WO2018160887A1

WO2018160887A1 - Pdl1-specific and beta-catenin-specific asymmetric interfering rna compositions, uses or preparation thereof

Info

Publication number: WO2018160887A1
Application number: PCT/US2018/020526
Authority: WO
Inventors: Chiang Jia Li; Youzhi Li; Yuan Gao; Wei Li; Xiangao Sun
Original assignee: Boston Biomedical, Inc.
Priority date: 2017-03-01
Filing date: 2018-03-01
Publication date: 2018-09-07

Abstract

The combination of PDL1-specific and β-catenin-specific asymmetric interfering RNAs (aiRNAs) is shown to silence β-Catenin/PDL1 mRNA and protein in a dose-dependent manner in a wide variety of murine tumor models, including subcutaneous human tumor xenografts, orthotopic human liver and lung tumors and syngeneic mouse colorectal, breast and lung tumors. Redistribution analysis of fluorescence-labeled PDL1-specific and β-catenin-specific aiRNAs demonstrated that the delivery of aiRNAs to xenograft tumors occurs within 5 minutes of aiRNA administration and lasts at least 8 hours. In all rodent tumor models tested, the administration of PDL1-specific and β-catenin-specific aiRNAs results in a synergistic inhibition of tumor growth not only in β-Catenin over-expressing colorectal tumor models, such as SW480 and Apc Min/+ , but also in tumor models expressing normal amounts of β-Catenin. The administration of PDL1-specific and β-catenin-specific asymmetric interfering RNAs (aiRNAs) is well tolerated and no signs of toxicity were observed even after repeated dosing.

Description

PDLl-SPECIFIC AND BETA-CATENIN-SPECIFIC ASYMMETRIC INTERFERING RNA COMPOSITIONS, USES OR PREPARATION THEREOF

BACKGROUND OF THE INVENTION

[0001] Immuno-oncology is a promising new area for cancer therapeutics that targets the specific immune evasion mechanisms that cancer cells use to avoid detection by the host immune system. These evasion mechanisms are the "checkpoints" of the immune system; specific cell-surface molecules that prevent the immune effectors from killing those cells that express them. The PD-1 immune checkpoint pathway is one such example of an immune checkpoint that has emerged as a critical mediator of immunosuppression in the local tumor microenvironment. The inhibitory co- receptor Programmed Death 1 (PD-1; also known as CD279), a member of the extended CD28/CTLA-4 family of T cell regulators, is expressed on immune cells, such as T, B and NK cells, whereas its ligand, the Programmed Cell Death Ligand 1 (PDL1, also known as CD274 or B7-H1) is a cell surface glycoprotein expressed on the surface of tumor cells of solid tumors as well as on human tumor associated antigen presenting cells (APCs), e.g., dendritic cells and macrophages. The interaction of the PDL1 ligand on tumor cells with the PD- 1 receptor on immune cells delivers an inhibitory signal to T lymphocytes that ultimately leads to T cell anergy and immune evasion.

[0002] The development of immune checkpoint inhibitors that prevent the activation of the PDLl/PD-1 immune checkpoint pathway has resulted in unprecedented and prolonged disease control in about 20-30% of cancer patients with melanoma, non-small cell lung cancer, renal cancer, or head/neck cancer (reviewed by Lipson et al., Semin. Oncol. (2015) 42(4):587-600; Zou et al. , Sci. Transl. Med. (2016) vol. 8, issue 328, pp. 328rv4). For example, ipilimumab, first approved in the United States in 2011, targets cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4); while nivolumab and pembrolizumab, both of which were first approved in the United States in 2014, target PD-1.

[0003] Despite this initial success, it remains unclear why 70-80% of cancer patients fail to respond to anti-PD-1 or anti-PDLl antibodies or why most patients with colorectal cancer, pancreatic cancer and other non-responsive tumor types are resistant to immune checkpoint inhibitors. In addition, immune-related adverse, sometimes lethal, events associated with monoclonal antibody therapies have been observed which casts a significant doubt about their safety for long term use (see, for example, Johnson et al. (2016). "Fulminant Myocarditis with Combination Immune Checkpoint Blockade." New England Journal of Medicine 375(18): 1749- 1755). Thus, there remains an unfulfilled need for efficient methods and compositions that achieve gene silencing, especially for two or more genes, as a viable treatment for cancer.

SUMMARY

[0004] The present disclosure addresses such needs by providing a composition that combines a PDL1 -specific and β-catenin-specific asymmetric interfering RNAs (aiRNAs) for inhibiting signaling by the immune checkpoint molecule, PD-1 and Wnt/p-catenin in cells (e.g., tumor cells). In certain embodiments, the aiRNAs are highly effective at silencing PDL1 and β-catenin mRNA and protein expression in vivo for several hours. The ensuing suppression of PDL1 expression in tumor cells prevents the activation of the PD-1 immune checkpoint pathway in T cells which is in large part responsible for the down regulation of tumor cell-specific T cell cytotoxicity and the concurrent breakdown of the immune surveillance for oncogenic cells in cancer patients. Thus, silencing of PDL1 in tumor cells and cancer stem cells restores tumor cell-specific immune responses whereas the simultaneous silencing of β-catenin, a key intermediate in sternness signaling pathways, is predicted to prevent proliferation and metastasis of cancer stem cells. The simultaneous administration of PDL1 -specific and β-catenin-specific aiRNAs exhibits surprisingly effective therapeutic synergy in the treatment of a variety of different cancers.

[0005] In certain embodiments, a composition is disclosed comprising a therapeutically effective amount of a β-catenin-specific asymmetric interfering RNA (aiRNA), and a therapeutically effective amount of a PDL1 -specific asymmetric interfering RNA (aiRNA), wherein the combination of the PDL1 -specific and β-catenin-specific aiRNAs is effective at producing therapeutic synergy in the treatment of cancer.

[0006] In certain embodiments, the β-catenin-specific asymmetric interfering RNA (aiRNA), comprises an antisense strand having 5 '-terminal and 3 '-terminal nucleotides that are 17, 18, 19, 20, or 21 nucleotides apart, and a sense strand comprising a 5 '-terminal nucleotide that is complementary to a nucleotide of the antisense strand other than its 3 '-terminal nucleotide and a 3 '-terminal nucleotide that is complementary to a nucleotide of the antisense strand, wherein the antisense strand is at least 70% complementary with the sense strand, and wherein 12, 13, 14, IS, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand are colinear with the corresponding complementary nucleotides in a target nucleotide sequence selected from SEQ ID NO. 180, 181, 182, 183, 184 or 185.

[0007] In certain embodiments, the PDL1 -specific asymmetric interfering RNA (aiRNA) comprises an antisense strand comprising 5 '-terminal and 3 '-terminal nucleotides that are 17, 18, 19, 20, or 21 nucleotides apart, and a sense strand comprising a 5 '-terminal nucleotide that is complementary to a nucleotide of the antisense strand other than its 3 '-terminal nucleotide and a 3 '-terminal nucleotide that is complementary to a nucleotide of the antisense strand, wherein the antisense strand is at least 70% complementary with the sense strand, and wherein 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand are colinear with the corresponding complementary nucleotides in a target nucleotide sequence selected from SEQ ID NO. 167, 168, 169, 170, 171, 172 or 173.

[0008] In certain embodiments, the sense or antisense strand of each asymmetric interfering RNA comprises at least one modified nucleotide or its analogue.

[0009] In certain embodiments, 2'-OH group of the at least one modified ribonucleotide or its analogue is replaced by H or a 2'-OMe group.

[00010] In certain embodiments, the at least one modified nucleotide or its analogue is a sugar-, backbone-, and/or base-modified ribonucleotide. For example, the backbone-modified ribonucleotide can be a modification in a phosphodiester linkage with another ribonucleotide, for example, the phosphodiester linkage may be modified to comprise a nitrogen or a sulfur heteroatom.

[00011] In certain embodiments, the at least one modified nucleotide or its analogue comprises a phosphothioate group, inosine or a tritylated base.

[00012] In certain embodiments, the at least one modified nucleotide or its analogue can be a sugar-modified ribonucleotide, wherein a 2 -OH group is replaced by H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, and wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, CI, Br, or I.

[00013] In certain embodiments, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) are contiguous and colinear with the corresponding complementary nucleotides in a target nucleotide sequence chosen from SEQ ID NO. 180, 181, 182, 183, 184 or 185.

[00014] In certain embodiments, the 5'-terminal nucleotide of the sense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the 3'-terminal nucleotide of the antisense strand.

[00015] In certain embodiments, the 3 '-terminal nucleotide of the sense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the 5'-terminal nucleotide of the antisense strand.

[00016] In certain embodiments, T the 5'-terminal and 3'-terminal nucleotides of the sense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) are 13 nucleotides apart.

[00017] In certain embodiments, the 5 '-terminal and 3 '-terminal nucleotides of the antisense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) are 19 nucleotides apart.

[00018] In certain embodiments, the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34% or about 35%.

[00019] In certain embodiments, the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 36%, about 37%, about 38?/o, about 39%, about 40%, about 41%, about 42%, about 43% or about 44%. [00020] In certain embodiments, the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57% or about 58%.

[00021] In certain embodiments, the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 32%.

[00022] In certain embodiments, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) are contiguous and colinear with the corresponding complementary nucleotides in a target nucleotide sequence chosen from SEQ ID NO. 167, 168, 169, 170, 171, 172 or 173.

[00023] In certain embodiments, the 5'-terminal nucleotide of the sense strand of the PDL1- specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the 3 '-terminal nucleotide of the antisense strand.

[00024] In certain embodiments, the 3 '-terminal nucleotide of the sense strand of the PDL1 - specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the 5'-terminal nucleotide of the antisense strand.

[00025] In certain embodiments, the 5 '-terminal and 3 '-terminal nucleotides of the sense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) are 13 nucleotides apart.

[00026] In certain embodiments, the 5 '-terminal and 3 '-terminal nucleotides of the antisense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) are 19 nucleotides apart.

[00027] In certain embodiments, the nucleotide sequence of the antisense strand of the PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34% or about 35%.

[00028] In certain embodiments, the nucleotide sequence of the antisense strand of the PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43% or about 44%.

[00029] In certain embodiments, the nucleotide sequence of the antisense strand of the PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57% or about 58%.

[00030] In certain embodiments, the nucleotide sequence of the antisense strand of the PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 30%.

[00031] In certain embodiments, the asymmetric interfering RNAs (aiRNA) comprise a modified ribonucleotide at one or more positions selected from:

• the 5 '-terminal nucleotide of the sense strand;

• the first nucleotide adjacent to the 5 '-terminal nucleotide of the sense strand;

• the 3 '-terminal nucleotide of the sense strand;

• the first nucleotide adjacent to the 3 '-terminal nucleotide of the sense strand;

• the second nucleotide adjacent to the 3 '-terminal nucleotide of the sense strand;

• the 5'-terminal nucleotide of the antisense strand;

• the second nucleotide adj acent to the 5 ' -terminal nucleotide of the antisense strand;

• the third nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand;

• the fourth nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand;

• the 3 '-terminal nucleotide of the antisense strand; or

• the first nucleotide adjacent to the 3 '-terminal nucleotide of the antisense strand. [00032] In certain embodiments, the at least one modified nucleotide or its analogue can be a sugar-modified ribonucleotide, wherein a 2 -OH group is replaced by H, OR, R, halo, SH, SR, NH₂, NHR, NR2, or CN, and wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, CI, Br, or I.

[00033] In certain embodiments, the modified ribonucleotide can be a 2'-methoxy- ribonucleotide.

[00034] In certain embodiments, the nucleotides of the sense and/or antisense strands are not connected to any adjacent nucleotide via phosphorothioate linkages.

[00035] In certain embodiments, the second nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand of the PDL1 -specific and β-catenin-specific aiRNAs is not a 2'- methoxy-ribonucleotide.

[00036] In certain embodiments, the second nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand of the PDL1 -specific and β-catenin-specific aiRNAs is a 2'- flouro-ribonucleotide.

[00037] In certain embodiments, the PDL1 -specific and/or β-catenin-specific aiRNA is bound to a peptide, an antibody, a polymer, a lipid, an oligonucleotide, cholesterol, or an aptamer.

[00038] In certain embodiments, the PDL1 -specific and β-catenin-specific aiRNAs are encapsulated in a nanoparticle. In certain embodiments, the nanoparticle may comprise an aqueous core comprising a magnesium salt, for example, magnesium phosphate.

[00039] In certain embodiments, the aqueous core is encapsulated in a lipid phase. For example, the lipid phase may comprise N41-(2,3-Dioleoyloxy) propyl ]-N,N,N- trimethylammonium chloride (DOTAP), cholesterol and l,2-distearoyl-sn-glycero-3- phosphoethanolamine- N-[amino(polyethylene glycol)-2000 (PEG2000-DSPE). [00040] In certain embodiments, the nanoparticle comprises RNA, magnesium phosphate, DOTAP-cholesterol and PEG2000-DSPE. In certain embodiments, the molar ratio of RNA: magnesium phosphate: DOTAP-cholesterol: PEG2000-DSPE is 1: 5.62: 0.78: 0.42.

[00041] In certain embodiments, the disclosure provides a pharmaceutical composition comprising PDL1 -specific and β-catenin-specific aiRNAs.

[00042] In certain embodiments, the pharmaceutical composition further comprises a lipid or a cholesterol molecule.

[00043] In certain embodiments, the lipid or cholesterol molecule is conjugated to the PDLl-specific and/or β-catenin-specific aiRNA.

[00044] In certain embodiments, the PDLl-specific and β-catenin-specific aiRNAs comprise a plurality of modified nucleotides or their analogues, each modified nucleotide or its analogue comprising: a 2'-0-methyl or a 2'-fluorine group, and/or a phosphothioate or phosphodiester backbone.

[00045] The disclosure also provides a kit for silencing PDL1 and β-catenin gene expression in tumor cells comprising PDLl-specific and β-catenin-specific aiRNAs of the compositions of any one of claims 1-35, a nanoparticle formulation and instructions for their use.

[00046] In certain embodiments, the disclosure provides an expression vector comprising a nucleic acid sequence encoding the PDLl-specific and/or β-catenin-specific aiRNAs. The expression vector can be a viral, a eukaryotic, or a bacterial expression vector.

[00047] In certain embodiments, the disclosure provides an isolated cell comprising the expression vector or a PDLl-specific and/or β-catenin-specific aiRNA. The cell can be a mammalian, avian, insect, yeast or bacterial cell.

[00048] A method for treating cancer in a subject in need thereof is also disclosed comprising administering an effective amount of the PDLl-specific and β-catenin-specific aiRNAs of one of claims 1-35 to the subject, wherein the combination of PDL1 -specific and β-catenin-specific aiRNAs exhibits therapeutic synergy in the treatment of cancer.

[00049] In certain embodiments, the PDL1 -specific and β-catenin-specific aiRNAs can be in a nanoparticle formulation.

[00050] In certain embodiments, the nanoparticle formulation comprises an aqueous core comprising a magnesium salt, e.g. magnesium phosphate.

[00051] In certain embodiments, the aqueous core is encapsulated in a lipid phase.

[00052] In certain embodiments, the lipid phase comprises N41-(2,3-Dioleoyloxy) propyl ]- Ν,Ν,Ν-trimethylammonium chloride (DOTAP), cholesterol and l,2-distearoyl-sn-glycero-3- phosphoethanolamine- N-[amino(polyethylene glycol)-2000 (PEG2000-DSPE).

[00053] In certain embodiments, the nanoparticle formulation comprises RNA, magnesium phosphate, DOTAP-cholesterol and PEG2000-DSPE.

[00054] In certain embodiments, the molar ratio of RNA: magnesium phosphate: DOTAP- cholesterol: PEG2000-DSPE is 1: 5.62: 0.78: 0.42.

[00055] In certain embodiments, the cancer is an AIDS-Related cancer, a breast cancer, a cancer of the digestive/gastrointestinal tract, an endocrine and neuroendocrine cancer, a cancer of the eye, a genitourinary cancer, a germ cell cancer, a gynecologic cancer, a head and neck cancer, a hematologic cancer, a musculoskeletal cancer, a neurologic cancer, a respiratory/thoracic cancer, a skin cancer, a childhood cancer or a cancer of unknown primary.

[00056] In certain embodiments, the cancer is metastatic, recurrent or resistant to chemotherapy and/or radiation.

