Showing papers on "Vector (molecular biology) published in 1995"

Efficient gene transfer into human hepatocytes by baculovirus vectors

Abstract: Viral vectors are the most efficient tools for gene delivery, and the search for tissue-specific infecting viruses is important for the development of in vivo gene therapy strategies. The baculovirus Autographa californica nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells, and its host specificity is supposed to be restricted to arthropods. Here we demonstrate that recombinant A. californica nuclear polyhedrosis virus is efficiently taken up by human hepatocytes via an endosomal pathway. High-level reporter gene expression from heterologous promoters was observed in human and rabbit hepatocytes in vitro. Mouse hepatocytes and some other epithelial cell types are targeted at a considerably lower rate. The efficiency of gene transfer by baculovirus considerably exceeds that obtained by calcium phosphate or lipid transfection. These properties of baculovirus suggest a use for it as a vector for liver-directed gene transfer but highlight a potential risk in handling certain recombinant baculoviruses.

Methods for construction of adenovirus vectors.

Abstract: Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.

Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells

Abstract: To determine which features of retroviral vector design most critically affect gene expression in hematopoietic cells in vivo, we have constructed a variety of different retroviral vectors which encode the same gene product, human adenosine deaminase (EC 3.5.4.4), and possess the same vector backbone yet differ specifically in transcriptional control sequences suggested by others to be important for gene expression in vivo. Murine bone marrow cells were transduced by each of the recombinant viruses and subsequently used to reconstitute the hematopoietic system of lethally irradiated recipients. Five to seven months after transplantation, analysis of the peripheral blood of animals transplanted with cells transduced by vectors which employ viral long terminal repeats (LTRs) for gene expression indicated that in 83% (77/93) of these animals, the level of human enzyme was equal to or greater than the level of endogenous murine enzyme. Even in bone marrow transplant recipients reconstituted for over 1 year, significant levels of gene expression were observed for each of the vectors in their bone marrow, spleen, macrophages, and B and T lymphocytes. However, derivatives of the parental MFG-ADA vector which possess either a single base mutation (termed B2 mutation) or myeloproliferative sarcoma virus LTRs rather than the Moloney murine leukemia virus LTRs led to significantly improved gene expression in all lineages. These studies indicate that retroviral vectors which employ viral LTRs for the expression of inserted sequences make it possible to obtain high levels of a desired gene product in most hematopoietic cell lineages for close to the lifetime of bone marrow transplant recipients.

Attenuated Shigella as a DNA Delivery Vehicle for DNA-Mediated Immunization

Abstract: Direct inoculation of DNA, in the form of purified bacterial plasmids that are unable to replicate in mammalian cells but are able to direct cell synthesis of foreign proteins, is being explored as an approach to vaccine development. Here, a highly attenuated Shigella vector invaded mammalian cells and delivered such plasmids into the cytoplasm of cells, and subsequent production of functional foreign protein was measured. Because this Shigella vector was designed to deliver DNA to colonic mucosa, the method is a potential basis for oral and other mucosal DNA immunization and gene therapy strategies.

Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector.

Abstract: Adenoviral vectors are widely used as highly efficient gene transfer vehicles in a variety of biological research strategies including human gene therapy. One of the limitations of the currently available adenoviral vector system is the presence of the majority of the viral genome in the vector, resulting in leaky expression of viral genes particularly at high multiplicity of infection and limited cloning capacity of exogenous sequences. As a first step to overcome this problem, we attempted to rescue a defective human adenovirus serotype 5 DNA, which had an essential region of the viral genome (L1, L2, VAI + II, pTP) deleted and replaced with an indicator gene. In the presence of wild-type adenovirus as a helper, this DNA was packaged and propagated as transducing viral particles. After several rounds of amplification, the titer of the recombinant virus reached at least 4 x 10(6) transducing particles per ml. The recombinant virus could be partially purified from the helper virus by CsCl equilibrium density-gradient centrifugation. The structure of the recombinant virus around the marker gene remained intact after serial propagation, while the pBR sequence inserted in the E1 region was deleted from the recombinant virus. Our results suggest that it should be possible to develop a helper-dependent adenoviral vector, which does not encode any viral proteins, as an alternative to the currently available adenoviral vector systems.

