Adaptive and Innate Immune Responses to Gene Transfer Vectors: Role of Cytokines and Chemokines in Vector Function

Gene Therapy (2003) 10, 991–998 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt REVIEW Adaptive and innate immune responses to gene transfer vectors: role of cytokines and chemokines in vector function

D Chen1, B Murphy2,3, R Sung2 and JS Bromberg1,2 1Carl C Ichan Center for Gene Therapy and Molecular Medicine, New York, NY, USA; 2Recanati/Miller Transplantation Institute, New York, NY, USA; and 3Division of Nephrology, Mount Sinai School of Medicine, New York, NY, USA

Gene Therapy (2003) 10, 991–998. doi:10.1038/sj.gt.3302031

Keywords: chemokine; cytokine; innate immunity; gene therapy

Introduction motifs. The character of the immune response depends on the type of vector, the route of administration, the The infection, transfection, or transduction of cells, parenchymal cell types involved, and the concurrent tissues, and organs by necessity involves an interaction disease state of the host. Thus, microbial pathogenesis, of gene transfer vectors or gene medicines with the when viewed from this perspective, is not simply the immune system of the recipient. In the simplest terms, immune response to foreign viral protein antigens. this immune interaction is often viewed as a variant of The section on Innate immunity of this review will viral or bacterial pathogenesis. Foreign microbial protein examine chemokines and innate immunity. Recent antigens, the components of the vectors, are taken up by evidence demonstrates powerful stereotyped responses professional or nonprofessional antigen-presenting cells of leukocytes and tissue parenchymal cells to viruses and (APCs), and the antigens presented in the context of host nucleic acids. These responses do not depend on classic major histocompatibility complex (MHC) antigens to T- or B-cell immunity. Among these responses are the CD4+ helper T cells. The activated helper T cells then production of a wide array of potent soluble mediators provide additional stimulatory signals to B cells to make termed chemokines. Other innate immune responses, antibodies, to induce CD8+ cytotoxic T cells, to activate which may lead to chemokine production, include the phagocytic macrophages, or to stimulate other CD4+ T activation of Toll receptors, activation of antigen non- cells. specific leukocytes, and induction of nucleic acid The resulting inflammatory milieu is then responsible sensitive intracellular second messenger pathways. Im- for removing free vector, cell-associated vector, vector- portantly, innate and adaptive immune responses inter- transduced cells, and transgene products. The concep- act, amplify, and regulate each other, which is the focus tual framework then for understanding these immune of the section on Interplay of innate and adaptive responses, so they can be manipulated and thereby immunity of this review. improve the performance of the vectors, has centered The section on Cytokine and chemokine regulation of around dissecting out how particular vectors interact vector function of this review will summarize specific with classical immune responses. Important components mechanisms of how cytokines and chemokines regulate of immunity are the soluble cytokines and chemokines vector function at the level of individual cells and tissues. produced by the multifarious interactions among vec- Recent reports demonstrate that it is not simply immune tors, leukocytes, and parenchymal cells. However, a clearance of vector, transgene product, or transduced study of these soluble mediators shows a far more varied cells that prevents successful gene transfer and expres- and complex series of events than those outlined above, sion; but rather more subtle effects on the pathways of and suggests not only many more levels of immune vector entry, transport, integration, and transcriptional complexity, but also opportunities to intervene in activation that determine transgene expression. Lastly, immune interactions to manipulate vector function. the review will draw conclusions about opportunities for The section on Adaptive immunity of this review will further investigation and directions for improving the focus on adaptive immune responses. Immunity is utility and efficacy of gene medicines. engendered not only in response to specific vector or transgene peptide antigens, but also in response to nonantigenic nucleic acid components, such as CpG Adaptive immunity

