INDUCIBLE RECOMBINASE SYSTEMS
20250304933 ยท 2025-10-02
Assignee
- Memorial Sloan-Kettering Cancer Center (New York, NY, US)
- SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH (New York, NY, US)
- MEMORIAL HOSPITAL FOR CANCER AND ALLIED DISEASES (New York, NY, US)
Inventors
- Ross Levine (New York, NY)
- Robert Lyle Bowman (New York, NY, US)
- Andrew Dunbar (New York, NY, US)
- Linde Miles (Cincinnati, OH, US)
Cpc classification
A01K2217/203
HUMAN NECESSITIES
A01K67/0275
HUMAN NECESSITIES
C12N9/22
CHEMISTRY; METALLURGY
C12N2800/30
CHEMISTRY; METALLURGY
C12N15/67
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
International classification
Abstract
Aspects of the disclosure provide compositions and methods for reversible expression of a target gene. In some aspects, the disclosure provides nucleic acids comprising multiple pairs of recombinase sites, each pair having first and second members flanking an inverted expression cassette encoding a target gene.
Claims
1. A nucleic acid comprising: i) a first nucleic acid segment comprising, in 5 to 3 order: a) a first member of a first pair of recombinase sites, each member of the first pair comprising a recognition sequence for a first recombinase; b) a first member of a second pair of recombinase sites, each member of the second pair comprising a recognition sequence for a second recombinase; c) a first member of a third pair of recombinase sites, each member of the third pair comprising a recognition sequence for the second recombinase; and d) a second member of the first pair of recombinase sites; ii) a second nucleic acid segment comprising an inverted expression cassette encoding a target gene; and iii) a third nucleic acid segment comprising, in 5 to 3 order: e) a second member of the second pair of recombinase sites; and f) a second member of the third pair of recombinase sites, wherein the nucleic acid comprises, in 5 to 3 order: (i), (ii), and (iii); or (iii), (ii), and (i).
2. The nucleic acid of claim 1, wherein (b)-(c) is not recombined upon exposure to the first recombinase.
3. The nucleic acid of claim 1 or 2, wherein (a) and (d) are separated from one another by a distance that prevents recombination of (b)-(c) upon exposure to the first recombinase.
4. The nucleic acid of any one of claims 1-3, wherein (a) and (d) are separated from one another by fewer than 90 contiguous nucleobases.
5. The nucleic acid of any one of claims 1-4, wherein (a) and (d) are separated from one another by fewer than 85 contiguous nucleobases.
6. The nucleic acid of any one of claims 1-5, wherein (a) and (d) are separated from one another by 82 contiguous nucleobases or fewer.
7. The nucleic acid of any one of claims 1-6, wherein the first and second members of each of the second and third pairs of recombinase sites are in an orientation opposite one another.
8. The nucleic acid of any one of claims 1-7, wherein the first and second members of each of the second and third pairs of recombinase sites are in forward and reverse orientations, respectively.
9. The nucleic acid of any one of claims 1-7, wherein the first and second members of each of the second and third pairs of recombinase sites are in reverse and forward orientations, respectively.
10. The nucleic acid of any one of claims 1-7, wherein: the first and second members of the second pair of recombinase sites are in forward and reverse orientations, respectively; and the first and second members of the third pair of recombinase sites are in reverse and forward orientations, respectively.
11. The nucleic acid of any one of claims 1-7, wherein: the first and second members of the second pair of recombinase sites are in reverse and forward orientations, respectively; and the first and second members of the third pair of recombinase sites are in forward and reverse orientations, respectively.
12. The nucleic acid of any one of claims 1-11, wherein: the first recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase; and the second recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase, provided that the first recombinase is different from the second recombinase.
13. The nucleic acid of any one of claims 1-12, wherein the first and second members of the first pair of recombinase sites are Lox sites, variant Lox sites, Frt sites, or variant Frt sites.
14. The nucleic acid of any one of claims 1-13, wherein: the first and second members of the second pair of recombinase sites are Rox sites, and the first and second members of the third pair of recombinase sites are variant Rox sites; or the first and second members of the second pair of recombinase sites are variant Rox sites, and the first and second members of the third pair of recombinase sites are Rox sites.
15. The nucleic acid of any one of claims 1-14, wherein the first and second members of each pair of recombinase sites are in an orientation opposite one another.
16. The nucleic acid of any one of claims 1-15, wherein the first and second members of the first pair of recombinase sites are in forward and reverse orientations, respectively.
17. The nucleic acid of any one of claims 1-15, wherein the first and second members of the first pair of recombinase sites are in reverse and forward orientations, respectively.
18. The nucleic acid of any one of claims 1-17, wherein the target gene encodes a protein of interest.
19. The nucleic acid of any one of claims 1-18, wherein the target gene encodes a protein comprising a disease-associated mutation.
20. The nucleic acid of any one of claims 1-14, wherein the second nucleic acid segment comprises: a first expression cassette encoding a first target gene; and a second expression cassette encoding a second target gene, wherein the first or second expression cassette is the inverted expression cassette.
21. The nucleic acid of claim 20, wherein: the first target gene encodes a wild-type protein, and the second target gene encodes a mutational variant of the wild-type protein; or the second target gene encodes a wild-type protein, and the first target gene encodes a mutational variant of the wild-type protein.
22. The nucleic acid of claim 20 or 21, wherein the first and second members of the first pair of recombinase sites are in the same orientation.
23. The nucleic acid of any one of claims 20-22, wherein the first and second members of the first pair of recombinase sites are variant Lox sites, each variant Lox site comprising a different sequence.
24. The nucleic acid of any one of claims 20-23, wherein the first pair of recombinase sites comprises a Lox66 site and a Lox71 site.
25. A cell comprising the nucleic acid of any one of claims 1-24.
26. The cell of claim 25, further comprising the first recombinase and/or a nucleic acid encoding the first recombinase.
27. The cell of claim 25 or 26, further comprising the second recombinase and/or a nucleic acid encoding the second recombinase.
28. The cell of any one of claims 25-27, further comprising an inducer that activates the first and/or second recombinase.
29. The cell of claim 28, wherein the inducer is a drug or chemical.
30. A non-human mammal comprising the nucleic acid of any one of claims 1-24 or the cell of any one of claims 25-29.
31. A nucleic acid comprising, in 5 to 3 order: a) a first member of a first pair of recombinase sites, each member of the first pair comprising a recognition sequence for a first recombinase; b) a first member of a second pair of recombinase sites, each member of the second pair comprising a recognition sequence for a second recombinase; c) a first member of a third pair of recombinase sites, each member of the third pair comprising a recognition sequence for the second recombinase; d) a second member of the first pair of recombinase sites; e) an inverted expression cassette encoding a target gene; f) a second member of the second pair of recombinase sites; and g) a second member of the third pair of recombinase sites, wherein the first and second members of each pair of recombinase sites are in an orientation opposite one another, wherein (b)-(c) is not inverted upon exposure to the first recombinase, and wherein (c)-(e) or (d)-(f) is inverted upon exposure to the second recombinase.
32. The nucleic acid of claim 31, wherein the first and second members of the first pair of recombinase sites are in the same orientation upon inversion of (c)-(e) or (d)-(f) by the second recombinase.
33. The nucleic acid of claim 31 or 32, wherein (a) and (d) are separated from one another by a distance that prevents inversion of (b)-(c) upon exposure to the first recombinase.
34. The nucleic acid of any one of claims 31-33, wherein (a) and (d) are separated from one another by fewer than 90 contiguous nucleobases, fewer than 85 contiguous nucleobases, or 82 contiguous nucleobases or fewer.
35. The nucleic acid of any one of claims 31-34, wherein the first member of each pair of recombinase sites is in a forward orientation, and the second member of each pair of recombinase sites is in a reverse orientation.
36. The nucleic acid of any one of claims 31-34, wherein the first member of each pair of recombinase sites is in a reverse orientation, and the second member of each pair of recombinase sites is in a forward orientation.
37. The nucleic acid of any one of claims 31-36, wherein: the first recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase; and the second recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase, provided that the first recombinase is different from the second recombinase.
38. The nucleic acid of any one of claims 31-37, wherein the first and second members of the first pair of recombinase sites are Lox sites, variant Lox sites, Frt sites, or variant Frt sites.
39. The nucleic acid of any one of claims 31-38, wherein: the first and second members of the second pair of recombinase sites are Rox sites, and the first and second members of the third pair of recombinase sites are variant Rox sites; or the first and second members of the second pair of recombinase sites are variant Rox sites, and the first and second members of the third pair of recombinase sites are Rox sites.
40. The nucleic acid of any one of claims 31-39, wherein the target gene encodes a protein of interest.
41. The nucleic acid of any one of claims 31-40, wherein the target gene encodes a protein comprising a disease-associated mutation.
42. A method for regulating expression of a target gene, the method comprising: contacting the nucleic acid of any one of claims 31-41 with the second recombinase, wherein the target gene is inverted to a sense orientation by the second recombinase.
43. The method of claim 42, further comprising contacting the nucleic acid with the first recombinase, wherein the target gene is excised from the nucleic acid by the first recombinase.
44. The method of claim 42 or 43, wherein the contacting is in a cell.
45. The method of claim 44, wherein the cell is in a non-human mammal.
46. A nucleic acid comprising, in 5 to 3 order: a) a first member of a first pair of recombinase sites, each member of the first pair comprising a recognition sequence for a first recombinase; b) a first member of a second pair of recombinase sites, each member of the second pair comprising a recognition sequence for the first recombinase; c) an inverted expression cassette encoding a target gene; d) a first member of a third pair of recombinase sites, each member of the third pair comprising a recognition sequence for a second recombinase; e) a second member of the first pair of recombinase sites; f) a second member of the second pair of recombinase sites; and g) a second member of the third pair of recombinase sites, wherein the first and second members of each pair of recombinase sites are in an orientation opposite one another, wherein (e)-(f) is not inverted upon exposure to the second recombinase, and wherein (b)-(d) or (c)-(e) is inverted upon exposure to the first recombinase.
47. The nucleic acid of claim 46, wherein the first and second members of the third pair of recombinase sites are in the same orientation upon inversion of (b)-(d) or (c)-(e) by the second recombinase.
48. The nucleic acid of claim 46 or 47, wherein (d) and (g) are separated from one another by a distance that prevents inversion of (e)-(f) upon exposure to the second recombinase.
49. The nucleic acid of any one of claims 46-48, wherein (d) and (g) are separated from one another by fewer than 90 contiguous nucleobases, fewer than 85 contiguous nucleobases, or 82 contiguous nucleobases or fewer.
50. The nucleic acid of any one of claims 46-49, wherein the first member of each pair of recombinase sites is in a forward orientation, and the second member of each pair of recombinase sites is in a reverse orientation.
51. The nucleic acid of any one of claims 46-49, wherein the first member of each pair of recombinase sites is in a reverse orientation, and the second member of each pair of recombinase sites is in a forward orientation.
52. The nucleic acid of any one of claims 46-51, wherein: the first recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase; and the second recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase, provided that the first recombinase is different from the second recombinase.
53. The nucleic acid of any one of claims 46-52, wherein the first and second members of the third pair of recombinase sites are Lox sites, variant Lox sites, Frt sites, or variant Frt sites.
54. The nucleic acid of any one of claims 46-53, wherein: the first and second members of the first pair of recombinase sites are Rox sites, and the first and second members of the second pair of recombinase sites are variant Rox sites; or the first and second members of the first pair of recombinase sites are variant Rox sites, and the first and second members of the second pair of recombinase sites are Rox sites.
55. The nucleic acid of any one of claims 46-54, wherein the target gene encodes a protein of interest.
56. The nucleic acid of any one of claims 46-55, wherein the target gene encodes a protein comprising a disease-associated mutation.
57. A method for regulating expression of a target gene, the method comprising: contacting the nucleic acid of any one of claims 46-56 with the first recombinase, wherein the target gene is inverted to a sense orientation by the first recombinase.
58. The method of claim 57, further comprising contacting the nucleic acid with the second recombinase, wherein the target gene is excised from the nucleic acid by the second recombinase.
59. The method of claim 57 or 58, wherein the contacting is in a cell.
60. The method of claim 59, wherein the cell is in a non-human mammal.
61. A nucleic acid comprising, in 5 to 3 order: a) a first member of a first pair of recombinase sites, each member of the first pair comprising a recognition sequence for a first recombinase; b) a first member of a second pair of recombinase sites, each member of the second pair comprising a recognition sequence for a second recombinase; c) a first member of a third pair of recombinase sites, each member of the third pair comprising a recognition sequence for the second recombinase; d) a second member of the first pair of recombinase sites; e) a first expression cassette encoding a first target gene; f) a second expression cassette encoding a second target gene, wherein the first or second expression cassette is an inverted expression cassette; g) a second member of the second pair of recombinase sites; and h) a second member of the third pair of recombinase sites, wherein the first and second members of the first pair of recombinase sites are in the same orientation, wherein the first and second members of each of the second and third pairs of recombinase sites are in an orientation opposite one another, wherein (b)-(c) is not excised from the nucleic acid upon exposure to the first recombinase, and wherein (c)-(f) or (d)-(g) is inverted upon exposure to the second recombinase.
62. The nucleic acid of claim 61, wherein the first and second members of the first pair of recombinase sites are in an orientation opposite one another upon inversion of (c)-(f) or (d)-(g) by the second recombinase.
63. The nucleic acid of claim 61 or 62, wherein (a) and (d) are separated from one another by a distance that prevents excision of (b)-(c) upon exposure to the first recombinase.
64. The nucleic acid of any one of claims 61-63, wherein (a) and (d) are separated from one another by fewer than 90 contiguous nucleobases, fewer than 85 contiguous nucleobases, or 82 contiguous nucleobases or fewer.
65. The nucleic acid of any one of claims 61-64, wherein the first and second members of the first pair of recombinase sites are in a forward orientation.
66. The nucleic acid of any one of claims 61-64, wherein the first and second members of the first pair of recombinase sites are in a reverse orientation.
67. The nucleic acid of any one of claims 61-66, wherein: the first recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase; and the second recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase, provided that the first recombinase is different from the second recombinase.
68. The nucleic acid of any one of claims 61-67, wherein the first and second members of the first pair of recombinase sites are Lox sites, variant Lox sites, Frt sites, or variant Frt sites.
69. The nucleic acid of any one of claims 61-68, wherein the first and second members of the first pair of recombinase sites are variant Lox sites, each variant Lox site comprising a different sequence.
70. The nucleic acid of any one of claims 61-69, wherein the first pair of recombinase sites comprises a Lox66 site and a Lox71 site.
71. The nucleic acid of any one of claims 61-70, wherein: the first and second members of the second pair of recombinase sites are Rox sites, and the first and second members of the third pair of recombinase sites are variant Rox sites; or the first and second members of the second pair of recombinase sites are variant Rox sites, and the first and second members of the third pair of recombinase sites are Rox sites.
72. The nucleic acid of any one of claims 61-71, wherein: the first target gene encodes a wild-type protein, and the second target gene encodes a mutational variant of the wild-type protein; or the second target gene encodes a wild-type protein, and the first target gene encodes a mutational variant of the wild-type protein.
73. The nucleic acid of any one of claims 61-72, wherein the first or second target gene encodes a protein comprising a disease-associated mutation.
74. A method for regulating expression of a target gene, the method comprising: contacting the nucleic acid of any one of claims 61-73 with the second recombinase, wherein the first or second target gene is inverted to a sense orientation by the second recombinase.
75. The method of claim 74, further comprising contacting the nucleic acid with the first recombinase, wherein the first or second target gene is inverted to an antisense orientation by the first recombinase.
