WO2023233290A1 - Rnai agents targeting pd-l1 - Google Patents

Rnai agents targeting pd-l1 Download PDF

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Publication number
WO2023233290A1
WO2023233290A1 PCT/IB2023/055528 IB2023055528W WO2023233290A1 WO 2023233290 A1 WO2023233290 A1 WO 2023233290A1 IB 2023055528 W IB2023055528 W IB 2023055528W WO 2023233290 A1 WO2023233290 A1 WO 2023233290A1
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Prior art keywords
rnai agent
nucleotides
modified
sense strand
antisense strand
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PCT/IB2023/055528
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French (fr)
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Yi Jin
Ellen Rosalie A VAN GULCK
Michael Martin Ollmann
Mahesh Ramaseshan
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Janssen Sciences Ireland Unlimited Company
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Publication of WO2023233290A1 publication Critical patent/WO2023233290A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins

Definitions

  • the disclosure relates generally to compounds, compositions and methods useful for treating a disease, such as a viral infection, more particularly a chronic viral infection, or such as a cancer.
  • the viral infection comprises a hepatitis B virus (HBV) infection, more particularly chronic HBV infection.
  • the viral infection, more particularly the chronic viral infection comprises a hepatitis D virus (HDV) infection, more particularly chronic HDV infection.
  • the cancer comprises a liver cancer, more particularly a hepatocellular carcinoma.
  • RNAi RNA interference
  • PD -LI is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9.
  • PD-L1 expression is involved in evasion of immune responses involved in chronic infection, e.g., chronic viral infections such as HBV (see, 2020; 11: 1037; and Sun, Y., et al., Am J Physiol Gastrointest Liver Physiol. 2020: 218(1): G162-G173).
  • PD-L1 expression has been detected in a number of tissues and cell types including T-cells, B-cells, macrophages, dendritic cells, and nonhematopoietic cells including endothelial cells, hepatocytes, muscle cells, and placenta.
  • T-cells T-cells
  • B-cells B-cells
  • macrophages dendritic cells
  • nonhematopoietic cells including endothelial cells, hepatocytes, muscle cells, and placenta.
  • nonhematopoietic cells including endothelial cells, hepatocytes, muscle cells, and placenta.
  • PD-L1 expression is also involved in suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immunologic clearance of tumors is rare. Part of this failure is due to immune suppression by the tumor microenvironment.
  • PD-L1 expression on many tumors is a component of this suppressive milieu
  • PD-L1 expression has been shown in situ on a wide variety of solid tumors including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck (Brown JA et al., 2003, Immunol. 170: 1257-66; Dong H et al. 2002. Nat. Med. 8:793-800; Hamanishi J, et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003. Cancer Res. 63:6501-5; Inman BA et al. 2007. Cancer 109: 1499-505; Konishi J et al.
  • Hepatitis B virus a member of the Hepadnaviridae family, is a noncytopathic hepatic DNA virus that only infects the liver of human and great apes (e.g., chimpanzee, orangutan, bonobo, gorilla).
  • the primary infection of adult humans with HBV causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood.
  • About 10-20% of adult patients are not able to overcome the virus infection and suffer a chronic disease progression over many years with increased risk of developing cirrhotic liver or liver cancer through the development of chronic hepatitis B virus (CHB) infection.
  • CHB chronic hepatitis B virus
  • HCC cirrhosis and hepatocellular carcinoma
  • HBV mini-chromosome covalently closed circular DNA can be transcriptionally silent for long periods.
  • HBV replication is controlled through effector arms of adaptive immunity, including HBV-specific CD4+ helper T cells, HBV-specific CD8+ cytotoxic T cells, appropriately primed B cells that can serve as antigen presenting cells, and cytokines such as interferon gamma and tumor necrosis factor (TNF) alpha.
  • HBV-specific T cells perform surveillance and kill the infected cells that are reactivated to keep the serum free of virus products, such as HBV DNA and HBV proteins.
  • HBV can take advantage of the tolerogenic liver environment and the changes in immune cell populations in CHB patients.
  • HBV hepatitis B surface antigen
  • HBeAg hepatitis B e-antigen
  • HBsAg is the most abundant HBV protein in the liver and peripheral blood of HBV infected patients. It is the first serologic marker appearing in the serum, and this is about 6 to 16 weeks following exposure to HBV. In self-resolving acute HBV infection, HBsAg usually disappears 1 to 2 months after the onset of symptoms. Patients with detectable serum HBsAg (with or without detectable serum e-antigen (HBeAg)) for more than 6 months are considered chronically infected.
  • HCC cirrhosis and hepatocellular carcinoma
  • Nucleoside analogs as inhibitors of reverse transcriptase activity are typically the first treatment option for many patients.
  • Long term administration of lamivudine, tenofovir, and/or entecavir has been shown to suppress hepatitis B virus replication, sometimes to undetectable levels, with improvement of liver function and reduction of liver inflammation typically seen as the most important benefits.
  • only few patients achieve complete and lasting remission after the end of treatment.
  • the hepatitis B virus develops drug resistance with increasing duration of treatment. This is especially difficult for patients super/co-infected with hepatitis B and human immunodeficiency virus (HIV). Both viruses are susceptible to nucleoside analogue drugs and may co-develop resistance.
  • Pegylated interferon-alpha has been used to treat mild to moderate chronic hepatitis B patients.
  • current treatment of chronic hepatitis B has limited efficacy (Erha et al., Gut. 2005 Jul; 54(7): 1009-1013).
  • the Asian genotype B gives very poor response rates.
  • Super/co-infection with hepatitis D virus (HDV) or human immunodeficiency virus has been shown to render interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy.
  • HDV hepatitis D virus
  • HBeAg-positive chronic HBV or HBeAg-negative hepatitis B have not established a benefit over monotherapy. Id.
  • the present disclosure generally relates to a ribonucleic acid interfering (RNAi) agent (useful for in inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) gene), wherein the RNAi agent comprises an antisense strand comprising a sequence of 15 to 23 nucleotides, wherein the nucleotide sequence of the antisense strand comprises at least 15 contiguous nucleotides, particularly 16 or more, 17 or more, 18 or more, or 19 or more contiguous nucleotides, of a sequence selected from SEQ ID NOs: 604-804 and wherein one or more nucleotides of the antisense strand is a modified nucleotide; and wherein the RNAi agent optionally further comprises a sense strand comprising a nucleotide sequence, which is of the same length as, or of a lower length than, the nucleotide sequence of the antisense strand, and wherein one or more nucleotides of the sense RNAi agent
  • the nucleotide sequence of the sense strand comprises a sequence of 15 to 21 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a sequence of the same length that is comprised in the sequence of the antisense strand. In some aspects, at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sequence of the antisense strand are modified nucleotides. In some aspects, all the nucleotides of the sequence of the antisense strand are modified nucleotides.
  • At least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sense strand are modified nucleotides. In some aspects, all the nucleotides of the sequence of the sense strand are modified nucleotides.
  • the antisense strand comprises a sequence selected from SEQ ID NOs: 604-804. In some aspects, the sense strand comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603. In some aspects, the sense strand comprises at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403- 603. In some aspects, the sense strand comprises a sequence selected from SEQ ID NOs: 403- 603. In some aspects, the number of nucleotides of the sense strand is 19 or 21.
  • the number of nucleotides of the antisense strand is 19, 21 or 23. In some aspects, the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 21; wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 21 ; wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 23; or wherein the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 19.
  • sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 805-1005; 1690; 1724; 1760; and 1839.
  • sequence of the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 403-603; 1689; 1723; 1759; 1799; and 1838.
  • sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table lb.
  • the modified nucleotide comprises a modified nucleoside and/or a modified phosphate and/or a modified intemucleotide linkage.
  • the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a 2’-deoxy-2’-fluoro-ribose (2’-F), a 2’ O-methyl ribose (2’ O-Me) or the acyclic sugar of an UNA nucleotide, for example, wherein the modified nucleotide is nucleotide).
  • the modified nucleotide comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate.
  • the modified nucleotide comprises a modified intemucleotide linkage selected from phosphorothioate and thiophosphoramidate linkages, more particularly wherein the modified intemucleotide linkage is a phosphorothioate linkage, optionally wherein the phosphorothioate
  • the RNAi agent further comprises one or more of invAb and targeting moieties, more particularly liver targeting moieties; wherein the liver targeting moieties are fatty acids, GalNAc, folic acid, cholesterol, tocopherol or palmitate, more particularly GalNAc, more particularly the liver targeting moieties.
  • all nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA.
  • the number of 2’F nucleotides in antisense strand 2, 4, 6, 9 or 10, more particularly 3, 4, 5 or 6;
  • the number of UNA nucleotides in the antisense strand is 1 UNA-U or 1-UNA-A;
  • the number of vinylphosphonate nucleotides in the antisense strand is 1 vinylphosphonate nucleotide in the antisense strand, more particularly 1 vinylphosphonate nucleotide at the 5 ’ end of the antisense strand;
  • the nucleotides in the antisense strand that are not modified by 2’F or by vinylphosphonate or that are not UNA are modified by 2’-0Me, optionally wherein all of said nucleotides are modified by 2’-0Me; and/or the number of 2’0-Me nucleotides is 10, 11,
  • the number of 2’F nucleotides in sense strand is 2, 4, 6, 9, or 10, more particularly 4, 5, 6 or 7; the nucleotides in the sense strand that are not modified by 2’F are modified by 2’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or the number of 2’0-Me nucleotides is 10, 11, 13, 15 or 17.
  • 1, 2 or 3 phosphorothioate linkages linking the 3’ end or 5’ end terminal nucleotides of the sense strand and the antisense strand more particularly 1, 2 or 3 phosphorothioate linkages linking (ntl and nt2), and/or (nt2 and nt3), and/or (nt3 and nt4) of the sense strand and antisense strand and/or the antisense strand.
  • the sequence of the antisense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
  • the sequence of the sense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
  • the sequences of the antisense and sense strands of the RNAi agent comprises the sequences of the duplex selected from the duplex of Tables 2, 2a, 5 and 7.
  • the RNAi agent is capable of inducing a PD-U1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%.
  • the RNAi agent is capable of inducing a PD-U1 knockdown at a KD of at least 60% and at an IC50 of less than 150 nM, more particularly less than lOOnM , for example, in a free uptake assay, such as on primary human hepatocytes (PHH).
  • the RNAi agent is subcutaneously or intravenously administered.
  • the RNAi agent is for use in combination with one or more agents chosen from among antiviral agents (e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators), immune checkpoints, immunomodulators (more particularly one or more TUR immunomodulators), vaccines (e.g., an anti-HBV therapeutic vaccine), anti-HBV siRNAs, anti- HBV ASOs and NAPs.
  • antiviral agents e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators
  • immune checkpoints e.g., immunomodulators (more particularly one or more TUR immunomodulators), vaccines (e.g.,
  • the present disclosure relates to a salt of an RNAi agent as described herein, more particularly a sodium salt.
  • the present disclosure relates to a LNP or liposome comprising an RNAi agent as described herein.
  • the present disclosure relates to an isolated cell comprising an RNAi agent as described herein.
  • the present disclosure relates to a non-human animal comprising an RNAi agent as described herein.
  • the present disclosure generally relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the RNAi agent as described herein and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.
  • the pharmaceutical composition is a liquid composition.
  • the liquid composition comprises water, saline, and/or buffer.
  • the pharmaceutical composition is a lyophilized composition.
  • the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use as described herein, for use in treating a viral infection, more particularly a chronic viral infection.
  • the viral infection comprises an HBV infection.
  • the viral infection comprises an HBV infection and an HDV infection.
  • the viral infection comprises a HIV infection.
  • the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use as described herein, for use in the treatment of hepatitis B, more particularly of chronic hepatitis B.
  • the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use in the treatment of hepatitis D, more particularly of chronic hepatitis D.
  • the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use in treating a cancer, for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma.
  • a cancer for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma.
  • any numerical value such as a % sequence identity or a % sequence identity range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a dosage of 10 mg includes 9 mg to 11 mg.
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein.
  • oligonucleotide (or “oligo”) has its usual meaning as understood by those skilled in the art and thus refers to a class of compounds that includes oligodeoxynucleotides, oligodeoxyribonucleotides and oligoribonucleotides.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, including reference to oligonucleotides composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) intemucleoside (backbone) linkages as well as “modified” or substituted oligonucleotides having non-naturally- occurring portions which function similarly, and/or including modifications such as GalNAc that increase circulation half-life and/or reduce degradation of the oligonucleotide, e.g., degradation by endonucleases, and increase cellular uptake.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • An “antisense” oligonucleotide is a synthetic oligonucleotide that recognizes or specifically anneals to a target RNA in a sequence-specific manner.
  • the sequence of the target RNA recognized by the ASO is defined by the nucleotide number of the protein-coding sequence of the mRNA, where the adenylate of the initiation codon (AUG) is designated nucleotide 1.
  • the sequences used in the present disclosure are of a length and sequence that can recognize the target with high specificity within a cell.
  • RNA has its usual meaning as understood by those skilled in the art and thus refers to a class of oligonucleotides that are referred to as small interfering RNA, short interfering RNA and/or silencing RNA. These compounds are non-coding doublestranded RNA molecules, typically 19-25 base pairs in length, that operate within the RNA interference (RNAi) pathway and interfere with the expression of specific genes that contain complementary nucleotide sequences to the anti-sense strand of the siRNA and mediate degradation of the mRNA. The reduced level of the mRNA will decrease translation and the amount of the target protein.
  • RNAi RNA interference
  • siRNA shall be understood to include reference to siRNA composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) intemucleoside (backbone) linkages as well as “modified” or substituted siRNA having non- naturally-occurring portions which function similarly, and/or including modifications such as GalNAc that increase circulation half-life and/or reduce degradation of the oligonucleotide, e.g., degradation by endonucleases, and increase cellular uptake.
  • one strand is guiding and complementary to the target RNA (antisense strand), and the other strand (sense strand) has the same or substantially identical sequence as the target RNA and hence is complementary to the guiding/antisense strand.
  • RNAi agent recognizing a target RNA through base pairing and have their usual meaning as understood by those skilled in the art to refer to a process by which the RNAi agent hybridizes to the target RNA and at least partially inhibits production of the RNA or protein to which it is targeted.
  • siRNA may cause silencing of a gene that encodes an mRNA by reducing the mRNA and thus decrease the products synthesized, including proteins, from that target RNA.
  • Various assay techniques may be used to determine the degree to which the RNAi agent at least partially inhibits production of the RNA or protein to which it is targeted.
  • virus molecule has its usual meaning as understood by those skilled in the art and thus refers to a class of molecules made from the genome of a virus.
  • a virus molecule is typically a viral protein, a viral DNA or a viral RNA. These molecules can be utilized or required by the virus to replicate their genome or regulate the outcome of the viral infection.
  • HBV molecule refers to a class of molecules made from the HBV genome. Examples of HBV molecules include S-antigen, E-antigen, Core-antigen, polymerase antigen, HBV RNA, and HBV DNA.
  • An “epitope” as used herein is a set of amino acid residues that form a site recognized by an immunoglobulin, T cell receptor or human leukocyte antigen (HLA) molecule.
  • the HLA proteins are encoded by clusters of genes that form a region located on chromosome 6 known as the Major Histocompatibility Complex (MHC), in recognition of the important role of the proteins encoded by the MHC loci in graft rejection. Accordingly, the HLA proteins are also referred to as MHC proteins.
  • HLA or MHC proteins are cell surface glycoproteins that bind peptides at intracellular locations and deliver them to the cell surface, where the combined ligand is recognized by a T cell. Class I MHC proteins are found on virtually all of the nucleated cells of the body.
  • the class I MHC proteins bind peptides present in the cytosol and form peptide-MHC protein complexes that are presented at the cell surface, where they are recognized by cytotoxic CD8+ T cells.
  • Class II MHC proteins are usually found only on antigen-presenting cells such as B lymphocytes, macrophages, and dendritic cells.
  • Each MHC Class I receptor consists of a variable a chain and a relatively conserved [32 -microglobulin chain.
  • Three different, highly polymorphic class I a chain genes have been identified. These are called HLA-A, HLA-B, and HLA-C. Variations in the a chain accounts for all of the different class I MHC genes in the population.
  • phrases “percent (%) sequence identity” or “% identity” or “% identical to” or “percent complementary to” when used with reference to a nucleotide sequence describe the number of matches (“hits”) of identical nucleotides of two or more aligned nucleotide sequences as compared to the number of nucleotide residues making up the overall length of the nucleotide sequences. In other terms, using an alignment, for two or more sequences the percentage of nucleotide residues that are the same (e.g.
  • sequences which are compared to determine sequence identity can thus differ by substitution(s), addition(s) or deletion(s) of amino acids or nucleotides. Suitable programs for aligning polynucleotide or protein sequences are known to the skilled person.
  • the percentage sequence identity of polynucleotide or protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g., using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).
  • the term “complementary” refers to the capacity for precise pairing between two nucleotides sequences with one another to form a duplex structure under certain conditions.
  • such conditions can be stringent conditions, such as the conditions of 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing.
  • Other conditions such as physiologically relevant conditions as can be encountered inside an organism, can apply.
  • the skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
  • An oligonucleotide is considered complementary to a target DNA or RNA sequence, when a sufficient number of nucleotides in the oligonucleotide can form hydrogen bonds with corresponding nucleotides in the target DNA or RNA to enable the formation of a stable complex.
  • the sequence of an siRNA compound need not be 100% complementary to its target nucleic acid.
  • the term “complementary” thus implies that the siRNA agent binds sufficiently strong and specific to the target molecule to provide the desired interference with the normal function of the target whilst leaving the function of non-target mRNAs unaffected.
  • an oligonucleotide that is “at least X% complementary” to another sequence means that at least X% of nucleotides in the oligonucleotide can form hydrogen bonds with corresponding nucleotides in the other sequence.
  • an oligonucleotide “at least X% complementary” to another sequence is at least X% identical to a sequence that is fully complementary to the other sequence.
  • RNAi agents e.g. , siRNA, e.g. , ASO
  • a therapeutic composition and an adjuvant e.g., alumimal sarcoma
  • Simultaneous administration can be administration of the two components at least within the same day.
  • a first therapy or component e.g. first RNAi agent
  • a second therapy or component e.g. second RNAi agent
  • a first therapy or component e.g. first RNAi agent
  • a second therapy or component e.g., e.g., second RNAi agent
  • a first therapy or component e.g. first RNAi agent
  • a second therapy or component e.g., second RNAi agent
  • a “non-naturally occurring” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature.
  • a “non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting.
  • a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment.
  • a “non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source.
  • a “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
  • operably linked refers to a linkage or a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing the transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest over a membrane.
  • subject means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the present disclosure.
  • mammal encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human.
  • a human subject can include a patient.
  • RNA interference agent refers to a composition that contains an RNA or RNA-hke (e.g. , chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner.
  • RNAi agents can operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA- induced silencing complex or RISC) of mammalian cells, or by any alternative mechanism(s) or pathway(s).
  • RNAi agents While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action.
  • RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short interfering RNAs (siRNAs), double-stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates.
  • RNAi agents of the application are preferably dsRNAs.
  • the antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted.
  • RNAi agents can be comprised of modified nucleotides and/or one or more non-phosphodiester linkages.
  • the term “PD-L1” refers to programmed death-ligand 1, also known as CD274, B7H1, B7-H, PDCD1L1 or PDCD1LG1.
  • the CD274 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog.
  • POLI also includes protein variants and recombinant PD-L1 or a fragment thereof. Unless specified as being from a non-human species, the term“PD-Ll” means human PD-L1.
  • the cDNA and protein sequences of PD-L1 can be obtained from public source, such as GenBank.
  • amino acid sequence of a full-length human PD-L1 is provided in GenBank as accession number NP 054862.1 and a cDNA sequence encoding the full-length human PD-L1 is provided in GenBank as accession number NM_014143.4.
  • double-stranded RNA refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure comprising two anti -parallel and substantially complementary nucleic acid strands.
  • the two strands forming the duplex structure can be different portions of one larger RNA molecule, or they can be separate RNA molecules.
  • the connecting RNA chain is referred to as a “hairpin loop.”
  • the connecting structure is referred to as a “linker.”
  • the RNA strands can have the same or a different number of nucleotides.
  • a dsRNA can comprise one or more nucleotide overhangs or can be blunt ended.
  • the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with oligomeric compounds, such as RNAi agents, described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.
  • oligomeric compounds such as RNAi agents
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
  • pharmaceutically acceptable salt refers to a salt of any of the compounds herein which are known to be non-toxic and are commonly used in the pharmaceutical literature.
  • the pharmaceutically acceptable salt of a compound retains the biological effectiveness of the compounds described herein and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66(1), 1-19.
  • Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.
  • Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2- hydroxyethylsulfonic acid, p-toluenesulfonic acid, stearic acid and salicylic acid.
  • Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
  • Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum.
  • Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.
  • therapeutically effective amount refers to that amount of a compound disclosed and/or described herein that is sufficient to affect treatment, as defined herein, when administered to a subject in need of such treatment.
  • the therapeutically effective amount will vary depending upon, for example, the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound, the dosing regimen to be followed, timing of administration, the manner of administration, all of which can readily be determined by one of ordinary skill in the art.
  • the therapeutically effective amount can be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability by one of ordinary skill in the art in view of the present disclosure.
  • a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or
  • a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a PD-L1 associated infection or disease, or a symptom associated therewith; (ii) reduce the duration of an PD-L1 associated infection or disease, or symptom associated therewith; (iii) prevent the progression of an PD-L1 associated disease, or symptom associated therewith; (iv) cause regression of an PD- L1 associated disease, or symptom associated therewith; (v) prevent the development or onset of an PD-L1 associated disease, or symptom associated therewith; (vi) prevent the recurrence of an PD-L1 associated disease or symptom associated therewith; (vii) reduce hospitalization of a subject having an PD-L1 associated disease; (viii) reduce hospitalization length of a subject having an PD-L1 associated disease; (ix) increase the survival of a subject
  • the application also relates to a vector comprising the first and/or second non- naturally occurring nucleic acid molecules.
  • a “vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs).
  • a vector is a DNA plasmid.
  • a vector can be a DNA vector or an RNA vector.
  • One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.
  • a vector of the application can be an expression vector.
  • expression vector refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed.
  • Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells.
  • Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADVAX, NTC8454, etc.
  • an expression vector is based on pVAX- 1, which can be further modified to optimize protein expression in mammalian cells.
  • pVAX-1 is commonly used plasmid in DNA vaccines, and contains a strong human intermediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH) -derived polyadenylation sequence (pA).
  • pVAX-1 further contains a pUC origin of replication and kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.
  • CMV-IE human intermediate early cytomegalovirus
  • bGH bovine growth hormone
  • pA
  • a vector of the application can also be a viral vector.
  • viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non- infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection.
  • viral vectors examples include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, Modified Vaccinia virus Ankara (MV A) vectors, lentiviral vectors, etc.
  • adenoviral vectors examples include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, Modified Vaccinia virus Ankara (MV A) vectors, lentiviral vectors, etc.
  • MV A Modified Vaccinia virus Ankara
  • viral vectors examples include, but are not limited to, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi- segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which comprise an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding an HBV antigen as described herein), arenavirus such as lymphocytic choriomeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain.
  • the vector can also be a non-viral vector.
  • a viral vector is an adenovirus vector, e.g. , a recombinant adenovirus vector.
  • a recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd).
  • an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc.
  • an adenovirus vector is a rhAd vector, e.g. , rhAd51, rhAd52 or rhAd53
  • ribonucleic acid molecules of the present disclosure described herein may contain particular components arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in ribonucleic acid molecules of the present disclosure.
  • the present disclosure contemplates use of any of the applicable components in any combination having any sequence that can be used in ribonucleic acid molecules of the present disclosure, whether or not a particular combination is expressly described.
  • PD-L1 (e.g., a full length 290 amino acid human PD-L1 shown in GenBank accession No. NP_054862) is a protein with extracellular IgV-like and IgC-like domains (e.g., amino acids 19-239 of the full-length human PD-L1), a transmembrane domain and an intracellular domain (e.g., approximately 30 amino acids of the full-length human PD-L1).
  • PD-L1 is constitutively expressed on many cells such as antigen presenting cells (e.g., dendritic cells, macrophages, and B-cells) and on hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and sites of immune privilege). PD-L1 is also expressed on a wide variety of tumors, and virally-infected cells and is a component of the immunosuppressive milieu (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of two T-cell co-inhibitors PD-1 and B7-1.
  • antigen presenting cells e.g., dendritic cells, macrophages, and B-cells
  • hematopoietic and non-hematopoietic cells e.g., vascular endothelial cells, pancreatic islets, and sites of immune privilege.
  • PD-L1
  • T-cell co-stimulatory and co-inhibitory molecules play a crucial role in regulating T-cell activation, subset differentiation, effector function and survival (Chen et al2013, Nature Rev. Immunol. 13 : 227-242).
  • co-signaling receptors co-localize with T-cell receptors at the immune synapse, where they synergize with TCR signaling to promote or inhibit T-cell activation and function (Flies et al. 2011, Yale J. Biol. Med. 84: 409-421).
  • the ultimate immune response is regulated by a balance between co-stimulatory and co-inhibitory signals (“immune checkpoints”) (Pardoll 2012, Nature 12: 252-264). While not wishing to be bound by theory, it is currently believed that PD-1 functions as one such ‘immune checkpoint’ in mediating peripheral T-cell tolerance and in avoiding autoimmunity. PD-1 binds to PD-L1 or PD-L2 and inhibits T-cell activation. The ability of PD-1 to inhibit T-cell activation is exploited by chronic viral infections and tumors to evade immune response.
  • PD-1 is highly expressed on virus-specific T-cells and these T-cells become “exhausted” with loss of effector functions and proliferative capacity (Freeman 2008, PNAS 105: 10275-1 0276).
  • the PD-1-PD-L1 system also plays an important role in induced T-regulatory (Treg) cell development and in sustaining Treg function (Francisco et al 2010, Immunol. Rev. 236:219-242).
  • RNAi Agents [0061] The present disclosure generally relates to a ribonucleic acid interfering (RNAi) agent useful for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) gene, wherein the RNAi agent comprises an antisense strand comprising a sequence of 15 to 23 nucleotides, wherein the nucleotide sequence of the antisense strand comprises at least 15 contiguous nucleotides (optionally 16 or more, 17 or more, 18 or more, or 19 or more contiguous nucleotides) from a sequence selected from SEQ ID NOs: 604-804 and wherein one or more nucleotides of the antisense strand is a modified nucleotide; and wherein the RNAi agent optionally further comprises a sense strand comprising a nucleotide sequence, which is of the same length as, or of a lower length than, the nucleotide sequence of the antisense strand, and wherein one or more nucleotides
  • each PD-L1 RNAi agent comprises a sense strand and an antisense strand.
  • the sense strand and the antisense strand each can be 15 to 23 nucleotides in length.
  • the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length.
  • the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length.
  • the sense strand of the RNAi agent comprises at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603.
  • the number of nucleotides of the sense strand of the RNAi agent is 19 or 21.
  • the number of nucleotides of the antisense strand of the RNAi agent is 19, 21 or 23.
  • the number of nucleotides of the sense strand of the RNAi agent is 19 and the number of nucleotides of the antisense strand of the RNAi agent is 21; the number of nucleotides of the sense strand of the RNAi agent is 21 and the number of nucleotides of the antisense strand of the RNAi agent is 21 ; the number of nucleotides of the sense strand of the RNAi agent is 21 and the number of nucleotides of the antisense strand of the RNAi agent is 23; or the number of nucleotides of the sense strand of the RNAi agent is 19 and the number of nucleotides of the antisense strand of the RNAi agent is 19.
  • a sense strand of the PD-L1 RNAi agents described herein includes a core stretch having at least about 85% identity to a nucleotide sequence of at least 15 consecutive nucleotides in an PD-L1 mRNA.
  • the sense strand core nucleotide stretch having at least about 85% identity to a sequence in an PD- LlmRNA is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • an antisense strand of a PD-L1 RNAi agent comprises a nucleotide sequence having at least about 85% complementary over a core stretch of at least 15 consecutive nucleotides to a sequence in an PD-LlmRNA and the corresponding sense strand.
  • the antisense strand core nucleotide sequence having at least about 85% complementarity to a sequence in an PD- LlmRNA or the corresponding sense strand is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
  • RNAi agents of the application can contain one or more mismatches to the target sequence. In a preferred embodiment, RNAi agents of the application contains no more than 13 mismatches. If the antisense strand of the RNAi agent contains mismatches to a target sequence, it is preferable that the area of mismatch not be located within nucleotides 2-7 of the 5’ terminus of the antisense strand. In another embodiment, it is preferable that the area of mismatch not be located within nucleotides 2-9 of the 5’ terminus of the antisense strand.
  • the PD-L1 RNAi agent sense and antisense strands anneal to form a duplex.
  • a sense strand and an antisense strand of a PD-L1 RNAi agent can be partially, substantially, or fully complementary to each other.
  • the sense strand core stretch sequence is at least about 85% complementary or 100% complementary to the antisense core stretch sequence.
  • the sense strand core stretch sequence contains a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least about 85% or 100% complementary to a corresponding 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense strand and antisense core stretch sequences of a PD-L1 RNAi agent have a region of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% base paired or 100% base paired).
  • the antisense strand of a PD-L1 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences described herein. In some embodiments, the sense strand of a PD-L1 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences described herein.
  • the sense strand and/or the antisense strand can optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3' end, the 5' end, or both the 3' and 5' ends of the core sequences.
  • the antisense strand additional nucleotides may or may not be complementary to the corresponding sequence in an PD-L1 mRNA.
  • the sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in an PD-L1 mRNA.
  • the antisense strand additional nucleotides, if present may or may not be complementary to the corresponding sense strand's additional nucleotides, if present.
  • an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and/or 3' end of the sense strand core stretch sequence and/or antisense strand core stretch sequence.
  • the extension nucleotides on a sense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding antisense strand.
  • the extension nucleotides on an antisense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding sense strand.
  • both the sense strand and the antisense strand of an RNAi agent contain 3' and 5' extensions.
  • a PD-L1 RNAi agent has an antisense strand having a 3' extension and a sense strand having a 5' extension. In some embodiments, a PD-L1 RNAi agent comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length.
  • a PD-LIRNAi agent comprises an antisense strand having a 3' extension of 1, 2, or 3 nucleotides in length.
  • one or more of the antisense strand extension nucleotides comprise uracil or thymidine nucleotides or nucleotides which are complementary to a corresponding PD-L1 mRNA sequence.
  • the 3' end of the antisense strand can include additional abasic nucleosides (Ab). In some embodiments, Ab or Ab Ab can be added to the 3' end of the antisense strand.
  • a PD-L1 RNAi agent comprises an antisense strand having a 5' extension of 1, 2, 3, 4, or 5 nucleotides in length. In other embodiments, a PD-L1 RNAi agent comprises an antisense strand having a 5' extension of 1 or 2 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprises uracil or thymidine nucleotides or nucleotides which are complementary to a corresponding PD-L1 mRNA sequence. An antisense strand can have any of the 3' extensions described above in combination with any of the 5' antisense strand extensions described, if present.
  • a PD-L1 RNAi agent comprises a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length.
  • one or more of the sense strand extension nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides which correspond to nucleotides in the PD-LlmRNA sequence.
  • the 3' end of the sense strand can include additional abasic nucleosides.
  • UUAb, UAb, or Ab can be added to the 3' end of the sense strand.
  • the one or more abasic nucleosides added to the 3' end of the sense strand can be inverted (invAb).
  • one or more inverted abasic nucleosides can be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent.
  • the inclusion of one or more inverted abasic nucleosides at or near the terminal end or terminal ends of the sense strand of an RNAi agent can allow for enhanced activity or other desired properties of an RNAi agent.
  • a PD-L1 RNAi agent comprises a sense strand having a 5' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length.
  • one or more of the sense strand extension nucleotides comprise uracil or adenosine nucleotides or nucleotides which correspond to nucleotides in the PD-L1 mRNA sequence.
  • the 5' end of the sense strand can include an additional abasic nucleoside (Ab) or nucleosides (AbAb).
  • the one or more abasic nucleosides added to the 5' end of the sense strand can be inverted (invAb).
  • one or more inverted abasic nucleosides can be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent.
  • the inclusion of one or more inverted abasic nucleosides at or near the terminal end or terminal ends of the sense strand of an RNAi agent can allow for enhanced activity or other desired properties of an RNAi agent.
  • the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a blunt end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form blunt ends. In some embodiments, neither end of an RNAi agent is blunt-ended.
  • a blunt end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base -pair).
  • the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a frayed end.
  • the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a frayed end.
  • both ends of an RNAi agent form a frayed end.
  • neither end of an RNAi agent is a frayed end.
  • a frayed end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands from a pair (i.e. do not form an overhang) but are not complementary (i.e. form a non-complementary pair).
  • an overhang is a stretch of one or more unpaired nucleotides at the end of one strand of a double stranded RNAi agent.
  • the unpaired nucleotides can be on the sense strand or the antisense strand, creating either 3' or 5' overhangs.
  • the RNAi agent contains: a blunt end and a frayed end, a blunt end and 5' overhang end, a blunt end and a 3' overhang end, a frayed end and a 5' overhang end, a frayed end and a 3' overhang end, two 5' overhang ends, two 3' overhang ends, a 5' overhang end and a 3' overhang end, two frayed ends, or two blunt ends.
  • a PD-L1 RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a PD-L1 RNAi agent is prepared as a sodium salt. Such forms are within the scope of the application disclosed herein.
  • the nucleotide sequence of the sense strand comprises a sequence of 15 to 23 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a sequence of the same length that is comprised in the sequence of the antisense strand. In some aspects, the nucleotide sequence of the sense strand comprises a sequence of 15 to 23 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% to a sequence of the same length that is comprised in the sequence complementary to the sequence of the antisense strand.
  • a PD-L1 RNAi agent is conjugated to one or more nonnucleotide groups including, but not limited to a targeting group, linking group, delivery polymer, or a delivery vehicle.
  • the non-nucleotide group can enhance targeting, delivery or attachment of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 6 of W02018027106.
