US20210348166A1

US20210348166A1 - Immunotherapy of cancer

Info

Publication number: US20210348166A1
Application number: US17/150,668
Authority: US
Inventors: Alexey WOLFSON; Alexey Eliseev; Taisia Shmushkovich
Original assignee: Phio Pharmaceuticals Corp
Current assignee: Phio Pharmaceuticals Corp
Priority date: 2013-12-02
Filing date: 2021-01-15
Publication date: 2021-11-11
Also published as: WO2015084897A2; RU2016126488A3; CN113151180A; US10934550B2; CN106061488A; RU2744194C2; EP3079707A2; JP2023067871A; JP6772062B2; EP3079707A4; RU2016126488A; JP2016540511A; CN106061488B; JP2021019608A; WO2015084897A3; US20160304873A1

Abstract

Immunogenic modulators and compositions comprising oligonucleotide agents capable of inhibiting suppression of immune response by reducing expression of one or more gene involved with an immune response suppression mechanism.

Description

CROSS REFERENCE

This application claims priority to U.S. Provisional Application No. 61/910,728, filed Dec. 2, 2013, herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to immunogenic compositions, method of making immunogenic compositions, and methods of using immunogenic compositions for the treatment of cell proliferative disorders or infectious disease, including, for example, cancer and autoimmune disorders.
More particularly, the invention provides cells that are treated with oligonucleotides specifically designed to modulate expression of target genes involved in tumor immune resistance mechanisms.

BACKGROUND

Immunotherapy is the “treatment of disease by inducing, enhancing, or suppressing an immune response”. Immunotherapies designed to elicit or amplify an immune response are activation immunotherapies, while immunotherapies that reduce or suppress immune response are classified as suppression immunotherapies.
Immunotherapy of cancer has become increasingly important in clinical practice over recent decades. The primary approach in today's standard of care is passive immunotherapy through the use of recombinant monoclonal antibodies (mAbs). MAbs act through a mechanisms relevant to the body's own humoral immune response, by binding to key antigens involved in the tumor development and causing moderate forms of cell-mediated immunity, such as antibody-dependent cell-mediated cytotoxicity (ADCC).
Another group of emerging immunotherapeutic approaches is based on the administration of cells capable of destroying tumor cells. The administered cells may be the patient's own tumor-infiltrating lymphocytes (TIL), isolated and expanded ex-vivo. In some cases, TIL are capable of recognizing a variety of tumor associated antigens (TAA), while in other cases TIL can be reactivated and expanded in vitro to recognize specific antigens. The TIL-based therapeutic approaches are commonly referred to as “adoptive cell transfer” (ACT).
Further developments of ACT involve genetic modifications of T-cells to express receptors that recognize specific tumor-associated antigens (TAA). Such modifications may induce the expression of a specific T-cell receptor (TCR) or of a chimeric antigen receptor (CAR) consisting of TAA-specific antibody fused to CD3/co-stimulatory molecule transmembrane and cytoplasmic domains.
The ACT methods may also be considered as passive immunotherapeutic approaches in that they act directly on the tumor cells without invoking an extended immune response. However, unlike mAbs, ACT agents are capable of fully destroying the tumor cells, as opposed to the blockade of selected receptors and moderate cellular responses such as ADCC.
There is ongoing development of numerous methods of active immunotherapy, which restore the ability of body's own immune system to generate antitumor response. Active immunotherapeutic agents are often called therapeutic cancer vaccines, or just cancer vaccines. Many cancer vaccines are currently in clinical trials, and sipuleucell-T has recently become the first such vaccine approved by the United States FDA.
There are several classes of cancer vaccines using different antigens and different mechanisms of generating cell-mediated immune response. One class of vaccines is based on peptide fragments of antigens selectively expressed by tumor cells. The peptides are administered alone or in combination with immune-stimulatory agents, which may include adjuvants and cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).
Another class of cancer vaccines is based on modified (e.g. sub-lethally irradiated) tumor cells used as antigens, also in combinations with immunostimulatory agents. Vaccines of this type currently in clinical trials are based both on autologous (e.g. OncoVAX, LipoNova) and allogeneic (e.g. Canvaxin, Onyvax-P, GVAX) tumor cell lines.
Yet another class of cancer vaccines uses dendritic cells. By their nature, dendritic cells (DC) are “professional” antigen-presenting cells capable of generating of a strong antigen-dependent cell-mediated immune response and eliciting therapeutic T-cells in vivo. DC-based cancer vaccines usually comprise DCs isolated from patients or generated ex vivo by culturing patient's hematopoietic progenitor cells or monocytes. DCs are further loaded with tumor antigens and sometimes combined with immune-stimulating agents, such as GM-CSF. A large number of DC-vaccines are now in clinical trials, and the first FDA-approved vaccine sipuleucell-T is based on DC.
Mechanisms of Immunosuppression and Therapeutic Approaches to its Mitigation
One of the key physiologic functions of the immune system is to recognize and eliminate neoplastic cells, therefore an essential part of any tumor progression is the development of immune resistance mechanisms. Once developed, these mechanisms not only prevent the natural immune system from effecting the tumor growth, but also limit the efficacy of any immunotherapeutic approaches to cancer. An important immune resistance mechanism involves immune-inhibitory pathways, sometimes referred to as immune checkpoints. The immune-inhibitory pathways play particularly important role in the interaction between tumor cells and CD8+ cytotoxic T-lymphocytes, including ACT therapeutic agents. Among important immune checkpoints are inhibitory receptors expressed on the T-cell surface, such as CTLA-4, PD1 and LAG3, among others.
The importance of the attenuation of immune checkpoints has been recognized by the scientific and medical community. One way to mitigate immunosuppression is to block the immune checkpoints by specially designed agents. The CTLA-4-blocking-antibody, ipilimumab, has recently been approved by the FDA. Several molecules blocking PD1 are currently in clinical development.
Immunosuppression mechanisms also negatively affect the function of dendritic cells and, as a consequence, the efficacy of DC-based cancer vaccines. Immunosuppressive mechanisms can inhibit the ability of DC to present tumor antigens through the MHC class I pathway and to prime naïve CD8+ T-cells for antitumor immunity. Among the important molecules responsible for the immunosuppression mechanisms in DC are ubiquitin ligase A20 and the broadly immune-suppressive protein SOCS1.
The efficacy of immunotherapeutic approaches to cancer can be augmented by combining them with inhibitors of immune checkpoints. Numerous ongoing preclinical and clinical studies are exploring potential synergies between cancer vaccines and other immunotherapeutic agents and checkpoint blocking agents, for example, ipilimumab. Such combination approaches have the potential to result in significantly improved clinical outcomes.
However, there are a number of drawbacks of using cancer immunotherapeutic agents in combination with checkpoint inhibitors. For example, immune checkpoint blockade can lead to the breaking of immune self-tolerance, thereby inducing a novel syndrome of autoimmune/auto-inflammatory side effects, designated “immune related adverse events,” mainly including rash, colitis, hepatitis and endocrinopathies (Corsello, et al. J. Clin. Endocrinol. Metab., 2013, 98:1361).
Reported toxicity profiles of checkpoint inhibitors are different than the toxicity profiles reported for other classes of oncologic agents. Those involve inflammatory events in multiple organ systems, including skin, gastrointestinal, endocrine, pulmonary, hepatic, ocular, and nervous system. (Hodi, 2013, Annals of Oncology, 24: Suppl, i7).
In view of the above, there is a need for new cancer therapeutic agents that can be used in combination with checkpoint inhibitors as well as other classes of oncolytic agents without risk of adverse inflammatory events in multiple organ systems previously reported for checkpoint inhibitors. The immunotherapeutic cells of the invention, prepared by treating cells with a combination oligonucleotide agents targeting genes associated with tumor or infections disease resistance mechanisms, as well as methods of producing such therapeutic cells and methods of treating disease with the produced therapeutic cells, satisfy this long felt need.

SUMMARY OF EMBODIMENTS OF THE INVENTION

The efficacy of immunotherapeutic approaches to cell proliferation disorders and infectious diseases can be augmented by combining them with inhibitors of immune checkpoints. Numerous synergies between cancer vaccines and other immunotherapeutic agents and checkpoint blocking agents provide opportunities for combination approaches that may significantly improve clinical outcomes for example, in proliferative cell disorders and immune diseases.
Various embodiments of the inventions disclosed herein include compositions comprising therapeutic cells obtained by treating cells ex vivo with oligonucleotides to modulate expression of target genes involved in immune suppression mechanisms. The oligonucleotide agent may be an antisense oliogonucleotide (ASO), including locked nucleic acids (LNAs), methoxyethyl gapmers, and the like, or an siRNA, miRNA, miRNA-inhibitor, morpholino, PNA, and the like. The oligonucleotide is preferably a self-delivered (sd) RNAi agent. The oligonucleotides may be chemically modified, for example, including at least one 2-O-methyl modification, 2′-Fluro modification, and/or phosphorothioate modification. The oligonucleotides may include one or more hydrophobic modification, for example, one or more sterol, cholesterol, vitamin D, Naphtyl, isobutyl, benzyl, indol, tryptophane, or phenyl hydrophobic modification. The oligonucleotide may be a hydrophobically-modified siRNA-antisense hybrid. The oligonucleotides may be used in combination with transmembrane delivery systems, such as delivery systems comprising lipids.
In an embodiment, the cells are obtained and/or derived from a cancer or infectious disease patient, and may be, for example, tumor infiltrating lymphocytes (TIL) and/or T-cells, antigen presenting cells such as dendritic cells, natural killer cells, induced-pluripotent stem cells, stem central memory T-cells, and the like. The T-cells and NK-cells are preferably genetically engineered to express high-affinity T-Cell receptors (TCR) and/or chimeric antibody or antibody-fragment—T-Cell receptors (CAR). In an embodiment, the chimeric antibody/antibody fragment is preferably capable of binding to antigens expressed on tumor cells. Immune cells may be engineered by transfection with plasmid, viral delivery vehicles, or mRNAs.
In an embodiment, the chimeric antibody or fragment is capable of binding CD19 receptors of B-cells and/or binding to antigens expressed on tumors, such as melanoma tumors. Such melanoma-expressed antigens include, for example, GD2, GD3, HMW-MAA, VEGF-R2, and the like.
Target genes identified herein for modification include: cytotoxic T-cell antigen 4 (CTLA4), programmed cell death protein 1 (PD1), tumor growth factor receptor beta (TGFR-beta), LAG3, TIM3, and adenosine A2a receptor; anti-apoptotic genes including, but not limited to: BAX, BAC, Casp8, and P53; A20 ubiquitine ligase (TNFAIP3, SOCS1 (suppressor of cytokine signaling), IDO (indolamine-2,3-dioxygenase; tryptophan-degrading enzyme), PD-L1 (CD274)(surface receptor, binder to PD1 on Tcells), Notch ligand Deltal (DLL1), Jagged 1, Jagged 2, FasL (pro-apoptotic surface molecule), CCL17, CCL22 (secreted chemokines that attract Treg cells), IL10 receptor (IL10RA), p38 (MAPK14), STAT3, TNFSF4 (OX40L), MicroRNA miR-155, miR-146a, anti-apoptotic genes including but not limited to BAX, BAC, Casp8 and P53, and the like genes, and combinations thereof. Representative target sequences are listed in Table 1.
The engineered therapeutic cells are treated with RNAi agents designed to inhibit expression of one or more of the targeted genes. The RNAi agent may comprise a guide sequence that hybridizes to a target gene and inhibits expression of the target gene through an RNA interference mechanism, where the target region is selected from the group listed in Table 1. The RNA agent can be chemically modified, and preferably includes at least one 2′-O-methyl, 2′-O-Fluoro, and/or phosphorothioate modification, as well as at least one hydrophobic modification such as cholesterol, and the like.
The immunogenic compositions described herein are useful for the treatment of proliferative disorders, including cancers, and/or infectious disease and are produced by the ex-vivo treatment of cells with oligonucleotides to modulate the expression of target genes involved in tumor immune resistance mechanisms. The ex vivo treatment of cells includes administering to the cells an oligonucleotide capable of targeting and inhibiting expression of a gene involved in a tumor suppressor mechanism, such as the genes listed in Table 1. The oligonucleotide can be used in combination with a transmembrane delivery system that may comprise one or more of: lipid(s) and vector, such as a viral vector.
The invention includes a method of treating a cell proliferative disorder or infectious disease by administering to a subject in need thereof, an immunogenic composition comprising cells that have been treated with one or more oligonucleotide to modulate the expression of one or more target gene involved in tumor immune resistance mechanisms, for example, one or more of the target genes of Table 1.
The invention preferably includes immunogenic cells treated with a plurality of oligonucleotide agents targeting a combination of target genes described herein. The combination may target a plurality of suppressor receptor genes, cytokine receptor genes, regulatory genes, and/or apoptotic factors in order to inhibit tumor immune resistance mechanisms.
The present invention is directed to novel immunotherapeutic cells, methods of generating the immunotherapeutic cells, and therapeutic methods employing such cells.
A new method of immune checkpoint inhibition is described herein, applicable to a broad variety of cell-based immunotherapies, including, but not limited to adaptive cell transfer, for example, based on TIL, TCR, CAR, and other cell types, as well as dendritic cell-based cancer vaccines. Self-deliverable RNAi technology provides efficient transfection of short oligonucleotides in any cell type, including immune cells, providing increased efficacy of immunotherapeutic treatments. In addition, the activated immune cells can be protected by preventing apoptosis via inhibition of key activators of the apoptotic pathway, such as BAC, BAX, Casp8, and P53, among others.
The activated immune cells modified by oligonucleotide transfer for a single therapeutic agent for administration to a subject, providing a number of advantages as compared to separately administered combinations of vaccines and immunotherapeutics and separately administered checkpoint inhibitors. These advantages include lack of side effects associated with the checkpoint inhibitors in a single therapeutic agent (activated immune cells modified by oligonucleotides targeting immune resistance genes).
The claimed immunotherapeutic cells, method of producing immunotherapeutic cells by introduction of oligonucleotide molecules targeting immune resistance pathways, and methods of treating proliferative and infectious disease, improves upon any known immunotherapeutic cells and methods of producing immunotherapeutic cells because it provides:

- 1) a single therapeutic composition providing a combination of checkpoint inhibitors and other immune resistance mechanism inhibitors;
- 2) with reduced toxicity; and
- 3) increased efficacy as compared with other compositions.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

FIG. 1 is a schematic diagram showing the structure of an sdRNA molecule.

FIG. 2 is a graph showing sdRNA-induced silencing of GAPDH and MAP4K4 in HeLa cells.

FIG. 3 is a graph showing sdRNA-induced knock-down of multiple targets using sdRNA agents directed to three genes in NK-92 cells.

FIG. 4 is a graph showing the knock-down of gene expression in Human Primary T cells by sdRNA agents targeting TP53 and MAP4K4.

FIG. 5 is a graph showing sdRNA-induced knock-down of CTLA4 and PD1 in Human Primary T cells.

FIG. 6 is a graph showing the reduction of PDCD1 and CTLA-4 surface expression by sdRNA in Human Primary T cells.

FIG. 7 is a graph showing MAP4K4-cy3 sdRNA delivery into T and B cells in human PBMCs.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The invention is defined by the claims, and includes oligonucleotides specifically designed and selected to reduce and/or inhibit expression of suppressors of immune resistance (inhibitory oligonucleotides), compositions comprising cells modified by treatment with such inhibitory oligonucleotides, methods of making such compositions, and methods of using the compositions to treat proliferation and/or infectious diseases. In particular, cells are treated with a combination of oligonucleotide agents, each agent particularly designed to interfere with and reduce the activity of a targeted immune suppressor.
Preferably, the combination of oligonucleotide agents targets multiple immune suppressor genes selected from checkpoint inhibitor genes such as CTLA4, PD-1/PD-1L, BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3, B7-H4 receptors, and TGF beta type 2 receptor; cytokine receptors that inactivate immune cells, such as TGF-beta receptor A and IL-10 receptor; regulatory genes/transcription factors modulating cytokine production by immune cells, such as STAT-3 and P38, miR-155, miR-146a; and apoptotic factors involved in cascades leading to cell death, such as p53 and Cacp8.
Most preferably the oligonucleotide agent is a self-deliverable RNAi agent, which is a hydrophobically modified siRNA-antisense hybrid molecule, comprising a double-stranded region of about 13-22 base pairs, with or without a 3′-overhang on each of the sense and antisense strands, and a 3′ single-stranded tail on the antisense strand of about 2-9 nucleotides. The oligonucleotide contains at least one 2′-O-Methyl modification, at least one 2′-O-Fluoro modification, and at least one phosphorothioate modification, as well as at least one hydrophobic modification selected from sterol, cholesterol, vitamin D, napthyl, isobutyl, benzyl, indol, tryptophane, phenyl, and the like hydrophobic modifiers (see FIG. 1). The oligonucleotide may contain a plurality of such modifications.

