Anti-VISTA antibody RNA or nucleic acid conjugates (ARC or ANC), compositions containing same and therapeutic uses thereof

Abstract

The present invention relates to anti-VISTA antibody RNA or nucleic acid conjugates (ARC or ANC) for the specific delivery of at least one nucleic acid, such as RNA, into immune cells, and the use of such ARC or ANC as a therapeutic agent, for example for the treatment of autoimmune, allergic and inflammatory disorders, such as autoimmune, allergic and inflammatory disorders. Or for treating cancer and / or for treating inflammatory symptoms associated with cancer caused by a specific immune cell type expressing VISTA.

Description

Related applications

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

[0002] Sequence Listing The contents of the electronic serial number (1143260_008613_SL.xml; size: 707,682 bytes; and creation date: June 3, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention disclosed herein relates to anti-VISTA antibody RNA or nucleic acid conjugates (ARC or ANC) that specifically deliver at least one nucleic acid (e.g., RNA or DNA or nucleic acid / protein complex) to immune cells, and the use of such ARC or ANC as therapeutic agents, for example for treating autoimmune and inflammatory conditions, or for treating cancers and / or symptoms associated with specific immune cell types. Background Technology

[0004] The regulation of RNA function is an emerging field with therapeutic value. Drugs affecting mRNA stability, such as antisense oligonucleotides and short interfering RNAs, are one way to regulate RNA function. Another group of oligonucleotides can regulate RNA function by altering the processing of pre-mRNA to include or exclude specific regions of the pre-mRNA in the final gene product (encoded protein). Therefore, oligonucleotide therapeutics represent a means of regulating protein expression in disease states and thus possess therapeutic utility.

[0005] In addition, RNA delivery to target cells is known through incorporation of ANCs or ARCs. However, existing ANCs or ARCs do not target immune cell types. Summary of the Invention

[0006] In one aspect, the present invention relates to anti-VISTA antibody RNA conjugates (ARCs) that can be used to specifically deliver nucleic acids (e.g., RNA or DNA or nucleic acid / protein complexes) into immune cells.

[0007] On the other hand, the present invention relates to the use of such ARCs as therapeutic agents, for example, for treating autoimmune, inflammatory and cancerous conditions.

[0008] In a specific aspect, the present invention provides antibody-RNA or antibody-nucleic acid conjugates (“ARC” or “ANC”) comprising (i) an antibody or antibody fragment that binds to an antigen specifically or primarily expressed by one or more immune cell types; directly or indirectly conjugated to (ii) one or more nucleic acids composed of wild-type or modified nucleotides, preferably RNA or DNA oligonucleotides (one or more “payloads”), said oligonucleotides specifically binding to a target gene (optionally an immunomodulatory gene) expressed by an immune cell or RNA encoded therefrom; and optionally (iii) a cleavable or non-cleavable linker or adaptor, such as a peptide between said (i) the antibody or antibody fragment and said (ii) one or more nucleic acids; wherein such ARC or ANC is internalized by immune cells upon contact with immune cells expressing an antigen bound by (i) the antibody or antibody fragment and provides release of said (ii) one or more nucleic acids into the immune cells; thereby optionally modulating the expression and / or function of a targeted immunomodulator.

[0009] In some specific aspects, any one of the foregoing aspects of the ARC or ANC comprises one or more payloads, the one or more payloads comprising one or more modified nucleotides, optionally at least one nucleotide modified with phosphonate and / or ribose, which facilitates direct or indirect linking of the one or more payloads to an antibody or antibody fragment, optionally via a peptide linker, further optionally via a cleavable or incleavable linker or adaptor, such as a peptide between (i) the antibody or antibody fragment and (ii) the payload.

[0010] In some specific aspects, any one of the foregoing aspects of the ARC or ANC includes a payload that is directly or indirectly conjugated to an antibody or antibody fragment via a reactive amine, the reactive amine optionally being contained on a lysine residue on the antibody or antibody fragment and / or (i) the antibody or antibody fragment being linked to (ii) one or more peptides of the payload.

[0011] In some specific aspects, any of the foregoing aspects of ARC or ANC comprises an antibody or antibody fragment that binds to VISTA, preferably human VISTA.

[0012] In some specific aspects, any one of the foregoing aspects of ARC or ANC comprises an antibody or antibody fragment binding to VISTA, preferably human VISTA, and comprises, with respect to, an antibody containing Figure 14 The same VH and VL CDRs as any anti-human VISTA antibody with the sequence in Appendix 1 or Appendix 3.

[0013] In any of the foregoing aspects, the antibody or antibody fragment in the ARC or ANC binds to VISTA, preferably human VISTA, and contains the same VH and / or VL regions and CDR as those described above: containing Figure 14 Any anti-human VISTA antibody containing the VH and / or VL sequences, or containing [specific sequences]. Figure 14 The antibody or antibody fragment having at least 90%, 95%, or 99% sequence identity in the VH and / or VL regions of any anti-human VISTA antibody, or containing the VH and / or VL sequences in Appendix 1 or Appendix 3; and the antibody or antibody fragment optionally containing a polypeptide with a constant IgG1, IgG2, IgG3, or IgG4 domain, further optionally containing a polypeptide with a constant IgG1 domain, and even more optionally containing a polypeptide with a sequence contained in Appendix 1 or 3.

[0014] In some specific aspects, the antibody or antibody fragment on the ANC or ARC contains 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 aspects, any one of the foregoing aspects of ARC or ANC includes one or more of the following: short interfering RNA (siRNA), antisense oligonucleotide (ASO), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterologous nuclear RNA (hnRNA).

[0016] In some specific aspects, any ARC or ANC in any of the foregoing aspects comprises a polynucleotide molecule with a length of 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; or it comprises a polynucleotide molecule with a length of 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.

[0017] In some specific aspects, any ARC or ANC in any of the foregoing aspects comprises a first polynucleotide and a second polynucleotide, optionally wherein the first polynucleotide is a sense strand or a transit strand and / or the second polynucleotide is an antisense strand or a guide strand.

[0018] In some specific aspects, any one of the foregoing aspects of ARC or ANC comprises siRNA, ASO, tRNA, rRNA, or mRNA.

[0019] In some specific aspects, any of the foregoing aspects of ARC or ANC comprises, or is encapsulated in or conjugated with lipid nanoparticles.

[0020] In some specific aspects, any one of the foregoing aspects of ARC or ANC comprises at least one payload that targets an immunomodulatory agent selected from any of the foregoing aspects, including cytokines, chemokines, interleukins, interferons, tumor necrosis factor, or receptors.

[0021] In some specific aspects, any one of the foregoing aspects of the ARC or ANC comprises a payload that 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 α (PIK3CA), or RAR-associated orphan receptor C (RORC), or has Figure 1 or Figure 2 Any molecule whose sequence is identified in the middle.

[0022] In some specific aspects, any one of the foregoing aspects of the ARC or ANC comprises an siRNA payload that targets RNA or DNA encoding GLUT3 or PIK3CA, optionally the sequence in Appendix 2 or 4.

[0023] In some specific aspects, any one of the foregoing aspects of ARC or ANC comprises an antibody or antibody fragment that binds to at least one immune cell, said immune cell being 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, mucosa-associated inertial 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 cells, etc. (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 aspects, any one of the foregoing aspects of ARC or ANC comprises an antibody or antibody fragment that binds to myeloid cells and / or T cells.

[0025] In some specific aspects, any one of the foregoing aspects of ARC or ANC comprises an antibody or antibody fragment that binds to T cells, T cell progenitor cells, or NK cells.

[0026] In some specific aspects, any ARC or ANC in any of the foregoing aspects comprises at least one nucleic acid payload, optionally RNA or DNA, further optionally siRNA or antisense RNA, said nucleic acid payload binding to a gene or nucleic acid encoding an antigen selected from the group consisting of: (1) 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 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, ADAM 15, ADAM 17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-L-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artesunate, anti-Id, ASPARTIC, atrial natriuretic factor, av / 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 osteoblasts, BMP-4, BMP-2b, BMP-5, BMP-6, Vgr-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, tadalafil, 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, CCL13CCL 14, CCL15, CCL16, CCL1 7. 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, CD 15. 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-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, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay accelerator factor, des(l-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, DNase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, Endothelin receptor, Enkephalinase, eNOS, Eot, eotaxinl, EpCAM, Ephrin B2 / EphB4EPO, 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, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-Stimulating Hormone, Fractal Chemokines, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, 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-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut 4, 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), HepB gpl20, heparinase, 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 gp120, HIV IIIB gp120 V3 ring, 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)-α, INF-β,INF-γ, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, Integrin α5 (αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, IP-10, 1-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, 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, luteinizing hormone, lymphotoxin β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, 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, Mucl, MUC18, Müllerian canal inhibitor, Mug, Musk, NAIP, NAP, NCAD, N-cadherin, NCA 90. NCAM, Enkephalin, Neurotrophic Factor-3, Neurotrophic Factor-4 or Neurotrophic Factor-6, Neuro-rank protein, Neuronal Growth Factor (NGF), NGFR, NGF-β, 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, PGJ2, PIN, PLA2, Placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin, Pro-relaxinProtein 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β panspecific, TGF-βRI (ALK-5), TGF-β RII, TGF-β Rllb, TGF-β RIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10A (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), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROY TAJ, 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 (CD40p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL Rl TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TLIA / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSFIA (TNF-α connexin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigens expressing Lewis Y-related carbohydrates, 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 antigen, 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 3) and hormone receptor; or (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, 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, TNFα, TGFβ, cytokine receptor, IL-2R, chemokines, chemokine receptors, growth factors, 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,CD9 3、CD94、CD95、CD96、CD97、CD98、CD99、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD108、CD109、CD110、CD111、CD112、CD114、CD 115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CDw121b、CD122、CD123、CD124、CD125、CD126、CD127、CDw128、CD129、CD130、CDw131、C D132、CD133、CD134、CD135、CDw136、CDw137、CD138、CD139、CD140a、b、CD141、CD142、CD143、CD144、CD145、CD146、CD147、CD148、CD149、CD150、C D151, CD152, CD153, CD154, CD155, CD156b, CD157, CD158, CD158a, CD159a, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167a, CD 168、CD169、CD170、CD171、CD172a、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD183、CD184、CD195、CDw197、CD 200、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) IL4ra, TNFa, BTK, RORgt, PIK3CA, JAK1, JAK3, TYK2 Any one of Glut1, Glut3, TAP1, CIITA, cGAS, IRF5, STAT3, STAT6, TAK1 (MAP3K7), HPK1; or any one of SOCS1, CD39, Cbl, or PTPN22; or (5) any one of Glut1, PI3K, BTK, TNF, or RORC; or (6) any one 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, and PVR.

[0027] In some specific aspects, any one of the foregoing aspects of the ARC or ANC comprises a nucleic acid payload, optionally RNA, further optionally siRNA or antisense RNA, having a payload comprising a sequence selected from those described in Appendix 2 or Appendix 4; or comprising INX-201 ARC selected from those described in Appendix 2, or comprising INX-201 ARC comprising an amino acid sequence and a payload sequence described in Appendix 4.

[0028] In some specific aspects, any one of the foregoing aspects of the ARC or ANC comprises at least two different RNA payloads that target the same or different immune regulatory genes or mRNAs, optionally as immune targets disclosed above.

[0029] In some specific aspects of ARC or ANC as described in any of the foregoing aspects, nucleic acids (optionally RNA payloads) are linked to antibodies or antibody fragments via cleavable or non-cleavable adapters.

[0030] In some specific aspects, any one of the foregoing aspects of ARC or ANC is used to deliver one or more gene-editing nucleic acids (e.g., CRISPR guide RNA (gRNA or sgRNA)) and optionally a CRISPR-associated endonuclease or a nucleic acid encoding a CRISPR-associated endonuclease.

[0031] In some specific aspects, any one of the foregoing aspects of 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 longer.

[0032] In some specific respects, any of the foregoing ARC or ANC does not induce any apparent toxicity to non-target cells.

[0033] In some specific aspects, the present invention provides a composition comprising any one of the foregoing aspects of ARC or ANC and a pharmaceutically acceptable carrier or excipient, wherein the ARC or ANC is optionally contained in or on lipid nanoparticles.

[0034] In some specific aspects, the present invention provides a method of treatment or prevention comprising administering to a subject in need any of the preceding aspects of ARC or ANC, or a composition containing the ARC or ANC.

[0035] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of neoplastic, proliferative, neurodegenerative, neuroinflammatory, infectious, autoimmune, allergic, or inflammatory conditions, or pathological symptoms associated with any of such conditions.

[0036] In some aspects, the present invention relates to administering any of the foregoing ARC or ANC, or a composition containing the ARC or ANC, to a subject in need for the treatment or prevention of autoimmune diseases, such as diseases involving myeloid cells or T cells.

[0037] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of neoplastic, proliferative, neurodegenerative, neuroinflammatory, infectious, autoimmune, or inflammatory diseases, and / or the prevention or suppression of at least one pathological symptom associated therewith.

[0038] In some aspects, the present invention relates to administering, to a subject in need, any of the foregoing ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of one or more autoimmune diseases selected from: acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson syndrome. Autoimmune syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglion disease (AAG) / autoimmune autonomic disorders | 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), autoimmune polyglandular syndrome, types I, II and III (APS type 1, APS type 2, APS type 3, APS type 4, APS type 5, APS type 6, APS type 7, APS type 8, APS type 9, APS type 1, APS type 1, APS type 1, APS type 2, APS type 3 ... Type 3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Barlow's disease, Behcet's disease, shotgun-like chorioretinopathy / shotgun-like uveitis, bullous pemphigoid, Kassman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Chag-Strauss syndrome / eosinophilic granulomatous polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome | localized cutaneous systemic sclerosis, Crohn's disease (CD), Canada-Kronka syndrome (CSS), cryptogenic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes Diabetes, discoid lupus, Desleser syndrome / post-myocardial infarction syndrome / post-pericardiotomy syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpassch syndrome / anti-GBM disease / anti-TBM disease, granulomatous polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid ophthalmopathy, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, allergic purpura / IgA vasculitis, hidradenitis suppurativa,Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Berge's disease, immune-mediated necrotizing myopathy (IMNM), immune thrombocytopenic purpura (ITP) / autoimmune thrombocytopenic purpura / autoimmune thrombocytopenic purpura, inclusion body myositis, IgG4-related sclerotic disease (ISD), interstitial cystitis, juvenile idiopathic arthritis / adult Still's disease, juvenile polymyositis / juvenile dermatomyositis / juvenile myositis, Kawasaki disease, Lambert-Eton myasthenic syndrome (LEMS), leukocytic clotting vasculitis, lichen planus, sclerosing lichen, woody conjunctivitis, linear IgA disease (LAD) / linear IgA bullous dermatosis (LABD), lupus Sootympanic nephritis, Lyme disease / chronic Lyme disease / treatment-induced 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), keratodermal ulcer, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devrwick disease, ocular cicatricial pemphigoid, oculoclonus-myoclonus syndrome (OMS), relapsing rheumatism, paraneoplastic cerebellum Degeneration, paraneoplastic pemphigus, Parsenne-Robbie syndrome (PRS) / hemispheric atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH), peripheral uveitis / parietal planus inflammation, PANS / PANDAS, Parsenne-Turner syndrome, pemphigus gestationis / herpes gestationis, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural 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 aplasia (PRCA), pyoderma gangrene, Rasmussen encephalitis, Raynaud's syndrome / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy syndrome (RSD) / complex regional pain syndrome (CRPS), relapsing polychondritis, restless legs syndrome (RLS) / Willis-Ekbom's disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome / autoimmune polyendocrine syndrome type II, scleritis, scleroderma, sclerosing mesenteric / mesenteric panniculitis, creeping choroidal lesions, Sjögren's syndrome, stiff-person syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE)Subacute cutaneous lupus erythematosus, Sussac syndrome, Siddenham's chorea, sympathetic ophthalmia, Takayasu arteritis (vasculitis), testicular autoimmune diseases (vasculitis, orchitis), Tolosa-Hunter syndrome, transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), pre / intermediate / post-uveitis, vasculitis, VEXAS syndrome, vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH), and / or prevention or suppression of at least one pathological symptom associated with them.

[0039] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, 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 thyroiditis, Sjögren's disease, asthma, type 2 diabetes and autoimmune type 1 diabetes, and / or prevention or suppression of at least one pathological symptom associated therewith.

[0040] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing the ARC or ANC, 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, herpes zoster, and vasculitis, and / or the prevention or suppression of at least one pathological symptom associated therewith.

[0041] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of neurodegenerative or neuroinflammatory diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, aphasia, Parkinson's disease, or spinal muscular atrophy, and / or prevention or inhibition of at least one pathological symptom associated therewith.

[0042] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of cancer or the prevention of cancer recurrence, and / or the suppression of at least one pathological symptom associated with a specific immune cell type.

[0043] In some aspects, the present invention relates to administering any of the foregoing ARC or ANC, or a composition containing the ARC or ANC, to a subject in need for the treatment or prevention of solid tumors, and / or the prevention or suppression of at least one pathological symptom associated therewith.

[0044] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing the ARC or ANC, for the treatment or prevention of hematologic malignancies, and / or the prevention or suppression of at least one pathological symptom associated therewith.

[0045] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of recurrent or refractory cancer or metastatic cancer, optionally recurrent or refractory solid tumor or metastatic solid tumor, recurrent or refractory hematologic malignancy or metastatic hematologic malignancy.

[0046] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of solid tumors selected from anal cancer, appendiceal cancer, biliary tract cancer (i.e., bile duct cancer), 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 adenoma, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer, and / or prevention or suppression of at least one associated pathological symptom.

[0047] In some aspects, the present invention relates to administering, to a subject in need, any of the foregoing ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of hematologic malignancies, optionally leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some cases, hematologic 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 macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and Burkitt lymphoma. Non-Burkita high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma or lymphomatoid granulomatosis, and / or prevention or suppression of at least one pathological symptom associated therewith.

[0048] In some aspects, the present invention relates to administering, to a subject in need, any of the foregoing ARC or ANC, or a composition containing such ARC or ANC, for the treatment or prevention of hematologic malignancies selected from 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), and Waldenstine lymphoma. Macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytic myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis. In some cases, hematologic malignancies are relapsed or refractory hematologic malignancies or metastatic hematologic malignancies, and / or prevention or suppression of at least one associated pathological symptom.

[0049] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, 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 thyroiditis, Sjögren's disease, asthma, type 2 diabetes and autoimmune type 1 diabetes, and / or the prevention or suppression of at least one pathological symptom associated therewith.

[0050] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, for the treatment of cancer, wherein the ARC or ANC comprises a nucleic acid, optionally an ASO or siRNA, that regulates or blocks 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.

[0051] In some aspects, the present invention relates to administering to a subject in need any of the preceding aspects an ARC or ANC, or a composition containing such ARC or ANC, to inhibit or treat disease- or aging-related immunosenescence, wherein the ARC or ANC optionally comprises a nucleic acid, and further optionally comprises an ASO or siRNA that regulates or blocks 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. Attached Figure Description

[0052] Figure 1 This list contains immunologically relevant siRNAs and ASO payloads covered by this invention and tested. In proof-of-concept experiments, K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and target knockdown was analyzed by qRTPCR using the ddct method, reported as a fold change relative to control. Scrambled siRNA or ASO controls were also purchased from IDT. Payloads marked with (*) were selected for conjugation with the delivery medium (anti-VISTA Mab INX201).

[0053] Figure 2 List of other identified and tested immunologically relevant siRNAs and ASO payloads. K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and target knockdown was analyzed by qRTPCR using the ddct method and reported as foldbacks relative to controls. Scrambled siRNA or ASO controls were also purchased from IDT. Payloads marked with (*) were selected for conjugation with the delivery medium (anti-VISTA Mab INX201). Only CD39 ARCs from second-round payloads are described in this report; conjugation of other ARCs is planned / in progress.

[0054] Figure 3 SDS-PAGE results containing the exemplary ARC (INX201 ARC) according to the present invention confirm effective RNA conjugation. INX201 ARC was resolved on reducing SDS-PAGE followed by silver staining. Lane markings: 201 - free INX201 anti-VISTA Mab; CD45 - ARC using CD45 siRNA; SOCS1 - ARC using SOCS1 ASO; A discrete distribution pattern of multiple conjugates was observed, confirming effective conjugation. Based on the effective payload molecular weight (approximately 17 kDa vs. approximately 6 kDa), the HC or LC shift of the siRNA was more significant than that of the ASO.

[0055] Figures 4A to 4B Exemplary ARC binding according to the present invention is shown, providing efficient internalization of siRNA from the surface and intracellular retention within K562-VISTA cells. Free INX201 (with circular lines) or eGFPARC (with square lines) were used at 200 nm. A) Time course of antibody binding and internalization. B) RNA accumulation assay measured by Cy5. MFI – mean fluorescence intensity; representing the two independent experiments shown.

[0056] Figures 5A to 5CThe exemplary ARC (INX201 ARC) knockdown protein expression according to the present invention is shown to be equivalent to in vitro transfection. K562-VISTA WT cells or eGFP+ cell pools were used in this study. A) Cells were treated with no drug (left column) or 200 nM eGFP ARC (right column) for 28 hours. The maximum level of eGFP protein knockdown (approximately 50%, measured at 24 hours) was established by transfecting the same cells with eGFP siRNA. ARC-mediated knockdown is similar to the maximum possible knockdown level (based on siRNA sequence). B) The dashed line represents the maximum level of CD45 protein knockdown expected based on the potency of the payload (siRNA), which is 50%, as measured at 48 hours by free transfected CD45 siRNA (right column); cells were treated with 200 nM eGFP ARC (middle column) or no drug (left column) for 72 hours. C) CD45 levels were measured in replicate experiments. Cells were treated for 72 hours with no drug (left column), free siRNA, no transfection (middle column), and 200 nM ARC (right column).

[0057] Figure 6 A to Figure 6 C illustrates an exemplary ARC (INX201 ARC) method according to the present invention to inhibit TNFα from PBMCs. Human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated with TNFα ARC (0-200 nM) or free RNA (200-1000 nM) for 48 h (LPS) or 72 h (beads). With ARC, TNFα levels decreased effectively in a dose-dependent manner, but not with free siRNA. C) qRTPCR of ARC-treated PBMCs collected at 72 h relative to untreated PBMCs confirmed effective target knockdown; no 0.1 nM ARC was observed (due to logarithmic scaling).

[0058] Figures 7A to 7BAn exemplary ARC (INX201 ARC) according to the present invention is shown to slow T cell proliferation. Human PBMCs were activated with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated for 72 hours with (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). With the use of ARC, newly proliferating T cells showed an effective dose-dependent reduction, but this was not the case with free siRNA treatment. Proliferation was analyzed by cell trace violet dilution and observed by flow cytometry; no ARC was observed at 0.1 nM (due to logarithmic scaling). Curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 9). Each concentration point was determined in parallel using a single technique.

