COMPOSITIONS AND METHODS FOR TARGETING TUMOR ASSOCIATED TRANSCRIPTION FACTORS
20230310623 · 2023-10-05
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
A61K31/7088
HUMAN NECESSITIES
C12N9/22
CHEMISTRY; METALLURGY
C12N2310/111
CHEMISTRY; METALLURGY
A61K38/465
HUMAN NECESSITIES
A61K47/64
HUMAN NECESSITIES
C12N2800/80
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
C07K19/00
CHEMISTRY; METALLURGY
C12N2740/15052
CHEMISTRY; METALLURGY
C12N2740/15043
CHEMISTRY; METALLURGY
A61K47/549
HUMAN NECESSITIES
C12N2310/113
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
International classification
A61K47/64
HUMAN NECESSITIES
C07K19/00
CHEMISTRY; METALLURGY
A61K31/7088
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
C12N15/90
CHEMISTRY; METALLURGY
Abstract
Described are compositions and methods for targeting tumor associated transcription factors (e.g., PU.1) using IncRNA, constructs comprising IncRNA, and CRISPR/Cas systems, and polynucleotides encoding IncRNA, constructs comprising IncRNA, and CRISPR/Cas systems, vectors containing the polynucleotides, viral or non-viral delivery vehicles containing the vectors, and compositions (e.g., pharmaceutical compositions) containing the same for use in methods treatment.
Claims
1. A polynucleotide comprising a sequence with at least 20 nucleotides of SEQ ID NO: 1, and variants thereof with at least 85% sequence identity thereto, wherein the polynucleotide has fewer than 2,381 nucleotides of SEQ ID NO: 1.
2. The polynucleotide of claim 1, wherein the variant of the polynucleotide has at least 90%, 95%, 97%, or 100% sequence identity to SEQ ID NO: 1.
3. The polynucleotide of claim 1 or 2, wherein the polynucleotide comprises a binding region for a Runt-related transcription factor 1 (RUNX1) protein or fragment thereof.
4. The polynucleotide of claim 3, wherein the binding region comprises all or at least 20 nucleotides of one or more transposable elements (TEs).
5. The polynucleotide of claim 4, wherein the one or more TEs comprise a nucleotide sequence with at least 85% sequence identity to at least 20 or more nucleotides of any one of SEQ ID NOs: 2-4.
6. The polynucleotide of claim 5, wherein the polynucleotide comprises two said TEs or three said TEs.
7. The polynucleotide of claim 6, wherein the polynucleotide comprises three said TEs, and wherein a first said TE comprises at least 20 nucleotides of SEQ ID NO: 2, a second said TE comprises at least 20 nucleotides of SEQ ID NO: 3, and a third said TE comprises at least 20 nucleotides of SEQ ID NO: 4.
8. The polynucleotide of claim 7, wherein the three said TEs comprise SEQ ID NOs: 2-4.
9. The polynucleotide of claim 7 or 8, wherein the first, second, and third TEs are present in the polynucleotide in order, 5′ to 3′, and wherein the TEs are linked directly or through a linker.
10. The polynucleotide of any one of claims 1-9, wherein the polynucleotide comprises at least 30 nucleotides of SEQ ID NO: 1.
11. The polynucleotide of any one of claims 1-10, wherein the polynucleotide comprises at least 40 nucleotides of SEQ ID NO: 1.
12. The polynucleotide of any one of claims 1-11, wherein the polynucleotide comprises at least 100 nucleotides of SEQ ID NO: 1.
13. The polynucleotide of any one of claims 1-12, wherein the polynucleotide comprises at least 500 nucleotides of SEQ ID NO: 1.
14. The polynucleotide of any one of claims 1-13, wherein the polynucleotide comprises at least 1700 nucleotides of SEQ ID NO: 1.
15. The polynucleotide of any one of claims 1-14, wherein the polynucleotide comprises at least 2000 nucleotides of SEQ ID NO: 1.
16. The polynucleotide of any one of claims 1-15, wherein the polynucleotide comprises at least 2300 nucleotides of SEQ ID NO: 1.
17. The polynucleotide of any one of claims 1-16, wherein the polynucleotide comprises at least 2350 nucleotides of SEQ ID NO: 1.
18. The polynucleotide of any one of claims 1-17, wherein the polynucleotide comprises at least 2375 nucleotides of SEQ ID NO: 1.
19. A construct comprising a RUNX1 protein, or fragment thereof, conjugated to at least one polynucleotide of any one of claims 1-18.
20. The construct of claim 19, wherein the construct comprises at least one said RUNX1 protein, or fragment thereof, bound to at least one said polynucleotide.
21. The construct of claim 19 or 20, wherein the RUNX1 protein, or fragment thereof, and the polynucleotide are bound through a covalent bond.
22. The construct of any one of claims 19-21, comprising the structure:
R-L-P (I) or P-L-R (II), wherein R is the RUNX1 protein or fragment thereof; P is the polynucleotide; and L is a linker.
23. The construct of claim 22, where the construct comprises the structure of R-L-P (I).
24. The construct of claim 22, wherein the construct comprises the structure of P-L-R (II).
25. The construct of any one of claims 22-24, wherein R comprises at least 100 amino acids of SEQ ID NO: 5, and variants thereof with at least 85% sequence identity thereto.
26. The construct of claim 25, wherein R has at least 90%, 95%, 97%, or 100% sequence identity to the sequence of SEQ ID NO: 5.
27. The construct of claim 26, wherein R polypeptide has the sequence of SEQ ID NO: 5.
28. The construct of any one of claims 22-27, wherein R polypeptide comprises at least one binding site for at least one polynucleotide regulatory element of PU.1.
29. The construct of claim 28, wherein the at least one PU.1 regulatory element has at least 85% sequence identity to the sequence of SEQ ID NO: 6.
30. The construct of claim 29, wherein the at least one PU.1 regulatory element has at least 90%, 95%, 97%, or 100% sequence identity to the sequence of SEQ ID NO: 6.
31. The construct of claim 30, wherein the at least one PU.1 regulatory element has the sequence of SEQ ID NO: 6.
32. The construct of claim 28, wherein the at least one PU.1 regulatory element is an upstream regulatory element (URE) and/or a proximal promoter region (PrPr).
33. The construct of claim 32, wherein the PrPr has at least 85% sequence identity to the sequence of SEQ ID NO: 7.
34. The construct of claim 33, wherein the PrPr has at least 90%, 95%, 97%, or 100% sequence identity to the sequence of SEQ ID NO: 7.
35. The construct of claim 34, wherein the PrPr has the sequence of SEQ ID NO: 7.
36. A polynucleotide encoding the construct of any one of claims 19-35.
37. A vector comprising the polynucleotide of any one of claims 1-18 or the polynucleotide of claim 36.
38. A composition comprising the polynucleotide of any one of claims 1-18, the construct of any one of claims 19-35, the polynucleotide of claim 36, or the vector of claim 37.
39. The composition of claim 38, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.
40. A kit comprising the polynucleotide of any one of claims 1-18, the construct of any one of claims 19-35, the polynucleotide of claim 36, the vector of claim 37, or the composition of claim 38 or 39, and a package insert comprising instructions for using the polynucleotide, construct, vector, or composition for treating a medical condition in a subject.
41. A method of treating a medical condition in a subject in need thereof comprising administering the polynucleotide of any one of claims 1-18.
42. The method of claim 41, wherein the medical condition is a cancer.
43. The method of claim 42, wherein the cancer is a blood cancer.
44. The method of claim 43, wherein the blood cancer is acute myeloid leukemia (AML).
45. The method of claim 43, wherein the blood cancer is myeloma.
46. The method of claim 42, wherein the cancer is liver cancer.
47. The method of claim 46, wherein the liver cancer is metastatic hepatocellular carcinoma (HCC).
48. A method of treating a medical condition in a subject in need thereof comprising administering the construct of any one of claims 19-35.
49. The method of claim 48, wherein the medical condition is a cancer.
50. The method of claim 49, wherein the cancer is a blood cancer.
51. The method of claim 50, wherein the blood cancer is acute myeloid leukemia (AML).
52. The method of claim 50, wherein the blood cancer is myeloma.
53. The method of claim 49, wherein the cancer is liver cancer.
54. The method of claim 53, wherein the liver cancer is metastatic hepatocellular carcinoma (HCC).
55. Use of the construct of any one of claims 19-35 in the preparation of a medicament for the treatment of a medical condition in a subject in need thereof.
56. A method of treating a medical condition in a subject, wherein the method comprises: a) delivering to a target cell a dCas activator system comprising: i) a plurality of first guide ribonucleic acids (gRNAs) directed to a first genomic site of an endogenous DNA molecule of the cell; and ii) a plurality of dCas fusion proteins; wherein the first gRNA forms a first complex with a first said dCas fusion protein at the first genomic site, and wherein the first complex promotes the expression of LOUP.
57. The method of claim 56, wherein the first guide gRNA specifically hybridizes to the first genomic site.
58. The method of claim 56 or 57, wherein the first genomic site and the target gene of interest are between 10-100,000 nucleotide base pairs apart.
59. The method of any one of claims 56-58, wherein the first genomic site comprises a protospacer adjacent motif (PAM) recognition sequence positioned upstream from said first genomic site.
60. The method of any one of claims 56-59, wherein the first guide RNA is a single guide RNA (sgRNA).
61. The method of any one of claims 56-60, wherein the dCas fusion protein is selected from a group comprising dCas9-VP64, dCas9-VPR, dCas9-SAM, dCas9-Scaffold, dCas9-Suntag, dCas9-P300, dCas9-VP160, and VP64-dCas9-BFP-VP64.
62. The method of claim 61, wherein the dCas fusion protein is dCas9-VP64.
63. The method of any one of claims 56-62, wherein the first target genomic site is associated with the medical condition.
64. The method of any one of claims 56-63, wherein the medical condition is a cancer.
65. The method of claim 64, wherein the cancer is a cancer associated with tumor suppressor gene PU.1.
66. The method of claim 65, wherein the cancer associated with tumor suppressor gene PU.1 is acute myeloid leukemia (AML), liver cancer, or myeloma.
67. The method of any one of claims 56-66, wherein the target gene of interest is tumor suppressor gene PU.1.
68. A nucleic acid comprising a polynucleotide comprising a nucleic acid sequence encoding dCas activator system.
69. The nucleic acid of claim 68, wherein the dCas activator system comprises a dCas fusion protein.
70. The nucleic acid of claim 68 or 69, further comprising a nucleic acid sequence encoding a first gRNA.
71. The nucleic acid of claim 70, wherein the first gRNA is directed to a first genomic site of an endogenous DNA molecule of a cell.
72. The nucleic acid of any one of claims 68-71, further comprising a promoter.
73. The nucleic acid of any one of claims 69-72, wherein the dCas fusion protein is selected from a group comprising dCas9-VP64, dCas9-VPR, dCas9-SAM, dCas9-Scaffold, dCas9-Suntag, dCas9-P300, dCas9-VP160, and VP64-dCas9-BFP-VP64.
74. A vector comprising the nucleic acid of any one of claims 68-73.
75. The vector of claim 74, wherein the vector is an expression vector or a viral vector.
76. The vector of claim 75, wherein the viral vector is a lentiviral vector.
77. A composition comprising: a) a plurality of first guide ribonucleic acids (gRNAs) directed to a first genomic site of an endogenous DNA molecule of the cell; and b) a plurality of dCas fusion proteins.
78. The composition of claim 77, wherein the first gRNA is in a first complex with a first said dCas fusion protein, wherein the first complex is configured to promote the expression of a target gene of interest.
79. The composition of claim 77 or 78, the dCas fusion protein is selected from a group comprising dCas9-VP64, dCas9-VPR, dCas9-SAM, dCas9-Scaffold, dCas9-Suntag, dCas9-P300, dCas9-VP160, and VP64-dCas9-BFP-VP64.
80. The composition of claim 79, wherein the dCas fusion protein is dCas9-VP64.
81. A pharmaceutical composition comprising the nucleic acid of any one of claims 68-76, or the composition of any one of claims 77-79, and a pharmaceutically acceptable carrier, excipient, or diluent.
82. A kit comprising the nucleic acid of any one of claims 68-76, the composition of any one of claims 77-79, or the pharmaceutical composition of claim 81, and a package insert comprising instructions for using the nucleic acid, composition, or pharmaceutical composition for treating a medical condition in a subject.
83. A method of treating a medical condition in a subject, wherein the method comprises: a) delivering to a target cell a gene editing system comprising: i) a plurality of first guide ribonucleic acids (gRNAs) directed to a first genomic site of an endogenous DNA molecule of the cell; and ii) a plurality of RNA programmable nucleases; wherein the first guide RNA forms a first complex with a first said RNA programmable nuclease at the first genomic site, and wherein the first complex promotes the inhibition of expression of LOUP.
84. The method of claim 83, wherein the first guide gRNA specifically hybridizes to the first genomic site.
85. The method of claim 83 or 84, wherein the first genomic site and the target gene of interest are between 10-100,000 nucleotide base pairs apart.
86. The method of any one of claims 83-85, wherein the first genomic site comprises a protospacer adjacent motif (PAM) recognition sequence positioned upstream from said first genomic site.
87. The method of any one of claims 83-86, wherein the first guide RNA is a single guide RNA (sgRNA).
88. The method of any one of claims 83-87, wherein the inhibition of expression of the target gene of interest is caused by non-homologous end-joining (NHEJ).
89. The method of any one of claims 83-88, wherein the first target genomic site is associated with the medical condition.
90. The method of any one of claims 83-89, wherein the medical condition is associated with tumor suppressor gene PU.1.
91. The method of claim 90, wherein the medical condition associated with PU.1 is Alzheimer's disease or asthma.
92. The method of any one of claims 83-91, wherein the target gene of interest is tumor suppressor gene PU.1.
93. The method of any one of claims 83-92, wherein the RNA program nuclease is a Cas RNA programmable nuclease.
94. The method of claim 93, wherein the Cas RNA programmable nuclease is a Cas9 RNA programmable nuclease.
95. A nucleic acid comprising a polynucleotide comprising a nucleic acid sequence encoding: a) a first gRNA directed to a first genomic site of an endogenous DNA molecule of a target cell; and b) an RNA-programmable nuclease; wherein the first genomic site is between 10-100,000 nucleotide base pairs from a target gene of interest comprising tumor suppressor gene PU.1.
96. The nucleic acid of claim 95, further comprising a promoter.
97. The nucleic acid molecule of claim 95 or 96, wherein the RNA programmable nuclease is a Cas RNA programmable nuclease.
98. The nucleic acid of claim 97, wherein the Cas RNA programmable nuclease is a Cas9 RNA programmable nuclease.
99. A vector comprising the nucleic acid of any one of claims 95-98.
100. The vector of claim 99, wherein the vector is an expression vector or a viral vector.
101. The vector of claim 100, wherein the viral vector is a lentiviral vector.
102. The polynucleotide of claim 1, wherein the polynucleotide comprises a binding region for a RUNX1 protein or fragment thereof.
103. The polynucleotide of claim 102, wherein the binding region comprises all or at least 20 nucleotides of one or more TEs.
104. The polynucleotide of claim 103, wherein the one or more TEs comprise a nucleotide sequence with at least 85% sequence identity to at least 20 or more nucleotides of any one of SEQ ID NOs: 2-4.
105. The polynucleotide of claim 104, wherein the polynucleotide comprises two said TEs or three said TEs.
106. The polynucleotide of claim 105, wherein the polynucleotide comprises three said TEs, and wherein a first said TE comprises at least 20 nucleotides of SEQ ID NO: 2, a second said TE comprises at least 20 nucleotides of SEQ ID NO: 3, and a third said TE comprises at least 20 nucleotides of SEQ ID NO: 4.
107. The polynucleotide of claim 106, wherein the three said TEs comprise SEQ ID NOs: 2-4.
108. The polynucleotide of claim 106, wherein the first, second, and third TEs are present in the polynucleotide in order, 5′ to 3′, and wherein the TEs are linked directly or through a linker.
109. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 30 nucleotides of SEQ ID NO: 1.
110. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 40 nucleotides of SEQ ID NO: 1.
111. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 100 nucleotides of SEQ ID NO: 1.
112. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 500 nucleotides of SEQ ID NO: 1.
113. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 1700 nucleotides of SEQ ID NO: 1.
114. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 2000 nucleotides of SEQ ID NO: 1.
115. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 2300 nucleotides of SEQ ID NO: 1.
116. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 2350 nucleotides of SEQ ID NO: 1.
117. The polynucleotide of claim 1, wherein the polynucleotide comprises at least 2375 nucleotides of SEQ ID NO: 1.
118. A construct comprising a RUNX1 protein, or fragment thereof, conjugated to at least one polynucleotide of claim 1.
119. The construct of claim 118, wherein the construct comprises at least one said RUNX1 protein, or fragment thereof, bound to at least one said polynucleotide.
120. The construct of claim 118, wherein the RUNX1 protein, or fragment thereof, and the polynucleotide are bound through a covalent bond.
121. The construct of claim 118, comprising the structure:
R-L-P (I) or P-L-R (II), wherein R is the RUNX1 protein or fragment thereof; P is the polynucleotide; and L is a linker.
122. The construct of claim 121, where the construct comprises the structure of R-L-P (I).
123. The construct of claim 121, wherein the construct comprises the structure of P-L-R (II).
124. The construct of claim 121, wherein R comprises at least 100 amino acids of SEQ ID NO: 5, and variants thereof with at least 85% sequence identity thereto.
125. The construct of claim 124, wherein R has at least 90%, 95%, 97%, or 100% sequence identity to the sequence of SEQ ID NO: 5.
126. The construct of claim 125, wherein R polypeptide has the sequence of SEQ ID NO: 5.
127. The construct of claim 121, wherein R polypeptide comprises at least one binding site for at least one polynucleotide regulatory element of PU.1.
128. The construct of claim 127, wherein the at least one PU.1 regulatory element has at least 85% sequence identity to the sequence of SEQ ID NO: 6.
129. The construct of claim 128, wherein the at least one PU.1 regulatory element has at least 90%, 95%, 97%, or 100% sequence identity to the sequence of SEQ ID NO: 6.
130. The construct of claim 129, wherein the at least one PU.1 regulatory element has the sequence of SEQ ID NO: 6.
131. The construct of claim 127, wherein the at least one PU.1 regulatory element is an upstream regulatory element (URE) and/or a proximal promoter region (PrPr).
132. The construct of claim 131, wherein the PrPr has at least 85% sequence identity to the sequence of SEQ ID NO: 7.
133. The construct of claim 132, wherein the PrPr has at least 90%, 95%, 97%, or 100% sequence identity to the sequence of SEQ ID NO: 7.
134. The construct of claim 133, wherein the PrPr has the sequence of SEQ ID NO: 7.
135. A polynucleotide encoding the construct of claim 118.
136. A vector comprising the polynucleotide of claim 1.
137. A composition comprising the polynucleotide of claim 1, a construct comprising a RUNX1 protein, or fragment thereof, conjugated to the polynucleotide, a polynucleotide encoding the construct, or a vector comprising the polynucleotide of claim 1.
