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author:("Ni, chaozheng")
1.  Directed Evolution Generates a Novel Oncolytic Virus for the Treatment of Colon Cancer 
PLoS ONE  2008;3(6):e2409.
Background
Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term “Directed Evolution” for creating highly potent oncolytic viruses.
Methodology/Principal Findings
Taking the “Directed Evolution” approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2–3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent.
Conclusions/Significance
Using the “Directed Evolution” methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies.
doi:10.1371/journal.pone.0002409
PMCID: PMC2423470  PMID: 18560559
2.  Evaluation of adenovirus vectors containing serotype 35 fibers for vaccination. 
In contrast to commonly used serotype 5 based adenovirus (Ad) vectors, Ads containing fibers derived from B-group serotype 35 (Ad5/35) efficiently transduce human DCs ex vivo and appear to target antigen-presenting cells after intravenous injection into baboons. Based on this, Ad5/35 vectors could be valuable tools for immunotherapy and vaccination. On the other hand, a number of studies indicate that signaling through the B-group Ad receptor, CD46, can cause tolerance or immuno-suppression. Since mice do not express CD46 in a human-like pattern, we studied the in vivo properties of Ad5/35 in transgenic mice that express CD46 in a pattern and at a level similar to humans. Hypersensitivity assays and analyses of frequencies of regulatory T-cells and T-cell responses did not indicate that Ad5/35 injection exerts detrimental effects on the host's immune system. An Ad5/35 vector expressing a model antigen was able to trigger a strong T-cell response against the test antigen after intramuscular injection. Overall, compared to Ad5 vectors, Ad5/35 vectors had a better safety profile, reflected by lower serum levels of pro-inflammatory cytokines.
doi:10.1016/j.ymthe.2005.12.008
PMCID: PMC1424671  PMID: 16461009
3.  Effect of adenovirus mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on anti-tumor immune responses 
Cancer research  2006;66(2):960-969.
Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen presenting cells. In this study we tested whether adenovirus (Ad)-mediated overexpression of secreted or membrane-associated forms of gp96 in tumor cells would stimulate an anti-tumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing subcutaneous tumors derived from syngeneic TC-1 cells, a cell line that expresses HPV16 E6 and E7 proteins. We found that secreted gp96 can induce protective and therapeutic anti-tumor immune responses. Our data also indicate that the anti tumor effect sgp96 expression appears to be limited by induction of suppressive regulatory T cells (Tregs). TC-1 tumor transplantation increased the number of splenic and tumor infiltrating Tregs. Importantly, treatment of mice with low-dose cyclophosphamide decreased the number Tregs and enhanced the immunostimulatory effect of sgp96 expression. We also tested whether an oncolytic vector (Ad.IR-E1A/TRAIL), that is able to induce tumor cell apoptosis and, potentially, release cryptic tumor epitopes in immunogenic form, can stimulate anti-tumor immune responses. While tumor cells infected ex vivo with Ad.IR-E1A/TRAIL had no anti-tumor effect when used as a vaccine alone, the additional treatment with low-dose cyclophosphamide resulted in elimination of pre-established tumors. This study gives a rationale for testing approaches that suppress Tregs in combination with oncolytic or immunostimulatory vectors.
doi:10.1158/0008-5472.CAN-05-2388
PMCID: PMC1360184  PMID: 16424031
adenovirus; oncolytic; heat shock protein; gp96; regulatory T-cells; cyclophosphamide
4.  Evaluation of Biodistribution and Safety of Adenovirus Vectors Containing Group B Fibers after Intravenous Injection into Baboons 
Human gene therapy  2005;16(6):664-677.
Vectors containing group B adenovirus (Ad) fibers are able to efficiently transduce gene therapy targets that are refractory to infection with standard Ad serotype 5 (Ad5) vectors, including malignant tumor cells, hematopoietic stem cells, and dendritic cells. Preliminary studies in mice indicate that, after intravenous injection, B-group fiber-containing Ads do not efficiently transduce most organs and cause less acute toxicity than Ad5 vectors. However, biodistribution and safety studies in mice are of limited value because the mouse analog of the B-group Ad receptor, CD46, is expressed only in the testis, whereas in humans, CD46 is expressed on all nucleated cells. Unlike mice, baboons have CD46 expression patterns and levels that closely mimic those in humans. We conducted a biodistribution and toxicity study of group B Ad fiber-containing vectors in baboons. Animals received phosphate-buffered saline, Ad5-bGal (a first-generation Ad5 vector), or B-group fiber-containing Ads (Ad5/35-bGal and Ad5/11-bGal) at a dose of 2 × 1012 VP/kg, and vector biodistribution and safety was analyzed over 3 days. The amount of Ad5/35-bGal and Ad5/11-bGal vector genomes was in most tissues one to three orders of magnitude below that of Ad5. Significant Ad5/35- and Ad5/11-mediated transgene (β-galactosidase) expression was seen only in the marginal zone of splenic follicles. Compared with the animal that received Ad5-bGal, all animals injected with B-group fiber-containing Ad vectors had lower elevations in serum proinflammatory cytokine levels. Gross and histopathology were normal in animals that received B-group Ad fiber-containing Ads, in contrast to the Ad5-infused animal, which showed widespread endothelial damage and inflammation. In a further study, a chimeric Ad5/35 vector carrying proapoptotic TRAIL and Ad E1A genes under tumor-specific regulation was well tolerated in a 30-day toxicity study. No major clinical, serologic, or pathologic abnormalities were noticed in this animal.
