Note: Descriptions are shown in the official language in which they were submitted.
WO 2021/247020
PCT/US2020/035972
1
METHODS FOR THE TREATMENT OF NUCLEOTIDE REPEAT EXPANSION
DISORDERS ASSOCIATED WITH MSH3 ACTIVITY
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0001] The content of the electronically submitted sequence
listing (Name:
4398 029PC00 Seqlisting ST25; Size: 154,840 bytes and Date of Creation: June
3,
2020) is incorporated herein by reference in its entirety.
BACKGROUND
[0002] Nucleotide repeat expansion disorders (e.g., trinucleotide
repeat expansion
disorders) are genetic disorders caused by nucleotide repeat expansions (e.g.,
trinucleotide repeats). Nucleotide repeat expansions (e.g., trinucleotide
repeat
expansions) are a type of genetic mutation where nucleotide repeats in certain
genes or
introns exceed the normal, stable threshold for that gene. The nucleotide
repeats (e.g.,
trinucleotide repeats) can result in defective or toxic gene products, impair
RNA
transcription, and/or cause toxic effects by forming toxic mRNA transcripts.
[0003] Nucleotide repeat expansion disorders (e.g., trinucleotide
repeat expansion
disorders) are generally categorized by the type of repeat expansion. For
example, Type
1 disorders such as Huntington's disease are caused by CAG repeats which
result in a
series of glutamine residues known as a polyglutamine tract, Type 2 disorders
are caused
by heterogeneous expansions that are generally small in magnitude, and Type 3
disorders
such as fragile X syndrome are characterized by large repeat expansions that
are generally
located outside of the protein coding region of the genes. Nucleotide repeat
expansion
disorders (e.g., trinucleotide repeat expansion disorders) are characterized
by a wide
variety of symptoms such as progressive degeneration of nerve cells that is
common in
the Type 1 disorders.
[0004] Subjects with a nucleotide repeat expansion disorder (e.g., a
trinucleotide repeat
expansion disorder) or those who are considered at risk for developing a
nucleotide repeat
expansion disorder (e.g., a trinucleotide repeat expansion disorder) have a
constitutive
nucleotide expansion in a gene associated with disease (i.e., the nucleotide
repeat
expansion is present in the gene during embryogenesis). Constitutive
nucleotide repeat
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
2
expansions (e.g., trinucleotide repeat expansions) can undergo expansion after
embryogenesis (i.e., somatic nucleotide repeat expansion). Both constitutive
ucleotide
repeat expansion and somatic nucleotide repeat expansion can be associated
with
presence of disease, age at onset of disease, and/or rate of progression of
disease.
SUMMARY OF THE DISCLOSURE
[0005] The present disclosure features useful compositions and methods
to treat
nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion
disorders), e.g.,
in a subject in need thereof. In some aspects, the compositions and methods
described
herein are useful in the treatment of disorders associated with IVISH3
activity.
Oligonttcleotides
[0006] Some aspects of the disclosure are related to a single-stranded
oligonucleotide of
15-30 linked nucleotides in length, wherein the oligonucleotide, or a portion
thereof, is at
least 95% complementary to at least 15 contiguous nucleobases at positions
2543-2573 of
SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide, or a portion thereof, is at least 98% complementary to at
least 15
contiguous nucleobases at positions 2543-2573 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
portion thereof, is at least 99% complementary to at least 15 contiguous
nucleobases at
positions 2543-2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
In some aspects, the oligonucleotide, or a portion thereof, is 100%
complementary to at
least 15 contiguous nucleobases at positions 2543-2573 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
portion thereof, is complementary to 17-23 contiguous nucleobases at positions
2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof. In some
aspects,
the oligonucleotide is complementary to 17-20 contiguous nucleobases at
positions 2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof. In some
aspects,
the 17-20 contiguous nucleobases begin at position 2543, 2544, 2545, 2546,
2547, 2548,
2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, or 2557 of SEQ ID NO: 614, or
a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is 17-20
linked nucleotides in length, or a pharmaceutically acceptable salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
3
[0007] In some aspects, the oligonucleotide, or a portion thereof, is
complementary to 20-
23 contiguous nucleobases at positions 2543-2573 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the 20-23
contiguous
nucleobases begin at position 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550,
2551,
2552, 2553, or 2554 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
In some aspects, the oligonucleotide is 20-23 linked nucleotides in length, or
a
pharmaceutically acceptable salt thereof.
[0008] In some aspects, the oligonucleotide, or a portion thereof, is
complementary to
positions 2543-2570 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
[0009] The disclosure also relates to single-stranded oligonucleotides
of 15-30 linked
nucleotides in length, wherein the oligonucleotide, or a portion thereof, is
at least 95%
complementary to at least 15 contiguous nucleobases at positions 2685-2714 of
SEQ ID
NO: 614, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a portion thereof, is at least 98% complementary to at
least 15
contiguous nucleobases at positions 2685-2714 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
portion thereof, is at least 99% complementary to at least 15 contiguous
nucleobases at
positions 2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
In some aspects, the oligonucleotide or a portion thereof, is 100%
complementary to at
least 15 contiguous nucleobases at positions 2685-2714 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
portion thereof is complementary to 17-23 contiguous nucleobases at positions
2685-2714
of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide, or a portion thereoff, is complementary to 17-20 contiguous
nucleobases
at positions 2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable
salt thereof
In some aspects, the oligonucleotide, or a portion thereof, is complementary
to 17-20
contiguous nucleobases beginning at position 2685, 2686, 2687, 2688, 2689,
2690, 2691,
2692, 2693, 2694, 2695, 2696, 2697, or 2698 of SEQ ID NO: 614, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide is 17-20 linked
nucleotides
in length, or a pharmaceutically acceptable salt thereof.
[0010] In some aspects, the oligonucleotide, or a portion thereof, is
complementary to 20-
23 contiguous nucleobases at positions 2685-2714 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
4
complementary to 20-23 contiguous nucleobases beginning at position 2685,
2686, 2687,
2688, 2689, 2690, 2691, 2692, 2693, 2694, or 2695 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is 20-23
linked nucleotides in length, or a pharmaceutically acceptable salt thereof.
In some
aspects, the oligonucleotide, or a portion thereof, is complementary to
positions 2685-
2714 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
100111 In some aspects of the above, the oligonucleotide is not any one
of Antisense
Oligo Nos. 1, 97, 193, or 289 of Table 3. In some aspects of the above, the
oligonucleotide does not have a nucleobase sequence consisting of any one of
SEQ ID
NOs: 1,97, 193, or 289.
[0012] In some aspects of the above disclosure, the
oligonucleotide comprises:
(a) a DNA core sequence comprising linked deoxyribonucleosides;
(b) a 5' flanking sequence comprising linked nucleosides; and
(c) a 3' flanking sequence comprising linked nucleosides;
wherein the DNA core comprises a region of at least 10 contiguous nucleobases
positioned between the 5' flanking sequence and the 3' flanking sequence,
wherein the 5'
flanking sequence and the 3' flanking sequence each comprises at least two
linked
nucleosides; and wherein at least one nucleoside of each flanking sequence
comprises an
alternative nucleoside, or a pharmaceutically acceptable salt thereof. In some
aspects of
the above disclosures, the oligonucleotide comprises at least one alternative
internucleoside linkage, or a pharmaceutically acceptable salt thereof. In
some aspects of
the above disclosures, the at least one alternative internucleoside linkage is
a
phosphorothioate internucleoside linkage. In some aspects of the above
disclosures, the
at least one alternative internucleoside linkage is a 2'-alkoxy
internucleoside linkage In
some aspects of the above disclosures, the at least one alternative
internucleoside linkage
is an alkyl phosphate internucleoside linkage.
[0013] In some aspects of the above disclosures, the oligonucleotide
comprises at least
one alternative nucleobase, or a pharmaceutically acceptable salt thereof In
some aspects
of the above disclosures, the alternative nucleobase is 5'-methylcytosine,
pseudouridine,
or 5-methoxyuridine. In some aspects of the above disclosures, the
oligonucleotide
comprises at least one alternative sugar moiety, or a pharmaceutically
acceptable salt
thereof. In some aspects, the alternative sugar moiety is 2'-0Me or a bicyclic
nucleic
acid.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
[0014] In some aspects of the above disclosures, the oligonucleotide
further comprises a
ligand conjugated to the 5' end or the 3' end of the oligonucleotide through a
monovalent
or branched bivalent or trivalent linker, or a pharmaceutically acceptable
salt thereof.
[0015] In some aspects of the above disclosures, the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs: 1-
384
and 390-613, or a pharmaceutically acceptable salt thereof. In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 2-96, 98-192, 194-288, 290-384, and 390-613, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide consists of a
nucleobase
sequence selected from the group consisting of any of SEQ ID NOs: 1-384, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
2-96,
98-192, 194-288, and 290-384, or a pharmaceutically acceptable salt thereof.
In some
aspects, the oligonucleotide consists of a nucleobase sequence selected from
the group
consisting of any of SEQ ID NOs: 1-96, or a pharmaceutically acceptable salt
thereof. In
some aspects, the oligonucleotide consists of a nucleobase sequence selected
from the
group consisting of any of SEQ ID NOs: 2-96, or a pharmaceutically acceptable
salt
thereof. In some aspects, the oligonucleotide consists of a nucleobase
sequence selected
from the group consisting of any of SEQ ID NOs: 97-192, or a pharmaceutically
acceptable salt thereof In some aspects, the oligonucleotide consists of a
nucleobase
sequence selected from the group consisting of any of SEQ ID NOs: 98-192, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
193-
288, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide
consists of a nucleobase sequence selected from the group consisting of any of
SEQ ID
NOs: 194-288, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 289-384, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of a nucleobase sequence selected from
the group
consisting of any of SEQ ID NOs: 288-384, or a pharmaceutically acceptable
salt thereof.
In some aspects, the oligonucleotide consists of a nucleobase sequence
selected from the
group consisting of any of SEQ ID NOs: 390-613, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of a nucleobase
sequence selected
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
6
from the group consisting of any of SEQ ID NOs: 390-480, or a pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide consists of a
nucleobase
sequence selected from the group consisting of any of SEQ ID NOs: 481-571, or
a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
572-
662, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide
consists of a nucleobase sequence selected from the group consisting of any of
SEQ ID
NOs: 663-613, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 1,6, 13, 17, 21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-
73, 75-76,
79-82, 84-86, 88-92, or 94-96, or a pharmaceutically acceptable salt thereof.
In some
aspects, the oligonucleotide consists of a nucleobase sequence selected from
the group
consisting of any of SEQ ID NOs: 6, 13, 17, 21, 24, 26, 29, 33-34, 37, 44, 49-
55, 57, 60-
73, 75-76, 79-82, 84-86, 88-92, or 94-96, or a pharmaceutically acceptable
salt thereof. In
some aspects, the oligonucleotide consists of a nucleobase sequence that is
SEQ ID NO:
1, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of a nucleobase sequence that is SEQ ID NO: 6, or a pharmaceutically
acceptable
salt thereof In some aspects, the oligonucleotide consists of a nucleobase
sequence
selected from the group consisting of any of SEQ ID NOs: 97, 100, 103, 105,
108, 110-
111, 113-117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148,
154-
155, 157-165, 168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically
acceptable
salt thereof In some aspects, the oligonucleotide consists of a nucleobase
sequence
selected from the group consisting of any of SEQ ID NOs: 100, 103, 105, 108,
110-111,
113-117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-
155,
157-165, 168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of a nucleobase
sequence that is
SEQ ID NO: 97, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 193-200, 202-230, 232-246, 248-253, 255, 258-261, 265, 270,
274-
276, or 285-286, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 194-200, 202-230, 232-246, 248-253, 255, 258-261, 265, 270,
274-
276, or 285-286, or a pharmaceutically acceptable salt thereof. In some
aspects, the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
7
oligonucleotide consists of a nucleobase sequence that is SEQ ID NO: 193, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
226-
227, 234, 240, or 243-244, or a pharmaceutically acceptable salt thereof. In
some aspects,
the oligonucleotide consists of a nucleobase sequence selected from the group
consisting
of any of SEQ ID NOs: 227, 234, 240, or 243-244, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence that is
SEQ ID NO: 226, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 289-290, 292, 305, 307, 313, 318, 323-324, 326, 329-330,
332, 338-
339, 341, 344, or 346, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 290, 292, 305, 307, 313, 318, 323-324, 326, 329-330, 332,
338-339,
341, 344, or 346, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of a nucleobase sequence that is SEQ ID NO: 289, or a
pharmaceutically acceptable salt thereof.
100161 Some aspects of the disclosure are directed to single-stranded
oligonucleotides,
wherein the nucleobase sequence of the oligonucleotide consists of any one of
SEQ ID
NOs: 1-384 and 390-613, or a pharmaceutically acceptable salt thereof In some
aspects,
the nucleobase sequence of the oligonucleotide consists of any one of SEQ ID
NOs: 2-96,
98-192, 194-288, 290-384, and 390-613, or a pharmaceutically acceptable salt
thereof. In
some aspects, the nucleobase sequence of the oligonucleotide consists of any
one of SEQ
ID NOs: 1-384, or a pharmaceutically acceptable salt thereof. In some aspects,
the
nucleobase sequence of the oligonucleotide consists of any one of SEQ ID NOs:
2-96, 98-
192, 194-288, or 290-384, or a pharmaceutically acceptable salt thereof. In
some aspects,
the oligonucleotide consists of the nucleobase sequence of any one of SEQ ID
NOs: 1-96,
or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of the nucleobase sequence of any one of SEQ ID NOs: 2-96, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 97-192, or a
pharmaceutically
acceptable salt thereof In some aspects, the oligonucleotide consists of the
nucleobase
sequence of any one of SEQ ID NOs: 96-192, or a pharmaceutically acceptable
salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
8
one of SEQ ID NOs: 193-288, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of the nucleobase sequence of any one of
SEQ ID
NOs: 194-288, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs:
289-384,
or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of the nucleobase sequence of any one of SEQ ID NOs: 290-384, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 390-613, or a
pharmaceutically
acceptable salt thereof In some aspects, the oligonucleotide consists of the
nucleobase
sequence of any one of SEQ ID NOs: 390-480, or a pharmaceutically acceptable
salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 481-571, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of the nucleobase sequence of any one of
SEQ ID
NOs: 572-662, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs:
663-613,
or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of the nucleobase sequence of any one of SEQ ID NOs: 1,6, 13, 17, 21,
24, 26,
29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96, or
a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 6, 13, 17, 21, 24, 26, 29,
33-34, 37,
44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide consists of the
nucleobase
sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of the nucleobase sequence of SEQ ID NO:
6, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 97, 100, 103, 105, 108, 110-
111,
113-117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-
155,
157-165, 168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 100, 103, 105, 108, 110-111, 113-117, 122-123, 127, 129-
130, 133-
136, 138-139, 141, 143-145, 147-148, 154-155, 157-165, 168-170, 172, 174-180,
184,
187, or 191, or a pharmaceutically acceptable salt thereof. In some aspects,
the
oligonucleotide consists of the nucleobase sequence of SEQ 11) NO: 97, or a
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
9
pharmaceutically acceptable salt thereof In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ TD NOs: 193-200, 202-230, 232-246,
248-
253, 255, 258-261, 265, 270, 274-276, or 285-286, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 194-200, 202-230, 232-246, 248-253, 255, 258-261, 265, 270,
274-
276, or 285-286, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NO:
193, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 226-227, 234, 240, or 243-
244, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 227, 234, 240, or 243-244,
or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of SEQ ID NO: 226, or a pharmaceutically acceptable
salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 289-290, 292, 305, 307, 313, 318, 323-324, 326, 329-330,
332, 338-
339, 341, 344, or 346, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs:
290,
292, 305, 307, 313, 318, 323-324, 326, 329-330, 332, 338-339, 341, 344, or
346, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of SEQ ID NO: 289, or a pharmaceutically acceptable
salt
thereof.
[0017] Some aspects of the disclosure are directed to nn
oligonucleotide selected from the
group consisting of Antisense Oligo Nos. 1-384 of Table 3 or 390-613 of Table
4, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 2-96, 98-192, 194-288, 290-
384 of
Table 3 and 390-613 of Table 4, or a pharmaceutically acceptable salt thereof.
In some
aspects, the oligonucleotide is selected from the group consisting of
Antisense Oligo Nos.
1-384 of Table 3, or a pharmaceutically acceptable salt thereof In some
aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
2-96, 98-
192, 194-288, and 290-384 of Table 3, or a pharmaceutically acceptable salt
thereof. In
some aspects, the oligonucleotide is selected from the group consisting of
Antisense
Oligo Nos. 1-96 of Table 3, or a pharmaceutically acceptable salt thereof In
some
aspects, the oligonucleotide is selected from the group consisting of
Antisense Oligo Nos.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
2-96 of Table 3, or a pharmaceutically acceptable salt thereof In some
aspects, the
oligonucleotide is selected from the group consisting of Anti sense Oligo Nos.
97-192 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
98-192 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
193-288 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
194-288 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
289-384 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
290-384 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
390-613 of
Table 4, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
390-480 of
Table 4, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
481-571 of
Table 4, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
1, 6, 13, 17,
21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92,
or 94-96 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
6, 13, 17,
21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92,
or 94-96 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is Antisense Oligo No. 1 of Table 3, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide is Antisense Oligo No. 6 of
Table 3, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 97, 100, 103, 105, 108, 110-
111, 113-
117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155,
157-
165, 168-170, 172, 174-180, 184, 187, or 191 of Table 3, or a pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide is selected from
the group
consisting of Antisense Oligo Nos. 100, 103, 105, 108, 110-111, 113-117, 122-
123, 127,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
11
129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155, 157-165, 168-170,
172,
174-180, 184, 187, or 191 of Table 3, or a pharmaceutically acceptable salt
thereof. In
some aspects, the oligonucleotide is Antisense Oligo No. 97 of Table 3, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 193-200, 202-230, 232-246,
248-253,
255, 258-261, 265, 270, 274-276, or 285-286 of Table 3, or a pharmaceutically
acceptable
salt thereof. In some aspects, the oligonucleotide is selected from the group
consisting of
Antisense Oligo Nos. 194-200, 202-230, 232-246, 248-253, 255, 258-261, 265,
270, 274-
276, or 285-286 of Table 3, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide is Antisense Oligo No. 193 of Table 3. In some
aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
226-227,
234, 240, or 243-244 of Table 3, or a pharmaceutically acceptable salt thereof
In some
aspects, the oligonucleotide is selected from the group consisting of Anti
sense Oligo Nos
227, 234, 240, or 243-244 of Table 3, or a pharmaceutically acceptable salt
thereof. In
some aspects, the oligonucleotide is Antisense Oligo No. 226 of Table 3, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 289-290, 292, 305, 307, 313,
318,
323-324, 326, 329-330, 332, 338-339, 341, 344, or 346 of Table 3, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide is selected from
the group
consisting of Antisense Oligo Nos. 290, 292, 305, 307, 313, 318, 323-324, 326,
329-330,
332, 338-339, 341, 344, or 346 of Table 3, or a pharmaceutically acceptable
salt thereof
In some aspects, the oligonucleotide is Antisense Oligo No. 289 of Table 3, or
a
pharmaceutically acceptable salt thereof.
[0018] In some aspects, the oligonucleotide, or a pharmaceutically
acceptable salt
thereof, described herein causes at least a 50% reduction in MSH3 mRNA
expression at
an oligonucleotide concentration of 10 nM. In some aspects, the
oligonucleotide, or a
pharmaceutically acceptable salt thereof, described herein causes at least a
60% reduction
in MSH3 mRNA expression at an oligonucleotide concentration of 10 nM. In some
aspects, the oligonucleotide, or a pharmaceutically acceptable salt thereof,
described
herein causes at least a 70% reduction in MSH3 mRNA expression at an
oligonucleotide
concentration of 10 nM. In some aspects, the oligonucleotide, or a
pharmaceutically
acceptable salt thereof, described herein causes at least an 80% reduction in
MSH3
mRNA expression at an oligonucleotide concentration of 10 nM.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
12
[0019] In some aspects, the oligonucleotide, or a pharmaceutically
acceptable salt
thereof, described herein causes at least a 50% reduction in MST-13 mRNA
expression at
an oligonucleotide concentration of 1 nM. In some aspects, the
oligonucleotide, or a
pharmaceutically acceptable salt thereof, described herein causes at least a
60% reduction
in MSH3 mRNA expression at an oligonucleotide concentration of 1 nM. In some
aspects, the oligonucleotide, or a pharmaceutically acceptable salt thereof,
described
herein causes at least a 70% reduction in MSH3 mRNA expression at an
oligonucleotide
concentration of 1 nM.
[0020] In some aspects, the MSH3 mRNA expression is evaluated in vitro.
In some
aspects, the MSH3 mRNA expression is evaluated in a cell based assay. In some
aspects,
the MSH3 mRNA expression is evaluated in HeLa cells. In some aspects, the MSH3
mRNA expression is determined by the quantitative reverse transcription
polymerase
chain reaction (RT-qPCR). In some aspects, the MSH3 mRNA is expression is
normalized to the mRNA expression of a reference gene. In some aspects, the
MSH3
mRNA expression is normalized to the mRNA expression of beta-glucuronidase
(GUSB).
In some aspects, the reduction in MSH3 mRNA expression is relative to a
control. In
some aspects, the control is the MSH3 mRNA expression in the absence of the
oligonucleotide, or pharmaceutically acceptable salt thereof. In some aspects,
the control
is the MSH3 mRNA expression in the absence of the oligonucleotide, or
pharmaceutically acceptable salt thereof, but in the presence of a control
oligonucleotide,
or salt thereof. In some aspects, the control oligonucleotide, or salt
thereof, is a scrambled
luciferase targeting oligonucleotide. In some aspects, the reduction in MSH3
mRNA
expression is calculated by a delta-delta Ct (AACT) method. In some aspects,
the delta-
delta Ct (AACT) method comprises the normalization of the MSH3 mRNA expression
to
the mRNA expression of a reference gene and to the MSH3 mRNA expression in the
absence of the oligonucleotide, or pharmaceutically acceptable salt thereof
but in the
presence of a control oligonucleotide, or salt thereof. In some aspects, the
reference gene
is beta-glucuronidase (GUSB) and/or the control oligonucleotide, or salt
thereof, is a
scrambled luciferase targeting oligonucleotide. In some aspects, the reduction
in MSH3
mRNA expression is determined by the method of Example 1. In some aspects, in
the
same assay, Antisense Oligo No. 1 causes approximately a 58% reduction in MSH3
mRNA expression at an oligonucleotide concentration of 10 nM. In some aspects,
in the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
13
same assay, Antisense Oligo No. 1 causes approximately a 14% reduction in MSH3
mRNA expression at an oligonucleotide concentration of 1 nM.
[0021] In some aspects disclosed herein, the oligonucleotide is
in the free base form.
[0022] In some aspects disclosed herein, the oligonucleotide is a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide is a sodium
salt.
Pharmaceutical Compositions and Methods of Treatment Using the Same
[0023] In some aspects, the application is directed to a pharmaceutical
composition
comprising one or more of the oligonucleotides, or pharmaceutically acceptable
salts
thereof, described herein and a pharmaceutically acceptable carrier or
excipient.
[0024] In some aspects, the application is directed to a composition
comprising one or
more of the oligonucleotides or pharmaceutically acceptable salts thereof,
described
herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex
nanoparticle, or a
liposome.
[0025] In some aspects, the application is directed to a method of
inhibiting transcription
of MSH3 in a cell, the method comprising contacting the cell with one or more
of the
oligonucleotides or pharmaceutically acceptable salts thereof, described
herein, a
pharmaceutical composition of one or more of the oligonucleotides or
pharmaceutically
acceptable salts thereof, described herein, or the composition of one or more
oligonucleotides or pharmaceutically acceptable salts thereof, described
herein and a lipid
nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome;
for a time
sufficient to obtain degradation of an mRNA transcript of a MSH3 gene,
inhibiting
expression of the MSH3 gene in the cell.
[0026] In some aspects, the application is directed to a method of
treating, preventing, or
delaying the progression a nucleotide repeat expansion disorder (e.g., a
trinucleotide
repeat expansion disorder) in a subject in need thereof, the method comprising
contacting
the cell with one or more of the oligonucleotides or pharmaceutically
acceptable salts
thereof, described herein, a pharmaceutical composition of one or more of the
oligonucleotides or pharmaceutically acceptable salts thereof, described
herein, or the
composition of one or more oligonucleotides or pharmaceutically acceptable
salts thereof,
described herein and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex
nanoparticle,
or a liposome; for a time sufficient to obtain degradation of an mRNA
transcript of a
MSII3 gene, inhibiting expression of the MSII3 gene in the cell.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
14
[0027] In some aspects, the application is directed to a method of
reducing the level
and/or activity of MSH3 in a cell of a subject identified as having a
nucleotide repeat
expansion disorder (e.g., a trinucleotide repeat expansion disorder), the
method
comprising contacting the cell with one or more of the oligonucleotides or
pharmaceutically acceptable salts thereof, described herein, a pharmaceutical
composition
of one or more of the oligonucleotides or pharmaceutically acceptable salts
thereof,
described herein, or the composition of one or more oligonucleotides, or
pharmaceutically
acceptable salts thereof, described herein and a lipid nanoparticle, a
polyplex
nanoparticle, a lipoplex nanoparticle, or a liposome, for a time sufficient to
obtain
degradation of an mRNA transcript of a MSH3 gene, inhibiting expression of the
MSH3
gene in the cell
[0028] In some aspects, the application is directed to a method for
inhibiting expression
of an MSH3 gene in a cell comprising contacting the cell with one or more of
the
oligonucleotides, or pharmaceutically acceptable salts thereof, described
herein, a
pharmaceutical composition of one or more of the oligonucleotides, or
pharmaceutically
acceptable salts thereof, described herein, or the composition of one or more
oligonucleotides, or pharmaceutically acceptable salts thereof, described
herein and a
lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a
liposome; for a
time sufficient to obtain degradation of an mRNA transcript of a MSH3 gene,
inhibiting
expression of the MSH3 gene in the cell, and maintaining the cell for a time
sufficient to
obtain degradation of a mRNA transcript of an MSH3 gene, thereby inhibiting
expression
of the MSH3 gene in the cell.
[0029] In some aspects, the application is directed to a method of
decreasing nucleotide
repeat expansion (e g , trinucleotide repeat expansion) in a cell, the method
comprising
contacting the cell with one or more of the oligonucleotides, or
pharmaceutically
acceptable salts thereof, described herein, a pharmaceutical composition of
one or more
of the oligonucleotides, or pharmaceutically acceptable salts thereof,
described herein, or
the composition of one or more oligonucleotides, or pharmaceutically
acceptable salts
thereof, described herein and a lipid nanoparticle, a polyplex nanoparticle, a
lipoplex
nanoparticle, or a liposome; for a time sufficient to obtain degradation of an
mRNA
transcript of a MSH3 gene, inhibiting expression of the MSH3 gene in the cell.
[0030] In some aspects, the cell is in a subject. In some aspects, the
subject is a human. In
some aspects, the cell is a cell of the central nervous system or a muscle
cell.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
[0031] In some aspects, the subject is identified as having a
nucleotide repeat expansion
disorder (e.g., a trinucleotide repeat expansion disorder). In some aspects,
the nucleotide
repeat expansion diorder is spinocerebellar ataxia type 36 or frontotemporal
dementia. In
some aspects, the nucleotide repeat expansion disorder is a trinucleotide
repeat expansion
disorder. In some aspects, the trinucleotide repeat expansion disorder is a
polyglutamine
disease. In some aspects, the polyglutamine disease is selected from the group
consisting
of dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar
muscular
atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2,
spinocerebellar
ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7,
spinocerebellar
ataxia type 17, and Huntington's disease-like 2. In some aspects, the
nucleotide repeat
expansion disorder (e.g., a trinucleotide repeat expansion disorder) is
Huntington's
disease.
[0032] In some aspects, the nucleotide repeat expansion disorder (e.g.,
a trinucleotide
repeat expansion disorder) is a non-polyglutamine disease. In some aspects,
the non-
polyglutamine disease is selected from the group consisting of fragile X
syndrome, fragile
X-associated tremor/ataxia syndrome, fragile XE mental retardation,
Friedreich's ataxia,
myotonic dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar
ataxia type 12,
oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian
failure,
FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.
In
some aspects, the nucleotide repeat expansion disorder (e.g., trinucleotide
repeat
expansion disorder) is Friedreich's ataxia. In some aspects, the nucleotide
repeat
expansion disorder (e.g., trinucleotide repeat expansion disorder) is myotonic
dystrophy
type 1.
[0033] In some aspects, the application is directed one or more of the
oligonucleotides, or
pharmaceutically acceptable salts thereof, described herein, a pharmaceutical
composition
of one or more of the oligonucleotides, or pharmaceutically acceptable salts
thereof,
described herein, or the composition of one or more oligonucleotides, or
pharmaceutically
acceptable salts thereof, described herein and a lipid nanoparticle, a
polyplex
nanoparticle, a lipoplex nanoparticle, or a liposome, for use in the
prevention or treatment
of a nucleotide repeat expansion disorder (e.g., a trinucleotide repeat
expansion disorder).
In some aspects, the one or more of the oligonucleotides, or pharmaceutically
acceptable
salts thereof, described herein, the pharmaceutical composition of one or more
of the
oligonucleotides, or pharmaceutically acceptable salts thereof, described
herein, or the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
16
composition of one or more oligonucleotides, or pharmaceutically acceptable
salts
thereof, described herein and a lipid nanoparticle, a polyplex nanoparticle, a
lipoplex
nanoparticle, or a liposome is administered intrathecally.
[0034] In some aspects, the one or more of the oligonucleotides, or
pharmaceutically
acceptable salts thereof, described herein, the pharmaceutical composition of
one or more
of the oligonucleotides, or pharmaceutically acceptable salts thereof,
described herein, or
the composition of one or more oligonucleotides, or pharmaceutically
acceptable salts
thereof, described herein and a lipid nanoparticle, a polyplex nanoparticle, a
lipoplex
nanoparticle, or a liposome is administered intraventricularly.
[0035] In some aspects, the one or more of the oligonucleotides, or
pharmaceutically
acceptable salts thereof, described herein, the pharmaceutical composition of
one or more
of the oligonucleotides, or pharmaceutically acceptable salts thereof,
described herein, or
the composition of one or more oligonucleotides, or pharmaceutically
acceptable salts
thereof, described herein and a lipid nanoparticle, a polyplex nanoparticle, a
lipoplex
nanoparticle, or a liposome is administered intramuscularly.
[0036] In some aspects, the application is directed to a method of
treating, preventing, or
delaying progression a disorder in a subject in need thereof wherein the
subject is
suffering from a nucleotide repeat expansion disorder (e.g., a trinucleotide
repeat
expansion disorder), comprising administering to said subject one or more of
the
oligonucleotides, or pharmaceutically acceptable salts thereof, described
herein, the
pharmaceutical composition of one or more of the oligonucleotides, or
pharmaceutically
acceptable salts thereof, described herein, or the composition of one or more
oligonucleotides, or pharmaceutically acceptable salts thereof, described
herein and a
lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a
liposome
[0037] In some aspects, the method of treating, preventing, or delaying
progression of a
disorder in a subject further comprises administering an additional
therapeutic agent. In
some aspects, the additional therapeutic agent is another oligonucleotide, or
pharmaceutically acceptable salt thereof, that hybridizes to an mRNA encoding
the
Huntingtin gene.
[0038] In some aspects, the method of treating, preventing, or delaying
progression of a
disorder in a subject progression delays progression of the nucleotide repeat
expansion
disorder (e.g., a trinucleotide repeat expansion disorder) by at least 120
days, for example,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
17
at least 6 months, at least 12 months, at least 2 years, at least 3 years, at
least 4 years, at
least 5 years, at least 10 years or more, when compared with a predicted
progression.
[0039] In some aspects, the application is directed to one or more of
the oligonucleotides,
or pharmaceutically acceptable salts thereof, described herein, the
pharmaceutical
composition of one or more of the oligonucleotides, or pharmaceutically
acceptable salts
thereof, described herein, or the composition of one or more oligonucleotides,
or
pharmaceutically acceptable salts thereof, described herein and a lipid
nanoparticle, a
polyplex nanoparticle, a lipoplex nanoparticle, or a liposome for use in
preventing or
delaying progression of a nucleotide repeat expansion disorder (e.g., a
trinucleotide repeat
expansion disorder) in a subject
Definitions
[0040] For convenience, the meaning of some terms and phrases used in
the specification,
examples, and appended claims are provided below. Unless stated otherwise, or
implicit
from context, the following terms and phrases include the meanings provided
below. The
definitions are provided to aid in describing particular aspects, and are not
intended to
limit the claimed technology, because the scope of the technology is limited
only by the
claims. Unless otherwise defined, all technical and scientific terms used
herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which this
technology belongs. If there is an apparent discrepancy between the usage of a
term in
the art and its definition provided herein, the definition provided within the
specification
shall prevail
[0041] In this application, unless otherwise clear from context, (i)
the term "a" can be
understood to mean "at least one-; (ii) the term "or" can be understood to
mean "and/or";
and (iii) the terms "including" and "comprising" can be understood to
encompass
itemized components or steps whether presented by themselves or together with
one or
more additional components or steps.
100421 As used herein, the terms -about" and -approximately" refer to a
value that is
within 10% above or below the value being described. For example, the term
"about 5
nM" indicates a range of from 4.5 to 5.5 nM.
[0043] The term "at least" prior to a number or series of numbers is
understood to include
the number adjacent to the term "at least", and all subsequent numbers or
integers that
could logically be included, as clear from context. For example, the number of
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
18
nucleotides in a nucleic acid molecule must be an integer. For example, "at
least 18
nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20,
or 21
nucleotides have the indicated property. When at least is present before a
series of
numbers or a range, it is understood that "at least" can modify each of the
numbers in the
series or range. "At least" is also not limited to integers (e.g., "at least
5% includes 5.0%,
5.1%, and 5.18% without consideration of the number of significant figures.
[0044] As used herein, "no more than" or "less than" is understood as
the value adjacent
to the phrase and logical lower values or integers, as logical from context,
to zero. For
example, an oligonucleotide with "no more than 3 mismatches to a target
sequence" has
3, 2, 1, or 0 mismatches to a target sequence. When "no more than" is present
before a
series of numbers or a range, it is understood that "no more than" can modify
each of the
numbers in the series or range.
[0045] As used herein, the term "administration" refers to the
administration of a
composition (e.g., a compound or a preparation that includes a compound as
described
herein) to a subject or system. Administration to an animal subject (e.g., to
a human) can
be by any appropriate route, such as one described herein.
[0046] As used herein, a -combination therapy- or -administered in
combination- means
that two (or more) different agents or treatments are administered to a
subject as part of a
defined treatment regimen for a particular disease or condition. The treatment
regimen
defines the doses and periodicity of administration of each agent such that
the effects of
the separate agents on the subject overlap. In some aspects, the delivery of
the two or
more agents is simultaneous or concurrent and the agents can be co-formulated.
In some
aspects, the two or more agents are not co-formulated and are administered in
a sequential
manner as part of a prescribed regimen In some aspects, administration of two
or more
agents or treatments in combination is such that the reduction in a symptom,
or other
parameter related to the disorder is greater than what would be observed with
one agent
or treatment delivered alone or in the absence of the other. The effect of the
two
treatments can be partially additive, wholly additive, or greater than
additive (e.g.,
synergistic). Sequential or substantially simultaneous administration of each
therapeutic
agent can be effected by any appropriate route including, but not limited to,
oral routes,
intraocular routes, subcutaneous routes, intra ci sterna magna routes,
intravenous routes,
intramuscular routes, and direct absorption through mucous membrane tissues.
The
therapeutic agents can be administered by the same route or by different
routes. For
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
19
example, one therapeutic agent of the combination can be administered by
intravenous
injection while an additional therapeutic agent of the combination can be
administered
orally.
[0047] As used herein, the term "MSH3" refers to MutS Homolog 3, a DNA
mismatch
repair protein, having an amino acid sequence from any vertebrate or mammalian
source,
including, but not limited to, human, bovine, chicken, rodent, mouse, rat,
porcine, ovine,
primate, monkey, and guinea pig, unless specified otherwise. The term also
refers to
fragments and variants of native MSH3 that maintain at least one in vivo or in
vitro
activity of a native MSH3. The term encompasses full-length unprocessed
precursor
forms of MSH3 as well as mature forms resulting from post-translational
cleavage of the
signal peptide. MSH3 is encoded by the MSH3 gene. The nucleic acid sequence of
an
exemplary Homo sapiens (human) MSH3 gene is set forth in NCBI Reference
NM 002439.4 or in SEQ ID NO: 385. The term "MSH3" also refers to natural
variants of
the wild-type MSH3 protein, such as proteins having at least 85% identity
(e.g., 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%
identity, or more) to the amino acid sequence of wild-type human MSH3, which
is set
forth in NCBI Reference No. NP 002430.3 or in SEQ ID NO: 386. The nucleic acid
sequence of an exemplary Mus musculus (mouse) MSH3 gene is set forth in NCBI
Reference No. NM 010829.2 or in SEQ ID NO: 387. The nucleic acid sequence of
an
exemplary Rattus noryegicus (rat) MSH3 gene is set forth in NCBI Reference No.
NM 001191957.1 or in SEQ ID NO: 388. The nucleic acid sequence of an exemplary
Macaca fascicularis (cyno) MSH3 gene is set forth in NCBI Reference No.
XM 005557283.2 or in SEQ ID NO: 389.
[0048] The term "MSH3" as used herein also refers to a particular
polypeptide expressed
in a cell by naturally occurring DNA sequence variations of the MSH3 gene,
such as a
single nucleotide polymorphism in the MSH3 gene. Numerous SNPs within the MSH3
gene have been identified and can be found at, for example, NCBI dbSNP (see,
e.g.,
www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the MSH3 gene
can
be found at, NCBI dbSNP Accession Nos.: rs1650697, rs70991108, rs10168,
rs26279 ,
rs26282, rs26779, rs26784, rs32989, rs33003, rs33008, rs33013, rs40139,
rs181747,
rs184967, rs245346, rs245397, rs249633, rs380691, rs408626, rs442767,
rs836802,
rs836808, rs863221, rs1105525, rs1428030, rs1478834, rs1650694, rs1650737,
rs1677626, rs1677658, rs1805355, rs2897298, rs3045983, rs3797897, rs4703819,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
rs6151627, rs6151640, rs6151662, rs6151670, rs6151735, rs6151838, rs7709909,
rs7712332, rs10079641, rs12513549, and rs12522132.
[0049] As used herein, "target sequence" refers to a contiguous portion
of the nucleotide
sequence of an mRNA molecule formed during the transcription of an MSH3 gene,
including mRNA that is a product of RNA processing of a primary transcription
product.
In one aspect, the target portion of the sequence will be at least long enough
to serve as a
substrate for oligonucleotide-directed (e.g., anti sense oligonucleotide (AS0)-
directed)
cleavage at or near that portion of the nucleotide sequence of an mRNA
molecule formed
during the transcription of a MSH3 gene. The target sequence can be, for
example, from
about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For
example,
the target sequence can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30
or from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-
23, 15-22,
15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25,
18-24, 18-
23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-
23, 19-22,
19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-
21, 21-
30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in
length.
Ranges and lengths intermediate to the above recited ranges and lengths are
also
contemplated.
[0050] "G," "C," "A," "T," and "U" each generally stand for a naturally-
occurring
nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as
a base,
respectively. However, it will be understood that the term "nucleotide" can
refer to an
alternative nucleotide, as further detailed below, or a surrogate replacement
moiety. The
skilled person is well aware that guanine, cytosine, adenine, and uracil can
be replaced by
other moieties without substantially altering the base pairing properties of
an
oligonucleotide comprising a nucleotide bearing such replacement moiety. For
example,
without limitation, a nucleotide comprising inosine as its base can base pair
with
nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides
containing uracil,
guanine, or adenine can be replaced in the nucleotide sequences of
oligonucleotides by a
nucleotide containing, for example, inosine. In another example, adenine and
cytosine
anywhere in the oligonucleotide can be replaced with guanine and uracil,
respectively to
form G-U Wobble base pairing with the target mRNA. Sequences containing such
replacement moieties are suitable for the compositions and methods featured
herein.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
21
[0051] The terms "nucleobase" and "base" include the purine (e.g.
adenine and guanine)
and pyrimi dine (e.g. uracil, thymine, and cytosine) moiety present in
nucleosides and
nucleotides which form hydrogen bonds in nucleic acid hybridization. The term
nucleobase also encompasses alternative nucleobases which can differ from
naturally-
occurring nucleobases, but are functional during nucleic acid hybridization.
In this
context, "nucleobase- refers to both naturally occurring nucleobases such as
adenine,
guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as
alternative
nucleobases. Such variants are for example described in Hirao et al (2012)
Accounts of
Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in
Nucleic
Acid Chemistry Suppl. 37 1.4.1.
[0052] The term "nucleoside" refers to a monomeric unit of an
oligonucleotide or a
polynucleotide having a nucleobase and a sugar moiety. A nucleoside can
include those
that are naturally-occurring as well as alternative nucleosides, such as those
described
herein. The nucleobase of a nucleoside can be a naturally-occurring nucleobase
or an
alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a
naturally-
occurring sugar or an alternative sugar.
[0053] The term -alternative nucleoside- refers to a nucleoside having
an alternative
sugar or an alternative nucleobase, such as those described herein.
[0054] In some aspects the nucleobase moiety is modified by changing
the purine or
pyrimidine into a modified purine or pyrimidine, such as substituted purine or
substituted
pyrimidine, such as an "alternative nucleobase" selected from isocytosine,
pseudoisocytosine, 5-methyl cytosine, 5-thiozolo-cytosine, 5-propynyl-
cytosine, 5-
propynyl-uridine, 5-bromouridine 5-thiazolo-uridine, 2-thio-uridine,
pseudouridine, 1-
methylpseudouridine, 5-methoxyuridine, 2'-thio-thymine, inosine,
diaminopurine, 6-
aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.
[0055] The nucleobase moieties can be indicated by the letter code for
each
corresponding nucleobase, e.g. A, T, G, C, or U, wherein each letter can
include
alternative nucleobases of equivalent function. In some aspects, e.g., for
gapmers, 5-
methyl cytosine LNA nucleosides can be used.