138. The composition of claim 137, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.
139. A kit comprising the polynucleotide of claim 1, a construct comprising a RUNX1 protein, or fragment thereof, conjugated to the polynucleotide, a polynucleotide encoding the construct, a vector comprising the polynucleotide of claim 1, or a composition comprising the polynucleotide of claim 1, and a package insert comprising instructions for using the polynucleotide, construct, vector, or composition for treating a medical condition in a subject.
140. A method of treating a medical condition in a subject in need thereof comprising administering the polynucleotide of claim 1.
141. The method of claim 140, wherein the medical condition is a cancer.
142. The method of claim 141, wherein the cancer is a blood cancer.
143. The method of claim 142, wherein the blood cancer is acute myeloid leukemia (AML).
144. The method of claim 142, wherein the blood cancer is myeloma.
145. The method of claim 141, wherein the cancer is liver cancer.
146. The method of claim 145, wherein the liver cancer is metastatic hepatocellular carcinoma (HCC).
147. A method of treating a medical condition in a subject in need thereof comprising administering the construct of claim 118.
148. The method of claim 147, wherein the medical condition is a cancer.
149. The method of claim 148, wherein the cancer is a blood cancer.
150. The method of claim 149, wherein the blood cancer is acute myeloid leukemia (AML).
151. The method of claim 149, wherein the blood cancer is myeloma.
152. The method of claim 148, wherein the cancer is liver cancer.
153. The method of claim 152, wherein the liver cancer is metastatic hepatocellular carcinoma (HCC).
154. Use of the construct of claim 118 in the preparation of a medicament for the treatment of a medical condition in a subject in need thereof.
155. The method of claim 56, wherein the first genomic site and the target gene of interest are between 10-100,000 nucleotide base pairs apart.
156. The method of claim 56, wherein the first genomic site comprises a protospacer adjacent motif (PAM) recognition sequence positioned upstream from said first genomic site.
157. The method of claim 56, wherein the first guide RNA is a single guide RNA (sgRNA).
158. The method of claim 56, wherein the dCas fusion protein is selected from a group comprising dCas9-VP64, dCas9-VPR, dCas9-SAM, dCas9-Scaffold, dCas9-Suntag, dCas9-P300, dCas9-VP160, and VP64-dCas9-BFP-VP64.
159. The method of claim 158, wherein the dCas fusion protein is dCas9-VP64.
160. The method of claim 56, wherein the first target genomic site is associated with the medical condition.
161. The method of claim 56, wherein the medical condition is a cancer.
162. The method of claim 161, wherein the cancer is a cancer associated with tumor suppressor gene PU.1.
163. The method of claim 162, wherein the cancer associated with tumor suppressor gene PU.1 is acute myeloid leukemia (AML), liver cancer, or myeloma.
164. The method of claim 56, wherein the target gene of interest is tumor suppressor gene PU.1.
165. The nucleic acid of claim 68, further comprising a nucleic acid sequence encoding a first gRNA.
166. The nucleic acid of claim 165, wherein the first gRNA is directed to a first genomic site of an endogenous DNA molecule of a cell.
167. The nucleic acid of claim 68, further comprising a promoter.
168. The nucleic acid of claim 69, wherein the dCas fusion protein is selected from a group comprising dCas9-VP64, dCas9-VPR, dCas9-SAM, dCas9-Scaffold, dCas9-Suntag, dCas9-P300, dCas9-VP160, and VP64-dCas9-BFP-VP64.
169. A vector comprising the nucleic acid of claim 68.
170. The vector of claim 169, wherein the vector is an expression vector or a viral vector.
171. The vector of claim 170, wherein the viral vector is a lentiviral vector.
172. The composition of claim 77, the dCas fusion protein is selected from a group comprising dCas9-VP64, dCas9-VPR, dCas9-SAM, dCas9-Scaffold, dCas9-Suntag, dCas9-P300, dCas9-VP160, and VP64-dCas9-BFP-VP64.
173. The composition of claim 79, wherein the dCas fusion protein is dCas9-VP64.
174. A pharmaceutical composition comprising the nucleic acid of claim 68, or a composition comprising (a) a plurality of first gRNAs directed to a first genomic site of an endogenous DNA molecule of the cell and (b) a plurality of dCas fusion proteins, and a pharmaceutically acceptable carrier, excipient, or diluent.
175. A kit comprising the nucleic acid of claim 68, a composition comprising (a) a plurality of first gRNAs directed to a first genomic site of an endogenous DNA molecule of the cell and (b) a plurality of dCas fusion proteins, or a pharmaceutical composition comprising the nucleic acid, and a package insert comprising instructions for using the nucleic acid, composition, or pharmaceutical composition for treating a medical condition in a subject.
176. The method of claim 83, wherein the first genomic site and the target gene of interest are between 10-100,000 nucleotide base pairs apart.
177. The method of claim 83, wherein the first genomic site comprises a protospacer adjacent motif (PAM) recognition sequence positioned upstream from said first genomic site.
178. The method of claim 83, wherein the first guide RNA is a single guide RNA (sgRNA).
179. The method of claim 83, wherein the inhibition of expression of the target gene of interest is caused by non-homologous end-joining (NHEJ).
180. The method of claim 83, wherein the first target genomic site is associated with the medical condition.
181. The method of claim 83, wherein the medical condition is associated with tumor suppressor gene PU.1.
182. The method of claim 181, wherein the medical condition associated with PU.1 is Alzheimer's disease or asthma.
183. The method of claim 83, wherein the target gene of interest is tumor suppressor gene PU.1.
184. The method of claim 83, wherein the RNA program nuclease is a Cas RNA programmable nuclease.
185. The method of claim 184, wherein the Cas RNA programmable nuclease is a Cas9 RNA programmable nuclease.
186. The nucleic acid of claim 95, wherein the RNA programmable nuclease is a Cas RNA programmable nuclease.
187. The nucleic acid of claim 186, wherein the Cas RNA programmable nuclease is a Cas9 RNA programmable nuclease.
188. A vector comprising the nucleic acid of claim 95.
189. The vector of claim 188, wherein the vector is an expression vector or a viral vector.
190. The vector of claim 189, wherein the viral vector is a lentiviral vector.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0106] Described herein are long non-coding RNA (e.g., LOUP RNA), polynucleotides encoding the lncRNA, vectors (e.g., viral vectors) containing polynucleotides encoding the lncRNA, constructs containing LOUP, methods of delivering LOUP, methods of increasing or decreasing LOUP expression using a gene editing system (e.g., a CRISPR/Cas system or CRISPRa), methods of altering PU.1 expression, methods of treating a disease (e.g., cancer (e.g., PU.1 associated cancer (e.g., AML, liver cancer, and myeloma)), Alzheimer's disease, or asthma), and methods of diagnosing treatment responsiveness (e.g., ATRA treatment) in a subject with cancer (e.g., AML, liver disease, or myeloma).
[0107] We discovered that an uncharacterized myeloid-specific lncRNA, termed “Long noncoding RNA Originating from the URE of PU.1”, or LOUP, induces gene-specific long-range transcription by modulating enhancer docking to a specific proximal promoter. LOUP is a product of unidirectional transcription, and undergoes splicing and polyadenylation, thereby exhibiting all the features of a 1d-eRNA. At single-cell resolution, LOUP and PU.1 expression is stringently associated with myeloid lineage identity. Both gain- and loss-of-function experiments demonstrated a LOUP-dependent expression of PU.1. We further discovered that LOUP associates with chromatin and induces interaction between the URE and the PrPr, resulting in the formation of an active chromatin loop at the PU.1 locus. Finally, we showed that LOUP recruits RUNX1 to its DNA-binding motifs at both the URE and the PrPr via a region embedded with transposable element (TE) variants. Collectively, these findings reveal an unanticipated role of a cell type-specific and TE-embedded 1d-eRNA in mediating gene-specific long-range transcription by cooperating with a ubiquitously expressed transcription factor.
[0108] The present disclosure relates to long non-coding RNA (e.g., LOUP RNA), polynucleotides encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), vectors (e.g., viral vectors) including polynucleotides encoding the lncRNA (or at least, e.g., 20 nucleotides or more, encoding the lncRNA), constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), a gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing system, vectors (e.g., viral vectors) including polynucleotides encoding the gene editing system and compositions including the same, and cells containing one or more of these compositions. The compositions disclosed herein can be used in methods of diagnosing, treating, and/or preventing conditions associated with PU.1 expression (e.g., cancer (e.g., AML, liver cancer, or myeloma), Alzheimer's disease, or asthma).
[0109] Polynucleotides
[0110] Featured polynucleotides include any nucleotide capable of inducing PU.1 expression. In some embodiments, the polynucleotide includes a binding region for Runt-related transcription factor 1 (RUNX1) protein, or fragment thereof. For example, the polynucleotide may include a nucleic acid sequence with at least about 20 nucleotides (e.g., at least about 25, at least about 40, at least about 60, at least about 80, at least about 100, at least about 150, at least about 300, at least about 500, at least about 900, at least about 1300, at least about 1700, at least about 2000, at least about 2300, at least about 2350, or at least about 2375) of SEQ ID NO: 1 and variants thereof with at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In some instances, the polynucleotide may include a nucleic acid sequence with between about 20 nucleotides and about 2380 nucleotides (e.g., between about 20 and about 100, between about 70 and about 300, between about 200 and about 500, between about 400 and about 800, between about 700 and about 1200, between about 1100 and about 1600, between about 1500 and about 2000, or between about 1900 and about 2380) or SEQ ID NO: 1, or variants thereof with at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In particular, the polynucleotide contains one or more transposable elements (TEs) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more TEs). The one or more transposable elements have a nucleic acid sequence of any one of SEQ ID NOs: 2-4 or a variant thereof with at least 85% (e.g., (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. The TE(s) of the polynucleotide may have a minimum length of at least about 50 nucleotides of the nucleotides of any one of SEQ ID NO: 2 or 3 (e.g., at least about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 or more nucleotides of SEQ ID NO: 2 or 3) or a variant thereof. In some embodiments, the polynucleotide includes two or three of the TEs or a variant thereof. For example, the polynucleotide includes a first TE of SEQ ID NO: 2, or a variant thereof, and a second TE of SEQ ID NO: 3 or 4, or a variant thereof (e.g., the polynucleotide includes TEs of SEQ ID NOs: 2 and 3, or variants thereof, or TEs of SEQ ID NOs: 2 and 4, or variants thereof). The polynucleotide may also include a first TE of SEQ ID NO: 3 and a second TE of SEQ ID NO: 4, or variants thereof.
[0111] Constructs
[0112] Featured constructs include a RUNX1 protein, or fragment thereof, conjugated to any polynucleotide capable of inducing PU.1 expression. In some embodiments, the RUNX1 protein, or fragment thereof, is bound (e.g., covalently bound) to any polynucleotide capable of inducing PU.1 expression. In some embodiments the constructs have the structure:
R-L-P (I) or P-L-R (II),
[0113] wherein R is the RUNX1 protein or fragment thereof;
[0114] P is the polynucleotide; and
[0115] L is a linker.
In some embodiments, the construct has the structure R-L-P (I). In other embodiments, the construct has the structure P-L-R (II). The RUNX1 protein may have at least 100 amino acids of SEQ ID NO: 5, or a variant thereof with at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. The RUNX1 protein may have at least one binding site (e.g., one, two, three, four, five, or more binding sites) for at least one polynucleotide regulatory element of PU.1 (e.g., at least one, two, three, four, five, or more regulatory elements of PU.1). In certain embodiments, the at least one PU.1 regulatory element has at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to, or the sequence of, SEQ ID NO: 6. In some embodiments, the at least one PU.1 regulatory element is an upstream regulatory element (URE) and/or a proximal promoter region (PrPr). In some embodiments, the at least one PU.1 regulatory element is an upstream regulatory element (URE). In some instances, the URE sequence has at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the sequence of SEQ ID NO: 6. In some instances, the URE has the sequence of SEQ ID NO: 6. In other embodiments, the at least one PU.1 regulatory element is a proximal promoter region (PrPr). In some instances, the PrPr sequence has at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the sequence of SEQ ID NO: 7. In some instances, the PrPr sequence has the sequence of SEQ ID NO: 7.
[0116] The polynucleotide of the construct may have a nucleic acid sequence with at least about 20 nucleotides (e.g., at least about 25, at least about 40, at least about 60, at least about 80, at least about 100, at least about 150, at least about 300, at least about 500, at least about 900, at least about 1300, at least about 1700, at least about 2000, at least about 2300, at least about 2350, or at least about 2375) of SEQ ID NO: 1 and variants thereof with at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. For example, the polynucleotide may include a nucleic acid sequence with between about 20 nucleotides and about 2380 nucleotides (e.g., between about 20 and about 100, between about 70 and about 300, between about 200 and about 500, between about 400 and about 800, between about 700 and about 1200, between about 1100 and about 1600, between about 1500 and about 2000, or between about 1900 and about 2380) of SEQ ID NO: 1, or a variant thereof with at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In particular, the polynucleotide contains one or more transposable elements (TEs) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more TEs). The one or more transposable elements have a nucleic acid sequence of any one of SEQ ID NOs: 2-4 or a variant thereof with at least 85% (e.g., (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. The TE(s) of the polynucleotide may have a minimum length of at least about 50 nucleotides of the nucleotides of any one of SEQ ID NO: 2 or 3 (e.g., at least about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 or more nucleotides of SEQ ID NO: 2 or 3) or a variant thereof with at least 85% (e.g., (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto. In some embodiments, the polynucleotide includes two or three of the TEs or a variant thereof. For example, the polynucleotide includes a first TE of SEQ ID NO: 2, or a variant thereof, and a second TE of SEQ ID NO: 3 or 4, or a variant thereof (e.g., the polynucleotide includes TEs of SEQ ID NOs: 2 and 3, or variants thereof, or TEs of SEQ ID NOs: 2 and 4, or variants thereof). The polynucleotide may also include a first TE of SEQ ID NO: 3 and a second TE of SEQ ID NO: 4, or variants thereof.
[0117] CRISPR/Cas
[0118] CRISPR/Cas systems may be used to alter the expression profile of anti-tumor proliferating gene PU.1. The CRISPR/Cas system may be designed to decrease the expression of LOUP. Alternatively, a CRISPR activating (CRISPRa) system may be used to increase the expression of LOUP, thereby increasing PU.1 expression.
[0119] The CRISPR/Cas system derives from a prokaryotic immune system that confers resistance to foreign genetic elements, such as those present within plasmids and phages. CRISPR itself comprises a family of DNA sequences in bacteria, which encode small segments of DNA from viruses that have previously been exposed to the bacterium. These DNA segments are used by the bacterium to detect and destroy DNA from similar viruses during subsequent attacks. In a palindromic repeat, the sequence of nucleotides is the same in both directions. Each repetition is followed by short segments of spacer DNA from previous exposures to foreign DNA (e.g., a virus or plasmid). Small clusters of Cas (CRISPR-associated system) genes are located next to CRISPR sequences. These observations form the basis of the CRISPR/Cas system in eukaryotic cells that allows for genome editing. By delivering an RNA programmable nuclease (e.g., a Cas9 nuclease) with one or more guide polynucleotides (e.g., one or more gRNAs) into a cell, the cell's genome can be edited at desired locations (e.g., coding or non-coding regions of a genome of a host cell), allowing an existing gene(s) to be modified and/or removed and/or new gene(s) to be added (e.g., a functional version of a defective gene). The Cas9-gRNA complex corresponds with the type II CRISPR/Cas RNA complex.
[0120] A number of bacteria express Cas9 protein variants that can be used in the featured methods (see, e.g., Tables 1 and 2). The Cas9 from Streptococcus pyogenes is presently the most commonly used. Several other Cas9 proteins have high levels of sequence identity with the S. pyogenes Cas9 and use the same guide RNAs. Still, others are more diverse, use different gRNAs, and recognize different PAM sequences as well (the 2-5 nucleotide sequence specified by the protein which is adjacent to the sequence specified by the RNA; see, e.g., Table 2). Chylinski et al. (RNA Biol. 10(5): 726-737, 2013) classified Cas9 proteins from a large group of bacteria, and a large number of Cas9 proteins are described herein. Additional Cas9 proteins that can be used in the featured gene editing system are described in, e.g., Esvelt et al. (Nat Methods 10(11): 1116-21, 2013) and Fonfara et al. (Nucleic Acids Res. 42(4): 2577-2590, 2013); incorporated herein by reference.
