279 Retroviral Vector Mediated In Vivo Transduction of Hematopoietic Stem Cells with Neonatal IV Injection Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������© ����������� �!����[.]
NOVEL APPROACHES FOR HEMATOPOIETIC GENE THERAPY D-luciferin preincubated BMPC Dilution of BMPC local concentration resulted in disappearance of signal between 36-72 hours Later observations demonstrated BMPC localization to the final hematopoietic sites: spleen, sternum, femur, tibia and spine No signal was identified in the control group Weak sources of photons associated with the projection of open skin/mucosa areas corresponded to the subinfrared thermal photon background PET imaging with [18F]FIAU is in progress Conclusions: Migration of BMPC can be efficiently monitored in vivo using highly sensitive BLI allowing the imaging of a small number of cells Lower limits for BMPC dose for BLI were identified Triple reporter-gene system reduced ex vivo BMPC culture maintenance time and provided several non-invasive imaging options including BLI, fluorescence and PET The 2nd patient received the transplantation of the MDR1transduced cells in October 2001 One week after the transplantation, 3.3 % of the peripheral blood mononuclear cells were P-gp-positive This patient was not treated with DTX for months The P-gppositive cells decreased, and almost disappeared before the start of DTX treatment Then the patient received cycles of consolidation chemotherapy with DTX After the DTX treatments, P-gp-positive cells were detected in the peripheral blood as determined by FACS and PCR These two patients are disease-free (clinical CR) and in good condition now No side effects associated with the transplantation of the MDR1-transduced cells were observed No symptom of lympho/myeloproliferative disease has been observed These results suggest the safety and feasibility of our MDR1 gene therapy 278 A Clinical Study of MDR1 Gene Therapy Against Breast Cancer Showed the In Vivo Expansion of the MDR1-Transduced Normal Hematopoietic Cells by Docetaxel 279 Retroviral Vector-Mediated In Vivo Transduction of Hematopoietic Stem Cells with Neonatal IV Injection Yoshikazu Sugimoto,1 Shunji Takahashi,2 Junko Mitsuhashi,2 Minoru Nakane,1 Satomi Tsukahara,1 Tetsuko Nagamine,2 Sayuri Minowa,2 Harumi Shibata,2 Yoshinori Ito,2 Keisuke Aiba,2 Kiyohiko Hatake,2 Takashi Tsuruo.3,4 Division of Molecular Biotherapy, Cancer Chemotherapy Center, Jpn Fdn Cancer Res., Tokyo, Japan; 2Division of Clinical Chemotherapy, Cancer Chemotherapy Center, Jpn Fdn Cancer Res., Tokyo, Japan; 3Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Jpn Fdn Cancer Res., Tokyo, Japan; 4Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan The MDR1 gene encodes the plasma membrane P-glycoprotein (P-gp) that acts as an ATP-dependent efflux pump for various antitumor agents Transduction of the hematopoietic progenitor/ stem cells of cancer patients with the MDR1 gene would be an attractive strategy to treat the patients with anticancer agents without life-threatening myelosuppression Here we show some promising results from our clinical study Two recurrent breast cancer patients were enrolled in this study After obtaining informed consent, peripheral blood stem cells were harvested from the patients and about one third of those cells were enriched for CD34+ cells The CD34+ cells were transduced with HaMDR retrovirus supernatant in the presence of cytokines (SCF, FL-ligand, IL-6, sIL-6R, TPO) and fibronectin fragment CH-296 (Takara Bio, Otsu, Japan) Transduction efficiencies ex vivo were 13-17 % in FACS analysis using MRK16 antibody In April 2001, the 1st patient received high dose chemotherapy and the transplantation of the MDR1-transduced CD34+ cells and unmodified peripheral blood stem cells The transplanted P-gppositive cells were 22 millions, which were % of whole transplanted CD34+ cells One week after the transplantation, 3-4 % of the peripheral blood mononuclear cells were P-gp-positive The ratio of P-gp-positive cells decreased to % in months Then the patient received 10 cycles of consolidation chemotherapy with docetaxel (DTX) every 3-6 weeks (1) In the 1st or 2nd cycles of DTX treatment, the ratios of P-gp-positive cells in the peripheral blood prior to the chemotherapy were % They increased to 2-3 % right after DTX treatment These increases were transient, and the P-gppositive cells decreased to the previous levels within weeks (2) In the 4th to 10th cycles of DTX treatment, the ratios of P-gp-positive cells prior to the chemotherapy were 2-4 % They increased to 5-10 % after DTX treatment, but they decreased to 2-4 % within weeks (3) Ten months after the 10th DTX treatment, 1-3 % of the peripheral white blood cells were still P-gp-positive These results suggest that MDR1-transduced cells were selectively enriched in vivo by DTX treatment S110 Lingfei Xu,1 Mark S Sands,2 Alex A Hofling,2 Katherine P Ponder.1 Internal Medicine, Washington University School of Medicine, St Louis, MO, United States; 2Internal Medicine and Genetics, Washington University School of Medicine, St Loius, MO, United States The purpose of this study was to