Recognition of Virus Infection and Innate Host Responses to Viral Gene Therapy Vectors

review

© The American Society of Gene & Cell Therapy

Recognition of Virus Infection and Innate Host Responses to Viral Gene Therapy Vectors Dmitry M Shayakhmetov1, Nelson C Di Paolo1 and Karen L Mossman2 Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington, USA; 2Institute for Infectious Disease Research, Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario, Canada 1

The innate immune and inflammatory response represents one of the key stumbling blocks limiting the ­efficacy of viral-based therapies. Numerous human diseases could be corrected or ameliorated if viruses were ­harnessed to safely and effectively deliver therapeutic genes to diseased cells and tissues in vivo. Recent ­studies have shown that host cells recognize viruses using an elaborate network of sensor proteins localized at the plasma membrane, in endosomes, or in the cytosol. Three classes of sensors have been implicated in sensing viruses in mammalian cells—Toll-like receptors (TLRs), retinoid acid-inducible gene (RIG)-I-like receptors (RLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs). The interaction of virus-­associated nucleic acids with these sensor molecules triggers a signaling cascade that activates the principal host defense program aimed to limit or eliminate virus infection and restore tissue homeostasis. In addition, recent data strongly ­suggest that host cells can mount innate immune responses to viruses without prior ­recognition of their nucleic acids. To deliver therapeutic genes into the nuclei of diseased cells, viral gene therapy vectors must be efficient at penetrating either the plasma or endosomal membrane. The therapeutic use of high numbers of virus particles disturbs cellular homeostasis, triggering cell damage and stress pathways, or “­sensing of modified self”. Accumulating data indicate that the sensing of modified self might ­represent a powerful framework explaining the innate immune response activation by viral gene therapy vectors. Received 1 April 2010; accepted 17 May 2010; published online 15 June 2010. doi:10.1038/mt.2010.124

Introduction Viruses are obligate intracellular pathogens that are highly ­efficient at infecting host cells that support their reproduction. Clinical studies indicate that a small number of virus particles is sufficient to initiate virus-associated disease.1 Moreover, viruses possess an array of factors and utilize strategies that allow them to evade or modulate the host immune system. These factors facilitate rapid completion of the viral reproductive cycle, contri­buting to the spread of infection. In response to the ever-present threat from viruses, hosts have evolved molecular mechanisms to control virus infection and restrict damage to self. A wealth of information has recently emerged on the molecular mechanisms mediating innate immune responses to viruses in mammalian cells.2 One of the fundamental principles of virus recognition by the host innate immune system is the sensing of virus-associated nucleic acids in infected cells by specialized classes of receptors—Tolllike receptors (TLRs), retinoid acid-inducible gene-I (­RIG-I)-like ­receptors (RLRs), and nucleotide oligomerization domain (NOD)like receptors (NLRs) (Figure 1). Recent evidence also indicates that host recognition of cell damage or stress, or “virus-inflicted modified self ”, induced by virus entry into cells is likely to be the second key principle for virus sensing, which does not rely on ­pathogen-associated nucleic acid recognition.

Molecular Basis For Cellular Recognition of Virus Infection The obligate nature of viral pathogens creates a significant challenge for the host to detect the onset and continuation of virus infection. Because viruses replicate in host cells and frequently utilize host protein and nucleic acid processing machineries, self/nonself discrimination becomes complicated based solely on pathogen-specific chemical moiety recognition. However, the natural diversity of virus-associated nucleic acids makes them a legitimate target for detection by the host’s pathogensensing machineries. Indeed, although mammalian cells under normal physiologic conditions possess only a limited number of nucleic acid types, such as double-stranded (ds) DNA with methylated CpG motifs and single-stranded (ss) RNA with a cap structure at the 5′-end of the molecule, the structure of virus-associated nucleic acids is vastly diverse. Linear dsDNA, circular dsDNA, ssDNA, ssRNA, and dsRNA, all with ­myriads of unique modifications and permutations of polarity and ­replicative intermediates, are present in cells infected with various viruses. The currently accepted paradigm suggests that the recognition of virus-associated nucleic acids by host cells is a fundamental principle for sensing virus infection in ­mammalian cells (Figure 1).