[0121] Cas molecules from a variety of species can be incorporated into the methods (e.g., the methods of treating a medical condition (e.g., a medical condition associated with PU.1 expression), compositions, and kits described herein. While the S. pyogenes Cas9 molecule is the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while much of the description herein refers to S. pyogenes Cas9 molecules, Cas9 molecules from the other species can replace them. Such species include those set forth in the following Table 1:
TABLE-US-00001 TABLE 1 Exemplary Cas9 nucleases GenBank Acc No. Bacterium 303229466 Veillonella atypica ACS-134-V-Col7a 34762592 Fusobacterium nucleatum subsp. vincentii 374307738 Filifactor alocis ATCC 35896 320528778 Solobacterium moorei F0204 291520705 Coprococcus catus GD-7 42525843 Treponema denticola ATCC 35405 304438954 Peptoniphilus duerdenii ATCC BAA-1640 224543312 Catenibacterium mitsuokai DSM 15897 24379809 Streptococcus mutans UA159 15675041 Streptococcus pyogenes SF370 16801805 Listeria innocua Clip11262 116628213 Streptococcus thermophilus LMD-9 323463801 Staphylococcus pseudintermedius ED99 352684361 Acidaminococcus intestini RyC-MR95 302336020 Olsenella uli DSM 7084 366983953 Oenococcus kitaharae DSM 17330 310286728 Bifidobacterium bifidum S17 258509199 Lactobacillus rhamnosus GG 300361537 Lactobacillus gasseri JV-V03 169823755 Finegoldia magna ATCC 29328 47458868 Mycoplasma mobile 163K 284931710 Mycoplasma gallisepticum str. F 363542550 Mycoplasma ovipneumoniae SC01 384393286 Mycoplasma canis PG 14 71894592 Mycoplasma synoviae 53 238924075 Eubacterium rectale ATCC 33656 116627542 Streptococcus thermophilus LMD-9 315149830 Enterococcus faecalis TX0012 315659848 Staphylococcus lugdunensis M23590 160915782 Eubacterium dolichum DSM 3991 336393381 Lactobacillus coryniformis subsp. torquens 310780384 Ilyobacter polytropus DSM 2926 325677756 Ruminococcus albus 8 187736489 Akkermansia muciniphila ATCC BAA-835 117929158 Acidothermus cellulolyticus 11B 189440764 Bifidobacterium longum DJO10A 283456135 Bifidobacterium dentium Bd1 38232678 Corynebacterium diphtheriae NCTC 13129 187250660 Elusimicrobium minutum Pei191 319957206 Nitratifractor salsuginis DSM 16511 325972003 Sphaerochaeta globus str. Buddy 261414553 Fibrobacter succinogenes subsp. succinogenes 60683389 Bacteroides fragilis NCTC 9343 256819408 Capnocytophaga ochracea DSM 7271 90425961 Rhodopseudomonas palustris BisB18 373501184 Prevotella micans F0438 294674019 Prevotella ruminicola 23 365959402 Flavobacterium columnare ATCC 49512 312879015 Aminomonas paucivorans DSM 12260 83591793 Rhodospirillum rubrum ATCC 11170 294086111 Candidatus Puniceispirillum marinum IMCC1322 121608211 Verminephrobacter eiseniae EF01-2 344171927 Ralstonia syzygii R24 159042956 Dinoroseobacter shibae DFL 12 288957741 Azospirillum sp- B510 92109262 Nitrobacter hamburgensis X14 148255343 Bradyrhizobium sp- BTAi1 34557790 Wolinella succinogenes DSM 1740 218563121 Campylobacter jejuni subsp. jejuni 291276265 Helicobacter mustelae 12198 229113166 Bacillus cereus Rock1-15 222109285 Acidovorax ebreus TPSY 189485225 uncultured Termite group 1 182624245 Clostridium perfringens D str. 220930482 Clostridium cellulolyticum H10 154250555 Parvibaculum lavamentivorans DS-1 257413184 Roseburia intestinalis L1-82 218767588 Neisseria meningitidis Z2491 15602992 Pasteurella multocida subsp. multocida 319941583 Sutterella wadsworthensis 3 1 254447899 gamma proteobacterium HTCC5015 54296138 Legionella pneumophila str. Paris 331001027 Parasutterella excrementihominis YIT 11859 34557932 Wolinella succinogenes DSM 1740 118497352 Francisella novicida U112
TABLE-US-00002 TABLE 2 Exemplary Cas nucleases and their associated PAM sequence Class and PAM Target SEQ Species/Variant of Cas Type Sequence Length ID NO SpCas9 Class II type II 3′ NGG 20 nt 11 Streptococcus pyogenes (SP) SpCas9 Class II type II 3′ NGG 20 nt 11 D1135E variant (3′NAG reduced 12 binding) SpCas9 Class II type II 3′ NGCG 20 nt 13 VRER variant SpCas9 Class II type II 3′ NGAG 20 nt 14 EQR variant SpCas9 Class II type II 3′ NGAN; or 20 nt 15 VQR variant 3′ NGNG 16 SaCas9 Class II type II 3′ NNGRRT or 20 to 24 nt 17 Staphylococcus aureus 3′ NNGRR(N) 18 (SA) SaCas9 Class II type II 3′ NNNRRT 21 nt 19 Staphylococcus aureus KKH variant Cas12a: Class Il type V 5′ TTTV 23, 24 nt 20 Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) Cas12a Class II type V 5′ TYCV 20 nt 21 AsCpf1 RR variant Cas12a Class II type V 5′ TYCV 20 nt 21 LbCpf1 RR variant Cas12a Class II type V 5′ TATV 20 nt 22 AsCpf1 RVR variant NmCas9 Class II type II 3′ NNNNGATT 23, 24 nt 23 Neisseria meningitidis (NM) StCas9 Class II type II 3′ NNAGAAW 19 to 20 nt 24 Streptococcus thermophilus1 (ST) StCas9 Class II type II 3′ NGGNG 19 nt 25 Streptococcus thermophilus3 TdCas9 Class II type II 3′ NAAAAC 20 nt 26 Treponema denticola (TD) Cas13a (C2c2) Class II type VI N/A N/A Leptotrichia buccalis Cas13a (C2c2) Class II type VI N/A N/A Leptotrichia shahii N/A - Cas13a have not been used in mammalian cells. The functional target length and PAM site remains unclear. For PAM sites: N can be any base; R can be A or G; V can be A, C, or G; W can be A or T; and Y can be C or T.
[0122] By way of example and not limitation, the methods described herein can include the use of any of the Cas proteins from Tables 1 and 2 and their corresponding guide polynucleotide(s) (e.g., guide RNA(s)) or other compatible guide RNAs. As an example, and not intended to be limiting in any way, the Cas9 from Streptococcus thermophilus LMD-9 CRISPR1 system has been shown to function in human cells (see, e.g., Cong et al. (2013, supra)). Cas9 orthologs from N. meningitides, which are described, e.g., in Hou et al. (Proc Natl Acad Sci USA. 110(39): 15644-9, 2013) and Esvelt et al. (2013, supra), can also be used in the compositions and methods described herein.
[0123] Guide Polynucleotides
[0124] The featured CRISPR/Cas protein complexes of the methods and compositions can be guided to a target site (e.g., a target genomic site, such as the genomic site associated with or encoding the lncRNA LOUP, described herein) using a guide polynucleotide (e.g., gRNA). Generally speaking, gRNAs come in two different systems: System 1, which uses separate crRNA and tracrRNAs that function together to guide cleavage by a Cas nuclease (e.g., Cas9), and System 2, which uses a chimeric crRNA-tracrRNA hybrid that combines the two separate guide RNAs in a single system (referred to as a single guide RNA or sgRNA: see also, e.g., Jinek et al. (2012, supra)). For System 2, gRNAs can be complementary to a target site region that is within about 100-800 base pairs (bp) upstream of a transcription start site of a gene, (e.g., within about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 150 bp, about 100 bp, or about 50 bp upstream of the transcription start site), includes the transcription start site, or is within about 100-800 bp downstream of a transcription start site (e.g., within about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 150 bp, about 100 bp, or about 50 bp downstream of the transcription start site). In particular embodiments, the target site region is within about 200-600 bp (e.g., 550 bp, 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, or 200 bp) upstream of LOUP's transcription start site, and the target site region. In some embodiments, vectors (e.g., viral vectors (e.g., lentiviral vectors)) encoding more than one gRNA can be used, e.g., vectors encoding, 2, 3, 4, 5, or more gRNAs directed to different target sites or target genomic sites in the same region of the target nucleic acid molecule (e.g., a gene or other site on a chromosome). In some instances, the genomic target site and the target gene of interest are between 10-100,000 nucleotide base pairs apart (e.g., between 50-150, between 100-800 (e.g., between 125-200, between 175-300, between 275-400, between 375-500, between 475-600, between 575-700, and between 675-800), between 700-2000, between 1000-5000, between 4000-10000, between 9000-20000, between 19000-30000, between 25000-50000, between 45000-75000, or between 70000-100000).
[0125] CRISPR/Cas protein complexes can be guided to specific 17-25 nt target sites (e.g., genomic target sites) bearing an additional PAM (e.g., sequence NGG for Cas9), using a guide RNA (e.g., a single gRNA or a tracrRNA/crRNA) bearing 17-25 nts at its 5′ end that are complementary to the complementary strand of a target nucleic acid molecule (e.g., genomic DNA at a target genomic site). Thus, the gene editing system can include the use of a single guide RNA comprising a crRNA fused to a normally trans-encoded tracrRNA, e.g., a single Cas guide RNA (such as those described in Mali et al. (2013, supra)), with a sequence at the 5′ end that is complementary to the target sequence, e.g., of 17-25, optionally 20 or fewer nucleotides (nts), e.g., 20, 19, 18, or 17 nts, preferably 17 or 18 nts, of the complementary strand to a target sequence immediately 5′ of a PAM.
[0126] Existing Cas-based nucleases use gRNA-DNA heteroduplex formation to guide targeting to genomic sites of interest. However, RNA-DNA heteroduplexes can form a more promiscuous range of structures than their DNA-DNA counterparts. In effect, DNA-DNA duplexes are more sensitive to mismatches, suggesting that a DNA-guided nuclease may not bind as readily to off-target sequences, making them comparatively more specific than RNA-guided nucleases. Thus, the guide RNAs featured in the compositions and methods described herein can be hybrids, e.g., wherein one or more deoxyribonucleotides, e.g., a short DNA oligonucleotide, replaces all or part of the gRNA, e.g., all or part of the complementarity region of a gRNA. This DNA-based molecule could replace either all or part of the gRNA in a single gRNA system or alternatively might replace all of part of the crRNA and/or tracrRNA in a dual crRNA/tracrRNA system. Such a system that incorporates DNA into the complementarity region can be used to target, e.g., an intended genomic DNA site due to the general intolerance of DNA-DNA duplexes to mismatching as compared to RNA-DNA duplexes. Methods for making such duplexes are known in the art (see, e.g., Barker et al. (BMC Genomics 6: 57, 2005) and Sugimoto et al. (Biochemistry 39(37): 11270-81, 2000)).
[0127] A guide polynucleotide (e.g., a gRNA) can be any polynucleotide having a nucleic acid sequence with sufficient complementarity with the sequence of a target polynucleotide (e.g., a polynucleotide within about 800 bp (e.g., within about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 150 bp, about 100 bp, or about 50 bp) upstream of the transcription start site of LOUP), a polynucleotide that includes the transcription start site of LOUP, a polynucleotide that is within about 100-800 bp (e.g., within about 500 bp, about 400 bp, about 300 bp, about 200 bp, about 150 bp, about 100 bp, or about 50 bp) downstream of a transcription start site of LOUP, or a polynucleotide within LOUP), such that the guide polynucleotide can specifically hybridize with the target polynucleotide (e.g., a polynucleotide associate with LOUP) and direct sequence-specific binding of a featured CRISPR/Cas protein complex to the target site. In some embodiments, the guide polynucleotide (e.g., gRNA) includes a sequence of ˜5-75 nucleotides that are complementary to a corresponding sequence of SEQ ID NO: 1 (e.g., SEQ ID NOs: 112-115 and 122-125). In some embodiments, the degree of complementarity between the sequence of a guide polynucleotide and corresponding sequence of the target site (e.g., a target site associated with LOUP), when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAST, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide polynucleotide (e.g., a gRNA) has about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide polynucleotide (e.g., a gRNA) has fewer than about 75, 50, 45, 40, 35, 30, 25, 20, 15, or 12 nucleotides. The ability of a guide polynucleotide to direct sequence-specific binding of a CRISPR complex to a target site may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR/Cas complex, including the guide polynucleotide to be tested, may be provided to a host cell having the corresponding target site sequence, such as by transfection with vectors encoding the components of the CRISPR/Cas complex, followed by an assessment of preferential cleavage within the sequence of the target site, such as by the incorporation of a reporter gene (e.g., a nucleic acid encoding enhanced green fluorescent protein (eGFP), or a nucleic acid encoding mCherry), or followed by an assessment of preferential gene expression, which are further described in the examples. Similarly, cleavage of a target site polynucleotide may be evaluated in a test tube by providing the target site, components of the featured CRISPR/Cas complex, including the guide polynucleotide to be tested and a control guide polynucleotide different from the test guide polynucleotide, and comparing binding or rate of cleavage at the target site between the test and control guide polynucleotide reactions. Other assay methods known to those skilled in the art can also be used.
Delivery Methods
[0128] Vectors
[0129] In addition to achieving high rates of transcription and translation, stable expression of an exogenous gene in a mammalian cell can be achieved by integration of the polynucleotide containing the gene into the nuclear genome of the mammalian cell. A variety of vectors for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed. Expression vectors are well known in the art and include, but are not limited to, viral vectors and plasmids.
[0130] Vectors for use in the compositions and methods described herein contain at least one polynucleotide encoding a featured polynucleotide (e.g., a polynucleotide including a nucleic acid sequence with at least 20 nucleotides of SEQ ID NO: 1, and variants thereof with at least 85% sequence identity thereto), constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), a gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing system, or fragment thereof (e.g., a fragment that retains the ability to form a complex with a guide polynucleotide (e.g., a gRNA) at a target site or target genomic site), and at least one guide polynucleotide (e.g., a gRNA). The vectors may also provide additional sequence elements used for the expression of these agents and/or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used for the expression of the featured polynucleotides (e.g., polynucleotides including a nucleic acid sequence with at least 20 nucleotides of SEQ ID NO: 1, and variants thereof with at least 85% sequence identity thereto), constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), and gene editing systems (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct transcription of the nucleic acid molecules encoding the featured components described herein. Other useful vectors for expression of the featured polynucleotides (e.g., polynucleotides including a nucleic acid sequence with at least 20 nucleotides of SEQ ID NO: 1, and variants thereof with at least 85% sequence identity thereto), constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), and gene editing systems (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression include polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5′ and 3′ untranslated regions, and/or a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, and blasticidin.
[0131] In vectors encoding a featured construct, linking sequences can encode random amino acids or can contain functional sites (e.g., a cleavage site).
[0132] In some embodiments, a vector encoding a featured polynucleotide (e.g., a polynucleotide including a nucleic acid sequence with at least 20 nucleotides of SEQ ID NO: 1, and variants thereof with at least 85% sequence identity thereto), construct including the lncRNA (e.g., a construct including a protein linked to a LOUP polynucleotide, and/or gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression can be codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of, or derived from, a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis.
[0133] Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura et al. (Nucl. Acids Res. 28:292, 2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a featured polynucleotides, constructs, CRISPR/Cas systems, and/or a gRNA, correspond to the most frequently used codon for a particular amino acid.
[0134] Viral Delivery Vehicles
[0135] Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (e.g., a lentiviral vector, see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127), adenovirus vectors, alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus), Ross River virus, adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655), vaccinia virus (e.g., Modified Vaccinia virus Ankara (MVA) or fowlpox), Baculovirus recombinant system, and herpes virus. Further examples of viral vectors for delivery of the featured polynucleotides (e.g., a polynucleotide including a nucleic acid sequence with at least 20 (or all) nucleotides of the lncRNA LOUP (SEQ ID NO: 1), and variants thereof with at least 85% sequence identity thereto), constructs including the polynucleotide (e.g., constructs including a protein linked to a LOUP polynucleotide), and/or gene editing systems (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus, replication deficient herpes virus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology (Third Edition) Lippincott-Raven, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the entire contents of which is hereby incorporated by reference.
[0136] Exemplary viral vectors include lentiviral vectors, AAVs, and retroviral vectors. Lentiviral vectors and AAVs can integrate into the genome without cell divisions, and both types have been tested in pre-clinical animal studies.
[0137] Methods for preparation of AAVs are described in the art, e.g., in U.S. Pat. Nos. 5,677,158, 6,309,634, and 6,683,058, the entire contents of each of which is incorporated herein by reference.
[0138] Methods for preparation and in vivo administration of lentiviruses are described in US 20020037281, the entire contents of which is hereby incorporated by reference. Lentiviral vectors (LVs) transduce a wide range of dividing and non-dividing cell types with high efficiency, conferring stable, long-term expression of the transgene. An overview of optimization strategies for packaging and transducing LVs is provided in Delenda (J. Gen Med 6: S125, 2004), the entire contents of which are incorporated herein by reference.
[0139] The use of lentivirus-based gene transfer techniques relies on the in vitro production of recombinant lentiviral particles carrying a highly deleted viral genome in which the transgene of interest is accommodated. In particular, the recombinant lentivirus are recovered through the in trans coexpression in a permissive cell line of (1) the packaging constructs, i.e., a vector expressing the Gag-Pol precursors together with Rev (alternatively expressed in trans); (2) a vector expressing an envelope receptor, generally of an heterologous nature; and (3) the transfer vector, consisting in the viral cDNA deprived of all open reading frames, but maintaining the sequences required for replication, incapsidation, and expression, in which the sequences to be expressed are inserted.
[0140] Enhancer elements can be used to increase expression of modified DNA molecules or increase the lentiviral integration efficiency. The LV used in the methods and compositions described herein may include a nef sequence. The LV used in the methods and compositions described herein may include a cPPT sequence which enhances vector integration. The cPPT acts as a second origin of the (+)-strand DNA synthesis and introduces a partial strand overlap in the middle of its native HIV genome. The introduction of the cPPT sequence in the transfer vector backbone strongly increased the nuclear transport and the total amount of genome integrated into the DNA of target cells. The LV used in the methods and compositions described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE to LV results in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo. The LV used in the methods and compositions described herein may include both a cPPT sequence and Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) sequence. The vector may also include an IRES sequence that permits the expression of multiple polypeptides from a single promoter.
[0141] The vector used in the methods and compositions described herein may include multiple promoters that permit expression of more than one polynucleotide and/or polypeptide. The vector used in the methods and compositions described herein may include a protein cleavage site that allows expression of more than one polypeptide. Examples of protein cleavage sites that allow expression of more than one polypeptide are described in, e.g., Klump et al. (Gene Ther 8:811 2001), Osborn et al. (Molecular Therapy 12:569, 2005), Szymczak and Vignali (Expert Opin Biol Ther. 5:627, 2005), and Szymczak et al. (Nat Biotechnol. 22:589, 2004), the disclosures of which are incorporated herein by reference. It will be readily apparent to one skilled in the art that other elements that permit expression of multiple polypeptides identified in the future are useful and may be utilized in the vectors suitable for use with the compositions and methods described herein.
[0142] The vector used in the methods and compositions described herein may be a clinical grade vector.
[0143] The viral vector may also include viral regulatory elements, which are components of delivery vehicles used to introduce nucleic acid molecules into a host cell. The viral regulatory elements are optionally retroviral regulatory elements. For example, the viral regulatory elements may be the LTR and gag sequences from HSC1 or MSCV. The retroviral regulatory elements may be from lentiviruses or they may be heterologous sequences identified from other genomic regions. One skilled in the art would also appreciate that as other viral regulatory elements are identified, these may be used with the viral vectors described herein.
[0144] Non-Viral Delivery Vehicles
[0145] Several non-viral vehicles can be used for delivery of the featured polynucleotides (e.g., a polynucleotide having a nucleic acid sequence with at least 20 (or all) nucleotides of the lncRNA LOUP (SEQ ID NO: 1), and variants thereof with at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%), sequence identity thereto), constructs including the lncRNA (e.g., a construct including a protein linked to a LOUP polynucleotide), and a gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression. These non-viral vectors include, e.g., prokaryotic and eukaryotic vectors (e.g., yeast- and bacteria-based plasmids), as well as plasmids for expression in mammalian cells. Methods of introducing the vectors into a host cell and isolating and purifying the expressed protein are also well known in the art (e.g., Molecular Cloning: A Laboratory Manual, second edition, Sambrook, et al. 1989, Cold Spring Harbor Press). Examples of host cells include, but are not limited to, mammalian cells, such as NS0, CHO cells, HEK and COS, and bacterial cells, such as E. coli.
[0146] Other non-viral delivery vehicles include polymeric, biodegradable microparticle, or microcapsule delivery devices known in the art. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
[0147] The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, in particular cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
[0148] Lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidyl-ethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoyl-phosphatidylcholine. The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Additional methods are known in the art and are described, for example in U.S. Patent Application Publication No. 20060058255.
[0149] Pharmaceutical Compositions
[0150] The disclosure also includes pharmaceutical compositions containing a polynucleotide described herein (e.g., all or at least about 20 or more nucleotides of the long non-coding RNA, LOUP (SEQ ID NO: 1), and variants thereof with at least 85% or more sequence identity thereto, a polynucleotide encoding the lncRNA (e.g., a polynucleotide encoding at least 20 nucleotides of SEQ ID NO: 1), a vector (e.g., a viral vector) including the lncRNA or a polynucleotide encoding the lncRNA, a construct including the lncRNA (e.g., a construct including a protein linked to a LOUP polynucleotide), a gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, a polynucleotide encoding the gene editing system, and a vector (e.g., a viral vector) including polynucleotides encoding the gene editing system, as described herein. The pharmaceutical composition can be prepared as a composition containing a pharmaceutically acceptable carrier, excipient, or stabilizer known in the art (Remington: The Science and Practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover). The compositions may also be provided in the form of a lyophilized formulation, as an aqueous solution, or as a pharmaceutical product suitable for direct administration.
