Anti-VISTA antibody RNA or nucleic acid conjugate (ARC or ANC), compositions containing the same, and therapeutic use thereof

Abstract

The present invention relates, in particular, to an anti-VISTA antibody RNA or nucleic acid conjugate (ARC or ANC) that delivers at least one nucleic acid, for example RNA, to immune cells, and to the use of such ARC or ANC as a therapeutic agent for treating, for example, autoimmune conditions, allergic conditions and inflammatory conditions, or for treating cancer and / or inflammatory symptoms associated with certain immune cell types that express VISTA.

Description

[Technical Field]

[0001] Related applications This PCT application claims priority to U.S. Provisional Application No. 63 / 506,177, filed on 5 June 2023, and U.S. Provisional Application No. 63 / 611,302, filed on 18 December 2023, the contents of which are incorporated in their entirety by reference.

[0002] Disclosure of sequence listings The contents of the electronic sequence listing (1143260_008613_SL.xml, size: 707,682 bytes, and creation date: June 3, 2024) are incorporated herein by reference in their entirety.

[0003] Field of Invention The present invention, as disclosed herein, relates in particular to anti-VISTA antibody RNA or nucleic acid conjugates (ARC or ANC) that deliver at least one nucleic acid, for example RNA or DNA, or a nucleic acid / protein complex, to immune cells, and to the use of such ARC or ANC as therapeutic agents for treating autoimmune and inflammatory conditions, or for treating cancer and / or related symptoms induced by certain immune cell types. [Background technology]

[0004] The modulation of RNA function is an area of ​​growing therapeutic interest. Drugs that affect mRNA stability, such as antisense oligonucleotides and small interfering RNAs, are one way to modulate RNA function. Another group of oligonucleotides can modulate RNA function by altering the processing of premRNA to include or exclude specific regions of the premRNA of the final gene product, i.e., the encoded protein. Therefore, oligonucleotide therapeutics are means of modulating protein expression in disease states and thus have therapeutic utility.

[0005] Furthermore, RNA delivery to target cells through integration into ANCs or ARCs is known. However, existing ANCs or ARCs do not target specific immune cell types. [Overview of the project]

[0006] In one embodiment, the present invention relates to an anti-VISTA antibody RNA conjugate (ARC), and more particularly to an anti-VISTA antibody RNA conjugate (ARC) that can be used to deliver nucleic acids, such as RNA or DNA or nucleic acid / protein complexes, to immune cells.

[0007] In another aspect, the present invention relates to the use of such ARC as a therapeutic agent for treating, for example, autoimmune conditions, inflammatory conditions, and cancerous conditions.

[0008] In certain embodiments, the present invention provides an antibody-RNA or antibody-nucleic acid conjugate ("ARC" or "ANC") comprising (i) an antibody or antibody fragment that binds specifically or primarily to an antigen expressed by one or more immune cell types; (ii) one or more nucleic acids, preferably RNA or DNA oligonucleotides ("payload"), which are directly or indirectly conjugated thereto, and which consist of wild-type or modified nucleotides, preferably RNA or DNA oligonucleotides, to which the oligonucleotide specifically binds to a target gene, optionally an immunomodulatory gene, or RNA encoded thereon; and optionally (iii) a cleavable or incleavable linker or adapter, for example, a peptide mediating the (i) antibody or antibody fragment and the (ii) one or more nucleic acids, wherein such an ARC or ANC, when brought into contact with an immune cell expressing the antigen to which the (i) antibody or antibody fragment binds, is internalized by the immune cell, resulting in the release of the (ii) one or more nucleic acids to the immune cell, thereby optionally modulating the expression and / or function of a targeted immunomodulator.

[0009] In some specific embodiments, any of the aforementioned ARCs or ANCs optionally include a peptide linker, and further optionally a cleavable or incleavable linker or adapter, and one or more payloads comprising one or more modified nucleotides, optionally at least one phosphonic acid and / or ribose-modified nucleotide, which facilitate the direct or indirect linking of the antibodies or antibody fragments of one or more payloads by (i) an antibody or antibody fragment and (ii) a peptide mediating the payload.

[0010] In some specific embodiments, any of the aforementioned ARCs or ANCs optionally include an antibody or antibody fragment and / or a payload that is directly or indirectly conjugated to the antibody or antibody fragment via a reactive amine contained in a lysine residue on a peptide that links the antibody or antibody fragment to one or more payloads.

[0011] In some specific embodiments, any of the aforementioned ARCs or ANCs include an antibody or antibody fragment that binds to VISTA, preferably human VISTA.

[0012] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises an antibody or antibody fragment that binds to VISTA, preferably human VISTA, and includes the same VH and VL CDRs as any one of the anti-human VISTA antibodies having the sequences of Figure 14 or Appendix 1 or Appendix 3.

[0013] In some specific embodiments of any of the foregoing, the antibody or antibody fragment in the ARC or ANC includes an antibody or antibody fragment that binds to VISTA, preferably human VISTA, and has the same VH and / or VL region and CDR as any one of the anti-human VISTA antibodies containing the VH and / or VL sequence of Figure 14, or an antibody or antibody fragment that has at least 90, 95, or 99% sequence identity with the VH and / or VL region as any one of the anti-human VISTA antibodies containing the VH and / or VL sequence of Figure 14, or an antibody or antibody fragment that contains the VH and / or VL sequence of Appendix 1 or Appendix 3, wherein the antibody or antibody fragment optionally includes an IgG1, IgG2, IgG3, or IgG4 constant domain polypeptide, further optionally an IgG1 constant domain polypeptide, and even further optionally an IgG1 constant domain polypeptide having a sequence included in Appendix 1 or 3.

[0014] In some specific embodiments, the antibody or antibody fragment on the ANC or ARC comprises a human Fc region, optionally human IgG1, IgG2, IgG3, or IgG4, and is further optionally modified to impair complement and / or FcR binding and / or enhance FcRn binding.

[0015] In some specific embodiments, any of the aforementioned ARCs or ANCs include one or more of the following: small interfering RNA (siRNA), antisense oligonucleotides (ASOs), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heteronuclear RNA (hnRNA).

[0016] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises a polynucleotide molecule that is about 10 to about 1000, 10 to about 500, 10 to about 400, 10 to about 300, 10 to about 200, 10 to about 150, 10 to about 100, 10 to about 50, about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length, or comprises a polynucleotide molecule that is about 50 nucleotides, about 45 nucleotides, about 40 nucleotides, about 35 nucleotides, about 30 nucleotides, about 25 nucleotides, about 20 nucleotides, about 19 nucleotides, about 18 nucleotides, about 17 nucleotides, about 16 nucleotides, about 15 nucleotides, about 14 nucleotides, about 13 nucleotides, about 12 nucleotides, about 11 nucleotides, or about 10 nucleotides in length.

[0017] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises a first polynucleotide and a second polynucleotide, optionally, the first polynucleotide is a sense strand or a passenger strand, and / or the second polynucleotide is an antisense strand or a guide strand.

[0018] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises siRNA, ASO, tRNA, rRNA, or mRNA.

[0019] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises lipid nanoparticles, or is encapsulated therein, or is conjugated thereto.

[0020] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises at least one payload that targets an immunomodulatory substance selected from cytokines, chemokines, interleukins, interferons, tumor necrosis factors, or any of the aforementioned receptors.

[0021] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises a payload targeting an RNA or DNA sequence encoding an immunomodulatory substance selected from any of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-22, IL-37, IL-1β, TGF-β, IFNα, IFNβ, IFNγ, TNF-α, TNF-β, GM-CSF, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), retinoic acid-related orphan receptor C (RORC), or a molecule having the sequence specified in FIG. 1 or FIG. 2.

[0022] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises a siRNA payload targeting RNA or DNA encoding GLUT3 or PIK3CA, optionally comprising the sequences of Appendix 2 or 4.

[0023] In some specific embodiments, any of the aforementioned ARCs or ANCs comprises an antibody or antibody fragment that binds to at least one immune cell selected from PMBC, T cells, T cell progenitor cells, CD4+ T cells, helper T cells, regulatory T cells, CD8+ T cells, naive T cells, effector T cells, memory T cells, stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, cytotoxic T cells, mucosal associated invariant T (MAIT) cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, and a / b T cells, g / d T cells, natural killer T (NKT) cells, cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, perforin-deficient cells, granzyme-deficient cells, B cells, myeloid cells, monocytes, macrophages, eosinophils, neutrophils, and dendritic cells.

[0024] In some specific embodiments, any of the aforementioned ARCs or ANCs include an antibody or antibody fragment that binds to bone marrow cells and / or T cells.

[0025] In some specific embodiments, any of the aforementioned ARCs or ANCs include an antibody or antibody fragment that binds to T cells or T cell progenitor cells or NK cells.

[0026] In some specific embodiments, any of the aforementioned ARCs or ANCs are antigens selected from the group consisting of: (1) 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, Al adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Adresin, aFGF, ALCAM, ALK, ALK-1, ALK-7, Alpha-l-antitrypsin, Alpha-V / Beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, a v / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte-stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, Osteogeny BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7(OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB(ALK-6), BRK-2, RPK-1, BMPR-II(BRK-3), BMP, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C 3a, C4, C5, C5a, CIO, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13,CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CC R, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33( p67タンパクquality), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, C D56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-l, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL 4. CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR 2. CXCR3, CXCR4, CXCR5, CXCR6, DAN, DCC, DcR3, DC-SIGN, collapse-promoting factor, des(l-3)-IGF-I (IGF-1), Dhh, Dゴキシン, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A. Etron receptor,Enkephalinase, eNOS, Eot, Eotaxin I, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor Ila, Factor VII, Factor VIIIc, Factor IX, Fibroblast-activating protein (FAP), Fas, FcRl, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-Alpha-1, GFR-Alpha-2, GFR-Alpha-3, GITR, Glucagon, Glut4, Glycoprotein Ilb / IIIa (GP HIV Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1RIL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, Interferon (INF)-alpha, INF-beta, INF-gamma, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin alpha 2, Integrin alpha 3, Integrin alpha 4, Integrin alpha 4 / beta 1, Integrin, Alpha 4 / beta 7, Integrin alpha 5 (Alpha V ), Integrin Alpha 5 / Beta 1, Integrin Alpha 5 / Beta 3, Integrin Alpha 6, Integrin Beta 1, Integrin Beta 2, Interferon Gamma, IP-10, 1-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein LI, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP(TGF-1), Latent TGF-1, Latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MH C(HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Mucl), MUC18, Müllerian duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, Neprilysin, Neurotrophin-3, 4,Alternatively, -6, Neurturin, Nerve Growth Factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDK-1, PECAM, PEM, PF 4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, Placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, Prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, Relaxin A chain, Relaxin B chain, Renin, Respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid Factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, Serum Albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (Tumor-Associated Glycoprotein-72), TARC, TCA-3, T Cell Receptor (e.g., T Cell Receptor Alpha / Beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, Testicular PLAP-like Alkaline Phosphatase -ze, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta panspecific, TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRIIb, TGF-betaRIIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT,TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSFllB(OPG OCIF、TR1)、TNFRSF12(TWEAK R). FN14) TNFRSF13B(TACI) TNFRSF13 C(BAFF R)、TNFRSF14(WHO ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR) TNFRSF19(TROY). CROWN、TRADE)、TNFRSF19L(RELT)、TNFRSFIA(TNF RI CD120a、p55-60)、TNFRSFIB(TNF RII CD120b、p75-80)、TNFRSF26(TNFRH3) TNFRSF3(LTbR TNF RIII) TNFC R), TNFRSF4(OX40 ACT35, TXGP1 R), TNFRSF 5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3). M68, TR6, TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB CD137, ILA), TNFRSF21(DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23(DcTRAIL Rl). TNFRH1) , TNFRSF25 ( DR3 Apo - 3 , LARD , TR - 3 , TRAMP , WSL - 1 ) , TNFSF10 ( TRAIL Apo-2 polymer TL2) TNFSF11(TRANCE / RANK dimer ODF) TNFSF12(TWEAK). Apo-3リンド, DR3リンド, TNFSF13(APRIL TALL2), TNFSF13B(BAFF LIGHT, TALL1, THANK, TNFSF20), TNFSF14(LIGHT HVEM diuretic LTg), TNFSF15(TLIA / VEGI), TNFSF18 (GITR diuretic AITR diuretic, TL6), TNFSFIA (TNF-a Links DIF, TNFSF2, TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), and TNFSF4 (OX40). gp34, TXGP1, TNFSF5(CD40-linked CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6(Fas-linked). Apo-1 factor, APT1 factor, TNFSF7 (CD27 factor CD70), TNFSF8 (CD30 factor CD153).TNFSF9 (4-1BB ligand, CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfering receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3 (T cell immunoglobulin and mucin protein-3), and hormone receptors; or (2) antigens selected from the group consisting of the following: BCMA, CTLA4 (cytotoxic T lymphocyte antigen-4) ), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene-3), TIM-3, CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIa (CD16), FcyRIIa (CD32a), FcyRIIb (CD32b), FcyRI (CD64), Toll-like receptor (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFβ, cytokine receptor, IL-2R, chemokine, chemokine receptor, growth factor, VEGF, and HGF; or (3) antigens selected from the following: CD1a, b, c, d; CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10,CD11a、b、c、d;CDw12、CD13、CD14、oCD15、CD15s、CD15u、CD16、CDw17、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD31、CD32、CD33、CD34、CD35、CD36、CD37、CD38、CD39、CD40、CD41、CD42a、b、c、d;CD43、CD44、CD45、CD45RO、CD45RA、CD45RB、CD46、CD47、CD48、CD49a、CD49b、CD49c、CD49d、CD49e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55m、CD56、CD57、CD58、CD59、CD60a、CD60b、CD61、CD61E、CD62L、CD62P、CD63、CD64、CD65、CD66a、CD66b、CD66c、CD66d、CD66e、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD75s、CD77、CD78、CD79α、β、CD80、CD81、CD82、CD83、CDw84、CD85、CD86、CD87、CD88、CD89、CD90、CD91、CD92、Cd92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD108、CD109、CD110、CD111、CD112、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CDw121b、CD122、CD123、CD124、CD125、CD126、CD127、CDw128、CD129、CD130、CDw131、CD132、CD133、CD134、CD135、CDw136、CDw137、CD138、CD139、CD140a、b、CD141、CD142、CD143、CD144、CD145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156b、CD157、CD158、CD158a、CD159a、CD160、CD161、CD162、CD162R、CD163、CD164, CD165, CD166, CD167a, CD168, CD169, CD170, CD171, CD172a, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD 183, CD184, CD195, CDw197, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CDw210, CD212, CD213a1, CD213a2, CDw217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, etc.; or (4) any of the following: IL4ra, TNFa, BTK, RORgt, PIK3CA, JAK1, JAK3, TYK2 The material comprises a gene or nucleic acid encoding Glut1, Glut3, TAP1, CIITA, cGAS, IRF5, STAT3, STAT6, TAK1 (MAP3K7), HPK1; or any of the following: SOCS1, CD39, Cbl, or PTPN22; or (5) any of the following: Glut1, PI3K, BTK, TNF, or RORC; or (6) any of the following: PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, LAG-3, HHLA2, TNFRSF12A, HLA-G, NECTIN2, TNFRSF25, TNFSF14, LAIR1, TNFSF15, TNFSF4, KIR2DL4, PDCD1, LGALS9, and PVR; at least one nucleic acid payload that optionally binds to RNA or DNA; optionally RNA or DNA; and optionally siRNA or antisense RNA.

[0027] In some specific embodiments, any of the aforementioned ARCs or ANCs may include a nucleic acid payload having a payload containing a sequence selected from those listed in Appendix 2 or Appendix 4, optionally RNA, and optionally siRNA or antisense RNA, or an INX-201 ARC selected from those listed in Appendix 2, or an INX-201 ARC selected from those containing an amino acid sequence and a payload sequence listed in Appendix 4.

[0028] In some specific embodiments, any of the aforementioned ARCs or ANCs include at least two different RNA payloads that target the same or different immunomodulatory genes or mRNAs, optionally, the immunotargets disclosed above.

[0029] In some specific embodiments of any of the aforementioned ARC or ANCs, a nucleic acid, optionally an RNA payload, is linked to the antibody or antibody fragment via a cleavable or incleavable linker.

[0030] In some specific embodiments, any of the aforementioned ARCs or ANCs are used to deliver one or more gene-editing nucleic acids (e.g., CRISPR guide RNA (gRNA or sgRNA)) and optionally a CRISPR-related endonuclease or a nucleic acid encoding a CRISPR-related endonuclease.

[0031] In some specific embodiments, any of the aforementioned ARC or ANC includes a PD of at least 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks or more.

[0032] In some specific embodiments, none of the aforementioned ARCs or ANCs induce toxicity that is recognizable to non-target cells.

[0033] In some specific embodiments, the present invention provides compositions comprising any of the aforementioned ARCs or ANCs and a pharmaceutically acceptable carrier or excipient, wherein the ARC or ANC is optionally contained in or on lipid nanoparticles.

[0034] In some specific embodiments, the present invention provides a method of treatment or prevention comprising administering any of the aforementioned ARCs or ANCs, or compositions containing them, to a subject requiring them.

[0035] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of a tumorous state, a proliferative state, a neurodegenerative state, a neuroinflammatory state, an infectious state, an autoimmune state, an allergic state, or an inflammatory state or a pathological condition related to any of the said.

[0036] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions comprising them, to subjects in need for the treatment or prevention of autoimmune diseases, such as bone marrow or T cell-related diseases.

[0037] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of neoplastic diseases, proliferative diseases, neurodegenerative diseases, neuroinflammatory diseases, infectious diseases, autoimmune diseases, or inflammatory diseases, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0038] In some embodiments, the present invention relates to acromegaly, acquired aplastic anemia, acquired hemophilia, agammaglobulinemia, primary alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) / fulminant antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic neuropathy (AAG) / autonomic neuropathy / autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis / acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, autoimmune hemolytic anemia (AIHA), and autoimmune Hepatitis epidemic (AIH), autoimmune hyperlipidemia, autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), polyglandular autoimmune syndrome, types I, II, and III (APS1, APS2, APS3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Baro's disease, Behçet's disease, scatter chorioretinopathy / scatter uveitis, bullous pemphigoid, Castleman disease Diseases such as celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome / regional systemic sclerosis, Crohn's disease (CD), Cronchite-Canada syndrome (CSS), idiopathic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, and uterine cancer. Erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome / anti-GBM / anti-TBM disease, granulomatosis with polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's disease / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schöne purpura / IgA vasculitis, hidradenitis suppurativa, Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia,IgA nephropathy / Berger's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, inclusion body myositis, IgG4-related sclerosis (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult Still's disease, juvenile polymyositis|juvenile dermatomyositis|juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenia dysthesia syndrome (LEMS), leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD)|linear IgA bullous dermatosis (LABD), lupus nephritis, Lyme disease / chronic Lyme disease / Post-treatment Lyme disease syndrome (PTLDS), lymphocytic colitis / microscopic colitis, lymphocytic hypophysitis / autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mollen's ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devic's disease, ocular scarring pemphigoid, ocular clonus-myoclonus syndrome (OMS), relapsing rheumatoid arthritis, paraneoplastic syndrome Cerebellar degeneration, paraneoplastic pemphigus, Parry-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis, PANS / PANDAS, Personage-Turner syndrome, bullous pemphigoid of pregnancy / herpes gestationis, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, orthostatic tachycardia syndrome (POTS), primary biliary cirrhosis (PBC) / primary biliary cholangitis, primary sclerosing cholangitis (PSC), psoriasis Palmoplantar pustulosis, psoriatic arthritis, idiopathic pulmonary fibrosis (IPF), pure red cell aneurysm (PRCA), pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud's disease / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy syndrome (RSD) / complex regional pain syndrome (CRPS), relapsing polychondritis, restless legs syndrome (RLS) / Willis-Ekbom disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, scleritis, scleroderma, sclerosing mesentericitis / mesenteral panniculitis, crawling choroidopathy, Sjögren's syndrome,This relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need of them, for the treatment or prevention of autoimmune diseases selected from one or more of the following: Generalized Rigidity Syndrome (SPS), Small Diameter Fiber Sensory Neuropathy, Systemic Lupus Erythematosus (SLE), Subacute Bacterial Endocarditis (SBE), Subacute Cutaneous Lupus Erythematosus, Suzak Syndrome, Sydenham's Chorea, Sympathetic Ophthalmitis, Takayasu's Arteritis (Vasculitis), Testicular Autoimmune Disease (Vasculitis, Orchitis), Tolosa-Hunt Syndrome, Transverse Myelitis (TM), Tubulointerstitial Nephritis-Uveitis Syndrome (TINU), Ulcerative Colitis (UC), Undifferentiated Connective Tissue Disease (UCTD), Uveitis | Anterior / Intermediate / Posterior, Vasculitis, VEXAS Syndrome, Vitiligo, and Vogt-Koyanagi-Harada Syndrome (VKH), and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0039] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of Addison's disease, arthritis, celiac disease, lupus, Graves' disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto's disease, Sjögren's disease, asthma, type 2 diabetes, and autoimmune type 1 diabetes, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0040] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of inflammatory diseases selected from the group consisting of fatty liver disease, endometriosis, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), asthma, rheumatoid arthritis, obesity, fibromyalgia, lupus, SLE, osteoarthritis, rheumatoid arthritis, shingles, herpes zoster, and vasculitis, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0041] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of neurodegenerative or neuroinflammatory diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, aphasia, Parkinson's disease, or spinal muscular atrophy, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0042] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of cancer, or for the prevention of cancer recurrence, and / or for the suppression of at least one pathological symptom associated with a particular immune cell type.

[0043] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of solid tumors and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0044] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of hematological malignancies and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0045] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of recurrent or refractory cancer, or metastatic cancer, optionally recurrent or refractory solid tumors, or metastatic solid tumors, recurrent or refractory hematological malignancies, or metastatic hematological malignancies.

[0046] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of solid tumors selected from anal cancer, appendiceal cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumors, breast cancer, cervical cancer, colon cancer, cancer of unknown primary origin (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumors, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0047] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of hematological malignancies, optionally leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some examples, hematological malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström hypergammaglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt hyper This includes preventing or suppressing at least one pathological condition of malignant grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prelymphoblastic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoplasmic myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis and / or associated therewith.

[0048] In some embodiments, the present invention relates to chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström hypergammaglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, and primary mediastinal B-cell lymphoma. This relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need of them, for the treatment or prophylaxis of hematological malignancies selected from PMBLs, immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prelymphoblastic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoplasm, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis. In some cases, the treatment or prophylaxis of hematological malignancies is to prevent or suppress relapsed or refractory hematological malignancies, or metastatic hematological malignancies and / or at least one pathological condition associated therewith.

[0049] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions containing them, to subjects in need for the treatment or prevention of autoimmune diseases selected from the group consisting of Addison's disease, arthritis, celiac disease, lupus, Graves' disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto's disease, Sjögren's disease, asthma, type 2 diabetes mellitus, and autoimmune type 1 diabetes mellitus, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

[0050] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions comprising them, to subjects in need for the treatment of cancer, wherein the ARC or ANC comprises nucleic acids that modulate or inhibit the expression of any of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, LAG-3, HHLA2, TNFRSF12A, HLA-G, NECTIN2, TNFRSF25, TNFSF14, LAIR1, TNFSF15, TNFSF4, KIR2DL4, PDCD1, LGALS9, VSIr(VISTA), and PVR, optionally comprising ASOs or siRNAs.

