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Biomedical subjects

T Friedmann

Publications and source records attributed to T Friedmann.

At least 109 records · Page 6Linked to original sources
PubMed1990

Animal models of Lesch-Nyhan syndrome.

In humans, deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) is associated with a disorder known as Lesch-Nyhan syndrome which includes severe neurobehavioral abnormalities. Several animal models which have been developed to examine the neurobiologic substrates of this disorder have suggested a role for abnormal function in purine/dopamine neurotransmission, but the relationship between HPRT-deficiency and these abnormalities remains unknown. Recently, HPRT-deficient mice have been produced which appear to have similar, though more subtle changes in brain dopamine function. These mice will be useful in elucidating the relationship between HPRT-deficiency and the neurological deficits observed in patients with this disorder.

Disease Models, Animal↗
PubMed1990

Gene therapy in the CNS: intracerebral grafting of genetically modified cells.

Grafting cells to the CNS has been suggested and applied as a potential approach to CNS therapy through the selective replacement of cells lost as a result of disease or damage. Independently, studies aimed at direct genetic therapy in model systems have recently begun to suggest conceptually new approaches to the treatment of several kinds of human genetic disease, especially those caused by single gene enzyme deficiencies. We suggest that a combination of these two approaches, namely the graftment into the CNS of genetically modified cells, may provide a new approach toward the restoration of some functions in the damaged or diseased CNS. We present evidence for the feasibility of this approach, including a description of some current techniques for mammalian cell gene transfer and CNS grafting, and several possible approaches to clinical applications. Specifically, we report that fibroblasts, genetically modified to secrete NGF by infection with a retroviral vector and implanted into the brains of rats with a surgical lesion of the fimbria-fornix, prevented the degeneration of cholinergic neurons that would die without treatment.

Animals↗
PubMed1990

The evolving concept of gene therapy.

The use of molecular techniques to correct human genetic diseases is a concept that was considered extremely remote by many investigators until quite recently. Several factors were responsible for changing the scientific community's attitude toward gene therapy: the development of recombinant DNA technology including the ability to clone disease-related genes; maturation of scientific and ethical reflection following apparent failures of early human experiments; and the development of efficient techniques for the transfer of genes into mammalian cells. Now is the time for the scientific and medical communities to come together and to cooperate to make human gene therapy a clinically useful procedure.

Biomedical Research↗
PubMed1990

Autocrine differentiation of PC12 cells mediated by retroviral vectors.

Rat pheochromocytoma PC12 cells have been modified genetically by the use of replication-defective retroviral vectors containing either the bacterial gene for beta-galactosidase (lac Z) or cDNAs for mouse beta-nerve growth factor (NGF) and the bacterial gene for neomycin resistance. Using the lac Z vector, clonal lines of PC12 cells were obtained in which almost 100% of cells stably expressed this histochemical marker. Infection of PC12 cells or the derived subclone PC12-BAG, which expresses beta-galactosidase, with the NGF vectors resulted in autocrine differentiation as assessed by extensive neurite formation, which occurred within hours after infection and was maintained for weeks in culture. Neurite formation could be partially blocked by antibodies to NGF. The percentage of cells expressing neurite outgrowth was greater than that of PC12 cells treated with exogenous NGF. PC12 cells infected with the NGF vectors were shown to release this trophic factor into the medium using a two-site enzyme immunoassay and a bioassay on 'naive' PC12 cells. PC12 cells genetically modified using these vectors provide a means to: follow the fate of the cells after transplantation into animals; test for delivery in vivo of NGF and catecholamines by grafted, autocrine-differentiated PC12 cells; and study the long-term actions of NGF on responsive cells without adding exogenous NGF.

Animals↗
PubMed1990

Post-transcriptional regulation of retroviral vector-transduced low density lipoprotein receptor activity.