[0151] Acceptable carriers, excipients, or stabilizers that can be used to prepare a pharmaceutical composition are considered to be non-toxic to a recipient, e.g., when included in the composition at therapeutic dosages and concentrations, and may include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, marmose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.
[0152] The compositions (e.g., when used in the methods described herein) generally include, by way of example and not limitation, an effective amount (e.g., an amount sufficient to mitigate disease, alleviate a symptom of disease and/or prevent or reduce the progression of disease) of a long non-coding RNA (e.g., a LOUP RNA), a polynucleotide encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), a vector (e.g., a viral vector) including a polynucleotide encoding the lncRNA, a construct including the lncRNA (e.g., a construct including a protein linked to a LOUP polynucleotide), a gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, a polynucleotide encoding the gene editing system, and/or a vector (e.g., a viral vector) including polynucleotides encoding the gene editing system, as described herein.
[0153] The composition may be formulated to include between about 1 μg/mL and about 1 g/mL of the long non-coding RNA (e.g., LOUP RNA), the polynucleotide encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), the vector (e.g., a viral vector) including the polynucleotide encoding the lncRNA, the construct including the lncRNA (e.g., a construct including a protein linked to a LOUP polynucleotide), the gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, the polynucleotide encoding the gene editing systems, and/or the vector (e.g., a viral vector) including the polynucleotide(s) encoding the gene editing system, or any combination thereof (e.g., between 10 μg/mL and 300 μg/mL, 20 μg/mL and 120 μg/mL, 40 μg/mL and 200 μg/mL, 30 μg/mL and 150 μg/mL, 40 μg/mL and 100 μg/mL, 50 μg/mL and 80 μg/mL, or 60 μg/mL and 70 μg/mL, or 10 mg/mL and 300 mg/mL, 20 mg/mL and 120 mg/mL, 40 mg/mL and 200 mg/mL, 30 mg/mL and 150 mg/mL, 40 mg/mL and 100 mg/mL, 50 mg/mL and 80 mg/mL, 60 mg/mL and 70 mg/mL, or 100 mg/ml and 1 g/ml (e.g., 150 mg/ml, 200 mg/ml, 250 mg/ml, 300 mg/ml, 350 mg/ml, 400 mg/ml, 450 mg/ml, 500 mg/ml, 550 mg/ml, 600 mg/ml, 650 mg/ml, 700 mg/ml, 750 mg/ml, 800 mg/ml, 850 mg/ml, 900 mg/ml, or 950 mg/ml).
[0154] A composition containing a non-viral vector of the disclosure may contain a unit dose containing a quantity of long non-coding RNA (e.g., LOUP RNA), polynucleotides encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), vectors (e.g., viral vectors) including polynucleotides encoding the lncRNA, constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing systems, and vectors (e.g., viral vectors) including polynucleotides encoding the gene editing system from 10 μg to 10 mg (e.g., from 25 μg to 5.0 mg, from 50 μg to 2.0 mg, or from 100 μg to 1.0 mg of polynucleotides, e.g., from 10 μg to 20 μg, from 20 μg to 30 μg, from 30 μg to 40 μg, from 40 μg to 50 μg, from 50 μg to 75 μg, from 75 μg to 100 μg, from 100 μg to 200 μg, from 200 μg to 300 μg, from 300 μg to 400 μg, from 400 μg to 500 μg, from 500 μg to 1.0 mg, from 1.0 mg to 5.0 mg, or from 5.0 mg to 10 mg of polynucleotides, e.g., about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 750 μg, about 1.0 mg, about 2.0 mg, about 2.5 mg, about 5.0 mg, about 7.5 mg, or about 10 mg of polynucleotides). The long non-coding RNA (e.g., LOUP RNA), polynucleotides encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), vectors (e.g., viral vectors) including polynucleotides encoding the lncRNA, constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing systems, and vectors (e.g., viral vectors) including polynucleotides encoding the gene editing system may be formulated in the unit dose above in a volume of 0.1 ml to 10 ml (e.g., 0.2 ml, 0.5 ml, 0.75 ml, 1 ml, 1.5 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml).
[0155] The compositions may also include a viral vector containing a nucleic acid sequence encoding a featured polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1), constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), and gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing systems or a composition containing a featured polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1), constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), and gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing systems. The compositions containing viral particles can be prepared in 1 ml to 10 ml (e.g., 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml) aliquots, having a viral titer of at least about 1×10.sup.6 pfu/ml (plaque-forming unit/milliliter), and, in general, not exceeding 1×10.sup.11 pfu/ml. Thus, the composition may contain, for example, about 1×10.sup.6 pfu/ml, about 2×10.sup.6 pfu/ml, about 4×10.sup.6 pfu/ml, about 1×10.sup.7 pfu/ml, about 2×10.sup.7 pfu/ml, about 4×10.sup.7 pfu/ml, about 1×10.sup.8 pfu/ml, about 2×10.sup.8 pfu/ml, about 4×10.sup.8 pfu/ml, about 1×10.sup.9 pfu/ml, about 2×10.sup.9 pfu/ml, about 4×10.sup.9 pfu/ml, about 1×10.sup.10 pfu/ml, about 2×10.sup.10 pfu/ml, about 4×10.sup.10 pfu/ml, and about 1×10.sup.11 pfu/ml. The composition can also contain a pharmaceutically acceptable carrier described herein. The pharmaceutically acceptable carrier can be, for example, a liquid carrier such as a saline solution, protamine sulfate (Elkins-Sinn, Inc., Cherry Hill, N.J.) or Polybrene (Sigma) as well as others described herein.
[0156] Methods for Diagnosing a Subject as a LOUP-Related Disease or Disorder
[0157] Also provided herein are methods of diagnosing a disease or disorder (e.g., a cancer (e.g., AML, liver cancer, or myeloma), Alzheimer's disease, or asthma) in a subject (e.g., a subject suspected of having a disease or disorder). The diagnostic method can be performed by determining a level of the transcription factor PU.1 in a subject or a level of LOUP expression in a subject.
[0158] For example, a sample (e.g., a tissue sample, a blood sample, a cell sample, or a fluidic sample) can be obtained from a subject (e.g., a subject suspected of having a disease or disorder) and analyzed for PU.1 expression. The level of PU.1 expression can be compared to a standard or reference level (e.g., a control sample, in which a known expression level of PU.1 has been linked to the presence or absence of the disease or disorder) or to a sample from a reference subject (e.g., a subject known to be healthy (e.g., to lack the disease or disorder) or a subject known to have the disease or disorder). Comparison of the PU.1 level to the standard or reference level can confirm the presence or absence of the disease or disorder in the subject being tested.
[0159] For example, a subject determined to have decreased expression of PU.1, as compared to a standard or reference, can be identified as having or at risk of developing a cancer (e.g., AML, liver cancer, or myeloma). Alternatively, a subject determined to have increased expression of PU.1, as compared to a standard or reference, can be identified as having or at risk of developing Alzheimer's disease or asthma.
[0160] For example, a sample (e.g., a tissue sample, a blood sample, a cell sample, or a fluidic sample) can be obtained from a subject (e.g., a subject suspected of having a disease or disorder) and analyzed for LOUP expression. The level of LOUP expression can be compared to a standard or reference level (e.g., a control sample, in which a known expression level of LOUP has been linked to the presence or absence of the disease or disorder) or to a sample from a reference subject (e.g., a subject known to be healthy (e.g., to lack the disease or disorder) or a subject known to have the disease or disorder). Comparison of the LOUP level to the standard or reference level can confirm the presence or absence of the disease or disorder in the subject being tested.
[0161] For example, a subject determined to have decreased expression of LOUP, as compared to a standard or reference, can be identified as having or at risk of developing a cancer (e.g., AML, liver cancer, or myeloma). Alternatively, a subject determined to have increased expression of LOUP, as compared to a standard or reference, can be identified as having or at risk of developing Alzheimer's disease or asthma.
[0162] Also provided are methods of diagnosing a subject as having a cancer (e.g., AML) that is susceptible to differentiation therapy with all-trans retinoic acid (ATRA) based on LOUP expression. A sample (e.g., a tissue sample, a blood sample, a cell sample, or a fluidic sample) from a subject (e.g., a subject having or suspected of having a cancer (e.g., AML)) can be analyzed for LOUP expression and compared to a standard or reference level (e.g., a control sample, in which a known expression level of LOUP has been linked to the presence or absence of the disease or disorder) or to a sample from a reference subject (e.g., a subject known to be healthy (e.g., to lack the disease or disorder) or a subject known to have the disease or disorder). Comparison of the LOUP level to the standard or reference level can be used to determine if the subject is likely to be sensitive to differentiation therapy with ATRA. For example, low levels of LOUP (relative to a standard or reference) would indicate resistance of the cancer to ATRA therapy.
[0163] Gene sequencing methods (e.g., next-generation gene sequencing methods, e.g., high-throughput sequencing, including but not limited to, Illumina sequencing, Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiD sequencing) can be used to analyze PU.1 and/or LOUP expression for the diagnosis of a disease or disorder.
[0164] Methods of Treatment
[0165] A subject in need of treatment for a disease or disorder associated with reduced expression of the transcription factor PU.1 (e.g., a cancer, such as AML, liver cancer, or myeloma) can be administered a composition described herein that increases expression of PU.1. Alternatively, a subject in need of treatment for a disease or disorder associated with increased expression of the transcription factor PU.1 (e.g., Alzheimer's disease or asthma) can be administered a composition described herein that decreases expression of PU.1. Each of these methods are described below.
[0166] For treatment of a disease or disorder associated with reduced expression of PU.1, generally, a composition containing the featured polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1) can be administered (e.g., intravenously) to a subject (e.g., a subject in need thereof, such as a human) as a medicament (e.g., for treating a medical condition (e.g., a cancer (e.g., a PU.1 associated cancer (e.g., AML, liver cancer, or myeloma)))). The featured polynucleotide described herein can be used to induce the expression of tumor suppressor gene PU.1, thereby treating the disease or disorder. In some embodiments, the featured polynucleotide can be delivered as a vector (e.g., a viral vector or non-viral vector) described herein. In certain embodiments, the featured polynucleotide can be delivered as a vector including a nucleic acid encoding the featured polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1) as described herein. In some embodiments, the vector is a viral vector (e.g., a lentiviral vector or an AAV vector). Gene sequencing methods (e.g., next-generation gene sequencing methods, e.g., high-throughput sequencing, including but not limited to, Illumina sequencing, Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiD sequencing) can be used to identify a subject in need thereof (e.g., a subject with a PU.1 associated cancer (e.g., AML, liver cancer, or myeloma)).
[0167] Alternatively, or in addition, a composition containing the featured gene editing system can be administered (e.g., intravenously) to a subject (e.g., a subject in need thereof, such as a human) as a medicament (e.g., for treating a medical condition (e.g., a PU.1 associated medical condition (e.g., a PU.1 associated cancer (e.g., AML, liver cancer, or myeloma)), or asthma)). In some embodiments, a composition including the featured gene editing system can be administered (e.g., intravenously or intracranially) to a subject (e.g., a subject in need thereof, such as a human) as a medicament (e.g., for treating a medical condition (e.g., a PU.1 associated medical condition (e.g., Alzheimer's Disease). In some embodiments, a composition including the featured gene editing system can be administered to a subject (e.g., a subject in need thereof, such as a human) as a medicament (e.g., for treating a medical condition (e.g., a PU.1 associated medical condition (e.g., a PU.1 associated cancer (e.g., AML, liver cancer, or myeloma)), Alzheimer's Disease, or asthma)) by any method that allows the featured gene editing system to target a genomic site associated with PU.1 expression. The gene editing system described herein can be used to efficiently target any of a number of genomic sites associated with a medical condition (e.g., a PU.1 associated medical condition). Gene sequencing methods (e.g., next-generation gene sequencing methods, e.g., high-throughput sequencing, including but not limited to, Illumina sequencing, Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiD sequencing) can be used to identify PU.1 or LOUP expression, which can identify the subject as one in need of treatment. The gene sequencing data can also be used to identify a suitable target site(s) or target genomic site(s) to be targeted by a guide polynucleotide(s) (e.g., a guide RNA(s) directed to a target site associated with LOUP) so as to limit any effect at off target sites. Target sites and target genomic sites will, preferably, but not necessarily, be uniquely associated with LOUP (e.g., a unique target site directing the CRISPR/Cas system to LOUP as described herein), and to the Cas nuclease of the featured CRISPR/Cas system.
[0168] The featured long non-coding RNA (e.g., LOUP RNA), polynucleotides encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), vectors (e.g., viral vectors) including polynucleotides encoding the lncRNA, constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing systems, and vectors (e.g., viral vectors) including polynucleotides encoding the gene editing system can be administered to a subject in need thereof (e.g., a human) to alter (e.g., increase or decrease) the expression of tumor associated gene PU.1. Compositions and methods for delivering the featured polynucleotides (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1) and/or CRISPR/Cas system or CRISPRa components include, e.g., a vector (e.g., a viral vector, such as a lentiviral vector particle), and non-vector delivery vehicles (e.g., nanoparticles), as discussed above. For example, the featured polynucleotides and CRISPR/Cas system described herein may be formulated for and/or administered to a subject in need thereof (e.g., a subject who has been diagnosed with a medical condition associated with anti-tumor proliferating gene PU.1 (e.g., a cancer (e.g., AML, liver cancer, or myeloma), Alzheimer's disease, or asthma)) by a variety of routes, such as local administration at or near the site affected by the medical condition (e.g., injection near a cancer, direct administration to the central nervous system (CNS) (e.g., intracranial, intracerebral, intraventricular, intrathecal, intracisternal, or stereotactic administration) for treating a neurological medical condition, such as Alzheimer's disease), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, topical, and oral administration. The most suitable route for administration in any given case may depend on the particular subject, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the subject's age, body weight, sex, severity of the disease being treated, the subject's diet, and the subject's excretion rate. Compositions may be administered once, or more than once (e.g., once annually, twice annually, three times annually, bi-monthly, monthly). For local administration, the featured polynucleotides (e.g., polynucleotides encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), constructs including a LOUP polynucleotide, gene editing system (e.g., CRISPR/Cas system or CRISPRa), and featured viral vectors containing nucleic acid sequences encoding the featured polynucleotides, constructs, or gene editing system may be administered by any means that places the polynucleotides, constructs, or gene editing system in a desired location, including catheter, syringe, shunt, stent, or microcatheter, pump. The subject can be monitored for PU.1 expression after treatment. Methods of monitoring the expression of PU.1 are discussed further below. The dosing regimen may be adjusted based on the monitoring results to ensure a therapeutic response.
[0169] Generally, the methods can include administering a composition containing the polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1), a construct including a LOUP polynucleotide, or the gene editing system (e.g., a CRISPR/Cas system), either incorporated as a nucleic acid molecule (e.g., in a vector, such as a viral vector) encoding the polynucleotide, construct, or the components of the gene editing system (e.g., Cas protein and guide polynucleotides (e.g., guide RNA)) to a subject in need thereof. Alternatively, the methods can include administering the gene editing system in protein form (e.g., as a composition containing a Cas protein in combination with one or more guide polynucleotide(s) (e.g., gRNA(s))). The compositions can be administered (e.g., intravenously or intracranially) to a subject (e.g., a subject in need thereof) as a medicament for the treatment of a medical condition associated with PU.1 expression.
Dosage and Administration
[0170] The pharmaceutical compositions described herein can be administered to a subject (e.g., a human) in a variety of ways. For example, the pharmaceutical compositions may be formulated for and/or administered orally, buccally, sublingually, parenterally, intravenously, subcutaneously, intramedullary, intranasally, as a suppository, using a flash formulation, topically, intradermally, subcutaneously, via pulmonary delivery, via intra-arterial injection, ophthalmically, optically, intrathecally, or via a mucosal route.
[0171] A viral vector, such as a lentiviral vector, can be administered in an amount effective to produce a therapeutic effect in a subject. The exact dosage of viral particles to be administered is dependent on a variety of factors, including the age, weight, and sex of the subject to be treated, and the nature and extent of the disease or disorder to be treated. The viral particles can be administered as part of a preparation having a titer of viral vectors of at least 1×10.sup.6 pfu/ml (plaque-forming unit/milliliter), and in general not exceeding 1×10.sup.11 pfu/ml, in a volume between about 0.5 ml to about 10 ml (e.g., 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, or about 10 ml). Thus, the administered composition may contain, for example, about 1×10.sup.6 pfu/ml, about 2×10.sup.6 pfu/ml, about 4×10.sup.6 pfu/ml, about 1×10.sup.7 pfu/ml, about 2×10.sup.7 pfu/ml, about 4×10.sup.7 pfu/ml, about 1×10.sup.8 pfu/ml, about 2×10.sup.8 pfu/ml, about 4×10.sup.8 pfu/ml, about 1×10.sup.9 pfu/ml, about 2×10.sup.9 pfu/ml, about 4×10.sup.9 pfu/ml, about 1×10.sup.10 pfu/ml, about 2×10.sup.10 pfu/ml, about 4×10.sup.10 pfu/ml, and about 1×10.sup.11 pfu/ml. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
[0172] Any of the non-viral vectors of the present invention can be administered to a subject in a dosage from about 10 μg to about 10 mg of polynucleotides (e.g., from 25 μg to 5.0 mg, from 50 μg to 2.0 mg, or from 100 μg to 1.0 mg of polynucleotides, e.g., from 10 μg to 20 μg, from 20 μg to 30 μg, from 30 μg to 40 μg, from 40 μg to 50 μg, from 50 μg to 75 μg, from 75 μg to 100 μg, from 100 μg to 200 μg, from 200 μg to 300 μg, from 300 μg to 400 μg, from 400 μg to 500 μg, from 500 μg to 1.0 mg, from 1.0 mg to 5.0 mg, or from 5.0 mg to 10 mg of polynucleotides, e.g., about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 600 μg, about 700 μg, about 750 μg, about 1.0 mg, about 2.0 mg, about 2.5 mg, about 5.0 mg, about 7.5 mg, or about 10 mg of polynucleotides) in a volume of a pharmaceutically acceptable carrier between about 0.1 ml to about 10 ml (e.g., about 0.2 ml, about 0.5 ml, about 1 ml, about 1.5 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, or about 10 ml).
[0173] Additionally, auxiliary substances, such as wetting or emulsifying agents, biological buffering substances, surfactants, and the like, may be present in such vehicles. A biological buffer can be virtually any solution which is pharmacologically acceptable and which provides the formulation with the desired pH, e.g., a pH in the physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank's buffered saline, and the like.
