Advances in adenovirus-mediated p53 cancer gene therapy

Introduction: The tumor suppressor p53 gene regulates diverse cellular processes, such as cell-cycle arrest, senescence, apoptosis and autophagy, and it is frequently inactivated by genetic alterations in ～ 50% of all types of human cancers. To restore wild-type p53 function in p53-inactivated tumors, adenovirus-mediated p53 gene therapy has been developed as a promising antitumor strategy in preclinical experiments and clinical studies.

Areas covered: This review focuses on the clinical relevance of replication-deficient adenovirus vectors that carry the wild-type p53 gene (Ad-p53; Advexin, Gendicine and SCH-58500) in clinical studies of patients with various cancers and the future perspectives regarding conditionally replicating adenovirus vectors expressing the wild-type p53 gene (CRAd-p53; AdDelta24-p53, SG600-p53, OBP-702) in preclinical experiments. Moreover, the recent advances in our understanding of the molecular basis for the p53-mediated tumor suppression network induced by Ad-p53 and CRAd-p53 vectors and the combination therapies for promoting the therapeutic potential of adenovirus-mediated p53 gene therapy are discussed.

Expert opinion: Exploration of the molecular mechanism underlying the p53-mediated tumor suppression network and the effective strategy for enhancing the p53-mediated cell death signaling pathway would provide novel insights into the improvement of clinical outcome in p53-based cancer gene therapy.     

Adenovirus p53 gene therapy

To date, dysfunctional tumour suppressor genes are the most common genetic lesions identified in human cancers. Functional copies of tumour suppressor genes can be introduced into cancer cells by gene transfer using adenoviral vectors. This approach has been extensively studied in the clinic with intratumoural injection of a replication-defective adenovirus that expresses p53 (Ad-p53). Overexpression of p53 in cancer cells induces growth arrest and apoptosis. Ad-p53 injections have an excellent safety profile, and have mediated tumour regression and growth arrest as monotherapy, or have overcome resistance or increased the effectiveness of radiation therapy and chemotherapy. Expression of the p53 transgene has occurred at high levels and is associated with the activation of other genes in the p53 pathway. These studies indicate proof-of-principle for tumour suppressor gene therapy and represent a new paradigm in targeted therapy.     

Failure of wild-type p53 gene therapy in human cancer cells expressing a mutant p53 protein.

The introduction of exogenous wild-type p53 into human cancer cells bearing p53 mutation does not necessarily result in inhibition of tumor growth. We have demonstrated this in MDA-MB468 breast cancer cells which are hemizygous for p53 mutation and also in KM12SM colorectal carcinoma cells which are heterozygous for p53 mutation. The wtp53 transfectants decreased three- to four-fold the number of colonies compared with controls. Most wtp53-expressing cells died by apoptosis at early passages, but some cells were able to form colonies and their proliferation rate was similar to control transfectants. This reversion was observed in three of the six MDA-MB-468 clones selected. When MDA-wtp53 transfectants were implanted orthotopically in nude mice only one clone showed prolonged tumor latency. No differences were found in either tumor proliferation or apoptosis in tumors. Integration and expression of exogenous wtp53 was assessed in early and late passages in vitro, and in tumors growing in vivo. Consistently, we found mutations in the exogenous wtp53 gene of MDA-MB468 transfectants. Excision of the exogenous gene was an alternative to abrogate the wtp53 function that was extremely efficient in KM12 cells, although they maintained resistance to geneticin. These results were corroborated by the functional assay in yeast. In conclusion, wtp53 is inactivated in these cancer cells by different mechanisms. The presence of mutated p53 may confer genome instability and mutator ability, which allows cells to escape the effects of the exogenous wtp53 and contributes to the failure of wtp53 gene therapy.     