[00057] In certain embodiments, the PDLl-specific and β-catenin-specific aiRNAs are administered systemically or locally. [00058] In certain embodiments, the PDL1 -specific and β-catenin-specific aiRNAs of the disclosure are effective at silencing PDL1 and β-catenin gene expression in a tumor, inhibiting tumor growth, inducing cancer stem cell death and/or enhancing an immune response against tumor cells.

[00059] In certain embodiments, the cancer comprises cells that express nuclear β-catenin.

[00060] In certain embodiments, the cancer comprises cells that do not express nuclear β-catenin.

[00061] In certain embodiments, the PDL1 -specific and β-catenin-specific aiRNAs of the disclosure can silence PDL1 and β-catenin gene expression for at least 8 hours.

[00062] In certain embodiments, the PDL1 -specific and β-catenin-specific aiRNAs of the disclosure can be delivered to tumors within 5 minutes of administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[00063] Without being limited to any particular theory or analysis and with respect to the exemplified RNA molecules, FIG. 1A shows an alignment of exemplary β-catenin target nucleotide sequences (e.g. SEQ ID NOs. 186-262) and their associated aiRNAs with an exemplary full-length β-catenin mRNA sequence (NM_001098209; SEQ ID NO. 179).

[00064] FIG. IB shows an exemplary full-length β-catenin mRNA (SEQ ID NO. 179) subdivided into 5 overlapping sequences (SEQ ID NOs.: 180-185), each of which can be targeted by a subset of the exemplary β-catenin-specific aiRNA molecules.

[00065] FIG. 1C depicts exemplary β-catenin target nucleotide sequences (e.g. SEQ ID NOs. 186-262) of the β-catenin aiRNA molecules together with their % GC content.

[00066] FIG. ID groups the exemplary β-catenin aiRNAs according to the % GC content (23-35%, 36-44% or 45-57%) of nucleotides in the antisense strand that are colinear with the corresponding complementary nucleotides of the β-catenin mRNA sequence (NM_001098209; SEQ ID NO. 179). [00067] FIG. IE compares exemplary β-catenin aiRNAs with respect to the length of sense and antisense strands, length of 3' and 5' overhangs, nucleotide sequence of the sense and antisense strands, total number of nucleotides in the antisense strand that are complimentary to the β-catenin target sequence (depicted with white letters), their % GC content and the length of the double stranded region.

[00068] FIG. IF ranks exemplary β-catenin-specific aiRNA molecules according to their RNAi activity in vitro at concentrations of 50nM, ΙΟΟρΜ, ΙΟρΜ and 5pM.

[00069] Without being limited to any particular theory or analysis and with respect to the exemplified RNA molecules, FIG. 2 A aligns exemplary PDL1 target nucleotide sequences (SEQ ID Nos.: 93-138) and associated PDL1 aiRNAs 1-46 with an exemplary full-length PDL1 mRNA sequence (Accession No.: ANM)014143; SEQ ID NO. 166).

[00070] FIG. 2B shows the exemplary full-length PDL1 mRNA (SEQ ID NO. 166) subdivided into 7 overlapping sequences (SEQ ID NOs.: 167-173), each of which can be targeted by a subset of the exemplary RNA molecules.

[00071] FIG. 2C depicts exemplary PDL1 target nucleotide sequences (SEQ ID Nos.: 93- 138) and their associated PDL1 aiRNAs 1-46.

[00072] FIG. 2D aligns the sense strands (SEQ ID NOs. 1-46) and antisense strands (SEQ ID NOs. 47-92) with exemplary PDL1 target mRNA sequences (SEQ ID NOs. 140-164) and provides the length of the sense and antisense strands, the number of nucleotides in the antisense strand that are colinear with the corresponding complementary nucleotides of the PDL1 mRNA sequence (shaded in dark grey), their %GC content and the length of the 5' and 3' overhangs of the antisense strand. FIG. 2D also portrays the exemplary PDL1 S'-terminal (SEQ ID NO. 139) and 3' terminal (SEQ ID NO. 165) mRNA sequences.

[00073] FIG. 2E groups the exemplary PDL1 aiRNAs according to the % GC content (23- 35%, 36-4494 or 45-57%) of nucleotides in the antisense strand that are colinear with the corresponding complementary nucleotides of the PDL1 mRNA sequence (NM_014143; SEQ ID NO. 166). [00074] FIG. 2F shows an exemplary embodiments of the RNAi activity of PDL1 -specific aiRNAs #1-41 on PDL1 mRNA levels in the DLD-1 human colorectal carcinoma cell line after stimulation with IFN-γ.

[00075] FIG. 2G shows an exemplary embodiment of the RNAi activity of PDL1 -specific aiRNAs #28 on PDL1 mRNA levels in the HCT-116 human colorectal carcinoma cell line.

[00076] FIG. 2H shows an exemplary embodiment of the RNAi activity of PDL1 -specific aiRNAs #42-46 on PDL1 protein levels in the HCT-116 human colorectal carcinoma cell line.

[00077] FIG. 21 shows an exemplary embodiment of the in vitro depletion of PDLI cell surface PDLI in vitro.

[00078] FIG. 3A provides an exemplary depiction of 2'-OMe and 2'-F modified β-catenin aiRNAs showing the location of nucleotides having a 2'-OMe or 2'-F substitution of their ribose moiety, e.g., 2'-OMe-modified Cat aiRNA #37 (Cat#37M2), Cat#57M2F6, Cat#57M2F6-CHO and Cat#57M2F10.

[00079] FIG. 3B shows an exemplary embodiment of the dose dependent depletion of β- catenin mRNA in the DLD-1 colorectal carcinoma cancer cell line transfected with increasing amounts of Cat#37M2, Cat#57M2, Cat#57M2F6 and Cat#57M2F10. ICso values were 1.6 pM, 1.8 pM. 0.6 pM and 0.5 pM respectively.

[00080] FIG. 3C shows an exemplary embodiment of the RNAi activity of 2'-OMe- modified β-Catenin-specific aiRNA #37 (Cat#37M2 or BCAT) after transfection into different cancer cell lines in vitro.

[00081] FIG. 3D depicts an exemplary embodiment of the durable silencing of β-catenin protein expression for at least 7 days after the transfection of DLD-1 cells with 2'-OMe-modified β-Catenin-specific aiRNA #37 (Cat#37M2; BCAT).

[00082] FIG. 3E shows an exemplary embodiment of induced cell death in DLD-1 cells transfected with lnM of 2'-OMe-modified β-catenin-specific aiRNA #37 (Cat#37M2; BCAT). [00083] FIG. 3F shows an exemplary embodiment of the inhibition of colony formation by DLD-1 human colorectal carcinoma cells and AGS gastric cancer cells after administration of 2'- OMe-modified β-catenin-specific and/or PDL1 -specific aiRNAs.

[00084] FIG. 4A shows an exemplary embodiment of a 2'-OMe-modified PDL1 aiRNA #22.

[00085] FIG. 4B shows an exemplary embodiment of a 2'-OMe-F-modified PDL1 aiRNA #2.

[00086] FIG.4C shows an exemplary embodiment of the dose dependent depletion of PDL1 mRNA in the RKO colorectal carcinoma cancer cell line transfected with increasing amounts of the 2'-OMe-modified PDL1 aiRNA #22 and the 2'-OMe-F-modified PDL1 aiRNA #2. ICso values were measured as 13.6 pM and 12.3 pM respectively.

[00087] FIG. 4D shows an exemplary embodiment of the restoration of <xCD28 antibody induced IL-2 expression in PD-1+ Jurkat cells after co-culture with HEK-293 cells co-transfected with an PDL1/TCR activator expression plasmid and the 2'-OMe-F-modified PDL1 aiRNA #2.

[00088] FIG.5 shows an exemplary embodiment of the effect of various exemplary 2'-OMe ribose modifications on the RNAi activity of β-catenin and PDL1 aiRNAs in vitro. FIG 5 A depicts the nucleotide sequences of the sense and antisense strand of β-catenin aiRNA #210. The red arrow indicates position 2 of the antisense strand, i.e. the second nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand. FIG. 5B provide a schematic of the locations of 2'-OMe ribose substitutions nos. 1-21 together with their activity on β-catenin protein expression as compared to mock or unmodified β-catenin aiRNA #210. R: non-modified nucleotide; M: 2'-OMe modified nucleotide.

[00089] FIG. 6 shows an exemplary embodiment of the 2'-OMe ribose substitutions at the second nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand of β-catenin aiRNAs #37 and #57 (FIG. 6A) and PDL1 aiRNAs # 2 and #22 (FIG. 6B). FIG. 6C shows an exemplary embodiment of the effect of the 2'-OMe ribose substitution at the second nucleotide adjacent to the 5' -terminal nucleotide of the antisense strand on the RNAi activity of the aforementioned β-catenin and PDL1 aiRNAs in vitro at a concentration of InM or ΙΟρΜ. [00090] FIG. 7A shows an exemplary embodiment of a method of generating DP3 nanoparticles comprising β-catenin and/or PDL1 aiRNAs according to the present disclosure.

[00091] FIG. 7B shows an exemplary embodiment of the inhibition of the volume of CT25 or SW40 xenograft tumors in response to the administration of mouse BCAT and PDL aiRNA DP3 NPs, mouse BCAT and PDL aiRNA LNPs, BCAT DP3 NPs or BCAT LNPs.

[00092] FIG. 8A shows an exemplary illustration of the incorporation of the aiRNA antisense strand but not sense strand into the RISC complex.

[00093] FIG. 8B shows an exemplary embodiment of the aiRNA components of the BBI- 801 pharmaceutical formulation comprising the 2'-OMe-modified Cat aiRNA #37 (Cat#37M2 or BCAT) and 2'-OMe-modified PDL1 aiRNA #22 (PDL#22M2 or PDL).

[00094] FIG. 8C and 8D shows an exemplary embodiment of a microarray analysis of BBI801, i.e. the DP3 nanoparticle formulation of BCAT and PDL aiRNAs. FIG. 8C shows an exemplary embodiment of a Volcano plot of sample BBI801 versus mock using standard selection criteria to identify differentially expressed genes established at log2 |fold change|≥ 1 and P-value < 0.001. FIG. 8D shows an exemplary embodiment of the genes whose expression is significantly changed by BBI801 (head map).

[00095] FIG. 8E and 8F shows an exemplary embodiment of the induction of cytokines by formulated RNA oligos in mice. FIG. 8E shows the experimental design where mouse blood were collected at 2, 6 and 24 hours after i.v. administration of either the positive control β-gal siRNA or BBI801 (both human and mouse version). FIG. 8F shows an exemplary embodiment of IFN-a, Π-6, TNF-a and IP- 10 protein levels measured in the blood samples by ELISA.

[00096] FIG. 9 shows an exemplary embodiment of the distribution of Alex Flour 555 labeled β-catenin aiRNA (red, aip-Cat) in subcutaneous SW480 tumors, 0 min, 5 min, 1 hour and 8 hours after a single intravenous dose of 3 mg/kg of the fluorescein labelled BCAT. β-CATENIN protein, COLLAGEN IV protein and DAPI were stained to outline tumor cells, vasculature and cell nuclei, respectively. Scale bar: 20μπι.

[00097] FIG. 10 shows an exemplary embodiment of β-catenin levels in S W480 xenografts probed via FISH for mRNA (FIG. 10A, red,) or for protein via immunofluorescence (IF; FIG. 10B, green) at 24, 48, 72, 96, 120 and 144 hours after one intravenous dose of 3 mg/kg BCAT. Counterstained with DAPI (blue). Scale bar: ΙΟμιη (A), 20μιη (B).

[00098] FIG. 11 shows an exemplary embodiment of the tumor volume/weight of subcutaneous (FIG. 11 A- 1 ID) or orthotopic (FIG. 1 IE, 1 IF) human cancer xenografts treated with 3mg/ kg aiP-Catenin (aiP-Cat) or scrambled aiRNA (aiScr) biweekly (FIG. 1 lA-11C) or triweekly (FIG. 1 lD-1 1 F). Data represent mean ± SEM of five tumors.

[00099] FIG. 12 shows an exemplary embodiment of the concurrent targeted knock down of β-Catenin and PDL1 protein levels in Apc**^'* intestinal tumors after administration of BBI-801 (BCAT + PDL) at 3mg/kg. Murine equivalent β-Catenin and PDL1 aiRNA sequences were used (see TABLE 6).

[000100] FIG. 13 shows an exemplary embodiment of BBI-801 's anti-tumor efficacy in murine syngeneic cancer models. Subcutaneous (FIG. 13 A; CRC CT26 and FIG. 13B; EMT6 breast cancer), spontaneous (FIG. 13C; CRC ApcMin/+) or orthotopic (FIG. 13D; lung cancer LL/2) murine syngeneic tumors were treated with individual aiRNA or BBI-801 at 3 mg/kg, biweekly. Murine equivalent β-Catenin and PDL1 aiRNA sequences were used. Data represent mean ± standard error of the mean (SEM) of at least five tumors. *p<0.05, ** p<0.01 , *** p<0.001 as compared to control.

[000101] The features and advantages of the present teachings may be more readily understood by those of ordinary skill in the art upon reading the following detailed description. It is to be appreciated that certain features of the present teachings that are, for clarity reasons, described above and below in the context of separate embodiments, may also be combined to form a single embodiment and that various features of the present teachings that are, for brevity reasons, described in the context of a single embodiment, may also be combined so as to form subcombinations thereof. Embodiments identified herein as exemplary or preferred are intended to be illustrative and not limiting.

[000102] The methods and techniques of the present teachings are generally performed according to conventional methods well known in the art and as described in certain general and more specific references that are cited and discussed throughout the present teachings unless otherwise indicated. See, e.g., M.R. Green and J. Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), Ausubel et al. , Current Protocols, John Wiley & Sons, Inc. (2000-2016), Antibodies: A Laboratory Manual, 2nd edition, edited by Edward A. Greenfield, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2014), and RNA: A Laboratory Manual by Rio et al. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2011), all of which are incorporated herein by reference.

[000103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present teachings, including definitions, will control. Methods and materials are described herein for use in the present teachings; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

[000104] As used herein, β-catenin, also referred to in the art as Catenin Beta 1, Catenin, Cadherin- Associated Protein Beta 1 (88kDa), CTNNB, Catenin Beta-1, Beta-Catenin, Armadillo or MRD19, is a structural component of cadherin-based adherens junctions that are necessary for the creation and maintenance of epithelial cell layers by regulating cell growth and adhesion between cells, β-catenin is also a key nuclear effector of canonical Wnt signaling in the nucleus (reviewed in Valenta et al. EMBO J. 2012 13; 31(12):2714-36). In the absence of Wnt, β-catenin forms a complex with AXIN1, AXIN2, APC, CSNK1A1 and GSK3B that promotes phosphorylation on its N-terminal Ser and Thr residues and the ubiquitination of β-catenin via BTRC which leads to its subsequent degradation by the proteasome. In the presence of Wnt ligand, β-catenin is not ubiquitinated and accumulates in the nucleus, where it acts as a coactivator for transcription factors of the TCF/LEF family, leading to the activation of Wnt responsive genes, β- catenin also blocks apoptosis of malignant kidney and intestinal epithelial cells and promotes their anchorage-independent growth by down-regulating DAPK2. Mutations in β-catenin are a cause of colorectal cancer (CRC), hepatocellular carcinoma, pilomatrixoma (PTR), meduUoblastoma (MDB), and ovarian cancer. Aberrant β-catenin/Wnt signaling often implicated in the survival and proliferation of multiple types of cancer cells. [000105] In certain embodiments, a β-catenin expressed nucleotide sequence (e.g., niRNA) refers to a nucleotide sequence comprising at least 25 nucleotides of Homo sapiens catenin beta 1 (CTNNB1) isoform 1, transcript variant 3 having the nucleotide sequence of Accession No. NM_001904.3 or transcript variant 2 having the nucleotide sequence of Accession No: NM_001098209.1 or transcript variant 3 having the nucleotide sequence of Accession No. NM 001098210.1. Transcript variants 1, 2 and 3 encode the same catenin beta-1 isoform 1. Transcript variant 1 represents the longest transcript. Transcript variants 2 and 3 differ in the 3' UTR compared to variant 1.

[000106] In certain embodiments, a β-catenin expressed nucleotide sequence (e.g., mRNA) refers to a nucleotide sequence comprising at least 25 nucleotides of Homo sapiens catenin beta 1 (CTNNBl) isoform 2, transcript variant 4 having the nucleotide sequence of Accession No. NM_001330729.1. Transcript variant 4 differs in the 5' and 3' UTRs and the 5' coding region and initiates translation at a downstream start codon, compared to variant 1. It encodes isoform 2, which has a shorter N-terminus, compared to isoform 1.