[0174] In some embodiments, the method may also include a step of assessing the subject for successful alteration in PU.1 expression (e.g., an increase or decrease in PU.1 expression). In some embodiments, the subject in need of a treatment (e.g., a human subject having a disease or disorder associated with PU.1 expression) is monitored for alleviation of the symptoms of the disease or disorder (e.g., cancer (e.g., AML, liver cancer, or myeloma), Alzheimer's disease, or asthma). In these instances, the subject will be monitored for a reduction or decrease in the side effects of a disease or disorder, such as those described herein, or the risk or progression of the disease or disorder, may be relative to a subject who did not receive treatment, e.g., a control, a baseline, or a known control level or measurement. The reduction or decrease may be, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or about 100% relative to a subject who did not receive treatment or a control, baseline, or known control level or measurement, or may be a reduction in the number of days during which the subject experiences the disease or disorder or associated symptoms (e.g., a reduction of 1-30 days, 2-12 months, 2-5 years, or 6-12 years). The results of monitoring a subject's response to a treatment can be used to adjust the treatment regimen.
[0175] In certain embodiments, the gene editing system can be used to introduce a genetic mutation (e.g., a missense mutation, a nonsense mutation, an insertion, a deletion, a duplication, a frameshift mutation, or a repeat expansion) or a gene of interest (e.g., a LOUP gene) into a genome of a target cell. In these instances, the mutation may be inserted to treat (e.g., in a human) a disease or disorder (e.g., Alzheimer's Disease or asthma) in a subject in need thereof. In these instances, the subject (e.g., a human subject) can be monitored for a change in the disease or disorder (e.g., a change in the progression of the disease or disorder or in a lessening of etiologies of the disease or disorder in a subject that has been treated, or, alternatively, in the production or increase in the etiologies of a disease or disorder in a subject (e.g., a research animal) that has had one or more cells edited to replicate the disease or disorder). The changes can be monitored relative to a subject who did not receive the treatment or editing modification, e.g., a control, a baseline, or a known control level or measurement. The change may be, e.g., by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or about 100% relative to a subject who did not receive treatment or editing modification or a control, baseline, or known control level or measurement, or may be a change in the number of days during which the subject experiences the disease or disorder or associated symptoms (e.g., a reduction of 1-30 days, 2-12 months, 2-5 years, or 6-12 years in a treated subject).
[0176] In certain embodiments, the treatment is monitored at the protein level. Successful expression of the featured gene editing system in a cell or tissue can be assessed by standard immunological assays, for example the ELISA (see, Ausubel et al. Current Protocols in Molecular Biology, Greene Publishing Associates, New York, V. 1-3, 2000; Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, the entire contents of which is hereby incorporated by reference).
[0177] Alternatively, the biological activity of LOUP and/or PU.1 can be measured directly by the appropriate assay, for example, the assays provided herein. The skilled artisan would be able to select and successfully carry out the appropriate assay to assess the biological activity of the gene product of interest in a particular sample. Such assays (e.g., real time PCR (qPCR)) might require removing a sample (e.g., cells or tissue) from the subject to use in the assay. Expression of the featured polynucleotides (e.g., polynucleotides encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1)) or successful gene editing using a gene editing system (e.g., CRISPR/Cas system) for delivering the same, may be monitored by any of a variety of detection methods available in the art, such as those described herein. For example, gene sequencing methods can be used to identify the successful insertion of the polynucleotide encoding the features polynucleotides using the gene editing system described herein. The subsequent expression of the target gene molecule (e.g., LOUPor PU.1) can be monitored.
Kits
[0178] Also featured are kits containing any one or more of the polynucleotides (e.g., polynucleotides including at least 20 nucleotides of SEQ ID NO: 1), constructs including, e.g., a protein and a polynucleotide (e.g., a LOUP polynucleotide), CRISPR/Cas system elements, or vectors comprising one or more of the polynucleotides, constructs, or CRISPR/Cas system elements disclosed in the above methods and compositions. Kits of the invention include one or more containers comprising, for example, one or more of a featured polynucleotide (e.g., polynucleotides including at least 20 nucleotides of SEQ ID NO: 1), or fragment thereof, construct including the lncRNA (e.g., a construct including a protein linked to a LOUP polynucleotide), CRISPR/Cas system or component thereof, one or more guide polynucleotide(s) (e.g., gRNAs), and/or one or more containers with nucleic acids encoding one or more of the polynucleotides, constructs, or CRISPR/Cas systems or components thereof, such as, e.g., a vector containing the nucleic acid molecules (e.g., a viral vector, such as a lentiviral vector, an adenoviral vector, or an AAV vector), and, optionally, instructions for use in accordance with any of the methods described herein.
[0179] Generally, these instructions comprise a description of administration or instructions for performance of an assay (e.g., a LOUP or PU.1 expression assay). The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also envisioned.
[0180] The kits may be provided in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
EXAMPLES
[0181] The following examples are put forth to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.
[0182] The following examples discuss identification and uses of long non-coding RNA (e.g., LOUP RNA) and polynucleotides encoding the same. Also described are vectors (e.g., viral vectors) including polynucleotides encoding the lncRNA and use of a gene editing system (e.g., a CRISPR/Cas system) to regulate PU.1 expression. Finally, examples are provided showing methods of diagnosing, treating, or preventing a disease (e.g., cancer (e.g., PU.1 associated cancer (e.g., AML, liver cancer, and myeloma)), Alzheimer's Disease, or asthma) associated with LOUP and/or PU.1 expression, as well as methods of diagnosing treatment (e.g., ATRA) responsiveness in a subject with cancer (e.g., AML, liver disease, or myeloma).
Example 1. Experimental Model and Subject Details
[0183] Long-range enhancer-promoter interactions result in dynamic expression patterns of lineage genes. How these communications occur in specific cell types and at specific gene loci remain elusive. Here we investigate whether RNAs coordinate with transcription factors to drive lineage gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA- and DNA-interactions with RUNX1 protein (described below), we identified a long noncoding RNA (lncRNA) arising from the upstream region of the myeloid master regulator PU.1. This myeloid-specific and polyadenylated lncRNA acts as a transcriptional inducer of PU.1 by modulating the formation of an active chromatin loop at the PU.1 locus. The lncRNA utilizes embedded transposable element variants to bind and recruit RUNX1 to both the enhancer and the promoter, resulting in the formation of the enhancer-promoter complex. These findings provide mechanistic insight, highlighting the important role of the interplay between cell type-specific RNAs and transcription factors in lineage-gene activation.
[0184] Cell Lines and Cell Culture
[0185] U937, HL-60, K562, HEK293T, RAW 264.7, NB4, Jurkat, Kasumi-1 and THP-1 cells were obtained from American Type Culture Collection (ATCC). U937, HL-60, NB4, Jurkat, Kasumi-1 and K562 cells were cultured in RPMI-1640 supplemented with 10% (vol/vol) fetal bovine serum (FBS; Cellgro) and 1% penicillin-streptomycin. THP-1 cells were cultured in the same medium supplemented with 2-mercaptoethanol to a final concentration of 0.05 mM. HEK293T and RAW 264.7 cells were cultured in DMEM supplemented with 10% (vol/vol) FBS and 1% penicillin-streptomycin. All cells were grown at 37° C. in 5% (vol/vol) CO2 and humidified incubators.
[0186] Lentiviral Generation
[0187] Lentiviral particles were generated following our optimized protocol (Trinh et al., J. Cell. Sci. 128: 3055-3067, 2015). Briefly, HEK293T cells were plated overnight to reach 80-85% confluency on the next day. Cells were then co-transfected with viral expression vector plus packaging plasmids (pMD2.G and psPAX2, Addgene) using Lipofectamine 2000 (Life Technologies). At 48 h and 72 h thereafter, culture supernatants were collected and filtered through a 0.45-mm PVDF filter (Millipore). Viruses were further concentrated using PEG-it® Virus Precipitation Solution (System Biosciences).
[0188] Plasmid Generation
[0189] LOUP cDNA in pCMV-SPORT6 plasmid (Dharmacon) was sub-cloned into the lentiviral pCDH-MSCV-MCS-EF1-copGFP expression vector that carries copGFP marker (System Biosciences).
[0190] Generation of CRISPR Knockout Cells (CRISPRko)
[0191] FUCas9Cherry (Aubrey et al., Cell Rep. 10: 1422-1432, 2015) (Addgene) was used as expression vector to generate mCherry-Cas9 lentiviral particles as described above. U937 cells were transduced with these particles using TRANSDUX® reagent (System Biosciences). Cas9-stable cells were then selected by several rounds of FACS sorting for mCherry positivity. LOUP-targeting sgRNAs were designed using Cas-Designer (Park et al., Bioinformatics 31:4014-4016, 2015) and cloned into pLVx U6se EF1a sfPac vector which carry eGFP. To avoid disruption of the URE, known to be critical for PU.S1 induction (Li et al. Blood 98: 2958-2965, 2001), single-guide RNAs (sgRNA) targeting two distinct regions of the LOUPgene: (1) the LOUP intronic area downstream of the URE, and (2) the intronic area right upstream of the second exon of the LOUP gene (˜15 kb downstream from the URE) were designed. Cas9-stable cells were then transduced with eGFP-sgRNA lentiviruses. Cells expressing high levels of both eGFP and mCherry were FACS sorted, one cell per well, into 96-well plates. Genomic DNA from cell clones were isolated using DNeasy Blood & Tissue Kit kit (QIAGEN) and used for P2R amplifying CRISPR/Cas9 target sites. PfR products were sequenced and indel profile were analyzed by ICE software (Hsiau, et al. BioRxiv 251 082 2018). Cell clones having homozygous indels were verified by Sanger sequencing. Primer and sgRNA sequences are provided in Table 3.
TABLE-US-00003 TABLE 3 Primer and sgRNA sequences SEQ ID Function Oligo name Description Sequence 5′ to 3′ NO: LOUP RT-PCR hLOUP F forward primer for GGCTTCAGCCTCCCT SEQ ID to check exon human LOUP RT-PCR AGACT NO: 28 junctions hLOUP R reverse primer for CTGGTCAGCAGGAAA SEQ ID human LOUP RT-PCR TTGGT NO: 29 mLOUP F forward primer for GAAGGAACACAGGC SEQ ID mouse LOUP RT-PCR CTCTCC NO: 30 mLOUP R reverse primer for GAGACCATGCCAGTC SEQ ID mouse LOUP RT-PCR TGGTT NO: 31 Primers to hLOUP F forward primer for GGCTTCAGCCTCCCT SEQ ID clone LOUP human LOUP RT-PCR, AGACT NO: 28 fragments used spliced LOUP to generate hLOUP R reverse primer for CTGGTCAGCAGGAAA SEQ ID RNA standard human LOUP RT-PCR, TTGGT NO: 29 curve spliced LOUP hLOUP F forward primer for GGCTTCAGCCTCCCT SEQ ID human LOUP RT-PCR, AGACT NO: 28 unspliced LOUP hLOUP R1 reverse primer for TCACCACAGGAAGCA SEQ ID human LOUP RT-PCR, TGTGT NO: 32 unspliced LOUP Strand- hLOUP F forward primer for GGCTTCAGCCTCCCT SEQ ID specific human LOUP RT-PCR AGACT NO: 28 RT-PCR hLOUP R reverse primer for CTGGTCAGCAGGAAA SEQ ID human LOUP RT-PCR TTGGT NO: 29 Un-related F forward primer for GGCAGAGTTCTCCCT SEQ ID CEBPA-AS1 RT-PCR GTGC NO: 33 Un-related R reverse primer for GTGGAGTCGCCGATT SEQ ID CEBPA-AS1 RT-PCR TTT NO: 34 qPCR hLOUP F forward primer for GGCTTCAGCCTCCCT SEQ ID mature human LOUP AGACT NO: 28 hLOUP R reverse primer for CTGGTCAGCAGGAAA SEQ ID mature human LOUP TTGGT NO: 29 hLOUP F1 forward primer for GTGGGCTAGTCTGTG SEQ ID immature human LOUP GAAGG NO: 35 hLOUP R reverse primer for CTGGTCAGCAGGAAA SEQ ID immature human LOUP TTGGT NO: 29 mLOUP F forward primer for GAAGGAACACAGGC SEQ ID mouse mature LOUP CTCTCC NO: 30 mLOUP R reverse primer for TTTCTGGCCTTGAAC SEQ ID mouse mature LOUP TGACA NO: 31 mLOUP F1 forward primer for CCACGAGACACTATC SEQ ID mouse LOUP CAGCA NO: 36 mLOUP R1 reverse primer for GAGACCATGCCAGTC SEQ ID mouse LOUP TGGTT NO: 31 hMALAT1F forward primer for GGTCTTTGGTGGGTT SEQ ID human MALAT1 GAACT NO: 37 hMALAT1R reverse primer for TTCCCACCCAGCATT SEQ ID human MALAT1 ACAGT NO: 38 mMALAT1F forward primer for GGTCTTTGGTGGGTT SEQ ID mouse MALAT1 GAACT NO: 37 mMALAT1R reverse primer for TTCCCACCCAGCATT SEQ ID mouse MALAT1 ACAGT NO: 38 RPPH1 F forward primer for CTAACAGGGCTCTCC SEQ ID human RPPH1 CTGAG NO: 39 RPPH1 R reverse primer for CAGCCATTGAACTCA SEQ ID human RPPH1 CTTCG NO: 40 mRPS18 F forward primer for CGGAAAATAGCCTTC SEQ ID mouse RPS18 GCCATCAC NO: 41 mRPS18 R reverse primer for ATCACTCGCTCCACC SEQ ID mouse RPS18 TCATCCT NO: 42 hPU.1 F forward primer for TGTTACAGGCGTGCA SEQ ID human PU.1 AAATGG NO: 43 hPU.1 R reverse primer for TGCGTTTGGCGTTGG SEQ ID human PU.1 TATAGA NO: 44 mm00488140_ Taqman set for mouse www.thermofisher.com/ m1 Spil Pu.1 (Purchased from taqman-gene- ThermoFisher) expression/product/Mm 00488140_m1?CID=&I CID=&subtype= GAPDH F forward primer for GTCTCCTCTGACTTC SEQ ID human GAPDH AACAGCG NO: 45 GAPDH R reverse primer for ACCACCCTGTTGCTG SEQ ID human GAPDH TAGCCAA NO: 46 URE F_3C forward primer for 3C GTGTCTGCTCCCTAG SEQ ID qPCR CTCCA NO: 47 Taqman_3C Taqman probe for 3C ATGGCGTGTGGTCAC SEQ ID qPCR CCAGA NO: 48 -8K R_3C reverse primer for GACAGTGCTACATGG SEQ ID measuring interaction GTGTGA NO: 49 with the -8K region by 3C Taqman qPCR -4K R_3C reverse primer for CTTTGGAGAGTCCCA SEQ ID measuring interaction AGTGC NO: 50 with the -4K region by 3C Taqman qPCR PrPr R_3C reverse primer for GAGCCATAGCGGTG SEQ ID measuring interaction AGTACG NO: 51 with the PrPr region by 3C Taqman qPCR Intergenic R_3C reverse primer for TTCTCCCTGGAGAGA SEQ ID measuring interaction CCTCA NO: 52 with intergenic region by 3C Taqman qPCR MYBPC3 R_3C reverse primer for GGTGTGCACCACCAT SEQ ID measuring interaction ACTTG NO: 53 with MYBPC3 gene by 3C Taqman qPCR URE F forward primer for GCCATGAAATGCTCT SEQ ID detecting URE by ChIRP GCTCT NO: 54 URE R reverse primer for CCTAGCCCTTGGAAG SEQ ID detecting URE by ChIRP GAGAC NO: 55 PrPr F forward primer for CAGCCCTTTGAGCAC SEQ ID detecting PrPr by ChIRP CAC NO: 56 PrPr R reverse primer for GAAGGGCCTGCCGC SEQ ID detecting PrPr by ChIRP TGGGAGATAG NO: 57 ACTBpro_F forward primer for AAAGGCAACTTTCGG SEQ ID detecting ACTB AACGG NO: 58 promoter by ChIRP ACTBpro_R reverse primer for TTCCTCAATCTCGCT SEQ ID detecting ACTB CTCGC NO: 59 promoter by ChIRP LOUP fRIP F1 forward primer for LOUP GGAGCCCCTTGAATC SEQ ID fRIP qPCR, amplicon #1 TTAGG NO: 60 LOUP fRIP R1 reverse primer for LOUP AAAGCAGGACAGGA SEQ ID fRIP qPCR, amplicon #1 AAGCAA NO: 61 LOUP fRIP F2 forward primer for LOUP CAGGTGGCACACATC SEQ ID fRIP qPCR, amplicon #2 CATAG NO: 62 LOUP fRIP R2 reverse primer for LOUP CATGCTTGGCCAGTT SEQ ID fRIP qPCR, amplicon #2 CTTTT NO: 63 LOUP fRIP F3 forward primer for LOUP TCAACAGATGGCTGT SEQ ID fRIP qPCR, amplicon #3 CTTGG NO: 64 LOUP fRIP R3 reverse primer for LOUP TCAGAAGCCTCATCC SEQ ID fRIP qPCR, amplicon #3 CCTTA NO: 65 URE ChIP F forward primer for URE CTGTGGTAATGGGCT SEQ ID ChIP qPCR GTTGG NO: 66 URE ChIP R reverse primer for URE CTCTGGGCAGGGTC SEQ ID ChIP qPCR ACAG NO: 67 PrPr