TLR9-independent CD8+ T cell responses in hepatic AAV gene transfer through IL-1R1-MyD88 signaling.

Upon viral infection of the liver, CD8+ T cell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+ T cells. Surprisingly, we find local initiation of a CD8+ T cell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1ß contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+ DCs, CD8+ T cells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral T cell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+ T responses against AAV capsid and transgene product.

Targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells in vivo using the engineered AVID adenovirus vector platform.

Targeted delivery and cell-type-specific expression of gene-editing proteins in various cell types in vivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of artificial vectors for intravascular delivery (AVIDs), an engineered adenovirus-based gene delivery platform that allows for highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) in vivo after intravenous vector administration. Due to a set of refined structural modifications, intravenous administration of AVIDs did not trigger cytokine storm, hepatotoxicity, or thrombocytopenia. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34+ cells, led to up to 20% transduction of CD34+CD38-CD45RA- HSPC subsets in the bone marrow. Importantly, targeted in vivo transduction of CD34+CD38-CD45RA-CD90-CD49f+ subsets, highly enriched for human hematopoietic stem cells (HSCs), reached up to 19%, which represented a 1,900-fold selectivity in gene delivery to HSC-enriched over lineage-committed CD34-negative cell populations. Because the AVID platform allows for regulated, cell-type-specific expression of gene-editing technologies as well as expression of immunomodulatory proteins to ensure persistence of corrected HSCs in vivo, the HSC-targeted AVID platform may enable development of curative therapies through in vivo gene correction in human HSCs after a single intravenous administration.

A paracrine circuit of IL-1ß/IL-1R1 between myeloid and tumor cells drives genotype-dependent glioblastoma progression.

Monocytes and monocyte-derived macrophages (MDMs) from blood circulation infiltrate glioblastoma (GBM) and promote growth. Here, we show that PDGFB-driven GBM cells induce the expression of the potent proinflammatory cytokine IL-1ß in MDM, which engages IL-1R1 in tumor cells, activates the NF-κB pathway, and subsequently leads to induction of monocyte chemoattractant proteins (MCPs). Thus, a feedforward paracrine circuit of IL-1ß/IL-1R1 between tumors and MDM creates an interdependence driving PDGFB-driven GBM progression. Genetic loss or locally antagonizing IL-1ß/IL-1R1 leads to reduced MDM infiltration, diminished tumor growth, and reduced exhausted CD8+ T cells and thereby extends the survival of tumor-bearing mice. In contrast to IL-1ß, IL-1α exhibits antitumor effects. Genetic deletion of Il1a/b is associated with decreased recruitment of lymphoid cells and loss-of-interferon signaling in various immune populations and subsets of malignant cells and is associated with decreased survival time of PDGFB-driven tumor-bearing mice. In contrast to PDGFB-driven GBM, Nf1-silenced tumors have a constitutively active NF-κB pathway, which drives the expression of MCPs to recruit monocytes into tumors. These results indicate local antagonism of IL-1ß could be considered as an effective therapy specifically for proneural GBM.

Structural Model for Factor X Inhibition of IgM and Complement-Mediated Neutralization of Adenovirus.

Adenovirus has strong therapeutic potential as an oncolytic virus and gene therapy vector. However, injecting human species C serotype 5 adenovirus, HAdv-C5, into the bloodstream leads to numerous interactions with plasma proteins that affect viral tropism and biodistribution, and can lead to potent immune responses and viral neutralization. The HAdv/factor X (FX) interaction facilitates highly efficient liver transduction and protects virus particles from complement-mediated neutralization after intravenous delivery. Ablating the FX interaction site on the HAdv-C5 capsid leaves the virus susceptible to neutralization by natural IgM followed by activation of the complement cascade and covalent binding of complement components C4b and C3b to the viral capsid. Here we present structural models for IgM and complement components C1, C4b, and C3b in complex with HAdv-C5. Molecular dynamics simulations indicate that when C3b binds near the vertex, multiple stabilizing interactions can be formed between C3b, penton base, and fiber. These interactions may stabilize the vertex region of the capsid and prevent release of the virally encoded membrane lytic factor, protein VI, which is packaged inside of the viral capsid, thus effectively neutralizing the virus. In a situation where FX and IgM are competing for binding to the capsid, IgM may not be able to form a bent conformation in which most of its Fab arms interact with the capsid. Our structural modeling of the competitive interaction of FX and IgM with HAdv-C5 allows us to propose a mechanistic model for FX inhibition of IgM-mediated virus neutralization. According to this model, although IgM may bind to the capsid, in the presence of FX it will likely retain a planar conformation and thus be unable to promote activation of the complement cascade at the virus surface.

