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M J Kadan

Publications and source records attributed to M J Kadan.

13 recordsLinked to original sources
PubMed2001

A versatile framework for the design of ligand-dependent, transgene-specific transcription factors.

The ability to regulate transgene expression will be essential for the safety and efficacy of many gene therapies. Various ligand-dependent transcription factors, including steroid hormone receptors, have been modified to enable transgene-specific regulation. To minimize effects on cellular gene expression, chimeric steroid receptors have been constructed by replacing their native DNA binding domain (DBD) with a heterologous DBD, like that from the yeast transcription factor GAL4. This approach has limitations for human gene therapy, including the potential immunogenicity of the GAL4 domain and the inability to discriminate between different GAL4-linked transgenes in the same cell. To address this, we have constructed chimeric regulators containing the human estrogen receptor (ER) ligand binding domain (LBD) and a Cys(2)-His(2)-type zinc finger DBD. Cys(2)-His(2) zinc finger domains are common among human DNA binding proteins and can be engineered to selectively bind different DNA sequences. We demonstrate over 500-fold drug-dependent transgene induction with these chimeric regulators in vitro and the ability to regulate an adenovirus-delivered transgene in mice. Two chimeras containing different Cys(2)-His(2) domains displayed highly sequence-specific binding and regulation. Incorporating a point mutation in the ER LBD that ablates estrogen binding enables selective in vivo regulation with the clinically useful anti-estrogen tamoxifen. These Cys(2)-His(2)-ER LBD chimeras represent a versatile framework for creating transgene-specific regulators potentially useful for human gene therapy applications.

Adenoviridae↗
PubMed2001

Generation and characterization of E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII.

The use of adenoviral vectors for gene therapy has been limited due to host immune responses directed toward the vector and/or transgene and vector toxicity. To decrease adenoviral vector immunogenicity and toxicity, we attenuated viral gene expression by eliminating E1, E2a, E3, and E4 early genes from the adenoviral backbone. Two highly attenuated, fourth-generation (Av4) E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII (FVIII) under the control of a liver-specific albumin promoter were generated. One Av4 vector (Av4DeltaE4FVIII) was deficient in the entire E4 coding region and the second vector contained a deletion of the E4 region, except for open reading frame 3 (orf 3; Av4orf3FVIII). The Av4 vectors were compared to an E1/E2a/E3-deficient third-generation vector (Av3H8101) containing an analogous transgene expression cassette in vitro and in vivo following intravenous administration in hemophiliac mice. In vitro transduction of Hep3B cells revealed at all three vectors expressed functional FVIII. However, the Av4DeltaE4FVIII vector could not be scaled-up for in vivo studies. Both Av3H8101 and Av4orf3FVIII initially expressed similar levels of FVIII in hemophiliac mice. However, at 3 months, animals treated with the Av4orf3FVIII vector no longer expressed FVIII while Av3H8101-treated mice displayed persistent FVIII expression. Liver enzyme analyses of plasma samples revealed that the Av4orf3FVIII vector was significantly less hepatotoxic than the Av3H8101 vector. These data demonstrate that further attenuation of the adenoviral vector backbone by removal of the majority of the E4 coding region significantly diminished vector toxicity; however, the duration of transgene expression was reduced.

Adenovirus E1 Proteins↗
PubMed2000

Evaluation of adenoviral vectors by flow cytometry.

Biological assays for adenoviral gene therapy vectors have included conventional procedures initially developed to detect wild-type adenoviruses. Standard virological assays to quantitate adenoviruses rely on the virus to infect and replicate in the host cell until a cytopathic effect is observed. The appearance of plaques, colonies of rounded, enlarged cells containing infectious virions, usually takes 2 to 3 weeks to reach an endpoint. We describe a flow cytometric bioassay for adenovirus which shortens the time from when the infection takes place to the time that biological titer is determined. A fluorescent focus-forming assay was one of the first rapid adenoviral bioassays developed. Virus titer was determined using fluorescence immunocytochemistry to detect adenovirus proteins and microscopy to count fluorescent foci in cultures of adenovirus-infected cells. In this study, we describe a flow cytometric assay performed on cells stained for adenovirus hexon capsid protein, where virus titer is determined based on the dose-dependent appearance of hexon-positive cells. Adenovirus hexon detection in infected cells can provide data to determine virus titer, inducible promoter function in vector-complementing cells, and vector replication in complementation-deficient cells.