[0059] Figures 8A to 8B An exemplary ARC (INX201-BTK ARC) of the present invention is shown to reduce PBMC activation. Human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated with BTK ARC (0-200 nM, triangular) or free RNA (200-1000 nM, star-shaped) or free INX201 (square) for 48 h (LPS) or 72 h (beads). Curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 9). CD69 levels were measured in A; new proliferating cells were measured in B; each concentration point was measured in parallel using a single technique; no ARC was observed at 0.1 nM (due to logarithmic scaling); unstimulated - unstimulated cells; MFI - mean fluorescence intensity.

[0060] Figures 9A to 9B An exemplary ARC R-induced cytokine production (INX201-Glut1ARC) according to the present invention is shown. Purified human T cells were activated with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated with Glut1 ARC (0-200 nM) for 72 hours. Each concentration point was measured in parallel using a single technique. Two human donors were tested: donor 1 – solid line, donor 2 – dashed line; no ARC was observed at 0.1 nM (due to logarithmic scaling). A) IFNg and B) IL17A were measured by Luminex.

[0061] Figures 10A to 10CAlso shown is an exemplary ARC (INX201-Glut1 ARC) for reducing cytokine production according to the invention. Purified human T cells were activated with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated with RORC ARC (0-200 nM) for 72 hours. Each concentration point was measured in parallel using a single technique. Two human donors were tested: donor 1 – solid line, donor 2 – dashed line; no ARC was observed at 0.1 nM (due to logarithmic scaling). A) IFNg, B) IL-6, and C) IL12p40 were measured by Luminex.

[0062] Figure 11 This demonstrates the successful targeting and enhancement of the immune response in human PBMCs by an exemplary ARC (INX201-CD39 ASO) according to the present invention. Human PBMCs were activated with anti-CD3 / CD28 beads and treated with CD39 ARC (0–200 nM, triangle) or free INX201 (0–200 nM, circle) for 72 hours. Curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 9). Each concentration point was determined in parallel using a single technique; no ARC was observed at 0.1 nM (due to logarithmic scaling).

[0063] Figures 12A to 12B This schematically illustrates how the ARC platform of the present invention can be optimized by using anti-VISTA Fab instead of Mab. A) The binding of INX201 Mab (square) and INX201 Fab (circle) to human VISTA ECD was compared by ELISA. B) A competitive assay based on K562-VISTA cells was performed, where increased pre-binding concentrations of either INX201 Mab (square) or INX201 Fab (circle) blocked the availability of VISTA on the cell surface (thus reducing VISTAMFI measurements of INX201-AF488); curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 9). Each concentration point was measured in parallel using n=2 techniques; no Ab at 0.0001 nM was observed (due to log scaling).

[0064] Figure 13An exemplary ARC according to the present invention is shown to not affect T cell viability. 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 by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 9). Each concentration point was determined in parallel using a single technique; no ARC was observed at 0.1 nM (due to logarithmic scaling); unstimulated–unstimulated cells.

[0065] Figure 14 The CDR and variable sequences of exemplary anti-human VISTA antibodies that can be used in the ARC or ANC according to the present invention are shown.

[0066] Figure 15 This study demonstrates that INX201 (anti-VISTA) PI3K ARC, rather than the allotype control IgG1 PI3K ARC, specifically knocked down PI3K expression. K562-VISTA cells were transfected with 40–200 nM INX201-PI3K ARC or allotype control IgG1-PI3K ARC, and target knockdown was analyzed by qRT-PCR using the ddct method and reported as target inhibition %. 0% inhibition was for INX201 alone; PI3K ARC: anti-human VISTA mAb conjugated with PI3K siRNA; PI3K allotype ARC: IgG control conjugated with PI3K siRNA; INX201, mAb unconjugated: naked anti-human VISTA mAb. Each concentration point was measured in parallel using a single technique.

[0067] Figures 16A to 16BHuman PI3K and GLUT3 ARC were demonstrated to be functional in vitro. Human PBMCs were activated for 72 hours with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 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 by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 10). IL5 / IL13 / TNFα / 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; CD25 levels were measured in live CD45+ / HLA-DR- / CD56- / CD3 cells. In A, single technique parallel determinations were used for each concentration point; in B, n=2 techniques parallel determinations were used for each concentration point; no ARC was observed at 0.1 nM (due to logarithmic scaling); MFI – mean fluorescence intensity. Where appropriate, data are expressed as mean ± SEM.

[0068] Figures 17A to 17C This study demonstrates that PI3K ARC reduced inflammatory cytokine responses in xenogeneic GvHD. 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 post-injection, mice were intraperitoneally stimulated with 0.5 mg / kg LPS. A) Xenogeneic GvHD LPS stimulation model. A schematic diagram of the experiment is shown. B) Changes in plasma human cytokine levels at day 7 (IFNg) or 4 hours (IL6, TNFα). Statistical analysis was performed using Student's t-test. Data are presented as mean ± SEM, *-p<0.05; **-p<0.01. C) A Z-score-based heatmap of cytokine levels at 4 hours post-LPS stimulation (n=6 per group).

[0069] Figure 18 PI3KARC was shown to reduce LPS-induced T cell proliferation in vivo. NSG mice were intravenously injected with either PI3KARC (triangular, n=6) or INX201 (circular, n=6) at 5 mg / kg, along with human PBMC transfer. Mice were intraperitoneally stimulated with 0.5 mg / kg LPS 18 hours post-injection. Changes in blood T cell counts for both CD4 and CD8 cells at day 14 are shown (n=6 mice per group). Statistical analysis was performed using Student's t-test. Data are expressed as mean ± SEM, *-p<0.05.

[0070] Figure 19The results show that PI3K ARC does not decrease the percentage of regulatory T cells in vivo. NSG mice were intravenously injected with either PI3K ARC (triangle, n=10) or INX201 (circle, n=8) at 5 mg / kg, along with human PBMC transfer (right panel). In the mouse-only groups, mice were intraperitoneally stimulated with 0.5 mg / kg LPS 18 hours after PBMC injection (for the INX201 and PI3K ARC groups, n=6, left panel). Blood samples were processed on day 28. The percentage change in Tregs is shown. Statistical analysis was performed using Student's t-test. Data are presented as mean ± SEM. ns- were not significant.

[0071] Figure 20 GLUT3 ARC was shown to reduce inflammatory cytokine responses in xenogeneic GvHD. NSG mice were intravenously injected with either PI3K ARC (triangular, n=10) or INX201 (circular, n=8) at 5 mg / kg, along with human PBMC transfer. Changes in plasma human cytokine levels on day 7 are shown. Statistical analysis was performed using Student's t-test. Data are presented as mean ± SEM, **-p<0.01; *-p<0.0001. Detailed Implementation

[0072] The present invention disclosed herein relates to anti-VISTA antibody oligonucleotide conjugates (ARC or ANC) that specifically deliver RNA to immune cells and their use as therapeutic agents, such as for the treatment of autoimmune and inflammatory conditions.

[0073] definition The following lists the definitions of various terms used to describe this disclosure. These definitions apply to terms used throughout this specification and claims, unless otherwise limited individually or as part of a larger group in particular cases.

[0074] The term “about” will not be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, when referring to measurable values ​​such as quantity, duration, etc., the term “about” is intended to cover a change of ±20% or ±10% compared to a specified value, including ±5%, ±1%, and ±0.1%, because such changes are suitable for performing the disclosed methods.

[0075] The terms “antibody-RNA conjugate” or “antibody-nucleic acid conjugate” or “ARC” or “ANC” as used herein generally refer to a conjugate comprising (i) an antibody or antibody fragment (such as Fab) that specifically or preferentially binds to one or more target immune cell types; and is directly or indirectly linked (e.g., via a cleavable or uncleavable peptide linker or other cleavable or uncleavable linker) to (ii) one or more nucleic acids, typically oligonucleotides, such as DNA or RNA, which may consist of wild-type or modified nucleotides, and which specifically target an immunomodulatory gene or RNA encoded therein and regulate the expression and / or activity of an immunomodulatory protein encoded therein. In a preferred embodiment, the antibody or antibody fragment is an internalizing antibody, i.e., when it binds to a target antigen on a target immune cell, it internalizes the immune cell and delivers the oligonucleotide cargo contained on the ARC or ANC to the immune cell. In an exemplary embodiment, the antibody or antibody fragment is an internalizing antibody or antibody fragment that specifically binds to VISTA, preferably human VISTA.

[0076] The term "alkyl" refers to a saturated straight-chain or branched hydrocarbon moiety containing one to six or one to eight carbon atoms in certain embodiments. Examples of Ci-6-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, α-butyl, tert-butyl, neopentyl, and n-hexyl moieties; and examples of Ci-s-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, α-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

[0077] The number of carbon atoms in an alkyl substituent can be represented by the prefix "C". x-y "" indicates that x is the minimum number of carbon atoms in the substituent and y is the maximum number of carbon atoms. Similarly, C x A chain refers to an alkyl chain containing x carbon atoms.

[0078] Unless otherwise stated, the term "heteroalkyl" on its own or in combination with another term means a stable straight-chain or branched alkyl group consisting of the stated 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. Heteratoms may be positioned anywhere on the heteroalkyl group, including between the remainder of the heteroalkyl group and the segment to which it is attached, and on the most distal carbon atom attached to the heteroalkyl group. Examples include: -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, -CH2-CH2-H-CH3, -CH2-S-CH2-CH3, and -CH2-CH2-S(=O)-CH3. At most two heteroatoms may be consecutive, such as -CH2-H-OCH or -CH2-CH2-SS-CH.

[0079] Unless otherwise stated, the term "aryl" used alone or in combination with other terms refers to a carbocyclic aromatic system containing one or more rings (typically one, two, or three rings), wherein such rings may be linked together in a side-chain manner, such as biphenyl, or may be fused, such as naphthalene. Examples of aryl groups include phenyl, anthracene, and naphthyl. In various embodiments, examples of aryl groups may include phenyl (e.g., C6-aryl) and biphenyl (e.g., Ci2-aryl). In some embodiments, the aryl group has six to sixteen carbon atoms. In some embodiments, the aryl group has six to twelve carbon atoms (e.g., C6-i2-aryl). In some embodiments, the aryl group has six carbon atoms (e.g., Ce-aryl).

[0080] As used herein, the terms "heteroaryl" or "heteroaromatic" refer to heterocycles that possess 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, excluding the number of heteroatoms. For instance, Ci-9-heteroaryl would contain one to four additional heteroatoms. Polycyclic heteroaryl groups can contain one or more partially saturated rings. Non-limiting examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (including, for example, 2-pyrimidinyl and 4-pyrimidinyl), pyridazinyl, thiopheneyl, furanyl, pyrroleyl (including, for example, 2-pyrroleyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (including, for example, 3-pyrazolyl 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.

[0081] Non-limiting examples of polycyclic heterocyclic compounds and heteroaryl groups include indolyl (including, for example, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, and 7-indolyl), indololinyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl (including, for example, 1-isoquinolinyl and 5-isoquinolinyl), 1,2,3,4-tetrahydroisoquinolinyl, cenolinyl, quinoxalinyl (including, for example, 2-quinoxalinyl and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthodioxinyl, 1,4-benzodioxane, coumarin, dihydrocoumarin, 1,5-naphthodioxinyl, and benzofuranyl (including, for example, 3-benzofuranyl). 2,3-Dihydrobenzofuranyl, 1,2-Benzisoxazolyl, benzothiophene (including, for example, 3-benzothiophene, 4-benzothiophene, 5-benzothiophene, 6-benzothiophene and 7-benzothiophene), benzoxazolyl, benzothiazolyl (including, for example, 2-benzothiazolyl and 5-benzothiazolyl), purine, benzimidazolyl (including, for example, 2-benzoimidazolyl), benzotriazolyl, thioxanyl, carbazolyl, carbazolinyl, acridinel, pyrrolizinyl and quinazinyl.

[0082] The term "protecting group" or "chemical protecting group" refers to a chemical moiety that blocks some or all of the reactive parts of a compound and prevents such moieties from participating in a chemical reaction until the protecting group is removed, such as those moieties listed and described in TW Greene, PGM Wuts, *Protective Groups in Organic Synthesis*, 3rd edition, John Wiley & Sons (1999). When different protecting groups are used, it may be advantageous that each (different) protecting group can be removed by different means. Cleavage of protecting groups under completely different reaction conditions allows for different removal of such protecting groups. For example, protecting groups can be removed by acid, base, and hydrogenolysis. Groups such as triphenylmethyl, monomethoxytriphenylmethyl, dimethoxytriphenylmethyl, acetal, and tert-butyldimethylsilyl are acid-labile and can be used to protect the carboxyl and hydroxyl reactive moieties in the presence of an amino group protected with a Cbz group and a base-labile Fmoc group, which can be removed by hydrogenolysis. The carboxylic acid moiety can be blocked by a base-indestructible group such as, but not limited to, methyl or ethyl, and the hydroxyl reactive moiety can be blocked by a base-indestructible group such as acetyl in the presence of an amine blocked by an acid-indestructible group such as tert-butyl carbamate or an amine blocked by an acid- and base-stable but hydrolyzable carbamate.

[0083] The reactive moiety of the carboxylic acid and hydroxyl group can also be blocked with a protecting group that can be hydrolyzed and removed, such as a benzyl group, while the amine group can be blocked with a base-unstable group, such as Fmoc. A particularly useful amine protecting group is trifluoroacetamide. The reactive moiety of the carboxylic acid can be blocked with a protecting group that can be oxidatively removed, such as 2,4-dimethoxybenzyl, while the coexisting amine group can be blocked with a fluoride-unstable silyl carbamate.

[0084] Allyl blocking groups are useful in the presence of both acid- and base-protecting groups, as the former are stable and can subsequently be removed by metal or π-acid catalysts. For example, allyl-blocked carboxylic acids can be protected by palladium(O)-catalyzed reactions in the presence of acid-instable tert-butyl carbamate or base-instable ammonium acetate protecting groups. Another form of protecting group is that which can be attached to a resin of a compound or intermediate. As long as the residue is attached to the resin, the functional group is blocked and cannot react. Once released from the resin, the functional group can be used for reaction.

[0085] The terms “nucleobase,” “base-pairing moiety,” “nucleobase-pairing moiety,” or “base” refer to the heterocyclic moiety of a nucleoside, nucleotide, and / or morpholino subunit. Nucleobases can be naturally occurring or can be modifications or analogs of these naturally occurring nucleobases; for example, one or more nitrogen atoms of a nucleobase can be independently substituted with carbon atoms each time it appears. Exemplary analogs include hypoxanthine (the base component of the nucleoside inosine); 2,6-diaminopurine; 5-methylcytosine; C5-propynyl-modified pyrimidines; 10-(9-(aminoethoxy)phenoxazine) (G-clamp), etc.

[0086] Other examples of base-pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine, and hypoxanthine (each with its amino group protected by an acyl protecting group), 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purines, xanthines, or hypoxanthines (the latter two being natural degradation products). Chiu and Rana are also envisioned. RNA , 2003, 9, 1034-1048; Limbach et al., Nucleic Acids Research ,1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry The modified nucleobases disclosed in Volume 7, 313 are incorporated herein by reference.

[0087] Other examples of base-pairing moieties include, but are not limited to, nucleobases in which one or more benzene rings have been added with size extensions. Glen Research catalog (www.glenresearch.com); 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 Nucleic acid base substitutions described in references 622-627, October 2006, are considered useful for the synthesis of the oligomers described herein, the contents of which are incorporated herein by reference.

[0088] The term "oligonucleotide" or "oligomery" 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 morpholine oligonucleotide.

[0089] The phrase "morpholino oligonucleotide" or "PMO" refers to a modified oligonucleotide having morpholino subunits linked together by an aminophosphate bond or a diaminophosphate bond, with the morpholino nitrogen of one subunit attached to the 5'-external carbon of the adjacent subunit. Each morpholino subunit contains a nucleobase-pairing portion that efficiently binds to nucleosides in a target via nucleobase-specific hydrogen bonds.

[0090] The terms “antisense oligomer,” “antisense compound,” and “antisense oligonucleotide” or “ASO” are used interchangeably and refer to subunit sequences, each containing a base-pairing portion linked by an intersubunit bond. This intersubunit bond allows the base-pairing portion to hybridize with a target sequence in a nucleic acid (usually RNA) via Watson-Crick base pairing to form a nucleic acid:oligomer heteroduplex within the target sequence. The oligomer can have exact (complete) or near (sufficient) sequence complementarity with the target sequence; sequence variations near the ends of the oligomer are generally superior to those within.

[0091] Such antisense oligomers can be programmed to block or inhibit the translation of mRNA or to inhibit / alter native or aberrant pre-mRNA splicing processing, and can be referred to as “targeting” or “targeting” the target sequence with which they hybridize. The target sequence is typically a region containing the AUG start codon of the mRNA, a splice site of the translation repressor oligomer or pretreated mRNA, or a splice repressor oligomer (SSO). The target sequence at the splice site can be an mRNA sequence downstream of the normal splice acceptor junction in the pretreated mRNA, having approximately 1 to 25 base pairs at its 5' end. In various embodiments, the target sequence can be any region of the pretreated mRNA that includes a splice site or is entirely contained within an exon coding sequence or spans a splice acceptor or donor site. When the oligomer targets the nucleic acid of the target as described above, it is more generally referred to as “targeting” a biologically relevant target, such as a protein, virus, or bacteria.

[0092] When a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen-bond with each other, the antisense oligonucleotide and the target RNA are complementary, resulting in stable and specific binding between the oligonucleotide and the target RNA. Therefore, "capable of specific hybridization" and "complementary" are terms used to indicate a sufficient degree of complementarity or precise pairing that allows for stable and specific binding between the oligonucleotide and the target. It should be understood in the art that the sequence of the oligonucleotide does not need to be 100% complementary to its target sequence for specific hybridization. Oligonucleotides are specifically hybridizable when the binding of the oligonucleotide to the target molecule interferes with the normal function of the target RNA and there is a sufficient degree of complementarity to prevent the antisense oligonucleotide from non-specifically binding to non-target sequences under conditions requiring specific binding (i.e., physiological conditions in in vivo assays or therapeutic treatments, and in in vitro assays, under the conditions under which the assay is performed).

[0093] Oligonucleotides may also contain nucleobase (generally referred to in the art simply as "base") modifications or substitutions. Oligonucleotides containing modified or substituted bases include oligonucleotides in which one or more of the most common purine or pyrimidine bases in the nucleic acid are replaced by less common or non-natural bases. In some embodiments, the nucleobase is covalently linked to the morpholine ring of the nucleotide or nucleoside at the N9 atom of the purine base or the N1 atom of the pyrimidine base.

[0094] Purine bases consist of a pyrimidine ring fused to an imidazole ring. Adenine and guanine are the two most common purine nucleobases in nucleic acids. These can be substituted by other naturally occurring purines, including but not limited to N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0095] Pyrimidine bases contain a six-membered pyrimidine ring. Cytosine, uracil, and thymine are the most common pyrimidine bases in nucleic acids. These can be substituted by other naturally occurring pyrimidines, including but not limited to 5-methylcytosine, 5-hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotide described herein contains a thymine base instead of uracil.

[0096] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, guanidine, lysine, 2-thiopyrimidine (e.g., 2-thiouracil, 2-thiothymidine), G-clamp and its derivatives, 5-substituted pyrimidines (e.g., 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazoguanine, 7-deazoadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazoguanine, 8-aza-7-deazoadenine, 8-aza-7-deazo-2,6-diaminopurine, Super G, Super A and N4-ethylcytosine, or their derivatives; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil, or their derivatives; and degenerate or universal bases, such as 2,6-difluorotoluene, or bases that are absent, such as debasing sites (e.g., 1-deoxyribose, 1,2-dideoxyribose, l-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the epoxide has been replaced by nitrogen (azaribose)). Pseudouracil is a naturally occurring isomerized form of uracil that has a C-glycoside instead of the conventional N-glycoside found in uridine.

[0097] It has been reported that certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of antisense oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. In various embodiments, the nucleobase may contain a 5-methylcytosine substitution, which has been shown to improve the stability of nucleic acid duplexes by 0.6–1.2 °C.

[0098] Modified or substituted nucleobases can also be used to facilitate the purification of antisense oligonucleotides. For example, antisense oligonucleotides may contain three or more (e.g., 3, 4, 5, 6, or more) consecutive guanine bases. In some antisense oligonucleotides, the sequence of three or more consecutive guanine bases can lead to oligonucleotide aggregation, complicating purification. In such antisense oligonucleotides, one or more guanine bases in the consecutive guanine bases can be substituted with hypoxanthine. The substitution of one or more guanine bases in the sequence of three or more consecutive guanine bases with hypoxanthine can reduce the aggregation of antisense oligonucleotides, thereby facilitating purification.

[0099] The oligonucleotides disclosed herein are synthetic and do not contain biologically derived antisense compositions. The molecules disclosed herein may also be mixed, encapsulated, conjugated, or otherwise associated with other molecules, molecular structures, or mixtures of compounds (e.g., liposomes, receptor-targeting molecules, oral, rectal, topical, or other formulations) to aid in uptake, distribution, or absorption, or combinations thereof.

[0100] The terms "complementary" and "complementarity" refer to oligonucleotides (i.e., nucleotide sequences) that are related by base pairing rules. For example, the sequence "TGA (5-3')" is complementary to the sequence "TCA (5'-3')". Complementarity can be "partial," where only some bases of the nucleic acids match according to the base pairing rules. Alternatively, there can be "complete," "full," or "perfect" (100%) complementarity between nucleic acids. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains. While perfect complementarity is generally required, some embodiments may include one or more, but preferably 6, 5, 4, 3, 2, or 1 mismatches relative to the target RNA. This hybridization can occur with "near" or "substantially" complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity. In some embodiments, the oligomer may hybridize to the target sequence with approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% complementarity. Variations at any position within the oligomer are also included. In some implementations, sequence variations near the ends of the oligomer are generally preferred over internal variations, and if present, are typically within about 6, 5, 4, 3, 2, or 1 nucleotides at the 5' end, 3' end, or both ends.

[0101] The term “peptide” refers to a compound containing multiple linked amino acids, which can be used to link a desired portion (e.g., an oligonucleotide) to an antibody or antibody fragment, typically an antibody or antibody fragment that specifically binds to one or more immune cell types (e.g., immune cells involved in autoimmune or inflammatory disease conditions).

[0102] In this invention, the term "internalized antibody or antibody fragment" generally refers to an antibody that, when it binds to its target antigen, internalizes an immune cell and thereby delivers one or more payloads (e.g., oligonucleotides (RNA or DNA composed of wild-type or modified nucleotides)) linked to it to the target immune cell. In an exemplary embodiment, the internalized antibody or antibody fragment is an antibody that binds to VISTA, preferably human VISTA.