  • the non-nucleotide group can be covalently linked to the 3' and/or 5' end of either the sense strand and/or the antisense strand.
  • a PD-L1 RNAi agent contains a non-nucleotide group linked to the 3' and/or 5' end of the sense strand.
  • a non-nucleotide group is linked to the 5' end of a PD-L1 RNAi agent sense strand.
  • a non-nucleotide group can be linked directly or indirectly to the RNAi agent via a linker/linking group.
  • a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.
  • the RNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art.
  • Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates (DPCs) (see, for example WO 2000/053722, WO 2008/022309, WO 2011/104169, and WO 2012/083185, each of which is incorporated herein by reference).
  • DPCs Dynamic Polyconjugates
  • a PD-L1 RNAi agent is delivered to target cells or tissues by covalently linking the RNAi agent to a targeting group (also referred to herein as a “targeting ligand”).
  • the targeting group can include a cell receptor ligand, such as an asialoglycoprotein receptor (ASGPr) ligand.
  • ASGPr asialoglycoprotein receptor
  • an ASGPr ligand includes or consists of a galactose derivative cluster.
  • a galactose derivative cluster includes an N-acetyl-galactosamine trimer or an N-acetyl- galactosamine tetramer.
  • a galactose derivative cluster is an N-acetyl- galactosamine trimer or an N-acetyl-galactosamine tetramer.
  • a targeting group can be linked to the 3' or 5' end of a sense strand or an antisense strand of a PD-L1 RNAi agent. In some embodiments, a targeting group is linked to the 3' or 5' end of the sense strand. In some embodiments, a targeting group is linked to the 5’ end of the sense strand. In some embodiments, a targeting group is linked to the RNAi agent via a linker (also referred to herein as a “linking group”).
  • a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.
  • Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are attached to improve cell-specific distribution and cellspecific uptake of the conjugate.
  • a targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency.
  • Representative targeting groups include, without limitation, compounds with affinity to cell surface molecule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules.
  • a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) groups.
  • a targeting group comprises a galactose derivative cluster.
  • the PD-L1 RNAi agents described herein can be synthesized having a reactive group, such as an amine group, at the 5 '-terminus. The reactive group can be used to subsequently attach a targeting moiety using methods typical in the art.
  • a targeting group comprises an asialoglycoprotein receptor ligand.
  • an asialoglycoprotein receptor ligand includes or consists of one or more galactose derivatives.
  • galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor that is equal to or greater than that of galactose.
  • Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetyl- galactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-iso- butanoylgalactos-amine (see for example: lobst, S.T. and Drickamer, K. J.B.C. 1996, 277, 6686).
  • Galactose derivatives, and clusters of galactose derivatives, that are useful for in vivo targeting of oligonucleotides and other molecules to the liver are known in the art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem, 257, 939- 945).
  • Galactose derivatives have been used to target molecules to hepatocvtes in vivo through their binding to the asialoglycoprotein receptor (ASGPr) expressed on the surface of hepatocytes.
  • ASGPr asialoglycoprotein receptor
  • ASGPr ligands can be monomeric (e.g., having a single galactose derivative) or multimeric (e.g., having multiple galactose derivatives).
  • the galactose derivative or galactose derivative cluster can be attached to the 3 Or 5' end of the RNAi polynucleotide using methods known in the art.
  • the preparation of targeting groups, such as galactose derivative clusters, is described in, for example, US20180064819 and US20170253875, the contents of both of which are incorporated herein in their entirety.
  • a galactose derivative cluster comprises a molecule having two to four terminal galactose derivatives. A terminal galactose derivative is attached to a molecule through its C-l carbon.
  • the galactose derivative cluster is a galactose derivative trimer (also referred to as tri-antennary galactose derivative or tri-valent galactose derivative).
  • the galactose derivative cluster comprises N-acetyl-galactosamines.
  • the galactose derivative cluster comprises three N-acetyl-galactosamines.
  • the galactose derivative cluster is a galactose derivative tetramer (also referred to as tetra-antennary galactose derivative or tetra-valent galactose derivative). In some embodiments, the galactose derivative cluster comprises four N-acetyl-galactosamines.
  • a galactose derivative trimer contains three galactose derivatives, each linked to a central branch point.
  • a galactose derivative tetramer contains four galactose derivatives, each linked to a central branch point.
  • the galactose derivatives can be attached to the central branch point through the C-l carbons of the saccharides.
  • the galactose derivatives are linked to the branch point via linkers or spacers.
  • the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (see, for example, U.S. Patent No. 5,885,968; Biessen et al. J. Med. Chem.
  • the PEG spacer is a PEG3 spacer.
  • the branch point can be any small molecule which permits attachment of three galactose derivatives and further permits attachment of the branch point to the RNAi agent.
  • An example of branch point group is a dilysine or di-glutamate. Attachment of the branch point to the RNAi agent can occur through a linker or spacer.
  • the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer.
  • the linker comprises a rigid linker, such as a cyclic group.
  • a galactose derivative comprises or consists of N-acetyl-galactosamine.
  • the galactose derivative cluster is comprised of a galactose derivative tetramer, which can be, for example, an N-acetyl- galactosamine tetramer.
  • a linking group is conjugated to the RNAi agent.
  • the linking group facilitates covalent linkage of the agent to a targeting group or delivery polymer or delivery vehicle.
  • the linking group can be linked to the 3 ' or the 5' end of the RNAi agent sense strand or antisense strand.
  • the linking group is linked to the RNAi agent sense strand.
  • the linking group is conjugated to the 5' or 3' end of an RNAi agent sense strand.
  • a linking group is conjugated to the 5' end of an RNAi agent sense strand.
  • linking groups include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleosides, ribitol (abasic ribose), and/or PEG groups.
  • a linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest via one or more covalent bonds.
  • a labile linkage contains a labile bond.
  • a linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer can further add flexibility and/or length to the linkage.
  • Spacers can include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description.
  • Targeting groups, modified nucleotides and linking groups include, but are not limited to, the following, for which their chemical structures are provided below in Table 1 : (PAZ), (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), (PAZ),
  • NAG in the structures provided in the above table is represented by the following structure:
  • Each (NAGx) can be attached to a PD-L1 RNAi agent via a phosphate group (e.g. , as in (NAG25), (NAG30), and (NAG31)), or a phosphorothioate group, (e.g., as is (NAG25)s, (NAG29)s, (NAG30)s, (NAG31)s, or (NAG37)s), or another linking group:
  • a phosphate group e.g. , as in (NAG25), (NAG30), and (NAG31)
  • a phosphorothioate group e.g., as is (NAG25)s, (NAG29)s, (NAG30)s, (NAG31)s, or (NAG37)s
  • another linking group e.g., as is (NAG25)s, (NAG29)s, (NAG30)s, (NAG31)s, or (NAG37)s
  • the RNAi agent comprises one or more from invAb and targeting moieties, more particularly liver targeting moieties; wherein the liver targeting moieties are fatty acids, GalNAc, folic acid, cholesterol, tocopherol or palmitate, more particularly GalNAc, more particularly
  • (GalNAc2) at the 3 ’ end of the sense strand optionally further comprising one or more invAb, more particularly 1 invAb at the 5’ end of the sense strand and/or 1 invAb at the 3’ end of the sense strand.
  • a delivery vehicle can be used to deliver an RNAi agent to a cell or tissue.
  • a delivery vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue.
  • a delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin- like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active poly amine.
  • the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art.
  • the RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference), or other delivery systems available in the art.
  • lipophilic compounds that have been conjugated to oligonucleotides include 1- pyrene butyric acid, l,3-bis-O-(hexadecyl)glycerol, and menthol.
  • a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor- mediated endocytosis. RNAi agents bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis.
  • ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides.
  • conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases.
  • Representative examples of cationic ligands are propylammonium and dimethylpropylammonium.
  • antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See Manoharan M, Antisense & Nucleic Acid Drug Development (2002) 12: 103 and references therein.
  • Additional modifications can also be made at other positions on the oligonucleotide, particularly the 3’ position of the sugar on the 3’ terminal nucleotide.
  • one additional modification of the ligand-conjugated oligonucleotides of the application involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties, such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci.
  • RNAi agent can be modified by a non-ligand group.
  • a number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the RNAi agent, and procedures for performing such conjugations are available in the scientific literature.
  • Such non-ligand moieties have included lipid moieties, such as cholesterol ( 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.
  • lipid moieties such as cholesterol ( 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.
  • RNAi agent conjugation protocols involve the synthesis of oligonucleotides 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 RNAi agent still bound to the solid support or following cleavage of the RNAi agent in solution phase. Purification of the RNAi agent conjugate by HPLC typically affords the pure conjugate.
  • the molecule being conjugated can be converted into a building block, such as a phosphoramidite, via an alcohol group present in the molecule or by attachment of a linker bearing an alcohol group that can be phosphorylated.
  • a building block such as a phosphoramidite
  • each of these approaches can be used for the synthesis of ligand conjugated RNAi agents.
  • Amino linked RNAi agents can be coupled directly with ligand via the use of coupling reagents or following activation of the ligand as an NHS or pentfluorophenolate ester.
  • Ligand phosphoramidites can be synthesized via the attachment of an aminohexanol linker to one of the carboxyl groups followed by phosphityation of the terminal alcohol functionality.
  • RNAi agents can also be utilized for conjugation to a chloroacetyl linker present on a synthesized oligonucleotide.
  • linkers such as cysteamine
  • cysteamine can also be utilized for conjugation to a chloroacetyl linker present on a synthesized oligonucleotide.
  • RNAi agents for the introduction of RNAi agents, means and methods have been provided.
  • targeted delivery by glycosylated and folate -modified molecules including the use of polymeric carriers with ligands, such as galactose and lactose or the attachment of folic acid to various macromolecules allows the binding of molecules to be delivered to folate receptors.
  • Targeted delivery by peptides and proteins other than antibodies, for example, including RGD- modified nanoparticles to deliver siRNA in vivo or multicomponent (nonviral) delivery systems including short cyclodextrins, adamantine- PEG are known.
  • Target directed delivery comprises, inter alia, hydrodynamic i.v. injection.
  • cholesterol conjugates of RNAi agents can be used for targeted delivery, whereby the conjugation to lipophilic groups enhances cell uptake and improve pharmacokinetics and tissue biodistribution of oligonucleotides.
  • cationic delivery systems are known, whereby synthetic vectors with net positive (cationic) charge to facilitate the complex formation with the polyanionic nucleic acid and interaction with the negatively charged cell membrane.
  • Such cationic delivery systems comprise also cationic liposomal delivery systems, cationic polymer and peptide delivery systems.
  • Other delivery systems for the cellular uptake of dsRNA/siRNA are aptamer-ds/si RNA.
  • gene therapy approaches can be used to deliver the described RNAi agents.
  • Such systems comprise the use of non-pathogenic virus, modified viral vectors, as well as deliveries with nanoparticles or liposomes.
  • Other delivery methods for the cellular uptake of RNAi agents are extracorporeal, for example ex vivo treatments of cells, organs or tissues.
  • RNAi agents and conjugates thereof are known in the art. Any such known methods can be used in the context of the present application to make and use RNAi agents and conjugates thereof for inhibiting the expression of an HBV gene. Methods of making and using RNAi agents and conjugates thereof are described, e.g., in US20130005793, W02013003520, W02018027106, US5218105, US5541307, US5521302, US5539082, US5554746, US5571902, US5578718, US5587361, US5506351, US5587469, US5587470, US5608046, US5610289, US6262241, WO9307883, all of which are incorporated herein by reference in their entirety.
  • A adenosine-3 '-phosphate
  • m any 2'-0Me modified nucleotide
  • fN any 2'-fluoro modified nucleotide
  • dN any 2'-deoxyribonucleotide
  • NuNA 2',3'-seco nucleotide mimics (unlocked nucleobase analogs)
  • NLNA locked nucleotide
  • NfANA 2'-F-Arabino nucleotide
  • UNA-N 2', 3 '-seco nucleotide (any unlocked nucleotide)
  • NM 2'-methoxyethyl nucleotide
  • AM 2'-methoxyethyladenosine-3'-phosphate
  • AMs 2'-methoxyethyladenosine-3 '-phosphorothioate
  • TM 2'-methoxyethylthymidine-3'-phosphate
  • TMs 2'-methoxyethylthymidine-3'-phosphorothioate
  • vpdN vinyl phosphonate deoxyribonucleotide
  • terminal nucleotide at the 3 ' end of a given oligonucleotide sequence would typically have a hydroxyl (- OH) group at the respective 3' position of the given monomer instead of a phosphate moiety ex vivo.
  • a “modified nucleotide” is a nucleotide other than a ribonucleotide
  • At least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides.
  • modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides, non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3 '-seco nucleotide mimics (unlocked nucleobase analogues), locked nucleotides, 3'-O-methoxy (2' intemucleoside linked) nucleotides, 2'-F- Arabino nucleotides, 5 '-Me, 2'-fluoro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucle
  • 2'-modified nucleotides include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2 -methoxylethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified.
  • ribonucleic acid molecule may be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.
  • Modified nucleobases include synthetic and natural nucleobases, such as 5 -substituted pyrimidmes, 6-azapyrimi dines and N-2, N-6 and 0-6 substituted purines, aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5 -methylcytosine (5-me-C), 5- hydroxymethyl cytosme, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6- ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2- ethyl, 2-isopropyl, or 2-n-butyl ) and other alkyl derivatives of adenine and guanine, 2- thiouracil.
  • synthetic and natural nucleobases such as 5 -substituted
  • 2-thiothymine 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6- azo uracil, 6-azo cytosine, 6-azo thymine, -uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-sulfhydiyl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoiOinethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-dea/aadenine.
  • 5-halo e.g., 5-bromo
  • RNAi agent all or substantially all of the nucleotides of an RNAi agent are modified nucleotides.
  • an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RN Ai agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides.
  • a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides.
  • an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides.
  • one or more nucleotides of an RNAi agent is a ribonucleotide.
  • sugar substituent group or “2 ’-substituent group” includes groups attached to the 2 ’-position of the ribofuranosyl moiety with or without an oxygen atom.
  • Sugar substituent groups include, but are not limited to, fluoro, O-alkyl, O-alkylamino, O- alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole and poly ethers of the formula (O-alkyl)m, wherein m is 1 to about 10.
  • polyethers linear and cyclic polyethylene glycols (PEGs), and (PEG)- containing groups, such as crown ethers and, inter alia, those which are disclosed by Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems (1992) 9:249). Further sugar modifications are disclosed by Cook (Anti-fibrosis Drug Design, (1991) 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O- alkylaminoalkyl, and alkyl amino substitution is described in U.S. Patent 6, 166,197, entitled “Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2’ and 5’ Substitutions.” hereby incorporated by reference in its entirety.
  • Additional sugar substituent groups amenable to the application include 2’-SR and 2‘- NR2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl.
  • 2 ’-SR Nucleosides are disclosed in US5670633, hereby incorporated by reference in its entirety. The incorporation of 2’ -SR monomer synthons is disclosed by Hamm et al. (J. Org. Chem., (1997) 62:3415-3420). 2’-NR nucleosides are disclosed by Thomson JB, J. Org.
  • E is C1-C10 alkyl, N(Q3)(Q4) or C(Q3)(Q4); each Q3 and Q4 is, independently, H, C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q3 and Q4, together, form a nitrogen protecting group or a ring structure optionally including at least one additional heteroatom selected from N and O; q 1 is an integer from 1 to 10; q2 is an integer from 1 to 10; q3 is 0 or 1; q4 is 0, 1 or 2; each Zl, Z2, and Z3 is, independently, C4-C7 cycloalkyl, C5-C14 aryl or C3- C15 heterocyclyl, where Z4 is OM1, SMI, or N(Ml)z; each Ml is, independently, H, Ci-Cs alkyl, Ci-Cs
  • Representative 2’-O-sugar substituent groups of formula I are disclosed in US6172209, entitled “Capped 2’-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety.
  • Representative cyclic 2’-O-sugar substituent groups of formula II are disclosed in US6271358, entitled “RNA Targeted 2’-Modified Oligonucleotides that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.
  • Oligonucleotides can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar.
  • sugar mimetics such as cyclobutyl moieties
  • Representative United States patents relating to the preparation of such modified sugars include, but are not limited to, US5359044, US5466786, US5519134, US5591722, US5597909, US5646,265, and US5700920, all of which are hereby incorporated by reference.
  • At least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sequence of the antisense strand of the RNAi agent are modified nucleotides. In some aspects, wherein all the nucleotides of the sequence of the antisense strand of the RNAi agent are modified nucleotides. In some aspects, at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sense strand of the RNAi agent are modified nucleotides. In some aspects, all the nucleotides of the sequence of the sense strand of the RNAi agent are modified nucleotides. In some aspects, the modified nucleotide comprises a modified nucleoside and/or a modified phosphate and/or a modified intemucleotide linkage.
  • the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a [0152] 2’-deoxy-2’-fluoro-ribose (
  • the modified nucleotide of the RNAi agent comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate .
  • a stabilized phosphonate mimic such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate .
  • the modified nucleotide comprises a modified intemucleotide linkage selected from
  • thiophosphoramidate linkages more particularly wherein the modified intemucleotide linkage is a phosphorothioate linkage.
  • the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NOs: 202-402. In some aspects, the sense strand of the RNAi agent comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-201. In some aspects, the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NOs: 604-804. In some aspects, the sense strand of the RNAi agent comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NOs: 403-603.
  • the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 805-1005; 1690; 1724; 1760; and 1839. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 403-603; 1689; 1723; 1759; 1799; and 1838. In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 240; 276; 280 and 283. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 642; 678; 682; and 685.
  • the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 441; 477; 481; and 484. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 39; 75; 79 and 82. In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 484; 843; 879; 883; 1690; 1724; and 1759. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 441; 477; 481; 886; 1689; 1723; and 1760.
  • sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table 1. In some aspects, the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table la. In some aspects, the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table lb. In some aspects, all of nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA. In some aspects, all of nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA, wherein optionally: a.
  • the number of 2’F nucleotides in antisense strand 2, 4, 6, 9 or 10, more particularly 3, 4, 5 or 6; b. optionally the number of UNA nucleotides in the antisense strand is 1 UNA-U or 1-UNA-A; c. optionally the number of vinylphosphonate nucleotides in the antisense strand is 1 vinylphosphonate nucleotide in the antisense strand, more particularly 1 vinylphosphonate nucleotide at the 5 ’ end of the antisense strand; d.
  • nucleotides in the antisense strand that are not modified by 2’F or by vinylphosphonate or that are not UNA are modified by 2 ’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or e. optionally the number of 2’0-Me nucleotides is 10, 11, 13, 15, 17 or 19.
  • all of nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA, wherein optionally: a. optionally number of 2’F nucleotides in sense strand is 2, 4, 6, 9, or 10, more particularly 4, 5, 6 or 7; b.
  • nucleotides in the sense strand that are not modified by 2’F are modified by 2’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or c. optionally the number of 2’0-Me nucleotides is 10, 11, 13, 15 or 17.
  • nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA, wherein optionally 1, 2 or 3 phosphorothioate linkages linking the 3’ end or 5’ end terminal nucleotides of the sense strand and the antisense strand, more particularly 1, 2 or 3 phosphorothioate linkages linking (ntl and nt2), and/or (nt2 and nt3), and/or (nt3 and nt4) of the sense strand and antisense strand and/or the antisense strand.
  • the RNAi agent comprises one or more of a sense strand of SEQ ID NO: 1855 and an antisense strand of SEQ ID NO: 1871 (duplex no. 39V9iv); a sense strand of SEQ ID NO: 1701 and an antisense strand of SEQ ID NO: 1717 (duplex no. 75V3B); a sense strand of SEQ ID NO: 1733 and an antisense strand of SEQ ID NO: 1750 (duplex no. 79V3A7); a sense strand of SEQ ID NO: 1700 and an antisense strand of SEQ ID NO: 1716 (duplex no.
  • duplex no. 75V3A a sense strand of SEQ ID NO: 1773 and an antisense strand of SEQ ID NO:
  • a sense strand of SEQ ID NO: 1762 and an antisense strand of SEQ ID NO: 1781 (duplex no. 82V 10b); a sense strand of SEQ ID NO: 1725 and an antisense strand of SEQ ID NO: 1724 (duplex no. 79V10b); a sense strand of SEQ ID NO: 1843 and an antisense sequence of SEQ ID NO: 1859 (duplex no. 39V10bul); a sense strand of SEQ ID NO: 1728 and an antisense strand of SEQ ID NO: 1745 (duplex no.
  • 79V10bul a sense strand of SEQ ID NO: 1850 and an antisense strand of SEQ ID NO: 1866 (duplex no. 39V3i); a sense strand of SEQ ID NO: 1699 and an antisense strand of SEQ ID NO: 1715 (duplex no. 75V3A7); a sense strand of SEQ ID NO: 1730 and an antisense strand of SEQ ID NO: 1747 (duplex no. 79V12c); a sense strand of SEQ ID NO: 1694 and an antisense strand of SEQ ID NO: 1710 (duplex no.
  • 75V10bul a sense strand of SEQ ID NO: 1693 and an antisense strand of SEQ ID NO: 1709 (duplex no. 75V10bu); a sense strand of SEQ ID NO: 1734 and an antisense strand of SEQ ID NO: 1751 (duplex no. 79V3A7v); a sense strand of SEQ ID NO: 1691 and an antisense strand of SEQ ID NO: 1707 (duplex no. 75V10b); a sense strand of SEQ ID NO: 1727 and an antisense strand of SEQ ID NO: 1744 (duplex no.
  • the RNAi agent comprises one or more of a sense strand of SEQ ID NO: 1855 and an antisense strand of SEQ ID NO: 1871 (duplex no. 39V9iv); a sense strand of SEQ ID NO: 1701 and an antisense strand of SEQ ID NO: 1717 (duplex no. 75V3B); a sense strand of SEQ ID NO: 1733 and an antisense strand of SEQ ID NO: 1750 (duplex no.
  • a sense strand of SEQ ID NO: 1700 and an antisense strand of SEQ ID NO: 1716 (duplex no. 75V3A7v); a sense strand of SEQ ID NO: 1697 and an antisense strand of SEQ ID NO: 1713 (duplex no. 75Vla); a sense strand of SEQ ID NO: 1848 and an antisense strand of SEQ ID NO: 1864 (duplex no. 39V3A7v); a sense strand of SEQ ID NO: 1736 and an antisense strand of SEQ ID NO: 1753 (duplex no.
  • 79V3i a sense strand of SEQ ID NO: 1732 and an antisense strand of SEQ ID NO: 1749 (duplex no. 79V3A); a sense strand of SEQ ID NO: 1698 and an antisense strand of SEQ ID NO: 1714 (duplex no. 75V3A); a sense strand of SEQ ID NO: 1773 and an antisense strand of SEQ ID NO: 1792 (duplex no. 82V3B); a sense strand of SEQ ID NO: 1842 and an antisense strand of SEQ ID NO: 1858 (duplex no.
  • the RNAi agent comprises one or more of a sense strand of SEQ ID NO: 1855 and an antisense strand of SEQ ID NO: 1871 (duplex no. 39V9iv); a sense strand of SEQ ID NO: 1701 and an antisense strand of SEQ ID NO: 1717 (duplex no. 75V3B); a sense strand of SEQ ID NO: 1733 and an antisense strand of SEQ ID NO: 1750 (duplex no.
  • the sequence of the antisense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
  • the sequence of the sense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
  • the sequences of the antisense and sense strands of the RNAi agent comprises the sequences of the duplex selected from the duplex of Tables 2, 2a, 5 and 7.
  • one or more nucleotides of the RNAi agent are linked by nonstandard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones).
  • a modified intemucleoside linkage is a non-phosphate-containing covalent intemucleoside linkage.
  • Modified intemucleoside linkages or backbones include, but are not limited to, 5’-phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3 '-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of
  • a modified intemucleoside linkage or backbone lacks a phosphoms atom.
  • Modified intemucleoside linkages lacking a phosphoms atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages.
  • modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, 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 other backbones having mixed N, O, S, and CEE components.
  • a sense strand of a PD-L1 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • an antisense strand of a PD-L1 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages.
  • a sense strand of a PD-L1 RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • an antisense strand of a PD-L1 RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages
  • both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.
  • a PD-L1 RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages.
  • the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand.
  • the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5' end of the sense strand.
  • a PD-L1 RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages.
  • the four phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the sense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24- 26 from the 5' end.
  • a PD-L1 RNAi agent contains at least two phosphorothioate intemucleoside linkages in the sense strand and three or four.
  • a PD-L1 RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2'- modified nucleoside is combined with modified intemucleoside linkage.
  • RNAi agents of the present application can also be chemically modified to enhance stability.
  • the nucleic acids of the application can be synthesized and/or modified by methods well established in the art. Chemical modifications can include, but are not limited to 2’ modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages, inverted deoxythymidines.
  • the integrity of the duplex structure is strengthened by at least one, and preferably two, chemical linkages.
  • Chemical linking can be achieved by any of a variety of well- known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues.
  • the chemical groups that can be used to modify the RNAi agents include, without limitation, methylene blue; bifunctional groups, preferably bis-(2- chloroethyl)amine; -acetyl-N’-(p- glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen.
  • the linker is a hexa-ethylene glycol linker.
  • the RNAi agents are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams DJ and Hall KB, Biochem. (1996) 35: 14665- 14670).
  • the 5 ’-end of the antisense strand and the 3 ’-end of the sense strand are chemically linked via a hexaethylene glycol linker.
  • at least one nucleotide of the RNAi agent comprises a phosphorothioate or phosphorodithioate groups.
  • the chemical bond at the ends of the RNAi agent is preferably formed by triple-helix bonds. Additional Agents
  • a combination therapy of the application may further comprise additional or other agent(s) that is(are) active against a disease to be treated, such as, for example, HBV.
  • anti-HBV agents suitable for use with the application include, but are not limited to small molecules, antibodies, and/or CAR-T therapies which bind HBV env (S-CAR cells), capsid assembly modulators, TLR agonists e.g., TLR7 and/or TLR8 agonists), cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir), and/or immune checkpoint inhibitors, etc.
  • the at least one anti-HBV agent can e.g., be chosen from among HBV DNA polymerase inhibitors; immunomodulators; toll-like receptor 7 modulators; toll-like receptor 8 modulators; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; modulators of IL- 10; HBsAg inhibitors; toll -like receptor 9 modulators; cyclophilin inhibitors; HBV prophylactic vaccines; HBV therapeutic vaccines; HBV viral entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; endonuclease modulators; inhibitors of ribonucleotide reductase; hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies
  • anti-HBV agents suitable for use with the application include more specifically (but are not limited to) adjuvants of the immune response, more specifically adjuvants that are potentially safe, well tolerated and effective in humans.
  • An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, and IL-7- hyFc.
  • immune checkpoint inhibitors e.g., anti-PDl, anti-TIM-3, etc.
  • toll-like receptor agonists e.g., TLR7 agonists and/or TLR8 agonists
  • Adjuvants can also e.g., be chosen from among the following anti-HBV agents: HBV DNA polymerase inhibitors; immunomodulators; toll-like receptor 7 modulators; toll-like receptor 8 modulators; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; modulators of IL- 10; HBsAg inhibitors; toll-like receptor 9 modulators; cyclophilin inhibitors; HBV prophylactic vaccines; HBV therapeutic vaccines; HBV viral entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; endonuclease modulators; inhibitors of ribonucleotide reductase; hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus;
  • the other additional agent may be administered to a subject in need thereof during any time period of the treatment.
  • the additional agent is administered to a subject in need thereof during a first treatment period.
  • the additional agent is administered to a subject in need thereof during a second treatment period.
  • the combinations described herein can be used in any methods or kits described below.
  • Hepatitis B Virus (HBV) HBV
  • hepatitis B virus or “HBV” refers to a virus of the hepadnaviridae family.
  • HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins.
  • the seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein.
  • HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest.
  • Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes.
  • PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion form infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein.
  • the HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome.
  • the nucleocapsid comprises 120 dimers of core protein and is covered by a capsid membrane embedded with the S, M, and L viral envelope or surface antigen proteins. After entry into the cell, the virus is uncoated and the capsid-containing relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus.
  • rcDNA relaxed circular DNA
  • phosphorylation of the Core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so-called importins.
  • importins mediate binding of the core protein to nuclear pore complexes upon which the capsid disassembles and polymerase/rcDNA complex is released into the nucleus.
  • the rcDNA becomes deproteinized (removal of polymerase) and is converted by host DNA repair machinery to a covalently closed circular DNA (cccDNA) genome from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein.
  • HBV pre-genomic RNA
  • pgRNA pre-genomic RNA
  • HBV HBV is divided into four serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome.
  • genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
  • HBV antigen As used herein, the terms “HBV antigen,” “antigenic polypeptide of HBV,” “HBV antigenic polypeptide,” “HBV antigenic protein,” “HBV immunogenic polypeptide,” and “HBV immunogen” all refer to a polypeptide capable of inducing an immune response against an HBV in a subject. The induced response can be a humoral and/or cellular mediated response.
  • the HBV antigen can be a polypeptide of HBV, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof.
  • an HBV antigen is capable of raising in a host a protective immune response, e.g, inducing an immune response against a viral disease or infection, and/or producing an immunity (i.e., vaccinates) a subject against a viral disease or infection, that protects the subject against the viral disease or infection.
  • an HBV antigen can comprise a polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), core protein, viral polymerase, or HBx protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G, and/or H, or combination thereof.
  • each of the terms “HBV core antigen,” “HBeAg,” and “core antigen” refers to an HBV antigen capable of inducing an immune response against an HBV core protein in a subject.
  • the induced immune response can be a humoral and/or cellular mediated response.
  • Each of the terms “core,” “core polypeptide,” and “core protein” refers to the HBV viral core protein.
  • Full-length core antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). The 34-residue nucleic acid binding domain is required for pre-genomic RNA encapsidation.
  • HBV core protein is dimeric in solution, with the dimers selfassembling into icosahedral capsids. Each dimer of core protein has four a-helix bundles flanked by an a-helix domain on either side. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids.
  • HBV polymerase antigen As used herein, the term “HBV polymerase antigen,” “HBV Pol antigen” or “HBV pol antigen” refers to an HBV antigen capable of inducing an immune response against an HBV polymerase in a subject.
  • the immune response can be a humoral and/or cellular mediated response.
  • polymerase polymerase polypeptide
  • Poly polymerase polypeptide
  • the HBV viral DNA polymerase has four domains, including, from the N terminus to the C terminus, a terminal protein (TP) domain, which acts as a primer for minus-strand DNA synthesis; a spacer that is nonessential for the polymerase functions; a reverse transcriptase (RT) domain for transcription; and an RNase H domain.
  • TP terminal protein
  • RT reverse transcriptase
  • each of the terms “HBV surface antigen,” “surface antigen,” “HBV envelope antigen,” “envelope antigen,” and “env antigen” refers to an HBV antigen capable of inducing or eliciting an immune response against one or more HBV surface antigens or envelope proteins in a subject.
  • the immune response can be a humoral and/or cellular mediated response.
  • Each of the terms “HBV surface protein,” “surface protein,” “HBV envelope protein” and “envelope protein” refers to HBV viral surface or envelope proteins.
  • HBV expresses three surface antigens, or envelope proteins.
  • Gene S is the gene of the HBV genome that encodes the surface antigens.
  • the surface antigen gene is one long open reading frame but contains three in frame “start” (ATG) codons that divide the gene into three sections, pre-Sl, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes called large (L) or L- surface antigen, middle (M) or M-surface antigen, and small (S) or S-surface antigen are produced, also named the HBV L, M and S envelope proteins. Two different promoters (PreSl and PreS2) drive transcription of the L, M, and S-surface antigen coding sequences resulting in three different translated proteins, the L, M and S envelope proteins.
  • the PreS2 promoter is sometimes referred to as the PreS2/S promoter since it is driving M-surface antigen and S-surface antigen transcription separately.
  • the amino acid sequence of the L-surface antigen is in-frame with the M and S-surface antigen sequences.
  • the L-surface antigen contains the M- and S- surface antigen domains and the M-surface antigen includes the S-surface antigen domain.
  • the L-, M- and S-surface antigen are co-C-terminal and share the entire S domain. Relative to S, M has an additional domain, pre-S2, at its N terminus, and relative to M, L has a pre-Sl domain.
  • an HBV antigen is an HBV PreSl antigen, which is encoded by a pre-Sl gene section and contains only the Pre-Sl domain of the L antigen.
  • the PreSl antigen can have various lengths, such as having 99 to 109 amino acids.
  • An HBV PreSl antigen of the present disclosure can contain the sequence of any naturally occurring Pre SI domain, and variants or derivatives thereof.
  • an HBV antigen is an HBV PreS2.S antigen, which is encoded by the pre-S2 and S gene sections and contains the PreS2 domain and the S domain.
  • the PreS2 domain can be about 55 amino acids long and the S-domain can contain about 226 amino acids.
  • An HBV PreS2.S antigen of the present disclosure can contain the sequences of any of the naturally occurring PreS2 and S domains, and variants or derivatives thereof.
  • an internal signal peptide of PreS2.S is left intact to facilitate secretion PreS2.S protein products of the HBV M and HBV S antigens.
  • an HBV PreS2.S antigen is an HBV M surface antigen.
  • an HBV PreS2.S antigen is an HBV S surface antigen.
  • an HBV PreS2.S antigen encompasses an HBV M surface antigen and an HBV S surface antigen.
  • the present disclosure also relates to compositions, pharmaceutical compositions, and therapeutic combinations comprising one or more RNAi agents according to the present disclosure. Any of the RNAi agents described herein can be used in the compositions, pharmaceutical compositions, and therapeutic combinations of the present disclosure.
  • compositions comprising any RNAi agent described herein, together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient.
  • Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings.
  • suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like.
  • suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like.
  • the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives.
  • the composition comprises a salt of RNAi agent, optionally a sodium salt.
  • the composition comprises LNP or liposome comprising the RNAi agent.
  • the composition comprises an isolated cell comprising the RNAi agent.
  • the composition comprises non-human animal comprising the RNAi agent.
  • the present disclosure generally relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the RNAi agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.
