METHODS OF TREATING DISORDERS
20210251988 · 2021-08-19
Inventors
- Qianhe ZHOU (Winchester, MA, US)
- Michael BOCKER (Cambridge, MA, US)
- Ho Man CHAN (Carlisle, MA, US)
- Luis SOARES (Cambridge, MA, US)
Cpc classification
A61K31/427
HUMAN NECESSITIES
A61K31/7088
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K38/465
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
A61K38/465
HUMAN NECESSITIES
A61K31/704
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/496
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/454
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K31/496
HUMAN NECESSITIES
A61K31/427
HUMAN NECESSITIES
A61K31/454
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/704
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present invention relates to methods and compositions for the treatment of BAF-related disorders such as cancers and viral infections.
Claims
1. A method of treating soft tissue sarcoma in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in the sarcoma.
2. A method of reducing tumor growth of a soft tissue sarcoma in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in the tumor.
3. A method of inducing apoptosis in a soft tissue sarcoma cell, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell.
4. A method of reducing the level and/or activity of BICRA in a soft tissue sarcoma cell, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell.
5. The method of claim 3 or 4, wherein the soft tissue sarcoma cell is in a subject.
6. The method of any one of claims 1 to 5, wherein the subject or cell has been identified as expressing SS18-SSX fusion protein or BICRA fusion protein.
7. The method of any one of claims 1 to 6, wherein the effective amount of the agent reduces the level and/or activity of BICRA by at least 5% as compared to a reference.
8. The method of any one of claims 1 to 7, wherein the effective amount of the agent reduces the level and/or activity of BICRA by at least 5% as compared to a reference for at least 12 hours.
9. The method of any one of claims 1 to 8, wherein the level and/or activity of SS18-SSX or BICRA fusion protein is reduced in the subject or cell.
10. The method of any one of claims 1 to 9, wherein the soft tissue sarcoma is adult soft tissue sarcoma.
11. The method of claim 10, wherein the adult soft tissue sarcoma is synovial sarcoma.
12. A method of modulating the activity of an SS18-SSX fusion protein, SS18 wild-type protein, or SSX wild-type protein in a cell, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell.
13. A method of modulating the level and/or activity of an SS18-SSX fusion protein, SS18 wild-type protein, or SSX wild-type protein in a cell or subject, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in a cell or subject.
14. The method of claim 12 or 13, wherein the cell is in a subject.
15. A method of treating a disorder related to an SS18-SSX fusion protein, SS18 wild-type protein, or SSX wild-type protein in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in an SS18-SSX fusion protein-expressing cell in the subject.
16. The method of any one of claims 12 to 15, wherein the subject has cancer.
17. The method of claim 16, wherein the cancer expresses SS18-SSX fusion protein and/or the cell or subject has been identified as expressing SS18-SSX fusion protein.
18. The method of any one of claims 15 to 17, wherein the disorder is synovial sarcoma or Ewing's sarcoma.
19. The method of claim 18, wherein the disorder is synovial sarcoma.
20. A method of modulating the activity of a BAF complex in a cell or subject, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell or subject.
21. A method of increasing the level and/or activity of BAF47 in a cell or subject, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell or subject.
22. A method of decreasing Wnt/β-catenin signaling in a cell or subject, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell or subject.
23. A method treating a disorder related to BAF47 in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in the subject.
24. The method of claim 23, wherein the disorder related to BAF47 is a cancer or viral infection.
25. The method of claim 24, wherein the cancer is a CD8+ T-cell lymphoma, endometrial carcinoma, ovarian carcinoma, bladder cancer, stomach cancer, pancreatic cancer, esophageal cancer, prostate cancer, renal cell carcinoma, melanoma, or colorectal cancer.
26. The method of claim 24, wherein the viral infection is an infection with a virus of the Retroviridae family, Hepadnaviridae family, Flaviviridae family, Adenoviridae family, Herpesviridae family, Papillomaviridae family, Parvoviridae family, Polyomaviridae family, Paramyxoviridae family, or Togaviridae family.
27. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in a cancer cell, wherein the cancer is a CD8+ T-cell lymphoma, endometrial carcinoma, ovarian carcinoma, bladder cancer, stomach cancer, pancreatic cancer, esophageal cancer, prostate cancer, renal cell carcinoma, melanoma, colorectal cancer, non-small cell lung cancer, stomach cancer, breast cancer, malignant rhabdoid tumor, multiple myeloma, or atypical teratoid rhabdoid tumor.
28. A method of reducing tumor growth of a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in a tumor cell, wherein the cancer is a CD8+ T-cell lymphoma, endometrial carcinoma, ovarian carcinoma, bladder cancer, stomach cancer, pancreatic cancer, esophageal cancer, prostate cancer, renal cell carcinoma, melanoma, colorectal cancer, non-small cell lung cancer, stomach cancer, breast cancer, malignant rhabdoid tumor, multiple myeloma, or atypical teratoid rhabdoid tumor.
29. A method of inducing apoptosis in a cancer cell, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell, wherein the cancer is a CD8+ T-cell lymphoma, endometrial carcinoma, ovarian carcinoma, bladder cancer, stomach cancer, pancreatic cancer, esophageal cancer, prostate cancer, renal cell carcinoma, melanoma, colorectal cancer, non-small cell lung cancer, stomach cancer, breast cancer, malignant rhabdoid tumor, multiple myeloma, or atypical teratoid rhabdoid tumor.
30. A method of reducing the level and/or activity of BICRA in a cancer cell, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell, wherein the cancer is a CD8+ T-cell lymphoma, endometrial carcinoma, ovarian carcinoma, bladder cancer, stomach cancer, pancreatic cancer, esophageal cancer, prostate cancer, renal cell carcinoma, melanoma, colorectal cancer, non-small cell lung cancer, stomach cancer, breast cancer, malignant rhabdoid tumor, multiple myeloma, or atypical teratoid rhabdoid tumor.
31. The method of any one of claims 27 to 30, wherein the cancer is a CD8+ T-cell lymphoma, endometrial carcinoma, ovarian carcinoma, bladder cancer, stomach cancer, pancreatic cancer, esophageal cancer, prostate cancer, renal cell carcinoma, melanoma, or colorectal cancer.
32. The method of any one of claims 27 to 31, wherein the cancer is non-small cell lung cancer, stomach cancer, breast cancer, malignant rhabdoid tumor, multiple myeloma, or atypical teratoid rhabdoid tumor.
33. A method of modulating the activity of a BICRA fusion protein in a cell or subject, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell or subject.
34. A method of modulating the level and/or activity of a BICRA fusion protein in a cell or subject, the method comprising contacting the cell with an effective amount of an agent that reduces the level and/or activity of BICRA in the cell or subject.
35. The method of claim 33 or 34, wherein the cell is in a subject.
36. A method of treating a disorder related to a BICRA fusion protein in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in a BICRA fusion protein-expressing cell.
37. The method of any one of claims 33 to 36, wherein the subject has cancer.
38. The method of claim 37, wherein the cancer expresses a BICRA fusion protein and/or the cell or subject has been identified as expressing a BICRA fusion protein.
39. The method of any one of claims 36 to 38, wherein the disorder related to a BICRA fusion protein is Ewing's sarcoma, lung cancer, or renal cancer.
40. The method of any one of claims 1 to 39, wherein the method further comprises administering to the subject or contacting the cell with an anticancer therapy.
41. The method of claim 40, wherein the anticancer therapy is a chemotherapeutic or cytotoxic agent or radiotherapy.
42. The method of claim 41, wherein the chemotherapeutic or cytotoxic agent is doxorubicin or ifosfamide.
43. The method of claim 41 or 42, wherein the anticancer therapy and the agent that reduces the level and/or activity of BICRA in a cell are administered within 28 days of each other and each in an amount that together are effective to treat the subject.
44. The method of any one of claims 1 to 43, wherein the subject or cancer has been identified as having an elevated level of an SS18-SSX fusion protein or a BICRA fusion protein as compared to a reference.
45. The method of any one of claims 1 to 44, wherein the subject or cancer has been identified as having a decreased level of SS18 wild-type protein or SSX wild-type protein as compared to a reference.
46. A method of treating a viral infection, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in a cell of the subject.
47. The method of claim 46, wherein the viral infection is an infection with a virus of the Retroviridae family, Hepadnaviridae family, Flaviviridae family, Adenoviridae family, Herpesviridae family, Papillomaviridae family, Parvoviridae family, Polyomaviridae family, Paramyxoviridae family, or Togaviridae family.
48. The method of any one of claims 1 to 47, wherein the agent that reduces the level and/or activity of BICRA in a cell is a small molecule compound, an antibody, an enzyme, and/or a polynucleotide.
49. The method of claim 48, wherein the agent that reduces the level and/or activity of BICRA in a cell is an enzyme.
50. The method of claim 49, wherein the enzyme is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a meganuclease.
51. The method of claim 50, wherein the CRISPR-associated protein is CRISPR-associated protein 9 (Cas9).
52. The method of claim 48, wherein the agent that reduces the level and/or activity of BICRA in a cell is a polynucleotide.
53. The method of claim 52, wherein the polynucleotide is an antisense nucleic acid, a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a micro RNA (miRNA), a CRISPR/Cas 9 nucleotide, or a ribozyme.
54. The method of claim 52, wherein the polynucleotide comprises a sequence having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-124.
55. The method of claim 54, wherein the polynucleotide comprises a sequence having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-68.
56. The method of claim 48, wherein the agent that reduces the level and/or activity of BICRA in a cell is a small molecule compound.
57. The method of claim 56, wherein the small molecule compound is a small molecule BICRA inhibitor.
58. The method of claim 56 or 57, wherein the small molecule compound is a degrader.
59. The method of claim 58, wherein the degrader has the structure of Formula I:
A-L-B Formula I wherein A is a BICRA binding moiety; L is a linker; and B is a degradation moiety.
60. The method of claim 59, wherein the degradation moiety is a ubiquitin ligase binding moiety.
61. The method of claim 60, wherein the ubiquitin ligase binding moiety comprises Cereblon ligands, IAP (Inhibitors of Apoptosis) ligands, mouse double minute 2 homolog (MDM2), or von Hippel-Lindau ligands, or derivatives or analogs thereof.
62. The method of claim 60 or 61, wherein the ubiquitin ligase binding moiety has the structure: ##STR00019## or is a derivative or an analog thereof.
63. The method of any one of claims 59 to 62, wherein the linker has the structure of Formula II:
A.sup.1-(B.sup.1).sub.f—(C.sup.1).sub.g—(B.sup.2).sub.h-(D)-(B.sup.3).sub.i—(C.sup.2).sub.j—(B.sup.4).sub.k-A.sup.2 Formula II wherein A.sup.1 is a bond between the linker and A; A.sup.2 is a bond between B and the linker; B.sup.1, B.sup.2, B.sup.3, and B.sup.4 each, independently, is selected from optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, O, S, S(O).sub.2, and NR.sup.N; R.sup.N is hydrogen, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; C.sup.1 and C.sup.2 are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; f, g, h, l, j, and k are each, independently, 0 or 1; and D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.f—(C.sup.1).sub.g—(B.sup.2).sub.h— to —(B.sup.3).sub.i—(C.sup.2).sub.j—(B.sup.4).sub.k-A.sup.2.
64. A method of treating cancer in a subject determined to have an elevated level of SS18-SSX fusion protein, SS18 wild-type protein, SSX wild-type protein, or a BICRA fusion protein, the method comprising administering to the subject an effective amount of an agent that reduces the level and/or activity of BICRA in the cell or subject.
65. The method of claim 64, wherein the level of SS18-SSX fusion protein, SS18 wild-type protein, SSX wild-type protein, or a BICRA fusion protein in the subject is measured in one or more cancer cells.
66. The method of claim 64 or 65, wherein the level of SS18-SSX fusion protein, SS18 wild-type protein, SSX wild-type protein, or a BICRA fusion protein in the subject is measured systemically.
67. A composition comprising an adult soft tissue sarcoma cell and an agent that reduces the level and/or activity of BICRA in a cell.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0117]
[0118]
DETAILED DESCRIPTION
[0119] The present inventors have found that depletion of BICRA in cancer cells inhibits cell growth and may result in the depletion of the SS18-SSX fusion protein and further inhibits the proliferation of the cancer cells.
[0120] Accordingly, the invention features methods and compositions useful for the inhibition of the activity of the SS18-SSX fusion proteins, e.g., for the treatment of cancer such as soft tissue sarcomas, e.g., adult soft tissue sarcomas. The invention further features methods and compositions useful for inhibition of the activity of the BICRA protein, e.g., for the treatment of cancer such as soft tissue sarcomas, e.g., in a subject in need thereof. Exemplary methods are described herein.
BICRA-Reducing Agents
[0121] Agents described herein that reduce the level and/or activity of BICRA in a cell may be an antibody, a protein (such as an enzyme), a polynucleotide, or a small molecule compound. The agents reduce the level of an activity related to BICRA, or a related downstream effect, or reduce the level of BICRA in a cell or subject.
[0122] In some embodiments, the agent that reduces the level and/or activity of BICRA in a cell is an enzyme, a polynucleotide, or a small molecule compound such as a degrader or small molecule BICRA inhibitor.
