Expansion of tropism of a feline parvovirus to target a human tumor cell line by display of an alpha(v) integrin binding peptide on the capsid.

The autonomous parvoviruses are small, non-enveloped, single strand DNA viruses. They occur in many species and they have oncolytic properties. We are modifying the capsid of feline panleukopenia virus (FPV), a parvovirus which normally infects feline cells, with the goal of targeting human tumor cells for potential cancer therapy. Using recombinant viruses transducing a luciferase reporter, we show that insertion of a cyclically constrained, integrin-binding peptide at an exposed position on the FPV capsid enables transduction of an alpha(v) integrin-expressing human rhabdomyosarcoma cell line (Rh18A). These cells were not transduced by virus with the unmodified FPV capsid. Transduction of Rh18A was specifically inhibited by an alpha(v) integrin blocking antibody. However, other human tumor lines expressing alpha(v) integrins were not transduced by virus with either the modified or unmodified capsid. We conclude that modification of the FPV capsid to bind alpha(v) integrins can contribute to, but is not generally sufficient for, redirecting infection to human tumor cells. The permissiveness of Rh18A cells presumably involves additional factors unique to this line among various human cell lines tested.

Promoter strength in adenovirus transducing vectors: down-regulation of the adenovirus E1A promoter in 293 cells facilitates vector construction.

Most adenovirus transducing vectors have the cytomegalovirus major immediate-early (CMV) or the Rous sarcoma virus long terminal repeat (RSV) promoter driving expression of the transgene. Both of these promoters are highly active in transfection and transduction assays in 293 cells, in which transducing vectors are constructed and grown, and in HeLa cells. The CMV promoter exhibits rapid activation while the RSV promoter exhibits a lag prior to the onset of viral DNA replication in transduction assays. While the use of very strong promoters facilitates expression of the transgene, high-level expression of certain gene products hinders virus construction and growth. For such genes, the use of the adenovirus type 5 E1A promoter offers advantages. The E1A promoter exhibits modest activity in HeLa cells after transfection or transduction, but very little activity in 293 cells, suggesting that the E1A promoter would permit construction and growth of vectors encoding deleterious gene products that could not be constructed with the CMV and RSV promoters. This idea was tested through attempts to construct viruses encoding the immunoglobulin loop 6 and transmembrane regions of the prostaglandin F2alpha receptor regulatory protein (FPRP), a product that inhibits adenovirus vector construction for reasons that are not clear. Only the E1A promoter permitted construction and growth of the transducing vector encoding the fragment of FPRP.

Expression of activated N-ras in a primary melanoma cell line counteracts growth inhibition by transforming growth factor-beta.

One critical factor in melanoma progression is the change from radial growth phase to vertical growth phase. We previously showed a high incidence of ras mutations in progressing but not early human melanomas. We also found that stable expression of activated Ras in a primary human melanoma cell line (WM35) led to enhanced proliferation, anchorage-independent survival, migration and invasion in vitro and enhanced subcutaneous tumor formation in vivo, transforming the melanoma phenotype from the radial growth phase to the vertical growth phase. Inhibitory cytokines, especially transforming growth factor-beta, are important in homeostasis of normal human melanocytes. Proliferation of early melanoma cells can be inhibited by transforming growth factor-beta, whereas more aggressive stages lose this response. Using a transforming growth factor-beta activated luciferase reporter transiently transfected into WM35, WM35N-ras, and WM35H-ras (WM35 transfected with mutant N-ras or H-ras genes), we demonstrated significant decreases (p < 0. 04) in transforming growth factor-beta induced reporter expression in both ras transfected cell lines. Transforming growth factor-beta also induced significant decreases (p < 0.002) in the proportion of WM35 cells in S-phase of the cell cycle; this effect was not observed in WM35N-ras cells. Furthermore, we demonstrated that an important controlling factor in transforming growth factor-beta inhibition of cell cycle progression, the phosphorylation of the Rb protein, was altered in WM35N-ras; transforming growth factor-beta caused a marked relative increase in hypophosphorylated pRb in WM35 cells, but not in WM35N-ras. These data suggest that activated Ras plays an important part in melanoma progression from the radial growth phase to the vertical growth phase by counteracting inhibition by cytokines such as transforming growth factor-beta, thus providing a growth advantage.

Positive tetracycline control of expression of p15INK4B from an Epstein-Barr autonomous plasmid in a human melanoma cell line.