  • the pharmaceutical composition is a liquid composition.
  • the liquid composition comprises water, saline, and/or buffer.
  • the pharmaceutical composition is a lyophilized composition.
  • compositions of the present disclosure can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections.
  • the parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection.
  • Pharmaceutical compositions of the present disclosure can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
  • compositions of the present disclosure are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
  • compositions for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g, an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS).
  • a pharmaceutically acceptable carrier e.g, an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the compositions and therapeutic combinations can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents.
  • a pharmaceutical composition of the present disclosure comprising a ribonucleic acid molecule can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier.
  • PBS phosphate buffered saline
  • the ribonucleic acid molecule can be administered for example at 1-1000 pg/dose.
  • an adjuvant is included in a pharmaceutical composition of the present disclosure or co-administered with a pharmaceutical composition of the present disclosure.
  • Use of an adjuvant is optional and can further enhance immune responses when the composition is used for vaccination purposes.
  • Adjuvants suitable for co-administration or inclusion in compositions in accordance with the present disclosure should preferably be ones that are potentially safe, well tolerated and effective in humans.
  • An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, and IL-7-hyFc.
  • the present disclosure also provides methods of making pharmaceutical compositions and therapeutic combinations of the present disclosure.
  • a method of producing a pharmaceutical composition or therapeutic combination comprises mixing an inhibitory oligonucleotide of the present disclosure with one or more pharmaceutically acceptable carriers.
  • One of ordinary skill in the art will be familiar with conventional techniques used to prepare such compositions.
  • RNAi component for use in combination with the other pharmaceutical compositions for treating an HBV infection or a disease or disorder associated with the HBV infection in a subject in need thereof.
  • a first, second, and/or third pharmaceutical compositions can be formulated together in one pharmaceutical composition. They can also be formulated as separate pharmaceutical compositions that can be packaged together or separately.
  • a pharmaceutical composition comprising one or more PD-L1 RNAi agents that can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, oral, subdermal e.g., via an implanted device), and intraparenchymal administration.
  • the pharmaceutical compositions described herein are preferably administered by subcutaneous injection.
  • methods described herein comprise one or more PD-L1 RNAi agents, wherein the one or more RNAi agents are prepared as pharmaceutical compositions or formulations.
  • pharmaceutical compositions include at least one PD-L1 RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism.
  • the pharmaceutical compositions can be used to treat a subject having a disease or disorder that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene.
  • the pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene.
  • the method includes administering a PD-L1 RNAi agent linked to a targeting ligand as described herein, to a subject to be treated.
  • one or more pharmaceutically acceptable excipients are added to the pharmaceutical compositions including a PD-L1 RNAi agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a human.
  • compositions that include a PD-L1 RNAi agent and methods disclosed herein may decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, or subject, including: administering to the subject a therapeutically effective amount of a herein described PD-L1 RNAi agent, thereby inhibiting the expression of a target mRNA in the subject.
  • the described pharmaceutical compositions including a PD-L1 RNAi agent are used for treating or managing clinical presentations associated with a PD-L1 associated disease, such as an HBV infection.
  • a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment, prevention, or management.
  • administration of any of the disclosed PD-L1 RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
  • the described pharmaceutical compositions including a PD-L1 RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of PD-L1 mRNA.
  • the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions including a PD-L1 RNAi agent thereby treating the symptom.
  • the subject is administered a prophylactically effective amount of one or more PD-L 1 RNAi agents, thereby preventing the at least one symptom.
  • the route of administration is the path by which a PD-L1 RNAi agent is brought into contact with the body.
  • methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein.
  • the PD-L1 RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route.
  • herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some embodiments, there herein described pharmaceutical compositions via subcutaneous injection.
  • compositions including a PD-L1 RNAi agent described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery technologies known in the art.
  • any suitable method recognized in the art for delivering a nucleic acid molecule in vitro or in vivo can be adapted for use with a herein described compositions.
  • delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.
  • the compositions are administered by subcutaneous or intravenous infusion or injection.
  • the herein described pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients.
  • the pharmaceutical compositions described herein can be formulated for administration to a subject.
  • a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., PD-L1 RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage.
  • Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use.
  • a pharmaceutically acceptable excipient may or may not be an inert substance.
  • Excipients include, but are not limited to: absorption enhancers, anti-adherents, antifoaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension.
  • Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
  • the active compounds can be prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • the PD-L1 RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • a pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions.
  • additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.).
  • anti-pruritics e.g., antihistamine, diphenhydramine, etc.
  • anti-inflammatory agents e.g., antihistamine, diphenhydramine, etc.
  • cells, tissues or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions.”
  • “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic, or preventive result.
  • one or more (e.g., at least two) PD-L1 RNAi agents described herein can be formulated into one single composition or separate individual compositions.
  • the PD-L1 RNAi agents in separate individual compositions can be formulated with the same or different excipients and carriers.
  • the PD-L1 RNAi agents in separate individual compositions agents can be administered through same or different administration routes.
  • the PD-L1 RNAi agents are administered subcutaneously.
  • the pharmaceutical compositions described herein including a PD-L1 RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and/or a vaccine.
  • a second or other RNAi agent a small molecule drug, an antibody, an antibody fragment, and/or a vaccine.
  • the described PD-L1 RNAi agents when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.
  • the pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials. Kits
  • kits comprising an effective amount of an RNAi component for use in a treatment of a disease, such as, for example, a viral infection, more particularly chronic viral infection, in a subject in need thereof, wherein the viral infection, more particularly the chronic viral infection, comprises hepatitis B virus (HBV) infection, more particularly chronic HBV infection, wherein the RNAi component as described herein.
  • a disease such as, for example, a viral infection, more particularly chronic viral infection
  • HBV hepatitis B virus
  • the kit further comprises a package insert including, without limitation, appropriate instructions for preparation and administration of the formulation, side effects of the formulation, and any other relevant information.
  • the instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, optical disc, or directions to internet-based instructions.
  • the kit for treating an individual who suffers from or is susceptible to the conditions described herein comprising a first container comprising a dosage amount of a composition or formulation as disclosed herein, and a package insert for use.
  • the container can be any of those known in the art and appropriate for storage and delivery of intravenous formulation.
  • the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for preparation of the formulation to be administered to the individual.
  • the kit comprises one or more doses of the RNAi agent .
  • the kit-of-parts or functional association comprises one or more doses of the RNAi component.
  • kits may also be provided that contain sufficient dosages of the compositions described herein (including pharmaceutical compositions thereof) to provide effective treatment for an individual for an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles or more.
  • one cycle of treatment is about 1-24 months, about 1-3 months, about 3-6 months, about 6-9 months, about 9-12 months, about 12-18 months, about 18-21 months or about 21-24 months.
  • one cycle of treatment is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months or about 24 months.
  • kits may also include multiple doses and may be packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • the kits may include a dosage amount of at least one composition as disclosed herein.
  • a method treating a disease such as a viral infection, more particularly a chronic viral infection, using the RNAi agent, salt thereof, LNP or liposome comprising, isolated cell comprising, non-human animal comprising, or pharmaceutical composition comprising, as described herein.
  • the viral infection comprises an HBV infection.
  • the viral infection comprises an HBV infection and an HDV infection.
  • the viral infection comprises a HIV infection.
  • Also provided herein is a method treating hepatitis D, more particularly of chronic hepatitis D, using the RNAi agent, salt thereof, LNP or liposome comprising, isolated cell comprising, non-human animal comprising, or pharmaceutical composition comprising, as described herein.
  • a method treating a cancer for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma, using the RNAi agent, salt thereof, LNP or liposome comprising, isolated cell comprising, non-human animal comprising, or pharmaceutical composition comprising, as described herein.
  • the subject to be treated has an HBV infection, more particularly a chronic HBV infection.
  • the subject has a further viral infection, more particularly a further chronic viral infection.
  • the subject may have an HBV infection (more particularly a chronic HBV infection) and a super-/co-infection with one or more from among hepatitis D virus (HDV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV), more particularly with at least HDV.
  • HBV infection hepatitis D virus
  • HCV hepatitis C virus
  • HCV human immunodeficiency virus
  • the RNAi component is formulated in a solid form, such as a tablet or capsule. In some embodiments, the RNAi component is formulated for subcutaneous injection. In some embodiments, the RNAi component is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized. [0216] In some aspects, the RNAi agent is capable of inducing a PD-L1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%.
  • the RNAi agent may be capable of inducing a PD-L1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%, at day 6 or 7 in mice C57/bl6 mice infected with AAV8-hPD-Ll firefly luciferase, for example as described in Example 5.
  • the RNAi agent is capable of inducing a PD-L1 knockdown at a KD of at least 60% and at an IC50 of less than 150 nM, more particularly less than lOOnM , for example, in a free uptake assay, such as on primary human hepatocytes (PHH).
  • the RNAi agent is for use in combination with one or more agents chosen from among antiviral agents (e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators), immune checkpoints, immunomodulators (more particularly one or more TLR immunomodulators), vaccines (e.g., an anti-HBV therapeutic vaccine), anti-HBV siRNAs, anti- HBV ASOs and NAPs.
  • antiviral agents e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators
  • immune checkpoints e.g., immunomodulators (more particularly one or more TLR immunomodulators), vaccines (e.g.,
  • Some embodiments described herein relate to a method of treating a disease, such as a viral infection (e.g., HBV infection) that can include administering to a subject identified as suffering from the viral infection an effective amount of an RNAi agent as described herein, or a pharmaceutical composition that includes an effective amount of an RNAi agent as described herein.
  • a disease such as a viral infection (e.g., HBV infection)
  • a pharmaceutical composition that includes an effective amount of an RNAi agent as described herein.
  • Some embodiments described herein relate to using an RNAi agent as described herein in the manufacture of a medicament for treating a disease such as a viral infection (e.g., HBV infection).
  • compositions and therapeutic combinations of the present disclosure will have a therapeutic aim to generate an immune response against a disease, such as a PD-L1 associated disease, such as, for example, HBV after HBV infection or development of symptoms characteristic of HBV infection.
  • a disease such as a PD-L1 associated disease
  • HBV HBV after HBV infection or development of symptoms characteristic of HBV infection.
  • an effective amount or “a therapeutically effective amount” refer to an amount of an RNAi agent or a composition comprising one or more RNAi agents sufficient to induce a desired immune effect or immune response in a subject in need thereof.
  • a therapeutically effective amount can be an amount sufficient to induce an immune response in a subject in need thereof.
  • a therapeutically effective amount can be an amount sufficient to produce immunity in a subject in need thereof, e.g., provide a therapeutic effect against a disease such as HBV infection.
  • a therapeutically effective amount can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; the particular application, e.g., providing protective immunity or therapeutic immunity; and the particular disease, e.g., viral infection, for which immunity is desired.
  • a therapeutically effective amount can readily be determined by one of ordinary skill in the art in view of the present disclosure.
  • a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or (xi
  • a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a PD-L1 associated infection or disease, or a symptom associated therewith; (ii) reduce the duration of an PD-L1 associated infection or disease, or symptom associated therewith; (iii) prevent the progression of an PD-L1 associated disease, or symptom associated therewith; (iv) cause regression of an PD- L1 associated disease, or symptom associated therewith; (v) prevent the development or onset of an PD-L1 associated disease, or symptom associated therewith; (vi) prevent the recurrence of an PD-L1 associated disease or symptom associated therewith; (vii) reduce hospitalization of a subject having an PD-L1 associated disease; (viii) reduce hospitalization length of a subject having an PD-L1 associated disease; (ix) increase the survival of a subject
  • a therapeutically effective amount when used with reference to a ribonucleic acid molecule can range from about 1 pg of ribonucleic acid molecule to about 1 mg of ribonucleic acid molecule, such as 1 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 p.g, 90 pig, 100 pig, 200 pig, 300 pig, 400 pig, 500 pig, 600 pig, 700 pig, 800 pig, 900 pig, or 1 mg.
  • a therapeutically effective amount of a ribonucleic acid molecule is about 10 pg to about 100 pg.
  • a therapeutically effective amount when used with reference to a ribonucleic acid molecule in a pharmaceutical composition can range from a concentration of about 0.001 mg/mL to about 1 mg/mL of a ribonucleic acid molecule total, such as 0.001 mg/mL, 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, or 1 mg/mL.
  • a therapeutically effective amount of a ribonucleic acid molecule is less than 1 mg/mL, more preferably less than 0.05 mg/mL.
  • a therapeutically effective amount can be from one ribonucleic acid molecule or from multiple ribonucleic acid molecules.
  • a therapeutically effective amount can be administered in a single composition, or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules or injectables, or any composition adapted to intradermal delivery, e.g., to intradermal delivery using an intradermal delivery patch), wherein the administration of the multiple capsules or injections collectively provides a subject with a therapeutically effective amount.
  • prime-boost regimen It is also possible to administer a therapeutically effective amount to a subject, and subsequently administer another dose of a therapeutically effective amount to the same subject, in a so-called prime-boost regimen.
  • This general concept of a primeboost regimen is well known to the skilled person in the vaccine field. Further booster administrations can optionally be added to the regimen, as needed.
  • a therapeutic combination comprising two RNAi agents can be administered to a subject by mixing both RNAi agents and delivering the mixture to a single anatomic site. Alternatively, two separate immunizations each delivering a single RNAi agent can be performed.
  • the first RNAi agent and the second RNAi agent can be administered in a ratio of 10: 1 to 1 : 10, by weight, such as 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1: 10, by weight.
  • the first and second RNAi agents are administered in a ratio of 1 : 1, by weight.
  • a subject to be treated according to the methods of the present disclosure is an HBV-infected subject, particularly a subject having chronic HBV infection.
  • Acute HBV infection is characterized by an efficient activation of the innate immune system complemented with a subsequent broad adaptive response (e.g, HBV-specific T-cells, neutralizing antibodies), which usually results in successful suppression of replication or removal of infected hepatocytes.
  • HBV-specific T-cells, neutralizing antibodies e.g., HBV-specific T-cells, neutralizing antibodies
  • HBV envelope proteins are produced in abundance and can be released in sub-viral particles in 1,000-fold excess to infectious virus.
  • Chronic HBV infection is described in phases characterized by viral load, liver enzyme levels (necroinflammatory activity), HBeAg, or HBsAg load or presence of antibodies to these antigens.
  • cccDNA levels stay relatively constant at approximately 10 to 50 copies per cell, even though viremia can vary considerably. The persistence of the cccDNA species leads to chronicity.
  • the phases of chronic HBV infection include: (i) the immune- tolerant phase characterized by high viral load and normal or minimally elevated liver enzymes; (ii) the immune activation HBeAg-positive phase in which lower or declining levels of viral replication with significantly elevated liver enzymes are observed; (iii) the inactive HBsAg carrier phase, which is a low replicative state with low viral loads and normal liver enzyme levels in the serum that may follow HBeAg seroconversion; and (iv) the HBeAg-negative phase in which viral replication occurs periodically (reactivation) with concomitant fluctuations in liver enzyme levels, mutations in the pre-core and/or basal core promoter are common, such that HBeAg is not produced by the infected cell.
  • chronic HBV infection refers to a subject having the detectable presence of HBV for more than 6 months.
  • a subject having a chronic HBV infection can be in any phase of chronic HBV infection.
  • Chronic HBV infection is understood in accordance with its ordinary meaning in the field.
  • Chronic HBV infection can for example be characterized by the persistence of HBsAg for 6 months or more after acute HBV infection.
  • a chronic HBV infection referred to herein follows the definition published by the Centers for Disease Control and Prevention (CDC), according to which a chronic HBV infection can be characterized by laboratory criteria such as: (i) negative for IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B virus DNA, or (ii) positive for HBsAg or nucleic acid test for HBV DNA, or positive for HBeAg two times at least 6 months apart.
  • a therapeutically effective amount refers to the amount of a composition or therapeutic combination of the present disclosure which is sufficient to treat chronic HBV infection.
  • a subject having chronic HBV infection is undergoing nucleoside analog (NUC) treatment and is NUC-suppressed.
  • NUC-suppressed refers to a subject having an undetectable viral level of HBV and stable alanine aminotransferase (ALT) levels for at least six months.
  • nucleoside/nucleotide analog treatment include HBV polymerase inhibitors, such as entacavir and tenofovir.
  • a subject having chronic HBV infection does not have advanced hepatic fibrosis or cirrhosis.
  • Such subject would typically have a METAVIR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa.
  • the METAVIR score is a scoring system that is commonly used to assess the extent of inflammation and fibrosis by histopathological evaluation in a liver biopsy of patients with hepatitis B.
  • the scoring system assigns two standardized numbers: one reflecting the degree of inflammation and one reflecting the degree of fibrosis.
  • HBV-induced diseases include, but are not limited to cirrhosis, cancer (e.g., hepatocellular carcinoma), and fibrosis, particularly advanced fibrosis characterized by a METAVIR score of 3 or higher for fibrosis.
  • a therapeutically effective amount is an amount sufficient to achieve persistent loss of HBsAg within 12 months and significant decrease in clinical disease (e.g., cirrhosis, hepatocellular carcinoma, etc.).
  • Methods according to embodiments of the present disclosure further comprises administering to the subject in need thereof another therapeutic or another anti-HBV agent in combination with a pharmaceutical composition of the present disclosure.
  • another anti-HBV agent or therapeutic agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, IL-7-hyFc; CAR-T which bind HBV env (S-CAR cells); capsid assembly modulators; cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir
  • compositions and therapeutic combinations of the present disclosure can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration.
  • parenteral administration e.g., intramuscular, subcutaneous, intravenous, or intradermal injection
  • oral administration e.g., oral administration
  • transdermal administration e.g., oral administration
  • nasal administration e.g., oral administration, transdermal administration, and nasal administration.
  • pharmaceutical compositions and therapeutic combinations are administered parenterally (e.g., by intramuscular injection or intradermal injection) or transdermally.
  • the RNAi agent is subcutaneously or intravenously administered.
  • administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection.
  • Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the ribonucleic acid molecules to cells.
  • electroporation refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane.
  • in vivo electroporation During in vivo electroporation, electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules from one side of a cellular membrane to the other.
  • in vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site.
  • transfection efficiency and immune response are significantly improved (e.g., up to 1,000-fold and 100-fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection.
  • electroporation is combined with intramuscular injection.
  • electroporation is also possible to combine electroporation with other forms of parenteral administration, e.g., intradermal injection, subcutaneous injection, etc.
  • Electroporation can be accomplished using an in vivo electroporation device.
  • Examples of electroporation devices and electroporation methods that can facilitate delivery of compositions and therapeutic combinations of the present disclosure, particularly those comprising RNAi agents, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Eigen electroporator (Inovio Pharmaceuticals, Inc.), Tri-GridTM delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121), and those described in U.S. Patent No. 7,664,545, U.S. Patent No. 8,209,006, U.S. Patent No. 9,452,285, U.S. Patent No. 5,273,525, U.S. Patent No. 6,110,161, U.S. Patent No. 6,261,281, U.S. Patent No. 6,958,060, and U.S. Patent No.
  • Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used.
  • Other methods of intracellular delivery contemplated by the methods of the present disclosure include, but are not limited to, liposome encapsulation, lipoplexes, nanoparticles, etc.
  • a ribonucleic acid molecule of the present disclosure can be formulated in a therapeutic composition that comprises one or more lipid molecules, preferably positively charged lipid molecules.
  • an RNAi agent of the disclosure can be formulated using one or more liposomes, lipoplexes, and/or lipid nanoparticles.
  • liposome or lipid nanoparticle formulations described herein can comprise a polycationic composition.
  • the formulations comprising a polycationic composition can be used for the delivery of the RNAi agent described herein in vivo and/or ex vitro.
  • the method of administration of the RNAi agent is a lipid composition, such as a lipid nanoparticle (UNP) or a liposome.
  • Uipid compositions preferably lipid nanoparticles or liposomes, that can be used to deliver a therapeutic product (such as one or more nucleic acid molecules of the invention), include, but are not limited to, liposomes or lipid vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers, or wherein the lipids coat an interior that comprises a therapeutic product; or lipid aggregates or micelles, wherein the lipid-encapsulated therapeutic product is contained within a relatively disordered lipid mixture.
  • the lipid composition can provide the therapeutic product (such as one or more RNAi agents, e.g., one or more RNAi agents comprising ribonucleic acid molecules) with full encapsulation, partial encapsulation, or both.
  • the therapeutic product is fully encapsulated in the lipid particle (e.g., to form an LNP).
  • Lipid compositions of this invention can comprise one or more lipids selected from cationic lipids, anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer conjugated lipids, phospholipids, glycolipids, and any combination of the foregoing.
  • the lipids can be saturated or unsaturated.
  • a mixture can comprise both saturated and unsaturated lipids.
  • the lipid compositions can be substantially free of liposomes or can contain liposomes. The use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail.
  • the lipids and nucleic acid molecules can be mixed and configured in any suitable structures.
  • the lipid compositions comprise a cationic lipid to encapsulate and/or enhance the delivery of an RNAi agent, such as an RNA molecule of the present disclosure, into the target cell.
  • the cationic lipid can be any lipid species that carries a net positive charge at a selected pH, such as physiological pH.
  • the cationic lipids such as ionizable amino lipids, promote self-assembly of the components into macromolecular nanoparticles that encapsulate the nucleic acid.
  • the nucleic acid-containing nanoparticles are efficiently taken up into target cells by endocytosis. Once inside the endosome, the positively-charged lipid nanoparticles interact with the negatively- charged endosome membrane, causing disruption of the compartment and release of the nucleic acid molecules into the cytoplasm, where the nucleic acid molecules can be expressed.
  • cationic lipids have been described in the literature, many of which are commercially available.
  • suitable cationic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane (DLinDMA), and 1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA).
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DLinDMA 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane
  • DLenDMA 1,2-Dilinolenyloxy-N,N- dimethylaminopropane
  • the pKa of formulated cationic lipids is correlated with the effectiveness of lipid particles for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)).
  • the preferred range of pKa is ⁇ 5 to ⁇ 7.
  • the cationic lipid is a compound of Formula (I):
  • Ri is a substituted alkyl consisting of 10 to 31 carbons
  • R2 is a linear alkyl, alkenyl or alkynyl consisting of 2 to 20 carbons
  • Rs is a linear or branched alkane consisting of 1 to 6 carbons
  • R4 and Rs are the same or different, each a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons
  • Li and L2 are the same or different, each a linear alkane of 1 to 20 carbons or a linear alkene of 2 to 20 carbons
  • Xi is S or O; or a salt or solvate thereof.
  • Exemplary compounds of formula (I), their synthesis and uses thereof are described in US2018/0169268, all of which are herein incorporated by reference.
  • the cationic lipid is a compound of formula (II):
  • Ri is a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons
  • Li is linear alkane of 1 to 15 carbons
  • R2 is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons or a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons
  • L2 is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons
  • X is O or S
  • Rs is a linear alkane of 1, 2, 3, 4, 5, or 6 carbons
  • R4and Rs are the same or different, each a linear or branched, noncyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons; or a pharmaceutically acceptable salt or solvate thereof.
  • Exemplary compounds of formula (II), their synthesis and uses thereof are described in US2018/0170866, all of which
  • the cationic lipid is a compound of formula (III), (IV) or (V):
  • R comprises a biologically active molecule
  • Li, L2, and L independently for each occurrence comprise a ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile; a pharmaceutically acceptable salt thereof; or a pharmaceutical composition thereof.
  • exemplary compounds of formula (III), (IV) and (V), their synthesis and uses thereof are described in US2017/0028074, all of which are herein incorporated by reference.
  • the cationic lipid is a compound of formula (VI):
  • the cationic lipid is a compound of formula (VII):
  • the cationic lipid is a compound of formula (VIII):
  • X is CR a ;
  • Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or
  • Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1;
  • R a is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-
  • Additional cationic lipids that can be used in compositions of the present disclosure include, but are not limited to, those described in W02019/036030, W02019/036028, W02019/036008, WO2019/036000, US2016/0376224, US2017/0119904, W02018/200943 and WO2018/191657, the relevant contents on the lipids, their synthesis and uses are herein incorporated by reference in their entireties.
  • the lipid nanoparticles can be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and polyethylene glycol (PEG) - modified, or pegylated, lipids, i.e. the lipid is modified by covalent atachment of a polyethylene glycol.
  • PEG provides the liposomes with a coat which can confer favorable pharmacokinetic characteristics c.g. it can increase stability and prevent non-specific adsorption of die liposomes.
  • the PEG has an average molecular mass of 1 kDa. to 12 kDa. such as 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, 11 or 12 kDa.
  • the length of the PEG can affect in vivo expression of encapsulated RNA, and that PEG with a molecular weight below I kDa (e.g. 500 or 750 Da) does not form stable liposomes. See, e.g., US2014/0255472, the relevant content of which is incorporated herein by reference.
  • the lipid formulations can include anionic lipids.
  • the anionic lipids can be any lipid species that carries a net negative charge at a selected pH, such as physiological pH.
  • the anionic lipids when combined with cationic lipids, are used to reduce the overall surface charge of LNPs and liposomes and to introduce pH-dependent disruption of the LNP or liposome bilayer structure, facilitating nucleotide release.
  • Several anionic lipids have been described in the literature, many of which are commercially available.
  • suitable anionic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, and palmitoyloleyolphosphatidylglycerol (POPG).
  • DOPE dioleoyl-sn-glycero-3- phosphoethanolamine
  • POPG palmitoyloleyolphosphatidylglycerol
  • the lipid formulations can also include a lipid bilayer stabilizing component.
  • Bilayer stabilizing components can be used to inhibit aggregation of LNPs, but bilayer stabilizing components are not limited to this function.
  • conjugated lipids such as PEG-lipid conjugates and cationic-polymer-lipid conjugates can be used to inhibit the aggregation of LNPs or liposomes.
  • concentration of the bilayer stabilizing component By controlling the composition and concentration of the bilayer stabilizing component, one can control the rate at which the bilayer stabilizing component exchanges out of the liposome and, in turn, the rate at which the liposome becomes fusogenic.
  • fusogenic refers to the ability of a liposome or other drug delivery system to fuse with membranes of a cell.
  • the rate at which the liposome becomes fusogenic can be varied, for example, by varying the concentration of the bilayer stabilizing component, by varying the molecular weight of the polyethyleneglycol, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide.
  • other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and/or control the rate at which the liposome becomes fusogenic. Other methods which can be used to control the rate at which the liposome becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.
  • LNPs and liposomes can be prepared using methods known in the art in view of the present disclosure.
  • the LNPs can be prepared using ethanol injection or dilution, thin film hydration, freeze-thaw, French press or membrane extrusion, diafiltration, sonication, detergent dialysis, ether infusion, and reverse phase evaporation.
  • One useful method of preparing liposomes involves mixing (i) an ethanolic solution of the lipids (ii) an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification.
  • Preferred liposomes of the invention e.g.
  • liposomes with a preferred diameter are obtainable by this mixing process.
  • mixing can be performed using a process in which two feed streams of aqueous nucleic acid solution are combined in a single mixing zone with one stream of an ethanolic lipid solution, all at the same flow rate e.g. in a microfluidic channel as described below.
  • compositions, and methods to create liposomes are described in US 2014/0255472, which is hereby incorporated by reference in its entirety.
  • lipids, lipid compositions, and methods to create lipid carriers for delivering active nucleic acid molecules are described in: US2017/0190661, US2006/0008910, US2015/0064242, US2005/0064595, WO/2019/036030, US2019/0022247, WO/2019/036028, WQ/2019/036008, WO/2019/036000, US2016/0376224, US2017/0119904, WO/2018/200943, WO/2018/191657, US2014/0255472, and US2013/0195968, the relevant content of each of which is hereby incorporated by reference in its entirety.
  • Liposomes are microscopic vesicles including at least one concentric lipid bilayer. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. In particular embodiments, liposomes provide a lipid composition that is an outer layer surrounding a porous nanoparticle.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PI phosphatidylinositol
  • SM sphingomyelin
  • DOPE dioleoylphosphatidylethanolamine
  • lipids capable of producing a stable liposome are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-
  • HSPC hydrogenated soy phosphati
  • Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine -lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, DDAB, dioctadecyl dimethyl ammonium chloride (DODAC), 1 ,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Choi, phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol, DOPG, and dicetylphosphate.
  • Embodiment 1 A ribonucleic acid interfering (RNAi) agent (useful for in inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) gene), wherein the RNAi agent comprises an antisense strand comprising a sequence of 15 to 23 nucleotides, wherein the nucleotide sequence of the antisense strand comprises at least 15 contiguous nucleotides, particularly 16 or more, 17 or more, 18 or more, or 19 or more contiguous nucleotides, from a sequence selected from SEQ ID NOs: 604-804 and wherein one or more nucleotides of the antisense strand is a modified nucleotide; and wherein the RNAi agent optionally further comprises a sense strand comprising a nucleotide sequence, which is of the same length as, or of a lower length than, the nucleotide sequence of the antisense strand, and wherein one or more nucleotides of the sense strand optionally
  • Embodiment 2 The RNAi agent of embodiment 1, wherein the nucleotide sequence of the sense strand comprises a sequence of 15 to 21 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a sequence of the same length that is comprised in the sequence of the antisense strand.
  • Embodiment 3 The RNAi agent of embodiment 1 or 2, wherein at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sequence of the antisense strand are modified nucleotides.
  • Embodiment 4 The RNAi agent of any one of the preceding embodiments, wherein all the nucleotides of the sequence of the antisense strand are modified nucleotides.
  • Embodiment 5 The RNAi agent of any one of the preceding embodiments, wherein at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sense strand are modified nucleotides.
  • Embodiment 6 The RNAi agent of any one of the preceding embodiments, wherein all the nucleotides of the sequence of the sense strand are modified nucleotides.
  • Embodiment 7 The RNAi agent of any one of the preceding embodiments, wherein the antisense strand comprises a sequence selected from SEQ ID NOs: 604-804.
  • Embodiment 8 The RNAi agent of any one of the preceding embodiments, wherein the sense strand comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603.
  • Embodiment 9 The RNAi agent of any one of the preceding embodiments, wherein the sense strand comprises at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603.
  • Embodiment 10 The RNAi agent of any one of the preceding embodiments, wherein the sense strand comprises a sequence selected from SEQ ID NOs: 403-603.
  • Embodiment 11 The RNAi agent of any one of the preceding embodiments, wherein the number of nucleotides of the sense strand is 19 or 21.
  • Embodiment 12 The RNAi agent of any one of the preceding embodiments, wherein the number of nucleotides of the antisense strand is 19, 21 or 23.
  • Embodiment 13 The RNAi agent of any one of the preceding claims, wherein: a. the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 21; b. wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 2; c. wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 23; or; d. wherein the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 19.
  • Embodiment 14 The RNAi agent of any one of the preceding embodiments, wherein the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 805-1005; 1690; 1724; 1760; and 1839.
  • Embodiment 15 The RNAi agent of any one of the preceding embodiments, wherein the sequence of the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 403-603; 1689; 1723; 1759; 1799; and 1838.
  • Embodiment 16 The RNAi agent of any one of the preceding embodiments, wherein the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table lb.
  • Embodiment 17 The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified nucleoside and/or a modified phosphate and/or a modified intemucleotide linkage.
  • Embodiment 18 The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a 2’-deoxy-2’-fluoro-ribose (2’-F), a 2’ O-methyl ribose (2’ O-Me) or the acyclic sugar of an UNA nucleotide, for example, wherein the modified nucleotide is nucleotide).
  • the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a 2’-deoxy-2’-fluoro-ribose (2’-F), a 2’ O-methyl ribose (2’ O-Me) or the acyclic sugar of an UNA nucleotide, for example, wherein the modified nucleotide is nucleotide).
  • Embodiment 19 The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate.
  • the modified nucleotide comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate.
  • Embodiment 20 The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified intemucleotide linkage selected from phosphorothioate and thiophosphoramidate linkages, more particularly wherein the modified intemucleotide linkage is a phosphorothioate linkage, optionally wherein the phosphorothioate
  • Embodiment 21 The RNAi agent of any one of the preceding embodiments, further comprising one or more from invAb and targeting moieties, more particularly liver targeting moieties; wherein the liver targeting moieties are fatty acids, GalNAc, folic acid, cholesterol, tocopherol or palmitate, more particularly GalNAc, more particularly the liver targeting moieties.
  • Embodiment 22 The RNAi agent of any one of the preceding embodiments, wherein all nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA.
  • nucleotides in the antisense strand that are not modified by 2’F or by vinylphosphonate or that are not UNA are modified by 2’-0Me, optionally wherein all of said nucleotides are modified by 2’-0Me; and/or e. optionally the number of 2’0-Me nucleotides is 10, 11, 13, 15, 17 or 19.
  • Embodiment 24 The RNAi agent of embodiment 22, wherein: a. optionally number of 2’F nucleotides in sense strand is 2, 4, 6, 9, or 10, more particularly 4, 5, 6 or 7; optionally the nucleotides in the sense strand that are not modified by 2’F are modified by 2’-0Me, optionally wherein all of said nucleotides are modified by 2’-0Me; and/or c. optionally the number of 2’0- Me nucleotides is 10, 11, 13, 15 or 17.
  • Embodiment 25 The RNAi agent of embodiment 22, wherein optionally 1, 2 or 3 phosphorothioate linkages linking the 3’ end or 5’ end terminal nucleotides of the sense strand and the antisense strand, more particularly 1, 2 or 3 phosphorothioate linkages linking (ntl and nt2), and/or (nt2 and nt3), and/or (nt3 and nt4) of the sense strand and antisense strand and/or the antisense strand.
  • Embodiment 26 The RNAi agent of any one of the preceding embodiments, wherein the sequence of the antisense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
  • Embodiment 27 The RNAi agent of any one of the preceding embodiments, wherein the sequence of the sense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
  • Embodiment 28 The RNAi agent of any one of the preceding embodiments, wherein the sequences of the antisense and sense strands of the RNAi agent comprises the sequences of the duplex selected from the duplex of Tables 2, 2a, 5 and 7.
  • Embodiment 29 The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is capable of inducing a PD-L1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%, e.g. , at day 6 or 7 in C57/bl6 mice infected with AAV8-hPD-Ll firefly luciferase for example as described in Example 5.
