TW201726919A - Asymmetric interfering RNAs, and compositions, use, or preparation thereof - Google Patents

Abstract

The present teachings provide RNA molecules, including asymmetric interfering RNAs (aiRNAs), that are capable of reducing or inhibiting cancer cell's immune-evasion mechanisms by modulating the expression of immune checkpoint proteins; compositions thereof; and methods of using thereof. For example, one of the RNA molecules includes an antisense strand and a sense strand, where the antisense strand consists of 19-23 nucleotides, the sense strand consists of 14-17 nucleotides, where the sense strand is complementary to the antisense strand; and where the antisense strand comprises a sequence that is complementary to a target nucleotide sequence in an mRNA that encodes a ligand to PD-1.

Description

Asymmetric interfering RNA and its constituents, uses or preparations

Immuno-oncology is a promising new field for cancer treatment. The immune system is capable of delicate adaptation and selective targeting, a procedure that is now being utilized and is aimed at advanced cancer. Treatment in this field manipulates the immune response against cancer in a number of different ways. Vaccines have been developed with the purpose of eliciting a cellular and humoral immune response against a particular cancer antigen, which is very similar to the author of a vaccine for microbial diseases. Other treatments target cancer cells to avoid specific immune evasion mechanisms detected by the host immune system. These escape mechanisms are "checkpoints" of the immune system; the immune-effector cells are prevented from killing the specific cell surface molecules of the cells that express them.

The recent clinical success of using antibodies targeting the Programmed Cell Death-1 Receptor (PD-1) and its ligands (PD-L1, PD-L2) has confirmed the following general concept: cancer cells can hijack immune checkpoints Genes destroy endogenous anti-cancer surveillance through the immune system. example For example, ipilimumab (which was first approved in the United States in 2011) targets cytotoxic T lymphocyte-associated antigen 4 (CTLA-4); whereas nivolumab and pemrolizumab ) (both approved in the US for the first time in 2014) aimed at PD-1. Despite the initial success of such immunological checkpoint inhibition antibodies, immune-related adverse events (sometimes fatal events) have been observed, which has led to significant doubts about their 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 unmet need for novel, safe and effective anti-cancer compounds, compositions, and their use in treating cancer, and in particular agents having activity against immune evasion mechanisms employed by cancer cells. .

The teachings herein address this need by providing PD-L1 specific asymmetric interfering RNA (aiRNA) that prevents the signaling through immunological checkpoint molecules (PD-1). In some embodiments, the aiRNA line is highly effective in PD-L1 expression in silent tumors. In some specific aspects, inhibition of PD-L1 expression in tumor cells prevents activation of the PD-1 immunological checkpoint pathway in T cells, which is largely responsible for the downregulation of tumor cell-specific T cell cytotoxicity in cancer patients. The immune surveillance of cancer cells collapses at the same time.

In a first aspect, the teachings herein provide an RNA molecule comprising an antisense strand and a sense strand comprising 5' ends and 3 of 17, 18, 19, 20, or 21 nucleotides apart a terminal nucleotide, the sense strand comprising a 5' terminal nucleotide complementary to a nucleotide other than the 3' terminal nucleotide of the antisense strand and a 3' end complementary to the nucleotide of the antisense strand a terminal nucleotide, wherein at least 14 nucleotide lines of the antisense strand are complementary to the sense strand, and wherein at least 14 nucleotide strands of the antisense strand are associated with a nucleotide sequence of interest selected from the group consisting of The corresponding complementary nucleotide in the linear correspondence: sequence identification number 167, 168, 169, 170, 171, 172 or 173.

In some embodiments, at least 14 nucleotides of the antisense strand of the RNA molecule are linearly associated with a corresponding complementary nucleotide in a nucleotide sequence of interest selected from: sequence identification number 140 , 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 or 164.

In some embodiments, the antisense strand of the RNA molecule comprises a nucleotide sequence of: sequence identifiers 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91 or 92.

In some embodiments, the antisense strand of the RNA molecule comprises at least 7 nucleotides that linearly correspond to the corresponding complementary nucleotides in the nucleotide sequence of interest selected from: sequence identification number 93 , 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 , 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138.

In some embodiments, at least 7 of at least 14 nucleotides of the antisense strand of the RNA molecule are contiguous with a corresponding complementary nucleotide in a nucleotide sequence of interest selected from: And linearly corresponding: sequence identification number 167, 168, 169, 170, 171, 172 or 173.

In some embodiments, the sense strand of the RNA molecule comprises at least 14 A nucleotide in a nucleotide sequence of interest selected from the group consisting of SEQ ID NO: 167, 168, 169, 170, 171, 172 or 173.

In some embodiments, the sense strand of the RNA molecule comprises at least 14 nucleotides in a nucleotide sequence of interest selected from the group consisting of: sequence identifiers 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 or 164.

In some embodiments, the sense strand of the RNA molecule comprises a nucleotide sequence selected from the group consisting of: sequence number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.

In some embodiments, the 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides of the antisense strand of the RNA molecule are associated with a nucleotide of interest selected from the group consisting of The corresponding linearity in the sequence is discontinuous compared to the complementary nucleotide: sequence identifier 167, 168, 169, 170, 171, 172 or 173.

In some embodiments, the 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides of the antisense strand of the RNA molecule are not associated with a target nucleoside selected from the group consisting of The corresponding linearity in the acid sequence corresponds to nucleotide complementation: sequence identifier 167, 168, 169, 170, 171, 172 or 173.

In some embodiments, at least 14 nucleotides of the sense strand of the RNA molecule are contiguous and linearly corresponding to the corresponding complementary nucleotide of the antisense strand.

In some embodiments, the 5' terminal nucleotide of the sense strand of the RNA molecule is complementary to the first, second or third nucleotide adjacent to the 3' terminal nucleotide of the antisense strand.

In some specific aspects, the 3' terminal nucleoside of the sense strand of the RNA molecule The acid is complementary to the first, second or third nucleotide adjacent to the 5' terminal nucleotide of the antisense strand.

In some embodiments, the 5' end of the sense strand of the RNA molecule is 13 nucleotides from the 3' end nucleotide.

In some embodiments, the 19, 20 or 21 nucleotides of the antisense strand of the RNA molecule correspond linearly to a corresponding complementary nucleotide in a nucleotide sequence of interest selected from the group consisting of: Identification number 167, 168, 169, 170, 171, 172 or 173.

In some embodiments, the nucleotide sequence of the antisense strand of the RNA molecule that corresponds linearly to the corresponding complementary nucleotide of the target nucleotide sequence has the following GC content: 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%.

In some embodiments, the nucleotide sequence of the antisense strand of the RNA molecule that corresponds linearly to the corresponding complementary nucleotide of the target nucleotide sequence has the following GC content: about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43% or about 44%.

In some embodiments, the nucleotide sequence of the antisense strand of the RNA molecule that corresponds linearly to the corresponding complementary nucleotide of the target nucleotide sequence has the following GC content: 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%.

In some embodiments, the nucleotide sequence of the antisense strand of the RNA molecule that corresponds linearly to the corresponding complementary nucleotide of the target nucleotide sequence has a GC content of about 33%.

In some embodiments, the teachings herein provide an RNA molecule selected from the group consisting of aiRNA 1-46.

In some embodiments, the meaning or antisense strand of the RNA molecule comprises at least A modified nucleotide or analog thereof.

In some embodiments, the 2'-OH group of the at least one modified ribonucleotide or analog thereof is replaced with an H or 2'-O-methyl group.

In some embodiments, the at least one modified nucleotide or analog thereof is a sugar, backbone, and/or base modified ribonucleotide. In some embodiments, the backbone modified ribonucleotide comprises a modification in the linkage to a phosphodiester of another ribonucleotide, for example, to comprise a nitrogen or sulfur heteroatom. In some embodiments, the at least one modified nucleotide or analog thereof comprises a phosphorothioate group, an inosine or a tritylated base. In some embodiments, the at least one modified nucleotide or analog thereof is a modified sugar ribonucleotide, wherein a 2'-OH group is H, OR, R, halo, SH, SR, NH ₂ , NHR, NR ₂ , or CN are substituted, and wherein each R is independently a C1-C6 alkyl, alkenyl or alkynyl group, and the halogen group is F, Cl, Br, or I.

In some embodiments, the 5' terminal nucleotide of the antisense strand and the first nucleotide adjacent to the 5' terminal nucleotide comprise an "AA" motif, a "UU" motif, and a "CC" The phantom, "AU" phantom, "AC" phantom, "UA" phantom, "UC" phantom, or "CA" phantom.

In some embodiments, the disclosed RNA molecules comprise deoxyribonucleotides.

In some embodiments, the first nucleotide of the adjacent 3' terminal nucleotide of the antisense strand is not dT.

In some embodiments, the PD-L1 expression nucleotide sequence is silenced by the RNA molecule to be more potent than the anti-PD-L1 antibody. In some embodiments, the RNA molecule is more potent than the anti-PD-L1 antibody in enhancing tumor-specific T cell cytotoxicity.

In some embodiments, administration of the RNA molecule to a subject in need thereof does not induce an interferon response.

In some embodiments, the disclosed embodiments comprise a composition comprising one or more of the RNA molecules. In some embodiments, the composition can be a nanoparticle composition.

In some embodiments, the disclosed is a kit comprising one or more of the RNA molecule or a composition comprising one or more of the RNA molecules.

In some embodiments, the disclosures comprise expression vectors comprising a nucleic acid sequence encoding the RNA molecule. The expression vector can be a viral, eukaryotic, or bacterial expression vector.

In some embodiments, the provided is an isolated cell comprising a performance vector encoding the RNA molecule or the RNA molecule. The cell can be a mammal, a bird, an insect, a yeast or a bacterial cell.

In a second aspect, the disclosure is a method for treating a disease or condition comprising administering a therapeutically effective amount of the RNA molecule to a subject in need thereof.

In a third aspect, the disclosure is a method for treating, preventing, or ameliorating cancer in a subject, comprising administering a therapeutically effective amount of the RNA molecule to a subject in need thereof. In some specific aspects, the cancer is associated with AIDS-related cancer, breast cancer, cancer of the digestive tract/gastrointestinal tract, endocrine and neuroendocrine cancer, cancer of the eye, genitourinary cancer, germ cell cancer, gynecological cancer, head and Cervical cancer, blood cancer, musculoskeletal cancer, neurological cancer, respiratory/thoracic cancer, skin cancer, childhood cancer or cancer of unknown origin. The RNA molecule can be administered systemically or locally. The cancer may be metastatic, recurrent or resistant to chemotherapy and/or radiation.

Features and advantages of the teachings herein will be more readily apparent to those of ordinary skill in the art in the <RTIgt; It should be appreciated that certain features of the teachings hereinabove described hereinbefore and in the following may be combined to form a single embodiment for clarity and for the sake of brevity. The various features of the teachings herein described above in the context of a single embodiment can also be combined to form a sub-combination. It is intended that the invention be considered as illustrative and not restrictive.

Unless otherwise indicated, the teachings and techniques of the teachings herein are generally known in the art, and are generally described in the general and more specific references cited and discussed throughout the teachings herein. carried out. See, for example, MR Green and J. Sambrook, Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012), Ausubel et al, Current Protocols, John Wiley & Sons, Inc. (2000-2016), Antibodies: A Laboratory Manual, Second Edition, edited by Edward A. Greenfield, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2014), and RNA by Rio et al.: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2011), the owner of each of which is incorporated herein by reference.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains, unless otherwise defined. In case of conflict, the teachings (including definitions) of this article should be used. Some methods and materials are described herein for use in the teachings herein; 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.

As used herein, PD-L1 is a ligand for PD-1, which is also known in the art as a CD274 molecule, a CD274 antigen, a B7 homolog, a planned cell death 1 ligand 1 , PDCD1 ligand, PDCD1LG1, PDCD1L1, PD-L1, B7H1, PDL1, program death ligand 1, B7-H1 or B7-H. The PD-L1 gene encodes an immunosuppressive receptor ligand 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 with immunoglobulin V similarity and C-like functional domains. The interaction of this ligand with its receptor inhibits T cell activation and interleukin production. This interaction is important in preventing autoimmunity by maintaining a constant immune response during infection or inflammation of normal tissues. In the tumor microenvironment, this interaction provides immune escape to tumor cells by inactivation of cytotoxic T cells. The expression of this gene in tumor cells is considered to be prognostic in many types of human malignancy, including colon cancer and renal cell carcinoma. Selective splicing resulted in at least 4 transcript variants. Other diseases associated with CD274 include, for example, lymphoid epithelial cancer and Paget's disease.

In certain embodiments, the PD-L1 expression nucleotide sequence is a nucleotide sequence comprising a Homo sapiens CD274 molecule (CD274) transcript variant 1 (3,691) having the sequence of SEQ ID NO:166 At least 25 nucleotides of bp linear mRNA; NCBI reference sequence: NM_014143.3). Transcript variant 1. This variant represents the longest transcript and encodes the longest isoform (a).

In some embodiments, the PD-L1 expression nucleotide sequence may be related to a nucleotide sequence comprising a Homo sapiens CD274 molecule (CD274) transcript variant 2 (3,349 bp linear mRNA; accession number: NM_001267706) .1) at least 25 nucleotides. Compared to variant 1, this variant lacks a selective in-frame exon in the 5' coding region, which results in a shorter protein (same b) than the isoform a.

In some embodiments, the PD-L1 expression nucleotide sequence may be related to a nucleotide sequence comprising a Homo sapiens CD274 molecule (CD274) transcript variant 3 (3,518 bp linearly transcribed RNA; accession number :NR_052005.1) at least 25 nucleotides.

In some embodiments, the PD-L1 expression nucleotide sequence may be related to a nucleotide sequence comprising a Homo sapiens CD274 molecule (CD274) transcript variant 4 (907 bp linear At least 25 nucleotides of mRNA; accession number: NM_001314029.1).

Unless otherwise specified, a singular may also include a plural. For example, "a" can relate to one or one or more.

As used herein, the phrase "and/or" is used in the teachings of the text and in the scope of the claims to be understood to mean "one or all" of the connected elements (ie, in some instances, In other examples, it is not a component that appears in conjunction.) Thus, as a non-limiting example, when used in connection with an open expression (such as "include"), reference to "A and/or B" in a particular aspect may relate to only A (optionally including B) In another embodiment, in relation to only B (optionally including elements other than A); and in yet another specific aspect, relating to both A and B (optionally included) Other components); and so on.

