Galpin/DA/N2 IIIBenv 
target cells with an efficiency at least comparable to that of the desired vector 45 - 46 . 
These observations emphasize that canine cell lines possess advantages for the 
production of murine retroviral vectors for human gene transfer therapies. 
(b) Split Structural Genes for gag/pol and env am : 
One of the most important safety concerns arises from the potential propensity of 
PCLs to generate RCR after introduction of a retroviral vector. This can occur as a 
result of homologous recombination between the provector DNA and the DNA 
encoding the MLV structural genes (gag/pol and env 1 ™ 1 ) present in the PCL 47 . This 
potential problem can be ameliorated by generating a PCL which contains genetically 
unlinked MLV structural genes (split genome). The split genome strategy was used in 
the construction of the canine packaging cell line, DA. However, it should be noted 
that the use of a split genome can not guarantee that recombination will not take place. 
(c) Reduced Sequence Homology Between Murine Retroviral Elements in the 
Producer Cell Line: 
There is reduced sequence homology between the MLV structural genes and N2 
IIIBenv retroviral DNA because the structural genes in the producer cell line use 
heterologous transcriptional promoter and termination signals instead of MoMLV 
LTRs. Multiple homologous recombination events within minimal sequence overlap 
would be required to generate a RCR (Figure 6). 
Approximately 700 base pairs of homologous sequence exist between the gag 
regions of the N2 IIIBenv vector and pSCVIO (the plasmid used to express MoMLV 
gag/pol in the PCL). However, only recombinational events upstream from the 
ATG->ATT mutation in N2 IIIBenv would result in RCR because a downstream 
recombinant could not make gag/pol protein. The functional overlap for homologous 
recombination is therefore less than 60 base pairs. 550 base pairs of apparent overlap 
45 Scolnick, et al„ J. Virol., 29:964-972, (1979). 
46 Scadden, et al.,7. Virol., 64:424427, (1990). 
47 Miller, Human Gene Therapy, 1:5-14, (1990). 
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