  • Embodiment 30 The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is capable of inducing a PD-L1 knockdown at a KD of at least 60% and at an IC50 of less than 150 nM, more particularly less than lOOnM , for example, in a free uptake assay, such as on primary human hepatocytes (PHH).
  • PHL primary human hepatocytes
  • Embodiment 31 The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is subcutaneously or intravenously administered.
  • Embodiment 32 The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is for use in combination with one or more agents chosen from among antiviral agents (e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators), immune checkpoints, immunomodulators (more particularly one or more TLR immunomodulators), vaccines (e.g., an anti-HBV therapeutic vaccine), anti-HBV siRNAs, anti-HBV ASOs and NAPs.
  • antiviral agents e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators
  • immune checkpoints e.g., immuno
  • Embodiment 33 A salt of the RNAi agent of any one of embodiments 1-32, more particularly a sodium salt.
  • Embodiment 34 A LNP or liposome comprising the RNAi agent of any one of embodiments 1-32.
  • Embodiment 35 An isolated cell comprising the RNAi agent of any one of embodiments 1-32.
  • Embodiment 36 A non-human animal comprising the RNAi agent of any one of embodiments 1-32.
  • Embodiment 37 A pharmaceutical composition comprising an effective amount of the RNAi agent of any one of embodiments 1-32 and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.
  • Embodiment 38 The pharmaceutical composition of embodiment 37, wherein the pharmaceutical composition is a liquid composition.
  • Embodiment 39 The pharmaceutical composition of embodiment 38, wherein the liquid composition comprises water, saline, and/or buffer.
  • Embodiment 40 The pharmaceutical composition of embodiment 37, wherein the pharmaceutical composition is a lyophilized composition.
  • Embodiment 41 The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiment 37-40, for use in treating a viral infection, more particularly a chronic viral infection.
  • Embodiment 42 The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of embodiment 41, wherein the viral infection comprises an HBV infection.
  • Embodiment 43 The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of embodiment 41 or embodiment 42, wherein the viral infection comprises an HBV infection and an HDV infection.
  • Embodiment 44 The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of any one of embodiments 41-43, wherein the viral infection comprises a HIV infection.
  • Embodiment 45 The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiments 37-40, for use in the treatment of hepatitis B, more particularly of chronic hepatitis B.
  • Embodiment 46 The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiments 37-40, for use in the treatment of hepatitis D, more particularly of chronic hepatitis D.
  • Embodiment 47 The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiments 37-40, for use in treating a cancer, for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma.
  • a cancer for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma.
  • the PD-L1 (CD274) human siRNAs (e.g., selected through in vitro screening) were conjugated to a GalNAc ligand (more particularly a GalNAc2 ligand; see below) at the 3 ’ end of the sense strand (Table 7).
  • DU145 cells were plated in 96-well plate (18,000 cells/well) in assay medium.
  • Cells were transfected with two doses of siRNA (10 or 2nM) or with dose response of siRNA using a reverse transfection method (Lipofectamine 2000, Invitrogen). After 24 hours the cells were lysed, and CD274 (PD-L1) mRNA level was determined using a bDNA assay (Quantigene 2.0) following the manufacturer’s instructions.
  • PD-L1 mRNA levels were normalized to GAPDH and shown as % of control (PBS-treated cells).
  • Frozen primary human hepatocytes were thawed and plated in 96-well plates (90,000 cells/well). GalNAc -conjugated siRNA duplexes were then added. 24 hours after plating, the cells were stimulated with 500 lU/ml IFN-g. 24 hours after stimulation, cells were lysed, and CD274 (PD-L1) mRNA level was determined using bDNA assay (Quantigene 2.0) by following the manufacturer’s instructions. PD-L1 mRNA levels were normalized to GAPDH and shown as % of control (PBS-treated cells).
  • Frozen PHH were thawed and plated in 384-well collagen coated cell carrier plate (Perkin Elmer, 25000cells/well), and Ipg/ml IFN-g (Gibco) was added together with the GalNAc conjugated siRNA duplexes.
  • the PHH were then incubated for one day at 37°C and 5% CO2. After 1 day, the medium was refreshed, and new medium was added containing Ipg/ml IFN-g. After the medium was refreshed and new medium added, the PHH were incubated for one day at 37°C and 5% CO2. Following incubation, the cells were fixed with 10% formaldehyde (Polysciences).
  • PD-L1 APC Prior to staining the cells with the primary antibody, PD-L1 APC (Miltenyi Biotec), cells were washed 3 times with PBS and blocked with Fc blocker (Biolegend). Cells were then incubated overnight at 4°C with the primary antibody. After the overnight incubation, cells were washed with PBS, and counter-staining mix was added, which mix contained Hoechst (ThermoFisher) and CELLMASKTM (Invitrogen). Following counter-staining, cells were incubated for 1 hour at room temperature. Images were then taken in the CV8000 (Wako) and analyzed by Phaedra software. [0006] Free uptake assay in primary human hepatocytes RNA knockdown by RT-QPCR
  • Frozen PHH were thawed and plated in collagen-coated 96-well plate (80,000 cells/well) together with GalNAc conjugated siRNA duplex and Ipg/ml IFN-g. After one day, the medium was replaced, the cells were washed, and new medium containing Ipg/ml IFN-g was added. One day following the medium replacement and addition, the cells were washed and then lysed with CELLS-TO-CTTM lysis buffer (Invitrogen) containing DNase for 5 minutes at room temperature. The lysis was subsequently stopped by adding a stopping solution.
  • CELLS-TO-CTTM lysis buffer Invitrogen
  • cDNA was synthesized.
  • lysate was mixed with water, PD-L1 pool, and B-actin 876 primer.
  • a second mix was added containing Buffer (EXPANDTM High Fidelity buffer (1 OX) with MgC12 - 11759 167 001 Roche), MgC12 (11 699 113 001 - Roche), dNTPs (lOpM each, 733-1363 - VWR), Protector RNase inhibitor (03 335 402 001 - Roche), and Expand Reverse Transcriptase (11785834001 - Roche).
  • cDNA synthesis was performed under the following conditions using a Thermocycler (Applied Biosystems): 30min at 42°C, 5min 95 °C, cool down to 4°C.
  • qPCR was performed using a LIGHTCYCLER® 480 II (5015278001 - Roche). qPCR detection was based on a duplex qPCR assay with Zen double quenched probes (Integrated DNA technologies) for both PD-L1 and B-actin.
  • the PD-L1 probe contains a FAM label and B-actin a Cy5 label.
  • the reagents used were the following: cDNA was mixed with water, LIGHTCYCLER® 480 Probes master 2X cone. (Roche - Cat. No. 04 887 301 001), each primer, and each probe.
  • cDNA and reaction mix were loaded in a LIGHTCY CLER® 480 Multiwell Plate 96, white (Roche - 4729692001) and sealed with Microseal B film (BioRad - MSB-1001). After briefly centrifuging, the plate was transferred to the qPCR platform (LC480II - Roche). The qPCR conditions used were 10 min at 95°C, followed by 45 cycles of 15 sec at 95°C, 1 min 60°C. After completion of the DNA amplification, the generated signals were analyzed with the second derivative method that calculates Cp values for both PD-L1 and B-actin. All Cp values were exported from the LC480 software and further analyzed. [0010] PD-L1 mRNA levels were normalized to B-actin and presented as percent of control (PBS treated cells).
  • mice were intravenously (i.v.) injected in the tail vein with AAV8-hPD-Ll- firefly luciferase (Vector Biolabs). 11 to 14 days after i.v. injection, mice were treated with GalNAc conjugated siRNA duplexes at 10 mg/kg. Both before treatment and after treatment, mice were injected intraperitoneal with 500mg/kg D-luciferin (Perkin Elmer). 10 minutes following administration of the substrate, mice were anesthetized with isoflurane. After anesthetization, mice were brought into the IVIS® to perform life imaging.
  • mice infected with AAV-hPD-Ll firefly luciferase were used, which were treated with saline or a control GalNac conjugated siRNA.
  • Each signal was normalized before starting treatment, and PD-L1 knockdown was followed over time. Results are shown from day 7 after start treatment in Table 11 below.
  • Example 1 PD-L1 siRNAs
  • Table 1 201 parent sequences of PD-L1 Human siRNA sequences
  • the 201 PD-L1 human siRNAs (see Table 1A) were synthesized at 0.2 imol scale with 2’-OMe and 2’-F chemical modifications as shown in Table 2. The siRNAs were then used for in vitro screening.
  • Example 2 As described in Example 1, a total of 201 human CD274/PD-L1 siRNA duplexes were synthesized at 0.2 pmol scale with 2’-0Me and 2’-F chemical modifications, as shown in Table 2 above. Following synthesis, a dual-dose in vitro screen was conducted in DU 145 cells. Briefly, as described above, DU145 cells were transfected with lOnM or 0.2nM of the siRNA duplexes. 24 hours following transfection, the cells are lysed, and PD-L1 knockdown was measured using branched DNA assays. The results obtained are summarized in Table 3 below. The data were normalized to GAPDH, and the data obtained are presented as the mean relative mRNA of quadruple wells plus the standard deviation.
  • Table 3 Dual Dose Screen of 201 siRNAs in DU145 cell line (mean of quadruplicate +/-SD)
  • the PD-L1 siRNA duplexes in Table 2 above were or are tested in the DU-145 cells in a dose response assay which started from 50nM, used a 1/6 dilution, and had 10 different doses.
  • Table 4 summarizes the results obtained for 48 siRNA duplexes, which results include EC50 and Max mRNA KD. Additional PD-L1 siRNA duplexes in Table 2 are also tested in the DU-145 cells in the dose response assay described above.
  • siRNAs of Table lb were (or are) synthesized following different patterns of 2’-F and 2’-0Me and were (or are) tested in DU 145-cells in dose responses as described above.
  • Table 5 shows examples of such siRNAs (duplex 16V, 30V, 39V, 42V, 75V, 79V, 82V, 143V, 160V and 187V of Table IB, with different patterns of 2’-F and 2’-0Me) and Table 6 below summarizes the EC50 and max mRNA KD results of the siRNAs of Table 5 and of corresponding siRNAs of Table 2.
  • siRNAs of Tables 2, 2a and 5 were (or are) conjugated to GalNAc and can be further evaluated in PHH assays by free uptake of the GalN Ac-conjugated siRNA duplexes.
  • Examples of such GalNAc conjugated siRNAs are provided in Table 7 below.
  • the siRNA duplexes activity was tested by a branched DNA assay.
  • the results obtained from the dual dose screen are summarized in Table 8, for which the data were normalized to GAPDH and which data are presented as the mean relative mRNA of quadruple wells plus the standard deviation.
  • GalNAc conjugated siRNAs were also evaluated in dose response assays starting from lOpM to 0.5nM in steps of 1 in 3. Table 9 summarizes the results obtained from these assays. The data were normalized to GAPDH, the IC50 values were calculated, and the maximal knockdown values are presented.
  • RNA knockdown In addition to the RNA knockdown, the protein knockdown also was evaluated on PHH after treatment with GalNAc conjugated siRNAs. PHH were treated with IFN-g and a dose response of the siRNAs as described above. Data were calculated as percent effect compared to PBS controls. EC50 values and max KD were calculated and are shown in Table 10. RT-PCR was also performed on cells from the same donor to evaluate knockdown on mRNA level. A summary of mean KD and EC50 are shown in Table 10.
  • mice C57/bl6 mice were infected with AAV8-hPD-Ll firefly luciferase. After 14 days, infection was established, and the mice were treated with one of the compounds. Knockdown of PD-L1 was followed in time by a non-invasive method. Mice were ip injected with a substrate, and cells that expressed huma PD-L1 were visualized during IVIS imaging. As a control, infected mice were treated with saline or irrelevant siRNA. In these mice luciferase expression in the liver was observed. For mice that were subcutaneously (s.c.) treated with compound, the knockdown was evaluated overtime. Table 12 presents results obtained 6 or 7 days after treatment. Every mouse used had its own control, and all data were normalized before treatment. In this manner percent knockdown was calculated.

Abstract

Described are RNA interference (RNAi) agents for inhibiting the expression of PD-L1, and compositions comprising the RNAi agents and methods of administering same. The RNAi agents are useful in the treatment of diseases, such as, for example, HBV infection, HDV infection, and/or cancer, more particularly chronic HBV infection.

Description

RNAI AGENTS TARGETING PD-L1
FIELD OF THE INVENTION
[0001] The disclosure relates generally to compounds, compositions and methods useful for treating a disease, such as a viral infection, more particularly a chronic viral infection, or such as a cancer. In some instances, the viral infection, more particularly the chronic viral infection, comprises a hepatitis B virus (HBV) infection, more particularly chronic HBV infection. In some instances, the viral infection, more particularly the chronic viral infection, comprises a hepatitis D virus (HDV) infection, more particularly chronic HDV infection. In some instances, the cancer comprises a liver cancer, more particularly a hepatocellular carcinoma. In particular, the application relates to RNA interference (RNAi) agents targeting PD-L1 for use in treating diseases, such as said viral infections or cancers.
BACKGROUND OF THE INVENTION
[0002] PD -LI is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is involved in evasion of immune responses involved in chronic infection, e.g., chronic viral infections such as HBV (see,
Figure imgf000002_0001
2020; 11: 1037; and Sun, Y., et al., Am J Physiol Gastrointest Liver Physiol. 2020: 218(1): G162-G173). PD-L1 expression has been detected in a number of tissues and cell types including T-cells, B-cells, macrophages, dendritic cells, and nonhematopoietic cells including endothelial cells, hepatocytes, muscle cells, and placenta. [0003] PD-L1 expression is also involved in suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immunologic clearance of tumors is rare. Part of this failure is due to immune suppression by the tumor microenvironment. PD-L1 expression on many tumors is a component of this suppressive milieu and acts in concert with other immunosuppressive signals. PD-L1 expression has been shown in situ on a wide variety of solid tumors including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymic epithelial, head, and neck (Brown JA et al., 2003, Immunol. 170: 1257-66; Dong H et al. 2002. Nat. Med. 8:793-800; Hamanishi J, et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003. Cancer Res. 63:6501-5; Inman BA et al. 2007. Cancer 109: 1499-505; Konishi J et al. 2004. Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007. Cancer Immunol. Immunother. 56: 1173-82; Nomi T et al. 2007. Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004. Proc. Natl. Acad. Sci. USA 101 : 17174-79; Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. 2006. Acta Histochem. 108: 19-24). In addition, the expression of the receptor for PD-L1, Programmed cell death protein 1 (also known as PD-1 and CD279) is upregulated on tumor infdtrating lymphocytes, and this also contributes to tumor immunosuppression (Blank C et al. 2003. Immunol . 171 :4574-81).
[0004] Hepatitis B virus (HBV), a member of the Hepadnaviridae family, is a noncytopathic hepatic DNA virus that only infects the liver of human and great apes (e.g., chimpanzee, orangutan, bonobo, gorilla). The primary infection of adult humans with HBV causes an acute hepatitis with symptoms of organ inflammation, fever, jaundice and increased liver transaminases in blood. About 10-20% of adult patients are not able to overcome the virus infection and suffer a chronic disease progression over many years with increased risk of developing cirrhotic liver or liver cancer through the development of chronic hepatitis B virus (CHB) infection. Perinatal vertical transmission from mothers with CHB to newborns also leads to chronic hepatitis in about 80% of cases. All patients with CHB are at increased risk of progression to cirrhosis and hepatocellular carcinoma (HCC), depending on host and viral factors (Lampertico et al., J Hepato , 2017, 67(2):370-398).
[0005] The HBV mini-chromosome covalently closed circular DNA (cccDNA) can be transcriptionally silent for long periods. During a period of viral latency, HBV replication is controlled through effector arms of adaptive immunity, including HBV-specific CD4+ helper T cells, HBV-specific CD8+ cytotoxic T cells, appropriately primed B cells that can serve as antigen presenting cells, and cytokines such as interferon gamma and tumor necrosis factor (TNF) alpha. HBV-specific T cells perform surveillance and kill the infected cells that are reactivated to keep the serum free of virus products, such as HBV DNA and HBV proteins. However, reactivation can occur in people who previously recovered from acute infection, even many years later, e.g., when under immune suppressive therapy. HBV induces suppressive function of the innate and adaptive immune cells in chronic HBV infection (see, e.g., Li et al., World J Gastroenterol. 2019 Jul 21; 25(27): 3527-3537, and references therein). How HBV regulates the innate and adaptive immune cells, leading to persistent virus infection and further consequences, continues to be an area of active research. For example, HBV can take advantage of the tolerogenic liver environment and the changes in immune cell populations in CHB patients. Also, long-term infection with HBV triggers the immune system by processing and presenting high levels of virions and sub-viral particles, which results in repetitive triggering of T-cell receptors and eventual T-cell exhaustion. Large quantities of circulating hepatitis B surface antigen (HBsAg) and hepatitis B e-antigen (HBeAg) have also been postulated to sabotage immunity directly by yet unidentified mechanisms.
[0006] HBsAg is the most abundant HBV protein in the liver and peripheral blood of HBV infected patients. It is the first serologic marker appearing in the serum, and this is about 6 to 16 weeks following exposure to HBV. In self-resolving acute HBV infection, HBsAg usually disappears 1 to 2 months after the onset of symptoms. Patients with detectable serum HBsAg (with or without detectable serum e-antigen (HBeAg)) for more than 6 months are considered chronically infected.
[0007] All patients with chronic HBV infection are at increased risk of progression to cirrhosis and hepatocellular carcinoma (HCC), depending on host and viral factors. The main goal of therapy is to improve survival and quality of life by preventing disease progression, and consequently HCC development. The induction of long-term suppression of HBV replication represents the main endpoint of current treatment strategies, while HBsAg loss is an optimal endpoint.
[0008] Nucleoside analogs as inhibitors of reverse transcriptase activity are typically the first treatment option for many patients. Long term administration of lamivudine, tenofovir, and/or entecavir has been shown to suppress hepatitis B virus replication, sometimes to undetectable levels, with improvement of liver function and reduction of liver inflammation typically seen as the most important benefits. However, only few patients achieve complete and lasting remission after the end of treatment. Furthermore, the hepatitis B virus develops drug resistance with increasing duration of treatment. This is especially difficult for patients super/co-infected with hepatitis B and human immunodeficiency virus (HIV). Both viruses are susceptible to nucleoside analogue drugs and may co-develop resistance.
[0009] Pegylated interferon-alpha (IFN) has been used to treat mild to moderate chronic hepatitis B patients. However, current treatment of chronic hepatitis B has limited efficacy (Erha et al., Gut. 2005 Jul; 54(7): 1009-1013). For example, the Asian genotype B gives very poor response rates. Super/co-infection with hepatitis D virus (HDV) or human immunodeficiency virus has been shown to render interferon-alpha therapy completely ineffective. Patients with strong liver damage and heavy fibrotic conditions are not qualified for interferon-alpha therapy. [0010] Several combinations have been tried in the treatment of HBV in various settings (Paul, Curr Hepat Rep . 2011 Jun; 10(2): 98-105). However, various combination therapies for HBeAg-positive chronic HBV or HBeAg-negative hepatitis B have not established a benefit over monotherapy. Id.
[0011] As such, there is a need for improved therapies targeting PD-L1 expression (or overexpression) and diseases associated with PD-L1 expression (or over-expression), such as, for example, improved HBV therapy or improved HDV therapy,, for example a therapy that can overcome at least one of the disadvantages of existing treatment options, such as toxicity, mutagenicity, lack of selectivity, poor efficacy, poor bioavailability, and difficulty of synthesis, and/or that can provide additional benefits such as increased potency, decreased immune suppression, decreased immune exhaustion, or an increased safety window. More specifically, there is a need for means that improve survival and health-related quality of life of HBV- infected patients and/or HDV-infected patients, by preventing disease progression, including advanced decompensated cirrhosis and development of HCC.
SUMMARY OF THE INVENTION
[0012] The present disclosure generally relates to a ribonucleic acid interfering (RNAi) agent (useful for in inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) gene), wherein the RNAi agent comprises an antisense strand comprising a sequence of 15 to 23 nucleotides, wherein the nucleotide sequence of the antisense strand comprises at least 15 contiguous nucleotides, particularly 16 or more, 17 or more, 18 or more, or 19 or more contiguous nucleotides, of a sequence selected from SEQ ID NOs: 604-804 and wherein one or more nucleotides of the antisense strand is a modified nucleotide; and wherein the RNAi agent optionally further comprises a sense strand comprising a nucleotide sequence, which is of the same length as, or of a lower length than, the nucleotide sequence of the antisense strand, and wherein one or more nucleotides of the sense strand optionally is a modified nucleotide. In some aspects, the nucleotide sequence of the sense strand comprises a sequence of 15 to 21 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a sequence of the same length that is comprised in the sequence of the antisense strand. In some aspects, at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sequence of the antisense strand are modified nucleotides. In some aspects, all the nucleotides of the sequence of the antisense strand are modified nucleotides. In some aspects, at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sense strand are modified nucleotides. In some aspects, all the nucleotides of the sequence of the sense strand are modified nucleotides.
[0013] In some aspects, the antisense strand comprises a sequence selected from SEQ ID NOs: 604-804. In some aspects, the sense strand comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603. In some aspects, the sense strand comprises at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403- 603. In some aspects, the sense strand comprises a sequence selected from SEQ ID NOs: 403- 603. In some aspects, the number of nucleotides of the sense strand is 19 or 21. In some aspects, the number of nucleotides of the antisense strand is 19, 21 or 23. In some aspects, the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 21; wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 21 ; wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 23; or wherein the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 19. In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 805-1005; 1690; 1724; 1760; and 1839. In some aspects, the sequence of the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 403-603; 1689; 1723; 1759; 1799; and 1838. In some aspects, the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table lb.
[0014] In some aspects, the modified nucleotide comprises a modified nucleoside and/or a modified phosphate and/or a modified intemucleotide linkage. In some aspects, the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a 2’-deoxy-2’-fluoro-ribose (2’-F), a 2’ O-methyl ribose (2’ O-Me) or the acyclic sugar of an UNA nucleotide, for example, wherein the modified nucleotide is
Figure imgf000006_0001
nucleotide). In some aspects, the modified nucleotide comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate. In some aspects, the modified nucleotide comprises a modified intemucleotide linkage selected from phosphorothioate and thiophosphoramidate linkages, more particularly wherein the modified intemucleotide linkage is a phosphorothioate linkage, optionally wherein the phosphorothioate
Figure imgf000007_0002
. In some aspects, the RNAi agent further comprises one or more of invAb and targeting moieties, more particularly liver targeting moieties; wherein the liver targeting moieties are fatty acids, GalNAc, folic acid, cholesterol, tocopherol or palmitate, more particularly GalNAc, more particularly
Figure imgf000007_0001
the
3 ’ end of the sense strand; optionally further comprising one or more invAb, more particularly 1 invAb at the 5’ end of the sense strand and/or 1 invAb at the 3’ end of the sense strand.
[0015] In some aspects, all nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA. In some aspects, the number of 2’F nucleotides in antisense strand = 2, 4, 6, 9 or 10, more particularly 3, 4, 5 or 6; the number of UNA nucleotides in the antisense strand is 1 UNA-U or 1-UNA-A; the number of vinylphosphonate nucleotides in the antisense strand is 1 vinylphosphonate nucleotide in the antisense strand, more particularly 1 vinylphosphonate nucleotide at the 5 ’ end of the antisense strand; the nucleotides in the antisense strand that are not modified by 2’F or by vinylphosphonate or that are not UNA are modified by 2’-0Me, optionally wherein all of said nucleotides are modified by 2’-0Me; and/or the number of 2’0-Me nucleotides is 10, 11, 13, 15, 17 or 19. In some aspects, the number of 2’F nucleotides in sense strand is 2, 4, 6, 9, or 10, more particularly 4, 5, 6 or 7; the nucleotides in the sense strand that are not modified by 2’F are modified by 2’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or the number of 2’0-Me nucleotides is 10, 11, 13, 15 or 17. In some aspects, 1, 2 or 3 phosphorothioate linkages linking the 3’ end or 5’ end terminal nucleotides of the sense strand and the antisense strand, more particularly 1, 2 or 3 phosphorothioate linkages linking (ntl and nt2), and/or (nt2 and nt3), and/or (nt3 and nt4) of the sense strand and antisense strand and/or the antisense strand.
[0016] In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7. In some aspects, the sequence of the sense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7. In some aspects, the sequences of the antisense and sense strands of the RNAi agent comprises the sequences of the duplex selected from the duplex of Tables 2, 2a, 5 and 7. In some aspects, the RNAi agent is capable of inducing a PD-U1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%. In some aspects, the RNAi agent is capable of inducing a PD-U1 knockdown at a KD of at least 60% and at an IC50 of less than 150 nM, more particularly less than lOOnM , for example, in a free uptake assay, such as on primary human hepatocytes (PHH). In some aspects, the RNAi agent is subcutaneously or intravenously administered. In some aspects, the RNAi agent is for use in combination with one or more agents chosen from among antiviral agents (e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators), immune checkpoints, immunomodulators (more particularly one or more TUR immunomodulators), vaccines (e.g., an anti-HBV therapeutic vaccine), anti-HBV siRNAs, anti- HBV ASOs and NAPs.
[0017] In some aspects, the present disclosure relates to a salt of an RNAi agent as described herein, more particularly a sodium salt. In some aspects, the present disclosure relates to a LNP or liposome comprising an RNAi agent as described herein. In some aspects, the present disclosure relates to an isolated cell comprising an RNAi agent as described herein. In some aspects, the present disclosure relates to a non-human animal comprising an RNAi agent as described herein.
[0018] Moreover, the present disclosure generally relates to a pharmaceutical composition comprising an effective amount of the RNAi agent as described herein and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof. In some aspects, the pharmaceutical composition is a liquid composition. In some aspects, the liquid composition comprises water, saline, and/or buffer. In some aspects, the pharmaceutical composition is a lyophilized composition.
[0019] In some aspects, the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use as described herein, for use in treating a viral infection, more particularly a chronic viral infection. In some aspects, the viral infection comprises an HBV infection. In some aspects, the viral infection comprises an HBV infection and an HDV infection. In some aspects, the viral infection comprises a HIV infection. In some aspects, the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use as described herein, for use in the treatment of hepatitis B, more particularly of chronic hepatitis B. In some aspects, the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use in the treatment of hepatitis D, more particularly of chronic hepatitis D. In some aspects, the present disclosure relates to an RNAi agent described herein, a salt thereof as described herein, a LNP or liposome as described herein, an isolated cell as described herein, a non-human animal as described herein, or a pharmaceutical composition for use in treating a cancer, for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma. [0020] Other aspects, features and advantages of the invention will be apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments and the appended claims.
DETAILED DESCRIPTION OF THE INVENTION [0021] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.
[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein.
[0023] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.
[0024] Unless otherwise stated, any numerical value, such as a % sequence identity or a % sequence identity range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a dosage of 10 mg includes 9 mg to 11 mg. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
[0025] As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” [0026] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention. [0027] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having.” Furthermore, as used herein the term “comprising” encompasses the term “consisting of.”
[0028] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any of the aforementioned terms of “comprising,” “containing,” “including,” and “having,” whenever used herein in the context of an aspect or embodiment of the invention can be replaced with the term “consisting of’ or “consisting essentially of’ to vary scopes of the disclosure.
[0029] As used herein the term “oligonucleotide” (or “oligo”) has its usual meaning as understood by those skilled in the art and thus refers to a class of compounds that includes oligodeoxynucleotides, oligodeoxyribonucleotides and oligoribonucleotides. Thus, “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, including reference to oligonucleotides composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) intemucleoside (backbone) linkages as well as “modified” or substituted oligonucleotides having non-naturally- occurring portions which function similarly, and/or including modifications such as GalNAc that increase circulation half-life and/or reduce degradation of the oligonucleotide, e.g., degradation by endonucleases, and increase cellular uptake.
[0030] An “antisense” oligonucleotide (ASO) is a synthetic oligonucleotide that recognizes or specifically anneals to a target RNA in a sequence-specific manner. The sequence of the target RNA recognized by the ASO is defined by the nucleotide number of the protein-coding sequence of the mRNA, where the adenylate of the initiation codon (AUG) is designated nucleotide 1. The sequences used in the present disclosure are of a length and sequence that can recognize the target with high specificity within a cell.
[0031] As used herein the term “siRNA” has its usual meaning as understood by those skilled in the art and thus refers to a class of oligonucleotides that are referred to as small interfering RNA, short interfering RNA and/or silencing RNA. These compounds are non-coding doublestranded RNA molecules, typically 19-25 base pairs in length, that operate within the RNA interference (RNAi) pathway and interfere with the expression of specific genes that contain complementary nucleotide sequences to the anti-sense strand of the siRNA and mediate degradation of the mRNA. The reduced level of the mRNA will decrease translation and the amount of the target protein. Reference herein to siRNA shall be understood to include reference to siRNA composed of naturally-occurring nucleobases, sugars and phosphodiester (PO) intemucleoside (backbone) linkages as well as “modified” or substituted siRNA having non- naturally-occurring portions which function similarly, and/or including modifications such as GalNAc that increase circulation half-life and/or reduce degradation of the oligonucleotide, e.g., degradation by endonucleases, and increase cellular uptake. In siRNA one strand is guiding and complementary to the target RNA (antisense strand), and the other strand (sense strand) has the same or substantially identical sequence as the target RNA and hence is complementary to the guiding/antisense strand.
[0032] As used herein the terms “target,” “targeting,” and similar terms, as used in the context of RNAi agent recognizing a target RNA through base pairing and have their usual meaning as understood by those skilled in the art to refer to a process by which the RNAi agent hybridizes to the target RNA and at least partially inhibits production of the RNA or protein to which it is targeted. For example, siRNA may cause silencing of a gene that encodes an mRNA by reducing the mRNA and thus decrease the products synthesized, including proteins, from that target RNA. Various assay techniques may be used to determine the degree to which the RNAi agent at least partially inhibits production of the RNA or protein to which it is targeted.
[0033] As used herein the term “virus molecule” has its usual meaning as understood by those skilled in the art and thus refers to a class of molecules made from the genome of a virus. A virus molecule is typically a viral protein, a viral DNA or a viral RNA. These molecules can be utilized or required by the virus to replicate their genome or regulate the outcome of the viral infection. For example, the term “HBV molecule” refers to a class of molecules made from the HBV genome. Examples of HBV molecules include S-antigen, E-antigen, Core-antigen, polymerase antigen, HBV RNA, and HBV DNA.
[0034] An “epitope” as used herein is a set of amino acid residues that form a site recognized by an immunoglobulin, T cell receptor or human leukocyte antigen (HLA) molecule.
[0035] The HLA proteins are encoded by clusters of genes that form a region located on chromosome 6 known as the Major Histocompatibility Complex (MHC), in recognition of the important role of the proteins encoded by the MHC loci in graft rejection. Accordingly, the HLA proteins are also referred to as MHC proteins. HLA or MHC proteins are cell surface glycoproteins that bind peptides at intracellular locations and deliver them to the cell surface, where the combined ligand is recognized by a T cell. Class I MHC proteins are found on virtually all of the nucleated cells of the body. The class I MHC proteins bind peptides present in the cytosol and form peptide-MHC protein complexes that are presented at the cell surface, where they are recognized by cytotoxic CD8+ T cells. Class II MHC proteins are usually found only on antigen-presenting cells such as B lymphocytes, macrophages, and dendritic cells. Each MHC Class I receptor consists of a variable a chain and a relatively conserved [32 -microglobulin chain. Three different, highly polymorphic class I a chain genes have been identified. These are called HLA-A, HLA-B, and HLA-C. Variations in the a chain accounts for all of the different class I MHC genes in the population.
[0036] The phrases “percent (%) sequence identity” or “% identity” or “% identical to” or “percent complementary to” when used with reference to a nucleotide sequence describe the number of matches (“hits”) of identical nucleotides of two or more aligned nucleotide sequences as compared to the number of nucleotide residues making up the overall length of the nucleotide sequences. In other terms, using an alignment, for two or more sequences the percentage of nucleotide residues that are the same (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full-length of the nucleotide sequences) can be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The same determination can be made for amino acid sequences. The sequences which are compared to determine sequence identity can thus differ by substitution(s), addition(s) or deletion(s) of amino acids or nucleotides. Suitable programs for aligning polynucleotide or protein sequences are known to the skilled person. The percentage sequence identity of polynucleotide or protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g., using the NCBI BLAST algorithm (Altschul SF, et al (1997), Nucleic Acids Res. 25:3389-3402).
[0037] As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides sequences with one another to form a duplex structure under certain conditions. For example, such conditions can be stringent conditions, such as the conditions of 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
[0038] An oligonucleotide is considered complementary to a target DNA or RNA sequence, when a sufficient number of nucleotides in the oligonucleotide can form hydrogen bonds with corresponding nucleotides in the target DNA or RNA to enable the formation of a stable complex. The sequence of an siRNA compound need not be 100% complementary to its target nucleic acid. The term “complementary” thus implies that the siRNA agent binds sufficiently strong and specific to the target molecule to provide the desired interference with the normal function of the target whilst leaving the function of non-target mRNAs unaffected.
[0039] As used herein, an oligonucleotide that is “at least X% complementary” to another sequence means that at least X% of nucleotides in the oligonucleotide can form hydrogen bonds with corresponding nucleotides in the other sequence. In some embodiments, an oligonucleotide “at least X% complementary” to another sequence is at least X% identical to a sequence that is fully complementary to the other sequence.
