IP Library › Granted Patent US 12,644,136
Granted Patent B2
US 12,644,136 · App. 15/619,737 · Granted Jun 2, 2026

Delivery, use and therapeutic applications of CRISPR systems and compositions for genome editing as to hematopoietic stem cells (HSCs)

Inventors: David Benjamin Turitz Cox (Cambridge, MA); James E. Dahlman (Cambridge, MA); Feng Zhang (Cambridge, MA)
Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology
C12N15/907A61K38/43C12N9/96C12N15/102C12N15/1082C12N15/111C12N15/63C12N15/85C12N9/52C12N2310/10C12N2310/20C12N2310/3519
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Quick Facts
Patent No.
US 12,644,136
App. No.
15/619,737
Granted
Jun 2, 2026
Kind
B2
Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

Claims (39)

1 . A method for modifying a single targeted genomic nucleotide sequence of an isolated primary CD34 + hematopoietic stem cell and progeny thereof, comprising

forming a pre-annealed Staphylococcus aureus CRISPR-Cas9 (SaCas9) ribonucleoprotein (RNP) complex by admixing a SaCas9 protein and a single guide RNA (sgRNA) at a 1:1 molar ratio,

generating a lipid nanoparticle formulation containing the pre-annealed SaCas9-sgRNA RNP complex with a lipid formulation comprising 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG), and cholesterol at a molar ratio of DOTAP: DMPC: PEG: cholesterol selected from 100:0:0:0, 90:0:10:0, or 90:0:5:5; and

transfecting the isolated CD34+ hematopoietic stem cells with the lipid nanoparticle formulation,

wherein the single guide RNA molecule (sgRNA) in each pre-annealed RNP complex contains an antisense nucleotide sequence that is capable of

hybridizing with the targeted genomic nucleotide sequence,

directing sequence-specific binding of the pre-annealed RNP complex to the targeted genomic nucleotide sequence, and

enabling gene editing of said targeted genomic nucleotide sequence.

2 . The method of claim 1 , wherein the single guide RNA is a chimeric single guide RNA that comprises, in a tandem arrangement,

a single guide sequence,

a tracr mate sequence; and

a tracr RNA molecule,

wherein the tracr RNA molecule can hybridize to the tracr mate sequence.

3 . The method of claim 1 , wherein the Staphylococcus aureus CRISPR-Cas9 (SaCas9) protein is codon-optimized for expression in the primary CD34+ hematopoietic stem cells and progeny thereof.

4 . The method of claim 1 , wherein the Staphylococcus aureus CRISPR-Cas9 (SaCas9) protein comprises one or more mutations at any or all amino acid residues corresponding to positions 10, 762, 840, 854, 863, or 986 of the Streptococcus pyogenes Cas9 (SpCas9) protein.

5 . The method of claim 1 , wherein the Staphylococcus aureus CRISPR-Cas9 (SaCas9) protein further comprises a nuclear localization sequence.

6 . The method of claim 1 , further comprising contacting the CD34 + hematopoietic stem cell with a population of nanoparticles containing a homology-directed repair (HDR) template.

7 . The method of claim 1 , wherein the CD34 + hematopoietic stem cells comprise CD34 + , CD59 + , Thy1/CD90 + , CD38 lo− , c-kit/CD117 + , and lin hematopoietic stem cells and progeny thereof.

8 . The method of claim 1 , wherein the targeted genomic nucleotide sequence comprises a disease-associated mutation.

9 . The method of claim 8 , wherein the mutation is associated with Hemophilia B, sickle cell anemia, SCID, SCID-X1, ADA-SCID, Hereditary tyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), HIV/AIDS, Krabbe Disease, Polycythemia vera (PCV), myeloproliferative neoplasm, Familial essential thrombocythaemia (ET), Alpha-mannosidosis, an Immunodeficiency disorder, Hematologic condition, a Leukodystrophy or genetic lysosomal storage disease.

10 . The method of claim 8 , wherein the targeted genomic nucleotide sequence comprises a nucleotide sequence of a PD-1 gene.

11 . The method of claim 1 , wherein the particle comprises a viral vector.

12 . The method of claim 11 , wherein the viral vector comprises an adeno-associated virus.

13 . The method of claim 1 , wherein the single guide RNA (sgRNA) molecule directs sequence-specific binding of the pre-annealed SaCas9 complex to the target genomic nucleotide sequence to facilitate modification of a methylation state of the target sequence.

14 . A method for modifying a single targeted genomic nucleotide sequence of an isolated primary CD34 + hematopoietic stem cell and progeny thereof, comprising delivering to the CD34 + hematopoietic stem cell

a first liposome particle comprising DOTAP, DMPC, PEG, and cholesterol at a molar ratio of DOTAP: DMPC: PEG: cholesterol selected from 100:0:0:0, 90:0:10:0, or 90:0:5:5, said particle containing a pre-annealed complex of a Staphylococcus aureus CRISPR-Cas9 (SaCas9)-derived protein with nicking enzyme activity and a first chimeric single guide RNA molecule (sgRNA), that directs cleavage of one strand of the targeted nucleotide sequence to generate a 5′-overhang, wherein the complex is formed by admixing the SaCas9 protein and the first sgRNA molecule at a 1:1 molar ratio; and

