HEMATOLOGIC-NEW ADVANCES >= 16.5 months after transduction Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells, and may be worthy of further investigation 1044 Radioprotective Gene Therapy through Retroviral Expression of Manganese Superoxide Dismutase Thomas D Southgate,1 Victoria Sheard,1 Michael D Milsom,1 Rob J Mairs,2 Marie Boyd,2 Leslie J Fairbairn.1 Gene Therapy, Paterson Institute for Cancer Research, Manchester, United Kingdom; 2Radiation Oncology, Cancer Research UK Beatson Laboratories, Glasgow, United Kingdom Overexpression of manganese superoxide dismutase (MnSOD, also known as SOD2) has been hypothesized to provide haemopoietic cells with radioprotection through the scavenging of oxygen radicals induced by ionizing radiation Here we demonstrate, using both biological and physical assays, that overexpression of SOD2 substantially protects both murine bone marrow and K562 cells from ionising radiation injury For this study the human SOD2 cDNA was cloned within a retroviral vector (SFβ91), optimised for initiation of transcription in primitive haemopoietic cells, along with eGFP as a marker Retroviral producer lines were established and used to infect murine bone marrow and the human erythroleukaemic cell line, K562 Cells were characterised by Western and Southern blotting and shown to be polyclonal by LAM PCR SOD enzymatic activity was assessed using a water-soluble tetrazolium salt microplate assay, whilst survival was determined by assessing colony forming ability after irradiation of cells at various doses The levels of DNA strand breaks were assessed using a COMET assay Cells transduced with the SFβ91-SOD2-eGFP vector exhibited expression of SOD2 by Western blot analysis, eGFP by FACS and a 2.5 fold increase in total SOD enzymatic activity In SOD2 transduced murine bone marrow and in K562 cells we have demonstrated a physical decrease in the levels of DNA strand breaks, whilst biologically we showed an approximately 2-fold survival advantage in SOD2-expressing cells over controls when exposed to varying doses of ionising radiation In conclusion, overexpression of SOD2 using a retroviral vector to infect cells of the haemopoietic compartment delivers both physical and biological protection against ionising radiation Our findings are encouraging for the development of SOD2 gene transfer for the protection against myelotoxic side effects of radiation 1045 Overexpression of an ABCG2 Multidrug Transporter Variant Does Not Effect Proliferation or Differentiation of Hematopoetic Stem Cells Olga Ujhelly,1 Nora Kucsma,1 Judith Cervenak,1 Linping Chen,2 Manuel Grez,2 Balazs Sarkadi,1 Katalin Nemet.1 Experimental Gene Therapy, National Medical Center, Institute of Haematolgy and Immunology, Budapest, Hungary; 2GeorgeSpeyer-Haus, Institute for Biomedical Research, Frankfurt, Germany Stem cell based gene therapy is often unsuccessful because of the low number of gene modified cells Growth advantage of the corrected cells may provide permanent recovery of the patient The multidrug resistance phenotype of haematopoietic stem cells (HSCs) allows the selection of gene-modified cells in vivo or in cancer patients, decreases the myelosuppressive side effect of chemotherapy In contrast with the potential benefit, some authors reported a harmfull side effect of several ABC transporters on the myeloid differentiation In the present work, overexpression of a substrat mutant of human ABCG2 (ABCG2-482G) was investigated in a mouse model Animals were transplanted with bone marrow cells transduced by a S404 retroviral vector encoding ABCG2-G Colony forming properties (CFU-C) of the transduced cells were investigated in vitro, while the repopulation of haematopoiesis was followed for 16 weeks Bisides ABCG2 expression, chimerism and linaege markers were detemined Our results show a highly detectable (45-60%), functionally active population of ABCG2-G positive progenitor cells, still the number of CFU-C was similar in the treated and control sample No significant difference was found in the hemopoietic tissues (bone marrow, spleen) nor in the mature white blood cell linaeges (WBC) of the transplanted mice Our data suggest that ABCG2 contribute in the protection of essential tissues and does not influance hematopoesis ABCG2482G is an ideal candidate for gene therapy application as an in vivo selectable marker 1046 Stable Correction of Hematopoietic Stem Cells with Non-Viral Gene Transfer Meenakshi Noll,1 Ryan Bennett,1 Stephen Yant,2 Mark A Kay,2 Markus Grompe.1 Dept of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR; 2Dept of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies and currently, allogeneic bone-marrow transplantation is the