Gene therapy for disorders of the CNS.
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Brain Neoplasms↗
Biomedical subjects
T Friedmann
Publications and source records attributed to T Friedmann.
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↗
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↗
Highly potent novel opioid receptor agonist in the 14-alkoxymetopon series.
The newly synthesized 14-alkoxymetopon derivatives, 14-methoxymetopon, 14-ethoxymetopon, 14-methoxy-5-methyl-morphinone, exhibit high affinity for the naloxone binding sites in rat brain. A substantial decrease in affinity was observed, in the presence of NaCl indicating a high degree of agonist activity. All three 14-alkoxymetopon derivatives displayed high affinity for [3H][D-Ala2,(Me)Phe4,Gly-ol5]enkephalin ([3H]DAMGO) binding sites, much less potency toward delta sites and were the least effective at kappa sites. Isolated tissue studies using the guinea pig ileum preparation confirmed their high agonist potency. Following administration the new compounds produced naloxone reversible antinociceptive effects and were 130-300 times more potent than morphine in the acetic acid induced abdominal constriction model in the mouse, and the hot plate and tail flick tests in the rat. The compounds also produced dose-dependent muscle rigidity, and potentiated barbiturate-induced narcosis. The in vivo apparent pA2 values for naloxone against 14-ethoxymetopon and morphine were similar in analgesia, suggesting an interaction with the same (mu) receptor site. The dependence liability of 14-alkoxymetopon derivatives in the withdrawal jumping test was less pronounced than that of morphine in either rats or mice, similar to tolerance to the their analgesic action. It is concluded that the 14-alkoxymetopon derivatives studied are selective and potent agonists at mu opioid receptors, with reduced dependence liability.
Analgesics↗
Provirus-anchored long-range (PAL) mapping of mammalian genomes.
To design a strategy for more efficient long-range physical mapping of large mammalian genomes, we have developed a method for directional mapping from anchor points of randomly integrated retroviral proviruses containing sequences that provide unique cleavage sites in the genome. Using this method, we have determined a physical map of approximately 1.6 Mb flanking an Mo MLV-proviral integration site in mouse NIH/3T3 cells. Our results indicate that this provirus-anchored long-range (PAL) mapping strategy, which uses only two proviral sequences as universal hybridization probes, can potentially allow rapid construction of physical maps for chromosomal regions devoid of other genetic markers.
3T3 Cells↗
Gene therapy--a new kind of medicine.
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Genetic Therapy↗
Gene therapy for disorders of the nervous system.
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Animals↗
Milestone and events in the early development of human gene therapy.
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Animals↗
Brain purines in a genetic mouse model of Lesch-Nyhan disease.
Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [14C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.
Adenine Nucleotides↗
Transduction of a drug-sensitive toxic gene into human leukemia cell lines with a novel retroviral vector.
To investigate the possibility of killing tumor cells by the expression of an exogenously introduced toxic gene, we have constructed a novel retroviral vector (LTRNL) which has the polyA signal deleted herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene. The vector becomes toxic by treating cells expressing HSV1-tk with the antiherpetic drugs acyclovir or ganciclovir (GCV). Cells of the human leukemia lines (K562, MEG-01) were infected with this vector and two transduced cell lines (K562/LTRNL, MEG-01/LTRNL) were established. Southern blot analysis confirmed the integration of the HSV1-tk transgene in these cells and Northern blot analysis exhibited the expression of 4.8-kb viral mRNA containing the HSV1-tk gene. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for the in vitro cytotoxic effects of GCV to these cells demonstrated that concentrations of about 2.5 microM for K562/LTRNL and 1.25 microM for MEG-01/LTRNL cells resulted in 50% inhibition of cell growth after 72 hr. Subcutaneous tumors of MEG-01/LTRNL in KSN nude mice, but not those of uninfected MEG-01 cells, showed durable regressions after exposure of the mice to 40 mg/kg of GCV given subcutaneously once a day for 15 days. This study indicates that the LTRNL-infected human leukemia cells exhibit inducible susceptibility to GCV.
Acyclovir↗
[Structure-activity relationship of synthetic and semisynthetic opioid agonists and antagonists].
This account presents the chemical structure-pharmacological activity relationships of semi-synthetic morphine and synthetic morphinan and benzomorphan derivatives. Substitution of nitrogen confers the analgesic or morphine antagonist activity. Opiate receptor selectivity of these compounds is also discussed. The importance of morphine agonists, and antagonists is mentioned with particular reference to the opiate receptor mapping.
Molecular Structure↗
Gene therapy of cancer through restoration of tumor-suppressor functions?
The tools and concepts of gene therapy are being applied to the development of effective new treatments for human cancer. Most human cancers are associated with multiple interacting and cooperating mutations in protooncogenes and tumor suppressor genes. In several model systems, some features of the tumor phenotype can be suppressed in vitro through the restoration of expression of tumor suppressor genes such as Rb and p53. Before this phenomenon can serve as the basis for gene therapy of cancer, many conceptual and technical problems must be solved. Because such genetically modified cells continue to contain and express other mutations, it is important to determine the mechanisms and frequency of reversion to the tumor phenotype. To be clinically useful, highly efficient and targeted gene delivery vectors must be developed. The experimental evidence for tumor suppression by restored gene expression and the pivotal role played by tumor suppressor genes in the regulation of cell replication suggests that restored expression of some tumor suppressor genes in some tumor cells will eventually play a role in cancer gene therapy.
