Enhancement of humoral immunity to SIVenv following simultaneous inoculation of mice by three recombinant adenoviruses encoding SIVenv/poliovirus chimeras, Tat and Rev.

A means of inducing gene expression by simultaneous infection with three recombinant adenoviruses (Ad) is described. The simian immunodeficiency virus (SIV) envelope-coding region was placed under the control of the human immunodeficiency virus type 1 (HIV-1) Tat and Rev proteins provided in trans by distinct Ad vectors (Ad-tat; Ad-rev). Coinfection of cells with the three recombinant adenoviruses led to induction of high levels of SIV env mRNA and protein synthesis, while inoculation of mice elicited anti-Env antibodies. Insertion of the poliovirus VP1 neutralization epitope (C3) in the V1 hypervariable region of SIV envelope not only proved to be highly immunogenic for the poliovirus epitope but also enhanced the kinetics of anti-SIV Env antibody production. By contrast, insertion in V4 elicited no anti-C3 response and only normal anti-Env responses.

Residual expression of reporter genes in constructs mimicking HIV genome organization.

Plasmids were constructed whereby the expression of a reporter gene, either the cDNA corresponding to the secreted form of human alkaline phosphatase (SEAP) or the herpes simplex virus type 1 (HSV1) thymidine kinase (tk) gene, was rendered dependent upon the expression of the human immunodeficiency virus type 1 (HIV1) tat and rev proteins. The SEAP or tk genes were placed between HIV1 splice donor and acceptor sites. One SEAP construct carried a series of alternating splice donor and acceptor sites. In all cases, the rev response element mapped within an intron. Despite such mimicry of the HIV1 genome, residual expression of the reporter gene in the absence of tat and rev was observed. These results, as well as non-specific T-cell recruitment, suggest limits to the specificity of using HIV-activated toxic gene expression to kill HIV-infected cells.

Polarity of secretion of alpha 1-antitrypsin by human respiratory epithelial cells after adenoviral transfer of a human alpha 1-antitrypsin cDNA.

alpha 1-Antitrypsin (alpha AT) deficiency, a hereditary cause of progressive emphysema, can potentially be treated by transfer of a functional human alpha 1AT gene to the respiratory epithelium. For such an approach to be successful, alpha 1AT must be provided to both the interstitium and the epithelial surface--that is, the alpha 1AT directed by the transferred gene must be secreted to both the apical and basolateral surfaces of the epithelial cells. To evaluate this concept, a recombinant, replication-deficient adenoviral vector (Ad-alpha 1AT) containing a human alpha 1AT cDNA driven by an adenovirus major late promoter was used to infect Bet-1A human respiratory epithelial cells. The infected cells expressed Ad-alpha 1AT-directed mRNA transcripts and synthesized and secreted functional human alpha 1AT as shown by [35S]methionine labeling and immunoprecipitation of a 52-kD glycosylated human alpha 1AT molecule capable of interacting with neutrophil elastase, its natural substrate. Bet-1A cells grown on microporous polycarbonate membranes formed tight junctions (resistance > 150 omega x cm2). After infection with Ad-alpha 1AT, [35S]methionine labeling and enzyme-linked immunoassay demonstrated that alpha 1AT was secreted into both the apical and basolateral compartments, with an average apical to basolateral ratio of 1.9 +/- 0.2. Thus, human respiratory epithelial cells infected with a recombinant adenoviral vector containing a human alpha 1AT cDNA secrete alpha 1AT across both the apical and basolateral cell membranes, suggesting that the respiratory epithelium could serve as a target for in vivo gene therapy of alpha 1AT deficiency.

[Adenovirus, a gene therapy vector: application to lung diseases]. / L'adénovirus, un vecteur de thérapie génique: application à des affections pulmonaires.

Due to their quiescent nature and spatial complexity, many target tissues for gene therapy will require novel strategies. An alternative to ex vivo gene transfer, providing many technical advantages and possibly allowing sufficient transfer of the therapeutic gene, is direct in vivo delivery of the vehicle. For a favorable outcome, this procedure is dependent on a high-titer vector, fully competent before post-mitotic cells. In view of the restrictions inherent in the use of retroviruses, we have investigated the potentials of adenovirus. Adenoviruses have as primary targets of infection the differentiated epithelial cell. The large DNA genome of the virus hints to a large cloning capacity. Furthermore, wildtype adenovirus and the precedent of administration to humans are suggestive of adenovirus-based gene therapy for diseases involving a variety of quiescent tissues. The use of a replication-defective adenovirus carrying a gene encoding a nuclearly-targeted beta-galactosidase demonstrated that replication-incompetent adenoviruses offer an efficient means to transfer a gene for extended periods of time to the liver, muscle, lung, and brain. Because of adenovirus' natural tropism for the lung epithelium, we have proposed that this virus be used as a CFTR gene delivery vector for the treatment of cystic fibrosis and lung cancer.

