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Project 8
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Project 8

Hildegard Büning

Inhibition of AAV infection by intracellular defense mechanisms in liver tissue

Clinic I for Internal Medicine, University of Cologne

Publications

Brief description in German:
Hepatozyten werden von rAAV2 und rAAV8 infiziert, ohne dass NFkappaB aktiviert wird, während die Interaktion mit humanen nicht-parenchymalen Zellen zur Induktion zellautonomer Immunreaktionen führt. In murinen nicht-parenchymalen Zellen wurde dagegen nur rAAV2 nachgewiesen. Nach einer HSPG-vermittelten Aufnahme akkumulierte dieser Serotyp in Abwesenheit von Serum in LSEC, während noch nicht identifizierte Serumkomponenten die intrazelluläre Degradation induzierten. Durch unsere Forschungsarbeit wollen wir nun den Mechanismus der intrazellulären Erkennung von AAV in nicht-parenchymalen Zellen aufgeklären und die Rolle der LSEC in der Auslösung von anti-AAV2 Immunantworten bestimmen.

Summary
rAAV2 and rAAV8 successfully infected human hepatocytes in the absence of NFkappaB activation, but induced a cell autonomous immune response in human non-parenchymal cells. In murine non-parenchymal cells only rAAV2 accumulated. Internalization and accumulation of rAAV2 in LSEC relied on AAV2’s ability to bind to HSPG, while yet unknown serum components mediated rAAV2’s degradation by this non-parenchymal cell type. We will now characterize the mechanism of AAV’s intracellular recognition in non-parenchymal cells and will determine the function of LSEC in the induction of anti-AAV2 host immune responses.

Host immune responses to adeno-associated viruses (AAV) are insufficiently characterized and pose a continued challenges for the clinical application of AAV-based vectors (rAAV) (Mingozzi and High, 2007; Mingozzi et al., 2009; Vandenberghe and Wilson, 2007). A main target for rAAV-mediated gene delivery is the liver, which is simultaneously an integral and unique component of the immune system.

We demonstrated that human non-parenchymal cells in contrast to hepatocytes activate NFkappaB and express cytokines upon interaction with rAAV of the serotype 2 and the serotype 8, which are used (rAAV2) or being developed (rAAV8) for liver-directed human gene therapy. Innate immune activation was transient and required high AAV particle numbers. Furthermore, compared to E. coli-mediated responses, the level of cell autonomous immune activation was low. Liver sinusoid endothelial cells (LSEC) can be activated by CpG oligonucleotides allowing to assume that TLR9 is a pattern recognition receptor (PRR) of this non-parenchymal cell type (Broxtermann and Protzer, unpublished). This PRR has been recently identified as sensor for rAAV2 in pDCs (Zhu et al., 2009). We confirmed this result for rAAV2 and demonstrated – in the HEK293-TLR9 assay system – that also rAAV8 is recognized by TLR9.

We will now characterize the function of TLR9 in the induction of anti-AAV immune responses in non-parenchymal cells. Furthermore, we will investigate whether the inflammasome system and/or the RIG-I system is capable of sensing rAAV infections. For both investigations the here characterized endosomal escape mutant rAAV2-76HD/AN (Stahnke et al., submitted) and its AAV8 analog, rAAV8-HD/AN, will be valuable tools.

In contrast to human non-parenchymal cells, which recognized rAAV2 and rAAV8, neither in murine Kupffer cells nor in murine LSEC significant amounts of rAAV8 were detectable, while rAAV2 accumulated with time. Accumulation of rAAV2 in LSEC relied on the ability to bind to heparan sulfate proteoglycan. In the presence of serum, however, rAAV2 was degraded. In line, intravenously injected rAAV2 as opposed to rAAV8 was efficiently degraded in murine livers. We now aim to identify the mechanism leading to rAAV2’s degradation in murine LSEC and to characterize the function of LSEC in host anti-AAV2 innate immune responses. For the latter purpose, we will determine cell autonomous immune responses elicit upon AAV2’s uptake into LSEC.

Furthermore, we will select by in vivo AAV peptide display for AAV2-based capsid variants which escape internalization by LSEC and efficiently transduce hepatocytes, and we will subsequently determine the consequences of this altered interaction with liver parenchymal and non-parenchymal cells for the induction of anti-AAV2 immune responses.

 

List of publications resulting from the project

Peer-reviewed publications:
Original work

Neerincx A, Jakobshagen K, Utermöhlen O, Büning H, Steimle V, Kufer TA. The N-terminal domain of NLRC5 confers transcriptional activity for MHC class I and II gene expression. J Immunol. 2014 193(6):3090-100

Hösel M, Lucifora J, Michler T, Holz G, Gruffaz M, Stahnke S, Zoulim F, Durantel D, Heikenwalder M, Nierhoff D, Millet R, Salvetti A, Protzer U, Büning H. Hepatitis B virus infection enhances susceptibility toward adeno-associated viral vector transduction in vitro and in vivo. Hepatology. 2014 59(6):2110-20.

Boucas, J., Lux, K., Huber, A., Schievenbusch, S., John von Feyend, M., Perabo, L., Quadt-Humme, S., Odenthal, M., Hallek, M., and Büning, H. (2009). Engineering adeno-associated virus serotype 2 based targeting vectors using a new insertion site – position 453 – and single point mutations. J. Gene Med. 11, 1103-13.

Hösel, M., Quasdorff, M., Wiegmann, K., Webb, D., Zedler, U., Broxtermann, M., Tedjokusumo, R., Esser, K., Arzberger, S., Kirschning, C. J., Langenkamp, A., Falk, C., Büning, H., Rose-John, S., and Protzer, U. (2009). Not interferon, but IL-6 controls early gene expression in Hepatitis B virus (HBV) infection. Hepatology 50, 1773-82.

Reviews
Büning H, Huber A, Zhang L, Meumann N, Hacker U. Engineering the AAV capsid to optimize vector-host-interactions. Curr Opin Pharmacol. 2015; 24:94-104

Mingozzi F, Büning H. Adeno-Associated Viral Vectors at the Frontier between Tolerance and Immunity. Front Immunol. 2015; 6:120

Büning, H., Perabo, L., Coutelle, O., Quadt-Humme, S., and Hallek, M. (2008). Recent developments in adeno-associated virus vector technology J. Gene Med. 10, 717–733.

Perabo, L., Huber, A., Märsch, S., Hallek, M., and Büning, H. (2008). Artificial Evolution with Adeno-Associated Viral libraries. Comb Chem High Throughput Screen. 11, 118-26.

Non peer-reviewed publications:

Perabo, L., Boucas, J., Coutelle, O., Hallek, M., and Büning, H. (2009). Engineering of viral capsids to evade the host immune system. In Gene Therapy Immunology (Editor: Roland W. Herzog). Wiley-Blackwell (ISBN 978-0-470-13406-1)

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