Transplantation to the rat brain of human neural progenitors that were genetically modified using adenoviruses.

Transplantations for neurological disorders are limited by the supply of human fetal tissue. To generate larger numbers of cells of appropriate phenotype, we investigated whether human neural progenitors expanded in vitro could be modified with recombinant adenoviruses. Strong expression of beta-galactosidase was obtained in vitro. Two or three weeks after transplantation of engineered cells to the rat brain, we observed a small percentage of surviving neuroblasts strongly expressing beta-galactosidase in four out of 13 rats. Thus human precursor cells that have been genetically modified using adenoviruses are a promising tool for ex vivo gene therapy of neurodegenerative diseases.

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.

Long-term correction of mouse dystrophic degeneration by adenovirus-mediated transfer of a minidystrophin gene.

Duchene muscular dystrophy (DMD) is a fatal progressive X-linked muscle disorder, caused by mutations in the dystrophin gene. We have investigated adenovirus-mediated transfer of a dystrophin minigene in a mutant mouse lacking dystrophin, the mdx mouse. We report here that six months after a single intramuscular injection of a recombinant adenovirus containing a human dystrophin minigene, a large number of dystrophin-positive fibres are still detected in the injected muscles. Moreover, although the minigene encodes a truncated protein, its expression is able to protect the fibres efficiently against the degeneration process that affects the dystrophin-deficient mdx myofibres.

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.

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.

Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo.

Cytotoxic T lymphocytes and natural killer cells are essential effectors of anti-tumor immune responses in vivo. Dendritic cells (DC) 'prime' tumor antigen-specific cytotoxic T lymphocytes; thus, we investigated whether DC might also trigger the innate, NK cell-mediated anti-tumor immunity. In mice with MHC class I-negative tumors, adoptively transferred- or Flt3 ligand-expanded DC promoted NK cell-dependent anti-tumor effects. In vitro studies demonstrated a cell-to-cell contact between DC and resting NK cells that resulted in a substantial increase in both NK cell cytolytic activity and IFN-gamma production. Thus, DC are involved in the interaction between innate and adaptive immune responses.

Efficient delivery of angiostatin K1-5 into tumors following insertion of an NGR peptide into adenovirus capsid.

Adenovirus (Ad)-mediated delivery of anti-angiogenic molecules into tumors constitutes an appealing approach for growth inhibition. However, lack of expression on tumors of Ad receptors leads to weak tumor transduction. Therefore, to provide Ad with a new entry pathway into tumors, an NGR peptide was inserted into either fiber (AdFNGR) or hexon (AdHNGR) capsid proteins. This strategy provided Ad with a very efficient entry pathway in both endothelial cells and tumor cells, with the highest efficacy observed for AdHNGR. Using pharmacological, biochemical and genetic approaches, AdHNGR and AdFNGR were shown to bind not only to CD13 receptor, but also to alphavbeta3 integrins. Both vectors were efficient tools to deliver angiostatin K1-5 cDNA into endothelial cells, thus leading to a dramatic inhibition of their proliferation and increased cell death. Although AdHNGR and Adwt were found to display similar gene transduction efficacy in Lewis lung carcinoma (LLC), pseudotyping AdHNGR with an Ad3-fiber unmasked the ability of NGR-peptide to target these tumors. As a result, delivery of angiostatin K1-5 cDNA into highly aggressive tumors translated into a stronger inhibition of their growth. Altogether, our results suggest that NGR-bearing Ad are valuable tools to realize the potential of this anti-angiogenic approach to anti-tumor therapy.

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.

Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo.

The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.

Reduction of brain metastases in plasminogen activator inhibitor-1-deficient mice with transgenic ocular tumors.

Plasminogen activator inhibitor-1 is known to play a paradoxical positive role in tumor angiogenesis, but its contribution to metastatic spread remains unclear. We studied the impact of plasminogen activator inhibitor (PAI)-1 deficiency in a transgenic mouse model of ocular tumors originating from retinal epithelial cells and leading to brain metastasis (TRP-1/SV40 Tag mice). PAI-1 deficiency did not affect primary tumor growth or vascularization, but was associated with a smaller number of brain metastases. Brain metastases were found to be differentially distributed between the two genotypes. PAI-1-deficient mice displayed mostly secondary foci expanding from local optic nerve infiltration, whereas wild-type animals displayed more disseminated nodules in the scissura and meningeal spaces. SuperArray GEarray analyses aimed at detecting molecules potentially compensating for PAI-1 deficiency demonstrated an increase in fibroblast growth factor-1 (FGF-1) gene expression in primary tumors, which was confirmed by reverse transcription-polymerase chain reaction and western blotting. Our data provide the first evidence of a key role for PAI-1 in a spontaneous model of metastasis and suggest that angiogenic factors, such as FGF-1, may be important for primary tumor growth and may compensate for the absence of PAI-1. They identify PAI-1 and FGF-1 as important targets for combined antitumor strategies.

Canstatin gene electrotransfer combined with radiotherapy: preclinical trials for cancer treatment.

