PDK1 controls upstream PI3K expression and PIP3 generation.

The PI3K/PDK1/Akt signaling axis is centrally involved in cellular homeostasis and controls cell growth and proliferation. Due to its key function as regulator of cell survival and metabolism, the dysregulation of this pathway is manifested in several human pathologies including cancers and immunological diseases. Thus, current therapeutic strategies target the components of this signaling cascade. In recent years, numerous feedback loops have been identified that attenuate PI3K/PDK1/Akt-dependent signaling. Here, we report the identification of an additional level of feedback regulation that depends on the negative transcriptional control of phosphatidylinositol 3-kinase (PI3K) class IA subunits. Genetic deletion of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or the pharmacological inhibition of its downstream effectors, that is, Akt and mammalian target of rapamycin (mTOR), relieves this suppression and leads to the upregulation of PI3K subunits, resulting in enhanced generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3). Apparently, this transcriptional induction is mediated by the concerted action of different transcription factor families, including the transcription factors cAMP-responsive element-binding protein and forkhead box O. Collectively, we propose that PDK1 functions as a cellular sensor that balances basal PIP3 generation at levels sufficient for survival but below a threshold being harmful to the cell. Our study suggests that the efficiency of therapies targeting the aberrantly activated PI3K/PDK1/Akt pathway might be increased by the parallel blockade of feedback circuits.

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.

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.

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.

In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted.

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response.

E1, E3-deleted, replication-deficient recombinant adenoviruses are widely studied as vectors for their capacity to transfer therapeutic genes in vivo. They can infect a wide variety of dividing and quiescent cells from different organs and possess a large packaging capacity. One of the major limitations in the use of these vectors for gene therapy is the transient expression of the transgene in vivo and the poor transduction efficiency when re-administered. Despite the deletion of the viral E1 region, low level of early and late viral genes are expressed in vivo. Thus, viral antigens plus those derived from transgene expression in transduced cells contribute to cellular immune responses leading to the destruction of these cells. Production of anti-adenovirus antibodies, the cellular immune response as well as the early non-specific clearance of the vectors, constitute barriers to successful gene therapy. New vectors have been derived with additional deletions in the E2a or the E4 regions. Such second generation vectors were evaluated in vivo. These studies have revealed the complexity of the immune mechanisms elicited by these vectors and the importance of several parameters in these evaluations (i.e. mouse strains, nature of the transgene, route of administration...). In order to inhibit the production of neutralizing antibodies to adenovirus that prevent from further readministration of the vectors, immunosuppressive strategies were undertaken. Treatment regimens with immunosuppressive drugs (cyclophosphamide, FK506) or with monoclonal antibodies that block either the T cell receptor or costimulation pathways allow prolonged transgene expression and/or readministration of adenoviral vectors. In addition, transduction efficiencies may be increased by transiently inhibiting non-specific immune mechanisms that lead to the dramatic early clearance of the vectors. Taken together, these strategies may improve further gene therapy protocols by decreasing the host immune response to adenoviral vectors.

Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli.

Despite recent technical improvements, the construction of recombinant adenovirus vectors remains a time-consuming procedure which requires extensive manipulations of the viral genome in both Escherichia coli and eukaryotic cells. This report describes a novel system based on the cloning and manipulation of the full-length adenovirus genome as a stable plasmid in E. coli, by using the bacterial homologous recombination machinery. The efficiency and flexibility of the method are illustrated by the cloning of the wild-type adenovirus type 5 genome, the insertion of a constitutive promoter upstream from the E3 region, the replacement of the E1 region by an exogenous expression cassette, and the deletion of the E1 region. All recombinant viral DNAS were shown to be fully infectious in permissive cells, and the modified E3 region or the inserted foreign gene was correctly expressed in the infected cells.

Targeting cell-specific gene expression with an adenovirus vector containing the lacZ gene under the control of the CFTR promoter.