[0123] Antibodies
[0124] The agent that reduces the level and/or activity of BICRA can be an antibody or antigen binding fragment thereof. For example, an agent that reduces the level and/or activity of BICRA described herein is an antibody that reduces or blocks the activity and/or function of BICRA through binding to BICRA. The making and use of therapeutic antibodies against a target antigen (e.g., BICRA) is known in the art. See, for example, the references cited herein above, as well as Zhiqiang An (Editor), Therapeutic Monoclonal Antibodies: From Bench to Clinic. 1st Edition. Wiley 2009, and also Greenfield (Ed.), Antibodies: A Laboratory Manual. (Second edition) Cold Spring Harbor Laboratory Press 2013, for methods of making recombinant antibodies, including antibody engineering, use of degenerate oligonucleotides, 5′-RACE, phage display, and mutagenesis; antibody testing and characterization; antibody pharmacokinetics and pharmacodynamics; antibody purification and storage; and screening and labeling techniques.
[0125] Polynucleotides
[0126] In some embodiments, the agent that reduces the level and/or activity of BICRA is a polynucleotide. In some embodiments, the polynucleotide is an inhibitory RNA molecule, e.g., that acts by way of the RNA interference (RNAi) pathway. An inhibitory RNA molecule can decrease the expression level (e.g., protein level or mRNA level) of BICRA. For example, an inhibitory RNA molecule includes a short interfering RNA (siRNA), short hairpin RNA (shRNA), and/or a microRNA (miRNA) that targets full-length BICRA. A siRNA is a double-stranded RNA molecule that typically has a length of about 19-25 base pairs. A shRNA is a RNA molecule including a hairpin turn that decreases expression of target genes via RNAi. A microRNA is a non-coding RNA molecule that typically has a length of about 22 nucleotides. miRNAs bind to target sites on mRNA molecules and silence the mRNA, e.g., by causing cleavage of the mRNA, destabilization of the mRNA, or inhibition of translation of the mRNA. Degradation is caused by an enzymatic, RNA-induced silencing complex (RISC).
[0127] In some embodiments, the agent that reduces the level and/or activity of BICRA is an antisense nucleic acid. Antisense nucleic acids include antisense RNA (asRNA) and antisense DNA (asDNA) molecules, typically about 10 to 30 nucleotides in length, which recognize polynucleotide target sequences or sequence portions through hydrogen bonding interactions with the nucleotide bases of the target sequence (e.g., BICRA). The target sequences may be single- or double-stranded RNA, or single- or double-stranded DNA.
[0128] In embodiments, the polynucleotide decreases the level and/or activity of a negative regulator of function or a positive regulator of function. In other embodiments, the polynucleotide decreases the level and/or activity of an inhibitor of a positive regulator of function.
[0129] A polynucleotide of the invention can be modified, e.g., to contain modified nucleotides, e.g., 2′-fluoro, 2′-o-methyl, 2′-deoxy, unlocked nucleic acid, 2′-hydroxy, phosphorothioate, 2′-thiouridine, 4′-thiouridine, 2′-deoxyuridine. Without being bound by theory, it is believed that certain modification can increase nuclease resistance and/or serum stability, or decrease immunogenicity. The polynucleotides mentioned above, may also be provided in a specialized form such as liposomes, microspheres, or may be applied to gene therapy, or may be provided in combination with attached moieties. Such attached moieties include polycations such as polylysine that act as charge neutralizers of the phosphate backbone, or hydrophobic moieties such as lipids (e.g., phospholipids, cholesterols, etc.) that enhance the interaction with cell membranes or increase uptake of the nucleic acid. These moieties may be attached to the nucleic acid at the 3′ or 5′ ends and may also be attached through a base, sugar, or intramolecular nucleoside linkage. Other moieties may be capping groups specifically placed at the 3′ or 5′ ends of the nucleic acid to prevent degradation by nucleases such as exonuclease, RNase, etc. Such capping groups include hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory action of the polynucleotide can be examined using a cell-line or animal based gene expression system of the present invention in vivo and in vitro. In some embodiments, the polynucleotide decreases the level and/or activity or function of BICRA. In embodiments, the polynucleotide inhibits expression of BICRA. In other embodiments, the polynucleotide increases degradation of BICRA and/or decreases the stability (i.e., half-life) of BICRA. The polynucleotide can be chemically synthesized or transcribed in vitro.
[0130] Inhibitory polynucleotides can be designed by methods well known in the art. siRNA, miRNA, shRNA, and asRNA molecules with homology sufficient to provide sequence specificity required to uniquely degrade any RNA can be designed using programs known in the art, including, but not limited to, those maintained on websites for Thermo Fisher Scientific, the German Cancer Research Center, and The Ohio State University Wexner Medical Center. Systematic testing of several designed species for optimization of the inhibitory polynucleotide sequence can be routinely performed by those skilled in the art. Considerations when designing interfering polynucleotides include, but are not limited to, biophysical, thermodynamic, and structural considerations, base preferences at specific positions in the sense strand, and homology. The making and use of inhibitory therapeutic agents based on non-coding RNA such as ribozymes, RNAse P, siRNAs, and miRNAs are also known in the art, for example, as described in Sioud, RNA Therapeutics: Function, Design, and Delivery (Methods in Molecular Biology). Humana Press 2010. Exemplary inhibitory polynucleotides, for use in the methods of the invention, are provided in Table 1, below. In some embodiments, the inhibitory polynucleotides have a nucleic acid sequence with at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the nucleic acid sequence of an inhibitory polynucleotide in Table 1. In some embodiments, the inhibitory polynucleotides have a nucleic acid sequence with at least 70% sequence identity (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% identity, or more) to the nucleic acid sequence of an inhibitory polynucleotide in Table 1.
[0131] Construction of vectors for expression of polynucleotides for use in the invention may be accomplished using conventional techniques which do not require detailed explanation to one of ordinary skill in the art. For generation of efficient expression vectors, it is necessary to have regulatory sequences that control the expression of the polynucleotide. These regulatory sequences include promoter and enhancer sequences and are influenced by specific cellular factors that interact with these sequences, and are well known in the art.
[0132] Gene Editing
[0133] In some embodiments, the agent that reduces the level and/or activity of BICRA is a component of a gene editing system. For example, the agent that reduces the level and/or activity of BICRA introduces an alteration (e.g., insertion, deletion (e.g., knockout), translocation, inversion, single point mutation, or other mutation) in BICRA. In some embodiments, the agent that reduces the level and/or activity of BICRA is a nuclease. Exemplary gene editing systems include the zinc finger nucleases (ZFNs), Transcription Activator-Like Effector-based Nucleases (TALENs), and the clustered regulatory interspaced short palindromic repeat (CRISPR) system. ZFNs, TALENs, and CRISPR-based methods are described, e.g., in Gaj et al., Trends Biotechnol. 31 (7):397-405 (2013).
[0134] CRISPR refers to a set of (or system including a set of) clustered regularly interspaced short palindromic repeats. A CRISPR system refers to a system derived from CRISPR and Cas (a CRISPR-associated protein) or other nuclease that can be used to silence or mutate a gene described herein. The CRISPR system is a naturally occurring system found in bacterial and archeal genomes. The CRISPR locus is made up of alternating repeat and spacer sequences. In naturally-occurring CRISPR systems, the spacers are typically sequences that are foreign to the bacterium (e.g., plasmid or phage sequences). The CRISPR system has been modified for use in gene editing (e.g., changing, silencing, and/or enhancing certain genes) in eukaryotes. See, e.g., Wiedenheft et al., Nature 482(7385):331-338 (2012). For example, such modification of the system includes introducing into a eukaryotic cell a plasmid containing a specifically-designed CRISPR and one or more appropriate Cas proteins. The CRISPR locus is transcribed into RNA and processed by Cas proteins into small RNAs that include a repeat sequence flanked by a spacer. The RNAs serve as guides to direct Cas proteins to silence specific DNA/RNA sequences, depending on the spacer sequence. See, e.g., Horvath et al., Science 327(5962):167-170 (2010); Makarova et al., Biology Direct 1:7 (2006); Pennisi, Science 341 (6148):833-836 (2013). In some examples, the CRISPR system includes the Cas9 protein, a nuclease that cuts on both strands of the DNA. See, e.g., Id.
[0135] In some embodiments, in a CRISPR system for use described herein, e.g., in accordance with one or more methods described herein, the spacers of the CRISPR are derived from a target gene sequence, e.g., from a BICRA sequence.
[0136] In some embodiments, the agent that reduces the level and/or activity of BICRA includes a guide RNA (gRNA) for use in a CRISPR system for gene editing. Exemplary gRNAs, for use in the methods of the invention, are provided in Table 1, below. In embodiments, the agent that reduces the level and/or activity of BICRA includes a ZFN, or an mRNA encoding a ZFN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) of BICRA. In embodiments, the agent that reduces the level and/or activity of BICRA includes a TALEN, or an mRNA encoding a TALEN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) of BICRA.
[0137] For example, the gRNA can be used in a CRISPR system to engineer an alteration in a gene (e.g., BICRA). In other examples, the ZFN and/or TALEN can be used to engineer an alteration in a gene (e.g., BICRA). Exemplary alterations include insertions, deletions (e.g., knockouts), translocations, inversions, single point mutations, or other mutations. The alteration can be introduced in the gene in a cell, e.g., in vitro, ex vivo, or in vivo. In some embodiments, the alteration decreases the level and/or activity of (e.g., knocks down or knocks out) BICRA, e.g., the alteration is a negative regulator of function. In yet another example, the alteration corrects a defect (e.g., a mutation causing a defect), in BICRA. In certain embodiments, the CRISPR system is used to edit (e.g., to add or delete a base pair) a target gene, e.g., BICRA. In other embodiments, the CRISPR system is used to introduce a premature stop codon, e.g., thereby decreasing the expression of a target gene. In yet other embodiments, the CRISPR system is used to turn off a target gene in a reversible manner, e.g., similarly to RNA interference. In embodiments, the CRISPR system is used to direct Cas to a promoter of a target gene, e.g., BICRA, thereby blocking an RNA polymerase sterically.
[0138] In some embodiments, a CRISPR system can be generated to edit BICRA using technology described in, e.g., U.S. Publication No. 20140068797; Cong et al., Science 339(6121):819-823 (2013); Tsai, Nature Biotechnd, 32(6):569-576 (2014); and U.S. Pat. Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359.
[0139] In some embodiments, the CRISPR interference (CRISPRi) technique can be used for transcriptional repression of specific genes, e.g., the gene encoding BICRA. In CRISPRi, an engineered Cas9 protein (e.g., nuclease-null dCas9, or dCas9 fusion protein, e.g., dCas9-KRAB or dCas9-SID4X fusion) can pair with a sequence specific guide RNA (sgRNA). The Cas9-gRNA complex can block RNA polymerase, thereby interfering with transcription elongation. The complex can also block transcription initiation by interfering with transcription factor binding. The CRISPRi method is specific with minimal off-target effects and is multiplexable, e.g., can simultaneously repress more than one gene (e.g., using multiple gRNAs). Also, the CRISPRi method permits reversible gene repression.
[0140] In some embodiments, CRISPR-mediated gene activation (CRISPRa) can be used for transcriptional activation, e.g., of one or more genes described herein, e.g., a gene that inhibits BICRA. In the CRISPRa technique, dCas9 fusion proteins recruit transcriptional activators. For example, dCas9 can be used to recruit polypeptides (e.g., activation domains) such as VP64 or the p65 activation domain (p65D) and used with sgRNA (e.g., a single sgRNA or multiple sgRNAs), to activate a gene or genes, e.g., endogenous gene(s). Multiple activators can be recruited by using multiple sgRNAs—this can increase activation efficiency. A variety of activation domains and single or multiple activation domains can be used. In addition to engineering dCas9 to recruit activators, sgRNAs can also be engineered to recruit activators. For example, RNA aptamers can be incorporated into a sgRNA to recruit proteins (e.g., activation domains) such as VP64. In some examples, the synergistic activation mediator (SAM) system can be used for transcriptional activation. In SAM, MS2 aptamers are added to the sgRNA. MS2 recruits the MS2 coat protein (MCP) fused to p65AD and heat shock factor 1 (HSF1). The CRISPRi and CRISPRa techniques are described in greater detail, e.g., in Dominguez et al., Nat. Rev. Mol. Cell Bid. 17(1):5-15 (2016), incorporated herein by reference.
