Generation and correction of a humanized mouse model with a deletion of dystrophin exon 44

Abstract

Duchenne muscular dystrophy (DMD), which affects 1 in 5,000 male births, is one of the most common genetic disorders of children. This disease is caused by an absence or deficiency of dystrophin protein in striated muscle. The major DMD deletion “hot spots” are found between exon 6 to 8, and exons 45 to 53. Here, a “humanized” mouse model is provided that can be used to test a variety of DMD exon skipping strategies. Among these are, CRISPR/Cas9 oligonucleotides, small molecules or other therapeutic modalities that promote exon skipping or micro dystrophin mini genes or cell based therapies. Methods for restoring the reading frame of exon 44 deletion via CRISPR-mediated exon skipping in the humanized mouse model, in patient-derived iPS cells and ultimately, in patients using various delivery systems are also contemplated. The impact of CRISPR technology on DMD is that gene editing can permanently correct mutations.

1 . A mouse whose genome comprises a deletion of exon 44 of the dystrophin gene resulting in an out of frame shift and a premature stop codon in exon 45. 
     
     
         2 . The mouse of  claim 1 , further comprising a reporter gene located downstream of and in frame with exon 79 of the dystrophin gene, and upstream of a dystrophin 3′-UTR, wherein the reporter gene is expressed when exon 79 is translated in frame with exon 43. 
     
     
         3 . The mouse of  claim 2 , wherein the reporter gene is luciferase. 
     
     
         4 .- 6 . (canceled) 
     
     
         7 . The mouse of  claim 1 , wherein the mouse is heterozygous for the deletion. 
     
     
         8 . The mouse of  claim 1 , wherein the mouse is homozygous for the deletion. 
     
     
         9 . The mouse of  claim 1 , wherein the mouse exhibits increased creatine kinase levels. 
     
     
         10 . The mouse of  claim 1 , wherein the mouse does not exhibit detectable dystrophin protein in heart or skeletal muscle. 
     
     
         11 .- 20 . (canceled) 
     
     
         21 . An isolated cell obtained from the mouse of  claim 1 . 
     
     
         22 .- 28 . (canceled) 
     
     
         29 . A mouse produced by a method comprising the steps of:
 (a) contacting a fertilized oocyte with CRISPR/Cas9 elements and two single guide RNA (sgRNA) targeting sequences flanking exon 44, thereby creating a modified oocyte, wherein deletion of exon 44 by CRISPR/Cas9 results in an out of frame shift and a premature stop codon in exon 45;   (b) transferring the modified oocyte into a recipient female.   
     
     
         30 . A method of screening a candidate substance for DMD exon-skipping activity comprising:
 (a) contacting a mouse according to  claim 1  with a candidate substance; and   (b) assessing in frame transcription and/or translation of exon 79,   
       wherein the presence of in frame transcription and/or translation of exon 79 indicates the candidate substance exhibits exon-skipping activity. 
     
     
         31 . The method of  claim 30 , wherein the mouse does not exhibit detectable dystrophin protein in heart or skeletal muscle. 
     
     
         32 .- 39 . (canceled) 
     
     
         40 . An isolated nucleic acid encoding a DMD guide RNA and comprising the sequence as set forth in any one of SEQ ID NO. 1-8 or 27-38. 
     
     
         41 - 44 . (canceled) 
     
     
         45 . A method of correcting a dystrophin gene defect in Exon 45 of the DMD gene in a subject comprising contacting a cell in the subject with Cpf1 or Cas9 and a DMD guide RNA as defined in claim R421140, resulting in selective skipping of a mutant DMD exon. 
     
     
         46 . The method of  claim 45 , wherein the cell is a muscle cell, a satellite cell, or an iPSC/iCM. 
     
     
         47 . The method of  claim 45 , wherein Cpf1 and/or DMD guide RNA are provided to the cell through expression from one or more expression vectors coding therefor. 
     
     
         48 . The method of  claim 47 , wherein the expression vector is a viral vector. 
     
     
         49 . The method of  claim 48 , wherein the viral vector is an adeno-associated viral vector. 
     
     
         50 . The method of  claim 47 , wherein the expression vector is a non-viral vector. 
     
     
         51 . The method of  claim 45 , wherein Cpf1 or Cas9 is provided to the cell as naked plasmid DNA or chemically-modified mRNA. 
     
     
         52 - 59 . (canceled) 
     
     
         60 . The method of  claim 45 , wherein the correction is permanent skipping of the mutant DMD exon. 
     
     
         61 .- 62 . (canceled)