Weekly- month to month 6 
PRN (at least monthly)- month 6 and beyond 
Liver biopsy -month 3 
Complete metabolic screen as described in the preliminary evaluation annually 
2. Molecular Analysis of Liver Biopsy 
Techniques for detecting gene transfer and recombinant gene expression in liver tissue 
from recipients of autologous, retroviral transduced hepatocytes, have been developed. 
Examples of the application of these technologies in the analysis of the WHHL experiments are 
summarized in section II. C. and presented in reprints contained in the Appendix section. The 
major problem with the analysis of the percutaneous biopsy specimen will be the limited 
amount of material that will be available. Based on our extensive experience in the WHHL 
rabbit we believe we can do the following analyses with the material provided. 
- In situ hybridization to detect RNA from fresh frozen tissue (See Ref. 16, 18) 
- Immunocytochemistry to detect LDL receptor protein from fresh frozen tissues 
(Linder development). 
- Routine histology from paraffin sections 
-Polymerase chain reaction to detect proviral DNA (See Ref. 16) 
-Polymerase chain reaction to detect proviral RNA (Routine in the laboratory) 
Additional assays are available if more liver tissue becomes available such as RNase 
protection analysis (16 and 18) or Western blot analysis (Ref. 18). 
The specific protocol will be as follows: two-thirds of the biopsy will be fresh frozen in 
OCT and cyrostat sections will be postfixed in paraformaldehyde for in situ hybridization and 
also fixed in acetone for immunocytochemistry. The remaining tissue will be divided in half and 
frozen for subsequent PCR studies or fixed in paraformaldehyde, embedded in paraffin, 
sectioned, and stained with hematoxylin and eosin. 
H. Evaluation of Rejection 
1. Identification of Possible Immunologic Consequences of 
Autologous Cell Transfer 
This proposal involves the harvest, transformation and reimplantation of autologous 
hepatocytes. This raises several concerns about the potential immune response of the recipient 
to these cells. The most favorable aspect of this protocol is the use of autologous hepatocytes for 
cell transfer. Because these are autologous cells, there is no risk of rejection on the basis of 
histo-incompatibility and no immunosuppressive therapy will be used. This markedly 
decreases the potential risks for the patients and also will reduce the possibility that 
inflammation from rejection episodes might damage the recipient's liver or spleen. It also 
makes it unlikely that the patients would develop cytotoxic HLA antibodies that could prevent a 
subsequent conventional therapy with an orthotopic liver transplant. Therefore, this protocol 
offers less immunologic risk to the patient than conventional liver transplantation and will not 
decrease the likelihood that patients could subsequently receive an orthotopic liver transplant, 
if necessary, to control their hypercholesterolemia. 
In contrast to the low risk of HLA sensitization, reasonable concerns about immune 
responses to the transferred cells focus on the introduction of new and unique proteins into an 
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