promoter produces a 3.4 kb transcript responsible for translation of the LDL receptor protein. 
The 3' LTR in the plasmid vector, which has been deleted of enhancer sequences, forms the 
template for both LTRs in the integrated provirus (94). The rationale for this modification is 
to minimize the LTR transcript and maximize the translated p-actin transcript in the infected 
cells. Both the 5.6 kb and 3.4 kb recombinant derived LDL receptor transcripts were detected 
in infected hepatocyte cultures from each donor. As expected, the smaller transcript initiated 
from the internal promoter was more abundant than the larger LTR initiated transcript. 
Hepatocyte cultures transduced with either the BAG or the LDL receptor virus were 
analyzed for recombinant gene expression using cytochemical analyses. Expression of E. coli p- 
galactosidase in the BAG transduced cells was detected with a histochemical stain using the 
chromogenic substrate X-gal that produces a diffuse blue cytoplasmic precipitate (91). This 
analysis demonstrated lacZ expression in approximately 10 to 25 % of cells thereby confirming 
the estimate of gene transfer provided by Southern blot analysis (0.3 proviral copies per cell). 
Expression of recombinant LDL receptor protein was analyzed using an in situ functional assay 
which detects uptake of fluorescent labeled LDL (14). Mock infected hepatocytes demonstrated 
low levels of fluorescence representing uptake via the endogenous receptors. Analysis of LDL 
receptor transduced hepatocytes revealed a subpopulation of highly fluorescent cells comprising 
approximately 20% of the culture. This result indicates that the recombinant derived receptor 
is functional and can be expressed at levels in far excess of the normal endogenous receptor. 
3. Ex Vivo Gene Therapy in Nonhuman Primates 
a) Aims. 
i) To demonstrate the feasibility of performing ex vivo gene therapy at a 
scale that would be used in humans. 
ii) Assess the short term toxicity and risks of the proposed procedures in 
a nonhuman primate. 
b ) Rationale and Experimental Strategy. 
These experiments were designed to exactly simulate the proposed human experiment 
using nearly identical reagents and methods. The only conceptual difference between the human 
experiments and the baboon studies relates to the potential immunological consequences of ex 
vivo gene therapy. In the former studies, human LDL receptor is expressed in a genetically 
deficient human, whereas, in the latter studies a human LDL receptor is expressed in a normal 
baboon. 
The basic design of the experiment is summarized in Table 2. The animal is subjected to 
a preoperative evaluation to identify any pathology that may confound the experiment. On day 0 
the animal is taken to the operating room where it undergoes a partial hepatectomy (resection of 
the left lateral segment) and placement of an indwelling catheter into the portal circulation. 
The catheter is inserted into the proximal segment of the inferior mesenteric vein and the vein 
is ligated distally. The distal end of the catheter is tunneled subcutaneously to a posterior 
position and exited percutaneously between the scapulae where it is inaccessible to the animal. 
The resected liver tissue is immediately taken to the laboratory where it is perfused 
with collagenase to release hepatocytes. The resulting cell suspension is plated in primary 
culture on tissue culture plastic at a density of 2 to 4 x 10 6 cells per 10 cm plate. Viral stocks 
from the human LDL receptor #132-10 are placed on the primary cultures for a 12-16 hr 
period approximately 48 hours after the initial seeding of hepatocytes. Following this exposure 
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Recombinant DNA Research, Volume 15 