76. The method of claim 74 or 75, wherein the contacting is in a cell.
77. The method of claim 76, wherein the cell is in a non-human mammal.
78. A nucleic acid comprising, in 5 to 3 order: a) a first member of a first pair of recombinase sites, each member of the first pair comprising a recognition sequence for a first recombinase; b) a first member of a second pair of recombinase sites, each member of the second pair comprising a recognition sequence for the first recombinase; c) a first expression cassette encoding a first target gene; d) a second expression cassette encoding a second target gene, wherein the first or second expression cassette is an inverted expression cassette; e) a first member of a third pair of recombinase sites, each member of the third pair comprising a recognition sequence for a second recombinase; f) a second member of the first pair of recombinase sites; g) a second member of the second pair of recombinase sites; and h) a second member of the third pair of recombinase sites, wherein the first and second members of each of the first and second pairs of recombinase sites are in an orientation opposite one another, wherein the first and second members of the third pair of recombinase sites are in the same orientation, wherein (f)-(g) is not excised from the nucleic acid upon exposure to the second recombinase, and wherein (b)-(e) or (c)-(f) is inverted upon exposure to the first recombinase.
79. The nucleic acid of claim 78, wherein the first and second members of the third pair of recombinase sites are in an orientation opposite one another upon inversion of (b)-(e) or (c)-(f) by the second recombinase.
80. The nucleic acid of claim 78 or 79, wherein (e) and (h) are separated from one another by a distance that prevents inversion of (f)-(g) upon exposure to the second recombinase.
81. The nucleic acid of any one of claims 78-80, wherein (e) and (h) are separated from one another by fewer than 90 contiguous nucleobases, fewer than 85 contiguous nucleobases, or 82 contiguous nucleobases or fewer.
82. The nucleic acid of any one of claims 78-81, wherein the first and second members of the third pair of recombinase sites are in a forward orientation.
83. The nucleic acid of any one of claims 78-81, wherein the first and second members of the third pair of recombinase sites are in a reverse orientation.
84. The nucleic acid of any one of claims 78-83, wherein: the first recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase; and the second recombinase is selected from Cre recombinase, Dre recombinase, and Flp recombinase, provided that the first recombinase is different from the second recombinase.
85. The nucleic acid of any one of claims 78-84, wherein the first and second members of the third pair of recombinase sites are Lox sites, variant Lox sites, Frt sites, or variant Frt sites.
86. The nucleic acid of any one of claims 78-85, wherein the first and second members of the third pair of recombinase sites are variant Lox sites, each variant Lox site comprising a different sequence.
87. The nucleic acid of any one of claims 78-86, wherein the third pair of recombinase sites comprises a Lox66 site and a Lox71 site.
88. The nucleic acid of any one of claims 78-87, wherein: the first and second members of the first pair of recombinase sites are Rox sites, and the first and second members of the second pair of recombinase sites are variant Rox sites; or the first and second members of the first pair of recombinase sites are variant Rox sites, and the first and second members of the second pair of recombinase sites are Rox sites.
89. The nucleic acid of any one of claims 78-88, wherein: the first target gene encodes a wild-type protein, and the second target gene encodes a mutational variant of the wild-type protein; or the second target gene encodes a wild-type protein, and the first target gene encodes a mutational variant of the wild-type protein.
90. The nucleic acid of any one of claims 78-89, wherein the first or second target gene encodes a protein comprising a disease-associated mutation.
91. A method for regulating expression of a target gene, the method comprising: contacting the nucleic acid of any one of claims 78-90 with the first recombinase, wherein the first or second target gene is inverted to a sense orientation by the first recombinase.
92. The method of claim 91, further comprising contacting the nucleic acid with the second recombinase, wherein the first or second target gene is inverted to an antisense orientation by the second recombinase.
93. The method of claim 91 or 92, wherein the contacting is in a cell.
94. The method of claim 93, wherein the cell is in a non-human mammal.
95. A cell comprising the nucleic acid of any one of claims 31-41, 46-56, 61-73, or 78-90.
96. The cell of claim 95, wherein the cell further comprises the first recombinase and/or a nucleic acid encoding the first recombinase.
97. The cell of claim 95 or 96, wherein the cell further comprises the second recombinase and/or a nucleic acid encoding the second recombinase.
98. The cell of any one of claims 95-97, wherein the first and/or second recombinase is activated by an inducer.
99. The cell of claim 98, wherein the inducer is a drug or chemical.
100. A non-human mammal comprising: the nucleic acid of any one of claims 31-41, 46-56, 61-73, or 78-90; or the cell of any one of claims 95-99.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.
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DETAILED DESCRIPTION
[0085] Among other aspects, the present disclosure relates to inducible recombinase systems that leverage certain principles of recombinase-mediated exchange to allow for temporal and spatial control of gene expression. Accordingly, in some aspects, the disclosure provides nucleic acid constructs configured for reversible activation of a target gene through recombinase-mediated exchange reactions. In some aspects, the nucleic acid constructs provided herein incorporate multiple pairs of recombinase sites in a configuration that precludes exchange by a first recombinase until after exchange by a second recombinase. In this way, target gene expression can be controlled by the manner and timing in which the nucleic acid constructs are exposed to the first and second recombinases.
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[0087] In some embodiments, nucleic acids of the present disclosure are configured such that expression of a target gene can be reversibly activated and inactivated in an inducible process mediated by recombinase activity. For example, in some embodiments, a nucleic acid construct comprises an inverted expression cassette encoding a target gene such that a protein of interest is not expressed from the target gene, and through inducible recombinase-mediated activity, the nucleic acid is reconfigured to activate expression of the protein of interest (e.g., a wild-type protein or a variant of a wild-type protein). In some embodiments, through further inducible recombinase-mediated activity, the target gene is excised from the nucleic acid to inactivate expression of the protein of interest.
[0088] In this example process, activation of target gene expression occurs through sequential inversion and deletion reactions mediated by Recombinase B. The inversion is a reversible reaction in which Recombinase B inverts the inverted expression cassette to a sense orientation through recombination at the second or third pair of recombinase sites. The recombination can occur at the second pair of recombinase sites to invert the intervening sequence between these sites to produce construct 100-1a, or the recombination can occur at the third pair of recombinase sites to invert the intervening sequence between these sites to produce construct 100-1b.
[0089] Moving forward in the process, Recombinase B can act on construct 100-1a by deletion of the intervening sequence between the third pair of recombinase sites to produce construct 100-2. Likewise, Recombinase B can act on construct 100-1b by deletion of the intervening sequence between the second pair of recombinase sites to produce construct 100-2. In some embodiments, the target gene can be expressed from construct 100-2, which comprises the expression cassette in a sense orientation. However, as each of constructs 100-1a, 100-1b, and 100-2 comprises the expression cassette in a sense orientation, it should be appreciated that any one of these constructs can express the target gene. In some embodiments, the use of two pairs of recombinase sites for Recombinase B advantageously promotes the formation of construct 100-2 by increasing the kinetic and/or thermodynamic favorability of the inversion reaction in the forward direction.
[0090] In some embodiments, construct 100 is configured such that Recombinase A cannot recombine at the first pair of recombinase sites. For example, in some embodiments, the first and second members of the first pair are separated by a distance on construct 100 that is less than a minimum distance required for recombination at these sites by Recombinase A. In some embodiments, recombination of construct 100 mediated by Recombinase B increases the separation distance between the first and second members of the first pair of recombinase sites. As such, in some embodiments, the first and second members of the first pair are separated by a distance on construct 100-2 that meets the minimum distance required for recombination at these sites by Recombinase A.
[0091] Following the recombination mediated by Recombinase B, expression of the target gene is activated. Expression of the target gene can be inactivated upon exposure of construct 100-2 to Recombinase A, which deletes the intervening sequence, including the expression cassette, between the first pair of recombinase sites to produce the inactivated construct 100-3. Accordingly, the configuration of construct 100 permits an inducible activation and inactivation of target gene expression.
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[0093] In some embodiments, the first nucleic acid segment (I) is upstream of (e.g., 5 to) the second nucleic acid segment (II), and the third nucleic acid segment (III) is downstream of (e.g., 3 to) the second nucleic acid segment (II), as illustrated by: constructs 100, 102, 104, and 106 (
[0094] In some embodiments, the first and second members of the first pair of recombinase sites in the first nucleic acid segment (I) are separated by a distance that prevents inversion of an intervening sequence between the first and second members of the first pair upon exposure to the first recombinase. In some embodiments, the first and second members of the first pair are separated from one another by an intervening sequence of fewer than 100 contiguous nucleobases or base pairs. In some embodiments, the first and second members of the first pair are separated from one another by an intervening sequence of fewer than 95, fewer than 90, fewer than 85, fewer than 84, fewer than 83, fewer than 82, fewer than 81, fewer than 80, fewer than 78, fewer than 76, or fewer than 74 contiguous nucleobases or base pairs. In some embodiments, the first and second members of the first pair are separated from one another by an intervening sequence of at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 contiguous nucleobases or base pairs (e.g., about 30-90, about 30-85, about 30-82, about 40-82, about 45-82, about 50-82, about 70-82, about 72-82, about 74-82, about 75-82 contiguous nucleobases or base pairs). In some embodiments, the intervening sequence between the first and second members can be determined based on the number of contiguous nucleobases or base pairs spanning the first nucleobase or base pair adjacent to the 3-most nucleotide of the first member and the first nucleobase or base pair adjacent to the 5-most nucleotide of the second member.
[0095] In some embodiments, the first and second members of each pair of recombinase sites are in an orientation opposite one another. In some embodiments, the first member of each pair of recombinase sites is in a forward orientation, and the second member of each pair of recombinase sites is in a reverse orientation, as illustrated by constructs 100 and 108. In some embodiments, the first member of each pair of recombinase sites is in a reverse orientation, and the second member of each pair of recombinase sites is in a forward orientation, as illustrated by constructs 106 and 114.
[0096] In some embodiments, the first members of the second and third pairs of recombinase sites are in the same orientation as one another, and the first member of the first pair of recombinase sites is in an opposite orientation relative to the first members of each of the second and third pairs, as illustrated by constructs 102, 104, 110, and 112. In some embodiments, the first members of the second and third pairs of recombinase sites are in an orientation opposite one another, as illustrated by the constructs shown in
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[0098] As described above, in some embodiments, nucleic acids of the present disclosure are configured such that expression of a target gene can be reversibly activated and inactivated in an inducible process mediated by recombinase activity. For example, in some embodiments, a nucleic acid construct is configured to express a first protein (e.g., a wild-type protein), and through inducible recombinase-mediated activity, the nucleic acid is reconfigured to activate expression of a second protein (e.g., a variant of the wild-type protein). In some embodiments, through further inducible recombinase-mediated activity, the nucleic acid is further reconfigured to inactivate expression of the second protein and return to a configuration that expresses the first protein.
[0099] For purposes of brevity, the target gene in this example refers to the second target gene, which can be activated and inactivated through inducible recombinase-mediated processes. However, it should be appreciated that in the context of this embodiment, when expression of the second target gene is inactive, expression of the first target gene is active. Accordingly, construct 150 is configured to express the first target gene (First Target), and the example process of
[0100] As shown, activation of second target gene expression occurs through sequential inversion and deletion reactions mediated by Recombinase B. The inversion is a reversible reaction in which Recombinase B inverts the first expression cassette encoding the first target gene to an antisense orientation, with concomitant inversion of the second expression cassette encoding the second target gene to a sense orientation, through recombination at the second or third pair of recombinase sites. The recombination can occur at the second pair of recombinase sites to invert the intervening sequence between these sites to produce construct 150-1a, or the recombination can occur at the third pair of recombinase sites to invert the intervening sequence between these sites to produce construct 150-1b.
[0101] Moving forward in the process, Recombinase B can act on construct 150-1a by deletion of the intervening sequence between the third pair of recombinase sites to produce construct 150-2. Likewise, Recombinase B can act on construct 150-1b by deletion of the intervening sequence between the second pair of recombinase sites to produce construct 150-2. In some embodiments, the second target gene can be expressed from construct 150-2, which comprises the second expression cassette in a sense orientation. However, as each of constructs 150-1a, 150-1b, and 150-2 comprises the second expression cassette in a sense orientation, it should be appreciated that any one of these constructs can express the second target gene. In some embodiments, the use of two pairs of recombinase sites for Recombinase B advantageously promotes the formation of construct 150-2 by increasing the kinetic and/or thermodynamic favorability of the inversion reaction in the forward direction.
[0102] In some embodiments, construct 150 is configured such that Recombinase A cannot recombine at the first pair of recombinase sites. For example, in some embodiments, the first and second members of the first pair are separated by a distance on construct 150 that is less than a minimum distance required for recombination at these sites by Recombinase A. In some embodiments, recombination of construct 150 mediated by Recombinase B increases the separation distance between the first and second members of the first pair of recombinase sites. As such, in some embodiments, the first and second members of the first pair are separated by a distance on construct 150-2 that meets the minimum distance required for recombination at these sites by Recombinase A.
[0103] Following the recombination mediated by Recombinase B, expression of the second target gene is activated. Expression of the second target gene can be inactivated, and expression of the first target gene reactivated, upon exposure of construct 150-2 to Recombinase A, which inverts the intervening sequence, including the first and second expression cassettes, between the first pair of recombinase sites to produce construct 150-3. Accordingly, the configuration of construct 150 permits an inducible process of switching between expression of the first target gene and the second target gene.
[0104]
[0105] In some embodiments, the first nucleic acid segment (I) is upstream of (e.g., 5 to) the second nucleic acid segment (II), and the third nucleic acid segment (III) is downstream of (e.g., 3 to) the second nucleic acid segment (II), as illustrated by: constructs 150, 152, 154, and 156 (
[0106] In some embodiments, the first and second members of the first pair of recombinase sites in the first nucleic acid segment (I) are separated by a distance that prevents inversion of an intervening sequence between the first and second members of the first pair upon exposure to the first recombinase. In some embodiments, the first and second members of the first pair are separated from one another by an intervening sequence of fewer than 100 contiguous nucleobases or base pairs. In some embodiments, the first and second members of the first pair are separated from one another by an intervening sequence of fewer than 95, fewer than 90, fewer than 85, fewer than 84, fewer than 83, fewer than 82, fewer than 81, fewer than 80, fewer than 78, fewer than 76, or fewer than 74 contiguous nucleobases or base pairs. In some embodiments, the first and second members of the first pair are separated from one another by an intervening sequence of at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 contiguous nucleobases or base pairs (e.g., about 30-90, about 30-85, about 30-82, about 40-82, about 45-82, about 50-82, about 70-82, about 72-82, about 74-82, about 75-82 contiguous nucleobases or base pairs). In some embodiments, the intervening sequence between the first and second members can be determined based on the number of contiguous nucleobases or base pairs spanning the first nucleobase or base pair adjacent to the 3-most nucleotide of the first member and the first nucleobase or base pair adjacent to the 5-most nucleotide of the second member.
[0107] In some embodiments, the first and second members of the second pair of recombinase sites are in an orientation opposite one another, and the first and second members of the third pair of recombinase sites are in an orientation opposite one another. In some embodiments, the first members of the second and third pairs are in a forward orientation, and the second members of the second and third pairs are in a reverse orientation, as illustrated by constructs 150, 152, 158, and 160. In some embodiments, the first members of the second and third pairs are in a reverse orientation, and the second members of the second and third pairs are in a forward orientation, as illustrated by constructs 154, 156, 162, and 164. In some embodiments, the first members of the second and third pairs of recombinase sites are in an orientation opposite one another, as illustrated by the constructs shown in
Recombinases
[0108] In some aspects, the disclosure provides a nucleic acid comprising multiple pairs of recombinase sites. A recombinase is a genetic recombination enzyme that catalyzes directionally sensitive nucleic acid (e.g., DNA) exchange reactions between a pair of recombinase sites that are specific to a particular recombinase. Depending on the position and orientation of the pair of recombinase sites, different outcomes can result from the exchange reactions. For example, if the first and second members of a pair of recombinase sites are in the same orientation (e.g., both members in a forward orientation or both members in a reverse orientation), the exchange results in an excision of the nucleic acid segment flanked by the first and second members. If the first and second members of a pair of recombinase sites are in an orientation opposite one another (e.g., one member in a forward orientation and the other member in a reverse orientation), the exchange results in an inversion of the nucleic acid segment flanked by the first and second members.