In vivo transfer of a marker gene to study motoneuronal development.

Adenovirus vectors containing a marker gene (lacZ from Escherichia coli) are potent for transferring the gene to neurones after intraparenchymal injections. Expression of the marker gene may lead to the synthesis of an enormous amount of beta-galactosidase which diffuses throughout the entire neurone, providing a 'Golgi-like' staining. This suggested that the technique may be used to study the morphology of specific neuronal populations. We have validated this hypothesis by analysing the postnatal development of motoneurones in the rat cervical cord. Injections of the viral suspension into one ventral horn were performed at different ages after birth. Histochemical staining using X-Gal revealed morphological changes occurring within the first 3 weeks with enlargement of the perikaryon and increased dendritic complexity. Immunoreactivity for CGRP was visualized in double-staining experiments. In vivo transfer of a marker gene therefore provides a new way to analyse neuronal morphology which allows selection of the cells to be studied and double-labelling with immunohistochemical markers.

In vivo correction of low density lipoprotein receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses.

A rabbit animal model of the human disease familial hypercholesterolemia (FH), which is the result of low density lipoprotein (LDL) receptor deficiency, was used to develop an in vivo approach to gene therapy based on recombinant adenoviruses. Recombinant, replication-defective adenoviruses expressing the lacZ gene under the control of different promoters were infused into the portal circulation of New Zealand White (NZW) rabbits. Expression of lacZ could be obtained in virtually all hepatocytes within 3 days post-infusion, but was undetectable by 3 weeks. This was not associated with liver pathology. An LDL receptor expressing adenovirus was constructed using the most active promoter and was infused into the portal vein of rabbits deficient in LDL receptor. Analysis of liver tissues harvested 3 days after virus infusion demonstrated human LDL receptor protein in the majority of hepatocytes that exceeded the levels found in human liver by at least 10-fold. Transgene expression was stable for 7-10 days and diminished to undetectable levels within 3 weeks. Infusion of LDL receptor expressing virus led to substantial reductions in serum cholesterol that returned to base line within 3 weeks; this acute reduction in serum cholesterol was associated with accumulations of lipid in hepatocytes. The development of neutralizing antibodies to the recombinant adenovirus markedly diminished the effectiveness of a second dose. These studies illustrate the advantages of recombinant adenoviruses for the treatment of liver metabolic diseases and define issues, such as viral genome instability and blocking immune response, that need to be overcome before the promise of this technology can be fully realized.

In vivo and in vitro analysis of electrical activity-dependent expression of muscle acetylcholine receptor genes using adenovirus.

Acetylcholine receptor (AChR) genes are repressed in extrajunctional domains of adult muscle fiber by neurally evoked electrical activity. Denervation elicits upregulation of AChR gene transcription in extrasynaptic areas. We have used an adenovirus (Ad)-based strategy to analyze in vitro and in vivo the electrical activity-dependent transcription of the chicken AChR alpha 1 subunit gene. The luciferase gene placed under the control of wild-type and mutated fragments of the alpha 1 subunit promoter was inserted in a defective Ad vector designed for the study of transcriptional regulation. Animals were infected by intramuscular injection and in vivo luciferase levels were normalized by coinfection with an Ad vector containing the chloramphenicol acetyltransferase gene driven by an electrical activity-insensitive promoter. Our results demonstrate that although both proximal MyoD binding sites of the alpha 1 promoter are required for muscle-specific expression of the alpha 1 gene, only one is necessary, albeit insufficient, to enhance alpha 1 promoter activity after denervation. Parallel results were obtained with cultured muscle cells in vitro following tetrodotoxin blocking of spontaneous electrical activity. These results substantiate a direct contribution of MyoD factors in electrical activity-dependent regulation of AChR expression and further indicate that Ad-based vectors constitute a powerful tool in the field of transcriptional regulation.

Adenovirus-mediated transfer of a human dystrophin gene to skeletal muscle of mdx mouse.

