Tolerizing immune-modifying nanoparticles to overcome immunogenicity of therapeutic vectors and proteins

Abstract

The present application is generally directed to tolerizing immune-mediating particles comprising gene therapy vector antigens for use in combination with gene therapy regimens to reduce immunogenicity to the gene therapy vector antigens and / or the transgene protein product expressed by the vector.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 336,754, filed April 29, 2022, and U.S. Provisional Patent Application No. 63 / 343,797, filed May 19, 2022, which are incorporated by reference in their entireties herein.

[0002] The present application is generally directed to tolerizing immune-mediating particles comprising gene therapy vector antigens for use in combination with gene therapy regimens to reduce immunogenicity to the gene therapy vector antigens and the transgene protein product expressed by the vector. [Background technology]

[0003] Advances in the design and development of gene delivery vectors (e.g., retroviral, lentiviral, adenoviral, and adeno-associated viral (AAV) vectors) have led to the emergence of gene therapy as an attractive treatment option for many diseases and conditions, including rare and inherited genetic disorders, autoimmune disorders, neurodegenerative conditions, and cancer. Gene therapy relies on vector-mediated delivery of exogenous DNA that encodes therapeutic proteins such as enzymes, cytokines, and antibodies. 1~6 The goal of gene therapy is to allow sustained, tissue-specific production of a therapeutic protein for long-term therapeutic efficacy.

[0004] In addition to producing proteins and enzymes for the treatment of rare and inherited genetic disorders (e.g., hemophilia, Pompe disease, Fabry disease, and mucopolysaccharidoses), gene therapy vectors have been used for tissue-specific expression of autoantigens and tolerance cytokines / chemokines for the treatment of autoimmune diseases, cancer vaccines, and the production of anti-tumor proteins, cytokines, chemokines, and antibodies for the treatment of cancer. 3、4、7 In addition, viral vectors called oncolytic viruses have been engineered to lyse tumor cells and induce antitumor immune responses for the treatment of cancer. 8、9.

[0005] Gene therapy vectors in use and currently under development for gene therapy applications are typically modified and / or engineered versions of naturally occurring retroviruses, lentiviruses, adenoviruses, and AAV. 10 , and several synthetic vectors have also been described in the literature. 11 To achieve sustained therapeutic efficacy, viral vectors must be periodically re-administered. 19、20 .

[0006] The immunogenicity of gene therapy vectors and the therapeutic proteins expressed from the vectors presents a significant clinical challenge for the periodic re-administration required for sustained therapeutic benefit, as both the vectors and the therapeutic proteins produced by the vectors are rapidly excreted after administration. In addition, a significant proportion of patients likely to benefit from gene therapy exhibit pre-existing immunity to gene therapy vectors and are ineligible for treatment. 12~14 Patients without pre-existing neutralizing antibodies are also subject to the immunogenicity of the vector and therapeutic proteins, as the initial vector administration induces an immune response against the vector proteins, thus preventing any re-administration required for sustained therapeutic benefit.

[0007] Current strategies aimed at preventing the immunogenicity of viral vectors rely on the concomitant administration of immunosuppressive regimens that are associated with significant side effects, including increased risk of infection and mortality. 15 . Summary of the Invention

[0008] Induction of antigen-specific T cell tolerance to gene therapy vectors and the transgene protein products produced by the vectors using TIMPs could potentially overcome the immunogenicity problems associated with gene therapy, enable re-administration, and result in improved therapeutic efficacy.

[0009] The present disclosure describes compositions and methods for inducing antigen-specific tolerance to gene therapy vectors and transgene protein products produced by the vectors. Provided herein are compositions comprising negatively charged carrier particles encapsulating an antigen, where the antigen is one or more gene therapy vector antigens and / or portions thereof, or a combination of gene therapy vector antigens or portions thereof. In various embodiments, the antigen is a therapeutic protein produced by the gene therapy vector and / or portions thereof, or one or more antigenic epitopes thereof. In various embodiments, the carrier particles comprise a polymer and have a negative zeta potential. In various embodiments, the polymer is a biodegradable polymer.

[0010] In various embodiments, the particles comprise a polymer selected from the group consisting of polyglycolic acid (PGA), polylactic acid (PLA), polysebacic acid (PSA), poly(lactic-co-glycolic) (PLGA), poly(lactic-co-sebacic) acid (PLSA), poly(glycolic-co-sebacic) acid (PGSA), polypropylene sulfide, poly(caprolactone), chitosan, a polysaccharide, or a lipid, polystyrene, diamond, liposome, PEG, cyclodextran, lipid, or a metal, such as iron (Fe), zinc (Zn), cadmium (Cd), gold, or silver, or a combination thereof.

[0011] In various embodiments, the polymer is a copolymer. In various embodiments, the copolymer has various molar ratios of the constituent polymers. In various embodiments, the molar ratio is 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0.

[0012] In various embodiments, the carrier particles comprise poly(lactic-co-glycolic acid) (PLGA). In various embodiments, the particles comprise about 50:50, about 80:20 to about 100:0 polylactic acid:polyglycolic acid, or about 50:50, about 80:20 to about 100:0 polyglycolic acid:polylactic acid. In various embodiments, the particles comprise 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particles comprise polylactic acid:polyglycolic acid of about 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 (including all values ​​and ranges therebetween).

[0013] In various embodiments, the particles have a negative zeta potential. In various embodiments, the zeta potential of the particles is about -100 mV to about 0 mV, about -100 mV to about -25 mV, about -100 to about -30 mV, about -80 mV to about -30 mV, about -75 mV to about -30 mV, about -70 mV to about -30 mV, about -75 to about -35 mV, about -70 to about -25 mV, about -60 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -30 mV. In various embodiments, the zeta potential is about -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV, including all values ​​and ranges therein. In various embodiments, the carrier particles have a negative zeta potential of -30mV to -80mV. In various embodiments, the carrier particles have a negative zeta potential of -30mV to -60mV. In various embodiments, the negative zeta potential is achieved by surface functionalization of the carrier particles. In various embodiments, the surface functionalization is carboxylation.

[0014] In various embodiments, the size, or diameter, of the carrier particles is from 0.05 μm to about 10 μm. In various embodiments, the diameter of the carrier particles is from 0.1 μm to about 10 μm. In various embodiments, the diameter of the carrier particles is from 0.1 μm to about 5 μm. In various embodiments, the diameter of the carrier particles is from 0.1 μm to about 3 μm. In various embodiments, the diameter of the carrier particles is from 0.3 μm to about 5 μm. In various embodiments, the diameter of the carrier particles is from about 0.3 μm to about 3 μm. In various embodiments, the diameter of the carrier particles is from about 0.3 μm to about 1 μm. In various embodiments, the diameter of the carrier particles is from about 0.4 μm to about 1 μm. In various embodiments, the carrier particles have a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 100-1,500 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm. In various embodiments, the carrier particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therein). In various embodiments, the negatively charged particles have a diameter between 400 nm and 800 nm.In various embodiments, the negatively charged particles have a diameter between 350 nm and 800 nm.

[0015] In various embodiments, the carrier particles have a homogeneous size distribution, with at least 90% of the particles having a diameter of 0.05 μm to about 10 μm, 0.1 μm to about 10 μm, 0.1 μm to about 5 μm, 0.1 μm to about 3 μm, 0.3 μm to about 5 μm, or 0.3 μm to about 3 μm, including all values ​​and ranges therein. In various embodiments, the carrier particles have a homogenous size distribution, with at least 90% of the particles having a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm (including all values ​​and ranges therein). In various embodiments, the carrier particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm, including all values ​​and ranges therein. In various embodiments, the carrier particles have a homogeneous size distribution, with at least 50% of the particles having a diameter of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, and about 0.3 μm to about 3 μm, including all values ​​and ranges therein. In various embodiments, the particles have a homogeneous size distribution, with at least 50% of the particles having a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm (including all values ​​and ranges therein). In various embodiments, the carrier particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therein).

[0016] In various embodiments, the carrier particles have a homogeneous size distribution, with at least 10% of the particles having a diameter of about 0.05 μm to about 10 μm, about 0.1 μm to about 10 μm, about 0.1 μm to about 5 μm, about 0.1 μm to about 3 μm, about 0.3 μm to about 5 μm, and about 0.3 μm to about 3 μm (including all values ​​and ranges therein). In various embodiments, the carrier particles have a homogenous size distribution, with at least 10% of the particles having a diameter of about 100-10,000 nm, about 100-5,000 nm, about 100-3,000 nm, about 100-2,000 nm, about 300-5,000 nm, about 300-3,000 nm, about 300-1,000 nm, about 300-800 nm, about 400-800 nm, or about 200-700 nm (including all values ​​and ranges therein). In various embodiments, the carrier particles have a diameter of about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therein).

[0017] In various embodiments, the particles encapsulate an antigen and have a negative zeta potential of -100 mV to 0 mV, and the particles are 100 to 1000 nm in diameter. In various embodiments, the particles encapsulate one or more antigens, including one or more gene therapy vector antigens, portions thereof, or combinations thereof, and the particles are 400 to 800 nm in size, and the particles have a negative zeta potential of -30 mV to -80 mV.

[0018] In various embodiments, the particles encapsulate an antigen and have a negative zeta potential of -100 mV to 0 mV, and the particles are 100 to 1000 nm in diameter. In various embodiments, the particles encapsulate one or more antigens including one or more protein therapeutics produced by a gene therapy vector, a portion or combination thereof, the particles are 400 to 800 nm in size, and the particles have a negative zeta potential of -30 mV to -80 mV.

[0019] In various embodiments, the particles encapsulate one or more gene therapy vector antigens, portions thereof, or combinations thereof, and one or more protein therapeutics produced by the gene therapy vector, portions thereof, or combinations thereof. In various embodiments, the antigens include one or more proteins, peptides, or antigenic epitopes thereof.

[0020] Also contemplated herein are compositions comprising liposomes encapsulating one or more gene therapy vector antigens, portions or combinations thereof.

[0021] In another embodiment, the present disclosure provides a composition comprising a liposome encapsulating a therapeutic protein produced by a gene therapy vector and / or a portion thereof.

[0022] In various embodiments, the liposomes are negatively charged. In various embodiments, the particles have a negative zeta potential. In various embodiments, the negative zeta potential is -100 mV to 0 mV. In various embodiments, the negatively charged liposomes have a zeta potential of about -100 mV to about 0 mV, about -100 mV to about -25 mV, about -100 to about -30 mV, about -80 mV to about -30 mV, about -75 mV to about -30 mV, about -70 mV to about -30 mV, about -75 to about -35 mV, about -70 to about -25 mV, about -60 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -30 mV. In various embodiments, the zeta potential is about -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV, including all values ​​and ranges therein. In various embodiments, the liposomes have a negative zeta potential of -30mV to -80mV. In various embodiments, the liposomes have a negative zeta potential of -30mV to -60mV.

[0023] In various embodiments, the liposomes are 100-1000 nm, or 300-800 nm, or 400-800 nm, In various embodiments, the liposomes are about 100 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

[0024] In various embodiments, the antigen comprises a gene therapy vector antigen or one or more portions thereof. In various embodiments, the gene therapy vector is a viral vector. In various embodiments, the viral vector is selected from the group consisting of adenovirus, adeno-associated virus (AAV), herpes simplex virus, hepatitis B virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, coxsackievirus, transfusion-transmitted virus, anerosome, human papillomavirus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, cowpea mosaic virus, cowpea chlorotic mottle virus, ground cherries mosaic virus, red clover necrotic mosaic virus, potato virus x, comovirus, chicken anemia virus, or cucumber mosaic virus. In various embodiments, the virus is an oncolytic virus. In various embodiments, the virus is a chimeric virus, a synthetic virus, a mosaic virus, or a pseudotyped virus.

[0025] In various embodiments, the particles encapsulate an antigen, and the antigen is one or more AAV vectors, portions or combinations thereof. In various embodiments, the AAV is selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-12, Anc80, synthetic AAV, combinations or engineered versions thereof. In various embodiments, the antigen is one or more AAV capsid proteins. In various embodiments, the AAV capsid proteins are VP-1, VP-2, and VP-3.

[0026] In various embodiments, the gene therapy vector is a bacterium, a bacteriophage, a yeast, an exosome, or a red blood cell.

