Using patient-derived iPSCs to identify new drug treatments for JNCL

Abstract

Purpose: Juvenile neuronal ceroid lipofuscinosis (JNCL) is an autosomal recessive lysosomal storage disorder that causes irreversible blindness, epilepsy, cognitive deficits and premature death. The most common mutation that causes JNCL is a one-kilobase genomic deletion in the gene CLN3. There is no cure for JNCL and treatments that are effective in slowing disease progression are limited. The purpose of this study was to generate patient-specific iPSC-derived retinal neurons stably expressing a caspase-sensitive fluorescent apoptosis reporter construct, Apoliner, for the purpose of high-throughput chemical screening and identification of therapeutic targets.

Methods: Skin biopsies were obtained from patients with molecularly confirmed CLN3-associated JNCL and from Cln3-associated mice (both Cln3ΔlacZ and Cln3Δex7-9). Pluripotent iPSCs were generated from cultured fibroblasts via viral transduction of the transcription factors OCT4, SOX2, KLF4 and c-Myc. To monitor the fate of diseased cells, iPSCs that stably-express the caspase-sensitive fluorescent apoptosis reporter, Apoliner, were engineered. iPSCs were subjected to retinal differentiation protocols to obtain retinal neurons. RT-PCR, Western blotting, and immunocytochemical analysis were performed to confirm presence of JNCL phenotype(s) and the function of the Apoliner construct.

Results: We have successfully generated iPSCs from patients harboring the most common genetic mutation in the gene CLN3 and from 2 separate mouse models of JNCL. As JNCL retinal neurons prematurely succumb to apoptotic cell death, the caspase-sensitive apoptosis fluorescent reporter, Apoliner (Bardet et.al., 2006, PNAS), was adopted for high-throughput screening purposes. The Apoliner construct was cloned into a lentiviral vector upstream of an antibiotic resistance cassette (LV-APO). Each of the above described iPSC lines were transduced with LV-APO and at 1-week post-transduction antibiotic resistant stable Apoliner-iPSC lines were clonally expanded. Stable lines were subjected to our previously developed stepwise differentiation protocol. Functionality of the Apoliner construct was confirmed in JNCL-specific neurons via confocal microscopy.

Conclusions: The iPSC reporter lines generated in this study provide the basis for future high-throughput small molecule/chemical screening studies focused on finding compounds capable of mitigating the JNCL phenotype.

Keywords: 721 stem cells • 695 retinal degenerations: cell biology • 638 pathology: human