TABLE-US-00001 TABLE 1 Exemplary Inhibitory Polynucleotides SEQ Type of ID Inhibitory NO. Polynucleotide Nucleic Acid Sequence 3 CRISPR gRNA GGAGGGCGCCCTGGTAGACA 4 CRISPR gRNA ATATCGGCTCCTGCTCCTGG 5 CRISPR gRNA TCCTGCTCCTGGAGGAGTCC 6 CRISPR gRNA GGCGCCCTGGTAGACATGGT 7 CRISPR gRNA TGCAGGGCGTCCTCAAAGGA 8 CRISPR gRNA GCAGCTGCTGAAACGCACCC 9 CRISPR gRNA GATCATTACCATCTCCGCTG 10 CRISPR gRNA CCTGCCCTACCATGTCTACC 11 CRISPR gRNA GAAGTCTAGGTCCACACTGG 12 CRISPR gRNA CCTGGTAGACATGGTAGGGC 13 CRISPR gRNA TCATTACCATCTCCGCTGAG 14 CRISPR gRNA GGTAGGGCAGGAGGCGATGC 15 CRISPR gRNA GCAGGGCGTCCTCAAAGGAG 16 CRISPR gRNA TCAGGGACCAGGTGGAGGGT 17 CRISPR gRNA TCTGCAGGGAGTCCTGAGTG 18 CRISPR gRNA CTGCCCTACCATGTCTACCA 19 CRISPR gRNA GTAGGGCAGGAGGCGATGCA 20 CRISPR gRNA AGGCCATGCTCAATAAATAT 21 CRISPR gRNA ACAGCTGGCCAAGGAGAAGC 22 CRISPR gRNA ATGCAGGGCGTCCTCAAAGG 23 CRISPR gRNA GCCTCCTCGGACCTTCCAGA 24 CRISPR gRNA AGAAGTCATTGAGGGCCTGT 25 CRISPR gRNA TCTCCTCCTGAATGAACATT 26 CRISPR gRNA TTCTGGAGGATGATTCCGGA 27 CRISPR gRNA GCAGGAAGGGCTGCACACTC 28 CRISPR gRNA GGGGCCTGGTGAGGTAGTGA 29 CRISPR gRNA CCTTGCTGGGCTGAAGCGTG 30 CRISPR gRNA ATCATTACCATCTCCGCTGA 31 CRISPR gRNA TTCCTTGCTGGGCTGAAGCG 32 CRISPR gRNA GACGGCCTTCCCCTCCTTTG 33 CRISPR gRNA GCTGGTGGCCTCGTCCACTT 34 CRISPR gRNA AAGTGGACGAGGCCACCAGC 35 CRISPR gRNA CAGCTGTTTATCCAAGGCAA 36 CRISPR gRNA GGTAGACATGGTAGGGCAGG 37 CRISPR gRNA GGAGCATTTGCACAAACACC 38 CRISPR gRNA TTCAGGAGGTGGACGCTCAT 39 CRISPR gRNA TCTAGGTCCACACTGGGGGC 40 CRISPR gRNA GCCCCAGGACGATCTTCTCC 41 CRISPR gRNA GACACACTCTGTGGCCGGGA 42 CRISPR gRNA CTTGGCCAGGAGCTGGGAGG 43 CRISPR gRNA GAGCTGTCCACCTGTGTGGG 44 CRISPR gRNA CGGAAGAGGCTGCGATGGGG 45 CRISPR gRNA GCGATGCAGGGCGTCCTCAA 46 CRISPR gRNA GTTCAGGAGGTGGACGCTCA 47 CRISPR gRNA TCCCCGCCGCCATGAACGTC 48 CRISPR gRNA CTTGTTCTGGAGGATGATTC 49 CRISPR gRNA GGCCTGGTGAGGTAGTGACG 50 CRISPR gRNA GGGCCTCCCCGGGATTATCC 51 CRISPR gRNA GTCCCCGTCACTACCTCACC 52 CRISPR gRNA CTTGTAGTCGGGGTGCAGGA 53 CRISPR gRNA CTCTGGGTTCAGGTGGTTGC 54 CRISPR gRNA GCTGCCTGGGAAGAGGGCTT 55 CRISPR gRNA CCTCCCCGGGATTATCCAGG 56 CRISPR gRNA TCGAGAAGAGCCTTCGGCTG 57 CRISPR gRNA GTTGTGACTGGAGGGTGGGT 58 CRISPR gRNA GATGAGCTGTCCACCTGTGT 59 CRISPR gRNA GCTGGAGGATGTCACAGGGC 60 CRISPR gRNA TGCCGGATCACAAGCTTGGT 61 CRISPR gRNA TCCCCCTCCAACCCTCCACC 62 CRISPR gRNA AGTGGACGAGGAGTTTGAGA 63 CRISPR gRNA GTAGTGACGGGGACGAAGAG 64 CRISPR gRNA GGGTGCAAGGGTGGCTCTGA 65 CRISPR gRNA GACCCCCTGGAGGACAGTGG 66 CRISPR gRNA GAGCATTTGCACAAACACCA 67 CRISPR gRNA GGGCCTGGTGAGGTAGTGAC 68 CRISPR gRNA GGGCCTTGTTGACCACGTCC 69 CRISPR gRNA GCCCAAAGTGCCTTCTATGA 70 CRISPR gRNA CAACATCACGGAGCAGACGC 71 CRISPR gRNA TGCCGGAAGCTTCTTGCACA 72 CRISPR gRNA CCGTGATGTTGGCCTCTTGG 73 CRISPR gRNA GCTCCGTGATGTTGGCCTCT 74 CRISPR gRNA CAGCGTCTGCTCCGTGATGT 75 CRISPR gRNA TGACCTCCTGGATAATCCCG 76 CRISPR gRNA GGCCTTGTTGACCACGTCCT 77 CRISPR gRNA CTGTGGCCACCACGCTCAAT 78 CRISPR gRNA TGACGGGCTGGCCCACCACC 79 miRNA CCACACAGCGGAGGGAGGCGGC 80 miRNA GGGGAGAGCGAGAGCCCGGCUG 81 miRNA CCUCCUUUCCGAGGGGCGUCGU 82 miRNA UUCUUCAGCGGACUCAGUUUGC 83 miRNA UGCACCGUCUCGACAGGCGCGG 84 miRNA UUAUCCAACCGAAGGGUGGUCU 85 miRNA UCUCUCACAGUCUUGUGCACAC 86 miRNA UGUCGAUGCGCCUCUGCAGGUG 87 miRNA UGCACACAGUGACACACACAGG 88 miRNA UGUCAAGCAAGUCGGAUCCAUG 89 miRNA UGCUCCAGCUUACAGGCUUCCU 90 miRNA UUGUGCACCGGCUCGCUGAGCC 91 miRNA UGUGCACAGUGACACACACACA 92 siRNA (guide GGAGAATTCTGTACATTTA strand) 93 siRNA (guide GTATAACGATTTTTTTAAA strand) 94 siRNA (guide CTTTGAAATCTGAGCAAAA strand) 95 siRNA (guide CTGTAAGATAAATTTTTTT strand) 96 siRNA (guide CATTTAGAACTCTTGTAAA strand) 97 siRNA (guide GTGATGACCTCCTGGATAA strand) 98 siRNA (guide GCATCTTTGTCATCCAAAA strand) 99 siRNA (guide CCCAGGCCATGCTCAATAA strand) 100 siRNA (guide GGCCATGCTCAATAAATAT strand) 101 siRNA (guide CCTCAGCGGAGATGGTAAT strand) 102 siRNA (guide CCACCCTTGCCTTGGATAA strand) 103 siRNA (guide CACCCCTCGACTTTGAAAT strand) 104 siRNA (guide CCGCGCCAGATTTTGAAAT strand) 105 siRNA (guide CCTGTTTCCTGGAGCATTT strand) 106 siRNA (guide GGCCCAAAGTGCCTTCTAT strand) 107 siRNA (guide CCAGGACGTTGACCAGATA strand) 108 siRNA (guide CTGTATTTATTGTGTATAA strand) 109 siRNA (guide CCGGAATCATCCTCCAGAA strand) 110 siRNA (guide CCAGGCCATGCTCAATAAA strand) 111 siRNA (guide CAGGCCATGCTCAATAAAT strand) 112 shRNA (loop GAGGATGGGAGATGCTTACTATCAAGAGTAGTAAGCATCTCCCATCCTC bolded) 113 shRNA (loop GGATGGGAGATGCTTACTAGATCAAGAGTCTAGTAAGCATCTCCCATCC bolded) 114 shRNA (loop GATGCTTACTAGACGTGATTTTCAAGAGAAATCACGTCTAGTAAGCATC bolded) 115 shRNA (loop GGATCCGAGAAGCTTGACAGTTCAAGAGACTGTCAAGCTTCTCGGATCC bolded) 116 shRNA (loop GAAGCTTGACAGTGATGACCTTCAAGAGAGGTCATCACTGTCAAGCTTC bolded) 117 shRNA (loop GGCCCAAAGTGCCTTCTATGATCAAGAGTCATAGAAGGCACTTTGGGCC bolded) 118 shRNA (loop GCCCAAAGTGCCTTCTATGAATCAAGAGTTCATAGAAGGCACTTTGGGC bolded) 119 shRNA (loop GCAAACTGAGTGGCCTGAAGATCAAGAGTCTTCAGGCCACTCAGTTTGC bolded) 120 shRNA (loop GGTAATGATCGACCGAATGTTTCAAGAGAACATTCGGTCGATCATTACC bolded) 121 shRNA (loop GCCAGGACGTTGACCAGATAATCAAGAGTTATCTGGTCAACGTCCTGGC bolded) 122 shRNA (loop GTGCACAAGTGAGTGAGAGATTCAAGAGATCTCTCACTCACTTGTGCAC bolded) 123 shRNA (loop GCACAAGTGAGTGAGAGATTTTCAAGAGAAATCTCTCACTCACTTGTGC bolded) 124 shRNA (loop GCTAGTCTTCCCTCTGTTCTTTCAAGAGAAGAACAGAGGGAAGACTAGC bolded)
[0141] Small Molecule Compounds
[0142] In some embodiments of the invention, the agent that reduces the level and/or activity of BICRA in a cell is a small molecule compound. In some embodiments, the small molecule compound is a structure of Formula I:
A-L-B Formula I
wherein A is a BICRA binding moiety; L is a linker; and B is a degradation moiety.
[0143] In some embodiments, the degradation moiety has the structure of:
##STR00013##
[0144] wherein X.sup.1 is CH.sub.2, O, S, or NR.sup.1, wherein R.sup.1 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; X.sup.2 is C═O, CH.sub.2, or
##STR00014##
R.sup.3 and R.sup.4 are, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; m is 0, 1, 2, 3, or 4; and each R.sup.2 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino,
[0145] or a pharmaceutically acceptable salt thereof;
##STR00015##
[0146] wherein each R.sup.4, R.sup.4′, and R.sup.7 is, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; R.sup.5 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.1-C.sub.6 alkyl C.sub.3-C.sub.10 carbocyclyl, or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl; R.sup.6 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.1-C.sub.6 alkyl C.sub.3-C.sub.10 carbocyclyl, or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl; n is 0, 1, 2, 3, or 4; each R.sup.8 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino; and each R.sup.9 and R.sup.10 is, independently, H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl, wherein R.sup.4′ or R.sup.5 comprises a bond to the linker, or a pharmaceutically acceptable salt thereof;
##STR00016##
[0147] wherein each R.sup.11, R.sup.13, and R.sup.15 is, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl; R.sup.12 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.1-C.sub.6 alkyl C.sub.3-C.sub.10 carbocyclyl, or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl; R.sup.14 is optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.1-C.sub.6 alkyl C.sub.3-C.sub.10 carbocyclyl, or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl; p is 0, 1, 2, 3, or 4; each R.sup.16 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino; q is 0, 1, 2, 3, or 4; and each R.sup.17 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino, or a pharmaceutically acceptable salt thereof; or
##STR00017##
[0148] wherein each R.sup.18 and R.sup.19 is, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.1-C.sub.6 alkyl C.sub.3-C.sub.10 carbocyclyl, or optionally substituted C.sub.1-C.sub.6 alkyl C.sub.6-C.sub.10 aryl; r1 is 0, 1, 2, 3, or 4; each R.sup.20 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino; r2 is 0, 1, 2, 3, or 4; and each R.sup.21 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino, or a pharmaceutically acceptable salt thereof.
[0149] In some embodiments, the linker has the structure of Formula II:
A.sup.1-(B.sup.1).sub.f—(C.sup.1).sub.g—(B.sup.2).sub.h-(D)-(B.sup.3).sub.i—(C.sup.2).sub.j—(B.sup.4).sub.k-A.sup.2 Formula II
[0150] wherein A.sup.1 is a bond between the linker and A; A.sup.2 is a bond between B and the linker; B.sup.1, B.sup.2, B.sup.3, and B.sup.4 each, independently, is selected from optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, O, S, S(O).sub.2, and NR.sup.N; R.sup.N is hydrogen, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; C.sup.1 and C.sup.2 are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; f, g, h, l, j, and k are each, independently, 0 or 1; and D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.f—(C.sup.1).sub.g—(B.sup.2).sub.h— to —(B.sup.3).sub.i—(C.sup.2).sub.j—(B.sup.4).sub.k-A.sup.2.
[0151] Linkers include, but are not limited to, the structure of:
##STR00018##
Pharmaceutical Uses
[0152] The compounds described herein are useful in the methods of the invention and, while not bound by theory, are believed to exert their desirable effects through their ability to modulate the level, status, and/or activity of a BAF complex, e.g., by inhibiting the activity or level of the BRG and BRM proteins in a cell within the BAF complex in a mammal.
[0153] An aspect of the present invention relates to methods of treating disorders related to BRG and BRM proteins such as cancer in a subject in need thereof. In some embodiments, the compound is administered in an amount and for a time effective to result in one of (or more, e.g., two or more, three or more, four or more of): (a) reduced tumor size, (b) reduced rate of tumor growth, (c) increased tumor cell death (d) reduced tumor progression, (e) reduced number of metastases, (f) reduced rate of metastasis, (g) decreased tumor recurrence (h) increased survival of subject, and (i) increased progression free survival of a subject.
[0154] Treating cancer can result in a reduction in size or volume of a tumor. For example, after treatment, tumor size is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) relative to its size prior to treatment. Size of a tumor may be measured by any reproducible means of measurement. For example, the size of a tumor may be measured as a diameter of the tumor.
