b. 
Laboratory studies of gene expression 
Is the added gene expressed? To what extent is expression only from 
the desired gene (and not from the surrounding DNA)? In what 
percentage of cells does expression from the added DNA occur? Is 
the product biologically active? What percentage of normal activity 
results from the inserted gene? Is the gene expressed in cells 
other than the target cells? If so. to what extent? 
Brief direct answers 
The inserted Neo R and human ADA genes are expressed in the hematopoietic 
system of monkeys at a low level. See detailed description in the attached 
manuscript. Kantoff et al.. J. Exp. Med. , in press (copy supplied in Appendix 
B). It appears that 1 in 100-200 cells is expressing significant levels of 
human ADA. which is biologically active, as well as detectable levels of NPT. 
At best 0.5% of the total ADA activity in the animal's hematopoietic system is 
composed of human ADA. There is no evidence, or indication, that DNA outside 
the provirus is contributing to NPT or ADA expression and there is no evidence, 
or indication, that any cells outside of the infected and re-infused bone 
marrow cells contain or express retrovirus vector genes. 
Overview: 
To study the expression of introduced genes using retroviral vectors, our 
laboratories have concentrated their efforts on vectors carrying a cDNA for 
human adenosine deaminase (ADA). Our primary vector, designated SAX, was 
derived from the parent vector. N2, which only carries the bacterial gene for 
R R 
neomycin resistance (Neo ). Expression of the Neo gene introduced by the N2 
vector has been demonstated in vitro in both human (Hock and Miller. 1986) and 
canine (Kwok et al . . 1986) hematopoietic cells. Gene transfer with the N2 
vector in murine bone marrow and subsequent expression in vivo following bone 
marrow transplantation has been achieved by our laboratory (Eglitis et al.. 
1985) and independently by Keller et al . (1985). The infection protocols used 
by our two groups varied slightly. Expression of the Neo R gene in the whole 
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Recombinant DNA Research, Volume 12 