[000107] In certain embodiments, a β-catenin expressed nucleotide sequence (e.g., mRNA) refers to a nucleotide sequence comprising at least 25 nucleotides of Homo sapiens catenin beta 1 (CTNNBl) transcript variant XI having the nucleotide sequence of Accession No. XM_005264886.2, transcript variant X2 having the nucleotide sequence of Accession No. XM_017005738.1, transcript variant X3 having the nucleotide sequence of Accession No. XM_006712983.1, transcript variant X4 having the nucleotide sequence of Accession No. XM_006712984.1 or transcript variant X5 having the nucleotide sequence of Accession No. XM_006712985.1.

[000108] Human β-catenin expressed nucleotide sequences (e.g. mRNAs) are transcribed from the Homo sapiens catenin beta 1 (CTNNBl) gene that is located on chromosome 3 with a nucleotide sequence of NCBI Reference Sequence: NG_013302.2.

[000109] As used herein, PDL1 refers to a ligand of PD-1, also referred to in the art as CD274 molecule, CD274 antigen, B7 homolog, Programmed Cell Death 1 Ligand 1, PDCD1 ligand, PDCD1LG1, PDCD1L1, PDL1, B7H1, PDL1, Programmed Death Ligand 1, B7-H1 or B7-H. The PDL1 gene encodes an immune inhibitory receptor ligand that is expressed by hematopoietic and non-hematopoietic cells, such as T cells and B cells and various types of tumor cells. The encoded protein is a type I transmembrane protein that has immunoglobulin V-like and C-like domains. Interaction of this ligand with its receptor inhibits T-cell activation and cytokine production. During infection or inflammation of normal tissue, this interaction is important for preventing autoimmunity by maintaining homeostasis of the immune response. In tumor microenvironments, this interaction provides an immune escape for tumor cells through cytotoxic T-cell inactivation. Expression of this gene in tumor cells is considered to be prognostic in many types of human malignancies, including colon cancer and renal cell carcinoma. Alternative splicing results in at least 4 transcript variants. Other diseases associated with CD274 include, for example, lymphoepithelioma-like carcinoma and Paget's disease.

[000110] In certain embodiments, a PDL1 expressed nucleotide sequence refers to a nucleotide sequence comprising at least 25 nucleotides of Homo sapiens CD274 molecule (CD274), transcript variant 1 (3,691 bp linear mRNA; NCBI Reference Sequence: NM_014143.3) having the sequence of SEQ ID No.: 166. Transcript variant 1. This variant represents the longest transcript and encodes the longest isoform (a).

[000111] In certain embodiments, a PDL1 expressed nucleotide sequence can refer to a nucleotide sequence comprising at least 25 nucleotides of Homo sapiens CD274 molecule (CD274), transcript variant 2 (3,349 bp linear mRNA; Accession: NM_001267706.1 ). This variant lacks an alternate in-frame exon in the 5' coding region, compared to variant 1 which results in a shorter protein (isoform b), compared to isoform a.

[000112] In certain embodiments, a PDL1 expressed nucleotide sequence can refer to a nucleotide sequence comprising at least 25 nucleotides of Homo sapiens CD274 molecule (CD274), transcript variant 3 (3,518 bp linear transcribed-RNA; Accession: NR_052005.1).

[000113] In certain embodiments, a PDL1 expressed nucleotide sequence can refer to a nucleotide sequence comprising at least 25 nucleotides of Homo sapiens CD274 molecule (CD274), transcript variant 4 (907 bp linear mRNA; Accession: NM_001314029.1 ).

[000114] Human PDL1 expressed nucleotide sequences (e.g. mRNAs) are transcribed from the Homo sapiens CD274 gene that is located on chromosome 9 with a nucleotide sequence of NC_000009.12 (REGION: 5450503.5470567 GPC_000001301). [000115] Unless specifically stated otherwise, references made in the singular may also include the plural. For example, "a" and "an" may refer to either one or one or more.

[000116] The phrase "and/or," as used herein in the present teachings and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a non- limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[000117] When a range of values is listed herein, it is intended to encompass each value and sub-range within that range. For example, "1-5 nucleotides" is intended to encompass 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 1-2 nucleotides, 1-3 nucleotides, 1-4 nucleotides, 1-5 nucleotides, 2-3 nucleotides, 2-4 nucleotides, 2-5 nucleotides, 3-4 nucleotides, 3- 5 nucleotides, or 4-5 nucleotides.

[000118] When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below those numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%, 10%, 5%, or 1%. In some embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of \0%. In certain embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of 5%. In certain embodiments, the term "about" is used to modify a numerical value above and below the stated value by a variance of 1%.

[000119] The term "RNA" as used herein refers to its generally accepted meaning in the art. Generally, the term RNA refers to a molecule comprising at least one ribofuranoside moiety. The term can include double-stranded RNA, single-stranded RNA, and isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non- nucleotide material, such as to the end(s) of the RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the present teachings can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[000120] The term "vector" as used herein refers to its meaning as is generally accepted in the art. The term vector generally refers to any nucleic acid- and/or viral-based expression system or technique used to deliver PDL1 and β-catenin aiRNA molecules to a targeted cell or organism.

[000121] The term "RNA interference" or term "RNAi" refer to the biological process of inhibiting or down regulating gene expression in a cell, as is generally known in the art, and which can be mediated by short interfering nucleic acid molecules or asymmetric interfering nucleic acid molecules of the present teachings. Additionally, the term RNAi is meant to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, transcriptional inhibition, or epigenetics. For example, aiRNA molecules of the present teachings can be used to epigenetically silence genes at either the post- transcriptional level or the pre-transcriptional level. In a non-limiting example, modulation of gene expression by aiRNA molecules of the present teachings can result from aiRNA mediated cleavage of RNA (either coding or non-coding RNA) via RISC, or via translational inhibition, as is known in the art or modulation can result from transcriptional inhibition.

[000122] The term "antisense strand" as used herein refers to what is generally accepted in the art. With reference to exemplary nucleic acid molecules of the present teachings, the term refers to a nucleotide sequence of an aiRNA molecule having at least partial complementarity to an expressed nucleic acid sequence of a target gene. In addition, the antisense region of an aiRNA molecule can optionally comprise a nucleic acid sequence having complementarity to a sense region of the aiRNA molecule. In certain embodiments, the antisense region of the aiRNA molecule is referred to as the antisense strand or guide strand. In certain embodiments, the antisense strand can have 10, 11, 12, 13, 14, IS, 16 or 17 nucleotides that base pair with the sense strand. In certain embodiments, the antisense strand can have 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides that are not contiguous with the corresponding colinear complementary nucleotides in a target nucleotide sequence. In certain embodiments, the antisense strand can have 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides that are not complementary to the corresponding colinear nucleotides in a target nucleotide sequence. In certain embodiments, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides of the antisense strand can be colinear with the corresponding complementary nucleotides in a target nucleotide sequence.

[000123] The terms "short interfering nucleic acid", "siNA", "short interfering RNA", "siRNA", "short interfering nucleic acid molecule", "short interfering oligonucleotide molecule", or "chemically modified short interfering nucleic acid molecule" refer to so-called "canonical" siRNA molecules that are capable of inhibiting or down regulating gene expression or viral replication by mediating RNA interference ("RNAi") or gene silencing in a sequence-specific manner and that includes an antisense strand and a sense strand, and the lengths of the two strands are the same. A canonical siRNAs are double-stranded nucleic acid molecules comprising self- complementary sense and antisense strands, wherein the antisense strand comprises a nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Canonical siRNAs are symmetrical interfering double stranded RNAs having a sense and antisense strands of 21 nucleotides each and two 3' overhangs of 2 nucleotides.

[000124] The terms "asymmetric interfering nucleic acid", "aiRNA", "asymmetric interfering oligonucleotide molecule", or "chemically modified asymmetric interfering nucleic acid molecule" refer to any nucleic acid molecule that has an antisense strand and a sense strand, where the lengths of the two strands can be different. These nucleic acid molecules can inhibit or down-regulate gene expression or viral replication by mediating RNA interference ("RNAi") or gene silencing in a sequence-specific manner. These terms can refer to both individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. The aiRNA can be a double-stranded RNA molecule or RNA duplex comprising at least partially complementary sense and antisense strands, wherein the antisense strand comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The aiRNA can also be a double-stranded nucleic acid molecule comprising complementary sense and antisense strands, wherein the antisense strand comprises a nucleotide sequence that is at least partially complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof that may have a nick (missing one nucleotide) or a gap (missing two or more nucleotides). In certain embodiments, an asymmetric interfering RNA as used here can have terminal overhangs on either end or both ends. In certain embodiments, the two strands of the duplex RNA can be linked through a chemical linker. Exemplary aiRNAs are disclosed in U.S. Patent No. 9,328,345, the content of which is hereby incorporated herein in its entirety.

[000125] All the PDL1 -specific and β-catenin-specific aiRNAs disclosed herein, including those recited in the present claims, belong to the same recognized chemical class of asymmetric interfering RNA molecules (aiRNAs) that all share a nucleic acid backbone as well as a common utility, i.e., modulating (silencing or attenuating) the accumulation of their respective target mRNAs, PDL1 and β-catenin respectively. Thus, while the individual sequences of the PDL1 aiRNAs differ, all of the sequences target the same PDL1 mRNA sequence (see FIG. 3 A) and thus share the common use of modulating or silencing PDL1 gene expression. Similarly, while the individual sequences of the β-catenin aiRNAs differ, all of the sequences target the same β-catenin mRNA sequence (see FIG. 1A) and thus share the common use of modulating or silencing PDL1 gene expression.

[000126] In certain embodiments, the PDL1 and β-catenin aiRNAs of the present disclosure exhibit significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less "off-target" gene silencing when compared to canonical siRNAs targeting the same target sequence.

[000127] As used herein, "off-target" gene silencing refers to unintended gene silencing due to, for example, spurious sequence homology between the antisense (guide) sequence and the unintended target mRNA sequence.

[000128] The term "specific," when used in connection with an aiRNA of the present disclosure, means that the interference or silencing of a target mRNA by such aiRNA is discriminatory. In certain embodiments, there is about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80% off-target interference or silencing. In certain embodiments, there is not greater than about 20%, not greater than about 15%, not greater than about 10%, not greater than about 5%, not greater than about 2%, not greater than about 1%, not greater than about 0.5%, or not greater than about 0.2% of off-target interference or silencing.

[000129] The term "complementarity" or "complementary" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the present teachings, the terms generally refer to the formation or existence of hydrogen bond(s) between one nucleic acid sequence and another nucleic acid sequence by traditional Watson-Crick, forming a base-paired, double-stranded region. In certain embodiments, base pairing i.e. the formation or existence of hydrogen bond(s) between one nucleic acid sequence and another nucleic acid sequence does not include non-traditional base-pairing (e.g. Hoogsteen base pairing) such as between complementary RNA and DNA sequences. In reference to the nucleic molecules of the present teachings, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. "Perfect complementarity" means that all the contiguous residues of a nucleic acid sequence can hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. "Partial complementarity" can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches, non-nucleotide linkers, or non- base paired nucleotides) within the nucleic acid molecule, which can result in bulges, loops, or overhangs between the sense strand or sense region and the antisense strand or antisense region of the nucleic acid molecule or between the antisense strand or antisense region of the nucleic acid molecule and a corresponding target nucleic acid molecule. Such partial complementarity can be represented by a % complementarity that is determined by the number of non-base paired nucleotides the total number of nucleotides. Thus for example, a first RNA sequence can have about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% base pair complementarity with second RNA sequence. Such partial complementarity is permitted to the extent that the nucleic acid molecule (e.g. aiRNA) maintains its function, for example the ability to mediate sequence specific RNAi. "Substantial complementarity" means that the sequences are sufficiently complementary to each other to hybridize under selected reaction conditions. Two substantially complementary strands can be, for example, perfectly complementary or can contain from 1 to many mismatches so long as the hybridization conditions are sufficient to allow, for example, discrimination between a pairing sequence and a non-pairing sequence. Accordingly, substantially complementary sequences can refer to sequences with base-pair complementarity of about 50%- 100% in a double-stranded region. The term "complementarity" in certain embodiments can refer to "perfect complementarity," "partial complementarity," or "substantial complementarity."

[000130] In double-stranded or duplex RNAs, one or more ribonucleotides of one strand stably associates with a complementary ribonucleotide in the other strand. The complementarity between the strands is brought about by the hydrogen bonds or "base pairing" between A and U, and between G and C (see, for example, TABLE 1 A).

[000131] In certain embodiments, an RNA duplex can have RNA strands that are either perfectly complimentary or partially complimentary, depending on the number of mismatched, i.e., non-base paired nucleotides present in the RNA duplex (see, for example, TABLE IB).

TABLE 1A

c C U A u A u G u G G u A G A SEQ ID NO. 174

G G A U A U A C A C C A U C U

TABLE IB

C C U u u A U u u G G U A G A SEQ ID NO. 176

G G A u A U A c A C C A U C U SEQ ID NO. 175

[000132] As used herein, the term "align" refers to the process of comparing the nucleotide sequence of two or more nucleotide sequences to assess their degree of sequence identity. As used herein, a "match" refers to the alignment of two or more nucleotide sequences having 100% sequence identity. Thus, for example, in TABLE 2 below, ROW 1 is aligned and matches ROW 2.

TABLE 2

[000133] Because the identity of a base on one strand of a RNA duplex can be used to infer the identity of the corresponding base on the other strand, the term "align" can also refer to the comparison between the nucleotide sequence of one strand (sense strand) and its complementary sequence in another RNA strand (antisense strand). Thus, for example, in TABLE 2, ROW 2 is aligned with ROW 3 because the complementary sequence of the nucleotide sequence of ROW 3 matches the nucleotide sequence in ROW 2. For example, in certain embodiments, the nucleotide sequence of an aiRNA's antisense strand can be aligned with its perfectly complementary or partially complementary nucleotide sequence in a target nucleotide sequence.

[000134] Generally, the term "contiguous" refers to those nucleotides that are immediately adjacent to each other in a polynucleotide chain. In certain embodiments, the term "contiguous" can refer to those nucleotides that are adjacent to each other in a polynucleotide chain that match the corresponding nucleotides in a second polynucleotide chain. Thus, for example in TABLE 3, the nucleotides from position 1 to 11 of RNA strand 2 are contiguous with the corresponding nucleotides 1 to 11 of RNA strand 1 because nucleotides 1-11 of RNA strand 2 match the nucleotides 1-11 of RNA strand 1 without any intervening mismatches.

[000135] In certain embodiments, the term "contiguous" can also refer to those nucleotides that are adjacent to each other in a first polynucleotide chain that align with perfectly complementary nucleotides in a second polynucleotide chain. Thus, for example in TABLE 3, the nucleotides of RNA strand 1 are contiguous with the nucleotides in RNA strand 3 because each nucleotide of RNA strand 1 aligns with the corresponding complimentary nucleotide in RNA strand 3 without any intervening mismatches.

[000136] Generally, the term "colinear" describes the 1 :1 relationship between the linear order of nucleotides in a first RNA strand and the linear order of nucleotides in a second RNA strand. Thus, for example in TABLE 3, RNA strand 2 is colinear with RNA strand 1 despite the lack of sequence identity at positions 12 and 13 because the linear order of nucleotides 1-11, 14 and 15 of RNA strand 2 matches the corresponding nucleotides of RNA strand 1. In certain embodiments, the term "colinear" also describes the 1:1 relationship between the linear order of nucleotides in a first RNA strand and the linear order of the corresponding complementary nucleotides in a second RNA strand. Thus, for example in TABLE 3, RNA strand 2 is colinear with RNA strand 3 despite the partial complimentary between the two strands because the linear order of nucleotides 1-11, 14 and IS of RNA strand 2 aligns with the corresponding complementary nucleotides at positions 1-11, 14 and 15 of RNA strand 3. In contrast, RNA strand 3 is not colinear with RNA strand 4 because the linear order of nucleotides in RNA strand 3 does not match the linear order of nucleotides in RNA strand 4 as a result of the insertion of an extra nucleotide at position 3. Similarly, the linear order of the nucleotides in RNA strand 2 is not colinear with the linear order of the corresponding complementary nucleotides in RNA strand 4 again because of the extra nucleotide at position 3.

