Sigrid Espenlaub

Hepatic activation of IKK/NFκB signaling induces liver fibrosis via macrophage-mediated chronic inflammation.

Liver damage in humans is induced by various insults including alcohol abuse, hepatitis B/C virus infection, autoimmune or metabolic disorders and, when persistent, leads to development of liver fibrosis. Because the nuclear factor‐κB (NF‐κB) system is activated in response to several of these stresses, we hypothesized that NF‐κB activation in hepatocytes may contribute to fibrosis development. To activate the NF‐κB signaling pathway in a time‐ and cell‐type‐specific manner in the liver, we crossed transgenic mice carrying the tetracycline‐responsive transactivator under the control of the liver activator protein promotor with transgenic mice carrying a constitutively active form of the Ikbkb gene (IKK2 protein [CAIKK2]). Double‐transgenic mice displayed doxycycline‐regulated CAIKK2 expression in hepatocytes. Removal of doxycycline at birth led to activation of NF‐κB signaling, moderate liver damage, recruitment of inflammatory cells, hepatocyte proliferation, and ultimately to spontaneous liver fibrosis development. Microarray analysis revealed prominent up‐regulation of chemokines and chemokine receptors and this induction was rapidly reversed after switching off the CAIKK2 expression. Turning off the transgene expression for 3 weeks reversed stellate cell activation but did not diminish liver fibrosis. The elimination of macrophages by clodronate‐liposomes attenuated NF‐κB‐induced liver fibrosis in a liver‐injury‐independent manner. Conclusion: Our results revealed that hepatic activation of IKK/NF‐κB is sufficient to induce liver fibrosis by way of macrophage‐mediated chronic inflammation. Therefore, agents controlling the hepatic NF‐κB system represent attractive therapeutic tools to prevent fibrosis development in multiple chronic liver diseases. (HEPATOLOGY 2012;56:1117–1128)

Adenoviral vectors coated with PAMAM dendrimer conjugates allow CAR independent virus uptake and targeting to the EGF receptor.

Adenovirus type 5 (Ad) is an efficient gene vector with high gene transduction potential, but its efficiency depends on its native cell receptors coxsackie- and adenovirus receptor (CAR) for cell attachment and α(v)β(3/5) integrins for internalization. To enable transduction of CAR negative cancer cell lines, we have coated the negatively charged Ad by noncovalent charge interaction with cationic PAMAM (polyamidoamine) dendrimers. The specificity for tumor cell infection was increased by targeting the coated Ad to the epidermal growth factor receptor using the peptide ligand GE11, which was coupled to the PAMAM dendrimer via a 2 kDa PEG spacer. Particles were examined by measuring surface charge and size, the degree of coating was determined by transmission electron microscopy. The net positive charge of PAMAM coated Ad enhanced cellular binding and uptake leading to increased transduction efficiency, especially in low to medium CAR expressing cancer cell lines using enhanced green fluorescent protein or luciferase as transgene. While PAMAM coated Ad allowed for efficient internalization, coating with linear polyethylenimine induced excessive particle aggregation, elevated cellular toxicity and lowered transduction efficiency. PAMAM coating of Ad enabled successful transduction of cells in vitro even in the presence of neutralizing antibodies. Taken together, this study clearly proves noncovalent, charge-based coating of Ad vectors with ligand-equipped dendrimers as a viable strategy for efficient transduction of cells otherwise refractory to Ad infection.

Capsomer-Specific Fluorescent Labeling of Adenoviral Vector Particles Allows for Detailed Analysis of Intracellular Particle Trafficking and the Performance of Bioresponsive Bonds for Vector Capsid Modifications

Adenoviral (Ad) vectors are widely used for gene therapy approaches. Because of the high abundance of the natural adenoviral receptors (coxsackievirus-adenovirus receptor and integrins) on a wide variety of cells, numerous methods have been developed to redirect the virions to specific receptors on target cell surfaces. Importantly, an increasing number of publications have shown that the success of targeting not only depends on receptor binding and cellular uptake, but also on intracellular trafficking processes. Therefore, improved knowledge about the intracellular fate of targeted Ad vector particles is mandatory for a rational design of targeted Ad vectors. However, the technologies currently available for fluorescent labeling of Ad vectors have significant limitations: (1) at present capsids are labeled all over the particle surface, and this imposes the risk of interference with particle infectivity; (2) capsomer-specific labeling requires extensive genetic modifications and has been demonstrated only at protein IX; and (3) two-color labeling approaches are not available. Here we present a novel, robust, and straightforward labeling procedure that overcomes these limitations. It allows for specific labeling of the capsomers fiber, protein IX, or hexon and permits two-color labeling. We demonstrate the potential of this labeling technology by analyzing two different bioresponsive bonds that can be used for the attachment of shielding or targeting moieties to the capsids: disulfide and hydrazone bonds. We demonstrate that in contrast to disulfide bonds, hydrazone bonds are quickly hydrolyzed after uptake of the virions and are thus favorable for the generation of bioresponsive vectors.