[0027] In various embodiments, the gene therapy vector is a therapeutic agent for treating cancer, autoimmune diseases, allergies, cardiovascular diseases, metabolic diseases, diabetes, enzyme deficiencies, protein deficiencies, cystic fibrosis, blood disorders, beta-thalassemia, sickle cell disease, hemophilia A, hemophilia B, lysosomal storage diseases, Fabry disease, Gaucher disease, Pompe disease, Niemann-Pick disease, Tay-Sachs disease, macular degeneration, mucopolysaccharidoses, venous thromboembolism, von Willebrand disease, purpura fulminans, growth hormone deficiency, gangliosidosis, hypoalkaline phosphatasia, cholesterol deficiency ... Sterol ester storage disease, hyperuricemia, Duchenne muscular dystrophy, Huntington's disease, Parkinson's disease, Alzheimer's disease, choroideremia, Stargardt disease, Batten disease, spinocerebellar ataxia, ALS, frontotemporal lobar degeneration, ornithine transcarbamylase deficiency, retinitis pigmentosa, RPE-65 mutation-related disease, epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, spinal muscular atrophy, phenylketonuria (PKU), X-linked myotubular myopathy, Crigler-Najjar syndrome, catecholamine-induced polymorphic ventricles Tachycardia, glycogen storage disease type 1, alpha-mannosidosis, fragile X syndrome, arginase deficiency, X-linked chronic granulomatous disease, adenosine deaminase deficiency, Leber congenital amaurosis, lipoprotein lipase deficiency, cerebral adrenoleukodystrophy, metachromatic leukodystrophy, Fanconi anemia, color vision disorders, scleroderma, osteogenesis imperfecta, coronary artery disease, tyrosinemia, peripheral sensory neuropathy, optic neuropathy, coronary artery disease, respiratory syncytial virus (RSV)-mediated lower respiratory tract disease, Danon disease, severe leukocyte adhesion deficiency, pyruvate kinase deficiency, Charcot-Marie-Tooth disease, Wiskott-Aldrich syndrome, alpha-synuclein tauopathy, refractory angina due to myocardial ischemia, myotonic dystrophy type I, claudication, peripheral arterial disease, methylmalonic acidemia, sucrase-isomaltase deficiency, Niemann-Pick disease type B, α1-PI deficiency, hereditary angioedema, fibrinogen deficiency, factor VIIa deficiency, factor X deficiency, factor XI deficiency, factor XII deficiency, protein C deficiency, antithrombin III deficiency, MPS I, MPS II, MPS III, MPS IV, MPS VI, MPS VII, MPSIt is used to treat IX, mineralization / ossification disorders, ENPP1 deficiency, ENPP3 deficiency, ABCC6 deficiency, aromatic L-amino acid decarboxylase deficiency, Angelman syndrome, hyperphenylalaninemia, dementia, and Rett syndrome and Usher syndrome.

[0028] In various embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, Addison's disease, ankylosing spondylitis, alopecia, osteoarthritis, psoriatic arthritis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, Raynaud's syndrome, Behcet's syndrome, Sjogren's syndrome, autoimmune uveitis, Eaton-Lambert disease, autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus, and / or rheumatoid arthritis. , neuromyelitis optica, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, myasthenia gravis, celiac disease, ulcerative colitis, Crohn's disease, erythema nodosum, glomerulonephritis, Goodpasture's syndrome, granulomas, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, Kawasaki disease, mixed connective tissue disease, multifocal motor neuropathy, peripheral biliary cirrhosis, polyangiitis overlap syndrome, scleroderma type 1, sclerosing cholangitis, stiff-man syndrome, Takayasu's arteritis, vitiligo, or granulomatosis with polyangiitis.

[0029] In various embodiments, the allergy is a food allergy. In various embodiments, the allergy is an environmental allergy. In various embodiments, the food allergy is a peanut allergy, a tree nut allergy, a milk allergy, an egg allergy, a fish allergy, a wheat allergy, a celery allergy, or a peach allergy. In various embodiments, the environmental allergy is a pollen allergy, a dust allergy, a pet dander allergy, or a mold allergy. In various embodiments, the pet allergy is a cat allergy or a dog allergy.

[0030] In various embodiments, the gene therapy vector is used to treat cancer, in various embodiments, a cancer selected from the group consisting of brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, melanoma, and mesothelioma.

[0031] In various embodiments, the therapeutic agent produced by the gene therapy vector is a protein, polypeptide, or peptide, hi various embodiments, the protein is a cytokine, chemokine, hormone, growth factor, enzyme, or antibody.

[0032] In various embodiments, the antibody is a monoclonal antibody. In various embodiments, the antibody is a recombinant monoclonal antibody. In various embodiments, the antibody is selected from the group consisting of an antibody protein fusion, an immunoadhesin, a monospecific antibody, a bispecific antibody, a trispecific antibody. In various embodiments, the antibody is selected from the group consisting of an antibody fragment, an antigen-binding fragment (Fab), a single chain variable fragment (scFv). In various embodiments, the antibody is a single domain antibody. In various embodiments, the antibody is IgA, IgD, IgE, IgM, and / or variants thereof. In various embodiments, the antibody is IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p70 , IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL -27b, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL2 5, CCL26, CCL27, CCL28, CXCL1, CXCL2(MCP-1), CXCL3(MIP-1α, CXCL4(MIP-1β, CXCL5(RANTES), CXCL6, CXCL7, C XCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, IFN-β, IFN-γ, T NF-α, TNF-α, TNF-β, TGF-β1, TGF-β2, TGF-β3, LT-β, ​​4-1BBL, APRIL, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22,CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD45RA, CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD161, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207, CD226, CD244, CD267, CD268, CD269, CD355, CD358, CRTH2, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, Integrin, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-10R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL-23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R, Cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1, PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR,LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, T L-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, Targeted to CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains, and / or combinations thereof.

[0033] In various embodiments, the antibody targets one or more immune cells, which in various embodiments are T cells, B cells, NK cells, NK-T cells, monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, basophils, TH1 cells, TH2a cells, Treg cells, Tr1 cells, and Breg cells.

[0034] In various embodiments, the antibody is selected from the group consisting of abciximab, adalimumab, alemtuzumab, avelumab, azetolizumab, basiliximab, bevacizumab, bezlotoxumab, blinatumomab, canakinumab, certolizumab, cetuximab, daclizumab, denosumab, durvalumab, efalizumab, emicizumab, etokimab, golimumab, inoximab, rituxim ... The antibody is selected from the group consisting of pilimumab, ixekizumab, infliximab, natalizumab, nivolumab, olaratumab, omalizumab, ofatimumab, palivizumab, panitumumab, pembrolizumab, ramucirumab, rituximab, tocilizumab, trastuzumab, tremelimumab, secukinumab, ustekinumab, and vedolizumab.

[0035] We have also updated our list of photos and videos. There is nothing wrong with that. The name IL-1α IL-1β IL-2 can be substituted for IL-2. 3、IL-4、FILL-5、FILL-6、FILL-7、FILL-8、FILL-9、FILL-10、FILL-11、FILL-12、FILL-12p70、FILL-13、FILL-14、FILL-15、FILL-16、FILL-16 -17、ART-18、IL-20、IL-21、IL-22、IL-23、IL-24、IL-25、IL-26、IL-27、IL-27b、IL-28、IL-29、IL-30、IL-30 -32、IL-33、IL-35、IL-36、CCL1、CCL2、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9、CCL10、CCL114、CCL11 CCL15, CCL16, CCL17, CCL18, CCL21, CCL21, CCL21 CXCL2(MCP-1)、CXCL3(MIP-1α、CXCL4(MIP-1β、CXCL5(RANTES)、CXCL6、CXCL7、CXCL8、CXCL9、CXCL10、CXCL11、 CXCL12. 、TGF-β2、TGF-β3、LT-β、4-1BBL、APRIL、GITRL、LIGHT、 OX40L、TALL-1、TRAIL TWEAK has been installed in TRANCE.

[0036] In various embodiments, the protein is adrenaline, melatonin, noradrenaline, norepinephrine, triiodthrienine, thyroxine, dopamine, prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin, anti-mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin, antidiuretic hormone, vasopressin, arginine vasopressin, calcitonin, cholecystokinin, corticotropin releasing hormone, cortistatin, enkephalin, endothelin, erythropoietin, follicle stimulating hormone, galanin, gastric inhibitory peptide, gastrin, ghrelin, glucagon, glucagon-like peptide 1, gonadotropin releasing hormone, growth releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, growth The hormone is selected from the group consisting of hormones, inhibin, insulin, insulin-like growth factor, leptin, lipotropin, luteinizing hormone, melanocyte-stimulating hormone, motilin, orexin, osteocalcin, oxytocin, pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, prolactin, prolactin-releasing hormone, relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, vasoactive intestinal peptide, guanylin, uroguanylin, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone, estriol, cortisol, progesterone, calcitriol, and calcisiol.

[0037] In various embodiments, the protein is adrenomedullin, angiopoietin, autocrine cell motility stimulating factor, bone morphogenetic protein, ciliary neurotrophic factor, leukemia inhibitory factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, epidermal growth factor, ephrin A1, ephrin A2, ephrin A3, ephrin A4, ephrin A5, ephrin B1, ephrin B2, ephrin B3, erythropoietin ... fibroblast growth factor 1, fibroblast growth factor 2, fibroblast growth factor 3, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblast growth factor 13, fibroblast growth factor 14, fibroblast growth factor 15, fibroblast growth factor 16, fibroblast growth factor and growth factors selected from the group consisting of neuregulin 17, fibroblast growth factor 18, fibroblast growth factor 19, fibroblast growth factor 20, fibroblast growth factor 21, fibroblast growth factor 22, fibroblast growth factor 23, fetal bovine somatotrophin, glial cell line-derived neurotrophic factor, neurturin, persephin, artemin, growth differentiation factor-9, hepatocyte growth factor, hepatoma-derived growth factor, insulin-like growth factor-1, insulin-like growth factor-2, keratinocyte growth factor, migration stimulating factor, macrophage stimulating protein, myostatin, neuregulin 1, neuregulin 2, neuregulin 3, neuregulin 4, brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, neurotrophin-4, placenta growth factor, platelet-derived growth factor, renalase, T cell growth factor, thrombopoietin, transforming growth factor, transforming growth factor alpha, transforming growth factor beta, tumor necrosis factor-alpha, and vascular endothelial growth factor. In various embodiments, the cytokine is selected from the group consisting of other hematopoietins, Epo, Tpo, Flt-3L, SCF, M-CSF, and MSP.

[0038] In various embodiments, the protein is a thrombolytic agent selected from the group consisting of tissue plasminogen activator, streptokinase, urokinase, anistreplase, reteplase, kabikikinase, tenecteplase, and rokinase.

[0039] In various embodiments, the therapeutic agent produced by the vector is an enzyme, hi various embodiments, the enzyme is selected from the group consisting of gene editing nuclease enzymes, meganucleases, homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases (TALENs), CRISPR-associated nucleases, and CRISPR-Cas9.

[0040] In various embodiments, the Cas protein is selected from the group consisting of Cas 3, Cas8a, Cas5, Cas8b, Cas8c, Cas 9, Cas10d, Csel, Cse2, Csyl, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csxll, Csx10, Csf1, Cas9, Csn2, Cas4, Cas12, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas14, C2c10, Cas12g, Cas12h, Cas12i, Cas12k, C2cr4, C2cr8, C2cr9, Cas13, Cas13a, Cas13b, Cas13c, Cas13d. In various embodiments, the TALEN is a FokI-based, PvuII-based, I-TevI-based, IAniI-based, I-OnuI-based, or MutH-based TALEN. In various embodiments, the enzyme is a nuclease selected from the group consisting of LAGLIDADG (meganuclease), GIY-YIG, His-Cys box, HNH, PD-(D / E)-xK, Vrs-like, megaTAL.

[0041] In various embodiments, the enzyme is imiglucerase, taliglucerase alpha, velaglucerase alpha, beta-glucocerebrosidase, alglucerase, agalsidase beta, agalsidase alpha, siberipase alpha, alpha-L-iduronidase, human iduronate-2-sulfatase, N-sulfoglucosamine sulfohydrolase, elosulfase alpha, galsulfase, alpha-glucosidase (GAA), human alpha mannosidase, factor VIII, factor IX, beta-galactosidase, arginase, dystrophy myotonica protein kinase, ornithine transcarbamylase, NADPH oxidase, NADH dehydrogenase 4, adenosine deaminase, lipoprotein lipase, beta-glucocerebrosidase, myotubularin, arylsulfatase A, DOPA decarboxylase, matrix metallopeptidase 1, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, pyruvate kinase, porphobilinogen deaminase, alkaline phosphatase, sucrase-isomaltase, acid sphingomyelinase, heparanthylsulfase sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, alpha-1 proteinase inhibitor, alpha-1-esterase inhibitor, fibrinogen, factor VIIa, factor X, factor XI, factor XII, protein C, antithrombin III, ectonucleotide pyrophosphatase / phosphodiesterase 1, ectonucleotide pyrophosphatase / phosphodiesterase 3, and aromatic L-amino acid decarboxylase.