Correspondence: Dr JS Bromberg, Mount Sinai School of Medicine, Box Adaptive immunity to vector antigens or transgene 1104, One Gustave L Levy Place, New York, NY 10029-6574, USA product can enhance or inhibit the intended therapeutic Adaptive and innate immune responses to gene transfer vectors D Chen et al 992 effect. Antivector immunity can enhance cell killing of inoculation of plasmid DNA into muscle or skin results target tissue or enhance the effectiveness of immuniza- in the activation of plasmid-containing DCs at the tion for gene therapy for infectious diseases. On the other injection site, in draining lymph nodes, and in systemic hand, vector persistence and transgene expression are circulation.16,17 These cells function as APC, can induce limited by antivector immunity, a disadvantage in specific proliferation of CD4+ T cells and generation of transplantation or metabolic diseases. Adenovirus vec- CD8+ CTLs, and can induce both primary and secondary tors are the most immunogenic and also the most adaptive immune responses.15,18 The direct pathway extensively studied of the gene transfer vectors, from appears to be the primary mode of antigen presentation an immunologic perspective; hence most of the discus- in this model, rather than indirect presentation of soluble sion that follows refers primarily to adenovirus. antigen by untransfected DC. Administration of first-generation, E1/E3-deleted ade- DCs infected in vitro with adenovirus encoding an novirus vectors generates well-characterized adaptive antigenic peptide can induce specific CTL responses that immune responses against viral capsid proteins.1,2 The can be augmented with repeated administration of development of cellular and humoral adaptive immunity infected DC.19 In contrast to direct immunization, which appears to be the primary mechanism by which generates neutralizing antibody that may limit repeat transgene expression is extinguished; the cellular arm administration of vector, low titers of neutralizing anti- effecting viral clearance, and the humoral response body are generated with this method. Thus, the capacity preventing effective readministration of vector. Each of of DCs to function as effective APCs may potentially be these immune response is associated with the production utilized for therapeutic benefits in designing vaccines or of a large number of key cytokines that facilitate T-cell– in tumor immunization. APC interactions. Transgene expression is generally Adaptive immunity to gene therapy vectors is tradi- enhanced when reagents or knockout mice, designed to tionally thought of as being primarily because of vector inhibit adaptive immunity, are utilized.3,4 Adeno-asso- antigens and transgene product. However, immunity can ciated virus (AAV) vectors, although less efficient than also be induced by vector nucleic acids. Bacterial DNA is adenovirus vectors, incite much less of an immune a powerful stimulator of polyclonal proliferation in B response. Consequently, expression by these vectors cells and cytokine production by monocytes and lym- results in greater vector persistence and transgene phocytes.20,21 These properties are attributed to un- expression.5 methylated CpG motifs present in plasmid DNA or Adaptive immunity to adenovirus has been attributed synthetic oligonucleotides, motifs which are much less to low levels of viral gene expression, or may reflect frequent in mammalian DNA, which is not immunosti- contamination by wild-type virus. In support of viral mulatory.22 These responses are sensitive to inhibitors of gene expression as a mechanism for antivector immunity endosomal acidification, which is coupled to the genera- is a diminution of Th2 responses and neutralizing tion of intracellular reactive oxygen species and the antibodies by E4-deleted vectors,6 and the absence of degradation of IkB.23,24 Indeed, inhibition of IkB degra- late inflammation in muscle when ‘gutted’ vectors dation suppresses the immunostimulatory effects of CpG lacking all viral genes are employed.7 However, vectors DNA.25 CpG DNA also induces the activation of c-Jun N- rendered biologically inactive can generate similar CD4+ terminal kinase and p38 MAPK, which lead to activation and CD8+ lymphocyte infiltration as do untreated of the transcription factor AP-1.26,27 The actions of CpG vectors, and antivector CTL responses can be generated motifs stimulate the secretion of cytokines such as TNFa in the absence of vector transcription.8,9 In addition, and IL-6, monocyte activation, and NK cell activity.23,28,29 empty adenoviral vectors can serve as potent adjuvants, The induction of type I interferons, IFNg, IL-12, and IL- implying that immunogenicity can be independent of 18, stimulates Th1 responses and enhance the develop- vector function.10 Therefore, cellular responses may not ment of adaptive immunity.30–33 Although CpG motifs necessarily require viral gene expression. Interestingly, are generally immunostimulatory to most cell types, both cellular and humoral immunity can be dramatically evidence exists that different sequences possess different diminished by modification of the vector with capsid cellular profiles of activity.22 Efforts to minimize im- polyethylene glycol.11 munostimulation by methylation of CpG sequences are A central component of the immunogenicity of effective in reducing cytokine induction, but impair adenovirus vectors appears to be their ability to infect vector efficiency because of methylation of promoter dendritic cells (DCs), the most potent of APC. Adeno- sequences.22 virus vectors efficiently infect both mature and immature DCs without altering their maturation or function, as 12 opposed to AAV vectors, which do not transfect DCs. Innate immunity In fact, APCs infected by adenovirus can be adoptively transferred to effect immune-mediated elimination of Gene transfer and vector transduction represent an either adenovirus- or AAV-infected cells. The inability of abortive infection of the target cell. The infection triggers AAV to transfect or effectively activate APCs may the host immune response, as discussed above, and account for their decreased immunogenicity.13,14 DCs, adaptive immunity plays an important role in vector- which represent potent APCs, play a prominent role in induced inflammation and clearance. However, as the adaptive immune responses to transgenes, via direct first line of host defense, innate immunity is also vector uptake and gene expression as well as by indirect stimulated by vector transduction. The innate immune presentation of soluble antigen. Much of the transgene system is a universal and ancient form of host defense produced by transfected DC can be presented directly, mechanism. Compared to the almost limitless diversity since depletion of transfected, antigen-bearing DC in of the adaptive immune system, innate immunity is draining lymph nodes prevents immunity.15 In addition, generally considered to be comprised of a limited

