inactivating the antibiotic. While amikacin may be inactivated by this enzyme, 
gentamicin and tobramycin and other aminoglycosides do not contain a 
hydroxyl at the 3’ position and are not inactivated. Therefore, introduction of 
the neo^ gene would not exclude the use of aminoglycosides or any other 
conventional antibiotic that may be needed in the clinical management of 
these patients. 
In short, the vectors to be used in this study are currently in clinical trial, and 
in extensive prior primate testing have not been associated with any adverse 
effects. 
5.0 PLAN OF CLINICAL STUDY 
5.1 Specific Inclusion £md Exclusion Criteria for Gene Transduction (general 
eligibility see Section 10) 
To ensure that a sufficient aliquot of marrow can be removed for transduction 
without potentially compromising the rate of reconstitution, the minimum 
quantity of marrow mononuclear cells to be obtained at harvest will be raised 
by 30% above the minimum value quoted in previous neuroblastoma protocols 
(2 X 10*/kg). Should this safety margin not be attainable, the patient will be 
ineligible for the transduction study. 
5.2 Preparation of Marrow 
Details of preparation and transduction are given in appendices. A marrow 
mononuclear cell fraction will be obtained using a Stericell separator. 30% of 
these cells will be used for cell free vector transduction. The remaining 
marrow will be processed and frozen as per standard techniques. The aliquot 
will be incubated with vector containing supernatants and will then be frozen. 
The patients will be treated with chemo-radiotherapy as per protocol and 
reinfusion with the transduced and non-transduced marrow will take place as 
indicated in the protocols. 
6.0 EVALUATION OF EFFECTS 
6.1 Relapse 
If relapse occurs then NB cells will be separated from the marrow by FACS 
analysis separating cells which are CD34‘, CD45’ and UJ13A'^. It will be 
determined by PCR whether or not NB cells contain the marker genes. We 
will also determine whether the relapse has occurred from a single clonogenic 
progenitor or whether many cells have the ability to re-populate the patient. 
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Recombinant DNA Research, Volume 14 