[0051] In some embodiments, the present invention relates to the administration of any of the aforementioned ARCs or ANCs, or compositions comprising them, to subjects in need for the suppression or treatment of disease- or age-related immunosenescence, wherein the ARC or ANC optionally comprises nucleic acids that modulate or inhibit the expression of any of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, LAG-3, HHLA2, TNFRSF12A, HLA-G, NECTIN2, TNFRSF25, TNFSF14, LAIR1, TNFSF15, TNFSF4, KIR2DL4, PDCD1, LGALS9, VSIr(VISTA), and PVR, and further optionally comprises ASOs or siRNAs. [Brief explanation of the drawing]

[0052] [Figure 1] This invention includes a list of immunologically relevant siRNA and ASO payloads that have been included in and tested. In proof-of-concept experiments, K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and targeted knockdown was analyzed by qRTPCR using the ddct method and reported as a factor relative to the control. Scrambled siRNA or ASO controls were also ordered from the IDT. Payloads marked with (*) were selected for conjugation to the delivery vehicle (anti-VISTA Mab INX201). [Figure 2]This report includes a list of other immunologically relevant siRNA and ASO payloads identified and tested. K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and targeted knockdown was analyzed by qRTPCR using the ddct method and reported as a factor relative to the control. Scrambled siRNA or ASO controls were also ordered from the IDT. Payloads marked with (*) were selected for conjugation to the delivery vehicle (anti-VISTA Mab INX201). This report describes only the CD39 ARC from the second round of payloads; conjugation of other ARCs is planned / in progress. [Figure 3] This report includes SDS-PAGE results for an exemplary ARC (INX201 ARC) according to the present invention, confirming effective RNA conjugation. INX201 ARC was isolated by reduced SDS-PAGE and subsequent silver staining. Lane labels: 201 - free INX201 anti-VISTA Mab; CD45 - ARC containing CD45 siRNA; SOCS1 - ARC containing SOCS1 ASO; distinct patterns of multiple conjugates were observed, confirming efficient conjugation. Based on payload molecular weight (approximately 17 vs. approximately 6 kDa), HC or LC shifts due to siRNA are more significant than those due to ASO. [Figure 4] This invention demonstrates that exemplary ARC efficiently binds to K562-VISTA cells, providing effective intrinsic translocation from the surface and intracellular retention of siRNA within K562-VISTA cells. Free INX201 (circled line) or eGFP ARC (squared line) was used at 200 nM. A) Time course of antibody binding and internalization. B) RNA accumulation assay measured by Cy5. MFI - mean fluorescence intensity, representative of two independent experiments shown. [Figure 5]This study demonstrates that the knockdown protein expression of an exemplary ARC (INX201 ARC) according to the present invention is equivalent to in vitro transfection. K562-VISTA WT cells or an eGFP+ cell pool were used in this study. A) Cells were treated for 28 hours with no drug (left bar) or with 200 nM eGFP ARC (right bar). The highest level of eGFP protein knockdown was established by transfecting the same cells with eGFP siRNA (approximately 50%, measured at 24 hours). ARC-mediated knockdown was similar to the maximum possible knockdown level (based on siRNA sequence). B) The dashed line represents the expected highest level of CD45 protein knockdown based on the potency of the payload (siRNA), which is 50% when measured from free transfected CD45 siRNA at 48 hours (right bar). Cells were treated for 72 hours with 200 nM eGFP ARC (center bar) or no drug (left bar). C) CD45 levels measured in repeated experiments. Cells were treated for 72 hours with no drug (left bar), free siRNA, no transfection (center bar), and 200 nM ARC (right bar). [Figure 6] This study demonstrates that an exemplary ARC (INX201 ARC) according to the present invention inhibits TNFα from PBMCs. Human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated with TNFα ARC (0-200 nM) or free RNA (200-1000 nM) for 48 hours (LPS) or 72 hours (beads). TNFα levels were efficiently reduced in a dose-dependent manner with ARC, but not with free siRNA. C) Efficient targeted knockdown was confirmed by qRTPCR performed on ARC-treated versus untreated PBMCs recovered at 72 hours. Levels without ARC are visualized as 0.1 nM (due to the logarithmic scale). [Figure 7]This study demonstrates that an exemplary ARC (INX201 ARC) according to the present invention slows T cell proliferation. Human PBMCs were activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated for 72 hours with either (A) TNFα ARC (0-200 nM) or free RNA (200-1000 nM) or (B) PI3K ARC (0-200 nM) or free RNA (200-1000 nM). ARC efficiently and dose-dependently reduced newly proliferating T cells, while free siRNA did not. Proliferation was analyzed by Cell Trace Violet dilution and visualized by flow cytometry. No ARC was visualized as 0.1 nM (due to the logarithmic scale). Curves were generated from three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). A single technical replication was used for each concentration point. [Figure 8] This study demonstrates that an exemplary ARC according to the present invention reduced PBMC activation (INX201-BTK ARC). Human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads (beads to T cell ratio 1:2) and treated with BTK ARC (0-200 nM, triangle) or free RNA (200-1000 nM, star) or free INX201 (square) for 48 hours (LPS) or 72 hours (beads). Curves were generated from a three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). CD69 levels were measured in A and newly proliferating cell percentage in B, with a single technical replication used for each concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale). Unstimulated-unstimulated cells; MFI-mean fluorescence intensity. [Figure 9] This example demonstrates that an exemplary ARC according to the present invention reduced cytokine production (INX201-Glut1 ARC). Purified human T cells were activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated with Glut1 ARC (0-200 nM) for 72 hours. A single technical replica was used for each concentration point. Two human donors were tested: Donor 1 - solid line, Donor 2 - dashed line. No ARC was visualized as 0.1 nM (due to logarithmic scale). A) IFNg and B) IL17A were measured by Luminex. [Figure 10] This example demonstrates that an exemplary ARC according to the present invention reduced cytokine production (INX201-Glut1 ARC). Purified human T cells were activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated with RORC ARC (0-200 nM) for 72 hours. A single technical replica was used for each concentration point. Two human donors were tested: Donor 1 - solid line, Donor 2 - dashed line. No ARC was visualized as 0.1 nM (due to the logarithmic scale). A) IFNg, B) IL-6, and C) IL12p40 were measured by Luminex. [Figure 11] This invention demonstrates that an exemplary ARC (INX201-CD39 ASO) successfully targets human PBMCs and enhances the immune response in human PBMCs. Human PBMCs were activated with anti-CD3 / CD28 beads and treated with CD39 ARC (0-200 nM, triangular) or free INX201 (0-200 nM, circular) for 72 hours. Curves were generated from three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). A single technical replica was used for each concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale). [Figure 12] This paper outlines how the ARC platform of the present invention can be optimized by using anti-VISTA Fab instead of Mab. A) Comparison of binding of INX201 Mab (square) and INX201 Fab (circle) to human VISTA ECD by ELISA. B) K562-VISTA cell-based competitive assay. Here, increasing concentrations of INX201 Mab (square) or INX201 Fab (circle) before binding blocked available VISTA on the cell surface (hence the decrease in VISTA MFI measurements by INX201-AF488). Curves were generated from a three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). Technical replication with n=2 per concentration point was used. Ab-free was visualized as 0.0001 nM (due to the logarithmic scale). [Figure 13]This study demonstrates that exemplary ARC according to the present invention does not affect T cell viability. Human PBMCs were activated with anti-CD3 / CD28 beads and treated with PI3K ARC (0-200 nM, triangular) or free INX201 (0-200 nM, square) for 72 hours. Curves were generated from 3-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). A single technical replica was used for each concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale). Unstimulated-unstimulated cells. [Figure 14] The CDR and variable sequence of an exemplary anti-human VISTA antibody that may be used in ARC or ANC according to the present invention are shown. [Figure 15] This study demonstrates that INX201 (anti-VISTA) PI3K ARC specifically knocks down PI3K expression, while isotype control IgG1 PI3K ARC does not. K562-VISTA cells were transfected with 40–200 nM and treated with 40–200 nM INX201-PI3K ARC or isotype control IgG1-PI3K ARC. Targeted knockdown was analyzed by qRTPCR using ddct and reported as target suppression %. Here, 0% suppression was the INX201-only sample. PI3K ARC: anti-human VISTA mAb conjugated with PI3K siRNA; PI3K isotype ARC: IgG control conjugated with PI3K siRNA; INX201: unconjugated mAb: naked anti-human VISTA mAb. Single technical replication was used per concentration point. [Figure 16]This study demonstrates the in vitro functionality of human PI3K and GLUT3 ARC. Human PBMCs were activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) for 72 hours and treated with (A) PI3K ARC or free INX201 (0-200 nM), or (B) GLUT3 ARC or free INX201 (0-200 nM). Curves were generated from three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism10). IL5 / IL13 / TNFa / IL17F cytokine levels were measured by Luminex. CD69 levels were measured in live CD45+ / HLA-DR- / CD56- / CD3+ / CD4+ / CD45RA- / CD27-T effector memory cells, and CD25 levels were measured in live CD45+ / HLA-DR- / CD56- / CD3 cells. In A, a single technical replica was used for each concentration point. In B, n=2 technical replicas were used per concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale). MFI - mean fluorescence intensity. Data are expressed as mean ± SEM, as needed. [Figure 17] This study demonstrates that PI3K ARC reduces the inflammatory cytokine response in heterologous GvHD. NSG mice were intravenously injected with 5 mg / kg of either PI3K ARC (triangle, n=6) or INX201 (circle, n=6) along with human PBMC transfer. Eighteen hours after injection, the mice were stimulated intraperitoneally with 0.5 mg / kg of LPS. A) Heterologous GvHD LPS-stimulated model. A schematic diagram of the experiment is shown. B) Changes in plasma human cytokine levels at day 7 (IFNg) or 4 hours (IL6, TNFa). Statistical analysis was performed by Student's t-test. Data are expressed as mean ± SEM. *-p<0.05;**-p<0.01. C) Heatmap based on Z-scores for cytokine levels 4 hours after LPS stimulation (n=6 per group). [Figure 18]This study demonstrates that PI3K ARC reduces LPS-induced T cell proliferation in vivo. NSG mice were intravenously injected with either PI3K ARC (triangle, n=6) or INX201 (circle, n=6) at 5 mg / kg, along with human PBMC transfer. Eighteen hours after injection, the mice were stimulated intraperitoneally with 0.5 mg / kg of LPS. Changes in serum T cell counts (n=6 mice per group) at day 14 are shown for both CD4 and CD8. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± SEM. *-p<0.05. [Figure 19] This study demonstrates that PI3K ARC does not reduce the percentage of regulatory T cells in vivo. NSG mice were intravenously injected with 5 mg / kg of either PI3K ARC (triangle, n=10) or INX201 (circle, n=8) along with human PBMC transfer (right graph). In each group of mice, 18 hours after PBMC injection, the mice were stimulated intraperitoneally with 0.5 mg / kg of LPS (n=6 for both the INX201 and PI3K ARC groups, left graph). Blood samples were processed on day 28. The graph shows the percentage change in Treg cells. Statistical analysis was performed by Student's t-test. Data are expressed as mean ± SEM. ns - not significant. [Figure 20] This study demonstrates that GLUT3 ARC reduces the inflammatory cytokine response in heterologous GvHD. NSG mice were intravenously injected with PI3K ARC (triangle, n=10) or INX201 (circle, n=8) at 5 mg / kg along with human PBMC transfer. Changes in plasma human cytokine levels are shown on day 7. Statistical analysis was performed by Student's t-test. Data are expressed as mean ± SEM. **-p<0.01;****-p<0.0001. [Modes for carrying out the invention]

[0053] The present invention, as disclosed herein, relates to anti-VISTA antibody oligonucleotide conjugates (ARC or ANC) that specifically deliver RNA to immune cells, and to their use as therapeutic agents for treating, for example, autoimmune and inflammatory conditions.

[0054] definition The following are definitions of various terms used to describe this disclosure. These definitions apply to the terms used throughout this specification and the claims, either individually or as part of a larger group, unless otherwise specified.

[0055] The term “approximately” will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. When used herein to refer to a measurable value such as quantity or duration, the term “approximately” means to include a variation of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from a particular value, such variation being appropriate for carrying out the method of this disclosure.

[0056] In this specification, the terms “antibody-RNA conjugate” or “antibody-nucleic acid conjugate” or “ARC” or “ANC” generally refer to a conjugate comprising (i) an antibody or antibody fragment (e.g., Fab) that specifically or preferentially binds to one or more target immune cell species, and (ii) one or more nucleic acids, generally oligonucleotides, e.g., DNA or RNA, which may consist of wild-type or modified nucleotides, and which specifically target immunomodulatory genes or RNA encoded by them, thereby modulating the expression and / or activity of immunomodulatory proteins encoded by them. In preferred embodiments, the antibody or antibody fragment is an endogenous antibody, i.e., when it binds to a target antigen on a target immune cell, it internalizes into the immune cell and delivers the oligonucleotide cargo contained in the ARC or ANC into the immune cell. In exemplary embodiments, the antibody or antibody fragment is an endogenous antibody or antibody fragment that specifically binds to VISTA, preferably human VISTA.

[0057] The term "alkyl" refers, in certain embodiments, to saturated, linear, or branched hydrocarbon moieties containing between 1 and 6 or 1 and 8 carbon atoms. Examples of Ci~6-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, <<-butyl, tert-butyl, neopentyl, and n-hexyl moieties. Examples of Ci-s-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, <<-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

[0058] The number of carbon atoms in an alkyl substituent is "C x~y This can be indicated by the prefix "x", where x is the minimum number of carbon atoms in the substituent and y is the maximum number. Similarly, C x The term "chain" refers to an alkyl chain containing x carbon atoms.

[0059] The term "heteroalkyl," either by itself or in combination with other terms, means, unless otherwise specified, a stable linear or branched alkyl group consisting of a specified number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, and S, wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) may be positioned at any position of the heteroalkyl group, including between the remainder of the heteroalkyl group and the fragment bonded to it, and may be bonded to the most distal carbon atom of the heteroalkyl group. Examples include -0-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-H-CH3, -CH2-S-CH2-CH3, and -CH2-CH2-S(=0)-CH3. For example, up to two heteroatoms may be consecutive, such as -CH2-H-OCH or -CH2-CH2-SS-CH.

[0060] When used alone or in combination with other terms, the term “aryl” means a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), unless otherwise indicated, where such rings may be linked together in a pendant-like manner, such as biphenyl, or condensed, such as naphthalene. Examples of aryl groups include phenyl, anthrasyl, and naphthyl. In various embodiments, examples of aryl groups include phenyl (e.g., C6 aryl) and biphenyl (e.g., Ci2 aryl). In some embodiments, the aryl group has between 6 and 16 carbon atoms. In some embodiments, the aryl group has between 6 and 12 carbon atoms (e.g., C6-i2 aryl). In some embodiments, the aryl group has 6 carbon atoms (e.g., Ce aryl).

[0061] As used herein, the terms “heteroaryl” or “heterocyclic aromatic” refer to a heterocycle having aromatic characteristics. Heteroaryl substituents can be defined by the number of carbon atoms; for example, Ci~9 heteroaryl indicates the number of carbon atoms in the heteroaryl group, not the number of heteroatoms. For example, Ci~9 heteroaryl may contain one to four additional heteroatoms. Polycyclic heteroaryls may contain one or more rings that are partially saturated. Non-limiting examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (e.g., including 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (e.g., including 2-pyrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (e.g., including 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

[0062] Non-limiting examples of polycyclic heterocyclic and heteroaryl compounds include indolyl (e.g., including 3-, 4-, 5-, 6-, and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (e.g., including 1- and 5-isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, synnolinyl, quinoxalinyl (e.g., including 2- and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthilidinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthilidinyl, benzofuryl (e.g., 3-, 4-, 5-) Examples include 2,3-dihydrobenzofuryl (including -, 6-, and 7-benzofuryl), 1,2-benzisoxazolyl, benzothienyl (e.g., including 3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl (e.g., including 2-benzothiazolyl and 5-benzothiazolyl), prinyl, benzimidazolyl (e.g., including 2-enzimidazolyl), benzotriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0063] The term “protecting group” or “chemical protecting group” refers to a chemical moiety that blocks some or all of the reactive moieties of a compound, preventing such moieties from participating in a chemical reaction until the protecting group is removed. For example, such moieties are listed and described in TW Greene, PGMWuts, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons (1999). When different protecting groups are used, it may be advantageous that each (different) protecting group is removable by different means. Protecting groups that are cleaved under completely different reaction conditions allow for differential removal of such protecting groups. For example, protecting groups may be removed by acid, base, and hydrolysis. Groups such as trityl, monomethoxytrityl, dimethoxytrityl, acetal, and tert-butyldimethylsilyl can be used to protect carboxy and hydroxy reactive moieties in the presence of an amino group protected by an acid-unstable and hydrolytically removable Cbz group, and a base-unstable Fmoc group. The carboxylic acid moiety may be blocked by a base-unstable group such as methyl or ethyl, but is not limited to the following; the hydroxy-reactive moiety may be blocked by an acid-unstable group such as tert-butyl carbamate, or by a base-unstable group such as acetyl in the presence of an amine that is stable to both acids and bases but blocked by a carbamate that can be removed by hydrolysis.

[0064] The carboxylic acid and hydroxyl reactive moieties may also be blocked by protecting groups that can be removed by hydrogenolysis, such as benzyl groups, while the amine group may be blocked by a base-unstable group such as Fmoc. A particularly useful amine protecting group is trifluoroacetamide. The carboxylic acid reactive moiety may also be blocked by protecting groups that can be removed by oxidation, such as 2,4-dimethoxybenzyl, while the coexisting amino group may be blocked by a fluoride-unstable silyl carbamate.

[0065] Allyl blocking groups are useful in the presence of acid and base protecting groups because the former are stable and can be later removed by metal or π-acid catalysts. For example, allyl-blocked carboxylic acids can be deprotected by palladium(O) catalytic reactions in the presence of acid-unstable t-butyl carbamate or base-unstable amine acetate protecting groups. Yet another form of protecting group is a resin to which a compound or intermediate can be bound. As long as the residue is bound to the resin, its functional group is blocked and cannot react. Once released from the resin, the functional group becomes reactive.

[0066] The terms “nucleic acid base,” “base pairing moiety,” “nucleic acid base pairing moiety,” or “base” refer to the heterocyclic portion of a nucleoside, nucleotide, and / or morpholino subunit. Nucleic acid bases may be naturally occurring, modified, or analogues of these naturally occurring nucleic acid bases, for example, one or more nitrogen atoms of a nucleic acid base may be independently substituted by carbon in each occurrence. Exemplary analogues include hypoxanthine (the base component of the nucleoside inosine), 2,6-diaminopurine, 5-methylcytosine, C5-propynyl-modified pyrimidine, and 10-(9-(aminoethoxy)phenoxadinyl) (G-clamp).

[0067] Further examples of base-pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil, in which each amino group is protected by an acyl protecting group, as well as other modified nucleic acid bases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being natural degradation products). Modified nucleic acid bases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al., Nucleic Acids Research, 1994, 22, 2183-2196, and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, whose contents are incorporated herein by reference, are also conceivable.

[0068] Further examples of base pairing moieties include, but are not limited to, enlarged nucleic acid bases to which one or more benzene rings are added. Nucleic acid base substitutions described in the Glen Research catalog (www.glenresearch.com), whose contents are incorporated herein by reference, are expected to be useful in the synthesis of the oligomers described herein. (Krueger AT et al, Acc. Chem. Res., 2007, 40, 141-150; Kool, ET, Acc. Chem. Res., 2002, 35, 936-943; Benner SA, et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, FE, et al, Curr. Opin. Chem. Biol, 2003, 7, 723-733; Hirao, L, Curr. Opin. Chem. Biol, 2006, 10, 622-627)

[0069] The term “oligonucleotide” or “oligomer” refers to a compound comprising multiple linked nucleosides, nucleotides, or combinations of both nucleosides and nucleotides. In the specific embodiments provided herein, the oligonucleotide is a morpholino oligonucleotide.

[0070] The term "morpholino oligonucleotide" or "PMO" refers to a modified oligonucleotide having morpholino subunits linked together by a phosphoramidate or phosphorodiamidate bond, in which the morpholino nitrogen of one subunit is linked to the 5'-external carbon of an adjacent subunit. Each morpholino subunit contains a nucleic acid base pairing moiety that is effective for binding to nucleic acid bases in a target via nucleic acid base-specific hydrogen bonds.

[0071] The terms “antisense oligomer,” “antisense compound,” and “antisense oligonucleotide,” or “ASO,” are used synonymously and refer to a sequence of subunits having a base-pairing portion linked by an inter-subunit bond, which enables the base-pairing portion to hybridize to a target sequence in a nucleic acid (generally RNA) via Watson-Crick base pairing, thereby forming a nucleic acid:oligomer heteroduplex within the target sequence. The oligomer may have exact (complete) or near (sufficient) sequence complementarity with respect to the target sequence. Sequence variations near the end of the oligomer are generally preferred over internal variations.

[0072] Such antisense oligomers may be designed to block or inhibit mRNA translation, or to inhibit / modify natural or abnormal premRNA splice processing, and can be said to be “directed to” or “targeted against” a target sequence into which they hybridize. The target sequence is generally the AUG start codon of mRNA, a translation repression oligomer, or a splice site, splice repression oligomer (SSO) region of pre-processing mRNA. A splice site target sequence may include an mRNA sequence having 1 to about 25 base pairs at the 5' end downstream of a normal splice acceptor junction in the pre-processing mRNA. In various embodiments, the target sequence may be any region of pre-processing mRNA that includes a splice site, is entirely contained within an exon coding sequence, or extends to a splice acceptor or donor site. More generally, when an oligomer is targeted against a target nucleic acid as described above, it is said to be “targeted against” a biologically relevant target such as a protein, virus, or bacterium.

[0073] Antisense oligonucleotides and target RNAs are complementary if a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen-bond to each other, thereby resulting in a stable and specific binding between the oligonucleotide and the target. Therefore, "specifically hybridizable" and "complementary" are terms used to indicate a sufficient degree of complementarity or precise pairing to result in a stable and specific binding between the oligonucleotide and the target. It is understood in the art that the sequence of an oligonucleotide does not need to be 100% complementary to the sequence of its target nucleic acid in order to be specifically hybridizable. If the binding of the oligonucleotide to the target molecule interferes with the normal function of the target RNA, the oligonucleotide is specifically hybridizable and has a sufficient degree of complementarity to avoid nonspecific binding of the antisense oligonucleotide to non-target sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic procedures, and under the conditions under which the assay is performed in the case of in vitro assays.

[0074] Oligonucleotides may also include modifications or substitutions of nucleic acid bases (often simply referred to as "bases" in the art). Oligonucleotides containing modified or substituted bases include oligonucleotides in which one or more of the most commonly found purine or pyrimidine bases in nucleic acids are substituted with less common or unnatural bases. In some embodiments, the nucleic acid base is covalently bonded to the morpholine ring of the nucleotide or nucleoside at the N9 atom of the purine base or at the N1 atom of the pyrimidine base.

[0075] Purine bases contain a pyrimidine ring fused to an imidazole ring. Adenine and guanine are the two most commonly found purine nucleic acid bases in nucleic acids. These may be substituted with other naturally occurring purines, including, but not limited to, N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0076] Pyrimidine bases contain a six-membered pyrimidine ring. Cytosine, uracil, and thymine are the most commonly found pyrimidine bases in nucleic acids. These may be substituted with other naturally occurring pyrimidines, including, but not limited to, 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain a thymine base instead of uracil.

[0077] Other modifications or substitutions of bases include 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, SuperT), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-dia Examples include, but are not limited to, minoprin, SuperG, SuperA, and N4-ethylcytosine or its derivatives; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil or its derivatives; and degenerate or universal base or base deletion such as 2,6-difluorotoluene (e.g., 1-deoxyribose, 1,2-dideoxyribose, l-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen is substituted with nitrogen (azalibose)). Pseudouracil is an isomerized version of naturally occurring uracil and has a C-glycoside instead of the usual N-glycoside like uridine.

[0078] Certain modified or substituted nucleic acid bases are reported to be particularly useful in increasing the binding affinity of antisense oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. In various embodiments, the nucleic acid bases may include 5-methylcytosine substitutions, which have been shown to increase the stability of nucleic acid double strands by 0.6–1.2°C.

[0079] Modified or substituted nucleic acid bases are also useful in facilitating the purification of antisense oligonucleotides. For example, an antisense oligonucleotide may contain three or more consecutive guanine bases (e.g., 3, 4, 5, 6, or more). In certain antisense oligonucleotides, a sequence of three or more consecutive guanine bases may lead to oligonucleotide aggregation, complicating purification. In such antisense oligonucleotides, one or more consecutive guanine bases may be substituted with hypoxanthine. By substituting one or more guanine bases in a sequence of three or more consecutive guanine bases with hypoxanthine, aggregation of the antisense oligonucleotide can be reduced, thereby facilitating purification.

[0080] The oligonucleotides disclosed herein are synthetic and do not contain antisense compositions of biological origin. The molecules of this disclosure may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, for example, as liposomes, receptor target molecules, or as oral, rectal, topical, or other formulations, to assist in uptake, distribution, or absorption, or a combination thereof.