When a range of numerical values is recited herein, it is intended to cover the various values and sub-ranges within the range. For example, "1-5 nucleotides" is intended to cover 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 1-2 nucleosides. Acid, 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.

When the term "about" is used in connection with a numerical range, it is modified by extending the boundaries above and below the numerical values. In general, the term "about" is used herein to modify a value to a variation of 20%, 10%, 5%, or 1% above or below the stated value. In some embodiments, the term "about" is used to modify a value to a variation of 10% above or below the stated value. In some embodiments, the term "about" is used to modify a value to a value of 5% above or below the stated value. In some embodiments, the term "about" is used to modify a value to a value of 1% above or below the stated value.

The term "acceptable" is to be compatible with the other ingredients of the formulation and is not deleterious to the patient.

As used herein, the term "acyclic nucleotide" is generally used with respect to any nucleoside having any acyclic ribose and, for example, one of ribose carbon/carbon or carbon/oxygen bonds, either independently or in combination. A nucleotide that is not present in the acid.

As used herein, the term "alkyl" is generally used with respect to saturated or unsaturated hydrocarbons which include straight chain, branched alkyl, alkenyl, alkynyl groups, and cyclic groups, but excludes aromatics. Group. In spite of the foregoing, alkyl groups are also related to non-aromatic heterocyclic groups. Preferably, the alkyl group has from 1 to 12 carbons. More preferably, it is a lower alkyl group of 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted, the substituent is preferably hydroxy, halo, cyano, C1-C4 alkoxy, =0, =S, NO ₂ , SH, NH ₂ or NR ₁ R ₂ wherein R ₁ and R ₂ may independently be H or a C1-C4 alkyl group. Preferably, the alkyl group is a C1-C4 alkyl group.

As used herein, the term "aryl" generally relates to an aromatic group having at least one ring having a conjugated π-electron system and comprising a carbocyclic aryl group, a heterocyclic aryl group, and a bisaryl group, The owners of them can be replaced as needed. Preferred substituents for the aryl group may be halogen, trihalomethyl, hydroxy, SH, OH, cyano, C1-C4 alkoxy, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 Alkynyl, NH ₂ and NR ₁ R ₂ , wherein R ₁ and R ₂ may independently be H or a C1-C4 alkyl group.

As used herein, the term "alkylaryl" is generally used with respect to an alkyl group (as described above) which is covalently bonded to an aryl group (as described above). The carbocyclic aryl group may be a group in which the ring atoms on the aromatic ring may all be carbon atoms. Its carbon atoms can be substituted as needed. The heterocyclic aryl group may be a group having from 1 to 3 hetero atoms as a ring atom in the aromatic ring and the remainder of the ring atom may be a carbon atom. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and examples of heterocyclic aryl groups having such heteroatoms include furyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl alkyl pyrrolido, Pyrimidinyl, pyridyl Base, imidazolyl and the like, all of which are optionally substituted.

As used herein, the term "guanamine" is generally referred to as -C(O)-NH-R, wherein R can be alkyl, aryl, alkylaryl, or hydrogen.

As used herein, the term "antisense region" is used with respect to the general recipients of the art. An exemplary nucleic acid molecule pertaining to the teachings herein is the nucleotide sequence of an aiRNA molecule that is complementary to the nucleic acid sequence represented by the gene of interest. Furthermore, the antisense region of an aiRNA molecule can optionally comprise a nucleic acid sequence that is complementary to the region of interest of the aiRNA molecule. In some embodiments, the antisense region of the aiRNA molecule is referred to as an antisense strand or a guide strand. In some embodiments, the antisense strand can have a nucleotide of 14, 15, 16, or 17 base pairs with the sense strand. In some embodiments, the antisense strand can have 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 complementary linear nucleosides corresponding to the corresponding nucleotide sequence in the target nucleotide sequence. The acid is a discontinuous nucleotide compared to the nucleotide. In some embodiments, the antisense strand can have 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 linear corresponding nucleosides that do not correspond to the target nucleotide sequence. An acid complementary nucleotide. In some embodiments, the 19, 20 or 21 nucleotides of the antisense strand can linearly correspond to a corresponding complementary nucleotide in the nucleotide sequence of interest.

The terms "asymmetric interfering nucleic acid", "aiRNA", "asymmetric interfering oligonucleotide molecule", or "chemically modified asymmetric interfering nucleic acid molecule" are for any antisense stock and meaning strand and the length of the two strands Can be different nucleic acid molecules. These nucleic acid molecules can be One-way mediates RNA interference ("RNAi") or gene silencing to inhibit or downregulate gene expression or viral replication. These terms may be with respect to individual nucleic acid molecules, plural such nucleic acid molecules, or pools of such nucleic acid molecules. The aiRNA may be a double-stranded nucleic acid molecule comprising a complementary sense and an antisense strand, wherein the antisense strand comprises a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule and the sense strand comprises a corresponding The nucleotide sequence of the target nucleic acid sequence or a portion thereof. The aiRNA may also be a double-stranded nucleic acid molecule comprising a complementary sense and an antisense strand, wherein the antisense strand comprises a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule and the sense strand comprises a corresponding The nucleotide sequence of the target nucleic acid sequence or a portion thereof has a gap (lack of a nucleotide) or a gap (lack of two or more nucleotides).

As used herein, the term "biological system" generally refers to materials in purified or unpurified form from biological sources including, but not limited to, humans or animals, wherein the system comprises the components required for RNAi activity. Thus, the term encompasses, for example, a cell, tissue, subject, or organism, or an extract thereof. The term also encompasses reconstituted materials from biological sources.

As used herein, the term "blunt end" is used in the sense that it is generally accepted in the art. An exemplary nucleic acid molecule pertaining to the teachings herein is directed to the ends of a double-stranded aiRNA molecule that does not have overhanging nucleotides. For example, two strands of a double-stranded aiRNA molecule having a blunt end are aligned with each other with an aligned base pair and no protruding nucleotides at the ends. The aiRNA double-stranded molecule of the teachings herein may be at one or both ends of the double strand (such as the end of the double strand at the 5' end of the antisense strand, the end of the 5' end of the sense strand, or both ends) Contains a blunt end. In some embodiments, the base of the 3' terminal nucleotide of the sense strand forms a blunt end when paired with the base of the 5' terminal nucleotide of the antisense strand.

As used herein, the term "cell" relates to it in the art. The meaning of being accepted. With respect to exemplary nucleic acid molecules of the teachings herein, the term can be used in its ordinary biological sense and is not related to the entire multicellular organism, such as, in particular, to humans. The cells may be present in an organism (eg, birds, plants, and mammals such as humans, cows, sheep, baboons, monkeys, pigs, dogs, and cats). The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell). The cell may be a cell of a somatic cell or a germ cell organ, a differentiated pluripotent or differentiated pluripotent cell, a dividing or non-dividing cell. The cell may also be derived from or may comprise a gamete or embryo, a stem cell, or a fully differentiated cell.

As used herein, the term "chemical modification" is used in the sense that it is generally accepted in the art. An exemplary nucleic acid molecule with respect to the teachings herein, is a modification of the chemical structure of any nucleotide that is substantially different from the nucleotide of the native nucleic acid. The term "chemical modification" encompasses, for example, the addition, substitution, or modification of a natural RNA to a sugar, base, or internucleotide linkage, as described herein or otherwise as known in the art. In some embodiments, the term "chemical modification" may refer to a form of RNA that occurs naturally in a biological system, such as a 2'-O-methyl modification or an inosine modification.

As used herein, the terms "complementarity" or "complementary" are used in the sense that they are generally accepted in the art. With respect to exemplary nucleic acid molecules of the teachings herein, the term is generally directed to the formation or presence of a conventional Watson-Crick or other non-conventional type of hydrogen bond that is bonded between a nucleic acid sequence and another nucleic acid sequence. (As described herein), a double-stranded region paired with one base is formed. With respect to the nucleic acid molecules of the teachings herein, the binding free energy of the nucleic acid molecule to its complementary sequence is sufficient to permit the associated function of the nucleic acid, such as RNAi activity. "Perfect complementarity" means that all consecutive residues of a nucleic acid sequence can be associated with a second nucleic acid sequence. The same number of consecutive residues are hydrogen bonded. "Partial complementarity" may include a variety of non-identical or base-unpaired nucleotides within a nucleic acid molecule (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more do not match) a non-nucleotide linker, or a nucleotide that is not paired with a base), which can result in an antisense strand or antisense between the sense strand or region of interest of the nucleic acid molecule and the antisense strand or antisense region or between the nucleic acid molecule A bulge, an inner loop, or a protrusion between the region and the corresponding target nucleic acid molecule. Such partial complementarity can be manifested by % complementarity, which is determined by the number of base unpaired nucleotides, ie, about 20%-99%, depending on the total number of nucleotides involved. The extent to which such partial complementarity can be allowed is the ability of the nucleic acid molecule (e.g., aiRNA) to maintain its function, e.g., to mediate sequence-specific RNAi. By "substantial complementarity" is meant that the sequences are complementary to each other sufficiently to be heterozygous under the selected reaction conditions. Two substantially complementary strands may, for example, be perfectly complementary or may contain from 1 to many mismatches, as long as the hybridization conditions are sufficient to allow, for example, to distinguish between the paired sequence and the unpaired sequence. Thus, a substantially complementary sequence may be related to a sequence having about 50% to 100% base pair complementarity in a double stranded region. In some specific contexts, the term "complementarity" may relate to "perfect complementarity," "partial complementarity," or "substantial complementarity."

A ribonucleotide consists of a phosphate group, a ribose group, a nucleobase which can be adenine (A), guanine (G), cytosine (C), or uracil (U). The RNA strand is a chain of ribonucleotides linked by a phosphodiester bond between the 5'-phosphate of one nucleotide and the 3' hydroxyl group of the next nucleotide. However, in some embodiments, the chain of ribonucleotides may comprise a phosphodiester bond other than the 5'-phosphate of one nucleotide and the 3' hydroxyl group of the next nucleotide. key.

In double-stranded or double-stranded RNA, one or more ribonucleotides are stably linked to complementary ribonucleotides in the other strand. The complementarity between these shares is between A and U Caused by interactions or "base pairing" between G and C (see, for example, Table IA).

In some embodiments, the RNA duplex can have perfectly complementary or partially complementary RNA strands, depending on the number of under-synchronizations (ie, nucleotides that are not paired with bases in the RNA duplex) (see ( For example) Table IB).

As used herein, the term "alignment ratio" is a procedure for comparing the nucleotide sequences of two or more nucleotide sequences to assess the degree of sequence identity of such sequences. As used herein, "anastomosis" relates to the ratio of two or more nucleotide sequences having 100% sequence identity.

Thus, for example, in Table II below, column 1 is aligned with column 2 and matches.

Since the identity of the bases on one strand of the RNA duplex can be used to infer the identity of the corresponding bases on the other strand, the term "alignment" can also refer to a nucleotide sequence in a strand of interest. Comparison with complementary sequences in another RNA strand (antisense strand). Thus, for example, in Table II, because the complementary sequence of the nucleotide sequence of column 3 coincides with the nucleotide sequence in column 2, column 2 is aligned with column 3.

For example, in some embodiments, the nucleotide sequence of an antisense strand of an aiRNA can be aligned with its perfectly complementary or partially complementary nucleotide sequence in the nucleotide sequence of interest.

In general, the term "continuous" relates to nucleotides that are adjacent to each other in a polynucleotide chain.

In some specific aspects, the term "continuous" may relate to a multicore

Nucleotides in the nucleotide chain that are contiguous with each other and that match a corresponding nucleotide in a second polynucleotide chain.

Thus, for example, in Table III, since nucleotides 1-11 of RNA strand 2 coincide with nucleotides 1-11 of RNA strand 1 without any intermediate inconsistency, positions 1 to 11 of RNA strand 2 The nucleotides are continuous with respect to the corresponding nucleotides 1 to 11 of the RNA strand 1.

In some embodiments, the term "continuous" may also refer to a nucleotide sequence that is equal to one another in a first polynucleotide chain and that is perfectly complementary to a second polynucleotide chain. Nucleotide.

Thus, for example, in Table III, the nucleotide phase of the RNA strand 1 is because there is no intermediate inconsistency between the individual nucleotides of the RNA strand 1 and the corresponding complementary nucleotides in the RNA strand 3. The nucleotide sequence in RNA strand 3 is contiguous.

In general, the term "linear correspondence" is used to describe 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 III, since the linear order of nucleotides 1-11, 14 and 15 of RNA strand 2 coincides with the corresponding nucleotide of RNA strand 1, the RNA strand 2 line linearly corresponds to RNA strand 1, Despite the lack of sequence identity at positions 12 and 13.

In some embodiments, the term "linear correspondence" also dictates between the linear order of nucleotides in a first RNA strand and the linear order of corresponding complementary nucleotides in a second RNA strand. 1:1 relationship.

Thus, for example, in Table III, because the linear order of nucleotides 1-11, 14 and 15 of RNA strand 2 is aligned with the corresponding complementary nucleotides at positions 1-111, 14 and 15 of RNA strand 3, The RNA strand 2 line corresponds linearly to the RNA strand 3, although the two strands are partially complementary.

In contrast, since the linear order of nucleotides in RNA strand 3 does not coincide with the linear order of nucleotides in RNA strand 4 due to insertion of additional nucleotides at position 3, RNA strand 3 does not interact with RNA. The stock 4 corresponds linearly. Likewise, the linear order of nucleotides in RNA strand 2 is again linearly corresponding to the linear order of the corresponding complementary nucleotides in RNA strand 4 because of the additional nucleotides at position 3.

In some embodiments, the term "linear correspondence" relates to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the antisense strand of an aiRNA. The linear order of more nucleotides is in a 1:1 relationship with the linear order of the corresponding complementary nucleotides in the target nucleotide sequence. In some embodiments, the antisense strand and the sense strand of the aiRNA may have perfect complementarity or partial complementarity.