ChIP F forward primer for PrPr GGCTGACTCCAGAAA SEQ ID ChIP qPCR GTGGA NO: 68 PrPr ChIP R reverse primer for PrPr GGGAGAACGTGTAG SEQ ID ChIP qPCR CTCTGC NO: 69 GD ChIP F forward primer for GENE GGCTAATCCTCTATG SEQ ID DESERT ChIP qPCR GGAGTCTGTC NO: 70 GD ChIP R reverse primer for GENE CCAGGTGCTCAAGGT SEQ ID DESERT ChIP qPCR CAACATC NO: 71 Identification P5_1F P5-splinkerette adapter AATGATACGGCGACC SEQ ID of ACCGAGATCTACACT NO: 72 5′ End of LOUP CTTTCCCTACACGAC trancript GCTCTTCCGATCT P5_2F P5 primer AATGATACGGCGACC SEQ ID ACCGAGATCT NO: 73 hLOUP R LOUP-specific nested CTGGTCAGCAGGAAA SEQ ID primer #1 TTGGT NO: 29 hLOUP R1 LOUP-specific nested CTGGTCAGCAGGAAA SEQ ID primer #2 TTGGT NO: 29 3′ RACE dTA_A Oligo dT-Anchor Primer GACCACGCGTATCGA SEQ ID Primers mix #1 TGTCGACTTTTTTTTT NO: 74 TTTTTTTA dTA_C Oligo dT-Anchor Primer GACCACGCGTATCGA SEQ ID mix #2 TGTCGACTTTTTTTTT NO: 75 TTTTTTTC dTA_G Oligo dT-Anchor Primer GACCACGCGTATCGA SEQ ID mix #3 TGTCGACTTTTTTTTT NO: 76 TTTTTTTG hLOUP F forward primer #1 for GGCTTCAGCCTCCCT SEQ ID LOUP 3′ RACE AGACT NO: 28 hLOUP F_a forward primer #2 for CTGTCTCCTTCCAAG SEQ ID LOUP 3′ RACE GGCTA NO: 77 hLOUP F_b forward primer #3 for CAGGTGGCACACATC SEQ ID LOUP 3′ RACE CATAG NO: 62 Anchor R Anchor reverse primer GACCACGCGTATCGA SEQ ID TGTCGAC NO: 78 ChIRP probes LOUP_01 LOUP-tiling oligo AAGGAGACAGGAGT SEQ ID CTAGGG/3BioTEG NO: 79 LOUP_02 LOUP-tiling oligo TCTGGTCAGCAGGAA SEQ ID ATTG/3BioTEG NO: 80 LOUP_03 LOUP-tiling oligo CAGAGCAAAAGAGG SEQ ID GGCAGA/3BioTEG NO: 81 LOUP_04 LOUP-tiling oligo AGAGGAGGGACAAC SEQ ID GAGGAG/3BioTEG NO: 82 LOUP_05 LOUP-tiling oligo CAGGACAAGAGGTG SEQ ID AGGAGG/3BioTEG NO: 83 LOUP_06 LOUP-tiling oligo GATCTCACATCACCA SEQ ID AGACA/3BioTEG NO: 84 LOUP_07 LOUP-tiling oligo CGGTTTGGTAATCCA SEQ ID TAACC/3BioTEG NO: 85 LOUP_08 LOUP-tiling oligo AGTACATCAGAAGCC SEQ ID TCATC/3BioTEG NO: 86 LOUP_09 LOUP-tiling oligo AGGGTCAATAACCTC SEQ ID TGGA/3BioTEG NO: 87 LOUP_10 LOUP-tiling oligo GCTCCAGGAGAAGG SEQ ID AAGATA/3BioTEG NO: 88 LOUP_11 LOUP-tiling oligo TGCTGGTTGTAAGCA SEQ ID AGGA/3BioTEG NO: 89 LOUP_12 LOUP-tiling oligo GCAAAGCAGGACAG SEQ ID GAAAGC/3BioTEG NO: 90 LOUP_13 LOUP-tiling oligo GAAAGCATGTCTGGC SEQ ID TGAG/3BioTEG NO: 91 LOUP_14 LOUP-tiling oligo GGTACACTTGGTCTC SEQ ID AAAG/3BioTEG NO: 92 LacZ_01 LacZ-tiling oligo CCAGTGAATCCGTAA SEQ ID TCATG/3BioTEG NO: 93 LacZ_02 LacZ-tiling oligo GTAGCCAGCTTTCAT SEQ ID CAACA/3BioTEG NO: 94 LacZ_03 LacZ-tiling oligo ATCTTCCAGATAACT SEQ ID GCCGT/3BioTEG NO: 95 LacZ_04 LacZ-tiling oligo ATAATTTCACCGCCG SEQ ID AAAGG/3BioTEG NO: 96 LacZ_05 LacZ-tiling oligo TTCATCAGCAGGATA SEQ ID TCCTG/3BioTEG NO: 97 LacZ_06 LacZ-tiling oligo TGATCACACTCGGGT SEQ ID GATTA/3BioTEG NO: 98 LacZ_07 LacZ-tiling oligo AAACGGGGATACTGA SEQ ID CGAAA/3BioTEG NO: 99 LacZ_08 LacZ-tiling oligo GTTATCGCTATGACG SEQ ID GAACA/3BioTEG NO: 100 LacZ_09 LacZ-tiling oligo TGTGAAAGAAAGCCT SEQ ID GACTG/3BioTEG NO: 101 LacZ 10 LacZ-tiling oligo GTAATCGCCATTTGA SEQ ID CCACT/3BioTEG NO: 102 DNA pull-down URE Runx1 wt F forward URE oligo [Btn]AGGGTGTGGCA SEQ ID assay containing Runx1 GGTGTGGACGT NO: 103 wildtype binding site URE Runx1 wt reverse URE oligo ACGTCCACACCTGCC SEQ ID R containing Runx1 ACACCCT NO: 104 wildtype binding site URE Runx1 mt forward URE oligo [Btn]AGGCTCTCACAG SEQ ID F containing Runx1 mutant CTCTCAACGT NO: 105 binding site URE Runx1 mt reverse URE oligo ACGTTGAGAGCTGTG SEQ ID R containing Runx1 mutant AGAGCCT NO: 106 binding site PrPr Runx1 wt F forward PrPr oligo [Btn]CAGTGGTGTGG SEQ ID containing Runx1 CAGAGCTAC NO: 107 wildtype binding site PrPr Runx1 wt R reverse PrPr oligo GTAGCTCTGCCACAC SEQ ID containing Runx1 CACTG NO: 108 wildtype binding site PrPr Runx1 mt F forward PrPr oligo [Btn]CAGTGCTCTCAC SEQ ID containing Runx1 mutant AGAGCTAC NO: 109 binding site PrPr Runx1 mt reverse PrPr oligo GTAGCTCTGTGAGAG SEQ ID R containing Runx1 mutant CACTG NO: 110 binding site Northern blot hLOUP F forward primer for GGCTTCAGCCTCCCT SEQ ID probe northern blot probe of AGACT NO: 28 LOUP hLOUP R reverse primer for CTGGTCAGCAGGAAA SEQ ID northern blot probe of TTGGT NO: 29 LOUP Oligos for #D1 sgRNA fwd forward single guide CACCGCAGGTGGTC SEQ ID cloning sgRNA RNA sequence insert for TCAGAGGTCGG NO: 111 into LOUP #D1 sgRNA CRISPR/Cas9 #D1 sgRNA rev reverse single guide AAACCCGACCTCTGA SEQ ID plasmids RNA sequence insert for GACCACCTGC NO: 112 LOUP #D1 sgRNA #D2 sgRNA fwd forward single guide CACCgCACAAGATCA SEQ ID RNA sequence insert for GGTAACAAGT NO: 113 LOUP #D2 sgRNA #D2 sgRNA rev reverse single guide AAACACTTGTTACCT SEQ ID RNA sequence insert for GATCTTGTGC NO: 114 LOUP #D2 sgRNA **control forward single guide AAACCCCACCAATAT SEQ ID sgRNA fwd RNA sequence insert for CAGTAATACC NO: 115 CRISPR/Cas9 non- targeting control **control reverse single guide AAACCCCACCAATAT SEQ ID sgRNA rev RNA sequence insert for CAGTAATACC NO: 116 CRISPR/Cas9 non- targeting control TA strata #D1 LOUP TA forward primer to amplify GAGCTGAGAGCCCA SEQ ID cloning of fwd amplicon containing #D1 GAAGAA NO: 117 LOUP LOUP CRISPR/Cas9 CRISPR/Cas9 target site targeted #D1 LOUP TA reverse primer to amplify CTCGGCCTTCTCGCA SEQ ID alleles rev amplicon containing #D1 AAGA NO: 118 LOUP CRISPR/Cas9 target site #D2 LOUP TA forward primer to amplify GACAGTGCTACATGG SEQ ID fwd amplicon containing #D2 GTGTGA NO: 119 LOUP CRISPR/Cas9 target site #D2 LOUP TA reverse primer to amplify AGGGACAACGAGGA SEQ ID rev amplicon containing #D2 GGTTTT NO: 120 LOUP CRISPR/Cas9 target site Oligos for #A1 sgRNA fwd single guide RNA CACCGAGAACTCCTA SEQ ID cloning sgRNA sequence insert for GCGGGACACT NO: 121 into CRISPR/dCas9-VP64 CRISPR/dCas9- targeting LOUP VP64 promoter region #A1 sgRNA rev single guide RNA AAACAGTGTCCCGCT SEQ ID sequence insert for AGGAGTTCTC NO: 122 CRISPR/dCas9-VP64 targeting LOUP promoter region #A2 sgRNA fwd single guide RNA CACCGATGGCTGAG SEQ ID sequence insert for GTTGATGGTTG NO: 123 CRISPR/dCas9-VP64 targeting LOUP promoter region #A2 sgRNA rev single guide RNA AAACCAACCATCAAC SEQ ID sequence insert for CTCAGCCATC NO: 124 CRISPR/dCas9-VP64 targeting LOUP promoter region Oligos for Sp6R1 fwd forward primer to amplify AATTTAGGTGACACT SEQ ID cloning LOUP LOUP R1-S ATAGAACTACAGGTG NO: 125 fragments into GCACACATCCA pSCAmpKan to R1 rv reverse primer to amplify GCTGGAGTGCAATG SEQ ID use in RNAP LOUP R1-S GCGTGATC NO: 126 assays Sp6R1-AS fwd forward primer to amplify AATTTAGGTGACACT SEQ ID LOUP R1-AS ATAGATCTTGGCCCA NO: 127 CTGTAGCCT R1-AS rev reverse primer to amplify ACTACAGGTGGCACA SEQ ID LOUP R1-AS CATCCAT NO: 128 Sp6R2 fwd forward primer to amplify AATTTAGGTGACACT SEQ ID LOUP R2-S ATAGAATACAATAAT NO: 129 TAGCTGGGCGTG R2 rv reverse primer to amplify GTTTCGCTCTTGTTG SEQ ID LOUP R2-S CCCAGGCTGG NO: 130 RR fwd forward primer to amplify AATTTAGGTGACACT SEQ ID LOUP RR ATAGAACAACCTCTA NO: 131 CGGAAAAGAGTATG RR rev reverse primer to amplify CCTTTCTTCTTTTCTC SEQ ID LOUP RR TCTTTTTCTTTTTC NO: 132 Italic amino acid residues are 5′ overhangs for cloning into CRISPR/Cas9 plasmids (pLVx U6se EF1a sfPac); **Addgene control oligo sequence www.addgene.org/80248/; Underlined amino acid residues are 5′ overhangs for cloning into CRISR/dCas9 plasmids (pXPR_502); Bold amino acid residues are 5′ overhangs containing sp6 promoter for in vitro transcription
[0192] Generation of CRISPR Activation Cells (CRISPRa)
[0193] sgRNAs targeting the 500 bp upstream region of LOUP's transcriptional start site were designed using Cas-Designer (Park et al., 2015, supra). The sgRNAs were then cloned into the pXR502 plasmid as previously described (Ran et al., Nat. Protoc. 8: 2281-2308, 2013). K562 cells stably expressing dCas9-VP64 were generated via lentiviral delivery of dCas9-VP64-Blast (Konermann et al., Nature 517: 583-588, 2015) and Blasticidin selection. dCas9-VP64 stable cells were transduced with lentiviruses that package the sgRNA-cloned pXR502 plasmids as previously described (Ran et al., 2013, supra). After one-day post-transduction, cells were selected with puromycin for 2-3 days before collection for analysis.
Method Details
[0194] Plasmid Transfections
[0195] K562 cells, in exponential growth, were electroporated with expression plasmids using program T16, kit V (Lonza). Electroporated cells were incubated at 37° C. overnight in a 5% CO2 incubator. The next day, cells were changed to fresh medium. Cells were harvested at 48 h after electroporation.
[0196] Cellular Fractionation, RNA Extraction, RT-PCR and qPCR Analysis
[0197] Cultured cells were washed with Phosphate-buffered saline (PBS). Total RNA was extracted with Trizol reagent (Invitrogen) or PURELINK™ RNA Mini Kit (Ambion) and treated with RNase-free DNase I (Roche) to remove contaminated genomic DNA. polyA− and polyA+ RNAs were isolated from total RNA using Poly(A)PURIST™ MAG Kit (Ambion) following manufactural procedure. Isolation of RNA from subcellular fractions was performed as previously described (Lee et al., Cell 164: 69-80, 2016) with modifications. Briefly, cells were lysed in cytosolic lysis solution (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5% NP40, 1 mM DTT plus protease and RNase inhibitors) for 10 min on ice. After centrifugation, the supernatant was collected as the cytoplasmic fraction for cytosol RNA isolation. After washing in cytosolic lysis solution, nuclear pellet was used for nuclear RNA isolation. To collect nucleoplasm and chromatin fractions, nuclear pellet was further lysed with nuclear lysis solution (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 450 nM NaCl, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, plus protease and RNase inhibitors). After centrifugation, nuclear-soluble fraction (nucleoplasm) was collected as supernatant and chromatin-associated fraction was collected as pellet. RNAs from collected fractions were extracted with Trizol reagent and treated with RNase-free DNase I (Roche).
[0198] For RT-PCR, RNA was reverse-transcribed by using SuperScript® III Reverse Transcriptase (Invitrogen). Red Taq Pro Complete (Denville Scientific) was used to amplify designated amplicons. For qPCR assays, cDNA was generated by QuantiTect Rev. Transcription Kit (Qiagen) which also includes additional DNA contamination removal. iQ SYBR Green Supermix (Biorad) was used for PCR quantitation in a RotorGene cycler (Corbett). Relative quantification was performed using the ddCt method. To calculate LOUP transcript numbers per cell, LOUP DNA fragments amplified by RT-PCR from HL-60 cDNA were cloned into pSCAmpKan plasmid (Agilent). LOUP RNA fragments were in vitro-transcribed by using MAXIscript™ Transcription Kit (Ambion). The RNA fragments were used to generate a standard curve for absolute quantification in qRT-PCR assays.
[0199] Fluorescence-Activated Cell Sorting and Analysis
[0200] Cell populations were isolated for RNA extraction as previously described (Zhang et al., Cancer Cell 24: 575-588, 2013). Briefly, mononuclear cells were isolated bone marrow, spleen and peripheral blood after lysing red blood cell with ACK lysis buffer (Zhang et al., Immunity 21: 853-863, 2004). Single cell suspension was stained with fluorochrome-conjugated antibodies (Biolegend and eBioscience) and FACS-sorted based on the following markers. LT-HSC: Lin-c-Kit+Sca-1+CD150+CD48−; ST-HSC: Lin-c-Kit+Sca-1+CD150−CD48+; LMPP: Lin-c-Kit+Sca-1+CD34+Flt3+; MEP: Lin-c-Kit+Sca-1-CD34−CD16/32−; CMP: Lin-c-Kit+Sca-1-CD34+CD16/32−; GMP: Lin-c-Kit+Sca-1-CD34+CD16/32+; Mac/Gr1:Mac1+Gr1+.
[0201] Myeloid surface marker staining and FACS analysis were performed following previously described procedure (Mueller et al., Blood 107: 3330-3338, 2006). Cells were stained with PACBLUE-CD11b (BioLegend). Stained cells were analyzed using LSRII flow cytometer (BD Biosciences) and FlowJo software (Tree Star).
[0202] Transcript Mapping by P5-Linker Ligation and 3′ RACE
[0203] The 5′ end of LOUP transcript was identified using P5-linker ligation method as described previously (Melo et al., Mol. Cell 49: 524-535, 2013). Briefly, single-stranded cDNAs were generated from HL-60 polyA+ RNA by using SuperScript III reverse transcriptase (Life Technologies) with LOUP-specific nested primer #1. Double-strand cDNAs were then synthesized from single-stranded cDNA using SUPERSCRIPT™ Double-Stranded cDNA Synthesis Kit (Life Technologies) and blunt-ended by NEBNext End Repair Enzym Module (New England Biolabs). After purification, these cDNAs were ligated with P5-splinkerette adapter and purified. All purification steps were done by using QiAquick PCR Purification Kit (QIAGEN). Ligated products were then purified and used as templates for PCR with P5 primer and LOUP-specific nested primers #1 and #2 with Phusion Hot Start DNA polymerase (Finnzymes). P5-linker ligation products were gel purified using QIAgen Gel Extraction Kit (QIAGEN) and sub-cloned into pSCAmpKan vector and transformed into competent bacteria using StrataClone Blunt PCR Cloning Kit (Agilent). 3′RACE assay was performed using 2nd Generation 5/3′ RACE Kit (Roche) according to manufacturer's instruction. Briefly, cDNA was generated from HL-60 polyA.sub.+ RNA using oligo dT-anchor primer mix. Overlapping RACE products were then amplified from cDNA using anchor primer and LOUP-specific primers. RACE products were sub-cloned into pSCAmpKan vector and transformed into competent bacteria using StrataClone Cloning Kit (Agilent). Plasmids containing p5-linker and RACE products were purified from bacteria, sequenced, and assembled.
[0204] Northern Blotting
[0205] 10 ug polyA− and polyA+ RNAs were dissolved and heat denatured in sample buffer containing formamide, MOPS and formaldehyde. Denatured RNAs were separated on a 1% denaturing agarose gel containing formaldehyde, MOPS and EtBr and transferred to Brightstar-plus positively charged nylon membrane (Life Technologies). LOUP probe was PCR amplified with primers described in Table 3 (Northern blot probe). PCR product was sub-cloned into cloned into pSCAmpKan vector using StrataClone PCR Cloning Kit (Agilent). Probe sequence was verified by Sanger sequencing. Probe was released from the vector by restriction enzyme digestion and gene purification. Probe was radiolabeled using the Random Primed DNA Labeling Kit (Roche). Northern blot was performed with EXPRESSHYB™ Hybridization Solution (Clontech) following manufacture protocol
[0206] Quantitative Chromosome Conformation Capture (3C-qPCR)
[0207] 3C-qPCR experiments were performed by adapting described methods (Deng and Blobel, Methods Mol. Biol. 1468: 51-62, 2017; Hagege et al., Nat. Protoc. 2: 1722-1733, 2007; Staber et al., 2013, supra). Briefly, 1×10.sup.6 cells were crosslinked using 1% formaldehyde in PBS at room temperature for 10 min. Crosslinking reaction was stopped by adding 0.125 M Glycine and incubated for 5 min at room temperature followed by 15 min on ice. Crosslinked cells were then washed with ice-cold PBS and lysed in 3C lysis buffer (10 mM Tris-HCl, pH 8.0; 10 mM NaCl; Igepal CA-630 0.2% (vol/vol); 1× protease inhibitor cocktail (Sigma)) with 15 Dounce homogenizer strokes. After centrifugation, nuclear pellets were washed in 1× restriction enzyme buffer before being lysed with 0.1% SDS in 1× restriction enzyme buffer at 65° C. for 10 min. After incubation, chromatin solution was supplemented with 1% Triton X-100 and digested by ApoI restriction enzyme (New England Biolabs) at 37° C. overnight with rotation. The following day, 1.5% SDS was added to the reaction and enzyme activity was inhibited by incubating at 65° C. for 30 min. Nearby DNA ends of digested chromatin were joined by T4-ligase (New England Biolabs) at 16° C. for 2 h. Bound proteins including histones were removed by proteinase K at 65° C. overnight. DNA library were extracted by phenol/chloroform using phase-lock gel tubes (SPRIME) and ethanol precipitation. RNA was removed by incubating 3C libraries with RNase A (Lucigen) at 37° C. for 15 min. TaqMan real-time PCR quantifications of ligation products were performed, using primers and probes as documented in Table 3.
