Note: Descriptions are shown in the official language in which they were submitted.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
1
COMPOSITIONS AND METHODS FOR INHIBITING GENE EXPRESSION
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. provisional applications
63/082,555
filed September 24, 2020 and 63/242,957 filed September 10, 2021. The contents
of the
aforementioned applications are hereby incorporated by reference in their
entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted
electronically in
ASCII format and is hereby incorporated by reference in its entirety. Said
ASCII copy, created on
September 17, 2021, is named 02057-7017W0_SL.txt and is 694,831 bytes in size.
BACKGROUND
Mis regulation of gene expression is the underlying cause of many diseases
(e.g., in mammals,
e.g., humans). While techniques exist that modulate gene expression for short
periods of time, the
treatment of many diseases calls for stable, long-term modulation of gene
expression. There is a need for
novel tools, systems, and methods to stably alter, e.g., decrease, expression
of disease associated genes.
SUMMARY
The disclosure provides, among other things, expression repressors and
expression repression
systems that may be used to modulate, e.g., decrease, expression of a target
gene.
In some aspects, an expression repressor comprises a DNA targeting moiety and
a repressor
domain capable of modulating (e.g., decreasing) the expression of a target
gene. In some embodiments, an
expression repressor comprises a DNA targeting moiety, a first repressor
domain, and a second repressor
domain. In some embodiments, the first repressor domain is different from the
second repressor domain.
In some embodiments, the first repressor domain is identical to the second
repressor domain.
In some aspects, the disclosure features an expression repression system
comprising a first
expression repressor comprising a first DNA-targeting moiety and a first
repressor domain, and a second
expression repressor comprising a second DNA-targeting moiety and a second
repressor domain. In some
embodiments, the first DNA-targeting moiety specifically binds a first DNA
sequence, and the second
DNA-targeting moiety specifically binds a second DNA sequence different from
the first DNA sequence.
In some embodiments, the first repressor domain is different from the second
repressor domain. In some
embodiments, an expression repression system comprises: (i) a first expression
repressor comprising a
first DNA-targeting moiety, a first repressor domain, and a second repressor
domain, and (ii) a second
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
2
expression repressor comprising a second DNA-targeting moiety and a first
repressor domain. In some
embodiments, all three of the repressor domains are different. In some
embodiments, at least two
repressor domains are identical. In some embodiments, an expression repression
system comprises: (i) a
first expression repressor comprising a first DNA-targeting moiety, a first
repressor domain, and a second
repressor domain, and (ii) a second expression repressor comprising a second
DNA-targeting moiety, a
first repressor domain and a second repressor domain. In some embodiments, all
four of the repressor
domains are different. In some embodiments, at least two repressor domains are
identical.
Generally, modulation of expression of a target gene by an expression
repression system involves
the binding of the first expression repressor and second expression repressor
to the first and second DNA
sequences, respectively. Binding of the first and second DNA sequences
localizes the functionalities of
the first and second repressor domains to those sites. Without wishing to be
bound by theory, in some
embodiments it is thought that employing the functionalities of both the first
and second repressor
domains stably represses expression of a target gene associated with or
comprising the first and/or second
DNA sequences, e.g., wherein the first and/or second DNA sequences are or
comprise sequences of the
target gene or one or more operably linked transcription control elements.
In some aspects, the disclosure provides an expression repressor or an
expression repression
system comprising: a targeting moiety that binds a genomic locus comprising at
least 14, 15, 16, 17, 18,
19, or 20 nucleotides of the sequence of SEQ ID NO: 1-21, wherein the
expression repressor or the
expression repression system is capable of decreasing the expression of a
target gene.
In some aspects, the disclosure provides an expression repressor comprising: a
DNA-targeting
moiety (wherein optionally the DNA-targeting moiety comprises a CRISPR/Cas
molecule, e.g., a
catalytically inactive CRISPR/Cas protein), that binds to a transcription
regulatory element (e.g., a
promoter or transcription start site (TSS)) operably linked to a target gene,
or a sequence proximal to said
transcription regulatory element; and a repressor domain. In some embodiments,
an expression repressor
comprises a DNA-targeting moiety (wherein optionally the DNA-targeting moiety
comprises a
CRISPR/Cas molecule, e.g., a catalytically inactive CRISPR/Cas protein), that
binds to a transcription
regulatory element (e.g., a promoter or transcription start site (TSS))
operably linked to a target gene, or a
sequence proximal to said transcription regulatory element, a first repressor
domain, and a second
repressor domain. In some embodiments, the first repressor domain is identical
to the second repressor
domain. In some embodiments, the first repressor domain is not identical to
the second repressor domain.
In some aspects, the disclosure provides an expression repression system
comprising: (i) a first
expression repressor comprising a first DNA-targeting moiety (wherein
optionally the DNA-targeting
moiety comprises a CRISPR/Cas molecule, e.g., a catalytically inactive
CRISPR/Cas protein), that binds
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
3
to a transcription regulatory element (e.g., a promoter or transcription start
site (TSS)) operably linked to
a target gene, or a sequence proximal to said transcription regulatory
element; and a first repressor
domain and a second expression repressor comprising a second DNA-targeting
moiety (wherein
optionally the DNA-targeting moiety comprises a CRISPR/Cas molecule, e.g., a
catalytically inactive
CRISPR/Cas protein), that binds to a transcription regulatory element (e.g., a
promoter or transcription
start site (TSS)) operably linked to a target gene, or a sequence proximal to
said transcription regulatory
element; and a first repressor domain. In some embodiments, the first
repressor domains of the first
expression repressor and the second expression repressors are identical. In
some embodiments, the first
repression domains of the first and the second expression pressors are
different. In some embodiments,
.. the first DNA-targeting moiety and the second DNA-targeting moiety are
identical. In some
embodiments, the first DNA-targeting moiety and the second DNA-targeting
moiety are different. In
some embodiments, an expression repression system comprises a first expression
repressor comprising a
first DNA-targeting moiety (wherein optionally the DNA-targeting moiety
comprises a CRISPR/Cas
molecule, e.g., a catalytically inactive CRISPR/Cas protein), that binds to a
transcription regulatory
element (e.g., a promoter or transcription start site (TSS)) operably linked
to a target gene, or a sequence
proximal to said transcription regulatory element, a first repressor domain,
and a second repressor domain
and a second expression repressor comprising a second DNA-targeting moiety
(wherein optionally the
DNA-targeting moiety comprises a CRISPR/Cas molecule, e.g., a catalytically
inactive CRISPR/Cas
protein), that binds to a transcription regulatory element (e.g., a promoter
or transcription start site (TSS))
operably linked to a target gene, or a sequence proximal to said transcription
regulatory element; a first
repressor domain and a second repressor domain. In some embodiments, the first
repressor domains of the
first expression repressor and the second expression repressors are identical.
In some embodiments, the
first repression domains of the first and the second expression pressors are
different. In some
embodiments, the second repressor domains of the first expression repressor
and the second expression
repressors are identical. In some embodiments, the second repression domains
of the first and the second
expression pressors are different.
In some aspects, the disclosure features a nucleic acid encoding an expression
repressor or a
component thereof (e.g., a gRNA). In some aspects, the disclosure is directed
to a nucleic acid encoding
the first expression repressor, second expression repressor, both, or a
component thereof (e.g., a gRNA).
In some aspects, the disclosure is directed to a vector comprising a nucleic
acid described herein. In some
aspects, the disclosure is directed to a cell comprising an expression
repressor, an expression repression
system, nucleic acid, or vector described herein. In some aspects, the
disclosure is directed to a lipid
nanoparticle comprising a vector, a nucleic acid, an expression repression
system, or an expression
repressor described herein. In some aspects, the disclosure is directed to a
reaction mixture comprising an
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
4
expression repressor, an expression repression system, a nucleic acid, a
vector, or a lipid nanoparticle
described herein. In some aspects, the disclosure is directed to a
pharmaceutical composition comprising
an expression repression system, nucleic acid, or vector described herein.
In some aspects, the disclosure is directed to a method of decreasing
expression of a target gene
comprising providing an expression repressor or an expression repression
system described herein and
contacting the target gene and/or one or more operably linked transcription
control elements with the
expression repressor or the expression repression system, thereby decreasing
expression of the target
gene.
In some aspects, the disclosure is directed to a method of treating a
condition associated with
over-expression of a target gene in a subject, comprising administering an
expression repressor or an
expression repression system, nucleic acid, or vector described herein to the
subject, thereby treating the
condition.
In some aspects, the disclosure is directed to a method of treating a
condition associated with mis-
regulation of a target gene in a subject, comprising administering an
expression repressor or an expression
repression system, nucleic acid, or vector described herein to the subject,
thereby treating the condition.
Additional features of any of the aforesaid methods or compositions include
one or more of the
following enumerated embodiments.
Those skilled in the art will recognize or be able to ascertain using no more
than routine
experimentation, many equivalents to the specific embodiments of the invention
described herein. Such
equivalents are intended to be encompassed by the following enumerated
embodiments.
All publications, patent applications, patents, and other references (e.g.,
sequence database
reference numbers) mentioned herein are incorporated by reference in their
entirety. For example, all
GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table
herein, are incorporated by
reference. Unless otherwise specified, the sequence accession numbers
specified herein, including in any
Table herein, refer to the database entries current as of September 23, 2019.
When one gene or protein
references a plurality of sequence accession numbers, all of the sequence
variants are encompassed.
ENUMERATED EMBODIMENTS
1. An expression repressor comprising:
a DNA-targeting moiety and
a repressor domain,
wherein the expression repressor is capable of decreasing expression of a
target gene.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
2. The expression repressor of embodiment 1, wherein the DNA-targeting moiety
binds to a transcription
regulatory element (e.g., a promoter, an enhancer, a super enhancer, or
transcription start site (TSS))
operably linked to the target gene or binds to a sequence proximal to said
transcription regulatory
element.
5
3. The expression repressor of embodiment 1 or 2, wherein the repressor domain
is covalently linked to
the DNA-targeting moiety.
4. The expression repressor of embodiments 1 or 2, wherein the repressor
domain is linked to the DNA-
targeting moiety via a linker.
5. The expression repressor of any of embodiment1-4, wherein the repressor
domain is C-terminal of the
DNA-targeting moiety.
6. The expression repressor of any of embodiments 1-4, wherein the repressor
domain is N-terminal of the
DNA-targeting moiety.
7. The expression repressor of any of embodiments1-6, wherein the repressor
domain is encoded by a
nucleotide sequence chosen from any of SEQ ID NOs: 47-56, or a sequence with
at least 80, 85, 90, 95,
99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 positions of difference thereto.
8. The expression repressor of any of embodiments1-7, wherein the repressor
domain comprises an amino
acid sequence according to any of SEQ ID NOs: 57-66, 90 or a sequence with at
least 80, 85, 90, 95, 99,
or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2, or 1 positions of difference thereto.
9. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is MQ1
or a functional variant or fragment thereof, e.g., wherein the repressor
domain comprises an amino acid
sequence of SEQ ID NO: 57 or 90 or a sequence with at least 80, 85, 90, 95,
99, or 100% identity thereto,
or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 positions of
difference thereto, wherein optionally the repressor domain is C-terminal of
the DNA-targeting moiety.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
6
10. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is
DNMT1 or a functional variant or fragment thereof, e.g., wherein the repressor
domain comprises an
amino acid sequence of SEQ ID NO: 58 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto, wherein optionally the repressor domain is C-
terminal of the DNA-
targeting moiety.
11. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is
DNMT3a/3L, or a functional variant or fragment thereof, e.g., wherein the
repressor domain comprises an
amino acid sequence of SEQ ID NO: 59 or 60 or a sequence with at least 80, 85,
90, 95, 99, or 100%
identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto, wherein optionally the repressor domain is C-
terminal of the DNA-
targeting moiety.
.. 12. The expression repressor of any of the preceding embodiments, wherein
the repressor domain is
KRAB, or a functional variant or fragment thereof, e.g., wherein the repressor
domain comprises an
amino acid sequence of SEQ ID NO: 61 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto, wherein optionally the repressor domain is C-
terminal of the DNA-
targeting moiety.
13. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is G9A,
or a functional variant or fragment thereof, e.g., wherein the repressor
domain comprises an amino acid
sequence of SEQ ID NO: 62 or a sequence with at least 80, 85, 90, 95, 99, or
100% identity thereto, or
.. having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 positions of
difference thereto, wherein optionally the repressor domain is N-terminal of
the DNA- targeting moiety.
14. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is
HDAC8, or a functional variant or fragment thereof, e.g., wherein the
repressor domain comprises an
amino acid sequence of SEQ ID NO: 63 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto, wherein optionally the repressor domain is C-
terminal of the DNA-
targeting moiety.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
7
15. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is
LSD1, or a functional variant or fragment thereof, e.g., wherein the repressor
domain comprises an amino
acid sequence of SEQ ID NO: 64 or a sequence with at least 80, 85, 90, 95, 99,
or 100% identity thereto,
or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 positions of
difference thereto, wherein optionally the repressor domain is C-terminal of
the DNA-targeting moiety.
16. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is
EZH2, or a functional variant or fragment thereof, e.g., wherein the repressor
domain comprises an amino
acid sequence of SEQ ID NO: 64 or a sequence with at least 80, 85, 90, 95, 99,
or 100% identity thereto,
or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 positions of
difference thereto, wherein optionally the repressor domain is N-terminal of
the DNA-targeting moiety.
17. The expression repressor of any of the preceding embodiments, wherein the
repressor domain is
FOG1, or a functional variant or fragment thereof, e.g., wherein the repressor
domain comprises an amino
acid sequence of SEQ ID NO: 66 or a sequence with at least 80, 85, 90, 95, 99,
or 100% identity thereto,
or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 positions of
difference thereto.
18. The expression repressor of any of the preceding embodiments, which
further comprises a second
repressor domain.
19. The expression repressor of embodiment 18, wherein the DNA-targeting
moiety is situated between
the repressor domain and the second repressor domain.
20. An expression repressor comprising:
a DNA-targeting moiety,
a first repressor domain and
a second repressor domain,
wherein the expression repressor is capable of decreasing expression of a
target gene.
21. The expression repressor of any preceding embodiment, wherein the DNA-
targeting moiety binds to a
transcription regulatory element (e.g., a promoter, an enhancer, a super
enhancer, or transcription start site
(TSS)) operably linked to the target gene or binds to a sequence proximal to
said transcription regulatory
element.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
8
22. The expression repressor of any of embodiments 18-21, wherein the first
repressor domain is identical
to the second repressor domain.
23. The expression repressor of any of embodiments 18-21, wherein the first
repressor domain is not
identical to the second repressor domain.
24. The expression repressor of any of embodiments 18-23, wherein the first
repressor domain and the
second repressor domain are covalently linked to the DNA-targeting moiety.
25. The expression repressor of any of embodiments18-24, wherein the first
repressor domain is linked to
the DNA-targeting moiety via a first linker and the second repressor domain is
linked to the DNA-
targeting moiety via a second linker.
26. The expression repressor of embodiment 25, wherein the first linker is
identical to the second linker.
27. The expression repressor of embodiment 25, wherein the first linker is not
identical to the second
linker.
28. The expression repressor of any of embodiments 18-27, wherein the first
repressor domain is N-
terminal of the DNA-targeting moiety.
29. The expression repressor of any of embodiments 18-28, wherein the second
repressor domain is C-
terminal of the DNA-targeting moiety.
30. The expression repressor of any of embodiments 25-29, wherein the C-
terminal end of the first
repressor domain is linked to the N-terminal end of the DNA-targeting moiety
via the first linker and the
N-terminal end of the second repressor domain is linked to the C-terminal end
of the DNA-targeting
moiety via the second linker.
31. The expression repressor of any of embodiments 18-30, wherein:
the first repressor domain is EZH2, or a functional variant or fragment
thereof, e.g., wherein the
first repressor domain comprises an amino acid sequence of SEQ ID NO: 64 or a
sequence with at least
80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19,
18, 17, 16, 15, 14, 13, 12, 11,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
9
10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the
first repressor domain is N-terminal of
the DNA-targeting moiety; and
the second repressor domain is KRAB, or a functional variant or fragment
thereof, e.g., wherein
the second repressor domain comprises an amino acid sequence of SEQ ID NO: 61
or a sequence with at
least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20,
19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and
the second repressor domain is C-
terminal of the DNA-targeting moiety.
32. The expression repressor of any of embodiments 18-30, wherein:
the first repressor domain is G9A, or a functional variant or fragment
thereof, e.g., wherein the
first repressor domain comprises an amino acid sequence of SEQ ID NO: 62 or a
sequence with at least
80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19,
18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the
first repressor domain is N-terminal of
the DNA-targeting moiety; and
the second repressor domain is KRAB, or a functional variant or fragment
thereof, e.g., wherein
the second repressor domain comprises an amino acid sequence of SEQ ID NO: 61
or a sequence with at
least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20,
19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and
the second repressor domain is C-
terminal of the DNA-targeting moiety.
33. The expression repressor of any of embodiments 18-30, wherein:
the first repressor domain is FOG1, or a functional variant or fragment
thereof, e.g., wherein the
first repressor domain comprises an amino acid sequence of SEQ ID NO: 66 or a
sequence with at least
80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20, 19,
18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and the
first repressor domain is N-terminal of
the DNA-targeting moiety; and
the second repressor domain is FOG1, or a functional variant or fragment
thereof, e.g., wherein
the second repressor domain comprises an amino acid sequence of SEQ ID NO: 66
or a sequence with at
least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20,
19, 18, 17, 16, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto, and
the second repressor domain is C-
terminal of the DNA-targeting moiety.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
34. The expression repressor of any of embodiments 1-33, wherein the DNA-
targeting moiety binds a
genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of
the sequence of any of SEQ
ID NO: 1-21.
5 35. The expression repressor of any of embodiments 1-34, wherein the DNA-
targeting moiety comprises
a CRISPR/Cas molecule (e.g., a catalytically inactive CRISPR/Cas protein), a
zinc finger domain, or a
TAL effector molecule.
36. The expression repressor of embodiment 35, wherein the CRISPR/Cas molecule
comprises one or
10 more mutations selected from DlOA, D839A, H840A, and N863A.
37. The expression repressor of any of embodiments 1-36, wherein the DNA-
targeting moiety binds to a
promoter region of a target gene, an enhancer region (e.g., a super enhancer
region), or an anchor
sequence.
38. The expression repressor of any of embodiments 1-37, wherein the repressor
domain comprises
DNMT1, DNMT3a/3L, MQ1, KRAB, G9A, HDAC8, LSD1, EZH2, or FOG1,
39. The expression repressor of any of embodiments 18-38, wherein the first
repressor domain
comprising, DNMT1, DNMT3a/31, MQ1, KRAB, G9A, HDAC8, LSD1, EZH2, or FOG1, and
the second
repressor domain comprises DNMT1, DNMT3a/31, MQ1, KRAB, G9A, HDAC8, LSD1,
EZH2, or
FOG1,
40. The expression repressor of any of embodiments 1-39, wherein the
expression repressor is encoded by
a nucleotide sequence chosen from any of SEQ ID NOs: 22-32, 92, 95 or a
sequence with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
41. The expression repressor of any of embodiments 1-40, wherein the
expression repressor comprises an
amino acid sequence chosen from any of SEQ ID Nos: 33-36, 38-44, 67-69, 93,
96, or a sequence with at
least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more than 20,
19, 18, 17, 16, 15, 14, 13,
12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
11
42. The expression repressor of any of embodiments1-41, wherein the DNA-
targeting moiety is encoded
by a nucleotide sequence chosen from any of SEQ ID NOs: 45 or 89 or a sequence
with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
43. The expression repressor of any of embodiments 1-42, wherein the DNA-
targeting moiety comprises
an amino acid sequence according to any of SEQ ID NOs: 46 or 88, or a sequence
with at least 80, 85, 90,
95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7,
6, 5, 4, 3, 2, or 1 positions of difference thereto.
44. The expression repressor of any of embodiments 18-43, wherein,
(i) the first repressor domain is encoded by a nucleotide sequence chosen from
any of SEQ ID
NOs: 47-56, 90 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having no more
than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto, and
(ii) the second repressor domain is encoded by a nucleotide sequence chosen
from any of SEQ ID
NOs: 47-56, 90 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having no more
than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
45. The expression repressor of any of embodiments 18-43, wherein,
(i) the first repressor domain comprises an amino acid sequence according to
any of SEQ ID
NOs: 57-66, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more
than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto, and
(ii) the second repressor domain comprises an amino acid sequence according to
any of SEQ ID
NOs: 57-66, or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more
than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
46. The expression repressor of any preceding embodiments, wherein the DNA-
targeting moiety
comprises a TAL effector molecule, a CRISPR/Cas molecule, a zinc finger
domain, a tetR domain, a
meganuclease domain, or an oligonucleotide.
47. The expression repressor of any preceding embodiments, wherein the DNA-
targeting moiety further
comprises a gRNA, e.g., a gRNA that binds a genomic locus comprising at least
14, 15, 16, 17, 18, 19, or
20 nucleotides of the sequence of any of SEQ ID NOs: 1-21, e.g., wherein the
gRNA comprises a
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
12
sequence that comprises at least 14, 15, 16, 17, 18, 19, or 20 nucleotides of
the sequence of any of SEQ
ID NOs: 1-21.
48. The expression repressor of any of embodiments 35-47, wherein the DNA-
targeting domain
comprises a CRISPR/Cas molecule, e.g., a catalytically inactive CRISPR/ Cas
protein, and a gRNA, e.g.,
a gRNA that binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19,
or 20 nucleotides of the
sequence of any of SEQ ID NOs: 1-21, e.g., wherein the gRNA comprises a
sequence that comprises at
least 14, 15, 16, 17, 18, 19, or 20 nucleotides of the sequence of any of SEQ
ID NOs: 1-21 and the
repressor domain comprises a moiety chosen from DNMT1, DNMt3a/31, MQ1, KRAB,
G9A, HDAC8,
LSD1, EZH2, or FOG1.
49. The expression repressor of embodiment 35-47, wherein the DNA-targeting
domain comprises a
CRISPR/Cas molecule, e.g., a catalytically inactive CRISPR/ Cas protein, and a
gRNA, e.g., a gRNA that
binds a genomic locus comprising at least 14, 15, 16, 17, 18, 19, or 20
nucleotides of the sequence of any
of SEQ ID NOs: 1-21, e.g., wherein the gRNA comprises a sequence that
comprises at least 14, 15, 16,
17, 18, 19, or 20 nucleotides of the sequence of any of SEQ ID NOs: 1-21, the
first repressor domain
comprises a moiety chosen from DNMT1, DNM T3a/3L, MQ1, KRAB, G9A, HDAC8, LSD1,
EZH2, or
FOG1 and the second domain comprises a moiety chosen from DNMT1, DNMt3a/3L,
MQ1, KRAB,
G9A, HDAC8, LSD1, EZH2, or FOG1.
50. The expression repressor of any of embodiments 35-49, wherein the
CRISPR/Cas molecule comprises
a Cas protein or Cpfl protein chosen from Table 1 or a variant (e.g., mutant)
of any thereof.
51. The expression repressor of any of embodiments 35-50, wherein the
CRISPR/Cas molecule comprises
a catalytically inactive CRISPR/Cas protein, e.g., dCas9.
52. The expression repressor of any of the preceding embodiments, which: (i)
comprises one or more
nuclear localization signal sequences (NLS), or (ii) does not comprise an NLS.
53. The expression repressor of any of the preceding embodiments, comprising a
first NLS at the N
terminus, e.g., wherein the first NLS has a sequence of SEQ ID NO: 86.
54. The expression repressor of any of the preceding embodiments, comprising
an NLS, e.g., a second
NLS, at the C terminus, e.g., having a sequence of SEQ ID NO: 87.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
13
55. The expression repressor of any of the preceding embodiments, wherein the
first and the second NLS
have the same sequence.
56. The expression repressor of any of embodiments 52-55, wherein the first
and the second NLS have
different sequences.
57. The expression repressor of any of the preceding embodiments, which
comprises an epitope tag.
58. The expression repressor of embodiment 57, wherein the epitope tag is an
HA tag, e.g., an HA tag
comprising an amnio acid seq of SEQ ID NO: 80.
59. The expression repressor of any of embodiments 37-58, wherein the anchor
sequence comprises the
sequence of SEQ ID NO: 81 or 82, or a sequence with no more than 8, 7, 6, 5,
4, 3, 2, or 1 alteration
relative thereto.
60. The expression repressor of any of embodiments 37-58, wherein the anchor
sequence comprises a
sequence according to SEQ ID NO: 83 or 84, or a sequence with no more than 8,
7, 6, 5, 4, 3, 2, or 1
alteration relative thereto.
61. The expression repressor of any of embodiments 37-60, wherein the anchor
sequence is on the same
chromosome as the gene.
62. The expression repressor of any of embodiments 37-61, wherein the anchor
sequence is upstream of
the target gene (e.g., upstream of the TSS or upstream of the promoter).
63. The expression repressor of any of embodiments 37-61 wherein the anchor
sequence is downstream of
the target gene (e.g., downstream of the TSS or upstream of the promoter).
64. The expression repressor of any of the preceding embodiments, wherein
binding of the expression
repressor to the target gene locus decreases expression of the target gene in
a cell by 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100% compared to expression of the target gene in the
absence of the expression
repressor.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
14
65. The expression repressor of any of the preceding embodiments, wherein
binding of the expression
repressor to the target gene locus appreciably decreases expression of the
target gene for a time period of
at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, or 22 days.
66. The expression repressor of any of embodiments 18-65, wherein the first
and/or second repressor
domain comprises a DNA methyltransferase, a histone methyltransferase, a
histone deacetylase, a histone
demethylase, or a recruiter of a histone modifying complex.
67. An expression repression system comprising:
a first expression repressor comprising a first DNA-targeting moiety and a
first repressor domain,
and
a second expression repressor comprising a second DNA-targeting moiety and a
second repressor
domain,
wherein the first DNA-targeting moiety specifically binds a first DNA
sequence, and the second
DNA-targeting moiety specifically binds a second DNA sequence different from
the first DNA sequence,
and
wherein the first repressor domain is different from the second repressor
domain.
68. An expression repression system comprising:
a first expression repressor comprising a first DNA-targeting moiety and a
first repressor domain,
and
a second expression repressor comprising a second DNA-targeting moiety and a
second repressor
domain,
wherein the first or second repressor domain comprises an MQ1 domain or
functional variant or
fragment thereof,
wherein the first repressor domain is different from the second repressor
domain.
69. An expression repression system of embodiment 67 or 68, wherein the first
expression repressor
further comprises a third repressor domain.
70. An expression repression system of any of embodiments 67-69, wherein the
second expression
repressor further comprises a fourth repressor domain.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
71. The expression repression system of any of embodiments 67-70, wherein the
first DNA-targeting
moiety specifically binds a first DNA sequence, and the second DNA-targeting
moiety specifically binds
a second DNA sequence different from the first DNA sequence.
5 72. The expression repression system of any of embodiments 67-70, wherein
the first DNA-targeting
moiety and the second DNA-targeting moiety specifically bind the same DNA
sequence.
73. The expression repression system of any of embodiments 67-72,
wherein the first DNA-targeting moiety comprises a first CRISPR/Cas molecule
comprising a
10 first CRISPR/Cas protein and first guide RNA, and the second DNA-
targeting moiety comprises a second
CRISPR/Cas molecule comprising a second CRISPR/Cas protein and a second guide
RNA.
74. The expression repression system of embodiment 73,
wherein the first CRISPR/Cas protein does not bind the second guide RNA, e.g.,
binds with a KD
15 of at least 10, 20, 50, 100, 1000, or 10,000 nM, and the second
CRISPR/Cas protein does not bind the
first guide RNA, e.g., binds with a KD of at least 10, 20, 50, 100, 1000, or
10,000 nM.
75. The expression repression system of either of embodiments 73 or 74:
wherein the first CRISPR/Cas protein comprises a different amino acid sequence
than the second
CRISPR/Cas protein.
76. The expression repression system of any of embodiments 67-75, wherein the
first or second DNA-
targeting moiety comprises a CRISPR/Cas molecule comprising a Cas protein or
Cpfl protein chosen
from Table 1 or a variant (e.g., mutant) of any thereof.
77. The expression repression system of any of embodiments 67-76, wherein the
first DNA-targeting
moiety comprises a first CRISPR/Cas molecule comprising a Cas protein or Cpfl
protein chosen from
Table 1 or a variant (e.g., mutant) of any thereof, and the second DNA-
targeting moiety comprises a
second CRISPR/Cas molecule comprising a different Cas protein or Cpfl protein
chosen from Table 1 or
a variant (e.g., mutant) of any thereof.
78. The expression repression system of any of embodiments 70-77, wherein the
first, second, third, or
fourth repressor domain comprises a histone methyltransferase activity.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
16
79. The expression repression system of embodiment 70-78, wherein the first,
second, third, or fourth
repressor domain comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e.,
G9A), EHMT1
(i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a
functional
variant or fragment of any thereof, e.g., a SET domain of any thereof.
80. The expression repression system of any of embodiments 70-79, wherein the
first, second, third, or
fourth repressor domain comprises a histone demethylase activity (e.g., a
lysine demethylase activity).
81. The expression repression system of any of embodiment 70-80, wherein the
first, second, third, or
fourth repressor domain comprises a protein chosen from KDM1A (i.e., LSD1),
KDM1B (i.e., LSD2),
KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066 (or a functional variant
or
fragment of any thereof.
82. The expression repression system of any of embodiments 70-81, wherein the
first, second, third, or
fourth repressor domain comprises a histone deacetylase activity.
83. The expression repression system of embodiment 70-82, wherein the first,
second, third, or fourth
repressor domain comprises a protein chosen from HDAC1, HDAC2, HDAC3, HDAC4,
HDAC5,
HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,
SIRT6, SIRT7, SIRT8, SIRT9, or a functional variant or fragment of any
thereof.
84. The expression repression system of any of embodiments 70-83, wherein the
first, second, second,
third, or fourth repressor domain comprises a DNA methyltransferase activity.
85. The expression repression system of embodiment 70-84, wherein the
first, second, third, or
fourth repressor domain comprises a protein chosen from MQ1, DNMT1, DNMT3A1,
DNMT3A2,
DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, or a functional
variant or fragment of any thereof.
86. The expression repression system of any of embodiments 70-85, wherein the
first, second, third, or
fourth repressor domain comprises a transcription repressor activity.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
17
87. The expression repression system of embodiment 70-86, wherein the first,
second, third, or fourth
repressor domain comprises a protein chosen from KRAB, MeCP2, HP1, RBBP4,
REST, FOG1, SUZ12,
or a functional variant or fragment of any thereof.
.. 88. The expression repression system of any of embodiments 67-87, wherein:
the first repressor domain comprises a protein chosen from SETDB1, SETDB2,
EHMT2 (i.e.,
G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1,
5UV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof, and
the second repressor domain comprises a different protein chosen from SETDB1,
SETDB2,
.. EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8,
SUV420H1,
5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B,
KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7,
HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7,
SIRT8,
SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4,
.. DNMT3B5, DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a
functional variant or fragment thereof.
89. The expression repression system of any of embodiments 69-88, wherein:
(i) the first repressor domain comprises a protein chosen from SETDB1, SETDB2,
EHMT2 (i.e.,
G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1,
5UV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof, and
(ii) the third repressor domain comprises a protein (e.g., a different
protein) chosen from
SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1,
5UV39H2,
SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A,
KDM2B,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
18
KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,
HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,
SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2,
DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST,
FOG1, SUZ12, or a functional variant or fragment thereof.
90. The expression repression system of any of embodiments 69-89, wherein:
(i) the first repressor domain comprises a protein chosen from SETDB1, SETDB2,
EHMT2 (i.e.,
G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1,
5UV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof,
(ii) the second repressor domain comprises a protein (e.g., different protein)
chosen from
SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1,
5UV39H2,
SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A,
KDM2B,
KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,
HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,
SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2,
DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST,
FOG1, SUZ12, or a functional variant or fragment thereof, and
(iii) the third repressor domain comprises a protein chosen from SETDB1,
SETDB2, EHMT2
(i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1,
5UV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
.. DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a
functional variant or
fragment thereof.
91. The expression repression system of any of embodiments 70-90, wherein:
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
19
(i) the first repressor domain comprises a protein chosen from SETDB1, SETDB2,
EHMT2 (i.e.,
G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1,
SUV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof,
(ii) the second repressor domain comprises a protein (e.g., a different
protein) chosen from
SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1,
5UV39H2,
SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A,
KDM2B,
KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,
HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,
SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2,
DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST,
FOG1, SUZ12, or a functional variant or fragment thereof,
(iii) the third repressor domain comprises a protein chosen from SETDB1,
SETDB2, EHMT2
(i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1,
5UV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof and
(iv) the fourth repressor domain comprises a protein (e.g., a different
protein) chosen from
SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1,
5UV39H2,
SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A,
KDM2B,
KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,
HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,
SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2,
DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST,
FOG1, SUZ12, or a functional variant or fragment thereof.
92. The expression repression system of any of embodiments 70-91, wherein:
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
(i) the second repressor domain comprises a protein chosen from SETDB1,
SETDB2, EHMT2
(i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1,
SUV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
5 HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8,
SIRT9, MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof, and
(ii) the fourth repressor domain comprises a protein (e.g., a different
protein) chosen from
10 SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2,
EZH1, 5UV39H2,
SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A,
KDM2B,
KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,
HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,
SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2,
15 DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, KRAB, MeCP2, HP1, RBBP4,
REST,
FOG1, SUZ12, or a functional variant or fragment thereof.
93. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone methyltransferase activity and the second repressor domain
comprises a histone
20 demethylase activity.
94. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone methyltransferase activity and the second repressor domain
comprises a histone
deacetylase activity.
95. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone methyltransferase activity and the second repressor domain
comprises a DNA
methyltransferase activity.
96. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone methyltransferase activity and the second repressor domain
comprises a DNA
demethylase activity.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
21
97. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone methyltransferase activity and the second repressor domain
comprises a transcription
repressor activity.
98. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone methyltransferase activity and the second repressor domain
comprises a histone
methyltransferase activity (e.g., wherein the histone methyltransferase
activities are the same or different
from each other).
99. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone demethylase activity and the second repressor domain
comprises a histone
deacetylase activity.
100. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone demethylase activity and the second repressor domain
comprises a DNA
methyltransferase activity.
101. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone demethylase activity and the second repressor domain
comprises a DNA demethylase
activity.
102. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone demethylase activity and the second repressor domain
comprises a transcription
repressor activity.
103. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone demethylase activity and the second repressor domain
comprises a histone
demethylase activity (e.g., wherein the histone demethylase activities are the
same or different from each
other).
104. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone deacetylase activity and the second repressor domain
comprises a DNA
methyltransferase activity.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
22
105. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone deacetylase activity and the second repressor domain
comprises a DNA demethylase
activity.
106. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone deacetylase activity and the second repressor domain
comprises a transcription
repressor activity.
107. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a histone deacetylase activity and the second repressor domain
comprises a histone deacetylase
activity (e.g., wherein the histone deacetylase activities are the same or
different from each other).
108. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a DNA methyltransferase activity and the second repressor domain
comprises a DNA
demethylase activity.
109. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a DNA methyltransferase activity and the second repressor domain
comprises a transcription
repressor activity.
110. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a DNA methyltransferase activity and the second repressor domain
comprises a DNA
methyltransferase activity (e.g., wherein the DNA methyltransferase activities
are the same or different
from each other).
111. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a DNA demethylase activity and the second repressor domain comprises
a transcription
repressor activity.
112. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a DNA demethylase activity and the second repressor domain comprises
a DNA demethylase
activity (e.g., wherein the DNA demethylase activities are the same or
different from each other).
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
23
113. The expression repression system of any of embodiments 67-92, wherein the
first repressor domain
comprises a transcription repressor activity and the second repressor domain
comprises a transcription
repressor activity (e.g., wherein the transcription repressor activities are
the same or different from each
other).
114. The expression repression system of any of embodiments 67-113, wherein:
the first repressor domain comprises a protein selected from: KRAB, a SET
domain (e.g., the
SET domain of SETDB1, EZH2, G9A, or SUV39H1), histone demethylase LSD1, FOG1
(e.g., the N-
terminal residues of FOG1), KAP1, or a functional variant or fragment of any
thereof; and
the second repressor domain comprises a protein selected from: DNMT3A (e.g.,
human
DNMT3A), DNMT3B, DNMT3L, DNMT3A/3L complex, bacterial MQ1, or a functional
variant or
fragment of any thereof.
115. The expression repression system of any of embodiments 67-114, wherein:
the first repressor domain comprises a protein selected from: EZH2, G9A,
SUV39H1, FOG1, or a
functional variant or fragment of any thereof; and
the second repressor domain comprises a protein selected from: DNMT3A (e.g.,
human
DNMT3A), DNMT3B, DNMT3L, DNMT3A/3L, DNMT1, LSD1, FOG1, KRAB, HDAC8, bacterial
MQ1, or a functional variant or fragment of any thereof.
116. The expression repression system of any of embodiments67-115, wherein:
(i) the first repressor domain comprises a protein selected from: KRAB, MeCP2,
HP1, RBBP4,
REST, FOG1, SUZ12, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B,
KDM5A,
KDM5B, KDM5C, KDM5D, KDM4B, N066, SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1
(i.e.,
GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1, 5UV420H2, KAP1, HDAC8,
MQ1,
DNMT1, DNMT3A, DNMT3a/31, or a functional variant or fragment of any thereof;
and
(ii) the second repressor domain comprises a protein selected from: KRAB,
FOG1, G9A, LSD1,
HDAC8, EZH2, DNMT3a/31, MQ1, DNMT1, DBMT3A, DNMT3A1, DNMT3A2, DNMT3B1,
DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L or a functional variant or
fragment of any thereof.
117. The expression repression system of any of embodiments 67-116, wherein
the first repressor domain
comprises KRAB, and the second repressor domain comprises DNMT3A (e.g., human
DNMT3A).
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
24
118. The expression repression system of any of embodiments 67-115, wherein
the first repressor domain
comprises G9A or a functional variant or fragment thereof, and the second
repressor domain comprises
KRAB or a functional variant or fragment thereof.
119. The expression repression system of any of embodiments 67-118, wherein
the first repressor domain
comprises LSD1 or a functional variant or fragment thereof and the second
repressor domain comprises
KRAB or a functional variant or fragment thereof.
120. The expression repression system of any of embodiments 67-119, wherein
the first repressor domain
comprises KRAB or a functional variant or fragment thereof and the second
repressor domain comprises
bacterial MQ1 or a functional variant or fragment thereof.
121. The expression repression system of any of embodiments 67-120, wherein:
the first repressor domain comprises G9A or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety; and
the second repressor domain comprises EZH2 or a functional variant or fragment
thereof, wherein
optionally the second repressor domain is N-terminal of the second DNA-
targeting moiety.
122. The expression repression system of any of embodiments 67-120, wherein:
the first repressor domain of comprises LSD1 or a functional variant or
fragment thereof, wherein
optionally the first repressor domain is C-terminal of the first DNA-targeting
moiety; and
the second repressor domain comprises G9A or a functional variant or fragment
thereof, wherein
optionally the second repressor domain is N-terminal of the second DNA-
targeting moiety.
123. The expression repression system of any of embodiments 67-120, wherein:
the first repressor domain comprises MQ1 or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is C-terminal of the first DNA-targeting
moiety; and
the second repressor domain comprises HDAC8 or a functional variant or
fragment thereof,
wherein optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
124. The expression repression system of any of embodiments 67-120, wherein:
the first repressor domain comprises LSD1 or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is C-terminal of the first DNA-targeting
moiety; and
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
the second repressor domain comprises HDAC8 or a functional variant or
fragment thereof,
wherein optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
125. The expression repression system of any of embodiments 67-120, wherein:
5 the first repressor domain comprises LSD1 or a functional variant or
fragment thereof, wherein
optionally the first repressor domain is C-terminal of the first DNA-targeting
moiety; and
the second repressor domain comprises EZH2 or a functional variant or fragment
thereof, wherein
optionally the second repressor domain is N-terminal of the second DNA-
targeting moiety.
10 126. The expression repression system of any of embodiments 67-120,
wherein:
the first repressor domain comprises EZH2 or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety; and
the second repressor domain comprises HDAC8 or a functional variant or
fragment thereof,
wherein optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
127. The expression repression system of any of embodiments 67-120, wherein:
the first repressor domain comprises G9A or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety; and
the second repressor domain comprises HDAC8 or a functional variant or
fragment thereof,
.. wherein optionally the second repressor domain is C-terminal of the second
DNA-targeting moiety.
128. The expression repression system of any of embodiments 69-120, wherein:
the first repressor domain comprises EZH2 or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety,
the third repressor domain comprises KRAB or a functional variant or fragment
thereof, wherein
optionally the third repressor domain is C-terminal of the first DNA-targeting
moiety; and
the second repressor domain comprises MQ1 or a functional variant or fragment
thereof, wherein
optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
.. 129. The expression repression system of any of embodiments 69-120,
wherein:
the first repressor domain comprises G9A or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety,
the third repressor domain comprises KRAB or a functional variant or fragment
thereof, wherein
optionally the third repressor domain is C-terminal of the first DNA-targeting
moiety, and
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
26
the second repressor domain comprises MQ1 or a functional variant or fragment
thereof, wherein
optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety
130. The expression repression system of any of embodiments 69-120, wherein:
the first repressor domain comprises EZH2 or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety,
the third repressor domain comprises KRAB or a functional variant or fragment
thereof, wherein
optionally the third repressor domain is C-terminal of the first DNA-targeting
moiety, and
the second repressor domain comprises HDAC8 or a functional variant or
fragment thereof,
wherein optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
131. The expression repression system of any of embodiments 69-120, wherein:
the first repressor domain comprises FOG1 or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety,
the third repressor domain comprises FOG1 or a functional variant or fragment
thereof, wherein
optionally the third repressor domain is C-terminal of the first DNA-targeting
moiety, and
the second repressor domain comprises KRAB or a functional variant or fragment
thereof,
wherein optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
132. The expression repression system of any of embodiments 69-120, wherein:
the first repressor domain comprises EZH2 or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety,
the third repressor domain comprises KRAB or a functional variant or fragment
thereof, wherein
optionally the third repressor domain is C-terminal of the first DNA-targeting
moiety, and
the second repressor domain comprises LSD1 or a functional variant or fragment
thereof, wherein
optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
133. The expression repression system of any of embodiments 69-130, wherein:
the first repressor domain comprises G9A or a functional variant or fragment
thereof, wherein
optionally the first repressor domain is N-terminal of the first DNA-targeting
moiety,
the third repressor domain comprises KRAB or a functional variant or fragment
thereof, wherein
optionally the third repressor domain is C-terminal of the first DNA-targeting
moiety, and
the second repressor domain comprises LSD1 or a functional variant or fragment
thereof, wherein
optionally the second repressor domain is C-terminal of the second DNA-
targeting moiety.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
27
134. The expression repression system of any of embodiments 67-133, wherein
the first DNA-targeting
moiety is selected from a TAL effector molecule, a CRISPR/Cas molecule, a zinc
finger domain, a tetR
domain, a meganuclease, or an oligonucleotide.
135. The expression repression system of any of embodiments 67-134, wherein
the second DNA-
targeting moiety is selected from a TAL effector molecule, a CRISPR/Cas
molecule, a zinc finger
domain, a tetR domain, a meganuclease, or an oligonucleotide.
136. The expression repression system of any of embodiments67-135, wherein the
first DNA-targeting
moiety is or comprises a CRISPR/Cas molecule comprising a CRISPR/Cas protein
(e.g., that is or
comprises a Cas protein or Cpfl protein chosen from Table 1 or a variant
(e.g., mutant) of any thereof)
and a guide RNA.
137. The expression repression system of any of embodiments67-136, wherein the
second DNA-targeting
moiety is or comprises a CRISPR/Cas molecule comprising a CRISPR/Cas protein
(e.g., that is or
comprises a Cas protein or Cpfl protein chosen from Table 1 or a variant
(e.g., mutant) of any thereof)
and a guide RNA.
138. The expression repression system of any of embodiments67-137, wherein:
the first DNA-targeting moiety is or comprises a CRISPR/Cas molecule
comprising a
CRISPR/Cas protein that is or comprises a Cas protein or Cpfl protein chosen
from Table 1 or a variant
(e.g., mutant) of any thereof), and
the second DNA-targeting moiety is or comprises a CRISPR/Cas molecule
comprising a different
CRISPR/Cas protein that is or comprises a Cas protein or Cpfl protein chosen
from Table 1 or a variant
(e.g., mutant) of any thereof).
139. The expression repression system of any of embodiments 135-138, wherein:
the first DNA-targeting moiety is or comprises a CRISPR/Cas molecule encoded
by a nucleic
acid sequence of SEQ ID NO: 45 or 89, and
the second DNA-targeting moiety is or comprises a CRISPR/Cas molecule encoded
by a nucleic acid
sequence of SEQ ID NO: 45 or 89.
140. The expression repression system of any of embodiments 135-138, wherein:
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
28
the first DNA-targeting moiety is or comprises a CRISPR/Cas molecule
comprising an amino
acid sequence of SEQ ID NO: 46 or 88, and
the second DNA-targeting moiety is or comprises a CRISPR/Cas molecule
comprising an amino acid
sequence of SEQ ID NO: 46 or 88.
141. The expression repression system of any of embodiments 67-140, wherein
the first or the second
DNA-targeting moiety specifically binds to a promoter sequence or to DNA
proximal to a promoter
sequence, e.g., a promoter sequence operably linked to a target gene.
142. The expression repression system of any of embodiments 67-141, wherein
the first or the second
DNA-targeting moiety specifically binds to an enhancer sequence or to DNA
proximal to an enhancer
sequence, e.g., an enhancer sequence that affects expression of and/or is
operably linked to a target gene.
143. The expression repression system of any of embodiments 67-142, wherein
the first or the second
DNA targeting moiety specifically binds to a target gene, e.g., sequence
comprised between the
transcription start site and stop codon of a target gene.
144. The expression repression system of any of embodiments 67-143, wherein
the first or the second
DNA targeting moiety specifically binds to the transcription start site of a
target gene.
145. The expression repression system of any of embodiments 67-144, wherein
the first or the second
DNA targeting moiety specifically binds to an intron, e.g., a splice site
associated with an intron, of a
target gene.
146. The expression repression system of any of embodiments 67-145, wherein
the first or the second
DNA targeting moiety specifically binds to an exon of a target gene.
147. The expression repression system of any of embodiments 67-146, wherein
first or the second DNA
targeting moiety specifically binds to an anchor sequence.
148. The expression repression system of embodiment 147, wherein the anchor
sequence comprises the
sequence of SEQ ID NO: 81 or 82, or a sequence with no more than 8, 7, 6, 5,
4, 3, 2, or 1 alterations
relative thereto.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
29
149. The expression repression system of embodiment 147, wherein the anchor
sequence comprises the
sequence of SEQ ID NO: 83 or 84, or a sequence with no more than 8, 7, 6, 5,
4, 3, 2, or 1 alterations
relative thereto.
150. The expression repression system of any of embodiments 147-149, wherein
the anchor sequence is
on the same chromosome as the gene.
151. The expression repression system of any of embodiments 147-149, wherein
the anchor sequence is
on a different chromosome as the gene.
152. The expression repression system of any of embodiments 147-151, wherein
the anchor sequence is
upstream of the target gene (e.g., upstream of the TSS or upstream of the
promoter).
153. The expression repression system of any of embodiments 148-151, wherein
the anchor sequence is
downstream of the target gene (e.g., downstream of the TSS or upstream of the
promoter).
154. The expression repression system of any of embodiments 67-141, wherein
the first DNA sequence is
a first site within or proximal to a promoter operably linked to a target gene
and the second DNA
sequence is a second site within or proximal to the promoter operably linked
to the target gene.
155. The expression repression system of any of embodiments 67-142, wherein
the first DNA sequence is
within or proximal to a promoter operably linked to a target gene and the
second DNA sequence is a
within or proximal to an enhancer that affects expression of or is operably
linked to the target gene.
156. The expression repression system of any of embodiments 67-142, wherein
the first DNA sequence is
within or proximal to an enhancer that affects expression of or is operably
linked to a target gene and the
second DNA sequence is a within or proximal to a promoter operably linked to
the target gene.
157. The expression repression system of any of embodiments 67-141 or 143,
wherein the first DNA
sequence comprises the transcription start site of a target gene and the
second DNA sequence is within or
proximal to a promoter operably linked to the target gene.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
158. The expression repression system of any of embodiments 67-140, 142 or
143, wherein the first DNA
sequence comprises the transcription start site of a target gene and the
second DNA sequence is within or
proximal to an enhancer that affects expression of or is operably linked to
the target gene.
5 159. The expression repression system of any of embodiments 67-141 or
143, wherein the first DNA
sequence within or proximal to a promoter operably linked to a target gene and
the second DNA sequence
is the transcription start site of the target gene.
160. The expression repression system of any of embodiments 67-140, 142, or
143, wherein the first
10 DNA sequence is within or proximal to an enhancer that affects
expression of or is operably linked to a
target gene and the second DNA sequence is the transcription start site of the
target gene.
161. The expression repression system of any of embodiments 67-160, wherein
the distance between the
first DNA sequence and the second DNA sequence is at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15,
15 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550,
600, 650, 700, 750, 800, 850,
900, 950, or 1000 base pairs or any size therebetween, e.g., 20-500 base
pairs.
20 .. 162.The expression repression system of any of embodiments 67-161,
wherein the distance between the
first DNA sequence and the second DNA sequence is no more than 100, 110, 120,
130, 140, 150, 160,
170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,
320, 330, 340, 350, 360, 370,
380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600,
650, 700, 750, 800, 850, 900,
950, or 1000 base pairs or any size therebetween, e.g., 20-500 base pairs.
163. The expression repression system of any of embodiments 67-162, wherein
the first DNA sequence
and the second DNA sequence are situated on different nucleic acids, e.g.,
different chromosomes.
164. The expression repression system of any of embodiments 67-163, wherein
the target gene is 13-2-
microglobulin, MYC, HSPA1B, GATA1, APOB, FOXP3, CXCL1, CXCL2, CXCL3, CXCL4,
CXCL5,
CXCL6, CXCL6, CXCL7, and/or CXCL8.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
31
165. The expression repression system of any of embodiments 73-164, wherein
the first guide RNA
comprises a sequence specific to, e.g., complementary to, the first DNA
sequence and the second guide
RNA comprises a sequence specific to, e.g., complementary to, the second DNA
sequence.
166. The expression repression system of any of embodiments 67-165, wherein
the first DNA-targeting
moiety does not appreciably bind to the second DNA sequence and the second DNA-
targeting moiety
does not appreciably bind to the first DNA sequence.
167. The expression repression system of any of embodiments 67-166, wherein
the first DNA-targeting
moiety binds the second DNA sequence with a KD of at least 500, 600, 700, 800,
900, 1000, 2000, 5000,
10,000, or 100,000 nM, and the second DNA-targeting moiety binds the first DNA
sequence with binds
with a KD of at least 500, 600, 700, 800, 900, 1000, 2000, 5000, 10,000, or
100,000 nM.
168. The expression repression system of any of embodiments 67-167, wherein
binding of the first
expression repressor to the first DNA sequence decreases expression of a
target gene, e.g., a target gene
operably linked to the first DNA sequence, in a cell.
169. The expression repression system of embodiment 168, wherein expression is
decreased by 10, 20,
30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of
the first expression
repressor, e.g., as measured by ELISA or as described in Examples 2-4.
170. The expression repression system of any of embodiments 67-169, wherein
binding of the first
expression repressor to the first DNA sequence appreciably decreases
expression of a target gene, e.g., a
target gene operably linked to the first DNA sequence, for a time period of at
least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days, or at
least 1,2, 3, 4, 5, 6,7, 8, 9, or 10
cell divisions, e.g., as measured by ELISA or as described in Examples 2-4.
171. The expression repression system of any of embodiments 67-170, wherein
binding of the second
expression repressor to the second DNA sequence decreases expression of a
target gene, e.g., a target
gene operably linked to the second DNA sequence, in a cell.
172. The expression repression system of embodiment 171, wherein expression is
decreased by 10, 20,
30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of
the second expression
repressor, e.g., as measured by ELISA or as described in Examples 2-4.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
32
173. The expression repression system of any of embodiments 67-172, wherein
binding of the second
expression repressor to the second DNA sequence appreciably decreases
expression of a target gene, e.g.,
a target gene operably linked to the second DNA sequence, for a time period of
at least 1, 2, 3, 4, 5, 6, 7,
8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days,
or at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 cell divisions, e.g., as measured by ELISA or as described in Examples 2-
4.
174. The expression repression system of any of embodiments 67-173, wherein
binding of the first
expression repressor to the first DNA sequence and the second expression
repressor to the second DNA
.. sequence decreases expression of a target gene, e.g., a target gene
operably linked to the first DNA
sequence and/or the second DNA sequence, in a cell.
175. The expression repression system of embodiment 174, wherein expression is
decreased by 10, 20,
30, 40, 50, 60, 70, 80, 90, or 100% compared to expression in the absence of
the first and second
expression repressors, e.g., as measured by ELISA or as described in Examples
2-4.
176. The expression repression system of any of embodiments 1-175, wherein
binding of the first
expression repressor to the first DNA sequence and the second expression
repressor to the second DNA
sequence appreciably decreases expression of a target gene, e.g., a target
gene operably linked to the first
DNA sequence, for a time period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
or 12 hours, or at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
or 25 days, or at least 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 cell divisions, e.g., as measured by ELISA or as described
in Examples 2-4.
177. The expression repression system of any of embodiments 174-176, wherein
the decrease in
.. expression resulting from the binding of the first expression repressor to
the first DNA sequence and the
second expression repressor to the second DNA sequence is greater than the
decrease in expression
resulting from the binding of the first expression repressor to the first DNA
sequence or the binding of the
second expression repressor to the second DNA sequence individually.
178. The expression repression system of embodiment 177, wherein the binding
of the first expression
repressor to the first DNA sequence and the second expression repressor to the
second DNA sequence
decreases expression 1.05x (i.e., 1.05 times), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x,
1.35x, 1.4x, 1.45x, 1.5x, 1.6x,
1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 50x, or 100x more
than either the binding of the
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
33
first expression repressor to the first DNA sequence or the binding of the
second expression repressor to
the second DNA sequence individually, e.g., as measured by ELISA or as
described in Examples 2-4.
179. The expression repression system of embodiment 174-178, wherein the
decrease in expression
resulting from the binding of the first expression repressor to the first DNA
sequence and the second
expression repressor to the second DNA sequence persists for a longer time
(e.g., more hours, days, or
cell divisions) than the decrease in expression resulting from the binding of
the first expression repressor
to the first DNA sequence or the binding of the second expression repressor to
the second DNA sequence
individually.
180. The expression repression system of embodiment 179, wherein the binding
of the first expression
repressor to the first DNA sequence and the second expression repressor to the
second DNA sequence
decreases expression 1.05x (i.e., 1.05 times), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x,
1.35x, 1.4x, 1.45x, 1.5x, 1.6x,
1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 50x, or 100x
longer (e.g., as measured in hours,
days, or cell divisions) than either the binding of the first expression
repressor to the first DNA sequence
or the binding of the second expression repressor to the second DNA sequence
individually, e.g., as
measured by ELISA or as described in Examples 2-4.
181. The expression repression system of any of embodiments 174-180, wherein
the cell is dividing at a
rate of no more than once per 24, 36, 48, 60, or 72 hours (and optionally, at
least once per 24, 36, 48, 60,
or 72 hours).
182. The expression repression system of any of embodiments 174-181, wherein
the cell is dividing at a
rate of at least once per 24, 36, 48, 60, or 72 hours (and optionally, no more
than once per 24, 36, 48, 60,
or 72 hours).
183. The expression repression system of any of embodiments 174-182, wherein
expression is
appreciably decreased indefinitely (e.g., for a time period greater than can
be experimentally measured).
184. The expression repression system of any of embodiments 67-183, wherein
the first DNA-targeting moiety comprises an S. pyo genes CRISPR/Cas protein
selected from
Table 1 or variant (e.g., mutant) thereof, e.g., an S. pyogenes dCas9, e.g., a
dCas9 comprising an amino
acid sequence of SEQ ID NO: 46; or a S. aureus CRISPR/Cas protein selected
from Table 1 or variant
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
34
(e.g., mutant) thereof, e.g., an S. aureus dCas9, e.g., a dCas9 comprising an
amino acid sequence of SEQ
ID NO: 88 and
the second DNA-targeting moiety comprises an S. pyogenes CRISPR/Cas protein
selected from
Table 1 or variant (e.g., mutant) thereof, e.g., an S. pyogenes dCas9, e.g., a
dCas9 comprising an amino
acid sequence of SEQ ID NO: 46; or a CRISPR/Cas molecule comprising a S.
aureus CRISPR/Cas
protein selected from Table 1 or variant (e.g., mutant) thereof, e.g., an S.
aureus dCas9, e.g., a dCas9
comprising an amino acid sequence of SEQ ID NO: 88.
185. The expression repression system of any of embodiments 67-184, wherein
the first DNA-targeting moiety comprises a CRISPR/Cas molecule comprising a
CRISPR/Cas
protein, e.g., a catalytically inactive dCas9, e.g., a dCas9, e.g., a dCas9
comprising one or more mutations
selected from D 10A, D839A, H840A, and N863A mutations; and
the second DNA-targeting moiety comprises a CRISPR/Cas molecule comprising a
CRISPR/Cas
protein, e.g., a catalytically inactive dCas9, e.g., a dCas9, e.g., a dCas9
comprising one or more mutations
selected from DlOA, D839A, H840A, and N863A mutations.
186. The expression repression system of any of embodiments 67-185, wherein
the first expression repressor comprises a first DNA-targeting moiety
comprising a CRISPR/Cas
molecule comprising an S. pyogenes CRISPR/Cas protein selected from Table 1 or
variant (e.g., mutant)
thereof, e.g., an S. pyogenes dCas9, and a first repressor domain comprising
KRAB or a functional variant
or fragment thereof; and
the second expression repressor comprises a second DNA-targeting moiety
comprising a
CRISPR/Cas molecule comprising a S. aureus CRISPR/Cas protein selected from
Table 1 or variant (e.g.,
mutant) thereof, e.g., an S. aureus dCas9, and a second repressor domain
comprising bacterial MQ1 or a
functional variant or fragment thereof.
187. A polypeptide comprising:
a DNA-targeting moiety; and
a repressor domain comprising bacterial MQ1 or a functional variant or
fragment thereof.
188. A polypeptide comprising:
a DNA-targeting moiety;
a first repressor domain comprising MQ1, DNMT1, DNMT3a/31, KRAB, G9A, HDAC8,
LSD1,
EZH2, FOG1 or a functional variant or fragment thereof, and
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
a second repressor domain comprising MQ1, DNMT1, DNMT3a/31, KRAB, G9A, HDAC8,
LSD1, EZH2, FOG1 or a functional variant or fragment thereof.
189. The polypeptide of embodiment 88a, wherein the DNA-targeting moiety is
situated between the first
5 repressor domain and the second repressor domain.
190. A nucleic acid encoding the first expression repressor of the expression
repression system of any of
embodiments 67-189.
10 191. A nucleic acid encoding the second expression repressor of the
expression repression system of any
of embodiments 67-189.
192. A nucleic acid encoding the expression repression system, e.g., the first
expression repressor and the
second expression repressor, of any of embodiments 67-189.
193. The nucleic acid of any of embodiments 190-192, which is an RNA, e.g., an
mRNA.
194. An expression repression system comprising:
a first nucleic acid comprising a sequence encoding the first expression
repressor of the
expression repression system of any of embodiments 67-189; and
a second nucleic acid comprising a sequence encoding the second expression
repressor of the
expression repression system of any of embodiments 67-189.
195. A nucleic acid encoding the polypeptide of any of embodiments 187-189.
196. The nucleic acid any of embodiments 190-193 or 195, wherein the nucleic
acid comprises mRNA.
197. A vector comprising the nucleic acid encoding the expression repressor or
the expression repression
system of any preceding embodiment.
198. A lipid nanoparticle comprising the expression repressor, expression
repression system, nucleic
acid, mRNA, or vector of any of the preceding embodiments.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
36
199. The lipid nanoparticle of embodiment 198 comprising an ionizable lipid,
e.g., a cationic lipid, e.g.,
MC3, SSOP.
200. The lipid nanoparticle of embodiment 198 or 199, further comprising one
or more of neutral lipids,
ionizable amine-containing lipids, biodegradable alkyn lipids, steroids,
phospholipids, polyunsaturated
lipids, structural lipids (e.g., sterols), PEG, cholesterol, or polymer
conjugated lipids.
