Note: Descriptions are shown in the official language in which they were submitted.
COMPOSITIONS AND METHODS FOR TREATING CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit
of the filing of U.S. Patent
Application No. 17/359,905, filed on June 28, 2021, titled "Compositions and
Methods for Treating
Cancer". The specification and claims thereof are incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing
which has been submitted
electronically in ASCII format and is hereby incorporated by reference in its
entirety. Said ASCII copy,
created on January 26, 2022, is named 32064-1035-PCT2_SL.txt and is 446,054
bytes in size.
BACKGROUND
[0003] A variety of cancer therapies and treatments exist
such as surgical resection of solid
tumors, radiation, and chemotherapy. While surgical resection and radiation
are used on localized
tumors, chemotherapy is often delivered systemically and impacts both cancer
and non-cancer cells,
leading to severe and even life-threatening side effects. Older cancer drugs,
including alkylators,
nucleotide antinnetabolites, and tubulin poisons, cause significant side
effects because they are
similarly toxic to normal cells as to cancer cells, especially those normal
cells undergoing routine cell
division in the intestine, scalp, and skin. For this reason, much of the
effort in contemporary cancer
drug discovery is devoted to finding targeted therapeutics which differentiate
between cancer cells
and normal cells (Neidle et al., (2014) Cancer Drug Design and Discovery).
This has led to drugs
which inhibit the function of oncolytic proteins that are mutated,
overexpressed, or abnormally
hyperactive in cancer but not in normal cells. Examples of such drugs include
kinase inhibitors,
histone deacetylase inhibitors, proteasonne inhibitors, nnTOR inhibitors, BCL2
inhibitors, and isocitrate
dehydrogenase inhibitors. Significant effort has also been devoted to
targeting cell surface antigens
which are differentially expressed in cancer cells compared to normal cells.
Monoclonal antibodies
and antibody-drug conjugates targeting cancer cell surface antigens have thus
been developed as
cancer therapeutics (Beck et al., (2017) Nat Rev Drug Disc 16, 315-337).
Another point of
differentiation between cancer cells and normal cells is metabolism. It was
discovered many years
ago that many cancer cells utilize glucose fermentation to generate ATP as
opposed to the process of
oxidative phosphorylation used by normal cells. A drug targeting isocitrate
dehydrogenase, involved
in abnormal glucose metabolism in cancer cells, was recently approved by the
FDA (Dhillon (2018)
Drugs 78, 1509-1516). Abnormalities in one-carbon metabolism, which
encompasses the folate and
nnethionine cycles and affects nucleotide synthesis and DNA nnethylation as a
way of controlling gene
expression, are strongly associated with some cancers (Fanidi et al., (2019)
Int J Cancer 145, 1499-
1503; Yang (2018) Front Oncol 8, 493). In this connection, it has been known
for a long time that
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certain synthetic analogs of folic acid (antifolates) can inhibit the growth
of cancer cells. It is also
known that some cancer cells are dependent for survival on the amino acid
methionine. If methionine
is restricted, the cancer cells die, while this has little effect on normal
cells. In recent years, evidence
has begun to emerge that some cancer cells might have an abnormal dependency
on vitamin B12.
The nature of this dependency is not understood but might, in part, involve
the use of vitamin B12 as
a catalytic cofactor by the enzyme methionine synthase in one-carbon
metabolism.
[0004] Vitamin B12 (cobalamin) is an essential micronutrient
in the human diet. It is a
cofactor for the metabolic enzymes methionine synthase and nnethylnnalonyl-CoA
nnutase (Fedosov et
al., (2012) Water Soluble Vitamins (book) 56, 347-367). After oral ingestion
and transport through the
intestine, cobalannin is almost completely protein bound in plasma to the
chaperone proteins
transcobalannin 1 (TCN1, haptocorrin, R-binder) (TC01_HUMAN) and
transcobalamin 2 (TCN2)
(TCO2_HUMAN). The TCN2-cobalamin complex (TCN2-Cbl) is taken up by most cells
using the
process of receptor-mediated endocytosis and has a plasma half-life of 1-15 h.
TCN2 has a high
affinity and specificity for cobalannin in its various dietary and nutritional
supplement forms, such as
methyl cobalannin, adenosyl cobalannin and cyanocobalannin (Fedosov et al.,
(2007) Biochenn 46,
6446-6458). TCN1 is a glycoprotein that exists in two different forms in
plasma (Marzolo and Farfan
(2011) Biol Res 44, 81-105). The most abundant form is sialylated and has a
plasma half-life of about
days (Bor (2004) Clin Chem 50, 1043-1049). A less abundant form is
desialylated and has a
plasma half-life of a few minutes. Unlike TCN2-Cbl, which can be taken up by
almost all cell types,
the transcobalannin 1-cobalannin complex (TCN1-Cb1) is quickly taken up by
certain liver cells, only in
its desialylated form, by receptor-mediated endocytosis.
[0005] CD320 and LRP2 are two receptors involved in the
uptake of cobalannin as TCN2-
Cbl. CD320, a member of the low-density lipoprotein receptor (LDLR) family, is
constitutively
expressed in most cells and is the receptor primarily responsible for the
uptake of cobalannin
(Quadros (2013) Biochinnie 95, 1008-1018). CD320 is overexpressed in some
types of cancer (Sycel
et al., (2013) Anticancer Res 33, 4203-4212; Amagasaki (1990) Blood 76, 1380-
1386). There is also
evidence that CD320 facilitates the transport of TCN2-Cbl through the blood-
brain barrier into the
brain (Lai et al.; (2013) FASEB 27, 2468-2475). LRP2 is another receptor in
the LDLR family. It is
expressed most highly in the kidney but also in other tissues. In addition to
cobalannin, LRP2 also
transports sundry proteins and small molecules, induding albumin, insulin and
vitamin D (Mazolo et
al., (2011) Biol Res 44, 89-105). In the liver, the asialoglycoprotein
receptor (ASGR) uptakes TCN1-
Cbl by receptor-mediated endocytosis so long as TCN1 is in its desialylated
form. Normal liver cells
and liver cancer cells express very high levels of ASGR (-50,000 receptors per
cell), making this
receptor attractive as a portal for delivering drugs to the liver (Luo et al.,
(2017) Biomedicine and
Pharnnacotherapy 88, 87-94; Stockert (1995) Physiological Rev 75, 595-609;
Soda et al., Blood
(1985) 65, 795-802).
[0006] After receptor mediated endocytosis, cobalannin is
sequestered in the endosonne,
where the endosonnal membrane prevents passive egress to the cytosol. A
specialized protein (cbIF)
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CA 03174172 2022- 9- 28
facilitates the transport of cobalannin through the endosonnal membrane to the
cytosol (Banerjee et al.,
(2009) Curr Opin Chem Bio 13,484-491).
BRIEF SUMMARY OF THE INVENTION
[0007] One embodiment of the present invention provides for
a double stranded RNA
interference (RNAi) agent comprising at least one of (i) a first double-
stranded ribonucleic acid
(dsRNA) for inhibiting the expression of a CD320 gene wherein the first dsRNA
comprises a sense
strand and an antisense strand forming a duplex, (ii) a second dsRNA for
inhibiting the expression of
a LRP2 gene wherein the second dsRNA comprises a sense strand and an antisense
strand forming
a duplex, or (iii) a cocktail of (i) and (ii) and wherein the sense strand of
the first dsRNA is at least
substantially complementary to the antisense strand of the first dsRNA and the
sense strand of the
second dsRNA is at least substantially complementary to the antisense strand
of the second dsRNA.
For example, the antisense strand of (i) the first dsRNA includes a region of
connplennentarity to a
CD320 RNA transcript and for example the sense strand of (i) the first dsRNA
is selected from Table
5. The antisense strand of (ii) the second dsRNA includes a region of
complementarity to an LRP2
RNA transcript and the sense strand of (ii) the second dsRNA are selected from
Table 6. In one
example, (i) the first dsRNA or (ii) the second dsRNA comprises a duplex
region which is 16-30
nucleotide pairs in length. In another example, (i) the first dsRNA or (ii)
the second dsRNA comprises
a duplex region which is 21-23 nucleotide pairs in length. In one embodiment,
the double stranded
RNAi agent includes at least one strand of: (i) the first dsRNA or (ii) the
second dsRNA which
comprises a 3 overhang of at least 2 nucleotides. Further still, in one
embodiment, the antisense
strand of (i) the first dsRNA, comprises the nucleotide sequence selected from
(5' 4
3'):CAGUUGCGCAGUUUCUUGUCAGUUCdTdT (SEQ ID NO: 17);
CAGUUGCGCAGUUUCUUGUCAGUUCdT*dT (SEQ ID NO 18);
mCmAmGmUmUmGmCmGmCmAmGmUmUmUmCmUmUmGmUmCmAmGmUmU
nnCdT*dT (SEQ ID NO 19);
mCmAmGmUmUmGmCmGmCmAmGmUmUmUmCmUmUmGmUmCmAmGmUmU
mC (SEQ ID NO 21);
mCmAmGmUmUmGmCmGmCmAmGmUmUmUmCmUmUmGmUmCmAmGmUmU
nnCdT*dT (SEQ ID NO 23);
mC2fAmG2fUmU2fGmC2fGmC2fAmG2fUmU2fUmC2fUmU2fGmU2fCmA2fGmU2fU
mCdT*dT (SEQ ID NO 24);
mC2fAmG2fUmU2fGmC2fGmC2fAmG2fUmU2fUmC2fUmU2fGmU2fCmA2fGmU2fU
nnC (SEQ ID NO 25);
2fCmA2fGmU2fUmG2fCmG2fCmA2fGmU2fUmU2fCmU2fUmG2fUmC2fAmG2fUmU
2fCdT*dT (SEQ ID NO 28);
2fCmA2fGmU2fUmG2fCmG2fCmA2fGmU2fUmU2fCmU2fUmG2fUmC2fAmG2fUmU
2fC (SEQ ID NO 29);
mC2fA2fG2fU2fU2fG2fC2fG2fC2fA2fG2fU2fU2fU2fC2fU2fU2fG2fU2fC2fA2fG2fU2fU
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2fCdT*dT (SEQ ID NO 30);
mC2fAmG2fUmU2fGmC2fGmC2fAmG2fUmU2fUmC2fUmU2fGmU2fCmA2fGmU2fU
mCdT*dT (SEQ ID NO 32);
mC2fAmG2fUmU2fGmC2fGmC2fAmG2fUmU2fUmC2fUmU2fGmU2fCmA2fGmU (SEQ ID NO 33);
mC2fAmG2fUmU2fGmC2fGmC2fAmG2fUmU2fUmC2fU2fU2fG2fU2fC2fA2fG2fU (SEQ ID NO 34);
wherein, mA, mC, mG, and mU are 2'-0-methyl adenosine, cytidine, guanosine, or
uridine,
respectively; 2fA, 2fC, 2fG, and 2fU are 2'-fluoro adenosine, cytidine,
guanosine, or uridine,
respectively; and * is a phosphorothioate linkage; and
the sense strand is at least substantially complementary to the antisense
strand.
[0008] Further still, in another embodiment, the double
stranded RNAi agent indudes the
antisense strand of (i) the first dsRNA, that comprises the nucleotide
sequence selected from (5' 4 3')
AAGAGCUCAGGUCUCUGAGGGdTdT (SEQ ID NO 64);
AAGAGCUCAGGUCUCUGAGGGdT*dT (SEQ ID NO 65);
mAmAmGmAmGmCmUmCmAmGmGmUmCmUmCmUmGmAnnGnnGnnGdT*dT (SEQ ID NO 66);
mAmAmGmAmGmCmUmCmAmGmGmUmCmUmCmUmGnnAnnGnnGnnG (SEQ ID NO 68);
mA2fAmG2fAmG2fCmU2fCmA2fGmG2fUmC2fUmC2fUmG2fAmG2fGmGdT*dT (SEQ ID NO 71);
mA2fAmG2fAmG2fCmU2fCmA2fGmG2fUmC2fUmC2fUmG2fAmG2fGmG (SEQ ID NO 72);
2fAmA2fGmA2fGmC2fUmC2fAmG2fGmU2fCmU2fCmU2fGmA2fGmG2fGdT*dT (SEQ ID NO 75);
2fAmA2fGmA2fGmC2fUmC2fAmG2fGmU2fCmU2fCmU2fGmA2fGmG2fG (SEQ ID NO 76);
mA2fA2fGmA2fGmC2fUmC2fAmG2fGmU2fCmU2fCmU2fGmA2fGmG2fG (SEQ ID NO 77);
mA2fA2fGmA2fGmC2fUmC2fAmG2fGmU2fCmU2fCmU2fGmA2fGmG2fGdT*dT (SEQ ID NO 78);
2fAmA2fGmA2fGmC2fUmC2fAmG2fGmU2fCmU2fCmU2fGmA2fGmG2fGdT*dT (SEQ ID NO 79);
2fAmA2fGmA2fGmC2fUmC2fAmG2fGmU2fCmU2fC2fU2fG2fA2fG2fG2fG (SEQ ID NO 81);
wherein, mA, mC, mG, and mU are 2'-0-methyl adenosine, cytidine, guanosine, or
uridine,
respectively; 2fA, 2fC, 2fG, and 2fU are 2'-fluoro adenosine, cytidine,
guanosine, or uridine,
respectively; and * is a phosphorothioate linkage; and
the sense strand is at least substantially complementary to the antisense
strand.
[0009] In another embodiment the double stranded RNAi agent
of (h) the second dsRNA
comprises the nucleotide sequence selected from (5' 4 3')
UUUGAUAGCACCAAACCUAGAGCCCdTdT (SEQ ID NO: 417);
UUUGAUAGCACCAAACCUAGAGCCCdT*dT (SEQ ID NO: 418);
mUm[mUmGmAmUmAmGmCmAmCmCmAmAmAmCmCmUmAmGmAmGmCmCmC
dT*dT (SEQ ID NO: 419);
mUmUmUmGmAmUmAmGmCmAmCmCmAmAmAmCmCmUmAmGmAmGmCmCmC (SEQ ID NO:
421);
mU2fUmU2fGmA2fUmA2fGmC2fAmC2fCmA2fAmA2fCmC2fUmA2fGmA2fGmC2fCmCdT*dT](SEQ
ID NO: 424);
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mU2fUmU2fGmA2fUmA2fGmC2fAmC2fCmA2fAmA2fCmC2fUmA2fGmA2fGmC2fCmC (SEQ ID NO:
425);
mU2fAmU2fCmA2fAmA2fCmC2fUmC2fGmA2fUmA2fGmC2fAmA2fCmA2fCmC2fGmC (SEQ ID NO:
429);
mU2fU2fU2fG2fA2fU2fA2fG2fC2fA2fC2fC2fA2fA2fA2fC2fC2fU2fA2fG2fA2fG2fC2fC2fCdT*dT
(SEQ
ID NO: 430);
mU2fUmU2fGmA2fUmA2fGmC2fAmC2fCmA2fAmA2fCmC2fUmA2fGmA2fGmC2fCmCdT*dT (SEQ
ID NO: 432);
mU2fUmU2fGmA2fUmA2fGmC2fAmC2fCmA2fAmA2fCmC2fUmA2fGmA2fGmC (SEQ ID NO: 433);
and
mU2fUmU2fGmA2fUmA2fGmC2fAmC2fCmA2fAmA2fC2fC2fU2fA2fG2fA2fG2fC (SEQ ID NO: 434)
wherein, mA, mC, mG, and mU are 2'-0-methyl adenosine, cytidine, guanosine, or
uridine,
respectively; 2fA, 2fC, 2fG, and 2fU are 2'-fluoro adenosine, cytidine,
guanosine, or uridine,
respectively; and * is a phosphorothioate linkage; and
the sense strand is at least substantially complementary to the antisense
strand.
[0010] In a further embodiment, the double stranded RNAi
agent antisense strand of (h) the
second dsRNA comprises the nucleotide sequence selected from (5' 4 3')
UUUGCAAUGACUCUCCUAUCAGUCCdTdT(SEQ ID NO: 448);
UUUGCAAUGACUCUCCUAUCAGUCCdT*dT (SEQ ID NO: 449);
mUmUmUmGmCmAmAmUmGmAmCmUmCmUmCmCmUmAmUmCmAnnGnnUnnCnnCdT*dT (SEQ
ID NO: 450);
mUmUmUmGmCmAmAmUmGmAmCmUmCmUmCmCmUmAmUmCmAmGmUmCmC (SEQ ID NO:
452);
mU2fUmU2fGmC2fAmA2fUmG2fAmC2fUmC2fUmC2fCmU2fAmU2fCmA2fGmU2fCmCdT*dT (SEQ
ID NO: 455);
mU2fUmU2fGmC2fAmA2fUmG2fAmC2fUmC2fUmC2fCmU2fAmU2fCmA2fGmU2fCmC (SEQ ID NO:
456);
mU2fUmU2fGmC2fAmA2fUmG2fAmC2fUmC2fUmC2fCmU2fAmU2fCmA2fCmU2fC
nnC (SEQ ID NO: 458);
2fUmU2fUmG2fCmA2fAmU2fGmA2fCmU2fCmU2fCmC2fUmA2fUmC2fAmG2fUmC2fCdT*dT (SEQ
ID NO: 459);
mU2fAmU2fCmC2fUmA2fAmG2fUmC2fAmC2fAmC2fGmU2fUmU2fGmA2fCmU2fGmC (SEQ ID NO:
460);
mU2fU2fU2fG2fC2fA2fA2fU2fG2fA2fC2fU2fC2fU2fC2fC2fU2fA2fU2fC2fA2fG2fU2fC2fCdT*dT
(SEQ
ID NO: 461);
mU2fUmU2fGmC2fAmA2fUmG2fAmC2fUmC2fUmC2fCmU2fAmU2fCmA2fGmU2fCmCdT*dT (SEQ
ID NO: 463);
mU2fUmU2fGmC2fAmA2fUmG2fAmC2fUmC2fUmC2fCmU2fAmU2fCmA2fGmU (SEQ ID NO: 464);
mU2fUmU2fGmC2fAmA2fUmG2fAmC2fUmC2fUmC2fC2fU2fA2fU2fC2fA2fG2fU (SEQ ID NO: 465)
CA 03174172 2022- 9- 28
wherein, mA, mC, mG, and mU are 2'-0-methyl adenosine, cytidine, guanosine, or
uridine,
respectively; 2fA, 2fC, 2fG, and 2fU are 2'-fluoro adenosine, cytidine,
guanosine, or uridine,
respectively; and * is a phosphorothioate linkage; and
the sense strand is at least substantially complementary to the antisense
strand.
[0011] For example, when the RNAi agent comprises (iii) the
combination of (i) the first
dsRNA and (ii) the second dsRNA, the antisense strand of (i) the first dsRNA
is selected from
CAGUUGCGCAGUUUCUUGUCAGUUCdTdT (SEQ ID NO: 17);
CAGUUGCGCAGUUUCUUGUCAGUUCdT*dT (SEQ ID NO 18);
AAGAGCUCAGGUCUCUGAGGGdTdT (SEQ ID NO 64); and
AAGAGCUCAGGUCUCUGAGGGdT*dT (SEQ ID NO 65); and
the antisense strand of (ii) the second dsRNA is selected from
UUUGAUAGCACCAAACCUAGAGCCCdTdT (SEQ ID NO: 417);
UUUGAUAGCACCAAACCUAGAGCCCdT*dT (SEQ ID NO: 418);
UUUGCAAUGACUCUCCUAUCAGUCCdTdT (SEQ ID NO: 448); and
UUUGCAAUGACUCUCCUAUCAGUCCdT*dT (SEQ ID NO: 449);
wherein * is a phosphorothioate linkage; and
the sense strand is at least substantially complementary to the antisense
strand.
[0012] In one embodiment, (i) the first dsRNA has the duplex
structure of (SEQ ID NOs: 17
and 110) or (SEQ ID NOs: 18 and 111). In another (ii) the second dsRNA has the
duplex structure of
(SEQ ID NOs: 417 and 808) or (SEQ ID NOs: 448 and 822).
[0013] Another embodiment provides for an isolated cell
comprising a double stranded RNAi
gent of (i), (ii) or (iii).
[0014] For example, the sense strand of (i) the first dsRNA
is no more than 30 nucleotides in
length, and the antisense strand of (i) the first dsRNA is no more than 30
nucleotides in length. For
example, the sense strand of (ii) the second dsRNA is no more than 30
nucleotides in length, and the
antisense strand is no more than 30 nucleotides in length.
[0015] Yet another embodiment provides a pharmaceutical
composition for inhibiting
expression of a CD320 gene, the pharmaceutical composition comprising a double
stranded RNAi
agent (i) or (iii). Further the pharmaceutical composition may include an
excipient.
[0016] Yet another embodiment provides a pharmaceutical
composition for inhibiting
expression of an LRP2 gene, the composition comprising a double stranded RNAi
agent (ii) or (iii).
Further the pharmaceutical composition may include an excipient.
[0017] Another embodiment of the present invention provides
a method for inhibiting
proliferation of a cancer cell (CC) comprising contacting of the CC with an
inhibitor of CD320 add/or
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LRP2 in an amount effective to inhibit proliferation of the CC. For example,
the CC may express
CD320 and/or LRP2 or both.
[0018] Another embodiment of the present invention provides
a method for treating a
therapeutically-resistant cancer in a subject who has previously received a
therapy, comprising
administering to the subject an inhibitor of CD320 add/or LRP2 in an amount
effective to inhibit or kill
cancer cells (CCs) present in the therapeutically-resistant cancer.
[0019] Another embodiment of the present invention provides
a method for treating cancer in
a subject who has recurring or relapsed cancer comprising administering to a
subject an inhibitor of
CD320 add/or LRP2 in an amount effective to inhibit or kill CCs in the cancer.
[0020] The CC is from a cancer selected from melanoma,
glioblastonna, lung carcinoma,
breast carcinoma, triple negative breast carcinoma, hepatocellular carcinoma,
renal carcinoma,
pancreatic carcinoma, ovarian carcinoma and prostate carcinoma.
[0021] The CD320 inhibitor is selected from an antibody that
binds CD320, a small molecule
inhibitor of CD320, and a RNAi agent that hybridizes to a nucleic acid
sequence encoding CD320.
[0022] Further, the method of inhibiting proliferation of a
CC, treating a therapeutically
resistive cancer in a subject or has a recurring or relapsed cancer comprises
administering a cancer
therapeutic in combination with an RNAi agent that hybridizes to an mRNA
encoding for CD320 or an
RNAi agent that hybridizes to an mRNA encoding for LRP2. For example, the
cancer therapeutic is
selected from the antifolate class, epigenetic modulatory class, or a small
molecule or protein inhibitor
of CD320 function or LRP2 function, such as an antibody for CD320 or an
antibody for LRP2. Further
still, the method further comprises administering mefformin. For example, the
RNAi agent comprises
an antisense strand of Table 5 or of Table 6.
[0023] The inhibitor is selected from the group consisting
of an antibody that binds LRP2, a
small molecule inhibitor of LRP2, and an RNAi agent that hybridizes to a
nucleic acid sequence
encoding LRP2. For example, the method further comprises administering a
cancer therapeutic
selected from the antifolate class, epigenetic modulatory class, or the small
molecule or protein
inhibitor of LRP2 function, such as an antibody, in combination with an RNAi
agent that hybridizes to
an mRNA encoding for LRP2.
[0024] The method further comprises administering a cancer
therapeutic in combination with
an RNAi agent that hybridizes to an mRNA encoding for LRP2.
[0025] One embodiment of the present invention provides for
a method for inhibiting
proliferation of a cancer cell (CC) comprising contacting of a CC with a
composition comprising an
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inhibitor of CD320 and an inhibitor of LRP2 in an amount effective to inhibit
proliferation of the CC.
For example, the composition is a cocktail comprising i) the CD320 inhibitor
selected from an antibody
that binds CD320, a small molecule inhibitor of CD320, and a RNAi agent that
hybridizes to a nucleic
acid encoding CD320 and any combination thereof, and ii) the LRP2 inhibitor
selected from an
antibody that binds LRP2, a small molecule inhibitor of LRP2, and a RNAi agent
that hybridizes to a
nucleic acid sequence encoding LRP2 and any combination thereof. Further, the
method further
comprises administering a cancer therapeutic selected from the antifolate
class and epigenetic
modulatory class. For example, the RNAi agent that hybridizes to the mRNA
encoding for CD320
comprises a first double-stranded ribonucleic acid (dsRNA) for inhibiting
expression of CD320,
wherein the first dsRNA comprises a sense strand and an antisense strand, the
antisense strand
comprising a region of complennentarity to a CD320 RNA transcript and the RNAi
agent that
hybridizes to the mRNA encoding for LRP2 comprises a second dsRNA for
inhibiting expression of
LRP2, wherein the second dsRNA comprises a sense strand and an antisense
strand, the antisense
strand comprising a region of complementarity to an LRP2 RNA transcript. In a
further example, the
antisense strand that is complementary to CD320 RNA transcript is selected
from Table 5 and the
antisense strand that is complementary to the RNA transcript for LRP2 is
selected from Table 6. The
method further comprises administering a cancer therapeutic selected from the
antifolate class and
epigenetic modulatory class. The method further comprises administering a
cancer therapeutic
selected from the innnnunonnodulatory class. Further still, the method further
comprises administering
nnefformin.
[0026] One aspect of one embodiment of the present invention
provides a method for the
inhibition of CD320 and LRP2 protein expression, such that the levels of these
proteins are reduced in
treated cells compared to their endogenous levels in untreated cells; this
inhibition may also be
referred to as the knockdown of CD320 and LRP2 expression. The method entails
the use of a
cocktail of small interfering RNA molecules, otherwise known as siRNAs, which
guide the mRNA
sequences encoding for either CD320 or LRP2 into an enzymatic complex which
leads to targeted
destruction of these mRNAs.
[0027] Another aspect of the present invention provides a
method for the individual or
concurrent inhibition of LRP2 and CD320 protein expression, which inhibits the
growth of many
cancer cells as compared to non-cancer (normal) cells. In some instances,
CD320 or LRP2 protein
knockdown alone is sufficient to severely inhibit cancer cell proliferation
compared to normal cells.
[0028] Another aspect of the present invention provides for
inhibition of cancer cell
proliferation by inhibiting LRP2 receptor expression.
[0029] Mechanistic investigations into the selectivity of
porphyrin uptake by cancer cells led
to several nonobvious compounds and methods of using the compound(s). It was
discovered that the
knockdown of the expression of either CD320 gene or LRP2 gene or the
simultaneous knockdown of
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the expression of CD320 gene and LRP2 gene caused cell death or inhibition of
cell growth in a panel
of lung cancer cell lines, compared to normal fibroblasts. The experimental
outline is illustrated in
FIG. 1. In these experiments, cells were plated on day 0. The next day (day
1), virus particles
encoding short hairpin RNAs (shRNAs) directed to the CD320 gene and the LRP2
gene or an
irrelevant shRNA control were added to the cell culture together with
protannine sulfate, a reagent that
facilitates cell entry of the virus particles.
[0030] Further investigations revealed that knockdown of the
expression of either the CD320
gene or LRP2 gene or the simultaneous knockdown of the expression of CD320 and
LRP2 genes
using small interfering RNAs (siRNAs) caused cell death or inhibition of cell
growth in a panel of
cancer cell lines that included lung cancer, prostate cancer, breast cancer,
glioblastonna and
melanoma, compared to normal fibroblasts (FIG. 9-10). It was also found that
that knockdown of one
gene, either CD320 or LRP2, led to increased expression of the other in some
cancer cell lines.
[0031] One aspect of the present invention provides for the
knockdown of the CD320
receptor, the LRP2 receptor or the simultaneous knockdown of both in vivo and
in vitro cancer cells
that express CD320 mRNA and/or LRP2 mRNA.
[0032] Another aspect of the present invention is a method
to inhibit cell growth or cause cell
death of cancer cells treated with a compound as described herein, while
leaving normal cells
unaffected or inhibiting cell growth to a lesser degree or producing less cell
death as compared to a
cancer cell treated with the same amount of the compound.
[0033] Another aspect of a first compound and method of use
is a selective therapy which
inhibits proliferation of cancer cells and/or kills cancer cells with an
inhibition of LRP2 Receptor while
leaving normal cells unharmed.
[0034] Another aspect of a second compound and method of use
is a selective therapy
which inhibits proliferation of cancer cells and/or kills cancer cells with an
inhibition of CD320
Receptor while leaving normal cells unharmed.
[0035] Another aspect of the present invention provides for
treating a cancer by
administering a therapy to selectively inhibit proliferation of a cancer
cell(s) and/or kill a cancer cell(s)
with one or more of the following, a first compound that is an inhibitor of
CD320 receptor, a second
compound that is an inhibitor of LRP2 receptor or a combination thereof.
DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
9
CA 03174172 2022- 9- 28
[0036] The accompanying drawings, which are incorporated
into and form a part of the
specification, illustrate one or more embodiments of the present invention
and, together with the
description, serve to explain the principles of the invention. The drawings
are only for the purpose of
illustrating one or more embodiments of the invention and are not to be
construed as limiting the
invention. In the drawings:
[0037] FIG. 1 illustrates an experimental design for
knocking down CD320 and LRP2 in a cell.
Cells were plated on day 0. The next day (day 1), virus particles encoding
short hairpin RNAs
(shRNAs) directed at the CD320 and LRP2 nnRNA or a non-targeting shRNA control
were added to
the cell culture together with protannine sulfate, a reagent that facilitates
cell entry of the virus
particles. Table 1 shows the sequences that were used. Each shRNA coding
sequence was also
combined with a unique drug resistance gene, which would allow for selecting
those cells that had
taken up the shRNA; cells that had not taken up the shRNA would not survive.
On day 2, drug
selection was started. On day 3, the cells were harvested and plated in a new
dish. Only the cells
with a drug resistance gene, i.e., those cells that had taken up shRNA virus
particles would survive
this re-plating procedure. From day 4 on, each culture was closely observed
for cell growth. Cells
infected with the non-targeting negative control shRNA continued growing ¨
data not shown. The
results for the cell lines that expressed the CD320+LRP2 shRNAs are shown in
Table 1.
[0038] FIG. 2 A-C illustrates sensitivity of cancer cell
lines to knockdown of CD320 and
LRP2. Normal cells (GM05659 fibroblasts) or cancer cells were infected with
lentiviruses expressing
shRNAs to control sequences or to shCD320 and shLRP2 as described in FIG. 1.
The cells were
grown as described in FIG. 1. On the ninth day after transfection with the
lentiviruses, pictures of the
cells were taken. The solid oval indicates healthy growth of normal fibroblast
infected with shRNAs to
CD320 and LRP2. The broken line ovals indicate unhealthy dying cancer cells
infected with shRNAs
targeting CD320 and LRP2 (FIG. 2A). The fields of cells in FIG. 2A were
counted and quantified and
illustrated in FIG. 2B. The data in FIG. 2B were normalized to the number of
control cells and
illustrated in FIG. 2C. FIG. 2C. shows that the cultures of cells infected
with lentivirus encoding the
shRNAs against CD320 and LRP2 (white bars) contain far fewer cells than the
cultures of cells
exposed to the shRNA control (black bar).
[0039] FIG. 3 A-F illustrate graphs of protein levels
resulting from transfection of HEK293,
MDA-MB-435S and MDA-MB-231 cells with siRNA to LRP2 and CD320. HEK293, MDA-MB-
435S
and MDA-MB-231 cells were transfected with 20 nM of indicated siRNAs and
incubated for 48 hours.
siRNAs targeting CD320 are designated OSC17 and 0SC47. siRNAs targeting LRP2
are designated
0SL245, 0SL47, OSL104, OSL90 and OSL119. Whole cell lysates were prepared and
immunoblotted for CD320 and LRP2 protein levels. The protein levels were
normalized to a
housekeeping control gene unaffected by the siRNA transfection. The graphs
FIG. 3 A-F represent
the fold change of protein levels compared to siScrannble (OSS1 or OSS2).
(Average +1- SEM is
shown, n=3).
CA 03174172 2022- 9- 28
[0040] FIG. 4 A-F illustrate a graph of cells after
transfection of LnCAP, MCF-7 and U251
cells with siRNA to LRP2 and CD320. LnCAP, MCF-7 and U251 cells were
transfected with 20 nM of
indicated siRNAs and incubated for 48 hours. siRNAs targeting CD320 are
designated OSC17 and
0SC47. siRNAs targeting LRP2 are designated 0SL245, 0SL47, OSL104, OSL90 and
OSL119).
Whole cell lysates were prepared and innnnunoblotted for CD320 and LRP2
protein levels. The protein
levels were normalized to a housekeeping control gene unaffected by the siRNA
transfection. The
graphs FIG. 4 A-F represent the fold change of protein levels compared to
siScrannble (OSS2).
[0041] FIG. 5A-C illustrate graphs of protein levels after
transfection of A172, DU145 and
GM05659 cells with siRNA to LRP2 and CD320. A172, DU145 and GM05659 cells were
transfected
with 20 nM of indicated siRNAs and incubated for 48 hours. siRNAs targeting
CD320 are designated
OSC17 and 0SC47. siRNAs targeting LRP2 are designated 0SL245, 0SL47, OSL104,
OSL90 and
OSL119). Whole cell lysates were prepared and innnnunoblotted for CD320. The
protein levels were
normalized to a housekeeping control gene unaffected by the siRNA
transfection. The graphs FIG.
5A-C represent the fold change of protein levels compared to siScrannble
(0552).
[0042] FIG. 6 illustrates a graph of relative LRP2 protein
expression in various cell lines ¨
Lysates were made from the cell lines indicated on the x-axis, and western
blot was performed to
determine LRP2 protein levels. The results represent the averages +/-SEM of
three independent
lysates.
[0043] FIG. 7 A-B illustrates graphs of the effect of
doxorubicin treatment on cell viability, as
measured by the CTG assay. A172 and HCC15 cells were plated at 1200 cells/well
in a 96 well plate.
The next day, cells were treated with doxorubicin at the indicated
concentrations. Four days after
doxorubicin treatment was initiated, the cells were assayed for viability
using the CTG assay. The
dashed line indicates the non-linear fitting of the data to calculate an IC50
value.
[0044] FIG. 8 is a schematic overview of the functional
assay for screening siRNA effects on
cell proliferation to facilitate quantification of the effects of knocking
down CD320 and LRP2 on cell
proliferation. Cells were plated in a 24-well plate. The next day, the cells
were transfected with
siRNAs targeting CD320 (OSC17, 0SC47) and/or targeting LRP2 (0SL231, 05L245),
or a control
siRNA (OSS2). The cell lines may require repeated transfections and/or time
for efficient toxicity (cell
line dependent). In this experimental set-up there is room for repeat
infection should some cell lines
require that for efficient toxicity. In addition, in a small subset of the
wells, cells were only treated with
doxorubicin as a positive control for toxicity. At the end of the study, the
cell lines are analyzed for cell
growth by the CTG assay.
[0045] FIG. 9 A-E illustrate graphs of the percent cell
survival of siCD320 and siLRP2 on cell
proliferation ¨ Cell lines representative of several types of cancers (lung,
brain) or normal fibroblasts
11
CA 03174172 2022- 9- 28
were transfected with individual or combinations of siRNAs targeting CD320
(OSC17, 0SC47) or
LRP2 (0SL231, 0SL245), individually at 20 nM or in combination (10 nM each),
or a negative control
siRNA (OSS2) (20 nM) as indicated. Cells were repeatedly transfected as
outlined in Table 9 for
efficient toxicity, then assayed for viability by the CTG assay. Doxorubicin-
treated cells served as a
positive control for cell toxicity in our assays (Table 8).
[0046] FIG. 10 A-E illustrate graphs of the effects of
siCD320 and siLRP2 on cell
proliferation ¨ Cell lines representative of several types of cancers (breast,
prostate, skin) were
transfected with individual or combinations of siRNAs targeting CD320 (OSC17,
0SC47) or LRP2
(0SL231, 0SL245) as indicated. Cells were repeatedly transfected as outlined
in Table 9 for efficient
toxicity, then assayed for viability by the CTG assay. Doxorubicin-treated
cells served as a positive
control for cell toxicity in our assays (Table 8).
[0047] FIG. 11A-B illustrate the effects of siCD320 and
siLRP2 molar proportions on cell
proliferation with different molar proportions of siRNA targeting CD320 and
siRNA targeting LRP2.
Cell lines representative of two types of cancers (breast, prostate) were
transfected with different
proportions of siRNAs targeting CD320 (OSC17) or LRP2 (0SL245) (0-20 nM) or a
negative control
siRNA (OSS2) as indicated. Cells were repeatedly transfected for efficient
toxicity then assayed for
viability by the CTG assay. Doxorubicin-treated cells served as a positive
control for cell toxicity in our
assays (Table 8).
[0048] FIG. 12A-B illustrate graphs of the duration of the
knockdown effect for siCD320 and
siLRP2 on MDA-MD-231 cells. A representative breast cancer cell line (MDA-MD-
231) was
transfected on Day 0 with 20 nM of an siRNA targeting CD320 (OSC17) or an
siRNA targeting LRP2
(0SL245) or a negative control siRNA (OSS2) and the percentage of protein
knockdown was
analyzed daily over a period of five days by western blot. Protein levels were
normalized to the
negative control (OSS2).
[0049] FIG. 13 is a schematic of polyethylinimine (PEI) and
siRNA complexation. PEI and
siRNAs are mixed together. Subsequently, polyplexes (a nanoparticle, broadly
speaking) of the PEI-
siRNA complex form, which are able to enter the cell.
[0050] FIG. 14 is a schematic that illustrates that siRNAs
are short RNA duplexes of
generally 16 to 30 nucleotides; the guide sequence of the siRNA is
complementary to a mRNA
expressed in the cell. Exogenous siRNA duplexes are introduced into the cell
via a method of
transfection. The siRNA duplexes are separated via the RISC/AGO (RNA-induced
silencing complex)
complex, whereby the guide strand of the siRNA hybridizes with its
complementary mRNA molecule.
The mRNA is degraded by the RISC/AGO complex, which has RNAse activity,
resulting in mRNA
degradation, and the protein encoded by the mRNA is not produced. This causes
the "knockdown"
effect or reduced protein levels of the gene targeted by the siRNA compared to
control treated cells.
12
CA 03174172 2022- 9- 28
[0051] FIG. 15 A-B illustrate graphs of A172 cell line or
MDA-MD-435S cell lines treated with
control siRNA (OSS1, OSS2) and siRNA directed to CD320 nnRNA (OSC17, 0SC47)
and siRNA
directed to LRP2 nnRNA (0SL231, 0SL245) to determine the effectiveness of
INTERFERin, a
polyethanolannine transfection reagent, in delivering siRNAs to cancer cells.
[0052] FIG. 16 A-D illustrate plated cells showing the
effects of siCD320 and siLRP2 on four
cell lines. Cell lines representative of four types of cancers (breast, two
prostate, skin) were
transfected with siRNAs targeting CD320 (OSC17) or LRP2 (0SL245) individually
at 20 nM or in
combination (10 nM each) or a negative control siRNA (OSS2) (20 nM) as
indicated. Cells were
repeatedly transfected for efficient toxicity as in Table 9 and then analyzed
by microscopy as
indicated.
[0053] FIG. 17 illustrates a graphical depiction of CD320
nnRNA. UTR references the
untranslated region, and the CDS references the protein coding sequence.
[0054] FIG. 18 illustrates a graphical depiction of LRP2
mRNA UTR references the
untranslated region, and the CDS references the protein coding sequence.
[0055] FIG. 19A-G illustrates the structures for unnatural
nudeotides which may be
incorporated within the sequence of an RNAi. "B" represents a natural (G, C,
A, U) RNA nudeobase,
a DNA nucleobase, or an unnatural nucleobase. FIG. 19A shows certain chemical
modifications to
the ribose 2'-position and phosphate moieties. FIG. 19B-D shows skeletal
modifications to the ribose
moiety that comprise bridging groups. FIG. 19E shows a deletion of the C2'-C3'
bond. FIG. 19F-G
shows other skeletal modifications to the ribose moiety wherein a six-membered
ring replaces the
five-membered ring.
[0056] FIG. 20 illustrates a schematic for the in vivo
nnurine xenograft model for breast
cancer. MDA-MB-231 cells were implanted into the flank of NSG mice and grown
to a volume of 70
mm3 after which siRNAs targeting CD320 (OSC17) and LRP2 (0SL245) were injected
intratunnorally
once every fourth day.
DETAILED DESCRIPTION OF THE INVENTION
[0057] One or more embodiment of the present invention
provides methods and RNAi
compounds for modulating the expression of a CD320 gene and/or an LRP2 gene in
a cell. In certain
embodiments, expression of a CD320 gene and/or a LRP2 gene is reduced or
inhibited using an
CD320 and/or LRP2 specific RNAi. Such inhibition can be useful in treating
disorders such as cancer
and/or creating cell lines that are useful for screening drugs that treat
cancer
13
CA 03174172 2022- 9- 28
[0058] The present invention also relates to a method for
knocking down (partially or
completely) the targeted genes.
[0059] One embodiment of the method of producing knockdown
cells and organisms
comprises introducing into a cell or organism in which a gene (referred to as
a targeted gene) to be
knocked down, an siRNA of about 16 to about 30 nucleotides (nt) that targets
the gene and
maintaining the resulting cell or organism under conditions under which RNAi
occurs, resulting in
degradation of the nnRNA of the targeted gene, thereby producing knockdown
cells or organisms.
Knockdown cells and organisms produced by the present method are also the
subject of embodiment
of the present invention.
[0060] An embodiment of the present invention also relates
to a method of examining or
assessing the function of a gene in a cell or organism. In one embodiment, RNA
of about 16 to about
30 nt which targets nnRNA of the gene for degradation is introduced into a
cell or organism in which
RNAi occurs. The cell or organism is referred to as a test cell or organism.
The cell or organism is
referred to as a test cell organism. The test cell or organism is maintained
under conditions under
which degradation of nnRNA of the gene occurs. The phenotype of the test cell
or organism is then
observed and compared to that of an appropriate control cell or organism, such
as a corresponding
cell or organism that is treated in the same manner except that the gene is
not targeted. A 16 to 30 nt
RNA that does not target the nnRNA for degradation can be introduced into the
control cell or
organism in place of the siRNA introduced into the test cell or organism,
although it is not necessary
to do so. A difference between the phenotypes of the test and control cells or
organisms provides
information about the function of the degraded nnRNA.
[0061] The RNA of about 16 to about 30 nucleotides is
isolated or synthesized and then
introduced into a cell or organism in which RNAi occurs (test cell or test
organism). The test cell or
test organism is maintained under conditions under which degradation of the
nnRNA occurs. The
phenotype of the test cell or organism is then observed and compared to that
of an appropriate
control, such as a corresponding cell or organism that is treated in the same
manner as the test cell or
organism except that the targeted gene is not targeted. A difference between
the phenotypes of the
test and control cells or organisms provides information about the function of
the targeted gene. The
information provided may be sufficient to identify (define) the function of
the gene or may be used in
conjunction with information obtained from other assays or analyses to do so.
[0062] An embodiment of the present invention also
encompasses a method of treating a
disease or condition associated with the presence of a protein in an
individual, comprising
administering to the individual RNA of from about 16 to about 30 nucleotides
which targets the nnRNA
of the protein (the nnRNA that encodes the protein) for degradation. As a
result, the protein is not
produced or is not produced to the extent it would be in the absence of the
treatment.
14
CA 03174172 2022- 9- 28
[0063] FIG. 14 shows that siRNAs are short RNA duplexes of
generally 16 to 30 nucleotides;
the sequence of the siRNA is complementary to a nnRNA expressed in the cell.
