individual (F.A.) came in contact with the cells prior to production of the 
Master Cell Bank and he avoided contact with transmissible agents or 
experimental animals during this period. Numerous vials were stored in liquid 
nitrogen in anticipation that a new vial would be thawed and used for each 
individual patient treated. 
c) S3A was assayed for helper virus and was found to have a 
replication-competent titer approximately 0.1% the level of SAX vector titer. 
Optimally, we want a helper-free producer cell line that remains helper-free 
after being passaged for at least 10 population doubling levels (PDL). 
d) The Integrity of the SAX vector was tested by functional assays. G418 
resistance and human ADA production. In addition. S3A was assayed for the copy 
number (one copy/cell) and Intactness of the inserted genome by restriction 
mapping. S3A cells after 10 PDL maintained insert stability by Southern blot 
analysis. 
e) Morphology of the S3A cells has not changed for 10 PDL. 
f) S3A was tested for mycoplasm and found to be negative. 
2. Retroviral Vector Supernatant (RVS) 
RVS should be assayed for pyrogen, as well as screened for bacteria, fungi 
and viruses prior to use In a clinical protocol. 
3. Assessing the recombination threat 
Attempts should be made to assess the homology of the Mo-MuLV retroviral 
LTRs with that of existing human DNA sequences by hybridization of the N2 
parent vector at various stingency conditions with human DNA. 
(2) If a non-retrovlral delivery system is used: What animal 
studies have been done to determine if there are pathological 
or other undesirable consequences of the protocol (including 
insertion of DNA Into cells other than those treated, 
particularly germ line cells)? How long have the animals been 
studied after treatment? What tests have been used and what is 
their sensitivity? 
Not applicable 
Recombinant DNA Research, Volume 12 
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