Adenoviral gene therapy vectors

Abstract

The present disclosure includes adenoviral vectors characterized by efficient transduction of hematopoietic cells (e.g., one or more specific hematopoietic cell types), for example, for in vivo or ex vivo gene therapy. The present disclosure includes, among others, Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, and Ad50 vectors and genomes. The Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, and Ad50 vectors and genomes of the present disclosure can include a therapeutic payload.

Description

[Technical Field]

[0001] Priority Application This application claims the benefit of U.S. Provisional Patent Application No. 63 / 175,249, filed April 15, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Many medical conditions are caused by genetic mutations and / or are treatable, at least in part, by gene therapy. Some conditions are particularly treatable by the modification of hematopoietic cells. Thus, there is a need for compositions and methods that target hematopoietic cells for gene therapy. Summary of the Invention

[0003] Gene therapy can treat many conditions with a genetic component, including, but not limited to, hemoglobinopathies, immunodeficiencies, and cancer. Hematopoietic cells are an important target in various gene therapies. However, current methods and compositions for modifying hematopoietic cells are limited. For example, there is a need to identify vectors that selectively target hematopoietic cells (e.g., one or more specific hematopoietic cell types). The present disclosure recognizes that certain adenoviral vectors selectively target hematopoietic cells (e.g., one or more specific hematopoietic cell types).

[0004] The present disclosure includes, inter alia, adenoviral vectors that selectively target various hematopoietic cell types provided herein. The present disclosure includes, inter alia, hematopoietic stem cells (HSCs, e.g., CD34 + Long-term (LT)-HSC and / or CD34 +Short-term (ST)-HSC), common lymphoid progenitor cells (CLP), T cells, NK cells, colony forming units (CFU)-pre-B cells, B cells, common myeloid progenitor cells (CMP), granulocyte-macrophage progenitor cells (GMP), CFU-M cells, monoblasts, monocytes, macrophages, CFU-G cells, myeloblasts, granulocytes, neutrophils, eosinophils, basophils, megakaryocyte-erythroid progenitor cells (MEP), BFU-E cells, CFU-E cells, erythroblasts, red blood cells, CFU-Mk cells, megakaryocytes, and / or platelets. + Adenoviral vectors that selectively target hematopoietic cells are included.

[0005] The present disclosure includes, among other things, Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors and Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genomes (e.g., "recombinant" or "engineered" adenoviral vectors and genomes) that selectively target one or more hematopoietic cell types. The Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors and genomes of the present disclosure can include various payloads. In various embodiments, the payloads can include one or more nucleic acid sequences encoding a CRISPR system, a base editing system, a prime editing system, or other expression products. The present disclosure includes, among other things, combinations of adenoviral vectors and adenoviral genomes that include nucleic acid sequences encoding multiple expression products that together contribute to the treatment of a disease or condition. The present disclosure includes, inter alia, Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 adenoviral vectors, as well as Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 adenoviral genomes for integrating a nucleic acid payload into a target cell genome. The present disclosure includes, inter alia, Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vectors, Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 adenoviral donor genomes, helper-dependent Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 adenoviral donor vectors, helper-dependent Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 adenoviral donor genomes, support vectors, support genomes, Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper vectors, and Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper genomes. For the avoidance of doubt, a list of serotypes such as "Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50" may alternatively be written as "Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, or Ad50."

[0006] In at least one aspect, the present disclosure provides a method of selectively targeting a hematopoietic cell type, the method comprising administering to a subject or system an adenoviral vector comprising: (a) a capsid comprising one or more viral polypeptides of an Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, or Ad50 serotype, wherein the one or more viral polypeptides comprise one or more of: (i) a fiber knob, (ii) a fiber shaft, (iii) a fiber tail, (iv) a penton, and (v) a hexon; and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload. In various embodiments, the genome further comprises (a) a 3'ITR and a 5'ITR, each of which is of a viral polypeptide serotype; and (b) a packaging sequence, each of which is of a viral polypeptide serotype.

[0007] In various embodiments, the hematopoietic cell type is or comprises a terminally differentiated cell type. In various embodiments, the hematopoietic cell type is or comprises a progenitor cell type. In various embodiments, the hematopoietic cell type is or comprises an HSC, a common lymphoid progenitor (CLP), a T cell, a NK cell, a colony forming unit (CFU)-pre-B cell, a B cell, a common myeloid progenitor (CMP), a granulocyte-macrophage progenitor (GMP), a CFU-M cell, a monoblast, a monocyte, a macrophage, a CFU-G cell, a myeloblast, a granulocyte, a neutrophil, an eosinophil, a basophil, a megakaryocyte-erythroid progenitor (MEP), a BFU-E cell, a CFU-E cell, an erythroblast, an erythrocyte, a CFU-Mk cell, a megakaryocyte, and / or a platelet, and optionally, the HSC is CD34 + Long-term hematopoietic stem cells (LT-HSC) and / or CD34 + Short-term (ST)-HSC.

[0008] In various embodiments, the method is a method of in vivo gene therapy. In various embodiments, the hematopoietic cell type is a mammalian hematopoietic cell type, and optionally, the mammalian hematopoietic cell type is a human hematopoietic cell type. In various embodiments, the subject is a mammalian subject, and optionally, the mammalian subject is a human subject. In various embodiments, the method includes mobilizing the subject's hematopoietic cells prior to administration of the adenoviral vector. In various embodiments, the method includes administering to the subject one or more immunosuppressive agents, and optionally, the administration of the one or more immunosuppressive agents is performed prior to the administration of the adenoviral vector.

[0009] In various embodiments, the method is a method of ex vivo gene therapy. In various embodiments, the hematopoietic cell type is a mammalian hematopoietic cell type, and optionally, the mammalian hematopoietic cell type is a human hematopoietic cell type. In various embodiments, the system is optionally a biological sample derived from or including a mammalian donor, where the mammalian donor is a human donor. In various embodiments, the heterologous nucleic acid payload includes a selectable marker, and optionally, the selectable marker is MGMT P140K is. In various embodiments, the method includes administering a selection agent to the subject, and optionally, the selection agent is O 6 BG and / or BCNU.

[0010] In various embodiments, the one or more viral polypeptides comprise (a) a fiber knob and a fiber shaft, (b) a fiber knob and a fiber tail, (c) a fiber knob and a penton, (d) a fiber knob and a hexon, (e) a fiber knob, a hexon, and a penton, (f) a fiber shaft and a fiber tail, (g) a fiber shaft and a penton, (h) a fiber shaft and a hexon, (i) a fiber shaft, a hexon, and a penton, (j) a fiber tail and a penton, (k) a fiber tail and a hexon, (l) a fiber tail, a hexon, and a penton, (m) a fiber knob, a fiber shaft, and a fiber tail, (n) a fiber knob, a fiber shaft, and a penton, (o) a fiber knob, a fiber shaft, and a hexon, (p) a fiber knob, a fiber shaft, a hexon, and a penton, (q) a fiber knob, a fiber shaft, a fiber tail, and a penton, (r) a fiber knob, a fiber shaft, a fiber tail, a penton, and a hexon, or (s) a penton and a hexon. In various embodiments, the fiber knob has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 15, 33, 51, 69, 87, 105, 123, 141, 159, 177, and 195. In various embodiments, the fiber shaft has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, and 194. In various embodiments, the fiber tail has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 18, 36, 54, 72, 90, 108, 126, 144, 162, 180, and 198. In various embodiments, the penton has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 16, 34, 52, 70, 88, 106, 124, 142, 160, 178, and 196. In various embodiments, the hexon has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 17, 35, 53, 71, 89, 107, 125, 143, 161, 179, and 197.In various embodiments, the adenoviral vector comprises a fiber of a serotype of the viral peptide. In various embodiments, the fiber has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, 157, 175, and 193. In various embodiments, the adenoviral vector is a chimeric vector characterized in that the capsid comprises at least one of a fiber knob, a fiber shaft, a fiber tail, a hexon, or a penton that is not a serotype of the viral peptide. In various embodiments, the adenoviral vector is a helper-dependent vector.

[0011] In various embodiments, the heterologous nucleic acid payload encodes a protein. In various embodiments, the heterologous nucleic acid payload encodes a chimeric antigen receptor (CAR), a T cell receptor (TCR), an antibody, or a small RNA, optionally wherein the small RNA is an shRNA. In various embodiments, the heterologous nucleic acid payload encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the hematopoietic cell type is or comprises a T cell. In various embodiments, the heterologous nucleic acid payload encodes an antibody, and the hematopoietic cell type is or comprises a B cell. In various embodiments, the heterologous nucleic acid payload encodes a gene editing enzyme or system, and the gene editing is selected from CRISPR editing, base editing, prime editing, and zinc finger nuclease editing.

[0012] In various embodiments, the heterologous nucleic acid payload is a nucleic acid payload that is associated with a disease such as adenosine deaminase deficiency (ADA), adrenoleukodystrophy (ALD), agammaglobulinemia, alpha-1 antitrypsin deficiency, congenital amegakaryocytic thrombocytopenia, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia-telangiectasia, Batten disease, Bernard-Soulier syndrome, CD40 / CD40L deficiency, chronic granulomatous disease, common variable immunodeficiency (CVID), congenital thrombotic thrombocytopenic purpura (cTTP), cystic fibrosis, Diamond-Blackfan anemia (DBA), DOCK8 deficiency, dyskeratosis congenita, Fabry disease, factor V deficiency, factor VII deficiency, factor X deficiency, or a combination thereof. Factor XI deficiency, Factor XII deficiency, Factor XIII deficiency, Familial apolipoprotein E deficiency and atherosclerosis (ApoE), Familial hemophagocytic lymphohistiocytosis, Fanconi anemia (FA), Friedreich's ataxia, Gaucher disease, Glanzmann thrombasthenia, Glycemia, Glycogen storage disease, Glycogen storage disease type I (GSDI), Gray platelet syndrome, Hemophilia, Hemophilia A, Hemophilia B, Hereditary hemochromatosis, Hurler syndrome, Hyper-IgM, Hypogammaglobulinemia, Krabbe disease, Major histocompatibility complex class II deficiency (MHC-II), Maple syrup urine disease, Metachromatic leukodystrophy (MLD), Mucopolysaccharidosis, Mucopolysaccharidosis type I (MPS) I), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morquio syndrome), MPS V, MPS VI (Maroteaux-Lamy syndrome), MPSVII (Sly syndrome), muscular dystrophy, Niemann-Pick disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pernicious anemia, phenylketonuria (PKU), Pompe disease, pulmonary alveolar proteinosis (PAP), pure red cell aplasia (PRCA), pyruvate kinase deficiency, refractory anemia, Shwachman-Diamond syndrome, selective IgA deficiency, severe aplastic anemia, severe combined immunodeficiency (SCID), severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) , sickle cell anemia, sickle cell disease, sickle cell trait, Tay-Sachs disease, thalassemia, thalassemia intermedia, von Gierke disease, von Willebrand disease, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked severe combined immunodeficiency (SCID-X1), Zellweger syndrome, alpha-mannosidosis, beta-mannosidosis, beta-thalassemia, and / or beta-thalassemia major.

[0013] In various embodiments, the capsid comprises one or more viral polypeptides of the Ad5, Ad7, Ad11, Ad16, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises monocytes. In various embodiments, the capsid comprises one or more viral polypeptides of the Ad11, Ad16, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises monocytes. In various embodiments, the capsid comprises one or more viral polypeptides of the Ad11, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises monocytes. In various embodiments, the monocytes are CD11+ / CD14+ monocytes.

[0014] In various embodiments, the capsid comprises one or more viral polypeptides of the Ad5, Ad7, Ad11, Ad16, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises T cells. In various embodiments, the capsid comprises one or more viral polypeptides of the Ad5, Ad16, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises T cells. In various embodiments, the capsid comprises one or more viral polypeptides of the Ad34 or Ad35 serotype, and the hematopoietic cell type is or comprises T cells. In various embodiments, the T cells are CD3+ T cells.

[0015] In various embodiments, the capsid comprises one or more viral polypeptides of the Ad5, Ad7, Ad11, Ad16, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises an NK cell. In various embodiments, the capsid comprises one or more viral polypeptides of the Ad11, Ad16, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises an NK cell. In various embodiments, the capsid comprises one or more viral polypeptides of the Ad11, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises an NK cell. In various embodiments, the NK cell is a CD3- / CD56+ NK cell.

[0016] In various embodiments, the capsid comprises one or more viral polypeptides of the Ad5, Ad7, Ad11, Ad16, Ad34, or Ad35 serotype, and the hematopoietic cell type is or comprises a B cell. In various embodiments, the capsid comprises one or more viral polypeptides of the Ad16 serotype, and the hematopoietic cell type is or comprises a B cell. In various embodiments, the B cell is a CD20+ B cell.

[0017] In at least one aspect, the present disclosure provides an adenoviral vector and a hematopoietic cell comprising an adenoviral vector genome, wherein the adenoviral vector comprises a capsid comprising one or more viral polypeptides of an Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, or Ad50 serotype, wherein the one or more viral polypeptides comprise one or more of: (i) a fiber knob, (ii) a fiber shaft, (iii) a fiber tail, (iv) a penton, and (v) a hexon, and wherein the adenoviral vector genome comprises a heterologous vector. and a double-stranded DNA genome comprising a seed nucleic acid payload, wherein the hematopoietic cells are HSCs, common lymphoid progenitors (CLPs), T cells, NK cells, colony forming units (CFUs)-pre-B cells, B cells, common myeloid progenitor (CMP) cells, granulocyte-macrophage progenitor (GMP) cells, CFU-M cells, monoblasts, monocytes, macrophages, CFU-G cells, myeloblasts, granulocytes, neutrophils, eosinophils, basophils, megakaryocyte-erythroid progenitor (MEP) cells, BFU-E cells, CFU-E cells, erythroblasts, erythrocytes, CFU-Mk cells, megakaryocytes, and / or platelets, and optionally the HSC cells are CD34 + Long-term hematopoietic stem cells (LT-HSC) and / or CD34 + Short-term (ST)-HSC.

[0018] In at least one aspect, the present disclosure provides a hematopoietic cell comprising an adenoviral vector genome, the adenoviral vector genome comprising: (a) a 3'ITR and a 5'ITR, wherein the 3'ITR and the 5'ITR are of the same serotype selected from Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, and Ad50, respectively; (b) a packaging sequence, wherein the packaging sequence is of the same serotype as the 3'ITR and the 5'ITR; and c) heterologous nucleic acid payloads, wherein the hematopoietic cells are HSCs, common lymphoid progenitors (CLPs), T cells, NK cells, colony forming units (CFUs)-pre-B cells, B cells, common myeloid progenitor (CMP) cells, granulocyte-macrophage progenitors (GMPs), CFU-M cells, monoblasts, monocytes, macrophages, CFU-G cells, myeloblasts, granulocytes, neutrophils, eosinophils, basophils, megakaryocyte-erythroid progenitor (MEP) cells, BFU-E cells, CFU-E cells, erythroblasts, erythrocytes, CFU-Mk cells, megakaryocytes, and / or platelets, and optionally the HSC cells are CD34 + Long-term hematopoietic stem cells (LT-HSC) and / or CD34 +In various embodiments, the cells are short-term (ST)-HSCs, and contain a heterologous nucleic acid payload. In various embodiments, the cells are selected from the group consisting of adenosine deaminase deficiency (ADA), adrenoleukodystrophy (ALD), agammaglobulinemia, alpha-1 antitrypsin deficiency, congenital amegakaryocytic thrombocytopenia, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia-telangiectasia, Batten disease, Bernard-Soulier syndrome, CD40 / CD40L deficiency, chronic granulomatous disease, common variable immunodeficiency (CVID), congenital thrombotic thrombocytopenic purpura (cTTP), cystic fibrosis, Diamond-Blackfan anemia (DBA), DOCK8 deficiency, dyskeratosis congenita, Fabry disease, factor V deficiency, factor VII deficiency, factor X deficiency, Factor XI deficiency, Factor XII deficiency, Factor XIII deficiency, Familial apolipoprotein E deficiency and atherosclerosis (ApoE), Familial hemophagocytic lymphohistiocytosis, Fanconi anemia (FA), Friedreich's ataxia, Gaucher disease, Glanzmann thrombasthenia, Glycemia, Glycogen storage disease, Glycogen storage disease type I (GSDI), Gray platelet syndrome, Hemophilia, Hemophilia A, Hemophilia B, Hereditary hemochromatosis, Hurler syndrome, Hyper-IgM, Hypogammaglobulinemia, Krabbe disease, Major histocompatibility complex class II deficiency (MHC-II), Maple syrup urine disease, Metachromatic leukodystrophy (MLD), Mucopolysaccharidosis, Mucopolysaccharidosis type I (MPS) I), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morquio syndrome), MPS V, MPS VI (Maroteaux-Lamy syndrome), MPSVII (Sly syndrome), muscular dystrophy, Niemann-Pick disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pernicious anemia, phenylketonuria (PKU), Pompe disease, pulmonary alveolar proteinosis (PAP), pure red cell aplasia (PRCA), pyruvate kinase deficiency, refractory anemia, Shwachman-Diamond syndrome, selective IgA deficiency, severe aplastic anemia, severe combined immunodeficiency (SCID), severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID), The cells are from a subject suffering from a condition selected from sickle cell anemia, sickle cell disease, sickle cell trait, Tay-Sachs disease, thalassemia, thalassemia intermedia, von Gierke disease, von Willebrand disease, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked severe combined immunodeficiency (SCID-X1), Zellweger syndrome, alpha-mannosidosis, beta-mannosidosis, beta-thalassemia, and / or beta-thalassemia major.

[0019] In at least one aspect, the present disclosure provides an in vivo gene therapy method in a mammalian subject, the method comprising administering to the subject an adenoviral vector comprising: (a) a capsid comprising one or more viral polypeptides of an Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad37, or Ad50 serotype, wherein the one or more viral polypeptides comprise one or more of: (i) a fiber knob, (ii) a fiber shaft, (iii) a fiber tail, (iv) a penton, and (v) a hexon; and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload. In various embodiments, the genome further comprises: (a) a 3' ITR and a 5' ITR, each of which is of a viral polypeptide serotype; and (b) a packaging sequence, wherein the packaging sequence is of the viral polypeptide serotype. In various embodiments, the method includes mobilizing the subject's hematopoietic stem cells prior to administration of the adenoviral vector. In various embodiments, the heterologous nucleic acid payload comprises a selectable marker, and optionally, the selectable marker is MGMT. P140K In various embodiments, the method includes administering a selection agent to the subject, and optionally, the selection agent is O 6 In various embodiments, the method includes administering one or more immunosuppressive agents to the subject, optionally, the administration of the one or more immunosuppressive agents occurs before administration of the adenoviral vector.

[0020] In at least one aspect, the present disclosure provides an adenoviral donor vector comprising: (a) a capsid comprising one or more viral polypeptides of an Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad37, or Ad50 serotype, wherein the one or more viral polypeptides comprise one or more of: (i) a fiber knob, (ii) a fiber shaft, (iii) a fiber tail, (iv) a penton, and (v) a hexon; and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload. In various embodiments, the genome further comprises: (a) a 3'ITR and a 5'ITR, each of which is of a viral polypeptide serotype; and (b) a packaging sequence, wherein the packaging sequence is of a viral polypeptide serotype. In various embodiments, the heterologous nucleic acid payload comprises a selectable marker, and optionally, the selectable marker is MGMT. P140K is.

[0021] In various embodiments, the one or more viral polypeptides comprise (a) a fiber knob and a fiber shaft, (b) a fiber knob and a fiber tail, (c) a fiber knob and a penton, (d) a fiber knob and a hexon, (e) a fiber knob, a hexon, and a penton, (f) a fiber shaft and a fiber tail, (g) a fiber shaft and a penton, (h) a fiber shaft and a hexon, (i) a fiber shaft, a hexon, and a penton, (j) a fiber tail and a penton, (k) a fiber tail and a hexon, (l) a fiber tail, a hexon, and a penton, (m) a fiber knob, a fiber shaft, and a fiber tail, (n) a fiber knob, a fiber shaft, and a penton, (o) a fiber knob, a fiber shaft, and a hexon, (p) a fiber knob, a fiber shaft, a hexon, and a penton, (q) a fiber knob, a fiber shaft, a fiber tail, and a penton, (r) a fiber knob, a fiber shaft, a fiber tail, a penton, and a hexon, or (s) a penton and a hexon. In various embodiments, the fiber knob has a sequence at least 80% identical to a sequence selected from SEQ ID NOs: 15, 33, 51, 69, 87, 105, 123, 141, and 159. In various embodiments, the fiber shaft has a sequence at least 80% identical to a sequence selected from SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, and 158. In various embodiments, the fiber tail has a sequence at least 80% identical to a sequence selected from SEQ ID NOs: 18, 36, 54, 72, 90, 108, 126, 144, and 162. In various embodiments, the penton has a sequence at least 80% identical to a sequence selected from SEQ ID NOs: 16, 34, 52, 70, 88, 106, 124, 142, and 160. In various embodiments, the hexon has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 17, 35, 53, 71, 89, 107, 125, 143, and 161. In various embodiments, the adenoviral vector comprises a serotype fiber of the virus peptide.In various embodiments, the fiber has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, and 157. In various embodiments, the adenoviral vector is a chimeric vector characterized in that the capsid includes at least one of a fiber knob, a fiber shaft, a fiber tail, a hexon, or a penton that is not a serotype of the viral peptide. In various embodiments, the adenoviral vector is a helper-dependent vector.

[0022] In at least one aspect, the present disclosure provides an adenoviral donor vector genome comprising: (a) a 3'ITR and a 5'ITR, wherein the 3'ITR and the 5'ITR are of the same serotype selected from Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad37, and Ad50, respectively; (b) a packaging sequence, wherein the packaging sequence is of an ITR serotype; and (c) a heterologous nucleic acid payload. In various embodiments, the heterologous nucleic acid payload comprises a selectable marker, and optionally, the selectable marker is MGMT. P140K is.

[0023] In various embodiments, the heterologous nucleic acid payload encodes a protein. In various embodiments, the heterologous nucleic acid payload encodes a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a small RNA, and optionally, the small RNA is an shRNA. In various embodiments, the heterologous nucleic acid payload encodes a gene editing enzyme or system, and the gene editing is selected from CRISPR editing, base editing, prime editing, or zinc finger nuclease editing. In various embodiments, the heterologous nucleic acid payload is a nucleic acid payload that is associated with a disease such as adenosine deaminase deficiency (ADA), adrenoleukodystrophy (ALD), agammaglobulinemia, alpha-1 antitrypsin deficiency, congenital amegakaryocytic thrombocytopenia, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia-telangiectasia, Batten disease, Bernard-Soulier syndrome, CD40 / CD40L deficiency, chronic granulomatous disease, common variable immunodeficiency (CVID), congenital thrombotic thrombocytopenic purpura (cTTP), cystic fibrosis, Diamond-Blackfan anemia (DBA), DOCK8 deficiency, dyskeratosis congenita, Fabry disease, factor V deficiency, factor VII deficiency, factor X deficiency, or a combination thereof. Factor XI deficiency, Factor XII deficiency, Factor XIII deficiency, Familial apolipoprotein E deficiency and atherosclerosis (ApoE), Familial hemophagocytic lymphohistiocytosis, Fanconi anemia (FA), Friedreich's ataxia, Gaucher disease, Glanzmann thrombasthenia, Glycemia, Glycogen storage disease, Glycogen storage disease type I (GSDI), Gray platelet syndrome, Hemophilia, Hemophilia A, Hemophilia B, Hereditary hemochromatosis, Hurler syndrome, Hyper-IgM, Hypogammaglobulinemia, Krabbe disease, Major histocompatibility complex class II deficiency (MHC-II), Maple syrup urine disease, Metachromatic leukodystrophy (MLD), Mucopolysaccharidosis, Mucopolysaccharidosis type I (MPS) I), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morquio syndrome), MPS V, MPS VI (Maroteaux-Lamy syndrome), MPSVII (Sly syndrome), muscular dystrophy, Niemann-Pick disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pernicious anemia, phenylketonuria (PKU), Pompe disease, pulmonary alveolar proteinosis (PAP), pure red cell aplasia (PRCA), pyruvate kinase deficiency, refractory anemia, Shwachman-Diamond syndrome, selective IgA deficiency, severe aplastic anemia, severe combined immunodeficiency (SCID), severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID) , sickle cell anemia, sickle cell disease, sickle cell trait, Tay-Sachs disease, thalassemia, thalassemia intermedia, von Gierke disease, von Willebrand disease, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked severe combined immunodeficiency (SCID-X1), Zellweger syndrome, alpha-mannosidosis, beta-mannosidosis, beta-thalassemia, and / or beta-thalassemia major.

[0024] In various embodiments, the present disclosure provides a pharmaceutical composition comprising an adenoviral vector of the present disclosure, wherein the pharmaceutical composition is formulated for injection into a subject in need thereof.

[0025] definition A, An, The: As used herein, "a," "an," and "the" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "(a) element" discloses embodiments of exactly one element as well as embodiments containing two or more elements.

[0026] About: As used herein, the term "about," when used in reference to a value, refers to a similar value in the context of the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the reasonable degree of variation encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0027] Administration: As used herein, the term "administration" typically refers to administering a composition to a subject or system to achieve delivery of an agent that is or is contained in the composition.

[0028] Adoptive cell therapy: As used herein, "adoptive cell therapy" or "ACT" involves the transfer of cells with therapeutic activity to a subject, e.g., a subject in need of treatment for a condition, disorder, or disease. In some embodiments, ACT involves the transfer of cells to a subject following ex vivo and / or in vitro manipulation and / or cell expansion.

[0029] Affinity: As used herein, "affinity" refers to the strength of the sum of non-covalent interactions between a particular binding agent (e.g., a viral vector) and / or its binding moiety and a binding target (e.g., a cell or cell type). Unless otherwise indicated, as used herein, "binding affinity" refers to a 1:1 interaction between a binding agent and its binding target (e.g., a target cell of a viral vector and the viral vector). Those skilled in the art will understand that a change in affinity can be described by comparison to a reference (e.g., increased or decreased compared to a reference) or can be described numerically. Affinity is determined by the equilibrium dissociation constant (K D ) and / or the equilibrium association constant (K A ) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D is k off / kon is the quotient of K A is k on / k off is the quotient of k on refers to the association rate constant between the viral vector and the target cell, and k off refers to, for example, the dissociation of a viral vector from a target cell. on and k off can be determined by techniques known to those skilled in the art.

[0030] Agent: As used herein, the term "agent" may refer to any chemical entity, including, but not limited to, any one or more of the following: atoms, molecules, compounds, amino acids, polypeptides, nucleotides, nucleic acids, proteins, protein complexes, liquids, solutions, sugars, polysaccharides, lipids, or combinations or complexes thereof.

[0031] Allogeneic: As used herein, the term "allogeneic" refers to any material derived from one subject and then introduced into another subject, for example, allogeneic HSC transplantation.

[0032] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising one or more standard immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). Thus, the term antibody includes, but is not limited to, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents comprising them. An antibody may be a naturally occurring immunoglobulin (e.g., produced by an organism in response to an antigen). Synthetic, non-natural, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0033] As is well known in the art, a typical human immunoglobulin is a tetrameric agent of approximately 150 kD, comprising two identical heavy (H) chain polypeptides (each approximately 50 kD) and two identical light (L) chain polypeptides (each approximately 25 kD), which associate with each other to form what is commonly referred to as a "Y-shaped" structure. Typically, each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain comprises three CH domains: CH1, CH2, and CH3. A short region known as the "switch" joins the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL), separated from each other by another "switch". Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complementarity-determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and / or light chains are typically understood to provide binding moieties capable of interacting with antigens. The constant domains can mediate antibody binding to various immune system cells (e.g., effector cells and / or cells mediating cytotoxicity), receptors, and elements of the complement system. The heavy and light chains can be linked to each other by a single disulfide bond, and two other disulfide bonds can link the heavy chain hinge regions together, thereby linking dimers to form tetramers. When a native immunoglobulin folds, the FR regions form beta sheets that provide the structural framework for the domain, and the CDR loop regions of both the heavy and light chains are packed in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure.

[0034] In some embodiments, the antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody). In some embodiments, the antibody comprises at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer comprising two heavy chain monomers and two light chain monomers. Furthermore, the term "antibody" (unless otherwise specified or clear from the context) refers to any art-known construct or format that utilizes antibody structural and / or functional features, including intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNARs or fragments thereof), camel antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affibodies, anti-idiotypic (anti-Id) antibodies (e.g., anti-anti-Id antibodies), and the like. Antibodies include, but are not limited to, small modular immunopharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAbs®), VHHs, Anticalins®, Nanobodies® minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, microproteins, Fynomers®, Centyrins®, and KALBITOR®, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0035] In various embodiments, an antibody comprises one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs) or variable domains. In some embodiments, an antibody may be a covalently modified ("conjugated") antibody (e.g., an antibody comprising a polypeptide comprising one or more standard immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently attached to one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, the antibody sequence elements are humanized, primatized, chimerized, etc., as known in the art.

[0036] Antibodies comprising a heavy chain constant domain can be of any known class of antibody, including, but not limited to, IgA, secretory IgA, IgG, IgE, and IgM, based on the heavy chain constant domain amino acid sequence (e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ)). IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. As used herein, "light chains" can be of different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the light chain constant domain. In some embodiments, antibodies have constant region sequences characteristic of murine, rabbit, primate, or human immunoglobulins. Naturally produced immunoglobulins are typically glycosylated on the CH2 domain. As is known in the art, the affinity and / or other binding properties of an Fc region for an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, an antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present invention comprise a glycosylated Fc domain, including, for example, an Fc domain that has been modified or engineered, e.g., with such glycosylation.

[0037] "Between" or "from": As used herein, the term "between" refers to the content between a stated upper and lower limit, or a first and second limit, inclusive of the limits. Similarly, when used in the context of a range of values, the term "from" indicates that the range includes the content between a stated upper and lower limit, or a first and second limit, inclusive of the limits.

