IP-10 mediates selective mononuclear cell accumulation and activation in response to intrapulmonary transgenic expression and during adenovirus-induced pulmonary inflammation.

CXC chemokines that lack the glutamine-leucine-arginine (ELR) motif, including interferon (IFN)-inducible protein 10 (IP-10 or CXCL10), have been shown to mediate the generation of type 1 immune responses. In this study, we found that the intrapulmonary transient transgenic expression of murine IP-10 in mice using adenoviral gene transfer resulted in the early accumulation of neutrophils, natural killer (NK) cells, and NK T cells within the lung, followed by the delayed accumulation of CD4+ T cells. Adenovirus-mediated transgenic expression of IP-10 also resulted in selective activation of mononuclear cells, including gamma(delta)-T cells and NK cells, as manifest by CD69 expression or induction of cell-associated IFN-gamma. Importantly, the intratracheal (i.t.) administration of a control human type 5 adenovirus also caused significant accumulation of NK, NK T, and CD4+ T cells, which was maximal at 7 days post vector administration and was associated with the induction of IP-10. Neutralization of endogenous IP-10 in animals receiving control adenovirus resulted in decreases in the numbers of NK, CD4+, and CD8+ T cells. These results indicate that IP-10 can direct the accumulation and activation of neutrophils and selected mononuclear cells to the lung and that adenovirus-induced IP-10 contributes to lung inflammatory cell recruitment/activation observed in response to adenoviral vectors used for gene therapy.     

Immune regulation of transgene expression in the brain: B cells regulate an early phase of elimination of transgene expression from adenoviral vectors

Cellular immune mechanisms that regulate viral gene expression within infected brain cells remain poorly understood. Previous work has shown that systemic immunization against adenovirus after vector delivery to the brain results in complete loss of brain cells infected by adenoviral vectors. Although T cells play an important role in this process, we demonstrate herein that B cells also significantly regulate transgene expression from the CNS. After the systemic immunization against adenovirus of animals injected via the brain with an adenoviral vector 30 days earlier, we uncovered substantial infiltration by CD19+ B cells of the area of the brain transduced by the virus. This suggests the involvement of B cells in the adaptive immune response-mediated loss of transduced cells from the brain. Confocal analysis of these brains demonstrated physical contacts between transduced brain cells and CD19+ cells. To test the hypothesis that B cells play a causal role in the loss of infected cells from the brain, we demonstrated that animals devoid of B cells were unable to eliminate transgene expression at early time points after immunization. This demonstrates that B cells play a necessary role in the loss of transgene expression at early, but not late, time points postimmunization. Thus, these data have important implications for our understanding of the role of B cells as immune effectors during the immune-mediated clearance of viral infections from the CNS, and also for understanding mechanisms operating in brain autoimmunity, as well as for the potential safety of clinical gene therapy for brain diseases.     

Recombinant adenovirus is an efficient and non-perturbing genetic vector for human dendritic cells

Recombinant adenoviral vectors have promise for human gene therapy because of efficient transgene expression in nondividing primary cell types. Dendritic cells (DC) have potential as adjuvants for immune therapy, since they are specialized to capture antigens to form MHC-peptide complexes, migrate to T cell areas in the lymph node, and activate T cells including CD4+ helpers and CD8+ cytotoxic T lymphocytes (CTL). We show that several current chemical and physical transfection methods allow < 2 % of DC to express reporter genes but that recombinant adenoviruses, encoding the reporter genes green fluorescent protein and LacZ, efficiently transfect monocyte-derived human DC. Immature DC, generated with IL-4 and GM-CSF, are transfected to 95% efficiency, while mature DC show reduced transfection (50%) and gene expression. Adenovirus-transfected, immature DC exhibit several critical functions. The DC can differentiate in the presence of lipopolysaccharide or a monocyte-conditioned medium to express the surface markers of mature, T cell stimulatory DC including CD25, CD83, and high levels of CD86 and HLA-DR. Transfected DC can also secrete high levels of IL-12 and are potent inducers of T cell growth. Transgene expression in DC is stable for at least 6 days in the presence of the DC survival factor, TRANCE. Therefore adenoviral infection does not perturb the maturation and function of DC. The efficiency of adenoviral-mediated gene transfer prompts the evaluation of this vector in studies of DC biology, including the expression of antigens for active immune therapy.     

