In vivo targeting of a variant causing vanishing white matter using CRISPR/Cas9

Molecular Therapy: Methods & Clinical Development

Vivi M. Heine¹  Tanja Rothgangl²  Martina Hruzova³  Marjo S. van der Knaap⁴  Anne E.J. Hillen⁴  Gerald Schwank⁴  Marianna Bugiani⁴  Marjolein Breur⁵ 

[1] Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Amsterdam Neuroscience, VU University, De Boelelaan 1085, 1081 Amsterdam, the Netherlands;Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland;Department of Biology, Institute for Molecular Health Sciences, ETH Zurich, Otto-Stern-Weg 7, 8093 Zurich, Switzerland;Department of Pediatrics and Child Neurology, Emma Children's Hospital, Amsterdam Neuroscience, Amsterdam UMC, De Boelelaan 1117, 1081 Amsterdam, the Netherlands;Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland;

关键词: vanishing white matter;    leukodystrophy;    CRISPR/Cas9;    gene therapy;    gene editing;   

DOI  :  

来源: DOAJ

【 摘 要 】

Vanishing white matter (VWM) is a leukodystrophy caused by recessive variants in subunits of eIF2B. At present, no curative treatment is available and patients often die at young age. Due to its monogenic nature, VWM is a promising candidate for the development of CRISPR/Cas9-mediated gene therapy. Here we tested a dual-AAV approach in VWM mice encoding CRISPR/Cas9 and a DNA donor template to correct a pathogenic variant in Eif2b5. We performed sequencing analysis to assess gene correction rates and examined effects on the VWM phenotype, including motor behavior. Sequence analysis demonstrated that over 90% of CRISPR/Cas9-induced edits at the targeted locus are insertion or deletion (indel) mutations, rather than precise corrections from the DNA donor template by homology-directed repair. Around half of the CRISPR/Cas9-treated animals died prematurely. VWM mice showed no improvement in motor skills, weight, or neurological scores at 7 months of age, and CRISPR/Cas9-treated controls displayed an induced VWM phenotype. In conclusion, CRISPR/Cas9-induced DNA double-strand breaks (DSBs) at the Eif2b5 locus did not lead to sufficient correction of the VWM variant. Moreover, indel formation in Eif2b5 induced an exacerbated VWM phenotype. Therefore, DSB-independent strategies like base- or prime editing might better suited for VWM correction.

【 授权许可】

Unknown