201. A reaction mixture comprising the expression repressor, expression
repression system, nucleic acid,
vector, or lipid nanoparticle of any of the preceding embodiments.
202. The reaction mixture of embodiment 201 further comprising a cell.
203. A cell comprising the expression repressor, expression repression system,
nucleic acid, mRNA,
vector, or nanoparticle of any of embodiments 1-202.
204. The cell of embodiment 203, wherein the cell is selected from a
hepatocyte, a neuronal cell, an
endothelial cell, a myocyte, and a lymphocyte.
205. A cell produced by a method comprising:
providing i) a cell and ii) an expression repressor, expression repression
system, polypeptide,
nucleic acid, vector, lipid nanoparticle, or reaction mixture of any of
embodiments 1-202; and
contacting the cell with the expression repressor, expression repression
system, polypeptide,
nucleic acid, vector, lipid nanoparticle, or reaction mixture.
206. A pharmaceutical composition comprising the expression repressor,
expression repression system,
nucleic acid, polypeptide, vector, lipid nanoparticle, or reaction mixture of
any of embodiments 1-202,
and at least one pharmaceutically acceptable excipient or carrier.
207. The expression repression system of any of embodiments 1-202, wherein the
first expression
repressor further comprises an additional moiety selected from: a tagging or
monitoring moiety, a
cleavable moiety (e.g., a cleavable moiety positioned between a DNA-targeting
moiety and a repressor
domain or at the N- or C-terminal end of a polypeptide), a small molecule, a
membrane translocating
polypeptide, or a pharmacoagent moiety.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
37
208. The expression repression system of any of embodiments 1-202, wherein the
second expression
repressor further comprises an additional moiety selected from: a tagging or
monitoring moiety, a
cleavable moiety (e.g., a cleavable moiety positioned between a DNA-targeting
moiety and a repressor
domain or at the N- or C-terminal end of a polypeptide), a small molecule, a
membrane translocating
polypeptide, or a pharmacoagent moiety.
209. A method of decreasing expression of a target genomic sequence in a cell,
comprising:
providing the expression repressor, expression repression system, nucleic
acid, polypeptide,
vector, lipid nanoparticle, reaction mixture, cell, or pharmaceutical
composition of any of embodiments 1-
204 or 206; and
contacting the cell with the expression repressor, expression repression
system, nucleic acid,
polypeptide, vector, lipid nanoparticle, reaction mixture, cell, or
pharmaceutical composition
thereby decreasing expression of the target genomic sequence.
210. A method of epigenetically modifying a target genomic sequence in a cell,
comprising:
providing an expression repressor, expression repression system, nucleic acid,
vector,
polypeptide, lipid nanoparticle, reaction mixture, cell, or pharmaceutical
composition of any of
embodiments 1-204 or 206; and
contacting the cell with the expression repressor, expression repression
system, nucleic acid,
.. vector, polypeptide, lipid nanoparticle, reaction mixture, cell, or
pharmaceutical composition,
thereby epigenetically modifying the target genomic sequence.
211. The method off embodiment 209 or 210, wherein the target genomic sequence
is:
a target gene (e.g., a site within a target gene, e.g., an exon, intron, or
splice site), e.g., the gene
encoding I3-2-microglobulin (I32M), the gene encoding MYC, the gene encoding
HSPA1B, or the gene
encoding GATA1,
a transcription control element operably linked to a target gene, or
an anchor sequence proximal to a target gene or associated with an anchor
sequence-mediated
conjunction operably linked to the target gene.
212. The method of embodiment211, wherein the epigenetic modification results
in a decrease in
expression of the target gene.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
38
213. The expression repressor, expression repression system, cell,
pharmaceutical composition, nucleic
acid, vector, or method of any of embodiments174-212, wherein the cell is a
mammalian cell, e.g., a
human cell.
214. The method of embodiment213, wherein the method is performed ex vivo
(e.g., in a sample from a
subject) or in vivo (e.g., by administering the expression repression system
to a subject).
215. The method of any of embodiments209-214, wherein expression is decreased
by 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100% compared to expression in an otherwise similar cell in
the absence of the
expression repression system.
216. The method of any of embodiments209-214, wherein expression is decreased
by 10, 20, 30, 40, 50,
60, 70, 80, 90, or 100% compared to expression in an otherwise similar cell
that has not been contacted
with the expression repression system.
217. The method of any of embodiments209-216, wherein expression is decreased
for a time period of at
least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, or 25 days, or at least
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cell divisions, e.g., as measured by ELISA or
as described in Examples 2-4.
218. The method of any of embodiments215-217, wherein the decrease in
expression comprises a
decrease in the level of RNA, e.g., mRNA, encoded by the target gene.
219. The method of any of embodiments215-218, wherein the decrease in
expression comprises a
decrease in the level of a protein encoded by the target gene.
220. The method of any of embodiments 209-219, wherein providing comprises
contacting the cell with a
nucleic acid, e.g., vector, encoding the expression repression system to the
cell comprising the target
gene.
221. The method of any of embodiments 209-220, wherein the nucleic acid
encoding the first expression
repressor is disposed on a first nucleic acid molecule and the nucleic acid
encoding the second expression
repressor is disposed on a second nucleic acid molecule.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
39
222. The method of any of embodiments 209-221, wherein the nucleic acid
encoding the first expression
repressor is disposed on the same nucleic acid molecule as the nucleic acid
encoding the second
expression repressor.
223. The method of any of embodiments 209-222, wherein administering comprises
contacting the cell
with the first nucleic acid molecule and the second nucleic acid molecule
together, e.g., simultaneously.
224. The method of any of embodiments 209-223, wherein administering comprises
contacting the cell
with the first nucleic acid molecule and the second nucleic acid molecule
separately, e.g., sequentially.
225. The method of any of embodiments 209-224, wherein providing comprises
contacting the cell with
a vesicle comprising the expression repression system or a nucleic acid, e.g.,
vector, encoding the
expression repression system.
226. The method of any of embodiments 209-225, wherein providing comprises
contacting the cell with a
lipid nanoparticle (LNP) comprising the expression repression system or a
nucleic acid, e.g., vector,
encoding the expression repression system.
227. The method of any of embodiments 209-226, wherein the expression
repression system comprises a
first expression repressor and a second expression repressor, and providing
comprises administering the
first expression repressor and, separately, administering the second
expression repressor.
228. The method of any of embodiments 209-227, wherein the expression
repression system comprises a
first expression repressor and a second expression repressor and providing
comprises administering the
first expression repressor and the second expression repressor together, e.g.,
simultaneously.
229. A method of treating a condition associated with over-expression of a
target gene in a subject,
comprising:
administering the expression repressor, expression repression system, nucleic
acid, vector,
polypeptide, lipid nanoparticle, reaction mixture, cell, or pharmaceutical
composition of any of
embodiments 1-204 or 206-208 to the subject,
thereby treating a condition associated with over-expression of a target gene
in a subject.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
230. A method of treating a condition associated with mis-regulation of a
target gene in a subject,
comprising:
administering the expression repressor, expression repression system, nucleic
acid, vector,
polypeptide, lipid nanoparticle, reaction mixture, cell, or pharmaceutical
composition of any of
5 embodiments 1-204 or 206-208 to the subject,
thereby treating a condition associated with mis-regulation of a target gene
in a subject.
231. A method of making a cell comprising an expression repression system, the
method comprising:
providing the expression repressor, expression repression system, nucleic
acid, vector,
10 polypeptide, or pharmaceutical composition of any of embodiments 1-201
or 206-208; and
contacting the cell with the expression repressor, expression repression
system, nucleic acid,
vector, or pharmaceutical composition,
thereby making a cell comprising an expression repression system.
15 232. A method of making a nucleic acid encoding an expression repression
system, the method
comprising:
providing a first nucleic acid encoding the first expression repressor of the
expression repression
system of any of embodiments 67-186, 207, or 208;
providing a second nucleic acid encoding the second expression repressor of
the expression
20 repression system of any of embodiments 67-186, 9207, or 208;
combining (e.g., ligating or recombining) the first nucleic acid and the
second nucleic acid into a
single nucleic acid molecule,
thereby making a nucleic acid encoding an expression repression system.
25 233. A cell treated or produced by the method of any of embodiments 209-
228 or 230.
234. The cell of embodiment 233, wherein the cell does not comprise the
expression repression system,
e.g., at least one component (e.g., all components) of the expression
repression system.
30 235. The cell of either of embodiments 233 or 234, wherein expression of
the target gene is decreased
relative to a cell not contacted, treated, or produced by the method of any of
embodiments 209-228 or
230.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
41
236. A kit comprising a container comprising a composition comprising an
expression repressor, an
expression repression system, one or more nucleic acids encoding said
expression repressor or an
expression repression system, a vector, a lipid nanoparticle, or a
pharmaceutical composition of any of
embodiments 1-208 and a set of instructions comprising at least one method for
modulating, e.g.,
.. decreasing the expression of a gene within a cell with said composition.
DEFINITIONS
Anchor Sequence: The term "anchor sequence" as used herein, refers to a
nucleic acid sequence
recognized by a nucleating agent that binds sufficiently to form an anchor
sequence-mediated
conjunction, e.g., a complex. In some embodiments, an anchor sequence
comprises one or more CTCF
binding motifs. In some embodiments, an anchor sequence is not located within
a gene coding region. In
some embodiments, an anchor sequence is located within an intergenic region.
In some embodiments, an
anchor sequence is not located within either of an enhancer or a promoter. In
some embodiments, an
anchor sequence is located at least 400 bp, at least 450 bp, at least 500 bp,
at least 550 bp, at least 600 bp,
at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least
850 bp, at least 900 bp, at least 950
bp, or at least lkb away from any transcription start site. In some
embodiments, an anchor sequence is
located within a region that is not associated with genomic imprinting,
monoallelic expression, and/or
monoallelic epigenetic marks. In some embodiments, the anchor sequence has one
or more functions
selected from binding an endogenous nucleating polypeptide (e.g., CTCF),
interacting with a second
anchor sequence to form an anchor sequence mediated conjunction, or insulating
against an enhancer that
is outside the anchor sequence mediated conjunction. In some embodiments of
the present disclosure,
technologies are provided that may specifically target a particular anchor
sequence or anchor sequences,
without targeting other anchor sequences (e.g., sequences that may contain a
nucleating agent (e.g.,
CTCF) binding motif in a different context); such targeted anchor sequences
may be referred to as the
"target anchor sequence". In some embodiments, sequence and/or activity of a
target anchor sequence is
modulated while sequence and/or activity of one or more other anchor sequences
that may be present in
the same system (e.g., in the same cell and/or in some embodiments on the same
nucleic acid molecule ¨
e.g., the same chromosome) as the targeted anchor sequence is not modulated.
In some embodiments, the
anchor sequence comprises or is a nucleating polypeptide binding motif. In
some embodiments, the
anchor sequence is adjacent to a nucleating polypeptide binding motif.
Anchor Sequence-Mediated Conjunction: The term "anchor sequence-mediated
conjunction" as
used herein, refers to a DNA structure, in some cases, a complex, that occurs
and/or is maintained via
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
42
physical interaction or binding of at least two anchor sequences in the DNA by
one or more polypeptides,
such as nucleating polypeptides, or one or more proteins and/or a nucleic acid
entity (such as RNA or
DNA), that bind the anchor sequences to enable spatial proximity and
functional linkage between the
anchor sequences (see, e.g. Figure 1).
Associated with: Two events or entities are "associated" with one another, as
that term is used
herein, if presence, level, form and/or function of one is correlated with
that of the other. For example, in
some embodiments, a particular entity (e.g., polypeptide, genetic signature,
metabolite, microbe, etc.) is
considered to be associated with a particular disease, disorder, or condition,
if its presence, level, form
and/or function correlates with incidence of and/or susceptibility to the
disease, disorder, or condition
(e.g., across a relevant population). In some embodiments, two or more
entities are physically
"associated" with one another if they interact, directly or indirectly, so
that they are and/or remain in
physical proximity with one another. In some embodiments, two or more entities
that are physically
associated with one another are covalently linked to one another; in some
embodiments, two or more
entities that are physically associated with one another are not covalently
linked to one another but are
.. non-covalently associated, for example by means of hydrogen bonds, van der
Waals interaction,
hydrophobic interactions, magnetism, and combinations thereof. In some
embodiments, a DNA sequence
is "associated with" a target genomic or transcription complex when the
nucleic acid is at least partially
within the target genomic or transcription complex, and expression of a gene
in the DNA sequence is
affected by formation or disruption of the target genomic or transcription
complex.
Domain: As used herein, the term "domain" refers to a section or portion of an
entity. In some
embodiments, a "domain" is associated with a particular structural and/or
functional feature of the entity
so that, when the domain is physically separated from the rest of its parent
entity, it substantially or
entirely retains the particular structural and/or functional feature.
Alternatively, or additionally, in some
embodiments, a domain may be or include a portion of an entity that, when
separated from that (parent)
entity and linked with a different (recipient) entity, substantially retains
and/or imparts on the recipient
entity one or more structural and/or functional features that characterized it
in the parent entity. In some
embodiments, a domain is or comprises a section or portion of a molecule
(e.g., a small molecule,
carbohydrate, lipid, nucleic acid, polypeptide, etc.). In some embodiments, a
domain is or comprises a
section of a polypeptide. In some such embodiments, a domain is characterized
by a particular structural
element (e.g., a particular amino acid sequence or sequence motif, alpha-helix
character, beta-sheet
character, coiled-coil character, random coil character, etc.), and/or by a
particular functional feature (e.g.,
binding activity, enzymatic activity, folding activity, signaling activity,
etc.).
Expression repressor: As used herein, the term "expression repressor" refers
to an agent or entity
with one or more functionalities that decrease expression of a target gene in
a cell and that specifically
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
43
binds to a DNA sequence (e.g., a DNA sequence associated with a target gene,
or a transcription control
element operably linked to a target gene). An expression repressor comprises
at least one DNA-targeting
moiety and at least one repressor domain.
Expression repression system: As used herein, the term "expression repression
system" refers to
a plurality of expression repressors which decrease expression of a target
gene in a cell. In some
embodiments, an expression repression system comprises a first expression
repressor and a second
expression repressor, wherein the first expression repressor and second
expression repressor (or nucleic
acids encoding the first expression repressor and second expression repressor)
are present together in a
single composition, mixture, or pharmaceutical composition. In some
embodiments, an expression
repression system comprises a first expression repressor and a second
expression repressor, wherein the
first expression repressor and second expression repressor (or nucleic acids
encoding the first expression
repressor and second expression repressor) are present in separate
compositions or pharmaceutical
compositions. In some embodiments, the first expression repressor and the
second expression repressor
are present in the same cell at the same time. In some embodiments, the first
expression repressor and the
second expression repressor are not present in the same cell at the same time,
e.g., they are present
sequentially. For example, the first expression repressor may be present in a
cell for a first time period,
and then the second expression repressor may be present in the cell for a
second time period, wherein the
first and second time periods may be overlapping or non-overlapping.
Genomic complex: As used herein, the term "genomic complex" is a complex that
brings
.. together two genomic sequence elements that are spaced apart from one
another on one or more
chromosomes, via interactions between and among a plurality of protein and/or
other components
(potentially including, the genomic sequence elements). In some embodiments,
the genomic sequence
elements are anchor sequences to which one or more protein components of the
complex binds. In some
embodiments, a genomic complex may comprise an anchor sequence-mediated
conjunction. In some
embodiments, a genomic sequence element may be or comprise a CTCF binding
motif, a promoter and/or
an enhancer. In some embodiments, a genomic sequence element includes at least
one or both of a
promoter and/or regulatory site (e.g., an enhancer). In some embodiments,
complex formation is
nucleated at the genomic sequence element(s) and/or by binding of one or more
of the protein
component(s) to the genomic sequence element(s). As will be understood by
those skilled in the art, in
some embodiments, co-localization (e.g., conjunction) of the genomic sites via
formation of the complex
alters DNA topology at or near the genomic sequence element(s), including, in
some embodiments,
between them. In some embodiments, a genomic complex comprises an anchor
sequence-mediated
conjunction, which comprises one or more loops. In some embodiments, a genomic
complex as described
herein is nucleated by a nucleating polypeptide such as, for example, CTCF
and/or Cohesin. In some
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
44
embodiments, a genomic complex as described herein may include, for example,
one or more of CTCF,
Cohesin, non-coding RNA (e.g., eRNA), transcriptional machinery proteins
(e.g., RNA polymerase, one
or more transcription factors, for example selected from the group consisting
of TFIIA, TFIIB, TFIID,
TFIIE, TFIIF, TFIIH, etc.), transcriptional regulators (e.g., Mediator, P300,
enhancer-binding proteins,
repressor-binding proteins, histone modifiers, etc.), etc. In some
embodiments, a genomic complex as
described herein includes one or more polypeptide components and/or one or
more nucleic acid
components (e.g., one or more RNA components), which may, in some embodiments,
be interacting with
one another and/or with one or more genomic sequence elements (e.g., anchor
sequences, promoter
sequences, regulatory sequences (e.g., enhancer sequences)) so as to constrain
a stretch of genomic DNA
into a topological configuration (e.g., a loop) that it does not adopt when
the complex is not formed.
Nucleic acid: As used herein, in its broadest sense, the term "nucleic acid"
refers to any
compound and/or substance that is or can be incorporated into an
oligonucleotide chain. In some
embodiments, a nucleic acid is a compound and/or substance that is or can be
incorporated into an
oligonucleotide chain via a phosphodiester linkage. As will be clear from
context, in some embodiments,
"nucleic acid" refers to an individual nucleic acid residue (e.g., a
nucleotide and/or nucleoside); in some
embodiments, "nucleic acid" refers to an oligonucleotide chain comprising
individual nucleic acid
residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some
embodiments, a "nucleic
acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises,
or consists of one or
more natural nucleic acid residues. In some embodiments, a nucleic acid is,
comprises, or consists of one
or more nucleic acid analogs. In some embodiments, a nucleic acid analog
differs from a nucleic acid in
that it does not utilize a phosphodiester backbone. For example, in some
embodiments, a nucleic acid is,
comprises, or consists of one or more "peptide nucleic acids", which are known
in the art and have
peptide bonds instead of phosphodiester bonds in the backbone, are considered
within the scope of the
present disclosure. Alternatively, or additionally, in some embodiments, a
nucleic acid has one or more
phosphorothioate and/or 5'-N-phosphoramidite linkages rather than
phosphodiester bonds. In some
embodiments, a nucleic acid is, comprises, or consists of one or more natural
nucleosides (e.g., adenosine,
thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy
guanosine, and
deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists
of one or more nucleoside
analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,
3 -methyl adenosine, 5-
methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,
C5-bromouridine, C5-
fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-
methylcytidine, 2-
aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-
oxoguanosine, 0(6)-
methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and
combinations thereof). In some
embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-
fluororibose, ribose, 2'-
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
deoxyribose, arabinose, and hexose) as compared with those in natural nucleic
acids. In some
embodiments, a nucleic acid has a nucleotide sequence that encodes a
functional gene product such as an
RNA or protein. In some embodiments, a nucleic acid includes one or more
introns. In some
embodiments, nucleic acids are prepared by one or more of isolation from a
natural source, enzymatic
5 synthesis by polymerization based on a complementary template (in vivo or
in vitro), reproduction in a
recombinant cell or system, and chemical synthesis. In some embodiments, a
nucleic acid is at least 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 110, 120, 130, 140,
150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,
475, 500, 600, 700, 800,
900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues
long. In some
10 embodiments, a nucleic acid is partly or wholly single stranded; in some
embodiments, a nucleic acid is
partly or wholly double stranded. In some embodiments a nucleic acid has a
nucleotide sequence
comprising at least one element that encodes, or is the complement of a
sequence that encodes, a
polypeptide. In some embodiments, a nucleic acid has enzymatic activity.
Operably linked: As used herein, the phrase "operably linked" refers to a
juxtaposition wherein
15 the components described are in a relationship permitting them to
function in their intended manner. A
transcription control element "operably linked" to a functional element, e.g.,
gene, is associated in such a
way that expression and/or activity of the functional element, e.g., gene, is
achieved under conditions
compatible with the transcription control element. In some embodiments,
"operably linked" transcription
control elements are contiguous (e.g., covalently linked) with coding
elements, e.g., genes, of interest; in
20 some embodiments, operably linked transcription control elements act in
trans to or otherwise at a
distance from the functional element, e.g., gene, of interest. In some
embodiments, operably linked
means two nucleic acid sequences are comprised on the same nucleic acid
molecule. In a further
embodiment, operably linked may further mean that the two nucleic acid
sequences are proximal to one
another on the same nucleic acid molecule, e.g., within 1000, 500, 100, 50, or
10 base pairs of each other
25 or directly adjacent to each other.
Peptide, Polyp eptide, Protein: As used herein, the terms "peptide,"
"polypeptide," and "protein"
refer to a compound comprised of amino acid residues covalently linked by
peptide bonds, or by means
other than peptide bonds. A protein or peptide must contain at least two amino
acids, and no limitation is
30 placed on the maximum number of amino acids that can comprise a
protein's or peptide's sequence.
Polypeptides include any peptide or protein comprising two or more amino acids
joined to each other by
peptide bonds or by means other than peptide bonds. As used herein, the term
refers to both short chains,
which also commonly are referred to in the art as peptides, oligopeptides, and
oligomers, for example, and
to longer chains, which generally are referred to in the art as proteins, of
which there are many types.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
46
Repressor domain: As used herein, the term "repressor domain" refers to a
domain with one or
more functionalities that decrease expression of a target gene in a cell when
appropriately localized in the
nucleus of a cell. In some embodiments, a repressor domain is or comprises a
polypeptide. In some
embodiments, a repressor domain is or comprises a polypeptide and a nucleic
acid. A functionality
associated with a repressor domain may directly affect expression of a target
gene, e.g., blocking
recruitment of a transcription factor that would stimulate expression of the
gene. A functionality
associated with a repressor domain may indirectly affect expression of a
target gene, e.g., introducing
epigenetic modifications or recruiting other factors that introduce epigenetic
modifications that induce a
change in chromosomal topology that inhibits expression of a target gene.
Specific binding: As used herein, the term "specific binding" refers to an
ability to discriminate
between possible binding partners in the environment in which binding is to
occur. In some
embodiments, a binding agent that interacts with one particular target when
other potential targets are
present is said to "bind specifically" to the target with which it interacts.
In some embodiments, specific
binding is assessed by detecting or determining degree of association between
the binding agent and its
.. partner; in some embodiments, specific binding is assessed by detecting or
determining degree of
dissociation of a binding agent-partner complex. In some embodiments, specific
binding is assessed by
detecting or determining ability of the binding agent to compete with an
alternative interaction between
its partner and another entity. In some embodiments, specific binding is
assessed by performing such
detections or determinations across a range of concentrations.
Substantially: As used herein, the term "substantially" refers to the
qualitative condition of
exhibiting total or near-total extent or degree of a characteristic or
property of interest. One of ordinary
skill in the art will understand that biological and chemical phenomena
rarely, if ever, go to completion
and/or proceed to completeness or achieve or avoid an absolute result. The
term "substantially" may
therefore be used in some embodiments herein to capture potential lack of
completeness inherent in many
biological and chemical phenomena.
Symptoms are reduced: As used herein, the phrase "symptoms are reduced" may be
used when
one or more symptoms of a particular disease, disorder or condition is reduced
in magnitude (e.g.,
intensity, severity, etc.) and/or frequency. In some embodiments, a delay in
the onset of a particular
symptom is considered one form of reducing the frequency of that symptom.
Target: An agent or entity is considered to "target" another agent or entity,
in accordance with
the present disclosure, if it binds specifically to the targeted agent or
entity under conditions in which they
come into contact with one another. In some embodiments, for example, an
antibody (or antigen-binding
fragment thereof) targets its cognate epitope or antigen. In some embodiments,
a nucleic acid having a
particular sequence targets a nucleic acid of substantially complementary
sequence.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
47
Target gene: As used herein, the term "target gene" means a gene that is
targeted for
modulation, e.g., of expression. In some embodiments, a target gene is part of
a targeted genomic
complex (e.g., a gene that has at least part of its genomic sequence as part
of a target genomic complex,
e.g. inside an anchor sequence-mediated conjunction), which genomic complex is
targeted by one or more
modulating agents as described herein. In some embodiments, modulation
comprises inhibition of
expression of the target gene. In some embodiments, a target gene is modulated
by contacting the target
gene or a transcription control element operably linked to the target gene
with an expression repression
system, e.g., expression repressor(s), described herein. In some embodiments,
a target gene is aberrantly
expressed (e.g., over-expressed) in a cell, e.g., a cell in a subject (e.g.,
patient).
DNA-targeting moiety: As used herein, in the context of expression repressors,
the term "DNA-
targeting moiety" means a domain that specifically binds to a DNA sequence
(e.g., a DNA sequence
associated with a target gene, or a transcription control element operably
linked to a target gene).
Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an
agent that, when
administered to a subject, has a therapeutic effect and/or elicits a desired
biological and/or
pharmacological effect. In some embodiments, a therapeutic agent is any
substance that can be used to
alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce
severity of, and/or reduce incidence
of one or more symptoms or features of a disease, disorder, and/or condition.
In some embodiments, a
therapeutic agent comprises an expression repression system, e.g., an
expression repressor, described
herein. In some embodiments, a therapeutic agent comprises a nucleic acid
encoding an expression
repression system, e.g., an expression repressor, described herein. In some
embodiments, a therapeutic
agent comprises a pharmaceutical composition described herein.
Therapeutically effective amount: As used herein, the term "therapeutically
effective amount"
means an amount of a substance (e.g., a therapeutic agent, composition, and/or
formulation) that elicits a
desired biological response when administered as part of a therapeutic
regimen. In some embodiments, a
therapeutically effective amount of a substance is an amount that is
sufficient, when administered to a
subject suffering from or susceptible to a disease, disorder, and/or
condition, to treat, diagnose, prevent,
and/or delay the onset of the disease, disorder, and/or condition. As will be
appreciated by those of
ordinary skill in this art, an effective amount of a substance may vary
depending on such factors as
desired biological endpoint(s), substance to be delivered, target cell(s) or
tissue(s), etc. For example, in
some embodiments, an effective amount of compound in a formulation to treat a
disease, disorder, and/or
condition is an amount that alleviates, ameliorates, relieves, inhibits,
prevents, delays onset of, reduces
severity of and/or reduces incidence of one or more symptoms or features of
the disease, disorder, and/or
condition. In some embodiments, a therapeutically effective amount is
administered in a single dose; in
some embodiments, multiple unit doses are required to deliver a
therapeutically effective amount.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
48
BRIEF DESCRIPTION OF THE DRAWING
The following detailed description of the embodiments of the invention will be
better understood
when read in conjunction with the appended drawings. For the purpose of
illustrating the invention, there
.. are shown in the drawings embodiments, which are presently exemplified. It
should be understood,
however, that the invention is not limited to the precise arrangement and
instrumentalities of the
embodiments shown in the drawings.
Figure 1 shows a Genome Browser view showing key features of the region near
the
transcription start site of I32M. RNAseq (top level) shows mRNA expression
from the I32M gene. A
previously described positive control (Cas9 targeting, GD-21547) is shown in
green. Blue guides (GD-
28228 and GD-28229) were used to target Sp-dCas9-KRAB and orange guides (GD-
28171, GD-28172
and GD-28173) were used to target Sa-dCas9-MQ1 to the CpG Island in the I32M
promoter region
(second level from the top). Guides were chosen based on hypomethylation of
the promoter region
(depicted in the CpG Methylation track, bottom level), where methylation runs
from 0 to 1 (0 to 100%)
and where higher methylation is depicted by red bars. Each bar represents a
CpG site.
Figure 2 shows a graph of percent change in I3-2-microglobulin expression
(mRNA) in HepG2
cells with treatment with a variety of expression repressors or controls. I32M
mRNA levels shown are
relative to the untreated control (gray). Points show biological replicates.
Bars show the average and error
bars indicate standard deviations. The guide ID number is shown on the x-axis.
Samples treated with
dCas9 are shown in magenta; dCas9-KRAB in green; sa-dCas9-MQ1 in light blue; a
combination of
dCas9-KRAB and sa-dCas9-MQ1 in dark blue. A 132M-knockout positive control
with Cas9 is shown in
orange.
Figure 3 shows a graph of percent change in I3-2-microglobulin mRNA with
treatment with a
variety of expression repressors or controls after 72 hours (left), and a
graph of flow cytometry data of 13-
2-microglobulin protein levels with treatment with a variety of expression
repressors or controls after 72
hours (right). (Left) Treatment with dCas9-KRAB (green) and Sa-dCas9-MQ1
(light blue) effectors,
separately and together (dark blue) results in a decrease in I32M mRNA
relative to untreated cells (grey)
and cells treated with dCas9 (magenta). (Right) Flow cytometry revealed
populations with decreased I32M
immunofluorescence in cells treated with dCas9-KRAB and Sa-dCas9-MQ1 relative
to untreated cells
.. and cells treated with dCas9.
Figure 4 shows a graph of the fold change in I3-2-microglobulin mRNA with
treatment with a
variety of expression repressors or controls over a 22-day period. Over a 22-
day period, non-transfected
(blue line) and dCas9 transfected (red line) cells don't change I32M
expression, whereas dCas9-KRAB
(green line) transfected cells have I32M repressed in the initial days post-
transfection, with maximum
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
49
effect on day 2, but the repression is lost after day 5. SadCas9-MQ1 (purple
line) transfected cells show
significant repression (up to 85% by day 4) and the repression is retained and
then slowly lost as seen by
line remaining just under the dotted line in the figure. dCas9-KRAB + SadCas9-
MQ1 combined
transfection (orange line) did not give any added repression than what is
already observed for SadCas9
alone (purple line).
Figure 5 shows a graph of the fold change in I3-2-microglobulin mRNA with
treatment with a
variety of expression repressors or controls over an 18-day period. Over an 18-
day period untreated cells
do not change I32M expression, whereas cells transfected with dCas9-MQ1, dCas9-
DNMT3a/3L (m),
dCas9-DNMT3a/3L (hs), dCas9-DNMT1 and dCas9-DNMT3B, show significant I32M
repression post-
.. transfection.
Figure 6 shows a graph of the fold change in MYC expression in K-562 cells
when treated with
different expression repressors or expression repression systems that target
the CTCF site located
upstream of MYC gene. The data showed that, at least, cells treated with dCas9-
DNMT3a/3L (h), dCas9-
KRAB, FOG1-dCas9-FOG1, G9A-dCas9 + EZH2-dCas9, dCas9-MQ1 + EZH2-dCas9-KRAB,
dCas9-
LSD1+G9A-dCas9, dCas9-MQ1+dCas9-HDAC8, and dCas9-MQ1+G9A-dCas9-KRAB showed
repression of MYC expression at 48 hour and/or 72-hour time point.
Figure 7 shows a graph of the fold change in I32M expression in K-562 cells
when treated with
different expression repressors or expression repression systems that target a
region upstream of the I32M
promoter. The data showed that, at least, cells treated with dCas9-HDAC8,
dCas9-LSD1, dCas9-MQ1,
FOG1-dCas9- FOG1, G9A-dCas9-KRAB, dCas9-DNMT3a/3L (h), G9A-dCas9, EZH2-dCas9-
KRAB,
dCas9-KRAB, EZH2-dCas9-KRAB + dCas9-HDAC8, dCas9-LSD1 + EZH2-dCas9-KRAB, dCas9-
LSD1 + dCas9-HDAC8, dCas9-KRAB + FOG1-dCas9- FOG1, dCas9-MQ1 + G9A-dCas9-KRAB,
G9A-
dCas9 + EZH2-dCas9, dCas9-MQ1 + EZH2-dCas9-KRAB, and dCas9-MQ1 + dCas9-HDAC8
showed
repression of I32M expression at 48 hour, 72 hour and/or 144 hour time point.
Figure 8 shows a graph of the fold change in HSPA1B expression in K-562 cells
when treated
with different expression repressors or expression repression systems that
target a region downstream of
the HSPA1B promoter. The data showed that, at least, cells treated with dCas9-
HDAC8, EZH2-dCas9,
dCas9-MQ1, dCas9-DNMT1, dCas9-DNMT3a/3L (h), dCas9-DNMT3a/3L (m), G9A-dCas9-
KRAB,
FOG1-dCas9-FOG1, G9A-dCas9, dCas9-KRAB, G9A-dCas9 + dCas9-HDAC8, dCas9-LSD1 +
G9A-
dCas9-KRAB, dCas9-LSD1+EZH2-dCas9, G9A-dCas9+EZH2-dCas9, dCas9-LSD1 + EZH2-
dCas9-
KRAB, EZH2-dCas9 +dCas9-HDAC8, dCas9-LSD1+dCas9-HDAC8, dCas9-MQ1 + EZH2-dCas9-
KRAB, dCas9-MQ1+dCas9-HDAC8, G9A-dCas9-KRAB+EZH2-dCas9-KRAB, dCas9-LSD1+G9A-
dCas9 and dCas9-MQ1+G9A-dCas9-KRAB showed repression of HSPA1B expression at
48 hour, 72
hour and/or 144 hour time point.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
Figure 9 shows a graph of the fold change in GATA1 expression in K-562 cells
when treated
with different expression repressors or expression repression systems. The
data showed that, at least, cells
treated with dCas9-HDAC8, dCas9-LSD1+dCas9-HDAC8, dCas9-MQ1+dCas9-HDAC8, and
G9A-
dCas9+dCas9-HDAC8 showed repression of GATA1 expression at 48-hour, 72 hour
and/or 144 hour
5 time point.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
10 The
present disclosure provides technologies for modulating, e.g., decreasing,
expression of a
target gene in cell, e.g., in a subject or patient, through the use of an
expression repressor or an expression
repression system. In some embodiments, an expression repressor comprises a
DNA binding moiety and
at least one repressor domain. In some embodiments, an expression repressor
comprises two repressor
domains. In some embodiments an expression repressor comprises two identical
repressor domains. In
15 some embodiments an expression repressor comprises two non-identical
repressor domains.
In some embodiments, an expression repression system comprises two or more
expression repressors,
each comprising a DNA-targeting moiety and at least one repressor domain. In
some embodiments, the
DNA-targeting moieties target two or more different DNA sequences (e.g., each
expression repressor may
target a different DNA sequence). Without wishing to be bound by theory, the
use of an expression
20 repression system comprising expression repressors targeting two or more
different DNA sequences may
be advantageous over similar systems targeting a single DNA sequence because
individual expression
repressors will not compete with one another or compete less with one another
for binding to their target
DNA sequences, thereby achieving superior localization of the various
functionalities of the repressor
domains. In some embodiments, it can be advantageous to use two different
DNA¨targeting moieties to
25 precisely target two different repressors to two different defined
locations, e.g., a first expression
repressor to a first location upstream of the transcription start site and a
second expression repressor to a
second location comprising the transcription start site. In some embodiments,
the expression repressors
comprise two or more different repressor domains (e.g., each expression
repressor comprises a different
repressor domain from each other expression repressor). Without wishing to be
bound by theory, the use
30 of an expression repression system comprising expression repressors
comprising two or more repressor
domains may be advantageous over similar systems comprising a single repressor
domain due to
synergistic effects on expression of a target gene associated with the
concerted action of the two or more
repressor domains.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
51
Expression Repressors
Expression repressors of the present disclosure may comprise a DNA-targeting
moiety and at
least one repressor domain. In some embodiments, an expression repressor may
comprise 1, 2, 3, 4, 5, 6,
or more repressor domains. In some embodiments, an expression repressor
targets two or more different
DNA sequences (e.g., a Pt and 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, V,--sth,
10th, 11th, 12th, and/or further DNA
sequence, and optionally no more than a 20th, 19th, 18th, 17th, 16th, 15th,
14th, 13th, 12th, llth, 10th, 9th, 8th,
6th, 5th, 4th, 3rd, or 2nd DNA sequence). In some embodiments, an expression
repressor comprises a DNA-
targeting moiety and a plurality of repressor domains (e.g., 1, 2, 3, 4, 5, 6,
7, 8,9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, or more repressor domains (and optionally, less than 20,
19, 18, 17, 16, 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 repressor domains)) each of which may be the
same or different from
another of the more than one repressor domains.
In some embodiments, an expression repressor comprises a first repressor
domain and a second
repressor domain, wherein the first repressor domain is not identical to the
second repressor domain. In
some embodiments, an expression repressor comprises a first repressor domain
and a second repressor
domain, wherein the first repressor domain is identical to the second
repressor domain. In some
embodiments, the DNA-targeting moiety is situated between the first repressor
domain and the second
repressor domain.
An expression repressor may comprise a plurality of repressor domains, where
each repressor
domain comprises a different functionality than the other repressor domains.
For example, an expression
repressor may comprise two repressor domains, where the first repressor domain
comprises DNA
methylase functionality and the second repressor domain comprises a
transcriptional repressor
functionality. In some embodiments, an expression repressor comprises
repressor domains whose
functionalities are complementary to one another with regard to decreasing
expression of a target gene,
where the functionalities together enable inhibition of expression and,
optionally, do not inhibit or
negligibly inhibit expression when present individually. In some embodiments,
an expression repressor
comprises a plurality of repressor domains, wherein each repressor domain
complements other repressor
domains, each repressor domain decreases expression of a target gene.
In some embodiments, an expression repressor comprises a combination of
repressor domains
whose functionalities synergize with one another with regard to decreasing
expression of a target gene.
Without wishing to be bound by theory, in some embodiments, epigenetic
modifications to a genomic
locus are cumulative, in that multiple transcription activating epigenetic
markers (e.g., multiple different
types of epigenetic markers and/or more extensive marking of a given type)
individually together inhibit
expression more effectively than individual modifications alone (e.g.,
producing a greater decrease in
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
52
expression and/or a longer-lasting decrease in expression). In some
embodiments, an expression repressor
comprises a plurality of repressor domains, wherein each repressor domain
synergizes with other
repressor domains, e.g., each repressor domain decreases expression of a
target gene. In some
embodiments, an expression repressor (comprising a plurality of repressor
domains which synergize with
one another) is more effective at inhibiting expression of a target gene than
an expression repressor
comprising an individual repressor domain. In some embodiments, an expression
repressor comprising
said plurality of repressor domains is at least 1.05x (i.e., 1.05 times),
1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x,
1.4x, 1.45x, 1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x,
2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x,
10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x as effective at
decreasing expression of a target
gene than an expression repressor comprising an individual repressor domain.
In some embodiments, an expression repressor comprises a DNA-targeting moiety
and a
repressor domain that are covalently linked, e.g., by a peptide bond. In some
embodiments, the DNA-
targeting moiety and the repressor domain are situated on the same polypeptide
chain, e.g., connected by
one or more peptide bonds and/or a linker. In some embodiments, the expression
repressor is or comprises
.. a fusion molecule, e.g., comprising the DNA-targeting moiety and the
repressor domain linked by a
peptide bond and/or a linker. In some embodiments, an expression repressor
comprises a targeting moiety
and a plurality of effector moieties, wherein the targeting moiety and the
plurality of effector moieties are
covalently linked, e.g., by peptide bonds (e.g., the targeting moiety and
plurality of effector moieties are
all connected by a series of covalent bonds, although each individual moiety
may not share a covalent
.. bond with every other effector moiety). In some embodiments, the expression
repressor comprises a
DNA-targeting moiety that is linked to the N-terminal of a repressor domain on
the same polypeptide
chain. In some embodiments, the expression repressor comprises a DNA-targeting
moiety that is linked to
the C-terminal of a repressor domain on the same polypeptide chain. In some
embodiments, the
expression repressor comprises a DNA-targeting moiety that is linked to the C-
terminal of a first
repressor domain and is linked to the N-terminal of a second repressor domain
on the same polypeptide
chain. In some embodiments, the expression repressor comprises a DNA-targeting
moiety that is disposed
N-terminal of a repressor domain on the same polypeptide chain. In some
embodiments, the expression
repressor comprises a DNA-targeting moiety that is disposed C-terminal of a
repressor domain on the
same polypeptide chain. In some embodiments, an expression repressor comprises
a DNA-targeting
moiety and a repressor domain that are covalently linked by a non-peptide
bond. In some embodiments, a
DNA-targeting moiety is conjugated to a repressor domain by a non-peptide
bond. In some embodiments,
an expression repressor comprises a DNA-targeting moiety and a plurality of
repressor domains, wherein
the DNA-targeting moiety and the plurality of repressor domains are covalently
linked, e.g., by peptide
bonds (e.g., the DNA-targeting moiety and plurality of repressor domains are
all connected by a series of
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
53
covalent bonds, although each individual domain or moiety may not share a
covalent bond with every
other domain or moiety).
In other embodiments, an expression repressor comprises a DNA-targeting moiety
and a
repressor domain that are not covalently linked, e.g., that are non-covalently
associated with one another.
In some embodiments, an expression repressor comprises a DNA-targeting moiety
that non-covalently
binds to a repressor domain or vice versa. In some embodiments, an expression
repressor comprises a
DNA-targeting moiety and a plurality of repressor domains, wherein the DNA-
targeting moiety and at
least one repressor domain are not covalently linked, e.g., are non-covalently
associated with one another,
and wherein the DNA-targeting moiety and at least one other repressor domain
are covalently linked, e.g.,
by a peptide bond.
In general, an expression repressor as described herein binds (e.g., via a DNA-
targeting moiety) a
genomic sequence element proximal to and/or operably linked to a target gene.
In some embodiments,
binding of the expression repressor to the genomic sequence element modulates
(e.g., decreases)
expression of the target gene. For example, binding of an expression repressor
comprising a repressor
domain that recruits or inhibits recruitment of components of the
transcription machinery to the genomic
sequence element may modulate (e.g., decrease) expression of the target gene.
As a further example,
binding of an expression repressor comprising a repressor domain with an
enzymatic activity (e.g., an
epigenetic modifying moiety) may modulate (e.g., decrease) expression of the
target gene) through the
localized enzymatic activity of the repressor domain. As a further example,
both binding of an expression
repressor to a genomic sequence element and the localized enzymatic activity
of an expression repressor
may contribute to the resulting modulation (e.g., decrease) in expression of
the target gene.
In some embodiments, an expression repressor comprises a repressor domain
wherein the
repressor domain comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e.,
G9A), EHMT1
(i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, KDM1A
(i.e.,
LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B,
N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10,
HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1,
DNMT1,
DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6,
DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof, and the second repressor domain comprises a different
protein chosen from SETDB1,
SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2,
SETD8,
SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B,
KDM5A,
KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,
HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,
SIRT7,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
54
SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3,
DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST,
FOG1, SUZ12, or a functional variant or fragment thereof.
In some embodiments, an expression repressor comprises a first repressor
domain wherein the
repressor domain comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e.,
G9A), EHMT1
(i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1, 5UV420H2, KDM1A
(i.e.,
LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B,
N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10,
HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1,
DNMT1,
DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6,
DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof, and the second repressor domain comprises a different
protein chosen from SETDB1,
SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2,
SETD8,
SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B,
KDM5A,
KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,
HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,
SIRT7,
SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3,
DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST,
FOG1, SUZ12, or a functional variant or fragment thereof and a second
repressor domain wherein the
repressor domain comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e.,
G9A), EHMT1
(i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1, 5UV420H2, KDM1A
(i.e.,
LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B,
N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10,
HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1,
DNMT1,
DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6,
DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof, and the second repressor domain comprises a different
protein chosen from SETDB1,
SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2,
SETD8,
SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B,
KDM5A,
KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,
HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,
SIRT7,
SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3,
DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST,
FOG1, SUZ12, or a functional variant or fragment thereof.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
In some embodiments, an expression repressor comprises a DNA-targeting moiety
and a
repressor domain wherein the C-terminal end of the repressor domain, e.g., a
repressor domain chosen
from EZH1, EZH2, G9A, SUV39H1, FOG1, SETDB1, or SETDB2, and the N-terminal end
of the DNA-
5 targeting moiety are covalently linked. In some embodiments, an
expression repressor comprises a DNA-
targeting moiety and a repressor domain wherein the N-terminal end of the
repressor domain, e.g., a
repressor domain chosen from LSD1, HDAC8, MQ1, DNMT1, DNMT3a/3L, FOG1, or
KRAB, and the
C-terminal end of the DNA-targeting moiety are covalently linked. In some
embodiments, an expression
repressor comprises a DNA-targeting moiety, a first repressor domain and
second repressor domain,
10 wherein, the C-terminal end of the first repressor domain, e.g., a
repressor domain chosen from EZH1,
EZH2, G9A, SUV39H1, FOG1, SETDB1, or SETDB2, and the N-terminal end of the DNA-
targeting
moiety are covalently linked and the C-terminal end of the DNA-targeting
moiety and the N-terminal end
of the second repressor domain, e.g., a repressor domain chosen from LSD1,
HDAC8, MQ1, DNMT1,
DNMT3a/3L, FOG1, or KRAB are covalently linked.
15 In some embodiments, an expression repressor as disclosed herein is
present in a composition,
pharmaceutical composition, or mixture.
While several of the embodiments herein describe systems comprising a
plurality of expression
repressors, it is understood that the present application also provides the
expression repressors
individually, e.g., for use as a single agent, or for use in a combination
with a generic second therapy that
20 need not be specified herein.
In some embodiments, an expression repressor described herein is part of an
expression
repression system, e.g., as described below.
Expression Repression Systems
25 Expression repression systems of the present disclosure may comprise two
or more expression
repressors. In some embodiments, an expression repression system comprises 2,
3, 4, 5, 6, 7, 8, 9, 10, 11,
12, or more expression repressors (and optionally no more than 15, 14, 13, 12,
11, 10, 9, 8, 7, 6, 5, 4, 3, or
2). In some embodiments, an expression repression system targets two or more
different DNA sequences
(e.g., a 1st and 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, V,--sth,
10th, 11th 12th, and/or further DNA sequence, and optionally
30 no more than a 20th, 19th, 18th, 17th, 16th, 15th, 14th, 13th, 12th,
llth, 10th, 9th, 8th, 6th, 5th, 4th, 3rd, or 2nd DNA
sequence). In some embodiments, an expression repression system comprises a
plurality of expression
repressors, wherein each member of the plurality of expression repressors does
not detectably bind, e.g.,
does not bind, to another member of the plurality of expression repressors. In
some embodiments, an
expression repression system comprises a first expression repressor and a
second expression repressor,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
56
wherein the first expression repressor does not detectably bind, e.g., does
not bind, to the second
expression repressor.
In some embodiments, an expression repression system of the present disclosure
comprises two
or more expression repressors, wherein the expression repressors are present
together in a composition,
pharmaceutical composition, or mixture. In some embodiments, an expression
repression system of the
present disclosure comprises two or more expression repressors, wherein one or
more expression
repressors is not admixed with at least one other expression repressor. For
example, an expression
repression system may comprise a first expression repressor and a second
expression repressor, wherein
the presence of the first expression repressor in the nucleus of a cell does
not overlap with the presence of
the second expression repressor in the nucleus of the same cell, wherein the
expression repression system
achieves a decrease in expression of a target gene via the non-overlapping
presences of the first and
second expression repressors.
In some embodiments, the expression repressors of an expression repression
system each
comprise a different DNA-targeting moiety (e.g., the first, second, third, or
further expression repressors
each comprise different DNA-targeting moieties from one another). For example,
an expression
repression system may comprise a first expression repressor and a second
expression repressor wherein
the first expression repressor comprises a first DNA-targeting moiety (e.g., a
Cas9 molecule, TAL
effector molecule, or Zn Finger domain), and the second expression repressor
comprises a second DNA-
targeting moiety (e.g., a Cas9 molecule, TAL effector molecule, or Zn Finger
domain) different from the
first DNA-targeting moiety. In some embodiments, different can mean comprising
distinct types of DNA-
targeting moiety, e.g., the first DNA-targeting moiety comprises a Cas9
molecule, and the second DNA-
targeting moiety comprises a TAL effector molecule. In other embodiments,
different can mean
comprising distinct variants of the same type of DNA-targeting moiety, e.g.,
the first DNA-targeting
moiety comprises a first Cas9 molecule (e.g., from a first species) and the
second DNA-targeting moiety
comprises a second Cas9 molecule (e.g., from a second species). In an
embodiment, when an expression
repression system comprises two or more DNA-targeting moieties of the same
type, e.g., two or more
Cas9 molecules, the DNA-targeting moieties specifically bind two or more
different DNA sequences. For
example, in an expression repression system comprising two or more Cas9
molecules, the two or more
Cas9 molecules may be chosen or altered such that they only appreciably bind
the gRNA corresponding
to their target DNA sequence (e.g., and do not appreciably bind the gRNA
corresponding to the target of
another Cas9 molecule). In a further example, in an expression repression
system comprising two or more
TAL effector molecules, the two or more TAL effector molecules may be chosen
or altered such that they
only appreciably bind to their target DNA sequence (e.g., and do not
appreciably bind the target DNA
sequence of another TAL effector molecule).
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
57
In some embodiments, an expression repression system comprises three or more
expression
repressors and two or more expression repressors comprise the same DNA-
targeting moiety. For example,
an expression repression system may comprise three expression repressors,
wherein the first and second
expression repressors both comprise a first DNA-targeting moiety and the third
expression repressor
.. comprises a second different DNA-targeting moiety. For a further example,
an expression repression
system may comprise four expression repressors, wherein the first and second
expression repressors both
comprise a first DNA-targeting moiety and the third and fourth expression
repressors comprises a second
different DNA-targeting moiety. For a further example, an expression
repression system may comprise
five expression repressors, wherein the first and second expression repressors
both comprise a first DNA-
targeting moiety, the third and fourth expression repressors both comprise a
second different DNA-
targeting moiety, and the fifth expression repressor comprises a third
different DNA-targeting moiety. As
described above, different can mean comprising different types of DNA-
targeting moieties or comprising
distinct variants of the same type of DNA-targeting moiety.
In some embodiments, the expression repressors of an expression repression
system each bind to
a different DNA sequence (e.g., the first, second, third, or further
expression repressors each bind DNA
sequences that are different from one another). For example, an expression
repression system may
comprise a first expression repressor and a second expression repressor
wherein the first expression
repressor binds to a first DNA sequence, and the second expression repressor
binds to a second DNA
sequence. In some embodiments, different can mean that: there is at least one
position that is not identical
between the DNA sequence bound by one expression repressor and the DNA
sequence bound by another
expression repressor, or that there is at least one position present in the
DNA sequence bound by one
expression repressor that is not present in the DNA sequence bound by another
expression repressor. For
example, a first expression repressor may bind to a first exemplary DNA
sequence 5'-
ATGATTGGATTTA-3' (SEQ ID NO: 97), and a second expression repressor may bind
to a second
exemplary DNA sequence 5'- TGATTGGATTTAG-3' (SEQ ID NO: 98); in said example,
the first and
second exemplary DNA sequences are different. For a further example, a first
expression repressor may
bind to a first exemplary DNA sequence 5'-ATGATTgGATTTA-3' (SEQ ID NO: 99),
and a second
expression repressor may bind to a second exemplary DNA sequence 5'-
ATGATTcGATTTA-3' (SEQ
ID NO: 100); in said example, the first and second exemplary DNA sequences are
different. In some
embodiments, the first DNA sequence may be situated on a first genomic DNA
strand and the second
DNA sequence may be situated on a second genomic DNA strand. In some
embodiments, the first DNA
sequence may be situated on the same genomic DNA strand as the second DNA
sequence.
In some embodiments, an expression repression system comprises three or more
expression
repressors and two or more expression repressors bind the same DNA sequence.
For example, an
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
58
expression repression system may comprise three expression repressors, wherein
the first and second
expression repressors both bind a first DNA sequence, and the third expression
repressor binds a second
different DNA sequence. For a further example, an expression repression system
may comprise four
expression repressors, wherein the first and second expression repressors both
bind a first DNA sequence
and the third and fourth expression repressors both bind a second DNA
sequence. For a further example,
an expression repression system may comprise five expression repressors,
wherein the first and second
expression repressors both bind a first DNA sequence, the third and fourth
expression repressors both
bind a second DNA sequence, and the fifth expression repressor binds a third
DNA sequence. As
described above, different can mean that there is at least one position that
is not identical between the
DNA sequence bound by one expression repressor and the DNA sequence bound by
another expression
repressor, or that there is at least one position present in the DNA sequence
bound by one expression
repressor that is not present in the DNA sequence bound by another expression
repressor.
In some embodiments, an expression repression system comprises two or more
(e.g., two)
expression repressors and a plurality (e.g., two) of the expression repressors
comprise DNA-targeting
moieties that bind to different DNA sequences. In such embodiments, a first
DNA-targeting moiety may
bind to a first DNA sequence and a second DNA-targeting moiety may bind to a
second DNA sequence,
wherein the first and the second DNA sequences are different and do not
overlap. In some such
embodiments, the first DNA sequence is separated from the second DNA sequence
by at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90,
95, or 100 base pairs (and optionally, no more than 500, 400, 300, 200, 100,
95, 90, 85, 80, 75, 70, 65, 60,
55, or 50 base pairs). In some such embodiments, the first DNA sequence is
separated from the second
DNA sequence by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs (and
optionally, no base pairs, e.g., the
first and second sequence are directly adjacent one another).
In some embodiments, the expression repressors of an expression repression
system each
comprise a different repressor domain (e.g., the first, second, third, or
further expression repressors each
comprise a different repressor domain from one another). For example, an
expression repression system
may comprise a first expression repressor and a second expression repressor
wherein the first expression
repressor comprises a first repressor domain (e.g., comprising a histone
methyltransferase or functional
fragment thereof), and the second expression repressor comprises a second
repressor domain (e.g.,
comprising a DNA methyltransferase or functional fragment thereof) different
from the first repressor
domain. In some embodiments, different can mean comprising distinct types of
repressor domain, e.g., the
first repressor domain comprises a histone methyltransferase and the second
repressor domain comprises
a DNA methyltransferase, or the first repressor domain comprises a histone
methyltransferase and the
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
59
second repressor domain comprises a small molecule inhibitor of an enzyme. In
other embodiments,
different can mean comprising distinct variants of the same type of repressor
domain, e.g., the first
repressor domain comprises a first histone methyltransferase (e.g., having a
first site specificity or amino
acid sequence) and the second repressor domain comprises a second histone
methyltransferase (e.g.,
.. having a second site specificity or amino acid sequence).
In some embodiments, an expression repression system comprises a first
expression repressor
comprising a first repressor domain and a second expression repressor
comprising a second repressor
domain, wherein the first repressor domain comprises a protein chosen from
SETDB1, SETDB2, EHMT2
(i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1,
SUV420H2,
.. KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a
.. functional variant or fragment thereof, and the second repressor domain
comprises a different protein
chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1,
EZH2, EZH1,
5UV39H2, SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2),
KDM2A,
KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4,
HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4,
.. SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1,
DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2,
HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment thereof.
In some embodiments, an expression repression system comprises a first
expression repressor
comprising a first repressor domain and a second expression repressor
comprising a second repressor
.. domain, wherein the first repressor domain comprises a protein chosen from
SETDB1, SETDB2, EHMT2
(i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1,
5UV420H2,
KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C,
KDM5D,
KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9,
HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1,
.. DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a
functional variant or fragment thereof, the second repressor domain comprises
a different protein chosen
from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2,
EZH1, 5UV39H2,
SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A,
KDM2B,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,
HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5,
SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2,
DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1,
5 RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment thereof,
and the third repressor domain
comprises a different protein chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A),
EHMT1 (i.e., GLP),
SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1),
KDM1B
(i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1,
HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1,
10 SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1,
DNMT3A1, DNMT3A2,
DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L,
KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or
fragment thereof.
In some embodiments, an expression repression system comprises: (i) a first
expression repressor
comprising a first repressor domain and a third repressor domain, and (ii) a
second expression repressor
15 comprising a second repressor domain and optionally a fourth repressor
domain, wherein the first
repressor domain comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e.,
G9A), EHMT1
(i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1, 5UV420H2, KDM1A
(i.e.,
LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B,
N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10,
20 HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9,
MQ1, DNMT1,
DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6,
DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a functional
variant or
fragment thereof, the second repressor domain comprises a different protein
chosen from SETDB1,
SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1, 5UV39H2,
SETD8,
25 SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B (i.e., LSD2), KDM2A,
KDM2B, KDM5A,
KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,
HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6,
SIRT7,
SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3,
DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST,
30 FOG1, SUZ12, or a functional variant or fragment thereof, the third
repressor domain comprises a
different protein chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e.,
GLP), SUV39H1,
EZH2, EZH1, 5UV39H2, SETD8, SUV420H1, 5UV420H2, KDM1A (i.e., LSD1), KDM1B
(i.e., LSD2),
KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, HDAC1, HDAC2, HDAC3,
HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
61
SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1, DNMT1, DNMT3A1, DNMT3A2,
DNMT3B1,
DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, KRAB, MeCP2,
HP1, RBBP4, REST, FOG1, SUZ12, or a functional variant or fragment thereof and
the fourth repressor
domain comprises a different protein chosen from SETDB1, SETDB2, EHMT2 (i.e.,
G9A), EHMT1 (i.e.,
GLP), SUV39H1, EZH2, EZH1, 5UV39H2, SETD8, SUV420H1, 5UV420H2, KDM1A (i.e.,
LSD1),
KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066,
HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10,
HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, MQ1,
DNMT1,
DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6,
.. DNMT3L, DNMT3a/3L, KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or a
functional variant or
fragment thereof.
In some embodiments, the first repressor domain comprises a histone
methyltransferase activity
(e.g., SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2,
EZH1, 5UV39H2,
SETD8, SUV420H1, 5UV420H2, or a functional variant or fragment of any thereof,
e.g., a SET domain
of any thereof) and the second repressor domain comprises a histone
demethylase activity (e.g., KDM1A
(i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D,
KDM4B,
N066, or a functional variant or fragment of any thereof). In some
embodiments, the first repressor
domain comprises a histone methyltransferase activity and the second repressor
domain comprises a
histone deacetylase activity (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,
HDAC7,
HDAC8, HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7,
SIRT8,
SIRT9, or a functional variant or fragment of any thereof). In some
embodiments, the first repressor
domain comprises a histone methyltransferase activity and the second repressor
domain comprises a DNA
methyltransferase activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1,
DNMT3B2,
DNMT3B3, DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, or a functional variant
or
fragment of any thereof). In some embodiments, the first repressor domain
comprises a histone
methyltransferase activity and the second repressor domain comprises a DNA
demethylase activity. In
some embodiments the first repressor domain comprises a histone
methyltransferase activity and the
second repressor domain comprises a transcription repressor activity (e.g.,
KRAB, MeCP2, HP1, RBBP4,
.. REST, FOG1, SUZ12, or a functional variant or fragment of any thereof). In
some embodiments, the first
repressor domain comprises a histone methyltransferase activity and the second
repressor domain
comprises a different histone methyltransferase activity. In some embodiments,
the first repressor domain
comprises a histone methyltransferase activity and the second repressor domain
comprises the same
histone methyltransferase activity. In some embodiments, the first repressor
domain comprises a histone
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
62
demethylase activity and the second repressor domain comprises a histone
deacetylase activity. In some
embodiments, the first repressor domain comprises a histone demethylase
activity and the second
repressor domain comprises a DNA methyltransferase activity. In some
embodiments, the first repressor
domain comprises a histone demethylase activity and the second repressor
domain comprises a DNA
demethylase activity. In some embodiments, the first repressor domain
comprises a histone demethylase
activity and the second repressor domain comprises a transcription repressor
activity. In some
embodiments, the first repressor domain comprises a histone demethylase
activity and the second
repressor domain comprises a different histone demethylase activity. In some
embodiments, the first
repressor domain comprises a histone demethylase activity and the second
repressor domain comprises
the same histone demethylase activity. In some embodiments, the first
repressor domain comprises a
histone deacetylase activity and the second repressor domain comprises a DNA
methyltransferase
activity. In some embodiments, the first repressor domain comprises a histone
deacetylase activity and the
second repressor domain comprises a DNA demethylase activity. In some
embodiments, the first
repressor domain comprises a histone deacetylase activity and the second
repressor domain comprises a
transcription repressor activity. In some embodiments, the first repressor
domain comprises a histone
deacetylase activity and the second repressor domain comprises a different
histone deacetylase activity. In
some embodiments, the first repressor domain comprises a histone deacetylase
activity and the second
repressor domain comprises the same histone deacetylase activity. In some
embodiments, the first
repressor domain comprises a DNA methyltransferase activity and the second
repressor domain
comprises a DNA demethylase activity. In some embodiments, the first repressor
domain comprises a
DNA methyltransferase activity and the second repressor domain comprises a
transcription repressor
activity. In some embodiments, the first repressor domain comprises a DNA
methyltransferase activity
and the second repressor domain comprises a different DNA methyltransferase
activity. In some
embodiments, the first repressor domain comprises a DNA methyltransferase
activity and the second
repressor domain comprises the same DNA methyltransferase activity. In some
embodiments, the first
repressor domain comprises a DNA demethylase activity and the second repressor
domain comprises a
transcription repressor activity. In some embodiments, the first repressor
domain comprises a DNA
demethylase activity and the second repressor domain comprises a different DNA
demethylase activity. In
some embodiments, the first repressor domain comprises a DNA demethylase
activity and the second
.. repressor domain comprises the same DNA demethylase activity. In some
embodiments, the first
repressor domain comprises a transcription repressor activity and the second
repressor domain comprises
a different transcription repressor activity. In some embodiments, the first
repressor domain comprises a
transcription repressor activity and the second repressor domain comprises the
same transcription
repressor activity.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
63
In some embodiments, an expression repression system comprises three or more
expression
repressors and two or more expression repressors comprise the same DNA-
targeting moiety. For example,
an expression repression system may comprise three expression repressors,
wherein the first and second
expression repressors both comprise a first repressor domain and the third
expression repressor comprises
a second different repressor domain. For a further example, an expression
repression system may
comprise four expression repressors, wherein the first and second expression
repressors both comprise a
first repressor domain and the third and fourth expression repressors
comprises a second different
repressor domain. For a further example, an expression repression system may
comprise five expression
repressors, wherein the first and second expression repressors both comprise a
first repressor domain, the
third and fourth expression repressors both comprise a second different
repressor domain, and the fifth
expression repressor comprises a third different repressor domain. As
described above, different can mean
comprising different types of repressor domain or comprising distinct variants
of the same type of
repressor domain.