The cryptic life style of adeno-associated virus

Abstract: Although 80-90% of adults are seropositive for antibodies against the human parvovirus adeno-associated virus (AAV), infection has not been associated with either symptoms or disease. In cell culture, AAV infection is not productive unless there is a coinfection with a helper virus, either adenovirus or any type of herpes virus; in the absence of a helper virus coinfection the viral genome is integrated into the genome, usually at a specific site on chromosome 19q13.3-qter. The integrated genome can be activated and rescued by subsequent super infection by a helper virus. The high frequency of site-specific integration by AAV and the lack of associated disease have encouraged the use of AAV as a vector for gene therapy. This review will focus on the molecular mechanisms involved in the establishment of, and rescue from, the latent state and their relevance to use of AAV as a vector.

Systemic gene therapy: biodistribution and long-term expression of a transgene in mice

Abstract: We have investigated the in vivo efficacy of a systemic gene transfer method, which combines a liposomal delivery system (DLS liposomes) with episomally replicative DNA plasmids to effect long-term expression of a transgene in cells. A single i.v. injection of a plasmid DNA vector containing the luciferase gene as a marker was administered with the DLS liposomes in BALB/c mice. The luciferase gene and its product were found in all mouse tissues tested as determined by PCR analysis and immunohistochemistry. Luciferase activity was also detected in all tissues tested and was present in lung, liver, spleen, and heart up to 3 months postinjection. In contrast to the nonepisomal vectors tested (pRSV-luc and pCMVintlux), human papovavirus (BKV)-derived episomal vectors showed long-term transgene expression. We found that these episomal vectors replicated extrachromosomally in lung 2 weeks postinjection. Results indicated that transgene expression in specific tissues depended on the promoter element used, DNA/liposome formulation, dose of DNA per injection, and route of administration.

Viral Vectors in Gene Therapy

Abstract: The use of DNA as a drug is both appeali ng and simple in concept. Indeed in many instances the feasibility of such an approach has been established using model systems. In practical terms, however, the delivery of DNA to human

AAV-mediated delivery of DNA to cells of the nervous system

Abstract: The invention relates to a method of delivering exogenous DNA to a target cell of the mammalian central nervous system using an adeno-associated virus (AAV)-derived vector. Also included in the invention are the AAV-derived vectors containing exogenous DNA which encodes a protein or proteins which prevent or treat nervous system disease, and a method of preventing or treating such disease.

Development and application of herpes simplex virus vectors for human gene therapy

Abstract: Advances in understanding the molecular basis of human disease and the development of recombinant DNA methods is rapidly creating new means of disease diagnosis and treatment. Among the most revolutionary developments are technologies for transfer of therapeutic genes to the human body to treat both inherited and acquired disease. Gene therapy offers considerable promise for ameliorating otherwise intractable diseases such as immunopathological conditions, cancer, heart disease, and various metabolic and neurodegenerative syndromes. To fulfill this promise, more efficient and effective methods of gene delivery and appropriate gene expression must be developed. The lack of such techniques is currently the most significant impediment to the use of genetic therapy. Both viral and nonviral delivery systems are under development for specific gene-therapy applications. Herpes simplex virus (HSV) represents a novel vector system for gene delivery to the nervous system and other tissues. HSV is able to establish latency in nondividing neuronal cells in which genomes persist long-term but do not integrate or alter host-cell metabolism and that carry a promoter system uniquely capable of escaping repression that shuts off the expression of HSV-lytic genes during latency. This review examines efforts to create defective HSV vectors that are safe, noncytotoxic, and applicable to the treatment of cancer and diseases affecting peripheral nerves. Perhaps the most important use of HSV vectors will be for the treatment of neurodegenerative diseases of the brain, but additional studies are required to improve the design of promoters to ensure regulatable or effective levels of therapeutic gene expression.

Nucleotide sequence encoding the enzyme i-scei and the uses thereof

Abstract: An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid.

