345. Gene Therapy for Wiskott-Aldrich Syndrome Using Lentiviral Vectors: Evidence for Efficacy and Safety after Transduction of Human T Cells and Hematopoietic Stem Cells

HEMATOLOGIC-DISEASE MODELS b-globin mRNA and protein in cell culture. In this study we incorporated the U7.623 antisense snRNA into lentiviral- and AAVvectors. Transient transfection of HeLa cell lines expressing IVS2654, and-705, with an AAV-U7 antisense snRNA plasmid, resulted in a restoration of both correctly spliced b-globin mRNA and fulllength b-globin polypeptide. The results show that antisense snRNAs shifted the splicing pathway from aberrant to correct and restored the correct mRNAs in a sequence specific and dosedependent manner. This was accomplished in model cell lines, in which the thalassemic b-globin intron was incorporated into the enhanced green fluorescence protein (EGFP) coding sequence and in HeLa cells that express thalassemic human b-globin genes. Transfection of EGFP-HeLa cell lines with IVS2-654, and -705U introns with AAV-U7.623 resulted in upregulation and expression of EGFP protein concomitant with expression of the of red fluorescence protein (RFP), an AAV viral reporter gene. Similar results, although less pronounced, were obtained in cells transiently transfected with lentiviral-U7.623 antisense snRNA construct. Interestingly, the restoration of correctly spliced mRNAs was faster in AAV than lentiviral vector treated cells. Based on this encouraging result, the AAV- and lentiviral-U7 antisense snRNA constructs will be evaluated in animal models for thalassemia. The snRNA antisensebased gene therapy may offer an attractive approach to cure thalassemia.

because T and NK cells will develop once the γc chain is introduced into patients’ CD34 positive cells in vitro. In addition, recent reports of retrovirus-based X-SCID gene therapy clinical trials from France and England demonstrated the efficacy of the gene therapy. Most of the patients who have received the gene therapy showed NK cell recovery and immunoglobulin production in addition to T cell reconstitution. However, a serious adverse effect was reported in three out of eleven patients treated in France. The complication was lymphoproliferative disease, which occurred about three years later after the gene therapy. Two of them were due to insertional mutagenesis of a proto-oncogene, LMO2 by retroviral vector. Therefore, development of a safer integrating vector is an urgent need. One of the approaches is to incorporate a suicide gene into a therapeutic retrovirus vector to offset the lymphoproliferative disease caused by the gene therapy. In this study, we constructed a retroviral vector encompassing a human γc chain cDNA and a suicide gene from the Herpes simplex virus thymidine kinase (HSVtk). For this purpose, a human γc cDNA was inserted into SFCMM3, a retroviral vector containing an HSVtk gene, which had already been applied in clinical trial. The resultant vector (SFCMM-gamma) was transfected into COS-7 cells and expression of the γc chain was confirmed. For recombinant retrovirus production, the SFCMMgamma was transfected into an amphotropic packaging cell line, ΨCRIP. 24 hours later after transfection, recombinant virusproducing ΨCRIP cells were irradiated (30Gy) and then co-cultured with EBV transformed B cell lines established from X-SCID patients for 48 hours. The infection cycle was repeated twice. Just prior and a week after starting the co-culture, expression of the γc chain of the EBV-B lines was assessed by flow cytometric analysis. After confirmation of the γc chain expression on the transduced EBV-B lines, the cells were treated with 0.1, 1.0, 10 and 100 mM of ganciclovir (GCV) for 24, 48, 72 and 96 hours. At the end of GCV treatment, expression of the γc chain was analyzed. Cells expressing the transduced γc chain were dramatically reduced within 48 hours after the addition of the GCV, and the γc chain could not be detected on the cell surface after 96 hours with 10 or 100 mM GCV. In contrast, untreated cells expressed the γc chain constantly throughout the experiment. Concentration of GCV up to 10 mM did not affect the growth of the EBV-B cell lines. To test the efficacy and safety feature of the gene therapy in vivo, a murine model of X-SCID was treated with the vector. Currently, immunologic reconstitution and efficacy of the suicide gene in this murine model were under investigation.

345. Gene Therapy for Wiskott-Aldrich Syndrome Using Lentiviral Vectors: Evidence for Efficacy and Safety after Transduction of Human T Cells and Hematopoietic Stem Cells

344. Application of a suicide Gene to X-SCID Gene Therapy Toru Uchiyama,1 Satoru Kumaki,1 Masahumi Onodera,2 Du Wei,1 Looi Chung Yeng,1 Yoichi Sasahara,1 Sigeru Tsuchiya.1 1 Department of Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan; 2Major of Medical Science, Graduate school of Comprehensive Human Science, Tsukuba University, Tsukuba, Japan. X-linked severe combined immunodeficiency (X-SCID) is a fatal disease characterized by the absence of humoral and cellular immunity due to mutations in the gene encoding the common gamma (γc) chain. X-SCID is a good candidate for the somatic gene therapy S134

Loïc Dupré,1 Samantha Scaramuzza,1 Francesco Marangoni,1,2 Sara Trifari,1 Silvana Martino,3 Shigeru Tsuchiya,4 Adrian Thrasher,5 Anne Galy,6 Luigi Naldini,1,2 Alessandro Aiuti,1 Maria Grazia Roncarolo.1,2 1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy; 2Vita-Salute San Raffaele University, San Raffaele Scientific Institute, Milan, Italy; 3 Department of Pediatrics, University of Turin, Turin, Italy; 4 Department of Pediatric Oncology, Tohoku University, Sendai, Japan; 5Molecular Immunology Unit, Institute of Child Health, London, United Kingdom; 6CNRS 8115, Généthon, Evry, France. Wiskott-Aldrich syndrome (WAS) is a X-linked primary immunodeficiency with a median survival of 14 years of age due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells (HSC) from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous HSC could Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

