非病毒载体系统在基因治疗中的应用

非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统.本文综述了目前基因治疗中所用的几种非病毒载体系统及其研究进展.     

HSV1-tk基因在肿瘤报告基因显像与治疗中的研究进展

自杀基因疗法是一种有潜在临床应用价值的肿瘤治疗策略。HSV1-tk基因兼具治疗基因和报告基因的功能,可用于基因显像、基因治疗及其疗效监测。近年来,无创伤性的肿瘤HSV1-tk基因显像发展很快,核素标记的显像底物有FIAU、FMAU、FHBG和FHPG等,使用的显像设备有PET、SPECT或γ照相机等,这些显像方法具有不同的敏感性和特异性,但均能监测动物体内表达HSV1-tk肿瘤部位的增殖情况和治疗效果,更复杂的HSV1-tk报告基因显像方案正在探索中。HSV1-tk基因治疗联合靶向治疗、放射治疗、药物治疗、免疫生物治疗、热疗及其他基因治疗具有疗效相加或协同效应,增强了对肿瘤的杀伤作用。     

MLL-mediated transcriptional gene regulation investigated by gene expression profiling

The human mixed lineage leukemia (MLL) gene is involved in about 50 different chromosomal translocations, associated with the disease phenotype of acute leukemia. However, the normal function of MLL is less understood. Homozygous knockouts of murine Mll were embryonal lethal, while heterozygous disruption led to aberrant hox gene expression associated with skeletal malformations, growth retardation, and impaired hematopoiesis. To understand MLL functions on the molecular level, gene expression profiling experiments were performed with a pair of murine cell lines (MLL(+/+) and MLL(-/-)). Microarray hybridization experiments revealed 197 potential target genes that are differentially expressed, providing new and important clues about MLL functions.     

Light-induced adenovirus gene transfer, an efficient and specific gene delivery technology for cancer gene therapy

A main issue for further clinical progress of cancer gene therapy is to develop technologies for efficient and specific delivery of therapeutic genes to tumor cells. In this work, we describe a photochemical treatment that substantially improves gene delivery by adenovirus, one of the most efficient gene delivery vectors known. Transduction of two different cell lines was studied by microscopy, flow cytometry, and an enzymatic assay, employing a beta-galactosidase-encoding adenovirus. The photochemical treatment induced a >20-fold increase in gene transduction, compared with conventional adenovirus infection, both when measured as the percentage of cells transduced, and when measured as the total beta-galactosidase activity in the cell population. The effect was most pronounced at lower virus doses, where in some cases the same transduction efficiency could be achieved with a 20 times lower virus dose than with conventional infection. Photochemical treatments are already in clinical use for cancer therapy, and generally are very specific and have few side effects. The photochemical internalization technology described thus has a clear potential for improving both the efficiency and the specificity of gene delivery in cancer gene therapy, making it possible to achieve efficient site-specific in vivo gene delivery by adenoviral vectors.     

bcr-abl和白介素-7共表达基因疫苗诱导小鼠免疫保护作用

目的探讨小鼠白介素-7（IL-7）基因对bcr-abl融合基因疫苗诱导机体免疫应答的影响。方法采用pVbcr-abl／mIL-7质粒免疫BALB／c小鼠,检测小鼠血清中bcr-abl特异性抗体水平。以转染bcr-abl融合基因片段的SF2／0细胞为靶细胞,用LDH释放实验检测免疫小鼠脾细胞特异性细胞毒活性。结果pVbcr-abl／mIL-7质粒能诱导小鼠产生血清特异性抗bcr-abl抗体,pVbcr-abl／mIL-7免疫组的血清特异性抗体滴度高于pVbcr-abl免疫组,脾细胞杀伤SP2／0／bcr-abl靶细胞的细胞毒活性明显增强。结论共表达bcr-abl和mIL-7的基因疫苗能在小鼠体内诱导较高的特异性体液免疫和细胞免疫水平,为慢性粒细胞白血病基因疫苗的临床前实验研究提供了依据。     

Saporin as a novel suicide gene in anticancer gene therapy

We used a non-viral gene delivery approach to explore the potential of the plant saporin (SAP) gene as an alternative to the currently employed suicide genes in cancer therapy. Plasmids expressing cytosolic SAP were generated by placing the region encoding the mature plant ribosome-inactivating protein under the control of cytomegalovirus (CMV) or simian virus 40 (SV40) promoters. Their ability to inhibit protein synthesis was first tested in cultured tumor cells co-transfected with a luciferase reporter gene. In particular, SAP expression driven by CMV promoter (pCI-SAP) demonstrated that only 10 ng of plasmid per 1.6 x 10(4) B16 cells drastically reduced luciferase activity to 18% of that in control cells. Direct intratumoral injection of pCI-SAP complexed with either lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) in B16 melanoma-bearing mice resulted in a noteworthy attenuation of tumor growth. This antitumor effect was increased in mice that received repeated intratumoral injections. A SAP catalytic inactive mutant (SAP-KQ) failed to exert any antitumor effect demonstrating that this was specifically owing to the SAP N-glycosidase activity. Our overall data strongly suggest that the gene encoding SAP, owing to its rapid and effective action and its independence from the proliferative state of target cells might become a suitable candidate suicide gene for oncologic applications.     

