Vector Replication Research Articles

Conditionally replicative adenovirus vector carrying TRAIL gene for enhanced oncolysis of human hepatocellular carcinoma

Conditionally replicative adenovirus (CRAd) mediated tumor specific gene therapy based on transcriptional control is considered a new direction for the treatment of cancer. Our previous studies showed that an HS4 insulator increased the alpha-fetoprotein (AFP) promoter-driven expression in the context of an adenovirus (Ad) vector, while retaining the highly specific gene expression in hepatoma cells in vitro and in vivo. In this study, we constructed two HS4-AFP promoter based CRAd vectors (Ad.HS4.AFP.E1a/TRAIL and Ad.HS4.AFP.E1a) with and without the expression cassette of TNF-related apoptosis-inducing ligand (TRAIL). The TRAIL-expressing virus vector, Ad.HS4.AFP.E1a/TRAIL, exhibited more obvious oncolytic effect than Ad.HS4.AFP.E1a in both high-AFP-producing HCC cell lines (Hep3B and HUH7) and a low-AFP-producing HCC cell line (PLC/PRF/5) examined, indicating endogenous TRAIL over-expression increased CRAd potency. The enhanced hepatoma cell death was mainly mediated through apoptotic mechanism, as evidenced by the activation of caspase-3, binding of annexin V and inhibition by caspase inhibitor z-vad-fmk. In s.c. xenograft of low-AFP-producing PLC/PRF/5 hepatoma model, the administration of Ad.HS4.AFP.E1a/TRAIL resulted in a more potent oncolytic effect compared with the same dose of Ad.HS4.AFP.E1a 28 days after virus exposure. This study demonstrated that the TRAIL in the context of a CRAd vector was able to increase the oncolytic activity in low-AFP-producing HCC cells in vitro and in vivo. Considering that oncolytic viruses destroy tumor cells expressing low levels of the tumor marker is a clinical concern, TRAIL might be a useful tool to improve the efficacy of these vectors.

Mechanisms of sphingosine and sphingosine 1-phosphate generation in human platelets

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.

Gene Therapy for ovarian cancer via a novel fiber modified conditionally replicating Adenovirus

Conditionally replicating adenoviruses (CRADs) are a novel approach for treatment of cancer. The oncolytic potency of CRADs is determined by their capability to infect tumor cells. Unfortunately, expression of the primary receptor for Ad5 is highly variable on ovarian cancer cells. Genetic fiber pseudotyping is an approach to circumvent this problem by using the alternative Ad serotype 3 receptor for entry into, and killing of ovarian cancer cells. Furthermore, restriction of the CRAD replication to tumor cells minimizes nontumor tissue injury. In this study, we constructed a fiber-modified CRAD containing the secretory leukoprotease inhibitor (SLPI) promoter to control viral replication via the E1A gene (Ad5/3SLPI). SLPI has been shown to be over-expressed in various tumors including ovarian cancer but minimally active in normal tissues. Viral replication and oncolysis were assessed in various ovarian cancer cell lines. Replication was also analyzed with quantitative PCR on three-dimensional primary tumor cell sheroids purified from fresh patient samples. Moreover, in a therapeutic orthotopic mouse model of peritoneal carcinomatosis, dramatically enhanced survival was noted with Ad5/3SLPI. To assess the selectivity of Ad5/3SLPI we evaluated the ability of the viruses to replicate in non-target liver cells in vivo. Liver pathology and viral E1A RNA levels were evaluated 48 hours after i.v. injection of viruses into immunocompetent mice. Little to no hepatotoxicity was seen with the non replicative E1-deleted vector whereas pronounced effects were seen with the replication competent Ad5/3wt vector. In summary, Ad5/3SLPI is a promising candidate for treating metastatic ovarian cancer and showed robust virus replication, oncolysis, and in vivo therapeutic efficacy. Ad5/3SLPI showed comparatively low liver toxicity and therefore holds great potential for clinical testing.

