Rooney to establish donor derived lymphoblastic cell lines. We will attempt 
to establish spontaneous EBV infected LCLs as described in Appendix I. 
These lines will take about a month to establish and will be expanded to 10 8 
cells and frozen. 
4.2 Cytotoxic T Cell Line Preparation 
At the time of pretransplant evaluation for MISMUD the ETNA protocol will 
be discussed with the patients and parents and consent obtained. Ten cc of 
peripheral blood will be obtained from the donor at the time of bone marrow 
harvest and used to generate EBV specific CTLs as described in Appendix I. 
After establishment the CTL lines will be checked for specificity and 
microbiological culture. 
4.3 Transduction With the Neomycin Resistance Gene 
CTL are transduced with the LNL6 or GINa vectors used in the previous St. 
Jude gene marking protocols, AMLREM, NEBREL and NEBREM. The 
efficiency of marking will be estimated by clonogenic growth in G418 and 
semi-quantitative PCR for the neomycin resistance gene. 
Both LNL6 and GINa have been extensively studied in rodent models and in 
non-human-primates and in man since 1989. The results of the first in vivo 
human studies on transduced cells have been reported, as have our own safety 
data using marrow cells as the vector target (29,30,31). The safety data for 
LNL-6 and Gl.Na are contained within the Drug Master Files it BB-MF-3886 
and BB-MF-4195 which were used for granting our institutional IND's 3838 
(LNL6) and 4272 (GIN). The current proposal will cross reference these 
data. The major safety concerns and their resolution have been dealt with at 
length in previous published protocols. 
4.4 Administration and Monitoring 
At Day 45 patients will be evaluated for eligibility by one of the principal 
investigators and consent obtained. CTLs will be administered weekly in 
escalating doses. Patients will be monitored for clinical toxicity by standard 
NIH criteria (Appendix 2) and for GVHD by the Seattle criteria (Appendix 
3). In addition we will monitor the kinetics of CTL survival by monitoring the 
presence of the marker gene in peripheral blood. We will also analyze 
immunological effects by monitoring lymphocyte phenotype, changes in 
repertoire of T cell receptors, and cytotoxic effector function in the BMT 
laboratory. Dr Rooney's laboratory will monitor the levels of EBV DNA in 
peripheral blood before and following infusion. 
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