only therapeutic option Despite the successful modeling of FA viral vector gene therapy in the mouse, application of the same approach to humans is complicated by the requirement for prolonged ex vivo manipulation and the scarcity of HSCs in this disease Here, we utilized the nonviral Sleeping Beauty (SB) transposon system to facilitate stable, gene transfer of the human FANCC cDNA without ex vivo culture This was achieved either by electroporation of naked DNA into whole bone marrow or by direct injection into the femur cavity Our studies demonstrate that a) hematopoietic stem cells from Fancc -/ - mice can be transduced by plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells (HSC) is possible; c) animal model was phenotypically corrected; and d) long-term therapeutic benefit could be achieved using this method Non-viral gene transfer may be a viable alternative for the treatment of hematopoietic diseases 1047 Chemoprotective Gene Therapy through Retroviral Expression of the X-Ray Cross Complementing Protein (XRCC1) Thomas D Southgate,1 Katherine Clarke,1 Leslie J Fairbairn.1 Gene Therapy, Paterson Institute for Cancer Research, Manchester, United Kingdom Administration of the chemotherapeutic camptothecin, a topoisomerase I inhibitor, causes a dose-limiting toxicitiy to rapidly renewing tissues leading to myelosuppression, diarrhoea and hemorrhagic cystitis Although the use of camptothecin deriviates, such as topotecan has reduced the urological toxicity associated with this class of compounds, they all still exhibit a marked toxicity to myeloid progenitors We have investigated whether retroviral transfer and expression of the x-ray cross complementing protein (XRCC1), using a vector (SFß91) which is optimised for transcription in primitive haemopoetic cells, could protect the haematopoietic compartment from camptothecin damage Although lacking any direct DNA repair activity, XRCC1 acts as a scaffolding protein facilitating the repair of both direct DNA strand breaks (such as those induced by camptothecin) and indirect DNA strand breaks (caused by agents such as the halogenated thymidine analogues) Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy GENE REGULATION: REGULATED SYSTEMS AND TISSUE SPECIFIC EXPRESS Here we demonstrate that cells transduced with SFß91 XRCC1 IRES eGFP acquire a fold increased resistance to camptothecin and 5-iodo-2’-deoxyuridine and display increased kinetics of repair Resistance to these agents could be reversed by addition of either the PARP inhibitor 3-aminobenzamide, or methoxyamine (an inhibitor of base excision repair) respectively Furthermore, cells transduced with XRCC1 demonstrate a significant increase in protection against ionising radiation In summary, retrovirallymediated transfer and expression XRCC1 to cells of the primary haemopoietic compartment delivers both physical and biological protection against the action of agents that cause either direct or indirect DNA strand breaks Our findings are encouraging for the development XRCC1 gene transfer for protection against the myelotoxic side effects of chemotherapy using camptothecin or its derivatives and we are currently evaluating this therapy in in vivo toxicity models 1048 Engineering and Development of I-AniI Homing Endonucleases for Gene Correction Applications Andrew M Scharenberg,1 David J Rawlings,1 Raymond J Monnat,3 Barry L Stoddard.2 Pediatrics, University of Washington, Seattle, WA; 2Basic Science, Fred Hutchinson Cancer Research Center, Seattle, WA; 3Genome Sciences, University of Washington, Seattle, WA Induction of a double strand break in DNA results in a large increase in the frequency of homologous recombination in the vicinity of the double strand break, raising the possibility of targeted gene correction if reagents were available for the simultaneous introduction of a site specific double strand break and a correcting DNA fragment Structural and molecular studies of homing endonucleases (HE’s) suggest that HE’s possess significant potential for engineering of their cut site specificities to diverse target sequences Here we discuss preliminary work on the I-AniI endonuclease with regards to its application to targeted gene correction of primary immune deficiencies Work to be discussed will focus on the engineering hurdles to be faced in applying I-AniI as a site specific DSB-inducing tool, and the development of model systems for the evaluation and benchmarking of HE’s and other DSB-inducing reagents in practical gene correction methods 1049 Serum Free Fedia Is the Best for Cord Blood Hematopoietic Cells Expansion Kamran Alimoghaddam,1 Mitra Khalili,1,2 Masoud Soleimani,3 Lili Moezi,1 Ardeshir Ghavamzadeh.1 