Preclinical evaluation of a prostate-targeted gene-directed enzyme prodrug therapy delivered by ovine atadenovirus.

Gene-directed enzyme prodrug therapy (GDEPT) based on the Escherichia coli enzyme, purine nucleoside phosphorylase (PNP), provides a novel strategy for treating slowly growing tumors like prostate cancer (CaP). PNP converts systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine, that kills PNP-expressing and nearby cells by inhibiting DNA, RNA and protein synthesis. Reporter gene expression directed by a hybrid prostate-directed promoter and enhancer, PSMEPb, was assayed after plasmid transfection or viral transduction of prostate and non-CaP cell lines. Androgen-sensitive (AS) LNCaP-LN3 and androgen-independent (AI) PC3 human CaP xenografts in nude mice were injected intratumorally with an ovine atadenovirus vector, OAdV623, that carries the PNP gene under PSMEPb, formulated with cationic lipid for enhanced infectivity. Fludarabine phosphate was then given intraperitoneally for 5 days at 75 mg/m2/day. PNP expression was evaluated by enzymic conversion of its substrate using reverse phase HPLC. OAdV623 showed excellent in vitro transcriptional specificity for CaP cells. In vivo, expression of PNP persisted for > 6 days after OAdV623 injection and a single treatment provided 100% increase in tumor doubling time and > 50% inhibition of tumor growth for both LNCaP-LN3 and PC3 lines, with increased tumor necrosis and apoptosis and decreased tumor cell proliferation. OAdV623 significantly suppressed the growth of AS and AI human CaP xenografts in mice.

Gene therapy for prostate cancer delivered by ovine adenovirus and mediated by purine nucleoside phosphorylase and fludarabine in mouse models.

A gene-directed enzyme pro-drug therapy (GDEPT) based on purine nucleoside phosphorylase (PNP), that converts the prodrug, fludarabine to 2-fluoroadenine, has been described, but studies are limited compared with other GDEPTs. We investigated the in vitro and in vivo efficacies of PNP-GDEPT for treating androgen-independent (AI) prostate cancer. The PNP gene controlled by Rous sarcoma virus (RSV) constitutive promoter was delivered using a recombinant ovine adenovirus vector (OAdV220) that uses a different receptor from human adenovirus type 5. In vitro, OAdV220 provided increased transgene expression over a comparable human Ad5 vector in infected AI, murine RM1 prostate cancer cells. Subsequent in vivo testing was therefore confined to OAdV220. Transduction of RM1 cells with OAdV220 before implantation in immunocompetent mice dramatically inhibited subcutaneous (s.c.) tumor growth when fludarabine phosphate was administered systemically and increased mouse survival in a dose-dependent manner. In tumor-bearing C57BL/6 mice, a single intratumoral injection of OAdV220 produced detectable PNP activity for at least 6 days and with prodrug, retarded the growth of aggressive RM1 s.c. tumors by 35% at day 14. There was a consistent trend to reduction of pre-established intraprostatic RM1 tumors. A similar regimen induced significant therapeutic efficacy in human PC3 xenografts. Thus, ovine adenovirus-mediated GDEPT using the PNP system was effective in vivo against AI prostate cancers, the aggressive murine RM1, and the human PC3 lines. Methods that improve viral dissemination and stimulate the immune system in vivo may further improve efficacy.

High level, tissue-specific expression of a modified calcitonin/calcitonin gene-related peptide promoter in a human medullary thyroid carcinoma cell line.

The efficient and high level expression of therapeutic genes in target cells is critical for effective gene therapy. We have developed a novel promoter by utilizing tandem repeats of a tissue-specific regulatory element from the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene placed in close proximity to a basal promoter, thereby removing interstitial sequences. This promoter drives expression of reporter genes at much higher levels than the natural promoter while significantly improving specificity in thyroid C cells.

Ovine adenovirus vectors mediate efficient gene transfer to skeletal muscle.

