Establishing a gene editing strategy for BBS7 using human retinal organoids

Abstract

Purpose : Variants in BBS7 result in an autosomal recessive ciliopathy with severe photoreceptor degeneration occurring by the first or second decade of life. Loss of BBS7 prevents assembly of the BBSome complex, which is critical for trafficking proteins and membrane components within the connecting cilium (CC) of photoreceptor cells. We aim to use an in vitro human retinal organoid model to optimize prime editing tools targeting BBS7 mutations as a therapeutic approach.

Methods : Human iPSCs were generated from a BBS7 patient (BBS7^G63R/Y354*), and pluripotency was confirmed using RT-PCR and immunocytochemistry with standard pluripotency markers: SOX2, OCT4, and NANOG. Karyotyping and Sanger sequencing were also performed on the newly-generated iPSC line. Using a previously optimized retinal differentiation protocol, 3D retinal organoids were generated from the BBS7 iPSC line and a control line. To test key markers, Western blotting (WB) and immunofluorescence were carried out on control retinal organoids at D240. Furthermore, transfection of control iPSCs with prime editor plasmids (PE2 and PEmax) was optimized to prepare for the development of a prime editing-based therapy targeting the BBS7 gene.

Results : We successfully generated a BBS7 iPSC line (BBS7^G63R/Y354*), which was fully characterized to verify pluripotency, normal karyotype, and presence of the mutations. The line is positive for OCT4, SOX2, and NANOG protein, and has a normal karyotype. Furthermore, the mutation on each allele was confirmed using Sanger sequencing. We have begun growing retinal organoids from the BBS7 iPSC line, as well as a control line, and so far, the organoids show typical morphology through day 45 (D45). Control organoids show immunolabeling for key markers outlining the CC, including BBS2 (in the CC), rootletin (below the CC), PRPH2 (above the CC in outer segments), and opsins (outer segments). In addition, control organoids exhibit BBS7 protein at D240 via WB. Finally, delivery of prime editor plasmids has been optimized in patient iPSCs.

Conclusions : Here, we established and fully characterized a patient-derived BBS7 iPSC line. This work forms the foundation for future experiments focused on BBS7 disease characterization and therapeutic development.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

BBS7 (A), BBS2 (red), PRPH2 (green), and rootletin (red) proteins are present in D240 control retinal organoids (B). BBS7^G63R/Y354* retinal organoids show normal morphology at D36 (C).