[0081] The terms “complementary” and “complementarity” refer to oligonucleotides (i.e., sequences of nucleotides) that are related by base pairing rules. For example, the sequence “TGA(5-3')” is complementary to the sequence “TCA(5'-3')”. Complementarity may be “partial,” where only a few bases of the nucleic acid match according to base pairing rules. Alternatively, there may be “complete,” “whole,” or “perfect” (100%) complementarity between nucleic acids. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. In many cases, perfect complementarity is desired, but some embodiments may include one or more mismatches with respect to the target RNA, preferably 6, 5, 4, 3, 2, or 1. Such hybridization may occur with “near” or “substantial” complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity. In some embodiments, the oligomer can hybridize to the target sequence with approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementarity. Mutations can be included at any position within the oligomer. In certain embodiments, mutations near the ends of the oligomer are generally preferred over internal mutations and, if present, are generally located within approximately 6, 5, 4, 3, 2, or 1 nucleotide at the 5' end, 3' end, or both ends.

[0082] The term "peptide" refers to a compound containing multiple linked amino acids, which can be used, for example, to link a desired portion, such as an oligonucleotide, to an antibody or antibody fragment, generally one or more immune cell types, such as immune cells involved in an autoimmune or inflammatory disease condition.

[0083] In the present invention, the term "internalized antibody or antibody fragment" generally refers to an antibody that, upon binding to a target antigen, migrates internally into an immune cell and delivers one or more bound payloads, such as oligonucleotides (RNA or DNA composed of wild-type or modified nucleotides), to the target immune cell. In exemplary embodiments, the internalized antibody or antibody fragment is an antibody that binds to VISTA, preferably human VISTA.

[0084] The terms "cell-permeable peptide" and "CPP" are used synonymously and refer to cationic cell-permeable peptides, also called transport peptides, carrier peptides, or peptide transduction domains. Such peptides have the ability to induce or enhance cell permeability in a given cell culture population.

[0085] The term “treatment” refers to the application of one or more specific procedures used to improve a disease. In certain embodiments, a specific procedure is the administration of one or more pharmaceuticals. “Treatment” of an organism (e.g., a mammal such as a human) or cells is any type of intervention used in an attempt to alter the natural course of the organism or cells. Treatments include, but are not limited to, the administration of pharmaceutical compositions and may be carried out either prophylactically or after the onset of a pathological event or contact with a pathogen. Treatments include any desired effect on the symptoms or pathology of a disease or condition, and may include, for example, a minimal change or improvement in one or more measurable markers of the disease or condition being treated. They also include “prophylactic” measures that may aim to slow the rate of progression of the disease or condition being treated, delay the onset of the disease or condition, or reduce the severity of its onset. “Effective dose” or “therapeutic effective dose” refers to the amount of a therapeutic compound, such as an antisense oligomer, administered to a mammalian subject, either as a single dose or as part of a series of doses, to produce a desired therapeutic effect.

[0086] The term “improvement” means a reduction in the severity of at least one indicator of a condition or disease. In certain embodiments, improvement includes a delay or slowing of the progression of one or more indicators of a condition or disease. The severity of an indicator may be determined by subjective or objective measures known to those skilled in the art.

[0087] As used herein, “pharmaceutically acceptable salt” means a derivative of the oligonucleotide of this disclosure in which the parent oligonucleotide has been modified by converting an existing acid or base moiety to its salt form. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

[0088] Nucleotides included in the ARC or ANC of the present invention The oligonucleotides herein generally refer to polynucleotide molecules that, when delivered into target immune cells, modulate the expression and / or activity of immune-modulating proteins whose genes are expressed by the immune cells. In some embodiments, the polynucleotide molecules described herein modulate the expression of immunomodulatory genes, which may be wild-type or may include one or more mutations, e.g., mutations correlated with disease conditions related to gene expression. In some examples, the polynucleotide molecule hybridizes to a target region of wild-type DNA or RNA encoding an immunomodulatory gene or a fragment thereof. In some examples, the polynucleotide molecule is a polynucleotide molecule that hybridizes to a target region of the DNA or RNA it encodes, which includes mutations (e.g., substitutions, deletions, or additions).

[0089] In some embodiments, the immunomodulatory genes and RNAs are selected from those identified in Figure 1 or Figure 2, or in Appendix 2 and Appendix 4.

[0090] In some embodiments, a polynucleic acid molecule hybridizes to a target region of DNA or RNA encoding a target immunomodulatory protein, which contains one or more mutations.

[0091] In some embodiments, the polynucleic acid molecule includes a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target gene sequences listed in Figure 1 or Figure 2. In some embodiments, the polynucleic acid molecule described herein includes RNA or DNA. In some cases, the polynucleic acid molecule includes RNA.

[0092] In some cases, RNA includes small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heteronuclear RNA (hnRNA). In some cases, RNA includes shRNA. In some cases, RNA includes miRNA. In some cases, RNA includes dsRNA. In some cases, RNA includes tRNA. In some cases, RNA includes rRNA. In some cases, RNA includes hnRNA. In some cases, RNA includes siRNA. In some cases, polynucleic acid molecules include siRNA.

[0093] In some embodiments, the polynucleic acid molecule is about 10 to about 50 nucleotides long. In some examples, the polynucleic acid molecule is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0094] In some embodiments, the polynucleotide molecule is about 50 nucleotides long. In some examples, the polynucleotide molecule is about 45 nucleotides long. In some examples, the polynucleotide molecule is about 40 nucleotides long. In some examples, the polynucleotide molecule is about 35 nucleotides long. In some examples, the polynucleotide molecule is about 30 nucleotides long. In some examples, the polynucleotide molecule is about 25 nucleotides long. In some examples, the polynucleotide molecule is about 20 nucleotides long. In some examples, the polynucleotide molecule is about 19 nucleotides long. In some examples, the polynucleotide molecule is about 18 nucleotides long. In some examples, the polynucleotide molecule is about 17 nucleotides long. In some examples, the polynucleotide molecule is about 16 nucleotides long. In some examples, the polynucleotide molecule is about 15 nucleotides long. In some examples, the polynucleotide molecule is about 14 nucleotides long. In some examples, the polynucleotide molecule is about 13 nucleotides long. In some examples, the polynucleotide molecule is about 12 nucleotides long. In some examples, the polynucleotide molecule is about 11 nucleotides long. In some cases, polynucleotide molecules are approximately 10 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 50 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 45 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 40 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 35 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 30 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 25 nucleotides long. In some cases, polynucleotide molecules are approximately 10 to 20 nucleotides long. In some cases, polynucleotide molecules are approximately 15 to 25 nucleotides long. In some cases, polynucleotide molecules are approximately 15 to 30 nucleotides long. In some cases, polynucleotide molecules are approximately 12 to 30 nucleotides long.

[0095] In some embodiments, the polynucleotide molecule comprises a first polynucleotide. In some examples, the polynucleotide molecule comprises a second polynucleotide. In some examples, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the first polynucleotide is a sense strand or passenger strand. In some examples, the second polynucleotide is an antisense strand or guide strand.

[0096] In some embodiments, the polynucleic acid molecule is a first polynucleotide. In some embodiments, the first polynucleotide is about 10 to about 50 nucleotides long. In some examples, the first polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0097] In some cases, the first polynucleotide is approximately 50 nucleotides long. In some cases, the first polynucleotide is approximately 45 nucleotides long. In some cases, the first polynucleotide is approximately 40 nucleotides long. In some cases, the first polynucleotide is approximately 35 nucleotides long. In some cases, the first polynucleotide is approximately 30 nucleotides long. In some cases, the first polynucleotide is approximately 25 nucleotides long. In some cases, the first polynucleotide is approximately 20 nucleotides long. In some cases, the first polynucleotide is approximately 19 nucleotides long. In some cases, the first polynucleotide is approximately 18 nucleotides long. In some cases, the first polynucleotide is approximately 17 nucleotides long. In some cases, the first polynucleotide is approximately 16 nucleotides long. In some cases, the first polynucleotide is approximately 15 nucleotides long. In some cases, the first polynucleotide is approximately 14 nucleotides long. In some cases, the first polynucleotide is approximately 13 nucleotides long. In some examples, the first polynucleotide is approximately 12 nucleotides long. In some examples, the first polynucleotide is approximately 11 nucleotides long. In some examples, the first polynucleotide is approximately 10 nucleotides long. In some examples, the first polynucleotide is approximately 10 to 50 nucleotides long. In some examples, the first polynucleotide is approximately 10 to 45 nucleotides long. In some examples, the first polynucleotide is approximately 10 to 40 nucleotides long. In some examples, the first polynucleotide is approximately 10 to 35 nucleotides long. In some examples, the first polynucleotide is approximately 10 to 30 nucleotides long. In some examples, the first polynucleotide is approximately 10 to 25 nucleotides long. In some examples, the first polynucleotide is approximately 10 to 20 nucleotides long. In some examples, the first polynucleotide is approximately 15 to 25 nucleotides long. In some examples, the first polynucleotide is approximately 15 to 30 nucleotides long.In some examples, the first polynucleotide is about 12 to 30 nucleotides long.

[0098] In some embodiments, the polynucleic acid molecule is a second polynucleotide. In some embodiments, the second polynucleotide is about 10 to about 50 nucleotides long. In some examples, the second polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides long.

[0099] In some cases, the second polynucleotide is approximately 50 nucleotides long. In some cases, the second polynucleotide is approximately 45 nucleotides long. In some cases, the second polynucleotide is approximately 40 nucleotides long. In some cases, the second polynucleotide is approximately 35 nucleotides long. In some cases, the second polynucleotide is approximately 30 nucleotides long. In some cases, the second polynucleotide is approximately 25 nucleotides long. In some cases, the second polynucleotide is approximately 20 nucleotides long. In some cases, the second polynucleotide is approximately 19 nucleotides long. In some cases, the second polynucleotide is approximately 18 nucleotides long. In some cases, the second polynucleotide is approximately 17 nucleotides long. In some cases, the second polynucleotide is approximately 16 nucleotides long. In some cases, the second polynucleotide is approximately 15 nucleotides long. In some cases, the second polynucleotide is approximately 14 nucleotides long. In some cases, the second polynucleotide is approximately 13 nucleotides long. In some examples, the second polynucleotide is approximately 12 nucleotides long. In some examples, the second polynucleotide is approximately 11 nucleotides long. In some examples, the second polynucleotide is approximately 10 nucleotides long. In some examples, the second polynucleotide is approximately 10 to 50 nucleotides long. In some examples, the second polynucleotide is approximately 10 to 45 nucleotides long. In some examples, the second polynucleotide is approximately 10 to 40 nucleotides long. In some examples, the second polynucleotide is approximately 10 to 35 nucleotides long. In some examples, the second polynucleotide is approximately 10 to 30 nucleotides long. In some examples, the second polynucleotide is approximately 10 to 25 nucleotides long. In some examples, the second polynucleotide is approximately 10 to 20 nucleotides long. In some examples, the second polynucleotide is approximately 15 to 25 nucleotides long. In some examples, the second polynucleotide is approximately 15 to 30 nucleotides long.In some examples, the second polynucleotide is approximately 12 to 30 nucleotides long.

[0100] In some embodiments, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some examples, the polynucleotide molecule further comprises a blunt end, an overhang, or a combination thereof. In some examples, the blunt end is a 5' blunt end, a 3' blunt end, or both. In some cases, the overhang is a 5' overhang, a 3' overhang, or both. In some cases, the overhang contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-paired nucleotides. In some cases, the overhang contains 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the overhang contains 1, 2, 3, or 4 non-base-paired nucleotides. In some cases, the overhang contains 1 non-base-paired nucleotide. In some cases, the overhang contains 2 non-base-paired nucleotides. In some cases, the overhang contains 3 non-base-paired nucleotides. In some cases, the overhang contains 4 non-base-paired nucleotides.

[0101] In some embodiments, the sequence of the polynucleotide molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to the target sequence. In some embodiments, the sequence of the polynucleotide molecule is at least 50% complementary to the target sequence. In some embodiments, the sequence of the polynucleotide molecule is at least 60% complementary to the target sequence. In some embodiments, the sequence of the polynucleotide molecule is at least 70% complementary to the target sequence. In some embodiments, the sequence of the polynucleotide molecule is at least 80% complementary to the target sequence. In some embodiments, the sequence of the polynucleotide molecule is at least 90% complementary to the target sequence. In some embodiments, the sequence of the polynucleotide molecule is at least 95% complementary to the target sequence. In some embodiments, the sequence of the polynucleotide molecule is at least 99% complementary to the target sequence. In some examples, the sequence of the polynucleotide molecule is 100% complementary to the target sequence.

[0102] In some embodiments, the sequence of the polynucleotide molecule has five or fewer mismatches with respect to the target sequence. In some embodiments, the sequence of the polynucleotide molecule has four or fewer mismatches with respect to the target sequence. In some examples, the sequence of the polynucleotide molecule has three or fewer mismatches with respect to the target sequence. In some examples, the sequence of the polynucleotide molecule has two or fewer mismatches with respect to the target sequence. In some examples, the sequence of the polynucleotide molecule has one or fewer mismatches with respect to the target sequence.

[0103] In some embodiments, the specificity of the polynucleic acid molecule hybridizing to the target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity of the polynucleic acid molecule to the target sequence. In some examples, hybridization is performed under highly stringent hybridization conditions.

[0104] In some embodiments, polynucleic acid molecules have reduced off-target effects. In some examples, “off-target” or “off-target effect” refers to any instance in which a polynucleic acid polymer has an unintended effect on a given target due to direct or indirect interaction with another mRNA sequence, DNA sequence, or cellular protein or other part. In some examples, “off-target effect” occurs when there is simultaneous degradation of another transcript due to partial homology or complementarity between the sense and / or antisense strands of the other transcript and the polynucleic acid molecule.

[0105] In some embodiments, polynucleic acid molecules include natural, synthetic, or artificial nucleotide analogs or bases. In some cases, polynucleic acid molecules include DNA, RNA, and / or combinations of nucleotide analogs. In some examples, synthetic or artificial nucleotide analogs or bases include modifications to one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0106] In some embodiments, nucleotide analogs or artificial nucleotide bases comprise nucleic acids having modifications to the 2' hydroxyl group of the ribose moiety. In some examples, the modifications include H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is the alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols, and oxygen. In some examples, the alkyl moiety further comprises modifications. In some examples, the modifications include azo groups, keto groups, aldehyde groups, carboxyl groups, nitro groups, nitroso groups, nitrile groups, heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) groups, isocyanate or cyanate groups, or sulfur-containing groups (e.g., sulfoxides, sulfones, sulfides, or disulfides). In some examples, the alkyl moiety further comprises heterosubstitutions. In some examples, the carbon atoms of the heterocyclic group are substituted with nitrogen, oxygen, or sulfur. Examples of heterocyclic substitutions include, but are not limited to, morpholinos, imidazoles, and pyrrolidinos.

[0107] In some cases, the modification at the 2'-hydroxyl group is either 2'-O-methyl modification or 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, 2'-O-methyl modification adds a methyl group to the 2'-hydroxyl group of the ribose moiety, while 2'-methoxyethyl modification adds a methoxyethyl group to the 2'-hydroxyl group of the ribose moiety.

[0108] In some examples, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, where an extended amine group containing a propyl linker attaches the amine group to the 2' oxygen. In some examples, this modification neutralizes the overall negative charge derived from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group for each sugar, thereby improving its cellular uptake properties due to its zwitterionic characteristics.

[0109] In some examples, the modification at the 2' hydroxyl group is a locked or cross-linked ribose modification (e.g., locked nucleic acid or LNA), in which the oxygen molecule bonded at the 2' carbon is linked to the 4' carbon by a methylene group, thereby forming a 2'-C,4'-C-oxymethylene-linked bicyclic ribonucleotide monomer. Exemplary representations of the chemical structure of LNA are known in the art.

[0110] In some examples, modifications at the 2' hydroxyl group involve ethylene nucleic acids (ENAs), such as 2'-4'-ethylene-bridged nucleic acids, which lock the sugar structure into a C3'-endoglycan puckering structure. ENAs are part of a class of bridged nucleic acids of modified nucleic acids, which also includes LNAs.

[0111] In some embodiments, additional modifications to the 2'-hydroxyl group include 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0112] In some embodiments, nucleotide analogs are not limited to, but include, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides having a modification at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halolysine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (e.g., 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azocytidine). Modified bases include, for example, midins, 5-methyl-2-thiouridine, other thio bases (e.g., 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, keuosin, alkaeosin, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (e.g., N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, or pyridine-2-one), phenyl and modified phenyl groups, for example, aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as a G-clamp nucleotide, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety, and nucleotides having a non-ribosyl sugar or its analogues. For example, the sugar portion may be mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocyclic or carbocyclic, or based thereon. The term nucleotide also includes what is known in the art as a universal base.Examples of universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularin.

[0113] In some embodiments, nucleotide analogs further include morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, 1',5'-anhydrohexitol nucleic acid (HNA), or combinations thereof. Morphorino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics that of natural nucleic acid structures but deviates from the usual sugar and phosphate structures. In some examples, a five-membered ribose ring is replaced by a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomer is linked by a phosphorodiamidate group instead of a phosphate group. In such cases, the skeletal modification removes all positive and negative charges, forming a morpholino-neutral molecule that can traverse the cell membrane without the aid of cell delivery agents, such as those used by charged oligonucleotides.

[0114] In some embodiments, peptide nucleic acids (PNAs) do not contain sugar rings or phosphate bonds, and the bases are attached to oligoglycine-like molecules, thereby appropriately spacing them out and removing the charge from the backbone.

[0115] In some embodiments, one or more modifications optionally occur in the nucleotide interbonding. In some examples, the modified nucleoside interbonding includes phosphorothioates; phosphorodithioates; methylphosphonates; 5'-alkylenephosphonates; 5'-methylphosphonates; 3'-alkylenephosphonates; borontrifluorideates; 3'-5' or 2'-5' linked boranophosphates and selenophosphates; phosphotriesters; thionoalkylphosphotriesters; hydrogen phosphonate bonds; alkylphosphonates; alkylphosphonothioates; arylphosphonothioates; phosphoroselenoates; phosphorodiselenoates; phosphinates; phosphoramidates; 3'-alkylphosphoramidates; aminoalkylphosphoramidates; thionophosphoramidates; phosphoropiperadates; phosphoranilothioates; phosphoro Anilides; ketones; sulfones; sulfonamides; carbonates; carbamates; methylene hydrazo; methylene dimethyl hydrazo; formacetals; thioformacetals; oximes; methylene iminos; methylene methyliminos; thioamidates; bonds containing a riboacetyl group; aminoethylglycine; silyl or siloxane bonds; alkyl or cycloalkyl bonds with or without heteroatoms, e.g., saturated or unsaturated and / or substituted and / or containing heteroatoms, alkyl or cycloalkyl bonds of 1 to 10 carbon atoms; bonds having morpholino structures, amides, or polyamides in which a base is directly or indirectly bonded to the aza nitrogen of the skeleton; and combinations thereof.

[0116] In some examples, the modifications are methyl or thiol modifications, such as methylphosphonate or thiolphosphonate modifications. Exemplary thiolphosphonate nucleotides and methylphosphonate nucleotides are known in the art, including 2'-fluoroN3-P5'-phosphoramidite. In some examples, the modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-anhydrohexitol nucleic acids (HNA)).

[0117] In some embodiments, one or more modifications may further optionally include modifications of the ribose moiety, phosphate backbone, and nucleoside, or modifications of nucleotide analogs at the 3' or 5' end. For example, the 3' end may optionally include a 3' cationic group or invert the 3' terminal nucleoside using a 3'-3' bond. In another alternative form, the 3' end may optionally be conjugated with an aminoalkyl group, e.g., 3'C5-aminoalkyldT. In yet another alternative form, the 3' end may optionally be conjugated with a debasing site, e.g., a depurine or depyrimidine base site. In some examples, the 5' end is conjugated with an aminoalkyl group, e.g., a 5'-O-alkylamino substituent. In some cases, the 5' end is conjugated with a debasing site, e.g., a depurine or depyrimidine base site.

[0118] In some embodiments, the polynucleic acid molecule comprises one or more of the artificial nucleotide analogs described herein. In some examples, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogs described herein. In some embodiments, artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof. In some examples, polynucleic acid molecules are 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2 The polynucleic acid molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more artificial nucleotide analogs selected from '-ON-methylacetamide (2'-O-NMA) modified nucleotides, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidites, or combinations thereof. In some examples, the polynucleic acid molecule contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more 2'-O-methyl-modified nucleotides.In some examples, polynucleic acid molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides. In some examples, polynucleic acid molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more thiol phosphonate nucleotides.

[0119] In some examples, polynucleic acid molecules contain at least one of the following modification levels: approximately 5% to approximately 100%, approximately 10% to approximately 100%, approximately 20% to approximately 100%, approximately 30% to approximately 100%, approximately 40% to approximately 100%, approximately 50% to approximately 100%, approximately 60% to approximately 100%, approximately 70% to approximately 100%, approximately 80% to approximately 100%, and approximately 90% to approximately 100%.

[0120] In some cases, polynucleic acid molecules contain at least one of the following modification levels: approximately 10% to approximately 90%, approximately 20% to approximately 90%, approximately 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, and approximately 80% to approximately 100%.

[0121] In some cases, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 80%, approximately 20% to approximately 80%, approximately 30% to approximately 80%, approximately 40% to approximately 80%, approximately 50% to approximately 80%, approximately 60% to approximately 80%, and approximately 70% to approximately 80%.

[0122] In some examples, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 70%, approximately 20% to approximately 70%, approximately 30% to approximately 70%, approximately 40% to approximately 70%, approximately 50% to approximately 70%, and approximately 60% to approximately 70%.

[0123] In some examples, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 60%, approximately 20% to approximately 60%, approximately 30% to approximately 60%, approximately 40% to approximately 60%, and approximately 50% to approximately 60%.

[0124] In some cases, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 50%, approximately 20% to approximately 50%, approximately 30% to approximately 50%, and approximately 40% to approximately 50%.

[0125] In some cases, polynucleic acid molecules contain at least one of the following modifications: approximately 10% to approximately 40%, approximately 20% to approximately 40%, and approximately 30% to approximately 40%.

[0126] In some cases, polynucleic acid molecules contain at least one of approximately 10% to 30% modifications and approximately 20% to 30% modifications.

[0127] In some cases, polynucleotide molecules contain approximately 10% to 20% modification. In other cases, polynucleotide molecules contain approximately 15% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% modification.

[0128] In further cases, the polynucleic acid molecule contains at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% modification.

[0129] In some embodiments, the polynucleic acid molecule contains at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, or more modifications.

[0130] In some examples, polynucleic acid molecules contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or more modified nucleotides.

[0131] In some examples, polynucleic acid molecules contain approximately 5% to approximately 100% of the artificial nucleotide analogs described herein. In some examples, polynucleic acid molecules contain approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the artificial nucleotide analogs. In some examples, polynucleic acid molecules contain approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the artificial nucleotide analogs. In some examples, polynucleic acid molecules contain approximately 5% of the artificial nucleotide analogs. In some cases, approximately 10% of polynucleotide molecules contain artificial nucleotide analogs.

[0132] In some embodiments, the polynucleic acid molecule is constructed from two distinct polynucleotides, one of which comprises a sense strand and the second polynucleotide comprising the antisense strand of the polynucleic acid molecule. In other embodiments, the sense strand is linked to the antisense strand via a linker molecule, which in some examples is a polynucleotide linker or a non-nucleotide linker.

[0133] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the pyrimidine nucleotides of the sense strand comprise 2'-O-methylpyrimidine nucleotides, and the purine nucleotides of the sense strand comprise 2'-deoxypurine nucleotides. In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the pyrimidine nucleotides present in the sense strand comprise 2'-deoxy-2'-fluoropyrimidine nucleotides, and the purine nucleotides present in the sense strand comprise 2'-deoxypurine nucleotides.

[0134] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide, when present in the antisense strand, is a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, when present in the antisense strand, is a 2'-O-methylpurine nucleotide.

[0135] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the pyrimidine nucleotide being a 2'-deoxy-2'-fluoropyrimidine nucleotide when present in the antisense strand, and the purine nucleotide being a 2'-deoxy-purine nucleotide when present in the antisense strand.

[0136] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand having terminal capping portions at its 5' end, 3' end, or both 5' and 3' ends. In other embodiments, the terminal capping portions are inverted deoxydecate portions.

