Materials and methods for treatment of autosomal dominant cone-rod dystrophy

Abstract

The present application provides materials and methods for treating a patient with autosomal dominant CORD, both ex vivo and in vivo; materials and methods for editing a GUCY2D gene in a human cell; and materials and methods for editing a R838H, R838C, or R838S mutation in a GUCY2D gene in a human cell. The present application also provides one or more gRNAs or sgRNAs for editing a GUCY2D gene; one or more gRNAs or sgRNAs for editing a R838H, R838C, or R838S mutation in a GUCY2D gene; and a therapeutic comprising at least one or more gRNAs or sgRNAs for editing a R838H, R838C, or R838S mutation in a GUCY2D gene. The present application provides a therapeutic for treating a patient with autosomal dominant CORD. The present application also provides a kit for treating a patient with autosomal dominant CORD. In addition, the present application provides a self-inactivating CRISPR-Cas system.

1 . A method for editing a guanylate cyclase 2D (GUCY2D) gene in a human cell, the method comprising:
 introducing into the human cell one or more deoxyribonucleic acid (DNA) endonucleases to effect one or more single-strand breaks (SSBs) or double-strand breaks (DSBs) within or near the GUCY2D gene or other DNA sequences that encode regulatory elements of the GUCY2D gene that results in a deletion, insertion, or correction thereby creating an edited human cell.   
     
     
         2 .- 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the one or more DNA endonucleases is a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination of the naturally occurring molecule thereof, codon-optimized thereof, or modified versions thereof, and combinations thereof. 
     
     
         6 . The method of  claim 5 , wherein the method comprises introducing into the cell one or more polynucleotides encoding the one or more DNA endonucleases. 
     
     
         7 . The method of  claim 5 , wherein the method comprises introducing into the cell one or more ribonucleic acids (RNAs) encoding the one or more DNA endonucleases. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the method further comprises: introducing into the cell one or more guide ribonucleic acids (gRNAs). 
     
     
         11 . The method of  claim 10 , wherein the one or more gRNAs are single-molecule guide RNA (sgRNAs). 
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 10  or  11 , wherein the one or more DNA endonucleases is pre-complexed with one or more gRNAs or one or more sgRNAs. 
     
     
         14 . The method of  claim 1 , further comprising: introducing into the cell a polynucleotide donor template comprising at least a portion of the wild-type GUCY2D gene, or cDNA. 
     
     
         15 .- 46 . (canceled) 
     
     
         47 . One or more gRNAs for editing a R838H, R838C, or R838S mutation in a GUCY2D gene in a cell from a patient with autosomal dominant Cone-Rod Dystrophy (CORD), the one or more gRNAs comprising a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5282-5313, 5398-5409, and 5434-5443 of the Sequence Listing. 
     
     
         48 . The one or more gRNAs of  claim 47 , wherein the one or more gRNAs are one or more sgRNAs. 
     
     
         49 .- 53 . (canceled) 
     
     
         54 . A therapeutic for treating a patient with autosomal dominant Cone-Rod Dystrophy (CORD) formed by a method comprising:
 introducing one or more DNA endonucleases;   introducing one or more gRNA or one or more sgRNA for editing a R838H, R838C, or R838S mutation in a GUCY2D gene;   optionally introducing one or more donor template;   wherein the one or more gRNAs or sgRNAs comprise a spacer sequence selected from the group consisting of nucleic acid sequences in SEQ ID NOs: 5282-5313, 5398-5409, 5434-5443 of the Sequence Listing.   
     
     
         55 .- 149 . (canceled)