[0103] The terms "cell-penetrating peptide" and "CPP" are used interchangeably and refer to cationic cell-penetrating peptides, also known as transport peptides, carrier peptides, or peptide transduction domains. These peptides have the ability to induce or enhance cell penetration in a given cell culture population.

[0104] The term "treatment" refers to the application of one or more specific procedures for improving a disease. In some embodiments, the specific procedure is the administration of one or more pharmaceutical agents. "Treatment" of an individual (e.g., a mammal, such as a human) or cell is any type of intervention intended to alter the natural processes of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be administered preventively or after the onset of a pathological event or exposure to a pathogen. Treatment includes any desired effect on the symptoms or pathology of a disease or condition and may include minimal change or improvement, such as in one or more measurable markers of the disease or condition being treated. "Preventative" treatment is also included, which may be aimed at reducing the rate of progression of the treated disease or condition, delaying the onset of the disease or condition, or reducing the severity of its onset. An "effective amount" or "therapeutic effective amount" refers to the amount of a therapeutic compound, such as an antisense oligomer, administered as a single dose or as part of a series of doses to a mammalian subject, which effectively produces the desired therapeutic effect.

[0105] The term "improvement" refers to a reduction in the severity of at least one indicator of a symptom or disease. In some embodiments, improvement includes a delay or slowing of the progression of one or more indicators of a symptom or disease. The severity of the indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0106] As used herein, “pharmaceutically acceptable salt” refers to a disclosed oligonucleotide derivative wherein the parent oligonucleotide is modified by converting an existing acid or base moiety into its salt form. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th edition, 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.

[0107] Oligonucleotides contained in the ARC or ANC of this invention In this document, oligonucleotides generally refer to polynucleotide molecules that, when delivered to target immune cells, regulate the expression and / or activity of proteins used to regulate immunity, specifically genes expressed by the immune cells. In some embodiments, the polynucleotide molecules described herein regulate the expression of an immunomodulatory gene, which may be wild-type or may contain one or more mutations, such as mutations associated with a disease condition related to gene expression. In some cases, the polynucleotide molecule hybridizes with a target region of wild-type DNA or RNA encoding an immunomodulatory gene or a fragment thereof. In some cases, the polynucleotide molecule is a polynucleotide molecule that hybridizes with a target region of DNA or RNA encoded by it that contains mutations (e.g., substitutions, deletions, or additions).

[0108] In some implementations, immune regulatory genes and RNA are selected from... Figure 1 or Figure 2 Or those identified in Appendix 2 and Appendix 4.

[0109] In some implementations, the polynucleotide molecule hybridizes with a target region of DNA or RNA encoding a targeted immunomodulatory protein containing one or more mutations.

[0110] In some implementations, the polynucleic acid molecule contains... Figure 1 or Figure 2 The target gene sequences listed herein have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some embodiments, the polynucleotide molecules described herein comprise RNA or DNA. In some cases, the polynucleotide molecules comprise RNA.

[0111] In some cases, RNA includes short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterologous nuclear 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, polynucleotide molecules contain siRNA.

[0112] In some implementations, the length of the polynucleotide molecule is about 10 to about 50 nucleotides. In some cases, the length of the polynucleotide 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.

[0113] In some implementations, the polynucleotide molecule is approximately 50 nucleotides long. In some cases, the polynucleotide molecule is approximately 45 nucleotides long. In some cases, the polynucleotide molecule is approximately 40 nucleotides long. In some cases, the polynucleotide molecule is approximately 35 nucleotides long. In some cases, the polynucleotide molecule is approximately 30 nucleotides long. In some cases, the polynucleotide molecule is approximately 25 nucleotides long. In some cases, the polynucleotide molecule is approximately 20 nucleotides long. In some cases, the polynucleotide molecule is approximately 19 nucleotides long. In some cases, the polynucleotide molecule is approximately 18 nucleotides long. In some cases, the polynucleotide molecule is approximately 17 nucleotides long. In some cases, the polynucleotide molecule is approximately 16 nucleotides long. In some cases, the polynucleotide molecule is approximately 15 nucleotides long. In some cases, the polynucleotide molecule is approximately 14 nucleotides long. In some cases, the polynucleotide molecule is approximately 13 nucleotides long. In some cases, the polynucleotide molecule is approximately 12 nucleotides long. In some cases, the polynucleotide molecule is approximately 11 nucleotides long. In some cases, the length of a polynucleotide molecule is about 10 nucleotides. In some cases, the length is about 10 to 50 nucleotides. In some cases, the length is about 10 to 45 nucleotides. In some cases, the length is about 10 to 40 nucleotides. In some cases, the length is about 10 to 35 nucleotides. In some cases, the length is about 10 to 30 nucleotides. In some cases, the length is about 10 to 25 nucleotides. In some cases, the length is about 10 to 20 nucleotides. In some cases, the length is about 15 to 25 nucleotides. In some cases, the length is about 15 to 30 nucleotides. In some cases, the length is about 12 to 30 nucleotides.

[0114] In some implementations, the polynucleotide molecule contains a first polynucleotide. In some cases, the polynucleotide molecule contains a second polynucleotide. In some cases, the polynucleotide molecule contains both a first polynucleotide and a second polynucleotide. In some cases, the first polynucleotide is a sense strand or a transit strand. In some cases, the second polynucleotide is an antisense strand or a guide strand.

[0115] In some embodiments, the polynucleotide molecule is the first polynucleotide. In some embodiments, the length of the first polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of 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.

[0116] In some cases, the first polynucleotide is about 50 nucleotides long. In some cases, the first polynucleotide is about 45 nucleotides long. In some cases, the first polynucleotide is about 40 nucleotides long. In some cases, the first polynucleotide is about 35 nucleotides long. In some cases, the first polynucleotide is about 30 nucleotides long. In some cases, the first polynucleotide is about 25 nucleotides long. In some cases, the first polynucleotide is about 20 nucleotides long. In some cases, the first polynucleotide is about 19 nucleotides long. In some cases, the first polynucleotide is about 18 nucleotides long. In some cases, the first polynucleotide is about 17 nucleotides long. In some cases, the first polynucleotide is about 16 nucleotides long. In some cases, the first polynucleotide is about 15 nucleotides long. In some cases, the first polynucleotide is about 14 nucleotides long. In some cases, the first polynucleotide is about 13 nucleotides long. In some cases, the first polynucleotide is about 12 nucleotides long. In some cases, the first polynucleotide is about 11 nucleotides long. In some cases, the length of the first polynucleotide is about 10 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 45 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 40 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 35 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 30 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 25 nucleotides. In some cases, the length of the first polynucleotide is about 10 to about 20 nucleotides. In some cases, the length of the first polynucleotide is about 15 to about 25 nucleotides. In some cases, the length of the first polynucleotide is about 15 to about 30 nucleotides. In some cases, the length of the first polynucleotide is about 12 to about 30 nucleotides.

[0117] In some embodiments, the polynucleotide molecule is a second polynucleotide. In some embodiments, the length of the second polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of 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.

[0118] 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 cases, the second polynucleotide is approximately 12 nucleotides long. In some cases, the second polynucleotide is approximately 11 nucleotides long. In some cases, the length of the second polynucleotide is about 10 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 50 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 45 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 40 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 35 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 30 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 25 nucleotides. In some cases, the length of the second polynucleotide is about 10 to about 20 nucleotides. In some cases, the length of the second polynucleotide is about 15 to about 25 nucleotides. In some cases, the length of the second polynucleotide is about 15 to about 30 nucleotides. In some cases, the length of the second polynucleotide is about 12 to about 30 nucleotides.

[0119] In some implementations, the polynucleotide molecule comprises a first polynucleotide and a second polynucleotide. In some cases, the polynucleotide molecule also comprises blunt ends, overhanging ends, or combinations thereof. In some cases, the blunt end is a 5' blunt end, a 3' blunt end, or both. In some cases, the overhanging end is a 5' overhang, a 3' overhang, or both. In some cases, the overhanging end comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base-paired nucleotides. In some cases, the overhanging end comprises 1, 2, 3, 4, 5, or 6 non-base-paired nucleotides. In some cases, the overhanging end comprises 1, 2, 3, or 4 non-base-paired nucleotides. In some cases, the overhanging end comprises 1 non-base-paired nucleotide. In some cases, the overhanging end comprises 2 non-base-paired nucleotides. In some cases, the overhanging end comprises 3 non-base-paired nucleotides. In some cases, the overhanging end comprises 4 non-base-paired nucleotides.

[0120] 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 cases, the sequence of the polynucleotide molecule is 100% complementary to the target sequence.

[0121] In some embodiments, the sequence of the polynucleotide molecule has five or fewer mismatches with the target sequence. In some embodiments, the sequence of the polynucleotide molecule has four or fewer mismatches with the target sequence. In some cases, the sequence of the polynucleotide molecule has three or fewer mismatches with the target sequence. In some cases, the sequence of the polynucleotide molecule has two or fewer mismatches with the target sequence. In some cases, the sequence of the polynucleotide molecule has one or fewer mismatches with the target sequence.

[0122] In some implementations, the specificity of the polynucleotide molecule hybridizing with the target sequence described herein is 95%, 98%, 99%, 99.5%, or 100% sequence complementarity between the polynucleotide molecule and the target sequence. In some cases, the hybridization is under highly stringent hybridization conditions.

[0123] In some implementations, polynucleotide molecules exhibit reduced off-target effects. In some cases, "off-target" or "off-target effect" refers to any situation where a polynucleotide polymer targeting a given target causes an unintended effect through direct or indirect interaction with another mRNA sequence, DNA sequence, or cellular protein or other part. In some cases, an "off-target effect" occurs when other transcripts are simultaneously degraded due to partial homology or complementarity between the sense and / or antisense strands of the polynucleotide molecule.

[0124] In some implementations, the polynucleotide molecule comprises a natural, synthetic, or artificial nucleotide analog or base. In some cases, the polynucleotide molecule comprises a combination of DNA, RNA, and / or nucleotide analogs. In some cases, the synthetic or artificial nucleotide analog or base comprises a modification at one or more of the ribose moiety, phosphate moiety, nucleoside moiety, or combinations thereof.

[0125] In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid having a modified 2' hydroxyl group at the ribose moiety. In some cases, the modification includes H, OR, R, a halogen, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary amines), amides, ethers, esters, alcohols, and oxygen. In some cases, the alkyl moiety also contains modifications. In some cases, modifications include azo groups, ketone groups, aldehyde groups, carboxyl groups, nitro groups, nitroso groups, nitrile groups, heterocyclic groups (e.g., imidazole, hydrazine, or hydroxyamino), isocyanate or cyanate groups, or sulfur-containing groups (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some cases, the alkyl moiety also contains heterosubstitution. In some cases, the carbon atom of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some cases, heterocyclic substitution includes, but is not limited to, morpholino, imidazole, and pyrrolidinyl.

[0126] 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'-O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety.

[0127] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification, which includes an extended amine group at the propyl linker that binds the amine group to the 2' oxygen. In some cases, this modification neutralizes the overall negative charge derived from the phosphate of the oligonucleotide molecule by introducing a positive charge from the amine group of each sugar, thereby improving cellular uptake properties due to its zwitterionic nature.

[0128] In some cases, the modification at the 2' hydroxyl group is a locked or bridged ribonucleotide modification (e.g., locked nucleic acid or LNA), in which an oxygen molecule bound at the 2' carbon is linked to the 4' carbon via a methylene group, thereby forming a 2'-C,4'-C-oxy-methylene-linked bicyclic ribonucleotide monomer. Exemplary representations of the chemical structure of LNA are known in the art.

[0129] In some cases, modifications at the 2' hydroxyl group include ethylene nucleic acids (ENAs), such as 2'-4'-ethylene bridging nucleic acids, which lock the sugar conformation to the C3'-inner sugar fold conformation. ENAs are part of a class of bridging nucleic acids that also contain LNAs.

[0130] In some embodiments, additional modifications at the 2' hydroxyl group include 2'-deoxy, T-deoxy-2'-fluorine, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA).

[0131] In some embodiments, the nucleotide analog comprises modified bases such as, but not limited to, 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 modified at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenine, 2-methyladenine, 3-methylcytidine, 6-methyluridine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 5-methylaminoethyluridine, 5-methyloxyuridine, denitronucleotides (such as 7-denitro-adenine, 6-methylaminoethyluridine, 5-methyloxyuridine), and denitronucleotides (such as 7-denitro-adenine, 6-methylaminoethyluridine, 5-methyloxyuridine, 5 ... -Azauridine, 6-azacytidine or 6-azathymidine), 5-methyl-2-thiouridine, other thiobases (such as 2-thiouridine and 4-thiouridine and 2-thiocytidine), dihydrouridine, pseudouridine, braided glycoside, archapurin, naphthyl and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one or pyridin-2-one), phenyl and modified phenyl such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosine acting as G-clamp nucleotides, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides modified relative to the sugar moiety, as well as nucleotides having a non-ribosyl sugar or analogue. For example, in some cases, the sugar moiety is or is based on mannose, arabinose, pyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbon rings. The term nucleotide also includes universal bases known in the art. For example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or cinnamic nucleotides.

[0132] In some embodiments, nucleotide analogs also comprise morpholino, peptide nucleic acid (PNA), methylphosphonate nucleotide, thiophosphonate nucleotide, 2'-fluoroN3-P5'-phosphamide, 1',5'-dehydrated hexadiol nucleic acid (HNA), or combinations thereof. Morpholino or phosphoryldiamine morpholino oligonucleotides (PMOs) comprise synthetic molecules whose structure mimics the structure of native nucleic acids but deviates from the normal sugar and phosphate structures. In some cases, the 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 phosphoryldiamine ester group instead of a phosphate group. In these cases, the backbone alteration removes all positive and negative charges, allowing the neutral morpholino molecule to cross the cell membrane without the aid of cell delivery agents (such as those used in charged oligonucleotides).

[0133] In some implementations, the peptide nucleic acid (PNA) does not contain sugar rings or phosphate bonds, and the bases are linked by oligoglycine-like molecules and appropriately spaced, thus eliminating the main chain charge.

[0134] In some embodiments, one or more modifications optionally occur at the internucleotide bond. In some cases, the modified internucleotide bond includes, but is not limited to, thiophosphates; dithiophosphates; methylphosphonates; 5'-alkylphosphonates; 5'-methylphosphonates; 3'-alkylphosphonates; trifluoroborates; boron phosphates and selenophosphates with 3'-5' or 2'-5' bonds; triphosphates; thioalkylphosphonates; phosphonate hydrogen bonds; alkylphosphonates; alkylthiophosphonates; arylthiophosphonates; selenophosphates; diselenophosphates; hypophosphonates; aminophosphates; 3'-alkylaminophosphates; aminoalkylaminophosphates; thioaminophosphates; piperazine phosphates; aniline. Thiophosphates; aniline phosphates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylene hydrazine; methylene dimethyl hydrazine; methyl acetal; thiomethyl acetal; oximes; methylene imino; methylene methyl imino; thioamide esters; bonds having a riboacetyl group; aminoethyl glycine; silyl or siloxane bonds; saturated or unsaturated and / or substituted and / or heteroatom-containing alkyl or cycloalkyl bonds having or not having heteroatoms, for example, 1 to 10 carbons; bonds having a morpholino structure, amide or polyamide, wherein the base is directly or indirectly attached to a nitrogen atom in the main chain; and combinations thereof.

[0135] In some cases, the modification is a methyl or thiol modification, such as a methylphosphonate or a thiophosphonate modification. Exemplary thiophosphonate nucleotides and methylphosphonate nucleotides are known in the art and include 2'-fluoroN3-P5'-phosphamide. In some cases, the modified nucleotides include, but are not limited to, hexitol nucleic acids (or 1',5'-dehydrated hexitol nucleic acids (HNA)).

[0136] In some embodiments, one or more modifications optionally include modifications to the ribose moiety, the phosphate backbone, and the nucleoside, or modifications to the 3' or 5' end of a nucleotide analog. For example, the 3' end optionally includes a 3' cationic group, or by reversing the 3'-terminal nucleoside with a 3'-3' bond. In another alternative, the 3' end is optionally conjugated to an aminoalkyl group, such as 3' C5-aminoalkyl dT. In yet another alternative, the 3' end is optionally conjugated to a debasement site, such as a depurinyl or depyrimidine site. In some cases, the 5' end is conjugated to an aminoalkyl group, such as a 5'-O-alkylamino substituent. In some cases, the 5' end is conjugated to a debasement site, such as a depurinyl or depyrimidine site.

[0137] In some embodiments, the polynucleotide molecule comprises one or more of the artificial nucleotide analogs described herein. In some cases, the polynucleotide 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, the artificial nucleotide analogues 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoroN3-P5'-phosphamide, or combinations thereof. In some cases, polynucleotide molecules contain 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, said artificial nucleotide analogs being selected from 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP) Modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA), LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoroN3-P5'-phosphinate, or combinations thereof. In some cases, polynucleotide molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more nucleotides modified with 2'-O-methyl. In some cases, polynucleotide molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more nucleotides modified with 2'-O-methoxyethyl (2'-O-MOE). In other cases, polynucleotide molecules contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25 or more thiophosphonate nucleotides.

[0138] In some cases, polynucleotide molecules contain at least one of the following: about 5% to about 100% modification, about 10% to about 100% modification, about 20% to about 100% modification, about 30% to about 100% modification, about 40% to about 100% modification, about 50% to about 100% modification, about 60% to about 100% modification, about 70% to about 100% modification, about 80% to about 100% modification, and about 90% to about 100% modification.

[0139] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 90% modification, about 20% to about 90% modification, about 30% to about 90% modification, about 40% to about 90% modification, about 50% to about 90% modification, about 60% to about 90% modification, about 70% to about 90% modification, and about 80% to about 100% modification.

[0140] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 80% modification, about 20% to about 80% modification, about 30% to about 80% modification, about 40% to about 80% modification, about 50% to about 80% modification, about 60% to about 80% modification, and about 70% to about 80% modification.

[0141] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 70% modification, about 20% to about 70% modification, about 30% to about 70% modification, about 40% to about 70% modification, about 50% to about 70% modification, and about 60% to about 70% modification.

[0142] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 60% modification, about 20% to about 60% modification, about 30% to about 60% modification, about 40% to about 60% modification, and about 50% to about 60% modification.

[0143] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 50% modification, about 20% to about 50% modification, about 30% to about 50% modification, and about 40% to about 50% modification.

[0144] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 40% modification, about 20% to about 40% modification, and about 30% to about 40% modification.

[0145] In some cases, polynucleotide molecules contain at least one of the following: about 10% to about 30% modification and about 20% to about 30% modification.

[0146] In some cases, polynucleotide molecules contain about 10% to about 20% of modifications. In other cases, polynucleotide molecules contain about 15% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% of modifications.

[0147] In other cases, polynucleotide molecules contain at least about 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of modifications.

[0148] In some implementations, the polynucleotide 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.

[0149] In some cases, polynucleotide molecules contain 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 modified nucleotides.

[0150] In some cases, approximately 5% to approximately 100% of the polynucleotide molecules contain the artificial nucleotide analogs described herein. In some cases, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the polynucleotide molecules contain artificial nucleotide analogs. In some cases, approximately 5% of the polynucleotide molecules contain artificial nucleotide analogs. In some cases, approximately 10% of the polynucleotide molecules contain artificial nucleotide analogs.

[0151] In some embodiments, the polynucleotide molecule is assembled from two separate polynucleotides, one polynucleotide containing the sense strand of the polynucleotide molecule and the second polynucleotide containing the antisense strand of the polynucleotide molecule. In other embodiments, the sense strand is linked to the antisense strand via a linker molecule, which in some cases is a polynucleotide linker or a non-nucleotide linker.

[0152] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide in the sense strand comprises a 2'-O-methylpyrimidine nucleotide and the purine nucleotide in the sense strand comprises a 2'-deoxypurine nucleotide. In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide present in the sense strand comprises a 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide present in the sense strand comprises a 2'-deoxypurine nucleotide.

[0153] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the pyrimidine nucleotide, when present in the antisense strand, is 2'-deoxy-2'-fluoropyrimidine nucleotide, and the purine nucleotide, when present in the antisense strand, is 2'-O-methylpurine nucleotide.

[0154] In some embodiments, the polynucleotide 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 wherein the purine nucleotide, when present in the antisense strand, comprises a 2'-deoxy-purine nucleotide.

[0155] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand includes a terminal cap portion at the 5' end, 3' end, or both the 5' and 3' ends. In other embodiments, the terminal cap portion is a reverse deoxygenated debasement portion.

[0156] In some implementations, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand contains a phosphate backbone modification at its 3' end. In some cases, the phosphate backbone modification is a thiophosphate ester.

[0157] In some implementations, the polynucleotide molecule contains a sense strand and an antisense strand, wherein the antisense strand contains a glycerol group modification at the 3' end of the antisense strand.

[0158] In some implementations, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises 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) phosphate-thioester nucleotide 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'-fluorine, and / or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universally modified nucleotides, and optionally a terminal cap molecule at the 3' end, 5' end, or both of the 3' end and 5' end of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphate thioester nucleotide 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'-fluorine, 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 a terminal cap molecule at the 3' end, 5' end, or both of the 3' end and 5' end of the antisense strand. In other embodiments, one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluorine nucleotides, with or without one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) phosphate thioester nucleotide internucleotide bonds present in the same or different strands and / or a terminal cap molecule at the 3' end, 5' end, or both of the 3' end and 5' end.

[0159] In some implementations, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises 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) phosphate-thioester nucleotide 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'-fluorine, 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 a terminal cap molecule at the 3' end, 5' end, or both of the 3' end and 5' end of the sense strand; and wherein the antisense strand comprises 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) phosphate thioester nucleotide 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'-fluorine, 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 a terminal cap molecule at the 3' end, 5' end or 3' end and 5' end of the antisense strand. In other embodiments, one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) pyrimidine nucleotides of sense and / or antisense strands are chemically modified with or without 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) phosphate thioester nucleotide inter-bonds and / or terminal cap molecules at the 3' end, 5' end or both the 3' end and 5' end in the same or different strands.

[0160] In some implementations, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises 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) phosphate-thioester nucleotide 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'-fluorine, 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 terminal cap molecules at the 3' end, 5' end, or both of the 3' end and 5' end of the sense strand; and wherein the antisense strand comprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate thioester nucleotide 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'-fluorine, 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 a terminal cap molecule at the 3' end, 5' end, or both of the 3' end and 5' end of the antisense strand. In other embodiments, 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) pyrimidine nucleotides of sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl and / or 2'-deoxy-2'-fluoronucleotides, with or without one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) phosphate thioester nucleotide inter-links and / or terminal cap molecules at the 3' end, 5' end or both the 3' end and 5' end, in the case of having or not having terminal cap molecules present in the same or different strands.

