Abstract:
HepaRG cells are a suitable in vitro human liver model for absorption, distribution, metabolism, and excretion (ADME) research and toxicity studies, as they express multiple cytochrome P450 (CYP) isozymes and exhibit several regulatory pathways in a comparable level to primary human hepatocytes. CRISPR/Cas9-mediated genome editing of HepaRG cells could thus be a promising tool for new investigations regarding interindividual differences in ADME processes. However, application of CRISPR/Cas9 genome editing to HepaRG cells could be challenging because of their non-clonal origin and the required differentiation process to develop hepatic properties.
Due to their polyclonal origin, it was necessary to investigate whether differentiated clonal HepaRG cells have a hepatic phenotype comparable to the parental cell line. However, clonal selection of HepaRG cells resulted in a heterogenous mixture of individual cell clones where many of them lost their differentiation capability. Further single cell selection of clonal HepaRG cell lines resulted in a slight stabilization of phenotype but no completely homogenous phenotype was detectable which would be necessary for investigation of phenotype-genotype relations in genome-edited cell clones.
As HepaRG cells are hard to transfect several CRISPR/Cas9 delivery methods had to be tested. Two effective working protocols for CRISPR/Cas9-induced genome editing in HepaRG cells were established in this thesis. Lentiviral delivery of Cas9 and sgRNA was shown to be an effective approach for genome editing in HepaRG cells. This approach was used to create a HepaRG cell line that constitutively expresses Cas9 and that retains the ability to differentiate into hepatocyte-like cells with CYP expression and activity profiles that are highly similar to those of the parent cell line. Transfection of sgRNAs into these cells can now be used to study the influence of various genes on drug metabolism and other hepatic functions in a metabolically competent human hepatic cell line.
As a first target for CRISPR/Cas9-induced gene knockout in HepaRG cells the NADPH cytochrome P450 reductase (POR), a ubiquitous flavoprotein localized in the endoplasmatic reticulum, was chosen. POR is required for the two-electron transfer from NADPH to microsomal CYPs and is therefore essential for CYP-mediated drug metabolism as well as for other CYP dependent endogenous processes. Genetic knockout of POR resulted in differential, isozyme-dependent effects on CYP activities. The seemingly weak impact of POR knockdown on CYP2C8 activity led to the unveiling of a general role of CYB5 as alternative NADH-dependent electron donor in HepaRG cells, in particular for CYP2C8-dependent amodiaquine N-desethylation. This was confirmed by CRISPR/Cas9-mediated genetic CYB5A single and POR/CYB5A double-knockout using transfection of sgRNAS in Cas9 expressing HepaRG cells.
To further characterize the impact of POR knockdown on a more global level, drug metabolizing CYP protein expression, mRNA expression of a selected panel of genes and bile acid secretion were analyzed. POR knockdown influences the mRNA expression of several transcriptional regulators of hepatic CYP expression, bile acid and lipid homeostasis leading to decreased expression of various CYPs involved in drug as well as endogenous metabolism. The measured changes in bile acid homeostasis could be responsible for the observed gene expression patterns. Moreover, additional knockout experiments of CYP27A1 were performed to further analyze the involvement of POR and CYP27A1 in bile and lipid homeostasis.
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