Adenoviral gene therapy vectors

Abstract

The present disclosure includes adenoviral vectors characterized by efficient transduction of HSCs, for example for in vivo gene therapy. The present disclosure includes, inter alia, Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad37, and Ad50 vectors and genomes. The Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, 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 / 129,233, filed December 22, 2020, the contents of which are incorporated by reference in their entirety herein. [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 modification of hematopoietic stem cells (HSCs). Thus, there is a need for compositions and methods for HSC 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. In various gene therapies, hematopoietic stem cells (HSCs) are an important target. However, current methods and compositions for modifying HSCs are limited. Some gene therapy vectors, such as lentiviruses, have relatively limited payload capacity. Others, such as adenovirus serotype 5, are characterized by significant payload capacity, but are widespread enough that a large percentage of humans have antibodies against the vector proteins, some of which may be neutralizing antibodies. Furthermore, various viral vectors are characterized by different transduction efficiencies for various cell types, such as HSCs. The present disclosure has identified adenovirus serotypes that are characterized by high payload capacity and high transduction efficiency for HSCs.

[0004] The present disclosure includes, among others, Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors and Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genomes (e.g., "recombinant" or "engineered" adenoviral vectors and genomes). The Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors and genomes of the present disclosure can include various payloads. In various embodiments, the payloads can include one or more of a nucleic acid sequence encoding a CRISPR system, a base editing system, a prime editing system, or other expression products. The present disclosure includes, among others, combinations of adenoviral vectors and adenoviral genomes that include a nucleic acid sequence encoding multiple expression products that together contribute to the treatment of a disease or condition. The present disclosure includes, inter alia, Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 adenoviral vectors and Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 adenoviral genomes for incorporating a nucleic acid payload into a target cell genome. The disclosure includes, inter alia, Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vectors, Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 adenovirus donor genomes, helper-dependent Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 adenovirus donor vectors, helper-dependent Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 adenovirus donor genomes, support vectors, supporting genomes, Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper vectors, and Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper genomes. For the avoidance of doubt, a list of serotypes such as "Ad3, 7, 11, 14, 16, 21, 34, 37, or 50" may alternatively be expressed as "Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad37, or Ad50."

[0005] In at least one aspect, the disclosure provides a method of in vivo gene therapy in a mammalian subject, comprising administering to the subject an adenoviral vector, wherein the adenoviral vector comprises: (a) a capsid comprising one or more viral polypeptides of an Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad37, or Ad50 serotype (e.g., having at least 80% sequence identity to a reference polypeptide of the 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 (e.g., has at least 80% sequence identity to a reference sequence of the same serotype as the viral polypeptide serotype); and (b) a packaging sequence, each of which is of a viral polypeptide serotype. In various embodiments, the method comprises mobilizing hematopoietic stem cells of the subject prior to administration of the adenoviral vector. In various embodiments, the heterologous nucleic acid payload comprises a selection marker, optionally, the selection marker is MGMTP140K. In various embodiments, the method comprises administering a selection agent to the subject, optionally, the selection agent comprises O6BG and / or BCNU. In various embodiments, the method comprises administering one or more immunosuppressants to the subject, optionally, the administration of the one or more immunosuppressants is prior to administration of the adenoviral vector.

[0006] In at least one aspect, the 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, where 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 the 3'ITR and the 5'ITR being of a viral polypeptide serotype; and (b) a packaging sequence, the packaging sequence being of a viral polypeptide serotype. In various embodiments, the heterologous nucleic acid payload comprises a selectable marker, optionally, the selectable marker is MGMTP140K.

[0007] In various embodiments of aspects described by the present disclosure, the one or more viral polypeptides are (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 (l) fiber tail, hexon, and penton, (m) fiber knob, fiber shaft, and fiber tail, (n) fiber knob, fiber shaft, and penton, (o) fiber knob, fiber shaft, and hexon, (p) fiber knob, fiber shaft, hexon, and penton, (q) fiber knob, fiber shaft, fiber tail, and penton, (r) fiber knob, fiber shaft, fiber tail, penton, and hexon, or (s) penton and hexon.