In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more nucleotide phases in the antisense strand of the aiRNA The corresponding complementary nucleotide sequence is contiguous compared to the nucleotide sequence of interest. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 or more nucleosides in the antisense strand of the aiRNA The acid phase is discontinuous compared to the corresponding complementary nucleotide sequence in the nucleotide sequence of interest.

An exemplary 5' overhang of aiRNA, which is a teaching of the teachings herein, is shown below, wherein the 3' terminal nucleotide of the sense strand is complementary to the third nucleotide of the 5' terminal nucleotide adjacent to its antisense strand:

An exemplary 5' overhang of aiRNA, which is a teaching of the teachings herein, is shown below, wherein the 3' terminal nucleotide of the sense strand is complementary to the second nucleotide of the 5' terminal nucleotide adjacent to its antisense strand:

An exemplary 5' overhang of aiRNA, which is a teaching of the teachings herein, is shown below, wherein the 3' terminal nucleotide of the sense strand is complementary to the first nucleotide adjacent to the 5' terminal nucleotide of its antisense strand:

An exemplary depiction of aiRNA, as taught herein, is shown below, wherein the 5' terminal nucleotide of the sense strand is complementary to the 3' terminal nucleotide of its antisense strand:

An exemplary 3' overhang of aiRNA, which is a teaching of the teachings herein, is shown below, wherein the 5' terminal nucleotide of the sense strand is complementary to the first nucleotide adjacent to the 3' terminal nucleotide of its antisense strand:

An exemplary 3' overhang of aiRNA, which is a teaching of the teachings herein, is shown below, wherein the 5' terminal nucleotide of the sense strand is complementary to the second nucleotide adjacent to the 3' terminal nucleotide of its antisense strand:

An exemplary 3' overhang of aiRNA, which is a teaching of the teachings herein, is shown below, wherein the 5' terminal nucleotide of the sense strand is complementary to the third nucleotide adjacent to the 3' terminal nucleotide of its antisense strand:

As used herein, the term "composition" or "mixture" is used in the sense that it is generally accepted in the art. Such terms are generally in relation to a composition or formulation, such as in a pharmaceutically acceptable carrier or diluent, and in a suitable form for administration (eg, systemic or topical administration) to a cell or subject ( Contains forms such as humans. The appropriate form depends, in part, on its use or route of entry (eg, oral, transdermal, inhaled, or by injection). Such a form should not prevent the composition or formulation from reaching the target cell (i.e., the cell to which the negatively charged nucleic acid is desired to be delivered). For example, the composition that is injected into the bloodstream should be soluble. Other factors are known in the art and include considerations such as toxicity and form that prevent the composition or formulation from exerting its efficacy. As used herein, pharmaceutical formulations comprise formulations for human and veterinary use. Non-limiting examples of agents suitable for use in formulations of nucleic acid molecules having the teachings herein include: lipid nanoparticles (see, eg, Semple et al, 2010, Nat Biotechnol., February; 28(2) 1726); - glycoprotein inhibitors (such as Pluronic P85); biodegradable polymers such as poly(DL lactide-co-glycolide) microspheres for sustained release delivery (Emerich, DF et al, 1999, Cell Transplant) , 8, 47-58); with loaded Nanoparticles, such as those made from polybutylcyanoacrylate. Non-limiting examples of other delivery strategies for nucleic acid molecules for use in the teachings herein include those described in Boado et al., 1998, J. Pharm Sci., 87, 1308-1315; Tyler et al., 1999. , FEBS Lett., 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.

As used herein, the term "cancer" in a subject relates to cells having uncontrolled proliferation, immortality, metastatic potential, rapid growth and increased proliferation rate, and certain morphological characteristics. Cancer cells can aggregate in the form of tumors or clumps and/or circulate in separate blood cells or lymphatic system.

The term "cancer" includes, for example, AIDS-related cancer, breast cancer, gastrointestinal/gastrointestinal cancer, endocrine and neuroendocrine cancer, cancer of the eye, genitourinary cancer, germ cell cancer, gynecological cancer, head and neck cancer, Hematological cancer, musculoskeletal cancer, neurological cancer, respiratory/thoracic cancer, skin cancer, childhood cancer, and cancer of unknown origin.

Exemplary AIDS-related cancers include, but are not limited to, AIDS-related lymphomas, primary central nervous system lymphomas, and Kaposi's sarcoma.

Exemplary breast cancers include, but are not limited to, in situ ductal carcinoma (DCIS), invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), triple-negative breast cancer (where tumor cells are for progesterone, estrus , with her2/neu negative system), inflammatory breast cancer, metastatic breast cancer, breast cancer during pregnancy, Paget's disease of nipple, phyllodes tumor, adenoid cystic (or cystic) carcinoma, low grade Adenosquamous carcinoma, myeloid carcinoma, ductal carcinoma, mastoid carcinoma, mucinous (colloidal) carcinoma, breast lymphoma, glandular muscle epithelioma, giant cell sarcoma of the breast, leiomyosarcoma of the breast, milk Angiosarcoma, phyllodes cystosarcoma, liposarcoma of the breast, breast cancer, acinar cell carcinoma, eosinophilic granulosa cell carcinoma (mammary epithelial eosinophilic granulosa), mucinous epithelial carcinoma, breast spinning Sputum cell carcinoma, squamous cell carcinoma of the breast, secretory carcinoma of the breast (adolescent secretory cancer), metaplastic carcinoma of the breast, invasive microemulsion of the breast, adenoid cystic carcinoma of the breast, sieve Carcinoma, breast myofibroblastoma (benign spindle stromal tumor of the breast) and hepatic glycogen-free clear cell carcinoma of the breast.

Exemplary gastrointestinal/gastrointestinal cancers include, but are not limited to, anal cancer, appendic cancer, gastrointestinal cancer, cholangiocarcinoma, carcinoid tumor, gastrointestinal cancer, colon cancer, esophageal cancer, gallbladder cancer, gastrointestinal stromal tumor (GIST) ), islet cell tumor, pancreatic neuroendocrine tumor, liver cancer, pancreatic cancer, rectal cancer, small intestine cancer, gastroesophageal junction (GEJ) cancer, and stomach (stomach) cancer.

Exemplary endocrine and neuroendocrine cancers include, but are not limited to, adrenocortical carcinoma, gastrointestinal cancer, islet cell tumor, pancreatic neuroendocrine tumor, Merkel's cell carcinoma, non-small cell lung neuroendocrine tumor, small cell lung neuroendocrine Tumor, parathyroid cancer, pheochromocytoma, pituitary tumor, and thyroid cancer.

Exemplary genitourinary cancers include, but are not limited to, bladder cancer, kidney (kidney cell) cancer, penile cancer, prostate cancer, renal pelvis and ureteral cancer, transitional cells, testicular cancer, urethral cancer, Wilms' tumor, and other children Kidney tumor.

Exemplary gynecological cancers include, but are not limited to, cervical cancer, endometrial cancer, fallopian tube cancer, gestational mesoderm tumor, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, primary peritoneal cancer, uterus Sarcoma, vaginal cancer and genital cancer.

Exemplary head and neck cancers include, but are not limited to, hypopharyngeal, laryngeal, lip and oral cancer, metastatic squamous neck cancer with unidentified primary site, oral cancer, nasopharyngeal cancer, oral cancer, and lips And oropharyngeal cancer, paranasal sinus and nasal cancer, parathyroid cancer, pharyngeal cancer, salivary gland cancer, laryngeal cancer and thyroid cancer.

Exemplary hematological cancers include, but are not limited to, leukemia, acute lymphoblastic leukemia, adult, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelosuposis Leukemia, hairy cell leukemia, lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma, adult Hodgkin's lymphoma, Hodgkin's lymphoma in children, Hodgkin's lymphoma during pregnancy, Verrucous granuloma, non-Hodgkin's lymphoma in children, adult non-Hodgkin's lymphoma, non-Hodgkin's lymphoma during pregnancy, primary central nervous system lymphoma, Sézary's syndrome, skin T Cellular lymphoma, Waddenstrom's macroglobulinemia, chronic myeloproliferative neoplasms, Langerhans cell histopathy, multiple myeloma/plasma neoplasms, myelodysplastic syndrome and myelodysplasia / myeloproliferative neoplasms.

Exemplary musculoskeletal cancers include, but are not limited to, bone cancer, You Ying's sarcoma, osteosarcoma, malignant fibrous histiocytoma of the bone, rhabdomyosarcoma of children and soft tissue sarcoma.

Exemplary neurological cancers include, but are not limited to, adult brain tumors, childhood brain tumors, astrocytomas, brain and spinal cord tumors, brainstem gliomas, atypical terats/striated muscle-like central nervous system tumors, embryonic central nervous system Tumor, germ cell central nervous system tumor, craniopharyngioma, ependymoma, neuroblastoma, pituitary tumor and primary central nervous system (CNS) lymphoma.

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

Exemplary skin cancers include, but are not limited to, cutaneous T-cell lymphoma, card Persian sarcoma, melanoma, Merkel's cell carcinoma, skin cancer, cutaneous T-cell lymphoma, verrucous granuloma and Sézary's syndrome.

Also included in the term "cancer" is a "solid tumor." As used herein, the term "solid tumor" relates to such conditions (such as cancer) that form abnormal tumor masses, such as sarcomas, carcinomas, and lymphomas. Examples of solid tumors include, but are not limited to, non-small cell lung cancer (NSCLC), neuroendocrine tumors, thymoma, fibroid tumors, metastatic colorectal cancer (mCRC), and the like. In some embodiments, the solid tumor disease can be an adenocarcinoma, a squamous cell carcinoma, a large cell carcinoma, and the like.

As used herein, with respect to exemplary nucleic acid molecules of the teachings herein, the term "corresponding" relates to the relationship between the sequence of the target nucleotide sequence and the sequence in the antisense strand of the aiRNA molecule of the teachings herein. The term may be modified by the words "partial," "substantially," or "completely" to indicate the degree of the relationship. In some embodiments, a portion correspondingly means that about 20-99% of the ribonucleotides A, U, G, and C lines in the antisense strand of the aiRNA sequence are complementary to their corresponding target nucleotide sequences. In some embodiments, substantially corresponding to about 50%-100% of the ribonucleotides A, U, G, and C lines in the antisense strand of the aiRNA sequence and the corresponding target nucleotide sequence Complementary. In some embodiments, the nucleotide sequence of interest can be a PD-L1 mRNA sequence. In some embodiments, the nucleotide sequence of interest can be the nucleotide sequence of SEQ ID NO: 166. In some embodiments, the nucleotide sequence of interest comprises a sequence selected from the sequence identification numbers 93-138.

The term "cytotoxic agent/cytostatic agent" relates to a compound which mainly causes cell death or inhibits cell proliferation by directly interfering with the function of cells or inhibiting or interfering with cell mitosis, and comprises an alkylating agent, a tumor necrosis factor, an intercalating agent. , hypoxia-activatable compounds, microtubule inhibitors/microtubule stabilizers, inhibitors of mitotic actin, tissue proteins to B Inhibitors of chymase, inhibitors of kinases involved in mitosis progression, antimetabolites; biological response modifiers; hormone/antihormone therapeutics, hematopoietic growth factors, monoclonal antibody target therapeutics, topoisomerase inhibition Agent, proteasome inhibitor and ubiquitin-binding enzyme inhibitor.

As used herein, the term "deoxyribonucleotide" is used in the sense that it is generally accepted in the art. The term is generally referred to as a nucleotide having a proton at the 2' position of the ?-D-deoxyribofuranosyl moiety. This term also encompasses any chemically modified deoxyribonucleotides. "dT" is about 2'-deoxythymidine.

As used herein, "polynucleotide" relates to a polymeric chain containing two or more nucleotides. "Polynucleotide" includes primers, oligonucleotides, nucleic acid strands, and the like. Polynucleotides can contain standard or non-standard nucleotides. Typically, the polynucleotide contains a 5' phosphate ("5' end") at one end of the strand and a 3' hydroxyl group ("3' end") at the other end. The most 5' nucleotide of a polynucleotide may be referred to herein as the "5' terminal nucleotide" of the polynucleotide. The most 3' nucleotide of a polynucleotide may be referred to herein as the "3' terminal nucleotide" of the polynucleotide.

As used herein, "double stranded RNA", "double stranded RNA" or "RNA duplex" is a double stranded RNA having at least one double stranded region and is contained within a double stranded region or at two adjacent There are, for example, at least one gap, gap, protrusion, and/or bubble of RNA molecules between the double-stranded regions. In some embodiments, a strand is considered to have multiple segments if there is a single-strand region with gaps or non-appropriate nucleotides between the two double-strand regions. In some embodiments, double-stranded RNA, as used herein, can have terminal projections at either or both ends. In some embodiments, the double strands of the double stranded RNA can be joined by a chemical linker.

As used herein, the term "effective amount" of an active agent is in an amount sufficient to induce a desired biological response. If it is to be understood by those of ordinary skill in the art, this article The effective amount of the RNA molecule of the teachings will vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the molecule, the disease to be treated, the mode of administration, and the patient.

An "effective amount" of an anti-cancer agent that reduces the growth of cancer cells means that the amount of growth and/or metastasis of some cancer or tumor cells can be reduced (to some extent). The term encompasses an amount that is capable of causing growth inhibition, cytostatic and/or cytotoxic efficacy, and/or apoptosis of a cancer or tumor cell.

As used herein, the terms "gene" or "target gene" or "target nucleotide sequence" are used in relation to what is generally accepted in the art. The term generally refers to a target nucleotide (eg, DNA or RNA) sequence comprising a coding sequence of a partial length or the entire length necessary for the production of a polypeptide (eg, PD-L1 or PD-L2). In some embodiments, the nucleotide sequence of interest may also comprise a non-translated region (UTR) or a non-coding region of the gene of interest. In some embodiments, a gene or a target gene may also encode a functional RNA (fRNA) or a non-coding RNA (ncRNA), such as a small transient RNA (stRNA), a small RNA (micro RNA, miRNA), Small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and its precursors. In certain embodiments, such non-coding RNA can serve as a target nucleotide sequence for aiRNA-mediated RNA interference that modulates the activity of fRNA or ncRNA involved in a functional or regulatory cellular program. Abnormal fRNA or ncRNA activity leading to disease can therefore be modulated by aiRNA molecules as taught herein. In some embodiments, aiRNA molecules targeting fRNA and ncRNA can also be used to intervene in cellular programs such as genetic imprinting, transcription, translation, or nucleic acid processing (eg, amino transfer, methylation, etc.). Manipulate or alter the genotype or phenotype of an object, organism, or cell. In some embodiments, the target gene can be derived from a cell A gene, an endogenous gene, a transgenic gene, or an exogenous gene, such as a gene of a pathogen (such as a virus), which occurs in a cell after infection by a cell. The cell containing the gene of interest may be derived or contained in any organism (eg, a plant, animal, protozoa, virus, bacterium, or fungus). Non-limiting examples of plants include monocots, dicots, or gymnosperms. Non-limiting examples of animals include vertebrate or invertebrates. Non-limiting examples of fungi include mold or yeast. For a review, see, for example, Snyder and Gerstein, 2003, Science, 300, 258-260.