In some embodiments, two repressor domains comprise a histone
methyltransferase activity (e.g.,
SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1 (i.e., GLP), SUV39H1, EZH2, EZH1,
SUV39H2,
SETD8, SUV420H1, SUV420H2, or a functional variant or fragment of any thereof,
e.g., a SET domain
of any thereof) and the other repressor domain comprises a histone demethylase
activity (e.g., KDM1A
(i.e., LSD1), KDM1B (i.e., LSD2), KDM2A, KDM2B, KDM5A, KDM5B, KDM5C, KDM5D,
KDM4B,
N066, or a functional variant or fragment of any thereof). In some
embodiments, two repressor domains
comprise a histone methyltransferase activity and the other repressor domain
comprises a histone
deacetylase activity (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7,
HDAC8,
HDAC9, HDAC10, HDAC11, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8,
SIRT9, or a
functional variant or fragment of any thereof). In some embodiments, two
repressor domains comprise a
histone methyltransferase activity and the other repressor domain comprises a
DNA methyltransferase
activity (e.g., MQ1, DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3,
DNMT3B4, DNMT3B5, DNMT3B6, DNMT3L, DNMT3a/3L, or a functional variant or
fragment of any
thereof). In some embodiments, two repressor domains comprise a histone
methyltransferase activity and
the other repressor domain comprises a DNA demethylase activity. In some
embodiments, two repressor
domains comprise a histone methyltransferase activity and the other repressor
domain comprises a
transcription repressor activity (e.g., KRAB, MeCP2, HP1, RBBP4, REST, FOG1,
SUZ12, or a
functional variant or fragment of any thereof). In some embodiments, two
repressor domains comprise a
histone methyltransferase activity and the other repressor domain comprises a
different histone
methyltransferase activity. In some embodiments, two repressor domains
comprise a histone
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
64
methyltransferase activity and the other repressor domain comprises the same
histone methyltransferase
activity. In some embodiments, two repressor domains comprise a histone
demethylase activity and the
other repressor domain comprises a histone deacetylase activity. In some
embodiments, two repressor
domains comprise a histone demethylase activity and the other repressor domain
comprises a DNA
methyltransferase activity. In some embodiments, two repressor domains
comprise a histone demethylase
activity and the other repressor domain comprises a DNA demethylase activity.
In some embodiments,
two repressor domains comprise a histone demethylase activity and the other
repressor domain comprises
a transcription repressor activity. In some embodiments, two repressor domains
comprise a histone
demethylase activity and the other repressor domain comprises a different
histone demethylase activity. In
some embodiments, two repressor domains comprise a histone demethylase
activity and the other
repressor domain comprises the same histone demethylase activity. In some
embodiments, two repressor
domains comprise a histone deacetylase activity and the other repressor domain
comprises a DNA
methyltransferase activity. In some embodiments, two repressor domains
comprise a histone deacetylase
activity and the other repressor domain comprises a DNA demethylase activity.
In some embodiments,
two repressor domains comprise a histone deacetylase activity and the other
repressor domain comprises a
transcription repressor activity. In some embodiments, two repressor domains
comprise a histone
deacetylase activity and the other repressor domain comprises a different
histone deacetylase activity. In
some embodiments, two repressor domains comprise a histone deacetylase
activity and the other repressor
domain comprises the same histone deacetylase activity. In some embodiments,
two repressor domains
comprise a DNA methyltransferase activity and the other repressor domain
comprises a DNA
demethylase activity. In some embodiments, two repressor domains comprise a
DNA methyltransferase
activity and the other repressor domain comprises a transcription repressor
activity. In some
embodiments, two repressor domains comprise a DNA methyltransferase activity
and the other repressor
domain comprises a different DNA methyltransferase activity. In some
embodiments, two repressor
domains comprise a DNA methyltransferase activity and the other repressor
domain comprises the same
DNA methyltransferase activity. In some embodiments, two repressor domains
comprise a DNA
demethylase activity and the other repressor domain comprises a transcription
repressor activity. In some
embodiments, two repressor domains comprise a DNA demethylase activity and the
other repressor
domain comprises a different DNA demethylase activity. In some embodiments,
two repressor domains
comprise a DNA demethylase activity and the other repressor domain comprises
the same DNA
demethylase activity. In some embodiments, two repressor domains comprise a
transcription repressor
activity and the other repressor domain comprises a different transcription
repressor activity. In some
embodiments, two repressor domains comprise a transcription repressor activity
and the other repressor
domain comprises the same transcription repressor activity.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
In some embodiments, two or more (e.g., all) expression repressors of an
expression repression
system are not covalently associated with each other, e.g., each expression
repressor is not covalently
associated with any other expression repressor. In another embodiment, two or
more expression
repressors of an expression repression system are covalently associated with
one another. In an
5 embodiment, an expression repression system comprises a first expression
repressor and a second
expression repressor disposed on the same polypeptide, e.g., as a fusion
molecule, e.g., connected by a
peptide bond and optionally a linker. In an embodiment, an expression
repression system comprises a first
expression repressor and a second expression repressor that are connected by a
non-peptide bond, e.g., are
conjugated to one another.
Linkers
An expression repressor or an expression repression system as disclosed herein
may comprise one
or more linkers. A linker may connect a DNA-targeting moiety to a repressor
domain, a repressor domain
to another repressor domain, or a DNA-targeting moiety to another DNA-
targeting moiety. A linker may
be a chemical bond, e.g., one or more covalent bonds or non-covalent bonds. In
some embodiments, a
linker is covalent. In some embodiments, a linker is non-covalent. In some
embodiments, a linker is a
peptide linker. Such a linker may be between 2-30, 5-30, 10-30, 15-30, 20-30,
25-30, 2-25, 5-25, 10-25,
15-25, 20-25, 2-20, 5-20, 10-20, 15-20, 2-15, 5-15, 10-15, 2-10, 5-10, or 2-5
amino acids in length, or
greater than or equal to 2, 5, 10, 15, 20, 25, or 30 amino acids in length
(and optionally up to 50, 40, 30,
25, 20, 15, 10, or 5 amino acids in length). In some embodiments, a linker can
be used to space a first
domain or moiety from a second domain or moiety, e.g., a DNA-targeting moiety
from a repressor
domain. In some embodiments, for example, a linker can be positioned between a
DNA-targeting moiety
and a repressor domain, e.g., to provide molecular flexibility of secondary
and tertiary structures. A
linker may comprise flexible, rigid, and/or cleavable linkers described
herein. In some embodiments, a
linker includes at least one glycine, alanine, and serine amino acids to
provide for flexibility. In some
embodiments, a linker is a hydrophobic linker, such as including a negatively
charged sulfonate group,
polyethylene glycol (PEG) group, or pyrophosphate diester group. In some
embodiments, a linker is
cleavable to selectively release a moiety (e.g., polypeptide) from a
modulating agent, but sufficiently
stable to prevent premature cleavage.
In some embodiments, one or more moieties and/or domains of an expression
repressor described
herein are linked with one or more linkers. In some embodiments, an expression
repression may
comprise a linker situated between the DNA-targeting moiety and the repressor
domain. In some
embodiments, an expression repressor may comprise a first linker situated
between the DNA-targeting
moiety and the first repressor domain, and a second linker situated between
the DNA-targeting moiety
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
66
and the second repressor domain. In some embodiments, the first and the second
linker may be identical.
In some embodiments, the first and the second linker may be different.
As will be known by one of skill in the art, commonly used flexible linkers
have sequences
consisting primarily of stretches of Gly and Ser residues ("GS" linker).
Flexible linkers may be useful for
joining domains/moieties that require a certain degree of movement or
interaction and may include small,
non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Incorporation
of Ser or Thr can also maintain
the stability of a linker in aqueous solutions by forming hydrogen bonds with
water molecules, and
therefore reduce unfavorable interactions between a linker and
moieties/domains.
Rigid linkers are useful to keep a fixed distance between domains/moieties and
to maintain their
independent functions. Rigid linkers may also be useful when a spatial
separation of domains is critical to
preserve the stability or bioactivity of one or more components in the fusion.
Rigid linkers may have an
alpha helix-structure or Pro-rich sequence, (XP)11, with X designating any
amino acid, preferably Ala,
Lys, or Glu.
Cleavable linkers may release free functional domains in vivo. In some
embodiments, linkers
may be cleaved under specific conditions, such as presence of reducing
reagents or proteases. In vivo
cleavable linkers may utilize reversible nature of a disulfide bond. One
example includes a thrombin-
sensitive sequence (e.g., PRS) between the two Cys residues. In vitro thrombin
treatment of CPRSC
results in the cleavage of a thrombin-sensitive sequence, while a reversible
disulfide linkage remains
intact. Such linkers are known and described, e.g., in Chen et al. 2013.
Fusion Protein Linkers: Property,
Design and Functionality. Adv Drug Deliv. Rev. 65(10): 1357-1369. /n vivo
cleavage of linkers in
fusions may also be carried out by proteases that are expressed in vivo under
certain conditions, in
specific cells or tissues, or constrained within certain cellular
compartments. Specificity of many
proteases offers slower cleavage of the linker in constrained compartments.
Examples of molecules suitable for use in linkers described herein include a
negatively charged
sulfonate group; lipids, such as a poly (--CH2--) hydrocarbon chains, such as
polyethylene glycol (PEG)
group, unsaturated variants thereof, hydroxylated variants thereof, amidated
or otherwise N-containing
variants thereof; noncarbon linkers; carbohydrate linkers; phosphodiester
linkers, or other molecule
capable of covalently linking two or more components of an expression
repressor. Non-covalent linkers
are also included, such as hydrophobic lipid globules to which the polypeptide
is linked, for example
through a hydrophobic region of a polypeptide or a hydrophobic extension of a
polypeptide, such as a
series of residues rich in leucine, isoleucine, valine, or perhaps also
alanine, phenylalanine, or even
tyrosine, methionine, glycine, or other hydrophobic residue. Components of an
expression repressor may
be linked using charge-based chemistry, such that a positively charged
component of an expression
repressor is linked to a negative charge of another component.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
67
Nucleic acids
In one aspect, the disclosure provides nucleic acid sequences encoding an
expression repressor,
an expression repression system, a DNA-targeting moiety and/or a repressor
domain as described herein.
A skilled artisan is aware that the nucleic acid sequences of RNA are
identical to the corresponding DNA
sequences, except that typically thymine (T) is replaced by uracil (U). It
will be understood that when a
nucleotide sequence is represented by a DNA sequence (e.g., comprising, A, T,
G, C), this disclosure also
provides the corresponding RNA sequence (e.g., comprising, A, U, G, C) in
which "U" replaces "T."
Conventional notation is used herein to describe polynucleotide sequences: the
left-hand end of a single-
stranded polynucleotide sequence is the 5'-end; the left-hand direction of a
double-stranded
polynucleotide sequence is referred to as the 5'-direction.
It will be appreciated by those skilled in the art that due to the degeneracy
of the genetic code, a
multitude of nucleotide sequences encoding an expression repressor comprising
DNA-targeting moiety
and/or a repressor domain as described herein may be produced, some of which
have similarity, e.g.,
90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleic acid sequences
disclosed herein. For instance,
codons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.
Thus, at every
position in the nucleic acid molecules of the disclosure where an arginine is
specified by a codon, the
codon can be altered to any of the corresponding codons described above
without altering the encoded
polypeptide.
In some embodiments a nucleic acid sequence encoding an expression repressor
comprising a
DNA-targeting moiety and/or one or more repressor domains may be part or all
of a codon-optimized
coding region, optimized according to codon usage in mammals, e.g., humans. In
some embodiments, a
nucleic acid sequence encoding a DNA-targeting moiety and/or one or more
repressor domains is codon
optimized for increasing the protein expression and/or increasing the duration
of protein expression. In
some embodiments, a protein produced by the codon optimized nucleic acid
sequence is at least 1%, at
least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%,
at least 40%, or at least 50%
higher compared to levels of the protein when encoded by a nucleic acid
sequence that is not codon
optimized.
In one aspect, the disclosure is directed to a polypeptide comprising one or
more (e.g., one) DNA-
targeting moiety and one or more repressor domain, e.g., wherein the repressor
domain is or comprises
MQ1, e.g., bacterial MQ1, or a functional variant or fragment thereof. In some
embodiments, MQ1 is
Spiroplasma monobiae MQ1, e.g., MQ1 from strain ATCC 33825 and/or
corresponding to Uniprot ID
P15840. In some embodiments, MQ1 repressor domain is encoded by a nucleotide
sequence of SEQ ID
NO: 47. In some embodiments, a nucleotide sequence described herein comprises
a sequence of SEQ ID
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
68
NO: 47 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more than
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1
positions of difference thereto.
AGCAAGGTGGAGAACAAGACCAAGAAGCTGCGGGTGTTCGAGGCCTTCGCCGGCATCGGCG
CCCAGCGGAAGGCCCTGGAGAAGGTGCGGAAGGACGAGTACGAGATCGTGGGCCTGGCCG
AGTGGTACGTGCCCGCCATCGTGATGTACCAGGCCATCCACAACAACTTCCACACCAAGCTG
GAGTACAAGAGCGTGAGCCGGGAGGAGATGATCGACTACCTGGAGAACAAGACCCTGAGCT
GGAACAGCAAGAACCCCGTGAGCAACGGCTACTGGAAGCGGAAGAAGGACGACGAGCTGA
AGATCATCTACAACGCCATCAAGCTGAGCGAGAAGGAGGGCAACATCTTCGACATCCGGGA
CCTGTACAAGCGGACCCTGAAGAACATCGACCTGCTGACCTACAGCTTCCCCTGCCAGGACC
TGAGCCAGCAGGGCATCCAGAAGGGCATGAAGCGGGGCAGCGGCACCCGGAGCGGCCTGCT
GTGGGAGATCGAGCGGGCCCTGGACAGCACCGAGAAGAACGACCTGCCCAAGTACCTGCTG
ATGGAGAACGTGGGCGCCCTGCTGCACAAGAAGAACGAGGAGGAGCTGAACCAGTGGAAG
CAGAAGCTGGAGAGCCTGGGCTACCAGAACAGCATCGAGGTGCTGAACGCCGCCGACTTCG
GCAGCAGCCAGGCCCGGCGGCGGGTGTTCATGATCAGCACCCTGAACGAGTTCGTGGAGCT
GCCCAAGGGCGACAAGAAGCCCAAGAGCATCAAGAAGGTGCTGAACAAGATCGTGAGCGA
GAAGGACATCCTGAACAACCTGCTGAAGTACAACCTGACCGAGTTCAAGAAAACCAAGAGC
AACATCAACAAGGCCAGCCTGATCGGCTACAGCAAGTTCAACAGCGAGGGCTACGTGTACG
ACCCCGAGTTCACCGGCCCCACCCTGACCGCCAGCGGCGCCAACAGCCGGATCAAGATCAA
GGACGGCAGCAACATCCGGAAGATGAACAGCGACGAGACCTTCCTGTACATCGGCTTCGAC
AGCCAGGACGGCAAGCGGGTGAACGAGATCGAGTTCCTGACCGAGAACCAGAAGATCTTCG
TGTGCGGCAACAGCATCAGCGTGGAGGTGCTGGAGGCCATCATCGACAAGATCGGCGGC
(SEQ ID NO: 47)
In some embodiments, MQ1 comprises an amino acid sequence of SEQ ID NO: 90. In
some
embodiments, MQ1 comprises an amino acid sequence of SEQ ID NO: 57. In some
embodiments, an
effector domain described herein comprises SEQ ID NO: 90 or 57, or a sequence
with at least 80, 85, 90,
95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7,
6, 5, 4, 3, 2, or 1 positions of difference thereto.
MSKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLE
YKSVSREEMIDYLENKTLSWNSKNPVSNGYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRT
LKNIDLLTYSFPCQDLS QQGIQKGMKRGSGTRSGLLWEIERALD S TEKNDLPKYLLMENVGALL
HKKNEEELNQWKQKLESLGYQNSIEVLNAADFGS S QARRRVFMISTLNEFVELPKGDKKPKSIK
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
69
KVLNKIVSEKDILNNLLKYNLTEFKKTKSNINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSR
IKIKDGSNIRKMNSDETFLYIGFDSQDGKRVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGG (SEQ
ID NO: 90)
SKVENKTKKLRVFEAFAGIGAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEY
KSVSREEMIDYLENKTLSWNSKNPVSNGYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTL
KNIDLLTYSFPCQDLSQQGIQKGMKRGSGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLH
KKNEEELNQWKQKLESLGYQNSIEVLNAADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKK
VLNKIVSEKDILNNLLKYNLTEFKKTKSNINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRI
KIKDGSNIRKMNSDETFLYIGFDSQDGKRVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGG (SEQ
ID NO: 57)
In some embodiments, MQ1 for use in a polypeptide described herein is a
variant, e.g., comprising one or
more mutations, relative to wildtype MQ1 (e.g., SEQ ID NO: 90 or SEQ ID NO:
57). In some
embodiments, an MQ1 variant comprises one or more amino acid substitutions,
deletions, or insertions
relative to wildtype MQ1. In some embodiments, an MQ1 variant comprises a
K297P substitution. In
some embodiments, an MQ1 variant comprises a N299C substitution. In some
embodiments, an MQ1
variant comprises a E301Y substitution. In some embodiments, an MQ1 variant
comprises a Q147L
substitution (e.g., and has reduced DNA methyltransferase activity relative to
wildtype MQ1). In some
embodiments, an MQ1 variant comprises K297P, N299C, and E301Y substitutions
(e.g., and has reduced
DNA binding affinity relative to wildtype MQ1). In some embodiments, an MQ1
variant comprises
Q147L, K297P, N299C, and E301Y substitutions (e.g., and has reduced DNA
methyltransferase activity
and DNA binding affinity relative to wildtype MQ1). In some embodiments, the
polypeptide comprises
one or more linkers described herein, e.g., connecting a moiety/domain to
another moiety/domain. In
some embodiments, the polypeptide comprises a DNA-targeting moiety that is or
comprises a
CRISPR/Cas molecule, e.g., comprising a CRISPR/Cas protein, e.g., a dCas9
protein. In some
embodiments, the polypeptide is a fusion protein comprising a repressor domain
that is or comprises
MQ1 and a DNA-targeting moiety that is or comprises a CRISPR/Cas molecule,
e.g., comprising a
CRISPR/Cas protein, e.g., a dCas9 protein. In some embodiments, the
polypeptide comprises an
additional moiety described herein. In some embodiments, the polypeptide
decreases expression of a
target gene (e.g., a target gene described herein). In some embodiments, the
polypeptide may be used in
methods of modulating, e.g., decreasing, gene expression, methods of treating
a condition, or methods of
epigenetically modifying a target gene or transcription control element
described herein, e.g., in place of
an expression repression system. In some embodiments, an expression repression
system comprises two
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
or more (e.g., two, three, or four) expression repressors, wherein the first
expression repressor comprises
a repressor domain comprising MQ1, e.g., bacterial MQ1, or a functional
variant or fragment thereof.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
5 .. comprising one or more (e.g., one) targeting moiety and one or more
effector moiety, wherein the effector
moiety is or comprises Krueppel-associated box (KRAB) e.g., as according to
NP_056209.2 or the
protein encoded by NM_015394.5 or a functional variant or fragment thereof. In
some embodiments,
KRAB is a synthetic KRAB construct. In some embodiments, KRAB comprises an
amino acid sequence
of SEQ ID NO: 61:
DAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVIL
RLEKGEEPWLVEREIHQETHPDSETAFEIKSSV (SEQ ID NO: 61)
In some embodiments, the KRAB repressor domain is encoded by a nucleotide
sequence of SEQ
ID NO: 51. In some embodiments, a nucleotide sequence described herein
comprises a sequence of SEQ
ID NO: 51 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more than
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1
positions of difference thereto.
GACGCCAAGAGCCTGACCGCCTGGAGCCGGACCCTGGTGACCTTCAAGGACGTGTTCGTGG
ACTTCACCCGGGAGGAGTGGAAGCTGCTGGACACCGCCCAGCAGATCCTGTACCGGAACGT
GATGCTGGAGAACTACAAGAACCTGGTGAGCCTGGGCTACCAGCTGACCAAGCCCGACGTG
ATCCTGCGGCTGGAGAAGGGCGAGGAGCCCTGGCTGGTGGAGCGGGAGATCCACCAGGAGA
CCCACCCCGACAGCGAGACCGCCTTCGAGATCAAGAGCAGCGTG (SEQ ID NO: 51)
In some embodiments, KRAB for use in a polypeptide or an expression repressor
described
herein is a variant, e.g., comprising one or more mutations, relative to the
KRAB sequence of SEQ ID
NO: 61. In some embodiments, an KRAB variant comprises one or more amino acid
substitutions,
deletions, or insertions relative to SEQ ID NO: 61.
In some embodiments, the polypeptide or the expression repressor is a fusion
protein comprising
a repressor domain that is or comprises KRAB and a DNA-targeting moiety. In
some embodiments, the
polypeptide or the expression repressor comprises an additional moiety
described herein. In some
embodiments, the polypeptide or the expression repressor decreases expression
of a target gene. In some
embodiments, the polypeptide or the expression repressor may be used in
methods of modulating, e.g.,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
71
decreasing, gene expression, methods of treating a condition, or methods of
epigenetically modifying a
target gene, e.g., a transcription control element described herein, e.g., in
place of an expression
repression system. In some embodiments, an expression repression system
comprises two or more (e.g.,
two, three, or four) expression repressors, wherein the first expression
repressor comprises a repressor
domain comprising the KRAB sequence of SEQ ID NO: 61, or a functional variant
or fragment thereof.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) DNA-targeting moiety and one or more
repressor domain, wherein the
repressor domain is or comprises DNMT1, e.g., human DNMT1, or a functional
variant or fragment
thereof In some embodiments, DNMT1 is human DNMT1, e.g., corresponding to Gene
ID 1786, e.g.,
corresponding to UniPort ID P26358.2. In some embodiments, DNMT1 comprises an
amino acid
sequence of SEQ ID NO: 58. In some embodiments, a repressor domain described
herein comprises a
sequence according to SEQ ID NO: 58 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto:
VDLRTLDVFSGCGGLSEGFHQAGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILL
KLVMAGETTNSRGQRLPQKGDVEMLCGGPPCQGFSGMNRFNSRTYSKFKNSLVVSFLSYCDYY
RPRFFLLENVRNFVSFKRSMVLKLTLRCLVRMGYQCTFGVLQAGQYGVAQTRRRAIILAAAPGE
KLPLFPEPLHVFAPRACQLSVVVDDKKFVSNITRLSSGPFRTITVRDTMSDLPEVRNGASALEISY
NGEPQSWFQRQLRGAQYQPILRDHICKDMSALVAARMRHIPLAPGSDWRDLPNIEVRLSDGTMA
RKLRYTHHDRKNGRSSSGALRGVCSCVEAGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLY
GRLEWDGFFSTTVTNPEPMGKQGRVLHPEQHRVVSVRECARSQGFPDTYRLFGNILDKHRQVG
NAVPPPLAKAIGLEIKLCMLAKARESASAKIKEEEAAKD (SEQ ID NO: 58)
In some embodiments, DNMT1 is encoded by a nucleotide sequence of SEQ ID NO:
48. In some
embodiments, a nucleic acid described herein comprises a sequence of SEQ ID
NO: 48 or a sequence
with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having no more
than 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 positions of difference
thereto:
GTGGATCTGAGGACACTCGACGTGTTTAGCGGATGCGGCGGACTCTCCGAAGGCTTCCACCA
AGCCGGAATTTCCGACACACTCTGGGCCATTGAGATGTGGGACCCCGCCGCTCAAGCCTTCA
GACTGAATAATCCCGGCTCCACCGTGTTCACCGAGGACTGCAACATTCTGCTGAAGCTGGTG
ATGGCTGGCGAAACCACCAACTCTAGAGGCCAGAGGCTGCCCCAGAAGGGAGATGTGGAAA
TGCTCTGTGGAGGCCCTCCTTGCCAAGGCTTCTCCGGCATGAACAGGTTCAACTCTAGAACA
TACAGCAAGTTCAAGAACTCTCTGGTCGTGAGCTTTCTGAGCTACTGCGACTACTATAGACC
TAGGTTCTTTCTGCTGGAGAACGTGAGAAATTTCGTGTCCTTCAAGAGGAGCATGGTGCTGA
AGCTGACACTGAGGTGTCTGGTGAGGATGGGCTACCAGTGCACATTCGGAGTGCTGCAAGCT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
72
GGCCAGTACGGCGTGGCCCAGACCAGAAGGAGGGCCATCATTCTGGCTGCTGCCCCCGGCG
AGAAACTCCCTCTGTTCCCCGAGCCCCTCCACGTGTTCGCCCCTAGAGCTTGCCAGCTGAGC
GTGGTGGTCGACGATAAGAAGTTCGTGAGCAACATCACAAGGCTGTCCAGCGGACCCTTCA
GAACCATTACCGTGAGGGATACCATGTCCGACCTCCCCGAGGTGAGGAATGGCGCCAGCGC
TCTGGAGATTTCCTACAACGGCGAACCTCAGAGCTGGTTCCAAAGGCAGCTGAGAGGCGCTC
AGTATCAGCCCATTCTGAGGGACCACATCTGCAAAGATATGAGCGCTCTGGTGGCCGCTAGA
ATGAGACATATTCCTCTGGCCCCCGGCAGCGACTGGAGAGATCTGCCCAATATTGAGGTGAG
ACTCAGCGACGGAACAATGGCTAGAAAACTGAGGTACACCCATCATGATAGAAAGAACGGA
AGGAGCAGCAGCGGCGCTCTGAGAGGAGTGTGTAGCTGCGTGGAAGCTGGCAAGGCTTGCG
ATCCCGCCGCTAGGCAGTTCAATACCCTCATCCCTTGGTGTCTGCCTCACACCGGCAACAGA
CACAATCATTGGGCTGGACTGTATGGAAGGCTCGAATGGGACGGCTTTTTCAGCACCACCGT
GACCAATCCCGAACCTATGGGCAAGCAAGGAAGGGTGCTCCACCCCGAGCAGCATAGAGTC
GTGTCCGTGAGAGAATGCGCTAGAAGCCAAGGCTTCCCCGACACCTATAGACTGTTCGGCAA
CATTCTGGATAAGCACAGACAAGTGGGAAATGCTGTCCCTCCTCCTCTGGCCAAGGCTATCG
GACTGGAGATCAAGCTGTGTATGCTCGCCAAAGCTAGGGAGAGCGCTTCCGCCAAGATTAA
GGAGGAGGAGGCCGCCAAGGAC (SEQ ID NO: 48)
In some embodiments, DNMT1 for use in a polypeptide or an expression repressor
described
herein is a variant, e.g., comprising one or more mutations, relative to a
DNMT sequence of SEQ ID NO:
58. In some embodiments, the effector domain comprises one or more amino acid
substitutions, deletions,
or insertions relative to wild type DNMT1. In some embodiments, the
polypeptide is a fusion protein
comprising a repressor domain that is or comprises DNMT1 and a targeting
moiety. In some
embodiments, an expression repression system comprises two or more (e.g., two,
three, or four)
expression repressors, wherein the first expression repressor comprises a
repressor domain comprising
DNMT1, or a functional variant or fragment thereof.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) DNA-targeting moiety and one or more
repressor domain, wherein the
repressor domain is or comprises DNMT3a/3L complex, or a functional variant or
fragment thereof. In
some embodiments, the DNMT3a/3L complex is a fusion construct. In some
embodiments the
DNMT3a/3L complex comprises DNMT3A, e.g., human DNMT3A, e.g., as according to
NP_072046.2
or the protein encoded by NM_022552.4). In some embodiments the DNMT3a/3L
complex comprises
mouse DNMT3A, e.g., as according to NP_031898 or the protein encoded by
NM_007872. In some
embodiments the DNMT3a/3L complex comprises human DNMT3L (e.g., as according
to NP_787063.1
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
73
or the protein encoded by NM_175867.3). In some embodiments the DNMT3a/3L
complex comprises
mouse DNMT3L (e.g., as according to NP_001075164 or the protein encoded by
NM_001081695). In
some embodiments, DNMT3a/3L comprises an amino acid sequence of SEQ ID NO:59
or 60. In some
embodiments, a repressor domain described herein comprises SEQ ID NO: 59 or
SEQ ID NO: 60, or a
sequence with at least 80, 85, 90, 95, 99, or 100% identity thereto, or having
no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5,4, 3, 2, or 1 positions of difference
thereto.
NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRH
QGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDA
RPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRP
LASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFG
FPVHYTDV SNMSRLARQRLLGRSWS VPVIRHLFAPLKEYFACVS SGNSNANSRGPSFS SGLVPLS
LRGSHMNPLEMFETVPVWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKD
VEEWGPFDLVYGATPPLGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDL
DVASRFLEMEPVTIPDVHGGSLQNAVRVWSNIPAIRSRHWALV SEEELSLLAQNKQS SKLAAKW
PTKLVKNCFLPLREYFKYFSTELTSSL (SEQ ID NO: 59)
NHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRH
QGKIMYVGDVRSVTQKHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDA
RPKEGDDRPFFWLFENVVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRP
LASTVNDKLELQECLEHGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFG
FPVHYTDV SNMSRLARQRLLGRSWS VPVIRHLFAPLKEYFACVS SGNSNANSRGPSFS SGLVPLS
LRGSHMGPMEIYKTVSAWKRQPVRVLSLFRNIDKVLKSLGFLESGSGSGGGTLKYVEDVTNVVR
RDVEKWGPFDLVYGSTQPLGS SCDRCPGWYMFQFHRILQYALPRQES QRPFFWIFMDNLLLTED
DQETTTRFLQTEAVTLQDVRGRDYQNAMRVWSNIPGLKSKHAPLTPKEEEYLQAQVRSRSKLD
APKVDLLVKNCLLPLREYFKYFSQNSLPL (SEQ ID NO: 60)
In some embodiments, DNMT3a/3L is encoded by a nucleotide sequence of SEQ ID
NO: 49 or
SEQ ID NO: 50. In some embodiments, a nucleic acid described herein comprises
a sequence of SEQ ID
NO: 49 or 50 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more
than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
AACCACGACCAGGAGTTCGACCCCCCCAAGGTGTACCCCCCCGTGCCCGCCGAGAAGCGGA
AGCCCATCCGGGTGCTGAGCCTGTTCGACGGCATCGCCACCGGCCTGCTGGTGCTGAAGGAC
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
74
CTGGGCATCCAGGTGGACCGGTACATCGCCAGCGAGGTGTGCGAGGACAGCATCACCGTGG
GCATGGTGCGGCACCAGGGCAAGATCATGTACGTGGGCGACGTGCGGAGCGTGACCCAGAA
GCACATCCAGGAGTGGGGCCCCTTCGACCTGGTGATCGGCGGCAGCCCCTGCAACGACCTG
AGCATCGTGAACCCCGCCCGGAAGGGCCTGTACGAGGGCACCGGCCGGCTGTTCTTCGAGTT
CTACCGGCTGCTGCACGACGCCCGGCCCAAGGAGGGCGACGACCGGCCCTTCTTCTGGCTGT
TCGAGAACGTGGTGGCCATGGGCGTGAGCGACAAGCGGGACATCAGCCGGTTCCTGGAGAG
CAACCCCGTGATGATCGACGCCAAGGAGGTGAGCGCCGCCCACCGGGCCCGGTACTTCTGG
GGCAACCTGCCCGGCATGAACCGGCCCCTGGCCAGCACCGTGAACGACAAGCTGGAGCTGC
AGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGTGCGGACCATCACCACCCG
GAGCAACAGCATCAAGCAGGGCAAGGACCAGCACTTCCCCGTGTTCATGAACGAGAAGGAG
GACATCCTGTGGTGCACCGAGATGGAGCGGGTGTTCGGCTTCCCCGTGCACTACACCGACGT
GAGCAACATGAGCCGGCTGGCCCGGCAGCGGCTGCTGGGCCGGAGCTGGAGCGTGCCCGTG
ATCCGGCACCTGTTCGCCCCCCTGAAGGAGTACTTCGCCTGCGTGAGCAGCGGCAACAGCAA
CGCCAACAGCCGGGGCCCCAGCTTCAGCAGCGGCCTGGTGCCCCTGAGCCTGCGGGGCAGC
CACATGAATCCTCTGGAGATGTTCGAGACAGTGCCCGTGTGGAGAAGGCAACCCGTGAGGG
TGCTGAGCCTCTTCGAGGACATTAAGAAGGAGCTGACCTCTCTGGGCTTTCTGGAATCCGGC
AGCGACCCCGGCCAGCTGAAACACGTGGTGGACGTGACCGACACAGTGAGGAAGGACGTGG
AAGAGTGGGGCCCCTTTGACCTCGTGTATGGAGCCACACCTCCTCTCGGCCACACATGCGAT
AGGCCTCCCAGCTGGTATCTCTTCCAGTTCCACAGACTGCTCCAGTACGCCAGACCTAAGCC
CGGCAGCCCCAGACCCTTCTTCTGGATGTTCGTGGACAATCTGGTGCTGAACAAGGAGGATC
TGGATGTGGCCAGCAGATTTCTGGAGATGGAACCCGTGACAATCCCCGACGTGCATGGCGG
CTCTCTGCAGAACGCCGTGAGAGTGTGGTCCAACATCCCCGCCATTAGAAGCAGACACTGGG
CTCTGGTGAGCGAGGAGGAACTGTCTCTGCTGGCCCAGAATAAGCAGTCCTCCAAGCTGGCC
GCCAAGTGGCCCACCAAGCTGGTGAAGAACTGCTTTCTGCCTCTGAGGGAGTATTTCAAGTA
TTTCAGCACCGAACTGACCAGCAGCCTG (SEQ ID NO: 49)
AACCACGACCAGGAGTTCGACCCCCCCAAGGTGTACCCCCCCGTGCCCGCCGAGAAGCGGA
AGCCCATCCGGGTGCTGAGCCTGTTCGACGGCATCGCCACCGGCCTGCTGGTGCTGAAGGAC
CTGGGCATCCAGGTGGACCGGTACATCGCCAGCGAGGTGTGCGAGGACAGCATCACCGTGG
GCATGGTGCGGCACCAGGGCAAGATCATGTACGTGGGCGACGTGCGGAGCGTGACCCAGAA
GCACATCCAGGAGTGGGGCCCCTTCGACCTGGTGATCGGCGGCAGCCCCTGCAACGACCTG
AGCATCGTGAACCCCGCCCGGAAGGGCCTGTACGAGGGCACCGGCCGGCTGTTCTTCGAGTT
CTACCGGCTGCTGCACGACGCCCGGCCCAAGGAGGGCGACGACCGGCCCTTCTTCTGGCTGT
TCGAGAACGTGGTGGCCATGGGCGTGAGCGACAAGCGGGACATCAGCCGGTTCCTGGAGAG
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
CAACCCCGTGATGATCGACGCCAAGGAGGTGAGCGCCGCCCACCGGGCCCGGTACTTCTGG
GGCAACCTGCCCGGCATGAACCGGCCCCTGGCCAGCACCGTGAACGACAAGCTGGAGCTGC
AGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGTGCGGACCATCACCACCCG
GAGCAACAGCATCAAGCAGGGCAAGGACCAGCACTTCCCCGTGTTCATGAACGAGAAGGAG
5 GACATCCTGTGGTGCACCGAGATGGAGCGGGTGTTCGGCTTCCCCGTGCACTACACCGACGT
GAGCAACATGAGCCGGCTGGCCCGGCAGCGGCTGCTGGGCCGGAGCTGGAGCGTGCCCGTG
ATCCGGCACCTGTTCGCCCCCCTGAAGGAGTACTTCGCCTGCGTGAGCAGCGGCAACAGCAA
CGCCAACAGCCGGGGCCCCAGCTTCAGCAGCGGCCTGGTGCCCCTGAGCCTGCGGGGCAGC
CACATGGGCCCCATGGAGATCTACAAGACCGTGAGCGCCTGGAAGCGGCAGCCCGTGCGGG
10 TGCTGAGCCTGTTCCGGAACATCGACAAGGTGCTGAAATCCCTGGGCTTCCTGGAGAGCGGC
AGCGGCAGCGGCGGCGGCACCCTGAAGTACGTGGAGGACGTGACCAACGTGGTGCGGCGGG
ACGTGGAGAAGTGGGGCCCCTTCGACCTGGTGTACGGCAGCACCCAGCCCCTGGGCAGCAG
CTGCGACCGGTGCCCCGGCTGGTACATGTTCCAGTTCCACCGGATCCTGCAGTACGCCCTGC
CCCGGCAGGAGAGCCAGCGGCCCTTCTTCTGGATCTTCATGGACAACCTGCTGCTGACCGAG
15 GACGACCAGGAGACCACCACCCGGTTCCTGCAGACCGAGGCCGTGACCCTGCAGGACGTGC
GGGGCCGGGACTACCAGAACGCCATGCGGGTGTGGAGCAACATCCCCGGCCTGAAATCCAA
GCACGCCCCCCTGACCCCCAAGGAGGAGGAGTACCTGCAGGCCCAGGTGCGGAGCCGGAGC
AAGCTGGACGCCCCCAAGGTGGACCTGCTGGTGAAGAACTGCCTGCTGCCCCTGCGGGAGT
ACTTCAAGTACTTCAGCCAGAACAGCCTGCCCCTG (SEQ ID NO: 50)
In some embodiments, DNMT3a/3L for use in a polypeptide or an expression
repressor described
herein is a variant, e.g., comprising one or more mutations, relative to the
DNMT3a/3L of SEQ ID NO:
59 or SEQ ID NO: 60. In some embodiments, an DNMT3a/3L variant comprises one
or more amino acid
substitutions, deletions, or insertions relative to SEQ ID NO: 59 or SEQ ID
NO: 60. In some
embodiments, the polypeptide or the expression repressor is a fusion protein
comprising a repressor
domain that is or comprises DNMT3a/3L and a DNA-targeting moiety. In some
embodiments, an
expression repression system comprises two or more (e.g., two, three, or four)
expression repressors,
wherein the first expression repressor comprises a repressor domain comprising
DNMT3a/3L, or a
functional variant or fragment thereof.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) DNA-targeting moiety and one or more
repressor domain, wherein the
repressor domain is or comprises DNMT3b, e.g., human DNMT3b, or a functional
variant or fragment
thereof. In some embodiments the DNMT3b is human DNMT3b e.g., as according to
NP_008823.1 or
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
76
A0X21819.1, or the protein encoded by NM_006892.4 or KX447429.) In some
embodiments, DNMT3b
comprises an amino acid sequence of SEQ ID NO: 85. In some embodiments, a
repressor domain
described herein comprises SEQ ID NO: 85, or a sequence with at least 80, 85,
90, 95, 99, or 100%
identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
DNMT3b
MKGDTRHLNGEEDAGGRED SILVNGAC SD QS SD SPPILEAIRTPEIRGRRS S SRLS KREVS SLLS YT
QDLTGDGDGEDGDGSDTPVMPKLFRETRTRSESPAVRTRNNNSVSSRERHRPSPRSTRGRQGRN
HVDESPVEFPATRS LRRRATAS AGTPWPSPPS S YLTIDLTDDTEDTHGTPQS S S TPYARLAQD S QQ
GGMESPQVEADSGDGDSSEYQVSADKLVALGLFSQHFNLATFNKLVSYRKAMYHALEKARVR
AGKTFPSSPGDSLEDQLKPMLEWAHGGFKPTGIEGLKPNNTQPENKTRRRTADDSATSDYCPAP
KRLKTNCYNNGKDRGDEDQSREQMASDVANNKSSLEDGCLSCGRKNPVSFHPLFEGGLCQTCR
DRFLELFYMYDDDGYQSYCTVCCEGRELLLCSNTSCCRCFCVECLEVLVGTGTAAEAKLQEPWS
CYMCLPQRCHGVLRRRKDWNVRLQAFFTSDTGLEYEAPKLYPAIPAARRRPIRVLSLFDGIATGY
LVLKELGIKVGKYVASEVCEESIAVGTVKHEGNIKYVNDVRNITKKNIEEWGPFDLVIGGSPCND
LSNVNPARKGLYEGTGRLFFEFYHLLNYSRPKEGDDRPFFWMFENVVAMKVGDKRDISRFLECN
PVMIDAIKVSAAHRARYFWGNLPGMNRPVIASKNDKLELQDCLEYNRIAKLKKVQTITTKSNSIK
QGKNQLFPVVMNGKEDVLWCTELERIFGFPVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAP
LKDYFACE (SEQ ID NO: 85)
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) targeting moiety and one or more effector
moiety, wherein the effector
moiety is or comprises G9A e.g., as according to NP_001350618.1 or the protein
encoded by
NM_001363689.1 or a functional variant or fragment thereof. In some
embodiments, G9A comprises an
amino acid sequence of SEQ ID NO: 62:
GNRAIRTEKIICRDVARGYENVPIPCVNGVDGEPCPEDYKYISENCETSTMNIDRNITHLQHCTCV
DDCSSSNCLCGQLSIRCWYDKDGRLLQEFNKIEPPLIFECNQACSCWRNCKNRVVQSGIKVRLQL
YRTAKMGWGVRALQTIPQGTFICEYVGELISDAEADVREDDSYLFDLDNKDGEVYCIDARYYG
NISRFINHLCDPNIIPVRVFMLHQDLRFPRIAFFSSRDIRTGEELGFDYGDRFWDIKSKYFTCQCGS
EKCKHSAEAIALEQSRLARLD (SEQ ID NO: 62)
In some embodiments, the G9A repressor domain is encoded by a nucleotide
sequence of SEQ ID
NO: 52. In some embodiments, a nucleotide sequence described herein comprises
a sequence of SEQ ID
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
77
NO: 52 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more than
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1
positions of difference thereto.
GGAAATAGGGCTATCAGAACCGAGAAGATCATCTGTAGGGACGTGGCTAGAGGCTACGAGA
ACGTGCCCATTCCTTGCGTGAATGGCGTGGATGGCGAACCTTGCCCCGAGGACTACAAATAC
ATCTCCGAGAACTGCGAAACCAGCACAATGAACATCGACAGAAACATCACCCACCTCCAGC
ACTGCACATGTGTGGATGACTGCTCCTCCAGCAACTGTCTGTGCGGCCAGCTCTCCATCAGA
TGCTGGTACGACAAGGACGGCAGACTGCTGCAAGAGTTCAACAAGATCGAACCCCCTCTCA
TCTTCGAGTGTAACCAAGCTTGCAGCTGCTGGAGAAACTGCAAGAATAGAGTGGTCCAGAG
CGGCATCAAGGTGAGACTGCAACTGTACAGAACCGCCAAGATGGGATGGGGAGTGAGGGCT
CTGCAAACCATTCCCCAAGGCACCTTCATCTGCGAATACGTGGGCGAACTGATCTCCGACGC
CGAAGCTGACGTGAGAGAGGACGACAGCTATCTCTTCGATCTGGACAATAAGGACGGCGAG
GTGTACTGCATCGACGCTAGATATTACGGCAACATCTCTAGATTCATCAACCACCTCTGCGA
TCCCAACATCATTCCCGTGAGGGTGTTCATGCTGCACCAAGATCTGAGGTTCCCTAGAATCG
CCTTCTTCAGCTCTAGAGACATCAGAACCGGCGAGGAGCTGGGCTTCGATTACGGCGATAGA
TTCTGGGACATCAAGTCCAAGTACTTCACATGCCAGTGCGGCAGCGAGAAGTGTAAGCACA
GCGCTGAGGCCATTGCTCTGGAGCAGTCTAGACTGGCCAGACTGGAT (SEQ ID NO: 52)
In some embodiments, G9A for use in a polypeptide or an expression repressor
described herein
is a variant, e.g., comprising one or more mutations, relative to the G9A
sequence of SEQ ID NO: 62. In
some embodiments, an G9A variant comprises one or more amino acid
substitutions, deletions, or
insertions relative to SEQ ID NO: 62.
In some embodiments, the polypeptide or the expression repressor is a fusion
protein comprising
a repressor domain that is or comprises G9A and a DNA-targeting moiety. In
some embodiments, the
polypeptide or the expression repressor comprises an additional moiety
described herein. In some
embodiments, the polypeptide or the expression repressor decreases expression
of a target gene. In some
embodiments, the polypeptide or the expression repressor may be used in
methods of modulating, e.g.,
decreasing, gene expression, methods of treating a condition, or methods of
epigenetically modifying a
target gene, e.g., a transcription control element described herein, e.g., in
place of an expression
repression system. In some embodiments, an expression repression system
comprises two or more (e.g.,
two, three, or four) expression repressors, wherein the first expression
repressor comprises a repressor
domain comprising the G9A sequence of SEQ ID NO: 62, or a functional variant
or fragment thereof.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
78
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide comprising one or
more (e.g., one) targeting moiety and one or more effector moiety, wherein the
effector moiety is or
comprises HDAC8, e.g., as according to NP_001159890 or the protein encoded by
NM_001166418 or a
functional variant or fragment thereof. In some embodiments, HDAC8 comprises
an amino acid sequence
of SEQ ID NO: 63:
EEPEEPADSGQSLVPVYIYSPEYVSMCDSLAKIPKRASMVHSLIEAYALHKQMRIVKPKVASMEE
MATFHTDAYLQHLQKVS QEGDDDHPD SIEYGLGYDCPATEGIFDYAAAIGGATITAAQCLID GM
CKVAINWSGGWHHAKKDEASGFCYLNDAVLGILRLRRKFERILYVDLDLHHGDGVEDAFSFTS
KVMTVSLHKFSPGFFPGTGDVSDVGLGKGRYYSVNVPIQDGIQDEKYYQICESVLKEVYQAFNP
KAVVLQLGADTIAGDPMCSFNMTPVGIGKCLKYILQWQLATLILGGGGYNLANTARCWTYLTG
VILGKTLSSEIPDHEFFTAYGPDYVLEITPSCRPDRNEPHRIQQILNYIKGNLKHVV (SEQ ID NO:
63)
In some embodiments, the HDAC8 repressor domain is encoded by a nucleotide
sequence of
SEQ ID NO: 53. In some embodiments, a nucleotide sequence described herein
comprises a sequence of
SEQ ID NO: 53 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no
more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
or 1 positions of difference
thereto.
GAGGAGCCCGAGGAGCCCGCCGATAGCGGACAATCTCTGGTGCCCGTCTACATCTACAGCC
CCGAATATGTGAGCATGTGTGATTCCCTCGCCAAGATCCCTAAGAGAGCCAGCATGGTGCAT
TCTCTGATCGAGGCCTACGCTCTGCATAAGCAAATGAGGATCGTGAAGCCCAAGGTCGCCAG
CATGGAAGAGATGGCCACCTTTCACACCGATGCCTACCTCCAACATCTCCAGAAGGTGTCCC
AAGAGGGCGACGACGACCACCCCGACTCCATTGAGTACGGACTGGGCTATGATTGCCCCGC
CACCGAGGGCATCTTTGACTATGCCGCCGCTATCGGCGGAGCTACCATCACAGCCGCCCAGT
GTCTGATTGATGGCATGTGCAAGGTCGCCATCAACTGGTCCGGAGGCTGGCATCATGCCAAG
AAGGATGAGGCCTCCGGCTTCTGTTATCTGAATGACGCCGTGCTGGGCATTCTGAGACTGAG
GAGGAAATTCGAGAGGATTCTGTACGTGGATCTGGATCTGCATCACGGAGATGGAGTCGAA
GATGCCTTCAGCTTCACCAGCAAGGTGATGACAGTCTCTCTGCACAAGTTCTCCCCCGGCTTC
TTTCCCGGAACCGGCGACGTGTCCGACGTGGGACTGGGCAAGGGAAGGTACTACAGCGTGA
ACGTGCCCATTCAAGACGGCATCCAAGACGAGAAGTACTACCAGATCTGCGAGTCCGTGCTC
AAGGAGGTCTACCAAGCCTTCAATCCTAAGGCTGTCGTGCTCCAACTGGGAGCTGATACCAT
TGCTGGCGATCCCATGTGCAGCTTCAATATGACACCCGTCGGAATCGGCAAGTGCCTCAAGT
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
79
ACATCCTCCAGTGGCAGCTCGCCACCCTCATTCTCGGAGGAGGCGGATACAATCTGGCTAAT
ACCGCCAGATGCTGGACCTATCTGACCGGCGTGATTCTGGGCAAAACACTGAGCAGCGAAA
TCCCCGACCACGAGTTTTTCACCGCTTACGGCCCCGACTACGTGCTGGAGATCACCCCCAGC
TGCAGACCCGATAGAAACGAACCCCATAGAATCCAGCAAATTCTGAACTATATCAAGGGCA
ACCTCAAGCACGTCGTG (SEQ ID NO: 53)
In some embodiments, HDAC8 for use in a polypeptide or an expression repressor
described
herein is a variant, e.g., comprising one or more mutations, relative to the
HDAC8 sequence of SEQ ID
NO: 63. In some embodiments, an HDAC8 variant comprises one or more amino acid
substitutions,
deletions, or insertions relative to SEQ ID NO: 63.
In some embodiments, the polypeptide or the expression repressor is a fusion
protein comprising
a repressor domain that is or comprises HDAC8 and a DNA-targeting moiety. In
some embodiments, the
polypeptide or the expression repressor comprises an additional moiety
described herein. In some
embodiments, the polypeptide or the expression repressor decreases expression
of a target gene. In some
embodiments, the polypeptide or the expression repressor may be used in
methods of modulating, e.g.,
decreasing, gene expression, methods of treating a condition, or methods of
epigenetically modifying a
target gene, e.g., a transcription control element described herein, e.g., in
place of an expression
repression system. In some embodiments, an expression repression system
comprises two or more (e.g.,
two, three, or four) expression repressors, wherein the first expression
repressor comprises a repressor
domain comprising the HDAC8 sequence of SEQ ID NO: 63, or a functional variant
or fragment thereof.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) targeting moiety and one or more effector
moiety, wherein the effector
moiety is or comprises LSD1 e.g., as according to NP_055828.2 or the protein
encoded by NM_015013.4
or a functional variant or fragment thereof. In some embodiments, KRAB
comprises an amino acid
sequence of SEQ ID NO: 64:
LSGKKAAAAAAAAAAAATGTEAGPGTAGGSENGSEVAAQPAGLSGPAEVGPGAVGERTPRKK
EPPRASPPGGLAEPPGSAGPQAGPTVVPGSATPMETGIAETPEGRRTSRRKRAKVEYREMDESLA
NLSEDEYYSEEERNAKAEKEKKLPPPPPQAPPEEENESEPEEPSGVEGAAFQSRLPHDRMTSQEA
ACFPDIISGPQQTQKVFLFIRNRTLQLWLDNPKIQLTFEATLQQLEAPYNSDTVLVHRVHSYLERH
GLINFGIYKRIKPLPTKKTGKVIIIGSGVSGLAAARQLQSFGMDVTLLEARDRVGGRVATFRKGN
YVADLGAMVVTGLGGNPMAVVSKQVNMELAKIKQKCPLYEANGQAVPKEKDEMVEQEFNRL
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
LEATSYLSHQLDFNVLNNKHVSLGQALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLNKM
VNLKEKIKELHQQYKEASEVKPPRDITAEFLVKSKHRDLTALCKEYDELAETQGKLEEKLQELEA
NPPSDVYLSSRDRQILDWHFANLEFANATPLSTLSLKHWDQDDDFEFTGSHLTVRNGYSCVPVA
LAEGLDIKLNTAVRQVRYTASGCEVIAVNTRSTSQTFIYKCDAVLCTLPLGVLKQQPPAVQFVPP
5 LPEWKTSAVQRMGFGNLNKVVLCFDRVFWDPSVNLFGHVGSTTASRGELFLFWNLYKAPILLA
LVAGEAAGIMENISDDVIVGRCLAILKGIFGSSAVPQPKETVVSRWRADPWARGSYSYVAAGSS
GNDYDLMAQPITPGPSIPGAPQPIPRLFFAGEHTIRNYPATVHGALLSGLREAGRIADQFLGAMYT
LPRQATPGVPAQQSPSM (SEQ ID NO: 64)
10 In some embodiments, the LSD1 repressor domain is encoded by a
nucleotide sequence of SEQ
ID NO: 54. In some embodiments, a nucleotide sequence described herein
comprises a sequence of SEQ
ID NO: 54 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more than
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1
positions of difference thereto.
15 CTCTCCGGAAAGAAAGCCGCCGCTGCCGCTGCCGCCGCTGCTGCTGCCGCTACCGGCACAGA
AGCTGGCCCCGGAACAGCTGGCGGAAGCGAAAACGGAAGCGAAGTGGCTGCCCAGCCCGCC
GGACTGTCCGGACCCGCCGAGGTGGGACCCGGCGCTGTCGGCGAAAGGACCCCCAGAAAAA
AGGAGCCTCCTAGAGCCAGCCCTCCCGGCGGACTCGCCGAACCTCCCGGCAGCGCTGGACCT
CAAGCCGGACCTACAGTGGTGCCCGGCAGCGCTACACCTATGGAGACCGGCATCGCTGAGA
20 CCCCCGAGGGAAGGAGAACCAGCAGAAGGAAGAGAGCCAAGGTGGAGTACAGAGAGATGG
ATGAGTCTCTGGCTAACCTCAGCGAGGACGAGTACTACTCCGAGGAGGAAAGGAATGCCAA
GGCCGAGAAGGAGAAGAAGCTGCCCCCTCCTCCCCCTCAAGCCCCCCCCGAGGAGGAGAAC
GAGTCCGAGCCCGAGGAACCCAGCGGAGTGGAAGGAGCCGCCTTTCAGTCCAGACTGCCCC
ACGACAGAATGACATCCCAAGAGGCCGCTTGCTTTCCCGACATTATTTCCGGCCCTCAGCAG
25 ACCCAGAAGGTGTTTCTGTTCATTAGAAATAGAACACTCCAGCTGTGGCTCGACAACCCCAA
GATCCAGCTGACCTTCGAGGCTACCCTCCAACAGCTGGAGGCCCCCTACAATAGCGATACCG
TGCTGGTGCACAGAGTGCACAGCTATCTGGAGAGGCACGGCCTCATTAACTTCGGCATTTAC
AAGCGGATCAAGCCCCTGCCCACCAAGAAAACAGGCAAGGTGATCATCATCGGCAGCGGCG
TGAGCGGCCTGGCCGCCGCCCGGCAGCTGCAGAGCTTCGGCATGGACGTGACCCTGCTGGA
30 GGCCCGGGACCGGGTGGGCGGCCGGGTGGCCACCTTCCGGAAGGGCAACTACGTGGCCGAC
CTGGGCGCCATGGTGGTGACCGGCCTGGGCGGCAACCCCATGGCCGTGGTGAGCAAGCAGG
TGAACATGGAGCTGGCCAAGATCAAGCAGAAGTGCCCCCTGTACGAGGCCAACGGCCAGGC
CGTGCCCAAGGAGAAGGACGAGATGGTGGAGCAGGAGTTCAACCGGCTGCTGGAGGCCACC
AGCTACCTGAGCCACCAGCTGGACTTCAACGTGCTGAACAACAAGCACGTGAGCCTGGGCC
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
81
AGGCCCTGGAGGTGGTGATCCAGCTGCAGGAGAAGCACGTGAAGGACGAGCAGATCGAGC
ACTGGAAGAAGATCGTGAAAACACAGGAGGAGCTGAAGGAGCTGCTGAACAAGATGGTGA
ACCTGAAGGAGAAGATCAAGGAGCTGCACCAGCAGTACAAGGAGGCCAGCGAGGTGAAGC
CCCCCCGGGACATCACCGCCGAGTTCCTGGTCAAAAGCAAGCACCGGGACCTGACCGCCCT
GTGCAAGGAGTACGACGAGCTGGCCGAGACCCAGGGCAAGCTGGAGGAGAAGCTGCAGGA
GCTGGAGGCCAACCCCCCCAGCGACGTGTACCTGAGCAGCCGGGACCGGCAGATCCTGGAC
TGGCACTTCGCCAACCTGGAGTTCGCCAACGCCACCCCCCTGAGCACCCTGAGCCTGAAGCA
CTGGGACCAGGACGACGACTTCGAGTTCACCGGCAGCCACCTGACCGTGCGGAACGGCTAC
AGCTGCGTGCCCGTGGCCCTGGCCGAGGGCCTGGACATCAAGCTGAACACCGCCGTGCGGC
AGGTGCGGTACACCGCCAGCGGCTGCGAGGTGATCGCCGTGAACACCCGGAGCACCAGCCA
GACCTTCATCTACAAGTGCGACGCCGTGCTGTGCACCCTGCCCCTGGGCGTGCTGAAGCAGC
AGCCCCCCGCCGTGCAGTTCGTGCCCCCCCTGCCCGAGTGGAAAACAAGCGCCGTGCAGCG
GATGGGCTTCGGCAACCTGAACAAGGTGGTGCTGTGCTTCGACCGGGTGTTCTGGGACCCCA
GCGTGAACCTGTTCGGCCACGTGGGCAGCACCACCGCCAGCCGGGGCGAGCTGTTCCTGTTC
TGGAACCTGTACAAGGCCCCCATCCTGCTGGCCCTGGTGGCCGGCGAGGCCGCCGGCATCAT
GGAGAACATCAGCGACGACGTGATCGTGGGCCGGTGCCTGGCCATCCTGAAGGGCATCTTC
GGCAGCAGCGCCGTGCCCCAGCCCAAGGAGACCGTGGTGAGCCGGTGGCGGGCCGACCCCT
GGGCCCGGGGCAGCTACAGCTACGTGGCCGCCGGCAGCAGCGGCAACGACTACGACCTGAT
GGCCCAGCCCATCACCCCCGGCCCCAGCATCCCCGGCGCCCCCCAGCCCATCCCCCGGCTGT
TCTTCGCCGGCGAGCACACCATCCGGAACTACCCCGCCACCGTGCACGGCGCCCTGCTGAGC
GGCCTGCGGGAGGCAGGACGGATCGCCGACCAGTTCCTGGGCGCCATGTACACCCTGCCCC
GGCAGGCCACCCCCGGCGTGCCCGCCCAGCAGAGCCCCAGCATG (SEQ ID NO: 54)
In some embodiments, LSD1 for use in a polypeptide or an expression repressor
described herein
is a variant, e.g., comprising one or more mutations, relative to the LSD1
sequence of SEQ ID NO: 64. In
some embodiments, an LSD1 variant comprises one or more amino acid
substitutions, deletions, or
insertions relative to SEQ ID NO: 64.
