Research Summary

Our goal is to develop new and effective treatments for diseases by uncovering the fundamental biological principles underlying development and disease. To accomplish our goals, we develop and deploy a broad range of in vitro and in vivo tools that reveal the fundamental mechanisms involved in human disease. These approaches include genetic editing in vitro and in vivo, human induced pluripotent cell (iPS cell) models of disease, haploid cells for genetic as well as compound screening paradigms, mouse and human organoid cultures, as well as genetically engineered mice. Our multidisciplinary techniques enable us to model and study the complexity of diseases.

 

Although our research falls under four broadly defined thematic areas below, these systems are subject to substantial crosstalk. As a result, our research in one field influences and informs another, leading to unexpected insight and advances that acknowledge and embrace the complexity of disease and biology.

 

Projects

Blood vessel engineering – The prevalence of diabetes is increasing constantly, resulting in a global epidemic. Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke or lower limb amputation; in large parts because of marked changes in blood vessels, defined by expansion of the basement membrane and a loss of vascular cells. Diabetes also impairs endothelial cell (EC) function and disturbs EC-pericyte communication. How endothelial/pericyte dysfunction leads to diabetic vasculopathy remains largely elusive. Here we report the development of self-organizing 3D human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycemia and inflammatory cytokines in vitro induced thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimic the microvascular changes in diabetic patients. Dll4-Notch3 were identified as key drivers of diabetic vasculopathy in human blood vessels. Thus, organoids derived from human stem cells faithfully recapitulate the structure and function of human blood vessels and are amenable to model and identify regulators of diabetic vasculopathy, affecting hundreds of millions of patients. (Wimmer et al. Nature 2019) 

BH4 – Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in monoamine neurotransmitter production, nitric oxide generation and pain. We now identify a fundamental role for BH4 in T cell biology. Genetic inactivation of GTP cyclohydrolase 1 (GCH1), the rate-limiting enzyme in the synthesis of BH4 and inhibition of sepiapterin reductase (SPR), the terminal enzyme in its synthetic pathway, severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T cell-mediated autoimmunity and allergic inflammation, while enhancing BH4 levels by GCH1 overexpression, augments CD4+ and CD8+ T cell responses increasing their anti-tumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands intra-tumoral effector T cells. Kynurenine, a tryptophan metabolite that blocks anti-tumour immunity, inhibits T cell proliferation in a manner that can be rescued by BH4.  Finally, developed a potent SPR antagonist for potential clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4, as critical regulators of T cell biology, which can be readily manipulated to either block autoimmunity or enhance anti-cancer immunity.  For me as trained T cell biologist, who wrote the first CTLA4 knock-out paper, it’s quite amazing to find an entirely unexpected new pathway required for T cell biology – blocking the pathway we can control e.g. multiple autoimmune diseases or allergic airway inflammation. (Cronin et al. Nature 2018).

Glycoproteomics in cancer – Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite its importance in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. We developed a novel quantitative approach to identify intact glycopeptides from comparative proteomic data-sets, allowing us to not only infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We applied this method to human and murine embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method was confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discovered proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins rendered cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our novel comparative glycoproteomics platform enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems. (Stadlmann et al. Nature 2017).

Haploid stem cells – The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. We used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid murine embryonic stem cell (mESC) lines targeting 16,950 genes with genetically bar-coded, conditional and reversible mutations. This Haplobank is the largest resource of hemi-/homozygous mutant mESCs to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We utilize Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mESCs, and to identify novel genes that control sprouting angiogenesis and blood vessel lineage specification. Further, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor required for cytotoxicity by rhinoviruses, which cause the common cold. Thus, Haplobank clones and revertible technologies enable high-throughput, reproducible functional annotation of the genome. (Elling et al. Nature 2017).

 

Collaborations

Clifford Woolf, Harvard Medical School – BH4
Hudson Freeze, San Diego – Glycoproteomics
Juergen Knoblich, IMBA, Vienna – tissue engineering
Jingson Li, Chinese Academy of Sciences – haploid stem cells

Bio

Dr. Josef Penninger is a Canada 150 Chair in Functional Genetics and the Director of the Life Sciences Institute at the University of British Columbia, since December 2018. Prior to this, from 2002-2018, he was the Scientific and Founding Director of IMBA, the Institute for Molecular Biotechnology of the Austrian Academy of Sciences, in Vienna, Austria. From 1994-2002, he worked as a lead researcher at the Amgen Research Institute in Toronto, affiliated with the University of Toronto and the Ontario Cancer Research Center.

