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At least 145 records · Page 8Linked to original sources

PubMed1991

Leukemia inhibitory factor improves survival of retroviral vector-infected hematopoietic stem cells in vitro, allowing efficient long-term expression of vector-encoded human adenosine deaminase in vivo.

Low recovery and poor retroviral vector infection efficiency of hematopoietic stem cells has hindered application of gene therapy for disease affecting blood-forming tissues. Developmental restriction (or death) of stem cells during ex vivo infection has contributed to these difficulties. In these studies we report that the cytokine leukemia inhibitory factor (LIF) directly or indirectly supported the survival of hematopoietic stem cells during culture of bone marrow with vector-producing fibroblasts, resulting in efficient recovery of stem cells able to compete for engraftment in irradiated recipient animals. The infection efficiency of hematopoietic stem cells recovered from these cultures was approximately 80%; and all recipients (20/20) of the LIF-treated marrow were stably engrafted with the progeny of provirus-bearing stem cells. Expression of vector-encoded human adenosine deaminase (hADA) was detected in all recipients at levels averaging 15-50% of endogenous murine ADA in all their hematolymphoid tissues. Survival of stem cells in untreated cultures was approximately 10% of that observed from LIF-treated cultures, resulting in poor engraftment of recipient animals with transplanted cells. The infection efficiency of the few stem cells recovered from untreated cultures, however, was high (approximately 80%), suggesting that LIF did not have an effect on infection efficiency per se, but acted at the level of stem cell survival. Consistent with the poor engraftment observed in the control animals, expression of vector-encoded ADA was only approximately 4-20% of the endogenous levels. These results support the postulated role of LIF as a regulator of hematopoiesis and suggest that cytokine stimulation can positively affect inefficient retroviral vector transduction in hematopoietic stem cells.

Adenosine Deaminase↗

PubMed2002

Adeno-associated viral vectors penetrate human solid tumor tissue in vivo more effectively than adenoviral vectors.

The transduction efficiencies of adeno-associated viral vectors (AAV, serotype 2) and adenovirus vectors (ADV, serotype 5) were examined in three different models of cancer. First, we used flow cytometry to quantitate AAV-GFP or ADV-GFP transduction in 13 cell lines derived from malignant tissue (6 gliomas, 6 mammary cancers, and 1 leukemia). These experiments showed variable transduction efficiency (0%-81%) between the cell lines, with ADV being more effective compared to AAV in 9 of 13 cell lines. Second, spheroids prepared from human glioblastomas were infected with ADV or AAV expressing GFP or lacZ cassettes, and after 2 weeks, uniform reporter gene expression was observed on the spheroid. Whereas AAV produced consistent transduction throughout the spheroids, ADV infection was mainly limited to the outer cell layers of the spheroids, suggesting that AAV were more efficient at penetrating solid tumor tissue. Third, human biopsies from glioblastoma multiforme patients were xenografted into nude rats and grown for 4 weeks followed by viral vector injection. Combined use of high-resolution magnetic resonance imaging (MRI) and histologic analysis allowed the identification of transduced cells and their spatial distribution within the tumors. AAV-mediated transgene expression was observed in cell clusters through the entire tumor, while ADV-mediated transduction was restricted to cells at the tumor periphery. Thus, while AAV and ADV vectors may infect tumor-derived cell lines to a similar degree, AAV penetrated glioblastoma spheroids and xenografts more efficiently compared to ADV vectors. These results suggest that AAV may be suitable for therapeutic gene delivery to malignant tumors.

Adenoviridae↗

PubMed2004

Trans-splicing adeno-associated viral vector-mediated gene therapy is limited by the accumulation of spliced mRNA but not by dual vector coinfection efficiency.

Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model for Duchenne muscular dystrophy. Two different AAV viruses were delivered to the mdx muscle. Similar to previous reports in normal muscle, coinfection efficiency reached approximately 90% in the diseased muscle. This result suggests that coinfection is not a hurdle in dystrophic muscle. Another critical step in the trans-splicing method is the transcription and splicing across the inverted terminal repeat (ITR) junction in the reconstituted genome. To test whether this represented a significant obstacle, we systematically evaluated the transcription, pre-mRNA stability and splicing, and translation in a synthetic lacZ construct that mimicked the reconstituted genome. Although inserting an intron in the lacZ gene had no effect on its expression, inclusion of the ITR junction in the intron reduced expression by 50%. In construct containing the ITR junction, the mRNA transcript level was significantly reduced. This mRNA level reduction was associated with decreased pre-mRNA stability. These data suggest that the accumulation of mRNA is a rate-limiting factor in trans-splicing vector-mediated gene therapy.

Animals↗

PubMed2006

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy.

