UCLA

Dominant optic atrophy (DOA) is the most prevalent genetic optic neuropathy, affecting roughly 1:12,000 to 1:50,000 individuals worldwide. DOA patients exhibit retinal ganglion cell (RGC) degeneration, which leads to progressive bilateral vision loss. The majority of DOA cases are caused by mutations in the nuclear gene optic atrophy 1 (OPA1), which encodes a dynamin-related GTPase that localizes to the inner mitochondrial membrane. Although OPA1 is ubiquitously expressed in all human tissues, RGCs appear to be the primary cell type affected by OPA1 mutations. It is therefore essential to study DOA in human RGCs to understand how an OPA1 deficiency renders them particularly susceptible to degeneration. To overcome the scarcity of human RGCs, we have focused on establishing in vitro DOA disease models using human pluripotent stem cell (PSC)-derived 3D retinal organoids (ROs) that spontaneously develop human RGCs. We have established isogenic, OPA1 mutant, human embryonic stem cell (ESC) lines using CRIPSR-Cas9 gene editing as well as induced pluripotent stem cell (iPSC) lines from DOA patients with distinct OPA1 mutations. To derive isogenic control iPSCs, we have also corrected one DOA patient’s mutation by performing CRISPR-Cas9-mediated homology directed repair (HDR). Western blot analysis demonstrates that wild-type (WT) and OPA1 heterozygous mutant PSCs have similar expression levels of the same five OPA1 protein isoforms and that the five isoforms are expressed relatively equally to one another. As expected, total OPA1 protein levels are reduced in PSCs that contain heterozygous OPA1 nonsense mutations and are restored to WT control levels in an isogenic corrected line. Additionally, OPA1 homozygous loss of function ESCs lack OPA1 expression. Structured illumination microscopy (SIM) reveals the OPA1 homozygous loss-of-function ESCs have an altered mitochondrial morphology from WT PSCs and from PSCs with OPA1 haploinsufficiency. Finally, cellular respiration assays show that OPA1 mutant PSCs have significantly lower oxygen consumption rates (OCR) and mitochondrial ATP production rates than isogenic WT control PSCs. As expected, heterozygous OPA1 mutant PSCs can derive 3D retinal organoids and develop RGCs. Like PSCs, WT and OPA1 mutant ROs express the same OPA1 protein isoforms, but unlike PSCs, ROs express certain OPA1 protein isoforms more strongly than others. Research is ongoing to characterize the OPA1 mutant ROs and RGCs in order to evaluate their potential use as reliable human models of DOA. Ultimately, when compared with ROs derived from their isogenic controls, these OPA1 mutant, human PSC-derived RO disease models can reveal novel insights regarding the molecular mechanisms underlying the RGC-specific degeneration observed in DOA patients and facilitate the development of therapies that preserve or rescue vision in these patients.