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Molecular Genetics Laboratory

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Sinja Kieninger

Surname Kieninger
First name Sinja
Position and Title PhD student
Project Gene-editing based therapy for OPA1-linked optic neuropathies

Business address

Molecular Genetics Laboratory
Institute for Ophthalmic Research
Centre for Ophthalmology,
University of Tübingen
Elfriede-Aulhorn-Strasse 7
D-72076 Tübingen,
Germany

Phone: +49 (0)7071 29-80931

E-mail: sinja.kieninger[at]med.uni-tuebingen.de

Project descriptions

Gene-editing based therapy for OPA1-linked optic neuropathies

OPA1-linked optic neuropathies (OPONs) represent a group of inherited neurodegenerative disorders, which are associated with mutations in the OPA1 gene. OPA1 encodes a mitochondrial dynamin-related GTPase that plays a key role in the fusion process of mitochondria and the maintenance of cristae junctions. Mutations in OPA1 lead therefore to mitochondrial dysfunction resulting eventually in the loss of retinal ganglion cells forming the optic nerve. Patients experience a progressive loss of vision due to the degeneration of the optic nerve. Patients of autosomal dominant optic atrophy plus (adOAplus) or Behr syndrome exhibit also additional extraocular symptoms such as hearing loss, muscle weakness, ataxia, spasticity and mental retardation. In multiple families, Behr syndrome and adOAplus are associated with deep intronic mutations (DIMs) in OPA1. These DIMs generate cryptic splice sites, which leads to an inclusion of a cryptic exon bearing a premature stop codon resulting in reduced OPA1 activity.

I intend to further develop and validate a therapeutic approach for OPA1-linked optic neuropathies using the CRISPR/Cas9 technology to target such cryptic splice sites and excise the resulting cryptic exon. The genome editing tool CRISPR/Cas9 consists of the Cas9 nuclease and single guide RNAs (sgRNAs). These sgRNAs contain targeting sequences and guide the Cas9 nuclease to cleave the target DNA. The resulting double-stranded breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous-directed repair (HDR).  In a former in vitro-assay, CRISPR/Cas9 was successfully used to rescue the splicing defect caused by the c.610+364G>A mutation in OPA1. I want to further follow this work and assay the splicing rescue of CRISPR/Cas9 treated cells. The most efficient sgRNA pair will be used for editing primary patient fibroblasts. Edited fibroblast cultures will be assayed for OPA1 protein and mRNA level and also be used to assess mitochondrial physiology.