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At least 1,387 records · Page 77Linked to original sources
PubMed2006

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Adenoviridae↗
PubMed1999

Viral gene delivery.

Experimental studies of viral gene delivery generally support the principle that virus-mediated gene transfer is indeed possible. However, the field of gene therapy has not yet been realised as a practicable clinical intervention. The delay in translation of laboratory work to clinical utility largely reflects the inability of gene delivery vectors to convey adequate genetic material to a desired location, with adequate durability and low enough toxicity to be effective. Current studies of viral gene therapy vehicles have focused on re-engineering viruses being tested as vectors at present, treating the host to facilitate viral gene transfer and the development of new vectors. Initial enthusiasm for oncoretroviral and adenoviral vectors has cooled, while adeno-associated virus and lentiviral vectors are attracting more interest. Experimental studies with modified SV40-based vectors have also been very promising. The future of gene therapy will probably entail using an array of gene delivery vehicles, each with its own strengths and weaknesses. The vector systems will probably be as diverse as the applications to which they will be put.

Journal Article↗
PubMed1994

Herpes simplex virus vectors and gene transfer to brain.

Experiments are in progress to engineer herpes simplex virus type 1 as a gene transfer vector for the nervous system. This virus is well suited for this purpose due to its ability to persist for long periods in a latent state in neurons wherein lytic genes are inactive and a unique viral promoter is capable of inducing transcription from the latent viral genome. The virus can be highly cytotoxic for neurons, however, and thus genes which initiate the lytic cycle must be deleted from the genome in order to force the virus into latency. Candidate genes include the immediately early functions ICP0, ICP27, ICP4 and the virus host shut-off function. The design of a suitable promoter regulator capable of expressing foreign genes during latency must also be developed. Many promoters including strong viral promoters and neuronal-specific cellular promoters have proved to be inadequate due to the transient nature of their activity from the viral vector genome and the viral latency promoter region appears to be weakly active in brain. Current efforts concern the development of auto-regulatable promoters which can remain active even if the viral genome is bound by chromatin.

Base Sequence↗
PubMed2006

[New option for gene-targeting vector construction Red/ET recombination].

The study of human new gene's function has become an increasingly active academic field basically relying on the gene knockout (KO) mouse. The construction of targeting vector economically and efficiently has turned into the key step to acquire a KO mouse because of the low efficiency of recombination with traditional constructed targeting vector. For study of the function of new gene-Resp18, we brought in a new DNA engineering platform-Red/ET recombination to construct Resp18 targeting vector. Red/ET recombineering differs from the conventional ways of vector construction (e.g., PCR, restriction enzyme digestion and ligation) and achieves genetic modification by acquisition, insertion, fusion or replacement of the target gene through small fragments mediated homologous recombination. Now Resp18 targeting vectors of three strategies were yielded successfully through two homologous recombination processes of retrieve and neo-targeting. Red/ET recombination has the advantage of getting longer homology regions without mutation, which makes it a new and reliable alternative to the construction of a targeting vector today.

Bacteriophage lambda↗
PubMed2004

Alternative gene therapy strategies for the repair of craniofacial bone defects.

Craniofacial anomalies, bone defects and cartilage lesions pose a difficult and challenging problem for both the doctor and for patients and their families. Conventional therapies focus on orthopaedic surgery, grafting with autogenous bone, allogenic bone grafts, or distraction osteogenesis. However, the efficacy of these techniques is limited by high cost, donor morbidity, scarcity of tissue resources, and alterations in volume [Marx & Morales, 1988]. On the basis of recent insights into the development, growth, and adaptation of bone, together with the significant advances in recombinant DNA technology, gene therapy is increasingly becoming recognised as an alternative technique for augmenting and promoting bone regeneration in vivo. It can be applied in craniofacial skeletal tissues by transferring genes encoding for specific growth factors such as BMPs in osteoblasts, chondrocytes or progenitor cells for the purpose of enhancing protein production [Scaduto & Lieberman, 1999]. It can be performed by either direct administration of gene delivery vectors, or by transplantation of genetically modified cells. This review will focus on recent advances in molecular mechanisms of bone formation, and development in various viral and non-viral vectors for direct in vivo therapeutic gene transfer and genetically engineered cells ex vivo therapy.

Adenoviridae↗
PubMed1995

Bicistronic retroviral vector reveals capacity of v-erbA to induce erythroleukemia and to co-operate with v-myb.