[0038] Binding: As used herein, the term "binding" refers to a non-covalent association between two or more agents. "Direct" binding involves physical contact between the agents, while indirect binding involves physical interaction through physical contact with one or more intermediate agents. Binding between two or more agents can occur and / or be assessed in any of a variety of contexts (e.g., while covalently or otherwise associated with a carrier agent and / or within a biological system or cell), including when the interacting agents are studied alone or in the context of a more complex system.

[0039] Biological sample: As used herein, the term "biological sample" refers to a sample obtained or derived from a biological source of interest (e.g., a tissue or organism or cell culture) as described herein. In some embodiments, the biological resource is or includes an organism, such as an animal or a human. In some embodiments, the biological sample is or includes a biological tissue or fluid. In some embodiments, the biological sample can be or include cells (e.g., hematopoietic cells), tissue, or bodily fluid (e.g., blood). A biological sample can be a "primary sample" obtained directly from a biological source, or can be a "processed sample" (e.g., a sample prepared from a primary sample). A biological sample may also be referred to as a "sample."

[0040] Cancer: As used herein, the term "cancer" refers to a condition, disorder, or disease in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in them exhibiting an abnormally elevated growth rate and / or an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer can comprise one or more tumors. In some embodiments, cancer can be or can comprise cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, cancer is or can comprise a solid tumor. In some embodiments, cancer is or can comprise a hematological tumor.

[0041] Chimeric Antigen Receptor: As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered protein that includes (i) an extracellular domain that includes a portion that binds to a target antigen, (ii) a transmembrane domain, and (iii) an intracellular signaling domain that sends an activating signal when the CAR is stimulated by binding of the extracellular binding portion to the target antigen. CARs are also known as chimeric T cell receptors or chimeric immune receptors.

[0042] Combination therapy: As used herein, the term "combination therapy" refers to the administration of two or more agents or regimens to a subject, such that the two or more agents or regimens together treat a condition, disorder, or disease in the subject. In some embodiments, the two or more therapeutic agents or regimens can be administered simultaneously, sequentially, or in overlapping administration regimens. Those skilled in the art will understand that combination therapy includes, but does not necessarily require, the two agents or regimens to be administered together in a single composition or simultaneously.

[0043] Control of expression or activity: As used herein, a first element (e.g., a protein such as a transcription factor, or a nucleic acid sequence such as a promoter) "controls" or "drives" the expression or activity of a second element (e.g., a protein or a nucleic acid encoding an agent such as a protein) when the expression or activity of the second element is completely or partially dependent on the state (e.g., presence, absence, conformation, chemical modification, interaction, or other activity) of the first element under at least one set of conditions. Control of expression or activity can be substantial, for example, in that a change in the state of the first element can result in a change in the expression or activity of the second element of at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold) compared to a reference control under at least one set of conditions.

[0044] "Corresponding to": As used herein, the term "corresponding to" may be used to indicate the location / identity of a structural element in a compound or composition through comparison to an appropriate reference compound or composition. For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding" to a residue in an appropriate reference polymer. For example, those skilled in the art will understand that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or labeled) according to the scheme of the associated reference sequence (e.g., even if such designation does not reflect the literal numbering of the provided sequence). By way of illustration, if a reference sequence contains a particular amino acid motif at positions 100-110 and a second, related sequence contains the same motif at positions 110-120, the motif position in the second, related sequence can be said to "correspond" to positions 100-110 of the reference sequence. Those skilled in the art will appreciate that corresponding positions can be readily identified, for example, by alignment of sequences, and that such alignment is generally accomplished by any of a variety of known tools, strategies, and / or algorithms, including, but not limited to, software programs such as BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE.

[0045] Dosage regimen: As used herein, the term "dosage regimen" can refer to a set of one or more identical or different unit doses administered to a subject, typically comprising the administration of multiple unit doses, where the administration of each unit dose is separated from the administration of other unit doses by a period of time. In various embodiments, one or more or all of the unit doses of a dosage regimen can be the same or can vary (e.g., increase over time, decrease over time, or adjust according to the subject's and / or physician's decision). In various embodiments, one or more or all of the periods between each dose can be the same or can vary (e.g., increase over time, decrease over time, or adjust according to the subject's and / or physician's decision). In some embodiments, a given therapeutic agent has a recommended dosing regimen, which can include one or more doses. Typically, at least one recommended dosing regimen for a marketed drug is known to those of skill in the art. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0046] Downstream and upstream: As used herein, the term "downstream" means that a first DNA region is closer to the C-terminus of a nucleic acid comprising the first and second DNA regions than a second DNA region. As used herein, the term "upstream" means that a first DNA region is closer to the N-terminus of a nucleic acid comprising the first and second DNA regions than a second DNA region.

[0047] Effective amount: An "effective amount" is the quantity of a composition (e.g., a formulation) needed to produce a desired physiological change in a subject. Effective amounts are often administered for research purposes.

[0048] "Engineered": As used herein, the term "engineered" refers to aspects that have been manipulated by the hand of man. For example, a polynucleotide is considered "engineered" if the hand of man has manipulated two or more sequences that are not inherently linked together in that order, such that they are directly linked to one another within the engineered polynucleotide. Those skilled in the art will understand that an "engineered" nucleic acid or amino acid sequence can be a recombinant nucleic acid or amino acid sequence and can be referred to as "genetically engineered." In some embodiments, an engineered polynucleotide includes coding and / or regulatory sequences that are found in nature operably linked to a first sequence but not naturally found operably linked to a second sequence, which are in the engineered polynucleotide operably linked to the second sequence by the hand of man. In some embodiments, a cell or organism is considered "engineered" or "genetically engineered" if it has been manipulated to change its genetic information (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution, deletion, or mating). As is common knowledge and understood by those of skill in the art, complete or incomplete progeny or copies of an engineered polynucleotide or cell are typically still referred to as "engineered," regardless of the direct manipulation of the previous entity.

[0049] Excipient: As used herein, "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc.

[0050] Expression: As used herein, "expression" refers individually and / or cumulatively to one or more biological processes that result in the production of an encoded agent, such as a protein, from a nucleic acid sequence. Expression specifically includes either or both of transcription and translation.

[0051] Contiguous: As used herein, a first element (e.g., a nucleic acid sequence or an amino acid sequence) present in a contiguous sequence with a second element and a third element is "flanked" by the second and third elements when the first and third elements are positioned in the contiguous sequence between the second and third elements. Thus, in such a configuration, the second and third elements can be referred to as "adjacent" to the first element. Flanking elements can be directly adjacent to the flanking element or separated from the flanking element by one or more related units. In various examples where the contiguous sequence is a nucleic acid or amino acid sequence and the related units are bases or amino acid residues, respectively, the number of units in the contiguous sequence between the flanked sequences and, independently, the first and / or second flanking elements can be, for example, 50 units or less, e.g., 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1 unit or less, or 0 units.

[0052] Fragment: As used herein, a "fragment" refers to a structure that comprises and / or consists of a discrete portion of a reference material (sometimes referred to as a "parent" material). In some embodiments, a fragment lacks one or more portions found in the reference material. In some embodiments, a fragment comprises or consists of one or more portions found in the reference material. In some embodiments, the reference material is a polymer such as a polynucleotide or a polypeptide. In some embodiments, a fragment of a polymer comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomer units (e.g., residues) of a reference polymer. In some embodiments, a fragment of a polymer comprises or consists of at least 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomer units (e.g., residues) found in the reference polymer. A fragment of a reference polymer is not necessarily identical to the corresponding portion of the reference polymer. For example, a fragment of a reference polymer may be a polymer having a sequence of residues that is at least 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the reference polymer. Fragments may or may not be generated by physical fragmentation of the reference material. In some cases, fragments are generated by physical fragmentation of the reference material. In some cases, fragments are not generated by physical fragmentation of the reference material, but instead may be generated, for example, by de novo synthesis or other means.

[0053] Gene, transgene: As used herein, the term "gene" refers to a DNA sequence that is or includes a coding sequence (i.e., a DNA sequence that encodes an expression product such as an RNA product and / or a polypeptide product), optionally together with some or all of the regulatory sequences that control expression of the coding sequence. In some embodiments, a gene includes non-coding sequences, such as, but not limited to, introns. In some embodiments, a gene can include both coding (e.g., exon) and non-coding (e.g., intron) sequences. In some embodiments, a gene includes a regulatory sequence that is a promoter. In some embodiments, a gene includes one or both of (i) DNA nucleotides extending a predetermined number of nucleotides upstream of the coding sequence in a reference context, such as a source genome, and (ii) DNA nucleotides extending a predetermined number of nucleotides downstream of the coding sequence in a reference context, such as a source genome. In various embodiments, the predetermined number of nucleotides can be 500 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 75 kb, or 100 kb. As used herein, "transgene" refers to a gene that is not endogenous or native to the reference construct in which it is present or into which it can be placed by manipulation.

[0054] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (pre- and / or post-processing) or the polypeptide (pre- and / or post-modification) encoded by the RNA transcribed from a gene.

[0055] Host cell, target cell: As used herein, "host cell" refers to a cell into which exogenous DNA, such as a transgene, has been introduced (recombinantly or otherwise). One of skill in the art will understand that a "host cell" can be the cell into which the exogenous DNA was originally introduced and / or its complete or incomplete progeny or copies. In some embodiments, a host cell comprises one or more viral genes or transgenes. In some embodiments, a host cell is a cell that has been transduced by a viral vector, e.g., a vector of the present disclosure, or its viral genome, e.g., a viral genome disclosed herein. In some embodiments, an intended or potential host cell may be referred to as a target cell. In some embodiments, a cell or cell type selectively transduced and / or selectively transduced by a viral vector of the present disclosure may be referred to as a target cell of the viral vector. In some embodiments, a host cell that has been transduced and / or transduced (e.g., selectively transduced and / or selectively transduced) by a viral vector of the present disclosure may be referred to as a target cell of the viral vector. In some embodiments, the term "host cell" or "target cell" includes progeny of cells that have been introduced and / or transduced (e.g., selectively introduced and / or selectively transduced) by a viral vector of the present disclosure, e.g., progeny that contain exogenous DNA sequences derived from the DNA sequences introduced by the viral vector.

[0056] In various embodiments, host or target cells are identified by the presence, absence, or expression level of various surface markers.

[0057] A statement that a cell or population of cells is "positive" for or expresses a particular marker refers to the detectable presence of the particular marker on or within the cells. When referring to a surface marker, the term can refer to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, where the staining is detectable by flow cytometry at a level substantially above that detected by performing the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a substantially similar level for cells known to be positive for the marker, and / or at a substantially higher level than cells known to be negative for the marker.

[0058] A statement that a cell or population of cells is "negative" for a particular marker or lacks expression of the marker refers to the substantial absence of detectable presence of the particular marker on or within the cells. When referring to a surface marker, the term can refer to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, where the staining is not detected by flow cytometry at a level substantially greater than that detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that of cells known to be positive for the marker, and / or at a level substantially similar to that of cells known to be negative for the marker.

[0059] Identity: As used herein, the term "identity" refers to the overall relatedness between polymers, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for calculating the percent identity between two provided sequences are known in the art. The term "% sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between a protein sequence and a nucleic acid sequence, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described below. Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A.M., and Griffin, H.G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.For example, calculating the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps and the length of each gap that may need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using computational algorithms such as BLAST (Base Local Alignment Search Tool). Sequence alignment and percent identity calculations may be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignments may also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989)) with default parameters (gap penalty = 10, gap length penalty = 10).Relevant programs include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990)), DNASTAR (DNASTAR, Inc., Madison, Wisconsin), and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, pp. 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). It can also be performed using the NIH Publication No. 2002 / 0100106, NIH Publication No. 2002 / 0100106, and ...

[0060] "Improve," "Increase," "Inhibit," or "Reduce": As used herein, the terms "improve," "increase," "inhibit," and "reduce," and their grammatical equivalents, indicate a qualitative or quantitative difference from a reference.

[0061] Isolated: As used herein, "isolated" refers to substances and / or entities that (1) have been separated from at least some of the components with which they were associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) have been designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of the other components with which they were originally associated. In some embodiments, isolated agents are greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may be considered "isolated" or even "pure" even after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the substance is calculated without such carriers or excipients. By way of example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is considered "isolated" if: a) by its origin or derivation, it is not associated with some or all of the components that naturally accompany it in its native state; b) it is substantially free from other polypeptides or nucleic acids of the same species from the species that naturally produces it; or c) it is expressed by or otherwise associated with components from cells or other expression systems other than the species that naturally produces it. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that which naturally produces it is considered to be an "isolated" polypeptide.Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from a) other components with which it is naturally associated, and / or b) other components with which it was associated when originally produced.

[0062] Operably linked: As used herein, "operably linked" or "operatively linked" refers to the association of at least a first element with a second element, such that the component elements are in a relationship permitting them to function in their intended manner. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence when the regulatory sequence and coding sequence are associated in a manner that allows control of expression of the coding sequence by the regulatory sequence. In some embodiments, an "operably linked" regulatory sequence is directly or indirectly covalently linked to the coding sequence (e.g., in a single nucleic acid). In some embodiments, the regulatory sequence controls expression of the coding sequence in trans, and inclusion of the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement for operably linked.

[0063] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable," when applied to one or more, or all, of the ingredients for the formulation of a composition disclosed herein, means that each ingredient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0064] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that facilitates the formulation of an agent (e.g., a pharmaceutical agent), alters the bioavailability of the agent, or facilitates the transport of the agent from one organ or part of a subject to another organ or part of a subject. Some examples of materials which can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0065] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.

[0066] Promoter: As used herein, a "promoter" or "promoter sequence" may be a DNA regulatory region involved directly or indirectly (e.g., via a promoter-binding protein or substance) in the initiation and / or processivity of transcription of a coding sequence. A promoter can initiate transcription of a coding sequence when one or more transcription factors and / or regulatory moieties bind to the promoter under appropriate conditions. A promoter involved in initiating transcription of a coding sequence can be "operably linked" to a coding sequence. In certain instances, a promoter can be or include a DNA regulatory region extending upstream (5' direction) from the transcription initiation site (at its 3' end), such that the sequence so designated contains one or both of the minimum number of bases or elements necessary to initiate a transcription event. A promoter can also be, include, be operably associated with, or be operably linked to expression control sequences such as enhancer and repressor sequences. In some embodiments, a promoter can be inducible. In some embodiments, a promoter can be a constitutive promoter. In some embodiments, a conditional (e.g., inducible) promoter can be unidirectional or bidirectional. A promoter may be or contain sequences identical to sequences known to be present in the genome of a particular species. In some embodiments, a promoter may be or contain a hybrid promoter in which sequences containing transcriptional regulatory regions can be obtained from one source and sequences containing transcriptional initiation regions can be obtained from a second source. Systems for linking regulatory elements to coding sequences within a transgene are well known in the art (general molecular biology and recombinant DNA techniques are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0067] Reference: As used herein, "reference" refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or a population thereof, or a measure or characteristic representative thereof, is compared to a reference, agent, sample, sequence, subject, animal, or individual, or a population thereof, or a measure or characteristic representative thereof. In some embodiments, the reference is a measured value. In some embodiments, the reference is an established standard or expected value. In some embodiments, the reference is a longitudinal reference. The reference may be quantitative or qualitative. Typically, as understood by those of skill in the art, the reference and the value to which it is compared represent measurements under comparable conditions. Those of skill in the art will understand when sufficient similarity exists to justify reliance and / or comparison. In some embodiments, a suitable reference may be an agent, sample, sequence, subject, animal, or individual, or a population thereof, under conditions that a skilled artisan would recognize as comparable, for example, for purposes of assessing one or more particular variables (e.g., the presence or absence of an agent or condition), or a measure or characteristic representative thereof. While not intending to be bound by any particular embodiment(s), in various embodiments, the reference sequence may be a sequence associated with a sequence accession number provided herein, certain of which are provided in the list of accession sequences below.

[0068] Regulatory sequence: As used herein in the context of expression of a nucleic acid coding sequence, a regulatory sequence is a nucleic acid sequence that controls the expression of the coding sequence. In some embodiments, a regulatory sequence can control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.).

[0069] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, rat, or mouse). In some embodiments, the subject is suffering from a disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is not suffering from a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject exhibits one or more features characteristic of susceptibility to, or risk for, a disease, disorder, or condition. In some embodiments, the subject is a subject being tested for and / or treated with therapy for a disease, disorder, or condition. In some embodiments, a human subject can be referred to interchangeably as a "patient" or an "individual."

[0070] Therapeutic Agent: As used herein, the term "therapeutic agent" refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of a model organism or a human population. In some embodiments, the appropriate population may be defined by various criteria, such as a particular age group, sex, genetic background, pre-existing clinical condition, etc. In some embodiments, a therapeutic agent is a substance that can be used to treat a disease, disorder, or condition. In some embodiments, a therapeutic agent is a drug that has been approved, or needs to be approved, by a government agency before being commercially available for administration to humans. In some embodiments, a therapeutic agent is a drug that requires a prescription for administration to humans.

[0071] Therapeutically effective amount: As used herein, "therapeutically effective amount" refers to an amount that produces a desired effect when administered. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to the disease, disorder, and / or condition in accordance with a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is one that reduces the occurrence and / or severity of, and / or delays the onset of, one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not, in fact, require that successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to patients in need of such treatment, results in a specific desired pharmacological response in a substantial number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount measured in one or more specific tissues (e.g., tissues affected by a disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will appreciate that in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose, hi some embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0072] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that results in the partial or complete alleviation, amelioration, relief, inhibition, delay in onset, less severity, and / or reduction in incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, or condition, or is administered with the intent to achieve any such result. In some embodiments, such treatment may be of subjects who do not exhibit symptoms of the associated disease, disorder, or condition and / or who exhibit only early symptoms of the disease, disorder, or condition. Alternatively or additionally, such treatment may be of subjects who exhibit one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects who have been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be of subjects known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, or condition. "Prophylactic treatment" includes treatment administered to a subject who does not exhibit signs or symptoms of the condition to be treated, or who exhibits only early signs or symptoms of the condition to be treated, such that the treatment is administered with the intent to reduce, prevent, or decrease the risk of developing the condition. Prophylactic treatment thus serves as a prophylactic treatment against the condition. "Therapeutic treatment" includes treatment administered to a subject who exhibits symptoms or signs of a condition, with the intent to reduce the severity or progression of the condition.

[0073] Unit dose: As used herein, the term "unit dose" refers to a single dose of a pharmaceutical composition and / or an amount administered in a physically discrete unit. In many embodiments, a unit dose contains a predetermined amount of an active agent, e.g., a predetermined viral titer (the number of viruses, virions, or viral particles in a given volume). In some embodiments, a unit dose contains an entire single dose of an agent. In some embodiments, two or more unit doses are administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required or expected to be required to achieve the intended effect. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic moieties, a predetermined amount of one or more therapeutic moieties in solid form, a sustained-release formulation or drug delivery device containing a predetermined amount of one or more therapeutic moieties, etc. It will be understood that a unit dose can be present in a formulation containing any of a variety of ingredients in addition to the therapeutic moiety(s). For example, it can include an acceptable carrier (e.g., a pharmaceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, etc. As will be appreciated by those skilled in the art, in many embodiments, a suitable total daily dose of a particular therapeutic agent can comprise a fraction or multiple of a unit dose and can be determined, for example, by a physician within the scope of sound medical judgment. In some embodiments, the specific effective dose level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific active compound used, the particular composition used, the age, weight, general health, sex, and diet of the subject, the time of administration and rate of excretion of the particular active compound used, the duration of treatment, drugs and / or additional therapies used in combination with or concurrently with the particular compound(s) used, and similar factors well known in the medical arts. [Brief explanation of the drawings]

[0074] [Figure 1]1 is a diagram showing the results of anti-hexon staining of CD34+ cells 3 hours after infection of the cells with the indicated adenovirus serotypes. Cells were infected with 5,000 virus particles per cell or 2,000 virus particles per cell. For each tested serotype, the diagram contains two replicates of data, with each replicate including the analysis results at 5,000 virus particles per cell and 2,000 virus particles per cell, in the order shown. Data represent infection efficiencies. [Figure 2] 1 is a diagram showing the results of qPCR analysis of adenoviral DNA in CD34+ cells infected with the indicated adenovirus serotypes. Cells were infected with 5,000 viral particles per cell or 2,000 viral particles per cell. For each tested serotype, the diagram contains two replicates of data, with each replicate including the analysis results at 5,000 viral particles per cell and 2,000 viral particles per cell, in the order shown. Data represent relative infection efficiencies. [Figure 3] FIG. 1 is a schematic diagram of hematopoietic cell differentiation, including hematopoietic stem cells, progenitor cells, and terminally differentiated cells. [Figure 4] 1 is a schematic diagram of a plasmid containing an E1-deleted Ad5 genome with an EGFP reporter construct. The schematic shows a single vector, but for representation purposes only, the adenoviral genome is shown in two sections with the hexon region contained in each section and the orientation of each section relative to each other is shown. [Figure 5] 1 is a schematic diagram of a plasmid containing an E1-deleted Ad7 genome with an EGFP reporter construct. The schematic shows a single vector, but for representation purposes only, the adenoviral genome is shown in two sections with the hexon region contained in each section and the orientation of each section relative to each other is shown. [Figure 6] 1 is a schematic diagram of a plasmid containing an E1-deleted Ad11 genome with an EGFP reporter construct. The schematic shows a single vector, but for representation purposes only, the adenoviral genome is shown in two sections with the hexon region contained in each section and the orientation of each section relative to each other is shown. [Figure 7] 1 is a schematic diagram of a plasmid containing an E1-deleted Ad16 genome with an EGFP reporter construct. The schematic shows a single vector, but for representation purposes only, the adenoviral genome is shown in two sections with the hexon region contained in each section and the orientation of each section relative to each other is shown. [Figure 8] 1 is a schematic diagram of a plasmid containing an E1-deleted Ad34 genome with an EGFP reporter construct. The schematic shows a single vector, but for representation purposes only, the adenoviral genome is shown in two sections with the hexon region contained in each section and the orientation of each section relative to each other is shown. [Figure 9] 1 is a schematic diagram of a plasmid containing an E1-deleted Ad35 genome with an EGFP reporter construct. The schematic shows a single vector, but for representation purposes only, the adenoviral genome is shown in two sections with the hexon region contained in each section and the orientation of each section relative to each other is shown. [Figure 10] Schematic diagram of a plasmid containing an E1-deleted Ad35++ genome with an EGFP reporter construct. The diagram shows a single vector, but for representation purposes only, the adenoviral genome is shown in two sections with the hexon region contained in each section and the orientation of each section relative to each other is shown. [Figure 11] 1 is an exemplary depiction of the gating used to analyze monocyte, T cell, NK cell, and B cell populations present in PBMCs using flow cytometry. The boxes indicate the gates used to define cell types. Arrows from one plot to another indicate that the gated population in the first plot is displayed in the second plot. The representative data shown in this figure corresponds to human PBMCs 48 hours after infection with an E1-deleted adenoviral vector of the present disclosure at an MOI of 2,000 viral particles per cell. [Figure 12]Figure 1 is a graph showing the results of GFP analysis of monocytes present in human PBMCs from donor 1 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenovirus serotypes. Monocytes were identified as CD14+ / CD11b+ myeloid cells. The percentage of cells that were GFP positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, Ad35++, and Ad16 (from left to right). Data represent infection efficiencies. [Figure 13] 1 is a graph showing the results of GFP analysis of T cells present in human PBMCs from donor 1 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenovirus serotypes. T cells were identified as CD3+ lymphoid cells. The percentage of cells that were GFP-positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, Ad35++, and Ad16 (from left to right). Data represent infection efficiencies. [Figure 14] Figure 1 is a graph showing the results of GFP analysis of NK cells present in human PBMCs from donor 1 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenoviral serotypes. NK cells were identified as CD3- / CD56+ lymphoid cells. The percentage of cells that were GFP positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, Ad35++, and Ad16 (from left to right). Data represent infection efficiencies. [Figure 15]1 is a graph showing the results of GFP analysis of B cells present in human PBMCs from donor 1 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenovirus serotypes. B cells were identified as CD20+ lymphoid cells. The percentage of cells that were GFP-positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, Ad35++, and Ad16 (from left to right). Data represent infection efficiencies. [Figure 16] Figure 1 is a graph showing the results of GFP analysis of monocytes present in human PBMCs from donor 2 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenoviral serotypes. Monocytes were identified as CD14+ / CD11b+ myeloid cells. The percentage of cells that were GFP positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, and Ad35++ (from left to right). Data represent infection efficiencies. [Figure 17] 1 is a graph showing the results of GFP analysis of T cells present in human PBMCs from donor 2 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenoviral serotypes. T cells were identified as CD3+ lymphoid cells. The percentage of cells that were GFP-positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, and Ad35++ (from left to right). Data represent infection efficiencies. [Figure 18]Figure 1 is a graph showing the results of GFP analysis of NK cells present in human PBMCs from donor 2 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenoviral serotypes. NK cells were identified as CD3- / CD56+ lymphoid cells. The percentage of cells that were GFP positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, and Ad35++ (from left to right). Data represent infection efficiencies. [Figure 19] 1 is a graph showing the results of GFP analysis of B cells present in human PBMCs from donor 2 48 hours after infection of the cells with E1-deleted adenoviral vectors of the indicated adenoviral serotypes. B cells were identified as CD20+ lymphoid cells. The percentage of cells that were GFP-positive is shown. Cells were infected at an MOI of 500, 2000, and 5000 viral particles per cell. At each MOI, data are shown for Ad5, Ad7, Ad11, Ad34, Ad35, and Ad35++ (from left to right). Data represent infection efficiencies. DETAILED DESCRIPTION OF THE INVENTION

[0075] The present disclosure includes compositions and methods for selectively targeting hematopoietic cells (e.g., one or more specific types of hematopoietic cells). In particular, the present disclosure includes viral vectors that selectively target one or more types of hematopoietic cells. In various embodiments, the viral vector that selectively targets one or more types of hematopoietic cells is an adenoviral vector of the present disclosure, such as the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector disclosed herein. Various hematopoietic stem cell types that can be targeted by the vectors of the present disclosure are disclosed herein, including hematopoietic stem cell types, hematopoietic progenitor cell types, and further differentiated hematopoietic cell types, including, but not limited to, terminally differentiated hematopoietic cell types.

[0076] Hematopoiesis refers to the process by which various types of blood cells are produced. Because diverse cell types are derived from hematopoietic stem and progenitor cells (HSPCs) through the process of differentiation, hematopoiesis is sometimes presented as a hierarchy. Hematopoietic stem cells (HSCs) are positioned at the "top" of this hierarchy (see Figure 3). Without intending to be bound by any particular scientific theory, HSCs are understood to be self-renewing and multipotent, differentiating into progenitor cells that further differentiate to produce mature and / or terminally differentiated blood cells. Differentiation stages are disclosed herein (including, for example, Figure 3), and further differentiation refers to increasing differentiation compared to HSCs or other chronologically preceding states and / or further change away from the HSC state, as shown in the differentiation lineages depicted in Figure 3 or otherwise disclosed herein. By some estimates, an adult human may contain tens of thousands of HSCs, which differentiate into precursor cells and ultimately give rise to hundreds of millions of progenitor cells that differentiate into mature effector cells. Thus, a population of multipotent self-renewing HSCs generates large numbers of differentiated progeny by amplification and progressive lineage restriction. As referred to herein, hematopoietic cell type refers to any and all cell types that are or are derived from hematopoietic stem cells and / or hematopoietic progenitor cells, including, but not limited to, the specific cell types disclosed herein.

[0077] Without intending to be bound by any particular scientific theory, HSCs can be divided into two subpopulations according to their CD34 expression: CD34 + Long-term (LT)-HSC and CD34 + Short-term (ST)-HSCs. LT-HSCs differentiate into ST-HSCs, which subsequently differentiate into multipotent progenitor cells (MPPs). In various embodiments, the hematopoietic cell type is CD34 + Being or comprising a hematopoietic cell.

[0078] Without intending to be bound by any particular scientific theory, it is understood that progenitor cells are characterized by restricted differentiation, in that they lack the capacity for self-renewal and can only give rise to cells of specific lineages. The precursors can be myeloid or lymphoid progenitor cells (termed common myeloid progenitors (CMP) and common lymphoid progenitors (CLP), respectively).

[0079] CMPs can differentiate into granulocyte-macrophage progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs). GMPs can differentiate into granulocytes (e.g., neutrophils, eosinophils, and basophils) and monocytes (which can differentiate into macrophages). MEPs can differentiate into megakaryocytes / platelets and erythrocytes. CLPs can differentiate into T, NK, and B cells.

[0080] Hematopoiesis further includes cell types named based on their identification in colony-forming unit assays. Cells that form hematopoietic colonies (so-called CFUs or CFCs) can represent steps or stages of hematopoietic differentiation between HSCs and more terminally differentiated cells. CFUs can be identified by culturing hematopoietic cells in semi-solid medium (typically methylcellulose or agar) supplemented with cytokines that promote localized proliferation and differentiation of hematopoietic cells in individual colonies. CFUs can be distinguished by factors including, but not limited to, the number of cells within the colony, the time required to produce the colony, and / or the cell types within the colony. Generally, without wishing to be bound by any particular scientific theory, progenitor cells can produce colonies containing, for example, at least 30,000 cells, including multiple lineage cell types, by, for example, 15-18 days of culture. In various embodiments, the culture can produce colonies that generate erythroid bursts (e.g., 5,000 cells), referred to as burst-forming units-erythroid (BFU-E). Other colony types can include granulomonocytic colonies (colony forming unit, granulomonocytic (CFU-GM)), and colonies of 50-200 cells that are, for example, erythroid cells (colony forming unit, erythroid (CFU-E)), granulocytic cells (CFU-G), or monocytic cells (CFU-M). These descriptions of colonies are for general illustration only, and methods and techniques for colony analysis and identification are known in the art.