Correspondence: Dmitry M Shayakhmetov, Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195-7720, USA. E-mail: [email protected] or Karen L Mossman, Institute for Infectious Disease Research, Department of Pathology & Molecular Medicine, McMaster University, Hamilton, Ontario L8N 3Z5, Canada. E-mail: [email protected]

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Innate Host Responses to Viral Gene Therapy Vectors

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Figure 1 Three classes of pattern recognition receptors implicated in viral nucleic acids recognition in mammalian cells. NLR, nucleotide oligomerization domain–like receptor; RLR, retinoid acid-inducible geneI-like receptor; TLR, Toll-like receptor.

TLR-dependent recognition of virus infection To date, TLRs are the best-studied family of receptors mediating pathogen recognition by the innate immune system. There are >10 distinct TLRs identified in mammals to date.3 In the cellular endosomal compartment virus-associated nucleic acids are recognized by TLR3, TLR7, and TLR9. TLR3 is activated by dsRNA,4 TLR7 is activated by ssRNA,5–7 and TLR9 is activated by unmethylated CpG DNA motifs.8 Although TLR3 is expressed in many cell types, TLR7 and TLR9 are predominantly expressed in plasma­ cytoid dendritic cells (pDCs).9 All TLR-initiated signaling converges to induce type I interferon (IFN-I) through the engagement of IFN regulatory factors (IRFs) and the early response inflammatory cytokine genes via activation of nuclear factor (NF)-κB10 (Figure  1). TLR3 plays a critical role in controlling the replication of murine cytomegalovirus in natural hosts and is required for induction of IFN-I in response to purified reovirus genomic dsRNA.4,11 In pDCs, vesicular stomatitis virus RNA is recognized by TLR7 upon autophagosome formation12 and IFN-I production in response to adenovirus (Ad) occurs in a TLR9-dependent manner.13,14 RLR-dependent recognition of virus infection Upon entry into host cells, many viral pathogens with lipid envelopes such as influenza and human immunodeficiency virus avoid exposure of their genomic nucleic acids to endosomal TLRs. However, activation of IFN-I in response to these viruses is afforded through a cytoplasmic detection of viral RNAs by the RLR family of receptors (Figure 1), consisting of RIG-I and melanoma differentiation-associated gene 5 (MDA5).15–17 Although RIG-I and MDA5 directly bind viral RNA via a helicase domain,18 MDA5 activates IFN-I production upon binding of long (>2 kb) dsRNA species,19 whereas RIG-I induces IFN-I upon binding of short dsRNA and 5′-triphosphate-containing ssRNA.20–22 Upon binding to viral RNA, RIG-I and MDA5 engage the adapter protein IFN-β promoter stimulator-1, also known as mitochondrial antiviral signaling protein, virus-induced signaling adapter, or caspase recruitment domain adapter inducing IFN-β.23–26 IFN-β promoter stimulator-1 recruits tumor necrosis factor (TNF) R-associated death domain protein which forms a complex with TRAF3 and receptor-interacting protein-1.27 TRAF3 mediates the Molecular Therapy vol. 18 no. 8 aug. 2010

activation of TBK1 and IKKi kinases that phosphorylate IRF3 and IRF7, leading to activation of IFN-I.28,29 RIG-I is critical for IFN-I activation in response to paramyxovirus, vesicular stomatitis virus, influenza virus, hepatitis C virus, and Japanese encephalitis virus infection.30–33 MDA5 is important for IFN-I production in response to reoviruses19 and picornaviruses, including encephalomyocarditis virus and Theiler’s virus.34 Mice that lack both MDA5 and RIG-I are highly susceptible to vesicular stomatitis virus and encephalomyocarditis virus virus infection.2,17 Recent studies provided structural evidence on how Ebolavirus antagonizes viral dsRNA recognition by cytosolic RLRs. Conserved basic residues in virus-encoded protein VP35 IFN inhibitory domain ­recognize the dsRNA backbone, whereas the dsRNA blunt ends are “end-capped” by a pocket of hydrophobic residues of VP35 that mimic RLR recognition of blunt-end dsRNA.35,36 Interactions of VP35 with genomic dsRNA lead to reduced IFN induction and, thus, contribute to immune evasion.35–37 Collectively, cytoplasmic detection of viral RNAs by RLRs is the key pathway of host innate immunity activation by viral pathogens with RNA genomes.

