The retinoblastoma tumor suppressor modifies the therapeutic response of breast cancer

The retinoblastoma tumor suppressor (RB) protein is functionally inactivated in the majority of human cancers and is aberrant in one-third of all breast cancers. RB regulates G(1)/S-phase cell-cycle progression and is a critical mediator of antiproliferative signaling. Here the specific impact of RB deficiency on E2F-regulated gene expression, tumorigenic proliferation, and the response to 2 distinct lines of therapy was investigated in breast cancer cells. RB knockdown resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the RB deficiency compromised the short-term cell-cycle inhibition following cisplatin, ionizing radiation, and antiestrogen therapy. In the context of DNA-damaging agents, this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of antiestrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of aberrations in RB function were recapitulated by ectopic E2F expression, indicating that control of downstream target genes was an important determinant of the observed responses. Specific analyses of an RB gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB pathway is a critical determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for directing therapy in the treatment of breast cancer.     

Persistent hepatic expression of human apo A-I after transfer with a helper-virus independent adenoviral vector

Gene transfer with 'gutted' vectors is associated with persistent transgene expression and absence of hepatotoxicity, but the requirement of helper viruses hampers efficient production and leads to contamination of viral batches with these helper-viruses. In the present study, gene transfer with a helper-virus independent E(1)/E(3)/E(4)-deleted adenoviral vector induced persistent expression of human apo A-I (200 +/- 16 mg/dl at day 35, 190 +/- 15 mg/dl at 4 months, 170 +/- 16 mg/dl at 6 months) and stable transgene DNA levels (3.5 +/- 0.60 at day 35, 3.3 +/- 0.39 at 4 months, 3.1 +/- 0.47 mg/dl at 6 months) in C57BL/6 mice in the absence of significant toxicity. The vector contained the 1.5 kb human alpha(1)-antitrypsin promoter in front of the genomic human apo A-I sequence and four copies of the human apo E enhancer (hAAT.gA-I.4xapoE) and was deleted in E(1), E(3) and E(4). Reintroduction of E(4) ORF 3 and E(4) ORF 4 in the viral backbone caused a more than four-fold decline of transgene DNA between day 35 and 4 months after transfer both in wild-type and in C57BL/6 SCID and C57BL/6 Rag-1(-/-) mice, indicating that the effect of E(4) ORF 3 and E(4) ORF 4 is independent of a cellular immune response against viral epitopes. Co-injection of an E(1)-deleted vector containing no expression cassette and the E(1)/E(3)/E(4)-deleted vector containing the hAAT.gA-I.4xapoE expression cassette indicated that E(4) gene products destabilize transgene DNA in trans. Gene transfer with an E(1)/E(3)/E(4)-deleted vector containing only E(4) ORF 3 and the hAAT.gA-I.4xapoE expression cassette was associated with transgene DNA decline, but not with hepatotoxicity, indicating that transgene DNA persistence and hepatotoxicity are dissociated processes. After transfer with E(1)/E(3)/E(4)-deleted vectors containing expression cassettes with a different promoter or a different position of the apo E enhancers, transgene DNA levels were less stable than after transfer with the vector containing hAAT.gA-I.4xapoE, indicating that the expression cassette is an important determinant of episomal stability. In conclusion, gene transfer with an E(1)/E(3)/E(4)-deleted vector containing the hAAT.gA-I.4xapoE expression cassette induces persistent expression of human apo A-I in the absence of hepatotoxicity. Transgene DNA turnover is independent of an adaptive cellular immune response against viral epitopes and of hepatotoxicity. E(1)/E(3)/E(4)-deleted vectors containing transgenes under control of the hAAT promoter in combination with four copies of the human apo E enhancer may be suitable for hepatocyte-specific overexpression of transgenes after gene transfer. doi:10.1038/sj.gt.3301824     

Reduced toxicity, attenuated immunogenicity and efficient mediation of human p53 gene expression in vivo by an adenovirus vector with deleted E1-E3 and inactivated E4 by GAL4-TATA promoter replacement.