[0155] Treating cancer may further result in a decrease in number of tumors. For example, after treatment, tumor number is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) relative to number prior to treatment. Number of tumors may be measured by any reproducible means of measurement, e.g., the number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification (e.g., 2×, 3×, 4×, 5×, 10×, or 50×).
[0156] Treating cancer can result in a decrease in number of metastatic nodules in other tissues or organs distant from the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to number prior to treatment. The number of metastatic nodules may be measured by any reproducible means of measurement. For example, the number of metastatic nodules may be measured by counting metastatic nodules visible to the naked eye or at a specified magnification (e.g., 2×, 10×, or 50×).
[0157] Treating cancer can result in an increase in average survival time of a population of subjects treated according to the present invention in comparison to a population of untreated subjects. For example, the average survival time is increased by more than 30 days (more than 60 days, 90 days, or 120 days). An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with the compound described herein. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with a pharmaceutically acceptable salt of a compound described herein.
[0158] Treating cancer can also result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. For example, the mortality rate is decreased by more than 2% (e.g., more than 5%, 10%, or 25%). A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with a pharmaceutically acceptable salt of a compound described herein. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with a pharmaceutically acceptable salt of a compound described herein.
Combination Therapies
[0159] A method of the invention can be used alone or in combination with an additional therapeutic agent, e.g., other agents that treat cancer or symptoms associated therewith, or in combination with other types of therapies to treat cancer. In combination treatments, the dosages of one or more of the therapeutic compounds may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)). In this case, dosages of the compounds when combined should provide a therapeutic effect.
[0160] In some embodiments, the second therapeutic agent is a chemotherapeutic agent (e.g., a cytotoxic agent or other chemical compound useful in the treatment of cancer). These include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopyyllotoxins, antibiotics, L-Asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog. Also included is 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed Engl. 33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®, cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Two or more chemotherapeutic agents can be used in a cocktail to be administered in combination with the first therapeutic agent described herein. Suitable dosing regimens of combination chemotherapies are known in the art and described in, for example, Saltz et al., Proc. Am. Soc. Clin. Oncol. 18:233a (1999), and Douillard et al., Lancet 355(9209):1041-1047 (2000).
[0161] In some embodiments, the second therapeutic agent is a therapeutic agent which is a biologic such a cytokine (e.g., interferon or an interleukin (e.g., IL-2)) used in cancer treatment. In some embodiments the biologic is an anti-angiogenic agent, such as an anti-VEGF agent, e.g., bevacizumab (AVASTIN®). In some embodiments the biologic is an immunoglobulin-based biologic, e.g., a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein or a functional fragment thereof) that agonizes a target to stimulate an anti-cancer response, or antagonizes an antigen important for cancer. Such agents include RITUXAN® (rituximab); ZENAPAX® (daclizumab); SIMULECT® (basiliximab); SYNAGIS® (palivizumab); REMICADE® (infliximab); HERCEPTIN® (trastuzumab); MYLOTARG® (gemtuzumab ozogamicin); CAMPATH® (alemtuzumab); ZEVALIN® (ibritumomab tiuxetan); HUMIRA® (adalimumab); XOLAIR® (omalizumab); BEXXAR® (tositumomab-I-131); RAPTIVA® (efalizumab); ERBITUX® (cetuximab); AVASTIN® (bevacizumab); TYSABRI® (natalizumab); ACTEMRA® (tocilizumab); VECTIBIX® (panitumumab); LUCENTIS® (ranibizumab); SOURIS® (eculizumab); CIMZIA® (certolizumab pegol); SIMPONI® (golimumab); ILARIS® (canakinumab); STELARA® (ustekinumab); ARZERRA® (ofatumumab); PROLIA® (denosumab); NUMAX® (motavizumab); ABTHRAX® (raxibacumab); BENLYSTA® (belimumab); YERVOY® (ipilimumab); ADCETRIS® (brentuximab vedotin); PERJETA® (pertuzumab); KADCYLA® (ado-trastuzumab emtansine); and GAZYVA® (obinutuzumab). Also included are antibody-drug conjugates.
[0162] The second agent may be a therapeutic agent which is a non-drug treatment. For example, the second therapeutic agent is radiation therapy, cryotherapy, hyperthermia, and/or surgical excision of tumor tissue.
[0163] The second agent may be a checkpoint inhibitor. In one embodiment, the inhibitor of checkpoint is an inhibitory antibody (e.g., a monospecific antibody such as a monoclonal antibody). The antibody may be, e.g., humanized or fully human. In some embodiments, the inhibitor of checkpoint is a fusion protein, e.g., an Fc-receptor fusion protein. In some embodiments, the inhibitor of checkpoint is an agent, such as an antibody, that interacts with a checkpoint protein. In some embodiments, the inhibitor of checkpoint is an agent, such as an antibody, that interacts with the ligand of a checkpoint protein. In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA4 antibody or fusion a protein such as ipilimumab/YERVOY® or tremelimumab). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of PD-1 (e.g., nivolumab/OPDIVO®; pembrolizumab/KEYTRUDA®; pidilizumab/CT-011). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of PDL1 (e.g., MPDL3280A/RG7446; MEDI4736; MSB0010718C; BMS 936559). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or Fc fusion or small molecule inhibitor) of PDL2 (e.g., a PDL2/Ig fusion protein such as AMP 224). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of B7-H3 (e.g., MGA271), B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands, or a combination thereof.
[0164] In some embodiments, the anti-cancer therapy is a T cell adoptive transfer (ACT) therapy. In some embodiments, the T cell is an activated T cell. The T cell may be modified to express a chimeric antigen receptor (CAR). CAR modified T (CAR-T) cells can be generated by any method known in the art. For example, the CAR-T cells can be generated by introducing a suitable expression vector encoding the CAR to a T cell. Prior to expansion and genetic modification of the T cells, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In some embodiments, the T cell is an autologous T cell. Whether prior to or after genetic modification of the T cells to express a desirable protein (e.g., a CAR), the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.
[0165] In any of the combination embodiments described herein, the first and second therapeutic agents are administered simultaneously or sequentially, in either order. The first therapeutic agent may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours up to 24 hours or up to 1-7, 1-14, 1-21 or 1-30 days before or after the second therapeutic agent.
[0166] Delivery of Anti-BICRA Agents
[0167] A variety of methods are available for the delivery of anti-BICRA agents to a subject including viral and non-viral methods.
[0168] Viral Delivery Methods
[0169] In some embodiments, the agent that reduces the level and/or activity of BICRA is delivered by a viral vector (e.g., a viral vector expressing an anti-BICRA agent). Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. 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. Examples of viral vectors 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 papillomavirus, 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 teachings of which are incorporated herein by reference.
[0170] 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. 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, each of which is incorporated herein by reference. Methods for preparation and in vivo administration of lentiviruses are described in US 20020037281 (incorporated herein by reference). Preferably, a lentiviral vector is a replication-defective lentivirus particle. Such a lentivirus particle can be produced from a lentiviral vector comprising a 5′ lentiviral LTR, a tRNA binding site, a packaging signal, a promoter operably linked to a polynucleotide signal encoding the fusion protein, an origin of second strand DNA synthesis and a 3′ lentiviral LTR.
[0171] Retroviruses are most commonly used in human clinical trials, as they carry 7-8 kb, and have the ability to infect cells and have their genetic material stably integrated into the host cell with high efficiency (see, e.g., WO 95/30761; WO 95/24929, each of which is incorporated herein by reference). Preferably, a retroviral vector is replication defective. This prevents further generation of infectious retroviral particles in the target tissue. Thus, the replication defective virus becomes a “captive” transgene stable incorporated into the target cell genome. This is typically accomplished by deleting the gag, env, and pol genes (along with most of the rest of the viral genome). Heterologous nucleic acids are inserted in place of the deleted viral genes. The heterologous genes may be under the control of the endogenous heterologous promoter, another heterologous promoter active in the target cell, or the retroviral 5′ LTR (the viral LTR is active in diverse tissues).
[0172] These delivery vectors described herein can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein (e.g., an antibody to a target cell receptor).
[0173] Reversible delivery expression systems may also be used. The Cre-loxP or FLP/FRT system and other similar systems can be used for reversible delivery-expression of one or more of the above-described nucleic acids. See WO2005/112620, WO2005/039643, US20050130919, US20030022375, US20020022018, US20030027335, and US20040216178. In particular, the reversible delivery-expression system described in US20100284990 can be used to provide a selective or emergency shut-off.
[0174] Non-Viral Delivery Methods
[0175] Several non-viral methods exist for delivery of anti-BICRA agents including polymeric, biodegradable microparticle, or microcapsule delivery devices known in the art. For example, a colloidal dispersion system may be used for targeted delivery an anti-BICRA agent described herein. 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.
[0176] The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially 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.
[0177] 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.
Pharmaceutical Compositions
[0178] The pharmaceutical compositions described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
[0179] The compounds described herein may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the methods described herein. In accordance with the methods of the invention, the described compounds or salts, solvates, or prodrugs thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds described herein may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, intratumoral, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
[0180] A compound described herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, a compound described herein may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. A compound described herein may also be administered parenterally. Solutions of a compound described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe. Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form includes an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter. A compound described herein may be administered intratumorally, for example, as an intratumoral injection. Intratumoral injection is injection directly into the tumor vasculature and is specifically contemplated for discrete, solid, accessible tumors. Local, regional, or systemic administration also may be appropriate. A compound described herein may advantageously be contacted by administering an injection or multiple injections to the tumor, spaced for example, at approximately, 1 cm intervals. In the case of surgical intervention, the present invention may be used preoperatively, such as to render an inoperable tumor subject to resection. Continuous administration also may be applied where appropriate, for example, by implanting a catheter into a tumor or into tumor vasculature.
[0181] The compounds described herein may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.
Dosages
[0182] The dosage of the compounds described herein, and/or compositions including a compound described herein, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds described herein are administered to a human at a daily dosage of, for example, between 0.05 mg and 3000 mg (measured as the solid form). Dose ranges include, for example, between 10-1000 mg (e.g., 50-800 mg). In some embodiments, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg of the compound is administered.
[0183] Alternatively, the dosage amount can be calculated using the body weight of the patient. For example, the dose of a compound, or pharmaceutical composition thereof, administered to a patient may range from 0.1-50 mg/kg (e.g., 0.25-25 mg/kg). In exemplary, non-limiting embodiments, the dose may range from 0.5-5.0 mg/kg (e.g., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/kg) or from 5.0-20 mg/kg (e.g., 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mg/kg).
Kits
[0184] The invention also features kits including (a) a pharmaceutical composition including an agent that reduces the level and/or activity of BICRA in a cell or subject described herein, and (b) a package insert with instructions to perform any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition including an agent that reduces the level and/or activity of BICRA in a cell or subject described herein, (b) an additional therapeutic agent (e.g., an anti-cancer agent), and (c) a package insert with instructions to perform any of the methods described herein.
EXAMPLES
Example 1—High Density Tiling sgRNA Screen Against Human BAF Complex Subunits in Synovial Sarcoma Cell Line SYO1
[0185] The following example shows that BICRA sgRNA inhibits cell growth in synovial sarcoma cells.
[0186] Procedure: To perform high density sgRNA tiling screen, an sgRNA library against BAF complex subunits was custom synthesized at Cellecta (Mountain View, Calif.). All BICRA-targeting sgRNAs used in this screen are listed in Table 2. Negative and positive control sgRNA were included in the library. Negative controls consisted of 200 sgRNAs that do not target human genome. The positive controls are sgRNAs targeting essential genes (CDC16, GTF2B, HSPA5, HSPA9, PAFAH1B1, PCNA, POLR2L, RPL9, and SF3A3). All positive and negative control sgRNAs are listed in Table 3. Procedures for virus production, cell infection, and performing the sgRNA screen were previously described (Tsherniak et al, Cell 170:564-576 (2017); Munoz et al, Cancer Discovery 6:900-913 (2016)). For each sgRNA, 50 counts were added to the sequencing counts and for each time point the resulting counts were normalized to the total number of counts. The log 2 of the ratio between the counts (defined as dropout ratio) at day 24 and day 1 post-infection was calculated. For negative control sgRNAs, the 2.5 and 97.5 percentile of the log 2 dropout ratio of all non-targeting sgRNAs was calculated and considered as background (grey box in the graph). Protein domains were obtained from PFAM regions defined for the UNIPROT identifier: Q9NZM4.
[0187] Results: As shown in
Example 2—High Density Tiling sgRNA Screen Against Human BAF Complex Subunits in Synovial Sarcoma Cell Line HS-SY-II
[0188] The following example shows that BICRA sgRNA inhibits cell growth in synovial sarcoma cells.
[0189] Procedure: To perform high density sgRNA tiling screen, an sgRNA library against BAF complex subunits was custom synthesized at Cellecta (Mountain View, Calif.). All BICRA-targeting sgRNAs used in this screen are listed in Table 2. Negative and positive control sgRNA were included in the library. Negative controls consisted of 200 sgRNAs that do not target human genome. The positive controls are sgRNAs targeting essential genes (CDC16, GTF2B, HSPA5, HSPA9, PAFAH1B1, PCNA, POLR2L, RPL9, and SF3A3). All positive and negative control sgRNAs are listed in Table 3. Procedures for virus production, cell infection, and performing the sgRNA screen were previously described (Tsherniak et al, Cell 170:564-576 (2017); Munoz et al, Cancer Discovery 6:900-913 (2016)). For each sgRNA, 50 counts were added to the sequencing counts and for each time point the resulting counts were normalized to the total number of counts. The log 2 of the ratio between the counts (defined as dropout ratio) at day 24 and day 1 post-infection was calculated. For negative control sgRNAs, the 2.5 and 97.5 percentile of the log 2 dropout ratio of all non-targeting sgRNAs was calculated and considered as background (grey box in the graph). Protein domains were obtained from PFAM regions defined for the UNIPROT identifier: Q9NZM4.