NLR-dependent recognition of virus infection The NLR family consists of a relatively large number of cytosolic proteins with a prototypic tripartite structure.38,39 The N-terminus of NLRs is composed of either a caspase recruitment domain or a Pyrin domain that are important for signal transduction. The central part of the NLRs is composed of a nucleotide-binding domain critical for oligomerization. The C-terminus is composed of a leucine-rich repeat domain, mediating ligand binding.40 Upon engagement of a leucine-rich repeat–specific ligand, the ­nucleotide-binding domain binds ATP, leading to NLR ­oligomerization and initiation of a signal transduction cascade via N-terminal domain binding specific adaptors, culminating in MAPK kinase and NF-κB activation. NLR oligomerization can also lead to the formation of a cytoplasmic supramolecular complex called the “inflammasome”41 that also includes the adapter protein apoptosis speck protein and inflammatory caspases such as caspase-1 (ref. 42; Figure 1). Upon inflammasome formation, pro-caspase-1 is cleaved into functionally active caspase-1, which processes pre-interleukin (IL)-1β into mature IL-1β, leading to its release from cells and activation of the IL-1R signaling pathway. There is abundant data demonstrating the critical role of NLRs in activating innate immune responses to microbial pathogens in mammalian cells.43 Recent studies indicate that the NLR family member NLRP3 (also called Cryopyrin) plays a role in sensing viral and microbial DNA in macrophages in vitro.44 Moreover, it was found that NLRP3 mediates recognition of influenza A virus infection both in vitro and in vivo.45,46 Recognition of cytoplasmic dsDNA by DAI and AIM2 Cytosolic detection of dsDNA represents an important mechanism for the detection of both viral and microbial pathogens. In spite of early evidence that IFN-I- and NF-κB-dependent inflammatory cytokines are rapidly activated in cells in vitro upon transfection with dsDNA,47 the molecular sensors of dsDNA in the cytoplasm remained unidentified until recently. Takaoka et al. showed that dsDNA activates IFN-I via a protein named DNA-dependent activator of IRFs (DAI).48 DAI, also known as Z-DNA-binding 1423

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protein ZBP-1 or DLM-1, directly binds dsDNA via its Zα and Zβ DNA-binding domains. Although DAI binds left-handed Z-form DNA with high affinity, it is also capable of binding B-form DNA, leading to the phosphorylation of IRF3.49 Inhibition of DAI using small-interfering RNA reduced IFN-I production in response to human herpes simplex virus 1 (HSV-1), suggesting its role in cytosolic sensing of DNA viruses. Recently, four groups reported the identification of protein absent in melanoma 2 (AIM2) as a specific sensor of dsDNA in the cytosol.50–53 AIM2 binds dsDNA via its C-terminal HIN-200 (hematopoietic IFN-inducible nuclear proteins with a 200–amino acid repeat) domain and this interaction leads to receptor oligo­ merization.54 The N-terminal Pyrin domain domain of AIM2 is capable of recruiting both apoptosis speck protein and caspase-1 inflammasome components and drives the activation of caspase-1, leading to IL-1β processing and release. AIM2 knockdown abrogates caspase-1 activation in response to cytoplasmic dsDNA and vaccinia virus in vitro.

Specifics of Innate Immune Responses To Viral Gene Therapy Vectors Upon interaction with cognate ligands, TLRs, NLRs, and RLRs initiate production of IFN-I and NF-κB-dependent proinflammatory cytokines and chemokines that restrict virus replication and dissemination. This host response creates a severe evolutionary pressure for the viruses to evolve effective evasion strategies and mechanisms allowing for the subversion of the host antiviral defense systems. Human hepatitis C virus encodes protein NS3/4A that cleaves IFN-β promoter stimulator-1 from the mitochondrial membrane and prevents IFN-I activation upon virus infection.55,56 The packaging of viral genomic nucleic acids in association with virus-encoded DNA- or RNA-binding proteins shields virus genomes from recognition by the host sensor proteins and may also represent a strategy to avoid virus detection in infected cells. To achieve the goal of correcting human diseases, viral gene therapy vectors must be able to safely and effectively deliver therapeutic genes into the nuclei of diseased cells. However, due to length constrains imposed on viral vector genomes by the physical size of the virus particle, the incorporation of therapeutic genes under the control of all the necessary regulatory elements often requires the deletion of viral genes that counteract host innate and adaptive immunity but are dispensable for vector production. Inevitably, this makes viral vectors more prone to induce strong innate and/or adaptive immune host antiviral responses. Moreover, it is important to emphasize that for majority of gene therapy applications, viral vectors are introduced into tissues and cell types that they would not normally access upon natural viral infection. Another important factor that plays a role in activating potent innate immune and inflammatory responses to viral gene therapy vectors is drastically higher doses of virus particles that are ­delivered in vivo for therapeutic gene transfer, compared to doses of the virus that initiate natural infection. Indeed, although a handful of wild-type Ad particles are sufficient to induce a respiratory disease in humans, in gene therapy clinical trials Ad vectors may be delivered at a single bolus dose in excess of 10 × 1012 virus particles.57 Therefore, it is not surprising that the exposure of host cells to such a massive number of virus particles triggers 1424

