Adenovirally transferred p16INK4/CDKN2 and p53 genes cooperate to induce apoptotic tumor cell death.

Repression of cell cycle progression by tumor suppressors might provide a means for tumor therapy. Here we demonstrate that ectopic overexpression of the p16INK4/CDKN2 tumor suppressor from an adenovirus vector in various cell lines results in block of cell division and, subsequently, in a gradual reduction of the levels of the product of retinoblastoma susceptibility gene, pRb. Overexpression of p53 and p16INK4/CDKN2, but not p53 on its own, induces apoptotic death only in tumor cells. Simultaneous adenoviral transfer of p16 and p53 genes leads to inhibition of tumor growth in nude mice. These results suggest that combined delivery of two cooperating genes like p16 and p53 could be the basis for the development of a new strategy for cancer gene therapy.

Reconstitution of mutant V2 vasopressin receptors by adenovirus-mediated gene transfer. Molecular basis and clinical implication.

Recent studies with transfected COS-7 cells have shown that functionally inactive mutant V2 vasopressin receptors (occurring in patients with nephrogenic diabetes insipidus) can be functionally rescued by coexpression of a carboxy-terminal V2 receptor fragment (V2-tail) spanning the region where various mutations occur [Schöneberg, T., J. Yun, D. Wenkert, and J. Wess. 1996. EMBO (Eur. Mol. Biol. Organ.) J. 15:1283-1291]. In this study, we set out to characterize the underlying molecular mechanism. Using a coimmunoprecipitation strategy and a newly developed sandwich ELISA system, a direct and highly specific interaction between the mutant V2 vasopressin receptor proteins and the V2-tail polypeptide was demonstrated. To study the potential therapeutic usefulness of these findings, Chinese hamster ovary (CHO) cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors were created and infected with a recombinant adenovirus carrying the V2-tail gene fragment. After adenovirus infection, vasopressin gained the ability to stimulate cAMP formation with high potency and efficacy in all CHO cell clones studied. Moreover, adenovirus-mediated gene transfer also proved to be a highly efficient method for achieving expression of the V2-tail fragment (as well as the wild-type V2 receptor) in Madin-Darby canine kidney tubular cells. Taken together, these studies clarify the molecular mechanisms by which receptor fragments can restore function of mutationally inactivated G protein-coupled receptors and suggest that adenovirus-mediated expression of receptor fragments may lead to novel strategies for the treatment of a variety of human diseases.

Liver-directed gene transfer and application to therapy.

The liver is an important and attractive target for the development of gene therapy strategies. Many genetic diseases are manifested in the liver, and both infectious and malignant diseases affect this organ. Retroviral and adenoviral vectors have been shown to infect hepatocytes with varying efficiently in vitro and in vivo. The presence of unique receptors at the cellular membrane of hepatocytes has stimulated the development of transfer strategies based on receptor targeting of vectors. The results of a first clinical trial for gene therapy in the liver based on ex vivo gene delivery has shown both the feasibility and the limits of current technology. This review discusses both existing vectors and strategies and prospective developments towards liver-directed gene therapy of genetic and malignant diseases.

Polyoma virus pseudocapsids as efficient carriers of heterologous DNA into mammalian cells.

Polyoma virus VP1 pseudocapsids, generated from a recombinant baculovirus, have been successfully used to transfer exogenous DNA stably into rodent (rat-2) cells. To evaluate the efficiency and biological usefulness of this route for introducing heterologous DNA into cells, the gene for a transforming deletion mutant of the middle T antigen of polyoma virus, dl8 MT, was used initially. Whereas the amount of DNA packaged together with pseudocapsids was found to be variable (2-30%), even at low efficiency its transfer as biologically functional information was high. The dl8 MT gene was stably transferred and integrated in low copy numbers into the host chromosome. Transformed cell lines (derived from single foci) were shown to produce high levels of the corresponding mutant protein, which was active in an in vitro protein kinase assay. In comparisons with the calcium phosphate DNA coprecipitation procedure (or lipofectin route), the VP1 pseudocapsid approach was shown to have many advantages in terms of maintenance of DNA fidelity and increased efficiency of gene expression. This system was also assessed for its ability to transfer into and express the chloramphenicol acetyl transferase (CAT) gene in a human liver cell line. Here again, the assay for functional CAT expression showed the pseudocapsid transfer procedure to compare favorably with lipofectin transfer. In another transient assay, a low-level endogenously expressed gene, p43, was complexed with pseudocapsids and transferred into human embryo lung fibroblasts, thereby increasing the expression levels. The ease of production of VP1 pseudocapsids, coupled with their efficient transfer of biologically useful information, should make this route of gene delivery an attractive proposition for further exploration with regard to gene therapy.