Publications
  1. Wimmer R.A., Leopoldi A., Aichinger M., Wick N., Hantusch B., Novatchkova M., Taubenschmid J., Hämmerle M., Esk C., Bagley J.A., Lindenhofer D., Chen G., Boehm M., Agu C.A., Yang F., Fu B., Zuber J., Knoblich J.A., Kerjaschki D., Penninger J.M. (2019). Human blood vessel organoids as a model of diabetic vasculopathyNature. 565(7740): 505-510.
  2. Cronin, S.J.F., Seehus, C., Weidinger, A., Talbot, S., Reissig, S., Seifert, M., Pierson, Y., McNeill, E., Longhi, M.S., Turnes, B.L., Kreslavsky, T., Kogler, M., Hoffmann, D., Ticevic, M., da Luz Scheffer, D., Tortola, L., Cikes, D., Jais, A., Rangachari, M., Rao, S., Paolino, M., Novatchkova, M., Aichinger, M., Barrett, L., Latremoliere, A., Wirnsberger, G., Lametschwandtner, G., Busslinger, M., Zicha, S., Latini, A., Robson, S.C., Waisman, A., Andrews, N., Costigan, M., Channon, K.M., Weiss, G., Kozlov, A.V., Tebbe, M., Johnsson, K., Woolf, C.J., Penninger, J.M. (2018). The metabolite BH4 controls T cell proliferation in autoimmunity and cancer. Nature. 563: 564–568.
  3. Stadlmann, J., Taubenschmid, J., Wenzel, D., Gattinger, A., Dürnberger, G., Dusberger, F., Elling, U., Mach, L., Mechtler, K., Penninger, J.M. (2017). Comparative glycoproteomics of stem cells identifies new players in ricin toxicity. Nature. 549(7673):538-542.
  4. Elling, U., Wimmer, R.A., Leibbrandt, A., Burkard, T., Michlits, G., Leopoldi, A., Micheler, T., Abdeen, D., Zhuk, S., Aspalter, I.M., Handl, C., Liebergesell, J., Hubmann, M., Husa, A.M., Kinzer, M., Schuller, N., Wetzel, E., van de Loo, N., Martinez, J.A.Z., Estoppey, D., Riedl, R., Yang, F., Fu, B., Dechat, T., Ivics, Z., Agu, C.A., Bell, O., Blaas, D., Gerhardt, H., Hoepfner, D., Stark, A., Penninger, J.M. (2017). A reversible haploid mouse embryonic stem cell biobank resource for functional genomics. Nature. 550(7674):114-118.
  5. Wirnsberger, G., Zwolanek, F., Asaoka, T., Kozieradzki, I., Tortola, L., Wimmer, R.A., Kavirayani, A., Fresser, F., Baier, G., Langdon, W.Y., Ikeda, F., Kuchler, K., Penninger, J.M. (2016). Inhibition of CBLB protects from lethal Candida albicans sepsis. Nat Med. 22(8):915-23.
  6. Sigl, V., Owusu-Boaitey, K., Joshi, P.A., Kavirayani, A., Wirnsberger, G., Novatchkova, M., Kozieradzki, I., Schramek, D., Edokobi, N., Hersl, J., Sampson, A., Odai-Afotey, A., Lazaro, C., Gonzalez-Suarez, E., Pujana, M.A., Cimba, F., Heyn, H., Vidal, E., Cruickshank, J., Berman, H., Sarao, R., Ticevic, M., Uribesalgo, I., Tortola, L., Rao, S., Tan, Y., Pfeiler, G., Lee, E.Y., Bago-Horvath, Z., Kenner, L., Popper, H., Singer, C., Khokha, R., Jones, L.P., Penninger, J.M. (2016). RANKL/RANK control Brca1 mutation-driven mammary tumors. Cell Res. 26(7):761-74.
  7. Paolino, M., Choidas, A., Wallner, S., Pranjic, B., Uribesalgo, I., Loeser, S., Jamieson, A.M., Langdon, W.Y., Ikeda, F., Fededa, J.P., Cronin, S.J., Nitsch, R., Schultz-Fademrecht, C., Eickhoff, J., Menninger, S., Unger, A., Torka, R., Gruber, T., Hinterleitner, R., Baier, G., Wolf, D., Ullrich, A., Klebl, B.M., Penninger, J.M. (2014). The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature. 507(7493):508-12.
  8. Hanada, T., Weitzer, S., Mair, B., Bernreuther, C., Wainger, B.J., Ichida, J., Hanada, R., Orthofer, M., Cronin, S.J., Komnenovic, V., Minis, A., Sato, F., Mimata, H., Yoshimura, A., Tamir, I., Rainer, J., Kofler, R., Yaron, A., Eggan, K.C., Woolf, C.J., Glatzel, M., Herbst, R., Martinez, J., Penninger, J.M. (2013). CLP1 links tRNA metabolism to progressive motor-neuron loss. Nature. 495(7442):474-80.
  9. Hashimoto, T., Perlot, T., Rehman, A., Trichereau, J., Ishiguro, H., Paolino, M., Sigl, V., Hanada, T., Hanada, R., Lipinski, S., Wild, B., Camargo, S.M., Singer, D., Richter, A., Kuba, K., Fukamizu, A., Schreiber, S., Clevers, H., Verrey, F., Rosenstiel, P., Penninger, J.M. (2012). ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation. Nature. 487(7408):477-81.
  10. Elling, U., Taubenschmid, J., Wirnsberger, G., O’Malley, R., Demers, SP., Vanhaelen, Q., Shukalyuk, A.I., Schmauss, G., Schramek, D., Schnuetgen, F., von Melchner, H., Ecker, J.R., Stanford, W.L., Zuber, J., Stark, A., Penninger, J.M. (2011). Forward and reverse genetics through derivation of haploid mouse embryonic stem cells. Cell Stem Cell. 9(6):563-74.
  11. Schramek, D., Leibbrandt, A., Sigl, V., Kenner, L., Pospisilik, J.A., Lee, H.J., Hanada, R., Joshi, P.A., Aliprantis, A., Glimcher, L., Pasparakis, M., Khokha, R., Ormandy, C.J., Widschwendter, M., Schett, G., Penninger, J.M. (2010). Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Nature. 468(7320):98-102

For a full list of publications please see: http://scholar.google.at/citations?user=OdlFlsMAAAAJ

 

 

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