Prostate cancer gene therapy-what have we learned and where are we going?

With recent advances in genetic engineering, tumor biology, and immunology, gene therapy has been recognized as a promising new treatment option for various cancers, including prostate cancer. Several clinical trials of prostate cancer gene therapy, using therapeutic genes which include suicide genes, immunomodulatory genes, tumor suppressor genes, and anti-oncogenes, are under way and preliminary reports have emerged. Although gene therapy for prostate cancer is still at an early stage and requires additional technological breakthroughs, new insights obtained from recent clinical trials indicate a promising potential for prostate cancer gene therapy. In this report, general concepts, current progress, and future prospects in prostate cancer gene therapy are summarized.     

Therapeutic genes for cancer gene therapy

Cancer still represents a disease of high incidence and is therefore one major target for gene therapy approaches. Gene therapy for cancer implies that ideally selective tumor cell killing or inhibition of tumor cell growth can be achieved using nucleic acids (DNA and RNA) as the therapeutic agent. Therefore, the majority of cancer gene therapy strategies introduce foreign genes into tumor cells which aim at the immunological recognition and destruction, the direct killing of the target cells or the interference with tumor growth. To achieve this goal for gene therapy of cancer, a broad variety of therapeutic genes are currently under investigation in preclinical and in clinical studies. These genes are of very different origin and of different mechanisms of action, such as human cytokine genes, genes coding for immunstimulatory molecules/antigens, genes encoding bacterial or viral prodrug-activating enzymes (suicide genes), tumor suppressor genes, or multidrug resistance genes.     

A microarray gene analysis of peripheral whole blood in normal adult male rats after long-term GH gene therapy

The main aims of this study were to determine the effects of GH gene abuse/misuse in normal animals and to discover genes that could be used as candidate biomarkers for the detection of GH gene therapy abuse/misuse in humans. We determined the global gene expression profile of peripheral whole blood from normal adult male rats after long-term GH gene therapy using CapitalBio 27 K Rat Genome Oligo Arrays. Sixty one genes were found to be differentially expressed in GH gene-treated rats 24 weeks after receiving GH gene therapy, at a two-fold higher or lower level compared to the empty vector group (p < 0.05). These genes were mainly associated with angiogenesis, oncogenesis, apoptosis, immune networks, signaling pathways, general metabolism, type I diabetes mellitus, carbon fixation, cell adhesion molecules, and cytokine-cytokine receptor interaction. The results imply that exogenous GH gene expression in normal subjects is likely to induce cellular changes in the metabolism, signal pathways and immunity. A real-time qRT-PCR analysis of a selection of the genes confirmed the microarray data. Eight differently expressed genes were selected as candidate biomarkers from among these 61 genes. These 8 showed five-fold higher or lower expression levels after the GH gene transduction (p < 0.05). They were then validated in real-time PCR experiments using 15 single-treated blood samples and 10 control blood samples. In summary, we detected the gene expression profiles of rat peripheral whole blood after long-term GH gene therapy and screened eight genes as candidate biomarkers based on the microarray data. This will contribute to an increased mechanistic understanding of the effects of chronic GH gene therapy abuse/misuse in normal subjects.     

A new strategy for cancer therapy based on a predictive indicator.

Tumor-associated genes have been analyzed at the molecular level in recent years, and using the analyses of these genes as a predictive indicator for cancer therapy has attracted attention. Among such genes, the actions of a tumor suppressor gene p53 are focused on the cancer therapy, and it is suggested that p53 genotypes can be used as a predictive indicator for radiotherapy, thermotherapy and chemotherapy. Transfection of wtp53 to p53-null cells increased radiation- or thermo-sensitivity and stimulated apoptosis induced by these therapies. Although therapy-induced apoptosis is suppressed in mp53 cells, apoptosis can be stimulated by glycerol treatment as a chemical chaperone. Therefore, a new strategy of combining p53-targeted gene therapy or chemical chaperone therapies is expected to improve the outcome and efficiency of cancer therapy in the future.     