Definitions

As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context requires otherwise:
Proliferative disease, as used herein, includes diseases and disorders characterized by excessive proliferation of cells and turnover of cellular matrix, including cancer, atherlorosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, cirrhosis of the liver, and the like. Cancers include but are not limited to, one or more of: small cell lung cancer, colon cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, pancreatic cancer, melanoma, hematological malignancy such as chronic myeloid leukemia, and the like cancers where immunotherapeutic intervention to suppress tumor related immune resistance is needed.
Immune target genes can be grouped into at least four general categories: (1) checkpoint inhibitors; (2) cytokine receptors that inactivate immune cells, (3) anti-apoptotic genes; and (4) regulator genes, for example, transcription factors.
Immune Checkpoint inhibitors (ICI), as used herein, include immunotherapeutic agents that bind to certain checkpoint proteins, such as cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) and its ligand PD-L1 to block and disable inhibitory proteins that prevent the immune system from attacking diseased cells such as cancer cells, liberating tumor-specific T cells to exert their effector function against tumor cells.
Tumor related immune resistance genes, as used herein, include genes involved in checkpoint inhibition of immune response, such as CTLA-4 and PD-1/PD-L1; TGF-beta, LAG3, Tim3, adenosine A2a receptor;
Regulator genes, as used herein, include transcription factors and the like that modulate cytokine production by immune cells, and include p38, STAT3, microRNAs miR-155, miR-146a;
Anti-apoptotic genes, as used herein, include BAX, BAC, Casp8, P53 and the like; and combinations thereof.
Infectious diseases, as used herein, include, but are not limited to, diseases caused by pathogenic microorganisms, including, but not limited to, one or more of bacteria, viruses, parasites, or fungi, where immunotherapeutic intervention to suppress pathogen related immune resistance and/or overactive immune response.
Immunogenic composition, as used herein, includes cells treated with one or more oligonucleotide agent, wherein the cells comprise T-cells. The T-cells may be genetically engineered, for example, to express high affinity T-cell receptors (TCR), chimeric antibody—T-cell receptors (CAR), where the chimeric antibody fragments are capable of binding to CD19 receptors of B-cells and/or to antigens expressed on tumor cells. In one embodiment, the chimeric antibody fragments bind antigens expressed on melanoma tumors, selected from GD2, GD3, HMW-MAA, and VEGF-R2.
Immunogenic compositions described herein include cells comprising antigen-presenting cells, dendritic cells, engineered T-cells, natural killer cells, stem cells, including induced pluripotent stem cells, and stem central memory T-cells. The treated cell also comprises one or a plurality of oligonucleotide agents, preferably sdRNAi agents specifically targeting a gene involved in an immune suppression mechanism, where the oligonucleotide agent inhibits expression of said target gene.
In one embodiment, the target gene is selected from A20 ubiquitin ligase such as TNFAIP3, SOCS1 (suppressor of cytokine signaling), Tyro3/Axl/Mer (suppressors of TLR signaling), IDO (indolamine-2,3-dioxygenase, tryptophan-degrading enzyme), PD-L1/CD274 (surface receptor, binds PD1 on T-cells), Notch ligand Delta (DLL1), Jagged 1, Jagged 2, FasL (pro-apoptotic surface molecule), CCL17, CCL22 (secreted chemokines that attract Treg cells), IL-10 receptor (IL10Ra), p38 (MAPK14), STAT3, TNFSF4 (OX40L), microRNA miR-155, miR-146a, anti-apoptotic genes, including but not limited to BAX, BAC, Casp8, and P53; and combinations thereof.
Particularly preferred target genes are those shown in Table 1.
Ex-vivo treatment, as used herein, includes cells treated with oligonucleotide agents that modulate expression of target genes involved in immune suppression mechanisms. The oligonucleotide agent may be an antisense oligonucleotide, including, for example, locked nucleotide analogs, methyoxyethyl gapmers, cyclo-ethyl-B nucleic acids, siRNAs, miRNAs, miRNA inhibitors, morpholinos, PNAs, and the like. Preferably, the oligonucleotide agent is an sdRNAi agent targeting a gene involved in an immune suppression mechanism. The cells treated in vitro by the oligonucleotide agent may be immune cells expanded in vitro, and can be cells obtained from a subject having a proliferative or infectious disease. Alternatively, the cells or tissue may be treated in vivo, for example by in situ injection and/or intravenous injection.
Oligonucleotide or oligonucleotide agent, as used herein, refers to a molecule containing a plurality of “nucleotides” including deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double-stranded form. The term encompasses nucleotides containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
Nucleotide, as used herein to include those with natural bases (standard), and modified bases well known in the art. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, for example, PCT Publications No. WO 92/07065 and WO 93/15187. Non-limiting examples of base modifications that can be introduced into nucleic acid molecules include, hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine and pseudouridine), propyne, and others. The phrase “modified bases” includes nucleotide bases other than adenine, guanine, cytosine, and uracil, modified for example, at the 1′ position or their equivalents.
As used herein, the term “deoxyribonucleotide” encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and/or to the linkages between deoxyribonucleotide in the oligonucleotide.
As used herein, the term “RNA” defines a molecule comprising at least one ribonucleotide residue. The term “ribonucleotide” defines a nucleotide with a hydroxyl group at the 2′ position of a □-D-ribofuranose moiety. The term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Nucleotides of the RNA molecules described herein may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
As used herein, “modified nucleotide” refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally-occurring that result from modification by enzymes that modify nucleotides, such as methyltransferases.
Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2′ modifications, e.g., 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2) 2-O-2′-bridge, 2′-LNA, and 2′-O-(N-methylcarbamate) or those comprising base analogs. In connection with 2′-modified nucleotides as described for the present disclosure, by “amino” is meant 2′-NH2 or 2′-O—NH2, which can be modified or unmodified. Such modified groups are described, for example, in U.S. Pat. Nos. 5,672,695 and 6,248,878.
As used herein, “microRNA” or “miRNA” refers to a nucleic acid that forms a single-stranded RNA, which single-stranded RNA has the ability to alter the expression (reduce or inhibit expression; modulate expression; directly or indirectly enhance expression) of a gene or target gene when the miRNA is expressed in the same cell as the gene or target gene. In one embodiment, a miRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a single-stranded miRNA. In some embodiments miRNA may be in the form of pre-miRNA, wherein the pre-miRNA is double-stranded RNA. The sequence of the miRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the miRNA is at least about 15-50 nucleotides in length (e.g., each sequence of the single-stranded miRNA is 15-50 nucleotides in length, and the double stranded pre-miRNA is about 15-50 base pairs in length). In some embodiments the miRNA is 20-30 base nucleotides. In some embodiments the miRNA is 20-25 nucleotides in length. In some embodiments the miRNA is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
Target gene, as used herein, includes genes known or identified as modulating the expression of a gene involved in an immune resistance mechanism, and can be one of several groups of genes, such as suppressor receptors, for example, CTLA4 and PD1; cytokine receptors that inactivate immune cells, for example, TGF-beta receptor, LAG3, Tim3, adenosine A2a receptor, and IL10 receptor; regulatory genes for example, STAT3, p38, mir155 and mir146a; and apoptosis factors involved in cascades leading to cell death, for example, P53, Casp8, BAX, BAC, and combinations thereof. See also preferred target genes listed in Table 1.
As used herein, small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, defines a group of double-stranded RNA molecules, comprising sense and antisense RNA strands, each generally of about 1022 nucletides in length, optionally including a 3′ overhang of 1-3 nucleotides. siRNA is active in the RNA interference (RNAi) pathway, and interferes with expression of specific target genes with complementary nucleotide sequences.
As used herein, sdRNA refers to “self-deliverable” RNAi agents, that are formed as an asymmetric double-stranded RNA-antisense oligonucleotide hybrid. The double stranded RNA includes a guide (sense) strand of about 19-25 nucleotides and a passenger (antisense) strand of about 10-19 nucleotides with a duplex formation that results in a single-stranded phosphorothiolated tail of about 5-9 nucleotides.
The RNA sequences may be modified with stabilizing and hydrophobic modifications such as sterols, for example, cholesterol, vitamin D, naphtyl, isobutyl, benzyl, indol, tryptophane, and phenyl, which confer stability and efficient cellular uptake in the absence of any transfection reagent or formulation. Immune response assays testing for IFN-induced proteins indicate sdRNAs produce a reduced immunostimulatory profile as compared other RNAi agents. See, for example, Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864.

Cell-Based Immunotherapeutics

In general, cells are obtained from subjects with proliferative disease such as cancer, or an infectious disease such as viral infection. The obtained cells are treated directly as obtained or may be expanded in cell culture prior to treatment with oligonucleotides. The cells may also be genetically modified to express receptors that recognize specific antigens expressed on the tumor cell surface (CAR) or intracellular tumor antigens presented on MHC class I (TCR).

Oligonucleotide Agents

Antisense Oligonucleotides
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a double stranded RNA molecule, generally 19-25 base pairs in length. siRNA is used in RNA interference (RNAi), where it interferes with expression of specific genes with complementary nucleotide sequences.
Double stranded DNA (dsRNA) can be generally used to define any molecule comprising a pair of complementary strands of RNA, generally a sense (passenger) and antisense (guide) strands, and may include single-stranded overhang regions. The term dsRNA, contrasted with siRNA, generally refers to a precursor molecule that includes the sequence of an siRNA molecule which is released from the larger dsRNA molecule by the action of cleavage enzyme systems, including Dicer.
sdRNA (self-deliverable) are a new class of covalently modified RNAi compounds that do not require a delivery vehicle to enter cells and have improved pharmacology compared to traditional siRNAs. “Self-deliverable RNA” or sdRNA is a hydrophobically modified RNA interfering-antisense hybrid, demonstrated to be highly efficacious in vitro in primary cells and in vivo upon local administration. Robust uptake and/or silencing without toxicity has been demonstrated in several tissues including dermal, muscle, tumors, alveolar macrophages, spinal cord, and retina cells and tissues. In dermal layer and retina, intradermal and intra-vitreal injection of sdRNA at mg doses induced potent and long lasting silencing.
While sdRNA is a superior functional genomics tool, enabling RNAi in primary cells and in vivo, it has a relatively low hit rate as compared to conventional siRNAs. While the need to screen large number of sequences per gene is not a limiting factor for therapeutic applications, it severely limits the applicability of sdRNA technology to functional genomics, where cost effective compound selection against thousands of genes is required. To optimize sdRNA structure, chemistry, targeting position, sequence preferences, and the like, a proprietary algorithm has been developed and utilized for sdRNA potency prediction. Availability of sdRNA reagents that are active in all cell types ex vivo and in vivo enables functional genomics and target stratification/validation studies.

Proprietary Algorithm

SdRNA sequences were selected based on a proprietory selection algorithm, designed on the basis of a functional screen of over 500 sdRNA sequences in the luciferase reporter assay of HeLa cells. Regression analysis of was used to establish a correlation between the frequency of occurrence of specific nucleotide and modification at any specific position in sdRNA duplex and its functionality in gene suppression assay. This algorithm allows prediction of functional sdRNA sequences, defined as having over 70% knockdown at 1 μM concentration, with a probability over 40%.
Table 1 shows predictive gene targets identified using the proprietary algorithm and useful in the cellular immunotherapeutic compositions and methods described herein.

Delivery of RNAi Agents

BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; Applic BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; ation of RNAi technology to functional genomics studies in prim BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; ary cells and in vivo is limited by requirements to formulate siRNAs into lipids or use of other cell delivery techniques. To circumvent delivery problems, the self-deliverable RNAi technology provides a method of directly transfecting cells with the RNAi agent, without the need for additional formulations or techniques. The ability to transfect hard-to-transfect cell lines, high in vivo activity, and simplicity of use, are characteristics of the compositions and methods that present significant functional advantages over traditional siRNA-based techniques. The sdRNAi technology allows direct delivery of chemically synthesized compounds to a wide range of primary cells and tissues, both ex-vivo and in vivo.
To enable BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; self-delivery, traditional siRNA molecules require a substantial reduction in size and the introduction of extensive chemical modifications which are not well tolerated by RNAi machinery, resulting in extremely low probability of finding active molecules (low hit rate). In contrast, the sdRNA technology allows efficient RNAi delivery to primary cells and tissues in vitro and in vivo, with demonstrated silencing efficiency in humans.
The general structure of sdRNA molecules is shown in FIG. 1. sdRNA are formed as hydrophobically-modified siRNA-antisense oligonucleotide hybrid structures, and are disclosed, for example in Byrne et al., December 2013, J. Ocular Pharmacology and Therapeutics, 29(10): 855-864.
Oligonucleotide Modifications: 2′-O-Methyl, 2′-O-Fluro, Phosphorothioate
The oligonucleotide agents preferably comprise one or more modification to increase stability and/or effectiveness of the therapeutic agent, and to effect efficient delivery of the oligonucleotide to the cells or tissue to be treated. Such modifications include at least one
BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; 2′-O-methyl modification, at least one 2′-O-Fluro modification, and at least one diphosphorothioate modification. Additionally, the oligonucleotide is modified to include one or more hydrophobic modification selected from sterol, cholesterol, vitamin D, naphtyl, isobutyl, benzyl, indol, tryptophane, and phenyl. The hydrophobic modification is preferably a sterol.
Delivery of Oligonucleotide Agents to Cells
The oligonucleotides may be delivered to the cells in combination with a transmembrane delivery system, preferably comprising lipids, viral vectors, and the like. Most preferably, the oligonucleotide agent is a self-delivery RNAi agent, that does not require any delivery agents.
Combination Therapy
Most preferred for this invention, e.g. particular combinations of elements and/or alternatives for specific needs. This objective is accomplished by determining the appropriate genes to be targeted by the oligonucleotide in order to silence immune suppressor genes and using the proprietary algorithm to select the most appropriate target sequence.
It is preferred that the immunotherapeutic cell be modified to include multiple oligonucleotide agents targeting a variety of genes involved in immune suppression and appropriate for the selected target disease and genes. For example, a preferred immunotherapeutic cell is a T-Cell modified to knock-down both CTLA-4 and PD-1
Additional combinations of oligonucleotides to related genes involved in immune suppression include varied combinations of the selected target sequences of Table 1.
BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; Preferred BTLA (B and T-lymphocyte attenuator), KIR (killer immunoglobulin-like receptors), B7-H3 and B7-H4 receptors and TGFbeta type 2 receptor; therapeutic combinations include cells engineered to knock down gene expression of the following target genes:

- a) CTLA4 and PD1
- b) STAT3 and p38
- c) PD1 and BaxPD1, CTLA4, Lag-1, ILM-3, and TP53
- d) PD1 and Casp8
- e) PD1 and IL10R

The therapeutic compositions described herein are useful to treat a subject suffering from a proliferation disorder or infectious disease. In particular, the immunotherapeutic composition is useful to treat disease characterized by suppression of the subjects immune mechanisms. The sdRNA agents described herein are specifically designed to target genes involved in diseases-associated immune suppression pathways.
Methods of treating a subject comprise administering to a subject in need thereof, an immunogenic composition comprising an sdRNAi agent capable of inhibiting expression of genes involved in immune suppression mechanisms, for example, any of the genes listed in Table 1 or otherwise described herein.

EXAMPLES

The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Example 1

Self-Deliverable RNAi Immunotherapeutic Agents

Immunotherapeutic agents described herein were produced by treating cells with particular sdRNA agents designed to target and knock down specific genes involved in immune suppression mechanisms. In particular, the following cells and cell lines have been successfully treated with sdRNA and were shown to knock down at least 70% of targeted gene expression in the specified human cells.
These studies demonstrated utility of these immunogenic agents to suppress expression of target genes in cells normally very resistant to transfection, and suggests the agents are capable of reducing expression of target cells in any cell type.

TABLE 2

Cell Type	Target Gene	sdRNA target sequence	% Knock Down

Primary human T-cells	TP53 (P53)	GAGTAGGACATACCAGCTTA (SEQ ID NO: 1001)	>70% 2 uM

Primary human T-cells	MAP4K4	AGAGTTCTGTGGAAGTCTA (SEQ ID NO: 1002)	>70% 2 uM

Jurkat T-Iymphoma cells	MAP4K4	AGAGTTCTGTGGAAGTCTA (SEQ ID NO: 1003)	100% 1 uM 72h

NK-92 cells	MAP4K4	AGAGTTCTGTGGAAGTCTA (SEQ ID NO: 1004)	80% 2 uM 72h

NK-92 cells	PPIB	ACAGCAAATTCCATCGTGT (SEQ ID NO: 1005)	>75% 2 uM 72h

NK-92 cells	GADPH	CTGGTAAAGTGGATATTGTT (SEQ ID NO: 1006)	>90% 2 uM 72h

HeLa Cells	MAP4K4	AGAGTTCTGTGGAAGTCTA (SEQ ID NO: 1007)	>80% 2 uM 72h

Example 2

Oligonucleotide Sequences for Inhibiting Expression of Target Genes

A number of human genes were selected as candidate target genes due to involvement in immune suppression mechanisms, including the following genes shown in Table 3:

TABLE 3

BAX (NM_004324)	BAK1 (NM_001188)	CASP8 (NM_001228)
ADORA2A (NM_000675)	CTLA4 (NM_005214)	LAG3 (NM002286)
PDCD1 (NM_NM005018)	TGFBR1 (NM-004612)	HAVCR2 (NM_032782)
CCL17 (NM_002987)	CCL22 (NM_002990)	DLL2 (NM_005618)
FASLG (NM_000639)	CD274 (NM_001267706)	IDO1 (NM_002164)
IL10RA (NM_001558)	JAG1 (NM_000214)	JAG2 (NM_002226)
MAPK14 (NM_001315)	SOCS1 (NM_003745)	STAT3 (NM_003150)
TNFA1P3 (NM_006290)	TNFSF4 (NM_003326)	TYRO2 (NM_006293)
TP53 (NM_000546)

Each of the genes listed above was analyzed using a proprietary algorithm to identify preferred sdRNA targeting sequences and target regions for each gene for prevention of immunosuppression of antigen-presenting cells and T-cells. Results are shown in Table 1.

Example 3

Knock-Down of Target Gene (GAPDH) by sdRNA in HeLa Cells

HeLa cells (ATCC CRM-CCL-2) were subcultured 24 hours before transfection and kept log phase. The efficacy of several GAPDH sdRNAs was tested by qRT-PCR, including G13 sdRNA listed in the Table 1.
Solutions of GAPDH, MAP4K4 (positive control) and NTC (non-targeting control) sdRNA with twice the required concentration were prepared in serum-free EMEM medium, by diluting 100 μM oligonucleotides to 0.2-4 μM.
The total volume of medium for each oligo concentration point was calculated as [50 μl/well]×[number of replicates for each serum point]. Oligonucleotides were dispensed into a 96 well plate at 50 μl/well.
Cells were collected for transfection by trypsinization in a 50 ml tube, washed twice with medium containing 10% FBS without antibiotics, spun down at 200×g for 5 minutes at room temperature and resuspended in EMEM medium containing twice the required amount of FBS for the experiment (6%) and without antibiotics. The concentration of the cells was adjusted to 120,000/ml to yield a final concentration of 6,000 cells/50 μl/well. The cells were dispensed at 50 μl/well into the 96-well plate with pre-diluted oligos and placed in the incubator for 48 hours.
Gene Expression Analysis in HeLa Cells Using qRT-PCR
RNA was isolated from transfected HeLa cells using the PureLink™ Pro96 total RNA purification Kit (Ambion, Cat. No. 12173-011A), with Quanta qScript XLT One-Step RT-qPCR ToughMix, ROX (VWR, 89236672). The isolated RNA was analyzed for gene expression using the Human MAP4K4-FAM (Taqman Hs0377405 ml) and Human GAPDH-VIC (Applied Biosystems, Cat. No. 4326317E) gene expression assays.
The incubated plate was spun down and washed once with 100 μl/well PBS and lysed with 60 μl/well buffer provided in the kit. RNA isolation was conducted according to the manufacturer's instructions, and the RNA was eluted with 100 μl RNase-free water, and used undiluted for one-step qRT-PCR.
Dilutions of non-transfected (NT) cells of 1:5 and 1:25 were prepared for the standard curve using RNase-free water. qRT-PCR was performed by dispensing 9 μl/well into a low profile PCR plate and adding 1 μl RNA/well from the earlier prepared RNA samples. After brief centrifugation, the samples were placed in the real-time cycler and amplified using the settings recommended by the manufacturer.
GAPDH gene expression was measured by qPCR, normalized to MAP4K4 and plotted as percent of expression in the presence of non-targeting sdRNA. The results were compared to the normalized according to the standard curve. As shown in FIG. 2, several sdRNA agents targeting GAPDH or MAP4K4 significantly reduced their mRNA levels leading to more than 80-90% knock-down with 1 μM sdRNA. (See FIG. 2).

Example 4

Silencing of Multiple Targets by sdRNA in NK-92 Cells

NK-92 cells were obtained from Conqwest and subjected to one-step RT-PCR analysis without RNA purification using the FastLane Cell Multiplex Kit (Qiagen, Cat. No. 216513). For transfection, NK-92 cells were collected by centrifugation and diluted with RPMI medium containing 4% FBS and IL2 1000 U/ml and adjusted to1,000,000 cells/ml.
Multiple sdRNA agents targeting MAP4K4, PPIB or GADPH were diluted separately in serum-free RPMI medium to 4 μM and individually aliquoted at 50 μl/well into a 96-well plate. The prepared cells were then added at 50 μl cells/well to the wells with either MAP4K4, PPIB or GAPDH sdRNAs. Cells were incubated for 24, 48, or 72 hours.
At the specified timepoints, the plated transfected cells were washed once with 100 μl/well PBS and once with FCW buffer. After removal of supernatant, cell processing mix of 23.5 μl FCPL and 1.5 μl gDNA wipeout solution was added to each well and incubated for five minutes at room temperature. Lysates were then transferred to PCR strips and heated at 75° C. for five minutes.
To setup qRT-PCR, the lysates were mixed with QuantiTect reagents from the FastLane Cell Multiplex Kit and with primer probe mix for MAP4K4-FAM/GAPDH-VIC or PPIB-FAM/GAPDH-VIC. The following Taqman gene expression assays were used: human MAP4K4-FAM (Taqman, Hs00377405_m1), human PPIB-FAM (Taqman, Hs00168719_m1) and human GAPDH-VIC (Applied Biosystems, cat. No 4326317E).
A volume of 9 μl/well of each reaction mix was dispensed into a low profile PCR plate. One μl lysate per well was added from the previously prepared lysates. The samples were amplified using the settings recommended by the manufacturer.
Results shown in FIG. 3 demonstrate significant silencing of each of the multiple targets, MAP4K4, PPIB, and GADPH by sdRNA agents transfected into NK-92 cells, including greater than 75% inhibition of expression of each target within 24 to 72 hours of incubation.