BACKGROUND: Adeno-associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long-term gene expression. OBJECTIVES: To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. METHODS AND RESULTS: AAV-based and lentiviral vectors were generated that express FIX from the same hepatocyte-specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra-physiological FIX levels (3000-6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self-limiting proinflammatory response (interleukin-6), which probably reflected their ability to efficiently interact with the innate immune system. CONCLUSIONS: AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.

Animals↗

PubMed2000

Q vectors, bicistronic retroviral vectors for gene transfer.

We have developed a retroviral vector that incorporates unique features of some previously described vectors. This vector includes: 3' long terminal repeats (LTRs) of the self-inactivating class; a 5' LTR that is a hybrid of the cytomegalovirus (CMV) enhancer and the mouse sarcoma virus promoter; an internal CMV immediate early region promoter to drive expression of the transduced gene and the neomycin phosphotransferase selectable marker; an expanded multiple cloning site and an internal ribosome entry site. An SV40 ori was introduced into the vector backbone to promote high copy number replication in packaging cell lines that express the SV40 large T antigen. We demonstrate that these retroviral constructs, designated Q vectors, can be used in applications where high viral titers and high level stable or transient gene expression are desirable.

Animals↗

PubMed2004

Why do we need new gene therapy viral vectors? Characteristics, limitations and future perspectives of viral vector transduction.

The use of viruses to transduce genes of interest into mammalian cells has been extremely revolutionary, both in terms of laboratory research and for clinical purposes. This approach has allowed expression and over-expression of proteins of interest as well as the understanding of both virus life cycles and eukaryotic cell mechanisms. Beginning in the late eighties gene transduction has been applied to clinical trials but mainly restricted to cancer treatment and genetic diseases. More recently it has been proposed for the cure of infectious diseases (AIDS), vascular diseases and others (Alzheimer's and Parkinson's disease). Viral vectors have been progressively modified in order to increase their transduction efficiency and to reduce their toxicity, immunogenicity and inflammatory potential. In this respect, much has been done in the last few years. By adding genes belonging to other viral species to the vectors' DNA, scientists were able to re-direct their tissue-specificity or to control protein expression. More recently, in the attempt of overcoming the limitations of each viral species, so-called chimeric viral vectors have been generated by combining favourable features of two or more different viruses into one. This review summarises the main characteristics of the most common viral vectors, including their advantages, limitations and possible future applications. It also briefly discusses development and evolution of chimeric vectors, treated in more details along this entire issue. Finally, we evaluate basic safety aspects, mandatory to consider for the clinical application of viral gene transduction.

Adenoviridae↗

PubMed2005

Nonviral vectors for cancer gene therapy: prospects for integrating vectors and combination therapies.

Gene therapy has the potential to improve the clinical outcome of many cancers by transferring therapeutic genes into tumor cells or normal host tissue. Gene transfer into tumor cells or tumor-associated stroma is being employed to induce tumor cell death, stimulate anti-tumor immune response, inhibit angiogenesis, and control tumor cell growth. Viral vectors have been used to achieve this proof of principle in animal models and, in select cases, in human clinical trials. Nevertheless, there has been considerable interest in developing nonviral vectors for cancer gene therapy. Nonviral vectors are simpler, more amenable to large-scale manufacture, and potentially safer for clinical use. Nonviral vectors were once limited by low gene transfer efficiency and transient or steadily declining gene expression. However, recent improvements in plasmid-based vectors and delivery methods are showing promise in circumventing these obstacles. This article reviews the current status of nonviral cancer gene therapy, with an emphasis on combination strategies, long-term gene transfer using transposons and bacteriophage integrases, and future directions.

Bacteriophages↗

PubMed2002

[Construction and expression of recombinant retrovirus vector carrying HBV vector].

BACKGROUND: To explore the possibility of using retrovirus vector to carry HBV vector, and to prove that replication defective HBV could be normally packaged. METHODS: Two kinds of full length of mutant HBV gene, which express dominant negative mutants, were inserted into retrovirus vector. After recombinant retroviruses were harvested, they were used to infect Hep G2 and 2.2.15 cell line. Then the expression of HBV core antigen in the Hep G2 cell was examined by immune fluorescence, and the existence of recombinant HB virion in the culture medium was examined by PCR. RESULTS: High titer of recombinant retroviruses were obtained in the culture medium of transfected PA317 cell line. Core antigen was detectable in the recombinant retrovirus infected Hep G2 cell. Recombinant HB virion was detectable in the culture medium of recombinant retrovirus infected 2.2.15 cell. CONCLUSIONS: The results suggested that recombinant retrovirus could carry HBV vector and express HBV products. When structural protein is offered by wt-HBV, the recombinant retrovirus may function as HBV vector, therefore it could be used in anti?HBV gene therapy.