Helper-dependent adenoviral vectors for gene therapy

Helper-dependent adenoviral vectors possess a number of characteristics that make them attractive gene therapy vectors. These vectors are completely devoid of viral coding sequences and are able to mediate high-efficiency transduction in vivo to direct sustain high-level transgene expression with negligible chronic toxicity. This review focuses on advances in helper-dependent adenoviral vector technology, selected examples of in vivo studies of particular interest, and the issue of vector-mediated acute toxicity.     

A model for optimizing adenoviral delivery in human cancer gene therapy trials

Optimization of adenoviral delivery to the target volume is required for adenovirus-mediated cancer gene therapy to reach its maximal potential. The purpose of these studies was to develop a model of gene expression to improve adenovirus-mediated cancer gene therapy in the clinic. We measured the distribution of gene expression after a single deposit of a replication-competent adenovirus carrying the human sodium iodide symporter (hNIS) reporter gene was delivered to naive canine prostate and to human tumor xenografts. We generated hypothetical treatment plans for two prospective prostate cancer patients, using standard brachytherapy algorithms. In both models, the gene expression distribution from a single adenoviral deposit could be accurately described by a Gaussian function. In the naive canine prostate, a 0.1-ml deposit of 3 x 10(11) viral particles (VP) resulted in a gene expression volume of 1.14 +/- 0.70 cm(3), indicating that a minimum of 40 adenoviral deposits would be required to cover a 40-cm(3) prostate with therapeutic gene expression. On a viral particle basis, the gene expression volume obtained in human tumor xenografts (7 x 10(-12) cm(3)/VP) was twice that (3.5 x 10(-12) cm(3)/VP) measured in the naive canine prostate. Hypothetical treatment plans for two prostates indicated that 26 and 57 0.1-ml adenoviral deposits would be required to cover, respectively, 24- and 49-cm(3) prostates with gene expression. Although our studies focused on prostate, we believe the methodology to model gene expression presented here has much broader application to optimize treatment plans in other solid tumor sites; this assertion should be confirmed experimentally.     

Head and neck cancer: gene therapy approaches. Part 1: adenoviral vectors

Treatment options for recurrent or refractory head and neck cancer are limited. The goal of gene therapy is to introduce new genetic material into cancer cells without affecting toxicity to surrounding malignant cells. The most common vehicles for delivery of genes are adenoviruses. Adenoviruses gain access to malignant and normal cell cytoplasm via viral ligand binding to a unique cell surface receptor (the coxsackie adenovirus receptor [CAR]). However, this receptor is not cancer specific. Genetic modification of adenoviral DNA can create cancer specific targeting. Adenoviruses can be modified to express cancer specific ligands thereby focusing binding to malignant tissue. Furthermore, adenoviral delivered genes can be put under cancer specific promoter control to further limit gene expression in malignant tissue. Increased antitumour activity from such modifications has been demonstrated preclinically and several clinical trials have been completed demonstrating safety and clinical activity of non-replicating and conditional replicating adenoviral vector thereby opening the door for gene delivery and cancer specific targeting.     

结直肠癌p53基因点突变和蛋白表达的研究

目的了解中国人结直肠癌ｐ５３基因的突变谱，探讨聚合酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术用于研究结直肠癌中ｐ５３基因突变的可行性。方法应用ＰＣＲ－ＳＳＣＰ银染技术检测４１例结直肠癌ｐ５３基因突变，以ＡＢＣ免疫组织化学染色检测结直肠癌Ｐ５３蛋白的表达。结果３４％（１４／４１）的病例显示有ｐ５３基因突变，其中外显子５，６，７和８各有４，１，５和４例突变。２０例有Ｐ５３蛋白异常表达，阳性率为４９％。结论导致Ｐ５３蛋白异常堆积的ｐ５３基因突变是结直肠癌的一种常见的分子结构改变，可能在结直肠癌的发生和发展中起着重要的作用。ＰＣＲ－ＳＳＣＰ银染技术是一简便、快速、有效的检测基因点突变的方法     

Epithelial defensins impair adenoviral infection: implication for adenovirus-mediated gene therapy

