Toca 511 gene transfer and 5-fluorocytosine in combination with temozolomide demonstrates synergistic therapeutic efficacy in a temozolomide-sensitive glioblastoma model.

Toca 511 (vocimagene amiretrorepvec), an amphotropic retroviral replicating vector (RRV), can successfully and safely deliver a functional, optimized cytosine deaminase (CD) gene to tumors in orthotopic glioma models. This agent, in conjunction with subsequent oral extended-release 5-fluorocytosine (5-FC) (Toca FC), is currently under investigation in patients with recurrent high-grade glioma . Temozolomide (TMZ) with radiation is the most frequently used first-line treatment for patients with glioblastoma, the most common and aggressive form of primary brain cancer in adults. However, subsets of patients with certain genetic alterations do not respond well to TMZ treatment and the overall median survival for patients who respond remains modest, suggesting that combinatorial approaches may be necessary to significantly improve outcomes. We show that in vitro TMZ delays but does not prevent RRV spread, nor interfere with Toca 511+5-FC-mediated cell killing in glioma tumor cells, and in vivo there is no significant hematologic effect from the combination of 5-FC and the clinically relevant dose of TMZ. A synergistic long-term survival advantage is observed in mice bearing an orthotopic TMZ-sensitive glioma after Toca 511 administration followed by coadministration of TMZ and 5-FC. These results provide support for the investigation of this novel combination treatment strategy in patients with newly diagnosed malignant glioma.

Simian immunodeficiency virus vector pseudotypes differ in transduction efficiency and target cell specificity in brain.

Lentiviral vectors have proven to be promising tools for transduction of brain cells in vivo and in vitro. In this study, we have examined the central nervous system (CNS) transduction efficiencies and patterns of a self-inactivating simian immunodeficiency virus (SIVmac)-derived lentiviral vector pseudotyped with glycoproteins from the vesicular stomatitis virus (VSV-G), the amphotropic murine leukemia virus (MLV4070Aenv), the lymphocytic choriomeningitis virus (LCMV-GP), the Ross River virus (RRV-GP) and the rabies virus (RV-G). All glycoproteins were efficiently incorporated into SIV virions, allowing efficient transduction of neuronal cell lines as well as of primary dissociated mouse brain cell cultures. After injection of highly concentrated vector stocks into the striatum of adult mice, quantitative analyses revealed high transduction efficiency with VSV-G pseudotypes, while LCMV-GP and RV-G pseudotypes exhibited moderate transduction efficiencies. MLV4070Aenv and RRV-GP pseudotypes, however, showed only weak levels of transduction after stereotactic injection into the brain. Regarding cell tropism in vivo, VSV-G-pseudotyped SIV vectors transduced neuronal as well as glial cells, whereas all other pseudotypes preferentially transduced neuroglial cells. In addition, we analyzed the influence of the central polypurine tract (cPPT) in context of the VSV-G-pseudotyped SIV transfer vector for infection of brain cells. Deletion of the cPPT sequence from the transfer vector decreased the in vivo transduction efficiency by fourfold, and, more importantly, this modification changed the transduction pattern, since these vectors were no longer able to infect neuronal cells in vivo. Vector injection into the brain did elicit a humoral immune response in the injected hemisphere; however, no gross signs of inflammation could be detected. Analysis of the biodistribution of the vector revealed that, besides the injected brain region, no vector-specific sequences could be detected in any of the organs evaluated. These data indicate SIV vectors as efficient gene delivery vehicles for the treatment of neurodegenerative diseases.

Effects of CD2 locus control region sequences on gene expression by retroviral and lentiviral vectors.

