HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
1. Clinical and laboratory tests (a summary of lab tests is in Appendix E) : i 
Engraftment will be monitored clinically by the patient's clinical course, by the :! 
appearance of liver-specific products (prealbumin, albumin, clotting factors (decrease jj 
PT, PTT) , and by serum levels of bilirubin, cholesterol, amino acids, and ammonia. | i 
Hepatic function will also be monitored using the lidocaine loading (MEGX) described * I 
above (Oellerich et al, 1987, 1989, 1990). Tests of hepatic function will be obtained,, 
when clinically indicated or at weekly intervals. Following discharge, these tests will.* I 
be repeated at monthly intervals for one year then as clinically indicated. | 
2. Histological tests: Biopsy samples obtained for clinical indications will be 
analyzed for the presence of cells transduced with the vector. If liver transplantation ■* 
is performed subsequent to HCT, the removed organ will be analyzed for the presence of j 
cells transduced with the vector. Current procedures call for liver biopsies to be [ 
performed ten days, one month, and one year following conventional OLT. We will perform ;i 
biopsies according to the same schedule. Additional biopsies will be performed when Ij! 
clinically indicated, or a yearly intervals for two years if evidence of engraftment Ij 
persists. If patients exhibit a long term remission of their hepatic disease an openf' 
biopsy will be performed to obtain a wedge biopsy. This biopsy will be used to evaluate 
the need to continue the immunosuppressive protocol. 
r 
3. Molecular tests: Several methods have been suggested for detecting - i| 
transplanted hepatocytes based on DNA polymorphism between donor and host cells. We , 
will evaluate the applicability of using PGR to identify VNTR sequence differences ■ ; 
between the donor and host. The presence of the donor cells should lead to a chimeric fcj 
pattern of VNTR detection. This method has been used successfully in assessing the *.ij 
success of bone marrow transplantation (Gasparini et al, 1989; Gatti et al, 1989). The f j 
sensitivity of this method may be limited by the amount of "stuttering" which creates ,1 
background in the amplified VNTR sequences. Conventional polymorphism might be .%!|| 
identified with greater sensitivity, particularly by PGR amplification across sequences ( 
after digestion with the polymorphic enzymes. Such methods, however, require 5 ;'i 
homozygosity for the restriction (+) phenotype in the host and at least one restriction | 
(-) allele in the donor and may be difficult to control properly in the range required !' !■ 
for this analysis due to the possibility of incomplete restriction digests and the 
sensitivity of the PGR reaction. Ij 
The purpose of genetically marking the donor hepatocytes is that the genetic marker®; 
provides additional opportunities to detect the transplanted cells. We will employ ft: 
multiple techniques to search for cells containing the NEO-R gene. Among the potential ft; 
techniques include: PGR for detection of proviral vector DNA in hepatic samples, fti 
enzymatic assay for the neomycin phosphoribosyl- transferase enzyme, in situ j 
hybridization, immunohistochemical staining using antibodies against the NEO-R gene, andftl 
hepatocellular isolation with selection in G418. None of these methods is ideal. ft 
The most powerful method is the use of PGR to identify rare proviral elements in DNA #'! 
extracts of liver samples. This method is sensitive and can detect the recombinant gene ^ 
in 10"^ cells (Rosenberg et al, 1990), is not dependent upon expression of the y 
recombinant gene, and can be employed in the small amounts of material available from • 
needle biopsy of the liver. PGR detection can be quantitated to some extent (at least 1 
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Recombinant DNA Research, Volume 14 
