Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR THE TREATMENT OF SUBJECTS HAVING A HEPATITIS B VIRUS
(HBV) INFECTION
RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Patent
Application No.
62/486,618, filed on April 18, 2017, U.S. Provisional Patent Application No.
62/553,358, filed on
September 1, 2017, U.S. Provisional Patent Application No. 62/646,978, filed
on March 23, 2018, and
U.S. Provisional Patent Application No. 62/655,862, filed on April 11, 2018.
The entire contents of
each of the foregoing provisional patent applications are incorporated herein
by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted
electronically
in ASCII format and is hereby incorporated by reference in its entirety. Said
ASCII copy, created
on April 12, 2018, is named 121301-07220_SL.txt and is 429,841 bytes in size.
BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) is an enveloped DNA virus that infects the liver that
causes
hepatocellular necrosis, inflammation, and is a major risk factor for
development of cirrhosis and
hepatocellular carcinoma (HCC). The World Health Organization (WHO) estimates
that there are 240
million chronically HBV infected individuals worldwide largely in low to
middle income countries
and that about 650,000 people will die annually of complications from HBV
infection. Although an
effective HBV vaccine is available and efforts to vaccinate infants at birth
have been effective in
reducing incidence and prevalence of HBV infection, such programs do not have
a demonstrable
effect on death rates for years (WHO Guidelines for prevention, care and
treatment of persons with
chronic Hepatitis B Infection, 2015 at
apps.who.inthris/bitstream/10665/154590/1/9789241549059_eng.pdf?ua=1&ua=1).
A number of therapeutic agents have been developed for the treatment of HBV
that
effectively reduce the disease burden of HBV infection, but they are not
typically curative as they do
not fully eliminate all replicative forms of the virus including the
covalently closed circular DNA
(cccDNA) that resides in the hepatocyte nucleus and becomes a template for
viral replication and
transcription of viral RNAs. Nucleotide and nucleoside analogs, typically
considered to be the gold
standard for treatment of chronic HBV infection due to their safety and
efficacy, are effective in
suppressing HBV replication, but they do not eliminate cccDNA, do not prevent
expression of viral
proteins, must be dosed chronically, and can result in the development of
resistance. Further,
treatment with nucleot(s)ide inhibitors does not fully mitigate the risk of
the development of
hepatocellular carcinoma (HCC) which remains significant (about 7% in 5-7
years despite treatment).
Interferon-based therapies can result in sero-conversion and cure of about 10-
15% of patients
allowing discontinuation of treatment, but the agents have severe side effects
and must be refrigerated
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for long term storage making them less desirable for use in countries where
HBV infection is most
prevalent.
Treatment of chronic HBV is further complicated by the ability of HBV to evade
or suppress
the immune response resulting in persistence of the infection. The HBV
proteins have immune-
inhibitory properties, with hepatitis B s antigen (HBsAg) comprising the
overwhelming majority of
HBV protein in the circulation of HBV infected subjects. Additionally, while
the removal (via
seroconversion) of hepatitis B e antigen (HBeAg) or reductions in serum
viremia are not correlated
with the development of sustained control of HBV infection off treatment, the
removal of serum
HBsAg from the blood (and seroconversion) in HBV infection is a well-
recognized prognostic
indicator of antiviral response to treatment which will lead to control of HBV
infection off treatment.
However, this only occurs in a small fraction of patients receiving
immunotherapy. Therefore, it is
possible, although rare, for patients to mount a sufficiently robust immune
response to suppress or
clear an HBV infection resulting in at least a functional cure of the disease.
SUMMARY OF THE INVENTION
The invention provides RNAi agents and HBV vaccines for use in treatment of
hepatitis B
virus (HBV) infection and methods for the treatment of a subject having a
hepatitis B virus (HBV)
infection, e.g., a chronic HBV infection, which includes a combination therapy
or treatment regimen
including an RNAi agent targeting at least one HBV transcript and a
therapeutic vaccination.
Accordingly, in one aspect, the present invention provides RNAi agents and HBV
vaccines
for use in treatment of hepatitis B virus (HBV) infection and methods for
treating a subject having a
hepatitis B virus (HBV) infection, e.g., a chronic HBV infection. The methods
include a regimen
which includes administering, e.g., sequentially administering, to the subject
having the HBV
infection, an RNAi agent that targets at least three HBV transcripts, wherein
the RNAi agent
comprises a sense strand and an antisense strand; a protein-based vaccine
comprising an HBV core
antigen (HBcAg) and an HBV surface antigen (HBsAg); and a nucleic acid-based
vaccine comprising
an expression construct encoding an HBcAg or an HBsAg, wherein the construct
encodes a protein
that shares an epitope with the protein-based vaccine, thereby treating the
subject.
In another aspect, the present invention provides a regimen for treating a
subject having a
hepatitis B virus (HBV) infection, e.g., a chronic HBV infection. The regimen
includes the use of an
RNAi agent that targets at least three HBV transcripts, wherein the RNAi agent
comprises a sense
strand and an antisense strand; a protein-based vaccine comprising an HBV core
antigen (HBcAg)
and an HBV surface antigen (HBsAg); and a nucleic acid-based vaccine
comprising an expression
construct encoding an HBcAg or an HBsAg, wherein the construct encodes a
protein that shares an
epitope with the protein-based vaccine.
In one embodiment, the HBcAg protein, or immunogenic fragment thereof, shares
an epitope
with the HBV core antigen (HBcAg) polypeptide, or immunogenic fragment
thereof, present in the
protein-based vaccine and/or the HBsAg protein, or immunogenic fragment
thereof, shares an epitope
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with the HBV surface antigen (HBsAg) polypeptide, or immunogenic fragment
thereof, present in the
protein-based vaccine.
In certain embodiments, the RNAi agent comprises at least one modified
nucleotide.
In certain embodiments, the nucleic acid-based vaccine comprises an expression
construct
encoding an HBcAg and an HBsAg.
In certain embodiments, the RNAi agent targeting HBV is administered to the
subject at least
two times.
In certain embodiments, the RNAi agent targeting HBV administered to the
subject decreases
HBsAg in the serum of the subject by at least 0.5 log 10 IU/ml. In certain
embodiments, the subject
has at least a 0.5 10g10 IU/ml decrease in HBsAg in serum prior to
administration of the first dose of
the protein based vaccine. In certain embodiments, the subject has at least a
1 10g10 IU/ml decrease
in HBsAg in serum prior to administration of the first dose of the protein
based vaccine. In certain
embodiments, the subject has an HBsAg of 2 log 10 IU/ml or less in serum prior
to administration of
the vaccine.
In certain embodiments, the RNAi agent is administered to the subject no more
than once per
week. In certain embodiments, the RNAi agent is administered to the subject no
more than once every
four weeks.
In certain embodiments, the RNAi ageint is administered to the subject at a
dose of 0.01
mg/kg to 10 mg/kg; or 0.5 mg/kg to 50 mg/kg; or 10 mg/kg to 30 mg/kg. In
certain embodiments, the
RNAi agent is administered to the subject at a dose selected from 0.5 mg/kg, 1
mg/kg, 1.5 mg/kg, 3
mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg. In some embodiments, a dose of the
RNAi agent is
administered to the subject about once per week; about one every two weeks;
about once every three
weeks; about once every four weeks; or a dose of the RNAi agent is
administered to the subject not
more than once per week; or a dose of the RNAi agent is administered to the
subject no more than
once every four weeks.
In certain embodiments, the protein-based vaccine comprises an amino acid
sequence of at
least one determinant of HBsAg and at least one determinant of HBcAg.
In certain embodiments, the nucleic acid-based vaccine comprising an
expression vector
construct encoding an HBcAg or an HBsAg encodes an amino acid sequence
comprising at least one
determinant of HBsAg or at least one determinant of HBcAg
In certain embodiments, the determinant of HBsAg comprises a sequence at least
90%
identical to amino acids 124 to 147 of SEQ ID NO: 22. In certain embodiments,
the determinant of
HBsAg comprises a sequence at least 90% identical to amino acids 99 to 168 of
SEQ ID NO: 23.
In certain embodiments, the determinant of HBcAg comprises a sequence
comprising amino
acid 80 of SEQ ID NO: 24. In certain embodiments, the sequence comprising
amino acid 80 of SEQ
ID NO: 24 comprises an amino acid sequence at lest 90% identical to at least
amino acids 70 to 90 of
SEQ ID NO: 24. In certain embodiments, the determinant of HBcAg comprises a
sequence
comprising amino acid 138 of SEQ ID NO: 24. In certain embodiments, the
sequence comprising
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amino acid 80 of SEQ ID NO: 14 comprises an amino acid sequence at lest 90%
identical to at least
amino acids 128 to 143 of SEQ ID NO: 24. In certain embodiments, the
determinant of HBcAg
comprises an amino acid sequence at least 90% identical to at least 40, 50,
60, 70, 80, 90, or 100
contiguous amino acids of SEQ ID NO: 24. In certain embodiments, the
determinant of HBcAg
comprises a sequence at least 90% identical to amino acids 18 to 143 of SEQ ID
NO: 24.
In certain embodiments, the protein-based vaccine administered to the subject
comprises a
dose of 0.1 lig to 1.0 mg of the HBcAg and a dose of 0.1 lig to 1.0 mg of
HBsAg. In some
embodiments, a dose of the protein-based vaccine is administered to the
subject about once per week;
about one every two weeks; about once every three weeks; about once every four
weeks; or a dose of
the protein-based vaccine is administered to the subject no more than once per
week; or a dose of the
protein-based vaccine is administered to the subject no more than once every
four weeks.
In certain embodiments, the HBcAg protein and the HBsAg protein are present in
a single
formulation. In certain embodiments, the HBcAg protein and the HBsAg protein
are not present in a
single formulation. In certain embodiments, the protein-based vaccine
comprises an adjuvant. In some
embodiments, the adjuvant stimulates a balanced Th1/Th2 response. In certain
embodiments, the
adjuvant is selected from monophosphoryl lipid A (MPL), poly(I:C), polyICLC
adjuvant, CpG DNA,
polyICLC adjuvant ,a STING agonist, c-di-AMP, c-di-GMP, c-di-CMP; short, blunt-
ended 5'-
triphosphate dsRNA (3pRNA) Rig-Iligand,
poly[di(sodiumcarboxylatoethylphenoxy)phosphazene]
(PCEP)), alum, virosomes, cytokines, IL-12, A502, A503, A504, MF59, ISCOMATRIX
, IC31 , or
Rig-I ligand. In certain embodiments, the adjuvant is selected from polyI:C
adjuvant, a CpG adjuvant,
a STING agonist, or a PCEP adjuvant.
In certain embodiments, the protein based vaccine is administered to the
subject at least two
times. In certain embodiments, the protein-based vaccine is administered to
the subject no more than
once every two weeks. In certain embodiments, the protein-based vaccine is
administered to the
subject no sooner than the day on which the final dose of the RNAi agent has
been administered to the
subject. In certain embodiments, the protein-based vaccine is administered to
the subject on the same
day on which the final dose of the RNAi agent has been administered to the
subject. In certain
embodiments, the protein based vaccine is administered to the subject no later
than one month after
the final dose of the RNAi agent has been administered to the subject. In
certain embodiments, the
protein based vaccine is administered to the subject no later than two months
after the final dose of
the RNAi agent has been administered to the subject. In certain embodiments,
the protein based
vaccine is administered to the subject no later than three months after the
final dose of the RNAi agent
has been administered to the subject.
In certain embodiments the methods of the invention further comprise
determining the serum
HBsAg level after administration of at least one dose of the RNAi agent and
prior to administration of
the protein based vaccine. That is, the serum HBsAg level is determined in the
subject after
administration of at least one dose of the RNAi agent and prior to
administration of the protein based
vaccine.
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In certain embodiments the methods of the invention further comprise
determining the serum
HBeAg level after administration of at least one dose of the RNAi agent and
prior to administration of
the protein based vaccine. That is, the serum HBeAg level is determined in the
subject after
administration of at least one dose of the RNAi agent and prior to
administration of the protein based
vaccine.
In certain embodiments the methods of the invention further comprise
determining the serum
HBsAg level and the HBeAg level after administration of at least one dose of
the RNAi agent and
prior to administration of the protein based vaccine. That is, the serum HBsAg
level and the serum
HBeAg level are determined in the subject after administration of at least one
dose of the RNAi agent
and prior to administration of the protein based vaccine.
In certain embodiments, the nucleic acid-based vaccine comprises at least one
expression
vector construct encoding both an HBcAg and an HBsAg. In certain embodiments,
the expression
construct promotes expression of HBcAg and HBsAg from a single promoter. In
other embodiments,
the expression construct promotes expression of HBcAg and HBsAg from separate
promoters.
In certain embodiments, at least one promoter is selected from a respiratory
syncytial virus
(RSV) promoter, a cytomegalovirus (CMV) promoter, a PH5 promoter, and an H1
promoter. In
certain embodiments, the expression construct comprises a viral vector. In
certain embodiments, the
viral vector is selected from adenovirus vector; retrovirus vector, lentiviral
vector, moloney murine
leukemia virus vector, adeno- associated virus vector; herpes simplex virus
vector; SV 40 vector;
polyoma virus vector; papilloma virus vector; picornavirus vector; pox virus
vector, orthopox virus
vector, vaccinia virus vector, modified vaccinia virus Ankara (MVA) vector,
avipox vector, canary
pox vector, fowl pox vector, adenovirus vector, and Epstein Barr virus vector.
In certain
embodiments, the viral vector is an MVA vector.
In certain emboidments, the nucleic acid-based vaccine administered to the
subject comprises
a tissue-culture infectious dose (TCID50) of 106 to 101 TCID50; or 106 to 109
TCID50; or 106 to 108
TCID50
In certain embodiments, the nucleic acid-based vector is administered to the
subject no sooner
than two weeks after administration of the final dose of the protein-based
vaccine is administered to
the subject.
In certain embodiments, the methods further comprise determining the serum
HBsAg level
after administration of at least one dose of the RNAi agent and prior to
administration of the nucleic
acid-based vaccine. That is, the serum HBsAg level is determined in the
subject after administration
of at least one dose of the RNAi agent and prior to administration of the
nucleic acid-based vaccine.
In certain embodiments, the nucleic acid-based vaccine is administered to the
subject after a further
decrease of at least 0.5 log 10 of serum HBsAg after at least one dose of the
protein-based vaccine is
administered to the subject. In certain embodiments of the regimen, a single
dose of the nucleic-acid
based vaccine is administered to the subject.
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In certain embodiments, the methods further comprise determining the serum
HBeAg level
after administration of at least one dose of the RNAi agent and prior to
administration of the nucleic
acid-based vaccine. That is, the serum HBeAg level is determined in the
subject after administration
of at least one dose of the RNAi agent and prior to administration of the
nucleic acid-based vaccine.
In certain embodiments, the nucleic acid-based vaccine is administered to the
subject after a further
decrease of at least 0.5 log 10 of serum HBeAg after at least one dose of the
protein-based vaccine is
administered to the subject. In certain embodiments of the regimen, a single
dose of the nucleic-acid
based vaccine is administered to the subject.
In certain embodiments, the methods further comprise determining the serum
HBsAg level
.. and the HBeAg level after administration of at least one dose of the RNAi
agent and prior to
administration of the nucleic acid-based vaccine. That is, the serum HBsAg
level and the serum
HBeAg level are determined in the subject after administration of at least one
dose of the RNAi agent
and prior to administration of the nucleic acid-based vaccine. In certain
embodiments, the nucleic
acid-based vaccine is administered to the subject after a further decrease of
at least 0.5 log 10 of
.. serum HBsAg and serum HBeAg after at least one dose of the protein-based
vaccine is administered
to the subject. In certain embodiments of the regimen, a single dose of the
nucleic-acid based vaccine
is administered to the subject.
In certain embodiments, the methods further comprise administering a
nucleot(s)ide analog to
the subject at least prior to administration of the iRNA targeted to HBV. In
certain embodiments, the
.. nucleot(s)ide analog is administered throughout the entire regimen. In
certain embodiments, the
nucleot(s)ide analog is selected from Tenofovir disoproxil fumarate (TDF),
Tenofovir alafenamide
(TAF), Lamivudine, Adefovir dipivoxil, Entecavir (ETV), Telbivudine, AGX-1009,
emtricitabine,
clevudine, ritonavir, dipivoxil, lobucavir, famvir, FTC, N-Acetyl-Cysteine
(NAC), PC1323,
theradigm-HBV, thymosin-alpha, and ganciclovir, besifovir (ANA-380/LB-80380),
and tenofovir-
exaliades (TLX/CMX157).
In certain embodiments, the subject has serum HBsAg below 3000 IU/ml prior to
administration of the RNAi agent. In certain embodiments, the subject has
serum HBsAg below 4000
IU/ml prior to administration of the RNAi agent. In certain embodiments,
subject has serum HBsAg
below 5000 IU/ml prior to administration of the RNAi agent.
In certain embodiments, the subject has a reduction of HBsAg level of at least
2 logio scales
after administration of the RNAi agent and prior to administration of the
first dose of a protein-based
vaccine. In certain embodiments, the subject has a reduction of HBeAg level of
at least 1 logio scale
after administration of the RNAi agent and prior to administration of the
first dose of a protein-based
vaccine. In certain embodiments, the subject has a reduction of HBsAg level of
at least 2 logio scales
.. and a reduction of HBeAg level of at least 1 logio scale after
administration of the RNAi agent and
prior to administration of the first dose of a protein-based vaccine.
In certain embodiments, the subject has a reduction of HBsAg level to 500
IU/ml or less after
administration of the RNAi agent and prior to administration of a first dose
of the protein based
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vaccine. In certain embodiments, the subject has a reduction of HBsAg level to
200 IU/ml or less after
administration of the RNAi agent and prior to administration of a first dose
of the protein based
vaccine. In certain embodiments, the subject has a reduction of HBsAg level to
100 IU/ml or less
after administration of the RNAi agent and prior to administration of a first
dose of the protein based
vaccine.
In certain embodiments, the subject has a reduction of HBeAg level to 500
IU/ml or less after
administration of the RNAi agent and prior to administration of a first dose
of the protein based
vaccine. In certain embodiments, the subject has a reduction of HBeAg level to
200 IU/ml or less after
administration of the RNAi agent and prior to administration of a first dose
of the protein based
vaccine. In certain embodiments, the subject has a reduction of HBeAg level to
100 IU/ml or less after
administration of the RNAi agent and prior to administration of a first dose
of the protein based
vaccine.
In certain embodiments, the subject has a reduction of HBsAg level and HBeAg
level to 500
IU/ml or less after administration of the RNAi agent and prior to
administration of a first dose of the
protein based vaccine. In certain embodiments, the subject has a reduction of
HBsAg level and
HBeAg level to 200 IU/ml or less after administration of the RNAi agent and
prior to administration
of a first dose of the protein based vaccine. In certain embodiments, the
subject has a reduction of
HBsAg level and HBeAg level to 100 IU/ml or less after administration of the
RNAi agent and prior
to administration of a first dose of the protein based vaccine.
In certain embodiments, the level of serum HBsAg and HBeAg in the subject are
decreased to
below the level of detection using a clinical assay for at least three months
after the end of the dose of
the nucleic acid-based vaccine. In certain embodiments, the level of serum
HBsAg and HBeAg in the
subject are decreased to below the level of detection using a clinical assay
for at least six months after
the end of the dose of the nucleic acid-based vaccine.
In certain embodiments, serum HBsAg in the subject is decreased to below the
level of
detection using a clinical assay for at least six months after the end of the
dose of the nucleic acid-
based vaccine.
In certain embodiments, the methods further comprise administration of an
immune
stimulator to the subject. In certain embodiments, the immune stimulator is
selected from the group
pegylated interferon alfa 2a (PEG-IFN-alpha-2a), Interferon alfa-2b, PEG-IFN-
alpha-2b, Interferon
lambda a recombinant human interleukin-7, and a Toll-like receptor 3, 7, 8 or
9 (TLR3, TLR7, TLR8,
TLR9) agonist, a viral entry inhibitor, Myrcludex, an oligonucleotide that
inhibits the secretion or
release of HBsAg, REP 9AC, a capsid inhibitor, Bay41-4109, NVR-1221, a cccDNA
inhibitor,
IHVR-25) a viral capsid, an MVA capsid, an immune checkpoint regulator, an
CTLA-4 inhibitor,
ipilimumab, a PD-1 inhibitor, Nivolumab, Pembrolizumab, BGB-A317 antibody, a
PD-Li inhibitor,
atezolizumab, avelumab, durvalumab, and an affimer biotherapeutic.
In certain embodiments of the regimen, the subject is human.
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In certain embodiments of the regimen, the RNAi agent targets four transcripts
of HBV. In
certain embodiments, the RNAi agent is selected from an iRNA in Appendix A. In
certain
embodiments, the RNAi agent is selected from any of the agents in any one of
Tables 2-11 in
Appendix A. In certain embodiments, the RNAi agent targets at least 15
contiguous nucleotides of
nucleotides 206-228, 207-229, 210-232, 212-234, 214-236, 215-237, 216-238, 226-
248, 245-267,
250-272, 252-274, 253-275, 254-276, 256-278, 258-280, 263-285, 370-392, 373-
395, 375-397, 401-
423, 405-427, 410-432, 411-433, 422-444, 424-446, 425-447, 426-448, 731-753,
734-756, 1174-
1196, 1250-1272, 1255-1277, 1256-1278, 1545-1567, 1547-1569, 1551-1571, 1577-
1597, 1579-1597,
1580-1598, 1806-1825, 1812-1831, 1814-1836, 1829-1851, 1831-1853, 1857-1879,
1864-1886, 2259-
2281, 2298-2320, or 2828-2850 of SEQ ID NO: 1 (NC_003977.1). In certain
embodiments, the
RNAi agent targets at least 15 contiguous nucleotides of nucleotides 1579-1597
or 1812-1831 of SEQ
ID NO: 1 (NC_003977.1). In certain embodiments, the RNAi agent targets
nucleotides 1579-1597 or
1812-1831 of SEQ ID NO: 1 (NC_003977.1).
In certain embodiments, the antisense strand of the RNAi agent comprises a
nucleotide
sequence of at least 15 contiguous nucleotides of UGUGAAGCGAAGUGCACACUU (SEQ
ID NO:
25) or AGGUGAAAAAGUUGCAUGGUGUU (SEQ ID NO: 26). In certain embodiments, the
antisense strand of the RNAi agent comprises a nucleotide sequence of at least
19 contiguous
nucleotides of UGUGAAGCGAAGUGCACACUU (SEQ ID NO: 25) or
AGGUGAAAAAGUUGCAUGGUGUU (SEQ ID NO: 26). In certain embodiments, the antisense
strand of the RNAi agent comprises a nucleotide sequence of
UGUGAAGCGAAGUGCACACUU
(SEQ ID NO: 25) or AGGUGAAAAAGUUGCAUGGUGUU (SEQ ID NO: 26).
In certain embodiments, the sense strand of the RNAi agent comprises a
nucleotide sequence
of at least 15 contiguous nucleotides of GUGUGCACUUCGCUUCACA (SEQ ID NO: 27)
or
CACCAUGCAACUUUUUCACCU (SEQ ID NO: 28). In certain embodiments, the sense
strand of
the RNAi agent comprises a nucleotide sequence of at least 19 contiguous
nucleotides of
GUGUGCACUUCGCUUCACA (SEQ ID NO: 27) or CACCAUGCAACUUUUUCACCU (SEQ ID
NO: 28). In certain embodiments, the sense strand of the RNAi agent comprises
a nucleotide sequence
of GUGUGCACUUCGCUUCACA (SEQ ID NO: 27) or CACCAUGCAACUUUUUCACCU (SEQ
ID NO: 28).
In certain embodiments, the antisense strand of the RNAi agent comprises a
nucleotide
sequence of at least 15 contiguous nucleotides of UGUGAAGCGAAGUGCACACUU (SEQ
ID NO:
25) and the sense strand comprises a nucleotide sequence of at least 15
contiguous nucleotides of
GUGUGCACUUCGCUUCACA (SEQ ID NO: 27). In certain embodiments, the antisense
strand of
the RNAi agent comprises a nucleotide sequence of at least 19 contiguous
nucleotides of
UGUGAAGCGAAGUGCACACUU (SEQ ID NO: 25) and the sense strand comprises a
nucleotide
sequence of at least 19 contiguous nucleotides of GUGUGCACUUCGCUUCACA (SEQ ID
NO: 27).
In certain embodiments, the antisense strand of the RNAi agent comprises a
nucleotide sequence of
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UGUGAAGCGAAGUGCACACUU (SEQ ID NO: 25) and the sense strand comprises a
nucleotide
sequence of GUGUGCACUUCGCUUCACA (SEQ ID NO: 27).
In certain embodiments, the antisense strand of the RNAi agent comprises a
nucleotide
sequence of at least 15 contiguous nucleotides of AGGUGAAAAAGUUGCAUGGUGUU (SEQ
ID
NO: 26) and the sense strand of the RNAi agent comprises a nucleotide sequence
of at least 15
contiguous nucleotides of CACCAUGCAACUUUUUCACCU (SEQ ID NO: 28). In certain
embodiments, the antisense strand of the RNAi agent comprises a nucleotide
sequence of at least 19
contiguous nucleotides of AGGUGAAAAAGUUGCAUGGUGUU (SEQ ID NO: 26) and the
sense
strand of the RNAi agent comprises a nucleotide sequence of at least 19
contiguous nucleotides of
CACCAUGCAACUUUUUCACCU (SEQ ID NO: 28). In certain embodiments, the antisense
strand
of the RNAi agent comprises a nucleotide sequence of AGGUGAAAAAGUUGCAUGGUGUU
(SEQ ID NO: 26) and the sense strand of the RNAi agent comprises a nucleotide
sequence of
CACCAUGCAACUUUUUCACCU (SEQ ID NO: 28).
In certain embodiments of the regimen, substantially all of the nucleotides of
said sense strand
and substantially all of the nucleotides of said antisense strand are modified
nucleotides, wherein said
sense strand is conjugated to a ligand attached at the 3'-terminus. In certain
embodiments, the ligand
is one or more GalNAc derivatives attached through a monovalent linker,
bivalent branched linker, or
trivalent branched linker. In certain embodiments, the at least one of said
modified nucleotides is
selected from the group consisting of a deoxy-nucleotide, a 3'-terminal deoxy-
thymine (dT)
nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified
nucleotide, a 2'-deoxy-modified
nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally
restricted nucleotide, a
constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified
nucleotide, a 2'-0-allyl-
modified nucleotide, 2' -C-alkyl-modified nucleotide, 2' -hydroxly-modified
nucleotide, a 2' -
methoxyethyl modified nucleotide, a 2'-0-alkyl-modified nucleotide, a
morpholino nucleotide, a
phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran
modified nucleotide, a
1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a
nucleotide comprising
a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a
nucleotide
comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.
In certain embodiments, at least one strand of the RNAi agent comprises a 3'
overhang of at
least 1 nucleotide. In certain embodiments, at least one strand if the RNAi
agent comprises a 3'
overhang of at least 2 nucleotides. In certain embodiments, the double-
stranded region of the RNAi
agent is 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length;
17-25 nucleotide pairs in
length; 23-27 nucleotide pairs in length; 19-21 nucleotide pairs in length; 21-
23 nucleotide pairs in
length.
In certain embodiments, each strand of the RNAi agent has 15-30 nucleotides.
In certain embodiments, each strand of the RNAi agent has 19-30 nucleotides.
In certain embodiments, the ligand is
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HO 1:DEI
HO Of,-NN 0
AcHN
0
HO OH
0
HO
AcHN
0 0 0
O
HO H
0
HO
AcHN
0
and the RNAi agent is optionally conjugated to the ligand as shown in the
following
schematic
3'
9
0
__________________________________________________ OH
s S.
HO PH
fLO
AcHN 0
Ho OH
(1)- H
N
AcHN 0 0 0' 0
HO PH
O
HO N
AcHN H H
0
wherein X is 0 or S.
In certain embodiments, the sense strand comprises the nucleotide sequence 5'-
gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 29) and the antisense strand
comprises the
nucleotide sequence 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 30); or
the sense
strand comprises the nucleotide sequence 5'-csasccauGfcAfAfCfuuuuucaccu-3'
(SEQ ID NO: 31) and
the antisense strand comprises the nucleotide sequence 5'-
asGfsgugAfaAfAfaguuGfcAfuggugsusu-3'
(SEQ ID NO: 32), wherein a, c, and u are 2'-0-methyladenosine-3'-phosphate, 2'-
0-methylcytidine-
3'-phosphate, and 2'-0-methyluridine-3'-phosphate, respectively; as, cs, gs,
and us are 2'-0-
methyladenosine-3'- phosphorothioate, 2'-0-methylcytidine-3'-
phosphorothioate, 2'-0-
methylguanosine-3'- phosphorothioate, and 2'-0-methyluridine-3'-
phosphorothioate, respectively; Af,
Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-
phosphate, 2'-
fluoroguanosine-3'-phosphate, and 2'-fluorouridine-3'-phosphate, respectively;
and Gfs is 2'-
fluoroguanosine-3'-phosphorothioate. In certain embodiments, the RNAi agent is
AD-66810 or AD-
66816.
In certain embodiments, the protein-based vaccine comprises epitopes present
in at least 4, 5,
6, 7, 8, 9, or 10 genotypes of HBV.
In certain embodiments, the nucleic acid-based vaccine comprises epitopes
present in at least
4, 5, 6, 7, 8, 9, or 10 genotypes of HBV.
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The present invention further provides uses of the RNAi agents and vaccines
provided herein
for treatment of subjects having a hepatitis B virus infection based on the
methods provided herein.
In certain embodiment, the RNAi agents and the HBV vaccines are used in the
manufacture of
medicaments for treatment of a subject with an HBV infection.
The present invention further provides kits comprising RNAi agents and
vaccines provided
herein and instructions providing treatment regimens for their use for
treatment of subjects having a
hepatitis B virus infection.
The present invention is further illustrated by the following detailed
description and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 schematically depicts the structure of the approximately 3.2 kb
double-stranded
HBV genome. Replication of the HBV genome occurs through an RNA intermediate
and produces 4
overlapping viral transcripts of about 3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb
(termination sites indicated by
arrows), and the common 3' end encoding seven viral proteins (pre-S1, pre-S2,
S, P, X, pre-C, and C)
that are translated across three reading frames.
Figures 2A-2E are graphs showing suppression of HBV by siRNA in a transgenic
mouse
model. The level of (2A) HBsAg, (2B) HBeAg, and (2C) HBV-DNA in serum of
HBV1.3-xfs mice
(n = 6 per group) after a single subcutaneous 3 mg/kg or 9 mg/kg dose of a
control GalNAc-siRNA
(an siRNA that does not target HBV), AD-66816, or AD-66810. The level of (2D)
total HBV RNA
and (2E) 3.5 kb HBV transcripts from liver lysates determined via RT-qPCR and
normalized to
expression of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Figure 3 is a schematic showing a dosing regimen for an experiment assaying
the effect of
pretreatment of HBV1.3-xfs mice with the nucleoside inhibitor, Entecavir, an
HBV-shRNA, a control
GalNAc-siRNA, AD-66816, or AD-66810 prior to administration of a therapeutic
vaccine against
HBV.
Figures 4A-4C are graphs showing the level of (4A) HBsAg, (4B) HBeAg, and (4C)
HBV-
DNA in serum of HBV1.3-xfs mice after treatment based on the dosing regimen
provided in Figure 3.
Figures 5A-5F are graphs showing T cell immune response in the liver against
peptides (5A)
HBs(S208), (5B) HBc(C93), and (5C) MVA(B8R) in HBV1.3-xfs mice after treatment
based on the
dosing regimen provided in Figure 3. Figures 5D-5F show a reanalysis of the
same data in Figures
5A-5C performed to accommodate for an insufficient exclusion of dead immune
cells and shows T
cell immune response in the liver against peptides (5D) HBs(S208), (5E)
HBc(C93), and (5F)
MVA(B8R).
Figures 6A-6C show the level of HBV RNA and protein levels in liver cells in
HBV1.3-xfs
mice after treatment based on the dosing regimen provided in Figure 3. Figure
6A shows total HBV
RNA. Figure 6B shows HBV 3.5 kb transcript relative to GAPDH as determined by
RT-qPCR.
Figure 6C shows the number of HBcAg positive cells per mm2 of liver section
detected by
immunohistochemical staining.
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Figure 7 is a schematic showing a dosing regimen for the experiment assaying
the effect of
pretreatment of HBV1.3-xfs mice with control siRNA or AD-66816 prior to
administration of a
therapeutic vaccine against HBV.
Figures 8A-8C are graphs showing the level of (8A) HBsAg, (8B) HBeAg, and (8C)
HBV-
DNA in serum of HBV1.3-xfs mice after treatment based on the dosing regimen
provided in Figure 7.
Figures 9A-9D are graphs showing T cell immune response in the liver against
peptides (9A)
HBs(S208), (9B) HBc(C93), (9C) HBc(Cpool) and (9D) MVA(B8R) in HBV1.3-xfs mice
after
treatment based on the dosing regimen provided in Figure 7.
Figures 10A-10C show the level of HBV RNA and protein levels in liver cells of
HBV1.3-xfs
mice after treatment based on the dosing regimen provided in Figure 7. Figure
10A shows total HBV
RNA. Figure 10B shows HBV 3.5 kb transcript relative to GAPDH as determined by
RT-qPCR.
Figure 10C shows the number of HBcAg positive cells per mm2 of liver section
detected by
immunohistochemical staining.
Figures 11A and 11B are graphs showing the level of (11A) HBsAg and (11B)
HBeAg in
serum of individual HBV1.3-xfs mice at week 7 after the first vaccine dose
after the 6 week regimen
in the dosing regimen provided in Figure 7.
Figures 11C and 11D are graphs showing (11C) anti-HBs antibody response and
(11D) T
cell immune response in the liver against HBs(S208) of individual HBV1.3-xfs
mice at week 7 after
the first vaccine dose after the 6 week regimen in the dosing regimen provided
in Figure 7.
Figure 12 is a schematic showing a dosing regimen for the experiment assaying
the effect of
pretreatment of mice infected with an AAV vector encoding HBV with control
siRNA or AD-66816
prior to administration of a therapeutic vaccine against HBV.
Figures 13A-13G are based on the dosing regimen provided in Figure 12. Figures
13A and
13B are graphs showing the level of (13A) HBsAg and (13B) HBeAg in serum of
HBV-AAV mice
(inset 13C is an exploded portion of the later time points in graph 13B).
Figure 13D shows the serum
HBV DNA level at week 22. Figures 13D and 13E show the number of copies per
liver cell of (13D)
total HBV DNA and (13E) AAV-DNA at week 22. Figures 13F and 13G show the
relative expression
in liver of (13F) HBV 3.5 RNA relative to GAPDH RNA and (13G) total HBV RNA
relative to
GAPDH RNA.
Figures 14A-14C are graphs showing (14A) anti-HBs antibody response throughout
the
course of the experiment and (14B) anti-HBe antibody response at day 116 of
the experiment based
on the dosing regimen provided in Figure 12. Figure 14C is an exploded portion
of the later time
points in graph 14B.
Figures 15A and 15B show (15A) ALT levels and (15B) body weights of the
animals
throughout the experiment based on the dosing regimen provided in Figure 12.
A formal sequence listing is provided herewith that forms part of the
specification.
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An Appendix A providing exemplary target sequences for siRNA targeting and
iRNA agents
is provided herewith and forms part of the specification.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides treatment regimens and methods of use of iRNA
agents which
effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA
transcripts of one or
more HBV genes (open reading frames/ transcripts) and hepatitis B vaccines to
stimulate an immune
response against one or more HBV proteins in the treatment of HBV infection.
The treatment
regimens and methods preferably provide a functional cure within a defined
period of time.
The following detailed description discloses how to make and use compositions
containing
iRNAs to inhibit the expression of an HBV gene as well as compositions, uses,
and methods for
treating subjects having diseases and disorders that would benefit from
inhibition or reduction of the
expression of an HBV gene.
I. Definitions
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.
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.
The term "including" is used herein to mean, and is used interchangeably with,
the phrase
"including but not limited to".
The term "or" is used herein to mean, and is used interchangeably with, the
term "and/or,"
unless context clearly indicates otherwise.
The term "about" is used herein to mean within the typical ranges of
tolerances in the art. For
example, "about" can be understood as within 2 standard deviations from the
mean. In certain
embodiments, "about" means +/-10%. In certain embodiments, "about" means +/-
5%. When about
is present before a series of numbers or a range, it is understood that
"about" can modify each of the
numbers in the series or range.
The term "at least" prior to a number or series of numbers is understood to
include the
number adjacent to the term "at least", and all subsequent numbers or integers
that could logically be
included, as clear from context. For example, the number of nucleotides in a
nucleic acid molecule
must be an integer. For example, "at least 18 nucleotides of a 21 nucleotide
nucleic acid molecule"
means that 18, 19, 20, or 21 nucleotides have the indicated property. When at
least is present before a
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series of numbers or a range, it is understood that "at least" can modify each
of the numbers in the
series or range.
As used herein, "no more than" or "less than" is understood as the value
adjacent to the
phrase and logical lower values or integers, as logical from context, to zero.
For example, a duplex
with an overhang of "no more than 2 nucleotides" has a 2, 1, or 0 nucleotide
overhang. When "no
more than" is present before a series of numbers or a range, it is understood
that "no more than" can
modify each of the numbers in the series or range. As used herein, ranges
include both the upper and
lower limit.
In the event of a conflict between a sequence and its indicated site on a
transcript or other
sequence, the nucleotide sequence recited in the specification takes
precedence.
Various embodiments of the invention can be combined as determined appropriate
by one of
skill in the art.
As used herein, "Hepatitis B virus," used interchangeably with the term "HBV"
refers to the
well-known non-cytopathic, liver-tropic DNA virus belonging to the
Hepadnaviridae family.
The HBV genome is partially double-stranded, circular DNA with overlapping
reading frames
(see, e.g., Figure 1).
There are four transcripts (that may be referred to herein as "genes" or "open
reading
frames") based on size, encoded by the HBV genome. These contain open reading
frames called C,
X, P, and S. The core protein is coded for by gene C (HBcAg). Hepatitis B e
antigen (HBeAg) is
produced by proteolytic processing of the pre-core (pre-C) protein. The DNA
polymerase is encoded
by gene P. Gene S is the gene that codes for the surface antigens (HBsAg). The
HBsAg gene is one
long open reading frame which contains three in frame "start" (ATG) codons
resulting in polypeptides
of three different sizes called large, middle, and small S antigens, pre-S1 +
pre-52 + S, pre-52 + S, or
S. Surface antigens in addition to decorating the envelope of HBV, are also
part of subviral particles,
which are produced at large excess as compared to virion particles, and play a
role in immune
tolerance and in sequestering anti-HBsAg antibodies, thereby allowing for
infectious particles to
escape immune detection. The function of the non-structural protein coded for
by gene X is not fully
understood, but it plays a role in transcriptional transactivation and
replication and is associated with
the development of liver cancer. Exemplary protein sequences are provided in
the attached sequence
listing (see, e.g., SEQ ID NOs:1, 3, 16, and 20).
HBV is one of the few DNA viruses that utilize reverse transcriptase in the
replication process
which involves multiple stages including entry, uncoating, and transport of
the virus genome to the
nucleus. Initially, replication of the HBV genome involves the generation of
an RNA intermediate
that is then reverse transcribed to produce the DNA viral genome.
Upon infection of a cell with HBV, the viral genomic relaxed circular DNA
(rcDNA) is
transported into the cell nucleus and converted into episomal covalently
closed circular DNA
(cccDNA), which serves as the transcription template for the viral mRNAs.
After transcription and
nuclear export, cytoplasmic viral pregenomic RNA (pgRNA) is assembled with HBV
polymerase and
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capsid proteins to form the nucleocapsid, inside which polymerase-catalyzed
reverse transcription
yields minus-strand DNA, which is subsequently copied into plus-strand DNA to
form the progeny
rcDNA genome. The mature nucleocapsids are then either packaged with viral
envelope proteins to
egress as virion particles or shuttled to the nucleus to amplify the cccDNA
reservoir through the
intracellular cccDNA amplification pathway. cccDNA is an essential component
of the HBV
replication cycle and is responsible for the establishment of infection and
viral persistence.
HBV infection results in the production of two different particles: 1) the
infectious HBV
virus itself (or Dane particle) which includes a viral capsid assembled from
the HBcAg and is covered
by an envelope consisting of a lipid membrane with HBV surface antigens, and
2) subviral particles
(or SVPs) which contain the small and medium forms of the hepatitis B surface
antigen HBsAg which
are non-infectious. For each viral particle produced, over 10,000 SVPs are
released into the blood. As
such, SVPs (and the HBsAg protein they carry) represent the overwhelming
majority of viral protein
in the blood. HBV infected cells also secrete a soluble proteolytic product of
the pre-core protein
called the HBV e-antigen (HBeAg).
Eight genotypes of HBV, designated A to H, have been determined, and two
additional
genotypes I and J have been proposed, each having a distinct geographical
distribution. The virus is
non-cytopathic, with virus-specific cellular immunity being the main
determinant for the outcome of
exposure to HBV- acute infection with resolution of liver diseases with 6
months, or chronic HBV
infection that is frequently associated with progressive liver injury.
The term "HBV" includes any of the genotypes of HBV (A to J). The complete
coding
sequence of the reference sequence of the HBV genome may be found in for
example, GenBank
Accession Nos. GI:21326584 (SEQ ID NO:1) and GI:3582357 (SEQ ID NO:3).
Antisense sequences
are provided in SEQ ID NO: 2 and 4, respectively. Amino acid sequences for the
C, X, P, and S
proteins can be found, for example at NCBI Accession numbers YP_009173857.1 (C
protein);
YP_009173867.1 and BAA32912.1 (X protein); YP_009173866.1 and BAA32913.1 (P
protein); and
YP_009173869.1, YP_009173870.1, YP_009173871.1, and BAA32914.1 (S protein)
(SEQ ID NOs:
5-13) . Protein and DNA sequences from HBV genotype D, strain ayw are provided
in SEQ ID NOs.:
14-17. Protein and DNA sequences from HBV genotype A, strain adw are provided
in SEQ ID NOs.:
18-21.
Additional examples of HBV mRNA sequences are readily available using publicly
available
databases, e.g., GenBank, UniProt, and OMIM. The International Repository for
Hepatitis B Virus
Strain Data can be accessed at http://www.hpa-
bioinformatics.org.uk/HepSEQ/main.php.
The term "HBV," as used herein, also refers to naturally occurring DNA
sequence variations
of the HBV genome, i.e., genotypes A-J and variants thereof.
As used herein, "epitope" also referred to as "an antigenic determinant," or
"determinant," is
understood as the part of a protein antigen that is recognized by the immune
system, specifically by
antibodies, B cells, and/or T cells. Epitopes include conformational epitopes
and linear epitopes.
Proteins share an epitope when they share an amino acid sequence of sufficient
length or size and
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antigenicity to be recognized by an antibody, B cell, and/or T cell. T cell
epitopes presented by MHC
class I molecules are typically peptides about 8 to 11 amino acids in length,
whereas MHC class II
molecules present longer peptides about 13 to 17 amino acids in length.
Conformational epitopes are
typically discontinuous and span a longer amino acid sequence.
As used herein, the term "nucelot(s)ide analog" or "reverse transcriptase
inhibitor" is an
inhibitor of DNA replication that is structurally similar to a nucleotide or
nucleoside and specifically
inhibits replication of the HBV cccDNA and does not significantly inhibit the
replication of the host
(e.g., human) DNA. Such inhibitors include Tenofovir disoproxil fumarate
(TDF), Tenofovir
alafenamide (TAF), Lamivudine, Adefovir dipivoxil, Entecavir (ETV),
Telbivudine, AGX-1009,
emtricitabine, clevudine, ritonavir, dipivoxil, lobucavir, famvir, FTC, N-
Acetyl-Cysteine (NAC),
PC1323, theradigm-HBV, thymosin-alpha, ganciclovir, besifovir (ANA-380/LB-
80380), and tenofvir-
exaliades (TLX/CMX157). In certain embodiments, the nucelot(s)ide analog is
Entecavir (ETV).
Nucleot(s)ide analogs are commercially available from a number of sources and
are used in the
methods provided herein according to their label indication (e.g., typically
orally administered at a
specific dose) or as determined by a skilled practitioner in the treatment of
HBV.
As used herein, "target sequence" refers to a contiguous portion of the
nucleotide sequence of
an mRNA molecule formed during the transcription of an HBV transcript,
including mRNA that is a
product of RNA processing of a primary transcription product. In one
embodment, the target portion
of the sequence will be at least long enough to serve as a substrate for iRNA-
directed cleavage at or
near that portion of the nucleotide sequence of an mRNA molecule formed during
the transcription of
an HBV transcript. In one embodiment, the target sequence is within the
protein coding region of an
HBV transcript.
The target sequence may be from about 9-36 nucleotides in length, e.g., about
15-30
nucleotides in length. For example, the target sequence can be from about 15-
30 nucleotides, 15-29,
15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,
15-17, 18-30, 18-29, 18-
28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-
28, 19-27, 19-26, 19-25,
19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25,
20-24,20-23, 20-22, 20-
21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22
nucleotides in length. Ranges
and lengths intermediate to the above recited ranges and lengths are also
contemplated to be part of
the invention.
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.
"G," "C," "A," "T," and "U" each generally stand for a nucleotide that
contains guanine,
cytosine, adenine, thymidine, and uracil as a base, respectively. However, it
will be understood that
the term "ribonucleotide" or "nucleotide" 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 can be replaced by other moieties without substantially
altering the base pairing
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properties of an oligonucleotide comprising a nucleotide bearing such
replacement moiety. For
example, without limitation, a nucleotide comprising inosine as its base can
base pair with nucleotides
containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil,
guanine, or adenine can
be replaced in the nucleotide sequences of dsRNA featured in the invention by
a nucleotide
containing, for example, inosine. In another example, adenine and cytosine
anywhere in the
oligonucleotide can be replaced with guanine and uracil, respectively to form
G-U Wobble base
pairing with the target mRNA. Sequences containing such replacement moieties
are suitable for the
compositions and methods featured in the invention.