[0208] Chromatin Isolation by RNA Purification (ChIRP)
[0209] ChIRP assays were performed as described (Chu et al., J. Vis. Exp. 25(61): pii: 3912, Trimarchi et al., Cell 158: 893-606, 2014) with additional modifications. Briefly, to preserve RNA-Chromatin interactions, cells were first crosslinked with 2 mM EGS at room temperature for 45 washing cells with ice-cold PBS, cells were further crosslinked with 3% paraformaldehyde for 15 min at room temperature after ice-cold PBS washing. The crosslinking reaction was quenched with 0.125 M glycine for 5 min at room temperature. Crosslinked cells were washed in ice-cold PBS and lysed in sonication buffer (20 mM Tris pH 8, 150 mM NaCl, 0.1% SDS, 1% Triton-X, 2 mM EDTA, 1 mM PMSF) supplemented with COMPLETE™, Mini Protease Inhibitor Cocktail (Sigma-Aldrich) and SUPERase In RNase Inhibitor (Invitrogen). After sonication and centrifugation, supernatant containing sheared chromatin was collected and incubated with biotinylated anti-sense DNA tiling probes in hybridization buffer (750 mM NaCl, 1% Triton, 0.1% SDS, 50 mM Tris-CI pH 7.0, 1 mM EDTA, 15% formamide, 1 mM PMSF) supplemented with COMPLETE™, Mini Protease Inhibitor Cocktail and SUPERase In RNase Inhibitor. Hybridized chromatin fragments were captured using DYNABEADS™ MYONE™ Streptavidin C1 (Invitrogen). From the isolated chromatin pellet, chromatin-bound RNA was extracted by Trizol reagent to quantitate chromatin-bound LOUP by RT-qPCR, and DNA was isolated to quantitate enrichment of the URE and the PrPr by qPCR. Probes used in the ChIRP assay were designed by using the online probe designer at singlemoleculefish.com and are listed in Table 3 (ChIRP probes).
[0210] DNA Pull-Down Assay (DNAP)
[0211] DNAP was performed as described previously with minor modifications (Trinh et al., Oncogene 30: 2718-2729, 2011). Briefly, nuclear extract was pre-cleared with DYNABEADS™ MYONE™ Streptavidin C1 for 30 min at 4° C. then incubated overnight with biotinylated oligonucleotide in binding buffer (10 mM HEPES pH 7.9; 100 mM KCl, 5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.5% NP-40, 1 mM DTT) supplemented with 1× protease inhibitor cocktail (Sigma-Aldrich). Beads were washed with binding buffer then added to the binding reaction. After 1 h incubation, beads were washed five times with binding buffer. DNA-bound proteins were eluted from beads and subjected to SDS-PAGE and immunoblotting.
[0212] RNA Pull-Down Assay (RNAP) and RNA-Protein Interaction Prediction
[0213] RNAP were performed essentially as described previously (Tsai et al., Science 329: 689-693, 2010) with few modifications. Briefly, biotinylated RNA was in vitro-transcribed using the MAXISCRIPT™ Transcription Kit (Ambion). DNA template was removed by DNAsel treatment and transcribed RNA was purified using RNeasy Mini Kit (QIAGEN). Purified RNA was denatured by heating to 90° C. for 2 min following incubation on ice for 2 min in RNA structure buffer (10 mM Tris pH 7, 0.1 M KCl, 10 mM MgCl2). Denatured RNA was then shifted to room temperature for 20 min to form proper secondary structure. Nuclear extract was treated with RNase-free DNase I (Roche) to remove genomic DNA and pre-cleared with DYNABEADS™ MYONE™ Streptavidin C1 or Streptavidin agarose beads (Invitrogen) in binding buffer I (150 mM KCl, 25 mM Tris pH 7.4, 0.5 mM DTT, 0.5% NP40, 1 mM PMSF) supplemented with COMPLETE™, Mini Protease Inhibitor Cocktail and SUPERase In RNase Inhibitor. Pre-cleared extracts were then incubated with biotinylated RNAs in binding buffer I for 1 h. Beads were washed with binding buffer I then added to the binding reaction. After 1 h incubation, beads were washed five times with binding buffer I. RNA-bound proteins were eluted from beads and subjected to SDS-PAGE and immunoblotting. For recombinant proteins, binding buffer II (50 mM Tris-CI 7.9, 10% Glycerol, 100 mM KCl, 5 mM MgCl2, 10 mM β-ME 0.1% NP-40) was used.
[0214] In silico prediction of RNA-Protein interaction was performed using catRAPID Fragments algorithm where protein-RNA interaction propensities were predicted based on calculation of secondary structure, hydrogen bonding and van der Waals contributions (Bellucci et al., Nat. Methods 8: 444-445, 2011).
[0215] Formaldehyde RNA Immunoprecipitation Sequencing and qPCR (fRIP-Seq and fRIP-qPCR)
[0216] fRIP was performed following a protocol reported by Hendrickson et al. (Genome Biol. 17: 28, 2016) with modifications. Briefly, cells were crosslinked in 0.1% formaldehyde at room temperature for 10 minutes. The crosslinking reaction was quenched for 5 min at room temperature with 0.125 M glycine. Crosslinked cells were washed with ice-cold PBS. Cell pellet was lysed in RIPA lysis buffer (50 mM Tris (pH 8), 150 mM KCl, 0.1% SDS, 1% Triton-X, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5 mM DTT) supplemented with protease inhibitor cocktail (Thermo Scientific) and 100 U/ml RNASEOUT™ (Invitrogen). After sonication, cell lysate was pre-cleared by incubating with DYNABEADS® Protein G (Invitrogen). Beads were then captured and removed using a magnet. Pre-cleared lysate was incubated with anti-RUNX1 antibody or IgG (Abcam) at 4° C. for 2 h before adding 50 μl of DYNABEADS® Protein G to capture antibodies. After washing, beads were kept at −20° C. or preceded to incubation with reverse-crosslinking buffer (3×PBS (without Mg or Ca), 6% N-lauroyl sarcosine, 30 mM EDTA, 15 mM DTT) supplemented with Proteinase K (Ambion) and RNASEOUT™ together with input sample. Captured RNAs were extracted by Trizol reagent. Extracted RNA was treated with DNAse from RNase-Free DNase Set (QIAGEN) then ribosomal RNA was removed using the RIBO-ZERO™ Magnetic Gold Kit (Epicentre). Treated RNA was purified using RNeasy MinElute Cleanup Kit (QIAGEN). RNA quality was determined using the RNA 6000 Pico Kit on a Bioanalyzer (Agilent). Purified RNA was used for qRT-PCR as described elsewhere and cDNA library construction with the Truseq stranded total RNA library prep kit (Illumina) according to manufacturer's protocol. The libraries were pooled together and subjected to pair-end sequencing on a Nextseq500 (Illumina) to achieve 2×40 bp reads.
[0217] Chromatin Immunoprecipitation and qPCR (ChIP-qPCR)
[0218] ChIP was performed as previously described (Mikkelsen et al., Nature 10: 553-560, 2007). Briefly, 2×10.sup.6 U937 cells were crosslinked with 1% formaldehyde (formaldehyde solution, freshly made: 50 mM HEPES-KOH; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 11% formaldehyde) for 10 min at room temperature. The crosslinking reaction was stopped by incubating with 0.125 M glycine for 5 min at room temperature. Crosslinked cells were washed twice with ice-cold PBS (freshly supplemented with 1 mM PMSF). Cell pellet was lysed for 10 min on ice and chromatin was fragmented by sonication (25 cycles, 30-s on, 60-s off, high power, Bioruptor). Chromatin solution was incubated with 10 μg antibody overnight at 4° C. Protein A magnetic beads (New England Biolabs) was used to capture antibody-bound chromatin. After washing, chromatin was reverse-crosslinked and treated with proteinase K 65° C. Beads were then removed using a magnet and chromatin solution was treated with treatment (Epicentre) for 30 min at 37° C. ChIP DNA was extracted with Phenol:chloroform:isoamyl Alcohol 25:24:1, pH:8 (Sigma-Aldrich) and then precipitated with equal volume of isopropanol in presence of glycogen. DNA pellet was dissolved in 30 μl of TE buffer for qPCR analyses. Fold enrichment was calculated using the formula 2.sup.(−ΔΔCt(ChIP/IgG)). Primer sets used for ChIP-qPCR are listed in Table 3 (qPCR).
fRIP-Seq and ChIP-Seq Data Analyses
[0219] fRIP-seq samples were de-multiplexed. Reads were deduplicated by Clumpify from the BBtools suite, (sourceforge.net/projects/bbmap/) with the parameters “dedupe spany addcount”. Adaptor quality trimming and filtering was performed by BBDuck from the BBtools suite with the parameters “ktrim=l hdist=2”. Low quality reads/bases were removed by Trimmomatic (Bolger et al., Bioinformatics 30: 2114-2120, 2014) with the parameters “LEADING:28 SLIDINGWINDOW:4:26 TRAILING:28 MINLEN:20”. The processed reads were then aligned to Human genome build 38 (hg38) by STAR aligner (Dobin et al., 2013) with the parameters “--outFilterScoreMinOverLread 0.05--outFilterMatchNminOverLread 0.05--outFilterMultimapNmax 30--outSAMprimaryFlag AllBestScore”. Coverage maps were generated using bamCoverage (part of the deepTools suite (Ramirez et al., Nucleic Acids Res. 44: W160-W165, 2016) with default parameters. Peak calling was performed using HOMER (v4.10) (Heinz et al., 2010). RUNX1 peaks with at least ten-fold over local region were selected for annotation using HOMER. Peaks were assigned to a gene locus by satisfying at least one of the following location criteria: a nearest transcription start site, on promoter, and on a transcript body. The latest version of ensemble 97 human gene CRCh38.p12 was used to retrieved gene annotation information through Biomart in Ensembl (Hunt et al., Ensembl variation resources Database (Oxford), 2018). For RUNX1 ChIP-seq data, raw reads in THP-1 cells (RUNX1: GSM2108052) were downloaded from GEO (GSE79899). Read quality were evaluated by FastQC (Andrews, Babraham Bioinformatics version 0115, 2016) before using for alignment and annotation as done for fRIP-seq data.
[0220] The following gene tracks are from published data that were deposited in GEO and processed via the Cistrome pipeline (Zheng et al., Nat. Commun. 8: 14049, 2019). H3K27Ac overlay track includes monocyte (GSM2679933), THP-1 (GSM2544236) and HL-60 (GSM2836486). H3K4Me1 overlay track includes monocyte (GSM1435532), HL-60 (GSM2836484) and THP-1 (GSM3514951). H3K4Me3 overlay track includes monocyte (GSM1435535), HL-60 (GSM945222) and THP-1 (GSM2108047). DNAse-seq overlay track includes monocyte (GSM701541) and HL-60 (GSM736595). RUNX1 ChIP-seq tracks includes CD34.sup.+ cells from healthy donors (GSM1097884), AML patient with FLT3-ITD and no other defined mutations (GSM1581788), AML patient with non-t(8;21) (GSM722708). The CAGE track (reverse strand and max counts) was imported from the FANTOM5 project (de Rie et al., Nat. Biotechnol. 35: 872-878, 2017).
[0221] RNA Sequencing Data Analysis (RNA-Seq)
[0222] Raw sequencing reads (FASTQ files) of the Human Body Map data set were downloaded from AEArrayExpress (E-MTAB-513). Read quality were assessed by FastQC (Andrews, 2016, supra). Reads with low-quality were trimmed by trim_galore (Krueger, Babraham Bioinformatics 045, 2017). LOUP transcript was integrated into the Ensembl human cDNA catalog GRCh38 and transcript levels were quantified against this catalog using Salmon (Patro et al., Nat. Methods 14: 417-419, 2017). For RNA-seq track visualization, the following RNA-seq raw data were downloaded from GEO: THP-1 (GSM1843218), HL-60 (GSM1843216), CD34.sub.+ HSPC (GSM1843222), Monocyte (GSM1843224) and Jurkat (GSM2260195). Read quality was assessed by FastQC (Andrews, 2016, supra). Where necessary, reads with low-quality were trimmed by trim_galore. Coverage maps were generated using bamCoverage (part of the deepTools suite (Ramirez et al., 2016, supra) with default parameters). BigWig files were uploaded and viewed via the UCSC genome browser.
[0223] Single-Cell RNA-Seq (scRNA-Seq) Data Analyses
[0224] Raw fastq files data of mononuclear cells isolated from peripheral blood and bone marrow were obtained from the 10× Genomics public datasets repository (www.10xqenomics.com/resources/datasets/) and pooled together. Transcripts were mapped to the human transcriptome using Cell Ranger (10× Genomics) with a custom hg38 gtf containing the LOUP transcript details. Subsequent analyses were performed in R (v3.6.2) using previously published Bioconductor workflow with minor modifications (Lun et al., F1000Res 3: 2122, 2016). Filtering criteria are as bellow. First, cells with library sizes more than three median absolute deviations (MADs) below the median library or four MAD's above the median library size were filtered out. Second, cells with a total number of expressed genes (>=1 read) more than three MADs below the median total number of expressed genes or four MAD's above the median total number of expressed genes were filtered out. Third, cells with a total percent of expressed genes originating from mitochondrial DNA more than eight MADs above the median were filtered out. A doublet score was then computed to estimate the percentage of barcodes for two or more cells as previously described (Wolock et al., Cell Syst. 8, 281-291 e289, 2019). Cells with a doublet score of 0.99 were excluded. Expression of each cell was normalized by a size factor approach as previously described (Lun et al., Genome Biol. 17: 75, 2016) resulting in log.sub.2(normalize_expression) values. Principle component and t-Distributed Stochastic Neighbor Embedding (tSNE) analyses revealed no significant batch effects to be regressed out for the samples. To account for dropouts which are being more frequent for genes with lower expression magnitude in scRNA-seq (Kharchenko et al., Nat. Methods 11: 740-742, 2014), cells with undetectable LOUP and PU.1 transcripts were referred as LOUP.sup.low/PU.1.sup.low and cells with detectable LOUP and PU.1 transcripts were referred as LOUP.sup.high/pU.1.sup.high Expression data visualization was performed using SPRING software (Weinreb et al., 2018). Briefly, a graph of cells connected to their nearest neighbors in gene expression space was determined. The data were then projected into two dimensions using a force-directed graph layout. Identity of each cell was inferred using Blueprint-Encode annotation which includes normalized expression values of 259 bulk RNA-seq samples generated from pure and defined cell populations (Consortium, Nature 489: 57-74, 2012; Martens and Stunnenberg, Haematologica 98: 1487-1489, 2013). This annotation was integrated in SingleR R package (Aran et al., Nat. Immunol. 20: 163-172, 2019). Annotated cells were grouped into major definitive cell lineages as described in the text. Gene Ontology (GO) analysis was performed using the Database for Annotation, Visualization and Integrated Discovery functional annotation tool (david.abcc.ncifcrf.gov). Significance of over-represented Gene Ontology biological processes was examined based on −log.sub.10 of corrected p-values from Bonferroni-corrected modified Fisher's exact test (Dennis et al., Genome Biol. 4: P3, 2003). A list of enriched genes in LOUP.sup.high/pU.1.sup.high group vs. LOUP.sup.low/PU.1.sup.low group was generated using SPRING software (Weinreb et al., Bioinformatics 34: 1246-1248, 2018). Upregulated genes (Z-score >1) was used for GO analysis.
[0225] Prediction of Coding Potential with PhyloCSF
[0226] The cross-species multiple sequence comparisons result of 46 species (i.e., multiz100way) was downloaded from the UCSC genome browser (genome.ucsc.edu). Guided by the GENCODE gene annotation (ver. 28), the alignment of the longest isoform of each gene was extracted from alignments of cross-species multiple sequence comparisons. The alignment was analyzed by PhyloCSF (Lin et al., 2011, supra) with 58mammals mode. All possible coding reading frames on the same strand were scanned. The maximal score was used.
[0227] Quantitation and Statistical Analysis
[0228] In general, quantitation and statistical tests were performed using GraphPad Prism 8.0 software (otherwise specified in respective figure legends). Data are shown as mean±SD, n>=3. Unpaired Two-tailed Student's t-test was used to calculate statistical significance of differences between two experimental groups. p≤0.05 was considered statistically significant.
[0229] Data and Software Availability
[0230] Data are available on the Gene Expression Omnibus database under GEO Series accession number GEO: GSE140459.
