DESCRIPTION (provided by applicant): The road toward curing genetic diseases by gene therapy has seen some success but also significant disappointment. In particular, gene addition via uncontrolled insertion of transgenes has been associated with insertional oncogenesis and silencing. Gene targeting, correction of the endogenous gene sequence, is preferable as it avoids both of these issues. While the spontaneous rate of gene targeting in most cell types is very low, zinc finger nucleases (ZFNs) have recently been shown to be an excellent tool for stimulating homologous recombination between an exogenous repair (donor) DNA template and the endogenous locus by introducing a double strand break in the locus of interest. ZFNs are artificial proteins consisting of an engineered DNA binding domain and nuclease domain. So far ZFNs have been used to stimulate gene targeting in a variety of somatic cell types and organisms; however, to bring their therapeutic benefit to fruition will require their successful application in stem cells. To establish clinically relevant ZFN-mediated gene targeting, very high absolute rates of gene targeting must first be attained, or methods for selecting and expanding rare targeted cells in vitro must be developed. This proposal addresses both of these approaches. Previously validated ZFNs will be used to stimulate gene targeting in spermatogonial stem cells (SSCs) derived from a mouse homozygous for a mutant GFP gene knocked-in to the Rosa26 locus. This mouse models an autosomal recessive disease. In Aim 1 ZFN-mediated gene targeting will be optimized in SSCs by testing parameters including gene delivery method, cell cycle and cell differentiation state. The completion of this aim should help to define the requirements for gene therapy in germline and somatic stem cells. In Aim 2 cultured SSCs will be used to model the therapeutic process of ex vivo ZFN-mediated gene correction, selection, amplification, molecular characterization and transplantation. In addition to testing methods for isolation and expansion of rare gene-corrected stem cells, the accomplishment of this aim provides proof of principle for transgenerational gene therapy at a chromosomal locus in mice; that is, the ability of germ cells to transmit a corrected gene to the next generation. Mouse and human spermatogonial stem cells (SSCs) have recently been shown to be capable of dedifferentiation into pluripotent embryonic stem - like cells. This exciting discovery has led to the proposal that the testis could be an excellent source of histocompatible cells for autologous transplantation in men. One day it may be possible to cure genetic diseases by deriving patient-specific pluripotent cells from a testicular biopsy, correcting a mutated gene in vitro, differentiating the cells into the desired cell type and then performing transplantation to replace the diseased cells. An important first step toward this ambitious goal is the development of technology for efficient gene targeting in SSCs as described in this proposal. PUBLIC HEALTH RELEVANCE: Correcting a mutated gene in stem cells and transplanting the corrected stem cells into a patient could be an effective way to cure certain genetic diseases. A promising new method to correct mutant genes is to cut the gene using molecular scissors (zinc finger nucleases), a process that then stimulates the exchange of the mutated gene sequence with an exogenously provided normal gene sequence. We propose to test and optimize this process of gene correction in cultured spermatogonial stem cells, cells with great therapeutic potential and that are highly related to pluripotent embryonic stem cells.

PUBLIC HEALTH RELEVANCE STATEMENT/NARRATIVE Correcting a mutated gene in stem cells and transplanting the corrected stem cells into a patient could be an effective way to cure certain genetic diseases. A promising new method to correct mutant genes is to cut the gene using molecular scissors (zinc finger nucleases), a process that then stimulates the exchange of the mutated gene sequence with an exogenously provided normal gene sequence. We propose to test and optimize this process of gene correction in cultured spermatogonial stem cells, cells with great therapeutic potential and that are highly related to pluripotent embryonic stem cells.