In some embodiments, the polypeptide or the expression repressor is a fusion
protein comprising
a repressor domain that is or comprises LSD1 and a DNA-targeting moiety. In
some embodiments, the
polypeptide or the expression repressor comprises an additional moiety
described herein. In some
embodiments, the polypeptide or the expression repressor decreases expression
of a target gene. In some
embodiments, the polypeptide or the expression repressor may be used in
methods of modulating, e.g.,
decreasing, gene expression, methods of treating a condition, or methods of
epigenetically modifying a
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
82
target gene, e.g., a transcription control element described herein, e.g., in
place of an expression
repression system. In some embodiments, an expression repression system
comprises two or more (e.g.,
two, three, or four) expression repressors, wherein the first expression
repressor comprises a repressor
domain comprising the LSD1 sequence of SEQ ID NO: 64, or a functional variant
or fragment thereof.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) targeting moiety and one or more effector
moiety, wherein the effector
moiety is or comprises EZH2, e.g., as according to NP-004447.2 or the protein
encoded by NM_004456.5
or a functional variant or fragment thereof. In some embodiments, EZH2
comprises an amino acid
sequence of SEQ ID NO: 65:
GQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFS SNRQKILERTEILNQEWKQRRI
QPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSPLQQNFMVEDETVLHNI
PYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALGQYNDDDDDDDGDDPEE
REEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEKYKELTEQQLPGALPPECT
PNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKRKNTETALDNKPCGPQCYQ
HLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNS SRPS TPTINVLESKDTD SDREAGTETGGEN
NDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEASMFRVLIGTYYDNFCAIARLI
GTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHCRKIQLKKDGSSNHVYNYQP
CDHPRQPCD S SCPCVIA QNFCEKFCQC S SECQNRFPGCRC KAQCNTKQCPCYLAVRECDPDLCLT
CGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDPVQKNEFISEYCGEIISQDEAD
RRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNCYAKVMMVNGDHRIGIFAKR
AIQTGEELFFDYRYSQADALKYVGIEREMEIP (SEQ ID NO: 65)
In some embodiments, the EZH2 repressor domain is encoded by a nucleotide
sequence of SEQ
ID NO: 55. In some embodiments, a nucleotide sequence described herein
comprises a sequence of SEQ
ID NO: 55 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more than
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1
positions of difference thereto.
GGCCAGACCGGCAAGAAGAGCGAGAAGGGCCCCGTGTGCTGGCGGAAGCGGGTGAAGAGC
GAGTACATGCGGCTGCGGCAGCTGAAGCGGTTCCGGCGGGCCGACGAGGTGAAGAGCATGT
TCAGCAGCAACCGGCAGAAGATCCTGGAGCGGACCGAGATCCTGAACCAGGAGTGGAAGCA
GCGGCGAATCCAGCCCGTGCACATCCTGACCAGCGTGAGCAGCCTGCGGGGCACCCGGGAG
TGCAGCGTGACCAGCGACCTGGACTTCCCCACCCAGGTGATCCCCCTAAAGACCCTGAACGC
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
83
CGTGGCCAGCGTGCCCATCATGTACAGCTGGAGCCCCCTGCAGCAGAACTTCATGGTGGAGG
ACGAGACCGTGCTGCACAACATCCCCTACATGGGCGACGAGGTGCTGGACCAGGACGGCAC
CTTCATCGAGGAGCTGATCAAGAACTACGACGGCAAGGTGCACGGCGACCGGGAGTGCGGC
TTCATCAACGACGAGATCTTCGTGGAGCTGGTGAACGCCCTGGGCCAGTACAACGACGACG
ACGACGACGACGACGGCGACGACCCCGAGGAGCGGGAGGAGAAGCAGAAGGACCTGGAGG
ACCACCGGGACGACAAGGAGAGCCGGCCCCCCCGGAAGTTCCCCAGCGACAAGATCTTCGA
GGCCATCAGCAGCATGTTCCCCGACAAGGGCACCGCCGAGGAGCTGAAGGAGAAGTACAAG
GAGCTGACCGAGCAGCAGCTGCCCGGCGCCCTGCCCCCCGAGTGCACCCCCAACATCGACG
GCCCCAACGCCAAGAGCGTGCAGCGGGAGCAGAGCCTGCACAGCTTCCACACCCTGTTCTG
CCGGCGGTGCTTCAAGTACGACTGCTTCCTGCACCCCTTCCACGCCACCCCCAACACCTACA
AGCGGAAGAACACCGAGACCGCCCTGGACAACAAGCCCTGCGGCCCCCAGTGCTACCAGCA
CCTGGAGGGCGCCAAGGAGTTCGCCGCCGCCCTGACCGCCGAGCGGATCAAGACCCCCCCC
AAGCGGCCCGGCGGCCGGCGGCGGGGCCGGCTGCCCAACAACAGCAGCCGGCCCAGCACCC
CCACCATCAACGTGCTGGAGAGCAAGGACACCGACAGCGACCGGGAGGCCGGCACCGAGA
CCGGCGGCGAGAACAACGACAAGGAGGAGGAGGAGAAGAAGGACGAGACCAGCAGCAGC
AGCGAGGCCAACAGCCGGTGCCAGACCCCCATCAAGATGAAGCCCAACATCGAGCCCCCCG
AGAACGTGGAGTGGAGCGGCGCCGAGGCCAGCATGTTCCGGGTGCTGATCGGCACCTACTA
CGACAACTTCTGCGCCATCGCCCGGCTGATCGGCACCAAGACCTGCCGGCAGGTGTACGAGT
TCCGGGTGAAGGAGAGCAGCATCATCGCCCCCGCCCCCGCCGAGGACGTGGACACCCCCCC
CCGGAAGAAGAAGCGGAAGCACCGGCTGTGGGCCGCCCACTGCCGGAAGATCCAGCTGAAG
AAGGACGGCAGCAGCAACCACGTGTACAACTACCAGCCCTGCGACCACCCCCGGCAGCCCT
GCGACAGCAGCTGCCCCTGCGTGATCGCCCAGAACTTCTGCGAGAAGTTCTGCCAGTGCAGC
AGCGAGTGCCAGAACCGGTTCCCCGGCTGCCGGTGCAAGGCCCAGTGCAACACCAAGCAGT
GCCCCTGCTACCTGGCCGTGCGGGAGTGCGACCCCGACCTGTGCCTGACCTGCGGCGCCGCC
GACCACTGGGACAGCAAGAACGTGAGCTGCAAGAACTGCAGCATCCAGCGGGGCAGCAAG
AAGCACCTGCTGCTGGCCCCCAGCGACGTGGCCGGCTGGGGCATCTTCATCAAGGACCCCGT
GCAGAAGAACGAGTTCATCAGCGAGTACTGCGGCGAGATCATCAGCCAGGACGAGGCCGAC
CGGCGGGGCAAGGTGTACGACAAGTACATGTGCAGCTTCCTGTTCAACCTGAACAACGACTT
CGTGGTGGACGCCACCCGGAAGGGCAACAAGATCCGGTTCGCCAACCACAGCGTGAACCCC
AACTGCTACGCCAAGGTGATGATGGTGAACGGCGACCACCGGATCGGCATCTTCGCCAAGC
GGGCCATCCAGACCGGCGAGGAGCTGTTCTTCGACTACCGGTACAGCCAGGCCGACGCCCT
GAAGTACGTGGGCATCGAGCGGGAGATGGAGATCCCC (SEQ ID NO: 55)
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
84
In some embodiments, EZH2 for use in a polypeptide or an expression repressor
described herein
is a variant, e.g., comprising one or more mutations, relative to the EZH2
sequence of SEQ ID NO: 65. In
some embodiments, an EZH2 variant comprises one or more amino acid
substitutions, deletions, or
insertions relative to SEQ ID NO: 65.
In some embodiments, the polypeptide or the expression repressor is a fusion
protein comprising a
repressor domain that is or comprises EZH2 and a DNA-targeting moiety. In some
embodiments, the
polypeptide or the expression repressor comprises an additional moiety
described herein. In some
embodiments, the polypeptide or the expression repressor decreases expression
of a target gene. In some
embodiments, the polypeptide or the expression repressor may be used in
methods of modulating, e.g.,
decreasing, gene expression, methods of treating a condition, or methods of
epigenetically modifying a
target gene, e.g., a transcription control element described herein, e.g., in
place of an expression
repression system. In some embodiments, an expression repression system
comprises two or more (e.g.,
two, three, or four) expression repressors, wherein the first expression
repressor comprises a repressor
domain comprising the EZH2 sequence of SEQ ID NO: 65, or a functional variant
or fragment thereof.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) targeting moiety and one or more effector
moiety, wherein the effector
moiety is or comprises FOG1 e.g., as according to NP_722520.2 or the protein
encoded by NM_153813.3
or a functional variant or fragment thereof. In some embodiments, FOG1
comprises an amino acid
sequence of SEQ ID NO: 66:
SRRKQSNPRQIKRSLGDMEAREEVQLVGASHMEQKATAPEAPSP (SEQ ID NO: 66)
In some embodiments, the FOG1 repressor domain is encoded by a nucleotide
sequence of SEQ
ID NO: 56. In some embodiments, a nucleotide sequence described herein
comprises a sequence of SEQ
ID NO: 56 or a sequence with at least 80, 85, 90, 95, 99, or 100% identity
thereto, or having no more than
20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or 1
positions of difference thereto.
AGCAGAAGGAAGCAGAGCAACCCCAGACAAATCAAGAGATCTCTGGGCGACATGGAGGCC
AGAGAGGAAGTGCAGCTGGTGGGCGCCAGCCACATGGAGCAGAAGGCTACAGCCCCCGAG
GCCCCCAGCCCC (SEQ ID NO: 56)
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
In some embodiments, FOG1 for use in a polypeptide or an expression repressor
described herein
is a variant, e.g., comprising one or more mutations, relative to the FOG 1
sequence of SEQ ID NO: 66.
In some embodiments, an FOG1 variant comprises one or more amino acid
substitutions, deletions, or
insertions relative to SEQ ID NO: 66.
5
In some embodiments, the polypeptide or the expression repressor is a fusion
protein comprising a
repressor domain that is or comprises FOG1 and a DNA-targeting moiety. In some
embodiments, the
polypeptide or the expression repressor comprises an additional moiety
described herein. In some
embodiments, the polypeptide or the expression repressor decreases expression
of a target gene. In some
10 embodiments, the polypeptide or the expression repressor may be used in
methods of modulating, e.g.,
decreasing, gene expression, methods of treating a condition, or methods of
epigenetically modifying a
target gene, e.g., a transcription control element described herein, e.g., in
place of an expression
repression system. In some embodiments, an expression repression system
comprises two or more (e.g.,
two, three, or four) expression repressors, wherein the first expression
repressor comprises a repressor
15 domain comprising the FOG1 sequence of SEQ ID NO: 66, or a functional
variant or fragment thereof.
In some embodiments, provided technologies are described as comprising a gRNA
that
specifically targets a target gene. In some embodiments, the target gene is an
oncogene, a tumor
suppressor, or a MYC mis-regulation disorder related gene. In some
embodiments, the target gene is
20 MYC. In some embodiments, the target gene is an MHC class I molecule,
e.g., I32M. In some
embodiments, the target gene encodes a heat shock protein, e.g., HSPA1B. In
some embodiments, the
target gene is a transcription factor, e.g., GATAl.
In some embodiments, technologies provided herein include methods of
delivering one or more
25 expression repressors or expression repression systems described herein
to a subject, e.g., to a nucleus of
a cell or tissue of a subject, by linking such a moiety to a DNA-targeting
moiety as part of a fusion
molecule.
In some embodiments, an expression repressor comprises a nuclear localization
sequence (NLS).
30 In some embodiments, the expression repressor comprises an NLS, e.g., an
5V40 NLS at the N-terminus.
In some embodiments, the expression repressor comprises an NLS, e.g., a
nucleoplasmin NLS at the C-
terminus. In some embodiments, the expression repressor comprises a first NLS
at the N-terminus and a
second NLS at the C-terminus. In some embodiments the first and the second NLS
have the same
sequence. In some embodiments, the first and the second NLS have different
sequences. In some
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
86
embodiments, the expression repression repressor comprises an SV40 NLS, e.g.,
the expression repressor
comprises a sequence according to PKKKRK (SEQ ID NO: 86). In some embodiments,
the expression
repressor comprises an epitope tag, e.g., an HA tag: YPYDVPDYA (SEQ ID NO:
80). For example, the
expression repressor may comprise two copies of the epitope tag.
While an epitope tag is useful in many research contexts, it is sometimes
desirable to omit an
epitope tag in a therapeutic context. Accordingly, in some embodiments, the
expression repressor lacks
an epitope tag. In some embodiments, an expression repressor described herein
comprises a sequence
provided herein (or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having no
more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2,
or 1 positions of difference
thereto), but lacking the HA tag of SEQ ID NO: 80. In some embodiments, a
nucleic acid described
herein comprises a sequence provided herein (or a sequence with at least 80,
85, 90, 95, 99, or 100%
identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12,
11, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto), but lacking a region encoding the HA tag of
SEQ ID NO: 80. In some
embodiments, the expression repressor comprises a nucleoplasmin NLS, e.g., the
expression repressor
comprises a sequence of KRPAATKKAGQAKKK (SEQ ID NO: 87). In some embodiments,
the
expression repressor does not comprise an NLS. In some embodiments, the
expression repressor does not
comprise an epitope tag. In some embodiments the expression repressor does not
comprise an HA tag. In
some embodiments, the expression repressor does not comprise an HA tag
sequence according to SEQ ID
NO: 80.
DNA-Targeting Moieties
DNA-targeting moieties may specifically bind a DNA sequence, e.g., a DNA
sequence associated
with a target gene, e.g., binds, a genomic sequence element (e.g., a promoter,
a TSS, or an anchor
sequence) in, proximal to, and/or operably linked to a target gene. Any
molecule or compound that
specifically binds a DNA sequence may be used as a DNA-targeting moiety.
In some embodiments, a DNA-targeting moiety targets, e.g., binds, a component
of a genomic
complex (e.g., ASMC). In some embodiments, a DNA-targeting moiety targets,
e.g., binds, an expression
control sequence (e.g., a promoter or enhancer) operably linked to a target
gene. In some embodiments, a
DNA-targeting moiety targets, e.g., binds, a target gene, or a part of a
target gene. The target of a DNA-
targeting moiety may be referred to as its targeted component. A targeted
component may be any
genomic sequence element operably linked to a target gene, or the target gene
itself, including but not
limited to a promoter, enhancer, anchor sequence, exon, intron, UTR encoding
sequence, a splice site, or
a transcription start site. In some embodiments, a DNA-targeting moiety binds
specifically to one or more
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
87
target anchor sequences (e.g., within a cell) and not to non-targeted anchor
sequences (e.g., within the
same cell).
In some embodiments, a DNA-targeting moiety may be or comprise a CRISPR/Cas
molecule, a
TAL effector molecule, a Zn finger domain, peptide nucleic acid (PNA) or a
nucleic acid molecule. In
some embodiments, an expression repressor comprises one DNA-targeting moiety.
In some
embodiments, an expression repression system comprises a plurality of
expression repressors, wherein
each member of the plurality of expression repressors comprises a DNA-
targeting moiety, wherein each
DNA-targeting moiety does not detectably bind, e.g., does not bind, to another
DNA-targeting moiety. In
some embodiments, an expression repression system comprises a first expression
repressor comprising a
first DNA-targeting moiety and a second expression repressor comprising a
second DNA-targeting
moiety, wherein the first DNA-targeting moiety does not detectably bind, e.g.,
does not bind, to the
second DNA-targeting moiety. In some embodiments, an expression repression
system comprises a first
expression repressor comprising a first DNA-targeting moiety and a second
expression repressor
comprising a second DNA-targeting moiety, wherein the first DNA-targeting
moiety does not detectably
bind, e.g., does not bind, to another first DNA-targeting moiety, and the
second DNA-targeting moiety
does not detectably bind, e.g., does not bind, to another second DNA-targeting
moiety. In some
embodiments, a DNA-targeting moiety for use in the compositions and methods
described herein is
functional (e.g., binds to a DNA sequence) in a monomeric, e.g., non-dimeric,
state.
In some embodiments, binding of a targeting moiety to a targeted component
decreases binding
affinity of the targeted component for another transcription factor, genomic
complex component, or
genomic sequence element. In some embodiments, a DNA-targeting moiety binds to
its target sequence
with a KD of less than or equal to 500, 450, 400, 350, 300, 250, 200, 150,
100, 50, 40, 30, 20, 10, 9, 8, 7,
6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08,
0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01,
0.005, 0.002, or 0.001 nM (and optionally, a KD of at least 50, 40, 30, 20,
10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9,
0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04,
0.03, 0.02, 0.01, 0.005, 0.002, or
0.001 nM). In some embodiments, a DNA-targeting moiety binds to its target
sequence with a KD of
0.001 nM to 500 nM, e.g., 0.1 nM to 5 nM, e.g., about 0.5 nM. In some
embodiments, a DNA-targeting
moiety binds to a non-target sequence with a KD of at least 500, 600, 700,
800, 900, 1000, 2000, 5000,
10,000, or 100,000 nM (and optionally, does not appreciably bind to a non-
target sequence). In some
embodiments, a DNA-targeting moiety does not bind to a non-target sequence.
In some embodiments, a DNA-targeting moiety comprises a nucleic acid sequence
complementary to a targeted component, e.g., a promoter of a target gene. In
some embodiments, a
targeting moiety comprises a nucleic acid sequence that is at least 10%, 15%,
20%, 25%, 30%, 35%,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
88
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%
complementary to a
targeted component.
In some embodiments, the DNA-targeting moiety of an expression repressor
comprises no more
than 100, 90, 80, 70, 60, 50, 40, 30, or 20 nucleotides (and optionally at
least 10, 20, 30, 40, 50, 60, 70,
80, or 90 nucleotides). In some embodiments, an expression repressor or a
repressor domain of a fusion
molecule, comprises no more than 2000, 1900, 1800, 1700, 1600, 1500, 1400,
1300, 1200, 1100, 1000,
900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids (and optionally at
least 50, 100, 200, 300, 400,
500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,
or 1900 amino acids). In
some embodiments, an expression repressor or the effector moiety of a fusion
molecule, comprises 100-
2000, 100-1900, 100-1800, 100-1700, 100-1600, 100-1500, 100-1400, 100-1300,
100-1200, 100-1100,
100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-
200, 200-2000, 200-
1900, 200-1800, 200-1700, 200-1600, 200-1500, 200-1400, 200-1300, 200-1200,
200-1100, 200-1000,
200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-2000, 300-
1900, 300-1800, 300-
1700, 300-1600, 300-1500, 300-1400, 300-1300, 300-1200, 300-1100, 300-1000,
300-900, 300-800, 300-
700, 300-600, 300-500, 300-400, 400-2000, 400-1900, 400-1800, 400-1700, 400-
1600, 400-1500, 400-
1400, 400-1300, 400-1200, 400-1100, 400-1000, 400-900, 400-800, 400-700, 400-
600, 400-500, 500-
2000, 500-1900, 500-1800, 500-1700, 500-1600, 500-1500, 500-1400, 500-1300,
500-1200, 500-1100,
500-1000, 500-900, 500-800, 500-700, 500-600, 600-2000, 600-1900, 600-1800,
600-1700, 600-1600,
600-1500, 600-1400, 600-1300, 600-1200, 600-1100, 600-1000, 600-900, 600-800,
600-700, 700-2000,
700-1900, 700-1800, 700-1700, 700-1600, 700-1500, 700-1400, 700-1300, 700-
1200, 700-1100, 700-
1000, 700-900, 700-800, 800-2000, 800-1900, 800-1800, 800-1700, 800-1600, 800-
1500, 800-1400, 800-
1300, 800-1200, 800-1100, 800-1000, 800-900, 900-2000, 900-1900, 900-1800, 900-
1700, 900-1600,
900-1500, 900-1400, 900-1300, 900-1200, 900-1100, 900-1000, 1000-2000, 1000-
1900, 1000-1800,
1000-1700, 1000-1600, 1000-1500, 1000-1400, 1000-1300, 1000-1200, 1000-1100,
1100-2000, 1100-
1900, 1100-1800, 1100-1700, 1100-1600, 1100-1500, 1100-1400, 1100-1300, 1100-
1200, 1200-2000,
1200-1900, 1200-1800, 1200-1700, 1200-1600, 1200-1500, 1200-1400, 1200-1300,
1300-2000, 1300-
1900, 1300-1800, 1300-1700, 1300-1600, 1300-1500, 1300-1400, 1400-2000, 1400-
1900, 1400-1800,
1400-1700, 1400-1600, 1400-1500, 1500-2000, 1500-1900, 1500-1800, 1500-1700,
1500-1600, 1600-
2000, 1600-1900, 1600-1800, 1600-1700, 1700-2000, 1700-1900, 1700-1800, 1800-
2000, 1800-1900, or
1900-2000 amino acids.
An expression repressor or an expression repression system as disclosed
herein, may comprise
nucleic acid, e.g., one or more nucleic acids. In some embodiments, a nucleic
acid is or comprises RNA;
in some embodiments, a nucleic acid is or comprises DNA. In some embodiments,
a nucleic acid is or
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
89
comprises more than 50% ribonucleotides. In some embodiments, a nucleic acid
is, comprises, or consists
of one or more natural nucleic acid residues. In some embodiments, a nucleic
acid is, comprises, or
consists of one or more nucleic acid analogs. In some embodiments, a nucleic
acid analog differs from a
nucleic acid in that it does not utilize a phosphodiester backbone. For
example, in some embodiments, a
nucleic acid is, comprises, or consists of one or more peptide nucleic acids.
Alternatively, or additionally,
in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-
N-phosphoramidite
linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid
is, comprises, or
consists of one or more natural nucleosides (e.g., adenosine, thymidine,
guanosine, cytidine, uridine,
deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some
embodiments, a nucleic
.. acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-
aminoadenosine, 2-
thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-
methylcytidine, C-5 propynyl-
cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-
fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-
aminoadenosine, 7-deazaadenosine, 7-
deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-
thiocytidine, methylated bases,
intercalated bases, and combinations thereof). In some embodiments, a nucleic
acid comprises one or
more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose,
arabinose, and hexose) as compared
with those in natural nucleic acids. In some embodiments, a nucleic acid has a
nucleotide sequence that
encodes a functional gene product such as an RNA or protein. In some
embodiments, a nucleic acid
includes one or more introns. In some embodiments, nucleic acids are prepared
by one or more of
.. isolation from a natural source, enzymatic synthesis by polymerization
based on a complementary
template (in vivo or in vitro), reproduction in a recombinant cell or system,
and chemical synthesis. In
some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
20, 225, 250, 275, 300, 325,
350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,
3000, 3500, 4000, 4500,
5000 or more residues long. In some embodiments, nucleic acids may have a
length from about 2 to
about 5000 nts, about 10 to about 100 nts, about 50 to about 150 nts, about
100 to about 200 nts, about
150 to about 250 nts, about 200 to about 300 nts, about 250 to about 350 nts,
about 300 to about 500 nts,
about 10 to about 1000 nts, about 50 to about 1000 nts, about 100 to about
1000 nts, about 1000 to about
2000 nts, about 2000 to about 3000 nts, about 3000 to about 4000 nts, about
4000 to about 5000 nts, or
any range therebetween. In some embodiments, a nucleic acid is partly or
wholly single stranded; in some
embodiments, a nucleic acid is partly or wholly double stranded. In some
embodiments a nucleic acid has
a nucleotide sequence comprising at least one element that encodes, or is the
complement of a sequence
that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic
activity.
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
In some embodiments, a DNA-targeting moiety comprises or is nucleic acid.
In some embodiments, a nucleic acid that may be included in a moiety may be or
comprise DNA,
RNA, and/or an artificial or synthetic nucleic acid or nucleic acid analog or
mimic. For example, in some
embodiments, a nucleic acid may be or include one or more of genomic DNA
(gDNA), complementary
5 DNA (cDNA), a peptide nucleic acid (PNA), a peptide-nucleic acid mixmer,
a peptide- oligonucleotide
conjugate, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a
polyamide, a triplex- forming
oligonucleotide, an antisense oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA,
siRNA or other
RNAi molecule (e.g., that targets a non-coding RNA as described herein and/or
that targets an expression
product of a particular gene associated with a targeted genomic complex as
described herein), etc. A
10 nucleic acid sequence may include modified oligonucleotides (e.g.,
chemical modifications, such as
modifications that alter backbone linkages, sugar molecules, and/or nucleic
acid bases) and/or artificial
nucleic acids. In some embodiments, a nucleic acid sequence includes, but is
not limited to, genomic
DNA, cDNA, peptide nucleic acids (PNA) or peptide oligonucleotide conjugates,
locked nucleic acids
(LNA), bridged nucleic acids (BNA), polyamides, triplex forming
oligonucleotides, modified DNA,
15 antisense DNA oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA,
gRNA, and siRNA or
other RNA or DNA molecules. In some embodiments, a nucleic acid may include
one or more residues
that is not a naturally occurring DNA or RNA residue, may include one or more
linkages that is/are not
phosphodiester bonds (e.g., that may be, for example, phosphorothioate bonds,
etc.), and/or may include
one or more modifications such as, for example, a 2'0 modification such as 2'-
0MeP. A variety of
20 nucleic acid structures useful in preparing synthetic nucleic acids is
known in the art (see, for example,
W02017/0628621 and W02014/012081) those skilled in the art will appreciate
that these may be utilized
in accordance with the present disclosure.
In some embodiments, a nucleic acid described herein comprises one or more
nucleoside analogs.
In some embodiments, a nucleic acid sequence may include in addition or as an
alternative to one or more
25 natural nucleosides, e.g., purines or pyrimidines, e.g., adenine,
cytosine, guanine, thymine, and uracil, one
or more nucleoside analogs. In some embodiments, a nucleic acid sequence
includes one or more
nucleoside analogs. A nucleoside analog may include, but is not limited to, a
nucleoside analog, such as
5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 4-
methylbenzimidazole, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethy1-2-thiouridine, 5-
30 carboxymethylaminomethyluracil, dihydrouracil, dihydrouridine, beta-D-
galactosylqueosine, inosine, N6-
isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-
methyladenine, 2-
methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine, 5-
methylaminomethyluracil, 5-methoxyaminomethy1-2-thiouracil, beta-D-
mannosylqueosine, 5'-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine, uracil-5-oxyacetic
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
91
acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-
thiouracil, 2-thiouracil, 4-
thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-
oxyacetic acid (v), 5-methy1-2-
thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-
diaminopurine, 3-nitropyrrole, inosine,
thiouridine, queuosine, wyosine, diaminopurine, isoguanine, isocytosine,
diaminopyrimidine, 2,4-
difluorotoluene, isoquinoline, pyrrolo[2,3-13]pyridine, and any others that
can base pair with a purine or a
pyrimidine side chain.
CRISPR/Cas Domains
In some embodiments, a DNA-targeting moiety is or comprises a CRISPR/Cas
molecule. A
CRISPR/Cas molecule comprises a protein involved in the clustered regulatory
interspaced short
palindromic repeat (CRISPR) system, e.g., a Cas protein, and optionally a
guide RNA, e.g., single guide
RNA (sgRNA). In some embodiments, the gRNA comprised by the CRISPR/Cas
molecule is
noncovalently bound by the CRISPR/Cas protein.
CRISPR systems are adaptive defense systems originally discovered in bacteria
and archaea.
CRISPR systems use RNA-guided nucleases termed CRISPR-associated or "Cas"
endonucleases (e. g.,
Cas9 or Cpfl) to cleave foreign DNA. For example, in a typical CRISPR/Cas
system, an endonuclease is
directed to a target nucleotide sequence (e. g., a site in the genome that is
to be sequence-edited) by
sequence-specific, non-coding "guide RNAs" that target single- or double-
stranded DNA sequences.
Three classes (I-III) of CRISPR systems have been identified. The class II
CRISPR systems use a single
Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR
system includes a type II Cas
endonuclease such as Cas9, a CRISPR RNA ("crRNA"), and a trans-activating
crRNA ("tracrRNA").
The crRNA contains a "guide RNA", typically about 20-nucleotide RNA sequence
that corresponds to a
target DNA sequence. crRNA also contains a region that binds to the tracrRNA
to form a partially
double-stranded structure which is cleaved by RNase III, resulting in a
crRNA/tracrRNA hybrid. A
.. crRNA/tracrRNA hybrid then directs Cas9 endonuclease to recognize and
cleave a target DNA sequence.
A target DNA sequence must generally be adjacent to a "protospacer adjacent
motif' ("PAM") that is
specific for a given Cas endonuclease; however, PAM sequences appear
throughout a given genome.
CRISPR endonucleases identified from various prokaryotic species have unique
PAM sequence
requirements; examples of PAM sequences include 5'-NGG (Streptococcus
pyogenes), 5'-NNAGAA
(Streptococcus thermophilus CRISPR1), 5' -NGGNG (Streptococcus thermophilus
CRISPR3), and 5'-
NNNGATT (Neisseria meningiditis). Some endonucleases, e.g., Cas9
endonucleases, are associated with
G-rich PAM sites, e. g., 5'-NGG, and perform blunt-end cleaving of the target
DNA at a location 3
nucleotides upstream from (5' from) the PAM site. Another class II CRISPR
system includes the type V
endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from
Acidaminococcus sp.)
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
92
and LbCpfl (from Lachnospiraceae sp.). Cpfl-associated CRISPR arrays are
processed into mature
crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system
requires only Cpfl
nuclease and a crRNA to cleave a target DNA sequence. Cpfl endonucleases, are
associated with T-rich
PAM sites, e. g., 5'-TTN. Cpfl can also recognize a 5'-CTA PAM motif. Cpfl
cleaves a target DNA by
introducing an offset or staggered double-strand break with a 4- or 5-
nucleotide 5' overhang, for example,
cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18
nucleotides downstream
from (3' from) from a PAM site on the coding strand and 23 nucleotides
downstream from the PAM site
on the complimentary strand; the 5-nucleotide overhang that results from such
offset cleavage allows
more precise genome editing by DNA insertion by homologous recombination than
by insertion at blunt-
end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759 ¨ 771.
A variety of CRISPR associated (Cas) genes or proteins can be used in the
technologies provided
by the present disclosure and the choice of Cas protein will depend upon the
particular conditions of the
method. Specific examples of Cas proteins include class II systems including
Casl, Cas2, Cas3, Cas4,
Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpfl, C2C1, or C2C3. In some embodiments,
a Cas protein, e.g., a
Cas9 protein, may be from any of a variety of prokaryotic species. In some
embodiments a particular Cas
protein, e.g., a particular Cas9 protein, is selected to recognize a
particular protospacer-adjacent motif
(PAM) sequence. In some embodiments, a DNA-targeting moiety includes a
sequence targeting
polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments a Cas
protein, e.g., a Cas9 protein,
may be obtained from a bacteria or archaea or synthesized using known methods.
In certain embodiments,
a Cas protein may be from a gram-positive bacterium or a gram-negative
bacterium. In certain
embodiments, a Cas protein may be from a Streptococcus (e.g., S. pyogenes, or
a S. thermophilus), a
Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an
Acidaminococcus (e.g., an
Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a
Cryptococcus, a Corynebacterium,
a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a
Marinobacter.
.. In some embodiments, a Cas protein requires a protospacer adjacent motif
(PAM) to be present in or
adjacent to a target DNA sequence for the Cas protein to bind and/or function.
In some embodiments, the
PAM is or comprises, from 5' to 3', NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV,
NTTN, or
NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for
A or G, and V stands
for A or C or G. In some embodiments, a Cas protein is a protein listed in
Table 1. In some
embodiments, a Cas protein comprises one or more mutations altering its PAM.
In some embodiments, a
Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous
substitutions to the amino
acids corresponding to said positions. In some embodiments, a Cas protein
comprises E782K, N968K,
and R1015H mutations or analogous substitutions to the amino acids
corresponding to said positions. In
some embodiments, a Cas protein comprises D1135V, R1335Q, and T1337R mutations
or analogous
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
93
substitutions to the amino acids corresponding to said positions. In some
embodiments, a Cas protein
comprises S542R and K607R mutations or analogous substitutions to the amino
acids corresponding to
said positions. In some embodiments, a Cas protein comprises S542R, K548V, and
N552R mutations or
analogous substitutions to the amino acids corresponding to said positions.
Table 1
Name Enzym Species # of PAM Mutations to alter Mutations
to make
AAs PAM recognition catalytically
dead
FnCas Cas9 Francisella 1629 5'-NGG- Wt
D11A/H969A/N995A
9 novicida 3'
FnCas Cas9 Francisella 1629 5'-YG-3' E1369R/E1449H/R1 D11A/H969A/N995A
9 RHA novicida 556A
SaCas Cas9 Staphylococc 1053 5'- Wt D1OA/H557A
9 us aureus NNGRR
T-3'
SaCas Cas9 Staphylococc 1053 5'- E782K/N968K/R10 D1OA/H557A
9 us aureus NNNRR 15H
KKH T-3'
SpCas Cas9 Streptococcu 1368 5'-NGG- Wt
D1OA/D839A/H840A/
9 s pyogenes 3' N863A
SpCas Cas9 Streptococcu 1368 5'-NGA- D1135V/R1335Q/T D1OA/D839A/H840A/
9 VQR s pyogenes 3' 1337R N863A
AsCpf Cpfl Acidaminoco 1307 5'-TYCV- S542R/K607R E993A
1 RR ccus sp. 3'
BV3L6
AsCpf Cpfl Acidaminoco 1307 5'-TATV- S542R/K548V/N552 E993A
1 RVR ccus sp. 3'
BV3L6
FnCpf Cpfl Francisella 1300 5'-NTTN- Wt
D917A/E1006A/D1255
1 novicida 3' A
NmCa Cas9 Neisseria 1082 5'- Wt
D16A/D587A/H588A/
s9 meningitidis NNNGA N611A
TT-3'
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
94
In some embodiments, the Cas protein is modified to deactivate the nuclease,
e.g., nuclease
deficient Cas9. Whereas wild-type Cas9 generates double-strand breaks (DSBs)
at specific DNA
sequences targeted by a gRNA, a number of CRISPR endonucleases having modified
functionalities are
available, for example: a "nickase" version of Cas9 generates only a single-
strand break; a catalytically
inactive Cas9 ("dCas9") does not cut target DNA. In some embodiments, dCas9
binding to a DNA
sequence may interfere with transcription at that site by steric hindrance. In
some embodiments, a DNA-
targeting moiety is or comprises a catalytically inactive Cas9, e.g., dCas9.
In some embodiments, a
DNA-targeting moiety is or comprises a catalytically inactive mutant Cas9,
e.g., Cas9m4. Many
catalytically inactive Cas9 proteins are known in the art. In some
embodiments, dCas9 comprises
mutations in each endonuclease domain of the Cas protein, e.g., DlOA and H840A
mutations.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9,
comprises a DllA
mutation or an analogous substitution to the amino acid corresponding to said
position. In some
embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a
H969A mutation or an
analogous substitution to the amino acid corresponding to said position. In
some embodiments, a
catalytically inactive Cas9 protein, e.g., dCas9, comprises a N995A mutation
or an analogous substitution
to the amino acid corresponding to said position. In some embodiments, a
catalytically inactive Cas9
protein, e.g., dCas9, comprises D11A, H969A, and N995A mutations or analogous
substitutions to the
amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9,
comprises a DlOA
mutation or an analogous substitution to the amino acid corresponding to said
position. In some
embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a
H557A mutation or an
analogous substitution to the amino acid corresponding to said position. In
some embodiments, a
catalytically inactive Cas9 protein, e.g., dCas9, comprises DlOA and H557A
mutations or analogous
substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9,
comprises a D839A
mutation or an analogous substitution to the amino acid corresponding to said
position. In some
embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a
H840A mutation or an
analogous substitution to the amino acid corresponding to said position. In
some embodiments, a
catalytically inactive Cas9 protein, e.g., dCas9, comprises a N863A mutation
or an analogous substitution
to the amino acid corresponding to said position. In some embodiments, a
catalytically inactive Cas9
protein, e.g., dCas9, comprises DlOA and D839A mutations or analogous
substitutions to the amino acids
corresponding to said positions. In some embodiments, a catalytically inactive
Cas9 protein, e.g., dCas9,
comprises DlOA, D839A, and H840A mutations or analogous substitutions to the
amino acids
corresponding to said positions. In some embodiments, a catalytically inactive
Cas9 protein, e.g., dCas9,
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
comprises DlOA, D839A, H840A, and N863A mutations or analogous substitutions
to the amino acids
corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9,
comprises a E993A
mutation or an analogous substitution to the amino acid corresponding to said
position.
5 In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9,
comprises a D917A
mutation or an analogous substitution to the amino acid corresponding to said
position. In some
embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a
E1006A mutation or an
analogous substitution to the amino acid corresponding to said position. In
some embodiments, a
catalytically inactive Cas9 protein, e.g., dCas9, comprises a D1255A mutation
or an analogous
10 substitution to the amino acid corresponding to said position. In some
embodiments, a catalytically
inactive Cas9 protein, e.g., dCas9, comprises D917A, E1006A, and D1255A
mutations or analogous
substitutions to the amino acids corresponding to said positions.
In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9,
comprises a D16A
mutation or an analogous substitution to the amino acid corresponding to said
position. In some
15 embodiments, a catalytically inactive Cas9 protein, e.g., dCas9,
comprises a D587A mutation or an
analogous substitution to the amino acid corresponding to said position. In
some embodiments, a
catalytically inactive Cas9 protein, e.g., dCas9, comprises a H588A mutation
or an analogous substitution
to the amino acid corresponding to said position. In some embodiments, a
catalytically inactive Cas9
protein, e.g., dCas9, comprises a N611A mutation or an analogous substitution
to the amino acid
20 corresponding to said position. In some embodiments, a catalytically
inactive Cas9 protein, e.g., dCas9,
comprises D16A, D587A, H588A, and N611A mutations or analogous substitutions
to the amino acids
corresponding to said positions.
In another aspect, the disclosure is directed to an expression repressor or a
polypeptide
comprising one or more (e.g., one) DNA-targeting moiety and one or more
repressor domain, wherein the
25 one or more DNA-targeting moiety is or comprises a CRISPR/Cas molecule
comprising a Cas protein,
e.g., catalytically inactive Cas9 protein, e.g., dCas9, e.g., dCas9m4, or a
functional variant or fragment
thereof. In some embodiments, dCas9 comprises an amino acid sequence of SEQ ID
NO: 46 or 88:
DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA
TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVA
30 YHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY
NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDL
AEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK
RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEE
LLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
96
ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFT
VYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV
EDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK
QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVS
GQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR
ERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD INRLSDYDVAAIV
PQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAER
GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDF
QFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKAT
AKYFFYS NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWD KGRDFATVRKVLSMPQVNIV KKTE
V QTGGFSKESILPKRNS D KLIARKKDWDPKKYGGFD SPTVAYSVLVVAKVEKGKSKKLKSVKEL
LGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALP
SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAY
NKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID
LSQLGGD (SEQ ID NO: 46)
AKRNYILGLAIGIT SVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRS KRGARRLKRRR
RHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLS QKLSEEEFSAALLHLAKRRGVHNVNEVE
EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKA
YHQLD QSFIDTYIDLLETRRTYYEGPGEGS PFGWKDIKEWYEMLMGHCTYFPEELRSV KYAYNA
DLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTG
KPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKG
YTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFI
QS IKVINAIIKKYGLPND IIIELAREKNS KDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEK
IKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDAIIPRS VS FDNS FNNKVLVKQEENSKKGNRTPF
QYLSS SD S KISYETFKKHILNLAKGKGRIS KTKKEYLLEERDINRFSV QKD FINRNLVDTRYATRG
LMNLLRS YFRVNNLDVKV KS INGGFTS FLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWK
KLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLI
NDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQY
GDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKP
YRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKIN
GELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVK
SKKHPQIIKKG (SEQ ID NO: 88)
In some embodiments, the dCas9 is encoded by a nucleic acid sequence of SEQ ID
NO: 45 or 89:
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
97
GACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCA
CCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAG
CATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACC
CGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGC
AGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGA
GAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTG
GACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGG
ACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTC
CGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGT
TCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGG
CGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTG
ATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCC
TGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCT
GAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTAC
GCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCG
GGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAG
CACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACA
AGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAG
CCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAG
CTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCA
GCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTC
TACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTA
CTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAG
GAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCT
TCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCA
CAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCG
AGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCT
GTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATC
GAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCA
CCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGA
GGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAG
GAGCGGCTGAAAACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGC
GGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCA
GAGCGGCAAGACCATCCTGGACTTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGC
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
98
AGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGG
CCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAG
GGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGC
CCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGA
ACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCC
TGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCT
GCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTAC
GACGTGGCCGCCATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGC
TGACCCGGAGCGACAAGGCCCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGA
AGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTT
CGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATC
AAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCC
GGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCT
GAAATCCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATC
AACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCA
AGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCG
GAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTAC
AGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGC
GGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTT
CGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTG
CAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCG
CCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTA
CAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAA
GGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTC
CTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACA
GCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAA
GGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACG
AGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACA
AGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGC
CGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGG
GAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTT
CAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGAC
GCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCT
GGGCGGCGAC (SEQ ID NO: 45)
CA 03196827 2023-03-24
WO 2022/067033 PCT/US2021/051945
99
GCCAAGCGGAACTACATCCTGGGCCTGGCCATCGGCATCACCAGCGTGGGCTACGGCATCAT
CGACTACGAGACCCGGGACGTGATCGACGCCGGCGTGCGGCTGTTCAAGGAGGCCAACGTG
GAGAACAACGAGGGCCGGCGGAGCAAGCGGGGCGCCCGGCGGCTGAAGCGGCGGCGGCGG
CACCGGATCCAGCGGGTGAAGAAGCTGCTGTTCGACTACAACCTGCTGACCGACCACAGCG
AGCTGAGCGGCATCAACCCCTACGAGGCCCGGGTGAAGGGCCTGAGCCAGAAGCTGAGCGA
GGAGGAGTTCAGCGCCGCCCTGCTGCACCTGGCCAAGCGGCGGGGCGTGCACAACGTGAAC
GAGGTGGAGGAGGACACCGGCAACGAGCTGAGCACCAAGGAGCAGATCAGCCGGAACAGC
AAGGCCCTGGAGGAGAAGTACGTGGCCGAGCTGCAGCTGGAGCGGCTGAAGAAGGACGGC
GAGGTGCGGGGCAGCATCAACCGGTTCAAGACCAGCGACTACGTGAAGGAGGCCAAGCAGC
TGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCATCGACACCTACATCGA
CCTGCTGGAGACCCGGCGGACCTACTACGAGGGCCCCGGCGAGGGCAGCCCCTTCGGCTGG
AAGGACATCAAGGAGTGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAGC
TGCGGAGCGTGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAA
CCTGGTGATCACCCGGGACGAGAACGAGAAGCTGGAGTACTACGAGAAGTTCCAGATCATC
GAGAACGTGTTCAAGCAGAAGAAGAAGCCCACCCTGAAGCAGATCGCCAAGGAGATCCTGG
TGAACGAGGAGGACATCAAGGGCTACCGGGTGACCAGCACCGGCAAGCCCGAGTTCACCAA
CCTGAAGGTGTACCACGACATCAAGGACATCACCGCCCGGAAGGAGATCATCGAGAACGCC
GAGCTGCTGGACCAGATCGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGG
AGGAGCTGACCAACCTGAACAGCGAGCTGACCCAGGAGGAGATCGAGCAGATCAGCAACCT
GAAGGGCTACACCGGCACCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAG
CTGTGGCACACCAACGACAACCAGATCGCCATCTTCAACCGGCTGAAGCTGGTGCCCAAGA
AGGTGGACCTGAGCCAGCAGAAGGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAG
CCCCGTGGTGAAGCGGAGCTTCATCCAGAGCATCAAGGTGATCAACGCCATCATCAAGAAG
TACGGCCTGCCCAACGACATCATCATCGAGCTGGCCCGGGAGAAGAACAGCAAGGACGCCC
AGAAGATGATCAACGAGATGCAGAAGCGGAACCGGCAGACCAACGAGCGGATCGAGGAGA
TCATCCGGACCACCGGCAAGGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGA
CATGCAGGAGGGCAAGTGCCTGTACAGCCTGGAGGCCATCCCCCTGGAGGACCTGCTGAAC
AACCCCTTCAACTACGAGGTGGACGCCATCATCCCCCGGAGCGTGAGCTTCGACAACAGCTT
CAACAACAAGGTGCTGGTGAAGCAGGAGGAGAACAGCAAGAAGGGCAACCGGACCCCCTT
CCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAGACCTTCAAGAAGCACATCCTG
AACCTGGCCAAGGGCAAGGGCCGGATCAGCAAGACCAAGAAGGAGTACCTGCTGGAGGAG
CGGGACATCAACCGGTTCAGCGTGCAGAAGGACTTCATCAACCGGAACCTGGTGGACACCC
GGTACGCCACCCGGGGCCTGATGAACCTGCTGCGGAGCTACTTCCGGGTGAACAACCTGGA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
100
CGTGAAGGTGaaatccATCAACGGCGGCTTCACCAGCTTCCTGCGGCGGAAGTGGAAGTTCAAG
AAGGAGCGGAACAAGGGCTACAAGCACCACGCCGAGGACGCCCTGATCATCGCCAACGCCG
ACTTCATCTTCAAGGAGTGGAAGAAGCTGGACAAGGCCAAGAAGGTGATGGAGAACCAGAT
GTTCGAGGAGAAGCAGGCCGAGAGCATGCCCGAGATCGAGACCGAGCAGGAGTACAAGGA
GATCTTCATCACCCCCCACCAGATCAAGCACATCAAGGACTTCAAGGACTACAAGTACAGCC
ACCGGGTGGACAAGAAGCCCAACCGGAAGCTGATCAACGACACCCTGTACAGCACCCGGAA
GGACGACAAGGGCAACACCCTGATCGTGAACAACCTGAACGGCCTGTACGACAAGGACAAC
GACAAGCTGAAGAAGCTGATCAACAAGAGCCCCGAGAAGCTGCTGATGTACCACCACGACC
CCCAGACCTACCAGAAGCTGAAGCTGATCATGGAGCAGTACGGCGACGAGAAGAACCCCCT
GTACAAGTACTACGAGGAGACCGGCAACTACCTGACCAAGTACAGCAAGAAGGACAACGGC
CCCGTGATCAAGAAGATCAAGTACTACGGCAACAAGCTGAACGCCCACCTGGACATCACCG
ACGACTACCCCAACAGCCGGAACAAGGTGGTGAAGCTGAGCCTGAAGCCCTACCGGTTCGA
CGTGTACCTGGACAACGGCGTGTACAAGTTCGTGACCGTGAAGAACCTGGACGTGATCAAG
AAGGAGAACTACTACGAGGTGAACAGCAAGTGCTACGAGGAGGCCAAGAAGCTGAAGAAG
ATCAGCAACCAGGCCGAGTTCATCGCCAGCTTCTACAAGAACGACCTGATCAAGATCAACG
GCGAGCTGTACCGGGTGATCGGCGTGAACAACGACCTGCTGAACCGGATCGAGGTGAACAT
GATCGACATCACCTACCGGGAGTACCTGGAGAACATGAACGACAAGCGGCCCCCCCACATC
ATCAAGACCATCGCCAGCAAGACCCAGAGCATCAAGAAGTACAGCACCGACATCCTGGGCA
ACCTGTACGAGGTGaaatccAAGAAGCACCCCCAGATCATCAAGAAGGGC (SEQ ID NO: 89)
Guide RNA (gRNA)
In some embodiments, a DNA-targeting moiety may comprise a Cas molecule
comprising or linked (e.g.,
covalently) to a gRNA. A gRNA is a short synthetic RNA composed of a
"scaffold" sequence necessary
for Cas-protein binding and a user-defined ¨20 nucleotide targeting sequence
for a genomic target. In
practice, guide RNA sequences are generally designed to have a length of
between 17 ¨ 24 nucleotides
(e.g., 19, 20, or 21 nucleotides) and be complementary to the targeted nucleic
acid sequence. Custom
gRNA generators and algorithms are available commercially for use in the
design of effective guide
RNAs. Gene editing has also been achieved using a chimeric "single guide RNA"
("sgRNA"), an
engineered (synthetic) single RNA molecule that mimics a naturally occurring
crRNA-tracrRNA complex
and contains both a tracrRNA (for binding the nuclease) and at least one crRNA
(to guide the nuclease to
the sequence targeted for editing). Chemically modified sgRNAs have also been
demonstrated to be
effective for use with Cas proteins; see, for example, Hendel et al. (2015)
Nature Biotechnol., 985 ¨991.
In some embodiments, a gRNA comprises a nucleic acid sequence that is
complementary to a
DNA sequence associated with a target gene. In some embodiments, the DNA
sequence is, comprises, or
overlaps an expression control element that is operably linked to the target
gene. In some embodiments, a
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
101
gRNA comprises a nucleic acid sequence that is at least 90, 95, 99, or 100%
complementary to a DNA
sequence associated with a target gene. In some embodiments, a gRNA for use
with a DNA-targeting
moiety that comprises a Cas molecule is an sgRNA.
In some embodiments, a gRNA for use with a CRISPR/Cas molecule specifically
binds a target
sequence associated with I3-2-microglobulin expression. Such a gRNA may
comprise a target-binding
sequence selected from:
GD-28228: TCTCCTTGGTGGCCCGCCGT (SEQ ID NO: 1),
GD-28229: GTCCCAAAGGCGCGGCGCTG (SEQ ID NO: 2),
GD-28171: CCCTGCTCCCCGCCGAAAGGG (SEQ ID NO: 3),
GD-28172: CTCTGGCTCCCCCAGCGCAGC (SEQ ID NO: 4), or
GD-28173: GTGAACGCGTGGAGGGGCGCT (SEQ ID NO: 5).
In some embodiments, a gRNA for use with a CRISPR/Cas domain specifically
binds a target sequence
associated with CTCF. In some embodiments, a gRNA for use with a CRISPR/Cas
domain specifically
binds a target sequence associated with the promoter. In some embodiments the
gRNA binds a target
sequence listed in Table 3.
Table 3: Exemplary gRNA sequences
Guide Target Target sequence Genomic SEQ ID NO:
Coordinates
GD- MYC chr8:128746082- 11
29639 AGGGTGATGTTCATTAGCAG 128746104
GD- MYC chr8:128746177- 12
29640 TGCAGAAGGTCCGAAGAAAG 128746199
GD- I32M chr15:45003632- 13
27634 GAGACAGGTGACGGTCCCTG 45003654
GD- I32M chr15:45003545- 14
29500 GAGTCTCGTGATGTTTAAGA 45003567
GD- HSPA1B chr6:31795352- 17
29542 CCTGCCCTCTGATTGGTCCA 31795374
GD- HSPA1B chr6:31795429- 18
29544 GCCTCATCGAGCTTGGTGAT 31795451
GD- GATA1 chrX:48641126- 19
29536 CTGTGGTGTCAAAAGCACCA 48641148
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
102
GD- GATA1 chrX:48641245- 20
29693 GAACCCCAGAAGATGCCAGG 48641267
GD- I32M GGCGCGCACCCCAGATCGGA chr15 :45003481- 21
29499 promoter 45003503
In some embodiments, an expression repressor system comprises a first
expression repressor
comprising a first DNA-targeting moiety and a second expression repressor
comprising a second DNA-
targeting moiety, wherein the first DNA-targeting moiety comprises or is a
first CRISPR/Cas molecule
and the second DNA-targeting moiety comprises or is a second CRISPR/Cas
molecule. In some
embodiments, the first CRISPR/Cas molecule comprises a first CRISPR/Cas
protein and first guide RNA,
and the second CRISPR/Cas molecule comprises a second CRISPR/Cas protein and a
second guide RNA.
In some embodiments, the first CRISPR/Cas protein does not appreciably bind
(e.g., does not bind) the
second guide RNA, e.g., binds with a KD of at least 10, 20, 50, 100, 1000, or
10,000 nM, and the second
.. CRISPR/Cas protein does not appreciably bind (e.g., does not bind) the
first guide RNA, e.g., binds with
a KD of at least 10, 20, 50, 100, 1000, or 10,000 nM.
TAL effector domains
In some embodiments, a DNA-targeting moiety is or comprises a TAL effector
molecule. A TAL
effector molecule, e.g., a TAL effector molecule that specifically binds a DNA
sequence, comprises a
plurality of TAL effector domains or fragments thereof, and optionally one or
more additional portions of
naturally occurring TAL effectors (e.g., N- and/or C-terminal of the plurality
of TAL effector domains).
Many TAL effectors are known to those of skill in the art and are commercially
available, e.g., from
Thermo Fisher Scientific.
TALEs are natural effector proteins secreted by numerous species of bacterial
pathogens
including the plant pathogen Xanthomonas which modulates gene expression in
host plants and facilitates
bacterial colonization and survival. The specific binding of TAL effectors is
based on a central repeat
domain of tandemly arranged nearly identical repeats of typically 33 or 34
amino acids (the repeat-
variable di-residues, RVD domain).
Members of the TAL effectors family differ mainly in the number and order of
their repeats. The
number of repeats ranges from 1.5 to 33.5 repeats and the C-terminal repeat is
usually shorter in length
(e.g., about 20 amino acids) and is generally referred to as a "half-repeat".
Each repeat of the TAL
effector feature a one-repeat-to-one-base-pair correlation with different
repeat types exhibiting different
base-pair specificity (one repeat recognizes one base-pair on the target gene
sequence). Generally, the
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
103
smaller the number of repeats, the weaker the protein-DNA interactions. A
number of 6.5 repeats has
been shown to be sufficient to activate transcription of a reporter gene
(Scholze et al., 2010).
Repeat to repeat variations occur predominantly at amino acid positions 12 and
13, which have
therefore been termed "hypervariable" and which are responsible for the
specificity of the interaction with
the target DNA promoter sequence, as shown in Table 2 listing exemplary repeat
variable di-residues
(RVD) and their correspondence to nucleic acid base targets.
Table 2 ¨ RVDs and Nucleic Acid Base Specificity
Target Possible RVD Amino Acid Combinations
A NI NN CI HI KI
NN GN SN VN LN DN QN EN HN RH NK AN FN
HD RD KD ND AD
NG HG VG IG EG MG YG AA EP VA QG KG RG
Accordingly, it is possible to modify the repeats of a TAL effector to target
specific DNA sequences.
Further studies have shown that the RVD NK can target G. Target sites of TAL
effectors also tend to
include a T flanking the 5' base targeted by the first repeat, but the exact
mechanism of this recognition is
not known. More than 113 TAL effector sequences are known to date. Non-
limiting examples of TAL
effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXa10 and AvrB
s3.
Accordingly, the TAL effector domain of the TAL effector molecule of the
present invention may
be derived from a TAL effector from any bacterial species (e.g., Xanthomonas
species such as the African
strain of Xanthomonas myzae pv. Oiyzae (Yu et al. 2011), Xanthomonas
campestris pv. raphani strain
756C and Xanthomonas myzae pv. olyzico/astrain BL5256 (Bogdanove et al. 2011).
As used herein, the
TAL effector domain in accordance with the present invention comprises an RVD
domain as well as
flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of
the RVD domain) also from
the naturally occurring TAL effector. It may comprise more or fewer repeats
than the RVD of the
naturally occurring TAL effector. The TAL effector molecule of the present
invention is designed to
target a given DNA sequence based on the above code and others known in the
art. The number of TAL
effector domains (e.g., repeats (monomers or modules)) and their specific
sequence are selected based on
the desired DNA target sequence. For example, TAL effector domains, e.g.,
repeats, may be removed or
added in order to suit a specific target sequence. In an embodiment, the TAL
effector molecule of the
present invention comprises between 6.5 and 33.5 TAL effector domains, e.g.,
repeats. In an embodiment,
TAL effector molecule of the present invention comprises between 8 and 33.5
TAL effector domains,
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
104
e.g., repeats, e.g., between 10 and 25 TAL effector domains, e.g., repeats,
e.g., between 10 and 14 TAL
effector domains, e.g., repeats.
In some embodiments, the TAL effector molecule comprises TAL effector domains
that
correspond to a perfect match to the DNA target sequence. In some embodiments,
a mismatch between a
repeat and a target base-pair on the DNA target sequence is permitted as along
as it allows for the
function of the expression repression system, e.g., the expression repressor
comprising the TAL effector
molecule. In general, TALE binding is inversely correlated with the number of
mismatches. In some
embodiments, the TAL effector molecule of an expression repressor of the
present invention comprises
no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3
mismatches, 2 mismatches, or
1 mismatch, and optionally no mismatch, with the target DNA sequence. Without
wishing to be bound by
theory, in general the smaller the number of TAL effector domains in the TAL
effector molecule, the
smaller the number of mismatches will be tolerated and still allow for the
function of the expression
repression system, e.g., the expression repressor comprising the TAL effector
molecule. The binding
affinity is thought to depend on the sum of matching repeat-DNA combinations.
For example, TAL
effector molecules having 25 TAL effector domains or more may be able to
tolerate up to 7 mismatches.
In addition to the TAL effector domains, the TAL effector molecule of the
present invention may
comprise additional sequences derived from a naturally occurring TAL effector.
The length of the C-
terminal and/or N-terminal sequence(s) included on each side of the TAL
effector domain portion of the
TAL effector molecule can vary and be selected by one skilled in the art, for
example based on the studies
of Zhang et al. (2011). Zhang et al., have characterized a number of C-
terminal and N-terminal truncation
mutants in Hax3 derived TAL-effector based proteins and have identified key
elements, which contribute
to optimal binding to the target sequence and thus activation of
transcription. Generally, it was found that
transcriptional activity is inversely correlated with the length of N-
terminus. Regarding the C-terminus,
an important element for DNA binding residues within the first 68 amino acids
of the Hax 3 sequence was
identified. Accordingly, in some embodiments, the first 68 amino acids on the
C-terminal side of the TAL
effector domains of the naturally occurring TAL effector is included in the
TAL effector molecule of an
expression repressor of the present invention. Accordingly, in an embodiment,
a TAL effector molecule
of the present invention comprises 1) one or more TAL effector domains derived
from a naturally
occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150,
170, 180, 190, 200, 220, 230,
240, 250, 260, 270, 280 or more amino acids from the naturally occurring TAL
effector on the N-terminal
side of the TAL effector domains; and/or 3) at least 68, 80, 90, 100, 110,
120, 130, 140, 150, 170, 180,
190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally
occurring TAL effector on the
C-terminal side of the TAL effector.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
105
Zn finger domains
In some embodiments, a DNA-targeting moiety is or comprises a Zn finger
domain. A Zn finger
domain comprises a Zn finger protein, e.g., a naturally occurring Zn finger
protein or engineered Zn
finger protein, or fragment thereof. Many Zn finger proteins are known to
those of skill in the art and are
commercially available, e.g., from Sigma-Aldrich.
In some embodiments, a Zn finger domain comprises a non-naturally occurring Zn
finger protein
that is engineered to bind to a target DNA sequence of choice. See, for
example, Beerli, et al. (2002)
Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-
340; Isalan, et al. (2001)
Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol.
12:632-637; Choo, et al.
(2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242;
6,534,261; 6,599,692; 6,503,717;
6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635;
7,253,273; and U.S. Patent
Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated
herein by reference in
their entireties.
An engineered Zn finger protein may have a novel binding specificity, compared
to a naturally
.. occurring Zn finger protein. Engineering methods include, but are not
limited to, rational design and
various types of selection. Rational design includes, for example, using
databases comprising triplet (or
quadruplet) nucleotide sequences and individual Zn finger amino acid
sequences, in which each triplet or
quadruplet nucleotide sequence is associated with one or more amino acid
sequences of zinc fingers
which bind the particular triplet or quadruplet sequence. See, for example,
U.S. Pat. Nos. 6,453,242 and
6,534,261, incorporated by reference herein in their entireties.
Exemplary selection methods, including phage display and two-hybrid systems,
are disclosed in
U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248;
6,140,466; 6,200,759; and
6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO
98/53057; WO 00/27878;
and WO 01/88197 and GB 2,338,237. In addition, enhancement of binding
specificity for zinc finger
.. proteins has been described, for example, in International Patent
Publication No. WO 02/077227.
In addition, as disclosed in these and other references, zinc finger domains
and/or multi-fingered
zinc finger proteins may be linked together using any suitable linker
sequences, including for example,
linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.
6,479,626; 6,903,185; and 7,153,949
for exemplary linker sequences 6 or more amino acids in length. The proteins
described herein may
.. include any combination of suitable linkers between the individual zinc
fingers of the protein. In addition,
enhancement of binding specificity for zinc finger binding domains has been
described, for example, in
co-owned International Patent Publication No. WO 02/077227.