[0056] A "sugar" or "sugar moiety," includes naturally occurring sugars
having a
furanose ring. A sugar also includes an "alternative sugar," defined as a
structure that is
capable of replacing the furanose ring of a nucleoside. In some aspects,
alternative sugars
are non-furanose (or 4'-substituted furanose) rings or ring systems or open
systems. Such
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
22
structures include simple changes relative to the natural furanose ring, such
as a six-
membered ring, or can be more complicated as is the case with the non-ring
system used
in peptide nucleic acid. Alternative sugars can include sugar surrogates
wherein the
furanose ring has been replaced with another ring system such as, for example,
a
morpholino or hexitol ring system. Sugar moieties useful in the preparation of
oligonucleotides having motifs include, without limitation, I3-D-ribose, I3-D-
2'-
deoxyribose, substituted sugars (such as 2', 5' and bis substituted sugars),
4'-S-sugars
(such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose),
bicyclic
alternative sugars (such as the 2'-O _______ CH2-4' or 2'-O __ (CH2)2-4'
bridged ribose derived
bicyclic sugars) and sugar surrogates (such as when the ribose ring has been
replaced with
a morpholino or a hexitol ring system). The type of heterocyclic base and
internucleoside
linkage used at each position is variable and is not a factor in determining
the motif. In
most nucleosides having an alternative sugar moiety, the heterocyclic
nucleobase is
generally maintained to permit hybridization.
[0057] A "nucleotide," as used herein, refers to a monomeric unit of an
oligonucleotide
or polynucleotide that comprises a nucleoside and an internucleosidic linkage.
The
internucleosidic linkage can include a phosphate linkage. Similarly, -linked
nucleosides"
can be linked by phosphate linkages. Many "alternative internucleosidic
linkages" are
known in the art, including, but not limited to, phosphate, phosphorothioate,
and
boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides
(BNAs)
(e.g., locked nucleosides (LNAs (e.g., A-LNA, 5mC-LNA, G-LNA, and T-LNA)) and
constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs),
phosphotriesters,
phosphorothionates, phosphoramidates, and other variants of the phosphate
backbone of
native nucleoside, including those described herein
[0058] An "alternative nucleotide," as used herein, refers to a
nucleotide having an
alternative nucleoside or an alternative sugar, and an internucleoside
linkage, which can
include alternative nucleoside linkages.
[0059] The terms "oligonucleotide" and "polynucleotide," as used
herein, are defined as
it is generally understood by the skilled person as a molecule comprising two
or more
covalently linked nucleosides. Such covalently bound nucleosides can be
referred to as
nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the
laboratory by solid-phase chemical synthesis followed by purification. When
referring to
a sequence of the oligonucleotide, reference is made to the sequence or order
of
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
23
nucleobase moieties, or modifications thereof, of the covalently linked
nucleotides or
nucleosides. The oligonucleotide can be man-made. For example, the
oligonucleotide can
be chemically synthesized, and be purified or isolated. Oligonucleotide is
also intended
to include (i) compounds that have one or more furanose moieties that are
replaced by
furanose derivatives or by any structure, cyclic or acyclic, that can be used
as a point of
covalent attachment for the base moiety, (ii) compounds that have one or more
phosphodiester linkages that are either modified, as in the case of
phosphoramidate or
phosphorothioate linkages, or completely replaced by a suitable linking moiety
as in the
case of formacetal or riboacetal linkages, and/or (iii) compounds that have
one or more
linked furanose-phosphodiester linkage moieties replaced by any structure,
cyclic or
acyclic, that can be used as a point of covalent attachment for the base
moiety. The
oligonucleotide can comprise one or more alternative nucleosides or
nucleotides (e.g.,
including those described herein). It is also understood that oligonucleotide
includes
compositions lacking a sugar moiety or nucleobase but are still capable of
forming a
pairing with or hybridizing to a target sequence. "Oligonucleotide" refers to
a short
polynucleotide (e.g., of 100 or fewer linked nucleosides).
[0060] As used herein, the term "oligonucleotide comprising a
nucleobase sequence"
refers to an oligonucleotide comprising a chain of nucleotides or nucleosides
that is
described by the sequence referred to using the standard nucleotide
nomenclature.
[0061] The term "contiguous nucleobase region" refers to the region of
the
oligonucleotide which is complementary to the target nucleic acid. The term
can be used
interchangeably herein with the term "contiguous nucleotide sequence" or
"contiguous
nucleobase sequence." In some aspects, all the nucleotides of the
oligonucleotide are
present in the contiguous nucleotide or nucleoside region In some aspects, the
oligonucleotide comprises the contiguous nucleotide region and can comprise
further
nucleotide(s) or nucleoside(s), for example a nucleotide linker region which
can be used
to attach a functional group to the contiguous nucleotide sequence. The
nucleotide linker
region can be complementary to the target nucleic acid. In some aspects, the
internucleoside linkages present between the nucleotides of the contiguous
nucleotide
region are all phosphorothioate internucleoside linkages. In some aspects, the
contiguous
nucleotide region comprises one or more sugar-modified nucleosides.
[0062] The term "gapmer," as used herein, refers to an oligonucleotide
which comprises a
region of RN ase H recruiting oligonucleotides (gap or DNA core) which is
flanked 5' and
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
24
3' by regions which comprise one or more affinity enhancing alternative
nucleosides
(wings or flanking sequence). Various gapmer designs are described herein.
Headmers
and tailmers are oligonucleotides capable of recruiting RNase H where one of
the flanks
is missing, i.e. only one of the ends of the oligonucleotide comprises
affinity enhancing
alternative nucleosides. For headmers the 3' flanking sequence is missing
(i.e. the 5'
flanking sequence comprises affinity enhancing alternative nucleosides) and
for tailmers
the 5' flanking sequence is missing (i.e. the 3' flanking sequence comprises
affinity
enhancing alternative nucleosides). A "mixed flanking sequence gapmer" refers
to a
gapmer wherein the flanking sequences comprise at least one alternative
nucleoside, such
as at least one DNA nucleoside or at least one 2' substituted alternative
nucleoside, such
as, for example, 2'-0-alkyl-RNA, 2'-0-methyl-RNA, 2'-alkoxy-RNA, 2'-0-
methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, 2'-F-ANA nucleoside(s),
or
bicyclic nucleosides (e.g., locked nucleosides or constrained ethyl (cEt)
nucleosides). In
some aspects the mixed flanking sequence gapmer has one flanking sequence
which
comprises alternative nucleosides (e.g. 5' or 3') and the other flanking
sequence (3' or 5'
respectfully) comprises 2' substituted alternative nucleoside(s).
[0063] A "linker" or "linking group" is a connection between two atoms
that links one
chemical group or segment of interest to another chemical group or segment of
interest
via one or more covalent bonds. The oligonucleotides disclosed herein can
comprise one
or more linkers capable of linking one or more oligonucleotides disclosed
herein to one or
more other oligonucleotides disclosed herein, and/or to any other
oligonucleotide, and/or
to any conjugate moiety. For example, a linker could be used to link an
oligonucleotide
disclosed herein to an oligonucleotide that targets the Huntingtin gene.
[0064] Linkers may be susceptible to cleavage ("cleavable linker")
thereby facilitating
release of the different oligonucleotides and/or different conjugate moieties
disclosed
herein. Such cleavable linkers may be susceptible, for example, to nuclease-
induced
cleavage, acid-induced cleavage, photo-induced cleavage, peptidase-induced
cleavage,
esterase-induced cleavage, and disulfide bond cleavage, at suitable
conditions. Suitable
cleavable linking groups for use in cleavable linkers include those which are
sufficiently
stable outside the cell, but which upon entry into a target cell is cleaved to
release the two
parts the linker is holding together.
[0065] Alternatively, linkers may be substantially resistant to
cleavage ("non-cleavable
linker"). Such non-cleavable linkers can be any chemical moiety capable of
linking one or
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
more different oligonucleotides disclosed herein to one or more other
oligonucleotides
disclosed herein, and/or to any conjugate moiety in a stable, covalent manner
and does
not fall off under the categories listed above for cleavable linkers Thus, non-
cleavable
linkers are substantially resistant to acid-induced cleavage, nuclease-induced
cleavage,
photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage
and
disulfide bond cleavage. Furthermore, non-cleavable refers to the ability of
the chemical
bond in the linker or adjoining to the linker to withstand cleavage induced by
an acid, a
nuclease, photolabile-cleaving agent, a peptidase, an esterase, or a chemical
or
physiological compound that cleaves a disulfide bond, at conditions under
which the
oligonucleotides disclosed herein do not lose their activity or intended
purpose.
[0066] Conjugate moieties can be attached to the oligonucleotide
directly or through a
linking moiety (e.g. linker or tether). Linkers serve to covalently connect a
third region,
e.g. a conjugate moiety to an oligonucleotide (e.g. the termini of region A or
C). In some
aspects, the conjugate or oligonucleotide conjugate can, comprise a linker
region which is
positioned between the oligonucleotide and the conjugate moiety. In some
aspects, the
linker between the conjugate and oligonucleotide is biocleavable.
Phosphodiester
containing biocleavable linkers are described in more detail in WO 2014/076195
(herein
incorporated by reference).
[0067] In some aspects, two or more linkers can be linked in tandem.
When multiple
linkers connect one or more oligonucleotides disclosed herein to one or more
other
oligonucleotides disclosed herein, and/or to any conjugate moiety, each of the
linkers can
be the same or different.
[0068] As used herein, and unless otherwise indicated, the term
"complementary," when
used to describe a first nucleotide or nucleoside sequence in relation to a
second
nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide
or
polynucleotide comprising the first nucleotide or nucleoside sequence to
hybridize and
form a duplex structure under certain conditions with an oligonucleotide or
polynucleotide comprising the second nucleotide sequence, as will be
understood by the
skilled person. Such conditions can, for example, be stringent conditions,
where stringent
conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 C, or
70
C, for 12-16 hours followed by washing (see, e.g., "Molecular Cloning: A
Laboratory
Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other
conditions, such as physiologically relevant conditions as can be encountered
inside an
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
26
organism, can be used. The skilled person will be able to determine the set of
conditions
most appropriate for a test of complementarity of two sequences in accordance
with the
ultimate application of the hybridized nucleotides or nucleosides.
[0069] "Complementary" sequences, as used herein, can include, or be
formed entirely
from, non-Watson-Crick base pairs and/or base pairs formed from non-natural
and
alternative nucleotides or nucleosides, in so far as the above requirements
with respect to
their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs
include, but are
not limited to, G:U Wobble or Hoogstein base pairing. Complementary sequences
between an oligonucleotide and a target sequence as described herein, include
base-
pairing of the oligonucleotide or polynucleotide comprising a first nucleotide
or
nucleoside sequence to an oligonucleotide or polynucleotide comprising a
second
nucleotide or nucleoside sequence over the entire length of one or both
nucleotide or
nucleoside sequences. Such sequences can be referred to as "fully
complementary" with
respect to each other herein. However, where a first sequence is referred to
as
"substantially complementary" with respect to a second sequence herein, the
two
sequences can be fully complementary, or they can form one or more, but
generally not
more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex
up to 30
base pairs, while retaining the ability to hybridize under the conditions most
relevant to
their ultimate application, e.g., inhibition of gene expression via an RNase H-
mediated
pathway. "Substantially complementary" can refer to a polynucleotide that is
substantially complementary to a contiguous portion of the mRNA of interest
(e.g., an
mRNA encoding MSH3). For example, a polynucleotide is complementary to at
least a
part of a MSH3 mRNA if the sequence is substantially complementary to a non-
interrupted portion of an mRNA encoding MSH3
[0070] As used herein, the term "region of complementarity" refers to
the region on the
oligonucleotide that is substantially complementary to all or a portion of a
gene, primary
transcript, a sequence (e.g., a target sequence, e.g., an MSH3 nucleotide
sequence), or
processed mRNA, so as to interfere with expression of the endogenous gene
(e.g.,
MSH3). Where the region of complementarity is not fully complementary to the
target
sequence, the mismatches can be in the internal or terminal regions of the
molecule.
Generally, the most tolerated mismatches are in the terminal regions, e.g.,
within 5, 4, 3,
or 2 nucleotides of the 5'- and/or 3'-terminus of the oligonucleotide.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
27
[0071] As used herein, an "agent that reduces the level and/or activity
of MSH3- refers to
any polynucleotide agent (e.g., an oligonucleotide, e.g., an ASO) that reduces
the level of
or inhibits expression of MSH3 in a cell or subject. The phrase "inhibiting
expression of
MSH3," as used herein, includes inhibition of expression of any MSH3 gene
(such as,
e.g., a mouse MSH3 gene, a rat MSH3 gene, a monkey MSH3 gene, or a human MSH3
gene) as well as variants or mutants of a MSH3 gene that encode a MSH3
protein. Thus,
the MSH3 gene can be a wild-type MSH3 gene, a mutant MSH3 gene, or a
transgenic
MSH3 gene in the context of a genetically manipulated cell, group of cells, or
organism.
[0072] By "reducing the activity of MSH3" is meant decreasing the level
of an activity
related to MSH3 (e.g., by reducing the amount of nucleotide repeats in a gene
associated
with a nucleotide repeat expansion disorder, e.g., a trinucleotide repeat
expansion
disorder, that is related to MSH3 activity). The activity level of MSH3 can be
measured
using any method known in the art (e.g., by directly sequencing a gene
associated with a
nucleotide repeat expansion disorder to measure the levels of nucleotide
repeats)
[0073] By "reducing the level of MSH3" is meant decreasing the level of
MSH3 in a cell
or subject, e.g., by administering an oligonucleotide, or pharmaceutically
acceptable salt
thereof, to the cell or subject. The level of MSH3 can be measured using any
method
known in the art (e.g., by measuring the levels of MSH3 mRNA or levels of MSH3
protein in a cell or a subject).
[0074] By "modulating the activity of a MutSI3 heterodimer comprising
MSH3" is meant
altering the level of an activity related to a MutSI3 heterodimer, or a
related downstream
effect. The activity level of a MutSI3 heterodimer can be measured using any
method
known in the art.
[0075] As used herein, the term "inhibitor" refers to any agent which
reduces the level
and/or activity of a protein (e.g., MSH3). Non-limiting examples of inhibitors
include
polynucleotides (e.g., oligonucleotide, e.g., AS0s). The term "inhibiting," as
used herein,
is used interchangeably with "reducing," "silencing," "downregulating,"
"suppressing,"
and other similar terms, and includes any level of inhibition.
[0076] The phrase "contacting a cell with an oligonucleotide," such as
an oligonucleotide,
as used herein, includes contacting a cell by any possible means. Contacting a
cell with
an oligonucleotide includes contacting a cell in vitro with the
oligonucleotide or
contacting a cell in vivo with the oligonucleotide. The contacting can be done
directly or
indirectly. Thus, for example, the oligonucleotide can be put into physical
contact with
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
28
the cell by the individual performing the method, or alternatively, the
oligonucleotide
agent can be put into a situation that will permit or cause it to subsequently
come into
contact with the cell.
[0077] Contacting a cell in vitro can be done, for example, by
incubating the cell with the
oligonucleotide. Contacting a cell in vivo can be done, for example, by
injecting the
oligonucleotide into or near the tissue where the cell is located, or by
injecting the
oligonucleotide agent into another area, e.g., the bloodstream or the
subcutaneous space,
such that the agent will subsequently reach the tissue where the cell to be
contacted is
located. For example, the oligonucleotide can contain and/or be coupled to a
ligand, e.g.,
GalNAc3, that directs the oligonucleotide to a site of interest, e.g., the
liver.
Combinations of in vitro and in vivo methods of contacting are also possible.
For
example, a cell can be contacted in vitro with an oligonucleotide and
subsequently
transplanted into a subject.
[0078] In one aspect, contacting a cell with an oligonucleotide
includes "introducing" or
"delivering the oligonucleotide into the cell" by facilitating or effecting
uptake or
absorption into the cell. Absorption or uptake of an ASO can occur through
unaided
diffusive or active cellular processes, or by auxiliary agents or devices.
Introducing an
oligonucleotide into a cell can be in vitro and/or in vivo. For example, for
in vivo
introduction, oligonucleotides can be injected into a tissue site or
administered
systemically. In vitro introduction into a cell includes methods known in the
art such as
electroporation and lipofection. Further approaches are described herein below
and/or are
known in the art.
[0079] As used herein, "lipid nanoparticle" or "LNP" is a vesicle
comprising a lipid layer
encapsulating a pharmaceutically active molecule, such as a nucleic acid
molecule, e.g.,
an oligonucleotide LNP refers to a stable nucleic acid-lipid particle. LNPs
typically
contain a cationic lipid, a non-cationic lipid, and a lipid that prevents
aggregation of the
particle (e.g., a PEG-lipid conjugate). LNPs are described in, for example,
U.S. Pat. Nos.
6,858,225; 6,815,432; 8,158,601; and 8,058,069, the entire contents of which
are hereby
incorporated herein by reference.
[0080] As used herein, the term "liposome" refers to a vesicle composed
of amphiphilic
lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of
bilayers.
Liposomes include unilamellar and multilamellar vesicles that have a membrane
formed
from a lipophilic material and an aqueous interior. The aqueous portion
contains the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
29
oligonucleotide composition. The lipophilic material isolates the aqueous
interior from
an aqueous exterior, which typically does not include the oligonucleotide
composition,
although in some examples, it can. Liposomes also include "sterically
stabilized"
liposomes, a term which, as used herein, refers to liposomes comprising one or
more
specialized lipids that, when incorporated into liposomes, result in enhanced
circulation
lifetimes relative to liposomes lacking such specialized lipids.
[0081] "Micelles" are defined herein as a particular type of molecular
assembly in which
amphipathic molecules are arranged in a spherical structure such that all the
hydrophobic
portions of the molecules are directed inward, leaving the hydrophilic
portions in contact
with the surrounding aqueous phase. The converse arrangement exists if the
environment
is hydrophobic.
[0082] The term "antisense," as used herein, refers to a nucleic acid
comprising an
oligonucleotide or polynucleoti de that is sufficiently complementary to all
or a portion of
a gene, primary transcript, or processed mRNA, so as to interfere with
expression of the
endogenous gene (e.g., MSH3). "Complementary" polynucleotides are those that
are
capable of base pairing according to the standard Watson-Crick complementarity
rules.
Specifically, purines will base pair with pyrimidines to form a combination of
guanine
paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the
case of
DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood
that two
polynucleotides can hybridize to each other even if they are not completely
complementary to each other, provided that each has at least one region that
is
substantially complementary to the other.
[0083] As used herein, the terms -effective amount," -therapeutically
effective amount,"
and "a "sufficient amount" of an agent that reduces the level and/or activity
of MSH3
(e.g., in a cell or a subject) described herein refer to a quantity sufficient
to, when
administered to the subject, including a human, effect beneficial or desired
results,
including clinical results, and, as such, an "effective amount" or synonym
thereto depends
on the context in which it is being applied. For example, in the context of
treating a
nucleotide repeat expansion disorder (e.g., a trinucleotide repeat expansion
disorder), it is
an amount of the agent that reduces the level and/or activity of MSH3
sufficient to
achieve a treatment response as compared to the response obtained without
administration
of the agent that reduces the level and/or activity of MSH3. The amount of a
given agent
that reduces the level and/or activity of MSH3 described herein that will
correspond to
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
such an amount will vary depending upon various factors, such as the given
agent, the
pharmaceutical formulation, the route of administration, the type of disease
or disorder,
the identity of the subject (e.g., age, sex, and/or weight) or host being
treated, and the
like, but can nevertheless be routinely determined by one of skill in the art.
Also, as used
herein, a "therapeutically effective amount" of an agent that reduces the
level and/or
activity of MSH3 of the present disclosure is an amount which results in a
beneficial or
desired result in a subject as compared to a control. As defined herein, a
therapeutically
effective amount of an agent that reduces the level and/or activity of MSH3 of
the present
disclosure can be readily determined by one of ordinary skill by routine
methods known
in the art. Dosage regimen can be adjusted to provide the optimum therapeutic
response.
[0084] "Prophylactically effective amount," as used herein, is intended
to include the
amount of an oligonucleotide that, when administered to a subject having or
predisposed
to have a nucleotide repeat expansion disorder (e.g., a trinucl eoti de repeat
expansion
disorder), is sufficient to prevent or ameliorate the disease or one or more
symptoms of
the disease. Ameliorating the disease includes slowing the course of the
disease or
reducing the severity of later-developing disease. The "prophylactically
effective
amount- can vary depending on the oligonucleotide, how the agent is
administered, the
degree of risk of disease, and the history, age, weight, family history,
genetic makeup, the
types of preceding or concomitant treatments, if any, and other individual
characteristics
of the patient to be treated. A prophylactically effective amount can refer
to, for example,
an amount of the agent that reduces the level and/or activity of MSH3 (e.g.,
in a cell or a
subject) described herein or can refer to a quantity sufficient to, when
administered to the
subject, including a human, delay the onset of one or more of the nucleotide
repeat
disorders (e g , trinucleotide repeat expansion disorders) described herein by
at least 120
days, for example, at least 6 months, at least 12 months, at least 2 years, at
least 3 years,
at least 4 years, at least 5 years, at least 10 years or more, when compared
with the
predicted onset.
[0085] A "therapeutically-effective amount" or "prophylactically
effective amount" also
includes an amount (either administered in a single or in multiple doses) of
an
oligonucleotide that produces some desired local or systemic effect at a
reasonable
benefit/risk ratio applicable to any treatment. Oligonucleotides employed in
the methods
herein can be administered in a sufficient amount to produce a reasonable
benefit/risk
ratio applicable to such treatment.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
31
[0086] As used herein, the term "region of complementarity" refers to
the region on the
ol igonucl eoti de that is substantially complementary to all or a portion of
a gene, primary
transcript, a sequence (e.g., a target sequence, e.g., an MSH3 nucleotide
sequence), or
processed mRNA, so as to interfere with expression of the endogenous gene
(e.g.,
MSH3). Where the region of complementarity is not fully complementary to the
target
sequence, the mismatches can be in the internal or terminal regions of the
molecule.
Generally, the most tolerated mismatches are in the terminal regions, e.g.,
within 5, 4, 3,
or 2 nucleotides of the 5'- and/or 3'-terminus of the oligonucleotide.
[0087] An "amount effective to reduce nucleotide repeat expansion" of a
particular gene
refers to an amount of the agent that reduces the level and/or activity of
MSH3 (e.g., in a
cell or a subject) described herein, orto a quantity sufficient to, when
administered to the
subject, including a human, to reduce the nucleotide repeat expansion of a
particular gene
(e.g., a gene associated with a nucleotide repeat expansion disorder, e.g., a
trinucleotide
repeat expansion disorder, described herein).
[0088] As used herein, the term "a subject identified as having a
nucleotide repeat
expansion disorder" refers to a subject identified as having a molecular or
pathological
state, disease or condition of or associated with a nucleotide repeat
expansion disorder,
such as the identification of a nucleotide repeat expansion disorder or
symptoms thereof,
or to identification of a subject having or suspected of having a nucleotide
repeat
expansion disorder who can benefit from a particular treatment regimen.
[0089] As used herein, "trinucleotide repeat expansion disorder" refers
to a class of
genetic diseases or disorders characterized by excessive trinucleotide repeats
(e.g.,
trinucleotide repeats such as CAG) in a gene or intron in the subject which
exceed the
normal, stable threshold, for the gene or intron. Nucleotide repeats are
common in the
human genome and are not normally associated with disease. In some cases,
however,
the number of repeats expands beyond a stable threshold and can lead to
disease, with the
severity of symptoms generally correlated with the number of repeats.
Nucleotide repeat
expansion disorders include "polyglutamine" and "non-polyglutamine" disorders.
[0090] By "determining the level of a protein" is meant the detection
of a protein, or an
mRNA encoding the protein, by methods known in the art either directly or
indirectly.
"Directly determining" means performing a process (e.g., performing an assay
or test on a
sample or "analyzing a sample" as that term is defined herein) to obtain the
physical
entity or value. -Indirectly determining" refers to receiving the physical
entity or value
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
32
from another party or source (e.g., a third-party laboratory that directly
acquired the
physical entity or value). Methods to measure protein level generally include,
but are not
limited to, western blotting, immunoblotting, enzyme-linked immunosorbent
assay
(ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence,
surface
plasmon resonance, chemiluminescence, fluorescent polarization,
phosphorescence,
immunohistochemical analysis, matrix-assisted laser desorption/ionization time-
of-flight
(MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry,
microcytometry, microscopy, fluorescence activated cell sorting (FACS), and
flow
cytometry, as well as assays based on a property of a protein including, but
not limited to,
enzymatic activity or interaction with other protein partners. Methods to
measure mRNA
levels are known in the art.
[0091] "Percent (%) sequence identity" with respect to a reference
polynucleotide or
polypepti de sequence is defined as the percentage of nucleic acids or amino
acids in a
candidate sequence that are identical to the nucleic acids or amino acids in
the reference
polynucleotide or polypeptide sequence, after aligning the sequences and
introducing
gaps (DNA core sequences), if necessary, to achieve the maximum percent
sequence
identity. Alignment for purposes of determining percent nucleic acid or amino
acid
sequence identity can be achieved in various ways that are within the
capabilities of one
of skill in the art, for example, using publicly available computer software
such as
BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine
appropriate parameters for aligning sequences, including any algorithms needed
to
achieve maximal alignment over the full length of the sequences being
compared. For
example, percent sequence identity values can be generated using the sequence
comparison computer program BLAST As an illustration, the percent sequence
identity
of a given nucleic acid or amino acid sequence, A, to, with, or against a
given nucleic
acid or amino acid sequence, B, (which can alternatively be phrased as a given
nucleic
acid or amino acid sequence, A that has a certain percent sequence identity
to, with, or
against a given nucleic acid or amino acid sequence, B) is calculated as
follows:
100 multiplied by (the fraction X/Y)
where X is the number of nucleotides or amino acids scored as identical
matches by a
sequence alignment program (e.g., BLAST) in that program's alignment of A and
B, and
where Y is the total number of nucleic acids in B. It will be appreciated that
where the
length of nucleic acid or amino acid sequence A is not equal to the length of
nucleic acid
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
33
or amino acid sequence B, the percent sequence identity of A to B will not
equal the
percent sequence identity of B to A.
[0092] By "level" is meant a level or activity of a protein, or mRNA
encoding the protein
(e.g., MSH3), optionally as compared to a reference. The reference can be any
useful
reference, as defined herein. By a "decreased level" or an "increased level"
of a protein is
meant a decrease or increase in protein level, as compared to a reference
(e.g., a decrease
or an increase by about 5%, about 10%, about 15%, about 20%, about 25%, about
30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about
150%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or
an
increase of more than 10%, 15%, 200/a, 50%, 75%, 100%, or 200%, as compared to
a
reference; a decrease or an increase by less than 0.01-fold, 0.02-fold, 0.1-
fold, 0.3-fold,
0.5-fold, 0.8-fold, or less; or an increase by more than 1.2-fold, 1.4-fold,
1.5-fold, 1.8-
fold, 20-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0-fold, 10-fold, 15-fold, 20-
fold, 30-fold, 40-
fold, 50-fold, 100-fold, 1000-fold, or more). A level of a protein can be
expressed in
mass/vol (e.g., g/dL, mg/mL, pg/mL, or ng/mL) or percentage relative to total
protein or
mRNA in a sample.
[0093] The term "pharmaceutical composition," as used herein,
represents a composition
containing a compound described herein formulated with a pharmaceutically
acceptable
excipient, and can be manufactured or sold with the approval of a governmental
regulatory agency as part of a therapeutic regimen for the treatment of
disease in a
mammal. Pharmaceutical compositions can be formulated, for example, for oral
administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap,
or syrup); for
topical administration (e.g., as a cream, gel, lotion, or ointment); for
intravenous
administration (e.g., as a sterile solution free of particulate emboli and in
a solvent system
suitable for intravenous use); for intrathecal injection; for
intracerebroventricular
injections; for intraparenchymal injection; for intraocular administration
(e.g., for
intravitreal or subretinal administration); or in any other pharmaceutically
acceptable
formulation.
[0094] A "pharmaceutically acceptable excipient," as used herein,
refers any ingredient
other than the compounds described herein (for example, a vehicle capable of
suspending
or dissolving the active compound) and having the properties of being
substantially
nontoxic and non-inflammatory in a patient. Excipients can include, for
example:
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
34
antiadherents, antioxidants, binders, coatings, compression aids,
disintegrants, dyes
(colors), emollients, emulsifiers, fillers (diluents), film formers or
coatings, flavors,
fragrances, glidants (flow enhancers), lubricants, preservatives, printing
inks, sorbents,
suspensing or dispersing agents, sweeteners, and waters of hydration.
Exemplary
excipients include, but are not limited to: butylated hydroxytoluene (BHT),
calcium
carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose,
crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose,
gelatin,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium
stearate,
maltitol, mannitol, methionine, methyl cellulose, methyl paraben,
microcrystalline
cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
pregelatinized starch,
propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium
carboxymethyl
cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn),
stearic acid,
sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.
[0095] As used herein, the term "pharmaceutically acceptable salt"
means any
pharmaceutically acceptable salt of the compound of any of the compounds
described
herein. For example, pharmaceutically acceptable salts of any of the compounds
described herein include those that are within the scope of sound medical
judgment,
suitable for use in contact with the tissues of humans and animals without
undue toxicity,
irritation, allergic response and are commensurate with a reasonable
benefit/risk ratio.
Pharmaceutically acceptable salts are well known in the art. For example,
pharmaceutically acceptable salts are described in: Berge et al., J.
Pharmaceutical
Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and
Use, (Eds.
P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in
situ
during the final isolation and purification of the compounds described herein
or separately
by reacting a free base group with a suitable organic acid.
[0096] The compounds described herein can have ionizable groups so as
to be capable of
preparation as pharmaceutically acceptable salts. These salts can be acid
addition salts
involving inorganic or organic acids or the salts can, in the case of acidic
forms of the
compounds described herein, be prepared from inorganic or organic bases.
Frequently,
the compounds are prepared or used as pharmaceutically acceptable salts
prepared as
addition products of pharmaceutically acceptable acids or bases. Suitable
pharmaceutically acceptable acids and bases and methods for preparation of the
appropriate salts are well-known in the art. Salts can be prepared from
pharmaceutically
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
acceptable non-toxic acids and bases including inorganic and organic acids and
bases.
Representative acid addition salts include acetate, adipate, alginate,
ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate,
citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
glucoheptonate, glycerophosphate, hemi sulfate, heptonate, hexanoate,
hydrobromide,
hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate,
lauryl 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, toluenesulfonate, undecanoate, and valerate salts.
Representative
alkali or alkaline earth metal salts include sodium, lithium, potassium,
calcium, and
magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine
cations,
including, but not limited to ammonium, tetramethyl ammonium, tetraethyl
ammonium,
methylamine, dimethylamine, trimethylamine, triethyl amine, and ethylamine.
[0097] By a "reference" is meant any useful reference used to compare
protein or mRNA
levels or activity. The reference can be any sample, standard, standard curve,
or level that
is used for comparison purposes. The reference can be a normal reference
sample or a
reference standard or level. A "reference sample" can be, for example, a
control, e.g., a
predetermined negative control value such as a "normal control" or a prior
sample taken
from the same subject; a sample from a normal healthy subj ect, such as a
normal cell or
normal tissue; a sample (e.g., a cell or tissue) from a subject not having a
disease; a
sample from a subject that is diagnosed with a disease, but not yet treated
with a
compound described herein; a sample from a subject that has been treated by a
compound
described herein; or a sample of a purified protein (e g , any described
herein) at a known
normal concentration. By "reference standard or level" is meant a value or
number
derived from a reference sample. A "normal control value" is a pre-determined
value
indicative of non-disease state, e.g., a value expected in a healthy control
subject.
Typically, a normal control value is expressed as a range ("between X and Y"),
a high
threshold ("no higher than X"), or a low threshold ('no lower than X"). A
subject having
a measured value within the normal control value for a particular biomarker is
typically
referred to as "within normal limits" for that biomarker. A normal reference
standard or
level can be a value or number derived from a normal subject not having a
disease or
disorder (e.g., a nucleotide or trinucleotide repeat expansion disorder); a
subject that has
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
36
been treated with a compound described herein. In some aspects, the reference
sample,
standard, or level is matched to the sample subject sample by at least one of
the following
criteria: age, weight, sex, disease stage, and overall health. A standard
curve of levels of
a purified protein, e.g., any described herein, within the normal reference
range can be
used as a reference.
[0098] As used herein, the term "subject" refers to any organism to
which a composition
can be administered, e.g., for experimental, diagnostic, prophylactic, and/or
therapeutic
purposes. Typical subjects include any animal (e.g., mammals such as mice,
rats, rabbits,
non-human primates, and humans). A subject can seek or be in need of
treatment, require
treatment, be receiving treatment, be receiving treatment in the future, or be
a human or
animal who is under care by a trained professional for a particular disease or
condition.
[0099] As used herein, the terms "treat," "treated," and "treating"
mean both therapeutic
treatment and prophylactic or preventative measures wherein the object is to
prevent or
slow down (lessen) an undesired physiological condition, disorder, or disease,
or obtain
beneficial or desired clinical results. Beneficial or desired clinical results
include, but are
not limited to, alleviation of symptoms, diminishment of the extent of a
condition,
disorder, or disease; stabilized (i.e., not worsening) state of condition,
disorder, or
disease; delay in onset or slowing of condition, disorder, or disease
progression;
amelioration of the condition, disorder, or disease state or remission
(whether partial or
total), whether detectable or undetectable; an amelioration of at least one
measurable
physical parameter, not necessarily discernible by the patient; or enhancement
or
improvement of condition, disorder, or disease. Treatment includes eliciting a
clinically
significant response without excessive levels of side effects. Treatment also
includes
prolonging survival as compared to expected survival if not receiving
treatment
[0100] As used herein, the terms "variant" and "derivative" are used
interchangeably and
refer to naturally-occurring, synthetic, and semi-synthetic analogues of a
compound,
peptide, protein, or other substance described herein. A variant or derivative
of a
compound, peptide, protein, or other substance described herein can retain or
improve
upon the biological activity of the original material.
[0101] The details of one or more aspects are set forth in the
description below. Other
features, objects, and advantages will be apparent from the description and
from the
claims.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
37
DETAILED DESCRIPTION
[0102] The present inventors have found that inhibition or depletion of
MSH3 level
and/or activity in a cell is effective in the treatment of a nucleotide repeat
expansion
disorder (e.g., a trinucleotide repeat expansion disorder). Accordingly,
useful
compositions and methods to treat nucleotide repeat expansion disorders (e.g.,
a
trinucleotide repeat expansion disorder), e.g., in a subject in need thereof
are provided
herein.
I. Nucleotide Repeat Expansion Disorders
[0103] Nucleotide repeat expansion disorders (e.g., trinucleotide
repeat expansion
disorders) are a family of genetic disorders characterized by the pathogenic
expansion of
a repeat region within a genomic region. In such disorders, the number of
repeats
exceeds that of a gene's normal, stable threshold, expanding into a diseased
range.
[0104] Nucleotide repeat expansion disorders (e.g., trinucleotide
repeat expansion
disorders) generally can be categorized as "polyglutamine" or "non-
polyglutamine."
Polyglutamine disorders, including Huntington's disease (HD) and several
spinocerebellar
ataxias, are caused by a CAG (glutamine) repeats in the protein-coding regions
of specific
genes. Non-polyglutamine disorders are more heterogeneous and can be caused by
CAG
nucleotide repeat expansions in non-coding regions, as in Myotonic dystrophy,
or by the
expansion of nucleotide repeats other than CAG that can be in coding or non-
coding
regions such as the CGG repeat expansion responsible for Fragile X Syndrome.
[0105] Nucleotide repeat expansion disorders (e.g., trinucleotide
repeat expansion
disorders) are dynamic in the sense that the number of repeats can vary from
generation-
to-generation, or even from cell-to-cell in the same individual. Repeat
expansion is
believed to be caused by polymerase "slipping" during DNA replication. Tandem
repeats
in the DNA sequence can "loop out" while maintaining complementary base
pairing
between the parent strand and daughter strands. If the loop structure is
formed from the
daughter strand, the number of repeats will increase.
[0106] Conversely, if the loop structure is formed from the parent
strand, the number of
repeats will decrease_ It appears that expansion is more common than reduction
In
general, the length of repeat expansion is negatively correlated with
prognosis; longer
repeats are correlated with an earlier age of onset and worsened disease
severity. Thus,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
38
nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion
disorders) are
subject to "anticipation," meaning the severity of symptoms and/or age of
onset worsen
through successive generations of affected families due to the expansion of
these repeats
from one generation to the next.
[0107] Nucleotide repeat expansion disorders (e.g., trinucleotide
repeat expansion
disorders) are well known in the art. For example, frontotemporal dementia
(FTD) is a
hexanucleotide repeat string of nucleotides GGGGCC that is repeated many more
times
in an individual than an individual without FTD. Additionally, an individual
having
spinocerebellar ataxia type 36 (SCA36) has many more GGCCTG repeats than an
individual without SCA36.
[0108] Exemplary trinucleotide repeat expansion disorders and
the trinucleotide repeats
of the genes commonly associated with them are included in Table 1.
Table 1: Exemplary Trinucleotide Repeat Expansion Disorders
Disease Gene Nucleotide
Repeat
ARX-nonsyndromic X-linked mental ARX GCG
retardation (XLMR)
Baratela-Scott Syndrome XYLT 1 GGC
Blcpharophimosis / Ptosis / Epicanthus FOXL2 GCG
inversus syndrome type II
Cleidocranial dysplasia (CCD) RUNX2 GCG
Congenital central hypoventilation PHOX-213 GCG
Congenital central hypoventilation PHOX2B GCG
syndrome (CCHS)
Creutzfeldt-Jakob disease PRNP
Dentatorubral-pallidoluysian atrophy ATN1 CAG
(DRPLA) / Haw River syndrome
Early infantile epileptic encephalopathy ARX GCG
(Ohtahara syndrome)
FRA2A syndrome AFF3 CGC
FRA7A syndrome ZNF71 3 CGG
Fragile X mental retardation (FRAX-E) AFF2 / FMR2 GCC
Fragile X Syndrome (FXS) FMR1 CGG
Fragile X-associated Primary Ovarian FMR1 CGG
Insufficiency (FXPOI)
Fragile X-associated Tremor Ataxia FMR1 CGG
Syndrome (FXTAS)
Fricdrcich ataxia (FRDA) FXN GAA
Fuchs' Corneal Endothelial Dystrophy TCF4 CTG
(FECD)
Hand-foot genital syndrome (HFGS) HOXA 13 GCG
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
39
Holoprosencephaly disorder (HPE) Z1C2 GCG
Huntington disease-like 2 (HDL2) JPH3 CTG
Huntington's Disease (HD) HTT CAG
Infantile spasm syndrome / West ARX GCG
syndrome (TS S)
Jacobsen syndrome
KCNN3 -associated (e .g KCNN3 CAG
schizophrenia)
Multiple Skeletal dysplasias COMP GAC
Myotonic Dystrophy type 1 (D1\41) DMPK CTG
Myotonic Dystrophy type 2 (DM2) CNBP CCTG
NCOA3-associated (e.g., increased risk NCOA3 CAG
of prostate cancer)
Neuronal intranuclear inclusion disease NOTCH2NLC GGC
(NI1D)
Oculopharyngeal Muscular Dystrophy PABPN1 GCG
(OPMD)
Spastic ataxia - Charlevoix-Saguenay
Spinal Muscular Bulbar Atrophy AR CAG
(SMBA)
Spinocerebellar ataxia type 1 (SCA1) ATXN1 CAG
Spinocerebellar ataxia type 10 (S CA 10) ATXN 1 0 ATTCT
Spinocercbellar ataxia type 12 (SCA12) PPP2R2B CAG
Spinocerebellar ataxia type 17 (SCA 17) TBP / ATXN 17 CAG
Spinocerebellar ataxia type 2 (SCA2) ATXN2 CAG
Spinocerebellar ataxia type 3 (SCA3) / ATXN3 CAG
Machado-Joseph Disease
Spinocerebellar ataxia type 45 (S CA45) FAT2 CAG
Spinocerebellar ataxia type 6 (SCA6) CACNA1A CAG
Spinocerebellar ataxia type 7 (SCA7) ATXN7 CAG
Spinocerebellar ataxia type 8 (SCA8) ATXN 8 CTG
Syndromic neurodevelopmental MAB21L1 CAG
disorder with cerebellar, ocular,
craniofacial, and genital features
(COFG syndrome)
Synpolydactyly (SPD I) HO XD 13 GCG
Synpolydactyly (SPD II) HO XD 12 GCG
[0109] The proteins associated with nucleotide repeat expansion
disorders (e.g.,
trinucleotide repeat expansion disorders) are typically selected based on an
experimental
association of the protein associated with a nucleotide repeat expansion
disorder (e.g., a
trinucleotide repeat expansion disorder) to a nucleotide repeat expansion
disorder. For
example, the production rate or circulating concentration of a protein
associated with a
nucleotide repeat expansion disorder (e.g., trinucleotide repeat expansion
disorder) can be
elevated or depressed in a population having a nucleotide repeat expansion
disorder (e.g.,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
a trinucleotide repeat expansion disorder) relative to a population lacking
the nucleotide
repeat expansion disorder (e.g., trinucleotide repeat expansion disorder).
Differences in
protein levels can be assessed using proteomic techniques including but not
limited to
Western blot, immunohistochemical staining, enzyme linked immunosorbent assay
(ELISA), and mass spectrometry. Alternatively, the proteins associated with
nucleotide
repeat expansion disorders (e.g., trinucleotide repeat expansion disorders)
can be
identified by obtaining gene expression profiles of the genes encoding the
proteins using
genomic techniques including, but not limited to, DNA microarray analysis,
serial
analysis of gene expression (SAGE), and quantitative real-time polymerase
chain reaction
(qPCR).
II Evidence for the Involvement of Mismatch Repair Pathway
in Nucleotide Repeat
Expansion
101101 There is growing evidence that DNA repair pathways, particularly
mismatch
repair (M1VIR), are involved in the expansion of nucleotide repeats (e.g.,
trinucleotide
repeats). A recent genome-wide association (GWA) analysis led to the
identification of
loci harboring genetic variations that alter the age at neurological onset of
Huntington's
disease (HD) (GEM-11D Consortium, Cell. 2015 Jul 30;162(3):516-26). The study
identified MLITI, the human homolog of the E. coil DNA mismatch repair gene
mull; A
subsequent GWA study in polyglutamine disease patients found significant
association of
age at onset when grouping all polyglutamine diseases (HD and SCAs) with DNA
repair
genes as a group, as well as significant associations for specific SNPs in
FAN] and PMS2
with the diseases (Bettencourt et al., (2016) Ann. Neural., 79: 983-990).
