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A6358 - Defining PiMZ AATD Liver Disease Susceptibility with CRISPR Targeted Syngeneic iPSCs
Author Block: J. Kaserman1, M. Higgins2, D. N. Kotton3, A. A. Wilson4; 1Center for Regenerative Medicine (CReM), Boston University School of Medicine, Boston, MA, United States, 2Center for Regenerative Medicine, Boston, MA, United States, 3Center for Regenerative Medicine, Boston Univ Sch of Med, Boston, MA, United States, 4Center for Regenerative Medicine (CReM), Boston Univ, Boston, MA, United States.
Objective: Individuals homozygous for the “Z” mutation in alpha-1 antitrypsin deficiency (AATD) have long been recognized as having an increased risk for lung and chronic liver disease (CLD) driven in part by misfolded proteotoxic ZAAT-polymers. However, whether a single Z allele is sufficient to confer increased risk of disease remains unproven. To address the question of relative MZ injury risk, we have utilized CRISPR/Cas9 technology to correct the Z mutation, creating syngeneic MZ and MM variants from ZZ patient iPSCs. By comparing cells that are genetically identical other than at the diseased locus we can isolate the effects that result from ZAAT. We hypothesize that subsets of ZZ and MZ individuals are at increased risk of liver injury based on undefined genetic cofactors that alter hepatic AAT processing, and that CRISPR-corrected MZ iPSC-hepatic cells created from ZZ patients with established CLD recapitulate this susceptibility via a relative increase in hepatocellular AAT retention.
Methods: We selected highly phenotyped patient-iPSCs previously characterized by our lab from AATD patients homozygous for the Z mutation. gRNA sequences were administered together with two ssODN repair templates containing the sequence for ZAAT or MAAT to accomplish simultaneous homozygous and heterozygous correction. The syngeneic iPSCs were then differentiated to the hepatic stage and analyzed for changes in intracellular AAT accumulation.
Results: Using this approach we have achieved bi-allelic and mono-allelic correction in iPSCs from three individuals representing distinct clinical phenotypes. Upon differentiation to the hepatic stage the syngeneic MM iPSC-hepatic cells demonstrated significant and uniform reduction in intracellular AAT accumulation with a concomitant increase in protein secretion and restoration of neutrophil elastase inhibition relative to their parental ZZ-lines. In contrast the MZ iPSC-hepatic cells had significantly more variation in intracellular ZAAT protein retention with the highest levels occurring in the MZ iPSC-hepatic cells derived from the patient phenotype with the most severe CLD.
Conclusion: CRISPR genomic editing utilizing mixed ssODN repair templates can efficiently generate heterozygous and homozygous syngeneic daughter lines from AATD iPSCs. Following successful targeting of the Z mutation we have demonstrated the PiMM iPSCs lose the pathognomonic AAT retention in a uniform manner, while the isogenic PiMZ iPSCs have significant differences that may reflect patient specific variation in ZAAT processing. Through this approach we hope to advance our understanding of CLD risk in MZ individuals, and have generated a platform from which we can test potential therapeutics on iPSC-hepatic cells with defined disease phenotypes.