Exogenous siRNA
duplexes are introduced into the cell via a method of transfection. The siRNA
duplexes are unwound
via the RNA-induced silencing complex (RISC), whereby the guide strand of the
siRNA hybridizes
with its complementary mRNA molecule. The nnRNA is degraded by the RISC/AGO
complex, which
has RNAse cleave activity. The end result is that the nnRNA targeted by the
siRNA is degraded, and
the protein encoded by the nnRNA is not produced. This causes the "knockdown"
effect or reduced
protein levels of the gene targeted by the siRNA compared to control treated
cells.
[0064] In one embodiment, at least one strand of the RNA
molecule has a 3 overhang from
about 1 to about 6 nucleotides (e.g., pyrinnidine nucleotides, purine
nucleotides) in length. In other
embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from
about 1 to about 3
nucleotides and from about 2 to about 4 nudeotides in length or, for example,
the overhang can be up
to 14 nucleotides if the guide strand were a 27-nner. In one embodiment the
RNA molecule is double
stranded, one strand has a 3' overhang and the other strand can be blunt-ended
or have an
overhang. In the embodiment in which the RNA molecule is double stranded and
both strands
comprise an overhang, the length of the overhangs may be the same or different
for each strand. In a
particular embodiment, the RNA of the present invention comprises 21-27
nucleotide strands which
are Watson-Crick paired and which have overhangs of from about 1 to about 3,
particularly about 2,
nucleotides on both 3' ends of the RNA. In order to further enhance the
stability of the RNA of the
present invention, the 3' overhangs can be stabilized against degradation. In
one embodiment, the
RNA is stabilized by induding purine nucleotides, such as adenosine or
guanosine nucleotides.
Alternatively, substitution of pyrinnidine nucleotides by unnatural
nucleotides, e.g., substitution of
uridine 2 nucleotide 3' overhangs by 2'-deoxythynnidine, is tolerated and does
not affect the efficiency
of RNAi. The absence of a 2' hydroxyl significantly enhances the nuclease
resistance of the overhang
in tissue culture medium. The 3'-overhangs can be further stabilized by
introduction of
phosphorothioate groups in place of the phosphodiesters.
[0065] The 16-30 nt RNA molecules of the present invention
can be obtained using a
number of techniques known to those of skill in the art. For example, the RNA
can be chemically
synthesized or reconnbinantly produced using methods known in the art.
[0066] In order that the present invention may be more
readily understood, certain terms are
first defined. In addition, it should be noted that whenever a value or range
of values of a parameter
are recited, it is intended that values and ranges intermediate to the recited
values are also intended
to be part of this invention.
[0067] The articles "a" and "an" are used herein to refer to
one or to more than one (i.e., to at
least one) of the grammatical object of the article. By way of example, "an
element" means one
element or more than one element, e.g., a plurality of elements.
CA 03174172 2022- 9- 28
[0068] The term "including" is used herein to mean, and is
used interchangeably with, the
phrase "including but not limited to".
[0069] The term "or" is used herein to mean, and is used
interchangeably with, the term
"and/or," unless context clearly indicates otherwise.
[0070] As used herein, "CD320 " refers to the gene or
protein. CD320 is also known as 8D6
antigen, CD320 antigen, 8D6A, transcobalalnnin receptor, FDC-SM-8D6, FDC-
Signaling Molecule
8D6, 8D6, TCBLR, TCbIR, TCN2R. The term CD320 includes human CD320, the amino
acid and
nucleotide sequence of which may be found in, for example, GenBank Accession
No. NM_016579.4
and NM_001165895.2; mouse CD320, the amino acid and nudeotide sequence of
which may be
found in, for example, GenBank Accession No. NM_019421.3; rat CD320, the amino
acid and
nucleotide sequence of which may be found in, for example, GenBank Accession
No.
NM_001014201.1. Additional examples of CD320 nnRNA sequences are readily
available using, e.g.,
GenBank. Additional information is found at FIG. 17.
[0071] The CD320 DNA sequence from honno sapiens is as
follows: >NM_016579.4 Homo
sapiens CD320 molecule (CD320), transcript variant 1, DNA
GTGCGCGTGCGCAGGGATAAGAGAGCGGTCTGGACAGCGCGTGGCCGGCGCCGCTGTGGGGACAGCATGA
GCGGCGGTTGGATGGCGCAGGTTGGAGCGTGGCGAACAGGGGCTCTGGGCCTGGCGCTGCTGCTGCTGCT
CGGCCTCGGACTAGGCCTGGAGGCCGCCGCGAGCCCGCTTTCCACCCCGACCTCTGCCCAGGCCGCAGGC
CCCAGCTCAGGCTCGTGCCCACCCACCAAGTTCCAGTGCCGCACCAGTGGCTTATGCGTGCCCCTCACCT
GGCGCTGCGACAGGGACTTGGACTGCAGCGATGGCAGCGATGAGGAGGAGTGCAGGATTGAGCCATGTAC
CCAGAAAGGGCAATGCCCACCGCCCCCTGGCCTCCCCTGCCCCTGCACCGGCGTCAGTGACTGCTCTGGG
GGAACTGACAAGAAACTGCGCAACTGCAGCCGCCTGGCCTGCCTAGCAGGCGAGCTCCGTTGCACGCTGA
GCGATGACTGCATTCCACTCACGTGGCGCTGCGACGGCCACCCAGACTGTCCCGACTCCAGCGACGAGCT
CGGCTGTGGAACCAATGAGATCCTCCCGGAAGGGGATGCCACAACCATGGGGCCCCCTGTGACCCTGGAG
AGTGTCACCTCTCTCAGGAATGCCACAACCATGGGGCCCCCTGTGACCCTGGAGAGTGTCCCCTCTGTCG
GGAATGCCACATCCTCCTCTGCCGGAGACCAGTCTGGAAGCCCAACTGCCTATGGGGTTATTGCAGCTGC
TGCGGTGCTCAGTGCAAGCCTGGTCACCGCCACCCTCCTCCTITTGTCCTGGCTCCGAGCCCAGGAGCGC
CTCCGCCCACTGGGGTTACTGGTGGCCATGAAGGAGTCCCTGCTGCTGTCAGAACAGAAGACCTCGCTGC
CCTGAGGACAAGCACTTGCCACCACCGTCACTCAGCCCTGGGCGTAGCCGGACAGGAGGAGAGCAGTGAT
GCGGATGGGTACCCGGGCACACCAGCCCTCAGAGACCTGAGCTCTTCTGGCCACGTGGAACCTCGAACCC
GAGCTCCTGCAGAAGTGGCCCTGGAGATTGAGGGTCCCTGGACACTCCCTATGGAGATCCGGGGAGCTAG
GATGGGGAACCTGCCACAGCCAGAACTGAGGGGCTGGCCCCAGGCAGCTCCCAGGGGGTAGAACGGCCCT
GTGCTTAAGACACTCCTGCTGCCCCGTCTGAGGGTGGCGATTAAAGTTGCTTCACATCCTCAAAAAAAAA
16
CA 03174172 2022- 9- 28
AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO. 935).
[0072] A protein sequence from CD320 derived from the nnRNA
sequence above is as
follows: >splQ9NPFOICD320_HUMAN CD320 antigen OS=Honno sapiens OX=9606
GN=CD320
PE=1 SV=1
MSGGWMAQVGAWRTGALGLALLLLLGLG LGLEAAASPLSTPTSAQAAGPSSGSCPPTKFQ
CRTSG LCVPLTW RCDRDLDCSDGSDEE ECRI EPCTQKGQCP P P PGLPCPCTGVSDCSGGT
DKKLRNCSRLACLAG ELRCTLSDDCI PLTWRCDG HP DCPDSSDE LGCGTN El LP EG DATT
MG PPVTLESVTSLR NATTM G P PVTLESVPSVG NATSSSAG DQSGSPTAYGVIAAAAVLSA
SLVTATLLLLSWLRAQERLRPLGLLVAMKESLLLSEQKTSLP (SEQ ID NO. 936)
[0073] The CD320 DNA sequence from honno sapiens is as
follows: > NM_001165895.2
Homo sapiens CD320 molecule (CD320), transcript variant 2, DNA
GCGTGCGCGTGCGCAGGGATAAGAGAGCGGTCTGGACAGCGCGTGGCCGGCGCCGCTGTGGGGACAGCAT
GAGCGGCGGTTGGATGGCGCAGGTTGGAGCGTGGCGAACAGGGGCTCTGGGCCTGGCGCTGCTGCTGCTGC
TCGGCCTCGGACTAGGCCTGGAGGCCGCCGCGAGCCCGCTITCCACCCCGACCTCTGCCCAGGCCGCAGGGA
TTGAGCCATGTACCCAGAAAGGGCAATGCCCACCGCCCCCTGGCCTCCCCTGCCCCTGCACCGGCGTCAGTGA
CTGCTCTGGGGGAACTGACAAGAAACTGCGCAACTGCAGCCGCCTGGCCTGCCTAGCAGGCGAGCTCCGTTG
CACGCTGAGCGATGACTGCATTCCACTCACGTGGCGCTGCGACGGCCACCCAGACTGTCCCGACTCCAGCGAC
GAGCTCGGCTGTGGAACCAATGAGATCCTCCCGGAAGGGGATGCCACAACCATGGGGCCCCCTGTGACCCTG
GAGAGTGTCACCTCTCTCAGGAATGCCACAACCATGGGGCCCCCTGTGACCCTGGAGAGTGTCCCCTCTGTCG
GGAATGCCACATCCTCCTCTGCCGGAGACCAGTCTGGAAGCCCAACTGCCTATGGGGTTATTGCAGCTGCTGC
GGTGCTCAGTGCAAGCCTGGTCACCGCCACCCTCCTCCTTTTGTCCTGGCTCCGAGCCCAGGAGCGCCTCCGCC
CACTGGGGTTACTGGTGGCCATGAAGGAGTCCCTGCTGCTGTCAGAACAGAAGACCTCGCTGCCCTGAGGAC
AAGCACTTGCCACCACCGTCACTCAGCCCTGGGCGTAGCCGGACAGGAGGAGAGCAGTGATGCGGATGGGT
ACCCGGGCACACCAGCCCTCAGAGACCTGAGCTCTTCTGGCCACGTGGAACCTCGAACCCGAGCTCCTGCAGA
AGTGGCCCTGGAGATTGAGGGTCCCTGGACACTCCCTATGGAGATCCGGGGAGCTAGGATGGGGAACCTGC
CACAGCCAGAACTGAGGGGCTGGCCCCAGGCAGCTCCCAGGGGGTAGAACGGCCCTGTGCTTAAGACACTCC
TGCTGCCCCGTCTGAGGGTGGCAATTAAAGTTGCTTCACATCCTC (SEQ ID NO. 937)
[0074] A protein sequence from CD320 derived from the DNA
sequence above is as follows:
>sp I Q9NPF0-2ICD320_HU MAN Isoform 2 of CD320 antigen OS=Homo sapiens OX=9606
GN=CD320
MSGGWMAQVGAWRTGALGLALLLLLGLG LG LEAAASPLSTPTSAQAAG I EPCTQKGQCPP PPG
LPCPCTGVSDC
SGGTDKKLRNCSR LACLAGELRCTLSDDCI PLTWRCDG HP DCP DSSD ELGCGTN El LP EG
DATTMGPPVTLESVTSL
RNATTM G P PVTLESVPSVG NATSSSAG DQSGSPTAYGVIAAAAVLSASLVTATLLLLSWLRAQE R LRP
LGLLVAM K
ESLLLSEQKTSLP (SEQ ID NO. 938)
17
CA 03174172 2022- 9- 28
[0075] Further, as used herein, "LRP2" refers to the gene or
protein. LRP2 is also known as
nnegalin, LRP-2, Glycoprotein 330, DBS, GP330, Gp330, Calcium Sensor Protein,
Heymann Nephritis
Antigen Honnolog, Low-Density Lipoprotein Receptor-Related Protein 2, EC
1.1.2.3, EC 3.4.21.9, LDL
receptor related protein 2. The term LRP2 includes human LRP2, the amino acid
and nucleotide
sequence of which may be found in, for example, GenBank Accession No.
NM_004525.3; mouse
LRP2, the amino acid and nucleotide sequence of which may be found in, for
example, GenBank
Accession No. NM_001081088.2; rat LRP2, the amino acid and nudeotide sequence
of which may be
found in, for example, GenBank Accession No. NM_030827.1. Additional examples
of LRP2 mRNA
sequences are readily available using, e.g., GenBank. Additional information
is found at FIG. 18.
[0076] One example of LRP2 is: >NM_004525.3 Homo sapiens LDL
receptor related protein
2 (LRP2), DNA:
GGTCTAAAGGGCTITATGCACTGTCTGGAGGGTGGGGACTGGCGCGGGTAGAAAACGGGATGCCTCGGGC
GTGGGGGCAGGCTITTGGCCACTAGGAGCTGGCGGAGGTGCAGACCTAAAGGAGCGTTCGCTAGCAGAGG
CGCTGCCGGTGCGGTGTGCTACGCGCGCCCACCTCCCGGGGAAGGAACGGCGAGGCCGGGGACCGTCGCG
GAGATGGATCGCGGGCCGGCAGCAGTGGCGTGCACGCTGCTCCTGGCTCTCGTCGCCTGCCTAGCGCCGG
CCAGTGGCCAAGAATGTGACAGTGCGCATTTTCGCTGTGGAAGTGGGCATTGCATCCCTGCAGACTGGAG
GTGTGATGGGACCAAAGACTGTTCAGATGACGCGGATGAAATTGGCTGCGCTGTTGTGACCTGCCAGCAG
GGCTATTTCAAGTGCCAGAGTGAGGGACAATGCATCCCCAACTCCTGGGTGTGTGACCAAGATCAAGACT
GTGATGATGGCTCAGATGAACGTCAAGATTGCTCACAAAGTACATGCTCAAGTCATCAGATAACATGCTC
CAATGGTCAGTGTATCCCAAGTGAATACAGGTGCGACCACGTCAGAGACTGCCCCGATGGAGCTGATGAG
AATGACTGCCAGTACCCAACATGTGAGCAGCTTACTTGTGACAATGGGGCCTGCTATAACACCAGTCAGA
AGTGTGATTGGAAAGTTGATTGCAGGGACTCCTCAGATGAAATCAACTGCACTGAGATATGCTTGCACAA
TGAGTITTCATGTGGCAATGGAGAGTGTATCCCTCGTGCTTATGTCTGTGACCATGACAATGATTGCCAA
GACGGCAGTGACGAACATGCTTGCAACTATCCGACCTGCGGTGGTTACCAGTTCACTTGCCCCAGTGGCC
GATGCATTTATCAAAACTGGGTTTGTGATGGAGAAGATGACTGTAAAGATAATGGAGATGAAGATGGATG
TGAAAGCGGTCCTCATGATGTTCATAAATGTTCCCCAAGAGAATGGTCTTGCCCAGAGTCGGGACGATGC
ATCTCCATTTATAAAGTTTGTGATGGGATTTTAGATTGCCCAGGAAGAGAAGATGAAAACAACACTAGTA
CCGGAAAATACTGTAGTATGACTCTGTGCTCTGCCTTGAACTGCCAGTACCAGTGCCATGAGACGCCGTA
TGGAGGAGCGTGITTTTGTCCCCCAGGTTATATCATCAACCACAATGACAGCCGTACCTGTGTTGAGTTT
GATGATTGCCAGATATGGGGAATTTGTGACCAGAAGTGTGAAAGCCGACCTGGCCGTCACCTGTGCCACT
GTGAAGAAGGGTATATCTTGGAGCGTGGACAGTATTGCAAAGCTAATGATTCCTTTGGCGAGGCCTCCAT
TATCTTCTCCAATGGTCGGGATTTGTTAATTGGTGATATTCATGGAAGGAGCTTCCGGATCCTAGTGGAG
TCTCAGAATCGTGGAGTGGCCGTGGGTGTGGCTITCCACTATCACCTGCAAAGAGTTITTTGGACAGACA
CCGTGCAAAATAAGGTTITTTCAGTTGACATTAATGGTTTAAATATCCAAGAGGTTCTCAATGTTTCTGT
18
CA 03174172 2022- 9- 28
TG AAACCCCAG AG AACCTG G CTGTG G ACTGG GTTAATAATAAAATCTATCTAGTGGAAACCAAG
GTCAAC
CG CATAGATATG GTAAATTTG GATGG AA G CTATCG GGTTACCCTTATAACTGAAAACTTGG G
GCATCCTA
G AG G AATTGCCGTGG AC CCAACTG TTG G TTATTTATTTTICTCAG ATTG GG AG AG CCTTTCTGGG
GAACC
TAAG CTGG AAA G G GCATTCATGGATGG CA G CAACCG TAAAG ACTTG GTGAAAACAAAG CTG G
GATGG CCT
G CTGG GGTAACTCTG GATATGATATCG AAG CGTG TTTA CTG G G TTG A CTCTCG G TTTG
ATTACATTG AAA
CTGTAACTTATGATGGAATTCAAAG G AAG ACTG TAG TTCATG GAG G CTCCCTCATTCCTCATCCCTTTGG
AG TAAG CTTATTTG AAG G TCA G G TG TTCTTTA CAG ATTG G ACAAAG ATG GC C GTG CTG
AA G G CAAACAAG
TTCACA G AG AC CAA CCCACAAG TG TA CTACCAG G CTTCC CTG A G G CC CTATG G AG TG
ACTGTTTACCATT
CC CTCAGACAG CCCTATG CTACCAATCCGTGTAAAGATAACAATGGGGGCTGTGAGCAG GTCTGTGTCCT
CA G C CACAG AACAGATAATGATG GTTTG GGTTTCCGTTGCAAGTG CACATTCG GCTTCCAACTG
GATACA
G ATG AG CG CCACTGCATTGCTGTTCAG AATTTCCTCATTITTTCATC CCAAG TTG CTATTCG TG G G
ATCC
CGTTCACCTTGTCTACCCAGG AA G ATGTCATG GTTCCAGTTTCG GG GAATCCTTCTITCTTTGTCGG GAT
TGATTTTGACGCCCAG GACAG CACTATC ______________ II 11111
CAGATATGTCAAAACACATGATTTTTAAG CAAAAG
ATTGATGG CACAG G AA G A GAAATTCTCG CAG CTAACAGGGTG G AAAATGTTG AAAG TTTG
GCTTTTGATT
G GATTTCAAAGAATCTCTATTG G A CAG ACTCTCATTACAAG AG TATCAG TGTCATG AG GCTAG
CTGATAA
AA CG AG A CG CACAG TA GTTCAG TATTTAAATAA CC CACG G TC G G TG GTAG TTCATC
CTTTTG CCG G G TAT
CTATTCTTCACTGATTG GTTCCGTCCTGCTAAAATTATG AG AG CATG G A GTG AC G G
ATCTCACCTCTTGC
CTGTAATAAACACTACTCTTGGATG GCCCAATGG CTTG GC CATC G ATTG G G CTG CTTCAC G ATTG
TA CTG
G GTAGATG CCTATTTTG ATAAAATTG AG CACAG CAC CTTTG ATG GTTTAG ACA G AA G AAG
ACTG GG CCAT
ATAG AG CAG ATGACACATCCGTTTGG ACTTGCCATCTTTGG AG AG CATTTATTTTTTACTGACTG GAGAC
TG G GTGCCATTATTCG AGTCAGG AAA G CA G ATG GTGG AG AAATG ACAGTTATCCG AAGTG
GCATTG CTTA
CATACTGCATTTG AAATCGTATG ATG TCAA CATC CAG A CTG GTTCTAACG CCTGTAATCAACCCACG
CAT
CCTAACGGTGACTG CAG CCACTTCTGCTTCCCG GTG CCAAATTTCCAG CG AG TG TG TG G
GTGCCCTTATG
G AATG AG G CTGGCTTCCAATCACTTGACATG CG AG GG G GACCCAACCAATG AACCACCCACAG AG
CAG TG
TG G CTTATTTTCCTTCCCCTGTAAAAATG GCAG ATGTGTG CC CAATTACTATCTCTG TG ATG G AG
TCG AT
G ATTG TCATG ATAACAGTGATG AG CAACTATGTGG CACACTTAATAATACCTGTTCATCTTCG GCGTTCA
CCTGTGGCCATG GGG AG TG CATTC CTG CA CACTG GCG CTGTG ACAAACGCAACG A CTGTGTG G
ATG GCAG
TG ATG AG CACAACTG CCCCACCCACG CACCTG CTTCCTG CCTTG ACACCCAATACACCTGTG
ATAATCAC
CA GTG TATCTCAAA G AA CTG G GTCTGTG A CACAG A CAATG ATTG TGG
GGATGGATCTGATGAAAAGAACT
G CAATTCG ACA G A G ACATG C CAA CCTAG TCAG TTTAATTG CCCCAATCATCGATGTATTG AC
CTATCGTT
TGTCTGTGATGGTGACAAGGATTGTGTTGATG GATCTG ATG AG GTTG GTTG TG TATTAAACTG TACTG
CT
TCTCAATTCAAGTGTG CCAGTGG GGATAAATGTATTG GCGTCACAAATCGTTGTGATG G TG TTTTTG ATT
G CAGTGACAACTCG GATGAAG CAG G CTG TC CAAC CA G G C CTCCTG GTATGTG
CCACTCAGATGAATTTCA
GTG CCAAGAAGATG GTATCTG CATC CC G AACTTCTG G GAATGTGATGGG CATCCAG
ACTGCCTCTATGG A
19
CA 03174172 2022- 9- 28
TCTGATGAGCACAATGCCTGTGTCCCCAAGACTTGCCCTTCATCATATTTCCACTGTGACAACGGAAACT
GCATCCACAGGGCATGGCTCTGTGATCGGGACAATGACTGCGGGGATATGAGTGATGAGAAGGACTGCCC
TACTCAGCCCTTTCGCTGTCCTAGTTG GCAATGG CAGTGTCTTG GCCATAACATCTGTGTGAATCTGAGT
GTAGTGTGTGATGG CATCTTTGACTG CCCCAATG G GACAGATGAGTCCCCACTTTGCAATG GGAACAG CT
GCTCAGATTTCAATGGTGGTTGTACTCACGAGTGTGTTCAAGAGCCCTTTGGGGCTAAATGCCTATGTCC
ATTGGGATTCTTACTTGCCAATGATTCTAAGACCTGTGAAGACATAGATGAATGTGATATTCTAGGCTCT
TGTAGCCAGCACTGTTACAATATGAGAGGTTCTTTCCGGTGCTCGTGTGATACAGGCTACATGTTAGAAA
GTGATGG GAG GACTTG CAAAGTTACAG CATCTGAGAGTCTG CTGTTACTTGTG GCAAGTCAGAACAAAAT
TATTGCCGACAGTGTCACCTCCCAGGTCCACAATATCTATTCATTGGTCGAGAATGGTTCTTACATTGTA
GCTGTTGATTTTGATTCAATTAGTGGTCGTATCTITTGGTCTGATGCAACTCAGGGTAAAACCTGGAGTG
CGTTTCAAAATG GAACG GACAGAAGAGTGGTATTTGACAGTAGCATCATCTTGACTGAAACTATTG CAAT
AGATTG GGTAG GTCGTAATCTTTACTG GACAGACTATG CTCTG GAAACAATTGAAGTCTCCAAAATTGAT
GGGAGCCACAGGACTGTGCTGATTAGTAAAAACCTAACAAATCCAAGAGGACTAGCATTAGATCCCAGAA
TGAATGAGCATCTACTGTTCTGGTCTGACTGGGGCCACCACCCTCGCATCGAGCGAGCCAGCATGGACGG
CAGCATGCGCACTGTCATTGTCCAGGACAAGATCTTCTGGCCCTGCGGCTTAACTATTGACTACCCCAAC
AGACTGCTCTACTTCATGGACTCCTATCTTGATTACATGGACTTTTGTGATTATAATGGACACCATCGGA
GACAGGTGATAGCCAGTGATTTGATTATACGGCACCCCTATGCCCTAACTCTCTTTGAAGACTCTGTGTA
CTGGACTGACCGTGCTACTCGTCGGGTTATGCGAGCCAACAAGTGGCATGGAGGGAACCAGTCAGTTGTA
ATGTATAATATTCAATGGCCCCTTGGGATTGTTGCGGTTCATCCTTCGAAACAACCAAATTCCGTGAATC
CATGTGCCTTITCCCGCTGCAGCCATCTCTGCCTGCTITCCTCACAGGGGCCTCATTITTACTCCTGTGT
TTGTCCTTCAGGATGGAGTCTGTCTCCTGATCTCCTGAATTGCTTGAGAGATGATCAACCTITCTTAATA
ACTGTAAGGCAACATATAATTTTTGGAATCTCCCTTAATCCTGAGGTGAAGAGCAATGATGCTATGGTCC
CCATAGCAGGGATACAGAATGGTTTAGATGTTGAATTTGATGATGCTGAGCAATACATCTATTGGGTTGA
AAATCCAG GTGAAATTCACAGAGTGAAGACAGATGGCACCAACAG GACAGTATTTG CTTCTATATCTATG
GTGGGGCCTTCTATGAACCTGGCCTTAGATTGGATTTCAAGAAACCITTATTCTACCAATCCTAGAACTC
AGTCAATCGAG GTTTTGACACTCCACG GAGATATCAGATACAGAAAAACATTGATTG CCAATGATG GGAC
AG CTCTTG GAGTTG GCTTTCCAATTGG CATAACTGTTGATCCTG CTCGTG GGAAG CTGTACTGGTCAGAC
CAAGGAACTGACAGTGGGGTTCCTGCCAAGATCGCCAGTGCTAACATGGATGGCACATCTGTGAAAACTC
TCTTTACTGG GAACCTCGAACACCTG GAGTGTGTCACTCTTGACATCGAAGAG CAGAAACTCTACTGGG C
AGTCACTGGAAGAGGAGTGATTGAAAGAGGAAACGTGGATGGAACAGATCGAATGATCCTGGTACACCAG
CTTTCCCACCCCTGGGGAATTGCAGTCCATGATTCTTTCCTTTATTATACTGATGAACAGTATGAGGTCA
TTGAAAGAGTTGATAAGGCCACTGGGGCCAACAAAATAGTCTTGAGAGATAATGTTCCAAATCTGAGGGG
TCTTCAAGTTTATCACAGACGCAATGCCGCCGAATCCTCAAATGGCTGTAGCAACAACATGAATGCCTGT
CAGCAGATTTGCCTGCCTGTACCAGGAGGATTGTITTCCTGCGCCTGTGCCACTGGATTTAAACTCAATC
CA 03174172 2022- 9- 28
CTGATAATCGGTCCTG CTCTCCATATAACTCTTTCATTGTTGTTTCAATG CTGTCTG CAATCA GA G GCTT
TA G CTTG G AATTGTCAGATCATTCAGAAACCATG GTGCCGGTGG CA G GC CAAG G A CG AAA CG
CACTG CAT
GTG GATGTGGATGTGTCCTCTGG CTTTATTTATTG GTGTGATTTTAGCAG CTCAGTG G CATCTGATAATG
CGATCCGTAGAATTAAACCAGATGGATCTTCTCTGATGAACATTGTGACACATG GAATAG G AG AAAATG G
AG TC CG GG GTATTGCAGTGGATTGG GTAGCAG GAAATCTTTATTTCACCAATG CCTTTGTTTCTGAAACA
CTGATAGAAGTTCTG CGGATCAATACTACTTACCG CCGTGTTCTTCTTAAAGTCACAGTGGACATG CCTA
G GCATATTGTTGTAGATCCCAAGAACAGATACCTCTTCTG G G CTG A CTATG GG CA G A G ACCAAAG
ATTG A
G CGTTCTTTCCTTG ACTGTACCAATCG AACAGTGCTTGTGTCAG AG G G CATTGTCACACCACG GG G
CTTG
G CAGTGG ACC G AAGTG ATG G CTACGTTTATTG GGTTGATGATTCTTTAGATATAATTGCAAGGATTCGTA
TCAATG G A G AG AA CTCTG AAGTGATTCGTTATGG CAGT CG TTAC C CAA CTC CTTATG G
CATCACTGTTTT
TGAAAATTCTATCATATGG GTAGATAG GAATTTGAAAAAGATCTTCCAAG CCAG CAA GG AA CCA G AG
AA C
ACAG AG CCACCCACAGTGATAAG AG ACAATATCAACTGGCTAAGAG ATGTGACCATCTTTG ACAAGCAAG
TC CAG C CC CG G TCAC CAG CAG AG GTCAACAACAACCCTTGCTTG GAAAACAATGGTG GGTG
CTCTCATCT
CTG CTTTG CTCTGCCTG GATTG CA CAC CC CAAAATGTG ACTGTG CCTTTGGG A CC CTG
CAAAGTGATG GC
AA G AATTG TG CCATTTCAA CA G AAAATTTCCTCATCTTTG C CTTGTCTAATTCCTTG AG AAG
CTTACACT
TG G AC CCTG AAAA CCATAG C CCA CCTTTC CAAACAATAAATG TG GAAAG AACTG TCATG
TCTCTAG ACTA
TGACAGTGTAAGTGATAGAATCTACTTCACACAAAATTTAG C CTCTG G A GTTG GACAGATTTCCTATG CC
AC CCTG TCTTCAG GGATCCATACTCCAACTGTCATTG CTTCAG GTATAG GGACTG CTGATG GCATTG
CCT
TTGACTGGATTACTAGAAGAATTTATTACAGTGACTACCTCAACCAGATGATTAATTCCATG GCTG AAG A
TG G GTCTAACCGCACTGTGATAG CCCG CGTTCCAAAACCAAG AG CAATTGTGTTAGATCCCTG CCAAG GG
TA CCTG TACTG G GCTGACTGGG ATACACATGCCAAAATCGAGAG AG CCACATTG G
GAGGAAACTTCCGCG
TA CC CATTG TG AA CAG CA GTCTG G TCATG CCCAGTG GG CTG A CTCTG G ACTATG AAG AG
G AC CTTCTCTA
CTG GGTGG ATG CTAG TCTG CAG A G G ATTG AACG CAG CACTCTG ACG GG CG TG G ATCGTG
AA G TCATTG TC
AATG CAG CCGTTCATGCTTTTG GCTTGACTCTCTATGG C CA GTATATTTACTG GACTG ACTTG TA
CACAC
AAAG AATTTACC G A G CTAACAAATATG AC G G GTCAGGTCAG ATTG CAATG AC CACAAATTTG
CTCTCCCA
G CCCAG GG GAATCAACACTGTTGTG AAG AAC CA G AAACAACAG TG TAACAATCCTTGTG AA CAG
TTTAAT
GGGGG CTGCAG CCATATCTGTG CAC CAG GTCCAAATG GTG CCG AG TG CCAGTGTCCACATG AG GG
CAA CT
G GTATTTGG CCAACAACA G G AA G CACTG CATTGTG GACAATG GTGAACGATGTGGTG
CATCTTCCTTCAC
CTG CTCCAATG GG CG CTG CATCTCG GAAG AGTGG AAG TGT GATAATG A CAAC G ACTG TG G
GGATG GCAGT
G ATG AG ATGG AAA GTG TCTGTG CACTTCACACCTGCTCACCGACAGCCTTCACCTGTG CCAATG GG CG
AT
GTGTCCAATACTCTTACCG CTGTGATTACTACAATGACTGTG GTGATG GCAGTG ATG AG G CA G GGTG
CCT
GTTCAG G GACTGCAATGCCACCACGGAGTTTATGTGCAATAACAGAAGGTGCATACCTCGTGAGTTTATC
TG CAATG G TG TAG ACAACTG CCATGATAATAACACTTCAG ATG AG AAAAATTG CC CTG ATC G CA
CTTG C C
AG TCTG GATACACAAAATGTCATAATTCAAATATTTGTATTCCTCG CGTTTATTTGTGTG AC G G A GA
CAA
21
CA 03174172 2022- 9- 28
TGACTGTG G A G ATAACAGTG ATG AAAA CCCTA CTTATTG CACCACTCACAC G TG CAG CAG CA G
TG A G TTC
CAATG CGCATCTGG G CGCTGTATTCCTCAACATTG GTATTG TG ATCAAG AAACAG ATTG TTTTG ATG
C CT
CTG ATG AA CCTG CCTCTTGTG GTCA CTCTG A G CGAACATG CCTAGCTG ATG AG TTCAAGTG TG
ATGGTG G
G AG G TG CATCC CAAG C G AATG G ATCTGTG A C G GTG ATAATG A CTGTG G GG ATATG A
GTG AC G AG G ATAAA
AG GCACCAGTGTCAGAATCAAAACTGCTCGG ATTCCG AGTTTCTCTGTGTAAATGACAGACCTCCGG ACA
G GAG GTGCATTCCCCAGTCTTGG GTCTGTGATG GCGATGTG GATTGTACTGACG G CTA CG ATG A G
AATCA
GAATTG CA CCA G G AG AACTTGCTCTG AAAATG AATTCACCTGTG
GTTACGGACTGTGTATCCCAAAGATA
TTCAGGTGTG AC CG G CA CAATG A CTG TG GTGACTATAG CG ACG AG AG G GG
CTGCTTATACCAGACTTGCC
AA CAG AATCAGTTTACCTGTCAG AA CG G G CGCTG CATTAG TAAAAC CTTCGTCTGTG ATG A G G
ATAATG A
CTGTG G A G ACG G ATCTG ATG A G CTGATG CAC CTG TG CCACAC CC CAG AAC CCAC
GTGTCCACCTCAC G AG
TTCAAG TGTG A CAATG G G CGCTG CATC G AG ATGATGAAACTCTG CAAC CA CCTA G ATG ACTG
TTTG GACA
ACAG CG ATG AG AAAGG CTGTG GCATTAATGAATG CCAT GA CC CTTCAATCAG TG GCTG
CGATCACAACTG
CA CAG ACACCTTAACCAGTTICTATTGTTCCTGTCGTCCTG GTTACAAG CTCATGTCTGACAAGCG GACT
TGTGTTGATATTG ATG AATG CACA G A G ATG CCTTTTGTCTGTAG CCAG AAGTGTG AG
AATGTAATAGG CT
CCTACATCTGTAAGTGTG CCC CA G G CTA CCTCCG A G AACCAG ATG G AAAG AC CTG CC G G
CAAAACAGTAA
CATCGAACCCTATCTCATTTTTAGCAACCGTTACTATTTGAG AAATTTAACTATAG ATGGCTATTTTTAC
TCCCTCATCTTGGAAG G A CTG GACAATGTTGTG G CATTAG ATTTTG ACC G AGTAG AG AAG AG
ATTGTATT
G GATTGATACACAG AG GCAAGTCATTGAGAGAATGTTTCTGAATAAG ACAAACAAGG AG ACAATCATAAA
CCACAGACTACCAG CTG CAG AAA GTCTG G CTG TAG ACTG GGTTTCCAGAAAG CTCTACTGGTTGGATG
CC
CG CCTG GATG G CCTCTTTG TCTCTG A CCTCAATG GTGGACACCG CC G CATG CTGG CCCAG CA
CTG TG TG G
ATGCCAACAACACCTTCTG CTTTGATAATCCCAG AG G ACTTG C CCTTCAC CCTC AATATG G G TA
CCTCTA
CTG GG CAGACTGG GGTCACCG CGCATACATTGG GAGAGTAGG CATG GATGG AAC CAA CAAG TCTGTG
ATA
ATCTCCACCAAGTTAG AG TG G CCTAATG G CATCACCATTG ATTA CAC CAATG ATCTA CTCTACTG G
G CA G
ATGCCCACCTGGGTTACATAG AG TACTCTG ATTTG GAG G GCCACCATCGACACACGGTGTATGATGGGGC
ACTG CCTCACCCTTTCG CTATTACCATTTTTGAAG ACACTATTTATTGG ACAG ATTGG AATACAAGG ACA
GTG GAAAAG GG AAACAAATATG ATGG ATCAAATA G ACAG A CACTG GTG AACA CAA CACACAG A
CCATTTG
ACATC CATG TG TA CCATCCATATAG G CAGCCCATTGTG AG CAATCC CTGTG G TA CCAACAATG
GTG G CTG
TTCTCATCTCTGCCTCATCAAG CCAGG AG GAAAAG GGTTCACTTG CG AGTGTCCAG ATG ACTTCC G
CAC C
CTTCAG CTG A GTG G CAG CACCTACTGCATG CCCATGTG CTC CA G CACC CA GTTCCTG TG C G
CTAACAATG
AAAAGTG CATTCCTATCTG GTG GAAATGTGATG G ACA G AAA G A CTG CTCAGATG G
CTCTGATGAACTGG C
CCTTTG CCCG CAG CG CTTCTG CCGACTGG G ACAGTTCCAG TGCA GTG ACG G CAA CTG CAC CAG
CC CG CA G
ACTTTATGCAATG CTCACCAAAATTG CC CTG ATG G GTCTGATGAAG ACC GTCTTCTTTGTG AG
AATCAC C
ACTGTGACTCCAATGAATGG CAG TG C G C CAA CAAACGTTG CATCC CA G AATCCTG G
CAGTGTGACACATT
TAACG A CTG TG AG GATAACTCAG ATG AAG ACAGTTCC CA CTGTG C CA G CA G G ACCTG CC
G G CC G G G CCAG
22
CA 03174172 2022- 9- 28
TTTCGGTGTGCTAATGGCCGCTGCATCCCGCAGGCCTGGAAGTGTGATGTGGATAATGATTGTGGAGACC
ACTCGGATGAGCCCATTGAAGAATGCATGAGCTCTGCCCATCTCTGTGACAACTTCACAGAATTCAGCTG
CAAAACAAATTACCGCTGCATCCCAAAGTGGGCCGTGTGCAATGGTGTAGATGACTGCAGGGACAACAGT
GATGAG CAAGGCTGTGAGGAGAGGACATGCCATCCTGTGGGGGATTTCCGCTGTAAAAATCACCACTG CA
TCCCTCTTCGTTGGCAGTGTGATGGGCAAAATGACTGTGGAGATAACTCAGATGAGGAAAACTGTGCTCC
CCGGGAGTGCACAGAGAGCGAGTTTCGATGTGTCAATCAGCAGTGCATTCCCTCGCGATGGATCTGTGAC
CATTACAACGACTGTGGGGACAACTCAGATGAACGGGACTGTGAGATGAGGACCTGCCATCCTGAATATT
TTCAGTGTACAAGTGGACATTGTGTACACAGTGAACTGAAATGCGATGGATCCGCTGACTGTTTGGATGC
GTCTGATGAAGCTGATTGTCCCACACGCTTTCCTGATGGTGCATACTGCCAGGCTACTATGTTCGAATGC
AAAAACCATGTTTGTATCCCGCCATATTGGAAATGTGATGGCGATGATGACTGTGGCGATGGTTCAGATG
AAGAACTTCACCTGTGCTTGGATGTTCCCTGTAATTCACCAAACCGTTTCCGGTGTGACAACAATCGCTG
CATTTATAGTCATGAG GTGTG CAATGGTGTGGATGACTGTG GAGATG GAACTGATGAGACAGAGGAG CAC
TGTAGAAAACCGACCCCTAAACCTTGTACAGAATATGAATATAAGTGTGGCAATGGGCATTGCATTCCAC
ATGACAATGTGTGTGATGATGCCGATGACTGTGGTGACTGGTCCGATGAACTGGGTTGCAATAAAGGAAA
AGAAAGAACATGTGCTGAAAATATATGCGAGCAAAATTGTACCCAATTAAATGAAGGAGGATTTATCTGC
TCCTGTACAGCTGGGTTCGAAACCAATGTITTTGACAGAACCTCCTGTCTAGATATCAATGAATGTGAAC
AATTTGGGACTTGTCCCCAGCACTGCAGAAATACCAAAGGAAGTTATGAGTGTGTCTGTGCTGATGGCTT
CACGTCTATGAGTGACCG CCCTG GAAAACGATGTG CAG CTGAG G GTAGCTCTCCTTTGTTG CTACTGCCT
GACAATGTCCGAATTCGAAAATATAATCTCTCATCTGAGAGGTTCTCAGAGTATCTTCAAGATGAGGAAT
ATATCCAAGCTGTTGATTATGATTGGGATCCCAAGGACATAGGCCTCAGTGTTGTGTATTACACTGTGCG
AGGGGAGGGCTCTAG GTTTGGTG CTATCAAACGTGCCTACATCCCCAACTTTGAATCCGGCCG CAATAAT
CTTGTGCAGGAAGTTGACCTGAAACTGAAATACGTAATGCAGCCAGATGGAATAGCAGTGGACTGGGTTG
GAAGGCATATTTACTGGTCAGATGTCAAGAATAAACGCATTGAGGTGGCTAAACTTGATGGAAGGTACAG
AAAGTGGCTGATTTCCACTGACCTGGACCAACCAGCTGCTATTGCTGTGAATCCCAAACTAGGGCTTATG
TTCTGGACTGACTGGGGAAAGGAACCTAAAATCGAGTCTGCCTGGATGAATGGAGAGGACCGCAACATCC
TGGTITTCGAGGACCTTGGTTGGCCAACTGGCCITTCTATCGATTATTTGAACAATGACCGAATCTACTG
GAGTGACTTCAAG GAG GACGTTATTGAAACCATAAAATATGATGG GACTGATAGGAGAGTCATTG CAAAG
GAAGCAATGAACCCTTACAGCCTGGACATCTTTGAAGACCAGTTATACTGGATATCTAAGGAAAAGGGAG
AAGTATGGAAACAAAATAAATTTGGGCAAGGAAAGAAAGAGAAAACGCTGGTAGTGAACCCTTGGCTCAC
TCAAGTTCGAATCTITCATCAACTCAGATACAATAAGTCAGTGCCCAACCITTGCAAACAGATCTGCAGC
CACCTCTGCCTTCTGAGACCTGGAGGATACAGCTGTGCCTGTCCCCAAGGCTCCAGCTITATAGAGGGGA
GCACCACTGAGTGTGATGCAGCCATCGAACTGCCTATCAACCTGCCCCCCCCATGCAGGTGCATGCACGG
AG GAAATTGCTATTTTGATGAGACTGACCTCCCCAAATGCAAGTGTCCTAGCGG CTACACCGGAAAATAT
TGTGAAATGGCGTTTTCAAAAGG CATCTCTCCAG GAACAACCGCAGTAG CTGTGCTGTTGACAATCCTCT
23
CA 03174172 2022- 9- 28
TG ATCGTCGTAATTG GAG CTCTGG CAATTG CAG GATTCTTCCACTATAGAAG GACCGG CTCCCTTTTGCC
TGCTCTGCCCAAGCTGCCAAGCTTAAGCAGTCTCGTCAAGCCCTCTGAAAATGGGAATGGGGTGACCTTC
AG ATCAG GG G CAG ATCTTAACATGG ATATTGGAGTGTCTG GTTTTGG ACCTG
AGACTGCTATTGACAGGT
CAATGGCAATGAGTGAAGACTTTGTCATGGAAATGGGGAAGCAGCCCATAATATTTGAAAACCCAATGTA
CTCAGCCAG AG ACAGTGCTGTCAAAGTG GTTCAGCCAATCCAG GTGACTGTATCTGAAAATGTG GATAAT
AAGAATTATGGAAGTCCCATAAACCCTTCTGAGATAGTTCCAGAGACAAACCCAACTTCACCAGCTGCTG
ATGGAACTCAGGTGACAAAATGGAATCTCTTCAAACGAAAATCTAAACAAACTACCAACTTTGAAAATCC
AATCTATG CACAGATGG AGAACG AG CAAAAG G AAAGTGTTGCTG CGACACCACCTCCATCACCTTCG CTC
CCTGCTAAGCCTAAGCCTCCTTCG AG AAG AGACCCAACTCCAACCTATTCTG CAACAGAAG ACACTTTTA
AAGACACCGCAAATCTTGTTAAAGAAGACTCTGAAGTATAGCTATACCAGCTATTTAGGGAATAATTAGA
AACACACTTTTGCACATATATTTTTTACAAACAGATGAAAAAAGTTAACATTCAGTACTTTATGAAAAAA
ATATATTMCCCTGITTGCCTATAGTTGGAGGTATCCTGTGTGR. __________ IIIIIII
ACTTATGCCGTCTCATA
TTTTTACAAATAATTATCACAATGTACTATATGTATATCTTTGCACTGAAGTTGTCTGAAGGTAATACTA
TAAATATATTGTATATTTGTAAATTTTGGAAAGATTATCCTGTTACTGAATTTGCTAATAAAGATGTCTG
CTGATTTG GTTG GTGATCATTATAGTAAATGATCCAACAAGAAAAG GAATTGACTGG GG AC CTTTAG CCG
TGTCTAAAGAAGAGGCACCACTCATATTTCCTATAAAATTATCTAGGAAAGGAATCCAGGCCCCGCTCTT
GGGTCCATTTTTACACATTAGCACTTAATTAATGTTCAATATTACATGTCAATTTGATTAATGGCTATGT
TG ATAG GG GCCACTATGTGTTGTATAG ACATCTG GACTTGACTGTAG ACTCCTCAGATAATACAG AAG GT
AG GAAAAGCAATTCAGTTTG GCCCTTCTGTGTGTTG GCATTGTCTAACCAG AACTCTCTGTTTCATGTGT
GTTCTCTCACTAGCTGCCAAGACAACATTTTTATTTGTGATGTCTATGAGGAAATCCCATATCATTAAGT
GCCAGTGTCCTGCATTGAGTTTGTGGTTAATTAAATGAGCTCTTCTGCTGATGGACCCTGGAGCAATTTC
TCCCCTCACCTGACATTCAAGGTG GTCACCTG CCCTAGTAGTTG GAG CTCAGTAGCTGAATTTCTGAAAC
CAAATCTGTGTCTTCATAAAATAAGGTGCAAAAAAAAAAAATACCAGTTAAGTAAAGCCTCAACTGGGTT
TTTGTTTCTATGAAAATATCATTATAATCACTATTTATTTCCTAAGTTGAACCTGAATAGAAAGGGAAAC
CATTCTTATTAAGL ___________ 11111 ATTAGGCCCTGTGGCTAAATGTGTACATTTATATTAG
AATGTACTG TACAG
TCCAGATCTITTCTTTAATTCTTATTGG _________ 1111111111111111111111
AGAGATGGAGTCTTGCTATA
TTGCCAAGGCTGATCTTGAAGTCCTGGGCTCAAGTGATCCTCCCACCTCAGCCTCCTGAGTGGTTGGGGT
TACG G GCGTG AG CCACTGTGCCTGG CTTCCAG CTCTCCTCTTAAATAGTG GGTATAGTCTGCACAACAG G
AACCATGGCAGGAATATACACTTTCCCATAGCAAATAGCATACCTGACTCTCTGTGCTAATATTGCACAT
TTGTTAAACAATGAATGAATGGATGGATGGATGGATGGATGAATGAATGAAACATATACTACTGATTATT
TTATTCCAGAGTTCTCAAAATATTTGTTGCTGATATTTTGAGTGCTGACTGTAATTACTTTGATTAGATA
AACAACTGGAAATAATGCTGCTGAAAAAGTTCTAATAAATGTGTATTTTATCAGA (SEQ ID NO. 939).
