organ, which allowed the continuous production of the therapeutic molecules. METHODS: The antiviral effects in vivo of 2F5 or sCD4-IgG or both compounds were evaluated in neo-organ-implanted SCID mice that were grafted with human CD4 CEM T cells and challenged with HIV-1 Lai or MN. RESULTS: In SCID mice implanted with 2F5 neo-organs, antibody plasma levels reached 500-2000 ng/ml. Viral loads after HIV-1 challenge were significantly reduced in neo-organ-implanted HIV-infected mice. Although 29 x 10(7) and 13 x 10(8) HIV-1-RNA copies/ml were detected at 12 days in the controls (mice injected with Lai and MN, respectively) less than 16.5 x 10(3) HIV-1-RNA copies/ml were observed in all implanted mice injected with either Lai or MN. The intracellular viral load was also reduced in CD4 cells recovered from the implanted mice. Comparable antiviral effects were obtained with CD4-IgG neo-organs. CONCLUSION: Our results confirm the anti-HIV properties of 2F5 and sCD4-IgG continuously produced in vivo after ex-vivo gene therapy in SCID mice.

Differential influence of the E4 adenoviral genes on viral and cellular promoters.

BACKGROUND: Strong and stable transgene expression is fundamental to the success of recombinant adenovirus vectors in human gene therapy. However, control of transgene expression is a complex process, involving both viral and cellular factors. In this study, the influence of the E4 adenoviral region on the activity of various promoters was investigated in vitro and in vivo. METHODS: Pairs of isogenic E1o and E1oE4o vectors were generated and compared. Levels of transgene expression were determined by Northern blot, ELISA and FACS analysis. Initiation of transcription was studied by nuclear run-on assays. RESULTS: Similar to the viral CMV and RSV promoters, the activity of the ubiquitous cellular PGK promoter required the presence of the E4 genes in vitro and in vivo. In contrast, transgene expression from selected liver- and tumor-specific promoters did not require E4 functions. CONCLUSION: Together with the reported low liver toxicity of E1oE4o vectors, the independence of E4 of liver-specific promoters renders such vectors interesting alternatives to the use of gutless vectors.

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.

Replication-defective bovine adenovirus type 3 as an expression vector.

Although recombinant human adenovirus (HAV)-based vectors offer several advantages for somatic gene therapy and vaccination over other viral vectors, it would be desirable to develop alternative vectors with prolonged expression and decreased toxicity. Toward this objective, a replication-defective bovine adenovirus type 3 (BAV-3) was developed as an expression vector. Bovine cell lines designated VIDO R2 (HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and 6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1 proteins) were developed and found to complement the E1A deletion in BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most of the E1A and E3 regions was constructed. This virus could be grown in VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results demonstrated that the E1 region of HAV-5 has the capacity to transform bovine retina cells and that the E1A region of HAV-5 can complement that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A similar replication-defective vector expressing the hemagglutinin-esterase gene of bovine coronavirus from the E3 region was isolated. Although these viruses grew less efficiently than the replication-competent recombinant BAV-3 (E3 deleted), they are suitable for detailed studies with animals to evaluate the safety, duration of foreign gene expression, and ability to induce immune responses. In addition, a replication-competent recombinant BAV-3 expressing green fluorescent protein was constructed and used to evaluate the host range of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of replication-defective BAV-3 vectors is a major technical advancement for defining the use of BAV-3 as vector for vaccination against diseases of cattle and somatic gene therapy in humans.

Gene therapy with cytokine-transfected xenogenic cells (Vero-IL-2) in patients with metastatic solid tumors: mechanism(s) of elimination of the transgene-carrying cells.

Eleven patients with advanced cancer were treated in a clinical gene therapy trial by repeated intra- tumoral injections with different doses of xenogenic fibroblasts secreting high amounts of human interleukin-2 (Vero-IL2). Treatments in a total of 14 courses were well tolerated and resulted in clinical responses and measurable biological effects. Together with increases in serum interleukin-2 (IL-2), modifications of the V-beta T cell receptor repertoire and induction of intratumoral T-cell infiltration were observed. When the intratumoral expression of endogenous cytokine genes and the persistence of the IL-2 transgene at the application site and in peripheral blood were investigated, rapid disappearance of the transgene at the application site appeared to be the most prominent biological effect. Tests detecting a single Vero-IL2 cell against a background of 10(5) non-transfected cells were not able to demonstrate significant expression of exogenous IL-2 (i.e. the transgene or transgene-carrying cells) in tumor biopsies or blood at different times. Therefore, further studies were performed to evaluate the mechanism(s) involved in the rapid disappearance of xenogenic carrier cells in more detail. We show here that significant in vitro cytotoxicity against transgene-carrying Vero cells can be observed in peripheral blood of all the patients before treatment as well as in healthy controls. "Cold" target inhibition shows that significant killing of Vero-IL2 cells is mediated by natural killer (NK) cells. This was confirmed by showing that established CD3(-)/CD16(+)/CD56(+) peripheral blood NK cell clones kill both K562 and Vero-IL2 target cells. The failure of other mechanisms (complement, antibody-dependent cell cytotoxicity or cytotoxic T lymphocytes) to destroy xenogenic, histoincompatible Vero cells in vitro suggests that NK cells also might be responsible for the killing of Vero-IL2 in vivo and for the failure to detect the transgene at the application site. These results might also be of importance for some aspects of the current discussion of xenotransplantation.

Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: requirement for E4 ORF3.

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol. 72:2022-2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV promoter was severely reduced in the absence of E4 functions but could be partially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

Development of a direct in situ RT-PCR method using labeled primers to detect cytokine mRNA inside cells.

We have developed an original protocol of direct in situ RT-PCR with biotinylated labeled primers to detect cytokine mRNA inside cells. This label improved the specificity of the technique compared with the use of digoxigenin or fluorescein-labeled primers. We found a reliable correlation between the known expression of cytokine mRNA in a given cell and a positive signal with in situ RT-PCR. Nuclear counterstaining demonstrated that the positive signal obtained was distributed in the cytoplasm in accordance with mRNA localization. In addition, direct demonstration of the presence of the expected PCR product in cell extracts without non-specific parasitic DNA amplification provided strong support for the specificity of the method. Designing the primers in order to prevent DNA amplification, the use of recombinant Thermus thermophilus (rTth) DNA polymerase and a decreased duration of each cycle of PCR by combining the annealing and hybridization steps improved the reproducibility and reliability of the technique and morphological preservation of the cells. Experiments in which different proportions of cytokine mRNA positive and negative cells were mixed argue against significant diffusion of PCR product into initially cytokine mRNA negative cells, thereby leading to false-positive results. In comparison with the direct incorporation of labeled dNTP during amplification, our procedure appears to ensure greater specificity and does not need DNAse treatment which is often difficult to standardize. Detection of IL-2 and IFNgamma mRNA induction after T cell activation using this direct in situ RT-PCR method showed that the technique may be helpful for monitoring cytokine gene expression at a single cell level.