As used herein, an "RNAi agent", an "iRNA agent", an "siRNA agent", and the
like, is a
double stranded RNA that preferably target regions in the HBV genome that are
conserved across all
serotypes of HBV and are preferably designed to inhibit all steps of the HBV
life cycle, e.g.,
replication, assembly, secretion of virus, and secretion of viral antigens, by
inhibiting expression of
more than one HBV transcript. In particular, since transcription of the HBV
genome results in
polycistronic, overlapping RNAs, an RNAi agent for use in the invention
targeting a single HBV
transcript preferably results in significant inhibition of expression of most
or all HBV transcripts. All
HBV transcripts are at least partly overlapping and share the same
polyadenylation signal. Therefore,
all viral transcripts have an identical 3-end and, thus, an RNAi agent of the
invention targeting the X
gene will target all viral transcripts and should result in inhibition of not
only X gene expression but
also the expression of all other viral transcripts, including pregenomic RNA
(pgRNA). Furthermore,
the RNAi agents of the invention have been designed to inhibit HBV viral
replication by targeting
pgRNA, HBV structural genes, polymerase, and the HBV X gene. In addition, they
have been
designed to mediate the silencing of SVP and other viral protiens that play a
role in immune
tolerance, thereby permitting a subject's immune system to detect and respond
to the presence of viral
antigens such that an immune response to control and to clear an HBV infection
is mounted. Without
intending to be limited by theory, it is believed that a combination or sub-
combination of the
foregoing properties and the specific target sites and/or the specific
modifications in these RNAi
agents confer to the RNAi agents of the invention improved efficacy,
stability, safety, potency, and
durability. Such agents are provided, for example, in PCT Publication Nos. WO
2016/077321, WO
2012/024170, WO 2017/027350, and WO 2013/003520, the entire contents of each
of which is
incorporated herein by reference. Exemplary target sites for RNAi agents and
exemplary RNAi agents
are provided in Appendix A, filed herewith, which forms a part of the
specification. The term RNAi
agents further includes shRNAs, e.g., adeno-associated virus (AAV) 8 vectors
for delivery of an
shRNA in an artificial mi(cro)RNA under a liver-specific promoter; optionally
co-delivered a decoy
("TuD") directed against the shRNA sense strand to curb off-target gene
regulation are provided in
Michler et al., 2016 (EMBO Mol. Med., 8:1082-1098, incorporated herein by
reference).
The majority of nucleotides of each strand of an iRNA agent may be
ribonucleotides, but as
described in detail herein, each or both strands can also include one or more
non-ribonucleotides, e.g.,
a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in
this specification, an
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"RNAi agent" may include ribonucleotides with chemical modifications; an RNAi
agent may include
substantial modifications at multiple nucleotides.
As used herein, the term "modified nucleotide" refers to a nucleotide having,
independently, a
modified sugar moiety, a modified internucleotide linkage, and/or modified
nucleobase. Thus, the
term modified nucleotide encompasses substitutions, additions or removal of,
e.g., a functional group
or atom, to internucleoside linkages, sugar moieties, or nucleobases. The
modifications suitable for
use in the agents of the invention 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.
As used herein, a "therapeutic HBV vaccine," and the like, can be a peptide
vaccine, a DNA
vaccine including a vector-based vaccine, or a cell-based vaccine that induces
an immune response,
preferably an effector T cell induced response, against one or more HBV
proteins. Preferably the
vaccine is a multi-epitope vaccine that is cross-reactive with multiple HBV
serotypes, preferably all
HBV serotypes. A number of therapeutic HBV vaccines are known in the art and
are at various stages
of pre-clinical and clinical development. Protein based vaccines include
hepatitis B surface antigen
(HBsAg) and core antigen (HBcAg) vaccines (e.g., Li et al., 2015, Vaccine.
33:4247-4254,
incorporated herein by reference). Exemplary DNA vaccines include HB-110
(Genexine, Kim et al.,
2008. Exp Mol Med. 40: 669-676.), pDPSC18 (PowderMed), INO-1800 (Inovio
Pharmaceuticals),
HBO2 VAC-AND (ANRS), and CVI-HBV-002 (CHA Vaccine Institute Co., Ltd.).
Exemplary
protein based vaccines include Theravax/DV-601 (Dynavax Technologies Corp.),
EPA-44
(Chongqing Jiachen Biotechnology Ltd.), and ABX 203 (ABIVAX S.A.). Exemplary
cell based
vaccines include HPDCs-T immune therapy (Sun Yat-Sen University). Combination
vaccines and
products are also known and include HepTcellTm (FP-02.2 vaccine (peptide) +
IC310 Adjuvant (a
combination peptide-oligonucleotide adjuvant),(see U.S. Patent Publication
Nos. 2013/0330382,
2012/0276138, and 2015/0216967, the entire contents of each of which is
incorporated herein by
reference)); GS-4774 (Gilead, a fusion protein S. core X vaccine + Tarmogen T
cell immune
stimulator), pSG2.HBs/MVA.HBs (protein prime/viral vector boost, Oxxon
Therapeutics), and a
protein-prime/modified vaccinia virus Ankara vector-boost (HBsAg and HBsAg
protein + HBcAg
and HBsAg in MVA expression vector, Backes et al., 2016, Vaccine. 34:923-32,
and
W02017121791, both of which are incorporated herein by reference).
As used herein, the term "adjuvant" is understood to be an agent that promotes
(e.g.,
enhances, accelerates, or prolongs) an immune response to an antigen with
which it is administered to
elicit long-term protective immunity. No substantial immune response is
directed at the adjuvant
itself. Adjuvants include, but are not limited to, pathogen components,
particulate adjuvants, and
combination adjuvants (see, e.g., www.niaid.nih.gov/research/vaccine-adjuvants-
types). Pathogen
components (e.g., monophosphoryl lipid A (MPL), poly(I:C), polyICLC adjuvant,
CpG DNA, c-di-
AMP, c-di-GMP, c-di-CMP; short, blunt-ended 5'-triphosphate dsRNA (3pRNA) RIG-
1 ligand, and
emulsions such as poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP))
can help trigger
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early non-specific, or innate, immune responses to vaccines by targeting
various receptors inside or on
the surface of innate immune cells. The innate immune system influences
adaptive immune responses,
which provide long-lasting protection against the pathogen that the vaccine
targets. Particulate
adjuvants (e.g., alum, virosomes, cytokines, e.g., IL-12) form very small
particles that can stimulate
the immune system and also may enhance delivery of antigen to immune cells.
Combination
adjuvants (e.g., AS02, AS03, and AS04 (all GSK); MF59 (Novartis); ISCOMATRIX
(CSL
Limited); and IC31 (Altimmune) elicit multiple protective immune responses.
Adjuvants that have
a modest effect when used alone may induce a more potent immune response when
used together.
In preferred embodiments of the invention, adjuvants for use in the invention
promote a
humoral as well as a cellular immune response. For this, a balanced Th1/Th2
helper T cell response is
desired to support neutralizing antibody responses as well as effector
cytotoxic T cell responses.
Preferably the adjuvant provides a balanced Th1/Th2 response. In certain
embodiments, the adjuvant
is one or more of a polyI:C adjuvant, a polyICLC adjuvant, a CpG adjuvant, a
STING agonist (a c-di-
AMP adjuvant, a c-di-GMP adjuvant, or a c-di-CMP adjuvant), an ISCOMATRIX
adjuvant, a
PCEP adjuvant, and a Rig-I-ligand adjuvant. In certain embodiments, the
adjuvant is a polyI:C
adjuvant, a CpG adjuvant, a STING agonist, or a PCEP adjuvant. In certain
embodiments, the
adjuvant is not alum.
As used herein, an "immune stimulator" is an agent that stimulates an immune
response that
may or may not be administered independently of an antigen. Immune stimulators
include, but are
not limited to, pegylated interferon alfa 2a (PEG-IFN-alpha-2a), interferon
alfa-2b, PEG-IFN-alpha-
2b, interferon lambda a recombinant human interleukin-7, and a Toll-like
receptor 3, 7, 8 or 9 (TLR3,
TLR7, TLR8, TLR9) agonist, a viral entry inhibitor (e.g., Myrcludex), an
oligonucleotide that inhibits
the secretion or release of HBsAg (e.g., REP 9AC), a capsid inhibitor (e.g.,
Bay41-4109 and NVR-
1221), a cccDNA inhibitor (e.g., IHVR-25). In certain embodiments, an immune
stimulator can
include a viral capsid, optionally an empty viral capsid, e.g., MVA capsid.
Immune stimulators can also include immune checkpoint regulators. Immune
checkpoint
regulators can be stimulatory or inhibitory. As used herein, immune checkpoint
regulators potentiate
an immune response. Immune checkpoint regulators include, but are not limited
to, CTLA-4
inhibitors, such as ipilimumab, PD-1 inhibitors, such as Nivolumab,
Pembrolizumab, and the BGB-
A317 antibody. PD-Li inhibitors include atezolizumab, avelumab, and
durvalumab, in addition to an
affimer biotherapeutic.
As used herein, a "subject" is an animal, such as a mammal, including a
primate (such as a
human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate
(e.g., a mouse model
or other animal model that can be infected with HBV). In an embodiment, the
subject is a human,
such as a human being treated or assessed for a disease, disorder, or
condition that would benefit from
reduction in HBV gene expression or replication. In certain embodiments, the
subject has a chronic
hepatitis B virus (HBV) infection. In certain embodiments, the subject has
both a chronic hepatitis B
virus (HBV) infection and a hepatitis D virus (HDV) infection.
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As used herein, the terms "treating" or "treatment" refer to a beneficial or
desired result
including, but not limited to, alleviation of one or more signs or symptoms in
a subject with HBV
infection including, but not limited to, the presence of serum HBV DNA or
liver HBV ccc DNA, the
presence of serum or liver HBV antigen, e.g., HBsAg or HBeAg. Diagnostic
criteria for HBV
infection are well known in the art. "Treatment" can also mean prolonging
survival as compared to
expected survival in the absence of treatment, or lower risk of HCC
development.
In certain embodiments, an HBV-associated disease is chronic hepatitis B
(CHB). In certain
embodiments, subjects have been infected with HBV for at least five years. In
certain embodiments,
subjects have been infected with HBV for at least ten years. In certain
embodiments, subjects became
infected with HBV at birth. Subjects having chronic hepatitis B disease are
immune tolerant, have an
active chronic infection accompanied by necroinflammatory liver disease, have
increased hepatocyte
turn-over in the absence of detectable necroinflammation, or have an inactive
chronic infection
without any evidence of active disease, and they are also asymptomatic.
Subjects having chronic
hepatitis B disease are HBsAg positive and have either high viremia (>104 HBV-
DNA copies / ml
blood) or low viremia (<103 HBV-DNA copies / ml blood). Patients with chronic
active hepatitis,
especially during the high replicative state, may have symptoms similar to
those of acute hepatitis.
The persistence of HBV infection in CHB subjects is the result of ccc HBV DNA
persistence. In
certain embodiments, a subject having CHB is HBeAg positive. In other
embodiments, a subject
having CHB is HBeAg negative.
In preferred embodiments, treatment of HBV infection results in a "functional
cure" of
hepatitis B. As used herein, the term "functional cure" is understood to be
clearance of circulating
HBsAg and is preferably accompanied by conversion to a status in which HBsAg
antibodies become
undetectable using a clinically relevant assay. For example, undetectable
antibodies can include a
signal lower than 10 mIU/m1 as measured by Chemiluminescent Microparticle
Immunoassay (CMIA)
or any other immunoassay, and may be called anti-HBs seroconversion.
Functional cure does not
require clearance of all replicative forms of HBV (e.g., cccDNA from the
liver). Anti-HBs
seroconversion occurs spontaneously in about 0.2-1% of chronically infected
patients per year.
However, even after anti-HBs seroconversion, low level persistence of HBV is
observed for decades
indicating that a functional rather than a complete cure occurs. Without being
bound to mechanism, it
is proposed that the immune system is able to keep HBV in check. A functional
cure permits
discontinuation of any treatment for the HBV infection. However, it is
understood that a "functional
cure" for HBV infection may not be sufficient to prevent or treat diseases or
conditions that result
from HBV infection, e.g., liver fibrosis, HCC, cirrhosis.
The term "lower" in the context of the level of HBV gene expression or HBV
replication in a
subject, or a disease marker or symptom, refers to a statistically significant
decrease in such level.
The decrease can be, for example, at least 70%, 75%, 80%, 85%, 90%, 95%, or
more. In monitoring
of HBV infection, a 10g10 scale is typically used to describe the level of
antigenemia (e.g., HBsAg
level in serum) or viremia (HBV DNA level in serum). It is understood that a 1
10g10 decrease is a
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90% decrease (10% remaining), a 2 10g10 decrease is a 99% decrease (1%
remaining), etc. In certain
embodiments, a disease marker is lowered to below the level of detection.
In certain embodiments, the expression of a disease marker is normalized,
i.e., decreased to a
level accepted as within the range of normal for an individual without such
disorder, e.g., the level of
.. a disease marker, such as, ALT or AST, is decreased to a level accepted as
within the range of normal
for an individual without such disorder. When the disease associated level is
elevated from the
normal level, the change is calculated from the upper level of normal (ULN).
When the disease
associated level is decreased from the normal level, the change is calculated
from the lower level of
normal (LLN). The lowering is the percent difference in the change between the
subject value and
the normal value. For example, a normal AST level can be reported as 10 to 40
units per liter. If,
prior to treatment, a subject has an AST level of 200 units per liter (i.e., 5
times the ULN, 160 units
per liter above the upper level of normal) and, after treatment, the subject
has an AST level of 120
units per liter (i.e., 3 times the ULN, 80 units per liter above the upper
level of normal), the elevated
AST would be lowered towards normal by 50% (80/160).
The term "inhibiting," as used herein, is used interchangeably with
"reducing," "silencing,"
"downregulating", "suppressing", and other similar terms, and includes any
level of inhibition.
Preferably inhibiting includes a statistically significant or clinically
significant inhibition.
The phrase "inhibiting expression of an HBV gene" is intended to refer to
knockdown of any
HBV transcript (e.g., 3.5 kb, 2.4 kb, 2.1 kb, or 0.7 kb transcript) encoding
one or more HBV viral
proteins (such as, e.g., preS1/2-S, preS, S, P, X, preC, and C), as well as
variants or mutants of an
HBV gene.
"Inhibiting expression of an HBV gene" includes any significant level of
inhibition of an
HBV gene or transcript, e.g., at least partial suppression of the expression
of an HBV gene S, P, X, or
C, or any combination thereof, e.g., S, P, and C. The expression of the HBV
gene may be assessed
based on the level, or the change in the level, of any variable associated
with HBV gene expression,
e.g., an HBV mRNA level, an HBV protein level, and/or an HBV cccDNA level.
This level may be
assessed in an individual cell or in a group of cells, including, for example,
a sample derived from a
subject, e.g., levels may be monitored in serum.
In some embodiments of the methods of the invention, expression of an HBV gene
is
inhibited by at least 70%, 75%, 80%, 85%, 90%, 95%, or to below the level of
detection of the assay.
In preferred embodiments, the inhibition of expression of an HBV gene results
in a clinically relevant
inhibition of the level of gene expression, e.g., sufficiently inhibited to
permit an effective immune
response to a vaccine against an HBV protein.
Inhibition of the expression of an HBV gene may be manifested by a reduction
of the amount
of RNA expressed by a first cell or group of cells (such cells may be present,
for example, in a sample
derived from a subject) in which an HBV gene is transcribed and which has or
have been treated (e.g.,
by contacting the cell or cells with an RNAi agent of the invention, or by
administering an RNAi
agent of the invention to a subject in which the cells are or were present)
such that the expression of
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an HBV gene is inhibited, as compared to a second cell or group of cells
substantially identical to the
first cell or group of cells but which has not or have not been so treated
(control cell(s)). In preferred
embodiments, the inhibition is assessed by the rtPCR method provided in
Example 2 of PCT
Publication No. WO 2016/077321 (the entire contents of which are incorporated
herein by reference),
with in vitro assays being performed in an appropriately matched cell line
with the duplex at a 10 nM
concentration, and expressing the level of mRNA in treated cells as a
percentage of the level of
mRNA in control cells, using the following formula:
(RNA in contd. c ¨ (RNA in treated celc) l
-
RNA in control cells
Alternatively, inhibition of the expression of an HBV gene may be assessed in
terms of a
reduction of a parameter that is functionally linked to HBV gene expression,
e.g., as described herein.
HBV gene silencing may be determined in any cell expressing an HBV gene,
either constitutively or
by genomic engineering, and by any assay known in the art.
Inhibition of the expression of an HBV protein may be manifested by a
reduction in the level
of an HBV protein that is expressed by a cell or group of cells (e.g., the
level of protein expressed in a
sample derived from a subject). As explained above, for the assessment of mRNA
suppression, the
inhibition of protein expression levels in a treated cell or group of cells
may similarly be expressed as
a percentage of the level of protein in a control cell or group of cells or in
serum.
A control cell or group of cells that may be used to assess the inhibition of
the expression of
an HBV gene includes a cell or group of cells that has not yet been contacted
with an RNAi agent of
.. the invention. For example, the control cell or group of cells may be
derived from an individual
subject (e.g., a human or animal subject) prior to treatment of the subject
with an RNAi agent. In
alternative embodiments, the level may be compared to an appropriate control
sample, e.g., a known
population control sample.
The level of HBV RNA that is expressed by a cell or group of cells, or the
level of circulating
HBV RNA, may be determined using any method known in the art for assessing
mRNA expression,
preferably using the rtPCR method provided in Example 2 of PCT Publication No.
WO
2016/077321,or Example 1 provided herein. In one embodiment, the level of
expression of an HBV
gene (e.g., total HBV RNA, an HBV transcript, e.g., HBV 3.5 kb transcript) in
a sample is determined
by detecting a transcribed polynucleotide, or portion thereof, e.g., RNA of
the HBV gene. RNA may
.. be extracted from cells using RNA extraction techniques including, for
example, using acid
phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA
preparation kits
(Qiagen0) or PAXgene (PreAnalytix, Switzerland). Typical assay formats
utilizing ribonucleic acid
hybridization include nuclear run-on assays, RT-PCR, RNase protection assays
(Melton et al., Nuc.
Acids Res. 12:7035), northern blotting, in situ hybridization, and microarray
analysis. Circulating
HBV mRNA may be detected using methods the described in PCT Publication No. WO
2012/177906,
the entire contents of which are hereby incorporated herein by reference.
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In one embodiment, the level of expression of an HBV gene is determined using
a nucleic
acid probe. The term "probe", as used herein, refers to any molecule that is
capable of selectively
binding to a specific HBV gene. Probes can be synthesized by one of skill in
the art, or derived from
appropriate biological preparations. Probes may be specifically designed to be
labeled. Examples of
molecules that can be utilized as probes include, but are not limited to, RNA,
DNA, proteins,
antibodies, and organic molecules.
Isolated RNA can be used in hybridization or amplification assays that
include, but are not
limited to, Southern or northern analyses, polymerase chain reaction (PCR)
analyses, and probe
arrays. One method for the determination of mRNA levels involves contacting
the isolated mRNA
with a nucleic acid molecule (probe) that can hybridize to an HBV mRNA. In one
embodiment, the
mRNA is immobilized on a solid surface and contacted with a probe, for example
by running the
isolated mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as
nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on
a solid surface and the
mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip
array. A skilled
artisan can readily adapt known mRNA detection methods for use in determining
the level of an HBV
mRNA.
An alternative method for determining the level of expression of an HBV gene
in a sample
involves the process of nucleic acid amplification or reverse transcriptase
(to prepare cDNA) of, for
example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set
forth in Mullis,
.. 1987, US Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc.
Natl. Acad. Sci. USA
88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc.
Natl. Acad. Sci. USA
87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc.
Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197),
rolling circle
replication (Lizardi et al., US Patent No. 5,854,033) or any other nucleic
acid amplification method,
followed by the detection of the amplified molecules using techniques well
known to those of skill in
the art. These detection schemes are especially useful for the detection of
nucleic acid molecules if
such molecules are present in very low numbers. In particular aspects of the
invention, the level of
expression of an HBV gene is determined by quantitative fluorogenic RT-PCR
(i.e., the TaqManTm
System), e.g., using the method provided herein.
The expression levels of an HBV RNA may be monitored using a membrane blot
(such as
used in hybridization analysis such as northern, Southern, dot, and the like),
or microwells, sample
tubes, gels, beads, or fibers (or any solid support comprising bound nucleic
acids). See U.S. Patent
Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, the entire
contents of each of which
are incorporated herein by reference. The determination of HBV expression
level may also comprise
using nucleic acid probes in solution.
In preferred embodiments, the level of RNA expression is assessed using real
time PCR
(qPCR). The use of these methods is described and exemplified in the Examples
presented herein.
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The level of HBV protein expression may be determined using any method known
in the art
for the measurement of protein levels. Such methods include, for example,
electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC), thin layer
chromatography (TLC),
hyperdiffusion chromatography, fluid or gel precipiting reactions, absorption
spectroscopy, a
colorimetric assays, spectrophotometric assays, flow cytometry,
immunodiffusion (single or double),
immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked
immunosorbent
assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays,
and the like.
In some embodiments, the efficacy of the methods of the invention can be
monitored by
detecting or monitoring a reduction in a symptom of an HBV infection. Symptoms
may be assessed
using any method known in the art.
As used herein, the term "Hepatitis B virus-associated disease" or "HBV-
associated disease,"
is a disease or disorder that is caused by, or associated with HBV infection
or replication. The term
"HBV-associated disease" includes a disease, disorder, or condition that would
benefit from reduction
in HBV gene expression or replication. Non-limiting examples of HBV-associated
diseases include,
for example, hepatitis D virus infection,; hepatitis delta; chronic hepatitis
B; liver fibrosis; end-stage
liver disease; cryoglobulinemia; hepatocellular carcinoma.
The term "sample," as used herein, includes a collection of similar fluids,
cells, or tissues
isolated from a subject or prepared therefrom, as well as fluids, cells, or
tissues present within a
subject. Examples of biological fluids include blood, serum, plasma, immune
cells, 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 the liver
(e.g., whole liver or
certain segments of liver or certain types of cells in the liver, such as,
e.g., hepatocytes, resident liver
immune cells). In preferred embodiments, a "sample derived from a subject"
refers to blood drawn
from the subject or plasma, serum, or selected cell pools derived therefrom
(e.g., populations of
immune cells). In further embodiments, a "sample derived from a subject"
refers to liver tissue (or
subcomponents thereof) obtained from the subject.
As used herein, "coding sequence" is understood to refer to a DNA sequence
that encodes for
a specific amino acid sequence. In certain embodiments, the DNA sequence can
be reverse
transcribed from an RNA sequence. In certain embodiments, an iRNA, e.g., an
shRNA, targets a
coding sequence.
The terms, "suitable regulatory sequences," and the like, are used herein is
to refer to
nucleotide sequences located upstream (5' non-coding sequences), within, or
downstream (3' non-
coding sequences) of a coding sequence, and which influence the transcription,
RNA processing or
stability, or translation of the associated coding sequence. Regulatory
sequences may include
promoters, translation leader sequences, introns, and polyadenylation
recognition sequences.
"Promoter" as used herein refers to a DNA sequence capable of controlling the
expression of
a coding sequence or functional RNA. In general, a coding sequence is located
3' to a promoter
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sequence. Promoters may be derived in their entirety from a native gene, or be
composed of different
elements derived from different promoters found in nature, or even comprise
synthetic DNA
segments. A promoter may be selected to promote expression of a coding
sequence in a particular cell
type or at different stages of development, or in response to different
environmental conditions. In
certain embodiments, the promoter is a promoter that is active in liver, e.g.,
a liver-specific promoter.
Many promoter sequences are known in the art and selection of an appropriate
promoter sequence for
a specific context is within the ability of those of skill in the art.
The term "operably linked" refers to the association of nucleic acid sequences
on a single
nucleic acid fragment so that the function of one is affected by the other.
For example, a promoter is
operably linked with a coding sequence when it is capable of affecting the
expression of that coding
sequence (i.e., the coding sequence is under the transcriptional control of
the promoter).
The term "expression" as used herein refers to the transcription and stable
accumulation of
sense (mRNA) or antisense RNA, or an RNAi agent (e.g., an shRNA) derived from
the nucleic acid
fragment of the subject technology. "Over-expression" refers to the production
of a gene product in
transgenic or recombinant organisms that exceeds levels of production in
normal or non-transformed
organisms.
"Expression vector" or "expression construct," as used herein, refers to a
nucleic acid in
which a coding sequence is operably linked to a promoter sequence to permit
expression of the coding
sequence under the control of the promoter. Expression vectors include, but
are not limited to, viral
vectors or plasmid vectors. Methods for delivery of expression vectors into
cells are known in the art.
Treatment Methods of the Invention
The present invention provides treatment regimens and methods for the
sequential use of an
agent to reduce the expression of an HBV gene, e.g., iRNA agents which effect
the RNA-induced
silencing complex (RISC)-mediated cleavage of one or more HBV transcripts, and
hepatitis B
vaccines to stimulate an immune response against one or more HBV proteins in
the treatment of HBV
infection. The treatment regimens and methods preferably provide a functional
cure of HBV within a
defined period of time.
The treatment regimens and methods provided herein include the ordered
administration of
therapeutic agents to provide treatment, and preferably a functional cure, for
HBV infection. The
agents used in the methods are known in the art. However, the agents alone
fail to consistently and
durably decrease HBV disease burden, e.g., reducing HBsAg levels to below 2
10g10, preferably 1
log10 IU/ml or to below the level of detection, in most subjects.
Significantly reducing HBV disease
burden, e.g., reducing HBsAg levels to below 2 10g10, preferably 1 10g10 IU/ml
or to below the level
of detection, will provide the opportunity of discontinuation of
administration of therapeutic agents
and provide a functional cure for HBV in a substantial number of subjects.
Without being bound by
mechanism, it is proposed that the treatment regimens and methods provided
herein, including
administration of an iRNA agent targeted to HBV, substantially reduces HBV
antigens and nucleic
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acid for a sufficient magnitude and duration in a subject to allow an
effective immune response
induced by administration of multiple doses of a therapeutic vaccine. The
regimens and methods,
provided herein, consistently provide a substantial reduction of disease
burden and a functional cure
in a significant number of subjects, preferably at least 30%, 40%, 50%, 60%,
or 70% of subjects.
A transgenic mouse model of HBV infection, HBV1.3 xfs was used to assess the
combination therapy provided herein. Primary studies demonstrated the efficacy
of two different
chemically modified GalNAc- iRNA agents targeted to HBV (AD-66816 and AD-
66810) to inhibit
the level of HBsAg and HBeAg proteins, and HBV DNA in serum for at least 21
days with a single
subcutaneous dose at 3 mg/kg or 9 mg/kg, with similar efficacy. No significant
knockdown was
observed with a non-HBV iRNA control (see Figure 2). Based on this result, the
lower dose of 3
mg/kg was selected for combination therapy studies.
In the first combination therapy trial (see Figure 3), mice were pretreated
with one of six
treatment regimens (n= 6 per group):
(1) No pretreatment;
(2) Entecavir at 1 ig/m1 in water throughout the course of the study beginning
on the first day
of Week 0;
(3) A 3 mg/kg dose on the first day of Weeks 0, 4, 8, and 12 of the control
iRNA agent.
(4) A single dose on the first day of Week 0 with an expression vector
encoding an shRNA
targeted to HBV (HBV-shRNA) (Michler et al., 2016); or
(5-6) A 3 mg/kg dose on the first day of Weeks 0, 4, 8, and 12 of AD-66816 or
AD-66810
(generically, HBV-siRNA).
On the first day of Weeks 12 and 14, a mixture of recombinantly expressed
yeast HBsAg (15
rig) and E. coli expressed HBcAg (15 rig) adjuvanted with 31.9 lig synthetic
phosphorothioated
CpGODN 1668 (CpG) and 25 lig polykli(sodiumcarboxylatoethyl-
phenoxy)phosphazene] (PCEP)
was subcutaneously administered to all mice as a protein prime vaccination
(Backes, 2016).
On the first day of week 16, a mixture of modified vaccinia virus Ankara
expressing HBsAg
or HBcAg (5 x 107 particles of each virus) was subcutaneously administered to
all mice as a boost
vaccination (Backes, 2016).
Blood samples were obtained on the first days of Week 0, 2, 4, 8, 12, 16, and
17 and were
assayed for levels of HBsAg, HBeAg, and HBV DNA. Results observed for HBsAg
and HBeAg
levels mice in groups 1, 2, and 3 (mock, Entecavir, control iRNA agent) were
similar (Figure 4A and
4B). The HBV-shRNA or HBV-siRNAs alone caused a significant decrease in HBsAg,
HBeAg, and
HBV DNA in serum. The three dose prime-boost vaccination scheme resulted in a
further decrease in
HBsAg in all groups, and reduced the level of HBsAg in at least some animals
in the HBV-shRNA
and HBV-siRNA groups to below the level of detection. However, vaccine
treatment did not decrease
HBeAg levels in any of the groups. HBV DNA levels were decreased to about the
lower limit of
quantitation with Entecavir alone so no effect of the three dose prime-boost
vaccine could be detected
(Figure 4C). Mock treatment and treatment with the HBV-shRNA, the HBV-siRNAs,
and control
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siRNA all decreased HBV DNA levels, and the level of HBV-DNA was further
decreased by the
prime-boost vaccine in all groups. These data demonstrate that RNAi is
superior to nucleot(s)ide
analog therapy in reducing viral antigens. Also, the combination of RNAi and
subsequent vaccination
have a greater effect on HBsAg and HBV DNA levels than either agent alone.
On the final day of the experiment (the first day of week 17), mice were
sacrificed and their
livers were harvested. Intrahepatic CD8+ T cell responses were assessed for
response to HBsAg,
HBcAg, and the MVA virus particle using the method provided in Backes, 2016.
Mice treated with
the HBV-shRNA or the HBV-siRNAs were able to generate a CD8+ immune response
against the
HBsAg and HBcAg (Figure 5A and 5B). No significant immune response against the
HBV antigens
was observed in the mock, Entecavir, or control siRNA groups. However, a
similar immune response
against the MVA virus was observed in all animals independent of pretreatment
or viral antigen levels
(Figure 5C) showing that vaccination had worked equally well in all animals,
demonstrating the
presence of a competent immune system. The data shown in Figure 5A-C were
reanalyzed to
accomodated for an insufficient exclusion of dead immune cells during the
first analysis. The data
obtained from the second analysis (shown in Figure 5 D-F) again shows that
only the mice pretreated
with the HBV-shRNA or the HBV-siRNAs were able to generate HBV-specific CD8+ T
cell
responses after therapeutic vaccination, but that MVA-specific responses were
not influenced by the
pretreatment. The reduced variances in the second analysis are attributed to
the more rigorous
exclussion of dead cells. These data demonstrate that RNAi treatment, in
contrast to the current
standard of care treatment with a nucleoside analog, can restore HBV-specific
T cell immunity and
enable the induction of HBV-specific CD8 T cell responses after therapeutic
vaccination.
Serum was also assessed for antibody immune response to HBV antigens. Although
the
vaccine was able to induce a T-cell immune response only in animals which had
received HBV-
siRNA or HBV-shRNA pretreatment, antibody responses against HBsAg and HBcAg
were similar
across all groups regardless of pretreatment at the time points evaluated
until week 17. No HBeAg
antibody responses could be detected. These data demonstrate that high HBV
antigen loads
preferentially inhibit HBV-specific T cell, not B cell responses.
Livers were also assessed for the presence of HBV transcripts by RT-qPCR and
normalized to
the liver GAPDH transcript. Mice treated with the HBV-shRNA or the HBV-siRNAs
demonstrated a
significant decrease in HBV transcript levels (Figure 6A and 6B). No
significant difference was
observed between the mock and Entecavir or control siRNA groups. Liver
sections were analysed by
immunohistochemical stainings for expression of core antigen to assess the
antiviral effect in the liver
(Figure 6C). In animals that were not pretreated before vaccination, there
were, on average, 83
hepatocytes per mm2 with cytoplasmic expression of HBc. This number was not
significantly changed
in animals that were pretreated with Entecavir or the control siRNA. However,
the number of
cytoplamic HBcAg positive cells was significantly reduced in AAV-shRNA or HBV
siRNA
pretreated animals. These data demonstrate that a combinatorial
RNAi/vaccination therapy suppresses
not only antigens in the serum, but also viral antigen expression in the
liver.
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Having demonstrated that suppression of expression of HBV antigens using shRNA
or
siRNA is effective at potentiating an immune response to an HBV vaccine
regimen, a study was
designed to determine if the duration of HBV antigen suppression had an effect
on the potentiation an
HBV immune response. Using the HBV 1.3xfs mouse model, mice were treated for
eight, six, or
three weeks with HBV-siRNA AD-66816, or the control siRNA for 8 weeks,
subcutaneously
administered at 3 mg/kg/dose, followed by administration of the prime-boost
vaccine regimen as set
forth above, except 10 lig c-di-AMP was used as an adjuvant rather than PCEP +
CPG (n= 6 per 8 and
3 group, n = 6 for 6 week group) (see Figure 7).
A significant decrease in the levels of each of HBsAg, HBeAg, and HBV DNA was
observed
after the first administration of AD-66816 (Figures 8A-8C). A further
significant decrease in HBsAg
was observed after treatment with the vaccine boost with the greatest decrease
observed in the 8 week
pretreatment group to below the level of detection of the assay, representing
a greater than 5 log10
decrease in HBsAg level in all treated animals. Immunization caused only
slight further reductions
(<0.5 10g10) of HBV DNA and no further reduction in HBeAg levels. These data
demonstrate that
efficacy of therapeutic vaccination correlates with duration of antigen
suppression before start of
vaccination. Reconstitution of HBV-specific CD8 T cell responses takes several
weeks, with a 6 or
preferably 8 week pretreatment resulting in more HBsAg knockdown than a 3 week
pretreatment.
Similarly, T-cell responses against HBsAg and HBcAg in liver corresponded with
the
duration of HBV antigen knockdown, with longer duration of HBV antigen
suppression resulting in
greater T-cell responses (Figure 9A-9C). Similar responses to MVA virus
antigens were observed
across all groups independent of pretreatment (Figure 9D). Antibody responses
were similar across
all groups.
Livers were also assessed for the presence of HBV transcripts by RT-qPCR and
normalized to
the liver GAPDH transcript. Longer pretreatment durations trended to higher
levels of HBV RNA
knockdown (Figure 10A and 10B).
Liver sections were also analysed by immunohistochemical staining for
cytoplasmic
expression of HBcAg to assess the antiviral effect of the treatment in the
liver (Figure 10C). In
animals pretreated with the control siRNA before vaccination, there were, on
average, 172
hepatocytes per mm2which showed cytoplasmic expression of HBc. With longer
duration of HBV
suppression before vaccination, there was a gradual decrease of cytoplasmic
HBcAg positive
hepatocytes with an average of only 12 cytoplasmic HBcAg positive hepatocytes
per mm2in the 8
week pretreatment group. These data demonstrate that the increasing HBV-
specific CD8+ T cell
response observed with longer antigen suppression before vaccination led to
decreased HBV antigen
expression in the liver. To assess the durability of response, blood samples
were collected from mice
(n = 6) pretreated with the AD-66816 HBV-siRNA for six weeks at 2 and 3 weeks
after
administration of the boost vaccination. In three of the six mice, HBsAg
levels continued to dropped
to below the level of detection of the assay (Figure 11A and 11B).
Specifically, the three animals
which had the highest HBsAg and HBeAg levels at start of vaccination (2, 4,
and 5) did show a
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decline of antigen titers after vaccination, but rebounded with antigen titers
towards the end of the
experiment. In contrast, the three animals with the lowest antigen titers at
start of vaccination (1, 3,
and 6) showed a further decline of HBsAg to below the detection limit. These
data demonstrate that a
functional cure is possible using the treatment regimens provided herein.
These data also suggest that
the antigen levels at start of vaccination can affect the response to
therapeutic vaccination. Finally,
without being bound by mechanism, these data suggest that the decline of
antigen levels after
therapeutic vaccination is mediated, at least in part, by CD8+ T cells. These
data demonstrate that
knockdown of circulating HBV antigens (e.g., HBsAg, HBcAg), but not inhibition
of HBV DNA
replication alone, potentiates immune response to HBV therapeutic vaccine,
e.g., a prime boost
therapeutic vaccination regimen. That is, an immune response can be
potentiated by pretreatment
with an siRNA, but not with nucleot(s)ide inhibitors alone as the immune
response is a CD8+ T-cell
based immune response.
The magnitude and duration of knockdown required depends on, for example, the
level of
disease burden in the subject. The higher the level of circulating antigen,
the greater the magnitude
and duration of HBV antigen knockdown required to potentiate an immune
response. Knockdown of
HBsAg in serum should be at least 0.5 log 10 (IU/ml), preferably llog 10
(IU/ml), 1.5 log 10 (IU/ml),
2 log 10 (IU/ml), or more from the level in the subject prior to treatment
with the therapeutic vaccine.
In certain embodiments, the level of serum HBsAg is no more than 2.5 log 10
(IU/ml), 2 log 10
(IU/ml), 1.5 log 10 (IU/ml) prior to vaccine administration.
Further, as demonstrated herein, a longer duration of HBV antigen knockdown
trended
towards a more robust immune response. Therefore, knockdown of serum HBsAg to
a sufficiently
low level for a duration of at least four weeks, six weeks, or eight weeks is
preferred prior to
administration of the vaccine.
A second series of experiments were designed to study the combination
siRNA/vaccination
treatment regimen in a mouse model of aquired HBV infection using an adeno-
associated virus
infection system. For these studies, wildtype C57/B16 mice (9 weeks of age)
were injected i.v. with
2x101 genome equivalents of Adeno-Associated-Virus Serotype 8 (AAV8) carrying
a 1.2-fold
overlength HBV genome of genotype D (AAV-HBV1.2) (see, e.g., Yang, et al.
(2014) Cell and Mol
Immunol 11:71). Starting 4 weeks after AAV-transduction (on day -28), animals
were treated with
three doses of either a control siRNA, or HBV siRNA (HBV AD-66816 or AD-66810)
on days 0, 29,
and 57, and subsequently half of the animals in each group were treated with a
vaccine regimen
consisting of prime protein vaccination with HBsAg, HbcAg, and 10 tig c-di-AMP
at days 57 and 70,
and boosted with MVA-HBs and MVA-HBc at day 84. The treatment regimen is shown
in Figure 12.
Following AAV-HBV1.2 transduction, HBsAg and HBeAg values rose to levels
comparable
to the levels seen in HBV transgenic mice (HBVxfs) (see, e.g., Figures 13A and
13B). Mice treated
with the HBV siRNAs showed mean reductions of HBsAg levels of about 2 logio
scales and of
HBeAg levels greater than 1 logio scale such that HBsAg and HBeAg levels
dropped to less than
about 500 IU/per ml, whereas the control siRNA had no effect on antigen
levels. Animals treated with
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one of the HBV siRNAs that did not receive the vaccine regimen showed slowly
rebounding antigen
levels after the last application of the siRNA. Antigenemia returned to
baseline levels after 18 weeks.
The combination treatment with the HBV-siRNA and vaccine regimen resulted in a
durable response
with a decrease in HBsAg and HBeAg to below the limit of detection through the
duration of the
experiment. A decrease in HBsAg and HBeAg levels was observed prior to the
administration of the
MVA boost, suggesting that the protein vaccination may be sufficient to affect
a cure. Both serum and
liver HBV DNA and RNA were significantly decreased after combination treatment
with the HBV-
siRNA and vaccine regimen (Figures 13D-13H). This demonstrates, that RNAi-
mediated suppression
can strongly reduce antigen expression, but that treatment with the vaccine
regimen extends the
durability of response.
The vaccine regimen alone in animals that received the control siRNA produced
a transient
decline of antigen levels which rebounded towards the end of the experiment.
In contrast, all animals
that received an HBV-siRNA and vaccination, HBsAg and HBeAg levels decreaased
to below the
detection limit after start of vaccination. Antigen levels remained largely
undetectable at all through
the last time point measured at least 22 weeks after the last siRNA
application (Figures 13A and 13B).
The durable loss of antigenemia in HBV siRNA pretreated animals, in contrast
to the antigen rebound
in control siRNA pretreated animals, further demonstrates that immune control
was only achieved in
animals which had lowered antigen titers before vaccination.
Coinciding with the loss of antigenemia, animals treated with HBV siRNA plus
the vaccine
regimen developed high titers of anti-HBs antibodies and resulted in anti-HBs
and anti-HBe
seroconversion in all vaccinated animals (Figure 14). siRNA-pretreated animals
developed 10-fold
higher and more constant anti-HBs titers and were able to completely and
persistently clear serum
HBsAg and HBeAg. Interestingly, 3/12 mice vaccinated after HBV siRNA treatment
showed a
transient drop in anti-HBe levels between week 15 and 22 resulting in a low-
level relapse of HBeAg
(Figure 13C) that was again controlled. Although anti-HBs antibodies could
also be measured in
animals that received the control siRNA plus the vaccine regimen, the levels
were lower. Further,
only animals that received HBV siRNA plus the vaccine regimen developed anti-
HBe antibodies.
Taken together, functional cure was not achieved by the siRNA treatement
regimen or the therapeutic
vaccination regimen alone. However, the loss of antigenemia, as well as
development of anti-HBs and
anti-HBe antibodies, demonstrates that the combination HBV siRNA plus vaccine
regimen can
achieve a functional cure.
Throughout the experiment, ALT and body weight of the animals were monitored.
The loss
of antigenemia concided with slight increases of ALT activity seen in
treatment groups which had
received HBV siRNA in conjunction with the vaccination regimen (Figure 15A).
These groups
showed significant but mild increases (both p>0.05 or smaller by repeated
measure two-way
ANOVA; only comparing time points after start of vaccination) as compared to
all other treatment
groups that did not receive the combination HBVsiRNA-vaccine regimen. Without
being bound by
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mechanism, it is suggested that the CD8+ T cells induced by the combinatorial
HBV siRNA-vaccine
regimen killed HBV-expressing hepatocytes resulting in elevated ALT.
The body weight of the animals was measured throughout the experiment to
assess
tolerability of the treatments. There was steady increases throughout the
experiment independent of
siRNA treatment. Animals that were vaccinated showed a slight and transient
decrease
(approximately 5%) of body weight after vaccination, but rebounded to normal
levels within nine
days, and subsequently gained weight comparable to the control groups (Figure
15B). Taken together,
both, ALT activity and body weight data demonstrate that all examined
treatments, including siRNA
only, vaccine only, as well as the combinatorial siRNA/vaccine regimen are
well tolerated.
The number and timing of doses of siRNA to knockdown HBsAg level in serum
depends, for
example, on the specific agent used. Due to the duration and potency of the
exemplary GalNAc
siRNAs used in the methods herein and provided, for example, in Appendix A and
in PCT Publication
No. WO 2016/077321 (the entire contents of which are incorporated herein by
reference), a single
dose of siRNA may be sufficient to provide the level and duration of knockdown
required prior to
administration of the therapeutic vaccine. As shown in Figure 4, a single dose
of an AAV vector
encoded shRNA was sufficient to provide durable knockdown of HBV antigens and
HBV DNA.
Those of skill in the art are able to monitor HBV disease status, e.g., by
measuring HBsAg levels in
blood, to determine the timing and level of siRNA and vaccine administration
appropriate for a
specific subject.
A number of therapeutic HBV vaccines are known in the art and discussed
herein. In
preferred embodiments, a prime-boost vaccination protocol, such as the
protocol that is used herein, is
preferred. However, the HBV antigen knockdown method provided herein can be
used in combination
with other therapeutic HBV vaccines known in the art, including those found to
be insufficient when
administered alone. Vaccinations include at least two doses of an antigen in
protein or nucleic acid
form. In certain embodiments, the vaccination includes three doses of a
protein-based vaccine. In
preferred embodiments, the methods include heterologous vaccine
administration, i.e., at least one
protein-based vaccine dose and at least one nucleic-acid based vaccine dose.
Exemplary
embodiments of vaccines and dosing regimens are provided, for example, in PCT
Publication No.
WO 2017/121791, the entire contents of which are incorporated herein by
reference.
The methods provided herein include the use of a nucleic acid-based vaccine
comprising an
expression vector construct encoding an HBcAg or an HBsAg, wherein the
construct encodes a
protein that shares an epitope with the protein-based vaccine. Therefore, it
is clearly understood that
neither the nucleic acid-based vaccine nor the protein-based vaccine are
required to provide the full
length protein. The nucleic acid-based vaccine and the protein-based vaccine
are required to provide
at least one shared epitope that is present in HBcAg or HBsAg, and does not
require that the full
length protein be provided. As noted above, epitopes may be relatively short,
MHC class I molecules
that are typically peptides about 8 to 11 amino acids in length, whereas MHC
class II molecules
present longer peptides about 13 to 17 amino acids in length, with
conformational epitopes being
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longer. However, it is understood that the use of protein antigens and coding
sequences for protein
antigens that encode multiple epitopes is preferred. Further, it is understood
that the antigens present
in the protein-based vaccine and encoded by the nucleic-acid based vaccine may
or may not be
identical. It is also obvious that antigens should also be selected for their
immunogenicity. Such
antigens are well known in the art.
In certain embodiments, the order of administration of the protein-based
vaccine and the
nucleic acid-based vaccine are reversed as compared to the order exemplified
in the methods provided
herein. That is, the nucleic acid-based vaccine is administered first and the
protein-based vaccine is
administered second. In certain embodiments, a total of three doses of vaccine
are administered, two
doses of the nucleic acid-based vaccine followed by a single dose of the
protein-based vaccine. In
alternative embodiments, a single dose of the nucleic acid-based vaccine is
followed by two doses of a
protein based vaccine. In other embodiments, one dose of each vaccine is
administered.
In preferred embodiments, the prime-boost vaccination method includes the use
of an
adjuvant with protein antigens. Appropriate adjuvants for use in the methods
promote a cell-based
response to the antigens. Adjuvants preferably provide a balanced Th1/Th2
response.