[0042] In various embodiments, the enzyme is a protease. In various embodiments, the protease is an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a serine protease, and / or a threonine protease. In various embodiments, the protease is selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, heparinase, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28.

[0043] In various embodiments, the therapeutic agent produced by the vector is a protein. In various embodiments, the protein is a light-activatable channelrhodopsin protein, tumor protein 53, HIV Gag protein, fragile X mental retardation protein, beta globin, fibroblast growth factor 4, human growth factor, Wiskott-Aldrich syndrome protein, motor neuron survival protein 1, motor neuron survival protein 2, retinal pigment epithelium (RPE) 65, vascular endothelial growth factor (VEGF), Rab escort protein (REP) 1, adrenoleukodystrophy protein, Fanconi anemia complementation group A protein, ATP-binding cassette transporter, collagen VII (COL7) protein, collagen type I, collagen types III and V, β2 integrin, lysosome-associated membrane protein, dystrophin, mini-dystrophin, insulin-like growth factor, T cell immunoregulatory factor 1 / ATPase H+ transporting V0 subunit A3 pancreatic and duodenal homeobox 1, MAF The protein is selected from the group consisting of BZIP transcription factor A, cystic fibrosis transmembrane conductance regulator protein, ATP-binding cassette subfamily C member 6, ATP-binding cassette subfamily A member 4, vascular endothelial growth factor A, serpin family A member 1, progranulin, microtubule-associated protein tau, C9orf72 protein, and gap junction β2.

[0044] The present disclosure provides a method of inducing antigen-specific immune tolerance using a TIMP that encapsulates an antigen, comprising administering to a subject a composition comprising negatively charged particles that encapsulate an antigen, wherein the antigen is one or more gene therapy vector antigens, and / or one or more transgene protein products produced by a gene therapy vector, a portion thereof, or a combination thereof. In various embodiments, the antigen is one or more gene therapy vector antigens, a portion thereof, or a combination thereof, and / or one or more transgene protein products produced by a vector, a portion thereof, or a combination thereof. In various embodiments, the TIMP that encapsulates one or more antigens induces antigen-specific tolerance when administered to a subject in need thereof.

[0045] Also provided is a method of inducing tolerance in a subject in need thereof, comprising administering to the subject a composition comprising a liposome as described herein.

[0046] In various embodiments, the subject is administered a TIMP encapsulating one or more gene therapy vector antigens in combination with a TIMP encapsulating one or more transgene protein products produced by the gene therapy vector. Administering a TIMP encapsulating a gene therapy vector antigen in combination with a TIMP encapsulating a transgene protein product produced by the gene therapy vector induces antigen-specific tolerance to both the gene therapy vector antigen and the transgene protein product produced by the gene therapy vector. In various embodiments, the subject is administered a TIMP encapsulating one or more gene therapy vector antigens before, simultaneously with, or after the subject is administered a TIMP encapsulating one or more transgene protein products produced by the gene therapy vector.

[0047] In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered 5, 10, 15, 30, 45, or 60 minutes prior to administration of the TIMP encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of the TIMP encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the TIMP encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months prior to administration of the TIMP encapsulating one or more introduced gene protein products produced by the gene therapy vector.

[0048] In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered 5, 10, 15, 30, 45, or 60 minutes after administration of the TIMP encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of the TIMP encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of the TIMP encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months following administration of the TIMP encapsulating one or more introduced gene protein products produced by the gene therapy vector.

[0049] In various embodiments, the TIMP is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally, hi various embodiments, the TIMP is administered prior to, simultaneously with, or after administration of the gene therapy vector.

[0050] In various embodiments, the TIMP is administered 5, 10, 15, 30, 45, or 60 minutes prior to administration of the gene therapy vector. In various embodiments, the TIMP is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of the gene therapy vector. In various embodiments, the TIMP is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the gene therapy vector. In various embodiments, the TIMP is administered 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months prior to administration of the gene therapy vector.

[0051] In various embodiments, the TIMP is administered 5, 10, 15, 30, 45, or 60 minutes after administration of the gene therapy vector. In various embodiments, the TIMP is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of the gene therapy vector. In various embodiments, the TIMP is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of the gene therapy vector. In various embodiments, the TIMP is administered 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after administration of the gene therapy vector.

[0052] In various embodiments, administering a TIMP to a subject in need thereof induces antigen-specific tolerance to the gene therapy vector antigen, a portion thereof, or a combination thereof. In various embodiments, administering a TIMP to a subject in need thereof induces antigen-specific tolerance to the transgene protein product, a portion thereof, or a combination thereof. In various embodiments, administering a TIMP in a subject reduces an inflammatory immune response to the gene therapy vector antigen and / or the transgene protein product expressed by the vector. In various embodiments, administering a TIMP in a subject reduces an inflammatory immune response to one or more encapsulated antigens. In various embodiments, administering a TIMP to a subject in need thereof reduces an inflammatory response to one or more antigens that are not encapsulated within the particle. Such an immunomodulatory response is referred to in the literature as "infectious tolerance." In various embodiments, the inflammatory immune response is a humoral immune response and / or an adaptive immune response. In various embodiments, the immune response is an antibody response. In various embodiments, the antibody response is an IgA, IgG, IgE, or IgM response. In various embodiments, the antibody response is a neutralizing antibody response, e.g., the formation of neutralizing antibodies against the gene therapy vector antigen and / or transgene protein produced by the vector. In various embodiments, the immune response is a T cell, B cell, NK cell monocyte, macrophage, eosinophil, or basophil response. In various embodiments, the immune response is reduced immune infiltration into tissues and / or organs.

[0053] In various embodiments, administering a TIMP to a subject in need thereof induces an antigen-specific immune modulator response. In various embodiments, administering a TIMP in a subject induces regulatory T cells, B cells, monocytes, and / or macrophages. In various embodiments, administering a TIMP in a subject induces antigen-specific regulatory T cells (Treg), Tr1, regulatory macrophages (Mreg), and / or regulatory B cells (Breg) cells.

[0054] In various embodiments, a subject is administered liposomes encapsulating one or more gene therapy vector antigens in combination with liposomes encapsulating one or more transgene protein products produced by a gene therapy vector. Administering liposomes encapsulating gene therapy vector antigens in combination with liposomes encapsulating one or more transgene protein products produced by a gene therapy vector induces antigen-specific tolerance to both the gene therapy vector antigens and the transgene protein products produced by the gene therapy vector. In various embodiments, a subject is administered liposomes encapsulating one or more gene therapy vector antigens before, simultaneously with, or after the subject is administered liposomes encapsulating one or more transgene protein products produced by a gene therapy vector.

[0055] In various embodiments, the liposomes encapsulating one or more gene therapy vector antigens are administered 5, 10, 15, 30, 45, or 60 minutes prior to administration of the liposomes encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the liposomes encapsulating one or more gene therapy vector antigens are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of the liposomes encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the liposomes encapsulating one or more gene therapy vector antigens are administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the liposomes encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, liposomes encapsulating one or more gene therapy vector antigens are administered 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months prior to administration of liposomes encapsulating one or more introduced gene protein products produced by the gene therapy vector.

[0056] In various embodiments, the liposomes encapsulating one or more gene therapy vector antigens are administered 5, 10, 15, 30, 45, or 60 minutes after administration of the liposomes encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the liposomes encapsulating one or more gene therapy vector antigens are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of the liposomes encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, the liposomes encapsulating one or more gene therapy vector antigens are administered 1, 2, 3, 4, 5, 6, or 7 days after administration of the liposomes encapsulating one or more transgene protein products produced by the gene therapy vector. In various embodiments, liposomes encapsulating one or more gene therapy vector antigens are administered for 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months following administration of liposomes encapsulating one or more introduced gene protein products produced by the gene therapy vector.

[0057] In various embodiments, the liposomes are administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally, hi various embodiments, the liposomes are administered prior to, simultaneously with, or after administration of the gene therapy vector.

[0058] In various embodiments, the liposomes are administered 5, 10, 15, 30, 45, or 60 minutes prior to administration of the gene therapy vector. In various embodiments, the liposomes are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours prior to administration of the gene therapy vector. In various embodiments, the liposomes are administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the gene therapy vector. In various embodiments, the liposomes are administered 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months prior to administration of the gene therapy vector.

[0059] In various embodiments, the liposomes are administered 5, 10, 15, 30, 45, or 60 minutes after administration of the gene therapy vector. In various embodiments, the liposomes are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 72, or 96 hours after administration of the gene therapy vector. In various embodiments, the liposomes are administered 1, 2, 3, 4, 5, 6, or 7 days after administration of the gene therapy vector. In various embodiments, the liposomes are administered 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after administration of the gene therapy vector.

[0060] In various embodiments, administering the liposomes to a subject in need thereof induces antigen-specific tolerance to the gene therapy vector antigen, a portion thereof, or a combination thereof. In various embodiments, administering the liposomes to a subject in need thereof induces antigen-specific tolerance to the transgene protein product, a portion thereof, or a combination thereof. In various embodiments, administering the liposomes in a subject reduces an inflammatory immune response to the gene therapy vector antigen and / or the transgene protein product expressed by the vector. In various embodiments, administering the liposomes in a subject reduces an inflammatory immune response to one or more encapsulated antigens. In various embodiments, administering the liposomes to a subject in need thereof reduces an inflammatory response to one or more antigens that are not encapsulated within the particle. Such an immunomodulatory response is referred to in the literature as "infectious tolerance." In various embodiments, the inflammatory immune response is a humoral immune response and / or an adaptive immune response. In various embodiments, the immune response is an antibody response. In various embodiments, the antibody response is an IgA, IgG, IgE, or IgM response. In various embodiments, the antibody response is a neutralizing antibody response, e.g., the formation of neutralizing antibodies against the gene therapy vector antigen and / or the transgene protein produced by the vector, hi various embodiments, the immune response is a T cell, B cell, NK cell monocyte, macrophage, eosinophil, or basophil response.

[0061] In various embodiments, administering the liposomes to a subject in need thereof induces an antigen-specific immune modulator response. In various embodiments, administering the liposomes in a subject induces regulatory T cells, B cells, monocytes, and / or macrophages. In various embodiments, administering the TIMPs in a subject induces antigen-specific Treg, Tr1, Mreg, and / or Breg cells.

[0062] Also contemplated are compositions as described herein comprising negatively charged particles encapsulating an antigen for use in inducing tolerance in a subject, wherein the antigen is one or more gene therapy vector antigens, and / or one or more transgene protein products produced by the gene therapy vector, a portion or combination thereof.

[0063] The present disclosure also provides for the use of a composition described herein comprising negatively charged particles encapsulating an antigen, in the preparation of a medicament for inducing tolerance in a subject, wherein the antigen is one or more gene therapy vector antigens, and / or one or more transgene protein products produced by a gene therapy vector, a portion or combination thereof.

[0064] In various embodiments, the negatively charged particle is a TIMP or a liposome as described herein.

[0065] It is understood that each feature or embodiment, or combination described herein is a non-limiting, illustrative example of any of the aspects of the invention, and as such is meant to be combinable with any other feature or embodiment, or combination described herein. For example, when a feature is described in language such as "one embodiment," "some embodiments," "particular embodiments," "further embodiments," "particular exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of the feature that is intended to be combined with any other feature or combination of features described herein, and it is not necessary to list all possible combinations. Such features or combinations of features apply to any of the aspects of the invention. When examples of values ​​that fall within a range are disclosed, any of these examples are contemplated as possible endpoints of the range, and any and all numerical values ​​between such endpoints are contemplated, and any and all combinations of the upper and lower endpoints are envisioned.