Development of a Serum-free Suspension Process for the Production of a Conditionally Replicating Adenovirus using A549 Cells

Conditionally replicating adenoviruses (CRAVs) are a group of recombinant human adenoviruses genetically engineered to replicate in selected tissues, such as tumors. These viruses could potentially offer significant medicinal benefits, since the restrictive replication of these viral vectors leads to the lysis of target cells without harm to the surrounding tissues. The in vitro propagation and amplification of the CRAV vectors often requires special host cells with deregulated growth control pathways. In order to develop an efficient cell culture process for the scaleable production of a CRAV vector, A549 cells, a human lung carcinoma cell line normally cultured in adherent culture, were adapted to suspension culture. CRAV production was demonstrated with the suspension-adapted A549 cells and a baseline production process was developed in shake flasks. The ability to scale-up virus production was confirmed in stirred tank bioreactors. Molecular characterization of the suspension-adapted A549 cells indicates no significant changes in cellular mechanisms related to adenovirus infection.

Targeting Gene-Virotherapy for Cancer

Gene therapy and viral therapy for cancer have therapeutic effects, but there has been no significant breakthrough in these two forms of therapy. Therefore, a new strategy called "targeting gene-virotherapy", which combines the advantages of gene therapy and viral therapy, has been formulated. This new therapy has stronger antitumor effects than either gene therapy or viral therapy. A tumor-specific replicative adenovirus vector ZD55 (E1B55KD deleted Adv.) was constructed and various single therapeutic genes were inserted into ZD55 to form ZD55-gene. These are the targeting gene-virotherapy genes. But experiments showed that a single gene was not effective in eliminating the tumor mass, and therefore two genes were separately inserted into ZD55. This strategy is called "targeting dual gene-virotherapy" (with PCT patent). Better results were obtained with this strategy, and all the xenograft tumor masses were completely eliminated in all mice when two suitable genes producing a synergetic or compensative effect were chosen. Twenty-six papers on these strategies have been published by researchers in our laboratory. Furthermore, an adenoviral vector with two targeting promoters harboring two antitumor genes has been constructed for cancer therapy. Promising results have been obtained with this adenoviral vector and another patent has been applied for. This antitumor strategy can be used to kill tumor cells completely with minimum damage to normal cells.

A tumor-specific conditionally replicative adenovirus vector expressing TRAIL for gene therapy of hepatocellular carcinoma

We constructed a novel hepatocellular carcinoma-specific conditionally replicative adenovirus (CRAd). This adenovirus, designated Ad.HS4.AFP.E1A/TRAIL, expresses E1A to mediate viral replication and TRAIL to enhance HCC-killing efficacy under the control of a modified AFP promoter. An insulator HS-4 was placed in front of the AFP promoter to enhance the fidelity of the heterologous promoter. This virus was shown to have specific cytolytic activity in AFP-expressing HCC cells in vitro. Furthermore, the replication efficiency of Ad.HS4.AFP.E1A/TRAIL correlated well with AFP expression of the host cells, showing a 100-fold and 1 000 000-fold decrease in the low-and non-AFP-expressing HCC cells, respectively, compared to the high AFP-expressing HCC cells. An increase in mRNA of TRAIL and the elevated Caspase-3 activity were also observed in Ad.HS4.AFP.E1A/TRAIL-infected HCC cells. These results indicated that TRAIL expression from the viral vector activated the Caspase-3 enzymatic capacity and the HCC cells were sensitive to TRAIL. In vivo, Ad.HS4.AFP.E1A/TRAIL effectively prevented the growth of low AFP-expressing BEL-7404 xenografts. These results indicate that Ad.HS4.AFP.E1A/TRAIL could provide a new strategy of gene therapy for HCC.

