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PubMed2002

Alphaviral gene transfer in neurobiology.

Semliki Forest virus (SFV), Sindbis virus (SIN), and Venezuelan equine encephalitis virus are simple, enveloped plus-strand RNA viruses belonging to the Alphavirus genus of the Togaviridae family. They have been developed into expression vectors that infect a wide host cell range and cause rapid and high-level transgene expression. Their easy and fast generation, classification into biosafety levels 1 and 2, and preferential transduction of neurons in cell and tissue cultures makes them an increasingly used gene transfer system. This review summarizes the alphaviral replication and expression, the replicon system, and its application in neurobiology. Alphaviral vectors can introduce multiple transgenes into host cells, and mutants with low or absent cytotoxicity and increased or decreased transgene expression levels are available. Temperature-dependent mutants permit to control the host cell specificity as well as the on- and offset of gene expression. These features, together with the transduction characteristics revealed in a direct comparison of alphaviral and other viral vectors in hippocampal slice cultures, make SFV and SIN vectors a powerful tool for neurobiological studies.

Alphavirus↗
PubMed2003

Gene transfer strategies to inhibit neointima formation.

Vascular smooth muscle cell (VSMC) proliferation after arterial injury results in neointima formation and plays an important role in the pathogenesis of restenosis after angioplasty, in-stent restenosis, vascular bypass graft occlusion, and allograft vasculopathy. Major progress has been made recently in elucidating the cellular and molecular mechanisms underlying neointima formation. However, no known curative treatment currently exists. In cases in which pharmacologic and surgical interventions have had limited success, gene therapy remains a potential strategy for the treatment of such vascular proliferative diseases. To date, recombinant adenoviral vectors continue to be the most efficient methods of gene transfer into the arterial wall. However, concerns over the safety of using viral vectors in a clinical situation have inspired the considerable progress that has been made in improving both viral and nonviral modes of gene transfer. This review discusses some of the recent insights and outstanding progress in vascular gene therapeutic approaches to inhibit neointima both from a biologic and therapeutic perspective.

Animals↗
PubMed2001

"Stealth" adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung.

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy.

Adenoviruses, Human↗
PubMed2005

Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV.

Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. This study examines the safety and immunogenicity of a replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) in BALB/c mice and rhesus monkeys. This novel Ad5/35 vector showed minimal hepatotoxicity after intramuscular administration with the novel Ad5/35 vector. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use.

AIDS Vaccines↗
PubMed2005

Increased utility in the CNS of a powerful neuron-specific tetracycline-regulatable adenoviral system developed using a post-transcriptional enhancer.

BACKGROUND: In previous studies we have found that the tetracycline (Tet)-regulatable system functions best in recombinant adenoviral (Ad) vectors when the Tet transactivators and the Tet-regulatable element (TRE) are incorporated into separate viral vectors. However, such a dual vector system is disadvantaged by the need to use relatively high titres that may elicit an immune response. Therefore, to develop a system that could be used at low titres while mediating strong, tightly regulatable gene expression in the central nervous system (CNS), we incorporated the woodchuck hepatitis virus post-transcriptional enhancer (WPRE) into a neuron-specific Tet-regulatable Ad system. METHODS: The WPRE was incorporated into Ad vectors encoding the Tet-Off (tTA) transactivator driven by the synapsin-1 and CMV promoters and encoding the TRE driving EGFP expression (TRE)-EGFP. RESULTS: The addition of the WPRE to the neuron-specific Tet-regulatable system mediated a greater than three-fold increase in transgene expression in primary hippocampal neurons with no loss of gene regulation. The results also showed that the addition of the WPRE enhanced transgene expression in the CNS without the loss of neuron specificity and without affecting the ability to regulate transgene expression. CONCLUSIONS: We have further developed a tetracycline-regulatable neuron-specific expression system such that it can now be used at low titres with no loss of transgene expression or ability to regulate transgene expression. It should therefore be of significant value to studies investigating neuronal gene function and to those seeking to develop effective neuronal gene therapy strategies.

Adenoviridae↗
PubMed2000

The construction of defective interfering rubella virus particles.

Two defective viral vectors containing nucleic acid sequences from rubella virus strains RJ and RS linked to the reporter gene luciferase have been constructed. The vector RNAs are shown to replicate and interfere with wildtype virus in co-infected cells. The interference is associated with a polymorphic nucleotide at nt34 in the 5' stem-loop. The use of these constructs as expression vectors is discussed.