Inactivation of the murine N-ras gene by gene targeting.

The mammalian ras genes have been implicated in the genesis of a wide variety of tumors. Although it is likely that they play an essential role in signal transduction, their specific function is still unknown. To initiate a genetic analysis of the ras genes in mammals we inactivated the N-ras gene in murine embryonic stem cells by gene targeting. The frequency of integration at the N-ras locus of our targeting vector being low (of the order of 1/5000 transfectants), we used a positive/negative selection followed by an analysis of individual colonies in order to minimize the in vitro manipulation of the embryonic stem cells. Using this approach, we isolated two clones of ES cells with one inactivated N-ras allele. These cells have no distinctive phenotype either in vitro or in vivo in chimeric mice.     

抗肿瘤基因疫苗

肿瘤基因疫苗的发展逐渐趋于成熟,常见基因疫苗主要有细胞因子类基因疫苗、多基因联合疫苗,转染基因的树突状细胞疫苗等.近些年肿瘤基因疫苗的发展逐渐趋向于寻找基凶疫苗佐剂或探索不同基凶的联合从而达到更加有效地治疗目的.     

Somatic gene therapy

Many problems obviously continue to exist in gene transfer using retroviruses as a means of inserting foreign DNA into hematopoietic stem cells, especially with regulated genes such as the human beta globin genes. First, it is unclear whether the available retroviral vectors will infect enough stem cells for gene transfer to be successful over the long term. Second, there may be sequences necessary for normal beta globin gene expression that may also inhibit the normal retroviral life cycle, thus decreasing the efficiency of gene transfer or gene expression. It seems clear that in order to optimize the success of gene transfer, the highest possible titer of viral production is necessary. New approaches are aimed at increasing viral titer. The transfect/infect method appears useful. Growth factors may also be useful by increasing stem cell proliferation. Single growth factors may not be sufficient to optimize stem cell cycling. To date, interleukin-3 seems to be the single most useful growth factor, although interleukin-3 with interleukin-6 or other combinations of growth factors including interleukin-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) appear to have potential. Future work also is required to optimize the number of marrow stem cells needed for successful transplantation. Long-term bone marrow culture and stromal cell cultures may provide new and improved marrow culture conditions for achieving this goal. Improvements in the efficiency of both gene transfer and gene expression are necessary before we can consider the concept of gene transfer for the treatment of various hematologic genetic diseases in humans.     

Cancer gene therapy

Cancer gene therapy is the transfer of genetic material to the cells of an individual with the goal of eradicating cancer cells, both in the primary tumor and metastases. Cancer gene therapy strategies exploit our expanding knowledge of the genetic basis of cancer, thereby allowing rationally targeted interventions at the molecular level. The successful implementation of cancer gene therapy in the clinic awaits the development of vectors capable of specific and efficient gene delivery to cancer cells. The first clinical applications of cancer gene therapy are likely to be in combination with conventional therapies, such as radiotherapy and immunotherapy.     

Adenoviral gene therapy

As of May 2001, 532 gene therapy protocols had been approved for evaluation in clinical trials; however, only five of those had been evaluated in phase III clinical trials. Among the most commonly used vectors for the delivery of genetic material into human cells are the adenoviruses. Remarkable progress has been made with these vectors in the last decade, but some shortcomings continue to challenge investigators. The newly acquired knowledge of the adenoviral life cycle and the positive outcomes from phase II clinical trials have led to the application of vectors engineered to selectively target tumor tissue under controlled promoters.     

Cancer gene therapy

The prognosis of patients with some kinds of cancers whose patients are often found unresectable upon diagnosis is still dismal. In these fields, development of a new therapeutic modality is needed and gene therapy represents one promising strategy. So far, numerous cancer gene therapy clinical trials based on these principles have been carried out and have shown the safety of such modalities, but have fallen short of the initial expectations to cure cancers. In this review, we would like to make a problem-oriented discussion of current status of cancer gene therapy research by using mainly gastrointestinal cancers as an example. In order to overcome obstacles for full realization of cancer gene therapy, numerous researches have been conducted by many researchers. Various cancer-selective and non-selective genes, as well as lytic viruses themselves have been employed for gene therapy. In the context of gene delivery method, different kinds of viral and non-viral strategies have been utilized. In addition, surrogate assays, such as soluble markers and imaging, have been developed for safer and more informative clinical trials. Many experiments and clinical trials to date have figured out current obstacles for the realization of an effective cancer gene therapy modality. Tireless efforts to overcome such hurdles and continuous infusion of novel concepts into this field should lead to break through technologies and the cure of the patients.     

