Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “vector engineering”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,495 records · Page 83Linked to original sources
PubMed1998

An enhanced packaging system for helper-dependent herpes simplex virus vectors.

Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types. The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (pac) elements have been deleted. Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles. Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector. The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in Escherichia coli from which the pac elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome. The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector. The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination. The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield.

Animals↗
PubMed1995

Comparison of genetically engineered herpes simplex viruses for the treatment of brain tumors in a scid mouse model of human malignant glioma.

Genetically engineered viruses and viral genes inserted into retroviral vectors are increasingly being considered for experimental therapy of brain tumors. A primary target of these viruses and vectors is human gliomas, the most frequently occurring primary human brain tumor. To investigate the potential of genetically engineered herpes simplex viruses (HSVs) in the therapy of these tumors, we compared the attributes of two viruses, a recombinant from which the gamma 1(34.5) gene had been deleted (R3616) and a recombinant in which the gamma 1(34.5) gene had been interrupted by a stop codon (R4009). Previous studies have shown that these recombinants were completely devoid of the ability to multiply in the central nervous system of rodents. To pursue these studies, we developed a scid mouse glioma model. Tumor cell response (survival) for 10(3), 10(4), and 10(5) implanted MT539MG glioma cells was 38, 23, and 15 days, respectively. The results were as follows: (i) both R3616 and R4009 replicate and cause cytolysis in diverse glioma cell lines of murine and human origin in vitro, and (ii) Winn-type assays 10(5) MT539MG cells coinoculated with R3616 or R4009 as compared to saline significantly prolonged survival in a dose-dependent fashion. Mice that received only tumor cells or the wild-type parent strain of the recombinants, HSV-1(F), died within 15 days. Survival was greatest with R4009. These experiments define both a model for screening oncolytic viruses and a genetically engineered virus of significant potential use as an oncolytic agent.

Animals↗
PubMed1995

Highly attenuated poxvirus vectors: NYVAC, ALVAC and TROVAC.

Three highly attenuated and efficacious poxvirus-based vectors, NYVAC, ALVAC and TROVAC, are available for targeted applications as recombinant vaccines in both human and veterinary medicine. The attenuated phenotype of the three vectors is consistent with safe use for vaccination purposes, for the vaccinee, for unvaccinated contacts, and for introduction into the environment. The precise deletion of virulence and host range genes in the NYVAC vector precludes reversion to the virulent phenotype by back mutation. Dissemination of recombinant vaccines based on the NYVAC, ALVAC and TROVAC vectors is highly diminished, because of the genetic engineering in NYVAC and the natural attenuated phenotype of ALVAC and TROVAC. Studies have demonstrated that these recombinant vectors are genetically and phenotypically stable after serial passage in vitro as well as in vivo. NYVAC, ALVAC and TROVAC vectors are the only three poxvirus-based vectors that are classified as BSL1 agents.

Animals↗
PubMed2000

Cartilage and bone regeneration using gene-enhanced tissue engineering.

Joint cartilage injury remains a major problem in orthopaedics with more than 500,000 cartilage repair procedures performed yearly in the United States at a cost of hundreds of millions of dollars. No consistently reliable means to regenerate joint cartilage currently exists. The technologies of gene therapy and tissue engineering were combined using a retroviral vector to stably introduce the human bone morphogenic protein-7 complementary deoxyribonucleic acid into periosteal-derived rabbit mesenchymal stem cells. Bone morphogenic protein-7 secreting gene modified cells subsequently were expanded in monolayer culture, seeded onto polyglycolic acid grafts, implanted into a rabbit knee osteochondral defect model, and evaluated for bone and cartilage repair after 4, 8, and 12 weeks. The grafts containing bone morphogenic protein-7 gene modified cells consistently showed complete or near complete bone and articular cartilage regeneration at 8 and 12 weeks whereas the grafts from the control groups had poor repair as judged by macroscopic, histologic, and immunohistologic criteria. This is the first report of articular cartilage regeneration using a combined gene therapy and tissue engineering approach.