Homozygous deletions in the region of chromosome 9p21 are frequent in human melanoma. Mutations in the p16INK4A cyclin-dependent kinase inhibitor (CDI) gene at this locus have implicated the product of this gene as a tumor suppressor. Less attention has been focused on the homologous, closely linked p15INK4B gene. To facilitate study of the phenotypic effects of restoring expression of the latter in aggressive melanoma cells lacking INK4 expression, we inserted the cDNA encoding p15INK4B into an autonomously maintained plasmid under positive tetracycline control ('TET ON' system). Similarly regulated luciferase and herpes thymidine kinase sequences were used as controls. We demonstrate that this system enabled efficient, and reasonably uniform, induction of p15INK4B expression in a human melanoma cell line exposed to the tetracycline derivative, doxycycline. Flow cytometry showed that this induction resulted in substantial accumulation of cells in the G0/G1 phase of the cell cycle. This system will facilitate detailed analysis of the cell cycle inhibitory mechanisms of this CDI in human melanoma cells.

Control of parvovirus DNA replication by a tetracycline-regulated repressor.

Autonomous parvoviruses are small, single strand DNA viruses which preferentially replicate in transformed and tumor cells, causing cell death by expression of the cytotoxic nonstructural protein, NS1. Several parvoviruses of the rodent group, including LuIII, efficiently infect human transformed cell lines. The potential for systemic use of these viruses in targeting metastases might be enhanced if NS1 expression and viral replication could be controlled by an innocuous drug such as tetracycline. We therefore substituted prokaryotic tetracycline operator sequences for part of P4 of LuIII, the promoter responsible for transcription of the mRNAs for nonstructural proteins. The resulting construct unexpectedly showed constitutive expression in transiently transfected cells, as indicated by efficient excision and amplification of viral replicative form (RF) DNA. This was apparently due to self-stimulatory transcriptional transactivation by NS1. This problem was overcome by cotransfection with a plasmid expressing a chimera of the repressor of the tetracycline operon with a KRAB transrepression domain. These conditions allowed efficient control of transcription and RF amplification by the tetracycline derivative, doxycycline. These observations form a basis for developing a therapeutic agent based on a drug-controlled parvovirus.

Overexpression of mutant ras in human melanoma increases invasiveness, proliferation and anchorage-independent growth in vitro and induces tumour formation and cachexia in vivo.

Malignant melanoma is the deadliest form of skin cancer. Previous studies have shown that the incidence of ras mutation increases with progression of melanoma, but that such mutations may not be present in the earliest radial growth phase melanomas. Recently it has been proposed that introduction of ras mutations into cells deficient in tumour suppressor genes such as p16 (INK4a) is sufficient to induce characteristics of cellular transformation such as anchorage-independent growth and tumour formation in vivo. To test this hypothesis in human melanoma, mutant N-ras, mutant H-ras or wild-type H-ras genes were transfected by electroporation into WM35 cells, a p16-deficient human melanoma cell line of low invasive potential. Increased expression of mutant ras p21 enhanced anchorage-dependent cell growth on tissue culture plastic. In addition, overexpression of mutant N-ras and H-ras, but not of wild-type H-ras, increased the experimental invasive potential, inducing anchorage-independent growth in soft agar, increasing cell motility measured by time-lapse video microscopy, and increasing invasiveness through reconstituted basement membranes. Finally, overexpression of mutant H-ras in melanoma cells was shown to increase tumorigenicity and to induce cachexia when H-ras transfected cell lines were injected subcutaneously in severe combined immunodeficiency (SCID) mice. Thus the addition of activating ras mutations to a melanoma cell line already deficient in p16 leads to enhanced proliferation, survival and migration in vitro and to enhanced subcutaneous tumour formation in vivo. This phenotype is typical of the behaviour of vertical growth phase (VGP) melanoma, and we propose that activation of the ras signalling pathway in the presence of deletions in p16 or related tumour suppressors can induce the VGP melanoma phenotype.

An adenovirus type 5 mutant with the preterminal protein gene deleted efficiently provides helper functions for the production of recombinant adeno-associated virus.

Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308DeltapTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308DeltapTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308DeltapTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function.

In vitro expression of the diphtheria toxin A-chain gene under the control of human chorionic gonadotropin gene promoters as a means of directing toxicity to ovarian cancer cell lines.

