1071 Lentiviral Mediated Gene Correction of Human Hematopoietic Progenitors from Fanconi Anemia Patients Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� �[.]
STEM CELL GENE THERAPY FOR GENETIC DISEASES for each gene was directly proportional to the amount of the specific virus added and, as expected for an MOI of 0.5, the total expression level for each mixture was 50% The degree of co-transduction was approximately equal to the product of the individual expression percentages, with an equal amount of both giving the highest level of co-transduction (25% AGT:25% GFP = 12% dual positive) Furthermore, upon treatment with 10 μM BG and 25 μM BCNU, the enrichment of co-transduced cells was higher in cells that were initially transduced with a low AGT:GFP MOI ratio (8.8 fold vs 2.5 fold for 0.05:0.45 and 0.25:0.25 AGT:GFP, respectively) With selection, the overall proportion of GFP expression remains constant since AGT only and dual positive cells are enriched while GFP only and untransduced cells are lost Similarly, drug selection of an XCGD cell line co-transduced with a low AGT to high gp91phox MOI ratio restored the oxidase positive cell population to approximately 80% of wildtype cells (enriched >50 fold), as measured by nitro blue tetrazolium (NBT) staining To mimic ex vivo transduction/in vivo selection conditions, co-transduced K562 cultures were diluted 50 fold into untransduced cells and treated with BG/BCNU After enrichment, the proportion of AGT singly positive to AGT/GFP dual positive cells remained constant for AGT:GFP MOI ratios of 0.05:0.45 to 0.45:0.05 Transplants of x 106 bone marrow cells cotransduced with AGT and GFP (MOI AGT:GFP = 5:5) into nonmyeloablated murine recipients resulted in undetectable expression However, after successive treatments with BG/BCNU, dual expressing cells were detected in bone marrow, spleen, and blood up to 11% These data demonstrate that the AGT-P140K vector coupled with a marker/therapeutic vector will allow enrichment of cell populations co-transduced with genes expressed from independent transcriptional cassettes This has important ramifications for linking stem cell selection to therapeutic gene expression 1070 An Improved Method for Gene Transfer to Murine Hematopoietic Progenitor Cells Using Retroviral Vectors Philip W Zoltick,1 Cesare Campagnoli,1 Shuichi Ashizuka,1 Alan W Flake.1 Children’s Institute for Surgical Science, Children’s Hospital of Philadelphia, Philadelphia, PA, United States Incubation with retroviral supernatants is a current method of transferring genes into bone marrow hematopoietic cells (BMs) Producing viral supernatants is rapid without requiring development of stable retrovirus-producing cell lines Viral supernatants result in at most 40% transduction efficiencies which can be significantly less than the efficiencies observed when BMs are cocultivated with retrovirus-producing cell lines We have modified previously described methods by using multiple, short-term exposures to viral supernatants at low multiplicity of infection (MOI) to achieve reproducible, highly efficient transduction of hematopoietic progenitor cells The MSCV-based vector contained an eGFP marker gene located downstream of an IRES Bicistronic vectors were derived by insertion of the murine CXCR4 or human HOXB4 cDNAs upstream of the IRES Vectors were generated by transient transfection in 293T cells and titered on NIH3T3 cells Primary hematopoietic cells were harvested from adult Balb/c or C57Bl/6 mice treated 48 hours before with 150mg/kg of 5-FU iv Mononuclear cells were cultured in media supplemented with interleukin (IL)-3, IL-6, and SCF for 48 hours and transferred to fibronectin coated plates with fresh media The retrovirus vector was added at an MOI of for 12-14 hours Nonadherent cells were removed, washed with PBS, and similarly replated with vector Following the third incubation with vector, cells were washed and replated in the absence of fibronectin for an additional 48 hours Transduction efficiency was determined by Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy FACS analysis and colony progenitor assays Data was expressed as mean ± S.D Statistical values were determined by Student’s t test At 48 hours after infection, the efficiency of primary hematopoietic cell transduction with eGFP (n =5), CXCR4-eGFP (n=1), and HOXB4-eGFP (n=5) vectors was determined by FACS analysis of eGFP+ cells as indicated in Table Cells transduced with the bicistronic vectors were assessed for the frequency of eGFP+ hematopoietic progenitors in colony assays Transduction with the HOXB4-eGFP vector resulted in a significantly (p = 0.02) greater number of colony forming cells (CFC) than controls, eGFP or mock infected cells (see Table 1) The eGFP+ CFC accounted for a significantly (p = 0.02) greater proportion of the progenitors