In vivo gene therapy requires the development of vectors able to deliver and express therapeutic genes preferentially into specific cell populations. This can be achieved by the manipulation of viral proteins mediating target-cell recognition, as well as by the introduction of tissue-specific promoters into viral vectors. As a first approach towards this goal, we describe here the construction and testing of a recombinant adenovirus expressing the lacZ gene encoding beta-galactosidase under the control of 2 kilobase pairs (kbp) of 5' untranslated DNA sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We show that such a recombinant virus directs beta-galactosidase expression in cell lines expressing CFTR, and in human and murine respiratory tract cells in vitro and in vivo. However, we were unable to demonstrate a cell-type specificity of expression strictly paralleling that of the endogenous CFTR gene. This data indicates that only part of the natural CFTR gene regulation is reconstituted in such a vector.

Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors.

Replication-defective E1-deleted adenoviruses are attractive vectors for gene therapy or live vaccines. However, manufacturing methods required for their pharmaceutical development are not optimized. For example, the generation of E1-deleted adenovirus vectors relies on the complementation functions present in 293 cells. However, 293 cells are prone to the generation of replication competent particles as a result of recombination events between the viral DNA and the integrated adenovirus sequences present in the cell line. We report here that human lung A549 cells transformed with constitutive or inducible E1-expression vectors support the replication of E1-deficient adenoviruses. E1A transcription was elevated in most of the cell lines, and E1A proteins were expressed at levels similar to those of 293 cells. However, the levels of expression of E1A did not correlate with the efficiencies of complementation of E1-deleted viruses in A549 clones, since some clones complemented replication in the absence of induction of E1A expression. In addition, complementation of E1-deficient adenoviruses did not require expression of the E1B 55-kDa protein. Although these cell lines contain the coding and cis-acting regulatory sequences of the structural protein IX gene, they are not able to complement viruses in which this gene has been deleted. In contrast to 293 cells, such new complementation cell lines do not contain the left end of the adenoviral genome and thus represent a significant improvement over the currently used 293 cells, in which a single recombination event is sufficient to yield replication competent adenovirus.

An efficient procedure to select and recover recombinant adenovirus vectors.

Adenoviruses are efficient gene transfer vectors for a variety of cell types. To date, the most widely used methods to construct recombinant adenoviruses involve either in vitro ligation or recombination between one-half of the virus genome, previously cloned in a plasmid vector and engineered to contain the desired expression cassette, and the other half of the virus genome prepared from virions. Although quite effective, these approaches produce viral progeny containing a mixture of recombinant and parental background virus. Thus the recovery of the recombinant virus can be difficult, especially when it grows more slowly than the parental virus. To improve selection and recovery of recombinant adenoviruses, we have constructed an adenovirus vector, AdTG6553, in which the E1 region has been replaced by the thymidine kinase (tk) gene of herpes simplex virus type 1. We show that infection of cells with AdTG6553 in the presence of the nucleoside analog ganciclovir (GCV) prevents viral replication. The conditional lethal phenotype introduced in AdTG6553 makes it a valuable tool to counter-select parental background virus in the presence of GCV and isolate replication-deficient recombinant adenoviruses in which the tk expression cassette has been replaced by a new gene.

Trans-complementation of E1-deleted adenovirus: a new vector to reduce the possibility of codissemination of wild-type and recombinant adenoviruses.

Treatment of cystic fibrosis by gene therapy will require the development of vectors capable of efficient and safe transfer of a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. To achieve this goal, replication-deficient (E1-) adenoviruses (Ad) are promising vectors. We have previously demonstrated efficient CFTR gene delivery to the airways of cotton rats and rhesus monkeys using a replication-deficient adenovirus, Ad-CFTR. Here, we have investigated an important safety issue, the interaction between the vector and wild-type virus which can provide the missing E1 function in trans. We show that Ad5 can mobilize the defective Ad-CFTR genome in vitro and in cotton rats. However, the extent of the complementation in vivo by wild-type virus is limited because no additional spreading or shedding of Ad-CFTR to trachea, lungs, and stools is elicited. To attenuate Ad-CFTR further, a mutation was introduced in the cis-acting regulatory sequences that control the encapsidation of the viral genome. We demonstrate that when cells are coinfected with wild-type virus and the new attenuated vector, the viral DNA containing the natural encapsidation sequences is preferentially packaged, leading to a rapid dilution of the recombinant virus.

