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At least 577 records · Page 32Linked to original sources
PubMed2006

Novel gene-directed enzyme prodrug therapies against prostate cancer.

There is no effective cure for late-stage hormone (androgen) refractory prostate cancer. Although chemotherapy offers palliation to these late-stage patients, it also leads to systemic toxicities leading to poor quality of life. Clearly, the focus is on the development and evaluation of novel biologically relevant alternatives such as cytoreductive gene-directed enzyme prodrug therapy (GDEPT). With the current limitations of effective gene delivery in vivo, the in situ amplification of cytotoxicity due to bystander effects of GDEPT has special attraction for patients with prostate cancer, the prostate being dispensable. This review focuses on the development, application and potential of various GDEPTs for treating prostate cancer. The current status of research related to the issues of enhancement of in situ GDEPT delivery and prostate cancer-specific targeting of vectors (especially viral vectors) is assessed. Finally, the scope and progress of synergies between GDEPT and other treatment modalities, both traditional and alternate, are discussed.

Animals↗
PubMed2002

Phagocytosis of synthetic particulate vaccine delivery systems to program dendritic cells.

Therapeutic prospects of particulates are increasingly recognized for vaccination purposes. Compared with biologic particulates, such as live or attenuated bacterial vectors and viral vectors, synthetic particulates may be expected to ease the hurdles of quality assurance and validation in vaccine development and production and shorten the time for approval and to the market. The ability of synthetic antigen-loaded particulates to elicit strong immune responses, even with low amounts of antigen and to weakly immunogenic epitopes, is suggested to be due to their efficient cross-talk with the most potent antigen-presenting cells, such as dendritic cells. Moreover, the potential of particulates for intracellular delivery and directing intracellular trafficking of antigens has evolved as a promising opportunity to target the major histocompatibility complex I pathway. In summary, synthetic particulate vaccine delivery systems are likely to play an increasingly active role in enhancing or even enabling the immunostimulating effect of antigens upon direct interaction with the target cells.

Adjuvants, Immunologic↗
PubMed2004

[Single cell DNA analysis: possibilities, methods, implications in relation to stem cell therapy].

In the analysis of circulating tumor cells or in the preimplantation genetic diagnosis it is frequently necessary to examine one single cell. Some methods are appropriate to isolate single cells: Laser Assisted Microdissection, magnetic cell separation or FACS. The use of the whole genome amplification methods are needful, because the amount of the DNA extracted from the isolated cells is very low and inappropriate for additional examinations. With different molecular biological methods (e.g. sequencing, chip-technology) it is possible to determine genetic alterations in the analysed cells, and to return the modified cells with in vitro gene technological methods (viral vectors, non viral methods). Our aim is to summarize the methods and the possible technical problems developing during the process of the single cell molecular biological analysis.

DNA Mutational Analysis↗
PubMed2006

Viral and non-viral methods for gene transfer into skeletal muscle.

Skeletal muscle is an important target for genetic manipulation and its stable post-mitotic nature allows the use of both integrating and non-integrating viral and non-viral vectors. Adeno-associated viral vectors and naked plasmid DNA are currently the vectors of choice for gene transfer into muscle. The last couple of years have seen major breakthroughs in the field of vector delivery systems, particularly those using the vascular route, such that gene therapy of muscular dystrophies and the use of muscle as a platform for the production of secreted proteins has become a clinical possibility.

Animals↗
PubMed1996

Gene transfer in hepatocarcinoma cell lines: in vitro optimization of a virus-free system.

Many approaches exist for hepatic gene delivery, including viral vectors and non-viral vectors. In this study, we tested a panel of liposomes to transfer pAGO, a plasmid containing one copy of herpes simplex virus (HSVtk) gene, and pYED11, a plasmid containing two copies of the HSVtk gene, into a murine hepatocarcinoma cell line (Hepa 1-6) and a human hepatocarcinoma cell line (Hep-G2). The efficiency of gene delivery and expression was characterized by beta-galactosidase staining, flow cytometric analysis and quantitative lacZ activity. Different combinations of liposomes and DNA and the ratio of the concentration of liposome to DNA were tested. The efficient transfer was shown with DOTAP followed by transfectam and lipofectamine. Under these conditions, we tested the cytotoxicity of ganciclovir (GCV) exposure on Hepa 1-6 and Hep-G2 transfected separately with liposome-pAGO and liposome-pYED11 complexes. This study demonstrates the in vitro efficacy of each liposome tested to transduce the HSVtk gene into hepatocarcinoma cell lines. The transfer of two copies of the HSVtk gene rendered cells 1.5 times more sensitive to GCV than cells transduced by pAGO as compared to controls. This was achieved most efficiently by the DOTAP-pYED11 complex. Thus, pYED11 may be considered as an alternative to pAGO as a gene transfer vector.