[0040] As used herein, the terms and phrases “in combination,” “in combination with,” “codelivery,” and “administered together with” in the context of the administration of two or more therapies or components to a subject refers to simultaneous administration of two or more therapies or components, such as RNAi agents, e.g. , siRNA, e.g. , ASO, or a therapeutic composition and an adjuvant. “Simultaneous administration” can be administration of the two components at least within the same day. When two components are “administered together with” or “administered in combination with,” they can be administered in separate compositions sequentially within a short time period, such as 24, 20, 16, 12, 8 or 4 hours, or within 1 hour, or they can be administered in a single composition at the same time. The use of the term “in combination with” does not restrict the order in which therapies or components are administered to a subject. For example, a first therapy or component (e.g. first RNAi agent) can be administered prior to (e.g., 5 minutes to one hour before), concomitantly with or simultaneously with, or subsequent to (e.g., 5 minutes to one hour after) the administration of a second therapy or component (e.g. , second RNAi agent). In some embodiments, a first therapy or component (e.g. first RNAi agent) and a second therapy or component (e.g., e.g., second RNAi agent) are administered in the same composition. In other embodiments, a first therapy or component (e.g. first RNAi agent) and a second therapy or component (e.g., second RNAi agent) are administered in separate compositions. [0041] As used herein, a “non-naturally occurring” nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not occur in nature. A “non-naturally occurring” nucleic acid or polypeptide can be synthesized, treated, fabricated, and/or otherwise manipulated in a laboratory and/or manufacturing setting. In some cases, a non-naturally occurring nucleic acid or polypeptide can comprise a naturally-occurring nucleic acid or polypeptide that is treated, processed, or manipulated to exhibit properties that were not present in the naturally-occurring nucleic acid or polypeptide, prior to treatment. As used herein, a “non-naturally occurring” nucleic acid or polypeptide can be a nucleic acid or polypeptide isolated or separated from the natural source in which it was discovered, and it lacks covalent bonds to sequences with which it was associated in the natural source. A “non-naturally occurring” nucleic acid or polypeptide can be made recombinantly or via other methods, such as chemical synthesis.
[0042] As used herein, the term “operably linked” refer to a linkage or a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence operably linked to a nucleic acid sequence of interest is capable of directing the transcription of the nucleic acid sequence of interest, or a signal sequence operably linked to an amino acid sequence of interest is capable of secreting or translocating the amino acid sequence of interest over a membrane.
[0043] As used herein, “subject” means any animal, preferably a mammal, most preferably a human, to whom will be or has been treated by a method according to an embodiment of the present disclosure. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, etc., more preferably a human. A human subject can include a patient.
[0044] As used herein, an “RNA interference agent,” “RNAi agent,” “RNA interference molecule” or “RNAi molecule” refer to a composition that contains an RNA or RNA-hke (e.g. , chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein, RNAi agents can operate through the RNA interference mechanism (i.e., inducing RNA interference through interaction with the RNA interference pathway machinery (RNA- induced silencing complex or RISC) of mammalian cells, or by any alternative mechanism(s) or pathway(s). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interference mechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short interfering RNAs (siRNAs), double-stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. RNAi agents of the application are preferably dsRNAs. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted. RNAi agents can be comprised of modified nucleotides and/or one or more non-phosphodiester linkages.
[0045] As used herein, the term “PD-L1” refers to programmed death-ligand 1, also known as CD274, B7H1, B7-H, PDCD1L1 or PDCD1LG1. The CD274 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog. The term “POLI” also includes protein variants and recombinant PD-L1 or a fragment thereof. Unless specified as being from a non-human species, the term“PD-Ll” means human PD-L1. The cDNA and protein sequences of PD-L1 can be obtained from public source, such as GenBank. For example, the amino acid sequence of a full-length human PD-L1 is provided in GenBank as accession number NP 054862.1 and a cDNA sequence encoding the full-length human PD-L1 is provided in GenBank as accession number NM_014143.4.
[0046] The term “double-stranded RNA,” “dsRNA molecule,” or “dsRNA,” as used herein, refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure comprising two anti -parallel and substantially complementary nucleic acid strands. The two strands forming the duplex structure can be different portions of one larger RNA molecule, or they can be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3 -end of one strand and the 5 ’ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3 ‘-end of one strand and the 5 ’ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands can have the same or a different number of nucleotides. In addition to the duplex structure, a dsRNA can comprise one or more nucleotide overhangs or can be blunt ended.
[0047] As used herein, the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or “knockdown” when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with oligomeric compounds, such as RNAi agents, described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated. [0048] By "optional" or "optionally" is meant that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.
[0049] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the pharmaceutical compositions.
[0050] The term "pharmaceutically acceptable salt" refers to a salt of any of the compounds herein which are known to be non-toxic and are commonly used in the pharmaceutical literature. In some embodiments, the pharmaceutically acceptable salt of a compound retains the biological effectiveness of the compounds described herein and are not biologically or otherwise undesirable. Examples of pharmaceutically acceptable salts can be found in Berge et al., Pharmaceutical Salts, J. Pharmaceutical Sciences, January 1977, 66(1), 1-19. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 2- hydroxyethylsulfonic acid, p-toluenesulfonic acid, stearic acid and salicylic acid.
Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines; and basic ion exchange resins. Examples of organic bases include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.
[0051] The term "therapeutically effective amount" or "effective amount" refers to that amount of a compound disclosed and/or described herein that is sufficient to affect treatment, as defined herein, when administered to a subject in need of such treatment. The therapeutically effective amount will vary depending upon, for example, the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound, the dosing regimen to be followed, timing of administration, the manner of administration, all of which can readily be determined by one of ordinary skill in the art. The therapeutically effective amount can be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability by one of ordinary skill in the art in view of the present disclosure.
[0052] In some embodiments of the application, a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy. In some embodiments of the application, a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a PD-L1 associated infection or disease, or a symptom associated therewith; (ii) reduce the duration of an PD-L1 associated infection or disease, or symptom associated therewith; (iii) prevent the progression of an PD-L1 associated disease, or symptom associated therewith; (iv) cause regression of an PD- L1 associated disease, or symptom associated therewith; (v) prevent the development or onset of an PD-L1 associated disease, or symptom associated therewith; (vi) prevent the recurrence of an PD-L1 associated disease or symptom associated therewith; (vii) reduce hospitalization of a subject having an PD-L1 associated disease; (viii) reduce hospitalization length of a subject having an PD-L1 associated disease; (ix) increase the survival of a subject with an HBV disease; (x) eliminate an PD-L1 associated disease in a subject; (xi) inhibit or reduce PD-L1 associated disease replication in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
[0053] The application also relates to a vector comprising the first and/or second non- naturally occurring nucleic acid molecules. As used herein, a “vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). Preferably, a vector is a DNA plasmid. A vector can be a DNA vector or an RNA vector. One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.
[0054] A vector of the application can be an expression vector. As used herein, the term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
[0055] Preferably, a DNA plasmid is an expression vector suitable for protein expression in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADVAX, NTC8454, etc. Preferably, an expression vector is based on pVAX- 1, which can be further modified to optimize protein expression in mammalian cells. pVAX-1 is commonly used plasmid in DNA vaccines, and contains a strong human intermediate early cytomegalovirus (CMV-IE) promoter followed by the bovine growth hormone (bGH) -derived polyadenylation sequence (pA). pVAX-1 further contains a pUC origin of replication and kanamycin resistance gene driven by a small prokaryotic promoter that allows for bacterial plasmid propagation.
[0056] A vector of the application can also be a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non- infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno- associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, Modified Vaccinia virus Ankara (MV A) vectors, lentiviral vectors, etc. Examples of viral vectors that can be used include, but are not limited to, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi- segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which comprise an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding an HBV antigen as described herein), arenavirus such as lymphocytic choriomeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain. The vector can also be a non-viral vector.
[0057] Preferably, a viral vector is an adenovirus vector, e.g. , a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g. , rhAd51, rhAd52 or rhAd53
[0058] In an attempt to help the reader of the present disclosure, the description has been separated in various paragraphs or sections or is directed to various embodiments of the present disclosure. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. For example, while embodiments of ribonucleic acid molecules of the present disclosure described herein may contain particular components arranged in a particular order, those having ordinary skill in the art will appreciate that the concepts disclosed herein may equally apply to other components arranged in other orders that can be used in ribonucleic acid molecules of the present disclosure. The present disclosure contemplates use of any of the applicable components in any combination having any sequence that can be used in ribonucleic acid molecules of the present disclosure, whether or not a particular combination is expressly described.
PD-L1
[0059] PD-L1 (e.g., a full length 290 amino acid human PD-L1 shown in GenBank accession No. NP_054862) is a protein with extracellular IgV-like and IgC-like domains (e.g., amino acids 19-239 of the full-length human PD-L1), a transmembrane domain and an intracellular domain (e.g., approximately 30 amino acids of the full-length human PD-L1). PD-L1 is constitutively expressed on many cells such as antigen presenting cells (e.g., dendritic cells, macrophages, and B-cells) and on hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and sites of immune privilege). PD-L1 is also expressed on a wide variety of tumors, and virally-infected cells and is a component of the immunosuppressive milieu (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of two T-cell co-inhibitors PD-1 and B7-1. [0060] T-cell co-stimulatory and co-inhibitory molecules (collectively named co-signaling molecules) play a crucial role in regulating T-cell activation, subset differentiation, effector function and survival (Chen et al2013, Nature Rev. Immunol. 13 : 227-242). Following recognition of cognate peptide-MHC complexes on antigen-presenting cells by the T-cell receptor, co-signaling receptors co-localize with T-cell receptors at the immune synapse, where they synergize with TCR signaling to promote or inhibit T-cell activation and function (Flies et al. 2011, Yale J. Biol. Med. 84: 409-421). The ultimate immune response is regulated by a balance between co-stimulatory and co-inhibitory signals (“immune checkpoints”) (Pardoll 2012, Nature 12: 252-264). While not wishing to be bound by theory, it is currently believed that PD-1 functions as one such ‘immune checkpoint’ in mediating peripheral T-cell tolerance and in avoiding autoimmunity. PD-1 binds to PD-L1 or PD-L2 and inhibits T-cell activation. The ability of PD-1 to inhibit T-cell activation is exploited by chronic viral infections and tumors to evade immune response. In chronic viral infections, PD-1 is highly expressed on virus-specific T-cells and these T-cells become “exhausted” with loss of effector functions and proliferative capacity (Freeman 2008, PNAS 105: 10275-1 0276). The PD-1-PD-L1 system also plays an important role in induced T-regulatory (Treg) cell development and in sustaining Treg function (Francisco et al 2010, Immunol. Rev. 236:219-242).
RNAi Agents [0061] The present disclosure generally relates to a ribonucleic acid interfering (RNAi) agent useful for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) gene, wherein the RNAi agent comprises an antisense strand comprising a sequence of 15 to 23 nucleotides, wherein the nucleotide sequence of the antisense strand comprises at least 15 contiguous nucleotides (optionally 16 or more, 17 or more, 18 or more, or 19 or more contiguous nucleotides) from a sequence selected from SEQ ID NOs: 604-804 and wherein one or more nucleotides of the antisense strand is a modified nucleotide; and wherein the RNAi agent optionally further comprises a sense strand comprising a nucleotide sequence, which is of the same length as, or of a lower length than, the nucleotide sequence of the antisense strand, and wherein one or more nucleotides of the sense strand optionally is a modified nucleotide.
[0062] In some aspects, each PD-L1 RNAi agent comprises a sense strand and an antisense strand. In some aspects, the sense strand and the antisense strand each can be 15 to 23 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides in length while the antisense strand is about 23 nucleotides in length. In some aspects, the sense strand of the RNAi agent comprises at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603. In some aspects, the number of nucleotides of the sense strand of the RNAi agent is 19 or 21. In some aspects, the number of nucleotides of the antisense strand of the RNAi agent is 19, 21 or 23. In some aspects, the number of nucleotides of the sense strand of the RNAi agent is 19 and the number of nucleotides of the antisense strand of the RNAi agent is 21; the number of nucleotides of the sense strand of the RNAi agent is 21 and the number of nucleotides of the antisense strand of the RNAi agent is 21 ; the number of nucleotides of the sense strand of the RNAi agent is 21 and the number of nucleotides of the antisense strand of the RNAi agent is 23; or the number of nucleotides of the sense strand of the RNAi agent is 19 and the number of nucleotides of the antisense strand of the RNAi agent is 19. In some aspects, a sense strand of the PD-L1 RNAi agents described herein includes a core stretch having at least about 85% identity to a nucleotide sequence of at least 15 consecutive nucleotides in an PD-L1 mRNA. In some embodiments, the sense strand core nucleotide stretch having at least about 85% identity to a sequence in an PD- LlmRNA is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some aspects, an antisense strand of a PD-L1 RNAi agent comprises a nucleotide sequence having at least about 85% complementary over a core stretch of at least 15 consecutive nucleotides to a sequence in an PD-LlmRNA and the corresponding sense strand. In some embodiments, the antisense strand core nucleotide sequence having at least about 85% complementarity to a sequence in an PD- LlmRNA or the corresponding sense strand is 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length.
[0063] In some aspects, RNAi agents of the application can contain one or more mismatches to the target sequence. In a preferred embodiment, RNAi agents of the application contains no more than 13 mismatches. If the antisense strand of the RNAi agent contains mismatches to a target sequence, it is preferable that the area of mismatch not be located within nucleotides 2-7 of the 5’ terminus of the antisense strand. In another embodiment, it is preferable that the area of mismatch not be located within nucleotides 2-9 of the 5’ terminus of the antisense strand.
[0064] In some aspects, the PD-L1 RNAi agent sense and antisense strands anneal to form a duplex. In some aspects, a sense strand and an antisense strand of a PD-L1 RNAi agent can be partially, substantially, or fully complementary to each other. Within the complementary duplex region, the sense strand core stretch sequence is at least about 85% complementary or 100% complementary to the antisense core stretch sequence. In some embodiments, the sense strand core stretch sequence contains a sequence of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least about 85% or 100% complementary to a corresponding 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the antisense strand core stretch sequence (i.e., the sense strand and antisense core stretch sequences of a PD-L1 RNAi agent have a region of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that is at least 85% base paired or 100% base paired).
[0065] In some embodiments, the antisense strand of a PD-L1 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences described herein. In some embodiments, the sense strand of a PD-L1 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences described herein.
[0066] In some aspects, the sense strand and/or the antisense strand can optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extension) at the 3' end, the 5' end, or both the 3' and 5' ends of the core sequences. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sequence in an PD-L1 mRNA. The sense strand additional nucleotides, if present, may or may not be identical to the corresponding sequence in an PD-L1 mRNA. The antisense strand additional nucleotides, if present, may or may not be complementary to the corresponding sense strand's additional nucleotides, if present.
[0067] In some aspects, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and/or 3' end of the sense strand core stretch sequence and/or antisense strand core stretch sequence. The extension nucleotides on a sense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding antisense strand. Conversely, the extension nucleotides on an antisense strand may or may not be complementary to nucleotides, either core stretch sequence nucleotides or extension nucleotides, in the corresponding sense strand. In some embodiments, both the sense strand and the antisense strand of an RNAi agent contain 3' and 5' extensions. In some embodiments, one or more of the 3' extension nucleotides of one strand base pairs with one or more 5' extension nucleotides of the other strand. In other embodiments, one or more of 3' extension nucleotides of one strand do not base pair with one or more 5' extension nucleotides of the other strand. In some embodiments, a PD-L1 RNAi agent has an antisense strand having a 3' extension and a sense strand having a 5' extension. In some embodiments, a PD-L1 RNAi agent comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, a PD-LIRNAi agent comprises an antisense strand having a 3' extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprise uracil or thymidine nucleotides or nucleotides which are complementary to a corresponding PD-L1 mRNA sequence. In some embodiments, the 3' end of the antisense strand can include additional abasic nucleosides (Ab). In some embodiments, Ab or Ab Ab can be added to the 3' end of the antisense strand.
[0068] In some embodiments, a PD-L1 RNAi agent comprises an antisense strand having a 5' extension of 1, 2, 3, 4, or 5 nucleotides in length. In other embodiments, a PD-L1 RNAi agent comprises an antisense strand having a 5' extension of 1 or 2 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprises uracil or thymidine nucleotides or nucleotides which are complementary to a corresponding PD-L1 mRNA sequence. An antisense strand can have any of the 3' extensions described above in combination with any of the 5' antisense strand extensions described, if present.
[0069] In some embodiments, a PD-L1 RNAi agent comprises a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide, or nucleotides which correspond to nucleotides in the PD-LlmRNA sequence. [0070] In some embodiments, the 3' end of the sense strand can include additional abasic nucleosides. In some embodiments, UUAb, UAb, or Ab can be added to the 3' end of the sense strand. In some embodiments, the one or more abasic nucleosides added to the 3' end of the sense strand can be inverted (invAb). In some embodiments, one or more inverted abasic nucleosides can be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic nucleosides at or near the terminal end or terminal ends of the sense strand of an RNAi agent can allow for enhanced activity or other desired properties of an RNAi agent. In some embodiments, a PD-L1 RNAi agent comprises a sense strand having a 5' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise uracil or adenosine nucleotides or nucleotides which correspond to nucleotides in the PD-L1 mRNA sequence.
[0071] In some embodiments, the 5' end of the sense strand can include an additional abasic nucleoside (Ab) or nucleosides (AbAb). In some embodiments, the one or more abasic nucleosides added to the 5' end of the sense strand can be inverted (invAb). In some embodiments, one or more inverted abasic nucleosides can be inserted between the targeting ligand and the nucleobase sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic nucleosides at or near the terminal end or terminal ends of the sense strand of an RNAi agent can allow for enhanced activity or other desired properties of an RNAi agent.
[0072] In some embodiments, the sense and antisense strands of the RNAi agents described herein contain the same number of nucleotides. In some embodiments, the sense and antisense strands of the RNAi agents described herein contain different numbers of nucleotides. In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a blunt end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form blunt ends. In some embodiments, neither end of an RNAi agent is blunt-ended. As used herein a blunt end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands are complementary (form a complementary base -pair). In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a frayed end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a frayed end. In some embodiments, both ends of an RNAi agent form a frayed end. In some embodiments, neither end of an RNAi agent is a frayed end. As used herein a frayed end refers to an end of a double stranded RNAi agent in which the terminal nucleotides of the two annealed strands from a pair (i.e. do not form an overhang) but are not complementary (i.e. form a non-complementary pair). As used herein, an overhang is a stretch of one or more unpaired nucleotides at the end of one strand of a double stranded RNAi agent. The unpaired nucleotides can be on the sense strand or the antisense strand, creating either 3' or 5' overhangs. In some embodiments, the RNAi agent contains: a blunt end and a frayed end, a blunt end and 5' overhang end, a blunt end and a 3' overhang end, a frayed end and a 5' overhang end, a frayed end and a 3' overhang end, two 5' overhang ends, two 3' overhang ends, a 5' overhang end and a 3' overhang end, two frayed ends, or two blunt ends.
[0073] In some embodiments, a PD-L1 RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. In some embodiments, a PD-L1 RNAi agent is prepared as a sodium salt. Such forms are within the scope of the application disclosed herein.
[0074] In some aspects, the nucleotide sequence of the sense strand comprises a sequence of 15 to 23 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a sequence of the same length that is comprised in the sequence of the antisense strand. In some aspects, the nucleotide sequence of the sense strand comprises a sequence of 15 to 23 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% to a sequence of the same length that is comprised in the sequence complementary to the sequence of the antisense strand.
Targeting Groups, Linking Groups, and Delivery Vehicles
[0075] In some embodiments, a PD-L1 RNAi agent is conjugated to one or more nonnucleotide groups including, but not limited to a targeting group, linking group, delivery polymer, or a delivery vehicle. The non-nucleotide group can enhance targeting, delivery or attachment of the RNAi agent. Examples of targeting groups and linking groups are provided in Table 6 of W02018027106. The non-nucleotide group can be covalently linked to the 3' and/or 5' end of either the sense strand and/or the antisense strand. In some embodiments, a PD-L1 RNAi agent contains a non-nucleotide group linked to the 3' and/or 5' end of the sense strand. In some embodiments, a non-nucleotide group is linked to the 5' end of a PD-L1 RNAi agent sense strand. A non-nucleotide group can be linked directly or indirectly to the RNAi agent via a linker/linking group. In some embodiments, a non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.
[0076] In some embodiments, the RNAi agents are delivered to target cells or tissues using any oligonucleotide delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, by encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, proteinaceous vectors or Dynamic Polyconjugates (DPCs) (see, for example WO 2000/053722, WO 2008/022309, WO 2011/104169, and WO 2012/083185, each of which is incorporated herein by reference). In some embodiments, a PD-L1 RNAi agent is delivered to target cells or tissues by covalently linking the RNAi agent to a targeting group (also referred to herein as a “targeting ligand”). In some embodiments, the targeting group can include a cell receptor ligand, such as an asialoglycoprotein receptor (ASGPr) ligand. In some embodiments, an ASGPr ligand includes or consists of a galactose derivative cluster. In some embodiments, a galactose derivative cluster includes an N-acetyl-galactosamine trimer or an N-acetyl- galactosamine tetramer. In some embodiments, a galactose derivative cluster is an N-acetyl- galactosamine trimer or an N-acetyl-galactosamine tetramer.
[0077] A targeting group can be linked to the 3' or 5' end of a sense strand or an antisense strand of a PD-L1 RNAi agent. In some embodiments, a targeting group is linked to the 3' or 5' end of the sense strand. In some embodiments, a targeting group is linked to the 5’ end of the sense strand. In some embodiments, a targeting group is linked to the RNAi agent via a linker (also referred to herein as a “linking group”).
[0078] In some embodiments, a non-nucleotide group enhances the pharmacokinetic or biodistribution properties of an RNAi agent or conjugate to which it is attached to improve cell- or tissue-specific distribution and cell-specific uptake of the conjugate. In some embodiments, a non-nucleotide group enhances endocytosis of the RNAi agent.
[0079] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate to which they are attached to improve cell-specific distribution and cellspecific uptake of the conjugate. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency. Representative targeting groups include, without limitation, compounds with affinity to cell surface molecule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity to cell surface molecules. In some embodiments, a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and/or ribitol (abasic ribose) groups. In some embodiments, a targeting group comprises a galactose derivative cluster. The PD-L1 RNAi agents described herein can be synthesized having a reactive group, such as an amine group, at the 5 '-terminus. The reactive group can be used to subsequently attach a targeting moiety using methods typical in the art. In some embodiments, a targeting group comprises an asialoglycoprotein receptor ligand. In some embodiments, an asialoglycoprotein receptor ligand includes or consists of one or more galactose derivatives. As used herein, the term galactose derivative includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor that is equal to or greater than that of galactose. Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetyl- galactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-iso- butanoylgalactos-amine (see for example: lobst, S.T. and Drickamer, K. J.B.C. 1996, 277, 6686). Galactose derivatives, and clusters of galactose derivatives, that are useful for in vivo targeting of oligonucleotides and other molecules to the liver are known in the art (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem, 257, 939- 945). Galactose derivatives have been used to target molecules to hepatocvtes in vivo through their binding to the asialoglycoprotein receptor (ASGPr) expressed on the surface of hepatocytes. Binding of ASGPr ligands to the ASGPr(s) facilitates cell-specific targeting to hepatocytes and endocytosis of the molecule into hepatocytes. ASGPr ligands can be monomeric (e.g., having a single galactose derivative) or multimeric (e.g., having multiple galactose derivatives). The galactose derivative or galactose derivative cluster can be attached to the 3 Or 5' end of the RNAi polynucleotide using methods known in the art. The preparation of targeting groups, such as galactose derivative clusters, is described in, for example, US20180064819 and US20170253875, the contents of both of which are incorporated herein in their entirety.
[0080] As used herein, a galactose derivative cluster comprises a molecule having two to four terminal galactose derivatives. A terminal galactose derivative is attached to a molecule through its C-l carbon. In some embodiments, the galactose derivative cluster is a galactose derivative trimer (also referred to as tri-antennary galactose derivative or tri-valent galactose derivative). In some embodiments, the galactose derivative cluster comprises N-acetyl-galactosamines. In some embodiments, the galactose derivative cluster comprises three N-acetyl-galactosamines. In some embodiments, the galactose derivative cluster is a galactose derivative tetramer (also referred to as tetra-antennary galactose derivative or tetra-valent galactose derivative). In some embodiments, the galactose derivative cluster comprises four N-acetyl-galactosamines.
[0081] As used herein, a galactose derivative trimer contains three galactose derivatives, each linked to a central branch point. As used herein, a galactose derivative tetramer contains four galactose derivatives, each linked to a central branch point. The galactose derivatives can be attached to the central branch point through the C-l carbons of the saccharides. In some embodiments, the galactose derivatives are linked to the branch point via linkers or spacers. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as a PEG group (see, for example, U.S. Patent No. 5,885,968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538- 1546). In some embodiments, the PEG spacer is a PEG3 spacer. The branch point can be any small molecule which permits attachment of three galactose derivatives and further permits attachment of the branch point to the RNAi agent. An example of branch point group is a dilysine or di-glutamate. Attachment of the branch point to the RNAi agent can occur through a linker or spacer. In some embodiments, the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some embodiments, the linker comprises a rigid linker, such as a cyclic group. In some embodiments, a galactose derivative comprises or consists of N-acetyl-galactosamine. In some embodiments, the galactose derivative cluster is comprised of a galactose derivative tetramer, which can be, for example, an N-acetyl- galactosamine tetramer.
[0082] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent linkage of the agent to a targeting group or delivery polymer or delivery vehicle. The linking group can be linked to the 3 ' or the 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of an RNAi agent sense strand. In some embodiments, a linking group is conjugated to the 5' end of an RNAi agent sense strand. Examples of linking groups, include, but are not limited to: reactive groups such a primary amines and alkynes, alkyl groups, abasic nucleosides, ribitol (abasic ribose), and/or PEG groups.
[0083] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer can further add flexibility and/or length to the linkage. Spacers can include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description. [0084] Targeting groups, modified nucleotides and linking groups include, but are not limited to, the following, for which their chemical structures are provided below in Table 1 : (PAZ), (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), (NAG39)s, vpdT, 5Me-Gf, cPrpTM, cPrpu, epTM, epTcPr, invAb, (invAb)s. Each sense strand and/or antisense strand can have any targeting groups or linking groups listed above, as well as other targeting or linking groups, conjugated to the 5' and/or 3' end of the sequence.
Table A: Structures Representing Various Modified Nucleotides, Targeting Groups, And Linking
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0002
[0085] wherein NAG in the structures provided in the above table is represented by the following structure:
Figure imgf000045_0001
(N -acetyl -galactosamine) .
[0086] Each (NAGx) can be attached to a PD-L1 RNAi agent via a phosphate group (e.g. , as in (NAG25), (NAG30), and (NAG31)), or a phosphorothioate group, (e.g., as is (NAG25)s, (NAG29)s, (NAG30)s, (NAG31)s, or (NAG37)s), or another linking group:
Figure imgf000046_0001
Phosphate group Phosphorothioate group
[0087] Other linking groups known in the art can be used.
In some aspects, the RNAi agent comprises one or more from invAb and targeting moieties, more particularly liver targeting moieties; wherein the liver targeting moieties are fatty acids, GalNAc, folic acid, cholesterol, tocopherol or palmitate, more particularly GalNAc, more particularly
Figure imgf000046_0002
(GalNAc2) at the 3 ’ end of the sense strand; optionally further comprising one or more invAb, more particularly 1 invAb at the 5’ end of the sense strand and/or 1 invAb at the 3’ end of the sense strand.
[0088] In some embodiments, a delivery vehicle can be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves delivery of the RNAi agent to a cell or tissue. A delivery vehicle can include, or consist of, but is not limited to: a polymer, such as an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin- like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active poly amine.
[0089] In some embodiments, the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO 2008/0022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO 2013/158141, each of which is incorporated herein by reference), or other delivery systems available in the art. [0090] Other lipophilic compounds that have been conjugated to oligonucleotides include 1- pyrene butyric acid, l,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor- mediated endocytosis. RNAi agents bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Attachment of folic acid to the 3 ‘-terminus of an oligonucleotide results in increased cellular uptake of the oligonucleotide (Li S, Deshmukh HM, and Huang L, Pharm. Res. (1998) 15: 1540). Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides. In certain instances, conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See Manoharan M, Antisense & Nucleic Acid Drug Development (2002) 12: 103 and references therein.
[0091] Additional modifications can also be made at other positions on the oligonucleotide, particularly the 3’ position of the sugar on the 3’ terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the application involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties, such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, (1989) 86:6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Lett., (1994) 4: 1053), athioether, 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 fhiochoiesterol (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: 1 1 1 ; 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). [0092] In certain instances, the RNAi agent can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the RNAi agent, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol ( 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: 1 1 1; 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). Typical conjugation protocols involve the synthesis of oligonucleotides 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 RNAi agent still bound to the solid support or following cleavage of the RNAi agent in solution phase. Purification of the RNAi agent conjugate by HPLC typically affords the pure conjugate.
[0093] Alternatively, the molecule being conjugated can be converted into a building block, such as a phosphoramidite, via an alcohol group present in the molecule or by attachment of a linker bearing an alcohol group that can be phosphorylated. Importantly, each of these approaches can be used for the synthesis of ligand conjugated RNAi agents. Amino linked RNAi agents can be coupled directly with ligand via the use of coupling reagents or following activation of the ligand as an NHS or pentfluorophenolate ester. Ligand phosphoramidites can be synthesized via the attachment of an aminohexanol linker to one of the carboxyl groups followed by phosphityation of the terminal alcohol functionality. Other linkers, such as cysteamine, can also be utilized for conjugation to a chloroacetyl linker present on a synthesized oligonucleotide. [0094] The person skilled in the art is readily aware of methods to introduce the RNAi agents of this application into cells, tissues or organisms. Corresponding examples have also been provided in the detailed description of the application above. For example, the RNAi agents can be introduced into cells or tissues by methods known in the art, like transfections etc.
[0095] Also for the introduction of RNAi agents, means and methods have been provided. For example, targeted delivery by glycosylated and folate -modified molecules, including the use of polymeric carriers with ligands, such as galactose and lactose or the attachment of folic acid to various macromolecules allows the binding of molecules to be delivered to folate receptors. Targeted delivery by peptides and proteins other than antibodies, for example, including RGD- modified nanoparticles to deliver siRNA in vivo or multicomponent (nonviral) delivery systems including short cyclodextrins, adamantine- PEG are known. Yet, also the targeted delivery using antibodies or antibody fragments, including (monovalent) Fab- fragments of an antibody (or other fragments of such an antibody) or single-chain antibodies are envisaged. Injection approaches for target directed delivery comprise, inter alia, hydrodynamic i.v. injection. Also, cholesterol conjugates of RNAi agents can be used for targeted delivery, whereby the conjugation to lipophilic groups enhances cell uptake and improve pharmacokinetics and tissue biodistribution of oligonucleotides. Also, cationic delivery systems are known, whereby synthetic vectors with net positive (cationic) charge to facilitate the complex formation with the polyanionic nucleic acid and interaction with the negatively charged cell membrane. Such cationic delivery systems comprise also cationic liposomal delivery systems, cationic polymer and peptide delivery systems. Other delivery systems for the cellular uptake of dsRNA/siRNA are aptamer-ds/si RNA. Also, gene therapy approaches can be used to deliver the described RNAi agents. Such systems comprise the use of non-pathogenic virus, modified viral vectors, as well as deliveries with nanoparticles or liposomes. Other delivery methods for the cellular uptake of RNAi agents are extracorporeal, for example ex vivo treatments of cells, organs or tissues. Certain of these technologies are described and summarized in publications, like Akhtar, Journal of Clinical Investigation (2007) 1 17:3623-3632, Nguyen et al, Current Opinion in Molecular Therapeutics (2008) 10: 158- 167, Zambon i, Clin Cancer Res (2005) 1 1 :8230- 8234 or Ikeda et al, Pharmaceutical Research (2006) 23 : 1631 -1640.
[0096] Methods of making and using RNAi agents and conjugates thereof are known in the art. Any such known methods can be used in the context of the present application to make and use RNAi agents and conjugates thereof for inhibiting the expression of an HBV gene. Methods of making and using RNAi agents and conjugates thereof are described, e.g., in US20130005793, W02013003520, W02018027106, US5218105, US5541307, US5521302, US5539082, US5554746, US5571902, US5578718, US5587361, US5506351, US5587469, US5587470, US5608046, US5610289, US6262241, WO9307883, all of which are incorporated herein by reference in their entirety.
Modified Nucleotides
[0097] As used herein, the following notations are used to indicate modified nucleotides, targeting groups, and linking groups. As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence, that when present in an oligonucleotide, the monomers are mutually linked by 5'-3'-phosphodiester bonds:
[0098] A = adenosine-3 '-phosphate
[0099] C = cytidine -3 '-phosphate
[0100] G = guanosine-3 '-phosphate
[0101] U = uridine-3 '-phosphate
[0102] m = any 2'-0Me modified nucleotide
[0103] a = 2'-o-methyladenosine-3'-phosphate
[0104] as = 2'-o-methyladenosine-3'-phosphorothioate
[0105] c = 2'-o-methylcytidine-3'-phosphate
[0106] cs = 2'-o-methylcytidine-3'-phosphorothioate
[0107] g = 2'-o-methylguanosine-3'-phosphate
[0108] gs = 2'-o-methylguanosine-3'-phosphorothioate
[0109] t = 2'-o-methyl-5-methyluridine-3 '-phosphate
[0110] ts = 2'-o-methyl-5-methyluridine-3'-phosphorothioate
[0111] u = 2'-o-methyluridine-3 '-phosphate
[0112] us = 2'-o-methyluridine-3'-phosphorothioate
[0113] fN = any 2'-fluoro modified nucleotide
[0114] fA = 2'-fluoroadenosine-3'-phosphate
[0115] fC = 2'-fluorocytidine-3 '-phosphate
[0116] fG = 2'-fluoroguanosine-3 '-phosphate
[0117] fT = 2'-fhioro-5'-methyhrridine-3'-phosphate
[0118] fU = 2'-fhiorouridine-3'-phosphate
[0119] dN = any 2'-deoxyribonucleotide
[0120] dT = 2'-deoxythymidine-3'-phosphate
[0121] NuNA = 2',3'-seco nucleotide mimics (unlocked nucleobase analogs) [0122] NLNA = locked nucleotide
[0123] NfANA = 2'-F-Arabino nucleotide
[0124] UNA-N = 2', 3 '-seco nucleotide (any unlocked nucleotide)
[0125] ps = phosphorothioate linkage
[0126] NM = 2'-methoxyethyl nucleotide
[0127] AM = 2'-methoxyethyladenosine-3'-phosphate
[0128] AMs = 2'-methoxyethyladenosine-3 '-phosphorothioate
[0129] TM = 2'-methoxyethylthymidine-3'-phosphate
[0130] TMs = 2'-methoxyethylthymidine-3'-phosphorothioate
[0131] R = ribitol
[0132] (invdN) = any inverted deoxyribonucleotide
[0133] (invAb) = inverted abasic deoxyribonucleotide
[0134] (invAb)s = inverted abasic deoxyribonucleotide-5'- phosphorothioate
[0135] (vinu) = vinylphosphonate
[0136] (GalNAc-2) = N-Acetylgalactosamine
Figure imgf000051_0001
[0137] (invn) = any inverted 2'-OMe nucleotide (3 '-3' linked nucleotide) s = phosphorothioate linkage
[0138] vpdN = vinyl phosphonate deoxyribonucleotide
[0139] (5Me-Nf) = 5'-Me, 2'-fluoro nucleotide
[0140] cPrp = cyclopropyl phosphonate
[0141] The person or ordinary skill in the art would readily understand that the terminal nucleotide at the 3 ' end of a given oligonucleotide sequence would typically have a hydroxyl (- OH) group at the respective 3' position of the given monomer instead of a phosphate moiety ex vivo.
