an ecotroplc (mouse and rat) host range and. therefore, cannot be used for 
human studies. PA-12 Is amphotroplc (all species), however, it is "leaky" In 
the sense that approximately 0.1* of viral particles produced with N2-type 
vectors are replication-competent. PA-317 was developed by Miller specifically 
to provide a helper-free amphotropic packaging line for the family of N2 
vectors such as SAX. Recent results suggest that PA-317 might also be leaky, 
however. 
See Appendix A for an extended discussion of packaging lines. 
2. Precllnlcal 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. 
a. Laboratory studies of the delivery system 
(1) What cells are the Intended recipients of gene therapy? If 
recipient cells are to be treated In vitro and returned to the 
patient, how will the cells be characterized before and after 
treatment? What Is the theoretical and practical basis for 
assuming that only the treated cells will act as recipients? 
Bone marrow precursor cells are the Intended recipients of the retroviral 
vectors. The methods of characterizing the recipient cells Include (a) 
detection of vector sequences utilizing DNA hybridization techniques, (b) 
detection of vector transcripts utilizing RNA hybridization techniques, (c) the 
acquisition of resistance to metabolic toxins by cells In tissue culture, and 
(d) the demonstration of specific enzyme activities In the treated cells. 
Bone marrow cells are first removed from the recipient animal and are then 
Infected with the retroviral vectors In vitro . Medium containing the viral 
particles Is washed away from the marrow cells before the cells are returned to 
the recipient animal. In this way only the Intended recipient cells, the bone 
marrow cells, are Infected by the retroviral vector. 
As a model for a possible clinical protocol, our non-human primate 
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