[0008] In various embodiments of the aspects described by this disclosure, the fiber knob has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, and 142. In various embodiments of the aspects described by this disclosure, the fiber shaft has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 125, and 141. In various embodiments of the aspects described by this disclosure, the fiber tail has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 157, 158, 159, 160, 161, 162, 163, 164, and 165. In various embodiments of the aspects described by the present disclosure, the penton has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 15, 31, 47, 63, 79, 95, 111, 127, and 143. In various embodiments of the aspects described by the present disclosure, the hexon has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, and 144. In various embodiments of the aspects described by the present disclosure, the adenoviral vector comprises a fiber of a serotype of a viral peptide. In various embodiments of the aspects described by the present disclosure, the fiber has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, and 140.

[0009] In various embodiments of the aspects described by the present disclosure, the adenoviral vector is a chimeric vector characterized in that the capsid contains at least one of a fiber knob, a fiber shaft, a fiber tail, a hexon, or a penton that is not of a serotype of the viral peptide.

[0010] In various embodiments of the aspects described by this disclosure, the adenoviral vector is a helper-dependent vector.

[0011] In at least one aspect, the disclosure provides an adenovirus donor vector genome comprising: (a) a 3'ITR and a 5'ITR, the 3'ITR and the 5'ITR being of the same serotype selected from Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad37, or Ad50 serotypes, respectively; (b) a packaging sequence, the packaging sequence being of an ITR serotype; and (c) a heterologous nucleic acid payload. In certain embodiments, the heterologous nucleic acid payload comprises a selection marker, optionally the selection marker is MGMTP140K.

[0012] In various embodiments of the aspects described by the present disclosure, the heterologous nucleic acid payload encodes a protein.In various embodiments of the aspects described by the present disclosure, the heterologous nucleic acid payload encodes a small RNA, and optionally, the small RNA is an shRNA.In various embodiments of the aspects described by the present disclosure, the heterologous nucleic acid payload encodes a gene editing enzyme or system, and wherein gene editing is selected from CRISPR editing, base editing, prime editing, or zinc finger nuclease editing.

[0013] In various embodiments of the aspects described by the present disclosure, the heterologous nucleic acid payload encodes an agent for the treatment of a condition selected from hemoglobinopathies, platelet disorders, Fanconi anemia, alpha-1 antitrypsin deficiency, sickle cell anemia, thalassemia, thalassemia intermedia, von Willebrand's disease, hemophilia A, hemophilia B, factor V deficiency, factor VII deficiency, factor X deficiency, factor XI deficiency, factor XII deficiency, factor XIII deficiency, Bernard-Soulier syndrome, gray platelet syndrome, mucopolysaccharidoses, cystic fibrosis, Tay-Sachs disease, and phenylketonuria. In various embodiments of the aspects described by the present disclosure, the heterologous nucleic acid payload is a nucleic acid payload that is capable of identifying or identifying a disease, such as Graves' disease, rheumatoid arthritis, pernicious anemia, multiple sclerosis (MS), inflammatory bowel disease, systemic lupus erythematosus (SLE), adenosine deaminase deficiency (ADA-SCID) or severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), adenosine deaminase 2 deficiency, familial idiopathies (FAD), rheumatoid arthritis ... rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, rheumato The compound encodes an agent for the treatment of a condition selected from Nkoni anemia (FA), Batten disease, adrenoleukodystrophy (ALD) or metachromatic leukodystrophy (MLD), muscular dystrophy, pulmonary alveolar proteinosis (PAP), pyruvate kinase deficiency, Shwachman-Diamond-Blackfan anemia, dyskeratosis congenita, cystic fibrosis, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (Lou Gehrig's disease).

[0014] In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad34. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad3. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad7. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad11. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad14. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad16. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad21. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad37. In various embodiments of the aspects described by the present disclosure, the serotype of the viral polypeptide is Ad50.

[0015] 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.

[0016] In various embodiments, the disclosure provides methods, vectors, genomes, or pharmaceutical compositions in which an adenoviral vector is infected and / or introduced into CD34+ cells, CD34+high cells, CD34+ / CD90+ cells, and / or CD34+high / CD90+ cells, optionally where the cells are hematopoietic cells.

[0017] 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, "an element" discloses embodiments that are exactly one element as well as embodiments containing more than one element.

[0018] About: As used herein, the term "about" when used in reference to a value refers to a value that is similar in context to the reference value. In general, a person skilled 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 a range of values ​​that are 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 reference value.

[0019] Administration: As used herein, the term "administration" typically refers to administering a composition to a subject or system to effect delivery of an agent that is or is contained in the composition.