OBJECTIVE: Our goal was to determine whether toxicity of the diphtheria toxin A-chain gene regulated by the human chorionic gonadotropin promoter can be directed to malignant ovarian cell lines. STUDY DESIGN: Plasmids containing diphtheria toxin A-chain gene linked to the regulatory elements of the metalloergothioneine and human chorionic gonadotropin promoters were transfected into the cell lines. Expression of diphtheria toxin A-chain gene was determined by the inhibition of a cotransfected luciferase reporter gene. RESULTS: Cytotoxicity of the diphtheria toxin A-chain gene is shown in a dose-responsive manner. Transfection of a plasmid expressing the diphtheria toxin A-chain gene controlled by a constitutive promoter readily inhibits protein synthesis. Specific inhibition of luciferase protein synthesis occurs in ovarian cancer cells transfected with the diphtheria toxin A-chain gene under the control of the human chorionic gonadotropin promoters when compared with normal ovarian epithelial cells or fibroblasts. CONCLUSIONS: These data demonstrate the preferential expression of the diphtheria toxin A-chain gene, regulated by the human chorionic gonadotropin promoter, to ovarian cancer cell lines. This provides an avenue for targeting such cells for suicide, toxin, or cytokine genes.

Tropic determinant for canine parvovirus and feline panleukopenia virus functions through the capsid protein VP2.

Canine parvovirus (CPV) can productively infect canine and feline cell lines whereas feline panleukopenia virus (FPV) is restricted to the latter. The major determinants of tropism are two amino acids in the sequence shared by the capsid proteins, VP1 and VP2. We have shown that a rodent parvovirus-derived transducing genome, containing the luciferase reporter, can be packaged by VP1 and VP2 from separate helper sources. Canine A72 cells and feline CFK cells were transduced with recombinant virions generated using VP1 and VP2 combinations from CPV and FPV. Both VP1 and VP2 were necessary for production of transducing virions. Efficient transduction of A72 cells required VP2 of CPV. Therefore, the capsid determinants of tropism for CPV and FPV are in VP2, although a source of VP1 is also necessary to produce infectious particles. The results extend similar observations on the tropic determinants of different strains of minute virus of mice.

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.

Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells.

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was approximately 250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3-6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3-6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.

Expression of parvovirus LuIII NS1 from a Sindbis replicon for production of LuIII-luciferase transducing virus.

In order to develop an alternative packaging system for recombinant parvoviruses, the gene for the major nonstructural protein (NS1) of parvovirus LuIII was inserted into a Sindbis replicon vector. Cells infected with recombinant SinNS1 virus produced NS1 RNA from the Sindbis 26S promoter and expressed NS1 protein which was able to transactivate a parvovirus P38 promoter. Co-transfections of Sindbis-NS1 RNA together with a packageable LuIII transducing genome and a coat protein expression plasmid generated detectable levels of LuIII-luciferase transducing virus. These levels could be increased by a capsid expression plasmid that was also capable of expressing NS2. These results show that a multi-functional parvovirus protein expressed from a Sindbis RNA molecule can be used to produce recombinant parvoviruses.

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.

Selective isolation of transiently transfected cells from a mammalian cell population with vectors expressing a membrane anchored single-chain antibody.

We present here a novel technology for the rapid selection of transiently transfected cells from total populations in culture. This system utilizes recombinant antibody technology to produce a 'molecular hook' by displaying a hapten-binding single-chain antibody (sFv) on the surface of transfected cells. Mammalian cell lines from several origins were transiently transfected with a plasmid (pHook-1) that encodes an sFv fused with a transmembrane anchor and found to express and display the functional hapten-binding sFv on their membranes. Transfected cells were selected from total populations in culture by virtue of their ability to bind to hapten-coated magnetic beads. Some cell lines were able to display sFv sufficient for selection as early as 2 h post-transfection. SK-BR-3 human breast carcinoma cells were co-transfected with pHook-1 and pCR31acZ (expresses beta-galactosidase), selected, and assayed for beta-galactosidase activity. The positive correlation between sFv and beta-galactosidase expression in these cells (95% of selected cells also expressed beta-galactosidase activity) suggests that pHook-1 will be useful in isolating cells co-expressing an exogenous gene of interest. Another vector was constructed in which a gene of interest may be expressed from the same plasmid as the sFv 'hook'. This construct (pHook-2) allows the selection of a homogenous population of cells expressing exogenous genes without co-transfection or the generation of stable transfectants. In experiments where the lacZ gene was co-expressed with the sFv 'hook' from this single plasmid, 100% of 293 human kidney cells and 100% of SK-BR-3 cells selected with antigen-coated magnetic beads stained positively for beta-galactosidase activity. We propose that this system will be a valuable tool for studying the acute and chronic effects of the expression of a variety of wild type and mutant proteins.