[0137] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having a phosphate backbone modification at its 3' end. In some examples, the phosphate backbone modification is a phosphorothioate.

[0138] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having a glyceryl modification at its 3' end.

[0139] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand having one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleoside bonds and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally at the 3' end, 5' end, or both of the 3' and 5' ends of the sense strand The antisense chain comprises a terminal cap molecule and includes approximately 1 to approximately 10 or more, particularly approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, phosphorothioate nucleoside interbondings and / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro and / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universally modified nucleotides, as well as optionally including a terminal cap molecule at the 3' end, 5' end, or both of the 3' and 5' ends of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoronucleotides, and have or do not have one or more phosphorothioate nucleoside interbonds and / or terminal cap molecules at the 3' end, 5' end, or both 3' and 5' ends present on the same or different strands.

[0140] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the sense strand having about 1 to about 25, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleoside bonds and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally at the 3' end, 5' end, or both of the 3' and 5' ends of the sense strand The antisense chain contains a terminal cap molecule and comprises approximately 1 to approximately 25 or more, e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleoside bonds and / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro and / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, and optionally contains a terminal cap molecule at the 3' end, 5' end, or both the 3' and 5' ends of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoronucleotides, and have or do not have about 1 to about 25 or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleoside interbondings and / or have or do not have terminal cap molecules at the 3' end, 5' end, or both 3' and 5' ends present on the same or different strands.

[0141] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having one or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleoside bonds and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universally modified nucleotides, as well as optionally the 3' and 5' ends of the sense strand. The antisense chain comprises, or includes terminal cap molecules at both the 3' and 5' ends, and the antisense chain comprises about 1 to about 10 or more, particularly about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, phosphorothioate nucleoside interbondings, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universally modified nucleotides, and optionally includes terminal cap molecules at the 3' end, 5' end, or both the 3' and 5' ends of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoronucleotides, and have or do not have one or more phosphorothioate nucleoside interbonds and / or terminal cap molecules at the 3' end, 5' end, or both 3' and 5' ends present on the same or different strands.

[0142] In some embodiments, the polynucleic acid molecule comprises a sense strand and an antisense strand, the antisense strand having about 1 to about 25 or more, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleoside bonds and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro and / or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, as well as optionally the 3' end, 5' end, or 3' and 5' ends of the sense strand. The antisense chain contains terminal cap molecules at both ends, and the antisense chain contains approximately 1 to approximately 25 or more, e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleoside bonds and / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro and / or one or more (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base-modified nucleotides, as well as optionally containing terminal cap molecules at the 3' end, 5' end, or both 3' and 5' ends of the antisense chain. In other embodiments, one or more pyrimidine nucleotides of the sense and / or antisense strands, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluoronucleotides, and have or do not have about 1 to about 5, e.g., about 1, 2, 3, 4, 5, or more phosphorothioate nucleoside interbondings and / or terminal cap molecules at the 3' end, 5' end, or both 3' and 5' ends present on the same or different strands.

[0143] In some embodiments, the polynucleic acid molecules described herein are chemically modified small interfering nucleic acid molecules having about 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate nucleotide interbondings in each chain of the polynucleic acid molecule.

[0144] In another embodiment, the polynucleic acid molecules described herein include 2'-5' nucleoside bonds. In some examples, the 2'-5' nucleoside bond(s) are located at the 3' end, 5' end, or both the 3' and 5' ends of one or both sequence strands. In further examples, the 2'-5' nucleoside bond(s) are located at various other positions within one or both sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides, including all nucleoside bonds, on one or both strands of the polynucleic acid molecule include 2'-5' nucleoside bonds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more purine nucleotides, including all nucleoside bonds, on one or both strands of the polynucleic acid molecule include 2'-5' nucleoside bonds.

[0145] In some embodiments, the polynucleic acid molecule is a single-stranded polynucleic acid molecule that mediates RNAi activity in cells or a replicated in vitro system, the polynucleic acid molecule comprises a single-stranded polynucleotide complementary to a target nucleic acid sequence, one or more pyrimidine nucleotides present in the polynucleic acid are 2'-deoxy-2'-fluoropyrimidine nucleotides (for example, all pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides, or multiple pyrimidine nucleotides alternately are 2'-deoxy-2'-fluoropyrimidine nucleotides), and one or more purine nucleotides present in the polynucleic acid are 2'-deoxypurine nucleotides. The polynucleotide is a nucleotide (for example, all purine nucleotides are 2'-deoxypurine nucleotides, or multiple purine nucleotides alternately are 2'-deoxypurine nucleotides), terminal cap modifications are optionally present at the 3' end, 5' end, or both the 3' and 5' ends of the antisense sequence, the polynucleotide molecule optionally further contains about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxypurine nucleotides at the 3' end of the polynucleotide molecule, the terminal nucleotides further contain one or more (e.g., 1, 2, 3, or 4) phosphorothioate nucleoside bonds, and the polynucleotide molecule optionally further contains terminal phosphate groups, for example, a 5' terminal phosphate group.

[0146] In some cases, one or more artificial nucleotide analogs are resistant to nucleases, such as ribonucleases, such as RNase H, deoxyribonucleases, such as DNase, or exonucleases, such as 5'-3' exonuclease and 3'-5' exonuclease, compared to natural polynucleic acid molecules. In some examples, artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoroN3-P5'-phosphoramidite, or combinations thereof, are nucleases, e.g., ribonucleases, e.g., RNases. It is resistant to RNase H, deoxyrib nucleases, e.g., DNase, or exonucleases, e.g., 5'-3' exonuclease and 3'-5' exonuclease. In some cases, 2'-O-methyl modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl modified polynucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-deoxy-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, T-deoxy-2'-fluoro-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-dimethylaminoethyl (2-O-DMAOE)-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, LNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, ENA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, HNA-modified polynucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, morpholino is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease).In some cases, PNA-modified polynucleic acid molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonate-modified polynucleic acid molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, thiolphosphonate-modified polynucleic acid molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidite are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, the 5' conjugate described herein inhibits 5'-3' exonuclease cleavage. In some cases, the 3' conjugate described herein inhibits 3'-5' exonuclease cleavage.

[0147] In some embodiments, one or more artificial nucleotide analogs exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. One or more artificial nucleotide analogs, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, or 2'-fluoroN3-P5'-phosphoramidite, exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules. In some cases, 2'-O-methyl modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-deoxy modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, T-deoxy-2'-fluoro modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-aminopropyl (2'-O-AP) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE)-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleic acid molecules.In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, LNA modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, ENA modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, PNA modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, HNA-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, morpholino-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, methylphosphonate-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, thiolphosphonate-modified polynucleic acid molecules exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, polynucleic acid molecules containing 2'-fluoroN3-P5'-phosphoramidite exhibit increased binding affinity to their mRNA targets compared to comparable natural polynucleic acid molecules. In some cases, the increase in affinity is indicated by a decrease in Kd, an increase in melting temperature (Tm), or a combination thereof.

[0148] In some embodiments, the polynucleic acid molecule is a chiralally pure (or sterically pure) polynucleic acid molecule, or a polynucleic acid molecule containing a single enantiomer. In some examples, the polynucleic acid molecule contains an L-nucleotide. In some examples, the polynucleic acid molecule contains a D-nucleotide. In some examples, the polynucleic acid molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less enantiomers. In some cases, the polynucleic acid molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less racemic mixtures.

[0149] In some embodiments, the polynucleic acid molecule may be further modified to include an aptamer conjugate moiety. In some examples, the aptamer conjugate moiety is a DNA aptamer conjugate moiety. In some examples, the aptamer conjugate moiety is an Alphamer (Centauri Therapeutics), which includes an aptamer moiety that recognizes a specific cell surface target and a moiety that presents a specific epitope for binding to a circulating antibody. In some examples, the polynucleic acid molecule described herein is further modified to include an aptamer conjugate moiety as described in U.S. Patents 8,604,184, 8,591,910, and 7,850,975.

[0150] In some embodiments, the polynucleic acid molecules described herein are modified to increase their stability. In some embodiments, the polynucleic acid molecule is RNA (e.g., siRNA), and the polynucleic acid molecule is modified to increase its stability. In some examples, the polynucleic acid molecule is modified by one or more of the above modifications to increase its stability. In some cases, the polynucleic acid molecule is modified at the 2' hydroxyl position by modifications such as 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA), or by locked or cross-linked ribose stereostructures (e.g., LNA or ENA). In some cases, polynucleotide molecules are modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, polynucleotide molecules also contain morpholino, PNA, HNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and / or 2'-fluoroN3-P5'-phosphoramidite to increase their stability. In some examples, polynucleotide molecules are chiralally pure (or sterically pure). In some examples, chirally pure (or sterically pure) polynucleotide molecules are modified to increase their stability. Appropriate modifications of RNA to increase stability against delivery will be obvious to those skilled in the art.

[0151] In some embodiments, the polynucleic acid molecules in the ARC or ANC according to the present invention have RNAi activity that modulates the expression of RNA encoded by a target immunomodulatory gene. In some examples, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of a target immunomodulatory protein, wherein one strand of the double-stranded siRNA molecule contains a nucleotide sequence complementary to the nucleotide sequence of the immunomodulatory gene or the RNA encoded by the immunomodulatory gene or a portion thereof, and the second strand of the double-stranded siRNA molecule contains a nucleotide sequence substantially similar to the nucleotide sequence of the immunomodulatory gene or the RNA encoded by the immunomodulatory gene or a portion thereof. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of an immunomodulatory gene, wherein each strand of the siRNA molecule contains about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, and each strand contains at least about 14, 17, or 19 nucleotides complementary to the nucleotides of the other strand. In some cases, the polynucleic acid molecules described herein are double-stranded siRNA molecules that downregulate the expression of immunomodulatory genes, where each strand of the siRNA molecule contains approximately 19 to approximately 23 nucleotides, and each strand contains at least approximately 19 nucleotides complementary to the nucleotides of the other strand. In some examples, RNAi activity occurs intracellularly. In other examples, RNAi activity occurs in a replicated in vitro system.

[0152] In some examples, the polynucleotide molecule in the ARC or ANC according to the present invention is a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some examples, the polynucleotide molecule is constructed from two distinct polynucleotides, in which case one strand is the sense strand and the other is the antisense strand, and the antisense and sense strands are self-complementary (for example, each strand comprises a nucleotide sequence complementary to the nucleotide sequence of the other strand, for example, if the antisense and sense strands form a double-stranded or double-stranded structure, for example, the double-stranded region has about 19, 20, 21, 22, 23 or more base pairs), wherein the antisense strand comprises a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, polynucleotide molecules are constructed from a single oligonucleotide, in which case the self-complementary sense and antisense regions of the polynucleotide molecule are linked by a nucleic acid-based or non-nucleic acid-based linker.

[0153] In some cases, the polynucleotide molecule in the ARC or ANC according to the present invention is a polynucleotide having a double helix, asymmetric double helix, hairpin or asymmetric hairpin secondary structure, and having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of an individual target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In other cases, the polynucleotide molecule is a cyclic single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence complementary to the nucleotide sequence of a target nucleic acid molecule or a portion thereof, and the sense region comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the cyclic polynucleotide is processed either in vivo or in vitro to produce an active polynucleotide molecule capable of mediating RNAi. In further cases, the polynucleic acid molecule also includes a single-stranded polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of the target nucleic acid molecule or a portion thereof (for example, such a polynucleic acid molecule does not need to be present in the polynucleic acid molecule of the nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), and the single-stranded polynucleotide further includes a terminal phosphate group, e.g., 5'-phosphate (see, e.g., Martinez et al., 2002, Cell, 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568) or 5',3'-diphosphate.

[0154] In some examples, an asymmetric double helix is ​​a linear polynucleotide molecule comprising an antisense region, a loop region containing nucleotides or non-nucleotides, and a sense region containing fewer nucleotides than the antisense region, within a range where the sense region has enough complementary nucleotides to base-pair with the antisense region and form a double helix with a loop. For example, an asymmetric hairpin polynucleotide molecule comprises an antisense region of sufficient length (e.g., about 19 to about 22 nucleotides) to mediate RNAi in a cell or in vitro system, a loop region containing about 4 to about 8 nucleotides, and a sense region having about 3 to about 18 nucleotides complementary to the antisense region. In some cases, an asymmetric hairpin polynucleotide molecule also contains a chemically modified 5' terminal phosphate group. In further cases, the loop region of an asymmetric hairpin polynucleotide molecule contains nucleotides, non-nucleotides, linker molecules, or conjugate molecules.

[0155] In some embodiments, an asymmetric double helix is ​​a polynucleic acid molecule having two distinct strands comprising a sense region and an antisense region, wherein the sense region contains fewer nucleotides than the antisense region, within a range where the sense region has enough complementary nucleotides to base-pair with the antisense region and form a double helix. For example, an asymmetric double-helix polynucleic acid molecule comprises an antisense region having a length sufficient to mediate RNAi in a cell or in vitro system (e.g., about 19 to about 22 nucleotides) and a sense region having about 3 to about 18 nucleotides complementary to the antisense region.

[0156] In some cases, universal bases refer to nucleotide base analogs that form base pairs with each of the natural DNA / RNA bases with little distinction between them. Non-limiting examples of universal bases, as known in the art (see, for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447), include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0157] Synthesis of polynucleic acid molecules for use in ARC or ANC of the present invention The target ARC or ANC comprises one or more polynucleic acid molecules, generally RNA, which can be synthesized as disclosed herein or by other known methods. In some embodiments, the polynucleic acid molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, the polynucleic acid molecules are chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the double helix formed between the polynucleic acid molecule and the target nucleic acid. Illustrative methods include those described below: U.S. Patents 5,142,047, 5,185,444, 5,889,136, 6,008,400, and 6,111,086, PCT Publication WO2009099942, or European Publication 1579015.Further exemplary methods include those listed below: Griffey et al., “2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides,” J.Med.Chem.39(26):5100-5109(1997)); Obika, et al., “Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering”. Tetrahedron Letters 38(50):8735(1997); Koizumi, M., “ENA oligonucleotides as therapeutics”. Current opinion in molecular therapeutics 8(2):144-149(2006); and Abramova et al., “Novel oligonucleotide analogues based on morpholino nucleoside subunits - antisense technologies: new chemical Possibilities, Indian Journal of Chemistry 48B:1721-1726 (2009). Alternatively, polynucleotide molecules can be biologically generated using expression vectors in which the polynucleotide molecule is subcloned in the antisense direction (i.e., the RNA transcribed from the inserted polynucleotide molecule is in the antisense direction relative to the target polynucleotide molecule of interest).

[0158] In some embodiments, polynucleic acid molecules are synthesized by a tandem synthesis method, in which both chains are synthesized as a single continuous oligonucleotide fragment or chain separated by a cleavable linker, and the linker is then cleaved to hybridize and provide separate fragments or chains that allow for the purification of the double hemisphere.

[0159] In some cases, polynucleic acid molecules are also constructed from two separate nucleic acid chains or fragments, in which case one fragment contains the molecule's sense region and the second fragment contains the molecule's antisense region.

[0160] For example, further modification methods for incorporating sugar, base, and phosphate modifications include: Eckstein et al., International Publication PCT WO92 / 07065; Perrault et al., Nature, 1990, 344, 565-568; Picken et al., Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci, 1992, 17, 334-339; Usman et al., International Publication PCT WO93 / 15187; Sproat, U.S. Patent No. 5,334,711 and Beigelman et al. al., 1995, J. Biol. Chem., 270, 25702; International PCT Publication No. WO97 / 26270 by Beigelman et al.; U.S. Patent No. 5,716,824 by Beigelman et al.; U.S. Patent No. 5,627,053 by Usman et al.; International PCT Publication No. WO98 / 13526 by Woolf et al.; U.S. Patent No. 60 / 082,404 filed on April 20, 1998 by Thompson et al.; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67, 99-134; and Burlina et al. al, 1997, Bioorg. Med. Chem., 5, 1999-2010. Such publications describe general methods and strategies for determining the incorporation sites of sugar, base, and / or phosphate modifications into nucleic acid molecules without regulating catalysis.

[0161] In some cases, chemical modification of nucleotide-nucleotide bonds in polynucleic acid molecules using phosphorothioates, phosphorodithioates, and / or 5'-methylphosphonate bonds improves stability, but excessive modification can lead to toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these nucleotide-nucleotide bonds is sometimes minimized. In such cases, reducing the concentration of these bonds reduces toxicity and increases the potency and specificity of these molecules.

[0162] Diseases treatable using ARC or ANC of the present invention In some embodiments, the ARC or ANC according to the present invention as described herein, or pharmaceutical compositions containing the same, are used to treat diseases or disorders, generally autoimmune disorders or inflammatory disorders, cancer, or conditions induced by certain immune cell types associated therewith.

[0163] In some cases, ARC or ANC or compositions containing them are used to treat autoimmune diseases, such as bone marrow or T cell-related diseases.

[0164] In some cases, ARC or ANC or compositions containing them are used to treat neoplastic diseases, proliferative disorders, neurodegenerative diseases, neuroinflammatory diseases, infectious diseases, autoimmune diseases or inflammatory diseases or their symptoms.

[0165] In some cases, ARC or ANC or compositions containing them are associated with acromegaly, acquired aplastic anemia, acquired hemophilia, agammaglobulinemia, primary alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) / fulminant antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic neuropathy (AAG) / autonomic neuropathy / autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis / acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, and autoimmune Autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), autoimmune hyperlipidemia, autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), polyglandular autoimmune syndrome, types I, II, and III (APS1, APS2, APS3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Baro's disease, Behçet's disease, shotgun chorioretinopathy / shotgun uvea Inflammation, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome / limited cutaneous systemic sclerosis, Crohn's disease (CD), Cronchite-Canada syndrome (CSS), idiopathic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic Esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome / anti-GBM / anti-TBM disease, granulomatosis with polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's disease / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schöne purpura / IgA vasculitis, hidradenitis suppurativa, Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE),Hypogammaglobulinemia, IgA nephropathy / Berge's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, inclusion body myositis, IgG4-related sclerosis (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult Still's disease, juvenile polymyositis / juvenile dermatomyositis / juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenia dysthenia syndrome (LEMS), leukocytosis-destructive vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD) / linear IgA bullous dermatosis (LABD), lupus nephritis, L Immune disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS), lymphocytic colitis / microscopic colitis, lymphocytic hypophysitis / autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mohlen's ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devic's disease, ocular scarring pemphigoid, ocular clonus-myoclonus syndrome (OMS), regression Rheumatoid arthritis, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, Parry-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / peripheral uveitis, PANS / PANDAS, Personage-Turner syndrome, bullous pemphigoid of pregnancy / herpes zoster of pregnancy, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural orthostatic tachycardia syndrome (POTS), primary biliary cirrhosis (PBC) / primary biliary cholangitis, primary sclerosing liver disease Cholangitis (PSC), psoriasis, palmoplantar pustulosis, psoriatic arthritis, idiopathic pulmonary fibrosis (IPF), pure red cell fistula (PRCA), pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud's disease / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy syndrome (RSD) / complex regional pain syndrome (CRPS), relapsing polychondritis, restless legs syndrome (RLS) / Willis-Ekbom disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, scleritis, scleroderma, sclerosing mesentericitis / mesenteral panniculitis, crawling choroidopathy,It is used to treat one or more autoimmune diseases selected from Sjögren's syndrome, generalized rigidity syndrome (SPS), small-diameter fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus erythematosus, Suzac syndrome, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis (vasculitis), testicular autoimmunity (vasculitis, orchitis), Tolosa-Hunt syndrome, transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis (anterior / intermediate / posterior), vasculitis, VEXAS syndrome, vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH).

[0166] In some cases, ARC or ANC is used to treat autoimmune diseases selected from the group consisting of Addison's disease, arthritis, celiac disease, lupus, Graves' disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto's disease, Sjögren's disease, asthma, type 2 diabetes, and autoimmune type 1 diabetes.

[0167] In some cases, ARC or ANC or compositions containing the same are used to treat inflammatory diseases selected from the group consisting of fatty liver disease, endometriosis, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), asthma, rheumatoid arthritis, obesity, fibromyalgia, lupus, SLE, osteoarthritis, rheumatoid arthritis, shingles (herpes zoster), and vasculitis.

[0168] In some cases, ANC or ARC or compositions containing them are used to treat neurodegenerative or neuroinflammatory diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body dementia, aphasia, Parkinson's disease, or spinal muscular atrophy.

[0169] In some cases, ARC or ANC or compositions containing them are used to treat symptoms associated with cancer or specific immune cell types.

[0170] In some embodiments, ARC or ANC or pharmaceutical compositions comprising the polynucleic acid molecules described herein are used to treat cancer. In some examples, the cancer is a solid tumor. In some examples, the cancer is a hematological malignancy. In some examples, the cancer is a recurrent or refractory cancer or a metastatic cancer. In some examples, the solid tumor is a recurrent or refractory solid tumor or a metastatic solid tumor. In some cases, the hematological malignancy is a recurrent or refractory hematological malignancy or a metastatic hematological malignancy.

[0171] In some embodiments, cancer is a solid tumor. Typical solid tumors include, but are not limited to, anal cancer, appendiceal cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of unknown primary origin (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0172] In some cases, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat solid tumors. In some cases, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat anal cancer, appendiceal cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumors, breast cancer, cervical cancer, colon cancer, cancer of unknown primary origin (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumors, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer. In some cases, the solid tumors are recurrent or refractory solid tumors, or metastatic solid tumors.

[0173] In some cases, cancer is a hematological malignancy. In some cases, hematological malignancies are leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some cases, hematological malignancies are chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's hypergammaglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma This includes peritoneum, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prelymphoblastic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytogenic myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis.

[0174] In some cases, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat hematological malignancies. In some cases, the polynucleic acid molecules or pharmaceutical compositions described herein are used to treat leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some cases, hematological malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's hypergammaglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, and nodal marginal zone B-cell lymphoma. This includes peritoneum, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prelymphoblastic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytogenic myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis. In some cases, hematological malignancies are relapsed or refractory hematological malignancies or metastatic hematological malignancies.

[0175] Pharmaceutical preparations In some embodiments, pharmaceutical formulations comprising ARC or ANC according to the present invention are administered to a target by multiple routes of administration, including but not limited to parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal. In some examples, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration. In other examples, the pharmaceutical compositions described herein are formulated for oral administration. In yet another example, the pharmaceutical compositions described herein are formulated for intranasal administration.

[0176] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, rapidly dissolving formulations, tablets, capsules, pills, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and mixed immediate and controlled-release formulations.

[0177] In some examples, the pharmaceutical formulation includes multi-particle formulations. In some examples, the pharmaceutical formulation includes nanoparticle formulations. In some examples, the nanoparticles include cMAP, cyclodextrin, or lipids. In some cases, the nanoparticles include solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Further exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, and quantum dots. In some examples, nanoparticles are nanoparticles of metals, such as scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium, lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium, potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, and combinations, alloys, or oxides thereof.

[0178] In some examples, nanoparticles include a core or a core and a shell, such as core-shell nanoparticles.

[0179] In some examples, the nanoparticles are further coated with molecules for binding to functional elements (e.g., one or more of the polynucleic acid molecules or binding sites described herein). In some examples, the coating includes chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginic acid, pectin, carrageenan, fucoidan, agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronic acid, glucosamine, galactosamine, chitin (or chitosan), polyglutamic acid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease, trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine, histone, protamine, ovalbumin, dextrin, or cyclodextrin. In some examples, the nanoparticles include graphene-coated nanoparticles.

[0180] In some cases, the nanoparticles have at least one of the following dimensions: approximately 500 nm, 400 nm, 300 nm, 200 nm, or less than 100 nm.

[0181] In some examples, nanoparticle formulations include paramagnetic nanoparticles, superparamagnetic nanoparticles, metallic nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as those having covalently bonded metal chelates), nanofibers, nanohorns, nanoonions, nanorods, nanoropes, or quantum dots. In some examples, the polynucleic acid molecules or binding sites described herein are directly or indirectly conjugated to the nanoparticles. In some examples, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more of the polynucleic acid molecules or binding sites described herein are directly or indirectly conjugated to the nanoparticles.

