Biomedical subjects

I H Maxwell

Publications and source records attributed to I H Maxwell.

At least 37 records · Page 2Linked to original sources

PubMed1995

The capsid determinant of fibrotropism for the MVMp strain of minute virus of mice functions via VP2 and not VP1.

The minute virus of mice, prototype strain MVMp, productively infects cultured murine fibroblasts but not T cells. The immunosuppressive strain, MVMi, shows the converse tropism. These reciprocal tropisms are mediated by the viral capsids, in which their determinants have been mapped to a few specific amino acids in the primary sequence shared by VP1 and VP2. Which of these proteins is relevant in presenting these determinants during infection is not known. We have approached this question using a recombinant parvovirus system in which a LuIII-derived transducing genome, containing the luciferase reporter in place of viral coding sequences, can be packaged by capsid proteins from separate helper sources. We generated transducing virions by using helper constructs expressing either VP1 or VP2, containing the MVMp or MVMi tropic determinant region, in various combinations. The virions were used to infect human NB324K cells and murine A9 fibroblasts. Transduction of the human cells (permissive for both MVMp and MVMi) required both VP1 and VP2 and was successful with all combinations of these proteins. In contrast, significant transducing activity for A9 cells was detected only with recombinant virions containing VP2 of MVMp, while the use of either source of VP1 had little effect. We conclude that VP2 from MVMp is necessary to enable infection of murine A9 fibroblasts.

Animals↗

PubMed1994

Gene therapy for B-cell lymphoma in a SCID mouse model using an immunoglobulin-regulated diphtheria toxin gene delivered by a novel adenovirus-polylysine conjugate.

Despite advances in conventional therapy, many lives continue to be lost to common forms of B-cell cancers, including leukemias, lymphomas and multiple myeloma. We propose a novel approach to therapy of such cancers using controlled expression of a diphtheria toxin gene (DT-A) to kill malignant cells. We have previously demonstrated selective killing of various cell types, in vitro and in vivo, by cell-specific, transcriptionally controlled expression of this gene. Organ-specific ablation in otherwise healthy transgenic mice has convincingly demonstrated the exquisite specificity achievable by this technique. In the studies now described, DT-A was delivered in vitro and in vivo using a novel gene delivery system employing DNA physically attached to the exterior of adenovirus. After demonstrating the efficacy of gene delivery to Epstein-Barr virus transformed human B-cells in vitro, in vivo work was performed using a SCID mouse model for B-cell lymphoma, in which protection against tumor was observed. The concepts of tissue-regulated toxin gene therapy, and this novel adenovirus gene delivery system are discussed.

Adenoviridae↗

PubMed1994

A modified plaque assay and infected cell hybridization assay for wild-type and recombinant LuIII autonomous parvovirus.

We describe a convenient infected cell hybridization assay for determining the infectious titer of a recombinant, replication-defective parvovirus. We previously generated recombinant derivatives of the autonomous parvovirus LuIII, transducing the luciferase reporter gene. Since luciferase expression is not readily detected at the single cell level, and since the recombinants cannot form plaques, we developed an alternative assay for infectious particles. Co-infection of NB324K cells with wild-type LuIII (multiplicity of infection ca. 5) and the recombinant virions allows amplification of the transducing DNA, which can be detected by hybridization with a probe for the reporter gene. Cell lysis and DNA transfer to a nylon membrane is performed in situ, in the culture dish, and hybridization is performed with a digoxigenin-labeled probe, using immunological detection. During this work we developed a conveniently modified plaque assay for wild-type LuIII that should be applicable to other lytic viruses. The modification employs a reduced volume of agarose overlay that is in turn overlaid with liquid medium, thus avoiding the need to maintain stocks of culture medium at higher than normal concentration.

Hybridization, Genetic↗

PubMed1993

Recombinant LuIII autonomous parvovirus as a transient transducing vector for human cells.