[0066] The headings herein are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments, and variations of the invention will be apparent from the detailed description and / or drawings and / or claims. [Brief description of the drawings]

[0067] [Figure 1] Effect of CNP-GFP+CNP-VP1 treatment on GFP expression after initial administration of AAV8-GFP via intramuscular injection. Female BALB / c mice (6-8 weeks old, n=10 / group) were intravenously injected with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded CNP (control) on days -7 and 0. Mice were administered rAAV8-eGFP via intramuscular injection in the left quadriceps on day 0. Mice were monitored by transcutaneous fluorescence spectroscopy for expression of the transgene eGFP in the primed left quadriceps on days 0, 3, 7, 14, 21, and 28. Treatment with CNP-GFP+CNP-VP1 resulted in significantly higher GFP expression compared to controls (*p<0.05, ***p<0.001, ****p<0.0001). [Figures 2A-2D]Effect of CNP-GFP+CNP-VP1 treatment on antigen-specific CD8 T cells after initial administration and re-administration of AAV8-GFP via intramuscular injection. Female BALB / c mice (6-8 weeks old, n=10 / group) were intravenously injected with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded CNP (control) on days -7 and 0. Mice were administered rAAV8-eGFP via intramuscular injection in the left quadriceps on day 0. On day 28, mice were re-administered intramuscularly with AAV8-GFP in the contralateral right quadriceps (N=5 from each group). Seven days after re-administration (day 35), mice were euthanized and both the left and right quadriceps were harvested for analysis. The frequency and total cell numbers of AAV8-VP1- and GFP-specific CD8+ T cells were assessed by flow cytometry using tetramer staining. Treatment with CNP-GFP+CNP-VP1 significantly reduced the number of VP1- and GFP-specific CD8+ T cells in both the initial tissue (left quadriceps) after AAV8-GFP administration (Figures 2A and 2B) and the tissue (right quadriceps) after AAV8-GFP re-administration (Figures 2C and 2D) (*p<0.05, **p<0.01). [Diagram 3]Effect of CNP-GFP+CNP-VP1 treatment on GFP expression after re-administration of AAV8-GFP via intramuscular injection. Female BALB / c mice (6-8 weeks old, n=10 / group) were intravenously injected with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded CNP (control) on days -7 and 0. Mice were administered rAAV8-eGFP via intramuscular injection in the left quadriceps on day 0. On day 35, mice in each treatment group (N=5 / group) were re-administered with AAV8-eGFP intramuscularly in the contralateral right quadriceps, and expression of eGFP in the right quadriceps was monitored by transcutaneous fluorescence spectroscopy. Mice treated with CNP-GFP+CNP-VP1 showed significantly higher eGFP expression in the right quadriceps compared to controls on days 14 and 21 after rechallenge with AAV8-GFP (**p<0.01, ****p<0.0001). Compared to control treatment, CNP-GFP+CNP-VP1 treatment resulted in a 58% increase in GFP expression from days 14 to 21. [Figure 4A-4C]Systemic administration of TIMPs encapsulating AAV8-GFP results in a tolerogenic phenotype. C57BL / 6 mice (6-8 weeks old, n=5) were primed with either PBS or empty AAV8 capsids on day 0. Mice were administered two intravenous doses of CNP-GFP (1.25 mg / mouse) + CNP-VP1 (1.25 mg / mouse) on days 14 and 21. Mice were challenged with AAV8-eGFP by IV administration on day 28. Mice were euthanized and a final readout was taken on day 53. Mice treated with CNP have reduced leukocytes in the heart compared to control mice (Figure 4A). CD4+ T cells display an increased regulatory phenotype in the spleen, liver, and heart (Figure 4B). Mice treated with CNP have increased regulatory T cells (CD4+Foxp3+ and CD4+Ctla4+) in the spleen and liver when compared to control-treated (PBS) mice (Figure 4B). Mice receiving CNP therapy also have increased numbers of CD4+ cells expressing the regulatory cytokine IL-10+ and CD44hi memory markers in the spleen (Figure 4B). Mice treated with CNP have increased memory CD4+ T cells (CD44hi IFN-gamma-) and increased regulatory cells (CD4+Foxp3+, CD4+PD-1+, CD4+Ctla4+, CD4+IL-10+) in the heart (Figure 4B). CD8+ T cells have a reduced effector phenotype (CD8+Granzyme+, CD8+PD-1+, CD8+CD69+) compared to the hearts of CNP-treated mice (Figure 4C). (*p<0.05, **p<0.01, ***p<0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Antigen-specific tolerance has been described as an attractive strategy to overcome the immunogenicity of gene therapy vectors and protein therapeutics produced by the vectors. Although several methods to induce antigen-specific tolerance have been described, their translation into the clinic has remained elusive. 16 There is a need for interventions that allow for the safe administration of multiple therapeutic viral vectors and overcome challenges associated with vector immunogenicity.

[0069] The present disclosure provides compositions of negatively charged particles that encapsulate one or more gene therapy vectors, portions thereof, or combinations thereof.The present disclosure also provides compositions of negatively charged particles that encapsulate one or more transgene protein products produced by gene therapy vectors, portions thereof, or combinations thereof.Also included are methods of inducing antigen-specific tolerance using the negatively charged particles described herein.

[0070] definition Unless otherwise stated, the following terms used in this Application, including the specification and claims, have the definitions given below.

[0071] As used in this specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural and singular referents unless the context clearly dictates otherwise.

[0072] The term "about" or "approximately" refers to a margin of error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" or "approximately" precedes the first number in a series of two or more numbers, it is understood that the term "about" or "approximately" applies to each one of the numbers in the series.

[0073] "Particle" as used herein refers to any non-tissue derived composition and may be a sphere or sphere-like entity, a bead, or a liposome. The terms "particle", "tolerizing immunomodulating particle", "carrier particle", and "bead" may be used interchangeably depending on the context. In addition, the term "particle" may be used to encompass beads and spheres.

[0074] As used herein, "negatively charged particles" refer to particles that have been modified to have a net surface charge that is less than zero.

[0075] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particle that has been modified to contain carboxyl groups on its surface. In some embodiments, the addition of carboxyl groups improves phagocyte / monocyte uptake of the particle from the circulation, for example, via interaction with scavenger receptors such as MARCO. Carboxylation of particles can be accomplished using any compound that adds carboxyl groups, including, but not limited to, poly(ethylene-maleic anhydride) (PEMA).

[0076] As used herein, the term "Th cells" or "helper T cells" refers to CD4 + Refers to cells. CD4 + T cells assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. T cells are activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs).

[0077] As used herein, the term "Th1 cells" refers to a subset of Th cells that produce proinflammatory mediators. Th1 cells secrete cytokines to promote immune responses and play a role in host defense against pathogens, in part by mediating the recruitment of neutrophils and macrophages to infected tissues. Th1 cells secrete cytokines including IFN-gamma, IL-2, IL-10, and TNF alpha / beta to orchestrate defense against intracellular pathogens such as viruses and some bacteria.

[0078] As used herein, the term "Th2 cells" refers to a subset of Th cells that mediate the activation and maintenance of antibody-mediated immune responses to extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing a variety of cytokines, such as IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25), which are involved in antibody production, eosinophil activation, and inhibition of several macrophage functions, thus providing a phagocyte-independent protective response.

[0079] "Polypeptide" and "protein" refer to polymers composed of amino acid residues, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof, linked via peptide bonds or peptide bond isosteres. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms "polypeptide" and "protein" are not limited to a minimum length of the product. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms "polypeptide" and "protein" also include post-expression modifications of the polypeptide or protein, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, a "polypeptide" can include "modifications" to the native sequence, such as deletions, additions, substitutions (which may be essentially conservative or may include substitutions with any of the 20 amino acids commonly found in human proteins, or with any other natural or non-natural or atypical amino acids), and chemical modifications (e.g., addition of or substitution with a peptidomimetic). These modifications may be deliberate, through site-directed mutagenesis or through chemical modification of amino acids to remove or attach chemical moieties, or may be accidental, such as through mutations that occur via the host cell producing the protein or through errors due to PCR amplification prior to host cell transfection.

[0080] As used herein, "antigenic moiety" or "antigen" refers to any moiety, e.g., peptide, that is recognized by the host's immune system. Examples of antigenic moieties include, but are not limited to, autoantigens, allergens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components.

[0081] As used herein, "gene therapy vector antigen" refers to a gene therapy vector, or a portion or fragment thereof, e.g., a surface protein, that produces an immune response against a protein or portion or fragment thereof. Gene therapy vector antigens can include the entire gene therapy vector (e.g., a virus, bacteria, bacteriophage, or other vector), or a protein portion of the gene therapy vector, e.g., a viral capsid protein, envelope protein, or other protein or portion associated with the vector that can elicit an immune response in a subject receiving the gene therapy vector.

[0082] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, and the like, such as phosphate buffered saline, 5% aqueous solution of dextrose, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, glidants, sweeteners, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection, or topical, transdermal, or transmucosal administration) or via inhalation.

[0083] "Pharmaceutically acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained, or with any components present outside or inside the individual's body.

[0084] As used herein, the term "subject" includes mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; farm animals such as cows, horses, sheep, goats, pigs, and the like; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or sex.

[0085] The term "epitope" refers to a portion of any molecule that can be recognized and bound by a selective binding agent at one or more of its antigen-binding regions. An epitope usually consists of a chemically active surface grouping of molecules, such as amino acids or carbohydrate side chains, and has specific three-dimensional structural and charge characteristics. As used herein, an epitope can be contiguous or non-contiguous. An epitope can also be a mimetic (mimotope) in that it contains a three-dimensional structure that is identical to the epitope used to generate antibodies, but that does not contain any, or only some, of the amino acid residues found in the target used to stimulate the antibody immune response. As used herein, a mimotope is not considered a different antigen than the epitope bound by the selective binding agent, and the selective binding agent recognizes the same three-dimensional structure of the epitope and the mimotope.

[0086] The term "therapeutically effective amount" is used herein to indicate an amount of an antigen-specific composition of the present disclosure effective to alleviate or reduce one or more symptoms or signs of the disease being treated.

[0087] The terms "treat," "treated," "treating," and "treatment" as used with respect to methods herein refer to eliminating, reducing, inhibiting, or alleviating, either temporarily or permanently, either partially or completely, the clinical symptoms, signs, or progression of an event, disease, or condition. Such treating need not be absolute to be useful.

[0088] particle Tolerizing immune-modifying particles (TIMPs) comprising one or more antigens have been previously described for the induction of antigen-specific tolerance to treat inflammatory conditions (e.g., autoimmune diseases and allergies) (WO20131319253 and WO2015023796, which are incorporated herein by reference). In several preclinical models of autoimmune diseases and allergies, TIMPs have shown efficacy in inducing antigen-specific tolerance and inhibition of pathological inflammatory immune responses.

[0089] The size and charge of the particles are important for tolerance induction. Although the particles vary in size and charge based on the antigen encapsulated within the particle, generally, the particles described herein are effective for inducing tolerance when they are about 100 nanometers to about 1500 nanometers and have a charge of 0 to about -100 mV. In various embodiments, the particles are 400 to 800 nanometers in diameter and have a charge of about -25 mV to -70 mV. In various embodiments, the particles are 400 to 800 nanometers in diameter and have a charge of about -30 mV to -80 mV. In various embodiments, the particles are 400 to 800 nanometers in diameter and have a charge of about -30 mV to -60 mV. The average particle size and charge of the particles may be slightly altered in the lyophilization process, and therefore both post-synthesis average and post-lyophilization average are described. As used herein, the terms "post-synthesis size" and "post-synthesis charge" refer to the size and charge of the particles before lyophilization. The terms "post-lyophilization size" and "post-lyophilization charge" refer to the size and charge of the particles after lyophilization.

[0090] In some embodiments, the particles are non-metallic. In these embodiments, the particles can be formed from polymers. In a preferred embodiment, the particles are biodegradable in the individual. In this embodiment, the particles can be provided in the individual over multiple doses without accumulation of the particles in the individual. Examples of suitable particles include polystyrene particles, PLGA particles, PLURONICS stabilized polypropylene sulfide particles, and diamond particles.

[0091] Preferably, the particle surface is composed of a material that minimizes non-specific or undesirable biological interactions. Interactions between the particle surface and the stroma may be a factor that plays a role in lymphatic uptake. The particle surface may be coated with a material that prevents or reduces non-specific interactions. Steric stabilization by coating the particle with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers, such as PLURONICS® (including copolymers of poly(ethylene glycol)-bl-poly(propylene glycol)-bl-poly(ethylene glycol)), may reduce non-specific interactions with interstitial proteins, as demonstrated by improved lymphatic uptake after subcutaneous injection. All of these facts indicate the relevance of the particle's physical properties in terms of lymphatic uptake. Biodegradable polymers may be used to make all or part of the polymer and / or particle and / or layer. Biodegradable polymers may degrade, for example, as a result of functional groups reacting with water in solution. The term "degradation" as used herein refers to becoming soluble, either by a reduction in molecular weight or by the conversion of hydrophobic groups to hydrophilic groups. Polymers bearing ester groups, such as polylactides and polyglycolides, are generally subject to spontaneous hydrolysis.

