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At least 109 records · Page 6Linked to original sources
PubMed2002

Immunology of viral-vector-mediated gene transfer into the brain: an evolutionary and developmental perspective.

The immune system imposes limitations on gene transfer into the brain. Viral vectors injected into the brain's ventricular system elicit innate and adaptive immune responses. However, when injected directly into the brain parenchyma, they elicit only transient inflammation owing to the absence of dendritic cells, which transport antigen to lymph nodes and present it to naive T cells to initiate adaptive immune responses. This article explores the evolutionary and developmental basis of brain immune responses and their implications for viral-vector-mediated neurological gene therapy. Elucidating the cellular and molecular basis of these differential reactions is essential to the long-term success of neurological gene therapy.

Animals↗
PubMed2002

Targeting functional subtypes of spinal motoneurons and skeletal muscle fibers in vivo by intramuscular injection of adenoviral and adeno-associated viral vectors.

We report that functional subtypes of spinal motoneurons and skeletal muscle fibers can be selectively transduced using replication-defective adenoviral (ADV) or adeno-associated (AAV) viral vectors. After intramuscular injection in adult rodents, ADV vectors transduced both fast-twitch and slow-twitch skeletal muscle fibers. Intramuscular injection of ADV vectors also caused transduction of spinal motoneurons and dorsal root ganglion cells. However, only neurons innervating the injected muscle were transduced, as shown by co-injection of a retrograde axonal tracer. In adult male rats it is therefore possible to transduce fast or slow spinal motoneurons and muscle fibers selectively since in these animals, the extensor digitorum longus and soleus muscles contain almost exclusively fast or slow motor units, respectively. In rats, AAV vectors transduced muscle fibers in the predominantly fast extensor digitorum longus but not in the predominantly slow soleus muscle. We did not observe any transduction of spinal motoneurons following intramuscular injection of AAV vectors. These results show that physiologically and clinically important subpopulations of cells in the neuromuscular system can be selectively transduced by viral vectors.

Adenoviruses, Human↗
PubMed2003

Toxicogenomics of non-viral vectors for gene therapy: a microarray study of lipofectin- and oligofectamine-induced gene expression changes in human epithelial cells.

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.

Annexin A5↗
PubMed2002

Evaluation of multicomponent non-viral vectors for liver directed gene delivery.

Multicomponent, non-viral gene delivery vehicles are designed to have as a minimum, a DNA binding component, and a cell recognition component for specific delivery to target cells. The DNA binding component cannot only bind, but also protect DNA from serum degradation, and tends to condense DNA to sizes that can be taken up by receptor-mediated processes of target cells. Generally, cationic peptides, single chained, e.g. poly-L-lysine or branched polymers or synthetic peptides with DNA binding properties are used for DNA binding components. Ligands for binding to receptors on cell surfaces can be covalently linked to the DNA binding component. Multicomponent, non-viral vectors have been successfully used to deliver genes into cells in vitro and in vivo. Improvements have been made to the non-viral carriers resulting in increased solubility of DNA/carrier complexes and longer survival in serum. Improvements have also been made by incorporating fusogenic/lysosomolytic components that enable DNA/carrier complexes to escape intracellular degradation and enhance the levels and duration of expression of genes in vitro and in vivo.

Animals↗
PubMed2005

Persistent liver expression of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1.

Plasma levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-l (apoA-l) are inversely related to risk for coronary heart disease. Overexpression of apoA-l inhibits atherosclerosis in animal models. A method of stably expressing apoA-l using somatic gene transfer would be of interest. Pseudotyped adeno-associated virus (AAV) vectors comprised of inverted terminal repeats from AAV serotype 2 have been used for liver-directed gene transfers. We hypothesized that liver-directed gene transfer of apoA-l using vectors based on AAV serotypes 1 and 5 would result in higher-level, prolonged expression of apoA-l and increased HDL-C. To test this hypothesis we injected apoA-l-/- mice via the tail vein with either AAV2, AAV1 or AAV5 vectors encoding the murine apoA-l cDNA driven by the liver-specific thyroxine binding globulin promoter. Plasma levels of murine apoA-l and HDL-C were highest in mice injected with the AAV1-based vector and lowest in mice injected with the AAV2-based vector. Expression of apoA-l was stable up to 1 year after vector injection. These results indicate that AAV5 and AAV1 are more effective vectors for achieving higher levels of stable transgene expression of apoA-l after liver-directed gene transfer than AAV2. Furthermore, AAV1-based vectors generate higher apoA-l levels than AAV5-based vectors. It is possible that the levels of expression achieved using these vectors will be therapeutic in preventing atherosclerosis.