Efficient inhibition of hepatitis B virus replication by hammerhead ribozymes delivered by hepatitis delta virus

Although it has been suggested that hepatitis delta virus (HDV) can be used as a vector to deliver biologically active RNAs into hepatocytes, modified HDV as a specific transporting and replicating vector in anti-viral research has not been investigated. In this study, we focused on the development of HDV as a replicative vector to deliver hammerhead ribozyme into hepatocytes and the study of the roles of delivered hammerhead ribozyme on the replication of hepatitis B virus (HBV). To investigate the effects of ribozyme delivered by HDV on HBV replication, we designed two hammerhead ribozymes that specifically target the hepatitis B virus genome. These two ribozymes were then inserted into the genome of hepatitis delta virus. Results showed that transfection of cells with tandem modified HDV cDNA resulted in the production of monomer form of sense and anti-sense genomic RNA indicating the recombinant HDV-ribozyme could replicate effectively. Our data also indicated that ribozymes delivered by the modified HDV had higher level of inhibition activity against HBV replication than that of ribozyme alone. This system provides a new approach for the study of mechanisms of HBV replication as well as for the potential treatment of HBV infection.

Triple layered rotavirus VLP production: Kinetics of vector replication, mRNA stability and recombinant protein production

Rotavirus infection causes diarrhoeal disease in infants, killing more than half million children each year. Virus-like particles (VLP) seem to be excellent vaccine candidates, since they are cheaper to produce than attenuated viral vaccines and safer, as they do not contain genetic material. The present work focus on a triple layered particle composed by three rotavirus structural proteins: VP2, VP6 and VP7, produced in an insect cell/baculovirus expressing system. Two strategies were evaluated for 2/6/7 VLP production: co-infection with three monocistronic baculovirus vectors or single-infection with a tricistronic multi-gene baculovirus vector; these strategies were followed at different levels: baculovirus DNA replication kinetics, mRNA stability, protein production and VLP formation. This study highlights some of the reasons why the tricistronic baculovirus strategy is more efficient for production of triple layered rotavirus 2/6/7 VLP than monocistronic co-infection, in particular: (i) the tricistronic vector presents higher DNA replication rates than the monocistronic vectors, (ii) the mRNA stability is invariant for all mRNAs corresponding to VP2, VP6 and VP7 and (iii) the tricistronic baculovirus strategy produces an excess of VP7 over VP6 when compared to the VP7/VP6 stoichiometric ratio in the native rotavirus. Although the co-infection strategy leads to protein production akin to the rotavirus VP7/VP6 stoichiometric ratio, the tricistronic vector strategy yields higher amounts of rotavirus-like particles.

Assessment of a Combined, Adenovirus-Mediated Oncolytic and Immunostimulatory Tumor Therapy

In this study, we identified murine breast cancer cell lines that support DNA replication of E1-deleted adenovirus vectors and which can be killed by an oncolytic adenovirus expressing adenovirus E1A and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) in a replication-dependent manner (Ad.IR-E1A/TRAIL). We showed that systemic or intratumoral (i.t.) injection of adenovirus vectors into mice increases plasma levels of proinflammatory cytokines and chemokines, including TNF-alpha, INF-gamma, and MCP-1, which are potent inducers of dendritic cell maturation. Furthermore, we showed that in vivo expression of Flt3L from an adenovirus vector increases the number of CD11b+ and CD11c+ cells (populations that include dendritic cells) in the blood circulation. Based on these findings, we tested whether Ad.IR-E1A/TRAIL induced killing of tumor cells in combination with dendritic cell mobilization by Ad.Flt3L or, for comparison, Ad.GM-CSF would have an additive antitumor effect. As a model, we used immunocompetent C3H mice with syngeneic s.c. tumors derived from C3L5 cells. We found that vaccination of mice with C3L5 cells that underwent viral oncolysis in combination with Flt3L or granulocyte-macrophage colony-stimulating factor (GM-CSF) expression induces a systemic antitumor immune response. I.t. injection of the oncolytic and Flt3L expressing vectors into established tumors delayed tumor growth but did not cause efficient tumor elimination. This study shows the effectiveness of a combined oncolytic/immunostimulatory tumor therapy approach.