Genetically modified dendritic cells for cancer immunotherapy

The ability to grow and differentiate dendritic cells (DC) ex vivo has allowed their genetic manipulation to enhance immune activation against tumor antigens. Gene engineering of DC can be achieved with a variety of physical methods and using different viral vectors. RNA or DNA transfection, either alone (naked), coated with liposomes or using electroporation or gene guns leads to T cell activation while transgene expression is frequently undetectable. Adenoviral and retroviral vectors have proven to be highly efficient in DC genetic modification, and have been widely used in preclinical models. Other vectors like lentivirus, poxvirus, herpes virus and adeno-associated virus (AAV) can also lead to foreign transgene expression in DC leading to immune cell activation. DC have been genetically engineered to provide constitutive and high level of tumor antigen expression or to introduce genes that further enhance their immune stimulatory ability. The promising results from preclinical animal models and from in vitro human immune cell culture systems have provided a strong rationale to initiate pilot clinical trials. Recently published or communicated clinical experiences and ongoing trials have used defined tumor antigen RNA transfection for prostate carcinoma and melanoma, liposome-encoated DNA transfection for breast or pancreatic cancer, adenoviral vector tumor antigen gene modification for melanoma and small cell lung cancer, and poxvirus-mediated expression of costimulatory molecules for colon carcinoma. These preliminary experiences suggest that genetically modified DC can safely induce T cell responses but few clinical responses.     

Toxicity and adaptive immune response to intracellular transgenes delivered by helper-dependent vs. first generation adenoviral vectors

The host immune response to intracellular transgenes delivered by helper-dependent (HDV) vs. first generation (FGV) adenoviral vectors has been relatively unstudied. Previous studies showed short-term correction of bovine and murine argininosuccinate synthetase (ASS) deficiency after first generation adenoviral-mediated liver gene therapy. To determine whether the host adaptive immune response against the intracellular transgene human ASS (hASS) contributed to loss of gene expression in this setting, the same vector (FGV-CAG-hASS) was injected into Rag-/- (immunodeficient) mice. As in wild-type C57BL/6 (B6) mice, Rag-/- mice also showed significant loss of hASS expression and vector by week 4 post-injection, with concomitant elevation of liver enzymes and disruption of liver architecture. Therefore, direct toxicity due to vector rather than adaptive immune response against hASS primarily accounted for loss of expression with FGVs. In contrast to hASS, beta-galactosidase is strongly immunogenic and activates the host adaptive immune response. Loss of transgene expression was observed in B6 mice with either a FGV or a HDV expressing beta-galactosidase. However, the drop in gene expression observed with the HDV was primarily due to the adaptive immune response, since both beta-galactosidase expression and vector genome were sustained in immunodeficient mice treated with HDV. As expected, with weakly immunogenic hASS, vector genome and hASS expression were sustained with a HDV in spite of ubiquitous expression of the transgene. Therefore, viral gene expression is a primary determinant of intermediate and chronic toxicities at day 3 and week 4 post-injection. However, even in the absence of viral gene expression, strongly immunogenic intracellular transgenes can stimulate clearance of transduced hepatocytes.     

