In vivo interleukin-2 gene therapy of established tumors with herpes simplex amplicon vectors

In vivo cytokine gene transfer may greatly simplify autologous tumor vaccine production. Herpes simplex viral amplicon vectors (HSV) are efficient gene-transfer vehicles and may overcome many limitations of prior gene-transfer methods. The interleukin-2 (IL-2) and β-galactosidase genes (lac) were inserted into an HSV amplicon vector and tested in a subcutaneous squamous cell carcinoma of lung origin to determine the efficiency of in vivo gene transfer and the utility of such a direct gene-transfer approach in cancer therapy. Gene transfer and expression were assessed by histochemical staining and enzyme-linked immunosorbent assay (ELISA). Growth of injected tumors as well as non-injected tumors remote from the site of injection was assessed. Assessment of lymphocytic infiltrates into tumors was performed by immunohistochemistry. Survival was recorded. Direct in vivo injection of established tumors with a HSVil2 resulted in efficient gene transfer and production of IL-2 in the injected tumor but not at tumors remote from the sites of injection. There was a significant suppression of growth of the tumors injected with HSVil2 (P < 0.01) when compared with tumors injected with HSV without il2. Of note, growth of tumors remote from sites of HSVil2 injection was also retarded and treatment was associated with a significant (P < 0.05) improvement in survival. Direct intratumoral administration of HSV amplicon vectors can result in efficient transfer of cytokine genes and have antitumor efficacy. HSV vectors are therefore potentially useful agents in such in vivo gene-therapy strategies and simplify cytokine antitumor gene-therapy strategies. Received: 19 June 1998 / Accepted: 25 September 1998     

Novel Therapeutic Approaches for Various Cancer Types Using a Modified Sleeping Beauty-Based Gene Delivery System

Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the host''s chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the non-viral SB-based gene delivery system still has limited therapeutic efficacy due to the lack of long-lasting gene expression potential and tumor cell specific gene transfer ability. These limitations could be overcome by modifying the SB system through the introduction of the hTERT promoter and the SV40 enhancer. In this study, a modified SB delivery system, under control of the hTERT promoter in conjunction with the SV40 enhancer, was able to successfully transfer the suicide gene (HSV-TK) into multiple types of cancer cells. The modified SB transfected cancer cells exhibited a significantly increased cancer cell specific death rate. These data suggest that our modified SB-based gene delivery system can be used as a safe and efficient tool for cancer cell specific therapeutic gene transfer and stable long-term expression.     

Use of bacteria in anti-cancer therapies

While a number of valid molecular targets have been discovered for tumours over the past decade, finding an effective way of delivering therapeutic genes specifically to tumours has proved more problematic. A variety of viral and non-viral delivery vehicles have been developed and applied in anti-cancer gene therapies. However, these suffer from either inefficient and/or short-lived gene transfer to target cells, instability in the bloodstream and inadequate tumour targeting. Recently, various types of non-pathogenic obligate anaerobic and facultative anaerobic bacteria have been shown to infiltrate and selectively replicate within solid tumours when delivered systemically. This has prompted the development of cancer gene therapy protocols that use such bacteria as gene delivery vehicles. Here, we review the evidence for the success of these in pre-clinical models and clinical trials, as single modality treatments and in combination with conventional cancer therapies.     

Site-specific transfer of an intact beta-globin gene cluster through a new targeting vector

The ideal gene-therapy vector for treating genetic disorders should deliver intact therapeutic genes and their essential regulatory elements into the specific "safe genomic site" and realize long-term, self-regulatory expression. For beta-thalassemia gene therapy, viral vectors have been broadly used, but the accompanying insertional mutation and immunogenicity remain problematic. Hence, we aimed to develop new non-viral vectors that are efficient and safe in treating diseases. As previous studies have demonstrated that physiological expression of beta-globin genes requires both a 5' locus control region and 3' specific elements, we constructed a new human chromosome-derived targeting vector to transfer the intact beta-globin gene cluster into K562 cells. The whole beta-globin gene cluster was precisely integrated into the target site and expressed in a self-regulatory pattern. The results proved that the human chromosome-derived vector was specifically targeted to the human genome and this could provide a novel platform for further gene therapy research.     

