Genome editing and gene replacement: towards the treatment of Batten disease.

Abstract

Purpose: Juvenile neuronal ceroid lipofuscinosis, or Batten disease, is a lysosomal storage disorder with an estimated prevalence of 1 in 100,000 live births. This disease is characterized by early onset blindness, subsequent motor deficits, seizures and premature death. The most common disease causing mutation observed is a 1 kb genomic deletion, which spans exons 7-8, in the gene CLN3. The purpose of this study was to develop gene augmentation and genome editing based approaches for the treatment of CLN3-associated disease.<br />

Methods: Skin biopsies were obtained from patients with CLN3-associated Batten disease and iPSCs generated from cultured keratinocytes via viral transduction of the transcription factors OCT4, SOX2, KLF4 and c-Myc. Lentiviral (LV) and adeno-associated viral (AAV) constructs carrying full-length CLN3 under the control of the cytomegalovirus promotor were cloned, packaged and transduced into iPSC-derived neurons. CLN3 expression was assessed via rt-PCR, Western blotting and immunofluorescence. CRISPR/Cas9 constructs harboring specific small guide RNAs targeting the 1 kb deletion in CLN3 were designed, cloned and tested. For correction of the CLN3 locus we also generated a donor construct carrying wild-type CLN3 spanning exons 7-8 to be co-delivered with CRISPR/Cas9 plasmids to induce homology directed repair.<br />

Results: IPSCs from three patients with CLN3-associated Batten disease have been generated. LV-CLN3 and AAV-CLN3 viral vectors both restored full-length CLN3 transcript and protein expression in iPSC-derived neurons. CRISPR/Cas9 constructs targeting CLN3 induced specific modification of the CLN3 locus. Patient-specific iPSCs, when transduced with CRISPR constructs along with the wild-type CLN3 donor construct, expressed restored full-length CLN3 following homology directed repair.<br />

Conclusions: We have demonstrated that viral-mediated delivery of CLN3 is sufficient to restore full-length CLN3 protein in iPSC-derived neurons from patients with Batten disease. Furthermore, we have genomically corrected diseased iPSCs using a CRISPR/Cas9-mediated homology directed repair strategy. The in vitro success of these treatment modalities pave the road for generation and testing of clinical-grade AAV-CLN3 and CRISPR-corrected iPSCs that will be produced in a clinical GMP facility.<br />