Due to their quiescent nature and spatial complexity, many target tissues for gene therapy will require novel strategies. An alternative to ex vivo gene transfer, providing many technical advantages and possibly allowing sufficient transfer of the therapeutic gene, is direct in vivo delivery of the vehicle. For a favorable outcome, this procedure is dependent on a high-titer vector, fully competent before post-mitotic cells. In view of the restrictions with the use of retroviruses, we investigated the potentials of adenovirus. Adenoviruses have as primary targets of infection the differentiated epithelial cell. The large DNA genome of the virus hints to a large cloning capacity. Furthermore, the wild type adenovirus has been largely used in man as a vaccine against adenovirus-induced respiratory disease. Taken together, the biological characteristics of adenovirus and the precedent of administration to humans are suggestive of adenovirus-based gene therapy for diseases involving a variety of quiescent tissues. The use of a replication-defective adenovirus carrying a gene encoding a nuclearly-targeted beta-galactosidase Ad.RSV beta gal demonstrated that replication-defective adenovirus offers an efficient means to transfer a gene for extended periods of time in the liver, muscle, lung and brain (1-6).

Assessment of recombinant adenoviral vectors for hepatic gene therapy.

Recombinant adenoviral vectors have recently been used to transfer genes into a number of different cell types in vitro and in vivo. A recombinant adenoviral vector bearing the Escherichia coli beta-galactosidase (beta-gal) gene was used to quantitate the frequency of hepatocyte transduction in the mouse after direct viral infusion into the portal vein. When 10(10) adenoviral particles were infused, over 95% of the hepatocytes were transduced in vivo as determined by x-gal staining. The transduction protocol is relatively safe in that there is no detectable helper virus production in transduced animals and that very few extrahepatic cells are transduced by this method. There is also no evidence of significant liver pathology unless substantially greater quantities of virus are used. However, the transduced hepatocytes do not appear to persist in vivo because the percentage of hepatocytes expressing beta-gal declined over time. Four months after the procedure, 0.5-10% of the hepatocytes contain detectable beta-gal activity in vivo. The change in beta-gal-positive cells correlates with decreasing amounts of adenoviral DNA. Thus, current recombinant adenoviral vectors may have clinical applications in gene therapy for acute hepatic disorders.

Direct gene transfer of human CFTR into human bronchial epithelia of xenografts with E1-deleted adenoviruses.

We describe the use of a human bronchial xenograft model for studying the efficiency and biology of in vivo gene transfer into human bronchial epithelia with recombinant E1 deleted adenoviruses. All cell types in the surface epithelium except basal cells efficiently expressed the adenoviral transduced recombinant genes, lacZ and CFTR, for 3-5 weeks. Stable transgene expression was associated with high level expression of the early adenoviral gene, E2a, in a subset of transgene expressing cells and virtually undetectable expression of the late adenoviral genes encoding the structural proteins, hexon and fiber. These studies begin to address important issues that relate to safety and in vivo efficacy of recombinant adenoviruses for gene delivery into the human airway.

Transfer of a foreign gene into the brain using adenovirus vectors.

The ability of a replication-deficient adenovirus vector to transfer a foreign gene into neural cells of adult rats in vivo has been analysed. A large number of neural cells (including neurons, astrocytes and ependymal cells) expressed an E. coli lacZ transgene for at least 45 days after inoculation of various brain areas. Injecting up to 3 x 10(5) pfu in 10 microliters did not result in any detectable cytopathic effects--these were only observed for very high titres of infection (> 10(7) pfu 10 microliters-1). Adenovirus vectors therefore appear to be a promising means for in vivo transfer of therapeutic genes into the central nervous system.

An adenovirus vector for gene transfer into neurons and glia in the brain.

The efficient introduction of genetic material into quiescent nerve cells is important in the study of brain function and for gene therapy of neurological disorders. A replication-deficient adenoviral vector that contained a reporter gene encoding beta-galactosidase infected rat nerve cells in vitro and in vivo. beta-Galactosidase was expressed in almost all sympathetic neurons and astrocytes in culture. After stereotactic inoculations into the rat hippocampus and the substantia nigra, beta-galactosidase activity was detected for 2 months. Infected cells were identified as microglial cells, astrocytes, or neurons with anatomical, morphological, and immunohistochemical criteria. No obvious cytopathic effect was observed.

Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer.