Impact of lysine-affinity chromatography on supercoiled plasmid DNA purification

Gene therapy and DNA vaccination cover a variety of applications using viral and non-viral vectors as vehicles of choice for treatment of genetic or acquired diseases. Recently, most therapeutic applications have been performed with non-viral biological agents preparations highly enriched in supercoiled plasmid molecules and it has been concluded that this isoform is more efficient at gene transfection than open circular isoform. This work describes for the first time a new strategy that uses lysine-chromatography to efficiently eliminate Escherichia coli impurities as well as other ineffective plasmid isoforms present in a complex clarified lysate to purify and obtain pharmaceutical-grade supercoiled plasmid DNA. The quality control tests indicated that the levels of impurities in the final plasmid product were below the generally accepted specifications. Furthermore, the delivery of the purified product to eukaryotic cells, the cell uptake and transfection efficiency were also analyzed. The results showed that the transfection efficiency reached with the application of the supercoiled plasmid conformation, purified with lysine-agarose, was higher than the values achieved for other plasmid topologies. Therefore, this study presents a new enabling technology to obtain the completely purified non-viral vector, able to act with good efficiency as gene therapy delivery vehicle in several diseases like cancer.     

Components of gene therapy experimentation that contribute to relative risk

Gene therapy is the purposeful delivery of genetic material to somatic cells for the purpose of treating disease or biomedical investigation. Either viral or non-viral vector methods can be used. The risk of collateral exposure of laboratory animal care personnel to gene therapy vectors is dependent on a number of factors. These factors are intrinsic to the gene therapy vector (the vehicle for genetic conveyance), product encoded by the genetic construct delivered, method of delivery, and immune status of the recipient. The component risks of gene therapy experiments can be analyzed to surmise the overall relative risk of the experiment. Knowledge of the components that contribute potential hazardous risk to a study can assist animal care staff in identifying area(s) where prudent practices should be focused. Gene therapy experiments involving viral vectors are generally performed at either biosafety level 2 or 3. The objective of this review is to report on various components of gene therapy experiments, focusing on characteristics of viral and non-viral vectors, to assist the laboratory animal science community in determining prudent biosafety practices.     

Enhancement of gene delivery to cancer cells by a retargeted adenovirus

The inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches that improve gene transfer efficiency have been described, but suffer from a number of limitations. Herein, a fiber-modified adenovirus, carrying the small peptide ligand on the capsid, was tested for the delivery of a transgene to cancer cells. The fiber-modified adenovirus was able to mediate the entry and expression of a beta-galactosidase into cancer cells with increased efficiency compared to the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to transferrin receptor overexpressing cancer cells, and could be used for future cancer gene therapy.     

A novel approach using transcomplementing adenoviral vectors for gene therapy of adrenocortical cancer.

Current therapies for adrenocortical carcinomas do not improve the life expectancy of patients. In this study, we tested whether a gene-transfer therapy based upon a suicide gene/prodrug system would be effective in an animal model of the disease. We employed E4- and E1A/B-depleted, herpes simplex virus-thymidine kinase-expressing adenoviral mutants that transcomplement each other within tumor cells, hereby improving transgene delivery and efficacy by viral replication in situ. Transcomplementation of vectors increased the fraction of transduced of tumor cells. This increase was accompanied by greater tumor volume reduction compared to non-transcomplementing approaches. Survival time improved with non-replicating vectors plus GCV compared to controls. However, transcomplementation/replication of vectors led to a further significant increment in anti-tumor activity and survival time (p < 0.02). In treated animals, we observed a high number of apoptotic nuclei both adjacent to and distant from injection sites and sites of viral oncolysis. Ultrastructural analyses exhibited nuclear inclusion bodies characteristic of virus production in situ, and provided further evidence that this therapy induced apoptotic cell death within tumor cells. We conclude that the efficacy of suicide gene therapy is significantly amplified by viral replication and, in combination with GCV, significantly reduces tumor burden and increases survival time.     

肿瘤基因治疗的靶向策略

对肿瘤组织的靶向性可以提高基因治疗的效果 ,避免对正常组织的损伤 ,并且能降低作为载体的微生物对机体的危害。对于瘤内注射的给药方法 ,靶向性似乎显得不是特别重要 ,但是如果要系统给药 ,靶向性是很关键的一个问题。靶向基因治疗肿瘤可以通过靶向基因导入和靶向基因表达来实现。近年来 ,在靶向基因导入方面的研究有很多进展 ,例如 ,用双亲性的桥连分子协助腺病毒和逆转录病毒靶向转导 ;在各种病毒载体的衣壳蛋白中插入靶向性的小肽或较大的多肽靶向结构域 ;增殖病毒作为一种很有前途的抗肿瘤制剂可有效地靶向杀伤肿瘤细胞。受体介导的DNA或DNA 脂质体复合物的靶向系统和其他一些靶向性的有疗效的载体 ,如细菌 ,也处于研究中。其中的一些载体已经进入临床实验。为了实现基因的靶向可调控表达 ,组织或肿瘤特异性的启动子和人工合成的可调控表达系统被用来调控治疗基因的表达。反义核酸、核酶以及脱氧核酶 (DNAzyme)被用来靶向抑制与肿瘤发生密切相关基因的表达。     

Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture

In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors.     

