HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
G. Surveillance for adverse consequences of retroviral gene transfer. 
We will institute surveillance measures intended to identify any unexpected 
consequences of the retroviral infection. The following tests will be performed to look 
for evidence of ongoing retroviral infection or recombination. 
1. Western blot assays will be performed to identify antibodies against MMLV 
retroviruses in patients receiving transplants with infected cells and in laboratory 
workers who are handling large amounts of the retroviral vector. The principle of this 
test will be identical to that used to detect exposure to HIV in which the patient's 
serum is used as a first antibody on western blots containing viral proteins. 
Antibodies against retroviral proteins on the western blot are then detected with a 
second, labelled antibody which reacts with the bound human IgG. 
2. We will perform direct infection and marker rescue assays on patient's serum, 
urine, saliva, or other effluents. NEO-R containing provirus will be assayed directly 
by infection and selection of NIH3T3 cells. Naturally recombinant, wild type virus will 
be assayed by a marker rescue assay described above. 
3. Histological examinations of biopsy tissues or autopsy material from patients 
will be performed using antibodies against MMLV proteins. Expression of these proteins 
would provide evidence for recombination with wild type virus. These studies will be 
particularly important if long-term follow-up reveals diseases which may or may not be 
related to the gene transfer protocol. 
4. Clinical surveillance for malignancies. Perhaps the most pronounced concern 
about retroviral gene transfer is that random insertion adjacent to proto-oncogenes or 
in other locations will induce malignancies. We consider the theoretical risk of 
retroviral induced malignancies to be small. 
It should be noted that there is a considerable risk of lymphoproliferative process 
associated with conventional immunosuppressive treatment for transplants of various 
organs (including liver). The incidence of lymphoproliferative disease has been 
reported to be 1.6% (Starzl, 1984; update quoted in: Nakazato et al , 1989) and as high 
as 4% in children (Ho et al, 1988). This lymphoproliferative process is commonly due 
to EBV (Hanto et al, 1983) and can sometimes be reversed by discontinuing 
immunosuppressive therapy (at the expense of the graft) (Starzl et al , 1984). We will 
be using a similar immunosuppressive protocol and might expect to see a similar 
incidence of lymphoproliferative disorders due to this drug even without retroviral 
transduction. We will analyze any such disorders for EBV as well as for any evidence 
of the recombinant provirus or other murine proviruses. 
We are also concerned about the possibility of hepatic malignancies following HCT 
itself. Several hepatic diseases including hepatitis and inborn errors of metabolism 
are associated with higher risk of hepatoblastoma, and it is possible that malignancies 
may be observed in patients where HCT successfully restores hepatic functions. 
Recombinant DNA Research, Volume 14 
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