Example 2. Identification of RUNX1-Interacting RNAs at Myeloid Gene Loci
[0231] A transcriptome-wide survey for RUNX1-interacting RNAs in the monocytic cell line THP-1 was performed using formaldehyde RNA immunoprecipitation sequencing (fRIP-seq) (Hendrickson et al. Genome Biol 17: 28, 2016; Zhao et al., Mol Cell 40: 939-953, 2010). RUNX1 transcriptome was captured by anti-RUNX1 antibody (
TABLE-US-00004 TABLE 4 List of myeloid genes Gene Gene description (HUGO Gene symbol Common protein name(s) Nomenclature) ABCC8 Sulfonylurea Receptor ATP binding cassette subfamily C member 8 ACP5 human purple acid phosphatase acid phosphatase 5, tartrate resistant ADGRE1 egf-like module containing, mucin-like, adhesion G protein-coupled receptor E1 hormone receptor-like 1 ALOX5 Leukotriene A4 Synthase arachidonate 5-lipoxygenase ALOX5AP MK-886-binding protein arachidonate 5-lipoxygenase activating protein ANPEP Myeloid Plasma Membrane Glycoprotein alanyl aminopeptidase, membrane CD13 AZU1 Neutrophil Azurocidin azurocidin 1 BTK Bruton tyrosine kinase Bruton tyrosine kinase CCL2 Monocyte Chemotactic and Activating Factor C-C motif chemokine ligand 2 CCL3 Macrophage Inflammatory Protein 1-Alpha C-C motif chemokine ligand 3 CD14 CD14 antigen, Myeloid Cell-Specific Leucine- CD14 molecule Rich Glycoprotein CD36 Glycoprotein IIIb, Leukocyte Differentiation CD36 molecule Antigen CD36) CD68 Macrophage Antigen CD68 CD68 molecule CEACAM8 CD66b CEA Cell Adhesion Molecule 8 CEBPA C/EBP-alpha CCAAT enhancer binding protein alpha CEBPB C/EBP-beta CCAAT enhancer binding protein beta CEBPE C/EBP-epsilon CCAAT enhancer binding protein epsilon CES1 human monocyte/macrophage serine carboxylesterase 1 esterase 1 CSF1R CD115, macrophage colony-stimulating factor colony stimulating factor 1 receptor receptor CSF2 granulocyte-macrophage colony stimulating colony stimulating factor 2 factor (GM-CSF) CSF2RA CD116, alpha-GM-CSF receptor colony stimulating factor 2 receptor alpha subunit CSF3 Granulocyte-colony stimulating colony stimulating factor 3 factor (G-CSF) CSF3R CD114, granulocyte colony-stimulating factor colony stimulating factor 3 receptor receptor (G-CSF-R) CTSG cathepsin G cathepsin G CUX1 Homeobox Protein Cut-Like 1 cut like homeobox 1 CXCL8 IL-8, Monocyte-Derived Neutrophil C-X-C motif chemokine ligand 8 Chemotactic Factor CXCL9 Small-Inducible Cytokine B9 C-X-C motif chemokine ligand 9 CXCR1 CD181, interleukin 8 receptor, alpha (IL8RA) C-X-C motif chemokine receptor 1 CYBB Neutrophil Cytochrome B 91 KDa cytochrome b-245 beta chain Polypeptide, NADPH oxidase 2 ELANE Neutrophil Elastase elastase, neutrophil expressed FCGR1A CD64, Fc Gamma Receptor Ia Fc fragment of IgG receptor Ia FCGR3A CD16a Antigen Fc fragment of IgG receptor IIIa FCGR3B CD16b Antigen Fc fragment of IgG receptor IIIb FES C-Fes/Fps Protein FES proto-oncogene, tyrosine kinase FGR Tyrosine-Protein Kinase Fgr FGR proto-oncogene, Src family tyrosine kinase FPR1 N-Formylpeptide Chemoattractant Receptor formyl peptide receptor 1 FPR2 Lipoxin A4 Receptor formyl peptide receptor 2 FPR3 N-Formyl Peptide Receptor 3 formyl peptide receptor 3 GATA1 GATA binding protein 1 GATA binding protein 1 GATA2 GATA binding protein 2 GATA binding protein 2 HCK Hemopoietic Cell Kinase HCK proto-oncogene, Src family tyrosine kinase HOXA10 homeobox A10 homeobox A10 IL18 interleukin 18, interferon-gamma-inducing interleukin 18 factor IL1B interleukin 1 beta interleukin 1 beta IL3 interleukin 3 interleukin 3 IL6 interleukin 6 interleukin 6 ITGAD CD11d Antigen integrin subunit alpha D ITGAM MAC-1, CD11b Antigen integrin subunit alpha M ITGAX CD11c Antigen, Myeloid Membrane Antigen, integrin subunit alpha X Alpha Subunit ITGB2 CD18, macrophage antigen 1 (mac-1) beta integrin subunit beta 2 subunit JUN C-Jun Jun proto-oncogene, AP-1 transcription factor subunit LTB4R G Protein-Coupled Receptor 16 leukotriene B4 receptor LTF Neutrophil Lactoferrin lactotransferrin LYZ lysozyme lysozyme MMP12 Macrophage Metalloelastase matrix metallopeptidase 12 MMP2 MMP-2, Neutrophil Gelatinase matrix metallopeptidase 2 MMP8 MMP-8, Neutrophil Collagenase matrix metallopeptidase 8 MPEG1 MPG1, Macrophage-Expressed Gene 1 macrophage expressed 1 Protein MPO myeloperoxidase myeloperoxidase MRC1 CD206, Macrophage Mannose Receptor 1 mannose receptor C-type 1 MSR1 CD204 Antigen, Macrophage Scavenger macrophage scavenger receptor 1 Receptor Type III MYB C-Myb MYB proto-oncogene, transcription factor MYC C-Myc, Myc Proto-Oncogene Protein MYC proto-oncogene, bHLH transcription factor MZF1 MZF-1, Myeloid Zinc Finger 1 myeloid zinc finger 1 NCF1 p47phox, Neutrophil NADPH Oxidase Factor 1 neutrophil cytosolic factor 1 NCF2 P67PHOX, neutrophil cytosolic factor 2 neutrophil cytosolic factor 2 PLAU Urokinase-Type Plasminogen Activator plasminogen activator, urokinase RUNX1 RUNX1, Acute Myeloid Leukemia 1 Protein runt related transcription factor 1 S100A9 Calgranulin B, Leukocyte L1 Complex Heavy S100 calcium binding protein A9 Chain SATB1 Special AT-Rich Sequence-Binding Protein 1 SATB homeobox 1 SERPINA1 Alpha-1 Protease Inhibitor serpin family A member 1 SIGLEC1 CD169 Antigen sialic acid binding Ig like lectin 1 SLC11A1 Natural Resistance-Associated Macrophage solute carrier family 11 member 1 Protein 1 SLPI Antileukoproteinase secretory leukocyte peptidase inhibitor SP1 Transcription Factor Sp1 Sp1 transcription factor SPI1 PU.1, Transcription Factor PU.1 Spi-1 proto-oncogene TNF tumor necrosis factor tumor necrosis factor TP53 p53, tumor protein p53 tumor protein p53
Example 3. LOUP is a 1d-eRNA that Arises from the Upstream Region of the PU.1 Locus
[0232] To map the RUNX1-interacting transcript(s), we inspected RNA expression and epigenetic landscapes at the upstream region of the PU.1 locus (
[0233] We next determined molecular features of the full-length URE-originating RNA. The RNA exhibited very low coding potential similar to that of other known lncRNAs (
Example 4. LOUP is Myeloid-Specific lncRNA that Correlates with PU.1 mRNA Levels
[0234] We sought to explore the LOUP expression landscape in normal tissues and cell types. By examining the LOUP transcript profile in different human tissue types from the Illumina Body Map dataset (Illumina), we noticed that this lncRNA was barely detectable in most tissues but elevated in leukocytes (
TABLE-US-00005 TABLE 5 List of enriched genes in LOUP.sup.high/PU.1.sup.high cells Gene symbol Gene Name LYPD2 LY6/PLAUR domain containing 2(LYPD2) SAT1 spermidine/spermine N1-acetyltransferase 1(SAT1) NEAT1 nuclear paraspeckle assembly transcript 1 (non-protein coding)(NEAT1) AIF1 allograft inflammatory factor 1(AIF1) S100A9 S100 calcium binding protein A9(S100A9) SPI1 PU.1, Spi-1 proto-oncogene(SPI1) SLC7A7 solute carrier family 7 member 7(SLC7A7) CFP complement factor properdin(CFP) WARS tryptophanyl-tRNA synthetase(WARS) APOBEC3A apolipoprotein B mRNA editing enzyme catalytic subunit 3A(APOBEC3A) SERPINA1 serpin family A member 1 (SERPINA1) FCGR3A Fc fragment of IgG receptor IIIa(FCGR3A) CFD complement factor D(CFD) PILRA paired immunoglobin like type 2 receptor alpha(PILRA) FTL ferritin light chain(FTL) MS4A7 membrane spanning 4-domains A7(MS4A7) C5AR1 complement C5a receptor 1(C5AR1) NCF2 neutrophil cytosolic factor 2(NCF2) LYZ lysozyme(LYZ) CST3 cystatin C(CST3) STXBP2 syntaxin binding protein 2(STXBP2) CTSS cathepsin S(CTSS) LRRC25 leucine rich repeat containing 25(LRRC25) IGSF6 immunoglobulin superfamily member 6(IGSF6) C1QA complement C1q A chain(C1QA) NPC2 NPC intracellular cholesterol transporter 2(NPC2) GPBAR1 G protein-coupled bile acid receptor 1(GPBAR1) HES4 hes family bHLH transcription factor 4(HES4) GRN granulin precursor(GRN) MNDA myeloid cell nuclear differentiation antigen(MNDA) VMO1 vitelline membrane outer layer 1 homolog(VMO1) LST1 leukocyte specific transcript 1(LST1) IFITM3 interferon induced transmembrane protein 3(IFITM3) IFI30 IF130, lysosomal thiol reductase(IFI30) TYMP thymidine phosphorylase(TYMP) CD68 CD68 molecule(CD68) FCN1 ficolin 1(FCN1) FCER1G Fc fragment of IgE receptor Ig(FCER1G) FGL2 fibrinogen like 2(FGL2) SLC31A2 solute carrier family 31 member 2(SLC31A2) TYROBP TYRO protein tyrosine kinase binding protein(TYROBP) CEBPB CCAAT/enhancer binding protein beta(CEBPB) LGALS3 galectin 3(LGALS3) PSAP prosaposin(PSAP) LGALS1 galectin 1(LGALS1) HCK HCK proto-oncogene, Src family tyrosine kinase(HCK) S100A11 S100 calcium binding protein A11(S100A11) ANXA5 annexin A5(ANXA5) COTL1 coactosin like F-actin binding protein 1(COTL1) CPVL carboxypeptidase, vitellogenic like(CPVL) ANXA2 annexin A2(ANXA2) CYBB cytochrome b-245 beta chain(CYBB) KLF4 Kruppel like factor 4(KLF4)
Example 5. LOUP Acts as a lncRNA Regulator of PU.1 Induction
[0235] To test our hypothesis that LOUP induces PU.1 expression, we investigated the impact of LOUP's loss-of-expression on PU.1 cellular levels. In order to deplete LOUP RNA transcripts, we employed CRISPR/Cas9 genome-editing technology to introduce small insertion and deletion (indel) mutations in LOUP gene via the non-homologous end-joining (NHEJ) DNA repair mechanism (Jiang et al., Nat. Biotechnol. 31: 233-239 2013; Jinek et al., Science 337: 816-821, 2012). The macrophage cell line U937 that expresses the high level of LOUP (
[0236] In converse experiments, transient in trans-overexpression of LOUP in K562 cells resulted in significant induction of PU.1 (
Example 6. LOUP Induces URE-PrPr Communication by Interacting with Chromatin at the PU.1 Locus
[0237] We have previously reported that the formation of a chromatin loop mediated by URE-PrPr interaction is crucial for PU.1 induction (Ebralidze et al., 2008, supra; Staber et al., 2013, supra). Because LOUP arises from the URE and extends toward the PrPr, we reasoned that LOUP drives long-range transcription of PU.1 by promoting URE-PrPr interaction. To elucidate this, we quantified the strength of URE interactions with the PrPr and surrounding viewpoints by chromosome conformation capture (3C) followed by qPCR (
Example 7. LOUP Coordinates Recruitment of RUNX1 to Both the URE and the PrPr
[0238] We next sought to gain a deeper mechanistic understanding of how LOUP modulates the chromatin structure in a gene specific manner. Point mutations abrogating the Runx binding sites in the URE are known to disrupt chromatin loop formation (Staber et al., 2014, supra). Additionally, we showed that LOUP interacts with RUNX1 at the PU.1 locus (
Example 8. LOUP Possesses Embedded TEs that Bind the Runt Domain of RUNX1
[0239] By aligning LOUP sequence with itself using the Basic Local Alignment Search Tool (BLAST), we unexpectedly uncovered a highly repetitive region (RR) of 670 bp near the 3′ end of LOUP (
Example 9. Diagnosis of a Disease or Disorder in a Subject
[0240] A subject can be diagnosed as having a disease or disorder associated with PU.1 expression (e.g., a cancer (e.g., AML, liver cancer, or myeloma), Alzheimer's disease, or asthma) as described herein. The diagnostic method can be performed by determining a level of the transcription factor PU. 1 in a subject or a level of LOUP expression in a subject as described herein.
[0241] For example, a sample (e.g., a tissue sample, a blood sample, a cell sample, or a fluidic sample) can be obtained from a subject (e.g., a subject suspected of having a disease or disorder) and analyzed for LOUP and/or PU.1 expression. The level of LOUP and/or PU.1 expression can be compared to a standard or reference level (e.g., a control sample, in which a known expression level of LOUP and/or PU.1 has been linked to the presence or absence of the disease or disorder) or to a sample from a reference subject (e.g., a subject known to be healthy (e.g., to lack the disease or disorder) or a subject known to have the disease or disorder). Comparison of the LOUP and/or PU.1 level to the standard or reference level can confirm the presence or absence of the disease or disorder in the subject being tested.
[0242] For example, a subject determined to have decreased expression of PU.1, as compared to a standard or reference, can be identified as having or at risk of developing a cancer (e.g., AML, liver cancer, or myeloma). Alternatively, a subject determined to have increased expression of PU.1, as compared to a standard or reference, can be identified as having or at risk of developing Alzheimer's disease or asthma.
[0243] For example, a subject determined to have decreased expression of LOUP, as compared to a standard or reference, can be identified as having or at risk of developing a cancer (e.g., AML, liver cancer, or myeloma). Alternatively, a subject determined to have increased expression of LOUP, as compared to a standard or reference, can be identified as having or at risk of developing Alzheimer's disease or asthma.
[0244] Gene sequencing methods (e.g., next-generation gene sequencing methods, e.g., high-throughput sequencing, including but not limited to, Illumina sequencing, Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiD sequencing) can be used to analyze PU.1 and/or LOUP expression for the diagnosis of a disease or disorder.
Example 10. Diagnosing a subject as susceptible to ATRA treatment
[0245] Also provided are methods of diagnosing a subject as having a cancer (e.g., AML) that is susceptible to differentiation therapy with all-trans retinoic acid (ATRA) based on LOUP expression. A sample (e.g., a tissue sample, a blood sample, a cell sample, or a fluidic sample) from a subject (e.g., a subject suspected of having a cancer) can be analyzed for LOUP expression and compared to a standard or reference level (e.g., a control sample, in which a known expression level of LOUP has been linked to the presence or absence of the disease or disorder) or to a sample from a reference subject (e.g., a subject known to be healthy (e.g., to lack the disease or disorder) or a subject known to have the disease or disorder). Comparison of the LOUP level to the standard or reference level can be used to determine if the subject is likely to be sensitive to differentiation therapy with ATRA. For example, low levels of LOUP (relative to a standard or reference) would indicate resistance of the cancer to ATRA therapy.
[0246] Gene sequencing methods (e.g., next-generation gene sequencing methods, e.g., high-throughput sequencing, including but not limited to, Illumina sequencing, Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiD sequencing) can be used to analyze PU.1 and/or LOUP expression for the diagnosis of a disease or disorder.
Example 11. Gene Editing Systems for Targeting LOUP Expression
[0247] A gene editing system, as described herein, can be used to target LOUP expression in a subject (e.g., a subject in need thereof) for the treatment of a PU.1 associated medical condition. As an example, a gene editing system can be designed to be directed to a target genomic site associated with LOUP (e.g., a LOUP transcription start site or the LOUP gene).
[0248] After identifying a target genomic site, deep gene sequencing methods can be used to identify suitable PAM sites to be used for targeting of the gene editing system. Methods of designing the sgRNA are described herein. A delivery vehicle can be developed that includes the CRISPR/Cas nuclease (e.g., an active CRISPR/Cas nuclease or a CRISPRa gene activating system) and the sgRNA that can be used to direct the CRISPR/Cas nuclease to the target genomic site of interest. Non-limiting examples of LOUP targeting are described below.
[0249] For treating a disease associated with decreased PU.1 expression (e.g., a cancer (e.g., AML, liver cancer, or myeloma)) a CRISPRa gene activating system can be designed to increase LOUP expression. Briefly, sgRNAs targeting the upstream region of LOUP's transcriptional start site can be designed using Cas-Designer (Park et al., 2015, supra). As described above, the CRISPRa gene activating system (e.g., a dCas9-VP64) can be incorporated into a delivery vehicle (e.g., a vector (e.g., a viral vector (e.g., a lentiviral vector))) along with the sgRNA, and, optionally, one or more promoters to induce expression of the gene editing system. The delivery vehicle can be administered to a subject in need thereof (e.g., a subject having a disease or disorder associated with a decreased PU.1 expression (e.g., a cancer (e.g., AML, liver cancer, or myeloma))) and provide the gene editing system to a target cell for LOUP activation.
[0250] Alternatively, for treating a disease associated with increase PU.1 expression (e.g., Alzheimer's disease or Asthma) it may be beneficial to decrease PU.1 expression by decreasing LOUP expression (e.g., “knocking out” LOUP). Briefly, LOUP-targeting sgRNAs can be designed as described herein using Cas-Designer (Park et al., Bioinformatics 31: 4014-4016, 2015). To avoid disruption of the URE, known to be critical for PU.1 induction (Li et al. Blood 98: 2958-2965, 2001), single-guide RNAs (sgRNA) targeting LOUP (e.g., two distinct regions of the LOUP gene: (1) the LOUP intronic area downstream of the URE, and (2) the intronic area right upstream of the second exon of the LOUP gene (˜15 kb downstream from the URE)) can be designed and cloned into a delivery vehicle (e.g., a vector (e.g., a lentiviral vector) also incorporating the CRISPR/Cas system. The delivery vehicle can be formulated for administration to a subject in need thereof (e.g., a subject having a disease or disorder associated with an increased PU.1 expression (e.g., Alzheimer's or asthma)) and provide the gene editing system to a target cell for LOUP knock out.
Example 12. Treating a Disease or Disorder Associated with Decreased PU.1 Expression
[0251] A subject in need of treatment for a disease or disorder associated identified as having reduced expression of the transcription factor PU.1 (e.g., a cancer, such as AML, liver cancer, or myeloma), as described herein, can be administered a composition including a featured polynucleotide that increases expression of PU.1.
[0252] For treatment of a disease or disorder associated with reduced expression of PU.1, generally, a composition containing the featured polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1) can be administered (e.g., intravenously) to a subject (e.g., a subject in need thereof, such as a human) as a medicament (e.g., for treating a medical condition (e.g., a cancer (e.g., a PU.1 associated cancer (e.g., AML, liver cancer, or myeloma)))). The featured polynucleotide described herein can be used to induce the expression of tumor suppressor gene PU.1, thereby treating the disease or disorder. The featured polynucleotide can be delivered as a vector (e.g., a viral vector or non-viral vector) described herein. In certain embodiments, the featured polynucleotide can be delivered as a vector including a nucleic acid encoding the featured polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1) as described herein. In some embodiments, the vector is a viral vector (e.g., a lentiviral vector or an AAV vector). Gene sequencing methods (e.g., next-generation gene sequencing methods, e.g., high-throughput sequencing, including but not limited to, Illumina sequencing, Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiD sequencing) can be used to identify a subject in need thereof (e.g., a subject with a PU.1 associated cancer (e.g., AML, liver cancer, or myeloma)).
Example 13. Altering PU.1 Expression in a Subject in Need Thereof
[0253] The featured long non-coding RNA (e.g., LOUP RNA), polynucleotides encoding the lncRNA (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1), vectors (e.g., viral vectors) including polynucleotides encoding the lncRNA, constructs including the lncRNA (e.g., constructs including a protein linked to a LOUP polynucleotide), gene editing system (e.g., a CRISPR/Cas system or CRISPRa) for regulating PU.1 expression, polynucleotides encoding the gene editing systems, and vectors (e.g., viral vectors) including polynucleotides encoding the gene editing system can be administered to a subject in need thereof (e.g., a human) to alter (e.g., increase or decrease) the expression of tumor associated gene PU.1. Compositions and methods for delivering the featured polynucleotides (e.g., a polynucleotide having at least 20 nucleotides of SEQ ID NO: 1) and/or CRISPR/Cas system components include, e.g., a vector (e.g., a viral vector, such as a lentiviral vector particle), and non-vector delivery vehicles (e.g., nanoparticles), as discussed above.
[0254] Generally, the methods can include administering a composition containing the polynucleotide (e.g., a polynucleotide including at least 20 nucleotides of SEQ ID NO: 1), a construct thereof, or the gene editing system (e.g., a CRISPR/Cas system CRISPRa), either incorporated as a nucleic acid molecule (e.g., in a vector, such as a viral vector) encoding the polynucleotide, construct, or the components of the gene editing system (e.g., Cas protein and guide polynucleotides (e.g., guide RNA)) to a subject in need thereof. Alternatively, the methods can include administering the gene editing system in protein form (e.g., as a composition containing a Cas protein in combination with one or more guide polynucleotide(s) (e.g., gRNA(s))). The compositions can be administered (e.g., intravenously or intracranially) to a subject (e.g., a subject in need thereof) as a medicament for the treatment of a medical condition associated with PU.1 expression.
OTHER EMBODIMENTS
[0255] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
[0256] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
[0257] Other embodiments are within the claims.