[000137] In certain embodiments, the term "colinear" refers to the 1:1 relationship between the linear order of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more nucleotides in an aiRNA's antisense strand and the linear order of the corresponding complementary nucleotides in a target nucleotide sequence. In certain embodiments, the aiRNA's antisense strand may have either perfect or partial complementarity with the sense strand.

[000138] In certain embodiments, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more nucleotides in the aiRNA's antisense strand are contiguous with the corresponding complementary nucleotides in a target nucleotide sequence. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more nucleotides in the aiRNA's antisense strand are not contiguous with the corresponding complementary nucleotides in a target nucleotide sequence. [000139] With reference to exemplary aiRNA molecules of the present teachings, the term "correspond" or "correspondence" as used herein refers to the relationship between a target nucleotide sequence and a sequence in the antisense strand of an aiRNA molecule of the present teachings. The term can be modified by the word "partially," "substantially," or "completely" to indicate the degree of the relationship. In certain embodiments, a partial correspondence means about 20-99% of the ribonucleotides A, U, G, and C in the antisense strand of aiRNA sequence is complementary to its corresponding target nucleotide sequence. In certain embodiments, a substantial correspondence means about 50%- 10094 of the ribonucleotides A, U, G, and C in the antisense strand of aiRNA sequence are complementary to the corresponding target nucleotide sequence. In certain embodiments, the target nucleotide sequence can comprise PDL1 or β-catenin mRNA sequences. In certain embodiments, the target PDL1 nucleotide sequence can be the nucleotide sequence of SEQ ID NO. 166 (PDL1) or any portion thereof. In certain embodiments, the target β-catenin nucleotide sequence can be the nucleotide sequence of or SEQ ID NO. 179 (β- catenin) or any portion thereof. In certain embodiments, the PDL1 target nucleotide sequence comprises a sequence chosen from SEQ ID NOs. 93-138 and the β-catenin target nucleotide sequence comprises a sequence chosen from SEQ ID NOs.186-262.

[000140] The term "non-base paired" refers to nucleotides that are not base paired between the sense strand or sense region and the antisense strand or antisense region of a double-stranded aiRNA molecule; and can include, but is not limited to, for example, mismatches, overhangs, single stranded loops, etc. In certain embodiments, non- Watson Crick base pairing, e.g. Hoogsteen base pairing, for example, as in RNA-DNA base pairing, will be understood to be non-base paired.

[000141] An exemplary 5' overhang of an aiRNA of the present teachings in which the 3'- terminal nucleotide of the sense strand is complementary to the third nucleotide adjacent to the 5'- terminal nucleotide of the antisense strand is shown below:

[000142] An exemplary 5' overhang of an aiRNA of the present teachings in which the 3'- terminal nucleotide of the sense strand is complementary to the second nucleotide adjacent to the 5'-terminal nucleotide of the antisense strand is shown below:

[000143] An exemplary 5' overhang of an aiRNA of the present teachings in which the 3'- terminal nucleotide of the sense strand is complementary to the first nucleotide adjacent to the 5'- terminal nucleotide of the antisense strand is shown below:

[000144] An exemplary depiction of an aiRNA of the present teachings in which the 5'- terminal nucleotide of the sense strand is complementary to the 3 '-terminal nucleotide of the antisense strand is shown below:

[000145] An exemplary 3' overhang of an aiRNA of the present teachings in which the 5'- terrninal nucleotide of the sense strand is complementary to the first nucleotide adjacent to the 3'- terminal nucleotide of the antisense strand is shown below:

[000146] An exemplary 3' overhang of an aiRNA of the present teachings in which the 5'- terminal nucleotide of the sense strand is complementary to the second nucleotide adjacent to the 3'-terminal nucleotide of the antisense strand is shown below:

[000147] An exemplary 3' overhang of an aiRNA of the present teachings in which the 5'- terminal nucleotide of the sense strand is complementary to the third nucleotide adjacent to the 3'- terminal nucleotide of the antisense strand is shown below:

[000148] The term "overhang" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary double stranded nucleic acid molecules, the term generally refers to the terminal portion of a nucleotide sequence that is not base paired between the two strands of a double-stranded nucleic acid molecule. In certain embodiments, an overhang can be single stranded. In certain embodiments, the nucleic acid molecules of the present teachings include two overhangs at the antisense strand (i.e., 3'- and 5' -overhangs), as exemplified below.

[000149] The term "blunt end" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the present teachings, the term refers to a terminus of a double-stranded aiRN A molecule having no overhanging nucleotides. For example, the two strands of a double-stranded aiRNA molecule having blunt ends align with each other without overhanging nucleotides at the termini. An aiRNA duplex molecule of the present teachings can comprise blunt ends at one or both termini of the duplex, such as the terminus located at the 5 '-end of the antisensc strand, the 5 '-end of the sense strand, or both termini of the duplex. In certain embodiments, a blunt end is formed when the 3'-terminal nucleotide of the sense strand base pairs with the S'-terminal nucleotide of the antisensc strand.

[000150] In certain embodiments, the nucleic acid molecules of the present teachings include one overhang and one blunt end (e.g., a 3'-overhang and a 5'-blunt end; or a 3'-blunt end and a 5'- overhang), as exemplified below.

[000151] In certain embodiments, the 3' overhang is not blunt.

[000152] Exemplary duplex RNAs having either two 3' overhangs or two 5' overhangs are depicted below.

[000153] The term "nucleotide" (or "nt") generally comprises a nucleobase, a sugar, and an internucleoside linkage, e.g., a phosphodiester bond. The base can be a natural base (standard), modified bases, or a base analog. Such bases can be generally located at the 1 '-position of a nucleotide sugar moiety. Additionally, the nucleotides can be unmodified or modified at the sugar, internucleoside linkage, and/or base moiety (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others). In certain embodiments, a nucleotide can be a ribonucleotide (which sometimes refers to as a RNA nucleotide). In certain embodiments, a nucleotide can be a deoxyribonucleotide (which sometimes refers to as a DNA nucleotide). In certain embodiments, the term nucleotide also includes a moiety having a nucleobase and a sugar. For example, when a nucleotide is located at one of the termini of a sense strand or an antisense strand, the nucleotide can include only a nucleobase and a sugar.

[000154] The term "ribonucleotide" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleotide with a hydroxyl group at the 2' position of a β-D-ribofuranose moiety. A ribonucleotide consists of a phosphate group, a ribose sugar group, and a nucleobase that can be either adenine (A), guanine (G), cytosine (C), or uracil (U). An RNA strand refers to a chain of ribonucleotides linked together by phosphodiester bonds between the 5'-phosphate of one nucleotide and the 3' hydroxyl group of the next nucleotide. However, in certain embodiments, the chain of ribonucleotides may comprise bonds other than phosphodiester bonds between the 5'-phosphate of one nucleotide and the 3' hydroxyl group of the next nucleotide.

[000155] The term "deoxyribonucleotide" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to a nucleotide with a proton at the T position of a β-D-deoxyribofuranose moiety. This term also includes any deoxyribonucleotides that are chemically modified. "dT" refers to 2'-deoxythymidine.

[000156] As used herein, a "polynucleotide" refers to a polymeric chain containing two or more nucleotides. "Polynucleotides" includes primers, oligonucleotides, nucleic acid strands, etc. A polynucleotide may contain standard or non-standard nucleotides. A polynucleotide has an end- to-end chemical orientation: the 5' end has a hydroxyl or phosphate group on the 5' carbon of its terminal sugar and the 3' end usually has a hydroxyl group on the 3' carbon of its terminal sugar. This directionality, plus the fact that synthesis proceeds 5' to 3', has given rise to the convention that polynucleotide sequences are written and read in the 5'— >3' direction (from left to right); for example, the sequence AUG is assumed to be (5^110(3·)." (see Molecular Cell Biology 4th edition by Harvey Lodish et al. © 2000, W. H. Freeman and Company).

[000157] Accordingly, as used herein, the nucleotide or base at the 5' end of a polynucleotide may be referred to herein as the "5'-terminal nucleotide" or "5'-terminal base" respectively. The nucleotide or base at the 3' end of a polynucleotide may be referred to herein as the "3 '-terminal nucleotide" or "3 '-terminal base" respectively.

[000158] As used herein, the "5'-terminal nucleotide" shall be understood to encompass a 5'- terrninal nucleoside having a 5'-terminal hydroxyl group or a 5'-terrninal nucleotide having 5'- terminal phosphate.

[000159] The term "modified nucleotide" as used herein refers to its meaning as is generally accepted in the art. The term generally refers a nucleotide, which contains a modification in the chemical structure of the base, sugar and/or phosphate of the unmodified (or natural) nucleotide as is generally known in the art.

[000160] The term "chemical modification" as used herein refers to its meaning as is generally accepted in the art. With reference to the exemplary PDL1 and β-catenin aiRNAs molecules of the present teachings, the term refers to any modification of the chemical structure of the nucleotides that differs from nucleotides of a native nucleic acid in general. The term "chemical modification" encompasses, for example, the addition, substitution, or modification of native RNA at the sugar, base, or internucleotide linkage, as described herein or as is otherwise known in the art. In certain embodiments, the term "chemical modification" can refer to certain forms of RNA that are naturally occurring in certain biological systems, for example 2'-0-methyl or 2'-fluorine modifications or inosine modifications.

[000161] The terms "internucleoside linkage" or "internucleoside linker" or "interaucleotide linkage" or "internucleotide linker" can be used herein interchangeably and refer to any linker or linkage between two nucleoside units, as is known in the art, including, for example, but not limited to, phosphate, analogs of phosphate, phosphonate, guanidine, hydroxylamine, hydroxythydrazinyl, amide, carbamate, alkyl, and substituted alkyl linkages. The term "phosphorothioate" refers to an internucleotide phosphate linkage comprising one or more sulfur atoms in place of an oxygen atom. Hence, the term phosphorothioate refers to both phosphorothioate and phosphorodithioate internucleotide linkages.

[000162] In certain embodiments, the PDL1 and β-catenin aiRNAs can be chemically modified to facilitate cellular uptake. For example, an aiRNA can be bound covalently via a linker or non-covalently to a positively-charged molecule, a peptide, a protein, a carbohydrate, a cholesterol, a lipid to improve cellular uptake. For example, conjugation of aiRNAs with cholesterol or octyl, dodecyl, and octadecyl residues or poly-L-lysine facilitates cellular uptake.

[000163] The terms "composition" or "formulation" as used herein generally refer to a composition or formulation, such as in a pharmaceutically acceptable carrier or diluent, in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including, for example, a human. Suitable forms, in part, depend upon the use or the route of entry, for example, oral, transdermal, inhalation, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged nucleic acid is desirable for delivery). For example, compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.

[000164] As used herein, pharmaceutical formulations include formulations for human and veterinary use. Non-limiting examples of agents suitable for formulation with the nucleic acid molecules of the present teachings include: lipid nanoparticles (see for example Semple et al. , 2010, NatBiotechnol., February; 28 (2): 172-6); P-glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made of polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for the nucleic acid molecules of the present teachings include material described in Boado et al. , 1998, J. Pharm Sci., %1, 1308-1315; Tyler et al. , 1999, FEBSLett., 421, 280-284; Pardridge et al. , 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada et al. , 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al. , 1999, PNAS USA., 96, 7053- 7058.

[000165] A "pharmaceutically acceptable salt" or "salt" of an RNA interfering agent, e.g. aiRNA, is a product of the disclosed RNA interfering agent that contains an ionic bond, and is typically produced by reacting the disclosed RNA interfering agent with either an acid or a base, suitable for administering to a subject. Pharmaceutically acceptable salt can include, but is not limited to, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates, acetates, benzoates, citrates, maleates, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Na, K, Li, alkali earth metal salts such as Mg or Ca, or organic amine salts.

[000166] A "pharmaceutical composition" is a formulation containing the disclosed RNA interfering agent, e.g. aiRNA, in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (e.g., a formulation of the disclosed duplex RNA molecule or salts thereof) in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a RNA interfering agent, e.g. aiRNA, of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active RNA interfering agent, e.g. aiRNA, is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[000167] The present disclosure provides a method of treatment comprising administering an effective amount of the pharmaceutical composition to a subject in need thereof. In some embodiments, the pharmaceutical composition is administered via a route selected from the group consisting of iv, sc, topical, po, and ip. In another embodiment, the effective amount can be SpM per day, 50pM per day or 500pM per day. In other embodiments, the effective amount can be 1 ng to 1 g per day, 100 ng to 1 g per day, or 1 μg to 1 mg per day.

[000168] The present disclosure also provides pharmaceutical formulations comprising a RNA interfering agent, e.g. aiRNA, of the present disclosure in combination with at least one pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA., which is incorporated by reference herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. 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 RNA interfering agent, e.g. aiRNA, use thereof in the compositions is contemplated. Supplementary active RNA interfering agents, e.g. aiRNAs, can also be incorporated into the compositions.

[000169] An RNA interfering agent, e.g. aiRNA, of the present disclosure is administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an efficacious level sufficient to achieve the desired therapeutic effect through inhibition of tumor growth, killing of tumor cells, treatment or prevention of cell proliferative disorders, etc.) of a RNA interfering agent, e.g. aiRNA, of the present disclosure (as an active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the disclosure). These procedures may involve mixing, granulating, compressing, or dissolving the ingredients as appropriate to attain the desired preparation. In another embodiment, a therapeutically effective amount of a RNA interfering agent, e.g. aiRNA, of the present disclosure is administered in a suitable dosage form without standard pharmaceutical carriers or diluents.

[000170] Pharmaceutically acceptable carriers include solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like. Other fillers, excipients, flavorants, and other additives such as are known in the art may also be included in a pharmaceutical composition according to this disclosure.

[000171] The term "solvate" represents an aggregate that comprises one or more molecules of a compound of the present disclosure with one or more molecules of a solvent or solvents. Solvates of the compounds of the present disclosure include, for example, hydrates.

[000172] The pharmaceutical compositions containing active RNA interfering agent, e.g. aiRNA, of the present disclosure may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and/or auxiliaries which facilitate processing of the active duplex RNA molecules into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.

[000173] An RNA interfering agent, e.g. aiRNA, or pharmaceutical composition of the disclosure can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment. For example, for treatment of cancers, a RNA interfering agent, e.g. aiRNA, of the present disclosure may be injected directly into tumors, injected into the blood stream or body cavities, taken orally, or applied through the skin with patches. [000174] In certain embodiments, aiRNA nanoparticles may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of nanoparticle appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. For any nanoparticle, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of nanoparticles can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., EDS0 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LDS0/EDS0. Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use.

[000175] Compositions suitable for parenteral administration may comprise at least one more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[000176] In various embodiments, a composition described herein includes an aiRNA composition and pharmaceutically acceptable salts and solvates thereof and one or more surfactants. In certain embodiments, the surfactant is sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS), or one or more polyoxylglycerides. For example, the polyoxylglyceride can be lauroyl polyoxylglycerides (sometimes referred to as Gelucire™) or linoleoyl polyoxylglycerides (sometimes referred to as Labrafil™). Examples of such compositions are disclosed in PCT Patent Application No. PCT/US2014/033566, the content of which is incorporated by reference herein in its entirety for any purpose. [000177] In certain embodiments, a kit is disclosed that comprises (1) one or more PDL1 aiRNAs and (2) one or more β-catenin aiRNAs, pharmaceutically acceptable salts of any of the foregoing, together with instructions for administration and/or use.

[000178] In certain embodiments, aiRNAs of the present disclosure conjugated to a natural ligand such as cholesterol, lipid, an aptamer, or bound non-covalently these ligands.

[000179] Other carrier choices include positive charged carriers (e.g., canonic lipids and polymers) and various protein carriers. In certain embodiments, the delivery of the aiRNA molecules of the present disclosure uses a ligand-targeted delivery system based on the cationic liposome complex or polymer complex systems (Woodle, et al. J Control Release 74: 309-311; Song, etal. Nat. Biotechnol. 23(6): 709-717 (2005); Morrissey et al. Nat Biotechnol. 23(8): 1002- 1007 (2005)).

[000180] In other embodiments, the aiRNA molecules of the present disclosure can be formulated with a collagen carrier, e.g., atelocollagen, for in vivo delivery. Atelocollagen has been reported to protect siRNA from being digested by RNase and to enable sustained release (Minakuchi et al. Nucleic Acids Res. 32: el09 (2004); Takei et al. Cancer Res. 64: 3365-3370 (2004)). In other embodiment, the aiRNA molecules of the present disclosure are formulated with nanoparticles or form a nanoemulsion, e.g., RGD peptide ligand targeted nanoparticles. It has been shown that different siRNA oligos can be combined in the same RGD ligand targeted nanoparticle to target several genes at the same time (Woodle et al. Materials Today 8 (suppl 1): 3441 (2005)).