Ovine adenovirus (OAV) vectors represent a promising tool for human gene therapy since these vectors overcome the problem of pre-existing immunity against human adenovirus vectors. In this report we investigated the in vivo characteristics of this novel vector system with respect to its potential for gene transfer into skeletal muscle. We found that moderate doses of an OAV-derived vector expressing the human alpha1-antitrypsin gene (OAVhaat) infected skeletal muscle in mice very efficiently resulting in high serum hAAT levels. The infection was restricted to skeletal muscle, but gene expression was transient and vector DNA was rapidly cleared. Vector clearance was also observed with a vector that lacked the transgene. The loss of vector DNA was accompanied by a cellular immune response in the infected muscle but was not connected with detectable expression of early or late genes of the viral backbone as analyzed by RT-PCR. A very low dose of OAVhaat (3x 10(7) infectious particles) was sufficient to produce reasonable amounts (>100 ng/ml) of serum hAAT, and this was accompanied by a weak immune response to the vector. Under these conditions, a second intramuscular injection of the same recombinant OAV vector was successful. Our study expands the known tissue tropism of OAV-derived vectors in vivo and points to the possible utility of the vector for muscle gene transfer and vaccination.

An ovine adenovirus vector lacks transforming ability in cells that are transformed by AD5 E1A/B sequences.

Adenoviruses of the Mastadenovirus and Aviadenovirus genera are able to transform certain cell types and induce tumor formation in susceptible animals. For the mastadenoviruses the E1A/B sequences are largely responsible for these properties but E4 sequences may also be involved. The transforming sequences of the aviadenoviruses, which lack E1A/B and E4 homologues, have not yet been fully identified. The recent proposal for a third genus of adenoviruses, which apparently lack an E1A homologue and have weak E1B homology, prompted an examination of the transforming properties of ovine adenovirus OAV287 (OAV), the prototype member of the new group. When OAV and human adenovirus type 5 (Ad5) were used to infect primary rat embryo cells, transformed foci developed in Ad5- but not in OAV-infected cultures. Similarly, after plasmid transfection, baby rat kidney cells were transformed by Ad5 E1A/B but not by OAV sequences. When CSL503 cells, an ovine cell line that is permissive for OAV, were transfected with Ad5 E1A/B sequences, transformed foci again appeared. However, plasmids or fragments containing complete or partial OAV genome sequences did not detectably transform CSL503 cells under the same conditions. When Ad5 E1A/B sequences were incorporated into the complete OAV genome and transfected, transformed clones were again obtained, showing that the gene dosage and transfection conditions were not limiting for transformation. The provision of Ad5 E1A and OAV sequences in combination marginally increased the number of morphologically altered foci in baby rat kidney cells but failed to induce multilayered focus formation. The data suggest that OAV lacks transforming functions in the cell types examined. Additional information suggesting that OAV may have a fundamentally distinct strategy for replication compared with other Ads is discussed.

Ovine adenovirus vectors overcome preexisting humoral immunity against human adenoviruses in vivo.

Recombinant human adenoviruses (hAd) have become widely used as tools to achieve efficient gene transfer. However, successful application of hAd-derived vectors in clinical trials is limited due to immunological and potential safety problems inherent in their human origin. In this study, we describe a recombinant ovine adenovirus (OAV) as an alternative vector for gene transfer in vivo. In contrast to an hAd vector, the OAV vector was not neutralized by human sera. An OAV vector which contained the cDNA of the human alpha1-antitrypsin (hAAT) gene linked to the Rous sarcoma virus promoter was generated and administered systemically to mice. The level and duration of hAAT gene expression was similar to that achieved with an hAd counterpart in both immunocompetent and immunodeficient mice. However, the tissue distribution of the OAV vector differed from that observed for hAd vectors in that the liver was not the dominant target. Significantly, we demonstrated efficient gene transfer with the OAV vector into mice immunized with hAd vectors and vice versa. We also confirm that the immune response to a transgene product can prevent its functional expression following sequential application of a vector. Our results suggest a possible solution to endemic humoral immunity against currently used hAd vectors and should therefore have an impact on the design of improved gene therapy protocols utilizing adenovirus vectors.

Altered tropism of an ovine adenovirus carrying the fiber protein cell binding domain of human adenovirus type 5.

