SBIR-STTR Award

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.

Thesaurus Terms:
Retroviridae, drug metabolism, genetic library, genome, sperm, technology /technique development, transposon /insertion element, zebrafish gene expression, nuclear transfer, transfection /expression vector biotechnology, cell line

At the present time it is not possible to rapidly and inexpensively obtain a vertebrate knockout in a gene of choice, or to readily perform whole genome forward genetic screening in a vertebrate. The goal of this Phase II SBIR application is to create the research tools necessary to perform these functional genomics experiments in a vertebrate by developing a zebrafish repository containing a tagged insertional mutation in every identifiable gene. The proposed research will focus on creating, storing, and managing a zebrafish sperm library, termed the Zenemark Library, containing retrovirally-mediated insertional mutations in each lene in the zebrafish genome. Each of these insertional mutations will be characterized, stored as multiple =liquots of frozen sperm, and entered in a databank using identifiers of flanking genomic sequence, zebrafish gene locus, and mammalian gene homologues. A collection of 1,000,000 independent inserts will create, on average, a tagged genetic disruption once every 1800 base pairs, essentially a mutation in every zebrafish gene. The database, and individual mutant strains derived from the library will be available to both commercial and academic researchers. Just as this resource will allow basic researchers to more readily study the genetics of biological processes in vertebrates, it will allow pharmaceutical researchers to apply whole genome forward genetics to the problems of drug and drug target discovery. In particular, the creation of the ZeneMark Library will allow pharmaceutical researchers to screen collections of mutant fish, such as fish with mutations in all druggable genes, to 1 ) identify novel drug targets, 2) identify zebrafish models of human disease that may be used directly for high-throughput in vivo drug discovery, and 3) identify the site(s) of action of known drugs, toxins, or other chemicals. This forward genetic screening will serve as the primary basis for commercialization of the library, and Znomics is already in the process of seeking both corporate partners as well as venture capital to fund Phase III of this program.

Thesaurus Terms:
Retroviridae, drug metabolism, gene mutation, genetic library, genetic registry /resource /referral center, genome, serial analysis of gene expression, sperm, technology /technique development, transposon /insertion element, zebrafish gene expression, nuclear transfer, transfection /expression vector biotechnology, cell line