738. Towards Gene Correction of X-Linked SCID Using Engineered Zinc Finger Nucleases and Integrase Defective Lentiviral Delivery
Authors: Angelo Lombardo, Christian Beausejour, Fyodor D. Urnov, Jeffrey C. Miller, Alessandro Aiuti, Philip D. Gregory, Michael C. Holmes, Luigi Naldini
Journal: Molecular Therapy (2006)

X-linked severe combined immunodeficiency (X-SCID), a fatal disorder caused by mutations in the IL2Rγ gene, has been 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, which allowed 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. An alternative approach to gene replacement is correction of the endogenous gene using engineered zinc finger protein-based nucleases (ZFNs) to specifically target a DNA double stranded break at the mutation site. 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 repair template during HR. By the combined delivery of 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 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 its possible adverse effects. However, for gene therapy applications, both ZFNs and donor DNA must be delivered into target cells with high efficiency and without perturbing their function, a challenge for gene transfer technologies. We have developed a delivery platform based on integrase-defective lentiviral vectors (IDLV), which takes advantage of the viral proficiency to both introduce donor DNA and transiently express ZFNs in the same cell. By using this approach in model cell lines, we achieved levels of gene correction that were higher than those observed with plasmid-based delivery. These vectors were further enhanced by optimizing the ZFN expression cassette and combining them with the donor DNA in the same vector. Remarkably, when these vectors targeting a mutation hotspot in the IL2Rγ gene were transduced into hematopoietic cell lines, we found up to 40% editing of the endogenous IL2Rγ gene. Importantly, HR with the donor vector accounted for 75% of the total editing. We then transduced a panel of immortalized B-lymphocytes from both normal donors and X-SCID patients. When the treated cells were analyzed for genome editing, we found up to 8% HR in the IL2Rγ locus. We are currently following-up these studies to assess functional restoration of IL2R signalling in patient cells, and extending the application of IDLV-ZFN mediated gene editing to human HSC. Our results provide proof of principle of a new gene delivery strategy reaching an unprecedented frequency of targeted gene correction and potentially improving efficacy and safety of gene therapy. Sangamo own ZFN technology and is developing its application.