As used herein, the term "homologous sequence" is used in the sense that it is generally accepted in the art. The term is generally directed to a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts, and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is encoded by two or more genes encoding related but different proteins (such as different members of a gene family, different protein epitopes, different protein isoforms or complete Different genes are shared. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as non-coding DNA or RNA, regulatory sequences, introns, and positions controlled or regulated by transcription. A homologous sequence can also comprise a region of a sequence that is shared by more than one polynucleotide sequence. Homology does not need to be a perfect consistency (100%), and this is because some homologous sequences are also considered and fall within the scope of the teachings herein (eg, at least about 95%, about 94%, about 93). %, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, About 80%, etc.). Percent homology is the number of nucleotides that are anastomosed between two sequences divided by the total length compared to multiplied by 100.

The term "improved RNAi activity" relates to in vitro and/or in vivo measurements An increase in RNAi activity, wherein the RNAi activity is a reflection of both the ability of aiRNA-mediated RNAi and the stability of the aiRNA as taught herein. In the teachings herein, the products of such activities can be increased in vitro and/or in vivo compared to siRNA, shRNA, or another RNA containing a plurality of ribonucleotides. In some instances, the activity or stability of the aiRNA molecule can be reduced (ie, less than ten fold) in vitro and/or in vivo, but the overall activity of the aiRNA molecule can be increased.

As used herein, the terms "inhibiting," "down," or "decreasing" are used in the sense that they are generally accepted in the art. With respect to an exemplary nucleic acid molecule of the teachings herein, the term generally refers to the expression of a gene, or the level of an RNA molecule or equivalent RNA molecule encoding one or more protein or protein subunits, or one or more protein or protein subunits. The decrease in activity is less than that observed in nucleic acid molecules (e.g., aiRNA) lacking the teachings herein. Down-regulation can also be associated with post-transcriptional silence, such as RNAi-mediated cleavage or by changes in DNA methylation patterns or DNA chromatin structure. The aiRNA molecule is used to inhibit, down-regulate or reduce an aiRNA molecule that can be associated with an inactive molecule, attenuated molecule, a scrambled sequence, or a non-analogous aiRNA molecule, or alternatively it can be associated with a system lacking the nucleic acid.

The terms "internucleoside linkage" or "internucleoside linkage" or "internucleotide linkage" or "nucleoside linkage" are used interchangeably herein and are meant to be between any two nucleoside units. Linkers or linkages, as known in the art, include, but are not limited to, for example, phosphates, phosphate analogs, phosphonates, hydrazines, hydroxylamines, hydroxy fluorenyls, hydrazines An amine, a carbamate, an alkyl group, attached to a substituted alkyl group. The internucleoside linkages constitute the backbone of the nucleic acid molecule.

As used herein, the term "isolated" or "purified" relates to a material. It is substantially or substantially free of the components normally associated with it in its natural state. Purity and homogeneity can typically be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

As used herein, the terms "mammalian" or "mammalian" are used in the sense that they are generally accepted in the art. The term is generally with respect to any warm-blooded vertebrate species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, domestic animals, and the like.

As used herein, the term "modulation" is used in the sense that it is generally accepted in the art. An exemplary nucleic acid molecule pertaining to the teachings herein, the term being related to the expression of a gene, or the level of one or more RNA molecules (encoded or non-coding), or one or more RNA molecules or protein or protein subunits When the activity is up-regulated or down-regulated, the performance, level, or activity is greater or less than that observed in the absence of the molecules that cause modulation. For example, the term "modulate" in certain instances may relate to, for example, inhibition of gene expression (eg, gene silencing) and in other specific aspects may be related to, for example, enhancement or up-regulation of gene expression. As used herein, "gene silencing" relates to reducing gene expression and may be about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% of the target gene, Approximately 90% or approximately 95% of the gene performance is reduced.

As used herein, the term "modified nucleotide" is used in the sense that it is generally accepted in the art. The term generally refers to a nucleotide comprising a chemical structure of a base of an unmodified (or native) nucleotide, a modification of a sugar and/or a phosphate, as generally known in the art. .

The term "base unpaired" relates to the stocks in the double-stranded aiRNA molecule. Or nucleotides in which the sense region is not paired with the antisense strand or the antisense region; and may include, but is not limited to, for example, a mismatch, a protrusion, a single inner loop, and the like.

The term "nucleotide" (or "nt") is used as generally recognized in the art. Nucleotides typically comprise nucleobases, sugars, and internucleoside linkages, such as phosphodiester linkages. The base can be a natural base (standard), a modified base, or a base analog. Such bases can generally be located at the 1&apos; position of the nucleotide sugar moiety. Furthermore, the nucleotide may be unmodified or modified in the sugar, internucleoside linkage, and/or base moieties (also referred to interchangeably as nucleotide analogs, modified nucleotides) , non-natural nucleotides, non-standard nucleotides and others). In some embodiments, the nucleotides can be ribonucleotides (sometimes referred to as RNA nucleotides). In some embodiments, the nucleotides can be deoxyribonucleotides (sometimes referred to as DNA nucleotides).

As used herein, the term "prominence" is used in the sense that it is generally accepted in the art. With respect to exemplary double-stranded nucleic acid molecules, the term is generally directed to the terminal portion of a nucleotide sequence that does not pair bases between two strands of a double-stranded nucleic acid molecule. In some specific aspects, the protrusions can be single-stranded. In some embodiments, the nucleic acid molecules of the teachings herein comprise two overhangs (i.e., 3&apos; and 5&apos; overhangs) in the antisense strand, as exemplified below.

In some embodiments, the nucleic acid molecules of the teachings herein comprise a projection and a blunt end (e.g., a 3&apos; projection and a 5&apos; blunt end; or a 3&apos; blunt end and a 5&apos; projection), as exemplified below.

In some embodiments, the 3' protrusion is not blunt.

An exemplary double-stranded RNA line with two 3' overhangs or two 5' overhangs is depicted below.

As used herein, the term "parenteral" is used in the sense that it is generally accepted in the art. The term is generally directed to a method or technique for administering a molecule, drug, agent, or compound other than through the digestive tract and comprising subcutaneous, subcutaneous, intravascular (eg, intravenous), intramuscular, or intrathecal injection. Or perfusion techniques and similar.

The term "administered" is used herein in its broadest sense. These terms relate to any method of delivering a compound or pharmaceutical composition described herein to a subject, cell or tumor by, for example, introducing a compound systemically, locally, or in situ. To the object. Thus, a compound of the teachings herein derived from a composition in a subject, whether or not the composition comprises the compound, is encompassed by the term. When the term is used in conjunction with the term "systemic" or "systemically", it is generally related to the systemic absorption or accumulation of the compound or composition in the bloodstream in vivo and its distribution throughout the body. In some embodiments, the term "administering" can be directed to, for example, the delivery of one or more recombinant vectors to a tumor cell, wherein the vector exhibits an RNA interference agent.

"Pharmaceutically acceptable composition" or "pharmaceutically acceptable formulation" may be a composition or formulation that allows the nucleic acid molecules of the teachings herein to be effectively distributed to the point where they are most suitable for their use. Active body position.

As used herein, the term "pharmaceutically acceptable excipient, carrier, or diluent" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, Forming agent, solvent or encapsulating material. Can be used as a pharmaceutically acceptable excipient Some examples of materials for the agent or diluent include: sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose, and derivatives thereof, such as sodium carboxymethylcellulose, ethylcellulose And cellulose acetate; powdered daun gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; a diol such as propylene glycol; a polyol such as glycerin, sorbitol, mannitol and polyethylene glycol; an ester such as ethyl oleate and ethyl laurate; agar; a buffer such as magnesium hydroxide and hydroxide Aluminum; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyoxyethylene-polyoxypropylene copolymers, as well as coloring agents, release agents, coating agents, sweeteners, flavorings and fragrances Agents, preservatives and antioxidants may also be present in the composition.

The term "phosphorothioate" relates to a mononucleotide acid phosphate linkage comprising one or more sulfur atoms in place of an oxygen atom. Thus, the term phosphorothioate relates to both the phosphorothioate and the phosphorodithioate nucleotide linkage.

As used herein, the term "ribonucleotide" is used in the sense that it is generally accepted in the art. The term is generally referred to as a nucleotide having a hydroxyl group at the 2' position of the β-D-nuclear furanosyl moiety. This term also encompasses any chemically modified ribonucleotide.

As used herein, the term "RNA" is used in the sense that it is generally accepted in the art. In general, the term RNA is directed to a molecule comprising at least one nucleofuranoside moiety. The term may encompass double stranded RNA, single stranded RNA, and isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and by addition, deletion, substitution, and/or Or altering one or more nucleotides with naturally occurring rRNA Different altered RNA. Such alterations can include the addition of a non-nucleotide material, such as to the end of the RNA or to the interior, such as one or more nucleotides of the RNA. Nucleotides of RNA molecules of the teachings herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically modified nucleotides or deoxynucleotides. Such altered RNA may be referred to as an analog or an analog of a naturally occurring rRNA.

The term "RNA interference" or the term "RNAi" is a biological procedure for inhibiting or downregulating the expression of a gene in a cell, as generally known in the art, and which may be comprised of short interfering nucleic acid molecules or Asymmetric interfering nucleic acid molecules are mediated. Furthermore, the term RNAi is intended to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational repression, transcriptional repression, or epigenetics. For example, the aiRNA molecules of the teachings herein can be used to epigenetically silence genes at post-transcriptional or pre-transcriptional levels. In a non-limiting example, modulation of gene expression of an aiRNA molecule through the teachings herein can be derived from AIRNA-mediated cleavage of RNA (coding or non-coding RNA) via RISC or via translational repression, as Known, or regulated, in the art may be derived from transcriptional inhibition.

As used herein, the term "meaning area" is used in the sense that it is generally accepted in the art. An exemplary nucleic acid molecule for the teachings herein relates to a nucleotide sequence of an aiRNA molecule that is complementary to an antisense region of the aiRNA molecule. Furthermore, a region of significance of an aiRNA molecule can comprise a nucleic acid sequence having homology or sequence identity to a target nucleotide sequence. In a specific aspect, the region of significance of the aiRNA molecule is also referred to as a sense strand or a passenger strand.

The terms "short interfering nucleic acid", "siNA", "short interfering RNA", "siRNA", "short" Interfering nucleic acid molecules, "short interfering oligonucleotide molecules", or "chemically modified short interfering nucleic acid molecules" are any of the following nucleic acid molecules that are capable of mediating RNA interference by sequence specificity ("RNAi" Or gene silencing to inhibit or down-regulate gene expression or viral replication, and it contains antisense strands and sense strands, and the lengths of the two strands are the same. This term may be with respect to individual nucleic acid molecules, a plurality of such nucleic acid molecules, or pools of such nucleic acid molecules. The siRNA may be a double-stranded nucleic acid molecule comprising a self-complementary sense and an antisense strand, wherein the antisense strand comprises a nucleotide sequence complementary to a nucleotide sequence or a portion thereof in the target nucleic acid molecule and the sense strand comprises A nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The siRNA can be a symmetric interfering RNA with two 3' overhangs.

As used herein, the term "object" is used in the sense that it is generally accepted in the art. The term generally refers to an organism to which a nucleic acid molecule of the teachings herein can be administered. The subject can be a mammalian or mammalian cell, including human or human cells. The term is also related to an organism which is the donor or recipient of the transplanted cells or the cells themselves. In some embodiments, the term "subject" relates to any animal (eg, a mammal), including but not limited to humans, mammals, and non-mammals, such as non-human primates, mice, rabbits, Sheep, dogs, cats, horses, cows, chickens, biogenic animals, fish, insects or reptiles, which are intended to be recipients of special treatments. In some instances, the terms "object" and "patient" are used interchangeably herein with respect to a human subject.

As used herein, the term "systemic administration" is used in the sense that it is generally accepted in the art. The term generally refers to the systemic absorption or accumulation of a drug in the bloodstream in vivo followed by the distribution throughout the body.

As used herein, the term "target position" or "target nucleotide sequence" is used to It is generally accepted in the art. The term is generally directed to a sequence within a target nucleic acid molecule (eg, RNA or DNA) that is "targeted." With respect to exemplary nucleic acid molecules of the teachings herein, the nucleotide sequence of interest can be the nucleotide sequence being expressed, ie, RNA synthesized by RNA polymerase targeted by aiRNA. In some embodiments, the nucleotide sequence of interest can be derived from mRNA transcribed from a target gene. In some embodiments, an antisense strand of an aiRNA can linearly correspond to its target nucleotide sequence. In some embodiments, an antisense strand of an aiRNA is perfectly complementary to at least 14 nucleotides of its target nucleotide sequence. In some embodiments, an antisense strand of an aiRNA is partially complementary to its target nucleotide sequence.

As used herein, the term "therapeutically effective amount" is used in the sense that it is generally accepted in the art. The term is generally directed to the amount of the compound or composition that will elicit the necessary biological or medical response in a cell, tissue, system, animal or human. For example, if a given clinical treatment is considered to be effective when there is at least a 25% reduction in measurable parameters associated with a disease or condition, then the therapeutically effective amount of the drug for the treatment of the disease or condition is Achieving at least about 25% of this parameter reduces the amount required.