Reductive amination as a strategy to reduce adenovirus vector promiscuity by chemical capsid modification with large polysaccharides.

Chemical capsid modification of adenovirus vectors with synthetic polymers has been shown to aid in overcoming typical barriers for adenovirus vector‐mediated gene transfer. Carbohydrate‐based polymers for covalent modification of adenovirus vectors have been largely neglected so far. We utilized a reductive amination strategy to generate a novel class of adenovirus‐based glycovectors with a mannan derivative.

Covalent decoration of adenovirus vector capsids with the carbohydrate epitope αGal does not improve vector immunogenicity, but allows to study the in vivo fate of adenovirus immunocomplexes

Adenovirus-based vectors are promising tools for genetic vaccination. However, several obstacles have to be overcome prior to a routine clinical application of adenovirus-based vectors as efficacious vectored vaccines. The linear trisaccharide epitope αGal (alpha-Gal) with the carbohydrate sequence galactose-α-1,3-galactosyl-β-1,4-N-acetylglucosamine has been described as a potent adjuvant for recombinant or attenuated vaccines. Humans and α-1,3-galactosyltransferase knockout mice do not express this epitope. Upon exposure of α-1,3-galactosyltransferase-deficient organisms to αGal in the environment, large amounts of circulating anti-Gal antibodies are produced consistently. Immunocomplexes formed between recombinant αGal-decorated vaccines and anti-Gal antibodies exhibit superior immunogenicity. We studied the effects of the trisaccharide epitope on CD8 T cell responses that are directed specifically to vector-encoded transgenic antigens. For that, covalently αGal-decorated adenovirus vectors were delivered to anti-Gal α-1,3-galactosyltransferase knockout mice. We generated replication-defective, E1-deleted adenovirus type 5 vectors that were decorated with αGal at the hexon hypervariable regions 1 or 5, at fiber knob, or at penton base. Surprisingly, none of the adenovirus immunocomplexes being formed from αGal-decorated adenovirus vectors and anti-Gal immunoglobulins improved the frequencies of CD8 T cell responses against the transgenic antigen ovalbumin. Humoral immunity directed to the adenovirus vector was neither increased. However, our data indicated that decoration of Ad vectors with the αGal epitope is a powerful tool to analyze the fate of adenovirus immunocomplexes in vivo.

47. A Model To Determine the In Vivo Fate of Defined Adenovirus Immune Complexes

Vectors based on Adenovirus type 5 (Ad5) are well suited for the purpose of genetic vaccination due to their high immunogenicity. However, it is known that immune complexes formed upon contact with vector-specific antibodies (Abs) can impede gene transfer and influence vector immune profiles. To model, analyze, and understand the effects of immune complex formation on in vivo biodistribution and vector immunogenicity, we established a novel model based on chemical tagging of Ad capsomers with a carbohydrate epitope. Of note, this model allows to direct antibodies to specific capsid regions and thus to generate highly defined Ad immune complexes independent of species and MHC restrictions.As an artificial immunogenic epitope, we chemically coupled alpha-Gal (Galactose-a1,3-Galactose-b1,4-N-Acetylglucosamine) to specific sites of Ad capsomers. We generated ΔE1 Ad5 vectors being alpha-Galylated at hexon HVR1, hexon HVR5, penton or fiber, and an unmodified control vector.We demonstrated that on account of the alpha-Gal epitope, anti-Gal IgG or IgM bound to selected tagged capsomers. Thus, we analyzed biodistribution and vector-induced immune responses in alpha-1,3-Galactosyltransferase knockout (a1,3-GT KO) mice, which harbor large amounts of anti-Gal Abs.First, to compare the effect of epitope tagging on vector biodistribution, we analyzed hepatic and splenic transgene expression after i.v. injection of alpha-Galylated vectors into anti-Gal-positive a1,3-GT KO mice. We observed that transgene expression from fiber-modified vectors was mildly decreased, while expression from hexon-modified vectors decreased drastically, in a way resembling neutralization. These data suggested that, in fact, our model is suitable to precisely analyze the role of capsomer-specific Ab binding on in vivo vector biodistribution and activity.Furthermore, we analyzed the effect of immune complexation on the induction of vector- and transgene product-directed immune responses after repetitive i.m. delivery of alpha-Galylated vectors into anti-Gal-positive a1,3-GT KO mice. Here, we found that the Ad immune complexes induced transgene product-directed immune responses, even upon repeated vector administration. In particular, the extents of hepatic and splenic transgene expression, and transgene-directed immune responses were inversely correlated. For hexon-modified vectors, in spite of weakest transgene expression, transgene-directed cellular immune responses were strongest.In summary, we showed that alpha-Galylation of Ad capsomers markedly influenced specific immune responses directed to the vector as well as the transgene product. Our concept shall be used as an in vivo model to characterize whether directing or diverting immune complexation to or from specific capsomer sites can improve future strategies for vaccination regimens using Ad vectors.