Gene therapy in rheumatoid arthritis: Strategies to select therapeutic genes

Significant advances have been achieved in recent years to ameliorate rheumatoid arthritis (RA) in animal models using gene therapy approaches rather than biological treatments. Although biological agents serve as antirheumatic drugs with suppressing proinflammatory cytokine activities, they are usually accompanied by systemic immune suppression resulting from continuous or high systemic dose injections of biological agents. Therefore, gene transfer approaches have opened an interesting perspective to deliver one or multiple genes in a target-specific or inducible manner for the sustained intra-articular expression of therapeutic products. Accordingly, many studies have focused on gene transferring methods in animal models by using one of the available approaches. In this study, the important strategies used to select effective genes for RA gene therapy have been outlined. Given the work done in this field, the future looks bright for gene therapy as a new method in the clinical treatment of autoimmune diseases such as RA, and by ongoing efforts in this field, we hope to achieve feasible, safe, and effective treatment methods.     

Prospects for gene therapy in human prostate cancer

Prostate cancer is the most common neoplasm in men and a significant cause of mortality in affected patients. Despite significant advances, current methods of treatment are effective only in the absence of metastatic disease. Gene therapy offers a renewed hope of using the differential characteristics of normal and malignant tissue in constructing treatment strategies. Several clinical trials in prostate cancer gene therapy are currently under way, using immunomodulatory genes, anti-oncogenes, tumor suppressor genes and suicide genes. A continued understanding of the etiological mechanisms involved in the establishment and progression of prostate cancer, along with advances in gene therapy technology, should make gene therapy for prostate cancer therapeutically valuable in the future.     

Gene therapy in cancer

Gene therapy, recently frequently investigated, is an alternative treatment method that introduces therapeutic genes into a cancer cell or tissue to cause cell death or slow down the growth of the cancer. This treatment has various strategies such as therapeutic gene activation or silencing of unwanted or defective genes; therefore a wide variety of genes and viral or nonviral vectors are being used in studies. Gene therapy strategies in cancer can be classified as inhibition of oncogene activation, activation of tumor suppressor gene, immunotherapy, suicide gene therapy and antiangiogenic gene therapy. In this review, we explain gene therapy, gene therapy strategies in cancer, approved gene medicines for cancer treatment and future of gene therapy in cancer. Today gene therapy has not yet reached the level of replacing conventional therapies. However, with a better understanding of the mechanism of cancer to determine the right treatment and target, in the future gene therapy, used as monotherapy or in combination with another existing treatment options, is likely to be used as a new medical procedure that will make cancer a controllable disease.     

Cancer immunotherapy using cells modified with cytokine genes

The ability of various cytokines to hamper tumor growth or to induce anti-tumor immune response has resulted in their study as antitumor agents in gene therapy approaches. In this review we will concentrate on the costimulation of antitumor immune responses using modification of various cell types by cytokine genes. Several strategies have emerged such as (i). modification of tumor cells with cytokine genes ex vivo (whole tumor cell vaccines), (ii). ex vivo modification of other cell types for cytokine gene delivery, (iii). delivery of cytokine genes into tumor microenvironment in vivo, (iv). modification of dendritic cells with cytokine genes ex vivo. Originally single cytokine genes were used. Subsequently, multiple cytokine genes were applied simultaneously, or in combination with other factors such as chemokines, membrane bound co-stimulatory molecules, or tumor associated antigens. In this review we discuss these strategies and their use in cancer treatment as well as the promises and limitations of cytokine based cancer gene therapy. Clinical trials, including our own experience, employing the above strategies are discussed.     

Gene delivery to adult neural stem cells

Neural stem cells may present an ideal route for gene therapy as well as offer new possibilities for the replacement of neurons lost to injury or disease. However, it has proved difficult to express ectopic genes in stem cells. We report methods to introduce genes into adult neural stem cells using viral and nonviral vectors in vitro and in vivo. Adenoviral and VSV-G-pseudotyped retroviral vectors are more efficient than plasmid transfection or VSV-G lentiviral transduction in vitro. We further show that adult neural stem cells can be directed to a neuronal fate by ectopic expression of neurogenin 2 in vitro. Plasmids can be delivered in vivo when complexed with linear polyethyleneimine, and gene expression can be targeted specifically to neural stem or progenitor cells by the use of specific promoters. These techniques may be utilized both to study the function of various genes in the differentiation of neural stem cells to specific cell fates and, ultimately, for gene therapy or to generate specific differentiated progeny for cell transplantation.     

