US 12,258,599 B2
Method to inactivate a mutant allele of an ELANE gene
David Baram, Tel Aviv (IL); Lior Izhar, Tel Aviv (IL); Asael Herman, Ness-Ziona (IL); Rafi Emmanuel, Ramla (IL); Liat Rockah, Rishon LeZion (IL); Nadav Marbach-Bar, Rehovot (IL); Michal Golan Mashiach, Ness-Ziona (IL); and Joseph Georgeson, Rehovot (IL)
Assigned to EmendoBio Inc., Wilmington, DE (US)
Filed by EmendoBio Inc., Wilmington, DE (US)
Filed on Nov. 5, 2020, as Appl. No. 17/090,814.
Claims priority of provisional application 62/931,655, filed on Nov. 6, 2019.
Prior Publication US 2021/0130804 A1, May 6, 2021
Int. Cl. C12N 9/22 (2006.01); C12N 9/64 (2006.01); C12N 15/11 (2006.01)
CPC C12N 9/6448 (2013.01) [C12N 9/22 (2013.01); C12N 15/111 (2013.01); C12N 2310/20 (2017.05)] 8 Claims
 
1. An ex vivo or in vitro method of modifying in a cell a mutant allele of the elastase, neutrophil expressed (ELANE) gene wherein the mutant allele has a mutation associated with severe congenital neutropenia (SCN) or cyclic neutropenia (CyN), the method comprising introducing to the cell a composition comprising:
(a) a CRISPR nuclease, or a DNA polynucleotide encoding said CRISPR nuclease, wherein said CRISPR nuclease comprises:
(i) the amino acid sequence of SEQ ID NO: 31123, 31124, 31125, or 31126; or
(ii) the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 31131 or 31132; and
(b) a first RNA molecule, or a DNA polynucleotide encoding the first RNA molecule, wherein said first RNA molecule comprises:
(i) a CRISPR RNA (crRNA) molecule and a transactivating CRISPR RNA (tracrRNA) molecule; or
(ii) a single-guide RNA (sgRNA) molecule,
wherein the first RNA molecule comprises (I) a guide sequence portion comprising 20, 21, or 22 contiguous nucleotides of the sequence set forth in SEQ ID NO: 5519; or (II) a guide sequence portion comprising SEQ ID NO: 5469, 5482, or 5519, wherein the CRISPR nuclease and the first RNA molecule form a complex, and wherein said complex binds to the mutant allele of the ELANE gene and introduces a double strand break in the mutant allele of the ELANE gene.