[0190] Results: As shown in
TABLE-US-00002 TABLE 2 BICRA sgRNA Library SEQ ID SEQ ID NO Nucleic Acid Sequence NO Nucleic Acid Sequence 125 GGAGGAGGCCACAGCAGAGG 354 GGCGTGTTGAGCGCCATGAC 126 GGTGGAGGATGGAGGGAGGT 355 GTTCAGGAGGTGGACGCTCA 127 CTTGGCCAGGAGCTGGGAGG 356 TCCGAGGGTCATCGGCTTCC 128 GCTGCCTGGGAAGAGGGCTT 357 CGGAAGAGGCTGCGATGGGG 129 ACTGAGCCTGGGCCCCGTGT 358 TGGCCACATTCAGGGTCGGG 130 CTTGGTGGGCAGGTGTGGAG 359 CTGCGTCTGTGCTGGTCAGT 131 CCACCCTCCAGTCACAACGG 360 CTGCTTGGCCAGGAGCTGGG 132 TACCTCACCAGGCCCCTCTG 361 GGTGAGCCCCTCAGCGGAGA 133 TCTGCAGGGAGTCCTGAGTG 362 CACGGGCCAGATGAACGGCA 134 GGTTGGGCTCTGGGTTCAGG 363 AACTCTGTGTTCGGAGGCGC 135 GCCCGGAGAAGATCGTCCTG 364 TGCTGCCTTGGTTCAGGAGG 136 ATCCTCCACCTCCTCTGCTG 365 GGAGGGCGCCCTGGTAGACA 137 AAGGCACTTTGGGCCTCCCC 366 GCCATGAACGTCAGGTTCTG 138 AAGTGGACGAGGCCACCAGC 367 TTGTTGACCACGTCCTGGGG 139 GACCCCCTGGAGGACAGTGG 368 TAAATATCGGCTCCTGCTCC 140 AGGGCCGAGGCCAGCTCCTT 369 CCCGACCCTGAATGTGGCCA 141 AGGGTGCAAGGGTGGCTCTG 370 CCCTCGGAAGAGGCTGCGAT 142 GGAGGCCACAGCAGAGGAGG 371 TACCCACCCTCCAGTCACAA 143 TAGCCCGGAGAAGATCGTCC 372 ACCCCCCGCTACCCTCAAGG 144 AAAGATGCCAGGCAGAGTTG 373 GGGGGCCCTTCAGCATACGC 145 GTGGAGGGTTGGAGGGGGAG 374 GCCCAAAGTGCCTTCTATGA 146 CCTCGGAAGAGGCTGCGATG 375 TCAGGGACCAGGTGGAGGGT 147 CATCCTCCAGAACAAGGCTG 376 GTCAGCTGGTAGAGCTTGGG 148 CCTGGAGGACGTCTCAGAGG 377 CTGCCTGGGAAGAGGGCTTG 149 TGTGCGATGCAGGATCACGT 378 TGCTCCTGGAGGAGTCCCGG 150 TGCCCACCAAGCTTGTGATC 379 CAGGCAGTAGGAACTGGTTC 151 GAAGCTGGCCTGGTCCACGT 380 GATGGCGCTCTGCAGGTGCT 152 GTCCAAGGAGGAGGCGGCAG 381 CTTCCTGGAAGATGACATCC 153 GGACCAGGTGGAGGGTTGGA 382 ACAGCTGGCCAAGGAGAAGC 154 ATCATTACCATCTCCGCTGA 383 CTGACCAGCACAGACGCAGC 155 TGGAGGATGGAGGGAGGTGG 384 GAAGTCTAGGTCCACACTGG 156 TGGAAGGTCCGAGGAGGCGG 385 ATGGGTTTGCTCAGGGCCCC 157 TGGGCAGCTGGAAGGGACCC 386 GATGACCTCCTGGATAATCC 158 GATCATTACCATCTCCGCTG 387 GTGCCTTCTATGAAGGTCCT 159 ACAGCAGAGGAGGTGGAGGA 388 CCCCACGGCCATCCTCACTC 160 TCCAGTTCCCACCCAGCCAG 389 AGCTTGTGATCCGGCACGGC 161 CTGAGTGAGGATGGCCGTGG 390 CCTCCTGGATAATCCCGGGG 162 TGACCTCCTGGATAATCCCG 391 CTACCTCACCAGGCCCCTCT 163 CTCTGCTGGAGGATGTCACA 392 CTCTGGGTTCAGGTGGTTGC 164 GCAAGGGTGGCTCTGAGGGA 393 TGCAAGGGTGGCTCTGAGGG 165 TGCCCCAGGACGATCTTCTC 394 CAAACGGCTTGGGCGTCAGC 166 CTCCTCTGCCGCCTCCTCCT 395 TTGCTGGGCTGAAGCGTGGG 167 GCCTCCTCGGACCTTCCAGA 396 TGGTCAACGTCCTGGCGCCG 168 AGAGGTTCTTCTGGATGACC 397 GGCGTCAGCTGGTAGAGCTT 169 TCTTCTCGAGAGATTTCACC 398 GTGCCGGATCACAAGCTTGG 170 AGCGTCCACCTCCTGAACCA 399 TGCAGGGCGTCCTCAAAGGA 171 TCTGCTTCCAGCCGAGAACA 400 GATCGACCGAATGTTCATTC 172 GGGCCTCCCCGGGATTATCC 401 CCTGGGGCAGGAACATCTGC 173 TGCCGGATCACAAGCTTGGT 402 CAGCGTCTGCTCCGTGATGT 174 AGAGTTCCCATTGAGCGTGG 403 TCCATGCAAGAAGTCATTGA 175 GGTGGCCTCGTCCACTTTGG 404 CAGGAAGTCTAGGTCCACAC 176 TTCCTGGAAGATGACATCCT 405 CCCCAGTGACTACCACAAAG 177 CTGCCCTACCATGTCTACCA 406 TGGCCTCTTGGAGGCTCTGC 178 TCCTGGGCTCTCCTGCGACA 407 TTCCTTGCTGGGCTGAAGCG 179 GCCCCAGGACGATCTTCTCC 408 GGGGGTGGTCACCATCTGGA 180 CTCGAGAAGAGCCTTCGGCT 409 CAGAACCAGTTCCTACTGCC 181 GGTAGTGACGGGGACGAAGA 410 CTGTGGCCACCACGCTCAAT 182 CGGTGAGCGTGTCATCCTGC 411 CAGCAAGGTCGTGCACAACA 183 GAAGAACCTCTCGGCCGCTG 412 GACGTTCATGGCGGCGGGGA 184 CTGCAGGGTGGGCAGCTGGA 413 AGTGGACGAGGAGTTTGAGA 185 TTGGCCAGGAGCTGGGAGGT 414 GGGACCAGGTGGAGGGTTGG 186 AGTGCCTTCTATGAAGGTCC 415 CTGTACCAGCGTATGCTGAA 187 TCCTGCTCCTGGAGGAGTCC 416 TCAGTGGGCCCGCCAGGTTC 188 GCCGTGCCGGATCACAAGCT 417 GGGCGCCAGGCAGTAGGAAC 189 CGGCTGGAAGCAGAGGAGGG 418 GGGGGTGTTGAGCATGGCCA 190 ACCCCCGTCGCCAAAGGAGC 419 GACCTTCATAGAAGGCACTT 191 GTCAGTGGGCAGGCCCCATC 420 TCTCGGCTGGAAGCAGAGGA 192 ACCCTCGGAAGAGGCTGCGA 421 GTCTGTGCTGGTCAGTGGGC 193 GGTGACCCCCTGGAGGACAG 422 GCTGGAAGTCGGATGGCGTA 194 GCAGGGCGTCCTCAAAGGAG 423 GTTGAGCATGGCCACGGCGC 195 CGGAGAGGATGTGCGCGCTG 424 AAGCTTGTGATCCGGCACGG 196 ACTACCTCACCAGGCCCCTC 425 GGTAGGGCAGGAGGCGATGC 197 TCTTGAGCTTGAGCCCGATG 426 GTCGGTGCTGCCTGGGAAGA 198 ATATCGGCTCCTGCTCCTGG 427 CATGGGTTTGCTCAGGGCCC 199 GTTGTGACTGGAGGGTGGGT 428 TGGGAACTGGAGCTGGAAGT 200 AGCGGCCTTGGCCACATTCA 429 GAGGCCAGCTCCTTTGGCGA 201 AGATGCCAGGCAGAGTTGGG 430 GTCACCATCTGGAAGGTCCG 202 GGCCTCCAAGGCCTGCCCAA 431 AATCTCTCGAGAAGAGCCTT 203 GGAAGGGGGTGGTCACCATC 432 AGTGAGGATGGCCGTGGGGG 204 ATGCAGGGCGTCCTCAAAGG 433 AAGATGCCAGGCAGAGTTGG 205 ATCCTCCAGAACAAGGCTGG 434 CCTGCAGATGTTCCTGCCCC 206 TTCTCGGCTGGAAGCAGAGG 435 GCAGGAAGCCGGGCTCAGCA 207 CAGCCCTTCCTGCAGCCTGT 436 CGGGCCTCGTAGGTCTTGAT 208 TGACCGAGGCAGGCACGGAG 437 GTCCTGAGTGAGGATGGCCG 209 CCTCGTAGGTCTTGATGGGC 438 ACCTACCGCGAGAACGTGGG 210 GCTGCAGTGTCACATTGCCC 439 CTCCAGTTCCCACCCAGCCA 211 ACGGGCATCTGGAAGAGCGC 440 AACTGGAGCTGGAAGTCGGA 212 GGGCCTTGTTGACCACGTCC 441 GGGAAGGCCGTCTTGTAGTC 213 AGCTTGGTGGGCAGGTGTGG 442 GGGGCCTGGTGAGGTAGTGA 214 GCTTGACAGTGATGACCTCC 443 ACCTGACGTTCATGGCGGCG 215 GCCTTGTTGACCACGTCCTG 444 GCCGTCGGGGGTGTTGAGCA 216 ACCAGGTGGAGGGTTGGAGG 445 GGTAGTCACTGGGGGAGGGG 217 GTCCTCGTCGGCGTCCAAGG 446 CTCCTCCTTGGACGCCGACG 218 ACCAAGCTTGTGATCCGGCA 447 ACTCTGTGTTCGGAGGCGCG 219 ATGACCTCCTGGATAATCCC 448 CGTAGGGGCTGGCAACCTGG 220 TCATCCTCCAGAACAAGGCT 449 CTTGTAGTCGGGGTGCAGGA 221 GGTGCGTTTCAGCAGCTGCG 450 CGTGCCGTTCATCTGGCCCG 222 TGCTGCTGCCTTGGTTCAGG 451 ATGACCAGGCCAGCCCCCTG 223 GCGATGCAGGGCGTCCTCAA 452 GTCCCCGTCACTACCTCACC 224 GGTCATCCAGAAGAACCTCT 453 AGAAGTCATTGAGGGCCTGT 225 CTTGGCCAGCTGTTTATCCA 454 AGCCCAGGATGTCATCTTCC 226 CGGGGCGCTGACTATGACCG 455 CTGAGAGCTGCTGCGGGAGC 227 CCTCCTCTGAGACGTCCTCC 456 AGCTGGAAGTCGGATGGCGT 228 AAGCCGATGACCCTCGGAAG 457 GTGGCCTCGTCCACTTTGGC 229 TCTAGGTCCACACTGGGGGC 458 GGTCGGTGCTGCCTGGGAAG 230 TCGGTGAGCGTGTCATCCTG 459 GGTTCTGGTTTGTGAGGATG 231 CCCATCGCAGCCTCTTCCGA 460 ACTGGAGGGTGGGTAGGCCT 232 GTGACACTGCAGCCCATCCC 461 GAACCCAGAGCCCAACCAGC 233 CAAGTCCGAGTCGCCCGACG 462 CCTGAGTGAGGATGGCCGTG 234 TCCCCCTCCAACCCTCCACC 463 ATTCAGGGTCGGGAGGTTGC 235 GCTCCGTGATGTTGGCCTCT 464 GCCTACCGTGCTGACCCACC 236 GCTGGTGGCCTCGTCCACTT 465 CAGTATGAGAGCAAACTGAG 237 ACCATCTGGAAGGTCCGAGG 466 AGGCCATGCTCAATAAATAT 238 CCGGGCCTCGTAGGTCTTGA 467 GGGCCTGGTGAGGTAGTGAC 239 GACCTACCGCGAGAACGTGG 468 CAGCATCCTGAACCTGCAGC 240 CCTCCCCGGGATTATCCAGG 469 GCCCCAGGACGTGGTCAACA 241 GGAGAGGATGTGCGCGCTGT 470 ATGAGCTGTCCACCTGTGTG 242 GGGTGCAAGGGTGGCTCTGA 471 TCTTGATGGGCGGGCGGTTG 243 TGAGCTGTCCACCTGTGTGG 472 CAGCCTCTTCCGAGGGTCAT 244 GCAGCTGCTGAAACGCACCC 473 CCATGAACGTCAGGTTCTGC 245 AGCCCGGAGAAGATCGTCCT 474 AAGTCGGATGGCGTAGGGGC 246 TCAGTGGGCAGGCCCCATCT 475 CGATGCTGCTGCCTTGGTTC 247 ACCTTCATAGAAGGCACTTT 476 TGGGCGTGGGTGTGCGATGC 248 CCTCCAAGAGGCCAACATCA 477 GGTAGGTCTTGCGCAGTGGC 249 ACCCAGGTCCAGCTCAGCCT 478 GGATCACAAGCTTGGTGGGC 250 TCCTTGCTGGGCTGAAGCGT 479 CTGGTACAGCTCGTCCTCCA 251 TGTCATCTTCCAGGAAGTCT 480 CCCCACGCTTCAGCCCAGCA 252 TTTGTCATCCAAAACCAGCT 481 ACCTTGAGGGTAGCGGGGGG 253 AGGCCCACAGGCTGCAGGAA 482 ACAAAGATGCCAGGCAGAGT 254 AGCCCGACAGCACCACGTTC 483 GACGGCCTTCCCCTCCTTTG 255 GCTGTGGCCACCACGCTCAA 484 CTTGCTGGGCTGAAGCGTGG 256 CAGGATCTGCCCGCCCACGT 485 TCTTCTCCGGGCTAGACGCC 257 GGTAGACATGGTAGGGCAGG 486 AGGCCAGCTCCTTTGGCGAC 258 CAACGTGGGCGGGCAGATCC 487 GGCGTCCTCAAAGGAGGGGA 259 CCTTGCCTTGGATAAACAGC 488 AGGAAGTCTAGGTCCACACT 260 TCGAGAAGAGCCTTCGGCTG 489 GGAGGGCGGGACACACTCTG 261 CCTTGCTGGGCTGAAGCGTG 490 CAGTGTGGACCTAGACTTCC 262 GGCCTGGTGAGGTAGTGACG 491 GTCACATTGCCCAGGCCCAC 263 TCTCGAGAAGAGCCTTCGGC 492 GCTTGGTGGGCAGGTGTGGA 264 ATTGAGCGTGGTGGCCACAG 493 GCCCTCAATGACTTCTTGCA 265 CTCGTCGGCGTCCAAGGAGG 494 CCACCGTTGTGACTGGAGGG 266 GCTGGCAAAAGCCTTGTTCT 495 TGACCAGCACAGACGCAGCG 267 CAGGAAGGGCTGCACACTCA 496 CAGCTGTTTATCCAAGGCAA 268 GATGAGCTGTCCACCTGTGT 497 TGACGGGCACCTGCTTGGCC 269 CAACATCACGGAGCAGACGC 498 CACAGAGTTCCCATTGAGCG 270 CAGGCCCACAGGCTGCAGGA 499 GGTCGTGCACAACACGGCCC 271 TGCAGGGTGGGCAGCTGGAA 500 TGCCATTGGGCAGGCCTTGG 272 CCTGCCCTACCATGTCTACC 501 CACCTGCTTGGCCAGGAGCT 273 AAAGTGGACGAGGCCACCAG 502 CAGGGAGTCCTGAGTGAGGA 274 CTCAATGGGAACTCTGTGTT 503 GGGCGTCAGCTGGTAGAGCT 275 TCGCGGTAGGTCTTGCGCAG 504 CTGTTTATCCAAGGCAAGGG 276 GGAAGGCCGTCTTGTAGTCG 505 GAAGGCACTTTGGGCCTCCC 277 GCCTTGGCCACATTCAGGGT 506 TTCTGGAGGATGATTCCGGA 278 GCAGAACCTGACGTTCATGG 507 AGTAGGAACTGGTTCTGGCC 279 GTCATCCTGCGGGCTGTCGC 508 AGGCCACCGTTGTGACTGGA 280 CCTCACAAACCAGAACCTGG 509 GGATGACGATGCTGCTGCCT 281 AACACGGGGCCCAGGCTCAG 510 CGGGCTGTCGCTGGAGAAGC 282 CATCCTCACAAACCAGAACC 511 GTGCACGACCTTGCTGAGCC 283 CTCCATGTGCAAGAAGCTTC 512 CTGGGGCAGGAACATCTGCA 284 AGGGGGAGTGGGGGACTTGT 513 GTCGCCAAAGGAGCTGGCCT 285 GCGCTGACTATGACCGAGGC 514 TCAAGATCAAGCAGGAAGCC 286 AAGCGGCCTTGGCCACATTC 515 ATCTGGAAGGTCCGAGGAGG 287 TTCAGGAGGTGGACGCTCAT 516 CTCGGCCACCTTGAGGGTAG 288 TTGTTCTCGGCTGGAAGCAG 517 CATGAACGTCAGGTTCTGCG 289 CTTCTCCAGCGACAGCCCGC 518 GGATGATTCCGGACGGCACC 290 CCTGGTAGACATGGTAGGGC 519 CCTCTTGGAGGCTCTGCTGG 291 GCTGGAGGATGTCACAGGGC 520 GAACTCTGTGTTCGGAGGCG 292 GGCGCCCTGGTAGACATGGT 521 ATATTTATTGAGCATGGCCT 293 GATGCCAGGCAGAGTTGGGG 