Ovine adenovirus OAV287 (OAV) is phylogenetically and serotypically distinct from human Ad5. OAV grows productively in CSL503 foetal ovine lung cells and abortively infects several human cell lines. OAV has a unique fiber and a penton protein that lacks a recognisable integrin-binding motif. It is not known whether a secondary receptor is required for infection. A hybrid virus was constructed in which the cell binding domain on the OAV fiber protein was exchanged for the equivalent region from human adenovirus type 5. The hybrid OAV grew to titres that were 1 to 2 log10 lower than wild-type OAV in permissive ovine cells. Human Ad5 also infected CSL503 cells but failed to compete with OAV for receptor binding sites on those cells. However, the hybrid virus did compete with Ad5, consistent with its use of the Ad primary receptor. The hybrid virus was also neutralised by Ad5 antiserum whereas OAV was not. Human 293 kidney and LNCaP prostate cell lines that were not detectably infected by OAV were infected by the hybrid virus and other human prostate and breast cancer cell lines showed greatly enhanced infectivity. Thus, modification of the fiber cell binding domain was sufficient to profoundly alter the tropism of OAV, suggesting that the interaction between the primary receptor and the virus particle is the major factor controlling virus entry during infection.

In vivo gene therapy for prostate cancer: preclinical evaluation of two different enzyme-directed prodrug therapy systems delivered by identical adenovirus vectors.

Advanced prostate cancer is invariably lethal once it becomes androgen independent (AI). With the aim of developing a new treatment we have used the human androgen-independent prostate cancer cell line, PC-3, to evaluate the effectiveness of two enzyme-directed prodrug therapy (EPT) systems as a novel means for promoting tumor cell destruction in vivo. We have confined our study to the use of a PSA promoter, in a preliminary attempt to achieve prostate specificity. The two EPT systems used were the HSVTK/GCV and PNP/6MPDR systems. These were chosen for their differential dependence on DNA replication for their mechanism of action. In the present work, either the HSVTK or PNP gene, each controlled by a PSA promoter fragment, was delivered by an E1-, replication-deficient human adenovirus (Ad5) into PC-3 tumors growing subcutaneously in BALB/c nude mice. Tumors were injected with a single dose of recombinant Ad5 and mice were treated intraperitoneally with the appropriate prodrug, twice daily, for 6 days thereafter. The growth of established PC-3 tumors was significantly suppressed and host survival increased with a single course of HSVTK/GCV or PNP/6MPDR treatment. HSVTK/GCV-treated PC-3 tumor growth was 80% less than that of control treatments on day 33, while PNP/6MPDR-treated tumor growth was approximately 75% less than that of control treatments on day 52. Survival data showed that 20% of HSVTK/GCV- or PNP/6MPDR-treated animals lived >45 and >448 days, respectively, longer than control animals. These results demonstrate that both HSVTK/GCV and PNP/6MPDR therapies interrupt the growth of an aggressive human prostate cancer cell line in vivo.

Identification of transcripts and promoter regions of ovine adenovirus OAV287.

The ovine adenovirus isolated OAV287 represents a new group of adenoviruses that are distinct from the Mast- and Aviadenoviruses by several criteria, including genome arrangement. The OAV major late promoter and some late transcripts were previously mapped. To better define the probable coding sequences and to identify the approximate location of early promoters a partial transcription map of the genome was elucidated using a PCR-based approach. This was possible because the complete nucleotide sequence of the genome was known. The strategy permitted the identification of transcription start sites and RNA splice junctions and allowed the approximate location of promoters in the lefthand end, IVa2, E2, P32K, and E4 regions to be deduced. The data showed that lefthand end and E4 regions are controlled by three and two temporally distinct promoters, respectively. The E2 region is controlled by a single promoter, in contrast to Mastadenoviruses, where E2 expression is controlled by the E2A and E2B promoters. The p32kDa structural protein at the lefthand end and the IVa2 protein are also expressed from their own promoters. These data contribute to the first overview of transcription from a non-Mastadenovirus genome.

Ovine adenovirus (OAV287) lacks a virus-associated RNA gene.

