EFFICIENT GENOME EDITING IN PRIMARY MYELOID CELLS

Abstract

Provided herein, inter alia, are compositions, methods, and systems for efficient genetic manipulation of myelod cells without the use of viral vectors. Further provided are strategies for gene disruption in primary myeloid cells (e.g. of human and murine origin) using electroporation-based delivery of Cas/ribonuclear proteins (RNPs). Methods provided herein including embodiments thereof can provide near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Cellular fitness and response to immunological stimuli may be unaffected by the gene editing process.

Description

Claims

1. A method for genetic modification of a myeloid cell, the method comprising transfecting the myeloid cell with a gene editing reagent targeting a genetic site of interest, wherein the myeloid cell is not transduced with a viral vector.

2. The method of claim 1, wherein the myeloid cell is a primary myeloid cell.

3. The method of claim 1, wherein the myeloid cell is a monocyte, macrophage, neutrophil, basophil, eosinophil, erythrocyte, dendritic cell, or megakaryocyte.

4. The method of claim 1, wherein the cell is transfected via electroporation, nucleofection, lipid-based transfection, or polymer-based transfection.

5-7. (canceled)

8. The method of claim 1, wherein the cell is not subjected to a selection step and/or an enrichment step after transfection.

9. The method of claim 1, wherein the gene editing reagent comprises an RNA-guided nuclease.

10-12. (canceled)

13. The method of claim 1, wherein the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, a guide RNA, and optionally a donor DNA.

14-15. (canceled)

16. The method of claim 1, wherein the gene editing reagent comprises transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an Argonaut endonuclease.

17. The method of claim 1, wherein the myeloid cell is contacted with an electroporation enhancer during transfection.

18. The method of claim 17, wherein the electroporation enhancer is selected from a carrier DNA, a single stranded DNA, a combination of single stranded and double stranded DNA, a polymeric additive, and an oligonucleotide.

19. The method of claim 18, wherein the carrier DNA is a single-stranded DNA oligonucleotide.

20. The method of claim 18, wherein the carrier DNA is nonhomologous to human, mouse, and/or rat genomes.

21. The method of claim 1, wherein the myeloid cell is differentiated prior to electroporation.

22. The method of claim 21, wherein the myeloid cell is differentiated into a dendritic cell.

23. (canceled)

24. The method of claim 1, wherein the myeloid cell is not activated prior to or during genetic modification.

25. The method of claim 1, wherein two or more distinct crispr RNAs (crRNA) to the site of interest are introduced into the myeloid cell.

26-28. (canceled)

29. The method of claim 25, wherein the crRNAs are single guide RNAs (sgRNAs).

30. The method of claim 1, wherein multiple genetic sites of interest are targeted.

31-32. (canceled)

33. A method for genetic modification of a plurality of myeloid cells, the method comprising transfecting the plurality of myeloid cells in the presence of a gene editing reagent targeting a site of interest, wherein the myeloid cells are not transduced with a viral vector.

34-36. (canceled)

37. The method of claim 33, wherein the site of interest is modified in at least 70% of the plurality of myeloid cells.

38. The method of claim 37, wherein the site of interest is modified in at least 80% of the plurality of myeloid cells.

39. The method of claim 37, wherein the site of interest is modified in at least 85% of the plurality of myeloid cells.

40. The method of claim 37, wherein the site of interest is modified in at least 90% of the plurality of myeloid cells.

41. The method of claim 33, wherein the plurality of myeloid cells comprises dendritic cells (DCs) and the site of interest is modified in at least 50% of the DCs.

42. The method of claim 33, wherein the viability of the plurality of myeloid cells after electroporation is at least 80%.

43. The method of claim 42, wherein the viability of the plurality of myeloid cells after electroporation is at least 90%.

44-51. (canceled)

52. The method of claim 9, wherein the RNA-guided nuclease comprises a guide RNA and a ribonucleoprotein (RNP), wherein the ratio of guide RNA to RNP is between 100:1 and 1:100.

53. The method of claim 52, wherein the ratio of guide RNA to RNP is less than or equal to about 3:1.

54. The method of claim 52, wherein the ratio of guide RNA to ribonucleoprotein (RNP) is less than or equal to about 2:1.

55. The method of claim 52, wherein the ratio of guide RNA to ribonucleoprotein (RNP) is about 3:1.

56. The method of claim 52, wherein the ratio of guide RNA to ribonucleoprotein (RNP) is about 2:1.

57. The method of claim 33, wherein at least 70% of the transfected cells are administered to a patient in need thereof, without selection or enrichment of the cells.

58. (canceled)

59. A method of genetically modifying a plurality of myeloid cells, comprising transfecting the myeloid cells with a gene editing reagent, wherein the myeloid cells are not transduced with a viral vector, and wherein the method does not comprise a selection step or enrichment step following myeloid cell transfection.

60-65. (canceled)

66. The method of claim 59, wherein the plurality of myeloid cells comprises dendritic cells (DCs) and the site of interest is modified in at least 50% of the DCs.

67-76. (canceled)

77. A system for genetically modifying a myeloid cell in the absence of a viral vector, the system comprising a chamber compatible with a transfection system, multiple myeloid cells within the chamber in a media compatible with electroporation, and at least one gene editing system designed to target at least one site of interest in the genome of myeloid cells.

78-96. (canceled)

97. A method of treating a disease treatable with a myeloid cell, comprising providing a genetically modified myeloid cell that has not been transduced with a virus, wherein the myeloid cell has been transfected with a gene editing reagent; and administering the myeloid cell to a patient in need thereof.

98-108. (canceled)

109. A composition comprising a plurality of myeloid cells, a gene editing reagent, a transfection buffer, and an electroporation enhancer, wherein the composition does not comprise a viral vector.

110-126. (canceled)

127. A genetically modified myeloid cell made by the method of claim 1 .

128. An assay for drug discovery comprising screening the effect of one or more compounds on the myeloid cell of claim 127.

129. A method for target validation of a compound, comprising contacting a myeloid cell of claim 127 with the compound and monitoring an effect on the cell.