[0161] In some embodiments, the polynucleotide molecule comprises a sense strand and an antisense strand, wherein the antisense strand comprises 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) phosphate-thioester nucleotide 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'-fluorine, 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 a terminal cap molecule at the 3' end, 5' end, or both of the 3' end and 5' end of the sense strand; and wherein the antisense strand comprises 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) phosphate thioester nucleotide 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'-fluorine, 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, terminal cap molecules at the 3' end, 5' end, or both the 3' and 5' ends of the antisense strand. In other embodiments, one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) pyrimidine nucleotides of the sense and / or antisense strands are chemically modified with 2'-deoxy, 2'-O-methyl, and / or 2'-deoxy-2'-fluorine nucleotides, with or without terminal cap molecules at the 3' end, 5' end, or both the 3' and 5' ends, present in the same or different strands.

[0162] In some embodiments, the polynucleotide molecules described herein are chemically modified short interfering nucleic acid molecules 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) phosphate thioester nucleotide bonds in each chain of the polynucleotide molecule.

[0163] In another embodiment, the polynucleotide molecule described herein comprises 2'-5' nucleotide interbonds. In some cases, the 2'-5' nucleotide interbonds are located at the 3' end, 5' end, or both the 3' and 5' ends of one or both strands of the sequence. In other cases, the 2'-5' nucleotide interbonds are present at various other locations within one or both strands of the sequence; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (inclusive) nucleotide interbonds of pyrimidine nucleotides in one or both strands of the polynucleotide molecule comprise 2'-5' nucleotide interbonds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (inclusive) nucleotide interbonds of purine nucleotides in one or both strands of the polynucleotide molecule comprise 2'-5' nucleotide interbonds.

[0164] In some embodiments, the polynucleotide molecule is a single-stranded polynucleotide molecule that mediates RNAi activity in cells or reconstructed in vitro systems, wherein the polynucleotide molecule comprises a single-stranded polynucleotide complementary to a target nucleic acid sequence, and wherein one or more pyrimidine nucleotides present in the polynucleotide are 2'-deoxy-2'-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides, or alternatively, multiple pyrimidine nucleotides are 2'-deoxy-2'-fluoropyrimidine nucleotides), and wherein one or more purine nucleotides present in the polynucleotide are 2'-deoxypurine nucleotides (e.g., whose...). All purine nucleotides in the polynucleotide are 2'-deoxypurine nucleotides, or alternatively, multiple purine nucleotides are 2'-deoxypurine nucleotides; and a terminal cap modification, optionally present at the 3' end, 5' end, or both 3' and 5' end of the antisense sequence, wherein the polynucleotide molecule optionally also includes about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal 2'-deoxynucleotides at the 3' end of the polynucleotide molecule, wherein the terminal nucleotides further include one or more (e.g., 1, 2, 3, or 4) phosphate thioester nucleotide interbonds, and wherein the polynucleotide molecule optionally also includes a terminal phosphate group, such as a 5' terminal phosphate group.

[0165] In some cases, one or more artificial nucleotide analogs exhibit resistance to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase, or exonucleases such as 5'-3' and 3'-5' exonucleases compared to natural polynucleotide molecules. In some cases, they contain 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), T0-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido. Artificial nucleotide analogs of (2'-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, 2'-fluoroN3-P5'-phosphamide, or combinations thereof, are resistant to nucleases such as ribonucleases like RNase H, deoxyribonucleases like DNase (e.g., deoxyribonuclease), deoxyribonucleases like DNase, or exonucleases like 5'-3' and 3'-5' exonucleases. In some cases, 2'-O-methyl modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules modified with 2'-O-methoxyethyl (2'-O-MOE) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules modified with 2'-O-aminopropyl are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules modified with 2'-deoxy are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules modified with T-deoxy-2'-fluorine are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules modified with 2'-O-aminopropyl (2'-O-AP) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In other cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyl (2-O-DMAOE) are also nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant).In some cases, polynucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules modified with TO-dimethylaminoethoxyethyl (2'-O-DMAEOE) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules modified with 2'-ON-methylacetamido (2'-O-NMA) are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, LNA-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, ENA-modified polynucleotide molecules are nuclease-resistant (e.g., resistant to RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, HNA-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, morpholinonucleotides are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, PNA-modified polynucleotide molecules are resistant to nucleases (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, methylphosphonic acid nucleotide-modified polynucleotide molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease). In some cases, polynucleotide molecules modified with thiophosphonic acid are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramide are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease resistant). In some cases, the 5' conjugates described herein inhibit cleavage by 5'-3' exonucleases. In some cases, the 3' conjugates described herein inhibit cleavage by 3'-5' exonucleases.

[0166] In some implementations, one or more artificial nucleotide analogs exhibit increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. One or more artificial nucleotide analogs comprising 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modification, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiophosphonate nucleotides, or 2'-fluoroN3-P5'-phosphamide exhibit increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. In some cases, 2'-O-methyl-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-O-methoxyethyl (2'-O-MOE)-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-O-aminopropyl-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-deoxy-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, T-deoxy-2'-fluoro-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, 2'-O-aminopropyl (2'-O-AP)-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminoethyl (2'-O-DMAOE) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-O-dimethylaminopropyl (2'-O-DMAP) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, polynucleotide molecules modified with TO-dimethylaminoethoxyethyl (2'-O-DMAEOE) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules. In some cases, polynucleotide molecules modified with 2'-ON-methylacetamido (2'-O-NMA) exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotide molecules.In some cases, LNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, ENA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, PNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, HNA-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, morpholino-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, methylphosphonate nucleotide-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, thiophosphonate nucleotide-modified polynucleotides exhibit increased binding affinity to their mRNA targets compared to equivalent natural polynucleotides. In some cases, polynucleotide molecules containing 2'-fluoroN3-P5'-phosphoramide exhibit increased binding affinity to their mRNA targets relative to equivalent natural polynucleotide molecules. In some cases, this increased affinity is described by lower Kd, higher melting temperature (Tm), or a combination thereof.

[0167] In some embodiments, the polynucleotide molecule is a chiral (or stereopure) polynucleotide molecule, or a polynucleotide molecule containing a single enantiomer. In some cases, the polynucleotide molecule contains an L-nucleotide. In some cases, the polynucleotide molecule contains a D-nucleotide. In some cases, the polynucleotide molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror-image enantiomers. In some cases, the polynucleotide molecule composition contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture.

[0168] In some embodiments, the polynucleotide molecule may be further modified to include an aptamer conjugate. In some cases, the aptamer conjugate is a DNA aptamer conjugate. In some cases, the aptamer conjugate is an alphamer (Centauri Therapeutics) that includes an aptamer portion that recognizes a specific cell surface target and a portion that presents a specific epitope for attachment to a circulating antibody. In some cases, the polynucleotide molecule described herein is further modified to include an aptamer conjugate, as described in U.S. Patent Nos. 8,604,184, 8,591,910, and 7,850,975.

[0169] In some embodiments, the polynucleotide molecules described herein are modified to increase their stability. In some embodiments, the polynucleotide molecule is RNA (e.g., siRNA), and the polynucleotide molecule is modified to increase its stability. In some cases, the polynucleotide molecule is modified to increase its stability by one or more of the modifications described above. In some cases, the polynucleotide molecule is modified at the 2' hydroxyl position, such as by 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modification, or by a locked or bridged ribose conformation (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, thiophosphonate nucleotides, and / or 2'-fluoroN3-P5'-phosphamide to increase their stability. In some cases, polynucleotide molecules are chiral (or stereopure) polynucleotide molecules. In some cases, chiral (or stereopure) polynucleotide molecules are modified to increase their stability. Appropriate modifications to RNA to increase delivery stability will be readily apparent to those skilled in the art.

[0170] In some embodiments, the polynucleotide molecule in the ARC or ANC according to the invention has RNAi activity that regulates the expression of RNA encoded by a target immunomodulatory gene. In some cases, the polynucleotide molecule described herein is a double-stranded siRNA molecule that downregulates 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 wherein 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 polynucleotide molecule described herein is a double-stranded siRNA molecule that downregulates 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 wherein each strand contains at least about 14, 17, or 19 nucleotides complementary to the nucleotides of the other strand. In some cases, the polynucleotide molecules described herein are double-stranded siRNA molecules that downregulate the expression of immunomodulatory genes, wherein each strand of the siRNA molecule contains about 19 to about 23 nucleotides, and wherein each strand contains at least about 19 nucleotides complementary to the nucleotides of the other strand. In some cases, RNAi activity occurs intracellularly. In other cases, RNAi activity occurs in reconstructed in vitro systems.

[0171] In some cases, the polynucleotide molecule in the ARC or ANC according to the invention is a double-stranded polynucleotide molecule comprising a self-complementary sense region and an antisense region, wherein the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. In some cases, the polynucleotide molecule is assembled from two separate polynucleotides, one strand being a sense strand and the other an antisense strand, wherein the antisense strand and the sense strand are self-complementary (e.g., each strand contains a nucleotide sequence complementary to a nucleotide sequence in the other strand; such as where the antisense strand and the sense strand form a double helix or double-stranded structure, for example, where the double-stranded region is about 19, 20, 21, 22, 23 or more base pairs); the antisense strand contains a nucleotide sequence complementary to a nucleotide sequence in the target nucleic acid molecule or a portion thereof, and the sense strand contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, multinucleic acid molecules are assembled from single oligonucleotides, wherein the complementary sense and antisense regions of the multinucleic acid molecule are linked by nucleic acid-based or non-nucleic acid-based linkers.

[0172] In some cases, the polynucleotide molecule in the ARC or ANC according to the invention is a polynucleotide having a double strand, an asymmetric double strand, a hairpin, or an asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region contains a nucleotide sequence complementary to a nucleotide sequence in a single target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a 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 containing self-complementary sense and antisense regions, wherein the antisense region contains a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof, and wherein the cyclic polynucleotide is processed in vivo or in vitro to generate an active polynucleotide molecule capable of mediating RNAi. In other cases, polynucleotide molecules also contain single-stranded polynucleotides having a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof (e.g., where the polynucleotide molecule does not require the presence of a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof within the polynucleotide molecule), wherein the single-stranded polynucleotide also contains a terminal phosphate group, such as 5'-phosphate (see, for example, Martinez et al., 2002). Cell , 110, 563-574 and Schwarz et al., 2002, Molecular Cell (, 10, 537-568) or 5',3'-bisphosphonic acid.

[0173] In some cases, asymmetric duplexes are linear polynucleotide molecules that contain an antisense region, a loop portion containing nucleotides or nonnucleotides, and a sense region containing fewer nucleotides than the antisense region, such that the sense region has enough complementary nucleotides to pair with the bases of the antisense region and form a duplex with the loop. For example, asymmetric hairpin polynucleotide molecules contain an antisense region (e.g., about 19 to 22 nucleotides) long enough to mediate RNAi in cellular or in vitro systems, a loop portion containing about 4 to 8 nucleotides, and a sense region containing about 3 to 18 nucleotides complementary to the antisense region. In some cases, asymmetric hairpin polynucleotide molecules also contain a chemically modified 5' phosphate group. In other cases, the loop portion of an asymmetric hairpin polynucleotide molecule contains nucleotides, nonnucleotides, linker molecules, or conjugate molecules.

[0174] In some implementations, an asymmetric duplex is a polynucleotide molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region contains fewer nucleotides than the antisense region, to the extent that the sense region has sufficient complementary nucleotides to pair with the bases of the antisense region and form a duplex. For example, an asymmetric duplex polynucleotide molecule comprises an antisense region (e.g., about 19 to about 22 nucleotides) of sufficient length to mediate RNAi in a cellular or in vitro system and a sense region of about 3 to about 18 nucleotides complementary to the antisense region.

[0175] In some cases, a universal base refers to a nucleotide base analog that forms a base pair with every natural DNA / RNA base, and there is little difference between them. Non-limiting examples of universal bases include C-phenyl, C-naphthyl and other aromatic derivatives, inosine, azolecarbamates, and nitrazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, for example, Loakes, 2001). Nucleic Acids Research , 29, 2437-2447).

[0176] Synthesis of polynucleic acid molecules used in the ARC or ANC of this invention Subject matter ARC or ANC comprises one or more polynucleotide molecules, typically RNA, which can be synthesized as disclosed herein or using other known methods. In some embodiments, the polynucleotide molecules described herein are constructed using chemical synthesis and / or enzymatic ligation reactions, employing procedures known in the art. For example, polynucleotide molecules are chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the double strand formed between the polynucleotide molecule and the target nucleic acid. Exemplary methods include those described in U.S. Patent Nos. 5,142,047, 5,185,444, 5,889,136, 6,008,400, and 6,111,086; PCT Publication No. WO2009099942; or European Publication No. 1579015. Further exemplary methods include Griffey et al., "2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides," J. Med. Chem39(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 Those described in 48B: 1721-1726 (2009). Alternatively, polynucleotide molecules are biologically generated using expression vectors into which polynucleotide molecules have been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted polynucleotide molecule will have an antisense orientation relative to the target polynucleotide molecule of interest).

[0177] In some embodiments, polynucleotide molecules are synthesized via a tandem synthesis approach, wherein two strands are synthesized as single, continuous oligonucleotide fragments or chains separated by a cleavable linker, which is then cleaved to provide hybridization and allow purification of the individual fragments or chains of the duplex.

[0178] In some cases, polynucleotide molecules are also assembled from two different nucleic acid chains or fragments, one of which contains the sense region of the molecule and the second fragment contains the antisense region of the molecule.

[0179] Other modification methods used for incorporation, such as sugar, base, and phosphate modification, include: Eckstein et al., International Publication PCT No. WO 92 / 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 No. WO 93 / 15187; Sproat, US Patent No. 5,334,711 and Beigelman et al., 1995, J. Biol. Chem ., 270, 25702; Beigelman et al., International PCT Publication No. WO 97 / 26270; Beigelman et al., U.S. Patent No. 5,716,824; Usman et al., U.S. Patent No. 5,627,053; Woolf et al., International PCT Publication No. WO 98 / 13526; Thompson et al., U.S. Serial No. 60 / 082,404, filed April 20, 1998; 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., 1997, Bioorg. Med. Chem ., 5, 1999–2010. These publications describe general methods and strategies for determining the locations of sugar, base, and / or phosphate modifications incorporated into nucleic acid molecules without modulating catalysis.

[0180] In some cases, while chemically modifying the internucleotide bonds of polynucleotide molecules with thiophosphates, dithiophosphates, and / or 5'-methylphosphonates improves stability, over-modification can sometimes lead to toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide bonds is minimized in some situations. In such cases, reducing the concentration of these bonds also reduces toxicity and increases the efficacy and specificity of the molecules.

[0181] Diseases that can be treated using the ARC or ANC of this invention In some embodiments, the ARC or ANC according to the present invention, or pharmaceutical compositions containing ARC or ANC as described herein, are used to treat diseases or conditions caused by specific immune cell types, typically autoimmune or inflammatory conditions, cancer, or related symptoms.

[0182] In some cases, ARC or ANC, or compositions containing ARC or ANC, are used to treat autoimmune diseases, such as those involving myeloid cells or T cells.

[0183] In some cases, ARC or ANC, or compositions containing ARC or ANC, are used to treat necrotic, proliferative, neurodegenerative, neuroinflammatory, infectious, autoimmune, or inflammatory diseases or their symptoms.

[0184] In some cases, ARC or ANC, or compositions containing ARC or ANC, are used to treat one or more of the following autoimmune diseases: acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglion disease (AAG) / autoimmune autonomic dysfunction | autoimmune 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), autoimmune polyglandular syndrome, types I, II, and III (APS type 1, APS type 2, APS type 3, APS type 4, APS type 5, APS type 6, APS type 7, APS type 8, APS type 9, APS type 1, APS type 1, APS type 1, APS type 2, APS type 3 ... Type 3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Barlow's disease, Behcet's disease, shotgun-like chorioretinopathy / shotgun-like uveitis, bullous pemphigoid, Kassman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Chag-Strauss syndrome / eosinophilic granulomatous polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome | localized cutaneous systemic sclerosis, Crohn's disease (CD), Canada-Kronka syndrome (CSS), cryptogenic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dresler syndrome / post-myocardial infarction syndrome / pericardiotomy Postoperative syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpassch syndrome / anti-GBM disease / anti-TBM disease, granulomatous polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, allergic purpura / IgA vasculitis, hidradenitis suppurativa, Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Berge's disease, immune-mediated necrotizing myopathy (IMNM).Immune thrombocytopenic purpura (ITP) / Autoimmune thrombocytopenic purpura / Autoimmune thrombocytopenic purpura, 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-Eton myasthenic syndrome (LEMS), leukocytic clotting 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 diseases Autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), keratitis erosiveus, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devrwick disease, ocular cicatricial pemphigoid, oculoclonus-myoclonus syndrome (OMS), relapsing rheumatism, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, Paroxysmal-Rossian syndrome (PRS) / hemispheric atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH) Peripheral uveitis / paris plana inflammation, PANS / PANDAS, Parsenaeus-Turner syndrome, pemphigus gestationis / herpes gestationis, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural 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 aplasia (PRCA), pyoderma gangrenosa, Rasmussen encephalitis, Raynaud's syndrome / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy Benign regional pain syndrome (RSD) / complex regional pain syndrome (CRPS), recurrent polychondritis, restless legs syndrome (RLS) / Willis-Ekbom disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, scleritis, scleroderma, sclerosing mesenteric / mesenteric panniculitis, creeping choroidal lesions, Sjögren's syndrome, stiff-person syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus erythematosus, Sussac syndrome, Siddenham's chorea, sympathetic ophthalmia, Takayasu arteritis (vasculitis), testicular autoimmune diseases (vasculitis, orchitis)Tolosa-Hunter syndrome, transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), pre- / middle / post-uveitis, vasculitis, VEXAS syndrome, vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH). In some cases, ARC or ANC is used to treat autoimmune diseases selected from a 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 thyroiditis, Sjögren's disease, asthma, type 2 diabetes, and autoimmune type 1 diabetes.

[0185] In some cases, ARC or ANC, or compositions containing ARC or ANC, 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, herpes zoster, and vasculitis.

[0186] In some cases, ANC or ARC, or compositions containing ANC or ARC, are used to treat neurodegenerative or neuroinflammatory diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, aphasia, Parkinson's disease, or spinal muscular atrophy.

[0187] In some cases, ARC or ANC, or compositions containing ARC or ANC, are used to treat cancers or their symptoms associated with specific immune cell types.

[0188] In some implementations, the ARC or ANC or pharmaceutical compositions comprising polynucleotide molecules described herein are used to treat cancer. In some cases, the cancer is a solid tumor. In some cases, the cancer is a hematologic malignancy. In some cases, the cancer is a relapsed or refractory cancer or a metastatic cancer. In some cases, the solid tumor is a relapsed or refractory solid tumor or a metastatic solid tumor. In some cases, the hematologic malignancy is a relapsed or refractory hematologic malignancy or a metastatic hematologic malignancy.

[0189] In some implementations, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, anal cancer, appendiceal cancer, biliary tract cancer (i.e., bile duct cancer), 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 adenoma, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0190] In some cases, the polynucleotide molecules or pharmaceutical compositions described herein are used to treat solid tumors. Specifically, they are used to treat anal cancer, appendiceal cancer, biliary tract cancer (i.e., bile duct cancer), 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 adenoma, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer. In some cases, the solid tumor is a recurrent or refractory solid tumor or a metastatic solid tumor.

[0191] In some cases, cancer is a blood malignancy. In others, blood malignancies include leukemia, lymphoma, myeloma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma. In still others, blood 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 macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, and nodal marginal zone B-cell lymphoma. Cellular lymphoma, Burkitt lymphoma, non-Burkkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis.

[0192] In some cases, the polynucleotide molecules or pharmaceutical compositions described herein are used to treat hematologic malignancies. In some cases, the polynucleotide molecules or pharmaceutical compositions described herein are used to treat leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some cases, hematologic 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 macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, and nodal marginal zone B-cell lymphoma. Hematologic malignancies include: Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis. In some cases, hematologic malignancies are relapsed or refractory hematologic malignancies or metastatic hematologic malignancies.

[0193] pharmaceutical preparations In some embodiments, the pharmaceutical formulation comprising the ARC or ANC according to the invention is administered to the subject via a variety of routes of administration, including but not limited to parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration. In some cases, the pharmaceutical compositions described herein are formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration. In other cases, the pharmaceutical compositions described herein are formulated for oral administration. In still other cases, the pharmaceutical compositions described herein are formulated for intranasal administration.

[0194] In some implementations, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersants, self-emulsifying dispersants, solid solutions, liposome dispersants, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations (e.g., nanoparticle formulations), and mixed immediate-release and controlled-release formulations.

[0195] In some cases, pharmaceutical formulations include multi-particulate formulations. In some cases, pharmaceutical formulations include nanoparticle formulations. In some cases, nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases, nanoparticles include solid lipid nanoparticles, polymer nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micelle solutions. Other exemplary nanoparticles include, but are not limited to, paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrites (such as metal chelates with covalent bonds), nanofibers, nanohorns, nanoonions, nanorods, nanowires, and quantum dots. In some cases, nanoparticles are metallic nanoparticles, such as those of 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.

[0196] In some cases, nanoparticles include a core or a core and a shell, such as in core-shell nanoparticles.

[0197] In some cases, the nanoparticles are further coated with molecules for attaching functional elements (e.g., using one or more polynucleotide molecules or binding moieties as described herein). In some cases, the coating comprises chondroitin sulfate, dextran sulfate, carboxymethyl dextran, alginate, pectin, carrageenan, fucoidan, agar pectin, alginic acid, arachidonic acid, 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 cases, the nanoparticles include graphene-coated nanoparticles.

[0198] In some cases, nanoparticles have at least one size smaller than about 500 nm, 400 nm, 300 nm, 200 nm or 100 nm.

[0199] In some cases, nanoparticle formulations comprise paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrites (such as metal chelates with covalent bonds), nanofibers, nanohorns, nanoonions, nanorods, nanotethers, or quantum dots. In some cases, the polynucleotide molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles. In some cases, at least 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleotide molecules or binding moieties described herein are directly or indirectly conjugated to the nanoparticles.

[0200] In some embodiments, the pharmaceutical formulation includes a delivery vector, such as a recombinant vector, for delivering polynucleotide molecules into cells. In some cases, the recombinant vector is a DNA plasmid. In other cases, the recombinant vector is a viral vector. Exemplary viral vectors include vectors derived from adeno-associated virus, retrovirus, adenovirus, or alphavirus. In some cases, the recombinant vector capable of expressing polynucleotide molecules provides stable expression in target cells. In other cases, a viral vector providing transient expression of polynucleotide molecules is used.