24
CA 03174172 2022- 9- 28
[0077] One example of a protein sequence from the above LRP2
DNA is:
>spIP98164ILRP2_HUMAN Low-density lipoprotein receptor-related protein 2
OS=Honno sapiens
OX=9606 GN=LRP2 PE=1 SV=3
MDRGPAAVACTLLLALVACLAPASGQECDSAHFRCGSGHCIPADWRCDGTKDCSDDADEI
GCAVVICQQGYFKCCISEGQCIPNSWVCDQDQDCDDGSDERQDCSCISTCSSHOITCSNGQC
IPSEYRCDHVRDCPDGADENDCQYPTCEQLTCDNGACYNTSQKCDWKVDCRDSSDEINCT
EICLHNEFSCGNGECIPRAYVCDHDNDCQDGSDEHACNYPTCGGYQFTCPSGRCIYQNWV
CDGEDDCKDNGDEDGCESGPHDVHKCSPREWSCPESGRCISIYKVCDGILDCPGREDENN
TSTGKYCSMTLCSALNCQYQCHETPYGGACFCPPGYIINHNDSRTCVEFDDCQIWGICDQ
KCESRPGRHLCHCEEGYILERGQYCKANDSFGEASIIFSNGRDLLIGDIHGRSFRILVES
QNRGVAVGVAFHYHLQRVFWTDTVQNKVFSVDINGLNIQEVLNVSVETPENLAVDWVNNK
IYLVETKVNRIDMVNLDGSYRVTLITENLGHPRGIAVDPTVGYLFFSDWESLSGEPKLER
AFMDGSNRKDLVKTKLGWPAGVTLDMISKRVYWVDSRFDYIETVTYDGIQRKTVVHGGSL
IPHPFGVSLFEGQVFFTDWTKMAVLKANKFTETNPQVYYQASLRPYGVTVYHSLRQPYAT
NPCKDNNGGCEQVCVLSHRTDNDGLGFRCKCTFGFQLDTDERHCIAVQNFLIFSSQVAIR
GIPFTLSTQEDVMVPVSGNPSFFVGIDFDAQDSTIFFSDMSKHMIFKQKIDGTGREILAA
NRVENVESLAFDWISKNLYWTDSHYKSISVMRLADKTRRTVVQYLNNPRSVVVHPFAGYL
FFTDWFRPAKIMRAWSDGSHLLPVINTTLGWPNGLAIDWAASRLYWVDAYFDKIEHSTFD
GLDRRRLGHIEQMTHPFGLAIFGEHLFFTDWRLGAIIRVRKADGGEMTVIRSGIAYILHL
KSYDVNIQTGSNACNQPTHPNGDCSHFCFPVPNFQRVCGCPYGMRLASNHLTCEGDPTNE
PPTEQCGLFSFPCKNGRCVPNYYLCDGVDDCHDNSDEQLCGTLNNTCSSSAFTCGHGECI
PAHWRCDKRNDCVDGSDEHNCPTHAPASCLDTQYTCDNHQCISKNWVCDTDNDCGDGSDE
KNCNSTETCQPSQFNCPNHRCIDLSFVCDGDKDCVDGSDEVGCVLNCTASQFKCASGDKC
IGVTNRCDGVFDCSDNSDEAGCPTRPPGMCHSDEFQCQEDGICIPNFWECDGHPDCLYGS
DEHNACVPKTCPSSYFHCDNGNCIHRAWLCDRDNDCGDMSDEKDCPTQPFRCPSWQWQCL
GHNICVNLSVVCDGIFDCPNGTDESPLCNGNSCSDFNGGCTHECVQEPFGAKCLCPLGFL
LANDSKTCEDIDECDILGSCSQHCYNMRGSFRCSCDTGYMLESDGRTCKVTASESLLLLV
ASQNKIIADSVTSQVHNIYSLVENGSYIVAVDFDSISGRIFWSDATQGKTWSAFQNGTDR
RVVFDSSIILTETIAIDWVGRNLYWTDYALETIEVSKIDGSHRTVLISKNLTNPRGLALD
PRMNEHLLFWSDWGHHPRIERASMDGSMRTVIVQDKIFWPCGLTIDYPNRLLYFMDSYLD
YMDFCDYNGHHRRQVIASDLIIRHPYALTLFEDSVYWTDRATRRVMRANKWHGGNQSVVM
YNIQWPLGIVAVHPSKQPNSVNPCAFSRCSHLCLLSSQGPHFYSCVCPSGWSLSPDLLNC
CA 03174172 2022- 9- 28
LRDDQPFLITVRQH I IFG ISLN PEVKSN DAMVPIAG IQN G LDVEFD DAEQYIYWVE N PG E
I H RVKTDGTN RTVFASISMVGPSM NLALDWISRN LYSTN PRTQSI EVLTLHGD I RYRKTL
IAN DGTALGVGFPIGITVD PARG KLYWSDQGTDSGVPAKIASAN M DGTSVKTLFTGN LEH
LECVTLDIEEQKLYWAVTG RGVI ERG NVDGTDRM I LVHQLSH PWG IAVH DSFLYYTDEQY
EVIERVDKATGANKIVLRDNVPN LRGLQVYH RRNAAESSNGCSNNM NACQQICLPVPGGL
FSCACATGFKLNPDNRSCSPYNSFIVVSM LSAIRGFSLELSDHSETMVPVAGQGRNALHV
DVDVSSGFIYWCDFSSSVASDNAIRRIKPDGSSLM NIVTHGIGENGVRGIAVDWVAGN LY
FTNAFVSETLIEVLRINTTYRRVLLKVTVDM PRH IVVDPKN RYLFWADYGQRPKI ERS FL
DCTN RTVLVSEG IVTPRGLAVDRSDGYVYWVDDSLD I IAR I RI NG E NSEVI RYGSRYPTP
YG ITVFENSIIWVDRN LKKI FQASKE PE NTE PPTVIRD N INWLRDVTIFDKQVQPRSPAE
VNNNPCLENNGGCSH LCFALPGLHTPKCDCAFGTLQSDGKNCAISTENFLIFALSNSLRS
LH LD PE N HSPPFQTI NVERTVMSLDYDSVSD RIYFTQN LASGVGQISYATLSSGIHTPTV
IASGIGTADGIAFDWITRRIYYSDYLNQM INSMAEDGSNRTVIARVPKPRAIVLDPCQGY
LYWADWDTHAKIERATLGGNFRVPIVNSSLVM PSG LTLDYE EDLLYWVDASLQRI E RSTL
TGVDREVIVNAAVHAFGLTLYGQYIYWTDLYTQR1YRANKYDGSGQIAMTTN LLSQPRG I
NTVVKNQKQQCNNPCEQFNGGCSH ICAPGPNGAECQCPH EG NWYLAN N RKHCIVD NG E RC
GASSFTCSNGRCISEEWKCDNDNDCGDGSDEM ESVCALHTCSPTAFTCANGRCVQYSYRC
DYYN DCG DGS DEAGCLFR DCNATTEF MCN N RRCIPR EFICNGVD NCH DN NTSDEKNCPDR
TCQSGYTKCH NSN ICI PRVYLCDGDN DCGD NSDE N PTYCTTHTCSSS EFQCASG RCI PQH
WYCDCIETDCFDASDEPASCGHSERTCLADEFKCDGGRCIPSEWICDG DN DCGDMSDE D KR
HQCQNQNCSDSEFLCVN DRPPDRRCIPQSWVCDG DVDCTDGYD ENQNCTRRTCSE N E FTC
GYG LCIPKIFRCD RH N DCG DYSDERGCLYQTCQQNQFTCQNGRCISKTFVCDEDNDCGDG
SD ELM H LCHTPEPTCPPHEFKCDNGRCIEM MKLCN HLDDCLDNSDEKGCGIN ECH DPSIS
GCDHNCTDTLTSFYCSCRPGYKLMSDKRTCVDIDECTEM PFVCSQKCENVIGSYICKCAP
GYLRE PDG KTCRQNS N I EPYLI FSN RYYLRN LTIDGYFYSLI LEG LDNVVALDFD RVE KR
LYWIDTQRQVI ER M FLNKTNKETIIN HRLPAAESLAVDWVSRKLYWLDARLDGLFVSDLN
GGH RRM LAQHCVDANNTFCFDNPRGLALH PQYGYLYWADWGH RAYIGRVGM DGTN KSVI I
STKLEWPNGITIDYTNDLLYWADAH LGYIEYSDLEGHH RHTVYDGALPH PFAITI FE DTI
YWTDWNTRTVE KG NKYDGSN RQTLVNTTH RPFDI HVYH PYRQPIVSN PCGTN NGGCSH LC
LI KPGG KG FTCECPD DFRTLQLSGSTYCM PMCSSTQFLCANNEKCIPIWWKCDGQKDCSD
GSDE LALCPQRFCRLGQFQCSDGNCTS PQTLCNAHCINCPDGSDE D MICE N H HCDSNEWQ
CANKRCI PESWQCDTFN DCEDNS DEDSSHCASRTCRPGQF RCANG RCIPQAWKCDVDN DC
26
CA 03174172 2022- 9- 28
GDHSDEPIEECMSSAHLCDNFTEFSCKTNYRCIPKWAVCNGVDDCRDNSDEQGCEERTCH
PVGDFRCKNHHCIPLRWQCDGQNDCGDNSDEENCAPRECTESEFRCVNQQCIPSRWICDH
YNDCGDNSDERDCEMRTCHPEYFQCTSGHCVHSELKCDGSADCLDASDEADCPTRFPDGA
YCQATMFECKNHVCIPPYWKCDGDDDCGDGSDEELHLCLDVPCNSPNRFRCDNNRCIYSH
EVCNGVDDCGDGTDETEEHCRKPTPKPCTEYEYKCGNGHCIPHDNVCDDADDCGDWSDEL
GCNKGKERTCAENICEQNCTQLNEGGFICSCTAGFETNVFDRTSCLDIN ECEQFGTCPQH
CRNTKGSYECVCADGFTSMSDRPGKRCAAEGSSPLLLLPDNVRIRKYNLSSERFSEYLQD
EEYIQAVDYDWDPKDIGLSVVYYTVRGEGSRFGAIKRAYIPNFESGRNN LVQEVDLKLKY
VMQPDGIAVDWVGRHIYWSDVKNKRIEVAKLDGRYRKWLISTDLDQPAAIAVNPKLGLMF
WTDWGKEPKIESAWMNGEDRNILVFEDLGWPTGLSIDYLNNDRIYWSDFKEDVIETIKYD
GTDRRVIAKEAMNPYSLDIFEDQLYWISKEKGEVWKQNKFGQGKKEKTLVVNPWLTQVRI
FHQLRYNKSVPNLCKQICSHLCLLRPGGYSCACPQGSSFIEGSTTECDAAIELPINLPPP
CRCMHGGNCYFDETDLPKCKCPSGYTGKYCEMAFSKGISPGTTAVAVLLTILLIVVIGAL
AIAGFFHYRRTGSLLPALPKLPSLSSLVKPSENGNGVTFRSGADLNMDIGVSGFGPETAI
DRSMAMSEDFVMEMGKQPIIFENPMYSARDSAVKVVQPIQVIVSENVDNKNYGSPINPSE
IVPETNPTSPAADGTQVTKWNLFKRKSKQTTNFENPIYAQMENEQKESVAATPPPSPSLP
AKPKPPSRRDPTPTYSATEDTFKDTANLVKEDSEV (SEQ ID NO. 940).
[0078] As used herein, "target sequence" refers to a
contiguous portion of the nucleotide
sequence of an nnRNA molecule formed during the transcription of a gene of
interest for example a
CD320 gene or an LRP2 gene, including mRNA that is a product of RNA processing
of a primary
transcription product.
[0079] As used herein, the term "strand comprising a
sequence" refers to an oligonucleotide
comprising a chain of nucleotides that is described by the sequence referred
to using the standard
nucleotide nomenclature.
[0080] "G," "C," "A" and "U" each generally stand for a
nucleotide that contains guanine,
cytosine, adenine, and uracil as a base, respectively. "T" and "dT" are used
interchangeably herein
and refer to a deoxyribonucleotide wherein the nucleobase is thynnine, e.g.,
deoxyribothymine, 2'-
deoxythymidine or thymidine. However, it will be understood that the term
"ribonucleotide" or
"nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide,
as further detailed
below, or a surrogate replacement moiety. The skilled person is well aware
that guanine, cytosine,
adenine, and uracil may be replaced by other moieties without substantially
altering the base pairing
properties of an oligonucleotide comprising a nudeotide bearing such
replacement moiety. For
example, without limitation, a nucleotide comprising inosine as its base may
base pair with
27
CA 03174172 2022- 9- 28
nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides
containing uracil, guanine, or
adenine may be replaced in the nucleotide sequences of the invention by a
nucleotide containing, for
example, inosine. Sequences comprising such replacement moieties are
embodiments of the
invention.
[0081] The term "siRNA" refers to a compound, cocktail,
composition or agent that contains
RNA as that term is defined herein, and which mediates the targeted cleavage
of an RNA transcript
via the RISC/AGO (RNA-induced silencing complex) complex, whereby the guide
strand of the siRNA
hybridizes with its complementary mRNA molecule. The nnRNA is degraded by the
RISC/AGO
complex, which has RNAse cleave activity, resulting in nnRNA degradation and
the protein encoded
by the nnRNA is not produced or is produced at a reduced level as compared to
untreated cell. This
causes the "knockdown" effect or reduced protein levels of the gene targeted
by the siRNA compared
to control treated cells. The siRNA modulates, e.g., inhibits, the expression
of CD320 in a cell or
LRP2 in a cell, e.g., a cell within a subject, such as a mammalian subject.
[0082] In one embodiment, an RNAi agent of the invention
indudes a single stranded RNA
that interacts with a target RNA sequence, e.g., a CD320 or LRP2 target nnRNA
sequence, to direct
the cleavage of the target RNA. Without wishing to be bound by theory, it is
believed that long double
stranded RNA introduced into cells is broken down into siRNA by a Type III
endonuclease known as
Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-Ill-like
enzyme, processes the
dsRNA into 19-23 base pair (bp) short interfering RNAs with characteristic two
base 3 overhangs
(Bernstein, et al., (2001) Nature 409:363). Initially, the siRNAs may consist
of two RNA strands, an
antisense (or guide) strand and a sense (or passenger) strand, which form a
duplex that varies in
length from 10-80 bp in length with or without a 3' nucleotide overhang. A
dsRNA can include one or
more single-stranded overhang(s) of one or more nucleotides. In one
embodiment, at least one end
of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1
or 2 nucleotides. In
another embodiment, the antisense strand of the dsRNA has 1-10 nudeotide
overhangs each at the
3' end and the 5' end over the sense strand. In further embodiments, the sense
strand of the dsRNA
has 1-10 nucleotide overhangs each at the 3' end and the 5' end over the
antisense strand.
[0083] The siRNA are then incorporated into an RNA-induced
silencing complex (RISC)
where one or more helicases unwind the siRNA duplex, enabling the
complementary antisense
(guide) strand to guide target recognition (Nykanen, et al., (2001) Cell
107:309). Upon binding to the
appropriate target nnRNA, one or more endonucleases within the RISC cleave the
target to induce
silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect
the invention relates to a
single stranded RNA (siRNA) generated within a cell and which promotes the
formation of a RISC
complex to effect silencing of the target gene, i.e., a CD320 or LRP2 gene.
Accordingly, the term
"siRNA" is also used herein to refer to an RNAi as described above.
28
CA 03174172 2022- 9- 28
[0084] In another embodiment, the RNAi agent may be a single-
stranded siRNA that is
introduced into a cell or organism to inhibit a target nnRNA. Single-stranded
RNAi agents bind to the
RISC endonuclease Argonaute 2, which then cleaves the target nnRNA. The single-
stranded siRNAs
are generally 15-80 nucleotides and may be chemically modified to improve
metabolic stability and
activity; wherein one or multiple pyrinnidine nucleotides could be modified as
2'-deoxy-2'-
fluoronucleotides, one or more purine nucleotides could be modified as 2'-
deoxypurine nucleotides
and, moreover, wherein terminal cap modifications could be present at the 3'
or 5' ends; particularly
by the introduction of one or more 2'-deoxythynnidine nucleotides, or by the
introduction of one or
more phosphorothioate groups linking any nudeotides in the sequence but
especially at the 3' and 5'
end . In addition, a 3'-terminal phosphate or vinylphosphonate group could be
introduced. Examples
of such modifications would include but not be limited to modifications to the
ribose moieties of the
nucleotides such as: 2'-deoxy, 2'-deoxyfluoro, 2'-nnethoxy (2'-0-methyl)
(Hutvanger et al., (2004)
PLOS Biol 2, 0465-0475; Janas et al., (2019) Nuc Acid Res 47, 3306-3320;
Jackson et al., (2006)
RNA 12, 1197-1205), and 2'-methoxyethyl, wherein it is understood that the
stereochennistry of the 2'-
substituent could be in the ribo- or arabino- orientation. Another
modification could be 2'-
trifluoronnethoxy. Other modifications to the ribose moieties could include
bridging modifications such
that the 2'-carbon of the sugar moiety is covalently linked to the 4'-carbon
of the sugar moiety by a
methylene or nnethoxynnethylene group to afford bridged nucleotides described
in the art as LNA and
(S)-cET, respectively (Corey et al., (2018) Nuc Acid Res 46; 1584-1600). In
addition, the sugar moiety
could be modified by removal of the bond between carbons C2' and C3' to afford
"open" chain
nucleotides analogous to those described in WO 2011/139843 A2. The ribose
moiety of the RNA
nucleotides could also be replaced by a nnorpholino group to afford PMO
nucleotides. Modifications to
the phosphate diester moieties of the nudeotides are also possible and could
include but not be
limited to replacement of the phosphodiester group by phosphorothioate and
thio-phosphorannidate
(Eckstein et al., (2014) Nuc Acid Therapeutics 24, 374-387). The ends of the
strand could be
modified with 2'-deoxynucleotides such as dT and, further, the dT nudeotides
could be modified by
phosphorothioate groups in place of diphosphate esters. The design and testing
of single-stranded
siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012)
Cell 150: 883-894, the
entire contents of each of which are hereby incorporated herein by reference.
Any of the antisense
nucleotide sequences described herein may be used as a single-stranded siRNA
as described herein
or as chemically modified by the methods described in Lima et al., (2012) Cell
150; 883-894.
[0085] In another embodiment, an "RNAi" for use in the
compositions, uses, and methods of
the invention is a double-stranded RNA and is referred to herein as a "double
stranded RNAi agent,"
"double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA". The term
"dsRNA" refers to
a complex of ribonucleic acid molecules, having a duplex structure comprising
two anti-parallel and
substantially complementary nucleic acid strands, referred to as having
"sense" (passenger) and
"antisense" (guide) orientations with respect to a target RNA, i.e., a CD320
gene or LRP2 gene. In
some embodiments of the invention, a double-stranded RNA (dsRNA) triggers the
degradation of a
29
CA 03174172 2022- 9- 28
target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing
mechanism referred to
herein as RNA interference or RNAi.
[0086] In general, the majority of nucleotides of each
strand of a dsRNA molecule are
ribonucleotides, but as described in detail herein, each or both strands can
also include one or more
non-ribonucleotides, e.g., a deoxyribonudeotide and/or a modified nucleotide.
In addition, as used in
this specification, an "RNAi agent" may include ribonucleotides with chemical
modifications (Corey et
al., (2018) Nuc Acid Res 46; 1584-1600); an RNAi agent may include substantial
modifications at
multiple nudeotides or at a single nucleotide. Such modifications may include
all types of
modifications disclosed herein or known in the art. Any such modifications, as
used in a siRNA type
molecule, are encompassed by "RNAi agent" for the purposes of this
specification and claims.
Examples of such modifications would indude but not be limited to
modifications to the ribose
moieties of the nudeotides such as: 2'-deoxy, 2'-deoxyfluoro, 2'-nnethoxy (2'-
0-methyl) (Hutvanger et
al., (2004) PLOS Biol 2, 0465-0475; Janas et al., (2019) Nuc Acid Res 47, 3306-
3320; Jackson et al.,
(2006) RNA 12, 1197-1205) , and 2'-methoxyethyl, wherein it is understood that
the stereochennistry
of the 2'-substituent could be in the ribo- or arabino- orientation. Another
modification could be 2'-
trifluoronnethoxy. Other modifications to the ribose moieties could include
bridging modifications such
that the 2'-carbon of the sugar moiety is covalently linked to the 4'-carbon
of the sugar moiety by a
methylene or nnethoxynnethylene group to afford bridged nucleotides described
in the art as LNA and
(S)-cET, respectively (Corey et al., (2018) Nuc Acid Res 46; 1584-1600). In
addition, the sugar
moiety could be modified by removal of the bond between carbons C2' and C3' to
afford "open" chain
nucleotides analogous to those described in WO 2011/139843 A2. The ribose
moiety of the RNA
nucleotides could also be replaced by a nnorpholino group to afford PMO
nucleotides. Modifications
to the phosphate diester moieties of the nucleotides are also possible and
could include but not be
limited to replacement of the phosphodiester group by phosphorothioate and
thio-phosphorannidate
(Eckstein et al., (2014) Nuc Acid Therapeutics 24, 374-387). The ends of the
sense and antisense
strands could be modified with 2'-deoxynucleotides such as dT and, further,
the dT nucleotides could
be modified by phosphorothioate groups in place of diphosphate esters (FIG.
19).
[0087] Chemical modifications to the ribonucleotides could
be made at any individual or
combination of nucleotides in the antisense and sense strands. In some cases,
all the nucleotides in
either the antisense or sense strand, or in both the antisense and sense
strands are chemically
modified (Allerson et al., (2005) J Med Chem 48, 901-904). In other cases,
only some of the
nucleotides in the antisense or sense strand, or in both the antisense and
sense strands are
chemically modified (Chiu et al., (2003) RNA 9, 1034-1048). In yet other
cases, the modifications
could follow a pattern of alternating 2'-nnethoxy and 2'-fluoro modifications
to either or both strands of
the siRNA and sometimes the complementary nucleotides of the antisense and
sense strands could
contain chemical modifications which are not identical, for example, where one
member of a
complementary nucleotide pair has a 2'-nnethoxy modification and the other
member has a 2'-fluoro
CA 03174172 2022- 9- 28
modification (Choung et al. (2006) Biochenn Biophys Res Connnnun 342, 919-927;
Hassler et al.,
(2018) Nucleic Acid Res 46, 2185-2196).
[0088] The two strands forming the duplex structure may be
different portions of one larger
RNA molecule, or they may be separate RNA molecules. Where the two strands are
part of one
larger molecule, and therefore are connected by an uninterrupted chain of
nucleotides between the 3'-
end of one strand and the 5'-end of the respective other strand forming the
duplex structure, the
connecting RNA chain is referred to as a "hairpin loop." Where the two strands
are connected
covalently by means other than an uninterrupted chain of nucleotides between
the 3'-end of one
strand and the 6-end of the respective other strand forming the duplex
structure, the connecting
structure is referred to as a "linker." The RNA strands may have the same or a
different number of
nucleotides. The maximum number of base pairs is the number of nucleotides in
the shortest strand
of the dsRNA minus any overhangs that are present in the duplex. In addition
to the duplex structure,
an RNAi agent may comprise one or more nucleotide overhangs.
[0089] In one embodiment, an RNAi agent of the invention is
a dsRNA of 20-30 nucleotides
that interacts with a target RNA sequence, e.g., a CD320 target mRNA sequence
or a LRP2 target
mRNA sequence, to direct the deavage of the target RNA.
[0090] The term "antisense strand" refers to the strand of a
double stranded RNAi agent
which includes a region that is substantially complementary to a target
sequence (e.g., a human
CD320 mRNA or a LRP2 mRNA). As used herein, the term "region complementary to
part of an
mRNA encoding CD320 or LRP2" refers to a region on the antisense strand that
is substantially
complementary to part of a mRNA sequence that codes for either CD320 or LRP2.
Where the region
of connplennentarity is not fully complementary to the target sequence, the
mismatches are most
tolerated in the terminal regions and, if present, are generally in a terminal
region or regions, e.g.,
within 6, 5, 4, 3, or 2 nucleotides of the 6 and/or 3 terminus. For example,
substantially
complementary can in certain embodiments mean that in a hybridized pair of
nucleobase sequences,
at least 85% but not all of the bases in a contiguous sequence of a first
polynucleotide will hybridize
with the same number of bases in a contiguous sequence of a second
polynudeotide.
[0091] The term "sense strand," as used herein, refers to
the strand of a dsRNA that
includes a region that is substantially complementary to a region of the
antisense strand.
[0092] As used herein, the term "cleavage region" refers to
a region that is located
immediately adjacent to the cleavage site. The cleavage site is the site on
the target at which
cleavage occurs. In some embodiments, the cleavage region comprises three
bases on either end of,
and immediately adjacent to, the cleavage site. In some embodiments, the
cleavage region
comprises two bases on either end of, and immediately adjacent to, the
cleavage site. In some
31
CA 03174172 2022- 9- 28
embodiments, the cleavage site specifically occurs at the site bound by
nucleotides 10 and 11 of the
antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.
[0093] As used herein, and unless otherwise indicated, the
term "complementary," when
used to describe a first nucleotide sequence in relation to a second
nucleotide sequence, refers to the
ability of an oligonucleotide or polynucleotide comprising the first nudeotide
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 may indude: 400 mM
NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 C. or 70 C for 12-16 hours followed
by washing. Other
conditions, such as physiologically relevant conditions as may be encountered
inside an organism,
can apply. For example, a complementary sequence is sufficient to allow the
relevant function of the
nucleic acid to proceed, e.g., RNAi. The skilled person will be able to
determine the set of conditions
most appropriate for a test of connplennentarity of two sequences in
accordance with the ultimate
application of the hybridized nucleotides.
[0094] Sequences can be "fully complementary" with respect
to each when there is base-
pairing of the nucleotides of the first nucleotide sequence with the
nucleotides of the second
nucleotide sequence over the entire length of the first and second nucleotide
sequences. 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 may
form one or more, but
generally not more than 4, 3 or 2 mismatched base pairs upon hybridization,
while retaining the ability
to hybridize under the conditions most relevant to their ultimate application.
However, where two
oligonucleotides are designed to form, upon hybridization, one or more single
stranded overhangs,
such overhangs shall not be regarded as mismatches with regard to the
determination of
complementarity. For example, a dsRNA comprising one oligonucleotide 21
nucleotides in length and
another oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a
sequence of 21 nucleotides that is fully complementary to the shorter
oligonucleotide, may yet be
referred to as "fully complementary" for the purposes described herein.
[0095] "Complementary" sequences, as used herein, may also
include, or be formed entirely
from, non-Watson-Crick base pairs and/or base pairs formed from non-natural
and modified
nucleotides, in as 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.
[0096] The terms "complementary," "fully complementary" and
"substantially
complementary" herein may be used with respect to the base matching between
the sense strand and
the antisense strand of a dsRNA, or between the antisense strand of a dsRNA
and a target sequence,
as will be understood from the context of their use.
32
CA 03174172 2022- 9- 28
[0097] As used herein, a polynucleotide that is
"substantially complementary to at least part
of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially
complementary to a
contiguous portion of the mRNA of interest (e.g., an mRNA encoding CD320 or an
mRNA encoding
LRP2) including a 5 UTR, an open reading frame (ORF), or a 3' UTR. For
example, a polynucleotide
is complementary to at least a part of a CD320 mRNA or LRP2 mRNA if the
sequence is substantially
complementary to a non-interrupted portion of an mRNA encoding CD320 or LRP2.
[0098] The term "inhibiting," as used herein, is used
interchangeably with "reducing,"
"silencing," "downregulating," "suppressing" and other similar terms, and
includes any level of
inhibition.
[0099] The phrase "inhibiting expression of a CD320,"
"inhibiting expression of a LRP2" as
used herein, includes inhibition of expression of any CD320 or LRP2 gene (such
as the identified
gene from, e.g., a mouse, a rat, a monkey, or a human) as well as variants,
(e.g., naturally occurring
variants), or mutants of the identified gene. Thus, the CD320 or LRP2 gene may
be a wild-type
CD320 or LRP2 gene, a mutant CD320 or LRP2 gene, or a transgenic CD320 or LRP2
gene in the
context of a genetically manipulated cell, group of cells, or organism.
[00100] "Inhibiting expression of a CD320 gene" or
"Inhibiting expression of a LRP2 gene"
includes any level of inhibition of a CD320 gene or a LRP2 gene, e.g., at
least partial suppression of
the expression of a CD320 or LRP2 gene, such as an inhibition of at least
about 5%, at least about
10%, at least about 15%, at least about 20%, at least about 25%, at least
about 30%, at least about
35%, at least about 40%, at least about 45%, at least about 50%, at least
about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about
85%, at least about 90%, at least about 91%, at least about 92%, at least
about 93%, at least about
94%. at least about 95%, at least about 96%, at least about 97%, at least
about 98%, or at least about
99%. In a preferred embodiment the inhibition is assessed by expressing the
level of CD320 or LRP2
protein in treated cells as a percentage of the level of mRNA in control
cells, using the following
formula:
Normalized protein level for treated cells/Normalized protein level for
control cells. The
control cells are the negative control siRNA. Normalized means the protein
level is normalized
to the level of a housekeeping protein.
[00101] The expression of a CD320 or LRP2 gene may be
assessed based on the level of
any variable associated with CD320 or LRP2 gene expression, e.g., CD320 or
LRP2 mRNA level,
CD320 or LRP2 protein level. Inhibition may 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 may be any
type of control level that is utilized in the art, e.g., a pre-dose baseline
level, or a level determined
33
CA 03174172 2022- 9- 28
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).
[00102] Contacting a cell with a RNAi agent, either ds or ss
as used herein, includes
contacting a cell by any possible means whether in vivo or in vitro.
Contacting a cell with a RNAi
agent includes contacting a cell in vitro with the RNAi agent or contacting a
cell in vivo with the RNAi
agent. The contacting may be done directly or indirectly. Thus, for example,
the RNAi agent may be
put into physical contact with the cell by the individual performing the
method, or alternatively, the
RNAi agent may be put into a situation that will permit or cause it to
subsequently come into contact
with the cell.
[00103] A "patient" or "subject," as used herein, is intended
to include either a human or non-
human animal, preferably a mammal, e.g., a monkey. Most preferably, the
subject or patient is a
human.
[00104] A "CD320-associated disease," as used herein, is
intended to include any disease
associated with a perturbation of the CD320 gene, or protein, polynnorphisnns,
single nucleotide
polymorphisms (SNPs) as well as epigenetic modifications of the CD320 gene.
Such a disease may
be caused, for example, by excess production of the CD320 protein, by CD320
gene mutations, by
abnormal cleavage of the CD320 protein, by abnormal folding of the CD320
protein, by abnormal
interactions between CD320 itself or with other proteins or other endogenous
or exogenous
substances. For example, cancer may be a CD320-associated disease. The degree
of inhibition of
protein expression may be measured by western blotting.
[00105] A "LRP2-associated disease," as used herein, is
intended to include any disease
associated with a perturbation of the LRP2 gene, protein, polynnorphisnns,
SNPs as well as epigenetic
modifications of the CD320 gene. Such a disease may be caused, for example, by
excess production
of the LRP2 protein, by LRP2 gene mutations, by abnormal cleavage of the LRP2
protein, by
abnormal folding of the LRP2 protein, by abnormal interactions between LRP2
molecules and other
proteins or other endogenous or exogenous substances. For example, cancer may
be a LRP2-
associated disease. The degree of inhibition of protein expression may be
measured by western
blotting.
[00106] "Therapeutically effective amount," as used herein,
is intended to include the amount
of an RNAi agent that, when administered to a cell or a patient for treating a
CD320 associated
disease or a LRP2 associated disease, is sufficient to effect treatment of the
disease (e.g., by
diminishing, ameliorating or maintaining the existing disease or one or more
symptoms of disease or
by preferentially causing the death of a disease cell as compared to a non-
disease cell). The
"therapeutically effective amount" may vary depending on the RNAi agent, how
the agent is
administered, the disease and its severity and the history, age, weight,
family history, genetic makeup,
34
CA 03174172 2022- 9- 28
stage of pathological processes mediated by CD320 or LRP2 expression, the
types of preceding or
concomitant treatments, if any, and other individual characteristics of the
patient to be treated.
[00107] "Prophylactically effective amount," as used herein,
is intended to include the amount
of an RNAi agent that, when administered to a subject who does not yet
experience or display
symptoms of a CD320 associated disease or a LRP2 associated disease, but who
may be
predisposed to the disease, 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" may vary
depending on the RNAi agent, 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.
[00108] A "therapeutically-effective amount" or
"prophylactically effective amount" also
includes an amount of an RNAi agent that produces some desired local or
systemic effect at a
reasonable benefit/risk ratio applicable to any treatment. RNAi agents
employed in the methods of
the present invention may be administered in a sufficient amount to produce a
reasonable benefit/risk
ratio applicable to such treatment.
Pharmaceutical Compositions
[00109] The methods described herein include administration
of a LRP2 inhibiting
composition and/or a CD320 inhibiting composition, e.g., a first siRNA
targeting a
CD320 gene and/or a second siRNA targeting a LRP2 gene. In some embodiments,
the LRP2
inhibiting composition and/or the CD320 inhibiting composition is a
pharmaceutical composition.
[00110] The methods described herein also include
administration of one or multiple LRP2
inhibiting compositions and/or one or multiple CD320 inhibiting compositions,
e.g., one or more
siRNAs targeting a CD320 gene and/or one or more siRNAs targeting an LRP2
gene. It is understood
that such compositions could be chemically modified in a variety of ways and
that such modifications
need not be identical in compositional mixtures. In some embodiments, the LRP2
inhibiting
composition and/or the CD320 inhibiting composition is a pharmaceutical
composition.
[00111] The pharmaceutical compositions of the present
invention may be administered in a
number of ways depending upon whether local or systemic treatment is desired
and upon the area to
be treated. Administration may be topical, pulmonary, e.g., by inhalation or
insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal, epidermal and
transdernnal, oral or
parenteral. Parenteral administration includes intravenous, intraarterial,
subcutaneous, intraperitoneal
or intramuscular injection or infusion; or intracranial, e.g.,
intraparenchymal, intrathecal or
intraventricular, administration.
CA 03174172 2022- 9- 28
[00112] The compositions can be delivered in a manner to
target a particular tissue, such as
the lung cells or breast cells or brain cells or bladder cells or uterine
cells or cervix cells or prostate
cells. Pharmaceutical compositions can be delivered by injection directly into
the brain. The injection
can be by stereotactic injection into a particular region of the brain (e.g.,
the substantia nigra, cortex,
hippocannpus, striatum, or globus pallidus), or the dsRNA can be delivered
into multiple regions of the
central nervous system (e.g., into multiple regions of the brain, and/or into
the spinal cord). The
dsRNA can also be delivered into diffuse regions of the brain (e.g., diffuse
delivery to the cortex of the
brain). In general siRNAs are administered 1) by intratunnoral injection, 2)
by systemic injection, 3) by
slow release from an implanted polymer. Other tissue specificity could be
achieved by antibody or
small molecule conjugation, or by a tissue-specific delivery device (e.g., a
catheter can be used to
deliver to the bladder).
[00113] In one embodiment, an RNAi targeting either LRP2 or
the CD320 can be delivered by
way of a cannula or other delivery device having one end implanted in a
tissue. The cannula can be
connected to a reservoir of the RNAi composition. The flow or delivery can be
mediated by a pump,
e.g., an osmotic pump or minipump. In one embodiment, a pump and reservoir are
implanted in an
area distant from the tissue, e.g., in the abdomen, and delivery is affected
by a conduit leading from
the pump or reservoir to the site of release.
[00114] Accordingly, in some embodiments, the pharmaceutical
compositions described
herein comprise one or more pharmaceutically acceptable excipients. The
pharmaceutical
compositions described herein are formulated for administration to a subject.
[00115] As used herein, a pharmaceutical composition or
medicament includes a
pharmacologically effective amount of at least one of the described RNAi
agents and one or more
pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients
(excipients) are
substances other than the Active Pharmaceutical Ingredient (API, therapeutic
product, e.g., CD320
RNAi agent or LRP2 RNAi agent) that are intentionally included in the drug
delivery system.
Excipients do not exert or are not intended to exert a therapeutic effect at
the intended dosage.
Excipients can act to a) aid in processing of the drug delivery system during
manufacture, b) protect,
support, or enhance stability, bioavailability or patient acceptability of the
API, c) assist in product
identification, and/or d) enhance any other attribute of the overall safety,
effectiveness, of delivery of
the API during storage or use. A pharmaceutically acceptable excipient may or
may not be an inert
substance.
[00116] Excipients include, but are not limited to:
absorption enhancers, anti-adherents, anti-
foaming agents, anti-oxidants, binders, buffering agents, carriers, coating
agents, colors, delivery
enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants,
emulsifiers, extenders, fillers,
flavors, glidants, hunnectants, lubricants, oils, polymers, preservatives,
saline, salts, solvents, sugars,
36
CA 03174172 2022- 9- 28
suspending agents, sustained release matrices, sweeteners, thickening agents,
tonicity agents,
vehicles, water-repelling agents, and wetting agents.
[00117] Pharmaceutical compositions suitable for injectable
use include sterile aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersions. For intravenous
administration, suitable
carriers indude physiological saline, bacteriostatic water, Cremophor®
ELTM (BASF, Parsippany,
N.J.) or phosphate buffered saline (PBS). The composition, understood to
include formulations and
drug delivery systems, should be stable under the conditions of manufacture
and storage and should
be preserved against the contaminating action of microorganisms such as
bacteria and fungi. The
carrier can be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol), and
suitable mixtures thereof.
The proper fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the
maintenance of the required particle size in the case of dispersion and by the
use of surfactants. In
many cases, it will be preferable to include isotonic agents, for example,
sugars, polyalcohols such as
mannitol, sorbitol, and sodium chloride in the composition. Prolonged
absorption of the injectable
compositions can be brought about by including in the composition an agent
which delays absorption,
for example, aluminum nnonostearate and gelatin.
[00118] Sterile injectable solutions can be prepared by
incorporating the active compound in
the required amount in an appropriate solvent with one or a combination of
ingredients enumerated
above, as required, followed by filter sterilization. Generally, dispersions
are prepared by
incorporating the active compound into a sterile vehicle, which contains a
basic dispersion medium
and the required other ingredients from those enumerated above. In the case of
sterile powders for
the preparation of sterile injectable solutions, methods of preparation
include vacuum drying and
freeze-drying which yields a powder of the active ingredient plus any
additional desired ingredient
from a previously sterile-filtered solution thereof.
[00119] Formulations suitable for intra-articular
administration can be in the form of a sterile
aqueous preparation of the drug that can be in nnicrocrystalline form, for
example, in the form of an
aqueous nnicrocrystalline suspension. Liposonnal formulations or biodegradable
polymer systems can
also be used to present the drug for both intra-articular and ophthalmic
administration.
[00120] The active compounds can be prepared with carriers
that will protect the compound
against rapid elimination from the body, such as a controlled release
formulation, including implants
and nnicroencapsulated delivery systems. Biodegradable, bioconnpatible
polymers can be used, such
as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic
acid. Methods for preparation of such formulations will be apparent to those
skilled in the art.
Liposonnal suspensions can also be used as pharmaceutically acceptable
carriers. These can be
37
CA 03174172 2022- 9- 28
prepared according to methods known to those skilled in the art, for example,
as described in U.S.
Pat. No. 4,522,811.
[00121] Dosage and Timing
The skilled artisan will appreciate that certain factors may influence the
dosage and timing required to
effectively treat a subject, including but not limited to the severity of the
disease or disorder, previous
treatments, the general health and/or age of the subject, and other diseases
present. Moreover,
treatment of a subject with a therapeutically effective amount of a
composition can include a single
treatment or a series of treatments. Estimates of effective dosages and in
vivo half-lives for the LRP2
inhibiting composition and or the CD320 -inhibiting compositions encompassed
by the invention can
be made using conventional methodologies or on the basis of in vivo testing
using an appropriate
animal model, as described elsewhere herein.