HEMATOLOGIC-DISEASE MODELS represent an alternative treatment applicable to all patients. We investigated the efficacy and safety of WAS gene transfer with HIVbased lentiviral vectors using untransformed T cells from WAS patients and HSC. We designed lentiviral vectors encoding the WAS protein (WASP) under the control of 3 different promoters: the ubiquitous PGK promoter and the hematopoietic-specific full-length (1.6 Kb) and minimal (0.5 Kb) WAS promoters. Transduction levels in WAS T cells were found to be comparable for each vector and to depend directly on vector concentrations used for transduction. The percentage of transduced T cells expressing WASP progressively increased during long-term in vitro culture indicating that WASP+ T cells have a selective growth advantage over WASP- T cells. WASP expression in transduced cells reached normal levels and no overexpression was observed. Correction of functional defects including TCR-driven proliferation and IL-2 production was achieved after transduction with all 3 vectors at a MOI of 100, leading to approximately 50% WASP+ T cells. Clones of transduced CD4+ T cells were generated to determine the relationship between number of integrated vectors, WASP expression levels and functional correction. One to two vector copies of the WASP-encoding vectors were sufficient to restore WASP expression and IL-2 production. No evidence for genotoxicity could be observed in long-term cultures of transduced clones or bulk cell lines. In parallel, CD34+ HSC were isolated from mobilized peripheral blood of healthy donors and transduced with the WASP-encoding lentiviral vectors using a previously optimized protocol. Following culture of the transduced HSC in the presence of cytokines in methyl-cellulose medium, the numbers of CFU-C were normal. This indicates that transduction with the WASP-encoding vectors had no detrimental impact on in vitro differentiation of HSC. The efficacy and safety of the vectors designed in this study will be further investigated in CD34+ HSC from WAS patients. The SCID-hu and the γ-chain-/- /RAG2-/- murine models will be used to study the in vivo differentiation of the transduced HSC. These studies will provide preclinical data for the implementation of a first gene therapy trial for WAS patients.

346. Gene Correction of X-Linked SCID Using Engineered Zinc Finger Nucleases and Integration Defective Lentiviral Delivery Angelo Lombardo,1,3 Christian Beausejour,2,3 Fyodor D. Urnov,2 Jeffrey C. Miller,2 Michael C. Holmes,2 Philip D. Gregory,2,4 Luigi Naldini.1,4 1 Telethon Institute for Gene Therapy, HSR, Milan, Italy; 2Sangamo BioSciences, Inc, Pt Richmond, CA; 3; 4Equal Contribution. X-linked severe combined immunodeficiency (X-SCID) is a fatal monogenic disorder caused by mutations in the IL2Rγ 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 Molecular Therapy Volume11, Supplement 1, May 2005 Copyright  The American Society of Gene Therapy

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. Here we developed a delivery platform based on late-generation integration-defective lentiviral vectors (IDLV), which takes advantage of the viral proficiency both to introduce donor DNA and transiently express ZFNs in the same cell. By optimizing the ZFNs expression cassette and the configuration of donor DNA, we reached significant levels of gene correction in an engineered reporter cell line. Remarkably, the correction frequency was higher than that observed with plasmid-based delivery. We also evaluated the ratio between HR and non-targeted integration in total and gene-corrected cells. By incorporating the donor sequence and ZFN expression cassette in the same IDLV, we enforced their combined delivery in all transduced cells, streamlining its application to primary hematopoietic cells. We are currently testing this improved system for correction of the most common IL2Rγ gene mutation in X-SCID cell lines and primary cells. Our results provide proof of principle of a new gene therapy strategy reaching an unprecedented frequency of targeted gene correction, and providing a unique combination of safety and potential efficacy.

347. Long-Term Gene Expression and Phenotypic Correction of FANCC Deficient Lymphoblastoid Cells Using the Sleeping Beauty Transposon System Shannon A. Wadman,1 Karl J. Clark,1 Perry B. Hackett,1,2 R Scott McIvor,1,2 Jeffrey J. Essner.1 1 Gene Therapy Section, Discovery Genomics, Inc., Minneapolis, MN; 2Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN. We are moving forward with development of our proprietary Sleeping Beauty Transposon (SBT) system for the treatment of Fanconi anemia (FA) via ex vivo electroporation of hematopoietic stem cells. FA is an inherited recessive disorder caused by deficiency in one of a number of genes whose products form a complex involved in DNA repair. The primary hematological hallmarks of the disease are aplastic anemia, bone marrow failure, and increased susceptibility to leukemias thought to be caused by defective cellular mechanisms for DNA repair. Allogeneic bone marrow transplantation is currently the only curative treatment for these aspects of the disease and despite improvements in clinical protocols over the years difficulties with allotransplants for FA still persist. The primary goal of this study is to evaluate the potential of SBT vectors carrying the FANCC gene to effectaffect transposition and establish long-term expression in FANCC-deficient cell populations after introduction by electroporation. Additionally, we show that integration of FANCC transposons can correct the sensitivity of lymphoblastoid cell lines (LCL) derived from FANCC patients to DNA damaging agents such as mytomycin C. We constructed a transposon containing a PGK promoter to regulate transcription of the normal human FANCC cDNA sequence. The vector containing the transposon also includes a ubiquitin promoter regulating expression of the SB transposase gene, encoded outside of the transposon. The SBT-FANCC vector was introduced via electroporation into FANCC deficient and normal control LCLs. Long-term maintenance of FANCC complementation in the FANCC-deficient cells was observed as a sustained reduction S135