Biosafety of adenoviral vectors

Adenoviral vectors can efficiently transduce a broad variety of different cell types and have been used extensively in preclinical and clinical studies. However, early generation of adenoviral vectors retained residual adenoviral genes that contribute to inflammatory immune responses and toxicity. In addition, these vectors often result in transient expression of the potentially therapeutic transgene. Some clinical trials based on early generation adenoviral vectors have been discontinued because of acute inflammatory responses and toxicity and even one patient has died as a direct consequence of adenoviral toxicity. The latest generation of high-capacity adenoviral vectors is devoid of viral genes, and is having a significantly improved safety profile and yielding more prolonged transgene expression compared to early generation vectors. Nevertheless, transgene expression gradually declines even when high-capacity adenoviral vectors are used, possibly due to the gradual loss of vector genomes. Despite their improved safety, high-capacity adenoviral vectors can still trigger transient toxic effects in animals and patients. Restricting the tropism of adenoviral vectors by immunologic or genetic re-targeting may further improve their therapeutic window. The safety of adenoviral vectors has been improved further through the development of safer packaging systems that eliminate the homologous overlap between vector and helper sequences and therefore prevent formation of replication-competent adenoviruses (RCA). RCA could exacerbate inflammatory responses and act as a helper to rescue adenoviral vectors, potentially increasing the effective vector dose. Conditionally replicating adenoviruses (CRAds) have been developed for cancer gene therapy, which replicate selectively in some cancer cells. The use of CRAds in combination with chemotherapy yielded therapeutic effects in patients suffering from cancer but dose-limiting toxicity was apparent. Although there appears to be a very low theoretical risk of malignancy that is predominantly associated with the occurrence of E1-positive recombinants, no malignancies have been reported that were associated with adenoviral vectors. Nevertheless, integrating adenoviral vectors carry a greater malignancy risk due to their ability to integrate randomly into the target genomes.     

Immune responses to adeno-associated virus vectors

One of the biggest challenges in optimizing viral vectors for gene therapy relates to the immune response of the host. Adeno-associated virus (AAV) vectors are associated with low immunogenicity and toxicity, resulting in vector persistence and long-term transgene expression. The inability of AAV vectors to efficiently transduce or activate antigen presenting cells (APCs) may account for their decreased immunogenicity. AAV mediated gene therapy however, leads to the development of antibodies against the vector capsid. Anti-AAV antibodies have neutralizing effects that decrease the efficiency of in vivo gene therapy and can prevent vector re-administration. Furthermore, recent studies have shown that AAV vectors can elicit both cellular and humoral immune responses against the transgene product. Both cell-mediated response and humoral response to the delivered gene depend on a number of variables; including the nature of the transgene, the promoter used, the route and site of administration, vector dose and host factors. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. It is therefore important to understand both, why AAV vectors are able to escape immunity and the circumstances and mechanisms that lead to the induction of immune responses. This review will summarize innate and adaptive immune responses to AAV vectors, discuss possible mechanisms and outline strategies, such as capsid modifications, use of alternative serotypes, or immunosuppression, which have been used to circumvent them.     

Centrifugation enhances integrin-mediated transduction of dendritic cells by conventional and RGD-modified adenoviral vectors

The level of antigen loading can impact on the capacity for dendritic cells (DC) to activate T cell responses. Several different approaches to adenoviral (Ad)-based transduction were therefore assessed for their effect on both transgene expression and T cell activation. While a conventional E1(-)/E3Delta Ad vector (Ad/GFP) produced a concentration-dependent expression of GFP, a modified vector expressing Arginine-Glycine-Aspartic Acid (RGD) sequence on its fiber knob (Ad-RGD/GFP) enhanced transgene expression by 9-20-fold at each MOI. The addition of centrifugal force (2000xg) during DC transduction with Ad/GFP also increased expression up to 20-fold. However, combining centrifugation with the Ad-RGD/GFP vector produced no effect on transduction rate and only a 1.5- to 2-fold increase in GFP expression, suggesting overlapping mechanisms of action. Consistent with this, exogenous RGD peptide blocked transduction regardless of the vector used, or the addition of centrifugal force, and transduction was primarily limited to DC expressing the CD51 integrin receptor. Ad vectors expressing ovalbumin (OVA) were used to assess transduced DC for their capacity to activate OVA-specific T cells. We observed a significant relationship between transgene expression and the capacity for T cell activation regardless of whether transgene expression was increased by using a higher MOI, an RGD-modified vector, or by employing centrifugal force. Furthermore, combining these approaches produced synergistic effects on T cell activation. We conclude that RGD-modified vectors and centrifugation both enhance DC transduction by increasing entry via integrin receptors and that the capacity for T cell activation can be optimized by combining approaches to achieve the highest possible level of transgene expression.     

