Immunogenicity of recombinant adenovirus serotype 35 vaccine in the presence of pre-existing anti-Ad5 immunity

The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4(+) T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.     

肿瘤基因治疗中腺病毒载体改造热点

在病毒介导的肿瘤基因治疗的研究中,腺病毒具有多种优点使其成为基因治疗中使用最多的载体之一.但腺病毒也存在许多问题迫使科学家们去不断的优化它,以达到更好的治疗效果.其中对腺病毒外壳蛋白的改造、对腺病毒基因组的改造以及使用化学修饰剂对腺病毒进行修饰改造一直是研究的热点,本文就对腺病毒改造的热点研究及取得的新成果作一评述.     

Photodynamic therapy boosts anti-glioma immunity in mice: a dependence on the activities of T cells and complement C3

Photodynamic therapy (PDT) involves the systemic administration of a tumor-specific photosensitizer and local irradiation of visible light, can generate highly cytotoxic molecular species in the tumor and kill malignant cells directly or by shutting down the tumor microvasculature. Collectively data show that anti-tumor immunity is an important mechanism that mediates the PDT-induced tumor-destroying effects in many types of cancers. However, it is unknown whether PDT also promotes anti-tumor immunity in gliomas in the central nervous system (CNS). Here we show that the PDT generates regional and systemic anti-tumor immunity in mice with G422 gliomas in the brain. The infiltration of immune cells and the release of inflammatory factors, such as TNF-α and IFN-γ, are increased in animals with G422 gliomas following PDT when compared with those without receiving PDT. The lymphocytes that are isolated from PDT-treated mice are able to induce anti-tumor immunity in nude mice. The anti-glioma immunity fostered by PDT is inhibited in complement C3 knockout mice and the nude mice indicate the requirement of the activities of complement C3 and T cells. Thus, T cells that produce cytokines, along with complement C3, may play crucial roles in mediating PDT-induced anti-glioma responses.     

HIV protease as a target for retrovirus vector-mediated gene therapy

The dimeric aspartyl protease of HIV has been the subject of intense research for almost a decade. Knowledge of the substrate specificity and catalytic mechanism of this enzyme initially guided the development of several potent peptidomimetic small molecule inhibitors. More recently, the solution of the HIV protease structure led to the structure-based design of improved peptidomimetic and non-peptidomimetic antiviral compounds. Despite the qualified success of these inhibitors, the high mutation rate associated with RNA viruses continues to hamper the long-term clinical efficacy of HIV protease inhibitors. The dimeric nature of the viral protease has been conducive to the investigation of dominant-negative inhibitors of the enzyme. Some of these inhibitors are defective protease monomers that interact with functional monomers to form inactive protease heterodimers. An advantage of macromolecular inhibitors as compared to small-molecule inhibitors is the increased surface area of interaction between the inhibitor and the target gene product. Point mutations that preserve enzyme activity but confer resistance to small-molecule inhibitors are less likely to have an adverse effect on macromolecular interactions. The use of efficient retrovirus vectors has facilitated the delivery of these macromolecular inhibitors to primary human lymphocytes. The vector-transduced cells were less susceptible to HIV infection in vitro, and showed similar levels of protection compared to other macromolecular inhibitors of HIV replication, such as RevM10. These preliminary results encourage the further development of dominant-negative HIV protease inhibitors as a gene therapy-based antiviral strategy.     

Production of adenovirus vector for gene therapy

The field of gene therapy is rapidly expanding with a major focus on the treatment of cancer. Replication-defective adenoviruses are vectors of choice for delivering corrective genes into human cells. Major efforts are directed to design new generations of adenoviral vectors that feature reduced immunogenicity and improved targeting ability. However, the production of adenoviral vectors for gene therapy applications faces a number of challenges that limit the availability of high quality material at the early stages of research and development in the gene therapy field. Moreover, very few papers have been published on the subject and information on large-scale production methods are only available through specialized conference proceedings. This review outlines the problems associated with mass production of adenovirus vectors and describes research efforts by a number of groups who have contributed to optimize production methods. Better understanding of the adenovirus infection and replication kinetics as well as better understanding of complementing cell line physiology and metabolism greatly contributed to improving vector titers and volumetric productivity at higher cell densities. Also, the critical aspect of viral vector quantitation is discussed.     

