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Biomedical subjects

T Friedmann

Publications and source records attributed to T Friedmann.

At least 37 records · Page 2Linked to original sources
PubMed1998

Enhanced gene transfer with fusogenic liposomes containing vesicular stomatitis virus G glycoprotein.

Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.

Culture Media↗
PubMed1998

In vitro cell-free conversion of noninfectious Moloney retrovirus particles to an infectious form by the addition of the vesicular stomatitis virus surrogate envelope G protein.

In the absence of envelope gene expression, retrovirus packaging cell lines expressing Moloney murine leukemia virus (MLV) gag and pol genes produce large amounts of noninfectious virus-like particles that contain reverse transcriptase, processed Gag protein, and viral RNA (gag-pol RNA particles). We demonstrate that these particles can be made infectious in an in vitro, cell-free system by the addition of a surrogate envelope protein, the G spike glycoprotein of vesicular stomatitis virus (VSV-G). The appearance of infectivity is accompanied by physical association of the G protein with the immature, noninfectious virus particles. Similarly, exposure in vitro of wild-type VSV-G to a fusion-defective pseudotyped virus containing a mutant VSV-G markedly increases the infectivity of the virus to titers similar to those of conventional VSV-G pseudotyped viruses. Furthermore, similar treatment of an amphotropic murine leukemia virus significantly allows infection of BHK cells not otherwise susceptible to infection with native amphotropic virus. The partially cell-free virus maturation system reported here should be useful for studies aimed at the preparation of tissue-targeted retrovirus vectors and will also aid in studies of nucleocapsid-envelope interactions during budding and of virus assembly and virus-receptor interactions during virus uptake into infected cells. It may also represent a potentially useful step toward the eventual development of a completely cell-free retrovirus assembly system.

Animals↗
PubMed1997

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents.

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 10(5) colony-forming units per ml.

Cell Line↗
PubMed1997

Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retroviruses, and adenoviruses.

We studied the efficiency of plasmid/liposome complexes, Moloney murine leukemia virus-derived (MMLV) retroviruses, pseudotyped vesicular stomatitis virus protein-G (VSV-G)-containing retroviruses, and adenoviruses in delivering genes into the rabbit carotid artery using a silastic collar applied to the adventitia. This method was used for gene transfer because (a) it provides a gene delivery reservoir; (b) no intraluminal manipulations are performed; (c) installation of the collar induces arterial smooth muscle cell (SMC) proliferation and enhances retroviral gene transfer efficiency where target cell proliferation is required. The transfer of the beta-galactosidase (lacZ) marker gene to the adventitia and media occurred with all gene transfer systems. Adenoviruses also transferred the beta-galactosidase gene to some endothelial cells. After 5 days, adenoviral vectors produced the highest gene transfer efficiency with up to 10%+/-6% of cells showing beta-galactosidase activity. Pseudotyped VSV-G retroviruses were also effective in achieving gene transfer in 0.05%+/-0.03% of cells in the adventitia and media. Plasmid/liposome complexes and MMLV retroviruses infected 0.05%+/-0.03% and <0.01%+/-0.01% of cells, respectively. It is concluded that replication-deficient adenoviruses, VSV-G pseudotyped retroviruses, and plasmid/liposome complexes can be used for gene transfer to the arterial wall using the collar method. Because the endothelium remains anatomically present throughout the experiments, the model may be useful for the gene transfer studies involving diffusible or secreted gene products that primarily act on the endothelium. Effects on medial SMC and even endothelium can be achieved from the adventitial side, suggesting an alternative route for the delivery of therapeutically useful genes into the arterial wall.

Adenoviridae↗
PubMed1997

The road toward human gene therapy--a 25-year perspective.

The concept of human gene therapy arose in the early 1970s and has now been the subject of many clinical trials in human patients. To date, there have been no truly convincing therapeutic successes, but any disappointment with that fact is due more to the exaggerated levels of our expectations than to any failure of concept. The concept of human gene therapy has been a startling success and it is now one of the most important driving forces in medicine. Current tools have simply not been adequate for the difficult job of efficient and stable gene delivery, faithful and regulated gene expression and clinical correction of a human disease. Nevertheless, progress toward human gene therapy is rapid and inexorable and the second phase of human gene therapy, the implementation phase, will soon also succeed.

Clinical Trials as Topic↗
PubMed1996

Adenovirus vector infection of chronic lymphocytic leukemia B cells.

