Effect of the E4 region on the persistence of transgene expression from adenovirus vectors

The utility of adenovirus vectors for gene therapy is limited by the transience of expression that has been observed in various in vivo models. Immunological responses to viral targets can eliminate transduced cells and cause the loss of transgene expression. We previously described the characterization of an E4 modified adenovirus, Ad2E4ORF6, which is replication defective in cotton rats. We reasoned that gene transfer vectors based on Ad2E4ORF6 would have a reduced potential for viral gene expression in vivo which might be beneficial for achieving persistence of transgene expression. E1 replacement vectors expressing the cystic fibrosis transmembrane regulator or beta-galactosidase were constructed as series of vectors that differed with respect to the E4 region. Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of expression. Results obtained with nude mice indicate that nonimmunological factors have a major influence on the longevity of transgene expression. Expression was transient from the E1a promoter with all vectors but persisted from the cytomegalovirus promoter only with a vector containing a wild-type E4 region. Transience of expression did not correlate with the disappearance of vector DNA, suggesting that promoter down-regulation may be involved. Coinfection studies indicate an E4 product(s) could be supplied in trans to allow persistent expression from the cytomegalovirus promoter. In summary, the choice of promoter is important for achieving persistence of expression; in addition, some promoters are highly influenced by the context of the vector backbone.     

Application of a Fas ligand encoding a recombinant adenovirus vector for prolongation of transgene expression

An adenovirus vector encoding murine Fas ligand (mFasL) under an inducible control was derived. In vivo ectopic expression of mFasL in murine livers induced an inflammatory cellular infiltration. Furthermore, ectopic expression of mFasL by myocytes did not allow prolonged vector-mediated transgene expression. Thus, ectopic expression of functional mFasL in vector-transduced cells does not appear to confer, by itself, an immunoprivileged site sufficient to mitigate adenovirus vector immunogenicity.     

The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures

Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry. AdV-mediated IL-12 expression was measured using a commercial ELISA. Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta3 and alpha(v)beta5 integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50. Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.     

Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus

The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies. However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression. Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ . We also show that psoralen-UV inactivated adenovirus significantly enhances transfection efficiency. The ability to deliver high molecular weight DNA using lipid-mediated transfection should expedite the analysis of large human genes contained within artificial chromosome vectors.     

The X-linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene

Mutations in the gene for Bruton's tyrosine kinase result in the B cell differentiation defects X-linked agammaglobulinemia in man and X-linked immunodeficiency in mice. Here we describe the generation of two yeast artificial chromosome (YAC)-transgenic mouse strains in which high-level expression of human Btk is provided by endogenous regulatory cis-acting elements that are present on a 340-kb transgene, Yc340-hBtk. The expression pattern of the transgenic human Btk was found to parallel that of the endogenous murine gene. When the Yc340-hBtk-transgenic mice were mated onto a Btk-deficient background, the xid B cell defects were fully corrected: conventional and CD5+ B-1 B cells were present in normal numbers, serum IgM and IgG3 levels as well as responses to T cell-independent type II antigens were in the normal ranges. In vivo competition experiments in Btk+/- female mice demonstrated that in the conventional B cell population the Yc340-hBtk transgene could fully compensate the absence of expression of endogenous murine Btk. We conclude that in the YAC-transgenic mice Btk is appropriately expressed in the context of native regulatory sequences.     

In vivo gene therapy for prostate cancer: preclinical evaluation of two different enzyme-directed prodrug therapy systems delivered by identical adenovirus vectors

Advanced prostate cancer is invariably lethal once it becomes androgen independent (AI). With the aim of developing a new treatment we have used the human androgen-independent prostate cancer cell line, PC-3, to evaluate the effectiveness of two enzyme-directed prodrug therapy (EPT) systems as a novel means for promoting tumor cell destruction in vivo. We have confined our study to the use of a PSA promoter, in a preliminary attempt to achieve prostate specificity. The two EPT systems used were the HSVTK/GCV and PNP/6MPDR systems. These were chosen for their differential dependence on DNA replication for their mechanism of action. In the present work, either the HSVTK or PNP gene, each controlled by a PSA promoter fragment, was delivered by an E1-, replication-deficient human adenovirus (Ad5) into PC-3 tumors growing subcutaneously in BALB/c nude mice. Tumors were injected with a single dose of recombinant Ad5 and mice were treated intraperitoneally with the appropriate prodrug, twice daily, for 6 days thereafter. The growth of established PC-3 tumors was significantly suppressed and host survival increased with a single course of HSVTK/GCV or PNP/6MPDR treatment. HSVTK/GCV-treated PC-3 tumor growth was 80% less than that of control treatments on day 33, while PNP/6MPDR-treated tumor growth was approximately 75% less than that of control treatments on day 52. Survival data showed that 20% of HSVTK/GCV- or PNP/6MPDR-treated animals lived >45 and >448 days, respectively, longer than control animals. These results demonstrate that both HSVTK/GCV and PNP/6MPDR therapies interrupt the growth of an aggressive human prostate cancer cell line in vivo.     

