Alpha-1 antitrypsin deficiency

Optimized bioassays for the preclinical screening of small molecules, siRNA and oligo therapeutic candidates and base editing approaches

A1ATD drug screening platform

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 introduced 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.

 

Compound response

 

 
ICC Endpoint HLCs
Figure 1. Intracellular accumulation of polymeric A1AT in A1ATD hepatocyte-like cells (HLCs) vs WT HLCs. A) Immunostaining with polymer-specific A1AT antibody shows the difference in polymer accumulation in A1ATD HLCs vs WT HLCs. B) Quantification of the intracellular levels of polymeric A1AT showing mean fluorescence intensity (MFI) per cell.
A1AT polymer accumulation consistently
Figure 2. Intracellular accumulation of polymeric A1AT in A1ATD Def-HEP cells decreases in a concentration dependent-manner when treated with Carbamazepine (CBZ). A) Immunostaining with polymer-specific A1AT antibody shows decreased levels of polymeric A1AT in cells treated with CBZ in a concentration-dependent manner. B) Quantification of the intracellular levels of polymeric A1AT showing mean fluorescence intensity (MFI) per cell (n=4). C) Heatmap showing quantification of polymeric A1AT across the central wells of a 96 well plate
Cells respond to CBZ
Figure 3. Intracellular accumulation of polymeric A1AT in A1ATD Def-HEP cells decreases in a concentration dependent-manner when treated with Carbamazepine (CBZ). A) Immunostaining with polymer-specific A1AT antibody shows decreased levels of polymeric A1AT in cells treated with CBZ in a concentration-dependent manner. B) Quantification of the intracellular levels of polymeric A1AT showing mean fluorescence intensity (MFI) per cell (n=4). C) Heatmap showing quantification of polymeric A1AT across the central wells of a 96 well plate

 

Related case studies:

Custom

Let us know your project specific requirements today
Contact us >
DefiniGEN-logo
Advance your scientific discoveries with our tailored disease modeling products and services.
Screening Services
Disease Modeling