Animals↗
PubMed2005

Nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery.

Nanotechnology, although not a new concept, has gained significant momentum in recent years. This stems partly from the realization that nanosystems have significantly different biological properties from large-sized systems (e.g. implants or microparticles) that could be used effectively to overcome problems in drug and gene therapy. In drug therapy, we face the problems of inefficacy or nonspecific effects; hence, nanosystems are being developed for targeted drug therapy. In gene therapy using non-viral systems, the main issues are relatively transient gene expression and lower efficiency than viral vectors. Research efforts have focused on understanding the barriers in gene delivery so that non-viral systems can be developed that are as effective as viral systems in gene transfection. Understanding the molecular mechanisms that underlie the interactions of nanosystems with the cell, their uptake properties and retention will be crucial for the successful development of these systems.

Animals↗
PubMed2004

Powerful and controllable angiogenesis by using gene-modified cells expressing human hepatocyte growth factor and thymidine kinase.

OBJECTIVES: This study investigated the possibility of achieving angiogenesis by using gene-modified cells as a vector. BACKGROUND: Although gene therapy for peripheral circulation disorders has been studied intensively, the plasmid or viral vectors have been associated with several disadvantages, including unreliable transfection and uncontrollable gene expression. METHODS: Human hepatocyte growth factor (hHGF) and thymidine kinase (TK) expression plasmids were serially transfected into NIH3T3 cells, and permanent transfectants were selected (NIH3T3 + hHGF + TK). Unilateral hindlimb ischemia was surgically induced in BALB/c nude mice, and cells were transplanted into the thigh muscles. All effects were assessed at four weeks. RESULTS: The messenger ribonucleic acid expression and protein production of hHGF were confirmed. Assay of growth inhibition by ganciclovir revealed that the 50% (median) inhibitory concentration of NIH3T3 + hHGF + TK was 1000 times lower than that of NIH3T3 + hHGF. The NIH3T3 + hHGF + TK group had a higher laser Doppler blood perfusion index, higher microvessel density, wider microvessel diameter, and lower rate of hindlimb necrosis, as compared with the plasmid- and adenovirus-mediated hHGF transfection groups or the NIH3T3 group. The newly developed microvessels were accompanied by smooth muscle cells, as well as endothelial cells, indicating that they were on the arteriolar or venular level. Laser Doppler monitoring showed that the rate of blood perfusion could be controlled by oral administration of ganciclovir. The transplanted cells completely disappeared in response to ganciclovir administration for four weeks. CONCLUSIONS: Gene-modified cell transplantation therapy induced strong angiogenesis and collateral vessel formation that could be controlled externally with ganciclovir.

3T3 Cells↗
PubMed2005

Pluronic Block Copolymers for Gene Delivery.

Amphiphilic block copolymers of poly(ethylene oxide) and poly(propylene oxide) called Pluronic or poloxamer are commercially available pharmaceutical excipients. They recently attracted considerable attention in gene delivery applications. First, they were shown to increase the transfection with adenovirus and lentivirus vectors. Second, they were shown to increase expression of genes delivered into cells using non-viral vectors. Third, the conjugates of Pluronic with polycations, were used as DNA-condensing agents to form polyplexes. Finally, it was demonstrated that they can increase regional expression of the naked DNA after its injection in the skeletal and cardiac muscles or tumor. Therefore, there is substantial evidence that Pluronic block copolymers can improve gene expression with different delivery routes and different types of vectors, including naked DNA. These results and possible mechanisms of Pluronic effects are discussed. At least in some cases, Pluronic can act as biological adjuvants by activating selected signaling pathways, such as NF-kappaB, and upregulating the transcription of the genes.