These results
were consistent with those from an earlier study comparing differences in
repeat
expansion in two different mouse models of Huntington's Disease, which
identified Mlhl
and M1h3 as novel critical modifiers of CAG instability (Pinto et al., (2013)
Mismatch
Repair Genes Mlhl andIVilh3 Modify CAG Instability in Huntington's Disease
Mice:
Genome-Wide and Candidate Approaches. PLoS Genet 9(10): e1003930). Another
member of the mismatch repair pathway, 8-oxo-guanine glycosylase (OGG1) has
also
been implicated in expansion, as somatic expansion was found to be reduced in
transgenic
mice lacking OGG/ (Kovtun T V et al (2007) Nature 447, 447-452) However,
another
study found that human subjects containing a Ser326Cys polymorphism in hOGG1,
which results in reduced OGG1 activity, results in increased mutant huntingtin
(Coppede
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
41
et al., (2009) ToxicoL, 278: 199-203). Likewise, complete inactivation of 1-
,ran], another
component of the DNA repair pathway, in a mouse HD model produces somatic CAG
expansions (Long et al. (2018)1 HUM Genet., 103: 1-9). MSH3, another component
of
the mismatch repair pathway, has been reported to be linked to somatic
expansion:
polymorphisms in Msh3 was associated with somatic instability of the expanded
CTG
trinucleotide repeat in myotonic dystrophy type 1 (DM1) patients (Morales et
al., (2016)
DNA Repair 40: 57-66). Furthermore, natural polymorphisms in Msh3 and Mlhl
have
been revealed as mediators of mouse strain specific differences in CTG-CAG
repeat
instability (Pinto et al. (2013) /bid; Tome et al., (2013) PLoS Genet. 9
e1003280).
Further evidence ofIVIsh2 and Msh3' s involvement in expansion repeats was
reported in a
study in which short hairpin RNA (shRNA) knockdown of either MSH2 or MSH3
slowed, and ectopic expression of either MSH2 or MSH3 induced GAA
trinucleotide
repeat expansion of the Friedreich Ataxia (FRDA) gene in fibroblasts derived
from
FRDA patients (Halabi et al., (2012) J. Biol. Chem. 287, 29958-29967). In
spite of some
inconsistent results provided above, there is strong evidence that the MNIR
pathway plays
some role in the expansion of trinucleotide repeats in various disorders.
Moreover, they
are the first to recognize that the inhibition of the MMR pathway provides for
the
treatment or prevention of these repeat expansion disorders; however, no
therapy is
currently available or in development which modulates MIVIR for purposes of
treating or
preventing these repeat expansion disorders.
Oligonucleotide Agents
[0111] Agents described herein that reduce the level and/or activity of
MSH3 in a cell can
be, for example, a polynucleotide, e.g., an oligonucleotide, or
pharmaceutically
acceptable salt thereof. These agents reduce the level of an activity related
to MSH3, or a
related downstream effect, or reduce the level of MSH3 in a cell or subject.
[0112] In some aspects, the agent that reduces the level and/or
activity of MSH3 is a
polynucleotide. In some aspects, the polynucleotide is a single-stranded
oligonucleotide,
e.g., that acts by way of an RNase H-mediated pathway. Oligonucleotides
include DNA
and DNA/RNA chimeric molecules, typically about 10 to 30 nucleotides in
length, which
recognize polynucleotide target sequences or sequence portions through
hydrogen
bonding interactions with the nucleotide bases of the target sequence (e.g.,
MSH3). An
oligonucleotide molecule can decrease the expression level (e.g., protein
level or mRNA
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
42
level) of MSH3. For example, an oligonucleotide includes oligonucleotides that
targets
full-length MSH3. In some aspects, the oligonucleotide molecule recruits an
RNase H
enzyme, leading to target mRNA degradation.
[0113] In some aspects, the oligonucleotide, or pharmaceutically
acceptable salt thereof,
decreases the level and/or activity of a positive regulator of function. In
other aspects, the
oligonucleotide, or pharmaceutically acceptable salt thereof, increases the
level and/or
activity of an inhibitor of a positive regulator of function. In some aspects,
the
oligonucleotide, or pharmaceutically acceptable salt thereof, increases the
level and/or
activity of a negative regulator of function.
[0114] In some aspects, the oligonucleotide, or pharmaceutically
acceptable salt thereof,
decreases the level and/or activity or function of MSH3. In some aspects, the
oligonucleotide, or pharmaceutically acceptable salt thereof, inhibits
expression of
MSH3. In other aspects, the oligonucleotide, or pharmaceutically acceptable
salt thereof,
increases degradation of MSH3 and/or decreases the stability (i.e., half-life)
of MSH3
The oligonucleotide, or pharmaceutically acceptable salt thereof, can be
chemically
synthesized.
[0115] An oligonucleotide, or pharmaceutically acceptable salt thereof,
can be
synthesized by standard methods known in the art as further discussed below,
e.g., by use
of an automated DNA synthesizer, such as are commercially available from, for
example,
Biosearch, Applied Biosystems, Inc.
[0116] The oligonucleotide, or pharmaceutically acceptable salt
thereof, compound can
be prepared using solution-phase or solid-phase organic synthesis or both.
Organic
synthesis offers the advantage that the oligonucleotide, or pharmaceutically
acceptable
salt thereof, comprising unnatural or alternative nucleotides can be easily
prepared A
single-stranded oligonucleotide, or pharmaceutically acceptable salt thereof,
can be
prepared using solution-phase or solid-phase organic synthesis or both.
[0117] Some aspects of the disclosure are related to a single-stranded
oligonucleotide of
15-30 linked nucleotides in length, wherein the oligonucleotide, or a portion
thereof, is at
least 95% complementary to at least 15 contiguous nucleobases at positions
2543-2573 of
SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide, or a portion thereof, is at least 98% complementary to at
least 15
contiguous nucleobases at positions 2543-2573 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
43
portion thereof, is at least 99% complementary to at least 15 contiguous
nucleobases at
positions 2543-2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
In some aspects, the oligonucleotide, or a portion thereof, is 100%
complementary to at
least 15 contiguous nucleobases at positions 2543-2573 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
portion thereof, is complementary to 17-23 contiguous nucleobases at positions
2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof. In some
aspects,
the oligonucleotide is complementary to 17-20 contiguous nucleobases at
positions 2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof. In some
aspects,
the 17-20 contiguous nucleobases begin at position 2543, 2544, 2545, 2546,
2547, 2548,
2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, or 2557 of SEQ ID NO: 614, or
a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is 17-20
linked nucleotides in length, or a pharmaceutically acceptable salt thereof.
[0118] In some aspects, the oligonucleotide, or a portion thereof, is
complementary to 20-
23 contiguous nucleobases at positions 2543-2573 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the 20-23
contiguous
nucleobases begin at position 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550,
2551,
2552, 2553, or 2554 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
In some aspects, the oligonucleotide is 20-23 linked nucleotides in length, or
a
pharmaceutically acceptable salt thereof.
[0119] In some aspects, the oligonucleotide, or a portion thereof, is
complementary to
positions 2543-2570 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
[0120] The disclosure also relates to single-stranded oligonucleotides
of 15-30 linked
nucleotides in length, wherein the oligonucleotide, or a portion thereof, is
at least 95%
complementary to at least 15 contiguous nucleobases at positions 2685-2714 of
SEQ ID
NO: 614, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide, or a portion thereof, is at least 98% complementary to at
least 15
contiguous nucleobases at positions 2685-2714 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
portion thereof, is at least 99% complementary to at least 15 contiguous
nucleobases at
positions 2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable salt
thereof.
In some aspects, the oligonucleotide or a portion thereof, is 100%
complementary to at
least 15 contiguous nucleobases at positions 2685-2714 of SEQ Ill NO: 614, or
a
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
44
pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide, or a
portion thereof is complementary to 17-23 contiguous nucleobases at positions
2685-2714
of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide, or a portion thereof, is complementary to 17-20 contiguous
nucleobases
at positions 2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable
salt thereof
In some aspects, the oligonucleotide, or a portion thereof, is complementary
to 17-20
contiguous nucleobases beginning at position 2685, 2686, 2687, 2688, 2689,
2690, 2691,
2692, 2693, 2694, 2695, 2696, 2697, or 2698 of SEQ ID NO: 614, or a
pharmaceutically
acceptable salt thereof In some aspects, the oligonucleotide is 17-20 linked
nucleotides
in length, or a pharmaceutically acceptable salt thereof.
[0121] In some aspects, the oligonucleotide, or a portion thereof, is
complementary to 20-
23 contiguous nucleobases at positions 2685-2714 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is
complementary to 20-23 contiguous nucleobases beginning at position 2685,
2686, 2687,
2688, 2689, 2690, 2691, 2692, 2693, 2694, or 2695 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is 20-23
linked nucleotides in length, or a pharmaceutically acceptable salt thereof.
In some
aspects, the oligonucleotide, or a portion thereof, is complementary to
positions 2685-
2714 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0122] In some aspects of the above, the oligonucleotide is not any one
of Antisense
Oligo Nos. 1, 97, 193, or 289 of Table 3. In some aspects of the above, the
oligonucleotide does not have a nucleobase sequence consisting of any one of
SEQ ID
NOs: 1, 97, 193, or 289.
[0123] In some aspects of the above disclosures, the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs: 1-
384
and 390-613, or a pharmaceutically acceptable salt thereof. In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 2-96, 98-192, 194-288, 290-384, and 390-613, or a
pharmaceutically
acceptable salt thereof In some aspects, the oligonucleotide consists of a
nucleobase
sequence selected from the group consisting of any of SEQ ID NOs: 1-384, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
2-96,
98-192, 194-288, and 290-384, or a pharmaceutically acceptable salt thereof In
some
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
aspects, the oligonucleotide consists of a nucleobase sequence selected from
the group
consisting of any of SEQ ID NOs: 1-96, or a pharmaceutically acceptable salt
thereof. In
some aspects, the oligonucleotide consists of a nucleobase sequence selected
from the
group consisting of any of SEQ ID NOs: 2-96, or a pharmaceutically acceptable
salt
thereof. In some aspects, the oligonucleotide consists of a nucleobase
sequence selected
from the group consisting of any of SEQ ID NOs: 97-192, or a pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide consists of a
nucleobase
sequence selected from the group consisting of any of SEQ ID NOs: 98-192, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
193-
288, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide
consists of a nucleobase sequence selected from the group consisting of any of
SEQ ID
NOs: 194-288, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 289-384, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of a nucleobase sequence selected from
the group
consisting of any of SEQ ID NOs: 288-384, or a pharmaceutically acceptable
salt thereof.
In some aspects, the oligonucleotide consists of a nucleobase sequence
selected from the
group consisting of any of SEQ ID NOs: 390-613, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of a nucleobase
sequence selected
from the group consisting of any of SEQ ID NOs: 390-480, or a pharmaceutically
acceptable salt thereof In some aspects, the oligonucleotide consists of a
nucleobase
sequence selected from the group consisting of any of SEQ ID NOs: 481-571, or
a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
572-
662, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide
consists of a nucleobase sequence selected from the group consisting of any of
SEQ ID
NOs: 663-613, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 1, 6, 13, 17, 21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-
73, 75-76,
79-82, 84-86, 88-92, or 94-96, or a pharmaceutically acceptable salt thereof.
In some
aspects, the oligonucleotide consists of a nucleobase sequence selected from
the group
consisting of any of SEQ ID NOs: 6, 13, 17, 21, 24, 26, 29, 33-34, 37, 44, 49-
55, 57, 60-
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
46
73, 75-76, 79-82, 84-86, 88-92, or 94-96, or a pharmaceutically acceptable
salt thereof. In
some aspects, the oligonucleotide consists of a nucleobase sequence that is
SEQ ID NO:
1, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of a nucleobase sequence that is SEQ ID NO: 6, or a pharmaceutically
acceptable
salt thereof In some aspects, the oligonucleotide consists of a nucleobase
sequence
selected from the group consisting of any of SEQ ID NOs: 97, 100, 103, 105,
108, 110-
111, 113-117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148,
154-
155, 157-165, 168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically
acceptable
salt thereof In some aspects, the oligonucleotide consists of a nucleobase
sequence
selected from the group consisting of any of SEQ ID NOs: 100, 103, 105, 108,
110-111,
113-117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-
155,
157-165, 168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of a nucleobase
sequence that is
SEQ ID NO: 97, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 193-200, 202-230, 232-246, 248-253, 255, 258-261, 265, 270,
274-
276, or 285-286, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 194-200, 202-230, 232-246, 248-253, 255, 258-261, 265, 270,
274-
276, or 285-286, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of a nucleobase sequence that is SEQ ID NO: 193, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
a nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
226-
227, 234, 240, or 243-244, or a pharmaceutically acceptable salt thereof. In
some aspects,
the oligonucleotide consists of a nucleobase sequence selected from the group
consisting
of any of SEQ ID NOs: 227, 234, 240, or 243-244, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence that is
SEQ ID NO: 226, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 289-290, 292, 305, 307, 313, 318, 323-324, 326, 329-330,
332, 338-
339, 341, 344, or 346, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of a nucleobase sequence selected from the group
consisting of
any of SEQ ID NOs: 290, 292, 305, 307, 313, 318, 323-324, 326, 329-330, 332,
338-339,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
47
341, 344, or 346, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of a nucleobase sequence that is SEQ ID NO: 289, or a
pharmaceutically acceptable salt thereof.
[0124] Some aspects of the disclosure are directed to single-stranded
oligonucleotides,
wherein the nucleobase sequence of the oligonucleotide consists of any one of
SEQ ID
NOs: 1-384 and 390-613, or a pharmaceutically acceptable salt thereof In some
aspects,
the nucleobase sequence of the oligonucleotide consists of any one of SEQ ID
NOs: 2-96,
98-192, 194-288, 290-384, and 390-613, or a pharmaceutically acceptable salt
thereof. In
some aspects, the nucleobase sequence of the oligonucleotide consists of any
one of SEQ
ID NOs: 1-384, or a pharmaceutically acceptable salt thereof. In some aspects,
the
nucleobase sequence of the oligonucleotide consists of any one of SEQ ID NOs:
2-96, 98-
192, 194-288, or 290-384, or a pharmaceutically acceptable salt thereof. In
some aspects,
the oligonucleotide consists of the nucleobase sequence of any one of SEQ ID
NOs: 1-96,
or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of the nucleobase sequence of any one of SEQ ID NOs: 2-96, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 97-192, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide consists of the
nucleobase
sequence of any one of SEQ ID NOs: 96-192, or a pharmaceutically acceptable
salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 193-288, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of the nucleobase sequence of any one of
SEQ ID
NOs: 194-288, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs:
289-384,
or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of the nucleobase sequence of any one of SEQ ID NOs: 290-384, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 390-613, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide consists of the
nucleobase
sequence of any one of SEQ ID NOs: 390-480, or a pharmaceutically acceptable
salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 481-571, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of the nucleobase sequence of any one of
SEQ ID
CA 03180981 2022- 12- 1
WO 2021/247020 PCT/US2020/035972
48
NOs: 572-662, or a pharmaceutically acceptable salt thereof In some aspects,
the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs:
663-613,
or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide
consists of the nucleobase sequence of any one of SEQ ID NOs: 1, 6, 13, 17,
21, 24, 26,
29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96, or
a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 6, 13, 17, 21, 24, 26, 29,
33-34, 37,
44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide consists of the
nucleobase
sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide consists of the nucleobase sequence of SEQ ID NO:
6, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 97, 100, 103, 105, 108, 110-
111,
113-117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-
155,
157-165, 168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 100, 103, 105, 108, 110-111, 113-117, 122-123, 127, 129-
130, 133-
136, 138-139, 141, 143-145, 147-148, 154-155, 157-165, 168-170, 172, 174-180,
184,
187, or 191, or a pharmaceutically acceptable salt thereof. In some aspects,
the
oligonucleotide consists of the nucleobase sequence of SEQ ID NO: 97, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 193-200, 202-230, 232-246,
248-
253, 255, 258-261, 265, 270, 274-276, or 285-286, or a pharmaceutically
acceptable salt
thereof In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ .. NOs: 194-200, 202-230, 232-246, 248-253, 255, 258-261, 265, 270,
274-
276, or 285-286, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NO:
193, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 226-227, 234, 240, or 243-
244, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of any one of SEQ ID NOs: 227, 234, 240, or 243-244,
or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of SEQ Ill NO: 226, or a pharmaceutically acceptable
salt
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
49
thereof. In some aspects, the oligonucleotide consists of the nucleobase
sequence of any
one of SEQ ID NOs: 289-290, 292, 305, 307, 313, 318, 323-324, 326, 329-330,
332, 338-
339, 341, 344, or 346, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs:
290,
292, 305, 307, 313, 318, 323-324, 326, 329-330, 332, 338-339, 341, 344, or
346, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
consists of
the nucleobase sequence of SEQ ID NO: 289, or a pharmaceutically acceptable
salt
thereof.
[0125] Some aspects of the disclosure are directed to nn
oligonucleotide selected from the
group consisting of Antisense Oligo Nos. 1-384 of Table 3 or 390-613 of Table
4, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 2-96, 98-192, 194-288, 290-
384 of
Table 3 and 390-613 of Table 4, or a pharmaceutically acceptable salt thereof.
In some
aspects, the oligonucleotide is selected from the group consisting of
Antisense Oligo Nos
1-384 of Table 3, or a pharmaceutically acceptable salt thereof. In some
aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
2-96, 98-
192, 194-288, and 290-384 of Table 3, or a pharmaceutically acceptable salt
thereof. In
some aspects, the oligonucleotide is selected from the group consisting of
Antisense
Oligo Nos. 1-96 of Table 3, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide is selected from the group consisting of
Antisense Oligo Nos.
2-96 of Table 3, or a pharmaceutically acceptable salt thereof In some
aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
97-192 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
98-192 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
193-288 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
194-288 of
Table 3, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
289-384 of
Table 3, or a pharmaceutically acceptable salt thereof In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
290-384 of
Table 3, or a pharmaceutically acceptable salt thereof In some aspects, the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
390-613 of
Table 4, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
390-480 of
Table 4, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
481-571 of
Table 4, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
1, 6, 13, 17,
21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92,
or 94-96 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
6, 13, 17,
21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92,
or 94-96 of
Table 3, or a pharmaceutically acceptable salt thereof. In some aspects, the
oligonucleotide is Anti sense Oligo No. 1 of Table 3, or a pharmaceutically
acceptable salt
thereof. In some aspects, the oligonucleotide is Antisense Oligo No. 6 of
Table 3, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 97, 100, 103, 105, 108, 110-
111, 113-
117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155,
157-
165, 168-170, 172, 174-180, 184, 187, or 191 of Table 3, or a pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide is selected from
the group
consisting of Antisense Oligo Nos. 100, 103, 105, 108, 110-111, 113-117, 122-
123, 127,
129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155, 157-165, 168-170,
172,
174-180, 184, 187, or 191 of Table 3, or a pharmaceutically acceptable salt
thereof. In
some aspects, the oligonucleotide is Antisense Oligo No. 97 of Table 3, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 193-200, 202-230, 232-246,
248-253,
255, 258-261, 265, 270, 274-276, or 285-286 of Table 3, or a pharmaceutically
acceptable
salt thereof. In some aspects, the oligonucleotide is selected from the group
consisting of
Antisense Oligo Nos. 194-200, 202-230, 232-246, 248-253, 255, 258-261, 265,
270, 274-
276, or 285-286 of Table 3, or a pharmaceutically acceptable salt thereof. In
some
aspects, the oligonucleotide is Antisense Oligo No. 193 of Table 3. In some
aspects, the
oligonucleotide is selected from the group consisting of Antisense Oligo Nos.
226-227,
234, 240, or 243-244 of Table 3, or a pharmaceutically acceptable salt thereof
In some
aspects, the oligonucleotide is selected from the group consisting of
Antisense Oligo Nos.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
51
227, 234, 240, or 243-244 of Table 3, or a pharmaceutically acceptable salt
thereof In
some aspects, the oligonucleotide is Anti sense Oligo No. 226 of Table 3, or a
pharmaceutically acceptable salt thereof. In some aspects, the oligonucleotide
is selected
from the group consisting of Antisense Oligo Nos. 289-290, 292, 305, 307, 313,
318,
323-324, 326, 329-330, 332, 338-339, 341, 344, or 346 of Table 3, or a
pharmaceutically
acceptable salt thereof. In some aspects, the oligonucleotide is selected from
the group
consisting of Antisense Oligo Nos. 290, 292, 305, 307, 313, 318, 323-324, 326,
329-330,
332, 338-339, 341, 344, or 346 of Table 3, or a pharmaceutically acceptable
salt thereof
In some aspects, the oligonucleotide is Antisense Oligo No. 289 of Table 3, or
a
pharmaceutically acceptable salt thereof.
[0126] In some aspects, the oligonucleotide, or a pharmaceutically
acceptable salt
thereof, described herein causes at least a 50% reduction in MSH3 mRNA
expression at
an oligonucleotide concentration of 10 nM In some aspects, the
oligonucleotide, or a
pharmaceutically acceptable salt thereof, described herein causes at least a
60% reduction
in MSH3 mRNA expression at an oligonucleotide concentration of 10 nM. In some
aspects, the oligonucleotide, or a pharmaceutically acceptable salt thereof,
described
herein causes at least a 70% reduction in MSH3 mRNA expression at an
oligonucleotide
concentration of 10 nM. In some aspects, the oligonucleotide, or a
pharmaceutically
acceptable salt thereof, described herein causes at least an 80% reduction in
MSH3
mRNA expression at an oligonucleotide concentration of 10 nM.
[0127] In some aspects, the oligonucleotide, or a pharmaceutically
acceptable salt
thereof, described herein causes at least a 50% reduction in MSH3 mRNA
expression at
an oligonucleotide concentration of 1 nM. In some aspects, the
oligonucleotide, or a
pharmaceutically acceptable salt thereof described herein causes at least a
60% reduction
in MSH3 mRNA expression at an oligonucleotide concentration of 1 nM. In some
aspects, the oligonucleotide, or a pharmaceutically acceptable salt thereof,
described
herein causes at least a 70% reduction in MSH3 mRNA expression at an
oligonucleotide
concentration of 1 nM.
[0128] The cell assay can comprise transfecting mammalian cells, such
as HEK293,
NIH3T3, or HeLa cells, with the desired a concentration of oligonucleotide
(e.g., 1 nM or
nM) using Lipofectamine 2000 (Invitrogen) and comparing MSH3 mRNA levels of
transfected cells to MSH3 levels of control cells. Control cells can be
transfected with
oligonucleotides not specific to MSH3 or mock transfected. mRNA levels can be
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
52
determined using RT-qPCR and MSH3 mRNA levels can be normalized to GAPDH
mRNA levels. The percent inhibition can be calculated as the percent of MSH3
mRNA
concentration relative to the MSH3 concentration of the control cells.
[0129] In some aspects, the MSH3 mRNA expression is evaluated in vitro.
In some
aspects, the MSH3 mRNA expression is evaluated in a cell based assay. In some
aspects,
the MSH3 mRNA expression is evaluated in HeLa cells. In some aspects, the MSH3
mRNA expression is determined by the quantitative reverse transcription
polymerase
chain reaction (RT-qPCR). In some aspects, the MSH3 mRNA is expression is
normalized to the mRNA expression of a reference gene. In some aspects, the
MSH3
mRNA expression is normalized to the mRNA expression of beta-glucuronidase
(GUSB).
In some aspects, the reduction in MSH3 mRNA expression is relative to a
control. In
some aspects, the control is the MSH3 mRNA expression in the absence of the
oligonucleotide, or pharmaceutically acceptable salt thereof. In some aspects,
the control
is the MSH3 mRNA expression in the absence of the oligonucleotide, or
pharmaceutically acceptable salt thereof, but in the presence of a control
oligonucleotide,
or salt thereof. In some aspects, the control oligonucleotide, or salt
thereof, is a scrambled
luciferase targeting oligonucleotide. In some aspects, the reduction in MSH3
mRNA
expression is calculated by a delta-delta Ct (AACT) method. In some aspects,
the delta-
delta Ct (AACT) method comprises the normalization of the MSH3 mRNA expression
to
the mRNA expression of a reference gene and to the MSH3 mRNA expression in the
absence of the oligonucleotide, or pharmaceutically acceptable salt thereof
but in the
presence of a control oligonucleotide, or salt thereof. In some aspects, the
reference gene
is beta-glucuronidase (GU SB) and/or the control oligonucleotide, or salt
thereof, is a
scrambled luciferase targeting oligonucleotide. In some aspects, the reduction
in MSH3
mRNA expression is determined by the method of Example 1 In some aspects, in
the
same assay, Antisense Oligo No. 1 causes approximately a 58% reduction in MSH3
mRNA expression at an oligonucleotide concentration of 10 nM. In some aspects,
in the
same assay, Antisense Oligo No. 1 causes approximately a 14% reduction in MSH3
mRNA expression at an oligonucleotide concentration of 1 nM.
[0130] In some aspects, the oligonucleotide, or contiguous nucleotide
region thereof, has
a gapmer design or structure also referred herein merely as "gapmer." In a
gapmer
structure the oligonucleotide comprises at least three distinct structural
regions a 5'-
flanking sequence (also known as a 5'-wing), a DNA core sequence (also known
as a
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
53
gap) and a 3'-flanking sequence (also known as a 3'-wing), in `5->3'
orientation. In this
design, the 5' and 3' flanking sequences comprise at least one alternative
nucleoside
which is adjacent to a DNA core sequence, and can, in some aspects, comprise a
contiguous stretch of 2-7 alternative nucleosides, or a contiguous stretch of
alternative
and DNA nucleosides (mixed flanking sequences comprising both alternative and
DNA
nucleosides).
101311 The length of the 5'- flanking sequence region can be at least
two nucleosides in
length (e.g., at least at least 2, at least 3, at least 4, at least 5, at
least 6, or more
nucleosides in length). The length of the 3'- flanking sequence region can be
at least two
nucleosides in length (e.g., at least 2, at least 3, at least at least 4, at
least 5, at least 6, or
more nucleosides in length). The 5' and 3' flanking sequences can be
symmetrical or
asymmetrical with respect to the number of nucleosides they comprise. In some
aspects,
the DNA core sequence comprises about 10 nucleosides flanked by a 5' and a 3'
flanking
sequence each comprising about 5 nucleosides. In some aspects, the DNA core
sequence
comprises about 11 nucleosides flanked by a 5' and a 3' flanking sequence each
comprising about 5 or about 6 nucleosides. In some aspects, the DNA core
sequence
comprises about 12 nucleosides flanked by a 5' sequence comprising about 5
nucleosides,
and a 3' flanking sequence comprising about 6 nucleosides. In some aspects,
the DNA
core sequence comprises about 12 nucleosides flanked by a 5' sequence
comprising about
6 nucleosides, and a 3' flanking sequence comprising about 5 nucleosides. In
some
aspects, the DNA core sequence comprises about 12 nucleosides flanked by a 5'
and a 3'
flanking sequence each comprising about 6 nucleosides.
101321 Consequently, the nucleosides of the 5' flanking sequence and
the 3' flanking
sequence which are adjacent to the DNA core sequence are alternative
nucleosides, such
as 2' alternative nucleosides. The DNA core sequence comprises a contiguous
stretch of
nucleotides which are capable of recruiting RNase H, when the oligonucleotide
is in
duplex with the MSH3 target nucleic acid. In some aspects, the DNA core
sequence
comprises a contiguous stretch of 5-16 DNA nucleosides. In other aspects, the
DNA core
sequence comprises a region of at least 10 contiguous nucleobases having at
least 80%
(e.g., at least 85%, at least 90%, at least 95%, or at least 99%)
complementarity to an
MSH3 gene. In some aspects, the gapmer comprises a region complementary to at
least
17 contiguous nucleotides, 19-23 contiguous nucleotides, or 19 contiguous
nucleotides of
a MSH3 gene. The gapmer is complementary to the MSH3 target nucleic acid, and
can
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
54
therefore be the contiguous nucleoside region of the oligonucleotide. In some
aspects, the
gapmer comprises a region complementary to at least 21 contiguous nucleotides,
20-25
contiguous nucleotides, or 23 contiguous nucleotides of a MSH3 gene. The
gapmer is
complementary to the MSH3 target nucleic acid, and can therefore be the
contiguous
nucleoside region of the oligonucleotide.
[0133] The 5' and 3' flanking sequences, flanking the 5' and 3' ends of
the DNA core
sequence, can comprise one or more affinity enhancing alternative nucleosides.
In some
aspects, the 5' and/or 3' flanking sequence comprises at least one 2'-0-
methoxyethyl
(MOE) nucleoside. In some aspects, the 5' and/or 3' flanking sequences,
contain at least
two MOE nucleosides. In some aspects, the 5' flanking sequence comprises at
least one,
at least two, at least three, at least four, at least five, or at least six or
more MOE
nucleosides. In some aspects, the 5' flanking sequence comprises at least one,
at least
two, at least three, at least four, at least five, or at least six or more MOE
nucleosides. In
some aspects, both the 5' and 3' flanking sequence comprise a MOE nucleoside.
In some
aspects, all the nucleosides in the flanking sequences are MOE nucleosides. In
other
aspects, the flanking sequence can comprise both MOE nucleosides and other
nucleosides
(mixed flanking sequence), such as DNA nucleosides and/or non-MOE alternative
nucleosides, such as bicyclic nucleosides (BNAs) (e.g., LNA nucleosides (e.g.,
A-LNA,
5mC-LNA, G-LNA, and T-LNA) or cET nucleosides), or other 2' substituted
nucleosides. In this case the DNA core sequence is defined as a contiguous
sequence of
at least 5 RNase H recruiting nucleosides (such as 5-16 DNA nucleosides)
flanked at the
5' and 3' end by an affinity enhancing alternative nucleoside, such as an MOE
nucleoside.
[0134] In other aspects, the 5' and/ or 3' flanking sequence comprises
at least one BNA
(e.g., at least one LNA nucleoside (e.g., A-LNA, 5mC-LNA, G-LNA, and T-LNA) or
cET nucleoside). In some aspects, 5' and/ or 3' flanking sequence comprises at
least 2
bicyclic nucleosides. In some aspects, the 5' flanking sequence comprises at
least one
BNA. In some aspects, both the 5' and 3' flanking sequence comprise a BNA. In
some
aspects, all the nucleosides in the flanking sequences are BNAs. In other
aspects, the
flanking sequence can comprise both BNAs and other nucleosides (mixed flanking
sequences), such as DNA nucleosides and/or non-BNA alternative nucleosides,
such as 2'
substituted nucleosides. In this case the DNA core sequence is defined as a
contiguous
sequence of at least five RNase H recruiting nucleosides (such as 5-16 DNA
nucleosides)
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
flanked at the 5' and 3' end by an affinity enhancing alternative nucleoside,
such as a
BNA, such as an LNA, such as beta-D-oxy-LNA.
[0135] The 5' flank attached to the 5' end of the DNA core sequence
comprises, contains,
or consists of at least one alternative sugar moiety (e.g., at least three, at
least four, at least
five, at least six, at least seven, or more alternative sugar moieties). In
some aspects, the
flanking sequence comprises or consists of from 1 to 7 alternative
nucleobases, such as
from 2 to 6 alternative nucleobases, such as from 2 to 5 alternative
nucleobases, such as
from 2 to 4 alternative nucleobases, such as from 1 to 3 alternative
nucleobases, such as
one, two, three or four alternative nucleobases. In some aspects, the flanking
sequence
comprises or consists of at least one alternative internucleoside linkage
(e.g., at least
three, at least four, at least five, at least six, at least seven, or more
alternative
internucleoside linkages).
[0136] The 3' flank attached to the 3' end of the DNA core sequence
comprises, contains,
or consists of at least one alternative sugar moiety (e.g., at least three, at
least four, at least
five, at least six, at least seven, or more alternative sugar moieties). In
some aspects, the
flanking sequence comprises or consists of from 1 to 7 alternative
nucleobases, such as
from 2 to 6 alternative nucleobases, such as from 2 to 5 alternative
nucleobases, such as
from 2 to 4 alternative nucleobases, such as from 1 to 3 alternative
nucleobases, such as
one, two, three, or four alternative nucleobases. In some aspects, the
flanking sequence
comprises or consists of at least one alternative internucleoside linkage
(e.g., at least
three, at least four, at least five, at least six, at least seven, or more
alternative
internucleoside linkages).
[0137] In an aspect, one or more or all of the alternative sugar
moieties in the flanking
sequence are 2' alternative sugar moieties_
[0138] In a further aspect, one or more of the 2' alternative sugar
moieties in the wing
regions are selected from 2'-0-alkyl-sugar moieties, 2'-0-methyl-sugar
moieties, 2'-
amino-sugar moieties, 2'-fluoro-sugar moieties, 2'-alkoxy-sugar moieties, MOE
sugar
moieties, LNA sugar moieties, arabino nucleic acid (ANA) sugar moieties, and
2'-fluoro-
ANA sugar moieties.
[0139] In one aspect, all the alternative nucleosides in the flanking
sequences are bicyclic
nucleosides. In a further aspect, the bicyclic nucleosides in the flanking
sequences are
independently selected from the group consisting of oxy-LNA, thio-LNA, amino-
LNA,
cET, and/or LNA, in either the beta-I) or alpha-L configurations or
combinations thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
56
[0140] In some aspects, the one or more alternative internucleoside
linkages in the
flanking sequences are phosphorothioate internucleoside linkages. In some
aspects, the
phosphorothioate linkages are stereochemically pure phosphorothioate linkages.
In some
aspects, the phosphorothioate linkages are Sp phosphorothioate linkages. In
other
aspects, the phosphorothioate linkages are Rp phosphorothioate linkages. In
some
aspects, the alternative internucleoside linkages are 2'-alkoxy
internucleoside linkages.
In other aspects, the alternative internucleoside linkages are alkyl phosphate
internucleoside linkages.
[0141] The DNA core sequence can comprise, contain, or consist of at
least 5-16
consecutive DNA nucleosides capable of recruiting RNase H. In some aspects,
all of the
nucleosides of the DNA core sequence are DNA units. In further aspects, the
DNA core
region can consist of a mixture of DNA and other nucleosides capable of
mediating
RNase H cleavage. In some aspects, at least 50% of the nucleosides of the DNA
core
sequence are DNA, such as at least 60%, at least 70% or at least 80%, or at
least 90%
DNA. In some aspects, all of the nucleosides of the DNA core sequence are RNA
units.
[0142] The oligonucleotide comprises a contiguous region which is
complementary to the
target nucleic acid. In some aspects, the oligonucleotide can further comprise
additional
linked nucleosides positioned 5' and/or 3' to either the 5' and 3' flanking
sequences.
These additional linked nucleosides can be attached to the 5' end of the 5'
flanking
sequence or the 3' end of the 3' flanking sequence, respectively. The
additional
nucleosides can, in some aspects, form part of the contiguous sequence which
is
complementary to the target nucleic acid, or in other aspects, can be non-
complementary
to the target nucleic acid.
[0143] The inclusion of the additional nucleosides at either, or both
of the 5' and 3'
flanking sequences can independently comprise one, two, three, four, or five
additional
nucleotides, which can be complementary or non-complementary to the target
nucleic
acid. In this respect the oligonucleotide, can in some aspects comprise a
contiguous
sequence capable of modulating the target which is flanked at the 5' and/or 3'
end by
additional nucleotides. Such additional nucleosides can serve as a nuclease
susceptible
biocleavable linker, and can therefore be used to attach a functional group
such as a
conjugate moiety to the oligonucleotide. In some aspects, the additional 5'
and/or 3' end
nucleosides are linked with phosphodiester linkages, and can be DNA or RNA. In
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
57
another aspect, the additional 5' and/or 3' end nucleosides are alternative
nucleosides
which can for example be included to enhance nuclease stability or for ease of
synthesis.
[0144] In other aspects, the oligonucleotides utilize "altimer" design
and comprise
alternating 2'-fluoro-ANA and DNA regions that are alternated every three
nucleosides.
Altimer oligonucleotides are discussed in more detail in Min, et al.,
Bioorganic &
Medicinal Chemistry Letters, 2002, 12(18): 2651-2654 and Kalota, et al., Nuc.
Acid Res.
2006, 34(2): 451-61 (herein incorporated by reference).
[0145] In other aspects, the oligonucleotides utilize "hemimer" design
and comprise a
single 2'-modified flanking sequence adjacent to (on either side of the 5' or
the 3' side of)
a DNA core sequence. Hemimer oligonucleotides are discussed in more detail in
Geary
et al., 2001, J. Pharm. Exp. Therap., 296: 898-904 (herein incorporated by
reference).
[0146] In some aspects, an oligonucleotide has a nucleic acid sequence
with at least 50%
(e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%,
at least 90%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%)
sequence
identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-384 and 390-
613. In
some aspects, an oligonucleotide has a nucleic acid sequence with at least 85%
sequence
identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-384 and 390-
613.
[0147] It will be understood that the nucleosides of the
oligonucleotide e.g., an
oligonucleotide, can comprise any one of the sequences set forth in any one of
SEQ ID
NOs: 1-384 that is an alternative nucleoside and/or conjugated or linked as
described in
detail below.
[0148] In some aspects, the oligonucleotide is an oligonucleotide
having at least 15
contiguous bases of the nucleobase sequence selected from the group consisting
of
Antisense Oligo Nos_ 1-384 of Table 3 or 390-613 of Table 4 In some aspects,
the
oligonucleotide is an oligonucleotide having at least 15 contiguous bases of
the
nucleobase sequence selected from the group consisting of Antisense Oligo Nos.
1-384 of
Table 3. In some aspects, the oligonucleotide is an oligonucleotide having at
least 15
contiguous bases of the nucleobase sequence selected from the group consisting
one of
Antisense Oligo Nos. 1-96 of Table 3. In some aspects, the oligonucleotide is
an
oligonucleotide having at least 15 contiguous bases of the nucleobase sequence
selected
from the group consisting of Antisense Oligo Nos. 97-192 of Table 3. In some
aspects,
the oligonucleotide is an oligonucleotide having at least 15 contiguous bases
of the
nucleobase sequence selected from the group consisting of Antisense Oligo Nos.
193-
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
58
288 of Table 3. In some aspects, the oligonucleotide is an oligonucleotide
having at least
15 contiguous bases of the nucleobase sequence selected from the group
consisting of
Antisense Oligo Nos. 289-384 of Table 3. In some aspects, the oligonucleotide
is an
oligonucleotide having at least 15 contiguous bases of the nucleobase sequence
selected
from the group consisting of Antisense Oligo Nos. 390-613 of Table 4. In some
aspects,
the oligonucleotide is an oligonucleotide having at least 15 contiguous bases
of the
nucleobase sequence selected from the group consisting of Antisense Oligo Nos.
390-
480 of Table 4. In some aspects, the oligonucleotide is an oligonucleotide
having at least
15 contiguous bases of the nucleobase sequence selected from the group
consisting of
Antisense Oligo Nos. 481-501 of Table 4. In some aspects, the oligonucleotide
is an
oligonucleotide having at least 15 contiguous bases of the nucleobase sequence
selected
from the group consisting of Antisense Oligo Nos. 502-592 of Table 4. In some
aspects,
the oligonucleotide is an oligonucleotide having at least 15 contiguous bases
of the
nucleobase sequence selected from the group consisting of Antisense Oligo Nos.
593-
613 of Table 4.
[0149] In some aspects, the oligonucleotide is an oligonucleotide
having at least 15
contiguous bases of the nucleobase sequence selected from the group consisting
of
Antisense Oligo Nos. 1, 6, 13, 17, 21, 24, 26, 29, 33-34, 37, 44, 49-55, 57,
60-73, 75-76,
79-82, 84-86, 88-92, or 94-96 of Table 3. In some aspects, the oligonucleotide
an
oligonucleotide having at least 15 contiguous bases of the nucleobase sequence
of
Antisense Oligo No. 6 of Table 3.
[0150] In some aspects, the oligonucleotide is an oligonucleotide
having at least 15
contiguous bases of the nucleobase sequence selected from the group consisting
of
Antisense Oligo Nos_ 97, 100, 103, 105, 108, 110-111, 113-117, 122-123, 127,
129-130,
133-136, 138-139, 141, 143-145, 147-148, 154-155, 157-165, 168-170, 172, 174-
180,
184, 187, or 191 of Table 3.
[0151] In some aspects, the oligonucleotide is an oligonucleotide
having at least 15
contiguous bases of the nucleobase sequence selected from the group consisting
of
Antisense Oligo Nos. 193-200, 202-230, 232-246, 248-253, 255, 258-261, 265,
270, 274-
276, or 285-286 of Table 3. In some aspects, the oligonucleotide is an
oligonucleotide
having at least 15 contiguous bases of the nucleobase sequence selected from
the group
consisting of Antisense Oligo Nos. 226-227, 234, 240, or 243-244 of Table 3.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
59
[0152] In some aspects, the oligonucleotide is an oligonucleotide
having at least 15
contiguous bases of the nucleobase sequence selected from the group consisting
of
Antisense Oligo Nos. 289-290, 292, 305, 307, 313, 318, 323-324, 326, 329-330,
332,
338-339, 341, 344, or 346 of Table 3.
[0153] An oligonucleotide agent as described herein can contain one or
more mismatches
to the target sequence. In one aspect, an oligonucleotide as described herein
contains no
more than 3 mismatches. If the oligonucleotide contains mismatches to a target
sequence,
in some aspects, the area of mismatch is not located in the center of the
region of
complementarity. If the oligonucleotide contains mismatches to the target
sequence, in
some aspects, the mismatch should be restricted to be within the last 5
nucleotides from
either the 5'- or 3'-end of the region of complementarity. For example, for a
30-linked
nucleoside oligonucleotide agent, the contiguous nucleobase region which is
complementary to a region of a MSH3 gene, generally does not contain any
mismatch
within the central 5-10 linked nucleosides. The methods described herein or
methods
known in the art can be used to determine whether an oligonucleotide
containing a
mismatch to a target sequence is effective in inhibiting the expression of a
MSH3 gene.
Consideration of the efficacy of oligonucleotides with mismatches in
inhibiting
expression of a MSH3 gene is important, especially if the particular region of
complementarity in a MSH3 gene is known to have polymorphic sequence variation
within the population.
[0154] Construction of vectors for expression of polynucleotides can be
accomplished
using conventional techniques which do not require detailed explanation to one
of
ordinary skill in the art. For generation of efficient expression vectors, it
is necessary to
have regulatory sequences that control the expression of the polynucleotide
These
regulatory sequences include promoter and enhancer sequences and are
influenced by
specific cellular factors that interact with these sequences, and are well
known in the art.
A. Alternative Oligonucleosides
[0155] In one aspect, one or more of the linked nucleosides or
internucleosidic linkages
of the oligonucleotide, is naturally occurring, and does not comprise, e.g.,
chemical
modifications and/or conjugations known in the art and described herein. In
another
aspect, one or more of the linked nucleosides or internucleosidic linkages of
an
oligonucleotide, is chemically modified to enhance stability or other
beneficial
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
characteristics. Without being bound by theory, it is believed that certain
modifications
can increase nuclease resistance and/or serum stability, or decrease
immunogenicity. For
example, oligonucleotides can contain nucleotides found to occur naturally in
DNA or
RNA (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine) or
can contain
alternative nucleosides or intemucleosidic linkages which have one or more
chemical
modifications to one or more components of the nucleotide (e.g., the
nucleobase, sugar, or
phospho-linker moiety). Oligonucleotides can be linked to one another through
naturally
occurring phosphodiester bonds, or can contain alternative linkages (e.g.,
covalently
linked through phosphorothioate (e.g., Sp phosphorothioate or Rp
phosphorothioate), 3 '-
methylenephosphonate, 5' -methylenephosphonate, 3 '-phosphoamidate, 2'-5'
phosphodiester, guanidinium, S-methylthiourea, 2'-alkoxy, alkyl phosphate, or
peptide
bonds).