[0081] CLPs may be referred to as CFU-L cells. In various embodiments, CFU-L cells can differentiate into CFU-B cells, which can differentiate into B lymphoblasts, which in turn differentiate into pre-B lymphocytes, which in turn differentiate into B lymphocytes. In various embodiments, CFU-L cells can differentiate into CFU-T cells, which can differentiate into T lymphoblasts, which in turn differentiate into pre-T lymphocytes, which in turn differentiate into T lymphocytes.

[0082] CMPs may be referred to as CFU-GEMM cells. GMPs may be referred to as CFU-GM cells. In various embodiments, CFU-GM cells can differentiate into CFU-M cells, which differentiate into monoblasts, and CFU-G cells, which differentiate into neutrophils (e.g., via myeloblasts and neutrophil myelocytes). MEPs can differentiate into BFU-E cells, which can differentiate into CFU-E cells, which can differentiate into erythroblasts (e.g., via proerythroblasts, normoblasts, and normochromatic erythroblasts). MEPs can differentiate into CFU-Mk cells, which differentiate into megakaryocytes. CFU-Gemm cells can also differentiate into CFU-Eo cells, which differentiate into eosinophils (e.g., via myeloblasts and eosinophilic myelocytes), and CFU-Baso cells, which differentiate into basophils (e.g., via myeloblasts and basophilic myelocytes). Megakaryocytic progenitor cells can include BFU-MK cells that differentiate into more mature progenitor cells called CFU-MK cells.

[0083] HSC self-renewal and hematopoietic differentiation are controlled by multiple positive and negative regulatory elements, through poorly understood mechanisms. Both intrinsic and extrinsic factors are likely involved, including epigenetic and microenvironmental factors, as well as endogenous transcription factors (TFs) and exogenous cytokines that contribute to the stepwise differentiation of HSCs into mature blood cells.

[0084] A variety of means for distinguishing between hematopoietic cell types are known in the art.

[0085] The present disclosure includes the recognition that gene therapy selectively targeting hematopoietic stem cells can be useful for long-term, propagable modification of hematopoietic cells. The present disclosure includes the recognition that gene therapy selectively targeting hematopoietic progenitor cells can be useful for long-term, propagable modification of hematopoietic cells. The present disclosure includes the recognition that gene therapy selectively targeting hematopoietic cells that are not stem cells and / or progenitor cells (e.g., terminally differentiated cells) can be useful for rapid therapeutic effects on one or more target cell types. For example, in various embodiments, differentiated cells can have a more immediate effect because they do not require time for differentiation into effector cells. The present disclosure includes the recognition that gene therapy selectively targeting hematopoietic cells that are not stem cells and / or progenitor cells (e.g., terminally differentiated cells) can be useful for transient modification of a target cell type population. For example, in various embodiments, differentiated cells do not produce or constitute a long-term reservoir. The present disclosure includes the recognition that gene therapy selectively targeting hematopoietic cells that are not stem cells and / or progenitor cells (e.g., terminally differentiated cells) can be useful for target cell type-specific modification. For example, in various embodiments, differentiated cells do not produce cells of multiple lineages. The present disclosure encompasses the recognition that gene therapy that selectively targets hematopoietic cells that are not stem cells and / or are not progenitor cells (e.g., terminally differentiated cells) can be useful to minimize the targeting of multiple different cell types, thereby minimizing the risk of complications such as genotoxicity.

[0086] The present disclosure provides methods and compositions comprising adenoviral vectors advantageous for gene therapy targeting hematopoietic cells (e.g., one or more specific types of hematopoietic cells). The methods and compositions of the present disclosure are based, at least in part, on the observation that adenoviral vectors of the Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, and Ad50 serotypes exhibit certain advantageous properties for gene therapy targeting hematopoietic cells (e.g., one or more specific types of hematopoietic cells) compared to one or more reference adenoviral vectors (e.g., Ad5 vectors or Ad5 / 35 vectors). Adenovirus (or, interchangeably, "adenoviral") vectors comprise viral particles characterized by one or more adenoviral protein sequences and, optionally, an adenoviral genome. An adenoviral genome comprises a nucleic acid sequence that (a) supports packaging of a nucleic acid sequence (including conditional packaging) into an adenoviral vector and (b) contains sufficient adenoviral sequences to express the coding sequence. An adenoviral genome can be a linear, double-stranded DNA sequence and / or molecule. As one skilled in the art will appreciate, a linear genome, such as an adenoviral genome, can reside in a circular plasmid, for example, for virus production purposes. The length of a native adenoviral genome ranges from 26 kb to 45 kb, depending on the serotype.

[0087] The present disclosure includes methods and compositions comprising engineered adenoviral vectors and adenoviral genomes. Adenoviral vectors include engineered adenoviral vectors that contain engineered adenoviral proteins or engineered adenoviral genomes. For example, the engineered adenoviral genome can be engineered to add or remove adenoviral genome sequences compared to a reference sequence.

[0088] In various embodiments, the adenoviral serotypes and / or vectors of the present disclosure exhibit increased infection of one or more hematopoietic cell type(s) compared to infection of the hematopoietic cell type(s) by one or more reference adenoviral serotypes and / or vectors (e.g., Ad5 and / or Ad5 / 35) and are therefore useful, for example, for targeting hematopoietic cell type(s) for therapeutic purposes. In various embodiments, the adenoviral serotypes and / or vectors of the present disclosure exhibit increased infection of one or more hematopoietic cell type(s) compared to infection of the one or more reference hematopoietic cell type(s) by the same serotypes and / or vectors and are therefore useful, for example, for targeting hematopoietic cell type(s). The methods and compositions of the present disclosure include adenoviral vectors of serotypes Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, and Ad50.

[0089] I. Gene Therapy Vectors 1(A). Adenovirus vector The present disclosure includes adenoviral vectors and adenoviral genomes useful for gene therapy. Adenoviruses are large, icosahedral, non-enveloped viruses. Natural adenoviral capsids contain three types of proteins: fiber, penton, and hexon. The hexon occupies the majority of the viral capsid and forms 20 triangular faces. Penton bases are located at each of the 12 vertices of the capsid, and a fiber (also called knobbed fiber) protrudes from each penton base. The pentons and fibers, especially the fiber knobs, are particularly important in receptor binding and internalization because they facilitate attachment of the capsid to host cells.

[0090] The adenoviral genome contains adenoviral DNA flanked on both ends by serotype-specific inverted terminal repeats (ITRs), which are understood to be cis-elements that contribute to or are necessary for viral genome replication and packaging. Depending on the serotype, the ITRs can be approximately 100 to 200 base pairs in length (e.g., approximately 160 base pairs), with the highest conservation at the nucleotide position closest to the end of the adenoviral genome (e.g., approximately 50 base pairs). The adenoviral genome also contains packaging sequences (e.g., conditional or non-conditional packaging sequences) that can facilitate packaging of the viral genome into a viral vector. The packaging sequence is located in the left portion of the genome.

[0091] The native adenovirus genome encodes several proteins, including the early transcription units E1, E2, E3, and E4, as well as the late transcription unit encoding the structural protein components of the adenoviral vector. Early (E) and late (L) transcription are separated by the initiation of viral genome replication. The E1 region (E1A and E1B) encodes proteins involved in regulating viral genome transcription. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral genome replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff. The products of the late genes, including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP is particularly efficient during the late stages of infection. mRNA transcribed using this promoter contains a 5'-triple leader (TPL) sequence that facilitates translation.

[0092] 1(B). Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, and 50 gene therapy vectors The present disclosure includes an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome. In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome comprises an ITR of an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector (e.g., the 5' ITR of SEQ ID NO: 1, 19, 37, 55, 73, 91, 109, 127, 145, 163, or 181 and the 5' ITR of SEQ ID NO: 2, 20, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 The 3' ITR of the nucleotide sequence of the present invention is a single- or double-stranded DNA sequence containing an ITR having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the 3' ITR of the nucleotide sequence of the present invention, or an ITR having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) thereto, individually and / or together. In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome is a single- or double-stranded DNA sequence that includes a packaging sequence of an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector (e.g., a packaging sequence set forth in SEQ ID NO: 3, 21, 39, 57, 75, 93, 111, 129, 147, 165, or 183), or a packaging sequence that has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to all or a portion thereof. In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome is a single- or double-stranded DNA sequence comprising a sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to all, a portion, a contiguous corresponding portion, or a non-contiguous corresponding portion of a reference Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome (e.g., SEQ ID NOs: 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, or 209).

[0093] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome is any nucleotide sequence that includes at least the ITRs of an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector (e.g., the 5' ITR of SEQ ID NO: 1, 19, 37, 55, 73, 91, 109, 127, 145, 163, or 181 and the 3' ITR of SEQ ID NO: 2, 20, 38, 56, 74, 92, 110, 128, 146, 164, or 182), or ITRs that individually and / or together have at least 75% sequence identity thereto (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity). In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome is an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome in which one or more nucleotides, coding sequences, and / or genes are completely or partially deleted compared to a reference sequence. For example, in some embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome can be a genome that does not contain one or more of E1, E2, E3, and E4. In certain embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome is a genome that does not contain any coding sequences of the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome (e.g., a "gutless" vector that contains ITRs with at least 75% sequence identity to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genomic ITRs, but does not contain any of the coding sequences present in the reference Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome).

[0094] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome comprises, does not comprise, or contains a deletion of all or a portion of the E1 sequence of SEQ ID NO: 4, 22, 40, 58, 76, 94, 112, 130, 148, 166, or 184, or a sequence having at least 75% sequence identity thereto (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0095] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome comprises, does not comprise, or contains a deletion of all or a portion of the E2 sequence of SEQ ID NO: 5, 23, 41, 59, 77, 95, 113, 131, 149, 167, or 185, or a sequence having at least 75% sequence identity thereto (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0096] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome comprises, does not comprise, or contains a deletion of all or a portion of the E3 sequence of SEQ ID NO: 6, 24, 42, 60, 78, 96, 114, 132, 150, 168, or 186, or a sequence having at least 75% sequence identity thereto (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0097] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome may or may not include a fiber-encoding sequence, and the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 7, 25, 43, 61, 79, 97, 115, 133, 151, 169, or 187.

[0098] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome may or may not include a sequence encoding a fiber shaft, and the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 9, 27, 45, 63, 81, 99, 117, 135, 153, 171, or 189.

[0099] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome may or may not include a sequence encoding a fiber knob, and the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 10, 28, 46, 64, 82, 100, 118, 136, 154, 172, or 190.

[0100] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome may or may not include a sequence encoding a fiber tail, and the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 8, 26, 44, 62, 80, 98, 116, 134, 152, 170, or 188.

[0101] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome may or may not include a sequence encoding a penton, and the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 11, 29, 47, 65, 83, 101, 119, 137, 155, 173, or 191.

[0102] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome may or may not include a sequence encoding hexon, and the sequence has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 12, 30, 48, 66, 84, 102, 120, 138, 156, 174, or 192.

[0103] The present disclosure includes Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors comprising a fiber with at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber (e.g., the fiber of SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, 157, 175, or 193).

[0104] The disclosure includes Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors comprising a fiber tail having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber tail (e.g., the fiber tail of SEQ ID NO: 18, 36, 54, 72, 90, 108, 126, 144, 162, 180, or 198, e.g., the fiber tail is the portion of the fiber comprising all amino acids N-terminal to the fiber shaft).

[0105] The present disclosure includes Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors comprising a fiber shaft having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber shaft (e.g., the fiber shaft of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, or 194).

[0106] The present disclosure includes Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors comprising a fiber knob having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber knob (e.g., the fiber knob of SEQ ID NO: 15, 33, 51, 69, 87, 105, 123, 141, 159, 177, or 195).

[0107] The present disclosure includes Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors comprising a penton with at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 penton (e.g., the penton of SEQ ID NO: 16, 34, 52, 70, 88, 106, 124, 142, 160, 178, or 196).

[0108] The present disclosure includes Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, 50 vectors comprising a hexon with at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 hexon (e.g., the hexon of SEQ ID NO: 17, 35, 53, 71, 89, 107, 125, 143, 161, 179, or 197).

[0109] In various embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector is any adenoviral vector that includes at least a fiber having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber (e.g., the fiber of SEQ ID NO: 13, 31, 49, 67, 85, 103, 121, 139, 157, 175, or 193).

[0110] In various embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector is any adenoviral vector that includes at least a fiber tail that has at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber tail (e.g., the fiber tail of SEQ ID NO: 18, 36, 54, 72, 90, 108, 126, 144, 162, 180, or 198, e.g., the fiber tail is the portion of the fiber that includes all amino acids at the N-terminus of the fiber shaft).

[0111] In various embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector is any adenoviral vector that includes at least a fiber shaft having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber shaft (e.g., the fiber shaft of SEQ ID NO: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, or 194).

[0112] In various embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector is any adenoviral vector comprising at least a fiber knob having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber knob (e.g., the fiber knob of SEQ ID NO: 15, 33, 51, 69, 87, 105, 123, 141, 159, 177, or 195).

[0113] In various embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector is any adenoviral vector comprising at least a penton having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 penton (e.g., the penton of SEQ ID NO: 16, 34, 52, 70, 88, 106, 124, 142, 160, 178, or 196).

[0114] In various embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector is any adenoviral vector that includes at least a hexon having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 hexon (e.g., the hexon of SEQ ID NO: 17, 35, 53, 71, 89, 107, 125, 143, 161, 179, or 197).

[0115] Thus, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector can be a chimeric adenovirus vector that includes at least a fiber knob having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber knob and at least one protein or portion thereof (e.g., fiber shaft, fiber tail, penton, or hexon) having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to a different adenovirus serotype.

[0116] The Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector can be a chimeric adenovirus vector comprising at least a fiber shaft having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber shaft and at least one protein or portion thereof (e.g., fiber knob, fiber tail, penton, or hexon) having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to a different adenovirus serotype.

[0117] The Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector can be a chimeric adenovirus vector that includes at least a fiber tail having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 fiber tail and at least one protein or portion thereof (e.g., fiber knob, fiber shaft, penton, or hexon) having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to a different adenovirus serotype.

[0118] The Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector can be a chimeric adenovirus vector that includes at least a penton having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 penton and includes at least one protein or portion thereof (e.g., fiber knob, fiber shaft, fiber tail, or hexon) having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to a different adenovirus serotype.

[0119] The Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector can be a chimeric adenovirus vector that includes at least a hexon having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 hexon and includes at least one protein or portion thereof (e.g., fiber knob, fiber shaft, fiber tail, or penton) having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to a different adenovirus serotype.

[0120] Exemplary sequences of Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 components (e.g., ITRs, packaging sequences, genes, and proteins) are provided in the table below. Viral polypeptides include proteins that are components of viral vectors and portions or fragments thereof, including, for example, fiber, fiber knob, fiber shaft, fiber tail, penton, or hexon.

[0121] In various embodiments, the Ad35 fiber knob of an Ad35 vector or chimeric Ad vector comprising an Ad35 fiber knob is a mutated Ad35 fiber knob. In certain embodiments, the mutated Ad35 fiber knob is an Ad35++ mutated fiber knob (alternatively referred to herein as an Ad35++ fiber knob). In various embodiments, the Ad35++ mutated fiber knob is an Ad35 fiber knob that has been mutated to increase affinity for CD46, for example, by 25-fold, e.g., to increase cell transduction efficiency at a lower multiplicity of infection (MOI) (Li and Lieber, FEBS Letters, 593(24):3623-3648, 2019). In various embodiments, the Ad35++ mutant fiber knob comprises at least one mutation selected from Ile192Val, Asp207Gly (or Glu207Gly in certain Ad35 sequences), Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, Ile256Leu, Ile256Val, Arg259Cys, and Arg279His. In various embodiments, the Ad35++ mutant fiber knob comprises each of the following mutations: Ile192Val, Asp207Gly (or Glu207Gly in certain Ad35 sequences), Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, Ile256Leu, Ile256Val, Arg259Cys, and Arg279His. In various embodiments, the amino acid numbering of the Ad35 fiber is according to GenBank Accession No. AP_000601 or a corresponding amino acid sequence, e.g., Glu or Asp at position 207. In various embodiments, the Ad35 fiber has an amino acid sequence according to GenBank Accession No. AP_000601. Further description of Ad35++ fiber knob mutations can be found in Wang 2008 J. Virol. 82(21):10567-10579, which is incorporated herein by reference in its entirety with respect to the fiber knob. The present disclosure includes, for example, recombinant Ad35 vectors having a mutated Ad35 fiber knob or Ad5 / 35 vectors having a mutated Ad35 fiber knob.

[0122] In various embodiments, the adenoviral vector or genome of the present disclosure may be the adenoviral vector and / or genome disclosed in WO2021 / 003432, which is incorporated herein by reference in its entirety, particularly with respect to adenoviral vectors and genomes.

[0123] The various sequences corresponding to the accession numbers disclosed herein (e.g., as shown in Tables 1-22, including the accession numbers referenced herein as SEQ ID NOs: 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, and / or 209) are provided in the list of accession sequences herein below. One of skill in the art will understand that such sequences, including those disclosed in the list of accession sequences below, may be referenced in whole (e.g., by accession number) or in part (e.g., by reference to a nucleotide position and / or set or range of nucleotide positions of the sequence and / or accession number). [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22]

[0124] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector or genome comprises a modification that reduces and / or eliminates viral replication in a recipient. Generally, there are three recognized "generations" of adenoviral vectors and genomes that have been engineered to reduce and / or eliminate viral replication in a recipient. The adenoviral vectors of the present disclosure can include vectors from any of these three generations.

[0125] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome differs from a reference Ad sequence (e.g., one or more standard, representative, exemplary, or wild-type sequences of an adenovirus of a desired serotype) in that at least the regulatory E1 genes (E1a and E1b) are removed from the Ad genome (a "first-generation" vector modification). A first-generation Ad vector containing an E1 deletion is an example of an E1-deleted vector. E1a and E1b are the first transcriptional regulators produced during the adenovirus replication cycle. The E1 deletion reduces or eliminates expression of certain viral genes controlled by E1, and E1-deleted helper viruses are replication-defective. Thus, first-generation Ad vectors are defective for replication in recipients. In some embodiments, first-generation adenoviral vectors are engineered to remove the E1 and E3 genes. The retained portion of the reference genome may be identical in sequence to the reference genome, or may have less than 100% identity to the reference genome, for example, at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity. Without these E1 (or E1 and E3) genes, the adenoviral vector cannot replicate on its own, but can be produced in a mammalian cell line that expresses E1 (e.g., of the same serotype) or another protein sufficient to restore expression of a specific viral gene. For example, if an E1-deleted Ad5 vector encodes Ad5 E4orf6, the helper vector can be propagated in a cell line that expresses Ad5 E1. In one exemplary cell type for adenoviral vector production, HEK293 cells express Ad5 E1b55k, which is known to form a complex with ORF6, the Ad5 E4 protein.

[0126] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome differs from a reference Ad sequence in that at least the E1 gene (E1a and E1b) and one or more of the nonstructural genes E2, E3, and / or E4 have been deleted (a "second-generation" modification). Second-generation Ads have a larger payload capacity than first-generation Ads and are more replication-deficient than first-generation viruses. In some aspects, in addition to the removal of E1 / E3, second-generation adenoviral vectors are engineered to remove the nonstructural genes E2 and E4, resulting in increased capacity and reduced immunogenicity. The retained portion of the reference genome may be identical in sequence to the reference genome or may have less than 100% identity to the reference genome, e.g., at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity.

[0127] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome differs from a reference Ad sequence at least in that it has been engineered to remove all viral coding sequences from the Ad genome, retaining only the ITRs and packaging sequences of the genome or a functional fragment thereof (a "third-generation" modification). Third-generation adenoviral vectors may be referred to as gutless, high-capacity adenoviral vectors, or helper-dependent adenoviral vectors (HdAds). The retained portion of the reference genome may be identical in sequence to the reference genome or may have less than 100% identity to the reference genome, for example, at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity.

[0128] Third-generation Ad genomes do not encode proteins necessary for viral production, making them helper-dependent; helper-dependent genomes can be packaged into vectors only when present in cells containing nucleic acid sequences that provide the viral proteins in trans. These helper-dependent vectors also feature greater capacity and reduced immunogenicity than first- and second-generation vectors. HDAd vectors do not express viral genes when used as vectors, reducing the risk of cytotoxic or interferon responses in recipients.

[0129] Helper-dependent adenoviral vectors (HDAd), engineered to lack all viral coding sequences, can efficiently transduce a wide variety of cell types, mediate long-term transgene expression, and exhibit negligible chronic toxicity. By deleting viral coding sequences and leaving only the cis-acting elements required for genome replication (ITRs) and packaging (ψ), cellular immune responses to Ad vectors are reduced. HDAd vectors possess a large cloning capacity, enabling the delivery of large payloads. These payloads can contain large therapeutic genes or even multiple transgenes and large regulatory elements, enhancing, extending, and modulating transgene expression. It has also been observed that certain HDAd vector genomes can be packaged most efficiently when the genome has at least a minimum total length, e.g., a minimum length of at least 20 kb relative to the total length (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 kb), which length can include, for example, a therapeutic payload and / or a "stuffer" sequence. If the payload does not utilize the large number of nucleotides that would cause the adenoviral genome to have at least the target length, a stuffer sequence can be used to achieve or exceed the target length. The present disclosure includes that a minimum length for efficient packaging is not required for beneficial use of the vectors provided herein, and thus, meeting any target length may be advantageous, but is not required, for use with the compositions and methods provided herein. Like other adenoviral vectors, typical HDAd genomes generally remain episomal and do not integrate with the host genome.

[0130] Because HDAd vectors do not encode the viral proteins necessary for viral particle production, viral proteins must be provided in trans, e.g., expressed within and / or by the cell in which the HDAd genome resides. In some HDAd vector systems, one viral genome (the helper genome) encodes all proteins required for replication (e.g., all structural viral proteins) but contains a conditional defect in the packaging sequence, making it less likely to be packaged into a vector under certain vector production conditions (e.g., in the presence of a drug that reduces the function of the conditionally defective packaging sequence). Thus, the HDAd donor viral genome contains (e.g., contains only) the Ad ITRs, a payload (e.g., a therapeutic payload), and a functional packaging sequence (e.g., a wild-type packaging sequence or a functional fragment thereof). This allows the HDAd donor viral genome to be selectively packaged into HDAd viral vectors produced from structural components expressed from the helper vector genome. In other words, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper vector can be used to produce an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector. Production of an HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vector can involve cotransfection of a plasmid containing the HDAd vector genome with a packaging-defective helper virus that provides the structural and nonstructural viral proteins. The helper virus genome can rescue the propagation of the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector, allowing the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector to be produced and isolated, for example, on a large scale.Various protocols are known in the art, for example, Palmer et al., 2009 Gene Therapy Protocols. Methods in Molecular Biology, Volume 433. Humana Press; Totowa, NJ: 2009. pp. 33-53. In some embodiments, the helper genome is E1-deleted.

[0131] In some HDAd vector systems, the helper genome utilizes a recombinase system (e.g., a Cre / loxP system) for conditional packaging. In certain such HDAd vector systems, the helper genome comprises a packaging sequence or a functional fragment thereof (e.g., a fragment of a packaging sequence sufficient for packaging, necessary for efficient packaging, or necessary for packaging of the Ad genome into capsids) flanked by recombinase (e.g., loxP) sites such that contact with a corresponding recombinase (e.g., Cre recombinase) cleaves the packaging sequence or a functional fragment thereof from the helper genome by recombinase-mediated (e.g., Cre-mediated) site-specific recombination between the recombinase sites (e.g., loxP sites). The present disclosure includes, inter alia, Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper vectors and genomes that contain two recombination sites flanking a packaging sequence or functional fragment thereof, wherein the two recombination sites are compatible with (i.e., directed to or acting on) the same recombinase.

[0132] In various embodiments, the helper genome can contain a deletion of E1, e.g., the helper genome contains all of the viral genes except E1, and the E1 expression product can be supplied by complementary expression from the genome of the producer cell line. In some embodiments, to prevent the generation of replication-competent Ad (RCA) as a result of homologous recombination between the helper and HDAd donor genomes present in the producer cell, a "stuffer" sequence can be inserted into the E3 region to render any recombinants too large to package and / or package efficiently.

[0133] For production of HDAd vectors, the HDAd donor genome can be delivered to cells expressing a recombinase to excise the conditional packaging sequence of the helper vector (e.g., 293 cells (HEK293) expressing Cre recombinase); optionally, the HDAd donor genome is delivered to the cells in a non-viral vector form, such as a bacterial plasmid form (e.g., when the HDAd donor genome is present in a bacterial plasmid (pHDAd) and / or released by restriction enzyme digestion). The same cells can be transduced with a helper genome containing the packaging sequence, or a functional fragment thereof, flanked by recombinase sites (e.g., loxP sites). Thus, producer cells can be transfected with an HDAd donor genome and transduced with a helper genome or functional fragment thereof having a packaging sequence or functional fragment thereof flanked by recombinase sites (e.g., loxP sites), and the cells express a recombinase (e.g., Cre) corresponding to the recombinase sites, such that excision of the packaging sequence or functional fragment thereof renders the helper virus genome deficient for packaging (e.g., non-packaging), yet still capable of providing all of the trans-acting factors necessary for production of an HDAd donor vector comprising the HDAd donor genome.

[0134] Similar HDAd production systems have been developed using FLP (e.g., FLPe) / frt site-specific recombination, in which FLP-mediated recombination between frt sites flanking the packaging sequence of the helper genome, or a functional fragment thereof, reduces or eliminates packaging of the helper genome in producer cells expressing FLP.

[0135] The HDAd vector, comprising the payload-containing donor vector genome, can be isolated from the producer cells. The HDAd donor vector can be further purified from the helper vector by physical means. In general, some contamination of the helper vector and / or helper genome in HDAd viral vectors and HDAd viral vector preparations can occur and can be tolerated.

[0136] HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, and 50 donor vectors, donor genomes, helper vectors, and helper genomes are also exemplary of the compositions provided herein and can be used in various methods of the disclosure. HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors or genomes are helper-dependent Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors or genomes. An Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper vector is a vector containing a helper genome that contains a conditionally expressed (e.g., flanked by frt or loxP sites) packaging sequence or a fragment thereof and encodes all of the trans-acting factors necessary for the production of Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 virions into which the donor genome can be packaged.

[0137] The present disclosure further includes HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector production systems, including cells comprising an HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor genome and an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper genome. In certain such cells, viral proteins encoded and expressed by the helper genome can be utilized to produce HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vectors packaged with the HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor genome. Thus, the present disclosure includes methods for producing HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vectors by culturing cells containing an HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor genome and an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper genome. In some embodiments, the cells encode and express a recombinase corresponding to the recombinase direct repeats flanking the packaging sequence of the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper vector. In some embodiments, the flanking packaging sequence of the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper genome is excised.

[0138] In some embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper genome encodes all of the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 coding sequence. In some embodiments, an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper genome encodes and / or expresses all of the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 coding sequence except for one or more of the E1 and / or E3 coding sequences and / or E4 coding sequences. In various embodiments, the helper genome does not encode and / or express the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 E1 gene and does not encode and / or express the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 E4 gene. In various embodiments, as will be appreciated by one of skill in the art, the cells of the compositions and methods of producing the HDAd donor vectors can be cells that express the E1 expression product.

[0139] The present disclosure includes, among other things, HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vectors and genomes that include an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 ITR (a 5' Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 ITR and a 3' ITR of the same serotype), e.g., two Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 ITRs flanking the packaging sequence and payload. The present disclosure includes, among other things, HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vectors and genomes that have a deletion of E1 or a fragment thereof. The present disclosure includes, inter alia, vectors and genomes of HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 in which E3 or a fragment thereof has been deleted.

[0140] In various embodiments, excision of the packaging sequence or a functional fragment thereof from the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 helper genome reduces vector propagation, e.g., by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% (e.g., reducing vector propagation by at least 20%, 30%, 40%, 50%, 60%, 70%, and 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%). or 100%), optionally measured as the number of viral particles produced by growth of an excised vector (a vector in which the recombinase site flanking sequences have been excised) compared to an intact vector (a vector in which the recombinase site flanking sequences have not been excised) or compared to a wild-type Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, 50 vector under equivalent conditions.

[0141] An additional optional engineering consideration may be engineering the helper genome to have a size that allows for separation of the helper vector from the HDAd3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector by centrifugation, e.g., CsCl ultracentrifugation. One means of achieving this result is to increase the size of the helper genome compared to a typical Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome. In particular, the adenoviral genome can be engineered to increase its length to at least 104% of its wild-type length. Certain helper vectors of the present disclosure can accommodate payload and / or stuffer sequences.

[0142] The present disclosure includes that, in various embodiments, the vectors or genomes of the disclosure can include a selection of components each selected from corresponding sequences of a single particular serotype or having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity). To provide an illustrative example, all components can correspond to (e.g., with at least 75% sequence identity to) Ad34, except for sequences indicated otherwise (e.g., payloads, e.g., heterologous payloads).

[0143] In various embodiments, the vectors of the present disclosure are HDAd5 / 35 vectors comprising Ad5 capsid proteins, except that the fiber is chimeric, comprising an Ad5 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob (see, e.g., Shayakhmetov et al. 2000 J. Virol 74(6):2567-2583), and optionally, the Ad35 fiber knob is mutated for increased affinity for CD46 (e.g., Ad5 / 35++). In certain embodiments, the Ad5 / 35++ vector is a chimeric Ad5 / 35 vector having a mutated Ad35++ fiber knob (see, e.g., Wang et al. 2008 J. Virol. 82(21):10567-79, incorporated herein by reference in its entirety, particularly with respect to the fiber knob mutation). In various embodiments, the Ad35++ mutant fiber knob is an Ad35 fiber knob that has been mutated to increase affinity for CD46, e.g., by 25-fold, e.g., the Ad35++ mutant fiber knob increases cell transduction efficiency, e.g., at a lower multiplicity of infection (MOI) (Li and Lieber, FEBS Letters, 593(24):3623-3648, 2019). In certain embodiments, the adenoviral vector is a chimeric "F35" vector in which all proteins are Ad5 proteins, except that the fiber is a chimera comprising an Ad5 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob (e.g., as described in Shayakhmetov et al. 2000 J.Virol 74(6):2567-2583), where the Ad35 fiber knob is a mutant Ad35 fiber knob containing mutations D207G and T245A that result in increased affinity for CD46 (see, e.g., Wang et al. 2008 J.Virol. 82(21):10567-79), and optionally the genome encoding the Ad5 / 35 vector contains an E1 deletion.