Locus control region (LCR) sequences are involved in the establishment of open chromosomal domains. To evaluate the possibility of exploiting the human CD2 LCR to regulate gene expression by Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors in T cells, it was included in vectors carrying the enhanced green fluorescence protein (EGFP) reporter gene; then transduction in vitro of lymphoid and nonlymphoid cell lines was performed. Deletion of the viral enhancer in the Mo-MLV long terminal repeat was necessary to detect LCR activity in the context of these retroviral vectors. It was found that a full-length (2.1 kb), but not a truncated (1.0 kb), CD2 LCR retained the ability to modulate reporter gene expression by Mo-MLV-derived retroviral vectors, leading to a homogeneous, unimodal pattern of EGFP expression that remained unmodified in culture over time, specifically in T-cell lines; on the other hand, viral titer was strongly reduced compared with vectors not carrying the LCR. Lentiviral vectors containing the CD2 LCR could be generated at higher titers and were used to analyze its effects on gene expression in primary T cells. Subcutaneous implantation of genetically modified cells in immunodeficient mice showed that retroviral vectors carrying the CD2 LCR conferred an advantage in terms of transgene expression in vivo, compared with the parental vector, by preventing the down-modulation of EGFP expression. These findings suggest a potential application of this LCR to increase gene expression by retroviral and lentiviral vectors in T lymphocytes.

Genetic reshuffling reconstitutes functional expression cassettes in retroviral vectors.

BACKGROUND: A major prerequisite for the design of retroviral vectors encoding cell toxic or harmful genes is the possibility to tightly control gene expression, thus limiting activity to the relevant target cells and protecting the packaging cell used for production of recombinant viral particles. METHODS: In the present study a system was developed in which genetic reshuffling during the retroviral life cycle is exploited, allowing reconstitution of functional expression cassettes from separate elements exclusively in transduced target cells. For construction of these murine leukaemia virus (MLV)-based reconstituting viral vectors (ReCon), a promoterless inverted enhanced green fluorescent protein (EGFP) reporter gene cassette was inserted in place of the U3 region of the 3' LTR. Subsequently, the human ubiquitin promoter was inserted in the inverse orientation into the R/U5 border of the 5' LTR of the vector. RESULTS: PA317 packaging cells stably transfected with ReCon vectors were established and EGFP expression was analysed by fluorescence-activated cell sorting (FACS). After detection of low-level background expression, an additional polyadenylation signal was introduced in antisense orientation into the 3' LTR at the R/U5 border to prevent accidental read-through transcription from neighbouring cellular promoters. Virus-containing cell culture supernatants were then used to infect NIH3T3 target cells. EGFP expression, recloning and sequencing of integrated proviruses demonstrated the correct reassembly of the transduced ubiquitin/EGFP transcription unit in these infected cells. CONCLUSIONS: This facile and convenient system should allow production of retroviral vectors encoding potentially toxic proteins, cell cycle inhibitors or inducers of apoptosis, all of which would interfere with vector production if expressed in the retroviral packaging cell.

In vivo perivascular implantation of encapsulated packaging cells for prolonged retroviral gene transfer.

Long-term benefits of coronary angioplasty remain limited by the treatment-induced renarrowing of arteries, termed restenosis. One of the mechanisms leading to restenosis is the proliferation of smooth muscle cells. Therefore, proliferating cells of the injured arterial wall, which can be selectively transduced by retroviruses, are potential targets for gene therapy strategies. A direct single-dose therapeutic application of retroviral vectors for inhibition of cell proliferation is normally limited by too low transduction efficiencies. Encapsulated retrovirus-producing cells release viral vectors from microcapsules, and may enhance the transduction efficiency by prolonged infection. Primary and immortal murine and porcine cells and murine retrovirus-producing cells were encapsulated in cellulose sulphate. Cell viability was monitored by analysing cell metabolism. Safety, stability, transfer efficiency and extent of restenosis using capsules were determined in a porcine restenosis model for local gene therapy using morphometry, histology, in situ beta-galactosidase assay and PCR. Encapsulation of cells did not impair cell viability. Capsules containing retrovirus-producing cells expressing the beta-galactosidase reporter gene were implanted into periarterial tissue or a pig model of restenosis. Three weeks following implantation, beta-galactosidase activity was detected in the pericapsular tissue with a transduction efficiency of approximately 1 in 500 cells. Adventitial implantation of vector-producing encapsulated cells for gene therapy may, therefore, facilitate successful targeting of proliferating vascular smooth muscle cells, and allow stable integration of therapeutic genes into surrounding cells. The encapsulation of vector-producing cells could represent a novel and feasible way to optimize local retroviral gene therapy.

Novel clinical strategies for the treatment of pancreatic carcinoma.