Previous studies have shown that v-erbA and v-myb can induce the proliferation of avian erythroid cells in culture. To study the combined effects of v-erbA and v-myb, the two oncogenes were engineered into a retrovirus bicistronic vector with an internal ribosomal entry site (IRES) or into a vector with a splice acceptor (SPL). This allowed coexpression of the two proteins and a comparison with the same vector containing either v-erbA or v-myb only. Both the erbA IRES and the erbA/myb IRES virus constructs transformed erythroid cells after infection of bone marrow or blastoderm cultures. The erbA/myb IRES virus exhibited a 5-10-fold higher transformed colony forming efficiency than the erbA IRES virus in the blastoderm assay. Surprisingly, when injected into chicken embryos in the presence of helper virus, both viruses induced an erythroleukemia in about half of the animals. In contrast, no leukemia was observed with a myb IRES virus, with spliced vectors containing v-erbA alone or v-erbA in combination with v-myb, nor with erbA IRES and erbA/myb IRES viruses produced in the absence of helper virus. The average latency of leukemia induction was shorter for the erbA/myb IRES virus (3.5 weeks) than for the erbA IRES virus (5 weeks). Nevertheless, for both viruses the leukemic blasts retained full factor dependence for growth. These results show that v-erbA is capable of inducing an erythroleukemia when expressed by a high titer bicistronic retrovirus under conditions of virus spreading and that its in vitro and in vivo transforming potential can be enhanced by v-myb.

Animals↗
PubMed1992

PCR and other test systems in human gene therapy.

Three protocols using retroviral-mediated gene transfer are currently undergoing clinical trials. The uniqueness of these protocols, which were the first approved experiments in which genetically engineered cells were administered to human beings, necessitated the development of novel biological and physical safety testing procedures. In addition to standard testing for contamination with exogenous pathogens and toxins, specific tests were developed to assay for the presence of recombinant helper virus in both retroviral vector preparations and in the engineered human cells. Two new assays were developed, an enhanced S+/L-virus assay called 3T3 amplification, and a specific polymerase chain reaction protocol of great sensitivity. The results of these safety tests, along with pre-clinical primate safety studies, and the accumulating clinical data, suggest that retroviral-mediated gene transfer does not pose any serious short-term safety problems for patients enrolled in gene therapy protocols.

Animals↗
PubMedDate not supplied

Continuous transforming growth factor beta1 secretion by cell-mediated gene therapy maintains chondrocyte redifferentiation.

One of the most important factors in the production of cartilage is transforming growth factor beta1 (TGF-beta1). To obtain sustained release of TGF-beta1, a cell-mediated gene therapy technique was introduced. We infected chondrocytes with a retroviral vector carrying the TGF-beta1 gene. The single clone derivative showed sustained TGF-beta1 secretion. It also showed constitutive type II collagen expression. Whereas the TGF-beta1 protein itself is unable to induce formation of cartilage in vivo, human chondrocytes engineered to express a retroviral vector encoding TGF-beta1 showed cartilage formation in vivo when cells were injected into nude mice intradermally. These data suggest that cell-mediated gene therapy using TGF-beta1 as a transgene would be a promising treatment for osteoarthritis.

Animals↗
PubMed1999

Interleukin-2 gene transfer into human transitional cell carcinoma of the urinary bladder.

Transitional cell carcinoma of the bladder is one of the human cancers most responsive to immunotherapy, and local interleukin-2 (IL-2) production appears to be an important requirement for immunotherapy to be effective. In this study, we engineered two human bladder cancer cell lines (RT112 and EJ) to constitutively release human IL-2 by retroviral vector-mediated gene transfer. Following infection and selection, stable and consistent production of biologically active IL-2 was demonstrated at both the mRNA and the protein level. Morphology, in vitro growth rate and proliferation, as well as other cytokine gene mRNA or membrane adhesion receptor expression, were not altered in IL-2 transduced cells as compared to their parental or control vector-infected counterparts. Moreover, IL-2 engineered cells lost their tumorigenicity into nu/nu mice and the mechanism of rejection appeared to involve multiple host effector cell populations, among which a prominent role was played by neutrophils and radiosensitive cells. These findings may offer support to the development of an IL-2-based gene therapy approach to human bladder cancer.

Animals↗
PubMed1990

BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells.