[0182] In some embodiments, the pharmaceutical formulation includes a delivery vector, such as a recombinant vector, for delivering polynucleic acid molecules into cells. In some examples, the recombinant vector is a DNA plasmid. In other examples, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. In some examples, the recombinant vector capable of expressing polynucleic acid molecules results in stable expression in target cells. In further examples, viral vectors that result in transient expression of polynucleic acid molecules are used.

[0183] In some embodiments, the pharmaceutical formulation includes a carrier or carrier material selected based on its compatibility with the compositions disclosed herein and the release profile characteristics of the desired dosage form. Exemplary carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. Examples of pharmaceutically compatible carrier materials include, but are not limited to, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, and pregelatinized starch. For example, see Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0184] In some examples, pharmaceutical formulations further contain pH adjusters or buffers, such as acids including acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases including sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers including citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0185] In some examples, a pharmaceutical formulation contains one or more salts in amounts necessary to bring the molar osmotic pressure concentration of the composition within an acceptable range. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, and preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0186] In some cases, diluents provide a more stable environment, and pharmaceutical formulations further include diluents used to stabilize compounds. Salts dissolved in buffer solutions (which also provide pH control or maintenance) are used as diluents in the art and include, but are not limited to, phosphate-buffered saline. In certain cases, diluents increase the volume of a composition to facilitate compression or to create a volume sufficient for homogeneous mixing for capsule filling. Examples of such compounds include lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, such as Avicel®, dicalcium phosphate, calcium hydrogen phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressed sugar, such as Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrose, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, and bentonite.

[0187] In some cases, pharmaceutical formulations contain disintegration agents (or disintegrants) to promote the breakdown or disintegration of substances. The term "disintegrate" includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starches, such as natural starches like cornstarch or potato starch; pregelatinized starches such as National 1551 or Amijel®; or sodium starch glycolate such as Promogel® or Explotab®; cellulose from wood products, such as methyl crystalline cellulose, such as Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, and Ming Examples include Tia (registered trademark), Solka-Floc (registered trademark), methylcellulose, croscarmellose, or cross-linked carboxymethylcellulose sodium (Ac-Di-Sol (registered trademark)), cross-linked carboxymethylcellulose, or cross-linked croscarmellose; cross-linked starches such as sodium starch glycolate; cross-linked polymers such as crospovidone; cross-linked polyvinylpyrrolidone; alginates such as alginic acid or alginic acid salts such as sodium alginate; clays such as Veegum (registered trademark) HV (aluminum magnesium silicate); gums such as agar, guar, carob, karaya, pectin, or tragacanth; sodium starch glycolate; bentonite; natural sponges; surfactants; resins such as cation exchange resins; citrus pulp; sodium lauryl sulfate; and sodium lauryl sulfate in combined starches.

[0188] In some examples, pharmaceutical formulations contain fillers such as lactose, calcium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, and polyethylene glycol.

[0189] The pharmaceutical formulations described herein may optionally include lubricants and flow promoters to prevent, reduce, or inhibit adhesion or friction of materials. Examples of lubricants include hydrocarbons such as stearic acid, calcium hydroxide, talc, sodium stearate fumarate, mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®), high fatty acids and their alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, and zinc, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000), or methoxypolyethylene glycol such as Carbowax®, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid® and Cab-O-Sil®, starches such as corn starch, silicone oils, and surfactants.

[0190] Plasticizers include compounds used to soften microencapsulated materials or film coatings to reduce their brittleness. Suitable plasticizers include, for example, polyethylene glycol such as PEG300, PEG400, PEG600, PEG1450, PEG3350, and PEG800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers also function as dispersants or wetting agents.

[0191] Examples of solubilizing agents include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycoflor, transktol, propylene glycol, and dimethyl isosorbide.

[0192] Examples of stabilizers include various compounds such as antioxidants, buffers, acids, and preservatives.

[0193] The suspending agents include polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30), vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol having molecular weights of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, and hydroxymethylcellulose acetate stearate. Examples of compounds include polysorbate-80, hydroxyethylcellulose, sodium alginate, gums (e.g., tragacanth gum and acacia gum, guar gum, xanthan gum, etc.), sugars, cellulose compounds (e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, etc.), polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, and povidone.

[0194] Examples of surfactants include sodium lauryl sulfate, sodium doxate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, polakixomer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, such as Pluronic® (BASF). Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol 10 and octoxynol 40. In some cases, surfactants are included to enhance physical stability or for other purposes.

[0195] Examples of thickening agents include methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, arginate, gum arabic, chitosan, and combinations thereof.

[0196] Examples of humectants include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium doxate, sodium oleate, sodium lauryl sulfate, sodium doxate (sodium doccusate), triacetin, Tween 80, vitamin E TPGS, and ammonium salts.

[0197] Treatment regimen In some embodiments, the pharmaceutical composition comprising ARC or ANC according to the present invention is administered for therapeutic purposes. In some embodiments, the pharmaceutical composition is administered once daily, twice daily, three times daily, or more frequently. The pharmaceutical composition is administered daily, daily, every other day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times a month, or more frequently. The pharmaceutical composition is administered for at least one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, two years, three years, or more.

[0198] In some embodiments, one or more pharmaceutical compositions in the ARC or ANC according to the present invention are administered simultaneously, sequentially, or at intervals. In some embodiments, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In further cases, one or more pharmaceutical compositions are administered at intervals (for example, the first administration of the first pharmaceutical composition is on day 1, followed by the administration of at least the second pharmaceutical composition after an interval of at least 1, 2, 3, 4, or 5 days).

[0199] In some embodiments, two or more different pharmaceutical compositions are co-administered. In some examples, two or more different pharmaceutical compositions are co-administered simultaneously. In some cases, two or more different pharmaceutical compositions are co-administered consecutively without intervals between administrations. In other cases, two or more different pharmaceutical compositions are co-administered consecutively with intervals of approximately 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or longer between administrations.

[0200] If the patient's condition improves, the physician may, at their discretion, continue administering the composition, or temporarily reduce or temporarily discontinue the dose of the administered composition for a specific period (i.e., a “drug-free period”). In some cases, the length of the drug-free period may vary between 2 days and 1 year, including, but are not limited to, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 28, 35, 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350, or 365 days. Dose reductions during drug-free periods range from 10% to 100%, including, but are not limited to, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0201] Once the patient's condition improves, a maintenance dose is administered as needed. Subsequently, depending on the symptoms, the dose, frequency, or both may be reduced as appropriate to maintain the improved disease, disability, or condition.

[0202] In some embodiments, the amount of a given drug corresponding to such a quantity varies depending on factors such as the specific compound, the severity of the disease, and the individual characteristics of the subject or host requiring treatment (e.g., body weight), but is still conventionally determined in ways known in the art, according to the specific circumstances surrounding the case, including, for example, the specific drug being administered, the route of administration, and the subject or host being treated. In some examples, the desired dose is conveniently provided as a single dose, or as divided doses administered simultaneously (or over a short period) or at appropriate intervals, for example, as two, three, four or more partial doses per day.

[0203] Given the large number of variables in individual treatment regimens and the frequent deviations from these recommendations, the aforementioned ranges are merely suggestions. Such dosages will vary depending on several variables, including, but not limited to, the activity of the compound used, the disease or condition being treated, the mode of administration, the requirements of the individual patient, the severity of the disease or condition being treated, and the physician's judgment.

[0204] In some embodiments, the toxicity and therapeutic effect of such a treatment regimen are determined by standard pharmaceutical procedures in cell culture or experimental animals, including, but not limited to, the determination of the LD50 (lethal dose for 50% of the population) and ED50 (therapeutic dose for 50% of the population). The dose-to-therapeutic ratio is the therapeutic index, which is expressed as the ratio between the LD50 and the ED50. Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used when formulating a range of doses for use in humans. Doses of such compounds are preferably within the range of blood concentrations containing the ED50 with minimal toxicity. Doses vary within this range depending on the dosage form used and the route of administration utilized.

[0205] Kit / Manufactured product In certain embodiments, one or more compositions and methods and kits and products for use are described herein. Such kits include a holder, package, or container divided to receive one or more containers, such as vials, tubes, etc., and each container(s) includes one of the individual elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials, such as glass or plastic.

[0206] The products provided herein include packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging materials suitable for the selected formulation and the intended mode of administration and treatment. For example, a container(s) may contain a nucleic acid molecule described herein that specifically binds to an immunomodulatory gene or RNA encoded thereby. Such a kit may optionally include an identification description or label or instruction manual relating to its use in the method described herein.

[0207] A kit typically includes a label listing the contents and / or instructions for use, as well as accompanying documentation containing instructions for use. A set of instructions is also typically included.

[0208] In one embodiment, the label is on the container or associated with the container. In one embodiment, the label is on the container if the letters, numbers, or other characters forming the label are attached to, molded, or etched onto the container itself, and the label is associated with the container, for example, as accompanying documentation, if it is located within a container or holder that also houses the container. In one embodiment, the label is used to indicate that the contents should be used for a particular therapeutic purpose. The label also indicates instructions for the use of the contents, such as in the methods described herein.

[0209] In certain embodiments, the pharmaceutical composition is provided as a pack or dispenser device containing one or more unit dosage forms comprising the compounds provided herein. For example, the pack includes metal or plastic foil, such as blister packaging. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accompanied by a notice associated with the container in a form prescribed by a government agency that regulates the manufacture, use, or sale of pharmaceuticals, the notice reflecting the government agency's approval of the form of the drug for human or veterinary administration. Such notice is, for example, a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. [Examples]

[0210] The examples shown below are for illustrative purposes only and are intended to illustrate certain specific embodiments of the present disclosure. However, the claims should not be limited in any way by the examples described herein. Various modifications and alterations to embodiments of the present disclosure will be apparent to those skilled in the art and include, but are not limited to, those relating to the chemical structures, substituents, derivatives, formulations, or methods of the present disclosure. Such modifications and alterations can be made without departing from the spirit of the present disclosure and the accompanying claims.

[0211] Example 1: Targeted RNA delivery to immune cells for the treatment of autoimmune diseases In this embodiment, the inventors provide a proof of concept that a direct antibody-RNA conjugate (ARC) platform can be used to deliver siRNA or antisense oligonucleotides (ASOs) to immune cells, such as myeloid cells and lymphocytes. These RNA-ARCs can be used to specifically inhibit mRNA related to key immune pathways, including PIK3CA and TNF, as well as other known and novel pathways involved in immunomodulation, tolerance, and metabolism. siRNAs that inhibit key immunological targets (Glut1, PI3K, BTK, TNF, RORC) delivered via ARC retain their function and inhibit the activation of their respective immune pathways, making them attractive targets in the therapeutic space for autoimmune diseases. Conversely, ASO-mediated inhibition of CD39, an ectonucleotidase involved in the production of extracellular adenosine by ATP hydrolysis, results in increased T cell activation and cytokine production [PMID:30871609]. CD39 delivered by ARC retains its activity and should be considered a promising approach in cancer treatment.

[0212] INX201, a humanized anti-human VISTA antibody on a human IgG1 / kappa skeleton having the L234A / L235A / E269R / K322A silencing mutation in the Fc region, was used as the payload delivery vehicle. In this example, the inventors demonstrate the following: 1) The inventors have found that by conjugating siRNA or ASO to anti-VISTA Mab, RNA can be efficiently delivered into target cells by VISTA binding and internalization. 2) ARC has similar binding and internalization properties to free INX201 Mab. 3) Known, publicly available siRNA payloads (https: / / www.idtdna.com / pages / products / functional-genomics / dsirnas-and-trifecta-rnai-kits) that are immunologically relevant and specific to knock down genes in a standard transfection setting (inhibiting target mRNA levels to <30%) will retain their function when conjugated to Mab and delivered into target cells. 4) siRNA conjugated with anti-VISTA Mab INX201 promoted siRNA accumulation and function within K562-VISTA cells. 5) siRNA conjugated with anti-VISTA Mab INX201 exhibited the function of reducing proliferation and cytokine production in human PBMCs and T cells. 6) CD39 ASO[PMID:30871609] conjugated to anti-VISTA Mab INX201 had the function of increasing cytokine production in human PBMCs.

[0213] Abbreviations used in this example PBS (phosphate-buffered saline) Fc antibody heavy chain constant region (hinge / CH2 / CH3) Mab monoclonal antibody Fab antibody fragment antigen-binding region min hr time RPMI culture medium (developed at Roswell Park Memorial Institute) PSG (Penicillin, Streptomycin, Glutamine) FBS (Fetal Bovine Serum) KO (Knockout) - Deficiency of a specific gene WT wild type ml (milliliter) μL (microliter) MFI average fluorescence intensity (a signal used in flow cytometry) EC50 (Median Effective Concentration) nM nanomolar concentration rpm (revolutions per minute) RT room temperature PBMC peripheral blood mononuclear cells Xeno-GvHD (Xenograft-versus-host disease) (animal model of autoimmune disease) VISTA is a V-domain Ig suppressor for T cell activation. ASO Antisense Oligonucleotides siRNA: Small interfering RNA (dsRNA of 20-24 nucleotides) ARC antibody RNA conjugate qRTPCR (Quantitative Reverse Transcription Polymerase Chain Reaction) LPS (Lipopolysaccharide) DAR drug-antibody ratio QC quality control

[0214] overview The applicant previously developed the IgG1 anti-VISTA Mab, INX201. This Mab is a humanized anti-human VISTA antibody on a human IgG1 / kappa skeleton with the L234A / L235A / E269R / K322A silencing mutation in the Fc region, and was used as a payload delivery vehicle.

[0215] In this example, the inventors describe relevant experiments to establish conjugation and functional proof-of-concept studies using an INX201 delivery vehicle and publicly available siRNA or ASO payloads (see Tables 1-2). Exemplary Ab sequences are shown in Appendix 1, and exemplary payload sequences used for conjugation are identified in Appendix 2. [Table 1] [Table 2]

[0216] Materials and methods K562-VISTA cell line assay: binding, internalization, transfection, qPCR material ● U-bottom 96-well plate (Falcon, #353077) ●K562-VISTA (autologously prepared from VISTA-expressing cells, WT K562, and ATCC CCL-243) ●IMDM medium (ATCC, 30-2005) + 10% FBS (ATCC, 30-2020) + P / S (Gibco, 15140-122) G418 added in a 1:100 ratio (selection for VISTA+ expression) ●Lonza Nucleofector 2b ●MACS Quant ●Cell line Nucleofector Kit V (Lonza, VCA-1003) ●DNA: pRP[Exp]-CAG>3xNLS / EGFP, Vector ID: VB900137-6122yyc (VectorBuilder, MaxH VB900137-6122yyc), for generating a GFP+ cell pool, the vector contains the PuroR puromycin resistance gene. ● Puromycin ● Anti-human IgG Fc secondary Ab (Biolegend, B172272) ●Test sample: Free INX201 or INX201 ARC, e.g., (INX201-eGFP_siRNA_Cy5) ●All payloads were synthesized by IDT and contained a 3'-sense strand Cy5 label.

[0217] K562-VISTA suspension cells 1. After reaching 0.5 - 1×10 6 cells / mL, passage the cells. Maintain the culture between 0.5 - 1×10 6 viable cells / mL. Seed at 1 - 2×10 5 cells / mL. Passage 2 days before nucleofection. Optimal density for nucleofection: 2 - 5×10 5 . 2. The basal medium for this cell line is ATCC formulated Iscove's Modified Dulbecco's Medium (Catalog No. 30 - 2005). To prepare the complete growth medium, add the following components to the basal medium: FBS at a final concentration of 10% 3. The culture can be maintained by adding or replacing fresh medium. Start a new culture at 1×10 5 viable cells / mL. Passage at 1×10 6 cells / ml.

[0218] Transfection Day 0 1. Add the co - reagent to the Nucleofector solution. a. The ratio of Nucleofector solution to co - reagent is 4.5:1. b. For a single reaction, use 82 μl of Nucleofector solution and 18 μl of co - reagent to make a total reaction volume of 100 μl. 2. Prepare a 12 - well plate by filling each well with 1.5 mL of medium and equilibrating the medium in the plate in the incubator (1×10 6 cells / sample). 3. Centrifuge the required number of cells (1×10 6 cells / sample) at 200×g for 10 minutes at room temperature in a 15 mL conical tube. Completely remove the supernatant. 4. Resuspend the cell pellet in 100 μL of room temperature Nucleofector solution per sample. a. Avoid leaving cells in the Nucleofector solution for extended periods (15 minutes or more). 5. Mix 100 μl of cell suspension with 2 μg of DNA (eGFP plasmid) or 20-200 nM of siRNA or ASO payload. 6. Transfer the cell / DNA or cell / RNA suspension to a certified cuvette (included in the kit). 7. Select program: K562: T-016 (or T-003). Add 8.0.5 mL of pre-equilibriumized culture medium to the cuvette and gently transfer the sample to a pre-equilibriumized 12-well plate (final volume of medium per well: 2 mL). Day 1 (24 hours) 1. Proceed to the qRTPCR protocol for RNA isolation and payload testing. Day 2 (Formation of a stable GFP+ cell pool) 1. Start antibiotic selection 48 hours after transfection (puromycin). a. K562: 0.5 μg / mL for 7-21 days 5 x 10 oz in 2.1% BSA / PBS 6 Resuspend the cells in 1 / mL and periodically test them by flow cytometry (eGFP signaling).

[0219] Resuspension of ASO and siRNA from IDT 1) Store the received items at -20°C (to keep them dry). 2) Centrifuge the tube in 500g for 5 minutes. 3) Resuspend the ASO in TE buffer, 100 μM stock solution (30 minutes). If precipitate remains, heat the oligo at 55°C for 1-5 minutes, then vortex thoroughly. 4) Resuspend the siRNA (double-stranded) in nuclease-free double-stranded buffer (or nuclease-free water) or 100 μM stock. 5) Vortex briefly, then heat at 94°C for 2 minutes, remove the tube from the heat source, and let it cool to room temperature, where the product is a resuspended double chain. 6) The resuspended ASO and siRNA should be stored at -20°C.

[0220] VISTA binding and internalization assay 1. Seed K562-VISTA cells in a U-bottom 96-well plate (see the table below for volume and cell count). [Table 3] 2. Preparation of the INX201-RNA dilution series (5× titration [2×] starting at 60 μg / mL) a.4 points +0Ab 3. Add Ab titration to the cells. a. INX201-eGFP-siRNA-Cy5 - 150 μL / well b.INX201 free Mab 4. Incubate on ice for 30 minutes. After 5.0 hours, collect 50 μL of aliquots, transfer them to a 96-well V-bottom plate maintained on ice, add 200 μL of ice-cold PBS, and centrifuge at 500 g for 2 minutes. a. Perform α-hIgG staining on the collected cells (Step 8). 6. Bring the plate containing the remaining cells to 37°C. 7. Collect 50 μL aliquots at 30 minutes, 1 hour, 2 hours, and 4 hours, transfer to a 96-well V-bottom plate maintained on ice, add 200 μL of ice-cold PBS, and centrifuge at 500 g for 2 minutes. 8. Perform α-hIgG staining at each time point (including 0 hours): a. Add 50 μL of secondary Ab mix (1:150 2× mix, 1:300 final). b. Stain at room temperature for 20 minutes. c. Wash with 200 μL of PBS. d. Centrifuge 500g at 4°C for 5 minutes to precipitate. e. Resuspend in 100 μL of PBS. 9. Perform flow cytometry and analyze the results. a. INX201-siRNA-Cy5 * - MACS Quant [Table 4] Analyze secondary Ab binding to INX201 or ARC as MFI from 10.0-hour aliquots (stained for 30 minutes on ice). 11. Analyze internalization based on the time course of the MFI signal.

[0221] Quantitative RT-PCR protocol material ●RNeasy Plus Mini Kit (Qiagen 74136) or NucleoSpin® RNA Plus (Macherey-Nagel 740984) ● TaqMan reverse transcription reagent (ThermoFisher N8080234) ● Taqman Master Mix 2 x Kit (ThermoFisher 4369016) ● Taqman probe for human GAPDH: Hs01922876_u1 (ThermoFisher 4331182) ● Taqman probe for human targets (ThermoFisher 4331182); see Table 5 below. The same protocol is used for K562 or immune cells (PBMCs). [Table 5]

[0222] RNA isolation RNeasy Plus Mini Kit (Qiagen 74136) ● The RNeasy Plus procedure isolates all RNA molecules longer than 200 nucleotides. This procedure results in mRNA enrichment. ● To obtain optimal RNA yield and purity, it is essential to use the correct amount of starting material (<2e6 large cells). ● Perform all steps of the procedure at room temperature. 1. Harvest the cells. Resuspend the cells in 375 μl of RLT buffer. ● Buffer RLT Plus may form precipitates during storage. If necessary, redissolve by warming and then place at room temperature. 2. Transfer 370 μl of the cell lysate to a gDNA Eliminator spin column placed in a 2 ml collection tube (supplied). Centrifuge at ≥8,000 × g for 30 seconds. Discard the column and save the flow-through. ● After centrifugation, ensure that no liquid remains on the column membrane. If necessary, repeat the centrifugation. 3. Add 350 μl of 70% ethanol to the flow-through and mix well by pipetting. ● If some lysate is lost during homogenization and DNA removal, adjust the amount of ethanol accordingly. When purifying RNA from specific cell lines, precipitates may be visible after ethanol addition. This does not affect the procedure. 4. Transfer a maximum of 700 μl of the sample, including any possible precipitates formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Gently close the lid and centrifuge at ≥8,000 × g for ǂ15 seconds. Discard the flow-through. 5. Add 700 μl of Buffer RW1 to the RNeasy spin column. Gently close the lid and centrifuge at ≥8,000 × g for 15 seconds to wash the spin column membrane. Discard the flow-through. 6. Add 500 μl of Buffer RPE to the RNeasy spin column. Gently close the lid and centrifuge at ≥8,000 × g for 15 seconds to wash the spin column membrane. Discard the flow-through. ● Buffer RPE is supplied as a concentrate. Add 4 volumes of ethanol (96 - 100%) as indicated on the bottle before first use. Add 7,500 μl of Buffer RPE to the RNeasy spin column. Gently close the lid and wash the spin column membrane by centrifugation at ≥8,000 × g for 2 minutes. 8. Place the RNeasy spin column into a new 2 ml collection tube (included) and discard the old collection tube containing the flow-through. Centrifuge at full speed for 1 minute. 9. Place the RNeasy spin column into a new 1.5 ml collection tube (included). Add 30-40 μl of RNase-free water directly to the spin column membrane. Gently close the lid and centrifuge at ≥8,000 × g for 1 minute to elute the RNA. 10. Evaluate the concentration using nanodrops.

[0223] Reverse transcription ● Especially when downstream priming efficiency is low, try using a random hexamer first for long reverse transcripts or reverse transcripts containing hairpins. ●The RNA segment that is transcribed and subsequently amplified may be at least 3kb long. ● Do not increase reverse transcriptase levels. For the synthesis of longer RNA transcripts, the incubation time may be increased to 60 minutes. ●When using DNA with a high G+C content, thawing at 97°C for the first few cycles helps generate a single-strand template for PCR amplification. 1. The reaction volume is 20 μl. 2. Thaw all reagents and keep them on ice. Mix the components and centrifuge briefly. Store the RNase inhibitor and MultiScribe RT in the freezer. 3,400 ng of RNA is separated and water is added to bring the final volume to 8.6 μl. 4. Combine the following ingredients and mix well. [Table 6] 5. Add 11.4 μl of the reaction mixture to the RNA. 6. Set the parameters: 10 minutes at 25°C and 37°C, 30 minutes at 99°C, and 5 minutes at 4°C.

[0224] Another method for reverse transcription GoScript reverse transcriptase (Promega) 1. The reaction volume is 20 μl. 2. Thaw all reagents and keep them on ice. Mix the components and centrifuge briefly. Store the RNase inhibitor and MultiScribe RT in the freezer. 3,800 ng of RNA is separated and water is added to bring the final volume to 10 μl. 4. Combine the following ingredients and mix well. [Table 7] 5. Add 10 μl of the reaction mixture to the RNA. 6. Set the parameters: 25°C, 5 minutes; 42°C, 60 minutes; 95°C, 5 minutes; 4°C, long time.

[0225] qPCR 1. The reaction volume is 20 μl. 2. Combine the following ingredients. [Table 8] Spin. Set parameters: 95°C, 10 minutes; 95°C, 15 seconds; 60°C, 1 minute, 40 cycles.