Example 5

Silencing of TP53 and MAP4K4 by sdRNA in Human Primary T-Cells

Primary human T-cells were obtained from AllCells (CA) and cultured in complete RPMI medium containing 1000 IU/ml IL2. Cells were activated with anti-CD3/CD28 Dynabeads (Gibco, 11131) according to the manufacturer's instructions for at least 4 days prior to the transfection. Cells were collected by brief vortexing to dislodge the beads from cells and separating them using the designated magnet.
sdRNA agents targeting TP53 or MAP4K4 were prepared by separately diluting the sdRNAs to 0.2-4 μM in serum-free RPMI per sample (well) and individually aliquoted at 100 μl/well of 96-well plate. Cells were prepared in RPMI medium containing 4% FBS and IL2 2000 U/ml at 1,000,000 cells/ml and seeded at 100 μl/well into the 96-well plate with pre-diluted sdRNAs.
At the end of the transfection incubation period, the plated transfected cells were washed once with 100 μl/well PBS and processed with FastLane Cell Multiplex Kit reagents essentially as described for the Example 4 and according to the manufacturer's instructions. Taqman gene expression assays were used in the following combinations: human MAP4K4-FAM/GAPDH-VIC or human TP53-FAM (Taqman, Hs01034249_m1)/GAPDH-VIC. A volume of 18 μl/well of each reaction mix was combined with 2 μl lysates per well from the previously prepared lysates. The samples were amplified as before (see Example 4).
Results shown in FIG. 4 demonstrate significant silencing of both MAP4K4 and TP53 by sdRNA agents transfected into T-cells, reaching 70-80% inhibition of gene expression with 1-2 μM sdRNA.

Example 6

Immunotherapeutic Combination of sdRNAs for Treating Melanoma

Melanomas utilize at least two particular pathways to suppress immune function of T-cells, and each involves both PD1 and CTLA4. Melanoma tumors expressing the PD1 ligand, PD1L, can be targeted with T-cells pretreated ex-vivo with sd-RNAi agents specifically designed to target PD1 and interfere with PD1 expression. PD1 is also known as PDCD1, and particular targeting sequences and gene regions identified and predicted to be particularly functional in sdRNA mediated suppression, are shown in Table 1 for PDCD1 (NM_005018) and for CTLA4 (NM005214).
Treatment of melanoma tumors can be effected by providing to melanoma cells T-cells, such as tumor-infiltrating lymphocytes, pretreated ex-vivo with a combination of sdRNAs targeting PD1/PDCD1 and CTLA4, for example, targeting one or more of the twenty target sequences listed for PD1/PDCD1 and/or CTLA4. A combination of sdRNAs targeting PD1/PDCD1 and FASLG (NM_000639) and/or CTLA4, can increase T-cell toxicity in tumors expressing both PD1L and FAS.
In addition to and in combination with anti-CTLA-4 and anti-PD1 sdRNAs, T-cells used for the immunotherapy of melanoma can also be treated with sdRNA targeting other genes implicated in immunosuppression by the tumor. These receptors include, but are not limited to TGF- beta type 1 and 2 receptors, BTLA (binder of herpes virus entry indicator (HVEM) expressed on melanoma cells), and receptors of integrins expressed by myeloid derived suppressor cells (MDSC), such as CD11b, CD18, and CD29.
For tumors whose profile of expressed suppressive proteins is unknown, any combination of sdRNAs targeting PD1/PDCD1 and any one of know suppressing receptors may be helpful to reduce immune suppression and increase therapeutic efficacy.

Example 7

Combination of sdRNAs for Mitigating Immune Cell Suppression

T-cell or dendritic cell suppression may be modulated by various cytokines, such as IL10 and/or TGF beta. Suppressing corresponding receptors in T-cells and dendritic cells may be beneficial for their activity. For example, providing a combination of anti-PD1 with anti-IL10R sdRNAs is exptected to mitigate cytokine induced suppression of T-cells and dendritic cells, as compared with anti-PD1 alone.

Example 8

Combination of sdRNAs for Mitigating Immune Cell Suppression

When the mechanism of tumor suppression of immune cells may be not known, use of sdRNA agents to suppress genes involved in apoptosis (programmed cell death), such as p53, Casp8 or other gene activating apoptosis may be beneficial to increase immune cell activity. Combination of an anti-receptor sdRNAs with sdRNAs against pro-apoptotic genes can additionally reduce death of immune cells and thus increase their activity. For example, combination of anti-PD1 with anti-p53 sdRNAs may additionally protect T-cells from suppression by blocking activation of apoptosis.

Example 9

Silencing of CTLA-4 and PDCD1 by sdRNA in Human Primary T-Cells

Primary human T-cells were cultured and activated essentially as described in Example 5. sdRNA agents targeting PDCD1 and CTLA-4 were prepared by separately diluting the sdRNAs to 0.4-4 μM in serum-free RPMI per sample (well) and aliquoted at 100 μl/well of 96-well plate. Cells were prepared in RPMI medium containing 4% FBS and IL2 2000 U/ml at 1,000,000 cells/ml and seeded at 100 μl/well into the 96-well plate with pre-diluted sdRNAs.
72 h later, the transfected cells were washed once with 100 μl/well PBS and processed with FastLane Cell Multiplex Kit reagents essentially as described for the Example 4 and according to the manufacturer's instructions. Taqman gene expression assays were used in the following combinations: human PDCD1-FAM (Taqman, Hs01550088_m1)/GAPDH-VIC or human CTLA4-FAM (Taqman, Hs03044418_m1)/GAPDH-VIC. A volume of 18 μl/well of each reaction mix was combined with 2 μl lysates per well from the previously prepared lysates. The samples were amplified as before (see Example 4).
Results shown in FIG. 5 demonstrate significant silencing of PDCD1 and CTLA-4 by using combined sdRNA agents delivered to T-cells, obtaining greater than 60-70% inhibition of gene expression with 2 μM sdRNA.

Example 10

Reduction of CTLA-4 and PDCD1 Surface Expression by sdRNA in Human Primary T-Cells

Primary human T-cells were cultured and activated essentially as described in Example 5.
sdRNA agents targeting CTLA-4 or PD1 were separately diluted to 5 μM in serum-free RPMI per sample (well) and aliquoted at 250 μl/well to 24-well plates. Cells mixed with magnetic beads were collected and adjusted to 500,000 cells in 250 μl RPMI medium containing 4% FBS and IL2 2000 IU/ml. Cells were seeded at 250 μl/well to the prepared plate containing pre-diluted sdRNAs. 24 hours later FBS was added to the cells to obtain 10% final concentration.
After 72 hours of incubation, the transfected cells were collected, separated from the activation beads using the magnet, as described in Example 5. Cells were washed with PBS, spun down and resuspended in blocking buffer (PBS with 3% BSA) at 200,000 cells/50 μl/sample.
Antibody dilutions were prepared in the blocking buffer. The antibodies were mixed in two combinations: anti-PD1/anti-CD3 (1:100 dilutions for both antibodies) and anti-CTLA4/anti-CD3 (10 μl/106 cells for anti-CTLA4; 1:100 for CD3). The following antibodies were used: rabbit monoclonal [SP7] to CD3 (Abcam, ab16669); mouse monoclonal [BNI3] to CTLA4 (Abcam, ab33320) and mouse monoclonal [NAT105] to PD1 (Abcam, ab52587). Cells were mixed with the diluted antibodies and incubated 30 minutes on ice. Cells were then washed twice with PBS containing 0.2% Tween-20 and 0.1% sodium azide.
Secondary antibodies were diluted in blocking buffer and mixed together resulting in a final dilution 1:500 for anti-mouse Cy5 (Abcam, ab97037) and 1:2000 for anti-rabbit Alexa-488 (Abcam, ab150077). Cells were mixed with the diluted antibodies at 1:1 ratio and incubated 30 minutes on ice. Cells were washed as before, and diluted in 500 μl PBS per tube. The data was acquired immediately on the Attune Acoustic Focusing Cytometer (Applied Biosystems).
As shown in FIG. 6, sdRNA efficiently reduced surface expression of CTLA-4 and PD1 in activated Human Primary T cells.

Example 11

MAP4K4 sdRNA Delivery into CD3- and CD19-Positive Subsets of Human Peripheral Blood Mononuclear Cells (PBMCs)

PBMCs were cultured in complete RPMI supplemented with 1.5% PHA solution and 500 U/ml IL2. For transfection, PBMCs were collected by centrifugation and diluted with RPMI medium containing 4% FBS and IL2 1000 U/ml and seeded to 24-well plate at 500,000 cells/well.
MAP4K4 sdRNA labeled with cy3 was added to the cells at 0.1 μM final concentration. After 72 hours of incubation, the transfected cells were collected, washed with PBS, spun down and diluted in blocking buffer (PBS with 3% BSA) at 200,000 cells/50 μl/sample.
Antibody dilutions were prepared in the blocking buffer as following: 1:100 final dilution anti-CD3 (Abcam, ab16669) and anti-CD19 at 10 μl/1,000,000 cells (Abcam, ab31947). Cells were mixed with the diluted antibodies and incubated 30 min on ice. Cells were then washed twice with PBS containing 0.2% Tween-20 and 0.1% sodium azide.
Secondary antibodies were diluted in the blocking buffer in a final dilution 1:500 for anti-mouse Cy5 (Abcam, ab97037) and 1:2000 for anti-rabbit Alexa-488 (Abcam, ab150077). Cells were mixed with the diluted antibodies at 1:1 ratio and incubated 30 min on ice. Cells were washed as before, and diluted in 500 μl PBS per tube. The data was acquired immediately on the Attune Acoustic Focusing Cytometer (Applied Biosystems).
FIG. 7 shows efficient transfection over 97% of CD3-positive (t cells) and over 98% CD19-positive (B-cells) subsets in Human Peripheral Blood Mononuclear Cells (PBMCs).

TABLE 1

Targeting sequences and gene regions of genes targeted with sdRNAs to prevent
immunosuppression of antigen-presenting cells and T-cells.

			SEQ ID		SEQ ID
Oligo_count	Oligo_ID	targeting sequence	NO:	Gene_region	NO:

Accession: NM_004324

HUGO gene symbol: BAX

1	BAX_NM_004324_	GAATTGCTCAAGTTCATTGA	1	CCTCCACTGCCTCTGGAATTGCTCAAG	21
	human_835			TTCATTGATGACCCTCTG

2	BAX_NM_004324_	TTCATCCAGGATCGAGCAGG	2	CTTTTGCTTCAGGGTTTCATCCAGGAT	22
	human_ 57			CGAGCAGGGCGAATGGGG

3	BAX_NM_004324_	ATCATCAGATGTGGTCTATA	3	TCTCCCCATCTTCAGATCATCAGATGT	23
	human_684			GGTCTATAATGCGTTTTC

4	BAX_NM_004324_	TACTTTGCCAGCAAACTGGT	4	GTTGTCGCCCTTTTCTACTTTGCCAGCA	24
	human_412			AACTGGTGCTCAAGGCC

5	BAX_NM_004324_	GGTTGGGTGAGACTCCTCAA	5	ATCCAAGACCAGGGTGGTTGGGTGAG	25
	human_538			ACTCCTCAAGCCTCCTCAC

6	BAX_NM_004324_	CTACTTTGCCAGCAAACTGG	6	GGTTGTCGCCCTTTTCTACTTTGCCAGC	26
	human_411			AAACTGGTGCTCAAGGC

7	BAX_NM_004324_	GCGTTTTCCTTACGTGTCTG	7	GATGTGGTCTATAATGCGTTTTCCTTA	27
	human_706			CGTGTCTGATCAATCCCC

8	BAX_NM_004324_	TACGTGTCTGATCAATCCCC	8	ATAATGCGTTTTCCTTACGTGTCTGATC	28
	human_7 16			AATCCCCGATTCATCTA

9	BAX_NM_004324_	TCAGGGTTTCATCCAGGATC	9	AGGGGCCCTTTTGCTTCAGGGTTTCAT	29
	human_ 50			CCAGGATCGAGCAGGGCG

10	BAX_NM_004324_	TGACGGCAACTTCAACTGGG	10	AGCTGACATGTTTTCTGACGGCAACTT	30
	human_ 72			CAACTGGGGCCGGGTTGT

11	BAX_NM_004324_	CAGCTGACATGTTTTCTGAC	11	TCTTTTTCCGAGTGGCAGCTGACATGT	31
	human_356			TTTCTGACGGCAACTTCA

12	BAX_NM_004324_	AGCTGACATGTTTTCTGACG	12	CTTTTTCCGAGTGGCAGCTGACATGTT	32
	human_357			TTCTGACGGCAACTTCAA

13	BAX_NM_004324_	CACTGTGACCTTGACTTGAT	13	AGTGACCCCTGACCTCACTGTGACCTT	33
	human_776			GACTTGATTAGTGCCTTC

14	BAX_NM_004324_	TCCTTACGTGTCTGATCAAT	14	GTCTATAATGCGTTTTCCTTACGTGTCT	34
	human_712			GATCAATCCCCGATTCA

15	BAX_NM_004324_	GATCAGAACCATCATGGGCT	15	CAAGGTGCCGGAACTGATCAGAACCA	35
	human_465			TCATGGGCTGGACATTGGA

16	BAX_NM_004324_	CTTCTGGAGCAGGTCACAGT	16	TCTGGGACCCTGGGCCTTCTGGAGCA	36
	human_642			GGTCACAGTGGTGCCCTCT

17	BAX_NM_004324_	TGAGCAGATCATGAAGACAG	17	GGGGCCCACCAGCTCTGAGCAGATCA	37
	human_117			TGAAGACAGGGGCCCTTTT

18	BAX_NM_004324_	TATAATGCGTTTTCCTTACG	18	TCATCAGATGTGGTCTATAATGCGTTT	38
	human_700			TCCTTACGTGTCTGATCA

19	BAX_NM_004324_	CCCATCTTCAGATCATCAGA	19	CAGTGGTGCCCTCTCCCCATCTTCAGA	39
	human_673			TCATCAGATGTGGTCTAT

20	BAX_NM_004324_	AGGTGCCGGAACTGATCAGA	20	AGGCCCTGTGCACCAAGGTGCCGGAA	40
	human_452			CTGATCAGAACCATCATGG

Accession: NM_001188

HUGO gene symbol: BAK1

1	BAK1_NM_001188	TGGTTTGTTATATCAGGGAA	41	ACAGGGCTTAGGACTTGGTTTGTTA	61
	_human_1813			TATCAGGGAAAAGGAGTAGG

2	BAK1_NM_001188	TGGTACGAAGATTCTTCAAA	42	TGTTGGGCCAGTTTGTGGTACGAAG	62
	_human_911			ATTCTTCAAATCATGACTCC

3	BAK1_NM_001188	TTATATCAGGGAAAAGGAGT	43	TTAGGACTTGGTTTGTTATATCAGG	63
	_human_1820			GAAAAGGAGTAGGGAGTTCA

4	BAK1_NM_001188	TCCCTTCCTCTCTCCTTATA	44	GTCCTCTCAGTTCTCTCCCTTCCTCTC	64
	_human_1678			TCCTTATAGACACTTGCT

5	BAK1_NM_001188	TCAAATCATGACTCCCAAGG	45	TGGTACGAAGATTCTTCAAATCATG	65
	_human_926			ACTCCCAAGGGTGCCCTTTG

6	BAK1_NM_001188	TGTTATATCAGGGAAAAGGA	46	GCTTAGGACTTGGTTTGTTATATCA	66
	_human_1818			GGGAAAAGGAGTAGGGAGTT

7	BAK1_NM_001188	ACGAAGATTCTTCAAATCAT	47	GGGCCAGTTTGTGGTACGAAGATTC	67
	_human_915			TTCAAATCATGACTCCCAAG

8	BAK1_NM_001188	GGTACGAAGATTCTTCAAAT	48	GTTGGGCCAGTTTGTGGTACGAAGA	68
	_human_912			TTCTTCAAATCATGACTCCC

9	BAK1_NM_001188	GAAGTTCTTGATTCAGCCAA	49	GGGGGTCAGGGGGGAGAAGTTCTT	69
	_human_2086			GATTCAGCCAAATGCAGGGAG

10	BAK1_NM_001188	CCTATGAGTACTTCACCAAG	50	CCACGGCAGAGAATGCCTATGAGTA	70
	_human_620			CTTCACCAAGATTGCCACCA

11	BAK1_NM_001188	TATCAGGGAAAAGGAGTAGG	51	GGACTTGGTTTGTTATATCAGGGAA	71
	_human_1823			AAGGAGTAGGGAGTTCATCT

12	BAK1_NM_001188	CTCTCCTTATAGACACTTGC	52	GTTCTCTCCCTTCCTCTCTCCTTATAG	72
	_human_1687			ACACTTGCTCCCAACCCA

13	BAK1_NM_001188	ACTTGGTTTGTTATATCAGG	53	ACTACAGGGCTTAGGACTTGGTTTG	73
	_human_1810			TTATATCAGGGAAAAGGAGT

14	BAK1_NM_001188	AAGATCAGCACCCTAAGAGA	54	ATTCAGCTATTCTGGAAGATCAGCA	74
	_human_1399			CCCTAAGAGATGGGACTAGG

15	BAK1_NM_001188	GTTTGAGAGTGGCATCAATT	55	GATTGCCACCAGCCTGTTTGAGAGT	75
	_human_654			GGCATCAATTGGGGCCGTGT

16	BAK1_NM_001188	GACTATCAACACCACTAGGA	56	TCTAAGTGGGAGAAGGACTATCAAC	76
	_human_1875			ACCACTAGGAATCCCAGAGG

17	BAK1_NM_001188	AGCTTTAGCAAGTGTGCACT	57	CCTCAAGAGTACAGAAGCTTTAGCA	77
	_human_1043			AGTGTGCACTCCAGCTTCGG

18	BAK1_NM_001188	TTCATCTGGAGGGTTCTAAG	58	AAAAGGAGTAGGGAGTTCATCTGG	78
	_human_1846			AGGGTTCTAAGTGGGAGAAGG

19	BAK1_NM_001188	AAGTTCTTGATTCAGCCAAA	59	GGGGTCAGGGGGGAGAAGTTCTTG	79
	_human_2087			ATTCAGCCAAATGCAGGGAGG

20	BAK1_NM_001188	GTTATATCAGGGAAAAGGAG	60	CTTAGGACTTGGTTTGTTATATCAG	80
	_human_1819			GGAAAAGGAGTAGGGAGTTC

Accession: NM_001228

HUGO gene symbol: CASP8

1	CASP8_NM_001228	TTAAATCATTAGGAATTAAG	121	TCTGCTTGGATTATTTTAAATCATTAG	141
	_human_2821			GAATTAAGTTATCTTTAA

2	CASP8_NM_001228	GAATTAAGTTATCTTTAAAA	122	ATTTTAAATCATTAGGAATTAAGTTAT	142
	_human_2833			CTTTAAAATTTAAGTATC

3	CASP8_NM_001228	AACTTTAATTCTCTTTCAAA	123	TGTTAATATTCTATTAACTTTAATTCT	143
	_human_2392			CTTTCAAAGCTAAATTCC

4	CASP8_NM_001228	GACTGAAGTGAACTATGAAG	124	TATTCTCACCATCCTGACTGAAGTGA	144
	_human_1683			ACTATGAAGTAAGCAACAA

5	CASP8_NM_001228	ATATTCTCCTGCCTTTTAAA	125	GGGAATATTGAGATTATATTCTCCTG	145
	_human_281			CCTTTTAAAAAGATGGACT

6	CASP8_NM_001228	AGTTATCTTTAAAATTTAAG	126	AATCATTAGGAATTAAGTTATCTTTA	146
	_human_2839			AAATTTAAGTATCTTTTTT

7	CASP8_NM_001228	TAGATTTTCTACTTTATTAA	127	TATTTACTAATTTTCTAGATTTTCTACT	147
	_human_2164			TTATTAATTGTTTTGCA

8	CASP8_NM_001228	CTGTGCCCAAATCAACAAGA	128	CATCCTGAAAAGAGTCTGTGCCCAAA	148
	_human_888			TCAACAAGAGCCTGCTGAA

9	CASP8_NM_001228	AGCTGGTGGCAATAAATACC	129	TTTGGGAATGTTTTTAGCTGGTGGCA	149
	_human_2283			ATAAATACCAGACACGTAC

10	CASP8_NM_001228	TCCTACCGAAACCCTGCAGA	130	GTGAATAACTGTGTTTCCTACCGAAA	150
	_human_1585			CCCTGCAGAGGGAACCTGG

11	CASP8_NM_001228	TATAAGAGCTAAAGTTAAAT	131	TGTTTTGCACTTTTTTATAAGAGCTAA	151
	_human_2200			AGTTAAATAGGATATTAA

12	CASP8_NM_001228	CACTATGTTTATTTACTAAT	132	ACTATTTAGATATAACACTATGTTTAT	152
	_human_2140			TTACTAATTTTCTAGATT

13	CASP8_NM_001228	ATTGTTATCTATCAACTATA	133	GGGCTTATGATTCAGATTGTTATCTA	153
	_human_2350			TCAACTATAAGCCCACTGT

14	CASP8_NM_001228	TAACTGTGTTTCCTACCGAA	134	GATGGCCACTGTGAATAACTGTGTTT	154
	_human_1575			CCTACCGAAACCCTGCAGA

15	CASP8_NM_001228	TAATTCTCTTTCAAAGCTAA	135	ATATTCTATTAACTTTAATTCTCTTTCA	155
	_human_2397			AAGCTAAATTCCACACT

16	CASP8_NM_001228	TATATGCTTGGCTAACTATA	136	TGCTTTTATGATATATATATGCTTGGC	156
	_human_2726			TAACTATATTTGCTTTTT

17	CASP8_NM_001228	CTCTGCTTGGATTATTTTAA	137	CATTTGCTCTTTCATCTCTGCTTGGAT	157
	_human_2805			TATTTTAAATCATTAGGA

18	CASP8_NM_001228	ATGCTTGGCTAACTATATTT	138	TTTTATGATATATATATGCTTGGCTAA	158
	_human_2729			CTATATTTGCTTTTTGCT

19	CASP8_NM_001228	ATAAGAGCTAAAGTTAAATA	139	GTTTTGCACTTTTTTATAAGAGCTAAA	159
	_human_2201			GTTAAATAGGATATTAAC

20	CASP8_NM_001228	ATCTTTAAAATTTAAGTATC	140	ATTAGGAATTAAGTTATCTTTAAAATT	160
	_human_2843			TAAGTATCTTTTTTCAAA

Accession: NM_000675

HUGO gene symbol: ADORA2A

1	ADORA2A_NM_00067	TAACTGCCTTTCCTTCTAAA	161	GTGAGAGGCCTTGTCTAACTGCC	181
	5_human_2482			TTTCCTTCTAAAGGGAATGTTT

2	ADORA2A_NM_00067	TTCCTTCTAAAGGGAATGTT	162	CTTGTCTAACTGCCTTTCCTTCTAA	182
	5_human_2491			AGGGAATGTTTTTTTCTGAG

3	ADORA2A_NM_00067	GCCTTTCCTTCTAAAGGGAA	163	AGGCCTTGTCTAACTGCCTTTCCT	183
	5_human_2487			TCTAAAGGGAATGTTTTTTTC

4	ADORA2A_NM_00067	TTTTCTGAGATAAAATAAAA	164	CTAAAGGGAATGTTTTTTTCTGAG	184
	5_human_2512			ATAAAATAAAAACGAGCCACA

5	ADORA2A_NM_00067	CATCTCTTGGAGTGACAAAG	165	TCTCAGTCCCAGGGCCATCTCTTG	185
	5_human_2330			GAGTGACAAAGCTGGGATCAA

6	ADORA2A_NM_00067	CATGGTGTACTTCAACTTCT	166	GGTCCCCATGAACTACATGGTGT	186
	5_human_987			ACTTCAACTTCTTTGCCTGTGT

7	ADORA2A_NM_00067	CTAACTGCCTTTCCTTCTAA	167	AGTGAGAGGCCTTGTCTAACTGC	187
	5_human_2481			CTTTCCTTCTAAAGGGAATGTT

8	ADORA2A_NM_00067	CTGATGATTCATGGAGTTTG	168	TGGAGCAGGAGTGTCCTGATGAT	188
	5_human_1695			TCATGGAGTTTGCCCCTTCCTA

9	ADORA2A_NM_00067	CTCAGAGTCCTCTGTGAAAA	169	CCTGGTTTCAGGAGACTCAGAGT	189
	5_human_264			CCTCTGTGAAAAAGCCCTTGGA

10	ADORA2A_NM_00067	AACGAGCCACATCGTGTTTT	170	CTGAGATAAAATAAAAACGAGCC	190
	5_human_2531			ACATCGTGTTTTAAGCTTGTCC

11	ADORA2A_NM_00067	TCCTTCTAAAGGGAATGTTT	171	TTGTCTAACTGCCTTTCCTTCTAAA	191
	5_human_2492			GGGAATGTTTTTTTCTGAGA

12	ADORA2A_NM_00067	CATGAACTACATGGTGTACT	172	TGAGGATGTGGTCCCCATGAACT	192
	5_human_978			ACATGGTGTACTTCAACTTCTT

13	ADORA2A_NM_00067	AACTGCCTTTCCTTCTAAAG	173	TGAGAGGCCTTGTCTAACTGCCTT	193
	5_human_2483			TCCTTCTAAAGGGAATGTTTT

14	ADORA2A_NM_00067	CAGATGTTTCATGCTGTGAG	174	TGGGTTCTGAGGAAGCAGATGTT	194
	5_human_1894			TCATGCTGTGAGGCCTTGCACC

15	ADORA2A_NM_00067	CCCATGAACTACATGGTGTA	175	TTTGAGGATGTGGTCCCCATGAA	195
	5_human_976			CTACATGGTGTACTTCAACTTC

16	ADORA2A_NM_00067	AGGCAGCAAGAACCTTTCAA	176	CGCAGCCACGTCCTGAGGCAGCA	196
	5_human_1384			AGAACCTTTCAAGGCAGCTGGC

17	ADORA2A_NM_00067	GTCCTGATGATTCATGGAGT	177	GGATGGAGCAGGAGTGTCCTGAT	197
	5_human_1692			GATTCATGGAGTTTGCCCCTTC

18	ADORA2A_NM_00067	GTACTTCAACTTCTTTGCCT	178	CATGAACTACATGGTGTACTTCAA	198
	5_human_993			CTTCTTTGCCTGTGTGCTGGT

19	ADORA2A_NM_00067	TGTAAGTGTGAGGAAACCCT	179	TTTTTCCAGGAAAAATGTAAGTGT	199
	5_human_2167			GAGGAAACCCTTTTTATTTTA

20	ADORA2A_NM_00067	CCTACTTTGGACTGAGAGAA	180	TGAGGGCAGCCGGTTCCTACTTT	200
	5_human_1815			GGACTGAGAGAAGGGAGCCCCA

Accession: NM_005214

HUGO gene symbol: CTLA4

1	CTLA4_NM_005214_	TGATTCTGTGTGGGTTCAAA	201	TCTATATAAAGTCCTTGATTCTGT	221
	human_61			GTGGGTTCAAACACATTTCAA

2	CTLA4_NM_005214_	TTATTTGTTTGTGCATTTGG	202	GCTATCCAGCTATTTTTATTTGTTT	222
	human_909			GTGCATTTGGGGGGAATTCA

3	CTLA4_NM_005214_	TGATTACATCAAGGCTTCAA	203	TCTTAAACAAATGTATGATTACAT	223
	human_1265			CAAGGCTTCAAAAATACTCAC

4	CTLA4_NM_005214_	GATGTGGGTCAAGGAATTAA	204	GGGATGCAGCATTATGATGTGGG	224
	human_1094			TCAAGGAATTAAGTTAGGGAAT

5	CTLA4_NM_005214_	CCTTTTATTTCTTAAACAAA	205	AAGTTAAATTTTATGCCTTTTATTT	225
	human_1241			CTTAAACAAATGTATGATTA

6	CTLA4_NM_005214_	GATTACATCAAGGCTTCAAA	206	CTTAAACAAATGTATGATTACATC	226
	human_1266			AAGGCTTCAAAAATACTCACA

7	CTLA4_NM_005214_	TCTGTGTGGGTTCAAACACA	207	TATAAAGTCCTTGATTCTGTGTGG	227
	human_65			GTTCAAACACATTTCAAAGCT

8	CTLA4_NM_005214_	TTGATAGTATTGTGCATAGA	208	TATATATATTTTAATTTGATAGTAT	228
	human_1405			TGTGCATAGAGCCACGTATG

9	CTLA4_NM_005214_	TGCCTTTTATTTCTTAAACA	209	TCAAGTTAAATTTTATGCCTTTTAT	229
	human_1239			TTCTTAAACAAATGTATGAT

10	CTLA4_NM_005214_	TCCATGAAAATGCAACAACA	210	TTTAACTCAATATTTTCCATGAAA	230
	human_1912			ATGCAACAACATGTATAATAT

11	CTLA4_NM_005214_	TTATTTCTTAAACAAATGTA	211	TAAATTTTATGCCTTTTATTTCTTA	231
	human_1245			AACAAATGTATGATTACATC

12	CTLA4_NM_005214_	TTAATGGTTTGAATATAAAC	212	GTTTTTGTGTATTTGTTAATGGTTT	232
	human_1449			GAATATAAACACTATATGGC

13	CTLA4_NM_005214_	ATGTGGGTCAAGGAATTAAG	213	GGATGCAGCATTATGATGTGGGT	233
	human_1095			CAAGGAATTAAGTTAGGGAATG

14	CTLA4_NM_005214_	AGCCGAAATGATCTTTTCAA	214	GTATGAGACGTTTATAGCCGAAA	234
	human_1208			TGATCTTTTCAAGTTAAATTTT

15	CTLA4_NM_005214_	GTTTGAATATAAACACTATA	215	GTGTATTTGTTAATGGTTTGAATA	235
	human_1455			TAAACACTATATGGCAGTGTC

16	CTLA4_NM_005214_	TATGCCTTTTATTTCTTAAA	216	TTTCAAGTTAAATTTTATGCCTTTT	236
	human_1237			ATTTCTTAAACAAATGTATG

17	CTLA4_NM_005214_	TTCCATGAAAATGCAACAAC	217	TTTTAACTCAATATTTTCCATGAA	237
	human_1911			AATGCAACAACATGTATAATA

18	CTLA4_NM_005214_	CATCTCTCTTTAATATAAAG	218	CATTTGGGGGGAATTCATCTCTCT	238
	human_937			TTAATATAAAGTTGGATGCGG

19	CTLA4_NM_005214_	GGAATTCATCTCTCTTTAAT	219	TTTGTGCATTTGGGGGGAATTCAT	239
	human_931			CTCTCTTTAATATAAAGTTGG

20	CTLA4_NM_005214_	ATCTATATAAAGTCCTTGAT	220	TCTGGGATCAAAGCTATCTATATA	240
	human_45			AAGTCCTTGATTCTGTGTGGG

Accession: NM_002286

HUGO gene symbol: LAG3

1	LAG3_NM_002286	GACTTTACCCTTCGACTAGA	241	ACTGGAGACAATGGCGACTTTACC	261
	_human_1292			CTTCGACTAGAGGATGTGAGC

2	LAG3_NM_002286	CAACGTCTCCATCATGTATA	242	CTACAGAGATGGCTTCAACGTCTC	262
	_human_1096			CATCATGTATAACCTCACTGT

3	LAG3_NM_002286	GTCCTTTCTCTGCTCCTTTT	243	TTTCTCATCCTTGGTGTCCTTTCTCT	263
	_human_1721			GCTCCTTTTGGTGACTGGA

4	LAG3_NM_002286	TCCAGTATCTGGACAAGAAC	244	GCTTTGTGAGGTGACTCCAGTATC	264
	_human_1465			TGGACAAGAACGCTTTGTGTG

5	LAG3_NM_002286	ATTTTCTGCCTTAGAGCAAG	245	GTGGCGACCAAGACGATTTTCTGC	265
	_human_1795			CTTAGAGCAAGGGATTCACCC

6	LAG3_NM_002286	TTTCACCTTTGGAGAAGACA	246	ACTGGAGCCTTTGGCTTTCACCTTT	266
	_human_1760			GGAGAAGACAGTGGCGACCA

7	LAG3_NM_002286	CATTTTGAACTGCTCCTTCA	247	AGCCTCCGACTGGGTCATTTTGAA	267
	_human_904			CTGCTCCTTCAGCCGCCCTGA

8	LAG3_NM_002286	TCATCACAGTGACTCCCAAA	248	CTGTCACATTGGCAATCATCACAGT	268
	_human_1398			GACTCCCAAATCCTTTGGGT

9	LAG3_NM_002286	GCTTTCACCTTTGGAGAAGA	249	TGACTGGAGCCTTTGGCTTTCACCT	269
	_human_1758			TTGGAGAAGACAGTGGCGAC

10	LAG3_NM_002286	CTTTGGCTTTCACCTTTGGA	250	TTTGGTGACTGGAGCCTTTGGCTTT	270