p38MAPK guards the integrity of endosomal compartments through regulating necrotic death.

Pathogens trigger activation of sensors of the innate immune system that initiate molecular signaling enabling appropriate host defense programs. Although recognition of pathogen-specific moieties or PAMPs by specialized receptors of the immune system is well defined for a great number of pathogens, the mechanisms of sensing of pathogen-induced functional perturbations to the host cell remain poorly understood. Here we show that the disruption of endosomal compartments in macrophages by a bacterium or fully synthetic nanoparticles activates stress-response p38MAPK kinase, which triggers execution of cell death of a necrotic type. p38MAPK-mediated necrosis occurs in cells with a compound homozygous deletion of pyroptosis-inducing caspases-1 and -11, apoptotic caspase-8, and necroptosis-inducing receptor-interacting protein kinase-3 (RIPK3), indicating that all of these principal cell death mediators are dispensable for p38MAPK-induced necrosis in response to endosome rupture. p38MAPK-mediated necrosis is suppressed by the receptor-interacting protein kinase 1, RIPK1, and degradation of RIPK1 sensitizes macrophages to necrotic death. Since pathogen-induced cell death of necrotic types is implicated in host defense against infection, our results indicate that functional perturbations in host cells are sensed as a component of the innate immune system.

Cytokine Responses to Adenovirus and Adenovirus Vectors.

The expression of cytokines and chemokines in response to adenovirus infection is tightly regulated by the innate immune system. Cytokine-mediated toxicity and cytokine storm are known clinical phenomena observed following naturally disseminated adenovirus infection in immunocompromised hosts as well as when extremely high doses of adenovirus vectors are injected intravenously. This dose-dependent, cytokine-mediated toxicity compromises the safety of adenovirus-based vectors and represents a critical problem, limiting their utility for gene therapy applications and the therapy of disseminated cancer, where intravenous injection of adenovirus vectors may provide therapeutic benefits. The mechanisms triggering severe cytokine response are not sufficiently understood, prompting efforts to further investigate this phenomenon, especially in clinically relevant settings. In this review, we summarize the current knowledge on cytokine and chemokine activation in response to adenovirus- and adenovirus-based vectors and discuss the underlying mechanisms that may trigger acute cytokine storm syndrome. First, we review profiles of cytokines and chemokines that are activated in response to adenovirus infection initiated via different routes. Second, we discuss the molecular mechanisms that lead to cytokine and chemokine transcriptional activation. We further highlight how immune cell types in different organs contribute to synthesis and systemic release of cytokines and chemokines in response to adenovirus sensing. Finally, we review host factors that can limit cytokine and chemokine expression and discuss currently available and potential future interventional approaches that allow for the mitigation of the severity of the cytokine storm syndrome. Effective cytokine-targeted interventional approaches may improve the safety of systemic adenovirus delivery and thus broaden the potential clinical utility of adenovirus-based therapeutic vectors.

Adenovirus-based vaccines-a platform for pandemic preparedness against emerging viral pathogens.

Zoonotic viruses continually pose a pandemic threat. Infection of humans with viruses for which we typically have little or no prior immunity can result in epidemics with high morbidity and mortality. These epidemics can have public health and economic impact and can exacerbate civil unrest or political instability. Changes in human behavior in the past few decades-increased global travel, farming intensification, the exotic animal trade, and the impact of global warming on animal migratory patterns, habitats, and ecosystems-contribute to the increased frequency of cross-species transmission events. Investing in the pre-clinical advancement of vaccine candidates against diverse emerging viral threats is crucial for pandemic preparedness. Replication-defective adenoviral (Ad) vectors have demonstrated their utility as an outbreak-responsive vaccine platform during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Ad vectors are easy to engineer; are amenable to rapid, inexpensive manufacturing; are relatively safe and immunogenic in humans; and, importantly, do not require specialized cold-chain storage, making them an ideal platform for equitable global distribution or stockpiling. In this review, we discuss the progress in applying Ad-based vaccines against emerging viruses and summarize their global safety profile, as reflected by their widespread geographic use during the SARS-CoV-2 pandemic.