Adenovirus E1 Proteins↗
PubMed1998

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy.

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.

Adenoviridae↗
PubMed1996

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy.

Adenoviridae↗
PubMed1993

Introduction of human genomic sequences renders CHO-K1 cells susceptible to infection by amphotropic retroviruses.

To learn more about the nature of the block to infection by amphotropic retroviruses exhibited by Chinese hamster cells (CHO-K1), CHO-K1 cells were made susceptible to amphotropic retrovirus infection by introducing genomic DNA from infectable human cells. A clone, designated CHO18, was obtained and shown to be infected as efficiently as NIH 3T3 fibroblasts. Susceptibility of CHO18 cells to infection was specific to retroviruses and vectors bearing an amphotropic envelope. By comparison to CHO-K1 cells, CHO18 cells may provide a useful model for analysis of the molecular events involved in the retrovirus-receptor interaction.

3T3 Cells↗
PubMed1992

Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments.

3T3 Cells↗
PubMed1991

Correlation of 125I-LSD autoradiographic labeling with serotonin voltage clamp responses in Aplysia neurons.

Autoradiographic receptor binding studies using 125I-LSD (2-[125I]lysergic acid diethyamide) revealed intense labelling on the soma of a symmetrically located pair of cells in the abdominal ganglion of Aplysia californica. This binding was blocked by micromolar concentrations of serotonin and lower concentrations of the serotonergic antagonists, cyproheptadine and mianserin (Kadan and Hartig, 1988). Electrophysiological investigation of responses to serotonin of neurons in the left upper quadrant, where one of the labeled neurons is located, revealed a range of serotonin responses. Cells L3 and L6 have a K+ conductance increase in response to serotonin that is not blocked by cyproheptadine or mianserin. Cells L2 and L4 have a biphasic response to serotonin: a Na+ conductance increase, which can be blocked by cyproheptadine and mianserin, followed by a voltage dependent Ca2+ conductance which is blocked by Co2+ but not the serotonergic antagonists. Cell L1, and its symmetrical pair, R1, have in addition to the Na+ and Ca2+ responses observed in L2 and L4, a Cl- conductance increase blocked by LSD, cyproheptadine and mianserin. LSD had little effect on the other responses. We conclude that the symmetrically located cells L1 and R1 have a Cl- channel linked to a cyproheptadine- and mianserin-sensitive serotonin receptor that is selectively labelled by 125I-LSD. This receptor has many properties in common with the mammalian serotonin 1C receptor.

Acetylcholine↗
PubMed1990

Infection with murine retrovirus confers resistance to the neurotoxin 1-methyl-4-phenylpyridinium ion in PC 12 cells.

High concentrations of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) are toxic to the catecholaminergic cell line PC12, derived from rat phenochromocytoma. Prolonged exposure of wild-type PC12 cells to 500 microM MPP+ yields toxin-resistant colonies at a frequency of 2 X 10(-4). These spontaneously arising MPP(+)-resistant cells are morphologically quite distinct from wild-type PC12 cells, and are lacking in most of their characteristic catecholaminergic properties. In contrast, among PC12 cells infected with the murine retrovirus ZIPNEOSV(X), 20% are resistant to the toxin MPP+, a resistance frequency approximately 1,000 times higher than for uninfected cells. The morphology and catecholaminergic phenotype of the virus-infected MPP+ resistant cells are quite similar to those of wild-type PC12 cells. The results presented in this study suggest a unique mechanism of MPP+ resistance in the infected PC12 cells which may be conferred by the presence and/or expression of the retrovirus ZIPNEOSV(X).

1-Methyl-4-phenylpyridinium↗
PubMed1988

Retroviral-mediated gene transfer. Applications in neurobiology.