Adenovirus vectors have several features that make them attractive for potential use in gene therapy, including a broad tissue tropism and an ability to infect quiescent or postmitotic cells. In light of this, we examined whether recombinant adenovirus vectors could transfer genes into neoplastic cells of patients with chronic lymphocytic leukemia (CLL), a leukemia of "resting" B cells. Using high-titer recombinant adenovirus vectors, we found we could transfer genes encoding beta-galactosidase or murine CD80 (B7-1) into the CLL B cells of all patients tested (n = 10). The efficiency of gene transduction into CLL B cells was approximately 100 to 1,000-fold lower than into HeLa cells at any given multiplicity of infection (MOI). At a MOI of 500, 10% to 70% of the CLL B cells from different patients were made to express the transgene, as assessed by multiparameter flow cytometric analysis. Sustained levels of expression with little loss in the percentage of infected cells were maintained for up to 9 days, at which point the analysis was stopped. We found that CLL B cells have markedly lower expression levels of integrins that facilitate internalization of adenovirus particles into target cells, perhaps accounting, in part, for the reduced efficiency of adenovirus-mediated gene transfer compared with that in HeLa cells. Although HeLa cells express high levels of alpha(v)beta5, and detectable amounts of alpha(v)beta3, we find CLL cells from all patients tested express only low amounts of alpha(v)beta3, and no detectable alpha(v)beta5. Activation of CLL cells via CD40 cross-linking enhances expression of alpha(v)beta3, and induces expression of alpha(v)beta5. This phenotypic change is associated with a fivefold increase in the efficiency of adenovirus-mediated gene transfer into such activated CLL B cells. This study demonstrates that adenovirus vectors can transduce genes into CLL B cells and that the efficiency of gene transduction is enhanced by activation via CD40 cross-linking. This is the first demonstration that high proportions of CLL B cells can be made to express a selected transgene, suggesting that such gene transfer methods may become useful for the study of the pathogenesis and/or treatment of this disease.

Adenoviridae↗
PubMed1996

Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.

Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.

Animals↗
PubMed1996

Generation of packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system.

We have previously shown that the G protein of vesicular stomatitis virus (VSV-G) can be incorporated into the virions of retroviruses. Since expression of VSV-G is toxic to most mammalian cells, development of stable VSV-G packaging cell lines requires inducible VSV-G expression. We have modified the tetracycline-inducible system by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, we show that VSV-G expression is tetracycline-dependent and can be modulated by beta-estradiol. Stable packaging cell lines can readily be established and high-titer pseudotyped retroviral vectors can be generated upon induction of VSV-G expression.

3T3 Cells↗
PubMed1996

Simplified plasmid rescue of host sequences adjacent to integrated proviruses.

We have previously described a Moloney murine leukemia retroviral (MoMLV) vector useful for the generation of anchored long-range maps of complex mammalian genomes. We now report the development of a modified vector carrying the ColE1 origin of replication and the chloramphenicol-resistance (CmR) gene to facilitate the recovery of genomic sequences adjacent to integrated proviruses. We demonstrate the utility of this new vector for the rescue in plasmid form of a 6-kb human fragment containing portions of an Alu element adjacent to the proviral 3'-LTR from an infected human primary fibroblast clone. We generated a sequence-tagged site (STS) from the derived sequence outside the Alu element, and used a somatic cell hybrid mapping panel to assign this STS to human chromosome 10 by a polymerase chain reaction-based assay. We suggest that this new CmR vector will be useful for recovering sequences of genes interrupted by proviral insertion, to generate directional maps with respect to the inserted provirus using the single cleavage sites within the vector, and to generate site-specific STS for defining and mapping the integration site.

Animals↗
PubMed1996

Retrovirol gene transfer in Xenopus cell lines and embryos.

A new class of retroviral vector pseudotypes have an expanded host species range and can be concentrated to high titers by ultracentrifugation. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein. We tested (a) the ability of pseudotyped (pantropic) and unmodified (amphotropic) vectors to stably infect three different Xenopus laevis cell lines, including one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate foreign gene expression in the embryonic CNS. Expression of the neomycin phosphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amphotropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stage 10, 20, and 25 embryos microinjected in the blastocoel or neural tube cavities with concentrated pantropic vector (10(8) cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector containing the coding sequence for the beta-galactosidase gene into the neural tube lumen of 24-h embryos yielded beta-galactosidase expressing cells in the brain. Thus, retroviral vectors provide an additional approach to existing strategies for gene transfer in Xenopus embryos and cell lines.

Animals↗
PubMed1996

The maturation of human gene therapy.

Human gene therapy is two things. It is the concept that human disease might be treated at the level of underlying genetic targets rather than at the level of aberrant metabolism, and it is the implementation of that concept toward a clinical reality. The conceptual aspect is established--gene therapy has become an accepted central driving force in medicine. The second aspect--that of converting the concepts into practical tools for human gene therapy--is maturing rapidly. Over the past several years, the level of expectation had risen to unrealistic proportions and recent initial clinical trials produced disappointment. These early clinical results should, however, be viewed not as failures, but rather as deliberate progress along the learning curve in this new and difficult field of biomedical science.

Clinical Trials as Topic↗
PubMed1996

Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system.