Bio and nanotechnological strategies for tumor-targeted gene therapy

Gene therapy is a new medical approach for the treatment of tumors. For safe and efficient gene therapy, therapeutic genes need to be delivered efficiently into the target tumor cells. Development of gene delivery systems to specifically recognize and target tumor cells and to distinguish them from normal cells, especially in the same tissue or organ, is one of the most important issues regarding the present gene delivery methodologies. The enhanced permeability and retention (EPR) effect using the characteristics of angiogenic tumor blood vessels, as well as gene delivery systems recognizing hyperactivated receptors or intracellular signals, is broadly applied to tumor-targeted gene therapy. In addition, bacterial vectors can be a useful means for targeting hypoxic or anoxic regions of a tumor.     

A novel approach for gene therapy: engraftment of fibroblasts containing the artificial chromosome expression system at the site of inflammation

BACKGROUND: Rheumatoid arthritis is characterized by inflammation of the synovial tissue. High systemic doses are necessary to achieve therapeutic levels of anti-rheumatic drugs in the joints. Gene transfer might provide a more efficient delivery system for genes encoding therapeutic proteins. METHODS: The artificial chromosome expression system (ACE System) is a new non-integrating, non-viral gene expression system which functions like a natural chromosome. This technology offers advantages over current expression systems because it allows stable and predictable expression of proteins encoded by single or multiple genes over long periods of time. We are developing ex vivo gene therapy using murine artificial chromosomes containing a reporter gene (LacZ and red fluorescent protein (RFP)) for local delivery of genes in rats with adjuvant arthritis (AA). RESULTS: The delivery of the intact ACE System into rat fibroblast-like synoviocytes (FLS) and rat skin fibroblasts (RSF) was detected within 24 to 48 h post-transfection. After growing cells under selection, clones expressing LacZ and RFP were identified. Furthermore, we investigated the feasibility of local delivery of a reporter gene to the joints of rats with AA by ex vivo gene therapy. This resulted in engraftment of the injected cells in the synovial tissue microarchitecture and expression of the reporter gene. CONCLUSIONS: This work demonstrates the potential feasibility of treating arthritis and other inflammatory diseases using fibroblasts containing the ACE System as a non-viral vector for gene therapy.     

Google goes cancer: improving outcome prediction for cancer patients by network-based ranking of marker genes

Predicting the clinical outcome of cancer patients based on the expression of marker genes in their tumors has received increasing interest in the past decade. Accurate predictors of outcome and response to therapy could be used to personalize and thereby improve therapy. However, state of the art methods used so far often found marker genes with limited prediction accuracy, limited reproducibility, and unclear biological relevance. To address this problem, we developed a novel computational approach to identify genes prognostic for outcome that couples gene expression measurements from primary tumor samples with a network of known relationships between the genes. Our approach ranks genes according to their prognostic relevance using both expression and network information in a manner similar to Google's PageRank. We applied this method to gene expression profiles which we obtained from 30 patients with pancreatic cancer, and identified seven candidate marker genes prognostic for outcome. Compared to genes found with state of the art methods, such as Pearson correlation of gene expression with survival time, we improve the prediction accuracy by up to 7%. Accuracies were assessed using support vector machine classifiers and Monte Carlo cross-validation. We then validated the prognostic value of our seven candidate markers using immunohistochemistry on an independent set of 412 pancreatic cancer samples. Notably, signatures derived from our candidate markers were independently predictive of outcome and superior to established clinical prognostic factors such as grade, tumor size, and nodal status. As the amount of genomic data of individual tumors grows rapidly, our algorithm meets the need for powerful computational approaches that are key to exploit these data for personalized cancer therapies in clinical practice.     