The siRNA + vaccine methods provided herein can be used in combination with
administration of nucleot(s)ide inhibitors which are the standard of care for
treatment of HBV. In
certain embodiments, subjects are treated with nucleot(s)ide inhibitors prior
to treatment with the
siRNA + vaccine treatment regimen. In certain embodiments, subjects are
treated throughout the
siRNA + vaccine treatment regimen with nucleot(s)ide inhibitors. In certain
embodiments, the
nucleot(s)ide inhibitor is dosed to reduce pre-existing inflammation
associated with HBV infection
prior to administration of the nucleic acid therapeutic targeted to HBV (e.g.,
siRNA, shRNA,
antisense oligonucleotide).
In certain embodiments, subjects may be pretreated, or concurrently treated,
with other agents
used for the treatment of HBV. Such agents include, but are not limited to an
immune stimulator
(e.g., pegylated interferon alfa 2a (PEG-IFN-a2a), Interferon alfa-2b, a
recombinant human
interleukin-7, and aToll-like receptor 7 (TLR7) agonist), a viral entry
inhibitor (e.g., Myrcludex), an
oligonucleotide that inhibits the secretion or release of HbsAg (e.g., REP
9AC), a capsid inhibitor
(e.g., Bay41-4109 and NVR-1221), a cccDNA inhibitor (e.g., IHVR-25), a Rig-I
ligand, or an
immune checkpoint regulator. In certain embodiments, the immune stimulator is
a Rig-I ligand or an
immune checkpoint regulator. A functional cure includes a sustained period of
at least 3 months,
preferably 6 months of HBsAg below 50 IU/ml, or a detectable antibody response
to HBsAg. In
preferred embodiments, a functional cure includes both a sustained period of
at least 3 months,
preferably 6 months of HBsAg below 50 IU/ml and a detectable antibody response
to HBsAg.
A. RNAi Agents For Use in the Methods of the Invention
The present invention includes the use of iRNAs, which inhibit the expression
of at least one
HBV transcript, and preferably three or four HBV transcripts (open reading
frames, sometimes
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referred to herein as genes). Due to the highly condensed structure of the HBV
genome, it is possible
to design single iRNAs that will inhibit the expression of three or four HBV
transcripts (see Figure 1).
For the sake of simplicity, the text herein refers to "an HBV transcript" or
"the HBV transcript." It is
understood that preferred embodiments include inhibition of more than one HBV
transcript (or open
.. reading frame), preferably at least three HBV transcripts (or open reading
frames). Further, it is
understood that there are eight HBV genotypes, and two proposed additional
genotypes, that may
further include mutations from published sequences. Therefore, certain iRNA
agents may inhibit
different numbers of genes based on the specific genotype and subject infected
with HBV.
In some embodiments, the iRNA agent includes double stranded ribonucleic acid
(dsRNA)
molecules for inhibiting the expression or decreasing the level of an HBV
transcript in a cell in a
subject with an HBV infection. The dsRNA includes an antisense strand having a
region of
complementarity, which is complementary to at least a part of an mRNA formed
in the expression of
an HBV transcript. The region of complementarity is about 30 nucleotides or
less in length (e.g.,
about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or
less in length). Upon contact
with a cell expressing the HBV gene, the iRNA selectively inhibits the
expression of at least one,
preferably three or four HBV genes. In preferred embodiments, inhibition of
expression is
determined by the qPCR method in an appropriate cell line as provided in the
examples. For in vitro
assessment of activity, percent inhibition is determined using the methods
provided in Example 2 of
PCT Publication No.WO 2016/077321 at a single dose at a 10 nM duplex final
concentration. For in
vivo studies, the level after treatment can be compared to, for example, an
appropriate historical
control or a pooled population sample control to determine the level of
reduction, e.g., when a
baseline value is not available for the subject. An appropriate control must
be carefully selected by
one of skill in the art.
A dsRNA includes two RNA strands that are complementary and hybridize to form
a duplex
structure under conditions in which the dsRNA will be used. One strand of a
dsRNA (the antisense
strand) includes a region of complementarity that is substantially
complementary, and generally fully
complementary, to a target sequence. The target sequence can be derived from
the sequence of an
mRNA formed during the expression of an HBV gene. As multiple HBV genotypes
are known,
iRNA agents are preferably designed to inhibit expression of HBV genes across
as many genotypes as
possible. It is understood that an siRNA that is perfectly complementary to
one or more HBV
genotypes will not be perfectly complementary to all genotypes. The other
strand (the sense strand)
includes a region that is complementary to the antisense strand, such that the
two strands hybridize
and form a duplex structure when combined under suitable conditions. As
described elsewhere herein
and as known in the art, the complementary sequences of a dsRNA can also be
contained as self-
.. complementary regions of a single nucleic acid molecule, as opposed to
being on separate
oligonucleotides.
Generally, the duplex structure is 15 and 30 base pairs in length, e.g., 15-
29, 15-28, 15-27,
15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,
18-29, 18-28, 18-27, 18-
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26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-
26, 19-25, 19-24, 19-23,
19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23,
20-22, 20-21, 21-30, 21-
29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.
Ranges and lengths
intermediate to the above recited ranges and lengths are also contemplated to
be part of the invention.
Similarly, the region of complementarity to the target sequence is 15 and 30
nucleotides in
length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21,
15-20, 15-19, 15-18, 15-
17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-
20, 19-30, 19-29, 19-28,
19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28,
20-27, 20-26, 20-25, 20-
24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-
23, or 21-22 nucleotides
in length. Ranges and lengths intermediate to the above recited ranges and
lengths are also
contemplated to be part of the invention.
In some embodiments, the dsRNA is about 15 to 20 nucleotides in length, or
about 25 to 30
nucleotides in length. In general, the dsRNA is long enough to serve as a
substrate for the Dicer
enzyme. For example, it is well-known in the art that dsRNAs longer than about
21-23 nucleotides in
length may serve as substrates for Dicer. As the ordinarily skilled person
will also recognize, the
region of an RNA targeted for cleavage will most often be part of a larger RNA
molecule, often an
mRNA molecule. Where relevant, a "part" of an mRNA target is a contiguous
sequence of an mRNA
target of sufficient length to allow it to be a substrate for RNAi-directed
cleavage (i.e., cleavage
through a RISC pathway).
One of skill in the art will also recognize that the duplex region is a
primary functional
portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g.,
about 10-36, 11-36, 12-36,
13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-
34, 11-34, 12-34, 13-
34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32,
11-32, 12-32, 13-32,
14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-
28, 15-27, 15-26, 15-
25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-
28, 18-27, 18-26, 18-25,
18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25,
19-24, 19-23, 19-22, 19-
21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-
21, 21-30, 21-29, 21-28,
21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one
embodiment, to the extent that it
becomes processed to a functional duplex, of e.g., 15-30 base pairs, that
targets a desired RNA for
cleavage, an RNA molecule or complex of RNA molecules having a duplex region
greater than 30
base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that
in one embodiment, a
miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring
miRNA. In
another embodiment, an iRNA agent useful to target HBV gene expression is not
generated in the
target cell by cleavage of a larger dsRNA.
The duplex region may be of any length that permits specific degradation of a
desired target
RNA through a RISC pathway, and may range from about 9 to 36 base pairs in
length, e.g., about 15-
30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such
as about 15-30, 15-29, 15-28,
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15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17,
18-30, 18-29, 18-28, 18-
27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-
27, 19-26, 19-25, 19-24,
19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-
23, 20-22, 20-21, 21-
30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in
length. Ranges and
lengths intermediate to the above recited ranges and lengths are also
contemplated to be part of the
invention.
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." A hairpin loop can comprise at
least one unpaired
nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at
least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least
20, at least 23 or more unpaired
nucleotides.
In some embodiments, a dsRNA agent of the invention comprises a tetraloop. As
used
herein, "tetraloop" in the context of a dsRNA refers to a loop (a single
stranded region) consisting of
four nucleotides that forms a stable secondary structure that contributes to
the stability of adjacent
Watson-Crick hybridized nucleotides. Without being limited to theory, a
tetraloop may stabilize an
adjacent Watson-Crick base pair by stacking interactions. In addition,
interactions among the four
nucleotides in a tetraloop include, but are not limited to, non-Watson-Crick
base pairing, stacking
interactions, hydrogen bonding, and contact interactions (Cheong et al.,
Nature 1990 Aug
16;346(6285):680-2; Heus and Pardi, Science 1991 Jul 12;253(5016): 191-4). A
tetraloop confers an
increase in the melting temperature (Tm) of an adjacent duplex that is higher
than expected from a
simple model loop sequence consisting of four random bases. For example, a
tetraloop can confer a
melting temperature of at least 55 C in 10mM NaHPO4 to a hairpin comprising a
duplex of at least 2
base pairs in length. A tetraloop may contain ribonucleotides,
deoxyribonucleotides, modified
nucleotides, and combinations thereof. Examples of RNA tetraloops include the
UNCG family of
tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the
CUUG tetraloop.
(Woese et al., Proc Natl Acad Sci U S A. 1990 Nov;87(21):8467-71 ; Antao et
al., Nucleic Acids Res.
1991 Nov 11;19(21):5901-5). Examples of DNA tetraloops include the d(GNNA)
family of tetraloops
(e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of
tetraloops, the d(CNNG)
family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)).
(Nakano et al. Biochemistry,
41(48), 14281 -14292, 2002; Shinji et al. Nippon Kagakkai Koen Yokoshu
vol.78th; no.2; page.731
(2000).)
In certain embodiments of the invention, tetraloop- and modified nucleotide-
containing
dsNAs are contemplated as described, e.g., as described in U.S. Patent
Publication No. 2011/0288147,
the entire contents of which are incorporated by reference herein. In certain
such embodiments, a
dsNA of the invention possesses a first strand and a second strand, where the
first strand and the
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second strand form a duplex region of 19-25 nucleotides in length, wherein the
first strand comprises
a 3' region that extends beyond the first strand-second strand duplex region
and comprises a tetraloop,
and the dsNA comprises a discontinuity between the 3' terminus of the first
strand and the 5' terminus
of the second strand.
Optionally, the discontinuity is positioned at a projected Dicer cleavage site
of the tetraloop-
containing double stranded nucleic acid (dsNA). It is contemplated that, as
for any of the other
dupexed oligonucleotides of the invention, tetraloop-containing duplexes of
the invention can possess
any range of modifications disclosed herein or otherwise known in the art,
including, e.g., 2'-0-
methyl, 2'- fluoro, inverted base, GalNAc moieties, etc. Typically, every
nucleotide on both strands of
the tetraloop-containing dsNA is chemically modified if the tetraloop-
containing dsNA is going to be
delivered without using lipid nanoparticles or some other delivery method that
protects the dsNA
from degradation during the delivery process. However, in certain embodiments,
one or more
nucleotides are not modified.
Where the two substantially complementary strands of a dsRNA are comprised by
separate
RNA molecules, those molecules need not, but can be covalently connected.
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 5'-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 may comprise one or more nucleotide overhangs.
A dsRNA as described herein can further include one or more single-stranded
nucleotide
overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one
nucleotide overhang can have
unexpectedly superior inhibitory properties relative to their blunt-ended
counterparts. A nucleotide
overhang can comprise or consist of a nucleotide/nucleoside analog, including
a
deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the
antisense strand or any
combination thereof. Furthermore, the nucleotide(s) of an overhang can be
present on the 5'-end, 3'-
end or both ends of either an antisense or sense strand of a dsRNA. In certain
embodiments, longer,
extended overhangs are possible.
A dsRNA can be synthesized by standard methods known in the art. iRNA
compounds of the
invention may be prepared using a two-step procedure. First, the individual
strands of the double
stranded RNA molecule are prepared separately. Then, the component strands are
annealed. The
individual strands of the siRNA compound can be prepared using solution-phase
or solid-phase
organic synthesis or both. In certain embodiments, the iRNA compound is
produced from an
expression vector delivered into a cell.
In one aspect, a dsRNA of the invention includes at least two nucleotide
sequences, a sense
sequence and an anti-sense sequence. The sense strand is selected from the
group of sequences
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provided in any one of the Tables in Appendix A, and the corresponding
antisense strand of the sense
strand is selected from the group of sequences of any one of the Tables in
Appendix A.
In some embodiments, the sense strand is selected from the group of sequences
provided in
any one of the Tables in Appendix A, and the corresponding antisense strand of
the sense strand is
.. selected from the group of sequences of any one of the Tables in Appendix
A. In this aspect, one of
the two sequences is complementary to the other of the two sequences, with one
of the sequences
being substantially complementary to a sequence of an mRNA generated in the
expression of an HBV
gene. As such, in this aspect, a dsRNA will include two oligonucleotides,
where one oligonucleotide
is described as the sense strand in any one of the Tables in Appendix A and
the second
oligonucleotide is described as the corresponding antisense strand of the
sense strand in any one of the
Tables in Appendix A. In one embodiment, the substantially complementary
sequences of the dsRNA
are contained on separate oligonucleotides. In another embodiment, the
substantially complementary
sequences of the dsRNA are contained on a single oligonucleotide.
It is understood that, although some of the sequences in the Tables in
Appendix A are
.. described as modified or conjugated sequences, the RNA of the iRNA of the
invention e.g., a dsRNA
of the invention, may comprise any one of the sequences set forth in the
Tables in Appendix A that is
un-modified, unconjugated, or modified or conjugated differently than
described therein. Additional
target sites are provided, for example, in PCT Publication Nos. WO
2016/077321, WO 2012/024170,
WO 2017/027350, and WO 2013/003520; and in Michler, 2016, the entire contents
of wach of which
are incorporated herein by reference.
The skilled person is well aware that dsRNAs having a duplex structure of
about 20 to 23
base pairs, e.g., 21, base pairs have been hailed as particularly effective in
inducing RNA interference
(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that
shorter or longer RNA
duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719;
Kim et al. (2005)
Nat Biotech 23:222-226). In the embodiments described above, by virtue of the
nature of the
oligonucleotide sequences provided in any one of the Tables in Appendix A,
dsRNAs described
herein can include at least one strand of a length of minimally 21
nucleotides. It can be reasonably
expected that shorter duplexes having one of the sequences of any one of the
Tables in Appendix A
minus only a few nucleotides on one or both ends can be similarly effective as
compared to the
dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16,
17, 18, 19, 20, or
more contiguous nucleotides derived from one of the sequences of any one of
the Tables in Appendix
A and differing in their ability to inhibit the expression of an HBV gene by
not more than 5, 10, 15,
20, 25, or 30 % inhibition from a dsRNA comprising the full sequence.
An iRNA as described herein can contain one or more mismatches to the target
sequence, or
to one or more HBV target sequences due, e.g., to sequence variations among
the HBV genotypes. In
one embodiment, an iRNA as described herein contains no more than 3
mismatches. If the antisense
strand of the iRNA contains mismatches to a target sequence, it is preferable
that the area of mismatch
is not located in the center of the region of complementarity. If the
antisense strand of the iRNA
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contains mismatches to the target sequence, it is preferable that the mismatch
be restricted to be
within the last 5 nucleotides from either the 5'- or 3'-end of the region of
complementarity. For
example, for a 23 nucleotide iRNA agent the strand which is complementary to a
region of an HBV
gene, generally does not contain any mismatch within the central 13
nucleotides. The methods
described herein or methods known in the art can be used to determine whether
an iRNA containing a
mismatch to a target sequence is effective in inhibiting the expression of an
HBV gene. Consideration
of the efficacy of iRNAs with mismatches in inhibiting expression of an HBV
gene is important,
especially if the particular region of complementarity in an HBV gene is known
to have polymorphic
sequence variation within various genotypes and the population.
i. Modified iRNAs For Use in the Methods of the Invention
In some embodiments, the RNA of the iRNA for use in the invention e.g., a
dsRNA, is un-
modified, and does not comprise, e.g., chemical modifications or conjugations
known in the art and
described herein, e.g., when produced from an expression vector. In other
embodiments, the RNA of
an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance
stability or other
beneficial characteristics. In certain embodiments of the invention,
substantially all of the
nucleotides of an iRNA of the invention are modified. In other embodiments of
the invention, all of
the nucleotides of an iRNA of the invention are modified. iRNAs of the
invention in which
"substantially all of the nucleotides are modified" are largely but not wholly
modified and can include
not more than 5, 4, 3, 2, or 1 unmodified nucleotides.
The nucleic acids featured in the invention can be synthesized or modified by
methods well
established in the art, such as those described in "Current protocols in
nucleic acid chemistry,"
Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA,
which is incorporated
herein by reference. Modifications include, for example, end modifications,
e.g., 5'-end
modifications (phosphorylation, conjugation, inverted linkages) or 3'-end
modifications (conjugation,
DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,
replacement with stabilizing
bases, destabilizing bases, or bases that base pair with an expanded
repertoire of partners, removal of
bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at
the 2'-position or 4'-
position) or replacement of the sugar; or backbone modifications, including
modification or
replacement of the phosphodiester linkages. Specific examples of iRNA
compounds useful in the
embodiments described herein include, but are not limited to RNAs containing
modified backbones or
no natural internucleoside linkages. RNAs having modified backbones include,
among others, those
that do not have a phosphorus atom in the backbone. For the purposes of this
specification, and as
sometimes referenced in the art, modified RNAs that do not have a phosphorus
atom in their
internucleoside backbone can also be considered to be oligonucleosides. In
some embodiments, a
modified iRNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones include, for example, phosphorothioates, chiral
phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and
other alkyl
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phosphonates including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,
and
boranophosphates having normal 3' -5' linkages, 2' -5' -linked analogs of
these, and those having
inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-
5' to 5'-3' or 2'-5' to 5'-
2'. Various salts, mixed salts and free acid forms are also included.
Representative US patents that teach the preparation of the above phosphorus-
containing
linkages include, but are not limited to, US Patent Nos. 3,687,808; 4,469,863;
4,476,301; 5,023,243;
5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;
5,399,676; 5,405,939;
5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316;
5,550,111; 5,563,253;
5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;
6,172,209; 6, 239,265;
6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035;
6,683,167; 6,858,715;
6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and US Pat
RE39464, the entire
contents of each of which are hereby incorporated herein by reference.
Modified RNA backbones that do not include a phosphorus atom therein have
backbones that
are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatoms and alkyl
or cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic
internucleoside linkages. These include those having morpholino linkages
(formed in part from the
sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and
sulfone backbones;
formacetyl and thioformacetyl backbones; methylene formacetyl and
thioformacetyl backbones;
alkene containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones; and others
having mixed N, 0, S
and CH2 component parts.
Representative US patents that teach the preparation of the above
oligonucleosides include,
but are not limited to, US Patent Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141;
5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;
5,489,677; 5,541,307;
5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360;
5,677,437; and, 5,677,439, the entire contents of each of which are hereby
incorporated herein by
reference.
In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs,
in which
both the sugar and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced
with novel groups. The base units are maintained for hybridization with an
appropriate nucleic acid
target compound. One such oligomeric compound, an RNA mimetic that has been
shown to have
excellent hybridization properties, is referred to as a peptide nucleic acid
(PNA). In PNA compounds,
the sugar backbone of an RNA is replaced with an amide containing backbone, in
particular an
aminoethylglycine backbone. The nucleobases are retained and are bound
directly or indirectly to aza
nitrogen atoms of the amide portion of the backbone. Representative US patents
that teach the
preparation of PNA compounds include, but are not limited to, US Patent Nos.
5,539,082; 5,714,331;
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and 5,719,262, the entire contents of each of which are hereby incorporated
herein by reference.
Additional PNA compounds suitable for use in the iRNAs of the invention are
described in, for
example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate
backbones
and oligonucleosides with heteroatom backbones, and in particular ¨CH2¨NH¨CH2-
, --CH2¨N(CH3)-
0¨CH24known as a methylene (methylimino) or MMI backbone], --CH2-0¨N(CH3)¨CH2--
, --CH2¨
N(CH3)¨N(CH3)¨CH2¨and ¨N(CH3)¨CH2¨CH24wherein the native phosphodiester
backbone is
represented as ¨0¨P¨O¨CH2--] of the above-referenced US Patent No. 5,489,677,
and the amide
backbones of the above-referenced US Patent No. 5,602,240. In some
embodiments, the RNAs
featured herein have morpholino backbone structures of the above-referenced US
Patent No.
5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The
iRNAs, e.g.,
dsRNAs, featured herein can include one of the following at the 2'-position:
OH; F; 0-, S-, or N-
alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or 0-alkyl-0-alkyl, wherein
the alkyl, alkenyl and
alkynyl can be substituted or unsubstituted Ci to Ci0 alkyl or C2 to Cio
alkenyl and alkynyl.
Exemplary suitable modifications include ORCH2).0] .CH3, 0(CH2).110CH3,
0(CH2).NH2, 0(CH2)
.CH3, 0(CH2).0NH2, and 0(CH2).0NRCH2).CH3)]2, where n and m are from 1 to
about 10. In other
embodiments, dsRNAs include one of the following at the 2' position: Ci to C10
lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, 0-alkaryl or 0-aralkyl, SH, SCH3,
OCN, Cl, Br, CN, CF3,
OCF3, SOCH3, 502CH3, 0NO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an
intercalator, a group for improving the pharmacokinetic properties of an iRNA,
or a group for
improving the pharmacodynamic properties of an iRNA, and other substituents
having similar
properties. In some embodiments, the modification includes a 2'-methoxyethoxy
(2'-0-
CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-M0E) (Martin et al.,
Hely. Chim. Acta,
1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification
is 2' -
dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMA0E,
as described in
examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art
as 2'-0-
dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O¨CH2-0¨CH2¨N(CH2)2.
Other modifications include 2' -methoxy (2'-OCH3), 2'-aminopropoxy (2'-
OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at
other positions on
the RNA of an iRNA, particularly the 3' position of the sugar on the 3'
terminal nucleotide or in 2'-5'
linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs can also
have sugar mimetics
such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Representative US patents that teach
the preparation of such modified sugar structures include, but are not limited
to, US Patent Nos.
4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;
5,514,785; 5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873;
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5,670,633; and 5,700,920, certain of which are commonly owned with the instant
application,. The
entire contents of each of the foregoing are hereby incorporated herein by
reference.
The RNA of an iRNA can also include nucleobase (often referred to in the art
simply as
"base") modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include
the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine
(T), cytosine (C) and
uracil (U). Modified nucleobases include other synthetic and natural
nucleobases such as deoxy-
thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-
aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-
propyl and other alkyl
derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-
thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,
5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-
substituted adenines and
guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-
substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
7-deazaguanine
and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases
include those
disclosed in US Patent No. 3,687,808, those disclosed in Modified Nucleosides
in Biochemistry,
Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed
in The Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.
L, ed. John Wiley
& Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991,
30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and
Applications, pages
289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these
nucleobases are
particularly useful for increasing the binding affinity of the oligomeric
compounds featured in the
invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and 0-6
substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-
propynylcytosine. 5-
methylcytosine substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2 C
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and
Applications, CRC Press,
Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more
particularly when
combined with 2'-0-methoxyethyl sugar modifications.
Representative US patents that teach the preparation of certain of the above
noted modified
nucleobases as well as other modified nucleobases include, but are not limited
to, the above noted US
Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;
5,432,272; 5,457,187;
5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,
5,596,091; 5,614,617;
5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;
6,380,368; 6,528,640;
6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents
of each of which are
hereby incorporated herein by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic
acids
(LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety
in which the ribose
moiety comprises an extra bridge connecting the 2' and 4' carbons. This
structure effectively "locks"
the ribose in the 3'-endo structural conformation. The addition of locked
nucleic acids to siRNAs has
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been shown to increase siRNA stability in serum, and to reduce off-target
effects (Elmen, J. et al.,
(2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc
Ther 6(3):833-
843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
In some embodiments, the iRNA of the invention comprises one or more monomers
that are
UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid,
wherein any of the
bonds of the sugar has been removed, forming an unlocked "sugar" residue. In
one example, UNA
also encompasses monomer with bonds between C1'-C4' have been removed (i.e.
the covalent carbon-
oxygen-carbon bond between the Cl' and C4' carbons). In another example, the
C2'-C3' bond (i.e. the
covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has
been removed (see
Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst.,
2009, 10, 1039 hereby
incorporated by reference).
The RNA of an iRNA can also be modified to include one or more bicyclic sugar
moities. A
"bicyclic sugar" is a furanosyl ring modified by the bridging of two atoms.
A"bicyclic
nucleoside"("BNA") is a nucleoside having a sugar moiety comprising a bridge
connecting two
carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In
certain embodiments, the
bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in
some embodiments an
agent of the invention may include one or more locked nucleic acids (LNA). A
locked nucleic acid is
a nucleotide having a modified ribose moiety in which the ribose moiety
comprises an extra bridge
connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide
comprising a bicyclic sugar
moiety comprising a 4'-CH2-0-2' bridge. This structure effectively "locks" the
ribose in the 3'-endo
structural conformation. The addition of locked nucleic acids to siRNAs has
been shown to increase
siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al.,
(2005) Nucleic Acids
Research 33(1):439-447; Mook, OR. Et al., (2007) Mol Canc Ther 6(3):833-843;
Grunweller, A. et
al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic
nucleosides for use in
the polynucleotides of the invention include without limitation nucleosides
comprising a bridge
between the 4' and the 2' ribosyl ring atoms. In certain embodiments, the
antisense polynucleotide
agents of the invention include one or more bicyclic nucleosides comprising a
4' to 2' bridge.
Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not
limited to 4'-(CH2)-0-2'
(LNA); 4'-(CH2)¨S-2'; 4'-(CH2)2-0-2' (ENA); 4'-CH(CH3)-0-2' (also referred to
as
"constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-0-2' (and analogs thereof;
see, e.g., US Patent
No. 7,399,845); 4'-C(CH3)(CH3)-0-2' (and analogs thereof; see e.g., US Patent
No. 8,278,283); 4'-
CH2¨N(OCH3)-2' (and analogs thereof; see e.g., US Patent No. 8,278,425); 4'-
CH2-0¨N(CH3)-
2' (see, e.g.,US20040171570); 4'-CH2¨N(R)-0-2', wherein R is H, C1-C12 alkyl,
or a protecting
group (see, e.g., US Patent No. 7,427,672); 4'-CH2¨C(H)(CH3)-2' (see, e.g.,
Chattopadhyaya et al.,
J. Org. Chem., 2009, 74, 118-134); and 4'-CH2¨C(=CH2)-2' (and analogs thereof;
see, e.g., US
Patent No. 8,278,426). The entire contents of each of the foregoing are hereby
incorporated herein by
reference.
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Additional representative US patents and US patent publications that teach the
preparation of
locked nucleic acid nucleotides include, but are not limited to, the
following: US Patent Nos.
6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207;
7,034,133;7,084,125;
7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425;
8,278,426; 8,278,283;
US 20080039618; and US 20090012281, the entire contents of each of which are
hereby incorporated
herein by reference.
Any of the foregoing bicyclic nucleosides can be prepared having one or more
stereochemical
sugar configurations including for example a-L-ribofuranose and I3-D-
ribofuranose (see WO
99/14226).
The RNA of an iRNA can also be modified to include one or more constrained
ethyl
nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a
locked nucleic acid
comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge. In one
embodiment, a
constrained ethyl nucleotide is in the S conformation referred to herein as "S-
cEt."
An iRNA of the invention may also include one or more "conformationally
restricted
nucleotides" ("CRN"). CRN are nucleotide analogs with a linker connecting the
C2' and C4' carbons
of ribose or the C3 and -05' carbons of ribose. CRN lock the ribose ring into
a stable conformation
and increase the hybridization affinity to mRNA. The linker is of sufficient
length to place the
oxygen in an optimal position for stability and affinity resulting in less
ribose ring puckering.
Representative publications that teach the preparation of certain of the above
noted CRN
include, but are not limited to, US 20130190383 and WO 2013036868, the entire
contents of each of
which are hereby incorporated herein by reference.
Potentially stabilizing modifications to the ends of RNA molecules can include
N-
(acetylaminocaproy1)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproy1-4-
hydroxyprolinol (Hyp-C6),
N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether),
N-
(aminocaproy1)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-
phosphate, inverted
base dT(idT) and others. Disclosure of this modification can be found in WO
2011005861.
Other modifications of the nucleotides of an iRNA of the invention include a
5' phosphate or
5' phosphate mimic, e.g., a 5'-terminal phosphate or phosphate mimic on the
antisense strand of an
RNAi agent. Suitable phosphate mimics are disclosed in, for example
U520120157511, the entire
contents of which are incorporated herein by reference.
In certain specific embodiments, the RNAi agent for use in the methods of the
invention is an
agent selected from the group of agents listed in any one of the Tables in
Appendix A. These agents
may further comprise a ligand.
ii. iRNAs Conjugated to Ligands
Another modification of the RNA of an iRNA of the invention involves
chemically linking to
the RNA one or more ligands, moieties or conjugates that enhance the activity,
cellular distribution, or
cellular uptake of the iRNA. Such moieties include but are not limited to
lipid moieties such as a
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cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:
6553-6556), cholic acid
(Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether,
e.g., beryl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al.,
Biorg. Med. Chem.
Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids
Res., 1992, 20:533-538), an
aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et
al., EMBO J, 1991,
10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et
al., Biochimie, 1993,
75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-
ammonium 1,2-di-O-hexadecyl-
rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-
3654; Shea et al.,
Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al.,
Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid
(Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta,
1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol
moiety (Crooke et
al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
In one embodiment, a ligand alters the distribution, targeting, or lifetime of
an iRNA agent
into which it is incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a
selected target, e.g., molecule, cell or cell type, compartment, e.g., a
cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a species absent
such a ligand. Preferred
ligands will not take part in duplex pairing in a duplexed nucleic acid.
Ligands can include a naturally occurring substance, such as a protein (e.g.,
human serum
albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate
(e.g., a dextran, pullulan,
chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic
acid); or a lipid. The
ligand can also be a recombinant or synthetic molecule, such as a synthetic
polymer, e.g., a synthetic
polyamino acid. Examples of polyamino acids include polyamino acid is a
polylysine (PLL),
poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride
copolymer, poly(L-lactide-
co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-
hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG),
polyvinyl alcohol
(PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide
polymers, or
polyphosphazine. Example of polyamines include: polyethylenimine, polylysine
(PLL), spermine,
spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,
dendrimer polyamine,
arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary
salt of a polyamine, or an
alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting
agent, e.g., a lectin,
glycoprotein, lipid, or protein, e.g., an antibody, that binds to a specified
cell type such as a kidney
cell. A targeting group can be a thyrotropin, melanotropin, lectin,
glycoprotein, surfactant protein A,
Mucin carbohydrate, multivalent lactose, monovalent or multivalent galactose,
N-acetyl-
galactosamine, N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,
glycosylated
polyaminoacids, transferrin, bisphosphonate, polyglutamate, polyaspartate, a
lipid, cholesterol, a
steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide
or RGD peptide mimetic.
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In certain embodiments, ligands include monovalent or multivalent galactose.
In certain
embodiments, ligands include cholesterol.
Other examples of ligands include dyes, intercalating agents (e.g. acridines),
cross-linkers
(e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin),
polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases
(e.g. EDTA), lipophilic
molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene
butyric acid,
dihydrotestosterone, 1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol,
borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic
acid,03-
(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or
phenoxazine)and peptide
conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,
phosphate, amino, mercapto,
PEG (e.g., PEG-40K), MPEG, [MPEG12, polyamino, alkyl, substituted alkyl,
radiolabeled markers,
enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g.,
aspirin, vitamin E, folic acid),
synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole
clusters, acridine-
imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl,
HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules
having a specific
affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a
specified cell type such as a
hepatic cell. Ligands can also include hormones and hormone receptors. They
can also include non-
peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors,
multivalent lactose,
monovalent or multivalent galactose, N-acetyl-galactosamine, N-acetyl-
gulucosamine multivalent
mannose, or multivalent fucose. The ligand can be, for example, a
lipopolysaccharide, an activator of
p38 MAP kinase, or an activator of NF-KB.
The ligand can be a substance, e.g., a drug, which can increase the uptake of
the iRNA agent
into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by
disrupting the cell's
microtubules, microfilaments, or intermediate filaments. The drug can be, for
example, taxon,
vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin
A, phalloidin, swinholide
A, indanocine, or myoservin.
In some embodiments, a ligand attached to an iRNA as described herein acts as
a
pharmacokinetic modulator (PK modulator). PK modulators include lipophiles,
bile acids, steroids,
phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc.
Exemplary PK
modulators include, but are not limited to, cholesterol, fatty acids, cholic
acid, lithocholic acid,
dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen,
ibuprofen, vitamin E,
biotin etc. Oligonucleotides that comprise a number of phosphorothioate
linkages are also known to
bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of
about 5 bases, 10 bases,
15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the
backbone are also
amenable to the present invention as ligands (e.g. as PK modulating ligands).
In addition, aptamers
that bind serum components (e.g. serum proteins) are also suitable for use as
PK modulating ligands
in the embodiments described herein.
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Ligand-conjugated oligonucleotides of the invention may be synthesized by the
use of an
oligonucleotide that bears a pendant reactive functionality, such as that
derived from the attachment of
a linking molecule onto the oligonucleotide (described below). This reactive
oligonucleotide may be
reacted directly with commercially-available ligands, ligands that are
synthesized bearing any of a
variety of protecting groups, or ligands that have a linking moiety attached
thereto.
The oligonucleotides used in the conjugates of the present invention may be
conveniently and
routinely made through the well-known technique of solid-phase synthesis.
Equipment for such
synthesis is sold by several vendors including, for example, Applied
Biosystems (Foster City, Calif.).
Any other means for such synthesis known in the art may additionally or
alternatively be employed. It
is also known to use similar techniques to prepare other oligonucleotides,
such as the
phosphorothioates and alkylated derivatives.
In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-
specific
linked nucleosides of the present invention, the oligonucleotides and
oligonucleosides may be
assembled on a suitable DNA synthesizer utilizing standard nucleotide or
nucleoside precursors, or
nucleotide or nucleoside conjugate precursors that already bear the linking
moiety, ligand-nucleotide
or nucleoside-conjugate precursors that already bear the ligand molecule, or
non-nucleoside ligand-
bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety,
the synthesis
of the sequence-specific linked nucleosides is typically completed, and the
ligand molecule is then
.. reacted with the linking moiety to form the ligand-conjugated
oligonucleotide. In some embodiments,
the oligonucleotides or linked nucleosides of the present invention are
synthesized by an automated
synthesizer using phosphoramidites derived from ligand-nucleoside conjugates
in addition to the
standard phosphoramidites and non-standard phosphoramidites that are
commercially available and
routinely used in oligonucleotide synthesis.
1. Lipid Conjugates
In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule.
Such a lipid or
lipid-based molecule preferably binds a serum protein, e.g., human serum
albumin (HSA). An HSA
binding ligand allows for distribution of the conjugate to a target tissue,
e.g., a non-kidney target
tissue of the body. For example, the target tissue can be the liver, including
parenchymal cells of the
liver. Other molecules that can bind HSA can also be used as ligands. For
example, naproxen or
aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance
to degradation of the
conjugate, (b) increase targeting or transport into a target cell or cell
membrane, or (c) can be used to
adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to inhibit, e.g., control the binding of the
conjugate to a
target tissue. For example, a lipid or lipid-based ligand that binds to HSA
more strongly will be less
likely to be targeted to the kidney and therefore less likely to be cleared
from the body. A lipid or
lipid-based ligand that binds to HSA less strongly can be used to target the
conjugate to the kidney.
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In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it
binds HSA with
a sufficient affinity such that the conjugate will be preferably distributed
to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that the HSA-
ligand binding cannot be
reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or
not at all, such
that the conjugate will be preferably distributed to the kidney. Other
moieties that target to kidney
cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up
by a target cell,
e.g., a proliferating cell. These are particularly useful for treating
disorders characterized by
unwanted cell proliferation, e.g., of the malignant or non-malignant type,
e.g., cancer cells.
Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins
include are B vitamin,
e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or
nutrients taken up by target cells
such as liver cells. Also included are HSA and low density lipoprotein (LDL).
2. Cell Permeation Agents
In another aspect, the ligand is a cell-permeation agent, preferably a helical
cell-permeation
agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide
such as tat or
antennopedia. If the agent is a peptide, it can be modified, including a
peptidylmimetic, invertomers,
non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical
agent is preferably an
alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred
to herein as
an oligopeptidomimetic) is a molecule capable of folding into a defined three-
dimensional structure
similar to a natural peptide. The attachment of peptide and peptidomimetics to
iRNA agents can
affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular
recognition and
absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids
long, e.g., about 5,
10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell permeation peptide,
cationic peptide,
amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of
Tyr, Trp or Phe). The
peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked
peptide. In another
alternative, the peptide moiety can include a hydrophobic membrane
translocation sequence (MTS).
An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid
sequence
AAVALLPAVLLALLAP (SEQ ID NO: 33). An RFGF analogue (e.g., amino acid sequence
AALLPVLLAAP (SEQ ID NO: 34) containing a hydrophobic MTS can also be a
targeting moiety.
The peptide moiety can be a "delivery" peptide, which can carry large polar
molecules including
peptides, oligonucleotides, and protein across cell membranes. For example,
sequences from the HIV
Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 35) and the Drosophila Antennapedia
protein
(RQIKIWFQNRRMKWKK (SEQ ID NO: 36) have been found to be capable of functioning
as
delivery peptides. A peptide or peptidomimetic can be encoded by a random
sequence of DNA, such
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as a peptide identified from a phage-display library, or one-bead-one-compound
(OBOC)
combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a
peptide or
peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for
cell targeting
purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A
peptide moiety can
range in length from about 5 amino acids to about 40 amino acids. The peptide
moieties can have a
structural modification, such as to increase stability or direct
conformational properties. Any of the
structural modifications described below can be utilized.
An RGD peptide for use in the compositions and methods of the invention may be
linear or
cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate
targeting to a specific
tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino
acids, as well as
synthetic RGD mimics. In addition to RGD, one can use other moieties that
target the integrin ligand.
Preferred conjugates of this ligand target PECAM-1 or VEGF.
A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial
cell, such as a
bacterial or fungal cell, or a mammalian cell, such as a human cell. A
microbial cell-permeating
peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or
Ceropin P1), a disulfide bond-
containing peptide (e.g., a -defensin, I3-defensin or bactenecin), or a
peptide containing only one or
two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation
peptide can also include a
nuclear localization signal (NLS). For example, a cell permeation peptide can
be a bipartite
amphipathic peptide, such as MPG, which is derived from the fusion peptide
domain of HIV-1 gp41
and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-
2724, 2003).
3. Carbohydrate Conjugates
In some embodiments of the compositions and methods of the invention, an iRNA
oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated
iRNA are
advantageous for the in vivo delivery of nucleic acids, as well as
compositions suitable for in vivo
therapeutic use, as described herein. As used herein, "carbohydrate" refers to
a compound which is
either a carbohydrate per se made up of one or more monosaccharide units
having at least 6 carbon
atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or
sulfur atom bonded to
each carbon atom; or a compound having as a part thereof a carbohydrate moiety
made up of one or
more monosaccharide units each having at least six carbon atoms (which can be
linear, branched or
cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom.
Representative
carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides
containing from about 4, 5, 6,
7, 8, or 9 monosaccharide units), and polysaccharides such as starches,
glycogen, cellulose and
polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5,
C6, C7, or C8)
sugars; di- and trisaccharides include sugars having two or three
monosaccharide units (e.g., C5, C6,
C7, or C8).
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In one embodiment, a carbohydrate conjugate for use in the compositions and
methods of the
invention is a monosaccharide. In another embodiment, a carbohydrate conjugate
for use in the
compositions and methods of the invention is selected from the group
consisting of:
OH
H0.7...._.
0 H H
HOOr.1\1,N 0
AcHN 0
HO OH
0,
0 H H
HO 0,(N,N1.0õ7"Nj
AcHN 0 0 ICI
O
HO H
0
HO 0NN0
AcHN H H
0 Formula II,
HO HO
HOT
0
HO HO H
HO1......\H
0,
0õ,...^Ø---...õ0õ..,..,,N_I---,....,0õ,...-i'l
HO HO HO CY
HOFic¨;......j4
0c)õ.0 ,N4
H Formula III,
OH
HO....\.....
0
HO 0()0
OH NHAc \----\
HO....\.... N-
0 --I
HO 0()0
NHAc Formula IV,
OH
HO,.....\.....
0
HO 00
NHAc
0
OH
H
H4HO 00.,¨/-0
NHAc Formula V,
HO OH
HO,...4.D...\ H
OrN
\
N
HO OHHAc 0
HO.-41\O1 NH/
NHAc 0 Formula VI,
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HO OH
HO OH NHAc
HOO,3
0
NHAc Ho oH
HO....\.C.)....)
NHAc Formula VII,
Bz2?_Boz
Bz0
i-----\
Bz0 __
B.z0.0Bz 0 OAc
f -0
Bz0
0 (Dlq,Formula VIII,
HC OH
.T...........\/
0
HO
H
NNy0
AcHN H 0
OH
HO
HO O ..:) N
.\/ 0
c H
NO
AcHN H T 0
OH
HO
0 0
0 oi___LI
HO-
NO
HO
AcHN H Formula IX,
O
HO H
0
HO 0c)ONO
AcHN H
OH
Ho e (=)
_c._2.\/
HO 0c)ON
AcHN H
0 0
OH
)
HO
0
0c),ON,0
HO
AcHN H Formula X,
Fi'03
H0(3)--\ - ?-1 H
___________ HO_- -' )
0
(1)Po; 1
...__Ot_Ho
HO \ - H
HO----
--\1 0
-33P
H 0
e
H c O ' ¨
0.,..,..--Ø-"-..-ON 0
H Formula XI,
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O PcT3
cHo
HO
HO
H H
P63 01,-NN)
!_?..._____!H 0
c...)._\
HO
HO 1Z)
H H
pr_ OrNN1Ø-,,µ,,,
_C2._:....OH 0 0 e
HO
0...................,...r.NNO
H H
0 Formula XII,
HO OH 0
.,r.(2.....\/0.,)i-,.. ----...--._ _ENI
HO N 0
_ ir \
AcHN H 0
HO OH 0
HO 0,=c H
m---......----.....----.....N 0.-----...----v
AcHN
H 11
0 ,----
HOv_OH 0 H 0
HO.,01¨NmNJLO---
AcHN H Formula XIII,
HO H
..:7-.._?.._\
HO\ 1.3H.f....... HO o 0
AcHN
0 0 0 -).L NH
HO
AcHN /\)LN/\/sr
H
0 Formula XIV,
HO\ __. H
HO ----r-?-o 0
HOµ 1_)_HT...... AcHN
0 0 0 =)NH
HO
AcHN
H
0 Formula XV,
HOµ _... H
H 0 -----r I-2--o 0
HO (:__Ir....-1_,....\ AcHN
u 0 0 .LNH
HO
H
0 Formula XVI,
OH
HO 1:2
"----___7...\0
OH HO 0
HO ).L
HOHO 0 0 ...r...... 0 -1\1H
HO
H
0 Formula XVII,
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OH
OH
H0 ,r2\0
HO 0
HO
'NH
HO
HO
0 Formula XVIII,
()H
OH H 1-1¨C-3--T9.-o 0
HO HO
II
HO
0 Formula XIX,
H0j-- OH
HOH-0
OH 0 0
HO
HO
0NH
HO
0 Formula XX,
HO OH
HO--11
HO
OH 0 0
HO
HO
0 NH
HO
0.LNPrPI
0 Formula XXI,
HO:-.\ 10H
HO11-10V---
OH 0 0
HO
HO
0NH
HO
0)LN'Pri
0 Formula XXII.
In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as
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HO OH
0
HO _-7O( 0
AcHN
0
HO OH
0
HO
AcHN
0 0 0
O
HO H
0
HOONNO
AcHN
0 Formula II.
Another representative carbohydrate conjugate for use in the embodiments
described herein
includes, but is not limited to,
H cp..c.TOH
HO
AcHN
H /OH 0 o
0
HO
AcHN H H
0
X0,
H /OH
0
L
0
HO
AcHN N
jcis(:0,Lo 0
0
(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a
hydrogen.
In certain embodiments of the invention, the GalNAc or GalNAc derivative is
attached to an
iRNA agent of the invention via a monovalent linker. In some embodiments, the
GalNAc or GalNAc
derivative is attached to an iRNA agent of the invention via a bivalent
linker. In yet other
embodiments of the invention, the GalNAc or GalNAc derivative is attached to
an iRNA agent of the
invention via a trivalent linker.
In one embodiment, the double stranded RNAi agents of the invention comprise
one GalNAc
or GalNAc derivative attached to the iRNA agent. In another embodiment, the
double stranded
RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6)
GalNAc or GalNAc
derivatives, each independently attached to a plurality of nucleotides of the
double stranded RNAi
agent through a plurality of monovalent linkers.
In some embodiments, for example, when the two strands of an iRNA agent of the
invention
are part of one larger molecule connected by an uninterrupted chain of
nucleotides between the 3'-end
of one strand and the 5'-end of the respective other strand forming a hairpin
loop comprising, a
plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin
loop may independently
comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The
hairpin loop may
also be formed by an extended overhang in one strand of the duplex.
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In some embodiments, the carbohydrate conjugate further comprises one or more
additional
ligands as described above, such as, but not limited to, a PK modulator or a
cell permeation peptide.
Additional carbohydrate conjugates suitable for use in the present invention
include those
described in WO 2014179620 and WO 2014179627, the entire contents of each of
which are
incorporated herein by reference.
4. Linkers
In some embodiments, the conjugate or ligand described herein can be attached
to an iRNA
oligonucleotide with various linkers that can be cleavable or non-cleavable.
The term "linker" or "linking group" means an organic moiety that connects two
parts of a
compound, e.g., covalently attaches two parts of a compound. Linkers typically
comprise a direct
bond or an atom such as oxygen or sulfur, a unit such as NR8, C(0), C(0)NH,
SO, SO2, SO2NH or a
chain of atoms, such as, but not limited to, substituted or unsubstituted
alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl,
arylalkenyl, arylalkynyl,
heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,
heterocyclylalkenyl,
heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl,
alkylarylalkyl,
alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl,
alkenylarylalkynyl,
alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,
alkylheteroarylalkyl, alkylheteroarylalkenyl,
alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl,
alkenylheteroarylalkynyl,
alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl,
alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,
alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl,
alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,
alkenylaryl, alkynylaryl,
alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more
methylenes can be
interrupted or terminated by 0, S, S(0), SO2, N(R8), C(0), substituted or
unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or unsubstituted
heterocyclic; where R8 is
hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the
linker is about 1-24 atoms,
2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16,
or 8-16 atoms.
