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PubMed1996

[Gene therapy in oncology: applications to lung cancer].

Gene therapy defines a new therapeutic avenue whose site of action is at the level of the gene itself; viral vectors (adenovirus, retrovirus, herpes virus) or non-viral (liposomes, plasmids) enable the transfer of a fraction of DNA (transgenic) to the target itself. In this review, we present recently acquired data on the mechanisms of oncogenesis and anti-tumor immunity which have enabled the application of several therapeutic strategies in oncology; the transfer of gene(s), coding for cytokines or for coactivation factors in order to develop active immunotherapy; the transfer of suicides genes; the transfer of multidrug resistance gene (MDR1); the transfer of tumor suppressor genes or of cDNA coding for antisense oligonucleotides in order to correct genomic anomalies which are responsible for the malign phenotype. The development of gene therapy demands the resolution of a number of technical difficulties such as vectorisation, targeting, and the expression of the stability of the trans-gene. Phase 1 trials in man have established the innocuity of certain vectors and have confirmed the expression of trans-genes (marker genes). Compared to monogenic hereditary diseases, the "molecular heterogenetic" of bronchial tumours, the consequence of the instability of the genome and the diversity of amplified oncogenes are a major difficulty. In addition, each one of these approaches prevents limiting factors: for example the exclusive targeting of malign cells is an indispensable pre-requisite for the transfer of suicide genes and in the same way the expression the tumour in antigens is the pre-requisite for the development of active immunotherapy. We report the overall results of applied trials for pulmonary carcinomas on murine models and present their applications which are underway in men.

Animals↗
PubMedDate not supplied

Cellular vaccines.

This project is devoted to the development of novel cellular vaccines designed to treat cancer patients. These cellular vaccines present and enhance immunogens, which will elicit a potent immune response. The goal is to achieve safe and effective immune reaction against the patient's own tumour. (1) Autologous cellular vaccines are prepared by processing circulating blood mononuclear cells outside of the patient's body (ex vivo) to differentiate them into antigen-presenting cells (APCs). Monocyte-derived APCs (MD-APCs) are then grown in the presence of exogenous target antigens (tumour cell debris, or apoptotic bodies) to become fully mature APCs. (2) Functionality for antigen presentation to T cells of ex vivo MD-APCs is evaluated in vivo. (3) Cellular vaccines are tested in selected rodent animal models. Efficiency and immune response are monitored in pertinent experimental systems for cancer. Pharmacological data are generated for clinical investigation. Tolerance and biologic effects are documented in primates. (4) The first clinical trials on cancer patients are taking place in 1998 on melanoma and prostate cancer to validate the concept. Specialized cell processors with dedicated software and standardized controls are being developed and used for the preparation of cellular vaccines. (5) The evaluation of new non-viral vectors and the validation of new non-viral transfection methods of mononuclear cells with marker genes is in progress and will lead to the ex vivo transfection of genes coding for immunostimulating cytokines or for tumour antigens in MD-APCs. Efficiency will be validated in vitro and in animal models. The ex vivo and animal model studies validate the clinical relevance of this new cellular immunotechnology. Clinical validation of individual autologous cellular vaccines in specific indications for which no treatment is presently available will allow the development of cellular and gene immunotherapy for other types of cancers.

Animals↗
PubMed1981

Expression of simian virus 40-rat preproinsulin recombinants in monkey kidney cells: use of preproinsulin RNA processing signals.

The complete rat preproinsulin gene I was cloned into a simian virus 30 (SV 40) vector. Most of the late region of the viral vector, including the SV40 intervening sequences (introns) and all of the major splice junctions, was deleted and replaced by the entire rat insulin gene. The recombinant molecules and a temperature-sensitive helper virus (tsA28) were inoculated into monkey kidney cultures. The formation of stable transcripts of the insulin insert was as efficient as the production of late SV40 mRNA. Analysis of these transcripts indicated that the rat preproinsulin gene nucleotide signals involved in RNA splicing and poly(A) addition were used. Examination of the 5' ends of the mRNAs showed several classes, one of which was the same size as the authentic rat insulinoma mRNA. This suggests that a portion of the transcripts may be initiated or processed faithfully, or both, at their 5' ends within rat insulin sequences. Significant quantities of a protein identified as rat proinsulin were synthesized. Detection of most of the proinsulin in the tissue culture medium suggests that this protein was secreted.

Animals↗
PubMed2002

Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: inhibition of primary tumor growth and metastatic spread.