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ's effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1ß and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

Systemic cancer therapy with engineered adenovirus that evades innate immunity.

Oncolytic virus therapy is a cancer treatment modality that has the potential to improve outcomes for patients with currently incurable malignancies. Although intravascular delivery of therapeutic viruses provides access to disseminated tumors, this delivery route exposes the virus to opsonizing and inactivating factors in the blood, which limit the effective therapeutic virus dose and contribute to activation of systemic toxicities. When human species C adenovirus HAdv-C5 is delivered intravenously, natural immunoglobulin M (IgM) antibodies and coagulation factor X rapidly opsonize HAdv-C5, leading to virus sequestration in tissue macrophages and promoting infection of liver cells, triggering hepatotoxicity. Here, we showed that natural IgM antibody binds to the hypervariable region 1 (HVR1) of the main HAdv-C5 capsid protein hexon. Using compound targeted mutagenesis of hexon HVR1 loop and other functional sites that mediate virus-host interactions, we engineered and obtained a high-resolution cryo-electron microscopy structure of an adenovirus vector, Ad5-3M, which resisted inactivation by blood factors, avoided sequestration in liver macrophages, and failed to trigger hepatotoxicity after intravenous delivery. Systemic delivery of Ad5-3M to mice with localized or disseminated lung cancer led to viral replication in tumor cells, suppression of tumor growth, and prolonged survival. Thus, compound targeted mutagenesis of functional sites in the virus capsid represents a generalizable approach to tailor virus interactions with the humoral and cellular arms of the immune system, enabling generation of "designer" viruses with improved therapeutic properties.

Innate immunity to adenovirus: lessons from mice.

Adenovirus is a highly evolutionary successful pathogen, as it is widely prevalent across the animal kingdom, infecting hosts ranging from lizards and frogs to dolphins, birds, and humans. Although natural adenovirus infections in humans rarely cause severe pathology, intravenous injection of high doses of adenovirus-based vectors triggers rapid activation of the innate immune system, leading to cytokine storm syndrome, disseminated intravascular coagulation, thrombocytopenia, and hepatotoxicity, which individually or in combination may cause morbidity and mortality. Much of the information on exactly how adenovirus activates the innate immune system has been gathered from mouse experimental systems. Intravenous administration of adenovirus to mice revealed mechanistic insights into cellular and molecular components of the innate immunity that detect adenovirus particles, activate pro-inflammatory signaling pathways and cytokine production, sequester adenovirus particles from the bloodstream, and eliminate adenovirus-infected cells. Collectively, this information greatly improved our understanding of mechanisms of activation of innate immunity to adenovirus and may pave the way for designing safer adenovirus-based vectors for therapy of genetic and acquired human diseases.

Tumour-associated macrophage-derived interleukin-1 mediates glioblastoma-associated cerebral oedema.