Localization of the cystic fibrosis transmembrane conductance regulator in airway secretory glands.

Cystic fibrosis (CF) is caused by mutations in the gene coding for the CF transmembrane conductance regulator (CFTR). From human normal tracheal submucosal gland cells in culture, we identified endogenous CFTR as a 170 kDa protein, consistent with that of fully glycosylated, mature CFTR molecule. This observation led to the hypothesis that airway secretory glands could be an important site for the CFTR expression. Using anti-human CFTR polyclonal and monoclonal antibodies, we examined the cellular and subcellular localization of the CFTR protein in airway submucosal glands from human and bovine tracheal tissues as well as in tracheal gland cell cultures. In human tracheal tissue, CFTR immunolabelling was present along both the apical and basolateral plasma membranes of glandular mucous cells. In contrast, CFTR was associated with the secretory granules of glandular serous cells. Using immunogold electron microscopy, we demonstrated that CFTR protein was more specifically associated with the membrane of serous cell secretory granules. In bovine tracheal tissue CFTR labelling was also identified in the secretory granules of glandular serous cells. In contrast, when bovine and human tracheal gland cells were cultured, no mature secretory granules were present, but a predominantly intracytoplasmic distribution of CFTR was observed. Our data thus suggest that in airway tissues, CFTR could be involved in intracellular processes of the mucus exocytosis in submucosal secretory glands.

[Emergency therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF)]. / Notfalltherapie mit Granulozyten-Makrophagen-Kolonien stimulierendem Faktor (GM-CSF).

Granulocyte-macrophage colony stimulating factor (GM-CSF) has been tested for tolerability and efficacy on a compassionate need case basis in 17 patients (5 females, 12 males aged 4-72 years, median 35 years). GM-CSF was given at the rate of 3.5-32 micrograms/kg for 2-64 days as a continuous infusion for the following indications: impending rejection following bone marrow transplantation (5 patients), severe neutropenia secondary to chemotherapy in tumor patients (5), severe aplastic anemia (3), immune granulocytopenia (2) and accidental overdose with cytostatic agents (2 patients). Tolerance of GM-CSF was good in regard to doses of up to 16 micrograms/kg. Fever, myalgia and eosinophilia were the most frequent side effects. The patient treated with 32 micrograms/kg developed thrombosis of the vena cava. Efficacy is more difficult to assess in this heterogenous population, but 11 of 17 patients showed increased granulocyte counts and 3 patients clearly recovered from severe neutropenia. The role of GM-CSF in this recovery, however, cannot be proven. The results further indicate that GM-CSF cannot reverse ongoing rejection following allogenic BMT and cannot correct immune neutropenia. The value of GM-CSF therapy in patients with severe aplastic anemia and in the context of chemotherapy still needs to be defined. It is certainly indicated in patients with an accidental overdose of chemotherapeutic agents.

Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines.

Transgenic mice were generated in which 5 kb of the 5' flanking promoter region of the human Factor IX (FIX) gene fused to various FIX constructs (gene, minigene and cDNA) were stably integrated in the germ line. Several transgenic mouse lines expressed high circulating levels of active and correctly processed recombinant human FIX. The presence of at least one FIX intron had a positive effect on the expression. The FIX transgenes were expressed in a tissue-specific manner in the liver of transgenic mice. By crossing transgenic mice synthesizing FIX with others prone to develop hepatoma, progeny which co-express the transgenes in hepatocytes were obtained. Hepatoma-derived cell lines were shown to have a differentiated phenotype and secrete active human FIX for many generations.

Chronic cyclosporine-associated nephrotoxicity in bone marrow transplant patients.