[0226] Isolation of human PBMCs and T cells Isolation of human PBMCs Human peripheral blood was obtained from apheresis cones provided by volunteer donors through the DHMC blood donor program. The cone blood was diluted 1:4 in PBS and carefully placed on 13 ml of Histopaque 1077. After centrifugation at 850 g for 20 minutes (room temperature, no brake on deceleration), mononuclear cells were collected from the Histopaque / PBS interface. After one wash in PBS, the PBMCs were frozen at 100 × 10⁶.6 at cells / ml (90% DMSO, 10% FBS), or resuspended at 10×10 6 cells / ml (complete RPMI medium).

[0227] Isolation of human T cells 1. T cells were isolated from human blood according to the manufacturer's instructions of the EasySep Direct Human T Cell Isolation Kit. a. Add 50 μL of the Isolation Cocktail of the EasySep Direct Human T Cell Isolation Kit (STEMCELL Technologies, catalog number 19661) (50 μL / mL of sample) to the blood. Invert the tube several times to mix well. b. Vortex the RapidSpheres for 30 seconds. Add 50 μL of the RapidSpheres to the blood and mix well. c. Incubate at room temperature for 5 minutes. d. Transfer the mixture to a 14 mL polystyrene round-bottom tube. e. Add D-PBS to make the volume 14 mL and gently mix using pipetting 2 - 3 times. f. Place the tube (without lid) in a magnet and incubate at room temperature (RT) for 5 minutes. g. Carefully pipette the concentrated cell suspension into a new 14 mL tube. h. Add 50 μL of the RapidSpheres blood to the concentrated cells and mix well. Incubate at RT for 5 minutes. i. Place the tube (without lid) in a magnet and incubate at room temperature (RT) for 5 minutes (second separation). j. Carefully pipette the concentrated cell suspension into a new 14 mL tube. k. Place the new 14 mL tube containing the second separation cells in a magnet and incubate at RT for 5 minutes. l. Carefully pipette the concentrated cell suspension into a new 50 mL tube. Supplement with D-PBS to 50 ml. Centrifuge 515g of m.mg at 10°C for 5 minutes to precipitate the cells, then discard the supernatant. Resuspend the cells in n.5 mL of D-PBS and count them. 2. Count T cells using a cell counter with AOPI. Add 1 × 10⁻⁶ units of D-PBS to the volume. 6 Adjust to reach cells / mL.

[0228] Labeling of PBMCs or T cells using Cell Trace Violet 1. Add 20 μl of DMSO to the Cell Trace (trademark) Violet staining solution vial. Add directly to 2.10 ml of cell suspension to a final concentration of 2 μM. 3. Incubate the cells in a 3.37°C water bath for 20 minutes. Add 4.40 ml of RPMI to the cells to allow them to absorb the unbound dye, incubate the cells for 3 minutes, centrifuge the cells at 300 x g for 5 minutes, and resuspend the cell pellet in pre-warmed complete RPMI.

[0229] Human PBMC and T cell activation and proliferation assay material ●RPMI1640 medium (Gibco, 11875-093) ● 10% FBS (ATCC, 30-2020) ● 100 U / ml penicillin / streptomycin (Gibco, 15140122) ● U-bottom 96-well plate ● INX201 and INX201 antibody RNA conjugate (ARC) using the following payloads: RORC, PIK3CA, TNFa, BTK, Glut1, CD45 (siRNA), CD39 ASO

[0230] Anti-CD3 / CD28 beads stimulate human PBMCs or T cells. Human T cells were activated for 3 days using CD3 / CD28 T cell activating dynabeads. a. Mix the DynaBeads by vortexing and transfer 2.5 μl / well of beads into a 2 ml Eppendorf tube. b. Add up to 2 ml of culture medium, vortex, and place the tube on a magnet. c. Once the beads have settled, remove the culture medium and resuspend the beads in a volume equivalent to the initial volume taken from the vial. d. Add beads directly to the cells, seed them in a U-bottom 96-well plate, and add 100 μl of culture medium to bring the total volume to 200 μl. e. Cell count: 500,000 PBMCs / well, 100,000 T cells / well. The ratio of beads to T cells is 1:2.

[0231] LPS stimulation of human PBMCs 1. PBMCs were stimulated with 10 ng / ml LPS (Ams bio). After 2.48 hours, the culture medium was collected for cytokine analysis, and the cells were collected for flow cytometry.

[0232] INX201 or ARC titration 1. Prepare INX201 or ARC in culture medium to 2× final concentration (200 nM as the maximum concentration) and generate serial dilutions. DAR was not quantified in these experiments; therefore, the Ab concentration was used in the ARC titration experiment.

[0233] Flow cytometry-based growth analysis 1. After incubation, centrifuge the plate at 515g for 4 minutes and collect the supernatant for cytokine analysis (freeze at -80°C until use). 2. Wash the cell pellet with PBS, centrifuge, and remove the supernatant. Cells were stained in 3.50 μl of the corresponding antibody mix (in PBS) with shaking (400 rpm) at room temperature for 30 minutes. In viability experiments, cells were stained in annexin buffer (Biolegend, 422201) and washed with PBS. Cells were resuspended in 100 μl of PBS and analyzed by flow cytometry. [Table 9] [Table 10] [Table 11]

[0234] result Selection of siRNA and ASO payloads The applicant selected a variety of immunologically relevant targets, including (1) existing approved drugs against (TNFa), (2) validated pathways with on-target toxicity concerns (PI3K), and (3) novel targets (Glut1). See Table 2. The first class of targets was used for proof-of-concept studies (TNFa), while the other classes of targets support the inherent advantages of targeted ARC / ANCs that preferentially target myeloid and lymphocytes. These immunotargeted RNA-ARCs should minimize or eliminate toxicity associated with the nature of the targets, such as non-immune tissue targeting (PI3K) and / or broad expression of the target (Glut1) or transcription factor (RORC), which have so far limited or hindered the success of developing biologics and / or small molecule inhibitors against these types of "difficult" targets (TNFa [PMID:34301319, PMID:2915 8574];BTK[PMID:27192942, PMID:31431692, PMID:33122850, PMID:36903645];PI3K[PMID:31928691, PMID:34127844, PM ID:26093105];RORC[PMID:30010338, PMID:34040108];Glut1[PMID:35878663, PMID:34018847];CD39[PMID:30871609]). [Table 12]

[0235] siRNA or ASO non-conjugated payloads (1-4 per target) were ordered from IDT (https: / / www.idtdna.com / pages / products / functional-genomics / dsirnas-and-trifecta-rnai-kits). K562-VISTA cells were transfected with 200 nM siRNA (or ASO) only, and targeted knockdown was analyzed by qRTPCR using the ddct method and reported as a factor relative to the control. Scrambled siRNA or ASO controls were also ordered from IDT (Figures 1-2). Payloads marked with (*) were selected for conjugation to the delivery vehicle (anti-VISTA Mab INX201).

[0236] As shown in Figure 1, immunologically relevant siRNA and ASO payloads were identified and tested. K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and targeted knockdown was analyzed by qRTPCR using the ddct method and reported as a factor relative to the control. Scrambled siRNA or ASO controls were also ordered from IDT. Payloads marked with (*) were selected for conjugation to the delivery vehicle (anti-VISTA Mab INX201).

[0237] As further shown in Figure 2, additional immunologically relevant siRNA and ASO payloads were tested in the ARCs of the present invention. In these experiments, K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and targeted knockdown was analyzed by qRTPCR using the ddct method and reported as a ratio relative to the control. Scrambled siRNA or ASO controls were also ordered from IDT. Payloads marked with (*) were selected for conjugation to the delivery vehicle (anti-VISTA Mab INX201). Results obtained in CD39 ARC and other ARCs, including payloads targeting other genes, are described herein.

[0238] Antibody-RNA conjugation and QC. The anti-VISTA Mab and payloads used for conjugation are listed in Tables 1-2 and Appendix 1-2. An oligoconjugation kit (Abcam, code ab218260) was used for all conjugations. All RNA oligos were purified by HPLC and resuspended at 100 μM. A widely used nonspecific conjugation strategy, lysine-based conjugation via an amine reactive group, was employed. All payloads were synthesized using IDT and contained a 3'-sense strand labeled with Cy5 and a 5'-amine on the antisense strand (see Appendix 2).

[0239] Reduced SDS-PAGE and silver staining. The level of ARC conjugation was analyzed by SDS-PAGE and subsequent silver staining for protein visualization. Briefly, ARC was mixed with reducing 2×Laemmli sample buffer (Bio-Rad, no. 1610737) and incubated at 80°C for 5 minutes. The reduced samples were separated using 4–15% Mini-Protean TGX Precast Gel (Bio-Rad, no. 4561083) according to the manufacturer's instructions. After electrophoresis, the gel was rinsed with Mili-Q water and the proteins were stained using SilverQuest reagent (Thermo Fisher, no. LC6070). CD45 siRNA ARC and SOCS1 ASO ARC were analyzed as representative ARCs for both siRNA and ASO payloads. Both showed the expected conjugation patterns (Figure 3). Nanodrop measurements performed for ASO ARC were 37.2 μM for RNA and 8.0 μM for protein. Therefore, the DAR was approximately 4.65.

[0240] Effective conjugation was confirmed by SDS-PAGE experiments using INX201 ARC, as shown in Figure 3. In these experiments, INX201 ARC was separated by reduced SDS-PAGE and subsequent silver staining. Lane labels: 201 - Free INX201 anti-VISTA Mab; CD45 - ARC containing CD45 siRNA; SOCS1 - ARC containing SOCS1 ASO; Different patterns of multiple conjugates were observed, confirming efficient conjugation. Based on the molecular weight of the payload (approximately 17 vs. approximately 6 kDa), the HC or LC shift by siRNA was more significant than that by ASO.

[0241] Binding and internalization (to the VISTA target) were compared between free INX201 and INX201 eGFP siRNA ARC. K562-VISTA cells were incubated with 200 nM of free Mab or ARC. Binding and internalization were measured over time. Binding to the target was comparable between the free antibody and ARC, as is evident from time zero (visualized as 0.1 hours, Figure 4A). Internalization was also similar between the free INX201 antibody and ARC, and was rapid and efficient, as previously described by the inventors. Specifically, within 30 minutes, more than 90% of the antibody was internalized in both ARC and free Ab samples. This is evident from the fact that the antibody was not detected on the cell surface in the time-course assay (Figure 4A).

[0242] Intracellular RNA was detected using Cy5 labeling present on siRNA (considering that antibodies are not detectable on the cell surface due to internalization in ARC samples, the inventors assumed that most Cy5 detection occurred intracellularly). As demonstrated by time-course experiments, Cy5 was readily detectable in K562-VISTA cells and maintained throughout the experimental period (24 hours).

[0243] The experimental results in Figure 4 demonstrate that exemplary ARC efficiently binds to K562-VISTA cells, translocates from the surface to the interior, and provides efficient and long-term retention of siRNA within K562-VISTA cells. Free INX201 (circled line) or eGFP ARC (squared line) was used at 200 nM. A) Time course of antibody binding and internalization. B) RNA accumulation assay measured by Cy5. MFI - mean fluorescence intensity, representative of the two independent experiments shown.

[0244] ARC function testing in K562-VISTA cells K562-VISTA cells were transfected with plasmid DNA expressing eGFP, and a GFP+ cell pool was used 14–28 days after transfection. The cell pool was treated with either no drug or eGFP ARC. eGFP or CD45 protein levels were measured by flow cytometry (Table 10). Maximum inhibition of eGFP protein levels of approximately 50% was achieved by ARC, which was similar to the eGFP mRNA knockdown levels measured by qPCR in the same cell pool treated with transfected eGFP free siRNA (Figure 5A).

[0245] K562-VISTA cells are naturally CD45+. Since CD45 is expressed on the cell surface, it presents an attractive target for proof-of-concept studies measuring targeted (CD45) knockdown. Cells were treated either untreated or transfected with siRNA or CD45 ARC (200 nM). In the transfection experiment, CD45 levels were measured at 48 hours by flow cytometry, and in the CD45 ARC experiment, CD45 levels were measured at 72 hours. Two independent experiments were performed, and the results were similar (Figures 5B-C). Approximately 50% knockdown of CD45 protein from the surface was observed in ARC-treated cells, but not in untreated cells. Transfected siRNA inhibited the protein to similar levels (Figure 5B), while free siRNA added to cells without transfection failed to inhibit CD45 levels (Figure 5C). Excess free siRNA was added to ensure that the knockdown observed by ARC was not attributable to residual free siRNA payload in the ARC conjugate. [Table 13]

[0246] As can be seen from the experimental results in Figures 5A-C, INX201 ARC knockdown protein expression is equivalent to in vitro transfection. K562-VISTA WT cells or an eGFP+ cell pool were used in this experiment. In the experiment in Figure 5A, cells were treated for 28 hours with no drug (left bar) or with 200 nM eGFP ARC (right bar). The highest level of eGFP protein knockdown was established by transfecting the same cells with eGFP siRNA (approximately 50%, measured at 24 hours). ARC-mediated knockdown was similar to the maximum possible knockdown level (based on siRNA sequence). In the experiment in Figure 5B, the dashed line represents the expected highest level of CD45 protein knockdown based on the potency of the payload (siRNA), which is 50% when measured from free transfected CD45 siRNA at 48 hours (right bar). Cells were treated for 72 hours with 200 nM eGFP ARC (center bar) or no drug (left bar). In the experiment shown in Figure 5C, CD45 levels were measured in replicate experiments. Cells were treated for 72 hours with no drug (left bar), free siRNA, no transfection (center bar), and 200 nM ARC (right bar).

[0247] ARC function testing in immune cells In further experiments shown in Figures 6A–C, human PBMCs were stimulated with LPS or anti-CD3 / CD28 DynaBeads as described in Materials and Methods. For cytokine analysis by Luminex, the culture medium was collected at 48 hours for LPS stimulation and at 72 hours for CD3 / CD28 stimulation. TNFa ARC was supplied at the time of stimulation at concentrations ranging from 200 nM to 0.32 nM. Free siRNA only (untransfected) was used as a control and applied at 200 to 1000 nM. TNFa protein, as measured by Luminex, was efficiently knocked down by TNFa ARC in a dose-dependent manner in both LPS-stimulated PBMCs (Figure 6A) and anti-CD3 / CD28-stimulated PBMCs (Figure 6B). The levels of knockdown were over 80% (LPS stimulation) and approximately 50% (anti-CD3 / CD28 stimulation). In anti-CD3 / CD28 stimulation, target TNFα was knocked down at the mRNA level using cells harvested at 72 hours for qRTPCR. In this case, approximately 60% targeted knockdown of RNA levels was observed in PBMCs, which correlated well with a 50% decrease in TNFα cytokine levels (Figure 6C).

[0248] More specifically, the experiments shown in Figures 6A-C demonstrate that INX201 ARC inhibits TNFα from PBMCs. Human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated with TNFα ARC (0-200 nM) or free RNA (200-1000 nM) for 48 hours (LPS) or 72 hours (beads). TNFα levels were efficiently reduced in a dose-dependent manner with ARC, but not with free siRNA. C) Efficient targeted knockdown was confirmed by qRTPCR performed on ARC-treated versus untreated PBMCs collected at 72 hours. Levels without ARC are visualized as 0.1 nM (due to the logarithmic scale).

[0249] In vitro PBMC or T cell proliferation assays demonstrate that blocking immunologically relevant targets with siRNA delivered via ARC (Glut1, PI3K, BTK, TNF, RORC) results in reduced cytokine production or T cell proliferation without affecting T cell viability (Figures 6–13). The reduction in proliferation is primarily demonstrated by a slowing of growth (the last two peaks of newly proliferating cells are affected), which highlights a key feature of siRNA-ARC therapy in controlling effector cell proliferation without acting as a systemic immunosuppressant.

[0250] Specifically, TNFα ARC slowed T cell proliferation, but free TNFα siRNA did not (Figure 7A). Similarly, PI3K ARC also slowed T cell proliferation in a dose-dependent manner, but free PI3K siRNA did not (Figure 7B).

[0251] Further experiments, shown in Figure 8, further demonstrated that INX201 ARC slows T cell proliferation. Human PBMCs were activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated for 72 hours with (A) TNFa ARC (0-200 nM) or free RNA (200-1000 nM) or (B) PI3K ARC (0-200 nM) or free RNA (200-1000 nM). As described above, exemplary ARCs efficiently dose-dependently reduced newly proliferating T cells, while free siRNA did not. Proliferation was analyzed by Cell Trace Violet dilution and visualized by flow cytometry. No ARC was visualized as 0.1 nM (due to the logarithmic scale). Curves were generated from three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). A single technical replication was used for each concentration point.

[0252] As described above, BTK ARC was tested in human PBMCs activated with LPS or anti-CD3 / CD28 DynaBeads. In LPS-stimulated experiments, BTK ARC, rather than free BTK siRNA, was able to reduce CD16+ monocyte activation, as measured by CD69 levels at 48 hours post-drug addition (Figure 8A). CD69 levels decreased to levels comparable to those of unstimulated cells. BTK ARC also delayed T cell proliferation, as measured at 72 hours post-stimulation (Figure 8B). Free INX201 antibody or free untransfected siRNA payloads did not affect these assays.

[0253] More specifically, the experiments shown in Figures 8A-B demonstrate the reduction of PBMC activation by INX201-BTK ARC. In these experiments, human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads (beads to T cell ratio 1:2) and treated with BTK ARC (0-200 nM, triangle) or free RNA (200-1000 nM, star) or free INX201 (square) for 48 hours (LPS) or 72 hours (beads). Curves were generated from a three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). CD69 levels were measured in A and newly proliferating cell percentage in B, with a single technical replication used for each concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale). Unstimulated-unstimulated cells; MFI-mean fluorescence intensity.

[0254] In the experiments shown in Figures 9A-B, Glut1 ARC was tested in purified human T cells stimulated with anti-CD3 / CD28 dynabeads for 72 hours. After incubation, the culture medium was collected, and cytokine levels were measured by Luminex for two human donors. Treatment of T cells with Glut1 ARC resulted in a dose-dependent reduction in IFNg and IL17A levels to 1 / 2 to 1 / 3 (Figures 9A-B).

[0255] More specifically, the experiments shown in Figures 9A-B demonstrate the reduction in cytokine production by INX201-Glut1 ARC, where purified human T cells were activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated with Glut1 ARC (0-200 nM) for 72 hours. A single technical replica was used for each concentration point. Two human donors were tested: Donor 1 - solid line, Donor 2 - dashed line. No ARC was visualized as 0.1 nM (due to the logarithmic scale). A) IFNg and B) IL17A were measured by Luminex.

[0256] In the experiments shown in Figures 10A-C, RORC ARC was also tested with purified human T cells stimulated with anti-CD3 / CD28 dynabeads for 72 hours. After incubation, the culture medium was collected, and cytokine levels were measured by Luminex for two human donors. Treatment of T cells with RORC ARC resulted in a dose-dependent reduction in IFNg, IL6, and IL12p40 levels to 1 / 2 to 1 / 4 (Figures 10A-C).

[0257] More specifically, in the experiments shown in Figures 10A-C, the reduction in cytokine production by INX201-Glut1 ARC was demonstrated using purified human T cells activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) and treated with RORC ARC (0-200 nM) for 72 hours. A single technical replica was used for each concentration point. Two human donors were tested: Donor 1 - solid line, Donor 2 - dashed line. No ARC was visualized as 0.1 nM (due to the logarithmic scale). A) IFNg, B) IL6, and C) IL12p40 were measured by Luminex.

[0258] Experiments shown in Figure 11 further demonstrated that CD39 knockdown using ARC enhances the immune response in stimulated PBMCs, in contrast to a decrease in T cell response. Specifically, these experiments showed a dose-dependent increase in IL-6 levels after PBMC treatment with CD39 ARC compared to free antibody INX201 (Figure 11).

[0259] More specifically, the experiment shown in Figure 11 demonstrated that INX201-CD39 ASO ARC targeting enhances the immune response in human PBMCs activated with anti-CD3 / CD28 beads and treated with CD39 ARC (0–200 nM, triangular) or free INX201 (0–200 nM, circular) for 72 hours. Curves were generated from a three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). A single technical replica was used for each concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale).

[0260] Delivery platform optimization: Mab and Fab To optimize the applicant's Ab delivery platform, the inventors designed INX201 Fab for use in conjugation to produce DAR2 antibody fragments with their respective payloads. To ensure that INX201 Fab functions correctly as a whole Mab, ELISA and cell-based competitive assays directly comparing INX201 Mab and Fab were performed (Figures 12A-B). Figure 12A shows that binding to the target (VISTA) is equivalent between INX201 Mab and Fab (EC50 of 0.1 nM vs. 0.7 nM, a 3-fold difference expected due to stoichiometry). In the competitive assay performed on the K562-VISTA cell line, cells were pre-incubated with increasing concentrations of unlabeled INX201 Mab or INX201 Fab to block targets on the cell surface, followed by detection of available residual targets on the surface using labeled INX201-AF488 antibody by flow cytometry. As is evident from the graph, both INX201 Mab and INX201 Fab can efficiently block VISTA targets on the cell surface in a dose-dependent manner (Figure 12B), confirming that INX201 Fab functionally behaves in exactly the same way as INX201 Mab.

[0261] Setting up VISTA ELISA. First, a 96-well flat-bottom plate (Thermo Scientific Nunc Immuno Maxisorp, catalog number 442404) was coated with 20 nM hIX50 (human VISTA ECD, manufactured by Aragen Bioscience for ImmuNext) in PBS for 1 hour at room temperature (RT). The wells were washed three times with PT (PBS containing 0.05% Tween20) and then blocked with PTB (PBS containing 0.05% Tween20 and 1% BSA) at room temperature for 30 minutes. INX201 Fab was diluted from 1000 nM to 0.02 nM in PTB and then added to the wells at room temperature for 1 hour, or INX201 Mab was diluted from 20 nM to 0.002 nM in PTB and then added to the wells at room temperature for 1 hour. After incubation, the wells were washed three times with PT, and then mouse anti-human kappa (Southern Biotech, catalog no. 9230-05) conjugated to HRP was used as the detection reagent at a 1 / 2000 dilution and incubated at room temperature for 1.5 hours. After three washes, TMB (Thermo Scientific, catalog no. 34028) was used as a colorimetric substrate to elucidate the ELISA reaction. After several minutes at room temperature, the reaction was stopped with 1 M H2SiO4. OD450 was read with a Molecular Devices Spectramax M3 plate reader and analyzed with SoftMaxPro software.

[0262] More specifically, Figure 12 shows platform optimization using anti-VISTA Fab instead of Mab. The experiment in Figure 12A compares the binding of INX201 Mab (square) and INX201 Fab (circle) to human VISTA ECD by ELISA. The experiment in Figure 12B shows the results of a K562-VISTA cell-based competitive assay, where increasing concentrations of INX201 Mab (square) or INX201 Fab (circle) before binding block available VISTA on the cell surface (hence the decrease in VISTA MFI measurements by INX201-AF488). Curves were generated from a three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). Technical replication with n=2 per concentration point was used. Ab-free was visualized as 0.0001 nM (due to the logarithmic scale).

[0263] Exemplary Test of the Effects of the Invention on T Cell Survival Rate (ARC) Human PBMCs were stimulated with anti-CD3 / CD28 DynaBeads as described in Materials and Methods. T cell viability was examined after 72 hours of stimulation using a dedicated experiment in which cells were stained with the antibody panel shown in Table 8. Live cells were defined as annexin V and live-dead negative. Apoptotic cells were double-positive for annexin V and live-dead, while necrotic cells were annexin V negative but live-dead positive. No difference was observed in cell viability or the percentage of apoptotic or necrotic T cells between INX201 ARC and free INX201, and no dose-dependent change in viability between Ab and ARC was observed (Figure 13).

[0264] More specifically, Figure 13 shows that exemplary ARCs did not affect T cell viability in experiments in which human PBMCs were activated with anti-CD3 / CD28 beads and treated with PI3K ARC (0-200 nM, triangle) or free INX201 (0-200 nM, square) for 72 hours. Curves were generated from a three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism9). A single technical replication was used for each concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale). Unstimulated-unstimulated cells.