[0144] In various embodiments, the adenoviral vector or genome of the present disclosure may be the adenoviral vector and / or genome disclosed in WO2021 / 003432, which is incorporated herein by reference in its entirety, particularly with respect to adenoviral vectors and genomes.

[0145] I(C).Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, and 50 gene therapy vector payloads The Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vectors and genomes of the present disclosure can comprise a variety of heterologous nucleic acid payloads, which can include any of one or more coding sequences encoding one or more expression products, one or more regulatory sequences operably linked to a coding sequence, one or more stuffer sequences, etc. In various embodiments, the payload is engineered to achieve a desired result, such as a therapeutic effect in a host cell or system, e.g., expression of a therapeutic protein, or expression of a gene editing system, e.g., a CRISPR / Cas system, a base editing system, or a prime editing system, to generate a therapeutic sequence modification, e.g., to correct a nucleic acid lesion.

[0146] In some embodiments, the payload can include a gene. A gene can include not only a coding sequence but also regulatory regions such as promoters, enhancers, termination regions, locus control regions (LCRs), termination and polyadenylation signal elements, splicing signal elements, silencers, and insulators. A gene can include introns and other DNA sequences spliced ​​from the expressed mRNA transcript, as well as variants resulting from alternative splice sites. A coding sequence can also include alternative synonymous codon usage compared to a reference sequence, for example, modified codon usage compared to the reference according to the codon preferences of a particular organism or target cell type.

[0147] A payload can comprise a single gene or multiple genes. A payload can comprise a single coding sequence or multiple coding sequences. A payload can comprise a single regulatory sequence or multiple regulatory sequences. A payload can comprise multiple coding sequences, for example, an endonuclease and a guide RNA, or where the individual expression products of the coding sequences function together independently, e.g., as two separate proteins that are not directly or indirectly linked. As will be understood by those skilled in the art, any payload or payload component (e.g., an expression product or regulatory sequence encoded by the payload) that is not encoded by the reference wild-type Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 genome can be referred to herein as a heterologous expression product.

[0148] For the avoidance of doubt, the present disclosure includes variants of the amino acid and nucleic acid sequences provided herein, including sequences having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the protein and nucleic acid sequences described or disclosed herein, wherein the variants exhibit substantially similar or improved biological function.

[0149] I(C)(i).Payload expression product The payload of the adenoviral donor vector or adenoviral donor genome of the present disclosure can include one or more coding sequences encoding any of a variety of expression products. Exemplary expression products include proteins, including, but not limited to, replacement therapy proteins for the treatment of diseases or conditions characterized by low expression or activity of a biologically active protein compared to reference levels. Exemplary expression products include CRISPR / Cas, base editor, and prime editor systems. Exemplary expression products include antibodies, CARs, and TCRs. Exemplary expression products include small RNAs. In various embodiments, integration of all or part of the donor vector payload into the host cell genome is not required to deliver the donor vector or genome to a target cell to produce an intended or targeted effect, for example, in certain instances where the intended or targeted effect includes editing the host cell genome with a CRISPR, base editor, or prime editor system. In various embodiments, integration of all or part of the donor vector payload is necessary or preferred for delivery of the donor vector or genome to a target cell to produce an intended or targeted effect, e.g., if expression of a payload-encoded expression product is desired in progeny cells of the transduced target cell. In various embodiments, the payload can include nucleic acid sequences ("integration elements") engineered for integration into the host cell genome, e.g., by recombination or transposition.

[0150] Gene sequences encoding one or more therapeutic proteins can be readily prepared synthetically or by recombinant methods from the relevant amino acid sequences. In certain embodiments, the gene sequences encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and / or 3' end of the coding sequence to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding another sequence. In certain embodiments, the gene sequence encoding the sequence can be codon-optimized for expression in mammalian cells.

[0151] Specific examples of therapeutic genes and / or expression products include γ-globin, Factor VIII, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, SLC46A1, FANC family genes (e.g., For example, FancA, FancB, FancC, FancD1 (BRCA2), FancD2, FancE, FancF, FancG, FancI, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), Fa ncP(SLX4), FancQ(ERCC4), FancR(RAD51), FancS(BRCA1), FancT(UBE2T), FancU(XRCC2), FancV(MAD2L2), and FancW(RFWD3)), soluble CD40, CTLA, Fas L, antibodies (e.g., those that specifically bind to CD4, CD5, CD7, CD52, IL1, IL2, IL6, TNF, P53, PTPN22, or DRB1*1501 / DQB1*0602), antibodies against TCRs specifically present in autoreactive T cells, IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, sTNFRI, sTNFRII, globin family genes, WAS, phox, dystrophin, pyruvate kinase These include, but are not limited to, CLN3, ABCD1, arylsulfatase A, SFTPB, SFTPC, NLX2.1, ABCA3, GATA1, ribosomal protein genes, TERT, TERC, DKC1, TINF2, CFTR, LRRK2, PARK2, PARK7, PINK1, SNCA, PSEN1, PSEN2, APP, SOD1, TDP43, FUS, ubiquilin 2, C9ORF72, and other therapeutic genes and / or expression products described herein.

[0152] Therapeutic genes can be selected to provide therapeutically effective responses to diseases related to red blood cells and coagulation. In certain embodiments, the disease is a hemoglobinopathy such as thalassemia, or sickle cell disease / trait. The therapeutic gene may be, for example, a gene that induces or increases the production of hemoglobin, a gene that induces or increases the production of β-globin, γ-globin, or α-globin, or a gene that increases the availability of oxygen to cells in the body. The therapeutic gene may be, for example, HBB or CYB5R3. Exemplary effective treatments may, for example, increase blood cell counts, improve blood cell function, or increase the oxygenation of the patient's cells. In another specific embodiment, the disease is hemophilia. A therapeutic gene can be, for example, a gene that increases the production of coagulation / clotting factor VIII or coagulation / clotting factor IX, a gene that causes the production of normal versions of coagulation factor VIII or coagulation factor IX, a gene that reduces the production of antibodies against coagulation / clotting factor VIII or coagulation / clotting factor IX, or a gene that causes the proper formation of blood clots. Exemplary therapeutic genes include F8 and F9. An exemplary effective treatment can, for example, increase or induce the production of coagulation / clotting factors VIII and IX, improve the function of coagulation / clotting factors VIII and IX, or shorten the clotting time in a subject.

[0153] In various embodiments of the present disclosure, the donor vector encodes a globin gene, and the globin protein encoded by the globin gene is selected from γ-globin, β-globin, and / or α-globin. The globin genes of the present disclosure can include one or more regulatory sequences, such as, for example, a promoter, operably linked to a nucleic acid sequence encoding the globin protein. As one skilled in the art will appreciate, γ-globin, β-globin, and / or α-globin are each components of fetal and / or adult hemoglobin and, therefore, are useful in various vectors disclosed herein.

[0154] In various embodiments, increasing the expression of a globin protein can refer to any one or more of: (i) increasing the amount, concentration, or expression (e.g., transcription or translation of an encoding nucleic acid) in a cell or system of a globin protein having a particular sequence; (ii) increasing the amount, concentration, or expression (e.g., transcription or translation of an encoding nucleic acid) in a cell or system of a particular type of globin protein (e.g., the total amount of all proteins identified by one of skill in the art or as described herein as γ-globin (or alternatively, β-globin or α-globin)), regardless of the sequence of the proteins relative to each other; and / or (iii) expressing in a cell or system a heterologous globin protein, e.g., a globin protein not encoded by the host cell prior to gene therapy.

[0155] The following references describe specific exemplary sequences of functional globin genes: References 1-4 relate to alpha-type globin sequences, and References 4-12 relate to beta-type globin sequences (including beta and gamma globin sequences), the sequences of which are incorporated herein by reference. (1) GenBank accession number Z84721 (March 19, 1997), (2) GenBank accession number NM_000517 (October 31, 2000), (3) Hardison et al., J. Mol. Biol. (1991) 222(2):233-249, (4) A Syllabus of Human Hemoglobin Variants (1996) by Titus et al., published by The Sickle Cell Anemia Foundation, Augusta, Ga. (available at globin.cse.psu.edu), (5) GenBank accession number J00179 (August 26, 1993), or U01317.1, (6) Tagle et al., Genomics (1992) 13(3):741-760, (7) Grovsfeld et al. al.,Cell(1987)51(6):975-985,(8)Li et al.,Blood(1999)93(7):2208-2216,(9)Gorman et al.,J.Biol.Chem.(2000)275(46):35914-35919,(10)Slightom et al. al., Cell (1980) 21 (3): 627-638, (11) Fritsch et al., Cell (1980) 19 (4): 959-972, (12) Marotta et al., J. Biol. Chem. (1977) 252 (14): 5040-5053. For further coding and non-coding regions of globin-encoding genes, see, e.g., Marotta et al., Prog. Nucleic Acid Res. Mol. Biol. 19, 165-175, 1976; Lawn et al., Cell 21(3), 647-651, 1980; and Sadelain et al. PNAS.; 92:6728-6732, 1995. In some embodiments, the globin gene encodes the G16D gamma globin variant.

[0156] An exemplary amino acid sequence of hemoglobin subunit β is provided, for example, at NCBI accession number P68871. An exemplary amino acid sequence of β-globin is provided, for example, at NCBI accession number NP_000509.

[0157] In addition to therapeutic genes and / or gene products, the transgene can also encode therapeutic molecules, such as checkpoint inhibitor reagents, chimeric antigen receptor molecules specific for one or more cancer antigens, and / or T cell receptors specific for one or more cancer antigens.

[0158] As another example, therapeutic genes can be selected to provide a therapeutically effective response to lysosomal storage diseases. In certain embodiments, the lysosomal storage disease is mucopolysaccharidosis (MPS) type I; MPS II or Hunter syndrome; MPS III or Sanfilippo syndrome; MPS IV or Morquio syndrome; MPS V; MPS VI or Maroteaux-Lamy syndrome; MPS VII or Sly syndrome; α-mannosidosis; β-mannosidosis; glycogen storage disease type I (also known as GSDI, von Gierke disease, or Tay-Sachs disease); Pompe disease; Gaucher disease; or Fabry disease. The therapeutic gene can be, for example, a gene encoding an enzyme, or inducing the production of an enzyme, or otherwise causing the degradation of mucopolysaccharides in lysosomes. Exemplary therapeutic genes include IDUA or iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, and HYAL1. Exemplary effective gene therapies for lysosomal storage diseases may, for example, encode or induce the production of enzymes involved in the breakdown of various substances in lysosomes, and may reduce, eliminate, prevent, or delay swelling of various organs, including the head (e.g., macrocephaly), liver, spleen, tongue, or vocal cords; may reduce fluid in the brain and reduce abnormalities of heart valves; may prevent or widen the airways and prevent associated upper respiratory tract diseases such as infections and sleep apnea; or may reduce, eliminate, prevent, or delay destruction of neurons and / or associated symptoms.

[0159] As another example, therapeutic genes can be selected to provide a therapeutically effective response to a hyperproliferative disease. In certain embodiments, the hyperproliferative disease is cancer. The therapeutic gene can be, for example, a tumor suppressor gene, a gene that induces apoptosis, a gene encoding an enzyme, a gene encoding an antibody, or a gene encoding a hormone. Exemplary therapeutic genes and gene products include (in addition to those listed elsewhere herein) 101F6, 123F2 (RASSF1), 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, ApoE, ATM, BAI-1, BDNF, beta*(BLU), bFGF, BLC1, BLC6, BRCA1, BRCA2, CBFA1, CBL, C-CAM, C NTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, Dp, DPC-4, E1A, E2F, EBRB2, erb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FCC, FGF, FGR, FHIT, fms, FOX, FUS1, FYN, G-CSF, GDAIF, gene 21 (NPRL2), gene 26 (CACNA2D2), GM-CSF , GMF, gsp, HCR, HIC-1, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, ING1, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, LUCA-1(HYAL1), LUCA-2(HYAL2), LYN, MADH4, MADR2, MCC, mda7, M These include DM2, MEN-I, MEN-II, MLL, MMAC1, MYB, MYC, MYCL1, MYCN, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NT5, OVCA1, p16, p21, p27, p57, p73, p300, PGS, PIM1, PL6, PML, PTEN, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ScFv, scFV ras, SEM A3, SRC, TALI, TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, and zac1.Exemplary effective gene therapies may suppress or eliminate tumors, reduce the number of cancer cells, reduce tumor size, slow or eliminate tumor growth, or alleviate symptoms caused by tumors.

[0160] As another example, therapeutic genes can be selected to provide a therapeutically effective response to an infectious disease. In a specific embodiment, the infectious disease is human immunodeficiency virus (HIV). The therapeutic gene may be, for example, a gene that renders immune cells resistant to HIV infection or a gene that enables immune cells to effectively neutralize the virus through immune reconstitution; a polymorphism in a gene encoding a protein expressed by an immune cell; an advantageous gene for fighting infection that is not expressed in the patient; a gene encoding an infectious factor, a receptor or co-receptor; a gene encoding a ligand for a receptor or co-receptor; a gene encoding viral and cellular genes essential for viral replication, including genes encoding ribozymes, antisense RNA, small interfering RNA (siRNA), or decoy RNA that block the action of specific transcription factors; a gene encoding a dominant-negative viral protein, an intracellular antibody, an intrakines, and a suicide gene. Exemplary therapeutic genes and gene products include α2β1, αvβ3, αvβ5, αvβ63, BOB / GPR15, Bonzo / STRL-33 / TYMSTR, CCR2, CCR3, CCR5, CCR8, CD4, CD46, CD55, CXCR4, aminopeptidase-N, HHV-7, ICAM, ICAM-1, PRR2 / HveB, HveA, α-dystroglycan, LDLR / α2MR / LRP, PVR, PRR1 / HveC, and laminin receptor. For example, a therapeutically effective amount for treating HIV may increase a subject's immunity to HIV, may alleviate symptoms associated with AIDS or HIV, or may induce an innate or adaptive immune response to HIV in a subject. An immune response to HIV includes antibody production and may result in the prevention of AIDS and / or alleviate the symptoms of AIDS or HIV infection in a subject, or reduce or eliminate the infectivity and / or virulence of HIV.

[0161] I(C)(i)(a). Binding domain, antibody, CAR, and TCR payload expression products The present disclosure includes payloads that can include sequences encoding any of a variety of binding domains, which can encode, for example, antibodies, chimeric antigen receptors, TCRs, or other binding polypeptides.

[0162] Antibodies and antibody fragments are exemplary of binding domains. The term "antibody" can refer to a polypeptide comprising one or more standard immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). Thus, the term antibody includes, but is not limited to, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents comprising them. An antibody can be a naturally occurring immunoglobulin (e.g., produced by an organism in response to an antigen). Synthetic, non-natural, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those skilled in the art.

[0163] As is well known in the art, a typical human immunoglobulin is a tetrameric agent of approximately 150 kD, comprising two identical heavy (H) chain polypeptides (each approximately 50 kD) and two identical light (L) chain polypeptides (each approximately 25 kD), which associate with each other to form what is commonly referred to as a "Y-shaped" structure. Typically, each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain comprises three CH domains: CH1, CH2, and CH3. A short region known as the "switch" joins the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain comprises a light chain variable domain (VL) and a light chain constant domain (CL), separated from each other by another "switch". Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complementarity-determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and / or light chains are typically understood to provide binding moieties capable of interacting with antigens. The constant domains can mediate binding of the antibody to various immune system cells (e.g., effector cells and / or cells mediating cytotoxicity), receptors, and elements of the complement system. The heavy and light chains are linked to each other by a single disulfide bond, and two other disulfide bonds link the heavy chain hinge regions together, linking dimers to form tetramers. When a native immunoglobulin folds, the FR regions form beta sheets that provide the structural framework for the domain, and the CDR loop regions of both the heavy and light chains are packed in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure.

[0164] In some embodiments, the antibody is a polyclonal, monoclonal, monospecific, or multispecific (including bispecific) antibody. In some embodiments, the antibody comprises at least one light chain monomer or dimer, at least one heavy chain monomer or dimer, at least one heavy chain-light chain dimer, or a tetramer comprising two heavy chain monomers and two light chain monomers. Furthermore, the term "antibody" (unless otherwise specified or clear from the context) refers to any art-known construct or format that utilizes antibody structural and / or functional features, including intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNARs or fragments thereof), camel antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affibodies, anti-idiotypic (anti-Id) antibodies (e.g., anti-anti-Id antibodies), and the like. Antibodies include, but are not limited to, small modular immunopharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAbs®), VHHs, Anticalins®, Nanobodies® minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, microproteins, Fynomers®, Centyrins®, and KALBITOR®, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0165] In various embodiments, an antibody comprises one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs) or variable domains. In some embodiments, an antibody may be a covalently modified ("conjugated") antibody (e.g., an antibody comprising a polypeptide comprising one or more standard immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently attached to one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, the antibody sequence elements are humanized, primatized, chimerized, etc., as known in the art.

[0166] Antibodies comprising a heavy chain constant domain can be of any known class of antibody, including, but not limited to, IgA, secretory IgA, IgG, IgE, and IgM, based on the heavy chain constant domain amino acid sequence (e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ)). IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. As used herein, "light chains" can be of different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the light chain constant domain. In some embodiments, antibodies have constant region sequences characteristic of murine, rabbit, primate, or human immunoglobulins. Naturally produced immunoglobulins are typically glycosylated on the CH2 domain. As is known in the art, the affinity and / or other binding properties of an Fc region for an Fc receptor can be modulated through glycosylation or other modifications. In some embodiments, an antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present invention comprise a glycosylated Fc domain, including, for example, an Fc domain that has been modified or engineered, e.g., with such glycosylation.

[0167] The term "antibody fragment" can refer to a portion of an antibody or antibody agent described herein, typically referring to an antigen-binding portion or portion comprising a variable region thereof. Antibody fragments can be generated by any means. For example, in some embodiments, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, antibody fragments can be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence). In some embodiments, antibody fragments can be wholly or partially synthetically produced. In some embodiments, antibody fragments (particularly antigen-binding antibody fragments) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, and in some embodiments, at least about 200 amino acids.

[0168] In some cases, it is beneficial for the binding domain to be derived from the same species as that in which it will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain to comprise a human antibody, a humanized antibody, or a fragment or engineered form thereof. Human-derived or humanized antibodies have reduced or no immunogenicity in humans and a reduced number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and engineered fragments thereof are generally selected to have reduced or no antigenicity in human subjects.

[0169] In various embodiments, the payload can encode a binding agent that is a checkpoint inhibitor, such as an antibody that specifically binds to an immune checkpoint protein. Several immune checkpoint inhibitors are known. Immune checkpoint inhibitors can include peptides, antibodies, nucleic acid molecules, and small molecules. Examples of immune checkpoints include PD-1, PD-L1, lymphocyte-activation gene-3 (LAG-3), and T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3).

[0170] The present disclosure further provides antibodies and other binding domains that bind to CD4, CD5, CD7, CD52, etc., antibodies against IL1, IL2, IL6, antibodies against TCRs specifically present on autoreactive T cells, IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, antibodies against TNF, ABCA3, ABCD1, ADA, AK2, APP, arginase, arylsulfatase A, A1AT, CD3D, CD3E, CD3G, CD3Z, CFTR, CHD7, chimeric antigen receptor (CAR), CIITA, CLN3, cofactors, CORO1A, CTLA, C1 inhibitor, C9ORF72, DCLRE1B, DCLRE1 C, decoy receptor, DKC1, DRB1*1501 / DQB1*0602, dystrophin, enzyme, factor VIII, FANC family genes (FancA, FancB, FancC, FancD1 (BRCA2), FancD2, FancE, FancF, FancG, FancI, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)), Fas These include L, FUS, GATA1, globin family genes (i.e., gamma-globin), F8, glutaminase, HBA1, HBA2, HBB, IL7RA, JAK3, LCK, LIG4, LRRK2, NHEJ1, NLX2.1, neutralizing antibodies, ORAI1, PARK2, PARK7, phox, PINK1, PNP, PRKDC, PSEN1, PSEN2, PTPN22, PTPRC, P53, pyruvate kinase, RAG1, RAG2, RFXANK, RFXAP, RFX5, RMRP, ribosomal protein genes, SFTPB, SFTPC, SOD1, soluble CD40, STIM1, sTNFRI, sTNFRII, SLC46A1, SNCA, TDP43, TERT, TERC, TINF2, ubiquilin 2, WAS, WHN, ZAP70, gamma C, and other therapeutic genes described herein.

[0171] Certain types of hematopoietic cells (e.g., T cells) can be engineered to encode and / or express chimeric antigen receptor (CAR) constructs. CARs can contain several different subcomponents that enable the cells to recognize and kill target cells, such as cancer cells. The subcomponents include at least an extracellular component and an intracellular component.

[0172] The extracellular CAR component can contain a binding domain that specifically binds to a marker that is preferentially present on the surface of unwanted cells. When the binding domain binds to such a marker, the intracellular component instructs the cell to destroy the bound cancer cell. The binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can be based on other formats that contain antibody-like antigen-binding sites.

[0173] The intracellular CAR component provides an activation signal based on the inclusion of an effector domain. First-generation CARs utilized the cytoplasmic region of CD3ζ as the effector domain. Second-generation CARs utilized CD3ζ in combination with cluster of differentiation 28 (CD28) or 4-1BB (CD137), whereas third-generation CARs utilized CD3ζ in combination with CD28 and 4-1BB in the intracellular effector domain.

[0174] The intracellular or other cytoplasmic signaling components of a CAR are involved in the activation of the cell in which the CAR is expressed. Thus, the term "intracellular signaling component" or "intracellular component" is meant to include any portion of the intracellular domain sufficient to transduce an activation signal. The intracellular component of an expressed CAR can include an effector domain. An effector domain is the intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response within the cell upon receiving an appropriate signal. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when bound, or directly binds to a target molecule that triggers a signal from the effector domain. An effector domain can directly promote a cellular response if it contains one or more signaling domains or motifs, such as immunoreceptor tyrosine-based activation motifs (ITAMs). In other embodiments, the effector domain indirectly promotes a cellular response by associating with one or more other proteins that directly promote a cellular response, such as a costimulatory domain.

[0175] The effector domain, upon binding to a cell marker expressed by a cancer cell, can result in activation of at least one function of the modified cell. Activation of the modified cell can include one or more of differentiation, proliferation, and / or activation, or other effector functions. In certain embodiments, the effector domain can include an intracellular signaling component comprising a T cell receptor and a costimulatory domain, which can include a cytoplasmic sequence derived from a co-receptor or costimulatory molecule.

[0176] The effector domain can comprise one, two, three or more receptor signaling domains, intracellular signaling components (e.g., cytoplasmic signaling sequences), costimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from the following: 4-1BB (CD137), CARD11, CD3γ, CD3δ, CD3ε, CD3ζ, CD27, CD28, CD79A, CD79B, DAP10, FcRα, FcRβ (FcεR1b), FcRγ, Fyn, HVEM (LIGHTR), ICOS, LAG3, LAT, Lck, LRP, NKG2D, NOTCH1, pTα, PTCH2, OX40, ROR2, Ryk, SLAMF1, Slp76, TCRα, TCRβ, TRIM, Wnt, Zap70, or any combination thereof. In certain embodiments, exemplary effector domains comprise signaling and costimulatory domains selected from the following: CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACA M1, CRTAM, Ly9(CD229), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0177] Intracellular signaling component sequences that act in a stimulatory manner can include ITAMs. Examples of ITAMs that contain primary cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, and the common FcRγ (FCER1G), FcγR11a, FcRβ (Fcε Rib), DAP10, and DAP12. In certain embodiments, variants of CD3ζ retain at least one, two, three, or all of the ITAM regions.

[0178] In certain embodiments, the effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or a signaling domain thereof, a protein comprising multiple ITAMs, a costimulatory domain, or any combination thereof.

[0179] Additional examples of intracellular signaling components include the cytoplasmic sequence of the CD3 zeta chain and / or co-receptors that act in concert to initiate signal transduction following engagement of the binding domains.

[0180] A costimulatory domain is a domain whose activation may be required for an efficient lymphocyte response to cell marker binding. Several molecules can be interchangeably referred to as intracellular signaling components or costimulatory domains. Examples of costimulatory domains include ligands that specifically bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. For example, CD27 costimulation has been demonstrated to enhance the proliferation, effector function, and survival of human CART cells in vitro, and to enhance the persistence and anti-cancer activity of human T cells in vivo (see Song et al. Blood. 2012;119(3):696-706). Further examples of such costimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11lc, ITGB1, and C. These include D29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0181] In certain embodiments, the amino acid sequence of the intracellular signaling component comprises a variant of CD3ζ and a portion of the 4-1BB intracellular signaling component.

[0182] In certain embodiments, the intracellular signaling component comprises (i) all or a portion of the signaling domain of CD3ζ, (ii) all or a portion of the signaling domain of 4-1BB, or (iii) all or a portion of the signaling domains of CD3ζ and 4-1BB.

[0183] Intracellular components may also be involved in the Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), the NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), the Hedgehog signaling pathway (e.g., PTCH or SMO), receptor tyrosine kinases (RTKs) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, and the like. The receptor family may include one or more proteins from the receptor family, the tyrosine kinase with immunoglobulin-like and EGF-like domain 1 (TIE) receptor family, the receptor tyrosine kinase-like orphan (ROR) receptor family, the discoidin domain (DDR) receptor family, the rearranged during transfection (RET) receptor family, the tyrosine protein kinase-like (PTK7) receptor family, the related receptor tyrosine kinase (RYK) receptor family, or the muscle-specific kinase (MuSK) receptor family, a G protein-coupled receptor, a GPCR (Frizzled or Smoothened), a serine / threonine kinase receptor (BMPR or TGFR); or a cytokine receptor (IL1R, IL2R, IL7R, or IL15R).

[0184] CARs also generally contain one or more linker sequences used for various purposes within the molecule. For example, a transmembrane domain can be used to link the extracellular component of the CAR to the intracellular component. A flexible linker sequence, often referred to as a spacer region, located membrane-proximal to the binding domain, can be used to create additional distance between the binding domain and the cell membrane. This can be beneficial for reducing steric hindrance to binding based on membrane proximity. A commonly used spacer region for this purpose is the IgG4 linker. Depending on the target cell marker, a more compact or longer spacer can be used. Other potential CAR subcomponents are described in more detail elsewhere herein. Here, the components of a CAR are described in additional detail as (a) the binding domain, (b) the intracellular signaling component, (c) the linker, (d) the transmembrane domain, (e) the junction amino acids, and (f) the control features, including the tag cassette.

[0185] The transmembrane domain in a CAR molecule often serves to connect extracellular and intracellular components across the cell membrane, and can anchor the expressed molecule to the membrane of the modified cell.

[0186] The transmembrane domain can be derived from either natural and / or synthetic sources. If the source is natural, the transmembrane domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can include at least the transmembrane region(s) of the α, β, or ζ chain of the T cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In certain embodiments, the transmembrane domain is selected from, for example, at least one of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA 1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The transmembrane domain(s) may include AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C. In certain embodiments, various human hinges may also be used, including human Ig (immunoglobulin) hinges (e.g., IgG4 hinges, IgD hinges), GS linkers (e.g., GS linkers described herein), KIR2DS2 hinges, or CD8a hinges.

[0187] TCR refers to a naturally occurring T cell receptor. The payload of the present disclosure can encode a TCR or a CAR / TCR hybrid that includes elements of a TCR and a CAR. For example, a CAR / TCR hybrid can have a naturally occurring TCR binding domain with an effector domain with which the TCR binding domain is not naturally associated. A CAR / TCR hybrid can have a mutated TCR binding domain and an ITAM signaling domain. A CAR / TCR hybrid can have a naturally occurring TCR with an inserted non-naturally occurring spacer region or transmembrane domain.

[0188] I(C)(i)(b). Gene editing systems and components In various embodiments, the payload of the present disclosure encodes at least one or all components of a gene editing system. Gene editing systems of the present disclosure include CRISPR systems, base editing, and prime editing systems. Generally, gene editing systems can include multiple components, including a gene editing enzyme selected from a CRISPR-associated RNA-guided endonuclease, a base editing enzyme, and a prime editing enzyme and at least one gRNA. Thus, the gene editing system of the present disclosure can include either: (i) in the case of a CRISPR system, a CRISPR enzyme that is a CRISPR-associated RNA-guided endonuclease and at least one guide RNA (gRNA); (ii) in the case of a base editing system, a base editing enzyme and at least one gRNA; or (iii) in the case of a prime editing system, and at least one prime editing gRNA. Nucleotide sequences encoding gene editing systems such as those disclosed herein are typically too large to be included in many volume-limited vector systems, but the large capacity of adenoviral vectors allows for the inclusion of such sequences in the adenoviral vectors and genomes of the present disclosure. A further advantage of the adenoviral vectors and genomes bearing payloads encoding the gene editing systems or components of the present disclosure is that the adenoviral genome does not naturally integrate into the host cell genome, facilitating transient expression of the gene editing systems and components, which may be desirable, for example, to avoid immunogenicity and / or genotoxicity.

[0189] In other embodiments, the gene editing system can include an engineered zinc finger nuclease (ZFN). For example, ZFNs are artificial endonucleases consisting of an engineered zinc finger protein (ZFP) fused to the cleavage domain of the FokI restriction enzyme. ZFNs can be redesigned to cleave new targets by developing ZFPs with new sequence specificities. For genome manipulation, ZFNs target selected genomic sequences for cleavage. ZFN-induced cleavage events trigger cellular repair processes, which then mediate efficient modification of the target locus. If ZFN-induced cleavage events are resolved by non-homologous end-joining, this can result in small deletions or insertions, effectively leading to gene knockout. If the cleavage is resolved through a homology-based process in the presence of a researcher-provided donor, small changes or entire transgenes can be transferred to a chromosome, often without selection. This can be referred to as "gene correction" and "gene addition," respectively.

