the gene transfer protocol will have serum samples removed for 
analysis as described in Section 9. PBL and serum will be stored 
frozen for further analysis and PCR. 
After treatment, the patients will be observed in the 
Clinical Research Center for 24 hours, and blood collections 
performed as described in Section 9. If there are no 
complications, the patient will be discharged after 24 hours. 
Should any abnormalities appear, the patient would be kept for 
further observation. 
6.2 Confirmation of Gene Transfer and Expression 
An aliquot will be processed to obtain DNA for PCR analysis. 
Limiting dilution PCR will be performed by standard methods. 
Reverse transcription PCR analysis will also be performed to 
confirm expression of the Rev M10 and ARev M10 transcripts. 
6.3 Analysis of Immune Parameters 
We will monitor immune parameters in these patients to 
determine whether the treatment exerts any therapeutic effects on 
the immune system. We will also monitor patients by an immune 
response to Rev M10 itself. To perform this analysis, a blood 
sample will be obtained prior to treatment to derive lymphocytes 
which will be immortalized using the Epstein-Barr virus. An 
aliquot of these cells will be further infected with an 
amphotropic Rev M10 retroviral vector (16) , and selected for G418 
resistance. Expression of Rev M10 will be confirmed by RNA 
analysis. These cells will subsequently be used in the 
laboratory as target cells for the cytolytic T cell assay. At no 
time will these cells be brought into the same building where the 
[18] 
Recombinant DNA Research, Volume 18 