A variety of pulmonary disorders, including cystic fibrosis, are potentially amenable to treatment in which a therapeutic gene is directly transferred to the bronchial epithelium. This is difficult to accomplish because the majority of airway epithelial cells replicate slowly and/or are terminally differentiated. Adenovirus vectors may circumvent this problem, since they do not require target cell proliferation to express exogenous genes. To evaluate the diversity of airway epithelial cell targets for in vivo adenovirus-directed gene transfer, a replication deficient recombinant adenovirus containing the Escherichia coli lacZ (beta-galactosidase [beta-gal]) gene (Ad.RSV beta gal) was used to infect lungs of cotton rats. In contrast to uninfected animals, intratracheal Ad.RSV beta gal administration resulted in beta-gal activity in lung lysate and cytochemical staining in all cell types forming the airway epithelium. The expression of the exogenous gene was dose-dependent, and the distribution of the beta-gal positive airway epithelial cells in Ad.RSV beta gal-infected animals was similar to the normal cell differential of the control animals. Thus, a replication deficient recombinant adenovirus can transfer an exogenous gene to all major categories of airway epithelial cells in vivo, suggesting that adenovirus vectors may be an efficient strategy for in vivo gene transfer in airway disorders such as cystic fibrosis.

Adenovirus-mediated in vivo gene transfer and expression in normal rat liver.

Replication deficient, recombinant adenovirus (Ad) vectors do not require target cell replication for transfer and expression of exogenous genes and thus may be useful for in vivo gene therapy in hepatocytes. In vitro, primary cultures of rat hepatocytes infected with a recombinant Ad containing a human alpha 1-antitrypsin cDNA (Ad-alpha 1AT) synthesized and secreted human alpha 1AT for 4 weeks. In rats, in vivo intraportal administration of a recombinant Ad containing the E. coli lacZ gene, was followed by expression of beta-galactosidase in hepatocytes 3 days after infection. Intraportal infusion of Ad-alpha 1AT produced detectable serum levels of human alpha 1AT for 4 weeks. Thus, targeted gene expression has been achieved in the liver, albeit at low levels, suggesting that adenovirus vectors may be a useful means for in vivo gene therapy in liver disorders.

Widespread long-term gene transfer to mouse skeletal muscles and heart.

Successful treatment of muscular disorders awaits an adapted gene delivery protocol. The clinically applicable technique used for hematopoietic cells which is centered around implantation of retrovirally modified cells may not prove sufficient for a reversal of phenotype when muscle diseases are concerned. We report here efficient, long-term in vivo gene transfer throughout mouse skeletal and cardiac muscles after intravenous administration of a recombinant adenovirus. This simple, direct procedure raises the possibility that muscular degenerative diseases might one day be treatable by gene therapy.

Adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells.

To evaluate the feasibility of using a replication-deficient recombinant adenovirus to transfer human genes to the human endothelium, human umbilical vein endothelial cells were infected in vitro with adenovirus vectors containing the lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA. After in vitro infection with the lacZ adenovirus vector, cultured endothelial cells expressed beta-galactosidase. In parallel studies with the alpha 1AT adenovirus vector, infected cells expressed human alpha 1AT transcripts, as evidenced by in situ hybridization and Northern analysis, and de novo synthesized and secreted glycosylated, functional alpha 1AT within 6 hr of infection, as shown by [35S]methionine labeling and immunoprecipitation. Quantification of the culture supernatants demonstrated 0.3-0.6 micrograms of human alpha 1AT secreted per 10(6) cells in 24 hr, for at least 14 days after adenovirus vector infection. To demonstrate the feasibility of direct transfer of genes into endothelial cells in human blood vessels, lacZ or alpha 1AT adenovirus vectors were placed in the lumen of intact human umbilical veins ex vivo. Histologic evaluation of the veins after 24 hr demonstrated transfer and expression of the lacZ gene specifically to the endothelium. alpha 1AT adenovirus infection resulted both in expression of alpha 1AT transcripts in the endothelium and in de novo synthesis and secretion of alpha 1AT. Quantification of alpha 1AT in the vein perfusates showed average levels of 13 micrograms/ml after 24 hr. These observations strongly support the feasibility of in vivo human gene transfer to the endothelium mediated by replication-deficient adenovirus vectors.

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium.

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.

Feasibility of adenovirus-mediated gene transfer in vivo.

We have evaluated the use of Adenovirus as a new vector for gene transfer in vivo. The capacity of this virus to express a foreign gene even in the absence of viral and cellular replication makes it attractive to deliver a gene to quiescent cells. We show here that organs like lung, liver and muscles are relevant to this strategy.