Gene Therapy Adaptive and innate immune responses to gene transfer vectors D Chen et al 993 number of stereotyped responses by few cell populations Table 1 Components of Innate Immunity (eg, natural killer (NK) cells, natural killer T (NKT) cells, macrophages, granulocytes, and DCs)34 and soluble Cell components Conventional NK, NKT, macrophages, components produced by these and other cells (eg, concept granulocytes, DCs Broadened sense All infected cells complement, proinflammatory cytokines, and chemo- Soluble Cytokines IFNa/b, IFNg,TNFa, kines). These proinflammatory cytokines and chemo- components IL-6, IL-12 kines not only play a direct role in clearing pathogens or Chemokines RANTES, IP-10, reducing their spread, but also help shape the down- MIP-2, MIP-1a stream adaptive immune responses.35 Before focusing Antiviral proteins 20-50-OAS/RnaseL, further on chemokines as the response of the innate PKR, DNA-PK Receptors TLRs, cell surface immune system to vector transduction, it is important to virus receptors examine the cellular and molecular elements that result in their induction. The interaction of a vector with cell surface receptors results in the initiation of multiple signal transduction reported that PKR and RNaseL suppress gene expression cascades in the target cells. In particular, many viruses from viral- and nonviral-based vectors, and RNaseLÀ/À and vectors bind to cell surface receptors, the cross- or PKRÀ/À murine embryonic fibroblasts express en- linking of which subsequently induce NF-kB,36 ATF-2/c- hanced levels of protein from transfected genes com- Jun,37 interferon regulatory factors (IRFs),38 and MAP pared to wild-type cells.56 kinases (p38, ERK, JNK).39 These pathways ultimately DNA-dependent protein kinase (DNA-PK) is a mem- converge in the nucleus to activate a diverse set of ber of the DNA repair machinery and has a pivotal role immunoregulatory genes, among them the inflammatory in the maintenance of genome stability.57 Similar to the cytokines IFNa/b, IFNg, IL-6 and IL-12,40–42 and the dsRNA-sensitive enzyme, DNA-PK also plays direct and chemokines RANTES IP-10 and MIP-2.43–46 Thus, while indirect roles in antiviral responses. It is activated in innate immunity was previously thought to be a response to virus infection and phosphorylates IRF-3 on nonspecific immune response characterized by engulf- Thr-135,58 inducing type I IFNs. It has also been reported ment and digestion of microorganisms, the elucidation of that DNA-PK inhibits retrovirus integration59 and that the molecular pathways involved in virus–cell interac- DNA-PK associated with adenovirus E4 gene products tions demonstrates that innate immunity also has inhibits concatamer formation of the adenovirus gen- considerable receptor and ligand specificity. ome.60 Toll-like receptors (TLRs) represent another set of Since RNaseL and DNA-PK are universally present in specific receptor–ligand interactions, an aspect of the all cell types, it is likely that all cells are able to activate innate immune recognition that has received significant inflammatory cytokine production and mount responses recent attention. The Toll protein was first identified as to clear vector nucleic acids. The finding that such an essential molecule for embryonic patterning in universal and primitive responses are involved in Drosophila.47 TLRs are mammalian homologues, ex- anti-viral immunity has significantly broadened our pressed at the surface of APCs, which are rapidly definition of innate immunity and should alter activated by pathogen components, such as lipids, future approaches to modify vector structure and carbohydrates, or nucleic acids, that bind to specific delivery to improve vector performance. The various TLRs.48 Distinct TLRs are key molecules in the selective components of innate responses are summarized in recognition of different pathogens.49 For example, while Table 1. not detectable in normal cells, but frequently produced as a result of viral replication, double-stranded RNA Chemokines induced by vectors (dsRNA) can trigger innate immunity through TLR3.36 The synthesis of antiviral cytokines and chemokines, not After ligand-mediated dimerization, TLRs recruit an only provides a first line of defense against infection by adapter protein MyD88. MyD88 then assembles a generating an intracellular environment that restricts signalsome containing IRAK, TRAF6, or ECSIT,50–52 and virus replication, but also signals the presence of viral together they mediate activation of NF-kB and pathogens to the adaptive immune system. Work from MAPKs, and lead to the production of cytokines and our laboratory further showed that cytokines and chemokines. chemokines induced by innate and adaptive antiadeno- dsRNAs enter additional signaling pathways to trigger virus vector immune responses negatively regulate antiviral responses. dsRNA-dependent protein kinase R transcription from the vector transgene.61,62 Thus, identi- (PKR) is activated upon binding to dsRNA.53 PKR in turn fying the cytokines and chemokines induced by a activates NF-kB and the MAP kinase signaling pathway, particular vector may provide a reasonable strategy to which leads to IFNa/b induction.54 dsRNA also activates improve the safety and efficacy of these agents for gene the 20–50-oligoadenylate synthase (OSA)/ RNaseL path- medicines. way.55 OAS is activated upon binding to viral dsRNA Chemokines are chemotactic cytokines that signal and produces an unusual nucleotide second messenger through G-protein linked, seven transmembrane span- 20–50 oligoadenylate. These oligonucleotides then activate ning receptors. Like cytokines, which have pleiotropic RNaseL, which destroys both viral and cellular RNA.55 effects, chemokines target multiple leukocyte and par- Therefore, in addition to induction of downstream enchymal cell subsets. They are essential for the inflammatory genes, dsRNA is able to mediate a direct trafficking of DCs and lymphocytes between the sites antiviral mechanism, which includes the cleavage of of infection and lymphoid organs. They enable antigen- viral RNA and induction of apoptosis in infected cells loaded DCs to home to lymph nodes to prime naı¨ve T because of cleavage of cellular RNA. Indeed, it was and B cells, and generate adaptive immune responses.