Pancreatic carcinoma ranks as the eighth most frequent type of solid tumour arising worldwide yet it represents the fourth most frequent cause of death. This discrepancy reflects the current lack of effective treatment available for the pancreatic cancer patient and highlights the urgent need for new therapeutic principles in this area. The last five years have seen an increasing number of novel approaches both in the pre-clinical area as well as in clinical trials for pancreatic cancer treatments. This review summarizes these new developments and attempts to rationalize the possibilities available for the patient at the beginning of the new millennium.

Necrotic, rather than apoptotic, cell death caused by cytochrome P450-activated ifosfamide.

Feline kidney cells were transfected with a vector overexpressing cytochrome P450 2B1 (CYP2B1). Transfected cells acquired a new specific biochemical activity, which could be demonstrated by a rapid CYP2B1 detection assay and showed selective sensitivity to the antitumorigenic prodrug ifosfamide (IFO). Further, the cell-killing effect was also mediated on nonmodified cells like feline kidney cells, mouse lymphoma, and human pancreatic cells in the vicinity of the CYP2B1-expressing cells due to the diffusible nature of the activated IFO metabolites. One of these, phosphoramide mustard, causes interstrand DNA cross-linking and it has been thought that the inability to repair this damage results in apoptosis. Surprisingly, our results clearly demonstrate a necrotic mechanism of IFO-induced cell death. This may have important implications for the activation of the immune system during CYP2B1/IFO suicide gene therapy of cancer.

Microencapsulated cell-mediated treatment of inoperable pancreatic carcinoma.

Pancreatic cancer can seldom be resected, and chemotherapy has only a limited effect on survival or tumour load. We did a phase I/II trial in 14 patients with pancreatic cancer to assess the safety of local activation of low-dose ifosfamide. We encapsulated genetically modified allogeneic cells, which expressed a cytochrome P450 enzyme, in cellulose sulphate and delivered them by supraselective angiography to the tumour vasculature. These cells locally activated systemically administered ifosfamide. The tumours of four patients regressed after treatment, and those of the other ten individuals who completed the study remained stable. Median survival was doubled in the treatment group by comparison with historic controls, and 1-year survival rate was three times better. Further studies of this cell-therapy-based treatment combined with chemotherapy for inoperable pancreatic cancer are warranted.

Influence of preassay and sequence variations on viral load determination by a multiplex real-time reverse transcriptase-polymerase chain reaction for feline immunodeficiency virus.

Determination of retroviral load is an important tool in the investigation of the success of therapeutic or vaccination trials in patients infected with lentiviruses such as HIV, or with their simian (SIV) or feline (FIV) counterparts. We have developed an one-tube quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay based on the ABI Prism 7700 Sequence Detection System (TaqMan) to quantify the viral load of FIV-infected cats. Two different primer/probe systems were designed to detect a broad range of clade A FIV isolates. Both systems are characterized by excellent reproducibility, high sensitivity, and a wide range of quantification. As a consequence of this improved precision in the quantitative RT-PCR, preassay variations have greater impact on the accuracy of the viral load estimation. To compensate for these variations, we improved the assay and developed a multiplex real-time RT-PCR, which allows simultaneous calculation of the viral copy number and the individual recovery rate in an one-tube reaction. This enables the rapid and accurate calculation of copy number independent of preassay variations. In further studies, two additional real-time RT-PCR assays were designed and used to investigate the influence of sequence variations in the binding regions for either the primers or probe. Sequence mismatches in this region had a significant effect (up to 4 logarithmic decades) on reaction efficiency. In view of the inherent variability of retroviral sequences, these results underline the necessity to check reaction efficiencies before determining viral load.

Modulation of Moloney leukemia virus long terminal repeat transcriptional activity by the murine CD4 silencer in retroviral vectors.

We investigated whether CD4 gene regulatory sequences might be useful for developing transcriptionally targeted Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors for gene expression specifically in CD4(+) cells. We could modulate Mo-MLV long terminal repeat (LTR) activity by inserting a 438-bp-long fragment containing the murine CD4 silencer in the LTR of the vector; both beta-galactosidase and green fluorescent protein reporter gene activities were strongly down-regulated in both murine and human CD8(+) cells, but not in CD4(+) lymphoid cell lines and freshly isolated lymphocytes transduced with this vector, compared with the findings using a control vector carrying wild-type LTRs. Titration experiments on NIH-3T3 cells revealed that inclusion of the CD4 silencer in the LTRs did not reduce the titer of the vectors. These findings indicate that a cellular silencer can be successfully included in retroviral vectors, where it maintains its transcription-regulatory function, thus suggesting a novel approach to transcriptional targeting.

