Recombinant DNA Advisory Committee - 12/3-4/92 
Dr. Kahn distributed a draft of the FDA's proposed recommendations for the testing of 
gene therapy products. These recommendations were formulated based on the FDA's 
scientific and regulatory knowledge and on the advice of experts in the field of 
retrovirology. 
The FDA recommendations are as follows: (1) testing of the master cell bank and the 
final production cells by an initial co-cultivation step with cells that are susceptible to a 
wide variety of murine retroviruses, namely Mus dunni cells, and subsequent passage of 
these cells for 4 weeks; (2) testing of the supernatant generated from the co-cultivation 
procedure for the presence of RCR; (3) testing of each individual production lot, since it 
is unknown at what passage RCR are generated; (4) testing of the final production lot 
supernatant by amplification in Mus dunni cells, because this cell line is susceptible to a 
wide range of murine retroviruses, except Moloney murine leukemia virus (MMLV); (5) 
testing for MMLV by amplification on NIH 3T3 cells; (6) testing the supernatants 
generated from the amplification procedures for RCR; (7) testing of the transduced 
target cells by an initial co-cultivation with Mus dunni cells followed by 
immunofluorescence or PCR assay; (8) testing of the supernatant generated from the 
transduced cells by the S + L‘ assay. Dr. Kahn stated that the aforementioned assays are 
those that the FDA recommends as currently being the most sensitive assays for the 
actual detection of RCR. 
The viable immunofluorescence assay can be performed on infected Mus dunni cells 
using a broadly reactive monoclonal antibody that will detect the envelope products of 
all 4 host range classes of retroviruses, ecotropic, xenotropic, amphotropic, and mink cell 
focus-inducing (MCF) viruses. 
The FDA recommends that the feline S + L‘ assay should be performed with the PG4 cell 
line for the detection of xenotropic and amphotropic viruses and the mouse S + L' should 
be performed using D56 cells for the detection of ecotropic viruses. DNA from infected 
Mus dunni cells and transduced target cells can be analyzed by PCR using murine 
leukemia virus (MuLV)-specific primers. 
Dr. Kahn described 2 assays that could be performed in addition to the aforementioned 
retrovirus assays; however, they are not as sensitive as the other assays. Transmission 
electron microscopy (TEM) can be used to examine transduced human cells, and the RT 
assay can be performed on supernatants generated from these cells. TEM and RT may 
be useful for the detection of potential human retroviruses or recombination in the 
transduced human cells, because there are no cell lines or assays that can be 
recommended at this point due to their unknown host range. 
Dr. Kahn stated that these recommendations are subject to modification with the 
development of more sensitive assays for the detection of RCR. 
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Recombinant DNA Research, Volume 16 
