DefiniGEN provide iPSC-derived disease model hepatocytes of Alpha-1 antitrypsin (A1AT) deficiency; associated with the PiZ variant of the Alpha-1 antitrypsin, caused by a (G>A) point mutation at codon 342 (Glu342Lys) in exon 5 of the SERPINA1 gene. Using CRISPR gene editing we successfully introduce the PiZ mutation into our in-house iPSC line, differentiated to hepatocytes using our Opti-Diff process and screened for key hepatic and disease markers using high content imaging.
The resulting cells were shown to have a strong hepatic phenotype and to display increase intracellular expression of polymeric A1AT which is characteristic of Alpha-1 antitrypsin deficiency. Subsequently we assessed the suitability of these cells for screening of small molecule and RNA-based therapies by transferring them to multiwell plates and treating with reference compounds (in particular Carbamazipine) or RNA-modulating molecules.
The cells were able to demonstrate dose-dependent restoration of function across a broad range of drug concentrations, these data confirm both sensitivity and specificity of response to treatment and therefore this disease model is highly applicable to candidate drug screening in A1ATD.
Figure 1. Sanger sequencing of the edited A1ATD line showing a homozygous single nucleotide change (GAG>AAG) leading to
a lysine to glutamate substitution at residue 342 (E342K).
Figure 2. Characterization of Def-HEP CRISPR A1ATD disease modelled hepatocytes. Gene expression of key hepatic markers: ALB (Albumin), A1AT (a1-Antitrypsin) and HNF4a (Hepatocyte Nuclear Factor 4 alpha).
Figure 3. A) Immunostaining with polymeric-specific A1AT antibody (2C1) shows increased intracellular polymeric A1AT. B) Quantification of the polymeric A1AT staining expressed as the mean fluorescence intensity (MFI) per cell and normalized to WT, set to 1. C) Number of globular inclusions of A1AT polymer per cell normalized to WT, set to 1. *** p<0.001.residue 342 (E342K).
Figure 4. Gene expression of Def-HEP with A1AT deficiency 48h after transfection with scramble siRNA (NTC) and two different combinations of siRNA targeting SERPINA1 (A1AT). Gene expression of A1AT dramatically decreases with both sets of siRNA (A) but other genes such as AFP (B) or HNF4a (C) remain consistently expressed.
Figure 5. Immunofluorescence for total A1AT. Representative pictures of A1AT GFP-immunofluorescence (green) staining using 2C1 antibody for patient-derived A1ATD hepatocytes transfected for 72h with Vehicle (A), Scrambled siRNA (B), and two different combinations of siRNA targeting SERPINA1 (C, D). Representative pictures of A1AT staining for CRISPR-derived A1ATD hepatocytes transfected for 72h with Vehicle (E), Scrambled siRNA (F), and two different combinations of siRNA targeting SERPINA1 (G, H). Quantification of A1AT staining in the different conditions (I)
Figure 6. Immunofluorescence measurement of polymeric A1AT foci by high content imaging. iPSC-derived hepatocytes from a patient with A1ATD or CRISPR editing of a wild type line were treated with function restoring siRNA 1, siRNA 2, a non function control (NTG) or left untreated. siRNA treatment significantly reduced the number of vesicles contain polymeric A1ATD.
Figure 7. Intracellular accumulation of polymeric A1AT in Z-AAT Def-HEP decreases in a concentration dependent-manner when treated with Carbamazepine (CBZ). A) Immunostaining with polymer-specific A1AT antibody (2C1) shows decreased levels of polymeric A1AT in cells treated with CBZ. B) Quantification of the intracellular levels of polymeric A1AT.
Figure 8. Intracellular accumulation of polymeric A1AT in Z-AAT Def-HEP decreases in a concentration dependent-manner when treated with other compounds that increase autophagy. A) Immunostaining with polymer-specific A1AT antibody shows decreased levels of polymeric A1AT in cells treated with two different compounds that induce autophagy. B) Quantification of the intracellular levels of polymeric A1AT in cells treated with different concentrations of compounds that induce autophagy.