[0265] Example 2: In vivo evaluation of anti-VISTA antibody RNA conjugates (ARCs) in GVHD models In this example, the efficacy of anti-VISTA antibody RNA conjugate (ARC) was evaluated in a GVHD model. INX201 was also used as the payload delivery vehicle in these experiments. These experiments provide further proof of concept that INX201 can be used as a delivery vehicle for the delivery of siRNA payloads to immune cells. In these proof-of-concept experiments, the siRNA payload targeted PIK3CA and GLUT3 (IDT). The Ab sequences used in these experiments are in Appendix 3, and the payload sequences used for conjugation are in Appendix 4.

[0266] Table 11 below shows the anti-VISTA and isotype control MAb, as well as the ARC, used for payload delivery. [Table 14]

[0267] As described above, the antisense strand of siRNA is an active drug. When delivered into the target cell by our VISTA delivery platform, it binds to its target mRNA, inducing its degradation, thereby reducing the levels of the target mRNA and protein.

[0268] Graft-versus-host disease (GvHD) model Because GVHD models are widely accepted autoimmune and inflammatory models, they were used to evaluate the efficacy of anti-VISTA antibody RNA conjugates (ARCs). Furthermore, humanized mouse models of xenograft-versus-host disease (GvHD) allow for in vivo studies of the effects of immunomodulatory compounds specific to human drug targets and inflammatory markers such as inflammatory cytokines. These GVHD models are based on the transfer of human peripheral blood mononuclear cells (PBMCs) into immunodeficient mouse strains.

[0269] The NSG (NOD-scid IL-2Rγnull) mouse GVHD model combines the features of a NOD / ShiLtJ background, a severe combined immunodeficiency mutation (scid), and IL2 receptor gamma chain deficiency. As a result, these mice lack mature T cells, B cells, or functional NK cells and have poor cytokine signaling, leading to better engraftment of human hematopoietic stem cells and peripheral blood mononuclear cells than any other publicly available mouse strain.

[0270] In the NSG model of heterologous GvHD, donor human T cells proliferate robustly in recipient mice, inducing anti-host cell reactivity and leading to skin tissue infiltration. Over time, the mice lose weight and, if left untreated, die from GvHD. One of the characteristic features of this disease is the massive production of measurable inflammatory cytokines in plasma [3]. The timeframe for disease progression can range from 3 to 7 weeks. Since both the GLUT3 and PI3K pathways have been described as important for the activation and proliferation of human T cells [4-8], heterologous GvHD is a particularly excellent model for testing the effects of GLUT3 or PI3K knockdown by our technology.

[0271] The materials and methods used in these in vivo experiments are described below.

[0272] Materials and methods Materials and Reagents 1. LPS, Chondrex (Catalog number 170031) 2. NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ Male, 8 weeks old (NSG mouse), Jackson Labs (Catalog No. 1557) 3. ACK lysis buffer, Gibco (catalog number A10492-01) 4. FACS lysis buffer (BD Biosciences, catalog number 349202) 5. PBS, Corning (Catalog No. 21-040-CV) 6. MILLIPLEX® MAP Human Cytokine / Chemokine / Growth Factor Panel A 48 Plex Premixed Magnetic Bead Panel - Immunology Multiplex Assay, EMD Millipore (Catalog No. 3HCYTA-60K-PX48) 7.eBioscience(TM) Foxp3 / Transcription Factor Staining Buffer Set, ThermoFisher, 00-5523-00 8. RPMI1640 medium (Gibco, 11875-093) 9.10% FBS (ATCC, 30-2020) Penicillin / streptomycin at 10,100 U / ml (Gibco, 15140122)

[0273] Isolation of human T cells Human T cells were isolated using the same procedure disclosed in Example 1.

[0274] Cell labeling of PBMCs or T cells using Cell Trace Violet PBMCs or T cells were labeled with Cell Trace Violet using the same procedure disclosed in Example 1.

[0275] Human PBMC and T cell activation and proliferation assay The same procedure disclosed in Example 1 was used for the activation and proliferation assay of human PBMCs and T cells.

[0276] INX201 or ARC titration INX201 and ARC were titrated using the same procedure as disclosed in Example 1.

[0277] Flow cytometry-based cell activation analysis The same procedure disclosed in Example 1 was used for flow cytometry-based cell activation analysis. The in vitro flow cytometry panel used for the analysis is shown in Table 12. [Table 15]

[0278] Heterogeneous GvHD models Test design The recipient mice were 8-week-old male NSG mice purchased from Jackson Laboratory. Unless otherwise specified, 10 mice were registered in each group.

[0279] On day 0, 10 × 6 Human PBMCs were administered intravenously (iv) to mice via tail vein injection. On day 14 (or at any other specific time point), blood was drawn from the mice, plasma was collected for cytokine analysis, and T cell counts were assessed using flow cytometry. Disease progression was monitored by measuring the mice's body weight three times a week, and mice were euthanized if their body weight fell below 75% of their initial body weight.

[0280] Isolation of human PBMCs Human peripheral blood was obtained from apheresis cones provided by volunteer donors in the DHMC blood donor program. The cone blood was diluted 1:4 in PBS and carefully placed on 13 ml of Histopaque 1077. After centrifugation at 850 g for 20 minutes (room temperature, no brake on deceleration), mononuclear cells were collected from the Histopaque / PBS interface. After one wash in PBS, the PBMCs were prepared for injection at a rate of 100 × 10⁶. 6 The cells were resuspended in PBS at a concentration of cells / ml.

[0281] Antibody administration INX201 alone or INX201 antibody RNA conjugate (ARC) was injected at a dose of 5 mg / kg (mixed with hPBMC). In all experiments described in this example, a single dose of ARC (or Ab) was used.

[0282] LPS Early Stimulation Model In several experiments, the early human PBMC cytokine response was evaluated in vivo after stimulation with LPS (0.5 mg / kg, ip injection) 18 hours after PBMC+ARC injection. In these early stimulation models, mice were injected with 10 million PBMCs per mouse. Plasma was collected at an early time point (4 hours to 7 days post-stimulation) for cytokine measurement using Luminex MILLIPLEX.

[0283] Evaluation of "absolute" immune cell changes in the blood On day 14 after PBMC cell transfer (or at other specific time points), the absolute number of human T cells was assessed by flow cytometry for posterior orbital hemorrhage. To obtain cell counts close to the absolute number, 100 μl of whole blood was stained by directly adding an antibody cocktail (Table 3) + mouse and human Fc blocks. After 30 minutes of room temperature incubation, 1 ml of BD FACS lysis solution was added to each sample. After 20 minutes of incubation, the samples were washed once with a large volume of PBS, then resuspended in 100 μl of PBS, and analyzed using the FlowJo program on a MACSQuant or Northern Lights (Cytek) flow cytometer.

[0284] The antibody cocktails that affected cell count in these experiments are shown in Table 13 below. [Table 16]

[0285] Evaluation of Treg cells in the blood (day 28 or as specified) The procedure used in these experiments to evaluate Treg cells in the blood (day 28 or as specified) is as follows: 1. Dispense 0.1 ml of blood into a 1.3 ml deep well plate. Add 2.1 ml of ACK and incubate for 10 minutes. Rotate 3.450g for 3 minutes, decant the supernatant, and if the pellet is still red, repeat the dissolution process for 5 minutes. 4. Wash with PBS (1.0 ml), rotate at 450 g for 3 minutes, decant the supernatant, and transfer to a V-bottom plate containing 200 μl of PBS. Agitate at 5.450g for 3 minutes, add 50ml of Ab mix (Table 4, excluding anti-FOXP3 and RORgt Ab), incubate at room temperature for 30 minutes, and wash once with PBS. Agitate at 450g for 3 minutes and decant the supernatant. 6. FoxP3 Fixation / Permeabilization was used according to the manufacturer's instructions for FOXP3 and RORgt staining. 7. After fixation and permeabilization, cells were resuspended in 50 μl of permeabilization buffer containing anti-FOXP3 and RORgt Ab (both diluted 1:50) at room temperature for 30 minutes. The cells were washed once with permeabilization buffer, then resuspended in 100 μl of PBS, and analyzed using the FlowJo program on a MACSQuant or Northern Lights (Cytek) flow cytometer.

[0286] The regulatory T cell antibody panel used in these experiments is shown in Table 14. [Table 17]

[0287] result PI3K and GLUT3 ARC conjugation and in vitro QC The anti-VISTA Mab, the payloads used for conjugation, and the ARCs are listed in Table 11 and Appendix 1-2. An oligoconjugation kit (Abcam, no. ab218260) was used for all conjugations. All RNA oligos were purified by HPLC, resuspended at 100 μM, and annealed. A lysine-based conjugation via an amine reactive group, one of the most widely used nonspecific conjugation strategies, was employed. All payloads were synthesized in IDT and contained a 3'-sense strand labeled with Cy5 and a 5'-amine on the antisense strand (see Appendix 2). The drug-to-antibody ratio (DAR) was assessed by UV / Vis spectroscopy (Nanodrop) and estimated to be 1.0 for both ARCs described.

[0288] K562-VISTA cells (described in ARC_01) were treated with 40-200 nM INX201-PI3K ARC or isotype-controlled IgG1-PI3K ARC (Table 1). PI3K knockdown was evaluated by qRTPCR at 24 hours. INX201-PI3K ARC achieved the target knockdown of 70%, while INX201 Ab alone or isotype-controlled PI3K ARC did not show specific PI3K knockdown (Figure 15).

[0289] As shown in the data in Figure 15, INX201 (anti-VISTA) PI3K ARC specifically knocks down PI3K, while the isotype control IgG1 PI3K ARC does not. In these experiments, K562-VISTA cells were transfected with 40–200 nM and treated with 40–200 nM INX201-PI3K ARC or isotype control IgG1-PI3K ARC. Targeted knockdown was analyzed by qRTPCR using the ddct method and reported as target suppression %. Here, 0% suppression was for INX201 alone. PI3K ARC: anti-human VISTA mAb conjugated with PI3K siRNA; PI3K isotype ARC: IgG control conjugated with PI3K siRNA; INX201, unconjugated mAb: naked anti-human VISTA mAb. Single technical replicates were used for each concentration point.

[0290] As described above, ARC was tested in vitro using human PBMCs activated with anti-CD3 / CD28 dynabeads for 72 hours. After incubation, the culture medium was collected and cytokine levels were measured by Luminex. PI3K ARC reduced inflammatory cytokine production, but free INX201 antibody did not. Specifically, PI3K ARC reduced IL5 and IL13 levels by 3-6 times and TNFα and IL17F levels by approximately 2 times compared to free INX201 levels (Figure 16A).

[0291] As can be seen in Figure 16A, GLUT3 ARC dose-dependently reduced T cell activation, while free INX201 Ab did not. Furthermore, GLUT3 ARC dose-dependently reduced the CD69 level of effector memory T cells (TEM) and the CD25 level of CD3 T cells, with IC50 values ​​of 1 and 6 nM, respectively (Figure 16B). Note that when DAR is 1.0, the amount of "drug" (siRNA) is equivalent to the amount of Ab delivery vehicle (nM).

[0292] More specifically, the experiments shown in Figures 16A-B demonstrated that human PI3K and GLUT3 ARC are functional in vitro. In these experiments, human PBMCs were activated with anti-CD3 / CD28 beads (bead-to-T cell ratio 1:2) for 72 hours and treated with (A) PI3K ARC or free INX201 (0-200 nM), or (B) GLUT3 ARC or free INX201 (0-200 nM). Curves were generated from three-parameter nonlinear regression analysis of inhibitor versus response (GraphPad Prism10). IL5 / IL13 / TNFa / IL17F cytokine levels were measured using Luminex. CD69 levels were measured in live CD45+ / HLA-DR- / CD56- / CD3+ / CD4+ / CD45RA- / CD27-T effector memory cells, and CD25 levels were measured in live CD45+ / HLA-DR- / CD56- / CD3 cells. In A, a single technical replica was used per concentration point. In B, n=2 technical replicas were used per concentration point. No ARC was visualized as 0.1 nM (due to the logarithmic scale). MFI - mean fluorescence intensity. Data are presented as mean ± SEM, where necessary.

[0293] PI3K ARC treatment reduces human cytokine response and T cell proliferation in vivo. In one experiment monitoring survival, no difference in weight loss or median survival (data not shown) was observed between PI3K ARC and INX201 alone in both classical heterologous GvHD and early LPS stimulation (data not shown). Therefore, as an alternative to the classical heterologous GvHD model in which human T cells proliferate and human cytokine levels increase over time, we considered using stimulation such as LPS to induce early cytokine production by human PBMCs within a short time (18 hours) after injection into the host (NSG mice). 18 hours after IV injection of hPBMC+Ab, mice were given ip injection. The response of LPS and serum cytokines was measured at 4 hours and on day 7 after LPS injection (Figure 17A). As can be seen from the figure, the peak levels of most cytokines were at 4 hours. PI3K ARC treatment reduced IL6 and TNFα production by up to 3 times compared to INX201 alone at 4 hours after LPS injection. PI3K ARC treatment also reduced the delay in the IFNg response on day 7 (Figure 17B). Overall, PI3K ARC had an overall effect on cytokine production, generally reducing most of them at an early time (4 hours) after LPS stimulation, as shown in Figure 17C. The reduction in inflammatory cytokines (Figure 3C) did not reach a statistically significant level due to response variability. PI3K ARC did not show any effect on human cytokine production in the group without LPS stimulation (data not shown).

[0294] The experiment in Figure 17 clearly demonstrates that PI3K ARC reduces the inflammatory cytokine response in heterologous GvHD mice. In the experiment shown, NSG mice were intravenously injected with PI3K ARC (triangle, n=6) or INX201 (circle, n=6) at 5 mg / kg, along with human PBMC transfer. Eighteen hours after injection, the mice were stimulated intraperitoneally with 0.5 mg / kg of LPS. Figure 17A shows a schematic diagram of the experiment in the heterologous GvHD LPS-stimulated model. Figure 17B shows the changes in plasma human cytokine levels at day 7 (IFNg) or 4 hours (IL6, TNFa). Statistical analysis was performed by Student's t-test. Data are expressed as mean ± SEM. *-p<0.05;**-p<0.01. Figure 17C includes a heatmap based on Z-scores for cytokine levels 4 hours after LPS stimulation (n=6 per group).

[0295] Results from the GVHD model further demonstrated that PI3K ARC treatment reduces LPS-stimulated T cell proliferation in heterologous GvHD, as indicated by the decrease in CD4 and CD8 T cell counts (more than half) in the PI3K ARC-treated group on day 14 (Figure 18). No difference in T cell counts was observed in classical heterologous GvHD without LPS stimulation (data not shown).

[0296] More specifically, the data in Figure 18 showed that PI3K ARC reduced LPS-induced T cell proliferation in vivo. In the experiment shown in the figure, NSG mice were intravenously injected with 5 mg / kg of either PI3K ARC (triangle, n=6) or INX201 (circle, n=6) along with human PBMC transfer. Eighteen hours after injection, the mice were stimulated intraperitoneally with 0.5 mg / kg of LPS. Changes in serum T cell counts on day 14 (n=6 mice per group) are shown for both CD4 and CD8. Statistical analysis was performed by Student's t-test. Data are expressed as mean ± SEM. *-p<0.05.

[0297] PI3K ARC does not reduce the percentage of regulatory T cells in vivo. To test whether PI3K ARC has any effect on regulatory T cell (Treg) count in a heterologous GvHD model, Treg cell levels in the blood of NSG mice of the same heterologous GvHD model were monitored. Again, 10 million hPBMCs (human INX201 alone or mixed with PI3K ARC at 5 mg / kg) were intravenously injected into the NSG mice. In individual arms of the experiment, some mice were stimulated with LPS 18 hours after PBMC+Ab or ARC injection. LPS was administered intravenously.

[0298] On day 28, human CD4 + FOXP3 + The frequency of (Treg) cells was measured in the blood. Compared to INX201, PI3K ARC treatment was observed not to reduce the frequency of Treg cells (Figure 19). In the KLPS-stimulated group, there was a tendency for the frequency of Treg cells to increase, but the difference was not statistically significant. No substantial amount of Th17 cells were detected in the blood (data not shown). In summary, the data indicate that PI3K ARC does not reduce the percentage of regulatory T cells in vivo.

[0299] More specifically, the experiment shown in Figure 19 revealed that PI3K ARC does not reduce the percentage of regulatory T cells in vivo. In this experiment, NSG mice were intravenously injected with 5 mg / kg of either PI3K ARC (triangle, n=10) or INX201 (circle, n=8) along with human PBMC transfer (right graph). In each group of mice, 18 hours after PBMC injection, the mice were stimulated intraperitoneally with 0.5 mg / kg of LPS (n=6 for both the INX201 and PI3K ARC groups, left graph). Blood samples were processed on day 28. The percentage change in Treg is shown. Statistical analysis was performed by Student's t-test. Data are expressed as mean ± SEM. ns - not statistically significant.

[0300] GLUT3 ARC treatment reduces the human cytokine response in vivo. One of the classic features of GVHD or heterologous GvHD disease progression is the massive production of measurable inflammatory cytokines in plasma. Based on this, we evaluated whether GLUT3 ARC could influence the serum cytokine response on day 7. GLUT3 ARC was found to reduce GMCSF, CCL4, and IL5 levels by approximately twofold and CXCL9 levels by threefold compared to INX201 Ab alone (Figure 20).

[0301] More specifically, the experiment in Figure 20 clearly demonstrates that GLUT3 ARC reduces the inflammatory cytokine response in heterologous GvHD. In these experiments, NSG mice were intravenously injected with PI3K ARC (triangle, n=10) or INX201 (circle, n=8) at 5 mg / kg along with human PBMC transfer. Changes in plasma human cytokine levels are shown on day 7. Statistical analysis was performed by Student's t-test. Data are expressed as mean ± SEM. **-p<0.01;****-p<0.0001.

[0302] conclusion Our experiments using heterologous GvHD models further support the ability of the anti-VISTA antibody RNA conjugate (ARC) according to the present invention to functionally deliver siRNA into immune cells and knock down the expression of target mRNA there, both in vitro and in vivo. In particular, these experimental data showed the following: ● PI3K and GLUT3 ARC, INX201 antibody RNA conjugates that deliver GLUT3 or PIK3CA siRNA to VISTA+ cells, are functional in vitro. ○ Reduction of T cell cytokine response ○ Reduction of T cell CD69 / CD25 activation profile ●GLUT3 and PI3K ARC are functional in vivo. ○GLUT3 treatment reduces the human cytokine response in vivo (heterogeneic GvHD). ○PI3K ARC treatment reduces the human cytokine response in vivo. ○PI3K ARC treatment reduces T cell proliferation in vivo. ○PI3K ARC does not reduce the percentage of regulatory T cells in vivo.

[0303] Therapeutic applications of ARC or ANS in the present invention To the best of the applicant's knowledge, no ARC or ANC has been described to date that specifically targets immune cells and delivers one or more nucleic acids into immune cells that modulate the expression and / or activity of specific immunomodulatory substances and / or immunomodulatory pathways expressed by specific immune cells. Therefore, the present invention provides a novel platform for treating diseases involving specific immune cell types without causing toxicity to non-target immune cells or other non-immune cell types.

[0304] Potential indications for the use of the target ARC as a therapeutic or prophylactic agent include, for example, autoimmune and inflammatory diseases, such as diseases with prominent bone marrow and / or T cell components (which, based on the site of VISTA expression, become the site of specific drug delivery). Potential indications include diseases in which limiting the activation and proliferation of autoresponsive effector T cells may be beneficial in controlling / reducing the disease. Our targeting technology may help eliminate on-target toxicity associated with target expression in non-immune tissues, which can support drugs against targets that were previously untargetable in novel drug development due to the broad expression profile of the target or the localization of the target (transcription factors). Specifically, rheumatoid arthritis, colitis, or systemic lupus erythematosus include autoimmune indications suitable for ARC treatment.

[0305] Similar to the CD39 knockdown approach described herein, the siRNA or ASO according to the present invention can be administered to act on targets such as immune checkpoint inhibitor proteins (e.g., PD-1, PD-L1, CTLA-4, B7-1, B7-2, LAG-3, HHLA2, TNFRSF12A, HLA-G, NECTIN2, TNFRSF25, TNFSF14, LAIR1, TNFSF15, TNFSF4, KIR2DL4, PDCD1, LGALS9, and PVR) that block or suppress the induction of an antitumor response in the host, so many cancer indications may also be treatable with the ARC of the present invention. By removing or inhibiting this block, the host is allowed to activate its immune system and innate antitumor immunity more efficiently.

[0306] conclusion In this patent application, the inventors have demonstrated the following: 1) The inventors have found that by conjugating siRNA or ASO to an anti-VISTA mab, RNA (e.g., siRNA and ASO) can be efficiently delivered into target cells by VISTA binding and internalization. 2) ARC has similar binding and internalization properties to free INX201 Mab. 3) Exemplary siRNA payloads specific to immunologically relevant targets are conjugated to anti-VISTA Mab and retain their function when delivered into target cells. 4) siRNA conjugated with anti-VISTA Mab INX201 promoted siRNA accumulation and function within K562-VISTA cells. 5) siRNA conjugated with anti-VISTA Mab INX201 was functional in human PBMCs and T cells, effectively reducing proliferation and cytokine production (siRNAs targeting five targets are described in this example). 6) CD39 ASO conjugated with anti-VISTA Mab INX201 functions in human PBMCs, i.e., increases cytokine production, which indicates that the ARC of the present invention is promising for cancer treatment. 7) Exemplary PI3K and GLUT3 ARC, which are INX201 antibody RNA conjugates that deliver GLUT3 or PIK3CA siRNA to VISTA+ cells, are functional in vitro, namely reducing the T cell cytokine response and reducing the T cell CD69 / CD25 activation profile. 8) Exemplary GLUT3 and PI3K ARC are functional in vivo; for example, GLUT3 treatment reduces the human cytokine response in vivo (heterogeneous GvHD), PI3K ARC treatment reduces the human cytokine response in vivo, PI3K ARC treatment reduces T cell proliferation in vivo, and PI3K ARC does not reduce the percentage of regulatory T cells in vivo.

[0307] Additional proof-of-concept research While the applicant has demonstrated the suitability of ARCs or ANCs containing exemplary anti-VISTA antibodies and siRNA payloads targeting GLUT3 or PIK3CA as in vitro and in vivo therapeutic agents in GVHD models, the ARCs or ANCs of the present invention have a much broader applicability. This can be supported by additional experiments such as the following: ● Optimize the payload sequence and test additional targets. ● Perform ARC conjugation with Cys via a maleimide or bromoacetimide reactive group. ● Generate functional data using other in vivo models

[0308] Addendum to the application Arrays that do not fit into the ST.26XML file due to their length Table A below lists the sequences found in Figure 14 of this application and in Figure 14 of the U.S. provisional priority applications (U.S. applications No. 63 / 506,177 and No. 63 / 611,302, identified in the section on related applications on page 1 above, all of which are incorporated herein by reference in their entirety), but due to the length of the sequences, they cannot be included in the 1143260_008613_SL.xml file submitted with this specification. [Table 18-1] [Table 18-2]

[0309] Appendix 1 Points to note ● Signal sequences are not included. ● Human IgG1 containing the INXLALA (L234A / L235A / E269R / K322A) Fc silencing mutation (Uniprot P01857) ○ Mutations are shown in blue. ●Hitokappa (Uniprot P01834) ●IgG1 and the kappa constant region are shown in bold. Antibody name:INX201 Light chain: >INX201_Kappa_LC [ka] Heavy chain: >INX201_INXLALA_HC [ka] FAB Name: INX201 FAB >INX201_FAB_HC [ka] >INX201_FAB_LC [ka]

[0310] [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4]

[0311] Appendix 3 Points to note ● Signal sequences are not included. ● Human IgG1 containing the INXLALA (L234A / L235A / E269R / K322A) Fc silencing mutation (Uniprot P01857) ○ Mutations are shown in blue. ●Hitokappa (Uniprot P01834) ●IgG1 and the kappa constant region are shown in bold. Antibody name:INX201 Light chain: >INX201_Kappa_LC [ka] Heavy chain: >INX201_INXLALA_HC [ka] FAB Name: INX201 FAB >INX201_FAB_HC [ka] >INX201_FAB_LC [ka]

[0312] [Table 20]

Claims

1. An antibody-RNA or antibody-nucleic acid conjugate ("ARC" or "ANC") comprising (i) an antibody or antibody fragment that binds specifically or primarily to an antigen expressed by one or more immune cell types, and (ii) one or more nucleic acids, preferably RNA or DNA oligonucleotides ("payload"), which are directly or indirectly conjugated thereto, and which consist of wild-type or modified nucleotides, the oligonucleotide which specifically binds to a target gene, optionally an immunomodulatory gene, or RNA encoded thereon, expressed by an immune cell, and optionally (iii) a cleavable or incleavable linker or adapter, for example, a peptide mediating the (i) antibody or antibody fragment and the (ii) one or more nucleic acids, wherein such an ARC or ANC, when brought into contact with an immune cell expressing the antigen to which the (i) antibody or antibody fragment binds, is internalized by the immune cell, resulting in the release of the (ii) one or more nucleic acids to the immune cell, thereby optionally modulating the expression and / or function of a targeted immune modulator.

