(2) Describe any other naterlal to be used In preparation of the 
naterlal 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? 
Calcium phosphate mediated DNA transfection and physical microinjection 
were developed in the 1970's in order to introduce genes into mammalian cells. 
These techniques resulted in more-or-less stable integration of transferred 
genes into the cellular genome but were not sufficiently efficient to be useful 
for clinical gene therapy (see review by Anderson. Science . 1984; copy in 
Appendix B). An alternative and more efficient method of introducing genes 
Into cells was developed: namely, retroviral -mediated gene transfer. The gene 
of Interest is substituted for the viral genes in a DNA clone of the proviral 
genome. Initially, DNA constructs were transfected into 3T3 cells which were 
then infected with wild type helper virus in order to produce viral particles 
containing the retroviral vector. The presence of large quantities of helper 
virus allows viral replication as well as infection by both the desired vector 
and possible wild type recombinants. Therefore, alternative methods of vector 
production were designed. 
To avoid Introduction of helper virus, several laboratories (Mulligan. 
Verma, Miller) have constructed "packaging cell lines" with a wide host range 
specificity. A packaging cell line contains an integrated provirus which 
encodes the viral proteins necessary for producing viral particles but the RNA 
transcript cannot be packaged because of a deletion in its packaging (i.e.. 
recognition) sequence. The construction of the packaging lines used in our 
studies: ^ 2 , PA-12, and PA-317, has been described (Mann et al.. 1984; Miller 
et al.. 1985; Miller et al., 1986; copies supplied in Appendix B). ^2 has only 
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Recombinant DNA Research, Volume 12 
