Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors

Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9-mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB. CONCLUSION: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV.     

Significance of Preexisting Vector Immunity and Activation of Innate Responses for Adenoviral Vector-Based Therapy

An adenoviral (AdV)-based vector system is a promising platform for vaccine development and gene therapy applications. Administration of an AdV vector elicits robust innate immunity, leading to the development of humoral and cellular immune responses against the vector and the transgene antigen, if applicable. The use of high doses (10¹¹–10¹³ virus particles) of an AdV vector, especially for gene therapy applications, could lead to vector toxicity due to excessive levels of innate immune responses, vector interactions with blood factors, or high levels of vector transduction in the liver and spleen. Additionally, the high prevalence of AdV infections in humans or the first inoculation with the AdV vector result in the development of vector-specific immune responses, popularly known as preexisting vector immunity. It significantly reduces the vector efficiency following the use of an AdV vector that is prone to preexisting vector immunity. Several approaches have been developed to overcome this problem. The utilization of rare human AdV types or nonhuman AdVs is the primary strategy to evade preexisting vector immunity. The use of heterologous viral vectors, capsid modification, and vector encapsulation are alternative methods to evade vector immunity. The vectors can be optimized for clinical applications with comprehensive knowledge of AdV vector immunity, toxicity, and circumvention strategies.     

Physical and biological barriers to viral vector-mediated delivery of genes to the airway epithelium

A gene therapy for cystic fibrosis (CF) lung disease by intralumenal delivery of therapeutic transgenes into the lung is a logical treatment strategy if efficient gene transfer can be achieved without detrimental effects to the patient. Indeed, pioneering work in the late 1980s showed that genetically engineered viruses could deliver the CF corrective transgene to cultured cells from patients with CF. However, after many attempts to deliver the corrective gene to the lungs of patients with CF in vivo and with the luxury of 20/20 hindsight, it is realized that although logical, the strategy to accomplish this task did not appreciate the evolution of the lung to resist invasion by pathogens such as viruses. It is now apparent that several levels of barriers exist that restrict exogenous gene delivery to the airway epithelium by commonly used viral vectors. Components of the innate and cell-mediated immune system collectively limit both the access to and duration of gene transfer vectors to the airway epithelium. Alternative viral vectors that have evolved to circumvent these barriers will require further development if gene transfer is ever to be considered a therapy for CF lung disease.     

线粒体抗病毒信号蛋白与RNA病毒的相互作用

天然免疫是宿主细胞在生物进化过程中形成的抵御外来侵袭的第一道防线。病毒感染后通过信号转导机制引发机体免疫反应,线粒体抗病毒信号蛋白(MAVS)作为天然免疫信号通路的重要接头蛋白,可以接收上游Toll样受体(TLR)和维甲酸诱导基因I(RIG-I)受体(RLRs)等识别RNA病毒并传递信号,活化下游NF-κB和IRF3/7相关信号通路,从而激活干扰素表达,介导天然免疫相关信号通路。RNA病毒在长期进化过程中针对MAVS也形成了一系列免疫逃逸策略。本综述介绍了MAVS的发现及结构,并对MAVS抗RNA病毒的作用及病毒对MAVS的负调控进行综述,旨在为相关研究提供帮助。     

In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance

Liver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNalphabeta response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNalphabeta, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type-specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.     