2. The ARC or ANC according to claim 1, wherein the one or more payloads optionally comprises a peptide linker, and optionally a cleavable or incleavable linker or adapter, one or more modified nucleotides, optionally at least one phosphonic acid and / or ribose-modified nucleotide, which facilitates direct or indirect linking of the one or more payloads to the antibody or antibody fragment by (i) the antibody or antibody fragment and (ii) the payload-mediated peptide.

3. The payload is optionally conjugated directly or indirectly to the antibody or antibody fragment via a reactive amine contained in a lysine residue on a peptide linking the antibody or antibody fragment to (i) one or more payloads, and / or the payload contains a 3'-sense chain labeled with Cy5 and a 5'-amine on the antisense chain, according to any of the prior claims, ARC or ANC.

4. The antibody or antibody fragment is bound to VISTA, preferably human VISTA, and is an ARC or ANC according to any of the prior claims.

5. The ARC or ANC according to any of the prior claims, wherein the antibody or antibody fragment is bound to VISTA, preferably human VISTA, and contains the same VH and VL CDR as any one of the anti-human VISTA antibodies having the sequence of Figure 14 or Appendix 1 or Appendix 3.

6. The antibody or antibody fragment is conjugated to VISTA, preferably human VISTA, and comprises the same VH and / or VL region and CDR as any one of the anti-human VISTA antibodies comprising the VH and / or VL sequence of Figure 14, or an antibody or antibody fragment comprising a VH and / or VL region having at least 90, 95, or 99% sequence identity with the VH and / or VL region as any one of the anti-human VISTA antibodies comprising the VH and / or VL sequence of Figure 14, or an antibody or antibody fragment comprising the VH and / or VL sequence of Appendix 1 or Appendix 3, wherein the antibody or antibody fragment optionally comprises an IgG1, IgG2, IgG3, or IgG4 constant domain polypeptide, further optionally an IgG1 constant domain polypeptide, and even further optionally an IgG1 constant domain polypeptide having a sequence included in Appendix 1 or 3, as described in any of the prior claims.

7. The ARC or ANC according to any of the prior claims, wherein the antibody or antibody fragment comprises a human Fc region, optionally human IgG1, IgG2, IgG3, or IgG4, and is further optionally modified to impair complement and / or FcR binding and / or enhance FcRn binding.

8. An ARC or ANC according to any of the prior claims, comprising one or more of the following: small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heteronuclear RNA (hnRNA).

9. It contains polynucleotide molecules with a length of approximately 10 to 5000, 10 to 4000, 10 to 3000, 10 to 2000, 10 to 1000, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 10 to 30, 15 to 30, 18 to 25, 18 to 24, 19 to 23, or 20 to 22 nucleotides, or approximately 50 nucleos An ARC or ANC according to any of the prior claims, comprising a polynucleic acid molecule having a length of approximately 45 nucleotides, approximately 40 nucleotides, approximately 35 nucleotides, approximately 30 nucleotides, approximately 25 nucleotides, approximately 20 nucleotides, approximately 19 nucleotides, approximately 18 nucleotides, approximately 17 nucleotides, approximately 16 nucleotides, approximately 15 nucleotides, approximately 14 nucleotides, approximately 13 nucleotides, approximately 12 nucleotides, approximately 11 nucleotides, or approximately 10 nucleotides.

10. An ARC or ANC according to any of the prior claims, comprising a first polynucleotide and a second polynucleotide, wherein optionally the first polynucleotide is a sense strand or a passenger strand, and / or the second polynucleotide is an antisense strand or a guide strand.

11. An ARC or ANC according to any of the prior claims, comprising siRNA, tRNA, rRNA, or mRNA.

12. An ARC or ANC according to any of the prior claims, comprising, encapsulated within, or conjugated therein, lipid nanoparticles.

13. An ARC or ANC according to any of the prior claims, wherein at least one payload targets an immunomodulator selected from cytokines, chemokines, interleukins, interferons, tumor necrosis factor, or any of the aforementioned receptors.

14. The payload targets an RNA or DNA sequence encoding an immunomodulator selected from IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-22, IL-37, IL-1β, TGF-β, IFNα, IFNβ, IFNγ, TNF-α, TNF-β, GM-CSF, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), RAR-associated orphan receptor C (RORC), or a molecule having a sequence identified in Figure 1 or Figure 2, as described in any of the prior claims, ARC or ANC.

15. An ARC or ANC according to any of the prior claims, which targets an RNA or DNA encoding GLUT3 or PIK3CA, and optionally comprises a sequence from Table 11, Appendix 2, or 4.

16. The antibody or antibody fragment may be PMBC, T cell, T cell progenitor cell, CD4+ T cell, helper T cell, regulatory T cell, CD8+ T cells, naive T cells, effector T cells, memory T cells, stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, terminally differentiated effector memory T cells, tumor invasion lymphocytes (TIL), immature T cells, mature T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, and a / b T cells, g / d An ARC or ANC according to any of the prior claims, which binds to at least one immune cell selected from T cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, perforin-deficient cells, granzyme-deficient cells, B cells, myeloid cells, monocytes, macrophages, eosinophils, neutrophils, and dendritic cells.

17. The antibody or antibody fragment binds to bone marrow cells and / or T cells, as described in any of the prior claims, ARC or ANC.

18. The antibody or antibody fragment binds to T cells or T cell progenitor cells or NK cells, as described in any of the prior claims, for the ARC or ANC.

19. Antigens selected from the following group: (1) 17-IA, 4-1BB, 4Dc, 6-keto-PGFla, 8-iso-PGF2a, 8-oxo-dG, Al-adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RUB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Adresin, aFGF, ALCAM, ALK, ALK-1, ALK-7, Alpha-l-antitrypsin, Alpha-V / Beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, Anti-Id, ASPARTIC, Atrial natriuretic factor, av / b3 integrin N, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte-stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bel, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BFM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3, Osteogenin, BMP-4, BMP-2b, BMP -5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMP, β-NGF, BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, Complement factor 3 (C3), C3a, C4, C5, C5a, CIO, CA125, CAD- 8. Calcitonin, cAMP, carcinoembryonic antigen (CEA), cancer-associated antigen, cathepsin A, cathepsin B, cathepsin C / DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L, cathepsin O, cathepsin S, cathepsin V, cathepsin X / Z / P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19,CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD10, CDlla, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCLXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, Cytokeratin tumor-related antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, E-cad, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, EN A, Endothelin receptor, Enkephalinase, eNOS, Eot, Eotaxin 1, EpCAMEphrin B2 / EphB4, EPO, ERCC, E-selectin, ET-1, Factor Ila, Factor VII, Factor VIIIc, Factor IX, Fibroblast-activating protein (FAP), Fas, FcRl, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF-3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, F ZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GFAP, GFRa-1, GFR-Alpha-1, GFR-Alpha-2, GFR-Alpha-3, GITR, Glucagon, Glut4, Glycoprotein Ilb / IIIa (GP Ilb / IIIa), GM-CSF, gpl30, gp72, GRO, growth hormone-releasing factor, hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gpl20, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gpl20, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R,IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, Interferon (INF)-alpha, INF-beta, INF-gamma, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin alpha 2, Integrin alpha 3, Integrin alpha 4, Integrin alpha 4 / beta 1, Integrin, Alpha 4 / beta 7, Integrin alpha 5 (Alpha V), Integrin alpha 5 / beta 1. Integrin Alpha 5 / Beta 3, Integrin Alpha 6, Integrin Beta 1, Integrin Beta 2, Interferon Gamma, IP-10, 1-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein LI, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpl, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, progesterone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MI G, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, Mucin (Mucl), MUC18, Müllerian duct inhibitor, Mug, MuSK, NAIP, NAP, NCAD, N-cadherin, NCA90, NCAM, Neprilysin, Neurotrophin-3, 4, or -6, Neuroturin, Nerve growth factor (NGF), NGFR,NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, pl50, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, P GJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid Factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, Serum Albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (Tumor-Associated Glycoprotein-72), TARC, TCA-3, T Cell Receptor (e.g., T Cell Receptor Alpha / Beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, Testicular PLAP-like Alkaline Phosphatase -ze, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta panspecific, TGF-betaRI (ALK-5), TGF-betaRII, TGF-betaRllb, TGF-betaRIIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, thrombin, thymus Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL Rl Apo-2, DR4), TNFRSFIOB (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2,4200)、48023001112 (228. 9002) 2)、48023007(907) 9390、421)、480230012(47452) 0814)、48023001333(4239)、4802301333) 2)、48023014(2657 4422、25、 2、LOVES S、SHO2)、SHOSES16(SYS) 07SYS、SYSYS17(SYSYS)、SYSYSYS18(JSYS). SHYSY、SYSYS10(SYS3 WHY、WHYWH、WHYWHYWHYWHYWHYWHYWHYWHYWHY 100000000000000000000000000000000000000000000000000000000000 H1200、F75806、SHOSH2 6(SYS3)、SYSYS3(SYS THIS SUCH、SHIS S6、SHOSCHS4(SYS)0 SIS33、SHIS1 S) 、WHYS 5(SH40 50)、SWHYS6 DA11、HAR1、DAY5)、DYSCHYS(DYS3 H68、S6)、S6S67(S027)、 FASHION(SHR30)、SHASHES(400) HR137、HARSH、SHRYSY21 DASH) 、SHASH22(DASH) 2 SHY2)、SYSYS23(SYSYS) SHYS1)、SHYSYS25(GY3 FASH3、SHASH、SHASH、SHASH 、SHAKE1)、SHASE100(SHARE). DAY2リガンド、LOY2)、SHAS11(SHAS SHASE RHリガンドDAY 、DAYリガンド)、DAYS12(SIR). DA3リガンド、DAY3リガンド)、DAYDAY13(SYS) LOVE2)、SIGNIFICANCE130 LOVE SY、 LOVE1 LOVE LOVE LOVE LOVE LOVE LOVE LIKEリガンド、THE)、SHASE15(30). SUCH SUCH 18(SUSHリガンドSUBJECTリガンド、CHASE)コネクチン、DYS2)、DYSYS1 LIKE、SYSYS1)、SYSYS(LOVE THIS、D33) THIS 4(S40リガンドW34、SH1)、SHOSIS5(CHA40リガンドH154、N39、NH10、13 、WATCHES、DAYSJS(THEリガンドDA11リガンド 、DA1リガンド) DASH 7(KS27リガンドCHR706、SHQS8(CHR33リガンドDA153)、DJSJN(401リガンドC137リガンド) CHA1、100、TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfering receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expression Lewis Y-related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VFM, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WN T8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, CTLA4 (cell (2) Antigens selected from the group consisting of: BCMA, CTLA4 (cytotoxic T lymphocyte antigen-4), PD1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), LAG-3 (lymphocyte activation gene 3), TIM-3 (T cell immunoglobulin and mucin protein 3), and hormone receptors; or (2) below: CD20, CD2, CD19, Her2, EGFR, EpCAM, FcyRIIIIa (CD16), FcyRIIIa (CD32a), FcyRIIIb (CD32b), FcyRI (CD64), Toll-like receptor (TLR), TLR4, TLR9, cytokines, IL-2, IL-5, IL-13, IL-6, IL-17, IL-12, IL-23, TNFa, TGFβ, cytokine receptor, IL-2R, chemokine, chemokine receptor, growth factor, VEGF, and HGF; or (3) antigens selected from the following: CD1a, b, c, d; CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, b, c, d; CDw12, CD13, CD14, oCD15, CD15s,CD15u、CD16、CDw17、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD31、CD32、CD33、CD34、CD35、CD36、CD37、CD38、CD39、CD40、CD41、CD42a、b、c、d;CD43、CD44、CD45、CD45RO、CD45RA、CD45RB、CD46、CD47、CD48、CD49a、CD49b、CD49c、CD49d、CD49e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55m、CD56、CD57、CD58、CD59、CD60a、CD60b、CD61、CD61E、CD62L、CD62P、CD63、CD64、CD65、CD66a、CD66b、CD66c、CD66d、CD66e、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD75s、CD77、CD78、CD79α、β、CD80、CD81、CD82、CD83、CDw84、CD85、CD86、CD87、CD88、CD89、CD90、CD91、CD92、Cd92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD108、CD109、CD110、CD111、CD112、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CDw121b、CD122、CD123、CD124、CD125、CD126、CD127、CDw128、CD129、CD130、CDw131、CD132、CD133、CD134、CD135、CDw136、CDw137、CD138、CD139、CD140a、b、CD141、CD142、CD143、CD144、CD145、CD146、CD147、CD148、CD149、CD150、CD151、CD152、CD153、CD154、CD155、CD156b、CD157、CD158、CD158a、CD159a、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171, CD172a, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD183, CD184, CD195, CDw197, CD200, CD20 1, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CDw210, CD212, CD213a1, CD213a2, CDw217, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240CE, CD240D, CD241, CD242, CD243, CD244, CD245, CD246, CD247, etc.; or (4) antigens selected from the following: IL4ra, TNFa, BTK, RORgt, PIK3CA, JAK1, JAK3, TYK2 Glut1, Glut3, TAP1, CIITA, cGAS, IRF5, STAT3, STAT6, TAK1 (MAP3K7), HPK1; or any of the following SOCS1, CD39, Cbl, or PTPN22; or (5) Glut1, PI3K, BTK, TNF, RORC, CD45, or CD39; or (6) any of the following PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, LAG-3, HH An ARC or ANC according to any of the prior claims, comprising a gene or nucleic acid encoding LA2, TNFRSF12A, HLA-G, NECTIN2, TNFRSF25, TNFSF14, LAIR1, TNFSF15, TNFSF4, KIR2DL4, PDCD1, LGALS9, and PVR, at least one nucleic acid payload optionally bound to RNA or DNA, optionally RNA, and further optionally siRNA or antisense RNA.

20. An ARC or ANC according to any of the prior claims, comprising a nucleic acid payload having a payload sequence selected from those having sequences listed in Table 11, Appendix 2, or Appendix 4, optionally comprising RNA, and further optionally comprising siRNA or antisense RNA, or comprising an INX-201 ARC having an amino acid sequence and payload sequence listed in Appendix 2, or comprising an INX-201 ARC having an amino acid sequence and payload sequence listed in Appendix 4.

21. An ARC or ANC according to any of the prior claims, comprising the same or different immunomodulatory genes or mRNAs, optionally comprising at least two different RNA payloads targeting immune targets enumerated in any of the prior claims.

22. The ARC or ANC according to any of the prior claims, wherein the nucleic acid, optionally an RNA payload, is linked to the antibody or antibody fragment via a cleavable or incleavable linker.

23. An ARC or ANC according to any of the prior claims, used for delivering one or more gene-editing nucleic acids (e.g., CRISPR guide RNA (gRNA or sgRNA)) and optionally a CRISPR-related endonuclease or a nucleic acid encoding a CRISPR-related endonuclease.

24. An ARC or ANC according to any of the prior claims, having a PD of at least one day, two days, three days, four days, five days, one week, two weeks, three weeks, four weeks, five weeks, six weeks or more.

25. An ARC or ANC according to any of the prior claims, which does not induce toxicity to non-target cells that can be recognized.

26. A composition comprising an ARC or ANC as described in any of the prior claims, and a pharmaceutically acceptable carrier or excipient, wherein the ARC or ANC is optionally contained in or on lipid nanoparticles.

27. A method of treatment or prevention comprising administering an ARC or ANC, or a composition containing the same, to a subject requiring the same, as described in any of the prior claims.

28. The method according to claim 27, wherein the treatment or prevention is for the treatment of a tumorous state, a proliferative state, a neurodegenerative state, a neuroinflammatory state, an infectious state, an autoimmune state, an allergic state, or an inflammatory state, or a pathological symptom associated with any of the aforementioned states.

29. The method according to claim 27, wherein the treatment or prevention is for the treatment or prevention of an autoimmune disease, for example, a disease relating to the bone marrow or T cells.

30. The method according to claim 27, wherein the treatment or prevention is for the treatment or prevention of neoplastic diseases, proliferative diseases, neurodegenerative diseases, neuroinflammatory diseases, infectious diseases, autoimmune diseases or inflammatory diseases, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

31. The aforementioned treatment or prevention applies to acromegaly, acquired aplastic anemia, acquired hemophilia, agammaglobulinemia, primary alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | fulminant antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic neuropathy (AAG) / autonomic neuropathy | autoimmune gastrointestinal motility disorder (AGID), autoimmune encephalitis | acute disseminated encephalomyelitis (ADEM), autoimmune gastritis, autoimmune hemolytic anemia (AIHA), autoimmune hepatitis (AIH), autoimmune hyperlipidemia, ＊ Autoimmune hypophysitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune myelofibrosis, autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis (AIP), polyglandular autoimmune syndromes, types I, II, and III (APS1, APS2, APS3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Baro's disease, Behçet's disease, scatter chorioretinopathy / scatter uveitis, bullous pemphigoid, Castleman disease, celiac disease, Chagas disease, chronic inflammation Demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Churg-Strauss syndrome / eosinophilic granulomatosis with polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CRESTO syndrome | focal systemic cutaneous scleroderma, Crohn's disease (CD), Cronchite-Canada syndrome (CSS), idiopathic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dressler syndrome / post-myocardial infarction / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulin Severe hemorrhagic thyroiditis, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome / anti-GBM / anti-TBM disease, granulomatosis with polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's disease / chronic lymphocytic thyroiditis / autoimmune thyroiditis, Henoch-Schönein purpura / IgA vasculitis, hidradenitis suppurativa, Haast's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Berger's disease, immune-mediated Necrotizing myopathy (IMNM), immune thrombocytopenia (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenia, inclusion body myositis, IgG4-related sclerosis (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult Still's disease, juvenile polymyositis | juvenile dermatomyositis | juvenile myositis, Kawasaki disease, Lambert-Eaton myasthenia dysthesia (LEMS), leukocytosis-destroying vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, linear IgA disease (LAD) | linear IgA bullous dermatosis (LABD), lupus nephritis, Lyme disease / chronic Lyme disease / post-treatment Lyme disease syndrome (PTLDS),Lymphocytic colitis / microscopic colitis, lymphocytic hypophysitis / autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), Mollen's ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devic's disease, ocular scarring pemphigoid, ocular clonus-myoclonus syndrome (OMS), relapsing rheumatoid arthritis, paraneoplastic cerebellar degeneration, tumors Parapemphigus, Parry-Romberg syndrome (PRS) / hemifacial atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / peripheral uveitis, PANS / PANDAS, Personage-Turner syndrome, bullous pemphigoid of pregnancy / herpes zoster of pregnancy, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, orthostatic tachycardia syndrome (POTS), primary biliary cirrhosis (PBC) / primary biliary cholangitis, primary sclerosing cholangitis (PSC), dry mouth Tinea, palmoplantar pustulosis, psoriatic arthritis, idiopathic pulmonary fibrosis (IPF), pure red cell fistula (PRCA), pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud's disease / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy syndrome (RSD) / complex regional pain syndrome (CRPS), relapsing polychondritis, restless legs syndrome (RLS) / Willis-Ekbom disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, scleritis, scleroderma, sclerosing mesentericitis / mesenteral panniculitis, crawling choroidopathy, Sjögren's syndrome, generalized rigidity syndrome (SPS), small-diameter fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus erythematosus, Suzak syndrome, Sydenham's chorea, sympathetic ophthalmitis, Takayasu's arteritis (vasculitis), testicular autoimmunity (vasculitis, orchitis), Tolosa-Hunt syndrome, transverse myelitis (TM), tubulointerstitial nephritis / uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis | anterior / intermediate / posterior, vasculitis, VEXAS syndrome, vitiligo,The method according to claim 27, for the treatment or prevention of an autoimmune disease selected from one or more of the following: and Vogt-Koyanagi-Harada syndrome (VKH), and / or for the prevention or suppression of at least one pathological symptom associated therewith.

32. The method according to claim 27, wherein the treatment or prevention is for the treatment or prevention of Addison's disease, arthritis, celiac disease, lupus, Graves' disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto's disease, Sjögren's disease, asthma, type 2 diabetes, and autoimmune type 1 diabetes, and / or for the prevention or suppression of at least one pathological condition associated therewith.

33. The method according to claim 27, wherein the treatment or prevention is for the treatment of an inflammatory disease selected from the group consisting of fatty liver disease, endometriosis, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), asthma, rheumatoid arthritis, obesity, fibromyalgia, lupus, SLE, osteoarthritis, rheumatoid arthritis, herpes zoster, and vasculitis, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

34. The method according to claim 27, wherein the treatment or prevention is for the treatment or prevention of neurodegenerative or neuroinflammatory diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body dementia, aphasia, Parkinson's disease, or spinal muscular atrophy, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

35. The method according to claim 27, wherein the treatment or prevention is for the treatment of cancer or for the prevention of cancer recurrence and / or for the suppression of at least one pathological symptom associated with a particular immune cell type.

36. The method according to claim 27, wherein the treatment or prevention is for the treatment of a solid tumor and / or for the prevention or suppression of at least one pathological symptom associated therewith.

37. The method according to claim 27, wherein the treatment or prevention is for the treatment of a hematological malignancy and / or for the prevention or suppression of at least one pathological symptom associated therewith.

38. The method according to claim 27, wherein the treatment or prevention is for recurrent or refractory cancer, or metastatic cancer, optionally recurrent or refractory solid tumor, or metastatic solid tumor, recurrent or refractory hematological malignancies, or metastatic hematological malignancies.

39. The method according to claim 27, wherein the treatment or prevention is for a solid tumor selected from anal cancer, appendiceal cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breast cancer, cervical cancer, colon cancer, cancer of unknown primary origin (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid disease, penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, pharyngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

40. The method according to claim 27, wherein the treatment or prevention is for hematological malignancies, optionally leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some cases, the aforementioned hematological malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström hypergammaglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, and non-Burkitt lymphoma. This includes preventing or suppressing high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prelymphoblastic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoplasmic myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis and / or at least one related pathological condition.

41. The aforementioned treatment or prevention applies to chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström's hypergammaglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, and non-Burkitt lymphoma. The method according to claim 27, for use against a hematological malignancy selected from high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, B-cell prelymphoblastic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoplasm, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis. In some cases, the hematological malignancies are intended to prevent or suppress recurrent or refractory hematological malignancies, or metastatic hematological malignancies and / or at least one associated pathological condition.

42. The method according to claim 27, wherein the treatment or prevention is for the treatment of an autoimmune disease selected from the group consisting of Addison's disease, arthritis, celiac disease, lupus, Graves' disease, myasthenia gravis, multiple sclerosis, ITP, rheumatoid arthritis, colitis, inflammatory bowel disease, pernicious anemia, Hashimoto's disease, Sjögren's disease, asthma, type 2 diabetes mellitus, and autoimmune type 1 diabetes mellitus, and / or for the prevention or suppression of at least one pathological symptom associated therewith.

43. The method according to claim 27, wherein the treatment or prevention is for the treatment of cancer, and the ARC or ANC comprises nucleic acids that regulate or inhibit the expression of any of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, LAG-3, HHLA2, TNFRSF12A, HLA-G, NECTIN2, TNFRSF25, TNFSF14, LAIR1, TNFSF15, TNFSF4, KIR2DL4, PDCD1, LGALS9, VSIr (VISTA), and PVR, optionally ASO or siRNA.

44. The method according to claim 27, wherein the treatment or prevention is for suppressing or treating immunosenescence associated with disease or aging, and the ARC or ANC optionally comprises nucleic acids that regulate or inhibit the expression of any of PD-1, PD-L1, PD-L2, CTLA-4, B7-1, B7-2, LAG-3, HHLA2, TNFRSF12A, HLA-G, NECTIN2, TNFRSF25, TNFSF14, LAIR1, TNFSF15, TNFSF4, KIR2DL4, PDCD1, LGALS9, VSIr (VISTA), and PVR, and optionally ASO or siRNA.

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