Nuclease-mediated genome editing of primary cells
In certain aspects, the present invention provides methods for inducing a stable gene modification of a target nucleic acid via homologous recombination in a primary cell, such as a primary blood cell and/or a primary mesenchymal cell. In certain other aspects, the present invention provides methods for enriching a population of genetically modified primary cells having targeted integration at a target nucleic acid. The methods of the present invention rely on the introduction of a DNA nuclease such as a Cas polypeptide and a homologous donor adeno-associated viral (AAV) vector into the primary cell to mediate targeted integration of the target nucleic acid. Also provided herein are methods for preventing or treating a disease in a subject in need thereof by administering to the subject any of the genetically modified primary cells or pharmaceutical compositions described herein to prevent the disease or ameliorate one or more symptoms of the disease.
1. A composition comprising a population of primary cells isolated from a subject, wherein primary cells of the population comprise:
(a) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to a target nucleic acid sequence in the genome of the primary cells and a second nucleotide sequence that interacts with a CRISPR-associated Cas polypeptide, wherein the first nucleotide sequence, the second nucleotide sequence, or both comprise a modified nucleotide;
(b) the Cas polypeptide, an mRNA encoding the Cas polypeptide, or a recombinant expression vector comprising a nucleotide sequence encoding the Cas polypeptide, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid sequence; and
(c) an adeno-associated viral (AAV) vector comprising a polynucleotide,
wherein the Cas polypeptide is capable of cleaving the target nucleic acid sequence, thereby generating a double-stranded break in the target nucleic acid sequence;
wherein the polynucleotide is capable of integrating into the double-stranded break in the target nucleic acid sequence, thereby producing a stable integration of the polynucleotide into the genome of the primary cells; and
wherein the polynucleotide is integrated into the genome of greater than 10% of primary cells in the population of primary cells.
2. The composition of claim 1 , wherein integration of the polynucleotide corrects a mutation in the target nucleic acid sequence that is associated with a disease.
3. The composition of claim 2 , wherein integration of the polynucleotide replaces a mutant allele in the target nucleic acid sequence with a wild-type allele of the target nucleic acid sequence.
4. The composition of claim 2 , wherein integration of the polynucleotide inserts an open reading frame (ORF) that corresponds to a wild-type cDNA of the target nucleic acid sequence.
5. The composition of claim 2 , wherein the disease is a hemoglobinopathy.
6. The composition of claim 5 , wherein the hemoglobinopathy is sickle cell disease, α-thalassemia, β-thalassemia, or δ-thalassemia.
7. The composition of claim 1 , wherein the target nucleic acid sequence is within the HBB gene.
8. The composition of claim 1 , wherein the target nucleic acid sequence is within the HBB gene, wherein integration of the polynucleotide corrects one or more disease-causing mutations in the HBB gene.
9. The composition of claim 8 , wherein the disease is sickle cell disease and the sickle cell disease-causing mutation in the HBB gene is E6V.
10. The composition of claim 1 , wherein the primary cells comprise primary blood cells, primary mesenchymal cells, or a combination thereof.
11. The composition of claim 1 , wherein the primary cells comprise primary stem cells, primary progenitor cells, or primary somatic cells.
12. The composition of claim 11 , wherein the stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and organ stem cells.
13. The composition of claim 11 , wherein the progenitor cells are selected from the group consisting of hematopoietic progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, multipotent progenitor cells, oligopotent progenitor cells, and lineage-restricted progenitor cells.
14. The composition of claim 11 , wherein the somatic cells are selected from the group consisting of fibroblasts, hepatocytes, heart cells, liver cells, pancreatic cells, muscle cells, skin cells, blood cells, neural cells, and immune cells.
15. The composition of claim 14 , wherein the immune cells are selected from the group consisting of T lymphocytes (T cells), B lymphocytes (B cells), small lymphocytes, natural killer cells (NK cells), natural killer T cells, macrophages, monocytes, monocyte-precursor cells, eosinophils, neutrophils, basophils, megakaryocytes, myeloblasts, mast cells and dendritic cells.
16. The composition of claim 1 , wherein the population of primary cells comprises a heterogeneous population of primary cells.
17. The composition of claim 1 , wherein the AAV vector is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12.
18. The composition of claim 1 , wherein the AAV vector is a recombinant AAV6 vector.
19. The composition of claim 1 , wherein the Cas polypeptide is a Cas9 polypeptide.
20. The composition of claim 19 , wherein the Cas9 polypeptide is a high fidelity Cas9 polypeptide having enhanced specificity with reduced off-target effects and improved on-target specificity.
21. The composition of claim 1 , wherein the modified sgRNA and the Cas polypeptide are formed in a ribonucleoprotein (RNP) complex.
22. The composition of claim 1 , wherein the modified nucleotide is selected from the group consisting of: a 2′-O-methyl nucleotide, a 2′-O-methyl 3′-phosphorothioate nucleotide, and a 2′-O-methyl 3′-thioPACE (MSP) nucleotide.
23. The composition of claim 1 , wherein the modified nucleotide comprises a 2′-O-methyl 3′-phosphorothioate nucleotide.
24. The composition of claim 1 , wherein a 5′ end, a 3′ end, or a combination thereof of the modified sgRNA comprises the modified nucleotide.
25. The composition of claim 1 , wherein the primary cells are human cells.
26. The composition of claim 1 , further comprising a culture medium comprising a cytokine cocktail.
27. The composition of claim 1 , further comprising a culture medium comprising UM171, any other pyrimidoindole derivative, a variant thereof, or a derivative thereof.
28. A composition comprising a population of primary hematopoietic stem and progenitor cells (HSPCs) isolated from a subject having sickle cell disease, wherein primary HSPCs of the population comprise:
(i) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to a target nucleic acid sequence in the HBB gene of the primary cells and a second nucleotide sequence that interacts with a CRISPR-associated Cas polypeptide, wherein the first nucleotide sequence, the second nucleotide sequence, or both comprise a modified nucleotide;
(ii) the Cas polypeptide, an mRNA encoding the Cas polypeptide, or a recombinant expression vector comprising a nucleotide sequence encoding the Cas polypeptide, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid sequence in the HBB gene; and
(iii) an adeno-associated viral (AAV) vector comprising a polynucleotide, wherein the polynucleotide comprises a corrective SNP for reverting an E6V mutation in the HBB gene;
wherein the Cas polypeptide is capable of cleaving the target nucleic acid sequence, thereby generating a double-stranded break in the target nucleic acid sequence;
wherein the polynucleotide is capable of integrating into the double-stranded break in the target nucleic acid sequence, thereby producing a stable integration of the polynucleotide into the genome of the primary cells and correcting an E6V mutation in the HBB gene;
wherein the polynucleotide is integrated into the genome of greater than 10% of primary HSPCs in the population of primary HSPCs.
29. The composition of claim 28 , wherein the modified nucleotide is selected from the group consisting of: a 2′-O-methyl nucleotide, a 2′-O-methyl 3′-phosphorothioate nucleotide, and a 2′-O-methyl 3′-thioPACE (MSP) nucleotide.
30. The composition of claim 28 , wherein the AAV vector is a recombinant AAV6 vector.