Gene Therapy Adaptive and innate immune responses to gene transfer vectors D Chen et al 994 Table 2 Classiﬁcations of chemokines and receptors

Classiﬁed by Families Chemokine receptor Chemokines Receptor-bearing cells

Structure CC CCR1 MIP-1a, RANTES, MCP-3, -4 NK, T, imDC, Mf, Ba,, Eo, Neu CCR2 MCP-1B4 NK, T, B, Mf, Neu CCR3 Eotaxin-1, -2, MCP2-4, RANTES, T, Ba,, Eo CCR4 TARC, MDC NK, T, imDC, thymocyte CCR5 MIP-1a, MIP-1b, RANTES, MCP-2 T, B, DC, Mf, thymocyte CCR6 LARC T, B, imDC CCR7 ELC, SLC T, B, maDC CCR8 I-309 T, B, thymocyte CCR9 TECK T, thymocyte CXC CXCR1 IL-8, ENA-78 Mf, Neu CXCR2 Groa/b/g, ENA-78, NAP-2, IL-8 NK,Mf, Eo, Neu CXCR3 MIG, IP-10, I-TAC T, B CXCR4 SDF-1 T, B, DC, Mf, Neu, thymocyte CXCR5 BCA-1 T, B C XCR1 Lymphotactin NK, T CX3C CX3CR1 Fractalkine NK, T, Mf

Inﬂammatory Function CCR1 MIP-1a, RANTES, MCP-3, -4 Effector T CCR2 MCP-1, 2, 3, 4 Effector T CCR3 Eotaxin-1,-2,RANTES, MCP-2,3,4, MEC Effector T CCR5 MIP-1a, MIP-1b, RANTES, MCP-2 Effector T CCR8 I309 Effector T CXCR2 IL-8, MIP-2, ENA78 Neu, NK CXCR3 MIG, IP-10, I-TAC Effector T CX3CR1 Fractalkine Effector T