A cleavable linking group is one which is sufficiently stable outside the
cell, but which upon
entry into a target cell is cleaved to release the two parts the linker is
holding together. In a preferred
embodiment, the cleavable linking group is cleaved at least about 10 times,
20, times, 30 times, 40
times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least
about 100 times faster in a
target cell or under a first reference condition (which can, e.g., be selected
to mimic or represent
intracellular conditions) than in the blood of a subject, or under a second
reference condition (which
can, e.g., be selected to mimic or represent conditions found in the blood or
serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox
potential or the
presence of degradative molecules. Generally, cleavage agents are more
prevalent or found at higher
levels or activities inside cells than in serum or blood. Examples of such
degradative agents include:
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redox agents which are selected for particular substrates or which have no
substrate specificity,
including, e.g., oxidative or reductive enzymes or reductive agents such as
mercaptans, present in
cells, that can degrade a redox cleavable linking group by reduction;
esterases; endosomes or agents
that can create an acidic environment, e.g., those that result in a pH of five
or lower; enzymes that can
hydrolyze or degrade an acid cleavable linking group by acting as a general
acid, peptidases (which
can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH.
The pH of
human serum is 7.4, while the average intracellular pH is slightly lower,
ranging from about 7.1-7.3.
Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have
an even more acidic
pH at around 5Ø Some linkers will have a cleavable linking group that is
cleaved at a preferred pH,
thereby releasing a cationic lipid from the ligand inside the cell, or into
the desired compartment of
the cell.
A linker can include a cleavable linking group that is cleavable by a
particular enzyme. The
type of cleavable linking group incorporated into a linker can depend on the
cell to be targeted. For
example, a liver-targeting ligand can be linked to a cationic lipid through a
linker that includes an
ester group. Liver cells are rich in esterases, and therefore the linker will
be cleaved more efficiently
in liver cells than in cell types that are not esterase-rich. Other cell-types
rich in esterases include
cells of the lung, renal cortex, and testis.
Linkers that contain peptide bonds can be used when targeting cell types rich
in peptidases,
such as liver cells and synoviocytes.
In general, the suitability of a candidate cleavable linking group can be
evaluated by testing
the ability of a degradative agent (or condition) to cleave the candidate
linking group. It will also be
desirable to also test the candidate cleavable linking group for the ability
to resist cleavage in the
blood or when in contact with other non-target tissue. Thus, one can determine
the relative
susceptibility to cleavage between a first and a second condition, where the
first is selected to be
indicative of cleavage in a target cell and the second is selected to be
indicative of cleavage in other
tissues or biological fluids, e.g., blood or serum. The evaluations can be
carried out in cell free
systems, in cells, in cell culture, in organ or tissue culture, or in whole
animals. It can be useful to
make initial evaluations in cell-free or culture conditions and to confirm by
further evaluations in
whole animals. In preferred embodiments, useful candidate compounds are
cleaved at least about 2,
4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell
(or under in vitro conditions
selected to mimic intracellular conditions) as compared to blood or serum (or
under in vitro conditions
selected to mimic extracellular conditions).
a. Redox cleavable linking groups
In one embodiment, a cleavable linking group is a redox cleavable linking
group that is
cleaved upon reduction or oxidation. An example of reductively cleavable
linking group is a
disulphide linking group (-S-S-). To determine if a candidate cleavable
linking group is a suitable
"reductively cleavable linking group," or for example is suitable for use with
a particular iRNA
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moiety and particular targeting agent one can look to methods described
herein. For example, a
candidate can be evaluated by incubation with dithiothreitol (DTT), or other
reducing agent using
reagents know in the art, which mimic the rate of cleavage which would be
observed in a cell, e.g., a
target cell. The candidates can also be evaluated under conditions which are
selected to mimic blood
or serum conditions. In one, candidate compounds are cleaved by at most about
10% in the blood. In
other embodiments, useful candidate compounds are degraded at least about 2,
4, 10, 20, 30, 40, 50,
60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro
conditions selected to mimic
intracellular conditions) as compared to blood (or under in vitro conditions
selected to mimic
extracellular conditions). The rate of cleavage of candidate compounds can be
determined using
standard enzyme kinetics assays under conditions chosen to mimic intracellular
media and compared
to conditions chosen to mimic extracellular media.
b. Phosphate-based cleavable linking groups
In another embodiment, a cleavable linker comprises a phosphate-based
cleavable linking
group. A phosphate-based cleavable linking group is cleaved by agents that
degrade or hydrolyze the
phosphate group. An example of an agent that cleaves phosphate groups in cells
are enzymes such as
phosphatases in cells. Examples of phosphate-based linking groups are -0-
P(0)(ORk)-0-, -0-
P(S)(0Rk)-0-, -0-P(S)(SRk)-0-, -S-P(0)(0Rk)-0-, -0-P(0)(0Rk)-S-, -S-P(0)(0Rk)-
S-, -0-
P(S)(ORk)-S-, -S-P(S)(ORk)-0-, -0-P(0)(Rk)-0-, -0-P(S)(Rk)-0-, -S-P(0)(Rk)-0-,
-S-P(S)(Rk)-0-,
-S-P(0)(Rk)-S-, -0-P(S)( Rk)-S-. Preferred embodiments are -0-P(0)(OH)-0-, -0-
P(S)(OH)-0-, -0-
P(S)(SH)-0-, -S-P(0)(OH)-0-, -0-P(0)(OH)-S-, -S-P(0)(OH)-S-, -0-P(S)(OH)-S-, -
S-P(S)(OH)-0-,
-0-P(0)(H)-0-, -0-P(S)(H)-0-, -S-P(0)(H)-0, -S-P(S)(H)-0-, -S-P(0)(H)-S-, -0-
P(S)(H)-S-. A
preferred embodiment is -0-P(0)(OH)-0-. These candidates can be evaluated
using methods
analogous to those described above.
c. Acid cleavable linking groups
In another embodiment, a cleavable linker comprises an acid cleavable linking
group. An
acid cleavable linking group is a linking group that is cleaved under acidic
conditions. In preferred
embodiments acid cleavable linking groups are cleaved in an acidic environment
with a pH of about
6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents
such as enzymes that can act
as a general acid. In a cell, specific low pH organelles, such as endosomes
and lysosomes can provide
a cleaving environment for acid cleavable linking groups. Examples of acid
cleavable linking groups
include but are not limited to hydrazones, esters, and esters of amino acids.
Acid cleavable groups
can have the general formula -C=NN-, C(0)0, or -0C(0). A preferred embodiment
is when the
carbon attached to the oxygen of the ester (the alkoxy group) is an aryl
group, substituted alkyl group,
or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates
can be evaluated using
methods analogous to those described above.
d. Ester-based linking groups
In another embodiment, a cleavable linker comprises an ester-based cleavable
linking group.
An ester-based cleavable linking group is cleaved by enzymes such as esterases
and amidases in cells.
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Examples of ester-based cleavable linking groups include but are not limited
to esters of alkylene,
alkenylene and alkynylene groups. Ester cleavable linking groups have the
general formula -C(0)0-,
or -0C(0)-. These candidates can be evaluated using methods analogous to those
described above.
e. Peptide-based cleaving groups
In yet another embodiment, a cleavable linker comprises a peptide-based
cleavable linking
group. A peptide-based cleavable linking group is cleaved by enzymes such as
peptidases and
proteases in cells. Peptide-based cleavable linking groups are peptide bonds
formed between amino
acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and
polypeptides. Peptide-based
cleavable groups do not include the amide group (-C(0)NH-). The amide group
can be formed
between any alkylene, alkenylene or alkynelene. A peptide bond is a special
type of amide bond
formed between amino acids to yield peptides and proteins. The peptide based
cleavage group is
generally limited to the peptide bond (i.e., the amide bond) formed between
amino acids yielding
peptides and proteins and does not include the entire amide functional group.
Peptide-based cleavable
linking groups have the general formula ¨ NHCHRAC(0)NHCHRBC(0)-, where RA and
RB are the
R groups of the two adjacent amino acids. These candidates can be evaluated
using methods
analogous to those described above.
In one embodiment, an iRNA of the invention is conjugated to a carbohydrate
through a
linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of
the compositions and
methods of the invention include, but are not limited to,
OH /OH
HO
AcHN II HO.to
cH OH 0
HO ----4===\,
0
AcHN
0 0 0
cH OH
HO
AcHN
0 (Formula XXIV),
HO\ OH
0
HO Nõ^õ,,N 0
HO,
AcHN 0
HO OH 0,
0
HO 0
AcHN 0 0 .CY
O
HO H
0
HOONNO
AcHN 0 (Formula XXV),
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HO OH
HO 0
H
...c N 0
N....,..õ---,..õ y 1.,...
AcHN H 0 X-01_
HO OH
0 0 H N
HO---r--P-_\/(k N
c ---.,-.---....-----....---
NON()1 1 \ O
AcHN H x 0 y
H 00 r
HO OH
HO ,, ,_,NNADJ y = 1-15
AcHN H (Formula XXVI),
HO OH 0
ON__T, H
N
HO0"...---..---"....- y \
AcHN H 0 X-R
HO OH 0 H 0 H N"
0 H
HO N N TO- N,Ir}l N (CD,4(nr N
AcHN
H 0 ,/ 0 H x 0 Y
HO OH
LA._:.,)._,
m N A0,- y = 1-15
HO __/ ./
AcHN H
(Formula XXVII),
HO eOH 0 H
0,1---. ... _ N 0
HO N __ __ __ y \ X-Ot
AcHN H 0
H ,O-Y
HO OH N
0
H H N,Lo
HO N NyO-N,..irHS¨S
AcHN 0 Y
H 0 0 x
HO OH x = 0-30
k-
, 0 H 0 y= 1-15
1---N m N Acy-
HO
AcHN H
(Formula XXVIII),
HO OH 0
__O/ H
01-..N---..õ---....õ---...- N yO\
HO X-R
AcHN H 0
HO OH
0
H H -(--
z 0 -)240
HO AcHN NH N y0,---N....rrHS¨S
Y
0 0 x
HO eOH x = 0-30
_________________ ,, 0 H 0 y= 1-15
k-,.)1.--N m NAG-- z = 1-20
HO--'
AcHN H
(Formula XXIX),
HO OH 0
?.s.\/ H
0-) i\J- Ny HO X-R
AcHN H 0 0, NH
H 0 ,,O-Y
HO OH N '
HO N N ...1 T0,4-1H0,/).r,,S¨S
AcHN x`' z 0 Y
0 r
HOZ H x = 1-30
0 y = 1-15
HO 0,---------1 L- ri....----....---------- NAcy--1 z = 1-20
AcHN H
(Formula XXX), and
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HO OH 0 H
HO-7-2-\=CLN y )._____. HO H X-0%
AcHN H 0
Y
H H N.4,-...yAo
HO 0 N7.N, 0-N--
_,H0.%)Ø.S----S(HThf
AcHN II Y
H N 0 i.-- 0 x z 0
HO eOH x = 1-30
= 1-15
HO INM N(`O' z =
AcHN H
(Formula XXXI),
when one of X or Y is an oligonucleotide, the other is a hydrogen.
In certain embodiments of the compositions and methods of the invention, a
ligand is one or
more "GalNAc" (N-acetylgalactosamine) derivatives attached through a bivalent
or trivalent branched
linker.
In one embodiment, a dsRNA of the invention is conjugated to a bivalent or
trivalent
branched linker selected from the group of structures shown in any of formula
(XXXII) - (XXXV):
Formula XXXII Formula XXXIII
.4. p2A_Q2A_R2A 1_2A 1-2A_L2A jp3A_Q3A_ 3A R3A1__T3A_L3A
q 41/' JUN. N q
1.,p2B_Q2B_R2B i2B -1-2B_L2B I\ p3B_Q3B_R3B I_3B T3B_L3B
q q
p5A-Q5A-R5A i_q5AT5A-L5A
p4A_Q4A_R4A 1 zi6, T4A_L4A
H:
q
p4B_Q4B_R4B 1_q4B -1-4B_L4B
1 pl5cp_5;5_cQ_R5BR5B 1_q5B
5_c1-5B_L5B
q 5T C-L5C
;
Formula XXXIV Formula XXXV
wherein:
q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for
each occurrence 0-20
and wherein the repeating unit can be the same or different;
p2A, p2B, p3A, p3B , p4A, p4B, p5A, p5B , p5C, T2A, T2B, T3A, T3B, T4A, T4B,
T4A, T5B, I,-.-,5C
are each
independently for each occurrence absent, CO, NH, 0, S, OC(0), NHC(0), CH2,
CH2NH or CH20;
Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, .-.5C
l,2 are independently for each occurrence absent, alkylene,
substituted alkylene wherin one or more methylenes can be interrupted or
terminated by one or more
of 0, S, S(0), SO2, N(RN), C(R')=C(R"), CEC or C(0);
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R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5c are each independently for each
occurrence absent, NH, 0,
0
HO¨L
H 1
S, CH, C(0)0, C(0)NH, NHCH(Ra)C(0), -C(0)-CH(Ra)-NH-, CO, CH=N-0,
0 S¨S
)_N ,N),,,, J. <¨s
H , ,,r-r'r/ \S" or
heterocyclyl;
L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5c represent the ligand; i.e. each
independently for
each occurrence a monosaccharide (such as GalNAc), disaccharide,
trisaccharide, tetrasaccharide,
oligosaccharide, or polysaccharide; andle is H or amino acid side
chain.Trivalent conjugating
GalNAc derivatives are particularly useful for use with RNAi agents for
inhibiting the expression of a
target gene, such as those of formula (XXXV):
Formula XXXV
p5A_Q5A_R5A i_T5A_L5A
'1111j-VE q5A
I p5B_Q5B_R5B 1_1-5B_L5B
q5B
Ip5C_Q5C_R5C 1771-5C_L5C
,
wherein L5A, L5B and L5c represent a monosaccharide, such as GalNAc
derivative.
Examples of suitable bivalent and trivalent branched linker groups conjugating
GalNAc
derivatives include, but are not limited to, the structures recited above as
formulas II, VII, XI, X, and
XIII.
Representative US patents that teach the preparation of RNA conjugates
include, but are not
limited to, US Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;
5,541,313; 5,545,730;
5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;
5,414,077; 5,486,603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;
4,789,737; 4,824,941;
4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;
5,082,830; 5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;
5,317,098; 5,371,241,
5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;
5,567,810; 5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and
5,688,941; 6,294,664;
6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire
contents of each of
which are hereby incorporated herein by reference.
It is not necessary for all positions in a given compound to be uniformly
modified, and in fact
more than one of the aforementioned modifications can be incorporated in a
single compound or even
at a single nucleoside within an iRNA. The present invention also includes
iRNA compounds that are
chimeric compounds.
"Chimeric" iRNA compounds or "chimeras," in the context of this invention, are
iRNA
compounds, preferably dsRNAs, which contain two or more chemically distinct
regions, each made
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up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA
compound. These iRNAs
typically contain at least one region wherein the RNA is modified so as to
confer upon the iRNA
increased resistance to nuclease degradation, increased cellular uptake, or
increased binding affinity
for the target nucleic acid. An additional region of the iRNA can serve as a
substrate for enzymes
capable of cleaving RNa:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular
endonuclease which cleaves the RNa strand of an RNA:DNA duplex. Activation of
RNase H,
therefore, results in cleavage of the RNA target, thereby greatly enhancing
the efficiency of iRNA
inhibition of gene expression. Consequently, comparable results can often be
obtained with shorter
iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs
hybridizing to
the same target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis
and, if necessary, associated nucleic acid hybridization techniques known in
the art.
In certain instances, the RNA of an iRNA can be modified by a non-ligand
group. A number
of non-ligand molecules have been conjugated to iRNAs in order to enhance the
activity, cellular
distribution or cellular uptake of the iRNA, and procedures for performing
such conjugations are
available in the scientific literature. Such non-ligand moieties have included
lipid moieties, such as
cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-
61; Letsinger et al.,
Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al.,
Bioorg. Med. Chem. Lett.,
1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann.
N.Y. Acad. Sci., 1992,
660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a
thiocholesterol (Oberhauser et
al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-
Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990,
259:327; Svinarchuk et
al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol
or triethylammonium 1,2-
di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron
Lett., 1995, 36:3651;
Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene
glycol chain (Manoharan
et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid
(Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim.
Biophys. Acta, 1995,
1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety
(Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents
that teach the
preparation of such RNA conjugates have been listed above. Typical conjugation
protocols involve
the synthesis of an RNAs bearing an aminolinker at one or more positions of
the sequence. The amino
group is then reacted with the molecule being conjugated using appropriate
coupling or activating
reagents. The conjugation reaction can be performed either with the RNA still
bound to the solid
support or following cleavage of the RNA, in solution phase. Purification of
the RNA conjugate by
HPLC typically affords the pure conjugate.
iii. Delivery of an iRNA of the Invention
The delivery of an iRNA of the invention to a cell e.g., a cell within a
subject, such as a
human subject infected with HBV can be achieved in a number of different ways.
Delivery may also
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be performed directly by administering a composition comprising an iRNA, e.g.,
a dsRNA, to a
subject. Alternatively, in vivo delivery may be performed indirectly by
administering one or more
vectors that encode and direct the expression of the iRNA. These alternatives
are discussed further
below.
In general, any method of delivering a nucleic acid molecule (in vitro or in
vivo) can be
adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian
RL. (1992) Trends Cell.
Biol. 2(5):139-144 and W09402595, which are incorporated herein by reference
in their entireties).
For in vivo delivery, factors to consider in order to deliver an iRNA molecule
include, for example,
biological stability of the delivered molecule, prevention of non-specific
effects, and accumulation of
the delivered molecule in the target tissue. For administering an iRNA
systemically for the treatment
of a disease, the RNA can be modified or alternatively delivered using a drug
delivery system; both
methods act to prevent the rapid degradation of the dsRNA by endo- and exo-
nucleases in vivo.
Modification of the RNA or the pharmaceutical carrier can also permit
targeting of the iRNA
composition to the target tissue and avoid undesirable off-target effects.
iRNA molecules can be
modified by chemical conjugation to lipophilic groups such as cholesterol to
enhance cellular uptake
and prevent degradation. In an alternative embodiment, the iRNA can be
delivered using drug
delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or
a cationic delivery
system. Positively charged cationic delivery systems facilitate binding of an
iRNA molecule
(negatively charged) and also enhance interactions at the negatively charged
cell membrane to permit
efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or
polymers can either be bound
to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim SH., et al
(2008) Journal of
Controlled Release 129(2):107-116) that encases an iRNA. The formation of
vesicles or micelles
further prevents degradation of the iRNA when administered systemically.
Methods for making and
administering cationic- iRNA complexes are well within the abilities of one
skilled in the art (see e.g.,
Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003)
Clin. Cancer Res.
9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are
incorporated herein by
reference in their entirety). Some non-limiting examples of drug delivery
systems useful for systemic
delivery of iRNAs include DOTAP (Sorensen, DR., et al (2003), supra; Verma,
UN., et al (2003),
supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS.,
et al (2006) Nature
441:111-114), cardiolipin (Chien, PY., et al (2005) Cancer Gene Ther. 12:321-
328; Pal, A., et al
(2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME., et al
(2008) Phann. Res. Aug
16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-
Gly-Asp (RGD)
peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia,
DA., et al (2007)
Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-
1804). In some
embodiments, an iRNA forms a complex with cyclodextrin for systemic
administration. Methods for
administration and pharmaceutical compositions of iRNAs and cyclodextrins can
be found in US
Patent No. 7,427,605, which is herein incorporated by reference in its
entirety. In certain
embodiments, the iRNA agents can be administered with amphipathic peptides to
facilitate pH-
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dependent endosomal escape (see, e.g., Bartz et al., 2011. Biochem. J. 435:475-
87, incorporated
herein by reference)
1. Vector encoded iRNAs for use in the Invention
iRNA targeting an HBV gene can be expressed from transcription units inserted
into DNA or
RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern,
A., et al., WO 00/22113,
WO 00/22114, and US Patent No. 6,054,299). Exemplary expression vectors for
expression of
shRNA targeted to HBV are provided in Michler et al., 2016 which discloses
adeno-associated virus
(AAV) 8 vectors for delivery included (i) embedded the shRNA in an artificial
mi(cro)RNA under a
liver-specific promoter; (ii) co-expressed Argonaute-2, a rate limiting
cellular factor whose saturation
with excess RNAi triggers can be toxic; or (iii) co-delivered a decoy ("TuD")
directed against the
shRNA sense strand to curb off-target gene regulation. The plasmids expressing
shRNAs shHBV4 to
7 that were used in the cell culture studies were cloned by direct insertion
of the respective shRNA-
encoding oligonucleotides into a self-complementary AAV vector plasmid
previously reported by
Grimm et al, 2006 (Nature 441: 537 ¨ 541), containing an H1 promoter followed
by two BbsI sites for
oligonucleotide insertion as well as an RSV promoter. Such constructs can also
be used for the
expression of vaccine antigens if appropriately sized for the expression
vector.
Expression can be transient (on the order of days to weeks) or sustained
(weeks to months, or
longer), depending upon the specific construct used and the target tissue or
cell type. These
transgenes can be introduced as a linear construct, a circular plasmid, or a
viral vector, which can be
an integrating or non-integrating vector. The transgene can also be
constructed to permit it to be
inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad.
Sci. USA (1995)
92:1292).
The individual strand or strands of an iRNA can be transcribed from a promoter
on an
expression vector. Where two separate strands are to be expressed to generate,
for example, a
dsRNA, two separate expression vectors can be co-introduced (e.g., by
transfection or infection) into
a target cell. Alternatively each individual strand of a dsRNA can be
transcribed by promoters both of
which are located on the same expression plasmid. In one embodiment, a dsRNA
is expressed as
inverted repeat polynucleotides joined by a linker polynucleotide sequence
such that the dsRNA has a
stem and loop structure.
iRNA expression vectors are generally DNA plasmids or viral vectors.
Expression vectors
compatible with eukaryotic cells, preferably those compatible with vertebrate
cells, can be used to
produce recombinant constructs for the expression of an iRNA as described
herein. Eukaryotic cell
expression vectors are well known in the art and are available from a number
of commercial sources.
Typically, such vectors are provided containing convenient restriction sites
for insertion of the desired
nucleic acid segment. Delivery of iRNA expressing vectors can be systemic,
such as by
subcutaneous, intravenous, or intramuscular administration. Such vectors can
also be used for
expression of viral antigens from nucleic acid-based vaccines.
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Viral vector systems which can be utilized with the methods and compositions
described
herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus
vectors, including but not
limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-
associated virus vectors;
(d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus
vectors; (g) papilloma virus
vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox,
e.g., vaccinia virus vectors
including modified vaccinia virus Ankara vector or avipox, e.g. canary pox or
fowl pox; and (j) a
helper-dependent or gutless adenovirus. Replication-defective viruses can also
be advantageous.
Different vectors will or will not become incorporated into the cells' genome.
The constructs can
include viral sequences for transfection, if desired. Alternatively, the
construct can be incorporated
into vectors capable of episomal replication, e.g. EPV and EBV vectors.
Constructs for the
recombinant expression of an iRNA or HBV antigen will generally require
regulatory elements, e.g.,
promoters, enhancers, etc., to ensure the expression of the iRNA in target
cells. Other aspects to
consider for vectors and constructs are further described below.
Such constructs and vectors can also be used for the expression of vaccine
antigens if
appropriately sized for the expression vector. Such limitations are well
understood by those of skill in
the art.
B. Therapeutic HBV Vaccines For Use in the Mehods of the Invention
Therapeutic HBV vaccines for use in the regimens and methods of the invention
can be a
peptide vaccine, a DNA vaccine including a vector-based vaccine, or cell-based
vaccine that induces
an immune response, preferably an effector T cell induced response, against
one or more HBV
proteins. Preferably the vaccine is a multi-epitope vaccine that is cross-
reactive with multiple HBV
serotypes, preferably all HBV serotypes.
A therapeutic vaccine is designed to activate the patient's immune system to
recognize and
control or eliminate an already established pathogen infection. This is
clearly distinct from a
prophylactic vaccination which is designed to promote rapid antibody-mediated
neutralization of an
invading pathogen. Control and elimination of persistent viruses such as
hepatitis, herpes, or
papilloma viruses requires multi-specific and poly-functional effector T cell
responses. These T cell
responses are ideally directed against continuously expressed viral antigens
to keep the pathogen in
check. Therapeutic vaccines are under development for a number of chronic
infections. Hepatitis B
virus infection is a candidate for treatment by therapeutic vaccination since
a spontaneous, immune-
mediated recovery of chronic hepatitis B and an elimination of the virus has
been observed in very
rare cases.
Robust T cell responses seem to be essential to achieve HBV cure. While HBV-
specific
CD4+ and CD8+ T cell responses are readily detectable in patients resolving
HBV infection, HBV-
specific T cells are scarce and functionally impaired in chronic hepatitis B
most likely due to high
amounts of circulating viral HBeAg and HBsAg. T cells eliminate HBV infected
cells by their
cytotoxic activity but also control HBV gene expression and replication in a
non-cytolytic fashion.
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To overcome immune tolerance in chronic hepatitis B different approaches have
been
investigated in preclinical models using DNA or peptide vaccines, or vector-
or cell-based vaccines to
induce an effector T cell response. Multi-epitope therapeutic vaccine
candidates that cover sufficient
different HBV genotypes and most frequent HLA types have been developed.
Although proper
peptide presentation was demonstrated, immunogenicity was limited and the
approach was not
translated into the clinics. A non-exhaustive list is provided in the table
below.
Therapeutic HBV Vaccine Tables
0
Vaccine Name Proteins/ coding Vaccine type/ Results available
Sponsor Development Clinical Trial References
oe
sequences Composition
Stage Reference No. (the entire contents of
each of which are
incorporated herein by
reference)
HB-110 HBsAgõ HBcAg, 3 Plasmid based Study completed,
Genexine Phase I NCT01813487 Kim et al., 2008. Exp Mol
human IL-12 DNA vaccine results not reported
NCT01641536 Med. 40: 669-676
NCT00513968
cs,
cs, HB-100 pGX10 S 5 Plasmid based
Yang et al., 2006. Gene
+pGX10 S1/52/X DNA vaccine ¨4
Therapy. 13:1110-1117
+pGX10 core antigens + human
+pGX10 Pol IL-12
+pGX10 hIL-
12N222L
ppdpSC18 DNA vaccine Study completed,
PowderMed Phase I NCT00277576
adjuvanted with results not reported
particle mediated
epidermal delivery
oe
INO-1800 HbsAg, HBcAg DNA plasmid Recruiting Inovio
Phase I NCT02431312 -a 5
oe
Pharmaceuticals
cr
ME1 27037625v.1
HB02 VAC- HB preS/S pCMV-S2.S DNA Well tolerated; No ANRS
Phase I/II NCT00536627 Mancini ¨ Bourgine et al.,
ADN vaccine; CMV change in relapse
2006. Vaccine 24:4482- 0
promoter, plasmid rate in HBV treated
4489
oe
vector patients or
decrease in
virological
breakthrough
CVI-HBV-002 HbsAg DNA+ L-pampo Recruiting CHA Vaccine
Phase I/II NCT02693652
Institute Co.,
Ltd.
Theravax (DV- HBsAg, HBcAg Protein + adjuvant Well tolerated;
Dynavax Phase Ib NCT01023230
601) anti-viral response
Technologies
cs,
observed in all Corp.
patients
HepTcellTm IC310 adjuvanted Recruiting Altimmune,
Phase I NCT02496897 US 20130330382
peptide Inc.
US 20120276138
US 20150216967
HBsAg/HBcAg HBsAg protein
Research Li et al., 2015, Vaccine.
HBcAg
33:4247-4254
GS-4774 Fusion protein Protein + Phase II naïve
Gilead Phase II NCT01943799 Gaggar et al., 2014.
HBsAg, HBcAg, Tamogen T cell group ongoing; no
NCT01779505 Vaccine. 32:4925-4931.
HBxAg stimulator significant viral
NCT02174276
oe
decrease in
oe
treatment-
ME1 27037625v.1
experience patients
EPA-44 Multi-peptide Some Chongqing
Phase II NCT00869778 0
vaccine seroconversion J aichen
NCT02862106
observed in all Biotechnology,
groups, no Ltd.
statistical analysis
ABX 203 HBsAg, HBcAg HBsAg, HBcAg Ongoing ABIVAX S.A.
Phase II/III NCT02249988
pSG2.HBs/ Protein prime- Well tolerated, but Oxxon
Phase ha ISRCTN Cavenaugh et al., 2011.
MVA.HBs viral vector boost did not control
Therapeutics 67270384 PLoS ONE 6: e14626.
HBV infection
HBsAg, HBcAg Adjuvanted
Research Backes, 2016, Vaccine;
protein prime-
W02017121791
OC cs,
viral vector boost
00
00
ME1 27037625v.1
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Existing prophylactic vaccines have been used to restore HBV-specific immunity
in
chronically infected patients, but have failed to provide a functional cure.
These subviral particle-
based vaccines were able to reduce HBV replication in animal models of chronic
hepadnaviral
infection, but have not been successful in patients with chronic hepatitis B.
An antigen-antibody
(HBsAg-HBIG) immunogenic complex therapeutic vaccine candidate with alum as
adjuvant first
showed promising results in a double-blind, placebo controlled, phase IIb
clinical trial, but results of a
phase III clinical trial including 450 patients were disappointing. This is
most likely due to the fact
that subviral particle vaccines with alum-based adjuvants are designed as
prophylactic vaccines and
preferentially induce antibodies but not cytotoxic T cell responses that would
be required for
therapeutic efficacy.
Alternatively, DNA vaccines encoding HBV envelope proteins were designed to
induce
HBV-specific T cells but also had limited success. Since DNA-based vaccines
hardly induce antibody
responses, they failed to achieve HBeAg or HBsAg seroconversion. An
alternative DNA prime,
poxvirus-boost vaccine encompassing the HBV preS/S region encoding for the HBV
envelope
.. proteins showed promising results in chimpanzees, but also failed in a
clinical phase Ha trial neither
inducing sustained T cell responses nor reducing viremia in chronic HBV
carriers. They may have
failed to induce T cell help or broad enough, multi-specific immune responses.
Any vaccines known in the art can be used in the methods and regimens provided
herein.
Preferred embodiments include the prime-boost vaccination scheme with protein
antigens
administered twice and a nucleic acid vaccine administered once as provided in
the Examples and
provided in PCT Publication No. WO 2017/121791 (the entire contents of which
are incorporated
herein by reference). As discussed above, the sequence of the protein antigen
in the vaccine and the
amino acid sequence encoded by nucleic acid-based vaccine need not be
identical. They simply must
share at least one epitope, preferably multiple epitopes, so that the
sequential administration of the
vaccines has the desired prime-boost effect. Further, as discussed herein, in
preferred embodiments,
the treatment regimen provided herein would provide a treatment regimen
effective across multiple, if
not all, HBV serotypes. Therefore, it is understood that the antigen delivered
to the subject may not
have antigen sequences identical to the antigens expressed by the HBV virus
that infected the subject.
It is known in the art that some portions of HBV antigens are the main targets
of antibodies
generated during the initial immune response to infection with HBV known as
determinants. For
example the HBsAg includes the "a" determinant epitope that is located at
amino acids 124 to 147
within the major hydrophilic region (MHR, amino acids 100 to 169) of the 226
amino acid S gene
(SEQ ID NO: 8 or 22). This "a" determinant is one of the main targets of anti-
HBs antibodies during
the course of the initial immune response in acute hepatitis B. In certain
embodiments, an
immunogenic fragment of HBsAg comprises compared to the full length protein at
least amino acids
99 to 168 corresponding to the amino acid positons of the small envelope
protein (SEQ ID NO: 23)
(see, e.g., Lada et al., J. Virol.(2006) 80:2968-2975, the entire contents of
which are incorporated
herein by reference).
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Similarly, determinants have been identified in HBcAg (see, e.g., Salfeld et
al., J. Virol.
(1989) 63:798-808, the entire contents of which are incorporated herein by
reference). The full-length
core protein is 183 amino acids in length and consists of an assembly domain
(amino acids 1 to 149)
and a nucleic acid-binding domain (amino acids 150 to 183). Three distinct
major determinants have
been characterized. The single conformational determinant responsible for HBc
antigenicity in the
assembled core (HBc) and a linear HBe-related determinant (HBel) were both
mapped to an
overlapping hydrophilic sequence around amino acid 80; a second HBe
determinant (HBe2) was
assigned to a location in the vicinity of amino acid 138 but found to require
for its antigenicity the
intramolecular participation of the extended sequence between amino acids 10
and 140. Typically,
such an immunogenic fragment comprises, compared to the full length core
protein, at least amino
acids 18 to 143 corresponding to the sequence positions set forth in SEQ ID
NO: 24. Analogous
sequences can be identified in SEQ ID NO: 10.
In preferred embodiments, the vaccines include an amino acid sequence or
encode an amino
acid sequence that includes at least one determinant of HBsAg or HBcAg.
Specifically, in certain
embodiments, a vaccine targeted to HBsAg includes at least the amino acid
sequence of amino acids
127 to 147 of HBsAg, e.g., includes at least amino acids 99 to 168 of the
amino acid sequence of the
small envelope protein (SEQ ID NO: 23). In certain embodiments, a vaccine
targeted to a hydrophilic
sequence at least around amino acid 80 of HBcAg or an amino acid sequence at
least around amino
acid 138, e.g., at least a 40 amino acid portion, at least a 50 amino acid
portion, at least a 60 amino
acid portion, at least a 70 amino acid portion, at least an 80 amino acid
portion, at least a 90 amino
acid portion, or at least a 100 amino acid portion of SEQ ID NO: 43 including
amino acid 80 or amino
acid 138, or a coding sequence therefor. In preferred embodiments, the antigen
amino sequence of the
antigen targeted to HBcAg includes at least 20 amino acids N-terminal and C-
terminal to amino acid
80 or 138 of SEQ ID NO: 24. In certain embodiments, a vaccine targeted to
HBcAg includes at least
the amino acid sequence of amino acids 10 to 140 or 18 to 143 of HBsAg.
In certain embodiments, the vaccine may comprise the entire amino acid
sequence or encode
the entire amino acid sequence of any one or more of SEQ ID NO: 22, 23, or 24.
It is understood that there are multiple serotypes of HBV with different
nucleic acid
sequences that encode different amino acid sequences. Therefore, it is
understood that the amino acid
sequence of a protein-based vaccine or the amino acid sequence encoded by a
nucleic acid-based
vaccine may not be 100% identical to the sequences provided in the SEQ ID NOs.
In certain
embodiments of the invention, the the amino acid sequence of a protein-based
vaccine or the amino
acid sequence encoded by a nucleic acid-based vaccine is at least 90%
identical, at least 91%
identical, at least 92% identical, at least 93% identical, at least 94%
identical, at least 95% identical, at
least 96% identical, at least 97% identical, or at least 98% identical to the
portion of SEQ ID NO: 22,
23, or 24. Additional exemplary HBsAg and HBcAg amino acid sequenences are
provided in the
sequence listing. Alignment methods to identify appropriate sequences
corresponding to the HBsAg
and HBcAg determinants in the sequences indicated above are known in the art.
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The vaccine preferably comprises MVA viruses in a concentration range of 104
to 109 tissue-
culture infectious dose (TCID)50/ml, preferably in a concentration range of
105 to 5x108TCID50/ml,
more preferably in a concentration range of 106 to 108 TCID50/ml, and most
preferably in a
concentration range of 107 to 108 TCID50/ml.
A preferred vaccination dose for humans comprises 106 to 109 TCID50, most
preferably a dose
of 106 TCID50 or 107 TCID50 or 108 TCID50.
The preferred methods of the invention include administration of both a
protein-based vaccine
and a nucleic acid-based vaccine. However, other methods include
administration of only protein
antigens. Less preferred embodiments include administration of only nucleic
acids encoding antigens.
i. Adjuvants
As used herein "adjuvant" is understood as an agent that promotes (e.g.,
enhances,
accelerates, or prolongs) an immune response to an antigen with which it is
administered to elicit
long-term protective immunity. No substantial immune response is directed at
the adjuvant itself.
Adjuvants include, but are not limited to, pathogen components, particulate
adjuvants, and
combination adjuvants (see, e.g., www.niaid.nih.gov/research/vaccine-adjuvants-
types). Pathogen
components (e.g., monophosphoryl lipid A (MPL), poly(I:C), CpG DNA, emulsions
such as
poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP)) can help trigger
early non-specific,
or innate, immune responses to vaccines by targeting various receptors inside
or on the surface of
innate immune cells. The innate immune system influences adaptive immune
responses, which
provide long-lasting protection against the pathogen that the vaccine targets.
Particulate adjuvants
(e.g., alum, virosomes, cytokines, e.g., IL-12) form very small particles that
can stimulate the immune
system and also may enhance delivery of antigen to immune cells. Combination
adjuvants (e.g.,
AS02, AS03 and AS04 (GSK); MF59 (Novartis); and IC31 (Altimmune) elicit
multiple protective
immune responses. Adjuvants that have a modest effect when used alone may
induce a more potent
immune response when used together. In certain embodiments, preferred
adjuvants include c-di-
AMP, c-di-GMP, c-di-CMP, PolyICLC, CpG, ISCOMATRIX , AS02, AS03, AS04, or a
RIG-I
ligand such as 5' 3P-RNA. In certain embodiments, a viral capsid, with or
without a nucleic acid
expressing an HBV antigen can be used as an adjuvant. For example, a vaccine
that preferentially
.. stimulates T cells such as an MVA-only or a DNA prime, MVA boost or an
adenovirus vector prime-
MVA boost can be used in the methods of the invention.
In preferred embodiments of the invention, adjuvants for use in the invention
promote a
humoral and a cellular immune response as discussed above. In certain
embodiments, adjuvants
provide a balanced Th1/Th2 response.
ii. Non-adjuvant immune stimulators and additional agents
Methods of the invention can further include administration of additional
agents used in the
treatment of HBV or to stimulate an immune response. Such agents can include
an immune
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stimulator (e.g., pegylated interferon alfa 2a (PEG-IFN-a2a), Interferon alfa-
2b, a recombinant human
interleukin-7, and aToll-like receptor 3, 7, 8, or 9 (TLR3, TLR7, TLR8, or
TLR9) agonist), a viral
entry inhibitor (e.g., Myrcludex), an oligonucleotide that inhibits the
secretion or release of HBsAg
(e.g., REP 9AC), a capsid inhibitor (e.g., Bay41-4109 and NVR-1221), a cccDNA
inhibitor (e.g.,
IHVR-25), a Rig-I-ligand, a STING agonist, an antibody based immune therapy
against HBV (mono-,
bi-, or trispecific antibody against HBV), or an immune checkpoint regulator.
Such agents are known
in the art.
C. Nucleotide and Nucleoside Analogs For Use in the Methods of the Invention
Nucleotide and nucleoside analogs are considered to be the standard of care
for HBV
infection as they are generally considered safe and inexpensive. However,
nucleotide and nucleoside
analogs cannot cure HBV infection, may cause the development of resistance,
and must be taken
indefinitely. Nucleotide analog and nucleoside analogs include, but are not
limited to, Tenofovir
disoproxil fumarate (TDF), Tenofovir alafenamide, Lamivudine, Adefovir
dipivoxil, Entecavir
(ETV), Telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil,
lobucavir, famvir,
FTC, N-Acetyl-Cysteine (NAC), PC1323, theradigm-HBV, thymosin-alpha,
ganciclovir, Besifovir
(ANA-380/LB-80380), and Tenofvir-Exalidex (TXL/CMX157) . In certain
embodiments, the
nucelot(s)ide analog is Entecavir (ETV) or Tenofovir or a derivative thereof.
In certain embodiments,
the nucleot(s)ide analog is not Lamivudine. Nucleot(s)ide analogs are
commercially available from a
number of sources and are used in the methods provided herein according to
their label indication
(e.g., typically orally administered at a specific dose) or as determined by a
skilled practitioner in the
treatment of HBV.
III. Antisense Oligonucleotides Targeting HBV
The present invention includes the use of iRNAs which promote cleavage of at
least one HBV
transcript, and preferably three or four HBV transcripts. Antisense
oligonucleotides can similarly be
used to promote cleavage of at least one HBV transcript, preferably three or
four HBV transcripts, in
the methods of the invention provided here. Exemplary antisense
oligonucleotides targeted to HBV
are provided, for example, in U.S. Patent Publication Nos. 2013/0035366,
2012/0207709, and
2004/0127446, the contents of each of which is incorporated by reference
herein in its entirety.
It is understood by those of skill in the art that conjugates, linkers, and
formulations for the
delivery of siRNAs as provided above can be used for the formulation and
delivery of antisense
oligonucleotide therapeutic agents to subjects.
IV. Pharmaceutical Compositions of the Invention
The present invention also includes pharmaceutical compositions and
formulations which
include the iRNAs or vaccines for use in the invention. It is understood that
approved therapeutic
agents are formulated and administered by the route indicated on their package
instructions.
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In some embodiments, provided herein are pharmaceutical compositions
containing an iRNA
or a vaccine, as described herein, and a pharmaceutically acceptable carrier.
The pharmaceutical
compositions containing the iRNA or vaccine are useful for treating subject
with an HBV infection.
Such pharmaceutical compositions are formulated based on the mode of delivery,
e.g., for systemic
administration via parenteral delivery, e.g., by subcutaneous (SC),
intramuscular (IM), or intravenous
(IV) delivery.
The pharmaceutical RNAi compositions for use in the invention may be
administered in
dosages sufficient to significantly reduce the level of at least one HBV
transcript. In general, a
suitable dose of an iRNA of the invention will be in the range of about 0.001
to about 200.0
milligrams per kilogram body weight of the recipient per day, generally in the
range of about 1 to 50
mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of
the invention will be
in the range of about 0.1 mg/kg to about 5.0 mg/kg, preferably about 0.3 mg/kg
and about 3.0 mg/kg.
A repeat-dose regimen may include administration of a therapeutic amount of
iRNA on a regular
basis, such as every other week or once a year. In certain embodiments, the
iRNA is administered
about once per month to about once per quarter (i.e., about once every three
months). In preferred
embodiments, the RNAi agent is administered subcutaneously.
Formulation and dosing of the vaccine will depend on the nature of the vaccine
administered.
In a protein prime-expression vector boost vaccination strategy, the protein-
based priming vaccines
are administered about 2, 3, or 4 weeks apart with the expression vector
vaccine boost being
administered about 2, 3, or 4 weeks after the second protein-based vaccine
dose. In certain
embodiments, it is about two weeks between the first and second doses of the
protein-based vaccine.
In certain embodiments, it is about two weeks between the second dose of the
protein based vaccine
and the DNA expression vector vaccine boost. In certain embodiments, the prime
and boost
vaccinations are administered by routes independently selected from
intramuscularly, intradermally,
or subcutaneously.
The pharmaceutical nucleic acid-based vaccines for use in the invention may be
administered
in dosages sufficient to promote an immune response, as either a prime agent
or a boost agent. The
amount of nucleic acid-based vaccine to be administered will depend, for
example, on the design of
the vaccine. As the regimens provided herein can include the use of existing
nucleic acid-based
vaccines, knowledge regarding appropriate dosages based on therapeutic
efficacy and safety should be
based on the specific agent used.
The pharmaceutical protein-based vaccines for use in the invention may be
administered in
dosages sufficient to promote an immune response, as either a prime or a boost
agent. The amount of
protein-based vaccine to be administered will depend, for example, on the
adjuvant used. Protein-
based vaccines can be dosed, for example, at about 5-100 mg/kg/dose, about 10-
50 mg/kg/dose, or
about 20-40 mg/kg/dose. As the regimens provided herein can include the use of
existing protein-
based vaccines, knowledge regarding appropriate dosages based on therapeutic
efficacy and safety
should be based on the specific agent used.
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After an initial treatment regimen, the treatments can be administered on a
less frequent basis.
In preferred embodiments, the treatment regimens and methods provided herein
result in a functional
cure allowing for discontinuation of treatment after completion of the regimen
or after diagnostic
criteria indicate a functional cure, e.g., decreased HBsAg levels preferably
to below the level of
detection of the methods provided herein and a detectable immune response to
HBsAg.
The skilled artisan will appreciate that certain factors can 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 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 individual therapeutic agent used in the methods and regimens invention
can be made using
conventional methodologies or on the basis of in vivo testing using an
appropriate animal model, as
described elsewhere herein and known in the art.
A. iRNA Formulations Comprising Membranous Molecular Assemblies
An iRNA for use in the compositions and methods of the invention can be
formulated for
delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As
used herein, the term
"liposome" refers to a vesicle composed of amphiphilic lipids arranged in at
least one bilayer, e.g.,
one bilayer or a plurality of bilayers. Liposomes include unilamellar and
multilamellar vesicles that
have a membrane formed from a lipophilic material and an aqueous interior. The
aqueous portion
contains the iRNA composition. The lipophilic material isolates the aqueous
interior from an aqueous
exterior, which typically does not include the iRNA composition, although in
some examples, it may.
Liposomes are useful for the transfer and delivery of active ingredients to
the site of action. Because
the liposomal membrane is structurally similar to biological membranes, when
liposomes are applied
to a tissue, the liposomal bilayer fuses with bilayer of the cellular
membranes. As the merging of the
liposome and cell progresses, the internal aqueous contents that include the
iRNA are delivered into
the cell where the iRNA can specifically bind to a target RNA and can mediate
iRNA. In some cases
the liposomes are also specifically targeted, e.g., to direct the iRNA to
particular cell types.
A liposome containing an iRNA agent can be prepared by a variety of methods.
In one
example, the lipid component of a liposome is dissolved in a detergent so that
micelles are formed
with the lipid component. For example, the lipid component can be an
amphipathic cationic lipid or
lipid conjugate. The detergent can have a high critical micelle concentration
and may be nonionic.
Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and
lauroyl sarcosine.
The iRNA agent preparation is then added to the micelles that include the
lipid component. The
cationic groups on the lipid interact with the iRNA agent and condense around
the iRNA agent to
form a liposome. After condensation, the detergent is removed, e.g., by
dialysis, to yield a liposomal
preparation of the iRNA agent.