Barley stripe mosaic virus (BSMV) possesses a tripartite genome composed of RNAs alpha, beta and gamma that have been cloned into in vitro transcription vectors from which infectious transcripts can be obtained. The BSMV genome has been engineered here to serve as an expression vector in plant protoplasts. Open reading frame (ORF) b of RNA beta, encoding a non-structural protein, was replaced by the firefly luciferase (luc) reporter gene to yield RNA beta 2-luc. In the presence of both RNAs alpha and gamma, RNA beta 2-luc mediated efficient expression of the luc gene upon transfection into tobacco and maize protoplasts. This expression ranged from 20- to 123-fold higher than the luciferase activity obtained from transfection with a luc gene mRNA. Replication of RNA beta and its derivatives i.e. 'minus' strand synthesis, was confirmed by Northern analysis, indicating that the high level of luc gene expression using RNA beta 2-luc resulted from RNA amplification. ORFa of RNA beta, encoding the coat protein, was also replaced by the luc gene to yield RNA beta 1-luc. Although transfection of RNA beta 1-luc alone produces luciferase efficiently, neither 'minus' strand synthesis nor further increase of luciferase activity was observed in the presence of RNAs alpha and gamma, or RNAs alpha, beta and gamma, suggesting that the deleted sequences within ORFa are cis-acting for replication of RNA beta. Our results demonstrate that a foreign gene can be expressed by replacement of a viral non-structural protein gene that is essential for virus multiplication in plants, leading to a potential strategy for virus 'containment' with use of 'disarmed' plant viral vectors.

Animals↗
PubMed2004

Engineering of tooth-supporting structures by delivery of PDGF gene therapy vectors.

Platelet-derived growth factor (PDGF) exerts potent effects on wound healing including the regeneration of tooth-supporting structures. Limitations of topical protein delivery to periodontal osseous defects include transient biological activity and the bioavailability of PDGF at the wound site. The objective of this investigation was to determine the feasibility of in vivo PDGF-B gene transfer to stimulate periodontal tissue regeneration in large tooth-associated alveolar bone defects in rats. Periodontal lesions (0.3 x 0.2 cm in size) were treated with a 2.6% collagen matrix alone or a matrix containing adenoviruses encoding luciferase (control), a dominant negative mutant of PDGF-A (PDGF-1308), or PDGF-B. Block biopsies were harvested at 3, 7, and 14 days post-gene delivery and descriptive histology and histomorphometric analyses were performed. The defects treated with Ad-PDGF-B demonstrated greater proliferating cell nuclear antigen positively stained cells and strong evidence of bone and cementum regeneration beyond that of Ad-luciferase and Ad-PDGF-1308 groups. Quantitative image analysis showed a nearly fourfold increase in bridging bone and sixfold increase in tooth-lining cemental repair in the Ad-PDGF-B-treated sites compared to lesions treated with Ad-luciferase or collagen matrix alone, which showed limited hard tissue neogenesis. In addition, the Xenogen In Vivo Imaging System revealed sustained and localized gene expression of the luciferase reporter at the periodontal lesions for up to 21 days after gene transfer. These results indicate that in vivo direct gene transfer of PDGF-B stimulates alveolar bone and cementum regeneration in large periodontal defects. Gene therapy utilizing PDGF-B may offer the potential for periodontal tissue engineering applications.

Adenoviridae↗
PubMed2006

Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain.

Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8 x 10(10) genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.

Animals↗
PubMed2005

Directed evolution of retrovirus envelope protein cytoplasmic tails guided by functional incorporation into lentivirus particles.

In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

Amino Acid Sequence↗
PubMed2001

Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Cre-dependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.

Animals↗
PubMed2005

NT-3 expression from engineered olfactory ensheathing glia promotes spinal sparing and regeneration.

Adenoviral (AdV) vectors encoding neurotrophin-3 (AdV-NT-3) or the bacterial marker enzyme beta-galactosidase (LacZ gene) were used to transduce olfactory ensheathing glia (OEG) cultures. AdV vector-transduced OEG expressed high levels of recombinant neurotrophin as shown by in situ hybridization and enzyme-linked immunosorbent assay techniques. The biological activity of vector-derived NT-3 was determined in a dorsal root ganglia neurite outgrowth assay. Engineered cell suspensions were then injected into adult Fischer 344 rat spinal cord immediately after unilateral cervical (C4) corticospinal tract (CST) transection. Transplanted animals received a total of 200,000 cells; either non-transduced OEG or OEG transduced with AdV vectors encoding NT-3 or LacZ, respectively. At 3 months after injury, lesion volumes were significantly smaller in all OEG-transplanted rats when compared with control (medium-injected) rats. Anterograde tracing of the lesioned CST projection, originating from the contralateral sensorimotor cortex, showed a significantly greater number of distal CST axons only in OEG-NT-3-transplanted rats. Behavioural analysis was performed on all rats using open field locomotion scoring, and a forelimb reaching task with Eshkol-Wachman movement notation. Analysis of behavioural tests revealed no significant differences in recovery between experimental groups, although movement analysis indicated that possible compensatory mechanisms were occurring after OEG implantation. The results demonstrate that OEG transplantation per se can promote tissue sparing after injury, but, after appropriate genetic modification, these olfactory-derived cells become far more effective in promoting long-distance maintenance/regeneration of lesioned adult CST axons.

Adenoviridae↗
PubMed2001

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability.

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen.

Bacterial Proteins↗