Local adenovirus-mediated CTLA4-immunoglobulin expression suppresses the immune responses to adenovirus vectors in the brain

The effect of local administration of two adenovirus vectors, one of which expressed CTLA4-immunoglobulin (AdCTLA), which blocks the B7-CD28 co-stimulatory pathway of T cell activation in the inflammatory response to adenovirus vectors was investigated. Mice injected with AdCTLA and an E1-deleted adenovirus vector that encodes the lacZ gene (AdRL) into the brain showed inflammatory cell infiltration from the early phase until day 6 after injection that was not different from that seen in control mice injected with an E1-deleted adenovirus vector containing no transgene (Ad0) and AdRL. After day 6 the inflammation in the control mice increased, peaked by day 15 and then decreased gradually but persisted until day 60. By contrast, in mice treated with AdCTLA and AdRL the inflammation, especially T cell infiltration, was suppressed after day 15. The anti-adenovirus antibody titer increased gradually until day 60 in the Ad0-AdRL control group, and whereas the mice injected with AdCTLA and AdRL showed lower anti-adenovirus antibody titers than the control group mice after day 15. Neutralizing antibody was not detected in either group. Expression of beta-galactosidase, the gene product of AdRL, at the injection site in the striatum and corpus callosum peaked on day 6 and remained until day 60 although it was very low in both groups; beta-galactosidase expression was similar in the two groups in spite of the difference in the degree and extent of the local immune response in the brain. This study demonstrated that the injection of an adenovirus vector expressing CTLA4-immunoglobulin into the brain suppressed not only local cell infiltration in the brain but also reduced the humoral immune response to adenovirus vectors.     

Immune responses to adenovirus and adeno-associated vectors used for gene therapy of brain diseases: the role of immunological synapses in understanding the cell biology of neuroimmune interactions

Researchers have conducted numerous pre-clinical and clinical gene transfer studies using recombinant viral vectors derived from a wide range of pathogenic viruses such as adenovirus, adeno-associated virus, and lentivirus. As viral vectors are derived from pathogenic viruses, they have an inherent ability to induce a vector specific immune response when used in vivo. The role of the immune response against the viral vector has been implicated in the inconsistent and unpredictable translation of pre-clinical success into therapeutic efficacy in human clinical trials using gene therapy to treat neurological disorders. Herein we thoroughly examine the effects of the innate and adaptive immune responses on therapeutic gene expression mediated by adenoviral, AAV, and lentiviral vectors systems in both pre-clinical and clinical experiments. Furthermore, the immune responses against gene therapy vectors and the resulting loss of therapeutic gene expression are examined in the context of the architecture and neuroanatomy of the brain immune system. The chapter closes with a discussion of the relationship between the elimination of transgene expression and the in vivo immunological synapses between immune cells and target virally infected brain cells. Importantly, although systemic immune responses against viral vectors injected systemically has thought to be deleterious in a number of trials, results from brain gene therapy clinical trials do not support this general conclusion suggesting brain gene therapy may be safer from an immunological standpoint.     