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the innate immune and inflammatory responses that are vastly distinct in magnitude and severity compared to those observed upon natural virus infection. One of the currently emerging concepts implies that due to the high vector doses that are needed to achieve therapeutic gene transfer and/or transgene expression, the activation of innate immunity to gene therapy vectors occurs due to the engagement of molecular mechanisms that are naturally responsible for the detection of host cell damage or stress. Indeed, accumulating evidence with gene therapy vectors suggests that incoming virus particles initiate innate immune responses ­independent of the classic nucleic acid sensing pathways.

Induction of Innate Immune Pathways By Enveloped Virus Vectors A consistent theme that emerges following studies of innate immune responses to enveloped virus vectors is the production of type I IFN. Although the effects of IFNs are mediated by IFNstimulated genes (ISGs), a subset of ISGs can be induced independent of IFN-I production, based on direct IRF3 promoter binding.58,59 Enveloped virus particles from a broad range of virus families induce a subset of ISGs in fibroblasts in the absence of detectable levels of viral replication and IFN-I production.60 The threshold of virus entry required for IRF3 activation is lower than that of NF-κB, explaining the induction of ISGs in the absence of IFN-I production.61 The rapid production of IFN-I following administration of enveloped virus gene therapy ­vectors likely reflects the high number of particles administered. Although initial studies using purified viral glycoproteins suggested that engagement of cell surface receptors is sufficient to induce ISGs,62,63 in response to intact virus particles, penetration of the physical virus particle into the cell is required.64,65 Although TLRs and RLRs are dispensable for mediating the antiviral state following enveloped virus particle entry, IRF3 is critical.60,61,66 Similar findings were reported following trypanosome infection of macro­phages and fibroblasts.67 Collectively, these findings suggest that the entry event activates IRF3 and elicits an innate antiviral response. Indeed, membrane fusion is sufficient to elicit IFN-I and ISG induction.68 In contrast, recognition of envelope glycoproteins by TLRs and entry of nonenveloped virus particles induce a ­proinflammatory cytokine response.69

HSV vectors HSV is a widespread human pathogen that typically causes lesions on oral and genital mucosal surfaces. HSV vectors ­demonstrate extensive host cell range, high efficiency gene transfer, and enhanced safety, as persistence of the genome as an episome decreases the likelihood of insertional mutagenesis.70 In particular, the tropism of HSV for neurons makes it an ideal vector system to target diseases of the central nervous system. Due to toxicity associated with viral gene expression,71,72 nonreplicating and amplicon vectors have been developed. HSV amplicons are eukaryotic expression vectors that harbor the HSV origin of replication and cleavage/packaging signals, along with up to 130 kbp of desired DNA sequences. Although first-generation amplicons required helper virus for their production, helper-free systems were rapidly developed as contaminating helper virus induced considerable inflammation in vivo.70 www.moleculartherapy.org vol. 18 no. 8 aug. 2010