Genetic Therapy↗

PubMed1992

Cell line issues related to specific expression vectors: retrovirus vectors.

Retroviruses have received attention as vectors for gene transfer because they can infect a variety of cell types, they integrate stably into the genome of the target cell and they have simple, well-defined structures. Insertion of the gene of interest into a retroviral vector results in the formation of replication-defective particles that can be produced in "packaging" cell lines. Due to the nature of the present generation of vectors and packaging cell lines, multiple recombination events would be needed to produce replication-competent particles. However, the concern still exists that replication-competent virus could arise either in the production of retroviral vector stocks or following their introduction into target cells. The current use of retrovirus vectors and the challenge they present to testing programmes are discussed.

Animals↗

PubMed1985

Comparison of the transient late region expression of SV40 DNA and SV40-based shuttle vectors: development of a new shuttle vector that is efficiently expressed.

Transient expression of an SV40-based shuttle vector similar in design to commonly used vectors is shown to be inefficient when compared to expression of SV40 viral DNA. To eliminate this problem, we have designed and constructed a new vector, pSVPiC, which contains a minimal noninterfering plasmid component, the 885-bp plasmid PiAN7, and two SV40 promoter/origin regions. Transient expression of the SV40 late region from pSVPiC is much more efficient than that from previously used vectors and even more efficient than that from SV40 viral DNA. When the gene for rabbit beta-globin is placed in the late region of pSVPiC, it is also expressed at high levels, indicating that this shuttle vector is generally useful for expressing eukaryotic genes.

DNA Replication↗

PubMed1996

Long-term persistence of defective HSV-1 vectors in the rat brain is demonstrated by reactivation of vector gene expression.

Wild-type HSV-1 is known to persist indefinitely in neurons in the latent state; however, defective HSV-1 vectors, or amplicons, contain only approximately 1% of the HSV-1 genome and persistence of these HSV-1 vectors has not been studied even semiquantitatively in the adult rat brain. Defective HSV-1 vectors contain both an HSV-1 origin of replication and a packaging site, and in the presence of helper virus can undergo DNA replication and packaging into HSV-1 particles. Our prototype defective HSV-1 vector, pHSVlac, uses the HSV-1 immediate-early (IE) promoter to regulate expression of the Escherichia coli lacZ gene. Using cultured neuronal cells, we have previously shown that expression from pHSVlac can be augmented by superinfection with a helper virus. In this study, pHSVlac was delivered into the adult rat striatum or hippocampus, and 2-3 months after gene transfer we utilized superinfection with several replication-incompetent HSV-1 mutants to reactivate expression from pHSVlac in approximately 30% of the number of cells observed at 4 days after gene transfer. Thus, HSV-1 plasmid vectors can persist for at least 2-3 months in at least approximately 30% of the cells which are initially infected.

Animals↗

PubMed1977

A global display of the heart vector (spherocardiogram). Applicability of vector-and polarcardiographic infarct criteria.

The spatial loop of the heart vector can be projected on the Aitoff projection of the globe. The complete information that is contained in the orthogonal leads in displayed on a single graph when the vector magnitudes are indicated by the diameter of circles drawn around the vector projection in short time intervals (spherocardiogram). Diagnostic vector- and polarcardiographic criteria were applied to the spherocardiograms of 68 survivors of inferior myocardial infarction and 30 healthy men. Sensitivity increased from 65% (standard 12-lead ECG) to 87% (VCG criteria) (P less than 0.001) to 94% (VCG and PCG criteria combined), with no false-positive diagnoses. The spherocardiogram can be obtained by use of an "intelligent plotter." In contrast to the vectorcardiogram (VCG) and the polarcardiogram (PCG), it permits the direct visual study of the spatial direction and the spatial magnitude of the heart vector and of their sequential changes during the heart cycle.

Adult↗

PubMedDate not supplied

[Bases for control of arthropod vectors: I--Definitions, bioecology of vectors (author's transl)].

Arthropoda form the most diversified and multitudinous phyllum of the animal kingdom. In this "arthropod world", the authors give the respective position of the arthropoda: a) detrimental to crops, b) venomous and noxious for human being, but mainly those who are vectors of human diseases, say about a hundred species. Biological, ecological and environmental main features of the most important arthropod vectors of human tropical diseases are reviewed. Various factors acting on the relation between pathological agent and vector and between vector and man are considered. Importance and complexity of entomological surveys are emphasized with, as a consequence, the necessity of specialized medical entomologists to manage them.

Acari↗

PubMed1998

A versatile prokaryotic cloning vector with six dual restriction enzyme sites in the polylinker facilitates efficient subcloning into vectors with unique cloning sites.