Gene transfer into hepatocytes and human liver tissue by baculovirus vectors.

Gene therapy of liver diseases requires the development of efficient vectors for gene transfer in vivo. Retroviral and adenoviral vectors have been shown to deliver genes efficiently into hepatocytes in vitro and in vivo. However, these vectors do not allow for exclusive infection of the liver which would be highly advantageous for in vivo gene therapy strategies. We have recently demonstrated that genetically modified baculoviruses (Autographa californica nuclear polyhedrosis virus) efficiently deliver genes into cultured cells and have a strong preference for hepatocytes of different origin. Baculoviral gene transduction efficiency into human hepatocytes was determined to approach 100% and expression levels are high, provided that gene expression is controlled by mammalian promoters. In this report, we present further properties of baculoviruses regarding their use for hepatocyte gene transfer. Baculovirus-mediated gene expression declines rapidly in the hepatocellular carcinoma cell line Huh7 and more slowly in primary cultures of mouse hepatocytes. Direct application of baculoviruses for gene delivery to the liver in vivo is hampered by serum components, presumably by complement. However, we demonstrate here that baculoviral gene transfer is feasible in ex vivo perfused human liver tissue. This result suggests the development of a strategy using baculoviral vectors for liver-directed gene therapy.

A phage T7 class-III promoter functions as a polymerase II promoter in mammalian cells.

A phage T7 class-III promoter (pT7), which is highly specific for T7 RNA polymerase in bacteria, was tested in mammalian cells for its specificity. After having shown that T7 RNA polymerase can transcribe from pT7 in the nucleus of stably transformed cells [Lieber et al., Nucleic Acids Res. 17 (1989) 8485-8493], we describe here that pT7 could also direct efficient intracellular gene expression in the absence of T7 RNA polymerase. Using the genomic human growth hormone-encoding gene and the firefly luciferase-encoding gene as reporters, we found expression levels comparable with those obtained with the Rous sarcoma viral promoter. Inhibition of expression with alpha-amanitin suggests that transcription is by RNA polymerase II. Binding studies with HeLa cell extracts clearly show that synthetic pT7 sequences are specifically bound (gel retardation) and that the promoter region is protected from DNase degradation. The experimental data, as well as the nucleotide sequence, suggest that pT7 has properties of an initiator element. Indeed, the activity of pT7 can be stimulated by the presence of an upstream element or an enhancer. These results have practical implications for the use of pT7 in mammalian expression vectors. Commercial pT7 plasmids can be used for both prokaryotic and eukaryotic expression systems.

Expression of the type I diabetes-associated gene LRP5 in macrophages, vitamin A system cells, and the Islets of Langerhans suggests multiple potential roles in diabetes.

LRP5 is a novel member of the low-density lipoprotein receptor family that is genetically associated with Type 1 diabetes. As a start to defining the normal function of LRP5 and to generate testable hypotheses of its potential role in Type 1 diabetes pathogenesis, we carried out an extensive expression analysis of this gene at the mRNA and protein levels in normal human, monkey, and mouse, as well as in non-obese diabetic (NOD) mice at several stages of diabetes development. In all species, expression of LRP5 was found in four functionally important cell types: the distributed mononuclear phagocyte system, the islets of Langerhans, vitamin A-metabolizing cells, and CNS neurons. Given the critical role of macrophages in the onset and progression of islet cell destruction in Type 1 diabetes and the hypothesized role of retinoids as modifiers of diabetes progression, these findings suggest that LRP5 may confer Type 1 diabetes risk by altering the normal functioning of one or more of these regulatory systems. Specifically, given that the LRP5 polymorphisms associated with diabetes are in the promoter region of the gene, alterations in LRP5 expression may be responsible for diabetes susceptibility and therefore may be potential targets for therapeutic intervention. (J Histochem Cytochem 48:1357-1368, 2000)

Mucin gene (MUC1) transfer into human dendritic cells by cationic liposomes and recombinant adenovirus.