	_human_1753			CACCTTTGGAGAAGACAGTG

11	LAG3_NM_002286	ATTTTGAACTGCTCCTTCAG	251	GCCTCCGACTGGGTCATTTTGAACT	271
	_human_905			GCTCCTTCAGCCGCCCTGAC

12	LAG3_NM_002286	CACATTGGCAATCATCACAG	252	GCTCAATGCCACTGTCACATTGGC	272
	_human_1387			AATCATCACAGTGACTCCCAA

13	LAG3_NM_002286	TTTCTGACCTCCTTTTGGAG	253	ACTGCCCCCTTTCCTTTTCTGACCTC	273
	_human_301			CTTTTGGAGGGCTCAGCGC

14	LAG3_NM_002286	CGACTGGGTCATTTTGAACT	254	ATCTCTCAGAGCCTCCGACTGGGT	274
	_human_895			CATTTTGAACTGCTCCTTCAG

15	LAG3_NM_002286	TACTTCACAGAGCTGTCTAG	255	CTTGGAGCAGCAGTGTACTTCACA	275
	_human_1625			GAGCTGTCTAGCCCAGGTGCC

16	LAG3_NM_002286	ATTGGCAATCATCACAGTGA	256	CAATGCCACTGTCACATTGGCAATC	276
	_human_1390			ATCACAGTGACTCCCAAATC

17	LAG3_NM_002286	CTGTTTCTCATCCTTGGTGT	257	GCAGGCCACCTCCTGCTGTTTCTCA	277
	_human_1703			TCCTTGGTGTCCTTTCTCTG

18	LAG3_NM_002286	TTGTGAGGTGACTCCAGTAT	258	CCTGGGGAAGCTGCTTTGTGAGGT	278
	_human_1453			GACTCCAGTATCTGGACAAGA

19	LAG3_NM_002286	TTTGGCTTTCACCTTTGGAG	259	TTGGTGACTGGAGCCTTTGGCTTTC	279
	_human_1754			ACCTTTGGAGAAGACAGTGG

20	LAG3_NM_002286	TGGAGACAATGGCGACTTTA	260	TGACCTCCTGGTGACTGGAGACAA	280
	_human_1279			TGGCGACTTTACCCTTCGACT

Accession: NM_005018

HUGO gene symbol: PDCD1

1	PDCD1_NM_005018	TATTATATTATAATTATAAT	281	CCTTCCCTGTGGTTCTATTATATTAT	301
	_human_2070			AATTATAATTAAATATGAG

2	PDCD1_NM_005018	TCTATTATATTATAATTATA	282	CCCCTTCCCTGTGGTTCTATTATATT	302
	_human_2068			ATAATTATAATTAAATATG

3	PDCD1_NM_005018	CATTCCTGAAATTATTTAAA	283	GCTCTCCTTGGAACCCATTCCTGAA	303
	_human_1854			ATTATTTAAAGGGGTTGGCC

4	PDCD1_NM_005018	CTATTATATTATAATTATAA	284	CCCTTCCCTGTGGTTCTATTATATT	304
	_human_2069			ATAATTATAATTAAATATGA

5	PDCD1_NM_005018	AGTTTCAGGGAAGGTCAGAA	285	CTGCAGGCCTAGAGAAGTTTCAGG	305
	_human_1491			GAAGGTCAGAAGAGCTCCTGG

6	PDCD1_NM_005018	TGTGGTTCTATTATATTATA	286	GGGATCCCCCTTCCCTGTGGTTCTA	306
	_human_2062			TTATATTATAATTATAATTA

7	PDCD1_NM_005018	TGTGTTCTCTGTGGACTATG	287	CCCCTCAGCCGTGCCTGTGTTCTCT	307
	_human_719			GTGGACTATGGGGAGCTGGA

8	PDCD1_NM_005018	CCCATTCCTGAAATTATTTA	288	GAGCTCTCCTTGGAACCCATTCCTG	308
	_human_1852			AAATTATTTAAAGGGGTTGG

9	PDCD1_NM_005018	TGCCACCATTGTCTTTCCTA	289	TGAGCAGACGGAGTATGCCACCAT	309
	_human_812			TGTCTTTCCTAGCGGAATGGG

10	PDCD1_NM_005018	AAGTTTCAGGGAAGGTCAGA	290	CCTGCAGGCCTAGAGAAGTTTCAG	310
	_human_1490			GGAAGGTCAGAAGAGCTCCTG

11	PDCD1_NM_005018	CTGTGGTTCTATTATATTAT	291	AGGGATCCCCCTTCCCTGTGGTTCT	311
	_human_2061			ATTATATTATAATTATAATT

12	PDCD1_NM_005018	TTCTATTATATTATAATTAT	292	CCCCCTTCCCTGTGGTTCTATTATA	312
	_human_2067			TTATAATTATAATTAAATAT

13	PDCD1_NM_005018	TTTCAGGGAAGGTCAGAAGA	293	GCAGGCCTAGAGAAGTTTCAGGG	313
	_human_1493			AAGGTCAGAAGAGCTCCTGGCT

14	PDCD1_NM_005018	CTTGGAACCCATTCCTGAAA	294	ACCCTGGGAGCTCTCCTTGGAACC	314
	_human_1845			CATTCCTGAAATTATTTAAAG

15	PDCD1_NM_005018	TCCCTGTGGTTCTATTATAT	295	ACAAGGGATCCCCCTTCCCTGTGG	315
	_human_2058			TTCTATTATATTATAATTATA

16	PDCD1_NM_005018	CCTGTGGTTCTATTATATTA	296	AAGGGATCCCCCTTCCCTGTGGTTC	316
	_human_2060			TATTATATTATAATTATAAT

17	PDCD1_NM_005018	TGGAACCCATTCCTGAAATT	297	CCTGGGAGCTCTCCTTGGAACCCA	317
	_human_1847			TTCCTGAAATTATTTAAAGGG

18	PDCD1_NM_005018	CCTTCCCTGTGGTTCTATTA	298	GGGACAAGGGATCCCCCTTCCCTG	318
	_human_2055			TGGTTCTATTATATTATAATT

19	PDCD1_NM_005018	TTCCCTGTGGTTCTATTATA	299	GACAAGGGATCCCCCTTCCCTGTG	319
	_human_2057			GTTCTATTATATTATAATTAT

20	PDCD1_NM_005018	CACAGGACTCATGTCTCAAT	300	CAGGCACAGCCCCACCACAGGACT	320
	_human_1105			CATGTCTCAATGCCCACAGTG

Accession: NM_004612

HUGO gene symbol: TGFBR1

1	TGFBR1_NM_004612	CCTGTTTATTACAACTTAAA	321	GTTAATAACATTCAACCTGTTTAT	341
	_human_5263			TACAACTTAAAAGGAACTTCA

2	TGFBR1_NM_004612	CCATTGGTGGAATTCATGAA	322	TTGCTCGACGATGTTCCATTGGTG	342
	_human_1323			GAATTCATGAAGATTACCAAC

3	TGFBR1_NM_004612	TTTTCCTTATAACAAAGACA	323	TTTAGGGATTTTTTTTTTTCCTTAT	343
	_human_6389			AACAAAGACATCACCAGGAT

4	TGFBR1_NM_004612	TGTATTACTTGTTTAATAAT	324	TTTTTATAGTTGTGTTGTATTACTT	344
	_human_3611			GTTTAATAATAATCTCTAAT

5	TGFBR1_NM_004612	TTATTGAATCAAAGATTGAG	325	TGCTGAAGATATTTTTTATTGAAT	345
	_human_3882			CAAAGATTGAGTTACAATTAT

6	TGFBR1_NM_004612	TTCTTACCTAAGTGGATAAA	326	GTTACAATTATACTTTTCTTACCTA	346
	_human_3916			AGTGGATAAAATGTACTTTT

7	TGFBR1_NM_004612	ATGTTGCTCAGTTACTCAAA	327	TAAAGTATGGGTATTATGTTGCTC	347
	_human_5559			AGTTACTCAAATGGTACTGTA

8	TGFBR1_NM_004612	ATATTTGTACCCCAAATAAC	328	GGTACTGTATTGTTTATATTTGTA	348
	_human_5595			CCCCAAATAACATCGTCTGTA

9	TGFBR1_NM_004612	TGTAAATGTAAACTTCTAAA	329	TTATGCAATCTTGTTTGTAAATGT	349
	_human_5222			AAACTTCTAAAAATATGGTTA

10	TGFBR1_NM_004612	AGAATGAGTGACATATTACA	330	AACCAAAGTAATTTTAGAATGAG	350
	_human_3435			TGACATATTACATAGGAATTTA

11	TGFBR1_NM_004612	CCATTTCTAAGCCTACCAGA	331	GTTGTTGTTTTTGGGCCATTTCTA	351
	_human_3709			AGCCTACCAGATCTGCTTTAT

12	TGFBR1_NM_004612	ATATTCCAAAAGAATGTAAA	332	ATTGTATTTGTAGTAATATTCCAA	352
	_human_5826			AAGAATGTAAATAGGAAATAG

13	TGFBR1_NM_004612	TTACTTCCAATGCTATGAAG	333	TATAATAACTGGTTTTTACTTCCA	353
	_human_3146			ATGCTATGAAGTCTCTGCAGG

14	TGFBR1_NM_004612	TCTTTATCTGTTCAAAGACT	334	TGTAAGCCATTTTTTTCTTTATCTG	354
	_human_2675			TTCAAAGACTTATTTTTTAA

15	TGFBR1_NM_004612	GTCTAAGTATACTTTTAAAA	335	CATTTTAATTGTGTTGTCTAAGTA	355
	_human_2529			TACTTTTAAAAAATCAAGTGG

16	TGFBR1_NM_004612	ATCTTTGGACATGTACTGCA	336	GAGATACTAAGGATTATCTTTGG	356
	_human_5079			ACATGTACTGCAGCTTCTTGTC

17	TGFBR1_NM_004612	GTGTTGTATTACTTGTTTAA	337	TTTGTTTTTATAGTTGTGTTGTATT	357
	_human_3607			ACTTGTTTAATAATAATCTC

18	TGFBR1_NM_004612	TGCTGTAGATGGCAACTAGA	338	CATGCCATATGTAGTTGCTGTAGA	358
	_human_5994			TGGCAACTAGAACCTTTGAGT

19	TGFBR1_NM_004612	TCTTTCACTTATTCAGAACA	339	GTATACTATTATTGTTCTTTCACTT	359
	_human_2177			ATTCAGAACATTACATGCCT

20	TGFBR1_NM_004612	GTATTTGTAGTAATATTCCA	340	TTTAAATTGTATATTGTATTTGTA	360
	_human_5814			GTAATATTCCAAAAGAATGTA

Accession: NM_032782

HUGO gene symbol: HAVCR2

1	HAVCR2_NM_032782	CTCATAGCAAAGAGAAGATA	361	TTTTCAAATGGTATTCTCATAGCA	381
	_human_937			AAGAGAAGATACAGAATTTAA

2	HAVCR2_NM_032782	GTATTCTCATAGCAAAGAGA	362	TTTAATTTTCAAATGGTATTCTCAT	382
	_human_932			AGCAAAGAGAAGATACAGAA

3	HAVCR2_NM_032782	TTGCTTGTTGTGTGCTTGAA	363	TGTATTGGCCAAGTTTTGCTTGTT	383
	_human_2126			GTGTGCTTGAAAGAAAATATC

4	HAVCR2_NM_032782	TATTCGTGGACCAAACTGAA	364	TCTGACCAACTTCTGTATTCGTGG	384
	_human_2171			ACCAAACTGAAGCTATATTTT

5	HAVCR2_NM_032782	ATTGTGGAGTAGACAGTTGG	365	GCTACTGCTCATGTGATTGTGGA	385
	_human_158			GTAGACAGTTGGAAGAAGTACC

6	HAVCR2_NM_032782	GTTGTGTGCTTGAAAGAAAA	366	GGCCAAGTTTTGCTTGTTGTGTGC	386
	_human_2132			TTGAAAGAAAATATCTCTGAC

7	HAVCR2_NM_032782	TGTTGTGTGCTTGAAAGAAA	367	TGGCCAAGTTTTGCTTGTTGTGTG	387
	_human_2131			CTTGAAAGAAAATATCTCTGA

8	HAVCR2_NM_032782	CCCTAAACTTAAATTTCAAG	368	TTGACAGAGAGTGGTCCCTAAAC	388
	_human_2313			TTAAATTTCAAGACGGTATAGG

9	HAVCR2_NM_032782	ACATCCAGATACTGGCTAAA	369	GATGTGAATTATTGGACATCCAG	389
	_human_489			ATACTGGCTAAATGGGGATTTC

10	HAVCR2_NM_032782	CATTTTCAGAAGATAATGAC	370	GGAGCAGAGTTTTCCCATTTTCAG	390
	_human_1272			AAGATAATGACTCACATGGGA

11	HAVCR2_NM_032782	CACATTGGCCAATGAGTTAC	371	TCTAACACAAATATCCACATTGGC	391
	_human_785			CAATGAGTTACGGGACTCTAG

12	HAVCR2_NM_032782	TGCTTGTTGTGTGCTTGAAA	372	GTATTGGCCAAGTTTTGCTTGTTG	392
	_human_2127			TGTGCTTGAAAGAAAATATCT

13	HAVCR2_NM_032782	GAGTAGACAGTTGGAAGAAG	373	GCTCATGTGATTGTGGAGTAGAC	393
	_human_164			AGTTGGAAGAAGTACCCAGTCC

14	HAVCR2_NM_032782	TTGTTGTGTGCTTGAAAGAA	374	TTGGCCAAGTTTTGCTTGTTGTGT	394
	_human_2130			GCTTGAAAGAAAATATCTCTG

15	HAVCR2_NM_032782	CGGCGCTTTAATTTTCAAAT	375	TCTGGCTCTTATCTTCGGCGCTTT	395
	_human_911			AATTTTCAAATGGTATTCTCA

16	HAVCR2_NM_032782	TTTGGCACAGAAAGTCTAAA	376	TGAAAGCATAACTTTTTTGGCACA	396
	_human_1543			GAAAGTCTAAAGGGGCCACTG

17	HAVCR2_NM_032782	GATCTGTCTTGCTTATTGTT	377	AGACGGTATAGGCTTGATCTGTC	397
	_human_2346			TTGCTTATTGTTGCCCCCTGCG

18	HAVCR2_NM_032782	GGTGTGTATTGGCCAAGTTT	378	GAAGTGCATTTGATTGGTGTGTA	398
	_human_2107			TTGGCCAAGTTTTGCTTGTTGT

19	HAVCR2_NM_032782	CCCATTTTCAGAAGATAATG	379	ATGGAGCAGAGTTTTCCCATTTTC	399
	_human_1270			AGAAGATAATGACTCACATGG

20	HAVCR2_NM_032782	TGGCACAGAAAGTCTAAAGG	380	AAAGCATAACTTTTTTGGCACAGA	400
	_human_1545			AAGTCTAAAGGGGCCACTGAT

Accession: NM_002987

HUGO gene symbol: CCL17

1	CCL17_NM_002987	AAATACCTGCAAAGCCTTGA	401	GTGAAGAATGCAGTTAAATACCTGC	421
	_human_385			AAAGCCTTGAGAGGTCTTGA

2	CCL17_NM_002987	TTTTGTAACTGTGCAGGGCA	402	CAGGGATGCCATCGTTTTTGTAACT	422
	_human_318			GTGCAGGGCAGGGCCATCTG

3	CCL17_NM_002987	AGAGTGAAGAATGCAGTTAA	403	GACCCCAACAACAAGAGAGTGAAG	423
	_human_367			AATGCAGTTAAATACCTGCAA

4	CCL17_NM_002987	AAGCCTTGAGAGGTCTTGAA	404	AGTTAAATACCTGCAAAGCCTTGAG	424
	_human_396			AGGTCTTGAAGCCTCCTCAC

5	CCL17_NM_002987	AATACCTGCAAAGCCTTGAG	405	TGAAGAATGCAGTTAAATACCTGCA	425
	_human_386			AAGCCTTGAGAGGTCTTGAA

6	CCL17_NM_002987	TGCAGTTAAATACCTGCAAA	406	CAAGAGAGTGAAGAATGCAGTTAA	426
	_human_378			ATACCTGCAAAGCCTTGAGAG

7	CCL17_NM_002987	CAACAACAAGAGAGTGAAGA	407	CATCTGTTCGGACCCCAACAACAAG	427
	_human_357			AGAGTGAAGAATGCAGTTAA

8	CCL17_NM_002987	CTGAATTCAAAACCAGGGTG	408	CTGCTGATGGGAGAGCTGAATTCAA	428
	_human_55			AACCAGGGTGTCTCCCTGAG

9	CCL17_NM_002987	ATACCTGCAAAGCCTTGAGA	409	GAAGAATGCAGTTAAATACCTGCAA	429
	_human_387			AGCCTTGAGAGGTCTTGAAG

10	CCL17_NM_002987	TTCCCCTTAGAAAGCTGAAG	410	ACTTCAAGGGAGCCATTCCCCTTAG	430
	_human_254			AAAGCTGAAGACGTGGTACC

11	CCL17_NM_002987	GGAGAGCTGAATTCAAAACC	411	CACCGCCTGCTGATGGGAGAGCTG	431
	_human_49			AATTCAAAACCAGGGTGTCTC

12	CCL17_NM_002987	GCAGTTAAATACCTGCAAAG	412	AAGAGAGTGAAGAATGCAGTTAAA	432
	_human_379			TACCTGCAAAGCCTTGAGAGG

13	CCL17_NM_002987	GAAGAATGCAGTTAAATACC	413	CAACAACAAGAGAGTGAAGAATGC	433
	_human_372			AGTTAAATACCTGCAAAGCCT

14	CCL17_NM_002987	ATGCAGTTAAATACCTGCAA	414	ACAAGAGAGTGAAGAATGCAGTTA	434
	_human_377			AATACCTGCAAAGCCTTGAGA

15	CCL17_NM_002987	CATTCCCCTTAGAAAGCTGA	415	GTACTTCAAGGGAGCCATTCCCCTT	435
	_human_252			AGAAAGCTGAAGACGTGGTA

16	CCL17_NM_002987	AGAGCTGAATTCAAAACCAG	416	CCGCCTGCTGATGGGAGAGCTGAAT	436
	_human_51			TCAAAACCAGGGTGTCTCCC

17	CCL17_NM_002987	GATGGGAGAGCTGAATTCAA	417	GTGTCACCGCCTGCTGATGGGAGA	437
	_human_45			GCTGAATTCAAAACCAGGGTG

18	CCL17_NM_002987	TGATGGGAGAGCTGAATTCA	418	AGTGTCACCGCCTGCTGATGGGAGA	438
	_human_44			GCTGAATTCAAAACCAGGGT

19	CCL17_NM_002987	ACTTTGAGCTCACAGTGTCA	419	GCTCAGAGAGAAGTGACTTTGAGCT	439
	_human_16			CACAGTGTCACCGCCTGCTG

20	CCL17_NM_002987	GAGTGAAGAATGCAGTTAAA	420	ACCCCAACAACAAGAGAGTGAAGA	440
	_human_368			ATGCAGTTAAATACCTGCAAA

Accession: NM_002990

HUGO gene symbol: CCL22

1	CCL22_NM_002990	GTATTTGAAAACAGAGTAAA	441	GCTGGAGTTATATATGTATTTGAA	461
	_human_2083			AACAGAGTAAATACTTAAGAG

2	CCL22_NM_002990	CAATAAGCTGAGCCAATGAA	442	GGTGAAGATGATTCTCAATAAGC	462
	_human_298			TGAGCCAATGAAGAGCCTACTC

3	CCL22_NM_002990	TACTTAAGAGGCCAAATAGA	443	TGAAAACAGAGTAAATACTTAAG	463
	_human_2103			AGGCCAAATAGATGAATGGAAG

4	CCL22_NM_002990	ATGTATTTGAAAACAGAGTA	444	AAGCTGGAGTTATATATGTATTTG	464
	_human_2081			AAAACAGAGTAAATACTTAAG

5	CCL22_NM_002990	TTCATACAGCAAGTATGGGA	445	TTGAGAAATATTCTTTTCATACAG	465
	_human_2496			CAAGTATGGGACAGCAGTGTC

6	CCL22_NM_002990	CTGCAGACAAAATCAATAAA	446	GAGCCCAGAAAGTGGCTGCAGAC	466
	_human_1052			AAAATCAATAAAACTAATGTCC

7	CCL22_NM_002990	TGCAGACAAAATCAATAAAA	447	AGCCCAGAAAGTGGCTGCAGACA	467
	_human_1053			AAATCAATAAAACTAATGTCCC

8	CCL22_NM_002990	GGCCAAATAGATGAATGGAA	448	AGTAAATACTTAAGAGGCCAAAT	468
	_human_2112			AGATGAATGGAAGAATTTTAGG

9	CCL22_NM_002990	AATAAGCTGAGCCAATGAAG	449	GTGAAGATGATTCTCAATAAGCT	469
	_human_299			GAGCCAATGAAGAGCCTACTCT

10	CCL22_NM_002990	AAGAGGCCAAATAGATGAAT	450	ACAGAGTAAATACTTAAGAGGCC	470
	_human_2108			AAATAGATGAATGGAAGAATTT

11	CCL22_NM_002990	AAATAGATGAATGGAAGAAT	451	AATACTTAAGAGGCCAAATAGAT	471
	_human_2116			GAATGGAAGAATTTTAGGAACT

12	CCL22_NM_002990	AAACAGAGTAAATACTTAAG	452	TATATATGTATTTGAAAACAGAGT	472
	_human_2091			AAATACTTAAGAGGCCAAATA

13	CCL22_NM_002990	AGCTGGAGTTATATATGTAT	453	TGACTTGGTATTATAAGCTGGAG	473
	_human_2067			TTATATATGTATTTGAAAACAG

14	CCL22_NM_002990	ACCTTTGACTTGGTATTATA	454	ATGGTGTGAAAGACTACCTTTGA	474
	_human_2047			CTTGGTATTATAAGCTGGAGTT

15	CCL22_NM_002990	AACCTTCAGGGATAAGGAGA	455	TGGCGTGGTGTTGCTAACCTTCA	475
	_human_238			GGGATAAGGAGATCTGTGCCGA

16	CCL22_NM_002990	GTGAAAGACTACCTTTGACT	456	AATTCATGCTATGGTGTGAAAGA	476
	_human_2037			CTACCTTTGACTTGGTATTATA

17	CCL22_NM_002990	CTATGGTGTGAAAGACTACC	457	ACAATCAAATTCATGCTATGGTGT	477
	_human_2030			GAAAGACTACCTTTGACTTGG

18	CCL22_NM_002990	CACTACGGCTGGCTAATTTT	458	ATTACAGGTGTGTGCCACTACGG	478
	_human_1682			CTGGCTAATTTTTGTATTTTTA

19	CCL22_NM_002990	GGAGTTATATATGTATTTGA	459	TTGGTATTATAAGCTGGAGTTATA	479