[0201] In some embodiments, the pharmaceutical formulation includes a carrier or carrier material selected based on 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, diluents, etc. Pharmaceutically compatible carrier materials include, but are not limited to, gum arabic, gelatin, colloidal silica, calcium glycerophosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurine, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sodium saccharide stearoyl lactylate, carrageenan, monoglycerides, diglycerides, pregelatinized starch, etc. See, for example, Remington: The Science and Practice of Pharmacy, 19th edition (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, 7th edition (Lippincott Williams & Wilkins 1999).

[0202] In some cases, pharmaceutical formulations also contain pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris(hydroxymethyl)aminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. These acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0203] In some cases, pharmaceutical preparations contain one or more salts in an amount required to achieve an acceptable osmolality of the composition. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0204] In some cases, pharmaceutical formulations also contain diluents to stabilize the compounds, as they provide a more stable environment. Salts dissolved in buffer solutions (which also provide pH control or maintenance) are used in the art as diluents, including but not limited to phosphate-buffered saline solutions. In some cases, diluents increase the volume of the composition to facilitate compression or to produce sufficient volume of a homogeneous blend for capsule filling. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, and microcrystalline cellulose such as Avicel. ® ; dicalcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars, such as Di-Pac ® (Amstar); Mannitol, Hydroxypropyl Methylcellulose, Hydroxypropyl Methylcellulose Acetate Stearate, Sucrose-based Diluent, Powdered Sugar; Calcium Sulfate Monohydrate, Calcium Sulfate Dihydrate; Calcium Lactate Trihydrate, Dextran; Hydrolyzed Cereal Solids, Amylose; Powdered Cellulose, Calcium Carbonate; Glycine, Kaolin; Mannitol, Sodium Chloride; Inositol, Bentonite, etc.

[0205] In some cases, pharmaceutical formulations contain disintegrants to facilitate the breakdown or disintegration of the substance. The term "disintegration" refers to the dissolution and dispersion of the dosage form upon contact with gastrointestinal fluids. Examples of disintegrants include starches, such as natural starches like corn starch or potato starch, and pregelatinized starches like National 1551 or Amijel. ® Or sodium glycolate starch such as Promogel ® Or Explotab ® Cellulose, such as wood products; methyl crystalline cellulose, such as Avicel ® Avicel ® PH101, Avicel ® PH102, Avicel ® PH105, Elcema ® P100, Emcocel ® Vivacel ® Ming Tia ® and Solka-Floc ® methylcellulose, croscarmellose cellulose, or croscarmellose such as sodium croscarmellose (Ac-Di-Sol) ® Cross-linked carboxymethyl cellulose or cross-linked carboxymethyl cellulose; cross-linked starch such as sodium glycolate starch; cross-linked polymers such as cross-povidone; cross-linked polyvinylpyrrolidone; alginates such as alginic acid or salts of alginic acid such as sodium alginate; clays such as Veegum ®HV (magnesium aluminum silicate); gels such as agar, guar gum, locust bean gum, ebony gum, pectin or tragacanth gum; sodium glycolate starch; bentonite, natural sponges; surfactants; resins such as cation exchange resins; citrus pomace; sodium lauryl sulfate; sodium lauryl sulfate in composite starches, etc.

[0206] In some cases, pharmaceutical preparations include fillers such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextran, dextran anhydride, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[0207] Lubricants and flow aids may also be optionally included in the pharmaceutical formulations described herein for preventing, reducing, or inhibiting adhesion or friction of materials. Exemplary lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearoyl fumarate, hydrocarbons such as mineral oil, or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex). ® ), higher fatty acids and their alkali metal and alkaline earth metal salts (such as aluminum, calcium, magnesium, zinc), stearic acid, sodium stearate, glycerol, talc, wax, Stearot ® Boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxy polyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil ® Starch, such as corn starch, silicone oil, surfactants, etc.

[0208] Plasticizers are compounds used to soften microencapsulated materials or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycol (such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800), stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. Plasticizers are also used as dispersants or wetting agents.

[0209] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl octanoate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropanol, cholesterol, bile salts, polyethylene glycol 200-600, tetraethylene glycol ether, diethylene glycol monoethyl ether, propylene glycol, dimethyl isosorbide, etc.

[0210] Stabilizers include compounds such as any antioxidants, buffers, acids, preservatives, etc.

[0211] The suspending agent comprises, for example, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30), vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol with a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, etc. Compounds of hydroxymethyl cellulose acetate stearate, polysorbate-80, hydroxyethyl cellulose, sodium alginate, gums (such as gum arabic and gum arabic, guar gum, xanthan gum, including xanthan gum), sugars, celluloses (such as sodium carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxyethyl cellulose), polysorbate-80, sodium alginate, polyethoxylated sorbitol monolaurate, polyethoxylated sorbitol monolaurate, povidone, etc.

[0212] Surfactants include sodium lauryl sulfate, sodium docusate, Tween 60 or 80, glyceryl triacetate, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, poloxamer, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide such as Pluronic. ® Compounds such as (BASF). Other 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 octylphenyl polyol 10 and octylphenyl polyol 40. Sometimes, surfactants are included to enhance physical stability or for other purposes.

[0213] Viscosity enhancers include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof.

[0214] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitol monooleate, sorbitol monolaurate, triethanolamine oleate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitol monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, glyceryl triacetate, Tween 80, vitamin ETPGS, ammonium salts, etc.

[0215] Treatment plan In some embodiments, a pharmaceutical composition comprising the ARC or ANC according to the invention is administered for therapeutic purposes. In some embodiments, the pharmaceutical composition is administered once daily, twice daily, three times daily, or more. The pharmaceutical composition may be administered daily, every other day, five days a week, once a week, once every other week, twice a month, three times a month, once a month, twice a month, three times a month, or more. The pharmaceutical composition may be administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or longer.

[0216] In some embodiments, one or more pharmaceutical compositions according to the ARC or ANC of 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 other cases, one or more pharmaceutical compositions are administered at intervals (e.g., the first administration of the first pharmaceutical composition is on day one, followed by administration at intervals of at least 1, 2, 3, 4, 5 days or more before administration of at least the second pharmaceutical composition).

[0217] In some embodiments, two or more different pharmaceutical compositions are administered co-administered. In some cases, two or more different pharmaceutical compositions are administered simultaneously. In some cases, two or more different pharmaceutical compositions are administered sequentially without a time interval between administrations. In other cases, two or more different pharmaceutical compositions are administered sequentially with an interval of about 0.5 hours, 1 hour, 2 hours, 3 hours, 12 hours, 1 day, 2 days, or longer between administrations.

[0218] If the patient's condition does improve, the composition may be continued as determined by the physician; alternatively, the dosage of the composition may be temporarily reduced or temporarily discontinued for a period of time (i.e., a "withdrawal period"). In some cases, the length of the withdrawal period varies from 2 days to 1 year, and by way of example only, includes 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. During the withdrawal period, the dose is reduced by 10%-100%, for example only, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0219] Once the patient’s condition improves, a maintenance dose may be administered if necessary. Subsequently, depending on the symptoms, the dose or frequency of administration, or both, may be reduced to the level required to maintain improvement in the disease, symptom, or condition.

[0220] In some embodiments, the amount of a given agent corresponding to this quantity varies depending on factors such as the specific compound, the severity of the disease, and the identity (e.g., weight) of the subject or host requiring treatment, but is still routinely determined in a manner known in the art based on the specific circumstances associated with the case, including, for example, the specific agent administered, the route of administration, and the subject or host being treated. In some cases, the desired dose is conveniently provided as a single dose or as a fractionated dose (e.g., as a sub-dose twice, three, four, or more times daily) administered simultaneously (or over a short period of time) or at appropriate intervals.

[0221] The aforementioned ranges are merely recommendations, as the number of variables relating to individual treatment regimens is vast, and significant deviations from these recommendations are not uncommon. Such dosages vary depending on many variables, including but not limited to the activity of the compound used, the disease or condition being treated, the method of administration, the individual subject's needs, the severity of the disease or condition being treated, and the physician's judgment.

[0222] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined using standard pharmaceutical procedures in cell cultures or laboratory animals, including but not limited to determining the LD50 (the dose that causes 50% of the population to die) and ED50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, expressed as the ratio between LD50 and ED50. Compounds exhibiting a high therapeutic index are preferred. A range of doses for human use is formulated using data obtained from cell culture assays and animal studies. The doses of such compounds are preferably within a range of cyclic concentrations that include the ED50 and have minimal toxicity. The dose varies within this range, depending on the dosage form and route of administration used.

[0223] reagent kits / products In some embodiments, these are kits and articles of manufacture used in conjunction with one or more compositions and methods described herein. Such kits include carriers, packages, or containers that are partitioned to accommodate one or more containers, such as vials, tubes, etc., each containing one of the individual elements to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from a variety of materials such as glass or plastic.

[0224] The products described herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, vials, and any packaging material suitable for the selected formulation and the intended method of administration and treatment. For example, containers may contain nucleic acid molecules described herein that specifically bind to immunomodulatory genes or RNA encoded by them. Such kits may optionally include identification descriptions or labels or instructions relating to their use in the methods described herein.

[0225] Kits typically include a label listing the contents and / or instructions for use, as well as a package insert with instructions for use. A separate instruction manual is also usually included.

[0226] In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other characters constituting the label are affixed, molded, or etched into the container itself; the label is associated with the container, such as as a packaging instruction, when the label is present within a holder or bracket that also supports the container. In one embodiment, the label is used to indicate that the contents will be used for a specific therapeutic application. The label also indicates instructions for use of the contents, such as those described herein.

[0227] In some embodiments, the pharmaceutical composition is contained in a packaging or dispensing device that includes one or more unit dosage forms comprising the compounds provided herein. The packaging may contain, for example, metal or plastic foil, such as blister packs. In one embodiment, the packaging or dispensing device is accompanied by instructions for use. In one embodiment, the packaging or dispenser also includes a container-related statement in the form prescribed by a government agency regulating the manufacture, use, or sale of the drug, reflecting that agency's approval of the form of the drug for human or veterinary use. For example, such a statement is a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product instruction.

[0228] Example The embodiments described below are for illustrative purposes and describe certain specific implementations of this disclosure. However, the embodiments described herein do not limit the scope of the claims in any way. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art, and such changes and modifications can be made without departing from the spirit of this disclosure and the scope of the appended claims, including but not limited to those related to the chemical structure, substituents, derivatives, formulations, or methods of this disclosure.

[0229] Example 1: Targeted RNA delivery to immune cells to treat autoimmune diseases In this embodiment, we provide a proof-of-concept for a direct antibody-RNA conjugate (ARC) platform that 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 mRNAs of key immune pathways, including PIK3CA and TNF, as well as other known and novel pathways involved in immune regulation, tolerance, and metabolism. siRNAs delivered via ARC that inhibit important immune targets (Glut1, PI3K, BTK, TNF, RORC) retain their functionality and inhibit activation of the corresponding immune pathways, making them attractive targets in the therapeutic space for autoimmune diseases. In contrast, ASO-mediated inhibition of CD39 (an exonuclease involved in the hydrolysis of ATP to produce exoadenosine) leads to T cell activation and increased cytokine production [PMID: 30871609]. ARC-delivered CD39 retains its activity and should be considered a promising approach for cancer therapy.

[0230] INX201 (a humanized anti-human VISTA antibody with a silent mutation of L234A / L235A / E269R / K322A in the Fc region on the human IgG1 / κ backbone) was used as the payload delivery medium. In this embodiment, we demonstrate that: 1) We can conjugate siRNA or ASO with anti-VISTA Mab and efficiently deliver the RNA to target cells via VISTA binding and internalization; 2) Compared to free INX201 Mab, ARC exhibits similar binding and internalization properties; 3) The publicly described siRNA payloads are known to be specific for immunologically relevant targets (https: / / www.idtdna.com / pages / products / functional-genomics / dsirnas-and-trifecta-rnai-kits), and their knockdown genes (repressing target mRNA levels to <30%) in a standard transfection setting retain their function when delivered to target cells with the Mab conjugate. 4) siRNA conjugated to anti-VISTA Mab INX201 promotes the accumulation and function of siRNA in K562-VISTA cells; 5) siRNA conjugated to anti-VISTA Mab INX201 is functional in human PBMCs and T cells, exhibiting reduced proliferation and cytokine production; and 6) CD39 ASO conjugated to anti-VISTA Mab INX201 [PMID: 30871609] is functional in human PBMCs, manifested as increased cytokine production.

[0231] The abbreviations used in this embodiment

[0232] Introduction The applicant has previously developed IgG1 anti-VISTA mAb INX201. This mab is a humanized anti-human VISTA antibody on the human IgG1 / κ backbone with a silent mutation in the Fc region of L234A / L235A / E269R / K322A, and is used as the payload delivery medium.

[0233] In this embodiment, we describe the experiments related to establishing conjugation and functional proof-of-concept studies using the INX201 delivery medium and publicly available siRNA or ASO payloads (see Tables 1 and 2). Exemplary Ab sequences are provided in Appendix 1; exemplary payload sequences used for conjugation are listed in Appendix 2.

[0234] Table 1. Anti-VISTA Mab and Fab for payload delivery.

[0235] Table 2. Exemplary Targets. In this embodiment, the payload of the targets shown in bold was verified to have ARC with defined functional activity.

[0236] Materials and methods K562-VISTA cell line assay: binding, internalization, transfection, qPCR Material • U-shaped bottom 96-hole plate (Falcon, #353077) • K562-VISTA (VISTA-expressing cells, prepared internally from WT K562, ATCC CCL-243) • IMDM medium (ATCC, 30-2005) + 10% FBS (ATCC, 30-2020) + P / S (Gibco, 15140-122) with G418 added at a 1:100 ratio (for selection of VISTA+ expression). • Lonza Nucleofector 2b • MACS Quant • Cell line nuclear transfection kit V (Lonza, VCA-1003) • DNA: pRP[Exp]-CAG>3xNLS / EGFP, vector ID: VB900137-6122yyc (VectorBuilder, MaxH VB900137-6122yyc), used to generate GFP+ cell pools; this vector contains the PuroR puromycin resistance gene. • Purinomycin • Anti-human IgG Fc secondary antibody (Biolegend, B172272) • Test product: Free INX201 or INX201 ARC, for example (INX201-eGFP_siRNA_Cy5) • All payloads were synthesized in IDT and contained a 3'-sense Cy5 tag. K562-VISTA suspension cells 1. At a level of 0.5-1×10 6 After passing cells at a density of 100 cells / mL, passage the cells. Maintain the culture at a concentration of 0.5–1 × 10⁶ cells / mL. 6 Between 1-2 × 10⁶ viable cells / mL. 5 Cells / mL. Passage culture 2 days before nuclear transfection. Optimal nuclear transfection density: 2-5 × 10⁻⁵ cells / mL. 5 .

[0237] 2. The basal medium for this cell line was Iscove-modified Dulbecco medium prepared by ATCC, catalog number 30-2005. To prepare the complete growth medium, the following components were added to the basal medium to achieve a final concentration of 10% FBS. 3. The culture can be maintained by adding or replacing fresh culture medium. Use 1×10⁻⁶ medium. 5 Begin a new culture with 1 × 10⁶ live cells / mL. 6 Cells / ml passage culture transfection Day 0 1. Add supplements to the Nucleofector solution.

[0238] a. The ratio of Nucleofector solution to supplement is 4.5:1.

[0239] b. For a single reaction, use 82 μl of Nucleofector solution plus 18 μl of supplement to prepare a total reaction volume of 100 μl.

[0240] 2. Prepare 12-well plates (1×10⁻⁶) by filling the wells with 1.5 mL of culture medium and equilibrating the medium in the plate in an incubator. 6 (cells / sample).

[0241] 3. In a 15 mL conical tube, at room temperature at 200... ×g Take the required number of cells (1×10) 6 Centrifuge (1 cell / sample) for 10 minutes. Completely remove the supernatant.

[0242] 4. Resuspend the cell pellet in 100 μL of room temperature Nucleofector solution for each sample.

[0243] a. Avoid leaving cells in Nucleofector solution for an extended period (15 minutes). .

[0244] 5. Mix 100 μl of cell suspension with 2 μg of DNA (eGFP plasmid) or 20-200 nM siRNA or ASO payload.

[0245] 6. Transfer the cell / DNA or cell / RNA suspension to a certified cuvette (included in the kit).

[0246] 7. Select program: K562: T-016 (or T-003).

[0247] 8. Add 0.5 mL of pre-equilibrated culture medium to the cuvette and gently transfer the sample to a pre-equilibrated 12-well plate (final volume: 2 mL of culture medium per well).

[0248] Day 1 (24 hours) 1. Perform RNA isolation and qRT-PCR protocol for payload testing. Day 2 (Stable GFP+ cell pool generation) 1. Antibiotic selection should begin 48 hours after transfection, using puromycin. a. K562: 0.5 µg / mL, for 7-21 days. 2. Place the cells at 5... × 10 6 / mL was resuspended in 1% BSA / PBS and the eGFP signal was periodically detected by flow cytometry. Resuspension of ASO and siRNA from IDT 1) Store the obtained items at -20℃ (dry). 2) Centrifuge tubes, 500 g, 5 minutes 3) Resuspend the ASO in TE buffer (30 minutes) and 100 µM stock solution. If any precipitation still occurs, try heating the oligomer at 55°C for 1-5 minutes, then vortex thoroughly. 4) Resuspend the siRNA (double strand) in nuclease-free double strand buffer (or nuclease-free water) or 100 µM stock solution. 5) Briefly vortex, then heat at 94°C for 2 minutes. Remove the tube from the heat source and allow it to cool to room temperature. The product is now a resuspended double chain. 6) Resuspended ASO and siRNA should be stored at -20°C. VISTA binding and internalization assay 1. Seed K562-VISTA cells in 96-well U-bottom plates (see table below for volume and cell count). Table 3. Details of VISTA binding and internalization assays.

[0249] 2. Prepare the INX201-RNA dilution series (5x titration starting from 60 µg / mL [2x]) a. 4 points + 0 Ab 3. Add Ab to the cells for titration. a. INX201-eGFP-siRNA-Cy5-150 µL / well b. INX201 free Mab 4. Incubate on ice for 30 minutes. 5. Collect 50 µL aliquots at 0 h, transfer to ice-cold 96-well V plates, add 200 µL of ice-cold PBS, and centrifuge at 500 g for 2 minutes. a. Stain the collected cells with α-hIgG (step 8) 6. Transfer 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 them to ice-preserved 96-well V plates, add 200 µL of ice-cold PBS, and centrifuge at 500 g for 2 minutes. 8. For each time point (including 0 hours), perform α-hIgG staining: a. Add 50 µL of the secondary antibody mixture (1:150 2x mixture, 1:30 0 final mixture). b. Stain at room temperature for 20 minutes c. Wash with 200 µL PBS d. Rotate at 500g, 4℃, for 5 minutes e. Resuspend in 100 µL PBS 9. Execution and analysis via flow cytometry a. INX201-siRNA-Cy5*-MACS Quant Table 4. VISTA combined with internalization flow cytometry group.

[0250] 10. Analyze the MFI binding of INX201 or ARC secondary antibodies in aliquots from 0-hour aliquots (stained on ice for 30 minutes). 11. Internalization through time-process analysis of MFI signals.

[0251] 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 Mixture 2X Kit (ThermoFisher 4369016) • Taqman probe for human GAPDH: Hs01922876_u1 (ThermoFisher 4331182) • Taqman probe (ThermoFisher 4331182) for human targets; see Table 5 below. The same protocol is used for K562 or immune cells (PBMCs). Table 5. Probes used for qRTPCR

[0252] RNA isolation RNeasy Plus Mini Reagent Kit (Qiagen 74136) • Use the RNeasy Plus program to isolate all RNA molecules longer than 200 nucleotides. This program provides... Enrichment of mRNA.

[0253] • Using the correct amount of starting material (<2e6 large cells) is crucial to obtaining optimal RNA yield and purity.

[0254] • Perform all steps of the procedure at room temperature.

[0255] 1. Harvest cells.

[0256] Resuspend the cells in 375 µl of RLT buffer.

[0257] • RLT Plus buffer may precipitate during storage. If necessary, redissolve by heating. Then place at room temperature.

[0258] 2. Transfer 370 µl of cell lysate into a gDNA Eliminator spinning column placed in a 2 ml collection tube (included). Use ≥8,000... × g Centrifuge for 30 seconds. Discard the column and retain the flow-through.

[0259] • Ensure no liquid residue remains on the column membrane after centrifugation. Repeat centrifugation if necessary.

[0260] 3. Add 350 µl of 70% ethanol to the flow solution and mix by pipetting.

[0261] • If some lysate is lost during homogenization and DNA removal, the volume of ethanol is adjusted accordingly. When purifying RNA from certain cell lines, precipitation may be visible after adding ethanol. This can indeed affect the procedure.

[0262] 4. Transfer up to 700 µl of sample (including any precipitate that may form) to an RNeasy spin column placed in a 2 ml collection tube (included). Gently cap the column and spray at ≥8,000. × g Centrifuge for 15 seconds. Discard the flowing liquid.

[0263] 5. Add 700 µl of buffer RW1 to the RNeasy spin column. Gently cap the column and spray with ≥8,000 ml of solution. × g Centrifuge for 15 seconds to wash the rotating column membrane. Discard the flow-through.

[0264] 6. Add 500 µl of buffer RPE to the RNeasy spin column. Gently cap the column and spray with ≥8,000... × g Centrifuge for 15 seconds to wash the rotating column membrane. Discard the flow-through.

[0265] • RPE buffer is provided as a concentrate. Before first use, add four times the volume of the concentrate as indicated on the bottle. Ethanol (96-100%).

[0266] 7. Add 500 µl of buffer RPE to the RNeasy spin column. Gently cap the column and spray with ≥8,000... × g Centrifuge for 2 minutes to wash the rotating column membrane.

[0267] 8. Place the RNeasy rotating 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.

[0268] 9. Place the RNeasy spin column into a new 1.5 ml collection tube (included). Add 30 to 40 µl of RNase-free water directly to the spin column membrane. Gently cap the column and add water at ≥8,000 ml / min. × g Centrifuge for 1 minute to wash out RNA.

[0269] 10. Evaluate concentration using nanodrop.

[0270] Reverse transcription • First, try using random hexamers, especially if downstream initiation is less efficient, or for long reverse transcripts or those containing... In the case of hairpin reverse transcripts.

[0271] • The RNA segment to be transcribed and subsequently amplified can be at least 3 kb in length.

[0272] • Do not add reverse transcriptase. For the synthesis of longer RNA transcripts, the incubation time can be increased to 60 minutes.

[0273] • For DNA with high G+C content, denaturation at 97°C in the first few cycles helps generate single-stranded DNA for PCR amplification. template.