[000181] In certain embodiments, the aiRNAs of the present disclosure are encapsulated in non-liposomal inorganic magnesium nanoparticles as described previously (see the published U.S. Patent Application No. 2016/0046936, the content of which is hereby incorporated herein in its entirety).

[000182] In certain embodiments, the aiRNAs of the present disclosure are encapsulated in a DP3 nanoparticle formulation as disclosed in Example 14.

[000183] In certain embodiments, the BBI801, BBI-801, BBI801-101 and BBI-801-101 can be used interchangeably to mean a formulation comprising the combination of aiPDLl and aip- catenin as disclosed herein. [000184] In certain embodiments, BBI801 refers to 2'-OMe-modified Cat aiRNA #37 (Cat#37M2; also referred herein as BCAT) DP3 nanoparticle formulation and a 2'-OMe-modified PDL1 aiRNA #22 (PDL#22M2; also referred herein as PDL) DP3 nanoparticle formulation.

[000185] In certain embodiments, BBI801 refers to a nanoparticle formulation comprising BCAT and PDL aiRNAs.

[000186] In certain embodiments, the molar ratio of BCAT to PDL in BBI801 formulations can be, for example, about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:50 or 1:100.

[000187] In certain embodiments, the molar ratio of PDL to BCAT in BBI801 formulations can be, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, 1:50 or 1:100.

[000188] The term "subject" as used herein refers to its meaning as is generally accepted in the art. The term generally refers an organism to which the nucleic acid molecules of the present teachings can be administered. A subject can be a mammal or mammalian cell, including a human or human cell. The term also refers to an organism, which is a donor or recipient of explanted cells or the cells themselves. In certain embodiments, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, mammals and non-mammals, such as a non- human primate, a mouse, a rabbit, sheep, a dog, a cat, a horse, a cow, a chicken, an amphibian, a fish, an insect or a reptile which is to be the recipient of a particular treatment. Under some circumstances, the terms "subject" and "patient" can be used interchangeably herein in reference to a human subject.

[000189] The term "cell" as used herein refers to its meaning as is generally accepted in the art. With reference to exemplary nucleic acid molecules of the present teachings, the term can be used in its usual biological sense, and does not refer to an entire multicellular organism, e.g., specifically does not refer to a human being. The cell can be present in an organism, e.g., birds, plants, and mammals, such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ line organ, totipotent or pluripotent, dividing or non-dividing. The cell can also be derived from or can comprise a gamete or embryo, a stem cell, or a fully differentiated cell. In certain embodiments, a cell can be a cancer stem cell. [000190] As used herein, the term "cancer" in a subject refers to cells having uncontrolled proliferation, immortality, metastatic potential, rapid growth and increased proliferation rate, as well as certain morphological features. Cancer cells can aggregate in the form of a tumors or masses and/or circulate in the blood stream or lymphatic system as independent cells.

[000191] The term "cancer" comprises, for example, AIDS-Related cancers, breast cancers, cancers of the digestive/gastrointestinal tract, endocrine and neuroendocrine cancers, cancers of the eye, genitourinary cancers, germ cell cancers, gynecologic cancers, head and neck cancers, hematologic cancers, musculoskeletal cancers, neurologic cancers, respiratory/thoracic cancers, skin cancers, childhood cancers as well as cancers of unknown primary.

[000192] In certain embodiments, the cancer comprises cancer stem cells that express a sternness gene, e.g. activated phosphorylated STAT3.

[000193] Exemplary AIDS-related cancers include, but are not limited to, AIDS-Related Lymphoma, Primary Central Nervous System Lymphoma and Kaposi Sarcoma.

[000194] Exemplary breast cancers include, but are not limited to, ductal carcinomas in situ (DOS), invasive ductal carcinomas (IDC), invasive lobular carcinoma (ILC), triple negative breast cancers (where the tumor cells are negative for progesterone, estrogen, and her2/neu receptors), inflammatory breast cancers, metastatic breast cancers, breast cancers during pregnancy, Paget disease of the nipple, Phyllodes tumor, adenoid cystic (or adenocystic) carcinoma, low-grade adenosquamous carcinoma, medullary carcinomas, tubular carcinomas, papillary carcinoma, mucinous (colloid) carcinomas, lymphoma of the breast, adenomyoepithelioma, giant cell sarcoma of the breast, leiomyosarcoma of the breast, angiosarcoma of the breast, cystosarcoma phylloides, and liposarcoma of the breast, carcinoid tumors of the breast, acinic cell carcinoma, oncocytic carcinoma (mammary epithelial oncocytoma), mucoepidermoid carcinoma, spindle cell carcinoma of the breast, squamous cell carcinoma of the breast, secretory carcinoma of the breast (juvenile secretory carcinoma), metaplastic carcinoma of the breast, invasive micropapillary carcinoma of the breast, adenoid cystic carcinoma of the breast, cribriform carcinoma, myofibroblastoma of the breast (benign spindle stromal tumor of the breast) and glycogen-rich clear cell carcinoma of the breast. [000195] Exemplary cancers of the digestive/gastrointestinal tract include, but are not limited to, anal cancer, appendix cancer, gastrointestinal carcinoid tumor, bile duct cancer, carcinoid tumor, gastrointestinal cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumors (GIST), islet cell tumors, pancreatic neuroendocrine tumors, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, gastro-esophageal junction (GEJ) cancer, and stomach (gastric) cancer.

[000196] Exemplary endocrine and neuroendocrine cancers include, but are not limited to, adrenocortical carcinomas, gastrointestinal carcinoid tumors, islet cell tumors, pancreatic neuroendocrine tumors, Merkel cell carcinomas, non-small cell lung neuroendocrine tumors, small cell lung neuroendocrine tumors, parathyroid cancers, pheochromocytomas, pituitary tumors, and thyroid cancers.

[000197] Exemplary genitourinary cancers include, but are not limited to, bladder cancer, kidney (renal cell) cancer, penile cancer, prostate cancer, renal pelvis and ureter cancer, transitional cell, testicular cancer, urethral cancer, Wilms tumor and other childhood kidney tumors.

[000198] Exemplary gynecologic cancers include, but are not limited to, cervical cancer, endometrial cancer, fallopian tube cancer, gestational trophoblastic tumor, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, primary peritoneal cancer, uterine sarcoma, vaginal cancer and vulvar cancer.

[000199] Exemplary head and neck cancers include, but are not limited to, hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous neck cancer with occult primary, mouth cancer, nasopharyngeal cancer, oral cavity cancer, lip and oropharyngeal cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, pharyngeal cancer, salivary gland cancer, throat cancer and thyroid cancer.

[000200] Exemplary hematologic cancers include, but are not limited to, leukemias, acute lymphoblastic leukemia, adult, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lymphomas, AIDS-related lymphoma, cutaneous T- cell lymphoma, adult Hodgkin lymphoma, childhood Hodgkin lymphoma, Hodgkin lymphoma during pregnancy, mycosis fungoides, childhood Non-Hodgkin lymphoma, adult Non-Hodgkin lymphoma, Non-Hodgkin lymphoma during pregnancy, primary central nervous system lymphoma, Sezary syndrome, cutaneous T-cell lymphoma, Waldenstrom macroglobulinaemia, chronic myeloproliferative neoplasms, Langerhans cell histiocytosis, multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

[000201] Exemplary musculoskeletal cancers include, but are not limited to, bone cancer, Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma of bone, childhood rhabdomyosarcoma and soft tissue sarcoma.

[000202] Exemplary neurologic cancers include, but are not limited to, adult brain tumor, childhood brain tumor, astrocytomas, brain and spinal cord tumors, brain stem glioma, atypical teratoid/rhabdoid central nervous system tumor, embryonal central nervous system tumors, germ cell central nervous system tumors, craniopharyngioma, ependymoma, neuroblastoma, pituitary tumor and primary central nervous system (CNS) lymphoma.

[000203] Exemplary respiratory/thoracic cancers include, but are not limited to, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, thymoma and thymic carcinoma.

[000204] Exemplary skin cancers include, but are not limited to, cutaneous T-cell lymphoma, Kaposi sarcoma, melanoma, Merkel cell carcinoma, skin cancer, cutaneous T-cell lymphoma, mycosis fungoides and Sezary syndrome.

[000205] Also included within the term "cancer" is "solid tumor." As used herein, the term "solid tumor" refers to those conditions, such as cancer, that form an abnormal tumor mass, such as sarcomas, carcinomas, and lymphomas. Examples of solid tumors include, but are not limited to, non-small cell lung cancer (NSCLC), neuroendocrine tumors, thyomas, fibrous tumors, metastatic colorectal cancer (mCRC), and the like. In certain embodiments, the solid tumor disease can be an adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and the like.

[000206] The terms "administer," "administering," or "administration" are used herein in their broadest sense. These terms refer to any method of delivering the composition described herein to a subject, cell or tumor by, for example, introducing the composition systemically, locally, or in situ to the subject. Thus, a compound of the present teachings produced in a subject from a composition (whether or not it includes the compound) is encompassed by these terms.

When these terms are used in connection with the term "systemic" or "systemically," they generally refer to in vivo systemic absorption or accumulation of the compound or composition in the blood stream and its distribution throughout the entire body. In certain embodiments, the terms "administer," "administering," or "administration" can refer to, for example, delivering one or more recombinant vectors to a tumor cell, wherein the vector expresses an RNA interfering agent.

[000207] The term "parenteral" as used herein refers to its meaning as is generally accepted in the art. The term generally refers to methods or techniques of administering the aiRNA composition described herein in a manner other than through the digestive tract, and includes epicutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.

[000208] The term "sensitize" means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy. In certain embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the cancer therapy. In certain embodiments, an increased sensitivity or a reduced sensitivity to a therapeutic treatment can be measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa et al. Cancer Res 1982; 42: 2159-2164) or cell death assays (Weisenthal et al. Cancer Res 1 84; 94: 161 - 173; Weisenthal et al. Cancer Treat Rep 1985; 69: 615-632; Weisenthal et al. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwcod Academic Publishers, 1993: 41 -432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivity or resistance may also be measured in animals by measuring the tumor size reduction over a period of time, for example, 6 months for humans and 4-6 weeks for mice. A composition or a method sensitizes cancer cells or tumor cells to a therapeutic treatment (e.g. inhibition of PD-l/PDLI signaling) if the increase in treatment sensitivity or the reduction in resistance is about 25% or more, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more, compared to treatment sensitivity or resistance in the absence of such composition or method. In certain embodiments, the increase in treatment sensitivity or the reduction in resistance is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold or more compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.

[000209] The term "synergy," "therapeutic synergy," "synergistic," "synergistically," or "enhanced" as used herein refers to an effect of the combination of PDL1 -specific and β-catenin- specific aiRNAs that is greater than the sum of their separate effects (or "additive effects"). A therapeutic synergistic effect may be attained when the PDL1 -specific and β-catenin-specific aiRNAs are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a therapeutic synergistic effect may be attained when the PDL1 -specific and β-catenin-specific aiRNAs are administered or delivered sequentially, e.g. in separate tablets, pills or capsules, or by different injections in separate syringes. A therapeutic synergistic anticancer effect denotes an anticancer effect which is greater than the predicted purely additive effects of the PDL1 -specific and β-catenin-specific aiRNAs administered separately.

[000210] A "therapeutically effective amount" of a combination of PDL1 -specific and β- catenin-specific aiRNAs in reference to the treatment of cancer, means an amount capable of invoking one or more of the following effects: (1) inhibition, to some extent, of cancer or tumor growth, including slowing down growth or complete growth arrest; (2) reduction in the number of cancer or tumor cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, slowing down, or complete stopping) of cancer or tumor cell infiltration into peripheral organs; (5) inhibition (i.e., reduction, slowing down, or complete stopping) of metastasis; (6) enhancement of anti-tumor immune response, which may, but is not required to, result in the regression or rejection of the tumor, or (7) relief, to some extent, of one or more measurable symptoms associated with the cancer or tumor. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual and the ability of one or more anti-cancer agents to elicit a desired response in the individual. A "therapeutically effective amount" is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. [000211] In certain embodiments, a subject is successfully "treated" according to the methods of the present teachings if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth reduced morbidity and mortality. In certain embodiments, the term "treating cancer," "treatment of cancer," or an equivalent thereof, means to decrease, reduce, or inhibit the replication of cancer cells; decrease, reduce or inhibit the spread (formation of metastases) of cancer; decrease tumor size; decrease the number of tumors (i.e. reduce tumor burden); lessen or reduce the number of cancerous cells in the body; prevent recurrence of cancer after surgical removal or other anticancer therapies; or ameliorate measurable treatment endpoints (i.e., outcomes). "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

[000212] As used herein, the terms "inhibiting", "to inhibit" and their grammatical equivalents, when used in the context of a bioactivity, refer to a down-regulation of the bioactivity, which may reduce or eliminate the targeted function, such as the production of a protein or the phosphorylation of a molecule. When used in the context of an organism (including a cell), the terms refer to a down-regulation of a bioactivity of the organism, which may reduce or eliminate a targeted function, such as the production of a protein or the phosphorylation of a molecule. In particular embodiments, inhibition may refer to a reduction, e.g., of about 10%, of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% of the targeted activity. When used in the context of a disorder or disease, the terms refer to success at preventing the onset of measurable symptoms, alleviating measurable symptoms, or eliminating the disease, condition or disorder.

[000213] For example, the administration of PDL1 -specific and β-catenin-specific aiRNAs to a tumor cell results in the simultaneous inhibition of PDL1 and β-catenin gene expression in the tumor cell including the level of PDL1 and β-catenin mRNA molecules or the level of PDL1 and β-catenin proteins or the activity of PDL1 and β-catenin proteins, below that observed in the absence of the nucleic acid molecules (e.g., aiRNA) of the present teachings or in the presence of an aiRNA having a random 'scrambled' nucleotide sequence. [000214] In certain embodiments, the exemplary PDL1 -specific and β-catenin-specific aiRNAs of the present teachings silence or inhibit the expression of PDL1 and β-catenin mRNAs by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% of the targeted gene.

[000215] In certain embodiments, the combination of the exemplary PDL1 -specific and β- catenin-specific aiRNAs of the present teachings inhibit tumor growth by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or about 100%.

[000216] The present disclosure provides compositions comprising PDL1 -specific and β- catenin-specific asymmetrical interfering RNAs (aiRNA), that can induce potent gene silencing of PDL1- and β-catenin gene expression in tumor cells. In one aspect, PDL1 -specific and β-catenin- specific asymmetrical interfering RNAs are characterized in the length asymmetry of the two RNA strands. This structural design can not only be functionally potent in effecting gene silencing but offer several advantages over the current state-of-art siRNAs. Among the advantages, the PDL1- specific and β-catenin-specific aiRNAs can have RNA duplex structure of much shorter length than previously reported siRNA designs, which reduces the cost of synthesis and abrogate/reduce the length-dependent triggering of nonspecific interferon-like responses. In addition, the asymmetry of the aiRNA structure abrogates and/or otherwise reduces the sense-strand mediated off-target effects. PDL1 -specific and β-catenin-specific aiRNA are therefore, in certain embodiments, more efficacious, more potent, with a more rapid-onset, and more durable at inducing gene silencing than any of the other RNA interfering agents.

[000217] In certain embodiments, PDL1 -specific and β-catenin-specific aiRNAs disclosed herein each comprises an antisense strand with a length from 18-23 nucleotides (nt) and a sense strand with a length from 12-17 nucleotides. In certain embodiments, the sense strand is substantially complementary to the antisense strand. In certain embodiments, the sense strand forms a double-stranded region with the antisense strand. In certain embodiments, the antisense strand can have a 3 '-overhang of 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides. In certain embodiments, the antisense strand can have a 5 -overhang from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. [000218] In certain embodiments, the antisense strand of the PDL1 -specific or β-catenin- specific aiRNA can be 18, 19, 20, 22, or 23 nucleotides long.

[000219] In certain embodiments, the sense strand of the PDL1 -specific or β-catenin- specific aiRNA can be 12, 13, 14, 15, 16, or 17 nucleotides long.

[000220] In certain embodiments, the 3'-overhang of the antisense strand is greater than 0 nucleotides in length.