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If necessary a carrier compound that assists in condensation can be added
during the
condensation reaction, e.g., by controlled addition. For example, the carrier
compound can be a
polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also
adjusted to favor
condensation.
Methods for producing stable polynucleotide delivery vehicles, which
incorporate a
polynucleotide/cationic lipid complex as structural components of the delivery
vehicle, are further
described in, e.g., WO 9637194, the entire contents of which are incorporated
herein by reference.
Liposome formation can also include one or more aspects of exemplary methods
described in Felgner,
P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; US Patent No.
4,897,355; US Patent No.
5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.
Biophys. Acta 557:9,
1979; Szoka, et al. Proc. Natl. Acad. Sci. USA 75: 4194, 1978; Mayhew, et al.
Biochim. Biophys. Acta
775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga,
et al. Endocrinol.
115:757, 1984. Commonly used techniques for preparing lipid aggregates of
appropriate size for use
as delivery vehicles include sonication and freeze-thaw plus extrusion (see,
e.g., Mayer, et al.
Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when
consistently small (50 to
200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim.
Biophys. Acta
775:169, 1984). These methods are readily adapted to packaging iRNA agent
preparations into
liposomes.
Liposomes fall into two broad classes. Cationic liposomes are positively
charged liposomes
which interact with the negatively charged nucleic acid molecules to form a
stable complex. The
positively charged nucleic acid/liposome complex binds to the negatively
charged cell surface and is
internalized in an endosome. Due to the acidic pH within the endosome, the
liposomes are ruptured,
releasing their contents into the cell cytoplasm (Wang et al., Biochem.
Biophys. Res. Commun., 1987,
147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids
rather than
complex with it. Since both the nucleic acid and the lipid are similarly
charged, repulsion rather than
complex formation occurs. Nevertheless, some nucleic acid is entrapped within
the aqueous interior of
these liposomes. pH-sensitive liposomes have been used to deliver nucleic
acids encoding the
thymidine kinase gene to cell monolayers in culture. Expression of the
exogenous gene was detected
in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-
274).
One major type of liposomal composition includes phospholipids other than
naturally-derived
phosphatidylcholine. Neutral liposome compositions, for example, can be formed
from dimyristoyl
phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic
liposome
compositions generally are formed from dimyristoyl phosphatidylglycerol, while
anionic fusogenic
liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
Another type of
liposomal composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC
and egg PC. Another type is formed from mixtures of phospholipid or
phosphatidylcholine or
cholesterol.
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Examples of other methods to introduce liposomes into cells in vitro and in
vivo include US
Patent Nos. 5,283,185 and 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024;
Felgner, J. Biol.
Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. USA 90:11307, 1993; Nabel,
Human Gene Ther.
3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO / 11:417, 1992.
Non-ionic liposomal systems have also been examined to determine their utility
in the
delivery of drugs to the skin, in particular systems comprising non-ionic
surfactant and cholesterol.
Non-ionic liposomal formulations comprising NovasomeTm I (glyceryl
dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeTm II
(glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver
cyclosporin-A into the
dermis of mouse skin. Results indicated that such non-ionic liposomal systems
were effective in
facilitating the deposition of cyclosporine A into different layers of the
skin (Hu et al. S.T.P.Phanna.
Sci., 1994, 4(6) 466).
Liposomes also include "sterically stabilized" liposomes, a term which, as
used herein, refers
to liposomes comprising one or more specialized lipids that, when incorporated
into liposomes, result
in enhanced circulation lifetimes relative to liposomes lacking such
specialized lipids. Examples of
sterically stabilized liposomes are those in which part of the vesicle-forming
lipid portion of the
liposome (A) comprises one or more glycolipids, such as monosialoganglioside
Gmi, or (B) is
derivatized with one or more hydrophilic polymers, such as a polyethylene
glycol (PEG) moiety.
While not wishing to be bound by any particular theory, it is thought in the
art that, at least for
sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-
derivatized lipids, the
enhanced circulation half-life of these sterically stabilized liposomes
derives from a reduced uptake
into cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et
al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art.
Papahadjopoulos
et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of
monosialoganglioside Gmi,
galactocerebroside sulfate and phosphatidylinositol to improve blood half-
lives of liposomes. These
findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA,
1988, 85, 6949). US
Patent No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes
comprising (1)
sphingomyelin and (2) the ganglioside Gmi or a galactocerebroside sulfate
ester. US Patent No.
5,543,152 discloses liposomes comprising sphingomyelin. Liposomes comprising
1,2-sn-
dimyristoylphosphatidylcholine are disclosed in WO 9713499 (Lim et al).
Cationic liposomes possess the advantage of being able to fuse to the cell
membrane. Non-
cationic liposomes, although not able to fuse as efficiently with the plasma
membrane, are taken up by
macrophages in vivo and can be used to deliver iRNA agents to macrophages.
Further advantages of liposomes include: liposomes obtained from natural
phospholipids are
biocompatible and biodegradable; liposomes can incorporate a wide range of
water and lipid soluble
drugs; liposomes can protect encapsulated iRNA agents in their internal
compartments from
metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms,"
Lieberman, Rieger and
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Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the
preparation of liposome
formulations are the lipid surface charge, vesicle size and the aqueous volume
of the liposomes.
A positively charged synthetic cationic lipid, N41-(2,3-dioleyloxy)propy1]-
N,N,N-
trimethylammonium chloride (DOTMA) can be used to form small liposomes that
interact
spontaneously with nucleic acid to form lipid-nucleic acid complexes which are
capable of fusing
with the negatively charged lipids of the cell membranes of tissue culture
cells, resulting in delivery of
iRNA agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA
8:7413-7417, 1987 and US
Patent No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can
be
used in combination with a phospholipid to form DNA-complexing vesicles.
LipofectinTM Bethesda
Research Laboratories, Gaithersburg, Md.) is an effective agent for the
delivery of highly anionic
nucleic acids into living tissue culture cells that comprise positively
charged DOTMA liposomes
which interact spontaneously with negatively charged polynucleotides to form
complexes. When
enough positively charged liposomes are used, the net charge on the resulting
complexes is also
positive. Positively charged complexes prepared in this way spontaneously
attach to negatively
charged cell surfaces, fuse with the plasma membrane, and efficiently deliver
functional nucleic acids
into, for example, tissue culture cells. Another commercially available
cationic lipid, 1,2-
bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim,
Indianapolis,
Indiana) differs from DOTMA in that the oleoyl moieties are linked by ester,
rather than ether
linkages.
Other reported cationic lipid compounds include those that have been
conjugated to a variety
of moieties including, for example, carboxyspermine which has been conjugated
to one of two types
of lipids and includes compounds such as 5-carboxyspermylglycine
dioctaoleoylamide ("DOGS")
(TransfectamTm, Promega, Madison, Wisconsin) and
dipalmitoylphosphatidylethanolamine 5-
carboxyspermyl-amide ("DPPES") (see, e.g., US Patent No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with
cholesterol ("DC-
Chol") which has been formulated into liposomes in combination with DOPE (See,
Gao, X. and
Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made
by conjugating
polylysine to DOPE, has been reported to be effective for transfection in the
presence of serum (Zhou,
X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these
liposomes containing
conjugated cationic lipids, are said to exhibit lower toxicity and provide
more efficient transfection
than the DOTMA-containing compositions. Other commercially available cationic
lipid products
include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine
(DOSPA) (Life
Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for
the delivery of
oligonucleotides are described in WO 98/39359 and WO 96/37194.
Liposomal formulations are particularly suited for topical administration.
Liposomes present
several advantages over other formulations. Such advantages include reduced
side effects related to
high systemic absorption of the administered drug, increased accumulation of
the administered drug at
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the desired target, and the ability to administer iRNA agent into the skin. In
some implementations,
liposomes are used for delivering iRNA agent to epidermal cells and also to
enhance the penetration
of iRNA agent into dermal tissues, e.g., into skin. For example, the liposomes
can be applied
topically. Topical delivery of drugs formulated as liposomes to the skin has
been documented (see,
e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du
Plessis et al., Antiviral
Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S.,
Biotechniques 6:682-690, 1988;
Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-
176, 1987; Straubinger,
R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and
Huang, L., Proc.
Natl. Acad. Sci. USA 84:7851-7855, 1987).
Liposomes that include iRNA can be made highly deformable. Such deformability
can enable
the liposomes to penetrate through pore that are smaller than the average
radius of the liposome. For
example, transfersomes are a type of deformable liposomes. Transferosomes can
be made by adding
surface edge activators, usually surfactants, to a standard liposomal
composition. Transfersomes that
include iRNA agent can be delivered, for example, subcutaneously by infection
in order to deliver
iRNA agent to keratinocytes in the skin. In order to cross intact mammalian
skin, lipid vesicles must
pass through a series of fine pores, each with a diameter less than 50 nm,
under the influence of a
suitable transdermal gradient. In addition, due to the lipid properties, these
transferosomes can be
self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-
repairing, and can frequently
reach their targets without fragmenting, and often self-loading.
Other formulations amenable to the present invention are described in WO
2008042973.
Surfactants find wide application in formulations such as emulsions (including
microemulsions) and liposomes. The most common way of classifying and ranking
the properties of
the many different types of surfactants, both natural and synthetic, is by the
use of the
hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also
known as the "head")
provides the most useful means for categorizing the different surfactants used
in formulations (Rieger,
in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, N.Y., 1988,
p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic
surfactant. Nonionic
surfactants find wide application in pharmaceutical and cosmetic products and
are usable over a wide
range of pH values. In general their HLB values range from 2 to about 18
depending on their
structure. Nonionic surfactants include nonionic esters such as ethylene
glycol esters, propylene
glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose
esters, and ethoxylated
esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols,
and ethoxylated/propoxylated block polymers are also included in this class.
The polyoxyethylene
surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or
dispersed in water,
the surfactant is classified as anionic. Anionic surfactants include
carboxylates such as soaps, acyl
lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl
sulfates and ethoxylated
alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl
isethionates, acyl taurates and
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sulfosuccinates, and phosphates. The most important members of the anionic
surfactant class are the
alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or
dispersed in water,
the surfactant is classified as cationic. Cationic surfactants include
quaternary ammonium salts and
ethoxylated amines. The quaternary ammonium salts are the most used members of
this class.
If the surfactant molecule has the ability to carry either a positive or
negative charge, the
surfactant is classified as amphoteric. Amphoteric surfactants include acrylic
acid derivatives,
substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has
been reviewed
(Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York,
N.Y., 1988, p. 285).
The iRNA for use in the methods of the invention can also be provided as
micellar
formulations. "Micelles" are defined herein as a particular type of molecular
assembly in which
amphipathic molecules are arranged in a spherical structure such that all the
hydrophobic portions of
the molecules are directed inward, leaving the hydrophilic portions in contact
with the surrounding
aqueous phase. The converse arrangement exists if the environment is
hydrophobic.
B. Lipid particles
iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in a lipid
formulation,
e.g., a LNP, or other nucleic acid-lipid particle. Expression vectors or RNAs
containing coding
sequences for viral antigens under the control of an appropriate promoter can
be formulated in lipid
particles for delivery.
As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle.
LNPs typically
contain a cationic lipid, a non-cationic lipid, and a lipid that prevents
aggregation of the particle (e.g.,
a PEG-lipid conjugate). LNPs are extremely useful for systemic applications,
as they exhibit extended
circulation lifetimes following intravenous (i.v.) injection and accumulate at
distal sites (e.g., sites
physically separated from the administration site). LNPs include "pSPLP,"
which include an
encapsulated condensing agent-nucleic acid complex as set forth in WO 0003683.
The particles of
the present invention typically have a mean diameter of about 50 nm to about
150 nm, more typically
about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most
typically about 70
nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic
acids when present in the
nucleic acid- lipid particles of the present invention are resistant in
aqueous solution to degradation
with a nuclease. Nucleic acid-lipid particles and their method of preparation
are disclosed in, e.g., US
Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; and 6,815,432;
US20100324120 and WO
9640964.
In some embodiments, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to
dsRNA ratio)
will be in the range of from about 1:1 to about 50:1, from about 1:1 to about
25:1, from about 3:1 to
about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or
about 6:1 to about 9:1.
Ranges intermediate to the above recited ranges are also contemplated to be
part of the invention.
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The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium
chloride
(DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3-
dioleoyloxy)propy1)-N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3-
dioleyloxy)propy1)-
N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethy1-2,3-
dioleyloxy)propylamine
(DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-
Dilinolenyloxy-N,N-
dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-
dimethylaminopropane (DLin-
C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (Dlin-DAC), 1,2-
Dilinoleyoxy-3-
morpholinopropane (DLin-MA), 1,2-Dilinoleoy1-3-dimethylaminopropane (DLinDaP),
1,2-
Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoy1-2-linoleyloxy-
3-
dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane
chloride salt
(DLin-TMA.C1), 1,2-Dilinoleoy1-3-trimethylaminopropane chloride salt (DLin-
TAP.C1), 1,2-
Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPz), or 3-(N,N-
Dilinoleylamino)-1,2-
propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-
Dilinoleyloxo-3-(2-N,N-
dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-
dimethylaminopropane
(DLinDMA), 2,2-Dilinoley1-4-dimethylaminomethy141,3]-dioxolane (DLin-K-DMA) or
analogs
thereof, (3aR,55,6aS)-N,N-dimethy1-2,2-di((9Z,12Z)-octadeca-9,12-
dienyl)tetrahydro-3aH-
cyclopent4d][1,3]dioxo1-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-
6,9,28,31-tetraen-19-y1
4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-
hydroxydodecyl)amino)ethyl)(2-
hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediy1)didodecan-2-ol (Tech
G1), or a mixture
thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol %
or about 40 mol %
of the total lipid present in the particle. In other embodiments, the compound
2,2-Dilinoley1-4-
dimethylaminoethy141,3]-dioxolane can be used to prepare lipid-siRNA
nanoparticles.
In some embodiments, the lipid-siRNA particle includes 40% 2, 2-Dilinoley1-4-
dimethylaminoethy141,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG
(mole
percent) with a particle size of 63.0 20 nm and a 0.027 siRNA/Lipid Ratio.
The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid
including, but not
limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine
(DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine
(DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE),
dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-
carboxylate (DOPE-
mal), dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-0-monomethyl PE, 16-0-dimethyl
PE, 18-1 -trans
PE, 1 -stearoy1-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a
mixture thereof. The
non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol
%, or about 58 mol %
if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles can be, for
example, a
polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-
diacylglycerol (DAG), a PEG-
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dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture
thereof. The
PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-
dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-
distearyloxypropyl (C]8). The
conjugated lipid that prevents aggregation of particles can be from 0 mol % to
about 20 mol % or
about 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes
cholesterol at, e.g.,
about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present
in the particle.
In one embodiment, the lipidoid ND98=4HC1 (MW 1487) (see US20090023673, which
is
incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-
Ceramide C16 (Avanti
Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01
particles). Stock
solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml;
Cholesterol, 25 mg/ml,
PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock
solutions
can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid
solution can be mixed
with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol
concentration is about
35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-
dsRNA
nanoparticles typically form spontaneously upon mixing. Depending on the
desired particle size
distribution, the resultant nanoparticle mixture can be extruded through a
polycarbonate membrane
(e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as
Lipex Extruder (Northern
Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol
removal and simultaneous
buffer exchange can be accomplished by, for example, dialysis or tangential
flow filtration. Buffer
can be exchanged with, for example, phosphate buffered saline (PBS) at about
pH 7, e.g., about pH
6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
0 N
0
N)NNNNrN
0
NO iCeN
ND98 Isomer I
Formula 1
LNP01 formulations are described, e.g., in WO 2008042973, which is hereby
incorporated by
reference.
Additional exemplary lipid-dsRNA formulations are described in Table 1.
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Table 1
cationic lipid/non-cationic
Ionizable/Cationic Lipid lipid/cholesterol/PEG-lipid
conjugate
Lipid:siRNA ratio
DLinDMA/DPPC/Cholesterol/PEG-cDMA
SNALP- 1,2-Dilinolenyloxy-N,N-
(57.1/7.1/34.4/1.4)
1 dimethylaminopropane (DLinDMA)
lipid:siRNA ¨ 7:1
XTC/DPPC/Cholesterol/PEG-cDMA
2,2-Dilinoley1-4-dimethylaminoethy141,3]-
2-XTC 57.1/7.1/34.4/1.4
dioxolane (XTC)
lipid:siRNA ¨ 7:1
XTC/DSPC/Cholesterol/PEG-DMG
2,2-Dilinoley1-4-dimethylaminoethy141,3]-
LNP05 57.5/7.5/31.5/3.5
dioxolane (XTC)
lipid:siRNA ¨ 6:1
XTC/DSPC/Cholesterol/PEG-DMG
2,2-Dilinoley1-4-dimethylaminoethy141,3]-
LNP06 57.5/7.5/31.5/3.5
dioxolane (XTC)
lipid:siRNA ¨ 11:1
XTC/DSPC/Cholesterol/PEG-DMG
2,2-Dilinoley1-4-dimethylaminoethy141,3]-
LNP07 60/7.5/31/1.5,
dioxolane (XTC)
lipid:siRNA ¨ 6:1
XTC/DSPC/Cholesterol/PEG-DMG
2,2-Dilinoley1-4-dimethylaminoethy141,3]-
LNP08 60/7.5/31/1.5,
dioxolane (XTC)
lipid:siRNA ¨ 11:1
XTC/DSPC/Cholesterol/PEG-DMG
2,2-Dilinoley1-4-dimethylaminoethy141,3]-
LNP09 50/10/38.5/1.5
dioxolane (XTC)
Lipid:siRNA 10:1
(3aR,55,6aS)-N,N-dimethy1-2,2-
di((9Z,12Z)-octadeca-9,12- ALN100/DSPC/Cholesterol/PEG-DMG
LNP10 dienyl)tetrahydro-3aH- 50/10/38.5/1.5
cyclopenta[d][1,3]dioxo1-5-amine Lipid:siRNA 10:1
(ALN100)
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMG
LNP11 tetraen-19-y1 4-(dimethylamino)butanoate 50/10/38.5/1.5
(MC3) Lipid:siRNA 10:1
1,1'-(2-(4-(2-((2-(bis(2- Tech Gl/DSPC/Cholesterol/PEG-DMG
LNP12
hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5
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hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1
yl)ethylazanediy1)didodecan-2-ol (Tech
Gl)
XTC/DSPC/Chol/PEG-DMG
LNP13 XTC 50/10/38.5/1.5
Lipid:siRNA: 33:1
MC3/DSPC/Chol/PEG-DMG
LNP14 MC3 40/15/40/5
Lipid:siRNA: 11:1
MC3/DSPC/Chol/PEG-DSG/Ga1NAc-PEG-
DSG
LNP15 MC3
50/10/35/4.5/0.5
Lipid:siRNA: 11:1
MC3/DSPC/Chol/PEG-DMG
LNP16 MC3 50/10/38.5/1.5
Lipid:siRNA: 7:1
MC3/DSPC/Chol/PEG-DSG
LNP17 MC3 50/10/38.5/1.5
Lipid:siRNA: 10:1
MC3/DSPC/Chol/PEG-DMG
LNP18 MC3 50/10/38.5/1.5
Lipid:siRNA: 12:1
MC3/DSPC/Chol/PEG-DMG
LNP19 MC3 50/10/35/5
Lipid:siRNA: 8:1
MC3/DSPC/Chol/PEG-DPG
LNP20 MC3 50/10/38.5/1.5
Lipid:siRNA: 10:1
C12-200/DSPC/Chol/PEG-DSG
LNP21 C12-200 50/10/38.5/1.5
Lipid:siRNA: 7:1
XTC/DSPC/Chol/PEG-DSG
LNP22 XTC 50/10/38.5/1.5
Lipid:siRNA: 10:1
DSPC: distearoylphosphatidylcholine
DPPC: dipalmitoylphosphatidylcholine
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PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg
mol wt of
2000)
PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol
wt of 2000)
PEG-cDMA: PEG-carbamoy1-1,2-dimyristyloxypropylamine (PEG with avg mol wt of
2000)
SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising
formulations are
described in International Publication No. WO 2009/127060, filed April 15,
2009, which is hereby
incorporated by reference.
XTC comprising formulations are described in PCT Publication No. WO
2010/088537, the
entire contents of which are hereby incorporated herein by reference.
MC3 comprising formulations are described, e.g., in U.S. Publication No.
2010/0324120, the
entire contents of which are hereby incorporated by reference.
ALNY-100 comprising formulations are described in PCT Publication No. WO
2010/054406,
the entire contents of which are hereby incorporated herein by reference.
C12-200 comprising formulations are described in PCT Publication No. WO
2010/129709,
the entire contents of which are hereby incorporated herein by reference.
Compositions and formulations for oral administration include powders or
granules,
microparticulates, nanoparticulates, suspensions or solutions in water or non-
aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents,
diluents, emulsifiers,
dispersing aids or binders can be desirable. In some embodiments, oral
formulations are those in
which dsRNAs featured in the invention are administered in conjunction with
one or more penetration
enhancer surfactants and chelators. Suitable surfactants include fatty acids
or esters or salts thereof,
bile acids or salts thereof. Suitable bile acids/salts include
chenodeoxycholic acid (CDCA) and
ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,
deoxycholic acid,
glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid,
taurodeoxycholic acid, sodium
tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty
acids include
arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid,
capric acid, myristic acid,
palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an
acylcholine, or a
monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof
(e.g., sodium). In some
embodiments, combinations of penetration enhancers are used, for example,
fatty acids/salts in
combination with bile acids/salts. One exemplary combination is the sodium
salt of lauric acid, capric
acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl
ether,
polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be
delivered orally, in granular
form including sprayed dried particles, or complexed to form micro or
nanoparticles. DsRNA
complexing agents include poly-amino acids; polyimines; polyacrylates;
polyalkylacrylates,
polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins,
starches, acrylates,
polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-
derivatized polyimines,
pollulans, celluloses and starches. Suitable complexing agents include
chitosan, N-trimethylchitosan,
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poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine,
polyvinylpyridine,
polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino),
poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate,
DEAE-hexylacrylate,
DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate,
poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol
(PEG). Oral formulations for dsRNAs and their preparation are described in
detail in US Patent Nos.
6,887,906 and 6,747,014, and US 20030027780, each of which is incorporated
herein by reference.
Pharmaceutical compositions of the present invention include, but are not
limited to,
solutions, emulsions, and liposome-containing formulations. These compositions
can be generated
from a variety of components that include, but are not limited to, preformed
liquids, self-emulsifying
solids and self-emulsifying semisolids. Particularly preferred are
formulations that target the liver
when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of the present invention, which can
conveniently be
presented in unit dosage form, can be prepared according to conventional
techniques well known in
the pharmaceutical industry. Such techniques include the step of bringing into
association the active
ingredients with the pharmaceutical carrier(s) or excipient(s). In general,
the formulations are
prepared by uniformly and intimately bringing into association the active
ingredients with liquid
carriers or finely divided solid carriers or both, and then, if necessary,
shaping the product.
V. Uses of the Methods of the Invention
The invention sets forth various methods and treatment regimens. It is
understood that the
methods can be provided as uses of the RNAi agents and vaccines provided
herein. That is, the
invention provides
a) an RNAi agent that targets at least three HBV transcripts, wherein the RNAi
agent
comprises a sense strand and an antisense strand;
b) a protein-based vaccine comprising an HBV core antigen (HBcAg) and an HBV
surface
antigen (HBsAg); and
c) a nucleic acid-based vaccine comprising an expression vector construct
encoding an
HBcAg or an HBsAg, wherein the construct encodes a protein that shares an
epitope with the protein-
based vaccine; thereby treating the subject
for use in methods of treating a subject having a hepatitis B infection.
The uses include all of the variations and exemplary RNAi agents, protein-
based vaccines,
and nucleic acid-based vaccines provided herein.
VI. Kits for Practicing the Methods of the Invention
The invention sets forth various methods, treatment regimensm and uses of
agents for the
treatment of a subject having a hepatitis B infection. It is understood that
agents for practicing the
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methods of the invention can be prepared based in the disclosure provided
herein. Such a kit would
include,
a) an RNAi agent that targets at least three HBV transcripts, wherein the RNAi
agent
comprises a sense strand and an antisense strand;
b) a protein-based vaccine comprising an HBV core antigen (HBcAg) and an HBV
surface
antigen (HBsAg);
c) a nucleic acid-based vaccine comprising an expression vector construct
encoding an
HBcAg or an HBsAg, wherein the construct encodes a protein that shares an
epitope with the protein-
based vaccine; thereby treating the subject; and
d) instructions for use in methods of treating a subject having a hepatitis B
infection.
The uses include all of the variations and exemplary RNAi agents, protein-
based vaccines,
and nucleic acid-based vaccines provided herein. The components of the kit may
be provided
together, e.g., in a box. In certain embodiments, the components of the
invention may be provided
separately, e.g., due to different storage requirements, but be provided for
use together, e.g., based on
.. package instructions for use.
This invention is further illustrated by the following examples which should
not be construed
as limiting. The entire contents of all references, patents and published
patent applications cited
.. throughout this application, as well as the Figures, Appendix A, and the
Sequence Listing, are hereby
incorporated herein by reference.
EXAMPLES
.. Example 1. Materials and methods
Exemplaiy iRNAs
Exemplary iRNA target sites and unmodified and modified siRNA sequences are
provided in
the tables in Appendix A.
The chemically modified HBV-siRNA duplexes used in the experiments below have
the
.. following sequences:
Unmodified sequences:
DuplexID Sense Sequence Unmodified (5' to 3') SEQ Antisense Sequence
Umodified (5' to 3') SEQ
ID:
ID:
AD-66810 GUGUGCACUUCGCUUCACA 27 UGUGAAGCGAAGUGCACACUU
25
AD-66816 CACCAUGCAACUUUUUCACCU 28 AGGUGAAAAAGUUGCAUGGUGUU 26
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Modified sequences:
DuplexID SEQ Antisense Sequence Modified (5' to 3') SEQ
Sense Sequence Modified (5' to 3')
ID:
ID:
AD-66810 gsusguGfcAfCfUfucgcuucacaL96 37 usGfsugaAfgCfGfaaguGfcAfcacsusu 30
AD-66816 csasccauGfcAfAfCfuuuuucaccuL96 38 asGfsgugAfaAfAfaguuGfcAfuggugsusu
32
Abbreviations for nucleotide monomers in modified nucleic acid sequences are
provided in
Table 1 of Appendix A.
The target site of AD-66810 is GTGTGCACTTCGCTTCACA (SEQ ID NO: 39) which is
nucleotides 1579-1597 of NC_003977.1 (SEQ ID NO: 1).
The target site of AD-66816 is CACCATGCAACTTTTTCACCT (SEQ ID NO: 40) which is
nucleotides 1812-1832 of NC_003977.1 (SEQ ID NO: 1).
Cell Culture Evaluation of HBV-siRNA
hNTCP-expressing HepG2 cells were infected (100 multiplicity of infection
(MOI)) HBV
particles/cell (subtype ayw)) in duplicate. At day 4 after infection, cells
were trypsinized and
reseeded into multiwell plates and transfected with control or HBV-siRNAs AD-
66810 (having the
chemical modifications shown in the table above and sense and antisense
sequences as set forth in
SEQ ID NOs: 37 and 30, respectively) or AD-66816 (having the chemical
modifications shown in the
table above and sense and antisense sequences as set forth in SEQ ID NOs: 38
and 32, respectively)
each delivered at 100 nM, 10 nM, or 1 nM using Lipofectamine RNAiMax.
Supernatant was
harvested at days 3, 6, 10, 13, and 17 after reseeding and HBeAg and HBsAg
levels were determined
relative to non-transfected control. HBsAg and HBeAg levels were determined
using a
chemiluminescent microparticle immunoassay (CMIA) measured in an Abbott
Architect
immunoassay analyzer (Abbott Laboratories, Abbott Park, IL, USA).
Mice, siRNA Administration, and Vaccinations
HBV-transgenic mice (StrainHBV1.3xfs (HBV genotype D, subtype ayw)), were
derived
from in-house breeding under specific pathogen-free conditions following
institutional guidelines.
For siRNA administration, mice were subcutaneously administered a 3 mg/kg or 9
mg/kg
dose of control siRNA or HBV-siRNA (modified AD-66810 or modified AD-66816);
or
intravenously administered by tail vein injection 1 x 10" AAV particles for
expression of the HBV-
shRNA as indicated in the Figures and Examples below.
For protein vaccinations, mice were immunized subcutaneously with recombinant
yeast
HBsAg and Escherichia coli HBcAg (APP Latvijas Biomedicinas, Riga, Latvia)
mixed with 31.91 lig
of synthetic phosphorothioated CpG ODN 1668 and 25 lig
poly[di(sodiumcarboxylatoethyl-
phenoxy)phosphazene] (PCEP) in 50 I.L1 PBS.
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Recombinant MVA were generated by homologous recombination and host range
selection as
described previously (Staib et al., 2003. Biotechniques. 34:694-700). The
entire HBcAg (genotype D,
subtype ayw) and HBsAg open reading frames (genotype A, subtype ayw or adw)
were cloned into
MVA transfer plasmids pIIIAHR-PH5 or pIIIAHR-P7.5, thereby placing the HBV
proteins under the
control of the early/late Vaccinia virus-specific promoters PH5 (HBcAg ayw
/HBsAg ayw/HBsAg
adw) or P7.5 (HBsAg ayw). After construction of each virus, gene expression,
sequence of inserted
DNA, and viral purity were verified. For generation of vaccine preparations,
MVA were routinely
propagated in CEF, purified by ultracentrifugation through sucrose,
reconstituted in 1 mM Tris-HC1
pH 9.0 and titrated following standard methodology (Staib et al., 2004.
Methods Mol Biol. 269:77-
.. 100). For MVA vaccination, mice were vaccinated intraperitoneally with 1 x
108 infectious units of
respective recombinant MVA in 500 I.L1 PBS.
Specific dosing regimens are provided in the Examples below and in Figures 3,
7, and 12.
Serological analysis
Serum levels of HBsAg and HBeAg were determined in 1:33 dilutions; and anti-
HBs levels
were determined in a 1:100 dilution using chemiluminescent microparticle
immunoassay (CMIA)
measured in an Abbott Architect immunoassay analyzer (Abbott Laboratories,
Abbott Park, IL, USA).
Quantification of serum HBV titers by real-time polymerase chain reaction and
determination of HBV
DNA levels in serum was performed as described in Untergasser et al., 2006
(Hepatology. 43:539-
47). The amount of HBV DNA was normalized to DNA level prior to treatment.
Levels of anti-HBc antibodies were determined in 1:20 dilution using the
Enzgnost anti-HBc
monoclonal test on the BEPIII platform (Both Siemens Healthcare, Eschborn,
Germany). If a sample
was measured outside the linear range, higher or lower dilutions were used as
appropiate.
Quantification of serum HBV titers by real-time polymerase chain reaction and
determination
of HBV DNA levels in serum was performed as described in Untergasser et al.,
2006 (Hepatology.
43:539-47). The amount of HBV DNA was normalized to DNA level prior to
treatment.
ALT activity was measured at the day of bleeding in a 1:4 dilution in
phosphate buffered
saline (PBS) using the Reflotron GPT/ALT test (Roche Diagnostics, Mannheim,
Germany).
Lymphocyte stimulation assay
Liver-associated lymphocytes (LAL) were isolated as described in Stross et
al., 2012
(Hepatology 56:873-83) and stimulated with H2-kb-or H-2Db-restricted peptides
(see Backes, 2016)
for 12 hours in the presence of 1 mg/ml Brefeldin A (Sigma-Aldrich,
Tauflcirchen,Germany). Cells
were live/dead-stained with ethidium monoazidebromide (Invitrogen , Karlsruhe,
Germany) and
blocked with anti-CD16/CD32-Fc-Block (BD Biosciences, Heidelberg, Germany).
Surface markers
were stained with PB-conjugated anti-CD8-alpha and PE-conjugated anti-CD4
(eBiosciences, Eching,
Germany). Intracellular cytokine staining (ICS) was performed with FITC anti-
IFN-gamma
(XMG1.2), PE-Cy7 anti-TNF-alpha and APC anti-IL-2 (eBiosciences, Ech-ing,
Germany) using the
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Cytofix/Cytoperm kit (BD Biosciences, Heidelberg, Germany) according to the
manufacturer's
recommendations. The same data were analyzed twice, the second time with a
more rigorous
exclusion of dead cells.
Immunohistochemical stainings for HBc-expressing hepatocytes
Livers were harvested, fixed in 4% paraformaldehyde, and paraffin-embedded.
The liver was
sectioned (2 [tin) and sections were stained with rabbit anti-HBcAg as the
primary antibody
(Diagnostic Biosystems, Pleasanton, CA; #RP 017; 1:50 dilution; retrieval at
100 C for 30 min
with EDTA) and a horseradish peroxide coupled secondary antibody. Incubation
in Ventana buffer
and staining were performed on a NEXES immunohistochemistry robot (Ventana
Instruments) using
an IVIEW DAB Detection Kit (Ventana Instruments) or on a Bond MAX (Leica
Biosystems). For
analysis, slides were scanned using a SCN 400 slide scanner and positive cells
were counted using the
integrated Tissue AT software (both Leica Biosystems).
HBV transcripts from liver lysate
For analysis of HBV RNA from liver lysate, RNA was extracted from 30 mg liver
tissue with
the RNeasy mini kit (Qiagen) and cDNA was synthesized with the Superscript III
kit (Thermo Fisher
Scientific). HBV transcripts were amplified with primers specific for only the
3.5 kb transcripts
(forward primer 5'- GAGTGTGGATTCGCACTCC-3' (SEQ ID NO: 41); reverese primer 5'-
GAGGCGAGGGAGTTCTTCT-3' (SEQ ID NO: 42)), or with primers binding to the common
3 end
of all HBV transcripts (forward primer 5'- TCACCAGCACCATGCAAC-3' (SEQ ID NO:
43);
reverse primer 5'- AAGCCACCCAAGGCACAG-3' (SEQ ID NO: 44)) (Denaturation: 95 C
5 min;
Amplification: 95 C 3s, 60 C 30s (40 cycles)) (Yan et al., 2012). Results were
normalised to murine
GAPDH expression (forward primer 5'- ACCAACTGCTTAGCCC-3' (SEQ ID NO: 45);
reverse
primer 5'- CCACGACGGACACATT-3' (SEQ ID NO: 46)) (Denaturation: 95 C 5 min;
Amplification: 95 C 15s, 60C 10s, 72 C 25s (45 cycles)). All PCR reactions
were performed on a
LightCycler 480 (Roche Diagnostics).
AAV-HBV mouse model
For the AAV mouse model, wildtype C57/B16 mice (9 weeks of age; 6 animals per
treatment
group) were injected i.v. with 2x101 genome equivalents (geq) of Adeno-
Associated-Virus Serotype
8 (AAV8) carrying a 1.2-fold overlength HBV genome of genotype D (AAV-HBV1.2)
(day -28) (see,
e.g., Yang, et al. (2014) Cell and Mol Immunol 11:71) . Starting 4 weeks after
AAV-transduction
(day 0), animals were treated with 3 injections (3 mg/kg bw, n=12 per group)
of either a control
siRNA, or HBV siRNA (AD-66816 or AD-66810) (days 0, 29, and 57). Each siRNA
treatment group
was divided into two groups (n = 6 per group) to be not treated or treated
with the vaccine regimen
consisting of recombinant HBsAg, HBcAg (15 tig of each) and 10 tig c-di-AMP
given at day 57 and
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70 and boosted with MVA-HBs and MVA-HBc (5x107 geq of each) at day 84. A
schematic showing
the treatment regimen is provided in Figure 12.
Example 2¨ Evaluation of HBV-siRNA in HepG2-NTCP Cell Culture and Dose Finding
Experiments in HBVxfs Mice
Anti-HBV siRNAs were evaluated for efficient knockdown of HBV antigens and DNA
in an
in vitro infection model using HBV-infected HepG2-NTCP cells treated with 1
nM, 10 nM, or 100
nM of one of modified AD-66810 or modified AD-66816 HBV-siRNA, or a control
siRNA as
provided above. Supernatants were collected at days 3, 6, 10, 13, and 17 days
after siRNA treatment
and assayed for HBeAg and HBsAg levels as compared to untransfected control.
Both HBV-siRNAs
were demonstrated to effectively knockdown expression of both HBeAg and HBsAg
with the highest
levels of knockdown observed at days 13 and 17. No significant knockdown was
observed with the
control siRNA. These data demonstrate that the AD-66810 or AD-66816 HBV-siRNAs
are effective
at knocking down expression of HBV antigens in a sustained manner in an in
vitro system of HBV
infection.
The HBV and control siRNAs were then tested in the HBVxfs transgenic model of
chronic
hepatitis B. The HBVxfs mice include an integrated HBV genome that is
expressed under the control
of a liver-specific promoter. At about 10 weeks of age, mice were administered
a 3 or 9 mg/kg dose of
one of AD-66810 or AD-66816 HBV-siRNA or a control siRNA (n = 6 per group).
Blood samples
were collected at days 6, 13, and 21 after siRNA treatment and serum was
prepared. HBeAg, HBsAg,
and HBV DNA levels in serum (Figures 2A-2C) were determined as provided above.
Further, RNA
was isolated from liver and total HBV RNA and HBV 3.5 kB transcript levels
were detected using the
method of Yan, 2012 and normalized to GAPDH expression (Figures 2D and 2E).
Both of the HBV-siRNAs at both doses were demonstrated to effectively
knockdown
expression of HBsAg and HBeAg and to decrease HBV DNA levels in serum as
compared to control
levels (see Figures 2A-2C). Futher, total liver HBV RNA relative to GAPDH DNA
and 3.5 kb HBV
RNA relative to GAPDH RNA levels were strongly decreased by both doses of the
HBV siRNAs as
compared to control levels (Figures 2D and 2E). Based on these results, the 3
mg/kg dose was
selected for use in further experiments.
Example 3 ¨ Comparison of Reducing Antigen Load with siRNA to Nucleot(s)ide
Analogs Prior to Therapeutic Vaccine Administration in an HBV Transgenic Mouse
Model
Having demonstrated that siRNA targeted to HBV can effectively knockdown
expression of
HBsAg and HBeAg and decrease HBV DNA levels in serum in the HBVxfs mouse
model, the effect
of treatment of mice with the nucleoside analog Entecavir or HBV-siRNA prior
to therapeutic vaccine
administration using a prime-boost regimen was tested. The treatment scheme is
shown in Figure 3.
Mice were pretreated with one of six treatment regimens prior to vaccination
using a prime-
boost regimen (n= 6 per group):
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(1) No pretreatment;
(2) Entecavir at 1 lig/m1 in water throughout the course of the study
beginning on the first day
of Week 0 (expected dose of about 4 mg/day based on calculations provided in
Liitgehetmann et al.,
2011, Gastroenterology.140:2074-83);
(3) A 3 mg/kg dose on the first day of Weeks 0, 4, 8, and 12 of the control
iRNA agent.
(4) A single dose on the first day of Week 0 with an expression vector
encoding an shRNA
targeted to HBV (HBV-shRNA) (Michler et al., 2016); or
(5-6) A 3 mg/kg dose on the first day of Weeks 0, 4, 8, and 12 of modified AD-
66816 or
modified AD-86610 (generically HBV-siRNA).
On the first day of Weeks 12 and 14, a mixture of recombinantly expressed
yeast HBsAg (15
rig) and E. coli expressed HBcAg (15 rig) adjuvanted with 31.9 lig synthetic
phosphorothioated
CpGODN 1668 (CpG) and 25 lig polykli(sodiumcarboxylatoethyl-
phenoxy)phosphazene] (PCEP)
was subcutaneously administered to all mice as a protein-prime vaccination
(Backes, 2016).
On the first day of week 16, a mixture of modified vaccinia viruses Ankara
expressing
HBsAg or HBcAg (5 x 107 particles of each virus) was subcutaneously
administered to all mice as a
boost vaccination (Backes, 2016).
Blood samples were obtained on the first days of Week 0, 2, 4, 8, 12, 16, and
17 and serum
samples prepared therefrom were assayed for levels of HBsAg, HBeAg, and HBV
DNA. Results are
shown in Figure 4.
HBsAg and HBeAg levels mice in groups 1, 2, and 3 (mock, Entecavir, control
iRNA agent)
were similar. The HBV-shRNA or HBV-siRNAs (AD-66816 or AD-86610) alone caused
a
significant decrease in HBsAg, HBeAg, and HBV DNA in serum (Figures 4A-4C).
The three dose
prime-boost vaccination scheme resulted in a further decrease in HBsAg in all
groups, and reduced
the level of HBsAg in at least some animals in the HBV-shRNA and HBV-siRNA
groups to below
the level of detection. However, vaccine treatment did not decrease HBeAg
levels in any of the
groups. Without being bound by mechanism, it is proposed that the decrease in
HBsAg, but not
HBeAg, results from the immune response induced by the vaccine against the s
antigen, but not the e
antigen, which is produced by proteolytic processing of the C protein (see
Figure 5 discussed below).
HBV DNA levels were decreased to about the lower limit of quantitation with
Entecavir alone
so no effect of the three dose prime-boost vaccine could be detected (Figure
4C). Mock treatment and
treatment with the HBV-shRNA, the HBV-siRNAs, and control siRNA all decreased
HBV DNA
levels and the level of HBV-DNA was further decreased by the prime-boost
vaccine in all groups. It
is unclear why the mock treatment and control siRNA decreased HBV DNA levels
in this experiment.
No decrease in HBV DNA was observed in response to treatment with control
siRNA in other
experiments (see, e.g., Figures 2C and 8C). These data demonstrate that RNAi
is superior to
nucleot(s)ide analog therapy in reducing viral antigens. Also, RNAi and
subsequent vaccination have
a combined effect on HBsAg and HBV DNA levels greater than either agent alone.
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On the final day of the experiment (first day of Week 17), mice were
sacrificed and their
livers harvested. Liver associated lymphocytes were isolated from liver and
after peptide stimulation
CD8+ T cell responses measured via intracellular cytokine staining.
Specifically, intrahepatic CD8+
T cell responses were assessed for response to HBsAg, HBcAg, and the MVA virus
particle using the
method provided in Backes, 2016. The data were analyzed twice using two
different thresholds for
the exclusion of dead immune cells as the exclusion of dead immune cells in
the first analysis
provided in Figures 5A-5C was determined to be insufficient. The second
analysis is presented in
Figures 5D-5F. The second analysis confirmed the conclusions from the first
analysis.
Mice pretreated with the HBV-shRNA or the HBV-siRNAs before vaccination were
able to
generate a CD8+ T cell immune response against the HBsAg and HBcAg (Figures
5A, 5B, 5D, and
5E) indicating that cytotoxic T cells able to clear HBV infection are induced
when HBV antigen
levels are suppressed prior to vaccination. No significant CD8+ T cell immune
response against the
HBV antigens was observed in the mock, Entecavir, or control siRNA groups. A
significant and
similar CD8+ T cell immune response against the MVA virus in all animals,
independent of
pretreatment or viral antigen levels, demonstrated that vaccination had worked
equally well in all
animals and was not influenced by HBV antigen levels, thereby demonstrating
the presence of a
competent immune system (Figures 5C and 5F). No significant differences in
antibody production
were observed between mock treated animals and any of the other groups
indicating that high HBV
antigen levels may induce T cell tolerance.
These data demonstrate that RNAi treatment, in contrast to the current
standard of care
treatment with a nucleoside analog, can restore HBV-specific T cell immunity
in the liver and enable
the induction of HBV-specific CD8+ T cell responses after therapeutic
vaccination. A robust CD8+
effector T cell response has been associated with viral clearance and
functional cure (see, e.g.,
Thimme et al., 2003. J. Virol. 77:68-76, and Backes et al., 2016. Vaccine.
34:923-932).
RNA was also isolated from liver and total HBV RNA and HBV 3.5 kB transcript
levels were
detected using the method of Yan, 2012 and normalized to GAPDH expression.
Treatment of mice
with HBV-siRNA or HBV-shRNA prior to vaccine administration resulted in a
significant decrease in
HBV total RNA and HBV 3.5 kb transcript as compared to the mock treated
control (Figures 6A and
6B). No significant change in HBV total RNA and HBV 3.5 kb transcript was
observed in the mice
treated with Entecavir or the control siRNA prior to vaccination as compared
to the mock treated
control. These data demonstrate that both HBV siRNAs and the shRNA, in
contrast to the control and
Entecavir groups, successfully led to a decrease in HBV transcript levels.
Further, expression of HBV antigens in the liver was analyzed by
immunohistochemical
staining for HBc of liver sections and counting of HBc positive cells per mm2
(Figure 6C). Only
groups of animals pre-treated with HBV siRNA or shRNA, but not Entecavir or
the control siRNA,
showed reduced HBc expression following vaccination.
Throughout the experiment, body weight and ALT levels were monitored. No
significant
differences were observed in any of the treatment groups as compared to mock
treated control.
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Example 4 ¨ Evaluation of the Effect of Duration of HBV Antigen Knockdown on
Response to
Immunization in an HBV Transgenic Mouse Model
Having demonstrated that suppression of expression of HBV antigens using shRNA
or siRNA
is effective at potentiating an immune response to an HBV vaccine regimen, a
study was designed to
determine if the length of time of HBV antigen suppression had an effect on
potentiation of an HBV
immune response. A treatment scheme is shown in Figure 7.
Using the HBV1.3xfs mouse model, mice were treated for eight, six, or three
weeks with
HBV-siRNA AD-66816 (modified) or the control siRNA for 8 weeks, administered
subcutaneously at
3 mg/kg/dose. siRNA administration was followed by administration of the prime-
boost vaccine
regimen as set forth above with the exception that c-di-AMP was used as an
adjuvant with protein
administration rather than PCEP + CPG (n= 6 per group). Therefore, mice in the
8 week group (n =
6) received three doses of siRNA with the third dose being administered on the
first day of vaccine
administration. The mice in the 6 week group (n = 12) received two doses of
siRNA with the second
dose being administered two weeks prior to the first day of vaccine
administration. Finally, the mice
in the 3 week group (n = 6) received one dose of siRNA with the dose being
administered three weeks
prior to the first day of vaccine administration.