BACKGROUND: Dendritic cells (DC) as antigen presenting cells play an important role in immunotherapy of cancer. Mucin, encoded by the gene MUC1, is a human tumor antigen expressed in breast, pancreatic and ovarian cancers. Therefore, MUC1-transfected DC would be an attractive tool in constructing cancer vaccines. MATERIALS AND METHODS: Using two different cationic liposome preparations and, for comparison, a recombinant adenovirus expressing mucin, we tested the efficiency of mucin gene transfer into DC by flow cytometry. We investigated if these transfected DC were able to specifically stimulate autologous peripheral blood lymphocytes (PBL) from healthy donors. RESULTS: Flow cytometry revealed that 5-20% of DC transfected with liposomes Lipofectin and 20-40% of DC transduced with adenovirus expressed the relevant mucin epitopes. The expression of mucin on DC was similar to the expression of mucin found on carcinoma cells. After antigen uptake, DC specifically stimulated autologous PBL. CONCLUSION: We have shown that cationic liposomal gene transfer into human DC was feasible. We could obtain antigen specific stimulation of PBL at a similar rate as with adenoviral MUC1-transduced DC.

Combined adenoviral transfer of tumor suppressor and cell-cycle genes for tumor-cell apoptosis.

The loss of control over the cell cycle and the disruption of cascade mechanisms for programmed cell death are major factors in tumorigenesis. Defects in thep53 gene and in the regulation of genes of the retinoblastoma pathway such as p16 or cyclin D1 occur in a large percentage of tumors and have been well studied. Reintroduction or overexpression of genes suppressing proliferation or promoting apoptosis offers a potential for selective suicide of tumor cells. Transfer of tumor suppressor genes into growing tumors with mutations in the respective gene can indeed reduce tumor growth (1). The reason for this effect is probably not just a reestablishement of the normal phenotype, but rather a severe disturbance of the cancer cell's regulatory balance of life and death, which can result in apoptosis. Retransfer of two or more cancer genes can have synergistic effects on apoptosis induction. The choice, which gene combination will be particularly efficient, depends on the pattern of mutated genes. We have recently reported that the cotransfer of p53 and p16 leads to a better induction of apoptosis and reduction of tumor growth than the transfer of either gene alone (2). In addition, it seems that normal cells with an intact genotype are more resistant to the action of tumor supressor genes than tumor cells with mutations in the respective genes. Several approaches are now underway to exploit those gene combinations which are the most efficient for tumor cell-specific apoptosis on a given genetic background.

New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: studies on growth, metabolism and virus propagation.

Few suspension cells can be used for vaccine manufacturing today as they either do not meet requirements from health regulatory authorities or do not produce high virus titres. Two new avian designer cell lines (AGE1.CR and AGE1.CR.pIX) that have been adapted to grow in suspension in serum-free medium were evaluated for their potential as host cells for influenza and modified vaccinia Ankara (MVA, wild type) vaccine production. Their metabolism was studied during growth in static (T-flasks) and dynamic cultivation systems (roller bottles, stirred tank reactor, wave bioreactor). High cell concentrations up to 5.8x10(6)cells/mL were obtained with doubling times of 23h for AGE1.CR and 35h for AGE1.CR.pIX, respectively. Both viruses were produced to high titres (3.5 logHA/100 microL for influenza virus, 3.2x10(8)pfu/mL for MVA). Hence, the CR cell lines are an appropriate substrate for pharmaceutical influenza and MVA production.

A mutant T7 phage promoter is specifically transcribed by T7-RNA polymerase in mammalian cells.

The phage T7 promoter/polymerase system is highly specific in bacteria in contrast to that observed in mammalian cells. A number of cell lines exhibit a considerable level of expression from the T7 promoter, even in the absence of T7-RNA polymerase. Here, we demonstrate that nuclear-factor-including components of the TFIID fraction, bind to the T7 promoter and inhibit transcription by T7-RNA polymerase. In order to increase the specificity of the promoter for T7-RNA polymerase and to abolish binding of nuclear factors, a novel strategy for the selection of randomly mutated promoters was established. The strategy involves adsorption of mutant promoters to HeLa extracts and binding of the free oligonucleotides to T7-RNA polymerase, cloning, and functional testing of the recombinants. After selection, the resulting mutant promoters showed an increase in specificity for transcription by T7-RNA polymerase.