Selective astrocytic transgene expression in vitro and in vivo from the GFAP promoter in a HSV RL1 null mutant vector--potential glioblastoma targeting

Due to the lack of any effective therapy, novel approaches are currently being explored for the treatment of primary brain tumours. It has previously been demonstrated that variants of HSV-1 which are deleted in the RL1 gene and fail to produce the virulence factor ICP34.5 are potential candidates for tumour therapy. The RL1 variant 1716 replicates selectively within tumour cells and has the potential to deliver a therapeutic or tumour killing gene directly to the site of tumour growth. As many intracerebral tumours are glial and predominantly astrocytic in origin, we have evaluated the ability of 1716 to deliver a reporter gene specifically to astrocytes in vivo and in vitro using a 2.2 kb fragment which controls expression of the glial fibrillary acidic protein (GFAP), an astrocyte specific protein. Two 1716 variants, 1774 and 1775, were constructed which contain the GFAP-promoter element linked to the E. coli beta-galactosidase gene, inserted into the HSV-1 UL43 and US5 loci, respectively. In primary cultures, human primary tumour cell lines and established tumour cell lines in vitro, 1774 and 1775 gave high levels of expression of beta-galactosidase specifically in astrocytes. In vivo following intracerebral inoculation, both viruses demonstrated high levels of beta-galactosidase expression predominantly in astrocytes. These results indicate that the GFAP promoter element could be used for efficient and selective transgene delivery to human gliomas.     

An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3

Human adenoviruses (Ads) are attracting considerable attention because of their potential utility for gene transfer and gene therapy, for development of live viral vectored vaccines, and for protein expression in mammalian cells. Engineering Ad vectors for these applications requires a variety of reagents in the form of Ads and bacterial plasmids containing viral DNA sequences and requires different strategies for construction of vectors for different purposes. To simplify Ad vector construction and develop a procedure with maximum flexibility, efficiency, and cloning capacity, we have developed a vector system based on use of Ad5 DNA sequences cloned in bacterial plasmids. Expanded deletions in early region 1 (3180 bp) and early region 3 (2690 or 3132 bp) can be combined in a single vector that should have a capacity for inserts of up to 8.3 kb, enough to accommodate the majority of cDNAs encoding proteins with regulatory elements. Genes can be inserted into either early region 1 or 3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. To illustrate the flexibility of the system, we have introduced a wild-type early region 3 into the vectors, and to illustrate the high capacity for inserts, we have isolated a vector with two genes totaling 7.8 kb.     

Activation of ET(A) receptors is partially responsible for the rapid increase in haematocrit induced by bacterial lipopolysaccharide in the rat

It is believed that changes in the production and release of endothelin-1 (ET-1) are mediated over prolonged periods, and, therefore, that it is unlikely that ET-1 mediates rapid responses within the circulation. Here we show that ET-1 is involved in the rapid changes produced by injection of LPS in vivo. In anaesthetised rats, a bolus of LPS induced an increase in haematocrit and a fall in blood pressure within 10 min. The increase in haematocrit was reduced by administration of antagonists selective for endothelin ET(A) receptors, while the accompanying decrease in MAP was potentiated. Thus, activation of ET(A) receptors is partially responsible for the rapid increase in haematocrit seen in this model. This clearly demonstrates that ET-1 can act as a rapid responder to acute cardiovascular challenges.     

Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization

The development of transfection technology for malaria parasites holds significant promise for a more detailed characterization of molecules targeted by vaccines or drugs. One asexual blood stage vaccine candidate, apical membrane antigen-1 (AMA-1) of merozoite rhoptries has been shown to be the target of inhibitory, protective antibodies in both in vitro and in vivo studies. We have investigated heterologous (trans-species) expression of the human malaria Plasmodium falciparum AMA-1 (PF83/AMA-1) in the rodent parasite Plasmodium berghei. Transfected P. berghei expressed correctly folded and processed PF83/AMA-1 under control of both pb66/ama-1 and dhfr-ts promoters. Timing of expression was highly promoter-dependent and was critical for subsequent subcellular localization. Under control of pb66/ama-1, PF83/AMA-1 expression and localization in P. berghei was limited to the rhoptries of mature schizonts, similar to that observed for PF83/AMA-1 in P. falciparum. In contrast the dhfr-ts promoter permitted PF83/AMA-1 expression throughout schizogony as well as in gametocytes and gametes. Localization was aberrant and included direct expression at the merozoite and gamete surface. Processing from the full-length 83-kDa protein to a 66-kDa protein was observed not only in schizonts but also in gametocytes, indicating that processing could be mediated outside of rhoptries by a common protease. Trans-species expressed PF83/AMA-1 was highly immunogenic in mice, resulting in a response against a functionally critical domain of the molecule.     

