Recombinant DNA Advisory Committee - 06/1-2/92 
assays offer the most sensitive measures of helper virus. Reverse transcriptase is not a 
very sensitive or useful assay. 
Dr. Dunbar explained that the most sensitive procedure for the detection of helper virus 
is to culture the producer lines for extended periods of time. Since helper virus is 
amplified quickly, high titers of contaminating virus will be detectable in culture. 
Mr. Capron asked if helper virus amplification occurred in vivo , would the only sensitive 
method of detection be if the animal or patient got sick? Dr. Dunbar explained that if 
amplification has not occurred in vitro , then there is no virus present during transduction. 
Mr. Capron inquired if there is no replication competent virus present during the 
transduction process than how is the 15 particle number derived? Dr. Dunbar said that 
no helper virus contamination was detected. However, based on statistical analysis of 
the number of milliliters tested and the sensitivity of the assay, it is not possible to say 
that there are less than 15 contaminating particles per liter. The producer cells are 
maintained in culture in order to detect amplified levels of contaminating helper virus 
particles and no helper virus has been detected. Therefore, the confidence level is 
probably much higher than 95%. Dr. Wivel suggested that if producer cells were 
coincubated with NIH 3T3 cells rather than supernatant, the sensitivity may be increased 
because of the fact that retroviruses are cell associated. 
Mr. Capron stated that if the absence of helper virus is an important criterion for 
approval of these protocols, then perhaps the FDA and the RAC should develop a 
standard regarding the acceptable level of confidence for helper virus contamination in 
these retroviral vector supernatants. 
Dr. D. Miller stated that 15 particles per liter is a reasonable standard. This standard is 
de facto anyway because the confidence level can be increased by growing the cells 
longer than is required to make clinical supernatant. Mr. Capron inquired as to what 
would be an acceptable time period for growing the producer cells? Dr. D. Miller 
responded that three weeks is probably an acceptable time frame; however, these 
standards would be more appropriately addressed by GTI. 
Dr. Dronamraju referenced a statement by the investigators that the transduction 
efficiency in myeloma and breast cancer cells has not been addressed because of 
difficulties encountered growing these cells in culture. Dr. Dunbar needs to expand on 
this statement. Dr. Dunbar responded that primary myeloma and breast cancer cells 
cannot be grown in culture long enough to determine transduction efficiency. Dr. 
Dunbar stated that she did not know if an interpretation can be drawn between 
transduction efficiencies in tumor cell lines versus primary tumor cells. 
Committee Motion 
Recombinant DNA Research, Volume 15 
[729] 
