Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10

Leber congenital amaurosis type 10 is a severe retinal dystrophy caused by mutations in the CEP290 gene1,2. We developed EDIT-101, a candidate genome-editing therapeutic, to remove the aberrant splice donor created by the IVS26 mutation in the CEP290 gene and restore normal CEP290 expression. Key to this therapeutic, we identified a pair of Staphylococcus aureus Cas9 guide RNAs that were highly active and specific to the human CEP290 target sequence. In vitro experiments in human cells and retinal explants demonstrated the molecular mechanism of action and nuclease specificity. Subretinal delivery of EDIT-101 in humanized CEP290 mice showed rapid and sustained CEP290 gene editing. A comparable surrogate non-human primate (NHP) vector also achieved productive editing of the NHP CEP290 gene at levels that met the target therapeutic threshold, and demonstrated the ability of CRISPR/Cas9 to edit somatic primate cells in vivo. These results support further development of EDIT-101 for LCA10 and additional CRISPR-based medicines for other inherited retinal disorders.In human cells, a humanized mouse model and non-human primates, CRISPR/Cas9 corrects the splicing defect in a gene associated with congenital blindness.

[1]  F. Coppieters,et al.  CEP290, a gene with many faces: mutation overview and presentation of CEP290base , 2010, Human mutation.

[2]  Jong-il Kim,et al.  Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells , 2015, Nature Methods.

[3]  D. G. Green,et al.  Effect on grating identification of sampling with degenerate arrays. , 1992, Journal of the Optical Society of America. A, Optics and image science.

[4]  E. Stone Leber congenital amaurosis - a model for efficient genetic testing of heterogeneous disorders: LXIV Edward Jackson Memorial Lecture. , 2007, American journal of ophthalmology.

[5]  Jeongbin Park,et al.  Digenome-seq web tool for profiling CRISPR specificity , 2017, Nature Methods.

[6]  T. Aleman,et al.  Centrosomal‐ciliary gene CEP290/NPHP6 mutations result in blindness with unexpected sparing of photoreceptors and visual brain: implications for therapy of Leber congenital amaurosis , 2007, Human mutation.

[7]  V. Myer,et al.  Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus , 2017, Nature Communications.

[8]  A. Swaroop,et al.  In-frame deletion in a novel centrosomal/ciliary protein CEP290/NPHP6 perturbs its interaction with RPGR and results in early-onset retinal degeneration in the rd16 mouse. , 2006, Human molecular genetics.

[9]  Morgan L. Maeder,et al.  UDiTaS™, a genome editing detection method for indels and genome rearrangements , 2018, BMC Genomics.

[10]  E. Stone,et al.  CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype , 2014, Gene Therapy.

[11]  Morgan L. Maeder,et al.  Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications , 2015, Genome Biology.

[12]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[13]  Peter M. G. Munro,et al.  Identification and Correction of Mechanisms Underlying Inherited Blindness in Human iPSC-Derived Optic Cups , 2016, Cell stem cell.

[14]  P. Sieving,et al.  Assessment of foveal cone photoreceptors in Stargardt's macular dystrophy using a small dot detection task , 1993, Vision Research.

[15]  A. J. Roman,et al.  Natural History of Cone Disease in the Murine Model of Leber Congenital Amaurosis Due to CEP290 Mutation: Determining the Timing and Expectation of Therapy , 2014, PloS one.

[16]  Marcel Martin Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[17]  F. Niesen,et al.  The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability , 2007, Nature Protocols.

[18]  Paul D. Gamlin,et al.  The human rhodopsin kinase promoter in an AAV5 vector confers rod- and cone-specific expression in the primate retina. , 2012, Human gene therapy.

[19]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[20]  Martin J. Aryee,et al.  Engineered CRISPR-Cas9 nucleases with altered PAM specificities , 2015, Nature.

[21]  I. Solovei,et al.  Quick and reliable method for retina dissociation and separation of rod photoreceptor perikarya from adult mice , 2015, MethodsX.

[22]  Jin-Soo Kim,et al.  Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases , 2014, Bioinform..

[23]  T. Meitinger,et al.  Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis. , 2006, American journal of human genetics.

[24]  J. Bennett,et al.  CEP290 and the primary cilium. , 2014, Advances in experimental medicine and biology.

[25]  Adam P. DeLuca,et al.  Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease. , 2017, Ophthalmology.

[26]  D. Scherman,et al.  AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation , 2012, Molecular therapy. Nucleic acids.

[27]  A. J. Roman,et al.  Cone photoreceptors are the main targets for gene therapy of NPHP5 (IQCB1) or NPHP6 (CEP290) blindness: generation of an all-cone Nphp6 hypomorph mouse that mimics the human retinal ciliopathy. , 2011, Human molecular genetics.

[28]  A. Smolyar,et al.  Atomic structure of an αβ T cell receptor (TCR) heterodimer in complex with an anti‐TCR Fab fragment derived from a mitogenic antibody , 1998, The EMBO journal.

[29]  R. Roepman,et al.  Unexpected CEP290 mRNA Splicing in a Humanized Knock-In Mouse Model for Leber Congenital Amaurosis , 2013, PloS one.

[30]  A. D. den Hollander,et al.  Antisense Oligonucleotide (AON)-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290 , 2012, Molecular therapy. Nucleic acids.

[31]  Martin J. Aryee,et al.  GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases , 2014, Nature Biotechnology.

[32]  E. Stone,et al.  Basal exon skipping and genetic pleiotropy: A predictive model of disease pathogenesis , 2015, Science Translational Medicine.