Ovine adenovirus OAV287 (OAV) is the prototype of a virus group which is phylogenetically distinct from the mastadenoviruses and aviadenoviruses. The genome arrangement of OAV showed that virus-associated (VA) RNA genes were not located between the reading frames for p52/55K and terminal protein as these overlapped. To determine whether VA genes were located elsewhere, several approaches were used. Nuclear extracts containing RNA polymerase III activity were used to transcribe OAV genome fragments in vitro. A product of approximately 120 bp was produced from two widely separated coding regions of the genome. However, when these were subcloned and used as radiolabelled probes to analyse RNA from OAV-infected cells, no VA-like RNA was detected, although late mRNAs that were transcribed from the regions were identified. In addition, 5' radiolabelling of small RNA species in control- and OAV-infected cells followed by gel analysis did not identify candidate VA RNAs. Radiolabelling of proteins in control- and OAV-infected cells showed that there was little preferential translation of viral proteins compared with host polypeptides, in contrast to the situation for adenovirus 5 (Ad5). In addition, the kinetics of OAV infection were slower than observed for human adenoviruses. Collectively, the data suggest that OAV is unique in that it does not produce VA RNA during infection. This conclusion is supported by a comparison of the genomes of the phylogenetically related OAV and egg drop syndrome viruses which shows that the VA gene identified in the latter is located in a region absent from OAV.

Sequential nucleic acid and recombinant adenovirus vaccination induces host-protective immune responses against Taenia ovis infection in sheep.

Sheep were immunized with a protective recombinant antigen (45W) from the cestode parasite Taenia ovis using three different vaccine delivery systems, either alone or in different combinations. The DNA encoding 45W was cloned into the expression plasmid pcDNA 3 and an ovine adenovirus to create nucleic acid and recombinant viral vector vaccines, respectively. Sheep received two vaccinations with various combinations of these two delivery systems and/or purified recombinant 45W protein in a conventional vaccine formulation containing Quil A as adjuvant (protein/Quil A vaccine). Sheep receiving two inoculations of either the nucleic acid or the recombinant adenovirus alone, demonstrated only low levels of 45W-specific antibody. However, immunization with either nucleic acid or recombinant adenovirus primed animals to mount an enhanced immune response after a subsequent vaccination with the protein/ Quil A vaccine. The most striking result was that sheep initially immunized with the nucleic acid vaccine and boosted with the recombinant adenovirus, mounted IgG1 responses > 65 fold higher than those of sheep receiving either vaccine alone. The level of antibody in these sheep was commensurate with that observed in animals vaccinated twice with the protein/Quil A adjuvanted vaccine. In both cases, host-protection from experimental challenge infection with T. ovis was obtained.

Construction of ovine adenovirus recombinants by gene insertion or deletion of related terminal region sequences.

An ovine adenovirus which may be the prototype for a new group of adenoviruses has been engineered as a gene transfer vector. One recombinant containing a 0.95-kb insertion expressed a sheep parasite antigen from the ovine adenovirus major late promoter and tripartite leader sequences. It was shown that insertions of at least 4.3 kb were tolerated at either one of two sites in the genome without the introduction of a compensating deletion. The unique structure of this viral genome was further emphasized by the discovery that four open reading frames at the right hand end show significant identity to each other but not to other sequences in the databases. Two other unrelated open reading frames were also present. RT-PCR analysis identified two transcripts in this region which were derived from a promoter which was located very close to, or within the ITR sequence. Splicing removed all but the first and last of the ORFs from these RNAs, suggesting that some sequences might be nonessential for replication in vitro. A approximately 2-kb deletion, which removed or truncated the internal reading frames was introduced into the region without affecting virus viability. The carrying capacity of OAV recombinants should therefore be at least 6.3 kb. The relative packaging capacity of OAV (114%) therefore exceeds that of Ad5 (105%), although a comparison of virus particle sizes by electron microscopy showed that OAV was smaller than Ad5. These studies improve the potential utility of OAV as a gene transfer vector.

Gene expression by atypical recombinant ovine adenovirus vectors during abortive infection of human and animal cells in vitro.