[0142] As used herein, a “modified nucleotide” is a nucleotide other than a ribonucleotide
(2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include, but are not limited to, deoxyribonucleotides, nucleotide mimics, abasic nucleotides, 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides, non-natural base-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3 '-seco nucleotide mimics (unlocked nucleobase analogues), locked nucleotides, 3'-O-methoxy (2' intemucleoside linked) nucleotides, 2'-F- Arabino nucleotides, 5 '-Me, 2'-fluoro nucleotide, morpholino nucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides. 2'-modified nucleotides (i.e. a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy nucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2 -methoxylethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in the ribonucleic acid molecule or even in a single nucleotide thereof. The ribonucleic acid molecule may be synthesized and/or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.
[0143] Modified nucleobases include synthetic and natural nucleobases, such as 5 -substituted pyrimidmes, 6-azapyrimi dines and N-2, N-6 and 0-6 substituted purines,
Figure imgf000052_0001
aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5 -methylcytosine (5-me-C), 5- hydroxymethyl cytosme, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6- ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2- ethyl, 2-isopropyl, or 2-n-butyl ) and other alkyl derivatives of adenine and guanine, 2- thiouracil. 2-thiothymine. 2-thiocytosine, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6- azo uracil, 6-azo cytosine, 6-azo thymine, -uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-sulfhydiyl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoiOinethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-dea/aadenine. 3- deazaguanine, and 3 -deazaadenine. In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RN Ai agent having four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides. As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. As used herein, an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is a ribonucleotide.
[0144] As used herein, the term “sugar substituent group” or “2 ’-substituent group” includes groups attached to the 2 ’-position of the ribofuranosyl moiety with or without an oxygen atom. Sugar substituent groups include, but are not limited to, fluoro, O-alkyl, O-alkylamino, O- alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole and poly ethers of the formula (O-alkyl)m, wherein m is 1 to about 10. Preferred among these polyethers are linear and cyclic polyethylene glycols (PEGs), and (PEG)- containing groups, such as crown ethers and, inter alia, those which are disclosed by Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems (1992) 9:249). Further sugar modifications are disclosed by Cook (Anti-fibrosis Drug Design, (1991) 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O- alkylaminoalkyl, and alkyl amino substitution is described in U.S. Patent 6, 166,197, entitled “Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2’ and 5’ Substitutions.” hereby incorporated by reference in its entirety.
[0145] Additional sugar substituent groups amenable to the application include 2’-SR and 2‘- NR2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl. 2 ’-SR Nucleosides are disclosed in US5670633, hereby incorporated by reference in its entirety. The incorporation of 2’ -SR monomer synthons is disclosed by Hamm et al. (J. Org. Chem., (1997) 62:3415-3420). 2’-NR nucleosides are disclosed by Thomson JB, J. Org. Chem., (1996) 61 :6273-6281 ; and Polushin et al., Tetrahedron Lett., (1996) 37:3227-3230. Further representative 2‘ -substituent groups amenable to the application include those having one of formula I or II:
Figure imgf000053_0001
[0146] wherein E is C1-C10 alkyl, N(Q3)(Q4) or C(Q3)(Q4); each Q3 and Q4 is, independently, H, C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q3 and Q4, together, form a nitrogen protecting group or a ring structure optionally including at least one additional heteroatom selected from N and O; q 1 is an integer from 1 to 10; q2 is an integer from 1 to 10; q3 is 0 or 1; q4 is 0, 1 or 2; each Zl, Z2, and Z3 is, independently, C4-C7 cycloalkyl, C5-C14 aryl or C3- C15 heterocyclyl, where Z4 is OM1, SMI, or N(Ml)z; each Ml is, independently, H, Ci-Cs alkyl, Ci-Cs haloalkyl, C(=NH)N(H)M2, C(=0)N(H)M2 or 0C(=0)N(H)M2; M2 is H or Ci-Cs alkyl; and in the heteroatom in said heterocyclyl group is selected from oxygen, nitrogen and sulfur; Z5 is C1-C10 alkyl, C1-C0 haloalkyl, C2-C10 alkenyl, C2-C10 alkynyl, Ce-Ci4 aryl, N(Q3)(Q4), OQ3, halo, SQ3 or CN.
[0147] Representative 2’-O-sugar substituent groups of formula I are disclosed in US6172209, entitled “Capped 2’-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety. Representative cyclic 2’-O-sugar substituent groups of formula II are disclosed in US6271358, entitled “RNA Targeted 2’-Modified Oligonucleotides that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.
[0148] Sugars having O -substitutions on the ribosyl ring are also amenable to the application. Representative substitutions for ring O include, but are not limited to, S, CH2, CHF, and CF2.
[0149] Oligonucleotides can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents relating to the preparation of such modified sugars include, but are not limited to, US5359044, US5466786, US5519134, US5591722, US5597909, US5646,265, and US5700920, all of which are hereby incorporated by reference.
[0150] In some aspects, at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sequence of the antisense strand of the RNAi agent are modified nucleotides. In some aspects, wherein all the nucleotides of the sequence of the antisense strand of the RNAi agent are modified nucleotides. In some aspects, at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sense strand of the RNAi agent are modified nucleotides. In some aspects, all the nucleotides of the sequence of the sense strand of the RNAi agent are modified nucleotides. In some aspects, the modified nucleotide comprises a modified nucleoside and/or a modified phosphate and/or a modified intemucleotide linkage.
[0151] In some aspects, the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a [0152] 2’-deoxy-2’-fluoro-ribose (
Figure imgf000055_0001
Base
[0153] a 2’ O-methyl ribose (2’ O-Me)
Figure imgf000055_0002
[0154] the acyclic sugar of an UNA nucleotide
Figure imgf000055_0003
[0155] In some aspects, the modified nucleotide of the RNAi agent comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate .
[0156] In some aspects, the modified nucleotide comprises a modified intemucleotide linkage selected from
[0157] phosphorothioate
Figure imgf000055_0004
Figure imgf000056_0001
[0158] thiophosphoramidate linkages, more particularly wherein the modified intemucleotide linkage is a phosphorothioate linkage.
[0159] In some aspects, the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NOs: 202-402. In some aspects, the sense strand of the RNAi agent comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-201. In some aspects, the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NOs: 604-804. In some aspects, the sense strand of the RNAi agent comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NOs: 403-603. In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 805-1005; 1690; 1724; 1760; and 1839. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 403-603; 1689; 1723; 1759; 1799; and 1838. In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 240; 276; 280 and 283. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 642; 678; 682; and 685. In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 441; 477; 481; and 484. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 39; 75; 79 and 82. In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 484; 843; 879; 883; 1690; 1724; and 1759. In some aspects, the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 441; 477; 481; 886; 1689; 1723; and 1760. In some aspects, the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table 1. In some aspects, the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table la. In some aspects, the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table lb. In some aspects, all of nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA. In some aspects, all of nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA, wherein optionally: a. the number of 2’F nucleotides in antisense strand = 2, 4, 6, 9 or 10, more particularly 3, 4, 5 or 6; b. optionally the number of UNA nucleotides in the antisense strand is 1 UNA-U or 1-UNA-A; c. optionally the number of vinylphosphonate nucleotides in the antisense strand is 1 vinylphosphonate nucleotide in the antisense strand, more particularly 1 vinylphosphonate nucleotide at the 5 ’ end of the antisense strand; d. optionally the nucleotides in the antisense strand that are not modified by 2’F or by vinylphosphonate or that are not UNA are modified by 2 ’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or e. optionally the number of 2’0-Me nucleotides is 10, 11, 13, 15, 17 or 19. In some aspects, all of nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA, wherein optionally: a. optionally number of 2’F nucleotides in sense strand is 2, 4, 6, 9, or 10, more particularly 4, 5, 6 or 7; b. optionally the nucleotides in the sense strand that are not modified by 2’F are modified by 2’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or c. optionally the number of 2’0-Me nucleotides is 10, 11, 13, 15 or 17. In some aspects, all of nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA, wherein optionally 1, 2 or 3 phosphorothioate linkages linking the 3’ end or 5’ end terminal nucleotides of the sense strand and the antisense strand, more particularly 1, 2 or 3 phosphorothioate linkages linking (ntl and nt2), and/or (nt2 and nt3), and/or (nt3 and nt4) of the sense strand and antisense strand and/or the antisense strand. In some aspects, the RNAi agent comprises one or more of a sense strand of SEQ ID NO: 1855 and an antisense strand of SEQ ID NO: 1871 (duplex no. 39V9iv); a sense strand of SEQ ID NO: 1701 and an antisense strand of SEQ ID NO: 1717 (duplex no. 75V3B); a sense strand of SEQ ID NO: 1733 and an antisense strand of SEQ ID NO: 1750 (duplex no. 79V3A7); a sense strand of SEQ ID NO: 1700 and an antisense strand of SEQ ID NO: 1716 (duplex no. 75V3A7v); a sense strand of SEQ ID NO: 1697 and an antisense strand of SEQ ID NO: 1713 (duplex no. 75Vla); a sense strand of SEQ ID NO: 1848 and an antisense strand of SEQ ID NO: 1864 (duplex no. 39V3A7v); a sense strand of SEQ ID NO: 1736 and an antisense strand of SEQ ID NO: 1753 (duplex no. 79V3i); a sense strand of SEQ ID NO: 1732 and an antisense strand of SEQ ID NO: 1749 (duplex no. 79V3A); a sense strand of SEQ ID NO: 1698 and an antisense strand of SEQ ID NO: 1714
(duplex no. 75V3A); a sense strand of SEQ ID NO: 1773 and an antisense strand of SEQ ID NO:
1792 (duplex no. 82V3B); a sense strand of SEQ ID NO: 1842 and an antisense strand of SEQ ID NO: 1858 (duplex no. 39V10bu); a sense strand of SEQ ID NO: 1726 and an antisense strand of SEQ ID NO: 1743 (duplex no. 79V10b-3B); a sense strand of SEQ ID NO: 1696 and an antisense strand of SEQ ID NO: 1712 (duplex no. 75V12c); a sense strand of SEQ ID NO: 1706 and an antisense strand of SEQ ID NO: 1722 (duplex no. 75V9iv); a sense strand of SEQ ID NO: 1762 and an antisense strand of SEQ ID NO: 1781 (duplex no. 82V 10b); a sense strand of SEQ ID NO: 1725 and an antisense strand of SEQ ID NO: 1724 (duplex no. 79V10b); a sense strand of SEQ ID NO: 1843 and an antisense sequence of SEQ ID NO: 1859 (duplex no. 39V10bul); a sense strand of SEQ ID NO: 1728 and an antisense strand of SEQ ID NO: 1745 (duplex no. 79V10bul); a sense strand of SEQ ID NO: 1850 and an antisense strand of SEQ ID NO: 1866 (duplex no. 39V3i); a sense strand of SEQ ID NO: 1699 and an antisense strand of SEQ ID NO: 1715 (duplex no. 75V3A7); a sense strand of SEQ ID NO: 1730 and an antisense strand of SEQ ID NO: 1747 (duplex no. 79V12c); a sense strand of SEQ ID NO: 1694 and an antisense strand of SEQ ID NO: 1710 (duplex no. 75V10bul); a sense strand of SEQ ID NO: 1693 and an antisense strand of SEQ ID NO: 1709 (duplex no. 75V10bu); a sense strand of SEQ ID NO: 1734 and an antisense strand of SEQ ID NO: 1751 (duplex no. 79V3A7v); a sense strand of SEQ ID NO: 1691 and an antisense strand of SEQ ID NO: 1707 (duplex no. 75V10b); a sense strand of SEQ ID NO: 1727 and an antisense strand of SEQ ID NO: 1744 (duplex no. 79V10bu); and/or a sense strand of SEQ ID NO: 1767 and an antisense strand of SEQ ID NO: 1786 (duplex no. 82V 12c). In some aspects, the RNAi agent comprises one or more of a sense strand of SEQ ID NO: 1855 and an antisense strand of SEQ ID NO: 1871 (duplex no. 39V9iv); a sense strand of SEQ ID NO: 1701 and an antisense strand of SEQ ID NO: 1717 (duplex no. 75V3B); a sense strand of SEQ ID NO: 1733 and an antisense strand of SEQ ID NO: 1750 (duplex no. 79V3A7); a sense strand of SEQ ID NO: 1700 and an antisense strand of SEQ ID NO: 1716 (duplex no. 75V3A7v); a sense strand of SEQ ID NO: 1697 and an antisense strand of SEQ ID NO: 1713 (duplex no. 75Vla); a sense strand of SEQ ID NO: 1848 and an antisense strand of SEQ ID NO: 1864 (duplex no. 39V3A7v); a sense strand of SEQ ID NO: 1736 and an antisense strand of SEQ ID NO: 1753 (duplex no. 79V3i); a sense strand of SEQ ID NO: 1732 and an antisense strand of SEQ ID NO: 1749 (duplex no. 79V3A); a sense strand of SEQ ID NO: 1698 and an antisense strand of SEQ ID NO: 1714 (duplex no. 75V3A); a sense strand of SEQ ID NO: 1773 and an antisense strand of SEQ ID NO: 1792 (duplex no. 82V3B); a sense strand of SEQ ID NO: 1842 and an antisense strand of SEQ ID NO: 1858 (duplex no. 39V10bu); and/or a sense strand of SEQ ID NO: 1726 and an antisense strand of SEQ ID NO: 1743 (duplex no. 79V10b-3B). In some aspects, the RNAi agent comprises one or more of a sense strand of SEQ ID NO: 1855 and an antisense strand of SEQ ID NO: 1871 (duplex no. 39V9iv); a sense strand of SEQ ID NO: 1701 and an antisense strand of SEQ ID NO: 1717 (duplex no. 75V3B); a sense strand of SEQ ID NO: 1733 and an antisense strand of SEQ ID NO: 1750 (duplex no. 79V3A7); and/or a sense strand of SEQ ID NO: 1700 and an antisense strand of SEQ ID NO: 1716 (duplex no. 75V3A7v). [0160] In some aspects, the sequence of the antisense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7. In some aspects, the sequence of the sense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7. In some aspects, the sequences of the antisense and sense strands of the RNAi agent comprises the sequences of the duplex selected from the duplex of Tables 2, 2a, 5 and 7.
Modified Internucleoside Linkages
[0161] In some embodiments, one or more nucleotides of the RNAi agent are linked by nonstandard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones). In some embodiments, a modified intemucleoside linkage is a non-phosphate-containing covalent intemucleoside linkage. Modified intemucleoside linkages or backbones include, but are not limited to, 5’-phosphorothioate groups (represented herein as a lower case “s”), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3 '-amino phosphoramidate, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified intemucleoside linkage or backbone lacks a phosphoms atom. Modified intemucleoside linkages lacking a phosphoms atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, 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 other backbones having mixed N, O, S, and CEE components. [0162] In some embodiments, a sense strand of a PD-L1 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of a PD-L1 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of a PD-L1 RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of a PD-L1 RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages. In some embodiments, a PD-L1 RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5' end of the sense strand. In some embodiments, a PD-L1 RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages. In some embodiments, the four phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the sense strand and between the nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24- 26 from the 5' end. In some embodiments, a PD-L1 RNAi agent contains at least two phosphorothioate intemucleoside linkages in the sense strand and three or four.
[0163] In some embodiments, a PD-L1 RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2'- modified nucleoside is combined with modified intemucleoside linkage.
Chemical Modifications
[0164] RNAi agents of the present application can also be chemically modified to enhance stability. The nucleic acids of the application can be synthesized and/or modified by methods well established in the art. Chemical modifications can include, but are not limited to 2’ modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages, inverted deoxythymidines. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and preferably two, chemical linkages. Chemical linking can be achieved by any of a variety of well- known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Preferably, the chemical groups that can be used to modify the RNAi agents include, without limitation, methylene blue; bifunctional groups, preferably bis-(2- chloroethyl)amine; -acetyl-N’-(p- glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one preferred embodiment, the linker is a hexa-ethylene glycol linker. In this case, the RNAi agents are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams DJ and Hall KB, Biochem. (1996) 35: 14665- 14670). In a particular embodiment, the 5 ’-end of the antisense strand and the 3 ’-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the RNAi agent comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the RNAi agent is preferably formed by triple-helix bonds. Additional Agents
[0165] In addition to the RNAi component, a combination therapy of the application may further comprise additional or other agent(s) that is(are) active against a disease to be treated, such as, for example, HBV.
[0166] For instance, examples of anti-HBV agents suitable for use with the application include, but are not limited to small molecules, antibodies, and/or CAR-T therapies which bind HBV env (S-CAR cells), capsid assembly modulators, TLR agonists e.g., TLR7 and/or TLR8 agonists), cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir), and/or immune checkpoint inhibitors, etc. The at least one anti-HBV agent can e.g., be chosen from among HBV DNA polymerase inhibitors; immunomodulators; toll-like receptor 7 modulators; toll-like receptor 8 modulators; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; modulators of IL- 10; HBsAg inhibitors; toll -like receptor 9 modulators; cyclophilin inhibitors; HBV prophylactic vaccines; HBV therapeutic vaccines; HBV viral entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; endonuclease modulators; inhibitors of ribonucleotide reductase; hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines, such as IL12; capsid assembly modulators, nucleoprotein inhibitors (HBV core or capsid protein inhibitors); nucleic acid polymers (NAPs); stimulators of retinoic acid-inducible gene 1; stimulators of N0D2; recombinant thymosin alpha- 1; hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; agonists of co-stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28, etc.; BTK inhibitors; other drugs for treating HBV; IDO inhibitors; arginase inhibitors; and KDM5 inhibitors. Such anti-HBV agents can be administered with the compositions and immunogenic combinations of the application simultaneously or sequentially.
[0167] Further examples of anti-HBV agents suitable for use with the application include more specifically (but are not limited to) adjuvants of the immune response, more specifically adjuvants that are potentially safe, well tolerated and effective in humans. An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, and IL-7- hyFc. Adjuvants can also e.g., be chosen from among the following anti-HBV agents: HBV DNA polymerase inhibitors; immunomodulators; toll-like receptor 7 modulators; toll-like receptor 8 modulators; toll-like receptor 3 modulators; interferon alpha receptor ligands; hyaluronidase inhibitors; modulators of IL- 10; HBsAg inhibitors; toll-like receptor 9 modulators; cyclophilin inhibitors; HBV prophylactic vaccines; HBV therapeutic vaccines; HBV viral entry inhibitors; antisense oligonucleotides targeting viral mRNA, more particularly anti-HBV antisense oligonucleotides; short interfering RNAs (siRNA), more particularly anti-HBV siRNA; endonuclease modulators; inhibitors of ribonucleotide reductase; hepatitis B virus E antigen inhibitors; HBV antibodies targeting the surface antigens of the hepatitis B virus; HBV antibodies; CCR2 chemokine antagonists; thymosin agonists; cytokines, such as IL12; capsid assembly modulators, nucleoprotein inhibitors (HBV core or capsid protein inhibitors); nucleic acid polymers (NAPs); stimulators of retinoic acid-inducible gene 1; stimulators of N0D2; recombinant thymosin alpha- 1; hepatitis B virus replication inhibitors; PI3K inhibitors; cccDNA inhibitors; immune checkpoint inhibitors, such as PD-L1 inhibitors, PD-1 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, Lag3 inhibitors, and CTLA-4 inhibitors; agonists of co-stimulatory receptors that are expressed on immune cells (more particularly T cells), such as CD27, CD28, etc.; BTK inhibitors; other drugs for treating HBV; IDO inhibitors; arginase inhibitors; and KDM5 inhibitors.
[0168] The other additional agent may be administered to a subject in need thereof during any time period of the treatment. For example, in one embodiment, the additional agent is administered to a subject in need thereof during a first treatment period. In another embodiment, the additional agent is administered to a subject in need thereof during a second treatment period. [0169] The combinations described herein can be used in any methods or kits described below. Hepatitis B Virus (HBV)
[0170] As used herein “hepatitis B virus” or “HBV” refers to a virus of the hepadnaviridae family. HBV is a small (e.g., 3.2 kb) hepatotropic DNA virus that encodes four open reading frames and seven proteins. The seven proteins encoded by HBV include small (S), medium (M), and large (L) surface antigen (HBsAg) or envelope (Env) proteins, pre-Core protein, core protein, viral polymerase (Pol), and HBx protein. HBV expresses three surface antigens, or envelope proteins, L, M, and S, with S being the smallest and L being the largest. The extra domains in the M and L proteins are named Pre-S2 and Pre-S 1, respectively. Core protein is the subunit of the viral nucleocapsid. Pol is needed for synthesis of viral DNA (reverse transcriptase, RNaseH, and primer), which takes place in nucleocapsids localized to the cytoplasm of infected hepatocytes. PreCore is the core protein with an N-terminal signal peptide and is proteolytically processed at its N and C termini before secretion form infected cells, as the so-called hepatitis B e-antigen (HBeAg). HBx protein is required for efficient transcription of covalently closed circular DNA (cccDNA). HBx is not a viral structural protein. All viral proteins of HBV have their own mRNA except for core and polymerase, which share an mRNA. With the exception of the protein preCore, none of the HBV viral proteins are subject to post-translational proteolytic processing. [0171] The HBV virion contains a viral envelope, nucleocapsid, and single copy of the partially double-stranded DNA genome. The nucleocapsid comprises 120 dimers of core protein and is covered by a capsid membrane embedded with the S, M, and L viral envelope or surface antigen proteins. After entry into the cell, the virus is uncoated and the capsid-containing relaxed circular DNA (rcDNA) with covalently bound viral polymerase migrates to the nucleus. During that process, phosphorylation of the Core protein induces structural changes, exposing a nuclear localization signal enabling interaction of the capsid with so-called importins. These importins mediate binding of the core protein to nuclear pore complexes upon which the capsid disassembles and polymerase/rcDNA complex is released into the nucleus. Within the nucleus the rcDNA becomes deproteinized (removal of polymerase) and is converted by host DNA repair machinery to a covalently closed circular DNA (cccDNA) genome from which overlapping transcripts encode for HBeAg, HBsAg, Core protein, viral polymerase and HBx protein. Core protein, viral polymerase, and pre-genomic RNA (pgRNA) associate in the cytoplasm and selfassemble into immature pgRNA-containing capsid particles, which further convert into mature rcDNA-capsids and function as a common intermediate that is either enveloped and secreted as infections virus particles or transported back to the nucleus to replenish and maintain a stable cccDNA pool. [0172] To date, HBV is divided into four serotypes (adr, adw, ayr, ayw) based on antigenic epitopes present on the envelope proteins, and into eight genotypes (A, B, C, D, E, F, G, and H) based on the sequence of the viral genome. The HBV genotypes are distributed over different geographic regions. For example, the most prevalent genotypes in Asia are genotypes B and C. Genotype D is dominant in Africa, the Middle East, and India, whereas genotype A is widespread in Northern Europe, sub-Saharan Africa, and West Africa.
HBV Antigens
[0173] As used herein, the terms “HBV antigen,” “antigenic polypeptide of HBV,” “HBV antigenic polypeptide,” “HBV antigenic protein,” “HBV immunogenic polypeptide,” and “HBV immunogen” all refer to a polypeptide capable of inducing an immune response against an HBV in a subject. The induced response can be a humoral and/or cellular mediated response. The HBV antigen can be a polypeptide of HBV, a fragment or epitope thereof, or a combination of multiple HBV polypeptides, portions or derivatives thereof. An HBV antigen is capable of raising in a host a protective immune response, e.g, inducing an immune response against a viral disease or infection, and/or producing an immunity (i.e., vaccinates) a subject against a viral disease or infection, that protects the subject against the viral disease or infection. For example, an HBV antigen can comprise a polypeptide or immunogenic fragment(s) thereof from any HBV protein, such as HBeAg, pre-core protein, HBsAg (S, M, or L proteins), core protein, viral polymerase, or HBx protein derived from any HBV genotype, e.g., genotype A, B, C, D, E, F, G, and/or H, or combination thereof.
(1) HBV Core Antigen
[0174] As used herein, each of the terms “HBV core antigen,” “HBeAg,” and “core antigen” refers to an HBV antigen capable of inducing an immune response against an HBV core protein in a subject. The induced immune response can be a humoral and/or cellular mediated response. Each of the terms “core,” “core polypeptide,” and “core protein” refers to the HBV viral core protein. Full-length core antigen is typically 183 amino acids in length and includes an assembly domain (amino acids 1 to 149) and a nucleic acid binding domain (amino acids 150 to 183). The 34-residue nucleic acid binding domain is required for pre-genomic RNA encapsidation. This domain also functions as a nuclear import signal. It comprises 17 arginine residues and is highly basic, consistent with its function. HBV core protein is dimeric in solution, with the dimers selfassembling into icosahedral capsids. Each dimer of core protein has four a-helix bundles flanked by an a-helix domain on either side. Truncated HBV core proteins lacking the nucleic acid binding domain are also capable of forming capsids. (2) HBV Polymerase Antigen
[0175] As used herein, the term “HBV polymerase antigen,” “HBV Pol antigen” or “HBV pol antigen” refers to an HBV antigen capable of inducing an immune response against an HBV polymerase in a subject. The immune response can be a humoral and/or cellular mediated response. Each of the terms “polymerase,” “polymerase polypeptide,” “Pol” and “pol” refers to the HBV viral DNA polymerase. The HBV viral DNA polymerase has four domains, including, from the N terminus to the C terminus, a terminal protein (TP) domain, which acts as a primer for minus-strand DNA synthesis; a spacer that is nonessential for the polymerase functions; a reverse transcriptase (RT) domain for transcription; and an RNase H domain.
(3) HBV Surface Antigens
[0176] As used herein, each of the terms “HBV surface antigen,” “surface antigen,” “HBV envelope antigen,” “envelope antigen,” and “env antigen” refers to an HBV antigen capable of inducing or eliciting an immune response against one or more HBV surface antigens or envelope proteins in a subject. The immune response can be a humoral and/or cellular mediated response. Each of the terms “HBV surface protein,” “surface protein,” “HBV envelope protein” and “envelope protein” refers to HBV viral surface or envelope proteins. HBV expresses three surface antigens, or envelope proteins. Gene S is the gene of the HBV genome that encodes the surface antigens. The surface antigen gene is one long open reading frame but contains three in frame “start” (ATG) codons that divide the gene into three sections, pre-Sl, pre-S2, and S. Because of the multiple start codons, polypeptides of three different sizes called large (L) or L- surface antigen, middle (M) or M-surface antigen, and small (S) or S-surface antigen are produced, also named the HBV L, M and S envelope proteins. Two different promoters (PreSl and PreS2) drive transcription of the L, M, and S-surface antigen coding sequences resulting in three different translated proteins, the L, M and S envelope proteins. The PreS2 promoter is sometimes referred to as the PreS2/S promoter since it is driving M-surface antigen and S-surface antigen transcription separately. The amino acid sequence of the L-surface antigen is in-frame with the M and S-surface antigen sequences. Thus, the L-surface antigen contains the M- and S- surface antigen domains and the M-surface antigen includes the S-surface antigen domain.. The L-, M- and S-surface antigen are co-C-terminal and share the entire S domain. Relative to S, M has an additional domain, pre-S2, at its N terminus, and relative to M, L has a pre-Sl domain.
[0177] In some embodiments, an HBV antigen is an HBV PreSl antigen, which is encoded by a pre-Sl gene section and contains only the Pre-Sl domain of the L antigen. The PreSl antigen can have various lengths, such as having 99 to 109 amino acids. An HBV PreSl antigen of the present disclosure can contain the sequence of any naturally occurring Pre SI domain, and variants or derivatives thereof.
[0178] In other embodiments, an HBV antigen is an HBV PreS2.S antigen, which is encoded by the pre-S2 and S gene sections and contains the PreS2 domain and the S domain. The PreS2 domain can be about 55 amino acids long and the S-domain can contain about 226 amino acids. An HBV PreS2.S antigen of the present disclosure can contain the sequences of any of the naturally occurring PreS2 and S domains, and variants or derivatives thereof. In some embodiments, an internal signal peptide of PreS2.S is left intact to facilitate secretion PreS2.S protein products of the HBV M and HBV S antigens. In one embodiment, an HBV PreS2.S antigen is an HBV M surface antigen. In another embodiment, an HBV PreS2.S antigen is an HBV S surface antigen. In yet another embodiment, an HBV PreS2.S antigen encompasses an HBV M surface antigen and an HBV S surface antigen.
Compositions
[0179] The present disclosure also relates to compositions, pharmaceutical compositions, and therapeutic combinations comprising one or more RNAi agents according to the present disclosure. Any of the RNAi agents described herein can be used in the compositions, pharmaceutical compositions, and therapeutic combinations of the present disclosure.
[0180] The present disclosure provides, for example, a pharmaceutical composition comprising any RNAi agent described herein, together with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is non-toxic and should not interfere with the efficacy of the active ingredient. Pharmaceutically acceptable carriers can include one or more excipients such as binders, disintegrants, swelling agents, suspending agents, emulsifying agents, wetting agents, lubricants, flavorants, sweeteners, preservatives, dyes, solubilizers and coatings. The precise nature of the carrier or other material can depend on the route of administration, e.g., intramuscular, intradermal, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut), intranasal or intraperitoneal routes. For liquid injectable preparations, for example, suspensions and solutions, suitable carriers and additives include water, glycols, oils, alcohols, preservatives, coloring agents and the like. For solid oral preparations, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. For nasal sprays/inhalant mixtures, the aqueous solution/suspension can comprise water, glycols, oils, emollients, stabilizers, wetting agents, preservatives, aromatics, flavors, and the like as suitable carriers and additives. In some aspects, the composition comprises a salt of RNAi agent, optionally a sodium salt. In some aspects, the composition comprises LNP or liposome comprising the RNAi agent. In some aspects, the composition comprises an isolated cell comprising the RNAi agent. In some aspects, the composition comprises non-human animal comprising the RNAi agent.
[0181] In some aspects, the present disclosure generally relates to a pharmaceutical composition comprising an effective amount of the RNAi agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof. In some aspects, the pharmaceutical composition is a liquid composition. In some aspects, the liquid composition comprises water, saline, and/or buffer. In some aspects, the pharmaceutical composition is a lyophilized composition.
[0182] Pharmaceutical compositions of the present disclosure can be formulated in any matter suitable for administration to a subject to facilitate administration and improve efficacy, including, but not limited to, oral (enteral) administration and parenteral injections. The parenteral injections include intravenous injection or infusion, subcutaneous injection, intradermal injection, and intramuscular injection. Pharmaceutical compositions of the present disclosure can also be formulated for other routes of administration including transmucosal, ocular, rectal, long acting implantation, sublingual administration, under the tongue, from oral mucosa bypassing the portal circulation, inhalation, or intranasal.
[0183] In a preferred embodiment of the present disclosure, pharmaceutical compositions of the present disclosure are formulated for parental injection, preferably subcutaneous, intradermal injection, or intramuscular injection, more preferably intramuscular injection.
[0184] According to embodiments of the present disclosure, pharmaceutical compositions for administration will typically comprise a buffered solution in a pharmaceutically acceptable carrier, e.g, an aqueous carrier such as buffered saline and the like, e.g., phosphate buffered saline (PBS). The compositions and therapeutic combinations can also contain pharmaceutically acceptable substances as required to approximate physiological conditions such as pH adjusting and buffering agents. For example, a pharmaceutical composition of the present disclosure comprising a ribonucleic acid molecule can contain phosphate buffered saline (PBS) as the pharmaceutically acceptable carrier. The ribonucleic acid molecule can be administered for example at 1-1000 pg/dose.
[0185] In certain embodiments, an adjuvant is included in a pharmaceutical composition of the present disclosure or co-administered with a pharmaceutical composition of the present disclosure. Use of an adjuvant is optional and can further enhance immune responses when the composition is used for vaccination purposes. Adjuvants suitable for co-administration or inclusion in compositions in accordance with the present disclosure should preferably be ones that are potentially safe, well tolerated and effective in humans. An adjuvant can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, and IL-7-hyFc.
[0186] The present disclosure also provides methods of making pharmaceutical compositions and therapeutic combinations of the present disclosure. A method of producing a pharmaceutical composition or therapeutic combination comprises mixing an inhibitory oligonucleotide of the present disclosure with one or more pharmaceutically acceptable carriers. One of ordinary skill in the art will be familiar with conventional techniques used to prepare such compositions.
[0187] Also provided is a pharmaceutical composition comprising the RNAi component for use in combination with the other pharmaceutical compositions for treating an HBV infection or a disease or disorder associated with the HBV infection in a subject in need thereof. In some aspects, a first, second, and/or third pharmaceutical compositions can be formulated together in one pharmaceutical composition. They can also be formulated as separate pharmaceutical compositions that can be packaged together or separately.
RNAi Agent Pharmaceutical Compositions
[0188] In another aspect, described herein are methods for therapeutic and/or prophylactic treatment of diseases/disorders that are associated with PD-L1, such as HBV infection or inhibition of expression of PD-L1 comprising administering a pharmaceutical composition comprising one or more PD-L1 RNAi agents that can be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration can be, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, oral, subdermal e.g., via an implanted device), and intraparenchymal administration. In some embodiments, the pharmaceutical compositions described herein are preferably administered by subcutaneous injection.
