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T Friedmann

Publications and source records attributed to T Friedmann.

At least 37 records · Page 2Linked to original sources
PubMed1995

Differentiation of PC12 cells by infection with an HSV-1 vector expressing nerve growth factor.

We have developed an HSV-1 vector with mutations in the viral IE 3 and VP16 genes that expresses mouse beta nerve growth factor (NGF) from a latency associated transcript (LAT) promoter modified by insertion of a Rous sarcoma virus (RSV) enhancer. The backbone double mutant vector has reduced cytotoxicity compared with a single mutant deleted for IE 3 and is able to express the reporter luciferase gene in rat pheochromocytoma (PC12) cells at low but relatively stable levels in vitro. Intracellular NGF levels of approximately 3 pg per mg of cellular protein were detected in infected PC12 cells for at least 7 days after infection. Furthermore, infected PC12 cells exhibited differentiated morphology marked by neurite outgrowth similar to that found after exposure of PC12 cells to purified 2.5S beta NGF. A persistent level of 0.2 to 0.3 pM of the expressed NGF was detected in the culture media. The NGF-expressing vector also showed reduced cytotoxicity compared with that of the parental virus in infected PC12 cells. In PC12 cells that overexpress the human proto-oncogene bcl-2, the cytotoxicity of both viruses was significantly reduced. These studies demonstrate that the reduced cytotoxicity of the IE 3-VP16 double mutant virus and the increased duration of transgene expression from the RSV-modified LAT promoter permit terminal differentiation of PC12 cells after infection with an NGF-expressing HSV-1 vector.

Animals↗
PubMed1995

Efficient in vivo transduction of the neonatal mouse liver with pseudotyped retroviral vectors.

Ideal methods for human gene therapy will eventually include direct gene transfer to defective tissues in a patient in vivo. Toward that goal, we have used high titer, pseudotyped retroviral vectors expressing genes for the Escherichia coli beta-galactosidase (lacZ) or hepatitis B virus surface antigen (HBsAg) to infect mouse liver by in vivo direct injection into the liver parenchyma. We have found that a single percutaneous injection of small volumes of vectors into the newborn mouse liver leads to transduction of at least 25-30% of the hepatocytes throughout the liver, as judged by in situ staining of liver sections for beta-gal activity at 4 weeks after injection. We have demonstrated that stable levels of HBsAg were also detected in the circulation of injected mice up to 4 months after HBsAg-vector injection. We suggest that the high efficiency of in vivo transduction in the neonatal liver and subsequent stable transgene expression by high-titer pseudotyped retroviral vectors in the absence of an invasive partial hepatectomy may effectively be applied to gene therapy studies in a number of human liver disease [corrected].

Animals↗
PubMed1994

A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes.

Retroviral vectors have been central components in many studies leading to human gene therapy. However, the generally low titers and inefficient infectivity of retroviral vectors in human cells have limited their use. We previously reported that the G protein of vesicular stomatitis virus can serve as the exclusive envelope protein component for one specific retroviral vector, LGRNL, that expresses vesicular stomatitis virus G. We now report a more useful general transient transfection scheme for producing very high-titer vesicular stomatitis virus G-enveloped pseudotypes from any Moloney murine leukemia-based retroviral vector without having to rely on the expression of the cytotoxic G protein from the retroviral vector itself. We also demonstrate very high efficiency of infection with a pseudotyped lacZ vector in primary mouse hepatocytes. We suggest that pseudotyped retroviral vectors carrying reporter genes will permit genetic studies in many previously inaccessible vertebrate and invertebrate systems. Furthermore, because these vectors represent retroviral vectors of sufficiently high titer to allow efficient direct retroviral-mediated in vivo gene transfer, we also suggest that pseudotyped vectors carrying potentially therapeutic genes will become useful to test the potential for in vivo gene therapy.

Animals↗
PubMed1994

Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish.