Animals↗
PubMed2004

Using sindbis viral vectors for specific detection and suppression of advanced ovarian cancer in animal models.

We studied the therapeutic value of Sindbis vectors for advanced metastatic ovarian cancer by using two highly reproducible and clinically accurate mouse models: a SCID xenograft model, established by i.p. inoculation of human ES-2 ovarian cancer cells, and a syngenic C57BL/6 model, established by i.p. inoculation of mouse MOSEC ovarian cancer cells. We demonstrate through imaging, histologic, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in the peritoneal cavity, leading to significant suppression of the carcinomatosis in both animal models. Use of two different bioluminescent genetic markers for the IVIS Imaging System permitted demonstration, for the first time, of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis vector infection and growth suppression of murine MOSEC tumor cells indicate that Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the Mr 67,000 laminin receptor. Immunohistochemical staining of tumor cells indicates that laminin receptor is elevated in tumor versus normal cells. Down-regulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. We show that incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer.

Animals↗
PubMed2006

Strategies for gene transfer to solid organs: viral vectors.

A major complication associated with transplantation of solid organs is immunological rejection, which is currently controlled pharmacologically with immunosuppressive drugs, which must be administered indefinitely and may have harmful side effects. Gene transfer to donor organs or recipient immune cells prior to transplantation could limit their use. The effects of transfer of candidate genes in experimental models of allograft rejection is outlined in this chapter, followed by a description of the features of an ideal gene-therapy vector. Finally, a brief overview of viral vector systems used commonly for gene transfer is presented.

Animals↗
PubMed2004

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications.

BACKGROUND: Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo. METHODS: Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo. RESULTS: Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days. CONCLUSIONS: FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.

Alkaline Phosphatase↗
PubMed2005

The use of viral vectors to produce hepatitis B virus core particles in plants.

The expression and assembly of the hepatitis B virus (HBV) nucleocapsid protein (HBcAg) were investigated in plants using viral vectors. Constructs based on either Potato virus X (PVX) or Cowpea mosaic virus (CPMV) containing the sequence of HBcAg were able to infect the appropriate host plants and remained genetically stable during infection. Analysis of HBcAg expression revealed that the protein can self-assemble into core-like particles and that the assembled material could be partially purified by differential centrifugation. Thus, the use of viral vectors can be considered a practical method for rapid production of assembled HBcAg particles in plants. This approach provides a means whereby a variety of chimaeric particles can be assessed quickly and cheaply for various diagnostic and vaccine applications.

Centrifugation↗
PubMed1998

Human gene targeting by viral vectors.

Stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. Here we report that vectors based on adeno-associated virus (AAV) can efficiently modify homologous human chromosomal target sequences. Both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by AAV vectors. Site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest targeting rates occurring in normal human fibroblasts. These results suggest that AAV vectors could be used to introduce specific genetic changes into the genomic DNA of a wide variety of mammalian cells, including therapeutic gene targeting applications.

Adenoviridae↗
PubMed2000

Dual-viral vector approach induced strong and long-lasting protective immunity against very virulent infectious bursal disease virus.

