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At least 505 records · Page 28Linked to original sources
PubMed2004

Nonviral gene therapy approaches to hemophilia.

The goal of hemophilia gene therapy is to obtain long-term therapeutic levels of factor VIII (FVIII) or factor IX (FIX) without stimulating an immune response against the transgene product or the vector. The success of gene therapy is largely dependent on the development of appropriate gene delivery vectors. Both viral vectors and nonviral vectors have been considered for the development of hemophilia gene therapy. In general, viral vectors are far more efficient than nonviral gene delivery approaches and resulted in long-term therapeutic levels of FVIII or FIX in preclinical animal models. However, there are several reasons why a nonviral treatment would still be desirable, particularly because some viral vectors are associated with inflammatory reactions, that render transgene expression transient, or with an increased risk of insertional oncogenesis when random integrating vectors are used. Nonviral vectors may obviate some of these concerns. Since nonviral vectors are typically assembled in cell-free systems from well-defined components, they have significant manufacturing advantages over viral vectors. The continued development of improved nonviral gene delivery approaches offers new perspectives for gene therapy of chronic diseases including hemophilia.

Animals↗
PubMed1998

Neuron-specific transduction in the rat septohippocampal or nigrostriatal pathway by recombinant adeno-associated virus vectors.

Viral vector-mediated gene transfer in brain can provide a means for gene therapy and functional studies. However, robust and persistent transgene expression in specific populations of the adult brain has been difficult to achieve. In an attempt to produce localized and persistent transduction in rat brain, we compared recombinant adeno-associated virus (rAAV) vectors incorporating either the immediate early cytomegalovirus (CMV) promoter or the neuron-specific enolase (NSE) promoter. Transduction in hippocampus resulting from the NSE promoter-containing construct was more efficient and persistent than that resulting from the CMV promoter-containing construct. Most hippocampal cells transduced with the NSE promoter had multipolar neuron morphology. Neurons with glutamatergic morphology were transduced weakly. In order to produce a local supply of neurotrophic factor to cells that degenerate under certain disease and experimental conditions, the NSE promoter was utilized to drive expression of brain-derived neurotrophic factor (BDNF) in medial septum or substantia nigra. In this construct, the NSE promoter drives dicistronic expression of BDNF and an enhanced version of green fluorescent protein (GFP). We estimated 3000-15,000 GFP-positive cells per injection of rAAV into septum or substantia nigra, a transduction ratio of 5-20 infectious virus particles per transduced cell. This frequency may be sufficient for trophic factor gene therapy as well as for investigating specific protein function in "topical (i.e., localized) transgenic" animals produced by rAAV.

Animals↗
PubMed2006

pH-responsive shielding of non-viral gene vectors.

PEG shielding of non-viral vectors reduces undesired interactions with the extracellular environment. Combination with cell-binding domains enables in vivo targeting via specific attachment to the target cells. Pegylation, however, also interferes with effective intracellular nucleic acid delivery. Consistently triggered removal of the PEG shield after reaching the target cell would make non-viral vectors more compatible with the intracellular delivery steps. Physiological triggers may include changes in pH, enzyme concentration or redox potential. This review focuses on pH-sensitive shielding strategies that exploit the endosomal acidification process after endocytosis for deshielding of the delivery system.

Drug Delivery Systems↗
PubMed2002

Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells.

Non-viral gene transfer into a wide range of human cells was examined in order to clarify the factors that affect the efficiency and safety of non-viral vectors and to optimize the conditions so that high efficiency and low toxicity could be achieved. Six non-viral vectors (Lipofectin, LipofectAMINE PLUS, SuperFect, Effectene, DMRIE-C and DOTAP) were used to transfect a mammalian expression plasmid pCMVbeta into 16 types of human primary cells and cultured cell lines. Transfection efficiency was quantified using a galactosidase assay. Cytotoxic effects were measured by lactate dehydrogenase (LDH) assay and WST-8 assay. In serum-free conditions, LipofectAMINE PLUS, Effectene and SuperFect, on average, transfected DNA more successfully than Lipofectin, DMRIE-C, and DOTAP, although the levels of gene expression with these vectors varied remarkably in different cells. The most effective vector also differed depending on the cell type. Serum was found to inhibit gene transfer and reduce the cytotoxicity of all of these vectors except Effectene. The efficiency and toxicity of the non-viral vectors used depended on the type of vector, the DNA/vector ratio, the type of cell, and the presence of serum. These results provided useful information for the optimization of transfer conditions of these non-viral vectors.