Electroporation-mediated gene transfer relies upon direct delivery of plasmids into cells permeabilized by electric fields, a method more efficient than transfer using nonviral vectors, although neither approaches the transfer efficiency of viral vectors. Here we studied electrotransfer of a gene encoding an angiogenesis inhibitor (endostatin) into primary tumors and muscle tissues, which would serve as a site of synthesis and secretion into the bloodstream of a therapeutic antimetastatic protein with systemic effects. Optimum electroporation conditions (voltage, number and duration of impulses, separation of caliper electrodes) were first established to maximize expression of a reporter gene transferred into murine Renca kidney carcinoma, B16(F10) melanoma, or skeletal muscle tissues. In neoplastic tissues, electrotransfer of plasmid DNA was far more efficient than electroporation with lipoplexes, but no differences between naked DNA and lipoplexes were found in case of electroporated muscles. We then studied the electrotransfer of plasmid DNA carrying the endostatin gene into pre-established experimental Renca tumors. A significant inhibition of tumor growth was observed in animals electroporated with this construct. Electrotransfer of the endostatin gene into muscle tissues resulted in reduced numbers of experimental B16(F10) metastases in the lungs. This study clearly shows that electroporation may be used to efficiently transfer antiangiogenic genes into both normal and neoplastic tissues.

Angiogenesis Inhibitors↗
PubMed1994

Somatic gene therapy for cystic fibrosis.

There is considerable potential to ameliorate the pulmonary disease in cystic fibrosis (CF) using somatic gene therapy. Even low levels of expression of the gene in airways epithelium may be beneficial. Adenoviral vectors, DNA-liposome complexes, adeno-associated viral vectors and DNA-ligand complexes have been used effectively in vitro and have been tested in animals to varying extent. Adenoviral vectors and DNA-liposome complexes are being used to deliver the CF gene to patient airways in phase I clinical trials. Transient correction of the electrophysiological defect in human CF nasal epithelium has been achieved. Major goals are (i) to demonstrate that expression of the CF gene in airways epithelium will ameliorate lung disease in CF patients, and (ii) to achieve long-term expression of the introduced gene either through a single delivery with persistent expression or through the ability to use a delivery system repetitively with safety and efficacy.

Cystic Fibrosis↗
PubMed2001

Prospects and problems of gene therapy: an update.

The gene therapy approach can vary from delivering extra copies of a gene, through modifications of a genome using the properties of ribozymes or chimeraplasts, to injection of modified cells. For the treatment of genetic deficits the ultimate goal would be the repair of the mutated gene in the target tissue(s). The techniques required for such an approach are emerging, albeit slowly. Therefore, delivery of an extra copy of a normal gene in a specific vector remains the predominant approach. Moreover, this method finds wider applications in gene therapy relating to disorders other than heritable defects, e.g., malignancies, cardiovascular diseases and infections. The major and most intensive areas of research are: i) vectors and delivery methods, ii) regulation of transgene expression and iii) stability of expression. Targeting of the therapeutic gene is being accomplished by using viral vectors or non-viral delivery systems, either ex vivo or in vivo. The choice of vectors and delivery routes depends on the nature of the target cells and the required levels and stability of expression. Although there have been the first positive clinical results and significant technical achievements over the past 2 years, there are still obstacles to the development of effective clinical products and these remain largely unchanged. The most important barriers are the low levels and stability of expression and immune responses to vectors and/or gene products. The safety aspects of gene therapy have become painfully evident with the first death conclusively linked to gene therapy. The progress in AAV and lentiviral vectors, improved regulation of transgene expression and advances in stem cell technology are among the recent most exciting developments.

Journal Article↗
PubMed2004

[Gene delivery system based on low molecular weight polyethylenimine and its transfection activity in the skin tissue].

Low molecular weight polyethylenimine (LMW-PEI) was linked to an expressing plasmid contains a green fluorescence protein (GFP) reporter gene and effective gene transfer was observed in CM7221 cell line tested. We examined the relationship among the molecular weight, structure of PEI and their transfection activity and cytotoxicity on CM7221 cell line. We also examined the position and continuance time of the GFP reporter gene expressed in the skin tissue of mouse. Results showed that LMW-PEI/DNA complexes led to high levels of expression in the CM7221 cell line (about 55%). However, with the increasing of PEI molecular weight, the transfection activity of PEI was decreasing. There was an increasing cytotoxicity with the larger PEI molecules. Further research showed that LMW-PEI induced a significant and long-lasting (7 days) expression of the GFP reporter gene in the hair vesicle, sweat, gland, sebaceous gland in the mouse skin tissues. The LMW-PEI described here is a new, highly efficient and non-cytotoxic vector. It would be a useful non-viral vector for gene delivery technology, particular useful as simple skin-specific vehicles of therapeutic genes.