Zn finger proteins and methods for design and construction of fusion proteins
(and
polynucleotides encoding same) are known to those of skill in the art and
described in detail in U.S. Pat.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
106
Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988;
6,013,453; and 6,200,759;
International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057;
WO 98/54311; WO
00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO
98/53060;
WO 02/016536; and WO 03/016496.
In addition, as disclosed in these and other references, Zn finger proteins
and/or multi-fingered Zn
finger proteins may be linked together, e.g., as a fusion protein, using any
suitable linker sequences,
including for example, linkers of 5 or more amino acids in length. See, also,
U.S. Pat. Nos. 6,479,626;
6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids
in length. The Zn finger
domains described herein may include any combination of suitable linkers
between the individual zinc
finger proteins and/or multi-fingered Zn finger proteins of the Zn finger
domain.
In certain embodiments, the DNA-targeting moiety comprises a Zn finger domain
comprising an
engineered zinc finger protein that binds (in a sequence-specific manner) to a
target DNA sequence. In
some embodiments, the Zn finger domain comprises one Zn finger protein or
fragment thereof. In other
embodiments, the Zn finger domain comprises a plurality of Zn finger proteins
(or fragments thereof),
e.g., 2, 3, 4, 5, 6 or more Zn finger proteins (and optionally no more than
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2
Zn finger proteins). In some embodiments, the Zn finger domain comprises at
least three Zn finger
proteins. In some embodiments, the Zn finger domain comprises four, five or
six fingers. In some
embodiments, the Zn finger domain comprises 8, 9, 10, 11 or 12 fingers. In
some embodiments, a Zn
finger domain comprising three Zn finger proteins recognizes a target DNA
sequence comprising 9 or 10
nucleotides. In some embodiments, a Zn finger domain comprising four Zn finger
proteins recognizes a
target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn
finger domain
comprising six Zn finger proteins recognizes a target DNA sequence comprising
18 to 21 nucleotides.
In some embodiments, a Zn finger domain comprises a two-handed Zn finger
protein. Two
handed zinc finger proteins are those proteins in which two clusters of zinc
finger proteins are separated
by intervening amino acids so that the two zinc finger domains bind to two
discontinuous target DNA
sequences. An example of a two-handed type of zinc finger binding protein is
SIP1, where a cluster of
four zinc finger proteins is located at the amino terminus of the protein and
a cluster of three Zn finger
proteins is located at the carboxyl terminus (see Remade, et al. (1999) EMBO
Journal 18(18):5073-5084).
Each cluster of zinc fingers in these proteins is able to bind to a unique
target sequence and the spacing
between the two target sequences can comprise many nucleotides.
Nucleic acid molecule
In some embodiments, a DNA-targeting moiety is or comprises a DNA-binding
domain from a nuclease.
For example, the recognition sequences of homing endonucleases and
meganucleases such as I-SceI, I-
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
107
CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-
CreI, I-TevI, I-TevII and I-
TevIII are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort, et
al. (1997) Nucleic Acids Res.
25:3379-3388; Dujon, et al. (1989) Gene 82:115-118; Perler, et al. (1994)
Nucleic Acids Res. 22:1125-
1127; Jasin (1996) Trends Genet. 12:224-228; Gimble, et al. (1996) J. Mol.
Biol. 263:163-180; Argast, et
al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. In
addition, the DNA-
binding specificity of homing endonucleases and meganucleases can be
engineered to bind non-natural
target sites. See, for example, Chevalier, et al. (2002) Molec. Cell 10:895-
905; Epinat, et al. (2003)
Nucleic Acids Res. 31:2952-2962; Ashworth, et al. (2006) Nature 441:656-659;
Paques, et al. (2007)
Current Gene Therapy 7:49-66; U.S. Patent Publication No. 2007/0117128.
In some embodiments, a DNA-targeting moiety comprises or is nucleic acid. In
some
embodiments, a nucleic acid that may be included in a DNA-targeting moiety,
may be or comprise DNA,
RNA, and/or an artificial or synthetic nucleic acid or nucleic acid analog or
mimic. For example, in some
embodiments, a nucleic acid may be or include one or more of genomic DNA
(gDNA), complementary
DNA (cDNA), a peptide nucleic acid (PNA), a peptide- oligonucleotide
conjugate, a locked nucleic acid
(LNA), a bridged nucleic acid (B NA), a polyamide, a triplex- forming
oligonucleotide, an antisense
oligonucleotide, tRNA, mRNA, rRNA, miRNA, gRNA, siRNA or other RNAi molecule
(e.g., that targets
a non-coding RNA as described herein and/or that targets an expression product
of a particular gene
associated with a targeted genomic complex as described herein), etc. In some
embodiments, a nucleic
acid may include one or more residues that is not a naturally occurring DNA or
RNA residue, may
include one or more linkages that is/are not phosphodiester bonds (e.g., that
may be, for example,
phosphorothioate bonds, etc.), and/or may include one or more modifications
such as, for example, a 2'0
modification such as 2'-0MeP. A variety of nucleic acid structures useful in
preparing synthetic nucleic
acids is known in the art (see, for example, W02017/0628621 and W02014/012081)
those skilled in the
art will appreciate that these may be utilized in accordance with the present
disclosure.
A nucleic acid suitable for use in an expression repressor, e.g., in the DNA-
targeting moiety, may
include, but is not limited to, DNA, RNA, modified oligonucleotides (e.g.,
chemical modifications, such
as modifications that alter backbone linkages, sugar molecules, and/or nucleic
acid bases), and artificial
nucleic acids. In some embodiments, a nucleic acid includes, but is not
limited to, genomic DNA, cDNA,
peptide nucleic acids (PNA) or peptide oligonucleotide conjugates, locked
nucleic acids (LNA), bridged
nucleic acids (BNA), polyamides, triplex forming oligonucleotides, modified
DNA, antisense DNA
oligonucleotides, tRNA, mRNA, rRNA, modified RNA, miRNA, gRNA, and siRNA or
other RNA or
DNA molecules.
In some embodiments, a DNA-targeting moiety comprises a nucleic acid with a
length from
about 15-200, 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200,
100-200, 110-200, 120-
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
108
200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 215-190,
20-190, 30-190, 40-190,
50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190,
140-190, 150-190, 160-
190, 170-190, 180-190, 15-180, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180,
80-180, 90-180, 100-
180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 15-170, 20-
170, 30-170, 40-170,
50-170, 60-170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170,
140-170, 150-170, 160-
170, 15-160, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160,
100-160, 110-160, 120-
160, 130-160, 140-160, 150-160, 215-150, 20-150, 30-150, 40-150, 50-150, 60-
150, 70-150, 80-150, 90-
150, 100-150, 110-150, 120-150, 130-150, 140-150, 15-140, 20-140, 30-140, 40-
140, 50-140, 60-140, 70-
140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 15-130, 20-130, 30-
130, 40-130, 50-130, 60-
130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 215-120, 20-120, 30-
120, 40-120, 50-120, 60-
120, 70-120, 80-120, 90-120, 100-120, 110-120, 15-110, 20-110, 30-110, 40-110,
50-110, 60-110, 70-
110, 80-110, 90-110, 100-110, 15-100, 20-100, 30-100, 40-100, 50-100, 60-100,
70-100, 80-100, 90-100,
15-90, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 15-80, 20-80, 30-80,
40-80, 50-80, 60-80, 70-80,
15-70, 20-70, 30-70, 40-70, 50-70, 60-70, 15-60, 20-60, 30-60, 40-60, 50-60,
15-50, 20-50, 30-50, 40-50,
15-40, 20-40, 30-40, 15-30, 20-30, or 15-20 nucleotides, or any range
therebetween.
Repressor Domains
Expression repressors of the present disclosure comprise one or more repressor
domains. A
repressor domain has one or more functionality that, when used as part of an
expressor repressor or an
expression repression system described herein, decreases expression of a
target gene in a cell.
In some embodiments, a repressor domain comprises a histone modifying
functionality, e.g., a histone
methyltransferase, histone demethylase, or histone deacetylase activity. In
some embodiments, a histone
methyltransferase functionality comprises H3K9 targeting methyltransferase
activity. In some
embodiments, a histone methyltransferase functionality comprises H3K56
targeting methyltransferase
activity. In some embodiments, a histone methyltransferase functionality
comprises H3K27 targeting
methyltransferase activity. In some embodiments, a histone methyltransferase
or demethylase
functionality transfers one, two, or three methyl groups. In some embodiments,
a histone demethylase
functionality comprises H3K4 targeting demethylase activity. In some
embodiments, a repressor domain
is or comprises a protein chosen from SETDB1, SETDB2, EHMT2 (i.e., G9A), EHMT1
(i.e., GLP),
SUV39H1, EZH2, EZH1, SUV39H2, SETD8, SUV420H1, SUV420H2, or a functional
variant or
fragment of any thereof, e.g., a SET domain of any thereof. In some
embodiments, a repressor domain is
or comprises a protein chosen from KDM1A (i.e., LSD1), KDM1B (i.e., LSD2),
KDM2A, KDM2B,
KDM5A, KDM5B, KDM5C, KDM5D, KDM4B, N066, or a functional variant or fragment
of any
thereof. In some embodiments, a repressor domain is or comprises a protein
chosen from HDAC1,
HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, SIRT1,
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
109
SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7, SIRT8, SIRT9, or a functional
variant or fragment of any
thereof.
In some embodiments, a repressor domain comprises an epigenetic modifying
moiety. In some
embodiments, a repressor domain comprises a DNA modifying functionality, e.g.,
a DNA
methyltransferase. In some embodiments, a repressor domain is or comprises a
protein chosen from MQ1,
DNMT1, DNMT3A1, DNMT3A2, DNMT3B1, DNMT3B2, DNMT3B3, DNMT3B4, DNMT3B5,
DNMT3B6, DNMT3L, DNMT3a/3L or a functional variant or fragment of any thereof.
In some embodiments, a repressor domain comprises a transcription repressor.
In some embodiments the
transcription repressor blocks recruitment of a factor that stimulates or
promotes transcription, e.g., of the
target gene. In some embodiments, the transcription repressor recruits a
factor that inhibits transcription,
e.g., of the target gene. In some embodiments, a repressor domain, e.g.,
transcription repressor, is or
comprises a protein chosen from KRAB, MeCP2, HP1, RBBP4, REST, FOG1, SUZ12, or
a functional
variant or fragment of any thereof.
In some embodiments a repressor domain promotes epigenetic modification, e.g.,
directly, or
indirectly. For example, a repressor domain can indirectly promote epigenetic
modification by recruiting
an endogenous protein that epigenetically modifies the chromatin. In some
embodiments, a repressor
domain can directly promote epigenetic modification by catalyzing epigenetic
modification, wherein the
repressor domain comprises enzymatic activity and directly places an
epigenetic mark on the chromatin.
In some embodiments, a repressor domain comprises a protein having a
functionality described
herein. In some embodiments, a repressor domain is or comprises a protein
selected from:
KRAB (e.g., as according to NP_056209.2 or the protein encoded by NM_015394.5,
e.g., as according to
SEQ ID NO: 61);
a SET domain (e.g., the SET domain of:
SETDB1 (e.g., as according to NP_001353347.1 or the protein encoded by
NM_001366418.1);
EZH2 (e.g., as according to NP-004447.2 or the protein encoded by NM_004456.5,
e.g., as according to
SEQ ID NO: 65);
G9A (e.g., as according to NP_001350618.1 or the protein encoded by
NM_001363689.1; e.g., as
according to SEQ ID NO: 62); or
SUV39H1 (e.g., as according to NP_003164.1 or the protein encoded by
NM_003173.4));
histone demethylase LSD1 (e.g., as according to NP_055828.2 or the protein
encoded by NM_015013.4,
e.g., as according to SEQ ID NO: 64);
FOG1 (e.g., the N-terminal residues of FOG1) (e.g., as according to
NP_722520.2 or the protein encoded
by NM_153813.3, e.g., as according to SEQ ID NO: 66);
KAP1 (e.g., as according to NP_005753.1 or the protein encoded by
NM_005762.3); or
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
110
HDAC8 (e.g., as according to NP_001159890 or the protein encoded by
NM_001166418, e.g., as
according to SEQ ID NO: 63);
a functional fragment or variant of any thereof, or
a polypeptide with a sequence that has at least 80, 85, 90, 91, 92, 93, 94,
95, 96, 97, 98, or 99% identity to
any of the above-referenced sequences. In some embodiments, a repressor domain
is or comprises a
protein selected from:
DNMT3A (e.g., human DNMT3A) (e.g., as according to NP_072046.2
or the protein encoded by NM_022552.4, e.g., as according to SEQ ID NO: 58);
DNMT3B (e.g., as according to or A0X21819.1
.. or the protein encoded by NM_006892.4 or I(X447429);
DNMT3L (e.g., as according to NP_787063.1
or the protein encoded by NM_175867.3);
DNMT3A/3L complex (e.g., as according to SEQ ID NO: 59 or 60),
bacterial MQ1 (e.g., as according to CAA35058.1 or P15840.3, e.g., as
according to SEQ ID NO: 57 or
90);
a functional fragment of any thereof, or
a polypeptide with a sequence that has at least 80, 85, 90, 91, 92, 93, 94,
95, 96, 97, 98, or 99% identity to
any of the above-referenced sequences.
.. In some embodiments, a repressor domain is or comprises a polypeptide. In
some embodiments, a
repressor domain has enzymatic activity.
In some embodiments, a DNA-binding domain comprises a helix-hairpin-helix
(HhH) motif.
DNA-binding proteins with a HhH structural motif may be involved in non-
sequence-specific DNA
.. binding that occurs via the formation of hydrogen bonds between protein
backbone nitrogens and DNA
phosphate groups.
In some embodiments, a DNA-binding domain comprises a helix-loop-helix (HLH)
motif. DNA-
binding proteins with an HLH structural motif are transcriptional regulatory
proteins and are principally
related to a wide array of developmental processes. An HLH structural motif is
longer, in terms of
residues, than HTH or HhH motifs. Many of these proteins interact to form homo-
and hetero-dimers. A
structural motif is composed of two long helix regions, with an N-terminal
helix binding to DNA, while a
complex region allows the protein to dimerize.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
111
In some embodiments, a DNA-binding domain comprises a leucine zipper motif. In
some
transcription factors, a dimer binding site with DNA forms a leucine zipper.
This motif includes two
amphipathic helices, one from each subunit, interacting with each other
resulting in a left-handed coiled-
coil super secondary structure. A leucine zipper is an interdigitation of
regularly spaced leucine residues
in one helix with leucines from an adjacent helix. Mostly, helices involved in
leucine zippers exhibit a
heptad sequence (abcdefg) with residues a and d being hydrophobic and other
residues being hydrophilic.
Leucine zipper motifs can mediate either homo- or heterodimer formation.
In some embodiments, a DNA-binding domain comprises a Zn finger domain, where
a Zn++ ion
is coordinated by 2 Cys and 2 His residues. Such a transcription factor
includes a trimer with the
stoichiometry 1313 'a. An apparent effect of Zn++ coordination is
stabilization of a small complex structure
instead of hydrophobic core residues. Each Zn-finger interacts in a
conformationally identical manner
with successive triple base pair segments in the major groove of the double
helix. Protein-DNA
interaction is determined by two factors: (i) H-bonding interaction between a-
helix and DNA segment,
mostly between Arg residues and Guanine bases. (ii) H-bonding interaction with
DNA phosphate
backbone, mostly with Arg and His. An alternative Zn-finger motif chelates
Zn++ with 6 Cys.
An exemplary repressor domain may include, but is not limited to: ubiquitin,
bicyclic peptides as
ubiquitin ligase inhibitors, transcription factors, DNA and protein
modification enzymes such as
topoisomerases, topoisomerase inhibitors such as topotecan, DNA
methyltransferases such as the DNMT
family (e.g., DNMT3A, DNMT3B, DNMT3L), protein methyltransferases (e.g., viral
lysine
methyltransferase (vSET), protein-lysine N-methyltransferase (SMYD2),
deaminases (e.g., APOBEC,
UG1), histone methyltransferases such as enhancer of zeste homolog 2 (EZH2),
PRMT1, histone-lysine-
N-methyltransferase (Setdbl), histone methyltransferase (SET2), euchromatic
histone-lysine N-
methyltransferase 2 (G9A), histone-lysine N-methyltransferase (SUV39H1), and
G9a), histone
deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8), enzymes with a role in DNA
demethylation (e.g.,
the TET family enzymes catalyze oxidation of 5-methylcytosine to 5-
hydroxymethylcytosine and higher
oxidative derivatives), protein demethylases such as KDM1A and lysine-specific
histone demethylase 1
(LSD1), transcription repressors (e.g., KRAB, FOG1), helicases such as DHX9,
deacetylases (e.g., sirtuin
1, 2, 3, 4, 5, 6, or 7), kinases, phosphatases, DNA-intercalating agents such
as ethidium bromide, SYBR
green, and proflavine, efflux pump inhibitors such as peptidomimetics like
phenylalanine arginyl 13-
naphthylamide or quinoline derivatives, nuclear receptor activators and
inhibitors, proteasome inhibitors,
competitive inhibitors for enzymes such as those involved in lysosomal storage
diseases, protein synthesis
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
112
inhibitors, nucleases (e.g., Cpfl, Cas9, zinc finger nuclease), fusions of one
or more thereof (e.g., dCas9-
DNMT, dCas9-APOBEC, dCas9-UG1), and specific domains from proteins, such as a
KRAB domain.
In some embodiments, a candidate domain may be determined to be suitable for
use as a
repressor domain by methods known to those of skill in the art. For example, a
candidate repressor
domain may be tested by assaying whether, when the candidate repressor domain
is present in the nucleus
of a cell and appropriately localized (e.g., to a target gene or transcription
control element operably linked
to said target gene, e.g., via a DNA-targeting moiety), the candidate
repressor domain decreases
expression of the target gene in the cell, e.g., decreases the level of RNA
transcript encoded by the target
gene (e.g., as measured by RNASeq or Northern blot) or decreases the level of
protein encoded by the
target gene (e.g., as measured by ELISA).
In some embodiments, an expression repression system comprises a plurality of
expression
repressors, wherein each member of the plurality of expression repressors
comprises a repressor domain,
wherein each repressor domain does not detectably bind, e.g., does not bind,
to another repressor domain.
In some embodiments, an expression repression system comprises a first
expression repressor comprising
a first repressor domain and a second expression repressor comprising a second
repressor domain,
wherein the first repressor domain does not detectably bind, e.g., does not
bind, to the second repressor
domain. In some embodiments, an expression repression system comprises a first
expression repressor
comprising a first repressor domain and a second expression repressor
comprising a second repressor
domain, wherein the first repressor domain does not detectably bind, e.g.,
does not bind, to another first
repressor domain, and the second repressor domain does not detectably bind,
e.g., does not bind, to
another second repressor domain. In some embodiments, a repressor domain for
use in the compositions
and methods described herein is functional in a monomeric, e.g., non-dimeric,
state.
In some embodiments, a repressor domain is or comprises an epigenetic
modifying moiety, e.g.,
that modulates the two-dimensional structure of chromatin (i.e., that modulate
structure of chromatin in a
way that would alter its two-dimensional representation).
Epigenetic modifying moieties useful in methods and compositions of the
present disclosure
include agents that affect epigenetic markers, e.g., DNA methylation, histone
methylation, histone
acetylation, histone sumoylation, histone phosphorylation, and RNA-associated
silencing. Exemplary
epigenetic enzymes that can be targeted to a genomic sequence element as
described herein include DNA
methylases (e.g., DNMT3a, DNMT3b, DNMTL), DNA demethylation (e.g., the TET
family), histone
methyltransferases, histone deacetylase (e.g., HDAC1, HDAC2, HDAC3), sirtuin
1, 2, 3, 4, 5, 6, or 7,
lysine-specific histone demethylase 1 (LSD1), histone-lysine-N-
methyltransferase (Setdbl), euchromatic
histone-lysine N-methyltransferase 2 (G9a), histone-lysine N-methyltransferase
(SUV39H1), enhancer of
zeste homolog 2 (EZH2), viral lysine methyltransferase (vSET), histone
methyltransferase (SET2), and
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
113
protein-lysine N-methyltransferase (SMYD2). Examples of such epigenetic
modifying agents are
described, e.g., in de Groote et al. Nuc. Acids Res. (2012):1-18.
In some embodiments, an expression repressor, e.g., comprising an epigenetic
modifying moiety,
useful herein comprises or is a construct described in Koferle et al. Genome
Medicine 7.59 (2015):1-
3incorporated herein by reference. For example, in some embodiments, an
expression repressor
comprises or is a construct found in Table 1 of Koferle et al., e.g., histone
deacetylase, histone
methyltransferase, DNA demethylation, or H3K4 and/or H3K9 histone demethylase
described in Table 1
(e.g., dCas9-p300, TALE-TET1, ZF-DNMT3A, or TALE-LSD1).
Additional Moieties
An expression repressor may further comprise one or more additional moieties
(e.g., in addition
to one or more DNA-targeting moieties and one or more repressor domains). In
some embodiments, an
additional moiety is selected from a tagging or monitoring moiety, a cleavable
moiety (e.g., a cleavable
moiety positioned between a DNA-targeting moiety and a repressor domain or at
the N- or C-terminal end
of a polypeptide), a small molecule, a membrane translocating polypeptide, or
a pharmacoagent moiety.
Exemplary Expression Repressors
The following exemplary expression repressors are presented for illustration
purposes only and
are not intended to be limiting.
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising
dCas9, e.g., an S. aureus dCas9, and a repressor domain comprising MQ1, e.g.,
bacterial MQ1. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NO: 91 (e.g., a
plasmid encoding the expression repressor), SEQ ID NO: 22 (e.g., a nucleic
acid (e.g., cDNA) encoding
the expression repressor) and/or SEQ ID NO: 92 (e.g., a nucleic acid (e.g.,
cDNA) encoding the
expression repressor). In some embodiments, a nucleic acid described herein
comprises a nucleic acid
sequence of SEQ ID NO: 22, 91 or 92 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto. In some embodiments, the DNA-targeting moiety
is encoded by the
nucleic acid sequence of SEQ ID NO: 45 or 89 and/or the repressor domain is
encoded by the nucleic acid
sequence of SEQ ID NO: 47.
dCas9-MQ1 (MR-28125) mRNA sequence:
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
114
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT
CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC
GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA
CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT
CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG
ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA
TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT
CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG
CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC
ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG
GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC
TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
115
GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG
CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG
TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT
GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG
GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA
CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC
GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC
AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG
GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG
GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG
CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC
GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA
GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC
TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG
CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG
CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA
GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC
GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA
GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA
GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC
AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG
TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT
GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG
GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC
GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG
CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG
GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG
AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT
GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC
CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA
CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC
ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAAGCGGCCCGCCG
CCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGCCCGGGACAGCAAGGTGGAGAACA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
116
AGACCAAGAAGCTGCGGGTGTTCGAGGCCTTCGCCGGCATCGGCGCCCAGCGGAAGGCCCT
GGAGAAGGTGCGGAAGGACGAGTACGAGATCGTGGGCCTGGCCGAGTGGTACGTGCCCGCC
ATCGTGATGTACCAGGCCATCCACAACAACTTCCACACCAAGCTGGAGTACAAGAGCGTGA
GCCGGGAGGAGATGATCGACTACCTGGAGAACAAGACCCTGAGCTGGAACAGCAAGAACCC
CGTGAGCAACGGCTACTGGAAGCGGAAGAAGGACGACGAGCTGAAGATCATCTACAACGCC
ATCAAGCTGAGCGAGAAGGAGGGCAACATCTTCGACATCCGGGACCTGTACAAGCGGACCC
TGAAGAACATCGACCTGCTGACCTACAGCTTCCCCTGCCAGGACCTGAGCCAGCAGGGCATC
CAGAAGGGCATGAAGCGGGGCAGCGGCACCCGGAGCGGCCTGCTGTGGGAGATCGAGCGG
GCCCTGGACAGCACCGAGAAGAACGACCTGCCCAAGTACCTGCTGATGGAGAACGTGGGCG
CCCTGCTGCACAAGAAGAACGAGGAGGAGCTGAACCAGTGGAAGCAGAAGCTGGAGAGCC
TGGGCTACCAGAACAGCATCGAGGTGCTGAACGCCGCCGACTTCGGCAGCAGCCAGGCCCG
GCGGCGGGTGTTCATGATCAGCACCCTGAACGAGTTCGTGGAGCTGCCCAAGGGCGACAAG
AAGCCCAAGAGCATCAAGAAGGTGCTGAACAAGATCGTGAGCGAGAAGGACATCCTGAAC
AACCTGCTGAAGTACAACCTGACCGAGTTCAAGAAAACCAAGAGCAACATCAACAAGGCCA
GCCTGATCGGCTACAGCAAGTTCAACAGCGAGGGCTACGTGTACGACCCCGAGTTCACCGG
CCCCACCCTGACCGCCAGCGGCGCCAACAGCCGGATCAAGATCAAGGACGGCAGCAACATC
CGGAAGATGAACAGCGACGAGACCTTCCTGTACATCGGCTTCGACAGCCAGGACGGCAAGC
GGGTGAACGAGATCGAGTTCCTGACCGAGAACCAGAAGATCTTCGTGTGCGGCAACAGCAT
CAGCGTGGAGGTGCTGGAGGCCATCATCGACAAGATCGGCGGCCCCAGCAGCGGCGGCAAG
CGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTAC
GACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTC
TGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTA
GGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 22)
Sa-dCas9-MQ1 (PL-27695) Plasmid DNA sequence:
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACA
GCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTG
GCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT
ATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGC
CATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAG
CTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT
CACGACGTTGTAAAACGACGGCCAGTGAATTGACGCGTATTGGGATGGTACCTAATACGACT
CACTATAAGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
117
CCCAAGAAGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGCCAAGCGGAACTACA
TCCTGGGCCTGGCCATCGGCATCACCAGCGTGGGCTACGGCATCATCGACTACGAGACCCGG
GACGTGATCGACGCCGGCGTGCGGCTGTTCAAGGAGGCCAACGTGGAGAACAACGAGGGCC
GGCGGAGCAAGCGGGGCGCCCGGCGGCTGAAGCGGCGGCGGCGGCACCGGATCCAGCGGG
TGAAGAAGCTGCTGTTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAA
CCCCTACGAGGCCCGGGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAGGAGTTCAGCGCC
GCCCTGCTGCACCTGGCCAAGCGGCGGGGCGTGCACAACGTGAACGAGGTGGAGGAGGACA
CCGGCAACGAGCTGAGCACCAAGGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAGGAGA
AGTACGTGGCCGAGCTGCAGCTGGAGCGGCTGAAGAAGGACGGCGAGGTGCGGGGCAGCA
TCAACCGGTTCAAGACCAGCGACTACGTGAAGGAGGCCAAGCAGCTGCTGAAGGTGCAGAA
GGCCTACCACCAGCTGGACCAGAGCTTCATCGACACCTACATCGACCTGCTGGAGACCCGGC
GGACCTACTACGAGGGCCCCGGCGAGGGCAGCCCCTTCGGCTGGAAGGACATCAAGGAGTG
GTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAGCTGCGGAGCGTGAAGTAC
GCCTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAACCTGGTGATCACCCGGG
ACGAGAACGAGAAGCTGGAGTACTACGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCA
GAAGAAGAAGCCCACCCTGAAGCAGATCGCCAAGGAGATCCTGGTGAACGAGGAGGACAT
CAAGGGCTACCGGGTGACCAGCACCGGCAAGCCCGAGTTCACCAACCTGAAGGTGTACCAC
GACATCAAGGACATCACCGCCCGGAAGGAGATCATCGAGAACGCCGAGCTGCTGGACCAGA
TCGCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAGGAGCTGACCAACCT
GAACAGCGAGCTGACCCAGGAGGAGATCGAGCAGATCAGCAACCTGAAGGGCTACACCGG
CACCCACAACCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTGTGGCACACCAAC
GACAACCAGATCGCCATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGAGCC
AGCAGAAGGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTGGTGAAGCG
GAGCTTCATCCAGAGCATCAAGGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAAC
GACATCATCATCGAGCTGGCCCGGGAGAAGAACAGCAAGGACGCCCAGAAGATGATCAACG
AGATGCAGAAGCGGAACCGGCAGACCAACGAGCGGATCGAGGAGATCATCCGGACCACCG
GCAAGGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAGGGCA
AGTGCCTGTACAGCCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAACCCCTTCAACTAC
GAGGTGGACGCCATCATCCCCCGGAGCGTGAGCTTCGACAACAGCTTCAACAACAAGGTGC
TGGTGAAGCAGGAGGAGAACAGCAAGAAGGGCAACCGGACCCCCTTCCAGTACCTGAGCAG
CAGCGACAGCAAGATCAGCTACGAGACCTTCAAGAAGCACATCCTGAACCTGGCCAAGGGC
AAGGGCCGGATCAGCAAGACCAAGAAGGAGTACCTGCTGGAGGAGCGGGACATCAACCGG
TTCAGCGTGCAGAAGGACTTCATCAACCGGAACCTGGTGGACACCCGGTACGCCACCCGGG
GCCTGATGAACCTGCTGCGGAGCTACTTCCGGGTGAACAACCTGGACGTGAAGGTGaaatccAT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
118
CAACGGCGGCTTCACCAGCTTCCTGCGGCGGAAGTGGAAGTTCAAGAAGGAGCGGAACAAG
GGCTACAAGCACCACGCCGAGGACGCCCTGATCATCGCCAACGCCGACTTCATCTTCAAGGA
GTGGAAGAAGCTGGACAAGGCCAAGAAGGTGATGGAGAACCAGATGTTCGAGGAGAAGCA
GGCCGAGAGCATGCCCGAGATCGAGACCGAGCAGGAGTACAAGGAGATCTTCATCACCCCC
CACCAGATCAAGCACATCAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGACAAGA
AGCCCAACCGGAAGCTGATCAACGACACCCTGTACAGCACCCGGAAGGACGACAAGGGCAA
CACCCTGATCGTGAACAACCTGAACGGCCTGTACGACAAGGACAACGACAAGCTGAAGAAG
CTGATCAACAAGAGCCCCGAGAAGCTGCTGATGTACCACCACGACCCCCAGACCTACCAGA
AGCTGAAGCTGATCATGGAGCAGTACGGCGACGAGAAGAACCCCCTGTACAAGTACTACGA
GGAGACCGGCAACTACCTGACCAAGTACAGCAAGAAGGACAACGGCCCCGTGATCAAGAA
GATCAAGTACTACGGCAACAAGCTGAACGCCCACCTGGACATCACCGACGACTACCCCAAC
AGCCGGAACAAGGTGGTGAAGCTGAGCCTGAAGCCCTACCGGTTCGACGTGTACCTGGACA
ACGGCGTGTACAAGTTCGTGACCGTGAAGAACCTGGACGTGATCAAGAAGGAGAACTACTA
CGAGGTGAACAGCAAGTGCTACGAGGAGGCCAAGAAGCTGAAGAAGATCAGCAACCAGGC
CGAGTTCATCGCCAGCTTCTACAAGAACGACCTGATCAAGATCAACGGCGAGCTGTACCGG
GTGATCGGCGTGAACAACGACCTGCTGAACCGGATCGAGGTGAACATGATCGACATCACCT
ACCGGGAGTACCTGGAGAACATGAACGACAAGCGGCCCCCCCACATCATCAAGACCATCGC
CAGCAAGACCCAGAGCATCAAGAAGTACAGCACCGACATCCTGGGCAACCTGTACGAGGTG
aaatccAAGAAGCACCCCCAGATCATCAAGAAGGGCAAGCGGCCCGCCGCCACCAAGAAGGCC
GGCCAGGCCAAGAAGAAGAAGGCCCGGGACAGCAAGGTGGAGAACAAGACCAAGAAGCTG
CGGGTGTTCGAGGCCTTCGCCGGCATCGGCGCCCAGCGGAAGGCCCTGGAGAAGGTGCGGA
AGGACGAGTACGAGATCGTGGGCCTGGCCGAGTGGTACGTGCCCGCCATCGTGATGTACCA
GGCCATCCACAACAACTTCCACACCAAGCTGGAGTACAAGAGCGTGAGCCGGGAGGAGATG
ATCGACTACCTGGAGAACAAGACCCTGAGCTGGAACAGCAAGAACCCCGTGAGCAACGGCT
ACTGGAAGCGGAAGAAGGACGACGAGCTGAAGATCATCTACAACGCCATCAAGCTGAGCGA
GAAGGAGGGCAACATCTTCGACATCCGGGACCTGTACAAGCGGACCCTGAAGAACATCGAC
CTGCTGACCTACAGCTTCCCCTGCCAGGACCTGAGCCAGCAGGGCATCCAGAAGGGCATGA
AGCGGGGCAGCGGCACCCGGAGCGGCCTGCTGTGGGAGATCGAGCGGGCCCTGGACAGCAC
CGAGAAGAACGACCTGCCCAAGTACCTGCTGATGGAGAACGTGGGCGCCCTGCTGCACAAG
AAGAACGAGGAGGAGCTGAACCAGTGGAAGCAGAAGCTGGAGAGCCTGGGCTACCAGAAC
AGCATCGAGGTGCTGAACGCCGCCGACTTCGGCAGCAGCCAGGCCCGGCGGCGGGTGTTCA
TGATCAGCACCCTGAACGAGTTCGTGGAGCTGCCCAAGGGCGACAAGAAGCCCAAGAGCAT
CAAGAAGGTGCTGAACAAGATCGTGAGCGAGAAGGACATCCTGAACAACCTGCTGAAGTAC
AACCTGACCGAGTTCAAGAAaACCAAGAGCAACATCAACAAGGCCAGCCTGATCGGCTACA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
119
GCAAGTTCAACAGCGAGGGCTACGTGTACGACCCCGAGTTCACCGGCCCCACCCTGACCGCC
AGCGGCGCCAACAGCCGGATCAAGATCAAGGACGGCAGCAACATCCGGAAGATGAACAGC
GACGAGACCTTCCTGTACATCGGCTTCGACAGCCAGGACGGCAAGCGGGTGAACGAGATCG
AGTTCCTGACCGAGAACCAGAAGATCTTCGTGTGCGGCAACAGCATCAGCGTGGAGGTGCT
GGAGGCCATCATCGACAAGATCGGCGGCCCCAGCAGCGGCGGCAAGCGGCCCGCCGCCACC
AAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTAC
GCCTGAGcggccgcttaattaagctgccttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctg
tacctcttggtctttgaataaa
gcctgagtaggaagtctagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaa
aaattgtcttcttcatcgcctgcagATCCCAATGGCGCGCCGAGCTTGGCTCGAGCATGGTCATAGCTGTTTC
CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGT
AAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGC
TTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA
GGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT
CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAG
GGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA
AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA
CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTG
GAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTC
TCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGG
TCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA
TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGC
CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG
TGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT
TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG
GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTG
ATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCAT
GAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAA
TCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGA
AACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAAT
GAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGA
TTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAA
GTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCT
TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAA
ACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
120
AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT
TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCAGGGATCGCAGTGGT
GAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAAT
TCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCA
TGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGA
TTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAA
TCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTA
TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAA
ATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAAC
CATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC (SEQ ID
NO: 91)
Sa-dCas9-MQ1 (MR-28126) Expressed mRNA sequence:
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGCCAAGCGGAACTACATCCTGGG
CCTGGCCATCGGCATCACCAGCGTGGGCTACGGCATCATCGACTACGAGACCCGGGACGTG
ATCGACGCCGGCGTGCGGCTGTTCAAGGAGGCCAACGTGGAGAACAACGAGGGCCGGCGGA
GCAAGCGGGGCGCCCGGCGGCTGAAGCGGCGGCGGCGGCACCGGATCCAGCGGGTGAAGA
AGCTGCTGTTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTAC
GAGGCCCGGGTGAAGGGCCTGAGCCAGAAGCTGAGCGAGGAGGAGTTCAGCGCCGCCCTGC
TGCACCTGGCCAAGCGGCGGGGCGTGCACAACGTGAACGAGGTGGAGGAGGACACCGGCA
ACGAGCTGAGCACCAAGGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAGGAGAAGTACG
TGGCCGAGCTGCAGCTGGAGCGGCTGAAGAAGGACGGCGAGGTGCGGGGCAGCATCAACC
GGTTCAAGACCAGCGACTACGTGAAGGAGGCCAAGCAGCTGCTGAAGGTGCAGAAGGCCTA
CCACCAGCTGGACCAGAGCTTCATCGACACCTACATCGACCTGCTGGAGACCCGGCGGACCT
ACTACGAGGGCCCCGGCGAGGGCAGCCCCTTCGGCTGGAAGGACATCAAGGAGTGGTACGA
GATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAGCTGCGGAGCGTGAAGTACGCCTACA
ACGCCGACCTGTACAACGCCCTGAACGACCTGAACAACCTGGTGATCACCCGGGACGAGAA
CGAGAAGCTGGAGTACTACGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGAAGAAG
AAGCCCACCCTGAAGCAGATCGCCAAGGAGATCCTGGTGAACGAGGAGGACATCAAGGGCT
ACCGGGTGACCAGCACCGGCAAGCCCGAGTTCACCAACCTGAAGGTGTACCACGACATCAA
GGACATCACCGCCCGGAAGGAGATCATCGAGAACGCCGAGCTGCTGGACCAGATCGCCAAG
ATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAGGAGCTGACCAACCTGAACAGCG
AGCTGACCCAGGAGGAGATCGAGCAGATCAGCAACCTGAAGGGCTACACCGGCACCCACAA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
121
CCTGAGCCTGAAGGCCATCAACCTGATCCTGGACGAGCTGTGGCACACCAACGACAACCAG
ATCGCCATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGAGCCAGCAGAAGG
AGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTGGTGAAGCGGAGCTTCATC
CAGAGCATCAAGGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCATCA
TCGAGCTGGCCCGGGAGAAGAACAGCAAGGACGCCCAGAAGATGATCAACGAGATGCAGA
AGCGGAACCGGCAGACCAACGAGCGGATCGAGGAGATCATCCGGACCACCGGCAAGGAGA
ACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAGGGCAAGTGCCTGTA
CAGCCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAACCCCTTCAACTACGAGGTGGAC
GCCATCATCCCCCGGAGCGTGAGCTTCGACAACAGCTTCAACAACAAGGTGCTGGTGAAGC
AGGAGGAGAACAGCAAGAAGGGCAACCGGACCCCCTTCCAGTACCTGAGCAGCAGCGACA
GCAAGATCAGCTACGAGACCTTCAAGAAGCACATCCTGAACCTGGCCAAGGGCAAGGGCCG
GATCAGCAAGACCAAGAAGGAGTACCTGCTGGAGGAGCGGGACATCAACCGGTTCAGCGTG
CAGAAGGACTTCATCAACCGGAACCTGGTGGACACCCGGTACGCCACCCGGGGCCTGATGA
ACCTGCTGCGGAGCTACTTCCGGGTGAACAACCTGGACGTGAAGGTGaaatccATCAACGGCGG
CTTCACCAGCTTCCTGCGGCGGAAGTGGAAGTTCAAGAAGGAGCGGAACAAGGGCTACAAG
CACCACGCCGAGGACGCCCTGATCATCGCCAACGCCGACTTCATCTTCAAGGAGTGGAAGA
AGCTGGACAAGGCCAAGAAGGTGATGGAGAACCAGATGTTCGAGGAGAAGCAGGCCGAGA
GCATGCCCGAGATCGAGACCGAGCAGGAGTACAAGGAGATCTTCATCACCCCCCACCAGAT
CAAGCACATCAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGACAAGAAGCCCAAC
CGGAAGCTGATCAACGACACCCTGTACAGCACCCGGAAGGACGACAAGGGCAACACCCTGA
TCGTGAACAACCTGAACGGCCTGTACGACAAGGACAACGACAAGCTGAAGAAGCTGATCAA
CAAGAGCCCCGAGAAGCTGCTGATGTACCACCACGACCCCCAGACCTACCAGAAGCTGAAG
CTGATCATGGAGCAGTACGGCGACGAGAAGAACCCCCTGTACAAGTACTACGAGGAGACCG
GCAACTACCTGACCAAGTACAGCAAGAAGGACAACGGCCCCGTGATCAAGAAGATCAAGTA
CTACGGCAACAAGCTGAACGCCCACCTGGACATCACCGACGACTACCCCAACAGCCGGAAC
AAGGTGGTGAAGCTGAGCCTGAAGCCCTACCGGTTCGACGTGTACCTGGACAACGGCGTGT
ACAAGTTCGTGACCGTGAAGAACCTGGACGTGATCAAGAAGGAGAACTACTACGAGGTGAA
CAGCAAGTGCTACGAGGAGGCCAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTCATC
GCCAGCTTCTACAAGAACGACCTGATCAAGATCAACGGCGAGCTGTACCGGGTGATCGGCG
TGAACAACGACCTGCTGAACCGGATCGAGGTGAACATGATCGACATCACCTACCGGGAGTA
CCTGGAGAACATGAACGACAAGCGGCCCCCCCACATCATCAAGACCATCGCCAGCAAGACC
CAGAGCATCAAGAAGTACAGCACCGACATCCTGGGCAACCTGTACGAGGTGaaatccAAGAAG
CACCCCCAGATCATCAAGAAGGGCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCA
AGAAGAAGAAGGCCCGGGACAGCAAGGTGGAGAACAAGACCAAGAAGCTGCGGGTGTTCG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
122
AGGCCTTCGCCGGCATCGGCGCCCAGCGGAAGGCCCTGGAGAAGGTGCGGAAGGACGAGTA
CGAGATCGTGGGCCTGGCCGAGTGGTACGTGCCCGCCATCGTGATGTACCAGGCCATCCACA
ACAACTTCCACACCAAGCTGGAGTACAAGAGCGTGAGCCGGGAGGAGATGATCGACTACCT
GGAGAACAAGACCCTGAGCTGGAACAGCAAGAACCCCGTGAGCAACGGCTACTGGAAGCG
GAAGAAGGACGACGAGCTGAAGATCATCTACAACGCCATCAAGCTGAGCGAGAAGGAGGG
CAACATCTTCGACATCCGGGACCTGTACAAGCGGACCCTGAAGAACATCGACCTGCTGACCT
ACAGCTTCCCCTGCCAGGACCTGAGCCAGCAGGGCATCCAGAAGGGCATGAAGCGGGGCAG
CGGCACCCGGAGCGGCCTGCTGTGGGAGATCGAGCGGGCCCTGGACAGCACCGAGAAGAAC
GACCTGCCCAAGTACCTGCTGATGGAGAACGTGGGCGCCCTGCTGCACAAGAAGAACGAGG
AGGAGCTGAACCAGTGGAAGCAGAAGCTGGAGAGCCTGGGCTACCAGAACAGCATCGAGG
TGCTGAACGCCGCCGACTTCGGCAGCAGCCAGGCCCGGCGGCGGGTGTTCATGATCAGCAC
CCTGAACGAGTTCGTGGAGCTGCCCAAGGGCGACAAGAAGCCCAAGAGCATCAAGAAGGTG
CTGAACAAGATCGTGAGCGAGAAGGACATCCTGAACAACCTGCTGAAGTACAACCTGACCG
AGTTCAAGAAaACCAAGAGCAACATCAACAAGGCCAGCCTGATCGGCTACAGCAAGTTCAA
CAGCGAGGGCTACGTGTACGACCCCGAGTTCACCGGCCCCACCCTGACCGCCAGCGGCGCC
AACAGCCGGATCAAGATCAAGGACGGCAGCAACATCCGGAAGATGAACAGCGACGAGACC
TTCCTGTACATCGGCTTCGACAGCCAGGACGGCAAGCGGGTGAACGAGATCGAGTTCCTGAC
CGAGAACCAGAAGATCTTCGTGTGCGGCAACAGCATCAGCGTGGAGGTGCTGGAGGCCATC
ATCGACAAGATCGGCGGCCCCAGCAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCG
GCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGcggccg
cttaattaagctgccttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtct
ttgaataaagcctgagtaggaagtcta
gaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aa (SEQ ID NO:
92)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 93,
33, 67, or 68. In some embodiments, an expression repressor described herein
comprises an amino acid
sequence of SEQ ID NO: 93, 33, 67, 68, or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
Sa-dCas9-MQ1 Protein sequence:
MAPKKKRKVGIHGVPAAAKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRR
SKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLA
KRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYV
KEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF
PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILV
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
123
NEEDIKGYRVT STGKPEFTNLKVYHDIKDITARKEIIENAELLD QIAKILTIY QS S EDIQEELTNLNS
ELT QEEIEQIS NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLS QQKEIPTTL
VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEII
RTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDAIIPRSVSFDNSFNNKVLV
.. KQEENS KKGNRTPFQYLSS SD S KISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFI
NRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS INGGFT SFLRRKWKFKKERNKGYKHHAED
ALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK
YSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHD
PQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYS KKDNGPVIKKIKYYGNKLNAHLDITDD
YPNS RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNS KCYEEAKKLKKIS NQ
AEFIAS FYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMND KRPPHIIKTIAS KT QSI
KKYSTDILGNLYEVKSKKHPQIIKKGKRPAATKKAGQAKKKKARDSKVENKTKKLRVFEAFAGI
GAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSW
NS KNPV SNGYWKRKKDDELKIIYNAIKLS EKEGNIFDIRDLYKRTLKNIDLLTYSFPC QDLS QQGI
QKGMKRGSGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLHKKNEEELNQWKQKLESLG
YQNSIEVLNAADFGS S QARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNL
TEFKKTKSNINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIG
FDS QDGKRVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGGPSSGGKRPAATKKAGQAKKKKGSY
PYDVPDYA (SEQ ID NO: 93)
dCas9-MQ1 amino acid sequence
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
124
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGD KRPAATKKAGQAKKKKARD S KVENKTKKLRVFEAFAGIGAQRKALE
KVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSV SREEMIDYLENKTLSWNS KNPVS N
GYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRG
SGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLHKKNEEELNQWKQKLESLGYQNSIEVLN
AADFGS SQARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKS
NINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDS QDGK
RVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGGPSSGGKRPAATKKAGQAKKKKGSYPYDVPDY
A (SEQ ID NO: 33)
Sa-dCas9-MQ1 without HA tag
MAPKKKRKVGIHGVPAAAKRNYILGLAIGITS VGYGIIDYETRDVIDAGVRLFKEANVENNEGRR
S KRGARRLKRRRRHRIQRV KKLLFDYNLLTDHSELS GINPYEARVKGLS QKLSEEEFSAALLHLA
KRRGVHNVNEVEEDTGNELSTKEQISRNS KALEEKYVAELQLERLKKDGEVRGSINRFKTSDYV
KEAKQLLKV QKAYHQLD QSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF
PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILV
NEEDIKGYRVT STGKPEFTNLKVYHDIKDITARKEIIENAELLD QIAKILTIY QS S EDIQEELTNLNS
ELT QEEIEQIS NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLS QQKEIPTTL
VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEII
RTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDAIIPRSVSFDNSFNNKVLV
KQEENS KKGNRTPFQYLSS SD S KISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFI
NRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS INGGFT SFLRRKWKFKKERNKGYKHHAED
ALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYK
YSHRVDKKPNRKLINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHD
PQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYS KKDNGPVIKKIKYYGNKLNAHLDITDD
YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQ
AEFIAS FYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMND KRPPHIIKTIAS KT QSI
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
125
KKYSTDILGNLYEVKSKKHPQIIKKGKRPAATKKAGQAKKKKARDSKVENKTKKLRVFEAFAGI
GAQRKALEKVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSW
NS KNPV SNGYWKRKKDDELKIIYNAIKLS EKEGNIFDIRDLYKRTLKNIDLLTYSFPC QDLS QQGI
QKGMKRGSGTRSGLLWEIERALDS TEKNDLPKYLLMENVGALLHKKNEEELNQWKQKLESLG
YQNSIEVLNAADFGS S QARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNL
TEFKKTKSNINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIG
FDS QDGKRVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGGPSSGGKRPAATKKAGQAKKKKGS
(SEQ ID NO: 67)
.. dCas9-MQ1 without HA tag
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGD KRPAATKKAGQAKKKKARD S KVENKTKKLRVFEAFAGIGAQRKALE
KVRKDEYEIVGLAEWYVPAIVMYQAIHNNFHTKLEYKSVSREEMIDYLENKTLSWNSKNPVSN
GYWKRKKDDELKIIYNAIKLSEKEGNIFDIRDLYKRTLKNIDLLTYSFPCQDLSQQGIQKGMKRG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
126
SGTRSGLLWEIERALDSTEKNDLPKYLLMENVGALLHKKNEEELNQWKQKLESLGYQNSIEVLN
AADFGSSQARRRVFMISTLNEFVELPKGDKKPKSIKKVLNKIVSEKDILNNLLKYNLTEFKKTKS
NINKASLIGYSKFNSEGYVYDPEFTGPTLTASGANSRIKIKDGSNIRKMNSDETFLYIGFDSQDGK
RVNEIEFLTENQKIFVCGNSISVEVLEAIIDKIGGPSSGGKRPAATKKAGQAKKKKGS (SEQ ID
NO: 68)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising KRAB, e.g., a
KRAB domain. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NOs: 94 (e.g.,
a plasmid encoding the expression repressor) and/or 95 (e.g., a nucleic acid
(e.g., cDNA) encoding the
expression repressor). In some embodiments, a nucleic acid described herein
comprises a nucleic acid
sequence of SEQ ID NO: 94 or 95 or a sequence with at least 80, 85, 90, 95,
99, or 100% identity thereto,
or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 positions of
difference thereto.
Sp-dCas9-KRAB (PL-27687) Plasmid DNA sequence:
TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACA
GCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTG
GCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT
ATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGC
CATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAG
CTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGT
CACGACGTTGTAAAACGACGGCCAGTGAATTGACGCGTATTGGGATGGTACCTAATACGACT
CACTATAAGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCC
CCCAAGAAGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGC
ATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGG
TGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCT
GATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACC
GCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCA
ACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGA
GGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTAC
CACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGG
CCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTG
ATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
127
AGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGC
CATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCC
GGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCA
ACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTA
CGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGG
CCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGAT
CACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTG
ACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCG
ACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTA
CAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTG
AACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGA
TCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAG
GACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCT
GGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGaaatccGAGGAGACCATCACCCCCTG
GAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACC
AACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGT
ACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCC
CGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGG
AAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCG
TGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCT
GAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGA
CATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAaACCT
ACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTG
GGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATC
CTGGACTTCCTGaaatccGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACA
GCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCA
CGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTG
AAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCG
AGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGA
AGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGG
AGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACAT
GTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTG
CCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGG
CCCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
128
GGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGC
CGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAG
ACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACG
ACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGaaatccAAGCTGGTGAGCG
ACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCAC
GACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGA
GCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGA
GCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCA
AGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGG
CGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTG
AGCATGCCCCAGGTGAACATCGTGAAGAAaACCGAGGTGCAGACCGGCGGCTTCAGCAAGG
AGAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCC
CAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAG
GTGGAGAAGGGCAAGAGCAAGAAGCTGaaatccGTGAAGGAGCTGCTGGGCATCACCATCATG
GAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGG
TGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCG
GAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGC
AAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGG
ACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGA
GCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTG
AGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACC
TGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGAC
CGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCA
CCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAAGCGGCCCGCCGC
CACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGCCAGCGACGCCAAGAGCCTGACCGC
CTGGAGCCGGACCCTGGTGACCTTCAAGGACGTGTTCGTGGACTTCACCCGGGAGGAGTGG
AAGCTGCTGGACACCGCCCAGCAGATCCTGTACCGGAACGTGATGCTGGAGAACTACAAGA
ACCTGGTGAGCCTGGGCTACCAGCTGACCAAGCCCGACGTGATCCTGCGGCTGGAGAAGGG
CGAGGAGCCCTGGCTGGTGGAGCGGGAGATCCACCAGGAGACCCACCCCGACAGCGAGACC
GCCTTCGAGATCAAGAGCAGCGTGAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCG
GCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGcggccg
cttaattaagctgccttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtct
ttgaataaagcctgagtaggaagtcta
gaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aattgtcttcttcatcgcc
tgcagATCCCAATGGCGCGCCGAGCTTGGCTCGAGCATGGTCATAGCTGTTTCCTGTGTGAAAT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
129
TGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGG
GTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGG
GAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCG
TATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCG
AGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCA
GGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG
CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCA
GAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTC
GTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA
AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC
AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTA
TCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA
GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA
CGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA
AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTT
TGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC
GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA
AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA
TATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTT
ATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA
ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGT
CCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATC
ACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTT
GTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTC
ATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAA
CAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGA
ATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCAGGGATCGCAGTGGTGAGTAACC
ATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAG
CCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAG
AAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGA
CATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGC
CTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTATTGAAGC
ATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
130
AATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTA
TCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC (SEQ ID NO: 94)
Sp-dCas9-KRAB (MR-28122) Expressed mRNA sequence:
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGaaatccGAGGAGACCATCACCCCCTGGAACTTCGA
GGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGAC
AAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCG
TGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTTCCT
GAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACC
GTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
131
GCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCAT
CAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTG
ACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAaACCTACGCCCACCT
GTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTG
AGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCC
TGaaatccGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTT
CAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACAT
CGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTG
GACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCC
GGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCG
AGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCC
AGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGA
CCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCCAGAGC
TTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCGGGGCA
AGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGC
TGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGG
CGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAG
ATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGAGAACG
ACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGaaatccAAGCTGGTGAGCGACTTCCGGA
AGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCCTA
CCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTC
GTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGA
TCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAG
ATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCG
GCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCC
CCAGGTGAACATCGTGAAGAAaACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATC
CTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGT
ACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAA
GGGCAAGAGCAAGAAGCTGaaatccGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAG
CAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAG
GACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGA
TGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGT
GAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAG
CAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
132
GCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTA
CAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACC
CTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCG
GTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTG
TACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAAGCGGCCCGCCGCCACCAAGA
AGGCCGGCCAGGCCAAGAAGAAGAAGGCCAGCGACGCCAAGAGCCTGACCGCCTGGAGCC
GGACCCTGGTGACCTTCAAGGACGTGTTCGTGGACTTCACCCGGGAGGAGTGGAAGCTGCTG
GACACCGCCCAGCAGATCCTGTACCGGAACGTGATGCTGGAGAACTACAAGAACCTGGTGA
GCCTGGGCTACCAGCTGACCAAGCCCGACGTGATCCTGCGGCTGGAGAAGGGCGAGGAGCC
CTGGCTGGTGGAGCGGGAGATCCACCAGGAGACCCACCCCGACAGCGAGACCGCCTTCGAG
ATCAAGAGCAGCGTGAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCC
AAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGcggccgcttaattaagctgc
cttctgcggggcttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtctttgaataaagcctg
agtaggaagtctagaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID
NO: 95)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 96 or
72. In some embodiments, an expression repressor described herein comprises a
an amino acid sequence
of SEQ ID NO: 72 or 96 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
Sp-dCas9-KRAB Protein sequence:
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
133
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDKRPAATKKAGQAKKKKASDAKSLTAWSRTLVTFKDVFVDFTREEWK
LLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIK
SSVSGGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 96)
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
134
SITGLYETRIDLSQLGGDKRPAATKKAGQAKKKKASDAKSLTAWSRTLVTFKDVFVDFTREEWK
LLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIK
SSVSGGKRPAATKKAGQAKKKKGS (SEQ ID NO: 72)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising DNMT1, e.g., a
DNMT1 domain. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NOs: 23 (e.g.,
a nucleic acid (e.g., cDNA) encoding the expression repressor). In some
embodiments, a nucleic acid
described herein comprises a nucleic acid sequence of SEQ ID NO: 23 or a
sequence with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
dCas9-DNMT1 (MR-29419)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
135
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT
CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC
GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA
CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT
CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG
ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA
TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT
CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG
CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC
ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG
GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC
TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT
GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG
CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG
TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT
GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG
GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA
CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC
GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC
AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG
GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG
GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG
CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC
GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA
GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC
TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG
CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG
CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA
GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC
GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
136
GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA
GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC
AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG
TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT
GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG
GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC
GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG
CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG
GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG
AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT
GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC
CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA
CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC
ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGGCGGCAAGC
GGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGTCGGGCGGGGGTGGCT
CAGTGGATCTGAGGACACTCGACGTGTTTAGCGGATGCGGCGGACTCTCCGAAGGCTTCCAC
CAAGCCGGAATTTCCGACACACTCTGGGCCATTGAGATGTGGGACCCCGCCGCTCAAGCCTT
CAGACTGAATAATCCCGGCTCCACCGTGTTCACCGAGGACTGCAACATTCTGCTGAAGCTGG
TGATGGCTGGCGAAACCACCAACTCTAGAGGCCAGAGGCTGCCCCAGAAGGGAGATGTGGA
AATGCTCTGTGGAGGCCCTCCTTGCCAAGGCTTCTCCGGCATGAACAGGTTCAACTCTAGAA
CATACAGCAAGTTCAAGAACTCTCTGGTCGTGAGCTTTCTGAGCTACTGCGACTACTATAGA
CCTAGGTTCTTTCTGCTGGAGAACGTGAGAAATTTCGTGTCCTTCAAGAGGAGCATGGTGCT
GAAGCTGACACTGAGGTGTCTGGTGAGGATGGGCTACCAGTGCACATTCGGAGTGCTGCAA
GCTGGCCAGTACGGCGTGGCCCAGACCAGAAGGAGGGCCATCATTCTGGCTGCTGCCCCCG
GCGAGAAACTCCCTCTGTTCCCCGAGCCCCTCCACGTGTTCGCCCCTAGAGCTTGCCAGCTG
AGCGTGGTGGTCGACGATAAGAAGTTCGTGAGCAACATCACAAGGCTGTCCAGCGGACCCT
TCAGAACCATTACCGTGAGGGATACCATGTCCGACCTCCCCGAGGTGAGGAATGGCGCCAG
CGCTCTGGAGATTTCCTACAACGGCGAACCTCAGAGCTGGTTCCAAAGGCAGCTGAGAGGC
GCTCAGTATCAGCCCATTCTGAGGGACCACATCTGCAAAGATATGAGCGCTCTGGTGGCCGC
TAGAATGAGACATATTCCTCTGGCCCCCGGCAGCGACTGGAGAGATCTGCCCAATATTGAGG
TGAGACTCAGCGACGGAACAATGGCTAGAAAACTGAGGTACACCCATCATGATAGAAAGAA
CGGAAGGAGCAGCAGCGGCGCTCTGAGAGGAGTGTGTAGCTGCGTGGAAGCTGGCAAGGCT
TGCGATCCCGCCGCTAGGCAGTTCAATACCCTCATCCCTTGGTGTCTGCCTCACACCGGCAA
CAGACACAATCATTGGGCTGGACTGTATGGAAGGCTCGAATGGGACGGCTTTTTCAGCACCA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
137
CCGTGACCAATCCCGAACCTATGGGCAAGCAAGGAAGGGTGCTCCACCCCGAGCAGCATAG
AGTCGTGTCCGTGAGAGAATGCGCTAGAAGCCAAGGCTTCCCCGACACCTATAGACTGTTCG
GCAACATTCTGGATAAGCACAGACAAGTGGGAAATGCTGTCCCTCCTCCTCTGGCCAAGGCT
ATCGGACTGGAGATCAAGCTGTGTATGCTCGCCAAAGCTAGGGAGAGCGCTTCCGCCAAGA
TTAAGGAGGAGGAGGCCGCCAAGGACGGAGGTGGCGGATCGGGAAAGCGGCCCGCCGCCA
CCAAGAAGGCCGGTCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTA
CGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCT
TCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAA (SEQ ID NO: 23)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 34 or
69. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 34 or 69, or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
dCas9-DNMT
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
RLSDYDVAAIVPQSFLKDD SIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLIT Q
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
138
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKSGGGGSVDLRTLDVFSGCGGLSEGFHQ
AGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILLKLVMAGETTNSRGQRLPQKGDVEML
CGGPPCQGFSGMNRFNSRTYSKFKNSLVVSFLSYCDYYRPRFFLLENVRNFVSFKRSMVLKLTLR
CLVRMGYQCTFGVLQAGQYGVAQTRRRAIILAAAPGEKLPLFPEPLHVFAPRACQLSVVVDDKK
FVSNITRLSSGPFRTITVRDTMSDLPEVRNGASALEISYNGEPQSWFQRQLRGAQYQPILRDHICK
DMSALVAARMRHIPLAPGSDWRDLPNIEVRLSD GTMARKLRYTHHDRKNGRS S SGALRGVC SC
VEAGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNPEPMGKQGRVL
HPEQHRVVSVRECARSQGFPDTYRLFGNILDKHRQVGNAVPPPLAKAIGLEIKLCMLAKARESAS
AKIKEEEAAKDGGGGSGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 34)
dCas9-DNMT without HA tag
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNL SDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
139
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKSGGGGSVDLRTLDVFSGCGGLSEGFHQ
AGISDTLWAIEMWDPAAQAFRLNNPGSTVFTEDCNILLKLVMAGETTNSRGQRLPQKGDVEML
CGGPPCQGFSGMNRFNSRTYSKFKNSLVVSFLSYCDYYRPRFFLLENVRNFVSFKRSMVLKLTLR
CLVRMGYQCTFGVLQAGQYGVAQTRRRAIILAAAPGEKLPLFPEPLHVFAPRACQLSVVVDDKK
FVSNITRLSSGPFRTITVRDTMSDLPEVRNGASALEISYNGEPQSWFQRQLRGAQYQPILRDHICK
DMSALVAARMRHIPLAPGSDWRDLPNIEVRLSD GTMARKLRYTHHDRKNGRS S SGALRGVC SC
VEAGKACDPAARQFNTLIPWCLPHTGNRHNHWAGLYGRLEWDGFFSTTVTNPEPMGKQGRVL
HPEQHRVV SVRECARS QGFPDTYRLFGNILDKHRQVGNAVPPPLAKAIGLEIKLCMLAKARESAS
AKIKEEEAAKDGGGGSGKRPAATKKAGQAKKKKGS (SEQ ID NO: 69)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising DNMT3a/3L. In
some embodiments, the
expression repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 24
(e.g., a nucleic acid
(e.g., cDNA) encoding a fully human expression repressor) and/or 25 (e.g., a
nucleic acid (e.g., cDNA)
encoding a chimeric expression repressor). In some embodiments, a nucleic acid
described herein
comprises a nucleic acid sequence of SEQ ID NO: 24 or 25 or a sequence with at
least 80, 85, 90, 95, 99,
or 100% identity thereto, or having no more than 20, 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2, or 1 positions of difference thereto.