140. Primary Adenovirus Specific Cytotoxic T Lymphocyte Response Occurs after Viral Clearance and Liver Enzyme Elevation

Top of pageAbstract The relative contribution of the specific cytotoxic T lymphocyte (CTL) response and innate immune response to the clearance of adenovirus (Ad) and its transgene in the context of gene therapy has been a subject of debate. Transient transgene expression was believed due to the elimination of Ad transduced cells by CTLs. To determine the relative contribution of specific CTL killing of Ad-infected hepatocytes, we have utilized a wild-type Ad5 to study viral clearance from the liver and its associated liver enzyme elevation during a primary immune response. Human Ad5 replicates poorly in mice due to species restriction but induces a dominant CTL response to the E1B peptide (AA192 VNIRNCCYI) associated with mouse class I MHC antigen, H2-Db, in C57BL/6 (B6) mice. Thus, Ad5 was used to mimic a low replicative Ad vectors used in humans and an MHC-I tetramer that contains the E1Bp, H2-Db and b2-microglubulin (Db-E1B), was developed and used to detect the Ad-specific CTLs in Ad5-primed mice. After Ad5 infection, the Db-E1B+CD8+T cells were undetectable in the spleen, liver and lung until day 5, and peaked at day 8. At the peak of the CTL response, 8 days post infection, 10% of CD8+ cells in the spleen. Surprisingly, 45% in the liver and 55% in the lung of the CD8+ T cells were Db-E1B+, indicating that most CD8+ T cells in target tissue are specifically recruited to these sites. Furthermore, nearly 100% of Db-E1B+CD8+ cells were IFN|[gamma]| and granzyme-B positive with intracellular staining, indicating that the recruited CD8+CTL exhibit potent killing activity. The E1B-specific in vivo CTL response was detected by the transfer of CFSE-labeled, E1B192 peptide-pulsed target cells from H-2b mice into Ad5-primed syngeneic mice. The magnitude of the in vivo CTL response correlated with the number of Db-E1B+ CTLs in the spleen and liver (r2=0.96, p<0.01). Interestingly, viral DNA genome and E1B mRNA peaked at day 2 and reduced rapidly by day 6 and then maintained at a stable level without significant change 200 days after infection. Viral clearance correlated with the peak liver enzyme response that occurred on day 4, before CTLs became detectable. In contrast, there was no further change in the level of viral DNA genome and E1B mRNA in the liver or lung as a result of the peak CTL response at day 8. Administration of Ad5 to athymic SCID mice induced no CD8 T cells or in vivo specific CTL response against E1B192 peptide, yet viral clearance and liver enzyme response in SCID mice were comparable to those of B6 mice. Taken together, our results suggest that the innate response, but not the CTL response, plays a dominant role in elimination of Ad during a primary response. Stable long-term expression of lower levels of virus and transgene persist for 200 days despite the presence of potent CD8+ CTLs, indicating that they reside in liver cells that are resistant to CTL elimination. These findings will be useful in developing efficient and safe Ad gene therapy protocols.

553. A Potent Replicative Delta Adenoviral Vector for HPV Tumors

Several human epithelial neoplasms are associated with human papillomavirus (HPV) infection, and there are 'high-risk' strains of HPV involved in common malignancies such as cervical, anorectal, head and neck carcinomas. The global impact of these tumors on public health is serious, and for some tumor types the current therapeutic tools are only palliative. Oncolytic adenoviral vectors have emerged as promising antineoplastic tools, which also trigger confined antitumor effects. The main strategies to generate these vectors are based on the control of the vector replication by a tissue-specific promoter or by mutations in the E1A/E1B adenoviral sequences that are essential for viral replication. We constructed a series of oncolytic vectors driven by the upstream regulatory promoter region (URR) of HPV-16, which contained different mutations at the E1A region (dl1015 and/or |[Delta]|24). These vectors were tested for their ability to produce replicative particles and to induce cytopathic effects in HPV+ and HPV|[ndash]| cell lines. Viral DNA replication and E1A expression were also assessed by quantitative polymerase chain reaction. The vector Ad-URR/E1A|[Delta]|24 displayed a potent cytopathic effect that was associated with high selectivity for HPV+ cell lines.