Gene therapy using anticancer drug-resistance genes

Myelosuppression is a major dose-limiting factor in cancer chemotherapy. Introduction of drug-resistance genes into bone marrow cells of cancer patients has been proposed to overcome this limitation. In theory, any gene whose expression protects cells against the toxic effects of chemotherapy should be useful in vivo for this purpose. Among such genes, human multidrug-resistance gene (MDR1) has been studied most extensively for this purpose, and clinical trials of drug-resistance gene therapy have been started in the US for cancer patients who undergo high-dose chemotherapy with autologous hematopoietic stem cell transplantation. In Japan, our clinical protocol of MDR1 gene therapy "A clinical study of drug-resistance gene therapy to improve the efficacy and safety of chemotherapy against breast cancer" has been submitted to the government. To improve the efficacy and safety of this drug-resistance gene therapy, we have constructed a series of MDR1-bicistronic retrovirus vectors using a retrovirus backbone of Harvey murine sarcoma virus and internal ribosome entry site (IRES) from picornavirus to co-express a second gene with the MDR1 gene. MDR1-MGMT bicistronic vectors can be used to protect bone marrow cells of cancer patients from combination chemotherapy with MDR1-related anticancer agents and nitrosoureas. In addition, MDR1-bicistronic retrovirus vectors can be designed to use the MDR1 gene as an in vivo selectable marker to enrich the transduced cells which express therapeutic genes, if disease is curable by the expression of a single-peptide gene in any types of bone marrow cells or peripheral blood cells.     

杆状病毒作为基因治疗载体的研究进展

杆状病毒是昆虫专一性的病原病毒.近来的研究表明杆状病毒能进入哺乳动物细胞, 但病毒自身不能在哺乳动物细胞中复制, 感染也不引起细胞病变.另外,已经证明杆状病毒能在体外或体内高效地转导许多类型哺乳动物细胞,并且能得到固定表达细胞系,显示了杆状病毒作为基因治疗载体有着良好的应用前景.综述了该领域的最新研究进展并探讨了其发展趋势.     

Non‐viral nucleic acid delivery to the central nervous system and brain tumors

Gene therapy is a rapidly emerging remedial route for many serious incurable diseases, such as central nervous system (CNS) diseases. Currently, nucleic acid medicines, including DNAs encoding therapeutic or destructive proteins, small interfering RNAs or microRNAs, have been successfully delivered to the CNS with gene delivery vectors using various routes of administration and have subsequently exhibited remarkable therapeutic efficiency. Among these vectors, non‐viral vectors are favorable for delivering genes into the CNS as a result of their many special characteristics, such as low toxicity and pre‐existing immunogenicity, high gene loading efficiency and easy surface modification. In this review, we highlight the main types of therapeutic genes that have been applied in the therapy of CNS diseases and then outline non‐viral gene delivery vectors.     

Protection of CHO cells by transfer of survivin driven by ovarian-specific promoter OSP-2

Chemotherapy is the major therapy for cancer in clinic. However, chemotherapeutic agents can harm the other tissues/organs besides cancer. Thus, there are great interests in protecting the innocents by the transfer of protective genes. There are two problems to be solved, one is the selection of protective genes and the other is the orientation of the exotic genes. Recent researches demonstrated that the principal mechanism of chemotherapeutics was through apoptosis. Hereby, introduction of anti-apoptosis genes might interrupt the processes of apoptosis to avoid side effect from chemotherapeutics. On the other hand, tissue-specific promoters, which control gene expression in a tissue-specific manner, might be an alternative tool to guarantee the location of target genes. In this research, we applied gene therapy to chemoprotection using anti-apoptosis gene survivin and ovarian-specific promoter OSP-2. The results showed that OSP-2 could specifically drive the expression of survivin in ovarian cells and survivin could protect cells via inhibiting apoptosis. This might put a light on the future of chemoprotective gene therapy.     

Gene therapy and metabolic engineering

Gene therapy involves the introduction of normal, healthy genes into cells to correct the underlying cause of a wide variety of inherited and acquired diseases. Future progress in developing effective clinical protocols involving gene therapy for the treatment of cellular dysfunction associated with disease may incorporate metabolic engineering. Metabolic engineering can be applied to gene therapy for the successful identification of disease genes; elucidation of disease pathways; development of safe and efficient gene-delivery systems; and regulation and control of gene expression. Cystic fibrosis, cancer, and diabetes are reviewed as examples of diseases where gene therapy approaches are being studied.     

Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-alphaR/NFkappaB pathway

BACKGROUND: Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease affecting children in the United States and Europe. Extensive work is being performed to develop both gene and drug therapies. The principal mutation causing CF is in the CFTR gene ([Delta F508]CFTR). This mutation causes the mutant protein to traffic poorly to the plasma membrane, and degrades CFTR chloride channel activity. CPX, a candidate drug for CF, binds to mutant CFTR and corrects the trafficking deficit. CPX also activates mutant CFTR chloride channel activity. CF airways are phenotypically inundated by inflammatory signals, primarily contributed by sustained secretion of the proinflammatory cytokine interleukin 8 (IL-8) from mutant CFTR airway epithelial cells. IL-8 production is controlled by genes from the TNF-alphaR/NFkappaB pathway, and it is possible that the CF phenotype is due to dysfunction of genes from this pathway. In addition, because drug therapy with CPX and gene therapy with CFTR have the same common endpoint of raising the levels of CFTR, we have hypothesized that either approach should have a common genomic endpoint. MATERIALS AND METHODS: To test this hypothesis, we studied IL-8 secretion and global gene expression in IB-3 CF lung epithelial cells. The cells were treated by either gene therapy with wild-type CFTR, or by pharmacotherapy with the CFTR-surrogate drug CPX. CF cells, treated with either CFTR or CPX, were also exposed to Pseudomonas aeruginosa, a common chronic pathogen in CF patients. cDNA microarrays were used to assess global gene expression under the different conditions. A novel bioinformatic algorithm (GENESAVER) was developed to identify genes whose expression paralleled secretion of IL-8. RESULTS: We report here that IB3 CF cells secrete massive levels of IL-8. However, both gene therapy with CFTR and drug therapy with CPX substantially suppress IL-8 secretion. Nonetheless, both gene and drug therapy allow the CF cells to respond with physiologic secretion of IL-8 when the cells are exposed to P. aeruginosa. Thus, neither CFTR nor CPX acts as a nonspecific suppressor of IL-8 secretion from CF cells. Consistently, pharmacogenomic analysis indicates that CF cells treated with CPX greatly resemble CF cells treated with CFTR by gene therapy. Additionally, the same result obtains in the presence of P. aeruginosa. Classical hierarchical cluster analysis, based on similarity of global gene expression, also supports this conclusion. The GENESAVER algorithm, using the IL-8 secretion level as a physiologic variable, identifies a subset of genes from the TNF-alphaR/NFkappaB pathway that is expressed in phase with IL-8 secretion from CF epithelial cells. Certain other genes, previously known to be positively associated with CF, also fall into this category. Identified genes known to code for known inhibitors are expressed inversely, out of phase with IL-8 secretion. CONCLUSIONS: Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells. The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway. The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF. From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences. This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery. Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases.     

Long term physiologic modification using rAAV in utero gene-therapy

BACKGROUND: Transfer of genes in utero via the amniotic fluid was shown previously with recombinant adeno-associated viruses (rAAV) to be highly efficient. Expression for over one year was demonstrated using reporter genes. In addition, it was shown previously that transgenes delivered by this method release protein into the general circulation. Given these results experiments were designed to test the hypothesis that in utero rAAV gene therapy could result in long term physiologic modification. METHODS: A rAAV recombinant expressing ciliary neurotrophic factor (cntf) and green fluorescent (gfp) in a polycistronic messenger was used to treat rat fetuses in utero. CNTF causes weight loss and decreased water consumption as a measurable physiologic effect. GFP was used as a marker of gene expression. RESULTS: In utero gene transfer with rAAV carrying human cntf and gfp resulted in long-term gene expression in rat. CNTF-specific physiologic effects of a decrease in weight and water intake were obtained. Expression of the GFP was documented in the treated animals at one year of age. CONCLUSION: Given this data, in utero gene therapy with rAAV into multipotential stem cells resulted in long term systemic physiologic modification of the treated animals by the transgene product. In utero rAAV gene therapy potentially could be used for gene replacement therapy in metabolic disorders.     

Osteogenic differentiation effects on rat bone marrow-derived mesenchymal stromal cells by lentivirus-mediated co-transfection of human BMP2 gene and VEGF165 gene

We proposed a novel combined gene therapy of human vascular endothelial growth factor 165 gene (hVEGF165) and human bone morphogenetic protein 2 gene (hBMP2) for bone regeneration by lentivirus-mediated co-transfection of both genes into rat bone marrow-derived mesenchymal stromal cells (MSCs). Both genes were successfully co-expressed in MSCs confirmed by real-time PCR and ELISA. And the alkaline phosphatase activity of MSCs was significantly augmented by the co-transfection with both genes than any single gene transfection (P < 0.01). These results demonstrated the feasibility of the combined gene therapy by using MSCs lentivirally co-transfected with hVEGF165 and hBMP2 for bone regeneration.