OVERVIEW SUMMARY
B-group Ad fiber-containing vectors are promising tools for gene therapy, for example, for the treatment of metastatic cancer or cardiovascular diseases, or for vaccination/immunotherapy. However, only a few studies of vectors containing B-group Ad fibers in mice have been conducted so far, and little is known about the mechanisms and effects of B-group Ad vector delivery in vivo. Before these vectors can be considered for clinical application, this knowledge gap must be filled. We performed biodistribution and safety studies after intravenous injection of chimeric Ad5 vectors containing Ad35 and Ad11 fibers into baboons. Our study suggests that Ad vectors possessing B-group Ad fibers have a better safety profile after intravenous injection than do conventional Ad5-based vectors.
doi:10.1089/hum.2005.16.664
PMCID: PMC1351080  PMID: 15960598
5.  Adenovirus-Platelet Interaction in Blood Causes Virus Sequestration to the Reticuloendothelial System of the Liver▿  
Journal of Virology  2007;81(9):4866-4871.
Intravenous (i.v.) delivery of recombinant adenovirus serotype 5 (Ad5) vectors for gene therapy is hindered by safety and efficacy problems. We have discovered a new pathway involved in unspecific Ad5 sequestration and degradation. After i.v. administration, Ad5 rapidly binds to circulating platelets, which causes their activation/aggregation and subsequent entrapment in liver sinusoids. Virus-platelet aggregates are taken up by Kupffer cells and degraded. Ad sequestration in organs can be reduced by platelet depletion prior to vector injection. Identification of this new sequestration mechanism and construction of vectors that avoid it could improve levels of target cell transduction at lower vector doses.
doi:10.1128/JVI.02819-06
PMCID: PMC1900148  PMID: 17301138
6.  Adenovirus Binding to Blood Factors Results in Liver Cell Infection and Hepatotoxicity 
Journal of Virology  2005;79(12):7478-7491.
Adenoviruses (Ad) are efficient vehicles for gene delivery in vitro and in vivo. Therefore, they are a promising tool in gene therapy, particularly in the treatment of cancer and cardiovascular diseases. However, preclinical and clinical studies undertaken during the last decade have revealed a series of problems that limit both the safety and efficacy of Ad vectors, specifically after intravenous application. Major obstacles to clinical use include innate toxicity and Ad sequestration by nontarget tissues. The factors and mechanisms underlying these processes are poorly understood. The majority of intravenously injected Ad particles are sequestered by the liver, which in turn causes an inflammatory response characterized by acute transaminitis and vascular damage. Here, we describe a novel pathway that is used by Ad for infection of hepatocytes and Kupffer cells upon intravenous virus application in mice. We found that blood factors play a major role in targeting Ad vectors to hepatic cells. We demonstrated that coagulation factor IX and complement component C4-binding protein can bind the Ad fiber knob domain and provide a bridge for virus uptake through cell surface heparan sulfate proteoglycans and low-density lipoprotein receptor-related protein. An Ad vector, Ad5mut, which contained mutations in the fiber knob domain ablating blood factor binding, demonstrated significantly reduced infection of liver cells and liver toxicity in vivo. This study contributes to a better understanding of adenovirus-host interactions for intravenously applied vectors. It also provides a rationale for novel strategies to target adenovirus vector to specific tissues and to reduce virus-associated toxicity after systemic application.
doi:10.1128/JVI.79.12.7478-7491.2005
PMCID: PMC1143681  PMID: 15919903
7.  Development and Assessment of Human Adenovirus Type 11 as a Gene Transfer Vector 
Journal of Virology  2005;79(8):5090-5104.
Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in Escherichia coli. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34+ hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism.
doi:10.1128/JVI.79.8.5090-5104.2005
PMCID: PMC1069572  PMID: 15795294
8.  Analysis of Adenovirus Sequestration in the Liver, Transduction of Hepatic Cells, and Innate Toxicity after Injection of Fiber-Modified Vectors 
Journal of Virology  2004;78(10):5368-5381.
After intravenous administration, adenovirus (Ad) vectors are predominantly sequestered by the liver. Delineating the mechanisms for Ad accumulation in the liver is crucial for a better understanding of Ad clearance and Ad-associated innate toxicity. To help address these issues, in this study, we used Ad vectors with different fiber shaft lengths and either coxsackievirus-Ad receptor (CAR)-interacting Ad serotype 9 (Ad9) or non-CAR-interacting Ad35 fiber knob domains. We analyzed the kinetics of Ad vector accumulation in the liver, uptake into hepatocytes and Kupffer cells, and induction of cytokine expression and release in response to systemic vector application. Immediately after intravenous injection, all Ad vectors accumulated equally efficiently in the liver; however, only genomes of long-shafted Ads were maintained in the liver tissue over time. We found that Kupffer cell uptake of long-shafted Ads was mediated by the fiber knob domain and was CAR independent. The short-shafted Ads were unable to efficiently interact with hepatocellular receptors and were not taken up by Kupffer cells. Moreover, our studies indicated that Kupffer cells were not the major reservoir for the observed accumulation of Ads (used in this study) in the liver within the first 30 min after virus infusion. The lower level of liver cell transduction by short-shafted Ads correlated with a significantly reduced inflammatory anti-Ad response as well as liver damage induced by the systemic administration of these vectors. This study contributes to a better understanding of the biology of systemically applied Ad and will help in designing safer vectors that can efficiently transduce target tissues.
doi:10.1128/JVI.78.10.5368-5381.2004
PMCID: PMC400378  PMID: 15113916
9.  A New Type of Adenovirus Vector That Utilizes Homologous Recombination To Achieve Tumor-Specific Replication 
Journal of Virology  2002;76(21):10994-11002.
We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.
doi:10.1128/JVI.76.21.10994-11002.2002
PMCID: PMC136641  PMID: 12368342

Results 1-9 (9)