(10®/kg). Should this safety margin not be attainable, the patient will be 
ineligible for the transduction study. 
5.2 Preparation of Marrow 
Details are provided in technical appendices A and B. In brief, a marrow 
mononuclear cell fraction will be obtained using a Stericell separator. Thirty 
percent 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 for 6hrs with vector containing supernatants and 
will then be frozen. The patients will be treated with chemo-radiotherapy as 
per protocols and reinfusion with the transduced and non-transduced marrow 
will take place as indicated. 
6.0 EVALUATION OF EFFECTS 
6.1 Relapse 
If relapse occurs, then it will be determined whether or not the relapse 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. This question will be answered by analyzing 
the site where the marker gene has inserted using Southern blotting and RFLP 
and by inverted PCR (see technical appendices C and D). We will also 
determine whether the marker gene present in the relapsed cells is present in 
any normal circulating hematopoietic/lymphoid cells and determine whether 
the insertion sites in these cells are identical to those in the malignant clone. 
6.2 Regeneration of Cryopreserved Autologous Marrow 
When marrow is obtained to assess engraftment and disease progression 
(relevant protocol dependent), additional cultures will analyze whether marker 
genes are present in progenitor cells and in what lineage. Patients will also be 
monitored at weekly intervals for 6 weeks, monthly intervals for 6 months and 
annually thereafter for 5 years for the presence of cells containing the marker 
genes in the peripheral blood (PB) using FACS sorted populations. PB will be 
separated into T cells , B cells, monocytes and granulocytes using appropriate 
MAb, and each lineage examined for gene expression and for marker gene 
insertion sites by PCR and Southern Blotting and/or inverted PCR. To exclude 
the possibility of false positive PCR by low level contamination of one lineage 
by another, the amplification will be undertaken as quantitatively as possible 
for purposes of comparing positivity in each lineage. 
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