With regard to the treatment of cancer, "therapeutically effective amount" means an amount that causes one or more of the following effects: (1) inhibiting (to some extent) cancer or tumor growth, including slowing growth or completely preventing growth; (2) Reduce the number of cancer or tumor cells; (3) reduce tumor size; (4) inhibit (ie reduce, slow, or completely stop) cancer or tumor cells infiltrate into surrounding organs; (5) inhibition (ie, reduce, slow, Or completely stop) metastasis; (6) increase anti-tumor immune response, which may (but not necessarily) cause tumor regression or rejection, or (7) alleviate (to some extent) one or more associated with cancer or tumor The measurable symptoms. The therapeutically effective amount can vary depending on factors such as the stage of the disease, the age, sex, and weight of the individual and one or more anti-cancer agents. The ability of an individual to induce a desired response. A "therapeutically effective amount" is also an amount over which any toxic or detrimental effect is therapeutically beneficial.

As used herein, terms such as "treatment" or "alleviation" relate to (1) a therapeutic means of curing a pathological condition or condition that is diagnosed, slowing the condition or condition, alleviating the symptoms of the condition or condition, and / or stop the progression of the condition or condition and (2) prophylactic or preventative means that prevent or slow the development of the pathological condition or condition being targeted ("prevention"). Accordingly, such treatment in need include such patients already suffering from the disease; such predispositions have patients with the disease; and such patients are in need of prevention.

In some embodiments, if a patient exhibits one or more of the following, the subject is successfully "treated" according to the teachings herein: the number of cancer cells is reduced or completely free of cancer cells; tumor size Decreased; infiltration of cancer cells into surrounding organs (including cancer spread into soft tissues and bones) is inhibited or absent; tumor metastasis is inhibited or metastasized; tumor growth is inhibited or tumor growth-free morbidity and mortality are reduced. "Treatment" can also mean prolonged survival (compared to the expected survival in the absence of treatment).

In some embodiments, the term "treating cancer" or equivalent thereto means reducing, reducing, or inhibiting replication of cancer cells; reducing, reducing or inhibiting the spread of cancer (formation of metastasis); reducing tumor size; The number of tumors (ie, reduced tumor burden); reduced or reduced the number of cancer cells in the body; prevention of recurrence of cancer after surgical removal or other anti-cancer treatment; or improved measurable treatment endpoint (ie, outcome).

As used herein, the term "upward" is used in the sense that it is generally accepted in the art. An exemplary nucleic acid molecule with respect to the teachings herein, the term being related to increasing the expression of a gene, or an RNA molecule encoding one or more protein or protein subunits or equivalent The level of the RNA molecule, or the activity of one or more RNA, protein or protein subunits, is higher than that observed in nucleic acid molecules (eg, aiRNA) lacking the teachings herein. In some embodiments, the gene expression by the aiRNA molecule is up-regulated or elevated above the level observed in the presence of an inactive or attenuated molecule. In some embodiments, the gene expression by the aiRNA molecule is up-regulated or elevated above the level observed in the presence of, for example, a scrambled sequence or a mismatched aiRNA molecule. In some embodiments, the up-regulation or elevation of the gene by the aiRNA molecule is observed to be higher than in the presence of, for example, siRNA molecules having the same, substantially identical, similar antisense strands. Level. In some embodiments, the gene expression of the aiRNA molecule is up-regulated or elevated by a biologically active molecule (eg, a small molecule, an antibody, or another having a similar activity in the performance of the target gene, for example). The level observed in the presence of a creature). In certain embodiments, the gene expression of a nucleic acid molecule using the teachings herein is up-regulated or elevated in the presence of the nucleic acid molecule greater than in the absence of the nucleic acid molecule. In some embodiments, the gene exhibits up-regulation or elevation of RNAi-mediated gene silencing (such as down-regulation, inhibition, or silencing of the expression of a target gene by RNAi-mediated cleavage or silencing) ) The suppression is related.

As used herein, the term "carrier" is used in the sense that it is generally accepted in the art. The term carrier is generally directed to any nucleic acid and/or virus based expression system or technique that is used to deliver one or more nucleic acid molecules to a target cell or organism.

In one aspect, the teachings herein provide an RNA molecule. In some embodiments, the RNA molecule of the present invention comprises a first strand and a second strand, wherein the second strand is complementary to the first strand and the first strand and the second strand form at least one double Share area. In some embodiments, the first strand of the RNA molecule is longer than the second strand of the RNA molecule (the length is not Said). RNA molecules having length asymmetry in the teachings herein are sometimes referred to as aiRNA.

In some embodiments, the first strand has a length of from 5 to 100 nucleotides (nt.). In some embodiments, the first strand has a length of 10-30 nt. In some embodiments, the first strand has a length of 12-30 nt. In some embodiments, the first strand has a length of 15-28 nt. In some embodiments, the first strand has a length of 17-26 nt. In some embodiments, the first strand has a length of 19-23 nt. In some embodiments, the first strand has a length of 19 nt. In some embodiments, the first strand has a length of 20 nt. In some embodiments, the first strand has a length of 21 nt. In some embodiments, the first strand has a length of 22 nt. In some embodiments, the first strand has a length of 23 nt. In some specific aspects, the first strand is an antisense stock. In some embodiments, the antisense strand can be 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides in length. In some embodiments, the antisense strands are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides can linearly correspond to the PD-L1 mRNA sequence. In some embodiments, the antisense strands are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or Thirty nucleotides correspond linearly to the PD-L1 mRNA sequence and are continuous compared to the PD-L1 mRNA sequence. In some embodiments, the antisense strands are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides compared to PD The -L1 mRNA sequence may be non-contiguous.

In some embodiments, the second strand has a length of from 3 to 30 nt. In some embodiments, the second strand has a length of 3-29 nt. In some embodiments, the second strand has a length of 10-26 nt. In some embodiments, the second strand has a length of 12-26 nt. In some embodiments, the second strand has a length of 13-22 nt. In some specific aspects, The second strand has a length of 14-19 nt. In some embodiments, the second strand has a length of 14-17 nt. In some embodiments, the second strand has a length of 14 nt. In some embodiments, the second strand has a length of 15 nt. In some embodiments, the second strand has a length of 16 nt. In some embodiments, the second strand has a length of 17 nt. In some embodiments, the second strand has a length of 18 nt. In some embodiments, the second strand has a length of 19 nt. In some specific aspects, the second strand is a stock. In some embodiments, the sense strand can be 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 nucleotides in length.

In some embodiments, the first strand is at least 1 nt longer than the second strand. In some embodiments, the first strand is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 longer than the second strand , 18, 19, or 20 nt. In some embodiments, the first strand is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nt longer than the second strand. In some embodiments, the first strand is 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt longer than the second strand. In some embodiments, the first strand is 4, 5, 6, 7, 8, or 9 nt longer than the second strand.

In some embodiments, the double-stranded region has a length of from 3 to 98 base pairs (bp). In some embodiments, the double-stranded region has a length of 3-28 bp. In some embodiments, the double-stranded region has a length of 3-19 bp. In some embodiments, the double-stranded region has a length of 14-19 bp. In certain embodiments in which the aiRNA comprises two or more double-stranded regions, each of the double-stranded regions has a length of 2-17 bp. In some embodiments, the double-stranded region has a length of 14, 15, 16, 17, 18, or 19 bp. For example, the double-stranded region can have a length of 14, 15, 16, 17, 18, or 19 bp. In some embodiments, the length of the double-stranded region can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp.

In some embodiments, the double-stranded region of the RNA molecule does not contain any mismatch or protrusion, and the two strands are perfectly complementary to each other in the double-strand region. In another embodiment, the double stranded region of the RNA molecule contains non-synchronous and/or raised regions.

In some embodiments, the aiRNA comprises at least one double-stranded region, and two ends independently selected from the group consisting of a 5' overhang, a 3' overhang, and a blunt end. In some embodiments, the aiRNA comprises at least one double stranded region, a 5&apos; projection, and a blunt end (3&apos; blunt end) at the 3&apos; end. In some embodiments, the aiRNA comprises at least one double stranded region, a 3&apos; projection, and a blunt end (5&apos; blunt end) at the 5&apos; end. In some embodiments, the aiRNA comprises at least one double stranded region, a 3&apos; projection, and a 5&apos; projection.

In some embodiments, the terminal projection (comprising a 3&apos; or 5&apos; projection) comprises from 1 to 15 nucleotides. In some embodiments, the terminus comprises 1-9 nucleotides. In some embodiments, the terminal overhang comprises 1-8 nucleotides. In some embodiments, the terminal highlight comprises 1-7 nucleotides. In some embodiments, the terminal highlight comprises 1-6 nucleotides. In some embodiments, the terminal overhang comprises 1-5 nucleotides. In some embodiments, the terminal highlight comprises 1, 2, 3, 4, 5, 6, or 7 nucleotides.

Thus, in some embodiments, the 3' overhang of the antisense strand of the RNA molecule of the teachings herein comprises from 1 to 15 nucleotides. In some embodiments, the 3&apos; overhang comprises 1-9 nucleotides. In some embodiments, the 3&apos; overhang comprises 1-8 nucleotides. In some embodiments, the 3&apos; overhang comprises 1-7 nucleotides. In some embodiments, the 3&apos; overhang comprises 1-6 nucleotides. In some embodiments, the 3&apos; overhang comprises 1-5 nucleotides. In some embodiments, the 3&apos; overhang comprises 1 nucleotide. In some embodiments, the 3&apos; overhang comprises 2 nucleotides. In some embodiments, the 3&apos; overhang comprises 3 nucleotides. to In some embodiments, the 3&apos; overhang comprises 4 nucleotides. In some embodiments, the 3&apos; overhang comprises 5 nucleotides.

In some embodiments, the aiRNA comprises a 3 nucleotide 5' overhang and/or a 3 nucleotide 3' overhang.

Thus, in some embodiments, the 3' end of the antisense strand of the RNA molecule of the teachings herein contains 0-15 nucleotides. In some embodiments, the 3&apos; end comprises 0-9 nucleotides. In some embodiments, the 3&apos; end comprises 0-8 nucleotides. In some embodiments, the 3&apos; end comprises 0-7 nucleotides. In some embodiments, the 3&apos; end comprises 0-6 nucleotides. In some embodiments, the 3&apos; end comprises 0-5 nucleotides.

Thus, in some embodiments, the 5' overhang of the antisense strand of the RNA molecule of the teachings herein comprises from 1 to 15 nucleotides. In some embodiments, the 5&apos; overhang comprises 1-9 nucleotides. In some embodiments, the 5&apos; overhang comprises 1-8 nucleotides. In some embodiments, the 5&apos; overhang comprises 1-7 nucleotides. In some embodiments, the 5&apos; overhang comprises 1-6 nucleotides. In some embodiments, the 5&apos; overhang comprises 1-5 nucleotides. In some embodiments, the 5&apos; overhang comprises 1 nucleotide. In some embodiments, the 5&apos; overhang comprises 2 nucleotides. In some embodiments, the 5&apos; overhang comprises 3 nucleotides. In some embodiments, the 5&apos; overhang comprises 4 nucleotides. In some embodiments, the 5&apos; overhang comprises 5 nucleotides.

Thus, in some embodiments, the 5' end of the antisense strand of the RNA molecule of the teachings herein contains 0-15 nucleotides. In some embodiments, the 5&apos; end comprises 0-9 nucleotides. In some embodiments, the 5&apos; end comprises 0-8 nucleotides. In some embodiments, the 5&apos; end comprises 0-7 nucleotides. In some specific aspects, the 5’ end The end contains 0-6 nucleotides. In some embodiments, the 5&apos; end comprises 0-5 nucleotides.

Any region of the RNA molecule (including any terminal protrusions and gaps between the two double-stranded regions) of the teachings herein can be stabilized against decomposition (through chemical modification or secondary structure). Such RNA strands may have nucleotides that are not coincident or imperfectly anastomosed. Each strand may have one or more gaps (incisions in the nucleic acid backbone), gaps (stranded strands of one or more missing nucleotides), and modified nucleotides or nucleotide analogs. Not only can any or all of the nucleotides in the RNA molecule be chemically modified, but each strand can be conjugated to one or more moieties to enhance its functionality (eg, with, for example, one or more peptides, antibodies, antibody fragments, Part of the aptamer, polymer, etc.).

In some embodiments, the first strand of aiRNA of the teachings herein may comprise ribonucleotides (or RNA nucleotides). In some embodiments, each of the ribonucleotides can be an unmodified nucleotide or a modified nucleotide. In some embodiments, the 3&apos; overhang, the double stranded region, or the 5&apos; overhang can comprise ribonucleotides. In some embodiments, the 3&apos; overhang or 3&apos; blunt end of the antisense strand can comprise a ribonucleotide. In some embodiments, the 3&apos; overhang of the antisense strand can comprise substantially ribonucleotides. For example, the 3&apos; overhang of the antisense strand may comprise only ribonucleotides.

In some embodiments, the first strand of the aiRNA of the teachings herein may comprise deoxyribonucleotides (or DNA nucleotides). In some embodiments, the 3&apos; overhang, the double stranded region, or the 5&apos; overhang can comprise a deoxyribonucleotide.

In some embodiments, the second strand of the aiRNA of the teachings herein may comprise ribonucleotides (or DNA nucleotides). In some embodiments, the second strand of the aiRNA of the teachings herein may comprise deoxyribonucleotides (or DNA nucleotides).

The aiRNA of the teachings herein may comprise one or more modified or unmodified nucleotides. Thus, in some embodiments, the aiRNA can comprise one or more unmodified nucleotides. In some embodiments, the first strand of aiRNA of the teachings herein may comprise one or more unmodified nucleotides. In some embodiments, the second strand of the aiRNA of the teachings herein may comprise one or more unmodified nucleotides.

In some embodiments, the first strand of the aiRNA of the teachings herein comprises one or more modified nucleotides. In some embodiments, the first strand can comprise from 1 to 21 modified nucleotides. In some embodiments, the first strand can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 modified nucleotides. In some embodiments, the first strand can comprise 1, 2, 3, 4, 5, 6, 7, 8, or 10 modified nucleotides. In some embodiments, the first strand can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 modified nucleotides.