[0109] In some embodiments, a recombinase site is a nucleotide sequence comprising a central spacer sequence, which determines the orientation (e.g., forward or reverse) of the recombinase site, flanked by two palindromic recognition sequences. Accordingly, in some embodiments, each member of a pair of recombinase sites comprises two recognition sequences, where each recognition sequence is specifically recognized and bound by a recombinase (e.g., a recombinase monomer or subunit). In a nucleic acid exchange reaction, each recognition sequence of a recombinase site is bound by a recombinase such that each member of a pair of recombinase sites is bound by two recombinase subunits, and the four recombinase subunits catalyze the exchange reaction.
[0110] A recombinase may be any recombinase known in the art. Non-limiting examples of recombinases include: Cre recombinase, Dre recombinase, Flp recombinase, Hin recombinase, Tre recombinase, Nigri recombinase, Panto recombinase, Vika recombinase, and phiC31 recombinase. A recombinase may be a wild-type recombinase or a variant recombinase. In some embodiments, a wild-type recombinase binds to a wild-type recognition sequence. In some embodiments, a recombinase is a variant recombinase. A variant recombinase comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to a wild-type recombinase. In some embodiments, the one or more substitutions comprise at least one mutation, which may be a conservative mutation or a non-conservative mutation. A conservative mutation occurs when an amino acid of a wild-type protein is replaced with another amino acid having similar properties (e.g., charge, hydrophobicity, size). A non-conservative mutation occurs when an amino acid of a wild-type protein is replaced with another amino acid having different properties (e.g., charge, hydrophobicity, size). Substitutions may be made such that a variant recombinase recognizes a variant recognition sequence and/or such that the variant recombinase protein has superior protein expression and/or recombination activity in a specific cell type (e.g., mammalian cell, bacterial cell) or environment (e.g., high pH, low pH).
[0111] In some embodiments, a recombinase is a Cre recombinase. Cre recombinase is a tyrosine recombinase enzyme from the P1 bacteriophage. Cre recombinase catalyzes DNA recombination without cofactors (e.g., ATP) or accessory bacterial proteins and is thus able to conduct DNA recombination in a variety of cellular environments. A Cre recombinase may be a wild-type Cre recombinase or a variant Cre recombinase.
[0112] Cre recombinase catalyzes site-specific recombination between two recombinase sites known as locus of crossing over (x) in P1 bacteriophage (loxP) sites. These two loxP sites may be on the same or different DNA strands. LoxP sites are 34-base pair (bp) sites consisting of two 13-bp palindromic recognition sequences that flank an asymmetric 8-bp spacer sequence. One Cre protein binds to each palindromic sequence of each member of a pair of loxP sites, and four Cre proteins form a tetramer to bring two loxP sites together for recombination. Recombination occurs within the spacer sequence of the loxP sites. During recombination, the spacer region undergoes single strand breaks, followed by strand exchange, strand migration, and resolution of the Cre-loxP complex. The asymmetric spacers ensure the unidirectionality of the Cre-loxP reaction.
[0113] The orientation of two loxP sites determines the products of Cre recombination. A DNA segment between two loxP sites oriented in the same direction will be excised as a circular loop of DNA, and a DNA segment between two loxP sites oriented in opposite directions will be inverted. Recombination between two loxP sites located on different DNA molecules produces DNA strand exchange and translocation. While there is no direct contact between Cre and the loxP spacer region, the homology of the spacer is required for efficient recombination between the two loxP recognition sequences.
[0114] In some embodiments, a Cre recombinase is a wild-type Cre recombinase. In some embodiments, a wild-type Cre recombinase binds to a wild-type loxP site (Table 1). In some embodiments, a Cre recombinase is a variant Cre recombinase. A variant Cre recombinase comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to a wild-type Cre recombinase. The one or more substitutions may be made such that the variant Cre recombinase recognizes a variant loxP recognition sequence and/or such that the variant Cre recombinase protein has superior protein expression and/or recombination activity in a specific cell type (e.g., mammalian cell, bacterial cell) or environment (e.g., high pH, low pH). A variant Cre recombinase may be any variant Cre recombinase known in the art. Non-limiting examples of variant Cre recombinases include: ALSHG Cre, LNSGG Cre (Baldwin et al., (2010), Chem Biol. 10(11): 1085-1094); D7, D7L.sup.A3, D7R.sup.B2, D7L.sup.K201R, D7R.sup.Q311R (Hoersten et al., (2022), Nucleic Acids Research, 50(2): 1174-1186); and Cre-A1 (wild-type+K25R, D29R, R32E, D33L, Q35R), Cre-B1 (wild-type+E69D, R72K, L76E), Cre-A2 (Cre-A1+R337E), Cre-B2 (Cre-B1+E308R), Cre-A3 (Cre-A2+E123L), Cre-B3 (Cre-B2+E123L) (Zhang, Engineering Cre Recombinase for Genome Engineering, Washington University Open Scholarship).
[0115] In some embodiments, a variant Cre recombinase binds to a variant loxP site. A variant loxP site comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to a wild-type loxP site. In some embodiments, the one or more substitutions comprise at least one mutation, which may be a conservative or non-conservative mutation. A mutation may occur in the upstream (5) recognition sequence, the spacer sequence, the downstream (3) recognition sequence, or some combination thereof. A variant loxP site may be any variant loxP site known in the art. Non-limiting examples of variant loxP sites and their sequences are provided in Table 1.
[0116] In some embodiments, a recombinase is a D6 site-specific DNA recombinase (Dre recombinase). Dre recombinase is a tyrosine recombinase enzyme from the P1 bacteriophage and is closely related to Cre recombinase, but has a distinct DNA specificity for a 32-bp recombinase site. Similar to Cre recombinase, Dre catalyzes DNA recombination without accessory bacterial proteins and can conduct DNA in mammalian cells. Dre recombinase exhibits no cross-reactivity with Cre recombinase for loxP sites. A Dre recombinase may be a wild-type Dre recombinase or a variant Dre recombinase.
[0117] Dre recombinase catalyzes site-specific recombination between two recombinase sites known as region of crossover (roxP) sites. These two roxP sites may be on the same or different DNA strands. Rox sites are 32-base pair (bp) sites consisting of two 14-bp palindromic recognition sequences flanking an asymmetric 4-bp spacer sequence. One Dre protein binds to each recognition sequence of each member of a pair of roxP sites, and four Dre proteins form a tetramer to bring two roxP sites together for recombination. Recombination occurs within the spacer sequence of the roxP sites. During recombination, the spacer region undergoes single strand breaks, followed by strand exchange, strand migration, and resolution of the Dre-roxP complex. The asymmetric spacers ensure the unidirectionality of the Dre-roxP reaction.
[0118] The orientation of the two roxP sites determines the products of Dre recombination. A DNA segment between two roxP sites oriented in the same direction will be excised as a circular loop of DNA, and a DNA segment between two loxP sites oriented in opposite directions will be inverted. Recombination between two roxP sites located on different DNA molecules produces DNA strand exchange and translocation.
[0119] In some embodiments, a Dre recombinase is a wild-type Dre recombinase. In some embodiments, a wild-type Dre recombinase binds to a roxP site (Table 1). In some embodiments, a Dre recombinase is a variant Dre recombinase. A variant Dre recombinase protein comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to a wild-type Dre recombinase. The one or more substitutions may be made such that the variant Dre recombinase recognizes a variant roxP recognition sequence and/or such that the variant Dre recombinase has superior protein expression and/or recombination activity in a specific cell type (e.g., mammalian cell, bacterial cell) or environment (e.g., high pH, low pH). A variant Dre recombinase may be any variant Dre recombinase known in the art. Non-limiting examples of variant Dre recombinases are described in Nguyen et al., 2015, IOVS, 56(7).
[0120] In some embodiments, a variant Dre recombinase binds to a variant roxP site. A variant roxP site comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to a wild-type roxP site. In some embodiments, the one or more substitutions comprise at least one mutation, which may be a conservative or non-conservative mutation. A mutation may occur in the upstream (5) recognition sequence, the spacer sequence, the downstream (3) recognition sequence, or some combination thereof. A variant roxP site may be any variant roxP site known in the art. Non-limiting examples of variant roxP sites and their sequences are provided in Table 1.
[0121] In some embodiments, a recombinase is a flippase (Flp) recombinase. Flp is a tyrosine recombinase enzyme from the 2 p plasmid of Saccharomyces cerevisiae. A Flp recombinase may be a wild-type Flp recombinase or a variant Flp recombinase.
[0122] Flp recombinase catalyzes site-specific recombination between two recombinase sites known as flippase recognition target (FRT) sites. These two FRT sites may be on the same (cis) or different (trans) DNA strands. FRT sites are 34-base pair (bp) sites consisting of two 13-bp palindromic sequences that flank an 8-bp asymmetric spacer sequence. One Flp protein binds to each palindromic sequence of each member of a pair of FRT sites, and four Flp proteins form a tetramer to bring two FRT sites together for recombination. Recombination occurs within the spacer sequence of the FRT sites. During recombination, the spacer region undergoes single strand breaks, followed by strand exchange, strand migration, and resolution of the Flp-FRT complex. The asymmetric spacers ensure the unidirectionality of the Flp-FRT reaction.
[0123] The orientation of these two FRT sites determines the products of Flp recombination. A DNA segment between two FRT sites oriented in the same direction will be excised as a circular loop of DNA, and a DNA segment between two FRT sites oriented in opposite directions will be inverted. Recombination between two FRT sites located on different DNA molecules produces DNA strand exchange and translocation.
[0124] In some embodiments, a Flp recombinase is a wild-type Flp recombinase. A wild-type Flp recombinase binds to a Frt site (Table 1). In some embodiments, a Flp recombinase is a variant Flp recombinase. A variant Flp recombinase protein comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to a wild-type Flp recombinase.
[0125] The one or more substitutions may be made such that the variant Flp recombinase recognizes a variant Frt recognition sequence and/or such that the variant Flp recombinase has superior protein expression and/or recombination activity in a specific cell type (e.g., mammalian cell, bacterial cell) or environment (e.g., high temperature, low temperature, high pH). A variant Flp recombinase may be any variant Flp recombinase known in the art. Non-limiting examples of variant Flp recombinases include: Flpe (Buchholz et al., 1998, Nat Biotechnol, 16(7): 657-662); Flpo (Kranz et al., 2010, Genesis, 48(8): 512-520); Flp.sup.H305P, Flp.sup.H305L, Flp.sup.R308G, Flp.sup.H305E (Parsons et al., 1988, Mol Cell Biol., 8(8): 330-3310); and FV1, FV2, FV3, FV4, FV5, FV6, FV7, FV8 (Bolusani et al., 2006, Nuc Acids Res., 34: 5259-5269).
[0126] In some embodiments, a variant Flp recombinase binds to a variant Frt site. A variant Flp site comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to a wild-type Frt site. In some embodiments, the one or more substitutions comprise at least one mutation, which may be a conservative or non-conservative mutation. A mutation may occur in the upstream (5) recognition sequence, the spacer sequence, the downstream (3) recognition sequence, or some combination thereof. A variant Frt site may be any variant Frt site known in the art. Non-limiting examples of variant Frt sites and their sequences are provided in Table 1.
TABLE-US-00001 TABLE1 ExampleRecombinaseSites First Second Recognition Spacer Recognition Name Sequence Sequence Sequence loxsites Wild-type ATAACTTCGTATA NNNTANNN TATACGAAGTTAT loxP (SEQIDNO:1) (SEQIDNO:2) lox511 ATAACTTCGTATA ATGTATAC TATACGAAGTTAT (SEQIDNO:3) (SEQIDNO:4) lox5171 ATAACTTCGTATA ATGTGTAC TATACGAAGTTAT (SEQIDNO:5) (SEQIDNO:6) lox2272 ATAACTTCGTATA AAGTATCC TATACGAAGTTAT (SEQIDNO:7) (SEQIDNO:8) lox71 TACCGTTCGTATA NNNTANNN TATACGAAGTTAT (SEQIDNO:9) (SEQIDNO:10) lox72 TACCGTTCGTATA NNNTANNN TATACGAACGGTA (SEQIDNO:11) (SEQIDNO:12) lox66 ATAACTTCGTATA NNNTANNN TATACGAACGGTA (SEQIDNO:13) (SEQIDNO:14) loxPsym ATAACTTCGTATA ATGTACAT TATACGAACGGTA (SEQIDNO:15) (SEQIDNO:16) loxF8 ATAAATCTGTGGA AACGCTGC CACACAATCTTAG (SEQIDNO:17) (SEQIDNO:18) loxF8L ATAAATCTGTGGA AACGCTGC TCCACAGATTTAT (SEQIDNO:19) (SEQIDNO:20) loxF8R CTAAGATTGTGTG AACGCTGC CACACAATCTTAG (SEQIDNO:21) (SEQIDNO:22) loxM1 ATAACTTCATATA GCATACAT TATATGAAGTTAT (SEQIDNO:23) (SEQIDNO:24) loxM2 ATAACTTCGTATA AGAAACCA TATACGAAGTTAT (SEQIDNO:25) (SEQIDNO:26) loxM3 ATAACTTCGTATA TAATACCA TATACGAAGTTAT (SEQIDNO:27) (SEQIDNO:28) loxM4 ATAACTTTGTATA GCATACAT TATACAAAGTTAT (SEQIDNO:29) (SEQIDNO:30) loxM5 ATAACTTCGTGCA GCATACAT TGCACGAAGTTAT (SEQIDNO:31) (SEQIDNO:32) loxM6 ATAACTCTGTATA GCATACAT TATACAGAGTTAT (SEQIDNO:33) (SEQIDNO:34) loxM7 ATAACTTCGTATA AGATAGAA TATACGAAGTTAT (SEQIDNO:35) (SEQIDNO:36) loxM8 ATAACTCTGTGTA GCATACAT TACACAGAGTTAT (SEQIDNO:37) (SEQIDNO:38) loxM11 TACCGTTCGTATA CGATACCA TATACGAAGTTAT (SEQIDNO:39) (SEQIDNO:40) roxsites roxP TAACTTTAAATA ATGCCAAT TATTTAAAGTTA (SEQIDNO:41) (SEQIDNO:42) rox7 TAACTTTAAATA AGGCCAGT TATTTAAAGTTA (SEQIDNO:43) (SEQIDNO:44) rox8 TAACTTTAAATA ACGCCTCT TATTTAAAGTTA (SEQIDNO:45) (SEQIDNO:46) rox12 TAACTTTAAATA AGGCCTGT TATTTAAAGTTA (SEQIDNO:47) (SEQIDNO:48) rox61 TAACTTTAAATA AGGCCCGT TATTTAAAGTTA (SEQIDNO:49) (SEQIDNO:50) rox85 TAACTTTAAATA AGGCCGGT TATTTAAAGTTA (SEQIDNO:51) (SEQIDNO:52) Frtsites Frt GAAGTTCCTATTC TCTAGAAA GTATAGGAACTTC (SEQIDNO:53) (SEQIDNO:91) Frt1 GAAGTTCCTATTC TCTAGATA GTATAGGAACTTC (SEQIDNO:55) (SEQIDNO:54) Frt2 GAAGTTCCTATTC TCTACTTA GTATAGGAACTTC (SEQIDNO:57) (SEQIDNO:56) Frt3 GAAGTTCCTATTC TTCAAATA GTATAGGAACTTC (SEQIDNO:59) (SEQIDNO:58) Frt4 GAAGTTCCTATTC TCTAGAAG GTATAGGAACTTC (SEQIDNO:61) (SEQIDNO:60) Frt5 GAAGTTCCTATTC TTCAAAAG GTATAGGAACTTC (SEQIDNO:63) (SEQIDNO:62) Frt3 GAAGTTCCTATTC TCATATAA GTATAGGAACTTC (SEQIDNO:65) (SEQIDNO:64) Frt14 GAAGTTCCTATTC TATCAGAA GTATAGGAACTTC (SEQIDNO:67) (SEQIDNO:66) Frt545 GAAGTTCCTATTC TCTAAAAA GTATAGGAACTTC (SEQIDNO:69) (SEQIDNO:68) mFRT11 GAAGTTCCTATAG TTTCTAGA CTATAGGAACTTC (SEQIDNO:71) (SEQIDNO:70) mFRT545 GAAGTTCCTATTC TCTAAAAA GTATAGGAACTTC (SEQIDNO:73) (SEQIDNO:72) mFRT11-71 GAAGTTTCTATAG TTTCTAGA CTATAGAAACTTC (SEQIDNO:75) (SEQIDNO:74) mFRT71 GAAGTTTCTATTC TCTAGAAA GTATAGAAACTTC (SEQIDNO:77) (SEQIDNO:76) FL-IL10A AGTGATTTGATAC TTACATGA GTAAAGGAATTAG (SEQIDNO:79) (SEQIDNO:78) FRTw2 GAAGTTCCTATAC TATCTACA GAATAGGAACTTC (SEQIDNO:81) (SEQIDNO:80)
[0127] In some embodiments, a recombinase is an inducible recombinase. In some embodiments, an inducible recombinase refers to a recombinase that is activated in the presence of a chemical, drug, or form of energy. For example, in some embodiments, a system (e.g., a cell, an organism) does not express a recombinase in the absence of an inducer. In some embodiments, the system comprises a nucleic acid encoding the recombinase, and the inducer activates expression of the recombinase. In some embodiments, the system does not comprise a nucleic acid encoding a recombinase, and the inducer is itself a nucleic acid encoding the recombinase. The activity of an inducible recombinase protein is therefore controlled by the presence (or absence) of an inducer. This permits the timing of a particular recombination event to be controlled. One may want to restrict the timing of recombination to: the desired step in a sequence of events, when favorable environmental conditions are present, when a target gene of interest is being expressed (or shut off), when the desired stage of development or evolution is occurring, or any combination thereof.