Gene therapy of cancer

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. In the meantime, cancer has become by far the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors is direct intratumoral injection with or without the aid of various techniques such as endoscopy or CT-guidance. At present, viral and non-viral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently in use in clinical trials comprise retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. None of the available vectors satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses ("gutless vectors") as well as completely "synthetic viral vectors" will gain more and more importance in the future. Non-viral gene therapy methods include liposomes, injection of vector-free DNA ("naked DNA"), protein-DNA complexes, delivery by "gene gun," calcium-phosphate precipitation, electroporation, and intracellular microinjection of DNA. The first clinical trial of gene therapy for cancer was performed in 1991 in patients with melanoma, and since then more than 5000 patients have been treated worldwide in more than 400 clinical protocols. With the exception of a case of fatal toxicity in a young man with hereditary liver disease treated intrahepatically with high doses of adenovirus, side effects have been rare and usually mild in all these studies and expression of the transgene could be demonstrated in patients in vivo. However, despite anecdotal reports of therapeutic responses in some patients, unequivocal proof of clinical efficacy is still lacking for most of the varied approaches to gene therapy in humans. As well as our only fragmentary understanding of the molecular pathophysiology of many diseases, the principal reason for the present lack of clinical success of gene therapy is the very low transduction and expression efficiency in vivo of available vectors. Despite the complexities of gene therapy for cancer, the numerous different approaches can be subdivided into three basic concepts: (1) strengthening of the immune response against a tumour, (2) repair of cell cycle defects caused by losses of tumour suppressor genes or inappropriate activation of oncogenes, and (3) suicide gene strategies. In addition, the importance of gene marker studies and gene therapeutic protection of normal tissue are briefly covered in this review.     

Current status of gene therapy for cystic fibrosis pulmonary disease

Cystic fibrosis (CF) is a common lethal genetic disorder that affects all ethnic populations; however, it is most prevalent in Caucasians. Intensive basic research over the last 20 years has resulted in a wealth of information regarding the CF gene, its protein product and the mutational basis of disease. This increased understanding has lead to the development of gene therapy for the treatment of CF pulmonary disease. Delivery of the CF gene to the airway requires direct in vivo transfer using vectors encoding for normal CF transmembrane regulator (CFTR) protein. Several vectors are currently available for CF gene transfer and include both viral (adenoviruses, adeno-associated viruses) and non-viral (liposomal) systems. Initial clinical trials with each of these vectors have demonstrated that gene transfer to the CF airway is possible. The efficiency of transfer and duration of expression, however, have been limited. The effects of gene transfer on correction of the basic ion transport defects have also been highly variable and inconsistent, irrespective of the vector. Currently, the risk of severe immunological reactions is the primary factor limiting the clinical advancement of gene therapy. Both the adenoviral and liposomal vectors are associated with significant acute inflammatory reactions. The adenoviruses and adeno-associated viruses also elicit humoral immune responses that significantly reduce the efficiency of transgene expression and increase the risk of readministration. Several strategies are under investigation to improve the efficiency of gene transfer to the CF airway. These include overcoming local barriers in the lung, circumventing the immune response and improving vector internalization and/or uptake. Application of gene transfer in the child and possibly the fetus are also potential future clinical applications of gene therapy. However, despite considerable research with gene therapy, there is little evidence to suggest that a well tolerated and effective gene transfer method is imminent and aggressive use of conventional pharmacological therapies currently offer the greatest promise in the treatment of patients with CF.     

Directed evolution of adeno-associated virus to an infectious respiratory virus

Respiratory viruses evolve to maintain infectivity levels that permit spread yet prevent host and virus extinction, resulting in surprisingly low infection rates. Respiratory viruses harnessed as gene therapy vectors have illustrated this limitation. We used directed evolution in an organotypic human airway model to generate a highly infectious adeno-associated virus. This virus mediated gene transfer more than 100-fold better than parental strains and corrected the cystic fibrosis epithelial Cl⁻ transport defect. Thus, under appropriate selective pressures, viruses can evolve to be more infectious than observed in nature, a finding that holds significant implications for designing vectors for gene therapy and for understanding emerging pathogens.     