[0092] The particles disclosed herein may also contain additional components. For example, the carrier may have a contrast agent incorporated or conjugated to the carrier. An example of a carrier nanosphere with a contrast agent that is currently commercially available is Kodak X-sight nanosphere. Inorganic quantum confined luminescent nanocrystals, known as quantum dots (QDs), have emerged as ideal donors in FRET applications: their high quantum yield and adjustable size-dependent Stokes shift allow them to emit different sizes from blue to infrared when excited by a single ultraviolet wavelength. (Bruchez, et al., Science, 1998, 281, 2013; Niemeyer, CM Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LE J Chem. Phys. 1993, 79, 5566.) Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers known as dendrimers, can be used in biological labeling, imaging, and optical biosensing systems. (Lemon, et al., J. Am. Chem. Soc. 2000, 122, 12886.) Unlike conventional synthesis of inorganic quantum dots, the synthesis of these hybrid quantum dot nanoparticles does not require high temperatures or highly toxic, unstable reagents. (Etienne, et al., Appl. Phys. Lett. 87, 181913, 2005.)

[0093] The particles can be formed from a wide range of materials. The particles are preferably composed of materials suitable for biological use. For example, the particles can be composed of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids. More generally, the carrier particles can be composed of polyesters of linear or branched chain, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of linear or branched chain, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. In addition, the carrier particles can be or be composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). Carrier particles containing mixtures of ester and anhydride linkages (e.g., copolymers of glycolic acid and sebacic acid) may also be used. For example, the carrier particles may include materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA or PLG; the terms are interchangeable), poly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable urethanes, as well as copolymers thereof having linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy or dicarboxylic acids.In addition, biologically important amino acids with reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, can be included in copolymers with any of the aforementioned materials to provide reactive groups and conjugate moieties for conjugation to antigenic peptides and proteins. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers. Biocompatible but non-biodegradable materials can also be used in the carrier particles of the present invention. For example, acrylates, ethylene-vinyl acetate, non-biodegradable polymers of acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon can be used.

[0094] In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0. Suitable copolymer ratios for the immunomodifying particles can be 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 81:19, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13, 88:12, 89:11, 90:10, 91:9, 92:8, 93:7, 94:6, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. In various embodiments, the particles are PLURONICS stabilized polypropylene sulfide particles, polyglycolic acid particles (PGA), polylactic acid particles (PLA), or poly(lactic-co-glycolic acid) particles. In various embodiments, the particles are carboxylated PLGA particles. In various embodiments, the particles have a copolymer ratio of polylactic acid / polyglycolic acid 80:20, polylactic acid / polyglycolic acid 90:10, or polylactic acid:polyglycolic acid / 50:50. In various embodiments, the particles are poly(lactic-co-glycolic acid) particles and have a copolymer ratio of polylactic acid:polyglycolic acid of about 50:50. In various embodiments, the particles comprise about 50:50, about 80:20 to about 100:0 polylactic acid:polyglycolic acid, or about 50:50, about 80:20 to about 100:0 polyglycolic acid:polylactic acid. In various embodiments, the particles comprise 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particles comprise polylactic acid:polyglycolic acid in a ratio of about 99:1 to about 1:99, e.g., about 99:1, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, and about 1:99, including all values ​​and ranges therebetween.

[0095] It is contemplated that the particles may further comprise a surfactant. The surfactant may be anionic, cationic, or nonionic. Poloxamer and poloxamine family surfactants are commonly used in particle synthesis. Surfactants that may be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, Pluronic L-63, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methylcellulose, lecithin, didodecyldimethylammonium bromide (DMAB) and poly(ethylene-alt-maleic acid) (PEMA). In addition, biodegradable and biocompatible surfactants include, but are not limited to, vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), polyamino acids (e.g., polymers of lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or their enantiomers), and sulfate polymers. In some embodiments, two surfactants are used. For example, if the particles are produced by a double emulsion method, the two surfactants may include a hydrophobic surfactant for the first emulsion, and a hydrophobic surfactant for the second emulsion.

[0096] In various embodiments, the polypeptide antigen is encapsulated in the particles by a single emulsion process. In further embodiments, the polypeptide antigen is more hydrophobic. Sometimes, the double emulsion process leads to the formation of large particles that can lead to leakage of the hydrophilic active ingredient and low entrapment efficiency. Coalescence and Ostwald ripening are two mechanisms that can destabilize double emulsion droplets, while diffusion of the hydrophilic active ingredient through the organic phase is the main mechanism responsible for low levels of entrapped active ingredient. In some embodiments, it can be beneficial to reduce the nanoparticle size. One strategy to achieve this is to apply a second strong shear rate. The leakage effect can be reduced by using high polymer concentration and high polymer molecular weight, with an increase in the viscosity of the internal aqueous phase and an increase in the surfactant molecular weight. In certain embodiments, the particles encapsulating the antigen are produced by nanoprecipitation, co-precipitation, inert gas condensation, sputtering, microemulsion, sol-gel method, layer-by-layer technique, or ionic gelation method. Several methods for producing nanoparticles have been described in the literature and are incorporated herein by reference. 17、18 .

[0097] In some embodiments, the particle is a liposome. Liposomes can be prepared from a variety of lipid materials, including, but not limited to, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, dicetyl phosphate, monosialoganglioside, polyethylene glycol, stearyl aluminium, ovolecithin and cholesterol lipids, and mixtures thereof in various stoichiometries. As used herein, liposomes can also be formed from non-lipid amphiphilic molecules, such as block copolymers of poly(oxyethylene-b-isoprene-b-oxyethylene). In preferred embodiments, liposomes are prepared from lipids or incorporate lipids that form negatively charged liposomes, such as those produced from phosphatidylserine, dicetyl phosphate, and dimyristoyl phosphatidic acid. In various embodiments, the negatively charged liposomes have a zeta potential of about -100 mV to about 0 mV, about -100 mV to about -25 mV, about -100 to about -30 mV, about -80 mV to about -30 mV, about -75 mV to about -30 mV, about -70 mV to about -30 mV, about -75 to about -35 mV, about -70 to about -25 mV, about -60 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -30 mV. In various embodiments, the zeta potential is about -25mV, -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV, including all values ​​and ranges therein. In various embodiments, the liposomes have a negative zeta potential of -30mV to -80mV. In various embodiments, the liposomes have a negative zeta potential of -30mV to -60mV.

[0098] In various embodiments, the liposomes encapsulate one or more gene therapy vector antigens and / or portions thereof, or a combination of gene therapy vector antigens or portions thereof. In various embodiments, the liposomes encapsulate a therapeutic protein produced by the gene therapy vector and / or a fragment or portion thereof. In various embodiments, the therapeutic protein produced by the gene therapy vector is a protein, polypeptide, or peptide. In various embodiments, the therapeutic protein is a cytokine, chemokine, hormone, growth factor, enzyme, or antibody. In various embodiments, the gene therapy vector antigen is an adenovirus, an adeno-associated virus (AAV), AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-12, Anc80, synthetic AAV, combinations or engineered versions thereof, AAV capsid protein VP1, AAV capsid protein VP2, AAV capsid protein VP3, herpes simplex virus, hepatitis B virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, coxsackievirus, transfusion-transmitted virus, or the like. In some embodiments, the virus is selected from the group consisting of an infectious virus, anellovirus, human papillomavirus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, cowpea mosaic virus, cowpea chlorotic mottle virus, ground cherries mosaic virus, red clover necrotic mosaic virus, potato virus x, comovirus, chicken anemia virus, cucumber mosaic virus, oncolytic virus, chimeric virus, synthetic virus, mosaic virus, or pseudotyped virus, bacteria, bacteriophage, yeast, exosome, or red blood cell.

[0099] In various embodiments, the liposomes encapsulate one or more gene therapy vector antigens, the liposomes have a size between 100 and 1000 nm in diameter, and the liposomes have a negative zeta potential between -100 mV and -0 mV. In various embodiments, the liposomes encapsulate one or more gene therapy vector antigens, portions thereof, or combinations thereof, the liposomes have a size between 400 and 800 nm, and the liposomes have a negative zeta potential between -30 mV and -80 mV.

[0100] In various embodiments, the liposomes encapsulate a therapeutic protein and / or a fragment or portion thereof produced by a gene therapy vector, the size of the liposomes is between 100 and 1000 nm, and the liposomes have a negative zeta potential between -100 mV and 0 mV. In various embodiments, the liposomes encapsulate a therapeutic protein and / or a fragment or portion thereof produced by a gene therapy vector, the size of the liposomes is between 400 and 800 nm, and the liposomes have a negative zeta potential between -30 mV and -80 mV. In various embodiments, the liposomes are used to induce tolerance in a subject in need thereof. In various embodiments, the administration of the liposomes is intravenous.

[0101] Gene Therapy Vectors Gene therapy vectors are designed to deliver genes encoding therapeutic products to a subject to alleviate the symptoms of a genetic disorder or disease, such as cancer. Gene therapy vectors include viral, bacterial, bacteriophage, yeast, red blood cell, or exosome vectors.

[0102] Viral vectors commonly used in human gene therapy include adenoviruses, adeno-associated viruses (AAV), and retroviruses, such as lentiviruses.

[0103] Adenoviral vectors are often derived from human serotypes HAd2, HAd5, HAd26 and HAd35, or engineered, such as "high-capacity" adenoviral vectors (HCAd) or oncolytic vectors, and can also be derived from non-human mammalian Ad viruses (Bulcha et al., Signal Transduction and Targeted Therapy volume 6, Article number: 53 (2021)). Adenoviral expression can induce immune responses against the early transcription genes E1 (E1A, E1B), E2, E3, and E4, the most abundant structural components on the capsid surface, hexon, and the 12 penton proteins located on the capsid apex, giving rise to the elongated fibers found on these viruses. Carrier particles containing these viral antigens are intended to induce tolerance to these gene therapy vector antigens.

[0104] AAV vectors are small viral vectors with an icosahedral capsid composed mainly of three proteins: VP1, VP2, and VP3. Twelve serotypes of AAV are known. (Bulcha et al., supra). Serotypes commonly used in gene therapy include AAV-1, AAV-2, AAV-5, AAV-9, Anc80, and other serotypes include AAV-3, AAV-4, AAV-6, AAV-7, AAV-8, AAV-10, AAV-12, and synthetic AAV. Natural and engineered capsid proteins have been used to generate AAV vectors for administration in gene therapy. Carrier particles containing wild-type or engineered AAV viral capsid proteins are contemplated herein to induce tolerance to these gene therapy vector antigens.

[0105] Lentiviruses are enveloped viruses with three primary gene sets, gag, pol and env genes, and may have auxiliary genes (tat, rev, vif, vpr, vpu, and nef) useful for improving growth and pathogenesis. The Gag gene encodes the structural envelope protein. However, engineered lentiviral vectors may contain the VSV-G protein instead of the envelope protein. (Bulcha et al., supra). Carrier particles containing wild-type or engineered lentiviral proteins are contemplated herein to induce tolerance to these gene therapy vector antigens.

[0106] Bacterial vectors have been generated to deliver genes or proteins to target cancer cells (Baban et al., Bioengineered Bugs 1:6,385-394,2010), including vectors derived from Salmonella, Shigella, Listeria, or E. coli. These vectors have been used to express human tumor antigens, cytokines, growth factors, enzymes, and therapeutic proteins. Another use of bacterial vectors is bactofection, which refers to the bacterial transfer of bacteriophage or plasmid DNA into mammalian cells. Carrier particles containing bacterial or plasmid antigens are contemplated to induce tolerance to these gene therapy vector antigens.

[0107] Yeast gene therapy vectors expressing tumor or HIV antigens have been shown to induce tumor-reactive responses in vivo 21 Exosomes and polyethylenimine matrices (EPMs) have been used to administer short interfering RNA (siRNA) or plasmid DNA (pDNA) to patients, and exosomes have been used to deliver CRISPR / Cas9 plasmids to cancer cells. 22、23 Erythrocytes, e.g., erythrocyte ghosts, have been shown to effectively deliver plasmid DNA into cells. 24 or can be loaded with proteins for delivery in vivo 25 .

[0108] antigen An antigen refers to a discrete portion of a molecule, such as a polypeptide or peptide sequence, a 3D structural formation of a polypeptide or peptide, a polysaccharide or a polynucleotide that can be recognized by a host immune cell. Antigen specificity refers to the ability of a subject's host cells to recognize and generate an immune response against the antigen alone or against a molecule that closely resembles the antigen, such as an epitope or mimotope.