Hematology, Oncology and BMT Research Center, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran; Molecuar Biology, Khatam University, Tehran, Islamic Republic of Iran; 3Hematology, Tarbiat Modaress university, Tehran, Islamic Republic of Iran Objectives: to find the best cell culture condition for expansion of cord bloods hematopoietic CD34+/CD38- ex vivo expansion and study the role of MIP/a in expansion potential Material and methods: CD34+/CD38- cells separated from cord bloods by Mini-MACS and cultured in different culture mediums including 50ng/ml of TPO,IL-6,SCF and flt3-ligand.for some samples we used RPMI+ 10% FCS or autologous cord blood plasma and for others we used serum free media(SF) Also we added MIP/a (1050ng/ml) to some samples We cultured the cells for weeks in CO2 incubator and studied expansion potential by counting of MNCs ,CFU-assay , LTC-IC and studied the number of CD34+/CD38cells before expansion days and 14 days after expansion Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy Results: expansion potential of cord blood hematopoietic cells was good and maximally expanded to 25 times Also we found that serum free media isbetter than FCS 10% and autologous cord blood plasma MIP/a did not changed expansion potential of hematopoietic cells Conclusion: serum free media is the best medium for expansion and from GMP points of view and MIP/a is useful for expansion to prevent maturation during expansion that may be useful for increasing transduction efficiency without induction of maturation GENE REGULATION: REGULATED SYSTEMS AND TISSUE SPECIFIC EXPRESSION 1050 Adenoviral Clostridial Light Chain Expression in the Medial Forebrain Bundle: A Reversible Model of Dopamine Depletion Mary E Garrity-Moses,1 Qingshan Teng,1 Jun Yang,1 Thais Federici,1 Erin Gilbert,2 Thyagarajan Subramanian,2 Nicholas M Boulis.1 Neurosurgery, The Cleveland Clinic Foundation, Cleveland, OH; Neuroscience, The Cleveland Clinic Foundation, Cleveland, OH We have previously demonstrated focal synaptic inhibition through neuronal expression of the light chain (LC) fragment of tetanus toxin in vivo The transient effects are spatially discrete lending them to application in deep brain nuclei This experiment examines feasibility in creating a rat model for Parkinsons disease through gene-based synaptic inhibition of the substantia nigra The present experiment examined the impact of nigral LC expression on apomorphine induced rotations Methods: Tetanus light chain (LC) was cloned into an adenoviral vector under control of the CMV promoter containing a GFP marker Next, the impact of unilateral nigral 6-OHDA on striatal dopamine and glutamate synapses was compared to unilateral nigral LC expression Rats received medial forebrain bundle (MFB) injections of either or 8µL of AdTeTxLC, 4µL of 6-OHDA or PBS Apomorphine-induced rotational behavior was assessed using a rotometer weekly for up to weeks Results: A significant increase in contralateral rotation was observed in the 6-OHDA positive control group and the 8µL TeTxLC group, in comparison to the 4µL TeTxLC and PBS groups 6-OHDA animals demonstrated an average of 7.84 rotations per minute (+/0.45 SEM) and rats receiving 8µL TeTxLC demonstrated an average of 4.39 rotations per minute (+/- 0.41 SEM) PBS rats demonstrated an average of 0.325 rotations per minute and rats receiving 4µL TeTxLC demonstrated an average of 0.708 rotations per minute Significance: This initial model proves the feasibility of dopamine depletion through nigral LC expression Because LC expression inhibits synaptic activity without killing neurons, this approach represents a strategy for transient dopamine depletion A subsequent experiment will apply an adeno-associated vector containing a Tet-on expression cassette (rAAV.Tet-on.LC) This latter vector will facilitate controlled, transient nigral suppression and will facilitate the study of behavioral recovery and normalization of striatal receptors following the recovery of striatal dopaminergic input Transient and controlled nigral inhibition may provide a superior model for studying striatal recovery and dopaminergic reinnervation S405 ... encouraging for the development XRCC1 gene transfer for protection against the myelotoxic side effects of chemotherapy using camptothecin or its derivatives and we are currently evaluating this therapy. .. expansion Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy Results: expansion potential of cord blood hematopoietic cells was good and maximally... a rat model for Parkinsons disease through gene- based synaptic inhibition of the substantia nigra The present experiment examined the impact of nigral LC expression on apomorphine induced rotations