Epithelial cells have been to participate actively in host defense by producing small cationic peptides called defensins. To investigate the biological activity of epithelial defensins in more detail, we expressed two defensins, hBD-1 and HD-5, in eukaryotic cell lines. Defensins were localized in the cytoplasm and in cell culture medium and exhibited strong microbicidal activity toward Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Moreover, our data indicate that the presence of defensins protected the cells from adenoviral infection. The presence of HD-5 or hBD-1 reduced the infectivity of Av1CF2 three- to fivefold. These results imply that defensins must be considered a serious obstacle whenever adenovirus is used to deliver genes to epithelial cells.     

Non-viral gene delivery for p53

Abnormality in the tumor suppressor gene p53 is one of the most common occurrences associated with human neoplasia. Consequently, restoration of wild-type p53 function is seen as a particularly promising approach for cancer gene therapy. In recent years, considerable research effort has centered upon developing and improving non-viral delivery systems as alternatives to viral vectors for gene delivery. These methods include the use of lipoplexes and polyplexes, and even delivery of naked DNA. Optimally effective cancer gene therapy requires treatment of metastatic as well as local disease, and to achieve this end, systemic delivery systems for therapeutic genes will be required. This review will discuss some of the recent advances in ways to improve targeting, transfection efficiency and stability for systemic, non-viral p53 gene therapy.     

Adenoviral p53 gene therapy in squamous cell cancer of the head and neck region

Treatment options for recurrent head and neck squamous cell carcinoma are limited. Morbidity and mortality are largely related to local recurrence. Experimental investigation of adenoviral p53 gene therapy is being explored as a new therapeutic modality for patients with recurrent squamous cell carcinoma in the head and neck region. The function of the p53 gene is to maintain the genetic integrity of the cell and induce apoptosis when DNA damage is irreparable. Phase I trial results indicate that multiple injections of up to 2.5 x 10(12) viral particles per injection administered during a 3-week cycle are well tolerated and demonstrate evidence of local regional control. Dose-related activity correlating with transient survival advantage is demonstrated in a meta-analysis of phase II trials. This review will summarize phase I and II trial results. Conclusions drawn from these studies justify the currently ongoing phase III investigation.     

Histone deacetylase inhibitor FK228 enhances adenovirus-mediated p53 family gene therapy in cancer models

Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy.     

Antiangiogenic gene therapy for cancer via systemic administration of adenoviral vectors expressing secretable endostatin

A growing number of antiangiogenesis strategies have been investigated for the treatment of cancer and other angiogenesis-dependent diseases. One of the most promising strategies is to systemically administer one or more antiangiogenic proteins frequently enough to achieve a sufficient long-term steady state level of the protein(s) to achieve the maximum beneficial effect. However, the utility of this strategy is limited because of many technical difficulties, including obtaining both the quantity and quality of the protein(s) necessary for optimal therapeutic benefit. To overcome these difficulties, we hypothesized that a single administration of a replication-defective adenoviral vector expressing a secretable antiangiogenic protein could achieve an optimal long-term systemic concentration. We constructed a recombinant adenoviral vector, Av3mEndo, which encodes a secretable form of murine endostatin. We demonstrated secretion of endostatin from several cell lines transduced with Av3mEndo. Partially purified endostatin secreted from Av3mEndo-transduced mammalian cells was shown to potently inhibit endothelial cell migration in vitro. A single intravenous administration of Av3mEndo in mice was shown to result in (1) prolonged and elevated levels of circulating endostatin, (2) partial inhibition of VEGF-induced angiogenesis in a VEGF implant angiogenesis model, and (3) prolonged survival and in 25% of mice the complete prevention of tumor growth in a prophylactic human colon/liver metastasis xenograft murine model. These results support our contention that adenoviral vector-mediated expression of an antiangiogenic protein(s) represents an attractive therapeutic approach to cancer and other angiogenesis-dependent diseases.