	_human_2071			TATGTATTTGAAAACAGAGTA

20	CCL22_NM_002990	ATATCAATACAGAGACTCAA	460	CCAAAAGGCAGTTACATATCAAT	480
	_human_1111			ACAGAGACTCAAGGTCACTAGA

Accession: NM_005618

HUGO gene symbol: DLL1

1	DLL1_NM_005618	CTGTTTTGTTAATGAAGAAA	481	TATTTGAGTTTTTTACTGTTTTGTTA	501
	_human_3246			ATGAAGAAATTCCTTTTTA

2	DLL1_NM_005618	TTGTATATAAATGTATTTAT	482	TGTGACTATATTTTTTTGTATATAAA	502
	_human_3193			TGTATTTATGGAATATTGT

3	DLL1_NM_005618	TGTTTTGTTAATGAAGAAAT	483	ATTTGAGTTTTTTACTGTTTTGTTAA	503
	_human_3247			TGAAGAAATTCCTTTTTAA

4	DLL1_NM_005618	AATTTTGGTAAATATGTACA	484	GTTTTTTATAATTTAAATTTTGGTAA	504
	_human_3141			ATATGTACAAAGGCACTTC

5	DLL1_NM_005618	AAATTTTATGAATGACAAAA	485	ATATTTTTCCAAAATAAATTTTATGA	505
	_human_3293			ATGACAAAAAAAAAAAAAA

6	DLL1_NM_005618	TTTATGGAATATTGTGCAAA	486	TTGTATATAAATGTATTTATGGAATA	506
	_human_3208			TTGTGCAAATGTTATTTGA

7	DLL1_NM_005618	TTACTGTTTTGTTAATGAAG	487	TGTTATTTGAGTTTTTTACTGTTTTGT	507
	_human_3243			TAATGAAGAAATTCCTTT

8	DLL1_NM_005618	TTCTTGAATTAGAAACACAA	488	TTATGAGCCAGTCTTTTCTTGAATTA	508
	_human_2977			GAAACACAAACACTGCCTT

9	DLL1_NM_005618	CAGTTGCTCTTAAGAGAATA	489	CCGTTGCACTATGGACAGTTGCTCTT	509
	_human_2874			AAGAGAATATATATTTAAA

10	DLL1_NM_005618	CAACTTCAAAAGACACCAAG	490	CGGACTCGGGCTGTTCAACTTCAAA	510
	_human_2560			AGACACCAAGTACCAGTCGG

11	DLL1_NM_005618	TCCAAAATAAATTTTATGAA	491	TTTTTAAAATATTTTTCCAAAATAAA	511
	_human_3285			TTTTATGAATGACAAAAAA

12	DLL1_NM_005618	GAACTGAATTACGCATAAGA	492	TATATTTAAATGGGTGAACTGAATT	512
	_human_2909			ACGCATAAGAAGCATGCACT

13	DLL1_NM_005618	GGATTTTGTGACAAACCAGG	493	TGTGATGAGCAGCATGGATTTTGTG	513
	_human_1173			ACAAACCAGGGGAATGCAAG

14	DLL1_NM_005618	TACTGTTTTGTTAATGAAGA	494	GTTATTTGAGTTTTTTACTGTTTTGTT	514
	_human_3244			AATGAAGAAATTCCTTTT

15	DLL1_NM_005618	TTTGGTAAATATGTACAAAG	495	TTTTATAATTTAAATTTTGGTAAATA	515
	_human_3144			TGTACAAAGGCACTTCGGG

16	DLL1_NM_005618	CCAAAATAAATTTTATGAAT	496	TTTTAAAATATTTTTCCAAAATAAAT	516
	_human_3286			TTTATGAATGACAAAAAAA

17	DLL1_NM_005618	ATAATTTAAATTTTGGTAAA	497	TGATGTTCGTTTTTTATAATTTAAAT	517
	_human_3133			TTTGGTAAATATGTACAAA

18	DLL1_NM_005618	AAATGGGTGAACTGAATTAC	498	AGAGAATATATATTTAAATGGGTGA	518
	_human_2901			ACTGAATTACGCATAAGAAG

19	DLL1_NM_005618	TTCGGGTCTATGTGACTATA	499	TATGTACAAAGGCACTTCGGGTCTA	519
	_human_3168			TGTGACTATATTTTTTTGTA

20	DLL1_NM_005618	ACTGTTTTGTTAATGAAGAA	500	TTATTTGAGTTTTTTACTGTTTTGTTA	520
	_human_3245			ATGAAGAAATTCCTTTTT

Accession: NM_000639

HUGO gene symbol: FASLG

1	FASLG_NM_000639	TAGCTCCTCAACTCACCTAA	521	GGTTCAAAATGTCTGTAGCTCCTC	541
	_human_1154			AACTCACCTAATGTTTATGAG

2	FASLG_NM_000639	ATGTTTTCCTATAATATAAT	522	TGTCAGCTACTAATGATGTTTTCC	542
	_human_1771			TATAATATAATAAATATTTAT

3	FASLG_NM_000639	TTTTCCTATAATATAATAAA	523	CAGCTACTAATGATGTTTTCCTAT	543
	_human_1774			AATATAATAAATATTTATGTA

4	FASLG_NM_000639	TTCCTATAATATAATAAATA	524	GCTACTAATGATGTTTTCCTATAA	544
	_human_1776			TATAATAAATATTTATGTAGA

5	FASLG_NM_000639	TGCATTTGAGGTCAAGTAAG	525	GAGGGTCTTCTTACATGCATTTGA	545
	_human_1086			GGTCAAGTAAGAAGACATGAA

6	FASLG_NM_000639	ATTGATTGTCAGCTACTAAT	526	TAGTGCTTAAAAATCATTGATTGT	546
	_human_1750			CAGCTACTAATGATGTTTTCC

7	FASLG_NM_000639	AAATGAAAACATGTAATAAA	527	ATGTGCATTTTTGTGAAATGAAAA	547
	_human_1820			CATGTAATAAAAAGTATATGT

8	FASLG_NM_000639	ATTGTGAAGTACATATTAGG	528	AGAGAGAATGTAGATATTGTGAA	548
	_human_1659			GTACATATTAGGAAAATATGGG

9	FASLG_NM_000639	GCTTTCTGGAGTGAAGTATA	529	CTATGGAATTGTCCTGCTTTCTGG	549
	_human_667			AGTGAAGTATAAGAAGGGTGG

10	FASLG_NM_000639	CATTTGGTCAAGATTTTGAA	530	GGAAAATATGGGTTGCATTTGGT	550
	_human_1692			CAAGATTTTGAATGCTTCCTGA

11	FASLG_NM_000639	GGCTTATATAAGCTCTAAGA	531	TCTCAGACGTTTTTCGGCTTATAT	551
	_human_986			AAGCTCTAAGAGAAGCACTTT

12	FASLG_NM_000639	ACCAGTGCTGATCATTTATA	532	GCAGTGTTCAATCTTACCAGTGCT	552
	_human_911			GATCATTTATATGTCAACGTA

13	FASLG_NM_000639	CCATTTAACAGGCAAGTCCA	533	GCTGAGGAAAGTGGCCCATTTAA	553
	_human_598			CAGGCAAGTCCAACTCAAGGTC

14	FASLG_NM_000639	AAGTACATATTAGGAAAATA	534	AATGTAGATATTGTGAAGTACAT	554
	_human_1665			ATTAGGAAAATATGGGTTGCAT

15	FASLG_NM_000639	TGTGTGTGTGTATGACTAAA	535	GTGTGTGTGTGTGTGTGTGTGTG	555
	_human_1625			TGTATGACTAAAGAGAGAATGT

16	FASLG_NM_000639	AAGAGGGAGAAGCATGAAAA	536	CTGGGCTGCCATGTGAAGAGGGA	556
	_human_1238			GAAGCATGAAAAAGCAGCTACC

17	FASLG_NM_000639	GTGTATGACTAAAGAGAGAA	537	TGTGTGTGTGTGTGTGTGTATGA	557
	_human_1632			CTAAAGAGAGAATGTAGATATT

18	FASLG_NM_000639	GTATTTCCAGTGCAATTGTA	538	CCTAACACAGCATGTGTATTTCCA	558
	_human_1581			GTGCAATTGTAGGGGTGTGTG

19	FASLG_NM_000639	CAACTCTAATAGTGCTTAAA	539	ATGCTTCCTGACAATCAACTCTAA	559
	_human_1726			TAGTGCTTAAAAATCATTGAT

20	FASLG_NM_000639	GTGTGTGTGTATGACTAAAG	540	TGTGTGTGTGTGTGTGTGTGTGT	560
	human_1626			GTATGACTAAAGAGAGAATGTA

Accession: NM_001267706

HUGO gene symbol: CD274

1	CD274_NM_001267706	ACCTGCATTAATTTAATAAA	561	ATTGTCACTTTTTGTACCTGCATTA	581
	_human_3222			ATTTAATAAAATATTCTTAT

2	CD274_NM_001267706	AACTTGCCCAAACCAGTAAA	562	GCAAACAGATTAAGTAACTTGCC	582
	_human_1538			CAAACCAGTAAATAGCAGACCT

3	CD274_NM_001267706	ATTTGCTCACATCTAGTAAA	563	ACTTGCTGCTTAATGATTTGCTCA	583
	_human_1218			CATCTAGTAAAACATGGAGTA

4	CD274_NM_001267706	CCTTTGCCATATAATCTAAT	564	TTTATTCCTGATTTGCCTTTGCCAT	584
	_human_1998			ATAATCTAATGCTTGTTTAT

5	CD274_NM_001267706	ATATAGCAGATGGAATGAAT	565	ATTTTAGTGTTTCTTATATAGCAG	585
	_human_2346			ATGGAATGAATTTGAAGTTCC

6	CD274_NM_001267706	GCCTTTGCCATATAATCTAA	566	ATTTATTCCTGATTTGCCTTTGCCA	586
	_human_1997			TATAATCTAATGCTTGTTTA

7	CD274_NM_001267706	GATTTGCCTTTGCCATATAA	567	ATTATATTTATTCCTGATTTGCCTT	587
	_human_1992			TGCCATATAATCTAATGCTT

8	CD274_NM_001267706	AATTTTCATTTACAAAGAGA	568	CTTAATAATCAGAGTAATTTTCAT	588
	_human_1905			TTACAAAGAGAGGTCGGTACT

9	CD274_NM_001267706	AGTGTTTCTTATATAGCAGA	569	ATTTTTATTTATTTTAGTGTTTCTT	589
	_human_2336			ATATAGCAGATGGAATGAAT

10	CD274_NM_001267706	GCTTTCTGTCAAGTATAAAC	570	GAACTTTTGTTTTCTGCTTTCTGTC	590
	_human_2656			AAGTATAAACTTCACTTTGA

11	CD274_NM_001267706	CATTTGGAAATGTATGTTAA	571	TCTAAAGATAGTCTACATTTGGAA	591
	_human_2235			ATGTATGTTAAAAGCACGTAT

12	CD274_NM_001267706	TTATTTTAGTGTTTCTTATA	572	CTTTGCTATTTTTATTTATTTTAGT	592
	_human_2329			GTTTCTTATATAGCAGATGG

13	CD274_NM_001267706	GTGGTAGCCTACACACATAA	573	CAGCTTTACAATTATGTGGTAGCC 593
	_human_1433			TACACACATAATCTCATTTCA

14	CD274_NM_001267706	ATGAGGAGATTAACAAGAAA	574	GGAGCTCATAGTATAATGAGGAG	594
	_human_1745			ATTAACAAGAAAATGTATTATT

15	CD274_NM_001267706	CAATTTTGTCGCCAAACTAA	575	TTGTAGTAGATGTTACAATTTTGT	595
	_human_1183			CGCCAAACTAAACTTGCTGCT

16	CD274_NM_001267706	TATATAGCAGATGGAATGAA	576	TATTTTAGTGTTTCTTATATAGCA	596
	_human_2345			GATGGAATGAATTTGAAGTTC

17	CD274_NM_001267706	AAATGCCACTAAATTTTAAA	577	CTGTCTTTTCTATTTAAATGCCACT	597
	_human_2069			AAATTTTAAATTCATACCTT

18	CD274_NM_001267706	TCTTTCCCATAGCTTTTCAT	578	TTTGTTTCTAAGTTATCTTTCCCAT	598
	_human_2414			AGCTTTTCATTATCTTTCAT

19	CD274_NM_001267706	TATATTCATGACCTACTGGC	579	GATATTTGCTGTCTTTATATTCAT	599
	_human_129			GACCTACTGGCATTTGCTGAA

20	CD274_NM_001267706	GTCCAGTGTCATAGCATAAG	580	TATTATTACAATTTAGTCCAGTGT	600
	_human_1783			CATAGCATAAGGATGATGCGA

Accession: NM_002164

HUGO gene symbol: IDO1

1	IDO1_NM_002164	ATTCTGTCATAATAAATAAA	601	AAAAAAAAAAGATATATTCTGTCA	621
	_human_1896			TAATAAATAAAAATGCATAAG

2	IDO1_NM_002164	TATCTTATCATTGGAATAAA	602	AAGTTTTGTAATCTGTATCTTATCA	622
	_human_1532			TTGGAATAAAATGACATTCA

3	IDO1_NM_002164	GTGATGGAGACTGCAGTAAA	603	TTTTGTTCTCATTTCGTGATGGAGA	623
	_human_578			CTGCAGTAAAGGATTCTTCC

4	IDO1_NM_002164	TTCTGTCATAATAAATAAAA	604	AAAAAAAAAGATATATTCTGTCAT	624
	_human_1897			AATAAATAAAAATGCATAAGA

5	IDO1_NM_002164	CTTGTAGGAAAACAACAAAA	605	AATACCTGTGCATTTCTTGTAGGAA	625
	_human_1473			AACAACAAAAGGTAATTATG

6	IDO1_NM_002164	ATAAAATGACATTCAATAAA	606	TATCTTATCATTGGAATAAAATGAC	626
	_human_1547			ATTCAATAAATAAAAATGCA

7	IDO1_NM_002164	CGTAAGGTCTTGCCAAGAAA	607	GGTCATGGAGATGTCCGTAAGGTC	627
	_human_412			TTGCCAAGAAATATTGCTGTT

8	IDO1_NM_002164	TCTTGTAGGAAAACAACAAA	608	AAATACCTGTGCATTTCTTGTAGGA	628
	_human_1472			AAACAACAAAAGGTAATTAT

9	IDO1_NM_002164	AACTGGAGGCACTGATTTAA	609	ACTGGAAGCCAAAGGAACTGGAG	629
	_human_1248			GCACTGATTTAATGAATTTCCT

10	IDO1_NM_002164	CAATACAAAAGACCTCAAAA	610	GTTTTACCAATAATGCAATACAAAA	630
	_human_1440			GACCTCAAAATACCTGTGCA

11	IDO1_NM_002164	TGCTTCTGCAATCAAAGTAA	611	GGTGGAAATAGCAGCTGCTTCTGC	631
	_human_636			AATCAAAGTAATTCCTACTGT

12	IDO1_NM_002164	AATGACATTCAATAAATAAA	612	TTATCATTGGAATAAAATGACATTC	632
	_human_1551			AATAAATAAAAATGCATAAG

13	IDO1_NM_002164	ATCATTGGAATAAAATGACA	613	TGTAATCTGTATCTTATCATTGGAA	633
	_human_1538			TAAAATGACATTCAATAAAT

14	IDO1_NM_002164	ACCAATAATGCAATACAAAA	614	ACTATGCAATGTTTTACCAATAATG	634
	_human_1430			CAATACAAAAGACCTCAAAA

15	IDO1_NM_002164	ATCTGTATCTTATCATTGGA	615	ACTAGAAGTTTTGTAATCTGTATCT	635
	_human_1527			TATCATTGGAATAAAATGAC

16	IDO1_NM_002164	ATCTTATCATTGGAATAAAA	616	AGTTTTGTAATCTGTATCTTATCAT	636
	_human_1533			TGGAATAAAATGACATTCAA

17	IDO1_NM_002164	CAGCTGCTTCTGCAATCAAA	617	TATTGGTGGAAATAGCAGCTGCTT	637
	_human_632			CTGCAATCAAAGTAATTCCTA

18	IDO1_NM_002164	GCAATACAAAAGACCTCAAA	618	TGTTTTACCAATAATGCAATACAAA	638
	_human_1439			AGACCTCAAAATACCTGTGC

19	IDO1_NM_002164	TCCTACTGTATTCAAGGCAA	619	TGCAATCAAAGTAATTCCTACTGTA	639
	_human_657			TTCAAGGCAATGCAAATGCA

20	IDO1_NM_002164	CAGAGCCACAAACTAATACT	620	CATTACCCATTGTAACAGAGCCAC	640
	_human_1398			AAACTAATACTATGCAATGTT

Accession: NM_001558

HUGO gene symbol: IL10RA

1	IL10RA_NM_001558_	TTGTTCATTTATTTATTGGA	641	CTTTATTTATTTATTTTGTTCATTT	661
	human_3364			ATTTATTGGAGAGGCAGCAT

2	IL10RA_NM_001558_	TTATTCCAATAAATTGTCAA	642	AGTGATACATGTTTTTTATTCCAA	662
	human_3626			TAAATTGTCAAGACCACAGGA

3	IL10RA_NM_001558_	TATTTTCTGGACACTCAAAC	643	AGATCTTAAGGTATATATTTTCTG	663
	human_2395			GACACTCAAACACATCATAAT

4	IL10RA_NM_001558_	TTTATTGGAGAGGCAGCATT	644	TATTTTGTTCATTTATTTATTGGAG	664
	human_3375			AGGCAGCATTGCACAGTGAA

5	IL10RA_NM_001558_	ACCTTGGAGAAGTCACTTAT	645	GTTTCCAGTGGTATGACCTTGGA	665
	human_3469			GAAGTCACTTATCCTCTTGGAG

6	IL10RA_NM_001558_	TTATTTATTTATTTTGTTCA	646	GTTCCCTTGAAAGCTTTATTTATTT	666
	human_3351			ATTTTGTTCATTTATTTATT

7	IL10RA_NM_001558_	CTCTTTCCTGTATCATAAAG	647	TCTCCCTCCTAGGAACTCTTTCCT	667
	human_2108			GTATCATAAAGGATTATTTGC

8	IL10RA_NM_001558_	CTGAGGAAATGGGTATGAAT	648	GGATGTGAGGTTCTGCTGAGGAA	668
	human_3563			ATGGGTATGAATGTGCCTTGAA

9	IL10RA_NM_001558_	GAATGTGCCTTGAACACAAA	649	TGAGGAAATGGGTATGAATGTGC	669
	human_3579			CTTGAACACAAAGCTCTGTCAA

10	IL10RA_NM_001558_	GGACACTCAAACACATCATA	650	AGGTATATATTTTCTGGACACTCA	670
	human_2403			AACACATCATAATGGATTCAC

11	IL10RA_NM_001558_	CTGTATCATAAAGGATTATT	651	CCTAGGAACTCTTTCCTGTATCAT	671
	human_2115			AAAGGATTATTTGCTCAGGGG

12	IL10RA_NM_001558_	TCACTTCCGAGAGTATGAGA	652	TGAAAGCATCTTCAGTCACTTCCG	672
	human_563			AGAGTATGAGATTGCCATTCG

13	IL10RA_NM_001558_	TCTCTGGAGCATTCTGAAAA	653	TCTCAGCCCTGCCTTTCTCTGGAG673
	human_3197			CATTCTGAAAACAGATATTCT

14	IL10RA_NM_001558_	TTATGCCAGAGGCTAACAGA	654	AAGCTGGCTTGTTTCTTATGCCAG	674
	human_2987			AGGCTAACAGATCCAATGGGA

15	IL10RA_NM_001558_	AGTGGCATTGACTTAGTTCA	655	AGGGGCCAGGATGACAGTGGCA	675
	human_1278			TTGACTTAGTTCAAAACTCTGAG

16	IL10RA_NM_001558_	TTTCTGGACACTCAAACACA	656	TCTTAAGGTATATATTTTCTGGAC	676
	human_2398			ACTCAAACACATCATAATGGA

17	IL10RA_NM_001558_	GCATTGCACAGTGAAAGAAT	657	TTTATTGGAGAGGCAGCATTGCA	677
	human_3390			CAGTGAAAGAATTCTGGATATC

18	IL10RA_NM_001558_	GACCTTGGAGAAGTCACTTA	658	TGTTTCCAGTGGTATGACCTTGGA	678
	human_3468			GAAGTCACTTATCCTCTTGGA

19	IL10RA_NM_001558_	TCACGTTCACACACAAGAAA	659	AGGTGCCGGGAAACTTCACGTTC	679
	human_610			ACACACAAGAAAGTAAAACATG

20	IL10RA_NM_001558_	ACTTTGCTGTTTCCAGTGGT	660	GAAATTCTAGCTCTGACTTTGCTG	680
	human_3446			TTTCCAGTGGTATGACCTTGG

Accession: NM_000214

HUGO gene symbol: JAG1

1	JAG1_NM_000214	TATTTGATTTATTAACTTAA	681	ATTAATCACTGTGTATATTTGATTT	701
	_human_4799			ATTAACTTAATAATCAAGAG

2	JAG1_NM_000214	GAAAAGTAATATTTATTAAA	682	TTGGCAATAAATTTTGAAAAGTAA	702
	_human_5658			TATTTATTAAATTTTTTTGTA

3	JAG1_NM_000214	ACTTTGTATAGTTATGTAAA	683	AATGTCAAAAGTAGAACTTTGTAT	703
	_human_4752			AGTTATGTAAATAATTCTTTT

4	JAG1_NM_000214	GAATACTTGAACCATAAAAT	684	TCTAATAAGCTAGTTGAATACTTGA	704
	_human_5418			ACCATAAAATGTCCAGTAAG

5	JAG1_NM_000214	TCTTGGCAATAAATTTTGAA	685	TCTTTGATGTGTTGTTCTTGGCAAT	705
	_human_5641			AAATTTTGAAAAGTAATATT

6	JAG1_NM_000214	TTTCTGCTTTAGACTTGAAA	686	TGTTTGTTTTTTGTTTTTCTGCTTTA	706
	_human_5150			GACTTGAAAAGAGACAGGC

7	JAG1_NM_000214	TATATTTATTGACTCTTGAG	687	GATCATAGTTTTATTTATATTTATT	707