[0190] In some embodiments, a gene editing system (e.g., a CRISPR system, a base editing system, or a prime editing system) is engineered to modify a nucleic acid sequence encoding gamma globin, e.g., to increase gamma globin expression. Hemoglobin F (HbF), the predominant fetal form of hemoglobin, is formed by pairing a gamma-globin polypeptide subunit with an alpha-globin polypeptide subunit. Human fetal gamma-globin genes (HBG1 and HBG2; two highly homologous genes produced by evolutionary duplication) are typically silenced around birth, whereas adult beta-globin gene expression (HBB and HBD) increases. Mutations that cause or enable sustained expression of fetal gamma-globin throughout life can alleviate the phenotype of beta-globin deficiency. Therefore, reactivation of the fetal gamma-globin gene may be therapeutically beneficial, particularly in subjects with beta-globin deficiency. Various mutations that cause increased expression of γ-globin are known in the art (see, e.g., Wienert, Trends in Genetics 34(12):927-940, 2018, which is incorporated herein by reference in its entirety regarding mutations that increase γ-globin expression). Certain such mutations are found in the HBG1 or HBG2 promoters.

[0191] In various embodiments, the gene editing system designed to increase γ-globin expression comprises an HBG1 / 2 promoter-targeting gRNA designed to increase expression of the γ-globin coding gene by modifying and / or inactivating a BCL11A repressor protein binding site. In various embodiments, the gene editing system designed to increase γ-globin expression comprises a bcl11a-targeting gRNA designed to increase γ-globin expression by modifying and / or inactivating an erythroid bcl11a enhancer to reduce expression of the BCL11A repressor protein in erythroid cells. In various embodiments, the gene editing system designed to increase γ-globin expression comprises a gRNA targeted to cause a loss-of-function mutation in the gene encoding BCL11A.

[0192] I(C)(i)(b)(1).CRISPR payload expression product The present disclosure includes, inter alia, CRISPR editing materials and systems, as well as nucleic acids encoding same, for example, when the nucleic acid is present in an adenoviral vector or genome. A CRISPR editing system can include a CRISPR editing enzyme and / or at least one gRNA as its components. The CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated proteins) nuclease system is an engineered nuclease system used for bacterial genetic engineering. It is based in part on the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, a segment of the invader's DNA is converted into CRISPR RNA (crRNA) by the bacterial "immune" response. The crRNA then associates with another type of RNA, called tracrRNA, through a region of partial complementarity, to guide the Cas nuclease to a region homologous to the crRNA within the target DNA, called the "protospacer." Cas nucleases cleave DNA to generate blunt ends at double-strand breaks at sites specified by 20-nucleotide complementary sequences contained within the crRNA transcript. In some cases, Cas nucleases require both the crRNA and tracrRNA for site-specific DNA recognition and cleavage.

[0193] A guide RNA (gRNA) is an example of an element that can target CRISPR editing. In its simplest form, a gRNA provides a sequence (e.g., crRNA) that targets a site within a genome based on complementarity. However, as described below, a gRNA can also include additional components. For example, in certain embodiments, a gRNA can include a targeting sequence (e.g., crRNA) and a component that links the targeting sequence to a cleavage element. This linking component can be a tracrRNA. In certain embodiments, a gRNA, including a crRNA and a tracrRNA, can be expressed as a single molecule, referred to as a single gRNA (sgRNA). A gRNA can also be linked to a cleavage element via other mechanisms, such as a nanoparticle, or through the expression or construction of a dual- or multi-purpose molecule. Those skilled in the art will understand that gRNAs or other targeting elements that can be used to generate corrections or modifications of selected nucleic acid sequences, for example, in host cells of the adenoviral donor vectors or genomes of the present disclosure, can be readily designed and implemented, for example, based on available sequence information.

[0194] In certain embodiments, a targeting element (e.g., gRNA) can include one or more modifications (e.g., base modifications, backbone modifications) to provide the nucleic acid with new or enhanced characteristics (e.g., improved stability). Modified backbones can include backbones that retain a phosphorus atom in the backbone and backbones that do not have a phosphorus atom in the backbone. Suitable modified backbones containing a phosphorus atom can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, e.g., 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoamidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenium phosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linkage analogs, and those in which one or more internuclear linkages are reversed in polarity, such as 3' to 3', 5' to 5', or 2' to 2' linkages. Suitable targeting elements with reverse polarity can include a single 3' to 3' linkage at the 3'-most internucleotide linkage (i.e., a single inverted nucleoside residue in which a nucleobase is missing or has a hydroxyl group instead). Various salts (e.g., potassium chloride or sodium chloride), mixed salts, and free acid forms can also be included.

[0195] Exemplary cleavage elements include nucleases. CRISPR-Cas loci contain over 50 gene families, with no strictly universal genes, demonstrating rapid evolution and extreme diversity in locus architecture. Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12, including, e.g., spCas9, dCas9, nCas9, and Cas9-SpRY), Cas10, Cas12 (e.g., Cas12a (e.g., LbCas12a, AsCas12a, FnCas12a, MB3Cas1), and Cas1B (e.g., Cas1B, Cas1C, Cas1D, Cas1E, Cas1F, Cas1F, Cas1G, Cas1H, Cas1H, Cas1I ... 2a, Cas12a-M11, Cas12a-M13 (e.g., Cas12a-M13-1), Cas12a-M26 (e.g., Cas12a-M26-1), Cas12a-M28 (e.g., Cas12a-M28-1), Cas12a-M29 (e.g., Cas12a-M29-1), Cas12a-M30 (e.g., Cas12a-M30-1), Cas12a-M31 (e.g., Cas12a-M31-1), Cas1 2a-M32 (e.g., Cas12a-M32-1), Cas12a-M57, Cas12a-M58, Cas12a-M59, Cas12a-M60 (e.g., Cas12a-M60-9), Cas12a-M61, or Cas12a-M62), Cas12b, Cas12c, Cas12g, Cas12h, or Cas12i), Cas-Phi, CasX, CasY, Cpf1, C2c3, C2c2, C2c1, Cs These include y1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and their variants.

[0196] There are three major types of Cas nucleases (type I, type II, and type III), with ten subtypes, including five type I, three type II, and two type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(1):58-66). Type II Cas nucleases include Cas1, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, the amino acid sequence of a wild-type Cas9 polypeptide from Streptococcus pyogenes is set forth, for example, in NCBI accession number NP_269215, and the amino acid sequence of a wild-type Cas9 polypeptide from Streptococcus thermophilus is set forth, for example, in NCBI accession number WP_011681470.

[0197] In certain embodiments, Cas9 refers to an RNA-guided double-stranded DNA-binding nuclease or nickase protein. Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cleave different DNA strands. Cas9 can induce double-stranded breaks in genomic DNA (target DNA) when both functional domains are active. In some embodiments, the Cas9 enzyme comprises one or more catalytic domains of Cas9 proteins derived from bacteria such as Corynebacter, Sutterella, Legionella, Treponema, Filifacter, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, the Cas9 is a fusion protein, e.g., the two catalytic domains are derived from different bacterial species.

[0198] In some embodiments, the crRNA and tracrRNA can be combined into a single molecule called a single gRNA (sgRNA). In this engineered approach, the sgRNA guides Cas to target any desired sequence (see, e.g., Jinek et al. Science 337:816-821, 2012; Jinek et al., eLife 2:e00471, 2013; Segal, eLife 2:e00563, 2013). Thus, the CRISPR / Cas system can be engineered to create a double-strand break at a desired target in a cell's genome and utilize the cell's endogenous mechanisms to repair the break induced by HDR or NHEJ. Certain embodiments described herein utilize homology arms to promote HDR at defined integration sites.

[0199] In various embodiments, the variant of the Cas9 nuclease is RuvC. ” or HNH ” Cas9 nucleases contain a single inactive catalytic domain, such as an enzyme or nickase. Cas9 nickases have only one active functional domain and, in some embodiments, cleave only one strand of the target DNA, thereby generating a single-strand break or nick. In some embodiments, a mutant Cas9 nuclease having at least a D10A mutation is a Cas9 nickase. In other embodiments, a mutant Cas9 nuclease having at least an H840A mutation is a Cas9 nickase. Other examples of mutations present in Cas9 nickases include N854A and N863A. When at least two DNA target RNAs targeting opposite DNA strands are used, a double-strand break is introduced using a Cas9 nickase. The double-nick-induced double-strand break is repaired by HDR or NHEJ. This gene editing strategy generally favors HDR and reduces the frequency of indel mutations at off-target DNA sites. In some embodiments, the Cas9 nuclease or nickase is codon-optimized for the target cell or organism.

[0200] I(C)(i)(b)(2). Base editor payload expression product The present disclosure includes, inter alia, base editing agents and systems, as well as nucleic acids encoding same, for example, when the nucleic acid is present in an adenoviral vector or genome. The base editing system can include a base editing enzyme and / or at least one gRNA as a component. The base editing system can utilize a deaminase (e.g., a base editing system) to edit a nucleic acid target. In certain embodiments, the base editing agents and / or base editing systems of the present disclosure are present in an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 adenoviral vector.

[0201] Deamination is the removal of amine groups from molecules such as nucleic acid nucleotides. Deamination of nucleotides can cause changes in the sequence of nucleic acids, and deaminases are useful for editing at least for that reason. Deamination of adenosine (A) results in inosine (I), which has the same base-pairing preference as guanosine in DNA and is therefore recognized by the cellular replication machinery as guanosine, resulting in an AT to GC transition. Deamination of cytosine (C) results in uridine (U), which is recognized by the cellular replication machinery as thymine, resulting in a CG to TA transition. Collectively, deamination of cytosine and adenosine can be used to cause A to G, T to C, C to T, or G to A transitions. Other deaminase activities are also known. For example, deamination of 5-methylcytosine results in thymine, and deamination of guanosine results in xanthine, which pairs with cytosine like guanosine. A deaminase that deaminates cytosine can be referred to as a cytosine deaminase. A deaminase that deaminates adenosine can be referred to as an adenosine deaminase.

[0202] In certain embodiments, the base editing enzyme comprises a cytidine deaminase domain or an adenine deaminase domain. Certain embodiments utilize a cytidine deaminase domain as the nucleobase deaminase enzyme. Certain embodiments utilize an adenine deaminase domain as the nucleobase deaminase enzyme.

[0203] Examples of cytosine deaminase enzymes (CBEs) include APOBEC1, APOBEC3A, APOBEC3G, CDA1, and AID. APOBEC1 specifically accepts single-stranded (ss) DNA as a substrate but cannot act on double-stranded (ds) DNA.

[0204] An exemplary adenosine deaminase capable of acting on DNA for adenine base editing as an adenosine base editor (ABE) is a mutant TadA adenosine deaminase (TadA*), which accepts DNA as its substrate. E. coli TadA typically acts as a homodimer, deaminating adenosine in transfer RNA (tRNA). TadA* deaminase catalyzes the conversion of the target "A" to "I" (inosine), which is treated as "G" by cellular polymerases. The original genomic AT base pair can then be converted to a GC pair. Because cellular inosine excision repair is less active than uracil excision, ABEs do not require additional inhibitory proteins, such as UGI, in CBEs. In some embodiments, the ABE can comprise one or more or all of three components, including a wild-type E. coli tRNA-specific adenosine deaminase (TadA) monomer, which may play a structural role during base editing; a TadA* mutant TadA monomer that catalyzes deoxyadenosine deamination; and / or a Cas nickase, such as Cas9(D10A). In certain embodiments, there is a linker disposed between TadA and TadA*, and in certain embodiments, there is a linker disposed between TadA* and the Cas nickase. In various embodiments, one or both linkers comprise at least six amino acids, e.g., at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids (e.g., with a lower limit of 5, 6, 7, 8, 9, 10, or 15 amino acids and an upper limit of 20, 25, 30, 35, 40, 45, or 50 amino acids). In various embodiments, one or both linkers comprise 32 amino acids, hi some embodiments, one or both linkers have the sequence set forth in (SGGS)2-XTEN-(SGGS)2 (SEQ ID NO: 213), or a sequence otherwise known to one of skill in the art.

[0205] In various embodiments, the editing system includes a deaminase associated with a DNA-binding domain, such as a catalytically impaired nuclease domain. In various embodiments, the DNA-binding domain can localize the deaminase to a target nucleic acid where one or more nucleotides are deaminated by the deaminase. A catalytically impaired nuclease domain is a polypeptide domain that has an amino acid sequence engineered from a reference nuclease domain sequence but has a reduced or no ability to cause double-strand breaks (DSBs) compared to the reference (e.g., a wild-type and / or fully functional nuclease). As referred to herein, a nickase refers to a catalytically impaired nuclease domain that, upon contact with a double-stranded nucleic acid substrate, cleaves one strand of the double-stranded nucleic acid (e.g., the target strand), but does not cleave both strands of the double-stranded nucleic acid. In various embodiments, upon contact with double-stranded nucleic acid substrates, the nickase cleaves one strand of the double-stranded nucleic acid in at least 70% of the contacted double-stranded nucleic acid substrates (e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the double-stranded nucleic acid substrates), but does not cleave both strands of the double-stranded nucleic acid.

[0206] A base editing system is an example of an editing system that includes a deaminase enzyme. The base editing enzyme includes a deaminase enzyme fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase, e.g., a nickase that nicks a single strand, e.g., a non-edited strand). The DNA binding domain of the base editing enzyme can be an RNA-guided DNA binding domain, in that the RNA guide can direct the DNA binding domain to a target nucleic acid sequence. The catalytically impaired nuclease domain of the base editing enzyme can bind to nucleic acid and localize the deaminase enzyme to the target nucleic acid.

[0207] Any nuclease in a CRISPR system can be engineered to produce a catalytically impaired nuclease domain (e.g., a nickase) and used within the base editing enzyme or system. Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12, including, for example, spCas9, dCas9, nCas9, and Cas9-SpRY), Cas10, Cas12 (e.g., Cas12a (e.g., LbCas12a, AsCas12a, FnCas12a, MB3C), and the like. Cas12a, Cas12a-M11, Cas12a-M13 (e.g., Cas12a-M13-1), Cas12a-M26 (e.g., Cas12a-M26-1), Cas12a-M28 (e.g., Cas12a-M28-1), Cas12a-M29 (e.g., Cas12a-M29-1), Cas12a-M30 (e.g., Cas12a-M30-1), Cas12a-M31 (e.g., Cas12a-M31-1) ), Cas12a-M32 (e.g., Cas12a-M32-1), Cas12a-M57, Cas12a-M58, Cas12a-M59, Cas12a-M60 (e.g., Cas12a-M60-9), Cas12a-M61, or Cas12a-M62), Cas12b, Cas12c, Cas12g, Cas12h, or Cas12i), Cas-Phi, CasX, C2c3, C2c2, C2c1, Csy 1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and their variants. Numerous forms and variants of Cas nucleases (e.g., spCas9, dCas9, nCas9, Cas9-SpRY, and Cas12a) are known in the art and can have different characteristics, including, for example, recognition of different PAMs and PAM positions.

[0208] In various embodiments, the catalytically impaired nuclease domain generates a single-stranded nick in the non-deaminated DNA strand and, using the deaminated strand as a template, induces the cell to repair the non-deaminated strand. To provide an example, nCas9 can create a nick in the target DNA by cleaving a single strand, which reduces the likelihood of deleterious indel formation compared to methods requiring double-stranded breaks.

[0209] Certain embodiments utilize nuclease-inactive Cas9 (dCas9) as the catalytically inactivated nuclease. However, any nuclease in the CRISPR system (many of which are described above) can be disabled and used within the base editing system. In certain embodiments, a high-fidelity Cas9 domain is selected, where the Cas9 domain exhibits reduced electrostatic interactions between the Cas9 domain and the sugar phosphate backbone of DNA compared to a wild-type Cas9 domain. In some embodiments, the Cas9 domain (e.g., a wild-type Cas9 domain) contains one or more mutations that reduce the association between the Cas9 domain and the sugar phosphate backbone of DNA. High-fidelity Cas9 domains are known to those skilled in the art. For example, high-fidelity Cas9 domains are described in Kleinstiver (2016 Nature 529:490-495) and Slaymaker (2015 Science 351:84-88).

[0210] Other DNA-binding nucleases can also be used as base editing enzymes. For example, base editing systems can utilize zinc finger nucleases (ZFNs) (see, for example, Urnov 2010 Nat Rev Genet.11(9):636-46) and transcription activator-like effector nucleases (TALENs) (see, for example, Joung 2013 Nat Rev Mol Cell Biol.14(1):49-55). For more information on DNA-binding nucleases, see, for example, US2018 / 0312825.

[0211] In various embodiments, the base editing enzyme comprises a DNA glycosylase inhibitor. A DNA glycosylase inhibitor can override natural DNA repair mechanisms that might otherwise repair the intended base edit. The DNA glycosylase inhibitor can be a uracil DNA glycosylase inhibitor protein (UGI). One exemplary UGI is described in Wang (1991 Gene 99:31-37). In certain embodiments, the base editing enzyme can comprise one or more DNA glycosylase inhibitor domains (e.g., UGI domains). In various embodiments, a base editing enzyme comprising two or more DNA glycosylase inhibitor domains (e.g., UGI domains) can generate fewer indels and / or deaminate target nucleic acids more efficiently than a base editing enzyme comprising one DNA glycosylase inhibitor domain (e.g., UGI domain) and / or no DNA glycosylase inhibitor domain (e.g., UGI domain). For example, in certain embodiments, dCas9 or Cas9 nickase may be fused to a cytidine deaminase domain, and dCas9 or Cas9 nickase may be fused to one or more UGI domains. In certain embodiments, the deaminase domain is associated with the N-terminus of the catalytically inactivated nuclease. In certain embodiments, the deaminase domain is associated with the N-terminus of the catalytically inactivated nuclease. In certain embodiments, one or more glycosylase inhibitors (e.g., UGI domains) can be associated with the C-terminus of the catalytically inactivated nuclease.

[0212] Components of a base editor can be fused directly (e.g., by direct covalent bonding) or via a linker. For example, a catalytically inactivated nuclease can be fused to a deaminase enzyme and / or a glycosylase inhibitor via a linker. Multiple glycosylase inhibitors can also be fused via linkers. As will be appreciated by those of skill in the art, linkers can be used to link any peptide or portion thereof.

[0213] Exemplary linkers include polymeric linkers (e.g., polyethylene, polyethylene glycol, polyamide, polyester); amino acid linkers; carbon-nitrogen bond amide linkers; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linkers; monomeric, dimeric, or polymeric aminoalkanoic acid linkers; aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, β-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid) linkers; monomeric, dimeric, or polymeric aminohexanoic acid (Ahx) linkers; carbocyclic moiety (e.g., cyclopentane, cyclohexane) linkers; aryl or heteroaryl moiety linkers; and phenyl ring linkers.

[0214] The linker may also include a functionalized moiety to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0215] In certain embodiments, the linker is in the range of 4 to 100 amino acids in length, hi certain embodiments, the linker is 4 amino acids, 9 amino acids, 14 amino acids, 16 amino acids, 32 amino acids, or 100 amino acids.

[0216] Various base editing enzymes are known in the art. Examples of base editing enzymes include BE1 (APOBEC1-16 amino acid (aa) linker-Sp dCas9(D10A,H840A) (see, e.g., Komor 2016 Nature 533:420-424)), BE2 (APOBEC1-16aa linker-Sp dCas9(D10A,H840A)-4aa linker-UGI (see, e.g., Komor 2016 Nature 533:420-424)), BE3 (APOBEC1-16aa linker-SpnCas9(D10A)-4aa linker-UGI (see, e.g., Komor 2016 Nature 533:420-424)), HF-BE2 (rAPOBEC1-HF2 nCas9-UGI), HF-BE3 (APOBEC1-16aa linker-HF nCas9(D10A)-4aa linker-UGI (see, e.g., Rees 2017 Nat. Commun. 8:15790)), BE4 (rAPOBEC1-Sp nCas9-UGI-UGI), BE4max (APOBEC1-32aa linker-Sp nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Koblan 2018 Nat. Biotechnol 36(9):843-846 and / or Komor 2017 Sci. Adv. 3(8):eaao4774)), BE4-GAM (Gam-16aa linker-APOBEC1-32aa linker-Sp nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8):eaao4774)), YE1-BE3 (APOBEC1(W90Y,R126E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475-480)), EE-BE3 (APOBEC1(R126E,R132E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35:475-480)), YE2-BE3 (APOBEC1(W90Y,R132E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475-480)), YEE-BE3 (APOBEC1(W90Y,R126E,R132E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475-480)), VQR-BE3 (APOBEC1-16aa linker-Sp VQR nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475-480)), 2017 Nat. Biotechnol. 35:475-480)), EQR-BE3 (rAPOBEC1-EQR SpnCas9-UGI), VRER-BE3 (APOBEC1-16aa linker-Sp VRER nCas9(D10A)-4aa linker-UGI (e.g., Kim 2017 Nat. Biotechnol. 35:475-480)), Sa-BE3 (APOBEC1-16aa linker-Sa nCas9(D10A)-4aa linker-UGI (e.g., Kim 2017 Nat. Biotechnol. 35:475-480)), SA-BE4 (APOBEC1-32aa linker-Sa nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8):eaao4774)), SaBE4-Gam (Gam-16aa linker-APOBEC1-32aa linker-Sa nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8):eaao4774)), SaKKH-BE3 (APOBEC1-16aa linker-Sa KKH nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol.35:475-480), FNLS-BE3 (rAPOBEC1-Sp nCas9-UGI), RA-BE3 (rAPOBEC1(RA)-Sp nCas9-UGI), Cas12a-BE (APOBEC1-16aa linker-dCas12a-14aa linker-UGI (e.g., Li 2018 Nat. Biotechnol. 36:324-327)), Target-AID (Sp nCas9(D10A)-100aa linker-CDA1-9aa linker-UGI (e.g., Nishida 2016 Science 353(6305):aaf8729)), Target-AID-NG (Sp nCas9(D10A)-NG-100aa linker-CDA1-9aa linker-UGI (e.g., Nishimasu 2018 Science 361(6408):1259-1262), xBE3 (APOBEC1-16aa linker-xCas9(D10A)-4aa linker-UGI (e.g., Hu 2018 Nature 556:57-63), eA3A-BE3 (APOBEC3A(N37G)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (e.g., Gehrke 2018 Nat. Biotechnol. 36(10):977-982), A3A-BE3 (hAPOBEC3A-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (e.g., Wang 2018 Nat. Biotechnol.36:946-949)), eA3A-HF1-BE3-2xUGI (APOBEC3A-HF1 Sp nCas9-UGI-UGI), eA3A-HypaBE3-2xUGI (APOBEC3A-Hypa Sp nCas9-UGI-UGI), hA3A-BE3 (hAPOBEC3A-Sp nCas9-UGI), hA3B-BE3 (hAPOBEC3B-Sp nCas9-UGI), hA3G-BE3 (hAPOBEC3G-Sp nCas9-UGI), hAID-BE3 (hAPOBEC3A-Sp nCas9-UGI), SaCas9-BE3 (rAPOBEC1-SanCas9-UGI), xCas9-BE3 (rAPOBEC1-xnCas9-UGI), ScCas9-BE3 (rAPOBEC1-ScnCas9-UGI), SniperCas9-BE3 (rAPOBEC1-SnipernCas9-UGI), iSpyMac-BE3 (rAPOBEC1-iSpyMacnCas9-UGI), CRISPR-X (Sp dCas9-MS2-hAID), TAM (Sp dCas9-hAID(P182X)), AncBE4-Max (rAPOBEC1-Sp nCas9-UGI-UGI), ABE7.8 / 9 / 10 (ecTadA-ecTadA*-Sp nCas9), xCas9-ABE7.10 (ecTadA-ecTadA*-nxCas9), VQR-ABE (ecTadA-ecTadA*-Sp VQR nCas9), Sa(KKH)-ABE ecTadA-ecTadA*-Sa KKH nCas9), ABEmax (ecTadA-ecTadA*-Sp nCas9), ABE7.10max (ecTadA-ecTadA*-SpnCas9), ABE8e )ecTadA-ecTadA*-SpnCas9), PE1 (dSpCas9-MMLV-RT), PE2 (dSpCas9-MMLV-RT), PE3 (nSpCas9-MMLV-RT), and BE-PLUS (10X GCN4-Sp nCas(D10A) / ScFv-rAPOBEC1-UGI (see, for example, Jiang 2018 Cell Res.28(8):855-861)). For additional examples of BE complexes containing adenine deaminase base editors, see, for example, Rees 2018 Nat.Rev Genet.19(12):770-788 and / or Kantor 2020 Int.J.Mol.Sci.21(17):6240.

[0217] Various base editors are "dual base editors" that can edit both adenine and cytosine. Dual base editor enzymes can be fusion polypeptides containing a cytosine deaminase domain and an adenine deaminase domain. For example, the dual base editor known as Target-ACEmax contains a codon-optimized fusion of cytosine deaminase PmCDA1, adenosine deaminase TadA, and Cas9 nickase (Target-ACEmax) (see, e.g., Sakata 2020 Nature Biotechnology, 38(7), 865-869). Other exemplary dual base editors include SPACE (Synchronous Programmable Adenine and Cytosine Editor). SPACE editing enzymes are fusion polypeptides that contain both the miniABEmax-V82G and Target-AID editing domains along with the Cas9 (SpCas9-D10A) nickase domain (see, e.g., Grunewald 2020 Nat. Biotechnol. 38:861-864). A dual base editor known as A&C-BEmax contains fusions of both cytidine and adenosine deaminase domains with the Cas9 nickase domain (see, e.g., Zhang 2020 Nat. Biotechnol. 38:856-860).

[0218] The base editing system can include a guide RNA (gRNA) that includes at least a fragment that base-pairs with a complementary target nucleic acid (e.g., at least 80% identity between the fragment and the complement of the target nucleic acid, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), where the fragment can be 10-40 nucleotides in length (e.g., equal to or approximately equal to 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, or 40 nucleotides in length, e.g., 17-24 or 17-20 nucleotides in length), e.g., where the target sequence is upstream of a suitable PAM site. In various embodiments, the fragment of the gRNA that is complementary to the target nucleic acid sequence is positioned at the 5' end of the gRNA or is 5' to one or more other fragments of the gRNA. In various embodiments, the gRNA comprises a sequence that forms a stem-loop structure and binds to and / or recruits a catalytically impaired nuclease domain of a base-editing enzyme. A gRNA that comprises both a fragment that base-pairs with a complementary target nucleic acid sequence and a fragment that forms a stem-loop structure and binds to and / or recruits a catalytically impaired nuclease domain of a base-editing enzyme may be referred to as a single guide RNA (sgRNA). The fragments of the sgRNA can be associated via a linker fragment.

[0219] The guide RNA (e.g., sgRNA) is thought to randomly match nucleic acids until it encounters a nucleic acid sufficiently complementary to the 5' fragment. Upon binding of the gRNA to a DNA nucleic acid target present in double-stranded DNA, base pairing between the gRNA and the target nucleic acid strand causes the transposition of a small segment of single-stranded DNA. In various embodiments, the gRNA recruits a catalytically impaired nuclease domain. Nucleotides of the displaced single-stranded DNA can be modified by a deaminase enzyme. The resulting base pair can then be repaired to a new base pair by the cellular mismatch repair machinery, or alternatively, in some cases, can be reversed by base excision repair mediated by uracil glycosylase. In various embodiments, a glycosylase inhibitor (e.g., UGI) reduces the occurrence of repair.

[0220] The present disclosure includes base editing enzymes and systems engineered to increase the editing window of base editing. For example, the present disclosure includes circularly permuted base editors, such as those described in Huang 2020 Nature Biotechnology, 37(6), 626-631 (incorporated herein with respect to base editing enzymes, base editing systems, and their editing windows). Circularly permuted base editing enzymes and systems can feature an increased range of target bases that can be modified within a protospacer, including, for example, at least 5, 6, 7, 8, or even up to 9 nucleotides. For example, certain base editing systems, including Cas9 variants containing cytosine and four adenine base editing enzymes, can deaminate nucleotides within an expanded window from about 4-5 nucleotides up to about 8-9 nucleotides, optionally with reduced by-product formation.

[0221] Base editing enzymes and systems can also target and / or modify RNA molecules. One advantage of using an RNA editing system is that no permanent changes are made to the genome. RNA base editors achieve similar changes using RNA base-modifying components. For example, adenosine deaminase can modify transcribed mRNA, replacing adenosine with inosine at target sites. In mammals, the most common case of post-transcriptional RNA editing is catalyzed by adenosine deaminase enzymes (ADARs). ADAR proteins are a highly conserved protein family containing a single deaminase domain (DD) and one or more double-stranded RNA (dsRNA)-binding domains (e.g., ADAR1 or ADAR2). ADARs bind to dsRNA and catalyze the conversion of adenosine to inosine (A-to-I), which is then read as guanosine by the cellular translation machinery. The ADAR1 and ADAR2 domains have been demonstrated to achieve RNA editing, for example, in HSCs (see, e.g., Harter 2009 Nat. Immunol. 10(1):109-115). Many catalytically inactive Cas proteins have also been used to target RNA molecules, including Cas9, Cas13a, Cas13b, and Cas13d.

[0222] REPAIR (Programmable RNA Editing for Adenosine to Inosine Substitution) is an RNA base editing system that includes a catalytically inactive Cas13 protein and the deaminase activity of ADAR2. Cas13 generally contains two HEPN (Higher Eukaryotic and Prokaryotic Nucleotide Binding) domains that contribute to RNA-targeted nucleotide degradation activity. Mutation of HEPN abolishes RNA cleavage activity while maintaining RNA-targeting activity, and this has been used to generate RNA base editing enzymes (e.g., REPAIR) (see, e.g., Cox 2017 Science 358:1019-1027). dCas13-ADAR2 DDA catalytically inactive dCas13 variant with the RNA deaminase ADAR2 (E488Q) was subsequently developed to perform RNA editing for programmable A-to-I (G) substitutions. RNA editing for specific C-to-U exchanges (RESCUE) was subsequently developed (see, e.g., Abudayyeh 2019 Science 365:382-386). For example, a gRNA for mRNA editing can include a fragment complementary to the target RNA and an ADAR-recruiting fragment, thereby achieving site-directed RNA editing by recruiting ADAR to the complementary target nucleic acid. The RNA-guided RNA-targeting CRISPR nuclease C2C2 (later named Cas13a) from Leptotrichia shahii was demonstrated (Abudayyeh 2016 Science 353:aaf5573).