A recombinant adenovirus with deleted E1 and E3, and E4-inactivated by replacing the E4 promoter with a synthetic promoter composed of a minimal TATA box and five consensus yeast GAL4-binding site elements was developed and used to express the human tumor suppresser gene p53. The toxicity and immunogenicity of this vector and vector-mediated p53 gene expression in vivo were studied in immunocompetent C3H and C57BL/6 mice. Expression of the late viral gene product, hexon protein, was observed in C3H and C57BL/6 mice injected with E4 wild-type adenovirus constructs Adv-cmv-beta-Gal (BG), Adv-cmv-hp53 (WT), and empty E1- vector Adv-E4 (EW) 3 to 28 days after injection, but was undetectable in mice treated with E4 modified empty E1- vector Adv-GAL4 (EG) or Adv-cmv-hp53-GAL4 (G4). Expression of the p53 gene was observed in both WT- and G4-injected C3H and C57BL/6 mouse livers from days 3 to 28. Ten weeks after injection, p53 gene expression was still detected in G4-treated C57BL/6 mice at similar levels, but was not detectable in WT-treated mice. Vector-induced liver toxicity was evaluated by analyzing serum transaminases (SGOT and SGPT) activities. In all cases, SGOT and SGPT activities were markedly decreased in EG-treated C3H and C57BL/6 mice compared with those in EW-treated mice on days 3, 7 and 14 after injection. In C57BL/6 mice, the total anti-adenoviral CTL activities were two- to three-fold higher in animals treated with EW vector than in those treated with EG vector. These results suggest that inactivation of the E4 promoter efficiently diminished the viral replication and the late viral gene expression, reduced host immune response and consequently reduced toxicity and prolonged the duration of transgene expression in vivo.     

Suicidal gene therapy in the effective control of primary human hepatocellular carcinoma as monitored by noninvasive bioimaging

Hepatocellular carcinoma (HCC) is usually refractory to the available treatments. For cancer gene therapy purposes, real-time imaging of therapeutic gene expression is of great importance because there are multiple factors that modulate the therapeutic gene expression in a complex tumor microenvironment. As a consequence, multiple doses of therapeutic viral vectors may be required for improved efficacy. In the present study, the luciferase reporter gene and the yeast cytosine deaminase (yCD) genes were bicistronically expressed using the foot-and-mouth disease virus 2A peptide under the regulation of the cytomegalovirus (CMV) promoter. The effectiveness of the yCD/5-FC (5-fluorocytosine) killing efficacy mediated by the herpes simplex virus type 1 (HSV-1) amplicon viral vector was shown using HCC and non-HCC cell lines in vitro. In addition, in vivo experiment also showed tumor regression of a primary HCC 26-1004 tumor xenograft in tumor expressing high levels of the yCD gene (as determined by noninvasive imaging) after intratumoral injection of 1.5 × 10(6)?TU HGCX-L2C HSV-1 amplicon viral vector and 5-FC administration. The HSV-1 amplicon viral vector coupled with the yCD/5-FC prodrug activated suicide gene could potentially be of use in clinical gene therapy for HCC.     

Comparison of gene expression efficiency and innate immune response induced by Ad vector and lipoplex.

Vectors for gene expression are the essential tools for both gene therapy and basic research. There are two groups of gene therapy vectors, viral and non-viral vectors. At present, toxicity triggered by vectors is one of the major concerns for clinical trials. In general, non-viral vectors, such as plasmid DNA-cationic liposome complex (lipoplex), are thought to be safer than viral vectors, such as adenovirus (Ad) vector, although lipoplex is less efficient in term of gene expression than the Ad vector. However, there has been no study directly comparing the gene expression efficiency and safety of viral and non-viral vectors. Here, we present evidence that the Ad vector shows much more efficient gene expression and is safer than lipoplex, at least with respect to the innate immune response. After being systemically administered to mice, the Ad vector showed a transduction efficiency that was 2 to 5 log orders higher than that of lipoplex, depending on the organ. On the other hand, surprisingly, the administration of lipoplex produced a greater amount of inflammatory cytokines such as interleukin-6, interleukin-12, and tumor necrosis factor-alpha than did the administration of the Ad vector, whereas a comparable level of hepatotoxicity was induced by these vectors. The production of inflammatory cytokines induced by the injection of lipoplex was reduced when the CpG motifs were removed completely from plasmid DNA. Thus, care should be taken to ensure the innate immune response induced by gene therapy vectors, especially lipoplex.     