Advances in helper-dependent adenoviral vector research

The immunogenicity and cytotoxicity associated with early generations of adenoviral vectors provided a strong incentive for the development of helper-dependent adenovirus, a last generation of adenoviral vectors that is devoid of all viral coding sequences. These vectors have shown to mediate longer high-level transgene expression in vivo with reduced toxicity and thus offer enormous potential for human gene therapy. In addition, they possess a considerably larger cloning capacity than conventional adenoviral vectors making the transfer of large cDNAs, multiple transgenes and longer tissue-specific or regulable promoters possible. In this article, we review the progress made with helper-dependent adenoviral vectors. The development and optimization of scalable production processes and strategies for helper removal will be presented. Current chromatography options available for vector purification and the new challenges facing researchers for the separation of empty particles and/or helper viruses will be discussed. Finally, we will describe recent advances made in our understanding of their interaction with the immune system and their potential as gene delivery vehicles in vivo for the treatment of diseases affecting liver, skeletal muscle and brain.     

Immune responses against a single CD8+-T-cell epitope induced by virus vector vaccination can successfully control Trypanosoma cruzi infection

In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFkappaB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.     

Novel adenovirus-based vaccines induce broad and sustained T cell responses to HCV in man

Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor-α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.     

IL-4 and IL-2 upregulate the expression of antigen B7, the B cell counterstructure to T cell CD28: an amplification mechanism for T-B cell interactions.

We recently generated mAb 104 which is specific for the B cell activation antigen Ag B7. With this we studied the regulation of Ag B7 expression on normal tonsillar B lymphocytes as well as the activities of B7+ and B7- activated B cells. SAC and to a lesser extent anti-IgM antibody upregulated Ag B7 and this was further enhanced by IL-2 and most notably IL-4. Ag B7 was expressed on virtually all sIgG+ and sIgA+ B cells and approximately half of the sIgD+ and sIgM+ B cells. SAC-stimulated B7+ B cells proliferated and produced IgM, IgG and IgA in response to IL-2 and IgM and IgG in response to IL-4. SAC-stimulated B7- B cells proliferated and produced only IgM in response to IL-2 and IL-4. Considering that Ag B7 has recently been shown to be the counterstructure of the T cell CD28 and that CD28 triggering strongly enhances cytokine production by T cells, it is likely that the CD28/B7 interaction represents an important amplification phenomenon in T-B cell interaction leading to humoral immune responses. The preferential expression of Ag B7 on IgG and IgA committed cells suggests that CD28/B7 interaction may be more specific to secondary antibody responses provided by memory T and B cells.     

Encapsidated adenovirus minichromosomes allow delivery and expression of a 14 kb dystrophin cDNA to muscle cells

Adenovirus-mediated gene transfer to muscle is a promising technology for gene therapy of Duchenne muscular dystrophy (DMD). However, currently available recombinant adenovirus vectors have several limitations, including a limited cloning capacity of approximately 8.5 kb, and the induction of a host immune response that leads to transient gene expression of 3-4 weeks in immunocompetent animals. Gene therapy for DMD could benefit from the development of adenoviral vectors with an increased cloning capacity to accommodate a full-length (approximately 14 kb) dystrophin cDNA. This increased capacity should also accommodate gene regulatory elements to achieve expression of transduced genes in a tissue-specific manner. Additional vector modifications that eliminate adenoviral genes, expression of which is associated with development of a host immune response, might greatly increase long-term expression of virally delivered genes in vivo. We have constructed encapsidated adenovirus minichromosomes theoretically capable of delivering up to 35 kb of non-viral exogenous DNA. These minichromosomes are derived from bacterial plasmids containing two fused inverted adenovirus origins of replication embedded in a circular genome, the adenovirus packaging signals, a beta-galactosidase reporter gene and a full-length dystrophin cDNA regulated by a muscle-specific enhancer/promoter. The encapsidated minichromosomes are propagated in vitro by trans-complementation with a replication-defective (E1 + E3 deleted) helper virus. We show that the minichromosomes can be propagated to high titer (> 10(8)/ml) and purified on CsCl gradients due to their buoyancy difference relative to helper virus. These vectors are able to transduce myogenic cell cultures and express dystrophin in myotubes. These results suggest that encapsidated adenovirus minichromosomes may be useful for gene transfer to muscle and other tissues.      