The zebrafish is rapidly becoming a popular model system for the study of vertebrate development because it is ideal for both embryological studies and genetic analysis. To determine if a retroviral vector pseudotyped with the envelope glycoprotein of the vesicular stomatitis virus could infect zebrafish embryos, and in particular, the cells destined to become the germ line, a pseudotyped virus was injected into blastula-stage zebrafish embryos. Fifty-one embryos were allowed to develop and eight transmitted proviral DNA to their progeny. Founders were mosaic, but as expected, transgenic F1's transmitted proviral DNA in a Mendelian fashion to the F2 progeny. Transgenic F1 fish inherited a single integrated provirus, and a single founder could transmit more than one viral integration to its progeny. These results demonstrate that this pantropic pseudotyped vector, originally developed for human gene therapy, will make the use of retroviral vectors in zebrafish possible.

Animals↗
PubMed1994

Analysis of the p53 gene and its expression in human glioblastoma cells.

Chromosome 17p has been shown to be an early and frequent target for loss of heterozygosity through mitotic recombination in astrocytomas. These losses are frequently accompanied by point mutations in the p53 gene of the remaining allele, resulting in loss of wild type p53 function. However, a fraction of astrocytomas retain constitutional heterozygosity and do not have p53 mutations; some of these lose wild type p53 activity through binding to the protein product of amplified mdm2 genes. To test whether loss of wild type p53 biological function is a necessary step in astrocytoma progression we analyzed p53 expression and biological function in 13 glioma cell lines. All the cell lines expressed a 2.8-kilobase p53 transcript and showed various amounts of p53 protein by immunoprecipitation, except for cell line LN-Z308 which had only a small truncated p53 mRNA and no protein expression. To test whether the p53 expressed in these cell lines was functionally wild type or mutant we transfected them with a plasmid construct harboring a chloramphenicol acetyltransferase (CAT) reporter gene under the control of transcriptional elements that are induced by wild type but not mutant p53. Four lines were shown to retain wild type p53 function. Sequencing of the p53 gene in two of these cell lines confirmed the wild type genotype. These results show that inactivation of the p53 gene is not an obligatory step in glioblastoma genesis. This suggests either that two pathways (p53 inactivation dependent or independent) may lead to a tumor group classified histologically as glioblastoma or that in some cases p53 mutations are bypassed due to the presence of mutations in downstream effector genes.

Animals↗
PubMed1994

Pantropic retroviral vector-mediated gene transfer, integration, and expression in cultured newt limb cells.

Limb regeneration is a unique developmental phenomenon restricted to certain urodeles in which limb cells dedifferentiate and produce the blastema and then redifferentiate into the tissues that compose the missing part. Genetic modification of the blastema cells would greatly facilitate understanding the programmed gene expression that results in the reconstitution of the limb. To test whether pantropic retroviral vectors pseudotyped with the vesicular stomatitis virus G glycoprotein could mediate gene transfer into blastema cells, we infected a stable newt limb cell line and demonstrated integration and expression of the provirus. Thus, pantropic retroviral vectors offer a new tool for the study of limb regeneration and other developmental phenomena in amphibia.

Animals↗
PubMed1994

Gene therapy for neurological disorders.

It is very likely that genetic therapies will soon be established for a number of diseases and injuries of the central nervous system. The targets for these therapies will not only be the underlying causative genes, but also neurotrophic factors that affect the survival and function of neurons as well as aberrant secondary metabolic and neurotransmitter functions.

Animals↗
PubMed1994

Generation of high-titer pseudotyped retroviral vectors with very broad host range.