Given as a prophylactic treatment, a single muscle electrogene transfer of plasmid coding canstatin fused to human serum albumin (CanHSA), slowed down the development of two xenografted human carcinomas from mammary (MDA-MB-231) and prostate origin (PC-3) in nude mice and delayed lung metastatic spreading of B16F10 melanoma cells in syngenic mice. No effect was observed with unfused canstatin. The long lasting circulating blood level of CanHSA (20 ng ml(-1)) resulted in a profound disorganization of the tumor blood vessel network. However, when used as a curative treatment, on well-established tumors, CanHSA electrogenetherapy was ineffective in reducing tumor growth. As radiation is known to increase the alpha v beta3 and alpha v beta5 integrins, which are canstatin receptors, to extend the use of CanHSA electrogenetherapy, as a curative treatment, we explored the combination of CanHSA and ionizing radiation. We demonstrated a better efficacy (P=0.01) of the bitherapy over irradiation alone, as a result of strong vessel disorganization and dramatic increase of tumor cells apoptosis. This extremely simple virus free curative protocol could open the door to potential clinical applications, especially for prostate cancer that often develops radioresistance.

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.

Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.

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.

Low-efficiency of percutaneous adenovirus-mediated arterial gene transfer in the atherosclerotic rabbit.

Recombinant adenoviruses are the most efficient vectors with which to perform arterial gene transfer. Previous in vivo studies of adenovirus-mediated arterial transfection, however, have been performed using normal or endothelium-denuded arteries. It is unclear whether these results can be extended to atherosclerotic arteries. Accordingly, this study was designed to (a) assess the feasibility of adenovirus-mediated gene transfer to atherosclerotic lesions, and (b) compare the transfection efficiency, anatomic distribution of transfected cells, and duration of transgene expression achieved in normal versus atherosclerotic arteries. A recombinant adenovirus including a nuclear-targeted beta-galactosidase gene was percutaneously delivered to the iliac artery of normal (n = 25) and atherosclerotic (n = 25) rabbits. Transgene expression, assessed by morphometric as well as chemiluminescent analyses, was documented in all normal and atherosclerotic arteries between 3 and 14 d after gene transfer, but was undetectable at later time points. Transfected cells were identified as smooth muscle cells located in the media of normal arteries, and in the neointima and the vasa-vasora of atherosclerotic arteries. Two percent of medial cells, but only 0.2% of medial and neointimal cells expressed the transgene in normal and atherosclerotic arteries, respectively (P = 0.0001). Similarly, nuclear beta-galactosidase activity was higher in normal than in atherosclerotic arteries (3.2 vs. 0.8 mU/mg protein, P = 0.02). These findings indicate that atherosclerosis reduces the transfection efficiency which can be achieved with adenoviral vectors, and thus constitutes a potential limitation to adenovirus-based, arterial gene therapy.

A gene transfer comparative study of HSA-conjugated antiangiogenic factors in a transgenic mouse model of metastatic ocular cancer.

Different antiangiogenic and antimetastatic recombinant adenoviruses were tested in a transgenic mouse model of metastatic ocular cancer (TRP1/SV40 Tag transgenic mice), which is a highly aggressive tumor, developed from the pigmented epithelium of the retina. These vectors, encoding amino-terminal fragments of urokinase plasminogen activator (ATF), angiostatin Kringles (K1-3), endostatin (ES) and canstatin (Can) coupled to human serum albumin (HSA) were injected to assess their metastatic and antiangiogenic activities in our model. Compared to AdCO1 control group, AdATF-HSA did not significantly reduce metastatic growth. In contrast, mice treated with AdK1-3-HSA, AdES-HSA and AdCan-HSA displayed significantly smaller metastases (1.19+/-1.19, 0.87+/-1.5, 0.43+/-0.56 vs controls 4.04+/-5.12 mm3). Moreover, a stronger inhibition of metastatic growth was obtained with AdCan-HSA than with AdK1-3-HSA (P=0.04). Median survival was improved by 4 weeks. A close correlation was observed between the effects of these viruses on metastatic growth and their capacity to inhibit tumor angiogenesis. Our study indicates that systemic antiangiogenic factors production by recombinant adenoviruses, particularly Can, might represent an effective way of delaying metastatic growth via inhibition of angiogenesis.

Phase I study of a recombinant adenovirus-mediated gene transfer in lung cancer patients.