Abstract: Vectors expressing thefirst 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals ( 2,000 mIU/ml). This is the first report of the use of DNA-based immunizationforthegenerationofimmuneresponsestoanHCVprotein.Inaddition,thesefindingsshowthat itispossibletoelicitresponsestoviralepitopesfromtwodistinctvirusesviaDNAimmunizationwithchimeric vectors. Hepatitis C virus (HCV) has been shown to be the major causeofparenterallyacquiredviralnon-A,non-Bhepatitis(1). In most cases, the virus causes a persistent infection, and previous studies clearly indicate an association between chronic infection with HCV and the development of chronic liver diseases,cirrhosis,andhepatocellularcarcinoma(20,33,38).The prevalence of the virus throughout the world varies between 0.4 and 2%, although levels as high as 10 to 20% have been reportedinEgypt(15).Improvementsindiagnostictechniques for the detection of HCV antibodies (Abs) have reduced the risk of infection through contaminated blood products. However, community-acquired infection is still common, particularly in high-risk groups, with a significant proportion of cases in the United States associated with no known risk factors (1). Therefore, a vaccine to protect against infection by HCV remains necessary. The genome of HCV (9.5 kb; positive-strand RNA), similar in organization to those of the flavi- and pestivirus groups, is expressed as a single polypeptide from a long open reading frame, which is subsequently cleaved by viral and cellular proteases. The structural proteins consist of the core, forming the nucleocapsid, and at least two glycosylated envelope proteins, E1 and E2 (5, 16). The sequence of the viral envelope exhibits both genotype and subtype variation at the amino acid level (17), with the presence of a hypervariable region in the E2 domain (31, 32, 46). This region, which contains linear Abbinding epitopes, appears to be a target for human immune responses and has been suggested as a possible site through which the selection of immune escape mutants can occur (47). By contrast, the nucleocapsid is well conserved between genotypes (3, 18), and both cytotoxic T-lymphocyte (CTL) and B-cell determinants have been mapped to this antigen (2, 21, 22, 30, 42). CTL generated against internal viral antigens are thought to be important for an effective immune response and can lead to cross-protection against different viral strains. Protective immunity has been successfully induced in animals by usingthenucleoproteinofhepatitisBvirus(HBV)(19,29)and rabies virus (11), suggesting that the HCV nucleocapsid could

Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response.

Abstract: Gene therapy for heart diseases requires availability of an efficient vector for gene transfer into myocardium. Recombinant adenovirus expressing the Escherichia coli beta-galactosidase (beta-Gal) gene was shown to infect rat cardiocytes efficiently in vivo. However, a time course of gene expression showed that transgene expression was maximal during the first week following injection, then declined and disappeared by day 21. An immunosuppressive treatment prolonged beta-Gal expression for at least 21 days. On the contrary, a preimmunization of the animals by two intraperitoneal injections of the vector led to a decreased transgene expression 48 hr after intramyocardial injection and to a barely detectable expression at the sixth day. Appearance of adenovirus neutralizing antibodies in preimmunized animals could have contributed to such a refractoriness to further adenoviral infection. Finally, a neonatal intrathymic injection of the vector was able to induce long-term LacZ expression for more than 2 months after heart injection, although neutralizing as well as anti-beta-Gal antibodies were detected in sera of the animals. These results indicate that an immune response against first-generation replication-defective adenoviral vectors is a major cause of transient transgene expression, a cellular response being most probably responsible for ablation of transgene expression in immunocompetent animals.

Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells.

Abstract: Immortalized cell lines have been used to study infection and replication of adeno-associated virus (AAV) in culture, but primary cells presumably provide a better model for AAV behavior in animals. Here, we have evaluated the ability of AAV vectors to transduce primary and immortalized strains of human epithelial cells and fibroblasts. Two AAV vectors were used, one that transduced an alkaline phosphatase gene (AAV-LAPSN), and one that transduced a beta-galactosidase/neomycin phosphotransferase fusion gene (AAV-L beta geo). The transduction efficiency of the AAV-LAPSN vector, quantitated by measurement of alkaline phosphatase-positive cell foci following infection, was 10 to 60 times greater in immortalized human cells than in primary cells, and total alkaline phosphatase activity in cell lysates was 40 to 50 times greater in immortalized cells. The AAV-L beta geo vector gave similar results. In contrast, the transduction efficiency of a retrovirus vector encoding alkaline phosphatase was equivalent in primary and immortalized cells. Analysis of the quantity and state of the AAV vector genomes in cells showed that primary and immortalized cells contained comparable numbers of vector copies per cell and that the vast majority of vector DNA was not integrated into the cell genome. Additionally, the level of AAV vector-derived message paralleled the transduction efficiency. These results indicate that the block to functional transduction in primary cells occurred after virus entry and limited the abundance of vector-derived message. Data from AAV transduction in cultures of human cells containing immortalizing genes suggest that cellular changes secondary to the introduction of immortalizing genes increased permissiveness for transduction by AAV vectors. In summary, our data demonstrate that AAV vectors transduce primary human cells much less efficiently than immortalized cells and indicate the importance of using primary cells to evaluate AAV vectors for gene therapy applications.

Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10.

Abstract: The application of gene transfer techniques to organ transplantation offers the potential for modulation of immunity directly within an allograft without systemic side effects. Expression vectors and promoter elements are important determinants of gene transfer and expression. In this study, various vectors (naked plasmid DNA, retroviral vector, herpes simplex viral vector, and adenoviral vector) with various promoters (RSV-LTR, SV40, MuLV-LTR, HCMVie1) were directly compared to demonstrate the successful gene transfer and expression of beta-galactosidase in murine myoblasts in vitro and within murine heterotopic, nonvascularized cardiac isografts or allografts in vivo. Expression of transferred genes was not toxic to cells and strength of expression varied according to the type of vector. Plasmid DNA was expressed in myocytes, retroviral vector was expressed in the graft infiltrating cells, and herpes simplex and adenoviral vectors were expressed in both myocytes and graft-infiltrating cells. Preliminary studies evaluated the ability of these vectors to deliver immunologically important signals. Allografts injected with pSVTGF-beta 1, a plasmid-encoding transforming growth factor beta 1 (TGF-beta 1) under the control of the SV40 promoter, showed significant prolongation of graft survival of 26.3 +/- 2.5 days compared with 12.6 +/- 1.1 days for untreated allografts, and 12.5 +/- 1.5 days for the allografts injected with control plasmid (P < 0.05). Allografts injected with MFG-vIL-10, a retroviral vector encoding viral interleukin-10 under the control of the MuLV-LTR, showed prolongation of graft survival of 36.7 +/- 1.3 days versus 12.6 +/- 1.1 days for the untreated allograft, and 13.5 +/- 2.0 days for the allografts injected with control retroviral vector (P < 0.001). Both vectors were transcriptionally active in vivo and did not appear to have toxic effects. Gene therapy for transplantation can induce transient expression of immunologically relevant molecules within allografts that impede immune activation while avoiding the systemic toxicity of conventional immunosuppression.

Retroviruses as vectors

Abstract: Recombinant retroviruses have long been used to deliver heterologous genes to mammalian cells. Convenient packaging cell lines and vector plasmids have been distributed widely and 'home-made' retroviral vectors have now become a useful research tool in many laboratories. Compared to more traditional methods of gene transfer, retroviral vectors are extraordinarily efficient gene delivery vehicles which cause no detectable harm as they enter their target cells. In the nucleus the retroviral necleic acid becomes integrated into chromosomal DNA, ensuring its long-term persistence and stable transmission to all future progeny of the transduced cell. Up to 8 kilobases of foreign gene sequence can be packaged in a retroviral vector and this is more than enough for most gene therapy applications. Retroviral vectors can also be manufactured in large quantities to meet very stringent safety specifications. They have therefore been selected as the vectors of choice in 80% of the clinical gene therapy trials that have been approved to date. So far there have been no reported short- or long-term toxicity problems associated with their use in human gene therapy trials, now dating back to 1989. However, despite this impressive record, there is still great scope (and need) for the development of new, improved retroviral vectors and packaging systems to fuel further advances in the field of human gene therapy. In the following discussion, existing retroviral vectors are reviewed and current areas of technological development are emphasised.

A sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo

Abstract: The direct intramuscular delivery of naked plasmid DNA has been demonstrated to allow expression of encoded heterologous genes in the target myocytes. The method has been employed to elicit immunization based upon delivery of antigen encoding plasmid DNA. For application in the context of achieving anti-tumor immunization against antigenic transforming oncoproteins, delivery of plasmid DNAs encoding these molecules would create significant potential safety hazards. As an alternative to DNA polynucleotide vectors, we explored the utility of mRNA vehicles for inducing foreign gene expression in muscle cells in vivo. Synthetic reporter-gene encoding mRNA transcripts were derived for this analysis. The Sindbis virus vector was also used to derive luciferase mRNA transcripts which possessed self-replication capacity. In these studies, it could be shown that the replicative vector was capable of directing significantly elevated levels of reporter gene expression in myocytes compared to a non-replicative mRNA species. In addition, the replicative species was capable of achieving significantly prolonged levels of in vivo gene expression compared to non-replicative mRNA. Both of these characteristics will make replicative mRNA vectors of utility for polynucleotide-based immunization protocols.

Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption

Abstract: The delivery of viral vectors to the brain for treatment of intracerebral tumors is most commonly accomplished by stereotaxic inoculation directly into the tumor. However, the small volume of distribution by inoculation may limit the efficacy of viral therapy of large or disseminated tumors. We have investigated mechanisms to increase vector delivery to intracerebral xenografts of human LX-1 small-cell lung carcinoma tumors in the nude rat. The distribution of Escherichia coli lacZ transgene expression from primary viral infection was assessed after delivery of recombinant virus by intratumor inoculation or intracarotid infusion with or without osmotic disruption of the blood-brain barrier (BBB). These studies used replication-compromised herpes simplex virus type 1 (HSV; vector RH105) and replication-defective adenovirus (AdRSVlacZ), which represent two of the most commonly proposed viral vectors for tumor therapy. Transvascular delivery of both viruses to intracerebral tumor was demonstrated when administered intraarterially (i.a.) after osmotic BBB disruption (n = 9 for adenovirus; n = 7 for HSV), while no virus infection was apparent after i.a. administration without BBB modification (n = 8 for adenovirus; n = 4 for HSV). The thymidine kinase-negative HSV vector infected clumps of tumor cells as a result of its ability to replicate selectively in dividing cells. Osmotic BBB disruption in combination with i.a. administration of viral vectors may offer a method of global delivery to treat disseminated brain tumors.

Retroviral vector particles displaying the antigen-binding site of an antibody enable cell-type-specific gene transfer.

Abstract: Retroviral vectors are the most efficient tool for stably introducing genes into vertebrate cells However, their use is limited by the host range of the retrovirus from which they are derived To alter the host range, we recently constructed retrovirus vector particles, derived from spleen necrosis virus, that display a single-chain antigen-binding site of an antibody (scA) on the viral surface (T-H T Chu, I Martinez, W Sheay, and R Dornburg, Gene Ther 1:292-299, 1994) Using a hapten (2,4-dinitrophenol) model system, we showed that such particles are competent for infection In this study, we repeated our experiments using an scA directed against a cell surface protein expressed on various human carcinoma cell lines We found that such scA-displaying particles can efficiently infect human cells that express the corresponding antigen Particles with wild-type spleen necrosis virus envelope are minimally infectious on such cells The addition of the original monoclonal antibody to the viral vector particle solution prior to infection inhibited infection This competition assay showed that the infection is mediated by the antibody moiety and, therefore, is antibody specific These data indicate that retroviral vectors with antibody-envelope fusion proteins may be a valuable tool for selectively introducing genes into any target cell

Gene expression from recombinant viral vectors in the central nervous system after blood-brain barrier disruption

Abstract: DIRECT INTRACEREBRAL INJECTION of recombinant adenoviral vectors within the brain parenchyma or the ventricular system results in a limited volume of distribution of virus, as demonstrated by transgene expression. Global delivery to the central nervous system may increase the use of these ve

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors.

Abstract: We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying the Neo r selectable marker and the Escherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%. Neo r and lacZ genes were transcribed in vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss

Abstract: We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid.

Replication-competent herpes simplex virus mediates destruction of neoplastic cells

Abstract: A method for killing malignant brain tumor cells in vivo entails providing replication competent herpes simplex virus vectors to tumor cells. A replication competent herpes simplex virus vector, with defective expression of the γ34.5 gene and the ribonucleotide reductase gene, specifically destroys tumor cells, is hypersensitive to anti-viral agents, and yet is not neurovirulent.