Encapsidation of the VSV G protein into the virions of MoMLV-derived retroviral vectors in the absence of other VSV-encoded proteins is shown to be an efficient process, although the exact mechanism for this process is currently unclear. Unlike the conventional retroviral vectors bearing the amphotropic envelope protein, the pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation. This property of the pseudotyped virus enables the generation of high-titer retroviral vector stocks and has potential application for in vivo gene therapy studies. We have found as many as four copies of a pseudotyped vector to integrate into the genome of a single cell when a high multiplicity of infection was used to infect the cells. Multiple integration events were not observed with amphotropic retroviral vectors, probably because of their low virus titers. In addition, when retroviral vectors are pseudotyped with the VSV G protein, they acquire the host range of VSV and are able to infect nonmammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera. Since techniques for efficient gene transfer in some of these nonmammalian systems are not currently available, retrovirus-mediated gene transfer described here should be useful for transgenic and other genetic studies in lower vertebrate species. The inability to establish a stable cell line expressing the VSV G protein, however, limits large-scale production of the pseudotyped retroviral vectors. Generation of stable packaging cell lines for the pseudotyped retroviral vectors is a major challenge for the future.

3T3 Cells↗
PubMed1994

High efficiency retroviral-mediated gene transduction into CD34+ cells purified from peripheral blood of breast cancer patients primed with chemotherapy and granulocyte-macrophage colony-stimulating factor.

The hematopoietic system has been one of the major targets for designing human gene therapy protocols. In the present system, we have transduced LNL6, one of the most commonly used retroviral vectors in gene therapy, into purified CD34+ cells from peripheral blood of patients primed with chemotherapy and granulocyte-macrophage colony-stimulating factor (GM-CSF). Purification of CD34+ cells was achieved by incubation with a murine anti-CD34 monoclonal antibody (9C5), and subsequently with paramagnetic microspheres (Dynal) coated with sheep anti-mouse IgG1 (Fc). The CD34+ cells were released from the beads by treatment with chymopapain. Flow cytometry analysis using the anti-CD34 antibody HPCA-2-FITC targeted at another epitope of CD34 showed that 78-97.5% of the cells thus purified were CD34+. After retroviral-mediated gene transfer, polymerase chain reaction (PCR) analysis revealed that 67-100% of the hematopoietic colonies contained the marker gene neo, indicating that CD34+ cells purified by immunomagnetic microsphere method from peripheral mononuclear cells primed with hematopoietic growth factors are highly susceptible to retroviral-mediated gene transfer. The expression of neo as determined by reverse transcription (RT)-PCR appeared to be unstable and not persistent. Taken together, our data suggest that LNL6 is a suitable vector for gene marking of hematopoietic progenitors but not for gene therapy protocols based on persistent gene expression.

Antigens, CD↗
PubMed1994

Improved cell survival by the reduction of immediate-early gene expression in replication-defective mutants of herpes simplex virus type 1 but not by mutation of the virion host shutoff function.

Derivatives of herpes simplex virus type 1 (HSV-1) have elicited considerable interest as gene transfer vectors because of their ability to infect a wide range of cell types efficiently, including fully differentiated neurons. However, it has been found that infection of many types of cell with vectors derived from replication-defective mutants of HSV-1 is associated with cytopathic effects (CPE). We have previously shown that viral gene expression played an important role in the induction of CPE caused by an HSV-1 mutant deleted for the essential immediate-early gene 3 (IE 3) (P.A. Johnson, A. Miyanohara, F. Levine, T. Cahill, and T. Friedmann, J. Virol. 66:2952-2965, 1992). We have investigated which viral genes might be responsible for CPE by comparing the ability of each of the individual genes expressed by an IE 3 deletion mutant during a nonproductive infection to inhibit biochemical transformation after cotransfection of BHK or CV-1 cells with a selectable marker gene. Transfection of IE genes 1,2, and 4 individually all caused a marked inhibition of colony formation, while transfection of IE 5 and the large subunit of ribonucleotide reductase had little effect. These results suggested that it would be necessary to mutate or reduce the expression of nearly all HSV-1 IE genes to reduce virus-induced CPE. Therefore, we have used VP16 mutants, which are unable to transinduce IE gene expression (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989), to derive two replication-defective strains: 14H delta 3, which is deleted for both copies of IE 3, and in 1850 delta 42, which has a deletion in the essential early gene UL42. The IE 3-VP16 double mutant, 14H delta 3, is significantly less toxic than a single IE 3 deletion mutant over a range of multiplicities of infection, as measured in a cell-killing assay, and has an enhanced ability to persist in infected cells in a biologically retrievable form. In contrast, the UL42-VP16 double mutant, in 1850 delta 42, showed reduced toxicity only at low multiplicities of infection. To test the role of the virion host shutoff function as an additional candidate to influence virus-induced CPE, we have introduced a large insertion mutation into the virion host shutoff gene of an IE 3 deletion mutant and the double mutant 14H delta 3.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease.