[0274] 1. The reaction volume is 20 µl.

[0275] 2. Thaw all reagents and store on ice. Mix and briefly centrifuge the components. Store the RNase inhibitor and MultiScribe RT in the refrigerator.

[0276] 3. Divide 400 ng RNA into equal portions and add water to bring the final volume to 8.6 µl.

[0277] 4. Mix the following components thoroughly.

[0278] 5. Add 11.4 µl of the reaction mixture to the RNA.

[0279] 6. Setting parameters: 25℃, 10 minutes ® 37℃, 30 minutes ® 99℃, 5 minutes ® 4℃.

[0280] Alternative methods to reverse transcription GoScript reverse transcriptase (Promega) 1. The reaction volume is 20 µl.

[0281] 2. Thaw all reagents and store on ice. Mix and briefly centrifuge the components. Store the RNase inhibitor and MultiScribe RT in the refrigerator.

[0282] 3. Divide 800 ng RNA into equal portions and add water to bring the final volume to 10 µl.

[0283] 4. Mix the following components thoroughly.

[0284] 5. Add 10 µl of the reaction mixture to the RNA.

[0285] 6. Set parameters: 25℃, 5 minutes; 42℃, 60 minutes; 95℃, 5 minutes; 4℃, constant.

[0286] qPCR 1. The reaction volume is 20 µl.

[0287] 2. Mix the following components.

[0288] Rotate. Parameter settings: 95°C, 10 minutes; ® 95°C, 15 seconds; 60°C, 1 minute, 40 cycles.

[0289] 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 Donor Program. The cone blood was diluted 1:4 in PBS and carefully deposited on top of 13 ml Histopaque 1077. After centrifugation at 850 g (room temperature, deceleration without braking) for 20 min, monocytes were collected from the Histopaque / PBS interface. After washing once in PBS, PBMCs were centrifuged at 100 × 10⁻⁶. 6 Cells / ml resuspended for freezing (90% DMSO, 10% FBS) or at 10×10⁻⁶ cells / ml. 6 Cells / ml resuspended for experiments (complete RPMI medium).

[0290] Isolation of human T cells 1. Isolate T cells from human blood according to the manufacturer's instructions for the EasySep Direct Human T Cell Isolation Kit: a. Add 50 µL (50 µL / mL sample) of the separation mixture from the EasySep Direct Human T Cell Separation Kit (STEMCELL Technologies, catalog number 19661) to the blood. Invert the tube several times to mix.

[0291] b. Vortex RapidSpheres for 30 seconds. Add 50 µL of RapidSpheres to the blood and mix well.

[0292] c. Incubate at room temperature for 5 minutes.

[0293] d. Transfer the mixture to a 14 mL polystyrene round-bottom tube.

[0294] e. Add D-PBS to a volume of 14 mL and gently mix up and down 2-3 times using a pipette.

[0295] f. Place the test tube (without the cap) in the magnet and incubate at room temperature (RT) for 5 minutes.

[0296] g. Carefully transfer the enriched cell suspension into a new 14 mL test tube.

[0297] h. Add 50 µL / ml RapidSpheres blood to the enriched cells and mix well. Incubate at room temperature for 5 minutes.

[0298] i. Place the test tube (without the cap) in the magnet and incubate at room temperature (RT) for 5 minutes (secondary separation).

[0299] j. Carefully transfer the enriched cell suspension into a new 14 mL test tube.

[0300] k. Place the new 14 mL test tube containing the secondary isolated cells in a magnet and incubate at room temperature for 5 minutes.

[0301] 1. Carefully transfer the enriched cell suspension into a new 50 mL test tube. Add D-PBS to a final volume of 50 mL.

[0302] m. Centrifuge the cells at 515g for 5 minutes at 10 degrees Celsius and discard the supernatant.

[0303] n. Resuspend the cells in 5 mL of D-PBS and count them.

[0304] 2. Count T cells using an AOPI cell counter. Adjust the volume of D-PBS to 1 x 10⁻⁶ cells / mL. 6 Cells / mL.

[0305] Label PBMCs or T cells with Cell Trace Violet. 1. Add 20 µl of DMSO to the vial of Cell Trace™ Violet staining solution.

[0306] 2. Add directly to 10 ml of cell suspension to a final concentration of 2 µM.

[0307] 3. Incubate the cells in a 37°C water bath for 20 minutes.

[0308] 4. Add 40 ml of RPMI to the cells to absorb any unbound dye, incubate the cells for 3 minutes, centrifuge the cells at 300 x g for 5 minutes, and resuspend the cell pellet in preheated whole RPMI.

[0309] Human PBMC and T cell activation and proliferation assay Material • RPMI 1640 medium (Gibco, 11875-093) • 10% FBS (ATCC, 30-2020) • 100 U / ml penicillin / streptomycin (Gibco, 15140122) • U-shaped bottom 96-hole plate • Use the following payloads for INX201 and INX201 antibody RNA conjugates (ARC): RORC, PIK3CA, TNFα, BTK, Glut1, CD45 (siRNA); CD39 ASO Anti-CD3 / CD28 beads stimulate human PBMCs or T cells Human T cells were activated for 3 days using Dynabeads to activate CD3 / CD28 T cells.

[0310] a. Mix Dynabeads by vortexing and aliquot 2.5 µl / well bead into a 2 ml Eppendorf tube.

[0311] b. Add 2 ml of culture medium, vortex, and place the test tube on a magnet.

[0312] c. When the beads settle, remove the culture medium and resuspend the beads in a volume equivalent to the initial volume taken from the vial.

[0313] d. Add the beads directly to the cells and place them in a U-bottom 96-well plate, then add 100 µl of culture medium to a total volume of 200 µl.

[0314] e. Cell count: 500,000 cells / well for PBMCs; 100,000 cells / well for T cells. The ratio of pearls to T cells is 1:2.

[0315] LPS stimulation of human PBMCs 1. Stimulate PBMCs with 10 ng / ml LPS (Ams bio) 2. At 48 hours, culture medium was collected for cytokine analysis, and cells were collected for flow cytometry. INX201 or ARC titration 1. Prepare INX201 or ARC in the culture medium to twice the final concentration (maximum concentration 200 nM) and perform serial dilutions. In these experiments, DAR was not quantified; therefore, we used the Ab concentration in the ARC titration experiments.

[0316] Proliferation analysis based on flow cytometry 1. After incubation, centrifuge the plate for 4 minutes, 515g, and collect the supernatant for cytokine analysis (freeze at -80°C until used).

[0317] 2. Wash the cell pellet with PBS, centrifuge, and remove the supernatant.

[0318] 3. Stain cells in 50 µl of the corresponding antibody mixture (in PBS) at room temperature with shaking (400 rpm) for 30 minutes. For viability assays, stain cells in annexin buffer (Biolegend, 422201) and wash in PBS. Resuspend cells in 100 µl of PBS and analyze on a flow cytometer.

[0319] Table 6. PBMC Group (LPS Stimulation)

[0320] Table 7. T cell group (CD3 / CD28 stimulation)

[0321] Table 8. Vitality Group (CD3 / CD28 Stimulation)

[0322] result siRNA and ASO payload selection The applicant has selected a variety of immunologically relevant targets, including (1) existing approved drugs targeting (TNFa); (2) empirically proven pathways with target toxicity issues (PI3K); and (3) novel targets (Glut1), see Table 2. The first category of targets was used for proof-of-concept studies (TNFa), while the other targets demonstrated the inherent advantages of the subject ARC / ANC, which preferentially targets myeloid cells and lymphocytes. These immune-targeting RNA-ARCs should minimize or eliminate toxicities associated with the widespread expression of non-immune tissue-targeting RNAs (PI3K) and / or targets (Glut1) or the nature of the targets, such as being transcription factors (RORC), whose toxicity has so far limited or excluded targets against these types of "difficult" targets (TNFα [PMID: 34301319, PMID: 29158574]; BTK [PMID: 27192942, PMID: 31431692, PMID: 33122850, PMID: 36903645]; PI3K [PMID: 31928691, PMID: 34127844, PMID: 26093105]; RORC [PMID: 30010338, PMID: 34040108]; Glut1 [PMID: 35878663, PMID: 34018847]; CD39 The successful development of biologics and / or small molecule inhibitors (PMID: 30871609).

[0323] Table 9. Exemplary Payload

[0324] Unconjugated siRNA or ASO payloads (1-4 per target) were purchased from IDT (https: / / www.idtdna.com / pages / products / functional-genomics / dsirnas-and-trifecta-rnai-kits). K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and target knockdown was analyzed by qRT-PCR using the ddct method, reported as a fold change relative to control. Scrambled siRNA or ASO controls were also purchased from IDT (https: / / www.idtdna.com / pages / products / functional-genomics / dsirnas-and-trifecta-rnai-kits). Figures 1 to 2 Select the payload marked with (*) for conjugation with the delivery medium (anti-VISTA Mab INX201).

[0325] like Figure 1As shown, immunologically relevant siRNA and ASO payloads were identified and tested. K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and target knockdown was analyzed by qRT-PCR using the ddct method, reported as foldbacks relative to controls. Scrambled siRNA or ASO controls were also purchased from IDT. Payloads marked with (*) were selected for conjugation with the delivery medium (anti-VISTA Mab INX201).

[0326] like Figure 2 As further shown, additional immunologically relevant siRNA and ASO payloads have been tested in the ARC of this invention. In these experiments, K562-VISTA cells were transfected with only 200 nM siRNA (or ASO), and target knockdown was analyzed by qRTPCR using the ddct method and reported as a fold change relative to the control. Scrambled siRNA or ASO controls were also purchased from IDT. Payloads marked with (*) were selected for conjugation with the delivery medium (anti-VISTA Mab INX201). Results obtained using the CD39 ARC and other ARCs containing payloads targeting other genes are described herein.

[0327] Antibody RNA conjugation and QC.

[0328] The anti-VISTA Mab and payloads used for conjugation are described in Tables 1 and 2 and Appendices 1 and 2. All conjugations were performed using an oligomer conjugation kit (Abcam, #ab218260). All RNA oligomers were purified by HPLC and resuspended at 100 μM. Lysine-based conjugations were performed via amine reactive groups, using a widely used nonspecific conjugation strategy. All payloads were synthesized in IDT and contained a 3'-sense chain Cy5 tag and a 5'-amine on the antisense chain (see Appendix 2).

[0329] Reducing SDS-PAGE and silver staining.

[0330] To visualize the proteins, the level of ARC conjugation was analyzed by SDS-PAGE and subsequent silver staining. Briefly, ARCs were mixed with reducing 2x Laemmli sample buffer (Bio-Rad, #1610737) and incubated at 80°C for 5 min. The reduced sample was resolved using a 4-15% Mini-Protean TGX pre-prepared gel (Bio-Rad, #4561083) according to the manufacturer's instructions. After electrophoresis, the gel was washed with Mil-Q water and the proteins were stained with SilverQuest reagent (Thermo Fisher, #LC6070). CD45 siRNA ARCs and SOCS1 ASO ARCs were analyzed as representative ARCs of the siRNA and ASO payloads, respectively. Both showed the expected conjugation patterns ( Figure 3 Nanodrop measurements of ASO ARC were: RNA 37.2 μM, protein 8.0 μM; therefore, the DAR was approximately 4.65.

[0331] Figure 3 SDS-PAGE experiments with INX201 ARC confirmed effective conjugation. In these experiments, INX201 ARC was resolved on reducing SDS-PAGE followed by silver staining. Lane markers: 201 - free INX201 anti-VISTA Mab; CD45 - ARC using CD45 siRNA; SOCS1 - ARC using SOCS1 ASO; a discrete distribution pattern of multiple conjugates was observed, confirming effective conjugation. Based on the effective payload molecular weight (approximately 17 kDa vs. approximately 6 kDa), the HC or LC shift of the siRNA was more significant than that of the ASO.

[0332] The binding (to the VISTA target) and internalization of free INX201 and INX201 eGFP siRNA ARC were compared. K562-VISTA cells were incubated with 200 nM free Mab or ARC. Binding and internalization were measured over time. The binding of free antibody and ARC to the target was equivalent, as shown at the zero-hour time point (displayed as 0.1 hours). Figure 4A As we previously stated, the internalization of free INX201 antibody and ARC was similar, and rapid and efficient. Specifically, over 90% of the antibody was internalized in both ARC and free Ab samples within 30 minutes. Clearly, the antibody detected on the cell surface was absent in the time-course assay. Figure 4A ).

[0333] Intracellular RNA was detected using Cy5 markers present on siRNA (given the lack of antibody detection on the cell surface due to internalization in ARC samples, we assumed that most Cy5 detection was intracellular). As demonstrated by time-course experiments, Cy5 was readily detectable in K562-VISTA cells and remained intact during the experiment (24 hours).

[0334] The experimental results in Figure 4 show that the exemplary ARC effectively binds to and internalizes from the surface, providing efficient and prolonged intracellular retention of siRNA in K562-VISTA cells. Free INX201 (circular line) or eGFP ARC (square line) were used at 200 nm. A) Time course of antibody binding and internalization. B) RNA accumulation assay, measured by Cy5. MFI – mean fluorescence intensity; representing the two independent experiments shown.

[0335] ARC function test in K562-VISTA cells K562-VISTA cells were transfected with plasmid DNA expressing eGFP, and GFP+ cell pools were used 14–28 days post-transfection. Cell pools were treated with either drug-free or eGFP ARC. eGFP or CD45 protein levels were measured by flow cytometry (Table 10). Maximum inhibition of approximately 50% of eGFP protein levels was achieved by ARC, which is similar to the eGFP mRNA knockdown levels measured by qPCR in the same cell pools treated with transfected eGFP-free siRNA. Figure 5A ).

[0336] K562-VISTA cells are natively CD45+. CD45 is expressed on the cell surface, thus providing an attractive target for proof-of-concept studies in measuring target (CD45) knockdown. Cells were either untreated or treated with either transfection or CD45 ARC (200 nM) siRNA. For transfection experiments, CD45 levels were measured by flow cytometry at 48 hours; for CD45 ARC experiments, CD45 levels were measured at 72 hours. Two independent experiments were performed, and the results were similar. Figures 5B to 5C Approximately 50% of CD45 protein was observed to be knocked down from the surface in ARC-treated cells, but this was not observed in untreated cells. Transfected siRNA repressed the protein to a similar level. Figure 5B Furthermore, siRNA freely added to cells without transfection did not inhibit CD45 levels. Figure 5C Excessive addition of free siRNA ensures that any knockdown observed via ARC is not due to any residual free siRNA payload in the ARC conjugate.

[0337] Table 10. Flow cytometry groups / channels for ARC QC in K562 cells.

[0338] from Figures 5A to 5C The experimental results show that the knockdown of INX201 ARC protein expression is comparable to that of in vitro transfection. K562-VISTA WT cells or eGFP+ cell pools were used in this experiment. Figure 5A In the experiments, cells were treated for 28 hours with either no drug (left column) or 200 nM eGFP ARC (right column). The maximum level of eGFP protein knockdown (approximately 50%, measured at 24 hours) was established by transfecting the same cells with eGFP siRNA. ARC-mediated knockdown was similar to the maximum possible knockdown level (based on the siRNA sequence). Figure 5B In the experiments shown, the dashed line represents the maximum expected CD45 protein knockdown level based on the potency of the payload (siRNA), which is 50%, as measured at 48 hours from free transfected CD45 siRNA (right column); cells were treated with 200 nM eGFP ARC (middle column) or no drug (left column) for 72 hours. Figure 5C In the experiments, CD45 levels were measured in repeated experiments. Cells were treated for 72 hours with no drug (left column), free siRNA, no transfection (middle column), and 200 nM ARC (right column).

[0339] ARC function test in immune cells exist Figure 6 A to Figure 6 In another experiment shown in C, human PBMCs were stimulated with LPS or anti-CD3 / CD28 dynabeads as described in Materials and Methods. For LPS stimulation, culture medium was collected at 48 h, and for CD3 / CD28 stimulation, at 72 h, for cytokine analysis by Luminex. TNFα ARC was administered at concentrations ranging from 200 nM to 0.32 nM at the stimulation time. Free siRNA alone (untransfected) served as a control and was applied at 200–1000 nM. For LPS-stimulated PBMCs ( Figure 6 A) and PBMCs that are anti-CD3 / CD28 stimulated (A) Figure 6B) TNFa protein, as measured by Luminex, was effectively knocked down by TNFa ARC in a dose-dependent manner. Knockdown levels were >80% (LPS stimulation) and approximately 50% (anti-CD3 / CD28 stimulation). For anti-CD3 / CD28 stimulation, we performed qRT-PCR on cells collected at 72 hours to verify that the target TNFa was knocked down at the mRNA level. In this case, we observed approximately 60% target knockout at the RNA level in PBMCs, which was strongly correlated with a 50% reduction in TNFa cytokine levels. Figure 6 C).

[0340] More specifically, Figure 6 A to Figure 6 The experiment shown in C demonstrates that INX201 ARC inhibits TNFα from PBMCs. Human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated with TNFα ARC (0–200 nM) or free RNA (200–1000 nM) for 48 h (LPS) or 72 h (beads). With ARC, TNFα levels decreased effectively in a dose-dependent manner, but not with free siRNA. qRTPCR of ARC-treated PBMCs collected at 72 h relative to untreated PBMCs confirmed effective target knockdown; no 0.1 nM ARC was observed (due to logarithmic scaling).

[0341] In vitro PBMC or T cell proliferation assays showed that siRNA delivered via ARC (Glut1, PI3K, BTK, TNF, RORC) blocked immunologically related targets, leading to a reduction in cytokine production or T cell proliferation, but without affecting T cell viability. Figures 6 to 13 The reduction in proliferation was mainly manifested as a slowdown in proliferation (the last two peaks of new proliferating cells were affected), indicating an important characteristic of siRNA-ARC therapy that controls the proliferation of effector cells without acting as an overall immunosuppressant.

[0342] Specifically, TNFα ARC, rather than free TNFα siRNA, slowed T cell proliferation. Figure 7A ); PI3K ARC, rather than free PI3K siRNA, also slowed T cell proliferation in a dose-dependent manner. Figure 7B ).

[0343] In a separate experiment shown in Figure 8, it was further confirmed that INX201 ARC slowed T cell proliferation. Human PBMCs were activated with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated for 72 hours with (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). It can be seen that with the exemplary ARC, newly proliferating T cells were effectively reduced in a dose-dependent manner, but this was not the case with free siRNA treatment. Proliferation was analyzed by cell trace violet dilution and observed by flow cytometry; no ARC was observed at 0.1 nM (due to logarithmic scaling). Curves were generated by inhibitor-to-response three-parameter nonlinear regression analysis (GraphPadPrism 9). Each concentration point was measured in parallel using a single technique.

[0344] As described above, BTK ARC was tested in human PBMCs activated with LPS or anti-CD3 / CD28 dynabeads. In LPS stimulation assays, BTK ARC, rather than free BTK siRNA, reduced CD16+ monocyte activation, as measured by CD69 levels 48 hours after drug addiction. Figure 8A CD69 levels decreased to levels comparable to unstimulated cells. BTK ARC also slowed T cell proliferation, as measured at 72 hours post-stimulation. Figure 8B Free INX201 antibody or free untransfected siRNA payload had no effect on these assays.

[0345] More specifically, Figures 8A to 8B The experiments shown demonstrate that INX201-BTKARC reduces PBMC activation. In these experiments, human PBMCs were activated with (A) 10 ng / ml LPS or (B) anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2, and treated with BTKARC (0–200 nM, triangular) or free RNA (200–1000 nM, star-shaped) or free INX201 (square) for 48 h (LPS) or 72 h (beads). Curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism9). CD69 levels were measured in A; new proliferating cells were measured in B; each concentration point was determined in parallel using a single technique; no ARC was observed at 0.1 nM (due to logarithmic scaling); unstimulated – unstimulated cells; MFI – mean fluorescence intensity.

[0346] exist Figures 9A to 9BIn the experiments, Glut1 ARC was tested on purified human T cells stimulated with anti-CD3 / CD28 dynabeads for 72 hours. After incubation, the culture medium was collected, and cytokine levels from two human donors were measured using Luminex. When T cells were treated with Glut1 ARC, IFNg and IL17A levels decreased to 1 / 3–1 / 2 in a dose-dependent manner. Figures 9A to 9B ).

[0347] More specifically, Figures 9A to 9B The experiments shown demonstrate that INX201-Glut1 ARC reduces cytokine production in purified human T cells activated with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated with Glut1 ARC (0–200 nM) for 72 hours. Each concentration point was measured in parallel using a single technique. Two human donors were tested: donor 1 – solid line, donor 2 – dashed line; no ARC was observed at 0.1 nM (due to logarithmic scaling). A) IFNg and B) IL17A were measured by Luminex.

[0348] exist Figures 10A to 10C In the experiments shown, RORC ARC was also tested on purified human T cells stimulated with anti-CD3 / CD28 dynabeads for 72 hours. After incubation, the culture medium was collected, and cytokine levels from two human donors were measured using Luminex. When T cells were treated with RORC ARC, IFNg, IL6, and IL12p40 levels decreased to 1 / 4–1 / 2 in a dose-dependent manner. Figures 10A to 10C ).

[0349] More specifically, in Figures 10A to 10C In the experiments shown, purified human T cells activated with anti-CD3 / CD28 beads demonstrated that INX201-Glut1 ARC reduced cytokine production at a bead-to-T cell ratio of 1:2, and were treated with RORC ARC (0–200 nM) for 72 hours. Each concentration point was measured in parallel using a single technique. Two human donors were tested: donor 1 – solid line, donor 2 – dashed line; no ARC was observed at 0.1 nM (due to logarithmic scaling). A) IFNg, B) IL6, and C) IL12p40 were measured by Luminex.

[0350] Figure 11 The experiments shown further revealed that, in contrast to a weakened T-cell response, CD39 knockdown using ARC enhanced the immune response in stimulated PBMCs. Specifically, the experiments demonstrated that IL-6 levels increased in a dose-dependent manner after PBMC treatment with CD39 ARC compared to free antibody INX201. Figure 11 ).

[0351] More specifically, in Figure 11 The experiments shown demonstrated that INX201-CD39 ASO ARC targeting enhanced the immune response in human PBMCs activated with anti-CD3 / CD28 beads and treated with CD39 ARC (0–200 nM, triangles) or free INX201 (0–200 nM, circles) for 72 hours. Curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 9). Each concentration point was determined in parallel using a single technique; no ARC was observed at 0.1 nM (due to logarithmic scaling).