Anti-tumor gene therapy

Gene therapy as an anti-tumor strategy is becoming a powerful tool for cytokine delivery to inhibit the growth of many tumors. Several delivery systems are being utilized and designed for the expression of specific genes to achieve a therapeutic result. Liposomes, retroviral vectors, and adenoviral vectors have all been used and eventual clinical application may depend on the type of tumor, the location, the specific gene carried, and the patients health status. Novel expression vectors may eventually achieve tissue-specific targeting and low immuno-reactivity. Inactivation of mutated oncogenes, such as ras, or re-expression of inactive suppressor genes, such as p53 have been used as strategies for anti-tumor therapy. Additionally, exogenous genes, su ch as viral thymidine kinase that metabolize chemotherapeutic agents to achieve local cytotoxicity have also been employed. Neuro-endocrine tumors are targets of these genetic strategies since they are often difficult to treat by conventional methods bec ause of their location (brain tumors) or because they have spread from the primary tumor (melanoma). Further advances in the design of these vectors may achieve safe targeting of a variety of malignant tumors.     

Viral gene therapy

Cancer is a multigenic disorder involving mutations of both tumor suppressor genes and oncogenes. A large body of preclinical data, however, has suggested that cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses. The ability for the delivery of therapeutic genes, made them desirable for engineering virus vector systems. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Because these vector systems have unique advantages and limitations, each has applications for which it is best suited. Retroviral vectors can permanently integrate into the genome of the infected cell, but require mitotic cell division for transduction. Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types, but immune elimination of infected cells often limits gene expressionin vivo. Herpes simplex virus can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression remain as obstacles. AAV also infects many non-dividing and dividing cell types, but has a limited DNA capacity. This review discusses current and emerging virus-based genetic engineering strategies for the delivery of therapeutic molecules or several approaches for cancer treatment. Supported by an unrestricted educational grant from AstraZeneca.     

bcr-ab1和白介素-7共表达基因疫苗诱导小鼠免疫保护作用

目的探讨小鼠白介素-7(IL-7)基因对bcr-abl融合基因疫苗诱导机体免疫应答的影响。方法采用pVbcr-abl/mIL-7质粒免疫BALB/c小鼠,检测小鼠血清中bcr-abl特异性抗体水平。以转染bcr-abl融合基因片段的SP2/0细胞为靶细胞,用LDH释放实验检测免疫小鼠脾细胞特异性细胞毒活性。结果pVbcr-abl/mlL-7质粒能诱导小鼠产生血清特异性抗bcr-abl抗体,pVbcr-abl/mIL-7免疫组的血清特异性抗体滴度高于pVbcr-abl免疫组,脾细胞杀伤SP2/0/bcr-abl靶细胞的细胞毒活性明显增强。结论共表达bcr-abl和mIL-7的基因疫苗能在小鼠体内诱导较高的特异性体液免疫和细胞免疫水平,为慢性粒细胞白血病基因疫苗的临床前实验研究提供了依据。     

p73和p51基因及其在消化系统肿瘤中的作用

p73和p51基因是肿瘤抑制基因p53家族新成员.研究发现p73和p51基因的异常与许多肿瘤的发生发展有关.在各种消化道肿瘤中,p73和p51基因的异常表达平均可达到60%.通过对其深入的研究将有助于人们进一步认识肿瘤的本质,从而为肿瘤的基因治疗提供新的靶点.     

Cancer cells as targets for lentivirus-mediated gene transfer and gene therapy

Lentiviruses have been used as gene transfer vectors for almost 10 years and their utility has been demonstrated in a variety of different applications. However, their value in cancer gene therapy has not been studied thoroughly. Here we show that VSV-G pseudotyped HIV-1-based lentiviruses are efficient vectors for human tumor cells in vitro and in vivo. Lentiviral gene transfer efficiency was demonstrated by transducing 42 different cell lines, representing 10 different human tumor types. It was shown that most of the cell lines were good or excellent targets for lentiviral transduction, allowing 50-95% gene transfer efficiency. These results were comparable to those obtained with an E1/E3 deleted, serotype 5 adenovirus vector. Analysis of lentivirus vector structure revealed that virus particles devoid of HIV-1 accessory proteins appeared to be more efficient, but the presence of enhancing elements cPPT and WPRE did not play a major role in transduction efficiency to four different human tumor cell lines. However, their effect on the gene expression level in these cells was apparent. To examine the impact of lentiviral gene expression level on suicide gene therapy approach, human osteosarcoma cells were transduced with lentivirus- or adenovirus vectors carrying the fusion gene HSV-TK-GFP and exposed to ganciclovir. Cell viability analysis after the treatment revealed that both vector types induced similar level of cytotoxicity, suggesting that lentiviral expression of a suicide gene is adequate for tumor cell destruction. Finally, in vivo transduction studies with subcutaneous tumors showed that lentivirus vectors can yield similar gene transfer efficiency than adenovirus vector, despite three orders of magnitude lower titer of the lentiviral preparation. In conclusion, these data show that lentiviruses are efficient gene transfer vehicles for human tumor cells and justify their use in further preclinical cancer gene therapy studies.