following transduction with the HOXB4-eGFP vector compared to that of the eGFP vector The method was effective in transducing various hematopoietic lineages including GM-CFC and BFU-e The presence of eGFP+ colonies in the GEMM-CFC and HPP-CFC assays suggest transduction of primitive progenitor cells These modifications in the gene transfer protocol decrease the exposure time between BMs and vector, not impair stem cell biology in colony and HPP-CFC assays, and produce highly efficient gene transfer to progenitor cells In vivo reconstitution studies are in progress to determine the efficiency of HSC transduction using this modified protocol Mock eGFP CXCR4-eGFP HoxB4-eGFP Table Results following transduction of BMs Fluorescent Number of Positive CFC/105 BMs (%) Cells 320.0 ± 40.0 49.1 ± 7.8 306.6 ± 11.5 52.4 340 48.1 ± 4.2 473.3 ± 50.3 Fluorescent Positive CFC (%) 44.3 ± 7.5 42.0 61.0 ± 3.6 1071 Lentiviral-Mediated Gene Correction of Human Hematopoietic Progenitors from Fanconi Anemia Patients Francesco Galimi,1 Yoshiyuki Kanazawa,1 Grover Bagby,2 John Wagner,3 Inder Verma.1 Laboratory of Genetics, The Salk Institute, La Jolla, CA; Oregon Cancer Center, Oregon Health Science University, Portland, OR; 3Pediatric BMT Program, University of Minnesota Medical School, Minneapolis, MN Fanconi Anemia (FA) is an inherited cancer susceptibility syndrome due to mutations in a DNA repair pathway including at least genes, named FANCA, FANCC, FANCB/D1, FANCD2, FANCE, FANCF, and FANCG The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and by high incidence of acute myelogenous leukemia and solid tumors Allogeneic bone marrow transplantation is a therapeutic option, but is burdened by the lack of HLA matched donors and serious treatment-related toxicities Although gene therapy holds great promise for FA in humans, oncoretroviral vectors have proven ineffective, given the impaired proliferation potential and limiting number of human FA hematopoietic progenitors (HPCs) and their consequent resistance to retroviral-mediated gene transfer We previously reported that Fanconi genes can be delivered with remarkable efficiency into quiescent stem cells obtained from FA knock-out mice, without any ex vivo expansion Preliminary data show that human hematopoietic progenitors purified from FA patients bone marrow can be phenotypically corrected by lentiviral vector transduction Human progenitors were purified from FA bone marrow samples, and transduced with a single exposure to a lentivector carrying the appropriate corrected gene Transduced human FA cells recovered the ability of in vivo engraftment, as shown by NOD/scid mice repopulation assays S413 STEM CELL GENE THERAPY FOR GENETIC DISEASES This preclinical study supports the rationale for the use of lentiviral vectors in human FA gene therapy have inserted the gene encoding the CID activable version of Jak2 (V’VJAK2) into this vector, and will test whether marrow cells transduced using the CD68 regulated vector exhibit monocyte/ macrophage specific responses to CID treatment These strategies may eventually provide a safe and effective means for achieving gene therapy for the lysosomal storage diseases CANCER APOPTOSIS 1073 Anti-Tumor Effect Produced by LowVoltage-Electroporation-Mediated Transfer of Angiostatin and Endostatin Gene in Mice Yoshio Gunji,1 Masaya Uesato,1 Takenori Ochiai,1 Hideaki Shimada,1 Hisahiro Matsubara,1 Shinichi Miyazaki,1 Hisahiro Nabeya.1 Department of Academic Surgery (M9), Graduate School of Medicine, Chiba University, Chiba, Chiba, Japan 1072 Selective Expansion of Genetically Modified Macrophage Progenitor Cells Kenji Ihara,1 Peter J Gough,2 Elaine W Raines,2 C Anthony Blau.1 Hematology, University of Washington, Seattle, WA, United States; 2Pathology, University of Washington, Harborview Medical Center, Seattle, WA, United States We have shown that chemical inducers of dimerization (CIDs) can be used in combination with genes encoding proliferationassociated signaling molecules to regulate the growth of genetically modified cells Previous studies have demonstrated that transfer of a gene encoding a CID-responsive “cell growth switch” into stem cells results in a CID-mediated expansion of all of the downstream lineages that express the growth switch Furthermore, unrestricted transgene expression in stem cells and their progeny raises concern regarding the potential for leukemia Here we examine strategies for restricting CID induced growth to cells belonging to one or two different lineages Since transplantation of monocytes/macrophages has been shown to be effective in animal models of lysosomal storage diseases, we focused on restricting the effects of CID treatment to monocyte/macrophage lineage cells We have undertaken two strategies for achieving lineage specific cell growth The first approach