522 CGACTCGGACTTGCGCCGCT 294 GGTCCACCGTGCCGTTCATC 523 CTCACAAACCAGAACCTGGC 295 CCTTGGCCACATTCAGGGTC 524 TTTGTGGTAGTCACTGGGGG 296 GCCATGCTCAACACCCCCGA 525 TCATTACCATCTCCGCTGAG 297 TGCTGTCGATGGCGCTCTGC 526 GAGCATTTGCACAAACACCA 298 GCCTCGTAGGTCTTGATGGG 527 TCGTCCACTTTGGCGGGCAG 299 GCAGGAAGGGCTGCACACTC 528 ATCCTGGGCTCTCCTGCGAC 300 CTGGAAGTCGGATGGCGTAG 529 GCATCTGGAAGAGCGCAGGC 301 GTAGGGCAGGAGGCGATGCA 530 CTCGCCCTGGATGGTGAGCG 302 TGGCGTAGGGGCTGGCAACC 531 TTGAGCATGGCCACGGCGCT 303 GCTCTGCTGGAGGATGTCAC 532 GGGCGTGTTGAGCGCCATGA 304 TCTGGCCCGGCAGCATGTGC 533 ATCCATGCAAGAAGTCATTG 305 GAGGGGGAGTGGGGGACTTG 534 TCGGCCACCTTGAGGGTAGC 306 AGGTAGTGACGGGGACGAAG 535 CCAGCTGTTTATCCAAGGCA 307 CAACCTCCCGACCCTGAATG 536 CTCCCAGCTCCTGGCCAAGC 308 GCTGGTTGGGCTCTGGGTTC 537 ACAAGCTTGGTGGGCAGGTG 309 GGGCCAGAACGTGGTGCTGT 538 GCTGTACCAGCGTATGCTGA 310 CACCGTTGTGACTGGAGGGT 539 CTTGGATAAACAGCTGGCCA 311 TCTCCTCCTGAATGAACATT 540 CGTAGGTCTTGATGGGCGGG 312 GCAGCCCTTCCTGCAGCCTG 541 TCCCCGCCGCCATGAACGTC 313 TCCTTGGACGCCGACGAGGA 542 GAGCCGATATTTATTGAGCA 314 GACCAGGTGGAGGGTTGGAG 543 GAGGCCACCGTTGTGACTGG 315 CAACCTGGAGGACGTCTCAG 544 AAGAAGTCATTGAGGGCCTG 316 CCGTGATGTTGGCCTCTTGG 545 CTCAAGATCAAGCAGGAAGC 317 TCCTGAGTGAGGATGGCCGT 546 CAGGTTCTGGTTTGTGAGGA 318 GGACACACTCTGTGGCCGGG 547 GTCAGGTTCTGCGGGGCCTT 319 ACTGACCAGCACAGACGCAG 548 CAAAGATGCCAGGCAGAGTT 320 TCCTCCAGAACAAGGCTGGG 549 GCAGGCTGCACCGTGAGGAC 321 GGAGCATTTGCACAAACACC 550 TGTTGAGCGCCATGACGGGC 322 ATCATCCTCCAGAACAAGGC 551 GTGCAAATGCTCCAGGAAAC 323 GGCCTTGTTGACCACGTCCT 552 AACCTGACGTTCATGGCGGC 324 GACACACTCTGTGGCCGGGA 553 CTTCCAGATGCCCGTGTCGC 325 GGGCCAGATGAACGGCACGG 554 TGCTGCCTGGGAAGAGGGCT 326 AGGTTCTGGTTTGTGAGGAT 555 ACACTCTGTGGCCGGGAGGG 327 AATGGGAACTCTGTGTTCGG 556 TCGGACTTGCGCCGCTTGGC 328 GCTCAAGCTCAAGATCAAGC 557 CTTGTTCTGGAGGATGATTC 329 GCACCTGCTTGGCCAGGAGC 558 GCAAAAGCCTTGTTCTCGGC 330 TTGTGGTAGTCACTGGGGGA 559 CGATGAGCTGTCCACCTGTG 331 GAAGCGTGGGGGGCTTCTTC 560 GCTGCCCGCCAAAGTGGACG 332 GTGCCGTTCATCTGGCCCGT 561 CCCCCCCAGCCTTGTTCTGG 333 CAGCGGCCGAGAGGTTCTTC 562 GCCGAGGCCACCGTTGTGAC 334 GACTATGACCGAGGCAGGCA 563 CGACCGAATGTTCATTCAGG 335 AGCTCCTTTGGCGACGGGGG 564 CTTCCTGCAGCCTGTGGGCC 336 GGACGCTCATGGGTTTGCTC 565 CCGCCAGGTTCTGGTTTGTG 337 CCGTCTTGTAGTCGGGGTGC 566 TCCAGTCACAACGGTGGCCT 338 CCCCATCGCAGCCTCTTCCG 567 GATATTTATTGAGCATGGCC 339 TGTGACACTGCAGCCCATCC 568 GGCTGCCATTGGGCAGGCCT 340 GGGGAAGGCCGTCTTGTAGT 569 TGGTGGGTCAGCACGGTAGG 341 CCCGCAGAACCTGACGTTCA 570 TTCCTGCAGCCTGTGGGCCT 342 CATCACGGAGCAGACGCTGG 571 GCGCCCTGGTAGACATGGTA 343 AAGCTGGCCTGGTCCACGTC 572 TGCCGGAAGCTTCTTGCACA 344 TGTGGTAGTCACTGGGGGAG 573 CCTCCAGCAGAGCCTCCAAG 345 GCTGCGTCTGTGCTGGTCAG 574 GTAGTCACTGGGGGAGGGGA 346 GGTGTTTGTGCAAATGCTCC 575 CGTCAGGTTCTGCGGGGCCT 347 GGAAGTCTAGGTCCACACTG 576 CAGGTTCAGGATGCTGTCGA 348 GAACCTGACGTTCATGGCGG 577 TGTCGCTGGAGAAGCTGGCC 349 GTAGTGACGGGGACGAAGAG 578 GGCCAGAACGTGGTGCTGTC 350 GACGCTCATGGGTTTGCTCA 579 GAGCTGTCCACCTGTGTGGG 351 CCTCCAGAACAAGGCTGGGG 580 GCTCCAGTTCCCACCCAGCC 352 CGGAATCATCCTCCAGAACA 581 GGCTGGGTGGGAACTGGAGC 353 GCCTGGTGGGTCAGCACGGT
TABLE-US-00003 TABLE 3 Control sgRNA Library SEQ ID NO. gRNA Label Gene Nucleic Acid Sequence 582 1|sg_Non_Targeting_Human_0001| Non-Targeting_Human GTAGCGAACGTGTCCGGCGT Non_Targeting_Human 583 1|sg_Non_Targeting_Human_0002| Non-Targeting_Human GACCGGAACGATCTCGCGTA Non_Targeting_Human 584 1|sg_Non_Targeting_Human_0003| Non-Targeting_Human GGCAGTCGTTCGGTTGATAT Non_Targeting_Human 585 1|sg_Non_Targeting_Human_0004| Non-Targeting_Human GCTTGAGCACATACGCGAAT Non_Targeting_Human 586 1|sg_Non_Targeting_Human_0005| Non-Targeting_Human GTGGTAGAATAACGTATTAC Non_Targeting_Human 587 1|sg_Non_Targeting_Human_0006| Non-Targeting_Human GTCATACATGGATAAGGCTA Non_Targeting_Human 588 1|sg_Non_Targeting_Human_0007| Non-Targeting_Human GATACACGAAGCATCACTAG Non_Targeting_Human 589 1|sg_Non_Targeting_Human_0008| Non-Targeting_Human GAACGTTGGCACTACTTCAC Non_Targeting_Human 590 1|sg_Non_Targeting_Human_0009| Non-Targeting_Human GATCCATGTAATGCGTTCGA Non_Targeting_Human 591 1|sg_Non_Targeting_Human_0010| Non-Targeting_Human GTCGTGAAGTGCATTCGATC Non_Targeting_Human 592 1|sg_Non_Targeting_Human_0011| Non-Targeting_Human GTTCGACTCGCGTGACCGTA Non_Targeting_Human 593 1|sg_Non_Targeting_Human_0012| Non-Targeting_Human GAATCTACCGCAGCGGTTCG Non_Targeting_Human 594 1|sg_Non_Targeting_Human_0013| Non-Targeting_Human GAAGTGACGTCGATTCGATA Non_Targeting_Human 595 1|sg_Non_Targeting_Human_0014| Non-Targeting_Human GCGGTGTATGACAACCGCCG Non_Targeting_Human 596 1|sg_Non_Targeting_Human_0015| Non-Targeting_Human GTACCGCGCCTGAAGTTCGC Non_Targeting_Human 597 1|sg_Non_Targeting_Human_0016| Non-Targeting_Human GCAGCTCGTGTGTCGTACTC Non_Targeting_Human 598 1|sg_Non_Targeting_Human_0017| Non-Targeting_Human GCGCCTTAAGAGTACTCATC Non_Targeting_Human 599 1|sg_Non_Targeting_Human_0018| Non-Targeting_Human GAGTGTCGTCGTTGCTCCTA Non_Targeting_Human 600 1|sg_Non_Targeting_Human_0019| Non-Targeting_Human GCAGCTCGACCTCAAGCCGT Non_Targeting_Human 601 1|sg_Non_Targeting_Human_0020| Non-Targeting_Human GTATCCTGACCTACGCGCTG Non_Targeting_Human 602 1|sg_Non_Targeting_Human_0021| Non-Targeting_Human GTGTATCTCAGCACGCTAAC Non_Targeting_Human 603 1|sg_Non_Targeting_Human_0022| Non-Targeting_Human GTCGTCATACAACGGCAACG Non_Targeting_Human 604 1|sg_Non_Targeting_Human_0023| Non-Targeting_Human GTCGTGCGCTTCCGGCGGTA Non_Targeting_Human 605 1|sg_Non_Targeting_Human_0024| Non-Targeting_Human GCGGTCCTCAGTAAGCGCGT Non_Targeting_Human 606 1|sg_Non_Targeting_Human_0025| Non-Targeting_Human GCTCTGCTGCGGAAGGATTC Non_Targeting_Human 607 1|sg_Non_Targeting_Human_0026| Non-Targeting_Human GCATGGAGGAGCGTCGCAGA Non_Targeting_Human 608 1|sg_Non_Targeting_Human_0027| Non-Targeting_Human GTAGCGCGCGTAGGAGTGGC Non_Targeting_Human 609 1|sg_Non_Targeting_Human_0028| Non-Targeting_Human GATCACCTGCATTCGTACAC Non_Targeting_Human 610 1|sg_Non_Targeting_Human_0029| Non-Targeting_Human GCACACCTAGATATCGAATG Non_Targeting_Human 611 1|sg_Non_Targeting_Human_0030| Non-Targeting_Human GTTGATCAACGCGCTTCGCG Non_Targeting_Human 612 1|sg_Non_Targeting_Human_0031| Non-Targeting_Human GCGTCTCACTCACTCCATCG Non_Targeting_Human 613 1|sg_Non_Targeting_Human_0032| Non-Targeting_Human GCCGACCAACGTCAGCGGTA Non_Targeting_Human 614 1|sg_Non_Targeting_Human_0033| Non-Targeting_Human GGATACGGTGCGTCAATCTA Non_Targeting_Human 615 1|sg_Non_Targeting_Human_0034| Non-Targeting_Human GAATCCAGTGGCGGCGACAA Non_Targeting_Human 616 1|sg_Non_Targeting_Human_0035| Non-Targeting_Human GCACTGTCAGTGCAACGATA Non_Targeting_Human 617 1|sg_Non_Targeting_Human_0036| Non-Targeting_Human GCGATCCTCAAGTATGCTCA Non_Targeting_Human 618 1|sg_Non_Targeting_Human_0037| Non-Targeting_Human GCTAATATCGACACGGCCGC Non_Targeting_Human 619 1|sg_Non_Targeting_Human_0038| Non-Targeting_Human GGAGATGCATCGAAGTCGAT Non_Targeting_Human 620 1|sg_Non_Targeting_Human_0039| Non-Targeting_Human GGATGCACTCCATCTCGTCT Non_Targeting_Human 621 1|sg_Non_Targeting_Human_0040| Non-Targeting_Human GTGCCGAGTAATAACGCGAG Non_Targeting_Human 622 1|sg_Non_Targeting_Human_0041| Non-Targeting_Human GAGATTCCGATGTAACGTAC Non_Targeting_Human 623 1|sg_Non_Targeting_Human_0042| Non-Targeting_Human GTCGTCACGAGCAGGATTGC Non_Targeting_Human 624 1|sg_Non_Targeting_Human_0043| Non-Targeting_Human GCGTTAGTCACTTAGCTCGA Non_Targeting_Human 625 1|sg_Non_Targeting_Human_0044| Non-Targeting_Human GTTCACACGGTGTCGGATAG Non_Targeting_Human 626 1|sg_Non_Targeting_Human_0045| Non-Targeting_Human GGATAGGTGACCTTAGTACG Non_Targeting_Human 627 1|sg_Non_Targeting_Human_0046| Non-Targeting_Human GTATGAGTCAAGCTAATGCG Non_Targeting_Human 628 1|sg_Non_Targeting_Human_0047| Non-Targeting_Human GCAACTATTGGAATACGTGA Non_Targeting_Human 629 1|sg_Non_Targeting_Human_0048| Non-Targeting_Human GTTACCTTCGCTCGTCTATA Non_Targeting_Human 630 1|sg_Non_Targeting_Human_0049| Non-Targeting_Human GTACCGAGCACCACAGGCCG Non_Targeting_Human 631 1|sg_Non_Targeting_Human_0050| Non-Targeting_Human GTCAGCCATCGGATAGAGAT Non_Targeting_Human 632 1|sg_Non_Targeting_Human_0051| Non-Targeting_Human GTACGGCACTCCTAGCCGCT Non_Targeting_Human 633 1|sg_Non_Targeting_Human_0052| Non-Targeting_Human GGTCCTGTCGTATGCTTGCA Non_Targeting_Human 634 1|sg_Non_Targeting_Human_0053| Non-Targeting_Human GCCGCAATATATGCGGTAAG Non_Targeting_Human 635 1|sg_Non_Targeting_Human_0054| Non-Targeting_Human GCGCACGTATAATCCTGCGT Non_Targeting_Human 636 1|sg_Non_Targeting_Human_0055| Non-Targeting_Human GTGCACAACACGATCCACGA Non_Targeting_Human 637 1|sg_Non_Targeting_Human_0056| Non-Targeting_Human GCACAATGTTGACGTAAGTG Non_Targeting_Human 638 1|sg_Non_Targeting_Human_0057| Non-Targeting_Human GTAAGATGCTGCTCACCGTG