[000221] In certain embodiments, the antisense strand comprises a sequence being substantially complementary to a target PDL1 and β-catenin rnRNA sequences. In certain embodiments, the antisense strand of the PDL1 -specific and β-catenin-specific aiRNAs comprise a sequence that is at least about 50%, about 60%, about 70 %, about 80%, about 90%, about 95%, about 99% or about 100% complementary to their corresponding target mRNA sequences.

[000222] In certain embodiments, the present disclosure provides a method of treating cancer comprising administering PDL1 and β-catenin aiRNAs, wherein the β-catenin aiRNA acts in synergy with the co-adrninistered PDL1 aiRNA to inhibit tumor growth.

[000223] In certain embodiments, the change in the efficacy of a PDL1 -specific aiRNA. as a result of the co-administration of a β-catenin-specific aiRNA, can be evaluated in subcutaneous tumor animal models at endpoints such as the percent test/control (%T/C) tumor weights calculated on each day that tumors are measured, tumor growth delay, net log cell kill, median days to a defined tumor weight or to a specified number of tumor doublings, and tumor regression. In certain embodiments, the lowest calculated %T/C seen over time can be defined as the optimal %T/C because it defines the greatest level of activity seen with β-catenin-specific aiRNA. The rate and duration of partial and complete tumor regressions can also be considered to be clinically relevant endpoints.

[000224] For example, a T/C = 0% means no rumor growth. A T/C = 100% means no antitumor activity, i.e., the treated and control tumors grew equally. A T/C equal to or less than 42% is considered significant antitumor activity by the Drug Evaluation Branch of the Division of Cancer Treatment (NCI). A T/C value < 10% is considered to indicate highly significant antitumor activity, and is the level used by NCI to justify a clinical trial if toxicity, formulation, and certain other requirements are met (termed DN- 2 level activity).

[000225] The present disclosure reports on the surprising discovery that a treatment combination of PDL1 -specific and β-catenin-specific aiRNAs have a greater effect in inhibiting tumor cell growth than the added effects of the PDL1 -specific aiRNA and β-catenin-specific aiRNAs acting alone.

EXAMPLES

Example 1: Preparation and characterization of exemplary PDLl-specific and β-catenin- specific aiRNAs

[000226] PDLl-specific or β-catenin-specific aiRNAs were synthesized in DMT-on mode. Following completion of the synthesis, the solid support was suspended in 600 μΐ EtOH/NH40H solution (prepared by mixing 1 volume of 200 proof ethanol with 3 volumes of 28% NH₄OH) and heated at 55 °C for 2 hours. After primary de-protection, EtOH/NH40H was evaporated and the RNA oligo was dried to a pellet. 100μΙ of RNA de-protection solution (NMP/TEA.3HF (3:2)) was added and the solution was heated at 65 °C for 1.5 hours. The reaction was then quenched with 400 μΐ of 1.5 M ammonium bicarbonate. Purification was performed with Clarity® QSP Cartridges (Phenomenex, USA). The sense and antisense oligonucleotides for each target sequence were then annealed by mixing equimolar amounts of each and heating to 94°C for 5 min followed by gradual cooling. The annealing of the resulting duplexes was confirmed on a 15% PAGE gel.

[000227] An alignment showing the location of mRNA sequences targeted by the exemplary β-catenin aiRNAs within the human catenin beta 1 (CTNNBl), Transcript variant 2, mRNA (ACCESSION No.: NM 001098209.1; SEQ ID NO: 179) is depicted in FIG. 1A. FIG. IB shows an exemplary full-length β-catenin mRNA (SEQ ID NO. 179) subdivided into 5 overlapping sequences (SEQ ID NOs.: 180-185), each of which can be targeted by a subset of the exemplary β-catenin-specific aiRNA molecules. FIG. 1C depicts exemplary β-catenin target nucleotide sequences (e.g. SEQ ID NOs. 186-262) together with their % GC content. FIG. ID groups the exemplary β-catenin aiRNAs according to the % GC content (23-35%, 36-44% or 45-57%) of nucleotides in the antisense strand that are colinear with the corresponding complementary nucleotides of the β-catenin mRNA sequence (NM_001098209; SEQ ID NO. 179). FIG. IE compares exemplary β-catenin aiRNAs with respect to the length of sense and antisense strands, length of 3 ' and 5 ' overhangs, nucleotide sequence of the sense and antisense strands, total number of nucleotides in the antisense strand that are complimentary to the β-catenin target sequence (depicted with white letters), their % GC content and the length of the double stranded region. [000228] Similarly, an alignment showing the location of mRNA sequences targeted by the exemplary PDL1 aiRNAs 1-46 within the PDL1 mRNA sequence (Transcript Variant 1) (Accession No.: NM 014143; SEQ ID NO.: 166) is shown in FIG. 2A. FIG. 2B shows the exemplary full-length PDL1 mRNA (SEQ ID NO. 166) subdivided into 7 overlapping sequences (SEQ ID NOs.: 167-173), each of which can be targeted by a subset of the exemplary RNA molecules. FIG. 2C depicts exemplary PDL1 target nucleotide sequences (SEQ ID Nos.: 93-138) with their associated PDL1 aiRNAs 1-46. FIG. 2D aligns the sense strands (SEQ ID NOs. 1-46) and antisense strands (SEQ ID NOs. 47-92) with an exemplary PDL1 target mRNA sequences (SEQ ID NOs. 140-164) and compares exemplary PDL1 aiRNAs with respect to the length of the sense and antisense strands, the number of nucleotides in the antisense strand that are colinear with the corresponding complementary nucleotides of the PDL1 mRNA sequence (shaded in dark grey), their % GC content and the length of the 5' and 3' overhangs. FIG. 2E groups the exemplary PDL1 aiRNAs according to the % GC content (23-35%, 36-44% or 45-57%) of nucleotides in the antisense strand that are colinear with the corresponding complementary nucleotides of the PDL1 mRNA sequence (NM_014143; SEQ ID NO. 166).

Example 2: RNAi Activity of exemplary unmodified β-Catenin-specific aiRNAs in vitro

[000229] DLD1 human colorectal carcinoma cells were plated on p60 plate and transfected with various concentrations (50nM, ΙΟΟρΜ, 50pM, ΙΟρΜ and 5pM) of unmodified candidate β- catenin aiRNAs using Lipofectamine® RNAiMAX (Thermo Fisher, USA) according to the manufacturer's instructions. After 48 hours of treatment, cell were collected using Accutase® cell detachment solution (Life Technologies) and the relative β-catenin mRNA level for each β-catenin aiRNA was measured by RT-qPCR. Values were standardized with the expression level of housekeeping gene (B2M). The RNAi activity of the exemplary β-catenin aiRNAs is summarized in FIG. IF.

Example 3: RNAi Activity of exemplary unmodified PDLl-specific aiRNAs in vitro

[000230] IFN-γ stimulated DLD1 cells, HCT-116 or RKO cells were seeded into p60 plates. After culture for 24 hours, the cells were then transfected with different concentrations of PDLl- specific aiRNAs (aiRNAs #1-41) or scrambled aiRNA (at a final concentration of 0.1, 1, 10 or 50nM) using Lipofectamine® RNAiMAX (Thermo Fisher, USA) according to the manufacturer's instructions. The aiRNAs and RNAiMAX were incubated for 20 minutes in serum free OPTI- MEM (Thermo Fisher, USA), before being added to the cells with culture medium. 24-48 hours after transfection, the cells were harvested using Accutase® cell detachment solution (Life Technologies) and the relative PDL1 mRNA level for each PDL1 aiRNA was measured by RT- qPCR using CD274 TaqMan Gene Expression Assays (Thermo Fisher). The relative RNAi activity of each of the PDL1 aiRNAs 1-41 at 4 different concentrations (O.lnM, InM, lOnM and 50nM) is shown in FIG. 2F.

[000231 ] FIG. 2G shows the activity of 0. InM, 1 nM, 1 OnM and 50nM of the PDL1 aiRNA #28 on PDL1 mRNA levels in the human colorectal adenocarcinoma cell line, HCT-116.

[000232] FIG. 2H shows PDL1 aiRNAs #42, #43, and #45 at a concentration of 50nM inhibited the PDL1 protein expression in HCT-116 cells by more than 95%.

[000233] FIG. 21 shows the depletion of cell surface expression of PDL1 after transfection with PDL1 aiRNA#22. RKO and MDA-MB-231 cells were plated into p60 plate and transfected with PDL1 aiRNA #22 using RNAiMAX (Life technologies) following the recommended protocol. After 48 hours of treatment, cells were collected using Accutase (Life technologies). Cell surface PDL1 expression was determined by flow cytometry analysis of transfected cells stained with PE conjugated anti-PDLl (Biolegend) using Flow-jo software. Isotype control was used as background control.

Example 4: 2'-OMe and/or 2'-F modified β-Catenin-specific aiRNAs

[000234] β-catenin-specific aiRNA #37 (aip-cat #37) and #57 (aip-cat #57) containing select 2'-hydroxyl substitutions on the ribose of selected nucleotides were generated using standard procedures. In certain embodiments, the phosphodiester bonds between the nucleotides were not modified. In other embodiments, the phosphodiester bond is replaced with a phosphothioate bond. The location of the 2'-methoxy groups (-OCH3 or -OMc) and/or 2'-fluoro groups (-F) substitutions in the exemplary β-catenin-specific aiRNAs #37 and #57 is shown in FIG. 3A. Example 5: The modified β-Catenin-specific aiRNAs are highly effective at silencing β- catenin expression in vitro with an ICso of 0.5 - 1.8 pM

[000235] The activity of each of the modified β-Catenin-specific aiRNAs was tested in DLD- 1 human colorectal adenocarcinoma cells. DLD-1 cells plated on p60 plates were trans fected with various concentrations of β-Cat aiRNA#37M22, β-Cat aiRNA#57M22, β-Cat aiRNA#57M22F6 and β-Cat aiRNA#57M22F10 using Lipofectamine® RNAiMAX (Thermo Fisher, USA) according to the manufacturer's instructions. After 48 hours treatment, the cells were collected and the amount of β-catenin mRNA was determined by real time RT-PCR analysis. Values were standardized to the expression level of a housekeeping gene $2-nucroglobulin (B2M)). ICso values were calculated using Prism software (GraphPad Software). As shown in FIG. 3B, the modified β-Catenin-specific aiRNAs #37 and #57 were all highly effective at silencing β-catenin mRNA expression in vitro in a dose dependent manner with an ICso of 0.5 - 1.8 pM.

Example 6: The 2'-OMe modified β-Catenin aiRNA #37 is highly effective at silencing β- catenin expression in vitro in a wide variety of cancer cell lines

[000236] FIG. 3C shows the RNAi activity of 2'-OMe-modified β-Catenin-specific aiRNA #37 (Cat#37M22) after transfection into different cancer cell lines in vitro (DLD-1 (ATCC® CCL- 221™; a colorectal adenocarcinoma cell line), MKN-45 (RRID:CVCL_0434; a gastric cancer cell line), SK-UT-1 (ATCC® HTB-114™; a leiomyosarcoma cell line), MDA-MB-468 (ATCC® HTB-132™; a breast cancer cell line), NCI-H358 (ATCC® CRL-5807™; a lung cancer cell line), SK-MEL-28 (ATCC® HTB-72™; a melanoma skin cancer cell line) and LN-18 (ATCC® CRL- 2610™; a glioblastoma cell line). After 48 hours, cells were collected and beta-Catenin mRNA or protein expression levels were measured using standard procedures, β-actin was used as a control.

[000237] The results show that 2'-OMe modified β-Catenin aiRNA #37 is highly effective at silencing β-catenin expression in vitro in a wide variety of cancer cell lines.

Example 7: The 2'-OMe modified β-Catenin aiRNA #37 induces durable target gene silencing in vitro

[000238] DLD1 human CRC cancer cells were plated in p60 plate and treated with lnM of the 2'-OMe-modified β-Catenin-specific aiRNA #37 (Ca1#37M22). After transfection, cells were collected from day 1 to day7 and β-Catenin protein expression was determined by Western Blot. Actin was used as loading control. FIG.3D shows that the transfection of the Cat#37M22 aiRNA silenced β-Catenin protein expression for at least 7 days.

Example 8: The 2'-OMe modified β-Catenin aiRNA #37 inhibits survival of cancer cells in vitro

[000239] DLD1 cells were transfected with InM of the 2'-OMe-modified β-Catenin-specific aiRNA #37 (Cat#37M2) for 24h. Cells were then trypsinized and re-plated on 6-well plates at 500- 2,000 cells/well to determine the colony formation ability of the cells. After 11-14 days, colonies were stained with Giemsa stain and the number of colonies was counted.

[000240] As shown in FIG. 3E, the 2'-OMe-modified β-Catenin-specific aiRNA #37 (Cat#37M22) markedly inhibited the survival of human colon cancer cells (DLD1) in vitro.

Example 9: The modified β-Catenin-specific aiRNAs are highly effective at inhibiting CSC colony formation in vitro

[000241] Wnt signal dependent DLD1 human CRC cancer cell line and AGS human gastric cell line were plated in p60 plate and transfected with β-Cat aiRNA#37M2 or β-Cat aiRNA#57M22F6 with or without aiPDLl #1481. After 48 hours treatment, cell were harvested and plated into 6 well plate at 500 cells/well. The number of colonies was counted after 9 days and normalized with those from control samples.

[000242] As shown in FIG. 3F, both β-Cat aiRNA#37M2 or β-Cat aiRNA#57M2F6 were highly effective at inhibiting colony formation by DLD-1 human colorectal carcinoma cells whereas β-Cat aiRNA#57M22F6 is effective at inhibiting colony formation by AGS human gastric cancer cells

Example 10: 2'-OMe and/or 2'-F modified aiPDLl-specific aiRNAs

[000243] PDL1 -specific aiRNA #22 and #2 containing select 2'-hydroxyl substitutions on the ribose of selected nucleotides were generated using standard procedures. The location of the 2'- methoxy groups (-OCH3 or -OMe) and/or 2'-fluoro groups (-F) substitutions in the exemplary PDL1 -specific aiRNAs #22 and #2 are depicted in FIGs. 4A and 4B. Example 11: The modified PDLl-specific aiRNAs are highly effective at silencing PDL1 expression in vitro with an ICso of 12 J - 13.6 pM

[000244] The activity of each of the modified PDLl-specific aiRNAs was tested in RKO human CRC cancer cells. RKO cells plated on p60 plates were transfected with various concentrations of 2'-OMe-modified PDL1 aiRNA #22 or 2'-OMe-F-modified PDL1 aiRNA #2 using Lipofectamine® RNAiMAX (Thermo Fisher, USA) according to the manufacturer's instructions. After 24 or 48 hours, the cells were collected using Accutase (Life technologies). The amount of PDL1 mRNA was determined by real time RT-qPCR analysis. Values were standardized to the expression level of a housekeeping gene (β2 -microglobulin (B2M)). ICso values were calculated using Prism software (GraphPad Software). ICso values were calculated using Prism software.

[000245] As shown in FIG. 4C, the 2'-OMe-modified PDL1 aiRNA #22 or 2'-OMe-F- modified PDL1 aiRNA #2 were both highly effective at silencing PDL1 mRNA expression in vitro in a dose dependent manner with an ICso of 13.6 pM and 12.3 respectively.

Example 12: Restoration of oCD28 antibody induced 1L-2 expression in PD-1⁺ Jurkat cells after co-culture with HEK-293 cells co-transfected with an PDL1/TCR activator expression plasmid and the 2'-OMe-F-modified PDL1 aiRNA #2

[000246] AiScramble or 2'-OMe-F-modified PDL1 aiRNA #2 was transfected into HEK- 293 cells (human embryonic kidney 293 cells; ATCC® CRL-3216™) together with a TCR- Activator and PDL1 expressing plasmid (BPS Bioscience). Jurkat T cells stably expressing PD-1 were co-cultured with the aiRNA transfected HEK- cells and stimulated with aCD28 antibody (Bio Legend) for 24 hours. IL-2 expression was then measured by ELISA. FIG. 4D shows that co-culture of 2'-OMe-F-modified PDL1 aiRNA #2 transfected HEK-293 cells with PD-1⁺ Jurkat T cells, but not aiScramble transfected HEK-293 cells, restored aCD28 antibody induced IL-2 expression in PD-1⁺ Jurkat.