[00122] In general, a suitable dose of a pharmaceutical
composition of the LRP2 inhibiting
composition and/or the CD320 -inhibiting composition will be in the range of
0.01 to 300.0 milligrams
per kilogram body weight of the recipient per day, generally in the range of 1
to 50 mg per kilogram
body weight per day.
[00123] For example, the LRP2 inhibiting composition and/or
the CD320 -inhibiting
composition can be an siRNA composition of one or more siRNAs, and can be
administered at, 0.01
mg/kg, 0.05 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7
mg/kg, 0.8 mg/kg, 0.9
mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.628
mg/kg, 2 mg/kg, 3
mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 100 mg/kg,
200 mg/kg, 400
mg/kg per single dose. In another embodiment, the dosage is between 0.15 mg/kg
and 0.3 mg/kg.
For example, the LRP2 and/or the CD320 -inhibiting composition can be
administered at a dose of
0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, or 0.3 mg/kg. In an embodiment, the LRP2
and/or the CD320 -
inhibiting composition is administered at a dose of 0.3 mg/kg.
[00124] The pharmaceutical composition may be administered
once daily, or once or twice
every 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. The
dosage unit can be
compounded for delivery over several days, e.g., using a conventional
sustained release formulation
which provides sustained release of the LRP2 inhibiting composition and/or the
CD320 -inhibiting
composition over a several day period. Sustained release formulations are well
known in the art and
are particularly useful for delivery of agents at a particular site, such as
could be used with the agents
of the present invention.
[00125] In an embodiment, the LRP2 -inhibiting composition
and/or the CD320 -inhibiting
composition is dependent upon the tumor cell line, and the dosage is 0.3
mg/kg, and wherein the
dose is administered once every 21 days. In another embodiment, the effective
amount is 0.3 mg/kg
and the effective amount is administered once every 21 days via a 70 minute
infusion of 1 mUmin for
38
CA 03174172 2022- 9- 28
15 minutes followed by 3 nnUnnin for 55 minutes. In another embodiment, the
effective amount is 0.3
mg/kg and the effective amount is administered at two doses every 21-28 days
via a 60 minute
infusion of 3.3 mUmin, or via a 70 minute infusion of 1.1 mUmin for 15 minutes
followed by 3.3
mUmin for 55 minutes
[00126] A dosage of a LRP2 -inhibiting composition and/or the
CD320 -inhibiting composition
can be adjusted for treatment
[00127] A LRP2 -inhibiting composition and/or the CD320 -
inhibiting composition can be
administered in combination with other known agents effective in treatment of
pathological processes
mediated by target gene expression.
[00128] In another embodiment, the pharmaceutical composition
is formulated for
administration according to a dosage regimen described herein, e.g., not more
than once every four
weeks, not more than once every three weeks, not more than once every two
weeks, or not more than
once every week. In another embodiment, the administration of the
pharmaceutical composition can
be maintained for a month or longer, e.g., one, two, three, or six months, or
one year or longer.
[00129] In embodiments of the pharmaceutical compositions
described herein, the RNAi (e.g.,
dsRNA) is administered with a buffer solution. In embodiments, the buffer
solution comprises acetate,
citrate, prolamine, carbonate, or phosphate or any combination thereof. In
embodiments, the buffer
solution is phosphate buffered saline (PBS).
[00130] In embodiments of the pharmaceutical compositions
described herein, the
composition is administered intravenously.
[00131] In embodiments of the pharmaceutical compositions
described herein, the
composition is administered subcutaneously.
[00132] In certain embodiments, a pharmaceutical composition,
e.g., a composition described
herein, includes a lipid formulation. In embodiments, the composition is
administered intravenously.
[00133] In some embodiments, a pharmaceutical composition,
e.g., a composition described
herein, includes a cationic polyannine formulation or nanoparticle (e.g.,
JetPEI). In some
embodiments, the composition is administered intravenously.
[00134] In another embodiment, the pharmaceutical composition
is formulated for
administration according to a dosage regimen described herein, e.g., not more
than once every four
weeks, not more than once every three weeks, not more than once every two
weeks, or not more than
39
CA 03174172 2022- 9- 28
once every week. In another embodiment, the administration of the
pharmaceutical composition can
be maintained for a month or longer, e.g., one, two, three, or six months, or
one year or longer.
[00135] In another embodiment, a composition containing an
RNAi agent featured in the
invention, e.g., a dsRNA targeting LRP2 or CD320, is administered with a non-
RNAi therapeutic
agent, such as an agent known to treat a cancer such as lung cancer. In
another embodiment, a
composition containing an RNAi agent featured in the invention, e.g., a dsRNA
targeting LRP2 and/or
CD320, is administered along with a non-RNAi therapeutic regimen, such as
radiation, chemotherapy,
imnnunotherapy, photodynannic therapy or a combination thereof.
[00136] In an aspect provided herein is a method of
inhibiting LRP2 and/or CD320 expression
in a cell, the method comprising: (a) introducing into the cell an RNAi agent
(e.g., a dsRNA) described
herein and (b) maintaining the cell of step (a) for a time sufficient to
obtain degradation of the nnRNA
transcript of an LRP2 gene and/or CD320 gene, thereby inhibiting expression of
the LRP2 gene
and/or CD320 gene in the cell.
[00137] In an aspect provided herein is a method for reducing
or inhibiting the expression of
LRP2 gene and/or CD320 genes in a cell. The method includes: (a) introducing
into the cell one or
more complimentary double-stranded ribonudeic acid (dsRNA) molecules, in which
one sequence is
designated the sense strand and the other sequence the anti-sense strand, and
wherein the anti-
sense strand has significant connplennentarity to a portion of mRNA encoding
for LRP2 or CD320. The
complimentary region is 15-30 nucleotides in length, and generally 19-24
nucleotides in length, and
the dsRNA, upon entering a cell expressing LRP2 and/or CD320, inhibits the
expression of the LRP2
protein and/or CD320 protein by at least 10%, e.g., at least 20%, at least
30%, at least 40% or more;
(b) single or repeated treatment of the cell with dsRNAs, as described in part
(a), so as to maintain the
inhibition of LRP2 and/or CD320 protein expression over a desired period of
time by at least 10%,
e.g., at least 20%, at least 30%, at least 40% or more.
[00138] In embodiments of the foregoing methods of inhibiting
LRP2 and/or CD320
expression in a cell, the cell is treated ex vivo, in vitro, or in vivo. In
embodiments, the cell is a
melanoma, glioblastonna, lung carcinoma, triple negative breast carcinoma,
renal carcinoma,
pancreatic carcinoma, hepatocellular carcinoma, ovarian carcinoma and prostate
carcinoma.
[00139] In some embodiments, the cell is present in a subject
in need of treatment, prevention
and/or management of a CD320-associated disease or a LRP2-associated disease.
[00140] In embodiments, the expression of LRP2 and/or CD320
is inhibited by at least 30%.
[00141] In embodiments, the RNAi (e.g., dsRNA) has an IC50 in
the range of 0.01-50 nM.
CA 03174172 2022- 9- 28
[00142] In embodiments, the RNAi (e.g., dsRNA) has an IC50 in
the range of 0.01-1 nM.
[00143] In certain embodiments, the cell is a mammalian cell
(e.g., a human, non-human
primate, or rodent cell).
[00144] In one embodiment, the cell is treated ex vivo, in
vitro, or in vivo (e.g., the cell is
present in a subject (e.g., a patient in need of treatment, prevention and/or
management of a disorder
related to LRP2 and/or CD320 expression).
[00145] In one embodiment, the subject is a mammal (e.g., a
human) at risk, or diagnosed
with a proliferation disorder.
[00146] In embodiments, the RNAi (e.g., dsRNA) is formulated
as an lipid nanoparticle (LNP)
polyplex (polyamine) formulation.
[00147] In embodiments, RNAi (e.g., dsRNA) is administered at
a dose of 0.05001-500.01
mg/kg.
[00148] In embodiments, the RNAi (e.g., dsRNA) is
administered at a concentration of 0.01
mg/kg-50.1 ring/kg bodyweight of the subject.
[00149] In embodiments, the RNAi (e.g., dsRNA) is formulated
as an LNP formulation and is
administered at a dose of 0.050.1-50.5 ring/kg.
[00150] In embodiments, the RNAi (e.g., dsRNA) has an IC50 in
the range of 0.01-10 nM.
[00151] In embodiments, the RNAi (e.g., dsRNA) or composition
comprising the RNAi is
administered according to a dosing regimen. In embodiments, the RNAi (e.g.,
dsRNA) or composition
comprising the RNAi is administered as a single dose or at multiple doses,
e.g., according to a dosing
regimen.
[00152] The term "sample," as used herein, includes a
collection of fluids, cells, or tissues
isolated from a subject, as well as fluids, cells, or tissues present within a
subject. Examples of
biological fluids include blood, serum and serosal fluids, plasma,
cerebrospinal fluid, ocular fluids,
lymph, urine, saliva, and the like. Tissue samples may include samples from
tissues, organs or
localized regions. For example, samples may be derived from particular organs,
parts of organs, or
fluids or cells within those organs. In certain embodiments, samples may be
derived from a tumor. In
preferred embodiments, a "sample derived from a subject" refers to blood or
plasma drawn from the
subject. In further embodiments, a "sample derived from a subject" refers to
tissue biopsy derived
from the subject.
41
CA 03174172 2022- 9- 28
[00153] In one embodiment, an RNAi (e.g., a dsRNA) featured
herein includes a first
sequence of a dsRNA that is selected from the group consisting of the sense
sequences of Table 1
and a second sequence that is selected from the group consisting of the
corresponding antisense
sequences of Table 1. It is understood that the suffix A (e.g., OSC17A)
represents the antisense
strand whereas the suffix S (e.g., OSC175) represents the sense strand. In
those instances when we
refer to an siRNA with no suffix (e.g., OSC17), we mean that to indicate the
dsRNA comprised of the
antisense and sense strands corresponding to that number (e.g., OSC17A paired
with OSC175).
[00154] In some embodiments the RNAi is from about 15 to
about 25 nucleotides in length,
and in other embodiments the RNAi is from about 25 to about 30 nucleotides in
length. An RNAi
targeting CD320, upon contact with a cell expressing CD320, inhibits the
expression of a CD320 gene
by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at
least 40% or more, such
as when assayed by a method as described herein. In one embodiment, the RNAi
targeting CD320 is
formulated in a stable nucleic acid lipid particle (SNALP).
[00155] In some embodiments the RNAi is from about 15 to
about 25 nucleotides in length,
and in other embodiments the RNAi is from about 25 to about 30 nucleotides in
length. An RNAi
targeting LRP2, upon contact with a cell expressing LRP2, inhibits the
expression of a LRP2 gene by
at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at
least 40% or more, such as
when assayed by a method as described herein. In one embodiment, the RNAi
targeting LRP2 is
formulated in a stable nucleic acid lipid particle (SNALP).
[00156] In some embodiments the RNAi is from about 15 to
about 25 nucleotides in length,
and in other embodiments the RNAi is from about 25 to about 30 nucleotides in
length. An RNAi
targeting CD320, upon contact with a cell expressing CD320, inhibits the
expression of a CD320 gene
by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at
least 40% or more, such
as when assayed by a method as described herein. In one embodiment, the RNAi
targeting CD320 is
formulated as a complex, which may exist as a nanoparticle, with a cationic
polyannine.
[00157] In some embodiments the RNAi is from about 15 to
about 25 nucleotides in length,
and in other embodiments the RNAi is from about 25 to about 30 nucleotides in
length. An RNAi
targeting LRP2, upon contact with a cell expressing LRP2, inhibits the
expression of a LRP2 gene by
at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at
least 40% or more, such as
when assayed by a method as described herein. In one embodiment, the RNAi
targeting LRP2 is
formulated as a complex, which may exist as a nanoparticle, with a cationic
polyannine.
[00158] Referring now to Table 1 ¨ DNA sequences are
illustrated, which are subsequently
transcribed into shRNA, which hence targets the CD320 or LRP2 mRNA for
destruction in the cell.
shRNA sequences used in lentiviral vectors illustrates the sequences that were
used to target the
CD320 sequence coding for the CD320 protein and the LRP2 sequence coding for
the LRP2 protein.
42
CA 03174172 2022- 9- 28
The Each vector that carried a shRNA coding sequence also contained a unique
drug resistance gene
which would allow for selecting those cells that had taken up the shRNA as
those cells that had not
taken up the shRNA having the unique drug resistance gene would not survive.
On day 2, drug
selection was started. On day 3, the cells were harvested and plated in a new
dish. Only the cells
with a drug resistance gene, i.e., those cells that had taken up shRNA virus
particles would survive
this re-plating procedure. From day 4 on, each culture was closely observed
for cell growth. The
cells that were infected with the irrelevant control shRNA kept on growing as
expected (since the
shRNA was essentially a non-functional shRNA) ¨ data not shown. The results
for the cell lines that
took up the CD320+LRP2 shRNAs are shown in Table 1.
Table 1
Name Target Sense Sequence Anti-Sense Sequence
Locatio
n in
DNA
shScramble non-targeting control CCTAAGGTTAAGTCGCCCTC
CGAGGGCGACTTAACCTTAG N/A
G (SEQ ID NO. 941) G (SEQ ID NO. 942)
shCD320-#27 C0320 CCCTCAGAGACCTGAGCTCT
AAGAGCTCAGGTCTCTGAGG 1006-
(NM_016579.3) T G (SEQ ID NO. 944)
1026
(SEQ ID NO. 943)
shLRP2-#89 LRP2 (NM_004525.2) CCTGTAATAAACACTACTCTT
AAGAGTAGTGTTTATTACAG 2800-
G (SEQ ID NO. 946)
2820
(SEQ ID NO. 945)
The preliminary studies show that cancer cells are selectively killed by CD320
and LRP2 knockdown,
while normal cells remain unaffected (Table 2).
[00159] Table 2 shows the effect of simultaneous knockdown of
CD320 and LRP2 on cell
viability.
Table 2
Outcome of CD320/LRP2
knockdown
Cell arrest/death Alive
Cancer (Lung) HCC15 -\I
H157 -\I
H358 -\I
H1999 -\I
Non-cancer Normal fibroblast -\I
43
CA 03174172 2022- 9- 28
LDLR mutant fibroblast
[00160] Additional cancer cell lines were also treated with
the compounds described herein to
determine whether cancer cell lines were more susceptible to growth inhibition
and toxicity as
compared to non-cancer cells of the same origin. Cell lines from skin,
prostate, and brain cancers
were screened similarly to the experimental outline in FIG. 1. Table 3
summarizes the effects of
simultaneous knockdown of CD320 and LRP2 in cancer and normal cells.
Table 3
Cell shSCR shCD320 shLRP2 shCD320+shLRP2 Comments
Normal cells
6M05659 +++ +++ +++ +++ no effect of knockdown
6M00701 +++ +++ +++ cells grow very slow;
hard to determine if any affect of knockdown
SAEC pending
Lung cells
HCC15 +++ cells strongly affected by
knockdown
H157 +++ ++ senescent phenotype
H358 +++ ++ ++ morphology changes; cells
rounded
H1993 +++ ++ cells rounded; morphology
change
Melanoma Cells
morphology change; cells strongly affected b double
MDA-MB-4353 +++ knockdown
Prostate cells
LncAP +++ ++ cells rounded; morphology
change
PC3 +++ +++ ++ +++ cells minimally to not
affected by knockdown
DU-145 +++ ++ cells modestly affected by
knockdown
Glioblastoma
A172 +++ 0 cells strongly affected by
knockdown
U251MG +++ ++ 0 cells strongly affected by
knockdown
U343 +++ +++ ++ +++ cells modestly affected
by LRP2 knockdown
T98G +++ +++ ++ ++ cells slightly affected by
knockdown
F++ cells unaffected compared to shSCR (control)
F+ cells modestly affected compared to shSCR (control)
+ cells significantly affected compared to shSCR (control)
0 vast majority of cells killed compared to shSCR (control)
[00161] The screening results showed that lung, prostate,
skin, and brain cancer cell lines
were growth-inhibited or killed by the simultaneous knockdown ("double
knockdown") of CD320 and
LRP2, while non-cancerous cells were unaffected.
[00162] Referring now to FIG. 2, representative pictures of
the cells were taken to record their
phenotypes after the double knockdown of CD320 and LRP2 and to illustrate the
sensitivity of cancer
cell lines to knockdown of the expression of CD320 and LRP2 proteins.
[00163] Normal cells (GM05659 fibroblasts) or cancer cells
were infected with lentiviruses
expressing shRNAs to control sequences or to shCD320 and shLRP2 as described
in FIG. 1. The
cells were grown as described in FIG. 1. On the ninth day after transfection
with the lentiviruses,
pictures of the cells were taken. The solid line ovals indicate healthy growth
of normal fibroblast
44
CA 03174172 2022- 9- 28
infected with shRNAs to CD320 and LRP2. The broken line ovals indicate
unhealthy dying cells of
cancer cell lines infected with shRNAs to CD320 and LRP2.
[00164] These results support use of the compounds as
therapeutics based upon decreasing
expression of CD320 and LRP2 protein preferentially resulting in detrimental
effects in cancer cells as
compared to non-cancer cells. The original experiments were conducted using
shRNAs delivered by
lentiviral vectors. Short inhibitory RNAs (siRNAs), having a sequence
complimentary to a portion of
the CD320 protein and/or the LRP2 protein were designed. The siRNAs can be
chemically modified
to increase their stability and potency and reduce their innnnunogenicity, and
multiple platforms exist
for their delivery in clinical applications.
[00165] siRNA sequences that efficiently knock down the
protein levels of LRP2 and/or
CD320 were designed and identified. Table 4 is a list of siRNA sequences
complementary to nnRNA
for CD320 or LRP2 that were tested for their ability to knock down CD320 or
LRP2 protein,
respectively (see FIG. 3, FIG. 4, FIG. 5, FIG. 12, and FIG. 15).
Table 4
ID Passenger Sequence Target Size Nucleotide
Location
start site
OSS1 CCUAAGGUUAAGUCGCCCUCG (SRI none 21 N/A N/A
ID NO. 947)
OSS2 UGGUUUACAUGUUGUGUGA (SEQ ID none 19 N/A N/A
NO. 948)
0SL231 GGGCUCUAGGUUUGGUGCUAU LRP2 25 12537 CDS
CAAA (SKI ID NO. 949) NM_004525.2
0SL245 GGACUGAUAGGAGAGU LRP2 25 12995 CDS
CAUUGCAAA (SRI ID NO. 950) NM_004525.2
0SL47 CCUGUAAUAAACACUACUCUU (SEQ LRP2 21 2800 CDS
ID NO. 951) NM_004525.2
0SL104 CCUUCUAUGAACCUGGCCUUA (SKI LRP2 21 5677 CDS
ID NO. 952)
NM_004525.2
0SL90 GUGAUUUGAUUAUACGGCA (SEQ ID LRP2 19 5126 CDS
NO. 953) NM_004525.2
OSL119 CCUCAAAUGGCUGUAGCAA (SEQ ID LRP2 19 6266 CDS
NO. 954) NM_004525.2
OSC17 GAACUGACAAGAAACUGCGCAACUG CD320 25 422 CDS
NM_016579.3
(SKI ID NO. 955)
0SC47 CCCUCAGAGACCUGAGCUCUU (SRI CD320 21 1006 3'-UTR
ID NO. 956) NM_016579.3
[00166] The list of all potential siRNA sequences is quite
large. We have identified 340
potential siRNA sequences to LRP2 and 59 potential siRNA sequences to CD320.
(See Table 5 and
CA 03174172 2022- 9- 28
Table 6 for the complete list and Table 5A and Table 6A identify the target
position and sequence
that is complementary for each antisense sequence identified). In addition,
chemical modifications
can be made to these siRNA sequences to improve their stability and reduce
their off-target effects.
siRNA molecules are vulnerable to metabolic degradation, for example by RNase
or DNase enzymes.
Chemical modification of siRNA molecules by incorporation of one or more
unnatural, that is,
manmade, nucleotides within the sequence can render siRNAs resistant to such
metabolic
degradation and increase their biological half-life in the cell or in plasma.
Moreover, the indusion of
manmade nucleotides at strategic locations within the siRNA sequence can
decrease the
immunogenicity of the siRNA and improve the selectivity for the guide strand
over the passenger
strand. Modified siRNA molecules may incorporate manmade nucleotides of a
single type or may
include multiple manmade nucleotides of different types. Manmade nucleotides
may include, but are
not limited to, those which contain chemical modifications to the ribose
moiety or to the phosphate
moieties (FIG. 19 and Table 7). Examples of manmade nucleotides indude, but
are not limited to, the
structures shown in the Table 7. Moreover, modification of multiple structural
elements may be
combined. In addition, modification may be made to the nucleobase B, which, in
addition to the
natural RNA nucleobases (G, C, A, U), may include unnatural bases, such as
those containing a
sulfur atom (e.g., thiouracil).
Table 7. Nucleotide modifications corresponding to FIG. 19A
Designationa Y XZ R R' B
(nucleobase)
[2fN] 0 0 0 F H G, C, A, U,
other
2'-FANA 0 0 0 H F G, C, A, U,
other
[mN] 0 0 0 OMe H G, C, A, U,
other
2'-MOE 0 0 0 CH2CH20Me H G, C, A, U,
other
2'-EA 0 0 0 CH2CH2NH2 H G, C, A, U,
other
2'-DMEA 0 0 0 CH2CH2NMe2 H G, C, A, U,
other
2'-DMAP 0 0 0 CH2CH2CH2NMe2 H G, C, A, U,
other
* as in N1*N2 0 S 0 OH H G, C, A, U,
other
** as in N1**N2 S S 0 OH H G, C, A, U,
other
2'-deoxy 0 0 0 H H G, C, A, U,
other
4'-S 0 0 S H H G, C, A, U,
other
F-SRNA 0 0 S F H G, C, A, U,
other
Me-SRNA 0 0 S OMe H G, C, A, U,
other
4'-S-FANA 0 0 S H F G, C, A, U,
other
a N designates an arbitrary ribonucleotide or deoxyribonucleotide or analogs
thereof.
In some embodiments, chemical modification is made to the phosphodiester group
which covalently
connects two nucleotides, such that, for example, one or two oxygen atoms in
that group are
substituted with sulfur atoms, as indicated by a single or double asterisk
between two nucleotides to
represent the replacement of one or two oxygen atoms with sulfur in the
phosphodiester (Table 7 and
FIG. 19A). In some embodiments, the siRNA sequences may include other manmade
nucleotides
wherein further structural modifications have been made to the ribose moiety,
such as the addition of
bridging atoms that covalently link carbons 2' and 5' of the ribose moiety
(FIG. 19B-C) or positions 1'
and 2' of the ribose moiety (FIG. 19D), or alternatively, changes to the size
of the sugar ring in a given
46
CA 03174172 2022- 9- 28
nucleotide, for example, deletion of the bond between carbons 2' and 3' of the
ribose moiety (FIG.
19E), or increasing the size of the sugar ring from five to six atoms (FIG.
19F-G).
TABLE 5 CD320
OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5'
TO 3')
OSC4A AUCCAACCGCCGCUCAUGCUG [dT][dT] (SEQ 05C45
CAGCAUGAGCGGCGGUUGGAU [dT] [dT]
ID NO 4) (SEQ ID NO: 97)
OSC8A ACUGGAACUUGGUGGG UGG GC [dT] [dT] (SEQ 05C85
GCCCACCCACCAAGUUCCAGU[dT][dT]
ID NO 8) (SEQ ID NO: 101)
OSC12A UCCUCAUCGCUGCCAUCGCUG [dT] [dT] (SEQ 05C125 CAGCGAUGGCAGCGAUGAGGA[dT]
[dT]
ID NO 12) (SEQ ID NO: 105)
OSC16A AGUUUCUUGUCAGUUCCCCCA[dT] [dT] (SEQ 05C165 UGGGGGAACUGACAAGAAACU[dT]
[dT]
ID NO 16) (SEQ ID NO: 109)
OSC31A UUCCAGACUGGUCUCCGGCAG [dT] [dT] (SEQ 05C315
CUGCCGGAGACCAGUCUGGAA[dT][dT]
ID NO 48) (SEQ ID NO: 141)
05C35A ACAAAAGGAGGAGGGUGGCGG[dT] [dT] (SEQ 05C355 CCGCCACCCUCCUCCUUUUGU [dT]
[dT]
ID NO 52) (SEQ ID NO: 145)
OSC40A UCUGUUCUGACAGCAGCAGGG[dT][dT] (SEQ 05C405 GGCUGCUGCUGUCAGAACAGA[dT][dT]
ID NO 57) (SEQ ID NO: 150)
05C46A UCUCUGAGGGCUGGUGUGCCC[dT][dT] (SEQ 05C465 GGGCACACCAGCCCUCAGAGA[dT][dT]
ID NO 63) (SEQ ID NO: 156)
05C57A AGUGUCUUAAGCACAGGGCCG [dT] [dT] (SEQ 05C575
CGGCCCUGUGCUUAAGACACU[dT][dT]
ID NO: 91) (SEQ ID NO: 184)
05C59A UUUUUUUGAGGAUGUGAAGCA[dT] [dT] 05C595
UGCUUCACAUCCUCAAAAAAA[dT][dT]
(SEQ ID NO: 93) (SEQ ID NO: 186)
OSC10A ACGCAUAAGCCACUGGUGCGG [dT] [dT] (SEQ OSC10S CCGCACCAGUGGCUUAUGCG
U[dT][dT]
ID NO 10) (SEQ ID NO: 103)
OSC11A UCCAAGUCCCUGUCGCAGCGC[dT][dT] (SEQ OSC11S GCGCUGCGACAGGGACUUGGA[dT][dT]
ID NO 11) (SEQ ID NO: 104)
OSC13A UCACUGACGCCGGUGCAGGGG [dT] [dT] (SEQ 05C135 CCCCUGCACCGGCGUCAGUGA[dT]
[dT]
ID NO 13) (SEQ ID NO: 106)
OSC14A UUGUCAGUUCCCCCAGAGCAG [dT] [dT] (SEQ 05C145
CUGCUCUGGGGGAACUGACAA[dT][dT]
ID NO 14) (SEQ ID NO: 107)
OSC15A UUCUUGUCAGUUCCCCCAGAG [dT] [dT] (SEQ 05C155 CUCUGGGGGAACUGACAAGAA[dT]
[dT]
ID NO 15) (SEQ ID NO: 108)
OSC17A- CAGUUGCGCAGUUUCUUGUCAGUUC[dT] [dT 05C175 GAACUGACAAGAAACUGCGCAACUG
1 ] (SEQ ID NO 17) -1 [dT][dT] (SEQ ID NO:
110)
47
CA 03174172 2022- 9- 28
OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5'
TO 3')
OSC17A- CAGUUGCGCAGUUUCUUGUCAGUUC[dT]*[d 05C175
2 T] (SEQ ID NO 18) -2
GAACUGACAAGAAACUGCGCAACUG[dT]*
[dT] (SEQ ID NO: 111)
OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] 05C175
[mG][mA][mA][mC][mU][mG][mA][mC][
3 [mA][mG][mU][mU][mU][mC][mU][mU][mG -3 ..
mA][mA][mG][mA][mA][mA][mC][mU][m
][mU][mC][mA][mG][mU][mU][mC][dT]*[dT]
G][mC][mG][mC][mA][mA][mC][mU][mG]
] (SEQ ID NO 19) [dT]*[dT] (SEQ ID
NO: 112)
OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] 05C175
4 [mA][mG][mU][mU][mU][mC][mU][mU][mG -4
[mG][mA][mA][mC][mU][mG][mA][mC][
][mU][mC][mA][mG][mU][mU][mC][dT]*[dT]
mA][mA][mG][mA][mA][mA][mC][mU][m
] (SEQ ID NO 20)
G][mC][mG][mC][mA][mA][mC][mU][mG]
(SEQ ID NO: 113)
OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] 05C175
[mG][mA][mA][mC][mU][mG][mA][mC][
[mA][mG][mU][mU][mU][mC][mU][mU][mG -5 mA][mA][mG][mA][mA][mA][mC][mU][m
][mU][mC][mA][mG][mU][mU][mC] ] (SEQ ID
G][mC][mG][mC][mA][mA][mC][mU][mG]
NO 21) [dT]*[dT] (SEQ ID
NO: 114)
OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] 05C175
[mG][mA][mA][mC][mU][mG][mA][mC][
6 [mA][mG][mU][mU][mU][mC][mU][mU][mG -6
mA][mA][mG][mA][mA][mA][mC][mU][m
][mU][mC][mA][mG][mU][mU][mC] ] (SEQ ID
G][mC][mG][mC][mA][mA][mC][mU][mG]
NO 22) (SEQ ID NO: 115)
OSC17A- [mC][mA][mG][mU][mU][mG][mC][mG][mC] 05C175
7 [mA][mG][mU][mU][mU][mC][mU][mU][mG -7
GAACUGACAAGAAACUGCGCAACUG[dT]*
][mU][mC][mA][mG][mU][mU][mC][dT]*[dT] [dT] (SEQ ID NO:
116)
] (SEQ ID NO 23)
OSC17A- [mC][2fA][mG][2fU][mU][2fG][mC][2fG][mC] 05C175
[2fG][mA][2fA][mC][2fU][mG][2fA][mC][2
8 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -8
fA][mA][2fG][mA][2fA][mA][2fC][mU][2f
][mU][2fC][mA][2fG][mU][2fU][mC][dT]*[dT]
G][mC][2fG][mC][2fA][mA][2fC][mU][2fG
(SEQ ID NO 24) ][dT]*[dT] (SEQ ID
NO: 117)
OSC17A- [mC][2fA][mG][2fU][mU][2fG][mC][2fG][mC] 05C175
[2fG][mA][2fA][mC][2fU][mG][2fA][mC][2
9 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -9
fA][mA][2fG][mA][2fA][mA][2fC][mU][2f
][mU][2fC][mA][2fG][mU][2fU][mC] (SEQ ID
G][mC][2fG][mC][2fA][mA][2fC][mU][2fG
NO 25) ][dT]*[dT] (SEQ ID
NO: 118)
OSC17A- [mC][2fA][mG][2fU][mU][2fG][mC][2fG][mC] 05C175
[2fG][mA][2fA][mC][2fU][mG][2fA][mC][2
[2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -10
fA][mA][2fG][mA][2fA][mA][2fC][mU][2f
][mU][2fC][mA][2fG][mU][2fU][mC][dT]*[dT]
G][mC][2fG][mC][2fA][mA][2fC][mU][2fG
(SEQ ID NO 26) ] (SEQ ID NO: 119)
OSC17A- [mC][2fA][mG][2fU][mU][2fG][mC][2fG][mC] 05C175
11 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -11
[2fG][mA][2fA][mC][2fU][mG][2fA][mC][2
