University of Heidelberg

RNAi - Projects

  • Title: Implementation of a novel AAV vector-based therapeutic modality for inhibition of HCV-associated miR-122
  • Summary: Hepatitis C virus (HCV) has evolved a curious mechanism of interaction with an endogenous miRNA whereby the liver-specific miR-122 binds to the HCV 5' end and thus protects the viral RNA from degradation in the infected hepatocyte. In a joint effort, two of our PhD students therefore devised and engineered a variety of constitutive or inducible AAV vector-based inhibitors of miR-122 and were able to show marked anti-HCV efficacy in replicons as well as in HCV-infected cells.
  • People: Nina Schürmann & Stefan Mockenhaupt (equal contributions), in collaboration with M. Binder & R. Bartenschlager (Dept. of Infectious Diseases / CellNetworks)
  • Title: Engineering of synthetic AAV vectors for expression of therapeutic anti-HIV or anti-HBV RNAi
  • Summary: HIV is a prototype pathogen that mutually interacts with host cell miRNAs to foster replication or maintain latency. We thus engineer synthetic T-cell-targeted AAV vectors to regulate miRNAs that are critical for HIV latency, to force HIV replication so that it can be attacked by conventional therapies and to ultimately purge viral reservoirs. In parallel, we evaluate a variety of AAV vectors expressing optimized anti-HBV shRNAs (less off-targeting and/or higher efficacy) in HBV-transgenic mice.
  • People: Stefan Mockenhaupt, Kathleen Börner, Stefanie Große, Thomas Michler (together with H.G. Kräusslich (Dept. of Infectious Diseases) & U. Protzer (TU Munich))
  • Title: Identification of intra- and extra-cellular miRNAs that associate with persistent human HIV-1 infection
  • Summary: Based on previous reports that HIV dysregulates cellular miRNAs, we conducted a comprehensive study in which we analysed potential HIV effects also on circulating miRNAs in human patients. Notably, we identified >30 miRNAs that were significantly dysregulated in HIV plasma versus healthy controls. These miRNAs may serve as future biomarkers of HIV infection and potentially also as novel anti-HIV targets. At present, we are expanding this analysis to multiple further human pathogens.
  • People: Kathleen Börner, Gabriella Cotugno (together with H.G. Kräusslich (Dept. of Infectious Diseases) & M. Hartmann (Dermatology Hospital Heidelberg)
  • Title: Molecular evolution-based dissection of the functions of human Ago1 to 4 proteins
  • Summary: Humans possess four Ago proteins amongst which Ago2 and Ago3 carry a Slicer/PIWI domain, yet only Ago2 is actually able to cleave a bound target mRNA. To resolve this curiosity, we applied our DNA family shuffling technology and created a library of Ago2/3 chimeras which we then screened for hybrids with a Slicer phenotype (increased RNAi, versus inhibition with Ago3). This allowed us to identify the Ago2 N-terminus as a novel critical determinant of human Ago slicing activity.
  • People: Nina Schürmann, Stefan Mockenhaupt, in collaboration with Christian Bender
  • Title: Robust RNAi enhancement via Ago2 overexpression from plasmids, vectors or cell lines
  • Summary: Our previous work showed that Ago2 rate-limits the efficacy of si or shRNAs and is a critical factor in RNAi cytotoxicities (Grimm et al., Nature 2006 & J. Clin. Invest. 2010). We thus devised a set of potent and broadly applicable novel strategies that permit shRNA and Ago2 co-expression from plasmid or AAV vector contexts, or in which Ago2 is stably encoded in human cell lines. All 3 strategies increase RNAi efficacies by up to 10-fold and concurrently improve si/shRNA target specificities.
  • People: Kathleen Börner, Gabriella Cotugno, in collaboration with H.G. Kräusslich and numerous CellNetworks groups (including RNAi screening facility)
  • Title: Detection of physiological and pathological RNAi processes in living cells using super-high-resolution microscopy
  • Summary: The real-time visualization of the formation and the activity of RISC complexes in living human cells is one of the holy grails in the RNAi field. Towards this aim, we engineered fusions of all four human Ago proteins as well as of associated factors with green or red fluorophores that are compatible with state-of-the art STORM and PALM microscopy. Moreover, we design novel molecular strategies for labeling, tracking and co-localization of mi and mRNAs in healthy or diseased human cells.
  • People: Kathrin Tegeler, Kathleen Börner, Stefan Mockenhaupt (together with the labs of H. Erfle, M. Heilemann, V. Starkuviene, U. Kummer, F. Hamprecht)
Contact: E-Mail (Last update: 29/02/2012)