To induce strong protective immunity against very virulent infectious bursal disease virus (vvIBDV) in chickens, two viral vector systems, Marek's disease and Fowlpox viruses expressing the vvIBDV host-protective antigen VP2 (rMDV, rFPV), were used. Most of chickens vaccinated with the rFPV or rMDV alone, or vaccinated simultaneously with both at their hatch (rMDV-rFPV(1d)), were protected against developing clinical signs and mortality; however, only zero to 14% of the chickens were protected against gross lesions. In contrast, gross lesions were protected in 67% of chickens vaccinated primarily with the rMDV followed by boosting with the rFPV 2 weeks later (rMDV-rFPV(14d)). Protection against the severe histopathological lesions of rFPV, rMDV, rMDV-rFPV(1d), and rMDV-rFPV(14d) vaccine groups were 33, 42, 53, and 73%, respectively. Geometric mean antibody titers to VP2 of chickens vaccinated with the rFPV, rMDV, rMDV-rFPV(1d), and rMDV-rFPV(14d) before the challenge were 110, 202, 254, and 611, respectively. Persistent infection of the rMDV in chickens after the booster vaccination with rFPV was suggested by detection of the rMDV genes from peripheral blood lymphocyte DNA at 28 weeks of age. These results indicate that the dual-viral vector approach is useful for quickly and safely inducing strong and long-lasting protective immunity against vvIBDV in chickens.

Animals↗
PubMed2002

Tightly regulated long-term erythropoietin expression in vivo using tet-inducible recombinant adeno-associated viral vectors.

Recombinant adeno-associated viral (rAAV) vectors containing an improved tetracycline (tet) system of transcriptional regulation are an efficient strategy for the control of long-term therapeutic gene expression. In vivo studies with the original tet-off and tet-on vectors, while promising, have failed to demonstrate complete repression in the uninduced state. To address this issue, we incorporated the tTS(kid) fusion of the tet repressor and a KRAB-derived transcriptional silencer into the tet-on system in the context of rAAV vectors. The tTS(kid) repressor and rtTA activator were expressed constituitively from a regulator vector, and the repressor and an erythropoietin (Epo) transgene were expressed inducibly via a second vector. Following intramuscular co-injection of these vectors, we observed repeated induction of serum Epo protein following drug administration and undetectable levels after its withdrawal. Four cycles of regulation were achieved over a 32-week period. Thus, the tet-on system plus the tTS(kid) repressor delivered via nonpathogenic rAAV vectors is a powerful tool for controlling the in vivo expression of therapeutic transgenes. In a clinical setting, the repressor could provide a mechanism for abolishing transgene expression if it were no longer needed or if the safety of a patient became compromised.

Animals↗
PubMed2002

Gene therapy with viral vectors.

A key factor in the success of gene therapy is the development of gene delivery systems that are capable of efficient gene transfer in a broad variety of tissues, without causing any pathogenic effect. Currently, viral vectors based on many different viruses have been developed, and their performance and pathogenicity has been evaluated in animal models. The results of these studies form the basis for the first clinical trials for correcting genetic disorders using retroviral, adenoviral, and adeno-associated viral vectors. Even though the results of these trials are encouraging, vector development is still required to improve and refine future treatment of hereditary disorders.

Gene Transfer Techniques↗
PubMed2001

Gene transfer by viral vectors into blood vessels in a rat model of retinopathy of prematurity.

AIMS: To test the feasibility of gene transfer into hyaloid blood vessels and into preretinal neovascularisation in a rat model of retinopathy of prematurity (ROP), using different viral vectors. METHODS: Newborn rats were exposed to alternating hypoxic and hyperoxic conditions in order to induce ocular neovascularisation (ROP rats). Adenovirus, herpes simplex, vaccinia, and retroviral (MuLV based) vectors, all carrying the beta galactosidase (beta-gal) gene, were injected intravitreally on postnatal day 18 (P18). Two sets of controls were also examined: P18 ROP rats injected with saline and P18 rats that were raised in room air before the viral vectors or saline were injected. Two days after injection, the rats were killed, eyes enucleated, and beta-gal expression was examined by X-gal staining in whole mounts and in histological sections. RESULTS: Intravitreal injection of the adenovirus and vaccinia vectors yielded marked beta-gal expression in hyaloid blood vessels in the rat ROP model. Retinal expression of beta-gal with these vectors was limited almost exclusively to the vicinity of the injection site. Injection of herpes simplex yielded a punctuate pattern of beta-gal expression in the retina but not in blood vessels. No significant beta-gal expression occurred in rat eyes injected with the retroviral vector. CONCLUSIONS: Adenovirus is an efficient vector for gene transfer into blood vessels in an animal model of ROP. This may be a first step towards utilising gene transfer as a tool for modulating ocular neovascularisation for experimental and therapeutic purposes.