[0128] Any chemical, drug, or form of energy used in the art as an inducer may be used to induce a recombinase. Non-limiting examples of inducers include: tamoxifen, mifepristone (RU-486), rapamycin, doxycycline, ultraviolet light illumination, tetracycline, near infrared light illumination, non-photodamaging UVA light illumination, blue light illumination, red/far-red illumination, dexamethasone, triamcinolone acetonide, RU38486, dihydrotestosterone, and OH-flutamide. In some embodiments, an inducer of a recombinase is a nucleic acid encoding the recombinase. For example, in some embodiments, an inducer is a synthetic or recombinant nucleic acid (e.g., a DNA vector, an RNA transcript) that may be exogenously introduced into a system (e.g., a cell, an organism).
Expression Cassettes
[0129] In some aspects, the disclosure provides a nucleic acid comprising an expression cassette (e.g., an inverted expression cassette) encoding a target gene. In some embodiments, an expression cassette is a segment of a nucleic acid comprising a target gene(s) and the necessary regulatory sequences for the target gene(s) to be expressed in a cell. In some embodiments, an expression cassette contains one or more of a promoter sequence, an open reading frame comprising a target gene(s), and a 3 untranslated region (UTR) downstream of the open reading frame.
[0130] A promoter sequence may be an endogenous promoter sequence for a target gene or an exogenous promoter sequence. An endogenous promoter sequence is a promoter sequence that drives expression of a target gene under endogenous (natural) conditions. In some embodiments, a promoter sequence is an endogenous promoter sequence for a given target gene. In some embodiments, a promoter sequence comprises one or more substitutions (e.g., mutations, insertions, deletions) relative to an endogenous promoter sequence, which can optimize the activity of the endogenous promoter in a specific organism (e.g., eukaryote, prokaryote).
[0131] An exogenous promoter sequence is a promoter sequence that does not drive expression of a target gene under endogenous circumstances. An exogenous promoter may be derived from the same organism as the target gene or derived from a different organism as the target gene. An exogenous promoter may be selected for a target gene if the exogenous promoter has favorable expression characteristics (e.g., constitutive, inducible). For example, in some embodiments, an exogenous promoter is adapted for expressing genes in a specific organism (e.g., eukaryote, prokaryote). An exogenous promoter may be any exogenous promoter known in the art. Non-limiting examples of exogenous promoters include: SV40, CAG, PGK, TRE, U6, EF1a, UAS, T7, Sp6, lac, araBad, trp, Ptac, Ubc, human beta actin, Ac5, polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, T7lac, pL, and T3.
[0132] A target gene may be any gene (or portion thereof) known in the art. A nucleic acid provided herein may contain an expression cassette comprising multiple target genes. Multiple target genes may be in tandem in an expression cassette or separated by intervening sequences. In some embodiments, an expression cassette contains 1-25 target genes, 2-24 target genes, 3-23 target genes, 4-22 target genes, 5-21 target genes, 6-20 target genes, 7-19 target genes, 8-18 target genes, 9-17 target genes, 10-16 target genes, 11-15 target genes, or 12-14 target genes. In some embodiments, an expression cassette contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more target genes.
[0133] An expression cassette provided herein, in some embodiments, comprises a 3 untranslated region (3UTR). In some embodiments, a 3UTR immediately follows a termination codon of a target gene (or final target gene). In some embodiments, a 3UTR comprises one or more regulatory regions that regulate target gene expression, including polyadenylation, translation efficiency, localization, and stability of the transcribed target gene. A 3UTR may also comprise binding sites for regulatory proteins and microRNAs (miRNAs). miRNAs decrease gene (e.g., target gene) expression of various target genes by either inhibiting their translation or directly causing degradation of a transcript of a target gene(s).
[0134] In some embodiments, a 3UTR comprises one or more regulatory sequences, including, but not limited to, AU-rich elements (AREs), a polyadenylation sequence, a poly(A) binding protein sequence, a cytoskeleton protein binding sequence, or a combination thereof. AREs are bound by proteins that regulate the stability or decay of transcripts (e.g., transcribed from target genes) to regulate translation initiation. A polyadenylation sequence (e.g., AAUAAA) directs the addition of several hundred adenine bases to the 3 end of a transcript (e.g., mRNA transcript of a target gene(s)) to form a poly(A) tail. A poly(A) tail contains a poly(A) binding protein (PABP) sequence. PABP binding to a poly(A) tail regulates mRNA translation, stability, and export. A cytoskeleton protein binding sequence is bound by a cytoskeleton protein (e.g., actin, myosin), which regulates transport and localization of a target gene transcript (e.g., mRNA transcript) within a cell.
Target Genes
[0135] Provided herein, in some aspects, is a nucleic acid comprising a nucleotide sequence encoding a target gene. In some embodiments, a target gene is a gene whose expression is regulated by nucleic acids and methods provided herein. A target gene may be a complete gene, a partial gene, multiple complete genes, multiple partial genes, or some combination thereof. In some embodiments, a target gene is an exon. In some embodiments, a target gene is a nucleotide sequence encoding a protein of interest.
[0136] In some embodiments, a target gene comprises a disease-associated mutation or multiple disease-associated mutations. For example, in some embodiments, a target gene encodes a protein that comprises one or more disease-associated mutations relative to a wild-type protein. A disease-associated mutation(s) is a mutation(s) in a target gene whose expression is associated with a disease. Expressing the disease-associated mutation may cause the disease to occur in an organism (e.g., non-human mammal) with the nucleic acid containing the target gene. In some embodiments, a disease-associated mutation is necessary to cause the disease. In some embodiments, a disease-associated mutation is sufficient to cause the disease. In some embodiments, a disease-associated mutation is necessary and sufficient to cause the disease.
[0137] A disease-associated mutation may be any disease-associated mutation known in the art. Non-limiting examples of disease-associated mutations include: Jak2.sup.V617F (myeloproliferative neoplasms: polycythemia vera, thrombocythemia, idiopathic myelofibrosis); Flt3.sup.ITD (clonal hematopoiesis, CH); KRAS.sup.G12D, KRAS.sup.G12A, KRAS.sup.G12V (pancreatic adenocarcinoma, colorectal adenocarcinoma, prostate adenocarcinoma, melanoma); TERT (glioblastoma multiforme, colorectal adenocarcinoma, thyroid adenocarcinoma, bladder cancer, melanoma); KRAS.sup.G12S, KRAS.sup.G12R, KRAS.sup.G12C (colorectal adenocarcinoma, pancreatic adenocarcinoma); BRAF.sup.V600E (colorectal adenocarcinoma, thyroid adenocarcinoma, melanoma); PIKCA.sup.E545Q, PIKCA.sup.E545K (colorectal adenocarcinoma, breast adenocarcinoma, bladder cancer, melanoma); TP53.sup.R175H (colorectal adenocarcinoma, pancreatic adenocarcinoma, breast adenocarcinoma, melanoma); TP53.sup.R248P, TP53.sup.R248Q (glioblastoma multiforme, pancreatic adenocarcinoma, breast adenocarcinoma, bladder cancer, lymphoid malignancy); TP53.sup.282W (prostate adenocarcinoma, pancreatic adenocarcinoma, breast adenocarcinoma, melanoma); TP53.sup.Y220C (pancreatic adenocarcinoma, breast adenocarcinoma, bladder cancer, lymphoid malignancy); TP53.sup.R248G, TP53.sup.R248W (colorectal adenocarcinoma, prostate adenocarcinoma, prostate adenocarcinoma, breast adenocarcinoma, bladder cancer); IDH1.sup.R132H (glioblastoma multiforme, prostate adenocarcinoma, lymphoid malignancy); VAV1.sup.D26G, VAV1.sup.F69V, VAV1.sup.E84D. VAV1.sup.V102E, VAV1.sup.E157K, VAV1.sup.Y174C, VAV1.sup.E175V, VAV1.sup.L177R, VAV1.sup.E234D, VAV1.sup.E234Q, VAV1.sup.D324Y, VAV1.sup.H399Y, VAV1.sup.K404R, VAV1.sup.Q498K, VAV1.sup.Q498R, VAV1.sup.E391D, VAV1.sup.R436S, VAV1.sup.M501L, VAV1.sup.M501R, VAV1.sup.M501V, VAV1.sup.E556D, VAV1.sup.E556K, VAV1.sup.A511D, VAV1.sup.Q524E, VAV1.sup.E633G, VAV1.sup.G648C, VAV1.sup.A673V, VAV1.sup.R678Q, VAV1.sup.R790C, VAV1.sup.D797N, VAV1.sup.R798P, VAV1.sup.R798Q, VAV1.sup.E850K, VAV1.sup.K815E, VAV1.sup.R822Q, VAV1.sup.R822L, VAV1.sup.Y826S, VAV1.sup.W831L, VAV1.sup.A798T, VAV1.sup.G816R,VAV1.sup.G819S (peripheral T-cell lymphoma); GFAP.sup.R79H, GFAP.sup.R239C, GFAP.sup.R239H, GFAP.sup.R88C, GFAP.sup.R88S, GFAP.sup.L76F, GFAP.sup.N77Y, (Alexander disease); SOD1.sup.A4V, SOD1.sup.A4T, SOD1.sup.V7E, SOD1.sup.V14M, SOD1.sup.E21K, SOD1.sup.G37R, SOD1.sup.L38V, SOD1.sup.G41S, SOD1.sup.G41D, SOD1.sup.H43R, SOD1.sup.H46R, SOD1.sup.H48E, SOD1.sup.L84V, SOD1.sup.G85R, SOD1.sup.D90A, SOD1.sup.G93A, SOD1.sup.G93C, SOD1.sup.G93R, SOD1.sup.G93D, SOD1.sup.E100G, SOD1.sup.D101N, SOD1.sup.D101G, SOD1 SOD1.sup.L106V, SOD1.sup.I112T, SOD1.sup.I113T, SOD1.sup.R115G, SOD1.sup.D125H, SOD1.sup.L144F, SOD1.sup.V148G, SOD1.sup.I149T (amyotrophic lateral sclerosis); TBP.sup.(CAG).sub.63 (ataxia); NLGN3.sup.R451C (autism spectrum disorders); NLGN4.sup.186T (autism spectrum disorders); NOTCH3.sup.W71C, NOTCH3.sup.R69C, NOTCH3.sup.R182C (CADASIL); PMP22.sup.G150C (Dejerine-Sottas); PSEN1.sup.M146L, PSEN1.sup.H163R, PSEN1.sup.A246E, PSEN1.sup.L286V, PSEN1.sup.C410Y, PSEN1.sup.M139V, PSEN1.sup.H163Y, PSEN1.sup.E280A, PSEN1.sup.E280G, PSEN1.sup.P267S, PSEN1.sup.E120D, PSEN1.sup.A426P, PSEN1.sup.M146I, PSEN1.sup.L250S, PSEN1.sup.R278T, PSEN1.sup.C92S, PSEN1.sup.G206A, PSEN1.sup.G266S, (Alzheimer disease); APP (early-onset dementia); PCDH19.sup.V441E, PCDH19.sup.E85T, PCDH19.sup.S671ter, PCDH19.sup.E48ter, PCDH19.sup.N557K (focal epilepsy); CACNA1A.sup.R192Q, CACNA1A.sup.T666M, CACNA1A.sup.V714A, CACNA1A.sup.I1811L, CACNA1A.sup.D715E, CACNA1A.sup.Y1385C, CACNA1A.sup.S218L, CACNA1A.sup.R583Q, CACNA1A.sup.V1457L, CACNA1A.sup.I710T, CACNA1A.sup.R1347Q, (hemiplegic migraine); SYNGAP1.sup.K138ter, SYNGAP1.sup.R579ter, SYNGAP1.sup.K138ter, SYNGAP1.sup.W362R, SYNGAP1.sup.P562L, SYNGAP1.sup.W267ter, SYNGAP1.sup.R143ter (intellectual disability); NF1.sup.L348P, NF1.sup.R365X, NF1.sup.C5242T, NF1.sup.C5260T, NF1.sup.C5839T, NF1.sup.AA5843del, NF1.sup.R1947X, (neurofibromatosis type II); NF2.sup.L360P, NF2.sup.L535, NF2.sup.E538P, NF2.sup.F96del, NF2.sup.E182ter, NF2.sup.R262ter, NF2.sup.E320ter, NF2.sup.R431ter, NF2.sup.E407ter, NF2.sup.E463ter, NF2.sup.R466ter, NF2.sup.E527ter, (neurofibromatosis type II); ATP1A3.sup.T613M, ATP1A3.sup.1274T, ATP1A3.sup.E227K, ATP1A3.sup.1758S, ATP1A3.sup.F780L, ATP1A3.sup.D801R, ATP1A3.sup.D923N (rapid onset dystonia Parkinsonism); MECP2.sup.R133C, MECP2.sup.F155S, MECP2.sup.806del, MECP2.sup.R270ter, MECP2.sup.T158M, MECP2.sup.R106W, MECP2.sup.R294ter, and MECP2.sup.R306C (Rett syndrome).
[0138] In some embodiments, a target gene is Jak2 and encodes the protein Janus kinase 2 (JAK2). JAK2 is a non-receptor tyrosine kinase that regulates cytokine and growth factor signaling. Activated JAK2 recruits and phosphorylates signal transducer and activator of transcription (STAT) proteins, which translocate to the nucleus and regulate gene transcription. Dysregulation of JAK2 activity produces increased cellular proliferation and myeloproliferative neoplasms (MPNs). Jak2 may be any Jak2 known in the art. Non-limiting examples of Jak2 include: human (Gene ID: 3717), mouse (Gene ID: 16452), rat (Gene ID: 24514), cow (Gene ID: 525246), chicken (Gene ID: 374199), dog (Gene ID: 484185), rabbit (Gene ID: 100358134), frog (Gene ID: 100036599), rhesus monkey (Gene ID: 694184), chimpanzee (Gene ID: 464979), and pig (Gene ID: 397201).
[0139] In some embodiments, a target gene is Jak2.sup.V617F or a corresponding mutation in a non-human. The Jak2.sup.V617F mutation is present in myeloproliferative neoplasms (MPNs), with up to 97% of patients with polycythemia vera, up to 57% of patients with thrombocythemia, and up to 50% of patients with idiopathic myelofibrosis (see, e.g., Baxter, et al., 2005, Lancet, 365: 1054-1061). This Jak2.sup.V617F mutation is predicted to dysregulate JAK2 kinase activity, resulting in increased cell proliferation.
