title: CRISPR/Cas9-Mediated Genome Editing as a Therapeutic Approach for Leber Congenital Amaurosis 10.
abstract: As the most common subtype of Leber congenital amaurosis (LCA), LCA10 is a severe retinal dystrophy caused by mutations in the CEP290 gene. The most frequent mutation found in patients with LCA10 is a deep intronic mutation in CEP290 that generates a cryptic splice donor site. The large size of the CEP290 gene prevents its use in adeno-associated virus (AAV)-mediated gene augmentation therapy. Here, we show that targeted genomic deletion using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system represents a promising therapeutic approach for the treatment of patients with LCA10 bearing the CEP290 splice mutation. We generated a cellular model of LCA10 by introducing the CEP290 splice mutation into 293FT cells and we showed that guide RNA pairs coupled with SpCas9 were highly efficient at removing the intronic splice mutation and restoring the expression of wild-type CEP290. In addition, we demonstrated that a dual AAV system could effectively delete an intronic fragment of the Cep290 gene in the mouse retina. To minimize the immune response to prolonged expression of SpCas9, we developed a self-limiting CRISPR/Cas9 system that minimizes the duration of SpCas9 expression. These results support further studies to determine the therapeutic potential of CRISPR/Cas9-based strategies for the treatment of patients with LCA10.
authors.affiliation: Rare Diseases, Sanofi Genzyme, Framingham, MA 01701, USA. Electronic address: guoxiang.ruan@genzyme.com.
authors.forename: Guo-Xiang
authors.initials: GX
authors.lastname: Ruan
authors.affiliation: Rare Diseases, Sanofi Genzyme, Framingham, MA 01701, USA.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Barry
authors.affiliation: Rare Diseases, Sanofi Genzyme, Framingham, MA 01701, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Yu
authors.affiliation: Rare Diseases, Sanofi Genzyme, Framingham, MA 01701, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Lukason
authors.affiliation: Rare Diseases, Sanofi Genzyme, Framingham, MA 01701, USA.
authors.forename: Seng H
authors.initials: SH
authors.lastname: Cheng
authors.affiliation: Rare Diseases, Sanofi Genzyme, Framingham, MA 01701, USA.
authors.forename: Abraham
authors.initials: A
authors.lastname: Scaria
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D064113:CRISPR-Cas Systems; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D005260:Female; D000072669:Gene Editing; D015870:Gene Expression; D023061:Gene Order; D018390:Gene Targeting; D056426:Genetic Loci; D007438:Introns; D057130:Leber Congenital Amaurosis / Q000235:genetics* / Q000628:therapy; D051379:Mice; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger / Q000235:genetics; D012160:Retina / Q000378:metabolism; D017384:Sequence Deletion; D052416:Targeted Gene Repair
chemical_list: D000951:Antigens, Neoplasm; D018797:Cell Cycle Proteins; C493903:Cep290 protein, human; D003598:Cytoskeletal Proteins; D009363:Neoplasm Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger
keywords: CEP290; CRISPR/Cas9; LCA10