Homeostatic CCR4 MDC, TARC Effector/memory T CCR6 LARC Effector/memory T, B CCR10 CTACK, MEC Memory CXCR4 SDF-1 Naı¨ve, memory T, B, thymocyte CXCR5 BCA-1 Follicular B helper T, B CCR7 SLC, ELC Naı¨ve, central memory T, B mature medullary thymocyte CCR9 TECK Memory T, B, immature thymocyte

Roles in inﬂammation Th1 CCR2 MCP1-4 CCR5 RANTES, MIP1-a, MIP1-b, MCP-2 CXCR3 MIG, IP-10, I-TAC Th2 CCR3 Eotaxin-1, -2,MCP2-4,RANTES CCR8 I-309 Th1/Th2 CCR4 TARC,MDC

Chemokines also amplify inflammation by recruiting according to chemical structure, biological function, and immune effector cells into sites of inflammation or roles in inflammation. infection.63 Most studies investigating vector induction of chemo- Chemokines can be divided according to their mole- kines have focused on adenovirus vectors, as shown in cular structure into four subgroups (CXC, CC, C, and Table 3, because of their high transduction efficiency and CX3C). A functional chemokine classification uses common usage. Muruve and his coworkers demon- physiological features, which include conditions and strated that after systemic administration of adenovirus locations of chemokine production, as well as cellular vectors, there is an early dose-dependent inflammatory distribution of chemokine receptors, to distinguish response associated with a consistent pattern of CXC and between ‘inflammatory’ and ‘homeostatic’ chemo- CC chemokine induction in murine liver. The neutrophil kines.64,65 Inflammatory chemokines are specialized in chemoattractant MIP-2 is first upregulated, followed by the recruitment of effector cells in response to physiolo- predominantly CC chemokines such as MCP-1, gical stress. In contrast, homeostatic chemokines are RANTES, and MIP-1b, which are mainly monocyte– involved in maintaining physiological trafficking and macrophage chemoattractants. The T-cell-specific che- positioning of cells during hematopoiesis, antigen mokine most highly expressed following infection with sampling, and immune surveillance. Since chemokines adenovirus vectors was IP-10.43 Additional studies by exert their biologic function through chemokine recep- the same group showed that the early induction of IP-10 tors, their functions can be reflected by the cellular and RANTES was directly mediated through capsid- distribution of these receptors. Table 2 shows alternate dependent activation of NF-kB,44,46 while the activation classifications of chemokines and the cellular distribution of p38 MAPK and ERK signaling were involved in the of their receptors on immune cells; the classifications are early induction of only IP-10.45

Gene Therapy Adaptive and innate immune responses to gene transfer vectors D Chen et al 995 Table 3 Chemokine induction by virus vector transduction

Vectors Chemokines Organs/vector administration pathway Reference(s)

Adenovirus MCP-1 Liver/i.v., lung/intratracheal, cardiac graft/direct inj. Muruve et al43, Chen et al66, Otake et al67, Zaiss et al69 MIP-1a liver/i.v., cardiac graft/direct inj. Muruve et al43, Chen et al66, Otake et al67 MIP-1b Lung/intratracheal, cardiac graft/direct inj. Chen et al66, Otake et al67 RANTES Liver/i.v., lung/intratracheal, cardiac graft/direct inj. Muruve et al43, Chen et al66, Otake et al67, Zaiss et al69 MIP-2 Liver/i.v., lung/intratracheal, cardiac graft/direct inj. Muruve et al43, Chen et al66, Otake et al67, Zaiss et al69 IP-10 Liver/i.v., cardiac graft/direct inj. Muruve et al43, Chen et al66, Zaiss et al69 MIG Cardiac graft/direct inj. Chen et al66 AAV MCP-1 Liver/i.v. Zaiss et al69 MIP-2 Liver/i.v. Zaiss et al69 IP-10 Liver/i.v. Zaiss et al69