[0156] In some aspects, substantially all of the nucleosides or
internucleosi di c linkages of'
an oligonucleotide are alternative nucleosides. In other aspects, all of the
nucleosides or
internucleosidic linkages of an oligonucleotide are alternative nucleosides.
Oligonucleotides in which "substantially all of the nucleosides are
alternative
nucleosides" are largely but not wholly modified and can include not more than
five, four,
three, two, or one naturally-occurring nucleosides. In still other aspects,
oligonucleotides
can include not more than five, four, three, two, or one alternative
nucleosides.
[0157] The nucleic acids can be synthesized and/or modified by methods
well established
in the art, such as those described in "Current protocols in nucleic acid
chemistry,"
Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA,
which is
hereby incorporated herein by reference. Alternative nucleotides and
nucleosides include
those with modifications including, for example, end modifications, e.g., 5'-
end
modifications (phosphorylation, conjugation, inverted linkages) or 3'-end
modifications
(conjugation, DNA nucleotides, inverted linkages, etc.); base modifications,
e.g.,
replacement with stabilizing bases, destabilizing bases, or bases that base
pair with an
expanded repertoire of partners, removal of bases (abasic nucleotides), or
conjugated
bases; sugar modifications (e.g., at the T-position or 4'-position) or
replacement of the
sugar; and/or backbone modifications, including modification or replacement of
the
phosphodiester linkages. The nucleobase can be an isonucleoside in which the
nucleobase is moved from the Cl position of the sugar moiety to a different
position (e.g.
C2, C3, C4, or C5). Specific examples of oligonucleotide compounds useful in
the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
61
aspects described herein include, but are not limited to alternative
nucleosides containing
modified backbones or no natural internucleoside linkages. Nucleotides and
nucleosides
having modified backbones include, among others, those that do not have a
phosphorus
atom in the backbone. For the purposes of this specification, and as sometimes
referenced in the art, alternative RNAs that do not have a phosphorus atom in
their
internucleoside backbone can be considered to be oligonucleosides. In some
aspects, an
oligonucleotide will have a phosphorus atom in its internucleoside backbone.
[0158] Alternative internucleoside linkages include, for example,
phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-
alkylene
phosphonates and chiral phosphonates, phosphinates, phosphoramidates including
3'-
amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and boronophosphates
having
normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having
inverted polarity
wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-
5 to 5'-2'.
Various salts, mixed salts, and free acid forms are also included.
[0159] Representative U.S. patents that teach the preparation of the
above phosphorus-
containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808;
4,469,863;
4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717;
5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;
5,519,126;
5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050;
6,028,188;
6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199;
6,346,614;
6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;
6,878,805;
7,015,315; 7,041,816; 7,273,933; 7,321,029; and US. Pat RE39464, the entire
contents
of each of which are hereby incorporated herein by reference.
[0160] Alternative internucleoside linkages that do not include a
phosphorus atom therein
have backbones that are formed by short chain alkyl or cycloalkyl
internucleoside
linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages,
or one or
more short chain heteroatomic or heterocyclic internucleoside linkages. These
include
those having morpholino linkages (formed in part from the sugar portion of a
nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones;
alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
62
backbones; sulfonate and sulfonamide backbones; amide backbones; and others
having
mixed N, 0, S, and CH2 component parts.
[0161] Representative U.S. patents that teach the preparation of the
above
oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506;
5,166,315;
5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938;
5,434,257;
5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,608,046;
5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,
5,677,439, the
entire contents of each of which are hereby incorporated herein by reference.
[0162] In other aspects, suitable oligonucleotides include those in
which both the sugar
and the internucleoside linkage, i.e., the backbone, of the nucleotide units
are replaced.
The base units are maintained for hybridization with an appropriate nucleic
acid target
compound. One such oligomeric compound, a mimetic that has been shown to have
excellent hybridization properties, is referred to as a peptide nucleic acid
(PNA). In PNA
compounds, the sugar of a nucleoside is replaced with an amide containing
backbone, in
particular an aminoethylglycine backbone. The nucleobases are retained and are
bound
directly or indirectly to aza nitrogen atoms of the amide portion of the
backbone.
Representative U.S. patents that teach the preparation of PNA compounds
include, but are
not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire
contents of
each of which are hereby incorporated herein by reference. Additional PNA
compounds
suitable for use in the oligonucleotides are described in, for example, in
Nielsen et al.,
Science, 1991, 254, 1497-1500.
[0163] Some aspects include oligonucleotides with phosphorothioate
backbones and
oligonucleotides with heteroatom backbones, and in particular -CH2-NH-CH2-, -
CH2-
N(CH3)-0-CH2-[known as a methylene (methylimino) or MMI backbone], -CH2-0-
N(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2-[wherein the native
phosphodiester backbone is represented as -0-P-O-CH2-] of the above-referenced
U.S.
Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat.
No.
5,602,240. In some aspects, the oligonucleotides featured herein have
morpholino
backbone structures of the above-referenced U.S. Pat. No. 5,034,506. In other
aspects,
the oligonucleotides described herein include phosphorodiamidate morpholino
oligomers
(PMO), in which the deoxyribose moiety is replaced by a morpholine ring, and
the
charged phosphodiester inter-subunit linkage is replaced by an uncharged
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
63
phophorodiamidate linkage, as described in Summerton, et al., Antisense
Nucleic Acid
Drug Dev. 1997, 7:63-70.
[0164] Alternative nucleosides and nucleotides can contain one or more
substituted sugar
moieties. The oligonucleotides, e.g., oligonucleotides, featured herein can
include one of
the following at the 2I-position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-
alkenyl; 0-, S- or
N-alkynyl; or 0-alkyl-ID-alkyl, wherein the alkyl, alkenyl and alkynyl can be
substituted
or unsubstituted Ci to Co alkyl or C2 to C to alkenyl and alkynyl. Exemplary
suitable
modifications include -0[(CH2)nO]mCH3, -0(CH2)nOCH3, -0(CH2)n-NH2, -
0(CH2)nCH3,
-0(CH2)n-ONH2, and -0(CH2)n-ON[(CH2)DCH3]2, where n and m are from 1 to about
10.
In other aspects, oligonucleotides include one of the following at the 2'
position: Ci to Cio
lower alkyl, substituted lower alkyl, alkaryl, aralkyl, 0-alkaryl or 0-
aralkyl, SH, SCH3,
OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, 0NO2, NO2, N3, NI-12,
heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkyl amino, substituted silyl, an
RNA cleaving
group, a reporter group, an intercalator, a group for improving the
pharmacokinetic
properties of an oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having similar
properties. In
some aspects, the modification includes a 2'-methoxyethoxy (2'-0-CH2CH2OCH3,
also
known as 2'-0-(2-methoxyethyl) or 21-M0E) (Martin et al., Hely. Chin. Acta,
1995,
78:486-504) i.e., an alkoxy-alkoxy group. MOE nucleosides confer several
beneficial
properties to oligonucleotides including, but not limited to, increased
nuclease resistance,
improved pharmacokinetics properties, reduced non-specific protein binding,
reduced
toxicity, reduced immunostimulatory properties, and enhanced target affinity
as compared
to unmodified oligonucleotides.
[0165] Another exemplary alternative contains 2'-
dimethylaminooxyethoxy, i.e., a -
0(CH2)20N(CH3)2 group, also known as 2'-DMA0E, as described in examples herein
below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-
dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0-(CH2)2-0-(CH2)2-N(CH3)2.
Further exemplary alternatives include: 5'-Me-2'-F nucleotides, 51-Me-2'-0Me
nucleotides, 5'-Me-2'-deoxynucleotides, (both R and S isomers in these three
families); 2'-
alkoxyalkyl; and 2'-N1VIA (N-methylacetamide).
[0166] Other alternatives include 2'-methoxy (2'-OCH3), 2'-aminopropoxy
(2'-
OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can be made at
other
positions on the nucleosides and nucleotides of an oligonucleotide,
particularly the 3'
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
64
position of the sugar on the 3' terminal nucleotide or in 2'-5' linked
oligonucleotides and
the 5 position of 5' terminal nucleotide. Oligonucleotides can have sugar
mimetics such
as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative
U.S. patents
that teach the preparation of such modified sugar structures include, but are
not limited to,
U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;
5,446,137;
5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;
5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain
of which
are commonly owned with the instant application. The entire contents of each
of the
foregoing are hereby incorporated herein by reference.
[0167] An oligonucleotide can include nucleobase (often referred to in
the art simply as
"base") alternatives (e.g., modifications or substitutions). Unmodified or
natural
nucleobases include the purine bases adenine (A) and guanine (G), and the
pyrimidine
bases thymine (T), cytosine (C) and uracil (U). Alternative nucleobases
include other
synthetic and natural nucleobases such as 5-methylcytidine, 5-
hydroxymethylcytidine, 5-
formylcytidine, 5-carboxycytidine, pyrrolocytidine, dideoxycytidine, uridine,
5-
methoxyuridine, 5-hydroxydeoxyuridine, dihydrouridine, 4-thiourdine,
pseudouridine, 1-
methyl-pseudouridine, deoxyuridine, 5-hydroxybutyn1-2'-deoxyuridine, xanthine,
hypoxanthine, 7-deaza-xanthine, thienoguanine, 8-aza-7-deazaguanosine, 7-
methylguanosine, 7-deazaguanosine, 6-aminomethy1-7-deazaguanosine, 8-
aminoguanine,
2,2,7-trimethylguanosine, 8-methyladenine, 8-azidoadenine, 7-methyladenine, 7-
deazaadenine, 3-deazaadenine, 2,6-diaminopurine, 2-aminopurine, 7-deaza-8-aza-
adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 2-
aminoadenine, 6-
methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl
derivatives of adenine and guanine, 2-thiouridine, 2-thiothymine and 2-
thiocytosine, 5-
halouracil and cytosine, 5-propynyl uridine and cytidine, 6-azo uridine,
cytidine and
thymine, 4-thiouridine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal
other 8-
substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-
trifluoromethyl and
other 5-substituted uridines and cytidines, 8-azaguanine and 8-azaadenine, and
3-
deazaguanine. Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808,
those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and
Medicine,
Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia
Of
Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John
Wiley &
Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie,
International
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, Antisense
Research
and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press,
1993.
Certain of these nucleobases are particularly useful for increasing the
binding affinity of
the oligonucleotide. These include 5-substituted pyrimidines, 6-
azapyrimidines, and N-2,
N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-
propynyluracil, and
5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase
nucleic
acid duplex stability by 0.6-1.2 C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu,
B., Eds.,
Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278)
and
are exemplary base substitutions, even more particularly when combined with 2'-
0-
methoxyethyl sugar modifications.
[0168] Representative U.S. patents that teach the preparation of
certain of the above
noted alternative nucleobases as well as other alternative nucleobases
include, but are not
limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30;
5,134,066;
5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;
5,525,711;
5,552,540, 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692;
6,015,886,
6,147,200, 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;
6,617,438,
7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are
hereby
incorporated herein by reference.
[0169] In other aspects, the sugar moiety in the nucleotide can be a
ribose molecule,
optionally having a 2'-0-methyl, 2'-0-M0E, 2'-F, 2' -amino, 2' -0-propyl, 2'-
aminopropyl, or 2'-OH modification.
[0170] An oligonucleotide can include one or more bicyclic sugar
moieties. A "bicyclic
sugar" is a furanosyl ring modified by the bridging of two atoms. A "bicyclic
nucleoside"
("BNA") is a nucleoside having a sugar moiety comprising a bridge connecting
two
carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In
some aspects,
the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus,
in some
aspects, an oligonucleotide can include one or more locked nucleosides. A
locked
nucleoside is a nucleoside having a modified ribose moiety in which the ribose
moiety
comprises an extra bridge connecting the 2' and 4' carbons. In other words, a
locked
nucleoside is a nucleoside comprising a bicyclic sugar moiety comprising a 4'-
CH2-0-2'
bridge. This structure effectively "locks" the ribose in the 3'-endo
structural
conformation. The addition of locked nucleosides to oligonucleotides has been
shown to
increase oligonucleotide stability in serum, and to reduce off-target effects
(Grunweller,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
66
A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of
bicyclic
nucleosides for use in the polynucleoti des include without limitation
nucleosides
comprising a bridge between the 4' and the 2' ribosyl ring atoms. In some
aspects, the
polynucleotide agents include one or more bicyclic nucleosides comprising a 4'
to 2'
bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but
are not limited
to 4'-(CH2)-0-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-0-2' (ENA); 4'-CH(CH3)-0-2'
(also
referred to as "constrained ethyl" or "cEt") and 4'CH(CH2OCH3)-0-2' (and
analogs
thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-0-2' (and analogs
thereof;
see e.g., U.S. Pat. No. 8,278,283); 4'CH2-N(OCH3)-2' (and analogs thereof; see
e.g., U.S.
Pat. No. 8,278,425); 4'-CH2-0-N(CH.3)2-2' (see, e.g., U.S. Patent Publication
No.
2004/0171570); 4'-CH2-N(R)-0-2', wherein R is H, Ci-C12 alkyl, or a protecting
group
(see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g.,
Chattopadhyaya et
al., J. Org. Chem., 2009, 74, 118-134); and 4'CH2-C(=CH2)-2' (and analogs
thereof; see,
e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing
are hereby
incorporated herein by reference.
[0171] Additional representative U.S. Patents and US Patent
Publications that teach the
preparation of locked nucleic acid nucleotides include, but are not limited
to, the
following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748;
6,794,499;
6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686;
7,741,457;
8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US
2009/0012281, the entire contents of each of which are hereby incorporated
herein by
reference.
[0172] Any of the foregoing bicyclic nucleosides can be prepared having
one or more
stereochemical sugar configurations including for example a-L-ribofuranose and
(3-D-
ribofuranose (see WO 99/14226).
[0173] An oligonucleotide can be modified to include one or more
constrained ethyl
nucleosides. As used herein, a "constrained ethyl nucleoside" or "cEt" is a
locked
nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2'
bridge. In
one aspect, a constrained ethyl nucleoside is in the S conformation referred
to herein as
"S-cEt."
[0174] An oligonucleotide can include one or more "conformationally
restricted
nucleosides" ("CRN"). CRN are nucleoside analogs with a linker connecting the
C2' and
C4' carbons of ribose or the C3 and --05' carbons of ribose. CRN lock the
ribose ring
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
67
into a stable conformation and increase the hybridization affinity to mRNA.
The linker is
of sufficient length to place the oxygen in an optimal position for stability
and affinity
resulting in less ribose ring puckering.
[0175] Representative publications that teach the preparation of
certain of the above
noted CRN include, but are not limited to, US Patent Publication No.
2013/0190383; and
PCT publication WO 2013/036868, the entire contents of each of which are
hereby
incorporated herein by reference.
[0176] In some aspects, an oligonucleotide comprises one or more
monomers that are
UNA (unlocked nucleoside) nucleosides. UNA is unlocked acyclic nucleoside,
wherein
any of the bonds of the sugar has been removed, forming an unlocked "sugar"
residue. In
one example, UNA also encompasses monomer with bonds between CI'-C4' have been
removed (i.e. the covalent carbon-oxygen-carbon bond between the Cl' and C4'
carbons).
In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond
between the
C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp.
Series, 52,
133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby
incorporated by
reference).
[0177] Representative U.S. publications that teach the preparation of
UNA include, but
are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos.
2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of
which
are hereby incorporated herein by reference.
[0178] The ribose molecule can be modified with a cyclopropane ring to
produce a
tricyclodeoxynucleic acid (tricyclo DNA). The ribose moiety can be substituted
for
another sugar such as 1,5,-anhydrohexitol, threose to produce a threose
nucleoside
(TNA), or arabinose to produce an arabino nucleoside_ The ribose molecule can
be
replaced with non-sugars such as cyclohexene to produce cyclohexene nucleoside
or
glycol to produce glycol nucleosides.
[0179] Potentially stabilizing modifications to the ends of nucleoside
molecules can
include N-(acetylaminocaproy1)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproy1-4-
hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-
0-
deoxythymidine (ether), N-(aminocaproy1)-4-hydroxyprolinol (Hyp-C6-amino), 2-
docosanoyl-uridine-3"-phosphate, inverted base dT(idT) and others. Disclosure
of this
modification can be found in PCT Publication No. WO 2011/005861.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
68
[0180] Other alternatives chemistries of an oligonucleotide include a
5' phosphate or 5'
phosphate mimic, e.g., a 5'-terminal phosphate or phosphate mimic of an
oligonucleotide.
Suitable phosphate mimics are disclosed in, for example US Patent Publication
No.
2012/0157511, the entire contents of which are incorporated herein by
reference.
[0181] Exemplary oligonucleotides comprise nucleosides with alternative
sugar moieties
and can comprise DNA or RNA nucleosides. In some aspects, the oligonucleotide
comprises nucleosides comprising alternative sugar moieties and DNA
nucleosides.
Incorporation of alternative nucleosides into the oligonucleotide can enhance
the affinity
of the oligonucleotide for the target nucleic acid. In that case, the
alternative nucleosides
can be referred to as affinity enhancing alternative nucleotides.
[0182] In some aspects, the oligonucleotide comprises at least 1
alternative nucleoside,
such as at least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9,
at least 10, at least 11, at least 12, at least 13, at least 14, at least 15
or at least 16
alternative nucleosides. In other aspects, the oligonucleotides comprise from
1 to 10
alternative nucleosides, such as from 2 to 9 alternative nucleosides, such as
from 3 to 8
alternative nucleosides, such as from 4 to 7 alternative nucleosides, such as
6 or 7
alternative nucleosides. In an aspect, the oligonucleotide can comprise
alternatives,
which are independently selected from these three types of alternatives
(alternative sugar
moiety, alternative nucleobase, and alternative internucleoside linkage), or a
combination
thereof. In one aspect, the oligonucleotide comprises one or more nucleosides
comprising
alternative sugar moieties, e.g., 2' sugar alternative nucleosides. In some
aspect, the
oligonucleotide comprises the one or more 2' sugar alternative nucleoside
independently
selected from the group consisting of 2'-0-alkyl-RNA, 2'-0-methyl-RNA, 2'-
alkoxy-
RNA, 2'-0-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabino nucleic acid
(ANA), 2'-fluoro-ANA, and BNA (e.g., LNA (e.g., A-LNA, 5mC-LNA, G-LNA, and T-
LNA)) nucleosides. Exemplary structures of the LNAs are as follows (wherein
the
protecting groups are removed.
CA 03180981 2022- 12- 1
WO 2021/247020 PCT/US2020/035972
69
= N
r-,
CO:ft.1 Milsanw.
i i)s;t= tura¨
.
le"
tr======(
[0183] In some aspects, the one or more alternative nucleoside
is a BNA
[0184] In some aspects, at least 1 of the alternative nucleosides is a
BNA (e.g., an LNA
(e.g., A-LNA, 5mC-LNA, G-LNA, and T-LNA)), such as at least 2, such as at
least 3, at
least 4, at least 5, at least 6, at least 7, or at least 8 of the alternative
nucleosides are
BNAs. In a still further aspect, all the alternative nucleosides are BNAs.
[0185] In a further aspect, the oligonucleotide comprises at
least one alternative
internucleoside linkage. In some aspects, the internucleoside linkages within
the
contiguous nucleotide sequence are phosphorothioate or boronophosphate
internucleoside
linkages. In some aspects, all the internucleotide linkages in the contiguous
sequence of
the oligonucleotide are phosphorothioate linkages. In some aspects, the
phosphorothioate
linkages are stereochemically pure phosphorothioate linkages. In some aspects,
the
phosphorothioate linkages are Sp phosphorothioate linkages. In other aspects,
the
phosphorothioate linkages are Rp phosphorothioate linkages.
[0186] In some aspects, the oligonucleotide comprises at least one
alternative nucleoside
which is a 2'-M0E-RNA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-M0E-RNA
nucleoside
units. In some aspects, the 2'-M0E-RNA nucleoside units are connected by
phosphorothioate linkages. In some aspects, at least one of said alternative
nucleoside is
2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro-DNA nucleoside
units. In
some aspects, the oligonucleotide comprises at least one BNA unit and at least
one 2'
substituted modified nucleoside. In some aspects, the oligonucleotide
comprises both 2'
sugar modified nucleosides and DNA units. In some aspects, the oligonucleotide
or
contiguous nucleotide region thereof is a gapmer oligonucleotide.
B. Oligonucleotides Conjugated to Ligands
[0187] Oligonucleotides can be chemically linked to one or more
ligands, moieties, or
conjugates that enhance the activity, cellular distribution, or cellular
uptake of the
oligonucleotide. Such moieties include but are not limited to lipid moieties
such as a
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86:
6553-6556),
cholic acid (Manoharan et al., (1994) Biorg. Med. Chem, Let., 4:1053-1060), a
thioether,
e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci.,
660:306-309;
Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a
thiocholesterol
(Oberhauser etal., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain,
e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) ElVIBO J,
10:1111-
1118; Kabanov etal., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993)
Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethyl-
ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al.,
(1995)
Tetrahedron Lett., 36:3651-3654; Shea etal., (1990) Nucl. Acids Res., 18:3777-
3783), a
polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides
&
Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995)
Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995)
Biochim.
Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-
carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther.,
277:923-
937).
[0188] In one aspect, a ligand alters the distribution, targeting, or
lifetime of an
oligonucleotide agent into which it is incorporated. In some aspects, a ligand
provides an
enhanced affinity for a selected target, e.g., molecule, cell or cell type,
compartment, e.g.,
a cellular or organ compartment, tissue, organ, or region of the body, as,
e.g., compared to
a species absent such a ligand.
[0189] Ligands can include a naturally occurring substance, such as a
protein (e.g.,
human serum albumin (HSA), low-density lipoprotein (LDL), or globulin);
carbohydrate
(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-
acetylglucosamine, N-
acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can be a
recombinant or
synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino
acid.
Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly
L-
aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer,
poly(L-
lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-
(2-
hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG),
polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-
isopropylacrylamide polymers, or polyphosphazine. Example of polyamines
include:
polyethylenimine, polylysine (PLL), spermine, spermi dine, polyamine,
pseudopeptide-
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
71
polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine,
protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine,
or an alpha
helical peptide.
[0190] Ligands can include targeting groups, e.g., a cell or tissue
targeting agent, e.g., a
lectin, glycoprotein, lipid or protein, e.g., an antibody, that bind to a
specified cell type
such as a kidney cell. A targeting group can be a thyrotropin, melanotropin,
lectin,
glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose,
multivalent
galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent galactose,
transferrin,
bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid,
bile acid,
folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide
mimetic.
[0191] Other examples of ligands include dyes, intercalating agents
(e.g. acridines),
cross-linkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin,
Sapphyrin),
polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine),
artificial
endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic
acid,
adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-
0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol,
menthol, 1,3-
propanediol, heptadecyl group, palmitic acid, myristic acid,03-
(oleoyl)lithocholic acid,
03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide
conjugates (e.g.,
antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,
mercapto, PEG
(e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl,
radiolabeled
markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators
(e.g., aspirin,
vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,
bisimidazole, histamine,
imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, I-1RP, or AP.
[0192] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,
molecules having a
specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds
to a specified
cell type such as a hepatic cell. Ligands can include hormones and hormone
receptors.
They can include non-peptidic species, such as lipids, lectins, carbohydrates,
vitamins,
cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine,
N-acetyl-
gulucosamine multivalent mannose, or multivalent fucose.
[0193] The ligand can be a substance, e.g., a drug, which can increase
the uptake of the
oligonucleotide agent into the cell, for example, by disrupting the cell's
cytoskeleton, e.g.,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
72
by disrupting the cell's microtubules, microfilaments, and/or intermediate
filaments. The
drug can be, for example, taxon, vincristine, vinblastine, cytochalasin,
nocodazole,
japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or
myoservin.
[0194] In some aspects, a ligand attached to an oligonucleotide as
described herein acts as
a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles,
bile
acids, steroids, phospholipid analogues, peptides, protein binding agents,
PEG, vitamins
etc. Exemplary PK modulators include, but are not limited to, cholesterol,
fatty acids,
cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,
phospholipids,
sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides
that comprise
a number of phosphorothioate linkages are also known to bind to serum protein,
thus
short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15
bases, or 20
bases, comprising multiple of phosphorothioate linkages in the backbone are
also
amenable as ligands (e.g. as PK modulating ligands). In addition, aptamers
that bind
serum components (e.g. serum proteins) are also suitable for use as PK
modulating
ligands in the aspects described herein.
[0195] Ligand-conjugated oligonucleotides can be synthesized by the use
of an
oligonucleotide that bears a pendant reactive functionality, such as that
derived from the
attachment of a linking molecule onto the oligonucleotide (described below).
This
reactive oligonucleotide can be reacted directly with commercially-available
ligands,
ligands that are synthesized bearing any of a variety of protecting groups, or
ligands that
have a linking moiety attached thereto.
[0196] The oligonucleotides used in the conjugates can be conveniently
and routinely
made through the well-known technique of solid-phase synthesis. Equipment for
such
synthesis is sold by several vendors including, for example, Applied
Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art can
additionally or
alternatively be employed. It is also known to use similar techniques to
prepare other
oligonucleotides, such as the phosphorothioates and alkylated derivatives.
[0197] In the ligand-conjugated oligonucleotides, such as the ligand-
molecule bearing
sequence-specific linked nucleosides, the oligonucleotides and
oligonucleosides can be
assembled on a suitable DNA synthesizer utilizing standard nucleotide or
nucleoside
precursors, or nucleotide or nucleoside conjugate precursors that already bear
the linking
moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear
the ligand
molecule, or non-nucleoside ligand-bearing building blocks.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
73
[0198] When using conjugate precursors that already bear a linking
moiety, the synthesis
of the sequence-specific linked nucleosides is typically completed, and the
ligand
molecule is then reacted with the linking moiety to form the ligand-conjugated
oligonucleotide. In some aspects, the oligonucleotides or linked nucleosides
are
synthesized by an automated synthesizer using phosphoramidites derived from
ligand-
nucleoside conjugates in addition to the standard phosphoramidites and non-
standard
phosphoramidites that are commercially available and routinely used in
oligonucleotide
synthesis.
i. Lipid Conjugates
[0199] In one aspect, the ligand or conjugate is a lipid or lipid-based
molecule. Such a
lipid or lipid-based molecule can bind a serum protein, e.g., human serum
albumin
(HSA). An HSA binding ligand allows for distribution of the conjugate to a
target tissue,
e.g., a non-kidney target tissue of the body. A lipid or lipid-based ligand
can (a) increase
resistance to degradation of the conjugate, (b) increase targeting or
transport into a target
cell or cell membrane, and/or (c) be used to adjust binding to a serum
protein, e.g., HSA.
[0200] In another aspect, the ligand is a moiety, e.g., a vitamin,
which is taken up by a
target cell, e.g., a proliferating cell. Exemplary vitamins include vitamin A,
E, and K.
Cell Permeation Agents
[0201] In another aspect, the ligand is a cell-permeation agent, such
as a helical cell-
permeation agent. In one aspect, the agent is amphipathic. An exemplary agent
is a
peptide such as tat or antennopedia. If the agent is a peptide, it can be
modified,
including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide
linkages, and
use of D-amino acids. In one aspect, the helical agent is an alpha-helical
agent, which
can havea lipophilic and a lipophobic phase.
[0202] The ligand can be a peptide or peptidomimetic. A peptidomimetic
(also referred
to herein as an oligopeptidomimetic) is a molecule capable of folding into a
defined three-
dimensional structure similar to a natural peptide. The attachment of peptide
and
peptidomimetics to oligonucleotide agents can affect pharmacokinetic
distribution of the
oligonucleotide, such as by enhancing cellular recognition and absorption. The
peptide or
peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10,
15, 20, 25,
30, 35, 40, 45, or 50 amino acids long
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
74
[0203] A peptide or peptidomimetic can be, for example, a cell
permeation peptide,
cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g.,
consisting primarily
of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide,
constrained peptide
or crosslinked peptide. In another alternative, the peptide moiety can include
a
hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic
MTS-containing peptide is RFGF having the amino acid sequence
AAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequence
AALLPVLLAAP containing a hydrophobic MTS can be a targeting moiety. The
peptide
moiety can be a "delivery" peptide, which can carry large polar molecules
including
peptides, oligonucleotides, and protein across cell membranes. For example,
sequences
from the HIV Tat protein (GRKKRRQRRRPPQ and the Drosophila Antennapedia
protein (RQIKIWFQNRRMIKWKK have been found to be capable of functioning as
delivery peptides. A peptide or peptidomimetic can be encoded by a random
sequence of
DNA, such as a peptide identified from a phage-display library, or one-bead-
one-
compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991).
Examples of a peptide or peptidomimetic tethered to an oligonucleotide agent
via an
incorporated monomer unit for cell targeting purposes is an arginine-glycine-
aspartic acid
(RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5
amino acids to about 40 amino acids. The peptide moieties can have a
structural
modification, such as to increase stability or direct conformational
properties. Any of the
structural modifications described below can be utilized.
[0204] An RGD peptide for use in the compositions and methods can be
linear or cyclic,
and can be modified, e.g., glycosylated or methylated, to facilitate targeting
to a specific
tissue(s) RGD-containing peptides and peptidiomimemtics can include D-amino
acids,
as well as synthetic RGD mimics. In addition to RGD, one can use other
moieties that
target the integrin ligand. Some conjugates of this ligand target PECAM-1 or
VEGF.
[0205] A cell permeation peptide is capable of permeating a cell, e.g.,
a microbial cell,
such as a bacterial or fungal cell, or a mammalian cell, such as a human cell.
A microbial
cell-permeating peptide can be, for example, an a-helical linear peptide
(e.g., LL-37 or
Ceropin P1), a disulfide bond-containing peptide (e.g., a-defensin,f3-
defensin, or
bactenecin), or a peptide containing only one or two dominating amino acids
(e.g., PR-39
or indolicidin). A cell permeation peptide can include a nuclear localization
signal
(NLS). For example, a cell permeation peptide can be a bipartite amphipathic
peptide,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and
the
NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31 : 271 7-2724,
2003).
Carbohydrate Conjugates
[0206] In some aspects of the compositions and methods described
herein, an
oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated
oligonucleotides are advantageous for the in vivo delivery of nucleic acids,
as well as
compositions suitable for in vivo therapeutic use, as described herein. As
used herein,
"carbohydrate" refers to a compound which is either a carbohydrate per se made
up of one
or more monosaccharide units having at least 6 carbon atoms (which can be
linear,
branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each
carbon atom;
or a compound having as a part thereof a carbohydrate moiety made up of one or
more
monosaccharide units each having at least six carbon atoms (which can be
linear,
branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each
carbon atom.
Representative carbohydrates include the sugars (mono-, di-, tri- and
oligosaccharides
containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and
polysaccharides such
as starches, glycogen, cellulose and polysaccharide gums. Specific
monosaccharides
include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides
include
sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
[0207] In one aspect, a carbohydrate conjugate for use in the
compositions and methods
described herein is a monosaccharide.
[0208] In some aspects, the carbohydrate conjugate further comprises
one or more
additional ligands as described above, such as, but not limited to, a PK
modulator and/or a
cell permeation peptide.
[0209] Additional carbohydrate conjugates (and linkers) suitable for
use include those
described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the
entire
contents of each of which are incorporated herein by reference.
iv. Linkers
[0210] In some aspects, the conjugate or ligand described herein can be
attached to an
oligonucleotide with various linkers that can be cleavable or non-cleavable.
[0211] Linkers typically comprise a direct bond or an atom such as
oxygen or sulfur, a
unit such as NR8, C(0), C(0)NH, SO, SO2, SO2NH or a chain of atoms, such as,
but not
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
76
limited to, substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl,
substituted or unsubstituted al kynyl, aryl alkyl, aryl al kenyl, aryl
alkynyl, heteroaryl alkyl,
heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,
heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl,
alkylaryl alkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,
alkenylarylalkenyl,
alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,
alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,
alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,
alkynylheteroaryl alkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,
alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,
alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
alkynylheterocyclylalkynyl,
alkyl aryl, alkenyl aryl, alkynyl aryl, al kyl heteroaryl, alkenylheteroaryl,
alkynylhereroaryl,
which one or more methylenes can be interrupted or terminated by 0, S, S(0),
S02,
N(R8), C(0), substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl,
substituted or unsubstituted heterocyclic, where R8 is hydrogen, acyl,
aliphatic or
substituted aliphatic. In one aspect, the linker is between about 1-24, 2-24,
3-24, 4-24, 5-
24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, 8-16 or 1,2, 3,4, 5, 6, 7,
8,9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, or 24 atoms.
[0212] A cleavable linking group is one which is sufficiently stable
outside the cell, but
which upon entry into a target cell is cleaved to release the two parts the
linker is holding
together. In somes aspects, the cleavable linking group is cleaved at least 10
times, 20
times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times,
or more, or at
least 100 times faster in a target cell or under a first reference condition
(which can, e_g ,
be selected to mimic or represent intracellular conditions) than in the blood
of a subject,
or under a second reference condition (which can, e.g., be selected to mimic
or represent
conditions found in the blood or serum).
[0213] Cleavable linking groups are susceptible to cleavage agents,
e.g., pH, redox
potential, or the presence of degradative molecules. Generally, cleavage
agents are more
prevalent or found at higher levels or activities inside cells than in serum
or blood.
Examples of such degradative agents include: redox agents which are selective
for
particular substrates or which have no substrate specificity, including, e.g.,
oxidative or
reductive enzymes or reductive agents such as mercaptans, present in cells,
that can
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
77
degrade a redox cleavable linking group by reduction; esterases; endosomes or
agents that
can create an acidic environment, e.g., those that result in a pH of five or
lower; enzymes
that can hydrolyze or degrade an acid cleavable linking group by acting as a
general acid,
peptidases (which can be substrate specific), and phosphatases.
[0214] A cleavable linkage group, such as a disulfide bond can be
susceptible to pH. The
pH of human serum is 7.4, while the average intracellular pH is slightly
lower, ranging
from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0,
and
lysosomes have an even more acidic pH at around 5Ø Some linkers will have a
cleavable linking group that is cleaved at a preferred pH, thereby releasing a
cationic lipid
from the ligand inside the cell, or into the desired compartment of the cell.
[0215] A linker can include a cleavable linking group that is cleavable
by a particular
enzyme. The type of cleavable linking group incorporated into a linker can
depend on the
cell to be targeted. For example, a liver-targeting ligand can be linked to a
cationic lipid
through a linker that includes an ester group. Liver cells are rich in
esterases, and
therefore the linker will be cleaved more efficiently in liver cells than in
cell types that are
not esterase-rich. Other cell-types rich in esterases include cells of the
lung, renal cortex,
and testis.
[0216] Linkers that contain peptide bonds can be used when targeting
cell types rich in
peptidases, such as liver cells and synoviocytes.
[0217] In general, the suitability of a candidate cleavable linking
group can be evaluated
by testing the ability of a degradative agent (or condition) to cleave the
candidate linking
group. It will also be desirable to test the candidate cleavable linking group
for the ability
to resist cleavage in the blood or when in contact with other non-target
tissue. Thus, one
can determine the relative susceptibility to cleavage between at least two
conditions,
where at least one condition is selected to be indicative of cleavage in a
target cell and
another condition is selected to be indicative of cleavage in other tissues or
biological
fluids, e.g., blood or serum. The evaluations can be carried out in cell free
systems, in
cells, in cell culture, in organ or tissue culture, or in whole animals. It
can be useful to
make initial evaluations in cell-free or culture conditions and to confirm by
further
evaluations in whole animals. In some aspects, useful candidate compounds are
cleaved
at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the
cell (or under in
vitro conditions selected to mimic intracellular conditions) as compared to
blood or serum
(or under in vitro conditions selected to mimic extracellular conditions).
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
78
a. 1?edox Cleavable Linking Groups
[0218] In one aspect, a cleavable linking group is a redox cleavable
linking group that is
cleaved upon reduction or oxidation. An example of reductively cleavable
linking group
is a disulphide linking group (--S--S--). To determine if a candidate
cleavable linking
group is a suitable "reductively cleavable linking group," or for example is
suitable for
use with a particular oligonucleotide moiety and particular targeting agent
one can look to
methods described herein. For example, a candidate can be evaluated by
incubation with
dithiothreitol (DTT), or other reducing agent using reagents know in the art,
which mimic
the rate of cleavage which would be observed in a cell, e.g., a target cell.
The candidates
can be evaluated under conditions which are selected to mimic blood or serum
conditions.
In one aspect, candidate compounds are cleaved by at most about 10% in the
blood. In
other aspects, useful candidate compounds are degraded at least 2, 4, 10, 20,
30, 40, 50,
60, 70, 80, 90, or 100 times faster in the cell (or under in itro conditions
selected to
mimic intracellular conditions) as compared to blood (or under in vitro
conditions
selected to mimic extracellular conditions). The rate of cleavage of candidate
compounds
can be determined using standard enzyme kinetics assays under conditions
chosen to
mimic intracellular media and compared to conditions chosen to mimic
extracellular
media.
b. Phosphate-Based Cleavable Linking Groups
[0219] In another aspect, a cleavable linker comprises a phosphate-
based cleavable
linking group. A phosphate-based cleavable linking group is cleaved by agents
that
degrade or hydrolyze the phosphate group. An example of an agent that cleaves
phosphate groups in cells are enzymes such as phosphatases in cells. Examples
of
phosphate-based linking groups are -0-P(0)(ORk)-0-, -0-P(S)(ORk)-0-, -0-
P(S)(SIth)-
0-, -S-P(0)(0Rk)-0-, -0-P(0)(ORk)-S-, -S-P(0)(0Rk)-S-, -0-P(S)(ORk)-S-, -S-
P(S)(0Rk)-0-, -0-P(0)(Rk)-0-, -0-P(S)(Rk)-0-, -S-P(0)(Rk)-0-, -S-P(S)(Rk)-0-
, -S-P(0)(Rk)-S-, -0-P(S)(Rk)-S-. These candidates can be evaluated using
methods
analogous to those described above.
c. Acid Cleavable Linking Groups
[0220] In another aspect, a cleavable linker comprises an acid
cleavable linking group.
An acid cleavable linking group is a linking group that is cleaved under
acidic conditions.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
79
In some aspects, acid cleavable linking groups are cleaved in an acidic
environment with
a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),
or by agents
such as enzymes that can act as a general acid. In a cell, specific low pH
organelles, such
as endosomes and lysosomes can provide a cleaving environment for acid
cleavable
linking groups. Examples of acid cleavable linking groups include but are not
limited to
hydrazones, esters, and esters of amino acids. Acid cleavable groups can have
the general
formula ¨C=NN--, C(0)0, or --0C(0). In one aspect, the carbon is attached to
the
oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl
group, or tertiary
alkyl group such as dimethyl pentyl or t-butyl. These candidates can be
evaluated using
methods analogous to those described above.
d. Ester-Based Linking Groups
[0221] In another aspect, a cleavable linker comprises an ester-based
cleavable linking
group. An ester-based cleavable linking group is cleaved by enzymes such as
esterases
and amidases in cells. Examples of ester-based cleavable linking groups
include but are
not limited to esters of alkylene, alkenylene and alkynylene groups. Ester
cleavable
linking groups have the general formula --C(0)0--, or --0C(0)--. These
candidates can
be evaluated using methods analogous to those described above.
e. Peptide-Based Cleaving Groups
[0222] In yet another aspect, a cleavable linker comprises a peptide-
based cleavable
linking group. A peptide-based cleavable linking group is cleaved by enzymes
such as
peptidases and proteases in cells. Peptide-based cleavable linking groups are
peptide
bonds formed between amino acids to yield oligopeptides (e.g., dipeptides,
tripeptides
etc.) and polypeptides. Peptide-based cleavable groups do not include the
amide group (-
C(0)NH-). The amide group can be formed between any alkylene, alkenylene, or
alkynelene. A peptide bond is a special type of amide bond formed between
amino acids
to yield peptides and proteins. The peptide based cleavage group is generally
limited to
the peptide bond (i.e., the amide bond) formed between amino acids yielding
peptides and
proteins and does not include the entire amide functional group. Peptide-based
cleavable
linking groups have the general formula -NHCHRAC(0)NHCHRBC(0)--, where RA and
RB are the R groups of the two adjacent amino acids. These candidates can be
evaluated
using methods analogous to those described above_
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
[0223] In one aspect, an oligonucleotide is conjugated to a
carbohydrate through a linker.
Linkers include bivalent and trivalent branched linker groups. Linkers for
oligonucleotide carbohydrate conjugates include, but are not limited to, those
described in
formulas 24-35 of PCT Publication No. WO 2018/195165.
[0224] Representative U.S. patents that teach the preparation of
oligonucleotide
conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;
4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584;
5,109,124;
5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;
4,587,044;
4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582;
4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;
5,245,022;
5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,
5,391,723;
5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and
5,688,941;
6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022,
the entire
contents of each of which are hereby incorporated herein by reference.
[0225] It is not necessary for all positions in a given compound to be
uniformly modified,
and in fact more than one of the aforementioned modifications can be
incorporated in a
single compound or even at a single nucleoside within an oligonucleotide.
Oligonucleotide compounds that are chimeric compounds are also
contemplated. Chimeric oligonucleotides typically contain at least one region
wherein
the RNA is modified so as to confer upon the oligonucleotide increased
resistance to
nuclease degradation, increased cellular uptake, and/or increased binding
affinity for the
target nucleic acid. An additional region of the oligonucleotide can serve as
a substrate
for enzymes capable of cleaving RNA:DNA By way of example, RNase H is a
cellular
endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of
RNase H, therefore, results in cleavage of the RNA target, thereby greatly
enhancing the
efficiency of oligonucleotide inhibition of gene expression. Consequently,
comparable
results can often be obtained with shorter oligonucleotides when chimeric
oligonucleotides are used, compared to phosphorothioate deoxy oligonucleotides
hybridizing to the same target region. Cleavage of the RNA target can be
routinely
detected by gel electrophoresis and, if necessary, associated nucleic acid
hybridization
techniques known in the art.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
81
[0226] In certain instances, the nucleotides of an oligonucleotide can
be modified by a
non-ligand group. A number of non-ligand molecules have been conjugated to
oligonucleotides in order to enhance the activity, cellular distribution, or
cellular uptake
of the oligonucleotide, and procedures for performing such conjugations are
available in
the scientific literature. Such non-ligand moieties have included lipid
moieties, such as
cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm, 2007, 365(1):54-61;
Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid
(Manoharan et
al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-
tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al.,
Bioorg. Med.
Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids
Res., 1992,
20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-
Behmoaras et
al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327;
Svinarchuk et
al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol
or
triethyl ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et
al.,
Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,
18:3777), a
polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &
Nucleotides,
1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.,
1995,
36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995,
1264:229), or
an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al.,
J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents
that teach
the preparation of such oligonucleotide conjugates have been listed above.
Typical
conjugation protocols involve the synthesis of an oligonucleotide bearing an
aminolinker
at one or more positions of the sequence. The amino group is then reacted with
the
molecule being conjugated using appropriate coupling or activating reagents.
The
conjugation reaction can be performed either with the oligonucleotide still
bound to the
solid support or following cleavage of the oligonucleotide, in solution phase.
Purification
of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.
/V. Pharmaceutical Uses
[0227] The oligonucleotide, or pharmaceutically acceptable salt
thereof, compositions
described herein are useful in the methods described herein, and, while not
bound by
theory, are believed to exert their desirable effects through their ability to
modulate the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
82
level, status, and/or activity of a MutS13 heterodimer comprising MSH3, e.g.,
by
inhibiting the activity or level of the MSH3 protein in a cell in a mammal.
[0228] An aspect relates to methods of treating disorders related to
DNA mismatch repair
such as nucleotide repeat expansion disorders (e.g., trinucleotide repeat
expansion
disorders) in a subject in need thereof. Another aspect includes reducing the
level of
MSH3 in a cell of a subject identified as having a nucleotide repeat expansion
disorder
(e.g., a trinucleotide repeat expansion disorder). Still another aspect
includes a method of
inhibiting expression of MSH3 in a cell in a subject. Further aspects include
methods of
decreasing nucleotide repeat expansion in a cell. The methods include
contacting a cell
with an oligonucleotide, or pharmaceutically acceptable salt thereof, in an
amount
effective to inhibit expression of MSH3 in the cell, thereby inhibiting
expression of
MSH3 in the cell.
[0229] Based on the above methods, an oligonucleotide, or
pharmaceutically acceptable
salt thereof, or a composition comprising such an oligonucleotide, or
pharmaceutically
acceptable salt thereof, for use in therapy, or for use as a medicament, or
for use in
treating disorders related to DNA mismatch repair such as repeat expansion
disorders in a
subject in need thereof, or for use in reducing the level of MSH3 in a cell of
a subject
identified as having a nucleotide repeat expansion disorder (e.g., a
trinucleotide repeat
expansion disorder), or for use in inhibiting expression of MSH3 in a cell in
a subject, or
for use in decreasing nucleotide repeat expansion (e.g., trinucleotide repeat
expansion) in
a cell is contemplated. The uses include the contacting of a cell with the
oligonucleotide,
or pharmaceutically acceptable salt thereof, in an amount effective to inhibit
expression
of MSH3 in the cell, thereby inhibiting expression of MSH3 in the cell.
Aspects
described below in relation to the methods described herein are also
applicable to these
further aspects.
[0230] Contacting of a cell with an oligonucleotide, or
pharmaceutically acceptable salt
thereof, can be done in vitro or in vivo. Contacting a cell in vivo with the
oligonucleotide,
or pharmaceutically acceptable salt thereof, includes contacting a cell or
group of cells
within a subject, e.g., a human subject, with the oligonucleotide, or
pharmaceutically
acceptable salt thereof Combinations of in vitro and in vivo methods of
contacting a cell
are also possible. Contacting a cell can be direct or indirect, as discussed
above.
Furthermore, contacting a cell can be accomplished via a targeting ligand,
including any
ligand described herein or known in the art. In some aspects, the targeting
ligand is a
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
83
carbohydrate moiety, e.g., a GalNAc3 ligand, or any other ligand that directs
the
oligonucleotide to a site of interest. Cells can include those of the central
nervous system,
or muscle cells.
10231] Inhibiting expression of a MSH3 gene includes any level of
inhibition of a MSH3
gene, e.g., at least partial suppression of the expression of a MSH3 gene,
such as an
inhibition by at least 200/o. In some aspects, inhibition is by at least 25%,
at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at
1east60%, at 1east65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least
91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or
at least 99%.
10232] The expression of a MSH3 gene can be assessed based on the level
of any variable
associated with MSH3 gene expression, e.g., MSH3 mRNA level or MSH3 protein
level.
10233] Inhibition can be assessed by a decrease in an absolute or
relative level of one or
more of these variables compared with a control level. The control level can
be any type
of control level that is utilized in the art, e.g., a pre-dose baseline level,
or a level
determined from a similar subject, cell, or sample that is untreated or
treated with a
control (such as, e.g., buffer only control or inactive agent control).
10234] In some aspects, surrogate markers can be used to detect
inhibition of MSH3. For
example, effective treatment of a nucleotide repeat expansion disorder (e.g.,
a
trinucleotide repeat expansion disorder), as demonstrated by acceptable
diagnostic and
monitoring criteria with an agent to reduce MSH3 expression can be understood
to
demonstrate a clinically relevant reduction in MSH3.
10235] In some aspects of the methods, expression of a MSH3 gene is
inhibited by at
least 20%, 25%, 30%, 350/0, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, or 95%, or to below the level of detection of the assay. In some aspects,
the
methods include a clinically relevant inhibition of expression of MSH3, e.g.,
as
demonstrated by a clinically relevant outcome after treatment of a subject
with an agent to
reduce the expression of MSH3.
[0236] Inhibition of the expression of a MSH3 gene can be manifested by
a reduction of
the amount of mRNA expressed by a first cell or group of cells (such cells can
be present,
for example, in a sample derived from a subject) in which a MSH3 gene is
transcribed
and which has or have been treated (e.g., by contacting the cell or cells with
an
oligonucleotide, or pharmaceutically acceptable salt thereof, or by
administering an
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
84
oligonucleotide, or pharmaceutically acceptable salt thereof, to a subject in
which the
cells are or were present) such that the expression of a MSH3 gene is
inhibited, as
compared to a second cell or group of cells substantially identical to the
first cell or group
of cells but which has not or have not been so treated (control cell(s) not
treated with an
oligonucleotide, or pharmaceutically acceptable salt thereof, or not treated
with an
oligonucleotide, or pharmaceutically acceptable salt thereof, targeted to the
gene of
interest). The degree of inhibition can be expressed in terms of:
(mRNA in control cells) ¨ (mRNA in treated cells)
(mRNA in control cells) x 100%
[0237] In other aspects, inhibition of the expression of a MSH3 gene
can be assessed in
terms of a reduction of a parameter that is functionally linked to MSH3 gene
expression,
e.g., MSH3 protein expression or MSH3 signaling pathways. MSH3 gene silencing
can
be determined in any cell expressing MSH3, either endogenous or heterologous
from an
expression construct, and by any assay known in the art.
[0238] Inhibition of the expression of a MSH3 protein can be manifested
by a reduction
in the level of the MSH3 protein that is expressed by a cell or group of cells
(e.g., the
level of protein expressed in a sample derived from a subject). As explained
above, for
the assessment of mRNA suppression, the inhibition of protein expression
levels in a
treated cell or group of cells can similarly be expressed as a percentage of
the level of
protein in a control cell or group of cells.
[0239] A control cell or group of cells that can be used to assess the
inhibition of the
expression of a MSH3 gene includes a cell or group of cells that has not yet
been
contacted with an oligonucleotide. For example, the control cell or group of
cells can be
derived from an individual subject (e.g., a human or animal subject) prior to
treatment of
the subject with an oligonucleotide.
[0240] The level of MSH3 mRNA that is expressed by a cell or group of
cells can be
determined using any method known in the art for assessing mRNA expression. In
one
aspect, the level of expression of MSH3 in a sample is determined by detecting
a
transcribed polynucleotide, or portion thereof, e.g., mRNA of the MSH3 gene.
RNA can
be extracted from cells using RNA extraction techniques including, for
example, using
acid phenol/guanidine isothi ocyanate extraction (RNA zol B; Biogenesis),
RNF.ASYTm
RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical
assay
formats utilizing ribonucleic acid hybridization include nuclear run-on
assays, RT-PCR,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
RNase protection assays, northern blotting, in situ hybridization, and
microarray analysis.
Circulating MST-I3 mRNA can be detected using methods the described in PCT
Publication W02012/177906, the entire contents of which are hereby
incorporated herein
by reference. In some aspects, the level of expression of MSH3 is determined
using a
nucleic acid probe. The term "probe," as used herein, refers to any molecule
that is
capable of selectively binding to a specific MSH3 sequence, e.g. to an mRNA or
polypeptide. Probes can be synthesized by one of skill in the art, or derived
from
appropriate biological preparations. Probes can be specifically designed to be
labeled.
Examples of molecules that can be utilized as probes include, but are not
limited to, RNA,
DNA, proteins, antibodies, and organic molecules.
[0241] Isolated mRNA can be used in hybridization or amplification
assays that include,
but are not limited to, Southern or northern analyses, polymerase chain
reaction (PCR)
analyses, and probe arrays. One method for the determination of mRNA levels
involves
contacting the isolated mRNA with a nucleic acid molecule (probe) that can
hybridize to
MSH3 mRNA. In one aspect, the mRNA is immobilized on a solid surface and
contacted
with a probe, for example by running the isolated mRNA on an agarose gel and
transferring the mRNA from the gel to a membrane, such as nitrocellulose. In
an
alternative aspect, the probe(s) are immobilized on a solid surface and the
mRNA is
contacted with the probe(s), for example, in an AFFYMETRIX gene chip array. A
skilled artisan can readily adapt known mRNA detection methods for use in
determining
the level of MSH3 mRNA.
[0242] An alternative method for determining the level of expression of
MSH3 in a
sample involves the process of nucleic acid amplification and/or reverse
transcriptase (to
prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the
experimental
aspect set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain
reaction (Barany
(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence
replication
(Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878),
transcriptional
amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-
1177), Q-
Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle
replication
(Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid
amplification method,
followed by the detection of the amplified molecules using techniques well
known to
those of skill in the art. These detection schemes are especially useful for
the detection of
nucleic acid molecules if such molecules are present in very low numbers. In
some
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
86
aspects, the level of expression of MSH3 is determined by quantitative
fluorogenic RI-
PCR (i.e., the TAQMANT1" System) or the DUAL-GLO Luciferase assay.
[0243] The expression levels of MSH3 mRNA can be monitored using a
membrane blot
(such as used in hybridization analysis such as northern, Southern, dot, and
the like), or
microwells, sample tubes, gels, beads or fibers (or any solid support
comprising bound
nucleic acids). See U.S. Pat. Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195;
and
5,445,934, which are incorporated herein by reference. The determination of
MSH3
expression level can comprise using nucleic acid probes in solution.
[0244] In some aspects, the level of mRNA expression is assessed using
branched DNA
(bDNA) assays or real time PCR (qPCR). The use of this PCR method is described
and
exemplified in the Examples presented herein. Such methods can be used for the
detection of MSH3 nucleic acids.
[0245] The level of MSH3 protein expression can be determined using any
method
known in the art for the measurement of protein levels. Such methods include,
for
example, electrophoresis, capillary electrophoresis, high performance liquid
chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion
chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a
colorimetric
assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or
double),
immunoelectrophoresis, western blotting, radioimmunoassay (MA), enzyme-linked
immunosorbent assays (ELISAs), immunofluorescent assays,
electrochemiluminescence
assays, and the like. Such assays can be used for the detection of proteins
indicative of
the presence or replication of MSH3 proteins.
[0246] In some aspects of the methods described herein, the
oligonucleotide, or
pharmaceutically acceptable salt thereof, is administered to a subject such
that the
oligonucleotide, or pharmaceutically acceptable salt thereof, is delivered to
a specific site
within the subject. The inhibition of expression of MSH3 can be assessed using
measurements of the level or change in the level of MSH3 mRNA or MSH3 protein
in a
sample derived from a specific site within the subject. In some aspects, the
methods
include a clinically relevant inhibition of expression of MSH3, e.g., as
demonstrated by a
clinically relevant outcome after treatment of a subject with an agent to
reduce the
expression of MSH3.
[0247] In other aspects, the oligonucleotide, or pharmaceutically
acceptable salt thereof,
is administered in an amount and for a time effective to result in one of (or
more, e.g., two
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
87
or more, three or more, four or more of): (a) decrease the number of repeats,
(b) decrease
the level of polyglutamine, (c) decreased cell death (e.g., CNS cell death
and/or muscle
cell death), (d) delayed onset of the disorder, (e) increased survival of
subject, and (f)
increased progression free survival of a subject.
[0248] Treating nucleotide repeat expansion disorders (e.g.,
trinucleotide repeat
expansion disorders) can result in an increase in average survival time of an
individual or
a population of subjects treated with an oligonucleotide, or pharmaceutically
acceptable
salt thereof, described herein in comparison to a population of untreated
subjects. For
example, the survival time of an individual or average survival time of a
population is
increased by more than 30 days (more than 60 days, 90 days, or 120 days). An
increase
in survival time of an individual or in average survival time of a population
can be
measured by any reproducible means. An increase in survival time of an
individual can
be measured, for example, by calculating for an individual the length of
survival time
following the initiation of treatment with the compound described herein. An
increase in
average survival time of a population can be measured, for example, by
calculating for
the average length of survival time following initiation of treatment with the
compound
described herein. An increase in survival time of an individual can be
measured, for
example, by calculating for an individual length of survival time following
completion of
a first round of treatment with a compound or pharmaceutically acceptable salt
of a
compound described herein. An increase in average survival time of a
population can be
measured, for example, by calculating for a population the average length of
survival time
following completion of a first round of treatment with a compound or
pharmaceutically
acceptable salt of a compound described herein.
[0249] Treating nucleotide repeat expansion disorders (e g
trinucleotide repeat
expansion disorders) can result in a decrease in the mortality rate of a
population of
treated subjects in comparison to an untreated population. For example, the
mortality rate
is decreased by more than 2% (e.g., more than 5%, 10%, or 25%). A decrease in
the
mortality rate of a population of treated subjects can be measured by any
reproducible
means, for example, by calculating for a population the average number of
disease-related
deaths per unit time following initiation of treatment with a compound or
pharmaceutically acceptable salt of a compound described herein. A decrease in
the
mortality rate of a population can be measured, for example, by calculating
for a
population the average number of disease-related deaths per unit time
following
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
88
completion of a first round of treatment with a compound or pharmaceutically
acceptable
salt of a compound described herein.
A. Delivery of anti-MSH3 Agents
[0250] The delivery of an oligonucleotide to a cell e.g., a cell within
a subject, such as a
human subject e.g., a subject in need thereof, such as a subject having a
nucleotide repeat
expansion disorder (e.g., a trinucleotide repeat expansion disorder) can be
achieved in a
number of different ways. For example, delivery can be performed by contacting
a cell
with an oligonucleotide, or pharmaceutically acceptable salt thereof, either
in vitro or in
vivo. In vivo delivery can be performed directly by administering a
composition
comprising an oligonucleotide, or pharmaceutically acceptable salt thereof, to
a subject.
These alternatives are discussed further below.
[0251] In general, any method of delivering a nucleic acid molecule (in
vitro or in vivo)
can be adapted for use with an oligonucleotide (see e.g., Akhtar S. and Julian
R L., (1992)
Trends Cell. Biol. 2(5):139-144 and W094/02595, which are incorporated herein
by
reference in their entireties). For in vivo delivery, factors to consider in
order to deliver
an oligonucleotide molecule include for example, biological stability of the
delivered
molecule, prevention of non-specific effects, and accumulation of the
delivered molecule
in the target tissue. The non-specific effects of an oligonucleotide can be
minimized by
local administration, for example, by direct injection or implantation into a
tissue or
topically administering the preparation. Local administration to a treatment
site
maximizes local concentration of the agent, limits the exposure of the agent
to systemic
tissues that can otherwise be harmed by the agent or that can degrade the
agent, and
permits a lower total dose of the oligonucleotide, or pharmaceutically
acceptable salt
thereof, to be administered.
[0252] For administering an oligonucleotide, or pharmaceutically
acceptable salt thereof,
systemically for the treatment of a disease, the oligonucleotide can include
alternative
nucleobases, alternative sugar moieties, and/or alternative internucleoside
linkages, or
alternatively delivered using a drug delivery system; both methods act to
prevent the
rapid degradation of the oligonucleotide by endo- and exo-nucleases in vivo.
Modification of the oligonucleotide, or the pharmaceutical carrier, can permit
targeting of
the oligonucleotide composition to the target tissue and avoid undesirable off-
target
effects. Oligonucleotide molecules can be modified by chemical conjugation to
lipophilic
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
89
groups such as cholesterol to enhance cellular uptake and prevent degradation.
In an
alternative aspect, the oligonucleotide can be delivered using drug delivery
systems such
as a nanoparticle, a lipid nanoparticle, a polyplex nanoparticle, a lipoplex
nanoparticle, a
dendrimer, a polymer, liposomes, or a cationic delivery system. Positively
charged
cationic delivery systems facilitate binding of an oligonucleotide molecule
(negatively
charged) and also enhance interactions at the negatively charged cell membrane
to permit
efficient uptake of an oligonucleotide by the cell. Cationic lipids,
dendrimers, or
polymers can either be bound to an oligonucleotide, or induced to form a
vesicle or
micelle that encases an oligonucleotide. The formation of vesicles or micelles
further
prevents degradation of the oligonucleotide when administered systemically. In
general,
any methods of delivery of nucleic acids known in the art may be adaptable to
the
delivery of the oligonucleotides described herein. Methods for making and
administering
cationic oligonucleotide complexes are well within the abilities of one
skilled in the art
(see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN.
et al.,
(2003) Clin. Cancer Res. 9:1291-1300; Arnold, A Set al., (2007) J. Hypertens.
25:197-
205, which are incorporated herein by reference in their entirety). Some non-
limiting
examples of drug delivery systems useful for systemic delivery of
oligonucleotides
include DOTAP (Sorensen, D R., et al (2003), supra; Verma, UN. et al., (2003),
supra),
Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T S. et al.,
(2006)
Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther.
12:321-
328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine
(Bonnet M E.
et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J.
Biomed.
Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm.
3:472-
487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem Soc. Trans.
35:61-67;
Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804) In some aspects, an
oligonucleotide
forms a complex with cyclodextrin for systemic administration. Methods for
administration and pharmaceutical compositions of oligonucleotides and
cyclodextrins
can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by
reference in its
entirety. In some aspects, the oligonucleotides described herein are delivered
by polyplex
or lipoplex nanoparticles. Methods for administration and pharmaceutical
compositions
of oligonucleotides and polyplex nanoparticles and lipoplex nanoparticles can
be found in
U.S. Patent Application Nos. 2017/0121454; 2016/0369269; 2016/0279256;
2016/0251478; 2016/0230189; 2015/0335764; 2015/0307554; 2015/0174549;
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
2014/0342003; 2014/0135376; and 2013/0317086, which are herein incorporated by
reference in their entirety.
1. Membranous Molecular Assembly Delivery Methods
[0253] The oligonucleotide, or pharmaceutically acceptable salt
thereof, can be delivered
using a variety of membranous molecular assembly delivery methods including
polymeric, biodegradable microparticle, or microcapsule delivery devices known
in the
art. For example, a colloidal dispersion system can be used for targeted
delivery of an
oligonucleotide agent described herein. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based
systems
including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
Liposomes
are artificial membrane vesicles that are useful as delivery vehicles in vitro
and in vivo. It
has been shown that large unilamellar vesicles (LUV), which range in size from
0.2-4.0
ium can encapsulate a substantial percentage of an aqueous buffer containing
large
macromolecules. Liposomes are useful for the transfer and delivery of active
ingredients
to the site of action. Because the liposomal membrane is structurally similar
to biological
membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses
with
bilayer of the cellular membranes. As the merging of the liposome and cell
progresses,
the internal aqueous contents that include the oligonucleotide, or
pharmaceutically
acceptable salt thereof, are delivered into the cell where the oligonucleotide
can
specifically bind to a target RNA and can mediate RNase H-mediated gene
silencing. In
some cases, the liposomes are also specifically targeted, e g , to direct the
oligonucleotide
to particular cell types. The composition of the liposome is usually a
combination of
phospholipids, usually in combination with steroids, especially cholesterol.
Other
phospholipids or other lipids can be used. The physical characteristics of
liposomes
depend on pH, ionic strength, and the presence of divalent cations.
[0254] A liposome containing an oligonucleotide, or pharmaceutically
acceptable salt
thereof, can be prepared by a variety of methods. In one example, the lipid
component of
a liposome is dissolved in a detergent so that micelles are formed with the
lipid
component. For example, the lipid component can be an amphipathic cationic
lipid or
lipid conjugate. The detergent can have a high critical micelle concentration
and can be
nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside,
deoxycholate,
and lauroyl sarcosine. The oligonucleotide, or pharmaceutically acceptable
salt thereof,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
91
preparation is then added to the micelles that include the lipid component.
The cationic
groups on the lipid interact with the oligonucleotide, or pharmaceutically
acceptable salt
thereof, and condense around the oligonucleotide, or pharmaceutically
acceptable salt
thereof, to form a liposome. After condensation, the detergent is removed,
e.g., by
dialysis, to yield a liposomal preparation of oligonucleotide, or
pharmaceutically
acceptable salt thereof.
[0255] If necessary, a carrier compound that assists in condensation
can be added during
the condensation reaction, e.g., by controlled addition. For example, the
carrier
compound can be a polymer other than a nucleic acid (e.g., spermine or
spermidine). The
pH can be adjusted to favor condensation.
[0256] Methods for producing stable polynucleotide delivery vehicles,
which incorporate
a polynucleotide/cationic lipid complex as a structural component of the
delivery vehicle,
are further described in, e.g., WO 96/37194, the entire contents of which are
incorporated
herein by reference. Liposome formation can include one or more aspects of
exemplary
methods described in Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA
8.7413-
7417, U.S. Pat. No. 4,897,355, U.S. Pat. No. 5,171,678, Bangham et al., (1965)
M. Mol.
Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al.,
(1978)
Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta
775:169;
Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984)
Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates
of
appropriate size for use as delivery vehicles include sonication and freeze-
thaw plus
extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161.
Microfluidization can be used when consistently small (50 to 200 nm) and
relatively
uniform aggregates are desired (Mayhew et al., (1984) Biochim Biophys. Acta
775:169).
These methods are readily adapted to packaging oligonucleotide, or
pharmaceutically
acceptable salt thereof, preparations into liposomes.
[0257] Liposomes fall into two broad classes. Cationic liposomes are
positively charged
liposomes which interact with the negatively charged nucleic acid molecules to
form a
stable complex. The positively charged nucleic acid/liposome complex binds to
the
negatively charged cell surface and is internalized in an endosome. Due to the
acidic pH
within the endosome, the liposomes are ruptured, releasing their contents into
the cell
cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
92
102581 Liposomes, which are pH-sensitive or negatively charged, entrap
nucleic acids
rather than complex with them Since both the nucleic acid and the lipid are
similarly
charged, repulsion rather than complex formation occurs. Nevertheless, some
nucleic
acid is entrapped within the aqueous interior of these liposomes. pH sensitive
liposomes
have been used to deliver nucleic acids encoding the thymidine kinase gene to
cell
monolayers in culture. Expression of the exogenous gene was detected in the
target cells
(Zhou et al. (1992) Journal of Controlled Release, 19:269-274).
[0259] One major type of liposomal composition includes phospholipids
other than
naturally-derived phosphatidylcholine. Neutral liposome compositions, for
example, can
be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed
from
dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed
primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of
liposom al
composition is formed from phosphatidylcholine (PC) such as, for example,
soybean PC,
and egg PC. Another type is formed from mixtures of phospholipid and/or
phosphatidylcholine and/or cholesterol.
[0260] Examples of other methods to introduce liposomes into cells in
vitro and in vivo
include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO
93/24640;
WO 91/16024; Feigner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc.
Natl. Acad.
Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem.
32:7143; and Strauss, (1992) EMEO J. 11:417.
[0261] Non-ionic liposomal systems have also been examined to determine
their utility in
the delivery of drugs to the skin, in particular systems comprising non-ionic
surfactant
and cholesterol Non-ionic liposomal formulations comprising NOVASOMETm I
(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and
NOVASOMETm
(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver
cyclosporin-A into the dermis of mouse skin. Results indicated that such non-
ionic
liposomal systems were effective in facilitating the deposition of
cyclosporine A into
different layers of the skin (Hu et al., (1994) S.T.P.Pharma. Sci., 4(6):466).
[0262] Liposomes can be sterically stabilized liposomes, comprising one
or more
specialized lipids that result in enhanced circulation lifetimes relative to
liposomes
lacking such specialized lipids. Examples of sterically stabilized liposomes
are those in
which part of the vesicle-forming lipid portion of the liposome (A) comprises
one or more
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
93
glycolipids, such as monosialoganglioside Gm', or (B) is derivatized with one
or more
hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not
wishing to
be bound by any particular theory, it is thought in the art that, at least for
sterically
stabilized liposomes containing gangliosides, sphingomyelin, or PEG-
derivatized lipids,
the enhanced circulation half-life of these sterically stabilized liposomes
derives from a
reduced uptake into cells of the reticuloendothelial system (RES) (Allen et
al., (1987)
FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).
[0263] Various liposomes comprising one or more glycolipids are known
in the art.
Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the
ability of
monosialoganglioside Gml, galactocerebroside sulfate, and phosphatidylinositol
to
improve blood half-lives of liposomes. These findings were expounded upon by
Gabizon
et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No.
4,837,028 and WO
88/04924, both to Allen et al., disclose liposomes comprising (1)
sphingomyelin and (2)
the ganglioside Gm' or a galactocerebroside sulfate ester. U.S. Pat. No.
5,543,152 (Webb
et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-
sn-
dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
[0264] In one aspect, cationic liposomes are used. Cationic liposomes
possess the
advantage of being able to fuse to the cell membrane. Non-cationic liposomes,
although
not able to fuse as efficiently with the plasma membrane, are taken up by
macrophages in
vivo and can be used to deliver oligonucleotides to macrophages.
[0265] Further advantages of liposomes include: liposomes obtained from
natural
phospholipids are biocompatible and biodegradable; liposomes can incorporate a
wide
range of water and lipid soluble drugs; liposomes can protect encapsulated
oligonucleotides in their internal compartments from metabolism and
degradation
(Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker
(Eds.), 1988,
volume 1, p. 245). Important considerations in the preparation of liposome
formulations
are the lipid surface charge, vesicle size and the aqueous volume of the
liposomes.
[0266] A positively charged synthetic cationic lipid, N-E1-(2,3-
dioleyloxy)propy1]-
N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes
that
interact spontaneously with nucleic acid to form lipid-nucleic acid complexes
which are
capable of fusing with the negatively charged lipids of the cell membranes of
tissue
culture cells, resulting in delivery of oligonucleotide (see, e.g., Feigner,
P. L. et al., (1987)
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
94
Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a
description of
DOTMA and its use with DNA).
[0267] A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane
(DOTAP)
can be used in combination with a phospholipid to form DNA-complexing
vesicles.
LIPOFECTINTm Bethesda Research Laboratories, Gaithersburg, Md.) is an
effective
agent for the delivery of highly anionic nucleic acids into living tissue
culture cells that
comprise positively charged DOTMA liposomes which interact spontaneously with
negatively charged polynucleotides to form complexes. When enough positively
charged
liposomes are used, the net charge on the resulting complexes is also
positive. Positively
charged complexes prepared in this way spontaneously attach to negatively
charged cell
surfaces, fuse with the plasma membrane, and efficiently deliver functional
nucleic acids
into, for example, tissue culture cells. Another commercially available
cationic lipid, 1,2-
bi s(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim,
Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked
by ester,
rather than ether linkages.
[0268] Other reported cationic lipid compounds include those that have
been conjugated
to a variety of moieties including, for example, carboxyspermine which has
been
conjugated to one of two types of lipids and includes compounds such as 5-
carboxyspermylglycine dioctaoleoylamide ("DOGS") (TRANSFECTAMTm, Promega,
Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide
("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).
[0269] Another cationic lipid conjugate includes derivatization of the
lipid with
cholesterol ("DC-Chol") which has been formulated into liposomes in
combination with
DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun.
179:280).
Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to
be
effective for transfection in the presence of serum (Zhou, X. et al., (1991)
Biochim.
Biophys. Acta 1065:8). For certain cell lines, these liposomes containing
conjugated
cationic lipids, are said to exhibit lower toxicity and provide more efficient
transfection
than the DOTMA-containing compositions. Other commercially available cationic
lipid
products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and
Lipofectamine
(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids
suitable for
the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
102701 Liposomal formulations are particularly suited for topical
administration,
liposomes present several advantages over other formulations Such advantages
include
reduced side effects related to high systemic absorption of the administered
drug,
increased accumulation of the administered drug at the desired target, and the
ability to
administer oligonucleotide into the skin. In some implementations, liposomes
are used
for delivering oligonucleotide to epidermal cells and also to enhance the
penetration of
oligonucleotide into dermal tissues, e.g., into skin. For example, the
liposomes can be
applied topically. Topical delivery of drugs formulated as liposomes to the
skin has been
documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol.
2,405-410
and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J.
and Fould-
Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene
56:267-276;
Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and
Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang,
L.,
(1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).
[0271] Non-ionic liposomal systems have also been examined to determine
their utility in
the delivery of drugs to the skin, in particular systems comprising non-ionic
surfactant
and cholesterol. Non-ionic liposomal formulations comprising NOVASOME I
(glyceryl
dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NOVASOME II
(glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver
a drug into
the dermis of mouse skin. Such formulations with oligonucleotides are useful
for treating
a dermatological disorder.
[0272] The targeting of liposomes is also possible based on, for
example, organ-
specificity, cell-specificity, and organelle-specificity and is known in the
art. In the case
of a liposomal targeted delivery system, lipid groups can be incorporated into
the lipid
bilayer of the liposome to maintain the targeting ligand in stable association
with the
liposomal bilayer. Various linking groups can be used for joining the lipid
chains to the
targeting ligand. Additional methods are known in the art and are described,
for example
in U.S. Patent Application Publication No. 20060058255, the linking groups of
which are
herein incorporated by reference.
[0273] Liposomes that include oligonucleotides can be made highly
deformable. Such
deformability can enable the liposomes to penetrate through pore that are
smaller than the
average radius of the liposome. For example, transfersomes are yet another
type of
liposomes, and are highly deformable lipid aggregates which are attractive
candidates for
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
96
drug delivery vehicles. Transfersomes can be described as lipid droplets which
are so
highly deformable that they are easily able to penetrate through pores which
are smaller
than the droplet. Transfersomes can be made by adding surface edge activators,
usually
surfactants, to a standard liposomal composition. Transfersomes that include
oligonucleotides can be delivered, for example, subcutaneously by infection to
deliver
oligonucleotides to keratinocytes in the skin. To cross intact mammalian skin,
lipid
vesicles must pass through a series of fine pores, each with a diameter less
than 50 nm,
under the influence of a suitable transdermal gradient. In addition, due to
the lipid
properties, these transfersomes can be self-optimizing (adaptive to the shape
of pores,
e.g., in the skin), self-repairing, and can frequently reach their targets
without
fragmenting, and often self-loading. Transfersomes have been used to deliver
serum
albumin to the skin. The transfersome-mediated delivery of serum albumin has
been
shown to be as effective as subcutaneous injection of a solution containing
serum
albumin.
[0274] Other suitable formulations are described in U.S. provisional
application Ser. No.
61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748,
filed Mar. 26,
2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8,2008. PCT
application No. PCT/US2007/080331, filed Oct. 3, 2007 also describes suitable.
Surfactants find wide application in formulations such as emulsions (including
microemulsions) and liposomes. The most common way of classifying and ranking
the
properties of the many different types of surfactants, both natural and
synthetic, is by the
use of the hydrophile/lipophile balance (111_,B). The nature of the
hydrophilic group (also
known as the "head") provides the most useful means for categorizing the
different
surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms,
Marcel
Dekker, Inc., New York, NY., 1988, p. 285).
[0275] If the surfactant molecule is not ionized, it is classified as a
nonionic surfactant.
Nonionic surfactants find wide application in pharmaceutical and cosmetic
products and
are usable over a wide range of pH values. In general, their HLB values range
from 2 to
about 18 depending on their structure. Nonionic surfactants include nonionic
esters such
as ethylene glycol esters, propylene glycol esters, glyceryl esters,
polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers
such as fatty alcohol ethoxylates, propoxylated alcohols, and
ethoxylated/propoxylated
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
97
block polymers are also included in this class. The polyoxyethylene
surfactants are the
most popular members of the nonionic surfactant class.
[0276] If the surfactant molecule carries a negative charge when it is
dissolved or
dispersed in water, the surfactant is classified as anionic. Anionic
surfactants include
carboxylates such as soaps, acyl lactylates, acyl amides of amino acids,
esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as
alkyl benzene
sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and
phosphates. The most
important members of the anionic surfactant class are the alkyl sulfates and
the soaps.
[0277] If the surfactant molecule carries a positive charge when it is
dissolved or
dispersed in water, the surfactant is classified as cationic. Cationic
surfactants include
quaternary ammonium salts and ethoxylated amines. The quaternary ammonium
salts are
the most used members of this class.
[0278] If the surfactant molecule has the ability to carry either a
positive or negative
charge, the surfactant is classified as amphoteric. Amphoteric surfactants
include acrylic
acid derivatives, substituted alkyl amides, N-alkylbetaines, and phosphatides.
[0279] The use of surfactants in drug products, formulations and in
emulsions has been
reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New
York,
N.Y., 1988, p. 285).
[0280] The oligonucleotides, or pharmaceutically acceptable salts
thereof, for use in the
methods can be provided as micellar formulations. Micelles are a particular
type of
molecular assembly in which amphipathic molecules are arranged in a spherical
structure
such that all the hydrophobic portions of the molecules are directed inward,
leaving the
hydrophilic portions in contact with the surrounding aqueous phase. The
converse
arrangement exists if the environment is hydrophobic
i. Lipid Nanoparticle-Based Delivery Methods
[0281] Oligonucleotides can be fully encapsulated in a lipid
formulation, e.g., a lipid
nanoparticle (LNP), or other nucleic acid-lipid particle. LNPs are extremely
useful for
systemic applications, as they exhibit extended circulation lifetimes
following intravenous
(i.v.) injection and accumulate at distal sites (e.g., sites physically
separated from the
administration site). LNPs include "pSPLP," which include an encapsulated
condensing
agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683.
The
particles typically have a mean diameter of about 50 nm to about 150 nm, more
typically
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
98
about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most
typically about 70 nm to about 90 nm, and are substantially nontoxic. In
addition, the
nucleic acids when present in the nucleic acid-lipid particles are resistant
in aqueous
solution to degradation with a nuclease. Nucleic acid-lipid particles and
their method of
preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501;
6,534,484;
6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication
No. WO
96/40964.
[0282] In one aspect, the lipid to drug ratio (mass/mass ratio) (e.g.,
lipid to
oligonucleotide ratio) will be in the range of from about 1:1 to about 50:1,
from about 1:1
to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1,
from about 5:1
to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above
recited ranges
are also contemplated.
[0283] Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-
dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium
bromide (DDAB), N--(I-(2,3-dioleoyloxy)propy1)-N,N,N-trimethylammonium
chloride
(DOTAP), N--(I-(2,3-dioleyloxy)propy1)-N,N,N-trimethylammonium chloride
(DOTMA), N,N-dimethy1-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-
N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-
dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-
dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-
(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane
(DLin-MA), 1,2-Dilinoleoy1-3-dimethylaminopropane (DLinDAP), 1,2-
Dilinoleylthio-3-
dimethylaminopropane (DLin-S-DMA), 1-Linoleoy1-2-linoleyloxy-3-
dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane
chloride salt (DLin-TMA.C1), 1,2-Dilinoleoy1-3-trimethylaminopropane chloride
salt
(DLin-TAP.C1), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or
3-
(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-
propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-
EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoley1-
4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof,
(3aR,5s,6aS)-N,N-dimethy1-2,2-di((9Z,12Z)-octadeca-9,12-dienyetetrahydro--3aH-
cyclopenta[d] [1,3 ] dioxo1-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-
6,9,28,31-
tetraen-19-y14-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
99
hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-
yeethylazanediyedidodecan-2-ol (Tech Cl), or a mixture thereof. The cationic
lipid can
comprise, for example, from about 20 mol % to about 50 mol % or about 40 mol %
of the
total lipid present in the particle.
[0284] The ionizable/non-cationic lipid can be an anionic lipid or a
neutral lipid
including, but not limited to, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine
(POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-
phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-
mal), dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine
(DMPE), di stearoyl-phosphati dyl -ethanol amine (DSPE), 16-0-monomethyl PE,
16-0-
dimethyl PE, 18-1-trans PE, 1-stearoy1-2-oleoyl-phosphatidyethanolamine
(SOPE),
cholesterol, or a mixture thereof The non-cationic lipid can be, for example,
from about 5
mol % to about 90 mol %, about 10 mol %, or about 60 mol % if cholesterol is
included,
of the total lipid present in the particle.
[0285] The conjugated lipid that inhibits aggregation of particles can
be, for example, a
polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-
diacylglycerol
(DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer),
or
a mixture thereof The PEG-DAA conjugate can be, for example, a PEG-
dilauryloxypropyl (Cu), a PEG-dimyristyloxypropyl (C14), a PEG-
dipalmityloxypropyl
(C16), or a PEG-distearyloxypropyl (Cu). The conjugated lipid that prevents
aggregation
of particles can be, for example, from 0 mol % to about 20 mol % or about 2
mol % of the
total lipid present in the particle.
[0286] In some aspects, the nucleic acid-lipid particle further
includes cholesterol at, e.g.,
about 10 mol % to about 60 mol A or about 50 mol % of the total lipid present
in the
particle.
B. Combination Therapies
[0287] An oligonucleotide, or pharmaceutically acceptable salt thereof,
can be used alone
or in combination with at least one additional therapeutic agent, e.g., other
agents that
treat nucleotide repeat expansion disorders (e.g., trinucleotide repeat
expansion disorders)
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
100
or symptoms associated therewith, or in combination with other types of
therapies to treat
nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion
disorders). In
combination treatments, the dosages of one or more of the therapeutic
compounds can be
reduced from standard dosages when administered alone. For example, doses can
be
determined empirically from drug combinations and permutations or can be
deduced by
isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)). In
this case,
dosages of the compounds when combined should provide a therapeutic effect.
[0288] In some aspects, the oligonucleotide, or pharmaceutically
acceptable salt thereof,
agents described herein can be used in combination with at least one
additional
therapeutic agent to treat a nucleotide repeat expansion disorder (e.g., a
trinucleotide
repeat expansion disorder) associated with gene having a nucleotide repeat
(e.g., any of
the trinucleotide repeat expansion disorders and associated genes having a
nucleotide
repeat listed in Table 1) In some aspects, at least one of the additional
therapeutic agents
can be an oligonucleotide (e.g., an ASO) that hybridizes with the mRNA of gene
associated with a nucleotide or trinucleotide repeat expansion disorder (e.g.,
any of the
genes listed in Table 1). In some aspects, the nucleotide repeat expansion
disorder (e.g., a
trinucleotide repeat expansion disorder) is Huntington's disease (HD). In some
aspects,
the gene associated with a nucleotide repeat expansion disorder (e.g., a
trinucleotide
repeat expansion disorder) is Huntingtin (HTT). Several allelic variants of
the Huntingtin
gene have been implicated in the etiology of Huntington's disease. In some
cases, these
variants are identified on the basis of having unique HD-associated single
nucleotide
polymorphisms (SNPs). In some aspects, the oligonucleotide hybridizes to an
mRNA of
the Huntingtin gene containing any of the HD-associated SNPs known in the art
(e.g., any
of the RD-associated SNPs described in Skotte et al., PLoS One 2014, 9(9).
e107434,
Carroll et al., Mol. Ther. 2011, 19(12): 2178-85, Warby et al., Am. J. Hum.
Gen. 2009,
84(3): 351-66 (herein incorporated by reference)). In some aspects, the
oligonucleotide
that is an additional therapeutic agent hybridizes to an mRNA of the
Huntingtin gene
lacking any of the HD-associated SNPs. In some of the aspects, the
oligonucleotide, or
pharmaceutically acceptable salt thereof, that is an additional therapeutic
agent hybridizes
to an mRNA of the Huntingtin gene having any of the SNPs selected from the
group of
rs362307 and rs365331. In some aspects, the oligonucleotide, or
pharmaceutically
acceptable salt thereof, that is an additional therapeutic agent can be a
modified
oligonucleotide (e.g., an oligonucleotide including any of the modifications
described
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
101
herein). In some aspects, the modified oligonucleotides that is an additional
therapeutic
agent comprise one or more phosphorothioate internucleoside linkages. In some
aspects,
the modified oligonucleotide comprises one or more 2'-MOE moieties. In some
aspects,
the oligonucleotide that is an additional therapeutic agent that hybridizes to
the mRNA of
the Huntingtin gene has a sequence selected from the SEQ ID NOs. 6-285 of US
Patent
No. 9,006,198; SEQ ID NOs. 6-8 of US Patent Application Publication No.
2017/0044539; SEQ ID NOs. 1-1565 of US Patent Application Publication
2018/0216108; and SEQ ID NOs. 1-2432 of PCT Publication WO 2017/192679, the
sequences of which are hereby incorporated by reference.
[0289] In some aspects, at least one of the additional
therapeutic agents is a
chemotherapeutic agent (e.g., a cytotoxic agent or other chemical compound
useful in the
treatment of a nucleotide repeat expansion disorder, e.g., a trinucleotide
repeat expansion
disorder).
[0290] In some aspects, at least one of the additional therapeutic
agents can be a
therapeutic agent which is a non-drug treatment. For example, at least one of
the
additional therapeutic agents is physical therapy.
[0291] In any of the combination aspects described herein, the two or
more therapeutic
agents are administered simultaneously or sequentially, in either order. For
example, a
first therapeutic agent can be administered immediately, up to 1 hour, up to 2
hours, up to
3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8
hours, up to 9
hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14
hours, up to
hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21
hours, up to
22 hours, up to 23 hours up to 24 hours or up to 1-7, 1-14, 1-21 or 1-30 days
before or
after one or more of the additional therapeutic agents
V. Pharmaceutical Compositions
[0292] The oligonucleotides, or pharmaceutically acceptable salt
thereof, described
herein are formulated into pharmaceutical compositions for administration to
human
subjects in a biologically compatible form suitable for administration in
vivo.
[0293] The compounds described herein can be used in the form of the
free base, in the
form of salts, solvates, and as prodrugs. All forms are within the methods
described
herein. In accordance with the methods described herein, the described
oligonucleotides
or salts, solvates, or prodrugs thereof can be administered to a patient in a
variety of
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
102
forms depending on the selected route of administration, as will be understood
by those
skilled in the art. The compounds described herein can be administered, for
example, by
oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal,
buccal, sublingual,
intraocular (subretinal, intravitreal), intra cisterna magna (ICM), nasal,
rectal, patch,
pump, or transdermal administration and the pharmaceutical compositions
formulated
accordingly. Parenteral administration includes intravenous, intraperitoneal,
subcutaneous, intramuscular, transepitheli al, nasal, intrapulmonary,
intrathecal,
intraocular, intracerebroventricular, intraparenchymal, rectal, and topical
modes of
administration. Parenteral administration can be by continuous infusion over a
selected
period of time.