[0109] "Anergy", "tolerance", or "antigen-specific tolerance" refers to the insensitivity of T cells to T cell receptor-mediated stimulation. Such insensitivity is generally antigen-specific and persists after exposure to antigenic peptides ceases. For example, anergy in T cells is characterized by a lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to an antigen and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (costimulatory signal). Under these conditions, re-exposure of the cells to the same antigen (even if re-exposure occurs in the presence of costimulatory molecules) results in failure to produce cytokines and subsequent failure to proliferate. Thus, failure to produce cytokines prevents proliferation. Anergy T cells, however, can proliferate when cultured with cytokines (e.g., IL-2).

[0110] It is contemplated that the tolerization therapy described herein is antigen-specific.For example, the TIMP administered as a tolerization therapy encapsulates one or more antigens related to the tolerization therapy and the associated disease or condition to be treated.It is contemplated that the TIMP used in the tolerization therapy comprises one or more gene therapy vector antigens, portions thereof, or combinations thereof, and / or transgene protein products produced by gene therapy vectors, portions thereof, or combinations thereof.

[0111] Exemplary gene therapy vectors include, but are not limited to, adenovirus, adeno-associated virus (AAV), herpes simplex virus, hepatitis B virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, coxsackievirus, transfusion-transmitted virus, anerosomes, human papillomavirus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, cowpea mosaic virus, cowpea chlorotic mottle virus, physalis mosaic virus, red clover necrotic mosaic virus, potato virus x, comovirus, chicken anemia virus, cucumber mosaic virus, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-12, Anc80, and synthetic AAV. It is contemplated that the particles may encapsulate viral vector capsid or envelope proteins, portions or fragments thereof, or combinations thereof.Examples of capsid proteins include AAV-Po1 VP1 (UNIPROT ID: C0LA97), AAV-Po1 VP2 (UNIPROT ID: C0LA98), AAV-Po1 VP3 (UNIPROT ID: COLA99), AAV-Po2 VP1 (UNIPROT ID: C0LAA0), AAV-Po3 (UNIPROT ID: C0LA95), AAV VP1 (UNIPROT ID: B4Y875), AAV-1 capsid protein (UNIPROT ID: Q9WBP8), AAV-2 VP1 (UNIPROT ID: P03135), AAV-3 capsid protein (UNIPROT ID: Q65311), AAV-3B VP1 (UNIPROT ID: O56139), AAV-4 capsid (UNIPROT ID: O41855), AAV6 VP1 (UNIPROT ID: O56137), AAV7 capsid protein (UNIPROT ID: Q8JQG0), AAV8 capsid protein (UNIPROT ID: Q8JQF8), AAV9 VP1 (UNIPROT ID: Q6JC40), AAV-10 capsid protein (UNIPROT ID: Q5Y9B4), AAV-11 capsid protein (UNIPROT ID: Q5Y9B2), AAV13 capsid protein (UNIPROT ID: B5SUY7), and AAV12 VP1 (UNIPROT ID: A9RAI0).

[0112] In certain embodiments, one, two, three, or more antigens or antigenic peptides are used in TIMPs. In certain embodiments, one or more antigens are encapsulated in TIMPs by covalent attachment to the inner surface of the particle (see, for example, US Patent Publication No. 2019 / 0282707, which is incorporated herein by reference). In certain embodiments, it is contemplated that the sequences of two or more antigens are linked in a fusion protein and encapsulated in the TIMPs described herein. Methods for making TIMPs with linked epitopes are described in US Patent Publication No. 2019 / 0365656, which is incorporated herein by reference.

[0113] Exemplary enzymes useful for treating enzyme disorders and which may be produced by gene therapy vectors include imiglucerase, taliglucerase alpha, velaglucerase alpha, β-glucocerebrosidase, alglucerase, agalsidase beta, agalsidase alpha, siberipase alpha, α-L-iduronidase, human iduronate-2-sulfatase, N-sulfoglucosamine sulfohydrolase, elosulfase alpha, galsulfase, alpha-glucosidase (GAA), human alpha mannosidase, factor VIII, factor IX, beta-galactosidase, arginase, dystrophy myotonica protein kinase, ornithine transcarbamylase, NADPH oxidase, NADH dehydrogenase 4, adenosine deaminase, lipoprotein lipase, beta-glucocerebrosidase, myoglobin kinase ... tubularin, arylsulfatase A, DOPA decarboxylase, matrix metallopeptidase 1, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, pyruvate kinase, porphobilinogen deaminase, alkaline phosphatase, sucrase-isomaltase, acid sphingomyelinase, heparanthylsulfase sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, alpha-1 proteinase inhibitor, alpha-1-esterase inhibitor, fibrinogen, factor VIIa, factor X, factor XI, factor XII, protein C, antithrombin III, ectonucleotide pyrophosphatase / phosphodiesterase 1, ectonucleotide pyrophosphatase / phosphodiesterase 3, and aromatic L-amino acid decarboxylase.

[0114] In certain embodiments, the protein therapeutic is an antibody, e.g., a monoclonal. It is also contemplated that the antibody may be a monospecific, bispecific, trispecific, or bispecific T cell engager. In various embodiments, the antibody targets a receptor tyrosine kinase (RTK), EGFR, VEGF, VEGFR, PDGF, PDGFR, HER2 / Neu, ER, PR, TGF-β1, TGF-β2, TGF-β3, SIRP-α, PD-1, PD-L1, CTLA-4, CD3, CD25, CD19, CD20, CD39, CD47, CD73, FAP, IL-1β, IL-12, IL-2R, IL-15, IL-15R, IL-23, IL-33, IL-2R, IL-4Rα, T cells, B cells, NK cells, macrophages, monocytes, and / or neutrophils. In various embodiments, the antibody is selected from the group consisting of abciximab, adalimumab, alemtuzumab, avelumab, azetolizumab, basiliximab, bevacizumab, bezlotoxumab, blinatumomab, canakinumab, certolizumab, cetuximab, daclizumab, denosumab, durvalumab, efalizumab, emicizumab, etokimab, golimumab, inoximab, rituxim ... The antibody is selected from the group consisting of pilimumab, ixekizumab, infliximab, natalizumab, nivolumab, olaratumab, omalizumab, ofatimumab, palivizumab, panitumumab, pembrolizumab, ramucirumab, rituximab, tocilizumab, trastuzumab, tremelimumab, secukinumab, ustekinumab, and vedolizumab.

[0115] How to use Provided herein is a method of inducing tolerance in a subject in need thereof, comprising administering to the subject a composition comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more gene therapy vector antigens, and / or one or more transgene protein products produced by the gene therapy vector, a portion or combination thereof.

[0116] In various embodiments, the subject has undergone gene therapy, is undergoing gene therapy, or may undergo gene therapy. In various embodiments, the subject has undergone one treatment of gene therapy and may undergo another gene therapy treatment regimen, i.e., re-administration of gene therapy.

[0117] In various embodiments, the subject is undergoing gene therapy to treat cancer, an autoimmune disease, an allergy, a cardiovascular disease, a metabolic disease, an enzyme deficiency, or a protein deficiency.

[0118] In various embodiments, the subject is afflicted with a rare, inherited genetic disorder and is undergoing gene therapy to provide a transgene protein product produced by a vector that is an enzyme. In various embodiments, the subject is diagnosed with hemophilia A, hemophilia B, lysosomal storage diseases, Fabry disease, Gaucher disease, Pompe disease, Niemann-Pick disease, Tay-Sachs disease, macular degeneration, mucopolysaccharidoses, venous thromboembolism, von Willebrand disease, purpura fulminans, growth hormone deficiency, gangliosidosis, hypoalkaline phosphatasia, cholesterol ester storage disease, hyperuricemia, Duchenne muscular dystrophy, Huntington's disease, Parkinson's disease, Alzheimer's disease, choroideremia, Stargardt disease, Batten disease, spinocerebellar ataxia, ALS, frontotemporal lobar degeneration, ornithine transcarbamylase deficiency, retinitis pigmentosa, RPE-65 mutation associated disease, epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, spinal muscular atrophy, phenylketonuria (PKU), X-linked myotubular myopathy, Crigler-Nagy syndrome, , catecholamine-induced polymorphic ventricular tachycardia, glycogen storage disease type 1, alpha-mannosidosis, fragile X syndrome, arginase deficiency, X-linked chronic granulomatous disease, adenosine deaminase deficiency, Leber congenital amaurosis, lipoprotein lipase deficiency, cerebral adrenoleukodystrophy, metachromatic leukodystrophy, Fanconi anemia, color vision disorders, scleroderma, osteogenesis imperfecta, coronary artery disease, tyrosinemia, peripheral The patient suffers from a rare inherited genetic disorder selected from the group consisting of: sexensory neuropathy, optic neuropathy, coronary artery disease, respiratory syncytial virus (RSV)-mediated lower respiratory tract disease, Danon disease, severe leukocyte adhesion deficiency, pyruvate kinase deficiency, Charcot-Marie-Tooth disease, Wiskott-Aldrich syndrome, alpha-synuclein tauopathy, refractory angina due to myocardial ischemia, myotonic dystrophy type I, claudication, peripheral arterial disease, methylmalonic acidemia, MPS I, MPS II, MPS III, MPS IV, MPS VI, MPS VII, MPS IX, calcification / ossification disorder, ENPP1 deficiency, ENPP3 deficiency, ABCC6 deficiency, aromatic L-amino acid decarboxylase deficiency, Angelman syndrome, hyperphenylalaninemia, dementia, and Rett syndrome and Usher syndrome.

[0119] In various embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, Addison's disease, ankylosing spondylitis, alopecia, osteoarthritis, psoriatic arthritis, scleroderma, type I diabetes, rheumatoid arthritis, thyroiditis, systemic lupus erythematosus, Raynaud's syndrome, Behcet's syndrome, Sjogren's syndrome, autoimmune uveitis, Eaton-Lambert disease, autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus, and / or rheumatoid arthritis. , neuromyelitis optica, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, myasthenia gravis, celiac disease, ulcerative colitis, Crohn's disease, erythema nodosum, glomerulonephritis, Goodpasture's syndrome, granulomas, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, Kawasaki disease, mixed connective tissue disease, multifocal motor neuropathy, peripheral biliary cirrhosis, polyangiitis overlap syndrome, scleroderma type 1, sclerosing cholangitis, stiff-man syndrome, Takayasu's arteritis, vitiligo, or granulomatosis with polyangiitis.

[0120] In various embodiments, the allergy is a food allergy. In various embodiments, the allergy is an environmental allergy. In various embodiments, the food allergy is a peanut allergy, a tree nut allergy, a milk allergy, an egg allergy, a fish allergy, a wheat allergy, a celery allergy, or a peach allergy. In various embodiments, the environmental allergy is a pollen allergy, a dust allergy, a pet dander allergy, or a mold allergy. In various embodiments, the pet allergy is a cat allergy or a dog allergy.

[0121] In various embodiments, the cancer is selected from the group consisting of brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, melanoma, and mesothelioma.

[0122] In various embodiments, the carrier particles comprise one or more gene therapy vector antigens. In various embodiments, the carrier particles comprise one or more transgene protein products produced by a gene therapy vector, a portion thereof, a combination thereof, or one or more antigenic epitopes thereof. In various embodiments, the carrier particles comprise both one or more gene therapy vector antigens and a transgene protein product or one or more antigenic epitopes thereof.

[0123] When a carrier particle containing a single antigen is administered in combination with another carrier particle containing a different antigen, or with a second agent, the particles and / or the second agent may be administered simultaneously or sequentially. The combination or simultaneous administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, so long as there is an overlap in the period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as well as administration on different days or weeks. It is further contemplated that the therapeutic agent refers to agents administered in separate formulations, administered simultaneously or in combination, and given within 30 minutes of each other at the same time. Pre-administration refers to administration of the therapeutic agent within a range of one week prior to treatment with the carrier particle and up to 30 minutes prior to administration of the carrier particle. Subsequent administration is meant to describe administration from 30 minutes after treatment to one week after administration.