[0140] In some embodiments, a target gene is Flt3 and encodes the protein fins related receptor tyrosine kinase 3 (FLT3). FLT3 is a class III receptor tyrosine kinase that regulates hematopoiesis. FLT3 is activated by binding to FLT3 ligand (FLT3L) and phosphorylates and activates multiple effectors in apoptosis, proliferation, and hematopoietic stem cell differentiation in bone marrow. Dysregulations due to mutations in Flt3 are associated with clonal hematopoiesis (CH) and leukemia. Flt3 may be any Flt3 known in the art. Non-limiting examples of Flt3 include: human (Gene ID: 2322), mouse (Gene ID: 14255), rat (Gene ID: 140635), cow (Gene ID: 512700), chicken (Gene ID: 100858422), dog (Gene ID: 486025), frog (Gene ID: 101733150), rhesus monkey (Gene ID: 721712), and chimpanzee (Gene ID: 452508).
[0141] In some embodiments, a target gene is Flt3.sup.ITD or a corresponding mutation in a non-human. The Flt3.sup.ITD mutation is an internal tandem duplication (ITD) in exons 14 and 15 and is present in leukemia (e.g., acute myeloid leukemia, AML). The Flt3.sup.ITD mutation ranges in size from 3 to more than hundreds of nucleotides and are thought to disrupt an autoinhibitory conformation of the FLT3 receptor, resulting in constitutive activation of downstream signaling.
[0142] In some embodiments, a target gene is TP53 and encodes the protein tumor protein p53 (TP53). TP53 is a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. TP53 responds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Dysregulations in TP53 are associated with cancers. TP53 may be any TP53 known in the art. Non-limiting examples of TP53 include: human (Gene ID: 7157), mouse (Gene ID: 22059), rat (Gene ID: 24842), dog (Gene ID: 403869), pig (Gene ID: 397276), chicken (Gene ID: 396200), cow (Gene ID: 281542), cat (Gene ID: 493847), rabbit (Gene ID: 10009292), rhesus monkey (Gene ID: 716170), and chimpanzee (Gene ID: 455214).
[0143] In some embodiments, a target gene comprises TP53.sup.R175H, TP53.sup.R248P, TP53.sup.R248Q, TP53.sup.282W, TP53.sup.Y220C, TP53.sup.R248G, and/or TP53.sup.PR248W.
[0144] In some embodiments, a target gene is KRAS and encodes the protein Kirsten rat sarcoma viral oncogene (KRAS). KRAS is an oncogene homolog that is a member of the small GTPase superfamily. A single amino acid mutation in KRAS may cause an activating mutation that is implicated in numerous cancers, including lung adenocarcinoma, mucinous adenocarcinoma, ductal carcinoma of the pancreas, and colorectal carcinoma. KRAS may be any KRAS known in the art. Non-limiting examples of KRAS include: human (Gene ID: 3845), mouse (Gene ID: 16653), rat (Gene ID: 24525), dog (Gene ID: 403871), cow (Gene ID: 541140), rhesus monkey (Gene ID: 707977), cat (Gene ID: 751104), frog (Gene ID: 100485959), chicken (Gene ID: 418207), chimpanzee (Gene ID: 473387), and pig (Gene ID: 100518251).
[0145] In some embodiments, a target gene comprises KRAS.sup.G12S, KRAS.sup.G12R, KRAS.sup.G12C, KRAS.sup.G12D, KRAS.sup.G12V, KRAS.sup.G13D, KRAS.sup.A59T, KRAS.sup.G60R, KRAS.sup.D1S3V, KRAS.sup.T58I, KRAS.sup.V141, KRAS.sup.P34R, KRAS.sup.V52G, KRAS.sup.D153V, KRAS.sup.G13R, KRAS.sup.F156L, KRAS.sup.G60S, KRAS.sup.Y71S, KRAS.sup.K147E, KRAS.sup.G13C, KRAS.sup.L19F, KRAS.sup.A146T and/or KRAS.sup.A146V.
[0146] In some embodiments, a target gene is ASXL1 and encodes the protein additional sex combs-like 1 (ASXL1). ASXL1 is a protein that is a member of the Polycomb group of proteins which are necessary for the maintenance of stable repression of gene loci.
[0147] Specifically, ASXL1 is thought to disrupt chromatin in localized areas, enhancing transcription of certain genes while repressing the transcription of other genes. Dysregulations in ASXL1 are associated with myelodysplastic syndromes and chronic myelomonocytic leukemia (CML). ASXL1 may be any ASXL1 known in the art. Non-limiting examples of ASXL1 include: human (Gene ID: 171023), mouse (Gene ID: 228790), rat (Gene ID: 311553), rhesus monkey (Gene ID: 711799), cow (Gene ID: 522091), chimpanzee (Gene ID: 458168), chicken (Gene ID: 428158), dog (Gene ID: 100688017), and frog (Gene ID: 100379981).
[0148] In some embodiments, a target gene comprises Asxl1.sup.Q925ter, Asxl1.sup.R404ter Asxl1.sup.Q773ter, and/or Asxl1.sup.R965ter.
[0149] In some embodiments, a target gene is DNMT3a and encodes the protein DNA methyltransferase 3 alpha (DNMT3A). DNMT3A is a protein that functions in de novo methylation rather than maintenance methylation. DNA methylation is required for mammalian development. DNMT3A localizes to the cytoplasm and the nucleus and its expression is developmentally regulated. Mutations in DNMT3A are associated with acute myeloid leukemia (AML). DNMT3A may be any DNMT3A known in the art. Non-limiting examples of DNMT3A include: human (Gene ID: 1788), mouse (Gene ID: 13435), rat (Gene ID: 444984), chicken (Gene ID: 421991), cow (Gene ID: 359716), pig (Gene ID: 100037301), rhesus monkey (Gene ID: 694822) frog (Gene ID: 100496503), chimpanzee (Gene ID: 739139), dog (Gene ID: 482996), and cat (Gene ID: 100510798).
[0150] In some embodiments, a target gene comprises DNMT3A.sup.889TGGdel, DNMT3A.sup.L648P, DNMT3A.sup.M548K, DNMT3A.sup.F902S, DNMT3A.sup.R882H, DNMT3A.sup.R882C, DNMT3A.sup.W330R, and/or DNMT3A.sup.D333N.
[0151] In some embodiments, a target gene is RUNX1 and encodes a runt-related transcription factor 1 (RUNX1) protein. RUNX1 is a protein that forms half of the heterodimeric transcription factor core binding factor (CBF) that binds to the core element of many enhancers and promoters. RUNX1 is thought to be involved in hematopoiesis, and translocations in RUNX1 are associated with leukemias. RUNX1 may be any RUNX1 known in the art. Non-limiting examples of RUNX1 include: human (Gene ID: 861), mouse (Gene ID: 12394), rat (Gene ID: 50662), pig (Gene ID: 100512633), cow (Gene ID: 529631), frog (Gene ID: 100485449), chicken (Gene ID: 396152), rhesus monkey (Gene ID: 696749), chimpanzee (Gene ID: 101058316), and dog (Gene ID: 487746).
[0152] In some embodiments, a target gene comprises RUNX1.sup.IVS3As, G-T, -1, RUNX1.sup.R201Q, RUNX1.sup.K83E, RUNX1.sup.Y260ter, RUNX1.sup.A107P, RUNX1.sup.H58N, RUNX1.sup.A129E, and/or RUNX1.sup.8-bp del, NT442.
Cells and Cellular Organisms
[0153] In some aspects, the disclosure provides a cell comprising a nucleic acid described herein. In some embodiments, the cell is a eukaryotic cell. For example, in some embodiments, the cell is a mammalian cell, a plant cell, a fungi cell, a reptilian cell, a protozoal cell, an excavata cell, or a diatom. In some embodiments, a cell is a prokaryotic cell. For example, in some embodiments, the cell is a bacterial cell, an archaeal cell, or a cyanobacterial cell.
[0154] A nucleic acid provided herein may be introduced into a cell (e.g., for expression, genomic integration) by any method known in the art. Non-limiting examples for methods of introducing a nucleic acid into a cell include, without limitation, transfection (e.g., DEAE-dextran, calcium phosphate, artificial lipids), electroporation, microinjection, viral infection (e.g., lentivirus, adeno-associated virus, adenovirus), dendrimer delivery, and lipid nanoparticle delivery.
[0155] A nucleic acid may be independent of or integrated into the genome of a cell. For example, in some embodiments, a nucleic acid is of a circular form that is separate and not expressed from the genome of a host cell. In some embodiments, a nucleic acid is integrated into and expressed from the genome of a host cell.
[0156] In some embodiments, a nucleic acid provided herein is integrated into the genome of a cell. Integration of a nucleic acid into a genome may be achieved by any method known in the art. Non-limiting methods of integrating a nucleic acid into the genome of a cell include, for example, utilizing targeted integrase proteins and utilizing programmable nuclease proteins. An integrase protein binds to an attachment sequence(s) in a genome and catalyzes the integration of an exogenous nucleic acid sequence into the genome at the attachment sequence(s). An integrase protein may be any integrase protein known in the art. Non-limiting examples of integrase proteins include, without limitation, Bxb1 bacteriophage, 31 bacteriophage, Bxb1 bacteriophage, BT.sub.1 bacteriophage, and R4 bacteriophage, HIV-1 (see, e.g., Xu et al., 2013, BMC Biotechnology, 13(87)). A programmable nuclease protein binds a nucleic acid (e.g., cell genome) at a target sequence, introduces cuts into the nucleic acid, and catalyzes the insertion (or deletion) of an exogenous nucleic acid sequence into the cut nucleic acid. A programmable nuclease protein may be any programmable nuclease protein known in the art. Non-limiting examples of programmable nuclease proteins include, without limitation, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nucleases, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) (see, e.g., Chandrasegaran et al., 2016, J Mol Biol., 428(5 Pt B); 963-989).
[0157] In some embodiments, a cell is a eukaryotic cell. A eukaryotic cell may be any eukaryotic cell. Non-limiting examples of eukaryotic cells include mammalian cells, plant cells, fungi cells, reptilian cells, protozoal cells, and diatoms. In some embodiments, a eukaryotic cell is a mammalian cell. Non-limiting examples of mammalian cells include human cells, rodent cells (e.g., mouse cells, rat cells, hamster cells), non-human primate cells (e.g., monkey cells, chimpanzee cells, orangutan cells, gibbon cells), domestic pet cells (e.g., dog cells, cat cells), and livestock animal cells (e.g., cow cells, pig cells, chicken cells, duck cells, goat cells, sheep cells).
[0158] In some aspects, the disclosure provides a non-human mammal comprising a cell described herein (e.g., a cell comprising a nucleic acid of the disclosure). A non-human mammal may be any non-human mammal provided in the art. Non-limiting examples of non-human mammals include rodents (e.g., mouse, rat, hamster, ferret), non-human primate (e.g., monkey, chimpanzee, orangutan, gibbon), domestic pets (e.g., dog, cat), and livestock animal cells (e.g., cow, pig, chicken, duck, goat, sheep).
[0159] In some embodiments, the disclosure provides a mouse comprising a cell described herein (e.g., a cell comprising a nucleic acid of the disclosure). In some embodiments, the disclosure provides a mouse comprising a nucleic acid described herein. A mouse may be any mouse known in the art. Non-limiting examples of mouse models include, without limitation, inbred mouse strains (e.g., BALB/c, C3H, C57BL/6, CBA. DBA/2, C57BL/10, AKR, A, 129, SJL), outbred mouse strains (e.g., Swiss Webster, CD-1, ICR), spontaneous mutant mouse strains (e.g., athymic nude, nonobese diabetic (NOD)), and immunodeficient mice (e.g., J:NU, NOD.Cg-Prkdc.sup.scid-Il2rg.sup.tm1wjl/SzJ (NSG), NOD.Cg-Prkdc.sup.scid-Il2rg.sup.tm1Wjl Tg(CMV-IL3, CSF2, KITLG)1Eav/MloSzj (NSG-SGM3); C57Bl/6-Tg(TcraTcrb)1100Mjb/J; NSG-CSF1; NSG-HLA-A2/HHD; NOD.Cg-Rag.sup.tm1Mom Il2rg.sup.tm1Wjl/SzJ; B6.129-Ptprc.sup.tm1Holm/J; Rag1.sup.tm1Mom Tg(TIE2-lacZ)182Sato/J; NSG-huIL-15; NOD.Cg-Flt3.sup.em2Mvw Prkdc.sup.scid Il2rgm.sup.tm1wjl Tg(FLT3LG)7Sz/SzJ; NOD.Cg-Prkdc.sup.scid Otc.sup.em18shep Il2rg.sup.tm1Wjl/J).
[0160] In some embodiments, a mouse provided herein is a transgenic mouse. A transgenic mouse is a mouse that expresses an exogenous nucleic acid sequence. An exogenous nucleic acid sequence is a nucleic acid sequence that is not derived from or found in a mouse. An exogenous nucleic acid sequence may be derived from any organism provided herein. In some embodiments, an exogenous nucleic acid sequence is derived from human.
[0161] Provided herein, in some embodiments, are methods of producing a non-human mammal. Non-limiting methods of producing a non-human mammal include, without limitation, pronucleus microinjection, embryonic stem cell recombination and transfer, Cre-lox genome recombination, viral vector infection, cytoplasmic injection, primordial germ cell recombination, nuclear transfer, and spermatogonial manipulation.
[0162] In some embodiments, a non-human mammal is produced using pronucleus microinjection. This method is well-known in the art (see, e.g., Weinstein, 2019, Mouse Modeling, Part 2: Breeding and Crossing Mice, Addgene.org, which is incorporated by reference herein). Briefly, pronucleus microinjection includes: injecting a pronucleus (e.g., mouse pronucleus) with a nucleic acid, integrating the nucleic acid into the pronuclear genome, implanting the pronuclear into a pseudopregnant female non-human mammal, sequencing the resulting non-human mammals for nucleic acid integration, and breeding non-human mammals containing the nucleic acid.
Methods of Use
Characterizing a Disease or Disorder
[0163] In some aspects, the disclosure provides methods of characterizing a disease or disorder using a nucleic acid, cell, and/or non-human mammal described herein. The nucleic acids, cells, and non-human mammals described herein are advantageous for characterizing a disease or disorder because induction of the disease or disorder may be controlled by knocking-in and knocking-out a target gene (e.g., comprising a disease-associated mutation). Non-limiting aspects of characterizing a disease or disorder may include: measuring gene expression, detecting cytokine production, measuring cell proliferation, and measuring cell survival. Measuring gene expression may be by any method known in the art including, but not limited to: RNA-sequencing (RNA-SEQ) and quantitative real-time PCR (qRT-PCR). Detecting cytokine production may be by any method known in the art including, but not limited to: immunohistochemistry and Western blotting. Measuring cell proliferation may be by any method known in the art including, but not limited to: immunostaining for cell proliferation proteins (e.g., PCNA, Ki-67) and nuclear staining. Measuring cell survival may be by any method known in the art including, but not limited to: tissue staining for apoptosis markers (e.g., caspase) and immunostaining for mitochondrial apoptosis proteins (e.g., Bax/Bak, caspase).
[0164] Any disease or disorder that may be induced by knocking-in and/or knocking-out a target gene may be characterized by methods provided herein. Non-limiting examples of diseases or disorders that may be characterized include: cancers, neurological diseases, autoimmune diseases, metabolic diseases, inflammatory diseases, cardiovascular diseases, mental disorders, gastrointestinal disorders, skeletomuscular diseases, genitourinary disorders, cell storage diseases, renal diseases, and hepatic diseases.