Intratumoral gene therapy of malignant brain tumor in a rat model with angiostatin delivered by adeno-associated viral (AAV) vector

We have utilized a recombinant adeno-associated viral (AAV) vector carrying the angiostatin gene as an anti-angiogenesis strategy to treat the malignant brain tumor in a C6 glioma/Wistar rat model. Angiostatin, as a potent angiogenesis inhibitor, shows high promises as an anti-cancer drug through the inhibition of tumor neovessel formation. However, sustained in vivo protein delivery is required to achieve the therapeutic effects. The AAV vector has been proven to be able to deliver sustained and high-level gene expression in vivo, and therefore, is well suited to such a purpose. In this study, we implanted 5 x 10(5) C6 glioma cells into the rat brain 7 days before gene therapy. Intratumoral injection of a high-titer AAV-angiostatin vector has rendered efficacious tumor suppression and resulted in long-term survival in 40% of the treated rats, whereas the control AAV-GFP vector did not have any therapeutic benefits. In addition, we have investigated the combined gene therapy of an adenoviral vector carrying the suicidal thymidine kinase gene along with the AAV-angiostatin vector. The combined therapy offered the best tumor-suppressive effects and increased long-term survival to 55% in the treated rats. Our study has demonstrated the potential of using AAV as a safe and effective vector for anti-angiogenic gene therapy of brain tumors.     

Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter.

For effective gene therapy of chronic disease, persistent transgene expression at therapeutic levels is required. Clinical studies of airway gene transfer in patients with cystic fibrosis (CF) have resulted in short-lived transgene expression. We used intra-nasal dosing of naked plasmid DNA to the murine lung as a model for investigating the duration of airway gene transfer from a series of reporter expression plasmids. Transgene expression was transient when mediated by the viral promoters CMV, RSV and SV40, falling to less than 10% of peak expression after 2 weeks, although the presence of the adenoviral E4ORF3 gene in cis, resulted in extended duration of reporter activity from the CMV promoter. Transient expression from these promoters was not due to loss of the vector as determined by quantitative TaqMan PCR analysis. However, use of the promoters from the human polybiquitin C (UbC) and the elongation factor 1alpha (EF1alpha) genes resulted in persistent gene expression in the mouse lung. The UbC promoter directed high-level reporter activity which was maintained for up to 8 weeks and was still detectable 6 months after a single administration. Such persistent airway transgene expression from a nonviral vector without the concomitant expression of a potential antigen has not been reported previously. Thus, despite the persistence of vector DNA in vivo, attenuation of promoter function may lead to silencing of transgene expression and careful selection of promoter sequences is recommended for in vivo gene transfer.     

DNA replication of first-generation adenovirus vectors in tumor cells

A major role of the early gene 1A and 1B products (E1A and E1B) in adenovirus infection is to create a cellular environment appropriate for viral DNA replication. This is, in part, achieved by inactivation of tumor suppressor gene products such as pRb or p53. The functions of these same cellular proteins are also frequently lost in tumor cells. Therefore, we hypothesized that tumor cell lines with deregulated p53 and/or pRb pathways might support replication of E1A/E1B-deleted, first-generation adenovirus vectors (AdE1(-)). Here, we analyzed the impact of virus uptake, cell cycling, and the status of cell cycle regulators on AdE1(-) DNA synthesis. Cellular internalization of AdE1(-) vectors varied significantly among different tumor cell lines, whereas nuclear import of incoming viral DNA appeared to be less variable. Replication assays performed under equalized infection conditions demonstrated that all analyzed tumor cell lines supported AdE1(-) synthesis to varying degrees. There was no obvious correlation between the efficiency of viral DNA replication and the status of p53, pRb, and p16. However, the amount of virus attached and internalized changed with the cell cycle, affecting the intracellular concentration of viral DNA and thereby the replication efficacy. Furthermore, infection with AdE1 - vectors caused a partial G(2)/M arrest or delay in cell cycle progression, which became more pronounced in consecutive cell cycles. Correspondingly, vector DNA replication was found to be enhanced in cells artificially arrested in G(2)/M. Our findings suggest that cell cycling and thus passing through G(2)/M supports AdE1(-) DNA replication in the absence of E1A/E1B. This has potential implications for the use of first-generation adenovirus vectors in tumor gene therapy.     

Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector

Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occurrence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppressor gene p53 for treatment of various cancers. Multiple preparations of the replication-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biological assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no substantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.     

小鼠甲状腺转录因子-2转基因动物表达载体的构建及其在骨髓间充质干细胞中的表达

目的构建小鼠甲状腺转录因子-2（TTF2）转基因动物表达载体（pBROAD3-TTF2）,观察其在小鼠骨髓间充质干细胞（BMSC）中的表达。方法从C57BL／6J小鼠肝脏组织中提取基因组DNA,利用聚合酶链式反应方法扩增出nF2基因1113bD开放阅读框,通过DNA重组技术将丌R2基因片段插入克隆载体pMDl8．T中,经测序正确后,再重组于pBROAD3．mcs中,构建转基因动物表达载体pBROAD3-TTF2,用酶切电泳分析对其进行鉴定。运用脂质体转染试剂将其转染BMSC后,蛋白质印迹法检测717F2基因的表达。结果①DNA测序证实目的基因序列正确无突变,酶切电泳分析得到相应的目的片段,大小与理论计算值一致,成功构建转基因动物表达载体pBROAD3-TTF-2。②蛋白质印迹法显示转染的BMSC高表达TTF-2蛋白。结论成功构建了pBROAD3-TTF2转基因动物表达载体,显示其转染BMSC后丌F2基因的表达,为下一步建立刀F2转基因小鼠模型奠定了基础。     

Efficient melanoma cell killing and reduced melanoma growth in mice by a selective replicating adenovirus armed with tumor necrosis factor-related apoptosis-inducing ligand

High mortality and therapy resistance of melanoma demand the development of new strategies, and overcoming apoptosis deficiency appears as particularly promising. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown high potential for apoptosis induction in melanoma cells and may be applicable for gene therapy because of its selective impact on tumor cells. We have constructed a conditional replication-competent adenoviral vector with TRAIL controlled by a tetracycline-inducible promoter (AdV-TRAIL). A variant E1A protein and the lack of E1B aimed at the restriction of viral replication to tumor cells. In particular, the replication gene E1A is controlled by a tyrosinase promoter with high selectivity for melanoma cells. AdV-TRAIL mediated strong expression of E1A and doxycycline-dependent induction of TRAIL selectively in melanoma cells, which resulted in tumor cell lysis and induction of apoptosis. In contrast, non-melanoma cells and normal human melanocytes appeared to be protected. Comparison of the AdV-TRAIL approach with a comparable CD95L vector revealed similar efficacy in vitro. In mouse xenotransplantation models, AdV-TRAIL demonstrated its activity by significant melanoma growth reduction. Melanoma cell killing by AdV-TRAIL was further improved in vitro by combinations with chemotherapeutics. We demonstrate that melanoma cells may be efficiently targeted by TRAIL-based gene therapy, and resistance may be overcome by combined chemotherapy.     

Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology

The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, 'high-speed jet injector' hand-held system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the beta-galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-alpha) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3--5 microl plasmid DNA (1 microg DNA/microl in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with penetration depths of 5--10 mm. Analysis of tumor cryosections revealed moderate LacZ or GFP expression at 48 h and strong reporter gene expression 72 h and 96 h after jet injection. The simultaneous jet injection of the TNF-alpha and LacZ carrying vectors demonstrated efficient expression and secretion of both the cytokine, as well as LacZ expression within the tumor 24 h, 48 h, 72 h, 96 h and 120 h after jet injection. These studies demonstrate the applicability of jet injection for the efficient in vivo gene transfer into tumors for nonviral gene therapy of cancer using minimal amounts of naked DNA.     

Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates.

Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken beta-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-DeltaCFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005x10(6) copies/mug DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24x10(5) copies/microg) and mRNA expression. Immunoprecipitation and (32)P phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.