选择性复制型腺病毒介导的自杀基因HSV -tk 在 肿瘤基因治疗中的应用

自杀基因治疗是肿瘤基因治疗的手段之一,治疗效果与自杀基因能否被高效、选择性的导入肿瘤细胞有关。肿瘤选择性复制型腺病毒（conditionally replication adenovirus,CRADs）可以特异性的在肿瘤细胞中复制,在复制的同时所携带的治疗基因也大量表达。由CRAds介导的自杀基因,实现了对肿瘤的病毒治疗和基因治疗的结合,提高了治疗效率和使用复制型腺病毒的安全性。     

The influence of innate and pre‐existing immunity on adenovirus therapy

Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre‐existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed. Furthermore, memory T cell and humoral responses to Ad5 are associated with increased toxicity, raising safety concerns for therapeutic adenovirus vectors in immunized hosts. Most preclinical studies have been performed in naïve animals; although pre‐existing immunity is among the greatest hurdles for adenovirus therapies, it is also one of the most neglected experimentally. Here we summarize findings using adenovirus vectors in naïve animals, in Ad‐immunized animals and in clinical trials, and review strategies proposed to overcome innate immune responses and pre‐existing immunity. J. Cell. Biochem. 108: 778–790, 2009. © 2009 Wiley‐Liss, Inc.     

Recent trends in non-viral vector-mediated gene delivery

Nucleic acids-based next generation biopharmaceuticals (i.e., pDNA, oligonucleotides, short interfering RNA) are potential pioneering materials to cope with various incurable diseases. However, several biological barriers present a challenge for efficient gene delivery. On the other hand, developments in nanotechnology now offer numerous non-viral vectors that have been fabricated and found capable of transmitting the biopharmaceuticals into the cell and even into specific subcellular compartments like mitochondria. This overview illustrates cellular barriers and current status of non-viral gene vectors, i.e., lipoplexes, liposomes, polyplexes, and nanoparticles, to relocate therapeutic DNA-based nanomedicine into the target cell. Despite the awesome impact of physical methods (i.e., ultrasound, electroporation), chemical methods have been shown to accomplish high-level and safe transgene expression. Further comprehension of barriers and the mechanism of cellular uptake will facilitate development of nucleic acids-based nanotherapy for alleviation of various disorders.     

Helper-dependent canine adenovirus vector-mediated transgene expression in a neurodegenerative lysosomal storage disorder

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a severe neurodegenerative lysosomal storage disorder caused by a deficiency of N-sulfoglucosamine sulfohydrolase (SGSH) activity with subsequent accumulation of partially-degraded heparan sulfate and other glycolipids. In this study, we have evaluated a gene therapy approach using a helper-dependent canine adenovirus vector that expresses human SGSH as a means of delivering sustained transgene expression to the brain. Initial testing in a mixed neural cell culture model demonstrated that the vector could significantly increase SGSH activity in transduced cells, resulting in near-normalization of heparan sulfate-derived fragments. While administration of vector by direct injection into the brain of adult MPS-IIIA mice enabled transgene expression for at least 8.5 months post-treatment, it was only in discrete areas of brain. Heparan sulfate storage was reduced in some regions following treatment, however there was no improvement in secondary neuropathological changes. These data demonstrate that helper-dependent canine adenovirus vectors are capable of neural transduction and mediate long-term transgene expression, but increased SGSH expression throughout the brain is likely to be required in order to effectively treat all aspects of the MPS-IIIA phenotype.     

Adenoviral vector-mediated glucagon-like peptide 1 gene therapy improves glucose homeostasis in Zucker diabetic fatty rats

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that plays an important role in glucose homeostasis. Its functions include glucose-stimulated insulin secretion, suppression of glucagon secretion, deceleration of gastric emptying, and reduction in appetite and food intake. Despite the numerous antidiabetic properties of GLP-1, its therapeutic potential is limited by its short biological half-life due to rapid enzymatic degradation by dipeptidyl peptidase IV. The present study aimed to demonstrate the therapeutic effects of constitutively expressed GLP-1 in an overt type 2 diabetic animal model using an adenoviral vector system. METHODS: A novel plasmid (pAAV-ILGLP-1) and recombinant adenoviral vector (Ad-ILGLP-1) were constructed with the cytomegalovirus promoter and insulin leader sequence followed by GLP-1(7-37) cDNA. RESULTS: The results of an enzyme-linked immunosorbent assay showed significantly elevated levels of GLP-1(7-37) secreted by human embryonic kidney cells transfected with the construct containing the leader sequence. A single intravenous administration of Ad-ILGLP-1 into 12-week-old Zucker diabetic fatty (ZDF) rats, which have overt type 2 diabetes mellitus (T2DM), achieved near normoglycemia for 3 weeks and improved utilization of blood glucose in glucose tolerance tests. Circulating plasma levels of GLP-1 increased in GLP-1-treated ZDF rats, but diminished 21 days after treatment. When compared with controls, Ad-ILGLP-1-treated ZDF rats had a lower homeostasis model assessment for insulin resistance score indicating amelioration in insulin resistance. Immunohistochemical staining showed that cells expressing GLP-1 were found in the livers of GLP-1-treated ZDF rats. CONCLUSIONS: These data suggest that GLP-1 gene therapy can improve glucose homeostasis in fully developed diabetic animal models and may be a promising treatment modality for T2DM in humans.     

Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.