Validated targets for drug development are lacking for many important human diseases. Many functional genomics methods, such as microarray, are currently being applied to this problem, however these methods do not directly identify genes that are functionally involved in disease processes. One goal of the research at Znomics, Inc. is to develop a vertebrate system with the capacity to directly identify genes and pathways involved in human disease. To accomplish this goal and identify new drug targets, we plan to apply whole genome forward genetics to validated disease models and assays in the zebrafish, Danio rerio. The zebrafish is currently the only vertebrate organism well suited to this purpose. Importantly, substantial evidence indicates conservation of many important molecules and physiological pathways across lower vertebrate systems such as the zebrafish. Znomics has already obtained two validated human disease models in the fish, and is currently in the process of creating six additional models. In order to repetitively screen the zebrafish genome for drug targets in an efficient way, we propose to create an aliquoted sperm library containing approximately 1,000,000 defined, randomly distributed, retroviral insertions, so as to effectively achieve saturation mutagenesis of the genome. In the first phase of this program, we propose to develop a set of retroviral vectors (Specific Aim 1) for use in the saturation mutagenesis procedure. These viruses will be designed to inactivate genes by insertional disruption and/or transcriptional termination, or to alter gene expression. We will develop and characterize high titer virus stock (Specific Aim 2) which will then be used in phase 2 to create the saturation mutagenesis sperm library, and characterize the insertion sites, and hence mutated genes, associated with each fish strain. This library resource, which we estimate may be used for several hundred in vitro fertilizations, can then be used for drug target identification in both large scale screens in disease models as well as in candidate gene testing. Furthermore, the library will provide a resource for both internal Znomics research as well as for collaborative research involving other companies as well as academic investigators.