The ability to regulate gene expression via exogenous stimuli will facilitate the study of gene functions in mammalian cells. In the present study, we modified the tetracycline-controlled inducible system by the addition of the ligand-binding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator. A single retroviral vector can transduce both the transactivator gene and the gene of interest controlled by the tTA-inducible promoter into mammalian cells. We show that cell lines expressing the transactivator can readily be established and that expression of the gene of interest depends on the removal of tetracycline and the addition of estrogen. By using this system, cell lines with inducible expression of the G protein of vesicular stomatitis virus, a potentially toxic gene product, were established. The combination of a powerful inducible system and retrovirus-mediated gene transfer can not only be used to study gene function but may also be applied in the future to clinical trials in human gene therapy.

Cell Line↗
PubMed1996

Effect of amniotic fluid on cationic lipid mediated transfection and retroviral infection.

In preparation for foetal gene therapy by intra-amniotic gene application, we have investigated the effect of amniotic fluid on several gene transfer systems. In vitro lipofection of embryonically derived 3T3 cells by several of the tested cationic lipids decreases in the presence of human amniotic fluid, while two formulations, Lipid 67 and Tfx-50, remain highly active. As some body fluids are known to inactivate most retroviral vectors, we investigated the influence of amniotic fluid on the efficiency of infection of 3T3 cells by murine leukaemia virus (MoMLV)-based vectors, including amphotropic and ecotropic retrovirus, and a vesicular stomatitis virus G (VSV-G) glycoprotein pseudotyped retroviral vector. All showed a decrease of infectivity from 21 to 56% in the presence of amniotic fluid. The ecotropic retrovirus is the most infectious under normal conditions as well as in amniotic fluid. Our results suggest that intra-amniotic injection may allow efficient gene transfer using either amniotic fluid-resistant cationic lipids or ecotropic retroviral vectors in a murine in vivo model for foetal gene therapy. The VSV-G-pseudotyped vector, although displaying a decrease of infectivity, remains of great interest for gene delivery, because of its broad host range and because of the high virus titers achievable. Finally, a baculovirus-based vector shows no decrease of its infectivity and does not seem to be affected by amniotic fluid but has only low infectivity on the tested foetal fibroblast cell line.

3T3 Cells↗
PubMed1995

Enhanced levels of lipoperoxides in low density lipoprotein incubated with murine fibroblast expressing high levels of human 15-lipoxygenase.

There is strong experimental evidence that oxidized low density lipoprotein (Ox-LDL) plays an important role in atherosclerosis. However, the mechanisms by which Ox-LDL is formed in vivo are unknown. To test whether 15-lipoxygenase (15-LO) could play a role in oxidation of LDL by cells, we expressed 15-LO activity in murine fibroblasts, which do not normally have 15-LO activity, and tested their ability to modify LDL. Using a retroviral vector, we prepared fibroblasts that expressed 2- to 20-fold more 15-LO activity than control fibroblasts infected with a vector containing beta-galactosidase (lacZ). Compared with LDL incubated with lacZ cells, LDL incubated with 15-LO-containing cells were enriched with lipid hydroperoxides. When these LDL samples were subsequently subjected to oxidative stress, they were more susceptible to further oxidative modification, as judged by increased conjugated diene formation and by increased ability to compete with 125I-Ox-LDL for uptake by macrophages. These findings establish that cellular 15-LO can contribute to oxidative modification of LDL, but the quantitative significance of these findings to the in vivo oxidation of LDL remains to be established.

Animals↗
PubMed1995

Single cell monitoring of growth arrest and morphological changes induced by transfer of wild-type p53 alleles to glioblastoma cells.

Mutation of the p53 tumor suppressor gene is one of the earliest identified genetic lesions during malignant progression of human astrocytomas. To assess the functional significance of these mutations, wild-type (WT) p53 genes were introduced into glioblastoma cell lines having mutant, WT, or null endogenous p53 alleles. Populations of cells with mutant or null endogenous p53 alleles and exogenous WT p53 were spontaneously selected in culture for cells expressing only mutant p53 or no p53, which then displayed a growth and tumorigenic phenotype identical to the parental cells. To determine the phenotypic consequences of WT p53 expression before the occurrence of mutations, we developed a single cell assay to monitor WT p53-dependent transcription activity. Transfer and expression of exogenous WT p53 genes to cells with endogenous mutant or deleted, but not WT, p53 alleles caused growth arrest and morphological changes, including increased cell size and acquisition of multiple nuclei. This supports the hypothesis that genetic lesions of the p53 gene play an important role in the genesis of astrocytomas. Furthermore, the high sensitivity of the episomal single cell reporter strategy developed here has potential clinical applications in the rapid screening of patients for germ-line mutations of the p53 gene or any other gene with known targets for transcriptional transactivation.

Alleles↗