Animals↗
PubMed2004

Viral vectors for gene transfer to striated muscle.

Recent progress has generated exciting results that are increasing the prospects for gene therapy of a variety of disorders of striated muscle, including the muscular dystrophies and myopathies, acquired and inherited diseases of cardiac muscle, and aging-associated muscle wasting. Numerous viral vector systems are being employed to transfer therapeutic genes to striated muscles, and advances in vector technology are leading to improved gene transfer efficiencies coupled with reduced immunological responses. The current technology in the field of viral vectors as tools for gene delivery to striated muscle is summarized, and recent developments related to gene therapies for skeletal and cardiac muscle disorders are discussed.

Genetic Therapy↗
PubMed1998

Viral vectors for gene therapy.

Viruses have evolved to become highly efficient at nucleic acid delivery to specific cell types while avoiding immunosurveillance by an infected host. These properties make viruses attractive gene-delivery vehicles, or vectors, for gene therapy. Several types of viruses, including retrovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus, have been modified in the laboratory for use in gene therapy applications. Because these vector systems have unique advantages and limitations, each has applications for which it is best suited. Retroviral vectors can permanently integrate into the genome of the infected cell, but require mitotic cell division for transduction. Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types, but immune elimination of infected cells often limits gene expression in vivo. Herpes simplex virus can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression remain as obstacles. AAV also infects many nondividing and dividing cell types, but has a limited DNA capacity. Alternatively, chimeric viral-vector systems that combine advantageous properties of two or more viral systems are also being explored. Although viral-mediated gene delivery has proved to be the most efficient means of gene transfer, nonviral means are also under development. Many of these nonviral systems incorporate portions of viral vectors to increase the efficiency of gene delivery or expression. Retrovirus, adenovirus, and AAV vectors are being evaluated currently in several Phase 1 clinical trials for treatment of diseases such as cancer, cystic fibrosis, Gaucher disease, and arthritis.

Cystic Fibrosis↗
PubMed2004

Non-viral vectors in cystic fibrosis gene therapy: progress and challenges.

Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.

Animals↗
PubMed1997

Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension.

Antisense oligodeoxynucleotides have been designed to inhibit the production of specific proteins. In models of hypertension, we have targeted the renin-angiotensin system at the level of synthesis (angiotensinogen) and the receptor (AT1 receptor). The design of antisense oligonucleotides requires choosing a site to inhibit mRNA processig or translation. The strategy we use is to make three oligonucleotides of antisense sequences, upstream and downstream from the AUG site and over the AUG site. The oligonucleotides are tested in a screening test. Antisense oligonucleotides to AT1-receptor mRNA and to angiotensinogen mRNA reduce blood pressure in spontaneously hypertensive rats when injected into the brain. They significantly reduce the concentration of the appropriate protein. The oligonucleotides are also effective when administered systemically. The decrease in blood pressure with antisense oligonucleotides delivered in blood or brain lasts 3 to 7 days. To prolong the action, direct injection of naked DNA and injection of DNA in liposome carriers have been tested. Viral vectors have been developed to deliver antisense DNA. The viral vectors available include retroviruses and adenovirus, but the adeno-associated virus (AAV) vector is the vector of choice for ultimate use in gene therapy. It offers safety because it is nonpathogenic, has longevity because it integrates into the genome, and has sufficient carrying capacity to carry up to 4.5 kb antisense or gene in a recombinant AAV. Using rAAV-antisense to AT1 mRNA, there is efficient transfection into cells and an inhibition of AT1 receptor number. In in vivo tests, rAAV-AS AT1-receptor when injected into the brains of SHR reduces blood pressure for more than 2 months. In young rats (3 weeks old), rAAV-AS AT1-receptor decreases blood pressure and slows the development of hypertension. While further experiments need to be done on dose-response relationships and on the cellular mechanisms of these effects, the results show the feasibility of AAV as a vector for antisense inhibition, which may ultimately be used in gene therapy for hypertension.

Adenoviridae↗