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Stereotactic injection of a helper virus-packaged HSV ­amplicon vector into the mouse striatum resulted in enhanced intercellular adhesion molecule-1 staining of endothelial cells and microglia at day 1.73 By day 5, intercellular adhesion molecule-1 levels remained elevated, indicative of a sustained inflammatory response. Indeed, increases in IL-1β, MCP-1, TNFα, interferon-inducible protein (IP)-10 and IFNγ were also observed at day 5. Consistent with these findings, neutrophil infiltration was observed at both time points, along with a large number of major histocompatibility complex class II+ cells at day 5. When a similar amplicon produced in a ­helper-free virus system, only transient induction of inter­ cellular adhesion molecule-1, IL-1β, MCP-1, TNFα and IP-10 was observed, and by day 5, there was a qualitative decrease in infiltrating neutrophils and a complete lack of major ­histocompatibility complex class II+ cells. In a similar study using a different ­helper-free HSV amplicon vector, transient induction of IFN-I was observed, along with the transient induction of TNFα, IP-10, and protein kinase R (PKR).74 In this study, however, levels of IFNγ and other chemokines remained elevated 6 days postinjection. When this same vector was injected into the tail vein of mice, a rapid and transient induction of IFN-I, cytokines, and chemokines was observed within the liver.75 Expression of these immune factors was dependent on the transcription factor STAT1, as limited induction of cytokines and chemokines was observed in the livers of STAT1 null mice. More recently, whole genome microarray analysis was performed following stereotactic injection of mice with a replicationdefective HSV vector.76 Half of the differentially regulated genes were immune response genes, with the greatest increase observed in the antigen presentation pathway. Increases were also observed in IFN pathway molecules (STAT1, STAT2, IFNβ, PKR, TLR3 and ISG9) and, to a lesser degree, chemokine and cytokine molecules. Collectively, these observations suggest that in the absence of virus gene expression, HSV gene therapy vectors induce a detectable, yet restricted, host innate immune response. This innate response ­limits transcription of the vector-encoded therapeutic gene, resulting in ­transient transgene expression in vivo.75

Baculovirus and lentivirus vectors Gene therapy vectors based on baculoviruses and lentiviruses are also being developed for therapeutic use. Despite an ­inability to replicate in mammalian cells, promoter elements of baculo­ viruses are functional. When injected into mice, baculovirus vectors induce a rapid and transient induction of both IFNα and IFNβ, which is largely responsible for the subsequent induction of IL-6, IFNγ, MIG, and IP-10 and activation of dendritic cells.77 Microarray analysis following baculovirus vector injection into the striatum of rats revealed an induction of genes within TLR, cytokine, IFN, and antigen-processing pathways.78 A similar induction of IFN-I was observed in mice following intravenous administration of a lentivirus vector.79 Within 4 hours of infection, IFN-I was detected within the serum, whereas the IFN-induced protein OAS, TNFα, and IL-1 were detected within the liver and spleen. Plasmacytoid DCs were found to be the predominant mediators of IFN-I production. Of interest, IFN-I production was not mediated through a specific pseudotyped envelope, but was dependent on cell entry, as a “bald” vector, which was not ­pseudotyped with a viral ­envelope, failed to induce IFN-I. Molecular Therapy vol. 18 no. 8 aug. 2010

