Somatic Gene Therapy of Mucopolysaccharidosis with CRISPR/Cas9 Genome Editing

Abstract

Purpose : When molecular cloning was first introduced in the 1970’s, it created an extraordinary expectation of gene therapy for curing congenital and acquired diseases. However, the use of gene therapy for treating diseases in human and animal models remains daunting. The newly developed genome editing technique CRISPR (clustered regularly interspaced short palindromic repeats) offers a novel possibility of gene therapy in curing diseases caused by congenital genetic mutation. In present studies, attempts were made to examine the possibility of using CRISPR to correct genetic mutation in postnatal Gusb mice (an autosomal point mutation of β-glucuronidase, β) that manifests symptoms of mucopolysaccharidosis type VII, also known as Sly syndrome.

Methods : sgRNA specifically targeting the Gusb mutant allele and the correcting donor DNA were designed and tested in Gusb fibroblasts derived from the homozygous mutant mice. The validated sgRNA and donor DNA, and SpCas9 endonuclease were packaged into AAV-DJ virus. Both AAV-DJ-SpCas9 and AAV-DJ-sgRNA-Donor were co-injected directly into cornea stroma and/or systemically into the body via tail vein infusion. The experimental mice were weighed and examined weekly by HRTII in vivo confocal microscopy.

Results : To test the effectiveness of CRISPR/Cas9 to correct the genetic mutation, Gusb fibroblasts were transfected with: 1) a pX458 plasmid (Addgene) containing the U6-promoter driven sgRNA including the CTTGTCTTAGGCCCGTACG target sequence that matched to Gusb mutant allele and the SpCas9 expression cassette; and 2) a synthetic single strand donor oligo containing the wild type sequence: TTAGGCCCCGTACG flanked by homologous arms of 50 and 66 nucleotides in length. The transfected Gusb fibroblasts changed to a healthier spindle shape with little lysosome accumulation (less LAMP2 positive immunostaining) and expressed β-glu activity, suggesting an effective somatic gene correction. Subsequently, AAV-DJ-SpCas9 and AAV-DJ-U6-sgRNA-3 kb wild type donor DNA were co-injected into the corneas and/or the tail vein of mutant mice. HRTII examination revealed less corneal haze in treated mice three week after injection, compared to untreated counterparts. The experimental mice also maintained the body

Conclusions : The observation suggests that the CRISPR/Cas system is effective in treating the mice with corneal dystrophy caused by mucopolysaccharidosis VII.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.