[0124] In various embodiments, the carrier particles are administered at a dose of about 0.001 to about 10 mg / kg, about 0.005 to about 12 mg / kg, about 0.01 to about 12 mg / kg, about 0.05 to about 12 mg / kg, about 0.1 to about 12 mg / kg, about 0.5 to 10 mg / kg, about 1 to 8 mg / kg, about 1.5 to 10 mg / kg, about 2 to 12 mg / kg, about 2 to 10 mg / kg, about 3 to 10 mg / kg, about 4 to 10 mg / kg, about 4 to 12 mg / kg, or about 5 to 12 mg / kg. Optionally, the carrier particles are administered at a dose of about 0.001 mg / kg, about 0.0025 mg / kg, about 0.005 mg / kg, about 0.01 mg / kg, about 0.025 mg / kg, about 0.05 mg / kg, 0.1 mg / kg, 0.25, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 6 mg / kg, 8.0 mg / kg, 10 mg / kg, or 12 mg / kg. Alternatively, the carrier particles are administered in a dose of about 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, or 800 mg. In another embodiment, the carrier particles are administered at a concentration of about 0.0005 mg / mL to about 50 mg / mL, about 0.05 mg / mL to about 50 mg / mL, optionally about 0.0005 mg / mL, 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12.5 mg / mL, 15 mg / mL, 17.5 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL.

[0125] In some embodiments, carrier particles containing one or more gene therapy vector antigens and / or one or more transgene protein products produced by the gene therapy vector are administered in combination with one or more therapeutic agents. In various embodiments, the therapeutic agent is adoptive transfer of Tregs, CAR Tregs, TCR Tregs. In various embodiments, the therapeutic agent blocks or inhibits innate immune responses, complement responses, B cell responses, and / or T cell responses to the gene therapy vector and / or the transgene protein produced by the gene therapy vector.

[0126] In various embodiments, the therapeutic agent is an FcRn inhibitor. In various embodiments, the FcRn inhibitor is selected from the group consisting of efgartigimod, rozanoruximumab, nipocalimab, batoclimab, or olinarolimab. In various embodiments, the therapeutic agent is an immunosuppressant, a complement inhibitor, a calcineurin inhibitor, plasmapheresis, an IgG protease, a proteasome inhibitor, and / or an inducer of regulatory T cells. In various embodiments, the immunosuppressant is selected from the group consisting of a corticosteroid (such as methylprednisolone, prednisolone, or prednisone), rapamycin / sirolimus, or cyclophosphamide, mycophenolate mofetil. In various embodiments, the complement inhibitor is selected from the group consisting of C1 inhibitors (such as Cinryze, Berinert, Ruconest, stimulimab (Enjaymo), or Haegarda), C3 inhibitors (Pegacetaclplan (Empaveli), Pegacetaclplan injection (Syfovre)), C5 inhibitors (such as eculizumab, ravulizumab, avacopan, pozelimab, nomakopan, zirucoplan, vilobelimab, clovalimab, zimra, semdiciran, tesidolumab, abdulalalimab), factor D inhibitors (danicopan, bemircopan), or other complement proteins (such as narsoplimab, iptacopan). In various embodiments, the therapeutic agent is an anti-CD20 CAR T cell therapy, an anti-CD19 CAR T cell therapy, or an anti-BCMA CAR T cell therapy. In various embodiments, the therapeutic agent is selected from the group consisting of an anti-CD20 antibody (e.g., rituximab), an anti-CD19 antibody, an anti-CD40 antibody, a CTLA4 Ig (e.g., abatacept). In various embodiments, the calcineurin inhibitor is cyclosporine or tacrolimus. In various embodiments, the proteasome inhibitor is selected from the group consisting of bortezomib, carfilzomib, arsenic trioxide, hydroxychloroquine, or MnTBAP. In some embodiments, the IgG protease is IdeZ or IdeS (imrifidase). In various embodiments, the Treg inducer is an IL-2 variant / mutein or a CDK8 / 19 inhibitor.

[0127] In various embodiments, one or more therapeutic agents are administered prior to, simultaneously with, or after administration of the carrier particles described herein. In various embodiments, the therapeutic agent is administered 0.5, 1, 2, 4, 5, 8, 10, 12, 16, or 24 hours (including all ranges and values ​​intervening between these ranges) prior to administration of the carrier particles. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days prior to administration of the carrier particles. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to administration of the carrier particles. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years prior to administration of the carrier particles. In various embodiments, the therapeutic agent is administered simultaneously with the carrier particles. In various embodiments, the therapeutic agent is administered 0.5, 1, 2, 4, 5, 8, 10, 12, 16, or 24 hours (including all ranges and values ​​intervening between these ranges) after administration of the carrier particles. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, or 7 days after administration of the carrier particles. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after administration of the carrier particles. In various embodiments, the therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after administration of the carrier particles described herein.

[0128] Screening Methods It is contemplated that the induction of immune tolerance, and its maintenance, may be monitored in subjects who have been or are about to be treated with the carrier particles described herein.

[0129] Methods for screening cell types, cytokines, or other tolerance measures from subjects undergoing the tolerization therapy described herein are known in the art. Methods for evaluating tolerance are performed using techniques such as flow cytometry, mass cytometry (CyTOF), ELISA, ELISPOT, in vivo / ex vivo cell stimulation assays (including but not limited to cell proliferation assays, basophil activation test (BAT), macrophage stimulation assays), measuring autoantibodies or measuring Ig serotypes, for example, by ImmunoCap assay.

[0130] Lists of human metabolites that can be assayed from biological samples can be found in the literature, including (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan;35(Database issue):D521-6, 2007), and the Human Metabalome Database (HMDB), which are incorporated herein by reference.

[0131] One aspect of a subject's immune tolerance status and immune signature is determined by analyzing one or more cell surface proteins from a biological sample, in various embodiments, including but not limited to CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, CD45, CD 45RA, CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD69, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, B LAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD127, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD161, CD163, CD1 72a, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS 5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, integrin, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, C SF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-10R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL-23R, IL-27Rα, IL-31Rα, OSMR,CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD -1, PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β 2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AIT R, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, FAS, FAS-L, FC, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1 α, β, γ, δ, and ε, H60a, H60b, H60c, GPR15, ST2, and / or combinations thereof. Integrins include α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. TCRs include α, β, γ, δ, ε, ζ chains, and / or combinations thereof. Several methods for assaying cell surface protein expression have been described in the literature, including flow cytometry and mass cytometry (CyTOF).

[0132] In certain embodiments, the tolerance state of the subject is determined by analyzing nucleic acids from a biological sample. In various embodiments, the nucleic acids are DNA and / or RNA, including, but not limited to, single-stranded DNA, double-stranded DNA, mRNA, rRNA, tRNA, siRNA, miRNA, long non-coding RNA (long ncRNA, lncRNA), and non-coding RNA (ncRNA), mitochondrial RNA. In various embodiments, the tolerance state of the subject is determined by assaying gene expression from a biological sample. In various embodiments, the tolerance state is determined by assaying gene expression associated with immune function, antibodies, foreign body response, metabolism, apoptosis, cell death, necrosis, ferroptosis, autophagy, cell migration, endocytosis, phagocytosis, pinocytosis, tight junction regulation, cell adhesion, differentiation, and / or combinations thereof. In various embodiments, the tolerance state is determined by assaying gene expression associated with immune suppression. In various embodiments, the tolerance state is determined by assaying gene expression associated with immune activation. In various embodiments, the immune tolerance state is determined by assaying gene expression associated with immune regulatory function. In various embodiments, nucleic acid analysis is used to generate an immune tolerance signature. Several methodologies for high-throughput gene expression analysis have been described in the literature, including RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), exome sequencing, and microarray-based analysis.

[0133] The biological sample is optionally assayed after in vivo and / or ex vivo stimulation with one or more stimuli, such as antigens, allergens, and one or more activating agents. It is contemplated that the T cells, B cells, and immunoglobulins used in the assay are antigen-specific. Exemplary T cells include effector memory T cells, antigen-specific T cells, activated antigen-specific T cells, Th1 cells, pathogenic Th2a+ cells, Th17 cells, T follicular helper (TFH) cells, TH0 cells, or other antigen-specific T cells. B cells include effector B cells, memory B cells, plasma cells, and regulatory B (Breg) cells.

[0134] In various embodiments, the subject's immune tolerance state is determined by obtaining one or more samples, such as whole blood, from the subject from before administration on the day of the first TIMP administration (day 1) and on the day after administration.The whole blood can then be processed to isolate peripheral blood mononuclear cells (PBMCs), basophils, neutrophils, plasma, and serum for downstream analysis.The cells isolated from the one or more samples taken from the subject are assayed and analyzed.

[0135] Pharmaceutical preparations The pharmaceutical compositions of the present disclosure containing the TIMPs and antigens described herein may contain pharma- ceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, pharma- ceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacryl, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, Vaseline®, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharma- ceutically acceptable surfactants, and the like. The additives used are selected from the above or combinations thereof as necessary depending on the dosage form of the present disclosure, but are not limited thereto.

[0136] The formulation of pharmaceutical compositions will vary according to the route of administration (e.g., solution, emulsion) selected. A suitable composition containing the therapeutic agent to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient, or electrolyte replenishers.

[0137] Various aqueous carriers, e.g., sterile phosphate buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, may contain other proteins for enhanced stability, such as albumin, lipoproteins, globulins, and the like, that have been subjected to minor chemical modifications, and the like.

[0138] Therapeutic formulations of the inhibitors are prepared for storage by mixing the inhibitors having the desired purity, in the form of a lyophilized formulation or aqueous solution, with any physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or other soluble amines. or proteins such as immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0139] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0140] Aqueous suspensions may contain the active compound mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents may be naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyl-enoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as polyoxyethylene monostearate sorbitol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, such as polyethylene sorbitan monostearate. Aqueous suspensions may also contain one or more preservatives, such as ethyl, or n-propyl, p-hydroxybenzoate.

[0141] TIMPs containing the antigens described herein can be lyophilized for storage and reconstituted in a suitable carrier prior to use.

[0142] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the modified particles are mixed with at least one inert, pharma- ceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate, and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium laurate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0143] kit The present disclosure also provides kits that include one or more compounds or compositions packaged in a manner that facilitates their use to carry out the methods of the present disclosure. In one embodiment, such kits include the compounds or compositions described herein (e.g., compositions that include TIMPs alone or in combination with another agent) packaged in a container, such as a sealed bottle or vessel, with a label attached to the container or included in the package that describes the use of the compound or composition in carrying out the method. Preferably, the compounds or compositions are packaged in unit dosage form. The kits may further include a suitable device for administering the composition according to a specific route of administration or for carrying out a screening assay. Preferably, the kits include a label that describes the use of the particle composition.

[0144] Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. EXAMPLES

[0145] Example 1 - Efficacy of TIMPs to improve expression after AAV8-GFP administration and allow re-administration of AAV8-GFP We investigated the efficacy of TIMPs encapsulating recombinant eGFP (CNP-GFP) and the AAV8 VP1 capsid protein (CNP-VP1) (CNP-GFP+CNP-VP1) in inducing antigen-specific tolerance and improving transgene expression upon initial administration and re-administration with rAAV8-GFP vector.

[0146] The particles used in this test had an average diameter of 400-800 nm and a zeta potential of -30 to -80 mV.

[0147] Female BALB / c mice (6-8 weeks old, n=10 / group) were intravenously injected with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded CNP (control) on days -7 and 0. Mice were administered rAAV8-eGFP via intramuscular injection in the left quadriceps on day 0. Mice were monitored for expression of the transgene eGFP in the primed left quadriceps by transcutaneous fluorescence spectroscopy on days 0, 3, 7, 14, 21, and 28. As shown in Figure 1, treatment with CNP-GFP+CNP-VP1 resulted in significantly higher GFP expression compared to the control (*p<0.05, ***p<0.001, ****p<0.0001).

[0148] On day 28, mice were re-injected intramuscularly with AAV8-GFP in the contralateral right quadriceps (N=5 from each group). Seven days after re-injection (day 35), mice were euthanized and both the left and right quadriceps were harvested for analysis. AAV8-VP1 and GFP-specific CD8 +The frequency and total cell number of T cells were evaluated. As shown in Figure 2, treatment with CNP-GFP+CNP-VP1 significantly decreased the number of VP1- and GFP-specific CD8+ T cells in both the initial tissue (left quadriceps) after AAV8-GFP administration and the tissue (right quadriceps) after AAV8-GFP re-administration (*p<0.05, **p<0.01).

[0149] On day 35, the remaining mice in each treatment group (N=5 / group) were re-administered intramuscularly with AAV8-GFP into the contralateral right quadriceps and eGFP expression in the right quadriceps was monitored by transcutaneous fluorescence spectroscopy. As shown in Figure 3, mice treated with CNP-GFP+CNP-VP1 showed significantly higher eGFP expression in the right quadriceps compared to controls on days 14 and 21 after re-administration of AAV8-GFP (**p<0.01, ****p<0.0001). Compared to control treatment, CNP-GFP+CNP-VP1 treatment resulted in a 58% increase in GFP expression from days 14 to 21.