530. Adenoviral and Hydrodynamic Gene Transfer of Replicating Episomes Result in Stable Transgene Expression after Partial Hepatectomy in Mice

Background: Integration of viral vectors can be associated with insertional oncogenesis. Episomal replicative vectors may constitute a safe alternative for integrating vectors. Most currently used episomal replicative systems are based on the Epstein-Barr virus origin of replication. Because the Epstein-Barr virus based system may be associated with oncogenesis, the objective of the current study was to develop an episomal replicative system functioning independent of a viral transactivator in hepatocytes. In contrast to the low degree of hepatocyte replication in adults, hepatocytes replicate in children and treatment of serious inborn errors of metabolism requires gene transfer at very young age.

Cooperative Effect of Gag Proteins p12 and Capsid during Early Events of Murine Leukemia Virus Replication

The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.

Telomerase-Dependent Gene Therapy

Adenovirus-mediated gene therapy approaches have evolved as promising means for cancer treatment during the last decade. Utilizing a broad spectrum of tumor-specific promoters, numerous oncotropic vectors have been created with exceptional properties regarding tumor-restricted specificity. The discovery of telomerase, its high prevalence in tumor tissues and the discovery of its transcriptional regulation via the hTERT promoter have extended the applicability of adenoviral gene therapy vectors to approximately 90% of all tumors. First generation adenoviral vectors expressing transgenes under the control of the hTERT promoter confirmed the therapeutic potential but were restricted to initially transduced cancer cells. Recently, telomerase-dependent conditionally replicative adenoviral vectors (CRADs) have been developed that combine the specificity of hTERT promoter based expression systems with the lytic efficacy of replicative viruses. To evaluate the potential for clinical applications, various efforts have been made to establish combinative strategies including systemic chemotherapy, radiotherapy and antiangiogenesis. This review highlights the rapid advances of telomerase-based gene therapy and gives insight into future prospects and future development of oncotropic vectors.

Hypoxia reduces adenoviral replication in cancer cells by downregulation of viral protein expression

Successful cancer therapy using replicating viral vectors relies on the spread of virus from infected to uninfected cells. To date, there has been limited clinical success in the use of replicating adenoviruses. In animal models, established xenograft tumors are rarely eliminated despite the persistence of high viral titers within the tumor. Hypoxia is a prevalent characteristic of solid tumors, whereas adenovirus naturally infects tissues exposed to ambient oxygen concentrations. Here, we report that hypoxia (1% oxygen) reduces adenoviral replication in H1299 and A549 lung cancer cells, BxPC-3 pancreatic cancer cells, LNCaP prostate cancer cells and HCT116 colon cancer cells. However, hypoxia does not reduce cell viability or restrict S-phase entry. Importantly, the production of E1a and fiber proteins under hypoxic conditions was substantially decreased at 24 and 48 h compared to room air controls. In contrast, Northern analysis showed similar levels of E1a mRNA in room air and hypoxic conditions. In conclusion, a level of hypoxia similar to that found within solid tumors reduces the replication of adenoviral vectors by reduction of viral protein expression without a reduction in mRNA levels. To further improve oncolytic therapy using a replicating adenovirus, it is important to understand the mechanism through which hypoxia and the virus interact to control expression of viral and cellular proteins, and consequently to develop means to overcome decreased viral production in hypoxic conditions.