determine if a Moloney murine leukemia-based retroviral vector (RV) could transduce hematopoietic cells in vivo after neonatal IV injection Homozygous C57BL/6bm1 mice (n=5) were injected IV with 1x1010 transducing units (TU)/kg of an RV expressing canine Factor IX (cFIX) at or days after birth These mice expressed cFIX at 154% of normal levels, and the liver was the major organ transduced with 1.09+/-0.06 (SEM) copies of the RV per cell at 10 months after transduction as determined by real-time PCR At 15 months after transduction, peripheral blood cells from these mice contained 0.024+/-0.006 copies/cell of RV DNA To further test if hematopoietic stem cells were transduced, bone marrow (BM) was harvested from three RV-treated mice at 18 months after transduction Unfractionated peripheral blood cells contained 0.03+/-0.01 copies/per cell, while unfractionated BM cells contained 0.04+/-0.01 copies/cell of RV DNA FACS-purified BM cells that were Gr1/Mac1-positive (granulocytes and monocytes), B220-positive (B lymophocytes), and TCR-positive (T lymphocytes) contained 0.06+/-0.03, 0.07+/-0.03 and 0.07+/-0.02 copies/cell of RV DNA, respectively (Table 1) Samples from nontransduced mice did not have any detectable RV DNA sequences Part of the BM from each mouse was used for bone marrow transplantation (BMT) MPS VII mice (C57BL/6bm1/mps) from the same colony were used as recipients to enable us to determine if engraftment occurred by staining for β-glucuronidase activity Twomonth-old MPS VII mice were irradiated with 1000 rads before BMT, and 3x106 BM cells from the RV-transduced donors were injected via the tail vein into each recipient The peripheral blood from the recipients contained 0.076+/-0.022 copies/cell of RV DNA at months after BMT (n=7) (Table 1) We conclude that IV injection of RV into neonatal mice can transduce pluripotent hematopoietic stem cells in vivo This could be a problem for liver-directed gene therapy for hemophilia, since unnecessary transduction of BM cells would increase the risk of insertional mutagenesis In this study, transduced cells did not appear to have a selective advantage, as peripheral blood cells from hemophilia B mice that were injected with the same dose of RV as neonates had a similar copy number of 0.03+/-0.01 copies/cell at months after transduction (n=14), although additional studies are necessary to address this possibility On the other hand, transduction of pluripotent stem cells after IV Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy NOVEL APPROACHES FOR HEMATOPOIETIC GENE THERAPY injection into newborns might be an effective way to get genes into BM cells, particularly in large animals where in vitro transduction has been inefficient RV DNA copy per cell in the blood and bone marrow RV DNA copies per cell in donors RV DNA copies per @18m after RV-transduction cell in recipients @3m after BMT Peripheral Whole Gr1/Mac1 B220 TCR Peripheral Blood BM Positive BM Positive Positive Blood BM BM C1 0.01 0.04 0.04 0.05 0.03 0.03+/-0.00 (n=2) C5 0.04 0.05 0.12 0.13 0.09 0.13+/-0.03 (n=3) C6 0.04 0.02 0.03 0.03 0.09 0.05+/-0.01 (n=2) 280 Transduction of Primary Human Macrophages and Dentritic Cells by Recombinant SV40 Vectors Pierre Cordelier, Sandra A Calarota, David S Strayer Monocyte-derived macrophages (MDM) and dentritic cells (DC) are unique targets in the genetic therapy of infectious diseases and cancer Gene transfer to MDM and DC has been difficult to achieve using conventional vector systems because these cells are terminally differentiated and non-dividing, and their phagocytic functions lead to degradation of the phagocyted vector particles Recombinant SV40-derived vectors (rSV40) generally transduce resting cells efficiently In addition, rSV40 enter cells via caveolae and are then transported to the nucleus via microtubules, bypassing the phagocytic process Because of these properties of SV40 vectors, we tested whether they could transduce MDM and DC effectively For this purpose, we used two different rSV40s: SV(nef/FLAG), in which the HIV-1 protein Nef carrying a carboxyl terminal FLAG epitope is driven by the CMV promoter This construct was used as a marker, to facilitate detection of effective gene delivery by immunostaining for FLAG We also used SV(2C7), which carries a single chain Fv antibody against the cell membrane CCR5 In SV(2C7), transgene expression is also driven by the CMV promoter DC and MDM were prepared from buffy coat peripheral blood monocytes Monocytes were isolated by magnetic sorting for CD11b These cells were cultured with GM-CSF and M-CSF for two weeks to produce MDM DC were prepared by sorting for CD14, and were induced to mature using TNF-a MDM were treated with either SV(Nef/FLAG) or SV(2C7) Control MDM were mocktransduced or transduced with SV(HBS), a control rSV40 carrying the hepatitis B surface antigen Expression of Nef/FLAG and 2C7 was assessed by immunostaining MDM expressed high levels of these transgenes after transduction with SV(Nef/FLAG) and SV(2C7), respectively We also tested the ability of rSV40 gene delivery to blood monocytes to survive the differentiation process into MDM