][mU][2fC][mA][2fG][mU][2fU][mC] (SEQ ID
fA][mA][2fG][mA][2fA][mA][2fC][mU][2f
NO 27)
G][mC][2fG][mC][2fA][mA][2fC][mU][2fG
] (SEQ ID NO: 120)
OSC17A- [2fC][mA][2fG][mU][2fU][mG][2fC][mG][2fC] 05C175
12 [mA][2fG][mU][2fU][mU][2fC][mU][2fU][mG -12
[mG][2fA][mA][2fC][mU][2fG][mA][2fC][
48
CA 03174172 2022- 9- 28
OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5'
TO 3')
][2fU][mC][2fA][mG][2fU][mU][2fC][dT]*[dT]
mA][2fA][mG][2fA][mA][2fA][mC][2fU][m
(SEQ ID NO 28)
G][2fC][mG][2fC][mA][2fA][mC][2fU][mG
][dT]*[dT] (SEQ ID NO: 121)
OSC17A- [2fC][mA][2fG][mU][2fU][mG][2fC][mG][2fC] 05C175
[mG][2fA][mA][2fC][mU][2fG][mA][2fC][
13 [mA][2fG][mU][2fU][mU][2fC][mU][2fU][mG -13
mA][2fA][mG][2fA][mA][2fA][mC][
][2fU][mC][2fA][mG][2fU][mU][2fC] (SEQ ID
2fU][mG][2fC][mG][2fC][mA][2fA][mC][2f
NO 29) U][mG] (SEQ ID NO:
122)
OSC17A- [mC][2fA][2fG][2fU][2fU][2fG][2fC][2fG][2fC] 05C175
[2fU][mG][2fA][mC][2fA][mA][2fG][mA][2
14 [2fA][2fG][2fU][2fU][2fU][2fC][2fU][2fU][2fG -14
fA][mA][2fC][mU][2fG][mC][2fG][mC][2fA
][2fU][2fC][2fA][2fG][2fU][2fU][2fC][dT]*[dT]
][mA][2fC][mU][2fG][dT]*[dT] (SEQ ID
(SEQ ID NO 30) NO: 123)
OSC17A- [mC][2fA][2fG][2fU][2fU][2fG][2fC][2fG][2fC] 05C175
15 [2fA][2fG][2fU][2fU][2fU][2fC][2fU][2fU][2fG -15
[2fG][mA][2fA][mA][2fC][mU][2fG][mC][2
][2fU][2fC][2fA][2fG][2fU][2fU][2fC][dT]*[dT]
fG][mC][2fA][mA][2fC][mU][2fG][dT]*[dT
(SEQ ID NO 31) ] (SEQ ID NO: 124)
OSC17A- [mC][2fA][mG][2fU][mU][2fG][mC][2fG][mC] 05C175
16 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -16
[2fG][mA][2fA][mA][2fC][mU][2fG][mC][2
][mU][2fC][mA][2fG][mU][2fU][mC][dT]*[dT]
fG][mC][2fA][mA][2fC][mU][2fG][dT]*[dT
(SEQ ID NO 32) ] (SEQ ID NO: 125)
OSC17A- [mC][2fA][mG][2fU][mU][2fG][mC][2fG][mC] 05C175
[2fG][mA][2fA][mA][2fC][mU][2fG][mC][2
17 [2fA][mG][2fU][mU][2fU][mC][2fU][mU][2fG -17
fG][mC][2fA][mA][2fC][mU][2fG] (SEQ ID
][mU][2fC][mA][2fG][mU] (SEQ ID NO 33) NO: 126)
OSC17A- [mC][2fA][mG][2fU][mU][2fG][mC][2fG][mC] 05C175
[2fG][mA][2fA][mA][2fC][mU][2fG][mC][2
18 [2fA][mG][2fU][mU][2fU][mC][2fU][2fU][2fG -18
fG][mC][2fA][mA][2fC][mU][2fG] (SEQ ID
][2fU][2fC][2fA][2fG][2fU] (SEQ ID NO 34) NO: 127)
OSC18A AUGCAGUCAUCGCUCAGCGUG[dT][dT] (SEQ 05C185 CACGCUGAGCGAUGACUGCAU[dT][dT]
ID NO 35) (SEQ ID NO: 128)
OSC19A AAUGCAGUCAUCGCUCAGCGU[dT][dT] (SEQ 05C195 ACGCUGAGCGAUGACUGCAUU[dT][dT]
ID NO 36) (SEQ ID NO: 129)
OSC20A UGGAAUGCAGUCAUCGCUCAG[dT][dT] (SEQ 05C205 CUGAGCGAUGACUGCAUUCCA[dT][dT]
ID NO 37) (SEQ ID NO: 130)
OSC21A AGUGGAAUGCAGUCAUCGCUC[dT][dT] (SEQ 05C215 GAGCGAUGACUGCAUUCCACU[dT][dT]
ID NO 38) (SEQ ID NO: 131)
05C22A ACAGUCUGGGUGGCCGUCGCA[dT][dT] (SEQ 05C225 UGCGACGGCCACCCAGACUGU[dT][dT]
ID NO 39) (SEQ ID NO: 132)
05C23A AUUGGUUCCACAGCCGAGCUC[dT][dT] (SEQ 05C235 GAGCUCGGCUGUGGAACCAAU[dT][dT]
ID NO 40) (SEQ ID NO: 133)
05C24A UCUCAUUGGUUCCACAGCCGA[dT][dT] (SEQ 05C245 UCGGCUGUGGAACCAAUGAGA[dT][dT]
ID NO 41) (SEQ ID NO: 134)
49
CA 03174172 2022- 9- 28
OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5'
TO 3')
05C25A AUCUCAUUGGUUCCACAGCCG [dT] [dT] (SEQ 05C255 CGGCUGUGGAACCAAUGAGAU[dT]
[dT]
ID NO 42) (SEQ ID NO: 135)
05C26A AGGAUCUCAU UGGU UCCACAG [dT] [dT] (SEQ 05C265 CUGUGGAACCAAUGAGAUCCU
[dT] [dT]
ID NO 43) (SEQ ID NO: 136)
05C27A UGAGAGAGGUGACACUCUCCA[dT] [dT] (SEQ 05C275 UGGAGAG
UGUCACCUCUCUCA[dT][dT]
ID NO 44) (SEQ ID NO: 137)
05C28A UGGUUGUGGCAUUCCUGAGAG [dT] [dT] 05C285
CUCUCAGGAAUGCCACAACCA[dT][dT]
(SEQ ID NO 45) (SEQ ID NO: 138)
05C29A UGGCAU UCCCGACAGAGGGGA[dT][dT] (SEQ 05C295
UCCCCUCUGUCGGGAAUGCCA[dT][dT]
ID NO 46) (SEQ ID NO: 139)
OSC30A AGGAUGUGGCAU UCCCGACAG [dT] [dT] (SEQ 05C305
CUGUCGGGAAUGCCACAUCCU[dT][dT]
ID NO 47) (SEQ ID NO: 140)
05C32A AUAACCCCAUAGGCAG UUGGG [dT] [dT] (SEQ 05C325 CCCAACUGCCUAUGGGGUUAU[dT]
[dT]
ID NO 49) (SEQ ID NO: 142)
05C33A UUGCACUGAGCACCGCAGCAG [dT] [dT] (SEQ 05C335 CUGCUGCGGUGCUCAGUGCAA[dT]
[dT]
ID NO 50) (SEQ ID NO: 143)
05C34A AAAAGGAGGAGGGUGGCGGUG [dT] [dT] (SEQ 05C345 CACCGCCACCCUCCUCCU UUU
[dT][dT]
ID NO 51) (SEQ ID NO: 144)
05C36A ACCAGUAACCCCAGUGGGCGG [dT] [dT] (SEQ 05C365 CCGCCCACUGGGG UUACUGG U[dT]
[dT]
ID NO 53) (SEQ ID NO: 146)
05C37A UUCAUGGCCACCAGUAACCCC[dT][dT] (SEQ 05C375 GGGG
UUACUGGUGGCCAUGAA[dT][dT]
ID NO 54) (SEQ ID NO: 147)
05C38A ACUCCUUCAUGGCCACCAGUA[dT] [dT] (SEQ 05C385 UACUGGUGGCCAUGAAGGAGU[dT]
[dT]
ID NO 55) (SEQ ID NO: 148)
05C39A UUCUGACAGCAGCAGGGACUC[dT][dT] (SEQ 05C395 GAGUCCCUGCUGCUG UCAGAA[dT]
[dT]
ID NO 56) (SEQ ID NO: 149)
OSC41A UCU UCUGUUCUGACAGCAGCA[dT][dT] (SEQ 05C415 UGCUGCUGUCAGAACAGAAGA[dT]
[dT]
ID NO 58) (SEQ ID NO: 151)
05C42A AGG UCU UCUGU UCUGACAGCA[dT][dT] (SEQ 05C425
UGCUGUCAGAACAGAAGACCU[dT][dT]
ID NO 59) (SEQ ID NO: 152)
05C43A UUGUCCUCAGGGCAGCGAGG U[dT][dT] (SEQ 05C435 ACCUCGCUGCCCUGAGGACAA[dT]
[dT]
ID NO 60) (SEQ ID NO: 153)
05C44A AAGUGCU UG U CCU CAGGGCAG [dT][dT] (SEQ 05C445 CU GCCCU GAGGACAAGCACU U
[dT] [dT]
ID NO 61) (SEQ ID NO: 154)
CA 03174172 2022- 9- 28
OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5'
TO 3')
05C45A UACCCAUCCGCAUCACUGCUC[dT][dT] (SEQ 05C455
GAGCAGUGAUGCGGAUGGGUA[dT][dT]
ID NO 62) (SEQ ID NO: 155)
05C47A- AAGAGCUCAGGUCUCUGAGGG[dT][dT] (SEQ 05C475 CCCUCAGAGACCUGAGCUCUU
[dT][dT]
1 ID NO 64) -1 (SEQ ID NO: 157)
05C47A- AAGAGCUCAGGUCUCUGAGGG [dT]*[dT] 05C475
CCCUCAGAGACCUGAGCUCUU[dT]*[dT]
2 (SEQ ID NO 65) -2 (SEQ ID NO: 158)
05C47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] 05C475
3 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -3
[mC][mC][mC][mU][mC][mA][mG][mA][
[mG][mG][mG][dT]*[dT] (SEQ ID NO 66)
mG][mA][mC][mC][mU][mG][mA][mG][m
C][mU][mC][mU][mU][dT]*[dT] (SEQ ID
NO: 159)
05C47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] 05C475
[mC][mC][mC][mU][mC][mA][mG][mA][
4 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -4
mG][mA][mC][mC][mU][mG][mA][mG][m
[mG][mG][mG][dT]*[dT] (SEQ ID NO 67) C][mU][mC][mU][mU]
(SEQ ID NO: 160)
05C47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] 05C475
[mC][mC][mC][mU][mC][mA][mG][mA][
[mG][mG][mU][mC][mU][mC][mU][mG][mA] -5 mG][mA][mC][mC][mU][mG][mA][mG][m
[mG][mG][mG] (SEQ ID NO 68)
C][mU][mC][mU][mU][dT]*[dT] (SEQ ID
NO: 161)
05C47A- [mA][mA][mG][mA][mG][mC][mU][mC][mA] 05C475
[mC][mC][mC][mU][mC][mA][mG][mA][
6 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -6
mG][mA][mC][mC][mU][mG][mA][mG][m
[mG][mG][mG] (SEQ ID NO 69) C][mU][mC][mU][mU]
(SEQ ID NO: 162)
05C47A- [mA] [mA][mG] [mA] [mG] [mC] [mU][mC] [mA] 05C475
CCCUCAGAGACCUGAGCUCUU[dT]*[dT]
7 [mG][mG][mU][mC][mU][mC][mU][mG][mA] -7 (SEQ ID NO:
163)
[mG][mG][mG][dT]*[dT] (SEQ ID NO 70)
05C47A- [mA][2fA][mG][2fA][mG][2fC][mU][2fC][mA] 05C475
[2fC][mC][2fC][mU][2fC][mA][2fG][mA][2
8 [2fG][mG][2fU][mC][2fU][mC][2fU][mG][2fA] -8
fG][mA][2fC][mC][2fU][mG][2fA][mG][2f
[mG][2fG][mG][dT]*[dT] (SEQ ID NO 71)
C][mU][2fC][mU][2fU][dT]*[dT] (SEQ ID
NO: 164)
05C47A- [mA][2fA][mG][2fA][mG][2fC][mU][2fC][mA] 05C475
[2fC][mC][2fC][mU][2fC][mA][2fG][mA][2
9 [2fG][mG][2fU][mC][2fU][mC][2fU][mG][2fA] -9
fG][mA][2fC][mC][2fU][mG][2fA][mG][2f
[mG][2fG][mG] (SEQ ID NO 72)
C][mU][2fC][mU][2fU][dT]*[dT] (SEQ ID
NO: 165)
05C47A- [mA][2fA][mG][2fA][mG][2fC][mU][2fC][mA] 05C475
[2fC][mC][2fC][mU][2fC][mA][2fG][mA][2
[2fG][mG][2fU][mC][2fU][mC][2fU][mG][2fA] -10
fG][mA][2fC][mC][2fU][mG][2fA][mG][2f
[mG][2fG][mG][dT]*[dT] (SEQ ID NO 73) C][mU][2fC][mU][2fU]
(SEQ ID NO: 166)
05C47A- [mA][2fA][mG][2fA][mG][2fC][mU][2fC][mA] 05C475
[2fC][mC][2fC][mU][2fC][mA][2fG]][mA][
11 [2fG][mG][2fU][mC][2fU][mC][2fU][mG][2fA] -11 2fG
[mA] [2fC] [mC][2fU][mG] [2fA] [mG] [2f
[mG][2fG][mG] (SEQ ID NO 74) C][mU][2fC][mU][2fU]
(SEQ ID NO: 167)
05C47A- [2fA][mA][2fG][mA][2fG][mC][2fU][mC][2fA] 05C475
12 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -12
[mC][2fC][mC][2fU][mC][2fA][mG][2fA][
[2fG][mG][2fG][dT]*[dT] (SEQ ID NO 75)
mG][2fA][mC][2fC][mU][2fG][mA][2fG][m
51
CA 03174172 2022- 9- 28
OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5'
TO 3')
C][2fU][mC][2fU][mU][dT][dT]* (SEQ ID
NO: 168)
05C47A- [2fA][mA][2fG][mA][2fG][mC][2fU][mC][2fA] 05C475
13 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -13
[2fC][mC][2fC][mU][2fC][mA][2fG][mA][2
[2fG][mG][2fG] (SEQ ID NO 76)
fG][mA][2fC][mC][2fU][mG][2fA][mG][2f
C][mU][2fC][mU][2fU]-LIG-LINKER (SEQ
ID NO: 169)
05C47A- [mA][2fA][2fG][mA][2fG][mC][2fU][mC][2fA] 05C475
14 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -14
[2fC][mC][2fC][mU][2fC][mA][2fG][mA][2
[2fG][mG][2fG] (SEQ ID NO 77)
fG][mA][2fC][mC][2fU][mG][2fA][mG][2f
C][mU][2fC][mU][2fU][dT]*[dT] (SEQ ID
NO: 170)
05C47A- [mA][2fA][2fG][mA][2fG][mC][2fU][mC][2fA] 05C475
15 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -15
[2fG][mA][2fG][mA][2fC][mC][2fU][mG][
[2fG][mG][2fG][dT]*[dT] (SEQ ID NO 78)
2fA][mG][2fC][mU][2fC][mU][2fU][dT]*[d
T] (SEQ ID NO: 171)
05C47A- [2fA][mA][2fG][mA][2fG][mC][2fU][mC][2fA] 05C475
[2fG][mA][2fG][mA][2fC][mC][2fU][mG][
16 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -16
2fA][mG][2fC][mU][2fC][mU][2fU][dT]*[d
[2fG][mG][2fG][dT]*[dT] (SEQ ID NO 79) T] (SEQ ID NO: 172)
05C47A- [2fA][mA][2fG][mA][2fG][mC][2fU][mC][2fA] 05C475
17 [mG][2fG][mU][2fC][mU][2fC][mU][2fG][mA] -17
[2fG][mA][2fG][mA][2fC][mC][2fU][mG][
[2fG][mG][2fG][dT]*[dT] (SEQ ID NO 80)
2fA][mG][2fC][mU][2fC][mU][2fU] (SEQ
ID NO: 173)
05C47A- [2fA][mA][2fG][mA][2fG][mC][2fU][mC][2fA] 05C475
[2fG][mA][2fG][mA][2fC][mC][2fU][mG][
18 [mG][2fG][mU][2fC][mU][2fC][2fU][2fG][2fA] -18
2fA][mG][2fC][mU][2fC][mU][2fU] (SEQ
[2fG][2fG][2fG] (SEQ ID NO 81) ID NO: 174)
05C48A AAGAGCUCAGGUCUCUGAGGG[dT][dT] (SEQ 05C485 CCCUCAGAGACCUGAGCUCUU[dT][dT]
ID NO 82) (SEQ ID NO: 175)
05C49A AGAAGAGCUCAGGUCUCUGAG[dT][dT] (SEQ 05C495 CUCAGAGACCUGAGCUCUUCU[dT][dT]
ID NO: 83) (SEQ ID NO: 176)
OSC50A AUAGGGAGUGUCCAGGGACCC[dT][dT] (SEQ 05C505 GGGUCCCUGGACACUCCCUAU[dT][dT]
ID NO: 84) (SEQ ID NO: 177)
OSC51A UCCAUAGGGAGUGUCCAGGGA[dT][dT] (SEQ 05C515 UCCCUGGACACUCCCUAUGGA[dT][dT]
ID NO: 85) (SEQ ID NO: 178)
05C52A AUCUCCAUAGGGAGUGUCCAG[dT][dT] (SEQ 05C525 CUGGACACUCCCUAUGGAGAU[dT][dT]
ID NO: 86) (SEQ ID NO: 179)
05C53A UCAGUUCUGGCUGUGGCAGGU[dT][dT] (SEQ 05C535 ACCUGCCACAGCCAGAACUGA[dT][dT]
ID NO: 87) (SEQ ID NO: 180)
05C54A UUCUACCCCCUGGGAGCUGCC[dT][dT] (SEQ 05C545 GGCAGCUCCCAGGGGGUAGAA[dT][dT]
ID NO: 88) (SEQ ID NO: 181)
05C55A AAGCACAGGGCCGUUCUACCC[dT][dT] (SEQ 05C555 GGGUAGAACGGCCCUGUGCUU[dT][dT]
ID NO: 89) (SEQ ID NO: 182)
52
CA 03174172 2022- 9- 28
OS ID Antisense Strand (5' TO 3') OSID Sense Strand (5'
TO 3')
05C56A UG UCUUAAGCACAGGGCCGU U [dT] [dT] (SEQ 05C565 AACGGCCCUGUGCU
UAAGACA[dT][dT]
ID NO: 90) (SEQ ID NO: 183)
05C58A UU U UU UGAGGAUGUGAAGCAA[dT] [dT] 05C585 UUGCU
UCACAUCCUCAAAAAA[dT][dT]
(SEQ ID NO: 92) (SEQ ID NO: 185)
TABLE 6 LRP2
OS ID Antisense Strand (5' TO 3') OS ID Sense Strand (5' TO
3')
05L27A
AAUAAAUAACCAACAGUUGGG[dThdT] (SEQ ID NO: 213) 051_275
CCCAACUGUUGGUUAUUUAUU[dThdT] (SEQ ID NO: 587)
05L44A UUUAAAUACUGAACUACUGUG[dT][dT] (SEQ ID NO: 230)
051_445 CACAGUAGUUCAGUAUUUAAA[dT][dT] (SEQ ID NO: 604)
05L82A AAUCUAUUGCAAUAGUUUCAG[dT][dT] (SEQ ID NO: 268)
051_825 CUGAAACUAUUGCAAUAGAUU[dT][dT] (SEQ ID NO: 642)
05L123A AUUGAAACAACAAUGAAAGAG[di][dT] (SEQ ID NO: 309)
051_1235 CUCUUUCAUUGUUGUUUCAAU [dll[dT] (SEQ ID NO: 683)
OSL141A UUUUCAAAUUCCUAUCUACCC[dT][dT] (SEQ ID NO: 327)
051_1415 GGGUAGAUAGGAAUUUGAAAA[dT][dT] (SEQ ID NO: 701)
05L197A AAUCCAAUACAAUCUCUUCUC[dT][dT] (SEQ ID NO: 383) 051_1975
GAGAAGAGAUUGUAUUGGAUU[dT][dT] (SEQ ID NO: 757)
05L209A ACACAAUGUCCACUUGUACAC[dT][dT] (SEQ ID NO: 395) 051_2095
GUGUACAAGUGGACAUUGUGU[dT][dT] (SEQ ID NO: 769)
05L217A UAUUCUGUACAAGGUUUAGGG[dTlidT] (SEQ ID NO: 403)
051_2175 CCCUAAACCUUGUACAGAAUA[dT][dT] (SEQ ID NO: 777)
05L297A AUUUUAUAGGAAAUAUGAGUG[dTlidT] (SEQ ID NO: 517)
051_2975 CACUCAUAUUUCCUAUAAAAU[dT][dT] (SEQ ID NO: 891)
05L324A UAUAAAUGUACACAUUUAGCC[dThdT] (SEQ ID NO: 544) 0513245
GGCUAAAUGUGUACAUUUAUA[dT][dT] (SEQ ID NO: 918)
OS ID Antisense Strand (5' TO 3') OS ID Sense Strand
(5' TO 3')
UACUU UGUGAGCAAUCUUGAC[dT] [dT] GUCAAGAU
UGCUCACAAAGUA[dT] [dT]
OSL1A (SEQ ID NO: 187) OSL1S (SEQ ID NO:
561)
AU UCACUUGGGAUACACUGAC[dT] [dT]
GUCAGUGUAUCCCAAGUGAAU[dT][dT]
OSL2A (SEQ ID NO: 188) 05L25 (SEQ ID NO:
562)
ACAUGAAAACUCAUUGUGCAA[dT][dT] UUGCACAAUGAGUUU
UCAUGU [dT] [dT]
OSL3A (SEQ ID NO: 189) 05L35 (SEQ ID NO:
563)
UCU UUACAGUCAUCUUCUCCA[dT][dT] UGGAGAAGAUGACUG
UAAAGAUA[dT] [d
OSL4A (SEQ ID NO: 190) 05L45 T] (SEQ ID NO:
564)
UAUCU U UACAGUCAUCU UCUC[dT] [dT]
GAGAAGAUGACUGUAAAGAUA[dT][dT]
OSL5A (SEQ ID NO: 191) 05L55 (SEQ ID NO:
565)
AACAUU UAUGAACAUCAUGAG [dT] [dT] CUCAUGAUGU UCAUAAAUGU
U[dT] [dT]
OSL6A (SEQ ID NO: 192) 05L65 (SEQ ID NO:
566)
UCACAAACUU UAUAAAUGGAG [dT] [dT] CUCCAU UUAUAAAGUU UG
UGA[dT] [dT]
OSL7A (SEQ ID NO: 193) 05L75 (SEQ ID NO:
567)
AU CACAAACU UUAUAAAUGGA[dT] [dT]
UCCAUUUAUAAAGUUUGUGAU[dT][dT]
OSL8A (SEQ ID NO: 194) 05L85 (SEQ ID NO:
568)
AUACUACAGUAUU UUCCGGUA[dT] [dT]
UACCGGAAAAUACUGUAGUAU[dT][dT]
OSL9A (SEQ ID NO: 195) 05L95 (SEQ ID NO:
569)
53
CA 03174172 2022- 9- 28
OSL10 UCAUACUACAGUAUUUUCCGG[dT][dT]
CCGGAAAAUACUGUAGUAUGA[dT][dT]
A (SEQ ID NO: 196) OSL1OS (SEQ ID NO: 570)
OSL11 ACAAAUUCCCCAUAUCUGGCA[dT][dT] (SEQ
UGCCAGAUAUGGGGAAUUUGU[dT][dT]
A ID NO: 197) OSL11S (SEQ ID NO: 571)
05L12 AAGAUAUACCCUUCUUCACAG[dT][dT] (SEQ
CUGUGAAGAAGGGUAUAUCUU[dT][dT]
A ID NO: 198) 051_125 (SEQ ID NO: 572)
05L13 UUAGCUUUGCAAUACUGUCCA[dT][dT]
UGGACAGUAUUGCAAAGCUAA[dT][dT]
A (SEQ ID NO: 199) 05L135 (SEQ ID NO: 573)
05L14 AUCAUUAGCUUUGCAAUACUG[dT][dT]
CAGUAUUGCAAAGCUAAUGAU[dT][dT]
A (SEQ ID NO: 200) 051_145 (SEQ ID NO: 574)
05L15 AAAGGAAUCAUUAGCUUUGCA[dT][dT]
UGCAAAGCUAAUGAUUCCUUU[dT][dT]
A (SEQ ID NO: 201) 051_155 (SEQ ID NO: 575)
05L16 AUGAAUAUCACCAAUUAACAA[dT][dT] (SEQ
UUGUUAAUUGGUGAUAUUCAU[dT][dT]
A ID NO: 202) 051_165 (SEQ ID NO: 576)
05L17 AAAAAACCUUAUUUUGCACGG[dT][dT]
CCGUGCAAAAUAAGGUUUUUU[dT][dT]
A (SEQ ID NO: 203) 051_175 (SEQ ID NO: 577)
05L18 UGAAAAAACCUUAUUUUGCAC[dT][dT]
GUGCAAAAUAAGGUUUUUUCA[dT][dT]
A (SEQ ID NO: 204) 051_185 (SEQ ID NO: 578)
05L19 AAUGUCAACUGAAAAAACCUU[dT][dT] (SEQ
AAGGUUUUUUCAGUUGACAUU[dT][dT]
A ID NO: 205) 051_195 (SEQ ID NO: 579)
05L20 UAAUGUCAACUGAAAAAACCU[dT][dT] (SEQ
AGGUUUUUUCAGUUGACAUUA[dT][dT]
A ID NO: 206) 051_205 (SEQ ID NO: 580)
05L21 UUAAACCAUUAAUGUCAACUG[dT][dT]
CAGUUGACAUUAAUGGUUUAA[dT][dT]
A (SEQ ID NO: 207) 051_215 (SEQ ID NO: 581)
05L22 UAUUUAAACCAUUAAUGUCAA[dT][dT]
UUGACAUUAAUGGUUUAAAUA[dT][dT]
A (SEQ ID NO: 208) 051_225 (SEQ ID NO: 582)
05L23 UAGAUUUUAUUAUUAACCCAG[dT][dT]
CUGGGUUAAUAAUAAAAUCUA[dT][dT]
A (SEQ ID NO: 209) 051_235 (SEQ ID NO: 583)
05L24 AUAGAUUUUAUUAUUAACCCA[dT][dT]
UGGGUUAAUAAUAAAAUCUAU[dT][dT]
A (SEQ ID NO: 210) 051_245 (SEQ ID NO: 584)
05L25 AAAUUUACCAUAUCUAUGCGG[dT][dT]
CCGCAUAGAUAUGGUAAAUUU[dT][dT]
A (SEQ ID NO: 211) 051_255 (SEQ ID NO: 585)
05L26 AAGUUUUCAGUUAUAAGGGUA[dT][dT]
UACCCUUAUAACUGAAAACUU[dT][dT]
A (SEQ ID NO: 212) 051_265 (SEQ ID NO: 586)
05L27 AAUAAAUAACCAACAGUUGGG[dT][dT]
CCCAACUGUUGGUUAUUUAUU[dT][dT]
A (SEQ ID NO: 213) 051_275 (SEQ ID NO: 587)
05L28 AGAAAAAUAAAUAACCAACAG[dT][dT] (SEQ
CUGUUGGUUAUUUAUUUUUCU[dT][dT]
A ID NO: 214) 051_285 (SEQ ID NO: 588)
05L29 AUAUCAUAUCCAGAGUUACCC[dT][dT] (SEQ
GGGUAACUCUGGAUAUGAUAU[dT][dT]
A ID NO: 215) 051_295 (SEQ ID NO: 589)
54
CA 03174172 2022- 9- 28
OSL30 AGUUUCAAUGUAAUCAAACCG[dT][dT]
CGGUUUGAUUACAUUGAAACU[dT][dT]
A (SEQ ID NO: 216) 051_305 (SEQ ID NO: 590)
05L31 UUACAGUUUCAAUGUAAUCAA[dT][dT]
UUGAUUACAUUGAAACUGUAA[dT][dT]
A (SEQ ID NO: 217) 051_315 (SEQ ID NO: 591)
05L32 AUAAGUUACAGUUUCAAUGUA[dT][dT]
UACAUUGAAACUGUAACUUAU[dT][dT]
A (SEQ ID NO: 218) 051_325 (SEQ ID NO: 592)
05L33 UCUUUACACGGAUUGGUAGCA[dT][dT]
UGCUACCAAUCCGUGUAAAGA[dT][dT]
A (SEQ ID NO: 219) 051_335 (SEQ ID NO: 593)
05L34 AAAAUCAAUCCCGACAAAGAA[dT][dT] (SEQ
UUCUUUGUCGGGAUUGAUUUU[dT][dT]
A ID NO: 220) 051_345 (SEQ ID NO: 594)
05L35 UCUGAAAAAAAGAUAGUGCUG[dT][dT]
CAGCACUAUCUUUUUUUCAGA[dT][dT]
A (SEQ ID NO: 221) 051_355 (SEQ ID NO: 595)
05L36 AUCUGAAAAAAAGAUAGUGCU[dT][dT]
AGCACUAUCUUUUUUUCAGAU[dT][dT]
A (SEQ ID NO: 222) 051_365 (SEQ ID NO: 596)
05L37 AAAAAUCAUGUGUUUUGACAU[dT][dT]
AUGUCAAAACACAUGAUUUUU[dT][dT]
A (SEQ ID NO: 223) 051_375 (SEQ ID NO: 597)
05L38 UUUGCUUAAAAAUCAUGUGUU[dT][dT]
AACACAUGAUUUUUAAGCAAA[dT][dT]
A (SEQ ID NO: 224) 051_385 (SEQ ID NO: 598)
05L39 AACUUUCAACAUUUUCCACCC[dT][dT] (SEQ
GGGUGGAAAAUGUUGAAAGUU[dT][dT]
A ID NO: 225) 051_395 (SEQ ID NO: 599)
05L40 UUGAAAUCCAAUCAAAAGCCA[dT][dT] (SEQ
UGGCUUUUGAUUGGAUUUCAA[dT][dT]
A ID NO: 226) 051_405 (SEQ ID NO: 600)
05L41 UUUGAAAUCCAAUCAAAAGCC[dT][dT] (SEQ
GGCUUUUGAUUGGAUUUCAAA[dT][dT]
A ID NO: 227) 051_415 (SEQ ID NO: 601)
05L42 UAGAGAUUCUUUGAAAUCCAA[dT][dT]
UUGGAUUUCAAAGAAUCUCUA[dT][dT]
A (SEQ ID NO: 228) 051_425 (SEQ ID NO: 602)
05L43 AUAGAGAUUCUUUGAAAUCCA[dT][dT]
UGGAUUUCAAAGAAUCUCUAU[dT][dT]
A (SEQ ID NO: 229) 051_435 (SEQ ID NO: 603)
05L44 UUUAAAUACUGAACUACUGUG[dT][dT]
CACAGUAGUUCAGUAUUUAAA[dT][dT]
A (SEQID NO: 230) 051_445 (SEQ ID NO: 604)
05L45 UAUUUAAAUACUGAACUACUG[dT][dT]
CAGUAGUUCAGUAUUUAAAUA[dT][dT]
A (SEQ ID NO: 231) 051_455 (SEQ ID NO: 605)
05L46 AUAGAUACCCGGCAAAAGGAU[dT][dT] (SEQ
AUCCUUUUGCCGGGUAUCUAU[dT][dT]
A ID NO: 232) 051_465 (SEQ ID NO: 606)
05L47 AAGAGUAGUGUUUAUUACAGG[dT][dT]
CCCCUGUAAUAAACACUACUC[dT][dT]
A (SEQ ID NO: 233) 051_475 (SEQ ID NO: 607)
05L48 AUCAAAAUAGGCAUCUACCCA[dT][dT] (SEQ
UGGGUAGAUGCCUAUUUUGAU[dT][dT]
A ID NO: 234) 051_485 (SEQ ID NO: 608)
05L49 UCAAUUUUAUCAAAAUAGGCA[dT][dT]
UGCCUAUUUUGAUAAAAUUGA[dT][dT]
A (SEQ ID NO: 235) 051_495 (SEQ ID NO: 609)
CA 03174172 2022- 9- 28
OSL50 UAAAUGCUCUCCAAAGAUGGC[dT][dT]
GCCAUCUUUGGAGAGCAUUUA[dT][dT]
A (SEQ ID NO: 236) 051_505 (SEQ ID NO: 610)
05L51 UCAAAUGCAGUAUGUAAGCAA[dT][dT]
UUGCUUACAUACUGCAUUUGA[dT][dT]
A (SEQ ID NO: 237) 051_515 (SEQ ID NO: 611)
05L52 UUCAAAUGCAGUAUGUAAGCA[dT][dT]
UGCUUACAUACUGCAUUUGAA[dT][dT]
A (SEQ ID NO: 238) 051_525 (SEQ ID NO: 612)
05L53 UGAUUACAGGCGUUAGAACCA[dT][dT]
UGGUUCUAACGCCUGUAAUCA[dT][dT]
A (SEQ ID NO: 239) 051_535 (SEQ ID NO: 613)
05L54 UGUUAUCAUGACAAUCAUCGA[dT][dT]
UCGAUGAUUGUCAUGAUAACA[dT][dT]
A (SEQID NO: 240) 051_545 (SEQ ID NO: 614)
05L55 UUAUCACAGGUGUAUUGGGUG[dT][dT]
CACCCAAUACACCUGUGAUAA[dT][dT]
A (SEQ ID NO: 241) 051_555 (SEQ ID NO: 615)
05L56 AUUAUCACAGGUGUAUUGGGU[dT][dT]
ACCCAAUACACCUGUGAUAAU[dT][dT]
A (SEQ ID NO: 242) 051_565 (SEQ ID NO: 616)
05L57 AGUUCUUUGAGAUACACUGGU[dT][dT]
ACCAGUGUAUCUCAAAGAACU[dT][dT]
A (SEQ ID NO: 243) 051_575 (SEQ ID NO: 617)
05L58 UCGAAUUGCAGUUCUUUUCAU[dT][dT]
AUGAAAAGAACUGCAAUUCGA[dT][dT]
A (SEQ ID NO: 244) 051_585 (SEQ ID NO: 618)
05L59 UCAAUACAUCGAUGAUUGGGG[dT][dT]
CCCCAAUCAUCGAUGUAUUGA[dT][dT]
A (SEQ ID NO: 245) 051_595 (SEQ ID NO: 619)
05L60 AACGAUAGGUCAAUACAUCGA[dT][dT] (SEQ
UCGAUGUAUUGACCUAUCGUU[dT][dT]
A ID NO: 246) 051_605 (SEQ ID NO: 620)
05L61 ACAAACGAUAGGUCAAUACAU[dT][dT] (SEQ
AUGUAUUGACCUAUCGUUUGU[dT][dT]
A ID NO: 247) 051_615 (SEQ ID NO: 621)
05L62 UCAAAAACACCAUCACAACGA[dT][dT] (SEQ
UCGUUGUGAUGGUGUUUUUGA[dT][dT]
A ID NO: 248) 051_625 (SEQ ID NO: 622)
05L63 UCACAUUCCCAGAAGUUCGGG[dT][dT]
CCCGAACUUCUGGGAAUGUGA[dT][dT]
A (SEQ ID NO: 249) 051_635 (SEQ ID NO: 623)
05L64 AUCACAUUCCCAGAAGUUCGG[dT][dT] (SEQ
CCGAACUUCUGGGAAUGUGAU[dT][dT]
A ID NO: 250) 051_645 (SEQ ID NO: 624)
05L65 UGAUGAAGGGCAAGUCUUGGG[dT][dT]
CCCAAGACUUGCCCUUCAUCA[dT][dT]
A (SEQ ID NO: 251) 051_655 (SEQ ID NO: 625)
05L66 AGAAUCAUUGGCAAGUAAGAA[dT][dT]
UUCUUACUUGCCAAUGAUUCU[dT][dT]
A (SEQ ID NO: 252) 051_665 (SEQ ID NO: 626)
05L67 UAUCACAUUCAUCUAUGUCUU[dT][dT]
AAGACAUAGAUGAAUGUGAUA[dT][dT]
A (SEQ ID NO: 253) 051_675 (SEQ ID NO: 627)
05L68 AAUAUCACAUUCAUCUAUGUC[dT][dT]
GACAUAGAUGAAUGUGAUAUU[dT][dT]
A (SEQ ID NO: 254) 051_685 (SEQ ID NO: 628)
05L69 AACAUGUAGCCUGUAUCACAC[dT][dT] (SEQ
GUGUGAUACAGGCUACAUGUU[dT][dT]
A ID NO: 255) 051_695 (SEQ ID NO: 629)
56
CA 03174172 2022- 9- 28
OSL70 UCACUUUCUAACAUGUAGCCU[dT][dT]
AGGCUACAUGUUAGAAAGUGA[dT][dT]
A (SEQ ID NO: 256) 051_705 (SEQ ID NO: 630)
05L71 AUCACUUUCUAACAUGUAGCC[dT][dT] (SEQ
GGCUACAUGUUAGAAAGUGAU[dT][dT]
A ID NO: 257) 051_715 (SEQ ID NO: 631)
05L72 AAUGUAAGAACCAUUCUCGAC[dT][dT] (SEQ
GUCGAGAAUGGUUCUUACAUU[dT][dT]
A ID NO: 258) 051_725 (SEQ ID NO: 632)
05L73 UACAAUGUAAGAACCAUUCUC[dT][dT] (SEQ
GAGAAUGGUUCUUACAUUGUA[dT][dT]
A ID NO: 259) 051_735 (SEQ ID NO: 633)
05L74 AAAAUCAACAGCUACAAUGUA[dT][dT] (SEQ
UACAUUGUAGCUGUUGAUUUU[dT][dT]
A ID NO: 260) 051_745 (SEQ ID NO: 634)
05L75 AUUGAAUCAAAAUCAACAGCU[dT][dT] (SEQ
AGCUGUUGAUUUUGAUUCAAU[dT][dT]
A ID NO: 261) 051_755 (SEQ ID NO: 635)
05L76 UAAUUGAAUCAAAAUCAACAG[dT][dT] (SEQ
CUGUUGAUUUUGAUUCAAUUA[dT][dT]
A ID NO: 262) 051_765 (SEQ ID NO: 636)
05L77 AAGAUACGACCACUAAUUGAA[dT][dT] (SEQ
UUCAAUUAGUGGUCGUAUCUU[dT][dT]
A ID NO: 263) 051_775 (SEQ ID NO: 637)
05L78 AGUUUCAGUCAAGAUGAUGCU[dT][dT]
AGCAUCAUCUUGACUGAAACU[dT][dT]
A (SEQ ID NO: 264) 051_785 (SEQ ID NO: 638)
05L79 AAUAGUUUCAGUCAAGAUGAU[dT][dT]
AUCAUCUUGACUGAAACUAUU[dT][dT]
A (SEQ ID NO: 265) 051_795 (SEQ ID NO: 639)
05L80 UAUUGCAAUAGUUUCAGUCAA[dT][dT]
UUGACUGAAACUAUUGCAAUA[dT][dT]
A (SEQ ID NO: 266) 051_805 (SEQ ID NO: 640)
05L81 UCUAUUGCAAUAGUUUCAGUC[dT][dT]
GACUGAAACUAUUGCAAUAGA[dT][dT]
A (SEQ ID NO: 267) 051_815 (SEQ ID NO: 641)
05L82 AAUCUAUUGCAAUAGUUUCAG[dT][dT]
CUGAAACUAUUGCAAUAGAUU[dT][dT]
A (SEQ ID NO: 268) 051_825 (SEQ ID NO: 642)
05L83 AUUUUGGAGACUUCAAUUGUU[dT][dT]
AACAAUUGAAGUCUCCAAAAU[dT][dT]
A (SEQ ID NO: 269) 051_835 (SEQ ID NO: 643)
05L84 UUAGGUUUUUACUAAUCAGCA[dT][dT]
UGCUGAUUAGUAAAAACCUAA[dT][dT]
A (SEQ ID NO: 270) 051_845 (SEQ ID NO: 644)
05L85 UCAUUCUGGGAUCUAAUGCUA[dT][dT]
UAGCAUUAGAUCCCAGAAUGA[dT][dT]
A (SEQ ID NO: 271) 051_855 (SEQ ID NO: 645)
05L86 UUCAUUCUGGGAUCUAAUGCU[dT][dT]
AGCAUUAGAUCCCAGAAUGAA[dT][dT]
A (SEQ ID NO: 272) 051_865 (SEQ ID NO: 646)
05L87 AGUAGAUGCUCAUUCAUUCUG[dT][dT]
CAGAAUGAAUGAGCAUCUACU[dT][dT]
A (SEQ ID NO: 273) 051_875 (SEQ ID NO: 647)
05L88 AUUAUAAUCACAAAAGUCCAU[dT][dT] (SEQ
AUGGACUUUUGUGAUUAUAAU[dT][dT]
A ID NO: 274) 051_885 (SEQ ID NO: 648)
05L89 UCCAUUAUAAUCACAAAAGUC[dT][dT] (SEQ
GACUUUUGUGAUUAUAAUGGA[dT][dT]
A ID NO: 275) 051_895 (SEQ ID NO: 649)
57
CA 03174172 2022- 9- 28
OSL90 UGCCGUAUAAUCAAAUCAC[dT][dT] (SEQ ID
GUGUGAUUUGAUUAUACGGCA[dT][dT]
A NO: 276) 05005 (SEQ ID NO: 650)
05L91 AUAUUAUACAUUACAACUGAC[dT][dT]
GUCAGUUGUAAUGUAUAAUAU[dT][dT]
A (SEQ ID NO: 277) 05015 (SEQ ID NO: 651)
05L92 AUUGAAUAUUAUACAUUACAA[dT][dT]
UUGUAAUGUAUAAUAUUCAAU[dT][dT]
A (SEQ ID NO: 278) 05025 (SEQ ID NO: 652)
05L93 AAUUUGGUUGUUUCGAAGGAU[dT][dT]
AUCCUUCGAAACAACCAAAUU[dT][dT]
A (SEQ ID NO: 279) 05035 (SEQ ID NO: 653)
05L94 ACGGAAUUUGGUUGUUUCGAA[dT][dT]
UUCGAAACAACCAAAUUCCGU[dT][dT]
A (SEQ ID NO: 280) 05045 (SEQ ID NO: 654)
05L95 UUACAGUUAUUAAGAAAGGUU[dT][dT]
AACCUUUCUUAAUAACUGUAA[dT][dT]
A (SEQ ID NO: 281) 05055 (SEQ ID NO: 655)
05L96 UCCAAAAAUUAUAUGUUGCCU[dT][dT]
AGGCAACAUAUAAUUUUUGGA[dT][dT]
A (SEQ ID NO: 282) 05065 (SEQ ID NO: 656)
05L97 UUCCAAAAAUUAUAUGUUGCC[dT][dT]
GGCAACAUAUAAUUUUUGGAA[dT][dT]
A (SEQ ID NO: 283) 05075 (SEQ ID NO: 657)
05L98 UCUAAACCAUUCUGUAUCCCU[dT][dT] (SEQ
AGGGAUACAGAAUGGUUUAGA[dT][dT]
A ID NO: 284) 05085 (SEQ ID NO: 658)
05L99 AUCUAAACCAUUCUGUAUCCC[dT][dT] (SEQ
GGGAUACAGAAUGGUUUAGAU[dT][dT]
A ID NO: 285) 05095 (SEQ ID NO: 659)
05L10 UUCAACAUCUAAACCAUUCUG[dT][dT] (SEQ 05L100 CAGAAUGGUUUAGAUGUUGAA[dT][dT]
OA ID NO: 286) 5 (SEQ ID NO: 660)
05L10 AUUUUCAACCCAAUAGAUGUA[dT][dT] 05L101
UACAUCUAUUGGGUUGAAAAU[dT][dT]
1A (SEQ ID NO: 287) 5 (SEQ ID NO: 661)
05L10 UAUAGAAGCAAAUACUGUCCU[dT][dT] 05L102
AGGACAGUAUUUGCUUCUAUA[dT][dT]
2A (SEQ ID NO: 288) 5 (SEQ ID NO: 662)
05L10 UAGAUAUAGAAGCAAAUACUG[dT][dT] 05L103
CAGUAUUUGCUUCUAUAUCUA[dT][dT]
3A (SEQ ID NO: 289) 5 (SEQ ID NO: 663)
05L10 UAAGGCCAGGUUCAUAGAAGG[dT][dT] 05L104
CCUUCUAUGAACCUGGCCU[dT][dT] (SEQ
4A (SEQ ID NO: 290) 5 ID NO: 664)
05L10 UCUUGAAAUCCAAUCUAAGGC[dT][dT] 05L105
GCCUUAGAUUGGAUUUCAAGA[dT][dT]
5A (SEQ ID NO: 291) 5 (SEQ ID NO: 665)
05L10 AUAAAGGUUUCUUGAAAUCCA[dT][dT] 05L106
UGGAUUUCAAGAAACCUUUAU[dT][dT]
6A (SEQ ID NO: 292) 5 (SEQ ID NO: 666)
05L10 UCAAAACCUCGAUUGACUGAG[dT][dT] 05L107
CUCAGUCAAUCGAGGUUUUGA[dT][dT]
7A (SEQ ID NO: 293) 5 (SEQ ID NO: 667)
05L10 UUCUGUAUCUGAUAUCUCCGU[dT][dT] 05L108
ACGGAGAUAUCAGAUACAGAA[dT][dT]
8A (SEQ ID NO: 294) 5 (SEQ ID NO: 668)
05L10 UUUCUGUAUCUGAUAUCUCCG[dT][dT] 05L109
CGGAGAUAUCAGAUACAGAAA[dT][dT]
9A (SEQ ID NO: 295) 5 (SEQ ID NO: 669)
58
CA 03174172 2022- 9- 28
05L11 UUUUUCUGUAUCUGAUAUCUC[dT][dT] 05L110
GAGAUAUCAGAUACAGAAAAA[dT][dT]
OA (SEQ ID NO: 296) 5 (SEQ ID NO: 670)
05L11 AUCAAUGUUUUUCUGUAUCUG[dT][dT] 05L111
CAGAUACAGAAAAACAUUGAU[dT][dT]
1A (SEQ ID NO: 297) 5 (SEQ ID NO: 671)
05L11 AUAAAGGAAAGAAUCAUGGAC[dT][dT] 05L112
GUCCAUGAUUCUUUCCUUUAU[dT][dT]
2A (SEQ ID NO: 298) 5 (SEQ ID NO: 672)
05L11 AAUAAAGGAAAGAAUCAUGGA[dT][dT] 05L113
UCCAUGAUUCUUUCCUUUAUU[dT][dT]
3A (SEQ ID NO: 299) 5 (SEQ ID NO: 673)
05L11 UCAGUAUAAUAAAGGAAAGAA[dT][dT] 05L114
UUCUUUCCUUUAUUAUACUGA[dT][dT]
4A (SEQ ID NO: 300) 5 (SEQ ID NO: 674)
05L11 UUUCAAUGACCUCAUACUGUU[dT][dT] 05L115
AACAGUAUGAGGUCAUUGAAA[dT][dT]
5A (SEQ ID NO: 301) 5 (SEQ ID NO: 675)
05L11 AUUUGGAACAUUAUCUCUCAA[dT][dT] 05L116
UUGAGAGAUAAUGUUCCAAAU[dT][dT]
6A (SEQ ID NO: 302) 5 (SEQ ID NO: 676)
05L11 AGAUUUGGAACAUUAUCUCUC[dT][dT] 05L117
GAGAGAUAAUGUUCCAAAUCU[dT][dT]
7A (SEQ ID NO: 303) 5 (SEQ ID NO: 677)
05L11 UCAGAUUUGGAACAUUAUCUC[dT][dT] 05L118
GAGAUAAUGUUCCAAAUCUGA[dT][dT]
8A (SEQ ID NO: 304) 5 (SEQ ID NO: 678)
05L11 UUGCUACAGCCAUUUGAGG[dT][dT] (SEQ 05L119
CCUCAAAUGGCUGUAGCAA[dT][dT] (SEQ
9A ID NO: 305) 5 ID NO: 679)
05L12 AUGAAAGAGUUAUAUGGAGAG[dT][dT] 05L120
CUCUCCAUAUAACUCUUUCAU[dT][dT]
OA (SEQ ID NO: 306) 5 (SEQ ID NO: 680)
05L12 ACAAUGAAAGAGUUAUAUGGA[dT][dT] 05L121
UCCAUAUAACUCUUUCAUUGU[dT][dT]
1A (SEQ ID NO: 307) 5 (SEQ ID NO: 681)
05L12 UGAAACAACAAUGAAAGAGUU[dT][dT] 05L122
AACUCUUUCAUUGUUGUUUCA[dT][dT]
2A (SEQ ID NO: 308) 5 (SEQ ID NO: 682)
05L12 AUUGAAACAACAAUGAAAGAG[dT][dT] 05L123 CUCUUUCAUUGUUGUUUCAAU
[dT][dT]
3A (SEQ ID NO: 309) 5 (SEQ ID NO: 683)
05L12 AGCUAAAGCCUCUGAUUGCAG[dT][dT] 05L124
CUGCAAUCAGAGGCUUUAGCU[dT][dT]
4A (SEQ ID NO: 310) 5 (SEQ ID NO: 684)
05L12 AAGCUAAAGCCUCUGAUUGCA[dT][dT] 05L125
UGCAAUCAGAGGCUUUAGCUU[dT][dT]
5A (SEQ ID NO: 311) 5 (SEQ ID NO: 685)
05L12 ACAAUUCCAAGCUAAAGCCUC[dT][dT] (SEQ 05L126 GAGGCUUUAGCUUGGAAUUGU[dT][dT]
6A ID NO: 312) 5 (SEQ ID NO: 686)
05L12 UGACAAUUCCAAGCUAAAGCC[dT][dT] (SEQ 05L127 GGCUUUAGCUUGGAAUUGUCA[dT][dT]
7A ID NO: 313) 5 (SEQ ID NO: 687)
05L12 UGAAUGAUCUGACAAUUCCAA[dT][dT] 05L128
UUGGAAUUGUCAGAUCAUUCA[dT][dT]
8A (SEQ ID NO: 314) 5 (SEQ ID NO: 688)
05L12 AUGUUCAUCAGAGAAGAUCCA[dT][dT] 05L129
UGGAUCUUCUCUGAUGAACAU[dT][dT]
9A (SEQ ID NO: 315) 5 (SEQ ID NO: 689)
59
CA 03174172 2022- 9- 28
05L13 UAUUCCAUGUGUCACAAUGUU[dT][dT] OSL130
AACAUUGUGACACAUGGAAUA[dT][dT]
OA (SEQ ID NO: 316) 5 (SEQ ID NO: 690)
05L13 ACUUCUAUCAGUGUUUCAGAA[dT][dT] 05L131
UUCUGAAACACUGAUAGAAGU[dT][dT]
1A (SEQ ID NO: 317) 5 (SEQ ID NO: 691)
05L13 UAUUGAUCCGCAGAACUUCUA[dT][dT] 05L132
UAGAAGUUCUGCGGAUCAAUA[dT][dT]
2A (SEQ ID NO: 318) 5 (SEQ ID NO: 692)
05L13 UGUUCUUGGGAUCUACAACAA[dT][dT] 05L133
UUGUUGUAGAUCCCAAGAACA[dT][dT]
3A (SEQ ID NO: 319) 5 (SEQ ID NO: 693)
05L13 AAAGAACGCUCAAUCUUUGGU[dT][dT] 05L134
ACCAAAGAUUGAGCGUUCUUU[dT][dT]
4A (SEQ ID NO: 320) 5 (SEQ ID NO: 694)
05L13 UAAACGUAGCCAUCACUUCGG[dT][dT] (SEQ 05L135 CCGAAGUGAUGGCUACGUUUA[dT][dT]
5A ID NO: 321) 5 (SEQ ID NO: 695)
05L13 AUCUAAAGAAUCAUCAACCCA[dT][dT] (SEQ 05L136 UGGGUUGAUGAUUCUUUAGAU[dT][dT]
6A ID NO: 322) 5 (SEQ ID NO: 696)
05L13 UUAUAUCUAAAGAAUCAUCAA[dT][dT] 05L137
UUGAUGAUUCUUUAGAUAUAA[dT][dT]
7A (SEQ ID NO: 323) 5 (SEQ ID NO: 697)