Blood samples were collected on the first day of -8 weeks, -6 weeks, -4 weeks,
-2 weeks, 0
weeks, 2 weeks, 4 weeks, and 6 weeks, before (negative numbers) and after the
first dose of vaccine
administration on the first day of Week 0. Serum was prepared and HBsAg,
HBeAg, and HBV DNA
levels were assessed as provided above.
A significant decrease in each HBsAg, HBeAg, and HBV DNA was observed after
the first
administration of AD-66816 (Figures 8A-8C). A further significant decrease in
HBsAg was observed
after treatment with the vaccine boost, with the greatest decrease observed in
the 8 week pretreatment
group to below the level of detection of the assay, representing a greater
than 5 10g10 decrease in
HBsAg level in all treated animals. Immunization caused only slight further
reductions (<0.5 10g10)
of HBV DNA which had been significantly decreased by the siRNA treatment. No
further decrease in
HBeAg levels was observed in response to the vaccination regimen.
These data demonstrate that efficacy of therapeutic vaccination correlates
with duration of
antigen suppression before start of vaccination. Reconstitution of HBV-
specific CD8+ T cell
responses takes several weeks, with a 6 or preferably 8 week pretreatment
rather than a 3 week
pretreatment.
On the final day of the experiment, on the first day of Week 6 after the start
of immunization,
mice were sacrificed and their livers harvested for six mice from each group.
Liver associated
lymphocytes were isolated and a lymphocyte stimulation assay was performed as
provided above.
Specifically, intrahepatic CD8+ T cell responses were assessed for response to
HBsAg, HBcAg, and
the MVA virus particle using the method provided in Backes, 2016. T-cell
responses against HBsAg
and HBcAg in liver corresponded with the duration of HBV antigen knockdown,
with a trend of
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higher levels of immune response observed with longer duration of HBV antigen
resulting in greater
T-cell response (see Figures 9A-9C). Similar responses to MVA virus antigens
were observed across
all groups, independent of pretreatment, showing that vaccination had worked
equally well in all
animals and was not influenced by HBV antigen levels demonstrating the
presence of a competent
immune system (Figure 9D). No significant differences in antibody production
were observed
between control siRNA treated animals and any of the other groups. This
demonstrates, that
reconstitution of HBV-specific CD8+ T cell responses does not occur
immediately, with stronger
responses seen after therapeutic vaccination if animals had lowered HBV
antigen titers for at least 6 or
8 weeks compared to only 3 weeks. It further confirms the previous finding
that, in contrast to T cell
responses, B cell immunity does not seem to be significant influenced by HBV
antigen titers.
RNA was also isolated from liver and total HBV RNA and HBV 3.5 lcB transcript
levels were
detected using the method of Yan, 2012 and normalized to GAPDH expression.
Treatment of mice
with HBV-siRNA AD-66816 prior to vaccine administration resulted in a
significant decrease in HBV
total RNA and HBV 3.5 kb transcript in liver lysates as compared to the
control siRNA treated control
(Figures 10A and 10B). Further, HBV antigen expression in the liver was
analyzed by
immunohistochemical staning and counting of HBc positive cells per mm2 (Figure
10C). Correlating
the observed increase in HBV-specific CD8 responses with increased duration of
siRNA pretreatment,
decreased numbers of HBc expressing cells where observed in the liver. These
results demonstrate
that the CD8+ T cell responses did prevent antigen expression in the liver..
To assess the durability of response, blood samples were collected from mice
pretreated with
the AD-66816 HBV-siRNA using the six week treatment regimen at 2 and 3 weeks
after
administration of the boost vaccination (Figures 11A-11D). In three of the six
mice, HBsAg levels
continued to drop to below the level of detection of the assay (Figure 11A).
No similar decrease in
HBeAg levels were observed during the course of the experiment (Figure 11B).
These data show that
the maximum effect by the siRNA-vaccination combinatorial therapy provided
herein is later than 1
week after the MVA vaccination, which was chosen as termination time point to
best asses CD8+ T
cell responses. Anti-HBs antibody response (Figure 11C) and T cell immune
response in the liver
against HBs(5208) (Figure 11D) at week 7 after the first vaccine dose after
the 6 week regimen in the
dosing regimen. The antibody response varied among animals.
These data demonstrate that a functional cure is possible using the treatment
regimens
provided herein. Further, these data suggest that a lower HBsAg and HBeAg
burden can result in a
greater level of immune clearance of HB antigens and potentiate an immune
response, at least within
the short time course of the experiment.
Throughout the experiment, body weight and ALT levels were monitored. No
significant
differences were observed in any of the treatment groups as compared to mock
treated control.
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Example 5 ¨ Evaluation of the Effect of Duration of HBV Antigen Knockdown on
Response to
Immunization in an AAV-HBV Mouse Model
Having demonstrated the efficacy of the siRNA-vaccine combination treatment
regimen in a
transgenic mouse model, an AAV-HBV infection mouse model was used to study the
efficacy of the
treatment regimen in acquired infection model (see, e.g., Yang, et al. (2014)
Cell Mol Immunol
11:71). This mouse model exhibits sustained HBV viremia after infection with a
recombinant adeno-
associated virus (AAV) carrying a replicable HBV genome.
There are a number of differences between the HBV-transgenic and AAV-HBV mouse
models. The HBV transgenic mice can express HBV antigens essentially from
birth, whereas the
AAV-HBV model allows for the introduction of the HBV genome at a later time in
the life of the
mice. This may have an effect on immune tolerance. Further, the HBV transgenic
mice carry the
transgene in every cell of the body, providing the possibility of "leaky"
extrahepatic expression.
Although the AAV8 serotype could infect cells outside of the liver, it has a
strong liver tropism.
Moreover, it is not possible to clear an HBV infection in a transgenic mouse.
When a transgenic
HBV expressing liver cell is killed, it is replaced by a new HBV expressing
cell. In the infection
model, if the infected cells are killed, the newly dividing cells are not
infected at the time of cell
division.
Nine week old C57/B16 mice were infected with AAV-HBV (- 28 days). Mice were
then
treated with one of control siRNA, or one of two HBV-siRNAs, modified AD-66816
or modified AD-
66810 at 3 mg/kg administered subcutaneously on days 0, 29, and 57 (i.e., 0
weeks, 4 weeks, 8 weeks)
(n=12 per group). Each siRNA treatment group was divided into two groups (n=6
per group). One
group was treated with the HBV vaccine protocol (protein prime on days 57 and
70, and MVA boost
on day 84, i.e., weeks 8, 10, and 12, respectively) and one group was not. A
schematic showing the
dosing regimen is provided in Figure 12. Mice were monitored throughout the
experiment for serum
HBsAg, HBeAg, anti-HBs antibodies, body weight, and ALT. Anti-HBe antibody
levels were also
periodically tested.
Figures 13A and 13B show an increase in HBsAg and HBeAg levels comparable to
that seen
in transgenic mice within two weeks of transduction with the AAV-HBV virus.
Mice treated only
with the control siRNA replicated HBV for greater than 8 months at levels
comparable to chronically
infected humans (HBsAg levels around 2,000 IU/ml, HBV viremia 106107 IU/ml).
HBV siRNAs
AD-66816 and AD-66810 reduced HBsAg by 2 and 2.5 logio, respectively, and
HBeAg by >1 logio.
The effect persisted for at least 4 weeks after stopping siRNA treatment
before antigenemia slowly
rebounded to baseline levels after 18 weeks (Figures 13A and13B).
Intrahepatic HBV DNA (Figure 13D) and AAV DNA (Figure 13E) levels were
determined at
week 22 by qPCR as described above. The relative expression in liver of HBV
3.5 RNA relative to
GAPDH RNA (Figure 13F) and total HBV RNA relative to GAPDH RNA (Figure 13G)
were
determined using rtPCR. Mice treated with the combined siRNA-vaccine protocol
demonstrated a
significant decrease in total HBV DNA and AAV DNA as compared to untreated
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vaccine treatement alone at the 22 week time point (Figures 13D and 13E).
Notably, total HBV DNA
levels dropped to less than one copy per cell as a result of the combination
treatment (Figure 13D).
Mice treated with the combined siRNA-vaccine protocol also demonstrated a
significant decrease
level of HBV 3.5 kb RNA expression relative to GAPDH RNA expression as
compared to all other
treatment regimens (Figure 13F). A significant decrease in total HBV RNA
relative to GAPDH RNA
expression was observed as compared to treatment with vaccine or siRNA alone
(Figure 13G). These
data suggest that a short course of administration of siRNA alone or a
therapeutic vaccine against
HBV is insufficient to durably suppress HBV infection. Immune mediated control
of HBV after
siRNA knockdown of HBV expression is long lasting. At 22 weeks after the last
siRNA dose, the
.. effect of the siRNA was waning as seen in the groups which had only
received the HBV siRNAs
without vaccination. Mice treated with the siRNA-vaccine protocol maintained
HBV DNA and RNA
suppression long after the end of siRNA administration.
No immune responses were observed in these fully immune competent mice under
siRNA
treatment alone, but vaccine treatment resulted in anti-HBs seroconversion in
all vaccinated animals
(Figures 14A and 14B). siRNA-pretreated animals, however, developed 10-fold
higher and more
constant anti-HBs titers and were able to completely and persistently clear
serum HBsAg and HBeAg.
In contrast, anti-HBe seroconversion was only observed in antimals pretreated
with HBV siRNAs.
Interestingly, three of the 12 mice vaccinated after HBV siRNA treatment
showed a transient relapse
of HBeAg between week 15 and 22 co-inciding with decreased levels of anti-HBe
(Figure 13C).
Without being bound by mechanism, it is proposed that theHBeAg relapse was
controlled by a
memory immune response induced by the vaccine. Taken together, suppression of
HBV antigen
expression by an siRNA in combination with a heterologous prime-boost vaccine
is sufficient to break
immune tolerance to HBV antigens. The sequential therapy achieved long-term
functional cure in a
mouse model of persistent HBV infection without causing significant liver
damage.
Figures 14A and 14B show that animals treated with HBV siRNA plus the vaccine
regimen
developed high titers of anti-HBs antibodies and anti-HBe antibodies. The
level of anti-HBs
antibodies continued to increase after the last vaccine dose. Although anti-
HBs antibodies could also
be measured in animals that received the control siRNA plus the vaccine
regimen, the levels were
significantly lower. Further, only animals that received HBV siRNA plus the
vaccine regimen
developed anti-HBe antibodies and achieved anti-HBe seroconversion. The
combinatorial therapy
using siRNA and vaccine appeared to be well tolerated. All mice equally gained
weight and only a
mild ALT elevation (<2-fold upper limit of normal) was observed (Figures 15A
and 15B). The loss
of antigenemia concided with slight increases of ALT activity seen in
treatment groups which had
received HBV siRNA in conjunction with the vaccination regimen (Figure 15A).
These groups
showed significant but mild increases (both p>0.05 or smaller by repeated
measure two-way
ANOVA; only comparing time points after start of vaccination) as compared to
all other treatment
groups that did not receive the combination HBVsiRNA-vaccine regimen. There
was a steady
increase in body weight in all animals throughout the experiment independent
of siRNA treatment.
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Animals that were vaccinated showed a slight and transient decrease
(approximately 5%) of body
weight after vaccination, but rebounded to normal levels within nine days and
subsequently gained
weight comparable to the control groups (Figure 15B).
Without being bound by mechanism, the data provided herein strongly suggest
that the high
level of HBV antigen expression routinely detected as circulating HBsAg and
HBeAg prevents HBV-
specific CD8+ T cell responses, which has far reaching consequences for future
immune therapy of
chronic hepatitis B. Using 2 different mouse models for chronic hepatitis B,
it is proposed that HBV-
specific immunomodulation can be reverted by suppressing HBV protein
expression in hepatocytes
using an RNAi-based therapy. Such reduction of HBV antigens by RNAi, in
contrast to standard-of-
care nucleo(t)side analogues, allows for induction of strong HBV-specific CD8+
T cell responses by
therapeutic vaccination that are required for control of HBV infection.
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APPENDIX A
Table 1. Abbreviations of nucleotide monomers used in nucleic acid sequence
representation. It will
be understood that, unless otherwise indicated, these monomers, when present
in an oligonucleotide,
are mutually linked by 5'-3'-phosphodiester bonds.
Abbreviation Nucleotide(s)
A Adenosine-3'-phosphate
Af 2'-fluoroadenosine-3' -phosphate
Afs 2'-fluoroadenosine-3'-phosphorothioate
As adenosine-3' -phosphorothioate
cytidine-3' -phosphate
Cf 2'-fluorocytidine-3' -phosphate
Cfs 2'-fluorocytidine-3'-phosphorothioate
Cs cytidine-3'-phosphorothioate
guanosine-3'-phosphate
Gf 2'-fluoroguanosine-3' -phosphate
Gfs 2'-fluoroguanosine-3'-phosphorothioate
Gs guanosine-3'-phosphorothioate
5'-methyluridine-3' -phosphate
Tf 2'-fluoro-5-methyluridine-3' -phosphate
Tfs 2'-fluoro-5-methyluridine-3'-phosphorothioate
Ts 5-methyluridine-3'-phosphorothioate
Uridine-3'-phosphate
Uf 2'-fluorouridine-3' -phosphate
Ufs 2'-fluorouridine -3'-phosphorothioate
Us uridine -3' -phosphorothioate
any nucleotide (G, A, C, T or U)
a 2'-0-methyladenosine-3' -phosphate
as 2'-0-methyladenosine-3'- phosphorothioate
2'-0-methylcytidine-3'-phosphate
cs 2'-0-methylcytidine-3'- phosphorothioate
2'-0-methylguanosine-3'-phosphate
gs 2'-0-methylguanosine-3'- phosphorothioate
2'-0-methyl-5-methyluridine-3' -phosphate
ts 2'-0-methyl-5-methyluridine-3'-phosphorothioate
2'-0-methyluridine-3'-phosphate
us 2'-0-methyluridine-3'-phosphorothioate
phosphorothioate linkage
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Abbreviation Nucleotide(s)
L96 N-Itris(Ga1NAc-alkyl)-amidodecanoy1)]-4-hydroxyprolinol
Hyp-(GalNAc-alky1)3
(dT) T -deoxythymidine-3 -phosphate
Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2'-
0Me
furanose)
Y44 2-hydroxymethyl-tetrahydrofurane-5-phosphate
(Agn) Adenosine-glycol nucleic acid (GNA)
(Tgn) Thymidine-glycol nucleic acid (GNA) S-Isomer
(Cgn) Cytidine-glycol nucleic acid (GNA)
P Phosphate
VP Vinyl-phosphate
99
Table 2. Exemplary Unmodified Sense and Antisense Strand Sequences of HBV
dsRNAs (Activity data available in W02016/077321, incorporated
herein by reference)
0
n.)
o
1-,
oe
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3') vD
Name Name ID NO Oligo Name
NO: NC_003977.1 vi
1-,
c:
AD-61522 A-123463 AGUUAUAUGGAUGAUGUGGUA 47 A-123464 UACCACAUCAUCCAUAUAACUGA
263 731_753 vi
AD-61547 A-123487 GGAUGUGUCUGCGGCGUUUUA 48 A-123488 UAAAACGCCGCAGACACAUCCAG
264 373_395
AD-63938 A-127896 ACUCGUGGUGGACUUCUCUCA 49 A-127897 UGAGAGAAGUCCACCACGAGUCU
265 250_272
AD-63939 A-127909 ACUCGUGGUGGACUUCUCUCA 50 A-127906 UGAGAGAAGUCCACCACGAGUCU
266 250_272
AD-63940 A-127917 ACUCGUGGUGGACUUCTCUCA 51 A-127906 UGAGAGAAGUCCACCACGAGUCU
267 250_272
AD-63941 A-127905 ACUCGUGGUGGACUUCUCUCA 52 A-127925 UGAGAGAAGUCCACCACGAGUCU
268 250_272
AD-63942 A-127933 UCGUGGUGGACUUCUCUCA 53 A-127934 UGAGAGAAGUCCACCACGAGU 269
252_274
P
AD-63943 A-127944 ACUCGUGGUGGACUUCUCUCA 54 A-127942 UGAGAGAAGUCCACCACGAGUCU
270 250_272
c,
AD-63945 A-127910 ACUCGUGGUGGACUUCUCUCA 55 A-127906 UGAGAGAAGUCCACCACGAGUCU
271 250_272
AD-63946 A-127918 ACUCGUGGUGGACUUCUCUCA 56 A-127906 UGAGAGAAGUCCACCACGAGUCU
272 250_272 .
c,
AD-63947 A-127905 ACUCGUGGUGGACUUCUCUCA 57 A-127926 UGAGAGAAGUCCACCACGAGUCU
273 250_272
,
,-,
AD-63948 A-127935 GUGGUGGACUUCUCUCA 58 A-127936
UGAGAGAAGUCCACCACGA 274 254_276 ,
c,
.3
AD-63949 A-127944 ACUCGUGGUGGACUUCUCUCA 59 A-127906 UGAGAGAAGUCCACCACGAGUCU
275 250_272
AD-63950 A-127900 UCGUGGUGGACUUCUCUCAUU 60 A-127901 UGAGAGAAGUCCACCACGAUU 276
252_274
AD-63951 A-127911 ACUCGUGGUGGACUUCUCUCA 61 A-127906 UGAGAGAAGUCCACCACGAGUCU
277 250_272
AD-63952 A-127905 ACUCGUGGUGGACUUCUCUCA 62 A-127919 UGAGAGAAGUCCACCACGAGUCU
278 250_272
AD-63953 A-127905 ACUCGUGGUGGACUUCUCUCA 63 A-127927 UGAGAGAAGUCCACCACGAGUCU
279 250_272
AD-63955 A-127945 ACUCGUGGUGGACUUCUCUCA 64 A-127940 UGAGAGAAGUCCACCACGAGUCU
280 250_272 Iv
n
AD-63956 A-127902 UCGUGGUGGACUUCUCUCA 65 A-127903 UGAGAGAAGUCCACCACGAUU 281
252_274 1-3
AD-63957 A-127912 ACUCGUGGUGGACUUCUCUCA 66 A-127906 UGAGAGAAGUCCACCACGAGUCU
282 250_272
cp
n.)
AD-63958 A-127905 ACUCGUGGUGGACUUCUCUCA 67 A-127920 UGAGAGAAGUCCACCACGAGUCU
283 250_272 o
1-,
oe
AD-63959 A-127905 ACUCGUGGUGGACUUCUCUCA 68 A-127928 UGAGAGAAGUCCACCACGAGUCU
284 250_272
t..)
oe
AD-63960 A-126619 UAUUUCCUAGGGUACAA 69 A-127938
UGAGAGAAGUCCACCACGA 285 254_276
1-,
c:
AD-63961 A-127945 ACUCGUGGUGGACUUCUCUCA 70 A-127942 UGAGAGAAGUCCACCACGAGUCU
286 250_272
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD -63962 A-127902 UCGUGGUGGACUUCUCUCA 71 A-127904
UGAGAGAAGUCCACCACGAUU 287 252_274 0
n.)
AD -63963 A-127913 ACUCGUGGUGGACUUCUCUCA 72 A-127906
UGAGAGAAGUCCACCACGAGUCU 288 250_272
oe
AD -63964 A-127905 ACUCGUGGUGGACUUCUCUCA 73 A-127921
UGAGAGAAGUCCACCACGAGUCU 289 250_272
vi
AD -63965 A-127905 ACUCGUGGUGGACUUCUCUCA 74 A-127929
UGAGAGAAGUCCACCACGAGUCU 290 250_272
c:
vi
AD -63966 A-127939 ACUCGUGGUGGACUUCUCUCA 75 A-127940
UGAGAGAAGUCCACCACGAGUCU 291 250_272
AD -63967 A-127945 ACUCGUGGUGGACUUCUCUCA 76 A-127906
UGAGAGAAGUCCACCACGAGUCU 292 250_272
AD -63968 A-127905 ACUCGUGGUGGACUUCUCUCA 77 A-127906
UGAGAGAAGUCCACCACGAGUCU 293 250_272
AD -63968 A-127905 ACUCGUGGUGGACUUCUCUCA 78 A-127906
UGAGAGAAGUCCACCACGAGUCU 294 250_272
AD -63968 A-127905 ACUCGUGGUGGACUUCUCUCA 79 A-127906
UGAGAGAAGUCCACCACGAGUCU 295 250_272
AD -63969 A-127914 ACUCGUGGUGGACUUCUCUCA 80 A-127906
UGAGAGAAGUCCACCACGAGUCU 296 250_272
AD -63970 A-127905 ACUCGUGGUGGACUUCUCUCA 81 A-127922
UGAGAGAAGUCCACCACGAGUCU 297 250_272 Q
AD -63971 A-127905 ACUCGUGGUGGACUUCUCUCA 82 A-127930
UGAGAGAAGUCCACCACGAGUCU 298 250_272
u,
AD -63972 A-127941 ACUCGUGGUGGACUUCUCUCA 83 A-127942
UGAGAGAAGUCCACCACGAGUCU 299 250_272 .
'-c->
.
AD -63973 A-127946 ACUCGUGGUGGACUUCUCUCA 84 A-127947
UGAGAGAAGTCCACCACGAGUCU 300 250_272 " ,
' AD -63975 A-127915 ACUCGUGGUGGACUUCTCUCA
85 A-127906 UGAGAGAAGUCCACCACGAGUCU
301 250_272 ,
.
,
AD -63976 A-127905 ACUCGUGGUGGACUUCUCUCA 86 A-127923
UGAGAGAAGUCCACCACGAGUCU 302 250_272 ' .3
AD -63977 A-127917 ACUCGUGGUGGACUUCTCUCA 87 A-127931
UGAGAGAAGUCCACCACGAGUCU 303 250_272
AD -63978 A-127943 ACUCGUGGUGGACUUCUCUCA 88 A-127906
UGAGAGAAGUCCACCACGAGUCU 304 250_272
AD -63979 A-127908 ACUCGUGGUGGACUUCUCUCA 89 A-127906
UGAGAGAAGUCCACCACGAGUCU 305 250_272
AD -63980 A-127916 ACUCGUGGUGGACUUCTCUCA 90 A-127906
UGAGAGAAGUCCACCACGAGUCU 306 250_272
AD -63981 A-127905 ACUCGUGGUGGACUUCUCUCA 91 A-127924
UGAGAGAAGUCCACCACGAGUCU 307 250_272
AD -63982 A-127917 ACUCGUGGUGGACUUCTCUCA 92 A-127932
UGAGAGAAGUCCACCACGAGUCU 308 250_272 Iv
n
AD -63983 A-127944 ACUCGUGGUGGACUUCUCUCA 93 A-127940
UGAGAGAAGUCCACCACGAGUCU 309 250_272
cp
AD -63985 A-127961 GUGGUGGACUUCUCUCAAUUU 94 A-127956
AAAUUGAGAGAAGUCCACCACGA 310 254_276 n.)
1¨,
AD -63986 A-127969 GUGGUGGACUUCUCUCAAUUU 95 A-127956
AAAUUGAGAGAAGUCCACCACGA 311 254_276 0 e
AD -63987 A-127955 GUGGUGGACUUCUCUCAAUUU 96 A-127977
AAAUUGAGAGAAGUCCACCACGA 312 254_276 n.)
oe
1¨,
AD -63988 A-127986 UGGACUUCUCUCAAUUU 97 A-127987
AAAUUGAGAGAAGUCCACC 313 258_280
cr
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD-63989 A-127996 GUGGUGGACUUCUCUCAAUUU 98 A-127992 AAAUUGAGAGAAGUCCACCACGA
314 254_276 0
n.)
AD-63990 A-127950 GGUGGACUUCUCUCAAUUUUU 99 A-127951 AAAUUGAGAGAAGUCCACCUU 315
256_278 1--,
oe
AD-63991 A-127962 GUGGUGGACUUCUCUCAAUUU 100 A-127956 AAAUUGAGAGAAGUCCACCACGA
316 254_276 1--,
vi
AD-63992 A-127955 GUGGUGGACUUCUCUCAAUUU 101 A-127970 AAAUUGAGAGAAGUCCACCACGA
317 254_276 1--,
c:
vi
AD-63993 A-127955 GUGGUGGACUUCUCUCAAUUU 102 A-127978 AAAUUGAGAGAAGUCCACCACGA
318 254_276
AD-63994 A-127984 GGUGGACUUCUCUCAAUUU 103 A-127988 AAAUUGAGAGAAGUCCACCAC 319
256_278
AD-63995 A-127996 GUGGUGGACUUCUCUCAAUUU 104 A-127993 AAAUUGAGAGAAGUCCACCACGA
320 254_276
AD-63996 A-127952 GGUGGACUUCUCUCAAUUU 105 A-127953 AAAUUGAGAGAAGUCCACCUU 321
256_278
AD-63997 A-127963 GUGGUGGACUUCUCUCAAUUU 106 A-127956 AAAUUGAGAGAAGUCCACCACGA
322 254_276
AD-63999 A-127955 GUGGUGGACUUCUCUCAAUUU 107 A-127979 AAAUUGAGAGAAGUCCACCACGA
323 254_276
AD-64000 A-127986 UGGACUUCUCUCAAUUU 108 A-127989
AAAUUGAGAGAAGUCCACC 324 258_280 Q
AD-64001 A-127996 GUGGUGGACUUCUCUCAAUUU 109 A-127994 AAAUUGAGAGAAGUCCACCACGA
325 254_276
u,
AD-64002 A-127952 GGUGGACUUCUCUCAAUUU 110 A-127954 AAAUUGAGAGAAGUCCACCUU 326
256_278 .
'-c->
.
t.) AD-64003 A-127964 GUGGUGGACUUCUCUCAAUUU 111 A-127956
AAAUUGAGAGAAGUCCACCACGA 327 254_276 " ,
' AD-64004 A-127955 GUGGUGGACUUCUCUCAAUUU 112 A-127972 AAAUUGAGAGAAGUCCACCACGA
328 254_276 ,
.
,
AD-64005 A-127955 GUGGUGGACUUCUCUCAAUUU 113 A-127980 AAAUUGAGAGAAGUCCACCACGA
329 254_276 .3
AD-64006 A-127990 GUGGUGGACUUCUCUCAAUUU 114 A-127991 AAAUUGAGAGAAGUCCACCACGA
330 254_276
AD-64007 A-127996 GUGGUGGACUUCUCUCAAUUU 115 A-127995 AAAUUGAGAGAAGUCCACCACGA
331 254_276
AD-64008 A-127955 GUGGUGGACUUCUCUCAAUUU 116 A-127956 AAAUUGAGAGAAGUCCACCACGA
332 254_276
AD-64008 A-127955 GUGGUGGACUUCUCUCAAUUU 117 A-127956 AAAUUGAGAGAAGUCCACCACGA
333 254_276
AD-64009 A-127965 GUGGUGGACUUCUCUCAAUUU 118 A-127956 AAAUUGAGAGAAGUCCACCACGA
334 254_276
AD-64010 A-127955 GUGGUGGACUUCUCUCAAUUU 119 A-127973 AAAUUGAGAGAAGUCCACCACGA
335 254_276 Iv
n
AD-64011 A-127955 GUGGUGGACUUCUCUCAAUUU 120 A-127981 AAAUUGAGAGAAGUCCACCACGA
336 254_276
cp
AD-64012 A-127990 GUGGUGGACUUCUCUCAAUUU 121 A-127992 AAAUUGAGAGAAGUCCACCACGA
337 254_276 n.)
1--,
AD-64013 A-127997 GUGGUGGACTTCUCUCAAUUU 122 A-127998 AAAUUGAGAGAAGTCCACCACGA
338 254_276 0 e
AD-64014 A-127957 GUGGUGGACUUCUCUCAAUUU 123 A-127958 AAAUUGAGAGAAGUCCACCACGA
339 254_276 n.)
oe
1--,
AD-64015 A-127966 GUGGUGGACUUCUCUCAAUUU 124 A-127956 AAAUUGAGAGAAGUCCACCACGA
340 254_276 1--,
cr
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD-64016 A-127955 GUGGUGGACUUCUCUCAAUUU 125 A-127974 AAAUUGAGAGAAGUCCACCACGA
341 254_276 0
n.)
AD-64017 A-127968 GUGGUGGACUTCUCUCAAUUU 126 A-127982 AAAUUGAGAGAAGTCCACCACGA
342 254_276 1--,
oe
AD-64018 A-127990 GUGGUGGACUUCUCUCAAUUU 127 A-127993 AAAUUGAGAGAAGUCCACCACGA
343 254_276 1--,
vi
AD-64019 A-127959 GUGGUGGACUUCUCUCAAUUU 128 A-127956 AAAUUGAGAGAAGUCCACCACGA
344 254_276 1--,
c:
vi
AD-64020 A-127967 GUGGUGGACUUCUCUCAAUUU 129 A-127956 AAAUUGAGAGAAGUCCACCACGA
345 254_276
AD-64021 A-127955 GUGGUGGACUUCUCUCAAUUU 130 A-127975 AAAUUGAGAGAAGUCCACCACGA
346 254_276
AD-64022 A-127968 GUGGUGGACUTCUCUCAAUUU 131 A-127983 AAAUUGAGAGAAGTCCACCACGA
347 254_276
AD-64023 A-127990 GUGGUGGACUUCUCUCAAUUU 132 A-127994 AAAUUGAGAGAAGUCCACCACGA
348 254_276
AD-64024 A-127960 GUGGUGGACUUCUCUCAAUUU 133 A-127956 AAAUUGAGAGAAGUCCACCACGA
349 254_276
AD-64025 A-127968 GUGGUGGACUTCUCUCAAUUU 134 A-127956 AAAUUGAGAGAAGUCCACCACGA
350 254_276
AD-64026 A-127955 GUGGUGGACUUCUCUCAAUUU 135 A-127976 AAAUUGAGAGAAGUCCACCACGA
351 254_276 Q
AD-64027 A-127984 GGUGGACUUCUCUCAAUUU 136 A-127985 AAAUUGAGAGAAGUCCACCAC 352
256_278
u,
AD-64028 A-127990 GUGGUGGACUUCUCUCAAUUU 137 A-127995 AAAUUGAGAGAAGUCCACCACGA
353 254_276 .
'-c->
.
w AD-64272 A-128001 GUGCACUUCGCUUCACCUCUG 138 A-128002 CAGAGGUGAAGCGAAGUGCACAC
354 1577_1599 " ,
' AD-64274 A-128363 GUUGACAAAAAUCCUCACAAU 139 A-128364 AUUGUGAGGAUUUUUGUCAACAA
355 215_237 ,
.
,
AD-64275 A-128377 UGUUGACAAAAAUCCUCACAA 140 A-128378 UUGUGAGGAUUUUUGUCAACAAG
356 214_236 ' .3
AD-64276 A-128393 GGUGGACUUCUCUCAAUUUUA 141 A-128394 UAAAAUUGAGAGAAGUCCACCAC
357 256_278
AD-64277 A-128407 UCUUUUGGAGUGUGGAUUCGA 142 A-128408 UCGAAUCCACACUCCAAAAGACA
358 2259_2281
AD-64277 A-128407 UCUUUUGGAGUGUGGAUUCGA 143 A-128408 UCGAAUCCACACUCCAAAAGACA
359 2259_2281
AD-64278 A-128423 ACUGUUCAAGCCUCCAAGCUA 144 A-128424 UAGCUUGGAGGCUUGAACAAGAC
360 1857_1879
AD-64279 A-128435 UCUGCCGAUCCAUACUGCGGA 145 A-128436 UCCGCAGUAUGGAUCGGCAGAGG
361 1255_1277
AD-64280 A-128379 AUGUGUCUGCGGCGUUUUAUA 146 A-128380 UAUAAAACGCCGCAGACACAUCC
362 375_397 Iv
n
AD-64281 A-128395 CCCCGUCUGUGCCUUCUCAUA 147 A-128396 UAUGAGAAGGCACAGACGGGGAG
363 1545_1567
cp
AD-64282 A-128409 GCCUAAUCAUCUCUUGUUCAU 148 A-128410 AUGAACAAGAGAUGAUUAGCGAG
364 1831_1853 n.)
1--,
AD-64283 A-128425 UCUAGACUCGUGGUGGACUUC 149 A-128426 GAAGUCCACCACGAGUCUAGACU
365 245_267 0 e
AD-64284 A-128437 CUGCCGAUCCAUACUGCGGAA 150 A-128438 UUCCGCAGUAUGGAUCGGCAGAG
366 1256_1278 n.)
oe
1--,
AD-64285 A-128365 UUUUUCUUGUUGACAAAAAUA 151 A-128366 UAUUUUUGUCAACAAGAAAAACC
367 207_229 1--,
cr
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD -64286 A-128381 AUCUUCUUGUUGGUUCUUCUA 152 A-128382
UAGAAGAACCAACAAGAAGAUGA 368 426_448 0
n.)
AD -64289 A-128367 GUUUUUCUUGUUGACAAAAAU 153 A-128368
AUUUUUGUCAACAAGAAAAACCC 369 206_228 1--,
oe
AD -64290 A-128383 CUGCCUAAUCAUCUCUUGUUA 154 A-128384
UAACAAGAGAUGAUUAGGCAGAG 370 1829_1851 1--,
vi
AD -64291 A-128399 UCCUCACAAUACCACAGAGUA 155 A-128400
UACUCUGUGGUAUUGUGAGGAUU 371 226_248 1--,
c:
vi
AD -64292 A-128413 CUUGUUGACAAAAAUCCUCAA 156 A-128414
UUGAGGAUUUUUGUCAACAAGAA 372 212_234
AD -64293 A-128439 GCAACUUUUUCACCUCUGCCU 157 A-128440
AGGCAGAGGUGAAAAAGUUGCAU 373 1814_1836
AD -64294 A-128369 GGGAACAAGAGCUACAGCAUA 158 A-128370
UAUGCUGUAGCUCUUGUUCCCAA 374 2828_2850
AD -64295 A-128385 CGUGGUGGACUUCUCUCAAUU 159 A-128386
AAUUGAGAGAAGUCCACCAGCAG 375 253_275
AD -64297 A-128415 CUGCUGCUAUGCCUCAUCUUA 160 A-128416
UAAGAUGAGGCAUAGCAGCAGGA 376 411_433
AD -64298 A-128427 GUUGGAUGUGUCUGCGGCGUU 161 A-128428
AACGCCGCAGACACAUCCAACGA 377 370_392
AD -64299 A-128441 UUCAUCCUGCUGCUAUGCCUA 162 A-128442
UAGGCAUAGCAGCAGGAUGAAGA 378 405_427 Q
AD -64300 A-128371 UUCUUGUUGACAAAAAUCCUA 163 A-128372
UAGGAUUUUUGUCAACAAGAAAA 379 210_232
u,
AD -64302 A-128417 UAUAUGGAUGAUGUGGUAUUA 164 A-128418
UAAUACCACAUCAUCCAUAUAAC 380 734_756 .
'-c->
.
-i. AD -64303 A-128429 UUCAUCCUGCUGCUAUGCCUC 165 A-128430
GAGGCAUAGCAGCAGGAUGAAGA 381 405_427 " ,
' AD -64304 A-128443 GUGCACUUCGCUUCACCUCUA
166 A-128444 UAGAGGUGAAGCGAAGUGCAC AC
382 1577_1599 ,
.
,
AD -64305 A-128373 UUGACAAAAAUCCUCACAAUA 167 A-128374
UAUUGUGAGGAUUUUUGUCAACA 383 216_238 ' .3
AD -64307 A-128403 AAGCCUCCAAGCUGUGCCUUA 168 A-128404
UAAGGCACAGCUUGGAGGCUUGA 384 1864_1886
AD -64308 A-128419 CCUCUUCAUCCUGCUGCUAUA 169 A-128420
UAUAGCAGCAGGAUGAAGAGGAA 385 401_423
AD -64309 A-128431 CCUGCUGCUAUGCCUCAUCUU 170 A-128432
AAGAUGAGGCAUAGCAGCAGGAU 386 410_432
AD -64310 A-128375 CAUCUUCUUGUUGGUUCUUCU 171 A-128376
AGAAGAACCAACAAGAAGAUGAG 387 425_447
AD -64311 A-128391 CCGUCUGUGCCUUCUCAUCUA 172 A-128392
UAGAUGAGAAGGCACAGACGGGG 388 1547_1569
AD -64312 A-128405 CCUCAUCUUCUUGUUGGUUCU 173 A-128406
AGAACCAACAAGAAGAUGAGGCA 389 422_444 Iv
n
AD -64313 A-128421 CCACCAAAUGCCCCUAUCUUA 174 A-128422
UAAGAUAGGGGCAUUUGGUGGUC 390 2298_2320
cp
AD -64314 A-128433 GCUCCUCUGCCGAUCCAUACU 175 A-128434
AGUAUGGAUCGGCAGAGGAGCCA 391 1250_1272 n.)
1--,
AD -64315 A-128363 GUUGACAAAAAUCCUCACAAU 176 A-128445
AUUGUGAGGAUUUUUGUCAACAA 392 215_237 0 e
AD -64316 A-128377 UGUUGACAAAAAUCCUCACAA 177 A-128453
UUGUGAGGAUUUUUGUCAACAAG 393 214_236 n.)
oe
1--,
AD -64317 A-128393 GGUGGACUUCUCUCAAUUUUA 178 A-128461
UAAAAUUGAGAGAAGUCCACCAC 394 256_278 1--,
cr
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD-64318 A-128407 UCUUUUGGAGUGUGGAUUCGA 179 A-128469 UCGAAUCCACACUCCAAAAGACA
395 2259_2281 0
n.)
AD-64318 A-128407 UCUUUUGGAGUGUGGAUUCGA 180 A-128469 UCGAAUCCACACUCCAAAAGACA
396 2259_2281 1--,
oe
AD-64319 A-128423 ACUGUUCAAGCCUCCAAGCUA 181 A-128477 UAGCUUGGAGGCUUGAACAAGAC
397 1857_1879 1--,
vi
AD-64320 A-128435 UCUGCCGAUCCAUACUGCGGA 182 A-128483 UCCGCAGUAUGGAUCGGCAGAGG
398 1255_1277 1--,
c:
vi
AD-64321 A-123463 AGUUAUAUGGAUGAUGUGGUA 183 A-128446 UACCACAUCAUCCAUAUAACUGA
399 731_753
AD-64322 A-128379 AUGUGUCUGCGGCGUUUUAUA 184 A-128454 UAUAAAACGCCGCAGACACAUCC
400 375_397
AD-64323 A-128395 CCCCGUCUGUGCCUUCUCAUA 185 A-128462 UAUGAGAAGGCACAGACGGGGAG
401 1545_1567
AD-64324 A-128409 GCCUAAUCAUCUCUUGUUCAU 186 A-128470 AUGAACAAGAGAUGAUUAGCGAG
402 1831_1853
AD-64325 A-128425 UCUAGACUCGUGGUGGACUUC 187 A-128478 GAAGUCCACCACGAGUCUAGACU
403 245_267
AD-64326 A-128437 CUGCCGAUCCAUACUGCGGAA 188 A-128484 UUCCGCAGUAUGGAUCGGCAGAG
404 1256_1278
AD-64328 A-128381 AUCUUCUUGUUGGUUCUUCUA 189 A-128455 UAGAAGAACCAACAAGAAGAUGA
405 426_448 Q
AD-64330 A-128411 UUCUCUCAAUUUUCUAGGGGA 190 A-128471 UCCCCUAGAAAAUUGAGAGAAGU
406 263_285
u,
AD-64331 A-127905 ACUCGUGGUGGACUUCUCUCA 191 A-127907 UGAGAGAAGUCCACCACGAGUCU
407 250_272 .
'-c->
.
(.., AD-64332 A-128001 GUGCACUUCGCUUCACCUCUG 192 A-128485
CAGAGGUGAAGCGAAGUGCACAC 408 1577_1599 " ,
' AD-64333 A-128367 GUUUUUCUUGUUGACAAAAAU 193 A-128448 AUUUUUGUCAACAAGAAAAACCC
409 206_228 ,
.
,
AD-64334 A-128383 CUGCCUAAUCAUCUCUUGUUA 194 A-128456 UAACAAGAGAUGAUUAGGCAGAG
410 1829_1851 .3
AD-64335 A-128399 UCCUCACAAUACCACAGAGUA 195 A-128464 UACUCUGUGGUAUUGUGAGGAUU
411 226_248
AD-64336 A-128413 CUUGUUGACAAAAAUCCUCAA 196 A-128472 UUGAGGAUUUUUGUCAACAAGAA
412 212_234
AD-64337 A-127955 GUGGUGGACUUCUCUCAAUUU 197 A-127958 AAAUUGAGAGAAGUCCACCACGA
413 254_276
AD-64338 A-128439 GCAACUUUUUCACCUCUGCCU 198 A-128486 AGGCAGAGGUGAAAAAGUUGCAU
414 1814_1836
AD-64339 A-128369 GGGAACAAGAGCUACAGCAUA 199 A-128449 UAUGCUGUAGCUCUUGUUCCCAA
415 2828_2850
AD-64341 A-128401 UCAUCUUCUUGUUGGUUCUUA 200 A-128465 UAAGAACCAACAAGAAGAUGAGG
416 424_446 Iv
n
AD-64342 A-128415 CUGCUGCUAUGCCUCAUCUUA 201 A-128473 UAAGAUGAGGCAUAGCAGCAGGA
417 411_433
cp
AD-64343 A-128427 GUUGGAUGUGUCUGCGGCGUU 202 A-128479 AACGCCGCAGACACAUCCAACGA
418 370_392 n.)
1--,
AD-64344 A-128441 UUCAUCCUGCUGCUAUGCCUA 203 A-128487 UAGGCAUAGCAGCAGGAUGAAGA
419 405_427 0 e
AD-64345 A-128371 UUCUUGUUGACAAAAAUCCUA 204 A-128450 UAGGAUUUUUGUCAACAAGAAAA
420 210_232 n.)
oe
1--,
AD-64347 A-123487 GGAUGUGUCUGCGGCGUUUUA 205 A-128466 UAAAACGCCGCAGACACAUCCAG
421 373_395 1--,
cr
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD-64348 A-128417 UAUAUGGAUGAUGUGGUAUUA 206 A-128474 UAAUACCACAUCAUCCAUAUAAC
422 734_756 0
n.)
AD-64349 A-128429 UUCAUCCUGCUGCUAUGCCUC 207 A-128480 GAGGCAUAGCAGCAGGAUGAAGA
423 405_427
oe
AD-64350 A-128443 GUGCACUUCGCUUCACCUCUA 208 A-128488 UAGAGGUGAAGCGAAGUGCACAC
424 1577_1599
vi
AD-64351 A-128373 UUGACAAAAAUCCUCACAAUA 209 A-128451 UAUUGUGAGGAUUUUUGUCAACA
425 216_238
c:
vi
AD-64352 A-128389 CCAAGUGUUUGCUGACGCAAA 210 A-128459 UUUGCGUCAGCAAACACUUGGCA
426 1174_1196
AD-64352 A-128389 CCAAGUGUUUGCUGACGCAAA 211 A-128459 UUUGCGUCAGCAAACACUUGGCA
427 1174_1196
AD-64353 A-128403 AAGCCUCCAAGCUGUGCCUUA 212 A-128467 UAAGGCACAGCUUGGAGGCUUGA
428 1864_1886
AD-64354 A-128419 CCUCUUCAUCCUGCUGCUAUA 213 A-128475 UAUAGCAGCAGGAUGAAGAGGAA
429 401_423
AD-64355 A-128431 CCUGCUGCUAUGCCUCAUCUU 214 A-128481 AAGAUGAGGCAUAGCAGCAGGAU
430 410_432
AD-64356 A-128375 CAUCUUCUUGUUGGUUCUUCU 215 A-128452 AGAAGAACCAACAAGAAGAUGAG
431 425_447
AD-64357 A-128391 CCGUCUGUGCCUUCUCAUCUA 216 A-128460 UAGAUGAGAAGGCACAGACGGGG
432 1547_1569 Q
AD-64358 A-128405 CCUCAUCUUCUUGUUGGUUCU 217 A-128468 AGAACCAACAAGAAGAUGAGGCA
433 422_444
u,
AD-64359 A-128421 CCACCAAAUGCCCCUAUCUUA 218 A-128476 UAAGAUAGGGGCAUUUGGUGGUC
434 2298_2320 .
'-c->
.
cs, AD-64360 A-128433 GCUCCUCUGCCGAUCCAUACU 219 A-128482
AGUAUGGAUCGGCAGAGGAGCCA 435 1250_1272 " ,
' AD-64700 A-129379 ACUCGUGGUGTACUUCUCUCA 220 A-127906 UGAGAGAAGUCCACCACGAGUCU
436 250_272 ,
.
,
AD-64701 A-127905 ACUCGUGGUGGACUUCUCUCA 221 A-129387 UGAGAGAAGTCCACCACGAGUCU
437 250_272 ' .3
AD-64702 A-127905 ACUCGUGGUGGACUUCUCUCA 222 A-129395 UGAGAGAAGUCCACCACGAGUCU
438 250_272
AD-64703 A-129376 ACUCGUGGUGGACUUCACUCA 223 A-129385 UGAGAGAAGTCCACCACGAGUCU
439 250_272
AD-64704 A-129381 ACUCGUGGTGTACUUCACUCA 224 A-129389 UGAGAGAAGUCCACCACGAGUCU
440 250_272
AD-64705 A-129380 ACUCGUGGUGTACUUCACUCA 225 A-127906 UGAGAGAAGUCCACCACGAGUCU
441 250_272
AD-64706 A-127905 ACUCGUGGUGGACUUCUCUCA 226 A-129388 UGAGAGAAGUCCACCACGAGUCU
442 250_272
AD-64707 A-127905 ACUCGUGGUGGACUUCUCUCA 227 A-129396 UGAGAGAAGTCCACCACGAGUCU
443 250_272 Iv
n
AD-64708 A-129382 ACUCGUGGTGGACUUCTCUCA 228 A-129385 UGAGAGAAGTCCACCACGAGUCU
444 250_272
cp
AD-64709 A-129373 ACUCGUGGUGGACUUCUCUCA 229 A-129391 UGAGAGAAGTCCACCACGAGUCU
445 250_272 n.)