[0189] In another aspect, methods described herein comprise one or more PD-L1 RNAi agents, wherein the one or more RNAi agents are prepared as pharmaceutical compositions or formulations. In some embodiments, pharmaceutical compositions include at least one PD-L1 RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism. The pharmaceutical compositions can be used to treat a subject having a disease or disorder that would benefit from reduction in the level of the target mRNA, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering a PD-L1 RNAi agent linked to a targeting ligand as described herein, to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions including a PD-L1 RNAi agent, thereby forming a pharmaceutical formulation suitable for in vivo delivery to a human.
[0190] The pharmaceutical compositions that include a PD-L1 RNAi agent and methods disclosed herein may decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, or subject, including: administering to the subject a therapeutically effective amount of a herein described PD-L1 RNAi agent, thereby inhibiting the expression of a target mRNA in the subject.
[0191] In some embodiments, the described pharmaceutical compositions including a PD-L1 RNAi agent are used for treating or managing clinical presentations associated with a PD-L1 associated disease, such as an HBV infection. In some embodiments, a therapeutically or prophylactically effective amount of one or more of pharmaceutical compositions is administered to a subject in need of such treatment, prevention, or management. In some embodiments, administration of any of the disclosed PD-L1 RNAi agents can be used to decrease the number, severity, and/or frequency of symptoms of a disease in a subject.
[0192] The described pharmaceutical compositions including a PD-L1 RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder that would benefit from reduction or inhibition in expression of PD-L1 mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions including a PD-L1 RNAi agent thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more PD-L 1 RNAi agents, thereby preventing the at least one symptom.
[0193] The route of administration is the path by which a PD-L1 RNAi agent is brought into contact with the body. In general, methods of administering drugs and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The PD-L1 RNAi agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally. In some embodiments, there herein described pharmaceutical compositions via subcutaneous injection. [0194] The pharmaceutical compositions including a PD-L1 RNAi agent described herein can be delivered to a cell, group of cells, tumor, tissue, or subject using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a herein described compositions. For example, delivery can be by local administration, (e.g., direct injection, implantation, or topical administering), systemic administration, or subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection. [0195] Accordingly, in some embodiments, the herein described pharmaceutical compositions may comprise one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions described herein can be formulated for administration to a subject.
[0196] As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical ingredient (API, therapeutic product, e.g., PD-L1 RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients may act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and/or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.
[0197] Excipients include, but are not limited to: absorption enhancers, anti-adherents, antifoaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.
[0198] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0199] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0200] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystalline form, for example, in the form of an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.
[0201] The active compounds can be prepared with carriers that will protect the compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
[0202] The PD-L1 RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
[0203] A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.). It is also envisioned that cells, tissues or isolated organs that express or comprise the herein defined RNAi agents may be used as “pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic, or preventive result.
[0204] In some instances, one or more (e.g., at least two) PD-L1 RNAi agents described herein can be formulated into one single composition or separate individual compositions. In some embodiments, the PD-L1 RNAi agents in separate individual compositions can be formulated with the same or different excipients and carriers. In some embodiments, the PD-L1 RNAi agents in separate individual compositions agents can be administered through same or different administration routes. In some embodiments, the PD-L1 RNAi agents are administered subcutaneously.
[0205] For treatment of disease or for formation of a medicament or composition for treatment of a disease, the pharmaceutical compositions described herein including a PD-L1 RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment including, but not limited to: a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, and/or a vaccine. [0206] The described PD-L1 RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers. The pharmaceutical compositions described herein may be packaged in pre-filled syringes or vials. Kits
[0207] Also provided herein is a kit comprising an effective amount of an RNAi component for use in a treatment of a disease, such as, for example, a viral infection, more particularly chronic viral infection, in a subject in need thereof, wherein the viral infection, more particularly the chronic viral infection, comprises hepatitis B virus (HBV) infection, more particularly chronic HBV infection, wherein the RNAi component as described herein.
[0208] In another aspect, the kit further comprises a package insert including, without limitation, appropriate instructions for preparation and administration of the formulation, side effects of the formulation, and any other relevant information. The instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, optical disc, or directions to internet-based instructions.
[0209] In another aspect, the kit for treating an individual who suffers from or is susceptible to the conditions described herein is provided, comprising a first container comprising a dosage amount of a composition or formulation as disclosed herein, and a package insert for use. The container can be any of those known in the art and appropriate for storage and delivery of intravenous formulation. In certain embodiments, the kit further comprises a second container comprising a pharmaceutically acceptable carrier, diluent, adjuvant, etc. for preparation of the formulation to be administered to the individual.
[0210] In some embodiments, the kit comprises one or more doses of the RNAi agent . In some embodiments, the kit-of-parts or functional association comprises one or more doses of the RNAi component.
[0211] In another aspect, kits may also be provided that contain sufficient dosages of the compositions described herein (including pharmaceutical compositions thereof) to provide effective treatment for an individual for an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles or more. In some embodiments, one cycle of treatment is about 1-24 months, about 1-3 months, about 3-6 months, about 6-9 months, about 9-12 months, about 12-18 months, about 18-21 months or about 21-24 months. In some embodiments, one cycle of treatment is about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 15 months, about 18 months, about 21 months or about 24 months.
[0212] In some embodiments, the kits may also include multiple doses and may be packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies. In certain embodiments the kits may include a dosage amount of at least one composition as disclosed herein.
Methods
[0213] Also provided herein is a method treating a disease, such as a viral infection, more particularly a chronic viral infection, using the RNAi agent, salt thereof, LNP or liposome comprising, isolated cell comprising, non-human animal comprising, or pharmaceutical composition comprising, as described herein. In some aspects, the viral infection comprises an HBV infection. In some aspects, the viral infection comprises an HBV infection and an HDV infection. In some aspects, the viral infection comprises a HIV infection. Also provided herein is a method treating hepatitis B, more particularly of chronic hepatitis B, using the RNAi agent, salt thereof, LNP or liposome comprising, isolated cell comprising, non-human animal comprising, or pharmaceutical composition comprising, as described herein. Also provided herein is a method treating hepatitis D, more particularly of chronic hepatitis D, using the RNAi agent, salt thereof, LNP or liposome comprising, isolated cell comprising, non-human animal comprising, or pharmaceutical composition comprising, as described herein. Also provided herein is a method treating a cancer, for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma, using the RNAi agent, salt thereof, LNP or liposome comprising, isolated cell comprising, non-human animal comprising, or pharmaceutical composition comprising, as described herein.
[0214] In some aspects, the subject to be treated has an HBV infection, more particularly a chronic HBV infection. In some embodiments, the subject has a further viral infection, more particularly a further chronic viral infection. For example, the subject may have an HBV infection (more particularly a chronic HBV infection) and a super-/co-infection with one or more from among hepatitis D virus (HDV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV), more particularly with at least HDV.
[0215] In some embodiments, the RNAi component is formulated in a solid form, such as a tablet or capsule. In some embodiments, the RNAi component is formulated for subcutaneous injection. In some embodiments, the RNAi component is formulated in in a liquid form, such as suspensions, solutions, emulsions, or syrups, or may be lyophilized. [0216] In some aspects, the RNAi agent is capable of inducing a PD-L1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%. For example, the RNAi agent may be capable of inducing a PD-L1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%, at day 6 or 7 in mice C57/bl6 mice infected with AAV8-hPD-Ll firefly luciferase, for example as described in Example 5. In some aspects, the RNAi agent is capable of inducing a PD-L1 knockdown at a KD of at least 60% and at an IC50 of less than 150 nM, more particularly less than lOOnM , for example, in a free uptake assay, such as on primary human hepatocytes (PHH).
[0217] In some aspects, the RNAi agent is for use in combination with one or more agents chosen from among antiviral agents (e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators), immune checkpoints, immunomodulators (more particularly one or more TLR immunomodulators), vaccines (e.g., an anti-HBV therapeutic vaccine), anti-HBV siRNAs, anti- HBV ASOs and NAPs.
[0218] Some embodiments described herein relate to a method of treating a disease, such as a viral infection (e.g., HBV infection) that can include administering to a subject identified as suffering from the viral infection an effective amount of an RNAi agent as described herein, or a pharmaceutical composition that includes an effective amount of an RNAi agent as described herein. Some embodiments described herein relate to using an RNAi agent as described herein in the manufacture of a medicament for treating a disease such as a viral infection (e.g., HBV infection).
Pharmaceutical Composition
[0219] Typically, the administration of pharmaceutical compositions and therapeutic combinations of the present disclosure will have a therapeutic aim to generate an immune response against a disease, such as a PD-L1 associated disease, such as, for example, HBV after HBV infection or development of symptoms characteristic of HBV infection.
[0220] As used herein, “an effective amount” or “a therapeutically effective amount” refer to an amount of an RNAi agent or a composition comprising one or more RNAi agents sufficient to induce a desired immune effect or immune response in a subject in need thereof. A therapeutically effective amount can be an amount sufficient to induce an immune response in a subject in need thereof. A therapeutically effective amount can be an amount sufficient to produce immunity in a subject in need thereof, e.g., provide a therapeutic effect against a disease such as HBV infection. A therapeutically effective amount can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; the particular application, e.g., providing protective immunity or therapeutic immunity; and the particular disease, e.g., viral infection, for which immunity is desired. A therapeutically effective amount can readily be determined by one of ordinary skill in the art in view of the present disclosure. [0221] In some embodiments, a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of an HBV infection or a symptom associated therewith; (ii) reduce the duration of an HBV infection or symptom associated therewith; (iii) prevent the progression of an HBV infection or symptom associated therewith; (iv) cause regression of an HBV infection or symptom associated therewith; (v) prevent the development or onset of an HBV infection, or symptom associated therewith; (vi) prevent the recurrence of an HBV infection or symptom associated therewith; (vii) reduce hospitalization of a subject having an HBV infection; (viii) reduce hospitalization length of a subject having an HBV infection; (ix) increase the survival of a subject with an HBV infection; (x) eliminate an HBV infection in a subject; (xi) inhibit or reduce HBV replication in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy. In some embodiments of the application, a therapeutically effective amount refers to the amount of a composition or therapeutic combination which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a PD-L1 associated infection or disease, or a symptom associated therewith; (ii) reduce the duration of an PD-L1 associated infection or disease, or symptom associated therewith; (iii) prevent the progression of an PD-L1 associated disease, or symptom associated therewith; (iv) cause regression of an PD- L1 associated disease, or symptom associated therewith; (v) prevent the development or onset of an PD-L1 associated disease, or symptom associated therewith; (vi) prevent the recurrence of an PD-L1 associated disease or symptom associated therewith; (vii) reduce hospitalization of a subject having an PD-L1 associated disease; (viii) reduce hospitalization length of a subject having an PD-L1 associated disease; (ix) increase the survival of a subject with an HBV disease; (x) eliminate an PD-L1 associated disease in a subject; (xi) inhibit or reduce PD-L1 associated disease replication in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.
[0222] As general guidance, a therapeutically effective amount when used with reference to a ribonucleic acid molecule can range from about 1 pg of ribonucleic acid molecule to about 1 mg of ribonucleic acid molecule, such as 1 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 p.g, 90 pig, 100 pig, 200 pig, 300 pig, 400 pig, 500 pig, 600 pig, 700 pig, 800 pig, 900 pig, or 1 mg. Preferably, a therapeutically effective amount of a ribonucleic acid molecule is about 10 pg to about 100 pg. A therapeutically effective amount when used with reference to a ribonucleic acid molecule in a pharmaceutical composition can range from a concentration of about 0.001 mg/mL to about 1 mg/mL of a ribonucleic acid molecule total, such as 0.001 mg/mL, 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, or 1 mg/mL. Preferably, a therapeutically effective amount of a ribonucleic acid molecule is less than 1 mg/mL, more preferably less than 0.05 mg/mL. A therapeutically effective amount can be from one ribonucleic acid molecule or from multiple ribonucleic acid molecules. A therapeutically effective amount can be administered in a single composition, or in multiple compositions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compositions (e.g., tablets, capsules or injectables, or any composition adapted to intradermal delivery, e.g., to intradermal delivery using an intradermal delivery patch), wherein the administration of the multiple capsules or injections collectively provides a subject with a therapeutically effective amount. It is also possible to administer a therapeutically effective amount to a subject, and subsequently administer another dose of a therapeutically effective amount to the same subject, in a so-called prime-boost regimen. This general concept of a primeboost regimen is well known to the skilled person in the vaccine field. Further booster administrations can optionally be added to the regimen, as needed.
[0223] A therapeutic combination comprising two RNAi agents can be administered to a subject by mixing both RNAi agents and delivering the mixture to a single anatomic site. Alternatively, two separate immunizations each delivering a single RNAi agent can be performed. In such embodiments, whether both RNAi agents are administered in a single immunization as a mixture of in two separate immunizations, the first RNAi agent and the second RNAi agent can be administered in a ratio of 10: 1 to 1 : 10, by weight, such as 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1: 10, by weight. Preferably, the first and second RNAi agents are administered in a ratio of 1 : 1, by weight.
[0224] Preferably, a subject to be treated according to the methods of the present disclosure is an HBV-infected subject, particularly a subject having chronic HBV infection. Acute HBV infection is characterized by an efficient activation of the innate immune system complemented with a subsequent broad adaptive response (e.g, HBV-specific T-cells, neutralizing antibodies), which usually results in successful suppression of replication or removal of infected hepatocytes. In contrast, such responses are impaired or diminished due to high viral and antigen load, e.g., HBV envelope proteins are produced in abundance and can be released in sub-viral particles in 1,000-fold excess to infectious virus.
[0225] Chronic HBV infection is described in phases characterized by viral load, liver enzyme levels (necroinflammatory activity), HBeAg, or HBsAg load or presence of antibodies to these antigens. cccDNA levels stay relatively constant at approximately 10 to 50 copies per cell, even though viremia can vary considerably. The persistence of the cccDNA species leads to chronicity. More specifically, the phases of chronic HBV infection include: (i) the immune- tolerant phase characterized by high viral load and normal or minimally elevated liver enzymes; (ii) the immune activation HBeAg-positive phase in which lower or declining levels of viral replication with significantly elevated liver enzymes are observed; (iii) the inactive HBsAg carrier phase, which is a low replicative state with low viral loads and normal liver enzyme levels in the serum that may follow HBeAg seroconversion; and (iv) the HBeAg-negative phase in which viral replication occurs periodically (reactivation) with concomitant fluctuations in liver enzyme levels, mutations in the pre-core and/or basal core promoter are common, such that HBeAg is not produced by the infected cell.
[0226] As used herein, “chronic HBV infection” refers to a subject having the detectable presence of HBV for more than 6 months. A subject having a chronic HBV infection can be in any phase of chronic HBV infection. Chronic HBV infection is understood in accordance with its ordinary meaning in the field. Chronic HBV infection can for example be characterized by the persistence of HBsAg for 6 months or more after acute HBV infection. For example, a chronic HBV infection referred to herein follows the definition published by the Centers for Disease Control and Prevention (CDC), according to which a chronic HBV infection can be characterized by laboratory criteria such as: (i) negative for IgM antibodies to hepatitis B core antigen (IgM anti-HBc) and positive for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), or nucleic acid test for hepatitis B virus DNA, or (ii) positive for HBsAg or nucleic acid test for HBV DNA, or positive for HBeAg two times at least 6 months apart. Preferably, a therapeutically effective amount refers to the amount of a composition or therapeutic combination of the present disclosure which is sufficient to treat chronic HBV infection.
[0227] In some embodiments, a subject having chronic HBV infection is undergoing nucleoside analog (NUC) treatment and is NUC-suppressed. As used herein, “NUC-suppressed” refers to a subject having an undetectable viral level of HBV and stable alanine aminotransferase (ALT) levels for at least six months. Examples of nucleoside/nucleotide analog treatment include HBV polymerase inhibitors, such as entacavir and tenofovir. Preferably, a subject having chronic HBV infection does not have advanced hepatic fibrosis or cirrhosis. Such subject would typically have a METAVIR score of less than 3 for fibrosis and a fibroscan result of less than 9 kPa. The METAVIR score is a scoring system that is commonly used to assess the extent of inflammation and fibrosis by histopathological evaluation in a liver biopsy of patients with hepatitis B. The scoring system assigns two standardized numbers: one reflecting the degree of inflammation and one reflecting the degree of fibrosis.
[0228] It is believed that elimination or reduction of chronic HBV may allow early disease interception of severe liver disease, including virus-induced cirrhosis and hepatocellular carcinoma. Thus, the methods of the present disclosure can also be used as therapy to treat HBV- induced diseases. Examples of HBV-induced diseases include, but are not limited to cirrhosis, cancer (e.g., hepatocellular carcinoma), and fibrosis, particularly advanced fibrosis characterized by a METAVIR score of 3 or higher for fibrosis. In such embodiments, a therapeutically effective amount is an amount sufficient to achieve persistent loss of HBsAg within 12 months and significant decrease in clinical disease (e.g., cirrhosis, hepatocellular carcinoma, etc.).
[0229] Methods according to embodiments of the present disclosure further comprises administering to the subject in need thereof another therapeutic or another anti-HBV agent in combination with a pharmaceutical composition of the present disclosure. For example, another anti-HBV agent or therapeutic agent can be a small molecule or antibody including, but not limited to, immune checkpoint inhibitors (e.g., anti-PDl, anti-TIM-3, etc.), toll-like receptor agonists (e.g., TLR7 agonists and/or TLR8 agonists), RIG-1 agonists, IL- 15 superagonists (Aitor Bioscience), mutant IRF3 and IRF7 genetic adjuvants, STING agonists (Aduro), FLT3L genetic adjuvant, IL-12 genetic adjuvant, IL-7-hyFc; CAR-T which bind HBV env (S-CAR cells); capsid assembly modulators; cccDNA inhibitors, HBV polymerase inhibitors (e.g., entecavir and tenofovir). The one or other anti-HBV active agents can be, for example, a small molecule, an antibody or antigen binding fragment thereof, a polypeptide, protein, or nucleic acid.
Methods of Delivery
[0230] Pharmaceutical compositions and therapeutic combinations of the present disclosure can be administered to a subject by any method known in the art in view of the present disclosure, including, but not limited to, parenteral administration (e.g., intramuscular, subcutaneous, intravenous, or intradermal injection), oral administration, transdermal administration, and nasal administration. Preferably, pharmaceutical compositions and therapeutic combinations are administered parenterally (e.g., by intramuscular injection or intradermal injection) or transdermally. In some aspects, the RNAi agent is subcutaneously or intravenously administered.
[0231] In some embodiments of the present disclosure in which a pharmaceutical composition or therapeutic combination comprises one or more ribonucleic acid molecules, administration can be by injection through the skin, e.g., intramuscular or intradermal injection, preferably intramuscular injection. Intramuscular injection can be combined with electroporation, i.e., application of an electric field to facilitate delivery of the ribonucleic acid molecules to cells. As used herein, the term “electroporation” refers to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane. During in vivo electroporation, electrical fields of appropriate magnitude and duration are applied to cells, inducing a transient state of enhanced cell membrane permeability, thus enabling the cellular uptake of molecules unable to cross cell membranes on their own. Creation of such pores by electroporation facilitates passage of biomolecules from one side of a cellular membrane to the other. For instance, in vivo electroporation for the delivery of DNA vaccines has been shown to significantly increase plasmid uptake by host cells, while also leading to mild-to-moderate inflammation at the injection site. As a result, transfection efficiency and immune response are significantly improved (e.g., up to 1,000-fold and 100-fold respectively) with intradermal or intramuscular electroporation, in comparison to conventional injection.
[0232] In a typical embodiment, electroporation is combined with intramuscular injection. However, it is also possible to combine electroporation with other forms of parenteral administration, e.g., intradermal injection, subcutaneous injection, etc.
[0233] Administration of a pharmaceutical composition, therapeutic combination of the present disclosure via electroporation can be accomplished using electroporation devices that can be configured to deliver to a desired tissue of a mammal a pulse of energy effective to cause reversible pores to form in cell membranes. The electroporation device can include an electroporation component and an electrode assembly or handle assembly. The electroporation component can include one or more of the following components of electroporation devices: controller, current waveform generator, impedance tester, waveform logger, input element, status reporting element, communication port, memory component, power source, and power switch. Electroporation can be accomplished using an in vivo electroporation device. Examples of electroporation devices and electroporation methods that can facilitate delivery of compositions and therapeutic combinations of the present disclosure, particularly those comprising RNAi agents, include CELLECTRA® (Inovio Pharmaceuticals, Blue Bell, PA), Eigen electroporator (Inovio Pharmaceuticals, Inc.), Tri-Grid™ delivery system (Ichor Medical Systems, Inc., San Diego, CA 92121), and those described in U.S. Patent No. 7,664,545, U.S. Patent No. 8,209,006, U.S. Patent No. 9,452,285, U.S. Patent No. 5,273,525, U.S. Patent No. 6,110,161, U.S. Patent No. 6,261,281, U.S. Patent No. 6,958,060, and U.S. Patent No. 6,939,862, U.S. Patent No. 7,328,064, U.S. Patent No. 6,041,252, U.S. Patent No. 5,873,849, U.S. Patent No. 6,278,895, U.S. Patent No. 6,319,901, U.S. Patent No. 6,912,417, U.S. Patent No. 8,187,249, U.S. Patent No. 9,364,664, U.S. Patent No. 9,802,035, U.S. Patent No. 6,117,660, and International Patent Application Publication WO2017172838, all of which are herein incorporated by reference in their entireties. Other examples of in vivo electroporation devices are described in International Patent Publication No. W02019/126120, entitled “Method and Apparatus for the Delivery of Hepatitis B Virus (HBV) Vaccines,” filed on December 18, 2018, the present disclosure of which are hereby incorporated by reference in their entireties. Also contemplated by the application for delivery of the compositions and therapeutic combinations of the present disclosure are use of a pulsed electric field, for instance as described in, e.g., U.S. Patent No. 6,697,669, which is herein incorporated by reference in its entirety.
[0234] Methods of delivery are not limited to the above described embodiments, and any means for intracellular delivery can be used. Other methods of intracellular delivery contemplated by the methods of the present disclosure include, but are not limited to, liposome encapsulation, lipoplexes, nanoparticles, etc. For example, a ribonucleic acid molecule of the present disclosure can be formulated in a therapeutic composition that comprises one or more lipid molecules, preferably positively charged lipid molecules. In some embodiments, an RNAi agent of the disclosure can be formulated using one or more liposomes, lipoplexes, and/or lipid nanoparticles. In some embodiments, liposome or lipid nanoparticle formulations described herein can comprise a polycationic composition. In some embodiments, the formulations comprising a polycationic composition can be used for the delivery of the RNAi agent described herein in vivo and/or ex vitro.
Uiposomes and Uipid Nanoparticles
[0235] In certain embodiments of the present disclosure, the method of administration of the RNAi agent is a lipid composition, such as a lipid nanoparticle (UNP) or a liposome. Uipid compositions, preferably lipid nanoparticles or liposomes, that can be used to deliver a therapeutic product (such as one or more nucleic acid molecules of the invention), include, but are not limited to, liposomes or lipid vesicles, wherein an aqueous volume is encapsulated by amphipathic lipid bilayers, or wherein the lipids coat an interior that comprises a therapeutic product; or lipid aggregates or micelles, wherein the lipid-encapsulated therapeutic product is contained within a relatively disordered lipid mixture.
[0236] The lipid composition can provide the therapeutic product (such as one or more RNAi agents, e.g., one or more RNAi agents comprising ribonucleic acid molecules) with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the therapeutic product is fully encapsulated in the lipid particle (e.g., to form an LNP).
[0237] Lipid compositions of this invention can comprise one or more lipids selected from cationic lipids, anionic lipids, zwitterionic lipids, neutral lipids, steroids, polymer conjugated lipids, phospholipids, glycolipids, and any combination of the foregoing. The lipids can be saturated or unsaturated. A mixture can comprise both saturated and unsaturated lipids. The lipid compositions can be substantially free of liposomes or can contain liposomes. The use of at least one unsaturated lipid for preparing liposomes is preferred. If an unsaturated lipid has two tails, both tails can be unsaturated, or it can have one saturated tail and one unsaturated tail. The lipids and nucleic acid molecules can be mixed and configured in any suitable structures.
[0238] In particular embodiments, the lipid compositions comprise a cationic lipid to encapsulate and/or enhance the delivery of an RNAi agent, such as an RNA molecule of the present disclosure, into the target cell. The cationic lipid can be any lipid species that carries a net positive charge at a selected pH, such as physiological pH. Without wishing to be bound by the theory, the cationic lipids, such as ionizable amino lipids, promote self-assembly of the components into macromolecular nanoparticles that encapsulate the nucleic acid. The nucleic acid-containing nanoparticles are efficiently taken up into target cells by endocytosis. Once inside the endosome, the positively-charged lipid nanoparticles interact with the negatively- charged endosome membrane, causing disruption of the compartment and release of the nucleic acid molecules into the cytoplasm, where the nucleic acid molecules can be expressed.
[0239] Several cationic lipids have been described in the literature, many of which are commercially available. For example, suitable cationic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- DiLinoleyloxy-,N,N-dimethylaminopropane (DLinDMA), and 1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA). The pKa of formulated cationic lipids is correlated with the effectiveness of lipid particles for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). The preferred range of pKa is ~5 to ~7.
[0240] In one embodiment, the cationic lipid is a compound of Formula (I):
Figure imgf000083_0001
Formula (I) wherein Ri is a substituted alkyl consisting of 10 to 31 carbons, R2 is a linear alkyl, alkenyl or alkynyl consisting of 2 to 20 carbons, Rs is a linear or branched alkane consisting of 1 to 6 carbons, R4 and Rs are the same or different, each a hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; Li and L2 are the same or different, each a linear alkane of 1 to 20 carbons or a linear alkene of 2 to 20 carbons, and Xi is S or O; or a salt or solvate thereof. Exemplary compounds of formula (I), their synthesis and uses thereof are described in US2018/0169268, all of which are herein incorporated by reference.
[0241] In another embodiment, the cationic lipid is a compound of formula (II):
Figure imgf000083_0002
Formula (II) wherein Ri is a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; Li is linear alkane of 1 to 15 carbons; R2 is a linear alkyl or alkenyl of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons or a branched, noncyclic alkyl or alkenyl of 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 21, or 22 carbons; L2is a linear alkane of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 carbons; X is O or S; Rs is a linear alkane of 1, 2, 3, 4, 5, or 6 carbons; and R4and Rs are the same or different, each a linear or branched, noncyclic alkyl of 1, 2, 3, 4, 5, or 6 carbons; or a pharmaceutically acceptable salt or solvate thereof. Exemplary compounds of formula (II), their synthesis and uses thereof are described in US2018/0170866, all of which are herein incorporated by reference.
[0242] In another embodiment, the cationic lipid is a compound of formula (III), (IV) or (V):
Figure imgf000084_0001
wherein R comprises a biologically active molecule, and Li, L2, and L independently for each occurrence comprise a ligand selected from the group consisting of a carbohydrate, a polypeptide, or a lipophile; a pharmaceutically acceptable salt thereof; or a pharmaceutical composition thereof. Exemplary compounds of formula (III), (IV) and (V), their synthesis and uses thereof are described in US2017/0028074, all of which are herein incorporated by reference. [0243] In another embodiment, the cationic lipid is a compound of formula (VI):
Figure imgf000084_0002
Formula (VI) wherein X is a linear or branched alkylene or alkenylene, monocyclic, bicyclic, or tricyclic arene or heteroarene; Y is a bond, an ethene, or an unsubstituted or substituted aromatic or heteroaromatic ring; Z is S or O; L is a linear or branched alkylene of 1 to 6 carbons; Ra and R4 are independently a linear or branched alkyl of 1 to 6 carbons; Ri and R2 are independently a linear or branched alkyl or alkenyl of 1 to 20 carbons; r is 0 to 6; and m, n, p, and q are independently 1 to 18; wherein when n=q, m=p, and RI=R2, then X and Y differ; wherein when X=Y, n=q, m=p, then Ri and R2 differ; wherein when X=Y, n=q, and RI=R2, then m and p differ; and wherein when X=Y, m=p, and RI=R2, then n and q differ; or a pharmaceutically acceptable salt thereof. Exemplary compounds of formula (VI), their synthesis and uses thereof are described in US2017/0190661, all of which are herein incorporated by reference.
[0244] In another embodiment, the cationic lipid is a compound of formula (VII):
Figure imgf000085_0001
Formula (VII) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — ,
— S(O)y-, — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— ,
— N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— , and the other of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)y-, — S — S‘, — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— , — N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Rais, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, Ci- C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2have, at each occurrence, the following structure, respectively:
Figure imgf000085_0002
a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2 are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 5 to 10; d1 and d2 are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
[0245] In another embodiment, the cationic lipid is a compound of formula (VIII):
Figure imgf000086_0001
Formula (VIII) or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — ,
— S(O)y-, — S— S— , — C(=O)S— , SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— ,
— N(Ra)C(=O)N(Ra)— , — OC(=O)N(Ra)— or — N(Ra)C(=O)O— , and the other of G1 or G2 is, at each occurrence, — O(C=O) — , — (C=O)O — , — C(=O) — , — O — , — S(O)y-,
— S— S— , — C(=O)S— , — SC(=O)— , — N(Ra)C(=O)— , — C(=O)N(Ra)— ,
— N(Ra)C(=O)N(Ra) — , — OC(=O)N(Ra) — or — N(Ra)C(=O)O — or a direct bond; L is, at each occurrence, ~O(C=O) — , wherein ~ represents a covalent bond to X; X is CRa; Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1 ; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Rais, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R1 and R2 have, at each occurrence, the following structure, respectively:
Figure imgf000086_0002
R' is, at each occurrence, independently H or C1-C12 alkyl; a1 and a2 are, at each occurrence, independently an integer from 3 to 12; b1 and b2are, at each occurrence, independently 0 or 1; c1 and c2 are, at each occurrence, independently an integer from 2 to 12; d1 and d2 are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a1, a2, c1, c2, d1 and d2 are selected such that the sum of aGcGd1 is an integer from 18 to 30, and the sum of a2+c2+d2 is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.
[0246] Exemplary compounds of formula (VII) and (VIII), their synthesis and uses thereof are described in US20190022247, all of which are herein incorporated by reference.
[0247] Additional cationic lipids that can be used in compositions of the present disclosure include, but are not limited to, those described in W02019/036030, W02019/036028, W02019/036008, WO2019/036000, US2016/0376224, US2017/0119904, W02018/200943 and WO2018/191657, the relevant contents on the lipids, their synthesis and uses are herein incorporated by reference in their entireties.
[0248] The lipid nanoparticles can be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and polyethylene glycol (PEG) - modified, or pegylated, lipids, i.e. the lipid is modified by covalent atachment of a polyethylene glycol. PEG provides the liposomes with a coat which can confer favorable pharmacokinetic characteristics c.g. it can increase stability and prevent non-specific adsorption of die liposomes. In certain embodiments, the PEG has an average molecular mass of 1 kDa. to 12 kDa. such as 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, 11 or 12 kDa. For example, it was reported that the length of the PEG can affect in vivo expression of encapsulated RNA, and that PEG with a molecular weight below I kDa (e.g. 500 or 750 Da) does not form stable liposomes. See, e.g., US2014/0255472, the relevant content of which is incorporated herein by reference.
[0249] The lipid formulations can include anionic lipids. The anionic lipids can be any lipid species that carries a net negative charge at a selected pH, such as physiological pH. The anionic lipids, when combined with cationic lipids, are used to reduce the overall surface charge of LNPs and liposomes and to introduce pH-dependent disruption of the LNP or liposome bilayer structure, facilitating nucleotide release. Several anionic lipids have been described in the literature, many of which are commercially available. For example, suitable anionic lipids for use in the compositions and methods of the invention include l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, and palmitoyloleyolphosphatidylglycerol (POPG).
[0250] The lipid formulations can also include a lipid bilayer stabilizing component. Bilayer stabilizing components can be used to inhibit aggregation of LNPs, but bilayer stabilizing components are not limited to this function. For example, conjugated lipids such as PEG-lipid conjugates and cationic-polymer-lipid conjugates can be used to inhibit the aggregation of LNPs or liposomes. By controlling the composition and concentration of the bilayer stabilizing component, one can control the rate at which the bilayer stabilizing component exchanges out of the liposome and, in turn, the rate at which the liposome becomes fusogenic. The term “fusogenic” refers to the ability of a liposome or other drug delivery system to fuse with membranes of a cell. For instance, when a polyethyleneglycol-phosphatidylethanolamine conjugate or a polyethyleneglycol-ceramide conjugate is used as the bilayer stabilizing component, the rate at which the liposome becomes fusogenic can be varied, for example, by varying the concentration of the bilayer stabilizing component, by varying the molecular weight of the polyethyleneglycol, or by varying the chain length and degree of saturation of the acyl chain groups on the phosphatidylethanolamine or the ceramide. In addition, other variables including, for example, pH, temperature, ionic strength, etc. can be used to vary and/or control the rate at which the liposome becomes fusogenic. Other methods which can be used to control the rate at which the liposome becomes fusogenic will become apparent to those of skill in the art upon reading this disclosure.
[0251] LNPs and liposomes can be prepared using methods known in the art in view of the present disclosure. For example, the LNPs can be prepared using ethanol injection or dilution, thin film hydration, freeze-thaw, French press or membrane extrusion, diafiltration, sonication, detergent dialysis, ether infusion, and reverse phase evaporation. One useful method of preparing liposomes involves mixing (i) an ethanolic solution of the lipids (ii) an aqueous solution of the nucleic acid and (iii) buffer, followed by mixing, equilibration, dilution and purification. Preferred liposomes of the invention, e.g. liposomes with a preferred diameter, are obtainable by this mixing process. To obtain liposomes with the desired diameter(s), mixing can be performed using a process in which two feed streams of aqueous nucleic acid solution are combined in a single mixing zone with one stream of an ethanolic lipid solution, all at the same flow rate e.g. in a microfluidic channel as described below. Further examples, compositions, and methods to create liposomes are described in US 2014/0255472, which is hereby incorporated by reference in its entirety.