Recently, using a murine cardiac transplant model and activation of these cells with subsequent upregulation of a cDNA array technique, we profiled the chemokine important costimulatory molecules, such as CD80 and expression patterns in adenovirus vector-transduced or CD86, and class II MHC, thereby maximizing the untransduced grafts, and found that adenovirus vector capacity of these cells to act as professional APCs in induces a broad panel of inflammatory genes, including adaptive immunity. Vector and viral components also the CC chemokines and receptors MCP-1, MIP-1a, MIP- induce proinflammatory cytokines in APC and DC such 1b, RANTES, and CCR5; and the CXC chemokines KC, as IL-1, IL-6, and TNFa, which can further cause local MIP-2, IP-10, and MIG. Among the potent inflammatory tissue injury, either directly or indirectly because of the Th1 chemokines such as IP-10 and MIG, their induction production of nitric oxygen or reactive oxygen species by vector occurs earlier and more strongly than after intermediates. Tissue injury can result in the further local alloantigenic stimulation alone.66 Similarly, increased expression of additional chemokines and chemokine expression of MIP-2, MCP-1, and MIP-1b has been receptors, with recruitment of neutrophils and macro- demonstrated in the murine respiratory epithelium phages and the activation of local endothelium or following intratracheal administration of a first-genera- epithelium. Activation of endothelium and epithelium tion adenoviral vector.67 results in the expression of adhesion molecules and AAV vectors are being developed for human gene additional chemokines. Activated endothelium can also therapy, because of their long-term gene expression, play a pivotal role in the direct recruitment of antigen- broad host range, and decreased immunogenicity.68 A dependent and -independent inflammatory cells to the comparison study of the differential activation of the site of inflammation. Stimulation of DCs by vector and innate immune response by adenovirus versus AAV viral components also induces IL-12, driving T-cell vectors was recently reported. In vitro, AAV did not differentiation towards a Th1 phenotype. Macrophages induce the same degree of expression of various and DCs thus serve as an important link between the chemokines in two epithelial-derived cell lines.69 In vivo, innate and adaptive immune responses, by contributing AAV vectors induced only transient chemokine and to the recruitment of antigen-specific T cells, and by cytokine expression and leukocyte recruitment in the undergoing enhancement of their capacity to act as liver, and this transient induction could be abolished by professional APCs, most notably for DCs that possess the the depletion of Kupffer cells, suggesting that AAV did very strong ability to activate naı¨ve T cells. Once not activate the target cells. Thus, the impression that activated, T cells then produce cytokines that act in AAV vectors are less immunogenic than adenovirus autocrine and paracrine fashions to amplify the adaptive vectors is supported by this type of analysis of the immune response. molecular and cellular components of innate immunity. It should be clear from the discussion that interactions These approaches demonstrate that it is possible to between innate and adaptive immunity are not unidirec- determine how vectors induce immunity, what precise tional, but bidirectional with one response amplifying components of the immune response are induced by and affecting the other. It is also clear that much of the individual vectors, how these components influence communication that occurs among various cell types and vector function, and how vectors may be altered or between innate and adaptive immunity is mediated by immunity circumvented to improve the performance of soluble chemokine and cytokine molecules. As is true for gene medicines. most other signaling interactions, there is not a single, well-defined linear pathway, but rather a highly inter- Interplay of innate and adaptive immunity dependent network with multiple ramifying interactions and a large number of nodes, representing potential The innate and adaptive immune responses are not points for positive and negative feedback, regulation, mutually exclusive processes, with the innate immune and interventional strategies. response in particular serving to direct and amplify the subsequent adaptive response. Thus, various stimuli such as allergens, infectious agents, or ischemia may Cytokine and chemokine regulation of vector each trigger specific components of the innate immune function response. In the context of gene transfer, recognition of vector components or viral particles by TLRs or other Expression by gene transfer vectors is influenced by the receptors expressed on macrophages and DCs induces type of promoter directing transgene expression. Most