[0352] Delivery platform optimization: Mab and Fab To optimize the applicant's antibody delivery platform, we designed INX201 Fab, which will be used for conjugation to prepare DAR2 antibody fragments with appropriate payloads. To ensure that INX201 Fab functions entirely as a complete mab, we performed direct comparisons of INX201 mab and Fab by ELISA and competitive cell-based assays. Figures 12A to 12B ). Figure 12A The results show that the binding of INX201 Mab and Fab to the target (VISTA) is comparable (0.1 nM vs. 0.7 nM EC50, a three-fold difference expected due to stoichiometry). In a competitive assay on the K562-VISTA cell line, cells were pre-incubated with increasing concentrations of unlabeled INX201 Mab or INX201 Fab to block the target on the cell surface, followed by flow cytometry detection of available residual targets on the surface using a labeled INX201-AF488 antibody. The figures clearly show that both INX201 Mab and INX201 Fab effectively block the VISTA target on the cell surface in a dose-dependent manner. Figure 12B This further proves that the INX201 Fab is functionally identical to the INX201 Mab.

[0353] Vista ELISA settings.

[0354] First, at room temperature (RT), 96-well flat-bottom plates (Thermo Scientific Nunc ImmunoMaxisorp, catalog number 442404) were coated for one hour with 20 nM hIX50 (human VISTA ECD, manufactured for ImmuNext by Arragen Bioscience) in PBS. The wells were washed three times with PT (PBS containing 0.05% Tween 20) and then blocked with PTB (PBS containing 0.05% Tween 20 and 1% BSA) at room temperature for 30 minutes. INX201 Fab was diluted from 1000 nM to 0.02 nM in PTB and added to the wells for one hour at room temperature, or INX201 Mab was diluted from 20 nM to 0.002 nM in PTB and added to the wells for one hour at room temperature. After incubation, the wells were washed three times with PT, and then mouse anti-human κ conjugated with HRP (SouthernBiotech, catalog number 9230-05) at a dilution of 1 / 2000 was used as the detection reagent, and incubation was performed at room temperature for 1.5 hours. After three washes, the ELISA reaction was visualized using TMB (Thermo Scientific, catalog number 34028) as the colorimetric substrate. The reaction was terminated with 1M H2SO4 after a few minutes at room temperature. OD450 was read on a Moleculardevices Spectramax M3 plate reader and analyzed using SoftMaxPro software.

[0355] More specifically, Figure 12 illustrates platform optimization using the VISTA Fab instead of the Mab. Figure 12A The experiment compared the binding of INX201 Mab (square) and INX201 Fab (circular) to human VISTA ECD using ELISA. Figure 12B The experiments presented in this paper demonstrate the results of a competitive assay based on K562-VISTA cells, where increased pre-binding concentrations of INX201 Mab (square) or INX201 Fab (circle) blocked the availability of VISTA on the cell surface (thus reducing VISTA MFI measurements of INX201-AF488); curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPadPrism 9). Each concentration point was measured in parallel using n=2 techniques; no Ab of 0.0001 nM was observed (due to logarithmic scaling).

[0356] Test Example: Effect of the ARC of the present invention on T cell viability Human PBMCs were stimulated with anti-CD3 / CD28 dynabeads as described in Materials and Methods. T cell viability was studied in a dedicated assay in which cells were stained with the antibody group 72 hours after stimulation, as shown in Table 8. Live cells were defined as negative for both annexin V and live-dead; apoptotic cells were double-positive for both annexin V and live-dead; and necrotic cells were annexin V negative but live-dead positive. No differences in cell viability or percentage of apoptotic or necrotic T cells were found between INX201 ARC and free INX201, nor were dose-dependent changes in viability of either the anti-CD3 or anti-CD28 dynabeads noted. Figure 13 ).

[0357] More specifically, Figure 13 The study demonstrates that in experiments involving activation of human PBMCs with anti-CD3 / CD28 beads followed by treatment with either PI3K ARC (0–200 nM, triangles) or free INX201 (0–200 nM, squares) for 72 hours, the exemplary ARC did not affect T cell viability. Curves were generated using a three-parameter nonlinear regression analysis of the inhibitor versus response (GraphPad Prism 9). Each concentration point was determined in parallel using a single technique; no ARC was observed at 0.1 nM (due to logarithmic scaling); unstimulated–unstimulated cells.

[0358] Example 2: In vivo evaluation of anti-VISTA antibody RNA conjugate (ARC) in GVHD model In this embodiment, the efficacy of the anti-VISTA antibody RNA conjugate (ARC) was evaluated in a GVHD model. These experiments again used INX201 as the payload delivery medium. These experiments provide further evidence that INX201 can be used as a delivery medium for delivering 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 included in Appendix 4.

[0359] Table 11 below shows the anti-VISTA and isotype control mAbs, as well as the ARC used for payload delivery.

[0360] Table 11. Anti-VISTA and isotype control mAbs, and ARC for payload delivery.

[0361] As described above, the antisense strand of siRNA represents the active drug. Once delivered into the cells of interest via our VISTA delivery platform, it binds to its target mRNA and triggers degradation, thereby reducing the levels of the target mRNA and protein.

[0362] Graft-versus-host disease (GvHD) model GVHD models are used to evaluate the efficacy of anti-VISTA antibody RNA conjugates (ARCs) because they are recognized models of autoimmunity and inflammation. Furthermore, humanized mouse models of xenograft-versus-host disease (GvHD) allow for in vivo studies of immunomodulatory compounds specific to human drug targets and their effects on inflammatory markers such as inflammatory cytokines. These GVHD models are based on the transfer of human peripheral blood mononuclear cells (PBMCs) from immunodeficient mouse strains.

[0363] The NSG (NOD-scid IL-2Rγnull) mouse GVHD model combines the characteristics of a NOD / ShiLtJ background, severe combined immunodeficiency mutation (scid), and IL2 receptor γ chain deficiency. Therefore, these mice lack mature T cells, B cells, or functional NK cells, and lack cytokine signaling, resulting in better transplantation of artificial hematopoietic stem cells and peripheral blood mononuclear cells than any other published mouse strain.

[0364] In the NSG model of xenogeneic GvHD, donor human T cells significantly proliferate in recipient mice and induce anti-host cell reactivity, leading to skin tissue infiltration. Over time, mice lose weight and die from GvHD without treatment. A hallmark of the disease is the massive production of pro-inflammatory cytokines, which can be measured in plasma [3]. The timeframe for disease progression can range from 3 to 7 weeks. Both the GLUT3 and PI3K pathways have been described as important for human T cell activation and proliferation [4–8], thus xenogeneic GvHD is a particularly excellent model for studying the effects of GLUT3 or PI3K knockout using our techniques.

[0365] The materials and methods used in these in vivo experiments are described below.

[0366] Materials and methods Materials and reagents 1. LPS, Chondrex (Catalog No. 170031) 2. NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ male 8-week-old (NSG mice), Jackson Labs (catalog number 1557) 3. ACK lysis buffer, Gibco (catalog number A10492-01) 4. FACS lysis buffer (BD Biosciences, catalog number 349202) 5. PBS, Corning (Catalogue No. 21-040-CV) 6. MILLIPLEX® MAP Human Cytokines / Chemokines / Growth Factors A 48-fold Premixed Magnetic Bead Assay - Immunological Multiplex Assay, EMD Millipore (Catalog No. 3 HCYTA-60K-PX48) 7. eBioscience™ Foxp3 / transcription factor staining buffer kit, ThermoFisher, 00-5523-00 8. RPMI 1640 medium (Gibco, 11875-093) 9. 10% FBS (ATCC, 30-2020) 10. 100 U / ml penicillin / streptomycin (Gibco, 15140122) Isolation of human T cells Human T cells were isolated using the same procedure disclosed in Example 1.

[0367] Cell Trace Violet was used to label PBMCs or T cells. PBMCs or T cells were labeled with Cell Trace Violet using the same procedure disclosed in Example 1.

[0368] Human PBMC and T cell activation and proliferation assay Human PBMCs and T cell activation and proliferation assays were performed using the same procedure disclosed in Example 1.

[0369] INX201 or ARC titration INX201 and ARC were titrated using the same procedure disclosed in Example 1.

[0370] Cell activation analysis based on flow cytometry Cell activation analysis based on flow cytometry was performed using the same procedure disclosed in Example 1. The in vitro flow cytometry groups used in the analysis are listed in Table 12.

[0371] Table 12. In vitro flow cytometry group.

[0372] Xenomorphic GvHD Model Research Design Recipient mice were 8-week-old male NSG mice purchased from Jackson Laboratory. Unless otherwise stated, 10 mice were enrolled in each group.

[0373] On day 0, mice were intravenously (iv) injected via tail vein with 10 × 10⁻⁶ premixed antibody in 200 µl PBS.6 Individual PBMCs. On day 14 (or another specified time point), mice were bled, and plasma was collected for cytokine analysis and T cell counts were assessed using flow cytometry. Mice were weighed three times a week to monitor disease progression, and euthanasia was performed if their body weight dropped to less than 75% of their initial body weight.

[0374] Human PBMC separation Human peripheral blood was obtained from apheresis cones provided by volunteer donors through the DHMC Donor Program. The cone blood was diluted 1:4 in PBS and carefully deposited on top of 13 ml Histopaque 1077 plasma. After centrifugation at 850 g (room temperature, deceleration without braking) for 20 min, monocytes were collected from the Histopaque / PBS interface. After washing once in PBS, PBMCs were centrifuged at 100 × 10⁻⁶. 6 Cells / ml were resuspended in PBS for injection.

[0375] Antibody administration INX201 or its antibody-RNA conjugate (ARC) was injected at 5 mg / kg (mixed with hPBMC). A single dose of ARC (or Ab) was used in all experiments described in this embodiment.

[0376] LPS early stimulation model In some experiments, we evaluated early human PBMC cytokine responses in vivo following stimulation with LPS (0.5 mg / kg, intraperitoneal injection) 18 hours after PBMC+ARC injection. In these early stimulation models, each mouse was injected with 10 million PBMCs. Plasma was collected at early time points (4 hours to 7 days post-stimulation) for cytokine measurements using Luminex Milliplex.

[0377] Evaluation of changes in "absolute" immune cells in the blood On day 14 post-PBMC cell transfer (or other designated time point), the absolute number of human T cells was assessed by flow cytometry of retro-orbital hemorrhage. To obtain near-absolute cell counts, 100 µl of whole blood was stained by direct addition of an antibody mixture (Table 3) plus mouse and human Fc blockers. After incubation at room temperature for 30 min, 1 ml of BD FACS lysis solution was added to each sample. After incubation for 20 min, the samples were washed once in a large volume of PBS, then resuspended in 100 µl of PBS and continuously run on a MACSQuant or Northern Lights (Cytek) flow cytometer and analyzed using the FlowJo program.

[0378] The antibody mixtures that affected cell counts in these experiments are listed in Table 13 below.

[0379] Table 13: Antibody mixtures used for absolute cell counting

[0380] Evaluation of Treg cells in the blood (day 28 or as specified) The procedures used to evaluate Treg cells in the blood (day 28 or as specified) in these experiments are as follows: 1. Divide 0.1 ml of blood into 1.3 ml deep-well plates.

[0381] 2. Add 1 ml of ACK and incubate for 10 minutes.

[0382] 3. Rotate at 450 g for 3 minutes, decant the supernatant. If the precipitate is still red, repeat the lysis for 5 minutes.

[0383] 4. Wash with PBS (1.0 ml), rotate at 450 g for 3 minutes, decant the supernatant, and transfer to 200 µl of PBS in a V-shaped plate. 5. Rotate at 450 g for 3 min, add 50 ml of the Ab mixture (Table 4, except for anti-FOXP3 and RorGT Ab), incubate at room temperature for 30 min, and wash once with PBS. Rotate at 450 g for 3 min and decant the supernatant.

[0384] 6. Use FoxP3 for fixation / permeation according to the manufacturer's instructions for staining with FOXP3 and RORgt.

[0385] 7. After fixation and permeabilization, resuspend the cells in 50 µl of permeabilization buffer containing anti-FOXP3 and anti-RORgt Ab (both diluted 1:50) and incubate at room temperature for 30 minutes. Wash the cells once with permeabilization buffer and resuspend them in 100 µl of PBS. Run the flow cytometer on a MACSQuant or Northern Lights (Cytek) and analyze using the FlowJo program.

[0386] The regulatory T cell antibody groups used in these experiments are shown in Table 14: Table 14: Regulatory T cell antibody group

[0387] result PI3K and GLUT3 ARC conjugation and in vitro QC Anti-VISTA Mab, the payloads used for conjugation, and ARC are described in Table 11 and Appendices 1-2. All conjugations were performed using an oligomer conjugation kit (Abcam, #ab218260). All RNA oligomers were purified by HPLC, resuspended in 100 μM, and annealed. Lysine-based conjugation via amine reactive groups was used, one of the most widely used nonspecific conjugation strategies. All payloads were synthesized in IDT and contained a 3'-sense chain Cy5 tag and a 5'-amine on the antisense chain (see Appendix 2). The drug-antibody ratio (DAR) was assessed by UV / Vis spectroscopy (Nanodrop) and estimated to be 1.0 for both ARCs.

[0388] K562-VISTA cells were treated with 40–200 nM INX201-PI3K ARC or allotype control IgG1-PI3K ARC (described in ARC_01) (Table 1). PI3K knockdown was assessed by qRT-PCR at 24 hours. INX201-PI3K ARC achieved 70% target knockdown, while INX201 Ab alone or allotype control PI3K ARC did not show PI3K knockdown. Figure 15 This indicates specificity.

[0389] like Figure 15 The data show that INX201 (anti-VISTA) PI3K ARC, rather than the allotype control IgG1 PI3K ARC, specifically knocked down PI3K. In these experiments, K562-VISTA cells were transfected with 40–200 nM of INX201-PI3K ARC or allotype control IgG1-PI3K ARC, and the knockdown of the target was analyzed by qRT-PCR using the ddct method and reported as the percentage of target inhibition, where 0% inhibition was for the INX201 sample alone; PI3K ARC: anti-human VISTA mAb conjugated with PI3K siRNA; PI3K allotype ARC: IgG control conjugated with PI3K siRNA; INX201, mAb unconjugated: naked anti-human VISTA mAb. Each concentration point was measured in parallel using a single technique.

[0390] 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 using Luminex. PI3K ARC, rather than free INX201 antibody, reduced the production of pro-inflammatory cytokines. Specifically, PI3K ARC reduced IL5 and IL13 levels to 1 / 6–1 / 3; and reduced TNFα and IL17F levels to approximately 1 / 2, both compared to free INX201 levels. Figure 16A ).

[0391] from Figure 16A It can be seen that GLUT3 ARC, rather than free INX201 Ab, reduces T cell activation in a dose-dependent manner. Furthermore, GLUT3 ARC reduces CD69 levels on effector memory T cells (TEM) and CD25 levels on CD3 T cells in a dose-dependent manner, with IC50 values ​​of 1 and 6 nM, respectively. Figure 16B It should be noted that when DAR is 1.0, the amount of "drug" (siRNA) is equivalent to the amount of Ab delivery medium (nM).

[0392] More specifically, in Figures 16A to 16B In the experiments, the in vitro functionality of human PI3K and GLUT3 ARC was confirmed. Human PBMCs were activated for 72 hours with anti-CD3 / CD28 beads at a bead-to-T cell ratio of 1:2 and treated with (A) PI3K ARC or free INX201 (0-200 nM) or (B) GLUT3 ARC or free INX201 (0-200 nM). The curves were generated by inhibitor-response three-parameter nonlinear regression analysis (GraphPad Prism 10). IL5 / IL13 / TNFα / 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; CD25 levels were measured in live CD45+ / HLA-DR- / CD56- / CD3 cells. In A, single technique parallel determinations were used for each concentration point; in B, n=2 techniques parallel determinations were used for each concentration point; no ARC was observed at 0.1 nM (due to logarithmic scaling); MFI – mean fluorescence intensity. Where appropriate, data are expressed as mean ± SEM.

[0393] PI3K ARC treatment reduced human cytokine response and T cell proliferation in vivo. In an experiment monitoring survival, we observed no difference in weight loss and median survival (data not shown) between classical xenogeneic GvHD and early LPS stimulation alone, with PI3K ARC and INX201 alone. Therefore, as an alternative to the classical xenogeneic GvHD model (where human T cells proliferate and human cytokine levels increase over time), we considered inducing early cytokine production in human PBMCs using stimulation such as LPS shortly after injection into the host (NSG mice) (18 hours). LPS was administered intraperitoneally to mice 18 hours after intravenous injection of hPBMC+Ab, and hematopoietic cytokine responses were measured at 4 hours and 7 days post-LPS injection. Figure 17A As can be seen, peak levels of most cytokines occurred at 4 hours. At 4 hours post-LPS injection, PI3K ARC treatment reduced IL-6 and TNFα production to as low as one-third compared to INX201 alone. PI3K ARC treatment also reduced delayed IFNg responses on day 7. Figure 17B PI3K ARC generally has a global effect on cytokine production, typically reducing the production of most cytokines at an early time point (4 hours) after LPS stimulation, such as... Figure 17C As shown. Due to the variability of the response, the reduction of pro-inflammatory cytokines ( Figure 3 C) Not statistically significant. In the group without LPS stimulation, PI3K ARC showed no effect on human cytokine production (data not shown).

[0394] The experiments in Figure 17 clearly demonstrate that PI3K ARC reduces inflammatory cytokine responses in xenogeneic GvHD mice. In the experiments shown in the figure, 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 post-injection, the mice were intraperitoneally stimulated with 0.5 mg / kg LPS. Figure 17A A schematic diagram of the experiment in the heterologous GvHD LPS stimulation model is shown. Figure 17B The changes in plasma human cytokine levels on day 7 (IFNg) or 4 hours (IL6, TNFα) are shown. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± SEM; *-p<0.05; **-p<0.01. Figure 17C A heatmap based on Z-scores shows cytokine levels 4 hours after LPS stimulation (n=6 per group).

[0395] Results in the GVHD model further revealed that PI3K ARC treatment reduced LPS-responsive T cell proliferation in xenogeneic GvHD, as evidenced by the reduced number of CD4 and CD8 T cells (less than 1 / 2) in the PI3K ARC-treated group on day 14. Figure 18 No difference in T cell numbers was found in classical xenogeneic GvHD without LPS stimulation (data not shown).

[0396] More specifically, Figure 18 Data in the figure indicate that PI3K ARC reduces LPS-induced T cell proliferation in vivo. In the experiments shown, 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 post-injection, mice were intraperitoneally stimulated with 0.5 mg / kg LPS. Changes in blood T cell counts for both CD4 and CD8 cells at day 14 are shown (n=6 mice per group). Statistical analysis was performed using Student's t-test. Data are presented as mean ± SEM, *-p<0.05.

[0397] PI3K ARC does not reduce the percentage of regulatory T cells in the body. To test whether PI3K ARC had any effect on the number of regulatory T cells (Tregs) in a xenogeneic GvHD model, we monitored Treg cell levels in the blood of NSG mice in the same xenogeneic GvHD model. NSG mice were again intravenously injected with 10 million hPBMCs (mixed with either human INX201 or PI3K ARC alone, at 5 mg / kg). In the experimental individual groups, some mice were stimulated with LPS 18 hours after PBMC+Ab or ARC injection. LPS was administered intraperitoneally.

[0398] On day 28, human CD4 levels were measured in the blood. + FOXP3 + (Treg) cell frequency. We observed that PI3K ARC treatment did not reduce the frequency of Treg cells compared to INX201. Figure 19 In the KLPS-stimulated group, the frequency of Tregs tended to increase, but the difference was not statistically significant. No large numbers of Th17 cells were detected in the blood (data not shown). In conclusion, our data suggest that PI3K ARC does not reduce the percentage of regulatory T cells in vivo.

[0399] More specifically, Figure 19The experiments revealed that PI3K ARC did not reduce the percentage of regulatory T cells in vivo. In the experiments, NSG mice were intravenously injected with either PI3K ARC (triangle, n=10) or INX201 (circle, n=8) at 5 mg / kg, along with human PBMC transfer (right panel). In the mouse-only groups, mice were intraperitoneally stimulated with 0.5 mg / kg LPS 18 hours after PBMC injection (for the INX201 and PI3K ARC groups, n=6, left panel). Blood samples were processed on day 28. The percentage change of Tregs is shown. Statistical analysis was performed by Student's t-test. Data are presented as mean ± SEM. ns- no statistical significance.

[0400] GLUT3 ARC treatment reduced human cytokine responses in vivo. A classic hallmark of GVHD or xenogeneic GvHD disease progression is the massive production of pro-inflammatory cytokines, which can be measured in plasma. Based on this, we evaluated whether GLUT3 ARC might affect the hematopoietic cytokine response on day 7. We found that, compared to INX201 Ab alone, GLUT3 ARC reduced GMCSF, CCL4, and IL5 levels by approximately 1 / 2, and CXCL9 levels by approximately 1 / 3. Figure 20 ).

[0401] More specifically, Figure 20 The experiments clearly demonstrated that GLUT3 ARC reduced the inflammatory cytokine response in xenogeneic GvHD. In these experiments, NSG mice were intravenously injected with either PI3K ARC (triangular, n=10) or INX201 (circular, n=8) at 5 mg / kg, along with human PBMC transfer. Changes in plasma human cytokine levels on day 7 are shown. Statistical analysis was performed using Student's t-test. Data are expressed as mean ± SEM, **-p<0.01; ****-p<0.0001.

[0402] in conclusion Experiments using a xenogeneic GvHD model further confirmed the ability of the anti-VISTA antibody RNA conjugate (ARC) according to the invention to functionally deliver siRNA into immune cells and knock down the expression of target mRNAs therein, both in vitro and in vivo. Specifically, experimental data showed that: • PI3K and GLUT3 ARC-INX201 antibody RNA conjugates delivered to VISTA+ cells via GLUT3 or PIK3CA siRNA are functional in vitro. ○ Reduces T cell cytokine response ○ Reduces the CD69 / CD25 activation profile of T cells • GLUT3 and PI3K ARC are functional in vivo ○ GLUT3 treatment reduced the amount of [something] in the body (Alien GvHD) Human cytokine response ○ PI3K ARC treatment reduced human cytokine response in vivo. ○ PI3K ARC treatment reduced T cell proliferation in vivo. ○ PI3K ARC does not reduce the percentage of regulatory T cells in the body. Therapeutic applications of ARC or ANC in this invention To the applicant's knowledge, no specific targeting of immune cells and delivery of one or more nucleic acids that regulate the expression and / or activity of specific immunomodulatory agents and / or immunomodulatory pathways expressed by specific immune cells to ARC or ANC within immune cells has been described to date. Therefore, this invention provides a novel platform for treating diseases involving specific immune cell types without toxicity to untargeted immune cell types or other non-immune cell types.

