346. Gene Correction of X-Linked SCID Using Engineered Zinc Finger Nucleases and Integration Defective Lentiviral Delivery

Abstract

X-linked severe combined immunodeficiency (X-SCID) is a fatal monogenic disorder caused by mutations in the IL2R|[gamma]| gene. Recently, the disease was successfully treated by retroviral vector (RV) mediated gene replacement in hematopoietic stem cells (HSC). A crucial factor for the success of this approach was the selective growth advantage of gene modified cells in vivo, which allowed full lymphoid repopulation from a small number of transduced HSC. Unfortunately, leukemia developed in a fraction of treated patients, and its origin was linked to RV insertional mutagenesis. Thus, although stable genetic modification is required for HSC gene therapy, the widespread non-random distribution of retroviral integration poses a challenge for the safe use of RV in HSC. An alternative approach to gene replacement is the correction of the endogenous gene using engineered zinc finger protein-based nucleases (ZFNs) to specifically target a DNA double stranded break at the site of mutation. One mechanism the cell uses to repair these breaks is homologous recombination (HR), and specific nucleotide substitution can be made in the genome by delivering an extrachromosomal donor molecule to serve as the repair template during HR. By the combined delivery of the cognate ZFNs and template DNA, mutations may be corrected and normal gene function restored. Although this only occurs in a fraction of treated cells, selection of the corrected cells may allow successful exploitation of this new technology for gene therapy. Moreover, because gene correction stably restores both gene function and its normal regulation, it overcomes the limitations of integrative gene replacement and eliminates its possible adverse effects. However, for gene therapy applications, both ZFNs and donor DNA must be delivered into HSCs with high efficiency and without perturbing their function, a major challenge for current non-viral gene transfer technologies.