Animals↗
Concepts and strategies for human gene therapy.
Methods of modern molecular genetics have been developed that allow stable transfer and expression of foreign DNA sequences in human and other mammalian somatic cells. It is therefore no surprise that the methods have been applied in attempts to complement genetic defects and correct disease phenotypes. Two decades of research have now led to the first clinically applicable attempts to introduce genetically modified cells into human beings to cure diseases caused at least partially by genetic defects. We discuss here some of the strategies being followed for both in vitro and in vivo application of therapeutic gene transfer and summarize some of the technical and conceptual difficulties associated with somatic-cell gene therapy.
Animals↗
Suppression of acute lymphoblastic leukemia by the human wild-type p53 gene.
Independent mutations in both alleles of the p53 tumor suppressor gene are a frequent finding in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines and in the cells of some T-ALL patients in relapse. One major goal of studying the status of p53 (and other tumor suppressor genes) in human cancer is to facilitate the suppression of the tumorigenic phenotype through the restoration of the expression of the wild-type allele. While the efficient insertion of a suppressor into all cells of solid/metastatic human tumors may at present be impossible, insertion into leukemia cells may be feasible due to the accessibility of the leukemia cells in the body. To examine the feasibility of suppressing the tumorigenicity of human T-leukemia cells, the human T-ALL cell line Be-13, which lacks endogenous p53 protein, was infected with a recombinant retrovirus encoding the wild-type allele of human p53 (hwtp53). Expression of p53 reduced the growth rate of infected Be-13 cells in vitro, suppressed colony formation in methylcellulose cultures, and abrogated their tumorigenic phenotype in nude mice in vivo. These results suggest that suppression of the leukemic phenotype of relapse T-ALL-derived Be-13 cells is feasible. Acute leukemia cell suppression via high-efficiency infection with retroviruses encoding wtp53 may be feasible and beneficial in T-ALL cases as part of a bone marrow transplantation regimen in an effort to reduce the frequency of posttransplantation relapse.
Animals↗
Circulating human or canine factor IX from retrovirally transduced primary myoblasts and established myoblast cell lines grafted into murine skeletal muscle.
We have used retroviral vectors to introduce human or canine factor IX cDNAs into cultured primary murine and canine myoblasts and into the established murine myoblast cell line C2C12. In all cases, the stably infected cells produced biologically active factor IX in culture and secreted detectable amounts into the culture medium both before and after differentiation of the cells into myotubes. Myoblasts and differentiated myotubes are therefore capable of performing all the posttranslational modifications of the coagulation factor required for biological activity. We have grafted the genetically modified myoblasts into skeletal muscles of nude mice and have detected stable levels of circulating human factor IX for up to two months after grafting. We propose that grafting genetically modified primary myoblasts or established myoblast cell lines into skeletal muscle may represent a useful approach to gene therapy for a variety of genetic diseases, including intrinsic muscle disease and defects in circulating proteins as in the hemophilias.
Animals↗
Effects of gene transfer into cultured CNS neurons with a replication-defective herpes simplex virus type 1 vector.
Vectors derived from herpes simplex virus type 1 (HSV-1) may provide useful tools for gene transfer to cells of the mammalian nervous system. We have studied the infection of cultured CNS neurons using a vector derived from an HSV-1 mutant deleted for the major HSV-1 transcriptional regulatory protein-encoding gene, IE 3. This vector, denoted Cgal delta 3, contains the E. coli lacZ gene driven by the strong promotor of the human cytomegalovirus major immediate-early gene inserted into a non-coding portion of the mutant viral genome. We studied the efficiency of Cgal delta 3 infection of rat CNS neurons at various times after cell preparation from embryonic rats, the effect of vector infection on the glia subpopulation of the neuronal cultures, and the stability of lacZ expression in infected neurons cultured under conditions optimized for neuronal differentiation and survival using an astrocyte feeder layer. Under these conditions, an HSV-derived vector is a highly efficient vehicle in vitro for short-term gene transfer to cells of the CNS. Despite the fact that this vector cannot undergo a lytic cycle, it was toxic to cultured CNS neurons and glia. Even with the use of an astrocyte feeder layer to support infected neurons, we have detected only transient expression of the lacZ gene, due either to loss of the infected cells and/or to shut off of transgene expression. Further improvements will be needed in the design of HSV vectors to allow long-term gene transfer to cultured neurons.
Animals↗
A brief history of gene therapy.
The concepts of gene therapy arose initially during the 1960s and early 1970s whilst the development of genetically marked cells lines and the clarification of mechanisms of cell transformation by the papaovaviruses polyoma and SV40 was in progress. With the arrival of recombinant DNA techniques, cloned genes became available and were used to demonstrate that foreign genes could indeed correct genetic defects and disease phenotypes in mammalian cells in vitro. Efficient retroviral vectors and other gene transfer methods have permitted convincing demonstrations of efficient phenotype correction in vitro and in vivo, now making gene therapy a broadly accepted approach to therapy and justifying clinically applied studies with human patients.
Animals↗