1¨,
AD-64710 A-129373 ACUCGUGGUGGACUUCUCUCA 230 A-127906 UGAGAGAAGUCCACCACGAGUCU
446 250_272 0 e
AD-64711 A-129381 ACUCGUGGTGTACUUCACUCA 231 A-127906 UGAGAGAAGUCCACCACGAGUCU
447 250_272 n.)
oe
1¨,
AD-64712 A-127905 ACUCGUGGUGGACUUCUCUCA 232 A-129389 UGAGAGAAGUCCACCACGAGUCU
448 250_272
cr
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD -64713 A-127905 ACUCGUGGUGGACUUCUCUCA 233 A-129397
UGAGAGAAGTCCACCACGAGUCU 449 250_272 0
n.)
AD -64714 A-129384 ACUCGUGGTGGACUUCACUCA 234 A-129385
UGAGAGAAGTCCACCACGAGUCU 450 250_272 1--,
oe
AD -64715 A-129376 ACUCGUGGUGGACUUCACUCA 235 A-129391
UGAGAGAAGTCCACCACGAGUCU 451 250_272 1--,
vi
AD -64716 A-129374 ACUCGUGGUGGACUUCUCUCA 236 A-127906
UGAGAGAAGUCCACCACGAGUCU 452 250_272 1--,
c:
vi
AD -64717 A-129382 ACUCGUGGTGGACUUCTCUCA 237 A-127906
UGAGAGAAGUCCACCACGAGUCU 453 250_272
AD -64718 A-127905 ACUCGUGGUGGACUUCUCUCA 238 A-129390
UGAGAGAAGUCCACCACGAGUCU 454 250_272
AD -64719 A-127917 ACUCGUGGUGGACUUCTCUCA 239 A-129385
UGAGAGAAGTCCACCACGAGUCU 455 250_272
AD -64720 A-129381 ACUCGUGGTGTACUUCACUCA 240 A-129385
UGAGAGAAGTCCACCACGAGUCU 456 250_272
AD -64721 A-129382 ACUCGUGGTGGACUUCTCUCA 241 A-129391
UGAGAGAAGTCCACCACGAGUCU 457 250_272
AD -64722 A-129375 ACUCGUGGUGGACUUCCUCA 242 A-127906
UGAGAGAAGUCCACCACGAGUCU 458 250_272
AD -64723 A-129383 ACUCGUGGUGGACUUCTCUCA 243 A-127906
UGAGAGAAGUCCACCACGAGUCU 459 250_272 Q
AD -64725 A-127917 ACUCGUGGUGGACUUCTCUCA 244 A-129398
UGAGAGAAGTCCACCACGAGUCU 460 250_272
u,
AD -64726 A-129373 ACUCGUGGUGGACUUCUCUCA 245 A-129389
UGAGAGAAGUCCACCACGAGUCU 461 250_272 .
'-c->
.
---.1 AD -64727 A-129384 ACUCGUGGTGGACUUCACUCA
246 A-129391 UGAGAGAAGTCCACCACGAGUCU
462 250_272 " ,
' AD -64728 A-129376 ACUCGUGGUGGACUUCACUCA
247 A-127906 UGAGAGAAGUCCACCACGAGUCU
463 250_272 ,
.
,
AD -64729 A-129384 ACUCGUGGTGGACUUCACUCA 248 A-127906
UGAGAGAAGUCCACCACGAGUCU 464 250_272 ' .3
AD -64730 A-127905 ACUCGUGGUGGACUUCUCUCA 249 A-129392
UGAGAGAAGTCCACCACGAGUCU 465 250_272
AD -64731 A-129399 ACUCGUGGUGGACUUCTCUCA 250 A-129385
UGAGAGAAGTCCACCACGAGUCU 466 250_272
AD -64732 A-129376 ACUCGUGGUGGACUUCACUCA 251 A-129389
UGAGAGAAGUCCACCACGAGUCU 467 250_272
AD -64733 A-129381 ACUCGUGGTGTACUUCACUCA 252 A-129391
UGAGAGAAGTCCACCACGAGUCU 468 250_272
AD -64734 A-129377 ACUCGUGGUGGACUUCCCUCA 253 A-127906
UGAGAGAAGUCCACCACGAGUCU 469 250_272
AD -64735 A-127905 ACUCGUGGUGGACUUCUCUCA 254 A-129385
UGAGAGAAGTCCACCACGAGUCU 470 250_272 Iv
n
AD -64736 A-127905 ACUCGUGGUGGACUUCUCUCA 255 A-129393
UGAGAGAAGTCCACCACGAGUCU 471 250_272
cp
AD -64737 A-129399 ACUCGUGGUGGACUUCTCUCA 256 A-129398
UGAGAGAAGTCCACCACGAGUCU 472 250_272 n.)
1--,
AD -64738 A-129382 ACUCGUGGTGGACUUCTCUCA 257 A-129389
UGAGAGAAGUCCACCACGAGUCU 473 250_272 0 e
AD -64739 A-129378 ACUCGUGGUGGACUUCGCUCA 258 A-127906
UGAGAGAAGUCCACCACGAGUCU 474 250_272 n.)
oe
1--,
AD -64740 A-127905 ACUCGUGGUGGACUUCUCUCA 259 A-129386
UGAGAGAAGTCCACCACGAGUCU 475 250_272 1--,
cr
ME1 27037625v.1
Duplex Sense Oligo SEQ Antisense
SEQ ID Position in
Sense Sequence (5' to 3')
Antisense Sequence (5' to 3')
Name Name ID NO Oligo Name
NO: NC_003977.1
AD-64741 A-127905 ACUCGUGGUGGACUUCUCUCA 260 A-129394 UGAGAGAAGTCCACCACGAGUCU
476 250_272 0
n.)
o
AD-64742 A-129373 ACUCGUGGUGGACUUCUCUCA 261 A-129385 UGAGAGAAGTCCACCACGAGUCU
477 250_272
oe
AD-64743 A-129384 ACUCGUGGTGGACUUCACUCA 262 A-129389 UGAGAGAAGUCCACCACGAGUCU
478 250_272
un
1¨,
c:
u,
Table 3. Exemplary Modified Sense and Antisense Strand Sequences of HBV dsRNAs
(Activity data available in W02016/077321, incorporated herein
by reference)
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo
Name
NO:
NO:
AD-61522 A-123463 AfsgsUfuAfuAfuGfGfAfuGfaUfgUfgGfuAfL96 479 A-123464
usAfscCfaCfaUfcAfuccAfuAfuAfaCfusgsa 694 P
AD-61547 A-123487 GfsgsAfuGfuGfuCfUfGfcGfgCfgUfuUfuAfL96 480 A-123488
usAfsaAfaCfgCfcGfcagAfcAfcAfuCfcsasg 695
u,
AD-63938 A-127896 Y44ACUCGUGGUGGACUUCUCUCA
481 A-127897 UGAGAGAAGUCCACCACGAGUCU
696 .
cc AD-63939 A-127909 ascsucGfuGfgUfGfGfaCfuucUfcucaL96
482 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 697
,
,
AD-63940 A-127917 ascsucguggugdGacuuc(Tgn)cucaL96
483 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 698 ,
,
AD-63941 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 484 A-127925
usGfsaGfagaAfguccaCfcAfcgaGfuscsu 699 .
.3
AD-63942 A-127933 uscsGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 485 A-127934
usGfsaGfaGfaAfgUfccaCfcAfcGfasgsu 700
AD-63943 A-127944 ascsucGfuGfguGfGfaCfuucucucaL96 486 A-127942
usGfsAfgaGfaAfgUfccaCfcAfcGfaguscsu 701
AD-63945 A-127910 ascsucguGfgUfGfGfaCfuucUfcucaL96
487 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 702
AD-63946 A-127918 ascsucguGfgUfGfGfacuuCfucucaL96
488 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 703
AD-63947 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 489 A-127926
usGfsaGfagaagUfccaCfcAfcgaGfuscsu 704
IV
AD-63948 A-127935 gsusGfgUfGfGfaCfuUfcUfcUfcAfL96 490 A-127936
usGfsaGfaGfaAfgUfccaCfcAfcsgsa 705 n
AD-63949 A-127944 ascsucGfuGfguGfGfaCfuucucucaL96
491 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 706
cp
AD-63950 A-127900 Y44UfcGfuGfgUfgGfaCfuUfcUfcUfcAfusuY44 492 A-127901
usGfsasGfaGfaAfgUfcCfaCfcAfcGfausu 707 n.)
o
1¨,
AD-63951 A-127911 ascsucguGfgUfGfGfaCfuucucucaL96
493 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 708 0 e
AD-63952 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 494 A-127919
usGfsaGfaGfaagUfccaCfcAfcGfaGfuscsu 709 n.)
oe
1¨,
AD-63953 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 495 A-127927
usGfsagagaAfgUfccaCfcAfcgaguscsu 710
cr
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo
Name
NO:
NO: 0
n.)
AD-63955 A-127945 ascsucgugguGfGfacuucucucaL96 496 A-127940
usGfsAfgAfgAfaGfuccaCfCfaCfgAfguscsu 711
1¨,
oe
AD-63956 A-127902 Y44uscsGfuGfgUfgGfaCfuUfcUfcUfcAfY44 497 A-127903
usGfsaGfaGfaAfgUfcCfaCfcAfcGfasusu 712
un
AD-63957 A-127912 ascsucguGfgUfGfGfacuucucucaL96 498 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 713
cA
un
AD-63958 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 499 A-127920
usGfsagaGfaAfgUfccaCfcAfcgaGfuscsu 714
AD-63959 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 500 A-127928
usGfsaGfagaAfguccaCfcAfcgaguscsu 715
AD-63960 A-126619 usasUfuUfCfCfuAfgGfgUfaCfaAfL96 501 A-127938
PusGfsaGfaGfaAfgUfccaCfcAfcsgsa 716
AD-63961 A-127945 ascsucgugguGfGfacuucucucaL96 502 A-127942
usGfsAfgaGfaAfgUfccaCfcAfcGfaguscsu 717
AD-63962 A-127902 Y44uscsGfuGfgUfgGfaCfuUfcUfcUfcAfY44 503 A-127904
PusGfsaGfaGfaAfgUfcCfaCfcAfcGfasusu 718
AD-63963 A-127913 ascsucguggUfgGfacuucucucaL96 504 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 719
AD-63964 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 505 A-127921
usGfsaGfaGfaAfgUfccaCfcAfcgaguscsu 720 P
AD-63965 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 506 A-127929
usGfsagaGfaaGfuccaCfcAfcgaguscsu 721 .
L.
u9
AD-63966 A-127939 ascsUfcGfugguGfGfaCfuuCfuCfucaL96 507 A-127940
usGfsAfgAfgAfaGfuccaCfCfaCfgAfguscsu 722 ..'
s:) AD-63967 A-127945 ascsucgugguGfGfacuucucucaL96 508 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 723
,
AD-63968 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 509 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 724 .
,
,
AD-63968 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 510 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 725 ,
.3
AD-63968 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 511 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 726
AD-63969 A-127914 ascsucguggugGfacuucucucaL96 512 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 727
AD-63970 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 513 A-127922
usGfsagaGfaagUfccaCfcAfcgaGfuscsu 728
AD-63971 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 514 A-127930
usGfsagaGfaaguccaCfcAfcgaguscsu 729
AD-63972 A-127941 ascsUfcGfuGfguGfGfaCfuuCfuCfucaL96 515 A-127942
usGfsAfgaGfaAfgUfccaCfcAfcGfaguscsu 730
AD-63973 A-127946 ascsucguggudGdGacuucucucaL96 516 A-127947
usdGsaGfaGfaAfgdTccadCcAfcGfaguscsu 731 IV
n
AD-63975 A-127915 ascsucguggUfgGfacuuc(Tgn)cucaL96 517 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 732 1-3
AD-63976 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 518 A-127923
usGfsagaGfaAfgUfccaCfcAfcgaguscsu 733 cp
n.)
o
AD-63977 A-127917 ascsucguggugdGacuuc(Tgn)cucaL96 519 A-127931
usdGsagagaaguccadCcacgaguscsu 734
oe
C-5
AD-63978 A-127943 ascsUfcGfuGfguGfGfaCfuUfcUfcUfcaL96 520 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 735 n.)
oe
1¨,
AD-63979 A-127908 ascsucGfuGfgUfGfGfaCfuucUfcucAfL96 521 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 736
cA
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo
Name
NO:
NO: 0
n.)
AD-63980 A-127916 ascsucguggugGfacuuc(Tgn)cucaL96 522 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 737
1¨,
oe
AD-63981 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 523 A-127924
usGfsaGfagaAfgUfccaCfcAfcgaGfuscsu 738
un
AD-63982 A-127917 ascsucguggugdGacuuc(Tgn)cucaL96 524 A-127932
PusdGsagagaaguccadCcacgaguscsu 739
cA
un
AD-63983 A-127944 ascsucGfuGfguGfGfaCfuucucucaL96 525 A-127940
usGfsAfgAfgAfaGfuccaCfCfaCfgAfguscsu 740
AD-63985 A-127961 gsusggugGfaCfUfUfcUfcucAfauuuL96 526 A-127956
asAfsaUfuGfaGfaGfaagUfcCfaCfcAfcsgsa 741
AD-63986 A-127969 gsusggugGfaCfUfUfcucuCfaauuuL96 527 A-127956
asAfsaUfuGfaGfaGfaagUfcCfaCfcAfcsgsa 742
AD-63987 A-127955 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 528 A-127977
asAfsaUfugagaGfaagUfcCfaccAfcsgsa 743
AD-63988 A-127986 usgsGfaCfUfUfcUfcUfcAfaUfuUfL96 529 A-127987
asAfsaUfuGfaGfaGfaagUfcCfascsc 744
AD-63989 A-127996 gsusgguggacUfUfcucucaauuuL96 530 A-127992
asAfsAfUfuGfaGfaGfaagUfcCfaCfcacsgsa 745
AD-63990 A-127950 Y44GfgUfgGfaCfuUfcUfcUfcAfaUfuUfusuY44 531 A-127951
asAfsasUfuGfaGfaGfaAfgUfcCfaCfcusu 746 P
AD-63991 A-127962 gsusggugGfaCfUfUfcUfcucaauuuL96 532 A-127956
asAfsaUfuGfaGfaGfaagUfcCfaCfcAfcsgsa 747 .
L.
u9
AD-63992 A-127955 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 533 A-127970
asAfsaUfuGfagaGfaagUfcCfaCfcAfcsgsa 748 ..'
.
.
AD-63993 A-127955 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 534 A-127978
asAfsauugaGfaGfaagUfcCfaccacsgsa 749
,
AD-63994 A-127984 gsgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 535 A-127988
PasAfsaUfuGfaGfaGfaagUfcCfaCfcsasc 750 .
,
,
AD-63995 A-127996 gsusgguggacUfUfcucucaauuuL96 536 A-127993
asAfsAfuuGfaGfaGfaagUfCfcaCfcacsgsa 751 ,
.3
AD-63996 A-127952 Y44gsgsUfgGfaCfuUfcUfcUfcAfaUfuUfY44 537 A-127953
asAfsaUfuGfaGfaGfaAfgUfcCfaCfcsusu 752
AD-63997 A-127963 gsusggugGfaCfUfUfcucucaauuuL96 538 A-127956
asAfsaUfuGfaGfaGfaagUfcCfaCfcAfcsgsa 753
AD-63999 A-127955 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 539 A-127979
asAfsaUfugaGfagaagUfcCfaccacsgsa 754
AD-64000 A-127986 usgsGfaCfUfUfcUfcUfcAfaUfuUfL96 540 A-127989
PasAfsaUfuGfaGfaGfaagUfcCfascsc 755
AD-64001 A-127996 gsusgguggacUfUfcucucaauuuL96 541 A-127994
asAfsAfUfuGfaGfaGfaagUfCfcaCfcacsgsa 756
AD-64002 A-127952 Y44gsgsUfgGfaCfuUfcUfcUfcAfaUfuUfY44 542 A-127954
PasAfsaUfuGfaGfaGfaAfgUfcCfaCfcsusu 757 IV
n
AD-64003 A-127964 gsusgguggaCfuUfcucucaauuuL96 543 A-127956
asAfsaUfuGfaGfaGfaagUfcCfaCfcAfcsgsa 758 1-3
AD-64004 A-127955 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 544 A-127972
asAfsaUfuGfaGfaGfaagUfcCfaccacsgsa 759 cp
n.)
o
AD-64005 A-127955 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 545 A-127980
asAfsauuGfagAfgaagUfcCfaccacsgsa 760
oe
C-5
AD-64006 A-127990 gsusGfgugGfaCfUfUfcUfcUfcAfaUfuuL96 546 A-127991
asAfsaUfuGfaGfaGfaagUfcCfaCfcacsgsa 761 n.)
oe
1¨,
AD-64007 A-127996 gsusgguggacUfUfcucucaauuuL96 547 A-127995
asAfsAfUfugaGfaGfaagUfCfcaCfcacsgsa 762
cA
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo Name
NO:
NO: 0
n.)
AD -64008 A-127955
GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 548 A-127956 asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a 763 o
1¨,
oe
AD -64008 A-127955
GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 549 A-127956 asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a 764
un
AD -64009 A-127965 gsusgguggacuUfcucucaauuuL96
550 A-127956 asAfs aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a
765
cA
un
AD -64010 A-127955
GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 551 A-127973 asAfs
auuGfagaGfaagUfcCfaccAfc sg s a 766
AD -64011 A-127955
GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 552 A-127981 asAfs
auuGfagagaagUfcCfacc acsg s a 767
AD -64012 A-127990
gsusGfgugGfaCfUfUfcUfcUfcAfaUfuuL96 553 A-127992
asAfsAfUfuGfaGfaGfaagUfcCfaCfcacs gs a 768
AD -64013 A-127997
gsusgguggacdTdTcucucaauuuL96 554 A-127998
asdAsAfuugaGfaGfaagdTdCc aCfc ac sg s a 769
AD -64014 A-127957
Y44GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 555 A-127958 P asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfcs gs a 770
AD -64015 A-127966
gsusgguggaCfuUfcucuc(Agn)auuuL96 556 A-127956 asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a 771
AD -64016 A-127955
GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 557 A-127974 asAfs
auuGfaGfaGfaagUfcCfacc ac sgs a 772 P
AD -64017 A-127968
gsusgguggacudTcucuc(Agn)auuuL96 558 A-127982
asdAsauugagagaagdTccaccacsgsa 773 .
L.
u9
AD -64018 A-127990
gsusGfgugGfaCfUfUfcUfcUfcAfaUfuuL96 559 A-127993
asAfsAfuuGfaGfaGfaagUfCfc aCfc ac sg s a 774 ..'
.
.
. AD -64019 A-127959
gsusggUfgGfaCfUfUfcUfcucAfauuUfL96 560 A-127956 asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a 775
,
AD -64020 A-127967
gsusgguggacuUfcucuc(Agn)auuuL96 561 A-127956 asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a 776 .
,
,
AD -64021 A-127955
GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 562 A-127975 asAfs aUfug
aGfaGfaagUfcCfaccAfc sg s a 777 ,
.3
AD -64022 A-127968
gsusgguggacudTcucuc(Agn)auuuL96 563 A-127983
PasdAsauugagagaagdTccaccacsgsa 778
AD -64023 A-127990
gsusGfgugGfaCfUfUfcUfcUfcAfaUfuuL96 564 A-127994
asAfsAfUfuGfaGfaGfaagUfCfcaCfc ac sg s a 779
AD -64024 A-127960
gsusggUfgGfaCfUfUfcUfcucAfauuuL96 565 A-127956 asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a 780
AD -64025 A-127968
gsusgguggacudTcucuc(Agn)auuuL96 566 A-127956 asAfs
aUfuGfaGfaGfaagUfcCfaCfcAfc sg s a 781
AD -64026 A-127955
GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 567 A-127976
asAfsaUfugaGfagaagUfcCfaccAfcsgsa 782
AD -64027 A-127984
gsgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 568 A-127985
asAfsaUfuGfaGfaGfaagUfcCfaCfcsasc 783 IV
n
AD -64028 A-127990
gsusGfgugGfaCfUfUfcUfcUfcAfaUfuuL96 569 A-127995
asAfsAfUfugaGfaGfaagUfCfc aCfc ac sg s a 784 1-3
AD -64272 A-128001
GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuGfL96 570 A-128002
csAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 785 cp
n.)
o
AD -64274 A-128363
GfsusUfgAfcAfaAfAfAfuCfcUfcAfcAfaUfL96 571 A-128364
asUfsuGfuGfaGfgAfuuuUfuGfuCfaAfcs as a 786
oe
C-5
AD -64275 A-128377
UfsgsUfuGfaCfaAfAfAfaUfcCfuCfaCfaAfL96 572 A-128378
usUfsgUfgAfgGfaUfuuuUfgUfcAfaCfas as g 787 n.)
oe
1¨,
AD -64276 A-128393
GfsgsUfgGfaCfuUfCfUfcUfcAfaUfuUfuAfL96 573 A-128394
usAfsaAfaUfuGfaGfagaAfgUfcCfaCfcsasc 788
cA
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo
Name
NO:
NO: 0
n.)
AD-64277 A-128407 UfscsUfuUfuGfgAfGfUfgUfgGfaUfuCfgAfL96
574 A-128408 usCfsgAfaUfcCfaCfacuCfcAfaAfaGfasc s a
789 o
1¨,
oe
AD-64277 A-128407 UfscsUfuUfuGfgAfGfUfgUfgGfaUfuCfgAfL96
575 A-128408 usCfsgAfaUfcCfaCfacuCfcAfaAfaGfasc s a
790
un
AD-64278 A-128423 AfscsUfgUfuCfaAfGfCfcUfcCfaAfgCfuAfL96 576 A-128424
usAfsgCfuUfgGfaGfgcuUfgAfaCfaAfgsasc 791
cA
un
AD-64279 A-128435 UfscsUfgCfcGfaUfCfCfaUfaCfuGfcGfgAfL96 577 A-128436
usCfscGfcAfgUfaUfggaUfcGfgCfaGfasgsg 792
AD-64280 A-128379 AfsusGfuGfuCfuGfCfGfgCfgUfuUfuAfuAfL96 578 A-128380
usAfsuAfaAfaCfgCfcgcAfgAfcAfcAfuscsc 793
AD-64281 A-128395 CfscsCfcGfuCfuGfUfGfcCfuUfcUfcAfuAfL96 579 A-128396
usAfsuGfaGfaAfgGfcacAfgAfcGfgGfgsasg 794
AD-64282 A-128409 GfscsCfuAfaUfcAfUfCfuCfuUfgUfuCfaUfL96 580 A-128410
asUfsgAfaCfaAfgAfgauGfaUfuAfgCfgsasg 795
AD-64283 A-128425 UfscsUfaGfaCfuCfGfUfgGfuGfgAfcUfuCfL96 581 A-128426
gsAfsaGfuCfcAfcCfacgAfgUfcUfaGfascsu 796
AD-64284 A-128437 CfsusGfcCfgAfuCfCfAfuAfcUfgCfgGfaAfL96 582 A-128438
usUfscCfgCfaGfuAfuggAfuCfgGfcAfgsasg 797
AD-64285 A-128365 UfsusUfuUfcUfuGfUfUfgAfcAfaAfaAfuAfL96 583 A-128366
usAfsuUfuUfuGfuCfaacAfaGfaAfaAfascsc 798 P
AD-64286 A-128381 AfsusCfuUfcUfuGfUfUfgGfuUfcUfuCfuAfL96
584 A-128382 usAfsgAfaGfaAfcCfaacAfaGfaAfgAfusg s a
799 .
L.
u9
AD-64289 A-128367 GfsusUfuUfuCfuUfGfUfuGfaCfaAfaAfaUfL96 585 A-128368
asUfsuUfuUfgUfcAfacaAfgAfaAfaAfcscsc 800 ..'
.
.
AD-64290 A-128383 CfsusGfcCfuAfaUfCfAfuCfuCfuUfgUfuAfL96 586 A-128384
usAfsaCfaAfgAfgAfugaUfuAfgGfcAfgsasg 801
,
AD-64291 A-128399 UfscsCfuCfaCfaAfUfAfcCfaCfaGfaGfuAfL96 587 A-128400
usAfscUfcUfgUfgGfuauUfgUfgAfgGfasusu 802 .
,
,
AD-64292 A-128413 CfsusUfgUfuGfaCfAfAfaAfaUfcCfuCfaAfL96
588 A-128414 usUfsgAfgGfaUfuUfuugUfcAfaCfaAfgs as a
803 ,
o
.3
AD-64293 A-128439 GfscsAfaCfuUfuUfUfCfaCfcUfcUfgCfcUfL96 589 A-128440
asGfsgCfaGfaGfgUfgaaAfaAfgUfuGfcsasu 804
AD-64294 A-128369 GfsgsGfaAfcAfaGfAfGfcUfaCfaGfcAfuAfL96
590 A-128370 usAfsuGfcUfgUfaGfcucUfuGfuUfcCfc s as a
805
AD-64295 A-128385 CfsgsUfgGfuGfgAfC11JfuCfuCfuCfaAfuUfL96
591 A-128386 asAfsuUfgAfgAfgAfaguCfcAfcCfaGfcs as g
806
AD-64297 A-128415 CfsusGfcUfgCfuAfUfGfcCfuCfaUfcUfuAfL96
592 A-128416 usAfs aGfaUfgAfgGfcauAfgCfaGfcAfg sg s a
807
AD-64298 A-128427 GfsusUfgGfaUfgUfG11JfcUfgCfgGfcGfuUfL96
593 A-128428 asAfscGfcCfgCfaGfacaCfaUfcCfaAfc sg s a
808
AD-64299 A-128441 UfsusCfaUfcCfuGfCfUfgCfuAfuGfcCfuAfL96
594 A-128442 usAfsgGfcAfuAfgCfagcAfgGfaUfgAfasg s a
809 IV
n
AD-64300 A-128371 UfsusCfuUfgUfuGfAfCfaAfaAfaUfcCfuAfL96
595 A-128372 usAfsgGfaUfuUfuUfgucAfaCfaAfgAfas as a
810 1-3
AD-64302 A-128417 UfsasUfaUfgGfaUfGfAfuGfuGfgUfaUfuAfL96 596 A-128418
usAfsaUfaCfcAfcAfucaUfcCfaUfaUfasasc 811 cp
n.)
o
AD-64303 A-128429 UfsusCfaUfcCfuGfC11JfgCfuAfuGfcCfuCfL96
597 A-128430 g sAfsgGfcAfuAfgCfagcAfgGfaUfgAfasg s a
812
oe
C-5
AD-64304 A-128443 GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuAfL96 598 A-128444
usAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 813 n.)
oe
1¨,
AD-64305 A-128373 UfsusGfaCfaAfaAfAfUfcCfuCfaCfaAfuAfL96
599 A-128374 usAfsuUfgUfgAfgGfauuUfuUfgUfcAfasc s a 814
cA
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo
Name
NO:
NO: 0
n.)
AD-64307 A-128403 AfsasGfcCfuCfcAfAfGfcUfgUfgCfcUfuAfL96
600 A-128404 usAfs aGfgCfaCfaGfcuuGfgAfgGfcUfusg s a
815
1¨,
oe
AD-64308 A-128419 CfscsUfcUfuCfaUfCfCfuGfcUfgCfuAfuAfL96
601 A-128420 usAfsuAfgCfaGfcAfgg aUfgAfaGfaGfg s as a
816
un
AD-64309 A-128431 CfscsUfgCfuGfcUfAfUfgCfcUfcAfuCfuUfL96 602 A-128432
asAfsgAfuGfaGfgCfauaGfcAfgCfaGfgsasu 817
cA
un
AD-64310 A-128375 CfsasUfcUfuCfuUfGfUfuGfgUfuCfuUfcUfL96
603 A-128376 asGfs aAfgAfaCfcAfac aAfgAfaGfaUfg s as g
818
AD-64311 A-128391 CfscsGfuCfuGfuGfCfCfuUfcUfcAfuCfuAfL96 604 A-128392
usAfsgAfuGfaGfaAfggcAfcAfgAfcGfgsgsg 819
AD-64312 A-128405 CfscsUfcAfuCfuUfCfUfuGfuUfgGfuUfcUfL96
605 A-128406 asGfs aAfcCfaAfcAfag aAfgAfuGfaGfg sc s a
820
AD-64313 A-128421 CfscsAfcCfaAfaUfGfCfcCfcUfaUfcUfuAfL96 606 A-128422
usAfsaGfaUfaGfgGfgcaUfuUfgGfuGfgsusc 821
AD-64314 A-128433 GfscsUfcCfuCfuGfCfCfgAfuCfcAfuAfcUfL96
607 A-128434 asGfsuAfuGfgAfuCfggcAfgAfgGfaGfc scs a
822
AD-64315 A-128363 GfsusUfgAfcAfaAfAfAfuCfcUfcAfcAfaUfL96
608 A-128445 P asUfsuGfuGfaGfgAfuuuUfuGfuCfaAfc s as a 823
AD-64316 A-128377 UfsgsUfuGfaCfaAfAfAfaUfcCfuCfaCfaAfL96
609 A-128453
PusUfsgUfgAfgGfaUfuuuUfgUfcAfaCfas as g 824 P
AD-64317 A-128393 GfsgsUfgGfaCfuUfCfUfcUfcAfaUfuUfuAfL96
610 A-128461 Pus Afs aAfaUfuGfaGfag
aAfgUfcCfaCfc s asc 825 .
L.
u9
AD-64318 A-128407 UfscsUfuUfuGfgAfGfUfgUfgGfaUfuCfgAfL96
611 A-128469
PusCfsgAfaUfcCfaCfacuCfcAfaAfaGfasc s a 826 ..'
.
.
w AD-64319 A-128423 AfscsUfgUfuCfaAfGfCfcUfcCfaAfgCfuAfL96
612 A-128477 Pus AfsgCfuUfgGfaGfgcuUfgAfaCfaAfgs asc 827
,
AD-64320 A-128435 UfscsUfgCfcGfaUfCfCfaUfaCfuGfcGfgAfL96 613 A-128483
PusCfscGfcAfgUfaUfggaUfcGfgCfaGfasgsg 828 .
,
,
AD-64321 A-123463 AfsgsUfuAfuAfuGfGfAfuGfaUfgUfgGfuAfL96
614 A-128446 Pus
AfscCfaCfaUfcAfuccAfuAfuAfaCfus gs a 829 ,
.3
AD-64322 A-128379 AfsusGfuGfuCfuGfCfGfgCfgUfuUfuAfuAfL96
615 A-128454 Pus AfsuAfaAfaCfgCfcgcAfgAfcAfcAfuscsc 830
AD-64323 A-128395 CfscsCfcGfuCfuG11JfGfcCfuUfcUfcAfuAfL96
616 A-128462 Pus AfsuGfaGfaAfgGfcacAfgAfcGfgGfg s asg 831
AD-64324 A-128409 GfscsCfuAfaUfcAfUfCfuCfuUfgUfuCfaUfL96 617 A-128470
PasUfsgAfaCfaAfgAfgauGfaUfuAfgCfgsasg 832
AD-64325 A-128425 UfscsUfaGfaCfuCfGfUfgGfuGfgAfcUfuCfL96 618 A-128478
PgsAfsaGfuCfcAfcCfacgAfgUfcUfaGfascsu 833
AD-64326 A-128437 CfsusGfcCfgAfuCfCfAfuAfcUfgCfgGfaAfL96 619 A-128484
PusUfscCfgCfaGfuAfuggAfuCfgGfcAfgsasg 834
AD-64328 A-128381 AfsusCfuUfcUfuGfUfUfgGfuUfcUfuCfuAfL96
620 A-128455 Pus
AfsgAfaGfaAfcCfaacAfaGfaAfgAfusg s a 835 IV
n
AD-64330 A-128411 UfsusCfuCfuCfaAfUfUfuUfcUfaGfgGfgAfL96 621 A-128471
PusCfscCfcUfaGfaAfaauUfgAfgAfgAfasgsu 836 1-3
AD-64331 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 622 A-127907
PusGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 837 cp
n.)
o
AD-64332 A-128001 GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuGfL96 623 A-128485
PcsAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 838
oe
C-5
AD-64333 A-128367 GfsusUfuUfuCfuUfGfUfuGfaCfaAfaAfaUfL96 624 A-128448
PasUfsuUfuUfgUfcAfacaAfgAfaAfaAfcscsc 839 n.)
oe
1¨,
AD-64334 A-128383 CfsusGfcCfuAfaUfCfAfuCfuCfuUfgUfuAfL96
625 A-128456 Pus Afs aCfaAfgAfgAfugaUfuAfgGfcAfg s as g 840
cA
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo
Name
NO:
NO: 0
n.)
AD-64335 A-128399 UfscsCfuCfaCfaAfUfAfcCfaCfaGfaGfuAfL96
626 A-128464 Pus AfscUfcUfgUfgGfuauUfgUfgAfgGfasusu 841
1¨,
oe
AD-64336 A-128413 CfsusUfgUfuGfaCfAfAfaAfaUfcCfuCfaAfL96
627 A-128472 PusUfsgAfgGfaUfuUfuugUfcAfaCfaAfg s as a 842
un
AD-64337 A-127955 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96
628 A-127958 P asAfs aUfuGfaGfaGfaagUfcCfaCfcAfcs gs a 843
cA
un
AD-64338 A-128439 GfscsAfaCfuUfuUfUfCfaCfcUfcUfgCfcUfL96 629 A-128486
PasGfsgCfaGfaGfgUfgaaAfaAfgUfuGfcsasu 844
AD-64339 A-128369 GfsgsGfaAfcAfaGfAfGfcUfaCfaGfcAfuAfL96
630 A-128449 Pus AfsuGfcUfgUfaGfcucUfuGfuUfcCfc s as a 845
AD-64341 A-128401 UfscsAfuCfuUfcUfUfGfuUfgGfuUfcUfuAfL96
631 A-128465 Pus Afs aGfaAfcCfaAfcaaGfaAfgAfuGfasg sg 846
AD-64342 A-128415 CfsusGfcUfgCfuA11JfGfcCfuCfaUfcUfuAfL96
632 A-128473 Pus Afs aGfaUfgAfgGfcauAfgCfaGfcAfg sg s a 847
AD-64343 A-128427 GfsusUfgGfaUfgUfG11JfcUfgCfgGfcGfuUfL96
633 A-128479 P asAfscGfcCfgCfaGfacaCfaUfcCfaAfcs g s a
848
AD-64344 A-128441 UfsusCfaUfcCfuGfCfUfgCfuAfuGfcCfuAfL96
634 A-128487 Pus AfsgGfcAfuAfgCfagcAfgGfaUfgAfas gs a 849
AD-64345 A-128371 UfsusCfuUfgUfuGfAfCfaAfaAfaUfcCfuAfL96
635 A-128450 Pus
AfsgGfaUfuUfuUfgucAfaCfaAfgAfas as a 850 P
AD-64347 A-123487 GfsgsAfuGfuGfuCfUfGfcGfgCfgUfuUfuAfL96
636 A-128466 Pus Afs
aAfaCfgCfcGfcagAfcAfcAfuCfc s asg 851 .
L.
u9
AD-64348 A-128417 UfsasUfaUfgGfaUfGfAfuGfuGfgUfaUfuAfL96
637 A-128474 Pus Afs
aUfaCfcAfcAfucaUfcCfaUfaUfas asc 852 ..'
.
.
AD-64349 A-128429 UfsusCfaUfcCfuGfC11JfgCfuAfuGfcCfuCfL96
638 A-128480 Pgs AfsgGfcAfuAfgCfagcAfgGfaUfgAfas gs a 853
,
AD-64350 A-128443 GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuAfL96
639 A-128488 Pus
AfsgAfgGfuGfaAfgcgAfaGfuGfcAfc s asc 854 .
,
,
AD-64351 A-128373 UfsusGfaCfaAfaAfAfUfcCfuCfaCfaAfuAfL96
640 A-128451 Pus AfsuUfgUfgAfgGfauuUfuUfgUfcAfasc s a 855
.3
AD-64352 A-128389 CfscsAfaGfuGfuUfUfGfcUfgAfcGfcAfaAfL96
641 A-128459 PusUfsuGfcGfuCfaGfc aaAfcAfcUfuGfg scs a 856
AD-64352 A-128389 CfscsAfaGfuGfuUfUfGfcUfgAfcGfcAfaAfL96
642 A-128459 PusUfsuGfcGfuCfaGfc aaAfcAfcUfuGfg scs a 857
AD-64353 A-128403 AfsasGfcCfuCfcAfAfGfcUfgUfgCfcUfuAfL96
643 A-128467 Pus Afs aGfgCfaCfaGfcuuGfgAfgGfcUfusg s a 858
AD-64354 A-128419 CfscsUfcUfuCfaUfCfCfuGfcUfgCfuAfuAfL96
644 A-128475 Pus AfsuAfgCfaGfcAfggaUfgAfaGfaGfg s as a 859
AD-64355 A-128431 CfscsUfgCfuGfcUfAfUfgCfcUfcAfuCfuUfL96 645 A-128481
PasAfsgAfuGfaGfgCfauaGfcAfgCfaGfgsasu 860
AD-64356 A-128375 CfsasUfcUfuCfuUfGfUfuGfgUfuCfuUfcUfL96 646 A-128452
PasGfsaAfgAfaCfcAfacaAfgAfaGfaUfgsasg 861 IV
n
AD-64357 A-128391 CfscsGfuCfuGfuGfCfCfuUfcUfcAfuCfuAfL96
647 A-128460 Pus
AfsgAfuGfaGfaAfggcAfcAfgAfcGfg sg sg 862 1-3
AD-64358 A-128405 CfscsUfcAfuCfuUfCfUfuGfuUfgGfuUfcUfL96
648 A-128468 P asGfs aAfcCfaAfcAfag
aAfgAfuGfaGfg scs a 863 cp
n.)
o
AD-64359 A-128421 CfscsAfcCfaAfaUfGfCfcCfcUfaUfcUfuAfL96
649 A-128476 Pus Afs aGfaUfaGfgGfgc aUfuUfgGfuGfgsusc 864
oe
C-5
AD-64360 A-128433 GfscsUfcCfuCfuGfCfCfgAfuCfcAfuAfcUfL96
650 A-128482 P
asGfsuAfuGfgAfuCfggcAfgAfgGfaGfc scs a 865 n.)
oe
1¨,
AD-64700 A-129379 ascsucguggugdTacuu(Cgn)ucucaL96 651 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 866
cA
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name Name Oligo
Name
NO:
NO: 0
n.)
AD-64701 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 652 A-129387
PusgsagagaagdTccadCcacgaguscsu 867 o
1¨,
oe
AD-64702 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 653 A-129395
usGsagadGaaguccaCcacgaguscsu 868
un
AD-64703 A-129376 ascsucguggugdGacuucdAcucaL96 654 A-129385
usdGsagagaagdTccadCcacgaguscsu 869
cA
un
AD-64704 A-129381 ascsucguggdTgdTacuucdAcucaL96 655 A-129389
usdGsagadGaaguccadCcacgaguscsu 870
AD-64705 A-129380 ascsucguggugdTacuucdAcucaL96 656 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 871
AD-64706 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 657 A-129388
usdGsadGagaaguccadCcacgaguscsu 872
AD-64707 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 658 A-129396
usgsagadGaagdTccadCcacgaguscsu 873
AD-64708 A-129382 ascsucguggdTgdGacuuc(Tgn)cucaL96 659 A-129385
usdGsagagaagdTccadCcacgaguscsu 874
AD-64709 A-129373 ascsucguggugdGacuu(Cgn)ucucaL96 660 A-129391
usdGsagadGaagdTccadCcacgaguscsu 875
AD-64710 A-129373 ascsucguggugdGacuu(Cgn)ucucaL96
661 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 876 P
AD-64711 A-129381 ascsucguggdTgdTacuucdAcucaL96
662 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 877 .
L.
u9
AD-64712 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 663 A-129389
usdGsagadGaaguccadCcacgaguscsu 878 ..'
.
.
AD-64713 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 664 A-129397
PusgsagadGaagdTccadCcacgaguscsu 879
,
AD-64714 A-129384 ascsucguggdTgdGacuucdAcucaL96 665 A-129385
usdGsagagaagdTccadCcacgaguscsu 880 .
,
,
AD-64715 A-129376 ascsucguggugdGacuucdAcucaL96 666 A-129391
usdGsagadGaagdTccadCcacgaguscsu 881 ,
.3
AD-64716 A-129374 ascsucguggugdGacuucu(Cgn)ucaL96
667 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 882
AD-64717 A-129382 ascsucguggdTgdGacuuc(Tgn)cucaL96
668 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 883
AD-64718 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 669 A-129390
usdGsagagadAguccadCcacgaguscsu 884
AD-64719 A-127917 ascsucguggugdGacuuc(Tgn)cucaL96 670 A-129385
usdGsagagaagdTccadCcacgaguscsu 885
AD-64720 A-129381 ascsucguggdTgdTacuucdAcucaL96 671 A-129385
usdGsagagaagdTccadCcacgaguscsu 886
AD-64721 A-129382 ascsucguggdTgdGacuuc(Tgn)cucaL96 672 A-129391
usdGsagadGaagdTccadCcacgaguscsu 887 IV
n
AD-64722 A-129375 ascsucguggugdGacuucY34cucaL96
673 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 888 1-3
AD-64723 A-129383 ascsucguggugdGdAcuuc(Tgn)cucaL96
674 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 889 cp
n.)
o
AD-64725 A-127917 ascsucguggugdGacuuc(Tgn)cucaL96 675 A-129398
PusdGsagagaagdTccadCcacgaguscsu 890
oe
C-5
AD-64726 A-129373 ascsucguggugdGacuu(Cgn)ucucaL96 676 A-129389
usdGsagadGaaguccadCcacgaguscsu 891 n.)
oe
1¨,
AD-64727 A-129384 ascsucguggdTgdGacuucdAcucaL96 677 A-129391
usdGsagadGaagdTccadCcacgaguscsu 892
cA
ME1 27037625v.1
SEQ
SEQ
Duplex Sense Oligo
Antisense
Sense Sequence (5' to 3') ID
Antisense Sequence (5' to 3') ID
Name Name Oligo
Name
NO:
NO: 0
n.)
AD-64728 A-129376 ascsucguggugdGacuucdAcucaL96 678 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 893 o
1¨,
oe
AD-64729 A-129384 ascsucguggdTgdGacuucdAcucaL96 679 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 894
un
AD-64730 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 680 A-129392
usGsagagaagdTccadCcacgaguscsu 895
cA
un
AD-64731 A-129399 Y34ascsucguggugdGacuuc(Tgn)cucaL96 681 A-129385
usdGsagagaagdTccadCcacgaguscsu 896
AD-64732 A-129376 ascsucguggugdGacuucdAcucaL96 682 A-129389
usdGsagadGaaguccadCcacgaguscsu 897
AD-64733 A-129381 ascsucguggdTgdTacuucdAcucaL96 683 A-129391
usdGsagadGaagdTccadCcacgaguscsu 898
AD-64734 A-129377 ascsucguggugdGacuucdCcucaL96 684 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 899
AD-64735 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 685 A-129385
usdGsagagaagdTccadCcacgaguscsu 900
AD-64736 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 686 A-129393
usdGsagagaagdTccaCcacgaguscsu 901
AD-64737 A-129399 Y34ascsucguggugdGacuuc(Tgn)cucaL96 687 A-129398
PusdGsagagaagdTccadCcacgaguscsu 902 P
AD-64738 A-129382 ascsucguggdTgdGacuuc(Tgn)cucaL96 688 A-129389
usdGsagadGaaguccadCcacgaguscsu 903 .
L.
u9
AD-64739 A-129378 ascsucguggugdGacuucdGcucaL96 689 A-127906
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 904 ..'
.
.
AD-64740 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 690 A-129386
usgsagagaagdTccadCcacgaguscsu 905
,
AD-64741 A-127905 AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 691 A-129394
usGsagagaagdTccaCcacgaguscsu 906 .
,
,
AD-64742 A-129373 ascsucguggugdGacuu(Cgn)ucucaL96 692 A-129385
usdGsagagaagdTccadCcacgaguscsu 907 ,
o
.3
AD-64743 A-129384 ascsucguggdTgdGacuucdAcucaL96 693 A-129389
usdGsagadGaaguccadCcacgaguscsu 908
Table 4. Unmodified Sense and Antisense Strand Sequences of HBV dsRNAs
(Activity data available in W02016/077321, incorporated herein by
reference)
Iv
SEQ
SEQ n
,-i
Duplex ID Sense Sequence (5' to 3') ID Antisense Sequence (5'
to 3') ID
NO:
NO: cp
n.)
o
AD-65369 UCGUGGUGGACUUCUCUCA 909 UGAGAGAAGUCCACCACGAUU 938
oe
CB
AD-65381 UCGUGGUGGACUUCUCUCA 910 UGAGAGAAGUCCACCACGAUU 939
n.)
oe
1¨,
AD-63962 UCGUGGUGGACUUCUCUCA 911 UGAGAGAAGUCCACCACGAUU 940
cA
ME1 27037625v.1
SEQ
SEQ
Duplex ID Sense Sequence (5' to 3') ID Antisense Sequence (5'
to 3') ID
NO:
NO: 0
t..)
AD-63938 ACUCGUGGUGGACUUCUCUCA 912 UGAGAGAAGUCCACCACGAGUCU 941
cio
AD-65561 UCGUGGUGGACUUCUCUCA 913 UGAGAGAAGUCCACCACGAUU 942
vD
u,
AD-65566 UCGUGGUGGACUUCUCUCA 914 UGAGAGAAGUCCACCACGAUU 943
u,
AD-63944 UCGUGGUGGACUUCUCUCAUU 915 UGAGAGAAGUCCACCACGAUU
944
AD-63968 ACUCGUGGUGGACUUCUCUCA 916 UGAGAGAAGUCCACCACGAGUCU 945
AD-65406 UCGUGGUGGACUUCUCUCA 917 UGAGAGAAGUCCACCACGAUU 946
AD-65396 ACUCGUGGUGGACUUCUCUCA 918 UGAGAGAAGUCCACCACGAGUUU 947
AD-65427 GUGCACUUCGCUUCACCUCUA 919 UAGAGGUGAAGCGAAGUGCACUU 948
AD-65573 GUGCACUUCGCUUCACCUCUA 920 UAGAGGUGAAGCGAAGUGCACAC 949
AD-65432 GCACUUCGCUUCACCUCUA 921 UAGAGGUGAAGCGAAGUGCAC 950
P
AD-64332 GUGCACUUCGCUUCACCUCUG 922 CAGAGGUGAAGCGAAGUGCACAC 951
.