Glioblastoma is the most common and uncompromising primary brain tumour and is characterized by a dismal prognosis despite aggressive treatment regimens. At the cellular level, these tumours are composed of a mixture of neoplastic cells and non-neoplastic cells, including tumour-associated macrophages and endothelial cells. Cerebral oedema is a near-universal occurrence in patients afflicted with glioblastoma and it is almost exclusively managed with the corticosteroid dexamethasone despite significant drawbacks associated with its use. Here, we demonstrate that dexamethasone blocks interleukin-1 production in both bone marrow-derived and brain resident macrophage populations following stimulation with lipopolysaccharide and interferon gamma. Additionally, dexamethasone is shown to inhibit downstream effectors of interleukin-1 signalling in both macrophage populations. Co-culture of bone marrow-derived macrophages with organotypic tumour slices results in an upregulation of interleukin-1 cytokines, an effect that is absent in co-cultured microglia. Genetic ablation of interleukin-1 ligands or receptor in mice bearing RCAS/tv-a-induced platelet-derived growth factor B-overexpressing glioblastoma results in reduced oedema and partial restoration of the integrity of the blood-brain barrier, respectively; similar to results obtained with vascular endothelial growth factor neutralization. We establish that tumours from dexamethasone-treated mice exhibit reduced infiltration of cells of the myeloid and lymphoid compartments, an effect that should be considered during clinical trials for immunotherapy in glioblastoma patients. Additionally, we emphasize that caution should be used when immune profiling and single-cell RNA sequencing data are interpreted from fresh glioblastoma patient samples, as nearly all patients receive dexamethasone after diagnosis. Collectively, this evidence suggests that interleukin-1 signalling inhibition and dexamethasone treatment share therapeutic efficacies and establishes interleukin-1 signalling as an attractive and specific therapeutic target for the management of glioblastoma-associated cerebral oedema.

Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways.

For decades, cationic polymer nanoparticles have been investigated for nucleic acid delivery. Despite promising in vitro transfection results, most formulations have failed to translate into the clinic due to significant in vivo toxicity - especially when delivered intravenously. To address this significant problem, we investigated the detailed mechanisms that govern the complex in vivo systemic toxicity response to common polymeric nanoparticles. We determined that the toxicity response is material dependent. For branched polyethylenimine (bPEI) nanoparticles - toxicity is a function of multiple pathophysiological responses - triggering of innate immune sensors, induction of hepatic toxicity, and significant alteration of hematological properties. In contrast, for chitosan-based nanoparticles - systemic toxicity is primarily driven through innate immune activation. We further identified that modification of primary amines to secondary and tertiary amines using the small molecule imidazole-acetic-acid (IAA) ameliorates in vivo toxicity from both nanocarriers by different, material-specific mechanisms related to Toll-like receptor 4 activation (for bPEI) and complement activation driven neutrophil infiltration (for chitosan), respectively. Our results provide a detailed roadmap for evaluating in vivo toxicity of nanocarriers and identifies potential opportunities to reduce toxicity for eventual clinical translation.

Influence of Fat on Differential Receptor Interacting Serine/Threonine Protein Kinase 1 Activity Leading to Apoptotic Cell Death in Murine Liver Ischemia Reperfusion Injury Through Caspase 8.

Current understanding is that receptor interacting serine/threonine protein kinase 1 (RIPK1) can lead to two distinct forms of cell death: RIPK3-mediated necroptosis or caspase 8 (Casp8)-mediated apoptosis. Here, we report that RIPK1 signaling is indispensable for protection from hepatocellular injury in a steatotic liver undergoing ischemia reperfusion injury (IRI) but not in the lean liver. In lean liver IRI, RIPK1-mediated cell death is operational, leading to protection in RIP1 kinase-dead knock-in (RIPK1K45A) mice and necrostatin-1s (Nec1s)-treated lean wild-type (WT) mice. However, when fed a high-fat diet (HFD), RIPK1K45A-treated and Nec1s-treated WT mice undergoing IRI demonstrate exacerbated hepatocellular injury along with decreased RIPK1 ubiquitylation. Furthermore, we demonstrate that HFD-fed RIPK3-/-/Casp8-/- mice show protection from IRI, but HFD-fed RIPK3-/-/Casp8-/+ mice do not. We also show that blockade of RIPK1 leads to increased Casp8 activity and decreases mitochondrial viability. Conclusion: Although more studies are required, we provide important proof of concept for RIPK1 inhibition leading to distinctive outcomes in lean and steatotic liver undergoing IRI. Considering the rising incidence of nonalcoholic fatty liver disease (NAFLD) in the general population, it will be imperative to address this critical difference when treating patients with RIPK1 inhibitors. This study also presents a new target for drug therapy to prevent hepatocellular injury in NAFLD.

Caspase-8 Collaborates with Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock.

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

Inflammasome components caspase-1 and adaptor protein apoptosis-associated speck-like proteins are important in resistance to Cryptosporidium parvum.

