Ian H. Maxwell

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.

Autonomous Parvovirus and Densovirus Gene Vectors

Publisher Summary Viruses have evolved as efficient natural vehicles of genetic information and they are logical systems to develop as gene vectors. Several members of the family Parvoviridae and Densovirus are being evaluated for their potential as gene delivery vehicles. Recombinant MVM and LuIII vectors bearing reporter genes have been used to transduce various transformed or untransformed cell lines. To extend the utility of the LuIII vectors, LuIII luciferase transducing genomes have been pseudotyped with virions from heterologous parvoviruses. The results suggest that packaging LuIII transducing genomes into heterologous virions can effectively alter the specificity of transduction and may allow delivery of genes to a wider variety of cell types. When specific tissues are to be targeted, it should be possible to restrict expression of the transgene to certain cell or tissue types with the use of transcriptional control elements. In the case of densovirus, initial results with several densoviruses indicate that they and their promoters will be useful for introducing and expressing foreign genes in insect cells and probably live insects. The β-galactosidase gene has been inserted into genomes of both JcDNV and AeDNV, and then packaged into particles able to deliver ZacZ to target cells in culture, indicating that it will also be possible to utilize these vectors for gene delivery.

Autonomous parvovirus vectors.

Parvoviruses are small, icosahedral viruses (approximately 25 nm) containing a single-strand DNA genome (approximately 5 kb) with hairpin termini. Autonomous parvoviruses (APVs) are found in many species; they do not require a helper virus for replication but they do require proliferating cells (S-phase functions) and, in some cases, tissue-specific factors. APVs can protect animals from spontaneous or experimental tumors, leading to consideration of these viruses, and vectors derived from them, as anticancer agents. Vector development has focused on three rodent APVs that can infect human cells, namely, LuIII, MVM, and H1. LuIII-based vectors with complete replacement of the viral coding sequences can direct transient or persistent expression of transgenes in cell culture. MVM-based and H1-based vectors with substitution of transgenes for the viral capsid sequences retain viral nonstructural (NS) coding sequences and express the NS1 protein. The latter serves to amplify the vector genome in target cells, potentially contributing to antitumor activity. APV vectors have packaging capacity for foreign DNA of approximately 4.8 kb, a limit that probably cannot be exceeded by more than a few percent. LuIII vectors can be pseudotyped with capsid proteins from related APVs, a promising strategy for controlling tissue tropism and circumventing immune responses to repeated administration. Initial success has been achieved in targeting such a pseudotyped vector by genetic modification of the capsid. Subject to advances in production and purification methods, APV vectors have potential as gene transfer agents for experimental and therapeutic use, particularly for cancer therapy.

Species specificity for transduction of cultured cells by a recombinant LuIII rodent parvovirus genome encapsidated by canine parvovirus or feline panleukopenia virus

We previously reported that a recombinant genome derived from the autonomous rodent parvovirus LuIII could be pseudotyped with capsids of the closely related viruses, H1 and minute virus of mice. To determine whether this was also possible with less related viruses, LuIII recombinant genomes containing a luciferase reporter were cotransfected into permissive cells together with plasmids expressing the capsid proteins of either feline panleukopenia virus (FPV) or its host range variant, canine parvovirus (CPV). We observed efficient packaging of the recombinant DNA into transducing virions that displayed the cell tropism of the virus that supplied the capsid. Thus, the FPV- and CPV-pseudotyped virions were able to transduce a feline cell line but they showed no transducing activity for the human NB324K line, which is permissive for LuIII. The transducing activity of the pseudotyped viruses was not inhibited by neuraminidase treatment of the permissive recipient cells, in contrast to that of virions packaged using LuIII capsid proteins. Furthermore, canine A72 cells (permissive for CPV but not FPV) were efficiently transduced by CPV-packaged but not by FPV-packaged LuIII recombinant genomes. Pseudotyped recombinants will be useful for elucidating parvovirus host range determinants since they enable the packaged DNA and each of the capsid proteins to be supplied independently. They should also facilitate control over the targeting of parvovirus vectors for gene transfer.