Combined chemotherapy of murine mammary tumors by local activation of the prodrugs ifosfamide and 5-fluorocytosine.

The success of chemotherapeutic intervention is limited because the necessary high local drug doses cannot be achieved without systemic toxicity. Application of suicide genes (SGs) and direct conversion of prodrugs (PDs) to toxic metabolites in situ by SGs may enhance the efficacy of chemotherapy. To evaluate this strategy in two murine breast cancer models, TS/A and GR, we injected cellulose sulfate capsules harboring cat kidney cells expressing the SGs cytosine deaminase and cytochrome P450 2B1 (CYP2B1) intratumorally. The PDs 5-fluorocytosine and ifosfamide were administered in 3-day intervals. The effect of in situ chemotherapy with each PD alone and the combination was analyzed over a period of 100 days. The results reveal that for TS/A tumors, the antitumoral effect mediated by CYP2B1 is more efficient than that of cytosine deaminase, whereas for GR tumors, both systems worked equally well. Furthermore, we find additive toxicity using both SG/PD systems for both TS/A and GR tumors.

Rapid and sensitive detection of enhanced green fluorescent protein expression in paraffin sections by confocal laser scanning microscopy.

Various forms of green fluorescent protein (GFP) have become important reporters of gene transfer and expression after transfection or infection of cells in cell culture. Frequently, molecular biological assays (Northern blots, PCR) are applied to detect reporter gene expression in target organs. However, these methods are not suitable for evaluation of tissue- or cell-specific expression which would be of great interest especially in case of using tissue-specific promoters. Therefore, organs of transgenic mice with the enhanced green fluorescent protein (EGFP) gene under control of the cytomegalovirus (CMV) promoter were processed for histology by formaldehyde fixation and embedding in paraffin. Sections were deparaffinized, mounted and evaluated for fluorescence in a confocal laser scanning microscope. This method combines the advantages of direct exploitation of tissue sections without further staining procedures with evaluable tissue-, cell-, and even subcellular-specific distribution patterns of EGFP expression in tissues. Results obtained by direct evaluation of EGFP fluorescence in paraffin sections were confirmed by immunohistochemical staining with anti-EGFP. In the present report, we demonstrate that application of confocal microscopy on routinely processed histological preparations is very suitable for determining gene transfer efficiency and promotor activities.

Xenotransplantation: is the risk of viral infection as great as we thought?

Two major hurdles remain before xenotransplantation can enter the clinic. The first is the more technical issue of being able to overcome the human immune response that leads to rejection of transplanted organs/cells from other species. The second, reviewed here, concerns the potential risk of inadvertent transfer of animal viruses present in the xenotransplant that are able to infect the human recipient. The threat from viruses is a particularly contentious topic because it poses a risk not only to those individuals who receive xenotransplants, but also to healthy individuals who come into contact, either directly or indirectly, with the xenotransplant recipient. In this review, we describe some of the virus types, in addition to the much discussed porcine endogenous retroviruses that might cross the species barrier, and assess the risk of such viruses causing disease in human hosts.

Accurate estimation of transduction efficiency necessitates a multiplex real-time PCR.

Transduction efficiency can be easily monitored during pre-clinical trials by inclusion of marker genes. However, the use of such marker genes should be avoided in the final clinical gene therapy application since their products are often immunogenic, making it difficult to monitor transduction, especially if the vector is applied in vivo. In these cases PCR-based methods like the real-time PCR might provide a powerful tool to estimate biodistribution. To investigate the accuracy of this method, we have developed and tested a real-time PCR assay for the quantification of the enhanced green fluorescent protein (EGFP) gene and compared the results with transduction efficiencies estimated by FACS analysis. Although our real-time PCR assay itself was characterized by a high precision over a wide dynamic range of quantification, significant differences in the transduction efficiency compared with FACS data were initially observed. Accurate determination could only be achieved using an optimized multiplex real-time PCR assay, which allows the simultaneous calculation of cell number and EGFP copy number in the same tube. In view of future needs for methods allowing precise and accurate analysis of biodistribution in gene therapy trials, our data highlight the necessity critically to check both parameters in the implemented assay.