Animals↗
PubMed2004

Oral administration of a mite allergen expressed by zucchini yellow mosaic virus in cucurbit species downregulates allergen-induced airway inflammation and IgE synthesis.

BACKGROUND: Sublingual-swallow immunotherapy in house dust mite-related asthma has a good safety profile and improves respiratory function and bronchial hyperreactivity. Zucchini yellow mosaic virus (ZYMV) is envisaged as a promising viral vector for expressing large quantity of foreign proteins in cucurbit species. OBJECTIVE: We sought to investigate whether oral feeding of dust mite allergen expressed by ZYMV in a cucurbit species can suppress allergen-induced inflammation and IgE synthesis. METHODS: An infectious plant virus clone, p35SZYMV2-26, that contains the full-length cDNA to the genomic RNA of a Taiwan isolate of ZYMV, driven by the cauliflower mosaic virus 35S promoter, was engineered as an in vivo viral vector to express Dermatophagoides pteronyssinus group 5 allergen (Der p 5) in cucurbit species. Female BALB/c mice were intraperitoneally sensitized with Escherichia coli bacteria-expressed Der p 5 and orally treated with the virus-expressed Der p 5 (vDer p 5) extracted from the recombinant virus-infected squash plants. Der p 5-specific immunoglobulins were measured by ELISA, and bronchoalveolar lavage assays were used to measure airway inflammation. RESULTS: Infectivity assays and immunoblotting revealed that large quantities of free-form vDer p 5 are produced in the recombinant virus-infected squash plants. The recombinant virus carried and expressed the Der p 5 allergen in squash plants for at least 1 year after numerous passages. In animal tests, squash extract containing vDer p 5 inhibited Der p 5-specific IgE synthesis and airway inflammation. CONCLUSION: Our results suggest that oral feeding with allergen produced by the plant viral vector provides a novel approach for the therapy of allergic asthma.

Administration, Oral↗
PubMed2014

Engineering adeno-associated viruses for clinical gene therapy.

Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

Capsid↗
PubMed2003

RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism.

Recombinant adeno-associated virus (AAV) has become an attractive vector system for a number of gene therapy paradigms. However, the utility of AAV vectors is often limited by the absence of heparan sulfate proteoglycan (HSPG), the virus's primary attachment receptor, on the desired target cell population. In order to achieve HSPG-independent gene delivery, several groups have shown that the endogenous tropism of AAV can be expand by genetically altering the viral capsid. However, the parameters of this developing technology have yet to be defined and it has not yet been determined if these modified vectors actually infect cells via these engineered interactions. Previously we constructed a series of insertion mutants spanning the AAV capsid protein gene and identified specific sites that can tolerate the insertion of small exogenous peptides. Here we describe a number of sites within the AAV capsid gene that can be used for the insertion of integrin-targeting peptide epitopes. Incorporation of an Arg-Gly-Asp (RGD)-containing peptide at these sites enables AAV to infect integrin-expressing cells independent of HSPG. Mutant AAV vectors displaying these peptide ligands can be produced to wild-type titer and have been shown to specifically interact with the targeted integrin receptors and mediate infection via this interaction. We report significant increases in gene transfer to Raji, K562, and SKOV-3 cell lines that express integrin, but little HSPG, suggesting that rAAV vectors displaying RGD peptides may be of great utility for treatment of neoplasms characterized by the deficiency of HSPG expression. We have also demonstrated that due to their expanded tropism, these novel vectors are capable of efficient transduction of AAV2-resistant tumors in vivo suggesting that they may offer significant therapeutic advantages.

Animals↗
PubMed2004

Tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography.

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 microg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.

Amino Acid Sequence↗
PubMed2005

Isolation of cell lines that show novel, murine leukemia virus-specific blocks to early steps of retroviral replication.