Journal Article↗
PubMed2006

Apoptosis mediated by lentiviral TRAIL transfer involves transduction-dependent and -independent effects.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, which selectively induces apoptosis in many transformed cells without apparent toxic side effects in normal tissue. We recently described the construction and characterization of a lentiviral vector for expression of TRAIL. In this report, we evaluate its suitability for therapeutic application. In vitro, we observed specific induction of apoptosis upon transduction in human lung cancer cells. Cell death was partially dependent on successful integration and TRAIL expression by the vectors, but was to some extent mediated by protein carryover, as we found TRAIL protein associated with virus particles. Transduction of subcutaneously growing lung tumors on nude mice with lentiviral TRAIL mediated a transient suppression of tumor growth. Analysis of tumor sections revealed that transduction efficiency of lentiviral control vector but not of lentiviral TRAIL vector was high. This was because of the direct cytotoxic activity of recombinant TRAIL present in viral particles, which prevented efficient tumor transduction. These data therefore suggest that enveloped viral vectors constitutively expressing TRAIL are well suited for ex vivo applications, such as the transduction of tumor-homing cells, but may have a lower effect when used directly for the transduction of tumor cells in vivo.

Animals↗
PubMedDate not supplied

Gene transfer for the therapy of erectile dysfunction: progress in the 21st century.

Gene transfer represents the next potential era of advancement in medicine for the prevention of the effects of aging or for treatment of genetic or acquired disease. For gene transfer to be a practical successor to today's oral and minimally invasive therapies, the product must have a high safety profile and a long duration of effectiveness to correct the need for on-demand administration. Several types of vectors have been used in preclinicals studies, but because of widely publicized adverse events, progress using viral vectors in humans has been limited. There is a current phase I human trial using naked DNA as the vector with the maxi-K gene to modify cellular contractility. Preliminary results in the safety trial thus far have shown no treatment-related adverse events, no transfer to the semen, and the possibility of efficacy in one participant.

Animals↗
PubMed2001

[Feasibility of inner ear gene transfer after middle ear administration of an adenovirus vector].

BACKGROUND: Several groups demonstrated in animal experiments that gene transfer is a feasible tool for inner ear intervention. Various approaches for inoculation of vectors have been successfully used for inner ear gene therapy. One possible way to reduce the risk of hearing loss following the opening of the cochlea for application of the vector into the perilymphatic space is to deliver vectors through the round window. This study was designed to determine whether middle ear application of an adenoviral vector is a feasible approach to inoculate vectors and lead to transduction of cells in the inner ear. METHODS: A unilateral middle ear application of an adenoviral vector was performed in 4 guinea pigs directly on the round window membrane (RWM) and in 4 additional animals by placing a cotton patch soaked with the vector solution on the RWM. The expression of a reporter gene (lacZ) was used to localize vector-transduced cells. RESULTS: Only one out of 8 animals showed cochlear expression of the reporter gene, whereas all 8 animals showed strong lacZ expression in the middle ear mucosa, in the RWM and in the mucosa surrounding the stapes. CONCLUSION: Our results indicate that the RWM presents a close barrier, almost completely preventing the adenovirus to diffuse into the perilymphatic space. Therefore middle ear application of an adenoviral vector cannot be used to induce inner ear gene transfer. However, middle ear application of a viral vector may be useful for developing treatment for diseases of the middle ear mucosa.

Adenoviruses, Human↗
PubMed1997

Gene therapy for cancer: what have we done and where are we going?

Gene-based therapies for cancer in clinical trials include strategies that involve augmentation of immunotherapeutic and chemotherapeutic approaches. These strategies include ex vivo and in vivo cytokine gene transfer, drug sensitization with genes for prodrug delivery, and the use of drug-resistance genes for bone marrow protection from high-dose chemotherapy. Inactivation of oncogene expression and gene replacement for tumor suppressor genes are among the strategies for targeting the underlying genetic lesions in the cancer cell. A review of clinical trial results to date, primarily in patients with very advanced cancers refractory to conventional treatments, indicates that these treatments can mediate tumor regression with acceptably low toxicity. Vector development remains a critical area for future research. Important areas for future research include modifying viral vectors to reduce toxicity and immunogenicity, increasing the transduction efficiency of nonviral vectors, enhancing vector targeting and specificity, regulating gene expression, and identifying synergies between gene-based agents and other cancer therapeutics.

Clinical Trials as Topic↗
PubMed2003

Targeting genetically modified macrophages to the glomerulus.

Macrophages are key players in the development of the majority of renal diseases and are therefore ideal cellular vectors for site specifically targeting gene therapy to inflamed glomeruli. Macrophages can be genetically modified using viral vectors ex vivo then re-introduced into the body where they can home to the diseased site. This review summarises current experience in efficiently targeting modified macrophages to the inflamed glomerulus focussing on the factors controlling macrophage localisation, macrophage gene transfer methods, in vivo gene delivery and results of recent investigations using modified macrophage gene therapy for glomerular disease.