Delivery systems for gene therapy

The structure of DNA was unraveled by Watson and Crick in 1953, and two decades later Arber, Nathans and Smith discovered DNA restriction enzymes, which led to the rapid growth in the field of recombinant DNA technology. From expressing cloned genes in bacteria to expressing foreign DNA in transgenic animals, DNA is now slated to be used as a therapeutic agent to replace defective genes in patients suffering from genetic disorders or to kill tumor cells in cancer patients. Gene therapy provides modern medicine with new perspectives that were unthinkable two decades ago. Progress in molecular biology and especially, molecular medicine is now changing the basics of clinical medicine. A variety of viral and non-viral possibilities are available for basic and clinical research. This review summarizes the delivery routes and methods for gene transfer used in gene therapy.     

Efficient stable gene transfer into human cells by the Sleeping Beauty transposon vectors

Transposable elements can be considered as natural, non-viral gene delivery vehicles capable of efficient genomic insertion. The plasmid-based transposon system of Sleeping Beauty (SB) combines the advantages of viruses and naked DNA molecules. In contrast to plasmid vectors, transposons integrate through a precise, recombinase-mediated mechanism into chromosomes, providing long-term expression of the gene of interest in cells. The advantages of transposons in comparison to viral systems include their simplicity and improved safety/toxicity profiles. In addition, the hyperactive SB100X is the first plasmid-based delivery system that overcomes the efficacy of non-viral delivery. The transposon delivery system consists of the transposase and the integration cassette, recognized by the transposase. The plasmid-based transposon delivery system can be combined with any non-viral delivery method. Here we provide two detailed protocols to apply SB-mediated, non-viral gene transfer in cultured cells. In our first example, we use a lipid-based delivery method in combination with the transposon-based integration system in an easy-to-transfect (HeLa) cell line. Second, we show how to achieve 40–50% stable expression of a transgene in clinically relevant, hard-to-transfect cells (hematopoetic stem cells, HSCs) by nucleofection. The given protocols are adaptable to any vertebrate cells in culture.     

基因治疗导入载体的研究进展

基因治疗在恶性肿瘤、癌症、遗传性疾病和心脑血管等疾病的治疗中开始应用,临床治疗效果明显。基因治疗中的关键技术是选用合适的载体将外源基因高效导入受体靶细胞,综述了基因治疗中病毒和非病毒载体的研究进展。     

Gene therapy in The Netherlands: highlights from the Low Countries

Gene therapy is an active research area in The Netherlands and Dutch scientists involved in fundamental and clinical gene therapy research significantly contribute to the progresses made in this field. This ranges from the establishment of the 293, 911 and PER.C6 cell lines, which are used worldwide for the production of replication-defective adenoviral vectors, to the development of targeted viral vectors and T lymphocytes as well as of non-viral vectors. Several milestones have been achieved in Dutch clinical gene therapy trials, including the first treatment worldwide of patients with adenosine deaminase deficiency with genetically corrected hematopoietic stem cells in collaboration with French and British scientists. Until now, about 230 patients with various diseases have been treated with viral and non-viral gene therapy in this country. Ongoing and upcoming Dutch clinical trials focus on the translation of new developments in gene therapy research, including the restoration of genetic defects other than SCID, and the use of oncolytic adenoviruses and targeted T cells for the treatment of cancer. The growing commercial interest in Dutch clinical gene therapy is reflected by the involvement of two Dutch companies in ongoing trials as well as the participation of Dutch clinical centres in large phase III international multicenter immuno-gene therapy trials on prostate cancer sponsored by an American company. Translational gene therapy research in The Netherlands is boosted at a governmental level by the Dutch Ministry of Health via a dedicated funding programme. This paper presents an overview on milestones in Dutch basic gene therapy research as well as on past, present and future clinical gene therapy trials in The Netherlands.     