[0150] Based on these results, treatment with CNP-GFP+CNP-VP1 can inhibit the production of anti-VP1 and anti-GFP antibodies. Inhibition of antibody responses is measured from blood using ELISA.

[0151] The results of this study show that TIMPs encapsulating AAV8 VP1 capsid protein and transgene products are effective in inducing antigen-specific tolerance resulting in improved transgene expression and allowing re-challenge of AAV8-GFP.

[0152] Example 2 - Intravenous administration of TIMPs encapsulating AAV8-GFP results in systemic tolerance. We investigated the efficacy of TIMPs encapsulating recombinant eGFP (CNP-GFP) and AAV8 VP1 capsid protein (CNP-VP1) in inducing antigen-specific tolerance upon intravenous administration in a therapeutic mouse model.

[0153] The particles used in this test had an average diameter of 400-800 nm and a zeta potential of -30 to -80 mV.

[0154] C57BL / 6 mice (6-8 weeks old, n=5) were primed with either PBS or empty AAV8 capsid on day 0. Mice received two intravenous doses of CNP-GFP (1.25 mg / mouse)+CNP-VP1 (1.25 mg / mouse) on days 14 and 21. Mice were challenged by intravenous AAV8-GFP administration on day 28. Mice were euthanized and final readouts were taken on day 53.

[0155] Mice treated with CNP have reduced immune infiltration in the heart compared to control mice (Figure 4A).

[0156] As shown in Figure 4B, CD4+ T cells obtained from mice treated with CNP show increased regulatory phenotype in the spleen, liver, and heart (Figure 4B). Mice treated with CNP have increased numbers of CD4+ T cells expressing regulatory markers (Foxp3+ and Ctla4+) in the spleen and liver compared to control-treated mice. Mice receiving CNP therapy also have increased numbers of CD4+ cells expressing the regulatory cytokine IL-10+ and CD44hi memory markers in the spleen (Figure 4B). Mice treated with CNP have increased CD4+ T cells with memory phenotype (CD44hi, CD44hi IFN-gamma, and CD44hi IFN-gamma-) and increased regulatory cells (CD4+Foxp3+, CD4+PD-1+, CD4+CTLA4+, CD4+IL-10+) in the heart. (*p<0.05, **p<0.01.)

[0157] As shown in Figure 4C, mice therapeutically treated with CNP and re-challenged with AAV had reduced numbers of effector CD8+ T cells (CD8+ granzyme+, CD8+ PD-1+, CD8+ CD69+) infiltrating the heart compared to control-treated mice (Figure 4C) (*p<0.05, **p<0.01, ***p<0.001).

[0158] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It is understood that the disclosed invention is not limited to the specific methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the appended claims.

[0159] Those skilled in the art will recognize, or be able to determine, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

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Claims

1. A composition comprising negatively charged particles that encapsulate an antigen, The antigen is one or more gene therapy vector antigens, a portion thereof, or a combination thereof, and / or one or more transgene protein products expressed from a gene therapy vector, a portion thereof, or a combination thereof. The particles have a diameter of 300 nm to 1000 nm and a zeta potential of -30 to -100 mV. composition.

2. The composition according to claim 1, wherein the particles comprise poly(lactide-co-glycolide) (PLG), polylactic acid (PLA), a copolymer of PLG and PLA (PLGA), polycaprolactone (PCL), polystyrene, polyethylene glycol (PEG), chitosan, polysaccharide, one or more lipids, iron, zinc, cadmium, gold, or silver.

3. The composition according to claim 1, wherein the particles (i) have a zeta potential of -30 mV to -100 mV or -30 to -80 mV, and / or (ii) have a diameter of 300 nm to 1000 nm or 400 to 800 nm.

4. The gene therapy vector is a viral vector, Optionally, the viral vector is adenovirus, adeno-associated virus (AAV), herpes simplex virus, hepatitis B virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, coxsackievirus, transfusion-transmitted virus, anestrosomal, human papillomavirus, poxvirus, vaccinia virus, modified Ankara virus, varicella stomatitis virus, picornavirus, tobacco mosaic virus, cowpea mosaic virus, cowpea chlorotic mottle virus, physalis mosaic virus, red clover necrotic mosaic virus, potato virus x, comovirus, chicken anemia virus, or cucumber mosaic virus. Arbitrarily, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-12, Anc80, Synthetic AAV, combinations thereof, manipulated or modified versions thereof, The composition according to claim 1.

5. The antigen is associated with one or more viral vector capsid proteins, peptides, and / or their antigenic epitopes. Optionally, the capsid protein is VP-1, VP-2, VP-3, a combination thereof, or a part thereof. The composition according to claim 1.

6. The composition according to claim 1, wherein one or more transgene protein products expressed from the vector are enzymes selected from the group consisting of gene editing nuclease enzymes, meganucleases, homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases (TALEN), and CRISPR-related nucleases.

7. A pharmaceutical composition comprising negatively charged particles encapsulating an antigen, to be administered to a subject for use in inducing tolerance in a subject requiring tolerance induction, wherein the antigen is one or more gene therapy vector antigens and / or one or more transgene protein products expressed from a gene therapy vector, a portion thereof or a combination thereof. Optionally, the composition may be administered in combination with one or more therapeutic agents. Pharmaceutical composition.

8. The negatively charged particles encapsulating one or more gene therapy vector antigens are administered in combination with negatively charged particles encapsulating one or more transgene protein products expressed from the gene therapy vector. Optionally, negatively charged particles encapsulating one or more gene therapy vector antigens are administered before, simultaneously with, or after the administration of negatively charged particles encapsulating one or more transgene protein products produced by the gene therapy vector. A pharmaceutical composition for use according to claim 7.

9. The pharmaceutical composition for use according to claim 7, wherein the particles comprise poly(lactide-co-glycolide) (PLG), polylactic acid (PLA), a copolymer of PLG and PLA (PLGA), polycaprolactone (PCL), polyethylene glycol (PEG), polystyrene, chitosan, polysaccharide or one or more lipids, iron, zinc, cadmium, gold or silver.

10. The particle has a negative zeta potential, and optionally, (i) The zeta potential is 0 mV to -100 mV or -30 mV to -80 mV, and / or (ii) The size of the particles is 100 nm to 1000 nm or 400 nm to 800 nm. A pharmaceutical composition for use according to claim 7.

11. The gene therapy vector is a viral vector, Optionally, the viral vector is adenovirus, adeno-associated virus (AAV), herpes simplex virus, hepatitis B virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, coxsackievirus, transfusion-transmitted virus, anestrosomal, human papillomavirus, poxvirus, vaccinia virus, modified Ankara virus, varicella stomatitis virus, picornavirus, tobacco mosaic virus, cowpea mosaic virus, cowpea chlorotic mottle virus, physalis mosaic virus, red clover necrotic mosaic virus, potato virus x, comovirus, chicken anemia virus, or cucumber mosaic virus. Optionally, the AAV is AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-12, Anc80, a composite AAV, a combination thereof, or an operated manner thereof. A pharmaceutical composition for use according to claim 7.

12. The aforementioned conditions include cancer, autoimmune diseases, allergies, cardiovascular diseases, metabolic diseases, diabetes, enzyme deficiencies, protein deficiencies, cystic fibrosis, blood disorders, beta-thalassemia, sickle cell disease, hemophilia A, hemophilia B, lysosomal storage disorders, Fabry disease, Gaucher disease, Pompe disease, Niemann-Pick disease, Tay-Sachs disease, macular degeneration, mucopolysaccharidosis, venous thromboembolism, von Willebrand disease, fulminant purpura, growth hormone deficiency, gangliosidosis, hypoalkaline phosphatasia, and cholesterol ester accumulation. Physiological conditions, hyperuricemia, Duchenne muscular dystrophy, Huntington's disease, Parkinson's disease, Alzheimer's disease, choroidemia, Stargardt disease, Batten disease, spinocerebellar ataxia, ALS, frontotemporal lobar degeneration, ornithine transcarbamylase deficiency, retinitis pigmentosa, RPE-65 mutation-related disorders, epidermolysis bullosa, recessive dystrophic epidermolysis bullosa, spinal muscular atrophy, phenylketonuria (PKU), X-linked myotubular myopathy, Crigler-Nadjar syndrome, catecholamine-induced polymorphic ventricular tachycardia, glycogen storage disease type 1 Alpha-mannosidosis, Fragile X syndrome, Arginase deficiency, X-linked chronic granulomatosis, Adenosine deaminase deficiency, Leber congenital amaurosis, Lipoprotein lipase deficiency, Cerebral adrenoleukodystrophy, Metachromatic leukodystrophy, Fanconi anemia, Color blindness, Scleroderma, Osteogenesis imperfecta, Coronary artery disease, Tyrosinemia, Peripheral sensory neuropathy, Optic neuropathy, Coronary artery disease, Respiratory syncytial virus (RSV)-mediated lower respiratory tract disease, Danon disease, Severe leukocyte adhesion disorder, Pyruvate kinase deficiency, Charcot-Marie-Tooth disease, Wiscot-Aldrich syndrome, alpha-synuclein tauopathy, refractory angina due to myocardial ischemia, myotonic dystrophy type I, claudication, peripheral artery disease, methylmalonic acidemia, sucrase-isomaltase deficiency, Niemann-Pick disease type B, α1-PI deficiency, hereditary angioedema, fibrinogen deficiency, factor VIIa deficiency, factor X deficiency, factor XI deficiency, factor XII deficiency, protein C deficiency, antithrombin III deficiency, MPS I, MPS II, MPS III, MPS IV, MPS VI, MPS VII, MPSA pharmaceutical composition for use according to claim 7, having IX, calcification / ossification disorders, ENPP1 deficiency, ENPP3 deficiency, ABCC6 deficiency, aromatic L-amino acid decarboxylase deficiency, Angelman syndrome, hyperphenylalaninemia, dementia, Rett syndrome, and Usher syndrome.

13. The pharmaceutical composition for use according to claim 7, wherein the composition is administered before, after, or concurrently with the gene therapy vector.

14. Administration of the composition reduces the immune response to the gene therapy vector and / or the transgene protein product expressed from the vector. Optionally, the immune response may be an inflammatory immune response, a humoral immune response, an adaptive immune response, an innate immune response, a T cell, B cell, monocyte, macrophage, neutrophil, basophil, or eosinophil response, or an antibody response. A pharmaceutical composition for use according to claim 7.

15. The pharmaceutical composition for use according to claim 7, wherein administration of the composition induces an immunomodulatory response to the gene therapy vector and / or the transgene protein product expressed from the vector, and optionally the immunomodulatory response is regulatory T cells, B cells, monocytes, macrophages, antigen-specific Treg, Tr1, Mreg, and / or Breg cells.

16. The pharmaceutical composition for use according to claim 7, wherein the composition is administered intravenously, intramuscularly, intraocularly, intraperitoneally, transdermally, transnasally, orally and / or subcutaneously.

17. A composition comprising liposomes encapsulating one or more gene therapy vector antigens, a portion thereof or a combination thereof, or liposomes encapsulating a therapeutic protein produced by a gene therapy vector and / or a portion thereof, wherein the liposomes have a diameter of 300 nm to 1000 nm and a zeta potential of -30 to -100 mV.

18. The composition according to claim 17, wherein the negative zeta potential is -80 mV to -30 mV.

19. A composition according to any one of claims 1 to 6 or 17 to 18, comprising negatively charged liposomes for encapsulating an antigen for use in inducing tolerance in a subject, wherein the antigen is one or more gene therapy vector antigens and / or one or more transgene protein products expressed from a gene therapy vector, a portion thereof or a combination thereof, and optionally, the composition is administered in combination with one or more therapeutic agents.

20. The therapeutic agent is selected from the group consisting of immunosuppressants, complement inhibitors, calcineurin inhibitors, plasmapheresis, IgG proteases, proteasome inhibitors, and / or regulatory T cell inducers, and optionally the therapeutic agent is an FcRn inhibitor, corticosteroid, rapamycin / sirolimus, cyclophosphamide, mycophenolate mofetil, anti-CD20 CAR T cell therapy, anti-CD19 CAR T cell therapy, anti-BCMA CAR T cell therapy, anti-CD20 antibody, anti-CD19 antibody, anti-CD40 antibody, or CTLA4 IgG. A pharmaceutical composition for use according to claim 19.

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