In order to fill the gap in studies performed in the mouse and heterologous systems, we generated human induced neuronal (iN) cells from induced pluripotent stem (iPS) cells derived from subjects carrying homozygous alleles for either MOR N40 or D40 in order to better dissect the role of MOR N40D in a physiologically relevant and human-specific model system. The aim of this project is to unravel the cellular/synaptic mechanism(s) of MOR N40D gene variants in a human neuronal cell context but we do not intend to elucidate the etiology of N40D MOR variants here because the main readout is patch clamp synaptic physiology (which is rather labor intensive and low in throughput). We found that MOR modulation of synaptic function is affected by N40D substitutions in human neurons in donor iPS cells (3 N40, 4 D40 homozygous subjects) as well as in two pairs of isogenic N40D neurons generated using CRISPR gene targeting. However, we believe an analysis of 3 vs 4 unrelated donors, varying at only a single SNP, will be difficult to justify any detectable effects at the