Non_Targeting_Human 639 1|sg_Non_Targeting_Human_0058| Non-Targeting_Human GTCGGTGATCCAACGTATCG Non_Targeting_Human 640 1|sg_Non_Targeting_Human_0059| Non-Targeting_Human GAGCTAGTAGGACGCAAGAC Non_Targeting_Human 641 1|sg_Non_Targeting_Human_0060| Non-Targeting_Human GTACGTGGAAGCTTGTGGCC Non_Targeting_Human 642 1|sg_Non_Targeting_Human_0061| Non-Targeting_Human GAGAACTGCCAGTTCTCGAT Non_Targeting_Human 643 1|sg_Non_Targeting_Human_0062| Non-Targeting_Human GCCATTCGGCGCGGCACTTC Non_Targeting_Human 644 1|sg_Non_Targeting_Human_0063| Non-Targeting_Human GCACACGACCAATCCGCTTC Non_Targeting_Human 645 1|sg_Non_Targeting_Human_0064| Non-Targeting_Human GAGGTGATCGATTAAGTACA Non_Targeting_Human 646 1|sg_Non_Targeting_Human_0065| Non-Targeting_Human GTCACTCGCAGACGCCTAAC Non_Targeting_Human 647 1|sg_Non_Targeting_Human_0066| Non-Targeting_Human GCGCTACGGAATCATACGTT Non_Targeting_Human 648 1|sg_Non_Targeting_Human_0067| Non-Targeting_Human GGTAGGACCTCACGGCGCGC Non_Targeting_Human 649 1|sg_Non_Targeting_Human_0068| Non-Targeting_Human GAACTGCATCTTGTTGTAGT Non_Targeting_Human 650 1|sg_Non_Targeting_Human_0069| Non-Targeting_Human GATCCTGATCCGGCGGCGCG Non_Targeting_Human 651 1|sg_Non_Targeting_Human_0070| Non-Targeting_Human GGTATGCGCGATCCTGAGTT Non_Targeting_Human 652 1|sg_Non_Targeting_Human_0071| Non-Targeting_Human GCGGAGCTAGAGAGCGGTCA Non_Targeting_Human 653 1|sg_Non_Targeting_Human_0072| Non-Targeting_Human GAATGGCAATTACGGCTGAT Non_Targeting_Human 654 1|sg_Non_Targeting_Human_0073| Non-Targeting_Human GTATGGTGAGTAGTCGCTTG Non_Targeting_Human 655 1|sg_Non_Targeting_Human_0074| Non-Targeting_Human GTGTAATTGCGTCTAGTCGG Non_Targeting_Human 656 1|sg_Non_Targeting_Human_0075| Non-Targeting_Human GGTCCTGGCGAGGAGCCTTG Non_Targeting_Human 657 1|sg_Non_Targeting_Human_0076| Non-Targeting_Human GAAGATAAGTCGCTGTCTCG Non_Targeting_Human 658 1|sg_Non_Targeting_Human_0077| Non-Targeting_Human GTCGGCGTTCTGTTGTGACT Non_Targeting_Human 659 1|sg_Non_Targeting_Human_0078| Non-Targeting_Human GAGGCAAGCCGTTAGGTGTA Non_Targeting_Human 660 1|sg_Non_Targeting_Human_0079| Non-Targeting_Human GCGGATCCAGATCTCATTCG Non_Targeting_Human 661 1|sg_Non_Targeting_Human_0080| Non-Targeting_Human GGAACATAGGAGCACGTAGT Non_Targeting_Human 662 1|sg_Non_Targeting_Human_0081| Non-Targeting_Human GTCATCATTATGGCGTAAGG Non_Targeting_Human 663 1|sg_Non_Targeting_Human_0082| Non-Targeting_Human GCGACTAGCGCCATGAGCGG Non_Targeting_Human 664 1|sg_Non_Targeting_Human_0083| Non-Targeting_Human GGCGAAGTTCGACATGACAC Non_Targeting_Human 665 1|sg_Non_Targeting_Human_0084| Non-Targeting_Human GCTGTCGTGTGGAGGCTATG Non_Targeting_Human 666 1|sg_Non_Targeting_Human_0085| Non-Targeting_Human GCGGAGAGCATTGACCTCAT Non_Targeting_Human 667 1|sg_Non_Targeting_Human_0086| Non-Targeting_Human GACTAATGGACCAAGTCAGT Non_Targeting_Human 668 1|sg_Non_Targeting_Human_0087| Non-Targeting_Human GCGGATTAGAGGTAATGCGG Non_Targeting_Human 669 1|sg_Non_Targeting_Human_0088| Non-Targeting_Human GCCGACGGCAATCAGTACGC Non_Targeting_Human 670 1|sg_Non_Targeting_Human_0089| Non-Targeting_Human GTAACCTCTCGAGCGATAGA Non_Targeting_Human 671 1|sg_Non_Targeting_Human_0090| Non-Targeting_Human GACTTGTATGTGGCTTACGG Non_Targeting_Human 672 1|sg_Non_Targeting_Human_0091| Non-Targeting_Human GTCACTGTGGTCGAACATGT Non_Targeting_Human 673 1|sg_Non_Targeting_Human_0092| Non-Targeting_Human GTACTCCAATCCGCGATGAC Non_Targeting_Human 674 1|sg_Non_Targeting_Human_0093| Non-Targeting_Human GCGTTGGCACGATGTTACGG Non_Targeting_Human 675 1|sg_Non_Targeting_Human_0094| Non-Targeting_Human GAACCAGCCGGCTAGTATGA Non_Targeting_Human 676 1|sg_Non_Targeting_Human_0095| Non-Targeting_Human GTATACTAGCTAACCACACG Non_Targeting_Human 677 1|sg_Non_Targeting_Human_0096| Non-Targeting_Human GAATCGGAATAGTTGATTCG Non_Targeting_Human 678 1|sg_Non_Targeting_Human_0097| Non-Targeting_Human GAGCACTTGCATGAGGCGGT Non_Targeting_Human 679 1|sg_Non_Targeting_Human_0098| Non-Targeting_Human GAACGGCGATGAAGCCAGCC Non_Targeting_Human 680 1|sg_Non_Targeting_Human_0099| Non-Targeting_Human GCAACCGAGATGAGAGGTTC Non_Targeting_Human 681 1|sg_Non_Targeting_Human_0100| Non-Targeting_Human GCAAGATCAATATGCGTGAT Non_Targeting_Human 682 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACGGAGGCTAAGCGTCGCAA 0101|Non_Targeting_Human 683 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCTTCCGCGGCCCGTTCAA 0102|Non_Targeting_Human 684 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ATCGTTTCCGCTTAACGGCG 0103|Non_Targeting_Human 685 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTAGGCGCGCCGCTCTCTAC 0104|Non_Targeting_Human 686 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CCATATCGGGGCGAGACATG 0105|Non_Targeting_Human 687 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TACTAACGCCGCTCCTACAG 0106|Non_Targeting_Human 688 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TGAGGATCATGTCGAGCGCC 0107|Non_Targeting_Human 689 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GGGCCCGCATAGGATATCGC 0108|Non_Targeting_Human 690 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TAGACAACCGCGGAGAATGC 0109|Non_Targeting_Human 691 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACGGGCGGCTATCGCTGACT 0110|Non_Targeting_Human 692 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCGGAAATTTTACCGACGA 0111|Non_Targeting_Human 693 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CTTACAATCGTCGGTCCAAT 0112|Non_Targeting_Human 694 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GCGTGCGTCCCGGGTTACCC 0113|Non_Targeting_Human 695 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGGAGTAACAAGCGGACGGA 0114|Non_Targeting_Human 696 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGAGTGTTATACGCACCGTT 0115|Non_Targeting_Human 697 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGACTAACCGGAAACTTTTT 0116|Non_Targeting_Human 698 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CAACGGGTTCTCCCGGCTAC 0117|Non_Targeting_Human 699 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CAGGAGTCGCCGATACGCGT 0118|Non_Targeting_Human 700 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TTCACGTCGTCTCGCGACCA 0119|Non_Targeting_Human 701 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTGTCGGATTCCGCCGCTTA 0120|Non_Targeting_Human 702 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CACGAACTCACACCGCGCGA 0121|Non_Targeting_Human 703 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCTAGTACGCTCCTCTATA 0122|Non_Targeting_Human 704 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TCGCGCTTGGGTTATACGCT 0123|Non_Targeting_Human 705 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CTATCTCGAGTGGTAATGCG 0124|Non_Targeting_Human 706 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AATCGACTCGAACTTCGTGT 0125|Non_Targeting_Human 707 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CCCGATGGACTATACCGAAC 0126|Non_Targeting_Human 708 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACGTTCGAGTACGACCAGCT 0127|Non_Targeting_Human 709 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCGACGACTCAACCTAGTC 0128|Non_Targeting_Human 710 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GGTCACCGATCGAGAGCTAG 0129|Non_Targeting_Human 711 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CTCAACCGACCGTATGGTCA 0130|Non_Targeting_Human 712 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGTATTCGACTCTCAACGCG 0131|Non_Targeting_Human 713 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CTAGCCGCCCAGATCGAGCC 0132|Non_Targeting_Human 714 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GAATCGACCGACACTAATGT 0133|Non_Targeting_Human 715 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACTTCAGTTCGGCGTAGTCA 0134|Non_Targeting_Human 716 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTGCGATGTCGCTTCAACGT 0135|Non_Targeting_Human 717 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCCTAATTTCCGGATCAAT 0136|Non_Targeting_Human 718 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGTGGCCGGAACCGTCATAG 0137|Non_Targeting_Human 719 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACCCTCCGAATCGTAACGGA 0138|Non_Targeting_Human 720 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AAACGGTACGACAGCGTGTG 0139|Non_Targeting_Human 721 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACATAGTCGACGGCTCGATT 0140|Non_Targeting_Human 722 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GATGGCGCTTCAGTCGTCGG 0141|Non_Targeting_Human 723 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ATAATCCGGAAACGCTCGAC 0142|Non_Targeting_Human 724 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCCGGGCTGACAATTAACG 0143|Non_Targeting_Human 