Recombinants based on the genome of the autonomous parvovirus, LuIII, were constructed by replacing the viral coding sequences in an infectious clone (pGLu883) by a luciferase or beta-galactosidase reporter, which was linked to the viral P4 promoter. In cells cotransfected with either of these constructs, together with a plasmid supplying LuIII nonstructural and capsid proteins, excision and replication of the recombinant genome occurred. Transducing virions accumulated in the culture medium of the cotransfected cells, as assayed by reporter activity in recipient cells exposed to this medium. Transducing activity could be neutralized by antiserum to LuIII. Production of replicative form DNA and transducing virions were observed following cotransfection of HeLa, 293, or NB324K cells, in increasing order of efficiency. When homology existed between the recombinant genome and sequences flanking the viral genes in the helper construct, concomitant production of replication-competent, cytopathic virus was sometimes observed. This could be minimized by removal of the left end homology from the helper; by this means, preparations of luciferase transducing virus were obtained free from replication-competent virus. With such preparations, we observed luciferase expression (declining after 3 days) for up to 7 days in recipient HeLa cells. Hybridization of the recombinant viral DNA with strand-specific luciferase probes indicated packaging of both strands (as reported for LuIII), but with a several-fold excess of the (-) strand. We suggest that transducing-autonomous parvoviruses will be useful in gene transfer applications, possibly including gene therapy when only transient expression is desired.

Blotting, Southern↗

PubMed1993

Encapsidation of a recombinant LuIII parvovirus genome by H1 virus and the fibrotropic or lymphotropic strains of minute virus of mice.

We previously constructed a recombinant LuIII parvovirus genome lacking viral coding sequences and used it to generate luciferase-transducing virions, by cotransfection of cells with a helper plasmid expressing LuIII viral proteins. Here, we describe similar cotransfections using alternative, replication-defective helpers encoding the non-structural and capsid proteins of parvovirus H1, or of either the fibrotropic or lymphotropic parvovirus strain of minute virus of mice [MVM(p) or MVM(i)]. Each cotransfection generated transducing virus which directed luciferase expression after infection of HeLa cells. The transducing activity of virus produced using either LuIII or H1 helper plasmids could be specifically neutralized by antiserum raised against the corresponding infectious virus. When the recombinant LuIII parvovirus was pseudotyped with MVM(p) or MVM(i), the resulting virions efficiently expressed luciferase after infection in human or murine cells known to be permissive for both MVM strains. The MVM(p) pseudotyped virus also expressed this reporter efficiently when infected into the murine A9 fibroblast line. In contrast, the recombinant virus generated with an MVM(i) helper gave luciferase expression that was barely detectable after infection of A9 cells which are highly restrictive for MVM(i) productive infection. These results support the notion that the allotropic determinant of these MVM strains functions through their capsid proteins. Pseudotyping of recombinant parvovirus genomes should be useful in controlling their host range as vectors, and in studying mechanisms influencing the permissiveness of parvovirus infections.

Capsid↗

PubMed1992

Inhibition of HIV production in cells containing an integrated, HIV-regulated diphtheria toxin A chain gene.

Construction of a DNA plasmid that expresses a diphtheria toxin A chain (DT-A) gene under control of human immunodeficiency virus (HIV-1) proteins Tat and Rev has been described. Here the generation of HeLa cell clones containing integrated, HIV-regulated DT-A sequences is reported. Five such clones were identified by their decreased expression of a luciferase reporter gene transiently cotransfected with Tat- and Rev-encoding plasmids. The decreased luciferase expression most probably was due to activation of the integrated DT-A gene because higher luciferase activity could be restored by introducing either DT antitoxin or a gene encoding a mutant, DT-resistant elongation factor 2 (the intracellular target for DT-A). Analysis by polymerase chain reaction (PCR) indicated that all clones expressed DT-A encoding RNA. The clones were then transfected with an HIV proviral clone and were tested for HIV production; all five clones demonstrated substantially impaired HIV production compared with parental HeLa cells, as shown by p24 assays of culture supernatants. Our success in generating these cell lines indicates that extremely low basal expression has been achieved in view of the high cellular lethality of DT-A. HIV-regulated expression of DT-A may be applicable as a gene therapy approach for the acquired immune deficiency syndrome (AIDS), to bring about selective suicide of HIV-infected cells before production of viral progeny.