Animals↗
PubMed2005

Site-specific integration with phiC31 integrase for prolonged expression of therapeutic genes.

Need of a site-specific integrating vector in gene therapy has become pressing, as recent work has shown that many of the current integrating vectors used preferentially integrate in the vicinity of genes. A site-specific integrating vector would reduce the risk of insertional mutagenesis posed by randomly integrating vectors, and a non-viral vector would reduce the safety and immunogenicity problems associated with viral vectors. The phiC31 integrase is a protein from Streptomyces phage phiC31 that has been developed as a non-viral site-specific gene therapy vector. The phiC31 integrase catalyzes the integration of a plasmid containing attB into pseudo attP sites in mammalian genomes. It has been shown to function in tissue culture cells as well as in mice. Vectors based on the phiC31 integrase were able to treat tyrosinemia type I in a mouse model and two forms of epidermolysis bullosa in keratinocytes from patients, demonstrating its effectiveness as a gene therapy vector. Development of phiC31 integrase-based vectors is still underway, but it has already been shown to provide long-term expression through site-specific integration.

Animals↗
PubMed2006

Toxicity of cationic lipids and cationic polymers in gene delivery.

Gene therapy, as a promising therapeutics to treat genetic or acquired diseases, has achieved exciting development in the past two decades. Appropriate gene vectors can be crucial for gene transfer. Cationic lipids and polymers, the most important non-viral vectors, have many advantages over viral ones as non-immunogenic, easy to produce and not oncogenic. They hold the promise to replace viral vectors to be used in clinic. However, the toxicity is still an obstacle to the application of non-viral vectors to gene therapy. For overcoming the problem, many new cationic compounds have been developed. This article provides a review with respect to toxicity of cationic lipids and polymers in gene delivery. We evaluate the structural features of cationic compounds and summarize the relationship of toxicity and structure and hope to provide available suggestions on the development of these cationic compounds.

Animals↗
PubMed2006

Immobilization of gene vectors on polyurethane surfaces using a monoclonal antibody for localized gene delivery.

BACKGROUND: Conventional strategies of gene therapy using viral vectors result in suboptimal localization and potentially dangerous distal spread of vector. We hypothesized that localized delivery of adenoviral gene vectors could be achieved from a polyurethane (PU) film through a mechanism involving anti-viral antibody tethering. METHODS: PU films were formulated with a collagen coating. Anti-adenoviral monoclonal antibodies were covalently bound to the collagen surface. These antibodies enabled tethering of replication-defective adenoviruses [Ad-GFP (encoding green fluorescent protein)] through highly specific antigen-antibody affinity. The binding stability and in vitro delivery of virus bound on PU films were investigated. Cell culture studies with rat arterial smooth muscle cells (A10) assessed transduction on or near the PU matrix. In vivo experiments with collagen-coated PU films investigated atrial epicardial implant and subdermal implant models in Yorkshire swine. RESULTS: We report for the first time successful PU film-based gene delivery using antibody-tethered adenovirus encoding the green fluorescent protein (GFP), demonstrating efficient and highly localized gene delivery to arterial smooth muscle cells in cell culture and pig implant. In comparison, direct injections of viral vectors into subcutaneous sites gave sparse, needle-track-oriented GFP expression patterns. CONCLUSION: We conclude that PU film is a suitable platform for a localizable viral vector delivery system that also prevents systemic spread of vector. Gene delivery using PU film-based anti-viral antibody tethering of vectors should be suitable for a wide array of single or multiple therapeutic gene strategies, and for further device-based gene delivery therapeutic strategies.

Adenoviridae↗
PubMedDate not supplied

Analysis of recombination in mammalian cells using SV40 and SV40-derived vectors.

Viruses and viral vectors have played a crucial role in our understanding of the pathways of homologous and non-homologous recombination in mitotically dividing mammalian cells. In particular, they have allowed the confirmation of the preponderance of non-homologous over homologous recombination events and led to schemes for the selection and isolation of homologous recombination products. These studies have allowed an examination of the properties of reciprocal and non-reciprocal homologous recombination events extrachromosomally, in the chromosome and between plasmids and chromosomes. They suggest that it is feasible now to direct DNA segments to predetermined chromosomal locations by homologous recombination.

Animals↗
PubMed2005

Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs.