In many instances, any nucleotide in the first strand of the aiRNA of the teachings herein may be a modified nucleotide. In some embodiments, the first nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the second nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the third nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fourth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fifth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the sixth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the seventh nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some specific states In this case, the eighth nucleotide leaving the 5' terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the ninth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the tenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the eleventh nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the twelfth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the thirteenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fourteenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fifteenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fifteenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the seventeenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the eighteenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the nineteenth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the twentieth nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the twenty-first nucleotide leaving the 5&apos; terminal nucleotide of the first strand is a modified nucleotide.

In some embodiments, the first nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the second nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the third nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fourth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. to In some embodiments, the fifth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the sixth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the seventh nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the eighth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the ninth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the tenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the eleventh nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the twelfth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the thirteenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fourteenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the fifteenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the sixteenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the seventeenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the eighteenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the nineteenth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the twentieth nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide. In some embodiments, the twenty-first nucleotide leaving the 3&apos; terminal nucleotide of the first strand is a modified nucleotide.

In some specific aspects, the second part of the aiRNA of the teachings of this article may be One or more modified nucleotides are included. In some embodiments, the second strand can comprise from 1 to 15 modified nucleotides. In some embodiments, the second strand can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modified nucleosides acid. In some embodiments, the first strand can comprise 1, 2, 3, 4, 5, 6, 7, 8, or 10 modified nucleotides. In some embodiments, the first strand can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modified nucleotides.

In certain instances, any of the nucleotides in the second strand of the aiRNA of the teachings herein may be modified nucleotides. In some embodiments, the first nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the second nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the third nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fourth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fifth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the sixth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the seventh nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the eighth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the ninth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the tenth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the eleventh nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the twelfth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some specific states In this case, the thirteenth nucleotide leaving the 5' terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fourteenth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fifteenth nucleotide leaving the 5&apos; terminal nucleotide of the second strand is a modified nucleotide.

In some embodiments, the first nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the second nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the third nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fourth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fifth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the sixth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the seventh nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the eighth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the ninth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the tenth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the eleventh nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the twelfth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the thirteenth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fourteenth nucleotide leaving the 3&apos; terminal nucleotide of the second strand is a modified nucleotide. In some embodiments, the fifteenth nucleotide leaving the 3' terminal nucleotide of the second strand is Modified nucleotides.

In some embodiments, any of the first strand of the aiRNA and the modified nucleotide of the second strand are independently linked to their nucleobase, their sugar, and their nucleosides. The inter-ligation, or a combination of the above, is modified. In some embodiments, the modified nucleotide is modified in its nucleobase. In some embodiments, the modified nucleotide is modified by its sugar. In some embodiments, the modified nucleotide may comprise a 2'-methoxy-saccharide (ie, wherein the sugar is methylated at its 2'-hydroxyl group, and such specific aspects The modified nucleotide in the molecule is sometimes referred to as a 2'-OMe modified nucleotide). In some embodiments, the modified nucleotide is modified by its internucleoside linkage. Without wishing to be bound by any particular theory, aiRNA containing modified nucleotides (including 2'-OMe modified nucleotides) may be compared to such unmodified or less modified nucleotides. With improved stability and activity.

In some embodiments, the first or antisense strand of the aiRNA of the teachings herein is complementary to a target RNA. In some embodiments, the 3' overhang of the antisense strand is complementary to a target RNA. In some embodiments, the double-strand region of the antisense strand is complementary to a target RNA. In some embodiments, the 5' overhang of the antisense strand is complementary to a target RNA. In some embodiments, the 3' overhang of the antisense strand is complementary to a double stranded region with a target RNA. In some embodiments, the double-stranded region and its 5&apos; overhang are complementary to a target RNA. In some embodiments, the complementarity between the antisense strand (or its 5' overhang, double stranded region, or 3' overhang) and the target RNA is partially complementary, substantially complementary, or Perfect complementarity. In some embodiments, the 3' overhang of the antisense strand is complementary, substantially complementary, or perfectly complementary to the target RNA portion. In some embodiments, the double stranded region of the antisense strand is complementary, substantially complementary, or perfectly complementary to the target RNA portion. In some specific cases, the antisense stock is 5’ Prominently complementary, substantially complementary, or perfectly complementary to the target RNA portion.

In some embodiments, the 3' overhang, the double stranded region, and the 5' overhang of the antisense strand are independent of the target RNA. In some embodiments, the 3' overhang of the antisense strand is independent of the target RNA. In some embodiments, the double-strand region of the antisense strand is independent of the target RNA. In some embodiments, the 3' blunt end of the antisense strand is independent of the target RNA. In some embodiments, the 5' blunt end of the antisense strand is independent of the target RNA. In some embodiments, the 5' overhang of the antisense strand is independent of the target RNA.

In some embodiments, one or more of the 3&apos;, double-stranded, and 5&apos; protrusions of the antisense strand independent of the target RNA can comprise A, U, or C. In some specific aspects, one or more of the 3' protrusion, the double-strand area, and the 5' protrusion of the anti-sense strand may include an "AA" motif, a "UU" motif, a "CC" motif, "AU" phantom, "AC" phantom, "UA" phantom, "UC" phantom, or "CA" phantom. In some specific aspects, the 5' protruding or 5' blunt end (or 5' end together) of the antisense strand may include an "AA" motif, a "UU" motif, a "CC" motif, and AU" phantom, "AC" phantom, "UA" phantom, "UC" phantom, or "CA" phantom. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise an "AA" motif. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise a "UU" motif. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise a "CC" motif. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise an "AU" motif. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise an "AC" motif. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise a "UA" motif. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise a "UC" motif. In some embodiments, the 5' overhang or 5' blunt end of the antisense strand can comprise a "CA" motif.

In some embodiments, the target RNA encodes an mRNA for a ligand of PD-1. In certain embodiments, the target RNA encodes mRNA for PD-L1 or PD-12. In some embodiments, the target RNA encodes mRNA for PD-L1. In some embodiments, the nucleotide sequence of interest is in mRNA encoding PD-L1.

In some specific aspects, the aiRNA of the teachings herein may include a sense strand and an antisense strand, wherein the sense strand forms a double strand region with the antisense strand, and the sense strand is selected from the sequence identifier 1-46. And the antisense strand is selected from the sequence identification number 47-92, as follows: * G, C, U, and A represent ribonucleotides with guano, cytosine, uracil, and adenine, respectively.

In some embodiments, the aiRNA has a sense strand having a sequence of sequence identifier 22 and an antisense strand having a sequence of sequence identifier 68. In some embodiments, the aiRNA is referred to as aiRNA #22 or aiPD-L1 #22.

In some embodiments, the aiRNA of the teachings herein modulates the expression of one or more genes associated with a ligand for the Programmed Cell Death-1 Receptor (PD-1) on the lymphocytes. In some embodiments, the lymphocyte T cell, helper T cell, or natural killer cell. In some embodiments, the lymphoid line is a cytotoxic T cell or a CD8 ⁺ cell. In some embodiments, the PD-1 coordination system PD-L1 or PD-L2. In some specific aspects, the coordination system PDL1. Thus, in certain embodiments, the aiRNA modulates the expression of PD-L1 or PDL2. In some embodiments, the aiRNA down-regulates, reduces, or inhibits the expression of PD-L1 or PD-L2. In some embodiments, the aiRNA down-regulates, reduces, or inhibits the expression of PD-L1.

In some embodiments, the aiRNA of the teachings herein can therefore silence genes associated with PD-L1 expression. Thus, in certain aspects, the teachings herein provide a PD-L1 inhibitor. As discussed in more detail herein, in certain aspects, the teachings herein provide a PD-L1 specific inhibitor. For ease of discussion only, the aiRNA of the teachings herein may sometimes be referred to as aiPD-L1.

In certain embodiments, the aiRNA of the teachings herein downregulates, reduces, or inhibits PD-L1 expression in cancer cells by mediating RNA interference or gene silencing in a sequence-specific manner. In many instances, specific gene silencing improves gene silencing and reduces Desirable events. In some embodiments, the specific sequence is referred to as a target position or a nucleotide sequence of interest.

In some embodiments, the nucleotide sequence of interest is a PD-L1 mRNA sequence or a fragment thereof. In some embodiments, the nucleotide sequence of interest of the teachings herein can be selected from the PD-L1 mRNA sequence of SEQ ID NO: 93-138.

** G, C, U, and A represent adenine nucleotides (adenosine 5'-monophosphate), guanine nucleotides (guanosine 5'-monophosphate), uracil Nucleotide (uridine 5'-monophosphate) and cytosine nucleotide (cytidine 5'-monophosphate). N can be, U, G or C.

In some embodiments, the target position of the aiRNA used in the teachings herein may be sequence identification number 114.

In one aspect, the teachings herein provide a composition of RNA molecules comprising the teachings herein. In some embodiments, the composition can comprise a PD-L1 inhibitor. In some embodiments, the composition can comprise aiPD-L1. In some specific aspects, the composition The object may comprise an aiRNA having a sense strand and an antisense strand, wherein the sense strand forms a double strand region with the antisense strand, the sense strand may be selected from the sequence identifier 1-46, and the antisense strand may be selected from The corresponding complementary nucleotide sequence of the following: SEQ ID NO: 47-92. In some embodiments, the composition can comprise an aiRNA having a sense strand having the sequence of SEQ ID NO: 22 and an antisense strand having the sequence of SEQ ID NO: 68. In some embodiments, the aiRNA is referred to as aiRNA #22 or aiPD-L1 #22.

In some embodiments, the compositions of the teachings herein can comprise an excipient, a carrier, or a diluent. In some embodiments, the compositions of the teachings herein may comprise a pharmaceutically acceptable excipient, carrier, or diluent.

In one aspect, the teachings herein provide methods for modulating gene expression in a cell. In some embodiments, the cell can be a cancer cell. In some embodiments, the gene encodes a ligand for PD-1. In some embodiments, the gene encodes PD-L1 or PD-L2. In some embodiments, the gene encodes PD-L1. In some embodiments, the method can comprise downregulating, reducing, or inhibiting the performance of a PD-1 ligand. In some embodiments, the method can include downregulating, reducing, or inhibiting the performance of PD-L1 or PD-l2. In some embodiments, the method can include downregulating, reducing, or inhibiting the performance of PD-L1.

In one aspect, the teachings herein provide methods for modulating the interaction between cells and lymphocytes. In some embodiments, the cell can be a cancer cell. In some embodiments, the lymphocytes can be T cells, helper T cells, or natural killer cells. In some embodiments, the lymphocytes can be cytotoxic T cells or CD8 ⁺ cells. In some embodiments, the interaction between the cell and the lymphocytes can be linked to the binding of a ligand in the cell to a receptor in the lymphocyte. In some embodiments, the interaction can be a combination of PD-L1 or PD-L2 and PD-1. In some embodiments, the interaction can be a combination of PD-L1 and PD-1.

In some embodiments, the method reduces or inhibits the amount of PD-L1 mRNA and/or PD-L1 in the cell by silently encoding the gene for PD-L1. In some embodiments, the method reduces or inhibits the interaction between the cell and the lymphocyte due to a decrease in the amount of PD-L1 mRNA and/or PD-L1. Thus further, in certain embodiments, the method activates or reactivates the lymphocytes (T cells, cytotoxic T cells, or CD8 ⁺ ) such that the lymphocytes recognize, attack, and kill the cells. In some embodiments, the method may also increase the infiltration of lymphocytes (T cells, cytotoxic T cells, or CD8 ⁺ ). In some embodiments, the cell can be a cancer cell.

In another aspect, the teachings herein provide immunotherapy. In some embodiments, the immunotherapy treats, prevents, or ameliorates a condition in a subject. In some embodiments, the immunotherapy can comprise providing a composition of the aiRNA of the teachings herein or a composition comprising aiRNA of the teachings herein to a subject in need thereof. In certain embodiments, the immunotherapy can comprise providing a therapeutically effective amount of the aiRNA of the teachings herein or a therapeutically effective amount of a composition comprising aiRNA of the teachings herein to a subject in need thereof.

In some embodiments, the subject can be a mammal, including an animal or a human. In some embodiments, the subject can be a human subject (or patient). In some embodiments, the subject can be a mammalian cell. In some embodiments, the subject can be a human cell. In some embodiments, the subject can be a cancer cell. In some embodiments, the subject can be diagnosed as positive for the performance of the ligand for PD-1. In some embodiments, the subject can be positive for PD-L1. In some embodiments, the subject can be PDL2 positive. This diagnosis can be carried out according to methods generally known in the art. For example, it can be based on relevant genetic material Or a test of immunochemical methods. An example of a diagnosis is discussed in Ohigashi et al., Clin. Cancer Res. 11(8): 2947-2953 (2005) and those of ordinary skill in the art will appreciate that other methods can be used to identify PD- L1 or PD-L2 positive subjects.

In some embodiments, the condition can be cancer. In some specific aspects, the cancer may be hepatobiliary cancer (including liver cancer (including hepatocellular carcinoma or cholangiocarcinoma), gallbladder cancer, biliary tract cancer, or pancreatic cancer), gastrointestinal cancer, urinary cancer (including bladder cancer) , prostate cancer, kidney cancer, testicular cancer, and the like), renal cell carcinoma, urothelial cancer, solid tumor, hepatocellular carcinoma, ovarian cancer, or cancer of the fallopian tube. In some embodiments, the cancer can be a hepatobiliary cancer (including liver cancer (including hepatocellular or cholangiocarcinoma), gallbladder cancer, biliary tract cancer, or pancreatic cancer). In some embodiments, the cancer can be gastrointestinal cancer. In some embodiments, the cancer can be a urinary system cancer (including bladder cancer, prostate cancer, kidney cancer, testicular cancer, and the like), renal cell carcinoma, or urothelial cancer. In some embodiments, the cancer can be a solid tumor. In some specific aspects, the solid tumor can be pancreatic cancer; bladder cancer; colorectal cancer; breast cancer; prostate cancer; kidney cancer; hepatocellular carcinoma; lung cancer; ovarian cancer; cervical cancer; gastric cancer; Head and neck cancer; melanoma; neuroendocrine cancer; CNS cancer; brain tumor; bone cancer; or soft tissue sarcoma. In some embodiments, the solid tumor can be lung cancer (non-small cell lung cancer, small cell lung cancer), colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, or breast cancer.