Cell Line↗
PubMed2001

Gene therapy for pulmonary diseases.

Gene therapy for pulmonary disease has attracted a great deal of attention since the first report of successful gene delivery 10 years ago. Potential indications for gene therapy include chronic illnesses such as cystic fibrosis and alpha(1)-antitrypsin deficiency, and acute illnesses such as acute transplant rejection and chemotherapy-induced lung injury. The key technological impediment to successful gene therapy is vector optimization. Viral vectors, including adenovirus and adeno-associated virus, have relatively low efficiency in vivo. In addition, adenovirus has been associated with a brisk inflammatory response and limited duration of expression in the lung. Nonviral vectors, particularly liposomes, have also been tried, with limited expression efficiency and some toxicity. Although work is ongoing to improve adenoviral and adeno-associated viral vectors and test other viral and nonviral vectors, an ideal vector has not yet been identified. Several important barriers to successful gene therapy, including the host inflammatory response, promotor down-regulation, tissue-specific targeting, and physical barriers to gene delivery in the airway, will need to be overcome. Despite these daunting problems, several human gene therapy trials have been completed, using adenovirus, adeno-associated virus, and liposomes. In general, these trials have been focused on safety, and have shown that there is dose-dependent inflammation in response to adenovirus. Adeno-associated virus appears to cause little inflammation. Demonstration of successful gene delivery and transcription has been quite variable in human trials. In general, the level of expression of transgene appears to be quite low. In summary, although there is great promise for gene therapy in the lung, significant challenges remain in translating this technology to successful human therapy.

Adenoviridae↗
PubMed2002

Progress and challenges in viral vector-mediated gene transfer to the brain.

Gene transfer into the brain allows the manipulation of transgene expression in both time and space. Recently developed gene transfer technologies allow transgenes to be expressed in any anatomically, biochemically or functionally distinct group of brain cells. Gene transfer has been used to alter the expression of neurotransmitter receptors, ion channels, signaling proteins, neuronal growth, differentiation and survival factors, and thus to modify brain anatomy, neuron physiology, behavior and pathology. However, challenges remain in making gene therapy a more widespread tool for the treatment of neurological disease. We have identified the following as areas needing development: access and delivery of viral vectors to the brain; diffusion of viral vectors and transgenes throughout large areas of brain tissue; viral vector side effects and toxicity, inflammatory and immune responses to vectors; long-term stable transgene expression; cell type-specific expression of transgenes; and the ability of the experimenter or physician to switch transgene expression 'on' and 'off' at will. In the last year, neuro-gene therapy has shown that brain defects in experimental disease models can be prevented and corrected, and that viral vectors and encoded transgenes can be made to diffuse over larger brain areas. In addition, the cause of vector-induced inflammation and immune responses have begun to be elucidated, so that rational approaches can be developed to avoid these complications. Further improvements in viral vectors will facilitate clinical trials in the near future.

Adenoviridae↗
PubMed2004

Sustained gene transfer and enhanced cell death following glucosylated-PEI-mediated p53 gene transfer with photochemical internalisation in p53-mutated head and neck carcinoma cells.