Improving adenovirus based gene transfer: strategies to accomplish immune evasion

Adenovirus (Ad) based gene transfer vectors continue to be the platform of choice for an increasing number of clinical trials worldwide. In fact, within the last five years, the number of clinical trials that utilize Ad based vectors has doubled, indicating growing enthusiasm for the numerous positive characteristics of this gene transfer platform. For example, Ad vectors can be easily and relatively inexpensively produced to high titers in a cGMP compliant manner, can be stably stored and transported, and have a broad applicability for a wide range of clinical conditions, including both gene therapy and vaccine applications. Ad vector based gene transfer will become more useful as strategies to counteract innate and/or pre-existing adaptive immune responses to Ads are developed and confirmed to be efficacious. The approaches attempting to overcome these limitations can be divided into two broad categories: pre-emptive immune modulation of the host, and selective modification of the Ad vector itself. The first category of methods includes the use of immunosuppressive drugs or specific compounds to block important immune pathways, which are known to be induced by Ads. The second category comprises several innovative strategies inclusive of: (1) Ad-capsid-display of specific inhibitors or ligands; (2) covalent modifications of the entire Ad vector capsid moiety; (3) the use of tissue specific promoters and local administration routes; (4) the use of genome modified Ads; and (5) the development of chimeric or alternative serotype Ads. This review article will focus on both the promise and the limitations of each of these immune evasion strategies, and in the process delineate future directions in developing safer and more efficacious Ad-based gene transfer strategies.     

Infection with highly pathogenic H7 influenza viruses results in an attenuated proinflammatory cytokine and chemokine response early after infection

Avian influenza A viruses of the H7 subtype have resulted in more than 100 cases of human infection since 2002. Highly pathogenic avian influenza (HPAI) H7 viruses have the capacity to cause severe respiratory disease and even death; however, the induction of the human innate immune response to H7 virus infection has not been well characterized. To better understand H7 virus pathogenesis in the human respiratory tract, we employed a polarized human bronchial epithelial cell model and primary human monocyte-derived macrophages. Here, we show that infection with HPAI H7 viruses resulted in a delayed and weakened production of cytokines, including the type I interferon response, compared with infections of other influenza A subtypes, including H7 viruses of low pathogenicity. These studies revealed that H7 viruses vary greatly in their ability to activate host innate responses and may contribute to the virulence of these viruses observed in humans.     

Alphaviruses in gene therapy

Alphaviruses are enveloped single stranded RNA viruses, which as gene therapy vectors provide high-level transient gene expression. Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan Equine Encephalitis (VEE) virus have been engineered as efficient replication-deficient and -competent expression vectors. Alphavirus vectors have frequently been used as vehicles for tumor vaccine generation. Moreover, SFV and SIN vectors have been applied for intratumoral injections in animals implanted with tumor xenografts. SIN vectors have demonstrated natural tumor targeting, which might permit systemic vector administration. Another approach for systemic delivery of SFV has been to encapsulate replication-deficient viral particles in liposomes, which can provide passive targeting to tumors and allow repeated administration without host immune responses. This approach has demonstrated safe delivery of encapsulated SFV particles to melanoma and kidney carcinoma patients in a phase I trial. Finally, the prominent neurotropism of alphaviruses make them attractive for the treatment of CNS-related diseases.     

按蚊抗疟原虫感染的先天性免疫防御反应

在疟原虫-蚊媒相互作用的基础上，研究按蚊抗疟原虫的先天性免疫防御反应机制，并最终利用其防御机制限制或杀死移行发育中的疟原虫，以期实现有成效的疟疾媒介控制策略。     

Evasion of Influenza A Viruses from Innate and Adaptive Immune Responses

The influenza A virus is one of the leading causes of respiratory tract infections in humans. Upon infection with an influenza A virus, both innate and adaptive immune responses are induced. Here we discuss various strategies used by influenza A viruses to evade innate immune responses and recognition by components of the humoral and cellular immune response, which consequently may result in reduced clearing of the virus and virus-infected cells. Finally, we discuss how the current knowledge about immune evasion can be used to improve influenza A vaccination strategies.     

