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.

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.

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.

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-1β 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-1β, the biogenesis of IL-1α and its distinctive role in the inflammatory process remain poorly defined.

Interdependence between Interleukin-1 and Tumor Necrosis Factor Regulates TNF-Dependent Control of Mycobacterium tuberculosis Infection.

The interleukin-1 receptor I (IL-1RI) is critical for host resistance to Mycobacterium tuberculosis (Mtb), yet the mechanisms of IL-1RI-mediated pathogen control remain unclear. Here, we show that without IL-1RI, Mtb-infected newly recruited Ly6G(hi) myeloid cells failed to upregulate tumor necrosis factor receptor I (TNF-RI) and to produce reactive oxygen species, resulting in compromised pathogen control. Furthermore, simultaneous ablation of IL-1RI and TNF-RI signaling on either stroma or hematopoietic cells led to early lethality, indicating non-redundant and synergistic roles of IL-1 and TNF in mediating macrophage-stroma cross-talk that was critical for optimal control of Mtb infection.

Recognition of human oncogenic viruses by host pattern-recognition receptors.

Human oncogenic viruses include Epstein-Barr virus, hepatitis B virus, hepatitis C virus, human papilloma virus, human T-cell lymphotropic virus, Kaposi's associated sarcoma virus, and Merkel cell polyomavirus. It would be expected that during virus-host interaction, the immune system would recognize these pathogens and eliminate them. However, through evolution, these viruses have developed a number of strategies to avoid such an outcome and successfully establish chronic infections.

IL-1α and complement cooperate in triggering local neutrophilic inflammation in response to adenovirus and eliminating virus-containing cells.

Inflammation is a highly coordinated host response to infection, injury, or cell stress. In most instances, the inflammatory response is pro-survival and is aimed at restoring physiological tissue homeostasis and eliminating invading pathogens, although exuberant inflammation can lead to tissue damage and death. Intravascular injection of adenovirus (Ad) results in virus accumulation in resident tissue macrophages that trigger activation of CXCL1 and CXCL2 chemokines via the IL-1α-IL-1RI signaling pathway.

The analysis of innate immune response to adenovirus using antibody arrays.

Even though natural infections with adenovirus (Ad) are largely harmless in humans, an intravenous Ad vector administration for gene delivery purposes, especially at high doses, stimulates strong innate and adaptive immune responses, and can be fatal to the host. In animal models, intravenous Ad administration has been shown to induce transcription and release in the serum of a great number of pro-inflammatory cytokines and chemokines. Macrophages, including tissue residential macrophages (e.

Interleukin-1 receptor 2 keeps the lid on interleukin-1α.

The molecular mechanisms that regulate functional activation of IL-1α remain elusive. In this issue of Immunity, Zheng et al. (2013) describe a molecular system implicating interleukin-1 receptor-2 (IL-1R2) as a principal cytosolic factor that controls functional IL-1α activation during necrosis.

Coagulation factor X activates innate immunity to human species C adenovirus.

Although coagulation factors play a role in host defense for "living fossils" such as horseshoe crabs, the role of the coagulation system in immunity in higher organisms remains unclear. We modeled the interface of human species C adenovirus (HAdv) interaction with coagulation factor X (FX) and introduced a mutation that abrogated formation of the HAdv-FX complex. In vivo genome-wide transcriptional profiling revealed that FX-binding-ablated virus failed to activate a distinct network of nuclear factor κB-dependent early-response genes that are activated by HAdv-FX complex downstream of TLR4/MyD88/TRIF/TRAF6 signaling.

Recognition of virus infection and innate host responses to viral gene therapy vectors.

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.

Adenovirus de-targeting from the liver.

Adenovirus (Ad) vectors have substantial potential as biological therapeutics for the treatment of human diseases. Evidence from preclinical studies and clinical trials indicated that several acquired and inherited diseases could be corrected or ameliorated with cell type-specific Ad targeting. One of the major barriers for in vivo Ad targeting is the sequestration of the blood-borne virus in the liver.

Virus binding to a plasma membrane receptor triggers interleukin-1 alpha-mediated proinflammatory macrophage response in vivo.

The recognition of viral components by host pattern-recognition receptors triggers the induction of the antiviral innate immune response. Toll-like receptor 9 (TLR9) and NLRP3 inflammasome were shown to be the principal specific sensors of viral double-stranded DNA. Here we present evidence that macrophages in vivo activated an innate immune response to a double-stranded DNA virus, adenovirus (Ad), independently of TLR9 or NLRP3 inflammasome.

Redundant and synergistic mechanisms control the sequestration of blood-born adenovirus in the liver.

Human adenovirus (Ad) is a ubiquitous pathogen causing a wide range of diseases. Although the interactions of human Ad serotype 5 (Ad5) with susceptible cells in vitro are known in great detail, host factors controlling the tissue specificity of Ad5 infection in vivo remain poorly understood. Here, we analyzed the mechanisms of sequestration by the liver for blood-born human Ads and Ad5-based vectors.

Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells.

Lymph nodes prevent the systemic dissemination of pathogens such as viruses that infect peripheral tissues after penetrating the body's surface barriers. They are also the staging ground of adaptive immune responses to pathogen-derived antigens. It is unclear how virus particles are cleared from afferent lymph and presented to cognate B cells to induce antibody responses.

Fiber shaft-chimeric adenovirus vectors lacking the KKTK motif efficiently infect liver cells in vivo.

The molecular mechanisms governing the infectivity of adenovirus (Ad) toward specific cell and tissue types in vivo remain poorly understood. The direct Ad binding to hepatic heparan sulfate proteoglycans via the KKTK motif within the fiber shaft domain was suggested to be the major mechanism of Ad liver cell infection in vivo. Here, we describe the generation and in vitro and in vivo infectivity studies of Ad5-based vectors possessing long Ad31- or Ad41-derived fiber shaft domains, which lack the KKTK motif.

The influence of blood on in vivo adenovirus bio-distribution and transduction.

Intravascular delivery of adenovirus (Ad) vectors is being developed for liver-directed gene therapy for targeting disseminated disease in cancer therapeutics and for targeting non-hepatic tissues and organs through vector engineering strategies. The utility of Ad vectors is not limited to serotype 5 (Ad5), and many alternate human serotypes and non-human serotypes of Ad are currently being investigated. Critical to intravascular delivery of Ad is the interaction of the virus with host blood cells and plasma proteins, because immediate contact is observed following injection.

Effect of adenovirus-mediated heat shock protein expression and oncolysis in combination with low-dose cyclophosphamide treatment on antitumor immune responses.

Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen-presenting cells. In this study, we tested whether adenovirus-mediated overexpression of secreted or membrane-associated forms of gp96 in tumor cells would stimulate an antitumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing s.