In order to identify cellular proteins required for early stages of retroviral replication, a high volume screening with mammalian somatic cells was performed. Ten pools of chemically mutagenized Chinese hamster ovary (CHO-K1) cells were challenged with a murine leukemia virus (MLV) vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G), and cells that failed to be transduced were enriched by cell sorting. Each pool yielded a clonally derived cell line with a 5-fold or greater resistance to virus infection, and five cell lines exhibited a >50-fold resistance. These five cell lines were efficiently infected by a human immunodeficiency virus vector pseudotyped with VSV-G. When engineered to express the TVA receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A), the five cell lines were resistant to infection with a MLV vector pseudotyped with the ASLV-A envelope protein but were fully susceptible to infection with an ASLV-A vector. Thus, the defect in these cells resides after virus-cell membrane fusion and, unlike those in other mutant cell lines that have been described, is specific for the MLV core. To identify the specific stages of MLV infection that are impaired in the resistant cell lines, real-time quantitative PCR analyses were employed and two phenotypic groups were identified. Viral infection of three cell lines was restricted before reverse transcription; in the other two cell lines, it was blocked after reverse transcription, nuclear localization, and two-long terminal repeat circle formation but before integration. These data provide genetic evidence that at least two distinct intracellular gene products are required specifically for MLV infection. These cell lines are important tools for the biochemical and genetic analysis of early stages in retrovirus infection.

Animals↗
PubMed2003

Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans.

Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat alpha-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.

Animals↗
PubMed1995

Experimental quantification of transmission of genetically engineered pseudorabies virus.

There is concern that live pseudorabies virus (PRV) vaccine or PRV vector vaccine strains may spread from vaccinated to unvaccinated pigs. Moreover, it is feared that recombining PRV vaccine strains with related vaccine or wild-type strains may lead to spread and survival of recombinant PRV. To learn more about to what extent different PRV vaccine strains could spread we used a previously described experimental model to study the transmission of intranasally inoculated PRV mutant strains under experimental conditions. We used PRV strains that lacked glycoprotein E (gE) or thymidine kinase (TK), and a PRV vector vaccine (gE-, TK-, gG-) that expresses the glycoprotein E1 (E1) of hog cholera virus. In addition, we investigated whether intranasally co-inoculated gE-negative and gE-positive PRV strains competed in transmission among pigs. The extent of transmission was estimated using the reproduction ratio R. This ratio has a threshold property; when R1, the infection can spread; when R < 1, the infection will disappear. We found that R for a gE-negative strain was 10.1, and R for a TK-negative strain was 5. Furthermore, the R for the vector vaccine (gE-, TK-, gG-) expressing E1 was 0.18, and did not differ significantly from the R for the control strain without E1. The R of gE-negative strain was significantly 1 (P = 0.0005). Co-inoculation with a gE-positive field strain did not prevent the transmission of a gE-negative strain. This study shows that a small-scale experiment can be used to estimate the transmission of genetically engineered organisms in their host species. The results of this study indicate that the deletion of gE alone or TK alone is not enough to prevent spread of PRV among susceptible pigs, and that transmission of gE-negative PRV is not firmly limited by co-presence of a gE-positive strain.

Administration, Intranasal↗
PubMedDate not supplied

Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21.

BACKGROUND: We modified a tetracycline-regulated system that can control the activity of individual genes quantitatively and reversibly in transgenic mammals. Despite these advances, there remained one problem in the intensive use of the tet-system: the limited range of acceptor cell lines, expressing a tetracycline-controlled transcriptional activator (tTA). This study describes in detail new vectors and a unifying strategy to generate tTA-expressing cell lines. METHOD: Two retroviral vectors pLNCtTA-hCMV and pLNCtTA-EF1alpha coding for the tTA were used to engineer cell lines to constitutively express tTA. New expression vectors pETE-Hyg and pETE-Bsd were also created that replicate in episomal form in human cells and facilitate tetracycline-regulated expression of targeted genes. RESULTS: The primate-tropic retroviruses efficiently delivered the regulatory tTA gene into 12 selected human cancer cell lines. Two candidate tumor suppressor genes from the human 3p21-p22 region MAPKAPK3 (3pK) and MLH1 were cloned into the episomal vector and transfected into engineered A9 and KRC/Y cells. The transfectants were subcutaneously grown in SCID mice, and the expression of the transgene was successfully controlled in vivo by tetracycline administered ad libitum in drinking water. The experiments demonstrated that both transgenes did not antagonize the tumorous growth of these cells. CONCLUSIONS: New retroviral and episomal vectors appear particularly suited for tight regulation of genes that cause suppression of cell growth. The generated cell lines can be used in various applications to study the effect of an inducible transgene in human cancer cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

[Antiviral vaccines].

Vaccination has been successful in controlling numerous diseases in man and animals. Smallpox has been eradicated and poliomyelitis is on the verge of being eradicated. The traditional immunization arsenal includes vaccines using live, attenuated, and inactivated organisms. DNA recombinant technology has added two new types of vaccines, i.e. subunit vaccines based on purified antigens produced by genetic engineering in bacterial, yeast, or animal-cell cultures and live recombinant vaccines based on attenuated bacterial or viral vectors. Currently the best known examples of these new vaccines are those using poxvirus vectors (vaccinia virus, canarypox virus, or fowlpox virus) but new vectors are under development. Another application for genetic engineering in the field of vaccinology is the development of DNA vaccines using naked plasmid DNA. This technique has achieved remarkable results in small rodents but its efficacy, safety, and feasibility in man has yet to be demonstrated. Numerous studies are now under way to improve the process. In the field of synthetic vaccines, lipopeptides have shown promise for induction of cell immune response. Development of vaccines for administration by the oral or nasal route may one day revolutionize vaccination techniques. However, effective vaccines against hepatitis C and HIV have stalled in the face of the complexity and pathophysiology of these diseases. These are the greatest challenges confronting scientists at the dawn of the new millennium.

AIDS Vaccines↗
PubMed2003

[Research advances in gene-enhanced tissue engineering].

Gene-enhanced engineering deals with the scientific and technologic endeavour to produce cultured cells or polymer matrices transduced with multiple gene vectors encoding cytokine cDNA by means of genetic engineering technique, to make transduced cells or gene activated matrices highly express according cytokine, and then to enhance certain abilities of the artificial tissue. Up to now, various genes encoding modulatory species of ribonucleic of proteins such as growth factors, receptors, and transcription factors have been used in the context of gene-enhanced tissue engineering and expressed within numerous tissues, including artificial blood vessels, bone, cartilage, skin and urinary system, etc. Many experiments in vitro or in vivo have begun to show good prospects and great potential application of the new approach. We believe great changes will take place in the research field of tissue engineering due to the induction the of genetic engineering, and the new approach will become a very promising and valuable tool for therapy.

Blood Vessel Prosthesis↗
PubMed1997

The physico-chemical factors that govern retrovirus-mediated gene transfer.

The most commonly used vehicle for gene transfer into human target cells is a replication incompetent retroviral vector. The efficiency of gene transfer with this type of vector has proven to be too low to implement effective gene therapy. To date much effort has gone into engineering the genetic and biochemical functionalities of retroviral vectors. Although progress has been achieved, high-efficiency reproducible gene transfer into human cells remains elusive. There are many important physico-chemical and systemic kinetic factors that govern the process of retrovirus-mediated gene transfer. These factors have gone mostly unrecognized to date. The former include the nature of the random Brownian motion of the retrovirus and the physico-chemical forces that determine the binding of the retroviral vector to the target cell. The latter arise from the kinetics of virus binding and entry into the target cell, as well as the kinetic interplay between cell-cycle and retroviral life-cycle events that determine the intracellular fate of the virus. This review describes these processes and how they constrain the efficiency of the gene transfer process.