p53 is frequently mutated in head-and-neck squamous cell carcinoma. Wild-type p53 gene transfer induces apoptosis in vitro and tumor regression in vivo and clinical investigations of p53 gene therapy have been reported, mostly using viral vectors. Non-viral vectors are increasingly being used as an alternative to viral vectors and photochemical internalisation (PCI) of non-viral vectors has been reported to yield high gene transfer efficiency. The p53-mutated status of FaDu human pharynx carcinoma cell line was first assessed by DNA sequencing and the cells were transfected using tetraglucosylated polyethylenimine (PEI-Glu4) in conjunction with photochemical internalisation (PCI). The green fluorescent protein (GFP) was used as a reporter for determination of the transgene expression kinetics with or without PCI. p53 gene transfer was performed in these optimised conditions, and subsequent induction of apoptosis was investigated by flow cytometric determination of the phosphatidylserine externalization. Long-term cell death was assessed using colony forming assays. DNA sequencing in FaDu cells showed a G/T point mutation at codon 248 in exon 7 of p53 gene, resulting in an arginine-to-leucine substitution. As a consequence, P53 was shown to be expressed in >90% of untreated cells using immunocytochemistry. Using PEI-Glu4 as vector, PCI was found to significantly enhance GFP gene transfer whatever the formulation solution. Transfection efficiency was significantly increased with PCI. GFP expression kinetics (24-144 h) demonstrates that PCI induces sustained transgene expression with >10% of cells remaining transfected after 144 h. In such conditions, p53 gene transfer using PEI-Glu4 and PCI, resulted in spontaneous induction of apoptosis. As a consequence, long-term cell death was significantly enhanced after wt-p53 gene transfer when PCI was used, reaching up to 50% cell death. Wild-type p53 gene transfer using PEI-Glu4/DNA complexes and PCI, yields sustained transgene expression and induces cell death in p53-mutated FaDu cells.

Apoptosis↗
PubMed2004

Conception and realization of a non-cationic non-viral DNA vector.

Cationic non-viral DNA vectors are very successful in in vitro transfections but less efficient in in vivo tests. This seems mainly due to the cationic nature of the molecules used to complex DNA. In this article, we describe the design and the route towards the realization of a non-viral non-cationic vector. The strategy follows three steps: first, the incorporation of DNA to a lamellar phase; second, the making of multilamellar vesicles containing a high loading of DNA by shearing the lamellar phase and, finally, the grafting of peptides onto the surface of the vesicles to target a specific receptor on the cells. Throughout this process, we had to overcome many obstacles; this review describes the present state of our work and summarizes the remaining steps.

Animals↗
PubMed2024

Adeno-Associated Virus Engineering and Load Strategy for Tropism Modification, Immune Evasion and Enhanced Transgene Expression.

Gene therapy aims to add, replace or turn off genes to help treat disease. To date, the US Food and Drug Administration (FDA) has approved 14 gene therapy products. With the increasing interest in gene therapy, feasible gene delivery vectors are necessary for inserting new genes into cells. There are different kinds of gene delivery vectors including viral vectors like lentivirus, adenovirus, retrovirus, adeno-associated virus et al, and non-viral vectors like naked DNA, lipid vectors, polymer nanoparticles, exosomes et al, with viruses being the most commonly used. Among them, the most concerned vector is adeno-associated virus (AAV) because of its safety, natural ability to efficiently deliver gene into cells and sustained transgene expression in multiple tissues. In addition, the AAV genome can be engineered to generate recombinant AAV (rAAV) containing transgene sequences of interest and has been proven to be a safe gene vector. Recently, rAAV vectors have been approved for the treatment of various rare diseases. Despite these approvals, some major limitations of rAAV remain, namely nonspecific tissue targeting and host immune response. Additional problems include neutralizing antibodies that block transgene delivery, a finite transgene packaging capacity, high viral titer used for per dose and high cost. To deal with these challenges, several techniques have been developed. Based on differences in engineering methods, this review proposes three strategies: gene engineering-based capsid modification (capsid modification), capsid surface tethering through chemical conjugation (surface tethering), and other formulations loaded with AAV (virus load). In addition, the major advantages and limitations encountered in rAAV engineering strategies are summarized.

Dependovirus↗
PubMed1997

A packaging system for SV40 vectors without viral coding sequences.

SV40 vectors have been used as expression vectors for mammalian cells since the early 1980s. More recently, they have been used as gene transfer vectors in mice and in human peripheral blood cells. Here we described a system for packaging SV40 vectors without viral coding sequences. Recombinant adenovirus-expressing SV40 capsids can effectively package plasmids that contain the SV40 replication origin. The final yield of infectious SV40 vector is about 3 x 10(5), with a SV40 to adenoviral vector ratio of about 1000:1. Helper adenoviruses can be effectively heat-inactivated with no effect on the infectivity of SV40 vectors.

Adenoviridae↗
PubMed1999

Swinepox virus as a vaccine vector for swine pathogens.