Gene Therapy Adaptive and innate immune responses to gene transfer vectors D Chen et al 996 common are viral promoters such as MMLV, RSV, SV40, with repetitive binding sites for NF-kB (which is or CMV, but many cellular promoters such as the b-actin transported into the nucleus) have dramatically in- promoter are also utilized. Promoter characteristics may creased uptake compared with unmodified vectors; in have dramatic influences on transgene expression. For addition, these binding sites also function as transcrip- example, the efficiency of the CMVie promoter varies tional enhancers.84 In this study, both NF-kB-mediated depending on whether the promoter is derived from functions could be regulated by agents that modulate the human or murine CMV, on the size of the vector intracellular distribution of NF-kB, thereby allowing the promoter fragment, and on the target cell type.70 The possibility of regulating vector function by externally nature of the promoter may determine interactions with modifying the cytokine environment. transcription factors and viral gene products that ultimately determine vector efficacy. For example, the RSV but not the CMV promoter can be activated by gene Conclusions and future directions products of the adenoviral E4 region.71 While the induction of cytokine and chemokines have The entry of gene transfer vectors into cells sets into far reaching effects on antivector immunity, they can also motion two broad categories of immune responses. First, affect vector function more directly. Interactions of the vectors enter both endocytic vesicles and cytoplasmic promoters with components of the immune system, such spaces and so are potentially available to enter MHC Class as cytokines and chemokines, represent an important II and Class I, respectively, antigen presentation pathways means by which transgene expression is attenuated. A and initiate classical components of microbial immunity. variety of cytokines have been demonstrated to inhibit or Second, the processes of receptor binding, internalization, augment vector promoter function at the transcriptional uncoating, cytoplasmic entry, nuclear transport, integra- level.72,73 The CMV promoter may be the most widely tion, and transcriptional activation are each capable of studied in this regard. While interferons and TNFa are activating receptors or generating second messengers that known to have a variety of antiviral activities, such as influence the expression of many gene products involved regulation of MCMV infection and inhibition of reactiva- in inflammation. Thus, the immune response to vectors is tion from latency, there is evidence that interferons inhibit not simply the response to foreign microbial antigens, but the onset of MCMVie gene transcription as well, implying is integral to the nature of gene transfer: the act of a more direct effect on promoter function.73–77 Inhibition introducing any vector or nucleic acid into a cell is capable of transgene expression by interferons at a post-transcrip- of stimulating adaptive and innate immunity. tional level has been demonstrated in HBV infection, and The adaptive immune response and the resultant in MMLV-based retroviral vectors.78,79 Promoter attenua- cytokines and chemokines produced will depend on a tion by IL-12 in vivo,mediatedbyIFNg,hasbeen number of variables. Among the most important are the demonstrated for promoters in nonviral vectors.80 CMV precise cell types(s) transduced by the vector (profes- promoter activity may also increase following adenovirus sional versus nonprofessional APC), the cell biology of vector administration, and is stimulated by the transcrip- the vector (which antigen presentation pathways it tion factor NF-kB.81 This may explain the early efficiency enters), and the nature of other ongoing inflammatory of CMV promoter-based vectors, as NF-kB activation can responses because of the disease process for which gene be stimulated by TNFa, IL-1, and IL-6, all of which are therapy is being administered (infection, cancer, genetic induced by vector administration. deficiency). Therefore, opportunities for research and Cytokines induced by immune responses to gene development of better vectors and vector performance transfer vectors may also exert competitive or synergistic may lie in preventing vector entry into antigen presenta- effects on transgene expression at the promoter level. We tion pathways, in particular, vector entry into profes- have shown that IFNg and TNFa in combination are sional APC, and modification of costimulatory and synergistic in inhibition of transgene expression by a coinflammatory signals that accompany specific disease variety of vectors and promoter types in vitro,andby entities. By their very nature, these maneuvers will likely adenovirus vectors in vivo.73 In contrast, NF-kB-dependent be highly specific for particular vectors, routes of HIV gene expression is stimulated by TNFa, but inhibited administration, and disease processes. Nonetheless, by IFNa by competitive binding of transcription factors general principles will probably emerge to deepen our induced by these cytokines to the NF-kBcoactivator understanding of the barriers that prevent successful p300.82 Whether an individual cytokine inhibits or gene transfer and also broaden the understanding of augments transgene expression may also be determined microbial pathogenesis. by vector promoter structure; for example, the deletion or Similarly, the innate immune response and the mutation of a transcription factor binding site may lead to cytokines and chemokines produced by innate immunity loss of inhibition by a given cytokine. Characterization of are integral to the nature of vector entry and transcrip- the interactions between cytokines, chemokines, and tional activation within virtually any cell type, including promoter elements may ultimately lead to the design of parenchymal cells, lymphocytes, and professional APC. gene therapy vectors with promoters that positively Research opportunities lie in elucidating the molecular interact with the milieu of the target tissue, resulting in interactions of vector components with cellular receptors augmentation of expression rather than attenuation. and second messenger pathways in cytoplasmic and Cytokines, growth factors, and their downstream nuclear domains. These investigations will determine the mediators may also affect vector replication, distribution, molecular pathways important for vector gene expres- and uptake. TNFa, IGF-1, and EGF, which trigger PI3K sion versus those that induce innate immunity. This activation, lead to adenovirus vector internalization, approach will likely improve vector efficiency, while whereas VGEF increases vector entry through effects on concomitantly inhibiting innate responses. It is also microvascular permeability.83,84 DNA vectors constructed possible that certain innate responses are inextricably

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