An bovine adenovirus, which is phylogenetically distinct from the Mastadeno- and Aviadenoviruses, was used to construct recombinants in which reporter genes were expressed from the OAV major late, or human cytomegalovirus promoters. It was demonstrated by transgene expression that OAV could infect bovine nasal turbinate and rabbit kidney cells as well as a range of human cell types, including lung and foreskin fibroblasts as well as liver, prostate, breast, colon, and retinal lines. Some human lines, e.g., 293 and LNCaP were not detectably infected. Infection occurred even though OAV has a fiber protein with a unique cell binding domain and a penton protein that lacks the integrin-binding Arg-Gly-Asp motif which facilitates entry by human adenoviruses. Most cell lines showed little or no ill effect for several days after infection but a prominent cytopathic effect appeared in fibroblasts after 3-4 days. However, no viral DNA synthesis was detected and replication was abortive. Viral promoter activity during infection of nonpermissive cell types was assayed by RT-PCR. Early promoter activity was detectable in some, but not all cell types. In a liver and a colon carcinoma cell line, none of the promoters examined was significantly active, even when a higher multiplicity of infection was used. Major late promoter activity was not detectable in any cell type. The lack of DNA replication and MLP function suggests that a critical transition from early to late gene expression does not occur during abortive infection by OAV.

Relative efficiency of tumor cell killing in vitro by two enzyme-prodrug systems delivered by identical adenovirus vectors.

Enzyme-prodrug therapy for the treatment of cancer is an experimental procedure that is under intensive investigation. However, the relative merits of the various systems for use under specific conditions are still being determined. We have compared the efficacy of cell killing by the herpesvirus thymidine kinase (HSVTK)/ganciclovir and the purine nucleoside phosphorylase (PNP)/9-(beta-M-2-deoxy-erythropentofuranosyl)6-methylpurine enzyme/prodrug systems. These were chosen because of their differential dependence on DNA replication for their mechanism of action. The HSVTK and PNP genes, expressed from the identical prostate-specific antigen promoter, were transduced into human prostate and breast cancers cells using the same human adenovirus vector. The kinetics of cell killing in the presence of the respective prodrugs was monitored using a nondestructive assay that measured total cell bioactivity. The PNP/9-(beta-D-2-deoxy-erythropentofuranosyl)6-methylpurine system was clearly superior in its ability to cause cell death in vitro. Cells were killed in about half the time and at a 5-10-fold lower input of virus relative to the HSVTK/ganciclovir system. The PNP system may offer advantages for the treatment of slow-growing tumors in which the daily proliferative rate is low or in situations in which gene delivery or expression is inefficient.

Nucleotide sequence of ovine adenovirus tripartite leader sequence and homologues of the IVa2, DNA polymerase and terminal proteins.

Ovine adenovirus OAV287 was previously isolated from sheep in Western Australia. Here we describe a portion of its genome between map units 10.3 and 31.7 which includes major ORFs for homologues of the IVa2 polypeptide and the DNA replication proteins, Terminal protein and DNA polymerase, as well as the N-terminal portion of the 52/55-kDa polypeptide. In addition, as a prelude to possible adaptation of this virus as a vector we have mapped the elements which make up the tripartite leader sequence of late mRNAs, thereby defining the probable location of the OAV major late promoter. In other human and animal adenovirus genomes, one or two VA RNA genes are encoded between the ORFs for Terminal protein and 52/55-kDa polypeptides. In OAV, these ORFs overlap, suggesting that if VA RNA genes are present, they may lie elsewhere in the OAV genome.

Unique genome arrangement of an ovine adenovirus: identification of new proteins and proteinase cleavage sites.