Viral vectors available for gene therapy are either inefficient or suffer from safety concerns for human applications. Foamy viruses are non-pathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. In this report, we describe the use of simian foamy virus type 1 (SFV-1) vector to examine the efficacy of therapeutic genes. Hairpin short-interfering RNA (siRNA) that targets the simian immunodeficiency virus (SIV) rev/env was placed under the control of the PolIII U6 snRNA promoter for expression and screened for silencing target genes using cognate target-reporter fusions. We have identified an effective siRNA (designated R2) which reduces the rev and env gene expression by 89% and 95%, respectively. Using the simian foamy virus type 1 (SFV-1) based vector, we delivered the PolIII expressed R2 siRNA into cultured cells and challenged with SIV. The results show that the R2 siRNA is a potent inhibitor of SIV replication as determined by p27 expression and reverse transcriptase assays. Vectors based on a non-pathogenic SFV-1 vector may provide a safe and efficient alternative to currently available vectors, and the SIV model will help devise protocols for effective anti-HIV gene therapy.

Animals↗
PubMed1997

Gene transfer into vascular cells using adeno-associated virus (AAV) vectors.

OBJECTIVES: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial beta-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. METHODS: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0 x 10(5) to 1.0 x 10(7). Expression of beta-galactosidase (beta-gal) in VSMC was evaluated by X-gal staining and a beta-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of beta-gal in the aortic segments was evaluated by X-gal staining. RESULTS: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the beta-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. CONCLUSIONS: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.

Aorta, Thoracic↗
PubMed2001

Introduction to retroviruses and retroviral vectors.

As various viral vector systems for gene transfer are developed, interest in using such systems in applied settings continues to grow. This Chapter is designed to provide background information for readers interested in learning about lentiviral vector systems for gene transfer applications but who lack a background in retrovirology. To assist those readers who are unfamiliar with retroviral vector systems, basic outlines of the retroviral replication cycle and of characteristics of retroviral vector systems are introduced here in order to present and define concepts and terms that are discussed in subsequent Chapters.

Gene Transfer Techniques↗
PubMed2006

Efficient immunization of rhesus macaques with an HCV candidate vaccine by heterologous priming-boosting with novel adenoviral vectors based on different serotypes.

Efficient vaccination against viral agents requires a strong T-cell-mediated immune response to clear viral-infected cells. Optimal vaccination can be achieved by administration of recombinant viral vectors encoding phatogen antigens. Adenoviral vectors have attracted considerable attention as potential viral vectors for genetic vaccination owing to their favorable safety profile and potent transduction efficiency following intramuscular injection. However, the neutralizing antibody response against adenoviral capsid proteins following adenoviral vectors injection limits the success of vaccination protocols based on multiple administrations of the same adenoviral serotype. In this work, we describe efficient immunization of rhesus macaques, the preferred model for preclinical assessment, with an HCV candidate vaccine by heterologous priming-boosting with adenoviral vectors based on different serotypes. The induced responses are broad and show significant cross-strain reactivity. Boosting can be delayed for over 2 years after priming, indicating that there is long-term maintenance of resting memory cells.

Adenoviridae↗
PubMed2001

Recent developments in retroviral gene delivery systems.

Since the first gene therapy clinical trial carried out by Rosenberg et al. in 1990 [1], recombinant retroviral vectors are still the most popular gene delivery tools for gene therapy purposes. According to the databases of the Journal of Gene Medicine [101] 35% of gene therapy protocols employ retroviruses, other vectors such as adenoviral vectors are used in 27%, pox viral vectors in 6%, adeno-associated viral vectors in 2% and herpes simplex based vectors in 0.5% of clinical trials. It has become clear that the early retroviral vectors based on simple retroviruses (e.g., murine leukaemia viruses (MLV) are characterised by relatively low viral titre, low transduction efficiency and difficulties in in vivo administration. On the other hand, lentiviral vectors (complex retroviruses) can be grown to a high titre, can stably transduce non-dividing cells and can effectively deliver genes to human progenitor cells (CD34+). This article aims to summarise the first year of the 21st century's developments in the retroviral gene delivery system.

Genetic Vectors↗
PubMed2006

Non-viral ocular gene therapy: potential ocular therapeutic avenues.

Non-viral vectors for potential gene replacement and therapy have been developed in order to overcome the drawbacks of viral vectors. The diversity of non-viral vectors allows for a wide range of various products, flexibility of application, ease of use, low-cost of production and enhanced "genomic" safety. Using non-viral strategies, oligonucleotides (ODNs) can be delivered naked (less efficient) or entrapped in cationic lipids, polymers or peptides forming slow release delivery systems, which can be adapted according to the organ targeted and the therapy purposes. Tissue and cell internalization can be further enhanced by changing by physical or chemical means. Moreover, a specific vector can be selected according to disease course and intensity of manifestations fulfilling specific requirements such as the duration of drug release and its level along with cells and tissues specific targeting. From accumulating knowledge and experience, it appears that combination of several non-viral techniques may increase the efficacy and ensure the safety of these evolving and interesting gene therapy strategies.

