THERAPEUTIC USES OF GENOME EDITING WITH CRISPR/Cas SYSTEMS

Patent metadata

Field	Value
Publication number	US-2020048659-A1
Application number	US-201916278645-A
Country	US
Kind code	A1
Filing date	Feb 18, 2019
Priority date	Jul 9, 2013
Publication date	Feb 13, 2020
Grant date	—

Abstract

Disclosed herein are methods, compositions, and kits for high efficiency, site-specific genomic editing of cells for treating or preventing genetic blood disorders.

First claim

1 .- 12 . (canceled)

13 . A method for altering a target beta thalassemia-associated polynucleotide sequence in a cell comprising contacting the beta thalassemia-associated polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and from one to two ribonucleic acids, wherein the ribonucleic acids direct Cas protein to and hybridize to a target motif of the target beta thalassemia-associated polynucleotide sequence, wherein the target beta thalassemia-associated polynucleotide sequence is cleaved, and wherein the efficiency of alteration is from about 50% to about 80%.

14 .- 18 . (canceled)

19 . The method according to claim 13 , wherein the Cas protein is Streptococcus pyogenes Cas9 protein or a functional portion thereof selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.

20 .- 21 . (canceled)

22 . The method according to claim 13 , wherein the Cas protein is Cas9 protein from any bacterial species or functional portion thereof selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain.

23 .- 24 . (canceled)

25 . The method according to claim 13 , wherein the Cas protein is complexed with the one to two ribonucleic acids.

26 .- 32 . (canceled)

33 . The method according to claim 13 , wherein the target motif is G(N)19NGG.

34 .- 41 . (canceled)

42 . The method according to claim 13 , wherein the alteration results in reduced expression of the target polynucleotide sequence, a knock out of the target polynucleotide sequence, or correction of the target polynucleotide sequence from an undesired sequence to a desired sequence.

43 .- 57 . (canceled)

58 . The method according to claim 13 , wherein the cell is selected from the group consisting of a peripheral blood cell, a stem cell, a pluripotent cell, a hematopoietic stem cell, a CD34+ cell, a CD34+ mobilized peripheral blood cell, a CD34+ cord blood cell, a CD34+ bone marrow cell, a CD34 + CD38-Lineage-CD90 + CD45RA − cell, a primary human cell, a non-transformed human cell, and combinations thereof.

59 .- 107 . (canceled)

108 . The method according to claim 13 , wherein the target polynucleotide sequence is HBB.

109 . The method according to claim 108 , wherein at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of SEQ ID NOS. 814-908, or at least a 12 nucleotide fragment thereof.

110 . The method according to claim 108 , wherein at least one of the one to two ribonucleic acids comprises a sequence with a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOS. 814-908, or at least a 12 nucleotide fragment thereof.

111 .- 173 . (canceled)

174 . The method according to claim 13 , wherein the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell.

175 .- 193 . (canceled)

194 . The method according to claim 13 , wherein at least one of the ribonucleic acids is a modified ribonucleic acid comprising one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thio-uridine, 5-methyluridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5,6-dihydrouridine-5′-triphosphate, and 5-azauridine-5′-triphosphate.

195 .- 272 . (canceled)

273 . The method according to claim 13 , wherein the cell was selected for Cas protein expression.

274 . The method according to claim 13 , wherein the target motif of the target beta thalassemia-associated polynucleotide sequence comprises nucleotides located between position 5246806 and position 5248263 of human chromosome 11.

Assignees

• Harvard College
• Childrens Medical Ct Corp

Inventors

• Musunuru Kiran
• Cowan Chad A
• Rossi Derrick J

Classifications

• A61P7/00

  Drugs for disorders of the blood or the extracellular fluid · CPC title

• A61P7/06

  Antianaemics · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61K38/465

  acting on ester bonds (3.1), e.g. lipases, ribonucleases · CPC title

• C12N15/907 Primary

  in mammalian cells · CPC title