We have examined several aspects of neurotransmitter function in the brains of mice carrying a deletion mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). During the first 6 weeks of postnatal development, dopamine levels in whole-brain extracts from the mutant mice (HPRT-) failed to increase at rates comparable to normal animals, resulting in 40% lower dopamine levels throughout adulthood. Regional analysis in adult animals showed the caudoputamen to be the most severely affected region, with dopamine deficits of 48-64%. Dopamine levels in other regions were normal or less severely affected. The decrease in dopamine was accompanied by a decrease in tyrosine hydroxylase (TH) activity, the rate-limiting step in dopamine synthesis. Kinetic analysis of TH extracted from the caudoputamen of normal and HPRT- mice demonstrated a 45% decrease in Vmax with an increased affinity for the tetrahydropterin cofactor in the mutants. Labeling of midbrain dopamine neurons using TH immunohistochemistry revealed no obvious deficits in the number of midbrain dopamine neurons, but quantitative autoradiographic studies revealed significant reductions in the binding of 3H-N-[1-(2-benzo(beta)thiophenyl)cyclohexyl]piperidine (3H-BTCP) to dopamine uptake sites in the forebrain of the mutants. In contrast to these abnormalities of the dopamine systems in the mutant mice, other neurotransmitter systems appeared relatively unaffected. Norepinephrine, 5-HT, tryptophan hydroxylase, and glutamic acid decarboxylase were present at normal levels in the brains of the mutants. ChAT activity was slightly lower than normal in the caudoputamen of the mutant animals, but was normal in all other brain regions examined. These results indicate that HPRT deficiency is associated with a relatively specific deficit in basal ganglia dopamine systems that emerges during the first 2 months of postnatal development.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Modulation of cell proliferation and gene expression by a p53-estrogen receptor hybrid protein.

We report that p53her, a chimeric protein consisting of the complete human wild-type p53 and the human estrogen receptor hormone-binding domain, strongly suppresses proliferation and induces characteristic morphological changes in Saos-2 human osteosarcoma cells when induced by 17 beta-estradiol. In contrast, p53her constitutively transactivates a p53-responsive promoter in transfection assays, so that transactivation is not regulated by estradiol. However, coexpression of p53her and oncoprotein MDM-2, which associates with and presumably inactivates p53, results in suppression of p53her-mediated transactivation in the absence, but not the presence, of estradiol. Similarly, p53her induces expression of an endogenous MDM-2 transcript only in the presence of estradiol. These results suggest a correlation between the growth suppressor function of p53her and release of a transactivation block mediated by MDM-2.

Amino Acid Sequence↗
PubMed1993

Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.

The restricted host-cell range and low titer of retroviral vectors limit their use for stable gene transfer in eukaryotic cells. To overcome these limitations, we have produced murine leukemia virus-derived vectors in which the retroviral envelope glycoprotein has been completely replaced by the G glycoprotein of vesicular stomatitis virus. Such vectors can be concentrated by ultracentrifugation to titers > 10(9) colony-forming units/ml and can infect cells, such as hamster and fish cell lines, that are ordinarily resistant to infection with vectors containing the retroviral envelope protein. The ability to concentrate vesicular stomatitis virus G glycoprotein pseudotyped vectors will facilitate gene therapy model studies and other gene transfer experiments that require direct delivery of vectors in vivo. The availability of these pseudotyped vectors will also facilitate genetic studies in nonmammalian species, including the important zebrafish developmental system, through the efficient introduction and expression of foreign genes.

Adenoviruses, Human↗