dCas9-DNMT 3a/3L (h) (MR-29414)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
140
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT
CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC
GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA
CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT
CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG
ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA
TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT
CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG
CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC
ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG
GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC
TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT
GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG
CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG
TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT
GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG
GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA
CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC
GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC
AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG
GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG
GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
141
CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC
GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA
GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC
TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG
CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG
CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA
GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC
GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA
GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA
GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC
AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG
TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT
GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG
GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC
GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG
CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG
GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG
AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT
GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC
CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA
CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC
ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGCCGGCGGCG
GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCCCCAAGAAGAAGCGGAAGG
TGGCCGCCGCCGGCAGCAACCACGACCAGGAGTTCGACCCCCCCAAGGTGTACCCCCCCGT
GCCCGCCGAGAAGCGGAAGCCCATCCGGGTGCTGAGCCTGTTCGACGGCATCGCCACCGGC
CTGCTGGTGCTGAAGGACCTGGGCATCCAGGTGGACCGGTACATCGCCAGCGAGGTGTGCG
AGGACAGCATCACCGTGGGCATGGTGCGGCACCAGGGCAAGATCATGTACGTGGGCGACGT
GCGGAGCGTGACCCAGAAGCACATCCAGGAGTGGGGCCCCTTCGACCTGGTGATCGGCGGC
AGCCCCTGCAACGACCTGAGCATCGTGAACCCCGCCCGGAAGGGCCTGTACGAGGGCACCG
GCCGGCTGTTCTTCGAGTTCTACCGGCTGCTGCACGACGCCCGGCCCAAGGAGGGCGACGAC
CGGCCCTTCTTCTGGCTGTTCGAGAACGTGGTGGCCATGGGCGTGAGCGACAAGCGGGACAT
CAGCCGGTTCCTGGAGAGCAACCCCGTGATGATCGACGCCAAGGAGGTGAGCGCCGCCCAC
CGGGCCCGGTACTTCTGGGGCAACCTGCCCGGCATGAACCGGCCCCTGGCCAGCACCGTGA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
142
ACGACAAGCTGGAGCTGCAGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGT
GCGGACCATCACCACCCGGAGCAACAGCATCAAGCAGGGCAAGGACCAGCACTTCCCCGTG
TTCATGAACGAGAAGGAGGACATCCTGTGGTGCACCGAGATGGAGCGGGTGTTCGGCTTCC
CCGTGCACTACACCGACGTGAGCAACATGAGCCGGCTGGCCCGGCAGCGGCTGCTGGGCCG
GAGCTGGAGCGTGCCCGTGATCCGGCACCTGTTCGCCCCCCTGAAGGAGTACTTCGCCTGCG
TGAGCAGCGGCAACAGCAACGCCAACAGCCGGGGCCCCAGCTTCAGCAGCGGCCTGGTGCC
CCTGAGCCTGCGGGGCAGCCACATGAATCCTCTGGAGATGTTCGAGACAGTGCCCGTGTGGA
GAAGGCAACCCGTGAGGGTGCTGAGCCTCTTCGAGGACATTAAGAAGGAGCTGACCTCTCT
GGGCTTTCTGGAATCCGGCAGCGACCCCGGCCAGCTGAAACACGTGGTGGACGTGACCGAC
ACAGTGAGGAAGGACGTGGAAGAGTGGGGCCCCTTTGACCTCGTGTATGGAGCCACACCTC
CTCTCGGCCACACATGCGATAGGCCTCCCAGCTGGTATCTCTTCCAGTTCCACAGACTGCTCC
AGTACGCCAGACCTAAGCCCGGCAGCCCCAGACCCTTCTTCTGGATGTTCGTGGACAATCTG
GTGCTGAACAAGGAGGATCTGGATGTGGCCAGCAGATTTCTGGAGATGGAACCCGTGACAA
TCCCCGACGTGCATGGCGGCTCTCTGCAGAACGCCGTGAGAGTGTGGTCCAACATCCCCGCC
ATTAGAAGCAGACACTGGGCTCTGGTGAGCGAGGAGGAACTGTCTCTGCTGGCCCAGAATA
AGCAGTCCTCCAAGCTGGCCGCCAAGTGGCCCACCAAGCTGGTGAAGAACTGCTTTCTGCCT
CTGAGGGAGTATTTCAAGTATTTCAGCACCGAACTGACCAGCAGCCTGAGCGGCGGCAAGC
GGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACG
ACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCT
GGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAG
GAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 24)
dCas9-DNMT3a/3L (m) (MR-28195)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
143
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT
CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC
GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA
CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT
CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG
ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA
TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT
CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG
CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC
ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG
GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC
TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT
GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG
CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG
TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT
GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG
GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA
CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC
GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC
AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
144
GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG
GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG
CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC
GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA
GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC
TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG
CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG
CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA
GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC
GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA
GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA
GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC
AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG
TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT
GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG
GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC
GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG
CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG
GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG
AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT
GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC
CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA
CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC
ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGCCGGCGGCG
GCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCCCCAAGAAGAAGCGGAAGG
TGGCCGCCGCCGGCAGCAACCACGACCAGGAGTTCGACCCCCCCAAGGTGTACCCCCCCGT
GCCCGCCGAGAAGCGGAAGCCCATCCGGGTGCTGAGCCTGTTCGACGGCATCGCCACCGGC
CTGCTGGTGCTGAAGGACCTGGGCATCCAGGTGGACCGGTACATCGCCAGCGAGGTGTGCG
AGGACAGCATCACCGTGGGCATGGTGCGGCACCAGGGCAAGATCATGTACGTGGGCGACGT
GCGGAGCGTGACCCAGAAGCACATCCAGGAGTGGGGCCCCTTCGACCTGGTGATCGGCGGC
AGCCCCTGCAACGACCTGAGCATCGTGAACCCCGCCCGGAAGGGCCTGTACGAGGGCACCG
GCCGGCTGTTCTTCGAGTTCTACCGGCTGCTGCACGACGCCCGGCCCAAGGAGGGCGACGAC
CGGCCCTTCTTCTGGCTGTTCGAGAACGTGGTGGCCATGGGCGTGAGCGACAAGCGGGACAT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
145
CAGCCGGTTCCTGGAGAGCAACCCCGTGATGATCGACGCCAAGGAGGTGAGCGCCGCCCAC
CGGGCCCGGTACTTCTGGGGCAACCTGCCCGGCATGAACCGGCCCCTGGCCAGCACCGTGA
ACGACAAGCTGGAGCTGCAGGAGTGCCTGGAGCACGGCCGGATCGCCAAGTTCAGCAAGGT
GCGGACCATCACCACCCGGAGCAACAGCATCAAGCAGGGCAAGGACCAGCACTTCCCCGTG
TTCATGAACGAGAAGGAGGACATCCTGTGGTGCACCGAGATGGAGCGGGTGTTCGGCTTCC
CCGTGCACTACACCGACGTGAGCAACATGAGCCGGCTGGCCCGGCAGCGGCTGCTGGGCCG
GAGCTGGAGCGTGCCCGTGATCCGGCACCTGTTCGCCCCCCTGAAGGAGTACTTCGCCTGCG
TGAGCAGCGGCAACAGCAACGCCAACAGCCGGGGCCCCAGCTTCAGCAGCGGCCTGGTGCC
CCTGAGCCTGCGGGGCAGCCACATGGGCCCCATGGAGATCTACAAGACCGTGAGCGCCTGG
AAGCGGCAGCCCGTGCGGGTGCTGAGCCTGTTCCGGAACATCGACAAGGTGCTGAAATCCC
TGGGCTTCCTGGAGAGCGGCAGCGGCAGCGGCGGCGGCACCCTGAAGTACGTGGAGGACGT
GACCAACGTGGTGCGGCGGGACGTGGAGAAGTGGGGCCCCTTCGACCTGGTGTACGGCAGC
ACCCAGCCCCTGGGCAGCAGCTGCGACCGGTGCCCCGGCTGGTACATGTTCCAGTTCCACCG
GATCCTGCAGTACGCCCTGCCCCGGCAGGAGAGCCAGCGGCCCTTCTTCTGGATCTTCATGG
ACAACCTGCTGCTGACCGAGGACGACCAGGAGACCACCACCCGGTTCCTGCAGACCGAGGC
CGTGACCCTGCAGGACGTGCGGGGCCGGGACTACCAGAACGCCATGCGGGTGTGGAGCAAC
ATCCCCGGCCTGAAATCCAAGCACGCCCCCCTGACCCCCAAGGAGGAGGAGTACCTGCAGG
CCCAGGTGCGGAGCCGGAGCAAGCTGGACGCCCCCAAGGTGGACCTGCTGGTGAAGAACTG
CCTGCTGCCCCTGCGGGAGTACTTCAAGTACTTCAGCCAGAACAGCCTGCCCCTGAGCGGCG
GCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACC
CCTACGACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTG
CCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTG
AGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 25)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 35,
36, 70, or 71. In some embodiments, an expression repressor described herein
comprises an amino acid
sequence of SEQ ID NO: 35, 36, 70, or 71 or a sequence with at least 80, 85,
90, 95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
dCas9-DNMT3a/3L (h)
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
146
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSNHDQEFDPPKVYPPVP
AEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQ
KHIQEWGPFDLVIGGSPCNDLS IVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFEN
VVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLE
HGRIAKFS KVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLAR
QRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMNPLEMFETVP
VWRRQPVRVLS LFEDIKKELT SLGFLESGS DPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPP
LGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPD
VHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQS SKLAAKWPTKLVKNCFLPLREYF
KYFSTELTSSLSGGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 35)
dCas9-DNMT3a/31 (m)
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
147
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSNHDQEFDPPKVYPPVP
AEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQ
KHIQEWGPFDLVIGGSPCNDLS IVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFEN
VVAMGVSDKRDISRFLESNPVMIDAKEVS AAHRARYFWGNLPGMNRPLASTVNDKLELQECLE
HGRIAKFS KVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLAR
QRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMNPLEMFETVP
VWRRQPVRVLS LFEDIKKELT SLGFLESGS DPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPP
LGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPD
VHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQS SKLAAKWPTKLVKNCFLPLREYF
KYFSTELTSSLSGGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 36)
dCas-DNMT3a/3L (h) without HA tag
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
148
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSNHDQEFDPPKVYPPVP
AEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQ
KHIQEWGPFDLVIGGSPCNDLS IVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFEN
VVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLE
HGRIAKFS KVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLAR
QRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMNPLEMFETVP
VWRRQPVRVLS LFEDIKKELT SLGFLESGS DPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPP
LGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPD
VHGGSLQNAVRVWSNIPAIRSRHWALVSEEELSLLAQNKQS SKLAAKWPTKLVKNCFLPLREYF
KYFSTELTSSLSGGKRPAATKKAGQAKKKKGS (SEQ ID NO: 70)
dCas9-DNMT3a/3L (m) without HA tag
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
149
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSAGGGGSGGGGSGGGGSGPKKKRKVAAAGSNHDQEFDPPKVYPPVP
AEKRKPIRVLSLFDGIATGLLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQ
KHIQEWGPFDLVIGGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFEN
VVAMGVSDKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLE
HGRIAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSRLAR
QRLLGRSWSVPVIRHLFAPLKEYFACVSSGNSNANSRGPSFSSGLVPLSLRGSHMNPLEMFETVP
VWRRQPVRVLSLFEDIKKELTSLGFLESGSDPGQLKHVVDVTDTVRKDVEEWGPFDLVYGATPP
LGHTCDRPPSWYLFQFHRLLQYARPKPGSPRPFFWMFVDNLVLNKEDLDVASRFLEMEPVTIPD
VHGGS LQNAVRVWS NIPAIRSRHWALV SEEELS LLAQNKQS SKLAAKWPTKLVKNCFLPLREYF
KYFSTELTSSLSGGKRPAATKKAGQAKKKKGS (SEQ ID NO: 71)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising G9A, e.g., a G9A
domain. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NOs: 26 (e.g.,
a nucleic acid (e.g., cDNA) encoding the expression repressor). In some
embodiments, a nucleic acid
described herein comprises a nucleic acid sequence of SEQ ID NO: 26 or a
sequence with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
150
G9A-dCas9 (MR-29387)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCTCGGGCGGGGGTGGCTCAGGAAA
TAGGGCTATCAGAACCGAGAAGATCATCTGTAGGGACGTGGCTAGAGGCTACGAGAACGTG
CCCATTCCTTGCGTGAATGGCGTGGATGGCGAACCTTGCCCCGAGGACTACAAATACATCTC
CGAGAACTGCGAAACCAGCACAATGAACATCGACAGAAACATCACCCACCTCCAGCACTGC
ACATGTGTGGATGACTGCTCCTCCAGCAACTGTCTGTGCGGCCAGCTCTCCATCAGATGCTG
GTACGACAAGGACGGCAGACTGCTGCAAGAGTTCAACAAGATCGAACCCCCTCTCATCTTCG
AGTGTAACCAAGCTTGCAGCTGCTGGAGAAACTGCAAGAATAGAGTGGTCCAGAGCGGCAT
CAAGGTGAGACTGCAACTGTACAGAACCGCCAAGATGGGATGGGGAGTGAGGGCTCTGCAA
ACCATTCCCCAAGGCACCTTCATCTGCGAATACGTGGGCGAACTGATCTCCGACGCCGAAGC
TGACGTGAGAGAGGACGACAGCTATCTCTTCGATCTGGACAATAAGGACGGCGAGGTGTAC
TGCATCGACGCTAGATATTACGGCAACATCTCTAGATTCATCAACCACCTCTGCGATCCCAA
CATCATTCCCGTGAGGGTGTTCATGCTGCACCAAGATCTGAGGTTCCCTAGAATCGCCTTCTT
CAGCTCTAGAGACATCAGAACCGGCGAGGAGCTGGGCTTCGATTACGGCGATAGATTCTGG
GACATCAAGTCCAAGTACTTCACATGCCAGTGCGGCAGCGAGAAGTGTAAGCACAGCGCTG
AGGCCATTGCTCTGGAGCAGTCTAGACTGGCCAGACTGGATGGAGGTGGCGGATCGGGAGA
CAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACC
GACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCA
TCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCG
GCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAG
GAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGA
GCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA
CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGAC
AGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCG
GGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTC
ATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCG
TGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGAT
CGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTG
GGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGA
GCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGC
CGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGG
TGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
151
CCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAG
GAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCC
AGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCT
GCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGC
ATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTA
CCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACT
ACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGA
GACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTC
ATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACA
GCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGA
GGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTG
TTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCG
AGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCAC
CTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAG
GACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGG
AGCGGCTGAAAACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCG
GCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAG
AGCGGCAAGACCATCCTGGACTTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCA
GCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGC
CAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGG
GCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCC
CGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAA
CAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCT
GAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTG
CAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACG
ACGTGGCCGCCATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCT
GACCCGGAGCGACAAGGCCCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAA
GAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTC
GACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCA
AGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCG
GATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTG
AAATCCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCA
ACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAA
GAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
152
AAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACA
GCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCG
GCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTC
GCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGC
AGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGC
CCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTAC
AGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAG
GAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCT
GGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGC
CTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGG
GCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAG
AAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAG
CACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCG
ACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGA
GCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCA
AGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGC
CACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTG
GGCGGCGACAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAG
AAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCT
GCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGG
TCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID
NO: 26)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 38 or
73. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 38 or 73 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
G9a-dCas9
MAPKKKRKVGIHGVPAASGGGGSGNRAIRTEKIICRDVARGYENVPIPCVNGVDGEPCPEDYKYI
SENCETSTMNIDRNITHLQHCTCVDDCS S SNCLCGQLSIRCWYDKDGRLLQEFNKIEPPLIFECNQ
ACSCWRNCKNRVVQSGIKVRLQLYRTAKMGWGVRALQTIPQGTFICEYVGELISDAEADVRED
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
153
D SYLFDLDNKDGEVYCIDARYYGNIS RFINHLCDPNIIPVRVFMLHQDLRFPRIAFFS S RD IRTGEE
LGFDYGDRFWDIKS KYFTCQCGSEKCKHSAEAIALEQSRLARLDGGGGSGD KKYS IGLAIGTNS V
GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY
LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD
KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS
ARLS KS RRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLS KDTYDDDLDNL
LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL
PEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG
SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP
WNFEEVVD KGASAQS FIERMTNFD KNLPNEKVLPKHS LLYEYFTVYNELTKVKYVTEGMRKPA
FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK
DFLDNEENED ILEDIVLTLTLFEDREMIEERLKTYAHLFDD KVMKQLKRRRYTGWGRLSRKLING
IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGD SLHEHIANLAGSPAIKK
GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
VENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVAAIVPQSFLKDD S IDNKVLTRSD KA
RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI
TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR
KKDWDPKKYGGFD S PTVAYSVLVVAKVEKGKS KKLKSVKELLGITIMERS SFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKGNELALPSKYVNFLYLASHYEKLKGSPE
DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT
NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS QLGGDSGGKRPAATKKAG
QAKKKKGSYPYDVPDYA (SEQ ID NO: 38)
G9A-dCas9 without HA tag
MAPKKKRKVGIHGVPAASGGGGSGNRAIRTEKIICRDVARGYENVPIPCVNGVDGEPCPEDYKYI
SENCETSTMNIDRNITHLQHCTCVDDCS SSNCLCGQLSIRCWYDKDGRLLQEFNKIEPPLIFECNQ
ACSCWRNCKNRVVQSGIKVRLQLYRTAKMGWGVRALQTIPQGTFICEYVGELISDAEADVRED
D SYLFDLDNKDGEVYCIDARYYGNIS RFINHLCDPNIIPVRVFMLHQDLRFPRIAFFS S RD IRTGEE
LGFDYGDRFWDIKS KYFTCQCGSEKCKHSAEAIALEQSRLARLDGGGGSGD KKYS IGLAIGTNS V
GWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICY
LQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTD
KADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILS
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
154
ARLS KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKS NFDLAEDAKLQLSKDTYDDDLDNL
LAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL
PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG
SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP
WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPA
FLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK
DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLING
IRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKK
GILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP
VENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVAAIVPQSFLKDD S IDNKVLTRSDKA
RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI
TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA
VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE
IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR
KKDWDPKKYGGFD S PTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERS SFEKNPIDFLEAKG
YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE
DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT
NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLS QLGGDSGGKRPAATKKAG
QAKKKKGS (SEQ ID NO: 73)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising HDAC8, e.g., a
HDAC8 domain. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NOs: 27 (e.g.,
a nucleic acid (e.g., cDNA) encoding the expression repressor). In some
embodiments, a nucleic acid
described herein comprises a nucleic acid sequence of SEQ ID NO: 27 or a
sequence with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
dCas9-HDAC8 (MR-29439)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
155
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT
CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC
GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA
CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT
CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG
ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA
TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT
CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG
CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC
ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG
GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC
TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT
GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG
CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG
TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
156
GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG
GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA
CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC
GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC
AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG
GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG
GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG
CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC
GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA
GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC
TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG
CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG
CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA
GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC
GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA
GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA
GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC
AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG
TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT
GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG
GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC
GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG
CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG
GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG
AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT
GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC
CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA
CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC
ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGGCGGCAAGC
GGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGTCGGGCGGGGGTGGCT
CAGAGGAGCCCGAGGAGCCCGCCGATAGCGGACAATCTCTGGTGCCCGTCTACATCTACAG
CCCCGAATATGTGAGCATGTGTGATTCCCTCGCCAAGATCCCTAAGAGAGCCAGCATGGTGC
ATTCTCTGATCGAGGCCTACGCTCTGCATAAGCAAATGAGGATCGTGAAGCCCAAGGTCGCC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
157
AGCATGGAAGAGATGGCCACCTTTCACACCGATGCCTACCTCCAACATCTCCAGAAGGTGTC
CCAAGAGGGCGACGACGACCACCCCGACTCCATTGAGTACGGACTGGGCTATGATTGCCCC
GCCACCGAGGGCATCTTTGACTATGCCGCCGCTATCGGCGGAGCTACCATCACAGCCGCCCA
GTGTCTGATTGATGGCATGTGCAAGGTCGCCATCAACTGGTCCGGAGGCTGGCATCATGCCA
AGAAGGATGAGGCCTCCGGCTTCTGTTATCTGAATGACGCCGTGCTGGGCATTCTGAGACTG
AGGAGGAAATTCGAGAGGATTCTGTACGTGGATCTGGATCTGCATCACGGAGATGGAGTCG
AAGATGCCTTCAGCTTCACCAGCAAGGTGATGACAGTCTCTCTGCACAAGTTCTCCCCCGGC
TTCTTTCCCGGAACCGGCGACGTGTCCGACGTGGGACTGGGCAAGGGAAGGTACTACAGCG
TGAACGTGCCCATTCAAGACGGCATCCAAGACGAGAAGTACTACCAGATCTGCGAGTCCGT
GCTCAAGGAGGTCTACCAAGCCTTCAATCCTAAGGCTGTCGTGCTCCAACTGGGAGCTGATA
CCATTGCTGGCGATCCCATGTGCAGCTTCAATATGACACCCGTCGGAATCGGCAAGTGCCTC
AAGTACATCCTCCAGTGGCAGCTCGCCACCCTCATTCTCGGAGGAGGCGGATACAATCTGGC
TAATACCGCCAGATGCTGGACCTATCTGACCGGCGTGATTCTGGGCAAAACACTGAGCAGCG
AAATCCCCGACCACGAGTTTTTCACCGCTTACGGCCCCGACTACGTGCTGGAGATCACCCCC
AGCTGCAGACCCGATAGAAACGAACCCCATAGAATCCAGCAAATTCTGAACTATATCAAGG
GCAACCTCAAGCACGTCGTGGGAGGTGGCGGATCGGGAAAGCGGCCCGCCGCCACCAAGAA
GGCCGGTCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGA
GCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCC
CTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAA (SEQ ID NO: 27)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 39 or
74. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 39 or 74 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
dCas9-HDAC8
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNL SDAILLSDILRVNT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
158
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKSGGGGSEEPEEPAD SGQSLVPVYIYS PE
YVSMCDSLAKIPKRASMVHSLIEAYALHKQMRIVKPKVASMEEMATFHTDAYLQHLQKVSQEG
DDDHPD S IEYGLGYDCPATEGIFDYAAAIGGATITAAQCLID GMCKVAINWSGGWHHAKKDEAS
GFCYLNDAVLGILRLRRKFERILYVDLDLHHGDGVEDAFSFTSKVMTVSLHKFSPGFFPGTGDVS
DVGLGKGRYYS VNVPIQDGIQDEKYYQICES VLKEVYQAFNPKAVVLQLGADTIAGDPMC SFN
MTPVGIGKCLKYILQWQLATLILGGGGYNLANTARCWTYLTGVILGKTLS SEIPDHEFFTAYGPD
YVLEITPSCRPDRNEPHRIQQILNYIKGNLKHVVGGGGSGKRPAATKKAGQAKKKKGSYPYDVP
DYA (SEQ ID NO: 39)
dCas9-HDAC8 without HA tag
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKV
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
159
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
.. QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGD S GGKRPAAT KKAGQAKKKKS GGGGSEEPEEPAD SGQSLVPVYIYS PE
YVSMCDSLAKIPKRASMVHSLIEAYALHKQMRIVKPKVASMEEMATFHTDAYLQHLQKVSQEG
DDDHPDSIEYGLGYDCPATEGIFDYAAAIGGATITAAQCLIDGMCKVAINWSGGWHHAKKDEAS
GFCYLNDAVLGILRLRRKFERILYVDLDLHHGDGVEDAFSFTSKVMTVSLHKFSPGFFPGTGDVS
DVGLGKGRYYSVNVPIQDGIQDEKYYQICESVLKEVYQAFNPKAVVLQLGADTIAGDPMCSFN
MTPVGIGKCLKYILQWQLATLILGGGGYNLANTARCWTYLTGVILGKTLSSEIPDHEFFTAYGPD
YVLEITPSCRPDRNEPHRIQQILNYIKGNLKHVVGGGGSGKRPAATKKAGQAKKKKGS (SEQ ID
NO: 74)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising LSD1, e.g., a
LSD1 domain. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NOs: 28 (e.g.,
a nucleic acid (e.g., cDNA) encoding the expression repressor). In some
embodiments, a nucleic acid
described herein comprises a nucleic acid sequence of SEQ ID NO: 28 or a
sequence with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
dCas9-LSD1 (MR-29129)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
160
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT
CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC
GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA
CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT
CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG
ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA
TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT
CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG
CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC
ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG
GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC
TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT
GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
161
CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG
TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT
GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG
GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA
CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC
GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC
AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG
GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG
GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG
CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC
GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA
GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC
TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG
CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG
CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA
GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC
GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA
GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA
GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC
AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG
TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT
GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG
GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC
GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG
CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG
GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG
AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT
GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC
CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA
CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC
ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGGCGGCAAGC
GGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGAGAGCGGCGGCGGCA
GCCTCTCCGGAAAGAAAGCCGCCGCTGCCGCTGCCGCCGCTGCTGCTGCCGCTACCGGCACA
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
162
GAAGCTGGCCCCGGAACAGCTGGCGGAAGCGAAAACGGAAGCGAAGTGGCTGCCCAGCCC
GCCGGACTGTCCGGACCCGCCGAGGTGGGACCCGGCGCTGTCGGCGAAAGGACCCCCAGAA
AAAAGGAGCCTCCTAGAGCCAGCCCTCCCGGCGGACTCGCCGAACCTCCCGGCAGCGCTGG
ACCTCAAGCCGGACCTACAGTGGTGCCCGGCAGCGCTACACCTATGGAGACCGGCATCGCT
GAGACCCCCGAGGGAAGGAGAACCAGCAGAAGGAAGAGAGCCAAGGTGGAGTACAGAGAG
ATGGATGAGTCTCTGGCTAACCTCAGCGAGGACGAGTACTACTCCGAGGAGGAAAGGAATG
CCAAGGCCGAGAAGGAGAAGAAGCTGCCCCCTCCTCCCCCTCAAGCCCCCCCCGAGGAGGA
GAACGAGTCCGAGCCCGAGGAACCCAGCGGAGTGGAAGGAGCCGCCTTTCAGTCCAGACTG
CCCCACGACAGAATGACATCCCAAGAGGCCGCTTGCTTTCCCGACATTATTTCCGGCCCTCA
GCAGACCCAGAAGGTGTTTCTGTTCATTAGAAATAGAACACTCCAGCTGTGGCTCGACAACC
CCAAGATCCAGCTGACCTTCGAGGCTACCCTCCAACAGCTGGAGGCCCCCTACAATAGCGAT
ACCGTGCTGGTGCACAGAGTGCACAGCTATCTGGAGAGGCACGGCCTCATTAACTTCGGCAT
TTACAAGCGGATCAAGCCCCTGCCCACCAAGAAAACAGGCAAGGTGATCATCATCGGCAGC
GGCGTGAGCGGCCTGGCCGCCGCCCGGCAGCTGCAGAGCTTCGGCATGGACGTGACCCTGC
TGGAGGCCCGGGACCGGGTGGGCGGCCGGGTGGCCACCTTCCGGAAGGGCAACTACGTGGC
CGACCTGGGCGCCATGGTGGTGACCGGCCTGGGCGGCAACCCCATGGCCGTGGTGAGCAAG
CAGGTGAACATGGAGCTGGCCAAGATCAAGCAGAAGTGCCCCCTGTACGAGGCCAACGGCC
AGGCCGTGCCCAAGGAGAAGGACGAGATGGTGGAGCAGGAGTTCAACCGGCTGCTGGAGG
CCACCAGCTACCTGAGCCACCAGCTGGACTTCAACGTGCTGAACAACAAGCACGTGAGCCT
GGGCCAGGCCCTGGAGGTGGTGATCCAGCTGCAGGAGAAGCACGTGAAGGACGAGCAGATC
GAGCACTGGAAGAAGATCGTGAAAACACAGGAGGAGCTGAAGGAGCTGCTGAACAAGATG
GTGAACCTGAAGGAGAAGATCAAGGAGCTGCACCAGCAGTACAAGGAGGCCAGCGAGGTG
AAGCCCCCCCGGGACATCACCGCCGAGTTCCTGGTCAAAAGCAAGCACCGGGACCTGACCG
CCCTGTGCAAGGAGTACGACGAGCTGGCCGAGACCCAGGGCAAGCTGGAGGAGAAGCTGCA
GGAGCTGGAGGCCAACCCCCCCAGCGACGTGTACCTGAGCAGCCGGGACCGGCAGATCCTG
GACTGGCACTTCGCCAACCTGGAGTTCGCCAACGCCACCCCCCTGAGCACCCTGAGCCTGAA
GCACTGGGACCAGGACGACGACTTCGAGTTCACCGGCAGCCACCTGACCGTGCGGAACGGC
TACAGCTGCGTGCCCGTGGCCCTGGCCGAGGGCCTGGACATCAAGCTGAACACCGCCGTGC
GGCAGGTGCGGTACACCGCCAGCGGCTGCGAGGTGATCGCCGTGAACACCCGGAGCACCAG
CCAGACCTTCATCTACAAGTGCGACGCCGTGCTGTGCACCCTGCCCCTGGGCGTGCTGAAGC
AGCAGCCCCCCGCCGTGCAGTTCGTGCCCCCCCTGCCCGAGTGGAAAACAAGCGCCGTGCA
GCGGATGGGCTTCGGCAACCTGAACAAGGTGGTGCTGTGCTTCGACCGGGTGTTCTGGGACC
CCAGCGTGAACCTGTTCGGCCACGTGGGCAGCACCACCGCCAGCCGGGGCGAGCTGTTCCTG
TTCTGGAACCTGTACAAGGCCCCCATCCTGCTGGCCCTGGTGGCCGGCGAGGCCGCCGGCAT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
163
CATGGAGAACATCAGCGACGACGTGATCGTGGGCCGGTGCCTGGCCATCCTGAAGGGCATC
TTCGGCAGCAGCGCCGTGCCCCAGCCCAAGGAGACCGTGGTGAGCCGGTGGCGGGCCGACC
CCTGGGCCCGGGGCAGCTACAGCTACGTGGCCGCCGGCAGCAGCGGCAACGACTACGACCT
GATGGCCCAGCCCATCACCCCCGGCCCCAGCATCCCCGGCGCCCCCCAGCCCATCCCCCGGC
TGTTCTTCGCCGGCGAGCACACCATCCGGAACTACCCCGCCACCGTGCACGGCGCCCTGCTG
AGCGGCCTGCGGGAGGCAGGACGGATCGCCGACCAGTTCCTGGGCGCCATGTACACCCTGC
CCCGGCAGGCCACCCCCGGCGTGCCCGCCCAGCAGAGCCCCAGCATGAGCGGCGGCAAGCG
GCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGA
CGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTG
GCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGG
AAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 28)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 40 or
75. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 40 or 75, or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
dCas9-LSD1
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDD SIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLIT Q
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
164
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLD KVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT STKEVLDATLIHQ
SITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKESGGGSLSGKKAAAAAAAAAAAATGT
EAGPGTAGGSENGSEVAAQPAGLSGPAEVGPGAVGERTPRKKEPPRASPPGGLAEPPGSAGPQA
GPTVVPGSATPMETGIAETPEGRRTS RRKRAKVEYREMDES LANLSEDEYYSEEERNAKAEKEK
KLPPPPPQAPPEEENESEPEEPSGVEGAAFQSRLPHDRMTS QEAACFPDIISGPQQTQKVFLFIRNR
TLQLWLDNPKIQLTFEATLQQLEAPYNSDTVLVHRVHSYLERHGLINFGIYKRIKPLPTKKTGKVI
IIGSGVSGLAAARQLQSFGMDVTLLEARDRVGGRVATFRKGNYVADLGAMVVTGLGGNPMAV
V SKQVNMELAKIKQKCPLYEANGQAVPKEKDEMVEQEFNRLLEATS YLSHQLDFNVLNNKHVS
LGQALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLNKMVNLKEKIKELHQQYKEASEVKPP
RD ITAEFLVKS KHRDLTALCKEYDELAETQGKLEEKLQELEANPPSDVYLS SRDRQILDWHFANL
EFANATPLS TLS LKHWD QDDDFEFTGSHLTVRNGYS CVPVALAEGLDIKLNTAVRQVRYTAS GC
EVIAVNTRSTSQTFIYKCDAVLCTLPLGVLKQQPPAVQFVPPLPEWKTSAVQRMGFGNLNKVVL
CFDRVFWDPS VNLFGHVGSTTAS RGELFLFWNLYKAPILLALVAGEAAGIMENIS DDVIVGRCLA
ILKGIFGSSAVPQPKETVVSRWRADPWARGSYSYVAAGS SGNDYDLMAQPITPGPSIPGAPQPIPR
LFFAGEHTIRNYPATVHGALLSGLREAGRIAD QFLGAMYTLPRQATPGVPA QQS PS MSGGKRPA
ATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 40)
dCas9-LSD1 without HA tag
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNS VGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYL QEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNL SDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
165
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKESGGGSLSGKKAAAAAAAAAAAATGT
EAGPGTAGGSENGSEVAAQPAGLSGPAEVGPGAVGERTPRKKEPPRASPPGGLAEPPGSAGPQA
GPTVVPGSATPMETGIAETPEGRRTSRRKRAKVEYREMDESLANLSEDEYYSEEERNAKAEKEK
KLPPPPPQAPPEEENESEPEEPSGVEGAAFQSRLPHDRMTS QEAACFPDIISGPQQTQKVFLFIRNR
TLQLWLDNPKIQLTFEATLQQLEAPYNSDTVLVHRVHSYLERHGLINFGIYKRIKPLPTKKTGKVI
IIGSGVSGLAAARQLQSFGMDVTLLEARDRVGGRVATFRKGNYVADLGAMVVTGLGGNPMAV
VSKQVNMELAKIKQKCPLYEANGQAVPKEKDEMVEQEFNRLLEATSYLSHQLDFNVLNNKHVS
LGQALEVVIQLQEKHVKDEQIEHWKKIVKTQEELKELLNKMVNLKEKIKELHQQYKEASEVKPP
RD ITAEFLVKSKHRDLTALCKEYDELAETQGKLEEKLQELEANPPSDVYLS SRDRQILDWHFANL
EFANATPLSTLSLKHWDQDDDFEFTGSHLTVRNGYSCVPVALAEGLDIKLNTAVRQVRYTASGC
EVIAVNTRSTSQTFIYKCDAVLCTLPLGVLKQQPPAVQFVPPLPEWKTSAVQRMGFGNLNKVVL
CFDRVFWDPSVNLFGHVGSTTASRGELFLFWNLYKAPILLALVAGEAAGIMENISDDVIVGRCLA
ILKGIFGSSAVPQPKETVVSRWRADPWARGSYSYVAAGSSGNDYDLMAQPITPGPSIPGAPQPIPR
LFFAGEHTIRNYPATVHGALLSGLREAGRIAD QFLGAMYTLPRQATPGVPA QQSPS MSGGKRPA
ATKKAGQAKKKKGS (SEQ ID NO: 75)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising EZH2, e.g., a
EZH2 domain. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NOs: 29 (e.g.,
a nucleic acid (e.g., cDNA) encoding the expression repressor). In some
embodiments, a nucleic acid
described herein comprises a nucleic acid sequence of SEQ ID NO: 29 or a
sequence with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
166
EZH2-dCas9 (MR-28938)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCGGCAGCGGCGGCAGCGGCCAGACCGGCAAGAAGAGCGAGAAGG
GCCCCGTGTGCTGGCGGAAGCGGGTGAAGAGCGAGTACATGCGGCTGCGGCAGCTGAAGCG
GTTCCGGCGGGCCGACGAGGTGAAGAGCATGTTCAGCAGCAACCGGCAGAAGATCCTGGAG
CGGACCGAGATCCTGAACCAGGAGTGGAAGCAGCGGCGAATCCAGCCCGTGCACATCCTGA
CCAGCGTGAGCAGCCTGCGGGGCACCCGGGAGTGCAGCGTGACCAGCGACCTGGACTTCCC
CACCCAGGTGATCCCCCTAAAGACCCTGAACGCCGTGGCCAGCGTGCCCATCATGTACAGCT
GGAGCCCCCTGCAGCAGAACTTCATGGTGGAGGACGAGACCGTGCTGCACAACATCCCCTA
CATGGGCGACGAGGTGCTGGACCAGGACGGCACCTTCATCGAGGAGCTGATCAAGAACTAC
GACGGCAAGGTGCACGGCGACCGGGAGTGCGGCTTCATCAACGACGAGATCTTCGTGGAGC
TGGTGAACGCCCTGGGCCAGTACAACGACGACGACGACGACGACGACGGCGACGACCCCGA
GGAGCGGGAGGAGAAGCAGAAGGACCTGGAGGACCACCGGGACGACAAGGAGAGCCGGCC
CCCCCGGAAGTTCCCCAGCGACAAGATCTTCGAGGCCATCAGCAGCATGTTCCCCGACAAGG
GCACCGCCGAGGAGCTGAAGGAGAAGTACAAGGAGCTGACCGAGCAGCAGCTGCCCGGCG
CCCTGCCCCCCGAGTGCACCCCCAACATCGACGGCCCCAACGCCAAGAGCGTGCAGCGGGA
GCAGAGCCTGCACAGCTTCCACACCCTGTTCTGCCGGCGGTGCTTCAAGTACGACTGCTTCC
TGCACCCCTTCCACGCCACCCCCAACACCTACAAGCGGAAGAACACCGAGACCGCCCTGGA
CAACAAGCCCTGCGGCCCCCAGTGCTACCAGCACCTGGAGGGCGCCAAGGAGTTCGCCGCC
GCCCTGACCGCCGAGCGGATCAAGACCCCCCCCAAGCGGCCCGGCGGCCGGCGGCGGGGCC
GGCTGCCCAACAACAGCAGCCGGCCCAGCACCCCCACCATCAACGTGCTGGAGAGCAAGGA
CACCGACAGCGACCGGGAGGCCGGCACCGAGACCGGCGGCGAGAACAACGACAAGGAGGA
GGAGGAGAAGAAGGACGAGACCAGCAGCAGCAGCGAGGCCAACAGCCGGTGCCAGACCCC
CATCAAGATGAAGCCCAACATCGAGCCCCCCGAGAACGTGGAGTGGAGCGGCGCCGAGGCC
AGCATGTTCCGGGTGCTGATCGGCACCTACTACGACAACTTCTGCGCCATCGCCCGGCTGAT
CGGCACCAAGACCTGCCGGCAGGTGTACGAGTTCCGGGTGAAGGAGAGCAGCATCATCGCC
CCCGCCCCCGCCGAGGACGTGGACACCCCCCCCCGGAAGAAGAAGCGGAAGCACCGGCTGT
GGGCCGCCCACTGCCGGAAGATCCAGCTGAAGAAGGACGGCAGCAGCAACCACGTGTACAA
CTACCAGCCCTGCGACCACCCCCGGCAGCCCTGCGACAGCAGCTGCCCCTGCGTGATCGCCC
AGAACTTCTGCGAGAAGTTCTGCCAGTGCAGCAGCGAGTGCCAGAACCGGTTCCCCGGCTGC
CGGTGCAAGGCCCAGTGCAACACCAAGCAGTGCCCCTGCTACCTGGCCGTGCGGGAGTGCG
ACCCCGACCTGTGCCTGACCTGCGGCGCCGCCGACCACTGGGACAGCAAGAACGTGAGCTG
CAAGAACTGCAGCATCCAGCGGGGCAGCAAGAAGCACCTGCTGCTGGCCCCCAGCGACGTG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
167
GCCGGCTGGGGCATCTTCATCAAGGACCCCGTGCAGAAGAACGAGTTCATCAGCGAGTACT
GCGGCGAGATCATCAGCCAGGACGAGGCCGACCGGCGGGGCAAGGTGTACGACAAGTACAT
GTGCAGCTTCCTGTTCAACCTGAACAACGACTTCGTGGTGGACGCCACCCGGAAGGGCAACA
AGATCCGGTTCGCCAACCACAGCGTGAACCCCAACTGCTACGCCAAGGTGATGATGGTGAA
CGGCGACCACCGGATCGGCATCTTCGCCAAGCGGGCCATCCAGACCGGCGAGGAGCTGTTC
TTCGACTACCGGTACAGCCAGGCCGACGCCCTGAAGTACGTGGGCATCGAGCGGGAGATGG
AGATCCCCAGCACCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCG
GCCGACCCGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGGGC
CGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGAC
CGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCG
AGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTG
CTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGC
TGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAA
CATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAG
CTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGAT
CAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGAC
AAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGC
CAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAG
AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCC
TGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCT
GCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGAC
CAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACAT
CCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTAC
GACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA
AGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGG
CGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACC
GAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACA
ACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGA
GGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGG
ATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAA
ATCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCC
CAGAGCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGC
CCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTA
CGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
168
GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCA
AGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAG
CCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAG
GAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGA
TGATCGAGGAGCGGCTGAAAACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCT
GAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGG
GACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAATCCGACGGCTTCGCCAACCGGA
ACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCA
GGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCC
ATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCC
GGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGG
GCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCA
GCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCT
GTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTG
AGCGACTACGACGTGGCCGCCATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACA
ACAAGGTGCTGACCCGGAGCGACAAGGCCCGGGGCAAGAGCGACAACGTGCCCAGCGAGG
AGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCA
GCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCC
GGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCC
TGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGG
TGATCACCCTGAAATCCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTG
CGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCG
CCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTA
CGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTA
CTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGA
TCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGG
CCGGGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAA
ACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACA
AGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCAC
CGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAA
ATCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCC
ATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGC
CCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGA
GCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
169
AGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGG
AGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGT
GATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAG
CCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCC
CGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAG
GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCT
GAGCCAGCTGGGCGGCGACAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCA
GGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGCGGCCGCT
TAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTG
TACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AA (SEQ ID NO: 29)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 41 or
76. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 41 or 76 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
EZH2-dCas9
MAPKKKRKVGGSGGSGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQK
ILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSP
LQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALG
QYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEK
YKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKR
KNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVL
ESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEAS
MFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHC
RKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCN
TKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDP
VQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIRFANHSVNPNC
YAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPSTGGSGGSGGSGG
SGGSGRPDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
170
VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ
TYNQLFEENPINASGVDAKAILSARLS KS RRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNF
DLAEDAKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM
IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGASQEEFYKFIKPILEKMDGT
EELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVG
PLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG
VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM
KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAI
VPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE
RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD
FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA
TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT
EV QTGGFS KESILPKRNS D KLIARKKDWDPKKYGGFD SPTVAYSVLVVAKVEKGKS KKLKSVKE
LLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLAS AGELQKGNELAL
PS KYVNFLYLAS HYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQIS EFS KRVILADANLD KVLS A
YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSGGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 41)
EZH2-dCas9 without HA tag
MAPKKKRKVGGSGGSGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFS SNRQK
ILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSP
LQQNFMVEDETVLHNIPYMGDEVLD QDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALG
QYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEK
YKELTEQQLPGALPPECTPNIDGPNAKS VQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKR
KNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNS SRPSTPTINVL
ES KDTD SDREAGTETGGENND KEEEEKKDET S S S SEANS RC QTPIKMKPNIEPPENVEWS GAEAS
MFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHC
RKIQLKKDGS SNHVYNYQPCDHPRQPCDS SCPCVIAQNFCEKFCQCS SEC QNRFPGCRCKAQCN
TKQCPCYLAVRECDPDLCLTCGAADHWD S KNV SC KNC SIQRGS KKHLLLAPSDVAGWGIFIKDP
V QKNEFISEYCGEIIS QDEADRRGKVYD KYMCS FLFNLNNDFVVDATRKGN KIRFANHSVNPNC
YAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPSTGGSGGSGGSGG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
171
SGGSGRPDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE
VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ
TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF
DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM
IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKFIKPILEKMDGT
EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVG
PLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG
VEDRFNAS LGTYHDLLKIIKDKDFLDNEENEDILED IVLTLTLFEDREMIEERLKTYAHLFDDKVM
KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAI
VPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE
RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD
FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA
TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT
EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE
LLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLAS AGELQKGNELAL
PSKYVNFLYLAS HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS EFS KRVILADANLDKVLS A
YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSGGKRPAATKKAGQAKKKKGS (SEQ ID NO: 76)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a first repressor domain comprising EZH2 e.g.,
a EZH2 domain and a
second repressor domain comprising KRAB, e.g., a KRAB domain. In some
embodiments, the expression
repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 30 (e.g., a
nucleic acid (e.g., cDNA)
encoding the expression repressor). In some embodiments, a nucleic acid
described herein comprises a
nucleic acid sequence of SEQ ID NO: 30 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
EZH2- dCas9-KRAB (MR-29448)
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
172
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCGGCAGCGGCGGCAGCGGCCAGACCGGCAAGAAGAGCGAGAAGG
GCCCCGTGTGCTGGCGGAAGCGGGTGAAGAGCGAGTACATGCGGCTGCGGCAGCTGAAGCG
GTTCCGGCGGGCCGACGAGGTGAAGAGCATGTTCAGCAGCAACCGGCAGAAGATCCTGGAG
CGGACCGAGATCCTGAACCAGGAGTGGAAGCAGCGGCGAATCCAGCCCGTGCACATCCTGA
CCAGCGTGAGCAGCCTGCGGGGCACCCGGGAGTGCAGCGTGACCAGCGACCTGGACTTCCC
CACCCAGGTGATCCCCCTAAAGACCCTGAACGCCGTGGCCAGCGTGCCCATCATGTACAGCT
GGAGCCCCCTGCAGCAGAACTTCATGGTGGAGGACGAGACCGTGCTGCACAACATCCCCTA
CATGGGCGACGAGGTGCTGGACCAGGACGGCACCTTCATCGAGGAGCTGATCAAGAACTAC
GACGGCAAGGTGCACGGCGACCGGGAGTGCGGCTTCATCAACGACGAGATCTTCGTGGAGC
TGGTGAACGCCCTGGGCCAGTACAACGACGACGACGACGACGACGACGGCGACGACCCCGA
GGAGCGGGAGGAGAAGCAGAAGGACCTGGAGGACCACCGGGACGACAAGGAGAGCCGGCC
CCCCCGGAAGTTCCCCAGCGACAAGATCTTCGAGGCCATCAGCAGCATGTTCCCCGACAAGG
GCACCGCCGAGGAGCTGAAGGAGAAGTACAAGGAGCTGACCGAGCAGCAGCTGCCCGGCG
CCCTGCCCCCCGAGTGCACCCCCAACATCGACGGCCCCAACGCCAAGAGCGTGCAGCGGGA
GCAGAGCCTGCACAGCTTCCACACCCTGTTCTGCCGGCGGTGCTTCAAGTACGACTGCTTCC
TGCACCCCTTCCACGCCACCCCCAACACCTACAAGCGGAAGAACACCGAGACCGCCCTGGA
CAACAAGCCCTGCGGCCCCCAGTGCTACCAGCACCTGGAGGGCGCCAAGGAGTTCGCCGCC
GCCCTGACCGCCGAGCGGATCAAGACCCCCCCCAAGCGGCCCGGCGGCCGGCGGCGGGGCC
GGCTGCCCAACAACAGCAGCCGGCCCAGCACCCCCACCATCAACGTGCTGGAGAGCAAGGA
CACCGACAGCGACCGGGAGGCCGGCACCGAGACCGGCGGCGAGAACAACGACAAGGAGGA
GGAGGAGAAGAAGGACGAGACCAGCAGCAGCAGCGAGGCCAACAGCCGGTGCCAGACCCC
CATCAAGATGAAGCCCAACATCGAGCCCCCCGAGAACGTGGAGTGGAGCGGCGCCGAGGCC
AGCATGTTCCGGGTGCTGATCGGCACCTACTACGACAACTTCTGCGCCATCGCCCGGCTGAT
CGGCACCAAGACCTGCCGGCAGGTGTACGAGTTCCGGGTGAAGGAGAGCAGCATCATCGCC
CCCGCCCCCGCCGAGGACGTGGACACCCCCCCCCGGAAGAAGAAGCGGAAGCACCGGCTGT
GGGCCGCCCACTGCCGGAAGATCCAGCTGAAGAAGGACGGCAGCAGCAACCACGTGTACAA
CTACCAGCCCTGCGACCACCCCCGGCAGCCCTGCGACAGCAGCTGCCCCTGCGTGATCGCCC
AGAACTTCTGCGAGAAGTTCTGCCAGTGCAGCAGCGAGTGCCAGAACCGGTTCCCCGGCTGC
CGGTGCAAGGCCCAGTGCAACACCAAGCAGTGCCCCTGCTACCTGGCCGTGCGGGAGTGCG
ACCCCGACCTGTGCCTGACCTGCGGCGCCGCCGACCACTGGGACAGCAAGAACGTGAGCTG
CAAGAACTGCAGCATCCAGCGGGGCAGCAAGAAGCACCTGCTGCTGGCCCCCAGCGACGTG
GCCGGCTGGGGCATCTTCATCAAGGACCCCGTGCAGAAGAACGAGTTCATCAGCGAGTACT
GCGGCGAGATCATCAGCCAGGACGAGGCCGACCGGCGGGGCAAGGTGTACGACAAGTACAT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
173
GTGCAGCTTCCTGTTCAACCTGAACAACGACTTCGTGGTGGACGCCACCCGGAAGGGCAACA
AGATCCGGTTCGCCAACCACAGCGTGAACCCCAACTGCTACGCCAAGGTGATGATGGTGAA
CGGCGACCACCGGATCGGCATCTTCGCCAAGCGGGCCATCCAGACCGGCGAGGAGCTGTTC
TTCGACTACCGGTACAGCCAGGCCGACGCCCTGAAGTACGTGGGCATCGAGCGGGAGATGG
AGATCCCCAGCACCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCG
GCCGACCCGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGGGC
CGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGAC
CGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCG
AGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTG
CTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGC
TGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAA
CATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAG
CTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGAT
CAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGAC
AAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGC
CAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAG
AACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCC
TGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCT
GCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGAC
CAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACAT
CCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTAC
GACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGA
AGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGG
CGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACC
GAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACA
ACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGA
GGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGG
ATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAA
ATCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCC
CAGAGCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGC
CCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTA
CGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTG
GACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCA
AGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
174
CCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAG
GAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGA
TGATCGAGGAGCGGCTGAAAACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCT
GAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGG
GACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAATCCGACGGCTTCGCCAACCGGA
ACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCA
GGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCC
ATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCC
GGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGG
GCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCA
GCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCT
GTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTG
AGCGACTACGACGTGGCCGCCATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACA
ACAAGGTGCTGACCCGGAGCGACAAGGCCCGGGGCAAGAGCGACAACGTGCCCAGCGAGG
AGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCA
GCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCC
GGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCC
TGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGG
TGATCACCCTGAAATCCAAGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTG
CGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCG
CCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTA
CGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTA
CTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGA
TCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGG
CCGGGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAA
ACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACA
AGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCAC
CGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAA
ATCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCC
ATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGC
CCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGA
GCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCC
AGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGG
AGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
175
GATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAG
CCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCC
CGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAG
GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCT
GAGCCAGCTGGGCGGCGACAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCA
GGCCAAGAAGAAGAAGTCGGGCGGGGGTGGCTCAGACGCTAAGTCTCTGACCGCTTGGAGC
AGAACACTGGTCACCTTCAAGGACGTGTTCGTCGACTTCACAAGAGAGGAGTGGAAACTGC
TGGACACCGCCCAGCAGATCCTCTATAGAAACGTCATGCTGGAGAACTACAAGAATCTGGT
GTCTCTGGGCTACCAGCTGACCAAGCCCGACGTGATTCTGAGGCTGGAGAAGGGCGAGGAG
CCTTGGCTGGTGGAGAGAGAGATCCACCAAGAAACCCACCCCGACAGCGAAACCGCCTTCG
AGATCAAGAGCAGCGTGGGAGGTGGCGGATCGGGAAAGCGGCCCGCCGCCACCAAGAAGG
CCGGTCAGGCCAAGAAGAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGC
GGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTT
GCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAA (SEQ ID NO: 30)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 42 or
77. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 42 or 77 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
EZH2-dCas9-KRAB
MAPKKKRKVGGSGGSGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFSSNRQK
ILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSP
LQQNFMVEDETVLHNIPYMGDEVLDQDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALG
QYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEK
YKELTEQQLPGALPPECTPNIDGPNAKSVQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKR
KNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNSSRPSTPTINVL
ESKDTDSDREAGTETGGENNDKEEEEKKDETSSSSEANSRCQTPIKMKPNIEPPENVEWSGAEAS
MFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHC
RKIQLKKDGSSNHVYNYQPCDHPRQPCDSSCPCVIAQNFCEKFCQCSSECQNRFPGCRCKAQCN
TKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDP
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
176
V QKNEFISEYCGEIIS QDEADRRGKVYD KYMCS FLFNLNNDFVVDATRKGN KIRFANHSVNPNC
YAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPSTGGSGGSGGSGG
SGGSGRPDKKYSIGLAIGTNSVGWAVITDEYKVPS KKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE
VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ
TYNQLFEENPINASGVDAKAILSARLS KS RRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNF
DLAEDAKLQLS KDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM
IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGASQEEFYKFIKPILEKMDGT
EELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVG
PLARGNSRFAWMTRKSEETITPWNFEEVVD KGASAQS FIERMTNFD KNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG
VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM
KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAI
VPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE
RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD
FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA
TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT
EV QTGGFS KESILPKRNS D KLIARKKDWDPKKYGGFD SPTVAYSVLVVAKVEKGKS KKLKSVKE
LLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLAS AGELQKGNELAL
PS KYVNFLYLAS HYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQIS EFS KRVILADANLD KVLS A
YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSGGKRPAATKKAGQAKKKKSGGGGSDAKSLTAWSRTLVTFKDVFVDFTREEWKL
LDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKS
SVGGGGSGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 42)
EZH2-dCas9-KRAB without HA tag
MAPKKKRKVGGSGGSGQTGKKSEKGPVCWRKRVKSEYMRLRQLKRFRRADEVKSMFS SNRQK
ILERTEILNQEWKQRRIQPVHILTSVSSLRGTRECSVTSDLDFPTQVIPLKTLNAVASVPIMYSWSP
LQQNFMVEDETVLHNIPYMGDEVLD QDGTFIEELIKNYDGKVHGDRECGFINDEIFVELVNALG
QYNDDDDDDDGDDPEEREEKQKDLEDHRDDKESRPPRKFPSDKIFEAISSMFPDKGTAEELKEK
YKELTEQQLPGALPPECTPNIDGPNAKS VQREQSLHSFHTLFCRRCFKYDCFLHPFHATPNTYKR
KNTETALDNKPCGPQCYQHLEGAKEFAAALTAERIKTPPKRPGGRRRGRLPNNS SRPSTPTINVL
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
177
ESKDTDSDREAGTETGGENNDKEEEEKKDETSSS SEANSRCQTPIKMKPNIEPPENVEWSGAEAS
MFRVLIGTYYDNFCAIARLIGTKTCRQVYEFRVKESSIIAPAPAEDVDTPPRKKKRKHRLWAAHC
RKIQLKKDGS SNHVYNYQPCDHPRQPCDS SCPCVIAQNFCEKFCQCS SECQNRFPGCRCKAQCN
TKQCPCYLAVRECDPDLCLTCGAADHWDSKNVSCKNCSIQRGSKKHLLLAPSDVAGWGIFIKDP
V QKNEFISEYCGEIIS QDEADRRGKVYD KYMCS FLFNLNNDFVVDATRKGNKIRFANHSVNPNC
YAKVMMVNGDHRIGIFAKRAIQTGEELFFDYRYSQADALKYVGIEREMEIPSTGGSGGSGGSGG
SGGSGRPDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA
EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE
VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ
TYNQLFEENPINAS GVDAKAILS ARLS KS RRLENLIAQLPGEKKNGLFGNLIALS LGLTPNFKSNF
DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM
IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKFIKPILEKMDGT
EELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVG
PLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG
VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVM
KQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV
SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNS
RERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVAAI
VPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAE
RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD
FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA
TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKT
EVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE
LLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLAS AGELQKGNELAL
PSKYVNFLYLAS HYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQIS EFS KRVILADANLD KVLS A
YNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSGGKRPAATKKAGQAKKKKSGGGGSDAKSLTAWSRTLVTFKDVFVDFTREEWKL
LDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKS
SVGGGGSGKRPAATKKAGQAKKKKGS (SEQ ID NO: 77)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a first repressor domain comprising G9A e.g.,
a G9A domain and a
second repressor domain comprising KRAB, e.g., a KRAB domain. In some
embodiments, the expression
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
178
repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 31 (e.g., a
nucleic acid (e.g., cDNA)
encoding the expression repressor). In some embodiments, a nucleic acid
described herein comprises a
nucleic acid sequence of SEQ ID NO: 31 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
G9A-dCas9-KRAB (MR-29442)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCTCGGGCGGGGGTGGCTCAGGAAG
CGCCGCTATCGCCGAAGTGCTGCTGAATGCCAGATGCGATCTGCATGCCGTGAACTACCACG
GCGACACCCCTCTGCATATCGCCGCTAGAGAGAGCTACCATGACTGTGTGCTGCTGTTTCTG
AGCAGAGGCGCCAATCCCGAGCTCAGAAACAAAGAGGGCGACACCGCTTGGGATCTGACAC
CCGAGAGATCCGACGTGTGGTTCGCTCTGCAACTGAATAGAAAACTGAGACTGGGCGTCGG
CAATAGGGCCATTAGAACCGAGAAGATCATCTGTAGGGACGTGGCTAGGGGCTACGAGAAC
GTGCCCATCCCTTGTGTGAACGGAGTGGATGGAGAGCCTTGCCCCGAGGATTACAAATACAT
CAGCGAGAACTGCGAAACCTCCACCATGAATATCGATAGAAACATTACACACCTCCAGCAC
TGTACATGCGTGGACGATTGCAGCAGCAGCAACTGTCTGTGCGGCCAACTGAGCATCAGATG
CTGGTACGACAAGGATGGCAGACTGCTGCAAGAGTTCAACAAGATCGAACCCCCTCTGATCT
TCGAGTGTAACCAAGCTTGCAGCTGTTGGAGGAACTGCAAGAATAGGGTCGTGCAGTCCGG
AATCAAGGTGAGACTGCAGCTGTATAGAACAGCTAAGATGGGATGGGGAGTCAGAGCTCTG
CAGACCATCCCCCAAGGCACATTCATCTGTGAGTACGTCGGCGAACTCATCAGCGACGCTGA
GGCCGATGTGAGGGAGGACGACAGCTATCTCTTCGACCTCGACAACAAGGACGGCGAGGTG
TACTGCATCGACGCTAGATATTACGGCAACATCAGCAGATTCATCAACCACCTCTGCGACCC
CAATATCATCCCCGTGAGAGTGTTCATGCTCCATCAAGATCTGAGATTCCCTAGGATCGCCTT
CTTCAGCTCTAGAGACATTAGAACCGGCGAGGAGCTGGGATTCGACTACGGCGACAGGTTCT
GGGACATCAAGAGCAAGTACTTCACATGCCAATGCGGCAGCGAGAAATGCAAGCATAGCGC
CGAGGCCATTGCTCTGGAGCAGTCTAGACTGGCTAGGCTGGACCCTCACCCCGAGCTGCTGC
CCGAACTGGGATCTCTGCCTCCCGTGAATACCGGAGGTGGCGGATCGGGAGACAAGAAGTA
CAGCATCGGCCTGGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTAC
AAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGA
ACCTGATCGGCGCCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCG
GACCGCCCGGCGGCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTC
AGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGG
TGGAGGAGGACAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
179
CTACCACGAGAAGTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGAC
AAGGCCGACCTGCGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTT
CCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTG
GTGCAGACCTACAACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCA
AGGCCATCCTGAGCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCT
GCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACC
CCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACA
CCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTC
CTGGCCGCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCG
AGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGA
CCTGACCCTGCTGAAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCT
TCGACCAGAGCAAGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTT
CTACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAG
CTGAACCGGGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACC
AGATCCACCTGGGCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTG
AAGGACAACCGGGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCC
CCCTGGCCCGGGGCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCAC
CCCCTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGG
ATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGT
ACGAGTACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCG
GAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACC
AACCGGAAGGTGACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCG
ACAGCGTGGAGATCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGA
CCTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTG
GAGGACATCGTGCTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGA
AAACCTACGCCCACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACAC
CGGCTGGGGCCGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAA
GACCATCCTGGACTTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCC
ACGACGACAGCCTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGA
CAGCCTGCACGAGCACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTG
CAGACCGTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAAC
ATCGTGATCGAGATGGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGG
GAGCGGATGAAGCGGATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAG
CACCCCGTGGAGAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
180
GCCGGGACATGTACGTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGC
CGCCATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGG
AGCGACAAGGCCCGGGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATG
AAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACC
TGACCAAGGCCGAGCGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCA
GCTGGTGGAGACCCGGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAAC
ACCAAGTACGACGAGAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCA
AGCTGGTGAGCGACTTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTA
CCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTAC
CCCAAGCTGGAGAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGA
TCGCCAAGAGCGAGCAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACAT
CATGAACTTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTG
ATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCG
TGCGGAAGGTGCTGAGCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGG
CGGCTTCAGCAAGGAGAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAG
AAGGACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGC
TGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGC
TGGGCATCACCATCATGGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGC
CAAGGGCTACAAGGAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTC
GAGCTGGAGAACGGCCGGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAAC
GAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCT
GAAGGGCAGCCCCGAGGACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTA
CCTGGACGAGATCATCGAGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCC
AACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGG
CCGAGAACATCATCCACCTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTAC
TTCGACACCACCATCGACCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCC
TGATCCACCAGAGCATCACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGG
CGACAGCGGCGGCAAGCGGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAA
GTCGGGCGGGGGTGGCTCAGACGCTAAGTCTCTGACCGCTTGGAGCAGAACACTGGTCACCT
TCAAGGACGTGTTCGTCGACTTCACAAGAGAGGAGTGGAAACTGCTGGACACCGCCCAGCA
GATCCTCTATAGAAACGTCATGCTGGAGAACTACAAGAATCTGGTGTCTCTGGGCTACCAGC
TGACCAAGCCCGACGTGATTCTGAGGCTGGAGAAGGGCGAGGAGCCTTGGCTGGTGGAGAG
AGAGATCCACCAAGAAACCCACCCCGACAGCGAAACCGCCTTCGAGATCAAGAGCAGCGTG
GGAGGTGGCGGATCGGGAAAGCGGCCCGCCGCCACCAAGAAGGCCGGTCAGGCCAAGAAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
181
AAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCT
GCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGG
TCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID
NO: 31)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 43 or
78. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 42 or 77 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
.. no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4,
3, 2, or 1 positions of difference
thereto.