Several small and large viruses (e.g., adenovirus, poxvirus, and herpesviruses) have been investigated as vaccine vectors. Each viral system has its advantages and disadvantages. One major advantage for viral vector vaccines is their ability to elicit a protective cell-mediated immunity as well as a humoral response to the antigen delivered by the vector. One major problem to using recombinant viruses as vaccines is the pathogenic potential of the parent virus. Therefore, it is important that along with the optimal expression of the foreign genes and ability to provide protection, the pathogenicity of the vector virus must be reduced during genetic manipulation without affecting its multiplication. The requirements to develop a viral vector, for example, swinepox virus, are a cell culture system that will support the growth of the virus, a suitable nonessential region(s) in the virus genome for insertion of foreign DNA so that virus replication is not affected, a foreign gene(s) that encodes for an immunogenic protein of a swine pathogen, strong transcriptional regulatory elements (promoters) necessary for optimal expression of the foreign genes, a procedure for delivering the foreign gene(s) into the nonessential locus, and a convenient method of distinguishing the recombinant viruses from the parent wild-type virus. Using this methodology, recombinant swinepox virus vaccines expressing pseudorabies virus antigens have been developed and shown to provide protection against challenge. These studies and evidence of local infection of the oral tract by swinepox virus indicate its potential as a recombinant vector for providing immunity against various swine pathogens including those that infect the respiratory and gastrointestinal tracts.

Animals↗
PubMedDate not supplied

In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers.

BACKGROUND: Gene transfer into the airways could be of importance for the treatment of chronic lung diseases such as cystic fibrosis. In the past few years several attempts have been made to effectively deliver DNA to the lung using different viral and non-viral vector systems. Viral vectors and cationic lipids have been tested intensively but the properties of cationic polymers such as polyethylenimine (PEI) 25 kDa and fractured polyamidoamine dendrimers to deliver DNA to the airways have not been studied. Surfactant preparations have been shown to influence pulmonary adenoviral and naked plasmid DNA mediated gene transfer in vivo. We investigated the gene delivery efficiency of branched PEI 25 kDa and fractured dendrimers to the murine lung in vivo and also examined the effect of surfactant on PEI 25 kDa mediated gene transfer to the lung. METHODS: Cationic polymer/DNA complexes were prepared in 25 mM HEPES buffer (pH = 7.4) or double distilled water and administered to the lungs of BALB/c mice via cannula intubation. The trachea, left and right lung, heart, liver and esophagus were examined for luciferase activity. Inflammation was assessed by performing standard histology. RESULTS: PEI/DNA complexes showed a high level of luciferase gene expression in the lung. Complexes formed in double distilled water exhibited higher gene expression than complexes formed in 25 mM HEPES buffer (pH 7.4). The optimal N/P ratio was found to be N/P = 10 in double distilled water. Luciferase activity was only detected in the lung and decreased rapidly in a time-dependent manner. The addition of a natural surfactant preparation, Alveofact, slightly reduced gene transfer of branched PEI 25 kDa. Luciferase gene expression obtained by using fractured dendrimers was very low. CONCLUSION: The present study demonstrates that PEI 25 kDa, but not polyamidoamine dendrimers, effectively mediates transient gene transfer to the murine lung after intratracheal intubation. In conclusion, branched PEI 25 kDa was found to be an effective vector for pulmonary gene delivery in vivo, being superior to fractured dendrimers.

Animals↗
PubMed2003

Therapeutic angiogenesis. Devising new strategies based on past experiences.

Therapeutic angiogenesis improves tissue ischemia by supplementing and supporting the intrinsic process of angiogenesis. Besides direct administration of angiogenic growth factor proteins, injection of naked DNA, non-viral vectors and viral vector constructs carrying angiogenic genes have been used. A novel approach is to achieve therapeutic angiogenesis through cell mediated gene transfer. Genetically modulated cells carrying exogenous genes encoding for angiogenic factors and the cells with inherent ability to secrete angiogenic cytokines, such as bone marrow stem cells, embryonic stem cells and endothelial progenitor cells, have been used to achieve revascularization. This review discusses proof of the concept pre-clinical studies and phase-I/II human trials using VEGF, and cellular angiogenesis at length in the light of the literature and analyzes the problems and considerations of these approaches as a treatment strategy in the clinical perspective for the treatment of ischemic heart disease.