Animals↗
PubMed1995

Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10.

The application of gene transfer techniques to organ transplantation offers the potential for modulation of immunity directly within an allograft without systemic side effects. Expression vectors and promoter elements are important determinants of gene transfer and expression. In this study, various vectors (naked plasmid DNA, retroviral vector, herpes simplex viral vector, and adenoviral vector) with various promoters (RSV-LTR, SV40, MuLV-LTR, HCMVie1) were directly compared to demonstrate the successful gene transfer and expression of beta-galactosidase in murine myoblasts in vitro and within murine heterotopic, nonvascularized cardiac isografts or allografts in vivo. Expression of transferred genes was not toxic to cells and strength of expression varied according to the type of vector. Plasmid DNA was expressed in myocytes, retroviral vector was expressed in the graft infiltrating cells, and herpes simplex and adenoviral vectors were expressed in both myocytes and graft-infiltrating cells. Preliminary studies evaluated the ability of these vectors to deliver immunologically important signals. Allografts injected with pSVTGF-beta 1, a plasmid-encoding transforming growth factor beta 1 (TGF-beta 1) under the control of the SV40 promoter, showed significant prolongation of graft survival of 26.3 +/- 2.5 days compared with 12.6 +/- 1.1 days for untreated allografts, and 12.5 +/- 1.5 days for the allografts injected with control plasmid (P < 0.05). Allografts injected with MFG-vIL-10, a retroviral vector encoding viral interleukin-10 under the control of the MuLV-LTR, showed prolongation of graft survival of 36.7 +/- 1.3 days versus 12.6 +/- 1.1 days for the untreated allograft, and 13.5 +/- 2.0 days for the allografts injected with control retroviral vector (P < 0.001). Both vectors were transcriptionally active in vivo and did not appear to have toxic effects. Gene therapy for transplantation can induce transient expression of immunologically relevant molecules within allografts that impede immune activation while avoiding the systemic toxicity of conventional immunosuppression.

Animals↗
PubMed2003

Molecular therapeutics of HBV.

The hepatitis B virus (HBV) infection is a public health problem worldwide, particularly in East Asia. The current therapy of HBV infection is mostly based on chemical agents and cytokines that have been shown to provide limited efficacy and are also toxic to the human body. Gene therapy is a new therapeutic strategy against HBV infection, involving the transmission of gene drugs into liver cells by specific delivery systems and methods. Although this new anti-HBV infection technique is under active investigation, various promising anti-HBV viral gene drugs have been developed for gene therapy, including antisense RNA and DNA, hammerhead ribozymes, dominant negative HBV core mutants, single chain antibody, co-nuclease fusion protein, and antigen. In order to optimize their antiviral effects and/or enhance anti-HBV immunity, various novel gene delivery systems have also been developed to (specifically) deliver such DNA constructs into liver cells; some of them are viral vectors, such as adenoviral vectors, retroviral vectors and poxviral vectors, and even hepatitis B viral for its hepatocellular specificity. Others are non-viral vectors, in which naked DNA and liposomes are frequently used for DNA vaccine or nucleotide analogs for inhibiting HBV DNA polymerase. This review addresses various aspects of gene therapy for HBV infection, including gene drugs, delivery methods, animal model, and liver transplantation with combination therapy. It also discusses the problems that remain to be solved.

Animals↗
PubMed2005

Packaging of heterologous RNAs by a minimal bovine leukemia virus RNA packaging signal into virus particles.

A minimal bovine leukemia virus (BLV) RNA packaging sequence (E) required for heterologous RNAs to be packaged into BLV particles was analyzed. The BLV E was inserted into a non-viral vector, pLacZ, in order to determine if packaging of the non-viral vector RNA would occur. The construct was transfected into cells chronically infected with BLV in order to produce virus particles. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of viral RNA from virus particles revealed that non-viral RNA containing the BLV E was packaged into BLV particles, indicating that the BLV E is necessary and sufficient to allow for packaging of a non-viral vector RNA. We also analyzed the ability of a chimeric murine leukemia virus (MLV) retroviral vector (pLN) containing BLV E to be packaged into BLV particles. Interestingly, it was found that pLNDelta (which does not possess psi+) could be packaged into BLV particles. This indicates that a MLV RNA region outside of psi+ allows for packaging of the MLV RNA into BLV particles.

Animals↗