Autonomous parvovirus transduction of a gene under control of tissue-specific or inducible promoters.

Several classes of viruses are in use, or are being developed, as gene therapy vectors. Viruses with small genomes containing few essential genes have the advantage of requiring only simple complementation systems to allow packaging of foreign DNA, substituted for the entire viral coding sequences. Retroviruses and the dependent parvovirus AAV (adeno-associated virus) have been used in this way, and both possess an efficient integration mechanism which should allow long-term expression of transduced genes. In some situations, however, long-term persistence may be undesirable and there is a need for small, non-integrating viral vectors. Autonomous parvoviruses, such as LuIII, have potential as such vectors for short-term expression of therapeutic genes. We previously described recombinants of LuIII that transduced reporter genes, expressed using the viral constitutive promoter, P4. We have now generated several recombinants containing regulated promoters. A virus including a liver-specific enhancer directed 10- to 20-fold preferential expression of the luciferase reporter in transduced human hepatoma (HepG2) versus HeLa cells. In additional LuIII recombinants, the luciferase reporter was linked with chimeric promoters containing binding sequences for either the yeast GAL4 protein or the bacterial tetracycline repressor. Luciferase expression was strongly activated when these viruses were used to infect cells containing a cognate trans-activator (GAL4 or tTA, a tetracycline repressor fusion with VP16 of herpes simplex), introduced by transfection. The response to tTA could be abolished, or reduced in a graded manner, by exposure of the infected cells to tetracycline. Further results suggested that an increase in basal expression, apparently mediated by the viral left terminal inverted repeat, could be minimized by interposing polyadenylation signals between this sequence and the promoter. These results confirm that appropriate transcriptional regulation can be achieved for genes transduced by an autonomous parvovirus vector. Such vectors therefore show promise for the delivery of therapeutic genes in situations requiring cell-specific, short-term expression, eg in targeting suicide genes for ablation of cancer cells.

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.

Symmetric-strand packaging of recombinant parvovirus LuIII genomes that retain only the terminal regions.

LuIII is an autonomous parvovirus which encapsidates either strand of its genome with similar efficiency in NB324K cells. Two parvoviruses closely related to LuIII, minute virus of mice (MVM) and H-1 virus, encapsidate primarily the minus strand of their genome when grown in the same cell type. It has been postulated that an AT-rich region unique to LuIII is responsible for symmetric encapsidation of plus- and minus-strand genomes by LuIII. To address this hypothesis, recombinant LuIII-luciferase genomes containing or lacking the AT-rich sequence (AT) were packaged into LuIII virions. Hybridization of strand-specific probes to DNA from these virions revealed that either strand of the genome was packaged regardless of the presence of AT. In addition, encapsidation of both strands of the AT+ LuIII-luciferase genome into MVM and H-1 virions was observed, suggesting that MVM and H-1 viral proteins are not responsible for the minus-strand packaging bias of these two viruses. Alignment of the published LuIII and MVMp sequences shows that AT exists as an insertion into an element that, in MVM, binds cellular proteins. We suggest that in LuIII, AT disrupts binding of these cellular proteins, allowing encapsidation of either strand.

Suppression of single and double nonsense mutations introduced into the diphtheria toxin A-chain gene: a potential binary system for toxin gene therapy.

We have previously shown that ablation of specific cells can be achieved through the transcriptionally regulated expression of the diphtheria toxin A-chain (DT-A) gene in both cell culture and transgenic mice. Such targeted toxin gene expression provides a novel approach to cancer and acquired immunodeficiency syndrome (AIDS) therapy. The use of mutants of DT-A with attenuated toxicity may allow targeting of cells for which only moderately selective gene regulatory elements are available. Alternatively, conditional mutants might be used to target cells in which conditions can be established for suppression of the mutation. We have investigated the effects of mutating selected serine codons to amber (TAG) nonsense codons in the DT-A coding sequence. In transient transfection of HeLa cells, DT-A activity was markedly reduced by the introduction of a single amber codon and was virtually eliminated by two amber mutations. Cotransfection of a serine inserting suppressor tRNA expression plasmid substantially restored DT-A expression from both single and double amber mutants. Expression of the same suppressor tRNA also suppressed a previously described amber mutation at the tyrosine codon 28 in DT-A. Thus, nonsense suppression can be used to control the expression of DT-A in mammalian cells, potentially allowing binary control over the targeting of tissues for selective ablation.

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.