[0223] Another example of an ADAR-containing RNA editing system involves removing the endogenous RNA targeting domain (dsRBMS) from human adenosine deaminase and replacing it with an antisense RNA oligonucleotide to produce a recombinant enzyme that can be directed to edit a selected RNA target. In certain embodiments, the ADAR2 deaminase domain is fused to an RNA-binding protein, and the sequence bound by the RNA-binding protein is associated with an antisense RNA guide oligonucleotide. In various embodiments, the RNA-binding protein is derived from the λ phage N protein-boxB RNA interaction, which normally regulates antitermination during transcription of λ phage mRNA. The λN peptide mediates N protein binding and is only 22 amino acids long, while the boxB RNA hairpin it recognizes is only 17 nucleotides long and can bind with nanomolar affinity. Thus, in various embodiments, the λN peptide can be fused to the deaminase domain of human ADAR2 (λN-DD). In various embodiments, a mutant ADAR2 DD(E488Q) can be used as the deaminase domain. In various embodiments, the editing enzyme can include an ADAR deaminase domain and two or more λN domains (e.g., 2, 3, 4, 5, or 6 λN domains). Examples of such editing enzymes and systems are described, for example, in Montiel-Gonzalez 2013 PNAS 110(45):18285-18290 and Montiel-Gonzalez 2016 Nuc.Acids.Res.44(2):e157, each of which is incorporated herein by reference with respect to editing systems.

[0224] Another example of an ADAR-containing editing system can involve utilizing endogenous ADARs for programmable editing of RNA (LEAPER) editing systems, which use short engineered ADAR recruiting RNAs (arRNAs) to recruit natural ADAR1 or ADAR2 deaminase enzymes to change specific adenosines to inosines. For example, in certain embodiments, an ADAR protein or its catalytic domain can be fused to a lambda N peptide. In certain embodiments, an ADAR protein or its catalytic domain can be fused to a lambda N peptide and a SNAP tag or a Cas protein (e.g., dCas13b). A gRNA can recruit the editing enzyme to a specific site. Further description of the LEAPER editing system can be found in Qu 2019 Nat. Biotech. 1059-1069, which is incorporated herein by reference with respect to the LEAPER editing system.

[0225] The base editing system can induce point mutations without generating double-strand breaks. The base editing system can induce point mutations without generating unwanted insertions and deletions (indels). For example, the base editing system can induce indels in less than 10%, 9%, 8%, 7%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of edited cells or editing events.

[0226] Those skilled in the art will understand that base-editing gRNAs (e.g., sgRNAs) or other targeting elements that generate selected nucleic acid sequence modifications in a target nucleic acid can be readily designed and implemented, for example, based on available sequence information.

[0227] The base editing system does not require double-stranded DNA breaks. The base editing system does not require a donor fragment or template. The base editing system provides precise control of the site at which the editing system modifies the target nucleic acid. The base editing system can be multiplexed to achieve editing of multiple targets, optionally including therapeutic targets, using a single editing enzyme. The present disclosure includes base editing systems that include multiple sgRNAs (e.g., two or more, e.g., two, three, four, or five) sgRNAs.

[0228] I(C)(i)(b)(3). Prime editor payload expression product The present disclosure includes, inter alia, prime editing materials and systems, as well as nucleic acids encoding same, for example, when the nucleic acid is present in an adenoviral vector or genome. A prime editing system can include a prime editing enzyme and / or at least one pegRNA as its components. Prime editing can introduce all possible types of point mutations, small insertions, and small deletions in a precise and targeted manner. The prime editing enzyme includes a reverse transcriptase fused to a DNA-binding domain that is a catalytically impaired nuclease domain (e.g., a nickase that nicks a single strand, e.g., the unedited strand). Reverse transcriptase is an enzyme that can synthesize DNA molecules from an RNA template. Reverse transcriptase generally produces DNA molecules complementary to the RNA template.

[0229] In certain embodiments, the editing enzyme comprises AMV reverse transcriptase, MLV reverse transcriptase, HIV-1 reverse transcriptase, or bacterial reverse transcriptase. Certain embodiments utilize an MLV reverse transcriptase domain. The reverse transcriptases of the present disclosure can have wild-type or engineered amino acid sequences.

[0230] Examples of reverse transcriptases include AMV reverse transcriptase (e.g., wild-type AMV reverse transcriptase (RNase H plus activity), eAMV™ (engineered, RNase H plus activity), or ThermoScript™ (engineered, reduced RNase H activity)), MLV reverse transcriptase (e.g., wild-type M-MLV reverse transcriptase, GoScript™, or MultiScribe™ (RNase H plus activity), AccuScript Hi-Fi (engineered, RNase H minus (3'-5' exonuclease activity), Affinity Script (engineered, E69K / E302R / W313F / L435G / N454K, non-specific RNase H activity), ArrayScript™ (engineered, non-specific RNase H activity), BioScript™ (engineered, reduced RNase H activity), CycleScript™ (engineered), EnzScript™ (engineered, RNase H Minus), EpiScript™ (Engineered, RNase H Minus), Expand™ Reverse Transcriptase (Engineered, RNase H Reduced), FIREScript (Engineered, RNase H Plus), GrandScript (Engineered, RNase H Plus), iScript™ (Engineered, RNase H Plus), Maxima™ RT (Engineered, RNase H Plus and Minus), MonsterScript™ (Engineered, RNase H Minus), PrimeScript™ (Engineered, RNase H Minus), PrimeScript™ II (Engineered, RNase H Minus), PrimeScript™ III (Engineered, RNase H Minus), PrimeScript™ IV (Engineered, RNase H Minus), ProtoScript® (Engineered, RNase H Plus), ProtoScript® II (Engineered, RNase H Reduced), qScript (Engineered, RNase H Plus), RevertAid™ (Engineered, RNase H plus and minus), ReverTra Ace® (engineered, RNase H minus), RevertUpII™ (engineered, RNase H minus), Rocketscript™ (engineered, RNase H plus and minus), Script (engineered, RNase H minus), SMART® (engineered), SMARTScribe™ (engineered, non-specific RNase H activity), SuperScript™ II (engineered, 524G / D583N / E562Q, RNase H reduced), SuperScript™ III (engineered, 204R / V223H / T306K / F309N / D524G / D583N / E562Q, RNase H reduced), SuperScript™ IV (engineered, RNase H reduced), or Transcriptor Reverse Transcriptase (engineered, RNase H plus)), HIV-1 reverse transcriptase (e.g., HIV-1 RT (wild type of group M subtype B, RNase H plus), Biotool High retrotranscriptase (engineered group O variant (K65R / V75I), RNase H plus), or Sunscript® (engineered group O variant with K358R / A359G / S360A alterations, RNase H plus and minus)), bacterial group II intron reverse transcriptase (e.g., Marathon RT (wild-type (Eubacterium rectale), lacking the RNase H domain) or TGIRT®-III RT (wild-type (Geobacillus stearothermophilus), lacking the RNase H domain), bacterial DNA polymerase (e.g., BcaBEST polymerase (engineered (Bacillus caldotenax DNA polymerase without 5'-3' and 3'-5' exonuclease activity), lacking the RNase H domain), Bst 3.0 DNA polymerase (G. stearothermophilus DNA polymerase I, large fragment, lacking 5'-3' and 3'-5' exonuclease activity, lacking RNase H domain), RapiDxFire™ reverse transcriptase (lacking RNase H domain), Volcano2G DNA polymerase (engineered Thermus aquaticusThese include Volcano3G DNA polymerase (an engineered T. aquaticus DNA polymerase lacking the RNase H domain), SOLIScript (engineered, RNase H reduced), Omniscript® (heterodimeric RT, RNase H plus), and SensiScript® (heterodimeric RT, RNase H plus).

[0231] In various embodiments, the reverse transcriptase is a retroviral reverse transcriptase. In various embodiments, the reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase (RT) (e.g., an engineered MLV RT). In various embodiments, the reverse transcriptase is a bacterial group II intron RT.

[0232] In various embodiments, the primed editing enzyme or system comprises a reverse transcriptase associated with a DNA-binding domain, such as a catalytically impaired nuclease domain, which in various embodiments is capable of localizing the reverse transcriptase to a target nucleic acid in which one or more nucleotides have been substituted, inserted, and / or deleted.

[0233] The DNA-binding domain of the prime editing enzyme can be an RNA-guided DNA-binding domain, in that the RNA guide can direct the DNA-binding domain to the target nucleic acid sequence. The catalytically impaired nuclease domain of the prime editing enzyme can bind to nucleic acid and localize the reverse transcriptase to the target nucleic acid where one or more nucleotides have been substituted, inserted, and / or deleted by the prime editing system.

[0234] Any nuclease in a CRISPR system can be engineered to create a catalytically impaired nuclease domain (e.g., a nickase) and used as a prime editing enzyme or in the system. Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12, including, e.g., spCas9, dCas9, nCas9, and Cas9-SpRY), Cas10, Cas12 (e.g., Cas12a (e.g., LbCas12a, AsCas12a, FnCas12a, MB3Ca), and Cas10. s12a, Cas12a-M11, Cas12a-M13 (e.g., Cas12a-M13-1), Cas12a-M26 (e.g., Cas12a-M26-1), Cas12a-M28 (e.g., Cas12a-M28-1), Cas12a-M29 (e.g., Cas12a-M29-1), Cas12a-M30 (e.g., Cas12a-M30-1), Cas12a-M31 (e.g., Cas12a-M31-1), C Cas12a-M32 (e.g., Cas12a-M32-1), Cas12a-M57, Cas12a-M58, Cas12a-M59, Cas12a-M60 (e.g., Cas12a-M60-9), Cas12a-M61, or Cas12a-M62), Cas12b, Cas12c, Cas12g, Cas12h, or Cas12i), Cas-Phi, CasX, CasY, C2c3, C2c2, C2c1, Cs These include y1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and their variants. Numerous forms and variants of Cas nucleases are known in the art (e.g., spCas9, dCas9, nCas9, Cas9-SpRY, and Cas12a) and can have different characteristics, including, for example, recognition of different PAMs and PAM positions.

[0235] Other DNA-binding nucleases can also be used as prime editing enzymes. For example, prime editing systems can utilize zinc finger nucleases (ZFNs) (see, for example, Urnov 2010 Nat Rev Genet.11(9):636-46) and transcription activator-like effector nucleases (TALENs) (see, for example, Joung 2013 Nat Rev Mol Cell Biol.14(1):49-55). For more information on DNA-binding nucleases, see, for example, US2018 / 0312825.

[0236] In various embodiments, the prime editing system comprises a prime editing gRNA (pegRNA), which specifies the target nucleic acid sequence and also specifies the sequence modification that the prime editing system introduces. The pegRNA comprises a sequence complementary to the target nucleic acid and recruits the prime editing enzyme to the target nucleic acid. The pegRNA comprises, from 5' to 3', (a) a fragment (sometimes referred to as a "spacer") that base pairs with a complementary target nucleic acid sequence (e.g., at least 80% identity, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity between the fragment and the complement of the target nucleic acid), wherein the fragment is 10-40 nucleotides in length (e.g., equal to or approximately equal to 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, or 40 nucleotides in length, e.g., 17-24 or 17-20 nucleotides in length). (b) a sequence that forms a stem-loop structure and binds to and / or recruits a catalytically impaired nuclease domain of a prime editing enzyme; (c) a fragment containing a sequence that contains one or more modifications (e.g., one or more substitutions, insertions, and / or deletions) relative to a target nucleic acid sequence (sometimes referred to as a "template sequence"), where the fragment is complementary to the same target nucleic acid strand (except for the modifications); and (d) a fragment containing a sequence complementary to a target sequence (sometimes referred to as a "binding region" or "primer binding site" (PBS)), e.g., where the target sequence is upstream of a suitable PAM site. In various embodiments, the PBS can be 5-20 nucleotides, e.g., 8-15 nucleotides in length. In various embodiments, the template sequence can be 10-20 nucleotides in length or longer. Because pegRNAs contain components characteristic of sgRNAs, they are sometimes described as extended sgRNAs. Any two fragments of a pegRNA can be associated independently, directly, or via a linker fragment.

[0237] The catalytically impaired nuclease domain of the prime editing enzyme can nick a target nucleic acid containing the appropriate PAM to expose a 3' flap and a 5' flap. After nicking the target nucleic acid, the released 3' flap hybridizes to the PEG-RNA PBS and primes reverse transcription of a template fragment of PEG-RNA containing the target sequence modification, allowing the modification to be directly introduced into the 3' flap of the target nucleic acid. The product of reverse transcription, the edited 3' flap, which is "redundant" to the 5' flap sequence produced by the nick (containing the original, unedited sequence of the target nucleic acid), can then compete with the original and redundant 5' flap sequences for reincorporation into the DNA duplex. While a perfectly complementary 5' flap is likely thermodynamically favored for hybridization to the unedited strand, the 5' flap is preferentially degraded by cellular endonucleases that are ubiquitous during lagging-strand DNA synthesis. After 5' flap resection and ligation of the edited strand, permanent installation of the edit occurs via DNA repair of the unedited strand, relying on the edited strand as a template. DNA repair of the unedited strand can be facilitated by contact with a secondary sgRNA, which directs nicking of the unedited strand. This additional nick stimulates resynthesis of the unedited strand using the edited strand as a template, resulting in a fully edited duplex. The primed editing system can introduce any of 12 types of point mutations (all possible nucleotide transitions and transversions), as well as one or more insertions and / or deletions.

[0238] In various embodiments, the prime editing system is engineered to disrupt a PAM site in the target nucleic acid. Disruption of the PAM site in the target nucleic acid can reduce the likelihood of repeated editing of a particular target nucleic acid. In various embodiments, disruption of the PAM site in the edited target nucleic acid can increase the efficiency of prime editing and / or gene therapy involving prime editing.

[0239] Exemplary prime editing systems include PE1, PE2, and PE3. Each of these prime editing enzymes contains a mutant Streptococcus pyogenes Cas9 nickase domain (H840A mutant) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (e.g., engineered to contain D200N / T306K / W313F / T330P / L603W). PE1 contains a pegRNA and a prime editing enzyme containing Cas9 H840A nickase and wild-type MLV RT. The Cas9 nickase acts only on the strand edited by the RT. PE2 contains a pegRNA and a prime editing enzyme containing Cas9 H840A nickase and an engineered MLV RT (D200N / T306K / W313F / T330P / L603W), which has been demonstrated to improve editing efficiency. PE3 contains the same prime editing enzyme as PE2 (as well as pegRNA), but additionally contains an sgRNA that targets the non-edited strand to nick 14–116 nucleotides away from the site of the pegRNA-induced nick (PE3), allowing the cellular mismatch repair pathway to repair the information introduced into the edited strand. Compared to PE2, the PE3b strategy has demonstrated improved editing efficiency and reduced levels of indel formation. A variant of the PE3 system, called PE3b, reduces the level of indel production by using a nicking sgRNA that targets only the edited sequence and prevents nicking of the non-edited DNA strand until the other strand has been converted to the edited sequence.

[0240] Those skilled in the art will understand that pegRNAs or other targeting elements for generating selected nucleic acid sequence modifications in target nucleic acids can be easily designed and implemented, for example, based on available sequence information. Various tools for designing pegRNAs are available. For example, pegFinder is a web-based tool for designing pegRNAs (see, for example, Chow 2020 Nat.Biomed.Eng.Doi:10.1038 / s41551-020-00622-8). Another example of a web-based tool for designing pegRNAs is PrimeDesign (see, for example, Hsu 2020 bioRxiv doi:10.1101 / 2020.05.04.077750).

[0241] The prime editing system does not require double-stranded DNA breaks. The prime editing system provides precise control of the site at which the editing system modifies the target nucleic acid. The prime editing system can be multiplexed to achieve editing of multiple targets using a single editing enzyme, optionally including therapeutic targets. The present disclosure includes that the prime editing system can include multiple pegRNAs (e.g., two or more, e.g., two, three, four, or five pegRNAs).

[0242] I(C)(i)(b)(4). Zinc finger nuclease The present disclosure includes zinc finger nucleases. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by associating sequence-targeting zinc finger DNA-binding units with a nuclease domain (e.g., a FokI nuclease domain) in a fusion protein. Each ZFN contains a nuclease domain (e.g., a FokI cleavage domain) linked to an array of three to six zinc finger (ZF) units. For example, a ZFN may contain several Cys2His2ZFs, each unit containing approximately 30 amino acids and specifically binding to approximately three nucleotides. The ZFs provide the ZFN with the ability to bind to a specific nucleic acid sequence. Because the FokI cleavage domain must dimerize to cleave DNA, monomers are not active and cleavage does not occur at a single binding site. Thus, for example, a ZFN containing three ZFs that bind together to a 9-bp target will function as a ZFN dimer that specifically binds 18 bp of DNA per cleavage site. In some embodiments, the ZFNs can comprise up to six ZFs per ZFN.

[0243] Cleavage of target nucleic acid by ZFN induces cellular repair processes that can mediate nucleic acid modification. ZFN-induced double-strand breaks can result in both targeted modification and targeted gene replacement. For example, when ZFN-induced breaks are resolved by non-homologous end joining, they can result in small deletions or insertions, which can lead to gene knockout. When ZFN-induced breaks are resolved by a homology-based process in the presence of provided donor nucleic acid, small changes (e.g., one or a few nucleotides) or more (e.g., up to the entire transgene) can be introduced into the target nucleic acid.

[0244] I(C)(i)(b)(5). TALENs for nucleic acid modification The present disclosure includes a transcription activator-like effector nuclease (TALEN) editing system. Various editing enzymes and systems can include a transcription activator-like (TAL) effector DNA binding domain and an endonuclease enzyme. An editing enzyme that includes a TAL effector DNA binding domain and an endonuclease can be referred to as a TALEN.

[0245] A TAL effector DNA-binding domain contains multiple monomers, each of which binds to one nucleotide in a target nucleic acid sequence. Each monomer contains 34 amino acids. In each monomer, positions 12 and 13 (referred to as a repeating variable dinucleotide, or RVD) are highly variable and contribute to the specific recognition of different nucleotides. The final monomer of a TAL effector DNA-binding domain, which binds to the nucleotide at the 3' end of the recognition site, can be only 20 amino acids long and is therefore sometimes referred to as a hemi-repeat. RVD sequences can be degenerate, since a particular RVD combination can bind, for example, to more than one nucleotide with distinct efficiencies. For example, RVDs include Asn and Ile (NI), Asn and Gly (NG), Asn and Asn (NN), and His and Asp (HD), which bind to A, T, G, and C nucleotides, respectively.

[0246] In various embodiments, the TAL effector DNA-binding domain is isolated from Xanthomonas spp. In various embodiments, the TALEN comprises an endonuclease domain (e.g., a FokI domain), e.g., C-terminal to the TAL effector DNA-binding domain.

[0247] TALENs function as pairs, with two members possessing target binding sites on opposite DNA strands of the target nucleic acid sequence, separated by a small fragment (e.g., 12–25 bp) that may be referred to as a spacer sequence. When a pair of TALENs binds to their target sites, the endonuclease (e.g., FokI) domains dimerize, creating a double-stranded break in the spacer sequence. Non-homologous end joining (NHEJ) to resolve DSBs involves direct ligation of DNA from either side of the double-stranded break, with little or no sequence overlap for annealing. This repair mechanism can cause indels (insertions or deletions) that can disrupt genes at the target nucleic acid sequence, or chromosomal rearrangements. Alternatively, DNA can be introduced into the genome through NHEJ in the presence of an exogenous double-stranded DNA fragment. Homologous recombination repair can also introduce foreign DNA into DSBs, as the transfected double-stranded sequence is used as a template for repair enzymes.

[0248] I(C)(i)(c). Small RNA payload expression product Small RNAs are short, non-coding RNA molecules that play a role in regulating gene expression. In certain embodiments, small RNAs are less than 200 nucleotides in length. In certain embodiments, small RNAs are less than 100 nucleotides in length. In certain embodiments, small RNAs are less than 50, 45, 40, 35, 30, 25, or 20 nucleotides in length. In certain embodiments, small RNAs are less than 20 nucleotides in length. In various embodiments, small RNAs have a length with a lower limit of 5, 10, 15, 20, 25, or 30 nucleotides and an upper limit of 20, 25, 30, 35, 40, 45, 50, 75, or 100 nucleotides. Small RNAs include, but are not limited to, microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), small nucleolar RNAs (snoRNAs), small tRNA-derived RNAs (tsRNAs), small rDNA-derived RNAs (srRNAs), and small nuclear RNAs. Additional classes of small RNAs continue to be discovered.

[0249] In certain embodiments, an interfering RNA molecule that is homologous to a target mRNA or that can hybridize with an interfering RNA can cause the degradation of the target mRNA molecule or a decrease in the translation of the target mRNA, a process known as RNA interference (RNAi) (Carthew, Curr. Opin. Cell. Biol. 13:244-248, 2001). RNAi occurs naturally in cells to remove foreign RNA (e.g., viral RNA). In some cases, natural RNAi proceeds via fragments cleaved from free double-stranded RNA (dsRNA), which directs the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be manufactured, for example, to silence the expression of a target gene. Exemplary RNAi molecules include small hairpin RNAs (shRNAs, also known as short hairpin RNAs) and small interfering RNAs (siRNAs).

[0250] Without limiting the disclosure and without being bound by theory, in nature and / or in some embodiments, RNA interference is typically a two-step process. In the first step, the initiation step, input dsRNA is digested into 21-23 nucleotide (nt) siRNAs, presumably by the action of Dicer, a member of the ribonuclease (RNase III) family of dsRNA-specific ribonucleases, which processes (cleaves) the dsRNA (directly or introduced via a transgene or virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA into 19-21 base pair (bp) duplexes (siRNAs), each with a 2-nucleotide 3' overhang.

[0251] In the second step, the effector step, the siRNA duplex binds to a nuclease complex to form an RNA-induced silencing complex (RISC). ATP-dependent unwinding of the siRNA duplex is required for RISC activation. The active RISC then targets homologous transcripts through base-pairing interactions and typically cleaves the mRNA into 12-nucleotide fragments from the 3' end of the siRNA. Studies have shown that each RISC contains a single siRNA and an RNase.

[0252] The remarkable potency of RNAi suggests an amplification step within the RNAi pathway. Amplification can occur through copying of the input dsRNA, which generates more siRNAs, or through replication of the siRNAs formed. Alternatively or additionally, amplification can be caused by multiple turnover events of RISC.

[0253] An shRNA is a single-stranded polynucleotide with a hairpin loop structure. The single-stranded polynucleotide has a loop segment connecting the 3' end of one strand of the double-stranded region and the 5' end of the other strand of the double-stranded region. The double-stranded region is formed from a first sequence capable of hybridizing to a target sequence, such as a polynucleotide encoding a transgene, and a second sequence complementary to the first sequence; thus, the first and second sequences form a double-stranded region whose ends are connected by a linking sequence to form a hairpin loop structure. The first sequence can be hybridizable to any part of a polynucleotide encoding a transgene. The double-stranded stem domain of the shRNA can contain a restriction endonuclease site.

[0254] Transcription of shRNAs is thought to initiate at the polymerase III (Pol III) promoter and terminate at position 2 of the 4-5 thymine transcription termination site. Upon expression, shRNAs are thought to fold into stem-loop structures with 3' UU overhangs, and the ends of these shRNAs are subsequently processed, converting the shRNAs into 21-23 nucleotide siRNA-like molecules.

[0255] The stem-loop structure of the shRNA can have an optional nucleotide overhang, e.g., a 2-bp overhang, e.g., a 3'UU overhang. Although variations may occur, the stem typically ranges from 15 to 49, 15 to 35, 19 to 35, 21 to 31, or 21 to 29 bp, and the loop can range from 4 to 30 bp, e.g., 4 to 23 bp. In certain embodiments, the shRNA sequence comprises 45 to 65 bp, 50 to 60 bp, or 51, 52, 53, 54, 55, 56, 57, 58, or 59 bp. In certain embodiments, the shRNA sequence comprises 52 or 55 bp. In certain embodiments, the siRNA has 15 to 25 bp. In certain embodiments, the siRNA has 16, 17, 18, 19, 20, 21, 22, 23, or 24 bp. In certain embodiments, the siRNA has 19 bp. However, those skilled in the art will understand that siRNA having a length of less than 16 nucleotides or more than 24 nucleotides can also function to mediate RNAi.Longer RNAi agents have been demonstrated to induce interferon or protein kinase R (PKR) response in certain mammalian cells, which may be undesirable.Preferably, RNAi agents do not induce PKR response (i.e., are sufficiently short in length).However, longer RNAi agents can be useful, for example, in situations where PKR response is downregulated or suppressed by alternative means.

[0256] In certain exemplary embodiments, the present disclosure includes an adenoviral vector payload encoding an shRNA that targets the gene encoding BCL11A, where the shRNA causes a decrease in the translation of BCL11A.

[0257] I(C)(ii). Payload regulatory sequence Promoters can include general promoters, tissue-specific promoters, cell-specific promoters, and / or cytoplasm-specific promoters. Promoters can include strong promoters, weak promoters, constitutive expression promoters, and / or inducible (conditional) promoters. Inducible promoters direct or control expression in response to specific conditions, signals, or cellular events. For example, a promoter can be an inducible promoter that requires a specific ligand, small molecule, transcription factor, hormone, or hormone protein to effect transcription from the promoter.

[0258] In various embodiments, the promoter sequence may be a native promoter sequence. A native promoter sequence, or a minimal promoter sequence, may refer to a sequence derived from a single contiguous sequence located 5' of a coding sequence in a reference genome. A native promoter sequence may include a core promoter and an associated 5'UTR. In certain embodiments, the 5'UTR may include an intron. In various embodiments, the promoter sequence may be a composite promoter sequence. In various embodiments, a composite promoter sequence may refer to a promoter sequence that includes portions derived from at least two different sources, e.g., two non-contiguous portions of a reference genome, two different genomes, or any two different source sequences. For example, in certain embodiments, a composite promoter sequence includes a sequence derived from a single contiguous sequence located 5' of a coding sequence in a reference genome and a sequence derived from another portion of the reference genome, e.g., an enhancer (e.g., a distal enhancer).

[0259] In certain embodiments, the promoter may be a sequence having one or more changes (e.g., one or more insertions, point mutations, or deletions) relative to a wild-type promoter sequence or a reference promoter. In certain embodiments, the promoter sequence differs from the wild-type or other reference promoter sequence by having one change per 20 nucleotide stretch, two changes per 20 nucleotide stretch, three changes per 20 nucleotide stretch, four changes per 20 nucleotide stretch, or five changes per 20 nucleotide stretch. In certain embodiments, the promoter sequence may differ from the wild-type or reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences. The promoter can have a length of, for example, 50 to 3,000 or more nucleotides, such as 100 to 1,000, 100 to 2,000, 100 to 3,000, 500 to 1,000, 500 to 2,000, 500 to 3,000, 1,000 to 2,000, or 1,000 to 3,000 nucleotides.

[0260] In various embodiments, the promoter is non-specific in that it causes the expression of the operably linked coding sequence in various types of cells or tissues.In various embodiments, the promoter is a ubiquitous promoter.In various embodiments, the ubiquitous promoter can be selected from, for example, a CMV promoter, an RSV promoter, or an SV40 promoter.

[0261] The coding sequences of the present disclosure can additionally be associated with sequences that increase the stability of the mRNA transcript, such as insulators and / or polyA tails.

[0262] I(C)(iii).Selection sequence In certain embodiments, the vector comprises a selection element that includes a selection cassette, which in certain embodiments comprises a promoter, a cDNA that confers or confers resistance to a selection agent, and a polyA sequence that allows for transcription of this independent transcription element to be turned off.

[0263] The selection cassette can encode one or more proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply important nutrients unavailable from complex media, e.g., a gene encoding Bacilli D-alanine racemase. Any number of selection systems may be used to recover transformed cell lines. In certain embodiments, the positive selection cassette comprises a resistance gene to neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blastidine, or viomycin. In certain embodiments, the positive selection cassette comprises a DHFR (dihydrofolate reductase) gene, which provides resistance to methotrexate, or a DHFR (dihydrofolate reductase) gene, which provides resistance to methotrexate. 6 MGMT is involved in resistance to BG / BCNU P140K The HAT selective medium contains the HPRT (hypoxanthine phosphoribosyltransferase) gene, which is involved in the transformation of specific bases (aminopterin, hypoxanthine, thymidine) present in the HAT selective medium, and other genes for detoxification of several drugs. In a specific embodiment, the selective agents include neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, viomycin, ampicillin, and 0. 6 Examples include BG / BCNU, methotrexate, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gln synthetase, or ADA.

[0264] In certain embodiments, the negative selection cassette contains a gene encoding an expression product that converts a substrate present (e.g., delivered) in a subject or system (e.g., medium) into a toxic substance, thereby sensitizing cells expressing the gene. In various embodiments, for example, the payload is engineered so that proper integration into the target genome disrupts expression of the negative selection gene. The negative selection cassette can include a gene encoding diphtheria toxin A fragment (DTA) (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221, 1999) or the thymidine kinase gene of herpesvirus (HSV TK) sensitive to the presence of ganciclovir or FIAU. In various embodiments, the negative selection cassette includes the HPRT gene for negative selection in the presence of 6-thioguanine (6TG).

[0265] In certain embodiments, the selection cassette comprises the MGMT gene as described in Olszko et al. (Gene Therapy 22:591-595, 2015). P140K In a specific embodiment, the selective agent is O 6 Includes BG / BCNU.

[0266] The MGMT gene encodes human alkylguanine transferase (hAGT), a DNA repair protein that confers resistance to the cytotoxic effects of alkylating agents such as nitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is an inhibitor of AGT that enhances the toxicity of nitrosoureas and is coadministered with TMZ to enhance the cytotoxic effects of this drug. Some mutant forms of MGMT, encoding variants of AGT, are highly resistant to inactivation by 6-BG but retain the ability to repair DNA damage (Maze et al. J. Pharmacol. Exp. Ther. 290:1467-1474, 1999). MGMT P140KDrug resistance gene therapy based on ribonucleotides has been shown to confer chemoprotection to mouse, dog, rhesus monkey, and human cells, particularly hematopoietic cells (Zielske et al. J. Clin. Invest. 112:1561-1570, 2003; Pollok et al., Hum. Gene Ther. 14:1703-1714, 2003; Gerull et al. Hum. Gene Ther. 18:451-456, 2007; Neff et al., Blood 105:997-1002, 2005; Larochelle et al. J. Clin. Invest. 119:1952-1963, 2009; Sawai et al., Mol. Ther. 3:78-87, 2001).

[0267] In certain embodiments, the combination with an in vivo selection cassette is a critical component of the disease without a selective advantage of the gene-corrected cells. For example, in SCID and some other immune deficiencies and FA, the corrected cells have an advantage, and transduction of a "small number" of HSPCs with a therapeutic gene is sufficient for therapeutic efficacy. For other diseases, such as hemoglobinopathies (i.e., sickle cell disease and thalassemia), where the therapeutically modified cells do not offer a competitive advantage, in vivo selection of the modified cells, e.g., MGMT P140K For expression of an in vivo selection cassette, such as , a small number of transduced HSPCs are selected, allowing for the expansion of gene-corrected cells to achieve therapeutic efficacy. This approach can also be applied to HIV by rendering HSPCs resistant to HIV in vivo rather than by ex vivo genetic modification.

[0268] I(C)(iv).Stuffer sequence In certain embodiments, the vector comprises a stuffer sequence. In certain embodiments, a stuffer sequence may be added to bring the genome to a size close to the wild-type length. Stuffer is a term generally recognized in the art that is intended to define a functionally inactive sequence intended to extend the length of the genome.

[0269] Stuffer sequences are used to achieve efficient packaging and vector stability, and in certain embodiments, stuffer sequences are used to reduce the genome size to 70% to 110% of that of the wild-type virus.

[0270] The stuffer sequence can be any DNA, preferably DNA of mammalian origin. In a preferred embodiment of the invention, the stuffer sequence is a non-coding sequence of mammalian origin, such as an intron fragment.

[0271] Stuffer sequences, when used to maintain the size of the vector, can be any non-coding sequence or sequences that allow the genome to remain stable in dividing or non-dividing cells. These sequences can be derived from other viral genomes (e.g., Epstein-Barr virus) or organisms (e.g., yeast). For example, these sequences can be functional portions of centromeres and / or telomeres.

[0272] I(C)(v). Payload integration and support vectors Gene therapy often requires the integration of a desired nucleic acid payload into the genome of a target cell. A variety of systems can be designed and / or used to integrate the payload into the host or target cell genome. Various such systems include certain payload sequence features and one or more of a support vector and a support genome (support genome).

[0273] One way to engineer adenoviral vectors to integrate payloads into the host cell genome is to create integrating viral hybrid vectors. Integrating viral hybrid vectors combine genetic elements of vectors that efficiently transduce target cells with genetic elements of vectors that stably integrate the vector payload. For example, integration elements of interest for use in combination with adenoviral vectors include bacteriophage integrase PHiC31, retrotransposons, retroviruses (e.g., LTR-mediated or retroviral integration-mediated), zinc finger nucleases, DNA-binding domain-retroviral integrase fusion proteins, AAV (e.g., AAV-ITR or AAV-Rep protein-mediated), and Sleeping Beauty (SB) transposase.

[0274] The Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 vectors described herein can optionally contain transposable elements, including a transposase and a transposon. The transposase can include an integrase derived from a retrotransposon or of retroviral origin, as well as an enzyme capable of transposition and a component of a functional nucleic acid-protein complex mediating transposition. The transposition reaction involves a transposon and a transposase or integrase enzyme. In certain embodiments, the efficiency of integration, the size of the DNA sequence that can be integrated, and the number of copies of the DNA sequence that can be integrated into the genome can be improved by using such transposable elements. A transposon comprises a short nucleic acid sequence with terminal repeats upstream and downstream of a larger segment of DNA. The transposase binds to the terminal repeats and catalyzes the movement of the transposon to another part of the genome.

[0275] Many transposases have been described in the art that facilitate the insertion of nucleic acids into the genomes of vertebrates, including humans. Examples of such transposases include Sleeping Beauty ("SB," e.g., from the genome of salmonid fish), PiggyBac (e.g., from lepidopterans and / or Myotis lucifugus), Mariner (e.g., from Drosophila), Frog Prince (e.g., from Rana pipiens), Tol1, Tol2 (e.g., from medaka fish), TcBuster (e.g., from the red flour beetle Tribolium castaneum), Helraiser, Himar1, Passport, Minos, Ac / Ds, PIF, Harbinger, Harbinger3-DR, HSmar1, and spinON.

[0276] PiggyBac (PB) transposase is a compact, functional transposase protein described, for example, in Fraser et al., Insect Mol. Biol., 1996, 5, 141-51; Mitra et al., EMBO J., 2008, 27, 1097-1109; Ding et al., Cell, 2005, 122, 473-83; and U.S. Patent Nos. 6,218,185, 6,551,825, 6,962,810, 7,105,343, and 7,932,088. Hyperactive piggyBac transposase is described in U.S. Patent No. 10,131,885.

[0277] Further information regarding DNA transposons can be found, for example, in Muñoz-Lopez & Garcia Perez, Curr Genomics, 11(2):115-128, 2010.

[0278] Sleeping Beauty is described in Ivics et al. Cell 91,501-510,1997, Izsvak et al., J. Mol. Biol.,302(1):93-102,2000, Geurts et al., Molecular Therapy,8(1):108-117,2003, Mates et al. Nature Genetics 41:753-761,2009, and U.S. Patent Nos. 6,489,458, 7,148,203, and 7,160,682, U.S. Publication Nos. 2011 / 117072, 2004 / 077572, and 2006 / 252140. In certain embodiments, the Sleeping Beauty transposase enzyme is a hyperactive Sleeping Beauty SB100x transposase enzyme. SB transposons are most efficiently transposed when present in circularized nucleic acid molecules (Yant et al., Nature Biotechnology, 20:999-1005, 2002).

[0279] Systematic mutagenesis studies have been conducted to increase the activity of SB transposase. For example, Yant et al. systematically replaced the 95 N-terminal AA residues of SB transposase with alanine (Mol. Cell Biol. 24:9239-9247, 2004). Ten of these substitutions resulted in a 200-400% increase in activity compared to the reference SB10. SB16, described by Baus et al. (Mol. Therapy 12:1148-1156, 2005), was reported to have a 16-fold increase in activity compared to SB10. Additional hyperactive SB variants have been described by Zayed et al. (Molecular Therapy 9(2):292-304, 2004) and in US Pat. No. 9,840,696.

[0280] SB transposase transposes the nucleic acid transposon payload located between SB ITR.Various SB ITRs are known in the art.In some embodiments, SB ITR is a 230bp sequence that includes a 32bp long imperfect direct repeat, which serves as a transposase recognition signal.

[0281] In various embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector or genome comprises a payload comprising an SB100x transposon inverted repeat flanked by integrational elements comprising at least one coding sequence encoding a β-globin expression product or a γ-globin expression product.

[0282] In various embodiments, the adenoviral transposition system includes an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector or genome containing integration elements flanked by transposon inverted repeats, and can further include an adenoviral support vector or genome. In various embodiments, the support vector includes (i) an adenoviral capsid and (ii) an adenoviral support genome containing a nucleic acid sequence encoding a transposase corresponding to the inverted repeats flanking the integration elements. Thus, in various embodiments, at least one function of the support vector or genome can be to encode, express, and / or deliver to a target cell a transposase for transposition of an integration element present in a donor vector administered to the target cell. For example, in some embodiments, the Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 donor vector or genome comprises an SB100x transposon inverted repeat flanked by integration elements comprising at least one coding sequence encoding a β-globin expression product or a γ-globin expression product, and the support vector or genome comprises a coding sequence encoding an SB100x transposase. In certain embodiments, the integration elements are flanked by recombinase direct repeats, e.g., the integration elements are flanked by transposon inverted repeats, and the transposon inverted repeats are flanked by recombinase direct repeats. In certain such embodiments, at least one function of the support vector or genome may be to encode, express, and / or deliver to a target cell a recombinase for recombination of recombinase sites present in the donor vector administered to the target cell. In various embodiments, the support vector or genome can encode, express, and / or deliver to the target cell a recombinase for recombination of recombinase sites present in a donor vector administered to the target cell, and can encode, express, and / or deliver to the target cell a transposase for transposition of integration elements present in a donor vector administered to the target cell.

[0283] Certain embodiments disclosed herein also use a site-specific recombinase system. In these embodiments, in addition to at least one therapeutic gene, the transposon containing a transposase-recognized inverted repeat also contains at least one recombinase-recognized site. Thus, in certain embodiments, the present disclosure also provides a method for integrating a therapeutic gene into a genome, comprising administering (a) a transposon containing a therapeutic gene, wherein the therapeutic gene is flanked by (i) an inverted repeat sequence recognized by the transposase and (ii) a recombinase-recognized site, and b) a transposase and a recombinase that serve to excise the therapeutic gene from a plasmid, episome, or transgene and integrate the therapeutic gene into the genome. In some embodiments, the protein(s) of (b) are administered as nucleic acids encoding the protein(s). In some embodiments, the transposon and the nucleic acids encoding the protein(s) of (b) are present on separate vectors. In some embodiments, the transposon and the nucleic acid encoding the protein(s) of (b) are present on the same vector. When present on the same vector, the portion of the vector encoding the protein(s) of (b) is located outside the portion carrying the transposon of (a). In other words, the transposase and / or recombinase coding region is located outside the region flanked by the inverted repeats and / or recombinase recognition sites. In the foregoing methods, the transposase protein recognizes the inverted repeats flanking the inserted nucleic acid, such as the nucleic acid to be inserted into the target cell genome. The use of a recombinase and recombinase recognition sites can further increase the size of the transposon that can be integrated into the genome.

[0284] Examples of recombinase systems include the Flp / Frt system, the Cre / loxP system, the Dre / rox system, the Vika / vox system, and the PhiC31 system. The Flp / Frt DNA recombinase system was isolated from Saccharomyces cerevisiae. The Flp / Frt system contains the recombinase Flp (flippase), which catalyzes DNA recombination at its Frt recognition site. Variants of the Flp protein include those with GenBank accession numbers ABD57356.1 and ANW61888.1.

[0285] The Cre / loxP system is described, for example, in EP02200009B1. Cre is a site-specific DNA recombinase isolated from bacteriophage P1. The recognition site for the Cre protein is a 34-base pair nucleotide sequence, the loxP site. Cre binds to 13-base pair inverted repeats and recombines the 34-bp loxP DNA sequence by catalyzing strand cleavage and religation within the spacer region. Staggered DNA cleavages made by Cre within the spacer region are separated by 6 base pairs, creating overlapping regions that act as homology sensors to ensure that only recombination sites with the same overlapping region are recombined. Variants of lox recognition sites that can be used include lox2272, lox511, lox66, lox71, loxM2, and lox5171. The VCr / VloxP recombinase system was isolated from the Vibrio plasmid p0908. The sCre / SloxP system is described in WO2010 / 143606. The Dre / rox system is described in US Pat. No. 7,422,889 and US Pat. No. 7,915,037B2. It generally comprises the DRE recombinase isolated from the Enterobacteriaceae phage D6 and a rox recognition site. The Vika / vox system is described in US Pat. No. 10,253,332. Furthermore, the PhiC31 recombinase recognizes the AttB / AttP binding site.

[0286] The amount of vector nucleic acid containing a transposon (including inverted repeats and / or recombinase recognition sites), and in various embodiments, the amount of vector nucleic acid encoding a transposase and / or recombinase, introduced into a cell is sufficient to provide the desired excision and insertion of the transposon nucleic acid into the target cell genome. Thus, the amount of vector nucleic acid introduced should result in a sufficient amount of transposase and / or recombinase activity and a sufficient copy number of the transposon desired to be inserted into the target cell genome. Particular embodiments include a transposon to transposase / recombinase ratio of 1:1, 1:2, or 1:3.

[0287] The subject methods result in stable integration of the nucleic acid into the genome of the target cell, meaning that the nucleic acid remains present in the genome of the target cell for more than a transient period of time and passes that portion of chromosomal genetic material to the progeny of the target cell.

[0288] As shown above, certain embodiments utilize homology arms to facilitate the targeted insertion of gene constructs using homologous recombination repair.Homology arms can have sufficient homology to the genome sequence at the cleavage site, for example, 70%, 80%, 85%, 90%, 95% or 100% homology with the nucleotide sequence adjacent to the cleavage site, for example, within 50 bases of the cleavage site or less, for example, within 30 bases, within 15 bases, within 10 bases, within 5 bases, or any length immediately adjacent to the cleavage site, to support HDR between HDR and the genome sequence with homology.Homology arms are generally identical to genome sequences, for example, the genome region where double-strand breaks (DSBs) occur.However, as shown, absolute identity is not required.

[0289] Certain embodiments can utilize homology arms having 25, 50, 100, or 200 nucleotides (nt), or greater than 200 nt of sequence homology (or any integer value between 10 and 200 nt, or greater) between the homology-directed repair template and the target genome sequence. In certain embodiments, the homology arms are 40 to 1000 nt in length. In certain embodiments, the homology arms are 500 to 2500 base pairs, 700 to 2000 base pairs, or 800 to 1800 base pairs. In certain embodiments, the homology arms comprise at least 800 base pairs or at least 850 base pairs. The length of the homology arms can be symmetric or asymmetric.

[0290] Certain embodiments may utilize first and / or second homology arms that each comprise at least 25, 50, 100, 200, 400, 600, 800, 1,000, 1,200, 1,400, 1,600, 1,800, 2,000, 2,500, or 3,000 or more nucleotides of sequence identity or homology to a corresponding fragment of the target genome. In some embodiments, the first and / or second homology arms each comprise a number of nucleotides having sequence identity or homology to a corresponding fragment of the target genome having a lower limit of 25, 50, 100, 200, 400, 600, 800, 1,000, 1,200, 1,400, 1,600, or 1,800 nucleotides and an upper limit of 1,000, 1,200, 1,400, 1,600, 1,800, 2,000, 2,500, or 3,000 nucleotides. In some embodiments, the first and / or second homology arms each comprise a number of nucleotides having sequence identity or homology to a corresponding fragment of the target genome that is between 40 and 1,000 nucleotides, between 500 and 2,500 nucleotides, between 700 and 2,000 nucleotides, or between 800 and 1,800 nucleotides, or that has a length of at least 800 nucleotides or at least 850 nucleotides. The first and second homology arms can have the same, similar, or different lengths.

[0291] For additional information regarding homology arms, see Richardson et al. Nat Biotechnol. 34(3):339-44, 2016.

[0292] In certain embodiments, a genetic construct (e.g., a gene leading to the expression of a therapeutic product in a cell) is precisely inserted within a genomic safe harbor. A genomic safe harbor site is an intragenic or extragenic region of the genome that can accommodate predictable expression of the newly integrated DNA without adversely affecting the host cell. A useful safe harbor must allow sufficient transgene expression to result in the desired level of the encoded protein. A genomic safe harbor site must also not alter cellular function. Methods for identifying genomic safe harbor sites are described in Sadelain et al., Nature Reviews 12:51-58, 2012 and Papapetrou et al., Nat Biotechnol. 29(1):73-8, 2011. In certain embodiments, a genomic safe harbor site meets one or more (one, two, three, four, or five) of the following criteria: (i) at least 50 kb away from the 5' end of any gene; (ii) at least 300 kb away from any cancer-associated gene; (iii) within an open / accessible chromatin structure (as measured by DNA cleavage with natural or engineered nucleases); (iv) location outside of gene transcription units; and (v) location outside of genomic ultraconserved regions (UCRs), microRNAs, or long non-coding RNAs.

[0293] In certain embodiments, to meet the criteria for genomic safe harbor, a chromatin site must be >150 kb away from a known cancer gene, >30 kb away from a known transcription start site, and not overlap with any coding mRNA. In certain embodiments, to meet the criteria for genomic safe harbor, a chromatin site must be >200 kb away from a known cancer gene, >40 kb away from a known transcription start site, and not overlap with any coding mRNA. In certain embodiments, to meet the criteria for genomic safe harbor, a chromatin site must be >300 kb away from a known cancer gene, >50 kb away from a known transcription start site, and not overlap with any coding mRNA. In certain embodiments, the genomic safe harbor meets the aforementioned criteria (>150 kb, >200 kb, or >300 kb away from a known transcription start site and does not overlap with any coding mRNA; >40 kb or >50 kb away from a known transcription start site and does not overlap with any coding mRNA) and additionally is 100% homologous between the relevant animal model animal and human genomes to enable rapid clinical application of relevant findings.

[0294] In certain embodiments, a genomic safe harbor meets the criteria described herein and further demonstrates that the locus does not affect the surrounding genetic material and demonstrates a 1:1 ratio of forward:reverse lentiviral integration.

[0295] Specific genomic safe harbor sites include CCR5, HPRT, AAVS1, Rosa, and albumin. For additional information and options regarding suitable genomic safe harbor integration sites, see also, e.g., U.S. Patent Nos. 7,951,925 and 8,110,379; U.S. Publication Nos. 2008 / 0159996, 2010 / 00218264, 2012 / 0017290, 2011 / 0265198, 2013 / 0137104, 2013 / 0122591, 2013 / 0177983, and 2013 / 0177960.

[0296] Various techniques known in the art can be used to direct the integration of integration elements at specific genomic loci, such as genomic safe harbors.For example, AAV-mediated gene targeting, and homologous recombination enhanced by the introduction of DNA double-strand breaks using site-specific endonucleases (zinc finger nucleases, meganucleases, transcription activator-like effector (TALE) nucleases), and CRISPR / Cas systems are all tools that can mediate the targeted insertion of foreign DNA at predetermined genomic loci, such as genomic safe harbors.

[0297] In certain embodiments, integration of an integration element at a specific genomic locus, such as a genomic safe harbor, can involve homology-directed integration using CRISPR enzyme-mediated cleavage of the target genome. The CRISPR enzyme (e.g., Cas9) cleaves double-stranded DNA at a site designated by a guide RNA (gRNA). The double-strand break can be repaired by homology-directed repair (HDR) in the presence of a donor template (such as an Ad3, 5, 7, 11, 14, 16, 21, 34, 35, 37, or 50 payload integration element containing left and right homology arms). In various such methods, the integration element is a "repair template" in that it contains left and right homology arms (e.g., 500-3,000 bp) for insertion into the cleaved target genome. CRISPR-mediated gene insertion can be several orders of magnitude more efficient than spontaneous recombination of DNA templates, demonstrating that CRISPR-mediated gene insertion can be an effective tool for genome editing. Exemplary methods for homology-directed integration of nucleic acid sequences into specific genomic loci are known in the art, e.g., Richardson et al. (Nat Biotechnol. 34(3):339-44, 2016).

[0298] II. Target Cell Population In various embodiments, the donor vectors and genomes of the present disclosure can selectively target (e.g., selectively enter and / or selectively transduce) one or more hematopoietic cell types disclosed herein. Selective targeting includes, but is not limited to, preferential targeting (e.g., binding, entry, transduction, and / or modification) of one or more cell types compared to one or more reference cell types. In various embodiments, the one or more preferentially targeted cell types are or comprise one or more of the hematopoietic cell types disclosed herein. In various embodiments, the one or more reference cell types are or comprise one or more of the hematopoietic cell types disclosed herein. In various embodiments, none of the reference cell types are the same as any of the preferentially targeted cell types. Thus, reference to a vector that selectively targets a hematopoietic cell type means or implies, but does not necessarily mean or imply, that the vector does not also target (e.g., selectively targets) one or more other hematopoietic cell types. In various embodiments, preferential targeting specifically refers to a comparison of one single hematopoietic cell type with a reference group comprising two or more hematopoietic cell types. In various embodiments, preferential targeting specifically refers to a comparison of a group comprising two or more hematopoietic cell types with a single reference hematopoietic cell type. In various embodiments, preferential targeting specifically refers to a comparison of a group comprising two or more hematopoietic cell types with a reference group comprising two or more hematopoietic cell types. In various embodiments, the hematopoietic cell type is a stem cell type, a progenitor cell type, or a further differentiated cell type (e.g., a terminally differentiated cell type). In various embodiments, the group of hematopoietic cell types can be stem cells, progenitor cells, or cells of a particular lineage, for example, a lineage identified by the least differentiated member of the identified cell group and including one or more or all more differentiated hematopoietic cells derived therefrom. Selective targeting includes, but does not require, preferential targeting of the preferentially targeted hematopoietic cell type(s) compared to all other hematopoietic cell types.In various embodiments, selective targeting comprises infection and / or transduction of at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells in a cell population of a preferentially targeted hematopoietic cell type.

[0299] Hematopoietic cell types (e.g., target hematopoietic cell types) of the present disclosure include hematopoietic cells of all lineages and stages of hematopoietic cell differentiation. Target cell types of the present disclosure include HSCs (e.g., CD34 + Long-term (LT)-HSC and / or CD34 + Hematopoietic cell types (e.g., target hematopoietic cell types) of the present disclosure include, but are not limited to, CD34, CD44+ ... + Contains hematopoietic cells.

[0300] HSCs can be targeted for in vivo gene repair by binding to CD46. HSCs or subsets thereof can also be distinguished by any of the following marker profiles: CD34+; Lin- / CD34+ / CD38- / CD45RA- / CD90+ / CD49f+ (HSC1); CD34+ / CD38- / CD45RA- / CD90- / CD49f+ / (HSC2). In various embodiments, human HSC1 can be distinguished by any of the following profiles: CD34+ / CD38- / CD45RA- / CD90+ or ​​CD34+ / CD45RA- / CD90+, and mouse LT-HSCs are Lin-Sca1+ckit+CD150+CD48-Flt3-CD34- (Lin represents the lack of expression of any markers of mature cells, including CD3, CD4, CD8, CD11b, CD11c, NK1.1, Gr1, and TER119). In certain embodiments, HSCs are identified by a CD164+ profile. In certain embodiments, HSCs are identified by a CD34+ / CD164+ profile. For more information regarding HSC marker profiles, see WO2017 / 218948.

[0301] The hematopoietic cells can advantageously encode and / or express a variety of payloads provided herein, including, but not limited to, TCRs and CARs (see, e.g., Gschweng et al. Immunol Rev. 2014 Jan;257(1):237-249).

[0302] Hematopoietic cell types that can be targeted by the vectors of the present disclosure include T cells. Several different subsets of T cells have been discovered, each with different functions. For example, the majority of T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains produced by independent T cell receptor alpha and beta (TCRα and TCRβ) genes, referred to as the α and β-TCR chains.

[0303] γδ T cells represent a small subset of T cells that have a different T cell receptor (TCR) on their surface. In γδ T cells, the TCR is composed of one γ chain and one δ chain. This T cell population is much rarer than αβ T cells (2% of all T cells).

[0304] CD3 is expressed on all mature T cells. Activated T cells express 4-1BB (CD137), CD69, and CD25. CD5 and transferrin receptors are also expressed on T cells.

[0305] T cells can be further classified into helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. T helper cells assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when peptide antigens are presented to them by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines, which regulate or support active immune responses.

[0306] Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize targets by binding to antigens associated with MHC class I, which is present on the surface of almost every cell in the body.

[0307] In certain embodiments, the CAR is genetically modified to be expressed in cytotoxic T cells.

[0308] "Central memory" T cells (or "T cells"), as used herein, refer to antigen-experienced CTLs that express CD62L or CCR7 and CD45RO on their surface, and do not express or have reduced expression of CD45RA compared to naive cells. In certain embodiments, central memory cells are positive for CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have reduced expression of CD45RA compared to naive cells.

[0309] "Effector memory" T cells (or "TEMs"), as used herein, refer to antigen-experienced T cells that do not express, or have reduced expression of, CD62L on their surface compared to central memory cells and do not express, or have reduced expression of CD45RA compared to naive cells. In certain embodiments, effector memory cells are negative for CD62L and CCR7 and have variable expression of CD28 and CD45RA compared to naive or central memory cells. Effector T cells are positive for granzyme B and perforin compared to memory or naive T cells.

[0310] "Naive" T cells, as used herein, refer to antigen-naive T cells that express CD62L and CD45RA, but do not express CD45RO compared to central or effector memory cells. In certain embodiments, naive CD8+ T lymphocytes are characterized by expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD127, and CD45RA.

[0311] Hematopoietic cell types that can be targeted by the vectors of the present disclosure include B cells. B cells are mediators of humoral responses and are responsible for the production and release of antigen-specific antibodies. There are several types of B cells that can be characterized by key markers. Generally, immature B cells express CD19, CD20, CD34, CD38, and CD45R, and as they mature, the key markers expressed are CD19 and IgM.

[0312] For the avoidance of doubt, in various embodiments, the vectors and genomes of the present disclosure can infect and / or transduce and / or selectively target CD11+ / CD14+ monocytes, CD3+ T cells, CD3- / CD56+ NK cells, and / or CD20+ B cells. In various embodiments, CD11+ / CD14+ monocytes and / or CD11+ / CD14+ phenotypes can refer to cells found to express CD11 and CD14, e.g., based on binding of the cells to a labeled anti-CD11 antibody and a labeled anti-CD14 antibody, e.g., as described in Example 10 and / or FIG. 14. In various embodiments, CD3+ T cells and / or CD3+ phenotypes can refer to cells found to express CD3, e.g., based on binding of the cells to a labeled anti-CD3 antibody, e.g., as described in Example 10 and / or FIG. 14. In various embodiments, CD3 / CD56+ NK cells and / or CD3 / CD56+ phenotypes can refer to cells found to express CD56 and not CD3, e.g., based on binding of the cells to a labeled anti-CD56 antibody and absence of binding of the cells to a labeled anti-CD3 antibody, e.g., as shown in Example 10 and / or FIG. 14. In various embodiments, CD20+ B cells and / or CD20+ phenotypes can refer to cells found to express CD20, e.g., based on binding of the cells to a labeled anti-CD20 antibody, e.g., as shown in Example 10 and / or FIG. 14. In various embodiments, labeling can be determined by any of a variety of methods known in the art, including, but not limited to, the relative presence of a label, such as the fluorescence of a fluorescent label. In various embodiments, labeling can be measured by techniques including methods such as fluorescence-activated cell sorting (FACS). Thus, in various embodiments, monocytes can refer to a population of cells determined to be CD11+ / CD14+ cells and / or have a CD11+ / CD14+ phenotype. In various embodiments, T cells can refer to a population of cells determined to be CD3+ cells and / or have a CD3+ phenotype.In various embodiments, NK cells can refer to a population of cells determined to be CD3- / CD56+ cells and / or have a CD3- / CD56+ phenotype. In various embodiments, B cells can refer to a population of cells determined to be CD20+ cells and / or have a CD20+ phenotype.

[0313] In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD11+ / CD14+ monocytes are vectors and genomes of the Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD3+ T cells are vectors and genomes of the Ad5, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD3− / CD56+ NK cells are vectors and genomes of the Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD20+ B cells are vectors and genomes of the Ad16 serotype.

[0314] In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD11+ / CD14+ monocytes are vectors and genomes of the Ad11, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD3+ T cells are vectors and genomes of the Ad34 and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD3- / CD56+ NK cells are vectors and genomes of the Ad11, Ad34, and / or Ad35 serotypes.

[0315] In various embodiments, the vectors and genomes of the present disclosure are capable of infecting and / or transducing and / or selectively targeting monocytes, T cells, NK cells, and / or B cells. In various embodiments, the vectors and genomes of the present disclosure that can infect and / or transduce and / or selectively target monocytes are vectors and genomes of the Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure that can infect and / or transduce and / or selectively target T cells are vectors and genomes of the Ad5, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure that can infect and / or transduce and / or selectively target NK cells are vectors and genomes of the Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure that are capable of infecting and / or transducing and / or selectively targeting B cells are Ad16 serotype vectors and genomes.

[0316] In various embodiments, the vectors and genomes of the present disclosure are capable of infecting and / or transducing and / or selectively targeting monocytes, T cells, and / or NK cells. In various embodiments, the vectors and genomes of the present disclosure that are capable of infecting and / or transducing and / or selectively targeting monocytes are vectors and genomes of the Ad11, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure that are capable of infecting and / or transducing and / or selectively targeting T cells are vectors and genomes of the Ad34 and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure that are capable of infecting and / or transducing and / or selectively targeting NK cells are vectors and genomes of the Ad11, Ad34, and / or Ad35 serotypes.

[0317] In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD11+ / CD14+ monocytes are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD3+ T cells are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting CD3- / CD56+ NK cells are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure that are capable of infecting and / or transducing and / or selectively targeting CD20+ B cells are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes.

[0318] In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting monocytes are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting T cells are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure capable of infecting and / or transducing and / or selectively targeting NK cells are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes. In various embodiments, the vectors and genomes of the present disclosure that are capable of infecting and / or transducing and / or selectively targeting B cells are vectors and genomes of the Ad5, Ad7, Ad11, Ad16, Ad34, and / or Ad35 serotypes.

[0319] III. Dosage, Formulation, and Administration The vector can be formulated to be pharmaceutically acceptable for administration to cells or animals, for example, humans. The vector can be administered in vitro, ex vivo, or in vivo. The adenoviral vectors described herein can be formulated for administration to a subject. The formulation includes an adenoviral vector encoding a therapeutic agent and one or more pharmaceutically acceptable carriers.

[0320] As disclosed herein, vectors can be in any form known in the art, including, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.