Promoter-controlled infectivity-enhanced conditionally replicative adenoviral vectors for the treatment of gastric cancer

Gastric cancer is the fourth most common malignancy worldwide. Adenoviral vectors (Ads) have been applied for gene therapy of various cancers because of their high transduction efficiency. However, the infectivity of gastrointestinal cancer cells is poor due to the limited expression of the Coxsackie-adenovirus receptor (CAR). In addition, few tumor-specific promoters (TSPs) have been characterized for this type of cancer. To overcome these problems, we proposed TSP-driven conditionally replicating adenoviruses (CRAds) with fiber modification for virotherapy of gastric cancer. We assessed the expression profile of eight TSPs in gastric cancer cell lines and evaluated promising candidates in the context of CRAd cytocidal effect. Next, infectivity enhancement by fiber modifications was analyzed in the gastric cancer cell lines. Finally, we combined the TSP-driven CRAds of choice with the fiber modifications to augment the killing effect. Out of the eight TSPs, the midkine (MK) and cyclooxygenase-2 (Cox-2M and Cox-2L) promoters showed high transcriptional activity in gastric cancer cells. When these promoters were used in a CRAd context, Cox-2 CRAds elicited the strongest cytocidal effect. The greatest infectivity enhancement was observed with adenoviral vectors displaying 5/3 chimeric fibers. Likewise, Cox-2 CRAds with 5/3 chimeric fibers showed the strongest cytocidal effect in gastric cancer cell lines. Therefore, Cox-2 CRAds with 5/3 chimeric fiber modification showed good selectivity and infectivity in gastric cancer cells to yield enhanced oncolysis. Cox-2 CRAds with 5/3 chimeric fiber modification are promising for virotherapy of gastric cancer.

A Novel Ex vivo Model System for Evaluation of Conditionally Replicative Adenoviruses Therapeutic Efficacy and Toxicity

Current animal tumor models are inadequate for the evaluation of toxicity and efficacy of conditionally replicative adenoviruses. A novel model system is needed that will provide insight into the anticipated therapeutic index of conditionally replicative adenoviruses preclinically. We endeavored to show a novel model system, which involves ex vivo evaluation of conditionally replicative adenovirus toxicity and therapeutic efficacy in thin, precision-cut slices of human primary tumor and liver. The Krumdieck thin-slice tissue culture system was used to obtain and culture slices of tumor xenografts of ovarian cancer cell lines, human primary ovarian tumors, and human liver. We determined the viability of slices in culture over a period of 36 to 48 hours by ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt)]) (MTS) assay. In vitro Hey cells, slices of Hey xenografts, and human ovarian tumor or human liver slices were infected with 500vp/cell of either replication competent wild-type adenovirus (Ad5/3wt), conditionally replicative adenovirus (Ad5/3cox-2), or the replication deficient adenovirus (Ad5/3luc1). At 12-, 24-, and 36-hour intervals, the replication of adenoviruses in these slices was determined by quantitative reverse transcription-PCR of adenoviral E4 copy number. Primary tumor slices were able to maintain viability for up to 48 hours after infection with nonreplicative virus (Ad5luc1). Infection of Hey xenografts with Ad5/3cox-2 showed replication consistent with that seen in Hey cells infected in an in vitro setting. Primary tumor slices showed replication of both Ad5/3wt and Ad5/3cox over a 36-hour time period. Human liver slices showed replication of Ad5/3wt but a relative reduction in replication of Ad5/3cox-2 indicative of conditional replication "liver off" phenotype, thus predicting lower toxicity. The thin-slice model system represents a stringent method of ex vivo evaluation of novel replicative adenoviral vectors and allows assessment of human liver replication relative to human tumor replication. This is the first study to incorporate this system for evaluation of therapeutic efficacy and replicative specificity of conditionally replicative adenoviruses. Also, the study is the first to provide a valid means for preclinical assay of potential conditionally replicative adenovirus-based hepatotoxicities, thus providing a powerful tool to determine therapeutic index for clinical translation of conditionally replicative adenoviruses.