Thus, peripheral blood monocytes received SV(Nef/ FLAG), SV(2C7), or control treatments, were induced to differentiate into macrophages as described, then tested for transgene expression We found that transgene expression continued to be strong in macrophages derived from rSV40-transduced monocytes In parallel, we tested the ability of rSV40 vectors to transduce primary DC, by immunostaining We observed that SV(Nef/FLAG) transduced both immature and mature DC cells with high efficiency Therefore, primary human MDM and DC and their more differentiated progeny may be efficiently transduced with rSV40 vectors These findings have important implications for approaches to immunization and treatment of many diseases that entail the ability to deliver transgenes to monocytes, macrophages and dentritic cells 281 Engineering Hematopoieitc Bone Marrow Cells Resistant to Thymidylate Synthase-Directed Cytoxic Inhibitors Marg Pena,1 Erin Morrey,2 H Trent Spencer.2 Biological Sciences, University of South Carolina, Columbia, SC, United States; 2Pediatrics; Division of Hematology/Oncology and BMT, Emory University School of Medicine, Atlanta, GA, United States The generation of genetically engineered drug resistant hematopoietic bone marrow cells can be used to decrease the myelosuppressive effects of anti-neoplastic chemotherapy agents and to preferentially increase the percentage of circulating genetically modified cells We generated MSCV-based retroviral constructs that encode the E coli thymidylate synthase (TS) enzyme and show that the cDNA optimized for expression in mammalian cells (optecTS) confers extremely high-level resistance against TS-directed antifolates Resistance can be conferred to several mouse and human cell lines as well as mouse hematopoietic progenitor cells Attempts to use the high-degree of resistance for in vivo protection were unsuccessful in mouse studies due to complications arising from circulating thymidine and folate levels and differences in the binding of inhibitors to mouse transporter proteins compared to human transporters However, it is shown that gene-modified cells can be selectively enriched ex vivo Similar to our earlier studies, transduction of mouse bone marrow cells with retroviral vectors encoding both optecTS and the green fluorescent protein confers resistance to BW1843U89 and raltitrexed at concentrations that completely inhibit growth of non-transduced cells, and all transduced cells growing in the presence of inhibitor are brightly fluorescent green, demonstrating that the proviral sequence confers drug resistance Also, mixing gene-modified mouse bone marrow cells with 70% non-modified cells and plating the mixture in methylcellulose with or without BW1843U89 selection, the number of GFP positive colonies are enriched from 20% to >75% In addition, hematopoietic stem cell protection was demonstrated using a competitive repopulation assay Bone marrow was harvested from 5-FU treated HW80 mice and co-cultured with MSCV/optecTS producer cells for three days On day transduced cells were mixed with an equal number of mock transduced C57Bl marrow and BW1843U89 with or without the nucleoside transport inhibitor dipyridamole The mixed population of cells were selected on days 3, and On day cells were washed and transplanted into a WWv recipient Eight weeks post transplant hemoglobin electrophoresis confirmed, i) optecTS protects hematopoietic stem and progenitor cells from BW1843U89 induced toxicity and ii) nucleoside transport inhibition is necessary for selection of primitive hematopoietic cells These results further show that the optecTS construct is a viable marker for selection of genetically engineered hematopoietic cells 282 T Cells Transfected with Ad-hSSTR2 into T Cells from hCARxTg71xGFP Triple Transgenic Mice Can Be Used To Determine T-Cell Activation and AICD In Vitro and In Vivo Huang-Ge Zhang,1,2 James M Mountz,3 Qi Wu,1 PingAr Yang,1 Hui-Chen Hsu,1 John D Mountz.1,2 Medicine, University of Alabama at Birmingham, Birmingham, AL, United States; 2Birmingham VAMC, Birmingham, AL, United States; 3Nuclear Medicine, University of Alabama at Birmingham, Birmingham, AL, United States The Db/H-Y T-cell receptor (TCR) transgenic (Tg71) mouse expresses high levels of a TCR that reacts with the Db/H-Y male antigen These T cells are autoreactive in H2b male mice but not H2b female mice, and tolerance is induced by clonal deletion and downregulation of CD8 To study these tolerance processes in vivo, we Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy S111 ... APPROACHES FOR HEMATOPOIETIC GENE THERAPY injection into newborns might be an effective way to get genes into BM cells, particularly in large animals where in vitro transduction has been inefficient... non-dividing, and their phagocytic functions lead to degradation of the phagocyted vector particles Recombinant SV40-derived vectors (rSV40) generally transduce resting cells efficiently In addition,... differences in the binding of inhibitors to mouse transporter proteins compared to human transporters However, it is shown that gene-modified cells can be selectively enriched ex vivo Similar