	_human_4526			GACTCTTGAGTTGTTTTTGT

8	JAG1_NM_000214	TATGATGACGTACAAGTAGT	688	TTTGTATATTGGTTTTATGATGACG	708
	_human_4566			TACAAGTAGTTCTGTATTTG

9	JAG1_NM_000214	GTGTTGTTCTTGGCAATAAA	689	AAATGCATCTTTGATGTGTTGTTCT	709
	_human_5634			TGGCAATAAATTTTGAAAAG

10	JAG1_NM_000214	CTGATCTAAAAGGGAATAAA	690	CCTTTTTCCATGCAGCTGATCTAAA	710
	_human_173			AGGGAATAAAAGGCTGCGCA

11	JAG1_NM_000214	TACGACGTCAGATGTTTAAA	691	GATGGAATTTTTTTGTACGACGTCA	711
	_human_5031			GATGTTTAAAACACCTTCTA

12	JAG1_NM_000214	AATAATCAAGAGCCTTAAAA	692	TTGATTTATTAACTTAATAATCAAG	712
	_human_4817			AGCCTTAAAACATCATTCCT

13	JAG1_NM_000214	GTATGAAAACATGGAACAGT	693	TTATTAAATTTTTTTGTATGAAAAC	713
	_human_5685			ATGGAACAGTGTGGCCTCTT

14	JAG1_NM_000214	TGGTTTTATGATGACGTACA	694	GTTGTTTTTGTATATTGGTTTTATG	714
	_human_4560			ATGACGTACAAGTAGTTCTG

15	JAG1_NM_000214	TTCTGCTTTAGACTTGAAAA	695	GTTTGTTTTTTGTTTTTCTGCTTTAG	715
	_human_5151			ACTTGAAAAGAGACAGGCA

16	JAG1_NM_000214	CTTGGCAATAAATTTTGAAA	696	CTTTGATGTGTTGTTCTTGGCAATA	716
	_human_5642			AATTTTGAAAAGTAATATTT

17	JAG1_NM_000214	TTTAATCTACTGCATTTAGG	697	GATTTGATTTTTTTTTTTAATCTACT	717
	_human_5377			GCATTTAGGGAGTATTCTA

18	JAG1_NM_000214	TGTATAGTTATGTAAATAAT	698	TCAAAAGTAGAACTTTGTATAGTTA	718
	_human_4756			TGTAAATAATTCTTTTTTAT

19	JAG1_NM_000214	ATTTATATTTATTGACTCTT	699	TTAGATCATAGTTTTATTTATATTTA	719
	_human_4523			TTGACTCTTGAGTTGTTTT

20	JAG1_NM_000214	CTTTTCACCATTCGTACATA	700	TGTAAATTCTGATTTCTTTTCACCAT	720
	_human_5325			TCGTACATAATACTGAACC

Accession: NM_002226

HUGO gene symbol: JAG2

1	JAG2_NM_002226	CGTTTCTTTAACCTTGTATA	721	AATGTTTATTTTCTACGTTTCTTTAA	741
	_human_4266			CCTTGTATAAATTATTCAG

2	JAG2_NM_002226	TAAATGAATGAACGAATAAA	722	GGCAGAACAAATGAATAAATGAAT	742
	_human_5800			GAACGAATAAAAATTTTGACC

3	JAG2_NM_002226	TCATTCATTTATTCCTTTGT	723	GGTCAAAATTTTTATTCATTCATTT	743
	_human_5450			ATTCCTTTGTTTTGCTTGGT

4	JAG2_NM_002226	GTAAATGTGTACATATTAAA	724	TGAAAGTGCATTTTTGTAAATGTGT	744
	_human_5021			ACATATTAAAGGAAGCACTC

5	JAG2_NM_002226	ACCCACGAATACGTATCAAG	725	AGTATAAAATTGCTTACCCACGAAT	745
	_human_5398			ACGTATCAAGGTCTTAAGGA

6	JAG2_NM_002226	GTTTTATAAAATAGTATAAA	726	AAACAGCTGAAAACAGTTTTATAA	746
	_human_5371			AATAGTATAAAATTGCTTACC

7	JAG2_NM_002226	CAACTGAGTCAAGGAGCAAA	727	TGAGGGGTAGGAGGTCAACTGAG	747
	_human_5691			TCAAGGAGCAAAGCCAAGAACC

8	JAG2_NM_002226	ATGTGTACATATTAAAGGAA	728	AGTGCATTTTTGTAAATGTGTACAT	748
	_human_5025			ATTAAAGGAAGCACTCTGTA

9	JAG2_NM_002226	TTCTTTAACCTTGTATAAAT	729	GTTTATTTTCTACGTTTCTTTAACCT	749
	_human_4269			TGTATAAATTATTCAGTAA

10	JAG2_NM_002226	ATTTTCTACGTTTCTTTAAC	730	AAAAACCAAATGTTTATTTTCTACG	750
	_human_4258			TTTCTTTAACCTTGTATAAA

11	JAG2_NM_002226	CAGTTTTATAAAATAGTATA	731	TAAAACAGCTGAAAACAGTTTTAT	751
	_human_5369			AAAATAGTATAAAATTGCTTA

12	JAG2_NM_002226	GCACAGGCAGAACAAATGAA	732	GAGTGAGGCTGCCTTGCACAGGCA	752
	_human_5780			GAACAAATGAATAAATGAATG

13	JAG2_NM_002226	TCAGGCTGAAAACAATGGAG	733	ATTATTCAGTAACTGTCAGGCTGA	753
	_human_4302			AAACAATGGAGTATTCTCGGA

14	JAG2_NM_002226	TAAAATTGCTTACCCACGAA	734	TTTTATAAAATAGTATAAAATTGCT	754
	_human_5387			TACCCACGAATACGTATCAA

15	JAG2_NM_002226	GTCAGGCTGAAAACAATGGA	735	AATTATTCAGTAACTGTCAGGCTG	755
	_human_4301			AAAACAATGGAGTATTCTCGG

16	JAG2_NM_002226	AAATGTGTACATATTAAAGG	736	AAAGTGCATTTTTGTAAATGTGTAC	756
	_human_5023			ATATTAAAGGAAGCACTCTG

17	JAG2_NM_002226	CAGTAACTGTCAGGCTGAAA	737	CTTGTATAAATTATTCAGTAACTGT	757
	_human_4293			CAGGCTGAAAACAATGGAGT

18	JAG2_NM_002226	GTATTCTCGGATAGTTGCTA	738	GCTGAAAACAATGGAGTATTCTCG	758
	_human_4321			GATAGTTGCTATTTTTGTAAA

19	JAG2_NM_002226	TCTCACACAAATTCACCAAA	739	AGGCGGAGAAGTTCCTCTCACACA	759
	_human_3994			AATTCACCAAAGATCCTGGCC

20	JAG2_NM_002226	TTGTTTTGCTTGGTCATTCA	740	CATTCATTTATTCCTTTGTTTTGCTT	760
	_human_5466			GGTCATTCAGAGGCAAGGT

Accession: NM_001315

HUGO gene symbol: MAPK14

1	MAPK14_NM_001315	TCATGCGAAAAGAACCTACA	761	ATTTCAGTCCATCATTCATGCGAAAA	781
	_human_670			GAACCTACAGAGAACTGCG

2	MAPK14_NM_001315	AAATGTCAGAAGCTTACAGA	762	CTGAACAACATTGTGAAATGTCAGA	782
	_human_833			AGCTTACAGATGACCATGTT

3	MAPK14_NM_001315	AAACATATGAAACATGAAAA	763	GAACTGCGGTTACTTAAACATATGA	783
	_human_707			AACATGAAAATGTGATTGGT

4	MAPK14_NM_001315	CAGTTCCTTATCTACCAAAT	764	ACAGATGACCATGTTCAGTTCCTTAT	784
	_human_863			CTACCAAATTCTCCGAGGT

5	MAPK14_NM_001315	TCCTGGTACAGACCATATTA	765	TGGAAGAACATTGTTTCCTGGTACA	785
	_human_1150			GACCATATTAACCAGCTTCA

6	MAPK14_NM_001315	TTCCTTATCTACCAAATTCT	766	GATGACCATGTTCAGTTCCTTATCTA	786
	_human_866			CCAAATTCTCCGAGGTCTA

7	MAPK14_NM_001315	TTCCTGGTACAGACCATATT	767	CTGGAAGAACATTGTTTCCTGGTAC	787
	_human_1149			AGACCATATTAACCAGCTTC

8	MAPK14_NM_001315	AAGTATATACATTCAGCTGA	768	ATTCTCCGAGGTCTAAAGTATATAC	788
	_human_896			ATTCAGCTGACATAATTCAC

9	MAPK14_NM_001315	CATTACAACCAGACAGTTGA	769	ATGCTGAACTGGATGCATTACAACC	789
	_human_1076			AGACAGTTGATATTTGGTCA

10	MAPK14_NM_001315	AGGGACCTAAAACCTAGTAA	770	GCTGACATAATTCACAGGGACCTAA	790
	_human_926			AACCTAGTAATCTAGCTGTG

11	MAPK14_NM_001315	CTCTGGAGGAATTCAATGAT	771	TTACACCTGCAAGGTCTCTGGAGGA	791
	_human_765			ATTCAATGATGTGTATCTGG

12	MAPK14_NM_001315	TAAACATATGAAACATGAAA	772	AGAACTGCGGTTACTTAAACATATG	792
	_human_706			AAACATGAAAATGTGATTGG

13	MAPK14_NM_001315	GATCTGAACAACATTGTGAA	773	CATCTCATGGGGGCAGATCTGAACA	793
	_human_815			ACATTGTGAAATGTCAGAAG

14	MAPK14_NM_001315	TCAGTTCCTTATCTACCAAA	774	TACAGATGACCATGTTCAGTTCCTTA	794
	_human_862			TCTACCAAATTCTCCGAGG

15	MAPK14_NM_001315	ATAATTCACAGGGACCTAAA	775	ATACATTCAGCTGACATAATTCACA	795
	_human_917			GGGACCTAAAACCTAGTAAT

16	MAPK14_NM_001315	CGAGGTCTAAAGTATATACA	776	ATCTACCAAATTCTCCGAGGTCTAA	796
	_human_887			AGTATATACATTCAGCTGAC

17	MAPK14_NM_001315	GAAATGTCAGAAGCTTACAG	777	TCTGAACAACATTGTGAAATGTCAG	797
	_human_832			AAGCTTACAGATGACCATGT

18	MAPK14_NM_001315	AGCTGTTGACTGGAAGAACA	778	GATGCATAATGGCCGAGCTGTTGAC	798
	_human_1125			TGGAAGAACATTGTTTCCTG

19	MAPK14_NM_001315	AAATTCTCCGAGGTCTAAAG	779	AGTTCCTTATCTACCAAATTCTCCGA	799
	_human_879			GGTCTAAAGTATATACATT

20	MAPK14_NM_001315	AATGTGATTGGTCTGTTGGA	780	CATATGAAACATGAAAATGTGATTG	800
	_human_725			GTCTGTTGGACGTTTTTACA

Accession: NM_003745

HUGO gene symbol: SOCS1

1	SOCS1_NM_003745	CTGCTGTGCAGAATCCTATT	801	TCTGGCTTTATTTTTCTGCTGTGCAGAA	821
	_human_1141			TCCTATTTTATATTTTT

2	SOCS1_NM_003745	GCTGTGCAGAATCCTATTTT	802	TGGCTTTATTTTTCTGCTGTGCAGAATC	822
	_human_1143			CTATTTTATATTTTTTA

3	SOCS1_NM_003745	TTAAAGTCAGTTTAGGTAAT	803	CCTATTTTATATTTTTTAAAGTCAGTTT	823
	_human_1170			AGGTAATAAACTTTATT

4	SOCS1_NM_003745	CTGTGCAGAATCCTATTTTA	804	GGCTTTATTTTTCTGCTGTGCAGAATCC	824
	_human_1144			TATTTTATATTTTTTAA

5	SOCS1_NM_003745	GTTTACATATACCCAGTATC	805	CTCCTACCTCTTCATGTTTACATATACC	825
	_human_1076			CAGTATCTTTGCACAAA

6	SOCS1_NM_003745	ATTTTGTTATTACTTGCCTG	806	CTGGGATGCCGTGTTATTTTGTTATTA	826
	_human_837			CTTGCCTGGAACCATGTG

7	SOCS1_NM_003745	TAACTGGGATGCCGTGTTAT	807	CCGTGCACGCAGCATTAACTGGGATG	827
	_human_819			CCGTGTTATTTTGTTATTA

8	SOCS1_NM_003745	TGTTATTACTTGCCTGGAAC	808	GATGCCGTGTTATTTTGTTATTACTTGC	828
	_human_841			CTGGAACCATGTGGGTA

9	SOCS1_NM_003745	TTTCTGCTGTGCAGAATCCT	809	GTCTCTGGCTTTATTTTTCTGCTGTGCA	829
	_human_1138			GAATCCTATTTTATATT

10	SOCS1_NM_003745	CGTGTTATTTTGTTATTACT	810	CATTAACTGGGATGCCGTGTTATTTTG	830
	_human_831			TTATTACTTGCCTGGAAC

11	SOCS1_NM_003745	TTTTAAAGTCAGTTTAGGTA	811	ATCCTATTTTATATTTTTTAAAGTCAGT	831
	_human_1168			TTAGGTAATAAACTTTA

12	SOCS1_NM_003745	TGCTGTGCAGAATCCTATTT	812	CTGGCTTTATTTTTCTGCTGTGCAGAAT	832
	_human_1142			CCTATTTTATATTTTTT

13	SOCS1_NM_003745	GGATGCCGTGTTATTTTGTT	813	ACGCAGCATTAACTGGGATGCCGTGTT	833
	_human_825			ATTTTGTTATTACTTGCC

14	SOCS1_NM_003745	TTTAAAGTCAGTTTAGGTAA	814	TCCTATTTTATATTTTTTAAAGTCAGTT	834
	_human_1169			TAGGTAATAAACTTTAT

15	SOCS1_NM_003745	TAAAGTCAGTTTAGGTAATA	815	CTATTTTATATTTTTTAAAGTCAGTTTA	835
	_human_1171			GGTAATAAACTTTATTA

16	SOCS1_NM_003745	TCTGCTGTGCAGAATCCTAT	816	CTCTGGCTTTATTTTTCTGCTGTGCAGA	836
	_human_1140			ATCCTATTTTATATTTT

17	SOCS1_NM_003745	ATATACCCAGTATCTTTGCA	817	CCTCTTCATGTTTACATATACCCAGTAT	837
	_human_1082			CTTTGCACAAACCAGGG

18	SOCS1_NM_003745	AGAATCCTATTTTATATTTT	818	ATTTTTCTGCTGTGCAGAATCCTATTTT	838
	_human_1150			ATATTTTTTAAAGTCAG

19	SOCS1_NM_003745	GGTTGTTGTAGCAGCTTAAC	819	CCTCTGGGTCCCCCTGGTTGTTGTAGC	839
	_human_1011			AGCTTAACTGTATCTGGA

20	SOCS1_NM_003745	CCCAGTATCTTTGCACAAAC	820	TCATGTTTACATATACCCAGTAT	840
	_human_1087			CTTTGCACAAACCAGGGGTTGG

Accession: NM_003150

HUGO gene symbol: STAT3

1	STAT3_NM_003150	ATATTGCTGTATCTACTTTA	841	TTTTTTTTTTTTGGTATATTGCTGT	861
	_human_4897			ATCTACTTTAACTTCCAGAA

2	STAT3_NM_003150	TGTTTGTTAAATCAAATTAG	842	GTTTCTGTGGAATTCTGTTTGTTA	862
	_human_4325			AATCAAATTAGCTGGTCTCTG

3	STAT3_NM_003150	TTTATCTAAATGCAAATAAG	843	TGTGGGTGATCTGCTTTTATCTAA	863
	_human_2730			ATGCAAATAAGGATGTGTTCT

4	STAT3_NM_003150	ATTTTCCTTTGTAATGTATT	844	TTTATAAATAGACTTATTTTCCTTT	864
	_human_3615			GTAATGTATTGGCCTTTTAG

5	STAT3_NM_003150	TATCAGCACAATCTACGAAG	845	GAGTCGAATGTTCTCTATCAGCAC	865
	_human_453			AATCTACGAAGAATCAAGCAG

6	STAT3_NM_003150	AGCTTAACTGATAAACAGAA	846	CTTCAGTACATAATAAGCTTAACT	866
	_human_4477			GATAAACAGAATATTTAGAAA

7	STAT3_NM_003150	GTTGTTGTTGTTCTTAGACA	847	CAGCTTTTTGTTATTGTTGTTGTTG	867
	_human_2870			TTCTTAGACAAGTGCCTCCT

8	STAT3_NM_003150	GTTGTTGTTCTTAGACAAGT	848	CTTTTTGTTATTGTTGTTGTTGTTC	868
	_human_2873			TTAGACAAGTGCCTCCTGGT

9	STAT3_NM_003150	TCTGTATTTAAGAAACTTAA	849	TATCAGCATAGCCTTTCTGTATTT	869
	_human_3096			AAGAAACTTAAGCAGCCGGGC

10	STAT3_NM_003150	TTATTTTCCTTTGTAATGTA	850	TTTTTATAAATAGACTTATTTTCCT	870
	_human_3613			TTGTAATGTATTGGCCTTTT

11	STAT3_NM_003150	TAACTGATAAACAGAATATT	851	AGTACATAATAAGCTTAACTGATA	871
	_human_4481			AACAGAATATTTAGAAAGGTG

12	STAT3_NM_003150	ACATTCTGGGCACAAACACA	852	GATCCCGGAAATTTAACATTCTGG	872
	_human_1372			GCACAAACACAAAAGTGATGA

13	STAT3_NM_003150	GTGATCTGCTTTTATCTAAA	853	AATGAGTGAATGTGGGTGATCTG	873
	_human_2720			CTTTTATCTAAATGCAAATAAG

14	STAT3_NM_003150	CAGACCCGTCAACAAATTAA	854	GCAGAATCTCAACTTCAGACCCGT	874
	_human_1044			CAACAAATTAAGAAACTGGAG

15	STAT3_NM_003150	GGAGCTGTTTAGAAACTTAA	855	GGAGGAGAGAATCGTGGAGCTG	875
	_human_1148			TTTAGAAACTTAATGAAAAGTGC

16	STAT3_NM_003150	ACCATTGGGTTTAAATCATA	856	GTGAGACTTGGGCTTACCATTGG	876
	_human_4523			GTTTAAATCATAGGGACCTAGG

17	STAT3_NM_003150	GGAGAATCTAAGCATTTTAG	857	AATAGGAAGGTTTAAGGAGAATC	877
	_human_3573			TAAGCATTTTAGACTTTTTTTT

18	STAT3_NM_003150	CCTTGCTGACATCCAAATAG	858	CATTGCACTTTTTAACCTTGCTGA	878
	_human_2987			CATCCAAATAGAAGATAGGAC

19	STAT3_NM_003150	AAATTAAGAAATAATAACAA	859	CCTAGGTTTCTTTTTAAATTAAGA	879
	_human_3041			AATAATAACAATTAAAGGGCA

20	STAT3_NM_003150	TTTTAAATTAAGAAATAATA	860	AAGCCCTAGGTTTCTTTTTAAATT	880
	_human_3037			AAGAAATAATAACAATTAAAG

Accession: NM_006290

HUGO gene symbol: TNFAIP3

1	TNFAIP3_NM_006290	AGCTTGAACTGAGGAGTAAA	881	ACTTCTAAAGAAGTTAGCTTGAAC	901
	_human_3451			TGAGGAGTAAAAGTGTGTACA

2	TNFAIP3_NM_006290	CCTTTGCAACATCCTCAGAA	882	AATACACATATTTGTCCTTTGCAA	902
	_human_916			CATCCTCAGAAGGCCAATCAT

3	TNFAIP3_NM_006290	TTCTTTCCAAAGATACCAAA	883	ACGAATCTTTATAATTTCTTTCCAA	903
	_human_4422			AGATACCAAATAAACTTCAG

4	TNFAIP3_NM_006290	TTATTTTATTACAAACTTCA	884	TGTAATTCACTTTATTTATTTTATT	904
	_human_3688			ACAAACTTCAAGATTATTTA

5	TNFAIP3_NM_006290	TATTTATACTTATTATAAAA	885	GTGAAAAAAAGTAATTATTTATAC	905
	_human_4536			TTATTATAAAAAGTATTTGAA

6	TNFAIP3_NM_006290	CATTTCAGACAAAATGCTAA	886	AAGGCCAATCATTGTCATTTCAGA	906
	_human_949			CAAAATGCTAAGAAGTTTGGA

7	TNFAIP3_NM_006290	ATGAAGGAGAAGCTCTTAAA	887	GATCCTGAAAATGAGATGAAGGA	907
	_human_1214			GAAGCTCTTAAAAGAGTACTTA

8	TNFAIP3_NM_006290	ATTTTGTGTTGATCATTATT	888	AGTTGATATCTTAATATTTTGTGT	908
	_human_4489			TGATCATTATTTCCATTCTTA

9	TNFAIP3_NM_006290	TTCATCGAGTACAGAGAAAA	889	TTTTGCACACTGTGTTTCATCGAG	909
	_human_2204			TACAGAGAAAACAAACATTTT

10	TNFAIP3_NM_006290	TTACTGGGAAGACGTGTAAC	890	AAAAATTAGAATATTTTACTGGGA	910
	_human_3394			AGACGTGTAACTCTTTGGGTT

11	TNFAIP3_NM_006290	TCATTGAAGCTCAGAATCAG	891	ACTGCCAGAAGTGTTTCATTGAA	911
	_human_2355			GCTCAGAATCAGAGATTTCATG

12	TNFAIP3_NM_006290	TTCCATTCTTAATGTGAAAA	892	TGTGTTGATCATTATTTCCATTCTT	912
	_human_4508			AATGTGAAAAAAAGTAATTA

13	TNFAIP3_NM_006290	TGAAGGATACTGCCAGAAGT	893	TGGAAGCACCATGTTTGAAGGAT	913
	_human_2332			ACTGCCAGAAGTGTTTCATTGA

14	TNFAIP3_NM_006290	CACAAGAGTCAACATTAAAA	894	ATAAATGTAACTTTTCACAAGAGT	914
	_human_4650			CAACATTAAAAAATAAATTAT

15	TNFAIP3_NM_006290	AATTATTTATACTTATTATA	895	AATGTGAAAAAAAGTAATTATTTA	915
	_human_4533			TACTTATTATAAAAAGTATTT

16	TNFAIP3_NM_006290	TTCGTGCTTCTCCTTATGAA	896	CATATTCATCGATGTTTCGTGCTT	916