05L13 AUAGAAUUUUCAAAAACAGUG[dT][dT] 05L138
CACUGUUUUUGAAAAUUCUAU[dT][dT]
8A (SEQ ID NO: 324) 5 (SEQ ID NO: 698)
05L13 UGAUAGAAUUUUCAAAAACAG[dT][dT] 05L139
CUGUUUUUGAAAAUUCUAUCA[dT][dT]
9A (SEQ ID NO: 325) 5 (SEQ ID NO: 699)
05L14 UUUCAAAUUCCUAUCUACCCA[dT][dT] (SEQ 05L140 UGGGUAGAUAGGAAUUUGAAA[dT][dT]
OA ID NO: 326) 5 (SEQ ID NO: 700)
05L14 UUUUCAAAUUCCUAUCUACCC[dT][dT] (SEQ 05L141 GGGUAGAUAGGAAUUUGAAAA[dT][dT]
1A ID NO: 327) 5 (SEQ ID NO: 701)
05L14 AUAUUGUCUCUUAUCACUGUG[dT][dT] 05L142
CACAGUGAUAAGAGACAAUAU[dT][dT]
2A (SEQ ID NO: 328) 5 (SEQ ID NO: 702)
05L14 UGAUAUUGUCUCUUAUCACUG[dT][dT] 05L143
CAGUGAUAAGAGACAAUAUCA[dT][dT]
3A (SEQ ID NO: 329) 5 (SEQ ID NO: 703)
05L14 UGAAAUGGCACAAUUCUUGCC[dT][dT] 05L144
GGCAAGAAUUGUGCCAUUUCA[dT][dT]
4A (SEQ ID NO: 330) 5 (SEQ ID NO: 704)
05L14 UGUUGAAAUGGCACAAUUCUU[dT][dT] 05L145
AAGAAUUGUGCCAUUUCAACA[dT][dT]
5A (SEQ ID NO: 331) 5 (SEQ ID NO: 705)
05L14 AAUUUUCUGUUGAAAUGGCAC[dT][dT] 05L146
GUGCCAUUUCAACAGAAAAUU[dT][dT]
6A (SEQ ID NO: 332) 5 (SEQ ID NO: 706)
05L14 AAAUUUUCUGUUGAAAUGGCA[dT][dT] 05L147
UGCCAUUUCAACAGAAAAUUU[dT][dT]
7A (SEQ ID NO: 333) 5 (SEQ ID NO: 707)
05L14 AUUAGACAAGGCAAAGAUGAG[dT][dT] 05L148
CUCAUCUUUGCCUUGUCUAAU[dT][dT]
8A (SEQ ID NO: 334) 5 (SEQ ID NO: 708)
05L14 ACAUUUAUUGUUUGGAAAGGU[dT][dT] 05L149
ACCUUUCCAAACAAUAAAUGU[dT][dT]
9A (SEQ ID NO: 335) 5 (SEQ ID NO: 709)
CA 03174172 2022- 9- 28
05L15 AUCACUUACACUGUCAUAGUC[dT][dT] (SEQ 05L150 GACUAUGACAGUGUAAGUGAU[dT][dT]
OA ID NO: 336) 5 (SEQ ID NO: 710)
05L15 UAGAUUCUAUCACUUACACUG[dT][dT] 05L151
CAGUGUAAGUGAUAGAAUCUA[dT][dT]
1A (SEQ ID NO: 337) 5 (SEQ ID NO: 711)
05L15 AGUAGAUUCUAUCACUUACAC[dT][dT] 05L152
GUGUAAGUGAUAGAAUCUACU[dT][dT]
2A (SEQ ID NO: 338) 5 (SEQ ID NO: 712)
05L15 UUUUGUGUGAAGUAGAUUCUA[dT][dT] 05L153 UAGAAUCUACUUCACACAAAA[dT][dT]
3A (SEQ ID NO: 339) 5 (SEQ ID NO: 713)
05L15 UAAAUUUUGUGUGAAGUAGAU[dT][dT] 05L154
AUCUACUUCACACAAAAUUUA[dT][dT]
4A (SEQ ID NO: 340) 5 (SEQ ID NO: 714)
05L15 UCUAGUAAUCCAGUCAAAGGC[dT][dT] 05L155
GCCUUUGACUGGAUUACUAGA[dT][dT]
5A (SEQ ID NO: 341) 5 (SEQ ID NO: 715)
05L15 AAUUCUUCUAGUAAUCCAGUC[dT][dT] 05L156
GACUGGAUUACUAGAAGAAUU[dT][dT]
6A (SEQ ID NO: 342) 5 (SEQ ID NO: 716)
05L15 UAAAUUCUUCUAGUAAUCCAG[dT][dT] 05L157
CUGGAUUACUAGAAGAAUUUA[dT][dT]
7A (SEQ ID NO: 343) 5 (SEQ ID NO: 717)
05L15 AUAAAUUCUUCUAGUAAUCCA[dT][dT] 05L158
UGGAUUACUAGAAGAAUUUAU[dT][dT]
8A (SEQ ID NO: 344) 5 (SEQ ID NO: 718)
05L15 AUAUACUGGCCAUAGAGAGUC[dT][dT] 05L159
GACUCUCUAUGGCCAGUAUAU[dT][dT]
9A (SEQ ID NO: 345) 5 (SEQ ID NO: 719)
05L16 UUCUU UGUGUGUACAAGUCAG [dT] [dT] 05L160
CUGACUUGUACACACAAAGAA[dT][dT]
OA (SEQ ID NO: 346) 5 (SEQ ID NO: 720)
05L16 AAUUCUUUGUGUGUACAAGUC[dT][dT] 05L161
GACUUGUACACACAAAGAAUU[dT][dT]
1A (SEQ ID NO: 347) 5 (SEQ ID NO: 721)
05L16 UCGGUAAAUUCUUUGUGUGUA[dT][dT] 05L162
UACACACAAAGAAUUUACCGA[dT][dT]
2A (SEQ ID NO: 348) 5 (SEQ ID NO: 722)
05L16 UGUUACACUGUUGUUUCUGGU[dT][dT] 05L163
ACCAGAAACAACAGUGUAACA[dT][dT]
3A (SEQ ID NO: 349) 5 (SEQ ID NO: 723)
05L16 AUUGUUACACUGUUGUUUCUG[dT][dT] 05L164
CAGAAACAACAGUGUAACAAU[dT][dT]
4A (SEQ ID NO: 350) 5 (SEQ ID NO: 724)
05L16 AAACUGUUCACAAGGAUUGUU[dT][dT] 05L165
AACAAUCCUUGUGAACAGUUU[dT][dT]
5A (SEQ ID NO: 351) 5 (SEQ ID NO: 725)
05L16 ACAUCGUUCACCAUUGUCCAC[dT][dT] (SEQ 05L166 GUGGACAAUGGUGAACGAUGU[dT][dT]
6A ID NO: 352) 5 (SEQ ID NO: 726)
05L16 UGUUAUUGCACAUAAACUCCG[dT][dT] 05L167
CGGAGUUUAUGUGCAAUAACA[dT][dT]
7A (SEQ ID NO: 353) 5 (SEQ ID NO: 727)
05L16 UCUGUUAUUGCACAUAAACUC[dT][dT] 05L168
GAGUUUAUGUGCAAUAACAGA[dT][dT]
8A (SEQ ID NO: 354) 5 (SEQ ID NO: 728)
05L16 UUAUGACAUUUUGUGUAUCCA[dT][dT] 05L169
UGGAUACACAAAAUGUCAUAA[dT][dT]
9A (SEQ ID NO: 355) 5 (SEQ ID NO: 729)
61
CA 03174172 2022- 9- 28
05L17 UGAAUUAUGACAUUUUGUGUA[dT][dT] OSL170
UACACAAAAUGUCAUAAUUCA[dT][dT]
OA (SEQ ID NO: 356) 5 (SEQ ID NO: 730)
05L17 UUUGAAUUAUGACAUU UUGUG [dT][dT] 05L171
CACAAAAUGUCAUAAUUCAAA[dT][dT]
1A (SEQ ID NO: 357) 5 (SEQ ID NO: 731)
05L17 UACAAAUAUUUGAAUUAUGAC[dT][dT] 05L172
GUCAUAAUUCAAAUAUUUGUA[dT][dT]
2A (SEQ ID NO: 358) 5 (SEQ ID NO: 732)
05L17 AAAUAAACGCGAGGAAUACAA[dT][dT] (SEQ 05L173 UUGUAUUCCUCGCGUUUAUUU[dT][dT]
3A ID NO: 359) 5 (SEQ ID NO: 733)
05L17 AAUAAGUAGGGUUUUCAUCAC[dT][dT] 05L174
GUGAUGAAAACCCUACUUAUU[dT][dT]
4A (SEQ ID NO: 360) 5 (SEQ ID NO: 734)
05L17 UCACAAUACCAAUGUUGAGGA[dT][dT] 05L175
UCCUCAACAUUGGUAUUGUGA[dT][dT]
5A (SEQ ID NO: 361) 5 (SEQ ID NO: 735)
05L17 UGUUUCUUGAUCACAAUACCA[dT][dT] 05L176
UGGUAUUGUGAUCAAGAAACA[dT][dT]
6A (SEQ ID NO: 362) 5 (SEQ ID NO: 736)
05L17 AACAAUCUGUUUCUUGAUCAC[dT][dT] 05L177
GUGAUCAAGAAACAGAUUGUU[dT][dT]
7A (SEQ ID NO: 363) 5 (SEQ ID NO: 737)
05L17 UUUACACAGAGAAACUCGGAA[dT][dT] (SEQ 05L178 UUCCGAGUUUCUCUGUGUAAA[dT][dT]
8A ID NO: 364) 5 (SEQ ID NO: 738)
05L17 AUUUACACAGAGAAACUCGGA[dT][dT] (SEQ 05L179 UCCGAGUUUCUCUGUGUAAAU[dT][dT]
9A ID NO: 365) 5 (SEQ ID NO: 739)
05L18 UUCUGAUUCUCAUCGUAGCCG[dT][dT] 05L180
CGGCUACGAUGAGAAUCAGAA[dT][dT]
OA (SEQ ID NO: 366) 5 (SEQ ID NO: 740)
05L18 AUUUUCAGAGCAAGUUCUCCU[dT][dT] 05L181
AGGAGAACUUGCUCUGAAAAU[dT][dT]
1A (SEQ ID NO: 367) 5 (SEQ ID NO: 741)
05L18 AUAUCUUUGGGAUACACAGUC[dT][dT] 05L182
GACUGUGUAUCCCAAAGAUAU[dT][dT]
2A (SEQ ID NO: 368) 5 (SEQ ID NO: 742)
05L18 AGGUAAACUGAUUCUGUUGGC[dT][dT] 05L183
GCCAACAGAAUCAGUUUACCU[dT][dT]
3A (SEQ ID NO: 369) 5 (SEQ ID NO: 743)
05L18 AUAGAAACUGGUUAAGGUGUC[dT][dT] 05L184
GACACCUUAACCAGUUUCUAU[dT][dT]
4A (SEQ ID NO: 370) 5 (SEQ ID NO: 744)
05L18 ACAAUAGAAACUGGUUAAGGU[dT][dT] 05L185
ACCUUAACCAGUUUCUAUUGU[dT][dT]
5A (SEQ ID NO: 371) 5 (SEQ ID NO: 745)
05L18 UCAUCAAUAUCAACACAAGUC[dT][dT] (SEQ 05L186 GACUUGUGUUGAUAUUGAUGA[dT][dT]
6A ID NO: 372) 5 (SEQ ID NO: 746)
05L18 AGAUGUAGGAGCCUAUUACAU[dT][dT] 05L187
AUGUAAUAGGCUCCUACAUCU[dT][dT]
7A (SEQ ID NO: 373) 5 (SEQ ID NO: 747)
05L18 UCGAUGUUACUGUUUUGCCGG[dT][dT] 05L188
CCGGCAAAACAGUAACAUCGA[dT][dT]
8A (SEQ ID NO: 374) 5 (SEQ ID NO: 748)
05L18 UUGCUAAAAAUGAGAUAGGGU[dT][dT] 05L189
ACCCUAUCUCAUUUUUAGCAA[dT][dT]
9A (SEQ ID NO: 375) 5 (SEQ ID NO: 749)
62
CA 03174172 2022- 9- 28
05L19 AUUUCUCAAAUAGUAACGGUU[dT][dT] OSL190
AACCGUUACUAUUUGAGAAAU[dT][dT]
OA (SEQ ID NO: 376) 5 (SEQ ID NO: 750)
05L19 AAUUUCUCAAAUAGUAACGGU[dT][dT] 05L191
ACCGUUACUAUUUGAGAAAUU[dT][dT]
1A (SEQ ID NO: 377) 5 (SEQ ID NO: 751)
05L19 AAAUUUCUCAAAUAGUAACGG[dT][dT] 05L192
CCGUUACUAUUUGAGAAAUUU[dT][dT]
2A (SEQ ID NO: 378) 5 (SEQ ID NO: 752)
05L19 AGUUAAAUUUCUCAAAUAGUA[dT][dT] 05L193
UACUAUUUGAGAAAUUUAACU[dT][dT]
3A (SEQ ID NO: 379) 5 (SEQ ID NO: 753)
05L19 AUCUAUAGUUAAAUUUCUCAA[dT][dT] 05L194
UUGAGAAAUUUAACUAUAGAU[dT][dT]
4A (SEQ ID NO: 380) 5 (SEQ ID NO: 754)
05L19 UAAAAAUAGCCAUCUAUAGUU[dT][dT] 05L195
AACUAUAGAUGGCUAUUUUUA[dT][dT]
5A (SEQ ID NO: 381) 5 (SEQ ID NO: 755)
05L19 AUCUAAUGCCACAACAUUGUC[dT][dT] (SEQ 05L196 GACAAUGUUGUGGCAUUAGAU[dT][dT]
6A ID NO: 382) 5 (SEQ ID NO: 756)
05L19 AAUCCAAUACAAUCUCUUCUC[dT][dT] (SEQ 05L197 GAGAAGAGAUUGUAUUGGAUU[dT][dT]
7A ID NO: 383) 5 (SEQ ID NO: 757)
05L19 ACAUUCUCUCAAUGACUUGCC[dT][dT] (SEQ 05L198 GGCAAGUCAUUGAGAGAAUGU[dT][dT]
8A ID NO: 384) 5 (SEQ ID NO: 758)
05L19 AUGAUUGUCUCCUUGUUUGUC[dT][dT] 05L199
GACAAACAAGGAGACAAUCAU[dT][dT]
9A (SEQ ID NO: 385) 5 (SEQ ID NO: 759)
05L20 AUUAUCACAGACUUGUUGGUU[dT][dT] 05L200
AACCAACAAGUCUGUGAUAAU[dT][dT]
OA (SEQ ID NO: 386) 5 (SEQ ID NO: 760)
05L20 UUCAAAAAUGGUAAUAGCGAA[dT][dT] 05L201
UUCGCUAUUACCAUUUUUGAA[dT][dT]
1A (SEQ ID NO: 387) 5 (SEQ ID NO: 761)
05L20 UCUUCAAAAAUGGUAAUAGCG[dT][dT] 05L202
CGCUAUUACCAUUUUUGAAGA[dT][dT]
2A (SEQ ID NO: 388) 5 (SEQ ID NO: 762)
05L20 AUUUGUUUCCCUUUUCCACUG[dT][dT] 05L203
CAGUGGAAAAGGGAAACAAAU[dT][dT]
3A (SEQ ID NO: 389) 5 (SEQ ID NO: 763)
05L20 AUUUGAUCCAUCAUAUUUGUU[dT][dT] 05L204
AACAAAUAUGAUGGAUCAAAU[dT][dT]
4A (SEQ ID NO: 390) 5 (SEQ ID NO: 764)
05L20 UAUAUGGAUGGUACACAUGGA[dT][dT] 05L205
UCCAUGUGUACCAUCCAUAUA[dT][dT]
5A (SEQ ID NO: 391) 5 (SEQ ID NO: 765)
05L20 AAAGAAGACGGUCUUCAUCAG[dT][dT] 05L206
CUGAUGAAGACCGUCUUCUUU[dT][dT]
6A (SEQ ID NO: 392) 5 (SEQ ID NO: 766)
05L20 UGAAUUCUGUGAAGUUGUCAC[dT][dT] 05L207
GUGACAACUUCACAGAAUUCA[dT][dT]
7A (SEQ ID NO: 393) 5 (SEQ ID NO: 767)
05L20 ACAAUGUCCACUUGUACACUG[dT][dT] (SEQ 05L208 CGGUGUACAAGUGGACAUUGU[dT][dT]
8A ID NO: 394) 5 (SEQ ID NO: 768)
05L20 ACACAAUGUCCACUUGUACAC[dT][dT] (SEQ 05L209 GUGUACAAGUGGACAUUGUGU[dT][dT]
9A ID NO: 395) 5 (SEQ ID NO: 769)
63
CA 03174172 2022- 9- 28
05L21 UUUUUGCAUUCGAACAUAGUA[dT][dT] 05L210
UACUAUGUUCGAAUGCAAAAA[dT][dT]
OA (SEQ ID NO: 396) 5 (SEQ ID NO: 770)
05L21 AUGGUUUUUGCAUUCGAACAU[dT][dT] 05L211
AUGUUCGAAUGCAAAAACCAU[dT][dT]
1A (SEQ ID NO: 397) 5 (SEQ ID NO: 771)
05L21 AUACAAACAUGGUUUUUGCAU[dT][dT] 05L212
AUGCAAAAACCAUGUUUGUAU[dT][dT]
2A (SEQ ID NO: 398) 5 (SEQ ID NO: 772)
05L21 AUCACAUUUCCAAUAUGGCGG[dT][dT] 05L213
CCGCCAUAUUGGAAAUGUGAU[dT][dT]
3A (SEQ ID NO: 399) 5 (SEQ ID NO: 773)
05L21 UGAAGUUCUUCAUCUGAACCA[dT][dT] 05L214
UGGUUCAGAUGAAGAACUUCA[dT][dT]
4A (SEQ ID NO: 400) 5 (SEQ ID NO: 774)
05L21 AUAAAUGCAGCGAUUGUUGUC[dT][dT] 05L215
GACAACAAUCGCUGCAUUUAU[dT][dT]
5A (SEQ ID NO: 401) 5 (SEQ ID NO: 775)
05L21 AUUCUGUACAAGGUUUAGGGG[dT][dT] 05L216
CCCCUAAACCUUGUACAGAAU[dT][dT]
6A (SEQ ID NO: 402) 5 (SEQ ID NO: 776)
05L21 UAUUCUGUACAAGGUUUAGGG[dT][dT] 05L217
CCCUAAACCUUGUACAGAAUA[dT][dT]
7A (SEQ ID NO: 403) 5 (SEQ ID NO: 777)
05L21 AUUCAUAUUCUGUACAAGGUU[dT][dT] 05L218
AACCUUGUACAGAAUAUGAAU[dT][dT]
8A (SEQ ID NO: 404) 5 (SEQ ID NO: 778)
05L21 UAUUCAUAUUCUGUACAAGGU[dT][dT] 05L219
ACCUUGUACAGAAUAUGAAUA[dT][dT]
9A (SEQ ID NO: 405) 5 (SEQ ID NO: 779)
05L22 UUAUAUUCAUAUUCUGUACAA[dT][dT] 05L220
UUGUACAGAAUAUGAAUAUAA[dT][dT]
OA (SEQ ID NO: 406) 5 (SEQ ID NO: 780)
05L22 UAUUGCAACCCAGUUCAUCGG[dT][dT] 05L221
CCGAUGAACUGGGUUGCAAUA[dT][dT]
1A (SEQ ID NO: 407) 5 (SEQ ID NO: 781)
05L22 AUAUUUUCAGCACAUGUUCUU[dT][dT] 05L222
AAGAACAUGUGCUGAAAAUAU[dT][dT]
2A (SEQ ID NO: 408) 5 (SEQ ID NO: 782)
05L22 UUAAUUGGGUACAAUUUUGCU[dT][dT] 05L223
AGCAAAAUUGUACCCAAUUAA[dT][dT]
3A (SEQ ID NO: 409) 5 (SEQ ID NO: 783)
05L22 AAAAACAUUGGUUUCGAACCC[dT][dT] (SEQ 05L224 GGGUUCGAAACCAAUGUUUUU[dT][dT]
4A ID NO: 410) 5 (SEQ ID NO: 784)
05L22 UGUCAAAAACAUUGGUUUCGA[dT][dT] 05L225
UCGAAACCAAUGUUUUUGACA[dT][dT]
5A (SEQ ID NO: 411) 5 (SEQ ID NO: 785)
05L22 UUCGAAUUCGGACAUUGUCAG[dT][dT] 05L226
CUGACAAUGUCCGAAUUCGAA[dT][dT]
6A (SEQ ID NO: 412) 5 (SEQ ID NO: 786)
05L22 AUUAUAUUUUCGAAUUCGGAC[dT][dT] 05L227
GUCCGAAUUCGAAAAUAUAAU[dT][dT]
7A (SEQ ID NO: 413) 5 (SEQ ID NO: 787)
05L22 AGAUUAUAUUUUCGAAUUCGG[dT][dT] 05L228
CCGAAUUCGAAAAUAUAAUCU[dT][dT]
8A (SEQ ID NO: 414) 5 (SEQ ID NO: 788)
05L22 UCAUCUUGAAGAUACUCUGAG[dT][dT] 05L229
CUCAGAGUAUCUUCAAGAUGA[dT][dT]
9A (SEQ ID NO: 415) 5 (SEQ ID NO: 789)
64
CA 03174172 2022- 9- 28
05L23 UAUAUUCCUCAUCUUGAAGAU[dT] [dT] 05L230
AUCUUCAAGAUGAGGAAUAUA[dT][dT]
OA (SEQ ID NO: 416) 5 (SEQ ID NO: 790)
05L23 UUUGAUAGCACCAAACCUAGAGCCC[dT] [dT 05L231 GGGCUCUAGGUUUGGUGCUAUCAAA[dT
1A-1 ] (SEQ ID NO: 417) 5-1 ][dT] (SEQ ID NO:
791)
05L23 UUUGAUAGCACCAAACCUAGAGCCC[dT]*[d 05L231 GGGCUCUAGGUUUGGUGCUAUCAAA[dT
1A-2 T] (SEQ ID NO: 418) 5-2 ]*[dT] (SEQ ID NO:
792)
[mU][mU][mU][mG][mA][mU][mA][mG][m
[mG][mG][mG][mC][mU][mC][mU][mA][
C][mA][mC][mC][mA][mA][mA][mC][mC][m
mG][mG][mU][mU][mU][mG][mG][mU][
05L23 U][mA][mG][mA][mG][mC][mC][mC][dT]*[d 05L231
mG][mC][mU][mA][mU][mC][mA][mA][
1A-3 T] (SEQ ID NO: 419) 5-3 mA][dT]*[dT] (SEQ ID
NO: 793)
[mU][mU][mU][mG][mA][mU][mA][mG][m
[mG][mG][mG][mC][mU][mC][mU][mA][
C][mA][mC][mC][mA][mA][mA][mC][mC][m
mG][mG][mU][mU][mU][mG][mG][mU][
05L23 U][mA][mG][mA][mG][mC][mC][mC][dT]*[d 05L231
mG][mC][mU][mA][mU][mC][mA][mA][
1A-4 T] (SEQ ID NO: 420) 5-4 mA] (SEQ ID NO: 794)
[mU][mU][mU][mG][mA][mU][mA][mG][m
C][mA][mC][mC][mA][mA][mA][mC][mC][m
[mG][mG][mG][mC][mU][mC][mU][mA][
U][mA][mG][mA][mG][mC][mC][mC] (SEQ
mG][mG][mU][mU][mU][mG][mG][mU][
05L23 ID NO: 421) 05L231
mG][mC][mU][mA][mU][mC][mA][mA][
1A-5 5-5 mA][dT]*[dT] (SEQ ID
NO: 795)
[mU][mU][mU][mG][mA][mU][mA][mG][m
[mG][mG][mG][mC][mU][mC][mU][mA][
C][mA][mC][mC][mA][mA][mA][mC][mC][m
mG][mG][mU][mU][mU][mG][mG][mU][
05L23 U][mA][mG][mA][mG][mC][mC][mC] (SEQ 05L231
mG][mC][mU][mA][mU][mC][mA][mA][
1A-6 ID NO: 422) 5-6 mA] (SEQ ID NO: 796)
[mU][mU][mU][mG][mA][mU][mA][mG][m
GGGCUCUAGGUUUGGUGCUAUCAAA[dT
C][mA][mC][mC][mA][mA][mA][mC][mC][m ]*[dT] (SEQ ID NO:
797)
05L23 U][mA][mG][mA][mG][mC][mC][mC][dT]*[d 05L231
1A-7 T] (SEQ ID NO: 423) 5-7
[mU][2fU][mU][2fG][mA][2fU][mA][2fG][m
[2fG][mG][2fG][mC][2fU][mC][2fU][mA][
C][2fA][mC][2fC][mA][2fA][mA][2fC][mC][2f
2fG][mG][2fU][mU][2fU][mG][2fG][mU][
05L23 U][mA][2fG][mA][2fG][mC][2fC][mC][dT]*[ 05L231
2fG][mC][2fU][mA][2fU][mC][2fA][mA][2
1A-8 dT] (SEQ ID NO: 424) 5-8 fA][dT]*[dT] (SEQ ID
NO: 798)
[mU][2fU][mU][2fG][mA][2fU][mA][2fG][m
[2fG][mG][2fG][mC][2fU][mC][2fU][mA][
C][2fA][mC][2fC][mA][2fA][mA][2fC][mC][2f
2fG][mG][2fU][mU][2fU][mG][2fG][mU][
05L23 U][mA][2fG][mA][2fG][mC][2fC][mC] (SEQ 05L231
2fG][mC][2fU][mA][2fU][mC][2fA][mA][2
1A-9 ID NO: 425) 5-9 fA][dT]*[dT] (SEQ ID
NO: 799)
[mU][2fU][mU][2fG][mA][2fU][mA][2fG][m
C][2fA][mC][2fC][mA][2fA][mA][2fC][mC][2f
[2fG][mG][2fG][mC][2fU][mC][2fU][mA][
U][mA][2fG][mA][2fG][mC][2fC][mC][dT]*[
2fG][mG][2fU][mU][2fU][mG][2fG][mU][
05L23 dT] (SEQ ID NO: 426) 05L231
2fG][mC][2fU][mA][2fU][mC][2fA][mA][2
1A-10 S-W fA] (SEQ ID NO: 800)
[mU][2fU][mU][2fG][mA][2fU][mA][2fG][m
[2fG][mG][2fG][mC][2fU][mC][2fU][mA][
C][2fA][mC][2fC][mA][2fA][mA][2fC][mC][2f
2fG][mG][2fU][mU][2fU][mG][2fG][mU][
05L23 U][mA][2fG][mA][2fC][mC][2fC][mC] (SEQ 05L231
2fG][mC][2fU][mA][2fU][mC][2fA][mA][2
1A-11 ID NO: 427) 5-11 fA] (SEQ ID NO: 801)
CA 03174172 2022- 9- 28
[2fU][mU][2fU][mG][2fA][mU][2fA][mG][2f
[mG][2fG][mG][2fC][mU][2fC][mU][2fA][
C][mA][2fC][mC][2fA][mA][2fA][mC][2fC][m
mG][2fG][mU][2fU][mU][2fG][mG][2Fu][
05L23 U][2fA][mG][2fA][mG][2fC][mC][2fC][dT]*[ 05L231
mG][2fC][mU][2fA][mU][2fC][mA][2fA][
1A-12 dT] (SEQ ID NO: 428) S-12 mA][dT]*[dT] (SEQ ID
NO: 802)
[mU][2fA][mU][2fC][mA][2fA][mA][2fC][mC
[2fG][mG][2fG][mC][2fU][mC][2fU][mA][
][2fU][mC][2fG][mA][2fU][mA][2fG][mC][2f
2fG][mG][2fU][mU][2fU][mG][2fG][mU][
05L23 A][mA][2fC][mA][2fC][mC][2fG][mC] (SEQ 05L231
2fG][mC][2fU][mA][2fU][mC][2fA][mA][2
1A-13 ID NO: 429) S-13 fALINKER-LIG (SEQ ID
NO: 803)
[mU][2fU][2fU][2fG][2fA][2fU][2fA][2fG][2f
C][2fA][2fC][2fC][2fA][2fA][2fA][2fC][2fC][2f
[2fU][mC][2fU][mA][2fG][mG][2fU][mU][
U][2fA][2fG][2fA][2fG][2fC][2fC][2fC][dT]*[
2fU][mG][2fG][mU][2fG][mC][2fU][mA][
05L23 dT] (SEQ ID NO: 430) 05L231
2fU][mC][2fA][mA][2fA][dT]*[dT] (SEQ
1A-14 S-14 ID NO: 804)
[mU][2fU][2fU][2fG][2fA][2fU][2fA][2fG][2f
C][2fA][2fC][2fC][2fA][2fA][2fA][2fC][2fC][2f
[2fU][mU][2fU][mG][2fG][mU][2fG][mC][
05L23 U][2fA][2fG][2fA][2fG][2fC][2fC][2fC][dT]*[ 05L231
2fU][mA][2fU][mC][2fA][mA][2fA][dT]*[
1A-15 dT] (SEQ ID NO: 431) 5-15 dT] (SEQ ID NO: 805)
[mU][2fU][mU][2fG][mA][2fU][mA][2fG][m
C][2fA][mC][2fC][mA][2fA][mA][2fC][mC][2f
[2fU][mU][2fU][mG][2fG][mU][2fG][mC][
05L23 U][mA][2fG][mA][2fG][mC][2fC][mC][dT]*[ 05L231
2fU][mA][2fU][mC][2fA][mA][2fA][dT]*[
1A-16 dT] (SEQ ID NO: 432) S-16 dT] (SEQ ID NO: 806)
[mU][2fU][mU][2fG][mA][2fU][mA][2fG][m
[2fU][mU][2fU][mG][2fG][mU][2fG][mC][
C][2fA][mC][2fC][mA][2fA][mA][2fC][mC][2f
2fU][mA][2fU][mC][2fA][mA][2fA] (SEQ
05L23 U][mA][2fG][mA][2fG][mC] (SEQ ID NO: 05L231 ID NO:
807)
1A-17 433) S-17
[mU][2fU][mU][2fG][mA][2fU][mA][2fG][m
[2fU][mU][2fU][mG][2fG][mU][2fG][mC][
C][2fA][mC][2fC][mA][2fA][mA][2fC][2fC] [2f
2fU][mA][2fU][mC][2fA][mA][2fA] (SEQ
05L23 U][2fA][2fG][2fA][2fG][2fC] (SEQ ID NO: 05L231 ID NO:
808)
1A-18 434) S-18
05L23 UCAAAGUUGGGGAUGUAGGCA[dT][dT] 05L232
UGCCUACAUCCCCAACUUUGA[dT][dT]
2A (SEQ ID NO: 435) 5 (SEQ ID NO: 809)
05L23 UCAGUUUCAGGUCAACUUCCU[dT][dT] 05L233
AGGAAGUUGACCUGAAACUGA[dT][dT]
3A (SEQ ID NO: 436) 5 (SEQ ID NO: 810)
05L23 UACGUAUUUCAGUUUCAGGUC[dT][dT] 05L234
GACCUGAAACUGAAAUACGUA[dT][dT]
4A (SEQ ID NO: 437) 5 (SEQ ID NO: 811)
05L23 UUACGUAUUUCAGUUUCAGGU[dT][dT] 05L235
ACCUGAAACUGAAAUACGUAA[dT][dT]
5A (SEQ ID NO: 438) 5 (SEQ ID NO: 812)
05L23 AGUUUAGCCACCUCAAUGCGU[dT][dT] 05L236
ACGCAUUGAGGUGGCUAAACU[dT][dT]
6A (SEQ ID NO: 439) 5 (SEQ ID NO: 813)
05L23 AACAUAAGCCCUAGUUUGGGA[dT][dT] 05L237
UCCCAAACUAGGGCUUAUGUU[dT][dT]
7A (SEQ ID NO: 440) 5 (SEQ ID NO: 814)
05L23 UCGAUUUUAGGUUCCUUUCCC[dT][dT] 05L238
GGGAAAGGAACCUAAAAUCGA[dT][dT]
8A (SEQ ID NO: 441) 5 (SEQ ID NO: 815)
66
CA 03174172 2022- 9- 28
05L23 UCGAAAACCAGGAUGUUGCGG[dT][dT] 05L239
CCGCAACAUCCUGGUUUUCGA[dT][dT]
9A (SEQ ID NO: 442) 5 (SEQ ID NO: 816)
05L24 UCAAAUAAUCGAUAGAAAGGC[dT][dT] 05L240
GCCUUUCUAUCGAUUAUUUGA[dT][dT]
OA (SEQ ID NO: 443) 5 (SEQ ID NO: 817)
05L24 UUGUUCAAAUAAUCGAUAGAA[dT][dT] 05L241
UUCUAUCGAUUAUUUGAACAA[dT][dT]
1A (SEQ ID NO: 444) 5 (SEQ ID NO: 818)
05L24 UUAUGGUUUCAAUAACGUCCU[dT][dT] 05L242
AGGACGUUAUUGAAACCAUAA[dT][dT]
2A (SEQ ID NO: 445) 5 (SEQ ID NO: 819)
05L24 UUUUAUGGUUUCAAUAACGUC[dT][dT] 05L243
GACGUUAUUGAAACCAUAAAA[dT][dT]
3A (SEQ ID NO: 446) 5 (SEQ ID NO: 820)
05L24 AUUUUAUGGUUUCAAUAACGU[dT][dT] 05L244
ACGUUAUUGAAACCAUAAAAU[dT][dT]
4A (SEQ ID NO: 447) 5 (SEQ ID NO: 821)
05L24 UUUGCAAUGACUCUCCUAUCAGUCC[dT][dT 05L245 GGACUGAUAGGAGAGUCAUUGCAAA[dT]
5A-1 ] (SEQ ID NO: 448) 5-1 [dT] (SEQ ID NO: 822)
UUUGCAAUGACUCUCCUAUCAGUCC[dT]*[d
05L24 T] (SEQ ID NO: 449) 05L245
GGACUGAUAGGAGAGUCAUUGCAA]A[dT
5A-2 S-2 ]*[dT] (SEQ ID NO:
823)
[mU][mU][mU][mG][mC][mA][mA][mU][m
[mG][mG][mA][mC][mU][mG][mA][mU][
G][mA][mC][mU][mC][mU][mC][mC][mU][
mA][mG][mG][mA][mG][mA][mG][mU][
05L24 mA][mU][mC][mA][mG][mU][mC][mC][dT]* 05L245
mC][mA][mU][mU][mG][mC][mA][mA][
5A-3 [dT] (SEQ ID NO: 450) S-3 mA][dT]*[dT] (SEQ ID
NO: 824)
[mU][mU][mU][mG][mC][mA][mA][mU][m
G][mA][mC][mU][mC][mU][mC][mC][mU][
[mG][mG][mA][mC][mU][mG][mA][mU][
mA][mU][mC][mA][mG][mU][mC][mC][dT]*
mA][mG][mG][mA][mG][mA][mG][mU][
05L24 [dT] (SEQ ID NO: 451) 05L245
mC][mA][mU][mU][mG][mC][mA][mA][
5A-4 S-4 mA](SEQ ID NO: 825)
[mU][mU][mU][mG][mC][mA][mA][mU][m
[mG][mG][mA][mC][mU][mG][mA][mU][
G][mA][mC][mU][mC][mU][mC][mC][mU][
mA][mG][mG][mA][mG][mA][mG][mU][
05L24 mA][mU][mC][mA][mG][mU][mC][mC] (SEQ 05L245
mC][mA][mU][mU][mG][mC][mA][mA][
5A-5 ID NO: 452) 5-5 mA][dT]*[dT](SEQ ID
NO: 826)
[mU][mU][mU][mG][mC][mA][mA][mU][m
[mG][mG][mA][mC][mU][mG][mA][mU][
G][mA][mC][mU][mC][mU][mC][mC][mU][
mA][mG][mG][mA][mG][mA][mG][mU][
05L24 mA][mU][mC][mA][mG][mU][mC][mC] (SEQ 05L245
mC][mA][mU][mU][mG][mC][mA][mA][
5A-6 ID NO: 453) S-6 mA] (SEQ ID NO: 827)
[mU][mU][mU][mG][mC][mA][mA][mU][m
GGACUGAUAGGAGAGUCAUUGCAAA[dT]
G][mA][mC][mU][mC][mU][mC][mC][mU][ *[dT] (SEQ ID NO:
828)
05L24 mA][mU][mC][mA][mG][mU][mC][mC][dT]* 05L245
5A-7 [dT] (SEQ ID NO: 454) S-7
[mU][2fU][mU][2fG][mC][2fA][mA][2fU][m
[2fG][mG][2fA][mC][2fU][mG][2fA][mU][
G][2fA][mC][2fU][mC][2fU][mC][2fC][mU][2
2fA][mG][2fG][mA][2fG][mA][2fG][mU][
05L24 fA][mU][2fC][mA][2fG][mU][2fC][mC][dT]*[ 05L245
2fC][mA][2fU][mU] [2fG][mC][2fA][mA] [2
5A-8 dT] (SEQ ID NO: 455) S-8 fA][dT]*[dT] (SEQ ID
NO: 829)
67
CA 03174172 2022- 9- 28
[mU][2fU][mU][2fG][mC][2fA][mA][2fU][m
[2fG][mG][2fA][mC][2fU][mG][2fA][mU][
G][2fA][mC][2fU][mC][2fU][mC][2fC][mU][2
2fA][mG][2fG][mA][2fG][mA][2fG][mU][
05L24 fA][mU][2fC][mA][2fG][mU][2fC][mC] (SEQ 05L245
2fC][mA][2fU][mU] [2fG][mC][2fA][mA] [2
5A-9 ID NO: 456) S-9 fA][dT]*[dT] (SEQ ID
NO: 830)
[mU][2fU][mU][2fG][mC][2fA][mA][2fU][m
[2fG][mG][2fA][mC][2fU][mG][2fA][mU][
G][2fA][mC][2fU][mC][2fU][mC][2fC][mU][2
2fA][mG][2fG][mA][2fG][mA][2fG][mU][
05L24 fA][mU][2fC][mA][2fG][mU][2fC][mC][dT]*[ 05L245
2fC][mA][2fU][mU] [2fG][mC][2fA][mA] [2
5A-10 dT] (SEQ ID NO: 457) S-W fA] (SEQ ID NO: 831)
[mU][2fU][mU][2fG][mC][2fA][mA][2fU][m
[2fG][mG][2fA][mC][2fU][mG][2fA][mU][
G][2fA][mC][2fU][mC][2fU][mC][2fC][mU][2
2fA][mG][2fG][mA][2fG][mA][2fG][mU][
05L24 fA][mU][2fC][mA][2fC][mU][2fC][mC] (SEQ 05L245
2fC][mA][2fU][mU] [2fG][mC][2fA][mA] [2
5A-11 ID NO: 458) 5-11 fA] (SEQ ID NO: 832)
[2fU][mU][2fU][mG][2fC][mA][2fA][mU][2f
[mG][2fG][mA][2fC][mU][2fG][mA][2fU][
G][mA][2fC][mU][2fC][mU][2fC][mC][2fU][
mA][2fG][mG][2fA][mG][2fA][mG][2fU][
05L24 mA][2fU][mC][2fA][mG][2fU][mC][2fC][dT]* 05L245
mC][2fA][mU][2fU][mG][2fC][mA][2fA][
5A-12 [dT] (SEQ ID NO: 459) S-12 mA][dT]*[dT] (SEQ ID
NO: 833)
[mU][2fA][mU][2fC][mC][2fU][mA][2fA][mG
[2fG][mG][2fA][mC][2fU][mG][2fA][mU][
][2fU][mC][2fA][mC][2fA][mC][2fG][mU][2f
2fA][mG][2fG][mA][2fG][mA][2fG][mU][
05L24 U][mU][2fG][mA][2fC][mU][2fG][mC] (SEQ 05L245
2fC][mA][2fU][mU] [2fG][mC][2fA][mA] [2
5A-13 ID NO: 460) S-13 fA] (SEQ ID NO: 834)
[mU][2fU][2fU][2fG][2fC][2fA][2fA][2fU][2f
[2fU][mG][2fA][mU][2fA][mG][2fG][mA][
G][2fA][2fC][2fU][2fC][2fU][2fC][2fC][2fU][2
2fG][mA][2fG][mU][2fC][mA][2fU][mU][
05L24 fA][2fU][2fC][2fA][2fG][2fU][2fC][2fC][dT]*[ 05L245
2fG][mC][2fA][mA][2fA][dT]*[dT] (SEQ ID
5A-14 dT] (SEQ ID NO: 461) S-14 NO: 835)
[mU][2fU][2fU][2fG][2fC][2fA][2fA][2fU][2f
[2fG][mA][2fG][mA][2fG][mU][2fC][mA][
G][2fA][2fC][2fU][2fC][2fU][2fC][2fC][2fU][2
2fU][mU][2fG][mC][2fA][mA][2fA][dT]*[
05L24 fA][2fU][2fC][2fA][2fG][2fU][2fC][2fC][dT]*[ 05L245 dT]
(SEQ ID NO: 836)
5A-15 dT] (SEQ ID NO: 462) 5-15
[mU][2fU][mU][2fG][mC][2fA][mA][2fU][m
[2fG][mA][2fG][mA][2fG][mU][2fC][mA][
G][2fA][mC][2fU][mC][2fU][mC][2fC][mU][2
2fU][mU][2fG][mC][2fA][mA][2fA][dT]*[
05L24 fA][mU][2fC][mA][2fG][mU][2fC][mC][dT]*[ 05L245 dT] (SEQ
ID NO: 837)
5A-16 dT] (SEQ ID NO: 463) S-16
[mU][2fU][mU][2fG][mC][2fA][mA][2fU][m
[2fG][mA][2fG][mA][2fG][mU][2fC][mA][
G][2fA][mC][2fU][mC][2fU][mC][2fC][mU][2
2fU][mU][2fG][mC][2fA][mA][2fA] (SEQ
05L24 fA][mU][2fC][mA][2fG][mU] (SEQ ID NO: 05L245 ID NO:
838)
5A-17 464) S-17
[mU][2fU][mU][2fG][mC][2fA][mA][2fU][m
ACCAGUUAUACUGGAUAUCUA[dT][dT]
G][2fA][mC][2fU][mC][2fU][mC][2fC][2fU][2 (SEQ ID NO: 840)
05L24 fA][2fU][2fC][2fA][2fG][2fU] (SEQ ID NO: 05L245
5A-18 465) S-18
05L24 UAGAUAUCCAGUAUAACUGGU[dT][dT] 05L246
GGGAGAAGUAUGGAAACAAAA[dT][dT]
6A (SEQ ID NO: 466) 5 (SEQ ID NO: 841)
05L24 UUUUGUUUCCAUACUUCUCCC[dT][dT] 05L247
AAGUAUGGAAACAAAAUAAAU[dT][dT]
7A (SEQ ID NO: 467) 5 (SEQ ID NO: 842)
68
CA 03174172 2022- 9- 28
05L24 AUUUAUUUUGUUUCCAUACUU[dT][dT] 05L248
UUCAUCAACUCAGAUACAAUA[dT][dT]
8A (SEQ ID NO: 468) 5 (SEQ ID NO: 843)
05L24 UAUUGUAUCUGAGUUGAUGAA[dT][dT] 05L249
CGGAGGAAAUUGCUAUUUUGA[dT][dT]
9A (SEQ ID NO: 469) 5 (SEQ ID NO: 844)
05L25 UCAAAAUAGCAAUUUCCUCCG[dT][dT] (SEQ 05L250 AGGAAAUUGCUAUUUUGAUGA[dT][dT]
OA ID NO: 470) 5 (SEQ ID NO: 845)
05L25 UCAUCAAAAUAGCAAUUUCCU[dT][dT] (SEQ 05L251 CACCGGAAAAUAUUGUGAAAU[dT][dT]
1A ID NO: 471) 5 (SEQ ID NO: 846)
05L25 AUUUCACAAUAUUUUCCGGUG[dT][dT] 05L252
UUGUGAAAUGGCGUUUUCAAA[dT][dT]
2A (SEQ ID NO: 472) 5 (SEQ ID NO: 847)
05L25 UUUGAAAACGCCAUUUCACAA[dT][dT] (SEQ 05L253 CAGGAUUCUUCCACUAUAGAA[dT][dT]
3A ID NO: 473) 5 (SEQ ID NO: 848)
05L25 UUCUAUAGUGGAAGAAUCCUG[dT][dT] 05L254
GGCAGAUCUUAACAUGGAUAU[dT][dT]
4A (SEQ ID NO: 474) 5 (SEQ ID NO: 849)
05L25 AUAUCCAUGUUAAGAUCUGCC[dT][dT] 05L255
GGCAAUGAGUGAAGACUUUGU[dT][dT]
5A (SEQ ID NO: 475) 5 (SEQ ID NO: 850)
05L25 ACAAAGUCUUCACUCAUUGCC[dT][dT] (SEQ 05L256 AUCUGAAAAUGUGGAUAAUAA[dT][dT]
6A ID NO: 476) 5 (SEQ ID NO: 851)
05L25 UUAUUAUCCACAUUUUCAGAU[dT][dT] 05L257
ACCAGUUAUACUGGAUAUCUA[dT][dT]
7A (SEQ ID NO: 477) 5 (SEQ ID NO: 840)
05L25 UCUUAUUAUCCACAUUUUCAG[dT][dT] 05L258
CUGAAAAUGUGGAUAAUAAGA[dT][dT]
8A (SEQ ID NO: 478) 5 (SEQ ID NO: 852)
05L25 UCCAUAAUUCUUAUUAUCCAC[dT][dT] 05L259
GUGGAUAAUAAGAAUUAUGGA[dT][dT]
9A (SEQ ID NO: 479) 5 (SEQ ID NO: 853)
05L26 UUCCAUAAUUCUUAUUAUCCA[dT][dT] 05L260
UGGAUAAUAAGAAUUAUGGAA[dT][dT]
OA (SEQ ID NO: 480) 5 (SEQ ID NO: 854)
05L26 UUUUCGUUUGAAGAGAUUCCA[dT][dT] 05L261
UGGAAUCUCUUCAAACGAAAA[dT][dT]
1A (SEQ ID NO: 481) 5 (SEQ ID NO: 855)
05L26 UAGAUUUUCGUUUGAAGAGAU[dT][dT] 05L262
AUCUCUUCAAACGAAAAUCUA[dT][dT]
2A (SEQ ID NO: 482) 5 (SEQ ID NO: 856)
05L26 UUUAGAUUUUCGUUUGAAGAG[dT][dT] 05L263
CUCUUCAAACGAAAAUCUAAA[dT][dT]
3A (SEQ ID NO: 483) 5 (SEQ ID NO: 857)
05L26 UUGUUUAGAUUUUCGUUUGAA[dT][dT] 05L264 UUCAAACGAAAAUCUAAACAA[dT][dT]
4A (SEQ ID NO: 484) 5 (SEQ ID NO: 858)
05L26 UAGUUUGUUUAGAUUUUCGUU[dT][dT] 05L265 AACGAAAAUCUAAACAAACUA[dT][dT]
5A (SEQ ID NO: 485) 5 (SEQ ID NO: 859)
05L26 UUUCAAAGUUGGUAGUUUGUU[dT][dT] 05L266
AACAAACUACCAACUUUGAAA[dT][dT]
6A (SEQ ID NO: 486) 5 (SEQ ID NO: 860)
05L26 AUUGGAUUUUCAAAGUUGGUA[dT][dT] 05L267
UACCAACUUUGAAAAUCCAAU[dT][dT]
7A (SEQ ID NO: 487) 5 (SEQ ID NO: 861)
69
CA 03174172 2022- 9- 28
05L26 AUAGAUUGGAUUUUCAAAGUU[dT][dT] 05L268 AACUUUGAAAAUCCAAUCUAU
[dT][dT]
8A (SEQ ID NO: 488) 5 (SEQ ID NO: 862)
05L26 UUAAAAGUGUCUUCUGUUGCA[dT][dT] 05L269
UGCAACAGAAGACACUUUUAA[dT][dT]
9A (SEQ ID NO: 489) 5 (SEQ ID NO: 863)
05L27 UCUUUAACAAGAUUUGCGGUG[dT][dT] 05L270
CACCGCAAAUCUUGUUAAAGA[dT][dT]
OA (SEQ ID NO: 490) 5 (SEQ ID NO: 864)
05L27 UCUUCUUUAACAAGAUUUGCG[dT][dT] 05L271
CGCAAAUCUUGUUAAAGAAGA[dT][dT]
1A (SEQ ID NO: 491) 5 (SEQ ID NO: 865)
05L27 UGGUAUAGCUAUACUUCAGAG[dT][dT] 05L272
CUCUGAAGUAUAGCUAUACCA[dT][dT]
2A (SEQ ID NO: 492) 5 (SEQ ID NO: 866)
05L27 AUUAUUCCCUAAAUAGCUGGU[dT][dT] 05L273
ACCAGCUAUUUAGGGAAUAAU[dT][dT]
3A (SEQ ID NO: 493) 5 (SEQ ID NO: 867)
05L27 UAAUUAUUCCCUAAAUAGCUG[dT][dT] 05L274
CAGCUAUUUAGGGAAUAAUUA[dT][dT]
4A (SEQ ID NO: 494) 5 (SEQ ID NO: 868)
05L27 AUAUAUGUGCAAAAGUGUGUU[dT][dT] 05L275
AACACACUUUUGCACAUAUAU[dT][dT]
5A (SEQ ID NO: 495) 5 (SEQ ID NO: 869)
05L27 AAAUAUAUGUGCAAAAGUGUG[dT][dT] 05L276
CACACUUUUGCACAUAUAUUU[dT][dT]
6A (SEQ ID NO: 496) 5 (SEQ ID NO: 870)
05L27 AACUUUUUUCAUCUGUUUGUA[dT][dT] 05L277
UACAAACAGAUGAAAAAAGUU[dT][dT]
7A (SEQ ID NO: 497) 5 (SEQ ID NO: 871)
05L27 UAAAGUACUGAAUGUUAACUU[dT][dT] 05L278
AAGUUAACAUUCAGUACUUUA[dT][dT]
8A (SEQ ID NO: 498) 5 (SEQ ID NO: 872)
05L27 UUUUUUUCAUAAAGUACUGAA[dT][dT] 05L279
UUCAGUACUUUAUGAAAAAAA[dT][dT]
9A (SEQ ID NO: 499) 5 (SEQ ID NO: 873)
05L28 UAUUUUUUUCAUAAAGUACUG[dT][dT] 05L280
CAGUACUUUAUGAAAAAAAUA[dT][dT]
OA (SEQ ID NO: 500) 5 (SEQ ID NO: 874)
05L28 AUUUGUAAAAAUAUGAGACGG[dT][dT] 05L281
CCGUCUCAUAUUUUUACAAAU[dT][dT]
1A (SEQ ID NO: 501) 5 (SEQ ID NO: 875)
05L28 ACAUUGUGAUAAUUAUUUGUA[dT][dT] 05L282
UACAAAUAAUUAUCACAAUGU[dT][dT]
2A (SEQ ID NO: 502) 5 (SEQ ID NO: 876)
05L28 AUACAUAUAGUACAUUGUGAU[dT][dT] 05L283
AUCACAAUGUACUAUAUGUAU[dT][dT]
3A (SEQ ID NO: 503) 5 (SEQ ID NO: 877)
05L28 AUAUACAUAUAGUACAUUGUG[dT][dT] 05L284
CACAAUGUACUAUAUGUAUAU[dT][dT]
4A (SEQ ID NO: 504) 5 (SEQ ID NO: 878)
05L28 AAAGAUAUACAUAUAGUACAU[dT][dT] 05L285
AUGUACUAUAUGUAUAUCUUU[dT][dT]
5A (SEQ ID NO: 505) 5 (SEQ ID NO: 879)
05L28 AUUACCUUCAGACAACUUCAG[dT][dT] (SEQ 05L286 CUGAAGUUGUCUGAAGGUAAU[dT][dT]
6A ID NO: 506) 5 (SEQ ID NO: 880)
05L28 UAUUUAUAGUAUUACCUUCAG[dT][dT] 05L287
CUGAAGGUAAUACUAUAAAUA[dT][dT]
7A (SEQ ID NO: 507) 5 (SEQ ID NO: 881)
CA 03174172 2022- 9- 28
05L28 UAAUCUUUCCAAAAUUUACAA[dT][dT] 05L288
UUGUAAAUUUUGGAAAGAUUA[dT][dT]
8A (SEQ ID NO: 508) 5 (SEQ ID NO: 882)
05L28 AGUAACAGGAUAAUCUUUCCA[dT][dT] 05L289
UGGAAAGAUUAUCCUGUUACU[dT][dT]
9A (SEQ ID NO: 509) 5 (SEQ ID NO: 883)
05L29 AUUCAGUAACAGGAUAAUCUU[dT][dT] 05L290
AAGAUUAUCCUGUUACUGAAU[dT][dT]
OA (SEQ ID NO: 510) 5 (SEQ ID NO: 884)
05L29 UAGCAAAUUCAGUAACAGGAU[dT][dT] 05L291
AUCCUGUUACUGAAUUUGCUA[dT][dT]
1A (SEQ ID NO: 511) 5 (SEQ ID NO: 885)
05L29 UUAGCAAAUUCAGUAACAGGA[dT][dT] 05L292
UCCUGUUACUGAAUUUGCUAA[dT][dT]
2A (SEQ ID NO: 512) 5 (SEQ ID NO: 886)
05L29 UCUUUAUUAGCAAAUUCAGUA[dT][dT] 05L293
UACUGAAUUUGCUAAUAAAGA[dT][dT]
3A (SEQ ID NO: 513) 5 (SEQ ID NO: 887)
05L29 AUCAUUUACUAUAAUGAUCAC[dT][dT] 05L294
GUGAUCAUUAUAGUAAAUGAU[dT][dT]
4A (SEQ ID NO: 514) 5 (SEQ ID NO: 888)
05L29 UUCUUGUUGGAUCAUUUACUA[dT][dT] 05L295
UAGUAAAUGAUCCAACAAGAA[dT][dT]
5A (SEQ ID NO: 515) 5 (SEQ ID NO: 889)