G9A-dCas9-KRAB
MAPKKKRKVGIHGVPAASGGGGSGSAAIAEVLLNARCDLHAVNYHGDTPLHIAARESYHDCVL
LFLSRGANPELRNKEGDTAWDLTPERSDVWFALQLNRKLRLGVGNRAIRTEKIICRDVARGYEN
VPIPCVNGVDGEPCPEDYKYISENCETSTMNIDRNITHLQHCTCVDDCSSSNCLCGQLSIRCWYD
KDGRLLQEFNKIEPPLIFECNQACSCWRNCKNRVVQSGIKVRLQLYRTAKMGWGVRALQTIPQG
TFICEYVGELISDAEAD VREDD SYLFDLDNKDGEVYCIDARYYGNISRFINHLCDPNIIPVRVFML
HQDLRFPRIAFFSSRDIRTGEELGFDYGDRFWDIKSKYFTCQCGSEKCKHSAEAIALEQSRLARLD
PHPELLPELGS LPPVNTGGGGS GDKKYSIGLAIGTNSVGWAVITDEYKVPS KKFKVLGNTDRHSI
KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE
DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL
NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG
NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD
ILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS Q
EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD
NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL
KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM
IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV
IEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYV
DQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLL
NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
182
VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY
DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT
VRKVLSMPQVNIVKKTEVQTGGFS KESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
AKVEKGKSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKR
MLASAGELQKGNELALPS KYVNFLYLAS HYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQIS EFS
KRVILADANLD KVLSAYNKHRD KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYT STKEVL
DATLIHQSITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKSGGGGSDAKSLTAWSRTLVT
FKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREI
HQETHPDSETAFEIKSSVGGGGSGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 43)
G9A-dCas9-KRAB without HA tag
MAPKKKRKVGIHGVPAASGGGGSGS AAIAEVLLNARCDLHAVNYHGDTPLHIAARESYHDCVL
LFLSRGANPELRNKEGDTAWDLTPERSDVWFALQLNRKLRLGVGNRAIRTEKIICRDVARGYEN
VPIPCVNGVDGEPCPEDYKYISENCETS TMNIDRNITHLQHCTCVDDC S S SNCLCGQLSIRCWYD
KDGRLLQEFNKIEPPLIFECNQACS CWRNC KNRVV QSGIKVRLQLYRTAKMGWGVRALQTIPQG
TFICEYVGELISDAEADVREDDSYLFDLDNKDGEVYCIDARYYGNISRFINHLCDPNIIPVRVFML
HQDLRFPRIAFFS SRDIRTGEELGFDYGDRFWDIKSKYFTCQCGSEKCKHSAEAIALEQSRLARLD
PHPELLPELGS LPPVNTGGGGS GD KKYSIGLAIGTNSVGWAVITDEYKVPS KKFKVLGNTDRHSI
KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE
DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDL
NPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG
NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSD
ILRVNTEITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGASQ
EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKD
NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQL
KEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM
IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV
IEMARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYV
DQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLL
NAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE
VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVY
DVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
183
VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVV
AKVEKGKSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKR
MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS
KRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKSGGGGSDAKSLTAWSRTLVT
FKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREI
HQETHPDSETAFEIKSSVGGGGSGKRPAATKKAGQAKKKKGS (SEQ ID NO: 78)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a first repressor domain comprising FOG1 e.g.,
a FOG1 domain and a
second repressor domain comprising FOG1, e.g., a FOG1 domain. In some
embodiments, the expression
repressor is encoded by the nucleic acid sequence of SEQ ID NOs: 32 (e.g., a
nucleic acid (e.g., cDNA)
encoding the expression repressor). In some embodiments, a nucleic acid
described herein comprises a
nucleic acid sequence of SEQ ID NO: 32 or a sequence with at least 80, 85, 90,
95, 99, or 100% identity
thereto, or having no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1
positions of difference thereto.
FOG1-dCas9-FOG1 (MR-29434)
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCTCGGGCGGGGGTGGCTCAAGCAG
AAGGAAGCAGAGCAACCCCAGACAAATCAAGAGATCTCTGGGCGACATGGAGGCCAGAGA
GGAAGTGCAGCTGGTGGGCGCCAGCCACATGGAGCAGAAGGCTACAGCCCCCGAGGCCCCC
AGCCCCGGAGGTGGCGGATCGGGAGACAAGAAGTACAGCATCGGCCTGGCCATCGGCACCA
ACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGT
GCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTGTTCGAC
AGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCGGCGGTACACCCGGC
GGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGA
CAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGG
CACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTA
CCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTACCTG
GCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGA
CAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAG
GAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGAGCGCCCGGCTGAGCA
AGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAACGGCCTGTT
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
184
CGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGG
CCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCT
GGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGAACCTGAGCGACGCC
ATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCA
GCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCG
GCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAGAACGGCTACGCC
GGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCATCAAGCCCATCCTGG
AGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAGGACCTGCTGCGGA
AGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCACGC
CATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCGGGAGAAGATCGAG
AAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGGGCAACAGCCGGTT
CGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTG
GACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGACAAGAACCTGC
CCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTACAACGA
GCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTTCCTGAGCGGCGAG
CAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTGACCGTGAAGCAGC
TGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATCAGCGGCGTGGA
GGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGATCATCAAGGACAAG
GACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTGCTGACCCTGACCC
TGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCCACCTGTTCGACGA
CAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCGGCTGAGCCGGAA
GCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGACTTCCTGAAATCC
GACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGA
GGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATCGCCAAC
CTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGGTGGTGGACGAGC
TGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGATGGCCCGGGAGAA
CCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCGGATCGAGGAGGG
CATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACCCAGCTGCAG
AACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTACGTGGACCAGGAGC
TGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCCAGAGCTTCCTGAAG
GACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCGGGGCAAGAGCGAC
AACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACG
CCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAGCGGGGCGGCCTGAG
CGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCCGGCAGATCACCAAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
185
CACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGAGAACGACAAGCTGA
TCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGACTTCCGGAAGGACTT
CCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAAC
GCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACG
GCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAGATCGGCA
AGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAAGACCGAGATCACC
CTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGCGAGACCGGCGAGA
TCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGAGCATGCCCCAGGT
GAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAGAGCATCCTGCCC
AAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCCAAGAAGTACGGC
GGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCA
AGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGGAGCAG
CTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAGGTGAAGAAGGAC
CTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCCGGAAGCGGATGC
TGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAA
CTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAACGAGCAG
AAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCG
AGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCTGAGCGCCTACAA
CAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTG
ACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGACCGGAAGCGGTA
CACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTAC
GAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGGCGGCAAGCGGCCCGCCGCCA
CCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGTCGGGCGGGGGTGGCTCAAGCAGAAGGA
AGCAGAGCAACCCCAGACAAATCAAGAGATCTCTGGGCGACATGGAGGCCAGAGAGGAAG
TGCAGCTGGTGGGCGCCAGCCACATGGAGCAGAAGGCTACAGCCCCCGAGGCCCCCAGCCC
CGGAGGTGGCGGATCGGGAAAGCGGCCCGCCGCCACCAAGAAGGCCGGTCAGGCCAAGAA
GAAGAAGGGCAGCTACCCCTACGACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGC
TGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTG
GTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID
NO: 32)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 44 or
79. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
186
SEQ ID NO: 44 or 79 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
FOG1-dCas9-FOG1
MAPKKKRKVGIHGVPAASGGGGSSRRKQSNPRQIKRSLGDMEAREEVQLVGASHMEQKATAPE
APS PGGGGS GD KKYSIGLAIGTNS VGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFD S G
ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI
VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ
LV QTYNQLFEENPINAS GVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFK
S NFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILL SD ILRVNTEIT KAPLS
ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM
DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL
YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSV
EISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDD
KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ
KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG
QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD
VAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT
KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDF
RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI
GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNI
VKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKL
KSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKG
NELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY
ETRIDLS QLGGDSGGKRPAATKKAGQAKKKKSGGGGS SRRKQSNPRQIKRSLGDMEAREEVQL
VGASHMEQKATAPEAPSPGGGGSGKRPAATKKAGQAKKKKGSYPYDVPDYA (SEQ ID NO: 44)
FOG1-dCas9-FOG1 without HA tag
MAPKKKRKVGIHGVPAASGGGGSSRRKQSNPRQIKRSLGDMEAREEVQLVGASHMEQKATAPE
APS PGGGGS GD KKYSIGLAIGTNS VGWAVITDEYKVPSKKFKVLGNTDRHS IKKNLIGALLFD S G
ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
187
VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQ
LVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFK
S NFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILL SD ILRVNTEIT KAPLS
ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKM
DGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY
YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL
YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSV
EISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDD
KVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ
KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG
QKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD
VAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT
KAERGGLSELDKAGFIKRQLVETRQITKHVAQILD SRMNTKYDENDKLIREVKVITLKS KLVS DF
RKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI
GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNI
VKKTEV QTGGFS KESILPKRNSDKLIARKKDWDPKKYGGFD S PTVAYSVLVVAKVEKGKSKKL
KSV KELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AGELQKG
NELALPS KYVNFLYLAS HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQIS EFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLY
ETRIDLS QLGGD S GGKRPAAT KKAGQAKKKKSGGGGS SRRKQSNPRQIKRS LGDMEAREEVQL
VGASHMEQKATAPEAPSPGGGGSGKRPAATKKAGQAKKKKGS (SEQ ID NO: 79)
In some embodiments, an expression repressor comprises a DNA-targeting moiety
comprising dCas9,
e.g., an S. pyogenes dCas9, and a repressor domain comprising DNMT, e.g., a
DNMT3b domain. In some
embodiments, the expression repressor is encoded by the nucleic acid sequence
of SEQ ID NOs: 15 (e.g.,
a nucleic acid (e.g., cDNA) encoding the expression repressor). In some
embodiments, a nucleic acid
described herein comprises a nucleic acid sequence of SEQ ID NO: 15 or a
sequence with at least 80, 85,
90, 95, 99, or 100% identity thereto, or having no more than 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, 10,9,
8, 7, 6, 5, 4, 3, 2, or 1 positions of difference thereto.
dCas9-DNMT3b
AGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCCCCAAGA
AGAAGCGGAAGGTGGGCATCCACGGCGTGCCCGCCGCCGACAAGAAGTACAGCATCGGCCT
GGCCATCGGCACCAACAGCGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGC
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
188
AAGAAGTTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGCG
CCCTGCTGTTCGACAGCGGCGAGACCGCCGAGGCCACCCGGCTGAAGCGGACCGCCCGGCG
GCGGTACACCCGGCGGAAGAACCGGATCTGCTACCTGCAGGAGATCTTCAGCAACGAGATG
GCCAAGGTGGACGACAGCTTCTTCCACCGGCTGGAGGAGAGCTTCCTGGTGGAGGAGGACA
AGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGCGGAAGAAGCTGGTGGACAGCACCGACAAGGCCGACCTG
CGGCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGG
CGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTAC
AACCAGCTGTTCGAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGA
GCGCCCGGCTGAGCAAGAGCCGGCGGCTGGAGAACCTGATCGCCCAGCTGCCCGGCGAGAA
GAAGAACGGCCTGTTCGGCAACCTGATCGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGA
GCAACTTCGACCTGGCCGAGGACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGA
CCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCCGCCAAGA
ACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGGGTGAACACCGAGATCACCAAGGC
CCCCCTGAGCGCCAGCATGATCAAGCGGTACGACGAGCACCACCAGGACCTGACCCTGCTG
AAGGCCCTGGTGCGGCAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCA
AGAACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTCTACAAGTTCAT
CAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAGCTGCTGGTGAAGCTGAACCGGGAG
GACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGG
GCGAGCTGCACGCCATCCTGCGGCGGCAGGAGGACTTCTACCCCTTCCTGAAGGACAACCG
GGAGAAGATCGAGAAGATCCTGACCTTCCGGATCCCCTACTACGTGGGCCCCCTGGCCCGGG
GCAACAGCCGGTTCGCCTGGATGACCCGGAAATCCGAGGAGACCATCACCCCCTGGAACTT
CGAGGAGGTGGTGGACAAGGGCGCCAGCGCCCAGAGCTTCATCGAGCGGATGACCAACTTC
GACAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCA
CCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAGGGCATGCGGAAGCCCGCCTT
CCTGAGCGGCGAGCAGAAGAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAGGTG
ACCGTGAAGCAGCTGAAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGA
TCAGCGGCGTGGAGGACCGGTTCAACGCCAGCCTGGGCACCTACCACGACCTGCTGAAGAT
CATCAAGGACAAGGACTTCCTGGACAACGAGGAGAACGAGGACATCCTGGAGGACATCGTG
CTGACCCTGACCCTGTTCGAGGACCGGGAGATGATCGAGGAGCGGCTGAAAACCTACGCCC
ACCTGTTCGACGACAAGGTGATGAAGCAGCTGAAGCGGCGGCGGTACACCGGCTGGGGCCG
GCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGAGCGGCAAGACCATCCTGGAC
TTCCTGAAATCCGACGGCTTCGCCAACCGGAACTTCATGCAGCTGATCCACGACGACAGCCT
GACCTTCAAGGAGGACATCCAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
189
CACATCGCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACCGTGAAGG
TGGTGGACGAGCTGGTGAAGGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAGAT
GGCCCGGGAGAACCAGACCACCCAGAAGGGCCAGAAGAACAGCCGGGAGCGGATGAAGCG
GATCGAGGAGGGCATCAAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAA
CACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGGGACATGTAC
GTGGACCAGGAGCTGGACATCAACCGGCTGAGCGACTACGACGTGGCCGCCATCGTGCCCC
AGAGCTTCCTGAAGGACGACAGCATCGACAACAAGGTGCTGACCCGGAGCGACAAGGCCCG
GGGCAAGAGCGACAACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCG
GCAGCTGCTGAACGCCAAGCTGATCACCCAGCGGAAGTTCGACAACCTGACCAAGGCCGAG
CGGGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTGGAGACCC
GGCAGATCACCAAGCACGTGGCCCAGATCCTGGACAGCCGGATGAACACCAAGTACGACGA
GAACGACAAGCTGATCCGGGAGGTGAAGGTGATCACCCTGAAATCCAAGCTGGTGAGCGAC
TTCCGGAAGGACTTCCAGTTCTACAAGGTGCGGGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTGGTGGGCACCGCCCTGATCAAGAAGTACCCCAAGCTGGAGAG
CGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAG
CAGGAGATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTCTTCAA
GACCGAGATCACCCTGGCCAACGGCGAGATCCGGAAGCGGCCCCTGATCGAGACCAACGGC
GAGACCGGCGAGATCGTGTGGGACAAGGGCCGGGACTTCGCCACCGTGCGGAAGGTGCTGA
GCATGCCCCAGGTGAACATCGTGAAGAAAACCGAGGTGCAGACCGGCGGCTTCAGCAAGGA
GAGCATCCTGCCCAAGCGGAACAGCGACAAGCTGATCGCCCGGAAGAAGGACTGGGACCCC
AAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTACAGCGTGCTGGTGGTGGCCAAGG
TGGAGAAGGGCAAGAGCAAGAAGCTGAAATCCGTGAAGGAGCTGCTGGGCATCACCATCAT
GGAGCGGAGCAGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAGGAG
GTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTCGAGCTGGAGAACGGCC
GGAAGCGGATGCTGGCCAGCGCCGGCGAGCTGCAGAAGGGCAACGAGCTGGCCCTGCCCAG
CAAGTACGTGAACTTCCTGTACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAG
GACAACGAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAGATCATCG
AGCAGATCAGCGAGTTCAGCAAGCGGGTGATCCTGGCCGACGCCAACCTGGACAAGGTGCT
GAGCGCCTACAACAAGCACCGGGACAAGCCCATCCGGGAGCAGGCCGAGAACATCATCCAC
CTGTTCACCCTGACCAACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGA
CCGGAAGCGGTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATC
ACCGGCCTGTACGAGACCCGGATCGACCTGAGCCAGCTGGGCGGCGACAGCGGCGGCAAGC
GGCCCGCCGCCACCAAGAAGGCCGGCCAGGCCAAGAAGAAGAAGTCGGGCGGGGGTGGCT
CAGTGGACGTGCTGAGGAGAAGGAAGGACTGGAACGTGAGACTGCAAGCCTTCTTCACCAG
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
190
CGACACCGGACTGGAATACGAGGCCCCTAAGCTGTACCCCGCCATTCCCGCCGCTAGACGCA
GACCCATTAGAGTGCTCTCTCTGTTTGACGGAATCGCCACCGGCTATCTGGTGCTGAAAGAG
CTGGGCATCAAGGTGGGCAAATACGTGGCCAGCGAGGTGTGCGAGGAGTCTATCGCCGTCG
GAACCGTGAAACACGAGGGAAACATCAAATACGTGAACGACGTGAGGAACATCACCAAGA
AAAACATCGAGGAGTGGGGCCCCTTCGATCTCGTGATCGGCGGCAGCCCTTGCAACGATCTC
TCCAATGTGAACCCCGCCAGAAAGGGACTGTACGAGGGCACCGGCAGACTGTTCTTCGAGTT
CTACCATCTGCTGAACTACAGCAGACCCAAGGAGGGCGACGACAGACCCTTCTTCTGGATGT
TCGAAAACGTGGTCGCCATGAAGGTGGGCGATAAGAGGGACATCAGCAGATTTCTGGAGTG
TAACCCCGTCATGATCGATGCCATTAAGGTCAGCGCCGCCCACAGAGCTAGGTACTTCTGGG
GAAATCTGCCCGGCATGAATAGACCCGTGATCGCTTCCAAGAACGACAAGCTGGAGCTGCA
AGACTGTCTGGAGTACAATAGAATCGCTAAGCTCAAGAAGGTCCAGACCATCACCACAAAA
TCCAACAGCATCAAGCAAGGCAAGAACCAGCTGTTCCCCGTGGTCATGAACGGCAAGGAGG
ATGTGCTCTGGTGCACCGAGCTGGAGAGGATCTTTGGCTTCCCCGTGCACTATACCGATGTG
TCCAACATGGGAAGAGGCGCTAGACAGAAACTGCTGGGAAGAAGCTGGAGCGTGCCCGTGA
TTAGACACCTCTTCGCCCCTCTGAAGGATTACTTCGCTTGCGAGGGAGGTGGCGGATCGGGA
AAGCGGCCCGCCGCCACCAAGAAGGCCGGTCAGGCCAAGAAGAAGAAGGGCAGCTACCCC
TACGACGTGCCCGACTACGCCTGAGCGGCCGCTTAATTAAGCTGCCTTCTGCGGGGCTTGCC
TTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAG
TAGGAAGTCTAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 15)
In some embodiments, an expression repressor comprises the amino acid sequence
of SEQ ID NOs: 16 or
37. In some embodiments, an expression repressor described herein comprises an
amino acid sequence of
SEQ ID NO: 16 or 37 or a sequence with at least 80, 85, 90, 95, 99, or 100%
identity thereto, or having
no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3,
2, or 1 positions of difference
thereto.
dCas9-DNMT3b
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNL SDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
191
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENT QLQNEKLYLYYLQNGRDMYVD QELD IN
RLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
S KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGDSGGKRPAATKKAGQAKKKKSGGGGSVDVLRRRKDWNVRLQAFFTS
DTGLEYEAPKLYPAIPAARRRPIRVLSLFDGIATGYLVLKELGIKVGKYVASEVCEESIAVGTVKH
EGNIKYVNDVRNITKKNIEEWGPFDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEFYHLLNYSR
PKEGDDRPFFWMFENVVAMKVGDKRDISRFLECNPVMIDAIKVS AAHRARYFWGNLPGMNRPV
IAS KND KLELQDCLEYNRIAKLKKV QTITT KS NSIKQGKNQLFPVVMNGKEDVLWCTELERIFGF
PVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACEGGGGSGKRPAATKKAGQAKK
KKGSYPYDVPDYA (SEQ ID NO: 16)
dCas9-DNMT3b without HA tag
MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI
GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKH
ERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNS
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILS ARLSKSRRLENLIAQLPGEKKNGLFGNLIALS
LGLTPNFKSNFDLAEDAKLQLS KDTYDDDLDNLLAQIGD QYADLFLAAKNLSDAILLSDILRVNT
EITKAPLS ASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFD QS KNGYAGYIDGGAS QEEFYKF
IKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEK
ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGAS AQSFIERMTNFDKNLPNEKV
LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFK
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
192
KIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT
YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSL
TFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMAREN
QTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN
RLSDYDVAAIVPQSFLKDD SIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLIT Q
RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLK
SKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI
AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERS SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS AG
ELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ
SITGLYETRIDLS QLGGD S GGKRPAAT KKAGQAKKKKS GGGGSVDVLRRRKDWNVRLQAFFTS
DTGLEYEAPKLYPAIPAARRRPIRVLSLFDGIATGYLVLKELGIKVGKYVASEVCEESIAVGTVKH
EGNIKYVNDVRNITKKNIEEWGPFDLVIGGSPCNDLSNVNPARKGLYEGTGRLFFEFYHLLNYSR
PKEGDDRPFFWMFENVVAMKVGDKRDISRFLECNPVMIDAIKVSAAHRARYFWGNLPGMNRPV
IASKNDKLELQDCLEYNRIAKLKKVQTITTKSNSIKQGKNQLFPVVMNGKEDVLWCTELERIFGF
PVHYTDVSNMGRGARQKLLGRSWSVPVIRHLFAPLKDYFACEGGGGSGKRPAATKKAGQAKK
KKGS (SEQ ID NO: 37)
Functional Characteristics
An expression repression system of the present disclosure can be used to
decrease expression of a
target gene in a cell. In some embodiments, decreasing expression comprises
decreasing the level of
RNA, e.g., mRNA, encoded by the target gene. In some embodiments, decreasing
expression comprises
decreasing the level of a protein encoded by the target gene. In some
embodiments, decreasing expression
comprises both decreasing the level of mRNA and protein encoded by the target
gene. In some
embodiments, the expression of a target gene in a cell contacted by or
comprising the expression
repression system is at least 1.05x (i.e., 1.05 times), 1.1x, 1.15x, 1.2x,
1.25x, 1.3x, 1.35x, 1.4x, 1.45x,
1.5x, 1.55x, 1.6x, 1.65x, 1.7x, 1.75x, 1.8x, 1.85x, 1.9x, 1.95x, 2x, 3x, 4x,
5x, 6x, 7x, 8x, 9x, 10x, 20x,
30x, 40x, 50x, 60x, 70x, 80x, 90x, or 100x lower than the level of expression
of the target gene in a cell
not contacted by or comprising the expression repression system. Expression of
a target gene may be
assayed by methods known to those of skill in the art, including RT-PCR,
ELISA, Western blot, and the
methods of Examples 2-4.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
193
An expression repression system of the present disclosure can be used to
decrease expression of a
target gene in a cell for a time period. In some embodiments, the expression
of a target gene in a cell
contacted by or comprising the expression repression system is appreciably
decreased for at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or
24 hours, or at least 1, 2, 3, 4, 5, 6,
7, 10, or 14 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1,2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 months,
or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, the
expression of a target gene in a cell
contacted by or comprising the expression repression system is appreciably
decreased for no more than
10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.
In some embodiments, the expression of a target in a cell contacted by or
comprising the
.. expression repressor or the system is appreciably decreased for at least 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 cell
divisions. An expression repressor or a system of the present disclosure can
be used to methylate CpG
nucleotides in a target region. In some embodiments, the methylation persists
for at least 1 days, at least 2
days, at least 5 days, at least 7 days, at least 10 days, at least 15 days, or
at least 22 days post-treatment
with an expression repressor or an expression repression system disclosed
herein.
An expression repression system may comprise a plurality of expression
repressors, where each
expression repressor comprises a repressor domain with a different
functionality than the repressor
domain of another expression repressor. For example, an expression repression
system may comprise two
expression repressors, where the first expression repressor comprises a first
repressor domain comprising
histone deacetylase functionality and the second expression repressor
comprises a second repressor
domain comprising DNA methyltransferase functionality. In some embodiments, an
expression
repression system comprises expression repressors comprising a combination of
repressor domains whose
functionalities are complementary to one another with regard to inhibiting
expression of a target gene,
e.g., where the functionalities together enable inhibition of expression and,
optionally, do not inhibit or
negligibly inhibit expression when present individually. In some embodiments,
an expression repression
system comprises a plurality of expression repressors, wherein each expression
repressor comprises a
repressor domain that complements the repressor domains of each other
expression repressor, e.g., each
repressor domain decreases expression of a target gene.
In some embodiments, an expression repression system comprises expression
repressors
.. comprising a combination of repressor domains whose functionalities
synergize with one another with
regard to inhibiting expression of a target gene. Without wishing to be bound
by theory, it is thought that
epigenetic modifications to a genomic locus are cumulative, in that multiple
repressive epigenetic markers
(e.g., multiple different types of epigenetic markers and/or more extensive
marking of a given type)
individually together reduce expression more effectively than individual
modifications alone (e.g.,
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
194
producing a greater decrease in expression and/or a longer-lasting decrease in
expression). In some
embodiments, an expression repression system comprises a plurality of
expression repressors, wherein
each expression repressor comprises a repressor domain that synergizes with
the repressor domains of
each other expression repressor, e.g., each repressor domain decreases
expression of a target gene. In
some embodiments, an expression repression system (comprising a plurality of
expression repressors
comprising a plurality of different repressor domains which synergize with one
another) is more effective
at inhibiting expression of a target gene than an individual expression
repressor, e.g., and its single
repressor domain. In some embodiments, an expression repression system is at
least 1.05x (i.e., 1.05
times), 1.1x, 1.15x, 1.2x, 1.25x, 1.3x, 1.35x, 1.4x, 1.45x, 1.5x, 1.55x, 1.6x,
1.65x, 1.7x, 1.75x, 1.8x,
1.85x, 1.9x, 1.95x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x,
60x, 70x, 80x, 90x, or 100x as
effective at inhibiting expression of a target gene than an individual
expression repressor. The level of an
epigenetic marker may be assayed by methods known to those of skill in the
art, including whole genome
bisulfite sequencing, reduced representation bisulfite sequencing, bisulfite
amplicon sequencing,
methylation arrays, pyrosequencing, ChIP-seq, or ChIP-qPCR.
Combinations of Repressors
In some embodiments, an expression repression system comprises a first
expression repressor
comprising a first repressor domain and a second expression repressor
comprising a second repressor
domain wherein the first repressor domain and second repressor domain are
different from one another. In
some embodiments, the first repressor domain is or comprises a first
epigenetic modifying moiety (e.g.,
that increases or decreases a first epigenetic marker) or functional fragment
thereof and the second
repressor domain is or comprises a second epigenetic modifying moiety (e.g.,
that increases or decreases a
second epigenetic marker) or functional fragment thereof. In some embodiments,
the first repressor
domain is or comprises a histone methyltransferase or functional fragment
thereof and the second
repressor domain is or comprises a DNA methyltransferase or functional
fragment thereof. In some
embodiments, the first repressor domain is or comprises a histone deacetylase
or functional fragment
thereof and the second repressor domain is or comprises a DNA
methyltransferase or functional fragment
thereof. In some embodiments, the first repressor domain is or comprises a
histone deacetylase or
functional fragment thereof and the second repressor domain is or comprises a
histone methyltransferase
or functional fragment thereof.
In some embodiments, the first repressor domain is or comprises KRAB (e.g., a
KRAB domain),
a SET domain (e.g., the SET domain of SETDB1 EZH2, G9A, or SUV39H1), histone
demethylase
LSD1, FOG1 (e.g., the N-terminal residues of FOG1), HDAC8, MQ1, DNMT1,
DNMT3a/31, or KAP1,
or a functional fragment of any thereof, and the second repressor domain is or
comprises KRAB (e.g., a
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
195
KRAB domain), a SET domain (e.g., the SET domain of SETDB1 EZH2, G9A, or
SUV39H1), histone
demethylase LSD1, FOG1 (e.g., the N-terminal residues of FOG1), DNMT3A (e.g.,
human DNMT3A),
DNMT3B, DNMT3L, DNMT3A/3L complex, or bacterial MQ1, or a functional fragment
of any thereof.
In some embodiments, the first repressor domain is or comprises KRAB or a
functional variant or
fragment thereof, and the second repressor domain is or comprises bacterial
MQ1 or a functional variant
or fragment thereof.
In some embodiments, the first repressor domain is or comprises KRAB or a
functional variant or
fragment thereof, and the second repressor domain is or comprises DNMT3A or a
functional variant or
fragment thereof.
In some embodiments, the first repressor domain is or comprises KRAB or a
functional variant or
fragment thereof, and the second repressor domain is or comprises DNMT3B or a
functional variant or
fragment thereof.
In some embodiments, the first repressor domain is or comprises KRAB or a
functional variant or
fragment thereof, and the second repressor domain is or comprises DNMT3L or a
functional variant or
fragment thereof.
In some embodiments, the first repressor domain is or comprises KRAB or a
functional variant or
fragment thereof, and the second repressor domain is or comprises DNMT3A/3L
complex or a functional
variant or fragment thereof.
In some embodiments, the first repressor domain is or comprises a SET domain
or a functional
variant or fragment thereof, and the second repressor domain is or comprises
bacterial MQ1 or a
functional variant or fragment thereof.
In some embodiments, the first repressor domain is or comprises LSD1 or a
functional variant or
fragment thereof, and the second repressor domain is or comprises bacterial
MQ1 or a functional variant
or fragment thereof.
In some embodiments, the first repressor domain is or comprises FOG1 or a
functional variant or
fragment thereof, and the second repressor domain is or comprises bacterial
MQ1 or a functional variant
or fragment thereof.
In some embodiments, the first repressor domain is or comprises KAP1 or a
functional variant or
fragment thereof, and the second repressor domain is or comprises bacterial
MQ1 or a functional variant
.. or fragment thereof.
In some embodiments, an expression repression system comprises a first
expression repressor,
wherein the first expression repressor is an expression repressor chosen from
dCas9-MQ1, dCas9-
DNMT1, dCas9-DNMT3a/31, G9A-dCas9, dCas9-HDAC8, dCas9-LSD1, EZH2-dCas9, EZH2-
dCas9-
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
196
KRAB, G9a-dCas9-KRAB, and Fogl-dCas9-Fogl, and a second expression repressor,
wherein the
second expression repressor is an expression repressor chosen from dCas9-MQ1,
dCas9-DNMT1, dCas9-
DNMT3a/31, G9A-dCas9, dCas9-HDAC8, dCas9-LSD1, EZH2-dCas9, EZH2-dCas9-KRAB,
G9a-
dCas9-KRAB, and Fogl-dCas9-Fogl.
In some embodiments, the expression repression system is encoded by a first
nucleic acid encoding the
first expression repressor, wherein expression is driven by a first promoter
or IRES, and a second nucleic
acid encoding the second expression repressor, wherein expression is driven by
a second promoter or
IRES.
Target Sites
Expression repression systems disclosed herein are useful for modulating,
e.g., decreasing,
expression of a target gene in cell, e.g., in a subject or patient. A target
gene may be any gene known to
those of skill in the art. In some embodiments, a target gene is associated
with a disease or condition in a
subject, e.g., a mammal, e.g., a human, bovine, horse, sheep, chicken, rat,
mouse, cat, or dog. A target
gene may include coding sequences, e.g., exons, and/or non-coding sequences,
e.g., introns, 3'UTR, or
5'UTR. In some embodiments, a target gene is operably linked to a
transcription control element.
A DNA-targeting moiety suitable for use in an expression repressor of an
expression repression
system described herein may bind, e.g., specifically bind, to any site within
a target gene, transcription
control element operably linked to a target gene, or anchor sequence (e.g., an
anchor sequence proximal
to a target gene or associated with an anchor sequence-mediated conjunction
operably linked to a target
gene (e.g., an anchor sequence-mediated conjunction is operably linked to a
target gene if disruption of
the conjunction alters expression of the target gene)).
In some embodiments, a DNA-targeting moiety binds to a target gene. In some
embodiments, a
DNA-targeting moiety binds to a site within an exon of a target gene. In some
embodiments, a DNA-
targeting moiety binds to a site within an intron of a target gene. In some
embodiments, a DNA-targeting
moiety binds to a site at the boundary of an exon and an intron, e.g., a
splice site, of a target gene. In some
embodiments, a DNA-targeting moiety binds to a site within the 5'UTR of a
target gene. In some
embodiments, a DNA-targeting moiety binds to a site within the 3'UTR of a
target gene. Target genes
include, but are not limited to, the gene encoding13-2-microglobulin, the gene
encoding MYC, the gene
encoding HSPA1B, the gene encoding GATA1, the gene encoding APOB, the gene
encoding FOXP3, the
gene encoding CXCL1, the gene encoding CXCL2, the gene encoding CXCL3, the
gene encoding
CXCL4, the gene encoding CXCL5, the gene encoding CXCL6, the gene encoding
CXCL7, and the gene
encoding CXCL8.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
197
In some embodiments, a DNA-targeting moiety binds to a transcription control
element operably
linked to a target gene, e.g., a promoter or enhancer. In some embodiments, a
DNA-targeting moiety
binds to a portion of or a site within a promoter operably linked to a target
gene. In some embodiments, a
DNA-targeting moiety binds to the transcription start site of a target gene.
In some embodiments, a DNA-
targeting moiety binds to a portion of or a site within an enhancer operably
linked to a target gene. A
promoter is, typically, a sequence element that initiates transcription of an
associated gene. Promoters are
typically near the 5' end of a gene, not far from its transcription start
site. As those of ordinary skill are
aware, transcription of protein-coding genes in eukaryotic cells is typically
initiated by binding of general
transcription factors (e.g., TFIID, TFIIE, TFIIH, etc.) and Mediator to core
promoter sequences as a
preinitiation complex that directs RNA polymerase II to the transcription
start site, and in many instances
remains bound to the core promoter sequences even after RNA polymerase escapes
and elongation of the
primary transcript is initiated. In some embodiments, a promoter includes a
sequence element such as
TATA, Inr, DPE, or BRE, but those skilled in the art are well aware that such
sequences are not
necessarily required to define a promoter. Those skilled in the art are
familiar with a variety of positive
.. (e.g., enhancers) or negative (e.g., repressors or silencers) transcription
control elements that are
associated with genes. In some embodiments, a transcription control element is
a transcription factor
binding site. Typically, when a cognate regulatory protein is bound to such a
transcription control
element, transcription from the associated gene(s) is altered (e.g., increased
or decreased).
In some embodiments, a DNA-targeting moiety binds to an anchor sequence, e.g.,
an anchor
sequence proximal to a target gene or associated with an anchor sequence-
mediated conjunction operably
linked to a target gene (e.g., an anchor sequence-mediated conjunction is
operably linked to a target gene
if disruption of the conjunction alters expression of the target gene). In
some embodiments, a DNA-
targeting moiety binds to or proximal to an anchor sequence of an anchor
sequence mediated conjunction
(ASMC) comprising a target plurality of genes, e.g., human CXCL1-8. In
general, an anchor sequence is
a genomic sequence element to which a genomic complex component binds
specifically. In some
embodiments, binding of a genomic complex component to an anchor sequence
nucleates complex
formation, e.g., anchor sequence-mediated conjunction formation. Each anchor
sequence-mediated
conjunction comprises one or more anchor sequences, e.g., a plurality. In some
embodiments, an anchor
sequence-mediated conjunction can be disrupted to alter, e.g., inhibit,
expression of a target gene. Such
disruptions may modulate gene expression by, e.g., changing topological
structure of DNA, e.g., by
modulating the ability of a target gene to interact with a transcription
control element (e.g., enhancing and
silencing/repressive sequences).
A DNA-targeting moiety suitable for use in an expression repressor of an
expression repression
system described herein may bind, e.g., specifically bind, to a site that is
proximal to a target gene (e.g.,
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
198
an exon, intron, or splice site within the target gene), proximal to a
transcription control element operably
linked to the target gene, or proximal to an anchor sequence, e.g., an anchor
sequence proximal to a target
gene or associated with an anchor sequence-mediated conjunction operably
linked to a target gene (e.g.,
an anchor sequence-mediated conjunction is operably linked to a target gene if
disruption of the
conjunction alters expression of the target gene). As used herein, proximal
refers to a closeness of two
sites, e.g., nucleic acid sites, such that binding of an expression repressor
at the first site and/or
modification of the first site by an expression repressor will produce the
same or substantially the same
effect as binding and/or modification of the other site. For example, a DNA-
targeting moiety may bind to
a first site that is proximal to an enhancer (the second site), and the
repressor domain associated with said
DNA-targeting moiety may epigenetically modify the first site such that the
enhancer's effect on
expression of a target gene is modified, substantially the same as if the
second site (the enhancer
sequence) had been bound and/or modified. In some embodiments, a site proximal
to a target gene (e.g.,
an exon, intron, or splice site within the target gene), proximal to a
transcription control element operably
linked to the target gene, or proximal to an anchor sequence is less than
5000, 4000, 3000, 2000, 1000,
900, 800, 700, 600, 500, 400, 300, 200, 100, 50, or 25 base pairs from the
target gene (e.g., an exon,
intron, or splice site within the target gene), transcription control element,
or anchor sequence (and
optionally at least 20, 25, 50, 100, 200, or 300 base pairs from the target
gene (e.g., an exon, intron, or
splice site within the target gene), transcription control element, or anchor
sequence).
A DNA-targeting moiety suitable for use in an expression repressor of an
expression repression
system described herein may bind, e.g., specifically bind, to a site
comprising at least 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, or 50 nucleotides or base pairs (and optionally no
more 50, 49, 48, 47, 46, 45,
44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26,
25, 24, 23, 22, 21, 20, 19, 18, 17,
16, 15, 14, 13, 12, 11, or 10 nucleotides or base pairs). In some embodiments,
a DNA-targeting moiety
binds to a site comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or
50 nucleotides or base pairs.
Expression repression systems of the present disclosure may comprise two or
more expression
repressors. In some embodiments, the expression repressors of an expression
repression system each
comprise a different DNA-targeting moiety.
In some embodiments, an expression repression system comprises a first
expression repressor
comprising a DNA-targeting moiety that binds a target gene, e.g., an exon,
intron, or exon intron
boundary (e.g., splice site), and second expression repressor comprising a DNA-
targeting moiety that
binds the target gene, e.g., an exon, intron, or exon intron boundary (e.g.,
splice site). In some
embodiments, an expression repression system comprises a first expression
repressor comprising a DNA-
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
199
targeting moiety that binds a target gene, e.g., an exon, intron, or exon
intron boundary (e.g., splice site),
and second expression repressor comprising a DNA-targeting moiety that binds
to a transcription control
element (e.g., promoter or enhancer) operably linked to the target gene. In
some embodiments, an
expression repression system comprises a first expression repressor comprising
a DNA-targeting moiety
that binds to a transcription control element (e.g., promoter or enhancer)
operably linked to a target gene,
and a second expression repressor comprising a DNA-targeting moiety that binds
to a transcription
control element (e.g., promoter or enhancer) operably linked to the target
gene. In some embodiments, an
expression repression system comprises a first expression repressor comprising
a DNA-targeting moiety
that binds to an anchor sequence proximal to a target gene or associated with
an anchor sequence-
mediated conjunction operably linked to a target gene, and a second expression
repressor comprising a
DNA-targeting moiety that binds to a transcription control element (e.g.,
promoter or enhancer) operably
linked to the target gene. In some embodiments, an expression repression
system comprises a first
expression repressor comprising a DNA-targeting moiety that binds to an anchor
sequence proximal to a
target gene or associated with an anchor sequence-mediated conjunction
operably linked to a target gene,
and a second expression repressor comprising a DNA-targeting moiety that binds
to the target gene, e.g.,
an exon, intron, or exon intron boundary (e.g., splice site). In some
embodiments, an expression
repression system comprises a first expression repressor comprising a DNA-
targeting moiety that binds to
an anchor sequence proximal to a target gene or associated with an anchor
sequence-mediated
conjunction operably linked to a target gene, and a second expression
repressor comprising a DNA-
targeting moiety that binds to an anchor sequence proximal to the target gene
or associated with an anchor
sequence-mediated conjunction operably linked to the target gene.
In some embodiments, an expression repression system comprises a first
expression repressor
comprising a DNA-targeting moiety that binds to a first site, e.g., in a
promoter operably linked to a target
gene, and a second expression repressor comprising a DNA-targeting moiety that
binds to a second site,
e.g., in the promoter operably linked to a target gene. The first site and
second site may be different and
non-overlapping sites, e.g., the first site and second site do not share any
sequence in common. The first
site and second site may be different but overlapping sites, e.g., the first
site and second site comprise
different sequences but share some sequence in common.
In some embodiments, a DNA-targeting moiety binds to a sequence selected from
SEQ ID NOs:
1-21 or a sequence with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1
alteration relative thereto.
ACGGCGGGCCACCAAGGAGA (SEQ ID NO: 6)
CAGCGCCGCGCCTTTGGGAC (SEQ ID NO: 7)
CCCTTTCGGCGGGGAGCAGGG (SEQ ID NO: 8)
GCTGCGCTGGGGGAGCCAGAG (SEQ ID NO: 9)
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
200
AGCGCCCCTCCACGCGTTCAC (SEQ ID NO: 10)
In some embodiments, the first DNA-targeting moiety binds to a sequence
comprising any one of
SEQ ID NOS: 1-21 and the second DNA-targeting moiety binds to a sequence
comprising any one of
SEQ ID NOS: 1-21, wherein the first and the second DNA-targeting moiety binds
to the same sequence.
In some embodiments, the first DNA-targeting moiety binds to a sequence
comprising any one of SEQ ID
NOS: 1-21 and the second DNA-targeting moiety binds to a sequence comprising
any one of SEQ ID
NOS: 1-21 wherein the first and the second targeting moiety binds to different
sequences.
In some embodiments, an expression repressor binds a genomic locus having a
sequence set forth
herein, e.g., any one of SEQ ID NOS: 1-21. It is understood that, in many
cases, the genomic locus being
bound comprises double stranded DNA, and this locus can be described by giving
the sequence of its
sense strand or its antisense strand. Thus, a gRNA having a given spacer
sequence may cause expression
repressor to bind to a particular genomic locus, wherein one strand of the
genomic locus has a sequence
similar or identical to the spacer sequence, and the other strand of the
genomic locus has the
complementary sequence. Typically, gRNA binding to the genomic locus will
involve some unwinding
of the genomic locus and pairing of the gRNA spacer with the strand to which
the spacer is
complementary.
In some embodiments, an anchor sequence comprises a nucleating polypeptide
binding motif,
e.g., a CTCF-binding motif:
N(T/C/G)N(G/A/T)CC(A/T/G)(C/G)(C/T/A)AG(G/A)(G/T)GG(C/A/T)(G/A)(C/G)(C/T/A)(G/A
/C)
(SEQ ID NO: 81), where N is any nucleotide.
A CTCF-binding motif may also be in an opposite orientation, e.g.,
(G/A/C)(C/T/A)(C/G)(G/A)(C/A/T)GG(G/T)(G/A)GA(C/T/A)(C/G)(A/T/G)CC(G/A/T)N(T/C/
G)N
(SEQ ID NO: 82). where N is any nucleotide
In some embodiments, an anchor sequence comprises SEQ ID NO: 81 or SEQ ID NO:
82 or a
sequence at least 75%, at least 80%, at least 90%, at least 95%, at least 96%,
at least 97%, at least 98%, at
least 99% identical to either SEQ ID NO: 81 or SEQ ID NO: 82.
In some embodiments, an anchor sequence comprises a nucleating polypeptide
binding motif,
e.g., a YY1-binding motif: CCGCCATNTT (SEQ ID NO: 83), where N is any
nucleotide.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
201
A YY1-binding motif may also be in an opposite orientation, e.g., AANATGGCGG
(SEQ ID
NO: 84), where N is any nucleotide.
A targeting moiety suitable for use in an expression repressor of an
expression repression system
described herein may bind, e.g., specifically bind, to a site comprising at
least 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50 nucleotides or base pairs (and optionally no more
50, 49, 48, 47, 46, 45, 44, 43,
42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,
23, 22, 21, 20, 19, 18, 17, 16, 15,
14, 13, 12, 11, or 10 nucleotides or base pairs). In some embodiments, a DNA-
targeting moiety binds to a
site comprising 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
nucleotides or base pairs.
Expression repression systems of the present disclosure may comprise two or
more expression
repressors. In some embodiments, the expression repressors of an expression
repression system each
comprise a different targeting moiety.
In some embodiments, an expression repression system comprises a first
expression repressor
comprising a DNA-targeting moiety that binds a target gene, e.g., an exon,
intron, or exon intron
boundary (e.g., splice site), and second expression repressor comprising a DNA-
targeting moiety that
binds the target gene, e.g., an exon, intron, or exon intron boundary (e.g.,
splice site). In some
embodiments, an expression repression system comprises a first expression
repressor comprising a DNA-
targeting moiety that binds a target gene, e.g., an exon, intron, or exon
intron boundary (e.g., splice site),
and second expression repressor comprising a DNA-targeting moiety that binds
to a transcription control
element (e.g., promoter or enhancer) operably linked to the target gene. In
some embodiments, an
expression repression system comprises a first expression repressor comprising
a DNA-targeting moiety
that binds to a transcription control element (e.g., promoter or enhancer)
operably linked to a target gene,
and a second expression repressor comprising a DNA-targeting moiety that binds
to a transcription
control element (e.g., promoter or enhancer) operably linked to the target
gene. In some embodiments, an
expression repression system comprises a first expression repressor comprising
a DNA-targeting moiety
that binds to an anchor sequence proximal to a target gene, e or associated
with an anchor sequence-
mediated conjunction operably linked to a target gene, and a second expression
repressor comprising a
DNA-targeting moiety that binds to a transcription control element (e.g.,
promoter or enhancer) operably
linked to the target gene,. In some embodiments, an expression repression
system comprises a first
expression repressor comprising a DNA-targeting moiety that binds to an anchor
sequence proximal to a
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
202
target gene, or associated with an anchor sequence-mediated conjunction
operably linked to a target gene,
and a second expression repressor comprising a DNA-targeting moiety that binds
to the target gene, e.g.,
an exon, intron, or exon intron boundary (e.g., splice site). In some
embodiments, an expression
repression system comprises a first expression repressor comprising a DNA-
targeting moiety that binds to
an anchor sequence proximal to a target gene, or associated with an anchor
sequence-mediated
conjunction operably linked to a target gene, and a second expression
repressor comprising a DNA-
targeting moiety that binds to an anchor sequence proximal to the target gene,
or associated with an
anchor sequence-mediated conjunction operably linked to the target gene.
Other Compositions
Nucleic acids and Vectors
The present disclosure is further directed, in part, to nucleic acids encoding
expression repressors
or expression repression systems described herein. In some embodiments, an
expression repression
system may be provided via a composition comprising a nucleic acid encoding
the expression repression
system, e.g., expression repressor(s) of the expression repression system,
wherein the nucleic acid is
associated with sufficient other sequences to achieve expression of the
expression repression system, e.g.,
expression repressor(s) of the expression repression system, in a system of
interest (e.g., in a particular
cell, tissue, organism, etc.).
In some particular embodiments, the present disclosure provides compositions
of nucleic acids that
encode an expression repression system, one or more expression repressors, or
polypeptide portion
thereof. In some such embodiments, provided nucleic acids may be or include
DNA, RNA, or any other
nucleic acid moiety or entity as described herein, and may be prepared by any
technology described
herein or otherwise available in the art (e.g., synthesis, cloning,
amplification, in vitro or in vivo
transcription, etc.). In some embodiments, provided nucleic acids that encode
an expression repression
system, one or more expression repressors, or polypeptide portion thereof may
be operationally associated
with one or more replication, integration, and/or expression signals
appropriate and/or sufficient to
achieve integration, replication, and/or expression of the provided nucleic
acid in a system of interest
(e.g., in a particular cell, tissue, organism, etc.). In some embodiments, a
composition for delivering an
expression repression system described herein is or comprises a vector, e.g.,
a viral vector, comprising
.. one or more nucleic acids encoding one or more components of an expression
repression system, e.g.,
expression repressor(s) of the expression repression system as described
herein.
In some embodiments, a composition for delivering an expression repressor or
an expression
repression system described herein is or comprises RNA, e.g., mRNA, comprising
one or more nucleic
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
203
acids encoding one or more components of an expression repressor or an
expression repression system,
e.g., expression repressor(s) of the expression repression system as described
herein.
Nucleic acids as described herein or nucleic acids encoding a protein
described herein, may be
incorporated into a vector. Vectors, including those derived from retroviruses
such as lentivirus, are
.. suitable tools to achieve long-term gene transfer since they allow long-
term, stable integration of a
transgene and its propagation in daughter cells. Examples of vectors include
expression vectors,
replication vectors, probe generation vectors, and sequencing vectors. An
expression vector may be
provided to a cell in the form of a viral vector. Viral vector technology is
well known in the art and
described in a variety of virology and molecular biology manuals. Viruses,
which are useful as vectors
.. include, but are not limited to, retroviruses, adenoviruses, adeno-
associated viruses, herpes viruses, and
lentiviruses. In general, a suitable vector contains an origin of replication
functional in at least one
organism, a promoter sequence, convenient restriction endonuclease sites, and
one or more selectable
markers.
Expression of natural or synthetic nucleic acids is typically achieved by
operably linking a
.. nucleic acid encoding the gene of interest to a promoter and incorporating
the construct into an expression
vector. Vectors can be suitable for replication and integration in eukaryotes.
Typical cloning vectors
contain transcription and translation terminators, initiation sequences, and
promoters useful for expression
of the desired nucleic acid sequence.
Additional promoter elements, e.g., enhancing sequences, may regulate
frequency of
.. transcriptional initiation. Typically, these sequences are located in a
region 30-110 bp upstream of a
transcription start site, although a number of promoters have recently been
shown to contain functional
elements downstream of transcription start sites as well. Spacing between
promoter elements frequently
is flexible, so that promoter function is preserved when elements are inverted
or moved relative to one
another. In a thymidine kinase (tk) promoter, spacing between promoter
elements can be increased to 50
.. bp apart before activity begins to decline. Depending on the promoter, it
appears that individual elements
can function either cooperatively or independently to activate transcription.
One example of a suitable promoter is the immediate early cytomegalovirus
(CMV) promoter
sequence. This promoter sequence is a strong constitutive promoter sequence
capable of driving high
levels of expression of any polynucleotide sequence operatively linked
thereto. In some embodiments of
.. a suitable promoter is Elongation Growth Factor-1a (EF-1a). However, other
constitutive promoter
sequences may also be used, including, but not limited to the simian virus 40
(5V40) early promoter,
mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long
terminal repeat
(LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-
Barr virus immediate
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
204
early promoter, a Rous sarcoma virus promoter, as well as human gene promoters
such as, but not limited
to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a
creatine kinase promoter.
The present disclosure should not interpreted to be limited to use of any
particular promoter or category
of promoters (e.g. constitutive promoters). For example, in some embodiments,
inducible promoters are
contemplated as part of the present disclosure. In some embodiments, use of an
inducible promoter
provides a molecular switch capable of turning on expression of a
polynucleotide sequence to which it is
operatively linked, when such expression is desired. In some embodiments, use
of an inducible promoter
provides a molecular switch capable of turning off expression when expression
is not desired. Examples
of inducible promoters include, but are not limited to a metallothionine
promoter, a glucocorticoid
promoter, a progesterone promoter, and a tetracycline promoter.
In some embodiments, an expression vector to be introduced can also contain
either a selectable
marker gene or a reporter gene or both to facilitate identification and
selection of expressing cells from
the population of cells sought to be transfected or infected through viral
vectors. In some aspects, a
selectable marker may be carried on a separate piece of DNA and used in a co-
transfection procedure.
Both selectable markers and reporter genes may be flanked with appropriate
transcriptional control
sequences to enable expression in the host cells. Useful selectable markers
may include, for example,
antibiotic-resistance genes, such as neo, etc.
In some embodiments, reporter genes may be used for identifying potentially
transfected cells
and/or for evaluating the functionality of transcriptional control sequences.
In general, a reporter gene is
a gene that is not present in or expressed by a recipient source (of a
reporter gene) and that encodes a
polypeptide whose expression is manifested by some easily detectable property,
e.g., enzymatic activity
or visualizable fluorescence. Expression of a reporter gene is assayed at a
suitable time after the DNA
has been introduced into the recipient cells. Suitable reporter genes may
include genes encoding
luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted
alkaline phosphatase, or the
green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-
82). Suitable expression
systems are well known and may be prepared using known techniques or obtained
commercially. In
general, a construct with a minimal 5' flanking region that shows highest
level of expression of reporter
gene is identified as a promoter. Such promoter regions may be linked to a
reporter gene and used to
evaluate agents for ability to modulate promoter-driven transcription.
Cells
The present disclosure is further directed, in part, to cells comprising an
expression repressor or
an expression repression system described herein. Any cell, e.g., cell line,
e.g., a cell line suitable for
expression of a recombinant polypeptide, known to one of skill in the art is
suitable to comprise an
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
205
expression repressor or an expression repression system described herein. In
some embodiments, a cell,
e.g., cell line, may be used to express an expression repressor or an
expression repression system, e.g.,
expression repressor(s), described herein. In some embodiments, a cell, e.g.,
cell line, may be used to
express or amplify a nucleic acid, e.g., a vector, encoding an expression
repressor or an expression
repression system, e.g., expression repressor(s), described herein. In some
embodiments, a cell comprises
a nucleic acid encoding an expression repressor or an expression repression
system, e.g., expression
repressor(s), described herein.
In some embodiments, a cell comprises a first nucleic acid encoding a first
component of an
expression repressor or an expression repression system, e.g., a first
expression repressor, and a second
nucleic acid encoding a second component of the expression repression system,
e.g., a second expression
repressor. In some embodiments, wherein a cell comprises nucleic acid encoding
an expression repression
system comprising two or more expression repressors, the sequences encoding
each expression repressor
are disposed on separate nucleic acid molecules, e.g., on different vectors,
e.g., a first vector encoding a
first expression repressor and a second vector encoding a second expression
repressor. In some
embodiments, the sequences encoding each expression repressor are disposed on
the same nucleic acid
molecule, e.g., on the same vector. In some embodiments, some or all of the
nucleic acid encoding the
expression repression system is integrated into the genomic DNA of the cell.
In some embodiments, the
nucleic acid encoding a first expression repressor of an expression repression
system is integrated into the
genomic DNA of a cell, and the nucleic acid encoding a second expression
repressor of an expression
repression system is not integrated into the genomic DNA of a cell (e.g., is
situated on a vector). In some
embodiments, the nucleic acid(s) encoding a first and a second expression
repressor of an expression
repression system are integrated into the genomic DNA of a cell, e.g., at the
same (e.g., adjacent or
colocalized) or different sites in the genomic DNA.
Examples of cells that may comprise and/or express an expression repression
system or
expression repressor described herein include, but are not limited to,
hepatocytes, neuronal cells,
endothelial cells, myocytes, and lymphocytes.
The present disclosure is further directed, in part, to a cell made by a
method or process described
herein. In some embodiments, the disclosure provides a cell produced by:
providing an expression
repression system described herein, providing the cell, and contacting the
cell with the expression
repression system (or a nucleic acid encoding the expression repression
system, or a composition
comprising said expression repression system or nucleic acid). Without wishing
to be bound by theory, a
cell contacted with an expression repression system described herein may
exhibit: a decrease in
expression of a target gene and/or a modification of epigenetic markers
associated with the target gene, a
transcription control element operably linked to the target gene, or an anchor
sequence proximal to the
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
206
target gene or associated with an anchor sequence-mediated conjunction
operably linked to the target
gene compared to a similar cell that has not been contacted by the expression
repression system. In some
embodiments, a cell exhibiting said decrease in expression of a target gene
and/or modification of
epigenetic markers does not comprise the expression repression system. The
decrease in expression
and/or modification of epigenetic markers may persist, e.g., for at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or at least 1, 2, 3,
4, 5, 6,7, 10, or 14 days, or at
least 1,2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or
12 months, or at least 1, 2, 3, 4, or
5 years (e.g., indefinitely) after contact with the expression repression
system. In some embodiments, a
cell previously contacted by an expression repression system retains the
decrease in expression and/or
modification of epigenetic markers after the expression repression system is
no longer present in the cell,
e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, or 24 hours, or
at least 1, 2, 3, 4, 5, 6, 7, 10, or 14 days, or at least 1, 2, 3, 4, or 5
weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely)
after the expression repression
system is no longer present in the cell.