This study describes the prevalence and degree of chronic cyclosporine-associated nephropathy and its risk factors. For this purpose we reviewed all available renal histology specimens in 169 bone marrow transplant recipients treated during an eight year period with cyclosporine for prevention of graft-versus-host-disease, and determined their pattern and degree of histomorphological changes. A total of 51 specimens obtained from 49 patients by biopsy (n = 12) or autopsy (n = 39) was evaluated. The pattern of histomorphological changes was compared with diagnosis, age, sex, and potential risk factors--such as cyclosporine dose, levels, duration of therapy, changes in serum creatinine and onset of hypertension. Morphological lesions of chronic cyclosporine-associated nephropathy were found in 67% of the specimens. They were more frequent and more severe with increasing duration of cyclosporine therapy, in patients with a higher increase in serum creatinine during the first 3 months and in patients given total-body irradiation for conditioning. These latter findings suggest that additional damage sensitizes the kidney to irreversible toxic effects of cyclosporine.

Treatment of thrombocytosis in myeloproliferative disorders with interferon alpha-2a.

In an open prospective pilot trial, we tested the effect of recombinant interferon alpha-2 a (rIFN alpha-2 a) on thrombocytosis in myeloproliferative disorders (MPD). Since October 1986, 13 patients with MPD (4 with chronic granulocytic leukemia, 4 with polycythemia vera, 3 with essential thrombocythemia and 2 with myeloid metaplasia) were treated with rIFN alpha-2 a. Platelet counts decreased in all treated patients within 2 to 10 weeks from a median value of 1,050 x 10(9)/l (range 610-1,940 x 10(9)/l) to 340 x 10(9)/l (range 230-495 x 10(9)/l). The response was dose-dependent. In 11 patients we observed a simultaneous reduction of the white blood cell count. Six patients still continue the IFN alpha-2 a therapy. In 7 treatment was discontinued, because of chronic side effects in 3, and because of noncompliance in one. In these patients, thrombocytosis recurred after discontinuation of the therapy. These results show that rIFN alpha-2 a is effective in controlling thrombocytosis in MPD. However, the long-term benefit of interferon in these disorders remains to be established.

Irradiated donor buffy coat following T cell-depleted bone marrow transplants.

Twenty patients aged 27-47 years (median, 35 years) with hematological malignancies, treated with T cell-depleted bone marrow transplantation received in a pilot study five donations of 15 Gy irradiated donor buffy coat cells at days +1, +3, +5, +7, +14 in order to prevent rejection and leukemic relapse. Patients were conditioned with etoposide, cyclophosphamide and 12 Gy fractionated total body irradiation and given cyclosporine postgrafting. Donor bone marrow was T cell-depleted (median 3% remaining T cells) by counterflow elutriation. All patients engrafted. Fourteen (70%) are alive. Two are living with relapse, 12 (60%) are alive and well without any signs of disease, 2-27 months (median, 9 months) post-transplant. Three patients died of interstitial pneumonitis and/or graft-versus-host-disease; three died of relapse. This pilot study supports previous animal data. Repeated infusions of 15 Gy irradiated donor buffy coat are feasible and do not appear to increase transplant related mortality. Whether this approach ultimately will reduce the rate of rejection and relapse following T cell depletion needs to be confirmed in a larger, prospective study.

Active gamma-carboxylated human factor IX expressed using recombinant DNA techniques.

Factor IX (Christmas factor), a vitamin K-dependent plasma protein made in the liver, functions in the middle phase of the intrinsic pathway of blood coagulation. A functional deficiency of factor IX underlies haemophilia B, a chromosome X-linked recessive disease for which the major therapeutic approach is replacement treatment using factor IX concentrates. The cloning and characterization of the gene for human factor IX would mean that human factor IX could be produced in greater yield and purity through using recombinant DNA techniques. We have now used a human factor IX cDNA clone, inserted into a vaccinia virus-derived vector, to infect human hepatoma cells which normally produce no factor IX, and mouse fibroblasts. Fully active factor IX was produced by the hepatoma cells, whereas the fibroblasts produced a protein less active than natural factor IX, even in the presence of high levels of vitamin K. Human factor IX is extensively post-translationally modified, and thus represents probably the most complex protein produced in active form by recombinant DNA techniques to date. Our study also illustrates the potential of vaccinia virus-based vectors for expressing significant amounts of complex, clinically useful proteins in eukaryotic cells, in addition to its already demonstrated usefulness for producing live recombinant vaccines.