Adenoviridae↗
PubMed2005

Pluronic block copolymers for gene delivery.

Amphiphilic block copolymers of poly(ethylene oxide) and poly(propylene oxide) called Pluronic or poloxamer are commercially available pharmaceutical excipients. They recently attracted considerable attention in gene delivery applications. First, they were shown to increase the transfection with adenovirus and lentivirus vectors. Second, they were shown to increase expression of genes delivered into cells using non-viral vectors. Third, the conjugates of Pluronic with polycations, were used as DNA-condensing agents to form polyplexes. Finally, it was demonstrated that they can increase regional expression of the naked DNA after its injection in the skeletal and cardiac muscles or tumor. Therefore, there is substantial evidence that Pluronic block copolymers can improve gene expression with different delivery routes and different types of vectors, including naked DNA. These results and possible mechanisms of Pluronic effects are discussed. At least in some cases, Pluronic can act as biological adjuvants by activating selected signaling pathways, such as NF-kappaB, and upregulating the transcription of the genes.

DNA↗
PubMed2005

HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector.

BACKGROUND: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4) and monocyte tropic (R5) viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. RESULTS: To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. CONCLUSIONS: Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

Journal Article↗
PubMed1999

Adenoviral nerve growth factor and beta-galactosidase transfer to spinal cord: a behavioral and histological analysis.

OBJECT: The present study characterizes the time course and loci of gene expression induced by the administration of adenoviral vectors into spinal cord. Although a marked inflammatory response to these vectors occurred, no effect on spinal cord function was seen in the 1st postoperative week. The expression of transgenic genes delivered by viral vectors is being exploited throughout the nervous system. The present study utilized adenoviral vectors containing the Rous sarcoma virus (RSV) promoter and a nuclear localization signal to achieve transgenic expression in mammalian spinal cord. METHODS: Initial experiments utilizing the vector Ad.RSVlacZ (10(12) particles/ml) injected into the region of the central canal resulted in viral gene expression stretching over approximately 1.2 cm of spinal cord. Gene expression was first detected 3 days following viral administration and lasted until postinjection Day 14 with peak expression at Day 7. A variety of cell types in both white and gray matter expressed lacZ. Transgenic expression of the neurotrophin nerve growth factor (NGF) was achieved using injections of Ad.RSVNGF. On histological examination mononuclear inflammatory infiltrate and gliosis were revealed surrounding the injection sites of spinal cords receiving adenovirus but not vehicle. To assess spinal cord function during viral gene expression, animals previously trained in an operant runway task were tested at 7 days postinjection (the peak of viral gene expression) and demonstrated no changes in spinal cord function. CONCLUSIONS: Results of this study using adenoviral neurotrophic gene transfer indicate that it provided an effective tool for the delivery of potentially therapeutic proteins to the injured or diseased spinal cord.

Adenoviridae↗
PubMed2003

Complete tumor prevention by engineered tumor cell vaccines employing nonviral vectors.

We report that 100% mice survival after tumor challenge is achieved with cytokine-engineered cells employing nonviral lipoplexes and without using viral vectors. We describe this effect with cytokine-secreting tumor cell vaccines, based on cell clones or fresh transfected cells. Tumor cells were transfected with murine granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-4 plasmids employing the cationic lipid DOTAP, were irradiated (150 Gy) and kept frozen until use. The transfection efficacy was analyzed by qRT-PCR and flow cytometry. Vaccination induced potent antitumor rejection, resulting in 100% mice survival. Furthermore, the antitumor immunity was long lasting, since a two-fold survival delay was observed in mice after tumor rechallenge (6 months later). While cell clones secreting GM-CSF were the most effective in wild-type tumor cell rejection, little or no effect was observed with clones secreting IL-4. We found similar antitumor efficacy employing fresh transfected cells by nonviral procedures, demonstrating that cells genetically modified by nonviral vectors (both clones and fresh transfected cells) are a safe and efficient tool for antitumor vaccines. These vaccines allow us to achieve the highest antitumor efficacy based on nonviral gene therapy techniques. In addition, the vaccination success with fresh transfected cells simplifies the procedure and provides new insights into the clinical application of nonviral gene therapy procedures.

Animals↗