[0294] A compound described herein can be orally administered, for
example, with an
inert diluent or with an assimilable edible carrier, or it can be enclosed in
hard or soft
shell gelatin capsules, or it can be compressed into tablets, or it can be
incorporated
directly with the food of the diet For oral therapeutic administration, a
compound
described herein can be incorporated with an excipient and used in the form of
ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and
wafers. A
compound described herein can be administered parenterally. Solutions of a
compound
described herein can be prepared in water suitably mixed with a surfactant,
such as
hydroxypropylcellulose. Dispersions can be prepared in glycerol, liquid
polyethylene
glycols, DMSO, and mixtures thereof with or without alcohol, and in oils.
Under
ordinary conditions of storage and use, these preparations can contain a
preservative to
prevent the growth of microorganisms. Conventional procedures and ingredients
for the
selection and preparation of suitable formulations are described, for example,
in
Remington's Pharmaceutical Sciences (2012, 22nd ed.) and in The United States
Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. The
pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile
injectable
solutions or dispersions. In all cases the form must be sterile and must be
fluid to the
extent that can be easily administered via syringe. Compositions for nasal
administration
can conveniently be formulated as aerosols, drops, gels, and powders. Aerosol
formulations typically include a solution or fine suspension of the active
substance in a
physiologically acceptable aqueous or non-aqueous solvent and are usually
presented in
single or multidose quantities in sterile form in a sealed container, which
can take the
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
103
form of a cartridge or refill for use with an atomizing device. Alternatively,
the sealed
container can be a unitary dispensing device, such as a single dose nasal
inhaler or an
aerosol dispenser fitted with a metering valve which is intended for disposal
after use.
Where the dosage form includes an aerosol dispenser, it will contain a
propellant, which
can be a compressed gas, such as compressed air or an organic propellant, such
as
fluorochlorohydrocarbon. The aerosol dosage forms can take the form of a pump-
atomizer. Compositions suitable for buccal or sublingual administration
include tablets,
lozenges, and pastilles, where the active ingredient is formulated with a
carrier, such as
sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal
administration
are conveniently in the form of suppositories containing a conventional
suppository base,
such as cocoa butter
[0295] The compounds described herein can be administered to an animal,
e.g., a human,
alone or in combination with pharmaceutically acceptable carriers, as noted
herein, the
proportion of which is determined by the solubility and chemical nature of the
compound,
chosen route of administration, and standard pharmaceutical practice.
Dosages
[0296] The dosage of the compositions (e.g., a composition including an
oligonucleotide,
or pharmaceutically acceptable salt thereof, described herein, can vary
depending on
many factors, such as the pharmacodynamic properties of the compound; the mode
of
administration; the age, health, and weight of the recipient; the nature and
extent of the
symptoms; the frequency of the treatment, and the type of concurrent
treatment, if any;
and the clearance rate of the compound in the animal to be treated. The
compositions
described herein can be administered initially in a suitable dosage that can
be adjusted as
required, depending on the clinical response. In some aspects, the dosage of a
composition (e.g., a composition including an oligonucleotide, or
pharmaceutically
acceptable salt thereof,) is a prophylactically or a therapeutically effective
amount.
VII Kits
[0297] Kits including (a) a pharmaceutical composition including an
oligonucleotide, or
pharmaceutically acceptable salt thereof, agent that reduces the level and/or
activity of
MSH3 in a cell or subject described herein, and (b) a package insert with
instructions to
perform any of the methods described herein are contemplated In some aspects,
the kit
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
104
includes (a) a pharmaceutical composition including an oligonucleotide, or
pharmaceutically acceptable salt thereof, agent that reduces the level and/or
activity of
MSH3 in a cell or subject described herein, (b) an additional therapeutic
agent, and (c) a
package insert with instructions to perform any of the methods described
herein.
EXAMPLE
Example I. Design and Selection of Antisense Oligonucleotides
[0298] Identification and selection of target transcripts: Target
transcript selection and
off-target scoring (below) utilized NCBI RefSeq sequences, downloaded from
NCBI 21
Nov. 2018. Experimentally validated "NM" transcript models were used except
for
cynomolgus monkey, which only has "XM" predicted models for the large majority
of
genes. The longest human, mouse, rat, and cynomolgus monkey MSH3 transcript
that
contained all mapped internal exons was selected (SEQ IDs 385, 386, 387, and
388 for
human, mouse, rat, and cynomolgus monkey, respectively, SEQ ID NO:389 is the
protein
sequence)
Knock Down by ASOs
[0299] ASO screen in Hela cells to identify the top ASO in Table 3 for
the MSH3 gene
was performed by Horizon.
[0300] In summary: ASO knockdown activity was evaluated in HeLa by
transfection at 1
nM and 10 nM. mRNA knockdown was analyzed by quantitative reverse
transcription
polymerase chain reaction (RT-qPCR) using TaqMan Gene Expression probes. mRNA
expression was calculated via delta-delta Ct (AACT) method where target
expression was
normalized to expression of the reference gene beta-glucuronidase (GUSB) and
to cells
treated with a scrambled luciferase targeting control ASO.
Transfection in HeLa cells
[0301] ASOs were resuspended in dH20 to 1000-fold their final assay
concentration (10
uM or 1 uM). ASOs were dispensed in quadruplicates and complexed with 5 ul of
Lipofectamine 3000 (Invitrogen) for 20 minutes before HeLa cells were added at
2,500
cells/well. Cells were cultured under standard culturing conditions for 24
hours. Cells
were processed for RT-qPCR readout using the Cells-to-CT 1-step TaqMan Kit
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
105
(1nvitrogen) according to manufacturer's instructions. TaqMan Gene Expression
probe
for MSH3 was 1-1s00989003 ml (Life Technologies Ltd) on a QuantStudio 6
(Applied
BioSystems).
Table 2: Key to Chemical Modifiers in Tables 3 and 4
"s" after base phosphorothioate linkage
"p" after base phosphodiester linkage
"o" before base moe (2'-0-methoxyethyl -RNA)
"d" before base deoxy (a DNA nucleoside)
ACTG core DNA bases: adenine; cytosine; thymine; and
guanine
methyl at position 5 on the nucleobase; all C (cytosine) are 5-methyl.
"moe synonymous with "moe T"
"moe T" synonymous with "moe II"
LNA (e.g., A-LNA, 5mC-LNA, G-LNA, and T-LNA)
[0302]
moeT can be substituted for one or more of the moeU nucleotides listed
in any of
the sequences below. Similarly, moeU can be substituted for one or more of the
modT
nucleotides listed in any of the sequences below.
[0303] In Table 3 below, the SEQ ID No. corresponds to the nucleobase
sequence of the
Antisense Oligo No. However, the specific Antisense Oligo No. (e.g., Antisense
Oligo
No. 1) includes the specified chemical modifications.
Table 3.
Mean % mRNA SEM %
mRNA
Remaining
Remaining
Antisense
Oligo No./
Chem Mod Seq 1 nM 10 nM 1 nM
10 nM
SEQ ID
NO:
1 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 86.09785 41.79781
10.956696 1.978577
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloA]
2 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 102.13406 40.30929
2.5403645 1.368438
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
106
3 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 86.89053 37.50409
4.95608 1.237848
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloA]
4 [oCsloUsloAploGploGOTOGsIdAsid 88.21922 46.14382 8.324711
2.068448
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloA]
[oCsloUsloAsloGsloGsIdTsIdGsIdAsId 88.06973 44.49033 7.0556625 1.637641
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloA]
6 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 66.27929 38.82585
7.304814 2.701934
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloA]
7 [oCsloUsloAsloGsloGsIdTsIdGspAsId 87.77148 39.91306
3.7650095 1.899814
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloA]
8 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 93.78302 39.9579
5.800865 2.591389
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloA]
9 [oCsloUsloAploGsloGOTOGsIdAsId 91.70171 46.43532 6.7248825
1.898713
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloA]
[oCsloUsloAsloGploGsIdTsIdGspAsId 90.12033 43.42065 11.205575 2.148471
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAl
11 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 99.38491 40.8259
7.1537125 4.453034
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloA]
12 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 92.85072 47.68991
9.7849105 0.732238
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloA]
13 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 82.76355 44.94987
2.3796215 1.213861
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloA]
14 [oCsloUsloAploGploGOTOGsIdAsid 100.30431 42.47324 7.2137325
3.762178
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloA]
[oCsloUsloAploGploGOTOGsIdAsid 90.00654 39.81399 10.431815 2.091189
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloA]
16 [oCsloUsloAploGploGsIdTsIdGsIdAsid 93.50777 41.81402
6.027847 2.177984
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloA]
17 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 78.87919 39.04837
4.3289815 3.888626
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloC]
18 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 89.8343 37.95063
6.6130055 2.550701
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
107
19 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 91.97128 45.99061
1.903787 2.837245
TsIdGs15mCsIdAs15mCsIdTsIdGsKs1
oUsloUsloUsloAsloC]
20 [oCsloUsloAploGploGOTOGsIdAsid 90.0287 45.36505
7.652128 3.536383
TsIdGs15mCsIdAs15mCsIdTsIdGsKs1
oUsloUsloUsloAsloC]
21 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 80.24857 37.65808
8.935247 5.090914
TsIdGs15mCsIdAs15mCsIdTsIdGs10Cs1
oUploUsloUsloAsloC]
22 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 101.45303 37.27497
23.097582 3.052247
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloC]
23 [oCsloUsloAsloGsloGsIdTsIdGspAsId 97.15427 41.07113
12.915649 3.502463
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloC]
24 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 80.67146 37.30983
3.1350315 4.460652
TsIdGs15mCsIdAs15mCsIdTsIdGs10Cs1
oUploUsloUsloAsloC]
25 [oCsloUsloAploGsloGOTOGsIdAsId 90.35424 39.93492 10.3572485
3.866769
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloC]
26 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 82.80083 43.89603
2.679553 3.062266
TsIdGs15mCsIdAs15mCsIdTsIdGsKs1
oUploUsloUsloAsloC]
27 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 88.08068 46.08224
6.516991 2.883583
TOGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloC]
28 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 100.38683 42.14532
7.9455835 .. 2.066183
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloC]
29 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 82.39148 42.89111
4.9474165 1.687775
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloC]
30 [oCsloUsloAploGploGOTOGsIdAsid 92.49721 41.87519 4.286409
1.419928
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloC]
31 [oCsloUsloAploGploGOTOGsIdAsid 88.1524 43.50525
4.0152095 1.165505
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloC]
32 1oCsloUsloAploGploGOTOGOAsid 95.59035 44.36914 6.098232
1.947992
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloC]
33 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 81.5396 46.37686
5.051866 2.340369
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloA]
34 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 75.50977 37.42608
6.55804 2.427113
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
108
35 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 86.89931 38.66673
1.6915285 0.403006
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloA]
36 [oCsloUsloAploGploGOTOGsIdAsid 92.05609 44.46799 6.699515
0.811156
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloA]
37 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 82.63172 39.09093
8.238769 2.549669
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloA]
38 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 102.24694 36.96398
14.530799 2.884441
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloA]
39 [oCsloUsloAsloGsloGsIdTsIdGspAsId 87.2629
39.3864 12.0735685 1.500768
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloA]
40 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 96.78945 37.19026
9.535124 1.52692
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloA]
41 [oCsloUsloAploGsloGOTOGsIdAsId 95.79925 45.78015 7.062957
5.396491
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloA]
42 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 87.43444 39.82331
10.1333325 1.186569
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloAl
43 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 93.03859 41.55274
8.5237595 1.542313
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloA]
44 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 79.29174 41.69657
9.4802245 3.7328
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloA]
45 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 97.84352 40.24762
4.024119 3.662828
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloA]
46 [oCsloUsloAploGploGOTOGsIdAsid 94.18266 36.69312 3.2339695
2.577563
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloA]
47 [oCsloUsloAploGploGOTOGsIdAsid 97.71451 49.27591 15.6053125
5.231299
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloA]
48 [oCsloUsloAploGploGsIdTsIdGsIdAsid 91.5412
44.77662 6.4424815 4.030398
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloA]
49 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 85.14544 43.97438
5.329607 1.787627
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloAsloC]
50 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 81.85128 40.2583
5.194841 1.498131
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
109
51 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 79.48116 41.09427
2.3949705 1.899097
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloAsloC]
52 [oCsloUsloAploGploGOTOGsIdAsid 82.36726 40.83192 5.178712
3.06104
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUsloUsloUsloAsloCsloAsloC]
53 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 73.45186 36.40204
4.508506 2.072795
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloAsloC]
54 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 83.26679 42.28042
9.2488475 3.422593
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloAsloC]
55 [oCsloUsloAsloGsloGsIdTsIdGspAsId 73.0733 44.80567
3.2515675 5.215558
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloAsloC]
56 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 86.11374 41.79482
6.120118 3.145374
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloAsloC]
57 [oCsloUsloAploGsloGOTOGsIdAsId 82.10891 41.5037 10.9839625
2.257748
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloAsloC]
58 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 89.70608 40.60014
3.8721065 0.87326
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloAsloC]
59 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 93.23447 43.85248
4.0394125 4.050107
TOGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloAsloC]
60 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 80.85138 43.55119
6.949772 3.486459
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloAsloC]
61 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 77.98907 37.1955
9.4067375 1.362401
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloAsloC]
62 [oCsloUsloAploGploGOTOGsIdAsid 71.11867 37.22296 7.9519255
2.922158
TsIdGs15mCsIdAs15mCsIdTsIdGsloCs1
oUploUsloUsloAsloCsloAsloC]
63 [oCsloUsloAploGploGOTOGsIdAsid 81.67628 40.47476 2.889408
2.316071
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUsloUsloUsloAsloCsloAsloC]
64 [oCsloUsloAploGploGsIdTsIdGsIdAsid 79.69746 38.81524
7.742031 0.204948
TsIdGs15mCsIdAs15mCsIdTsIdGsloCp
oUploUsloUsloAsloCsloAsloC]
65 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 69.99261 32.0998
5.4826715 3.037028
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUsloUsloUsloAsloC]
66 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 75.98915 34.73835
3.798421 3.084744
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUsloUsloUsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
110
67 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 83.04812 42.68424
4.758469 4.679494
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUsloUsloUsloAsloC]
68 [oCsloUsloAploGploGOTOGsIdAsid 74.06207 38.55632 9.9099225
7.981079
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUsloUsloUsloAsloC]
69 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 81.32662 38.51888
1.5401265 2.565575
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUploUsloUsloAsloC]
70 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 79.67053 27.90351
2.039101 1.648429
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PloUsloUsloUsloAsloC]
71 [oCsloUsloAsloGsloGsIdTsIdGspAsId 72.32385 34.23619
4.994041 3.100586
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
ploUPloUsloUsloAsloC]
72 [oCsloUsloAploGsloGOTOGsIdAsId 73.88174 35.08737 3.987852
3.319998
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUploUsloUsloAsloC]
73 [oCsloUsloAploGsloGOTOGsIdAsId 73.84298 39.89966 6.744107
3.444095
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PloUsloUsloUsloAsloC]
74 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 87.48091 31.76795
4.7688275 2.561686
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUploUsloUsloAsloC]
75 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 85.5695 37.95998
7.860177 4.320679
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
ploUsloUsloUsloAsloC]
76 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 82.15887 38.7112
6.4298045 2.851738
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PloUPloUsloUsloAsloC]
77 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 92.45412 41.20665
7.931752 5.873654
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PloUPloUsloUsloAsloC]
78 [oCsloUsloAploGploGOTOGsIdAsid 87.20785 34.56143 6.5475275
3.904361
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sloUploUsloUsloAsloC]
79 [oCsloUsloAploGploGOTOGsIdAsid 77.67484 47.25886 4.025454
9.140193
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PloUsloUsloUsloAsloC]
80 [oCsloUsloAploGploGsIdTsIdGsIdAsid 84.26525 44.90753
2.9419125 3.931427
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PloUPloUsloUsloAsloC]
81 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 74.15488 28.91957
4.4416545 2.079845
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTsloUsloUsloAsloCsloA]
82 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 83.52985 34.76393
7.7233095 5.276733
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTsloUsloUsloAsloCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
111
83 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 87.34228 34.26426
2.8872435 3.491405
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTsloUsloUsloAsloCsloA]
84 [oCsloUsloAploGploGOTOGsIdAsid 85.67873 35.6361 5.8302875
2.544952
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTsloUsloUsloAsloCsloA]
85 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 75.35312 32.86732
10.951265 3.621277
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTploUsloUsloAsloCsloA]
86 [oCsloUsloAsloGsloGsIdTsIdGsIdAsId 79.96271 34.14079
9.351093 1.427892
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PldTsloUsloUsloAsloCsloA]
87 [oCsloUsloAsloGsloGsIdTsIdGspAsId 100.34146 32.93465
7.409104 1.965485
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PldTPloUsloUsloAsloCsloA]
88 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 76.43184 29.11159
1.9009085 1.712673
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTploUsloUsloAsloCsloA]
89 [oCsloUsloAploGsloGOTOGsIdAsId 84.95746 33.1336 6.0100595
0.819655
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PldTsloUsloUsloAsloCsloA]
90 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 77.40614 34.79199
2.737082 1.090979
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTploUsloUsloAsloCsloA]
91 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 75.61531 41.57976
2.6137425 2.594538
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
pldTsloUsloUsloAsloCsloA]
92 [oCsloUsloAploGsloGsIdTsIdGsIdAsId 81.40445 36.37792
7.319196 0.14288
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PldTPloUsloUsloAsloCsloA]
93 [oCsloUsloAsloGploGsIdTsIdGsIdAsId 87.14688 41.27511
2.983684 3.624091
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PldTPloUsloUsloAsloCsloA]
94 [oCsloUsloAploGploGOTOGsIdAsid 83.39023 40.63864 1.431887
1.205991
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
sIdTploUsloUsloAsloCsloA]
95 [oCsloUsloAploGploGOTOGsIdAsid 78.10296 40.32743 4.682538
1.18152
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PldTsloUsloUsloAsloCsloA]
96 1oCsloUsloAploGploGOTOGsIdAsid 78.36184 41.53986 3.2471535
4.451705
TsIdGs15mCsIdAs15mCsIdTsIdGs15mC
PldTPloUsloUsloAsloCsloA]
97 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 73.53306 32.22054
5.7741956 1.828104
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloU]
98 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 85.30286 28.26088
7.1938475 1.170224
GspAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloC sloUsloU]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
112
99 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 80.72095 31.14705
6.94178635 0.847079
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloU]
100 [oUs oGsloCploUploAsIdGsIdGs dTsld 68.59723 30.44545
5.6598163 1.925702
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloU]
101 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 78.30012 29.94689
6.2378524 4.046731
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloU]
102 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 78.25236 30.06093
5.9663877 1.79938
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oU]
103 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 69.03511 35.56989
2.59911095 2.403012
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGPloCsloUsloU]
104 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 73.74024 30.77626
1.62019145 0.873961
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloU]
105 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 72.2508
33.81869 4.46870435 3.070223
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oU]
106 [oUs oGsloCsloUploAsIdGsIdGsIdTsId 80.18669 27.94099
3.6700273 0.683295
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUl
107 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 74.09831 34.42556
6.01870395 1.988182
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oU]
108 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 70.94729 35.1242
1.85488915 3.987895
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
10GPloCsloUsloU1
109 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 78.33754 34.87067
4.7590703 1.027952
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGploCsloUsloU]
110 [oUs oGsloCploUploAsIdGsIdGs dTsld 62.32599 33.30946
3.7307496 2.867815
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloU]
111 [oUs oGsloCploUploAsIdGsIdGs dTsld 70.12462 35.35927
3.28310255 2.413138
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oU]
112 [oUs oGsloCploUploAsIdGsIdGs dTsld 76.05292 32.20627
7.8049074 3.219622
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGPloCsloUsloUl
113 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 65.23498 32.17334
3.72245235 1.859401
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloU]
114 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 69.29675 32.68384
2.39326015 4.127576
GspAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloC sloUsloUsloU]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
113
115 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 70.21315 28.19354
5.9917878 1.260233
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloU]
116 [oUs oGsloCploUploAsIdGsIdGs dTsld 61.38246 27.89351
2.31272875 0.940955
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloU]
117 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 67.38278 29.83921
4.699614 3.62231
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloU]
118 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 74.8669
33.51358 4.66523895 3.23944
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
10GsloCsloUs oUsloU]
119 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 75.87658 33.86225
3.36764485 2.561053
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GPloCsloUsloUsloU]
120 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 74.89974 30.42127
5.3692428 1.390075
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloU]
121 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 77.25051 35.01204
10.42313865 2.358081
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GsloCsloUs oUsloU]
122 [oUs oGsloCsloUploAsIdGsIdGsIdTsId 68.7315 29.79101
4.2400331 3.545545
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUl
123 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 71.42427 34.17864
4.1743313 2.028014
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oUsloU]
124 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 86.14101 31.27527
4.48923135 2.071295
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GPloCsloUsloUsloU]
125 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 77.77647 31.7119
4.05551285 1.114638
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGploCsloUsloUsloU]
126 [oUs oGsloCploUploAsIdGsIdGs dTsld 78.6535
37.61556 1.55106765 2.362156
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloU]
127 [oUs oGsloCploUploAsIdGsIdGs dTsld 67.14048 33.39059
1.7917388 1.42932
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GsloCsloUs oUsloU]
128 [oUs oGsloCploUploAsIdGsIdGs dTsld 86.42336 39.03898
2.597663 2.239799
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGploCsloUsloUsloU]
129 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 68.63405 35.13624
1.53173715 2.451205
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloA]
130 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 69.38908 31.9245
4.0133836 3.413571
GspAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
114
131 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 82.64957 31.6315
10.9520439 1.487167
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloA]
132 [oUs oGsloCploUploAsIdGsIdGs dTsld 76.22312 31.57458
5.88891735 2.253033
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloA]
133 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 73.47365 27.08401
3.77061195 0.92052
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloA]
134 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 70.30414 33.09977
4.27095005 3.045878
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
10GsloCsloUs oUsloUsloA]
135 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 72.20899 30.08256
6.4512196 1.841916
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GPI0CsloUsloUsloUsloA]
136 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 67.44648 31.14491
2.3933433 2.847244
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloA]
137 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 73.72042 31.65609
3.1864744 1.736871
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GsloCsloUs oUsloUsloA]
138 [oUs oGsloCsloUploAsIdGsIdGsIdTsId 67.00788 29.57105
4.13148395 0.996282
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloAl
139 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 67.67345 29.52463
2.84221865 1.821037
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oUsloUsloA]
140 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 74.02419 34.75728
4.05854245 2.515477
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GPloCsloUsloUsloUsloA]
141 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 70.79225 32.87075
1.72015605 1.818799
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGploCsloUsloUsloUsloA]
142 [oUs oGsloCploUploAsIdGsIdGs dTsld 80.55014 28.38316
4.20708315 1.230702
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloA]
143 [oUs oGsloCploUploAsIdGsIdGs dTsld 62.97423 33.06359
2.1320595 3.346436
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
19GsloCsloUs oUsloUsloA]
144 [oUs oGsloCploUploAsIdGsIdGs dTsld 70.79163 33.67681
5.18027175 3.32755
GsIdAsIdTsIdGs15mCsIdAs15mCsloUp
loGploCsloUsloUsloUsloA]
145 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 61.07007 32.76229
1.578968 2.337807
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloAsloC]
146 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 77.42478 34.61478
7.0884636 3.366066
GspAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
115
147 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 67.36325 38.44457
3.158251 2.192247
GslclAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloAsloC]
148 [oUs oGsloCploUploAsIdGsIdGs dTsld 69.55673 34.56908
2.1775117 2.805347
GslclAsIdTsIdGs15mCsIdAs15mCsloUsl
oGsloCsloUsloUsloUsloAsloC]
149 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 82.73055 33.15298
3.68306915 2.319368
GslclAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloAsloC]
150 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 74.0629 34.61137
4.9070243 1.082019
GslclAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oUsloUsloAsloC]
151 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 86.07845 39.51103
3.76062145 3.134823
GslclAsIdTsIdGs15mCsIdAs15mCsloUp
10GPloCsloUsloUsloUsloAs oC]
152 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 82.2174 29.35929
3.78661745 1.69256
GslclAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloAsloC]
153 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 75.98421 35.81497
2.16191275 2.093193
GslciAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oUsloUsloAsloC]
154 [oUs oGsloCsloUploAsIdGsIdGsIdTsId 68.75699 32.99567
4.0256092 1.228557
GslclAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloAsloC]
155 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 65.0077 33.91453
4.8970901 1.843589
GslclAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oUsloUsloAsloC]
156 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 81.28814 36.71857
4.66906275 3.495077
GslclAsIdTsIdGs15mCsIdAs15mCsloUp
10GPloCsloUsloUsloUsloAs oC]
157 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 69.80645 66.34306
2.97949155 17.28836
GslclAsIdTsIdGs15mCsIdAs15mCsloUp
loGploCsloUsloUsloUsloAs oC]
158 [oUs oGsloCploUploAsIdGsIdGs dTsld 68.37275 72.33517
4.17040095 18.58276
GsIdAsIdTsIdGs15mCsIdAs15mCsloUsl
oGploCsloUsloUsloUsloAsloC]
159 [oUs oGsloCploUploAsIdGsIdGs dTsld 72.61681 47.70773
2.2537468 6.897452
GslclAsIdTsIdGs15mCsIdAs15mCsloUp
loGsloCsloUs oUsloUsloAsloC]
160 [oUs oGsloCploUploAsIdGsIdGs dTsld 68.78353 84.24789
3.0864697 31.8067
GslclAsIdTsIdGs15mCsIdAs15mCsloUp
loGploCsloUsloUsloUsloAs oC]
161 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 69.3108
75.14277 5.38158025 25.56809
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGsloCsloUsloUsloU]
162 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 72.9714 69.75189
5.9076121 32.69531
GslclAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGsloC sloUsloUsloU]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
116
163 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 59.9413
33.69336 2.7716944 2.300999
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGsloCsloUsloUsloU]
164 [oUs oGsloCploUploAsIdGsIdGs dTsld 65.88331 42.9639
6.38280315 2.950032
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGsloCsloUsloUsloU]
165 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 60.39611 41.07525
4.74049725 1.401234
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGploCsloUsloUsloU]
166 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 75.65893 53.6832
6.7614234 2.709928
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
oGsloCsloUsloUsloU]
167 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 78.27304 68.71663
3.6412356 20.69837
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
oGploCsloUsloUsloU]
168 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 69.66589 40.46645
4.8223807 1.966849
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGploCsloUsloUsloU]
169 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 65.01222 51.18193
2.0901017 5.432837
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTpl
oGsloCsloUsloUsloU]
170 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 64.20334 44.9343
3.03968285 2.38631
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGploCsloUsloUsloUl
171 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 74.50087 46.7009
7.1516196 3.17865
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
oGsloCsloUsloUsloU]
172 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 66.0219 51.29474
5.5452825 7.02709
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTpl
oGploCsloUsloUsloU]
173 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 88.90626 43.66599
7.76517935 4.598041
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
oGploCsloUsloUsloU]
174 [oUs oGsloCploUploAsIdGsIdGs dTsld 70.63885 81.73461
10.1429891 39.6005
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
oGploCsloUsloUsloU]
175 [oUs oGsloCploUploAsIdGsIdGs dTsld 69.65344 57.02863
4.09660005 13.08032
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTpl
oGsloCsloUsloUsloU]
176 [oUs oGsloCploUploAsIdGsIdGs dTsld 71.0292 88.44799
3.57082955 37.50085
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
oGploCsloUsloUsloU]
177 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 66.62139 41.51005
2.37850925 4.804473
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
dGsloCsloUsloUsloUsloA]
178 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 72.29763 44.50319
6.87317685 2.677576
GspAsIdTsIdGs15mCsIdAs15mCsidTs1
dGsloCsloUsloUsloUsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
117
179 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 68.20743 47.68644
5.01288235 7.92615
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
dGsloCsloUsloUsloUsloA]
180 [oUs oGsloCploUploAsIdGsIdGs dTsld 68.99648 42.6724
0.11444645 1.915543
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
dGsloCsloUsloUsloUsloA]
181 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 74.55986 75.31866
4.8690439 16.158
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
dGploCsloUsloUsloUsloA]
182 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 90.17217 57.85726
6.4500736 10.25964
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
dGsloCsloUsloUsloUsloA]
183 [oUs oGsloCsloUsloAsIdGsIdGsIdTsld 79.6849
44.0499 4.4692627 2.659523
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
dGploCsloUsloUsloUsloA]
184 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 72.7416 46.00267
6.4985466 1.990908
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
dGploCsloUsloUsloUsloA]
185 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 78.36759 57.88874
8.4461547 7.916173
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTpl
dGsloCsloUsloUsloUsloA]
186 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 77.17065 41.09619
4.06900365 5.266146
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
dGploCsloUsloUsloUsloAl
187 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 71.80721 47.54313
4.0522398 4.300292
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
dGsloCsloUsloUsloUsloA]
188 [oUs oGsloCploUsloAsIdGsIdGsIdTsld 83.68087 47.27023
11.6233265 2.662367
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTpl
dGploCsloUsloUsloUsloA]
189 [oUs oGsloCsloUploAsIdGsIdGsIdTsld 90.45532 46.05918
4.6439005 3.872029
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
dGploCsloUsloUsloUsloA]
190 [oUs oGsloCploUploAsIdGsIdGs dTsld 89.19433 34.62675
9.5661226 4.858138
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTs1
dGploCsloUsloUsloUsloA]
191 [oUs oGsloCploUploAsIdGsIdGs dTsld 64.72372 43.20547
3.7289791 2.557415
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTpl
dGsloCsloUsloUsloUsloA]
192 [oUs oGsloCploUploAsIdGsIdGs dTsld 92.70258 41.35463
12.14039325 0.531077
GsIdAsIdTsIdGs15mCsIdAs15mCsIdTP1
dGploCsloUsloUsloUsloA]
193 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 41.82279 25.79387
2.85807925 1.785736
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGsloCsloAsloA]
194 [oUs oGsloAploUs oCs15mCsIdTsIdGs 34.50835 24.88784
2.37599485 2.49983
IdTs1dTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
118
195 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 34.48394 27.68268
3.0471537 1.050805
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloA]
196 [oUs oGsloAploUploCs15mCsIdTsIdG 35.91642 27.09371
0.5861107 3.367908
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGsloCsloAsloA]
197 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 40.41755 30.50893
3.2331104 1.618927
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGploCsloAsloA]
198 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 37.18947 26.7628
1.96874185 1.810888
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGsloCsloAsloA]
199 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 41.58604 32.4533
2.0955341 3.083019
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGploCs oAsloA]
200 [oUs oGsloAploUs oCs15mCsIdTsIdGs 41.50165 29.4431
1.99516615 2.336989
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloA]
201 [oUs oGsloAploUs oCs15mCsIdTsIdGs 42.01393 23.06252
1.3193855 4.944764
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10APloGsloCsloAsloA]
202 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 41.74463 27.31196
3.3971388 1.186669
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloA]
203 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 40.8989
27.94533 2.5002774 2.019763
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAploGsloCsloAsloA]
204 [oUs oGsloAploUs oCs15mCsIdTsIdGs 39.77508 30.17107
2.68253975 3.295311
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10AP10GPloCsloAsloA1
205 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 40.56325 26.69679
1.7302717 0.745227
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10API0GPloCsloAsloA]
206 [oUs oGsloAploUploCs15mCsIdTsIdG 39.89604 19.42737
3.86707555 1.982879
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGploCsloAsloA]
207 [oUs oGsloAploUploCs15mCsIdTsIdG 40.47438 25.35393
2.3847392 1.251586
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGsloCsloAsloA]
208 [oUs oGsloAploUploCs15mCsIdTsIdG 39.84144 26.46836
2.76645765 1.184643
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGploCsloAsloA]
209 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 36.38624 24.02288
1.92817845 1.780277
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGsloCsloAsloAsloC]
210 [oUs oGsloAploUs oCs15mCsIdTsIdGs 36.27817 23.59247
1.9107749 1.785808
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
119
211 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 40.23379 26.02175
3.7752339 0.579919
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloAsloC]
212 [oUs oGsloAploUploCs15mCsIdTsIdG 38.73671 25.57041
2.1198962 0.563413
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGsloCsloAsloAsloC]
213 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 35.00403 27.29374
0.97998025 2.42817
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGploCsloAsloAsloC]
214 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 35.15246 27.73562
3.7541248 1.408177
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGsloCsloAsloAsloC]
215 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 39.43029 24.36535
0.35631305 2.03814
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGploCs oAsloAsloC1
216 [oUs oGsloAploUs oCs15mCsIdTsIdGs 38.4821
24.80134 2.7737859 0.915852
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloAsloC]
217 [oUs oGsloAploUs oCs15mCsIdTsIdGs 38.86511 22.24657
1.93345215 1.178782
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10APloGsloCsloAsloAsloC]
218 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 40.69835 26.7572
2.8425562 2.117941
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloAsloC]
219 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 39.97252 26.31039
1.18627875 1.508108
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAploGsloCsloAsloAsloC]
220 [oUs oGsloAploUs oCs15mCsIdTsIdGs 40.94544 24.39298
2.69640305 1.085099
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10AP10GPloCsloAsloAsloC]
221 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 39.59504 26.25572
2.87140485 1.887767
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAploGploCsloAsloAsloC]
222 [oUs oGsloAploUploCs15mCsIdTsIdG 34.32736 25.9504 1.1160027
1.377325
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGploCsloAsloAsloC]
223 [oUs oGsloAploUploCs15mCsIdTsIdG 40.65856 25.62251
1.38290445 2.322099
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGsloCsloAsloAsloC]
224 [oUs oGsloAploUploCs15mCsIdTsIdG 38.35045 24.81935
1.78788745 5.896552
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGploCsloAsloAsloC]
225 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 38.20957 22.48775
0.8970076 2.000011
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGsloCsloAsloAsloCsloA]
226 [oUs oGsloAploUs oCs15mCsIdTsIdGs 33.35769 27.38264
1.58936325 1.493528
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloAsloCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
120
227 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 30.72978 24.56478
1.12605755 0.803606
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloAsloCsloA]
228 [oUs oGsloAploUploCs15mCsIdTsIdG 41.09495 22.65474
2.4141379 1.912592
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGsloCsloAsloAsloCsloA]
229 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 36.5236 24.84599
1.6011969 1.293078
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGploCsloAsloAsloCsloA]
230 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 34.54957 26.61084
1.41653565 1.233035
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGsloCsloAsloAsloCsloA]
231 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 46.03036 34.13517
3.10815325 2.788523
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGploCs oAsloAsloCsloA]
232 [oUs oGsloAploUs oCs15mCsIdTsIdGs 35.25195 23.45108
1.14545295 0.875176
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloAsloCsloA]
233 [oUs oGsloAploUs oCs15mCsIdTsIdGs 39.59948 27.15972
1.1364332 1.671372
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10APloGsloCsloAsloAsloCsloA]
234 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 29.84471 24.57841
1.1966453 0.580119
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloAsloCsloAl
235 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 35.45558 25.43503
3.1614937 2.281794
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAploGsloCsloAsloAsloCsloA]
236 [oUs oGsloAploUs oCs15mCsIdTsIdGs 33.98368 25.36152
2.2232316 0.753668
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10AP10GPloCsloAsloAsloCsloA]
237 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 35.20734 26.34259
2.31304315 1.232096
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10API0GPloCsloAsloAsloCsloA]
238 [oUs oGsloAploUploCs15mCsIdTsIdG 34.15353 22.53496
1.9007468 1.47269
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGploCsloAsloAsloCsloA]
239 [oUs oGsloAploUploCs15mCsIdTsIdG 34.30311 25.10675
3.04473305 1.325174
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGsloCsloAsloAsloCsloA]
240 [oUs oGsloAploUploCs15mCsIdTsIdG 32.7831 26.79337
0.780257 1.496388
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGploCsloAsloAsloCsloA]
241 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 36.83221 25.878
1.45957465 1.202151
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGsloCsloAsloAsloCsloAsloC]
242 [oUs oGsloAploUs oCs15mCsIdTsIdGs 35.39125 26.34255
2.0733604 1.939231
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloAsloCsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
121
243 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 31.25962 26.87411
1.1648233 1.9468
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGsloCs oAsloAsloCsloAsloC]
244 [oUs oGsloAploUploCs15mCsIdTsIdG 33.7126 20.71987
2.985748 2.347571
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGsloCsloAsloAsloCsloAs oC]
245 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 37.45041 27.92649
2.2914196 1.775925
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGploCsloAsloAsloCsloAsloC]
246 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 37.40703 27.91025
0.37894275 2.801681
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGsloCsloAsloAsloCsloAsloC]
247 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 44.1762
24.73343 3.02169625 0.96668
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAploGploCs oAsloAsloCsloAsloC]
248 [oUs oGsloAploUs oCs15mCsIdTsIdGs 38.30889 23.31712
1.4411529 1.489325
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloAsloCsloAsloC]
249 [oUs oGsloAploUs oCs15mCsIdTsIdGs 38.31735 24.56753
2.5346059 1.77217
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10APloGsloCsloAsloAsloCsloAsloC]
250 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 35.17502 26.1372
1.8152499 2.083902
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAsloGPloCsloAsloAsloCsloAsloC]
251 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 36.24617 24.66592
1.38376155 0.821536
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAploGsloCsloAsloAsloCsloAsloC]
252 [oUs oGsloAploUs oCs15mCsIdTsIdGs 38.74019 26.77353
1.6343742 1.579313
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
10API0GPloCsloAsloAsloCsloAsloC]
253 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 38.33121 30.17397
1.0889853 2.797118
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
loAploGploCsloAsloAsloCsloAsloC]
254 [oUs oGsloAploUploCs15mCsIdTsIdG 44.32188 27.49314
2.67802585 1.31818
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAsloGploCsloAsloAsloCsloAsloC]
255 [oUs oGsloAploUploCs15mCsIdTsIdG 38.70488 30.24362
2.4257059 2.598832
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGsloCsloAsloAsloCsloAsloC]
256 [oUs oGsloAploUploCs15mCsIdTsIdG 43.28825 28.25934
3.05476895 1.497906
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sloAploGploCsloAsloAsloCsloAsloC]
257 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 45.77041 30.01057
2.41725105 2.688685
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dAsloGsloCsloAsloAsloC]
258 [oUs oGsloAploUs oCs15mCsIdTsIdGs 37.50516 29.19368
3.2609215 1.306107
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAsloGsloCs oAsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
122
259 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 37.61875 28.41851
4.79014865 1.914106
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAsloGsloCs oAsloAsloC]
260 [oUs oGsloAploUploCs15mCsIdTsIdG 39.57219 25.95941
1.0745636 2.611124
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdAsloGsloCsloAsloAsloC]
261 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 40.85794 29.04845
2.10254735 0.910299
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dAsloGploCsloAsloAsloC]
262 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 48.57987 31.96517
3.30537215 3.77167
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dAploGsloCsloAsloAsloC]
263 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 42.44673 32.35736
1.9875946 2.639212
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dAploGploCs oAsloAsloC1
264 [oUs oGsloAploUs oCs15mCsIdTsIdGs 43.82073 27.45321
2.0919192 1.819505
idTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAsloGPloCsloAsloAsloC]
265 [oUs oGsloAploUs oCs15mCsIdTsIdGs 40.81426 34.44821
3.2274531 3.246932
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAPloGsloCsloAsloAsloC]
266 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 44.58453 32.33173
4.3552897 2.179554
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
idAsloGPloCsloAsloAsloC]
267 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 45.58382 28.39511
0.9009167 1.911982
idTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAploGsloCsloAsloAsloC]
268 [oUs oGsloAploUs oCs15mCsIdTsIdGs 42.5512
29.79543 1.03173485 1.860662
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAPI0GPloCsloAsloAsloC]
269 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 44.6866
30.01983 2.5231542 4.434048
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAploGploCsloAsloAsloC]
270 [oUs oGsloAploUploCs15mCsIdTsIdG 38.02311 32.80756
3.4793287 4.749247
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdAsloGploCsloAsloAsloC]
271 [oUs oGsloAploUploCs15mCsIdTsIdG 48.88263 27.15217
3.5851377 1.816405
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdAploGsloCsloAsloAsloC1
272 [oUs oGsloAploUploCs15mCsIdTsIdG 46.69598 30.73665
2.0490884 2.855452
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdAploGploCsloAsloAsloC]
273 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 42.70873 30.68745
3.65148505 3.255575
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dAsIdGsloCsloAsloAsloCsloA]
274 [oUs oGsloAploUs oCs15mCsIdTsIdGs 40.32396 26.35517
3.335113 2.701905
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAsIdGsloCs oAsloAsloCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
123
275 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 40.74281 29.94494
0.6334559 2.557136
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAsIdGsloCs oAsloAsloCsloA]
276 [oUs oGsloAploUploCs15mCsIdTsIdG 40.29221 27.33965
0.8763938 2.574619
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdAsIdGsloCsloAsloAsloCsloA]
277 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 47.44893 29.86629
2.4738361 2.55434
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dAsIdGploCsloAsloAsloCsloA]
278 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 46.17489 33.16617
3.1173163 1.327764
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dApIdGsloCsloAsloAsloCsloA]
279 [oUs oGsloAsloUsloCs15mCsIdTsIdGs1 46.58545 33.39568
3.2287529 1.864188
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
dApIdGploCs oAsloAsloCsloA]
280 [oUs oGsloAploUs oCs15mCsIdTsIdGs 43.27573 27.28916
2.31554215 1.262593
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAsIdGPloCsloAsloAsloCsloA]
281 [oUs oGsloAploUs oCs15mCsIdTsIdGs 47.10235 32.83978
1.53071395 4.229922
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAPIdGsloCsloAsloAsloCsloA]
282 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 47.70091 29.60736
3.7604108 2.052869
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAsIdGPloCsloAsloAsloCsloAl
283 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 49.28544 34.66856
1.59176345 3.017275
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdApIdGsloCsloAsloAsloCsloA]
284 [oUs oGsloAploUs oCs15mCsIdTsIdGs 49.84421 32.28262
4.79490775 5.858741
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
IdAPIdGPloCsloAsloAsloCsloA]
285 [oUs oGsloAsloUp oCs15mCsIdTsIdGs 34.59283 22.87145
3.0164757 2.082604
IdTsIdTs15mCsIdTs15mCs15mCs15mCs
idAP1dGPloCsloAsloAsloCsloA]
286 [oUs oGsloAploUploCs15mCsIdTsIdG 39.38162 26.12473
1.8902058 1.570875
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdAsIdGploCsloAsloAsloCsloA]
287 [oUs oGsloAploUploCs15mCsIdTsIdG 50.21733 30.06642
3.10898985 1.234999
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdApIdGsloCsloAsloAsloCsloA]
288 [oUs oGsloAploUploCs15mCsIdTsIdG 50.69014 32.61033
2.79327395 1.497337
sIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdApIdGploCsloAsloAsloCsloA]
289 [oUs oUsloGsloAsloUsl5mCs15mCsId 65.03387 39.71818
3.2453458 1.232538
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloA]
290 [oUs oUsloGploAs oUsl5mCs15mCs d 61.89021 44.17769
3.0819054 2.818603
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
124
291 [oUs oUsloGsloAp oUsl5mCs15mCs d 70.81215 39.23055
5.9432651 2.293842
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloA]
292 [oUs oUsloGploAploUsl5mCs15mCsId 64.28917 35.27306
4.4374966 2.084275
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloA]
293 [oUs oUsloGsloAsloUsl5mCs15mCsId 75.30924 41.75287
6.3677188 3.571926
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloA]
294 [oUs oUsloGsloAsloUsl5mCs15mCsId 72.21538 40.63977
9.45384065 2.395694
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloA]
295 [oUs oUsloGsloAsloUsl5mCs15mCsId 65.43466 40.60514
4.9966345 4.010523
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloA]
296 [oUs oUsloGploAs oUsl5mCs15mCs d 70.68577 41.62804
4.61698135 0.639112
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloA]
297 [oUs oUsloGploAs oUsl5mCs15mCs d 68.70839 43.19171
4.0611799 2.66127
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloA]
298 [oUs oUsloGsloAp oUsl5mCs15mCs d 68.74235 42.8638
6.63886525 2.69242
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloA]
299 [oUs oUsloGsloAp oUsl5mCs15mCs d 65.56432 41.13655
2.8508521 1.098556
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloA]
300 [oUs oUsloGploAs oUsl5mCs15mCs d 73.25913 44.19704
0.3218877 3.006132
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloA]
301 [oUs oUsloGsloAp oUsl5mCs15mCs d 66.80031 44.33691
5.58574735 2.190286
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloA]
302 [oUs oUsloGploAploUsl5mCs15mCsId 67.79988 43.25911
3.47248465 2.245861
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloA]
303 [oUs oUsloGploAploUsl5mCs15mCsId 69.0764
39.9134 2.2580266 2.514186
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloA]
304 [oUs oUsloGploAploUsl5mCs15mCsId 68.85722 43.2033
2.66821415 1.577173
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloA]
305 [oUs oUsloGsloAsloUsl5mCs15mCsId 64.32531 36.98857
6.6048679 1.862941
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloA]
306 [oUs oUsloGploAs oUsl5mCs15mCs d 79.43795 38.19117
10.61625475 3.700974
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
125
307 [oUs oUsloGsloAp oUsl5mCs15mCs d 65.05429 35.73871
5.07137215 2.026283
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloA]
308 [oUs oUsloGploAploUsl5mCs15mCsId 67.69046 37.80261
3.3970135 1.359287
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloA]
309 [oUs oUsloGsloAsloUsl5mCs15mCsId 72.9064 39.22807
2.6173443 1.983172
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloA]
310 [oUs oUsloGsloAsloUsl5mCs15mCsId 65.42524 41.63383
1.64477605 2.287743
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloA]
311 [oUs oUsloGsloAsloUsl5mCs15mCsId 79.57865 47.30341
3.2793354 4.879421
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloA]
312 [oUs oUsloGploAs oUsl5mCs15mCs d 74.90458 37.7908 7.9156018
2.492285
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloA]
313 [oUs oUsloGploAs oUsl5mCs15mCs d 64.17809 41.25918
3.60546685 2.451177
TsIdGsIdTsIdTsISHICsIdTs15mCs SmC
sloCploAsloGsloCsloAsloA]
314 [oUs oUsloGsloAp oUsl5mCsI5mCs d 77.79264 36.3895
5.16839925 1.47233
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAl
315 [oUs oUsloGsloAp oUsl5mCs15mCs d 69.27439 40.78583
0.70050225 3.765485
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloA]
316 [oUs oUsloGploAs oUsl5mCs15mCs d 66.79254 41.42264
4.07791885 2.517419
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloA]
317 [oUs oUsloGsloAp oUsI5mCs15mCs d 69.95295 38.70855
5.1765897 1.322
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloA]
318 [oUs oUsloGploAploUsl5mCs15mCsId 64.59882 36.2354
1.94088075 2.245535
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloA]
319 [oUs oUsloGploAploUsl5mCs15mCsId 71.61855 40.11992
4.8955589 1.16262
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloA]
320 [oUs oUsloGploAploUsl5mCs15mCsId 73.74875 40.87392
1.9294505 0.764434
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloA]
321 [oUs oUsloGsloAsloUsl5mCs15mCsId 69.14922 42.64199
6.9911749 2.990965
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloC]
322 [oUs oUsloGploAs oUsl5mCs15mCs d 68.03041 40.82452
3.81307025 3.211419
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
126
323 [oUs oUsloGsloAp oUsl5mCs15mCs d 64.06085 38.06864
3.2673432 2.579056
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloC]
324 [oUs oUsloGploAploUsl5mCs15mCsId 64.06883 40.38284
2.58838335 2.66873
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloC]
325 [oUs oUsloGsloAsloUsl5mCs15mCsId 73.59074 39.51843
5.5155268 1.608472
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloC]
326 [oUs oUsloGsloAsloUsl5mCs15mCsId 64.14324 38.43417
2.9521007 1.560155
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloC]
327 [oUs oUsloGsloAsloUsl5mCs15mCsId 67.68504 45.94997
5.2482586 2.096218
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloC]
328 [oUs oUsloGploAs oUsl5mCs15mCs d 74.0999 38.3912
0.5712221 1.732884
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloC]
329 [oUs oUsloGploAs oUsl5mCs15mCs d 59.85657 40.50332
4.4957246 1.479709
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloC]
330 [oUs oUsloGsloAp oUsl5mCs15mCs d 62.98679 36.72381
9.8570092 0.854923
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloC]
331 [oUs oUsloGsloAp oUsl5mCs15mCs d 66.22289 38.29377
7.80879995 2.702458
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloC]
332 [oUs oUsloGploAs oUsl5mCs15mCs d 64.84554 38.62897
2.9279718 1.019158
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloC]
333 [oUs oUsloGsloAp oUsl5mCs15mCs d 72.02838 42.5541 4.6483644
3.24576
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloC]
334 [oUs oUsloGploAploUsl5mCs15mCsId 73.13125 37.42697
3.52337675 2.273121
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloC]
335 [oUs oUsloGploAploUsl5mCs15mCsId 65.10868 38.41509
2.69335365 3.463081
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloC]
336 [oUs oUsloGploAploUsl5mCs15mCsId 74.0986
41.06858 5.59980225 3.735298
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloC]
337 [oUs oUsloGsloAsloUsl5mCs15mCsId 68.10988 33.22407
2.04506795 1.219344
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloCsloA]
338 [oUs oUsloGploAs oUsl5mCs15mCs d 61.51268 39.42608
3.2210901 1.60843
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
127
339 [oUs oUsloGsloAp oUsl5mCs15mCs d 61.62901 38.10779
5.0486966 1.469955
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloCsloA]
340 [oUs oUsloGploAploUsl5mCs15mCsId 68.70971 37.9342
9.15707545 2.987791
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAsloGsloCsloAsloAsloCsloA]
341 [oUs oUsloGsloAsloUsl5mCs15mCsId 64.63658 37.92174
8.0800218 2.867291
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloCsloA]
342 [oUs oUsloGsloAsloUsl5mCs15mCsId 71.59294 36.10313
4.23800025 2.212339
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloCsloA]
343 [oUs oUsloGsloAsloUsl5mCs15mCsId 67.77124 42.50938
4.24974715 0.606911
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloCsloA]
344 [oUs oUsloGploAs oUsl5mCs15mCs d 62.77385 38.9199
1.20627285 2.566854
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloCsloA]
345 [oUs oUsloGploAs oUsl5mCs15mCs d 65.30274 37.50644
3.3490132 1.120349
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloCsloA]
346 [oUs oUsloGsloAp oUsl5mCs15mCs d 59.99562 43.52686
2.17603655 3.501488
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloCsloAl
347 [oUs oUsloGsloAp oUsl5mCs15mCs d 76.91818 36.71682
5.11543135 1.003503
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloCsloA]
348 [oUs oUsloGploAs oUsl5mCs15mCs d 74.07843 40.52268
2.68814825 1.670121
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloCsloA]
349 [oUs oUsloGsloAp oUsl5mCs15mCs d 66.80045 41.82373
5.23486615 1.955829
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloCsloA]
350 [oUs oUsloGploAploUsl5mCs15mCsId 71.27418 43.95148 3.022233
1.912625
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCsloAploGsloCsloAsloAsloCsloA]
351 [oUs oUsloGploAploUsl5mCs15mCsId 68.26772 40.87145
1.67750485 1.489503
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAsloGsloCsloAsloAsloCsloA]
352 [oUs oUsloGploAploUsl5mCs15mCsId 74.70355 46.98739
4.10748375 3.361829
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sloCploAploGsloCsloAsloAsloCsloA]
353 [oUs oUsloGsloAsloUsl5mCs15mCsId 82.69998 45.53944
2.7748001 5.248864
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAs oGsloCsloAsloA]
354 [oUs oUsloGploAs oUsl5mCs15mCs d 75.2964
41.58329 4.28755645 1.604165
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAs oGsloCsloAsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
128
355 [oUs oUsloGsloAp oUsl5mCs15mCs d 78.25078 38.04859
2.1756527 2.812668
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAs oGsloCsloAsloA]
356 [oUs oUsloGploAploUsl5mCs15mCsId 75.66047 46.42479
5.32824395 3.512293
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAs oGsloCsloAsloA]
357 [oUs oUsloGsloAsloUsl5mCs15mCsId 70.8279
39.60585 4.17139065 1.315399
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAploGsloCsloAs oA]
358 [oUs oUsloGsloAsloUsl5mCs15mCsId 88.71474 41.43116
2.63982225 2.837026
Ts1c1GsIdTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAsloGsloCsloAs oA]
359 [oUs oUsloGsloAsloUsl5mCs15mCsId 94.02871 55.61287
3.28254665 5.31701
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAploGs oCsloAsloA1
360 [oUs oUsloGploAs oUsl5mCs15mCs d 81.20155 48.93426
2.9797871 3.293206
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAploGsloCsloAs oA]
361 [oUs oUsloGploAs oUsl5mCs15mCs d 81.56161 43.33641
5.9890201 2.745271
Tsk1G-sidTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAsloGsloCsloAs oA]
362 [oUs oUsloGsloAp oUsl5mCs15mCs d 77.46717 40.6332
6.53245465 2.851351
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAploGsloCsloAs oA]
363 [oUs oUsloGsloAp oUsl5mCs15mCs d 91.06332 37.09205
6.16485235 1.515162
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAsloGsloCsloAs oA]
364 [oUs oUsloGploAs oUsl5mCs15mCs d 87.55428 42.57427
3.1016107 2.002001
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAploGs oCsloAsloA]
365 [oUs oUsloGsloAp oUsl5mCs15mCs d 90.76046 46.77878
4.93885515 3.608661
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAploGs oCsloAsloA]
366 [oUs oUsloGploAploUsl5mCs15mCsId 76.47815 41.28109
4.02130685 3.175797
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sl5mCsloAploGsloCsloAs oA]
367 [oUs oUsloGploAploUsl5mCs15mCsId 81.30346 46.78571
5.4938338 2.280535
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAsloGsloCsloAs oA]
368 [oUs oUsloGploAploUsl5mCs15mCsId 84.12098 44.30416
5.38885525 2.935461
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCploAploGs oCsloAsloA]
369 [oUs oUsloGsloAsloUsl5mCs15mCsId 70.44063 38.60541
6.7004305 3.620983
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
sl5mCsIclAs oGsloCsloAsloAsloC]
370 [oUs oUsloGploAs oUsl5mCs15mCs d 74.17394 36.98506
2.4084315 1.467456
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsIclAs oGsloCsloAsloAsloC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
129
371 [oUs oUsloGsloAp oUsl5mCs15mCs d 74.77409 37.54156
7.2673398 1.490426
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsIclAs oGsloCsloAsloAsloC]
372 [oUs oUsloGploAploUsl5mCs15mCsId 78.93023 38.38505
5.56282815 3.943036
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsIclAs oGsloCsloAsloAsloC]
373 [oUs oUsloGsloAsloUsl5mCs15mCsId 71.77732 36.26151
4.19725075 2.479808
TsIdGsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsIclAploGsloCsloAs oAsloC]
374 [oUs oUsloGsloAsloUsl5mCs15mCsId 75.98736 46.94162
3.67475295 2.387602
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
si5mCplclAsloGsloCsloAs oAsioC]
375 [oUs oUsloGsloAsloUsl5mCs15mCsId 83.01221 48.4748
1.83557915 3.954731
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCpIclAploGs oCsloAsloAsloC]
376 [oUs oUsloGploAs oUsl5mCs15mCs d 73.40898 45.08328
1.55749415 2.879961
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsIclAploGsloCsloAs ()AOC]
377 [oUs oUsloGploAs oUsl5mCs15mCs d 81.18265 43.25494
2.32470145 2.898854
Tsk1G-sidTsIdTs15inCsIdTs15mCs 5mC
sl5mCpIclAsloGsloCsloAs oAsloC]
378 [oUs oUsloGsloAp oUsl5mCs15mCs d 85.8249 35.81711
7.0913574 1.999337
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCsIclAploGsloCsloAs oAsloC]
379 [oUs oUsloGsloAp oUsl5mCs15mCs d 85.9207
39.07233 8.2451204 1.716324
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCpIclAsloGsloCsloAs AOC]
380 [oUs oUsloGploAs oUsl5mCs15mCs d 89.28203 44.69979
3.57026875 2.48225
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCpIclAploGs oCsloAsloAsloC]
381 [oUs oUsloGsloAp oUsl5mCs15mCs d 80.47781 43.39312
1.49203405 1.667053
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCpIclAploGs oCsloAsloAsloC]
382 [oUs oUsloGploAploUsl5mCs15mCsId 84.37027 35.88873
4.3911855 0.578314
TsIdGsIdTsIdTs15mCsIdTs15mCs 5mC
si5mCsIclAploGsloCsloAs AOC]
383 [oUs oUsloGploAploUsl5mCs15mCsId 78.04549 35.55957
3.76452225 3.138921
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCpIclAsloGsloCsloAs AOC]
384 [oUs oUsloGploAploUsl5mCs15mCsId 86.50257 42.97401
7.1309543 0.436476
Ts1c1GsidTsIdTs15mCsIdTs15mCs 5mC
sl5mCpIclAploGs oCsloAsloAsloC]
Table 4.
Antisense Variant Sequence
Oligo
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
130
No./SEQ
ID NO:
[oUsILGslo5mCsloUsloAsloGsloGsloUsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsIdGs1
390
5mCsloUsloU]
[oUsloGs1L5mCsloUsloAsloGsloGsloUsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsIdGs1
391
5mCsloUsloU]
[oUsloGs105mCsILTsloAsloGsloGsloUsIdGsIdAsIdTsIdGs15mCsIdAs 392
5mCsIdTsIdGs1
5mCsloUsloU]
[oUsloGslo5mCsloUsILAsloGsloGsloUsIdGsIdAsIdTsIdGs15mCsIdAs15mCsidTsIdGs1
393
5mCsloUsIOLT]
[oUsloGslo5mCsloUsloAsILGsloGsloUsIdGsIdAsIdTsIdGs15mCsIdAs15mCsidTsIdGs1
394
5mCsloUsloU]
roUsloGslo5mCsloUsloAsloGsILGsloUsIdGsIdAsIdTsIdGs15mCsIdAs15mCsidTsIdGs1
395
5mCsloUsloU]
396
,[oUsloGs15mCsIdTsIdAsIdGOGOTsIdGsIdAsIdTs dGslo5mCs oAslo5mCsloUsloGs
195mCsILTsloU]
[oUsloGs15mCsIdTsIdAsIdGsIdGsIdTsIdGsIdAsIdTs 397 dGslo5mCs
oAslo5mCsloUsloGs
IL5mCsloUsloU]
[oUsloGs15mCsIdTsIdAsIdGsIdGsIdTsIdGsIdAsIdTs 398 dGslo5mCs
oAslo5mCsloUsILGs
105mCsloUsloU]
[oUsloGs15mCsIdTsIdAsIdGsIdGsIdTsIdGsIdAsIdTs 399 dGslo5mCs
oAslo5mCsILTsloGs
105mCsloUsloU]
[oUsloGs15mCsIdTsIdAsIdGsIdGsIdTsIdGsIdAsIdTs 400 dGslo5mCs
oAs1L5mCsloUsloGs
195mCsloUsloU]
[oUsloGs15mCsIdTsIdAsIdGsIdGsIdTsIdGsIdAsIdTs 401
dGslo5mCsLAslo5mCsloUsloGs
195mCsloUsloU]
[oUsILGs105mCsloUsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 402
5mCsIdTsIdGs1
o5mCsloUsloU]
[oUsloGs1L5mCsloUsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 5mCsIdTsIdGs1
403
o5mCsloUsloU]
[oUsloGslo5mCsILTsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsIdGs1
404
o5mCsloUsloU]
[oUsloGslo5mCsloUsILAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 405
5mCsIdTsIdGs1
o5mCsloUsloU]
toUsloGs105mCsloUsloAsILGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 406
5mCsIdTsIdGs1
o5mCsloUsloU]
toUsloGslo5mCsIdTsIdAsIdGsIdGsIdTs 407 dGslclAsIdTsIdGs15mCs
oAslo5mCsloUsloGs
105mCsILTsloU]
[oUsloGslo5mCsIdTsIdAsIdGsIdGsIdTs 408 dGslclAsIdTsIdGs15mCs
oAslo5mCsloUsloGs
IL5mCsloUsloU]
[oUsloGslo5mCsIdTsIdAsIdGsIdGsIdTs 409 dGslclAsIdTsIdGs15mCs
oAslo5mCsloUsILGs
195mCsloUsloU]
[oUsloGslo5mCsIdTsIdAsIdGsIdGsIdTs 410 dGslclAsIdTsIdGs15mCs
oAslo5mCsILTsloGs
195mCsloUsloU]
[oUsloGslo5mCsIdTsIdAsIdGsIdGsIdTs 411 dGslidAsIdTsIdGs15mCs
oAs1L5mCsloUsloGs
195mCsloUsloU]
[oUsILGslo5mCsloUsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 412
5mCsIdTsIdGs1
5mCsloUsloU]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
131
[oUsloGs1L5mCsloUsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 413
5mCsIdTsIdGs1
5mCsloUsloU]
[oUsloGslo5mCsILTsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsIdGs1
414
5mCsloUslotil
[oUsloGs105mCsloUsILAsloGsloGslciTsIdGsIdAsIdTsIdGs15mCsIdAs 415
5mCsIdTsIdGs1
5mCsloUsloU]
[oUsloGs105mCsloUsloAsILGsloGslciTsIdGsIdAsIdTsIdGs15mCsIdAs 416
5mCsIdTsIdGs1
5mCsloUsloU]
[oGslo5mCsIdTsIdAsIdGsIdGsIdTsIdGs 417 dAsIdTsIdGs15mCsloAs
o5mCsloUsloGslo5
mCs LTsloU]
toGslo5mCsIdTsIdAsIdGsIdGsIdTsIdGs 418 dAsIdTsIdGs15mCsloAs
o5mCsloUsloGs1L5
mCs oUsloU]
[oGslo5mCsIdTsIdAsIdGsIdGsIdTsIdGs 419 dAsIdTsIdGs15mCsloAs
o5mCsloUsILGslo5
mCs oUsloU]
[oGslo5mCsIcITsIdAsIdGsIdGsIdTsIdGs 420 dAskiTsIdGs15mCsloAs
o5mCsILTsloGslo5
mCs oUsloU]
[oGslo5mCsIdTsIdAsIdGsIdGsIdTsIdGs 421
dAsIdTsIdGs15mCsloAsL5mCsloUsloGslo5
mCs oUsloU]
[oUsILGslo5mCsloUsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 422
5mCsIdTsloGs1
o5mCsloUsloU]
[oUsloGs1L5mCsloUsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 5mCsIdTsloGs1
423
o5mCsloUsloU]
[oUsloGslo5mCsILTsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsloGs1
424
o5mCsloUsloUl
[oUsloGs105mCsloUsILAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 425
5mCsIdTsloGs1
o5mCsloUsloU]
[oUsloGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGs
426
lo5mCsILTsloU]
[oUsloGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGs
427
1L5mCsloUsloU]
[oUsloGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsILG
428
slo5mCsloUsloU]
toUsloGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsILTsloGs
429
105mCsloUsloU]
[oUsILGs105mCsloUsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 430
5mCsIdTsloGs1
o5mCsloU1
[oUsloGs1L5mCsloUsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 431
5mCsIdTsloGs1
o5mCslolil
[oUsloGslo5mCsILTsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsloGs1
432
o5mCslotT]
[oUsloGslo5mCsloUsILAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 5mCsIdTsloGs1
433
o5mCslolil
toGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGslo5
434
mCs LTsloU]
[oGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGsIL5
435
mCs oUsloU]
[oGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsILGs 436
05
mCs oUsloUl
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
132
[oGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsILTsloGslo5
437
mCs oUsloU]
[oUsILGs105mCsloUsloAsloGsIdGslciTsIdGsIdAsIdTsIdGs15mCsIdAs 438
5mCsIdTsloGs1
o5mC]
[oUsloGs1L5mCsloUsloAsloGsIdGslciTsIdGsIdAsIdTsIdGs15mCsIdAs 439
5mCsIdTsloGs1
o5mC]
[oUsloGslo5mCsILTsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsloGs1
440
o5mC]
[oUsloGs105mCsloUsILAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 441
5mCsIdTsloGs1
o5mC]
to5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGslo5mCs1
442
LTsloU]
[o5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGs11-5mCs1
443
oUsloU]
[o5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsILGslo5mCs1
444
oUsloU]
[o5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsILTsloGslo5mCs1
445
oUsloU]
[oUsILGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 446
5mCsloUsloGsl
o5mCsloUsloU]
[oUsloGs1L5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 447
5mCsloUsloGsl
o5mCsloUsloU]
[oUsloGslo5mCsILTsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
448
o5mCsloUsloUl
[oUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
449
o5mCs11_,TsloU]
[oUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
450
L5mCsloUsloU]
[oUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsILGs1
451
o5mCsloUsloU]
[oUsILGslo5mCsloUsloAsIdGsIdGslciTsIclGsIdAsIdTsIdGs15mCsIdAs 452
5mCsloUsloGsl
o5mCsloU]
toUsloGs1L5mCsloUsloAsIdGsIdGslciTsIdGslciAsIdTsIdGs15mCsIdAs 5mCsloUsloGsl
45-3
o5mCsloU]
[oUsloGslo5mCsILTsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
454
o5mCsloU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5m
455
CaIOU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGsIL5m
456
CsloUsloU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsILGslo5m
457
CsloUsloU]
toUsILGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 458
5mCsloUsloGsl
o5mC]
[oUsloGs1L5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 459
5mCsloUsloGsl
o5mC]
[oUsloGslo5mCsILTsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs1511r1CsIdAs15mCsloUsloGs1
460
o5mC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
133
[o5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdG-s15mCsIdAs15mCsloUsloG-slo5mCsIL
461
TsloU]
[o5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGsIL5mCslo
462
UsloU]
[o5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsILGslo5mCslo
463
UsloU]
464
[oUsILGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs 5mCsloUsloG]
465 [oUs oGs1L5mCs oUs oAs dGs dGs dTs dGs dAs dTs dGs 5mCs
dAs 5mCs oUs oG]
466 [oUs oGs
o5mCsILTsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloG]
467 [oUs oAs
dGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5mCs LTs oU]
468 [oUs oAs dGs dGs dTs dGs dAs dTs dGs 5mCs dAs 5mCs oUs oGs
L5mCsloUsloU]
469 [oUs oAs dGs dGs dTs dGs dAs dTs dGs 5mCs dAs 5mCs oUs
LGslo5mCs oUs oU]
[oGs1L5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCs 470
oUsloGslo5m
CsloUsloU]
[oGslo5mCsILTsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5m
471
CsloUsloU]
[oUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
472
L5mCsloU]
roUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsILGs1
473
o5mCsloU]
roGs1L5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCs 474
oUsloGslo5m
CsloU]
[oGslo5mCsILTsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5m
475
CsloU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGsL5m
476
CsloU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsLGslo5m
477
CsloU]
[oGs1L5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCs 478
oUsloGslo5m
Cl
479
[oGslo5mCsILTsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5m
C]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsLGslo5m
480
C]
[oUsloGslo5naCsloUsloAsloGsloGsloUsIdGsIdAsIdTsIdGs15mCsIdAs 481
5mCsIdTsIdGs1
5mCsloUsloU]
[oUsloGs15mCsIdTsIdAsIdGsIdGsIdTsIdGsIdAsIdTs 482 dGslo5mCs
oAslo5mCsloUsloGs
lo5mCsloUsloT_T]
[oUsloGslo5mCsloUsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsIdGs1
483
o5mCsloUsloU]
roUsloGslo5mCsIdTsIdAsIdGsIdGsIdTs 484
dGsIdAsIdTsIdGs15mCs oAslo5mCsloUsloGs
105mCsloUsloU]
485
roUsloGslo5mCsloUsloAsloGsloGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsIdGs1
5mCsloUsloU]
[oGslo5mCsIdTsIdAsIdGsIdGsIdTsIdGs 486 dAsIdTsIdGs15mCsloAs
o5mCsloUsloGslo5
mCs oUsloU]
[oUsloGslo5mCsloUsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsloGs1
487
o5mCsloUsloU]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
134
[oUsloGslo5mCsloUsIdAsIdGOGOTOGOAsIdTsIdGs15mCsIdAslo5mCsloUsloGs
488
105mCsloUsloU]
[oUsloGslo5mCsloUsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsloGs1
489
o5mCsloU]
[oGslo5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGslo5
490
mCs oUsloU]
[oUsloGslo5mCsloUsloAsloGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsIdTsloGs1
491
o5mC]
[o5mCsloUsIdAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAslo5mCsloUsloGslo5mCs1
492
oUsloU]
[oUsloGslo5mCsloUsloAsIdGsIdGslcITOGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
493
o5mCsloUsloU]
foUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
494
o5mCsloU]
[oUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGs1
495
o5mC]
[o5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5mCslo
496
UsloU]
497
[oUsloGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloG]
498 [oUs oAs
dGs1c1GsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5mCs oUs oU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5m
499
CsloUsloU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5m
500
CsloU]
[oGslo5mCsloUsloAsIdGsIdGsIdTsIdGsIdAsIdTsIdGs15mCsIdAs15mCsloUsloGslo5m
501
Cl
502 [oUsILTsloGsloAs
oUslo5mCslo5mCsloUsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
slclAsIdGslo5mCsloA]
[oUsloUsILGsloAsloUslo5mCslo5mCsloUsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
503
slclAsIdGslo5mCsloA]
[oUsloUsloGsILAsloUslo5mCslo5mCsloUsIdGslcasIdTs15mCsIdTs15mCs15mCs15mC
504
slclAsIdGslo5mCsloA]
505 [oUsloUsloGsloAs
LTslo5mCslo5mCsloUsIdGOTsIdTs15mCsIdTs15mCs15mCs15mC
slclAsIdGslo5mCsloA]
[oUsloUsloGsloAsloUsIL5mCslo5mCsloUsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
506
slclAsIdGslo5mCsloA]
[oUsloUsloGsloAsloUslo5mCsIL5mCsloUsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
507
slclAsIdGslo5mCsloA]
roUsloUsIdGsIdAs dTs15mCs15mCsIciTsIdGsIdTsIdTs15mCsloUslo5mCslo5mCs o5m
08
CsloAsloGs1L5mCsloA]
roUsloUsIdGsIdAs dTs15mCs15mCsIciTsIdGsIdTsIdTs15mCsloUslo5mCslo5mCs o5m
509
CsloAsILGslo5mCsloA]
[oUsloUsIdGsIdAs dTs15mCs15mCsIciTsIdGsIdTsIdTs15mCsloUslo5mCslo5mCs o5m
510
CsILAsloGslo5mCsloA]
[oUsloUsIdGsIdAs dTs15mCs15mCsIciTsIdGsIdTsIdTs15mCsloUslo5mCslo5mCsL5m
511
CsloAsloGslo5mCsloA]
512 [oUsloUsIdGsIdAs
dTs15mCs15mCsIciTsIdGsIdTsIdTs15mCsloUslo5mCsIL5mCslo5m
CSIOASIOGS105MCSIOA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
135
[oUsloUsIdGsIdAs dTs15mCs15mCsIcITsIdGsIdTsIdTs15mCsloUsIL5mCslo5mCslo5m
51.3
CsloAsloGslo5mCsloA]
[oUsILTsloGsloAs oUslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs 5mC
514
sIdAsloGslo5mCsloA]
[oUsloUsILGsloAsloUslo5mCslo5mCsIdTsIdGsk1TsIdTs15mCsIdTs15mCs15mCs15mC
515
sIdAsloGslo5mCsloA]
[oUsloUsloGsILAsloUslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
516
sIdAsloGslo5mCsloA]
[oUsloUsloGsloAs LTslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs 5mC
517
sIdAsloGslo5mCsloA]
[oUsloUsloGsloAsloUsIL5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
518
slclAsloGslo5mCsloA]
519 roUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
o5mCslo5mCslo5m
CsloAsloGs1L5mCsloA]
520 [oUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
o5mCslo5mCslo5m
CsloAsILGslo5mCsloA]
521 [oUsloUsloGsIdAs dTs15mCs15mCsIcITsIdGsIdTsIdTs15mCsIdTs
o5mCslo5mCslo5m
CsILAsloGslo5mCsloA]
522 [oUsloUsloGsIdAs dTs15mCs15mCsIciTsIdGsIdTsIdTs15mCsIdTs
o5mCslo5mCs1L5m
CsloAsloGslo5mCsloA]
523 [oUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
o5mCs1L5mCs o5m
CsloAsloGslo5mCsloA]
[oUsILTsloGsloAs oUslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs 5mC
524
sIdAsloGslo5mC1
[oUsloUsILGsloAsloUslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
525
sIdAsloGslo5mC]
[oUsloUsloGsILAsloUslo5mCslo5mCsIdTsIdGslc1TsIdTs15mCsIdTs15mCs15mCs15mC
526
sIdAsloGslo5mC]
[oUsloUsloGsloAs LTslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs 5mC
527
sIdAsloGslo5mC]
[oUsloUsloGsloAsloUsIL5mCslo5mCsIdTsIdGOTsIdTs15mCsIdTs15mCs15mCs15mC
528
slcIAsloGslo5mC]
[oUsloGsIdAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTslo5mCslo5mCslo5mCslo
529
AsloGs1L5mCsloA]
toUsloGsIdAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTslo5mCslo5mCslo5mCslo
530
As11_,Gslo5mCsloA]
toUsloGslclAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTslo5mCslo5mCslo5mCsIL
531
AsloGslo5mCsloA]
[oUsloGsIdAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTslo5mCslo5mCsIL5mCslo
532
AsloGslo5mCsloA]
[oUsloGsIdAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTslo5mCsIL5mCslo5mCslo
533
AsloGslo5mCsloA]
534 [oUsILTsloGsloAs
oUslo5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGslo5mCsloA]
[oUsloUsILGsloAsloUslo5mCs15mCsIdTsIdGs dTsIdTs15mCsIdTs15mCs15mCs15mCs1
535
oAsloGslo5mCsloA]
536 [oUsloUsloGsILAsloUslo5mCs15mCsIdTsIdGs
dTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGslo5mCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
136
537 [oUsloUsloGsloAs
LTslo5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGslo5mCsloA]
538 ,[oUsloUsloGsloAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCslo5mCs
loAsloGs1L5mCsloA]
539 ,[oUsloUsloGsloAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCslo5mCs
loAsILGslo5mCsloA]
540 ,[oUsloUsloGsloAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCslo5mCs
ILAsloGslo5mCsloA]
541 [oUsloUsloGsloAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCs1L5mC
sloAsloGslo5mCsloA]
542 [oUsILTsloGsloAs
oUslo5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGslo5mC]
roUsloUsILGsloAsloUslo5mCs15mCsIdTsIdGs dTsIdTs15mCsIdTs15mCs15mCs15mCs1
543
oAsloGslo5mC]
[oUsloUsloGsILAsloUslo5mCs15mCsIdTsIdGs dTsIdTs15mCsIdTs15mCs15mCs15mCs1
544
oAsloGslo5mC]
545 [oUsloUsloGsloAs
LTslo5mCs15mCsIdTsIdGOTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGslo5mC]
546 ,[oUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCslo5mCs
loAsloGs1L5mCsloA]
547 [oUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTs1dTs15mCsIdTs
5mCslo5mCslo5mCs
loAsILGslo5mCsloA]
548 [oUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCslo5mCs
1LAsloGslo5mCsloA]
549 [oUsloUsloGsloAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCs1L5mC
sloAsloGslo5mCsloA]
550 [oUsILTsloGsloAs
oUslo5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloG]
[oUsloUsILGsloAsloUslo5mCs15mCsIdTsIdGs dTsIdTs15mCsIdTs15mCs15mCs15mCs1
551
oAsloG]
[oUsloUsloGsILAsloUslo5mCs15mCsIdTsIdGs dTsIdTs15mCsIdTs15mCs15mCs15mCs1
552
oAsloG]
[oUsloUsloGsloAs LTslo5mCs15mCsIdTsIdGOTsIdTs15mCsIdTs15mCs15mCs15mCs1
553
oAsloG]
toGsloAsIdTs15mCs15mCsIdTsIdGOTOTs15mCsIdTs15mCslo5mCslo5mCsloAsloGs
554
1L5mCsloA]
toGsloAsIdTs15mCs15mCsIdTsIdGOTOTs15mCsIdTs15mCslo5mCslo5mCsloAsILG
555 slo5mCsloA]
[oGsloAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCslo5mCslo5mCsILAsloG
556
slo5mCsloA]
[oGsloAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCslo5mCsIL5mCsloAsloG
557
slo5mCsloA]
558 [oUsILTsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsloGslo5mCsloA]
[oUsloUsILGsloAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 559
5mCslo5mCs1
oAsloGslo5mCsloA]
[oUsloUsloGsILAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 560
5mCslo5mCs1
oAsloGslo5mCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
137
561 [oUsloUsloGsloAs oUs15mCs15mCs
dTsliz1GsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsloGs1L5mCsloAi
562 [oUsloUsloGsloAs oUsl5mCs15mCs
dTs1c1GsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsILGslo5mCsloA]
563 [oUsloUsloGsloAs oUsl5mCs15mCs
dTs1c1GsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
LAsloGslo5mCsloA]
564 [oUsILTsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsloGslo5mC]
[oUsloUsILGsloAsloUsISmCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 565
5mCslo5mCs1
oAsloGslo5mC]
[oUsloUsloGsILAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 566
5mCslo5mCs1
oAsloGslo5mC]
567 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
oGs1L5mCsloA]
568 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
LGslo5mCsloA]
569 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsILAs1
oGslo5mCsloA]
570 [oUsILTsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsloG]
[oUsloUsILGsloAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 571
5mCslo5mCs1
oAsloG]
[oUsloUsloGsILAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 572
5mCslo5mCs1
oAsloG]
573
[oGsloAsloUsl5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAsloGs1
L5mCsloA]
[oGsloAsloUsl5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAsILGs1
574
o5mCsloA]
[oGsloAsloUsl5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsILAsloGs1
575
o5mCsloA]
576 [oUsILTsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oA]
[oUsloUsILGsloAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 577
5mCslo5mCs1
oA]
toUsloUsloGsILAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs 578
5mCslo5mCs1
oA]
roAsloUs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs 579
oAsloGs1L5m
CsloA]
[oAsloUsl5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs 580
oAsILGsloSm
CsloA]
[oAsloUsl5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsILAsloGsloSm
581
CsloA]
[oUsIL,GsloAsloUs15mCs15mCsIdTs dGs1c1Tslc1Ts15mCsIdTs15mCs15mCslo5mCsloAs1
582
oGslo5mCsloA]
[oUsloGsILAsloUs15mCs15mCsIdTs dGslcasIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
583
oGslo5mCsloA]
584 [oUsloUsloGsloAs oUsl5mCs15mCs
dTs1c1Gslc1TsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsILGslo5mC]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
138
585 [oUsloUsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
LAsloGslo5mC]
[oUsILGsloAsloUsl5mCs15mCsIdTs dGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
586
oGslo5mC]
[oUsloGsILAsloUs15mCs15mCsIdTs dGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
587
oGslo5mC]
588 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
LGslo5mC]
589 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsILAs1
oGslo5mC]
[oUsILGsloAsloUsl5mCs15mCsIdTs dGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
590
oG]
roUsloGsILAsloUs15mCs15mCsIdTs dGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
591
oG]
592 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsILAs1
oG]
593 [oUsloUsloGsloAs
oUslo5mCslo5mCsloUsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mC
sIdAsIdGslo5mCsloA]
[oUsloUsIdGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsloUslo5mCslo5mCs o5m
594
CsloAsloGslo5mCsloA]
[oUsloUsloGsloAs oUslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs 5mC
sIdAsloGslo5mCsloA]
596 [oUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
o5mCslo5mCslo5m
CsloAsloGslo5mCsloA]
[oUsloUsloGsloAs oUslo5mCslo5mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs 5mC
597
sIdAsloGslo5mC]
[oUsloGsIdAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTslo5mCslo5mCslo5mCslo
598
AsloGslo5mCsloA]
599 [oUsloUsloGsloAs
oUslo5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGslo5mCsloA]
600 [oUsloUsloGsloAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCslo5mCs
loAsloGslo5mCs oA]
601 [oUsloUsloGsloAs
oUslo5mCs15mCsIdTsIdGOTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloGslo5mC]
602 toUsloUsloGsIdAs dTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs
5mCslo5mCslo5mCs
loAsloGslo5mCs oA]
603 toUsloUsloGsloAs
oUslo5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCs15mCs1
oAsloG]
[oGsloAsIdTs15mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCslo5mCslo5mCsloAsloGs
604
105mCsloA]
605 [oUsloUsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsloGslo5mCsloA]
606 [oUsloUsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsloGslo5mC]
607 [oUsloUsloGsloAs oUsl5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oAsloG]
608
[oGsloAsloUsl5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAsloGs1
o5mCsloA]
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
139
609 [oUsloUsloGsloAs oUs1.5mCs15mCs
dTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs1
oA]
[oAsloUsl5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCs 610
oAsloGslo5m
CsloA]
611 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
oGslo5mCsloA]
612 [oUsloGsloAsloUs
5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
oGslo5mC]
[oUsloGsloAsloUs 5mCs15mCsIdTsIdGsIdTsIdTs15mCsIdTs15mCs15mCslo5mCsloAs1
613
oG]
[0304] The mRNA sequence of reference MSH3 mRNA NM 002439.4 (SEQ ID NO:
614) (https://wwvv.ncbi.nlm.nih.goy/nuccore/NM 002439.4, incorporated herein
by
reference), is provided below.