Activation of the uncoupling protein by fatty acids is modulated by mutations in the C-terminal region of the protein

The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C-terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2-bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate. It is revealed that substitution of Cys304 by non-charged residues alters the response of UCP to fatty acids. The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half-maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half-maximal concentration. We conclude that the C-terminal region participates in the fatty acid regulation of UCP activity. The observed correlation between yeast growth rates in the presence of bromopalmitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants.     

Non-cell-autonomous photoreceptor degeneration in rds mutant mice mosaic for expression of a rescue transgene

The inherited retinal dystrophies represent a large and heterogenous group of hereditary neurodegenerations, for many of which, the molecular defect has been defined. However, the mechanism of cell death has not been determined for any form of retinal degeneration. The retinal degeneration slow (rds-/-) mutation of mice is associated with nondevelopment of photoreceptor outer segments and gradual death of photoreceptor cell bodies, attributed to the absence of the outer segment protein rds/peripherin. Here, we examined the effects of a transgene encoding normal rds/peripherin that had integrated into the X-chromosome in male and female rds-/- mutant retinas. In 2-month-old transgenic males and homozygous-transgenic females on rds-/-, we observed virtually complete rescue of both the outer segment nondevelopment and photoreceptor degeneration. In contrast, hemizygous-transgenic rds-/- female littermates showed patchy distributions of the transgene mRNA, by in situ hybridization analysis, and of photoreceptor cells that contain outer segments. This pattern is consistent with random inactivation of the X-chromosome and mosaic expression of the transgene. Surprisingly, we observed significant photoreceptor cell loss in both transgene-expressing and nonexpressing patches in hemizygous female retinas. These observations were supported by nuclease protection analysis, which showed notably lower than predicted levels of transgene mRNA in retinas from hemizygous females compared with male and homozygous female littermates. This phenotype suggests an important component of non-cell-autonomous photoreceptor death in rds-/- mutant mice. These results have significance to both the etiology and potential treatment of human inherited retinal degenerations.     

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.     

Characterization of hemoglobin adducts from a 4, 4'-methylenedianiline metabolite evidently produced by peroxidative oxidation in vivo

4,4'-Methylenedianiline (MDA) is a widely used mutagenic and carcinogenic industrial chemical. It is also a metabolite of 4, 4'-methylenediphenyl diisocyanate (MDI), which is used in the manufacturing of polyurethane foams. Biomonitoring of MDA, like other aromatic amines, is mainly carried out by GC/MS measurement of cysteine adducts in Hb from the nitroso metabolite, released by alkaline hydrolysis. In the present study it was investigated whether the formation of Hb adducts from non-nitroso metabolites of MDA can be used for the dosimetry of MDA. The study was carried out by treatment of mice with MDA and tritiated MDA or deuterated MDA and by identification of their products of reaction with Hb, after enzymatic hydrolysis of the globin and enrichment of the adducts. The main adduct, about 50% of the total amount of MDA associated with Hb, was characterized by MS and was shown to be a reaction product of MDA and the amino group of N-terminal valine in Hb, the derived structure being 1-[(4-imino-2, 5-cyclohexadien-1-ylidene)methyl]benzene-4-azo-2-isovaleric acid. It is likely that this quinonoid MDA imine adduct to valine was formed by an attack of a metabolite formed through peroxidative oxidation of MDA, in analogy with earlier observed oxidation of some other aromatic amines, e.g., benzidine. The reactive intermediate is suggested to be [(4-imino-2, 5-cyclohexadien-1-ylidene)methyl]-4-aminobenzene. The formation of the adduct was confirmed by incubating MDA with valine methyl ester in vitro in the presence of H2O2 and lactoperoxidase. Further, the same adduct was detected in MDI-exposed and control rats, the level in the exposed animals being about 60 times higher than in the controls. This study indicates that, at least in the mouse, extrahepatic peroxidative metabolism is an important pathway for the bioactivation of MDA, possibly leading to a genotoxic reactive intermediate. This study also demonstrates the usefulness of Hb adduct analysis for the identification of reactive intermediates in vivo.