大鼠CD4+CD25-T细胞的Foxp3基因转染及其表达***◆

背景：调节性T细胞在维持机体免疫应答稳态和免疫耐受方面具有非常重要的作用,但外周血中CD4+CD25+调节性T细胞含量极低,且增殖能力较差。 目的：以携带Foxp3基因的慢病毒EGFP+载体转染大鼠CD4+CD25- T细胞,观察其在大鼠CD4+CD25- T细胞中的表达。 方法：以免疫磁珠两步法分选大鼠CD4+CD25- T细胞,用携带大鼠Foxp3基因的慢病毒载体体外转染分选的细胞,以转染Foxp3基因的CD4+CD25- T细胞为实验组,EGFP空白质粒组及CD4+CD25- T细胞为阴性对照组,CD4+CD25+ T细胞为阳性对照组。荧光显微镜和RT-PCR分别从蛋白和mRNA水平检测Foxp3基因的表达。 结果与结论：成功完成了免疫磁珠的分选,获得了纯度较高的CD4+CD25-T细胞和CD4+CD25+ T细胞,细胞存活率为(94±2)%,慢病毒转染的CD4+CD25- T细胞高表达Foxp3基因。表明以携带Foxp3基因的慢病毒载体系统可有效介导Foxp3基因在大鼠CD4+CD25- T细胞中高表达。     

Requirement of CD28-CD86 co-stimulation in the interaction between antigen-primed T helper type 2 and B cells

The interaction between CD28 and its ligands, CD80 and CD86, is crucial for an optimal activation of antigen-specific T cells. However, the requirement of CD80 or CD86 co-stimulation in Th2 cell differentiation and activation is controversial. Freshly isolated murine CD4+ and CD8+ T cells were incubated with P815 transfectants expressing a similar level of either CD80 or CD86 in the presence of anti-CD3 mAb. Both CD80 and CD86 co-stimulated the proliferation of CD4+ and CD8+ T cells at comparable time-kinetics and magnitude, but CD86 alone was able to co- stimulate IL-4 and especially IL-10 production in CD4+ T cells. In typical Th2-dependent immune responses elicited by Nippostrongylus brasillensis infection, the anti-CD86 mAb treatment but not the anti- CD80 mAb treatment efficiently inhibited antigen-specific IgE and IgG1 production, which was accompanied with the reduced IL-4 production. Our results suggest that CD86 co-stimulation plays a dominant role not only in the primary activation of Th2 cells but also in the secondary interaction between antigen-primed Th2 cells and B cells.      

Current strategies and future directions for eluding adenoviral vector immunity

Adenoviral (Ad) vectors can efficiently transduce a broad range of cell types and have been used extensively in preclinical and clinical studies for gene delivery applications. The presence of preexisting Ad immunity in the majority of human population and a rapid development of immune response against the Ad vector backbone following the first inoculation with the vector have impeded clinical use of these vectors. In addition, a number of animal inoculation studies have demonstrated that high systemic doses of Ad vectors invariably lead to initiation of acute inflammatory responses. This is mainly due to activation of innate immunity by vector particles. In general, vector and innate immune responses drastically limit the vector transduction efficiency and the duration of transgene expression. In order to have a predictable response with Ad vectors for gene therapy applications, the above limitations must be overcome. Strategies that are being examined to circumvent these drawbacks of Ad vectors include immunosuppression, immunomodulation, serotype switching, use of targeted Ad vectors, microencapsulation of Ad vectors, use of helper-dependent (HD) Ad vectors, and development of nonhuman Ad vectors. Here we review the current understanding of immune responses to Ad vectors, and recent advances in the strategies for immune evasion to improve the vector transduction efficiency and the duration of transgene expression. Development of novel strategies for targeting specific cell types would further boost the utility of Ad vectors by enhancing the safety, efficacy and duration of transgene expression.