a second liposome particle comprising DOTAP, DMPC, PEG, and cholesterol at a molar ratio of DOTAP: DMPC: PEG: cholesterol selected from 100:0:0:0, 90:0:10:0, or 90:0:5:5, said particle containing a pre-annealed complex of the SaCas9 protein with nicking enzyme activity and a second chimeric single guide RNA molecule (sgRNA) that directs cleavage of a strand opposite the targeted genomic nucleotide sequence to generate a 3′-overhang, wherein the complex is formed by admixing the SaCas9 protein and the second sgRNA molecule at a 1:1 molar ratio,

wherein the first and second chimeric single guide RNA molecules (sgRNAs) comprise a nucleotide sequence that is complementary to the targeted genomic nucleotide sequence,

wherein the targeted genomic nucleotide is repaired by homologous recombination with an exogenous template polynucleotide that results in the generation of a modified CD34 + hematopoietic stem cell comprising an insertion, deletion, or substitution of one or more nucleotides at said targeted genomic nucleotide sequence.

15 . The method of claim 14 , wherein the SaCas9 protein with nicking enzyme activity comprises one or more mutations in a catalytic domain.

16 . The method of claim 15 , wherein the one or more mutations in the catalytic domain with reference to the SaCas9 protein are selected from the group consisting of D10A, E762A, H840A, N854A, N863A, and D986A.

17 . The method of claim 14 , further comprising expanding the modified CD34 + hematopoietic stem cells to obtain a modified CD34 + hematopoietic stem cell population; and administering the modified CD34 + hematopoietic stem cell population to a host organism from which the CD34 + hematopoietic stem cells were harvested.

18 . A method for modifying a targeted genomic nucleotide sequence of an isolated primary CD34 + hematopoietic stem cell and progeny thereof, comprising delivering to the CD34 + hematopoietic stem cell

a first particle comprising DOTAP, DMPC, PEG, and cholesterol at a molar ratio of DOTAP:DMPC:PEG:cholesterol selected from 100:0:0:0, 90:0:10:0, or 90:0:5:5, said particle containing a pre-annealed complex of a first mutant Staphylococcus aureus CRISPR-Cas9 (SaCas9) protein having a complementary strand nickase activity, and a first chimeric single guide RNA (sgRNA) molecule, wherein the complex is formed by admixing the first mutant SaCas9 protein and the first sgRNA molecule at a 1:1 molar ratio,

wherein said first chimeric single guide RNA sgRNA molecule contains a first antisense nucleotide sequence that can hybridize with the targeted genomic nucleotide sequence and directs cleavage of the complementary strand to generate a 5′-overhang, and

a second particle comprising DOTAP, DMPC, PEG, and cholesterol at a molar ratio of DOTAP: DMPC: PEG: cholesterol selected from 100:0:0:0, 90:0:10:0, or 90:0:5:5, said particle containing a pre-annealed complex of a second mutant Staphylococcus aureus CRISPR-Cas9 (SaCas9) protein having a non-complementary strand nickase activity, and a second chimeric single guide RNA (sgRNA) molecule, wherein the complex is formed by admixing the second mutant SaCas9 protein and the second sgRNA molecule at a 1:1 molar ratio,

wherein the second chimeric single guide RNA (sgRNA) molecule contains a second antisense nucleotide sequence that can hybridize with the targeted genomic nucleotide sequence and directs the cleavage of the non-complementary strand of the targeted nucleotide sequence to generate a 3′-overhang,

wherein the targeted genomic nucleotide sequence is repaired by homologous recombination with an exogenous template polynucleotide that results in the generation of a modified CD34+ hematopoietic stem cell comprising an insertion, deletion, or substitution of one or more nucleotides at said targeted genomic nucleotide sequence.

19 . The method of claim 1 , wherein the hematopoietic stem cell is CD133+.

Assignments (9)
CORRECTIVE ASSIGNMENT TO CORRECT THE RECEIVING PARTY NAME PREVIOUSLY RECORDED AT REEL: 42675 FRAME: 081. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded May 15, 2019
From: DAHLMAN, JAMES E.
To: THE BROAD INSTITUTE, INC.
Reel/Frame 049189/0565 →
CORRECTIVE ASSIGNMENT TO CORRECT THE CONVEYING PARTY PREVIOUSLY RECORDED AT REEL: 046306 FRAME: 0097. ASSIGNOR(S) HEREBY CONFIRMS THE ASSIGNMENT. Recorded Feb 19, 2019
From: ZHANG, FENG
To: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 048373/0857 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 10, 2018
From: ZHANG, FENG
To: THE BROAD INSTITUTE INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 046306/0097 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 10, 2018
From: DAHLMAN, JAMES E.
To: THE BROAD INSTITUTE INC.
Reel/Frame 046306/0152 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 4, 2017
From: COX, DAVID BENJAMIN TURITZ
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 044288/0691 →
CONFIRMATORY LICENSE Recorded Jul 27, 2017
From: BROAD INSTITUTE, INC.
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 043350/0386 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 12, 2017
From: DAHLMAN, JAMES E.
To: THE BROAD INSTITUTE INC.
Reel/Frame 042675/0081 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 12, 2017
From: COX, DAVID BENJAMIN TURITZ
To: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 042675/0201 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 12, 2017
From: ZHANG, FENG
To: THE BROAD INSTITUTE INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Reel/Frame 042675/0164 →
Continuity (3)
Continuation In Part PCTUS2015065405 · Dec 11, 2015
Provisional Application 62091461 · Dec 12, 2014
Related Publication 20170349914A1 · Dec 7, 2017
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