[0403] Potential indications for ARC as a therapeutic or preventative agent include, for example, autoimmune and inflammatory diseases, such as those with significant myeloid and / or T-cell components (based on the location of VISTA expression, thus indicating the site where the drug will be specifically delivered). Potential indications also include diseases where limiting the activation and growth of autoreactive effector T cells may be beneficial in controlling / reducing the disease. Our targeting technology can help eliminate in-target toxicity associated with target expression in non-immune tissues; it can help target targets that were previously untreatable due to their overly broad expression profile or the localization of the target (transcription factor). Specifically, rheumatoid arthritis, colitis, or systemic lupus erythematosus include autoimmune indications suitable for ARC therapy.

[0404] Many cancer indications can also potentially be treated with the ARC of this invention because, similar to the CD39 knockdown method illustrated herein, siRNA or ASO according to the invention can be administered to act on targets that block or inhibit anti-tumor responses in the host, 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). Removing or inhibiting this blockade will enable the host to more effectively activate the immune system and innate anti-tumor immunity.

[0405] in conclusion In the subject patent application, we have demonstrated that: 1) We can conjugate siRNA or ASO with anti-VISTA Mab and efficiently deliver RNA (e.g., siRNA and ASO) to target cells via VISTA binding and internalization; 2) Compared to free INX201 Mab, ARC exhibits similar binding and internalization properties; 3) Exemplary siRNA payloads specific to immunologically relevant targets retain their function when delivered to target cells in combination with anti-VISTA Mab conjugates; 4) siRNA conjugated to anti-VISTA Mab INX201 promotes the accumulation and function of siRNA in K562-VISTA cells; 5) The siRNA conjugated to anti-VISTA Mab INX201 is functional in human PBMCs and T cells and effectively reduces proliferation and cytokine production (siRNAs for five targets are described in the subject examples); 6) CD39 ASO conjugated with anti-VISTA Mab INX201 functions in human PBMCs, i.e., it provides increased cytokine production, demonstrating the potential of the ARC of the present invention for cancer treatment; 7) Exemplary PI3K and GLUT3 ARC-INX201 antibody-RNA conjugates delivered to VISTA+ cells via GLUT3 or PIK3CA siRNA delivery are functional in vitro, namely reducing T cell cytokine responses and decreasing the T cell CD69 / CD25 activation profile; and 8) Exemplary GLUT3 and PI3K ARC are functional in vivo. For example, GLUT3 treatment reduces in vivo (xenogeneic GvHD) human cytokine response, PI3K ARC treatment reduces in vivo human cytokine response, PI3K ARC treatment reduces in vivo T cell proliferation, and PI3K ARC does not reduce the percentage of regulatory T cells in vivo.

[0406] Other proof-of-concept studies While the applicant has demonstrated the suitability of ARC or ANC comprising an exemplary anti-VISTA antibody and an siRNA payload targeting GLUT3 or PIK3CA as in vitro and in vivo therapeutic agents in GVHD models, the ARC or ANC of the present invention has broader applicability. This can be confirmed in other experiments such as the following: • Optimize payload sequences and test other targets • ARC conjugation with Cys via maleimide or bromoacetimide reactive groups • Use other in vivo models to generate functional data Application Appendix Sequences not entered into the ST.26 XML file due to sequence length Table A below lists the contents of this application. Figure 14 And the U.S. provisional priority applications (U.S. application numbers 63 / 506,177 and 63 / 611,302, mentioned in the Relevant Applications section on page 1 above, all of which are incorporated herein by reference in their entirety) Figure 14 The sequence provided was not included in the 1143260_008613_SL.xml file submitted with this submission due to the length of the sequence.

[0407] Appendix 1 Notes • Excluding signal sequences • Individuals with the INXLA (L234A / L235A / E269R / K322A) Fc silent mutation (IgG1 from Uniprot P01857) ○ Mutations are shown in blue. • Human κ (from Uniprot P01834) • IgG1 and κ constant regions are shown in bold. Antibody name: INX201 Light chain: >INX201_κ_LC DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNIYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C Heavy chain: >INX201_INXLALA_HC QVQLVQSGAEVKKPGASVKVSCKASGYTFANYLIGWVRQAPGQRLEWMGDIYPGGGFISYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRFDYGGYFFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHT C PP C PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHRDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FAB Name: INX201 FAB >INX201_FAB_HC QVQLVQSGAEVKKPGASVKVSCKASGYTFANYLIGWVRQAPGQRLEWMGDIYPGGGFISYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRFDYGGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC >INX201_FAB_LC DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNIYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Appendix 2

[0408] Appendix 3 Notes • Excluding signal sequences • Individuals with the INXLA (L234A / L235A / E269R / K322A) Fc silent mutation (IgG1 from Uniprot P01857) ○ Mutations are shown in blue. • Human κ (from Uniprot P01834) • IgG1 and κ constant regions are shown in bold. Antibody name: INX201 Light chain: >INX201_κ_LC DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNIYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C Heavy chain: >INX201_INXLALA_HC QVQLVQSGAEVKKPGASVKVSCKASGYTFANYLIGWVRQAPGQRLEWMGDIYPGGGFISYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRFDYGGYFFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHT C PP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHRDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FAB Name: INX201 FAB >INX201_FAB_HC QVQLVQSGAEVKKPGASVKVSCKASGYTFANYLIGWVRQAPGQRLEWMGDIYPGGGFISYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARRFDYGGYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC >INX201_FAB_LC DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNIYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Appendix 4

Claims

1. An antibody-RNA or antibody-nucleic acid conjugate ("ARC" or "ANC") comprising (i) an antibody or antibody fragment that binds to an antigen specifically or primarily expressed by one or more immune cell types; directly or indirectly conjugated to (ii) one or more nucleic acids composed of wild-type or modified nucleotides, preferably RNA or DNA oligonucleotides (one or more "payloads") that specifically bind to a target gene, optionally an immunomodulatory gene, or RNA encoded by an immune cell; and optionally (iii) a cleavable or non-cleavable adapter or linker, such as a peptide between the (i) antibody or antibody fragment and the (ii) one or more nucleic acids; wherein such ARC or ANC is internalized by an immune cell expressing an antigen bound by the (i) antibody or antibody fragment and provides release of the (ii) one or more nucleic acids into the immune cell; thereby optionally modulating the expression and / or function of a targeted immunomodulator.

2. The ARC or ANC of claim 1, wherein the one or more payloads comprise one or more modified nucleotides, optionally at least one nucleotide modified with phosphonate and / or ribose, which facilitates direct or indirect linking of the one or more payloads to the antibody or antibody fragment, optionally via a peptide linker, further optionally via a cleavable or incleavable linker or adaptor, such as a peptide between the (i) antibody or antibody fragment and (ii) the payload.

3. The ARC or ANC as claimed in any of the preceding claims, wherein the payload is directly or indirectly conjugated to the antibody or antibody fragment via a reactive amine, the reactive amine optionally being contained on a lysine residue on the antibody or antibody fragment and / or the (i) antibody or antibody fragment being linked to (ii) one or more peptides of the payload, and / or the payload comprising a 3'-sense chain Cy5 tag and a 5'-amine on the antisense chain.

4. The ARC or ANC as claimed in any of the preceding claims, wherein the antibody or antibody fragment binds to VISTA, preferably human VISTA.

5. The ARC or ANC as claimed in any of the preceding claims, wherein the antibody or antibody fragment binds to VISTA, preferably human VISTA, and comprises the same VH and VL CDR as any anti-human VISTA antibody comprising the sequences in FIG14 or Appendix 1 or Appendix 3.

6. The ARC or ANC as claimed in any of the preceding claims, wherein the antibody or antibody fragment binds to VISTA, preferably human VISTA, and comprises the same VH and / or VL regions and CDR as: any anti-human VISTA antibody comprising the VH and / or VL sequences in FIG14, 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 regions of any anti-human VISTA antibody comprising the VH and / or VL sequences in FIG14; or an antibody or antibody fragment comprising the VH and / or VL sequences in Appendix 1 or Appendix 3; and the antibody or antibody fragment optionally comprises a polypeptide with a constant domain of IgG1, IgG2, IgG3, or IgG4, further optionally comprising a polypeptide with a constant domain of IgG1, and even more optionally comprising a polypeptide with a constant domain of IgG1 having the sequences contained in Appendix 1 or 3.

7. The ARC or ANC as claimed in any of the preceding 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. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises one or more of short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterologous nuclear RNA (hnRNA).

9. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises lengths of about 10 to about 5000, 10 to about 4000, 10 to about 3000, 10 to about 2000, 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 2 3 or about 20 to about 22 nucleotides of polynucleotide molecules; or the ARC or ANC comprises polynucleotide molecules of 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.

10. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises a first polynucleotide and a second polynucleotide, optionally wherein the first polynucleotide is a sense strand or a guest strand and / or the second polynucleotide is an antisense strand or a guide strand.

11. The ARC or ANC as described in any of the preceding claims, wherein the ARC or ANC comprises siRNA, tRNA, rRNA, or mRNA.

12. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises, is encapsulated in or conjugated with lipid nanoparticles.

13. The ARC or ANC as described in any of the preceding claims, wherein at least one payload is an immunomodulatory agent that targets a cytokine, chemokine, interleukin, interferon, tumor necrosis factor, or receptor selected from any of the preceding claims.

14. The ARC or ANC as claimed in any of the preceding claims, wherein the payload targets and encodes an RNA or DNA sequence of 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 α (PIK3CA), RAR-associated orphan receptor C (RORC), or any molecule having a sequence identified in Figure 1 or Figure 2.

15. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises an siRNA payload that targets RNA or DNA encoding GLUT3 or PIK3CA, optionally a sequence in Table 11, Appendix 2 or Appendix 4.

16. The ARC or ANC as described in any of the preceding claims, wherein the antibody or antibody fragment binds to at least one immune cell, said immune cell being 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, mucosa-associated constant 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 cells. (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 ARC or ANC as claimed in any of the preceding claims, wherein the antibody or antibody fragment binds to myeloid cells and / or T cells.

18. The ARC or ANC as claimed in any of the preceding claims, wherein the antibody or antibody fragment binds to T cells or T cell progenitor cells or NK cells.

19. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises at least one nucleic acid payload, optionally RNA, further optionally siRNA or antisense RNA, said nucleic acid payload binding to a gene or nucleic acid encoding an antigen selected from the group consisting of: (1) 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 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, ADAM 15, ADAM 17 / T ACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7, α-L-antitrypsin, α-V / β-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, artesunate, anti-Id, ASPARTIC, atrial natriuretic factor, av / 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 osteogen, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-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, dermal glycosides, 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.CCL 14, CCL15, CCL16, CCL1 7. 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, CD 15. 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-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, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay accelerator factor, des(l-3)-IGF-I (Brain IGF-1), Dhh, Digoxin, DNAM-1, DNase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, Endothelin receptor, Enkephalinase, eNOS, Eot, eotaxinl, EpCAM, Ephrin B2 / EphB4EPO, 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, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-Stimulating Hormone, Fractal Chemokines, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, 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-α1, GFR-α2, GFR-α3, GITR, glucagon, Glut 4, 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, heparinase, 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 gp120, HIV IIIB gp120 V3 ring, 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)-α, INF-β,INF-γ, Inhibin, iNOS, Insulin A chain, Insulin B chain, Insulin-like growth factor 1, Integrin α2, Integrin α3, Integrin α4, Integrin α4 / β1, Integrin α4 / β7, Integrin α5 (αV), Integrin α5 / β1, Integrin α5 / β3, Integrin α6, Integrin β1, Integrin β2, Interferon γ, IP-10, 1-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, 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, luteinizing hormone, lymphotoxin β receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinases, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-α, 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, Mucl, MUC18, Müllerian canal inhibitor, Mug, Musk, NAIP, NAP, NCAD, N-cadherin, NCA 90. NCAM, Enkephalin, Neurotrophic Factor-3, Neurotrophic Factor-4 or Neurotrophic Factor-6, Neuro-rank protein, Neuronal Growth Factor (NGF), NGFR, NGF-β, 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, PGJ2, PIN, PLA2, Placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin, Pro-relaxinProtein 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 receptors (e.g., T cell receptor α / β), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, TGF-β panspecific, TGF-β RI (ALK-5), TGF-β RII, TGF-β Rllb, TGF-β RIII, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, thrombin, thymic Ck-1, thyroid-stimulating hormone, Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-α, TNF-αβ, TNF-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10A (TRAIL R2) DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcRl, LIT, TRID), TNFRSF10D (TRAIL R4DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSFllB (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEMATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF 18 (GITRAITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSFIA (TNF RI CD120a, p55-60),TNFRSFIB (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF 5 (CD40 p50), TNFRSF6 (FasApo-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 ligand, TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TLIA / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSFIA (TNF-α connexin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfer receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigens expressing Lewis Y-related carbohydrates, 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 antigen, 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 3) and hormone receptor; or (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, 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, TNFα, TGFβ, cytokine receptor, IL-2R, chemokines, chemokine receptors, growth factors, 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,CD9 3、CD94、CD95、CD96、CD97、CD98、CD99、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD108、CD109、CD110、CD111、CD112、CD114、CD 115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CDw121b、CD122、CD123、CD124、CD125、CD126、CD127、CDw128、CD129、CD130、CDw131、C D132、CD133、CD134、CD135、CDw136、CDw137、CD138、CD139、CD140a、b、CD141、CD142、CD143、CD144、CD145、CD146、CD147、CD148、CD149、CD150、C D151, CD152, CD153, CD154, CD155, CD156b, CD157, CD158, CD158a, CD159a, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167a, CD 168、CD169、CD170、CD171、CD172a、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD183、CD184、CD195、CDw197、CD 200、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) 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 one of SOCS1, CD39, Cbl, or PTPN22; or (5) Glut1, PI3K, BTK, TNF, RORC, CD45 or CD39; or (6) 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 and PVR.

20. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises a nucleic acid payload, optionally RNA, further optionally siRNA or antisense RNA having a payload sequence selected from those having the sequences described in Table 11, Appendix 2 or Appendix 4; or the ARC or ANC comprises an INX201 ARC having the amino acid sequence and payload sequence described in Appendix 2, or the ARC or ANC comprises an INX201 ARC having the amino acid sequence and payload sequence described in Appendix 4.

21. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC comprises at least two different RNA payloads that target the same or different immune regulatory genes or mRNAs, optionally as claimed in any of the preceding claims, for immune targets.

22. The ARC or ANC as claimed in any of the preceding claims, wherein the nucleic acid, optionally the RNA payload, is linked to the antibody or antibody fragment via a cleavable or non-cleavable adapter.

23. The ARC or ANC as described in any of the preceding claims, wherein the ARC or ANC is used to deliver one or more gene-editing nucleic acids (e.g., CRISPR guide RNA (gRNA or sgRNA)) and optionally a CRISPR-associated endonuclease or a nucleic acid encoding a CRISPR-associated endonuclease.

24. The ARC or ANC as claimed in any of the preceding claims, wherein the ARC or ANC has 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, 6 weeks or longer.

25. The ARC or ANC as described in any of the preceding claims, wherein the ARC or ANC does not cause any apparent toxicity to non-target cells.

26. A composition comprising ARC or ANC as described in any one of the preceding 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, the method comprising administering to a subject in need an ARC or ANC as described in any of the preceding claims, or a composition containing said ARC or ANC.

28. The method of claim 27, wherein the treatment or prevention is for treating a neoplastic, proliferative, neurodegenerative, neuroinflammatory, infectious, autoimmune, allergic, or inflammatory condition, or pathological symptoms associated with any of said conditions.

29. The method of claim 27, wherein the treatment or prevention is for treating or preventing autoimmune diseases, such as diseases involving myeloid cells or T cells.

30. The method of claim 27, wherein the treatment or prevention is for treating or preventing a neoplastic, proliferative, neurodegenerative, neuroinflammatory, infectious, autoimmune, or inflammatory disease, and / or preventing or suppressing at least one pathological symptom associated therewith.

31. The method of claim 27, wherein the treatment or prevention is for the treatment or prevention of one or more autoimmune diseases selected from: acromegaly, acquired aplastic anemia, acquired hemophilia, primary agammaglobulinemia, alopecia areata, ankylosing spondylitis (AS), anti-NMDA receptor encephalitis, antiphospholipid syndrome (APS) | catastrophic antiphospholipid syndrome (CAPS) / Asherson syndrome, arteriosclerosis, autoimmune Addison's disease (AAD), autoimmune autonomic ganglion disease (AAG) / autoimmune autonomic dysfunction | autoimmune 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), autoimmune polyglandular syndrome, types I, II, and III (APS type 1, APS type 2, APS type 3, APS type 4, APS type 5, APS type 6, APS type 7, APS type 8, APS type 9, APS type 1, APS type 1, APS type 1, APS type 2, APS type 3 ... Type 3, APECED), autoimmune progesterone dermatitis, autoimmune retinopathy (AIR), autoimmune sudden sensorineural hearing loss (SNHL), Barlow's disease, Behcet's disease, shotgun-like chorioretinopathy / shotgun-like uveitis, bullous pemphigoid, Kassman's disease, celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic urticaria (CU), Chag-Strauss syndrome / eosinophilic granulomatous polyangiitis (EGPA), Cogan syndrome, cold agglutinin disease, CREST syndrome | localized cutaneous systemic sclerosis, Crohn's disease (CD), Canada-Kronka syndrome (CSS), cryptogenic organizing pneumonia (COP), herpetiform dermatitis, dermatomyositis, type 1 diabetes, discoid lupus, Dresler syndrome / post-myocardial infarction syndrome / pericardiotomy Postoperative syndrome, eczema / atopic dermatitis, endometriosis, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, primary mixed cryoglobulinemia, Evans syndrome, fibrotic alveolitis / idiopathic pulmonary fibrosis (IPF), giant cell arteritis / temporal arteritis / Houghton's disease, giant cell myocarditis, glomerulonephritis, Goodpassch syndrome / anti-GBM disease / anti-TBM disease, granulomatous polyangiitis (GPA) / Wegener's granulomatosis, Graves' disease / thyroid eye disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis / chronic lymphocytic thyroiditis / autoimmune thyroiditis, allergic purpura / IgA vasculitis, hidradenitis suppurativa, Hearst's disease / acute hemorrhagic leukoencephalitis (AHLE), hypogammaglobulinemia, IgA nephropathy / Berge's disease, immune-mediated necrotizing myopathy (IMNM).Immune thrombocytopenic purpura (ITP) / Autoimmune thrombocytopenic purpura / Autoimmune thrombocytopenic purpura, 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-Eton myasthenic syndrome (LEMS), leukocytic clotting 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 diseases Autoimmune hypophysitis, Meniere's disease, microscopic polyangiitis (MPA) / ANCA-associated vasculitis, mixed connective tissue disease (MCTD), keratitis erosiveus, Mucha-Habermann disease, multifocal motor neuropathy, multiple sclerosis (MS), myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS), myasthenia gravis (MG), narcolepsy, neuromyelitis optica / Devrwick disease, ocular cicatricial pemphigoid, oculoclonus-myoclonus syndrome (OMS), relapsing rheumatism, paraneoplastic cerebellar degeneration, paraneoplastic pemphigus, Paroxysmal-Rossian syndrome (PRS) / hemispheric atrophy (HFA) / progressive hemifacial atrophy, paroxysmal nocturnal hemoglobinuria (PNH) Peripheral uveitis / paris plana inflammation, PANS / PANDAS, Parsenaeus-Turner syndrome, pemphigus gestationis / herpes gestationis, pemphigus foliaceus, pemphigus vulgaris, pernicious anemia, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, postural 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 aplasia (PRCA), pyoderma gangrenosa, Rasmussen encephalitis, Raynaud's syndrome / phenomenon, reactive arthritis / Reiter's syndrome, reflex sympathetic dystrophy Benign regional pain syndrome (RSD) / complex regional pain syndrome (CRPS), recurrent polychondritis, restless legs syndrome (RLS) / Willis-Ekbom disease, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome / autoimmune polyendocrine syndrome type II, scleritis, scleroderma, sclerosing mesenteric / mesenteric panniculitis, creeping choroidal lesions, Sjögren's syndrome, stiff-person syndrome (SPS), small fiber sensory neuropathy, systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), subacute cutaneous lupus erythematosus, Sussac syndrome, Siddenham's chorea, sympathetic ophthalmia, Takayasu arteritis (vasculitis), testicular autoimmune diseases (vasculitis, orchitis)Tolosa-Hunter syndrome, transverse myelitis (TM), tubulointerstitial nephritis-uveitis syndrome (TINU), ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), pre / intermediate / post-uveitis, vasculitis, VEXAS syndrome, vitiligo, and Vogt-Koyanagi-Harada syndrome (VKH), and / or prevention or suppression of at least one associated pathological symptom.

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

33. The method of claim 27, wherein the treatment or prevention is for treating 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 preventing or inhibiting at least one pathological symptom associated therewith.

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

35. The method of claim 27, wherein the treatment or prevention is for treating cancer or preventing cancer recurrence, and / or suppressing at least one pathological symptom associated with a specific immune cell type.

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

37. The method of claim 27, wherein the treatment or prevention is for treating hematologic malignancies and / or preventing or suppressing at least one pathological symptom associated therewith.

38. The method of 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 hematologic malignancy or metastatic hematologic malignancy.

39. The method of claim 27, wherein the treatment or prevention is for a solid tumor selected from anal cancer, appendix cancer, biliary tract cancer (i.e., bile duct cancer), 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 adenoma, prostate cancer, rectal cancer, skin cancer, gastric cancer, testicular cancer, laryngeal cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvar cancer, and / or to prevent or suppress at least one pathological symptom associated therewith.

40. The method of claim 27, wherein the treatment or prevention is for hematologic malignancies, optionally leukemia, lymphoma, myeloma, non-Hodgkin lymphoma, or Hodgkin lymphoma. In some cases, hematologic 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 macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, and Burkitt lymphoma. Non-Burkita high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-cell lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma or lymphomatoid granulomatosis, and / or prevention or suppression of at least one pathological symptom associated therewith.

41. The method of claim 27, wherein the treatment or prevention is for hematologic malignancies selected from 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 macroglobulinemia, multiple myeloma, extranodal malignancy... Marginal zone B-cell lymphoma, nodal-marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, or lymphomatoid granulomatosis. In some cases, hematologic malignancies are relapsed or refractory hematologic malignancies or metastatic hematologic malignancies, and / or prevention or suppression of at least one associated pathological symptom.

42. The method of claim 27, wherein the treatment or prevention is for treating 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 thyroiditis, Sjögren's disease, asthma, type 2 diabetes and autoimmune type 1 diabetes, and / or preventing or suppressing at least one pathological symptom associated therewith.

43. The method of claim 27, wherein the treatment or prevention is for treating cancer, and the ARC or ANC comprises a nucleic acid that regulates or blocks the expression of any one 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 of claim 27, wherein the treatment or prevention is for inhibiting or treating immune senescence associated with disease or aging, and the ARC or ANC optionally comprises nucleic acid, further optionally comprising ASO or siRNA that regulates or blocks the expression of any one 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.

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