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells in the intestinal tract and cause a flu-like diarrheal illness. Innate immunity is key to limiting the expansion of parasitic stages early in infection. One mechanism in which it does this is through the generation of early cytokines, such as IL-18. The processing and secretion of mature IL-18 (and IL-1ß) is mediated by caspase-1 which is activated within an inflammasome following the engagement of inflammasome-initiating sensors. We examined how the absence of caspase-1 and caspase-11, the adapter protein Asc, and other inflammasome components affects susceptibility to cryptosporidial infection by these and other key cytokines in the gut. We found that Casp-11-/-Casp-1-/- knockout mice have increased susceptibility to Cryptosporidium parvum infection as demonstrated by the 35-fold higher oocyst production (at peak infection) compared to wild-type mice. Susceptibility correlated with a lack of IL-18 in caspase-1 and caspase1/11 knockout mice, whereas IL-18 is significantly elevated in wildtype mice. IL-1ß was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1ß knockout mice. We also show that the adapter protein Asc is important to susceptibility, and that the caspase-1 canonical inflammasome signaling pathway is the dominant pathway in C. parvum resistance.

Adenovirus sensing by the immune system.

The host immune system developed multiple ways for recognition of viral pathogens. Upon disseminated adenovirus infection, the immune system senses adenovirus invasion from the moment it enters the bloodstream. The soluble blood factors, FX, antibodies, and complement, can bind and activate plethora of host-protective immune responses. Adenovirus binding to the cellular ß3 integrin and endosomal membrane rupture trigger activation of IL-1α/IL-1R1 proinflammatory cascade leading to attraction of cytotoxic immune cells to the site of infection. Upon cell entry, adenovirus exposes its DNA genome in the cytoplasm and triggers DNA sensors signaling. Even when inside the nucleus, the specialized cellular machinery that recognizes the double-strand DNA breaks become activated and triggers viral DNA replication arrest. Thus, the host employs very diverse mechanisms to prevent viral dissemination.

Interleukin 1α and the inflammatory process.

Inflammation occurs after disruption of tissue homeostasis by cell stress, injury or infection and ultimately involves the recruitment and retention of cells of hematopoietic origin, which arrive at the affected sites to resolve damage and initiate repair. Interleukin 1α (IL-1α) and IL-1ß are equally potent inflammatory cytokines that activate the inflammatory process, and their deregulated signaling causes devastating diseases manifested by severe acute or chronic inflammation. Although much attention has been given to understanding the biogenesis of IL-1ß, the biogenesis of IL-1α and its distinctive role in the inflammatory process remain poorly defined. In this review we examine key aspects of IL-1α biology and regulation and discuss its emerging importance in the initiation and maintenance of inflammation that underlie the pathology of many human diseases.

Immune Complexes Indirectly Suppress the Generation of Th17 Responses In Vivo.

The precise context in which the innate immune system is activated plays a pivotal role in the subsequent instruction of CD4+ T helper (Th) cell responses. Th1 responses are downregulated when antigen is encountered in the presence of antigen-IgG immune complexes. To assess if Th17 responses to antigen are subject to similar influences in the presence of immune complexes we utilized an inflammatory airway disease model in which immunization of mice with Complete Freund's Adjuvant (CFA) and ovalbumin (Ova) induces a powerful Ova-specific Th1 and Th17 response. Here we show that modification of that immunization with CFA to include IgG-Ova immune complexes results in the suppression of CFA-induced Th17 responses and a concurrent enhancement of Ova-specific Th2 responses. Furthermore, we show the mechanism by which these immune complexes suppress Th17 responses is through the enhancement of IL-10 production. In addition, the generation of Th17 responses following immunization with CFA and Ova were dependent on IL-1α but independent of NLRP3 inflammasome activation. Together these data represent a novel mechanism by which the generation of Th17 responses is regulated.

Hepatocyte Heparan Sulfate Is Required for Adeno-Associated Virus 2 but Dispensable for Adenovirus 5 Liver Transduction In Vivo.

UNLABELLED: Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. IMPORTANCE: Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents.