Tolerant immunomodified nanoparticles for overcoming immunogenicity of therapeutic carriers and proteins

Abstract

The present application generally relates to tolerant immune-mediating particles comprising gene therapy vector antigens for use in combination with a gene therapy regimen in order to reduce immunogenicity against the gene therapy vector antigens and / or transgenic gene protein products expressed by the vector.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 336,754 filed on April 29, 2022 and U.S. Provisional Patent Application No. 63 / 343,797 filed on May 19, 2022, which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application generally relates to tolerogenic 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 transgenic gene protein products expressed by the vector. Background Art

[0004] Advances in the design and development of gene delivery vectors, such as retroviral, lentiviral, adenoviral, and adeno-associated viral (AAV) vectors, have made gene therapy an attractive treatment option for a variety of diseases and conditions, including rare and inherited genetic disorders, autoimmune diseases, neurodegenerative conditions, and cancer. Gene therapy relies on vector-mediated delivery of exogenous DNA encoding therapeutic proteins, such as enzymes, cytokines, and antibodies. 1-6 The goal of gene therapy is to enable sustained tissue-specific production of therapeutic proteins to achieve long-term therapeutic efficacy.

[0005] In addition to the production of proteins and enzymes for the treatment of rare and inherited genetic disorders (e.g., hemophilia, Pompe Disease, Fabry Disease, and mucopolysaccharidoses), gene therapy vectors are also used for tissue-specific expression of self-antigens and tolerogenic 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 anti-tumor immune responses for the treatment of cancer. 8,9 .

[0006] Gene therapy vectors in use and currently in development for gene therapy applications are typically modified and / or engineered versions of naturally occurring retroviruses, lentiviruses, adenoviruses, and AAVs. 10 Several synthetic vectors have also been described in the literature 11 To achieve sustained therapeutic efficacy, viral vectors must be re-administered periodically 19,20 .

[0007] The immunogenicity of gene therapy vectors and therapeutic proteins expressed from the vectors presents a significant clinical challenge to the periodic re-administration required for sustained therapeutic benefit, since both the vectors and the therapeutic proteins produced by the vectors are rapidly cleared following administration. In addition, a significant portion of patients who could benefit from gene therapy exhibit pre-existing immunity to the gene therapy vectors, making them ineligible for treatment. 12-14 Patients without pre-existing neutralizing antibodies are also affected by the immunogenicity of the vector and therapeutic protein, as the initial vector administration induces an immune response to the vector protein that interferes with any re-administration required for sustained therapeutic benefit.

[0008] Current strategies aimed at preventing the immunogenicity of viral vectors rely on the concurrent administration of immunosuppressive regimens that have significant side effects, such as increased risk of infection and even death. 15 . Summary of the invention

[0009] The use of TIMPs to induce antigen-specific T cells tolerant to gene therapy vectors and transgene protein products produced by the vectors could potentially overcome the immunogenicity problems associated with gene therapy, enabling re-administration and resulting in improved therapeutic efficacy.

[0010] The present disclosure describes compositions and methods for inducing antigen-specific tolerance to gene therapy vectors and transgenic protein products produced by the vectors. The compositions provided herein comprise negatively charged carrier particles encapsulating antigens, wherein the antigens are one or more gene therapy vector antigens and / or portions thereof, or combinations of gene therapy vector antigens or portions thereof. In various embodiments, the antigens are therapeutic proteins and / or portions thereof or one or more antigenic epitopes thereof produced by the gene therapy vectors. In various embodiments, the carrier particles comprise polymers and have a negative zeta potential. In various embodiments, the polymers are biodegradable polymers.

[0011] 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 acid) (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, liposomes, PEG, cyclodextran, a lipid or a metal such as iron (Fe), zinc (Zn), cadmium (Cd), gold or silver or a combination thereof.

[0012] In various embodiments, the polymer is a copolymer. In various embodiments, the copolymer has constituent polymers in different molar ratios. 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.

[0013] 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 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 polylactic acid:polyglycolic acid, including all values ​​and ranges therebetween.

[0014] 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 -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, -50 mV, -55 mV, -60 mV, -65 mV, -70 mV, -75 mV, -80 mV, -85 mV, -90 mV, -95 mV, or -100 mV, including all values ​​and ranges therein. In various embodiments, the negative zeta potential of the carrier particles is between -30 mV and -80 mV. In various embodiments, the negative zeta potential of the carrier particles is between -30 mV and -60 mV. In various embodiments, the negative zeta potential is achieved by surface functionalization of the carrier particles. In various embodiments, the surface functionalization is carboxylation.

[0015] In various embodiments, the size or diameter of the carrier particles is between 0.05 μm and about 10 μm. In various embodiments, the diameter of the carrier particles is between 0.1 μm and about 10 μm. In various embodiments, the diameter of the carrier particles is between 0.1 μm and about 5 μm. In various embodiments, the diameter of the carrier particles is between 0.1 μm and about 3 μm. In various embodiments, the diameter of the carrier particles is between 0.3 μm and about 5 μm. In various embodiments, the diameter of the carrier particles is about 0.3 μm to about 3 μm. In various embodiments, the diameter of the carrier particles is between about 0.3 μm and about 1 μm. In various embodiments, the diameter of the carrier particles is between about 0.4 μm and about 1 μm. In various embodiments, the diameter of the carrier particle is about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000 nm, about 100 to 1500 nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm. In various embodiments, the diameter of the carrier particle is 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 diameter of the negatively charged particles is between 400 nm and 800 nm. In various embodiments, the diameter of the negatively charged particles is between 350 nm and 800 nm.

[0016] In various embodiments, the carrier particles have a uniform size distribution. In various embodiments, the carrier particles have a uniform size distribution, wherein at least 90% of the particles have a diameter between 0.05 μm and about 10 μm, between 0.1 μm and about 10 μm, between 0.1 μm and about 5 μm, between 0.1 μm and about 3 μm, between 0.3 μm and about 5 μm, between 0.3 μm and about 3 μm, and between 0.3 μm and about 3 μm, including all values ​​and ranges therein. In various embodiments, the carrier particles have a uniform size distribution, wherein at least 90% of the particles have a diameter of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000 nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm, including all values ​​and ranges therein. In various embodiments, the diameter of the carrier particles is 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 uniform size distribution, wherein at least 50% of the particles have a diameter between about 0.05 µm and about 10 µm, between about 0.1 µm and about 10 µm, between about 0.1 µm and about 5 µm, between about 0.1 µm and about 3 µm, between about 0.3 µm and about 5 µm, and between about 0.3 µm and about 3 µm, including all values ​​and ranges therein. In various embodiments, the particles have a uniform size distribution, wherein at least 50% of the particles have a diameter of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000 nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm, including all values ​​and ranges therein. In various embodiments, the diameter of the carrier particles is 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 carrier particles have a uniform size distribution, wherein at least 10% of the particles have a diameter between about 0.05 µm and about 10 µm, between about 0.1 µm and about 10 µm, between about 0.1 µm and about 5 µm, between about 0.1 µm and about 3 µm, between about 0.3 µm and about 5 µm, and between about 0.3 µm and about 3 µm, including all values ​​and ranges therein. In various embodiments, the carrier particles have a uniform size distribution, wherein at least 10% of the particles have a diameter of about 100 to 10000 nm, about 100 to 5000 nm, about 100 to 3000 nm, about 100 to 2000 nm, about 300 to 5000 nm, about 300 to 3000 nm, about 300 to 1000 nm, about 300 to 800 nm, about 400 to 800 nm, or about 200 to 700 nm, including all values ​​and ranges therein. In various embodiments, the diameter of the carrier particles is 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.

[0018] In various embodiments, the particle encapsulates an antigen, has a negative zeta potential between -100 mV and 0 mV, and wherein the diameter of the particle is between 100 and 1000 nm. In various embodiments, the particle encapsulates one or more antigens comprising one or more gene therapy vector antigens, portions thereof, or combinations thereof, and wherein the size of the particle is between 400 and 800 nm, and the particle has a negative zeta potential between -30 mV and -80 mV.

[0019] In various embodiments, the particle encapsulates an antigen, has a negative zeta potential between -100 mV and 0 mV, and wherein the diameter of the particle is between 100 and 1000 nm. In various embodiments, the particle encapsulates one or more antigens comprising one or more protein therapeutics produced by a gene therapy vector, a portion thereof, or a combination thereof, and wherein the size of the particle is between 400 and 800 nm, and the negative zeta potential of the particle is between -30 mV and -80 mV.

[0020] In various embodiments, the particles encapsulate one or more gene therapy vector antigens, portions or combinations thereof, and one or more protein therapeutics produced by the gene therapy vector, portions or combinations thereof. In various embodiments, the antigens comprise one or more proteins, peptides, or antigenic epitopes thereof.

[0021] Also contemplated herein is a composition comprising liposomes encapsulating one or more gene therapy vector antigens, portions thereof, or combinations thereof.

[0022] In another embodiment, the present disclosure provides a composition comprising liposomes encapsulating a therapeutic protein and / or a portion thereof produced by a gene therapy vector.

[0023] In various embodiments, the liposome is negatively charged. In various embodiments, the liposome has a negative zeta potential. In various embodiments, the negative zeta potential is between -100 mV and 0 mV. In various embodiments, the zeta potential of the negatively charged liposome 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 -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, -50 mV, -55 mV, -60 mV, -65 mV, -70 mV, -75 mV, -80 mV, -85 mV, -90 mV, -95 mV or -100 mV, including all values ​​and ranges therein. In various embodiments, the negative zeta potential of the liposome is between -30 mV and -80 mV. In various embodiments, the negative zeta potential of the liposome is between -30 mV and -60 mV.

[0024] In various embodiments, the liposome is between 100-1000 nm, or between 300-800 nm, or between 400-800 nm. In various embodiments, the liposome is 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.

[0025] In various embodiments, the antigen comprises a gene therapy vector antigen or one or more parts 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, anellovirus, 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 x virus, cowpea mosaic virus, 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.

[0026] In various embodiments, the particle encapsulates an antigen, wherein the antigen is one or more AAV vectors, portions thereof, 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 thereof, 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.

[0027] In various embodiments, the gene therapy vector is a bacterium, a bacteriophage, a yeast, an exosome, or an erythrocyte.

[0028] In various embodiments, the gene therapy vector is used to treat 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, mucopolysaccharidosis, venous thrombosis, von Willebrand disease, purpura fulminans, growth hormone deficiency, gangliosidosis, alkaline hypophosphatasia, cholesterol ester storage disease, hyperuricemia, Duchenne Muscular Dystrophy, Dystrophy, Huntington's disease, Parkinson's, 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), Syndymodulin myopathy, Crigler-Najjar syndrome syndrome, catecholamine-sensitive polymorphic ventricular tachycardia, glycogen storage disease type 1, alpha-mannosidosis, fragile X syndrome, arginase deficiency, X-linked chronic granulomatous disease, adenosine deaminase deficiency, Leber's congenital amaurosis, lipoprotein lipase deficiency, cerebral adrenoleukodystrophy, metachromatic leukodystrophy, Fanconi anemia, achromatopsia, scleroderma, osteogenesis imperfecta, coronary artery disease, tyrosinemia, peripheral neuropathy, optic neuropathy, coronary artery disease, respiratory syncytial virus (RSV)-mediated lower respiratory tract disease, Danon disease, severe leukocyte adhesion defect, pyruvate kinase deficiency, Charcot Marie Tooth disease, Wiskott Aldrich syndromesyndrome), alpha-synuclein tauopathy, refractory angina due to myocardial ischemia, myotonic dystrophy type I, claudication, peripheral arterial disease, methylmalonic acidemia, sucrase-isomaltase deficiency, Niemann-Pick type B disease, alpha-1-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, MPS IX, calcification / ossification disorders, ENPP1 deficiency, ENPP3 deficiency, ABCC6 deficiency, aromatic L-amino acid decarboxylase deficiency, Angelman syndrome, hyperphenylalaninemia, dementia, and Rett syndrome and Usher syndrome.

[0029] 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, Reynaud's syndrome, Behcet's syndrome, Sjorgen's syndrome, autoimmune uveitis, Eaton Lambert's disease, autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus, neuromyelitis optica, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, myasthenia gravis, celiac disease, ulcerative colitis, Crohn's disease, disease, erythema nodosum, glomerulonephritis, Goodpasture's syndrome, granulomatosis, Grave's disease, Guillain-Barre syndrome, Hashimoto disease, hemolytic anemia, Kawasaki disease, mixed connective tissue disease, multifocal motor neuropathy, peripheral biliary cirrhosis, polyangiitis overlap syndrome, scleroderma type 1, sclerosing cholangitis, stiff-person syndrome, Takayasu arteritis, vitiligo, or Wegeners granulomatosis.

[0030] 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 dairy 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.

[0031] In various embodiments, the gene therapy vector is used to treat cancer. 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.

[0032] In various embodiments, the therapeutic agent produced by the gene therapy vector is a protein, polypeptide or peptide. In various embodiments, the protein is a cytokine, chemokine, hormone, growth factor, enzyme or antibody.

[0033] 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: antibody-protein fusions, immunoadhesins, monospecific antibodies, bispecific antibodies, trispecific antibodies. In various embodiments, the antibody is selected from the group consisting of: antibody fragments, antigen binding fragments (Fab), single chain variable fragments (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 antibodies target 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, CCL1 5. CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2(MCP-1), CXCL3 (MIP-1α, CXCL4 (MIP-1β, CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, G M-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, TNF-a, TNF-β, TGF-β1, TGF-β2, TGF-β3, LT-β, ​​4-1BBL, APRIL, GITRL, LIGH T, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, C D16, 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, SLAMF2OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, A ITR, 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, RAE1α, 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 alpha, beta, gamma, delta, epsilon, zeta chains and / or combinations thereof. ,

[0034] In various embodiments, the antibody targets one or more immune cells. In various embodiments, the immune cells 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.

[0035] In various embodiments, the antibody is selected from the group consisting of abciximab, adalimumab, alemtuzumab, avelumab, atezolizumab, basiliximab, bevacizumab, bezlotoxumab, blinatumomab, canakinumab, certolizumab, cetuximab, daclizumab, denosumab, durvalumab, efalizumab, emicizumab, etokimab, golimumab, imumab, ipilimumab, ixekizumab, infliximab, natalizumab, nivolumab, olaratumab, omalizumab, ofatumumab, palivizumab, panitumumab, pembrolizumab, ramucirumab, rituximab, tocilizumab, trastuzumab, tremelimumab, secukinumab, ustekinumab, and vedolizumab.

[0036] In various embodiments, the therapeutic agent produced by the vector is a cytokine or a chemokine. In various embodiments, the cytokines and chemokines are selected from the group consisting of 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-2 8. 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, CCL 12. CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 (MIP-1α, CXCL4 (MIP-1β, CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, GM-CSF, IFN-α, IFN-β , IFN-γ, TNF-α, TNF-a, TNF-b, TGF-β1, TGF-β2, TGF-β3, LT-β, ​​4-1BBL, APRIL, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK and TRANCE.

[0037] In various embodiments, the protein is a hormone selected from the group consisting of: epinephrine, melatonin, noradrenaline, norepinephrine, triiodothyronine, thyroxine, dopamine, prostaglandins, leukotrienes, prostaglandins, thromboxanes, amylin, anti-Müllerianhormone, adiponectin, adrenocorticotropic hormone, angiotensinogen, angiotensin, 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 hormone, 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 polypeptide, guanylin, uroguanylin, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone, estriol, cortisol, progesterone, calcitriol, and calcifediol.

[0038] In various embodiments, the protein is a growth factor selected from the group consisting of adrenomedullin, angiogenin, autocrine motility 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, 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 17, fibroblast growth factor 18, fibroblast growth factor 19, fibroblast growth factor 20, fibroblast growth factor 21, fibroblast growth factor 22, In some embodiments, the cytokine is selected from the group consisting of: miscellaneous hematopoietic cells, Epo, Tpo, Flt-3L, SCF, M-CSF, and MSP.

[0039] 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.

[0040] In various embodiments, the therapeutic agent produced by the vector is an enzyme. In various embodiments, the enzyme is selected from the group consisting of: gene editing nucleases, meganucleases, homing endonucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), CRISPR-associated nucleases, and CRISPR-Cas9.

[0041] In various embodiments, the Cas protein is selected from the group consisting of: Cas 3, Cas8a, Cas5, Cas8b, Cas8c, Cas 9, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, 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.

[0042] In various embodiments, the enzyme is selected from the group consisting of: imiglucerase, taliglucerase alfa, velaglucerase alfa, β-glucocerebrosidase, alglucerase, agalsidase beta, agalsidase alpha, sebelipase alpha, alpha-L-iduronidase, human iduronate-2-sulfatase, N-sulfoglucosamine sulfohydrolase, elosulfase alpha. alpha), galsulfase, alpha-glucosidase (GAA), human alpha-mannosidase, factor VIII, factor IX, beta-galactosidase, arginase, myotonic 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, heparan sulfate sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, alpha-1 proteinase inhibitor, 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.

[0043] In various embodiments, the enzyme is a protease. In various embodiments, the protease is an aspartic 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, ADAM20, 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.

[0044] In various embodiments, the therapeutic agent produced by the vector is a protein. In various embodiments, the protein is selected from the group consisting of: light-activatable channel rhodopsin, tumor protein 53, HIV Gag protein, fragile X chromosome mental retardation protein, beta globulin, fibroblast growth factor 4, human growth factor, Wisckott Aldrich syndrome protein, survival motor neuron protein 1, survival motor neuron 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, type I collagen, type III and type V collagen, β2 integrin, lysosomal-associated membrane protein, dystrophin, mini-dystrophin, insulin-like growth factor, T-cell immunomodulatory factor 1 / ATPase H+ transport V0 subunit A3 pancreatic and duodenal homeobox 1, MAFBZIP transcription factor A, cystic fibrosis transmembrane conductance regulator, ATP-binding cassette family C member 6, ATP-binding cassette family A member 4, vascular endothelial growth factor A, serine protease inhibitor family A member 1, progranulin, microtubule-associated protein tau, C9 or f72 protein, and gap junction b2.

[0045] The present disclosure provides a method for inducing antigen-specific immune tolerance using TIMP-encapsulated antigens, the method comprising administering to a subject a composition of negatively charged particles comprising encapsulated antigens, wherein the antigen is one or more gene therapy vector antigens and / or one or more transgenic 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 a transgenic protein produced by a vector, a portion thereof, or a combination thereof. In various embodiments, administering to a subject in need thereof a TIMP encapsulating one or more antigens induces antigen-specific tolerance.

[0046] Also provided is a method of inducing tolerance in a subject in need thereof, the method comprising administering to the subject a composition comprising a liposome as described herein.

[0047] In various embodiments, a TIMP that encapsulates one or more gene therapy vector antigens is administered to a subject in combination with a TIMP that encapsulates one or more transgene protein products produced by a gene therapy vector. Administration of a TIMP that encapsulates a gene therapy vector antigen in combination with a TIMP that encapsulates a transgene protein product produced by a 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, a TIMP that encapsulates one or more gene therapy vector antigens is administered to a subject before, simultaneously with, or after administration of a TIMP that encapsulates one or more transgene protein products produced by a gene therapy vector to a subject.

[0048] 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 administering 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 administering 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 administering 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 one week, two weeks, three weeks, four 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 transgene protein products produced by the gene therapy vector.

[0049] In various embodiments, the TIMP encapsulating one or more gene therapy vector antigens is administered 5, 10, 15, 30, 45, or 60 minutes after the 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 the 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 the 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 one week, two weeks, three weeks, four 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 TIMP encapsulating one or more transgene protein products produced by the gene therapy vector.

[0050] In various embodiments, the TIMP is administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally. In various embodiments, the TIMP is administered prior to, concurrently with, or after administration of the gene therapy vector.

[0051] In various embodiments, the TIMP is administered 5, 10, 15, 30, 45, or 60 minutes prior to the 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 the administration of the gene therapy vector. In various embodiments, the TIMP is administered 1, 2, 3, 4, 5, 6, or 7 days prior to the administration of the gene therapy vector. In various embodiments, the TIMP is administered one week, two weeks, three weeks, four 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 the administration of the gene therapy vector.

[0052] In various embodiments, the TIMP is administered 5, 10, 15, 30, 45, or 60 minutes after the 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 the administration of the gene therapy vector. In various embodiments, the TIMP is administered 1, 2, 3, 4, 5, 6, or 7 days after the administration of the gene therapy vector. In various embodiments, the TIMP is administered one week, two weeks, three weeks, four 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 the administration of the gene therapy vector.

[0053] In various embodiments, the administration of TIMP induces antigen-specific tolerance to gene therapy vector antigens, portions thereof, or combinations thereof in subjects in need. In various embodiments, the administration of TIMP induces antigen-specific tolerance to transgenic protein products, portions thereof, or combinations thereof in subjects in need. In various embodiments, the administration of TIMP reduces the inflammatory immune response to gene therapy vectors and / or transgenic protein products expressed by vectors in subjects. In various embodiments, the administration of TIMP reduces the inflammatory immune response to one or more encapsulated antigens in subjects. In various embodiments, the administration of TIMP reduces the inflammatory response to one or more antigens not encapsulated in particles in subjects in need. This type of immunomodulatory response is referred to as 'infectious tolerance' in the literature. 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, such as the formation of neutralizing antibodies against gene therapy vector antigens and / or transgenic proteins 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 a reduction in immune infiltration of tissues and / or organs.

[0054] In various embodiments, administration of TIMP induces antigen-specific immunomodulatory factor responses in subjects in need thereof. In various embodiments, administration of TIMP induces regulatory T cells, B cells, monocytes and / or macrophages in subjects. In various embodiments, administration of TIMP induces antigen-specific regulatory T cells (Treg), Tr1, regulatory macrophages (Mreg) and / or regulatory B cells (Breg) cells in subjects.

[0055] In various embodiments, liposomes encapsulating one or more gene therapy vector antigens are administered to a subject in combination with liposomes encapsulating one or more transgene protein products produced by a gene therapy vector. Administration of liposomes encapsulating gene therapy vector antigens in combination with liposomes encapsulating transgene protein products produced by a gene therapy vector induces antigen-specific tolerance to both gene therapy vector antigens and transgene protein products produced by a gene therapy vector. In various embodiments, liposomes encapsulating one or more gene therapy vector antigens are administered to a subject before, simultaneously with, or after administration of liposomes encapsulating one or more transgene protein products produced by a gene therapy vector to a subject.

[0056] In various embodiments, liposomes encapsulating one or more gene therapy vector antigens are administered 5, 10, 15, 30, 45, or 60 minutes prior to administering liposomes encapsulating one or more transgene protein products produced by a gene therapy vector. In various embodiments, 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 administering liposomes encapsulating one or more transgene protein products produced by a gene therapy vector. In various embodiments, liposomes encapsulating one or more gene therapy vector antigens are administered 1, 2, 3, 4, 5, 6, or 7 days prior to administering liposomes encapsulating one or more transgene protein products produced by a gene therapy vector. In various embodiments, liposomes encapsulating one or more gene therapy vector antigens are administered one week, two weeks, three weeks, four 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 transgene protein products produced by the gene therapy vector.

[0057] In various embodiments, the liposomes encapsulating one or more gene therapy vector antigens are administered 5, 10, 15, 30, 45, or 60 minutes after the 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 the 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 the 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 one week, two weeks, three weeks, four 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 liposomes encapsulating one or more transgene protein products produced by the gene therapy vector.

[0058] In various embodiments, the liposomes are administered intravenously, subcutaneously, intramuscularly, intraperitoneally, intranasally, or orally. In various embodiments, the liposomes are administered before, simultaneously with, or after administration of the gene therapy vector.

[0059] In various embodiments, the liposomes are administered 5, 10, 15, 30, 45, or 60 minutes before the 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 before the administration of the gene therapy vector. In various embodiments, the liposomes are administered 1, 2, 3, 4, 5, 6, or 7 days before the administration of the gene therapy vector. In various embodiments, the liposomes are administered one week, two weeks, three weeks, four 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 before the administration of the gene therapy vector.

[0060] In various embodiments, the liposomes are administered 5, 10, 15, 30, 45, or 60 minutes after the 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 the administration of the gene therapy vector. In various embodiments, the liposomes are administered 1, 2, 3, 4, 5, 6, or 7 days after the administration of the gene therapy vector. In various embodiments, the liposomes are administered one week, two weeks, three weeks, four 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 the administration of the gene therapy vector.

[0061] In various embodiments, the administration of liposomes induces antigen-specific tolerance to gene therapy vector antigens, portions thereof, or combinations thereof in subjects in need. In various embodiments, the administration of liposomes induces antigen-specific tolerance to transgenic protein products, portions thereof, or combinations thereof in subjects in need. In various embodiments, the administration of liposomes reduces the inflammatory immune response to gene therapy vectors and / or transgenic protein products expressed by vectors in subjects. In various embodiments, the administration of liposomes reduces the inflammatory immune response to one or more encapsulated antigens in subjects. In various embodiments, the administration of liposomes reduces the inflammatory response to one or more antigens not encapsulated in particles in subjects in need. This type of immunomodulatory response is referred to as 'infectious tolerance' in the literature. 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, such as the formation of neutralizing antibodies against gene therapy vector antigens and / or transgenic proteins produced by the vector. In various embodiments, the immune response is a T cell, B cell, NK cell monocyte, macrophage, eosinophil or basophil response.

[0062] In various embodiments, administration of liposomes induces antigen-specific immunomodulatory factor responses in subjects in need thereof. In various embodiments, administration of liposomes induces regulatory T cells, B cells, monocytes and / or macrophages in subjects. In various embodiments, administration of TIMPs induces antigen-specific Treg, Tr1, Mreg and / or Breg cells in subjects.

[0063] Also contemplated are compositions described herein 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 a gene therapy vector, portions thereof, or combinations thereof, for inducing tolerance in a subject.

[0064] The present disclosure also provides for the use of the compositions described herein comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more gene therapy vector antigens and / or one or more transgenic gene protein products produced by a gene therapy vector, a portion thereof, or a combination thereof, for the preparation of a medicament for inducing tolerance in a subject.

[0065] In various embodiments, the negatively charged particle is a TIMP or a liposome as described herein.

[0066] It should be understood that each feature or embodiment or combination described herein is a non-limiting illustrative example of any one of the aspects of the present invention, and therefore means that it can be combined with any other feature or embodiment or combination described herein. For example, in the case of describing features with language such as "one embodiment", "some embodiments", "certain embodiments", "further embodiments", "specific exemplary embodiments" and / or "another embodiment", each of these types of embodiments is a non-limiting example of a feature, and the feature is intended to be combined with any other feature or combination of features described herein without necessarily listing every possible combination. These features or combinations of features apply to any one of the aspects of the present invention. In the case of disclosing examples of values ​​falling within a range, any of these examples is considered as a possible endpoint of the range, any and all numerical values ​​between these endpoints are considered, and any and all combinations with upper and lower endpoints are envisioned.

[0067] The headings herein are for the convenience of the reader and are not intended to be limiting.Other 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

[0068] Figure 1 : Effect of CNP-GFP+CNP-VP1 treatment on GFP expression after initial administration of AAV8-GFP via intramuscular injection. On days -7 and 0, female BALB / c mice (6-8 weeks old, n=10 per group) were intravenously injected with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded CNP (control group). On day 0, rAAV8-eGFP was administered to mice via intramuscular injection in the left quadriceps. By transcutaneous fluorescence spectroscopy, the expression of transgenic eGFP in the pre-sensitized left quadriceps of mice was monitored on days 0, 3, 7, 14, 21 and 28. When compared to the control group, treatment with CNP-GFP+CNP-VP1 caused significantly higher GFP expression ( p<0.05; p<0.001; p<0.0001).

[0069] Figures 2A to 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. On days -7 and 0, female BALB / c mice (6-8 weeks old, n=10 per group) were intravenously injected with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded CNP (control group). On day 0, rAAV8-eGFP was administered to mice via intramuscular injection in the left quadriceps. On day 28, AAV8-GFP was re-administered intramuscularly to mice at the contralateral right quadriceps (N=5 in each group). Seven days after re-administration (day 35), mice were killed and right and left quadriceps were harvested for analysis. AAV8-VP1 and GFP-specific CD8 + The frequency and total cell count of T cells were assessed by flow cytometry using tetramer staining. Treatment with CNP-GFP + CNP-VP1 resulted in the initial tissue (left quadriceps) after administration of AAV8-GFP ( Figure 2A , Figure 2B ) and tissues re-administered with AAV8-GFP (right quadriceps) ( Figure 2C , Figure 2D )The number of VP1- and GFP-specific CD8+ T cells in both p<0.05; p<0.01).

[0070] Figure 3 : Effect of CNP-GFP+CNP-VP1 treatment on GFP expression after re-administration of AAV8-GFP via intramuscular injection. On days -7 and 0, female BALB / c mice (6-8 weeks old, n=10 per group) were intravenously injected with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded CNP (control group). On day 0, rAAV8-eGFP was administered to mice via intramuscular injection in the left quadriceps. On day 35, AAV8-eGFP was re-administered intramuscularly to each treatment group of mice (N=5 per group) at the contralateral right quadriceps, and eGFP expression in the right quadriceps was monitored by transcutaneous fluorescence spectroscopy. When compared to the control group, mice treated with CNP-GFP+CNP-VP1 showed significantly higher eGFP expression in the right quadriceps 14 and 21 days after re-administration of AAV8-GFP ( p<0.01; p<0.0001). CNP-GFP+CNP-VP1 treatment caused a 58% increase in GFP expression from day 14 to day 21 relative to control treatment.

[0071] Figures 4A to 4C : Systemic administration of TIMPs encapsulating AAV8-GFP induces a tolerogenic phenotype. On day 0, C57BL / 6 mice (6-8 weeks old, n=5) were pre-sensitized with PBS or empty AAV8 capsid. On days 14 and 21, mice were administered 2 intravenous doses of CNP-GFP (1.25 mg / mouse) + CNP-VP1 (1.25 mg / mouse). On day 28, mice were challenged with AAV8-eGFP by IV administration. On day 53, mice were sacrificed and final readings were taken. Compared with control mice, the hearts of CNP-treated mice had a decrease in leukocytes ( Figure 4A CD4+ T cells show an increased regulatory phenotype in the spleen, liver, and heart ( Figure 4B When compared to control-treated (PBS) mice, CNP-treated mice had increased regulatory T cells (CD4+Foxp3+ and CD4+Ctla4+) in the spleen and liver ( Figure 4B ). Expression of regulatory cytokines IL-10+ and CD44 in the spleen of mice receiving CNP therapy hi The number of memory marker CD4+ cells also increased ( Figure 4B ). CNP-treated mice had increased memory CD4+ T cells (CD44hi IFN-γ-) and increased regulatory cells (CD4+Foxp3+, CD4+PD-1+, CD4+Ctla4+, CD4+IL-10+) in the hearts ( Figure 4B ). Compared with the hearts of CNP-treated mice, the effector phenotype of CD8+ T cells was reduced (CD8+ granzyme+, CD8+ PD-1+, CD8+ CD69+) ( Figure 4C ). p<0.05; p<0.01; p<0.001). DETAILED DESCRIPTION

[0072] 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. Several methods for inducing antigen-specific tolerance have been described, but their translation into the clinic has been difficult. 16 Interventions are necessary that allow safe multiple administrations of therapeutic viral vectors and overcome challenges associated with vector immunogenicity.

[0073] The present disclosure provides compositions of negatively charged particles encapsulating one or more gene therapy vectors, parts thereof or combinations thereof. The present disclosure also provides compositions of negatively charged particles encapsulating one or more transgenic protein products produced by gene therapy vectors, parts thereof or combinations thereof. Also included are methods of inducing antigen-specific tolerance using the negatively charged particles described herein.

[0074] definition

[0075] Unless stated otherwise, the following terms used in this application, including the specification and claims, have the definitions given below.

[0076] As used in this specification and the appended claims, the indefinite articles "a," "an," and "the" include plural as well as singular referents, unless the context clearly dictates otherwise.

[0077] The term "about" or "approximately" means the acceptable error of 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. When the term "about" or "approximately" precedes the first value in a series of two or more numerical values, it is understood that the term "about" or "approximately" applies to each of the numerical values ​​in the series.

[0078] As used herein, "particle" refers to any non-tissue derived composition of matter, which may be a sphere or sphere-like entity, a bead, or a liposome. Depending on the context, the term "particle", the term "tolerogenic immune modification particle", the term "carrier particle", and the term "bead" may be used interchangeably. In addition, the term "particle" may be used to encompass beads and spheres.

[0079] As used herein, "negatively charged particles" refer to particles that have been modified to have a net surface charge of less than zero.

[0080] "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 enhances uptake of the particle from the circulation by phagocytes / monocytes, for example via interaction with scavenger receptors such as MARCO. Carboxylation of the particle can be achieved using any compound that adds carboxyl groups, including but not limited to poly(ethylene-maleic anhydride) (PEMA).

[0081] As used herein, the term "Th cell" or "helper T cell" refers to a CD4 + Cells. CD4 + T cells assist other white blood cells in the immune process, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. T cells become activated when they present peptide antigens through MHC class II molecules expressed on the surface of antigen presenting cells (APCs).

[0082] As used herein, the term "Th1 cell" refers to a subset of Th cells that produce pro-inflammatory 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-γ, IL2, IL10, and TNF α / β to coordinate defense against intracellular pathogens (such as viruses and some bacteria).

[0083] As used herein, the term "Th2 cells" refers to a subset of Th cells that mediate activation and maintenance of antibody-mediated immune responses against extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing various cytokines, such as IL-4, IL-5, IL-6, IL-9, IL-13, and IL-17E (IL-25), which are responsible for producing antibodies, activating eosinophils, and inhibiting several macrophage functions, thereby providing a protective response that is independent of phagocytes.

[0084] "Polypeptide" and "protein" refer to polymers consisting of amino acid residues connected via peptide bonds or peptide bond isosteres, related naturally occurring structural variants, and synthetic analogs thereof that are not naturally occurring. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The terms "polypeptide" and "protein" are not limited to the minimum length of the product. The term "protein" generally refers to a larger polypeptide. The term "peptide" generally refers to a shorter polypeptide. Therefore, peptides, oligopeptides, dimers, multimers, and the like are included in the definition. The definition encompasses full-length proteins and their fragments. The terms "polypeptide" and "protein" also include post-expression modifications of polypeptides or proteins, such as glycosylation, acetylation, phosphorylation, and similar modifications thereof. In addition, for the purposes of this disclosure, "polypeptides" may also include "modifications" to natural sequences, such as deletions, additions, substitutions (which may be conservative in nature or may include substitutions with any of the 20 amino acids that are commonly present in human proteins, or any other naturally or non-naturally occurring or atypical amino acids) and chemical modifications (e.g., addition of peptide mimetics or substitutions with peptide mimetics). These modifications may be deliberate, such as 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 arising from a host cell producing the protein or through errors caused by PCR amplification prior to transfection of host cells.

[0085] As used herein, "antigenic portion" or "antigen" refers to any portion, such as a peptide, that is recognized by the host's immune system. Examples of antigenic portions include, but are not limited to, self-antigens, allergens, enzymes, and / or bacterial or viral proteins, peptides, drugs, or components.

[0086] As used herein, "gene therapy vector antigen" refers to a gene therapy vector, or a portion or fragment thereof, such as a surface protein, that induces an immune response against a protein or a portion or fragment thereof. Gene therapy vector antigens may include a whole gene therapy vector (e.g., a virus, bacteria, bacteriophage or other vector) or a protein portion of a gene therapy vector, such as a viral capsid protein, an envelope protein, or other vector-related protein or portion, which may induce an immune response in a subject receiving the gene therapy vector.

[0087] "Pharmaceutically acceptable carrier" refers to any of standard pharmaceutical carriers, buffers, and the like, such as phosphate buffered saline, 5% dextrose in water, 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 edition (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.

[0088] "Pharmaceutically acceptable" or "pharmacologically acceptable" means a substance that is not biologically or otherwise undesirable, i.e., a substance that can be administered to a subject without causing any undesirable biological effect or without interacting in a deleterious manner with any component of the composition in which it is contained or with any component present on or in the subject's body.

[0089] As used herein, the term "subject" encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class mammals: humans, non-human primates (such as chimpanzees and other apes and monkey species); farm animals, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc. The term does not indicate a specific age or sex.

[0090] 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 antigen binding regions. An epitope is usually composed of chemically active surface groups of a molecule (e.g., amino acids or carbohydrate side chains) and has specific three-dimensional structural characteristics and charge-to-mass ratio characteristics. As used herein, epitopes may be adjacent or non-adjacent. In addition, an epitope may be a mimetic (mimetope) because it contains a three-dimensional structure consistent with an epitope used to generate an antibody, but does not contain or contains only some amino acid residues found in the target for stimulating an antibody immune response. As used herein, a mimetope is not considered to be an antigen different from an epitope bound by a selective binding agent; a selective binding agent recognizes the same three-dimensional structure of an epitope and a mimetope.

[0091] As used herein, the term "therapeutically effective amount" refers to an amount of an antigen-specific composition of the present disclosure that is effective in improving or alleviating one or more symptoms or signs of the disease to be treated.

[0092] The terms "treat", "treated", "treating" or "treatment" as used herein with respect to the methods herein refer to temporarily or permanently, partially or completely eliminating, reducing, inhibiting or ameliorating clinical symptoms, manifestations or progression of an event, disease or condition. Such treatment may not necessarily be absolutely useful.

[0093] particle

[0094] Tolerogenic immune modified particles (TIMPs) comprising one or more antigens have been previously described for inducing antigen-specific tolerance to treat inflammatory conditions such as autoimmune diseases and allergies (WO20131319253 and WO2015023796, which are incorporated herein by reference). In several preclinical models of autoimmune diseases and allergies, TIMPs have demonstrated efficacy in inducing antigen-specific tolerance and inhibiting pathological inflammatory immune responses.

[0095] The size and charge of the particles are crucial to tolerance induction. Although the particles will differ in size and charge based on the antigen encapsulated therein, in general, when the particles described herein are between about 100 nanometers and about 1500 nanometers and have a charge between 0 and about -100 mV, they are effective in inducing tolerance. In various embodiments, the diameter of the particle is 400-800 nanometers and has a charge between about -25 mV to -70 mV. In various embodiments, the diameter of the particle is 400-800 nanometers and has a charge between about -30 mV and -80 mV. In various embodiments, the diameter of the particle is 400-800 nanometers and has a charge between about -30 mV and -60 mV. The average particle size and charge of the particles may change slightly during the freeze-drying process, therefore, the average value after synthesis and the average value after freeze-drying are described. As used herein, the terms "size after synthesis" and "charge after synthesis" refer to the size and charge of the particles before freeze-drying. The terms "post-lyophilization size" and "post-lyophilization charge" refer to the size and charge of the particles after lyophilization.

[0096] In some embodiments, the particle is non-metallic. In these embodiments, the particle can be formed by a polymer. In a preferred embodiment, the particle can be biodegradable in an individual. In this embodiment, particles spanning multiple doses can be provided in an individual without the presence of particle accumulation in the individual. Examples of particles that are suitable include polystyrene particles, PLGA particles, PLURONICS stable polypropylene sulfide particles and diamond particles.

[0097] Preferably, the particle surface is made of a material that minimizes nonspecific or non-desirable biological interactions. The interaction between the particle surface and the stroma may be a factor that plays a role in lymphatic absorption. The particle surface may be coated with a material to prevent or reduce nonspecific interactions. Spatial stabilization is achieved by coating the particles with a hydrophilic layer such as poly(ethylene glycol) (PEG) and its copolymers such as PLURONICS® (including copolymers of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) to reduce nonspecific interactions with stroma proteins, as indicated by improved lymphatic absorption after subcutaneous injection. All these facts indicate the relevance of the physical properties of particles in terms of lymphatic absorption. Biodegradable polymers may be used to prepare all or some polymers and / or particles and / or layers. Biodegradable polymers may, for example, cause degradation by functional groups that react with water in a solution. As used herein, the term "degradation" refers to becoming soluble by reducing molecular weight or by converting a hydrophobic group into a hydrophilic group. Polymers with ester groups generally undergo spontaneous hydrolysis, such as polylactide and polyglycolide.

[0098] The particles disclosed herein may also contain additional components. For example, the carrier may have an imaging agent incorporated into or bound to the carrier. An example of a carrier nanosphere with a currently commercially available imaging agent is the Kodak X-sight nanosphere. Inorganic quantum-confined luminescent nanocrystals, called quantum dots (QDs), have emerged as ideal donors in FRET applications: their high quantum yield and tunable size-dependent Stokes shift when excited at a single ultraviolet wavelength permit emission of different sizes from blue to infrared. (Bruchez et al., Science, 1998, 281, 2013; Niemeyer, C. M. Angew. Chem. Int. Ed. 2003, 42, 5796; Waggoner, A. Methods Enzymol. 1995, 246, 362; Brus, LEJ. Chem. Phys. 1993, 79, 5566). Quantum dots, such as hybrid organic / inorganic quantum dots based on a class of polymers called dendrimers, can be used in biolabeling, imaging, and optical biosensing systems. (Lemon et al., J. Am. Chem. Soc. 2000, 122, 12886). Unlike the traditional 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).

[0099] 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 the following: polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxy hydroxy acids; or linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked alkyl, haloalkyl, sulfanyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl or alkoxy dicarboxylic acid polyanhydrides. In addition, the carrier particles can be quantum dots or composed of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22: 1810-6). Carrier particles comprising mixtures of ester and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) may also be used. For example, carrier particles may include materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic acid-co-glycolic acid) copolymers (PLGA or PLG; the terms are interchangeable), poly(lactic acid-co-sebacic acid) copolymers (PLSA), poly(glycolic acid-co-sebacic acid) copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers suitable for use in the present invention include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, and degradable carbamates, as well as copolymers of these with linear or branched, substituted or unsubstituted alkyl, haloalkyl, sulfanyl, aminoalkyl, alkenyl, or aromatic hydroxycarboxylic acids or dicarboxylic acids. In addition, biologically important amino acids (such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine ​​or its enantiomers) with reactive side chain groups can be included in copolymers with any of the aforementioned substances to provide reactive groups for binding to antigenic peptides and proteins or binding moieties. 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, acyl-substituted cellulose acetate, non-degradable carbamates, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON ® (DuPont, Wilmington, Del.) and nonbiodegradable polymers of nylon.

[0100] In certain embodiments, the particles are copolymers having a molar ratio of about 80:20 to about 100:0. Suitable copolymer ratios of the immune modified particles of the invention may 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 acid-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 acid-co-glycolic acid) particles and have a copolymer ratio of about 50:50 polylactic acid:polyglycolic acid. 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 about 99:1 to about 1:99 polylactic acid:polyglycolic acid, for example, 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 in between these values.

[0101] It is contemplated that the particles may further comprise a surfactant. The surfactant may be anionic, cationic or nonionic. Surfactants in the poloxamer and poloaxamine families 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, didodecyl dimethylammonium bromide (DMAB) and poly (ethylene-alternative-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 process, the two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

[0102] In various embodiments, polypeptide antigens are encapsulated in particles by a single emulsion method. In another embodiment, the polypeptide antigen is more hydrophobic. Sometimes, the double emulsion method causes the formation of larger particles, which can cause leakage and low interception efficiency of hydrophilic active components. Coagulation and Ostwald ripening are two mechanisms that can make double emulsion droplets unstable, and diffusion through the organic phase of the hydrophilic active component is the main mechanism that causes low levels of encapsulated active components. In some embodiments, reducing the size of nanoparticles can be beneficial. One strategy to achieve this is to apply a second strong shear rate. The leakage effect can be reduced by using high polymer concentrations and high polymer molecular weights, accompanied by an increase in the viscosity of the internal aqueous phase and an increase in the molecular mass of the surfactant. In certain embodiments, the particles of the encapsulated antigen are manufactured by nanoprecipitation, coprecipitation, inert gas condensation, sputtering, microemulsion, sol-gel method, layer-by-layer technology or ion gelation method. Several methods for making nanoparticles have been described in the literature and incorporated herein by reference 17,18 .

[0103] In some embodiments, the particle is a liposome. Liposomes can be prepared from a variety of lipid materials, including, but not limited to, lipids of phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, dicetyl phosphate, monosialoganglioside, polyethylene glycol, stearylamine, lecithin-modified lipids and cholesterol, and mixtures of these in different stoichiometric amounts. Liposomes as used herein can also be formed from non-lipid amphiphilic molecules, such as block copolymers of poly(ethylene oxide-block-isoprene-block-ethylene oxide) and their analogs. In a preferred embodiment, liposomes are prepared from lipids or incorporate lipids that will form negatively charged liposomes, such as liposomes produced from phosphatidylserine, dicetyl phosphate and dimyristoylphosphatidic 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 -25 mV, -30 mV, -35 mV, -40 mV, -45 mV, -50 mV, -55 mV, -60 mV, -65 mV, -70 mV, -75 mV, -80 mV, -85 mV, -90 mV, -95 mV or -100 mV, including all values ​​and ranges therein. In various embodiments, the negative zeta potential of the liposome is between -30 mV and -80 mV. In various embodiments, the negative zeta potential of the liposome is between -30 mV and -60 mV.

[0104] 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 therapeutic proteins and / or fragments or portions thereof produced by the gene therapy vector. 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 selected from the group consisting of: adenovirus, 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, alpha virus, Flavivirus, rhabdovirus, measles virus, Newcastle disease virus, coxsackievirus, transfusion transmitted virus, anellovirus, 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 x virus, cowpea mosaic virus, chicken anemia virus, cucumber mosaic virus, oncolytic virus, chimeric virus, synthetic virus, mosaic virus, or pseudotyped virus, bacteria, bacteriophage, yeast, cytosome or erythrocyte.

[0105] In various embodiments, the liposomes encapsulate one or more gene therapy vector antigens, wherein the size of the liposomes is between 100 and 1000 nm in diameter, and the negative zeta potential of the liposomes is between -100 mV and -0 mV. In various embodiments, the liposomes encapsulate one or more gene therapy vector antigens, portions thereof, or combinations thereof, wherein the size of the liposomes is between 400 and 800 nm, and the negative zeta potential of the liposomes is between -30 mV and -80 mV.

[0106] In various embodiments, the liposomes encapsulate therapeutic proteins and / or fragments or portions thereof produced by a gene therapy vector, wherein the size of the liposomes is between 100 and 1000 nm, and the negative zeta potential of the liposomes is between -100 mV and 0 mV. In various embodiments, the liposomes encapsulate therapeutic proteins and / or fragments or portions thereof produced by a gene therapy vector, wherein the size of the liposomes is between 400 and 800 nm, and the negative zeta potential of the liposomes is 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 liposomes are administered intravenously.

[0107] Gene therapy vectors

[0108] Gene therapy vectors are designed to deliver genes encoding therapeutic products to subjects to alleviate the symptoms of a disease, such as a genetic disorder or cancer. Gene therapy vectors include viral, bacterial, bacteriophage, yeast, erythrocyte, or exosome vectors.

[0109] Viral vectors commonly used in human gene therapy include adenovirus, adeno-associated virus (AAV), and retroviruses such as lentivirus.

[0110] 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, Vol. 6, Article No.: 53 (2021)). Adenoviral expression can induce immune responses to the early transcribed genes E1 (E1A, E1B), E2, E3, and E4, hexons (which are the most abundant structural components on the capsid surface), and 12 penton proteins located at the vertices of the capsid, and give rise to the extended fibers found on these viruses. Vector particles containing these viral antigens are considered for inducing tolerance to these gene therapy vector antigens.

[0111] AAV vectors are small viral vectors with an icosahedral capsid composed mainly of three proteins VP1, VP2 and VP3. Twelve AAV serotypes are known. (Burcha et al., supra). Common serotypes used for gene therapy include AAV-1, AAV-2, AAV-5, AAV-9, Anc80, while other serotypes include AAV-3, AAV-4, AAV-6, AAV-7, AAV-8, AAV-10, AAV-12 and synthetic AAV. Native and engineered capsid proteins have been used to produce AAV vectors for administration in gene therapy. Vector particles containing wild-type or engineered AAV viral capsid proteins are contemplated herein for inducing tolerance to these gene therapy vector antigens.

[0112] Lentiviruses are enveloped viruses with three major gene sets, gag, pol, and env genes, and may have accessory genes (tat, rev, vif, vpr, vpu, and nef) useful for improving proliferation and pathogenesis. The gag gene encodes the structural envelope protein. However, engineered lentiviral vectors may contain VSV-G protein in place of the envelope protein. (Bulcha et al., supra). Vector particles containing wild-type or engineered lentiviral proteins are contemplated herein for inducing tolerance to these gene therapy vector antigens.

[0113] Bacterial vectors have been produced to deliver genes or proteins to target cancer cells (Baban et al., Bioengineered Bugs 1:6, 385-394, 2010) and include 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 transfer of bacteriophage or plasmid DNA to mammalian cells by bacteria. Vector particles containing bacterial or plasmid antigens are contemplated herein for inducing tolerance to these gene therapy vector antigens.

[0114] Yeast gene therapy vectors expressing tumor or HIV antigens were 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) in patients, and exosomes have been used to deliver CRISPR / Cas9 plasmids to cancer cells. 22,23 Erythrocytes, such as erythrocyte ghosts, have been shown to effectively deliver plasmid DNA to cells 24 , or can be loaded with proteins for in vivo delivery. 25

[0115] antigen

[0116] Antigen refers to a discrete part of a molecule that can be recognized by host immune cells, such as a polypeptide or peptide sequence, a 3D structure of a polypeptide or peptide, a polysaccharide or a polynucleotide. Antigen specificity refers to the ability of a subject's host cells to recognize a single antigen or a molecule that is extremely similar to the antigen (such as an epitope or mimotope) and to generate an immune response against it.

[0117] "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 cessation of exposure to antigenic peptides. For example, the anergy of T cells is characterized by the lack of cytokine (e.g., IL-2) production. When T cells are exposed to antigens and receive a first signal (T cell receptor or CD-3-mediated signal) in the absence of a second signal (co-stimulatory signal), T cell anergy occurs. Under these conditions, re-exposure of the cells to the same antigen (even if re-exposed in the presence of co-stimulatory molecules) still fails to produce cytokines and therefore fails to proliferate. Therefore, the inability to produce cytokines hinders proliferation. However, if cultured with cytokines (e.g., IL-2), anergic T cells can still proliferate.

[0118] It is contemplated that the tolerotherapy described herein is antigen-specific. For example, the TIMP administered as a tolerotherapy encapsulates one or more antigens associated with the tolerotherapy and the associated disease or condition being treated. It is contemplated that the TIMP used in tolerotherapy comprises one or more gene therapy vector antigens, portions thereof, or combinations thereof, and / or transgenic gene protein products produced by a gene therapy vector, portions thereof, or combinations thereof.

[0119] 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, anellovirus, 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 x virus, cowpea mosaic virus, 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 particle may encapsulate a viral vector capsid or envelope protein, a portion or fragment thereof, or a combination thereof. Capsid proteins include, for example, 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: O41856), AAV-6 VP2 (UNIPROT ID: O41857), AAV-7 capsid protein (UNIPROT ID: O41858), AAV-8 capsid protein (UNIPROT ID: O41859), AAV-9 capsid protein (UNIPROT ID: O41857), AAV-10 capsid protein (UNIPROT ID: O41857), AAV-11 capsid protein (UNIPROT ID: O41858), AAV-12 capsid protein (UNIPROT ID: O41859), AAV-13 capsid protein (UNIPROT ID: O41859), AAV-14 capsid protein (UNIPROT ID: O41858), AAV-15 capsid protein (UNIPROT ID: O41858), AAV-16 capsid protein (UNIPROT ID: O41858), AAV-17 capsid protein (UNIPROT ID: O41858 (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).

[0120] 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 bonding to the inner surface of the particle (see, e.g., U.S. Patent Publication US20190282707, which is incorporated herein by reference). In certain embodiments, it is contemplated that the sequences of two or more antigens are linked to a fusion protein and encapsulated in the TIMPs described herein. Methods for preparing TIMPs with linked epitopes are described in U.S. Patent Publication US20190365656, which is incorporated herein by reference.

[0121] Exemplary enzymes useful for treating enzyme diseases and produced by gene therapy vectors include: imiglucerase, talisidase alpha, velaglucerase alpha, β-glucocerebrosidase, arabinocerebrosidase, agalsidase β, agalsidase alpha, cybelase alpha, α-L-iduronidase, human iduronic acid-2-sulfatase, N-sulfoglucosamine sulfohydrolase, elosulfase alpha, sulfase, α-glucosidase (GAA), human α-mannosidase, factor VIII, factor IX, β-galactosidase, arginase, myotonic protein kinase, ornithine transcarbamylase, NADPH oxidase, NADH dehydrogenase 4, adenosine deaminase, lipoprotein lipase, β-glucocerebrosidase, Lipase, myotubularin, arylsulfatase A, DOPA decarboxylase, matrix metallopeptidase 1, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, pyruvate kinase, porphobilinogen deaminase, alkaline phosphatase, sucrase-isomaltase, acid sphingomyelinase, heparan sulfate sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, alpha-1 proteinase inhibitor, 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.

[0122] In certain embodiments, the protein therapeutic agent is an antibody, such as a monoclonal antibody. It is also contemplated that the antibody is a monospecific, bispecific, trispecific or bispecific T cell engagement molecule. In various embodiments, the antibody targets 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, atezolizumab, basiliximab, bevacizumab, bezlotoxumab, blinatumomab, canakinumab, certolizumab, cetuximab, daclizumab, denosumab, durvalumab, efalizumab, emicizumab, etokimab, golimumab, imumab, ipilimumab, ixekizumab, infliximab, natalizumab, nivolumab, olaratumab, omalizumab, ofatumumab, palivizumab, panitumumab, pembrolizumab, ramucirumab, rituximab, tocilizumab, trastuzumab, tremelimumab, secukinumab, ustekinumab, and vedolizumab.

[0123] How to use

[0124] 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 transgenic gene protein products produced by the gene therapy vector, a portion thereof, or a combination thereof.

[0125] In various embodiments, the subject is a subject who has received gene therapy, is receiving gene therapy, or will receive gene therapy. In various embodiments, the subject has received one gene therapy treatment and can undergo another gene therapy treatment regimen, i.e., re-administration of gene therapy.

[0126] In various embodiments, the subject is receiving gene therapy to treat cancer, autoimmune disease, allergy, cardiovascular disease, metabolic disease, enzyme deficiency, or protein deficiency.

[0127] In various embodiments, the subject suffers from a rare and inherited genetic disorder and receives gene therapy to obtain a transgene protein product produced by the vector, the transgene protein product being an enzyme. In various embodiments, the subject suffers from a rare and inherited genetic disorder selected from the group consisting of hemophilia A, hemophilia B, lysosomal storage disease, Fabry disease, Gaucher disease, Pompe disease, Niemann-Pick disease, Tay-Sachs disease, macular degeneration, mucopolysaccharidosis, venous thrombosis, von Wille disease, purpura fulminans, growth hormone deficiency, gangliosidosis, alkaline hypophosphatasia, cholesterol ester storage disease, hyperuricemia, Duchenne Hindsight 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), Syndymodulin myopathy, Gram-negative Fruit-Nagell syndrome, catecholamine-sensitive 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, achromatopsia, scleroderma, osteogenesis imperfecta, coronary artery disease disease, tyrosinemia, peripheral neuropathy, optic neuropathy, coronary artery disease, respiratory syncytial virus (RSV)-mediated lower respiratory tract disease, Danon disease, severe leukocyte adhesion defect, pyruvate kinase deficiency, Chuck-Marie-Doucet disease, Weill-Willi 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 disorders, ENPP1 deficiency, ENPP3 deficiency, ABCC6 deficiency, aromatic L-amino acid decarboxylase deficiency, Angelman syndrome, hyperphenylalaninemia, dementia, Rett syndrome, and Usher syndrome.

[0128] 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, Sjögren's syndrome, autoimmune uveitis, Eaton-Lambert disease, autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus, neuromyelitis optica, idiopathic thrombocytopenic purpura, blood Thrombotic thrombocytopenic purpura, membranous nephropathy, bullous pemphigoid, pemphigus vulgaris, myasthenia gravis, celiac disease, ulcerative colitis, Crohn's disease, erythema nodosum, glomerulonephritis, Guillain-Barre syndrome, granulomatosis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, Kawasaki disease, mixed connective tissue disease, multifocal motor neuropathy, peripheral biliary cirrhosis, polyangiitis overlap syndrome, type 1 scleroderma, sclerosing cholangitis, stiff-man syndrome, Takayasu's arteritis, vitiligo, or Wegener's granulomatosis.

[0129] 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 dairy 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.

[0130] 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.

[0131] In various embodiments, the vector particles comprise one or more gene therapy vector antigens. In various embodiments, the vector particles comprise one or more transgenic protein products produced by a gene therapy vector, portions thereof, combinations thereof, or one or more antigenic epitopes thereof. In various embodiments, the vector particles comprise one or more gene therapy vector antigens and transgenic protein products or one or more antigenic epitopes thereof.

[0132] If a carrier particle comprising a single antigen is administered in combination with another carrier particle or a second agent comprising a different antigen, the particles and / or the second agent may be administered concurrently or sequentially. Concomitant or concurrent administration of two therapeutic agents does not require the agents to be administered simultaneously or by the same route, as long as there is an overlap in the time period during which the agents exert their therapeutic effects. Concomitant or sequential administration is contemplated, such as administration on different days or weeks. Upon further consideration, the therapeutic agents are administered in the form of separate formulations and concurrently or concomitantly, wherein concurrently refers to administration of the agents within 30 minutes of each other. Prior administration refers to administration of the therapeutic agent within a range of one week prior to treatment with the carrier particles to 30 minutes prior to administration of the carrier particles. Subsequent administration means describing administration within 30 minutes after treatment to one week after administration.

[0133] 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 at 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 between about 0.0005 mg / mL and about 50 mg / mL, between about 0.05 mg / mL and 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.

[0134] In some embodiments, carrier particles comprising one or more gene therapy vector antigens and / or one or more transgenic protein products produced by a gene therapy vector are administered in combination with one or more therapeutic agents. In various embodiments, the therapeutic agent is adoptive transfer of Treg, CAR Treg, TCR Treg. In various embodiments, the therapeutic agent blocks or inhibits innate immune responses, complement responses, B cell responses, and / or T cell responses to gene therapy vectors and / or transgenic proteins produced by the gene therapy vectors.

[0135] In various embodiments, the therapeutic agent is an FcRn inhibitor. In various embodiments, the FcRn inhibitor is selected from the group consisting of: efgartigimod, rozanoluximumab, nipocalimab, batoclimab or orilanolimab. 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: a C1 inhibitor (e.g., Cinryze, Berinert, Ruconest, sutimlimab (Enjaymo), or Haegarda), a C3 inhibitor (e.g., Pegacetaclplan (Empaveli), Syfovre), a C5 inhibitor (e.g., eculizumab, ravulizumab, avacopan, Pazer), In some 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 some embodiments, the therapeutic agent is 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 (imlifidase). In various embodiments, the Treg inducer is an IL2 variant / mutant or a CDK8 / 19 inhibitor.

[0136] In various embodiments, one or more therapeutic agents are administered before, 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 prior to administration of the carrier particles, including all ranges and values ​​between those ranges. 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, 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 concurrently 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 after administration of the carrier particles, including all ranges and values ​​between those ranges. 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, or 4 weeks 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.

[0137] Screening methods

[0138] It is contemplated that the induction and maintenance of immune tolerance is monitored in subjects that have been treated or are to be treated with vector particles as described herein.

[0139] Methods for screening cell types, cytokines or other tolerance measures in subjects undergoing tolerance therapy as described herein are known in the art. Methods for assessing tolerance are performed using techniques such as flow cytometry, mass cytometry (CyTOF), ELISA, ELISPOT, in vitro / 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, such as by ImmunoCap analysis.

[0140] Lists of human metabolites that can be analyzed from biological samples can be found in the literature, including in (Psychogios et al., 2011), (Wishart et al., HMDB: the Human Metabolome Database. Nucleic Acids Res. 2007 Jan; 35(Database Number):D521-6, 2007) and the Human Metabalome Database (HMDB) and are incorporated herein by reference.

[0141] One aspect of the immune tolerance state of a subject and the immune signature is determined by analyzing one or more cell surface proteins from a biological sample. In various embodiments, the cell surface proteins include 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 ... 5RO, 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, LT βR, 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, APR IL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SI GLEC, 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 analyzing cell surface protein expression, including flow cytometry and mass cytometry (CyTOF), have been described in the literature. ,

[0142] In certain embodiments, the tolerance state of the subject is determined by analyzing nucleic acids from biological samples. In various embodiments, 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 immune tolerance state of the subject is determined by analyzing gene expression from biological samples. In various embodiments, the immune tolerance state is determined by analyzing gene expression related to immune function, antibodies, foreign body reaction, metabolism, apoptosis, cell death, necrosis, ferroptosis, autophagy, cell migration, endocytosis, phagocytosis, pinocytosis, tight junction regulation, cell adhesion, differentiation and / or its combination. In various embodiments, the immune tolerance state is determined by analyzing gene expression related to immunosuppression. In various embodiments, the immune tolerance state is determined by analyzing gene expression related to immune activation. In various embodiments, the immune tolerance state is determined by analyzing gene expression related to immunomodulatory function. In various embodiments, nucleic acid analysis is used to generate immune tolerance tags. Several methods have been described in the literature for high-throughput gene expression analysis, including RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), exome sequencing, and microarray-based analysis.

[0143] The biological sample is optionally analyzed after in vivo and / or ex vivo stimulation with one or more stimulants (such as antigens, allergens, and one or more activators). It is considered that the T cells, B cells, and immunoglobulins used for analysis 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 cells (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.

[0144] In various embodiments, the immune tolerance state of a subject is determined by obtaining one or more samples (e.g., whole blood) from the subject before administration on the day of the first administration of the TIMP (Day 1) and on a certain day after administration. The whole blood can then be processed to separate peripheral blood mononuclear cells (PBMC), basophils, neutrophils, plasma, and serum for downstream analysis. Cells isolated from one or more samples collected from the subject and analyzed are analyzed.

[0145] Drug formulations

[0146] The pharmaceutical compositions of the present disclosure containing the TIMPs and antigens described herein may contain pharmaceutically acceptable carriers or additives, depending on the route of administration. Examples of these carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerol, glycerol, propylene glycol, polyethylene glycol, Vaseline®, paraffin, stearyl alcohol, stearic acid, human serum albumin (human serum albumin, HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, etc. Depending on the dosage form of the present disclosure, the additives used are selected from (but not limited to) the above or a combination thereof as needed.

[0147] The formulation of the pharmaceutical composition will vary according to the selected route of administration (e.g., solution, emulsion). Suitable compositions comprising the administered therapeutic agent can be prepared in the form of a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles may include various additives, preservatives or fluids, nutrition or electrolyte supplements.

[0148] There are various aqueous carriers, such as sterile phosphate buffered saline solution, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and may include other proteins for enhanced stability, such as albumin, lipoproteins, globulins, etc., which undergo mild chemical modifications, etc.

[0149] Therapeutic formulations of the inhibitor in the form of a lyophilized formulation or an aqueous solution are prepared by mixing the inhibitor having the desired purity with an optional physiologically acceptable carrier, excipient or stabilizer for storage (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexahydroxyquaternium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol 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; proteins proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including 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 nonionic surfactants, such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[0150] Formulations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0151] Aqueous suspensions may contain the active compound in admixture 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 arabic; dispersants or wetting agents may be naturally occurring phospholipids 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 heptadecaethyleneoxyhexadecanoic acid, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate.

[0152] The TIMPs described herein can be stored lyophilized and reconstituted in a suitable carrier prior to use.

[0153] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the modified particles are mixed with: at least one inert, pharmaceutically 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 carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrants 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 promoters such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) adsorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0154] Reagent test kit

[0155] The present disclosure also provides a kit comprising one or more compounds or compositions packaged in a manner that facilitates their use in practicing the methods of the present disclosure. In one embodiment, such a kit includes a compound or composition described herein (e.g., a composition comprising a TIMP alone or in combination with another agent) packaged in a container (e.g., a sealed bottle or container), wherein a label is attached to the container or included in the package, describing the use of the compound or composition in the practice method. Preferably, the compound or composition is packaged in unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration or suitable for practicing a screening assay. Preferably, the kit contains a label describing the use of the particle composition.

[0156] Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.

[0157] Examples

[0158] Example 1 - Efficacy of TIMPs in enhancing expression of AAV8-GFP following administration and enabling re-administration of AAV8-GFP

[0159] The efficacy of TIMPs encapsulating recombinant eGFP (CNP-GFP) and AAV8 VP1 capsid protein (CNP-VP1) (CNP-GFP +CNP-VP1) in inducing antigen-specific tolerance and enhancing transgene expression upon initial administration and after re-administration with rAAV8-GFP vector was examined.

[0160] The particles used in this study had an average diameter of 400-800 nm and a zeta potential between -30 and -80 mV.

[0161] Female BALB / c mice (6-8 weeks old, n=10 per group) were injected intravenously with CNP-GFP (1.25 mg / mouse) and CNP-VP1 (1.25 mg / mouse) or unloaded with CNP (control group) on days -7 and 0. On day 0, mice were administered rAAV8-eGFP via intramuscular injection in the left quadriceps muscle. The expression of the transgenic eGFP in the pre-sensitized left quadriceps muscle of mice was monitored by transcutaneous fluorescence spectroscopy on days 0, 3, 7, 14, 21, and 28. Figure 1 As shown in Figure , treatment with CNP-GFP+CNP-VP1 resulted in significantly higher GFP expression when compared to the control group ( p<0.05; p<0.001; p<0.0001).

[0162] On day 28, mice were re-administered intramuscularly with AAV8-GFP in the contralateral right quadriceps muscle (N=5 per group). Seven days after re-administration (day 35), mice were sacrificed and right and left quadriceps muscles were harvested for analysis. + The frequency and total cell counts of T cells were assessed by flow cytometry using tetramer staining. As shown in Figure 2, treatment with CNP-GFP + CNP-VP1 resulted in a significant decrease in the number of VP1 and GFP-specific CD8+ T cells in both the initial tissue (left quadriceps) and the tissue (right quadriceps) to which AAV8-GFP was administered ( p<0.05; p<0.01).

[0163] On day 35, the remaining mice in each treatment group (N=5 per group) were re-administered AAV8-GFP intramuscularly in the contralateral right quadriceps muscle, and eGFP expression in the right quadriceps muscle was monitored by transcutaneous fluorescence spectroscopy. Figure 3 As shown in , mice treated with CNP-GFP+CNP-VP1 showed significantly higher eGFP expression in the right quadriceps muscle 14 and 21 days after re-administration of AAV8-GFP when compared to the control group ( p<0.01; p<0.0001). CNP-GFP+CNP-VP1 treatment caused a 58% increase in GFP expression from day 14 to day 21 relative to control treatment.

[0164] Based on these results, treatment with CNP-GFP+CNP-VP1 inhibited the production of anti-VP1 and anti-GFP antibodies. The inhibition of antibody response was measured from blood using ELISA.

[0165] The results of this study demonstrate that TIMPs encapsulating AAV8 VP1 capsid protein and the transgene product can effectively induce antigen-specific tolerance, leading to increased transgene expression and enabling re-administration of AAV8-GFP.

[0166] Example 2 - Intravenous administration of TIMPs encapsulating AAV8-GFP induces systemic tolerance

[0167] The efficacy of TIMPs encapsulating recombinant eGFP (CNP-GFP) and AAV8 VP1 capsid protein (CNP-VP1) in inducing antigen-specific tolerance following intravenous administration in a therapeutic mouse model was examined.

[0168] The particles used in this study had an average diameter of 400-800 nm and a zeta potential between -30 and -80 mV.

[0169] C57BL / 6 mice (6-8 weeks old, n=5) were pre-sensitized with PBS or empty AAV8 capsid on day 0. Mice were administered 2 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 administration of AAV8-GFP on day 28. On day 53, mice were sacrificed and final readings were taken.

[0170] Compared with control mice, the immune infiltration in the hearts of CNP-treated mice was reduced ( Figure 4A ).

[0171] like Figure 4B As shown in , CD4+ T cells obtained from CNP-treated mice showed an increase in the regulatory phenotype in the spleen, liver, and heart ( Figure 4B ). When compared to control-treated mice, the number of CD4+ T cells expressing regulatory markers (Foxp3+ and Ctla4+) was increased in the spleen and liver of CNP-treated mice. The number of CD4+ T cells expressing the regulatory cytokine IL-10+ and the CD44hi memory marker was also increased in the spleen of mice receiving CNP therapy ( Figure 4B). The hearts of CNP-treated mice had an increase in CD4+ T cells with a memory phenotype (CD44hi, CD44hi IFN-γ+, and CD44hi IFN-γ-) and an increase in regulatory cells (CD4+Foxp3+, CD4+PD-1+, CD4+CTLA4+, CD4+IL-10+). ( p<0.05; p<0.01).

[0172] like Figure 4C As shown in Figure 2, when compared to control-treated mice, mice treated with CNP and re-administered with AAV had a reduced number of effector CD8+ T cells (CD8+ granzyme+, CD8+ PD-1+, CD8+ CD69+) infiltrating the heart ( Figure 4C ). p<0.05; p<0.01; p<0.001).

[0173] All publications, patents and patent applications discussed and cited herein are hereby incorporated by reference in their entirety. It should be understood that the disclosed invention is not limited to the specific methods, protocols and materials described, as these may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the appended claims.

[0174] Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0175] References

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Claims

1. A composition comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more gene therapy vector antigens, parts thereof, or combinations thereof and / or one or more transgenic gene protein products produced by a gene therapy vector, parts thereof, or combinations thereof.

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

3. The composition of claim 1 or 2, wherein the zeta potential of the particles is between 0 mV and -100 mV.

4. The composition of claim 3, wherein the zeta potential of the particles is between -30 and -80 mV.

5. The composition according to any one of claims 1 to 4, wherein the particle size is between 100 nm and 1000 nm.

6. The composition of claim 5, wherein the particle size is between 400 and 800 nm.

7. The composition of any one of claims 1 to 5, wherein the antigen comprises one or more proteins, peptides or antigenic epitopes thereof.

8. The composition according to any one of claims 1 to 7, wherein the gene therapy vector is a viral vector.

9. The composition of 8, wherein 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, anellovirus, human papillomavirus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, cowpea mosaic virus, cowpea chlorotic mottle virus, tomato mosaic virus, red clover necrotic mosaic virus, potato x virus, cowpea mosaic virus, chicken anemia virus or cucumber mosaic virus.

10. The composition of claim 9, wherein 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, synthetic AAV, combinations, engineered or modified versions thereof.

11. The composition according to any one of claims 1 to 10, wherein the antigen is associated with one or more viral vector capsid proteins, peptides and / or antigenic epitopes thereof.

12. The composition of claim 11, wherein the capsid protein is VP-1, VP-2, VP-3, a combination or a portion thereof.

13. The composition according to any one of claims 1 to 12, wherein the transgene protein product is a cytokine, chemokine, hormone, growth factor, enzyme or antibody.

14. The composition of claim 13, wherein the antibody is a monoclonal antibody.

15. 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 transgenic gene protein products produced by the gene therapy vector, a portion thereof, or a combination thereof.

16. The method of claim 15, wherein 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 produced by the gene therapy vector.

17. The method of claim 16, wherein the negatively charged particles encapsulating one or more gene therapy vector antigens are administered before, simultaneously with, or after administration of negatively charged particles encapsulating one or more transgene protein products produced by the gene therapy vector.

18. The method of any one of claims 15 to 17, wherein the particles comprise poly(lactide-co-glycolide) (PLG), polylactic acid (PLA), copolymers of PLG and PLA (PLGA), polycaprolactone (PCL), polyethylene glycol (PEG), polystyrene, chitosan, a polysaccharide, or one or more lipids, iron, zinc, cadmium, gold, or silver.

19. A method according to any one of claims 15 to 18, wherein the particles have a negative zeta potential.

20. The method of any one of claims 15 to 19, wherein the zeta potential of the particles is between 0 mV and -100 mV.

21. The method of any one of claims 15 to 20, wherein the zeta potential of the particles is between -30 mV and -100 mV.

22. The method according to any one of claims 15 to 21, wherein the size of the particles is between 100 nm and 1000 nm.

23. The method according to any one of claims 15 to 22, wherein the size of the particles is between 400 nm and 800 nm.

24. The method of any one of claims 15 to 23, wherein the antigen comprises a protein, a peptide, or one or more antigenic epitopes thereof.

25. The method of any one of claims 15 to 24, wherein the gene therapy vector is a viral vector.

26. The method of claim 25, wherein the viral vector is an adenovirus, an adeno-associated virus (AAV), a herpes simplex virus, a hepatitis B virus, a lentivirus, a retrovirus, an alphavirus, a flavivirus, a rhabdovirus, a measles virus, a Newcastle disease virus, a coxsackievirus, a transfusion-transmitted virus, an anelovirus, a human papillomavirus, a poxvirus, a vaccinia virus, a modified Ankara virus, a vesicular stomatitis virus, a picornavirus, a tobacco mosaic virus, a cowpea mosaic virus, a cowpea chlorotic mottle virus, a plant mosaic virus, a red clover necrotic mosaic virus, a potato virus x, a cowpea mosaic virus, a chicken anemia virus, or a cucumber mosaic virus.

27. The method of claim 26, wherein 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 thereof, or engineered versions thereof.

28. The method of any one of claims 15 to 27, wherein the antigen is associated with one or more viral vector capsid proteins, peptides and / or antigenic epitopes thereof.

29. The method of claim 28, wherein the capsid protein is VP-1, VP-2, VP-3, a combination or a portion thereof.

30. The method of any one of claims 15 to 29, wherein the subject has cancer, an autoimmune disease, an allergy, a cardiovascular disease, a metabolic disease, diabetes, an enzyme deficiency, a protein deficiency, cystic fibrosis, a blood disorder, 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, mucopolysaccharidosis, venous thrombosis, von Willebrand disease, purpura fulminans, growth hormone deficiency, gangliosidosis, alkaline hypophosphatasia, cholesterol ester storage disease, hyperuricemia, Duchenne Muscular Dystrophy, Huntington's disease), Parkinson's, 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), Myotubular myopathy, Crigler-Najjar syndrome, Catecholamine-sensitive polymorphic ventricular tachycardia, Glycogen storage disease type 1, Alpha-mannosidosis, Fragile X syndrome, Arginase deficiency, X-linked chronic granulomatous disease, Adenosine deaminase deficiency, Leber's congenital amaurosis amaurosis, lipoprotein lipase deficiency, cerebral adrenoleukodystrophy, metachromatic leukodystrophy, Fanconi anemia, achromatopsia, scleroderma, osteogenesis imperfecta, coronary artery disease, tyrosinemia, peripheral neuropathy, optic neuropathy, coronary artery disease, respiratory syncytial virus (RSV)-mediated lower respiratory tract disease, Danon disease, severe leukocyte adhesion defect, pyruvate kinase deficiency, Charcot Marie Tooth disease, Wiskott Aldrich syndromesyndrome), alpha-synuclein tauopathy, refractory angina due to myocardial ischemia, myotonic dystrophy type I, claudication, peripheral arterial disease, methylmalonic acidemia, sucrase-isomaltase deficiency, Niemann-Pick type B disease, alpha-1-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, MPS 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.

31. The method of claim 30, wherein the subject has 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.

32. The method of claim 30, wherein the subject has an autoimmune disease 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, Reynaud's syndrome, Behcet's syndrome, Sjorgen's syndrome, autoimmune uveitis, Eaton-Lambert disease, Lambert's disease), autoimmune myocarditis, inflammatory bowel disease, amyotrophic lateral sclerosis (ALS), systemic lupus erythematosus, 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, granulomatous disease, Grave's disease, Guillain-Barre syndrome, Hashimoto disease, hemolytic anemia, Kawasaki disease Disease, mixed connective tissue disease, multifocal motor neuropathy, peripheral biliary cirrhosis, polyangiitis overlap syndrome, scleroderma type 1, sclerosing cholangitis, stiff-person syndrome, Takayasu arteritis, vitiligo, or Wegeners granulomatosis.

33. The method of claim 30, wherein the subject suffers from an allergy selected from the group consisting of peanut allergy, tree nut allergy, dairy allergy, egg allergy, fish allergy, wheat allergy, celery allergy, or peach allergy; in various embodiments, the environmental allergy is pollen allergy, dust allergy, pet dander allergy, or mold allergy.

34. The method of any one of claims 15 to 33, wherein the composition is administered before, after, or simultaneously with the gene therapy vector.

35. The method of any one of claims 15 to 34, wherein administering the composition reduces an immune response to the gene therapy vector and / or the transgene protein product produced by the vector.

36. The method of claim 35, wherein the immune response is an inflammatory immune response.

37. The method of any one of claims 35 to 36, wherein the immune response is a humoral immune response.

38. The method of any one of claims 35 to 36, wherein the immune response is an adaptive immune response.

39. The method of any one of claims 35 to 36, wherein the immune response is an innate immune response.

40. The method of any one of claims 35 to 39, wherein the immune response is a T cell, B cell, monocyte, macrophage, neutrophil, basophil or eosinophil response.

41. The method of any one of claims 35 to 40, wherein the immune response is an antibody response.

42. The method of claim 41, wherein the antibody response is the formation of neutralizing antibodies against the gene therapy vector and / or the transgene protein produced by the vector.

43. The method of any one of claims 15 to 42, wherein administration of the composition induces an immunomodulatory response to the gene therapy vector and / or the transgene protein product produced by the vector.

44. The method of claim 43, wherein the immunomodulatory response is regulatory T cells, B cells, monocytes and / or macrophages.

45. The method of claim 43, wherein the immunoregulatory response is antigen-specific Treg, Tr1, Mreg and / or Breg cells.

46. ​​The method of any one of claims 15 to 45, wherein the composition is administered intravenously, intramuscularly, ocularly, intraperitoneally, transdermally, nasally, orally and / or subcutaneously.

47. A composition comprising liposomes encapsulating one or more gene therapy vector antigens, portions thereof, or combinations thereof.

48. A composition comprising liposomes encapsulating a therapeutic protein and / or a portion thereof produced by the gene therapy vector.

49. according to the composition described in any one of claim 47 to 48, wherein said liposome has negative zeta potential.

50. The composition of claim 49, wherein the negative zeta potential is between -100 mV and 0 mV.

51. The composition of claim 50, wherein the negative zeta potential is between -80 mV and -30 mV.

52. The composition of any one of claims 47 to 51, wherein the gene therapy vector antigen is selected from the group consisting of: adenovirus, 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, anellovirus, 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, cowpea mosaic virus, chicken anemia virus, cucumber mosaic virus, oncolytic virus, chimeric virus, synthetic virus, mosaic virus, pseudotyped virus, bacteria, bacteriophage, yeast, cytosomes, or erythrocytes.

53. The composition of any one of claims 47 to 52, wherein the therapeutic protein and / or portion thereof produced by the gene therapy vector is selected from the group consisting of a cytokine, a chemokine, a hormone, a growth factor, an enzyme or an antibody.

54. A composition according to any one of claims 47 to 53, wherein the liposomes are between 100 and 1000 nm.

55. A method of inducing tolerance in a subject in need thereof, comprising administering to the subject a composition according to any one of claims 47 to 54.

56. A composition according to any one of claims 1 to 14 or 47 to 54, comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more gene therapy vector antigens and / or one or more transgenic gene protein products produced by the gene therapy vector, a portion thereof or a combination thereof, for inducing tolerance in a subject.

57. Use of a composition according to any one of claims 1 to 14 or 47 to 54, said composition comprising negatively charged particles encapsulating an antigen, wherein said antigen is one or more gene therapy vector antigens and / or one or more transgenic gene protein products produced by said gene therapy vector, a portion thereof or a combination thereof, said composition being used for the preparation of a medicament for inducing tolerance in a subject.

58. A method of inducing tolerance in a subject in need thereof, comprising administering to the subject a composition according to any one of claims 1 to 14 or 47 to 54 in combination with one or more therapeutic agents, the composition comprising negatively charged particles encapsulating an antigen, wherein the antigen is one or more gene therapy vector antigens and / or one or more transgenic gene protein products produced by the gene therapy vector, portions thereof, or a combination thereof.

59. The method of claim 58, wherein the therapeutic agent blocks or inhibits an innate immune response, complement response, B cell response, and / or T cell response to the gene therapy vector and / or a transgene protein produced by the gene therapy vector.

60. The method of claim 58 or 59, wherein the therapeutic agent is selected from the group consisting of an immunosuppressant, a complement inhibitor, a calcineurin inhibitor, plasmapheresis, an IgG protease, a proteasome inhibitor, and / or an inducer of regulatory T cells.

61. The method of any one of claims 58 to 60, wherein the therapeutic agent is an FcRn inhibitor.

62. The method of any one of claims 58 to 60, wherein the therapeutic agent is a corticosteroid, rapamycin / sirolimus, cyclophosphamide, or mycophenolate mofetil.

63. The method of any one of claims 58 to 60, wherein the therapeutic agent is anti-CD20 CAR T cell therapy, anti-CD19 CAR T cell therapy, or anti-BCMA CAR T cell therapy.

64. The method of any one of claims 58 to 60, wherein the therapeutic agent is an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD40 antibody, or CTLA4 Ig.

65. The method of any one of claims 58 to 64, wherein the one or more therapeutic agents are administered before, simultaneously with, or after administration of a composition according to any one of claims 1 to 14 or 47 to 54, wherein the composition comprises negatively charged particles encapsulating an antigen, wherein the antigen is one or more gene therapy vector antigens and / or one or more transgenic gene protein products produced by the gene therapy vector, a portion thereof, or a combination thereof.

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