725 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGTCGCCATATGCCGGTGGC 0144|Non_Targeting_Human 726 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGGGCCTATAACACCATCGA 0145|Non_Targeting_Human 727 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCCGTTCCGAGATACTTGA 0146|Non_Targeting_Human 728 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGGGACGTCGCGAAAATGTA 0147|Non_Targeting_Human 729 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TCGGCATACGGGACACACGC 0148|Non_Targeting_Human 730 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AGCTCCATCGCCGCGATAAT 0149|Non_Targeting_Human 731 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ATCGTATCATCAGCTAGCGC 0150|Non_Targeting_Human 732 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TCGATCGAGGTTGCATTCGG 0151|Non_Targeting_Human 733 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CTCGACAGTTCGTCCCGAGC 0152|Non_Targeting_Human 734 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGGTAGTATTAATCGCTGAC 0153|Non_Targeting_Human 735 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TGAACGCGTGTTTCCTTGCA 0154|Non_Targeting_Human 736 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGACGCTAGGTAACGTAGAG 0155|Non_Targeting_Human 737 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CATTGTTGAGCGGGCGCGCT 0156|Non_Targeting_Human 738 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CCGCTATTGAAACCGCCCAC 0157|Non_Targeting_Human 739 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AGACACGTCACCGGTCAAAA 0158|Non_Targeting_Human 740 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TTTACGATCTAGCGGCGTAG 0159|Non_Targeting_Human 741 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TTCGCACGATTGCACCTTGG 0160|Non_Targeting_Human 742 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GGTTAGAGACTAGGCGCGCG 0161|Non_Targeting_Human 743 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CCTCCGTGCTAACGCGGACG 0162|Non_Targeting_Human 744 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TTATCGCGTAGTGCTGACGT 0163|Non_Targeting_Human 745 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TACGCTTGCGTTTAGCGTCC 0164|Non_Targeting_Human 746 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCGGCCCACGCGTCATCGC 0165|Non_Targeting_Human 747 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AGCTCGCCATGTCGGTTCTC 0166|Non_Targeting_Human 748 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AACTAGCCCGAGCAGCTTCG 0167|Non_Targeting_Human 749 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCAAGGTGTCGGTAACCCT 0168|Non_Targeting_Human 750 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CTTCGACGCCATCGTGCTCA 0169|Non_Targeting_Human 751 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TCCTGGATACCGCGTGGTTA 0170|Non_Targeting_Human 752 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ATAGCCGCCGCTCATTACTT 0171|Non_Targeting_Human 753 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTCGTCCGGGATTACAAAAT 0172|Non_Targeting_Human 754 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TAATGCTGCACACGCCGAAT 0173|Non_Targeting_Human 755 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TATCGCTTCCGATTAGTCCG 0174|Non_Targeting_Human 756 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTACCATACCGCGTACCCTT 0175|Non_Targeting_Human 757 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TAAGATCCGCGGGTGGCAAC 0176|Non_Targeting_Human 758 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTAGACGTCGTGAGCTTCAC 0177|Non_Targeting_Human 759 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TCGCGGACATAGGGCTCTAA 0178|Non_Targeting_Human 760 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AGCGCAGATAGCGCGTATCA 0179|Non_Targeting_Human 761 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTTCGCTTCGTAACGAGGAA 0180|Non_Targeting_Human 762 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GACCCCCGATAACTTTTGAC 0181|Non_Targeting_Human 763 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACGTCCATACTGTCGGCTAC 0182|Non_Targeting_Human 764 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTACCATTGCCGGCTCCCTA 0183|Non_Targeting_Human 765 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TGGTTCCGTAGGTCGGTATA 0184|Non_Targeting_Human 766 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TCTGGCTTGACACGACCGTT 0185|Non_Targeting_Human 767 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGCTAGGTCCGGTAAGTGCG 0186|Non_Targeting_Human 768 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AGCACGTAATGTCCGTGGAT 0187|Non_Targeting_Human 769 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AAGGCGCGCGAATGTGGCAG 0188|Non_Targeting_Human 770 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ACTGCGGAGCGCCCAATATC 0189|Non_Targeting_Human 771 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGTCGAGTGCTCGAACTCCA 0190|Non_Targeting_Human 772 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TCGCAGCGGCGTGGGATCGG 0191|Non_Targeting_Human 773 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human ATCTGTCCTAATTCGGATCG 0192|Non_Targeting_Human 774 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TGCGGCGTAATGCTTGAAAG 0193|Non_Targeting_Human 775 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CGAACTTAATCCCGTGGCAA 0194|Non_Targeting_Human 776 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GCCGTGTTGCTGGATACGCC 0195|Non_Targeting_Human 777 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human TACCCTCCGGATACGGACTG 0196|Non_Targeting_Human 778 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human CCGTTGGACTATGGCGGGTC 0197|Non_Targeting_Human 779 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human GTACGGGGCGATCATCCACA 0198|Non_Targeting_Human 780 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AAGAGTAGTAGACGCCCGGG 0199|Non_Targeting_Human 781 1|sg_Non_Targeting_Human_GA_ Non-Targeting_Human AAGAGCGAATCGATTTCGTG 0200|Non_Targeting_Human 782 3|sg_hCDC16_CC_1|CDC16 CDC16 TCAACACCAGTGCCTGACGG 783 3|sg_hCDC16_CC_2|CDC16 CDC16 AAAGTAGCTTCACTCTCTCG 784 3|sg_hCDC16_CC_3|CDC16 CDC16 GAGCCAACCAATAGATGTCC 785 3|sg_hCDC16_CC_4|CDC16 CDC16 GCGCCGCCATGAACCTAGAG 786 3|sg_hGTF2B_CC_1|GTF2B GTF2B ACAAAGGTTGGAACAGAACC 787 3|sg_hGTF2B_CC_2|GTF2B GTF2B GGTGACCGGGTTATTGATGT 788 3|sg_hGTF2B_CC_3|GTF2B GTF2B TTAGTGGAGGACTACAGAGC 789 3|sg_hGTF2B_CC_4|GTF2B GTF2B ACATATAGCCCGTAAAGCTG 790 3|sg_hHSPA5_CC_1|HSPA5 HSPA5 CGTTGGCGATGATCTCCACG 791 3|sg_hHSPA5_CC_2|HSPA5 HSPA5 TGGCCTTTTCTACCTCGCGC 792 3|sg_hHSPA5_CC_3|HSPA5 HSPA5 AATGGAGATACTCATCTGGG 793 3|sg_hHSPA5_CC_4|HSPA5 HSPA5 GAAGCCCGTCCAGAAAGTGT 794 3|sg_hHSPA9_CC_1|HSPA9 HSPA9 CAATCTGAGGAACTCCACGA 795 3|sg_hHSPA9_CC_2|HSPA9 HSPA9 AGGCTGCGGCGCCCACGAGA 796 3|sg_hHSPA9_CC_3|HSPA9 HSPA9 ACTTTGACCAGGCCTTGCTA 797 3|sg_hHSPA9_CC_4|HSPA9 HSPA9 ACCTTCCATAACTGCCACGC 798 3|sg_hPAFAH1B1_CC_1|PAFAH1B1 PAFAH1B1 CGAGGCGTACATACCCAAGG 799 3|sg_hPAFAH1B1_CC_2|PAFAH1B1 PAFAH1B1 ATGGTACGGCCAAATCAAGA 800 3|sg_hPAFAH1B1_CC_3|PAFAH1B1 PAFAH1B1 TCTTGTAATCCCATACGCGT 801 3|sg_hPAFAH1B1_CC_4|PAFAH1B1 PAFAH1B1 ATTCACAGGACACAGAGAAT 802 3|sg_hPCNA_CC_1|PCNA PCNA CCAGGGCTCCATCCTCAAGA 803 3|sg_hPCNA_CC_2|PCNA PCNA TGAGCTGCACCAAAGAGACG 804 3|sg_hPCNA_CC_3|PCNA PCNA ATGTCTGCAGATGTACCCCT 805 3|sg_hPCNA_CC_4|PCNA PCNA CGAAGATAACGCGGATACCT 806 3|sg_hPOLR2L_CC_1|POLR2L POLR2L GCTGCAGGCCGAGTACACCG 807 3|sg_hPOLR2L_CC_2|POLR2L POLR2L ACAAGTGGGAGGCTTACCTG 808 3|sg_hPOLR2L_CC_3|POLR2L POLR2L GCAGCGTACAGGGATGATCA 809 3|sg_hPOLR2L_CC_4|POLR2L POLR2L GCAGTAGCGCTTCAGGCCCA 810 3|sg_hRPL9_CC_1|RPL9 RPL9 CAAATGGTGGGGTAACAGAA 811 3|sg_hRPL9_CC_2|RPL9 RPL9 GAAAGGAACTGGCTACCGTT 812 3|sg_hRPL9_CC_3|RPL9 RPL9 AGGGCTTCCGTTACAAGATG 813 3|sg_hRPL9_CC_4|RL9 RPL9 GAACAAGCAACACCTAAAAG 814 3|sg_hSF3A3_CC_1|SF3A3 SF3A3 TGAGGAGAAGGAACGGCTCA 815 3|sg_hSF3A3_CC_2|SF3A3 SF3A3 GGAAGAATGCAGAGTATAAG 816 3|sg_hSF3A3_CC_3|SF3A3 SF3A3 GGAATTTGAGGAACTCCTGA 817 3|sg_hSF3A3_CC_4|SF3A3 SF3A3 GCTCACCGGCCATCCAGGAA 818 3|sg_hSF3B3_CC_1|SF3B3 SF3B3 ACTGGCCAGGAACGATGCGA 819 3|sg_hSF3B3_CC_2|SF3B3 SF3B3 GCAGCTCCAAGATCTTCCCA 820 3|sg_hSF3B3_CC_3|SF3B3 SF3B3 GAATGAGTACACAGAACGGA 821 3|sg_hSF3B3_CC_4|SF3B3 SF3B3 GGAGCAGGACAAGGTCGGGG
Other Embodiments
[0191] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety 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. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.
[0192] While the invention has been described in connection with specific embodiments thereof, it will be understood that invention 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 present disclosure 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.
[0193] Other embodiments are in the claims.