Innate Host Responses to Viral Gene Therapy Vectors

Induction of the Innate Responses By Nonenveloped Virus Vectors Ad vectors Ad vectors are the most frequently used vectors in clinical ­trials worldwide.80 Even though natural infections with Ads are largely harmless in humans, intravenous Ad vector delivery for gene transfer purposes, especially at high doses, stimulates strong innate and adaptive immune responses and can be fatal for the host.57,81–84 Upon systemic Ad administration in rodents, rhesus monkeys, and humans, a rapid liver-mediated vector removal from the circulation was observed.84–90 This removal of Ad from the bloodstream temporally coincides with the induction of transcription and release into the blood of proinflammatory cyto­ kines and chemokines, including IFN-I, IL-6, TNF-α, RANTES, IP-10, IL-8, MIP-1α, MIP-1β, and MIP-2.91–97 Macrophages throughout the body, including tissue residential macrophages (e.g., Kupffer cells in the liver), and dendritic cells are the principal source of these cytokines and chemokines.98 In human gene therapy ­trials, serum levels of IL-6, IL-10, and IL-1 were elevated after intravenous Ad injection at high doses (2 × 1012–6 × 1013 virus particles).99–102 Histological evaluation of tissues, including lung, liver, and spleen, revealed areas of leukocyte and neutrophil infiltration and local necrosis,94,103 indicating that most, if not all, tissues in the body respond to intravenous Ad injection in a proinflammatory manner. Using microarray technology, it was demonstrated that Ad entry into cells deregulates expression of up to 15% of all mRNA transcripts in mouse liver tissue or in human epithelial cells.104,105 In mouse and nonhuman primate preclinical animal models, Wilson et al. showed activation of innate responses by transcriptionally defective Ad particles,92,93 indicating that the induction of innate immune and inflammatory responses occurs shortly after intravenous Ad vector delivery, but before initiation of viral gene expression. In later studies, a severe acute inflammatory response was also observed in a nonhuman primate model after the intravenous injection of a helper-dependent Ad vector that lacked all viral genes.57 These data provide clear evidence that the dose-­dependent activation of innate immune and inflammatory responses to Ad occurs primarily due to Ad particle interaction with host cells and does not require viral gene expression. The involvement of TLRs in the induction of anti-Ad host responses was analyzed in wild-type mice and mice deficient for MyD88. These studies showed that IFN-I secretion is activated in response to Ad infection in a MyD88-dependent and MyD88independent manner. Importantly, although pDCs produced IFN-I in a TLR9- and MyD88-dependent manner, conventional DCs and macrophages produced IFN-I via a MyD88-independent pathway.13,14,106 In a very thorough study, Nociari et al. provide clear evidence that IFN-I production in macrophages after Ad infection occurs in a MyD88-independent manner. Based on this data, the authors proposed the hypothesis that Ad genomic dsDNA is likely to trigger the activation of an as yet unidentified sensor of nucleic acids in the cytosol.107 Furthermore, this group provided compelling evidence that the Ad genomic dsDNA can indeed induce phosphorylation of IRF3. Recent studies by Fejer et al. showed that pDCs are the principal source of IFN-I in vivo, which is activated 2–4  hours after Ad administration.108 Using mice deficient for 1425

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Plasma membrane fusion, forced outside-in integrin signaling

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Figure 2 The viral gene therapy vectors trigger host innate immune responses through conventional molecular mechanisms implicated in sensing the virus-associated nucleic acids and through a new class of sensors activated by “modified self”. Ad, adenovirus; eTLR, endosomal compartment-localized TLRs; HSV, herpes simplex virus; IFN, interferon; IL, interleukin; NLR, nucleotide oligomerization domain–like receptor; pTLR, plasma membrane–localized TLRs; RLR, retinoid acid-inducible gene-I-like receptor; TLR, Toll-like receptor; TNF, tumor necrosis factor.

transcription factors IRF3 and IRF7, these authors defined IRF7 as a critical mediator of Ad-induced IFN-I activation in vivo. Using a set of mice deficient for critical mediators of innate immunity and inflammation, Di Paolo et al. showed that ­macrophage-derived IL-1α is the principal activator of the innate immune response to Ad in vivo.109 Activation of IL-1α did not require MyD88-, TRIF-, or TRAF6-signaling and occurred in mice deficient for IL-1β, IFN-IR or the inflammasome components caspase-1, apoptosis speck protein, or NLRP3. Furthermore, this study also showed that the IL-1α-mediated response critically depends on Ad RGD motif-mediated binding to macrophage β3 integrins, which occurs before the internalization of the virus into the cell.110,111 Although a ts1 mutant Ad, deficient at the endosome rupture step of infection, induced both the transcription and synthesis of IL-1α, it failed to trigger a full-scale activation of proinflammatory chemokines, suggesting that Ad-mediated cell damage is an important factor in triggering activation of the innate immune response to Ad in vivo. Based on this study one can also propose an intriguing speculation that forced outside–in integrin signaling might be recognized by the host cell as a universal ­danger signal, leading to the activation of proinflammatory cytokine and chemokine genes. Because many viral pathogens, including human immunodeficiency virus, herpesvirus, rotavirus, reovirus, foot-and-mouth disease virus, and adeno-associated virus (AAV) utilize integrins to gain entry into host cells, it remains to be shown whether integrin clustering induced by these virus pathogens is sufficient to trigger the activation of innate antiviral immunity.