Animals↗
PubMed2002

Oncolytic adenoviruses for treatment of brain tumours.

Standard therapies are not capable of curing patients with malignant glioma; more than 90% of patients die within 2 years after diagnosis. Gene therapy appeared as a promising new approach for this disease. However, results of clinical trials with replication deficient viral vectors were disappointing. The main reasons being poor transduction efficiency of adenovirus towards glioma cells and limited spread and distribution of the vector in the tumour. With the increasing knowledge of viral genetics and its functions, an attractive alternative tool to kill malignant glioma cells has been developed: Replicating adenovirus as an oncolytic agent. This type of therapy, also referred to as virotherapy, has the potential to overcome some of the limitations connected with replication deficient adenoviral vectors. In this review the authors describe the latest developments in strategies that are being used to create a tumour- or glioma-selective replicating adenovirus. Special attention is given to the methods of viral delivery to an infiltrating tumour in the brain, regarding optimal dose and toxicity. Furthermore, the role of conventional antitumour treatments, such as irradiation and chemotherapy, in enhancing the effect of virotherapy is being emphasised.

Adenoviridae↗
PubMed2005

Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector.

This work addresses the problem of efficient control of gene expression in the context of viral vectors, which still represents a difficult challenge. A number of lentiviral vectors incorporating the different elements of regulatable transcriptional systems have been described, but they fail to perform satisfactorily either because of a poor dynamic range of transcription levels or because they display high background activities in the uninduced state and mediocre inducer response. We report here on the systematic comparison of vector designs containing the elements of the doxycycline-inducible Tet-on system in their most advanced versions (rtTA2S-M2 transactivator and tTS(Kid) repressor). We show that a simple "all-in-one" vector can be obtained and used for efficient control of transgene expression in long-term tissue culture and in the hematopoietic system of mice following bone marrow transplantation. Using this vector, the uninduced state can be kept at background levels and induction factors of 100-fold are repeatedly obtained over months both in tissue culture and in vivo. Interestingly, the low background activity of the all-in-one vector renders the use of the tTS repressor dispensable, avoiding the problem of progressive loss of inducibility over time associated with irreversible modifications of the chromatin surrounding proviral sequences.

Animals↗
PubMed2006

[Progress in research of chitosan as a non-viral gene delivery vector].

Efficiency of non-viral gene delivery based on chitosan and chitosan derivatives as DNA condensing carrier is dependent on a series of factors, such as complex size, the charge ratio of chitosan/DNA, molecular mass of chitosan, the degree of chitosan deacetylation, pH and serum concentration of the transfection medium. Through modifying the chitosan in a certain extent, we can change the efficiency of transfection. Studies on transfection condition, efficiency and mechanism using chitosan and chitosan derivatives as transfection agents are reviewed.

Animals↗
PubMed2004

Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein.

BACKGROUND: Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles. METHODS: Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice. RESULTS: High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model. CONCLUSIONS: Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.

Animals↗
PubMed2006

Targeted in utero delivery of a retroviral vector for gene transfer in the rodent brain.

In vivo application of viral vectors for gene transfer is a commonly used tool in anatomical and functional studies, as well as in development of neuroprotective and restorative strategies for therapy. Although the most common route of administration is via direct injection into the brain parenchyma in adult animals, a number of short-term studies have been performed in the developing central nervous system. Here we investigated the long-term transgene expression following in utero delivery of a retroviral vector encoding for the green fluorescent protein (GFP) marker gene at embryonic days 14.5-17.5 using an ultrasound-guided injection system. Intraparenchymal injections of the ganglionic eminence were compared with vector delivery to the intracerebroventricular space. Injections into the ganglionic eminences resulted in a predominantly unilateral transduction localized to the forebrain, giving rise to GFP-positive (GFP+) neurons and astrocytes in the striatum, olfactory bulb, cortex and hippocampus. When the vector was injected into the lateral ventricle, on the other hand, widespread expression of GFP was seen throughout the brain. The total number of GFP+ cells in the striatum was estimated to be between 20,000 and 50,000 cells using a computerized stereological quantification tool. Phenotypic characterization of these transduced cells using confocal microscopical analysis showed that 64% were NeuN+ neurons, 14% APC+ oligodendrocytes and 15% glial cells labelled with GFAP, S100beta and Iba1, when the vector injection was performed at E14.5. Delivery into later embryos resulted in a reduction in neuronal profiles with a reciprocal increase in glial cells.