1 ccgcagacgc ctgggaactg cggccgcggg ctcgcgctcc tcgccaggcc ctgccgccgg
61 gctgccatcc ttgccctgcc atgtctcgcc ggaagcctgc gtcgggcggc ctcgctgcct
121 ccagctcagc ccctgcgagg caagcggttt tgagccgatt cttccagtct acgggaagcc
181 tgaaatccac ctcctcctcc acaggtgcag ccgaccaggt ggaccctggc gctgcagcgg
241 ctgcagcggc cgcagcggcc gcagcgcccc cagcgccccc agctcccgcc ttcccgcccc
301 agctgccgcc gcacatagct acagaaattg acagaagaaa gaagagacca ttggaaaatg
361 atgggcctgt taaaaagaaa gtaaagaaag tccaacaaaa ggaaggagga agtgatctgg
421 gaatgtctgg caactctgag ccaaagaaat gtctgaggac caggaatgtt tcaaagtctc
481 tggaaaaarr gaaagaattc rgcrgcgarr crgcccrrcc rcaaagtaga grccagacag
541 aatctctgca ggagagattt gcagttctgc caaaatgtac tgattttgat gatatcagtc
601 ttctacacgc aaagaatgca gtttcttctg aagattcgaa acgtcaaatt aatcaaaagg
661 acacaacact ttttgatctc agtcagtttg gatcatcaaa tacaagtcat gaaaatttac
721 agaaaactgc ttccaaatca gctaacaaac ggtccaaaag catctatacg ccgctagaat
781 tacaatacat agaaatgaag cagcagcaca aagatgcagt tttgtgtgtg gaatgtggat
841 ataagtatag attctttggg gaagatgcag agattgcagc ccgagagctc aatatttatt
901 gccatttaga tcacaacttt atgacagcaa gtatacctac tcacagactg tttgttcatg
961 tacgccgcct ggtggcaaaa ggatataagg tgggagttgt gaagcaaact gaaactgcag
1021 cattaaaggc cattggagac aacagaagtt cactcttttc ccggaaattg actgcccttt
1081 atacaaaatc tacacttatt ggagaagatg tgaatcccct aatcaagctg gatgatgctg
1141 taaatgttga tgagataatg actgatactt ctaccagcta tcttctgtgc atctctgaaa
1201 ataaggaaaa tgttagggac aaaaaaaagg gcaacatttt tattggcatt gtgggagtgc
1261 agcctgccac aggcgaggtt gtgtttgata gtttccagga ctctgcttct cgttcagagc
1321 tagaaacccg gatgtcaagc ctgcagccag tagagctgct gettecttcg gccttgtccg
1381 agcaaacaga ggcgctcatc cacagagcca catctgttag tgtgcaggat gacagaattc
1441 gagtcgaaag gatggataac atttattttg aatacagcca tgctttccag gcagttacag
1501 agttttatgc aaaagataca gttgacatca aaggttctca aattatttct ggcattgtta
1561 acttagagaa gcctgtgatt tgctctttgg ctgccatcat aaaatacctc aaagaattca
1621 acttggaaaa gatgctctcc aaacctgaga attttaaaca gctatcaagt aaaatggaat
1681 ttatgacaat taatggaaca acattaagga atctggaaat cctacagaat cagactgata
CA 03180981 2022- 12- 1
N/11/3 20211247020
PCT/US2020/035972
140
1741 tgaaaaccaa aggaagtttg ctgtgggttt tagaccacac taaaacttca tttgggagac
1801 ggaagttaaa gaagtgggtg acccagccac tccttaaatt aagggaaata aatgcccggc
1861 ttgatgctgt atcggaagtt ctccattcag aatctagtgt gtttggtcag atagaaaatc
1921 atctacgtaa attgcccgac atagagaggg gactctgtag catttatcac aaaaaatgtt
1981 ctacccaaga gttcttcttg attgtcaaaa ctttatatca cctaaagtca gaatttcaag
2041 caataatacc tgctgttaat tcccacattc agtcagactt gctccggacc gttattttag
2101 aaattcctga actcctcagt ccagtggagc attacttaaa gatactcaat gaacaagctg
2161 ccaaagttgg ggataaaact gaattattta aagacctttc tgacttccct ttaataaaaa
2221 agaggaagga tgaaattcaa ggtgttattg acgagatccg aatgcatttg caagaaatac
2281 gaaaaatact aaaaaatcct tctgcacaat atgtgacagt atcaggacag gagtttatga
2341 tagaaataaa gaactctgct gtatcttgta taccaactga ttgggtaaag gttggaagca
2401 caaaagctgt gagccgcttt cactctcctt ttattgtaga aaattacaga catctgaatc
2461 agctccggga gcagctagtc cttgactgca gtgctgaatg gcttgatttt ctagagaaat
2521 tcagtgaaca ttatcactcc ttgtgtaaag cagtgcatca cctagcaact gttgactgca
2581 ttttctccct ggccaaggtc gctaagcaag gagattactg cagaccaact gtacaagaag
2641 aaagaaaaat tgtaataaaa aatggaaggc accctgtgat tgatgtgttg ctgggagaac
2701 aggatcaata tgtcccaaat aatacagatt tatcagagga ctcagagaga gtaatgataa
2761 ttaccggacc aaacatgggt ggaaagagct cctacataaa acaagttgca ttgattacca
2821 tcatggctca gattggctcc tatgttcctg cagaagaagc gacaattggg attgtggatg
2881 gcattttcac aaggatgggt gctgcagaca atatatataa aggacagagt acatttatgg
2941 aagaactgac tgacacagca gaaataatca gaaaagcaac atcacagtcc ttggttatct
3001 tggatgaact aggaagaggg acgagcactc atgatggaat tgccattgcc tatgctacac
3061 ttgagtattt catcagagat gtgaaatcct taaccctgtt tgtcacccat tatccgccag
3121 tttgtgaact agaaaaaaat tactcacacc aggtggggaa ttaccacatg ggattcttgg
3181 tcagtgagga tgaaagcaaa ctggatccag gcgcagcaga acaagtccct gattttgtca
3241 ccttccttta ccaaataact agaggaattg cagcaaggag ttatggatta aatgtggcta
3301 aactagcaga tgttcctgga gaaattttga agaaagcagc tcacaagtca aaagagctgg
3361 aaggattaat aaatacgaaa agaaagagac tcaagtattt tgcaaagtta tggacgatgc
3421 ataatgcaca agacctgcag aagtggacag aggagttcaa catggaagaa acacagactt
3481 ctcttcttca ttaaaatgaa gactacattt gtgaacaaaa aatggagaat taaaaatacc
3541 aactgtacaa aataactctc cagtaacagc ctatctttgt gtgacatgtg agcataaaat
3601 tatgaccatg gtatattcct attggaaaca gagaggtttt tctgaagaca gtctttttca
3661 agtttctgtc ttcctaactt ttctacgtat aaacactctt gaatagactt ccactttgta
3721 attagaaaat tttatggaca gtaagtccag taaagcctta agtggcagaa tataattccc
3781 aagcttttgg agggtgatat aaaaatttac ttgatatttt tatttgtttc agttcagata
3841 attggcaact gggtgaatct ggcaggaatc tatccattga actaaaataa ttttattatg
3901 caaccagttt atccaccaag aacataagaa ttttttataa gtagaaagaa ttggccaggc
3961 atggtggctc atgcctgtaa tcccagcact ttgggaggcc aaggtaggca gatcacctga
4021 ggtcaggagt tcaagaccag cctggccaac atggcaaaac cccatcttta ctaaaaatat
4081 aaagtacatc tctactaaaa atacgaaaaa attagctggg catggtggcg cacacctgta
4141 gtcccagcta ctccggaggc tgaggcagga gaatctcttg aacctgggag gcggaggttg
4201 caatgagccg agatcacgtc actgcactcc agcttgggca acagagcaag actccatctc
4261 aaaaaaaaaa aaagaaaaaa gaaaagaaat agaattatca agcttttaaa aactagagca
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
141
4321 cagaaggaat aaggtcatga aatttaaaag gttaaatatt gtcataggat taagcagttt
4381 aaagattgtt ggatgaaatt atttgtcatt cattcaagta ataaatattt aatgaatact
4441 tgctataaaa aaaaaaaaaa aaaaaaaaaa aa
OTHER ASPECTS
[0305] All publications, patents, and patent applications mentioned in
this specification
are incorporated herein by reference in their entirety to the same extent as
if each
individual publication, patent, or patent application was specifically and
individually
indicated to be incorporated by reference in its entirety. Where a term in the
present
application is found to be defined differently in a document incorporated
herein by
reference, the definition provided herein is to serve as the definition for
the term.
[0306] While the invention has been described in connection with
specific aspects
thereof, it will be understood that invention is capable of further
modifications and this
application is intended to cover any variations, uses, or adaptations of the
invention
following, in general, the principles of the invention and including such
departures from
the present disclosure that come within known or customary practice within the
art to
which the invention pertains and can be applied to the essential features
hereinbefore set
forth, and follows in the scope of the claimed.
[0307] In addition to the various aspects described herein, the present
disclosure includes
the following aspects numbered El through. This list of aspects is presented
as an
exemplary list and the application is not limited to these particular.
[0308] El. A single-stranded oligonucleotide of 15-30 linked
nucleotides in length,
wherein the oligonucleotide, or a portion thereof, is at least 95%
complementary to at
least 15 contiguous nucleobases at positions 2543-2573 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof
[0309] E2. The oligonucleotide of El, wherein the oligonucleotide, or a
portion thereof,
is at least 98% complementary to at least 15 contiguous nucleobases at
positions 2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0310] E3. The oligonucleotide of El, wherein the oligonucleotide, or a
portion thereof,
is at least 99% complementary to at least 15 contiguous nucleobases at
positions 2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
142
[0311] E4. The oligonucleotide of El, wherein the oligonucleotide, or a
portion thereof,
is 100% complementary to at least 15 contiguous nucleobases at positions 2543-
2573 of
SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0312] E5. The oligonucleotide of any one of El-E5, wherein the
oligonucleotide, or a
portion thereof, is complementary to 17-23 contiguous nucleobases at positions
2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0313] E6. The oligonucleotide of any one of E1-E5, wherein the
oligonucleotide is
complementary to 17-20 contiguous nucleobases at positions 2543-2573 of SEQ ID
NO:
614, or a pharmaceutically acceptable salt thereof.
[0314] E7. The oligonucleotide of E6, wherein the 17-20 contiguous
nucleobases begin at
position 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553,
2554, 2555,
2556, or 2557 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof
[0315] E8. The oligonucleotide of any one of E1-E7, wherein the
oligonucleotide is 17-
20 linked nucleotides in length, or a pharmaceutically acceptable salt
thereof.
[0316] E9. The oligonucleotide of any one of E1-E5, wherein the
oligonucleotide, or a
portion thereof, is complementary to 20-23 contiguous nucleobases at positions
2543-
2573 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0317] E10. The oligonucleotide of E9, wherein the 20-23 contiguous
nucleobases begin
at position 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2552, 2553,
or 2554
of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0318] Eli. The oligonucleotide of any one of El-E10, wherein the
oligonucleotide is
20-23 linked nucleotides in length, or a pharmaceutically acceptable salt
thereof.
[0319] E12. The oligonucleotide of any one of El-Eli, wherein the
oligonucleotide, or a
portion thereof, is complementary to positions 2543-2570 of SEQ ID NO: 614, or
a
pharmaceutically acceptable salt thereof.
[0320] E13. A single-stranded oligonucleotide of E15-E30 linked
nucleotides in length,
wherein the oligonucleotide, or a portion thereof, is at least 95%
complementary to at
least 15 contiguous nucleobases at positions 2685-2714 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof.
[0321] E14. The oligonucleotide of E13, wherein the oligonucleotide, or
a portion
thereof, is at least 98% complementary to at least 15 contiguous nucleobases
at positions
2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
143
[0322] E15. The oligonucleotide of E13, wherein the oligonucleotide, or
a portion
thereof, is at least 99% complementary to at least 15 contiguous nucleobases
at positions
2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0323] E16. The oligonucleotide of E13, wherein the oligonucleotide or
a portion thereof,
is 100% complementary to at least 15 contiguous nucleobases at positions 2685-
2714 of
SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0324] E17. The oligonucleotide of any one of claims E13-E16, wherein
the
oligonucleotide, or a portion thereof is complementary to 17-23 contiguous
nucleobases
at positions 2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable
salt thereof
[0325] E18. The oligonucleotide of any one of claims E13-E17, wherein
the
oligonucleotide, or a portion thereof, is complementary to 17-20 contiguous
nucleobases
at positions 2685-2714 of SEQ ID NO: 614, or a pharmaceutically acceptable
salt thereof
[0326] E19. The oligonucleotide of El 8, wherein the oligonucleotide,
or a portion
thereof, is complementary to 17-20 contiguous nucleobases beginning at
position 2685,
2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693, 2694, 2695, 2696, 2697, or
2698 of
SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0327] E20. The oligonucleotide of any one of E13-E19, wherein the
oligonucleotide is
17-20 linked nucleotides in length, or a pharmaceutically acceptable salt
thereof.
[0328] E21. The oligonucleotide of any one of E13-E16, wherein the
oligonucleotide, or
a portion thereof, is complementary to 20-23 contiguous nucleobases at
positions 2685-
2714 of SEQ ID NO: 614, or a pharmaceutically acceptable salt thereof.
[0329] E22. The oligonucleotide of E21, wherein the oligonucleotide is
complementary
to 20-23 contiguous nucleobases beginning at position 2685, 2686, 2687, 2688,
2689,
2690, 2691, 2692, 2693, 2694, or 2695 of SEQ lD NO: 614, or a pharmaceutically
acceptable salt thereof.
[0330] E23. The oligonucleotide of any one of E13-E22, wherein the
oligonucleotide is
20-23 linked nucleotides in length, or a pharmaceutically acceptable salt
thereof.
[0331] E24. The oligonucleotide of E13-E23, wherein the
oligonucleotide, or a portion
thereof, is complementary to positions 2685-2714 of SEQ ID NO: 614, or a
pharmaceutically acceptable salt thereof
[0332] E25. The oligonucleotide of any one of El-E24, wherein the
oligonucleotide is not
any one of Antisense Oligo Nos. 1,97, 193, or 289 of Table 3.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
144
[0333] E26. The oligonucleotide of any one of El-E25, wherein the
oligonucleotide does
not have a nucleobase sequence consisting of any one of SEQ ID NOs. 1, 97,
193, or 289.
[0334] E27. The oligonucleotide of any one of El-E26, wherein the
oligonucleotide
comprises:
(a) a DNA core sequence comprising linked deoxyribonucleosides;
(b) a 5' flanking sequence comprising linked nucleosides; and
(c) a 3' flanking sequence comprising linked nucleosides;
wherein the DNA core comprises a region of at least 10 contiguous nucleobases
positioned between the 5' flanking sequence and the 3' flanking sequence;
wherein the 5'
flanking sequence and the 3' flanking sequence each comprises at least two
linked
nucleosides; and wherein at least one nucleoside of each flanking sequence
comprises an
alternative nucleoside, or a pharmaceutically acceptable salt thereof
[0335] E28. The oligonucleotide of any one of El-E27, wherein the
oligonucleotide
comprises at least one alternative internucleoside linkage, or a
pharmaceutically
acceptable salt thereof.
[0336] E29. The oligonucleotide of E28, wherein the at least one
alternative
internucleoside linkage is a phosphorothioate internucleoside linkage.
[0337] E30. The oligonucleotide of E28, wherein the at least one
alternative
internucleoside linkage is a 2'-alkoxy internucleoside linkage.
[0338] E31. The oligonucleotide of E28, wherein the at least one
alternative
internucleoside linkage is an alkyl phosphate internucleoside linkage.
[0339] E32. The oligonucleotide of any one of claims El- E31, wherein
the
oligonucleotide comprises at least one alternative nucleobase, or a
pharmaceutically
acceptable salt thereof.
[0340] E33. The oligonucleotide of claim E32, wherein the alternative
nucleobase is 5'-
methylcytosine, pseudouridine, or 5-methoxyuridine.
[0341] E34. The oligonucleotide of any one of El- E33, wherein the
oligonucleotide
comprises at least one alternative sugar moiety, or a pharmaceutically
acceptable salt
thereof.
[0342] E35. The oligonucleotide of E34, wherein the alternative sugar
moiety is 2'-0Me
or a bicyclic nucleic acid.
[0343] E36. The oligonucleotide of any one of El- E35, wherein the
oligonucleotide
further comprises a ligand conjugated to the 5' end or the 3' end of the
oligonucleotide
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
145
through a monovalent or branched bivalent or trivalent linker, or a
pharmaceutically
acceptable salt thereof.
[0344] E37. The oligonucleotide of any one of El- E36, wherein the
oligonucleotide
consists of a nucleobase sequence selected from the group consisting of any of
SEQ ID
NOs: 1-384 and 390-613, or a pharmaceutically acceptable salt thereof
[0345] E38. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs: 2-
96, 98-
192, 194-288, 290-384, and 390-613, or a pharmaceutically acceptable salt
thereof
[0346] E39. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs: 1-
384, or
a pharmaceutically acceptable salt thereof.
[0347] E40. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs: 2-
96, 98-
192, 194-288, and 290-384, or a pharmaceutically acceptable salt thereof.
[0348] E41. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs: 1-
96, or
a pharmaceutically acceptable salt thereof.
[0349] E42. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs: 2-
96, or
a pharmaceutically acceptable salt thereof.
[0350] E43. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
97-192,
or a pharmaceutically acceptable salt thereof.
[0351] E44. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
98-192,
or a pharmaceutically acceptable salt thereof.
[0352] E45. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
193-288,
or a pharmaceutically acceptable salt thereof.
[0353] E46. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
194-288,
or a pharmaceutically acceptable salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
146
[0354] E47. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
289-384,
or a pharmaceutically acceptable salt thereof.
[0355] E48. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
288-384,
or a pharmaceutically acceptable salt thereof.
[0356] E49. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
390-
613, or a pharmaceutically acceptable salt thereof.
[0357] E50. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
390-480,
or a pharmaceutically acceptable salt thereof.
[0358] E51. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
481-571,
or a pharmaceutically acceptable salt thereof.
[0359] E52. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
572-662,
or a pharmaceutically acceptable salt thereof.
[0360] E53. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
663-613,
or a pharmaceutically acceptable salt thereof.
[0361] E54. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
1, 6, 13,
17, 21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-
92, or 94-96,
or a pharmaceutically acceptable salt thereof.
[0362] E55. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
6, 13, 17,
21, 24, 26, 29, 33-34, 37, 44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92,
or 94-96, or a
pharmaceutically acceptable salt thereof.
[0363] E56. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence that is SEQ ID NO: 1, or a pharmaceutically acceptable
salt thereof.
[0364] E57. The oligonucleotide of claim E37, wherein the
oligonucleotide consists of a
nucleobase sequence that is SEQ ID NO: 6, or a pharmaceutically acceptable
salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
147
[0365] E58. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
97, 100,
103, 105, 108, 110-111, 113-117, 122-123, 127, 129-130, 133-136, 138-139, 141,
143-
145, 147-148, 154-155, 157-165, 168-170, 172, 174-180, 184, 187, or 191, or a
pharmaceutically acceptable salt thereof.
[0366] E59. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
100, 103,
105, 108, 110-111, 113-117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-
145,
147-148, 154-155, 157-165, 168-170, 172, 174-180, 184, 187, or 191, or a
pharmaceutically acceptable salt thereof.
[0367] E60. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence that is SEQ ID NO: 97, or a pharmaceutically acceptable
salt
thereof.
[0368] E61. The oligonucleotide of E 37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
193-200,
202-230, 232-246, 248-253, 255, 258-261, 265, 270, 274-276, or 285-286, or a
pharmaceutically acceptable salt thereof.
[0369] E62. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
194-200,
202-230, 232-246, 248-253, 255, 258-261, 265, 270, 274-276, or 285-286, or a
pharmaceutically acceptable salt thereof.
[0370] E63. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence that is SEQ ID NO: 193, or a pharmaceutically acceptable
salt
thereof.
[0371] E64. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
226-227,
234, 240, or 243-244, or a pharmaceutically acceptable salt thereof.
[0372] E65. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
227, 234,
240, or 243-244, or a pharmaceutically acceptable salt thereof
[0373] E66. The oligonucleotide of E37, wherein oligonucleotide
consists of the
nucleobase sequence that is SEQ ID NO: 226, or a pharmaceutically acceptable
salt
thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
148
103741 E67. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
289-290,
292, 305, 307, 313, 318, 323-324, 326, 329-330, 332, 338-339, 341, 344, or
346, or a
pharmaceutically acceptable salt thereof.
[0375] E68. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence selected from the group consisting of any of SEQ ID NOs:
290, 292,
305, 307, 313, 318, 323-324, 326, 329-330, 332, 338-339, 341, 344, or 346, or
a
pharmaceutically acceptable salt thereof.
[0376] E69. The oligonucleotide of E37, wherein the oligonucleotide
consists of a
nucleobase sequence that is SEQ ID NO: 289, or a pharmaceutically acceptable
salt
thereof.
[0377] E70. A single-stranded oligonucleotide, wherein the nucleobase
sequence of the
oligonucleotide consists of any one of SEQ ID NOs: 1-384 and 390-613, or a
pharmaceutically acceptable salt thereof.
10378] E71. The oligonucleotide of E70, wherein the nucleobase sequence
of the
oligonucleotide consists of any one of SEQ ID NOs: 2-96, 98-192, 194-288, 290-
384, and
390-613, or a pharmaceutically acceptable salt thereof
[0379] E72. The oligonucleotide of E70, wherein the nucleobase sequence
of the
oligonucleotide consists of any one of SEQ ID NOs: 1-384, or a
pharmaceutically
acceptable salt thereof.
[0380] E73. The oligonucleotide of E70, wherein the nucleobase sequence
of the
oligonucleotide consists of any one of SEQ ID NOs: 2-96, 98-192, 194-288, or
290-384,
or a pharmaceutically acceptable salt thereof.
[0381] E74. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 1-96, or a pharmaceutically
acceptable
salt thereof.
[0382] E75. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 2-96, or a pharmaceutically
acceptable
salt thereof.
[0383] E76. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 97-192, or a pharmaceutically
acceptable salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
149
[0384] E77. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 96-192, or a pharmaceutically
acceptable salt thereof.
[0385] E78. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 193-288, or a pharmaceutically
acceptable salt thereof.
[0386] E79. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 194-288, or a pharmaceutically
acceptable salt thereof.
[0387] E80. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 289-384, or a pharmaceutically
acceptable salt thereof.
[0388] E81. The oligonucleotide of claim E70, wherein the
oligonucleotide consists of
the nucleobase sequence of any one of SEQ ID NOs: 290-384, or a
pharmaceutically
acceptable salt thereof.
[0389] E82. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 390-613, or a pharmaceutically
acceptable salt thereof.
[0390] E83. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 390-480, or a pharmaceutically
acceptable salt thereof
[0391] E84. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 481-571, or a pharmaceutically
acceptable salt thereof.
[0392] E85. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 572-662, or a pharmaceutically
acceptable salt thereof.
[0393] E86. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 663-613, or a pharmaceutically
acceptable salt thereof.
[0394] E87. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 1, 6, 13, 17, 21, 24, 26, 29, 33-
34, 37,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
150
44, 49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96, or a
pharmaceutically
acceptable salt thereof.
[0395] E88. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 6, 13, 17, 21, 24, 26, 29, 33-
34, 37, 44,
49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96, or a pharmaceutically
acceptable
salt thereof
[0396] E89. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of SEQ ID NO: 1, or a pharmaceutically acceptable salt
thereof
[0397] E90. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of SEQ ID NO: 6, or a pharmaceutically acceptable salt
thereof
[0398] E91. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 97, 100, 103, 105, 108, 110-111,
113-
117, 122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155,
157-
165, 168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically acceptable
salt
thereof.
[0399] E92. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 100, 103, 105, 108, 110-111, 113-
117,
122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155, 157-
165,
168-170, 172, 174-180, 184, 187, or 191, or a pharmaceutically acceptable salt
thereof.
[0400] E93. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of SEQ ID NO: 97, or a pharmaceutically acceptable salt
thereof
[0401] E94. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 193-200, 202-230, 232-246, 248-
253,
255, 258-261, 265, 270, 274-276, or 285-286, or a pharmaceutically acceptable
salt
thereof.
[0402] E95. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 194-200, 202-230, 232-246, 248-
253,
255, 258-261, 265, 270, 274-276, or 285-286, or a pharmaceutically acceptable
salt
thereof.
[0403] E96. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NO: 193, or a pharmaceutically
acceptable
salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
151
[0404] E97. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 226-227, 234, 240, or 243-244,
or a
pharmaceutically acceptable salt thereof.
[0405] E98. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 227, 234, 240, or 243-244, or a
pharmaceutically acceptable salt thereof.
[0406] E99. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of SEQ ID NO: 226, or a pharmaceutically acceptable salt
thereof.
[0407] E100. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 289-290, 292, 305, 307, 313,
318, 323-
324, 326, 329-330, 332, 338-339, 341, 344, or 346, or a pharmaceutically
acceptable salt
thereof.
[0408] E101. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of any one of SEQ ID NOs: 290, 292, 305, 307, 313, 318,
323-324,
326, 329-330, 332, 338-339, 341, 344, or 346, or a pharmaceutically acceptable
salt
thereof.
[0409] E102. The oligonucleotide of E70, wherein the oligonucleotide
consists of the
nucleobase sequence of SEQ ID NO: 289, or a pharmaceutically acceptable salt
thereof.
[0410] E103. An oligonucleotide selected from the group consisting of
Antisense Oligo
Nos. 1-384 of Table 3 or 390-613 of Table 4, or a pharmaceutically acceptable
salt
thereof.
[0411] E104. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 2-96, 98-192, 194-288, 290-384 of
Table 3
and 3 90 -6 1 3 of Table 4, or a pharmaceutically acceptable salt thereof.
[0412] E105. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 1-384 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0413] E106. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 2-96, 98-192, 194-288, and 290-
384 of
Table 3, or a pharmaceutically acceptable salt thereof.
[0414] E107. The oligonucleotide E103, wherein the oligonucleotide is
selected from the
group consisting of Antisense Oligo Nos. 1-96 of Table 3, or a
pharmaceutically
acceptable salt thereof.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
152
[0415] E108. The oligonucleotide E103, wherein the oligonucleotide is
selected from the
group consisting of Anti sense Oligo Nos. 2-96 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0416] E109. The oligonucleotide E103, wherein the oligonucleotide is
selected from the
group consisting of Antisense Oligo Nos. 97-192 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0417] E110. The oligonucleotide E103, wherein the oligonucleotide is
selected from the
group consisting of Antisense Oligo Nos. 98-192 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0418] E111. The oligonucleotide E103, wherein the oligonucleotide is
selected from the
group consisting of Antisense Oligo Nos. 193-288 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0419] E112. The oligonucleoti de E103, wherein the oligonucleotide is
selected from the
group consisting of Antisense Oligo Nos. 194-288 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0420] E113. The oligonucleotide E103, wherein the oligonucleotide is
selected from the
group consisting of Antisense Oligo Nos. 289-384 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0421] E114. The oligonucleotide E103, wherein the oligonucleotide is
selected from the
group consisting of Antisense Oligo Nos. 290-384 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0422] E115. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 390-613 of Table 4, or a
pharmaceutically
acceptable salt thereof.
[0423] E116. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 390-480 of Table 4, or a
pharmaceutically
acceptable salt thereof.
[0424] E117. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 481-571 of Table 4, or a
pharmaceutically
acceptable salt thereof.
[0425] E118. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 1, 6, 13, 17, 21, 24, 26, 29, 33-
34, 37, 44,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
153
49-55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0426] E119. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 6, 13, 17, 21, 24, 26, 29, 33-34,
37, 44, 49-
55, 57, 60-73, 75-76, 79-82, 84-86, 88-92, or 94-96 of Table 3, or a
pharmaceutically
acceptable salt thereof.
[0427] E120. The oligonucleotide of E103, wherein the
oligonucleotide is Antisense
Oligo No. 1 of Table 3, or a pharmaceutically acceptable salt thereof.
[0428] E121. The oligonucleotide of E103, wherein the
oligonucleotide is Antisense
Oligo No. 6 of Table 3, or a pharmaceutically acceptable salt thereof.
[0429] E122. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 97, 100, 103, 105, 108, 110-111,
113-117,
122-123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155, 157-
165,
168-170, 172, 174-180, 184, 187, or 191 of Table 3, or a pharmaceutically
acceptable salt
thereof.
[0430] E123. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 100, 103, 105, 108, 110-111, 113-
117, 122-
123, 127, 129-130, 133-136, 138-139, 141, 143-145, 147-148, 154-155, 157-165,
168-
170, 172, 174-180, 184, 187, or 191 of Table 3, or a pharmaceutically
acceptable salt
thereof.
[0431] E124. The oligonucleotide of E103, wherein the
oligonucleotide is Antisense
Oligo No. 97 of Table 3, or a pharmaceutically acceptable salt thereof.
[0432] E125. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos 193-200, 202-230, 232-246, 248-
253, 255,
258-261, 265, 270, 274-276, or 285-286 of Table 3, or a pharmaceutically
acceptable salt
thereof.
[0433] E126. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 194-200, 202-230, 232-246, 248-
253, 255,
258-261, 265, 270, 274-276, or 285-286 of Table 3, or a pharmaceutically
acceptable salt
thereof.
[0434] E127. The oligonucleotide of E103, wherein the oligonucleotide
is Antisense
Oligo No. 193 of Table 3.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
154
[0435] E128. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Anti sense Oligo Nos 226-227, 234, 240, or 243-244 of
Table 3,
or a pharmaceutically acceptable salt thereof.
[0436] E129. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 227, 234, 240, or 243-244 of
Table 3, or a
pharmaceutically acceptable salt thereof.
[0437] E130. The oligonucleotide of E103, wherein the oligonucleotide
is Antisense
Oligo No. 226 of Table 3, or a pharmaceutically acceptable salt thereof.
[0438] E131. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos. 289-290, 292, 305, 307, 313, 318,
323-324,
326, 329-330, 332, 338-339, 341, 344, or 346 of Table 3, or a pharmaceutically
acceptable salt thereof.
[0439] E132. The oligonucleotide of E103, wherein the oligonucleotide
is selected from
the group consisting of Antisense Oligo Nos 290, 292, 305, 307, 313, 318, 323-
324, 326,
329-330, 332, 338-339, 341, 344, or 346 of Table 3, or a pharmaceutically
acceptable salt
thereof.
[0440] E133. The oligonucleotide of E103, wherein the oligonucleotide
is Antisense
Oligo No. 289 of Table 3, or a pharmaceutically acceptable salt thereof.
[0441] E134. The oligonucleotide of any one of El- E133, wherein the
oligonucleotide,
or a pharmaceutically acceptable salt thereof, causes at least a 50% reduction
in MSH3
mRNA expression at an oligonucleotide concentration of 10 nM.
[0442] E135. The oligonucleotide of any one of El- E133, wherein the
oligonucleotide,
or a pharmaceutically acceptable salt thereof, causes at least a 60% reduction
in MSH3
mRNA expression at an oligonucleotide concentration of 10 nM.
[0443] E136. The oligonucleotide of any one of El- E133, wherein the
oligonucleotide,
or a pharmaceutically acceptable salt thereof, causes at least a 70% reduction
in MSH3
mRNA expression at an oligonucleotide concentration of 10 nM.
[0444] E137. The oligonucleotide of any one of El- E133, wherein the
oligonucleotide,
or a pharmaceutically acceptable salt thereof, causes at least an 80%
reduction in MSH3
mRNA expression at an oligonucleotide concentration of 10 nM.
[0445] E138. The oligonucleotide of any one of El- E133, wherein the
oligonucleotide,
or a pharmaceutically acceptable salt thereof, causes at least a 50% reduction
in MSH3
mRNA expression at an oligonucleotide concentration of 1 nM.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
155
[0446] E139. The oligonucleotide of any one of El- E133, wherein the
oligonucleotide,
or a pharmaceutically acceptable salt thereof, causes at least a 600/0
reduction in MS143
mRNA expression at an oligonucleotide concentration of 1 nM.
[0447] E140. The oligonucleotide of any one of El- E133, wherein the
oligonucleotide,
or a pharmaceutically acceptable salt thereof, causes at least a 70% reduction
in MSH3
mRNA expression at an oligonucleotide concentration of 1 nM.
[0448] E141. The oligonucleotide of any one of E135- E140, wherein the
MSH3 mRNA
expression is evaluated in vitro.
[0449] E142. The oligonucleotide of E141, wherein the MSH3 mRNA
expression is
evaluated in a cell based assay.
[0450] E143. The oligonucleotide of E142, wherein the MSH3 mRNA
expression is
evaluated in HeLa cells.
[0451] E144. The oligonucleotide of any one of E134- E143, wherein the
MSH3 mRNA
expression is determined by the quantitative reverse transcription polymerase
chain
reaction (RT-qPCR).
[0452] E145. The oligonucleotide of any one of E134- E144, wherein the
MSH3 mRNA
is expression is normalized to the mRNA expression of a reference gene.
[0453] E146. The oligonucleotide of E145, wherein the MSH3 mRNA
expression is
normalized to the mRNA expression of beta-glucuronidase (GUSB).
[0454] E147. The oligonucleotide of any one of E134- E147, wherein the
reduction in
MSH3 mRNA expression is relative to a control.
[0455] E148. The oligonucleotide of E 147, wherein the control is the
MSH3 mRNA
expression in the absence of the oligonucleotide, or pharmaceutically
acceptable salt
thereof
[0456] E149. The oligonucleotide of E148, wherein the control is the
MSH3 mRNA
expression in the absence of the oligonucleotide, or pharmaceutically
acceptable salt
thereof, but in the presence of a control oligonucleotide, or salt thereof
[0457] E150. The oligonucleotide of E149, wherein the control
oligonucleotide, or salt
thereof, is a scrambled luciferase targeting oligonucleotide.
[0458] E151. The oligonucleotide of any one of E134- E150, wherein the
reduction in
MSH3 mRNA expression is calculated by a delta-delta Ct (AACT) method.
[0459] E152. The oligonucleotide of any one of EIS 1, wherein the delta-
delta Ct (AACT)
method comprises the normalization of the MSH3 mRNA expression to the mRNA
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
156
expression of a reference gene and to the MSH3 mRNA expression in the absence
of the
oligonucleotide, or pharmaceutically acceptable salt thereof but in the
presence of a
control oligonucleotide, or salt thereof.
[0460] E153. The oligonucleotide of E152, wherein the reference gene is
beta-
glucuronidase (GUSB) and/or the control oligonucleotide, or salt thereof, is a
scrambled
luciferase targeting oligonucleotide.
[0461] E154. The oligonucleotide of any one of E134- E153,
wherein the reduction in
MSH3 mRNA expression is determined by the method of Example 1.
[0462] E155. The oligonucleotide of any one of E134- E137 and E141-
E154, wherein in
the same assay, Antisense Oligo No. 1 causes approximately a 58% reduction in
MSH3
mRNA expression at an oligonucleotide concentration of 10 nM.
[0463] E156. The oligonucleotide of any one of E138- E154, wherein in
the same assay,
Anti sense Oligo No. 1 causes approximately a 14% reduction in MSH3 mRNA
expression at an oligonucleotide concentration of 1 nM.
[0464] E157. The oligonucleotide of any one of El- E156, wherein the
oligonucleotide is
in the free base form.
[0465] E158. The oligonucleotide of any one of El- E156, wherein the
oligonucleotide is
a pharmaceutically acceptable salt thereof.
[0466] E159. The oligonucleotide of E158, wherein the
oligonucleotide is a sodium salt.
[0467] E160. A pharmaceutical composition comprising one or more of the
oligonucleotides, or pharmaceutically acceptable salts thereof, of any one of
El- E159
and a pharmaceutically acceptable carrier or excipient.
[0468] E161. A composition comprising one or more of the
oligonucleotides, or
pharmaceutically acceptable salts thereof, of any one of El- El 59 and a lipid
nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.
[0469] E162. A method of inhibiting transcription of MSH3 in a cell,
the method
comprising contacting the cell with one or more of the oligonucleotides, or
pharmaceutically acceptable salts thereof, of any one of El- El 59, the
pharmaceutical
composition of E160, or the composition of E161 for a time sufficient to
obtain
degradation of an mRNA transcript of a MSH3 gene, inhibits expression of the
MSH3
gene in the cell.
[0470] E163. A method of treating, preventing, or delaying the
progression a nucleotide
repeat expansion disorder in a subject in need thereof, the method comprising
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
157
administering to the subject one or more of the oligonucleotides, or
pharmaceutically
acceptable salts thereof, of any one of El- E159, the pharmaceutical
composition of
E160, or the composition of E161.
[0471] E164. A method of reducing the level and/or activity of MSH3 in
a cell of a
subject identified as having a nucleotide repeat expansion disorder, the
method
comprising contacting the cell with one or more of the oligonucleotides, or
pharmaceutically acceptable salts thereof, of any one of El- E159, the
pharmaceutical
composition of E160, or the composition of E161.
[0472] E165. A method for inhibiting expression of an MSH3 gene in a
cell comprising
contacting the cell with one or more of the oligonucleotides, or
pharmaceutically
acceptable salts thereof, of any one of El- E159, the pharmaceutical
composition of
E160, or the composition of E161 and maintaining the cell for a time
sufficient to obtain
degradation of a mRNA transcript of an MSH3 gene, thereby inhibiting
expression of the
MSH3 gene in the cell.
[0473] E166. A method of decreasing nucleotide repeat expansion in a
cell, the method
comprising contacting the cell with one or more of the oligonucleotides, or
pharmaceutically acceptable salts thereof, of any one of El- E159, the
pharmaceutical
composition of E160, or the composition of E161.
[0474] E167. The method of E162 and E164- E166, wherein the cell
is in a subject.
[0475] E168. The method of E163 or E164, wherein the subject is
a human.
[0476] E169. The method of E168, wherein the cell is a cell of the
central nervous system
or a muscle cell.
[0477] E170. The method of E163, wherein the subject is identified as
having a
nucleotide repeat expansion disorder.
[0478] E171. The method of E170, wherein the nucleotide repeat
expansion disorder is
spinocerebellar ataxia type 36 or frontotemporal dementia.
[0479] E172. The method of E170, wherein the nucleotide repeat
expansion disorder is a
trinucleotide repeat expansion disorder.
[0480] E173. The method of E172, wherein the trinucleotide repeat
expansion disorder is
a polyglutamine disease.
[0481] E174. The method of E173, wherein the polyglutamine disease is
selected from
the group consisting of dentatorubropallidoluysian atrophy, Huntington's
disease, spinal
and bulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar
ataxia type 2,
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
158
spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar
ataxia type 7,
spinocerebellar ataxia type 17, and Huntington's disease-like 2.
[0482] E175. The method of E172, wherein the trinucleotide repeat
expansion disorder is
a non-polyglutamine disease.
[0483] E176. The method of E175, wherein the non-polyglutamine disease
is selected
from the group consisting of fragile X syndrome, fragile X-associated
tremor/ataxia
syndrome, fragile XE mental retardation, Friedreich' s ataxia, myotonic
dystrophy type 1,
spinocerebellar ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal
muscular
dystrophy, Fragile X-associated premature ovarian failure, FRA2A syndrome,
FRA7A
syndrome, and early infantile epileptic encephalopathy.
[0484] E177. One or more of the oligonucleotides, or pharmaceutically
acceptable salts
thereof, of any one of El- E159, the pharmaceutical composition of E160, or
the
composition of El 61 for use in the prevention or treatment of a nucleotide
repeat
expansion disorder.
[0485] E178. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition for the use of E177, wherein the
nucleotide
repeat expansion disorder is spinocerebellar ataxia type 36 or frontotemporal
dementia.
[0486] E179. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition for the use of E177, wherein the
nucleotide
repeat expansion disorder is a trinucleotide repeat expansion disorder.
[0487] E180. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition for the use of E179, wherein the
trinucleotide repeat expansion disorder is selected from the group consisting
of
dentatorubropallidoluysian atrophy, Huntington's disease, spinal and bulbar
muscular
atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2,
spinocerebellar
ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7,
spinocerebellar
ataxia type 17, Huntington's disease-like 2, fragile X syndrome, fragile X-
associated
tremor/ataxia syndrome, fragile XE mental retardation, Friedreich' s ataxia,
myotonic
dystrophy type 1, spinocerebellar ataxia type 8, spinocerebellar ataxia type
12,
oculopharyngeal muscular dystrophy, Fragile X-associated premature ovarian
failure,
FRA2A syndrome, FRA7A syndrome, and early infantile epileptic encephalopathy.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
159
[0488] E181. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition for the use of E179 or El 80,
wherein the
trinucleotide repeat expansion disorder is Huntington's disease.
[0489] E182. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of E179 or E180, wherein the
trinucleotide
repeat expansion disorder is Friedreich's ataxia.
[0490] E183. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition for the use of E179 or E180,
wherein the
trinucleotide repeat expansion disorder is myotonic dystrophy type 1.
[0491] E184. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of any of E177- E183, wherein the
oligonucleotide, pharmaceutical composition, or composition is administered
intrathecally, intraventricularly, intracerebroventricularly, intraocularly,
subcutaneously,
intravenously, intra cisterna magnally, intramuscularly, or orally.
[0492] E185. A method of treating, preventing, or delaying the
progression a disorder in a
subject in need thereof wherein the subject is suffering from nucleotide
repeat expansion
disorder, comprising administering to said subject one or more of the
oligonucleotides, or
pharmaceutically acceptable salts thereof, of any one of El- El 59, the
pharmaceutical
composition of E160, or the composition of E161.
[0493] E186. The method of E185, further comprising administering an
additional
therapeutic agent.
[0494] E187. The method of E185, wherein the additional therapeutic
agent is another
oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.
[0495] E188 A method of preventing or delaying the progression of a
nucleotide repeat
expansion disorder in a subject, the method comprising administering to the
subject one
or more of the oligonucleotides, or pharmaceutically acceptable salts thereof,
of any one
of El- E159, the pharmaceutical composition of E160, or the composition of
E161 or the
composition of E159 in an amount effective to delay progression of a
nucleotide repeat
expansion disorder of the subject.
[0496] E189. The method of E188, wherein the nucleotide repeat
expansion disorder is
spinocerebellar ataxia type 36 or frontotemporal dementia.
[0497] E190. The method of E188, wherein the nucleotide repeat
expansion disorder is a
trinucleotide repeat expansion disorder.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
160
[0498] E191. The method of E190, wherein the trinucleotide repeat
expansion disorder is
selected from the group consisting of dentatorubropallidoluysi an atrophy,
Huntington's
disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia type 1,
spinocerebellar
ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6,
spinocerebellar
ataxia type 7, spinocerebellar ataxia type 17, Huntington's disease-like 2,
fragile X
syndrome, fragile X-associated tremor/ataxia syndrome, fragile XE mental
retardation,
Friedreich's ataxia, myotonic dystrophy type 1, spinocerebellar ataxia type 8,
spinocerebellar ataxia type 12, oculopharyngeal muscular dystrophy, Fragile X-
associated
premature ovarian failure, FRA2A syndrome, FRA7A syndrome, and early infantile
epileptic encephalopathy.
[0499] E192. The method of E190 or E191, wherein the trinucleotide
repeat expansion
disorder is Huntington's disease.
[0500] E193. The method of E190 or El 91, wherein the trinucleotide
repeat expansion
disorder is Friedrich's ataxia.
[0501] E194. The method of E190 or E191, wherein the trinucleotide
repeat expansion
disorder is myotonic Dystrophy type 1.
[0502] E195. The method of E190 or E191, further comprising
administering an
additional therapeutic agent.
[0503] E196. The method of E195, wherein the additional therapeutic
agent is an
oligonucleotide that hybridizes to an mRNA encoding the Huntingtin gene.
[0504] E197. The method of any of E188- E196, wherein progression of
the nucleotide
repeat expansion disorder is delayed by at least 120 days, for example, at
least 6 months,
at least 12 months, at least 2 years, at least 3 years, at least 4 years, at
least 5 years, at
least 10 years or more, when compared with a predicted progression
[0505] E198. One or more of the oligonucleotides, or pharmaceutically
acceptable salts
thereof, of any one of El- E159, the phaimaceutical composition of E160, or
the
composition of E161 for use in preventing or delaying progression of a
nucleotide repeat
expansion disorder in a subject.
[0506] E199. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of E198, wherein the nucleotide
repeat
expansion disorder is spinocerebellar ataxia type 36 or frontotemporal
dementia.
CA 03180981 2022- 12- 1
WO 2021/247020
PCT/US2020/035972
161
105071 E200. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of El 98, wherein the nucleotide
repeat
expansion disorder is a trinucleotide repeat expansion disorder.
[0508] E201. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of E200, wherein the trinucleotide
repeat
expansion disorder is selected from the group consisting of
dentatorubropallidoluysian
atrophy, Huntington' s disease, spinal and bulbar muscular atrophy,
spinocerebellar ataxia
type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3,
spinocerebellar ataxia
type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17,
Huntington' s
disease-like 2, fragile X syndrome, fragile X-associated tremor/ataxia
syndrome, fragile
XE mental retardation, Friedreich's ataxia, myotonic dystrophy type 1,
spinocerebellar
ataxia type 8, spinocerebellar ataxia type 12, oculopharyngeal muscular
dystrophy,
Fragile X-associated premature ovarian failure, FRA2A syndrome, FRA7A
syndrome,
and early infantile epileptic encephalopathy.
[0509] E202. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of E200 or E201, wherein the
trinucleotide
repeat expansion disorder is Huntington's disease.
[0510] E203. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of E200 or E201, wherein the
trinucleotide
repeat expansion disorder is Friedrich' s ataxia.
[0511] E204. The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of E200 or E201, wherein the
trinucleotide
repeat expansion disorder is myotonic Dystrophy type 1.
[0512] E205 The oligonucleotide, or pharmaceutically acceptable salt
thereof,
pharmaceutical composition, or composition of any one of E198- E204, wherein
progression of the nucleotide repeat expansion disorder is delayed by at least
120 days,
for example, at least 6 months, at least 12 months, at least 2 years, at least
3 years, at least
4 years, at least 5 years, at least 10 years or more, when compared with a
predicted
progression.
CA 03180981 2022- 12- 1
