Evaluation of Relative Promoter Strength in Primary Hepatocytes Using Optimized Lipofection

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Abstract
For most genetic deficiencies manifested in the liver, maximization of gene expression in hepatocytes will be an important factor in achieving successful gene therapy. A rapid, highly efficient, and nontoxic method for transfecting DNA into hepatocytes was used to compare directly promoter strengths of various cellular and viral promoters. Conditions are described here for transfecting 5–10% of primary hepatocytes using the positively charged liposomes, Lipofectin. Cells are not damaged by this method as they continue to transcribe genes controlled by liver specific promoters and can survive for over 2 weeks in culture. We find that the cytomegalovirus, SRα, and β-actin promoters are more active than the SV40, RSV, RNA polymerase II, albumin, α1-antitrypsin, or phosphoenolpvriivatc carboxykinase promoters. A simple TK promoter and a TK promoter with the polyoma enhancer (MCI) were almost completely inactive. This information will be useful in the construction of vectors designed to express genes efficiently in primary hepatocytes for purposes of gene therapy, although the stability of expression from these promoters will need to be demonstrated in hepatocytes in vivo. For most genetic deficiencies manifested in the liver, the ability of an individual liver cell to express the therapeutic gene at high levels will be critical for success of gene therapy. A variety of viral and cellular promoters were tested for their relative efficiency of expression in primary hepatocytes in order to identify those that exhibit the highest level. Further experiments will need to be performed to verify that a similar hierarchy of promoter strength will be observed from vectors after transfer into hepatocytes in vivo.