Innate responses to AAV vectors Through a series of preclinical studies in animal models and human gene therapy trials it has been demonstrated that viral vectors based on AAV are relatively safe when delivered via an intravascular route and do not induce severe innate immune and inflammatory responses.112,113 However, the host innate immune system plays a key role in shaping the outcome of therapeutic gene delivery using AAV-based vector systems.114 Earlier ­studies clearly show that when injected intravenously into mice at high doses, AAV-based vectors induce transcriptional activation of inflammatory cytokine and chemokine genes, including TNF-α, RANTES, MIP-1β, MIP-2, MCP-1, and IP-10. Moreover, 1426

the absolute amounts of these cytokines and chemokines in the liver 1 hour after virus injection were comparable for AAV-based ­vectors and vectors based on Ad.115 Although the expression of these proinflammatory genes greatly receded by 6 hours after AAV (but not Ad) injection, these data indicate that the AAV cell entry process and/or early interactions with host cells triggers the activation of signaling pathways that initiate a stereotypic proinflammatory host response. Furthermore, the same study showed that Kupffer cells were primarily responsible for the activation of these proinflammatory cytokine and chemokine genes in the liver after ­intravenous AAV injection.115 Because adaptive immunity represents a principal barrier for successful gene transfer using AAV-based vectors,114 significant effort was made to decipher the molecular details of AAV interaction with professional antigen-presenting cells, specifically DCs. In a recent and elegant study, Zhu et al. show that AAV is recognized by pDCs via TLR9- and MyD88-dependent pathways.116 It is noteworthy that in this specialized cell type, the TLR-MyD88 pathway is coupled to IRF7 activation that triggers IFN-I production. Zhu et al. further showed that AAV-mediated induction of IFN-I in pDCs is completely dependent on TLR9 and MyD88 in vitro. Moreover, they showed that the TLR9-MyD88 pathway is critical for the activation of CD8+ T-cell responses to and neutralizing antibodies against both the transgene product and the AAV capsid in vivo.116 Collectively, these data are fundamental for our understanding of AAV-based recognition by the host cells in vivo and for the development of approaches to modulate the innate immune system to reduce vector recognition and improve therapeutic transgene expression in the target cells.

Conclusions The wealth of new data on cellular sensors of viral infection that emerged over recent years created an opportunity for the development of conceptual frameworks to explain the recognition of virus infection in mammalian cells. One of the fundamental principles of self/nonself discrimination by the innate immune system in the context of virus infection appears to rely on recognition of virusassociated nucleic acids by the specialized families of receptors (TLRs, RLRs, and NLRs). Although TLRs function at the plasma membrane and within cellular endosomal compartments, RLR and NLRs function within the cytosol. Although the engagement of TLRs and RLRs triggers the activation of IFN-I and NF-κBdependent proinflammatory cytokines and chemokines, NLRs are critical for the processing of inflammatory caspases, e.g. caspase-1, and inflammasome-dependent cytokines IL-1β, IL-18, and IL-33. The activation of innate immune and inflammatory responses to viral gene therapy vectors also occurs via the engagement of TLRs, RLRs, and NLRs. However, due to high vector doses ­frequently used in gene transfer protocols, host cells may also ­recognize viral vectors via molecular mechanisms tailored to sense cell damage or stress, or “modified self ” (Figure 2). Indeed, the mere fusion of viral envelop with plasma membrane (for enveloped virus vectors) may lead to the activation of IFN-I; forced outside–in integrin signaling for viruses utilizing integrins for internalization can trigger the activation of proinflammatory cytokine expression and synthesis; the endosome rupture step of virus infection can initiate signaling triggering cell death; the www.moleculartherapy.org vol. 18 no. 8 aug. 2010

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exposure of vector genomic DNA or RNA to cytosolic sensors can activate host antiviral responses; and finally, the translocation of vector DNA into the nucleus can trigger a DNA damage response. The host innate immune responses initiated at all of these steps of virus cell entry are critical for the efficacy of, and create a unique safety profile for, any given virus-based gene therapy vector. Future studies should focus on unraveling the cell type–specific signaling pathway in vivo that ultimately shape the multifaceted innate immune and inflammatory responses observed in preclinical studies and human gene therapy clinical trials. From these studies, one should be able to define the underlying principles on how early virus–host cell interactions lead to clinical signs of gene therapy vector-induced systemic toxicity. This information will be critical for the development of new approaches to selectively ­modulate these responses and ultimately to improve the safety of viral vector–based gene transfer for the therapy of numerous genetic and acquired human diseases. ACKNOWLEDGMENTS This work was supported by funding from the US National Institutes of Health grants AI065429 and CA141439 (D.M.S.), a Grand Challenges Explorations grant from the Bill and Melinda Gates Foundation (D.M.S.) and the Canadian Institutes for Health Research (K.L.M.).

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