	_human_3907			CTCCTTATGAAACTCCAGCTA

17	TNFAIP3_NM_006290	TATTTTATTACAAACTTCAA	897	GTAATTCACTTTATTTATTTTATTA	917
	_human_3689			CAAACTTCAAGATTATTTAA

18	TNFAIP3_NM_006290	TATTACAAACTTCAAGATTA	898	TCACTTTATTTATTTTATTACAAAC	918
	_human_3694			TTCAAGATTATTTAAGTGAA

19	TNFAIP3_NM_006290	CTCTTAAAGTTGATATCTTA	899	TGTTTTCATCTAATTCTCTTAAAGT	919
	_human_4467			TGATATCTTAATATTTTGTG

20	TNFAIP3_NM_006290	TTCCAAAGATACCAAATAAA	900	ATCTTTATAATTTCTTTCCAAAGAT	920
	_human_4426			ACCAAATAAACTTCAGTGTT

Accession: NM_003326

HUGO gene symbol: TNFSF4

1	TNFSF4_NM_003326	AATTTGACTTAGCCACTAAC	921	GAGATCAGAATTTTAAATTTGACT	941
	_human_2984			TAGCCACTAACTAGCCATGTA

2	TNFSF4_NM_003326	GATATTAATAATATAGTTAA	922	GAGAGTATTAATATTGATATTAAT	942
	_human_3422			AATATAGTTAATAGTAATATT

3	TNFSF4_NM_003326	CTGTGAATGCACATATTAAA	923	TGCTTACAGTGTTATCTGTGAATG	943
	_human_3119			CACATATTAAATGTCTATGTT

4	TNFSF4_NM_003326	GTTTTCTATTTCCTCTTAAG	924	GGATTTTTTTTTCCTGTTTTCTATT	944
	_human_2208			TCCTCTTAAGTACACCTTCA

5	TNFSF4_NM_003326	AAATAGCACTAAGAAGTTAT	925	ATTCAATCTGATGTCAAATAGCAC	945
	_human_1727			TAAGAAGTTATTGTGCCTTAT

6	TNFSF4_NM_003326	CCAATCCCGATCCAAATCAT	926	AATGCTTAAGGGATTCCAATCCC	946
	_human_3311			GATCCAAATCATAATTTGTTCT

7	TNFSF4_NM_003326	CTATTTAGAGAATGCTTAAG	927	TTAGTTAGATATTTTCTATTTAGA	947
	_human_3286			GAATGCTTAAGGGATTCCAAT

8	TNFSF4_NM_003326	CAGTTTGCATATTGCCTAAA	928	AGGTTAAATTGATTGCAGTTTGCA 948
	_human_1222			TATTGCCTAAATTTAAACTTT

9	TNFSF4_NM_003326	CTCGAATTCAAAGTATCAAA	929	TATCACATCGGTATCCTCGAATTC	949
	_human_326			AAAGTATCAAAGTACAATTTA

10	TNFSF4_NM_003326	ATCTGTGAATGCACATATTA	930	TATGCTTACAGTGTTATCTGTGAA	950
	_human_3117			TGCACATATTAAATGTCTATG

11	TNFSF4_NM_003326	TTTGTGGGAAAAGAATTGAA	931	TATACATGGCAGAGTTTTGTGGG	951
	_human_2938			AAAAGAATTGAATGAAAAGTCA

12	TNFSF4_NM_003326	ATTGACCATGTTCTGCAAAA	932	ATTTCACTTTTTGTTATTGACCATG	952
	_human_2537			TTCTGCAAAATTGCAGTTAC

13	TNFSF4_NM_003326	GATTCTTCATTGCAAGTGAA	933	GGTGGACAGGGCATGGATTCTTC	953
	_human_776			ATTGCAAGTGAAGGAGCCTCCC

14	TNFSF4_NM_003326	GATGTCAAATAGCACTAAGA	934	TATCAAATTCAATCTGATGTCAAA	954
	_human_1721			TAGCACTAAGAAGTTATTGTG

15	TNFSF4_NM_003326	GTATACAGGGAGAGTGAGAT	935	AAGAGAGATTTTCTTGTATACAG	955
	_human_1459			GGAGAGTGAGATAACTTATTGT

16	TNFSF4_NM_003326	GTTGCTATGAGTCAAGGAGT	936	AATGTCTATGTTCTTGTTGCTATG	956
	_human_3152			AGTCAAGGAGTGTAACCTTCT

17	TNFSF4_NM_003326	TAGTTGAAATGTCCCCTTAA	937	GTATCCCCTTATGTTTAGTTGAAA	957
	_human_1882			TGTCCCCTTAACTTGATATAA

18	TNFSF4_NM_003326	CTCTGTGCCAAACCTTTTAT	938	GATGATTTGTAACTTCTCTGTGCC	958
	_human_1980			AAACCTTTTATAAACATAAAT

19	TNFSF4_NM_003326	CTCTGTCTAGAAATACCATA	939	ATGAAAAATAATGATCTCTGTCTA	959
	_human_1770			GAAATACCATAGACCATATAT

20	TNFSF4_NM_003326	GGTTTCAAGAAATGAGGTGA	940	CACAGAAACATTGCTGGTTTCAA	960
	_human_1680			GAAATGAGGTGATCCTATTATC

Accession: NM_006293

HUGO gene symbol: TYRO3

1	TYRO3_NM_006293	AGTTGCTGTTTAAAATAGAA	961	CATTTCCAAGCTGTTAGTTGCTGTT	981
	_human_3927			TAAAATAGAAATAAAATTGA

2	TYRO3_NM_006293	CTGTTTAAAATAGAAATAAA	962	CCAAGCTGTTAGTTGCTGTTTAAAA	982
	_human_3932			TAGAAATAAAATTGAAGACT

3	TYRO3_NM_006293	GGCATCAGCGATGAACTAAA	963	ACATTGGACAGCTTGGGCATCAGC	983
	_human_1731			GATGAACTAAAGGAAAAACTG

4	TYRO3_NM_006293	AATATCCTAAGACTAACAAA	964	GCTACCAAATCTCAAAATATCCTAA	984
	_human_3699			GACTAACAAAGGCAGCTGTG

5	TYRO3_NM_006293	GTTGCTGTTTAAAATAGAAA	965	ATTTCCAAGCTGTTAGTTGCTGTTT	985
	_human_3928			AAAATAGAAATAAAATTGAA

6	TYRO3_NM_006293	AAAATAGAAATAAAATTGAA	966	TGTTAGTTGCTGTTTAAAATAGAAA	986
	_human_3938			TAAAATTGAAGACTAAAGAC

7	TYRO3_NM_006293	CTGTGAAGCTCACAACCTAA	967	GAGCACCATGTTTTCCTGTGAAGC	987
	_human_842			TCACAACCTAAAAGGCCTGGC

8	TYRO3_NM_006293	TTGAAGACTAAAGACCTAAA	968	AAAATAGAAATAAAATTGAAGACT	988
	_human_3953			AAAGACCTAAAAAAAAAAAAA

9	TYRO3_NM_006293	TCCTAAGACTAACAAAGGCA	969	CCAAATCTCAAAATATCCTAAGACT	989
	_human_3703			AACAAAGGCAGCTGTGTCTG

10	TYRO3_NM_006293	GGACATTTCCAAGCTGTTAG	970	GGTCCTAGCTGTTAGGGACATTTC	990
	_human_3909			CAAGCTGTTAGTTGCTGTTTA

11	TYRO3_NM_006293	ATGTTTCCATGGTTACCATG	971	AGGAGTGGGGTGGTTATGTTTCCA	991
	_human_3190			TGGTTACCATGGGTGTGGATG

12	TYRO3_NM_006293	TAGTTGCTGTTTAAAATAGA	972	ACATTTCCAAGCTGTTAGTTGCTGT	992
	_human_3926			TTAAAATAGAAATAAAATTG

13	TYRO3_NM_006293	AAAATTGAAGACTAAAGACC	973	GTTTAAAATAGAAATAAAATTGAA	993
	_human_3949			GACTAAAGACCTAAAAAAAAA

14	TYRO3_NM_006293	AGCTGTTAGGGACATTTCCA	974	CATGGGGCGGGTCCTAGCTGTTAG	994
	_human_3900			GGACATTTCCAAGCTGTTAGT

15	TYRO3_NM_006293	GAGGACGTGTATGATCTCAT	975	CCTCCGGAGTGTATGGAGGACGTG	995
	_human_2511			TATGATCTCATGTACCAGTGC

16	TYRO3_NM_006293	TTTTAGGTGAGGGTTGGTAA	976	CCTTGTAATATTCCCTTTTAGGTGA	996
	_human_3400			GGGTTGGTAAGGGGTTGGTA

17	TYRO3_NM_006293	AGCTGACATCATTGCCTCAA	977	TGTGAAGATGCTGAAAGCTGACAT	997
	_human_1895			CATTGCCTCAAGCGACATTGA

18	TYRO3_NM_006293	AAATCTCAAAATATCCTAAG	978	TCTGAGCACGCTACCAAATCTCAA	998
	_human_3690			AATATCCTAAGACTAACAAAG

19	TYRO3_NM_006293	AAGCTGTTAGTTGCTGTTTA	979	GTTAGGGACATTTCCAAGCTGTTA	999
	_human_3919			GTTGCTGTTTAAAATAGAAAT

20	TYRO3_NM_006293	TCCTTGTAATATTCCCTTTT	980	AGTCACAAAGAGATGTCCTTGTAA	1000
	_human_3384			TATTCCCTTTTAGGTGAGGGT

Accession: NM_000546

HUGO gene symbol: TP53

1	TP53_NM_000546_	TGTTTGGGAGATGTAAGAAA	81	TTTTACTGTGAGGGATGTTTGGG	101
	human_1630			AGATGTAAGAAATGTTCTTGCA

2	TP53_NM_000546_	GCATTGTGAGGGTTAATGAA	82	CCTACCTCACAGAGTGCATTGTGA	102
	human_1808			GGGTTAATGAAATAATGTACA

3	TP53_NM_000546_	TCGATCTCTTATTTTACAAT	83	TATCCCATTTTTATATCGATCTCTT	103
	human_2538			ATTTTACAATAAAACTTTGC

4	TP53_NM_000546_	TGTGAGGGTTAATGAAATAA	84	CCTCACAGAGTGCATTGTGAGGG	104
	human_1812			TTAATGAAATAATGTACATCTG

5	TP53_NM_000546_	GAGTATTTGGATGACAGAAA	85	GGAAATTTGCGTGTGGAGTATTT	105
	human_812			GGATGACAGAAACACTTTTCGA

6	TP53_NM_000546_	GGATGTTTGGGAGATGTAAG	86	GGTTTTTACTGTGAGGGATGTTTG	106
	human_1627			GGAGATGTAAGAAATGTTCTT

7	TP53_NM_000546_	GAAATGTTCTTGCAGTTAAG	87	GTTTGGGAGATGTAAGAAATGTT	107
	human_1646			CTTGCAGTTAAGGGTTAGTTTA

8	TP53_NM_000546_	ATGTACATCTGGCCTTGAAA	88	AGGGTTAATGAAATAATGTACAT	108
	human_1831			CTGGCCTTGAAACCACCTTTTA

9	TP53_NM_000546_	AGAAATGTTCTTGCAGTTAA	89	TGTTTGGGAGATGTAAGAAATGT	109
	human_1645			TCTTGCAGTTAAGGGTTAGTTT

10	TP53_NM_000546_	GGTGAACCTTAGTACCTAAA	90	GTCTGACAACCTCTTGGTGAACCT	110
	human_2015			TAGTACCTAAAAGGAAATCTC

11	TP53_NM_000546_	TAACTTCAAGGCCCATATCT	91	CTGTTGAATTTTCTCTAACTTCAA	111
	human_1753			GGCCCATATCTGTGAAATGCT

12	TP53_NM_000546_	CTTATCCGAGTGGAAGGAAA	92	GCCCCTCCTCAGCATCTTATCCGA	112
	human_782			GTGGAAGGAAATTTGCGTGTG

13	TP53_NM_000546_	ATGATCTGGATCCACCAAGA	93	CATCTCTTGTATATGATGATCTGG	113
	human_2086			ATCCACCAAGACTTGTTTTAT

14	TP53_NM_000546_	AATTTTCTCTAACTTCAAGG	94	TGTCCCTCACTGTTGAATTTTCTCT	114
	human_1744			AACTTCAAGGCCCATATCTG

15	TP53_NM_000546_	TCTCTTATTTTACAATAAAA	95	CCATTTTTATATCGATCTCTTATTT	115
	human_2542			TACAATAAAACTTTGCTGCC

16	TP53_NM_000546_	TTATTTTACAATAAAACTTT	96	TTTTATATCGATCTCTTATTTTACA	116
	human_2546			ATAAAACTTTGCTGCCACCT

17	TP53_NM_000546_	GCCTTGAAACCACCTTTTAT	97	AATAATGTACATCTGGCCTTGAAA	117
	human_1842			CCACCTTTTATTACATGGGGT

18	TP53_NM_000546_	TATATCGATCTCTTATTTTA	98	TTTATATCCCATTTTTATATCGATC	118
	human_2534			TCTTATTTTACAATAAAACT

19	TP53_NM_000546_	CCTTAGTACCTAAAAGGAAA	99	CAACCTCTTGGTGAACCTTAGTAC	119
	human_2021			CTAAAAGGAAATCTCACCCCA

20	TP53_NM_000546_	CATTGTGAGGGTTAATGAAA	100	CTACCTCACAGAGTGCATTGTGA	120
	human_1809			GGGTTAATGAAATAATGTACAT

Claims

1. An immune modulator comprising an sdRNA, wherein the sdRNA comprises a guide strand and a passenger strand, wherein the guide strand is about 19-25 nucleotides long, and the passenger strand is about 10-19 nucleotides long, wherein the sdRNA includes a double stranded region and a single stranded region, wherein the single stranded region includes 5-9 phosphorothioate modifications, wherein the sdRNA is chemically modified, including at least one 2′-O-methyl modification or 2′-fluoro modification, and wherein the sdRNA is capable of suppressing expression of HAVCR2.

2. An immunogenic composition comprising an sdRNA, wherein the sdRNA comprises a guide strand and a passenger strand, wherein the guide strand is about 19-25 nucleotides long, and the passenger strand is about 10-19 nucleotides long, wherein the sdRNA includes a double stranded region and a single stranded region, wherein the single stranded region includes 5-9 phosphorothioate modifications, wherein the sdRNA is chemically modified, including at least one 2′-O-methyl modification or 2′-fluoro modification, wherein the sdRNA is capable of suppressing expression of HAVCR2, and wherein the immunogenic composition further comprises immune cells modified by the sdRNA to suppress expression of HAVCR2.

3. The immunogenic composition of claim 2, wherein the immune cells within the composition are further modified to suppress expression of a plurality of immune checkpoint genes.

4. The immunogenic composition of claim 2, wherein said cells are modified to suppress expression of at least one immune checkpoint gene, and at least one gene selected from the group consisting of: an anti-apoptosis gene, a cytokine receptor gene, and a regulator gene.

5-8. (canceled)

9. The immunogenic composition of claim 3, wherein said modified cells further comprise one or more sdRNA capable of suppressing expression of at least one target gene selected from the group consisting of: CTLA4, PD1/PDCD1, TGFBR1, TGFRBR2, IL10RA, TP53, BAX, BAK1, CASP8, ADORA2A, LAG3, CCL17, CCL22, DLL2, FASLG, CD274, IDO1, ILIORA, JAG1, JAG2, MAPK14, SOCS1, STAT3, TNFAIP3, TYRO3, BTLA, KIR, B7-H3 receptor, and B7-H4 receptor.

10. The immunogenic composition of claim 9, wherein said cells are selected from the group consisting of T-cells, NK-cells, antigen-presenting cells, dendritic cells, stem cells, induced pluripotent stem cells, and/or stem central memory T-cells.

11. The immunogenic composition of claim 9, wherein said cells are T-cells comprising one or more transgene expressing high affinity T-cell receptors (TCR) and/or chimeric antibody-T-cell receptors (CAR).

12. (canceled)

13. The immunogenic composition of claim 9, wherein said cells comprise one or more sdRNA targeting one or more anti-apoptotic genes selected from the group consisting of BAX, BAC, TP53, and Casp8.

14. (canceled)

15. The immunogenic composition of claim 9, wherein said sdRNA comprises at least one 2′-O-methyl modification and/or at least one 2′-O-Fluoro modification, and at least one phosphorothioate modification.

16. The immunogenic composition of claim 9, wherein said sdRNA comprises at least one hydrophobic modification.

17. The immunogenic composition of claim 9, wherein said sdRNA is modified to comprises at least one cholesterol molecule.

18. The immunogenic composition of claim 9, wherein said sdRNA targets a sequence selected from SEQ ID NOs: 1-360 and 381-1000.

19. A method of producing the immunogenic composition of claim 3, said method comprising transforming at least one cell with one or more sdRNA capable of targeting and suppressing expression of one or more immune target genes, wherein at least one sdRNA interferes with HAVCR2 expression.

20. The method of claim 19, wherein the at least one cell comprises an sdRNA that inhibits expression of one or more genes selected from the group consisting of: CTLA4, PD1/PDCD1, TGFBR1, TGFRBR2, IL10RA, TP53, BAX, BAK1, CASP8, ADOARA2A, LAG3, CCL17, CCL22, DLL2, FASLG, CD274, IDO1, ILIORA, JAG1, JAG2, MAPK14, SOCS1, STAT3, TNFAIP3, TYRO3, BTLA, KIR, B7-H3 receptor, and B7-H4 receptor.

21. The method of claim 19, wherein said cells are selected from the group consisting of T-cells, NK-cells, antigen-presenting cells, dendritic cells, stem cells, induced pluripotent stem cells, and/or stem central memory T-cells.

22-24. (canceled)

25. A method for treating a subject having proliferative disease or infectious disease, the method comprising administering to the subject the immune modulator of claim 1.

26. The method of claim 25, wherein said proliferative disease is cancer.

27. The method of claim 25, wherein said infectious disease is a pathogen infection.

28. (canceled)

29. The immune modulator of claim 1, wherein at least one sdRNA targets a sequence selected from SEQ ID NOs: 361-380.

30. The immunogenic composition of claim 2, wherein at least one sdRNA targets a sequence selected from SEQ ID NOs: 361-380.