US 11,987,809 B2
Methods for the treatment of corneal dystrophies
Tara Moore, Ballyclare (GB); Andrew Nesbit, Coleraine (GB); Gene Lee, Burlingame, CA (US); Sun-young Cho, Seoul (KR); and Larry DeDionisio, Oakland (CA)
Assigned to Avellino Lab USA, Inc., Menlo Park, CA (US)
Filed by Avellino Lab USA, Inc., Menlo Park, CA (US)
Filed on May 11, 2018, as Appl. No. 15/977,565.
Application 15/977,565 is a continuation of application No. PCT/US2016/061893, filed on Nov. 14, 2016.
Claims priority of provisional application 62/255,310, filed on Nov. 13, 2015.
Prior Publication US 2019/0085288 A1, Mar. 21, 2019
Int. Cl. C12N 5/079 (2010.01); A61K 35/30 (2015.01); A61L 27/36 (2006.01); A61L 27/54 (2006.01); A61P 27/02 (2006.01); C12N 9/22 (2006.01); C12N 15/113 (2010.01); C12N 15/85 (2006.01); C12Q 1/68 (2018.01); C12Q 1/6883 (2018.01)
CPC C12N 5/0621 (2013.01) [A61K 35/30 (2013.01); A61P 27/02 (2018.01); C12N 9/22 (2013.01); C12N 15/113 (2013.01); C12N 15/85 (2013.01); C12Q 1/68 (2013.01); C12Q 1/6883 (2013.01); A61L 27/3604 (2013.01); A61L 27/3666 (2013.01); A61L 27/3695 (2013.01); A61L 27/54 (2013.01); C12N 2510/00 (2013.01); C12N 2533/92 (2013.01); C12Q 2600/156 (2013.01)] 20 Claims
 
1. A method of ameliorating or treating corneal dystrophy in a subject in need thereof, the method comprising:
manipulating a nucleic acid mutation in a corneal dystrophy target nucleic acid in a limbal epithelial stem cell to correct the nucleic acid mutation, thereby forming a manipulated limbal epithelial stem cell, wherein the manipulating comprises introducing an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system into the limbal epithelial stem cell, wherein the CRISPR/Cas9 system comprises at least one vector comprising a nucleotide molecule encoding Cas9 nuclease and an single guide RNA (sgRNA), and the Cas9 nuclease and said sgRNA do not naturally occur together, wherein the sgRNA comprises (i) CRISPR targeting RNA (crRNA) sequence, and (ii) a trans-activating crRNA (tracrRNA) sequence, wherein the tracrRNA comprises a nucleotide sequence having the nucleotide sequence of SEQ ID NO: 6, wherein the crRNA sequence and tracrRNA sequence do not naturally occur together; and
transplanting the manipulated limbal epithelial stem cell into the subject.