Using stem cells to develop gene therapy for Batten Disease.

Abstract

Purpose: Batten Disease, or juvenile neuronal ceroid lipofuscinosis (JNCL), is a devastating autosomal recessive lysosomal storage disorder characterized by early onset retinal degenerative blindness, epilepsy, cognitive decline and premature death. The most common mutation that causes JNCL is a one-kilobase genomic deletion in the gene CLN3. The purpose of this study is use patient-specific induced pluripotent stem cells (iPSCs) as a model system to investigate the pathophysiology of CLN3-associated JNCL and to develop gene and autologous cell-based therapeutic approaches for the treatment of this devastating disease.

Methods: iPSCs were generated via transduction of human dermal fibroblasts obtained from patients with molecularly confirmed CLN3-associated JNCL using the transcription factors Oct4, Sox2, c-Myc and KLF4. iPSC potency was characterized via rt-PCR, Western blotting, immunocytochemistry (ICC), and embryoid body formation. Pluripotent iPSCs were differentiated into retinal neurons using our previously published protocol consisting of the stepwise addition of neurotrophic growth factors, and inhibitors of Wnt, Notch and BMP. A combination of rt-PCR, Western blotting, ICC, and confocal microscopy were used to evaluate disease phenotype and efficacy of the developed gene therapeutic approach.

Results: Patient-specific iPSCs were generated from dermal fibroblasts obtained from two separate patients with CLN3-associated Batten disease. After 60 days of differentiation, retinal neurons were identified via immunocytochemical analysis targeted against OTX2, BRN3, TUJ1, NF200, MAP2, recoverin and cone opsins. As observed in vivo, differentiated retinal neurons harbored the one-kilobase deletion in CLN3 and loss of normal full-length transcript and protein. Accumulation of autofluorescent lysosomal storage material was accompanied by intense Lysosomal-Associated Membrane Protein-1 (LAMP-1) expression. Full-length CLN3 was cloned and packaged into lentiviral vectors and tested for the ability to drive expression of full-length CLN3 and reversion of JNCL phenotype.

Conclusions: Retinal neurons generated from JNCL patients recapitulate cardinal aspects of the disease in vitro. Genetic correction of the disease phenotype in patient-specific iPSCs will pave the way for combined gene and autologous cell replacement-based therapeutic trials for the treatment of Batten disease.

Keywords: 696 retinal degenerations: hereditary • 721 stem cells • 538 gene transfer/gene therapy