[0321] The selection or use of a particular form may depend, in part, on the intended mode of administration and therapeutic application. For example, compositions containing compositions intended for systemic or local delivery may be in the form of an injectable or infusible solution. Thus, vectors may be formulated for administration via parenteral modes (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration generally refers to modes of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, intrapulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, intrathecal, intraspinal, epidural, intracerebral, intracranial, intracarotid, and intracisternal injection and infusion. Parenteral administration routes may be, for example, by injection, intranasal, pulmonary, or transdermal. Administration can be systemic or local, by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0322] In various embodiments, the vectors of the present invention can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of the compositions described herein in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compositions described herein into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying, which yield a powder of the compositions described herein plus any additional desired ingredients (see below) from a previously sterile-filtered solution thereof. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0323] Vectors can be parenterally administered in the form of injectable formulations, including sterile solutions or suspensions in water or another pharmaceutically acceptable liquid. For example, vectors can be formulated by appropriately combining therapeutic molecules with a pharmaceutically acceptable vehicle or medium, such as sterile water and saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, and binders, followed by mixing in a unit dosage form required in accordance with generally accepted pharmaceutical practice. The amount of vector contained in the pharmaceutical formulation is such that an appropriate dosage within the specified range is provided. Non-limiting examples of oily liquids include sesame oil and soybean oil, which may be combined with benzyl benzoate or benzyl alcohol as a solubilizer. Other items that may be included are buffers such as phosphate buffer or sodium acetate buffer, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injections can be packaged in appropriate ampoules.

[0324] In various embodiments, subcutaneous administration can be achieved by a device such as a syringe, a pre-filled syringe, an auto-injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an ambulatory syringe infusion pump with a subcutaneous infusion set, or other device for subcutaneous infusion.

[0325] In some embodiments, the vectors described herein can be therapeutically delivered to a subject by local administration. As used herein, "local administration" or "local delivery" can refer to transport or vector-independent delivery of a vector to its intended target tissue or site via the vascular system. For example, a vector may be delivered by injection or implantation of a composition or agent, or by injection or implantation of a device containing the composition or agent. In certain embodiments, after local administration near the target tissue or site, the composition or agent, or one or more components thereof, may diffuse to the intended target tissue or site other than the site of administration.

[0326] In some embodiments, the compositions provided herein are in unit dosage form, and this unit dosage form can be suitable for self-administration.Such unit dosage form can be provided in a container, typically for example, vial, cartridge, pre-filled syringe or disposable pen.Dosage device such as the dosage device described in US6,302,855 can also be used with, for example, injection system as described herein.

[0327] Pharmaceutical forms of vector preparations suitable for injection can include sterile aqueous solutions or dispersions. The preparations can be sterile and must be fluid to allow proper flow in and out of a syringe. The preparations can also be stable under the conditions of manufacture and storage. The carrier can be a solvent or dispersion medium, including, for example, water and physiological saline or aqueous buffer solutions. Preferably, isotonic agents, such as sugars or sodium chloride, can be used in the preparations.

[0328] The appropriate dose of the vectors described herein may depend on various factors, including, for example, the age, sex, and weight of the subject being treated, the condition or disease being treated, and the particular vector used. Other factors that affect the dose administered to a subject include, for example, the type or severity of the condition or disease. Other factors include, for example, other medical disorders simultaneously or previously affecting the subject, the subject's general health condition, the subject's genetic makeup, diet, time of administration, excretion rate, drug combinations, and any other additional therapeutic agents administered to the subject. The appropriate means of administering the vector can be selected based on the condition or disease being treated and the age and condition of the subject. The dose and method of administration can vary depending on the patient's weight, age, condition, etc., and can be appropriately selected as needed by one of skill in the art. The specific dose and treatment regimen for any particular subject can be adjusted based on the physician's judgment.

[0329] In various examples, the vector can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The compositions of the present invention can include pharmaceutically acceptable salts, such as acid addition salts or base addition salts.

[0330] Exemplary commonly used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffers, bulking agents or fillers, chelating agents, coating agents, disintegration agents, dispersion media, gels, isotonicity agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0331] In various embodiments, compositions containing the vectors described herein, such as sterile preparations for injection, can be formulated according to conventional pharmaceutical practice using distilled water for injection as a vehicle. For example, isotonic solutions containing saline or glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as aqueous solutions for injection, optionally in combination with suitable solubilizers, such as alcohols such as ethanol and polyalcohols such as propylene glycol or polyethylene glycol, and non-ionic surfactants such as polysorbate 80™ and HCO-50.

[0332] The formulations disclosed herein can be formulated for administration, for example, by injection. For injection, the formulations can be formulated as aqueous solutions, such as Hank's solution, Ringer's solution, or buffers containing saline, or in media such as Iscove's modified Dulbecco's medium (IMDM). Aqueous solutions can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the formulations can be lyophilized and / or in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0333] Any formulation disclosed herein can advantageously include any other pharmaceutically acceptable carrier, including those that do not produce significant adverse, allergic, or other unpleasant reactions that outweigh the benefits of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the U.S. FDA's Office of Biological Standards and / or other relevant foreign regulatory authorities.

[0334] A therapeutically effective amount of an adenoviral vector associated with a therapeutic gene is, for example, 1×10 7 ~50×10 8 Infectious units (IU) or 5 x 10 7 ~20×10 8 In other examples, the dose can be in the range of 5 x 10 7 IU, 6×10 7 IU, 7×10 7 IU, 8×10 7 IU, 9×10 7 IU, 1×10 8 IU, 2×10 8 IU, 3×10 8 IU, 4×10 8 IU, 5×10 8 IU, 6×10 8 IU, 7×10 8 IU, 8×10 8 IU, 9×10 8 IU, 10 x 10 8 In certain embodiments, the therapeutically effective dose of an adenoviral vector associated with a therapeutic gene can comprise 4×10 IU, or more. 8In certain embodiments, the therapeutically effective amount of the adenoviral vector associated with a therapeutic gene can be administered subcutaneously or intravenously.In certain embodiments, the therapeutically effective amount of the adenoviral vector associated with a therapeutic gene can be administered after administration with one or more mobilizing factors.

[0335] In various embodiments of the present disclosure, in vivo gene therapy involves administering to a subject at least one viral gene therapy vector in combination with at least one immunosuppressive regimen. In in vivo gene therapy involving two or more vector species, such as a first vector that is a supporting viral gene therapy vector combined with a second vector that is a supporting vector, the first and second vectors can be administered in a single formulation or dosage form or in two separate formulations or dosage forms. In various embodiments, the first and second vectors can be administered simultaneously or at different times, e.g., during the same hourly period or during non-overlapping hourly periods. In various embodiments, the first and second vectors can be administered simultaneously or at different times, e.g., on the same day or on different days. In various embodiments, the first and second vectors can be administered at the same dose or at different doses, e.g., doses measured as total viral particles or viral particles per kilogram of subject. In various embodiments, the first and second vectors can be administered at a predetermined ratio. In various embodiments, this ratio ranges from 2:1 to 1:2, e.g., 1:1.

[0336] In various embodiments, the vector is administered to the subject in a single total dose per day. In various embodiments, the vector is administered in two, three, four, or more unit doses that together constitute the total dose. In various embodiments, one unit dose of the vector is administered to the subject on each of one, two, three, four, or more consecutive days per day. In various embodiments, two unit doses of the vector are administered to the subject on each of one, two, three, four, or more consecutive days per day. Thus, in various embodiments, a daily dose can refer to the dose of the vector received by the subject over a single day. In various embodiments, the term day refers to a 24-hour period, such as the 24-hour period from midnight on the first calendar day to midnight on the next calendar day.

[0337] In various embodiments, the unit dose, daily dose, or total dose of a vector, such as a viral gene therapy vector or a support vector, or the total combined dose of a viral gene therapy vector and a support vector, can be at least 1E8, 5E8, 1E9, 5E9, 1E10, 5E10, 1E11, 5E11, 1E12, 5E12, 1E13, 5E13, 1E14, or 1E15 viral particles per kilogram (vp / kg). In various embodiments, the unit dose, daily dose, or total dose of a vector, such as a viral gene therapy vector or a support vector, or the total combined dose of a viral gene therapy vector and a support vector, can fall within a range having a lower limit selected from 1E8, 5E8, 1E9, 5E9, 1E10, 5E10, 1E11, 5E11, 1E12, 5E12, 1E13, 5E13, 1E14, or 1E15 vp / kg, and an upper limit selected from 1E8, 5E8, 1E9, 5E9, 1E10, 5E10, 1E11, 5E11, 1E12, 5E12, 1E13, 5E13, 1E14, or 1E15 vp / kg.

[0338] In various embodiments, the viral gene therapy vector is administered at a unit dose, daily dose, or total dose of at least 1E10, 5E10, 1E11, 5E11, 1E12, 5E12, 1E13, 5E13, 1E14, or 1E15 vp / kg, and the support vector is administered at a unit dose, daily dose, or total dose of at least 1E8, 5E8, 1E9, 5E9, 1E10, 5E10, 1E11, and 5E11 vp / kg, optionally with the viral gene therapy vector unit dose, daily dose, or total dose being within the range of 1E10, 5E10, 1E11, 5E11, 1E12, and 5E12 vp / kg, with the lower limit selected from 1E11, 5E11, 1E12, 5E12, 1E13, 5E13, 1E14, and 1E15 vp / kg, and / or the unit dose, daily dose, or total dose of the support vector is within a range having a lower limit selected from 1E8, 5E8, 1E9, 5E9, 1E10, and 5E10 vp / kg and an upper limit selected from 1E9, 5E9, 1E10, 5E10, 1E11, and 5E11 vp / kg.

[0339] In...

Claims

1. A composition for use in a method of in vivo gene therapy in a mammalian subject, comprising: the composition comprises an adenoviral vector; The method includes administering to a subject the composition and selectively targeting a hematopoietic cell type; The adenoviral vector is (a) a capsid comprising one or more viral polypeptides of the Ad16, Ad7, Ad11, Ad34, Ad3, Ad14, Ad21, Ad37, or Ad50 serotype, wherein the one or more viral polypeptides are (i) fiber knob, (ii) a fiber shaft; (iii) fiber tails; (iv) pentons, and (v) a hexon; and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload.

2. The genome comprises: (a) a 3' ITR and a 5' ITR, each of which is of a viral polypeptide serotype; 2. The composition of claim 1, further comprising: (b) a packaging sequence, said packaging sequence being of said viral polypeptide serotype.

3. The hematopoietic cell type is (i) is or comprises a terminally differentiated cell type; (ii) is or comprises a progenitor cell type; or (iii) is or comprises a T cell, a NK cell, a common lymphoid progenitor (CLP), a colony forming unit (CFU)-pre-B cell, a B cell, a common myeloid progenitor (CMP), a granulocyte-macrophage progenitor (GMP), a CFU-M cell, a monoblast, a monocyte, a macrophage, a CFU-G cell, a myeloblast, a granulocyte, a neutrophil, an eosinophil, a basophil, a megakaryocyte-erythroid progenitor (MEP), a BFU-E cell, a CFU-E cell, an erythroblast, an erythrocyte, a CFU-Mk cell, a megakaryocyte, and / or a platelet; The composition according to claim 1 or 2. (i) the hematopoietic cell type is a mammalian hematopoietic cell type, and optionally, the mammalian hematopoietic cell type is a human hematopoietic cell type; (ii) the mammalian subject is a human subject; (iii) the method comprises mobilizing hematopoietic cells of the subject prior to administration of the composition; (iv) the method comprises administering one or more immunosuppressive agents to the subject, optionally prior to administration of the composition; or (v) any combination of the above is satisfied; The composition according to any one of claims 1 to 3.

5. A method of ex vivo gene therapy, the method comprising administering to a system an adenoviral vector and selectively targeting a hematopoietic cell type; The adenoviral vector is (a) a capsid comprising one or more viral polypeptides of the Ad16, Ad7, Ad11, Ad34, Ad3, Ad14, Ad21, Ad37, or Ad50 serotype, wherein the one or more viral polypeptides are (i) fiber knob, (ii) a fiber shaft; (iii) fiber tails; (iv) pentons, and (v) a hexon; and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload. (i) the hematopoietic cell type is a mammalian hematopoietic cell type, optionally the mammalian hematopoietic cell type is a human hematopoietic cell type; and / or 6. The method of claim 5, wherein (ii) the system is or comprises a biological sample derived from a mammalian donor, optionally wherein the mammalian donor is a human donor.

7. The heterologous nucleic acid payload comprises a selectable marker, and optionally, the selectable marker is MGMT. Ｐ１４０Ｋ and Optionally, the method comprises administering a selection agent to the subject or the system; 7. The composition or method of any one of claims 1 to 6, optionally wherein the selection agent comprises O 6 BG and / or BCNU.

8. The one or more viral polypeptides (a) a fiber knob and a fiber shaft; (b) a fiber knob and a fiber tail; (c) fiber knobs and pentons; (d) fiber knob and hexon; (e) fiber knobs, hexons, and pentons; (f) a fiber shaft and a fiber tail; (g) fiber shafts and pentons; (h) fiber shaft and hexon; (i) fiber shafts, hexons, and pentons; (j) fiber tails and pentons; (k) fiber tail and hexon; (l) fiber tails, hexons, and pentons; (m) a fiber knob, a fiber shaft, and a fiber tail; (n) fiber knobs, fiber shafts, and pentons; (o) fiber knobs, fiber shafts, and hexons; (p) fiber knobs, fiber shafts, hexons, and pentons; (q) fiber knobs, fiber shafts, fiber tails, and pentons; (r) fiber knobs, fiber shafts, fiber tails, pentons, and hexons, or A composition or method according to any one of claims 1 to 7, comprising (s) pentons and hexons. (i) the fiber knob has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 15, 33, 51, 69, 87, 105, 123, 141, 159, 177, and 195; (ii) the fiber shaft has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, 158, 176, and 194; (iii) the fiber tail has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 18, 36, 54, 72, 90, 108, 126, 144, 162, 180, and 198; (iv) the penton has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 16, 34, 52, 70, 88, 106, 124, 142, 160, 178, and 196; (v) the hexon has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 17, 35, 53, 71, 89, 107, 125, 143, 161, 179, and 197; or (vi) any combination of the above is satisfied; A composition or method according to any one of claims 1 to 8. (i) the adenoviral vector comprises a fiber of the serotype of the viral polypeptide, optionally wherein the fiber has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, and 157; (ii) the adenoviral vector is a chimeric vector, characterized in that the capsid comprises at least one of a fiber knob, a fiber shaft, a fiber tail, a hexon, or a penton that is not of said serotype of the viral polypeptide; (iii) the adenoviral vector is a helper-dependent vector; or (iv) any combination of the above is satisfied; A composition or method according to any one of claims 1 to 9.

11. (i) the heterologous nucleic acid payload encodes a protein; (ii) the heterologous nucleic acid payload encodes a chimeric antigen receptor (CAR), a T cell receptor (TCR), an antibody, or a small RNA, and optionally, the small RNA is an shRNA. (iii) the heterologous nucleic acid payload encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR) and the hematopoietic cell type is or comprises a T cell; (iv) the heterologous nucleic acid payload encodes an antibody and the hematopoietic cell type is or comprises a B cell; (v) the heterologous nucleic acid payload encodes a gene editing enzyme or system, and the gene editing is selected from CRISPR editing, base editing, prime editing, and zinc finger nuclease editing; (vi) the heterologous nucleic acid payload is selected from the group consisting of adenosine deaminase deficiency (ADA), adrenoleukodystrophy (ALD), agammaglobulinemia, alpha-1 antitrypsin deficiency, congenital amegakaryocytic thrombocytopenia, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia telangiectasia, Batten disease, Bernard-Soulier syndrome, CD40 / CD40L deficiency, chronic granulomatous disease, common variable immunodeficiency (CVID), congenital thrombotic thrombocytopenic purpura (cTTP), cystic fibrosis, Diamond Blackfan anemia (DBA), DOCK8 deficiency, dyskeratosis congenita, Fabry disease, factor V deficiency, factor VII deficiency, and factor X deficiency. , Factor XI deficiency, Factor XII deficiency, Factor XIII deficiency, Familial apolipoprotein E deficiency and atherosclerosis (ApoE), Familial hemophagocytic lymphohistiocytosis, Fanconi anemia (FA), Friedreich's ataxia, Gaucher disease, Glanzmann thrombasthenia, Glycemia, Glycogen storage disease, Glycogen storage disease type I (GSDI), Grey platelet syndrome, Hemophilia, Hemophilia A, Hemophilia B, Hereditary hemochromatosis, Hurler syndrome, Hyper-IgM, Hypogammaglobulinemia, Krabbe disease, Major histocompatibility complex class II deficiency (MHC-II), Maple syrup urine disease, Metachromatic leukodystrophy (MLD), Mucopolysaccharidosis, Mucopolysaccharidosis type I (MPS I), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morquio syndrome), MPS V, MPS VI (Maroteaux-Lamy syndrome), MPSVII (Sly syndrome), muscular dystrophy, Niemann-Pick disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pernicious anemia, phenylketonuria (PKU), Pompe disease, pulmonary alveolar proteinosis (PAP), pure red cell aplasia (PRCA), pyruvate kinase deficiency, refractory anemia, Shwachman-Diamond syndrome, selective IgA deficiency, severe aplastic anemia, severe combined immunodeficiency (SCID), severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID), sickle cell or encoding an agent for the treatment of a condition selected from: erythrocyte anemia, sickle cell disease, sickle cell trait, Tay-Sachs disease, thalassemia, thalassemia intermedia, von Gierke disease, von Willebrand disease, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked severe combined immunodeficiency (SCID-X1), Zellweger syndrome, α-mannosidosis, β-mannosidosis, β-thalassemia, and / or β-thalassemia major; or (vii) satisfying any combination of the above; A composition or method according to any one of claims 1 to 10.

12. (a) the capsid comprises one or more viral polypeptides of the Ad16, Ad7, Ad11, or Ad34 serotype, and the hematopoietic cell type is or comprises a monocyte, optionally, the monocyte is a CD11+ / CD14+ monocyte; (b) said capsid comprises one or more viral polypeptides of the Ad16, Ad7, Ad11, or Ad34 serotype, and said hematopoietic cell type is or comprises a T cell, and optionally said monocyte is a CD3+ T cell; (c) the capsid comprises one or more viral polypeptides of the Ad16, Ad7, Ad11, or Ad34 serotype, and the hematopoietic cell type is or comprises an NK cell, optionally wherein the NK cell is a CD3- / CD56+ NK cell; or (d) the capsid comprises one or more viral polypeptides of the Ad16, Ad7, Ad11, or Ad34 serotype, and the hematopoietic cell type is or comprises a B cell, optionally wherein the B cell is a CD20+ B cell; A composition or method according to any one of claims 1 to 11.

13. 1. An adenoviral vector and a hematopoietic cell comprising the adenoviral vector genome, The adenoviral vector comprises a capsid that includes one or more viral polypeptides of the Ad16, Ad7, Ad11, Ad34, Ad3, Ad14, Ad21, Ad37, or Ad50 serotype, the one or more viral polypeptides being (i) fiber knob, (ii) a fiber shaft; (iii) fiber tails; (iv) pentons, and (v) hexon, the adenoviral vector genome comprises a double-stranded DNA genome comprising a heterologous nucleic acid payload; The hematopoietic cell is a T cell, a NK cell, a common lymphoid progenitor (CLP), a colony forming unit (CFU)-pre-B cell, a B cell, a common myeloid progenitor (CMP) cell, a granulocyte-macrophage progenitor (GMP) cell, a CFU-M cell, a monoblast, a monocyte, a macrophage, a CFU-G cell, a myeloblast, a granulocyte, a neutrophil, an eosinophil, a basophil, a megakaryocyte-erythroid progenitor (MEP) cell, a BFU-E cell, a CFU-E cell, an erythroblast, an erythrocyte, a CFU-Mk cell, a megakaryocyte, and / or a platelet.

14. A hematopoietic cell comprising an adenoviral vector genome, the adenoviral vector genome comprises: (a) a 3'ITR and a 5'ITR, wherein the 3'ITR and the 5'ITR are of the same serotype selected from Ad16, Ad7, Ad11, Ad34, Ad3, Ad14, Ad21, Ad37, or Ad50, respectively; (b) a packaging sequence, wherein the packaging sequence is of the same serotype as the 3'ITR and the 5'ITR; and (c) a heterologous nucleic acid payload; The hematopoietic cell is a T cell, a NK cell, a common lymphoid progenitor (CLP), a colony forming unit (CFU)-pre-B cell, a B cell, a common myeloid progenitor (CMP) cell, a granulocyte-macrophage progenitor (GMP) cell, a CFU-M cell, a monoblast, a monocyte, a macrophage, a CFU-G cell, a myeloblast, a granulocyte, a neutrophil, an eosinophil, a basophil, a megakaryocyte-erythroid progenitor (MEP) cell, a BFU-E cell, a CFU-E cell, an erythroblast, an erythrocyte, a CFU-Mk cell, a megakaryocyte, and / or a platelet.

15. The hematopoietic cells are selected from the group consisting of adenosine deaminase deficiency (ADA), adrenoleukodystrophy (ALD), agammaglobulinemia, alpha-1 antitrypsin deficiency, congenital amegakaryocytic thrombocytopenia, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia telangiectasia, Batten disease, Bernard-Soulier syndrome, CD40 / CD40L deficiency, chronic granulomatous disease, common variable immunodeficiency (CVID), congenital thrombotic thrombocytopenic purpura (cTTP), cystic fibrosis, Diamond Blackfan anemia (DBA), DOCK8 deficiency, dyskeratosis congenita, Fabry disease, factor V deficiency, factor VII deficiency, factor X deficiency, factor XI deficiency, and the like. Factor deficiency, Factor XII deficiency, Factor XIII deficiency, Familial apolipoprotein E deficiency and atherosclerosis (ApoE), Familial hemophagocytic lymphohistiocytosis, Fanconi anemia (FA), Friedreich's ataxia, Gaucher disease, Glanzmann thrombasthenia, Glycemia, Glycogen storage disease, Glycogen storage disease type I (GSDI), Grey platelet syndrome, Hemophilia, Hemophilia A, Hemophilia B, Hereditary hemochromatosis, Hurler syndrome, Hyper-IgM, Hypogammaglobulinemia, Krabbe disease, Major histocompatibility complex class II deficiency (MHC-II), Maple syrup urine disease, Metachromatic leukodystrophy (MLD), Mucopolysaccharidoses, Mucopolysaccharidoses type I (MPS I), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morquio syndrome), MPS V, MPS VI (Maroteaux-Lamy syndrome), MPSVII (Sly syndrome), muscular dystrophy, Niemann-Pick disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pernicious anemia, phenylketonuria (PKU), Pompe disease, pulmonary alveolar proteinosis (PAP), pure red cell aplasia (PRCA), pyruvate kinase deficiency, refractory anemia, Shwachman-Diamond syndrome, selective IgA deficiency, severe aplastic anemia, severe combined immunodeficiency (SCID), severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID), sickle cell anemia, sickle 15. The hematopoietic cell of claim 13 or 14, which is a cell of a subject suffering from a condition selected from nephropathy, sickle cell trait, Tay-Sachs disease, thalassemia, thalassemia intermedia, von Gierke disease, von Willebrand disease, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked severe combined immunodeficiency (SCID-X1), Zellweger syndrome, α-mannosidosis, β-mannosidosis, β-thalassemia, and / or β-thalassemia major.

16. 1. An adenoviral donor vector, (a) a capsid comprising one or more viral polypeptides of the Ad16, Ad7, Ad11, Ad34, Ad3, Ad14, Ad21, Ad37, or Ad50 serotype, wherein the one or more viral polypeptides are (i) fiber knob, (ii) a fiber shaft; (iii) fiber tails; (iv) pentons, and (v) a hexon; and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload.

17. The genome comprises: (a) a 3' ITR and a 5' ITR, each of which is of a viral polypeptide serotype; 17. The vector of claim 16, further comprising: (b) a packaging sequence, said packaging sequence being of the viral polypeptide serotype.

18. The heterologous nucleic acid payload comprises a selectable marker, and optionally, the selectable marker is MGMT. Ｐ１４０Ｋ 18. The vector according to claim 16 or 17,

19. The one or more viral polypeptides (a) a fiber knob and a fiber shaft; (b) a fiber knob and a fiber tail; (c) fiber knobs and pentons; (d) fiber knob and hexon; (e) fiber knobs, hexons, and pentons; (f) a fiber shaft and a fiber tail; (g) fiber shafts and pentons; (h) fiber shaft and hexon; (i) fiber shafts, hexons, and pentons; (j) fiber tails and pentons; (k) fiber tail and hexon; (l) fiber tails, hexons, and pentons; (m) a fiber knob, a fiber shaft, and a fiber tail; (n) fiber knobs, fiber shafts, and pentons; (o) fiber knobs, fiber shafts, and hexons; (p) fiber knobs, fiber shafts, hexons, and pentons; (q) fiber knobs, fiber shafts, fiber tails, and pentons; (r) fiber knobs, fiber shafts, fiber tails, pentons, and hexons, or The vector according to any one of claims 16 to 18, comprising (s) a penton and a hexon.

20. (i) the fiber knob has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 15, 33, 51, 69, 87, 105, 123, 141, and 159; (ii) the fiber shaft has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 14, 32, 50, 68, 86, 104, 122, 140, and 158; (iii) the fiber tail has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 18, 36, 54, 72, 90, 108, 126, 144, and 162; (iv) the penton has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 16, 34, 52, 70, 88, 106, 124, 142, and 160; (v) the hexon has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 17, 35, 53, 71, 89, 107, 125, 143, and 161; or (vi) any combination of the above is satisfied; The vector according to any one of claims 16 to 19.

21. (i) the adenoviral vector comprises a fiber of the serotype of the viral polypeptide, optionally, the fiber having a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 13, 31, 49, 67, 85, 103, 121, 139, and 157; (ii) the adenoviral vector is a chimeric vector, characterized in that the capsid comprises at least one of a fiber knob, a fiber shaft, a fiber tail, a hexon, or a penton that is not of said serotype of the viral polypeptide; (iii) the adenoviral vector is a helper-dependent vector; or (iv) any combination of the above is satisfied; The vector according to any one of claims 16 to 20.

22. 1. An adenovirus donor vector genome, (a) a 3'ITR and a 5'ITR, wherein the 3'ITR and the 5'ITR are of the same serotype selected from Ad16, Ad7, Ad11, Ad34, Ad3, Ad14, Ad21, Ad37, or Ad50, respectively; (b) a packaging sequence, wherein the packaging sequence is of the ITR serotype; and (c) a heterologous nucleic acid payload.

23. The heterologous nucleic acid payload comprises a selectable marker, and optionally, the selectable marker is MGMT. Ｐ１４０Ｋ The genome of claim 22.

24. (i) the heterologous nucleic acid payload encodes a protein; (ii) the heterologous nucleic acid payload encodes a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a small RNA, and optionally, the small RNA is an shRNA; (iii) the heterologous nucleic acid payload encodes a gene editing enzyme or system, and the gene editing is selected from CRISPR editing, base editing, prime editing, or zinc finger nuclease editing; (iv) the heterologous nucleic acid payload is selected from the group consisting of adenosine deaminase deficiency (ADA), adrenoleukodystrophy (ALD), agammaglobulinemia, alpha-1 antitrypsin deficiency, congenital amegakaryocytic thrombocytopenia, amyotrophic lateral sclerosis (Lou Gehrig's disease), ataxia telangiectasia, Batten disease, Bernard-Soulier syndrome, CD40 / CD40L deficiency, chronic granulomatous disease, common variable immunodeficiency (CVID), congenital thrombotic thrombocytopenic purpura (cTTP), cystic fibrosis, Diamond Blackfan anemia (DBA), DOCK8 deficiency, dyskeratosis congenita, Fabry disease, factor V deficiency, factor VII deficiency, and factor X deficiency. , Factor XI deficiency, Factor XII deficiency, Factor XIII deficiency, Familial apolipoprotein E deficiency and atherosclerosis (ApoE), Familial hemophagocytic lymphohistiocytosis, Fanconi anemia (FA), Friedreich's ataxia, Gaucher disease, Glanzmann thrombasthenia, Glycemia, Glycogen storage disease, Glycogen storage disease type I (GSDI), Grey platelet syndrome, Hemophilia, Hemophilia A, Hemophilia B, Hereditary hemochromatosis, Hurler syndrome, Hyper-IgM, Hypogammaglobulinemia, Krabbe disease, Major histocompatibility complex class II deficiency (MHC-II), Maple syrup urine disease, Metachromatic leukodystrophy (MLD), Mucopolysaccharidosis, Mucopolysaccharidosis type I (MPS I), MPS II (Hunter syndrome), MPS III (Sanfilippo syndrome), MPS IV (Morquio syndrome), MPS V, MPS VI (Maroteaux-Lamy syndrome), MPSVII (Sly syndrome), muscular dystrophy, Niemann-Pick disease, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), pernicious anemia, phenylketonuria (PKU), Pompe disease, pulmonary alveolar proteinosis (PAP), pure red cell aplasia (PRCA), pyruvate kinase deficiency, refractory anemia, Shwachman-Diamond syndrome, selective IgA deficiency, severe aplastic anemia, severe combined immunodeficiency (SCID), severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID), sickle cell or encoding an agent for the treatment of a condition selected from: erythrocyte anemia, sickle cell disease, sickle cell trait, Tay-Sachs disease, thalassemia, thalassemia intermedia, von Gierke disease, von Willebrand disease, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked severe combined immunodeficiency (SCID-X1), Zellweger syndrome, α-mannosidosis, β-mannosidosis, β-thalassemia, and / or β-thalassemia major; or (v) any combination of the above is satisfied; A vector or genome according to any one of claims 16 to 23.

25. The vector or genome according to any one of claims 16 to 24, wherein the serotype of the viral polypeptide is Ad34.

26. A pharmaceutical composition comprising the adenoviral vector of any one of claims 16 to 25, wherein the pharmaceutical composition is formulated for injection into a subject in need thereof.

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