Angiogenesis Inducing Agents↗
PubMed2001

Hepatocyte-based gene therapy.

Hepatocyte-based gene therapy may be used to replace a missing gene product, confer proliferating ability to cultured hepatocytes, prevent allograft rejection, massively repopulate the host liver, or grow xenogeneic hepatocytes in mammalian liver. Gene transfer into isolated hepatocytes can be accomplished via nonviral or viral vectors, the viral vectors being more useful at this time. Common recombinant viruses that integrate into the host genome include murine leukemia retroviruses and lentiviruses, adenoassociated virus, and the T-antigen-deleted SV40 virus. Episomal viruses, such as adenoviruses, permit efficient gene transfer, but the transgene is lost upon proliferation of the transplanted hepatocyte in the host. Hybrid viruses that combine the high transduction efficiency of adenoviral vectors and the integrative capacity of other vectors, such as adenoassociated viruses, have been designed. Massive repopulation of the liver by transplanted hepatocytes can be achieved if a mitotic stimulus to the transplanted cells is combined with prevention of proliferation of the host hepatocytes. Treatment with a plant alkaloid or retrorsine, or preparative irradiation of the liver can be used to inhibit host hepatocellular proliferation, while partial hepatectomy, expression of Fas ligand, or thyroid hormone administration can be used as a mitotic stimulus to the transplanted cells.

Adenoviridae↗
PubMed2005

Gene therapy using non-viral peptide vector in a canine systemic lupus erythematosus model.

Although viral vectors are commonly used for therapeutic gene delivery, their applications are limited due to their specific cell membrane receptor-mediated infection and host immune response. In the present study, we constructed a non-viral peptide vector and applied it in the treatment of experimentally induced systemic lupus erythematosus-like disease in dogs. For therapeutic gene construction, the extracellular domain of canine CTLA-4, and the CH2-CH3 domains of canine immunoglobulin alpha constant region were inserted between the cytomegalovirus promoter and poly-adenylation sequence of bovine growth hormone. The constructed therapeutic gene was ligated to the non-viral synthetic peptide vector and was applied to systemic lupus erythematosus-like disease induced dogs. After gene therapy, clinical signs of systemic lupus erythematosus were reduced dramatically: the anti-nuclear antibody titers and urine protein/creatinine ratios were recovered to normal values, and the skin regained its normal histological features. The peptide vector did not show either tissue specific tropism or host induced immune response.

Amino Acid Sequence↗
PubMed2000

Gene transfer into neurones for the molecular analysis of behaviour: focus on herpes simplex vectors.

The use of viral vectors to transfect genes into specific brain-cell populations is a novel approach that can be used to investigate the molecular and cellular basis of brain function. Ideal vectors should be targetable and capable of regulated transgene expression. From the viral vectors developed so far, this article focuses on herpes simplex virus 1 (HSV-1)-based vectors. HSV-1 vectors can be engineered for gene transfer to the brain, which makes them suitable for neuroscience research applications. In particular, genetic manipulations of the virus can almost eliminate toxicity and allow expression of multiple transgenes simultaneously. In some instances, transfection of selected neuronal populations is also possible. Specific alterations in behaviour and in disease models have been described after the viral-vector-mediated expression of specific genes within highly localized brain regions.

Animals↗
PubMed2006

Increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector.

An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy.

Adenoviridae↗
PubMed2005

Cellular factors influencing Semliki Forest Virus vector biology.

Viral vectors are currently the best tools for gene delivery in a therapeutic setting, especially for in vivo use. Alphaviruses, a family of positive singlestranded RNA viruses, have been engineered to allow the formation of a highly efficient replicon. Using these replicons, it is possible to generate recombinant particles. Parental viruses and recombinant vectors share certain pathways while interacting with their target cells. In this review, we describe the consecutive events leading to transduction, and transgene expression, in view of the cellular factors that affect each individual step. Classical virology will benefit from the knowledge accumulated studying vectors, and such work will shed light on crosstalk between intruding viruses and their hosts. Ultimately, these data should help the design of vectors adapted to specific target cells.

Cells↗