[0020] Adoptive Cell Therapy: As used herein, "adoptive cell therapy" or "ACT" involves the transfer of therapeutically active cells 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 expansion of the cells.

[0021] 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 moieties and a binding target (e.g., a cell). 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 viral vector and a target cell of the viral vector). One of skill in the art will understand that a change in affinity can be described by comparison to a reference (e.g., an increase or decrease relative to a reference) or can be described numerically. Affinity can be measured by, but is not limited to, the equilibrium dissociation constant (K D ) and / or the equilibrium association constant (K A ) can be measured and / or expressed in a variety of ways known in the art. D koff / k on quotient of, while K A k on / k off where k on For example, k refers to the association rate constant between the viral vector and the target cell. 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.

[0022] Agent: As used herein, the term "agent" may refer to any chemical entity, including, but not limited to, any one or more of an atom, a molecule, a compound, an amino acid, a polypeptide, a nucleotide, a nucleic acid, a protein, a protein complex, a liquid, a solution, a sugar, a polysaccharide, a lipid, or a combination or complex thereof.

[0023] Allogeneic: As used herein, the term "allogeneic" refers to any material that is derived from one subject and then introduced into another subject (eg, allogeneic HSC transplantation).

[0024] Between or from: As used herein, the term "between" refers to the content that falls between (and including) the upper and lower boundaries, or first and second boundaries, as indicated. Similarly, the term "from," when used in the context of a range of values, indicates that the range includes the content that falls between (and including) the upper and lower boundaries, or first and second boundaries, as indicated.

[0025] 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, whereas 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, including when the interacting agents are tested alone or in the context of a more complex system (e.g., covalently bound or otherwise associated with a carrier agent and / or in a biological system or cell).

[0026] Cancer: As used herein, the term "cancer" refers to a condition, disorder, or disease in which cells exhibit relatively abnormal, unregulated, and / or autonomous growth, thereby exhibiting an abnormally high proliferation rate and / or an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer may include one or more tumors. In some embodiments, cancer may be or include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, cancer may be or include a solid tumor. In some embodiments, cancer may be or include a hematological tumor.

[0027] 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 moiety that binds to a target antigen, (ii) a transmembrane domain, and (iii) an intracellular signaling domain that delivers an activation signal when the CAR is stimulated by binding of the extracellular binding moiety to a target antigen. CARs are also known as chimeric T cell receptors or chimeric immune receptors.

[0028] Combination therapy: As used herein, the term "combination therapy" refers to administering two or more agents or regimens to a subject so that the two or more agents or regimens together treat the subject's condition, disorder, or disease. In some embodiments, 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 have to, that the two agents or regimens are administered together in a single composition, or at the same time.

[0029] Control 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 depends in whole or in part on the state (e.g., presence, absence, conformation, chemical modification, interaction, or other activity) of the first element, at least under a certain set of conditions. Control of expression or activity can be substantial control or activity, 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, at least under a certain set of conditions.

[0030] Corresponding: As used herein, the term "corresponding" may be used to designate the position / identity of a structural element of a compound or composition by comparison to an appropriate reference compound or composition. For example, in some embodiments, a monomeric 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, one of skill 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 of the second related sequence may 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 can generally be accomplished by any of a variety of known tools, strategies, and / or algorithms, including, but not limited to, software programs such as, for example, 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.

[0031] Dosing regimen: As used herein, the term "dosing regimen" may refer to a series of one or more same or different unit doses administered to a subject, typically including administration of multiple unit doses, each of which is spaced apart in time from administration of other unit doses. In various embodiments, one or more or all unit doses of a dosing regimen may be the same or different (e.g., increasing over time, decreasing over time, or adjusting according to the subject's and / or medical practitioner's decision). In various embodiments, one or more or all periods between each administration may be the same or different (e.g., increasing over time, decreasing over time, or adjusting according to the subject's and / or medical practitioner's decision). In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may include one or more administrations. Typically, a marketed drug will have at least one recommended dosing regimen known to those skilled in the art. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered to a relevant population (i.e., is a therapeutic dosing regimen).

[0032] 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 relative to 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 relative to a second DNA region.

[0033] Effective amount: An "effective amount" is the amount of a formulation required to produce a desired physiological change in a subject. Effective amounts are often administered for research purposes.

