M.J. Welsh and A.E. Smith, RAC Application 
Other proteins contaminating the virus preparation are likely to be from the 293 cells - that 
is, of human origin. Human proteins contaminating therapeutic agents derived from human 
sources are usually not problematic (121). In this case, however, we plan to test the 
production lot for transforming factors. Such factors could be activities of contaminating 
human proteins or of the Ad2/CFTR-1 vector or other contaminating agents. For the test, 
we propose to infect 10 dishes of Rat 1 cells containing 2 x 10 6 cells (the number of target 
cells in the patient) with 4 times the highest human dose of Ad2/CFTR-1 (2 x 10 8 PFU). 
Following infection, the cells will be plated out in agar and examined for the appearance of 
transformed foci for 2 weeks. Wild-type adenovirus will be used as a control. 
Transformation assays are done routinely at Genzyme (122). 
The bulk of nucleic acids and proteins would be expected to be separated from purified 
virus preparations upon equilibrium density centrifugation. Furthermore, we do not expect 
the human 293 cells to contain mouse VL30 sequences. Biologically active nucleic acids 
should be detected in the Rat 1 transformation assay. In addition the PCR analysis will 
reveal El sequences derived from the 293 cells. 
Because only a very small proportion of a production lot will be required for human use, in 
excess of 95% of the preparation will be available for testing. Thus, although it is difficult 
to access the sensitivity of some of the assays described above, we are in a position to test 
samples greatly in excess of the highest human dose. 
Bl.b.(l)(c) If co-cultivation is employed, what kinds of cells are being used for co- 
cultivation? What steps are being taken (and assays used with their sensitivity) to 
detect and eliminate any contaminating materials? Specifically, what test are being 
done to assess the material to be returned to the patient for the presence of live or 
killed donor cells or other non-vector materials (for example. VL30 sequences) 
ori ginating from those cells? 
Not applicable. 
B.l.b.(l)(d) If methods other than those covered bv (l)-(c) are used to introduce new 
genetic information into target cells, what steps are being taken to detect and 
eliminate any contaminating materials? What are possible sources of contamination? 
What is the sensitivity of tests used to monitor contamination? 
Not applicable. 
B.l.b.(2) Describe any other material to be used in preparation of the material to be 
administered to the patient. For example, if a viral vector is proposed, what is the 
nature of the helper virus or cell line? If carrier particles are to be used, what is the 
nature of these? 
The origin, growth, and characterization of the human 293 cell is described above. 
B.2. Preclinical studies, including risk-assessment studies. 
Describe the experimental basis (derived from tests in cultured cells and animals) for 
claims about the efficacy and safety of the proposed system for gene delivery and 
explain why the model(s) chosen is (are) the most appropriate. 
B.2.a. Laboratory studies of the delivery system . 
Recombinant DNA Research, Volume 16 
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