Evaluation of HBV promoters for use in hepatic gene therapy.

Strategies for in vivo hepatic gene therapy will require regulatory elements which allow for long-term expression of therapeutic genes and restriction of expression to hepatocytes. This study investigates the suitability of promoters derived from hepatitis B virus (HBV) for liver-specific gene expression in vectors for hepatic gene therapy. We provide three hepatocyte-specific promoters, the HBV core promoter, the HBV core promoter linked directly to the HBV enhancer I, and a hybrid promoter containing the HBV enhancer II and a basic CMV promoter, which are hepatocyte-specific and allow for increasing levels of reporter gene expression. Moreover, in long-term expression studies using our promoter constructs in the context of an EBV based expression system we found that expression from these promoters remained nearly unchanged over a period of at least two months in hepatocyte-derived cell lines.

Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: involvement of NFkappaB.

The cytomegalovirus (CMV) major immediate-early promoter/enhancer is active in many cell culture systems and is considered to be one of the strongest promoters in vitro. However, when this promoter was used in in vivo approaches to gene therapy, it was silenced within a few weeks in several organs including the liver. In this study, we demonstrated transcriptional inactivation of the CMV promoter in mouse liver. In contrast to the CMV promoter, a hybrid promoter consisting of a minimal CMV promoter and the enhancer II of hepatitis B virus was active for at least 11 weeks in mouse liver. While investigating the reason for the shutdown of the CMV promoter, we did not find evidence for methylation of adenovirus DNA in the region of transgene insertion, but we could show that the silenced CMV promoter was reactivated after lipopolysaccharide treatment of mice or partial hepatectomy. Both stimuli are known to activate the transcription factor NFkappaB, which binds to four sites in the CMV promoter/enhancer. We show that expression from the CMV promoter in hepatocyte-derived cell lines in vitro depends on NFkappaB. In vivo experiments demonstrate that NFkappaB, which is not present in mouse hepatocytes in vivo, is activated after infection with recombinant adenoviruses and that the time course of NFkappaB activation parallels that of CMV promoter-dependent expression. Moreover, adenovirus infection of transgenic mice carrying a CMV promoter-driven lacZ gene leads to strong activation of the expression of this gene in the liver. Thus, NFkappaB is involved in the activation of the CMV promoter in the liver.

Hepatocyte-specific binding of L/S-HBV particles expressed in insect cells.

The genome of hepatitis B virus (HBV) codes for three surface antigen proteins. Two of them are essential components of infectious viral particles. Whereas expression of the small (S) antigen led to formation of virus-like particles in different systems so far, secretion of neither the large antigen nor budding of virus-like particles containing both antigens could be observed. Using modified large antigen genes in dual expression vectors we were able to demonstrate secretion of virus-like particles in the baculovirus insect cell system. N-terminal fusion of an insect protein (melittin) derived signal sequence and destruction of the myristylation site resulted in secretion of the large antigen. Particles consisting of about 95% small and 5% large antigen bind specifically to hepatocytes. These pseudovirions could serve as a HBV vaccine and as a useful component of future hepatocyte-specific gene transfer vehicles.

Effect of amniotic fluid on cationic lipid mediated transfection and retroviral infection.

In preparation for foetal gene therapy by intra-amniotic gene application, we have investigated the effect of amniotic fluid on several gene transfer systems. In vitro lipofection of embryonically derived 3T3 cells by several of the tested cationic lipids decreases in the presence of human amniotic fluid, while two formulations, Lipid 67 and Tfx-50, remain highly active. As some body fluids are known to inactivate most retroviral vectors, we investigated the influence of amniotic fluid on the efficiency of infection of 3T3 cells by murine leukaemia virus (MoMLV)-based vectors, including amphotropic and ecotropic retrovirus, and a vesicular stomatitis virus G (VSV-G) glycoprotein pseudotyped retroviral vector. All showed a decrease of infectivity from 21 to 56% in the presence of amniotic fluid. The ecotropic retrovirus is the most infectious under normal conditions as well as in amniotic fluid. Our results suggest that intra-amniotic injection may allow efficient gene transfer using either amniotic fluid-resistant cationic lipids or ecotropic retroviral vectors in a murine in vivo model for foetal gene therapy. The VSV-G-pseudotyped vector, although displaying a decrease of infectivity, remains of great interest for gene delivery, because of its broad host range and because of the high virus titers achievable. Finally, a baculovirus-based vector shows no decrease of its infectivity and does not seem to be affected by amniotic fluid but has only low infectivity on the tested foetal fibroblast cell line.