Human somatic cell gene therapy

The prelude to successful human somatic gene therapy, i.e. the efficient transfer and expression of a variety of human genes into target cells, has already been accomplished in several systems. Safe methods have been devised to do this using non-viral and viral vectors. Potentially therapeutic genes have been transferred into many accessible cell types, including hematopoietic cells, hepatocytes and cancer cells, in several different approaches to ex vivo gene therapy. Successful in vivo gene therapy requires improvements in tissuetargeting and new vector design, which are already being sought. Gene-transfer protocols have been approved for human use in inherited diseases, cancer and acquired disorders. Althouth the results of these trials to date have been somewhat disappointing, human somatic cell gene therapy promises to be an effective addition to the arsenal of approaches to the therapy of many human diseases in the 21st century if not sooner.     

Co-transduction of Sleeping Beauty Transposase and Donor Plasmid via a Cell-penetrating Peptide: A simple one step Method

Transposable elements have emerged as a promising candidate for human non-viral gene-therapy. The Tc1/mariner transposon Sleeping Beauty is to date one of the most efficient transposons in mammals. Sleeping Beauty transposase has so far mostly been delivered to cells via a DNA source. This might cause spontaneous integration of the transposase gene and cause fatal damage to the affected cell. Hence, it would be advantageous to employ a non-genetic source for the transposase. We here show that a novel Cell-penetrating peptide, M918, has the ability to facilitate cellular delivery of both the transposase Sleeping Beauty as a protein and a transposon donor-plasmid carrying an antibiotic resistance gene in vitro. The technique is a simple and straightforward one-step method that might render a safe and efficient delivery platform for Sleeping Beauty mediated gene therapy.     

An N-terminal 78 amino acid truncation of REIC/Dkk-3 effectively induces apoptosis

Overexpression of REIC/Dkk-3 (a tumor suppressor gene) induces cancer cell apoptosis through endoplasmic reticulum (ER) stress. Therefore, the identification of the portion of REIC/Dkk-3 that causes ER stress may be essential for the development of cancer treatment based on REIC/Dkk-3. Here, we made several truncated forms of REIC/Dkk-3 and investigated their therapeutic potentials against prostate cancer. Among three truncated forms, a variant comprising the N-terminal 78 amino acid region of REIC/Dkk-3 (^1-78REIC/Dkk-3) most strongly induced ER stress and apoptosis in human prostate cancer cells (PC3). For in vivo gene expression, we coupled a biodegradable polymer with naked DNA, which attained robust trans-gene expression in PC3-derived subcutaneous tumor. In therapeutic experiments, we demonstrated that multiple direct injections of polymer-conjugated ^1-78REIC/Dkk-3 plasmid provoke ER stress and significantly reduced the subcutaneous tumor volume compared with the control group. We suggest this non-viral strategy may be an effective alternative to viral gene therapy.     

Recombinant mussel adhesive protein as a gene delivery material

Efficient target gene delivery into eukaryotic cells is important for biotechnological research and gene therapy. Gene delivery based on proteins, including histones, has recently emerged as a powerful non-viral DNA transfer technique. Here, we investigated the potential use of a recombinant mussel adhesive protein, hybrid fp-151, as a gene delivery material, in view of its similar basic amino acid composition to histone proteins, and cost-effective and high-level production in Escherichia coli. After confirming DNA binding affinity, we transfected mammalian cells (human 293T and mouse NIH/3T3) with foreign genes using hybrid fp-151 as the gene delivery carrier. Hybrid fp-151 displayed comparable transfection efficiency in both mammalian cell lines, compared to the widely used transfection agent, Lipofectamine 2000. Our results indicate that this mussel adhesive protein may be used as a potential protein-based gene-transfer mediator.     

Sporoplex: A hypothetical complex of fungal spore with oligonucleotide as a gene delivery vector

Various types of vehicles have been applied for gene delivery, including viral and non-viral vectors. The authors would like to hypothesize that fungal spores also potentially could be used as a gene delivery vehicle, which we call “Sporoplex”.     

Design,preparation and application of nucleic acid delivery carriers

Gene delivery vectors must deliver their cargoes into the cytosol or the nucleus, where DNA or siRNA functions in vivo. Therefore it is crucial for the rational design of the nucleic acid delivery carriers. Compared with viral vectors, non-viral vectors have overcome some fatal defections in gene therapy. Whereas the most important issue for the non-viral vectors is the low transfection efficiency, which hinders the progress of non-viral carriers. Sparked by the structures of the virus and understanding of the process of virus infection, various biomimic structures of non-viral carriers were designed and prepared to improve the transfection issues in vitro and in vivo. However, less impressive results are achieved. In this review, we will investigate the evolution of the virus-mimicking carriers of nucleic acids for gene therapy, especially in cancer therapy; explore and discuss the relationship between the structures, materials and functions of the carriers, to provide guidance for establishing safe and highly efficient non-viral carriers for gene therapy.