05L29 UCAAUUCCUUUUCUUGUUGGA[dT][dT] 05L296
UCCAACAAGAAAAGGAAUUGA[dT][dT]
6A (SEQ ID NO: 516) 5 (SEQ ID NO: 890)
05L29 AUUUUAUAGGAAAUAUGAGUG[dT][dT] 05L297
CACUCAUAUUUCCUAUAAAAU[dT][dT]
7A (SEQ ID NO: 517) 5 (SEQ ID NO: 891)
05L29 UAAUUUUAUAGGAAAUAUGAG[dT][dT] 05L298
CUCAUAUUUCCUAUAAAAUUA[dT][dT]
8A (SEQ ID NO: 518) 5 (SEQ ID NO: 892)
05L29 UGCUAAUGUGUAAAAAUGGAC[dT][dT] 05L299
GUCCAUUUUUACACAUUAGCA[dT][dT]
9A (SEQ ID NO: 519) 5 (SEQ ID NO: 893)
05L30 UUGAACAUUAAUUAAGUGCUA[dT][dT] 05L300
UAGCACUUAAUUAAUGUUCAA[dT][dT]
OA (SEQ ID NO: 520) 5 (SEQ ID NO: 894)
05L30 AUUGAACAUUAAUUAAGUGCU[dT][dT] 05L301
AGCACUUAAUUAAUGUUCAAU[dT][dT]
1A (SEQ ID NO: 521) 5 (SEQ ID NO: 895)
05L30 AUAUUGAACAUUAAUUAAGUG[dT][dT] 05L302
CACUUAAUUAAUGUUCAAUAU[dT][dT]
2A (SEQ ID NO: 522) 5 (SEQ ID NO: 896)
05L30 AAAUUGACAUGUAAUAUUGAA[dT][dT] 05L303
UUCAAUAUUACAUGUCAAUUU[dT][dT]
3A (SEQ ID NO: 523) 5 (SEQ ID NO: 897)
05L30 AUCAACAUAGCCAUUAAUCAA[dT][dT] (SEQ 05L304 UUGAUUAAUGGCUAUGUUGAU[dT][dT]
4A ID NO: 524) 5 (SEQ ID NO: 898)
05L30 UCUAUACAACACAUAGUGGCC[dT][dT] (SEQ 05L305 GGCCACUAUGUGUUGUAUAGA[dT][dT]
5A ID NO: 525) 5 (SEQ ID NO: 899)
05L30 AUGUCUAUACAACACAUAGUG[dT][dT] 05L306
CACUAUGUGUUGUAUAGACAU[dT][dT]
6A (SEQ ID NO: 526) 5 (SEQ ID NO: 900)
05L30 ACUGAAUUGCUUUUCCUACCU[dT][dT] 05L307
AGGUAGGAAAAGCAAUUCAGU[dT][dT]
7A (SEQ ID NO: 527) 5 (SEQ ID NO: 901)
71
CA 03174172 2022- 9- 28
OSL30 AAAUAAAAAUGUUGUCUUGGC[dT] [dT] 05L308
GCCAAGACAACAUUUUUAUUU[dT] [dT]
8A (SEQ ID NO: 528) 5 (SEQ ID NO: 902)
05L30 AUCACAAAUAAAAAUGUUGUC[dT][dT] 05L309
GACAACAUUUUUAUUUGUGAU[dT][dT]
9A (SEQ ID NO: 529) 5 (SEQ ID NO: 903)
05L31 AAUGAUAUGGGAUUUCCUCAU[dT][dT] 05L310
AUGAGGAAAUCCCAUAUCAUU[dT][dT]
OA (SEQ ID NO: 530) 5 (SEQ ID NO: 904)
05L31 AUUAACCACAAACUCAAUGCA[dT][dT] (SEQ 05L311 UGCAUUGAGUUUGUGGUUAAU[dT][dT]
1A ID NO: 531) 5 (SEQ ID NO: 905)
05L31 UUUAAUUAACCACAAACUCAA[dT][dT] (SEQ 05L312 UUGAGUUUGUGGUUAAUUAAA[dT][dT]
2A ID NO: 532) 5 (SEQ ID NO: 906)
05L31 UUUGGUUUCAGAAAUUCAGCU[dT][dT] 05L313
AGCUGAAUUUCUGAAACCAAA[dT][dT]
3A (SEQ ID NO: 533) 5 (SEQ ID NO: 907)
05L31 UUAUGAAGACACAGAUUUGGU[dT][dT] 05L314
ACCAAAUCUGUGUCUUCAUAA[dT][dT]
4A (SEQ ID NO: 534) 5 (SEQ ID NO: 908)
05L31 UUUCAUAGAAACAAAAACCCA[dT][dT] (SEQ 05L315 UGGGUUUUUGUUUCUAUGAAA[dT][dT]
5A ID NO: 535) 5 (SEQ ID NO: 909)
05L31 AUGAUAUUUUCAUAGAAACAA[dT][dT] 05L316
UUGUUUCUAUGAAAAUAUCAU[dT][dT]
6A (SEQ ID NO: 536) 5 (SEQ ID NO: 910)
05L31 UAUAAUGAUAUUUUCAUAGAA[dT][dT] 05L317
UUCUAUGAAAAUAUCAUUAUA[dT][dT]
7A (SEQ ID NO: 537) 5 (SEQ ID NO: 911)
05L31 UGAUUAUAAUGAUAUUUUCAU[dT][dT] 05L318
AUGAAAAUAUCAUUAUAAUCA[dT][dT]
8A (SEQ ID NO: 538) 5 (SEQ ID NO: 912)
05L31 AUAAAUAGUGAUUAUAAUGAU[dT][dT] 05L319
AUCAUUAUAAUCACUAUUUAU[dT][dT]
9A (SEQ ID NO: 539) 5 (SEQ ID NO: 913)
05L32 AAAAGCUUAAUAAGAAUGGUU[dT][dT] 05L320
AACCAUUCUUAUUAAGCUUUU[dT][dT]
OA (SEQ ID NO: 540) 5 (SEQ ID NO: 914)
05L32 AAAAAGCUUAAUAAGAAUGGU[dT][dT] 05L321
ACCAUUCUUAUUAAGCUUUUU[dT][dT]
1A (SEQ ID NO: 541) 5 (SEQ ID NO: 915)
05L32 UAAAUGUACACAUUUAGCCAC[dT][dT] (SEQ 05L322 GUGGCUAAAUGUGUACAUUUA[dT][dT]
2A ID NO: 542) 5 (SEQ ID NO: 916)
05L32 AUAAAUGUACACAUUUAGCCA[dT][dT] 05L323
UGGCUAAAUGUGUACAUUUAU[dT][dT]
3A (SEQ ID NO: 543) 5 (SEQ ID NO: 917)
05L32 UAUAAAUGUACACAUUUAGCC[dT][dT] 05L324
GGCUAAAUGUGUACAUUUAUA[dT][dT]
4A (SEQ ID NO: 544) 5 (SEQ ID NO: 918)
05L32 UUCUAAUAUAAAUGUACACAU[dT][dT] 05L325
AUGUGUACAUUUAUAUUAGAA[dT][dT]
5A (SEQ ID NO: 545) 5 (SEQ ID NO: 919)
05L32 AAGAAUUAAAGAAAAGAUCUG[dT][dT] 05L326
CAGAUCUUUUCUUUAAUUCUU[dT][dT]
6A (SEQ ID NO: 546) 5 (SEQ ID NO: 920)
05L32 AAUAAGAAUUAAAGAAAAGAU[dT][dT] 05L327
AUCUUUUCUUUAAUUCUUAUU[dT][dT]
7A (SEQ ID NO: 547) 5 (SEQ ID NO: 921)
72
CA 03174172 2022- 9- 28
05L32 AAACCAAUAAGAAUUAAAGAA[dT][dT] (SEQ 05L328 UUCUUUAAUUCUUAUUGGUUU[dT][dT]
8A ID NO: 548) 5 (SEQ ID NO: 922)
05L32 ACUAUACCCACUAUUUAAGAG[dT][dT] (SEQ 05L329 CUCUUAAAUAGUGGGUAUAGU[dT][dT]
9A ID NO: 549) 5 (SEQ ID NO: 923)
05L33 ACAAAUGUGCAAUAUUAGCAC[dT][dT] 051330
GUGCUAAUAUUGCACAUUUGU[dT][dT]
OA (SEQ ID NO: 550) 5 (SEQ ID NO: 924)
05L33 AACAAAUGUGCAAUAUUAGCA[dT][dT] 05L331
UGCUAAUAUUGCACAUUUGUU[dT][dT]
1A (SEQ ID NO: 551) 5 (SEQ ID NO: 925)
05L33 AUGUUUCAUUCAUUCAUCCAU[dT][dT] 05L332
AUGGAUGAAUGAAUGAAACAU[dT][dT]
2A (SEQ ID NO: 552) 5 (SEQ ID NO: 926)
05L33 AGUAGUAUAUGUUUCAUUCAU[dT][dT] 05L333
AUGAAUGAAACAUAUACUACU[dT][dT]
3A (SEQ ID NO: 553) 5 (SEQ ID NO: 927)
05L33 AAUCAGUAGUAUAUGUUUCAU[dT] [dT] 05L334
AUGAAACAUAUACUACUGAUU[dT][dT]
4A (SEQ ID NO: 554) 5 (SEQ ID NO: 928)
05L33 AAAUAAUCAGUAGUAUAUGUU[dT] [dT] 05L335
AACAUAUACUACUGAUUAUUU[dT][dT]
5A (SEQ ID NO: 555) 5 (SEQ ID NO: 929)
05L33 AAUCAAAGUAAUUACAGUCAG[dT][dT] 05L336
CUGACUGUAAUUACUUUGAUU[dT][dT]
6A (SEQ ID NO: 556) 5 (SEQ ID NO: 930)
05L33 AUCUAAUCAAAGUAAUUACAG[dT][dT] 05L337
CUGUAAUUACUUUGAUUAGAU[dT][dT]
7A (SEQ ID NO: 557) 5 (SEQ ID NO: 931)
05L33 UUAUUUCCAGUUGUUUAUCUA[dT][dT] 05L338
UAGAUAAACAACUGGAAAUAA[dT][dT]
8A (SEQ ID NO: 558) 5 (SEQ ID NO: 932)
05L33 UUAUUAGAACUUUUUCAGCAG[dT][dT] 05L339
CUGCUGAAAAAGUUCUAAUAA[dT][dT]
9A (SEQ ID NO: 559) 5 (SEQ ID NO: 933)
05L34 UUUAUUAGAACUUUUUCAGCA[dT][dT] 051340
UGCUGAAAAAGUUCUAAUAAA[dT][dT]
OA (SEQ ID NO: 560) 5 (SEQ ID NO: 934)
TABLE 5A CD320 ANTISENSE TARGET
ID target start
target sequence Location Size SEQ
position position ID
NO:
OSC1 2-24 2 TGCGCGTGCGCAGGGATAAGAGA 5 UTR 21 996
OSC2 4-26 4 CGCGTGCGCAGGGATAAGAGAGC 5 UTR 21 997
OSC3 48-70 48 GCGCCGCTGTGGGGACAGCATGA 5 UTR 21 998
OSC4 63-85 63 CAGCATGAGCGGCGGTTGGATGG 5'UTR - 21 999
CDS
OSC5 164-186 164 CCGCCGCGAGCCCGCTTTCCACC CDS 21
1000
OSC6 222-244 222 CTCGTGCCCACCCACCAAGTTCC CDS 21
1001
73
CA 03174172 2022- 9- 28
OSC7 225-247 225 GTGCCCACCCACCAAGTTCCAGT CDS 21
1002
OSC8 227-249 227 GCCCACCCACCAAGTTCCAGTGC CDS 21
1003
OSC9 244-266 244 CAGTGCCGCACCAGTGGCTTATG CDS 21
1004
OSC10 249-271 249 CCGCACCAGTGGCTTATGCGTGC CDS 21
1005
OSC11 282-304 282 GCGCTGCGACAGGGACTTGGACT CDS 21
1006
OSC12 306-328 306 CAGCGATGGCAGCGATGAGGAGG CDS 21
1007
OSC13 390-412 390 CCCCTGCACCGGCGTCAGTGACT CDS 21
1008
OSC14 411-433 411 CTGCTCTGGGGGAACTGACAAGA CDS 21
1009
OSC15 414-436 414 CTCTGGGGGAACTGACAAGAAAC CDS 21
1010
OSC16 417-439 417 TGGGGGAACTGACAAGAAACTGC CDS 21
1011
OSC17 422-466 422 GAACTGACAAGAAACTGCGCAACTG CDS 25
1012
OSC18 483-505 483 CACGCTGAGCGATGACTGCATTC CDS 21
1013
OSC19 484-506 484 ACGCTGAGCGATGACTGCATTCC CDS 21
1014
OSC20 487-509 487 CTGAGCGATGACTGCATTCCACT CDS 21
1015
OSC21 489-511 489 GAGCGATGACTGCATTCCACTCA CDS 21
1016
0SC22 520-542 520 TGCGACGGCCACCCAGACTGTCC CDS 21
1017
0SC23 556-578 556 GAGCTCGGCTGTGGAACCAATGA CDS 21
1018
0SC24 560-582 560 TCGGCTGTGGAACCAATGAGATC CDS 21
1019
0SC25 561-583 561 CGGCTGTGGAACCAATGAGATCC CDS 21
1020
0SC26 564-586 564 CTGTGGAACCAATGAGATCCTCC CDS 21
1021
0SC27 626-648 626 TGGAGAGTGTCACCTCTCTCAGG CDS 21
1022
0SC28 641-663 641 CTCTCAGGAATGCCACAACCATG CDS 21
1023
0SC29 689-711 689 TCCCCTCTGTCGGGAATGCCACA CDS 21
1024
OSC30 695-717 695 CTGTCGGGAATGCCACATCCTCC CDS 21
1025
OSC31 719-741 719 CTGCCGGAGACCAGTCTGGAAGC CDS 21
1026
0SC32 741-763 741 CCCAACTGCCTATGGGGTTATTG CDS 21
1027
0SC33 767-789 767 CTGCTGCGGTGCTCAGTGCAAGC CDS 21
1028
0SC34 795-817 795 CACCGCCACCCTCCTCCTTTTGT CDS 21
1029
0SC35 797-819 797 CCGCCACCCTCCTCCTTTTGTCC CDS 21
1030
0SC36 843-865 843 CCGCCCACTGGGGTTACTGGTGG CDS 21
1031
74
CA 03174172 2022- 9- 28
0SC37 852-874 852 GGGGTTACTGGTGGCCATGAAGG CDS 21
1032
0SC38 857-879 857 TACTGGTGGCCATGAAGGAGTCC CDS 21
1033
0SC39 874-896 874 GAGTCCCTGCTGCTGTCAGAACA CDS 21
1034
OSC40 878-900 878 CCCTGCTGCTGTCAGAACAGAAG CDS 21
1035
OSC41 881-903 881 TGCTGCTGTCAGAACAGAAGACC CDS 21
1036
0SC42 884-906 884 TGCTGTCAGAACAGAAGACCTCG CDS 21
1037
0SC43 901-923 901 ACCTCGCTGCCCTGAGGACAAGC CDS 21
1038
0SC44 907-929 907 CTGCCCTGAGGACAAGCACTTGC CDS - 21
1039
3'UTR
0SC45 971-993 971 GAGCAGTGATGCGGATGGGTACC 3 UTR 21
1040
0SC46 995- 995 GGGCACACCAGCCCTCAGAGACC 3' UTR 21
1041
1017
0SC47 1006- 1006 CCCTCAGAGACCTGAGCTCTT 3' UTR 21
1393
1026
0SC48 1006- 1006 CCCTCAGAGACCTGAGCTCTTCT 3' UTR 21
1042
1028
0SC49 1008- 1008 CTCAGAGACCTGAGCTCTTCTGG 3' UTR 21
1043
1030
OSC50 1082- 1082 GGGTCCCTGGACACTCCCTATGG 3' UTR 21
1044
1104
OSC51 1085- 1085 TCCCTGGACACTCCCTATGGAGA 3' UTR 21
1045
1107
0SC52 1088- 1088 CTGGACACTCCCTATGGAGATCC 3' UTR 21
1046
1110
0SC53 1129- 1129 ACCTGCCACAGCCAGAACTGAGG 3' UTR 21
1047
1151
0SC54 1163- 1163 GGCAGCTCCCAGGGGGTAGAACG 3' UTR 21
1048
1185
0SC55 1176- 1176 GGGTAGAACGGCCCTGTGCTTAA 3' UTR 21
1049
1198
0SC56 1182- 1182 AACGGCCCTGTGCTTAAGACACT 3' UTR 21
1050
1204
0SC57 1184- 1184 CGGCCCTGTGCTTAAGACACTCC 3' UTR 21
1051
1206
0SC58 1237- 1237 TTGCTTCACATCCTCAAAAAAAA 3' UTR 21
1052
1259
0SC59 1238- 1238 TGCTTCACATCCTCAAAAAAAAA 3' UTR 21
1053
1260
CA 03174172 2022- 9- 28
TABLE 6A LRP2 ANTISENSE TARGET
ID Target Start Target sequence Location Size SEQ
ID
position position NO:
OSL1 512-534 512 GTCAAGATTGCTCACAAAGTACA CDS 21
1054
OSL2 566-588 566 GTCAGTGTATCCCAAGTGAATAC CDS 21
1055
OSL3 763-785 763 TTGCACAATGAGTTTTCATGTGG CDS 21
1056
OSL4 939-961 939 TGGAGAAGATGACTGTAAAGATA CDS 21
1057
OSL5 941-963 941 GAGAAGATGACTGTAAAGATAAT CDS 21
1058
OSL6 992- 992 CTCATGATGTTCATAAATGTTCC CDS 21 1059
1014
OSL7 1053- 1053 CTCCATTTATAAAGTTTGTGATG CDS 21 1060
1075
OSL8 1054- 1054 TCCATTTATAAAGTTTGTGATGG CDS 21 1061
1076
OSL9 1119- 1119 TACCGGAAAATACTGTAGTATGA CDS 21 1062
1141
OSL10 1121- 1121 CCGGAAAATACTGTAGTATGACT CDS 21 1063
1143
OSL11 1267- 1267 TGCCAGATATGGGGAATTTGTGA CDS 21 1064
1289
OSL12 1329- 1329 CTGTGAAGAAGGGTATATCTTGG CDS 21 1065
1351
OSL13 1356- 1356 TGGACAGTATTGCAAAGCTAATG CDS 21 1066
1378
OSL14 1360- 1360 CAGTATTGCAAAGCTAATGATTC CDS 21 1067
1382
OSL15 1366- 1366 TGCAAAGCTAATGATTCCTTTGG CDS 21 1068
1388
OSL16 1423- 1423 TTGTTAATTGGTGATATTCATGG CDS 21 1069
1445
OSL17 1541- 1541 CCGTGCAAAATAAGGTTTTTTCA CDS 21 1070
1563
OSL18 1543- 1543 GTGCAAAATAAGGTTTTTTCAGT CDS 21 1071
1565
OSL19 1552- 1552 AAGGTTTTTTCAGTTGACATTAA CDS 21 1072
1574
OSL20 1553- 1553 AGGTTTTTTCAGTTGACATTAAT CDS 21 1073
1575
OSL21 1562- 1562 CAGTTGACATTAATGGTTTAAAT CDS 21 1074
1584
0SL22 1565- 1565 TTGACATTAATGGTTTAAATATC CDS 21 1075
1587
76
CA 03174172 2022- 9- 28
OS L23 1638- 1638 CTGGGTTAATAATAAAATCTATC CDS 21
1076
1660
OS L24 1639- 1639 TGGGTTAATAATAAAATCTATCT CDS 21
1077
1661
OS L25 1680- 1680 CCGCATAGATATGGTAAATTTGG CDS 21
1078
1702
OS L26 1719- 1719 TACCCTTATAACTGAAAACTTGG CDS 21
1079
1741
OS L27 1767- 1767 CCCAACTGTTGGTTATTTATTTT CDS 21
1080
1789
OS L28 1772- 1772 CTGTTGGTTATTTATTTTTCTCA CDS 21
1081
1794
OS L29 1895- 1895 GGGTAACTCTGGATATGATATCG CDS 21
1082
1917
OS L30 1942- 1942 CGGTTTGATTACATTGAAACTGT CDS 21
1083
1964
OS L31 1946- 1946 TTGATTACATTGAAACTGTAACT CDS 21
1084
1968
OS L32 1951- 1951 TACATTGAAACTGTAACTTATGA CDS 21
1085
1973
OS L33 2187- 2187 TGCTACCAATCCGTGTAAAGATA CDS 21
1086
2209
OS L34 2437- 2437 TTCTTTGTCGGGATTGATTTTGA CDS 21
1087
2459
OS L35 2469- 2469 CAGCACTATCTTTTTTTCAGATA CDS 21
1088
2491
OS L36 2470- 2470 AGCACTATCTTTTTTTCAGATAT CDS 21
1089
2492
OS L37 2491- 2491 ATGTCAAAACACATGATTTTTAA CDS 21
1090
2513
OS L38 2498- 2498 AACACATGATTTTTAAGCAAAAG CDS 21
1091
2520
OS L39 2558- 2558 GGGTGGAAAATGTTGAAAGTTTG CDS 21
1092
2580
OS L40 2579- 2579 TGGCTTTTGATTGGATTTCAAAG CDS 21
1093
2601
OS L41 2580- 2580 GGCTTTTGATTGGATTTCAAAGA CDS 21
1094
2602
OS L42 2589- 2589 TTGGATTTCAAAGAATCTCTATT CDS 21
1095
2611
OS L43 2590- 2590 TGGATTTCAAAGAATCTCTATTG CDS 21
1096
2612
OS L44 2670- 2670 CACAGTAGTTCAGTATTTAAATA CDS 21
1097
2692
77
CA 03174172 2022- 9- 28
OS L45 2672- 2672 CAGTAGTTCAGTATTTAAATAAC CDS 21
1098
2694
OS L46 2714- 2714 ATCCTTTTGCCGGGTATCTATTC CDS 21
1099
2736
OS L47 2800- 2800 CCTGTAATAAACACTACTCTT CDS 21
1394
2820
OS L48 2869- 2869 TGGGTAGATGCCTATTTTGATAA CDS 21
1100
2891
OS L49 2877- 2877 TGCCTATTTTGATAAAATTGAGC CDS 21
1101
2899
OS L50 2971- 2971 GCCATCTTTGGAGAGCATTTATT CDS 21
1102
2993
OS L51 3074- 3074 TTGCTTACATACTGCATTTGAAA CDS 21
1103
3096
OS L52 3075- 3075 TGCTTACATACTGCATTTGAAAT CDS 21
1104
3097
OS L53 3120- 3120 TGGTTCTAACGCCTGTAATCAAC CDS 21
1105
3142
OS L54 3356- 3356 TCGATGATTGTCATGATAACAGT CDS 21
1106
3378
OS L55 3546- 3546 CACCCAATACACCTGTGATAATC CDS 21
1107
3568
OS L56 3547- 3547 ACCCAATACACCTGTGATAATCA CDS 21
1108
3569
OS L57 3569- 3569 ACCAGTGTATCTCAAAGAACTGG CDS 21
1109
3591
OS L58 3629- 3629 ATGAAAAGAACTGCAATTCGACA CDS 21
1110
3651
OS L59 3681- 3681 CCCCAATCATCGATGTATTGACC CDS 21
1111
3703
OS L60 3690- 3690 TCGATGTATTGACCTATCGTTTG CDS 21
1112
3712
OS L61 3693- 3693 ATGTATTGACCTATCGTTTGTCT CDS 21
1113
3715
OS L62 3828- 3828 TCGTTGTGATGGTGTTTTTGATT CDS 21
1114
3850
OS L63 3945- 3945 CCCGAACTTCTGGGAATGTGATG CDS 21
1115
3967
OS L64 3946- 3946 CCGAACTTCTGGGAATGTGATGG CDS 21
1116
3968
OS L65 4015- 4015 CCCAAGACTTGCCCTTCATCATA CDS 21
1117
4037
OS L66 4348- 4348 TTCTTACTTGCCAATGATTCTAA CDS 21
1118
4370
78
CA 03174172 2022- 9- 28
0SL67 4379- 4379 AAGACATAGATGAATGTGATATT CDS 21 1119
4401
0SL68 4381- 4381 GACATAGATGAATGTGATATTCT CDS 21 1120
4403
0SL69 4455- 4455 GTGTGATACAGGCTACATGTTAG CDS 21 1121
4477
OSL70 4464- 4464 AGGCTACATGTTAGAAAGTGATG CDS 21 1122
4486
OSL71 4465- 4465 GGCTACATGTTAGAAAGTGATGG CDS 21 1123
4487
0SL72 4597- 4597 GTCGAGAATGGTTCTTACATTGT CDS 21 1124
4619
0SL73 4600- 4600 GAGAATGGTTCTTACATTGTAGC CDS 21 1125
4622
0SL74 4612- 4612 TACATTGTAGCTGTTGATTTTGA CDS 21 1126
4634
0SL75 4620- 4620 AGCTGTTGATTTTGATTCAATTA CDS 21 1127
4642
0SL76 4622- 4622 CTGTTGATTTTGATTCAATTAGT CDS 21 1128
4644
0SL77 4635- 4635 TTCAATTAGTGGTCGTATCTTTT CDS 21 1129
4657
0SL78 4732- 4732 AGCATCATCTTGACTGAAACTAT CDS 21 1130
4754
0SL79 4735- 4735 ATCATCTTGACTGAAACTATTGC CDS 21 1131
4757
OSL80 4741- 4741 TTGACTGAAACTATTGCAATAGA CDS 21 1132
4763
OSL81 4743- 4743 GACTGAAACTATTGCAATAGATT CDS 21 1133
4765
0SL82 4745- 4745 CTGAAACTATTGCAATAGATTGG CDS 21 1134
4767
0SL83 4806- 4806 AACAATTGAAGTCTCCAAAATTG CDS 21 1135
4828
0SL84 4847- 4847 TGCTGATTAGTAAAAACCTAACA CDS 21 1136
4869
0SL85 4883- 4883 TAGCATTAGATCCCAGAATGAAT CDS 21 1137
4905
0SL86 4884- 4884 AGCATTAGATCCCAGAATGAATG CDS 21 1138
4906
0SL87 4896- 4896 CAGAATGAATGAGCATCTACTGT CDS 21 1139
4918
0SL88 5077- 5077 ATGGACTTTTGTGATTATAATGG CDS 21 1140
5099
79
CA 03174172 2022- 9- 28
OS L89 5080- 5080 GACTTTTGTGATTATAATGGACA CDS 21
1141
5102
OS L90 5126- 5126 GTGATTTGATTATACGGCA CDS 19
1142
5144
OS L91 5241- 5241 GTCAGTTGTAATGTATAATATTC CDS 21
1143
5263
OS L92 5246- 5246 TTGTAATGTATAATATTCAATGG CDS 21
1144
5268
OS L93 5291- 5291 AT CCTTC GAAACAACCAAATTC C CDS 21
1145
5313
OS L94 5295- 5295 TTCGAAACAACCAAATTCCGTGA CDS 21
1146
5317
OS L95 5447- 5447 AACCTTTCTTAATAACTGTAAGG CDS 21
1147
5469
OS L96 5467- 5467 AGGCAACATATAATTTTTGGAAT CDS 21
1148
5489
OS L97 5468- 5468 GGCAACATATAATTTTTGGAATC CDS 21
1149
5490
OS L98 5538- 5538 AGGGATACAGAATGGTTTAGATG CDS 21
1150
5560
OS L99 5539- 5539 GGGATACAGAATGGTTTAGATGT CDS 21
1151
5561
OS L100 5545- 5545 CAGAATGGTTTAGATGTTGAATT CDS 21
1152
5567
OS L101 5584- 5584 TACATCTATTGGGTTGAAAATCC CDS 21
1153
5606
OS L102 5644- 5644 AGGACAGTATTTGCTTCTATATC CDS 21
1154
5666
OS L103 5648- 5648 CAGTATTTGCTTCTATATCTATG CDS 21
1155
5670
OS L104 5677- 5677 CCTTCTATGAACCTGGCCTTA CDS 21
1156
5697
OS L105 5692- 5692 GCCTTAGATTGGATTTCAAGAAA CDS 21
1157
5714
OSL106 5701- 5701 TGGATTTCAAGAAACCTTTATTC CDS 21
1158
5723
OS L107 5738- 5738 CTCAGTCAATCGAGGTTTTGACA CDS 21
1159
5760
OS L108 5765- 5765 ACGGAGATATCAGATACAGAAAA CDS 21
1160
5787
OS L109 5766- 5766 CGGAGATATCAGATACAGAAAAA CDS 21
1161
5788
OS L110 5768- 5768 GAGATATCAGATACAGAAAAACA CDS 21
1162
5790
CA 03174172 2022- 9- 28
OSL111 5775- 5775 CAGATACAGAAAAACATTGATTG CDS 21
1163
5797
OSL112 6115- 6115 GTCCATGATTCTTTCCTTTATTA CDS 21
1164
6137
OSL113 6116- 6116 TCCATGATTCTTTCCTTTATTAT CDS 21
1165
6138
OSL114 6123- 6123 TTCTTTCCTTTATTATACTGATG CDS 21
1166
6145
OSL115 6146- 6146 AACAGTATGAGGTCATTGAAAGA CDS 21
1167
6168
OSL116 6202- 6202 TTGAGAGATAATGTTCCAAATCT CDS 21
1168
6224
OSL117 6204- 6204 GAGAGATAATGTTCCAAATCTGA CDS 21
1169
6226
OSL118 6206- 6206 GAGATAATGTTCCAAATCTGAGG CDS 21
1170
6228
OSL119 6266- 6266 CCTCAAATGGCTGTAGCAA CDS 19
1171
6284
OSL120 6387- 6387 CTCTCCATATAACTCTTTCATTG CDS 21
1172
6409
OSL121 6390- 6390 TCCATATAACTCTTTCATTGTTG CDS 21
1173
6412
0SL122 6397- 6397 AACTCTTTCATTGTTGTTTCAAT CDS 21
1174
6419
0SL123 6399- 6399 CTCTTTCATTGTTGTTTCAATGC CDS 21
1175
6421
0SL124 6425- 6425 CTGCAATCAGAGGCTTTAGCTTG CDS 21
1176
6447
0SL125 6426- 6426 TGCAATCAGAGGCTTTAGCTTGG CDS 21
1177
6448
0SL126 6434- 6434 GAGGCTTTAGCTTGGAATTGTCA CDS 21
1178
6456
0SL127 6436- 6436 GGCTTTAGCTTGGAATTGTCAGA CDS 21
1179
6458
0SL128 6445- 6445 TTGGAATTGTCAGATCATTCAGA CDS 21
1180
6467
0SL129 6603- 6603 TGGATCTTCTCTGATGAACATTG CDS 21
1181
6625
OSL130 6619- 6619 AACATTGTGACACATGGAATAGG CDS 21
1182
6641
OSL131 6711- 6711 TTCTGAAACACTGATAGAAGTTC CDS 21
1183
6733
0SL132 6725- 6725 TAGAAGTTCTGCGGATCAATACT CDS 21
1184
6747
81
CA 03174172 2022- 9- 28
0SL133 6797- 6797 TTGTTGTAGATCCCAAGAACAGA CDS 21
1185
6819
0SL134 6849- 6849 ACCAAAGATTGAGCGTTCTTTCC CDS 21
1186
6871
0SL135 6939- 6939 CCGAAGTGATGGCTACGTTTATT CDS 21
1187
6961
0SL136 6961- 6961 TGGGTTGATGATTCTTTAGATAT CDS 21
1188
6983
0SL137 6965- 6965 TTGATGATTCTTTAGATATAATT CDS 21
1189
6987
0SL138 7062- 7062 CACTGTTTTTGAAAATTCTATCA CDS 21
1190
7084
0SL139 7064- 7064 CTGTTTTTGAAAATTCTATCATA CDS 21
1191
7086
0SL140 7087- 7087 TGGGTAGATAGGAATTTGAAAAA CDS 21
1192
7109
OSL141 7088- 7088 GGGTAGATAGGAATTTGAAAAAG CDS 21
1193
7110
0SL142 7152- 7152 CACAGTGATAAGAGACAATATCA CDS 21
1194
7174
0SL143 7154- 7154 CAGTGATAAGAGACAATATCAAC CDS 21
1195
7176
0SL144 7348- 7348 GGCAAGAATTGTGCCATTTCAAC CDS 21
1196
7370
0SL145 7351- 7351 AAGAATTGTGCCATTTCAACAGA CDS 21
1197
7373
0SL146 7358- 7358 GTGCCATTTCAACAGAAAATTTC CDS 21
1198
7380
0SL147 7359- 7359 TGCCATTTCAACAGAAAATTTCC CDS 21
1199
7381
0SL148 7381- 7381 CTCATCTTTGCCTTGTCTAATTC CDS 21
1200
7403
0SL149 7443- 7443 ACCTTTCCAAACAATAAATGTGG CDS 21
1201
7465
OSL150 7486- 7486 GACTATGACAGTGTAAGTGATAG CDS 21
1202
7508
OSL151 7494- 7494 CAGTGTAAGTGATAGAATCTACT CDS 21
1203
7516
0SL152 7496- 7496 GTGTAAGTGATAGAATCTACTTC CDS 21
1204
7518
0SL153 7506- 7506 TAGAATCTACTTCACACAAAATT CDS 21
1205
7528
0SL154 7510- 7510 ATCTACTTCACACAAAATTTAGC CDS 21
1206
7532
82
CA 03174172 2022- 9- 28
OS L155 7627- 7627 GCCTTTGACTGGATTACTAGAAG CDS 21
1207
7649
OS L156 7633- 7633 GACTGGATTACTAGAAGAATTTA CDS 21
1208
7655
OS L157 7635- 7635 CTGGATTACTAGAAGAATTTATT CDS 21
1209
7657
OS L158 7636- 7636 TGGATTACTAGAAGAATTTATTA CDS 21
1210
7658
OS L159 8007- 8007 GACTCTCTATGGCCAGTATATTT CDS 21
1211
8029
OS L160 8036- 8036 CTGACTTGTACACACAAAGAATT CDS 21
1212
8058
OS L161 8038- 8038 GACTTGTACACACAAAGAATTTA CDS 21
1213
8060
OS L162 8044- 8044 TACACACAAAGAATTTACCGAGC CDS 21
1214
8066
OS L163 8150- 8150 ACCAGAAACAACAGTGTAACAAT CDS 21
1215
8172
OS L164 8152- 8152 CAGAAACAACAGTGTAACAATCC CDS 21
1216
8174
OS L165 8167- 8167 AACAATCCTTGTGAACAGTTTAA CDS 21
1217
8189
OS L166 8293- 8293 GTGGACAATGGTGAACGATGTGG CDS 21
1218
8315
OS L167 8564- 8564 CGGAGTTTATGTGCAATAACAGA CDS 21
1219
8586
OS L168 8566- 8566 GAGTTTATGTGCAATAACAGAAG CDS 21
1220
8588
OS L169 8685- 8685 TGGATACACAAAATGTCATAATT CDS 21
1221
8707
OS L170 8689- 8689 TACACAAAATGTCATAATTCAAA CDS 21
1222
8711
OSL171 8691- 8691 CACAAAATGTCATAATTCAAATA CDS 21
1223
8713
OS L172 8699- 8699 GTCATAATTCAAATATTTGTATT CDS 21
1224
8721
OS L173 8715- 8715 TTGTATTCCTCGCGTTTATTTGT CDS 21
1225
8737
OS L174 8768- 8768 GTGATGAAAACCCTACTTATTGC CDS 21
1226
8790
OS L175 8844- 8844 TCCTCAACATTGGTATTGTGATC CDS 21
1227
8866
OS L176 8854- 8854 TGGTATTGTGATCAAGAAACAGA CDS 21
1228
8876
83
CA 03174172 2022- 9- 28
0SL177 8861- 8861 GTGATCAAGAAACAGATTGTTTT CDS 21
1229
8883
OS L178 9063- 9063 TTCCGAGTTTCTCTGTGTAAATG CDS 21
1230
9085
OS L179 9064- 9064 TCCGAGTTTCTCTGTGTAAATGA CDS 21
1231
9086
OS L180 9153- 9153 CGGCTACGATGAGAATCAGAATT CDS 21
1232
9175
OS L181 9181- 9181 AGGAGAACTTGCTCTGAAAATGA CDS 21
1233
9203
0SL182 9221- 9221 GACTGTGTATCCCAAAGATATTC CDS 21
1234
9243
OS L183 9308- 9308 GCCAACAGAATCAGTTTACCTGT CDS 21
1235
9330
OS L184 9595- 9595 GACACCTTAACCAGTTTCTATTG CDS 21
1236
9617
OS L185 9598- 9598 ACCTTAACCAGTTTCTATTGTTC CDS 21
1237
9620
OS L186 9657- 9657 GACTTGTGTTGATATTGATGAAT CDS 21
1238
9679
OS L187 9719- 9719 ATGTAATAGGCTCCTACATCTGT CDS 21
1239
9741
OS L188 9786- 9786 CCGGCAAAACAGTAACATCGAAC CDS 21
1240
9808
OS L189 9807- 9807 ACCCTATCTCATTTTTAGCAACC CDS 21
1241
9829
OS L190 9826- 9826 AACCGTTACTATTTGAGAAATTT CDS 21
1242
9848
OS L191 9827- 9827 ACCGTTACTATTTGAGAAATTTA CDS 21
1243
9849
OS L192 9828- 9828 CCGTTACTATTTGAGAAATTTAA CDS 21
1244
9850
OS L193 9832- 9832 TACTATTTGAGAAATTTAACTAT CDS 21
1245
9854
OS L194 9838- 9838 TTGAGAAATTTAACTATAGATGG CDS 21
1246
9860
OS L195 9849- 9849 AACTATAGATGGCTATTTTTACT CDS 21
1247
9871
OS L196 9892- 9892 GACAATGTTGTGGCATTAGATTT CDS 21
1248
9914
OS L197 9925- 9925 GAGAAGAGATTGTATTGGATTGA CDS 21
1249
9947
OS L198 9956- 9956 GGCAAGTCATTGAGAGAATGTTT CDS 21
1250
9978
84
CA 03174172 2022- 9- 28
0SL199 9987- 9987 GACAAACAAGGAGACAATCATAA CDS 21
1251
10009
OS L200 10272- 10272 AACCAACAAGTCTGTGATAATCT CDS 21
1252
10294
OS L201 10444- 10444 TTCGCTATTACCATTTTTGAAGA CDS 21
1253
10466
OS L202 10446- 10446 CGCTATTACCATTTTTGAAGACA CDS 21
1254
10468
OS L203 10499- 10499 CAGTGGAAAAGGGAAACAAATAT CDS 21
1255
10521
OS L204 10513- 10513 AACAAATATGATGGATCAAATAG CDS 21
1256
10535
OS L205 10574- 10574 TCCATGTGTACCATCCATATAGG CDS 21
1257
10596
OS L206 10958- 10958 CTGATGAAGACCGTCTTCTTTGT CDS 21
1258
10980
OS L207 11246- 11246 GTGACAACTTCACAGAATTCAGC CDS 21
1259
11268
OS L208 11623- 11623 CAGTGTACAAGTGGACATTGTGT CDS 21
1260
11645
OS L209 11625- 11625 GTGTACAAGTGGACATTGTGTAC CDS 21
1261
11647
OS L210 11745- 11745 TACTATGTTCGAATGCAAAAACC CDS 21
1262
11767
OS L211 11749- 11749 ATGTTCGAATGCAAAAACCATGT CDS 21
1263
11771
OS L212 11757- 11757 ATGCAAAAACCATGTTTGTATCC CDS 21
1264
11779
OS L213 11779- 11779 CCGCCATATTGGAAATGTGATGG CDS 21
1265
11801
OS L214 11820- 11820 TGGTTCAGATGAAGAACTTCACC CDS 21
1266
11842
OS L215 11887- 11887 GACAACAATCGCTGCATTTATAG CDS 21
1267
11909
OS L216 11984- 11984 CCCCTAAACCTTGTACAGAATAT CDS 21
1268
12006
OS L217 11985- 11985 CCCTAAACCTTGTACAGAATATG CDS 21
1269
12007
OS L218 11990- 11990 AACCTTGTACAGAATATGAATAT CDS 21
1270
12012
OS L219 11991- 11991 ACCTTGTACAGAATATGAATATA CDS 21
1271
12013
OS L220 11994- 11994 TTGTACAGAATATGAATATAAGT CDS 21
1272
12016
CA 03174172 2022- 9- 28
OS L221 12083- 12083 CCGATGAACTGGGTTGCAATAAA CDS 21
1273
12105
OS L222 12114- 12114 AAGAACATGTGCTGAAAATATAT CDS 21
1274
12136
OS L223 12140- 12140 AGCAAAATTGTACCCAATTAAAT CDS 21
1275
12162
OS L224 12193- 12193 GGGTTCGAAACCAATGTTTTTGA CDS 21
1276
12215
OS L225 12197- 12197 TCGAAACCAATGTTTTTGACAGA CDS 21
1277
12219
OS L226 12389- 12389 CTGACAATGTCCGAATTCGAAAA CDS 21
1278
12411
OS L227 12397- 12397 GTCCGAATTCGAAAATATAATCT CDS 21
1279
12419
OS L228 12399- 12399 CCGAATTCGAAAATATAATCTCT CDS 21
1280
12421
OS L229 12435- 12435 CTCAGAGTATCTTCAAGATGAGG CDS 21
1281
12457
OS L230 12443- 12443 ATCTTCAAGATGAGGAATATATC CDS 21
1282
12465
OS L231 12537- 12537 GGGCTCTAGGTTTGGTGCTATCAAA CDS 25
1283
12561
OS L232 12564- 12564 TGCCTACATCCCCAACTTTGAAT CDS 21
1284
12586
OS L233 12608- 12608 AGGAAGTTGACCTGAAACTGAAA CDS 21
1285
12630
OS L234 12616- 12616 GACCTGAAACTGAAATACGTAAT CDS 21
1286
12638
OS L235 12617- 12617 ACCTGAAACTGAAATACGTAATG CDS 21
1287
12639
OS L236 12705- 12705 ACGCATTGAGGTGGCTAAACTTG CDS 21
1288
12727
OS L237 12792- 12792 TCCCAAACTAGGGCTTATGTTCT CDS 21
1289
12814
OS L238 12825- 12825 GGGAAAGGAACCTAAAATCGAGT CDS 21
1290
12847
OS L239 12870- 12870 CCGCAACATCCTGGTTTTCGAGG CDS 21
1291
12892
OS L240 12911- 12911 GCCTTTCTATCGATTATTTGAAC CDS 21
1292
12933
OS L241 12915- 12915 TTCTATCGATTATTTGAACAATG CDS 21
1293
12937
OS L242 12965- 12965 AGGACGTTATTGAAACCATAAAA CDS 21
1294
12987
86
CA 03174172 2022- 9- 28
OS L243 12967- 12967 GACGTTATTGAAACCATAAAATA CDS 21
1295
12989
OS L244 12968- 12968 AC GTTATTGAAAC CATAAAATAT CDS 21
1296
12990
OS L245 12995- 12995
GGACTGATAGGAGAGTCATTGCAAA CDS 21 1297
13019
OS L246 13058- 13058 AC CAGTTATACTGGATATCTAAG CDS 25 1298
13080
OS L247 13086- 13086 GGGAGAAGTATGGAAACAAAATA CDS 21
1299
13108
OS L248 13091- 13091 AAGTATGGAAACAAAATAAATTT CDS 21
1300
13113
OS L249 13175- 13175 TTCATCAACTCAGATACAATAAG CDS 21
1301
13197
0SL250 13368- 13368 CGGAGGAAATTGCTATTTTGATG CDS 21 1302
13390
0SL251 13371- 13371 AGGAAATTGCTATTTTGATGAGA CDS 21 1303
13393
0SL252 13428- 13428 CACCGGAAAATATTGTGAAATGG CDS 21 1304
13450
0SL253 13440- 13440 TTGTGAAATGGCGTTTTCAAAAG CDS 21 1305
13462
0SL254 13541- 13541 CAGGATTCTTCCACTATAGAAGG CDS 21 1306
13563
0SL255 13659- 13659 GGCAGATCTTAACATGGATATTG CDS 21 1307
13681
0SL256 13725- 13725 GGCAATGAGTGAAGACTTTGTCA CDS 21 1308
13747
0SL257 13842- 13842 ATCTGAAAATGTGGATAATAAGA CDS 21 1309
13864
0SL258 13844- 13844 CTGAAAATGTGGATAATAAGAAT CDS 21 1310
13866
0SL259 13852- 13852 GTGGATAATAAGAATTATGGAAG CDS 21 1311
13874
0SL260 13853- 13853 TGGATAATAAGAATTATGGAAGT CDS 21 1312
13875
0SL261 13951- 13951 TGGAATCTCTTCAAACGAAAATC CDS 21 1313
13973
0SL262 13955- 13955 ATCTCTTCAAACGAAAATCTAAA CDS 21 1314
13977
0SL263 13957- 13957 CTCTTCAAACGAAAATCTAAACA CDS 21 1315
13979
0SL264 13960- 13960 TTCAAACGAAAATCTAAACAAAC CDS 21 1316
13982
87
CA 03174172 2022- 9- 28
OS L265 13964- 13964 AACGAAAATCTAAACAAACTACC CDS 21
1317
13986
OS L266 13976- 13976 AACAAACTACCAACTTTGAAAAT CDS 21
1318
13998
OS L267 13983- 13983 TACCAACTTTGAAAATCCAATCT CDS 21
1319
14005
OS L268 13987- 13987 AACTTTGAAAATCCAATCTATGC CDS 21
1320
14009
OS L269 14121- 14121 TGCAACAGAAGACACTTTTAAAG CDS 21
1321
14143
OS L270 14145- 14145 CACCGCAAATCTTGTTAAAGAAG CDS 21
1322
14167
OS L271 14148- 14148 CGCAAATCTTGTTAAAGAAGACT CDS 21
1323
14170
OS L272 14169- 14169 CTCTGAAGTATAGCTATACCAGC CDS/3'- 21
1324
14191 UTR
OS L273 14186- 14186 ACCAGCTATTTAGGGAATAATTA 3'-UTR 21
1325
14208
OS L274 14188- 14188 CAGCTATTTAGGGAATAATTAGA 3'-UTR 21
1326
14210
OS L275 14211- 14211 AACACACTTTTGCACATATATTT 3'-UTR 21
1327
14233
OS L276 14213- 14213 CACACTTTTGCACATATATTTTT 3'-UTR 21
1328
14235
OS L277 14236- 14236 TACAAACAGATGAAAAAAGTTAA 3'-UTR 21
1329
14258
OS L278 14252- 14252 AAGTTAACATTCAGTACTTTATG 3'-UTR 21
1330
14274
OS L279 14261- 14261 TTCAGTACTTTATGAAAAAAATA 3'-UTR 21
1331
14283
OS L280 14263- 14263 CAGTACTTTATGAAAAAAATATA 3'-UTR 21
1332
14285
OS L281 14341- 14341 CCGTCTCATATTTTTACAAATAA 3'-UTR 21
1333
14363
OS L282 14355- 14355 TACAAATAATTATCACAATGTAC 3'-UTR 21
1334
14377
OS L283 14366- 14366 ATCACAATGTACTATATGTATAT 3'-UTR 21
1335
14388
OS L284 14368- 14368 CACAATGTACTATATGTATATCT 3'-UTR 21
1336
14390
OS L285 14372- 14372 ATGTACTATATGTATATCTTTGC 3'-UTR 21
1337
14394
OS L286 14396- 14396 CTGAAGTTGTCTGAAGGTAATAC 3'-UTR 21
1338
14418
88
CA 03174172 2022- 9- 28
0SL287 14406- 14406 CTGAAGGTAATACTATAAATATA 3'-
UTR 21 1339
14428
0SL288 14437- 14437 TTGTAAATTTTGGAAAGATTATC 3'-
UTR 21 1340
14459
0SL289 14447- 14447 TGGAAAGATTATCCTGTTACTGA 3'-
UTR 21 1341
14469
0SL290 14451- 14451 AAGATTATCCTGTTACTGAATTT 3'-
UTR 21 1342
14473
0SL291 14457- 14457 ATCCTGTTACTGAATTTGCTAAT 3'-
UTR 21 1343
14479
0SL292 14458- 14458 TCCTGTTACTGAATTTGCTAATA 3'-
UTR 21 1344
14480
0SL293 14464- 14464 TACTGAATTTGCTAATAAAGATG 3'-
UTR 21 1345
14486
0SL294 14503- 14503 GTGATCATTATAGTAAATGATCC 3'-
UTR 21 1346
14525
0SL295 14513- 14513 TAGTAAATGATCCAACAAGAAAA 3'-
UTR 21 1347
14535
0SL296 14523- 14523 TCCAACAAGAAAAGGAATTGACT 3'-
UTR 21 1348
14545
0SL297 14579- 14579 CACTCATATTTCCTATAAAATTA 3'-
UTR 21 1349
14601
0SL298 14581- 14581 CTCATATTTCCTATAAAATTATC 3'-
UTR 21 1350
14603
0SL299 14633- 14633 GTCCATTTTTACACATTAGCACT 3'-
UTR 21 1351