Cloning, Molecular↗

PubMed1992

Inhibition of human immunodeficiency virus-1 production resulting from transduction with a retrovirus containing an HIV-regulated diphtheria toxin A chain gene.

Expression of a gene encoding the diphtheria toxin A (DT-A) chain, under the control of human immunodeficiency virus-1 (HIV-1) proteins Tat and Rev, has previously been shown to confer on cells an impaired ability to produce HIV. That work was done in HeLa cell lines that had stably integrated the regulated DT-A gene in a plasmid context. To increase the efficiency with which the HIV-regulated DT-A gene could be introduced into cells, we studied a recombinant, amphotropic murine leukemia virus containing the HIV-regulated DT-A transcription unit. Here we demonstrate that such recombinant retroviruses can be packaged, for both wild-type DT-A and an attenuated version, tox 176. In transient transfection assays, the proviral constructs exhibited similar basal and trans-activated levels of DT-A expression to the parental plasmids. Transduced H9 cells expressed the integrated DT-A gene upon transfection with plasmids encoding Tat and Rev, as assayed by decreased expression of a cotransfected luciferase reporter gene. Furthermore, the transduced H9 cells were substantially impaired in their ability to produce HIV, as demonstrated by p24 assays of culture supernatants following either transfection with an HIV proviral clone or infection with HIV-IIIB. These data demonstrate that basal expression of the regulated DT-A gene has been reduced to a tolerable level, both in packaging cells and transduced H9 cells. The use of HIV-regulated retroviruses encoding the highly lethal DT-A product may eventually be applicable as a gene therapy approach for the acquired immunodeficiency syndrome (AIDS).

Cell Line↗

PubMed1992

Expression of diphtheria toxin A-chain in mature B-cells: a potential approach to therapy of B-lymphoid malignancy.

Specifically targeted expression of a toxin gene potentially represents a novel approach to cancer therapy. With a view to the ablation of B-cell malignancies, we have constructed a plasmid, designated pTHA71, which expresses the A-chain of diphtheria toxin (DT-A) with high efficiency and specificity in transfected, mature B-lymphoid cells. The construction incorporated immunoglobulin (Ig) kappa light chain gene regulatory sequences, including a kappa promoter, small intron, partial constant region exon, and 3'-flanking sequence (but lacking a known enhancer). These sequences conferred substantially more efficient expression of DT-A in mature B-cells than was seen from constructs that included only Ig promoters and enhancers. When transfected into the 70Z/3 murine pre-B-cell line, pTHA71 was only expressed efficiently if the cells were induced to express their endogenous, rearranged Ig kappa gene by prior exposure to lipopolysaccharide. The insertion of the enhancer from the Ig kappa large intron into pTHA71, generating pTHA81, did not markedly influence the level of DT-A expression in 70Z/3 cells. The observed absence of expression in pre-B-cells suggests that DT-A constructs similar to pTHA71 might be used for the therapeutic ablation of malignant B-cells of mature stages, while sparing normal progenitor cells.

Animals↗

PubMed1991

Expression of the diphtheria toxin A-chain coding sequence under the control of promoters and enhancers from immunoglobulin genes as a means of directing toxicity to B-lymphoid cells.

Previous results have shown that cells can be killed by the expression of an introduced gene encoding diphtheria toxin A-fragment (DT-A) and that killing can be targeted using tissue-specific transcriptional regulatory elements. Here, we describe expression plasmids containing the DT-A gene linked with promoters and enhancers from immunoglobulin heavy chain or kappa-light chain genes. When these plasmids were transfected into cultured cells, DT-A was expressed in B-lymphoid cells but not detectably in HeLa cells or fibroblasts. A high specificity for B-cells was confirmed by assaying for luciferase reporter gene expression from a plasmid containing an analogous combination of immunoglobulin heavy chain regulatory elements. A plasmid containing an immunoglobulin-kappa promoter and enhancer was substantially less active in expressing DT-A in a pre-B-cell line than in B-lymphoma cells, suggesting the possibility of targeting DT-A expression to mature, malignant B-cells while sparing normal B-cell progenitors. By means of viral delivery vehicles, the constructs described might be applied in gene therapy for B-cell leukemias or lymphomas.

