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S. Verzeletti et al., in preparation.
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Verzeletti, S.1
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1842383754
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note
-
6 cells per 2-ml well in 24-well tissue culture plates containing RPMI 1640 with 5% human serum in the presence of recombinant human interleukin-2 (IL-2) (Euro-Cetus Italy S.r.I., Milan, Italy). For gene transfer, donor lymphocytes were cocultured with the irradiated packaging cell line for more than 48 hours in the presence of Polybrene (8 μg.ml), beginning the first day of polyclonal stimulation with phytohemagglutinin (2 μg/ml). No modification of culture conditions was introduced until the end of the procedure (13, 14). After gene transfer, transduced cells were selected for the expression of the cell surface marker ΔLNGFR by means of specific immunobeads. Cells were incubated with the murine monoclonal antibody 20.4 to human ΔLNGFR (American Type Culture Collection). After 30 min cells were washed and incubated with immunomagnetic beads coupled to a rat antibody to mouse immunoglobulin G1. After 45 min, positive cells were separated by use of magnetic immunobeads and incubated overnight at 37°C to remove antibody-coated beads. After one round of selection, the proportion of transduced cells was assessed by cytofluorimetric analysis (13). Up to this point of the study, a second round of selection was never required to achieve the indicated levels of purity. Cells were then cryopreserved for future infusions. In vitro ganciclovir sensitivity of HSV-TK-transduced lymphocytes (15), as well as acyclovir resistance (10), have been described previously. Before clinical use, cells were required to meet the specifications already in use in other clinical human gene therapy experiments. In particular, cells were tested for replication-competent retroviruses, IL-2-independent growth, and for adventitious infectious agents. Cells were infused by intravenous injection in saline with 4% human serum albumin at the time of diagnosis of complication (Table 1) and subsequently at monthly intervals (14). In some patients (Figs. 2 to 4) the schedule was adjusted in accord with clinical status and nature of the complication.
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-
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28
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1842398450
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note
-
Small amounts of peripheral blood in preservative-free heparin were obtained immediately before and after each lymphocyte infusion, weekly for the first 3 months after infusion, and monthly for the remainder of the first year and were used for the following immunological and gene marking studies: (i) cytofluorimetric analysis for the frequency of cells expressing the cell surface marker gene and for the ex vivo characterization of transduced cells; (ii) confirmation of the presence of transduced donor lymphocytes by PCR; (iii) G418 selection for detection and expansion of neo-transduced cells; (iv) ex vivo analysis of the persistence of ganciclovir sensitivity; and (v) ex vivo detection of immunity against transduced donor lymphocytes, by standard mixed lymphocyte culture and cytotoxic assays (25). Any diagnostic biopsy, including biopsies performed to detect GvHD, were used for the following studies: (i) immunohistochemical assays for the expression of the cell surface marker in infiltrating lymphocytes; (ii) analysis for vector gene presence by PCR; and (iii) immunophenotype of infiltrating lymphocytes. Serum, plasma, cells, and biopsies collected at specific time points were frozen for future analysis. After the administration of ganciclovir, peripheral blood samples were obtained daily for the first week and weekly thereafter. The in vivo selective elimination of transduced donor lymphocytes was monitored by means of the same immunological and gene marking criteria reported above.
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-
-
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30
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1842382775
-
-
note
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Patients were monitored weekly for the first month and monthly thereafter tor detailed clinical history, complete physical examination (including skin examination for the presence of GvHD), and general laboratory evaluation [including complete blood count and differential, urinalysis, blood urea nitrogen creatinine, bilirubin, aspartate transaminase (AST), alanine transferase (ALT), alkaline phosphatase, Na, K, Cl, albumin, total protein, glucose, and radiographic evaluation] as indicated. Disease status was assessed through examination of marrow aspirates and biopsy, cytogenetic analysis, and molecular analysis.
-
-
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31
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1842323040
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C. Bonini and S. Rossini, data not shown
-
C. Bonini and S. Rossini, data not shown.
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32
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1842351914
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M. Ponzoni and L. Ruggieri, data not shown
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M. Ponzoni and L. Ruggieri, data not shown.
-
-
-
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33
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1842356872
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-
note
-
If grade II GvHD or higher occurred, patients who had previously received transduced donor lymphocytes were treated with ganciclovir (10 mg/kg per day) for 7 days or less if all the GvHD signs and symptoms regressed. Patents who had previously received infusions of both transduced and untransduced donor lymphocytes were initially treated with ganciclovir at 10 mg/kg per day, to down-regulate GvHD.
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34
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1842271150
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personal communication
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H. J. Kolb, personal communication.
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49
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1842263320
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note
-
We thank N. Nobili, D. Maggioni, L. Parma, and G. Torriani for technical assistance; F. Candotti, H. J. Kolb, and P. Panina for help and support; and the nurses and clinical staff of the Gene Therapy and Bone Marrow Transplantation Unit. Supported by grants from Telethon, the Italian Association for Cancer Research (AIRC), the European Community (Bio 4-CT 95-0284), and Boehringer Mannheim (Penzberg, Germany).
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