[0252] Some examples of lipids, lipid compositions, and methods to create lipid carriers for delivering active nucleic acid molecules, such as those of this invention, are described in: US2017/0190661, US2006/0008910, US2015/0064242, US2005/0064595, WO/2019/036030, US2019/0022247, WO/2019/036028, WQ/2019/036008, WO/2019/036000, US2016/0376224, US2017/0119904, WO/2018/200943, WO/2018/191657, US2014/0255472, and US2013/0195968, the relevant content of each of which is hereby incorporated by reference in its entirety.
[0253] Liposomes are microscopic vesicles including at least one concentric lipid bilayer. Vesicle-forming lipids are selected to achieve a specified degree of fluidity or rigidity of the final complex. In particular embodiments, liposomes provide a lipid composition that is an outer layer surrounding a porous nanoparticle.
[0254] Liposomes can be neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other type of bipolar lipids including dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or more double C=C bonds. Examples of lipids capable of producing a stable liposome, alone, or in combination with other lipid components are phospholipids, such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebro sides, distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane- 1 -carboxylate (DOPE-mal). Additional non-phosphorous containing lipids that can become incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine -lauryl sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, DDAB, dioctadecyl dimethyl ammonium chloride (DODAC), 1 ,2-dimyristoyl-3-trimethylammonium propane (DMTAP), DOTAP, DOTMA, DC-Choi, phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol, DOPG, and dicetylphosphate. In particular embodiments, lipids used to create liposomes disclosed herein include cholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, the derivatized vesicleforming lipid PEG-DSPE.
[0255] Methods of forming liposomes are described in, for example, US Patent Nos. 4,229,360; 4,224,179; 4,241,046; 4,737,323; 4,078,052; 4,235,871; 4,501,728; and 4,837,028, as well as in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip. 40:89 (1986).
Embodiments
[0256] Embodiment 1. A ribonucleic acid interfering (RNAi) agent (useful for in inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) gene), wherein the RNAi agent comprises an antisense strand comprising a sequence of 15 to 23 nucleotides, wherein the nucleotide sequence of the antisense strand comprises at least 15 contiguous nucleotides, particularly 16 or more, 17 or more, 18 or more, or 19 or more contiguous nucleotides, from a sequence selected from SEQ ID NOs: 604-804 and wherein one or more nucleotides of the antisense strand is a modified nucleotide; and wherein the RNAi agent optionally further comprises a sense strand comprising a nucleotide sequence, which is of the same length as, or of a lower length than, the nucleotide sequence of the antisense strand, and wherein one or more nucleotides of the sense strand optionally is a modified nucleotide.
[0257] Embodiment 2. The RNAi agent of embodiment 1, wherein the nucleotide sequence of the sense strand comprises a sequence of 15 to 21 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a sequence of the same length that is comprised in the sequence of the antisense strand.
[0258] Embodiment 3. The RNAi agent of embodiment 1 or 2, wherein at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sequence of the antisense strand are modified nucleotides.
[0259] Embodiment 4. The RNAi agent of any one of the preceding embodiments, wherein all the nucleotides of the sequence of the antisense strand are modified nucleotides.
[0260] Embodiment 5. The RNAi agent of any one of the preceding embodiments, wherein at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sense strand are modified nucleotides.
[0261] Embodiment 6. The RNAi agent of any one of the preceding embodiments, wherein all the nucleotides of the sequence of the sense strand are modified nucleotides.
[0262] Embodiment 7. The RNAi agent of any one of the preceding embodiments, wherein the antisense strand comprises a sequence selected from SEQ ID NOs: 604-804. [0263] Embodiment 8. The RNAi agent of any one of the preceding embodiments, wherein the sense strand comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603.
[0264] Embodiment 9. The RNAi agent of any one of the preceding embodiments, wherein the sense strand comprises at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603.
[0265] Embodiment 10. The RNAi agent of any one of the preceding embodiments, wherein the sense strand comprises a sequence selected from SEQ ID NOs: 403-603.
[0266] Embodiment 11. The RNAi agent of any one of the preceding embodiments, wherein the number of nucleotides of the sense strand is 19 or 21.
[0267] Embodiment 12. The RNAi agent of any one of the preceding embodiments, wherein the number of nucleotides of the antisense strand is 19, 21 or 23.
[0268] Embodiment 13. The RNAi agent of any one of the preceding claims, wherein: a. the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 21; b. wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 2; c. wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 23; or; d. wherein the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 19.
[0269] Embodiment 14. The RNAi agent of any one of the preceding embodiments, wherein the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 805-1005; 1690; 1724; 1760; and 1839.
[0270] Embodiment 15. The RNAi agent of any one of the preceding embodiments, wherein the sequence of the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 403-603; 1689; 1723; 1759; 1799; and 1838.
[0271] Embodiment 16. The RNAi agent of any one of the preceding embodiments, wherein the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table lb.
[0272] Embodiment 17. The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified nucleoside and/or a modified phosphate and/or a modified intemucleotide linkage.
[0273] Embodiment 18. The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a 2’-deoxy-2’-fluoro-ribose (2’-F), a 2’ O-methyl ribose (2’ O-Me) or the acyclic sugar of an UNA nucleotide, for example, wherein the modified nucleotide is
Figure imgf000092_0001
nucleotide).
[0274] Embodiment 19. The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate.
[0275] Embodiment 20. The RNAi agent of any one of the preceding embodiments, wherein the modified nucleotide comprises a modified intemucleotide linkage selected from phosphorothioate and thiophosphoramidate linkages, more particularly wherein the modified intemucleotide linkage is a phosphorothioate linkage, optionally wherein the phosphorothioate
Figure imgf000092_0002
[0276] Embodiment 21. The RNAi agent of any one of the preceding embodiments, further comprising one or more from invAb and targeting moieties, more particularly liver targeting moieties; wherein the liver targeting moieties are fatty acids, GalNAc, folic acid, cholesterol, tocopherol or palmitate, more particularly GalNAc, more particularly
Figure imgf000093_0001
the
3 ’ end of the sense strand; optionally further comprising one or more invAb, more particularly 1 invAb at the 5’ end of the sense strand and/or 1 invAb at the 3’ end of the sense strand.
[0277] Embodiment 22. The RNAi agent of any one of the preceding embodiments, wherein all nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA.
[0278] Embodiment 23. The RNAi agent of embodiment 22, wherein: a. optionally the number of 2’F nucleotides in antisense strand = 2, 4, 6, 9 or 10, more particularly 3, 4, 5 or 6; b. optionally the number of UNA nucleotides in the antisense strand is 1 UNA-U or 1-UNA-A; c. optionally the number of vinylphosphonate nucleotides in the antisense strand is 1 vinylphosphonate nucleotide in the antisense strand, more particularly 1 vinylphosphonate nucleotide at the 5’ end of the antisense strand; d. optionally the nucleotides in the antisense strand that are not modified by 2’F or by vinylphosphonate or that are not UNA are modified by 2’-0Me, optionally wherein all of said nucleotides are modified by 2’-0Me; and/or e. optionally the number of 2’0-Me nucleotides is 10, 11, 13, 15, 17 or 19.
[0279] Embodiment 24. The RNAi agent of embodiment 22, wherein: a. optionally number of 2’F nucleotides in sense strand is 2, 4, 6, 9, or 10, more particularly 4, 5, 6 or 7; optionally the nucleotides in the sense strand that are not modified by 2’F are modified by 2’-0Me, optionally wherein all of said nucleotides are modified by 2’-0Me; and/or c. optionally the number of 2’0- Me nucleotides is 10, 11, 13, 15 or 17.
[0280] Embodiment 25. The RNAi agent of embodiment 22, wherein optionally 1, 2 or 3 phosphorothioate linkages linking the 3’ end or 5’ end terminal nucleotides of the sense strand and the antisense strand, more particularly 1, 2 or 3 phosphorothioate linkages linking (ntl and nt2), and/or (nt2 and nt3), and/or (nt3 and nt4) of the sense strand and antisense strand and/or the antisense strand.
[0281] Embodiment 26. The RNAi agent of any one of the preceding embodiments, wherein the sequence of the antisense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
[0282] Embodiment 27. The RNAi agent of any one of the preceding embodiments, wherein the sequence of the sense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7.
[0283] Embodiment 28. The RNAi agent of any one of the preceding embodiments, wherein the sequences of the antisense and sense strands of the RNAi agent comprises the sequences of the duplex selected from the duplex of Tables 2, 2a, 5 and 7.
[0284] Embodiment 29. The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is capable of inducing a PD-L1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%, e.g. , at day 6 or 7 in C57/bl6 mice infected with AAV8-hPD-Ll firefly luciferase for example as described in Example 5.
[0285] Embodiment 30. The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is capable of inducing a PD-L1 knockdown at a KD of at least 60% and at an IC50 of less than 150 nM, more particularly less than lOOnM , for example, in a free uptake assay, such as on primary human hepatocytes (PHH).
[0286] Embodiment 31. The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is subcutaneously or intravenously administered.
[0287] Embodiment 32. The RNAi agent of any one of the preceding embodiments, wherein the RNAi agent is for use in combination with one or more agents chosen from among antiviral agents (e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators), immune checkpoints, immunomodulators (more particularly one or more TLR immunomodulators), vaccines (e.g., an anti-HBV therapeutic vaccine), anti-HBV siRNAs, anti-HBV ASOs and NAPs.
[0288] Embodiment 33. A salt of the RNAi agent of any one of embodiments 1-32, more particularly a sodium salt.
[0289] Embodiment 34. A LNP or liposome comprising the RNAi agent of any one of embodiments 1-32. [0290] Embodiment 35. An isolated cell comprising the RNAi agent of any one of embodiments 1-32.
[0291] Embodiment 36. A non-human animal comprising the RNAi agent of any one of embodiments 1-32.
[0292] Embodiment 37. A pharmaceutical composition comprising an effective amount of the RNAi agent of any one of embodiments 1-32 and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.
[0293] Embodiment 38. The pharmaceutical composition of embodiment 37, wherein the pharmaceutical composition is a liquid composition.
[0294] Embodiment 39. The pharmaceutical composition of embodiment 38, wherein the liquid composition comprises water, saline, and/or buffer.
[0295] Embodiment 40. The pharmaceutical composition of embodiment 37, wherein the pharmaceutical composition is a lyophilized composition.
[0296] Embodiment 41. The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiment 37-40, for use in treating a viral infection, more particularly a chronic viral infection.
[0297] Embodiment 42. The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of embodiment 41, wherein the viral infection comprises an HBV infection.
[0298] Embodiment 43. The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of embodiment 41 or embodiment 42, wherein the viral infection comprises an HBV infection and an HDV infection.
[0299] Embodiment 44. The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of any one of embodiments 41-43, wherein the viral infection comprises a HIV infection.
[0300] Embodiment 45. The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiments 37-40, for use in the treatment of hepatitis B, more particularly of chronic hepatitis B. [0301] Embodiment 46. The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiments 37-40, for use in the treatment of hepatitis D, more particularly of chronic hepatitis D.
[0302] Embodiment 47. The RNAi agent of any one of embodiments 1-32, the salt of embodiment 33, the LNP or liposome of embodiment 34, the isolated cell of embodiment 35, the non-human animal of embodiment 36, or the pharmaceutical composition of any one of embodiments 37-40, for use in treating a cancer, for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma.
EXAMPLES
[0303] Materials and Methods
[0304] The parent sequences of 201 siRNAs targeting the huma PD-L1 (CD274) (see Table
1) were designed as 19-nt long sense and antisense strands which provide improved specificity to huma PD-L1, reduced targeting of human SNPs and reduced cross-reactivity with rhesus monkey, cynomolgus monkey, mouse and rat PD-L1. These parent sequences were synthesized at 0.2 pmol scale and were provided with chemical modifications, e.g., as shown in Table 2 below. The single strand identity was +/- 0.05% of calculated mass as measured by LC/MS, and the single strand purity was >85% full length oligonucleotide as measured by HPLC. Duplex purity was >90% as measured by non-denaturing HPLC.
[0305] The sequences of Table 2 were further used as parent sequences for the design of further (variant) sequences, see Table 5 below.
[0306] The PD-L1 (CD274) human siRNAs (e.g., selected through in vitro screening) were conjugated to a GalNAc ligand (more particularly a GalNAc2 ligand; see below) at the 3 ’ end of the sense strand (Table 7).
Figure imgf000097_0001
GalNAc2 structure
[0307] Transfection based screening assay
[0001] DU145 cells were plated in 96-well plate (18,000 cells/well) in assay medium. Cells were transfected with two doses of siRNA (10 or 2nM) or with dose response of siRNA using a reverse transfection method (Lipofectamine 2000, Invitrogen). After 24 hours the cells were lysed, and CD274 (PD-L1) mRNA level was determined using a bDNA assay (Quantigene 2.0) following the manufacturer’s instructions. PD-L1 mRNA levels were normalized to GAPDH and shown as % of control (PBS-treated cells).
[0002] Free uptake assay in primary human hepatocytes, bDNA read-out
[0003] Frozen primary human hepatocytes (PHH) were thawed and plated in 96-well plates (90,000 cells/well). GalNAc -conjugated siRNA duplexes were then added. 24 hours after plating, the cells were stimulated with 500 lU/ml IFN-g. 24 hours after stimulation, cells were lysed, and CD274 (PD-L1) mRNA level was determined using bDNA assay (Quantigene 2.0) by following the manufacturer’s instructions. PD-L1 mRNA levels were normalized to GAPDH and shown as % of control (PBS-treated cells).
[0004] Free uptake assay in primary human hepatocytes, protein knockdown by high content imaging
[0005] Frozen PHH were thawed and plated in 384-well collagen coated cell carrier plate (Perkin Elmer, 25000cells/well), and Ipg/ml IFN-g (Gibco) was added together with the GalNAc conjugated siRNA duplexes. The PHH were then incubated for one day at 37°C and 5% CO2. After 1 day, the medium was refreshed, and new medium was added containing Ipg/ml IFN-g. After the medium was refreshed and new medium added, the PHH were incubated for one day at 37°C and 5% CO2. Following incubation, the cells were fixed with 10% formaldehyde (Polysciences). Prior to staining the cells with the primary antibody, PD-L1 APC (Miltenyi Biotec), cells were washed 3 times with PBS and blocked with Fc blocker (Biolegend). Cells were then incubated overnight at 4°C with the primary antibody. After the overnight incubation, cells were washed with PBS, and counter-staining mix was added, which mix contained Hoechst (ThermoFisher) and CELLMASK™ (Invitrogen). Following counter-staining, cells were incubated for 1 hour at room temperature. Images were then taken in the CV8000 (Wako) and analyzed by Phaedra software. [0006] Free uptake assay in primary human hepatocytes RNA knockdown by RT-QPCR
[0007] Frozen PHH were thawed and plated in collagen-coated 96-well plate (80,000 cells/well) together with GalNAc conjugated siRNA duplex and Ipg/ml IFN-g. After one day, the medium was replaced, the cells were washed, and new medium containing Ipg/ml IFN-g was added. One day following the medium replacement and addition, the cells were washed and then lysed with CELLS-TO-CT™ lysis buffer (Invitrogen) containing DNase for 5 minutes at room temperature. The lysis was subsequently stopped by adding a stopping solution.
[0008] Following cell lysis, cDNA was synthesized. For cDNA synthesis, lysate was mixed with water, PD-L1 pool, and B-actin 876 primer. After denaturing the RNA, a second mix was added containing Buffer (EXPAND™ High Fidelity buffer (1 OX) with MgC12 - 11759 167 001 Roche), MgC12 (11 699 113 001 - Roche), dNTPs (lOpM each, 733-1363 - VWR), Protector RNase inhibitor (03 335 402 001 - Roche), and Expand Reverse Transcriptase (11785834001 - Roche). cDNA synthesis was performed under the following conditions using a Thermocycler (Applied Biosystems): 30min at 42°C, 5min 95 °C, cool down to 4°C.
[0009] qPCR was performed using a LIGHTCYCLER® 480 II (5015278001 - Roche). qPCR detection was based on a duplex qPCR assay with Zen double quenched probes (Integrated DNA technologies) for both PD-L1 and B-actin. The PD-L1 probe contains a FAM label and B-actin a Cy5 label. The reagents used were the following: cDNA was mixed with water, LIGHTCYCLER® 480 Probes master 2X cone. (Roche - Cat. No. 04 887 301 001), each primer, and each probe. cDNA and reaction mix were loaded in a LIGHTCY CLER® 480 Multiwell Plate 96, white (Roche - 4729692001) and sealed with Microseal B film (BioRad - MSB-1001). After briefly centrifuging, the plate was transferred to the qPCR platform (LC480II - Roche). The qPCR conditions used were 10 min at 95°C, followed by 45 cycles of 15 sec at 95°C, 1 min 60°C. After completion of the DNA amplification, the generated signals were analyzed with the second derivative method that calculates Cp values for both PD-L1 and B-actin. All Cp values were exported from the LC480 software and further analyzed. [0010] PD-L1 mRNA levels were normalized to B-actin and presented as percent of control (PBS treated cells).
[0011] Evaluation of knockdown ofhuma PD-Ll in reporter mice
[0308] C57/BL6 mice were intravenously (i.v.) injected in the tail vein with AAV8-hPD-Ll- firefly luciferase (Vector Biolabs). 11 to 14 days after i.v. injection, mice were treated with GalNAc conjugated siRNA duplexes at 10 mg/kg. Both before treatment and after treatment, mice were injected intraperitoneal with 500mg/kg D-luciferin (Perkin Elmer). 10 minutes following administration of the substrate, mice were anesthetized with isoflurane. After anesthetization, mice were brought into the IVIS® to perform life imaging. As a negative control, non-infected mice were used, and as a positive control, mice infected with AAV-hPD-Ll firefly luciferase were used, which were treated with saline or a control GalNac conjugated siRNA. Each signal was normalized before starting treatment, and PD-L1 knockdown was followed over time. Results are shown from day 7 after start treatment in Table 11 below.
[0309] Example 1: PD-L1 siRNAs
[0310] The parent sequences of the 201 huma PD-L1 human siRNA sequences (see Table 1) were tested in dual dose screen as described in Example 2.
Table 1: 201 parent sequences of PD-L1 Human siRNA sequences
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Table 1A: Modified PD-L1 siRNAs
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Table IB: Modified PD-L1 siRNAs
Figure imgf000110_0002
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
[0311] The 201 PD-L1 human siRNAs (see Table 1A) were synthesized at 0.2 imol scale with 2’-OMe and 2’-F chemical modifications as shown in Table 2. The siRNAs were then used for in vitro screening.
Table 2: PD-L1 siRNAs with Chemical Modifications
Figure imgf000116_0002
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Table 2a: Further PD-L1 siRNAs with Chemical Modifications
Figure imgf000133_0002
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
[0312] Example 2: In Vitro Dual Dose Screening of siRNAs
[0313] As described in Example 1, a total of 201 human CD274/PD-L1 siRNA duplexes were synthesized at 0.2 pmol scale with 2’-0Me and 2’-F chemical modifications, as shown in Table 2 above. Following synthesis, a dual-dose in vitro screen was conducted in DU 145 cells. Briefly, as described above, DU145 cells were transfected with lOnM or 0.2nM of the siRNA duplexes. 24 hours following transfection, the cells are lysed, and PD-L1 knockdown was measured using branched DNA assays. The results obtained are summarized in Table 3 below. The data were normalized to GAPDH, and the data obtained are presented as the mean relative mRNA of quadruple wells plus the standard deviation.
Table 3: Dual Dose Screen of 201 siRNAs in DU145 cell line (mean of quadruplicate +/-SD)
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
[0314] Example 3: In Vitro Dose Response Screen of siRNAs
[0315] The PD-L1 siRNA duplexes in Table 2 above were or are tested in the DU-145 cells in a dose response assay which started from 50nM, used a 1/6 dilution, and had 10 different doses. Table 4 below summarizes the results obtained for 48 siRNA duplexes, which results include EC50 and Max mRNA KD. Additional PD-L1 siRNA duplexes in Table 2 are also tested in the DU-145 cells in the dose response assay described above.
Table 4: Dose Response in Transfection-based Assay for 48 siRNAs
Figure imgf000156_0002
Figure imgf000157_0001
[0316] Example 3a: In Vitro Dose Response Screen of siRNAs
[0317] siRNAs of Table lb were (or are) synthesized following different patterns of 2’-F and 2’-0Me and were (or are) tested in DU 145-cells in dose responses as described above. Table 5 below shows examples of such siRNAs (duplex 16V, 30V, 39V, 42V, 75V, 79V, 82V, 143V, 160V and 187V of Table IB, with different patterns of 2’-F and 2’-0Me) and Table 6 below summarizes the EC50 and max mRNA KD results of the siRNAs of Table 5 and of corresponding siRNAs of Table 2.
Table 5: siRNAs
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Table 6: Dose Response in DU145 Cells
Figure imgf000169_0002
Figure imgf000170_0001
[0318] Example 4: Free uptake assay in PHH
[0319] The siRNAs of Tables 2, 2a and 5 were (or are) conjugated to GalNAc and can be further evaluated in PHH assays by free uptake of the GalN Ac-conjugated siRNA duplexes.
[0320] Examples of such GalNAc conjugated siRNAs are provided in Table 7 below. In first instance the siRNA duplexes activity was tested by a branched DNA assay. The results obtained from the dual dose screen are summarized in Table 8, for which the data were normalized to GAPDH and which data are presented as the mean relative mRNA of quadruple wells plus the standard deviation.
[0321] The GalNAc conjugated siRNAs were also evaluated in dose response assays starting from lOpM to 0.5nM in steps of 1 in 3. Table 9 summarizes the results obtained from these assays. The data were normalized to GAPDH, the IC50 values were calculated, and the maximal knockdown values are presented.
[0322] In addition to the RNA knockdown, the protein knockdown also was evaluated on PHH after treatment with GalNAc conjugated siRNAs. PHH were treated with IFN-g and a dose response of the siRNAs as described above. Data were calculated as percent effect compared to PBS controls. EC50 values and max KD were calculated and are shown in Table 10. RT-PCR was also performed on cells from the same donor to evaluate knockdown on mRNA level. A summary of mean KD and EC50 are shown in Table 10.
Table 7: GalNac Conjugated Compounds
Figure imgf000171_0001
Figure imgf000172_0001
Table 8 Free uptake assay PHH dual dose, mRNA Knockdown Evaluated by bDNA assay
Figure imgf000172_0002
Figure imgf000173_0001
Table 9 Free uptake assay PHH dose response, mRNA knockdown evaluated by bDNA assay
Figure imgf000173_0002
Table 10: Free uptake assay PHH dose response, protein knockdown evaluated by confocal microscopy
Figure imgf000173_0003
[0323] Example 5: Evaluation of GalNac-conjugated siRNA duplexes in huma PD-L1 reporter mice
[0324] Compounds from the free uptake assays were or are tested in an in vivo mouse model for knockdown evaluation. C57/bl6 mice were infected with AAV8-hPD-Ll firefly luciferase. After 14 days, infection was established, and the mice were treated with one of the compounds. Knockdown of PD-L1 was followed in time by a non-invasive method. Mice were ip injected with a substrate, and cells that expressed huma PD-L1 were visualized during IVIS imaging. As a control, infected mice were treated with saline or irrelevant siRNA. In these mice luciferase expression in the liver was observed. For mice that were subcutaneously (s.c.) treated with compound, the knockdown was evaluated overtime. Table 12 presents results obtained 6 or 7 days after treatment. Every mouse used had its own control, and all data were normalized before treatment. In this manner percent knockdown was calculated.
Table 12: Percent PD-L1 knockdown at day 7 after start treatment in AAV-hPD-Ll reporter mice
(A) > 70%, (B) 50-70% and (C) <50% of knockdown
Figure imgf000174_0001
Figure imgf000175_0001
[0325] It will be evident to one skilled in the art that the present disclosure is not limited to the foregoing illustrative examples, and that it can be embodied in other specific forms without departing from the essential attributes thereof. It is therefore desired that the examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing examples, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
[0326] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
[0327] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains.
[0328] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. For example, if X is described as selected from the group consisting of bromine, chlorine, and iodine, claims for X being bromine and claims for X being bromine and chlorine are fully described. Other embodiments are within the following claims.

Claims

CLAIMS We claim:
1. A ribonucleic acid interfering (RNAi) agent (useful for in inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) gene), wherein the RNAi agent comprises an antisense strand comprising a sequence of 15 to 23 nucleotides, wherein the nucleotide sequence of the antisense strand comprises at least 15 contiguous nucleotides, particularly 16 or more, 17 or more, 18 or more, or 19 or more contiguous nucleotides, of a sequence selected from SEQ ID NOs: 604-804 and wherein one or more nucleotides of the antisense strand is a modified nucleotide; and wherein the RNAi agent optionally further comprises a sense strand comprising a nucleotide sequence, which is of the same length as, or of a lower length than, the nucleotide sequence of the antisense strand, and wherein one or more nucleotides of the sense strand optionally is a modified nucleotide.
2. The RNAi agent of claim 1, wherein the nucleotide sequence of the sense strand comprises a sequence of 15 to 21 nucleotides, which is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a sequence of the same length that is comprised in the sequence of the antisense strand.
3. The RNAi agent of claim 1 or 2, wherein at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sequence of the antisense strand are modified nucleotides.
4. The RNAi agent of any one of the preceding claims, wherein all the nucleotides of the sequence of the antisense strand are modified nucleotides.
5. The RNAi agent of any one of the preceding claims, wherein at least 80%, more particularly at least 85%, more particularly at least 90%, more particularly at least 95% of the nucleotides of the sense strand are modified nucleotides.
6. The RNAi agent of any one of the preceding claims, wherein all the nucleotides of the sequence of the sense strand are modified nucleotides.
7. The RNAi agent of any one of the preceding claims, wherein the antisense strand comprises a sequence selected from SEQ ID NOs: 604-804. The RNAi agent of any one of the preceding claims, wherein the sense strand comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603. The RNAi agent of any one of the preceding claims, wherein the sense strand comprises at least 16 contiguous nucleotides, or at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides, or at least 19 contiguous nucleotides of a sequence selected from SEQ ID NOs: 403-603. The RNAi agent of any one of the preceding claims, wherein the sense strand comprises a sequence selected from SEQ ID NOs: 403-603. The RNAi agent of any one of the preceding claims, wherein the number of nucleotides of the sense strand is 19 or 21. The RNAi agent of any one of the preceding claims, wherein the number of nucleotides of the antisense strand is 19, 21 or 23. The RNAi agent of any one of the preceding claims, wherein: a. the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 21 ; b. wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 21 ; c. wherein the number of nucleotides of the sense strand is 21 and the number of nucleotides of the antisense strand is 23; or d. wherein the number of nucleotides of the sense strand is 19 and the number of nucleotides of the antisense strand is 19. The RNAi agent of any one of the preceding claims, wherein the sequence of the antisense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 805- 1005; 1690; 1724; 1760; and 1839. The RNAi agent of any one of the preceding claims, wherein the sequence of the sense strand of the RNAi agent comprises a sequence selected from SEQ ID NO: 403-603; 1689; 1723; 1759; 1799; and 1838. The RNAi agent of any one of the preceding claims, wherein the sequences of the sense and antisense strands of the RNAi agent are selected from the sequence duplex of Table lb. The RNAi agent of any one of the preceding claims, wherein the modified nucleotide comprises a modified nucleoside and/or a modified phosphate and/or a modified intemucleotide linkage. The RNAi agent of any one of the preceding claims, wherein the modified nucleotide comprises a modified nucleoside having a modified ribose, optionally wherein the modified sugar is a 2’-deoxy-2’-fluoro-ribose (2’-F), a 2’ O-methyl ribose (2’ O-Me) or the acyclic sugar of an UNA nucleotide, for example, wherein the modified nucleotide is
Figure imgf000179_0001
nucleotide). The RNAi agent of any one of the preceding claims, wherein the modified nucleotide comprises a modified nucleoside having a modified phosphate, optionally wherein the modified phosphate is a stabilized phosphonate mimic, such as vinylphosphonate or a cyclopropyl, more particularly vinylphosphonate. The RNAi agent of any one of the preceding claims, wherein the modified nucleotide comprises a modified intemucleotide linkage selected from phosphorothioate and thiophosphoramidate linkages, more particularly wherein the modified intemucleotide linkage is a phosphorothioate linkage, optionally wherein the phosphorothioate linkage is
Figure imgf000180_0002
The RNAi agent of any one of the preceding claims, further comprising one or more of invAb and targeting moieties, more particularly liver targeting moieties; wherein the liver targeting moieties are fatty acids, GalNAc, folic acid, cholesterol, tocopherol or palmitate, more particularly GalNAc, more particularly
Figure imgf000180_0001
(GalNAc2) at the 3 ’ end of the sense strand; optionally further comprising one or more invAb, more particularly 1 invAb at the 5 ’ end of the sense strand and/or 1 invAb at the 3’ end of the sense strand. The RNAi agent of any one of the preceding claims, wherein all nucleotides of the RNAi agent are chemically modified, more particularly chemically modified by F, OMe, or UNA. The RNAi agent of claim 22, wherein: a. the number of 2’F nucleotides in antisense strand = 2, 4, 6, 9 or 10, more particularly 3, 4, 5 or 6; b. the number of UNA nucleotides in the antisense strand is 1 UNA-U or 1-UNA-A; c. the number of vinylphosphonate nucleotides in the antisense strand is 1 vinylphosphonate nucleotide in the antisense strand, more particularly 1 vinylphosphonate nucleotide at the 5’ end of the antisense strand; d. the nucleotides in the antisense strand that are not modified by 2’F or by vinylphosphonate or that are not UNA are modified by 2’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or e. the number of 2’0-Me nucleotides is 10, 11, 13, 15, 17 or 19. The RNAi agent of claim 22, wherein: a. the number of 2’F nucleotides in sense strand is 2, 4, 6, 9, or 10, more particularly 4, 5, 6 or 7; b. the nucleotides in the sense strand that are not modified by 2’F are modified by 2’-OMe, optionally wherein all of said nucleotides are modified by 2’-OMe; and/or c. the number of 2’0-Me nucleotides is 10, 11, 13, 15 or 17. The RNAi agent of claim 22, wherein 1, 2 or 3 phosphorothioate linkages linking the 3’ end or 5 ’ end terminal nucleotides of the sense strand and the antisense strand, more particularly 1, 2 or 3 phosphorothioate linkages linking (ntl and nt2), and/or (nt2 and nt3), and/or (nt3 and nt4) of the sense strand and antisense strand and/or the antisense strand. The RNAi agent of any one of the preceding claims, wherein the sequence of the antisense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7. The RNAi agent of any one of the preceding claims, wherein the sequence of the sense strand of the RNAi agent comprises a sequence selected from the sequences of antisense strands that are listed in Tables 2, 2a, 5 and 7. The RNAi agent of any one of the preceding claims, wherein the sequences of the antisense and sense strands of the RNAi agent comprises the sequences of the duplex selected from the duplex of Tables 2, 2a, 5 and 7. The RNAi agent of any one of the preceding claims, wherein the RNAi agent is capable of inducing a PD-L1 knockdown of greater than 50%, more particularly of greater than 60%, more particularly of greater than 70%. The RNAi agent of any one of the preceding claims, wherein the RNAi agent is capable of inducing a PD-L1 knockdown at a KD of at least 60% and at an IC50 of less than 150 nM, more particularly less than lOOnM , for example, in a free uptake assay, such as on primary human hepatocytes (PHH). The RNAi agent of any one of the preceding claims, wherein the RNAi agent is subcutaneously or intravenously administered. The RNAi agent of any one of the preceding claims, wherein the RNAi agent is for use in combination with one or more agents chosen from among antiviral agents (e.g., NUC agents for example, tenofovir, entecavir, tenofovir alafenamide, tenofovir disoproxil, or lamivudine; e.g., Capsid Assembly Modulators), immune checkpoints, immunomodulators (more particularly one or more TLR immunomodulators), vaccines (e.g., an anti-HBV therapeutic vaccine), anti-HBV siRNAs, anti-HBV ASOs and NAPs. A salt of the RNAi agent of any one of claims 1-32, more particularly a sodium salt. A LNP or liposome comprising the RNAi agent of any one of claims 1-32. An isolated cell comprising the RNAi agent of any one of claims 1-32. A non-human animal comprising the RNAi agent of any one of claims 1-32. A pharmaceutical composition comprising an effective amount of the RNAi agent of any one of claims 1-32 and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof. The pharmaceutical composition of claim 37, wherein the pharmaceutical composition is a liquid composition. The pharmaceutical composition of claim 38, wherein the liquid composition comprises water, saline, and/or buffer. The pharmaceutical composition of claim 37, wherein the pharmaceutical composition is a lyophilized composition. The RNAi agent of any one of claims 1-32, the salt of claim 33, the LNP or liposome of claim 34, the isolated cell of claim 35, the non-human animal of claim 36, or the pharmaceutical composition of any one of claims 37-40, for use in treating a viral infection, more particularly a chronic viral infection. The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of claim 41, wherein the viral infection comprises an HBV infection. The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of claim 41 or claim 42, wherein the viral infection comprises an HBV infection and an HDV infection. The RNAi agent, the salt, the LNP or liposome, the isolated cell, the non-human animal, or the pharmaceutical composition for use of any one of 41-43, wherein the viral infection comprises a HIV infection. The RNAi agent of any one of claims 1-32, the salt of claim 33, the LNP or liposome of claim 34, the isolated cell of claim 35, the non-human animal of claim 36, or the pharmaceutical composition of any one of claims 37-40, for use in the treatment of hepatitis B, more particularly of chronic hepatitis B. The RNAi agent of any one of claims 1-32, the salt of claim 33, the LNP or liposome of claim 34, the isolated cell of claim 35, the non-human animal of claim 36, or the pharmaceutical composition of any one of claims 37-40, for use in the treatment of hepatitis D, more particularly of chronic hepatitis D. The RNAi agent of any one of claims 1-32, the salt of claim 33, the LNP or liposome of claim 34, the isolated cell of claim 35, the non-human animal of claim 36, or the pharmaceutical composition of any one of claims 37-40, for use in treating a cancer, for example a carcinoma, more particularly a liver cancer, for example a hepatocellular carcinoma.
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