There are now many examples of the successful expression of genes transduced by retroviruses in studies from outside the field of neuroscience. Retroviruses will undoubtedly also prove to be effective tools for neuro-scientists interested in expressing cloned neurotransmitter and receptor genes. There are also other less obvious applications of retroviruses, such as their insertional mutagenic effects, which may be useful in studies of the genetic factors and biochemical mechanisms involved in, for example, neurotoxicity. Strong cellular promoters have been identified by retroviral infection and subsequent rescue of the flanking genomic DNA. Retroviruses can be employed again to reintroduce these regulatory sequences back into cells. In this way the complexities of gene expression in the many subpopulations of neurons may be unraveled. Retroviruses can also serve as very useful genetic markers in studies of development and lineage relationships. Retroviruses may be used to efficiently transfer oncogenes into neuronal cells to create new cell lines. This application exploits one of the natural traits of retroviruses--oncogenesis--which led to their original discovery. Finally, there are neurotropic retroviruses that could serve as important vectors for delivering genes into neurons. Studying these retroviruses may lead to an understanding of how they cause neuropathologic changes in the CNS.

Animals↗
PubMed1988

Autoradiographic localization and characterization of [125I]lysergic acid diethylamide binding to serotonin receptors in Aplysia.

The sensitive serotonergic radioligand 2-[125I]lysergic acid diethylamide was used to study the distribution and pharmacological binding properties of serotonin receptors in Aplysia californica. The high specific activity of this radioligand allowed us to develop a methodology for the investigation of receptor binding properties and receptor distribution in a single ganglion. [125I]Lysergic acid diethylamide labels a population of high-affinity serotonergic sites (Kd = 0.41 nM) in Aplysia ganglia whose regional distribution matches that expected from previous electrophysiological and immunohistochemical studies. The properties of [125I]lysergic acid diethylamide binding sites in Aplysia are in general agreement with previous studies on [3H]lysergic acid diethylamide binding in this system but these sites differ from the serotonergic receptor subtypes described in the mammalian brain. Guanine nucleotides were shown to modulate agonist but not antagonist affinity for the [125I]lysergic acid diethylamide binding site in Aplysia, suggesting that this site is coupled to a G-protein. Images of serotonin receptor distribution in the Aplysia nervous system were obtained from autoradiograms of [125I]lysergic acid diethylamide binding. Serotonin receptors in ganglia tissue sections are located primarily within the neuropil. In addition, a subset of neuronal soma are specifically labeled by [125I]lysergic acid diethylamide. These studies indicate that [125I]lysergic acid diethylamide binds to sites in the Aplysia nervous system which display a regional distribution, pharmacological binding properties and evidence of coupling to a G-protein consistent with labeling of a subset of functional serotonin receptors. In addition, the techniques used in this investigation provide a general approach for rapidly characterizing the pharmacological properties and anatomical distribution of receptor binding sites in single invertebrate ganglia. Individual neurons containing these receptor subtypes can be identified by these methods and correlated with physiological responses in the same cell.

Animals↗
PubMed1984

Characterization of 125I-lysergic acid diethylamide binding to serotonin receptors in rat frontal cortex.

125I-Lysergic acid diethylamide (125I-LSD) is the first 125I-labeled ligand for serotonin receptor studies. Its binding to rat frontal cortex membranes is saturable, reversible, and stereospecific. Specific binding is linearly dependent on tissue concentration and represents 70-80% of the total binding. Scatchard plots of the binding data are linear with a KD of 1.5 nM, a Bmax of 12.4 fmol/mg wet weight tissue, and a Hill slope of 1.02. The binding kinetics are highly temperature-dependent. At 37 degrees C the bimolecular association rate constant is 1.28 X 10(8) min-1 M-1 and the dissociation rate constant is 0.087 min-1 (t 1/2 = 8.0 min). At 0 degrees C less than 4% dissociation occurs over 40 min and the association rate is similarly depressed. Inhibition of 125I-LSD binding by a variety of serotonergic, dopaminergic, and adrenergic ligands reveals a 5-hydroxytryptamine2 (5-HT2) serotonergic profile for this binding site. Regional distribution studies of 125I-LSD binding in rat brain show that areas with the highest levels of binding include the cortex and striatum. Iodinated radioligands can be synthesized with specific activities exceeding 2,000 Ci/mmol, which makes them approximately 75-fold more sensitive than tritiated radioligands. This high specific activity, coupled with the selectivity of 125I-LSD for 5-HT2 sites, makes this ligand a sensitive new probe for 5-HT2 serotonin receptors.

Animals↗