Gene therapy: prospects for treatment of liver disease.

Gene therapy in the liver for treatment of metabolic diseases is a great challenge. The development of vectors for gene transfer to hepatocytes in vitro and to the liver in vivo has advanced very rapidly within the last few years. However, none of the existing vectors would allow for expression of therapeutic levels of a gene product for a longer period of time. Our laboratory is developing alternative strategies for gene transfer to the liver in vivo which are based on composite vectors consisting of envelopes and promoters derived from hepatitis B virus, an EBV-derived replicon, and the chromosomal protein HMG1. Hepatocyte specificity of both, binding of the particles and expression of foreign genes, was demonstrated.

HBV-derived promoters direct liver-specific expression of an adenovirally transduced LDL receptor gene.

In vivo approaches to liver gene therapy will require restriction of transgene expression to hepatocytes. Since targeting of viral vectors exclusively to the liver is not easy to achieve, use of liver-specific promoters for driving expression of therapeutic genes is an interesting alternative. We have shown previously that regulatory elements of the hepatotrophic hepatitis B virus (HBV) are strong and liver-specific in vitro and therefore might be useful in hepatic gene therapy. Here we describe recombinant adenoviruses in which the human LDL receptor gene is under the transcriptional control of the HBV core promoter, the core promoter linked directly to HBV enhancer I, or a HBV-CMV hybrid promoter, respectively. These viruses allowed for a moderate to strong expression of the LDL receptor gene in vitro in a hepatocyte-specific manner when compared with the CMV immediate-early promoter. In vivo experiments demonstrated that the promoter gave rise to an expression level comparable to that from the CMV promoter in mouse liver, but was very weak in lung and skeletal muscle. Thus, the HBV-CMV hybrid promoter is strong and hepatocyte specific both in vitro and in vivo even in the adenoviral context and would be a good choice for driving a therapeutic gene in liver gene therapy.

Influence of DNA binding on the degradation of oxidized histones by the 20S proteasome.

The 20S proteasome is localized in the cytosol and nuclei of mammalian cells. Previous work has shown that the cytosolic 20S proteasome is largely responsible for the selective recognition and degradation of oxidatively damaged cytosolic proteins. Since nuclear proteins are also susceptible to oxidative damage (e.g., from metabolic free radical production, ionizing radiation, xenobiotics, chemotherapy) we investigated the degradation of oxidatively damaged histones, in the presence and in the absence of DNA, by the 20S proteasome. We find that both soluble histones and DNA-bound histones are susceptible to selective proteolytic degradation by the 20S proteasome following mild oxidative damage. In contrast, more severe oxidative damage actually decreases the proteolytic susceptibility of histones. Soluble H1 showed the highest basal and maximal absolute proteolytic rates. Histone fraction H4 exhibited the greatest relative increase in proteolytic susceptibility following oxidation, almost 14-fold, and this occurred at a peroxide exposure of 5 mM. At the other end of the spectrum, histone H2A exhibited a maximal proteolytic response to H2O2 of only 6-fold, and this required an H2O2 exposure of 15 mM. An oxidation of reconstituted linear DNA plasmid-histone complex makes up to 95% of the histones bound to DNA susceptible to degradation, whereas undamaged protein-DNA complexes are not substrates for the proteasome. Severe oxidation by high concentrations of H2O2 appears to decreases the proteolytic susceptibility of histones due to the formation of cross-linked histone-DNA aggregates which appear to inhibit the proteasome. We conclude that the degradation of nuclear proteins is highly selective and requires prior damage of the substrate protein, such as that caused by oxidation.