aims to achieve lineage specificity by targeting lineage committed progenitors, rather than stem cells, for gene transfer and transplantation This approach is supported by our previous observation that in some cases, lineage committed progenitors can retain a substantial capacity for self-renewal Mouse marrow cells were transduced with an MSCV-based vector encoding green fluorescence protein (GFP) and a CID activable version of Jak2 (GV’VJAK2) which we have shown to complement either c-kit ligand (KL) or flt-3 ligand (FL) in supporting the self-renewal of primary multipotential hemopoietic cells We used antibodies directed against the ER-MP12 and ER-MP20 antigens to immunomagnetically select cells committed to the monocyte/ macrophage lineage, and then tested these cells in the presence or absence of CID ER-MP12-selected cells exhibited a significant (5fold) albeit transient rise in GFP (+) macrophage-like cells in the presence of CID, and the effect was lost by day 10 of culture These results suggested that CID-mediated activation of JAK2 could support the differentiation of transduced monocyte/macrophage progenitors, but not their self renewal In contrast, ER-MP20selected cells failed to grow in response to the CID Our second approach to achieving monocyte/macrophage-specific expression of V’VJAK2 capitalizes on the recent description of a selfinactivating (SIN) vector incorporating a fragment of the 5’ flanking region and the first intron of the CD68 gene that can direct high level monocyte/macrophage gene expression in transplanted mice We S414 Electroporation facilitates transfer of chemicals or plasmid DNA from extracellular milieu into cells by increasing the permeability of the cell membrane Delivery of electric pulses to established tumor thereby can improve the susceptibility of tumors to an anti-cancer agent administered Recently low-voltage electrochemotherapy(CUY 21: electric fields ranging from 25 to 150 V/cm, TR TECH, Tokyo, Japan)has been reported to be effective on anti-tumor growth with convenience in clinical use We examined whether low-voltage electroporatin-mediated transfer of angiostatin and endostatin gene into solid tumors could produce anti-tumor effects in the tumor bearing mice Colon 26 tumor cells(1x106 ) were subcutaneouly injected into syngeneic Balb/c mice and were allowed to grow for days up to about 8-10 mm in diameter for the experiments Plasmid DNA containing mouse angiostatin(pBLAST-mAngio), and mouse endostatin ( p-BLAST-mEndo XV ) genes were injected into tumor nodules followed by electroporation Eight 100-ms electric pulses were delivered via the inserted needle electrodes, using a square electroporator The nominal applied field strengths (Voltahge to electrode spacing ration) of the pulses were experimental variables that ranged from 25 to 100 V/cm Inhibition of tumor growth was observed when 50 μg of pBLAST-mAngio, or p-BLAST-mEndo XV gene alone was administered into tumor nodule followed with low-voltage electroporation at a nominal electric field strengts of 50 V/cm However, no impact on the survival of mice was observed when either gene alone was delivered into tumor nodules When, a combined treatment of both genes was performed under low-voltage electroporation, it produced a significant inhibition of tumor growth There was also a significant difference in survival between mice treated with both genes in combination and control mice In vivo transfection of angiostatin, and endostatin genes with low-voltage electroporation could be a possible therapeutic strategy for established solid tumors when both genes were applied in combination 1074 Enhanced Paclitaxel Cytotoxicity and Prolonged Animal Survival Rate by Non-Viral Mediated Systemic Delivery of E1A Gene in Orthotopic Xonograft Human Breast Cancer1 Yong Liao,1 Yi-Yu Zou,1 Wei-Ya Xia,1 Mien-Chie Hung.1 Department of Mollecular & Cellular Oncology, U.T M.D Anderson Cancer Center, Houston, TX Taxol is a promising frontline chemotherapeutic agent for the treatment of breast and ovarian cancers To test whether E1A could sensitize low Her-2/neu-expressing MDA-MB-231 (p53 mutated) and MCF-7 (p53 wild-type) cell lines to Taxol-induced killing, we Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy ...STEM CELL GENE THERAPY FOR GENETIC DISEASES This preclinical study supports the rationale for the use of lentiviral vectors in human FA gene therapy have inserted the gene encoding the... combination with genes encoding proliferationassociated signaling molecules to regulate the growth of genetically modified cells Previous studies have demonstrated that transfer of a gene encoding... expression of V’VJAK2 capitalizes on the recent description of a selfinactivating (SIN) vector incorporating a fragment of the 5’ flanking region and the first intron of the CD68 gene that can