In large and complex vectors a single restriction enzyme recognition site may be available for introduction of additional DNA requiring the development of linker fragments to create compatible insertion sites. This technology can be time consuming and costly. We describe the construction of a simple phagemid, pSFI, with a polylinker that contains six pairs of dual, rare-cutting, restriction enzyme recognition sites (NotI, SpeI, EcoRV, PstI, SacII, EagI) with multiple unique sites between each pair. This has permitted rapid subcloning of DNA with creation of single flanking restriction enzyme sites. pSFI was used to expedite transfer of viral genes to a LacZ-inducible expression vector and to an adenovirus expression cassette for production of replication-defective virus. The use of this phagemid has facilitated complex vector manipulations and is a valuable adjunct to the family of multifunctional cloning vectors.

Bacteriophages↗

PubMed2001

Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors.

Gene therapy aims to complement or, ideally, correct defective genes. The broad clinical application of this emerging technology requires the development of safe high-capacity gene delivery vehicles that combine efficient transduction of dividing as well as quiescent cells with sustained transgene expression. Here we present a new hybrid vector system that unites favorable attributes of adenoassociated virus (AAV) and adenovirus (Ad) vectors in a single particle. This was achieved by inclusion of Ad packaging elements in different sized recombinant AAV genomes. In the presence of AAV replicative functions and a recombinant helper Ad, AAV/Ad hybrid particles were generated via encapsidation of AAV-dependent replicative intermediates into Ad capsids. In stringent in vitro models based on transduction of proliferating cells we show that AAV/Ad hybrid vectors are superior to Ad vectors in establishing prolonged transgene expression and can be used to deliver DNA fragments of at least 27 kb.

Adenoviruses, Human↗

PubMedDate not supplied

Approaches to vector control: new and trusted. 5. The epidemiological context of vector control.

This paper discusses 2 prominent, contemporary issues in the epidemiological context of insect vector control: (i) the magnitude of the control problem, and (ii) non-linear processes influencing vector control. It concludes that we still cannot reliably measure the scale of some important control problems; e.g., there is considerable uncertainty about the basic reproduction number of malaria. The emergence of new concepts such as strain-specific immunity, and a growing emphasis on disease control as distinct from infection control, mean that some quantitative problems are being redefined more quickly than they are being solved. Population biologists have urged exploration of density-dependent processes which may help or hinder new methods of vector control. This brief review of non-linear phenomena such as facilitation and limitation finds little evidence that they will significantly influence, e.g., the introduction of some novel refractory mechanism into a vector population.

Animals↗

PubMed1989

Avian retroviral vectors derived from avian defective leukemia virus: role of the translational context of the inserted gene on efficiency of the vectors.

We have constructed retroviral vectors derived from the genome of avian erythroblastosis virus ES4 (AEV ES4). The neo selectable gene was substituted for the original v-erbA or v-erbB oncogenes of AEV, either in the same or in a different reading frames. Recombinant retrovirus were rescued and used to infect chicken embryo fibroblasts or quail QT6 cells. When the neo gene was inserted in the same reading frame as the original oncogene, we obtained (1) a high level of expression of the neo gene, (2) a balanced ration of both genomic and subgenomic RNAs, and (3) high titer recombinant viruses. Conversely, when the neo gene was inserted in a reading frame different from that of the original oncogene, we observed (1) a very low level of expression of the neo protein, (2) a predominance of the viral transcript used as translational template for the neo protein synthesis, and (3) low titer recombinant viruses. One of the vectors was used to transfer a human delta-globin gene into avian cells in culture without detectable rearrangement of this gene, but exhibited a deletion within the conserved noncoding region located between the two original oncogenes. Our data provide information for further construction of double expression vectors. Furthermore, three of the vectors would provide helpful tools to identify genetic elements of the virus genome involved in splicing regulation.

Alpharetrovirus↗

PubMed1996

pUCS75, a stable high-copy-number Streptomyces--Escherichia coli shuttle vector which facilitates subcloning from pUC plasmid and M13 phage vectors.

A new Streptomyces-Escherichia coli shuttle vector, pUCS75, has been constructed to permit facile subcloning of DNA from the multiple cloning sites of the pUC plasmid and M13 phage vectors. In contrast to other commonly used shuttle vectors, pUCS75 retains the primary site for second-strand synthesis (ssi) of the parental streptomycete replicon, pIJ101. This sequence can not only enhance structural stability of the plasmid, but also confers on it an elevated copy number when replicated in Streptomyces. Consequently, the vector is useful for cloning sequences containing repeat structures and for allowing the high-level expression of cloned genes.

Bacteriophage M13↗