The completed sequence and genome organization of OAV287, a serologically distinct ovine adenovirus, is described. The genome of 29,544 bp has inverted terminal repeats that are only 46 bp in length. Many OAV genes are identified by their homology with other adenovirus (Ad) sequences but three groups of reading frames show little homology. One group at the left-hand end of the genome probably represents the E1A/E1B regions. Two others, on the complementary strand at the right-hand end of the genome, are tentatively proposed as the E4 and E3 regions. They are separated by approximately 1 kb of A/T-rich sequence of unknown function with E3 being adjacent to the terminus. Structural proteins V and IX of human Ads are absent from the OAV genome but a new, processed, 28-kDa virion polypeptide is encoded on the strand complementary to the proposed E1A region. The coding sequences for two other structural proteins are unidentified. The OAV penton protein lacks the region containing an Arg/Gly/Asp sequence that, in human adenoviruses, is thought to interact with cellular integrins to facilitate virus entry. Analysis of proteins and peptides in purified OAV identified several cleavage sites utilized by the Ad proteinase. Some of these were previously identified in human Ad proteins, but new sites, some of which did not conform to the known specificity of the human Ad proteinase, were also identified. The data emphasize that this ovine virus differs significantly from other known human and animal adenoviruses.

Construction and transfection of ovine adenovirus genomic clones to rescue modified viruses.

The genome of ovine adenovirus OAV287 has an arrangement which is unique among known adenoviruses. To facilitate further experimentation on the structure and function of this genome, plasmids containing a complete clone of the genome were constructed. The cloned viral genome was released from plasmids by restriction enzyme digestion as an intact linear molecule with authentic 5' termini. Transfection of the linear DNA into cells which supported replication produced infectious virus. Mutation of a unique SalI site at the right-hand end of the genome disrupted reading frames of unknown function without affecting virus rescue, identifying this region as nonessential for replication in vitro. A 20-bp oligonucleotide was also inserted into the short intergenic region between the pVIII and the fiber sequences, identifying a second site for gene insertion. These studies will facilitate the development of OAV as a gene transfer vector.

Membrane binding and endoplasmic reticulum retention sequences of rotavirus VP7 are distinct: role of carboxy-terminal and other residues in membrane binding.

The sequences responsible for binding rotavirus glycoprotein VP7 to the membrane of the endoplasmic reticulum (ER) have not been identified. Here we show that the sequences which promote membrane binding in vitro are distinct from the N-terminal sequences which promote retention of VP7 in the ER in vivo. The role of the C-terminal region in membrane binding was also examined by using truncation mutants. Membrane binding in vitro was reduced but not abolished by removing up to 102 residues from the C terminus. The data suggest that the last 36 residues of VP7 may be present in the membrane or translocation pore, possibly with the C terminus protruding into the cytoplasm, since these residues contribute to, but do not account for, membrane binding. Surprisingly, modified forms of VP7 which are secreted from transfected cells showed the same membrane-binding properties in vitro as the protein retained in the ER membrane. Thus, secreted VP7 may not be present as a soluble polypeptide in the ER. A model to explain these results is presented. Previously published data are consistent with the idea that the highly conserved C terminus of nascent VP7 could have a cytoplasmic orientation which is important for assembly of mature virus particles.

Investigation of promoter function in human and animal cells infected with human recombinant adenovirus expressing rotavirus antigen VP7sc.

Human adenovirus (Ad) vectors are being used increasingly for a variety of applications in vaccination and gene therapy. The ability of vectors to enter cells and the efficiency of promoters expressing the therapeutic gene or vaccine antigen are critical to the outcome of such experiments. To identify promoters which might be suitable for use under a variety of conditions we have investigated the expression of a rotavirus antigen, VP7sc, employing several commonly used promoters carried in E1-substituted Ad vectors both in cell types which support virus replication and in cells which do not. Although not all gene constructions were identical, wide variations in promoter function were evident even in human 293 cells which support virus replication. The simian virus type 40 (SV40) early and beta-actin promoters expressed poorly; the SV40 late promoter was somewhat better. The human IE94 cytomegalovirus (CMV) promoter and a modified Ad major late promoter were best, functioning equally well but with different kinetics. In other human cell lines the CMV promoter was more versatile, generally providing sustained expression at a significant level, in one case for at least 6 days. In addition, as mouse, rabbit and pig models of rotavirus infection are under investigation and VP7sc is a vaccine antigen, we also investigated the ability of the recombinant adenovirus to infect cells from these and other sources. VP7sc expression was detected in several heterologous cell types, illustrating the ubiquity of the human Ad receptor and the versatility of human Ad as vectors when suitable promoters are used.