14655
OSL300 14649- 14649 TAGCACTTAATTAATGTTCAATA 3'-
UTR 21 1352
14671
OSL301 14650- 14650 AGCACTTAATTAATGTTCAATAT 3'-
UTR 21 1353
14672
0SL302 14652- 14652 CACTTAATTAATGTTCAATATTA 3'-
UTR 21 1354
14674
0SL303 14665- 14665 TTCAATATTACATGTCAATTTGA 3'-
UTR 21 1355
14687
0SL304 14684- 14684 TTGATTAATGGCTATGTTGATAG 3'-
UTR 21 1356
14706
0SL305 14708- 14708 GGCCACTATGTGTTGTATAGACA 3'-
UTR 21 1357
14730
0SL306 14711- 14711 CACTATGTGTTGTATAGACATCT 3'-
UTR 21 1358
14733
0SL307 14767- 14767 AGGTAGGAAAAGCAATTCAGTTT 3'-
UTR 21 1353
14789
0SL308 14856- 14856 GCCAAGACAACATTTTTATTTGT 3'-
UTR 21 1360
14878
89
CA 03174172 2022- 9- 28
OS L309 14861- 14861 GACAACATTTTTATTTGTGATGT 3'-UTR 21
1361
14883
OS L310 14886- 14886 ATGAGGAAATCCCATATCATTAA 3'-UTR 21
1362
14908
OS L311 14921- 14921 TGCATTGAGTTTGTGGTTAATTA 3'-UTR 21
1363
14943
OS L312 14925- 14925 TTGAGTTTGTGGTTAATTAAATG 3'-UTR 21
1364
14947
OS L313 15034- 15034 AGCTGAATTTCTGAAACCAAATC 3'-UTR 21
1365
15056
OS L314 15049- 15049 ACCAAATCTGTGTCTTCATAAAA 3'-UTR 21
1366
15071
OS L315 15115- 15115 TGGGTTTTTGTTTCTATGAAAAT 3'-UTR 21
1367
15137
OS L316 15122- 15122 TTGTTTCTATGAAAATATCATTA 3'-UTR 21
1368
15144
OS L317 15126- 15126 TTCTATGAAAATATCATTATAAT 3'-UTR 21
1369
15148
OS L318 15130- 15130 ATGAAAATATCATTATAATCACT 3'-UTR 21
1370
15152
OS L319 15138- 15138 ATCATTATAATCACTATTTATTT 3'-UTR 21
1371
15160
OS L320 15188- 15188 AACCATTCTTATTAAGCTTTTTA 3'-UTR 21
1372
15210
OS L321 15189- 15189 ACCATTCTTATTAAGCTTTTTAT 3'-UTR 21
1373
15211
OS L322 15220- 15220 GTGGCTAAATGTGTACATTTATA 3'-UTR 21
1374
15242
OS L323 15221- 15221 TGGCTAAATGTGTACATTTATAT 3'-UTR 21
1375
15243
OS L324 15222- 15222 GGCTAAATGTGTACATTTATATT 3'-UTR 21
1376
15244
OS L325 15228- 15228 ATGTGTACATTTATATTAGAATG 3'-UTR 21
1377
15250
OS L326 15263- 15263 CAGATCTTTTCTTTAATTCTTAT 3'-UTR 21
1378
15285
OS L327 15266- 15266 ATCTTTTCTTTAATTCTTATTGG 3'-UTR 21
1373
15288
OS L328 15271- 15271 TTCTTTAATTCTTATTGGTTTTT 3'-UTR 21
1380
15293
OS L329 15438- 15438 CTCTTAAATAGTGGGTATAGTCT 3'-UTR 21
1381
15460
OS L330 15524- 15524 GTGCTAATATTGCACATTTGTTA 3'-UTR 21
1382
15546
CA 03174172 2022- 9- 28
0SL331 15525- 15525 TGCTAATATTGCACATTTGTTAA 3'-
UTR 21 1383
15547
0SL332 15575- 15575 ATGGATGAATGAATGAAACATAT 3'-
UTR 21 1384
15597
0SL333 15583- 15583 ATGAATGAAACATATACTACTGA 3'-
UTR 21 1385
15605
0SL334 15587- 15587 ATGAAACATATACTACTGATTAT 3'-
UTR 21 1386
15609
0SL335 15591- 15591 AACATATACTACTGATTATTTTA 3'-
UTR 21 1387
15613
0SL336 15655- 15655 CTGACTGTAATTACTTTGATTAG 3'-
UTR 21 1388
15677
0SL337 15659- 15659 CTGTAATTACTTTGATTAGATAA 3'-
UTR 21 1389
15681
0SL338 15675- 15675 TAGATAAACAACTGGAAATAATG 3'-
UTR 21 1390
15697
0SL339 15698- 15698 CTGCTGAAAAAGTTCTAATAAAT 3'-
UTR 21 1391
15720
0SL340 15699- 15699 TGCTGAAAAAGTTCTAATAAATG 3'-
UTR 21 1392
15721
Table 11
Additional table of siRNA sequences
SEQ OSID antisense sequence (5 to 3') OSID
sense sequence (3' to 5) SEQ ID
ID
NO:S
957 OSC17C-1 CAGUUGCGCAGUUUCUUGUC OSC17B-1 [dT][dT]GUCAACGCGUCA 973
AGUUC[dT][dT]
AAGAACAGUCAAG
958 OSC17C-2 CAGUUGCGCAGUUUCUUGU[nn OSC17B-2 [dT][dTrG*UCAACGCGUC 974
ClinnA]GUUC[dT][dT] AAAGAACAGUCAAG
959 OSC17C-3 CAGUUGCGCAGUUUCU[nnU][nn OSC17B-3 [dT][dTrG*UCAACGCGUC 975
G]UCAGUUC[dT][dT]
AAAGAACAG[mU]CAAG
960 OSC17C-4 CAGUUGCGCAGUUUCU[nnU][nn OSC17B-4 [dT][dTrG*UCAACGCGUC 976
G]U[nnC][nnA]GUUC[dT][dT]
AAAGAA[mC]AGUCAAG
OSC17B-5 [dT][dTrG*UCAACGCGUC 977
AAAGAA[nnC]AG[nnU]CAA
G
OSC17B-6 [dT][dT]*G*UCAA[nnC]GCG 978
UCAAAGAA[mC]AG[nnU]C
AAG
961 OSC47C-1 AAGAGCUCAGGUCUCUGAGG OSC47B-1 [dT][dT]UUCUCGAGUCCA 979
G[dT][dT] GAGACUCCC
962 OSC47C-2 AAGAGCUCAGGUCUC[nnU]GA OSC47B-2 [dT][dTrU*UCUCGAGUCC 980
GGG[dT][dT] AGAGACUCCC
963 OSC47C-3 AAGAGCUCAGGUCUC[nnU][nnG] OSC47B-3
[dT][dTrU*UCUCGAGUCC 981
AGGG[dT][dT] AGAGA[mC]UCCC
OSC47B-4 [dT][dTrU*UCUCGAGUCC 982
AGAG[nnA][nnC]UCCC
OSC47B-5 [dT][dTrU*UCUCGAG[nnU] 983
CCAGAG[nnA][mC]UCCC
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964 OSL231C-1 UUUGAUAGCACCAAACCUAGA OSL231B-1 [dT][dT]AAACUAUCGUGG 984
GCCC[dT][dT] UUUGGAUCUCGGG
965 OSL231C-2 UUUGA[nnU]AG[nnC]ACCAAACC[ OSL231B-2 [dT][dTrA*AACUAUCGUG 985
nnU]AGAGCCC[dT][dT] GUUUGGAUCUCGGG
966 OSL231C-3 UUUGAUAGCACCAAACC[nnU][ OSL231B-3 [dT][dT]*A*AACUA[nnU]CG[ 986
nnA]GAGCCC[dT][dT]
nnU]GGUUUGGA[nnU]CUC
GGG
967 OSL231C-4 UUUGAUAG[nnC]ACCAAACC[m OSL231B-4 [dT][dTrA*AACU[mA][mU] 987
U]AGAGCCC[dT][dT]
C[nnG][nnU]GGUUUGG[nnA]
[nnU]CUCGGG
968 OSL231C-5 UUUGAUAGCACCAAACC[nnU]A OSL231B-5 [dT][dTrA*AACUGUCGUG 988
GAGCCC[dT][dT]
GUUUGGA[nnU]CUCGGG
OSL231B-6 [dT][dTrA*AACUGUCG[nn 989
U]GGUUUGGA[nnU]CUCG
GG
OSL231B-7 [dT][dTrA*AACUAUCGUG 990
G[nnU]UUGGAUCUCGGG
969 0SL245C-1 UUUGCAAUGACUCUCCUAUCA 0SL245B-1 [dT][dT]AAACGUUACUGA 991
GUCC[dT][dT] GAGGAUAGUCAGG
970 0SL245C-2 UUUGCAA[nnU][nnG]ACUCUCC[ 0SL245B-2 [dT][dTrA*AACG[nnU]UA[nn 992
nnU][nnA]UCAGUCC[dT][dT]
C]UGAGAGGA[nnU]AGUC
AGG
971 0SL245C-3 UUUGCAAUGACUCUCC[nnU][nn 0SL245B-3 [dT][dTrA*AACGUUACUG 993
A]UCAGUCC[dT][dT]
AGAGGA[nnU]AGUCAGG
972 0SL245C-4 UUUGCAA[nnU][nnG]ACUCUCCU 0SL245B-4 [dT][dTrA*AACGUUA[nnC] 994
AUCAGUCC[dT][dT] UGAGAGGA[nn U]AG
UCAG
G
0SL245B-5 [dT][dTrA*AACGUUACUG 995
AGAGGAUAGUCAGG
Key to modifications
[dT] = DNA base (T) within RNA oligo
[mA], [mG], [mC], [mU] = 2'0-Methyl RNA
* = Phosphorothioate linkages
[00167] In one embodiment, an RNAi (e.g., a dsRNA) featured
herein includes a first
sequence of a dsRNA that is selected from the group including the sense
sequences of any table
herein and a second sequence that is selected from the group consisting of the
corresponding
antisense sequences of any table herein. A corresponding antisense sequence is
a nucleotide
sequence within the OSID family for example OSC17. In those instances when we
refer to an siRNA
with no suffix (e.g., OSC17), we mean that to indicate the dsRNA comprised of
the antisense and
sense strands corresponding to that number (e.g., OSC17A paired with OSC17S or
OSC17C-(n)
paired with OSC17B-(n) where "n" is any number of the OSC17 family).
[00168] Unless otherwise specified, the compounds provided
herein may be enantiomerically
pure, such as a single enantionner or a single diastereonner, or be
stereoisonneric mixtures, such as a
mixture of enantionners, e.g., a racennic mixture of two enantionners; or a
mixture of two or more
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CA 03174172 2022- 9- 28
diastereonners. Conventional techniques for the preparation/isolation of
individual enantionners include
synthesis from a suitable optically pure precursor, asymmetric synthesis from
achiral starting
materials, or resolution of an enantionneric mixture, for example, chiral
chromatography,
recrystallization, resolution, diastereonneric salt formation, or
derivatization into diastereonneric
adducts followed by separation. It is understood that the phosphorothioate
group, designated by an
asterisk (*), constitutes a stereogenic center, and the presence of each such
group in a sequence
engenders two diastereoisonners. The number of such diastereoisonners in a
double stranded RNAi
agent may be calculated by the formula 2^n, wherein n represents the number of
phosphorothioate
groups in a sequence comprised of a double stranded siRNA.
[00169] In some embodiments, the antisense strand (identified
with "A" in the OS ID name)
and/or the sense strand (identified with "S" in the OS ID name) of an RNAi
agent comprises or
consists of a nucleobase sequence, for example, "OSC17A-1"
CAGUUGCGCAGUUUCUUGUCAGUUC[dT][dT] (SEQ ID NO: 17), and the nucleobase sequence
may include at least one or more nucleotides as a modified nudeotide, and
wherein SEQ ID NO: 17 is
located at positions 1 to 25 (5'4 3') of the antisense strand and forms a
duplex with the corresponding
sense strand (identified as OSC17S-1. In some embodiments, the antisense
strand of an RNAi agent
comprises or consists of a nucleobase sequence for example
CAGUUGCGCAGUUUCUUGUCAGUUC[dT][dT] (SEQ ID NO: 17), wherein all or
substantially all or
1, 2, 3, 4 or 5 of the nucleotides are modified nucleotides (see for example
SEQ ID NO. 24), and
wherein SEQ ID NO: 24 is located at positions 1 to 27 (5 4 3') of the
antisense strand. For any
antisense or sense strand disclosed herein, in some embodiments, the antisense
strand of an RNAi
agent comprises or consists of the sequence (5 '4 3') wherein * is a
phosphorothioate linkage
between deoxy thynnine [dT]; and/or wherein nnC, nnA, mG, mU are 2'-0-methyl
cytidine, 2'-0-methyl
adenine, 2'-0-methyl guanosine, 2'-0-methyl uridine respectively; and/or
wherein 2fA, 2fU, 2fG, 2fC
are 2'-fluoro adenine, 2'-fluoro uridine, 2'-fluoro guanosine, and 2'-fluoro
cytosine respectively. The
antisense target on the nnRNA is identified with the same name but without the
notation of "A" or "S"
after the name. An antisense sequence with the same name, for example OSC17A-1
through
OSC17A-18 binds to the same nucleotide target sequence.
[00170] Sequences shown in Table 4 were transfected into HEK
293 (human embryonic
kidney) and MDA-MB-4355 (human melanoma) cell lines to determine their ability
to reduce the
protein expression of LRP2 and CD320 gene/protein. These two cell lines were
chosen because of
their relatively high expression levels of LRP2 as noted in the Human Protein
Atlas at world wide
web.proteinatlas.org and the NCI-60 gene expression profiles at
discover.nci.nih.gov/cellminer/ so
that a change in protein expression for LRP2 was easy to detect.
[00171] Referring now to FIG. 3A-B and FIG. 3D-E, HEK293 and
MDA-MB-231 cells were
transfected with 20nM of indicated siRNAs and incubated for 48 hours. Whole
cell lysates were
prepared and innnnunoblotted for CD320 and LRP2 protein levels. The protein
levels were normalized
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CA 03174172 2022- 9- 28
to a housekeeping control gene unaffected by the siRNA transfection. The
graphs represent the fold
change of protein levels compared to the scrambled siRNA control (OSS1).
(Average -1+ SEM is
shown, n=3).
[00172] CD320 and LRP2 protein levels were determined by
western blot and quantified by
Image Studio Software (LiCor Company), relative to a control protein that is
not affected by CD320 or
LRP2 knockdown. To determine the efficacy of knockdown, protein levels of
CD320 (FIG. 3 A-B) and
LRP2 (FIG. 3 D-E) on the samples that were exposed to siRNA sequences against
the nnRNA of
either gene, were compared to that in the untreated and scrambled controls
(black and gray bars,
respectively, in all graphs of FIG. 3). We found that both siRNA sequences
directed against CD320
(05C17 and 05C47) almost completely abrogated CD320 expression (circles in
FIG. 3 A-B). siLRP2
sequences resulted in variable efficiency in reducing LRP2 protein. Two
sequences (05L231 and
05L245) consistently reduced LRP2 levels 75% or more in both cell lines
(circles FIG 3 B, E).
[00173] Referring now to FIG. 6, lysates were made from
transformed (HEK293) and
representative cancer cell lines, and western blot was performed to determine
LRP2 protein levels.
The cancer cells screened have low levels of LRP2 expression. The results
represent the averages
+SEM of three independent lysates. The data suggests that the cancer cells
screened have very low
levels of LRP2 expression.
[00174] We transfected a panel of LRP2 and CD320 siRNAs into
cancer cell lines derived
from multiple tissues and analyzed the levels of LRP2 protein and CD320
protein in the cell line.
Representative cell lines from prostate, breast and glioblastonna, and normal
fibroblasts were exposed
to CD320 and LRP2 siRNAs in an experimental set-up similar to that described
for HEK293 and MDA-
MB-4355 cells. The results are shown in Fig 3 C, F, and FIG. 4.
[00175] Referring now to FIG. 3 C, F; and FIG. 4, MDA-MB-231
LnCAP, MCF-7 and U251
cells were exposed to siRNA sequences to knockdown CD320 (FIG. 3C, and FIG. 4
A-C) and LRP2
(FIG. 3 F, and FIG. 4 D-F), in a similar fashion as described for the data
represented in FIG. 3 A, B,
D, and E. CD320 protein knockdown FIG. 3C, and FIG. 4 A-C, compared to the
untreated or
scrambled controls, is more than 90% for all cell lines tested. LRP2 knockdown
is accomplished in all
cell lines too. However, the level of knockdown is less in the LnCAP cells
compared to the other cell
lines and the sequences that are effective may differ as well (FIG. 3 F, and
FIG. 4 D-F).
[00176] Referring now to FIG. 5, an experimental set up
similar to that described in FIG. 3
was employed. Additional prostate and brain cancer cell lines, as well as
normal fibroblast, were
exposed to siRNAs directed against CD320. Levels of CD320 were nearly
abrogated in DU-145
(prostate) cells, whereas the levels of knockdown in A172 brain cells and
normal fibroblasts were
21%-33% and 25%-28%, respectively (FIG. 5 A-C).
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[00177] From these studies we can conclude that two siRNAs to
CD320 (OSC17 and 0SC47)
are very effective in knocking down CD320 protein levels (80% or more), in
nearly every cell line
tested. While LRP2 is theoretically harder to knock down because of its size,
we have identified two
siRNAs, 0SL231 and 0SL245, that consistently knock down LRP2 in most cell
lines in which we can
detect LRP2.
[00178] In addition, LRP2 protein expression levels are very
high in HEK 293 cells and easily
detectable by western blot. Cancer cell lines have much lower expression of
LRP2 compared to
HEK293 cells as measured by western blot (FIG. 6), and some cell lines may
contain LRP2 at levels
below reliable detection.
[00179] Referring now to FIG. 7, the effects of doxorubicin
treatment on cell viability, as
measured by the CTG assay, are illustrated. A172 and HCC15 cells were plated
at 1200 cells/well in
a 96 well plate. The next day, cells were treated with doxorubicin at the
indicated concentrations.
Four days after the doxorubicin exposure was initiated, the cells were assayed
for viability using the
CTG assay. The line indicates the non-linear fitting of the data to calculate
an IC50 value. Instead of
visually assessing the effect of CD320/LRP2 gene expression knockdown on cell
proliferation (as
shown for shRNA-mediated CD320/LRP2 knockdown in FIG. 2), a functional assay
for quantitating
the effect on cell viability of the simultaneous knockdown of LRP2 and CD320
by siRNA was
developed. A widely used assay for the measurement of cell viability is the
Pronnega Cell-titer GLO
platform (CTG), which quantifies ATP levels in the cell (live cells produce
ATP, dead cells do not).
After incubating the cells with the CTG reagent, ATP levels can be indirectly
measured as light
production using the TECAN luminescence plate reader. As a first step,
toxicity of a known
chemotherapeutic drug, doxorubicin, was assayed on the cell lines of interest.
Doxorubicin was used
as a positive control for cell toxicity in our assay. Representative data from
A172 brain cancer cells
(FIG. 7A) and HCC15 lung cancer cells (FIG. 7B) exposed to doxorubicin are
shown in FIG. 7. From
this data, the IC50 of doxorubicin treatment on these cell lines was
determined: 132 nm for A172 cells
and 167 nnn for HCC15 cells. Based upon these findings, a larger screen was
initiated to determine
the IC50 of doxorubicin in several cancer and non-cancer cell lines, to
determine the doxorubicin dose
to use when cell lines are used in the viability assay to test the
simultaneous knockdown of CD320
and LRP2. The results of the cell lines tested are summarized in Table 8 IC50
determination of
doxorubicin.
[00180] To quantify the effects of knocking down CD320 and
LRP2 on cell proliferation, cells
are plated in a 24-well plate. The next day, the cells are transfected with
siRNAs to CD320 and/or
LRP2. The cell lines may require repeated transfections and/or time for
efficient toxicity (cell line
dependent). In this experimental set-up there is room for repeat infection
should some cell lines
require that for efficient toxicity. At the end of the study, the cell lines
are analyzed for cell growth by
the CTG assay. A schematic of this experimental setup is presented in FIG. 8.
CA 03174172 2022- 9- 28
Table 8
Cancer type Cell line IC50 (nM)
Glioblastoma A172 132
U251 24
Breast MDA-MB-231 43
MCF7 121
Prostate DU145 248
PC3 387
Lung NCI-H460 56
A549 126
HCC15 167
Melanoma MDA-MB-435S 325
Other GM05659 267
HEK293 29
[00181] The cells lines were plated at 1,000 to 4,000
cells/well in a 96-well plate and treated
with doxorubicin the following day. CTG activity was measured 4 days after
treatment. IC50 values
were calculated by GraphPad Prism Software. Results are tabulated in Table 8.
[00182] These data show that doxorubicin works efficiently on
this CTG platform (i.e.,
doxorubicin kills cancer cells) and can thus be used as a positive control in
the in vitro assay to
compare the cytotoxic effects of siRNA-knockdown of CD320 and LRP2. In this
latter assay, normal
or cancer cells are transfected with individual or combinations of siRNAs
sequences that are targeting
CD320 or LRP2 specifically or control siRNAs, similar to the experiments that
provided the data for
FIG, 3, 4, and 5. In FIG. 3, 4, and 5, protein levels are measured, but in the
in vitro assay, cell
viability is measured.
[00183] Referring now to FIG. 8, an overview of a functional
assay for screening (ds) siRNA
effects on cell proliferation is illustrated. To quantify the effects of
knocking down CD320 and LRP2
on cell proliferation, cells are plated in a 24-well plate. The next day, the
cells are transfected with
siRNAs to CD320 and/or LRP2. The cell lines may require repeated transfections
and/or time for
efficient toxicity (cell line dependent). In this experimental set-up there is
room for repeat infection
should some cell lines require that for efficient toxicity. At the end of the
study, the cell lines were
analyzed for cell growth by the CTG assay.
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[00184] Now, referring to FIG. 10B, MDA-MB-231 triple
negative breast cancer cells were
plated in a 24-well plate at 20,000 cells/well. Cells were transfected the
next day with an siRNA
selected from the group of OSC17, 0SC47, 0SL231, and 0SL245 at 20nM. Cells
were also
transfected with combinations of two siRNAs each of 10nM, one of these
targeting CD320 and the
other LRP2, with the siRNAs targeting CD320 selected from the group of OSC17
and 0SC47, and the
LRP2 targeting siRNAs selected from the group of 0SL231 and 0SL245, each dosed
at 10nM. Cells
were repeated transfected 4 times over the course of 11 days as indicated in
Table 9. At day 11,
cells were analyzed for cell growth by the CTG assay. The percent cell
survival compared to the non-
targeting control (OSS2) is shown. The data represented is the average of 6
experiments -/+ SEM.
[00185] Now, referring to FIG. 11, MDA-MB-231 and DU-145
cells were transfected with 20
nM of the negative control siRNA (OSS2), 20 nM siRNA targeting CD320 (OSC17),
or 20 nM siRNA
targeting LRP2 (05L245). Cells were also transfected with a combination of a
CD320 targeting
siRNA (OSC17) and LRP2 targeting siRNA (05L24), over a range of concentrations
(2-20 nM), so the
concentration of the two siRNAs equaled 20 nM total siRNA transfected, as
indicated in FIG. 11.
Cells were repeatedly transfected as indicated in Table 9, and the percent
cell survival is shown.
[00186] Now, referring to FIG. 12, MDA-MB-231 breast cancer
cells were transfected with 20
nM of the negative control siRNA (OSS2), 20 nM siRNA targeting CD320 (OSC17),
or 20 nM siRNA
targeting LRP2 (05L245). Each day, over five days, lysates were prepared.
Western blotting was
performed on the lysates for CD320 protein levels (FIG.12A) or LRP2 protein
levels (FIG. 12B).
[00187] Referring now to FIG. 9 and FIG. 10, data quantifying
the effects of knocking down
CD320 and LRP2 in various cell lines is represented. Cell lines representative
of several types of
cancers or normal fibroblasts were transfected with individual or combinations
of siRNAs to CD320 or
LRP2 as indicated. Cells were repeatedly transfected as outlined in Table 9
for efficient toxicity, then
assayed for viability by the CTG assay. Doxorubicin treated cells served as a
positive control for cell
toxicity in our assays.
[00188] The data of the individual experiments presented in
FIG. 9 and FIG. 10 and additional
cell lines we have screened are summarized in Table 9. These experiments show
the broad
applicability of siCD320 and siLRP2 toxicity in a variety of cancer types.
[00189] Referring now to FIG. 13, a schematic of PEI and
siRNA complexes is illustrated.
PEI and siRNAs are mixed together. Subsequently, polyplexes (a nanoparticle,
broadly speaking)
form of the PEI-siRNA complex, which are able to enter the cell via an
endocytotic or pinocytotic
mechanism.
[00190] Referring now to FIG. 14, siRNAs are short RNA
duplexes of generally 16 to 30
nucleotides; the sequence of the siRNA is complementary to a nnRNA expressed
in the cell.
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CA 03174172 2022- 9- 28
Exogenous siRNA duplexes are introduced into the cell via a method of
transfection. The siRNA
duplexes are unwound via the RISC (RNA-induced silencing complex) complex,
whereby the guide
strand of the siRNA hybridizes with its complementary nnRNA molecule. The
nnRNA is degraded by
the RISC/AGO complex, which has RNAse cleave activity. The end result is that
the nnRNA targeted
by the siRNA is degraded, and the protein encoded by the nnRNA is not
produced. This causes the
"knockdown" effect or reduced protein levels of the gene targeted by the siRNA
compared to control-
treated cells.
[00191] Referring now to FIG. 15 effectiveness of INTERFERin
in delivering siRNAs to cancer
cells is illustrated. 2 nM of indicated siRNAs were transfected into A172 and
MDA-MB-4355 cells as
per the manufacturers protocol. Cell lysates were prepared 3 days post-
infection and analyzed by
western blot for CD320 protein levels. OSS1 and OSS2 are non-targeting siRNA
controls. In this
experiment, both sequences were tested. CD320 protein levels were knocked down
to 9% to 18% for
A172 cells and 26% to 48% for MDA-MB-4355 cells, compared to OSS1. Much more
efficient
knockdown of CD320 is observed when the siRNAs were delivered with other
transfection reagents
(e.g. RNAiMAX, Viromer Blue) that were used in the experiments described
previously particularly for
MDA-MB-4355 cells. The Polyplus INTERFERin platform has been tested in vitro
in our laboratory in
a proof of principle experiment, whereby the platform is able to deliver
siRNAs to the target cells in
vitro.
[00192] Referring now to FIG. 16, treatment of breast,
prostate, and skin cancer cells with an
inhibitor of CD320 receptor or an inhibitor of LRP2 receptor or a combination
of both in an amount
effective to inhibit proliferation of the cancer cells as compared to the
control cells treated with control
siRNA is illustrated. MDA-MB-231, DU145, LnCAP, and MDA-MB-4355 cells were
plated at 20,000
cells per well in a 24-well plate. The next day, the cells were transected
with 20 nM of indicated
siRNAs to knock down CD320, LRP2, or scrambled control. For the combination of
siRNAs, cells
were treated with 10 nM of each siRNA for 20 nM total treatments. Cells were
repeatedly transfected
as in Table 9 for the length of time indicated in Table 9. The indicated
pictures of the cells were taken
at the end of the experiment.
[00193] Table 9. Summary of functional siRNA data screening
n Single siRNA knockdown Double siRNA
knockdown DOX
Cell line
# of Days (siCD320) (siLRP2) 05C17 +
05C47 +
txns expt
OSC- OSC- OSL- OSL- OSL OSL OSL OSL
47 245
17 231 231 245 231 245
Normal
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GM05659 3 4 7 133 104 96 103 111 129 97 107 39
Lung
HCC15 3 2 7 86 109 68 92 81 93 38 105 2
H157 3 5 8 7 9 116 33 119 39 31 15 20
Melanoma
MDA-MB-435S 4 5 10 119 108 92 31 122 56
103 70 51
Prostate
LnCAP 3 4 7 52 42 72 60 68 51 52 57 38
DU-145 6 4 7 39 82 71 44 80 44 90 76 40
Glioblastoma
A172 3 5 7 94 17 50 101 73 107 19 51 8
U251 4 6 8 94 50 77 97 99 94 66 87 51
Breast
MCF-7 3 2 7 61 69 32 68 31 52 26 48 5
MDA-MB-231 6 4 11 66 81 130 77 71 44
81 61 7
Note: The numbers represent percent survival compared to negative control
OSS2.
[00194] A murine human tumor xenograft model was established
using triple-negative breast
cancer cells (MDA-MB-231) injected into the flanks of nude mice to test the
efficacy of combined
dosing of OSC17 and 0SL245. The administration of the drug is by repeated
dosing over a range of
drug concentrations using intratunnoral, iv, ip or specialized route of
administration. The dosing
schedule is based on pilot studies to determine the tolerability of the
delivery vehicle and the drug and
will incorporate ranges that are taught in the art. Among the delivery
platforms are nanoparticles,
liposonnes, micelles, polymers, small molecule conjugates, aptanners and
antibody conjugates. Hybrid
technologies containing elements of the aforementioned delivery systems are
also known.
[00195] The manufacturing process consists of synthesizing
the two single strand
oligonucleotides of the duplex by conventional solid phase oligonudeotide
synthesis. After
purification, the two oligonucleotides are annealed into the duplex.
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CA 03174172 2022- 9- 28
[00196] In vivo JetPEI is a cationic polymer delivery system
that binds the negatively
charged siRNA molecules to the cationic polyannine polymer. Its use has been
reported in xenograft
models using MCF-7 (breast), MDA-MB-231 (breast) and A549 (lung) cell lines
both ip and
intratunnoral. This delivery system is currently used in seven human clinical
trials (Table 10). The
formulated siRNAs are reported to be very stable.
Table 10 ¨ Clinical trial use of in vivo-jetPEI
Organization Type of study Phase
Cancer Targeting Imaging and cancer Pre-clinical
Systems therapy
Benitec Lung metastases Pre-clinical
Avena Blood-brain barrier Pre-clinical
BiOncoTech Melanoma Phase 1
immunotherapy
Ottawa Hospital Research Acute myocardial Phase 1
Institute infarction gene therapy
CHU-Toulouse, Rangueil Pancreatic cancer gene Phase 2
Hospital therapy
BioCancell Bladder cancer gene Phase 3
therapy
[00197] Note that in the specification and claims, "about" or
"approximately" means within
twenty percent (20%) of the numerical amount cited. Although the invention has
been described in
detail with particular reference to these embodiments, other embodiments can
achieve the same
results. For example, antisense oligonucleotides that are complimentary to the
target nnRNA can
inhibit expression of the protein of interest even though the antisense
oligonucleotide is not provided
as a dsRNA and may not bind to RISC/AGO complex. Variations and modifications
of the present
invention will be obvious to those skilled in the art and it is intended to
cover in the appended claims
all such modifications and equivalents. The entire disclosures of all
references, applications, patents,
and publications cited above are hereby incorporated by reference.
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