Inherited inhibition of inborn immunity - an integrated molecular genetic approach

Inborn errors of the myeloid cell system comprise a heterogeneous group of rare diseases affecting neutrophils and monocytes. Patients suffer from severe and recurrent bacterial infections and may have an increased susceptibility to develop leukemia. NEUTRONET is a collaboration of five laboratories leading in their fields to tackle this disease by highly novel and integrative approaches. Embedded in this network, the laboratory of Dr. Josef Penninger proposes to undertake two crucial parts in the identification of new causes and modeling neutropenia in order to set the basis for the development of innovative gene-based therapeutic strategies. As the first pillar of the concept we propose to use Drosophila melanogaster, the best developed genetic model system of developmental biology. Such an approach is feasible since hematopoiesis displays surprisingly strong similarities between humans and fruit fly, especially within the myeloid lineage. Recent innovations in the system allow genome wide in vivo RNAi knockdown approaches in a time- and tissue-specific manner. We propose to utilize these new advances in high throughput genetics initially developed at our institute, IMBA, to screen all Drosophila genes with human orthologs to identify multiple new genes affecting hematopoesis. An integrative genomic view comparing all genes identified in flies and the syntenic regions of a genetic data set from human patients generated by the collaborators within the network offers a unique chance to rapidly identify novel gene defects in patients suffering from myeloid disorders. We propose to identify several novel mutations manifesting in neutropenia and related syndromes within the timeframe of this proposal. NEUTRO-NET further will be able to generate and provide the ideal genetically designed model for mechanistic studies. Our laboratory will provide to NEUTRO-NET the rapid generation of murine models to test the effects on mutations identified in human sequencing and fly screening and ultimately proof an identified mutation as causative. Due to an established pipeline of an ES cell targeting facility, 8 cell injection as well as the largest and most modern mouse facility in Austria Europe (www.austromouse.at) we have the ideal setup to achieve this goal in minimum time. Our extensive experience in immunology and hematopoiesis together with the complete technical infrastructure for analysis will enable rapid validation in these mammalian models. Generation of such animal models also represents an ideal platform for future drug design as well as a test system for gene-based therapies.

The lab of Josef Penninger performed a genome wide in vivo RNAi screen in fruit flies to identify genes involved in hematopoiesis. By overlapping the datasets with human GWAS association studies we were able to identify cross-species conservation in hematopoiesis. These results were published in nature (doi: 10.1038/nature10659, doi: 10.1038/nature11677)Furthermore, based on funding by the FWF project I 434-B13, developed haploid embryonic stem cells. These cells like all embryonic stem cells - have the potential to differentiate into most or all mature cell types in vivo and in vitro, the definition of embryonic stem cells. Due to the parthenogenic derivation protocol used, these cells only contain the maternal chromosome set. Surprisingly, even though living on a half chromosome set, the cells are indeed viable. The lab of Josef Penninger uses these cells for high throuput functional genetic assays since recessive mutations introduced using insertional, random mutagenesis will always manifest due to the lack of a second allele.In a proof of principle screen, the cells were used to unravel the genetics of ricin intoxication. Fucosylation as well as the novel cellular receptor Gpr107 were identified to be required for cellular toxicity of this bioweapon. Blocking these pathways therefore is a potential means of neutralizing ricin toxicity and thus an entry point for drug development (doi: 10.1016/j.stem.2011.10.012).