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- Title
1040. Towards Gene Correction Therapy for Wiskott-Aldrich Syndrome Using Designed Zinc Finger Endonucleases.
- Authors
Davis, Brian R.; Prokopishyn, Nicole L.; Rosenberg, Jonathan; DiCola, Michael; Brosius, Jennifer; Michael Blaese, R.; Metz, Richard A.; Zhang, Lei; Xia, Danny; Miller, Jeffrey C.; Kim, Kenneth; Urnov, Fyodor D.; Gregory, Philip D.; Holmes, Michael C.
- Abstract
Wiskott-Aldrich Syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from mutations in a hematopoietic- specific and developmentally-regulated gene. WAS is a prime candidate for treatment by autologous hematopoietic stem cell (HSC) gene editing since this disorder is caused by a single gene defect and corrected cells should have a strong selective advantage in vivo, especially at the level of T lymphocyte precursors and perhaps even at the level of HSCs.Based on the efficient gene editing demonstrated by chimeric Zinc Finger Nucleases (ZFNs) at the IL2Rg locus (Urnov et al., 2005), ZFNs engineered to target exon 10 of the human WASP gene were similarly developed. These ZFNs were shown to efficiently bind and cleave the intended target sequences in vitro. In order to assess the efficiency of the WAS-ZFNs to bind their intended target site and mediate double strand DNA break formation in cells, we generated an integrated reporter system in HEK-293 cells. These cells contain a single integrated copy of a defective GFP reporter gene, disrupted by a portion of the WAS exon 10 sequence (GFP- WAS). We observed efficient repair ( 1%) of the GFP-WAS reporter by co-delivering the ZFN/donor constructs by lipofection.Examination of potential factors that influence the efficiency of gene correction revealed that delivery method and cell cycle status are key factors. Direct nuclear microinjection of the ZFNs/donor constructs into the GFP-WAS cells increased the repair rate by several fold (6%). We have subsequently delivered ZFNs/donors to normal human dermal fibroblasts (NHDFs) and primary hematopoietic CD34+ cells via microinjection. We are currently analyzing gene editing at the endogenous WAS locus in these cells. We have also developed an optimized reversible cell cycle synchronization procedure to allow for increased gene editing and improved cell expansion. Administration of either Vinblastine or Nocodazole (G2/M arrest agents) to the GFP-WAS cells significantly increased the gene correction efficiency (5-10 fold), but only Nocodazole treatment was fully reversible. We have also confirmed the reversible Nocodazole cell cycle synchronization in primary NHDFs.We now have available for these studies T (HTLV-I transformed) and B (EBV transformed) cell lines derived from a WAS patient. Experiments examining the efficiency of gene correction in these two patient lines are in progress.Molecular Therapy (2006) 13, S398–S399; doi: 10.1016/j.ymthe.2006.08.1136
- Subjects
IMMUNOLOGICAL deficiency syndromes; GENE therapy; ZINC-finger proteins; ENDONUCLEASES; GENETIC mutation; STEM cells
- Publication
Molecular Therapy, 2006, Vol 13, pS398
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.1136