BACKGROUND: Despite vigorous efforts at curbing tobacco consumption and aggressive combined-modality treatment programs, both the incidence of and the mortality from lung cancer have remained virtually unchanged in the last 10 years. More effective innovative therapies are clearly needed. The direct transfer into tumor cells of tumor suppressor genes or toxic gene products that specifically promote tumor cell death and spare nonmalignant cells is a potentially novel anticancer treatment approach that should be investigated. PURPOSE: On the basis of compelling preclinical data, we initiated a phase I study involving six patients with inoperable lung cancer and an endobronchial lesion accessible by bronchoscopy. Our purpose was to evaluate the feasibility, tolerance, and clinical, biologic, and immunologic effects of the intratumoral administration of a recombinant, replication-deficient adenovirus (rAd.RSV beta-gal), using the Rous sarcoma virus promoter to drive transcription of the Escherichia coli lacZ marker gene that encodes for the bacterial enzyme beta-galactosidase (beta-gal). METHODS: From June 1994 through April 1995, six patients (five males and one female) were enrolled in the trial. A single dose of recombinant virus suspension containing 10(7) or 10(8) plaque-forming units (PFU) was injected intratumorally into two successive cohorts of three patients. Eligible patients received concomitant chemotherapy. Patients were kept under isolation conditions from 3 days before the injection was given until virus excretion was undetectable. Biopsy specimens of the tumor and surrounding mucosa were collected on the 8th day and at 1, 2, and 3 months after injection. They were analyzed by cell culture, polymerase chain reaction (PCR), and beta-gal expression for the presence of recombinant adenovirus. So that the risk of virus recombination or complementation could be minimized, wildtype adenovirus carriers among the hospital staff (identified by PCR) were excluded from contact with patients who were potentially excreting recombinant virus. RESULTS: beta-gal was expressed in tumor biopsy specimens of three patients (one who received the 10(7) PFU dose level and two who received 10(8)). Bronchoalveolar lavage specimens collected immediately after injection were positive for recombinant adenovirus when analyzed in culture and by PCR. All biologic fluids were negative for recombinant virus as judged by PCR after day 12, with the exception of bronchoalveolar lavage specimens (positive PCR up to 90 days in two of three patients treated with 10(8) PFU). The blood samples obtained from the three patients treated with 10(8) PFU showed positive PCR results immediately after virus injection. Patients were kept in isolation for a median of 17 days. The most common toxic effects were moderate bleeding (occurring in two patients) during bronchoscopy and fever (seen in four patients). Endoscopic and clinically objective antitumor responses were seen in four patients, including one patient who showed a complete response by pathologic evaluation. The median survival for the patients was 12.5 months (range, 3-16+ months). Throughout the study, hospital staff remained negative for recombinant adenovirus infection. CONCLUSIONS: This ongoing phase I study has demonstrated that a recombinant adenovirus-mediated marker gene, such as rAd.RSV beta-gal, can be safely introduced into humans and that the gene product is expressed by lung tumor cells of the host.

BRCA1 and BRCA2 are necessary for the transcription-coupled repair of the oxidative 8-oxoguanine lesion in human cells.

The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are likely to participate in DNA lesion processing. Oxidative lesions, such as 8-oxoguanine, occur in DNA after endogenous or exogenous oxidative stress. We show that deficiency for either BRCA1 or BRCA2 in human cancer cells leads to a block of the RNA polymerase II transcription machinery at the 8-oxoguanine site and impairs the transcription-coupled repair of the lesion, leading to a high mutation rate. Expression of wild-type BRCA1 from a recombinant adenovirus fully complements the repair defect in BRCA1-deficient cells. These results represent the first demonstration of the essential contribution of BRCA1 and BRCA2 gene products in the repair of the 8-oxoguanine oxidative damage specifically located on the transcribed strand in human cells. This suggests that cells from individuals predisposed to breast and/or ovarian cancer may undergo a high rate of mutations because of the deficiency of this damage repair pathway after oxidative stress.

Adenovirus-mediated transfer of the thyroid sodium/iodide symporter gene into tumors for a targeted radiotherapy.

The Na+/I- symporter (NIS) present in the membranes of thyroid cells is responsible for the capacity of the thyroid to concentrate iodide. This allows treatment of thyroid cancers with 131I. We propose to enlarge this therapeutic strategy to nonthyroid tumors by using an adenoviral vector to deliver the NIS gene into the tumor cells. We constructed a recombinant adenovirus encoding the rat NIS gene under the control of the cytomegalovirus promoter (AdNIS). Infection of SiHa cells (human cervix tumor cells) with AdNIS resulted in perchlorate-sensitive 125I uptake by these cells to a level 125-225 times higher than that in noninfected cells. Similar results were obtained for other human tumor cell lines, including MCF7 and T-47D (mammary gland), DU 145 and PC-3 (prostate), A549 (lung), and HT-29 (colon), demonstrating that the AdNIS vector can function in tumor cells of various origins. In addition, AdNIS-infected tumor cells were selectively killed by exposure to 131I, as revealed by clonogenic assays. To assess the efficiency of this cancer gene therapy strategy in vivo, we injected the AdNIS vector in human tumors (SiHa or MCF7 cells) established s.c. in nude mice. Immunohistological analysis confirmed the expression of the NIS protein in the tumor. Three days after intratumoral injection, AdNIS-treated tumors could specifically accumulate 125I or 123I, as revealed by kinetics and imaging experiments. A quantitative analysis demonstrated that the uptake in AdNIS-injected tumors was 4-25 times higher than that in nontreated tumors. On average, 11% of the total amount of injected 125I could be recovered per gram of AdNIS-treated tumor tissue. Altogether, these data indicate that AdNIS is very efficient in triggering significant iodide uptake by a tumor, outlining the potential of this novel cancer gene therapy approach for a targeted radiotherapy.

Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis.

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.