Animals↗

PubMed1991

Activation of a diphtheria toxin A gene by expression of human immunodeficiency virus-1 Tat and Rev proteins in transfected cells.

Expression of a gene encoding the diphtheria toxin A (DT-A) fragment, controlled by tissue specific regulatory elements, has previously been used to kill selected cell populations. Here, we have examined the feasibility of controlling DT-A expression using regulatory systems from the human immunodeficiency virus (HIV-1) genome. Plasmids were constructed which express either DT-A or, as a model system, the luciferase (luc) reporter gene, under control of HIV-1 long terminal repeat (LTR) sequences (-167 to +80). While trans-activation by expression of the viral protein Tat was demonstrated, significant basal expression was observed. To reduce basal expression, cis-acting negative regulatory elements from the env region of the HIV-1 genome were inserted in the 3' untranslated region of both the luc and DT-A constructs. This dramatically reduced basal expression from the HIV LTR, and now both viral regulatory proteins Tat and Rev were required for maximal trans-activation. Such regulation of DT-A expression might be therapeutically applied to selectively kill HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC).

Diphtheria Toxin↗

PubMed1991

Inefficiency of expression of luciferase reporter from transfected murine leukaemia proviral DNA may be partially overcome by providing a strong polyadenylation signal.

HepG2 human hepatoma cells were transfected with the luciferase reporter gene, linked with a liver-specific enhancer plus a minimal promoter, contained within either pBR/pUC or Moloney murine leukaemia virus (MMLV) proviral plasmid contexts. Reporter expression from the proviral plasmid was decreased 10- to 20-fold, regardless of whether or not the orientation within the proviral DNA was appropriate for the use of the poly(A) signal in the 3' long terminal repeat (LTR). Efficient reporter expression was restored when the proviral transcription unit was provided with a simian virus 40 poly(A) signal. These results imply that the MMLV LTR poly(A) signal is inefficient. Therefore, strategies to maximize expression of internal transcription units from retroviral vectors should include the provision of an efficient (unidirectional) poly(A) signal (its requiring insertion in the reverse orientation to that of viral transcription).

Carcinoma, Hepatocellular↗

PubMed1991

Construction and expression of plasmids containing mutated diphtheria toxin A-chain-coding sequences.

We previously demonstrated that cells can be killed through transfection of an expression plasmid that encodes the diphtheria toxin A-chain fragment (DT-A). This report describes the construction of expression plasmids containing three mutant DT-A-coding sequences substituting glutamic acid 148 with aspartic acid, serine, or glutamine which are known to have 100- to 300-fold-reduced ADP-ribosylation activity measured in vitro. The toxicity of these constructs was determined in cotransfection experiments using HeLa and 293 cells with a luciferase expression plasmid as the reporter. Dose responses were compared for the three new DT-A mutant plasmids and for the corresponding plasmids containing wild-type DT-A and the previously characterized tox 176 mutant. The dose required to produce 50% inhibition of control luciferase expression in 293 embryonic kidney cells for the five plasmids ranged from 0.01 micrograms for wild-type DT-A to 1.2 micrograms for the least toxic plasmid, which replaces glutamic acid 148 with glutamine. In conclusion, a wide range of DT-A toxicity can be achieved by using plasmid expression vectors that encode different DT-A mutations.

Diphtheria Toxin↗

PubMed1990

Sequence of cDNA for rat cystathionine gamma-lyase and comparison of deduced amino acid sequence with related Escherichia coli enzymes.

