Gene Therapy of Mucopolysaccharidosis Type VII (MPS VII) with CRISPR/Cas9 Genome Editing

Abstract

Purpose : Currently, there is no effective gene therapy treatment for lysosomal storage diseases. Lysosomal enzymes and transporting proteins are secreted via extracellular vesicles (EV). We thus hypothesize that CRISPR genome editing will correct a genetic defect in a fraction of somatic cells ameliorating the pathology via the secretion of EV containing functional enzyme/protein. Gusb mice caused by a single base nonsense deletion mutation in β-glucuronidase (β-Glu/Gusb) manifest a MPS phenotype similar to the human MPS VII (Sly syndrome). We aim to develop a strategy to treat congenital cornea disease using CRISPR genome editing.

Methods : Conventional techniques are used to prepare AAV2-DJ vectors for a binary AAV2-DJ-SpCas9 and AAV2-DJ-sgRNA/DNA-donor and a mono AAV2-DJ-SaCas9/sgRNA/DNA-donor. AAV2-DJ vectors at 10¹¹ viral particles/ml are administered to the experimental mice via tail vein or retro orbital intravenous infusion (ROIV). The experimental mice are subjected to HRTII in vivo confocal microscopy, survival rate and β-Glu histochemistry.

Results : Gusb mice have an age-related increase of corneal haze and an average life span of about 4 months. The onset of death commences around 8 weeks of age. Treatment with binary AAV2-DJ vectors via tail vein infusion produced limited improvements in the reduction of corneal haze and a delayed onset of death by 10 weeks. To improve the treatment efficacy, a mono AAV2-DJ was generated taking the advantage that SaCas9 plasmid DNA is about 500 bp smaller than that of SpCas9 allowing for the insertion of a U6-sgRNA plasmid and a 300 bp DNA donor for repairing the Gusb allele by homology directed repair. The administration of the single AAV2-DJ-SaCas9/sgRNA/DNA-donor via ROIV greatly improved corneal transparency and survival (treated mice are still alive at 28 weeks). Small amounts of β-glu is detected in the liver of treated Gusb mice.

Conclusions : These results suggest that the CRISPR/Cas9 system is an effective treatment for mouse corneal dystrophy e.g., MPS VII. The mono AAV2-DJ-SACas9/sgRNA/DNA-donor vector yielded a better therapeutic efficacy in terms of survival rate and reduction of corneal haze.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.