Superantigen expression is driven by both mouse mammary tumor virus long terminal repeat-associated promoters in transgenic mice.

In addition to the usual retroviral promoter, the mouse mammary tumor virus (MMTV) long terminal repeat carries a second promoter located in the U3 region. Here we show that both of these promoters are independently able to give rise to superantigen activity in transgenic mice. The ability of multiple MMTV promoters to drive superantigen expression underscores its importance in the virus life cycle.

Green fluorescent protein retroviral vector : generation of high-titer producer cells and virus supernatant.

Genes-encoding marker proteins, which are easily assayable, are useful to monitor cell lineage, gene expression, or promoter activities. In gene-transfer technology such marker genes allow a direct and simple detection of successfully transduced cells. The detection of marker gene products such as ß-galactosidase (ß-gal), chloramphenicol acetyltransferase (CAT), alkaline phosphatase, or luciferase involves either cell fixation, which kills the cells or antibody-mediated detection, which is time consuming. Drug-resistance genes such as neomycin, puromycin, hygromycin, or zeocin allow a positive selection of transduced cells, but require days to weeks of growth in selective media. Moreover, these genes can change the growth characteristics of the transduced cells through terminal differentiation or can interfere with the expression of the gene of interest (1). Therefore, a marker gene system that provides timely, accurate, and nontoxic detection of successfully transduced living cells would be of great advantage. One interesting candidate gene that fulfills these requirements is the gene-encoding green fluorescent protein (GFP). It was originally isolated from the jellyfish Aquorea victoria. The GFP cDNA consists of 730 bp, which encode a 238 amino acid protein with a molecular weight of 27 kD (2). Wild-type GFP emits a vibrant green fluorescence upon exposure to blue light (450-490 nm). The signal is detectable by fluorescence microscopy and fluorescence-activated cell sorting (FACS) (3). Because the fluorescence of wild-type GFP after excitation is not strong enough for many applications, different variants of GFP have been developed. In one such variant, a point mutation was introduced at amino acid 65 (GFP-S65T) leading to a "red-shifted" excitation maximum with an approximately five-fold stronger fluorescent intensity (4). In a further variant, the "red-shifted" GFP was "humanized" by the introduction of numerous silent mutations that alter the codons to those more commonly used in human genes resulting in the improved translation of the gene (5-7). An additional point mutation at amino acid 64 in which phenylalanine was altered to leucine (F64L) further enhances gene expression (8). GFP has been expressed without cytotoxic effects in different organisms and is of special interest as a marker for monitoring cell lines and gene expression (3). The application of GFP in gene-transfer protocols allows the simple detection of transduced cells and offers the possibility for immediate enrichment of viable transduced cells by FACS (3,9,10). This is of great interest in gene transfer into poorly transducable cells, e.g., hematopoietic stem and progenitor cells.

Protection of MLV vector particles from human complement.

Murine cell-derived MLV vector particles usually are highly sensitive to human complement-mediated lysis. Expression of the human complement inhibitor CD59 on murine packaging cells resulted in partial protection of these cells from lysis caused by human complement proteins. Furthermore, CD59 was incorporated into MLV vector particles released by these packaging cells, leading to an improved resistance of the virions against human complement-mediated inactivation.

Development of cellulose sulfate-based polyelectrolyte complex microcapsules for medical applications.

Microencapsulation, as a tool for immunoisolation for allogenic or xenogenic implants, is a rapidly growing field. However most of the approaches are based on alginate/polylysine capsules, despite this system's obvious disadvantages such as its pyrogenicity. Here we report a different encapsulation system based on sodium cellulose sulfate and polydiallyldimethyl ammonium chloride for the encapsulation of mammalian cells. We have characterized this system regarding capsule formation, strength and size of the capsules as well as viability of the cells after encapsulation. In addition, we demonstrate the efficacy of these capsules as a "microfactory" in vitro and in vivo. Using encapsulated hybridoma cells we were able to demonstrate long-term release of antibodies up to four months in vivo. In another application we could show the therapeutic relevance of encapsulated genetically modified cells as an in vivo activation center for cytostatic drugs during tumor therapy.