Improved production of recombinant AAV by transient transfection of NB324K cells using electroporation

Adeno-associated virus (AAV) is useful as an integrating vector for gene transfer. AAV recombinants are generally produced by transient co-transfection methods since it has proven difficult to generate stable packaging cell lines. Acceptable titers of transducing recombinants should be obtainable by optimizing conditions for transient co-transfection. Here, using a luciferase reporter derivative of the AAV infectious plasmid psub201, we show that substantially higher yields of transducing virus can be obtained using electroporation, rather than calcium phosphate transfection. Furthermore, we observed that electroporation of NB324K cells (an SV40-transformed human cell line) with the helper plasmid, pAAV/Ad, with concomitant adenovirus dl309 infection, gave yields of luciferase transducing recombinant AAV equal or superior to those obtained from the more commonly employed 293 cells. NB324K cells are easier to manipulate and show increased cell-association of the recombinant virus (facilitating its concentration and purification). We also adapted an in situ infected cell hybridization procedure, using a digoxigenin labeled probe, as a general method for determining infectious titer. Titers thus estimated were similar for luciferase-transducing and for alkaline phosphatase-transducing AAV vectors: the estimated titer of the latter agreed with that determined by in situ expression of alkaline phosphatase. We also describe a multiple cloning site derivative of psub201 which should facilitate generation of further AAV recombinants.

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.

Targeting diphtheria toxin A-chain transcription to activated endothelial cells using an E-selectin promoter.

Targeting the transcription of a toxin gene to activated endothelial cells might be used for inhibiting angiogenesis in solid tumors. As a model, we transiently transfected human endothelial cells (HUVEC) in culture with expression plasmids for the toxic A-chain of diphtheria toxin (DT-A), using electroporation (achieving ≈70% transfection efficiency). Protein synthesis in HUVEC was highly sensitive to DT-A expression from constitutive viral promoters. E-selectin is strongly expressed on HUVEC activated by TNFα or TPA. We therefore tested a human E-selectin promoter (−547 to +33) for targeting transcription of DT-A or reporter genes to HUVEC. Luciferase reporters were efficiently expressed in HUVEC from this promoter, with or without an enhancer responsive to Ets-1. Expression was increased by TNFα or TPA. DT-A showed highly preferential expression (increased by TNFα or TPA) in HUVEC, compared with WI38 human fibroblasts. HUVEC expressing DT-A were killed via apoptosis. Overall expression levels were influenced by alternative ‘backbone’ sequences used in the expression plasmids. We propose that delivery of transcriptionally regulated expression plasmids for DT-A in vivo, using cationic lipids that show preferential accumulation in activated or proliferating endothelium, may offer a novel means of inhibiting undesired angiogenesis.

Nucleotide sequence of porcine rotavirus (OSU strain) gene segments 7, 8, and 9

A cDNA library was prepared from a mixture of genomic RNA segments 7, 8, and 9 of the OSU strain of porcine rotavirus (serotype 5). Genoraic RNA was Isolated from virus which had been plaque-purified a minimum of three times 1n HA104 cells. Multiple cDNA libraries were constructed using several distinct RNA preparations, and each was screened for the presence of full-length clones. Individual cDNA clones were characterized using both chemical and dideoxy sequencing procedures. For the gene segment encoding the serotype-specific glycoprotein VP7, sequence data was also obtained by direct dideoxy sequencing of genomic RNA using oligonucleotide primers.

Use of a recombinant parvovirus to facilitate screening for human melanoma cell clones expressing tetracycline-responsive transactivators

The tetracycline regulatory (TET) system provides a useful means of controlling foreign gene expression in mammalian cells. Exploiting this system in cultured cells requires the prior isolation, from the cells of interest, of transfectant clones expressing the necessary TET transactivator, tTA, or reverse transactivator, rtTA. We describe a simple screening procedure for identifying transfectant clones expressing a properly regulated transactivator, and the application of this method to isolating clones of human melanoma cells expressing either tTA or rtTA. Clones in multi-well plates are transduced by exposure to a recombinant parvovirus containing a luciferase reporter, under control of a promoter responsive to the TET system transactivators. Transactivation of reporter expression in the presence or absence of doxycycline (DOXY) is determined after one to two days, using a rapid luciferase assay. Screening is easier and more reproducible with this transduction method than with conventional transient transfection of analogous reporter plasmids. Clones of two human melanoma cell lines showing >100-200-fold transactivation after transfection with either tTA or rtTA were readily identified using this method.