Fusion Protein Increases Marking Following. Transplant into Wild Type Mice. Uimook Choi,1 Lanise Cardwell,1 Harry L. Malech.1. 1Laboratory of Host Defence, ...
HEMATOLOGIC leukemia, and compared them with control infections of HeLa cells using similar MFG-B2-γ-chain vectors. Although we did observe frequent reversion of the B2 mutation, as expected, the data do not support the hypothesis that cells with B2 mutated vector genomes improve reconstitution significantly. In fact, B2 mutant genomes constituted approximately 30-45% of the total, suggesting that the B2 mutated genomes may have been occasionally selected against during hematopoietic reconstitution.
recipients (P=0.0003). Finally, there was better survival of the ex vivo selected mice as compared to mice undergoing subsequent in vivo selection. Further experiments are ongoing to determine if combinations of ex vivo and in vivo selection may achieve results superior to either alone.
661. Ex Vivo Pre-Selection of Transgenic Mouse Cells Expressing Green Fluorescent Protein-Methylguanine Methyltransferase P144K Fusion Protein Increases Marking Following Transplant into Wild Type Mice
Mehreen Hai,1 Thomas R. Bauer, Jr.,1 Laura M. Tuschong,1 Yuchen Gu,1 Robert A. Sokolic,1 Dennis D. Hickstein.1 1 Experimental Transplant and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD.
Uimook Choi,1 Lanise Cardwell,1 Harry L. Malech.1 1 Laboratory of Host Defence, NIAID NIH, Bethesda, MD. Of all drug resistance strategies to enhance numbers of transduced cells in vivo, the most promising studies have used a mutant form of methylguanine methyltransferase (MGMT*) which is resistant to inactivation by the irreversible inhibitor, O6-benzylguanine (BG) while still retaining DNA repair activity after alkylation of DNA from 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU). MGMT* allows the use of in vivo selection regimens that combine BG with BCNU or Temozolomide to enhance gene marking. We have previously shown that the GFP-MGMT* fusion protein retains MGMT* activity and expresses GFP at high fluorescence allowing for easier tracking, and is thus a convenient model system to explore optimization of in vivo selection (Exp. Hematol. 32 (2004) 709). We subsequently engineered a GFP-MGMT* transgenic mouse where T-cells, B-cells, granulocytes, and Sca-1+ cells express 40%95% green fluorescence and used this model to demonstrate in vivo selection with BG/BCNU in wild type C57B6 mice following transplant of transgenic bone marrow (BM). However, we observed significant toxicity to mice associated with the most effective in vivo selection regimens particularly in animals starting out with the lowest percent of GFP-MGMT* transgenic cells, suggesting that higher starting levels of primary engraftment of GFP-MGMT* cells serves to protect the animals from selection regimen toxicity. To enhance the level of primary engraftment of cells expressing GFPMGMT*, we have begun to explore pre-transplant ex vivo selection methods. Using a 1:4 transgenic to wild-type mixture of BM exposed ex vivo to 20µM BG for one hour followed by addition of 40µM BCNU for 2 additional hours (cells washed before transplant) we transplanted 1x10exp6 BM cells into wild type recipients. Different levels of conditioning regimens were tested: myeloablative 900 cGy, subablative 600cGy, and non-myeloablative 300cGy with or without rapamycin (3mg/kg/d for 30 days), given the possibility of rejection of the GFP expressing cells with low dose radiation. Ex vivo selected recipient mice showed 5 to 8 fold enrichment of gene marking compared to the control mice receiving untreated cells (1.90±0.85%, 4.94±1.11%, 20.27±5.59%, and 76.42±7.29% of GFP positive BM cells vs. 0.38±0.18%, 1.01±0.57%, 2.42±0.79%, 11.13±1.60% in 300 cGy, 300 cGy with rapamycin, 600 cGy, and 900 cGy irradiated mice respectively). There was no statistically significant difference in peripheral blood counts between the two groups. Notably, the effect of rapamycin was also significant with greater marking in the mice receiving the immunosuppressant in the ex vivo selection
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
662. Retroviral Insertion Site Analysis Following Therapeutic Gene Transfer of CD18 in a Canine Model of Leukocyte Adhesion Deficiency
Retroviral insertion site analysis following the infusion of genecorrected hematopoietic stem cells has become increasingly important following the development of leukemia in three children with common γ-chain severe combined immunodeficiency treated with retroviral gene therapy. This analysis is of particular importance in human gene therapy trials and in gene-transfer studies in diseasespecific, large-animal models. We have conducted pre-clinical gene therapy in canine leukocyte adhesion deficiency (CLAD), the canine equivalent of leukocyte adhesion deficiency, type 1 (LAD-1) in humans. Both diseases are characterized by life-threatening bacterial infections and are due to mutations in the leukocyte integrin CD18. We have demonstrated reversal of the disease phenotype in six dogs with CLAD following infusion of autologous CD34+ bone marrow hematopoietic stem cells transduced with the retroviral vector PG13/ MSCV-cCD18 after non-myeloablative conditioning with either 200 cGy TBI or 10 mg/kg busulfan. In the six CLAD dogs who were long-term survivors, the level of CD18+ gene corrected leukocytes ranged from 0.72% to 8.37% at 1 - 1.5 years post-transplant. Although no adverse events have occurred during this period of observation, demonstration of polyclonality in the gene-corrected cells and characterization of the genomic sites of retroviral integration remain critical safety issues. To this end, we have demonstrated the polyclonality of contributing retrovirus-transduced cells in all six dogs using linear-amplification mediated PCR (LAM-PCR) with DNA obtained from peripheral blood leukocytes up to 18 months following infusion of gene-corrected cells. Multiple clones were shown to contribute to hematopoiesis; no predominant clones emerged over time. Retroviral insertion sites in four of the six dogs were identified using either LAM-PCR or ligation-mediated PCR (LM-PCR) followed by sequencing of the genomic sequence adjacent to the integrated viral LTR. Mapping of insertion sites was facilitated by the recent completion of the annotated dog genome sequence. To date, we have identified 60 unique retroviral insertion sites from four treated CLAD dogs. The insertions sites appear to be distributed almost equally across all 39 dog chromosomes, with a slight preference for chromosomes 2, 10 and 25. 43% of the sites were located within gene-coding regions of the genome (introns or exons). These observations match previous reports of preferential integration of MLV retroviral vectors near coding sequences. We are continuing to sequence and characterize a large number of retroviral insertion sites from the gene-corrected CLAD dogs. Analysis of these insertion sites should provide considerable insight into the risk of genotoxicity through insertional mutagenesis in a disease-specific large-animal model of a human hematopoietic disease.
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