In some specific aspects, the cancer may be acute myeloid leukemia, astrocytoma, bladder cancer, bone cancer, brain cancer, breast cancer, chronic myeloid leukemia, colon cancer, colorectal cancer, stomach cancer, gastrointestinal cancer, Genitourinary cancer, glioblastoma, glioma, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, Kaposi's sarcoma, lung cancer, melanoma, myeloproliferative disorders, non-Hodgkin's disease Lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney thin Cell carcinoma, squamous cell carcinoma, or thyroid cancer.

Thus, in some embodiments, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering a therapeutically effective amount of an aiRNA or composition of the teachings herein to a subject in need thereof, The cancer is hepatobiliary cancer (including liver cancer (including hepatocellular carcinoma or cholangiocarcinoma), gallbladder cancer, biliary tract cancer, or pancreatic cancer), gastrointestinal cancer, urinary cancer (including bladder cancer, prostate cancer, kidney cancer, testicular) Cancer, and similar), renal cell carcinoma, urothelial cancer, solid tumor, hepatocellular carcinoma, ovarian cancer, or cancer of the fallopian tube. In some embodiments, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering a therapeutically effective amount of an aiRNA or composition of the teachings herein to a subject in need thereof and the cancer system Liver cancer, gallbladder cancer, biliary tract cancer, or pancreatic cancer. In some embodiments, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering a therapeutically effective amount of an aiRNA or composition of the teachings herein to a subject in need thereof and the cancer It is gastrointestinal cancer, bladder cancer, prostate cancer, kidney cancer, testicular cancer, renal cell carcinoma, urothelial cancer, or hepatocellular carcinoma. In some embodiments, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering a therapeutically effective amount of an aiRNA or composition of the teachings herein to a subject in need thereof and the cancer A solid tumor. In some embodiments, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering a therapeutically effective amount of an aiRNA or composition of the teachings herein to a subject in need thereof and the cancer Pancreatic cancer; bladder cancer; colorectal cancer; breast cancer; prostate cancer; kidney cancer; hepatocellular carcinoma; lung cancer; ovarian cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer; melanoma; neuroendocrine cancer; CNS cancer; brain tumor; bone cancer; or soft tissue sarcoma.

In some specific aspects, the teachings herein provide for treatment of cancer in a subject. A method comprising administering an aiRNA or a composition of the teachings herein to a cancer cell, wherein the cancer is hepatobiliary cancer (including liver cancer (including hepatocellular carcinoma or cholangiocarcinoma), gallbladder cancer, biliary tract cancer, Or pancreatic cancer), gastrointestinal cancer, urinary cancer (including bladder cancer, prostate cancer, kidney cancer, testicular cancer, and the like), renal cell carcinoma, urothelial cancer, solid tumor, hepatocellular carcinoma, ovarian cancer, Or cancer of the fallopian tube. In some embodiments, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering to the cancer cell a cancer or liver cancer, gallbladder cancer, the aiRNA or composition of the teachings herein. Biliary cancer, or pancreatic cancer. In some embodiments, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering to the cancer cell an aiRNA or composition of the teachings herein and the cancer being a gastrointestinal cancer, bladder cancer , prostate cancer, kidney cancer, testicular cancer, renal cell carcinoma, urothelial cancer, or hepatocellular carcinoma. In certain aspects, the teachings herein provide a method of treating cancer in a subject, wherein the method comprises administering to the cancer cell a tissue or composition of the teachings herein and the cancer is a solid tumor. In some embodiments, the teachings herein provide methods of treating cancer in a subject, wherein the method comprises administering aiRNA or a composition of the teachings herein to a cancer cell and the cancer is a pancreatic cancer; bladder Cancer; colorectal cancer; breast cancer; prostate cancer; kidney cancer; hepatocellular carcinoma; lung cancer; ovarian cancer; cervical cancer; gastric cancer; esophageal cancer; head and neck cancer; melanoma; neuroendocrine cancer; CNS cancer; brain tumor; Skeletal cancer; or soft tissue sarcoma. The administration can be in a test tube or in vivo. Those of ordinary skill in the art will appreciate that the method can be practiced by aiRNA, a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable solvate thereof, a prodrug thereof, or the like of the teachings herein. To the object to achieve. In the case of a prodrug, the compound can be administered to cancer cells when the prodrug is converted to the compound in vivo.

In some embodiments, each of the cancers discussed herein can be advanced, relapsing, refractory to standard cancer treatment, and/or metastatic. The standard cancer treatment can include surgery, chemotherapy, and/or radiation therapy.

Example

The aiRNA of the teachings herein can be prepared using methods described in the art that are generally known to those of ordinary skill in the art or that are equivalent to the PCT Patent Application Publication No. WO2009029688, the entire disclosure of which is incorporated herein by reference.

Example 1: Expansion of CMV-specific CTL

PBMCs classified as HLAs were purchased from Cellular Technology, Ltd. PBMC were diluted in 5 x 10 ⁶ cells/mL in medium (RPMI-1640 supplemented with 10% inactivated human serum, 50 μM 2-indole ethanol) and seeded into 24-well plates (5 x 10 ⁶ cells / mL / slot). HLA-A*02:01 CMV pp65 peptide (NLVPMVATV) (IBA-Lifesciences, USA) was added on day 0 to a final concentration of 5 μM and 25 IU/mL IL-2 and 5 ng/mL IL-15 were added. This was supplemented every 3-4 days with fresh medium containing 10 μM HLA-A*02:01 CMV pp65 peptide, 50 IU/mL IL-2 (R&D Systems, USA) and 10 ng/mL IL-15 (R&D Systems, USA). Cultures. CD8 ⁺ T cells isolated using the kit (Miltenyi Biotec, Germany) were isolated from PBMC CD8 ⁺ T cells according to the manufacturer's guidelines.

Example 2: Transfection of PD-L1 and GFP aiRNA into luciferase-MDA-MB-231 cells

Luciferase-expressing MDA-MB-231 cells (Luc-MDA-MB-231) were purchased from Cell Biolabs, Inc. and grown in Dulbecco supplemented with 10% inactivated fetal bovine serum (FCS). Modified in Eagle's medium (DMEM). The cells were seeded into 6-well plates (5 x 10 ⁴ cells / 2 mL / well) 24 h before transfection. Cells were then transfected with PD-L1 aiRNA (aiPD-L1 #22) or GFP aiRNA at a final concentration of 1 nM using Lipoofectamine® RNAiMAX (Thermo Fisher, USA) according to the manufacturer's instructions. AiRNA was incubated with RNAiMAX in serum-free OPTI-MEM (Thermo Fisher, USA) for a total of 20 minutes prior to addition to the medium. The cells were harvested 48 hours after transfection using Accutase® cell separation solution (Sigma Aldrich, USA). GFP aiRNA was used as a control group. PD-L1 mRNA levels were measured by RT-PCR and PD-L1 protein expression was determined by flow cytometry.

As shown in Figures 2-4, 1 nM of aiPD-L1 #22 reduced PD-L1 mRNA in the Luc-MDA-MB-231 cell line by 77.8% and aiPD-L1 #22 decreased in LucMDA-MB- Surface PD-L1 expression on 231 cells. Figure 3 is a histogram and Figure 4 shows the average fluorescence intensity (C).

Example 3: Cytotoxicity analysis

Luca-MDA-MB-231 cells transfected with aiPD-L1 #22 were incubated with or without HLA-A*02:01 CMV pp65 peptide (NLVPMVATV) for 2 hours and then with 37%, 5% CO2. Wash twice with PBS. Luc-MDA-MB-231 cells loaded with CMV peptide or without CMV peptide were plated into 96-well plates (2000 cells/well). CMV-specific CD8 ⁺ T cells were then added to the 96-well plate at a 50:1 effector:target cell (E/T) ratio and further cultured for a total of 24 hours. Anti-human PD-L1 Ab (Proliferation: MIH1, functional grade purified) (Affymetrix eBioscience, USA) at 10 μg/mL (final concentration) was used as a control group. The intracellular luciferase activity of Luc-MDA-MB-231 cells was then measured using XenoLight D-luciferin K+ salt (PerkinElmer, USA). The percentage lysis line was calculated as the luminescence of Luc-MDA-MB-231 pulsed with CMV peptide or the luminescence of Luc-MDA-MB-231 pulsed with no CMV peptide.

As shown in Figure 5, the aiPD-L1 #22 tube increased the antigen-specific cytotoxicity of CD8 ⁺ T cells to equal (if not greater than) the level of anti-human PDL1 antibody.

Example 4: Serum stability of aiPD-L1 modified by 2'-OMe

Unmodified aiPD-L1 or 2'-OMe modified aiPD-L1 in 100% human serum (10 modifications at the 3' and 5' ends of the first strand and in the second strand There are 5 modifications at the 3' end and 5' end) for a total of 3 hours. The aiRNA decomposition in each sample was then evaluated by running a 15% PAGE gel. As shown in Figure 6, the 2'-OMe modified aiPD-L1 was significantly more stable than the unmodified aiPD-L1 line.

Example 5: Efficacy of aiPD-L1 modified by 2'-OMe

The 2'-OMe modified aiPD-L1 of Example 4 was transfected into RKO cells at a concentration of 50 pM, 250 pM, and 500 pM. Western blot analysis of PD-L1 performance was performed two days after transfection. Immunoblotting analysis of total cell lysates was then performed using anti-PD-L1 and anti-beta actin antibodies. As shown in Figure 7, the 2'-OMe modified aiPD-L1 was effective in inhibiting PD-L1 protein expression even at pM concentrations.

Example 6: Cytotoxicity of aiPD-L1 modified by 2'-OMe

To determine the cytotoxicity of the modified aiPD-L1, 1 nM of the 2'-OMe modified aiPD-L1 or GFP control aiRNA of Example 4 was transfected into MDAMD-231 cells expressing luciferase. The expanded CD8 ⁺ T cells in the mixed lymphocyte-peptide culture (MIPC) were isolated by MACS separation. The peptide-pulsed/unpeptide-pulsed MDA-MB-231 and CD8 ⁺ T cells were then co-cultured overnight and the number of viable cells was measured by luminescence.

As shown in Figure 8, the 2'-OMe modified aiPD-L1 tube increased the antigen-specific cytotoxicity of CD8 ⁺ T cells.

Example 7: Co-culture of breast cancer cells transfected with aiPD-L1/TCR activator increased IL-2 expression in PD-1 ⁺ Jurkat cells treated with α CD28 antibody

The shuffling or ai aiPD-L1 RNA with TCR- activator plasmid (BPS Bioscience) together with transfected into MDA-MB-231 cells (human breast cancer cell lines; ATCC® HTB-26 ^TM) in. Jurkat T cells stably expressing PD-1 were co-cultured with aiRNA-transfected MDA-MB-231 cells and stimulated with α CD28 antibody for 24 hours. IL-2 performance was measured by ELISA.

Figure 9 shows that co-cultured aiPD-L1 transfected MDA-MB-231 cells (but not ai-transfected MDA-MB-231 cells) reverted to α-CD28 antibody in PD-1 ⁺ Jurkat Induced IL-2 manifestations.

Those of ordinary skill in the art will recognize (or be able to determine, by routine experimentation) many equivalents of the particular embodiments of the teachings herein. Such equivalents are intended to be covered by the scope of the following patent application.

Without wishing to be bound by any particular theory or analysis and with respect to the exemplified RNA molecules, Figure 1A shows the alignment stock (sequence number 1-46) and the antisense strand (sequence number 47-92) and the exemplary PD-L1 target mRNA sequence (SEQ ID NO: 140-164); depicting nucleotides that are linearly corresponding to the corresponding complementary nucleotides of the PD-L1 mRNA sequence in the antisense strand and the length of the antisense strand (in dark gray) The number of home shades) and the % GC content, and the antisense strands exceed the length of the 5' and 3' overhang of the sense strand; and depict the exemplary PD-L1 5' end (sequence ID 139) and the 3' end (SEQ ID NO: 165) mRNA sequence.

Without wishing to be bound by any particular theory or analysis and with respect to the exemplified RNA molecules, FIG. 1B classifies exemplary RNA molecules 1-46 according to %GC content, and Figure 1C aligns PD-L1 target nucleotide sequences (eg, sequences) Identification numbers 140, 141, 144, 147, 150, 155, 156, 158, 161 and 164) and exemplary full-length PD-L1 (sequence identification number 166); Figure 1D shows exemplary full-length PD-L1 mRNA (sequence identification number) 166) and subdivided into seven overlapping sequences (sequence identification number: 167-173), each of which can be targeted by a subset of exemplary RNA molecules.

Figure 2 shows relative PD-L1 mRNA levels in MDA-MB-231 cell lines treated with exemplary RNA molecules or aiGFP control groups according to specific aspects of the teachings herein.

3 is a histogram of an exemplary flow cytometry analysis of PD-L1 expression in an MDA-MB-231 cell line treated with an exemplary RNA molecule or an aiGFP control group, according to a specific aspect of the teachings herein. .

Figure 4 shows an example of PD-L1 expression in an MDA-MB-231 cell line treated with an exemplary RNA molecule or an aiGFP control group according to a specific aspect of the teachings herein. The average fluorescence intensity of the analyzed flow cytometry analysis. The asterisk (*) indicates a significant difference (p=0.0004) with respect to the control group calculated using the Dunnett's multiple comparison test.

Figure 5 shows relative tumor cell-specific T cell cytotoxicity following treatment of tumor cells with exemplary RNA molecules or PD-L1 antibodies, according to specific aspects of the teachings herein.

Figure 6 shows the relative serum stability of an exemplary modified RNA molecule compared to an unmodified RNA molecule in accordance with a particular aspect of the teachings herein.

Figure 7 shows the efficacy of an exemplary modified RNA molecule in accordance with a particular aspect of the teachings herein.

Figure 8 shows relative tumor cell-specific T cell cytotoxicity following treatment of tumor cells with exemplary modified RNA molecules in accordance with specific aspects of the teachings herein.

Figure 9 shows exemplary specific aspects of IL-2 expression in PD-1 ⁺ Jurkat co-cultured with aiScramble or aiPD-L1 transfected MDA-MB-231 cells.

