Authors :
Presenting Author: Arvin Sarkissian, BA – Emory University
Simren Kochhar, BS – Emory University
Cindy Lee, BS – Emory University
Charlotte Adams, BS – Emory University
Kaori Graybeal, MS – Emory University
Fikri Birey, PhD – Emory University
Rationale:
Variants in CACNA1A (encoding the α1A subunit of P/Q‑type CaV2.1 calcium channels) span gain and loss of function leading to diverse epilepsy phenotypes, yet human, region-specific disease mechanisms during early brain network formation remain unclear. Isogenic brain organoids combined with multimodal phenotyping assays permit evaluation of variant-class effects across cellular, synaptic, and network scales.Methods:
We differentiated an isogenic control and homozygous CACNA1A A713T (gain-of-function) iPSC line pair into cortical and subpallial organoids. Regional identity and synaptic programs were quantified by bulk RNA sequencing, cell-type composition and state by single-cell RNA sequencing, neuronal activity by 3D high-density MEA, calcium dynamics by live imaging, and synaptic structure by puncta quantification. Separately, prime editing was used to install A713T (gain-of-function) and Q680fs (frameshift; loss-of-function) across four iPSC backgrounds after pegRNA and nick-sgRNA screening, followed by isolation, expansion, and validation of heterozygous clones.
Results:
Guided differentiation of brain organoids carrying a homozygous CACNA1A A713T gain-of-function mutation produced appropriately patterned early cortical and subpallial fates assessed via bulk RNA sequencing. In cortical organoids, A713T homozygotes exhibited altered firing rates on 3D high-density MEAs and increased calcium event frequency and amplitude relative to isogenic control. Synaptic puncta density was preserved, whereas bulk RNA sequencing indicated reduced expression of synaptic genes, including CACNA1A and canonical markers (GPHN, SYN1, SYN2). Single-cell analyses of mature cortical and subpallial organoids revealed cell-type-specific phenotypes, suggesting unique vulnerabilities and adaptation mechanisms in excitatory and inhibitory neurons. Together, these multimodal data indicate early network hyperactivity with remodeled synaptic gene programs in A713T cortical organoids. To enable variant-class comparisons, we heterozygously installed CACNA1A A713T and Q680fs into 4 iPSC lines—establishing a set of 12 isogenic iPSC lines for downstream organoid phenotyping.
Conclusions:
This work demonstrates the potential for human brain organoids to model CACNA1A variant–linked alterations in neuronal activity with transcriptional changes in synaptic gene programs during early development. Functional and transcriptomic phenotypes of CACNA1A variants in isogenically controlled human brain organoids can be used to test epilepsy therapeutic candidates for patients.
Funding:
NIH F31 (NS139599). Orphan Disease Center MDBR (2024). CACNA1A Foundation.