We have reported the use of a retroviral vector to introduce the low density lipoprotein (LDL) receptor gene into receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbit fibroblasts (Miyanohara, A., M. F. Sharkey, D. Steinberg, J. L. Witztum, and T. Friedmann. 1988. Proc. Natl. Acad. Sci. USA. 85: 6538-6542). Because the cDNA for the LDL receptor did not contain the 5' sterol regulatory element that confers sterol-mediated inhibition of LDL receptor transcription, we did not anticipate that LDL receptor activity transduced by this vector would be sterol-responsive. However, we now demonstrate sterol-mediated down-regulation of receptor protein in the infected cells by a mechanism that appears to be mediated at a post-transcriptional level. Down-regulation of LDL receptor activity occurred when infected WHHL cells were preincubated with either LDL or cholesterol plus 25-hydroxycholesterol. Identically organized vectors bearing cDNAs encoding irrelevant genes such as firefly luciferase or bacterial beta-galactosidase exhibited no sterol regulation of reporter gene activity. Insulin receptor activity in WHHL fibroblasts and in WHHL fibroblasts infected with the LDL receptor retroviral vector was also unchanged by sterol. Transfection of LDL receptor-deficient Chinese hamster ovary (CHO) cells with a nonretroviral vector containing the same LDL receptor cDNA also resulted in sterol responsiveness of the transduced LDL receptor. These experiments suggest that the effect of sterol was specific for the LDL receptor transcript. Transgene LDL receptor mRNA levels from the infected cells were unaffected by sterol, indicating that the sterol-mediated reduction in LDL receptor activity did not result from alterations in steady state mRNA levels. These data suggest the existence of post-transcriptional level mechanisms that are responsible for sterol regulation of expression of the transduced LDL receptor gene.

Animals↗
PubMed1990

The human genome project--some implications of extensive "reverse genetic" medicine.

Impressive progress has been made during the past several decades in understanding the pathogenesis of human genetic disease. The tools of molecular biology have allowed the isolation of many disease-related genes by forward and a few by reverse genetics, and the imminent completion of a complete human genetic linkage map will accelerate the genetic characterization of many more genetic diseases. The major impacts of the molecular characterization of human genetic diseases will be 1. To increase markedly the number of human diseases that we recognize to have major genetic components. We already understand that genetic diseases are not rare medical curiosities with negligible societal impact, but rather constitute a wide spectrum of both rare and extremely common diseases responsible for an immense amount of suffering in all human societies. The characterization of the human genome will lead to the identification of genetic factors in many more human diseases, even those that now seem too multifactorial or polygenic for ready understanding. 2. To allow the development of powerful new approaches to diagnosis, detection, screening and even therapy of these disorders aimed directly at the mutant genes rather than at the gene products. This should eventually allow much more accurate and specific management of human genetic disease and the genetic factors in many human maladies. The preparation of a fine-structure physical map of the entire human genome together with an overlapping contiguous set of clones spanning entire chromosomes or large portions of chromosomes is rapidly becoming feasible, and the information that will flow from this effort promises eventually to affect the management of many important genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Mapping↗
PubMed1989

Progress toward human gene therapy.

Current therapies for most human genetic diseases are inadequate. In response to the need for effective treatments, modern molecular genetics is providing tools for an unprecedented new approach to disease treatment through an attack directly on mutant genes. Recent results with several target organs and gene transfer techniques have led to broad medical and scientific acceptance of the feasibility of this "gene therapy" concept for disorders of the bone marrow, liver, and central nervous system; some kinds of cancer; and deficiencies of circulating enzymes, hormones, and coagulation factors. The most well-developed models involve alteration of mutant target genes by gene transfer with recombinant pathogenic viruses in order to express new genetic information and to correct disease phenotypes--the conversion of the swords of pathology into the plowshares of therapy.

Animals↗
PubMed1989

Factors affecting long-term stability of Moloney murine leukemia virus-based vectors.

We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells. We have used Moloney murine leukemia virus-based vectors expressing human HPRT, firefly luciferase (luc), and Escherichia coli beta-galactosidase (lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance (neo) gene as selectable markers. All vectors, whether single or double gene, yielded both stable and unstable clones. Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure. Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity. Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-driven neo gene. In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus. In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown. The design of effective retroviral vectors and their possible use in vivo will require further characterization of these determinants of provirus stability.

Cell Line↗
PubMed1989

Grafting genetically modified cells into the rat brain: characteristics of E. coli beta-galactosidase as a reporter gene.

The utility of grafting genetically modified cells to the mammalian brain was examined using the E. coli beta-galactosidase gene (lacZ) as a reporter gene in retroviral infection. Following implantation of the infected cells to the brain, lacZ continued to be expressed in vivo and could be detected easily with enzyme histochemistry. However, beta-galactosidase-positive cells were also observed in control grafts which had not been infected with the virus. This false-positive staining was found to be endogenous lysosomal activity associated with macrophage infiltration presumably induced by the damage associated with grafting. The E. coli gene product was distinguished from cellular lysosomal beta-galactosidase by using immunohistochemical staining with an antibody specific for E. coli beta-galactosidase. With this antibody, retrovirus-infected cells could be distinguished in the brain, and no false positives were observed in non-infected cells. We conclude that E. coli beta-galactosidase is a useful reporter gene for determining the fate of implanted cells to the brain if appropriate caution is taken to distinguish it from cellular beta-galactosidase by immunocytochemical procedures.