[0165] In some embodiments, a disease or disorder that is characterized by methods provided herein is a cancer. A cancer may be a primary cancer or a recurrent cancer. Non-limiting examples of cancers that may be characterized by methods provided herein include: adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telegiectasia, Beckwith-Wiedeman syndrome, bile duct cancer, Birt-Hogg-Dube syndrome, bladder cancer, bone cancer, brain stem glioma, brain cancer, breast cancer, Carney complex, cervical cancer, colorectal cancer, Cowden Syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV/AIDS-related cancer, juvenile polyposis syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, leukemia, Li-Fraumeni syndrome, liver cancer, lung cancer, lymphoma, Lynch syndrome, mastocytosis, medullablastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH-associated polyposis, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, non-melanoma skin cancer, small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Waldenstrom Macroglobulinemia, Werner syndrome, Wilms tumor, and xeroderma pigmentosum.
Identifying Inhibitors and Treatments
[0166] In some aspects, methods provided herein comprise identifying inhibitors or treatments for a disease or disorder. A disease or disorder may be any disease or disorder provided herein. In some embodiments, an inhibitor is a compound that halts the progression or prevents the development of a disease or disorder. Non-limiting examples of types of inhibitors include: small molecule compounds, antibodies, peptides, nucleic acids, and proteins. Methods of identifying an inhibitor may depend on the type of disease or disorder, and multiple inhibitors may be utilized simultaneously. In some embodiments, a method for identifying an inhibitor of a disease or disorder comprises: (i) inverting or knocking-in a target gene comprising a disease-associated mutation in a cell or a non-human mammal comprising the cell; (ii) detecting onset of the disease or disorder; (iii) contacting the cell or non-human mammal with a putative inhibitor; and (iv) measuring proliferation or survival of the disease or disorder in the contacted cell or non-human mammal compared to a control. In some embodiments, the control is a cell or a non-human mammal in which the disease-associated mutation has been knocked-out or is otherwise absent. In some embodiments, the control is a cell or non-human mammal that has not been contacted with the putative inhibitor. If the putative inhibitor prevents or halts the progression or development of the disease or disorder, the putative inhibitor may be identified as an inhibitor of the disease or disorder.
[0167] In some embodiments, methods provided herein comprise identifying a treatment for a disease or disorder in a non-human mammal. A treatment is a compound that alleviates one or more symptoms of a disease or disorder provided herein. Non-limiting examples of symptoms that may be alleviated include: increased cell proliferation, increased cell survival, altered microenvironment (e.g., hypoxia, decreased extracellular glucose concentration, decreased extracellular amino acid concentration), decreased body weight, decreased energy, and decreased survival, all of which are relative to a non-human mammal that does not have the disease or disorder. Non-limiting examples of types of treatments may include: small molecule compounds, antibodies, peptides, nucleic acids, and proteins. Methods of identifying a treatment may depend on the type of disease or disorder, and multiple treatments may be utilized simultaneously. In some embodiments, a method for identifying a treatment for a disease or disorder comprises: (i) inverting or knocking-in a target gene with a disease-associated mutation in a non-human mammal; (ii) detecting onset of the disease or disorder; (iii) contacting the non-human mammal with a putative treatment; and (iv) measuring proliferation or survival of the disease or disorder in the contacted non-human mammal compared to a control non-human mammal in which the disease-associated mutation has been knocked-out and the non-human mammal has not been contacted with the putative treatment. If the putative treatment alleviates at least one symptom of the disease or disorder, the putative treatment may be considered a treatment of the disease or disorder.
EXAMPLES
Example 1: a Conditional Knock-In, Knock-Out Model of Jak2.SUP.V617F .Myeloproliferative Neoplasm (MPN)
[0168] To assess dependency of MPN maintenance on JAK.sup.V617F oncogenic signaling, a Dre-rox, Cre-lox dual recombinase Jak2.sup.V617F knock-in/knock-out mouse model (Jak2.sup.Rox/Lox/Jak2.sup.RL) was generated by gene targeting in mouse embryonic stem cells (
[0169] To assess the reversibility of the Jak2.sup.RL construct, Dre-electroporated, lineage-negative, tamoxifen-inducible Ubc:CreER-Jak2.sup.RL cells isolated from donor mice were cultured with active MPN ex vivo with increasing doses of 4-hydroxy-tamoxifen (40HT) cultured over bone marrow endothelial cells (BMECs) (
[0170] The phenotypic impact of reversible Jak2.sup.V617F expression in vivo was evaluated. Three or 12 weeks post-transplant, secondary recipient mice transplanted with Dre-electroporated Ubc:CreER-Jak2.sup.RL whole bone marrow in competition with CD45.1 marrow and exhibiting MPN were administered tamoxifen to delete Jak2.sup.V617F (
Example 2: Jak2.SUP.V617F .Reversal Impairs the Fitness of MPN Cells, Including MPN Stem Cells
[0171] The effects of Jak2.sup.V617F deletion on peripheral blood and bone marrow mutant cell fitness were evaluated in the chimeric transplant system (
[0172] The transcriptional changes seen when Jak2.sup.V617F is acutely reversed were characterized. RNA sequencing (RNA-Seq) analysis of purified HSPCs 3 and 7 days following Jak2.sup.V617F deletion (n=3-4) compared to controls (n=3-4) was performed. Transcriptional analysis of sorted, Jak2.sup.V617F-deleted LSK and MEP populations revealed near-complete loss of expression of STAT5 target genes as early as 3 days post-deletion (LSK: NES 1.77, padj=0.002; MEP: NES 1.53, padj=0.0065) indicating immediate disengagement from disease-defining pathway signaling (
Example 3: Differential Efficacy of Jak2.SUP.V617F .Deletion Compared to JAK Inhibitor Therapy
[0173] Given the limited ability of current JAK2 inhibitors to achieve disease modification and/or clonal remissions in polycythemia vera and myelofibrosis, the phenotypic and transcriptional effects of JAK inhibitor therapy with ruxolitinib were compared to the effects of Jak2.sup.V617F reversal. Similar RNA-seq was performed on Jak2.sup.V617F LSKs and MEPs following 7 days of ruxolitinib treatment (n=3) and compared to the effects of Jak2.sup.V617F deletion (n=3). Analysis revealed that JAK-STAT target gene expression and erythroid pathway gene expression were much less potently inhibited with ruxolitinib than with Jak2.sup.V617F deletion. Specifically, while similar reduced expression of negative regulators of JAK/STAT signaling (e.g. Socs2/3, Pim2, Cish) with ruxolitinib treatment were observed, the degree of down-regulation of STAT5 target gene expression was less pronounced as compared to Jak2.sup.V617F deletion (RUX NES 0.913, p=0.84 vs. TAM NES 1.51, p=0.003) (
[0174] To evaluate the phenotypic effects of Jak2.sup.V617F deletion in direct comparison to JAK kinase inhibition, an in vivo trial lasting 6 weeks comparing ruxolitinib to Jak2.sup.V617F deletion was performed (
[0175] Recent studies have suggested that MAPK signaling has an important role in MPN disease cell survival in the setting of type I JAK inhibitor therapy, and recent work has implicated the MAPK-dependent factor YBX1 as a critical mediator of JAK2.sup.V617F-mutant cell persistence. Here, distinct effects on MAPK activity by RNA-Seq with ruxolitinib treatment vs. Jak2.sup.V617F deletion in comparison to vehicle treated mice were observed, with MAPK activation in response to ruxolitinib (NES 1.65, padj=0.0003) and reduced MAPK output with Jak2.sup.V617F deletion (NES 1.35, padj=0.043) (
Example 4: Jak2.SUP.V617F .Dependency with Cooperative TET2.SUP.f/f .Loss
[0176] Previous studies of mutational order in primary MPN cells have shown that cooperating mutations in epigenetic regulators, including TET2, can precede the acquisition of JAK2.sup.V617F and that antecedent TET2 mutations can alter the in vitro sensitivity to ruxolitinib. In addition, in vitro/in vivo studies have shown that concurrent TET2 and JAK2.sup.V617F mutations promote enhanced mutant HSC fitness and increased risk of disease progression. The Jak2.sup.RL system allows for the assessment of Jak2.sup.V617F dependency in the setting of co-occurring mutant allele activation/inactivation, including in the context of antecedent mutations in epigenetic regulators. Therefore, the impact of Jak2.sup.V617F activation in concert with pre-existing Tet2 loss with the reversible Jak2.sup.RL allele was assessed (
[0177] The effects of Jak2.sup.V617F deletion on Jak2.sup.V617F/Tet2.sup.f/f mutant cell fitness in vivo in competition with CD45.1 marrow were evaluated. Treatment with tamoxifen at 9 weeks post-transplant resulted in normalization of hematologic parameters (p<0.005) and reductions in peripheral blood mutant cell fraction of double-mutant cells to a similar extent observed with Jak2.sup.V617F deletion in single-mutant Jak2.sup.RL transplanted mice (
[0178] Mutated kinases occur frequently in cancer and are amenable to targeted inhibition; however, mechanisms which mediate acquired resistance have been observed for most targeted therapies. By contrast, current JAK inhibitors fail to eliminate Jak2.sup.V617F-mutant clones in MPN suggesting inadequate target inhibition and/or other genetic/non--genetic factors mediate Jak2.sup.V617F-mutant cell persistence in the setting of JA K inhibitor therapy. Here, preclinical models show that there is an absolute requirement for Jak2.sup.V617F in MPN cells and that mutant-specific targeting of Jak2.sup.V617F abrogates MPN features, reduces mutant cell fraction, and extends overall survival with concomitant depletion of disease-sustaining stem cells within the HSPC compartment. Further, the data suggest that Jak2.sup.V617F dependency persists even in the setting of antecedent mutations in epigenetic regulators. These data support the notion that improved targeting of JAK2 signaling and downstream effectors offers greater therapeutic potential than current JAK kinase inhibitors, and Jak2.sup.V617F-mutant-selective inhibition represents a potential curative strategy for the treatment of MPN patients. Moreover, the feasibility of the dual-recombinase system to evaluate oncogenic signaling dependencies in vivo has been demonstrated, and a similar approach will allow for the assessment of oncogenic dependencies and mechanisms of mutant-mediated transformation across a spectrum of malignant contexts.
Example 5: Methods for Examples 1-4
[0179] Experimental animals. All animal experiments were performed in accordance with the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee-approved animal protocol. Animal care was in strict compliance with institutional guidelines established by MSKCC and the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences 1996). Mice with an intact immune system were used for all experiments. All animals were drug and test nave and not involved in previous procedures, maintained on a 12 hour light-dark cycle, and with access to water and standard chow ad libitum. Veterinary staff conducted regular husbandry procedures and provided routine care and monitoring. Animals were monitored daily for signs of disease or morbidity, failure to thrive, weight loss, open skin lesions, bleeding, infection, or fatigue and sacrificed immediately if they exhibited any signs of the above. Mice harboring the Jak2.sup.RL allele were generated by Ingenious Targeting Laboratory (Ronkonoma, NY) in a C57BL/6 background. Conditional Jak2.sup.V617F knock-in mice, RC::RLTG reporter mice, Tet2.sup.f/f conditional knock-out mice, Cre- TdTomato reporter mice, and UbcCreER+ mice have been described previously..sup.6,8,17,24 6-8 week old female and male Jak2.sup.RL or Jak2.sup.RL/Tet2.sup.f/f donor mice were used for Dre electroporation knock-in experiments. Age-matched 6-10 week old female mice were used as donors for all transplant experiments (Ly5.1 CD45.1 competitive or C57BL/6J non-competitive).
Mouse Genotyping.
[0180] DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD). Jak2.sup.V617F knock-in genotyping was carried out with the following primers: FWD: 5-GCCATCTTTCCAGCCTAAAATTAG-3 (SEQ ID NO: 83); REV: 5-TCCAAAGAGTCTGTAAGTACAGAACT-3 (SEQ ID NO: 84) and with the following reaction conditions: 94 C. for 3 minutes followed by 15 cycles of 94 C. for 15s, 65 C. for 15s, and 72 C. for 30s decreasing by 1 C. per cycle, and then followed by an additional 25 cycles of 94 C. for 15s, 50 C. for 15s, and 72 C. for 30s. Jak2.sup.V617F knock-out genotyping was carried out using the following primers: FWD: 5-GCCATCTTTCCAGCCTAAAATTAG-3 (SEQ ID NO: 83); REV: 5-ACCAGTTGCTCCAGGGTTACACG-3 (SEQ ID NO: 85) and with the following reaction conditions: 94 C. for 2 minutes followed by 30 cycles of 94 C. for 30s, 53 C. for 30s, and 72 C. for 30s. Sequencing of the unrecombined Rox-lox locus was carried out using the following primers: FWD: 5-AGGAGCATCGATGACTACATGATGAG-3 (SEQ ID NO: 86); REV: 5-AGACTCTCCACGGTCTCATCTACG-3 (SEQ ID NO: 87) and with the following reaction conditions: 98 C. for 30 seconds followed by 35 cycles of 98 C. for 10s, 65 C. for 15s, and 72 C. for 30s. Tet2 primers were carried out using the following primers/conditions: FWD: 5-AAGAATTGCTACAGGCCTGC-3 (SEQ ID NO: 88); REV: 5-TTCTTTAGCCCTTGCTGAGC-3 (SEQ ID NO: 89); ExR: 5-TAGAGGGAGGGGGCATAAGT-3 (SEQ ID NO: 90) and with the following reaction conditions: 94 C. for 2 minutes followed by 39 cycles of 94 C. for 35s, 58 C. for 45s, and 72 C. for 55s. Annotation of PCR genotyping results was carried out on a QIAxcel Advanced System (Qiagen) and analyzed using QIAxcel ScreenGel software (Qiagen).
Dre mRNA Electroporation.
[0181] Dre mRNA was purchased from TriLink Biotechnologies (San Diego, CA) and electroporation carried out using the Neon Transfection System (ThermoScientific) per the manufacturer's protocol. Specifically, bone marrow donor cells were isolated from limb bones into PBS (pH 7.2) containing 2% fetal calf serum via centrifugation. After red blood cell lysis, single-cell suspensions were depleted of lineage-committed hematopoietic cells using a Lineage Cell Depletion Kit according to manufacturer's protocol (EasySep, StemCell Technologies, Inc.). 2.5-3.010.sup.6 lineage-depleted bone marrow was then washed in PBS and then resuspended in 135 uL Buffer T to which 15 L of Dre mRNA (at 1 g/L) was quickly added and electroporated at the following conditions: 1700V for 20 ms1 pulse. The cells were then pipetted into penicillin-streptomycin free StemSpan SFEM medium with thrombopoietin (TPO; 20 ng/mL; PeproTech) and stem cell factor (SCF; 20 ng/mL; PeproTech), cultured for two hours, and then subsequently harvested and washed/resuspended in phosphate buffered saline (PBS) and transplanted via lateral tail vein injection into lethally irradiated (900cGy) 6-8 week old C57BL/6J recipient mice at approximately 410.sup.5 cells per recipient along with 50,000 un-electroporated whole bone marrow support cells. Double-mutant Jak2.sup.RL/Tet2.sup.f/f electroporations were carried out as above, except donor mice were dosed with tamoxifen (100 mg/kg by oral gavage daily 4; purchased from MedChemExpress) 6-8 weeks prior to harvest, and excision confirmed prior to Dre electroporation.
Transplantation Assays and In Vivo Experiments.