Age Factors↗
PubMedDate not supplied

Viral-mediated gene transfer to study the molecular physiology of the Mammalian inner ear.

Several classes of viral vectors including adenovirus, adeno-associated virus, herpes simplex virus, lentivirus and vaccinia virus have been reported to infect cells of the inner ears of mammals and may be useful for protein manipulation and therapeutic purposes. We have screened a few of these for use as vectors to mediate gene transfer into the sensory hair cells of organotypic cultures from the neonatal mouse cochlea and utricle. Recombinant, replication-deficient adenovirus has emerged as a useful vector for several reasons: ease of vector generation at high titer; efficient hair cell specific infection; robust expression of reporter genes and minimal toxicity. Previously, we characterized adenovirus infected hair cells using a vector that carried the gene for green fluorescent protein (GFP). We screened GFP-positive cells electrophysiologically and found that although hair cells survive adenoviral vector infection, their mechanosensitivity was compromised. Until recently this has limited the scope of adenovirus application to the problems of inner ear physiology and pathophysiology. However, a modified adenoviral vector, now available, has been reported to have reduced ototoxicity in vivo. The modifications include the deletion of the adenoviral genes E1, E3, the viral polymerase, and the preterminal protein. We are currently working to characterize viral-mediated gene transfer into hair cells of the cultured mouse utricle using this new modified adenoviral vector. We have found that hair cells infected with the modified vector have intact hair bundles and robust mechanotransduction.

Adenoviridae↗
PubMed1997

Gene therapy: panacea or placebo? I. Strategies and limitations of gene therapy.

Progress in genetics and molecular medicine has led to characterization of many disorders at the level of specific genes. Techniques for reliable diagnosis of these disorders have been developed in parallel. Attempts are currently being made to develop DNA-based therapeutic procedures to correct genetic diseases. These procedures are known under a common name of gene therapy. The initial step in gene therapy is the delivery of the gene of interest into target cells. There are several conceptually different approaches to achieve this, e.g. targeting can be accomplished by using viral vectors (transduction) or DNA-mediated routes (transformation) either ex vivo or in vivo. The selection of vector systems and the choice of delivering genes either into isolated cells or directly in the organism depend on factors like: the nature of target cell or organ, the levels of expression required, stability and/or regulation of expression, safety, etc. Many viruses have been adapted for use as vectors for gene therapy, according to their specific properties. Viral genomes have been modified to remove their ability to replicate and to increase the cloning capacity. Non-viral gene delivery systems rely on cellular mechanisms to import DNA into the cell nucleus and different methods to enhance DNA uptake have been attempted. Despire many significant achievements there are still obstacles to the development of effective clinical products. Most significant are the low levels and stability of expression of introduced genes and immune responses to vectors and/or gene products.

Animals↗
PubMed2006

Effect of medium sugar source on the production of retroviral vectors for gene therapy.

The production of gene therapy retroviral vectors presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high titers of infectious viral vectors. The objective of this work is to increase the production titers of retroviral vectors by manipulating the sugar carbon sources used in bioreaction. Four sugars were tested (glucose, galactose, sorbitol, and fructose) on an established Moloney murine leukemia virus (MoMLV) producer cell line. Galactose and sorbitol did not support cell growth or vector production. Glucose supplemented at 25 mM supported the highest cell growth; however, the use of glucose or fructose at 83 and 140 mM have shown to improve the infectious vector titer three to fourfold. The reasons for the titer improvements were further analyzed and, although, the cell-specific productivity in viral transgene RNA and reverse transcriptase were augmented 5- and 6-fold for glucose at 140 mM and 14- and 16-fold for fructose at 140 mM, comparing with glucose at 25 mM, these increases did not seem sufficient to account for the 14- (140 mM glucose) and 32- (140 mM fructose) fold increment obtained for the infectious particles-specific productivity. Further accounting the enhancement in the titers was the improvement in the viral stability, the half-life of the vectors was enhanced by 30-60%. This resulted in a product quality with a superior ratio of infectious to total particles, thus reducing the most problematic contaminant in the production of retroviral vectors, non-infectious retroviral particles.

Carbohydrates↗
PubMed2005

Biocompatible polymer enhances the in vitro and in vivo transfection efficiency of HVJ envelope vector.