Animals↗
PubMed1989

Grafting fibroblasts genetically modified to produce L-dopa in a rat model of Parkinson disease.

Rat fibroblasts were infected with a retroviral vector containing the cDNA for rat tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. A TH-positive clone was identified by biochemical assay and immunohistochemical staining. When supplemented in vitro with pterin cofactors required for TH activity, these cells produced L-dopa and released it into the cell culture medium. Uninfected control cells and fibroblasts infected with the TH vector were grafted separately to the caudate of rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. Only grafts containing TH-expressing fibroblasts were found to reduce rotational asymmetry. These results have general implications for the application of gene therapy to human neurological disease and specific implications for Parkinson disease.

Animals↗
PubMed1989

Retrovirus vector-mediated gene transfer into hepatocytes.

The introduction and stable expression of foreign genes in mammalian hepatocytes have recently been demonstrated by several techniques, including the use of physical approaches such as direct injection of a DNA calcium phosphate precipitate, electroporation of plasmid DNA and the exposure to liposome-erythrocyte ghost complexes as well as the biological approach of infection of primary hepatocyte cultures with retrovirus vectors. Retrovirus-mediated transduction has proven to be highly advantageous in many in vitro gene transfer studies of mammalian cells, and recent results with primary rat liver cultures have begun to define the conditions under which foreign genes can be transduced into hepatocytes in vitro. Fully differentiated hepatocytes are poorly susceptible, if at all, to infection with retroviruses, a phenomenon due at least in part to the fact that cells must undergo replication in order to retroviral integration and gene expression to occur. Hepatocytes are largely resting cells, arrested in G0. Nevertheless, primary cultures of hepatocytes are known to demonstrate a partial de-differentiation in vitro and undergo several rounds of replication. During a narrow period of time early in primary culture correlated roughly with the de-differentiation, adult hepatocytes do become susceptible to efficient infection with retrovirus vectors. In infected cells, gene expression remains relatively stable for the several week duration of the primary culture. It is not known if the restriction of virus infection in hepatocytes is a function of the state of cellular differentiation, of the availability of viral receptors or of other factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Suppression of the neoplastic phenotype by replacement of the RB gene in human cancer cells.

Mutational inactivation of the retinoblastoma susceptibility (RB) gene has been proposed as a crucial step in the formation of retinoblastoma and other types of human cancer. This hypothesis was tested by introducing, via retroviral-mediated gene transfer, a cloned RB gene into retinoblastoma or osteosarcoma cells that had inactivated endogenous RB genes. Expression of the exogenous RB gene affected cell morphology, growth rate, soft agar colony formation, and tumorigenicity in nude mice. This demonstration of suppression of the neoplastic phenotype by a single gene provides direct evidence for an essential role of the RB gene in tumorigenesis.

Cell Division↗
PubMed1988

Grafting genetically modified cells to the damaged brain: restorative effects of NGF expression.

Fibroblasts were genetically modified to secrete nerve growth factor (NGF) by infection with a retroviral vector and then implanted into the brains of rats that had surgical lesions of the fimbria-fornix. The grafted cells survived and produced sufficient NGF to prevent the degeneration of cholinergic neurons that would die without treatment. In addition, the protected cholinergic cells sprouted axons that projected in the direction of the cellular source of NGF. These results indicate that a combination of gene transfer and intracerebral grafting may provide an effective treatment for some disorders of the central nervous system.

Acetylcholinesterase↗
PubMed1988

Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit fibroblasts in vitro.

Familial hypercholesterolemia is caused by a genetic deficiency of the low density lipoprotein (LDL) receptor. The Watanabe heritable hyperlipidemic (WHHL) rabbit, which is also defective in LDL receptor activity, provides an excellent animal model of homozygous familial hypercholesterolemia. As a step toward development of effective gene therapy for familial hypercholesterolemia, we have constructed a transmissible retroviral vector containing a full-length human cDNA for the LDL receptor. WHHL fibroblasts infected in vitro expressed the human receptor efficiently, as indicated by RNA and ligand blotting studies. Infected fibroblasts bound and degraded a monoclonal antibody specific for the human LDL receptor (IgGC7) in a manner comparable to that seen with normal human fibroblasts. Human LDL was also degraded by infected WHHL cells and promoted cholesterol esterification to the same degree as seen in normal human fibroblasts. Although technical problems remain to be solved, these studies show that, in principle, gene therapy may be possible for familial hypercholesterolemia patients.

Animals↗