[0182] Jak2.sup.RL and Jak2.sup.RL/Tet2.sup.f/f lines were crossed to UbcCreER tamoxifen-inducible Cre lines and RLTG dual-recombinase reporter lines..sup.17,24 Primary recipient mice transplanted with Dre mRNA-recombined UbcCreER-Jak2.sup.RL or UbcCreER-Jak2.sup.RL/Tet2.sup.f/f bone marrow cells were bled every 3-4 weeks post-transplant to monitor disease status. Peripheral blood was isolated by submandibular bleeds. Complete blood counts were assessed on a ProCyte Dx (IDEXX Laboratories, Westbrook, ME) per manufacturer's instruction. For competitive repopulation assays, 1.210.sup.6 whole bone marrow from primary transplant recipient mice exhibiting MPN was harvested 6-8 weeks post-transplant and combined with age-matched 0.810.sup.6 CD45.1 (Jackson Laboratories, Bar Harbor, ME) whole bone marrow and transplanted into 6-8 week old lethally irradiated CD45.1 secondary recipient mice. To induce Cre and delete Jak2.sup.V617F, mice were treated with tamoxifen (TAM; purchased from MedChemExpress) 100 mg/kg daily (dissolved in corn oil) by oral gavage 4 followed by 14 days of TAM chow approx. 80 mg/kg daily (ENVIGO). Tamoxifen control studies were carried out using similar dosing schedules on 45.1 mice transplanted in competition with Dre-electroporated, Cre-negative Jak2.sup.RL MPN bone marrow cells. For terminal tissue isolation, mice were euthanized with CO.sub.2 asphyxiation, and tissues were dissected and fixed with 4% paraformaldehyde for histopathological analysis. For whole bone marrow isolation, the femurs, hips, and tibias were dissected and cleaned. Cells were isolated by centrifugation at 8000G for 1 minute followed by RBC lysis (BioLegend). Bone marrow cell numbers were determined via an automatic cell counter (ViCell Blu, Beckman Coulter). Single cell suspensions were generated from crushed whole spleen and filtered through a 70 uM filter. RBC lysis was performed (BioLegend) and cells were prepared for downstream processing or frozen.
In Vivo Drug Studies.
[0183] For in vivo inhibitor studies, approximately 8 weeks following transplant, secondary transplant cohorts of lethally-irradiated mice transplanted with UbcCreER-Jak2.sup.RL bone marrow in competition with CD45.1 marrow (as above) and exhibiting active MPN were bled and cohorted based on peripheral blood CD45.2 chimerism and total WBC count to achieve congruency across treatment arms. Mice were then treated with ruxolitinib (60 mg/kg P.O. twice daily; dissolved in 20% Captisol in PBS; purchased from MedChemExpress), CHZ868 (30 mg/kg P.O. daily; dissolved in 0.5% methylcellulose +0.5% Tween-80 in dH2O; purchased from MedChemExpress), tamoxifen to delete Jak2.sup.V617F (as above; purchased from MedChemExpress), or vehicle. Investigators were not blinded to the identity of mice or samples. Mice were treated for a total of 6 weeks before timed sacrifice and marrow/spleen harvested as above.
Bone Marrow Endothelial Cell (BMEC) Culture.
[0184] Bone marrow cells were isolated from limb bones into FACS buffer (phosphate buffered saline (PBS)+2% fetal bovine serum) via centrifugation. After red blood cell lysis, single-cell suspensions were depleted of lineage-committed hematopoietic cells using a Lineage Cell Depletion Kit according to manufacturer's protocol (EasySep, StemCell Technologies, Inc.). Subsequently, 50,000 of the resulting lineage.sup. cells were plated on a confluent monolayer of BMECs in a single well of a 12-well plate. Each well had 1 mL StemSpan SFEM (StemCell Technologies, Inc.) with 20 ng/mL recombinant murine SCF (PeproTech) in addition to the corresponding drug treatment: either 4--hydroxytamoxifen (4-OHT; Sigma Aldrich; stock concentration:13 mM) or its vehicle, appropriately diluted in media to its final concentration (i.e., 0.01% (v/v) of ethanol, or 200 nM, 400 nM or 1 uM of 4-OHT) (three replicates/condition). The BMECs were seeded two days before plating the lineage- cells at a density of 100,000 cells/well. Co-cultures were maintained for a total of 7 days at 37 C. and 5% CO.sub.2, with media being completely refreshed with the original SCF and drug/vehicle concentrations. 4OHT or ethanol EtOH vehicle as added to the culture on day 1 and again on day 4. On day 7, total cells were harvested with Accutase (Biolegend) and cell numbers were determined via an automatic cell counter (ViCell Blu, Beckman Coulter). Cells were then stained with the desired antibody cocktail and phenotyped by flow cytometry.
Flow Cytometry and Western Blot.
[0185] Following single cell preparation, murine peripheral blood, whole bone marrow, or spleen mononuclear cells were lysed for 10 minutes with RBC lysis buffer (BioLegend, San Diego, CA) and washed twice with FACS buffer (phosphate buffered saline (PBS)+2% fetal bovine serum). Cells were then resuspended in Fe (CD16/32) block for 15 minutes and then subsequently stained with a cocktail comprised of antibodies targeting CD3 (17A2), CD45R/B220 (RA3-6B2), Gr-1 (RB6-8C5), CD11b (M1/70), CD45.2 (104), and CD45.1 (A20) for 30 minutes. For hematopoietic stem/progenitor cell analysis, lysed bone marrow was stained with a cocktail of lineage markers along with antibodies against c-Kit (2B8), Sca-1 (D7), FcRII/III (2.4G2), CD34 (RAM34), CD150 (9D1), CD105, CD71 (R17217), CD41 (MWReg30), and CD48 (HM48-1). Erythroid progenitor flow was carried out on unlysed bone marrow with the following antibodies: All FACS antibodies were purchased from BD, BioLegend, or eBioscience. Following antibody incubation, cells were washed with FACS buffer and resuspended in a DAPI-containing FACS buffer solution for analysis and sorting. Samples were run on a Fortessa (Becton Dickinson) using FACSDiva software and analyzed with FlowJo (Treestar, Ashland, OR, USA). For Western blot analysis, whole-cell protein extracts from harvested splenocytes were prepared using RIPA buffer (ThermoScientific, Rockford, IL) containing a protease/phosphatase inhibitor cocktail (Thermo Scientific). Protein quantification was performed using the Pierce BCA protein assay kit (ThermoScientific) and analyzed on a Cytation 3 plate reader (BioTek). Proteins were separated by NuPAGE 4-12% Bis-Tris Gel and transferred to a nitrocellulose membrane. The following antibodies were used: -actin (Cell Signaling 4970S), STAT5 (Cell Signaling 94205S), and pSTAT5 (Cell Signaling, 9359S). Images were obtained using the ChemiDoc Imaging System (BioRad) and analyzed using ImageLab software (BioRad).
Histology Staining and Immunohistochemistry, Photography.
[0186] Tibia and spleen samples were fixed in 4% paraformaldehyde for over 24 hours and then embedded in paraffin. Paraffin sections were cut on a rotary microtome (Mikrom International AG), mounted on microscope slides (ThermoScientific), and air-dried in an oven at 37 C. overnight. After drying, tissue section slides were processed either automatically for hematoxylin and eosin (H&E) staining (COT20 stainer, Medite), or manually for reticulin staining. The following antibodies were used for immunohistochemistry: MacI (Cedarlane CL8942B, 1:100), Ter119 (BDBioscience, 550565 1:200), and phospho-ERK (Cell Signaling 4376, 1:100). Pictures were taken at 100, 200 and 400 (H&E and reticulin) magnification using an Olympus microscope and analyzed with Olympus Cellsens software. Tissue sections were formally evaluated by a hematopathologist (W. Xiao), including reticulin scoring.
Assessment of Cell Growth, Apoptosis, and Viability.
[0187] Apoptosis was measured by flow cytometry on a Fortessa (Becton Dickinson) cytometer with Annexin V PerCPCy5.5 antibody (BioLegend) and Annexin binding buffer (BioLegend) at 1:50 dilution in combination with DAPI as live/dead cell stain.
Colony Forming Assays.
[0188] To assess colony formation and serial replating capacity, RBC lysed 50,000 whole bone marrow cells were seeded in 1.5 mL MethoCult M3434 (Stem Cell Technologies) in triplicate on 6 well plates and scored on day 8. For replating, cells were harvested and pooled and then re-seeded once more at 50,000 cells/well in 1.5 mL MethoCult M3434 in triplicate.
Serum Cytokine Profiling.
[0189] Serum samples were diluted two-fold with PBS (pH 7.2) and stored at 80 C. until analysis. Cytokine levels were measured in duplicates (62.5 uL each) by Eve Technologies Corporation (Mouse Cytokine Array/Chemokine Array 44-Plex, Calgary, AB, Canada).
RNA-Seq and Data Analysis.
[0190] For gene expression analysis, secondary cohorts of mice transplanted with UbcCreER-Jak2.sup.RL bone marrow 8 weeks post-transplant and exhibiting MPN were treated with ruxolitinib (60 ng/g P.O. BID), tamoxifen (100 mg/kg daily 4 followed by 80 mg/kg daily of TAM chow 3) for 7 days and then sacrificed along with an untreated MPN control cohort. Lineage-depleted marrow was isolated and then TdTonmato+ (Jak2.sup.RL knock-in) or GFP+ (Jak2.sup.RL knock-out) LSKs and MEPs were sorted on a FACSAria III directly into Trizol LS (Invitrogen) and stored at 80 C. until processing. RNA was subsequently isolated using the Direct-Zol Microprep Kit (Zymo Research, R2061) according to manufacturer's protocol and quantified using the Agilent High Sensitivity RNA ScreenTape (Agilent 5067-5579) on an Agilent 2200 TapeStation. cDNA was generated from 1 ng of input RNA using the SMART-Seq HT Kit (Takara 634455) at half reaction volume followed by Nextera XT (Illumina FC-131-1024) library preparation. cDNA and tagmented libraries were quantified using High Sensitivity D5000 ScreenTape (5067-5592) and High Sensitivity D1000 ScreenTape respectively (5067-5584). Libraries were sequenced on a NovaSeq at the Integrated Genomics Operation (IGO) at MSKCC.
ATAC-Seq and Data Analysis.
[0191] Chromatin accessibility assays utilizing the bacterial Tn5 transposase were performed as described. Briefly, 5.010.sup.4 sorted reporter-positive TdTomato+ (Jak2.sup.RL knock-in) or GFP+ (Jak2.sup.RL knock-out) cKit+ bone marrow cells from mice treated for 7 days with ruxolitinib (60 mg/kg P.O BID), tamoxifen, or an untreated MPN control cohort were sorted on a FACSAria III into PBS and subsequently lysed and incubated with transposition reaction mix containing PBS, Tagment DNA buffer, 1% Digitonin, 10% Tween-20, and Transposase (Illumina). Samples were then incubated for 30 minutes at 37 C. in a thermomixer at 1000 rpm. Prior to amplification, samples were concentrated with the DNA Clean and Concentrator Kit-5 (Zymo). Samples were eluted in 20 L of elution buffer and PCR-amplified using the NEBNext 2 Master Mix (NEB) for 11 cycles and sequenced on a NextSeq 500 (Illumina). Libraries were sequenced on a NovaSeq at the Integrated Genomics Operation (IGO) at MSKCC.
Quantitative Real-Time PCR.
[0192] Total RNA was extracted from magnetic-bead isolated cKit+ bone marrow (Miltenyi Biotec) using the Direct-zol RNA extraction kit (Zymo) per manufacturers' protocols respectively. Complementary DNA was then reverse transcribed using the Verso cDNA Synthesis kit (ThermoFisher Scientific). Ybx1 expression was evaluated by quantitative reverse-transcription (qRT) PCR using Taqman probes purchased from ThermoFisher (Mm00850878_g1) on the RealPlex thermocycler (ThermoFisher Scientific, Fairlawn, NJ).
Statistical Analysis.
[0193] Statistical analyses were performed using Student's t-test (normal distribution) using GraphPad Prism version 6.Oh (GraphPad Software, San Diego, CA). Kaplan-Meier curves were determined using the log-rank test. P<0.05 was considered statistically significant. The number of animals, cells and experimental replication can be found in the respective figure legends. FASTQ files were demultiplexed using a java script from Takara. FASTQ files were mapped and transcript counts were enumerated using STAR (genome version mm10 and transcript version ensembl XX). Counts were input into R and RNA-sequencing analysis using DESeq2. Gene set enrichment analysis was performed using the fgsea package with genesets extracted from the msigdbr package. Single sample gene set enrichment analysis was performed using the gsva package. Figures were generated using ggplot2 and tidyhetamps packages.
Example 6: Inducible Mouse Model of Flt3 Internal Tandem Duplication
[0194] In order to model Flt3 mutations as late hits in leukemogenesis, an endogenously targeted, Flp-inducible Fft3.sup.Frt-ITD allele which could be inverted by a tamoxifen (TAM) inducible FlpoERT2 allele was developed (
[0195] To determine how Flt3.sup.Frt-ITD mutations changed the transcriptional landscape of HSPCs, low pass, multiplexed 3 RNA-sequencing was performed on purified LSKs and GMPs from Flt3.sup.ITD mice 24, 6 or 8 weeks after TAM administration, and from Flt3.sup.WT mice 4 weeks after TAM (
[0196] To understand the functional consequences of these gene expression alterations on cellular fitness, competitive transplantation between Flt3.sup.Frt-ITD and Cd45.1 WT cells was performed. Activation of Flt3.sup.Frt-ITD with TAM 4 weeks after transplantation resulted in robust, sustained competitive outgrowth of Flt3.sup.Frt-ITD compared to WT competitor cells (mean Cd45.2% at 18w 88.89% vs 25.8% p6.5410.sup.4). (
Example 7: Cooperating Mutations License Mutant-Flt3 for Transformation
[0197] Flt3.sup.Frt-ITD mice were crossed with a Npm1.sup.c-Frt mutation for simultaneous activation with a TAM-inducible FlpoERT2. Primary Flt3.sup.Frt-ITD mice showed a median survival 48 weeks, Npm1.sup.c-Frt Flt3.sup.Frt-ITD mice rapidly succumbed to disease within 5 weeks following TAM administration (
[0198] Mutant Flt3 was evaluated in a sequential mutagenesis model with a common CH mutation Dnmt3a.sup.R878H-Lox. Cre mRNA was administered to activate the underlying Dnmt3a.sup.R878H-Lox allele, and TAM was used to activate Flt3.sup.Frt-ITD four weeks after engraftment (
[0199] Gene expression analysis revealed that Npm1.sup.c-Frt Flt3.sup.Frt-ITD LSKs were enriched for genes which were acutely activated with Flt3.sup.Frt-ITD alone, reinforcing the idea that Npm1.sup.c might lock Flt3-mutant cells into an early activation state (
Example 8: Expanding the Sequential Mutagenesis Toolkit with Orthogonal Recombinases
[0200] To extend these models of sequential mutagenesis, a third recombinase, Dre, a Cre-homologue capable of recombining Rox sites was used. Additionally, dual recombinase alleles capable of activating and inactivating oncogenes at their endogenous loci were generated. A strategy termed GOLDI-Lox for governing oncogenic loci by dre inversion and lox deletion (GOLDI-Lox) was developed (
[0201] To benchmark the Flt3.sup.GL-ITD allele, it was crossed to the Ubc:CreER allele and a panel of Cre responsive alleles including Npm1.sup.C-Lox, Idh2.sup.R140Q-Lox and Tet2.sup.flox/flox mice. The cooperating allele was (in)activated with TAM; HSPCs were then isolated for Dre nRNA electroporation ex vivo (
Example 9: Sequential Mutagenesis Reveals Stage and Mutant-Specific Alterations in Differentiation
[0202] While Npm1.sup.C-Flt3.sup.ITD combinations potently induce AML in mice, most patients present with an antecedent mutation, the most common of which is found in DNMT3A, generating a triad of mutations which confers dismal prognosis for patients. This combination of alleles was modeled using 3 separably inducible alleles Dnmt3a.sup.R878H-Lox, Npm1.sup.c-Frt, and Flt3.sup.GL-ITD (
[0203] To model a Flt3 mutation as a late hit in leukemogenesis, Dre mRNA was electroporated into HSPCs at transplant to generate 4 new experimental groups (
Example 10: Inducible Recombinases Allow for Facile Disease Development
[0204] To enable sequential mutagenesis without relying upon transduction at transplant, orthogonal chemical tools were implemented to activate the recombinases orthogonal to existing tamoxifen inducible ER fusion proteins employed above. Previous studies have implemented RU486 inducible PR fusions for Dre, however these studies have been limited by in vivo inducibility. FKBP12-Dre fusions displayed excessive background recombination in the presence of the stabilizing ligand Shield1. A Stabilized Peptide Linkage (StaPL) approach was implemented by splitting Dre with an HCV-derived NS3 protease leading to constitutive autoproteolysis of Dre (
[0205] To complement existing Flpo and Cre tools, two new DreStaPL-ODC mouse lines were generated, targeting the fusion recombinase to either the endogenous Vav1 locus, or to the Hip11 safe harbor under the control of a synthetic CAG promoter (
[0206] Given the ability of limiting dilutions of TAM to generate an AML in the Npm1.sup.c-Frt Flt3.sup.Frt-ITD model (
[0207] Finally, it was sought to eliminate any reliance upon transplant for the induction of disease. Four allele mice were generated harboring Ubc:CreER, Vav1:DreStaPL-ODC, Flt3.sup.GL-ITD) and either Cre-inducible Npm1.sup.c-Lox or Idh2.sup.R140Q-lox mutations. Mice were administered tamoxifen to activate either Npm1.sup.c-Lox or Idh2.sup.R140Q-lox, and then 2 weeks later treated with GZV to induce Flt3.sup.GL-ITD (
Example 11: Evaluating Oncogene Dependency of Mutant Flt3
[0208] It was next sought to perturb disease evolution through the genetic deletion of mutant Flt3. Leukemias from the models in
[0209] Pathological analysis of the bone marrow revealed a decrease in cellularity, and marked differentiation with nuclear condensation (
[0210] This increase in phenotypic LSKs suggests either a maintenance of a stem-like compartment or a dramatic increase in inflammation and upregulation of Seal. Gene expression profiling on HSPCs from the Npm1.sup.c-Lox-Flt3.sup.GL-ITD model revealed TAM treatment was associated with a robust inflammatory gene signature driven by an interferon gamma response and STAT3/IL6 signaling (
[0211] Long term treatment with TAM resulted in an increase in overall survival, with a subset of Npm1.sup.c-Lox-Flt3.sup.GL-ITD (2/10) near eradication of leukemic cells (
[0212] Advances in single cell genomics have offered refined resolution into the evolutionary processes underlying cancer development and response to therapy. While GEMMs have served as a robust means to model disease, spatiotemporal control of stepwise somatic alterations remains elusive. Here, chemically orthogonal, multi-recombinase models of somatic event control which complement existing alleles in hematopoietic malignancy were employed. These studies identified new insights in acute and chronic phase activation of mutant Flt3, the most commonly mutated gene in acute myeloid leukemia. It was found that while Flt3 was insufficient to cause leukemia, cooperating mutations licensed Flt3-iTD for transformation, with different mutational combinations altering disease kinetics, transcriptional cooperativity, and immunophenotypic output.