A cDNA clone for cystathionine gamma-lyase was isolated from a rat cDNA library in lambda gt11 by screening with a monospecific antiserum. The identity of this clone, containing 600 bp proximal to the 3'-end of the gene, was confirmed by positive hybridization selection. Northern-blot hybridization showed the expected higher abundance of the corresponding mRNA in liver than in brain. Two further cDNA clones from a plasmid pcD library were isolated by colony hybridization with the first clone and were found to contain inserts of 1600 and 1850 bp. One of these was confirmed as encoding cystathionine gamma-lyase by hybridization with two independent pools of oligodeoxynucleotides corresponding to partial amino acid sequence information for cystathionine gamma-lyase. The other clone (estimated to represent all but 8% of the 5'-end of the mRNA) was sequenced and its deduced amino acid sequence showed similarity to those of the Escherichia coli enzymes cystathionine beta-lyase and cystathionine gamma-synthase throughout its length, especially to that of the latter.

Amino Acid Sequence↗

PubMed1990

Genetic ablation in transgenic mice with an attenuated diphtheria toxin A gene.

We have previously generated microphthalmic mice lacking lens fiber cells by targeting the expression of the diphtheria toxin A (DT-A) gene in transgenic mice with regulatory sequences associated with the mouse gamma 2-crystallin gene. Because of the extreme toxicity of DT to animal cells and the potential leakiness of many tissue-specific regulatory regions, we investigated whether there might be an experimental advantage in using a mutant, attenuated form of the DT-A gene (tox-176) fused to gamma 2-crystallin regulatory sequences to ablate fiber cells in the ocular lens. In contrast to the microphthalmia observed in transgenic animals carrying the native DT-A gene, independent lines of mice transgenic for the gamma 2tox176 construct displayed predominantly cataracts or clinical anophthalmia. These contrasting phenotypes were transmitted within each pedigree, although for some lines some phenotypic heterogeneity among offspring was noted. The difference in phenotype between cataractous and clinically anophthalmic transgenic lines could not be ascribed to differences in the transgene copy number. Instead, the results suggest that transgene expression and hence the extent of genetic ablation are modulated by the site of chromosomal integration and, to a lesser extent, by epigenetic events. They also suggest that the attenuated gamma 2tox176 construct can integrate into chromosomal regions that are particularly favorable for expression without compromising embryological development and therefore that the tox-176 gene may be more versatile and effective than the wild-type DT-A gene for achieving genetic ablation with a broad range of cell- or tissue-specific regulatory sequences.

Animals↗

PubMed1989

A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription.

A head-to-tail trimer of the SV40 Bcl I-Bam H1 DNA fragment, specifying polyadenylation of RNA transcripts, was cloned as a cassette flanked by multiple restriction sites. Insertion of the trimer into several expression vectors efficiently prevented spurious expression of reporter genes resulting from transcriptional initiation in prokaryotic plasmid sequences in transfected mammalian cells.

DNA, Viral↗

PubMed1988

Electroporation of mammalian cells with a firefly luciferase expression plasmid: kinetics of transient expression differ markedly among cell types.

Electroporation in combination with the luciferase reporter system provides a rapid and sensitive means of determining transient expression from plasmid constructs in various mammalian cells. Conditions are described allowing efficient electroporation of several cell types with a commercial electroporation device. Measurements on extracts prepared at various times after electroporation showed a rapid rise in luciferase activity for up to 12 hr, which was followed, in certain cell types, by a rapid decline. With such cells, determinations made after 48-72 hr, as is conventional, would greatly underestimate expression of the luciferase reporter gene. Therefore, to observe optimum activity, the kinetics of expression must be determined for each cell type.

Cell Line↗

PubMed1987

Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.

Animals↗

PubMed1987

Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene.

A method of deleting specific cell lineages has been developed that entails microinjection into fertilized eggs of a chimeric gene in which a cell-specific enhancer/promoter is used to drive the expression of a toxic gene product. We show that microinjection of a construct in which the elastase I promoter/enhancer is fused to a gene for diphtheria toxin A polypeptide results in birth of mice lacking a normal pancreas because of expression of the toxin in pancreatic acinar cells. A small pancreatic rudiment, containing islet and duct-like cells, was observed in some of the transgenic mice. This method provides a new approach for studying cell-lineage relationships and for analyzing cellular interactions during development.

Animals↗