AD-64322 AUGUGUCUGCGGCGUUUUAUA 923 UAUAAAACGCCGCAGACACAUCC 952
.
.
.
AD-64272 GUGCACUUCGCUUCACCUCUG 924 CAGAGGUGAAGCGAAGUGCACAC 953
,
AD-65583 GCACUUCGCUUCACCUCUA 925 UAGAGGUGAAGCGAAGUGCUU 954
.
,
,
,
AD-63994 GGUGGACUUCUCUCAAUUU 926 AAAUUGAGAGAAGUCCACCAC 955
.
.3
AD-65370 CGUGGUGGACUUCUCUCAAUU 927 AAUUGAGAGAAGUCCACCAGCAG 956
AD-65265 GUGGUGGACUUCUCUCAAUUU 928 AAAUUGAGAGAAGUCCACCACGA 957
AD-65407 CGUGGUGGACUUCUCUCAAUU 929 AAUUGAGAGAAGUCCACCAGCAG 958
AD-64027 GGUGGACUUCUCUCAAUUU 930 AAAUUGAGAGAAGUCCACCAC 959
AD-65266 GUGGUGGACUUCUCUCAAUUU 931 AAAUUGAGAGAAGUCCACCACGA 960
AD-65389 UGGUGGUCTUCUCUAAAUU 932 AAUUGAGAGAAGUCCACCAUU 961
od
n
AD-64008 GUGGUGGACUUCUCUCAAUUU 933 AAAUUGAGAGAAGUCCACCACGA 962
AD-65377 CGUGGUGGUCTUCUCUAAAUU 934 AAUUGAGAGAAGUCCACCAGCUU 963
cp
t..)
o
AD-65409 GGUGGACUUCUCUCAAUUUUA 935 UAAAAUUGAGAGAAGUCCACCAC 964
00
-a-,
AD-65403 GGUGGACUUCUCUCAAUUUUA 936 UAAAAUUGAGAGAAGUCCACCAC 965
w
cio
AD-65385 UGGACUACTCUCAAAUUUA 937 UAAAAUUGAGAGAAGUCCAUU 966
cr
ME1 27037625v.1
Table 5. Exemplary Modified Sense and Antisense Strand Sequences of HBV dsRNAs
(Activity data available in W02016/077321, incorporated herein
by reference)
0
t.)
o
1¨,
oe
SEQ
SEQ 1¨
DuplexID Sense Sequence (5' to 3') ID Antisense
Sequence (5' to 3') ID vi
1¨,
NO:
NO: c7,
u,
AD-65369 uscsguGfgUfGfGfacuuCfUfcucaL96 967
PusGfsagaGfaAfGfuccaCfcAfcgasusu 996
AD-65381 uscsguGfgUfGfGfacuucucucaL96 968
PusGfsagaGfaAfGfuccaCfcAfcgasusu 997
AD-63962 Y44uscsGfuGfgUfgGfaCfuUfcUfcUfcAfY44 969
PusGfsaGfaGfaAfgUfcCfaCfcAfcGfasusu 998
AD-63938 Y44ACUCGUGGUGGACUUCUCUCA 970
UGAGAGAAGUCCACCACGAGUCU 999
AD-65561 uscsguGfgUfGfGfacuuCfUfcucaL96 971
UfsGfsagaGfaAfGfuccaCfcAfcgasusu 1000
AD-65566 uscsguGfgUfGfGfacuucucucaL96 972
UfsGfsagaGfaAfGfuccaCfcAfcgasusu 1001
AD-63944 Y44ucGuGGuGGAcuucucucAusuY44 973
UfGfagAfgAfAfGUfccaCfCAfcgAusu 1002 P
AD-63968
AfscsUfcGfuGfgUfGfGfaCfuUfcUfcUfcAfL96 974
usGfsaGfaGfaAfgUfccaCfcAfcGfaGfuscsu 1003 u9
..'
. AD-65406 uscsguGfgUfGfGfacuuCfUfcucaL96 975
usGfsagaGfaAfGfuccaCfcAfcgasusu 1004 .
oc
,,
AD-65396 ascsucguGfgUfGfGfacuucucucaL96 976
usGfsagaGfaaguccaCfcAfcgagususu 1005 ,9
,
AD-65427 gsusgcacUfuCfGfCfuucaccucuaL96 977
PusAfsgagGfugaagegAfaGfugcacsusu 1006 ,
,
AD-65573 gsusgcacUfuCfGfCfuucaCfCfucuaL96
978 UfsAfsgagGfuGfAfagegAfaGfugcacsasc 1007 .3
AD-65432 gscsacUfucGfCfuucacCfucuaL96 979
PusAfsgagGfuGfAfagegAfaGfugcsasc 1008
AD-64332
GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuGfL96 980
PcsAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 1009
AD-64322
AfsusGfuGfuCfuGfCfGfgCfgUfuUfuAfuAfL96 981
PusAfsuAfaAfaCfgCfcgcAfgAfcAfcAfuscsc 1010
AD-64272
GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuGfL96 982
csAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 1011
AD-65583 gscsacuucgdCuucac(Cgn)ucuaL96 983
usdAsgagdGugaagcgdAagugcsusu 1012
Iv
AD-63994 gsgsUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 984
PasAfsaUfuGfaGfaGfaagUfcCfaCfcsasc 1013
n
,-i
AD-65370 csgsugguGfgAfCfUfucucUfCfaauuL96
985 asAfsuugAfgAfGfaaguCfcAfccagcsasg 1014
cp
t.)
AD-65265 gsusggugGfaCfUfUfcUfcucaauuuL96 986
asAfsaUfugagaGfaagUfcCfaccAfcsgsa 1015 =
1¨,
oe
AD-65407 csgsugguGfgAfCfUfucucUfCfaauuL96
987 asAfsuugAfgAfgAfaguCfcAfccagcsasg 1016 'a
t.)
AD-64027 gsgsUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 988
asAfsaUfuGfaGfaGfaagUfcCfaCfcsasc 1017
oe
1¨,
1¨,
AD-65266 gsusggugGfaCfUfUfcucuCfaauuuL96 989
asAfsaUfugagaGfaagUfcCfaccAfcsgsa 1018 cr
ME1 27037625v.1
SEQ
SEQ
DuplexID Sense Sequence (5' to 3') ID Antisense
Sequence (5' to 3') ID
NO:
NO: 0
n.)
AD-65389 usgsgudGgucdTucucuaaauuL96 990
asdAsuugagagdAagudCcaccasusu 1019 =
1¨,
oe
AD-64008 GfsusGfgUfgGfaCfUfUfcUfcUfcAfaUfuUfL96 991
asAfsaUfuGfaGfaGfaagUfcCfaCfcAfcsgsa 1020
AD-65377 csgsuggudGgucdTucucuaaauuL96 992
asdAsuugagagdAagudCcaccagcsusu 1021 vi
1¨,
c:
vi
AD-65409 gsgsuggaCfuUfCfUfcucaAfUfuuuaL96 993
PusAfsaaaUfuGfAfgagaAfgUfccaccsasc 1022
AD-65403 gsgsuggaCfuUfCfUfcucaAfUfuuuaL96 994
usAfsaaaUfuGfAfgagaAfgUfccaccsasc 1023
AD-65385 usgsgacuacdTcucaaauuuaL96 995
usdAsaaauugadGagadAguccasusu 1024
Table 6. Exemplary Unmodified Sense and Antisense Strand Sequences of HBV
dsRNAs (Activity data available in W02016/077321, incorporated
herein by reference)
P
SEQ
u,
ID Antisense
SEQ ID .
-f5 Duplex ID Sense ID Sense Sequence Unmodified (5' to 3') NO:
ID Antisense Sequence Unmodified (5' to 3')
NO: " ,
,
,
AD -65381 A-130366 UCGUGGUGGACUUCUCUCA 1025 A-131904
UGAGAGAAGUCCACCACGAUU 1036 .
,
.3
AD -66019 A-130366 UCGUGGUGGACUUCUCUCA 1026 A-131904
UGAGAGAAGUCCACCACGAUU 1037
AD -65375 A-130366 UCGUGGUGGACUUCUCUCA 1027 A-130364
UGAGAGAAGUCCACCACGAUU 1038
AD -65427 A-130441 GUGCACUUCGCUUCACCUCUA 1028 A-131905
UAGAGGUGAAGCGAAGUGCACUU 1039
1-d
AD -66110 A-130441 GUGCACUUCGCUUCACCUCUA 1029 A-131905
UAGAGGUGAAGCGAAGUGCACUU 1040 n
,-i
AD -65421 A-130441 GUGCACUUCGCUUCACCUCUA 1030 A-130442
UAGAGGUGAAGCGAAGUGCACUU 1041 cp
t.)
o
1¨
oe
AD -65407 A-130371 CGUGGUGGACUUCUCUCAAUU 1031 A-130372
AAUUGAGAGAAGUCCACCAGCAG 1042 'a
t.)
oe
1-
1¨
AD -65377 A-130384 CGUGGUGGUCTUCUCUAAAUU 1032 A-130748
AAUUGAGAGAAGUCCACCAGCUU 1043 cr
ME1 27037625v.1
SEQ
ID Antisense
SEQ ID
Duplex ID Sense ID Sense Sequence Unmodified (5' to 3') NO:
ID Antisense Sequence Unmodified (5' to 3')
NO: 0
n.)
o
1¨,
AD -65409 A-130388 GGUGGACUUCUCUCAAUUUUA 1033 A-131906
UAAAAUUGAGAGAAGUCCACCAC 1044 oe
1¨,
vo
un
1¨,
AD -66111 A-130388 GGUGGACUUCUCUCAAUUUUA 1034 A-131906
UAAAAUUGAGAGAAGUCCACCAC 1045 c:
un
AD -65403 A-130388 GGUGGACUUCUCUCAAUUUUA 1035 A-130389
UAAAAUUGAGAGAAGUCCACCAC 1046
Table 7. Exemplary Modified Sense and Antisense Strand Sequences of HBV dsRNAs
(Activity data available in W02016/077321, incorporated herein
by reference)
P
SEQ
SEQ
Antisense 2
Duplex ID Sense ID Sense Sequence (5 to 3' ID ) ID
Antisense Sequence (5' to 3') ID .
u,
NO:
NO: .
AD-65381 A-130366 uscsguGfgUfGfGfacuucucucaL96
1047 A-131904 PusGfsagaGfaAfGfuccaCfcAfcgasusu 1058
,
,
,
AD-66019 A-130366 uscsguGfgUfGfGfacuucucucaL96
1048 A-131904 VPusGfsagaGfaAfGfuccaCfcAfcgasusu
1059
.3
AD-65375 A-130366 uscsguGfgUfGfGfacuucucucaL96 1049 A-130364
usGfsagaGfaAfGfuccaCfcAfcgasusu 1060
AD-65427 A-130441 gsusgcacUfuCfGfCfuucaccucuaL96 1050 A-131905
PusAfsgagGfugaagcgAfaGfugcacsusu 1061
AD-66110 A-130441 gsusgcacUfuCfGfCfuucaccucuaL96 1051 A-131905
VPusAfsgagGfugaagcgAfaGfugcacsusu 1062
IV
AD-65421 A-130441 gsusgcacUfuCfGfCfuucaccucuaL96 1052
A-130442 us Afs gagGfugaagcgAfaGfugc ac susu 1063 n
c 4
AD-65407 A-130371 csgsugguGfgAfCfUfucucUfCfaauuL96 1053 A-130372
as AfsuugAfgAfgAfaguCfcAfccagc s as g 1064
n.)
o
1¨,
oe
AD-65377 A-130384 csgsuggudGgucdTucucuaaauuL96 1054 A-130748
asdAsuugagagdAagudCcaccagcsusu 1065 n.)
oe
1¨,
1¨,
cr
ME1 27037625v.1
SEQ SEQ
AMisense
Duplex ID Sense ID Sense Sequence (5' to 3') ID
Antisense Sequence (5' to 3') ID
ID
NO:
NO: 0
n.)
AD-65409 A-130388 gsgsuggaCfuUfCfUfcucaAfUfuuuaL96 1055 A-131906
PusAfs aaaUfuGfAfgagaAfgUfcc aces as c 1066
oe
1¨,
o
vi
AD-66111 A-130388 gsgsuggaCfuUfCfUfcucaAfUfuuuaL96 1056 A-131906
VPusAfsaaaUfuGfAfgagaAfgUfccaccsasc 1067
o
vi
AD-65403 A-130388 gsgsuggaCfuUfCfUfcucaAfUfuuuaL96 1057 A-130389
us AfsaaaUfuGfAfg ag aAfgUfccaccsasc 1068
Table 8. Exemplary Unmodified Sense and Antisense Strand Sequences of HBV
dsRNAs (Activity data available in W02016/077321, incorporated
herein by reference)
P
SEQ
SEQ .
Sense Oligo Antisense
.
DuplexID Sense Sequence (5' to 3') ID Antisense
Sequence (5' to 3') ID u,
Name OligoName
.
NO:
NO: .
AD-65776 A-131859 UGUGCACUUCGCUUCACCUCU 1069 A-131860
AGAGGUGAAGCGAAGUGCACACG 1115 ' ,
,
AD-65782 A-131877 UGCACUUCGCUUCACCUCUGA 1070 A-131878
UCAGAGGUGAAGCGAAGUGCACA 1116 ,
,
.
AD-65792 A-131865 GUGUGCACUUCGCUUCACCUA 1071 A-131866
UAGGUGAAGCGAAGUGCACACGG 1117 '
AD-65781 A-131861 CGUGUGCACUUCGCUUCACCU 1072 A-131862
AGGUGAAGCGAAGUGCACACGGU 1118
AD-64304 A-128443 GUGCACUUCGCUUCACCUCUA 1073 A-128444
UAGAGGUGAAGCGAAGUGCACAC 1119
AD-65771 A-131857 CCGUGUGCACUUCGCUUCACA 1074 A-131858
UGUGAAGCGAAGUGCACACGGUC 1120
AD-65758 A-131867 CACUUCGCUUCACCUCUGCAA 1075 A-131868
UUGCAGAGGUGAAGCGAAGUGCA 1121
AD-65777 A-131875 ACUUCGCUUCACCUCUGCACA 1076 A-131876
UGUGCAGAGGUGAAGCGAAGUGC 1122
IV
AD-61567 A-123525 GGCUGUAGGCAUAAAUUGGUA 1077 A-123526 UACCAAUUUAUGCCUACAGCCUC
1123 n
AD-65772 A-131873 UUCGCUUCACCUCUGCACGUA 1078 A-131874
UACGUGCAGAGGUGAAGCGAAGU 1124
cp
AD-65767 A-131871 UCGCUUCACCUCUGCACGUCA 1079 A-131872
UGACGUGCAGAGGUGAAGCGAAG 1125 n.)
o
1¨,
oe
AD-65763 A-131869 CUUCGCUUCACCUCUGCACGU 1080 A-131870
ACGUGCAGAGGUGAAGCGAAGUG 1126
k ..,
AD-64281 A-128395 CCCCGUCUGUGCCUUCUCAUA 1081 A-128396
UAUGAGAAGGCACAGACGGGGAG 1127 c'e
1¨,
1¨,
AD-64311 A-128391 CCGUCUGUGCCUUCUCAUCUA 1082 A-128392
UAGAUGAGAAGGCACAGACGGGG 1128 cr
ME1 27037625v.1
SEQ
SEQ
Sense Oligo Antisense
DuplexID Sense Sequence (5' to 3') ID
Antisense Sequence (5 to 3') ID
Name OligoName
NO:
NO: 0
n.)
AD-65790 A-131837 CCAGCACCAUGCAACUUUUUA 1083 A-131838
UAAAAAGUUGCAUGGUGCUGGUG 1129 o
1-,
oe
AD-65761 A-131841 CACCAGCACCAUGCAACUUUU 1084 A-131842
AAAAGUUGCAUGGUGCUGGUGCG 1130
vi
AD-65786 A-131849 CACCAUGCAACUUUUUCACCU 1085 A-131850
AGGUGAAAAAGUUGCAUGGUGCU 1131
c:
vi
AD-65785 A-131835 CAAUGUCAACGACCGACCUUA 1086 A-131836
UAAGGUCGGUCGUUGACAUUGCA 1132
AD-65787 A-131863 CGCUUCACCUCUGCACGUCGA 1087 A-131864
UCGACGUGCAGAGGUGAAGCGAA 1133
AD-65770 A-131845 ACCUUGAGGCAUACUUCAAAG 1088 A-131846
CUUUGAAGUAUGCCUCAAGGUCG 1134
AD-65766 A-131843 CCGACCUUGAGGCAUACUUCA 1089 A-131844
UGAAGUAUGCCUCAAGGUCGGUC 1135
AD-61555 A-123521 GACCUUGAGGCAUACUUCAAA 1090 A-123522
UUUGAAGUAUGCCUCAAGGUCGG 1136
AD-65762 A-131855 ACCGACCUUGAGGCAUACUUA 1091 A-131856
UAAGUAUGCCUCAAGGUCGGUCG 1137
AD-65755 A-131827 UCGCAUGGAGACCACCGUGAA 1092 A-131828
UUCACGGUGGUCUCCAUGCGACG 1138 P
AD-65788 A-131811 UUACAUAAGAGGACUCUUGGA 1093 A-131812
UCCAAGAGUCCUCUUAUGUAAGA 1139 .
u,
AD-65768 A-131803 UCUUACAUAAGAGGACUCUUA 1094 A-131804
UAAGAGUCCUCUUAUGUAAGACC 1140 .
t.) AD-61561 A-123523 ACUUCAAAGACUGUUUGUUUA 1095 A-123524
UAAACAAACAGUCUUUGAAGUAU 1141
,
AD-65764 A-131801 UACUUCAAAGACUGUUUGUUU 1096 A-131802
AAACAAACAGUCUUUGAAGUAUG 1142 .
,
,
,
AD-65753 A-131799 AUACUUCAAAGACUGUUUGUU 1097 A-131800
AACAAACAGUCUUUGAAGUAUGC 1143 .
.3
AD-65765 A-131817 UUGUUUAAAGACUGGGAGGAA 1098 A-131818 UUCCUCCCAGUCUUUAAACAAAC
1144
AD-65769 A-131819 GCAUACUUCAAAGACUGUUUA 1099 A-131820
UAAACAGUCUUUGAAGUAUGCCU 1145
AD-65759 A-131815 CAAAGACUGUUUGUUUAAAGA 1100 A-131816
UCUUUAAACAAACAGUCUUUGAA 1146
AD-65774 A-131831 AGACUGUUUGUUUAAAGACUA 1101 A-131832 UAGUCUUUAAACAAACAGUCUUU
1147
AD-65778 A-131807 GUUUGUUUAAAGACUGGGAGA 1102 A-131808
UCUCCCAGUCUUUAAACAAACAG 1148
AD-65773 A-131805 GGGGGAGGAGAUUAGAUUAAA 1103 A-131806 UUUAAUCUAAUCUCCUCCCCCAA
1149 Iv
n
AD-65789 A-131825 GGGGAGGAGAUUAGAUUAAAG 1104 A-131826
CUUUAAUCUAAUCUCCUCCCCCA 1150 1-3
AD-65783 A-131809 GUUGGGGGAGGAGAUUAGAUU 1105 A-131810 AAUCUAAUCUCCUCCCCCAACUC
1151 cp
n.)
o
AD-65754 A-131813 UUGGGGGAGGAGAUUAGAUUA 1106 A-131814 UAAUCUAAUCUCCUCCCCCAACU
1152
-,-:--,
AD-65779 A-131821 GGGAGGAGAUUAGAUUAAAGA 1107 A-131822 UCUUUAAUCUAAUCUCCUCCCCC
1153 n.)
oe
1-,
AD-65791 A-131851 UUAGAUUAAAGGUCUUUGUAA 1108 A-131852 UUACAAAGACCUUUAAUCUAAUC
1154
cr
ME1 27037625v.1
SEQ
SEQ
Sense Oligo Antisense
DuplexID Sense Sequence (5' to 3') ID
Antisense Sequence (5' to 3') ID
Name OligoName
NO:
NO: 0
n.)
AD-65760 A-131829 UAGAUUAAAGGUCUUUGUACU 1109 A-131830
AGUACAAAGACCUUUAAUCUAAU 1155 o
1-
oe
AD-65784 A-131823 AUUAGAUUAAAGGUCUUUGUA 1110 A-131824
UACAAAGACCUUUAAUCUAAUCU 1156 1-
o
vi
AD-65757 A-131853 GAGGAGAUUAGAUUAAAGGUA 1111 A-131854 UACCUUUAAUCUAAUCUCCUCCC
1157 1-
o
vi
AD-65775 A-131847 GGACUCUUGGACUCUCUGCAA 1112 A-131848
UUGCAGAGAGUCCAAGAGUCCUC 1158
AD-65780 A-131833 ACUCUUGGACUCUCUGCAAUA 1113 A-131834
UAUUGCAGAGAGUCCAAGAGUCC 1159
AD-65756 A-131839 AGAUUAAAGGUCUUUGUACUA 1114 A-131840
UAGUACAAAGACCUUUAAUCUAA 1160
Table 9. Exemplary Unmodified Sense and Antisense Strand Sequences of HBV
dsRNAs (Activity data available in W02016/077321, incorporated
herein by reference)
P
.
Sense SEQ Antisense
u,
SEQ ID
..'
Duplex ID Oligo Sense Sequence (5 to 3') ID Oligo
Antisense Sequence (5' to 3') .
NO:
w Name NO: Name
."
,
AD-65776 A-131859 UfsgsUfgCfaCfuUfCfGfcUfuCfaCfcUfcUfL96 1161 A-131860 as Gfs
aGfgUfgAfaGfcgaAfgUfgCfaCfasc sg 1207 ' ,
,
AD-65782 A-131877 UfsgsCfaCfuUfcGfCfUfuCfaCfcUfcUfgAfL96 1162 A-131878 usCfs
aGfaGfgUfgAfageGfaAfgUfgCfascs a 1208 2
AD-65792 A-131865 GfsusGfuGfcAfcUfUfCfgCfuUfcAfcCfuAfL96 1163 A-131866
usAfsgGfuGfaAfgCfgaaGfuGfcAfcAfcsgsg 1209
AD-65781 A-131861 CfsgsUfgUfgCfaCfUfUfcGfcUfuCfaCfcUfL96 1164 A-131862 as
GfsgUfgAfaGfc GfaagUfgCfaCfaCfgsgsu 1210
AD-64304 A-128443 GfsusGfcAfcUfuCfGfCfuUfcAfcCfuCfuAfL96 1165 A-128444
usAfsgAfgGfuGfaAfgcgAfaGfuGfcAfcsasc 1211
AD-65771 A-131857 CfscsGfuGfuGfcAfCfUfuCfgCfuUfcAfcAfL96 1166 A-131858 us
GfsuGfaAfgCfgAfaguGfcAfcAfcGfgsusc 1212
AD-65758 A-131867 CfsasCfuUfcGfcUfUfCfaCfcUfcUfgCfaAfL96 1167 A-131868
usUfsgCfaGfaGfgUfgaaGfcGfaAfgUfgsc s a 1213
AD-65777 A-131875 AfscsUfuCfgCfuUfCfAfcCfuCfuGfcAfcAfL96 1168 A-131876 us
GfsuGfcAfgAfgGfugaAfgCfgAfaGfusgsc 1214 Iv
n
1-i
AD-61567 A-123525 GfsgsCfuGfuAfgGfCfAfuAfaAfuUfgGfuAfL96 1169 A-123526
usAfscCfaAfuUfuAfugcCfuAfcAfgCfcsusc 1215
cp
AD-65772 A-131873 UfsusCfgCfuUfcAfCfCfuCfuGfcAfcGfuAfL96 1170 A-131874
usAfscGfuGfcAfgAfgguGfaAfgCfgAfasgsu 1216 t.)
o
1-,
AD-65767 A-131871 UfscsGfcUfuCfaCfCfUfcUfgCfaCfgUfcAfL96 1171 A-131872 us Gfs
aCfgUfgCfaGfaggUfgAfaGfcGfas asg 1217 0 e
AD-65763 A-131869 CfsusUfcGfcUfuCfAfCfcUfcUfgCfaCfgUfL96 1172 A-131870
asCfsgUfgCfaGfaGfgugAfaGfcGfaAfgsusg 1218 t.)
oe
1-,
AD-64281 A-128395 CfscsCfcGfuCfuGfUfGfcCfuUfcUfcAfuAfL96 1173 A-128396
usAfsuGfaGfaAfgGfcacAfgAfcGfgGfgsasg 1219
cr
ME1 27037625v.1
Sense SEQ Antisense
SE Q ID
Duplex ID Oligo Sense Sequence (5' to 3') ID Oligo
Antisense Sequence (5' to 3')
NO:
0
Name NO: Name
t.)
o
AD-64311 A-128391 CfscsGfuCfuGfuGfCfCfuUfcUfcAfuCfuAfL96 1174 A-128392
usAfsgAfuGfaGfaAfggcAfcAfgAfcGfgsgsg 1220
oe
AD-65790 A-131837 CfscsAfgCfaCfcAfUfGfcAfaCfuUfuUfuAfL96 1175 A-131838
usAfsaAfaAfgUfuGfcauGfgUfgCfuGfgsusg 1221
vi
AD-65761 A-131841 CfsasCfcAfgCfaCfCfAfuGfcAfaCfuUfuUfL96 1176 A-131842
asAfsaAfgUfuGfcAfuggUfgCfuGfgUfgscsg 1222
c:
vi
AD-65786 A-131849 CfsasCfcAfuGfcAfAfCfuUfuUfuCfaCfcUfL96 1177 A-131850 as
GfsgUfgAfaAfaAfguuGfcAfuGfgUfgscsu 1223
AD-65785 A-131835 CfsasAfuGfuCfaAfCfGfaCfcGfaCfcUfuAfL96 1178 A-131836 usAfs
aGfgUfc GfgUfcguUfgAfcAfuUfgscs a 1224
AD-65787 A-131863 CfsgsCfuUfcAfcCfUfCfuGfcAfcGfuCfgAfL96 1179 A-131864
usCfsgAfc GfuGfcAfgagGfuGfaAfgCfgs as a 1225
AD-65770 A-131845 AfscsCfuUfgAfgGfCfAfuAfcUfuCfaAfaGfL96 1180 A-131846
csUfsuUfgAfaGfuAfugcCfuCfaAfgGfuscsg 1226
AD-65766 A-131843 CfscsGfaCfcUfuGfAfGfgCfaUfaCfuUfcAfL96 1181 A-131844 us Gfs
aAfgUfaUfgCfcucAfaGfgUfcGfgsusc 1227
AD-61555 A-123521 GfsasCfcUfuGfaGfGfCfaUfaCfuUfcAfaAfL96 1182 A-123522
usUfsuGfaAfgUfaUfgccUfcAfaGfgUfcsgsg 1228
AD-65762 A-131855 AfscsCfgAfcCfuUfGfAfgGfcAfuAfcUfuAfL96 1183 A-131856
usAfsaGfuAfuGfcCfucaAfgGfuCfgGfuscsg 1229 P
AD-65755 A-131827 UfscsGfcAfuGfgAfGfAfcCfaCfcGfuGfaAfL96 1184 A-131828
usUfscAfcGfgUfgGfucuCfcAfuGfcGfascsg 1230 2
2
AD-65788 A-131811 UfsusAfcAfuAfaGfAfGfgAfcUfcUfuGfgAfL96 1185 A-131812
usCfscAfaGfaGfuCfcucUfuAfuGfuAfasgs a 1231
..'.
-I" AD-65768 A-131803 UfscsUfuAfcAfuAfAfGfaGfgAfcUfcUfuAfL96 1186 A-131804
usAfsaGfaGfuCfcUfcuuAfuGfuAfaGfascsc 1232
,
AD-61561 A-123523 Afsc sUfuCfaAfaGfAfCfuGfuUfuGfuUfuAfL96 1187 A-123524
usAfsaAfcAfaAfcAfgucUfuUfgAfaGfusasu 1233
,
AD-65764 A-131801 UfsasCfuUfcAfaAfGfAfcUfgUfuUfgUfuUfL96 1188 A-131802
asAfsaCfaAfaCfaGfucuUfuGfaAfgUfasusg 1234 2
AD-65753 A-131799 AfsusAfcUfuCfaAfAfGfaCfuGfuUfuGfuUfL96 1189 A-131800
asAfscAfaAfcAfgUfcuuUfgAfaGfuAfusgsc 1235
AD-65765 A-131817 UfsusGfuUfuAfaAfGfAfcUfgGfgAfgGfaAfL96 1190 A-131818
usUfscCfuCfcCfaGfucuUfuAfaAfcAfasasc 1236
AD-65769 A-131819 GfscsAfuAfcUfuCfAfAfaGfaCfuGfuUfuAfL96 1191 A-131820
usAfsaAfcAfgUfcUfuugAfaGfuAfuGfcscsu 1237
AD-65759 A-131815 CfsasAfaGfaCfuGfUfUfuGfuUfuAfaAfgAfL96 1192 A-131816
usCfsuUfuAfaAfcAfaacAfgUfcUfuUfgs as a 1238
AD-65774 A-131831 AfsgsAfcUfgUfuUfGfUfuUfaAfaGfaCfuAfL96 1193 A-131832
usAfsgUfcUfuUfaAfacaAfaCfaGfuCfususu 1239
Iv
AD-65778 A-131807 GfsusUfuGfuUfuAfAfAfgAfcUfgGfgAfgAfL96 1194 A-131808
usCfsuCfcCfaGfuCfuuuAfaAfcAfaAfcsasg 1240 n
AD-65773 A-131805 GfsgsGfgGfaGfgAfGfAfuUfaGfaUfuAfaAfL96 1195 A-131806
usUfsuAfaUfcUfaAfucuCfcUfcCfcCfc sas a 1241
cp
AD-65789 A-131825 GfsgsGfgAfgGfaGfAfUfuAfgAfuUfaAfaGfL96 1196 A-131826 c
sUfsuUfaAfuCfuAfaucUfcCfuCfcCfc sc s a 1242 t.)
o
1¨,
AD-65783 A-131809 GfsusUfgGfgGfgAfGfGfaGfaUfuAfgAfuUfL96 1197 A-131810
asAfsuCfuAfaUfcUfccuCfcCfcCfaAfcsusc 1243 0 e
AD-65754 A-131813 UfsusGfgGfgGfaGfGfAfgAfuUfaGfaUfuAfL96 1198 A-131814
usAfsaUfcUfaAfuCfuccUfcCfcCfcAfascsu 1244 t.)
oe
1¨,
AD-65779 A-131821 GfsgsGfaGfgAfgAfUfUfaGfaUfuAfaAfgAfL96 1199 A-131822
usCfsuUfuAfaUfcUfaauCfuCfcUfcCfcscsc 1245
cr
ME1 27037625v.1
Sense SEQ Antisense
SEQ ID
Duplex ID Oligo Sense Sequence (5' to 3') ID
Oligo Antisense Sequence (5' to 3')
NO:
0
Name NO: Name
t..)
o
AD-65791 A-131851 UfsusAfgAfuUfaAfAfGfgUfcUfuUfgUfaAfL96 1200 A-131852
usUfsaCfaAfaGfaCfcuuUfaAfuCfuAfasusc 1246
oe
AD-65760 A-131829 UfsasGfaUfuAfaAfGfGfuCfuUfuGfuAfcUfL96 1201 A-131830
asGfsuAfcAfaAfgAfccuUfuAfaUfcUfasasu 1247
yD
vi
AD-65784 A-131823 AfsusUfaGfaUfuAfAfAfgGfuCfuUfuGfuAfL96 1202 A-131824
usAfscAfaAfgAfcCfuuuAfaUfcUfaAfuscsu 1248
c:
vi
AD-65757 A-131853 GfsasGfgAfgAfuUfAfGfaUfuAfaAfgGfuAfL96 1203 A-131854
usAfscCfuUfuAfaUfcuaAfuCfuCfcUfcscsc 1249
AD-65775 A-131847 GfsgsAfcUfcUfuGfGfAfcUfcUfcUfgCfaAfL96 1204 A-131848
usUfsgCfaGfaGfaGfuccAfaGfaGfuCfcsusc 1250
AD-65780 A-131833 AfscsUfcUfuGfgAfCfUfcUfcUfgCfaAfuAfL96 1205 A-131834
usAfsuUfgCfaGfaGfaguCfcAfaGfaGfuscsc 1251
AD-65756 A-131839 AfsgsAfuUfaAfaGfGfUfcUfuUfgUfaCfuAfL96 1206 A-131840
usAfsgUfaCfaAfaGfaccUfuUfaAfuCfusasa 1252
Table 10. Exemplary Unmodified HBV X ORF Sense and Antisense Sequences.
(Activity data available in W02016/077321, incorporated herein by P
reference)
u9
..'
DuplexID Sense Sequence Unmodified (5' to 3') SEQ ID NO:
Antisense Sequence Umodified (5' to 3') SEQ ID NO:
.
,
,
AD-66808 GUCUGUGCCUUCUCAUCUA 1253
UAGAUGAGAAGGCACAGACUU 1263
.3
AD-66809 GUCUGUGCCUUCUCAUCUA 1254
UAGAUGAGAAGGCACAGACUU 1264
AD-66810 GUGUGCACUUCGCUUCACA 1255
UGUGAAGCGAAGUGCACACUU 1265
AD-66811 GUGUGCACUUCGCUUCACA 1256
UGUGAAGCGAAGUGCACACUU 1266
AD-66812 UGUGCACUUCGCUUCACCUCU 1257
AGAGGUGAAGCGAAGUGCACAUU 1267
AD-66813 UGUGCACUUCGCUUCACCUCU 1258
AGAGGUGAAGCGAAGUGCACAUU 1268 Iv
n
AD-66814 CACCAGCACCAUGCAACUUUU 1259
AAAAGUUGCAUGGUGCUGGUGUU 1269
AD-66815 CACCAGCACCAUGCAACUUUU 1260
AAAAGUUGCAUGGUGCUGGUGUU 1270 cp
t..)
o
1¨,
AD-66816 CACCAUGCAACUUUUUCACCU 1261
AGGUGAAAAAGUUGCAUGGUGUU 1271 oe
-a-,
t..,
AD-66817 CACCAUGCAACUUUUUCACCU 1262
AGGUGAAAAAGUUGCAUGGUGUU 1272 c'e
1¨,
1¨,
c:
ME1 27037625v.1
Table 11. Exemplary Modified HBV X ORF Sense and Antisense Sequences.
(Activity data available in W02016/077321, incorporated herein by
reference)
0
t.)
o
1¨,
oe
1¨,
DuplexID SEQ ID Antisense
Sequence Modified (5' to 3') SEQ ID
u,
Sense Sequence Modified (5' to 3')
,..,
NO:
NO: c7,
u,
AD-66808 gsuscuGfuGfCfCfuucucaucuaL96 1273
usAfsgauGfaGfAfaggcAfcAfgacsusu 1283
AD-66809 gsuscuGfuGfCfCfuucucaucuaL96 1274
UfsAfsgauGfaGfAfaggcAfcAfgacsusu 1284
AD-66810 gsusguGfcAfCfUfucgcuucacaL96 1275
usGfsugaAfgCfGfaaguGfcAfcacsusu 1285
AD-66811 gsusguGfcAfCfUfucgcuucacaL96 1276
UfsGfsugaAfgCfGfaaguGfcAfcacsusu 1286
AD-66812 usgsugcaCfuUfCfGfcuucaccucuL96 1277
asGfsaggUfgAfAfgcgaAfgUfgcacasusu 1287
P
AD-66813 usgsugcaCfuUfCfGfcuucaccucuL96 1278
AfsGfsaggUfgAfAfgcgaAfgUfgcacasusu 1288 .
u9
AD-66814 csasccagCfaCfCfAfugcaacuuuuL96 1279
asAfsaagUfuGfCfauggUfgCfuggugsusu 1289 ..'
cs, AD-66815 csasccagCfaCfCfAfugcaacuuuuL96 1280
AfsAfsaagUfuGfCfauggUfgCfuggugsusu 1290 " ,
AD-66816 csasccauGfcAfAfCfuuuuucaccuL96 1281
asGfsgugAfaAfAfaguuGfcAfuggugsusu 1291 ,
,
,
AD-66817 csasccauGfcAfAfCfuuuuucaccuL96 1282
AfsGfsgugAfaAfAfaguuGfcAfuggugsusu 1292 .3
Iv
n
,¨i
cp
t..,
=
oe
-c-:--,
t..,
oe
c7,
ME1 27037625v.1
CA 03059446 2019-10-08
WO 2018/195165 PCT/US2018/028116
Table 12: HBV Target Sequences, noting target sites on Accession No. X02763.1.
(Activity data and
exemplary chemical modifications available at W02012/024170, incorporated
herein by reference)
Target Sequence Target Site SEQ ID NO:
UCGUGGUGGACUUCUCUCA 1663 1293
GUGGUGGACUUCUCUCAAU 1665 1294
GCCGAUCCAUACUGCGGAA 2669 1295
CCGAUCCAUACUGCGGAAC 2670 1296
CAUCCUGCUGCUAUGCCUC 1818 1297
UGCUGCUAUGCCUCAUCUU 1823 1298
GGUGGACUUCUCUCAAUUU 1667 1299
UGGUGGACUUCUCUCAAUU 1666 1300
UAGACUCGUGGUGGACUUC 1658 1301
UCCUCUGCCGAUCCAUACU 2663 1302
UGCCGAUCCAUACUGCGGA 2668 1303
UGGAUGUGUCUGCGGCGUU 1783 1304
CGAUCCAUACUGCGGAACU 2671 1305
CGCACCUCUCUUUACGCGG 2934 1306
CUGCCGAUCCAUACUGCGG 2667 1307
CGUGGUGGACUUCUCUCAA 1664 1308
CUGCUGCUAUGCCUCAUCU 1822 1309
CCUGCUGCUAUGCCUCAUC 1821 1310
CUAGACUCGUGGUGGACUU 1657 1311
UCCUGCUGCUAUGCCUCAU 1820 1312
GACUCGUGGUGGACUUCUC 1660 1313
AUCCAUACUGCGGAACUCC 2673 1314
CUCUGCCGAUCCAUACUGC 2665 1315
GAUCCAUACUGCGGAACUC 2672 1316
127
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Target Sequence Target Site SEQ ID NO:
GAAGAACUCCCUCGCCUCG 567 1317
AAGCCUCCAAGCUGUGCCU 54 1318
AGAAGAACUCCCUCGCCUC 566 1319
GGAGUGUGGAUUCGCACUC 455 1320
CCUCUGCCGAUCCAUACUG 2664 1321
CAAGCCUCCAAGCUGUGCC 53 1322
UCCAUACUGCGGAACUCCU 2674 1323
CAGAGUCUAGACUCGUGGU 1651 1324
AAGAAGAACUCCCUCGCCU 565 1325
GAGUGUGGAUUCGCACUCC 456 1326
UCUAGACUCGUGGUGGACU 1656 1327
GCUGCUAUGCCUCAUCUUC 1824 1328
AGUCUAGACUCGUGGUGGA 1654 1329
CUCCUCUGCCGAUCCAUAC 2662 1330
UGGCUCAGUUUACUAGUGC 2077 1331
GUCUAGACUCGUGGUGGAC 1655 1332
UUCAAGCCUCCAAGCUGUG 51 1333
CUAUGGGAGUGGGCCUCAG 2047 1334
CUCGUGGUGGACUUCUCUC 1662 1335
CCUAUGGGAGUGGGCCUCA 2046 1336
AAGAACUCCCUCGCCUCGC 568 1337
UCUGCCGAUCCAUACUGCG 2666 1338
AGAGUCUAGACUCGUGGUG 1652 1339
GAAGAAGAACUCCCUCGCC 564 1340
UCAAGCCUCCAAGCUGUGC 52 1341
AGCCUCCAAGCUGUGCCUU 55 1342
128
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Target Sequence Target Site SEQ ID NO:
AGACUCGUGGUGGACUUCU 1659 1343
Table 13. Various HBV siNA sense and antisense sequences corresponding to the
identified target
sequences in Table la. (Activity data and exemplary chemical modifications
available at
W02012/024170, incorporated herein by reference)
E. S Q ID SEQ ID
Target Site Sense Sequence Anti sense Sequence
NO: NO:
1663 1344 UCGUGGUGGACUUCUCUCA UGAGAGAAGUCCACCACGA 1395
1665 1345 GUGGUGGACUUCUCUCAAU AUUGAGAGAAGUCCACCAC 1396
2669 1346 GCCGAUCCAUACUGCGGAA UUCCGCAGUAUGGAUCGGC 1397
2670 1347 CCGAUCCAUACUGCGGAAC GUUCCGCAGUAUGGAUCGG 1398
1818 1348 CAUCCUGCUGCUAUGCCUC GAGGCAUAGCAGCAGGAUG 1399
1823 1349 UGCUGCUAUGCCUCAUCUU AAGAUGAGGCAUAGCAGCA 1400
1667 1350 GGUGGACUUCUCUCAAUUU AAAUUGAGAGAAGUCCACC 1401
1666 1351 UGGUGGACUUCUCUCAAUU AAUUGAGAGAAGUCCACCA 1402
1658 1352 UAGACUCGUGGUGGACUUC GAAGUCCACCACGAGUCUA 1403
2663 1353 UCCUCUGCCGAUCCAUACU AGUAUGGAUCGGCAGAGGA 1404
2668 1354 UGCCGAUCCAUACUGCGGA UCCGCAGUAUGGAUCGGCA 1405
1783 1355 UGGAUGUGUCUGCGGCGUU AACGCCGCAGACACAUCCA 1406
2671 1356 CGAUCCAUACUGCGGAACU AGUUCCGCAGUAUGGAUCG 1407
2934 1357 CGCACCUCUCUUUACGCGG CCGCGUAAAGAGAGGUGCG 1408
2667 1358 CUGCCGAUCCAUACUGCGG CCGCAGUAUGGAUCGGCAG 1409
1664 1359 CGUGGUGGACUUCUCUCAA UUGAGAGAAGUCCACCACG 1410
1822 1360 CUGCUGCUAUGCCUCAUCU AGAUGAGGCAUAGCAGCAG 1411
1821 1361 CCUGCUGCUAUGCCUCAUC GAUGAGGCAUAGCAGCAGG 1412
1657 1362 CUAGACUCGUGGUGGACUU AAGUCCACCACGAGUCUAG 1413
1820 1363 UCCUGCUGCUAUGCCUCAU AUGAGGCAUAGCAGCAGGA 1414
1660 1364 GACUCGUGGUGGACUUCUC GAGAAGUCCACCACGAGUC 1415
129
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E. S Q ID SEQ
ID
Target Site Sense Sequence Antisense Sequence
NO: NO:
2673 1365 AUCCAUACUGCGGAACUCC GGAGUUCCGCAGUAUGGAU 1416
2665 1366 CUCUGCCGAUCCAUACUGC GCAGUAUGGAUCGGCAGAG 1417
2672 1367 GAUCCAUACUGCGGAACUC GAGUUCCGCAGUAUGGAUC 1418
567 1368 GAAGAACUCCCUCGCCUCG CGAGGCGAGGGAGUUCUUC 1419
54 1369 AAGCCUCCAAGCUGUGCCU AGGCACAGCUUGGAGGCUU 1420
566 1370 AGAAGAACUCCCUCGCCUC GAGGCGAGGGAGUUCUUCU 1421
455 1371 GGAGUGUGGAUUCGCACUC GAGUGCGAAUCCACACUCC 1422
2664 1372 CCUCUGCCGAUCCAUACUG CAGUAUGGAUCGGCAGAGG 1423
53 1373 CAAGCCUCCAAGCUGUGCC GGCACAGCUUGGAGGCUUG 1424
2674 1374 UCCAUACUGCGGAACUCCU AGGAGUUCCGCAGUAUGGA 1425
1651 1375 CAGAGUCUAGACUCGUGGU ACCACGAGUCUAGACUCUG 1426
565 1376 AAGAAGAACUCCCUCGCCU AGGCGAGGGAGUUCUUCUU 1427
456 1377 GAGUGUGGAUUCGCACUCC GGAGUGCGAAUCCACACUC 1428
1656 1378 UCUAGACUCGUGGUGGACU AGUCCACCACGAGUCUAGA 1429
1824 1379 GCUGCUAUGCCUCAUCUUC GAAGAUGAGGCAUAGCAGC 1430
1654 1380 AGUCUAGACUCGUGGUGGA UCCACCACGAGUCUAGACU 1431
2662 1381 CUCCUCUGCCGAUCCAUAC GUAUGGAUCGGCAGAGGAG 1432
2077 1382 UGGCUCAGUUUACUAGUGC GCACUAGUAAACUGAGCCA 1433
1655 1383 GUCUAGACUCGUGGUGGAC GUCCACCACGAGUCUAGAC 1434
51 1384 UUCAAGCCUCCAAGCUGUG CACAGCUUGGAGGCUUGAA 1435
2047 1385 CUAUGGGAGUGGGCCUCAG CUGAGGCCCACUCCCAUAG 1436
1662 1386 CUCGUGGUGGACUUCUCUC GAGAGAAGUCCACCACGAG 1437
2046 1387 CCUAUGGGAGUGGGCCUCA UGAGGCCCACUCCCAUAGG 1438
568 1388 AAGAACUCCCUCGCCUCGC GCGAGGCGAGGGAGUUCUU 1439
2666 1389 UCUGCCGAUCCAUACUGCG CGCAGUAUGGAUCGGCAGA 1440
1652 1390 AGAGUCUAGACUCGUGGUG CACCACGAGUCUAGACUCU 1441
564 1391 GAAGAAGAACUCCCUCGCC GGCGAGGGAGUUCUUCUUC 1442
130
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E. S Q ID SEQ
ID
Target Site Sense Sequence Antisense Sequence
NO: NO:
52 1392 UCAAGCCUCCAAGCUGUGC GCACAGCUUGGAGGCUUGA 1443
55 1393 AGCCUCCAAGCUGUGCCUU AAGGCACAGCUUGGAGGCU 1444
1659 1394 AGACUCGUGGUGGACUUCU AGAAGUCCACCACGAGUCU 1445
131
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EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more
than routine
experimentation, many equivalents to the specific embodiments and methods
described herein. Such
equivalents are intended to be encompassed by the scope of the following
claims.
132