[0213] While clonal hematopoiesis is a common somatic event in an aged population and is assessed with an increased probability for myeloid malignancy, transformation to AML remains a rare event. The models described here build off of previous research to understand this transition from a premalignant state towards leukemic development. While previous studies have enabled progressive mutation acquisition with CRISPR/Cas9 or viral overexpression, none have permitted stepwise sequential mutagenesis in the setting of native hematopoiesis. Given the selective pressure introduced with irradiation-based conditioning for transplantation, consolidation of somatic mutation systems in the absence of transplantation will be a critical tool to understand the mechanism underlying clonal dominance and its eventual emergence to leukemic transformation.
[0214] While doxycycline inducible systems have been used to assess oncogene-dependency, endogenous expression levels remain a critical concern, particularly for mutant-oncogenes with restricted cell type expression patterns such as Flt3. Other gene-trap approaches have retained locus specific expression levels but have thus far been largely deployed for loss of function alleles. Here, Cre and Dre recombinase were paired in the GOLDI-Lox framework to allow for both inducible antecedent events followed inducible activation and reversion of the Flt3 oncogene. This setup allowed for the performance of three functions with two recombinases, decreasing the genetic burden necessary to establish the models. With this approach, it was demonstrated that genetic inactivation of mutant-Flt3 results in immediate regression of leukemia albeit with distinct immunophenotypic consequences depending upon the cooperating mutations. Despite this robust early response, the majority of mice eventually succumbed to disease escaping genetic deletion. This can be explained, in part, through insufficient Cre activity with evidence of residual mutant Flt3. Even with robust FLT3 deletion, there was insufficient residual hematopoiesis from either remaining WT cells or Npm1 only mutant cells to recover from leukemic insult. These findings mirror the clinical scenario where the FLT3 tyrosine kinase inhibitor gilteritinib robustly generated complete remissions, yet increases in overall survival are only significantly established in patients who underwent hematopoietic stem cell transplant and continued gilteritinib therapy.
Example 12: Materials and Methods for Examples 6-11
Mouse Model Generation.
[0215] The Flt3.sup.Frt-ITD and Flt3.sup.GL-ITD mice were generated at Ingenious targeting labs. The targeting construct for Flt3.sup.Frt-ITD was generated A 9.8 kb genomic DNA used to construct the targeting vector was first subcloned from a positively identified C57BL/6 BAC clone (RP23-280N16). The region was designed such that the long homology arm (LA) extends 7 kb 5 to the 5 FRT cassette, and the short homology arm (SA) extends about 2 kb 3 to the insertion of the inversion cassette. The inversion cassette is flanked by mutant F3 sites that point away from each other. The 3 FRT site is placed right before the 3 F3 site. The inversion cassette consists of exon 13-mutant exon 14* (humanized Flt3-IDT)-exon 15 and the flanking genomic sequences from upstream of exon 13 to downstream of exon 15 for correct splicing (Inv.saE13-14*-E15Sd). This cassette was inserted in the reverse direction downstream of exon 15. The Lox2272-flanked Neo cassette was inserted immediately upstream of the inversion cassette and is 235 bp away from wt exon 15. The targeting region is 985 bp containing exons 13-15. Ten micrograms of the targeting vector was linearized and then transfected by electroporation of C57Bl/6 (B6) embryonic stem cells. After selection with G418 antibiotic, surviving clones were expanded for PCR analysis to identify recombinant ES clones. After successful clone identification, the neomycin cassette was removed with a transient pulse of Cre recombinase and clones were reconfirmed following expansion. Finally, ES cells were injected in C57B6 mice via tetraploid complementation (NYU).
Targeting Construct.
[0216] The targeting construct for Flt3.sup.GL-ITD, was generated as follows: A 9.8 kb genomic DNA used to construct the targeting vector was first subcloned from a positively identified C57BL/6 BAC clone (RP23-280N16). The region was designed such that the long homology arm (LA) extends 5.7 kb 5 to the cluster of Lox2272-Rox-Rox12-Lox2272 sites, and the short homology arm (SA) extends about 1.9 kb 3 to the Neo cassette and 3 Rox12 site. The inversion cassette is in between the second set of Lox2272 and Rox sites, and it consists of exons 12-15 with the IDT mutation engineered in exon 14 (Flt3-IDT) and the flanking genomic sequences from upstream of exon 12 to downstream of exon 15 for correct splicing (SaE12-W51-15Sd). The inversion cassette replaces wt exons 12-15 and the same flanking genomic sequences included in the cassette. The deleted region is 2.19 kb. The FRT-flanked Neo cassette is inserted immediately downstream of the inversion cassette. Each pair of the recombination sites are in opposite direction. Ten micrograms of the targeting vector was linearized and then transfected by electroporation of FLP C57Bl/6 (BF1) embryonic stem cells. After selection with G418 antibiotic, surviving clones were expanded for PCR analysis to identify recombinant ES clones. The Neo cassette in targeting vector has been removed during ES clone expansion. Targeted iTL BF1 (C57BL/6 FLP) embryonic stem cells were microinjected into Balb/c blastocysts. Resulting chimeras with a high percentage black coat color were mated to C57BL/6 WT mice to generate Germline Neo Deleted mice. Tail DNA was analyzed as described below from pups with black coat color.
Cloning.
[0217] Codon optimized versions of Cre, CreERT2 and Dre were synthesized (Thermo) and subcloned into pENTR using the directional TOPO cloning kit. Flpo was PCR amplified from a FlpoERT2 construct provided by Dr. Alex Joyner at MSKCC, and then TOPO cloned into pENTR using the same approach. DreStaPL and DreStaPL-ODC were generated by a gibson reaction with the backbone generated through inverse PCR of the pENTR vector, and the StaPL containing insert synthesized as a gBlock for IDT. A similar approach was used for inserting DHFR downstream of Flpo. These pENTR constructs were then cloned into a MSCV:IRES GFP destination vector using a gateway cloning approach (Thermo: LR Clonase II).
Virus Generation and Viral Infection.
[0218] MSCV retroviral vectors were transfected with pCL-Eco packaging vector into 293T-17 cells using JetPRIME following the manufacturers protocol (5 ug each, 1:1). Conditioned media was collected 72 hours later and passed through a 0.45 uM filter. Virus was either used fresh or stored at 80 C for later usage. For viral infection in
Tissue Harvest and Bleeds.
[0219] Peripheral blood was isolated by submandibular bleeds. Complete blood counts were assessed on a ProCyte by IDEXX. For flow cytometry analysis whole blood was lysed with RBC lysis buffer (Biolegend). For terminal tissue isolation, mice were euthanized with CO2 asphyxiation, tissues were dissected and fixed with 10% Zinc formalin for histopathological analysis. For whole bone marrow was isolation, the femur, hip and tibia were dissected and cleaned. Cells were isolated by centrifugation at 8000G for 1 minute. Single cell suspension were generated from crushed whole spleen and filtered through a 70 uM filter. RBC lysis was performed (Biolegend) and cells were prepared for downstream processing or frozen in 10% DMSO+90% fetal bovine serum.
Flow Cytometry and Sorting.
[0220] Freshly isolated or cryopreserved cells were washed twice with FACS buffer (phosphate buffered saline (PBS)+2% fetal bovine serum). Cells were incubated with antibody cocktails for 15 minutes at 4 C. Complete antibody details can be found in Supplemental table X. Following antibody incubation, cells were washed with FACS buffer and resuspended in a DAPI containing FACS buffer solution for analysis and sorting. Flow cytometry analysis was performed on a BD LSRFortessa with analysis using FACSDiva and FlowJo (v10.9). Cell isolation with FACS was performed on a Sony SH800.
Nucleic Acid Isolation and RNA-Sequencing Library Generations.
[0221] DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen). RNA was isolated using the Direct-zol RNA Microprep Kit (Zymo Research, R2061) and quantified using the Agilent High Sensitivity RNA ScreenTape (Agilent 5067-5579) on an Agilent 2200 TapeStation. 3 multiplexed RNA-sequencing was performed with the Takara SMART-Seq v4 3 DE Kit (Takara 635040) followed by Nextera XT (Illumina FC-131-1024) library preparation. For full length RNA-sequencing, cDNA was generated from 1 ng of input RNA using the SMART-Seq HT Kit (Takara 634455) at half reaction volume. cDNA and tagmented libraries were quantified using High Sensitivity D5000 ScreenTape (5067-5592) and High Sensitivity D1000 ScreenTape respectively (5067-5584). Libraries were sequenced on a NovaSeq at the Integrated Genomics Operation (IGO) at MSKCC.
Mass Cytometry and Data Analysis.
[0222] Cryopreserved mouse whole bone marrow or spleen cells were thawed and counted. Approximately 1 million from each mouse analyzed were used. Live cells were rested for 2 hours in RPMI+10% FBS (Gibco) (cRPMI) and then stained for viability with 5 M Cell-ID Cisplatin (Fluidigm 201198, 201194) for 2.5 minutes and washed in cRPMI. Cells were fixed with paraformaldehyde at a final concentration of 1.6% for 10 mins at RT in the dark and washed with Maxpar PBS (Fluidigm). Palladium mass-tag barcoding was performed as previously described using combinations of 6 palladium isotopes with Cell-ID 20-plex Pd barcoding kit (Fluidigm).1,2 All barcoded cells were then combined and washed with Maxpar cell staining buffer (CSB) (Fluidigm) and stained first with anti-CD16/32-159Tb for 30 min, followed by the rest of the surface antibody cocktail for 30 mins (Supplemental Table X). Cells were then washed with PBS, and permeabilized with ice cold 100% methanol (Fischer Scientific) for at least 30 mins. Following permeabilization, cell were washed and pelleted twice with CSB followed by intracellular antibody staining for 30 mins. Just prior to data collection, cells were stained with 250 nM Cell-ID Iridium intercalator (Fluidigm) in PBS with 1.6% PFA for 30 mins at 4 C. and then washed per protocol. Cells were then washed and rehydrated in double deionized water and collected on a Helios mass cytometer (Fluidigm). Barcorded .fcs files were normalized and debarcoded with Fluidigm Debarcoder software. Files were downloaded and gated in Cytobank (BeckmanCoulter) for single intercalator positive cells. CD45.2-positive cells were gated and files were downloaded and loaded into R for use by the CATALYST package.3,4 Analysis and plots were created using this following standard workflows.
mRNA Electroporation.
[0223] Lineage negative bone marrow cells were isolated using the Mouse Hematopoietic Progenitor Cell Isolation Kit (StemCell 19856) and electroporated with a custom Dre mRNA (Tri-Link) using the Thermo Neon electroporation device according to the manufacturer's protocol (100 uL kit, Thermo MPK10025). In brief, lineage negative bone marrow cells were resuspended in 153 ul of buffer T and 17 ul of Dre mRNA (1 ug/ul) was added and mixed by pipetting. Immediately after RNA addition, cells were electroporated using one pulse of 1700V for 20 ms. Following electroporation, cells were added to serum free media (StemSpan) with 50 ng SCF and 10 ng IL3 without antibiotics. For transplantation experiments, cells were incubated at 37 degrees for 1 hour and then injected into mice as described above. For extended culture, antibiotics were added 4 hours after electroporation.
Bioinformatic Analysis
[0224] FASTQ files were demultiplexed using a java script from Takara. FASTQ files were mapped and transcript counts were enumerated using STAR (genome version mm10 and transcript version ensembl XX). Counts were input into R and RNA-sequencing analysis using DESeq2. Gene set enrichment analysis was performed using the fgsea package with genesets extracted from the msigdbr package. Single sample gene set enrichment analysis was performed using the gsva package. Figures were generated using ggplot2 and tidyheatmaps packages.
Example 13: GOLDI-Lox On/Off Dre/Cre Npm1 Mouse
[0225]
[0226] The WT/mutant/WT functionality of the Npm1GOLDI-Lox allele was validated. Expression of mutant Npm1 has been previously shown to induce upregulation of the Hox/Meis loci. Additionally, the mutant Npm1 cDNA and protein harbor a unique C-terminal sequence which can be distinguished from Npm1 WT. Quantitative RT-PCR (qRT-PCR) was performed to determine expression levels of HoxA10, HoxB8, Meis1 (
[0227]
[0228]
EQUIVALENTS AND SCOPE
[0229] In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
[0230] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.
[0231] The phrase and/or, as used herein in the specification and in the claims, should be understood to mean either or both of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with and/or should be construed in the same fashion, i.e., one or more of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the and/or clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to A and/or B, when used in conjunction with open-ended language such as comprising can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
[0232] As used herein in the specification and in the claims, or should be understood to have the same meaning as and/or as defined above. For example, when separating items in a list, or or and/or shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as only one of or exactly one of, or, when used in the claims, consisting of, will refer to the inclusion of exactly one element of a number or list of elements. In general, the term or as used herein shall only be interpreted as indicating exclusive alternatives (i.e. one or the other but not both) when preceded by terms of exclusivity, such as either, one of, only one of, or exactly one of. Consisting essentially of, when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0233] As used herein in the specification and in the claims, the phrase at least one, in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase at least one refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, at least one of A and B (or, equivalently, at least one of A or B, or, equivalently at least one of A and/or B) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
[0234] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.
[0235] In the claims, as well as in the specification above, all transitional phrases such as comprising, including, carrying, having, containing, involving, holding, composed of, and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases consisting of and consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., comprising) are also contemplated, in alternative embodiments, as consisting of and consisting essentially of the feature described by the open-ended transitional phrase. For example, if the application describes a composition comprising A and B, the application also contemplates the alternative embodiments a composition consisting of A and B and a composition consisting essentially of A and B.
[0236] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
[0237] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
[0238] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.
[0239] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.