Animals↗
PubMed1985

A pBR322-derived vector for cloning blunt-ended cDNA: its use to detect molecular clones of low-abundance mRNAs.

In place of the unique Pst I site in pBR322, we have engineered by GC tailing a unique Sma I site bracketed by Pst I sites. The resulting vector, pDE61, and an improved derivative with greater symmetry around the Sma I site, pDE613, have been used to clone blunt-ended duplex cDNA molecules in Escherichia coli in an efficient manner (5 X 10(5) clones from 1 microgram of double-stranded cDNA). When DNA is cloned into the Sma I site, the ability of both vectors to confer ampicillin resistance is lost. Evidence suggests that functional beta-lactamase is made only after the GC-rich sequence containing the Sma I site is deleted: an insert in the Sma I site prevents this. Libraries in either vector, with single or multiple inserts, can be used to generate amplified amounts of cloned heterogeneous cDNA for screening other "target" libraries in a non-homologous vector (e.g., a Bacillus subtilis vector) for cDNA clones of low-abundance mRNAs. Species as infrequent as 0.003% can be readily detected by colony hybridization.

Cloning, Molecular↗
PubMed2002

Engineered Listeria monocytogenes as an AIDS vaccine.

Listeria monocytogenes (Lm) is an attractive vector to elicit T cell immunity because it infects antigen-presenting cells and because infection originates at the mucosa. Lm expressing HIV gag elicits sustained high levels of gag-specific CTL in mice. Since Lm causes disease in immunocompromised hosts, a highly attenuated strain of Lm that requires D-Ala for viability was produced. Attenuated bacteria expressing HIV-1 gag (Lmdd-gag) are as efficient as wild-type recombinants at stimulating gag-specific murine CTL when administered with D-Ala and at boosting human CTL in vitro. Lmdd-gag immunization protects mice from vaccinia-gag challenge and induces mucosal CTL, even after systemic immunization.

AIDS Vaccines↗
PubMed1995

[Gene therapy. A new prospect in the treatment of liver tumors].

To be effective, gene therapy requires three essential elements: a gene to transfer, a vector to carry the gene,and a target cell (here the cancer cell). One type of gene action of particular interest is the suicide gene. When introduced into the cell, the expression of this gene leads to cell death by production of pro-drugs. For example the gene for thymidine kinase from the type 1 herpes simplex virus (HSV1-TK) transforms nucleoside analogues such as ganciclovir into triphosphates which inhibit polymerases. When incorporated into deoxyribonucleic acid during cell division, DNA synthesis is arrested causing cell death. Thus only cells containing the HSV1-TK gene are sensitive to ganciclovir. The most widely used vectors are retroviruses. When these vectors infect a cell, the genes they carry are incorporated into the cell genome and, in the case of suicide genes, lead to cell death. These retrovirus vectors must however be transformed by genetic engineering to remove their capacity for replication so that no viral replication occurs, limiting the effect to the infected cell alone. In cancer therapy, retrovirus appears to be a choice vector since only cells undergoing active division, such as malignant tumour cells, are affected by the gene transfer. Primary or secondary liver tumours are a choice target for gene therapy for several reasons. First, cell division in these tumours is permanent and rapid while the surrounding healthy tissue is in a quiescent state. Secondly, gene therapy could be possible in man since extra-hepatic diffusion of the retrovirus could be avoided by temporary exclusion of the hepatic circulation during perfusion of the retrovirus. Although the long-term risks of using retrovirus vectors for gene therapy (effect on germ cells, oncogenesis), the effect on multiple or large tumours of the liver and the proliferative potential of residual tumour cells remain to be determined, gene therapy for liver cancer is now entering the stage where promising clinical applications may soon be proposed. We have applied gene therapy in in vivo experimental animal models with promising results suggesting that liver cancer may be one of the first applications of this new therapeutic tool in man.

Animals↗