BACKGROUND: Vector development is critical for the advancement of human gene therapy. However, the use of viral vectors raises many safety concerns and most non-viral methods are less efficient for gene transfer. One of the breakthroughs in vector technology is the combination of the vector with various polymers. METHODS: HVJ (hemagglutinating virus of Japan) envelope vector (HVJ-E) has been developed as a versatile gene transfer vector. In this study, we combined HVJ-E with cationized gelatin to make it a more powerful tool and assessed its transfection efficiency in vitro and in vivo. In addition, we investigated the mechanism of the gene transfer by means of the inhibition of fusion or endocytosis. RESULTS: The combination of both protamine sulfate and cationized gelatin with HVJ-E, referred to as PS-CG-HVJ-E, further enhanced the in vitro transfection efficiency. In CT26 cells, the luciferase gene expression of PS-CG-HVJ-E was approximately 10 times higher than that of the combination of protamine sulfate with HVJ-E or the combination of cationized gelatin with HVJ-E, referred to as PS-HVJ-E or CG-HVJ-E, respectively. Furthermore, the luciferase gene expression in liver mediated by intravenous administration of CG-HVJ-E was much higher than the luciferase gene expression mediated by PS-HVJ-E or PS-CG-HVJ-E and approximately 100 times higher than that mediated by HVJ-E alone. CONCLUSIONS: Cationized gelatin-conjugated HVJ-E enhanced gene transfection efficiency both in vitro and in vivo. These results suggest that low molecular weight cationized gelatin may be appropriate for complex formation with various envelope viruses, such as retrovirus, herpes virus and HIV.

Androstadienes↗
PubMed2000

Liver bypass significantly increases the transduction efficiency of recombinant adenoviral vectors in the lung, intestine, and kidney.

Recombinant adenoviruses have great potential as gene delivery systems because of their ability to infect a wide range of target cells. However, systemic delivery of viral vectors to tissues other than liver and spleen has been inefficient because of the rapid clearance of the circulating virus by the liver. In the present study we tested the hypothesis that a systemic administration of E1-deleted recombinant adenovirus vectors that bypasses the hepatic circulation will lead to enhanced expression of these vectors in extrahepatic tissues. The portal vein and hepatic artery in B6/129 F1 mice were clamped and an E1-deleted recombinant adenovirus carrying the beta-galactosidase gene (Ad.CBlacZ) was then administered through the retroorbital venous plexus. The clamp was released 30 min after viral injection with no major chronic ischemic consequences noted. High levels of LacZ expression were detected predominantly in the vessels and capillaries of the lung, intestinal wall, and renal glomeruli 7 days after viral infusion. The transgene expression persisted for at least 21 days. Intense LacZ staining was also observed in the liver, suggesting that liver infection occurred after the portal clamp was released. A retroorbital infusion of anti-adenovirus neutralizing antibodies 5 min before the release of the portal clamp significantly reduced postclamp viral infection to the liver, while LacZ expression in lung and intestine persisted after the antibody treatment. Taken together, these results suggest that liver bypass can significantly improve the transduction efficiency in the other target organs. This method could be used to develop animal models of human diseases that predominantly affect the vessels of the lung, intestine, and kidney.

Adenoviridae↗
PubMed2000

SV40-based gene therapy vectors: turning an adversary into a friend.

For gene delivery to be of use, a situation suitable for delivery of genetic material, a specific genetic construct to be delivered and the appropriate means to deliver it are required. Simian virus-40 (SV40) gene therapy vectors for gene transfer may be an important advance in the latter category. While other vectors are variably limited for example by immunogenicity, difficulties in production, restricted specificity, low titers, poor transduction efficiency, etc., recombinant viral vectors based on SV40 (rSV40) should not be similarly constrained. They are easily manipulated and produced at very high titer, stable, apparently lacking in immunogenicity, and capable of providing sustained high levels of transgene expression in almost any cell type, whether resting or dividing. The major limitation of SV40-derived vectors is packaging capacity, which restricts insert sizes. The rationale for developing SV40 as a gene therapy vector is reviewed, based on what is known of wild-type SV40. Studies with rSV40 gene transfer have focused mostly on hematopoietic progenitor cells (CD34+) and their derivatives, and on gene delivery to the liver. In both settings, in vitro and in vivo, SV40 has been very effective. It is therefore a highly promising gene delivery vehicle that may complement other vectors that are currently in use or that are being developed.

Animals↗