Pathogenesis of filoviral haemorrhagic fevers

The filoviruses, marburgvirus and ebolavirus, cause epidemics of haemorrhagic fever with high case-fatality rates. The severe illness results from a complex of pathogenetic mechanisms that enable the virus to suppress innate and adaptive immune responses, infect and kill a broad variety of cell types, and elicit strong inflammatory responses and disseminated intravascular coagulation, producing a syndrome resembling septic shock. Most experimental data have been obtained on Zaire ebolavirus, which causes uniformly lethal disease in experimentally infected non-human primates but produces a broader range of outcomes in naturally infected human beings. 10-30% of patients can survive the illness by mobilising adaptive immune responses, and there is limited evidence that mild or symptomless infections also occur. The other filoviruses that have caused human disease, Sudan ebolavirus, Ivory Coast ebolavirus, and marburgvirus, produce a similar illness but with somewhat lower case-fatality rates. Variations in outcome during an epidemic might be due partly to genetically determined differences in innate immune responses to the viruses. Recent studies in non-human primates have shown that blocking of certain host responses, such as the coagulation cascade, can result in reduced viral replication and improved host survival.     

Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus

Delivering transgenes to human cells through transduction with viral vectors constitutes one of the most encouraging approaches in gene therapy. Lentivirus-derived vectors are among the most promising vectors for these approaches. When the genetic modification of the cell must be performed in vivo, efficient specific transduction of the cell targets of the therapy in the absence of off-targeting constitutes the Holy Grail of gene therapy. For viral therapy, this is largely determined by the characteristics of the surface proteins carried by the vector. In this regard, an important property of lentiviral vectors is the possibility of being pseudotyped by envelopes of other viruses, widening the panel of proteins with which they can be armed. Here, we discuss how this is achieved at the molecular level and what the properties and the potentialities of the different envelope proteins that can be used for pseudotyping these vectors are.     

Gene therapy for hemophilia

PURPOSE OF REVIEW: This review will highlight the progress achieved in the past 2 years on using gene therapy to treat hemophilia in animals and humans. RECENT FINDINGS: There has been substantial progress in using gene therapy to treat animals with hemophilia. Novel approaches for hemophilia A in mice include expression of Factor VIII in blood cells or platelets derived from ex-vivo transduced hematopoietic stem cells, or in-vivo transfer of transposons expressing Factor VIII into endothelial cells or hepatocytes. Advances in large-animal models include the demonstration that neonatal administration of a retroviral vector expressing canine Factor VIII completely corrected hemophilia A in dogs, and that double-stranded adeno-associated virus vectors resulted in expression of Factor IX that is 28-fold that obtained using single-stranded adeno-associated virus vectors. In humans, one hemophilia B patient achieved 10% of normal activity after liver-directed gene therapy with a single-stranded adeno-associated virus vector expressing human Factor IX. Expression fell at 1 month, however, which was likely due to an immune response to the modified cells. SUMMARY: Gene therapy has been successful in a patient with hemophilia B, but expression was unstable due to an immune response. Abrogating immune responses is the next major hurdle for achieving long-lasting gene therapy.     

Foamy Virus Vectors for HIV Gene Therapy

Highly active antiretroviral therapy (HAART) has vastly improved outcomes for patients infected with HIV, yet it is a lifelong regimen that is expensive and has significant side effects. Retroviral gene therapy is a promising alternative treatment for HIV/AIDS; however, inefficient gene delivery to hematopoietic stem cells (HSCs) has so far limited the efficacy of this approach. Foamy virus (FV) vectors are derived from non-pathogenic viruses that are not endemic to the human population. FV vectors have been used to deliver HIV-inhibiting transgenes to human HSCs, and they have several advantages relative to other retroviral vectors. These include an attractive safety profile, broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. In addition, the titers of FV vectors are not reduced by anti-HIV transgenes that affect the production of lentivirus (LV) vectors. Thus FV vectors are very promising for anti-HIV gene therapy. This review covers the advantages of FV vectors and describes their preclinical development for anti-HIV gene therapy.