Example 13: 2'-OMe Modification of the ribose of the first nucleotide adjacent to the 5'- terminal nucleotide of the antisense strand reduces RNAi activity

[000247] 2'-OMe modified nucleotides were introduced at select positions of the sense and/or antisense strand of the aiRNAs by replacing phosphoramidites with 2'-OMe phosphoramidites at the appropriate time during oligo synthesis. The effect of a 2'-OMe modified nucleotide at position 2 (at position of red arrow, FIG. 5 A) of the antisense strand of Cat#210 aiRNA was then tested in vitro. R: non-modified nucleotide; M: 2'~OMe modified nucleotide.

[000248] The 2'-OMe modified Cat#210 aiRNAs were transfected into DLD-1 cells using Lipofectamine® RNAiMAX (Thermo Fisher, USA) according to the manufacturer's instructions. After 24 or 48 hours, the cells were collected using Accutase (Life technologies) and the level of β-catenin rnRNA expression was assessed by qPCR. As shown in FIG. 5B, 2'-OMe modification at position 2 of the antisense strand of Cat#210 aiRNA consistently resulted in a decrease in RNAi activity i.e. an increase in the amount of β-catenin expression. For example, a comparison of constructs 15 and 16 shows that addition of just one 2'-OMe at position 2 of the antisense strand leads to the abolition of Cat#210 RNAi activity.

[000249] The RNAi activity of 2'-OMe modified ribose at position 2 of the antisense strand of different aiRNAs (Cat#37, Cat#57, PDL#2 and PDL#22; FIG. 6 A) was also compared to that of their corresponding non-modified aiRNAs.

[000250] The aiRNA constructs depicted in FIG. 6A were transfected into DLD- 1 cells using Lipofectamine® RNAiMAX (Thermo Fisher, USA) at a concentration of lOpM or InM. After 24 hours, the cells were harvested and the amount of target mRNA levels were measured by QPCR As shown in FIG. 6B, 2'-OMe modification of the ribose of the first nucleotide adjacent to the 5 - terminal nucleotide of the antisense strand consistently reduced the RNAi activity of β-catenin or PDL1 aiRNAs. Substitution of the 2ΌΗ of the ribose of the first nucleotide adjacent to the 5'- terminal nucleotide of the antisense strand with a 2'fluoro group did not however have any detrimental effect on RNAi activity (see Cat#57M2F6 in FIG. 3B).

Example 14: Preparation of PDLl-specific and β-catenin-specific aiRNA nanoparticles

[000251] aiRNAs are synthesized in DMT-on mode. Following completion of the synthesis, the solid support is suspended in 600 μΐ EtOH/NRtOH solution (prepared by mixing 1 volume of 200 proof ethanol with 3 volumes of 28% NH40H) and heated at 55 °C for 2 hours. After primary de-protection, EtOH/NH40H is evaporated and the RNA oligo is dried to a pellet. ΙΟΟμΙ of RNA de-protection solution (NMP/TEA.3HF (3:2)) is added and the solution is heated at 65 °C for 1.5 hours. The reaction is then quenched with 400 μΐ of 1.5M ammonium bicarbonate. Purification is performed with Clarity® QSP Cartridges (Phenomenex, USA). The annealing of the resulting duplexes is confirmed on 15% PAGE gel.

[000252] 100ml of the active pharmaceutical ingredient (API) solution comprising BCAT and PDL aiRNAs and lipid-EtOH solutions were prepared as shown in TABLE 4 below:

[000253] The lipids were dissolved in absolute EtOH at room temperature. After complete dissolution of the lipids, the solution was filtered through a 0.2μιη pore size filter into the preparation system. The API solution was also filtered at room temperature through a 0.2μιη pore size filter. Liposomal nanoparticles were then generated using crossflow injection technology

(Polymun; see FIG. 7A). Liposomes formed at the site of injection when lipid solution and API solution were combined. The generated liposomes (intermediate volume) were collected in an intermediate sterile liposome collection bottle (TV bottle). Free aiRNA and EtOH were then removed by ultra-/diafiltration through a hollow fiber membrane (100 kDa MWCO). During the ultrafiltration step, the sample was concentrated down to the target volume (to achieve the target aiRNA concentration). During diafiltration, 10 volume exchanges were performed with aqua purificata to ensure complete removal of EtOH and free oligo-nuclcotide and to exchange the outer buffer. Finally, liposomes were 0.2 μιη filtered using a syringe filter and filled into sterile plastic vials under laminar flow hood. The vials remained sealed and stored at 2-8°C protected from light.

[000254] The liposome nanoparticle "DP3" formulations were then evaluated as follows.

[000255] Measurement for size/Pdl determination of liposomes was performed by Dynamic- Laser-Light-Scattering (DLS) using a Malvern Nano ZS. This system was equipped with a 4 mW Helium/Neon Laser at 633 nm wavelength and measured the liposome samples with the noninvasive backscatter technology at a detection angle of 173°C. Liposomes were diluted in PBS in ratio 1:19 and the experiments were carried out at 25°C.

[000256] The zeta potential of liposomes was measured using a Malvern Nano ZS. Liposomes were diluted in PBS at a ratio 1:19 and the experiments were carried out at 25°C.

[000257] Quantification of aiRNA was done by spectrophotometer at OD260 nm. The samples of the stock solution were measured in aqua purificata. The samples of the final bulk product were first diluted with aqua purificata and then with methanol/chloroform to lyse the liposomes and release the amount of encapsulated aiRNA. Lipid concentration in the samples was measured from the bulk volume using a generic HPLC method. Encapsulation efficiency of siRNA into liposomes was determined using a Quant-iT™ RiboGreen® RNA Assay. The RiboGreen® RNA reagent is one of the most sensitive detection dyes for the quantitation of RNA in solution, with linear fluorescence detection in the range of 1-200 ng of RNA. The exemplary results of these assays for defined test parameters are shown in TABLE 5 below:

TABLE 5

[000258] In certain embodiments, the final composition (weight ratio) of the DP3 nanoparticlc formulation, i.e., RNA: magnesium phosphate: DOTAP-cholesterol: PEG2000- DSPE was 1: 5.62: 0.78: 0.42.

[000259] LNPs were prepared using Lipid Mix LNP - LM02 (Precision NanoSystems Inc.) according to the manufacturer's instructions available on the Precision NanoSystems web site (www.precisionnanosystems.com/wp-content/uploads/2015/12/PNI-SOP-NA-005-EXT-Rev- 002-siRNA-DOTAP-LNP-Standard-Protocol.pdf).

[000260] The composition of the LNP formulation was:

DLin-MC3-DMA: DSPC: Choi: DMG-PEG at a molar ratio of 50: 10: 38.5: 1.5.

DLin-MC3-DMA: heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate DSPC: 1 ,2-distearoyl-sn-glycero-3-phosphocholine

Choi: cholesterol

DMG-PEG: polyethylene glycol-dimyristolglycerol

[000261] The composition of LNPs is also described in the published U.S. patent application 2016/0022580, the contents of which is incorporated herein in its entirety.

Example 15: The DP3 nanoparticle formulation is more efficient at delivering the BBI801 aiRNA formulation in vivo than a commercially available LNP formulation

[000262] SW480 and CT26 colorectal carcinoma cells were inoculated subcutaneously into male athymic nude mice (8 x 10⁶ cells/mouse) and syngeneic immunocompetent BALB/c mice respectively and allowed to form palpable tumors. Once the tumors reached approximately 200 mm³, the xenografted mice were randomized into the following treatment groups (10 mice/group):

• Group 1: Control

• Group 2: mouse aiPDL-1 + mouse aip-catenin DP3 NP formulation

• Group 3: mouse aiPDL-1 + mouse aip-catenin LNP formulation

[000263] The 2'-OMe modified mouse β-catenin and PDL1 aiRNAs had the following nucleotide sequences:

TABLE 6

[000264] Nanoparticle (NP) preparations comprising either a control, mouse PDL1 -specific and β-catenin-specific aiRNA DP3 NPs (BBI-801 DP3) or mouse PDL1 -specific and β-catenin- specific aiRNA LNPs (BBI-801 LNP) were administered to the appropriate group as indicated above by intravenous injection starting on day 1. The aiRNA injections were then repeated either every week (qw; CRC syngeneic CT26 model) or every other day (q2d; SW48 CRC xenograft) for 12 days, i.e., at a dose of 1.5 mg/kg β-Cat aiRNA#37M2 aiRNA + 1.5 mg/kg 2'-OMe-F- modified PDL1 aiRNA on days 1, 3, 5, 8, and 10.

[000265] Xenografted animals were periodically weighed to assess the effect of treatment on tumor burden. The treatment was well tolerated with minimal adverse side effects. Experimental groups contain 10 mice/group were representative of at least 2 independent experiments.

[000266] Administration of ai-PDLl and aip-catenin NP resulted in a delay in tumor progression as compared to tumor progression in subjects treated with the control. As shown in FIG. 7B, the BBI-801 DP3 NPs were significantly more effective at delivering the mouse PDL1- specific and β-catenin-specific aiRNAs to the xenograft tumors than the BBI-801 LNP as evidenced by the greater reduction in tumor volume.

Example 16: The combination of β-Cat aiRNA#37M2 and 2 '-OMe-F-modified

PDL16aiRNA #2 have improved specificity

[000267] To ensure selectivity of human β-Cat aiRNA#37M2 (hereinafter BCAT) and human 2 '-OMe-F-modified PDL1 (hereinafter PDL), Basic Local Alignment Search Tool (BLAST) analysis was conducted for these two aiRNAs. As shown in Table 1 BLAST analyses show that target genes (CTNNB1 and CD274) are the only human genes that are 100% complementary to the sequence of BCAT and PDL, respectively.

[000268] There were also no human genes with a single or 2 base-pair mismatch. This result indicated that BCAT and PDL sequences are predicted not to bind to any other human mRNA other than their target mRNA (CTNNBl and CD274, respectively).

TABLE 7: BLAST analysis showed BCAT and PDL target sequences are specific to their target genes

Example 17: BCAT and PDL aiRNAs have limited off-target effects in a microarray analysis

[000269] Off-target activities often complicate the interpretation of phenotypic effects in gene-silencing experiments and can potentially lead to unwanted toxicities of siRNAs. aiRNAs are reported to have no significant off target effects primarily because the sense strand is not incorporated into the RISC complex (see FIG. 8A). In vitro microarray experiments were conducted to confirm the limited off-target effects of BCAT and PDL (see FIG. 8C and 8D).

[000270] RKO cells were transfected with InM BCAT and PDL in 6-well plates using RNAimax (Invitrogen). RNA was isolated 12 h following transfection. The mRNA profiles were analyzed using Human OneArray Plus (Phalanx Biotech) containing nucleotides corresponding to ~ 28,000 human genes. The results in FIG. 8C and 8D showed that significantly less signature genes displayed a difference in expression level (p < 0.001) relative to mock-transfected cells.

Example 18: BCAT and PDL aiRNAs do not induce an immune response in vivo

[000271] Short interfering RNAs that mediate specific gene silencing through RNA interference (RNAi) can stimulate innate cytokine response in mammals. This could significantly influence the therapeutic application of RNA oligo owing to off-target effect and toxicities associated with immune stimulation. The ability of BCAT and PDL aiRNAs to induce an immune response in vivo was therefore assessed. As show in FIG. 8£ and 8F, there was a significant induction of plasma IFN-a, 11-6, TNF-a and IP- 10 upon administration of β-gal siRNA (served as a positive control) at 2-24 hours samples. In contrast BBI-801 DP3 NP formulation comprising human or mouse BCAT and PDL aiRNAs only induced IL10 at the 2 hour time point. No other cytokine induction was detected.

Example 19: Biodistribution analysis of fluorescence-labeled β-catenin-specific aiRNA in a

SW480 colon cancer xenograft tumor after systemic delivery [000272] Approximately, 6 X 10⁶ SW480 colon cancer cells are inoculated subcutaneously into male athymic nude mice and allowed to form palpable tumors. DP3 nanoparticle (NP) preparations comprising Alex Flour 555 labeled β-catenin-spccific aiRNAs were administered to the xenografted mice by intravenous injection starting at a dose of 3 mg/kg. Delivery of the fluorescently labeled aip-catenin to xenograft tumors occurred within 5 minutes of aiRNA administration and lasted at least 8 hours without any apparent adverse side effects (see FIG. 9).

Example 20: Gene silencing by β-Catenin aiRNA in human CRC xenograft, SW480

[000273] Approximately, 6 X 10⁶ SW480 colon cancer cells were inoculated subcutaneously into male athymic nude mice and allowed to form palpable tumors. Nanoparticle (NP) preparations comprising β-catenin-specific aiRNAs were administered to the xenografted mice by intravenous injection starting at a dose of 3 mg/kg. β-Catenin levels in SW480 xenografts were then probed via FISH for mRNA (FIG. 1 OA, red,) or for protein via immunofluorescence (IF) (FIG. 10B, green) after one intravenous dose of 3 mg/kg ai β-Cat. Counterstained with DAPI (blue). Scale bar: ΙΟμιη (A), 20μιη (B). The results show that the systemic delivery of βΐβ-ϋΒίβηΐη led to significant silencing of β-catenin expression in xenografted tumors that persisted for at least 120 hours post inoculation.

Example 21: Therapeutic efficacy of β-Catenin-aiRNA in various human xenograft tumor models

[000274] Several different mouse models were used to demonstrate anti-tumor activities of the DP3 NP aiRNA formulations.

[000275] SW480 (ATCC® CCL228™) human colon cancer cells were inoculated subcutaneously into female athymic nude mice (5xl0⁶ cells/mouse) and allowed to form palpable tumors. Once the tumors reached approximately 200 mm³, the animals were given scrambled control aiRNA or BCAT at 3 mg/kg intravenously (iv). All regimens were administered three times a week (tiw) (A) or once a week (qw) (B). Tumor size was evaluated periodically during treatment. Each point represents the mean + SEM of five tumors.

[000276] As shown in Figure 11 A, animals dosed with aiRNA β-Catenin intravenously as a monotherapy potently inhibited tumor growth. Tumor growth inhibitions of BCAT were calculated to be 76% at tiw or 56% at qw dosing and were statistically significant (p<0.001). There was no significant change in body weight due to intravenous administration of the scrambled control aiRNA or BCAT at 3 mg/kg.

[000277] As shown in Figure 11B-D, treatment with BCAT also potently inhibited tumor growth of NSCLC-A549 (A549 ATCC® CCL-185™) human lung cancer, TNBC MDA-MB-231 (ATCC ® TCP- 1003™) human triple-negative breast cancer and MKN-45 (RRID: CVCL0434) gastric cancer xenografts, with tumor growth inhibition of 70%, 93Vo, and 97%, respectively, all with a p< 0.0001. There was no significant change in body weight due to iv administration of BCAT at 3 mg/kg tiw for three weeks. These data indicate that BCAT can be safely dosed in a regimen that is effective in broad types of human cancer xenograft models.

[000278] BCAT anti-tumor activity was also evaluated in several orthotopic models.

[000279] In human pancreatic cancer orthotopic model, PDAC Mia-Paca-2 (ATCC ® CRL- 1420™), as shown in Figure 1 IE, treatment with BCAT potently inhibited tumor growth. Tumor growth inhibition of BCAT was calculated to be 74.5% and was statistically significant (p<0.001). There were no signs of toxicity due to intravenous administration of scrambled control aiRNA or BCAT at 3 mg/kg. These data suggest that BCAT can be safely administered in a regimen that is effective in this orthotopic model of human pancreatic cancer.

[000280] In the human liver cancer orthotopic model, HCC Hep G2 (ATCC® HB-8065™), BCAT inhibited tumor growth (Figure 1 IF). Tumor growth inhibition of BCAT was calculated to be 70% and was statistically significant (p<0.001). There were no signs of toxicity due to intravenous administration of scrambled control aiRNA or BCAT at 3 mg/kg. These data suggest that BCAT can be safely administered in a regimen that is effective in this orthotopic model of human liver cancer.

Example 22: Therapeutic efficacy of mouse β-Catenin and PDL1 aiRNAs in Ape*¹*¹* intestinal tumors

[000281] Intestinal tumors were isolated and β-Catenin (green) or PD-L1 (red) proteins were detected via immunofluorescence staining of FFPE intestinal tumors (Scale bar, 20μηι). As shown in Figure 12, in comparison to pre-dose animals or to animals that received a placebo scrambled aiRNA, the administration of BCATm or PDLm resulted in significant tumor target protein knockdown. The administration of mBBI801 resulted in similar levels of target protein knockdown as the respective monotherapies. Murine equivalent β-Catenin and PDL1 aiRNA sequences were used (see TABLE 6).