Methods of Making Expression Repression Systems and/or Expression Repressors
In some embodiments, an expression repressor comprises or is a protein and may
thus be
produced by methods of making proteins. In some embodiments, an expression
repression system, e.g.,
the expression repressor(s) of an expression repression system, comprise one
or more proteins and may
thus be produced by methods of making proteins. As will be appreciated by one
of skill, methods of
making proteins or polypeptides (which may be included in modulating agents as
described herein) are
routine in the art. See, in general, Smales & James (Eds.), Therapeutic
Proteins: Methods and Protocols
(Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar &
Meibohm (Eds.),
Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).
A protein or polypeptide of compositions of the present disclosure can be
biochemically
synthesized by employing standard solid phase techniques. Such methods include
exclusive solid phase
synthesis, partial solid phase synthesis methods, fragment condensation,
classical solution synthesis.
These methods can be used when a peptide is relatively short (e.g., 10 kDa)
and/or when it cannot be
produced by recombinant techniques (i.e., not encoded by a nucleic acid
sequence) and therefore involves
different chemistry.
Solid phase synthesis procedures are well known in the art and further
described by John Morrow
Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses, 2nd Ed.,
Pierce Chemical Company,
1984; and Coin, I., et al., Nature Protocols, 2:3247-3256, 2007.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
207
For longer peptides, recombinant methods may be used. Methods of making a
recombinant
therapeutic polypeptide are routine in the art. See, in general, Smales &
James (Eds.), Therapeutic
Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press
(2005); and Crommelin,
.. Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and
Applications, Springer
(2013).
Exemplary methods for producing a therapeutic pharmaceutical protein or
polypeptide involve
expression in mammalian cells, although recombinant proteins can also be
produced using insect cells,
yeast, bacteria, or other cells under control of appropriate promoters.
Mammalian expression vectors may
comprise non transcribed elements such as an origin of replication, a suitable
promoter, and other 5' or 3'
flanking non transcribed sequences, and 5' or 3' non-translated sequences such
as necessary ribosome
binding sites, a polyadenylation site, splice donor and acceptor sites, and
termination sequences. DNA
sequences derived from the 5V40 viral genome, for example, 5V40 origin, early
promoter, splice, and
polyadenylation sites may be used to provide other genetic elements required
for expression of a
heterologous DNA sequence. Appropriate cloning and expression vectors for use
with bacterial, fungal,
yeast, and mammalian cellular hosts are described in Green & Sambrook,
Molecular Cloning: A
Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press
(2012).
In cases where large amounts of the protein or polypeptide are desired, it can
be generated using
techniques such as described by Brian Bray, Nature Reviews Drug Discovery,
2:587-593, 2003; and
Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic
Press, NY, Section
VIII, pp 421-463.
Various mammalian cell culture systems can be employed to express and
manufacture
recombinant protein. Examples of mammalian expression systems include CHO
cells, COS cells, HeLA
and BHK cell lines. Processes of host cell culture for production of protein
therapeutics are described in
Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics
Manufacturing (Advances in
Biochemical Engineering/Biotechnology), Springer (2014). Compositions
described herein may include a
vector, such as a viral vector, e.g., a lentiviral vector, encoding a
recombinant protein. In some
embodiments, a vector, e.g., a viral vector, may comprise a nucleic acid
encoding a recombinant protein.
Compositions described herein may include a lipid nanoparticle encapsulating a
vector, such as a viral
vector, e.g., a lentiviral vector, encoding a recombinant protein. In some
embodiments, a lipid
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
208
nanoparticle encapsulating a vector, e.g., a viral vector, may comprise a
nucleic acid encoding a
recombinant protein.
Proteins comprise one or more amino acids. Amino acids include any compound
and/or substance
that can be incorporated into a polypeptide chain, e.g., through formation of
one or more peptide bonds.
In some embodiments, an amino acid has the general structure H2N¨C(H)(R)¨COOH.
In some
embodiments, an amino acid is a naturally occurring amino acid. In some
embodiments, an amino acid is
a non-natural amino acid; in some embodiments, an amino acid is a D-amino
acid; in some embodiments,
an amino acid is an L-amino acid. "Standard amino acid" refers to any of the
twenty standard L-amino
acids commonly found in naturally occurring peptides. "Nonstandard amino acid"
refers to any amino
acid, other than the standard amino acids, regardless of whether it is
prepared synthetically or obtained
from a natural source. In some embodiments, an amino acid, including a carboxy-
and/or amino-terminal
amino acid in a polypeptide, can contain a structural modification as compared
with the general structure
above. For example, in some embodiments, an amino acid may be modified by
methylation, amidation,
acetylation, pegylation, glycosylation, phosphorylation, and/or substitution
(e.g., of the amino group, the
carboxylic acid group, one or more protons, and/or the hydroxyl group) as
compared with the general
structure. In some embodiments, such modification may, for example, alter the
circulating half-life of a
polypeptide containing the modified amino acid as compared with one containing
an otherwise identical
unmodified amino acid. In some embodiments, such modification does not
significantly alter a relevant
activity of a polypeptide containing the modified amino acid, as compared with
one containing an
otherwise identical unmodified amino acid. As will be clear from context, in
some embodiments, the
term "amino acid" may be used to refer to a free amino acid; in some
embodiments it may be used to refer
to an amino acid residue of a polypeptide.
Purification of protein therapeutics is described in Franks, Protein
Biotechnology: Isolation,
Characterization, and Stabilization, Humana Press (2013); and in Cutler,
Protein Purification Protocols
(Methods in Molecular Biology), Humana Press (2010). Formulation of protein
therapeutics is described
in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to
formulation in the
Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).
Pharmaceutical Compositions, Formulation, Delivery, and Administration
The present disclosure is further directed, in part, to pharmaceutical
compositions comprising an
expression repressor or an expression repression system, e.g., expression
repressor(s), described herein, to
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
209
pharmaceutical compositions comprising nucleic acids encoding the expression
repressor or the
expression repression system, e.g., expression repressor(s), described herein,
and/or to and/or
compositions that deliver an expression repressor or an expression repression
system, e.g., expression
repressor(s), described herein to a cell, tissue, organ, and/or subject.
As used herein, the term "pharmaceutical composition" refers to an active
agent (e.g., an
expression repressor or nucleic acids of the expression receptor, e.g., an
expression repression system,
e.g., expression repressor(s) of an expression repression system, or nucleic
acid encoding the same),
formulated together with one or more pharmaceutically acceptable carriers
(e.g., pharmaceutically
acceptable carriers known to those of skill in the art). In some embodiments,
active agent is present in
unit dose amount appropriate for administration in a therapeutic regimen that
shows a statistically
significant probability of achieving a predetermined therapeutic effect when
administered to a relevant
population. In some embodiments, a pharmaceutical composition comprising an
expression repressor of
the present disclosure comprises an expression repressor or nucleic acid(s)
encoding the same. In some
embodiments, a pharmaceutical composition comprising an expression repression
system of the present
disclosure comprises or each of the expression repressors of the expression
repression system or nucleic
acid(s) encoding the same (e.g., if an expression repression system comprises
a first expression repressor
and a second expression repressor, the pharmaceutical composition comprises
the first and second
expression repressor). In some embodiments, a pharmaceutical composition
comprises less than all of the
expression repressors of an expression repression system comprising a
plurality of expression repressors.
For example, an expression repression system may comprise a first expression
repressor and a second
expression repressor, and a first pharmaceutical composition may comprise the
first expression repressor
or nucleic acid encoding the same and a second pharmaceutical composition may
comprise the second
expression repressor or nucleic acid encoding the same.
In some embodiments, pharmaceutical compositions may be specially formulated
for
administration in solid or liquid form, including those adapted for the
following: oral administration, for
example, drenches (aqueous or non-aqueous solutions or suspensions), tablets,
e.g., those targeted for
buccal, sublingual, and systemic absorption, boluses, powders, granules,
pastes for application to the
tongue; parenteral administration, for example, by subcutaneous,
intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or sustained-
release formulation; topical
application, for example, as a cream, ointment, or a controlled-release patch
or spray applied to the skin,
lungs, or oral cavity; intravaginally or intrarectally, for example, as a
pessary, cream, or foam;
sublingually; ocularly; transdermally; or nasally, pulmonary, and/or to other
mucosal surfaces.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
210
As used herein, the term "pharmaceutically acceptable" refers to those
compounds, materials,
compositions, and/or dosage forms which are, within the scope of sound medical
judgment, suitable for
use in contact with the tissues of human beings and animals without excessive
toxicity, irritation, allergic
response, or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
As used herein, the term "pharmaceutically acceptable carrier" means a
pharmaceutically
acceptable material, composition, or vehicle, such as a liquid or solid
filler, diluent, excipient, or solvent
encapsulating material, involved in carrying or transporting the subject
compound from one organ, or
portion of the body, to another organ, or portion of the body. Each carrier
must be "acceptable" in the
sense of being compatible with the other ingredients of the formulation and
not injurious to the patient.
In some embodiments, for example, materials which can serve as
pharmaceutically-acceptable
carriers include: sugars, such as lactose, glucose and sucrose; starches, such
as corn starch and potato
starch; cellulose, and its derivatives, such as sodium carboxymethyl
cellulose, ethyl cellulose and
cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such
as cocoa butter and
suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and
soybean oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar;
buffering agents, such as
magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline;
Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,
polycarbonates and/or polyanhydrides;
and other non-toxic compatible substances employed in pharmaceutical
formulations.
As used herein, the term "pharmaceutically acceptable salt", refers to salts
of such compounds
that are appropriate for use in pharmaceutical contexts, i.e., salts which
are, within the scope of sound
medical judgment, suitable for use in contact with the tissues of humans and
lower animals without undue
toxicity, irritation, allergic response, and the like, and are commensurate
with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art. For
example, S. M. Berge, et al.
describes pharmaceutically acceptable salts in detail in J. Pharmaceutical
Sciences, 66: 1-19 (1977). In
some embodiments, pharmaceutically acceptable salts include, but are not
limited to, nontoxic acid
addition salts, which are salts of an amino group formed with inorganic acids
such as hydrochloric acid,
hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with
organic acids such as acetic
acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid
or by using other methods used
in the art such as ion exchange. In some embodiments, pharmaceutically
acceptable salts include, but are
not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,
benzoate, bisulfate, borate,
butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,
gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,
lactate, laurate, lauryl
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
211
sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,
nicotinate, nitrate, oleate,
oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-
toluenesulfonate, undecanoate, valerate
salts, and the like. Representative alkali or alkaline earth metal salts
include sodium, lithium, potassium,
calcium, magnesium, and the like. In some embodiments, pharmaceutically
acceptable salts include,
when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations
formed using
counterions such as halide, hydroxide, carboxylate, sulfate, phosphate,
nitrate, alkyl having from 1 to 6
carbon atoms, sulfonate, and aryl sulfonate.
In various embodiments, the present disclosure provides pharmaceutical
compositions described
herein with a pharmaceutically acceptable excipient. Pharmaceutically
acceptable excipient includes an
excipient that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic, and
desirable, and includes excipients that are acceptable for veterinary use as
well as for human
pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in
the case of an aerosol
composition, gaseous.
Pharmaceutical preparations may be made following conventional techniques of
pharmacy
involving milling, mixing, granulation, and compressing, when necessary, for
tablet forms; or milling,
mixing, and filling for hard gelatin capsule forms. When a liquid carrier is
used, a preparation can be in
the form of a syrup, elixir, emulsion or an aqueous or non-aqueous solution or
suspension. Such a
liquid formulation may be administered directly per os.
In some embodiments, pharmaceutical compositions may be formulated for
delivery to a cell
and/or to a subject via any route of administration. Modes of administration
to a subject may include
injection, infusion, inhalation, intranasal, intraocular, topical delivery,
intercannular delivery, or ingestion.
Injection includes, without limitation, intravenous, intramuscular, intra-
arterial, intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid,
intraspinal, intracerebrospinal, and
intrasternal injection and infusion. In some embodiments, administration
includes aerosol inhalation, e.g.,
with nebulization. In some embodiments, administration is systemic (e.g.,
oral, rectal, nasal, sublingual,
buccal, or parenteral), enteral (e.g., system-wide effect, but delivered
through the gastrointestinal tract), or
local (e.g., local application on the skin, intravitreal injection). In some
embodiments, one or more
compositions is administered systemically. In some embodiments, administration
is non-parenteral and a
therapeutic is a parenteral therapeutic. In some embodiments, administration
may be bronchial (e.g., by
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
212
bronchial instillation), buccal, dermal (which may be or comprise, for
example, one or more of topical to
the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-
arterial, intradermal, intragastric,
intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal,
intravenous, intraventricular, within
a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal,
subcutaneous, sublingual, topical, tracheal
(e.g., by intratracheal instillation), vaginal, vitreal, etc. In some
embodiments, administration may be a
single dose. In some embodiments, administration may involve dosing that is
intermittent (e.g., a
plurality of doses separated in time) and/or periodic (e.g., individual doses
separated by a common period
of time) dosing. In some embodiments, administration may involve continuous
dosing (e.g., perfusion)
for at least a selected period of time.
Pharmaceutical compositions according to the present disclosure may be
delivered in a therapeutically
effective amount. A precise therapeutically effective amount is an amount of a
composition that will
yield the most effective results in terms of efficacy of treatment in a given
subject. This amount will vary
depending upon a variety of factors, including but not limited to
characteristics of a therapeutic compound
(including activity, pharmacokinetics, pharmacodynamics, and bioavailability),
physiological condition of
a subject (including age, sex, disease type and stage, general physical
condition, responsiveness to a given
dosage, and type of medication), nature of a pharmaceutically acceptable
carrier or carriers in
a formulation, and/or route of administration.
In some aspects, the present disclosure provides methods of delivering a
therapeutic comprising
administering a composition as described herein to a subject, wherein a
genomic complex modulating
agent is a therapeutic and/or wherein delivery of a therapeutic causes changes
in gene expression relative
to gene expression in absence of a therapeutic.
Methods as provided in various embodiments herein may be utilized in any some
aspects
delineated herein. In some embodiments, one or more compositions is/are
targeted to specific cells, or
one or more specific tissues.
For example, in some embodiments one or more compositions is/are targeted to
epithelial,
connective, muscular, and/or nervous tissue or cells. In some embodiments a
composition is targeted to a
cell or tissue of a particular organ system, e.g., cardiovascular system
(heart, vasculature); digestive
system (esophagus, stomach, liver, gallbladder, pancreas, intestines, colon,
rectum and anus); endocrine
system (hypothalamus, pituitary gland, pineal body or pineal gland, thyroid,
parathyroids, adrenal
glands); excretory system (kidneys, ureters, bladder); lymphatic system
(lymph, lymph nodes, lymph
vessels, tonsils, adenoids, thymus, spleen); integumentary system (skin, hair,
nails); muscular system
(e.g., skeletal muscle); nervous system (brain, spinal cord, nerves);
reproductive system (ovaries, uterus,
mammary glands, testes, vas deferens, seminal vesicles, prostate); respiratory
system (pharynx, larynx,
trachea, bronchi, lungs, diaphragm); skeletal system (bone, cartilage); and/or
combinations thereof.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
213
In some embodiments, a composition of the present disclosure crosses a blood-
brain-barrier, a placental
membrane, or a blood-testis barrier.
In some embodiments, a pharmaceutical composition as provided herein is
administered
systemically.
In some embodiments, administration is non-parenteral and a therapeutic is a
parenteral
therapeutic.
In some embodiments, a pharmaceutical composition of the present disclosure
has improved
PK/PD, e.g., increased pharmacokinetics or pharmacodynamics, such as improved
targeting, absorption,
or transport (e.g., at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%,
90% improved or
more) as compared to an active agent alone. In some embodiments, a
pharmaceutical composition has
reduced undesirable effects, such as reduced diffusion to a nontarget
location, off-target activity, or toxic
metabolism, as compared to a therapeutic alone (e.g., at least 5%, 10%, 15%,
20%, 30%, 40%, 50%, 60%,
75%, 80%, 90% or more reduced, as compared to an active agent alone). In some
embodiments, a
composition increases efficacy and/or decreases toxicity of a therapeutic
(e.g., at least 5%, 10%, 15%,
20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more) as compared to an active agent
alone.
Pharmaceutical compositions described herein may be formulated for example
including a carrier, such as
a pharmaceutical carrier and/or a polymeric carrier, e.g., a liposome or
vesicle, and delivered by known
methods to a subject in need thereof (e.g., a human or non-human agricultural
or domestic animal, e.g.,
cattle, dog, cat, horse, poultry). Such methods include transfection (e.g.,
lipid-mediated, cationic
polymers, calcium phosphate); electroporation or other methods of membrane
disruption (e.g.,
nucleofection) and viral delivery (e.g., lentivirus, retrovirus, adenovirus,
AAV). Methods of delivery are
also described, e.g., in Gori et al., Delivery and Specificity of CRISPR/Cas9
Genome Editing
Technologies for Human Gene Therapy. Human Gene Therapy. July 2015, 26(7): 443-
451.
doi:10.1089/hum.2015.074; and Zuris et al. Cationic lipid-mediated delivery of
proteins enables efficient
protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2014 Oct
30;33(1):73-80.
Liposomes are spherical vesicle structures composed of a uni- or multilamellar
lipid bilayer surrounding
internal aqueous compartments and a relatively impermeable outer lipophilic
phospholipid bilayer.
Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible,
nontoxic, can deliver both
hydrophilic and lipophilic drug molecules, protect their cargo from
degradation by plasma enzymes, and
transport their load across biological membranes and the blood brain barrier
(BBB) (see, e.g., Spuch and
Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages,
2011.
doi:10.1155/2011/469679 for review).
Vesicles can be made from several different types of lipids; however,
phospholipids are most
commonly used to generate liposomes as drug carriers. Vesicles may comprise
without limitation
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
214
DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield DOTMA
and cholesterol,
DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.
Methods for preparation
of multilamellar vesicle lipids are known in the art (see for example U.S.
Pat. No. 6,693,086, the
teachings of which relating to multilamellar vesicle lipid preparation are
incorporated herein by
reference). Although vesicle formation can be spontaneous when a lipid film is
mixed with an aqueous
solution, it can also be expedited by applying force in the form of shaking by
using a homogenizer,
sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of
Drug Delivery, vol. 2011,
Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
Extruded lipids can be
prepared by extruding through filters of decreasing size, as described in
Templeton et al., Nature Biotech,
15:647-652, 1997, the teachings of which relating to extruded lipid
preparation are incorporated herein by
reference.
Methods and compositions provided herein may comprise a pharmaceutical
composition
administered by a regimen sufficient to alleviate a symptom of a disease,
disorder, and/or condition. In
some aspects, the present disclosure provides methods of delivering a
therapeutic by administering
compositions as described herein.
Uses
The present disclosure is further directed to uses of the expression
repression systems disclosed
herein. Among other things, in some embodiments such provided technologies may
be used to achieve
modulation, e.g., repression, of target gene expression and, for example,
enable control of target gene
activity, delivery, and penetrance, e.g., in a cell. In some embodiments, a
cell is a mammalian, e.g.,
human, cell. In some embodiments, a cell is a somatic cell. In some
embodiments, a cell is a primary cell.
For example, in some embodiments, a cell is a mammalian somatic cell. In some
embodiments, a
mammalian somatic cell is a primary cell. In some embodiments, a mammalian
somatic cell is a non-
embryonic cell.
Dosage
The dosage of the administered agent or composition can vary based on, e.g.,
the condition being treated,
the severity of the disease, the subject's individual parameters, including
age, physiological condition, size
and weight, duration of treatment, the type of treatment to be performed (if
any), the particular route of
administration and similar factors. Thus, the dose administered of the agents
described herein can depend
on such various parameters. The dosage of an administered composition may also
vary depending upon
other factors as the subject's sex, general medical condition, and severity of
the disorder to be treated. It
may be desirable to provide the subject with a dosage of an expression
repressor or an expression
repression system or combination of expression repressors disclosed herein as
circumstances dictate. In
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
215
some embodiments, the subject is provided with a dosage of an expression
repressor, or an expression
repression system or combination of expression repressors disclosed herein as
circumstances dictate.
Pharmaceutical compositions according to the present disclosure may be
delivered in a therapeutically
effective amount. A precise therapeutically effective amount is an amount of a
composition that will
yield the most effective results in terms of efficacy of treatment in a given
subject. This amount will vary
depending upon a variety of factors, including but not limited to
characteristics of a therapeutic compound
(including activity, pharmacolcinetics, pharmacodynamics, and
bioavailability), physiological condition of
a subject (including age, sex, disease type and stage, general physical
condition, responsiveness to a given
dosage, and type of medication), nature of a pharmaceutically acceptable
carrier or carriers in a
formulation, and/or route of administration.
Lipid Nanoparticles
Expression repressors or expression repression systems as described herein can
be delivered using any
biological delivery system/formulation including a particle, for example, a
nanoparticle delivery system.
Nanoparticles include particles with a dimension (e.g. diameter) between about
1 and about 1000
nanometers, between about 1 and about 500 nanometers in size, between about 1
and about 100 nm,
between about 30 nm and about 200 nm, between about 50 nm and about 300 nm,
between about 75 nm
and about 200 nm, between about 100 nm and about 200 nm, and any range
therebetween. A nanoparticle
has a composite structure of nanoscale dimensions. In some embodiments,
nanoparticles are typically
spherical although different morphologies are possible depending on the
nanoparticle composition. The
portion of the nanoparticle contacting an environment external to the
nanoparticle is generally identified
as the surface of the nanoparticle. In some embodiments, nanoparticles have a
greatest dimension ranging
between 25 nm and 200 nm. Nanoparticles as described herein comprise delivery
systems that may be
provided in any form, including but not limited to solid, semi-solid,
emulsion, or colloidal nanoparticles.
A nanoparticle delivery system may include but not limited to lipid-based
systems, liposomes, micelles,
micro-vesicles, exosomes, or gene gun. In one embodiment, the nanoparticle is
a lipid nanoparticle
(LNP). In some embodiments, the LNP is a particle that comprises a plurality
of lipid molecules
physically associated with each other by intermolecular forces.
In some embodiments, an LNP may comprise multiple components, e.g., 3-4
components. In one
embodiment, the expression repressor or a pharmaceutical composition
comprising said expression
repressor (or a nucleic acid encoding the same, or pharmaceutical composition
comprising said expression
repressor nucleic acid) is encapsulated in an LNP. In one embodiment, the
expression repression system
or a pharmaceutical composition comprising said expression repression system
(or a nucleic acid
encoding the same, or pharmaceutical composition comprising said expression
repression system nucleic
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
216
acid) is encapsulated in an LNP. In some embodiments, the nucleic acid
encoding the first expression
repressor and the nucleic acid encoding the second expression repressor are
present in same LNP. In some
embodiments, the nucleic acid encoding the first expression repressor and the
nucleic acid encoding the
second expression repressor are present in different LNPs. Preparation of LNPs
and the modulating agent
encapsulation may be used/and or adapted from Rosin et al, Molecular Therapy,
vol. 19, no. 12, pages
1286-2200, December 2011). In some embodiments, lipid nanoparticle
compositions disclosed herein are
useful for expression of protein encoded by mRNA. In some embodiments, nucleic
acids, when present in
the lipid nanoparticles, are resistant in aqueous solution to degradation with
a nuclease.
In some embodiments, the LNP formulations may include a CCD lipid, a neutral
lipid, and/or a
helper lipid. In some embodiments, the LNP formulation comprises an ionizable
lipid. In some
embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic
lipid, or an amine-
containing lipid that can be readily protonated. In some embodiments, the
lipid is a cationic lipid that
can exist in a positively charged or neutral form depending on pH. In some
embodiments, the cationic
lipid is a lipid capable of being positively charged, e.g., under
physiological conditions. In some
embodiments, the lipid particle comprises a cationic lipid in formulation with
one or more of neutral
lipids, ionizable amine-containing lipids, biodegradable alkyn lipids,
steroids, phospholipids including
polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol,
and polymer conjugated lipids.
In some embodiments, LNP formulation (e.g., MC3 and/or SSOP) includes
cholesterol, PEG, and/or a
helper lipid. The LNPs may be, e.g., microspheres (including unilamellar and
multilamellar vesicles,
lamellar phase lipid bilayers that, in some embodiments, are substantially
spherical.
In some embodiments, the LNP can comprise an aqueous core, e.g., comprising a
nucleic acid encoding
an expression repressor or a system as disclosed herein. In some embodiments
of the present disclosure,
the cargo for the LNP formulation includes at least one guide RNA. In some
embodiments, the cargo,
e.g., a nucleic acid encoding an expression repressor, or a system as
disclosed herein, may be adsorbed to
the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some
embodiments, the cargo, e.g., a
nucleic acid encoding an expression repressor, or a system as disclosed herein
may be associated with the
LNP. In some embodiments, the cargo, e.g., a nucleic acid encoding an
expression repressor, or a system
as disclosed herein, may be encapsulated, e.g., fully encapsulated and/or
partially encapsulated in an LNP.
In some embodiments, an LNP comprising a cargo may be administered for
systemic delivery, e.g.,
delivery of a therapeutically effective dose of cargo that can result in a
broad exposure of an active agent
within an organism. Systemic delivery of lipid nanoparticles can be by any
means known in the art
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
217
including, for example, intravenous, intraarterial, subcutaneous, and
intraperitoneal delivery. In some
embodiments, systemic delivery of lipid nanoparticles is by intravenous
delivery. In some embodiments,
an LNP comprising a cargo may be administered for local delivery, e.g.,
delivery of an active agent
directly to a target site within an organism.
The LNPs may be formulated as a dispersed phase in an emulsion, micelles, or
an internal phase in a
suspension. In some embodiments, the LNPs are biodegradable. In some
embodiments, the LNPs do not
accumulate to cytotoxic levels or cause toxicity in vivo at a therapeutically
effective dose. In some
embodiments, the LNPs do not accumulate to cytotoxic levels or cause toxicity
in vivo after repeat
administrations at a therapeutically effective dose. In some embodiments, the
LNPs do not cause an
innate immune response that leads to a substantially adverse effect at a
therapeutically effective dose.
In some embodiments, the LNP used, comprises the formula (6Z,9Z,28Z,31Z)-
heptatriacont-6,9,28,31-
tetraene-19-y1 4-(dimethylamino) butanoate or ssPalm0-phenyl-P4C2 (ssPalmO-
Phe, SS-OP). In some
embodiments, the LNP formulation comprises the formula, (6Z,9Z,28Z,31Z)-
heptatriacont-6,9,28,31-
tetraene-19-y1 4-(dimethylamino)butanoate (MC3), 1,2-dioleoyl-sn-glycero-3-
phosphocholine (DOPC),
Cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
(PEG2k-DMG), e.g., MC3
LNP or ssPalm0-phenyl-P4C2 (ssPalmO-Phe, SS-OP), 1,2-dioleoyl-sn-glycero-3-
phosphocholine
(DOPC), Cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-
2000 (PEG2k-DMG),
e.g., SSOP-LNP.
Liposomes are spherical vesicle structures composed of a uni- or multilamellar
lipid bilayer surrounding
internal aqueous compartments and a relatively impermeable outer lipophilic
phospholipid bilayer.
Liposomes may be anionic, neutral, or cationic. Liposomes are biocompatible,
nontoxic, can deliver both
hydrophilic and lipophilic drug molecules, protect their cargo from
degradation by plasma enzymes, and
transport their load across biological membranes and the blood brain barrier
(BBB) (see, e.g., Spuch and
Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages,
2011.
doi:10.1155/2011/469679 for review).
Vesicles can be made from several different types of lipids; however,
phospholipids are most
commonly used to generate liposomes as drug carriers. Vesicles may comprise
without limitation
DOTMA, DOTAP, DOTIM, DDAB, alone or together with cholesterol to yield DOTMA
and cholesterol,
DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.
Methods for preparation
of multilamellar vesicle lipids are known in the art (see for example U.S.
Pat. No. 6,693,086, the
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
218
teachings of which relating to multilamellar vesicle lipid preparation are
incorporated herein by
reference). Although vesicle formation can be spontaneous when a lipid film is
mixed with an aqueous
solution, it can also be expedited by applying force in the form of shaking by
using a homogenizer,
sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of
Drug Delivery, vol. 2011,
Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
Extruded lipids can be
prepared by extruding through filters of decreasing size, as described in
Templeton et al., Nature Biotech,
15:647-652, 1997, the teachings of which relating to extruded lipid
preparation are incorporated herein by
reference.
Methods and compositions provided herein may comprise a pharmaceutical
composition
administered by a regimen sufficient to alleviate a symptom of a disease,
disorder, and/or condition. In
some aspects, the present disclosure provides methods of delivering a
therapeutic by administering
compositions as described herein.
Modulating Gene Expression
The present disclosure is further directed, in part, to a method of
modulating, e.g., decreasing,
expression of a target gene, comprising providing an expression repressor (or
a nucleic acid encoding the
same, or pharmaceutical composition comprising said expression repressor
nucleic acid) or an expression
repression system described herein (or a nucleic acid encoding the same, or
pharmaceutical composition
comprising said expression repression system or nucleic acid), and contacting
the target gene, and/or
operably linked transcription control element(s) with the expression repressor
or the expression repression
system. In some embodiments, modulating, e.g., decreasing expression of a
target gene, comprises
modulation of transcription of a target gene, as compared with a reference
value, e.g., transcription of a
target gene, in absence of the expression repressor or the expression
repression system. In some
embodiments, the method of modulating, e.g., decreasing, expression of a
target gene, are used ex vivo,
e.g., on a cell from a subject, e.g., a mammalian subject, e.g., a human
subject. In some embodiments, the
method of modulating, e.g., decreasing, expression of a target gene, are used
in vivo, e.g., on a
mammalian subject, e.g., a human subject. In some embodiments, the method of
modulating, e.g.,
decreasing, expression of a target gene, are used in vitro, e.g., on a cell or
cell line described herein.
The present disclosure is further directed, in part to a method of treating a
condition associated
with over-expression of a target gene in a subject, comprising administering
to the subject an expression
repression system described herein (or a nucleic acid encoding the same, or
pharmaceutical composition
comprising said expression repression system or nucleic acid). Conditions
associated with over-
expression of particular genes are known to those of skill in the art. Such
conditions include, but are not
limited to, metabolic disorders, neuromuscular disorders, cancer (e.g., solid
tumors), fibrosis, diabetes,
urea disorders, immune disorders, inflammation, and arthritis.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
219
The present disclosure is further directed, in part to a method of treating a
condition associated with mis-
regulation of the expression of a target gene in a subject, comprising
administering to the subject an
expression repression system described herein (or a nucleic acid encoding the
same, or pharmaceutical
composition comprising said expression repression system or nucleic acid).
Without wishing to be bound
by theory, it is thought that an expression repression system may be used to
target (e.g., decrease
expression of) a gene which modulates the expression of a target gene, thus
altering expression of the
target gene by altering expression of the modulating gene. Conditions
associated with mis-regulation of
the expression of particular genes are known to those of skill in the art.
Such conditions include, but are
not limited to metabolic disorders, neuromuscular disorders, cancer (e.g.,
solid tumors), fibrosis, diabetes,
urea disorders, immune disorders, inflammation, and arthritis.
Methods and compositions as provided herein may treat a condition associated
with over-
expression or mis-regulation of a target gene by stably or transiently
altering (e.g., decreasing)
transcription of a target gene. In some embodiments, such a modulation
persists for at least about 1 hr to
about 30 days, or at least about 2 hrs, 6 hrs, 12 hrs, 18 hrs, 24 hrs, 2 days,
3, days, 4 days, 5 days, 6 days,
7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days,
16 days, 17 days, 18 days, 19
days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days,
28 days, 29 days, 30 days,
or longer or any time therebetween. In some embodiments, such a modulation
persists for at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or
24 hours, or at least 1, 2, 3, 4, 5, 6,
or 7 days, or at least 1, 2, 3, 4, or 5 weeks, or at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, or 12 months, or at
least 1, 2, 3, 4, or 5 years (e.g., indefinitely). Optionally, such a
modulation persists for no more than 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 years.
In some embodiments, a method or composition provided herein may decrease
expression of a
target gene in a cell by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%
(and optionally up to 100%)
relative to expression of the target gene in a cell not contacted by the
composition or treated with the
method.
In some embodiments, a method provided herein may modulate, e.g., decrease,
expression of a
target gene by disrupting a genomic complex, e.g., an anchor sequence-mediated
conjunction, associated
with said target gene. A gene that is associated with an anchor sequence-
mediated conjunction may be at
least partially within a conjunction (that is, situated sequence-wise between
a first and second anchor
sequences), or it may be external to a conjunction in that it is not situated
sequence-wise between a first
and second anchor sequences, but is located on the same chromosome and in
sufficient proximity to at
least a first or a second anchor sequence such that its expression can be
modulated by controlling the
topology of the anchor sequence-mediated conjunction. Those of ordinary skill
in the art will understand
that distance in three-dimensional space between two elements (e.g., between
the gene and the anchor
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
220
sequence-mediated conjunction) may, in some embodiments, be more relevant than
distance in terms of
base pairs. In some embodiments, an external but associated gene is located
within 2 Mb, within 1.9 Mb,
within 1.8 Mb, within 1.7 Mb, within 1.6 Mb, within 1.5 Mb, within 1.4 Mb,
with 1.3 Mb, within 1.3 Mb,
within 1.2 Mb, within 1.1 Mb, within 1 Mb, within 900 kb, within 800 kb,
within 700 kb, within 500 kb,
within 400 kb, within 300 kb, within 200 kb, within 100 kb, within 50 kb,
within 20 kb, within 10 kb, or
within 5 kb of the first or second anchor sequence.
In some embodiments, modulating expression of a gene comprises altering
accessibility of a
transcriptional control sequence to a gene. A transcriptional control
sequence, whether internal or external
to an anchor sequence-mediated conjunction, can be an enhancing sequence or a
silencing (or repressive)
sequence.
Epigenetic Modification
The present disclosure is further directed, in part, to a method of
epigenetically modifying a target
gene, a transcription control element operably linked to a target gene, or an
anchor sequence (e.g., an
anchor sequence proximal to a target gene or associated with an anchor
sequence-mediated conjunction
operably linked to a target gene), the method comprising providing an
expression repression system (e.g.,
expression repressor(s)), or nucleic acid encoding the same or pharmaceutical
composition comprising
said expression repression system or nucleic acid; and contacting the target
gene or a transcription control
element operably linked to the target gene with the expression repression
system, thereby epigenetically
modifying the target gene or a transcription control element operably linked
to the target gene.
In some embodiments, a method of epigenetically modifying a target gene or a
transcription
control element operably linked to a target gene comprises increasing or
decreasing DNA methylation of
the target gene or a transcription control element operably linked to a target
gene. In some embodiments,
a method of epigenetically modifying a target gene or a transcription control
element operably linked to a
target gene comprises increasing or decreasing histone methylation of a
histone associated with the target
gene or a transcription control element operably linked to a target gene. In
some embodiments, a method
of epigenetically modifying a target gene or a transcription control element
operably linked to a target
gene comprises decreasing histone acetylation of a histone associated with the
target gene, or a
transcription control element operably linked to a target gene. In some
embodiments, a method of
epigenetically modifying a target gene or a transcription control element
operably linked to a target gene
.. comprises increasing or decreasing histone sumoylation of a histone
associated with the target gene or a
transcription control element operably linked to a target gene. In some
embodiments, a method of
epigenetically modifying a target gene or a transcription control element
operably linked to a target gene
comprises increasing or decreasing histone phosphorylation of a histone
associated with the target gene or
a transcription control element operably linked to a target gene.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
221
In some embodiments, a method of epigenetically modifying a target gene or a
transcription
control element operably linked to a target gene may decrease the level of the
epigenetic modification by
at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% (and optionally up to
100%) relative to the level of the
epigenetic modification at that site in a cell not contacted by the
composition or treated with the method.
In some embodiments, a method of epigenetically modifying a target gene or a
transcription control
element operably linked to a target gene may increase the level of the
epigenetic modification by at least
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300, 400,
500, 600, 700, 800, 900, or
1000% (and optionally up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, or
2000%) relative to the level
of the epigenetic modification at that site in a cell not contacted by the
composition or treated with the
method. In some embodiments epigenetic modification of a target gene or a
transcription control element
operably linked to a target gene may modify the level of expression of the
target gene, e.g., as described
herein.
In some embodiments, an epigenetic modification produced by a method described
herein persists
for at least about 1 hr to about 30 days, or at least about 2 hrs, 6 hrs, 12
hrs, 18 hrs, 24 hrs, 2 days, 3, days,
4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13
days, 14 days, 15 days, 16
days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days,
25 days, 26 days, 27 days,
28 days, 29 days, 30 days, or longer or any time therebetween. In some
embodiments, such a modulation
persists for at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, or 24 hours,
or at least 1, 2, 3, 4, 5, 6, or 7 days, or at least 1,2, 3, 4, or 5 weeks, or
at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10,
11, or 12 months, or at least 1, 2, 3, 4, or 5 years (e.g., indefinitely).
Optionally, such a modulation
persists for no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years.
In some embodiments, an expression repression system for use in a method of
epigenetically
modifying a target gene or a transcription control element operably linked to
a target gene comprises an
expression repressor comprising a repressor domain that is or comprises an
epigenetic modifying moiety.
For example, a repressor domain may be or comprise an epigenetic modifying
moiety with DNA
methyltransferase activity, and an endogenous or naturally occurring target
sequence (e.g. a target gene or
transcription control element) may be altered to increase its methylation
(e.g., decreasing interaction of a
transcription factor with a portion of target gene or transcription control
element, decreasing binding of a
nucleating protein to an anchor sequence, and/or disrupting or preventing an
anchor sequence-mediated
conjunction), or may be altered to decrease its methylation (e.g., increasing
interaction of a transcription
factor with a portion of a target gene or transcription control element,
increasing binding of a nucleating
protein to an anchor sequence, and/or promoting or increasing strength of an
anchor sequence-mediated
conjunction).
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
222
Kits
The present disclosure further directed, in part, to a kit comprising an
expression repressor or an
expression repression system, e.g., expression repressor(s), described herein.
In some embodiments, a kit
comprises an expression repressor or an expression repression system (e.g.,
the expression repressor(s) of
the expression repression system) and instructions for the use of said
expression repressor or expression
repression system. In some embodiments, a kit comprises a nucleic acid
encoding the expression
repressor or the expression repression system or a component thereof (e.g.,
the expression repressor(s) of
the expression repression system) and instructions for the use of said nucleic
acid and/or said expression
repressor or the expression repression system. In some embodiments, a kit
comprises a cell comprising a
nucleic acid encoding the expression repressor or the expression repression
system or a component
thereof (e.g., the expression repressor(s) of the expression repression
system) and instructions for the use
of said cell, nucleic acid, and/or said expression repression system.
In some embodiments, a kit comprises a unit dosage of an expression repressor
or an expression
repression system, e.g., expression repressor(s), described herein, or a unit
dosage of a nucleic acid, e.g., a
vector, encoding an expression repressor or an expression repression system,
e.g., expression repressor(s),
described herein.
In some embodiments the kit further comprises b) a set of instructions
comprising at least
one method for treating a disease or modulating, e.g., decreasing the
expression of target gene within a
cell with said composition. In some embodiments, the kits can optionally
include a delivery vehicle for
said composition (e.g., a lipid nanoparticle). The reagents may be provided
suspended in the excipient
and/or delivery vehicle or may be provided as a separate component which can
be later combined with the
excipient and/or delivery vehicle. In some embodiments, the kits may
optionally contain additional
therapeutics to be co-administered with the compositions to affect the desired
target gene expression.
While the instructional materials typically comprise written or printed
materials, they are not limited to
such. Any medium capable of storing such instructions and communicating them
to an end user is
contemplated. Such media include but are not limited to electronic storage
media (e.g., magnetic discs,
tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such
media may include addresses
to internet sites that provide such instructional materials.
In some embodiments, a kit comprises a unit dosage of an expression repressor
an expression
repression system, e.g., expression repressor(s), described herein, or a unit
dosage of a nucleic acid, e.g., a
vector, encoding an expression repression system, e.g., expression
repressor(s), described herein.
The following examples are provided to further illustrate some embodiments of
the present disclosure but
are not intended to limit the scope of the disclosure; it will be understood
by their exemplary nature that
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
223
other procedures, methodologies, or techniques known to those skilled in the
art may alternatively be
used.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
224
EXAMPLES
Example 1: Target Selection and Guide Design
The I3-2-microglobulin (I32M) gene was targeted for down-regulation using an
expression
repression system of the disclosure. A first expression repressor comprising
an S. pyo genes dCas9 and a
KRAB domain (Sp-dCas9-KRAB, corresponding to SEQ ID NOs: 91-93) and a second
expression
repressor comprising S. aureus dCas9 and MQ1 (Sa-dCas9-MQ1, corresponding to
SEQ ID Nos: 94-96)
were used. Guide RNAs (comprising target-binding sequences corresponding to
SEQ ID NOs: 1-5)
targeted each expression repressor to an area of CpG hypomethylation in the
promoter region of the I32M
gene (Figure 1), where methylation runs from 0 to 1 (0 to 100%) and where
higher methylation is
depicted by red bars. Each bar represents a CpG site. Blue guides (GD-28228
and GD-28229) were used
to target Sp-dCas9-KRAB and orange guides (GD-28171, GD-28172 and GD-28173)
were used to target
Sa-dCas9-MQ1 to the CpG Island in the I32M promoter region.
Example 2: I32M Expression Repression Using One or Two Expression Repressors
The guide RNAs of Example 1 were used to target the Sp-dCas9-KRAB expression
repressor, the
Sa-dCas9-MQ1 expression repressor, or both to the promoter region of I32M and
the effects on I32M
expression monitored by qPCR. mRNA encoding the expression repressor(s) and
guide RNA(s) were
delivered to cells in lipid nanoparticles (LNPs).
HepG2 cells were seeded in a 96 well plate at a density of 30,000 cells/well
in 100 tit RPMI (+10% FBS,
+Penn/Strep). Cells were allowed to attach for 24 hours prior to treatment
with 0.125 ug RNA (1:1
mRNA: sgRNA by weight) per guide. RNA was delivered by LNP formulated with
COATSOME SS-OP
ionizable lipid (NOF America). LNPs were formulated with one mRNA and one
guide, with the
exception of the samples noted as "co-formulated" which were prepared with two
guides, maintaining the
same mRNA: guide weight ratio. Cells were incubated with the LNPs for 72
hours, following which the
cells were harvested for I32M mRNA analysis by qPCR (Figure 2), with I32M mRNA
levels given relative
to untreated control cells.
dCas9 alone targeted to the I32M promoter did not decrease I32M mRNA levels.
The data shows that Sp-
dCas9-KRAB or Sa-dCas9-MQ1 alone decreased I32M mRNA levels, with some
variation in the decrease
depending on the guide RNA(s) used. Sp-dCas9-KRAB and Sa-dCas9-MQ1 together
decreased I32M
mRNA levels on average more than either expression repressor alone, again with
some variation in the
decrease depending on the guide RNA(s) used. Use of an expression repression
system comprising one or
two expression repressors decreased expression of a target gene The expression
repression system
comprising two expression repressors appeared to decrease expression of the
target gene more than a
single expression repressor.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
225
Example 3: I32M Expression Repression Across Doses and Using Flow Cytometry
Guide RNA of Example 1 (GD-28228 for Sp-dCas9 and Sp-dCas9-KRAB; GD-28172 for
Sa-dCas9-
MQ1; or GD-28228 and GD-28172 for Sp-dCas9-KRAB and Sa-dCas9-MQ1) was used to
target the Sp-
dCas9-KRAB expression repressor, the Sa-dCas9-MQ1 expression repressor, or
both to the promoter
region of I32M and the effects on I32M expression monitored by qPCR and also
by flow cytometry (using
a fluorescent I32M to monitor I32M protein levels). mRNA encoding the
expression repressor(s) and guide
RNA(s) were delivered to cells in LNPs at different dosages and formulations.
HepG2 cells were seeded in a 12-well plate at a density of 330,000 cells/well
1.5 mL RPMI (+10% FBS,
+Penn/Strep). Cells were allowed to attach for 24 hours prior to treatment
with 1.25 ug or 2.5 ug RNA
(1:1 mRNA: sgRNA by weight) per guide in 1 mL media. RNA was delivered by LNP
formulated with
COATSOME SS-OP ionizable lipid (NOF America). LNPs were formulated with one
mRNA and one
guide, with the exception of the samples noted as "co-formulated" which were
prepared with two guides,
maintaining the same mRNA: guide weight ratio. Cells were incubated with the
LNPs for 72 hours,
following which the cells were harvested for FACS and mRNA analysis by qPCR.
As seen in Example 2, dCas9 alone targeted to the I32M promoter did not
decrease I32M mRNA levels.
Sp-dCas9-KRAB or Sa-dCas9-MQ1 alone, and Sp-dCas9-KRAB and Sa-dCas9-MQ1
together decreased
I32M mRNA levels (Figure 3). Both 1.25 ug/ml and 2.5 ug/ml doses of the
expression repressors (alone or
together) decreased I32M mRNA levels, with the higher dose of Sp-dCas9-KRAB
appearing to give a
greater decrease than the lower dose. FACS monitoring of I32M protein levels
showed that Sp-dCas9-
KRAB or Sa-dCas9-MQ1 treated cells showed a decrease in the population of
cells having the untreated
level of I32M protein and the appearance of a new population of cells with a
decreased level of I32M
protein. These data are consistent with those of Example 2, showing that use
of an expression repression
system comprising one or two expression repressors decreases expression of a
target gene, and that
individual expression repressors decrease the levels of a protein encoded by
the target gene too.
Example 4: I32M Expression Repression Over Time
Guide RNA (GD-28228 for Sp-dCas9 and Sp-dCas9-KRAB; GD-28172 for Sa-dCas9-MQ1;
or
GD-28228 and GD-28172 for Sp-dCas9-KRAB and Sa-dCas9-MQ1) of Example 1 was
used to target the
Sp-dCas9-KRAB expression repressor, the Sa-dCas9-MQ1 expression repressor, or
both to the promoter
region of I32M and the effects on I32M expression monitored by qPCR. Cells
were collected at different
days after transfection to extract total RNA and probe for expression changes.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
226
K562 cells were plated in a 24-well plate and transfected with MC3-based LNP
formulation enclosing:
dCas9 mRNA with I32M targeting guide RNA, Sp-dCas9-KRAB mRNA with I32M
targeting guide RNA,
Sa-dCas9-MQ1 mRNA with I32M targeting guide RNA, or a mixture of Sp-dCas9-KRAB
and Sa-dCas9-
MQ1 mRNAs with I32M targeting guide RNAs. Cells were collected at different
days after transfection to
extract total RNA and probe for expression changes in I32M mRNA (Figure 4).
Dotted lines indicate
average untreated I32M mRNA levels over time (top) and average knockdown
levels of I32M mRNA 24
hours post transfection (bottom).
Over a 22-day period, non-transfected (blue line) and dCas9 transfected (red
line) cells don't change I32M
expression, whereas Sp-dCas9-KRAB (green line) transfected cells have lower
I32M mRNA levels in the
initial days post-transfection, with maximum effect on day 2, but the
repression is lost after day 5. Sa-
dCas9-MQ1 (purple line) transfected cells show significant repression (up to
85% by day 4) of I32M
mRNA levels and the repression is retained and then slowly the repression
decreases somewhat as seen by
the line remaining just under the dotted line in the figure at the end of the
test period. Sp-dCas9-KRAB
and Sa-dCas9-MQ1 combined transfection (orange line) did not give any added
repression than what is
already observed for Sa-dCas9-MQ1 alone (purple line).
These data show that use of an expression repression system comprising one or
two expression
repressors decreases expression of a target gene. Individual expression
repressors such as those
comprising MQ1 can repress expression of a target gene for an extended
duration, and expression
repression systems comprising more than one expression repressor, e.g., one of
which is an expression
repressor comprising MQ1, can repress expression of a target gene for an
extended duration as well.
Example 5: Durable repression of B2M expression after promoter methylation by
DNA
methyltransferases
This example demonstrates methylation of B2M promoter region by treatment with
LNPs
containing various DNMTs and LNPs containing a guide targeting the dCas9
fusions to the B2M
promoter region cause durable repression of B2M expression.
K-562 cells were seeded in 24 well plates at a density of 200,000 cells per
well in 750 tit of
growth medium (RPMI + 10% FBS + pen/strep). mRNAs encoding effectors, dCas9-
MQ1(SEQ ID NO:
33), dCas9-DNMT3a/3L (m) ("m" denoting that the 3a region has the human
sequence but the 3L region
has the mouse sequence) (SEQ ID NO:36), dCas9-DNMT3a/3L (h ("h" denoting that
both the 3a region
and the 3L region have the human sequences) (SEQ ID NO:35), dCas9-DNMT1(SEQ ID
NO: 35), and
dCas9-DNMT3B were formulated in MC3 lipids using a Precision NanoSystems
NanoAssemblr Spark
instrument. Single guide RNAs (sgRNAs) of SEQ ID NO: 21 were formulated in the
same lipids in the
same instrument in separate formulations. Stock LNP concentrations were 100
tig/mL. Cells were treated
with LNPs encapsulating mRNAs and LNPs encapsulating sgRNAs in a 1:1 ratio at
a final lipid
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
227
concentration of 1.25 tig/mL and incubated thereafter. Each effector and guide
combination were
transfected in quadruplicate. At different timepoints after transfection, 200
tiL of cells were removed and
processed to extract total RNA and probe for expression changes and 200 tit of
cells were moved into
550 tit of fresh media. This procedure continued until 18 days after the
initial transfection. RNA was
extracted using the RNeasy 96 Plus kits (Qiagen), converted to cDNA using
LunaScript RT SuperMix
(New England Biolabs) and used for RT-qPCR using a B2M specific TaqMan
primer/probe set Assay
with and the TaqMan' Fast Advanced Master Mix (Thermo Fisher Scientific). Data
is presented as fold
change compared to un-transfected control using the ddCT method.
Results show, all the constructs described in this Example were able to
repress B2M expression
ranging from about 35% to 60%, 4 days after treatment relative to an untreated
sample (Figure 5). All the
constructs were able to repress B2M expression ranging from about 15% to 30%
at the end of 8 days
relative to an untreated sample. At the end of 13 days, all effectors were
able to repress B2M expression
by about 45-55%, and at the end of 18 days, all effectors were able to repress
B2M expression by about
30-40% relative to an untreated sample (Figure 5).
Example 6: Identification of durable repressors or repressor systems that
downregulate MYC
expression by targeting a CTCF site located upstream of MYC gene in K-562
cells
This example demonstrates identification of individual expression repressors
or expression repression
systems that downregulate MYC expression by targeting the CTCF site located
upstream of MYC gene.
K-562 cells were grown in 96 well plates at a density of 1 x106 cell/mL in 100
tiL of growth
medium (RPMI + 10% FBS + pen/strep). A sgRNA pool was prepared by mixing GD-
29639 (SEQ ID
NO: 11) and GD-29640 (SEQ ID NO: 12) in 1:1 ratio. A repression master plate
was prepared by adding
5 iu.1 of mRNAs encoding effectors or effector combinations from Table 4 in
each well. After addition,
17.5 ng of each mRNA or combination of mRNAs were present in each well. After
plating the master
plates were stored frozen until the time of transfection.
Table 4: List of Expression repressors or expression repression systems tested
Expression repressors or expression repression systems
1 No effector (Untreated)
2 dCas9
3 dCas9-DNMT1
4 dCas9-DNMT3B
5 G9A-dCas9
6 dCas9-MQ1
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
228
7 dCas9-HDAC8
8 dCas9-DNMT3a/3L (h)
9 dCas9-DNMT3a/3L (m)
EZH2-dCas9
11 dCas9-LSD1
12 dCas9-KRAB
13 EZH2-dCas9-KRAB
14 G9A-dCas9-KRAB
FOG1-dCas9- FOG1
16 dCas9-KRAB + FOG1-dCas9- FOG1
17 EZH2-dCas9-KRAB + dCas9-HDAC8
18 dCas9-LSD1 + dCas9-HDAC8
19 G9A-dCas9-KRAB + EZH2-dCas9-KRAB
G9A-dCas9 + dCas9-HDAC8
21 EZH2-dCas9 + dCas9-HDAC8
22 dCas9-LSD1 + G9A-dCas9-KRAB
23 dCas9-LSD1 + EZH2-dCas9-KRAB
24 dCas9-LSD1 + EZH2-dCas9
G9A-dCas9-KRAB + dCas9-HDAC8
26 G9A-dCas9 + EZH2-dCas9
27 dCas9-MQ1 + EZH2-dCas9-KRAB
28 dCas9-LSD1 + G9A-dCas9
29 dCas9-MQ1 + dCas9-HDAC8
dCas9-MQ1 + G9A-dCas9-KRAB
Prior to transfection, 5 .1 of sgRNA pool were added in each well so that
8.75 ng of each sgRNA were
present in each well. The effector mRNA-sgRNA mix were diluted with Opti-MEM
media (Thermo
5 Fisher) to a total volume of 40 .1 in each well. A transfection master
mix of Lipofectamine'
MessengarMAX' (Thermo Fisher) and Opti-MEM were prepared using a ratio of 0.3
tiL of
Lipofectamine MessengarMAX' per 100 ng of total RNA as described in Table 5.
Immediately before
transfection, 35 tiL of transfection master mix were added to each well
containing RNA to bring the final
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
229
volume to 75 il and were incubated for 5 minutes at room temperature and was
used to transfect K-562
cells. After transfection the cells were incubated at 37 C with 5% CO2 in
standard incubator.
Table 5
Reagent Per well Per 96-well plate Per 2, 96-
well Per 4, 96-well
plates plates
MessengerMax 4.2 189 378 756
( L)
Optimem ( L) 30.8 1386 2772 5544
24 hours after transfection, 100 tiL of fresh media was added to each well.
Sample collection started 48
hours post transfection and continued until 6 days. Cell suspension samples
were collected at 48 hours, 72
hours, and 144-hour time point. Each time samples were removed for analysis
equivalent volumes of
fresh media was added and the incubations were continued. RNA was extracted
using the Rneasy Plus
96 kits (Qiagen), converted to cDNA using LunaScript RT SuperMix (New England
Biolabs) and used
for RT-qPCR using a MYC specific Taqman primer/probe set assay with the
Taqman' Fast Advanced
Master Mix (Thermo Scientific). Data was presented as fold change compared to
dCas9 treated control
using the ddCT method. The untreated and dCas9 samples were used as
calibrators.
The experiment showed that, at least, cells treated with dCas9-DNMT3a/3L (h),
dCas9-KRAB,
FOG1-dCas9-FOG1, G9A-dCas9 + EZH2-dCas9, dCas9-MQ1 + EZH2-dCas9-KRAB, dCas9-
LSD1+G9A-dCas9, dCas9-MQ1+dCas9-HDAC8, and dCas9-MQ1+G9A-dCas9-KRAB showed
repression of MYC expression at 48 hour and/or 72-hour time point (Figure 6).
Example 7: Identification of durable repressors or repression systems that
downregulate I32M
expression by targeting a region upstream of the I32M transcription start site
in K-562 cells
This example demonstrates identification of individual expression repressors
or expression repression
systems that downregulate B2M expression by targeting the upstream region of
I32M promoter.
K-562 cells were grown in 96 well plates at a density of lx106 cell/mL in 100
tiL of growth
medium (RPMI + 10% FBS + pen/strep). A sgRNA pool was prepared by mixing GD-
27634 (SEQ ID
NO: 13) and GD-29500 (SEQ ID NO: 14) in 1:1 ratio. The effectors as described
in Table X and sgRNA
pool mix was used to transfect the cells following the protocol described in
Example 6. Cell suspension
samples were collected at 48 hours, 72 hours, 144-hour, 192 hours, and 240
hour time point and were
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
230
processed using the protocol described in Example 6. Data was presented as
fold change compared to
dCas9 treated control using the ddCT method. The untreated and dCas9 samples
were used as calibrators.
The data showed that, at least, cells treated with dCas9-HDAC8, dCas9-LSD1,
dCas9-MQ1,
FOG1-dCas9- FOG1, G9A-dCas9-KRAB, dCas9-DNMT3a/3L (h), G9A-dCas9, EZH2-dCas9-
KRAB,
dCas9-KRAB, EZH2-dCas9-KRAB + dCas9-HDAC8, dCas9-LSD1 + EZH2-dCas9-KRAB, dCas9-
LSD1 + dCas9-HDAC8, dCas9-KRAB + FOG1-dCas9- FOG1, dCas9-MQ1 + G9A-dCas9-KRAB,
G9A-
dCas9 + EZH2-dCas9, dCas9-MQ1 + EZH2-dCas9-KRAB, and dCas9-MQ1 + dCas9-HDAC8
showed
repression of I32M expression at 48 hour, 72 hour and/or 144 hour time point
(Figure 7).
Example 8: Identification of durable repressors or repression systems that
downregulate HSPA1B
expression by targeting a region downstream of the HSPA1B transcription start
site in K-562 cells
This example demonstrates identification of individual expression repressors
or expression repression
systems that downregulate HSPA1B expression by targeting the downstream region
of HSPA1B
promoter.
K-562 cells were grown in 96 well plates at a density of 1x106 cell/mL in 100
tiL of growth
medium (RPMI + 10% FBS + pen/strep). A sgRNA pool was prepared by mixing GD-
29542 (SEQ ID
NO: 17) and GD-29544 (SEQ ID NO: 18) in 1:1 ratio. The effectors as described
in Table 4 and sgRNA
pool mix was used to transfect the cells following the protocol described in
Example 6. Cell suspension
samples were collected at 48 hours, 72 hours, and 144-hour time point and were
processed using the
protocol described in Example 6. Data was presented as fold change compared to
dCas9 treated control
using the ddCT method. The untreated and dCas9 samples were used as
calibrators.
The data showed that, at least, cells treated with dCas9-HDAC8, EZH2-dCas9,
dCas9-MQ1,
dCas9-DNMT1, dCas9-DNMT3a/3L (h), dCas9-DNMT3a/3L (m), G9A-dCas9-KRAB, FOG1-
dCas9-
FOG1, G9A-dCas9, dCas9-KRAB, G9A-dCas9 + dCas9-HDAC8, dCas9-LSD1 + G9A-dCas9-
KRAB,
dCas9-LSD1+EZH2-dCas9, G9A-dCas9+EZH2-dCas9, dCas9-LSD1 + EZH2-dCas9-KRAB,
EZH2-
dCas9 +dCas9-HDAC8, dCas9-LSD1+dCas9-HDAC8, dCas9-MQ1 + EZH2-dCas9-KRAB, dCas9-
MQ1+dCas9-HDAC8, G9A-dCas9-KRAB+EZH2-dCas9-KRAB, dCas9-LSD1+G9A-dCas9, and
dCas9-
MQ1+G9A-dCas9-KRAB showed repression of HSPA1B expression at 48 hour, 72 hour
and/or 144 hour
time point (Figure 8).
Example 9: Identification of durable repressors or repression systems that
downregulate GATA1
expression in K-562 cells
This example demonstrates identification of individual expression repressors
or expression repression
systems that downregulate GATA1 expression in K-562 cells.
CA 03196827 2023-03-24
WO 2022/067033
PCT/US2021/051945
231
K-562 cells were grown in 96 well plates at a density of 1 x106 cell/mL in 100
L of growth
medium (RPMI + 10% FBS + pen/strep). A sgRNA pool was prepared by mixing GD-
29536 (SEQ ID
NO: 19) and GD-29693 (SEQ ID NO: 20) in 1:1 ratio. The effectors as described
in Table 4 and sgRNA
pool mix was used to transfect the cells following the protocol described in
Example 6. Cell suspension
samples were collected at 48 hours, 72 hours, and 144-hour time point and were
processed using the
protocol described in Example 6. Data was presented as fold change compared to
dCas9 treated control
using the ddCT method. The untreated and dCas9 samples were used as
calibrators.
The data showed that, at least, cells treated with dCas9-HDAC8, dCas9-
LSD1+dCas9-HDAC8,
dCas9-MQ1+dCas9-HDAC8, and G9A-dCas9+dCas9-HDAC8 showed repression of GATA1
expression
at 48-hour, 72 hour and/or 144 hour time point (Figure 9).
EQUIVALENTS
It is to be understood that while the invention has been described in
conjunction with the detailed
description thereof, the foregoing description is intended to illustrate and
not limit the scope of the
invention, which is defined by the scope of the appended claims. Some aspects,
advantages, and
modifications are within the scope of the following claims.