Viral and host determinants of RNA virus vector replication and expression

Positive-strand RNA viruses have proven to be valuable vectors for delivery and expression of antigens for direct vaccination of animals and vaccine production in plants. However, optimal use of these viruses as vectors for vaccine and other purposes is limited by incomplete understanding of their replication pathways and associated constraints on inserted foreign genes. Further insights into RNA virus vector design and optimization are emerging from recent advances on the function of viral RNA replication factors, the nature of the viral RNA replication complex as a membrane-bounded compartment sequestering replication components from competing processes and host defenses, and identification of surprisingly diverse host genes contributing to many virus replication steps.

Biology of adenovirus and its use as a vector for gene therapy.

ADENOVIRUSES WERE FIRST DISCOVERED half a century ago by Rowe and colleagues, who were trying to culture adenoid tissue in the laboratory (Rowe et al., 1953). Since that time, nonhuman adenoviruses have been isolated from a number of species including chimpanzees, pig, mouse, and dog, as well as other mammalian and avian species (Shenk, 1996). Although human adenoviruses cause significant levels of respiratory, ocular, and gastrointestinal disease, they have been the object of intense study over the years mainly as a model system for basic eukaryotic cellular processes such as transcription, RNA processing, DNA replication, translation, and oncogenesis. Early on, it was shown that the virus could recombine during growth in culture (Lewis et al., 1966a,b; Pierce et al., 1968; Lewis and Rowe, 1970), ultimately setting the stage for the use of the virus as a vector for gene delivery into cells, animals, and humans. Many features of adenovirus make it well suited for gene delivery, including the ability to grow recombinant viruses to high titers, a relatively high capacity for transgene insertion, and efficient transduction of both quiescent and actively dividing cells, usually without incorporation of viral DNA into the host cell genome. These characteristics, as well as the development of many methods for manipulating the viral genome, have made adenovirus a popular choice as a gene delivery vehicle. This is evidenced by the fact that adenovirus is currently being used in roughly one-quarter of all gene therapy clinical trials, making it second only to the use of retroviral vectors (Journal of Genetic Medicine Website, www. wiley.co.uk/genmed/clinical). In this review, we summarize our current understanding of the life cycle of the human adenoviruses and then discuss how the structure and function of viral genes impact the use of the virus as a gene delivery vehicle. Readers desiring a more comprehensive coverage of the biology of the virus are directed to the volumes edited by Doerfler and Bohm (2003, 2004). VIRUS STRUCTURE

ComX activity ofStreptococcus mutansgrowing in biofilms

In many streptococci, including Streptococcus mutans, genetic competence is regulated by a quorum sensing system mediated by a competence stimulating peptide (CSP) pheromone, encoded by the comC gene. In Streptococcus pneumoniae, a central component of this system is ComX, which acts as an alternative sigma factor to activate competence genes involved in DNA uptake and processing. The quorum sensing system responsible for genetic competence induction in S. mutans has been linked to biofilm formation and the acid tolerance response. To examine the response of comX to CSP in S. mutans, a transcriptional fusion of the comX promoter (pcomX) with lacZ was constructed to generate reporter vector pcomx::pALH122 (replicative vector) and transformed into S. mutans UA159 comC−, which is unable to produce endogenous CSP. CSP was added and pcomX::lacZ relative expression index (REI) examined, revealing a 2-fold increase in maximal β-gal activity 5 and 10 min after CSP addition. The effect of endogenous CSP on pcomX::lacZ expression was also examined by measuring REI in cells grown as a biofilm; peak pcomX activity was observed at 3 h. To determine the temporal pattern of transformation frequency, pMA2, a Spr shuttle vector, was transformed into biofilm-grown cells, with maximal transformation frequency observed at 3 h. Confocal microscopy was performed to examine pcomX activity using a similarly constructed green fluorescent protein reporter vector, pcomX::gfp, in a 4-h biofilm, revealing active pcomX activity in high cell density areas within the biofilm population. These results demonstrated a positive correlation between pcomX activity, natural transformation and competence development in biofilms.