831. Evaluation of Lentiviral Vectors for Gene Therapy of Wiskott-Aldrich Syndrome

Abstract

Retroviral vector-mediated activation of the LMO2 oncogene led to the onset of leukemia in two X-SCID patients who underwent gene therapy (Science 302: 415, 2003). This has necessitated the development of safer vectors for use in a therapeutic setting. Many pre-clinical studies have now incorporated the use of insulator elements that have boundary and/or enhancer blocking functions to isolate the transcriptional unit within the genome. The chicken beta-globin 5' DNase I hypersensitive site 4 (5'HS4) insulator possesses both of these properties and has been used to overcome positional variegation effects on transgene expression. Characterizing this aspect of insulator function is essential, but it is also of interest to know if there is any differential effect on the pattern of retroviral integration. We are developing lentiviral vectors for gene therapy of Wiskott-Aldrich syndrome (WAS) because of their greater efficiency at transducing repopulating stem cells (Blood 103:4062, 2004) compared to oncoretroviral vectors. WAS is a rare X-linked recessive disorder caused by mutations in the WASP gene which affect cells of the hematopoietic lineage. We have previously demonstrated correction of the T-cell proliferation defect, partial correction of the cytokine secretion defect and improvement in the impaired secondary response to influenza virus in Wasp- mice using MSCV onco-retroviral vectors (Blood 102:3108, 2003). Due to the modest selective advantage of gene-corrected lymphocytes and the variability in the level of gene expression, we are now exploring the use of lentiviral vectors. The efficiency of stem cell-targeted gene transfer achieved with lentiviral vectors and the consistency of gene expression are crucial factors that will determine the success of any therapeutic application for the treatment of WAS. Towards this goal, we are evaluating various vector designs in mice using GFP as a marker gene. To examine the effect of the 5'HS4 insulator within the context of our lentiviral backbone, we have generated advanced third generation vectors containing a modified MSCV promoter (Mp) driving GFP with or without the 5'HS4 insulator. Several single- and multiple-copy T-cell (Jurkat) clones have been isolated for each construct and their expression characterized by flow cytometry. Inclusion of the 5'HS4 insulator did not significantly affect the average mean fluorescence intensity (MFI = 370|[plusmn]|221 versus 329|[plusmn]|120[5'HS4+]), but did reduce the variability of expression amongst individual clones as indicated by a decrease in the co-efficient of variation from 57|[plusmn]|26 to 40|[plusmn]|3. To determine if this insulator has an effect on the position or frequency of insertion, the specific integration sites in clones containing the proviral genomes are being characterized by ligation-mediated (LM)-PCR.