<110> Boston Biomedical Company Li Jiaqiang Sun Xiangao Shanchuan 絵里奈-古贺

<120> Asymmetric interfering RNA and its constituents, uses or preparations

<130> 1525P2/PCT

<150> US 62263684

<151> 2015-12-06

<150> US 62288061

<151> 2016-01-28

<160> 178

<170> PatentIn version 3.5

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<223> Antisense stock aiRNA sequence

<400> 81

<210> 82

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 82

<210> 83

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 83

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 84

<210> 85

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 85

<210> 86

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 86

<210> 87

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 87

<210> 88

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 88

<210> 89

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 89

<210> 90

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 90

<210> 91

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 91

<210> 92

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Antisense stock aiRNA sequence

<400> 92

<210> 93

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 93

<210> 94

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<220>

<221> misc_feature

<222> (20)..(20)

<223> N=A, U, G or C

<400> 94

<210> 95

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 95

<210> 96

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 96

<210> 97

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 97

<210> 98

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 98

<210> 99

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 99

<210> 100

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 100

<210> 101

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 101

<210> 102

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 102

<210> 103

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 103

<210> 104

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 104

<210> 105

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 105

<210> 106

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 106

<210> 107

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 107

<210> 108

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 108

<210> 109

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 109

<210> 110

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 110

<210> 111

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 111

<210> 112

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 112

<210> 113

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 113

<210> 114

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 114

<210> 115

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 115

<210> 116

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 116

<210> 117

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<220>

<221> misc_feature

<222> (20)..(20)

<223> N=A, U, G or C

<400> 117

<210> 118

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 118

<210> 119

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 119

<210> 120

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 120

<210> 121

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<220>

<221> misc_feature

<222> (20)..(20)

<223> n is a, c, g or u

<400> 121

<210> 122

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 122

<210> 123

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 123

<210> 124

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 124

<210> 125

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 125

<210> 126

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 126

<210> 127

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<220>

<221> misc_feature

<222> (20)..(20)

<223> N=A, U, G or C

<400> 127

<210> 128

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 128

<210> 129

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 129

<210> 130

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 130

<210> 131

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 131

<210> 132

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<220>

<221> misc_feature

<223> n is a, c, g or u

<222> (20)..(20)

<220>

<221> misc_feature

<222> (21)..(21)

<223> N=A, U, G or C

<400> 132

<210> 133

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 133

<210> 134

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 134

<210> 135

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 135

<210> 136

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 136

<210> 137

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 137

<210> 138

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<223> Target nucleotide sequence

<400> 138

<210> 139

<211> 33

<212> RNA

<213> Homo sapiens

<400> 139

<210> 140

<211> 33

<212> RNA

<213> Homo sapiens

<400> 140

<210> 141

<211> 33

<212> RNA

<213> Homo sapiens

<400> 141

<210> 142

<211> 33

<212> RNA

<213> Homo sapiens

<400> 142

<210> 143

<211> 33

<212> RNA

<213> Homo sapiens

<400> 143

<210> 144

<211> 33

<212> RNA

<213> Homo sapiens

<400> 144

<210> 145

<211> 33

<212> RNA

<213> Homo sapiens

<400> 145

<210> 146

<211> 33

<212> RNA

<213> Homo sapiens

<400> 146

<210> 147

<211> 33

<212> RNA

<213> Homo sapiens

<400> 147

<210> 148

<211> 33

<212> RNA

<213> Homo sapiens

<400> 148

<210> 149

<211> 33

<212> RNA

<213> Homo sapiens

<400> 149

<210> 150

<211> 33

<212> RNA

<213> Homo sapiens

<400> 150

<210> 151

<211> 33

<212> RNA

<213> Homo sapiens

<400> 151

<210> 152

<211> 33

<212> RNA

<213> Homo sapiens

<400> 152

<210> 153

<211> 33

<212> RNA

<213> Homo sapiens

<400> 153

<210> 154

<211> 33

<212> RNA

<213> Homo sapiens

<400> 154

<210> 155

<211> 33

<212> RNA

<213> Homo sapiens

<400> 155

<210> 156

<211> 33

<212> RNA

<213> Homo sapiens

<400> 156

<210> 157

<211> 33

<212> RNA

<213> Homo sapiens

<400> 157

<210> 158

<211> 33

<212> RNA

<213> Homo sapiens

<400> 158

<210> 159

<211> 33

<212> RNA

<213> Homo sapiens

<400> 159

<210> 160

<211> 33

<212> RNA

<213> Homo sapiens

<400> 160

<210> 161

<211> 33

<212> RNA

<213> Homo sapiens

<400> 161

<210> 162

<211> 33

<212> RNA

<213> Homo sapiens

<400> 162

<210> 163

<211> 33

<212> RNA

<213> Homo sapiens

<400> 163

<210> 164

<211> 33

<212> RNA

<213> Homo sapiens

<400> 164

<210> 165

<211> 33

<212> RNA

<213> Homo sapiens

<400> 165

<210> 166

<211> 3691

<212> RNA

<213> Homo sapiens

<400> 166

<210> 167

<211> 314

<212> RNA

<213> Homo sapiens

<400> 167

<210> 168

<211> 112

<212> RNA

<213> Homo sapiens

<400> 168

<210> 169

<211> 203

<212> RNA

<213> Homo sapiens

<400> 169

<210> 170

<211> 119

<212> RNA

<213> Homo sapiens

<400> 170

<210> 171

<211> 347

<212> RNA

<213> Homo sapiens

<400> 171

<210> 172

<211> 503

<212> RNA

<213> Homo sapiens

<400> 172

<210> 173

<211> 2433

<212> RNA

<213> Homo sapiens

<400> 173

<210> 174

<211> 15

<212> RNA

<213> Artificial sequence

<220>

<223> Oligonucleotide sequence

<400> 174

<210> 175

<211> 15

<212> RNA

<213> Artificial sequence

<220>

<223> Oligonucleotide sequence

<400> 175

<210> 176

<211> 15

<212> RNA

<213> Artificial sequence

<220>

<223> Oligonucleotide sequence

<400> 176

<210> 177

<211> 15

<212> RNA

<213> Artificial sequence

<220>

<223> Oligonucleotide sequence

<400> 177

<210> 178

<211> 16

<212> RNA

<213> Artificial sequence

<220>

<223> Oligonucleotide sequence

<400> 178

Claims (45)

An RNA molecule comprising an antisense strand comprising a 5' end and a 3' end nucleotide separated by 17, 18, 19, 20, or 21 nucleotides, and a sense strand, comprising a 5' terminal nucleotide complementary to a nucleotide other than a 3' terminal nucleotide thereof and a 3' terminal nucleotide complementary to a nucleotide of the antisense strand, wherein the antisense strand At least 14 nucleotide lines are complementary to the sense strand, and wherein at least 14 nucleotide strands of the antisense strand are linearly corresponding to a corresponding complementary nucleotide in a nucleotide sequence of interest selected from: Sequence identification number 167, 168, 169, 170, 171, 172 or 173. The RNA molecule according to claim 1, wherein at least 14 nucleotide lines of the antisense strand correspond linearly with a corresponding complementary nucleotide in a target nucleotide sequence selected from the group consisting of: a sequence identifier 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 or 164. The RNA molecule according to claim 1, wherein the antisense strand comprises a nucleotide sequence of: sequence identifiers 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 or 92. The RNA molecule according to claim 1, wherein the antisense strand comprises at least 7 nucleotides linearly corresponding to a corresponding complementary nucleotide in a nucleotide sequence of interest selected from the group consisting of: a sequence identifier 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 or 138. The RNA molecule according to claim 1, wherein at least 7 of at least 14 nucleotides of the antisense strand is compared to a corresponding complementary nucleotide in a target nucleotide sequence selected from the group consisting of Consecutive and linearly corresponding: sequence identification number 167, 168, 169, 170, 171, 172 or 173. An RNA molecule according to claim 1, wherein at least 14 nucleotide lines of the sense strand correspond linearly with a target nucleotide sequence in: sequence identifiers 167, 168, 169, 170, 171, 172 or 173. An RNA molecule according to claim 1, wherein at least 14 nucleotide lines of the sense strand correspond linearly with a target nucleotide sequence in: sequence identifiers 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 or 164. The RNA molecule according to claim 1, wherein the sense strand comprises a nucleotide sequence selected from the group consisting of: sequence number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46. An RNA molecule according to claim 1, wherein the antisense strand has 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides with a target nucleoside selected from the group consisting of The corresponding linearity in the acid sequence is discontinuous compared to the complementary nucleotide: sequence identifier 167, 168, 169, 170, 171, 172 or 173. An RNA molecule according to claim 1, wherein 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides of the antisense strand are not associated with a target core selected from the group consisting of The corresponding linear corresponding nucleotide complement in the nucleotide sequence: sequence identifier 167, 168, 169, 170, 171, 172 or 173. An RNA molecule according to claim 1 wherein at least 14 nucleotides of the sense strand are contiguous and linearly corresponding to the corresponding complementary nucleotide of the antisense strand. An RNA molecule according to claim 1, wherein the 5' terminal nucleotide of the sense strand is complementary to the first, second or third nucleotide adjacent to the 3' terminal nucleotide of the antisense strand . An RNA molecule according to claim 11 wherein the 3' terminal nucleotide of the sense strand is complementary to the first, second or third nucleotide adjacent to the 5' terminal nucleotide of the antisense strand . An RNA molecule according to the first aspect of the patent application, wherein the 5' end of the sense strand is separated from the 3' terminal nucleotide by 13 nucleotides. An RNA molecule according to claim 13 wherein the 19, 20 or 21 nucleotides of the antisense strand are linearly corresponding to a corresponding complementary nucleotide in a nucleotide sequence of interest selected from the group consisting of: Sequence identification number 167, 168, 169, 170, 171, 172 or 173. The RNA molecule according to claim 1, wherein the nucleotide sequence of the antisense strand that linearly corresponds to the corresponding complementary nucleotide in the target nucleotide sequence has the following GC content: 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%. The RNA molecule according to claim 1, wherein the nucleotide sequence of the antisense strand that linearly corresponds to the corresponding complementary nucleotide in the target nucleotide sequence has the following GC-containing Amount: about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, or about 44%. The RNA molecule according to claim 1, wherein the nucleotide sequence of the antisense strand that linearly corresponds to the corresponding complementary nucleotide in the target nucleotide sequence has the following GC content: 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%. The RNA molecule according to claim 1, wherein the nucleotide sequence of the antisense strand that linearly corresponds to the corresponding complementary nucleotide in the target nucleotide sequence has a GC content of about 33%. An anti-PD-L1 aiRNA 1-46. The RNA molecule of any one of claims 1 to 20, wherein the sense strand or the antisense strand comprises at least one modified nucleotide or analog thereof. The RNA molecule according to claim 21, wherein the 2'-OH group of the at least one modified ribonucleotide or analogue thereof is substituted with H or a 2'-O-methyl group. The RNA molecule according to claim 22, wherein the at least one modified nucleotide or analog thereof is a sugar, a backbone, and/or a base modified ribonucleotide. An RNA molecule according to claim 23, wherein the modified ribonucleotide of the main chain comprises a modification in linkage with a phosphodiester of another ribonucleotide. An RNA molecule according to claim 24, wherein the phosphodiester linkage is modified to comprise a nitrogen or sulfur heteroatom. An RNA molecule according to claim 24, wherein the at least one modified nucleotide or analogue thereof comprises a phosphothioate group. An RNA molecule according to claim 24, wherein the at least one modified nucleotide or analogue thereof comprises inosine or a tritylated base. The RNA molecule according to claim 24, wherein the at least one modified nucleotide or analog thereof is a modified sugar ribonucleotide, wherein the 2'-OH group is H, OR, R, Halogen, SH, SR, NH ₂ , NHR, NR ₂ , or CN, and wherein each R is independently a C1-C6 alkyl, alkenyl or alkynyl group, and the halo group is F, Cl, Br, or I . The RNA molecule according to any one of the preceding claims, wherein the 5' terminal nucleotide of the antisense strand and the first nucleotide of the adjacent 5' terminal nucleotide comprise an "AA" motif, "UU" Mold, "CC" phantom, "AU" phantom, "AC" phantom, "UA" phantom, "UC" phantom, or "CA" phantom. An RNA molecule according to any one of the preceding claims, wherein the RNA molecule comprises a deoxyribonucleotide. An RNA molecule according to any one of the preceding claims, wherein the first nucleotide of the adjacent 3' terminal nucleotide of the antisense strand is not dT. The RNA molecule according to any one of the preceding claims, wherein the silencing of the nucleotide sequence by PD-L1 by the RNA molecule is therapeutically more effective than the anti-PD-L1 antibody in enhancing tumor-specific T cell cytotoxicity. The RNA molecule according to any one of the preceding claims, wherein the administration of the RNA molecule to a subject in need thereof does not induce an interferon response. A composition comprising the RNA molecule according to item 1 of the scope of the patent application. The composition according to claim 34, wherein the composition comprises nanoparticle having one or more PD-L1 aiRNA. A kit comprising the RNA molecule according to item 1 of the patent application or the composition according to item 34 of the patent application. A performance vector comprising a nucleic acid sequence encoding an RNA molecule according to claim 1 of the scope of the patent application. A performance vector according to claim 37, wherein the expression vector is a viral, eukaryotic, or bacterial expression vector. An isolated cell comprising a performance vector according to claim 37 of the scope of the patent application. An isolated cell comprising an RNA molecule according to item 1 of the scope of the patent application. A cell according to claim 40, wherein the cell is a mammal, a bird, an insect, a yeast or a bacterial cell. A method for treating a disease or condition, comprising administering a therapeutically effective amount of an RNA molecule according to any one of claims 1 to 33 to a subject in need thereof. A method for treating, preventing, or ameliorating cancer in a subject, comprising administering a therapeutically effective amount of the RNA molecule according to any one of claims 1 to 33 to a subject in need thereof, wherein the cancer AIDS-related cancer, breast cancer, gastrointestinal/gastrointestinal cancer, endocrine and neuroendocrine cancer, cancer of the eye, genitourinary cancer, germ cell cancer, gynecological cancer, head and neck cancer, blood cancer, musculoskeletal cancer, Neurological cancer, respiratory/thoracic cancer, skin cancer, childhood cancer or cancer of unknown origin. The method of claim 42, wherein the RNA molecule is administered systemically or locally. The method according to claim 44, wherein the cancer is metastatic, recurrent or resistant to chemotherapy and/or radiation.