Example 23: Anti-Tumor efficacy of mouse p-Cateuin and PDL1 aiRNAs in murine syngeneic cancer models

[000282] To evaluate the efficacy of PDL and BBI801 (the combination of BCAT and PDL) in an immunocompetent host, the antitumor activity of mouse surrogate sequences of BCAT (BCATm) and PDL (PDLm) (see TABLE 6) was tested in colorectal, breast and lung cancers syngeneic mouse models.

Syngeneic Colorectal cancer CT26 model

[000283] CT26 cells were inoculated subcutaneously into female Balb/C mice (0.3x10⁶ cells/mouse) and allowed to form palpable tumors. Dosing began when the tumors reached approximately 100 mm³. Animals were treated iv with BCATm, or PDLm at 3 mg/kg qw. The combination group was treated with BBI801-101m (BCATm plus PDLm) 3mg/kg each (total of 5 doses). Each point represents the mean ± SEM of five tumors.

[000284] As shown in Figure 13A, treatment with BCATm, PDLm, or the BBI801m at qw 3mg/kg each aiRNA, the TGIs were 48% (p-value, 0.02), 65% (p-value, 0.003), and 86% (p-value, 0.0001), respectively. aiRNAs administration did not cause significant changes in body weight.

Syngeneic Lung cancer LL2 model

In addition, the efficacy of BCATm in combination with PDLm was also tested in immunocompetent animals with tumors locating at other tissues, an orthotopic murine syngeneic lung cancer model, LL2.

[000285] LL2 cells were inoculated intravenously into female Balb/C mice (0.3x10⁶cells/mousc). In this study, treatment started three days after inoculation. Animals were given scrambled aiRNA at 3 mg/kg, BCATm at 3 mg/kg, PDLm at 3 mg/kg, or the combination of BCATm and PDLm at 1.5 mg/kg each intravenously. All regimens were administered three times a week (tiw). The animals received treatment for 30 days or until they succumb to their tumors. Mice were euthanized when they become moribund or when body weight loss is over 15%. [000286] As shown in Figure 13D, the median survival times for control animals or animals that received aiRNA Scrambled were 14 days; the median survival times for animals that received BCATm or PDLm as monotherapies were 26 and 25.5 days, respectively, and were statistically significant compared to the control groups (PO.0001 ). By post implantation day-40, ten days after the last dose was given, animals that received BBI801-101m did not show any sign of disease nor significant change in body weight due to iv administration of aiRNAs. These data indicate that BBI801m can be safely dosed in a regimen that is effective in this murine lung cancer model.

Syngeneic Breast cancer EMT6 model

[000287] Treatment with BCATm and/or PDLm was tested in the murine syngeneic subcutaneous breast cancer model, EMT6 (FIG. 13C).

[000288] EMT6 cells were inoculated subcutaneously into female Balb/C mice (lxl 0⁶ cells/mouse) and allowed to form palpable tumors. Once the tumors reached approximately 50 mm3, the animals were given aiRNA Scrambled at 3 mg/kg, BCATm at 3 mg/kg, PDLm at 3 mg/kg, or the combination of BCATm and PDLm at 3 mg/kg each intravenously. All regimens were administered twice a week. The animals received a total of 5 doses of aiRNA. Tumor size was evaluated periodically during treatment. Each point represents the mean + SEM of five tumors.

[000289] BCATm and/or PDLm were effective at inhibiting tumor growth. Tumor growth inhibitions of BCATm or PDLm were calculated to be 46.2% and 46.3%, respectively (p<0.001). Treatment with BBI801-101m further inhibited tumor growth. Tumor growth inhibition of the combination treatment at 3.0 mg/kg each was calculated to be 83.7% (p<0.001). There were no signs of toxicity due to i.v. administration of the aiRNA. Therefore, BBI801-101m can be safely dosed in a regimen that is effective in this breast cancer model.

Syngeneic Ape**" *'- CRC model

[000290] In the Apc^^47" genetically engineered mouse CRC model (Figure 13C), control animals had an average of 60.2 intestinal tumors per mouse at 11-13 weeks of ages. Treatments with BCATm, or PDLm as monotherapies reduced the average number of tumors per mouse to

27.6 or 35.6, respectively (p<0.01 , p<0.05, respectively). Treatment with BBI801m, the combination, further reduced the average number of tumors per mouse to 12.4 (p<0.001), with no signs of toxicity due to i.v. administration of the aiRNAs.

Claims

1. A composition comprising

a therapeutically effective amount of a β-catenin-specific asymmetric interfering RNA (aiRNA), and

a therapeutically effective amount of a PDL1 -specific asymmetric interfering RNA (aiRNA),

wherein the combination of the PDL1 -specific and β-catenin-specific aiRNAs is effective at producing therapeutic synergy in the treatment of cancer.

2. The composition of claim 1, wherein the β-catenin-specific asymmetric interfering RNA (aiRNA) comprises

an antisense strand comprising 5 '-terminal and 3 '-terminal nucleotides that are 17, 18, 19, 20, or 21 nucleotides apart, and

a sense strand comprising a 5 '-terminal nucleotide that is complementary to a nucleotide of the antisense strand other than its 3 '-terminal nucleotide and a 3 '-terminal nucleotide that is complementary to a nucleotide of the antisense strand,

wherein the antisense strand is at least 70% complementary with the sense strand, and

wherein 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand are colinear with the corresponding complementary nucleotides in a target nucleotide sequence selected from SEQ ID NO. 180, 181, 182, 183, 184 or 185.

3. The composition of any one of claims 1 or 2, wherein the PDL1 -specific asymmetric interfering RNA (aiRNA) comprises

wherein the antisense strand is at least 70% complementary with the sense strand, and wherein 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand are colinear with the corresponding complementary nucleotides in a target nucleotide sequence selected from SEQ ID NO. 167, 168, 169, 170, 171, 172 or 173.

4. The composition of any one of claims 1, 2 or 3, wherein the sense or antisense strand of each asymmetric interfering RNA comprises at least one modified nucleotide or its analogue.

5. The composition of claim 4, wherein a 2 -OH group of the at least one modified ribonucleotide or its analogue is replaced by H or a 2'-OMe group.

6. The composition of claim 4, wherein the at least one modified nucleotide or its analogue is a sugar-, backbone-, and/or base-modified ribonucleotide.

7. The composition of claim 6, wherein the backbone-modified ribonucleotide comprises a modification in a phosphodiester linkage with another ribonucleotide.

8. The composition of claim 7, wherein the phosphodiester linkage is modified to comprise a nitrogen or a sulfur heteroatom.

9. The composition of claim 4, wherein the at least one modified nucleotide or its analogue comprises a phosphothioate group.

10. The composition of claim 4, wherein the at least one modified nucleotide or its analogue comprises inosine or a tritylated base.

11. The composition of claim 4, wherein the at least one modified nucleotide or its analogue is a sugar-modified ribonucleotide, wherein a 2 -OH group is replaced by H, OR, R, halo, SH, SR, NH₂, NHR, NR2, or CN, and wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, CI, Br, or I.

12. The composition of claim 2, wherein at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) are contiguous and colinear with the corresponding complementary nucleotides in a target nucleotide sequence chosen from SEQ ID NO. 180, 181, 182, 183, 184 or 185.

13 The composition of claim 2, wherein the 5 '-terminal nucleotide of the sense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the 3 '-terminal nucleotide of the antisense strand.

14. The composition of claim 2, wherein the 3 '-terminal nucleotide of the sense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the S'-terminal nucleotide of the antisense strand.

15. The composition of claim 2, wherein the 5 '-terminal and S'-terminal nucleotides of the sense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) are 13 nucleotides apart.

16. The composition of claim 15, wherein the 5 '-terminal and 3 '-terminal nucleotides of the antisense strand of the β-catenin-specific asymmetric interfering RNA (aiRNA) are 19 nucleotides apart.

17. The composition of claim 2, wherein the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34% or about 35%.

18. The composition of claim 2, wherein the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43% or about 44%.

19. The composition of claim 2, wherein the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57% or about 58%.

20. The composition of claim 2, wherein the nucleotide sequence of the antisense strand of the β-catenin-specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 32%.

21. The composition of claim 3, wherein at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides of the antisense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) are contiguous and colinear with the corresponding complementary nucleotides in a target nucleotide sequence chosen from SEQ ID NO. 167, 168, 169, 170, 171 , 172 or 173.

22. The composition of claim 3, wherein the 5'-terminal nucleotide of the sense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the 3 '-terminal nucleotide of the antisense strand.

23. The composition of claim 3, wherein the 3 '-terminal nucleotide of the sense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) is complementary to the first, second or third nucleotide adjacent to the 5'-terminal nucleotide of the antisense strand.

24. The composition of claim 3, wherein the 5 '-terminal and 3 '-terminal nucleotides of the sense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) are 13 nucleotides apart.

25. The composition of claim 3, wherein the 5 '-terminal and 3 '-terminal nucleotides of the antisense strand of the PDL1 -specific asymmetric interfering RNA (aiRNA) are 19 nucleotides apart.

26. The composition of claim 3, wherein the nucleotide sequence of the antisense strand of the

PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34% or about 35%.

27. The composition of claim 3, wherein the nucleotide sequence of the antisense strand of the PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43% or about 44%.

28. The composition of claim 3 , wherein the nucleotide sequence of the antisense strand of the PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57% or about 58%.

29. The composition of claim 3, wherein the nucleotide sequence of the antisense strand of the PDL1 -specific asymmetric interfering RNA that is colinear with the corresponding complementary nucleotides in the target nucleotide sequence has a GC content of about 30%.

30. The composition of any one of claims 2 or 3, wherein the asymmetric interfering RNAs (aiRNA) comprise a modified ribonucleotide at one or more positions selected from:

• the 5 '-terminal nucleotide of the sense strand;

• the 3 '-terminal nucleotide of the sense strand;

• the 5'-terminal nucleotide of the antisense strand;

• the 3 '-terminal nucleotide of the antisense strand; or the first nucleotide adjacent to the 3 '-terminal nucleotide of the antisense strand.

31. The composition of claim 30, wherein the at least one modified nucleotide or its analogue is a sugar-modified ribonucleotide, wherein a 2 -OH group is replaced by H, OR, R, halo, SH, SR, NH₂, NHR, NR2, or CN, and wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, CI, Br, or I.

32. The composition of claim 30, wherein the modified ribonucleotide is a 2'-methoxy- ribonucleotide.

33. The composition of claim 30, wherein the nucleotides of the sense and/or antisense strands are not connected to any adjacent nucleotide via phosphorothioate linkages.

34. The composition of any one of claims 2-30, wherein the second nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand is not a 2'-methoxy-ribonucleotide.

35. The composition of any one of claims 2-30, wherein the second nucleotide adjacent to the 5 '-terminal nucleotide of the antisense strand is a 2'-flouro-ribonucleotide.

36. The composition of any one of the preceding claims, wherein the PDL1 -specific and/or β-catenin-specific aiRNA is bound to a peptide, an antibody, a polymer, a lipid, an oligonucleotide, cholesterol, or an aptamer.

37. The composition of any one of the preceding claims, wherein the PDL1 -specific and β-catenin-specific aiRNAs are encapsulated in a nanoparticle.

38. The composition of claim 37, wherein the nanoparticle comprises an aqueous core comprising a magnesium salt.

39. The composition of claim 38, wherein the magnesium salt is magnesium phosphate.

40. The composition of claim 38, wherein the aqueous core is encapsulated in a lipid phase.

41. The composition of claim 40, wherein the lipid phase comprises N41-(2,3-Dioleoyloxy) propyl ]-N,N,N-trimethylammonium chloride (DOTAP), cholesterol and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)-2000 (PEG2000- DSPE).

42. The composition of claim 37, wherein the nanoparticle comprises RNA, magnesium phosphate, DOTAP-cholesterol and PEG2000-DSPE.

43. The composition of claim 42, wherein the molar ratio of RNA: magnesium phosphate:

DOTAP-cholesterol: PEG2000-DSPE is 1: 5.62: 0.78: 0.42.

44. A pharmaceutical composition comprising the PDL1 -specific and β-catenin-specific aiRNAs of the compositions of any one of claims 1-35.

45. The pharmaceutical composition of claim 44, further comprising a lipid or a cholesterol molecule.

46. The pharmaceutical composition of claim 45 , wherein the lipid or cholesterol molecule is conjugated to the PDLl-specific and/or β-catenin-specific aiRNA.

47. The pharmaceutical composition of claim 44, wherein the PDLl-specific and β-catenin-specific aiRNAs comprise a plurality of modified nucleotides or their analogues, each modified nucleotide or its analogue comprising:

a 2'-0-methyl or a 2'-fluorine group, and/or

a phosphothioate or phosphodiester backbone.

48. A kit for silencing PDL1 and β-catenin gene expression in tumor cells comprising PDLl- specific and β-catenin-specific aiRNAs of the compositions of any one of claims 1-35, a nanoparticle formulation and instructions for their use.

49. An expression vector comprising a nucleic acid sequence encoding the PDLl-specific and/or β-catenin-specific aiRNAs of the compositions of any one of claims 2 or 3.

50. The expression vector of claim 49, wherein the expression vector is a viral, a eukaryotic, or a bacterial expression vector.

51. An isolated cell comprising the expression vector of claim 49.

52. An isolated cell comprising the PDL1 -specific and β-catenin-specific aiRNAs of any one of claims 1-35.

53. The cell of claim 52, wherein the cell is a mammalian, avian, insect, yeast or bacterial cell.

54. A method for treating cancer in a subject in need thereof comprising administering an effective amount of the PDL1 -specific and β-catenin-specific aiRNAs of one of claims 1- 35 to the subject, wherein the combination of PDL1 -specific and β-catenin-specific aiRNAs exhibits therapeutic synergy in the treatment of cancer.

55. The method of claim 54, wherein the PDL1 -specific and β-catenin-specific aiRNAs are in a nanoparticle formulation.

56. The method of claim 55, wherein the nanoparticle formulation comprises an aqueous core comprising a magnesium salt.

57. The method of claim 56, wherein the magnesium salt is magnesium phosphate.

58. The method of claim 56, wherein the aqueous core is encapsulated in a lipid phase.

59. The method of claim 58, wherein the lipid phase comprises N41-(2,3-Dioleoyloxy) propyl ]-N,N,N-trimethylammonium chloride (DOTAP), cholesterol and 1,2-distearoyl- sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)-2000 (PEG2000- DSPE).

60. The method of claim 55, wherein the nanoparticle formulation comprises RNA, magnesium phosphate, DOTAP-cholesterol and PEG2000-DSPE.

61. The method of claim 60, wherein the molar ratio of RNA: magnesium phosphate: DOTAP- cholesterol: PEG2000-DSPE is 1: 5.62: 0.78: 0.42.

62. The method of claim 54, wherein the cancer is an AIDS-Related cancer, a breast cancer, a cancer of the digestive/gastrointestinal tract, an endocrine and neuroendocrine cancer, a cancer of the eye, a genitourinary cancer, a germ cell cancer, a gynecologic cancer, a head and neck cancer, a hematologic cancer, a musculoskeletal cancer, a neurologic cancer, a respiratory/thoracic cancer, a skin cancer, a childhood cancer or a cancer of unknown primary.

63. The method of claim 54, wherein the cancer is metastatic, recurrent or resistant to chemotherapy and/or radiation.

64. The method of claim 54, wherein the PDL1 -specific and β-catenin-specific aiRNAs are administered systemically or locally.

65. The method of claim 54, wherein the PDL 1 -specific and β-catenin-specific aiRNAs of the compositions of any one of claims 1 -35 are effective at silencing PDL1 and β-catenin gene expression in a tumor.

66. The method of claim 54, wherein the PDL1 -specific and β-catenin-specific aiRNAs are effective at inhibiting tumor growth.

67. The method of claim 54, wherein the PDL1 -specific and β-catenin-specific aiRNAs are effective at inducing cancer stem cell death.

68. The method of claim 54, wherein the PDL1 -specific and β-catenin-specific aiRNAs are effective at enhancing an immune response against tumor cells.

69. The method of claim 54, wherein the cancer comprises cells that express nuclear β-catenin.

70. The method of claim 54, wherein the cancer comprises cells that do not express nuclear β-catenin.

71. The method of claim 54, wherein the PDL1 -specific and β-catenin-specific aiRNAs silence PDL1 and β-catenin gene expression for at least 8 hours.

72. The method of claim 55, wherein the PDL1 -specific and β-catenin-specific aiRNAs are delivered to tumors within 5 minutes of administration.