[0034] 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 to be "engineered" when two or more sequences that are not naturally linked together in that order are directly linked to each other to become a polynucleotide that has been engineered by the hand of man. One of skill in the art will understand that an "engineered" nucleic acid or amino acid sequence may be a recombinant nucleic acid or amino acid sequence and may also be referred to as "genetically engineered." In some embodiments, an engineered polynucleotide includes coding and / or regulatory sequences that are found to be operably linked to a first sequence in nature, but not found to be operably linked to a second sequence in nature, and that are operably linked to the second sequence to become a polynucleotide that has been engineered by the hand of man. In some embodiments, a cell or organism is considered to be "engineered" or "genetically engineered" if it has been manipulated such that its genetic information is altered (e.g., new genetic material not previously present is introduced, e.g., by transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is altered or removed, e.g., by substitution, deletion, or mating). As is common practice and understood by those of skill in the art, progeny or copies of an engineered polynucleotide or cell, whether complete or incomplete, and regardless of the actual manipulation of the prior entity.

[0035] Excipient: As used herein, "excipient" refers to a non-therapeutic agent that can be included in a pharmaceutical composition, for example, to provide or impart a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients can 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, and the like.

[0036] Expression: As used herein, "expression" refers to one or more biological processes that individually and / or cumulatively 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.

[0037] Flanking: As used herein, a first element (e.g., a nucleic acid sequence or an amino acid sequence) that is present in a contiguous sequence that includes a second element and a third element is "flanked" by the second element and the third element when it is located in the contiguous sequence between the second element and the third element. Thus, in such an arrangement, the second element and the third element can be referred to as "flanking" the first element. The flanking elements may be directly adjacent to the flanking elements or the flanking elements may be separated by one or more relevant units. In various examples where the contiguous sequence is a nucleic acid or amino acid sequence and the relevant units are bases or amino acid residues, respectively, the number of units in the contiguous sequence between the flanking elements and, independently, the first and / or second flanking elements may be, for example, 50 units or less, e.g., only 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, or 0 units.

[0038] Fragment: As used herein, a "fragment" refers to a structure that comprises and / or consists of a discrete portion of a reference agent (sometimes referred to as a "parent" agent). In some embodiments, a fragment lacks one or more portions found in the reference agent. In some embodiments, a fragment comprises or consists of one or more portions found in the reference agent. In some embodiments, the reference agent is a macromolecule, 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 monomeric 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 monomeric 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 macromolecule can be a macromolecule having a sequence of residues that have 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 identity to the reference macromolecule. The fragment may or may not be generated by physical fragmentation of the reference agent. In some cases, the fragment is generated by physical fragmentation of the reference agent. In some cases, the fragment is not generated by physical fragmentation of the reference agent, but may be generated, for example, by de novo synthesis or other means.

[0039] 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 that extend a predetermined number of nucleotides upstream of the coding sequence in a reference context, such as a source genome, and (ii) DNA nucleotides that extend 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 a reference context in which it resides or in which it may be replaced by manipulation.

[0040] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or to a polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0041] Host cell, target cell: As used herein, "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise), such as a transgene, has been introduced. 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 progeny or copies, complete or incomplete. In some embodiments, a host cell contains one or more viral genes or transgenes. In some embodiments, an intended or potential host cell may also be referred to as a target cell.

[0042] In various embodiments, host or target cells are identified by the presence, absence, or expression levels of various surface markers.

[0043] 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 cell. When referring to a surface marker, the term can refer to the presence of surface expression detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, where the staining is detected by flow cytometry at a level substantially greater than that detected by performing the same procedure under otherwise identical conditions using an isotype-matched control, and / or at a level substantially similar to the staining of cells known to be positive for the marker, and / or at a level substantially greater than the staining of cells known to be negative for the marker.

[0044] 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 cell. In the context of a surface marker, the term can refer to the absence of surface expression detected by flow cytometry, e.g., by staining with and detecting an antibody that specifically binds to the marker, where the staining is not detected by flow cytometry at a level substantially greater than that detected by performing the same procedure under otherwise identical conditions using an isotype-matched control, and / or at a level substantially less than that of cells known to be positive for the marker, and / or at a level comparable to that of cells known to be negative for the marker.

[0045] Identity: As used herein, the term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., between DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for calculating percent identity between two sequences provided 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 protein and nucleic acid sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") may be readily calculated by known methods, including those described in 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 for determining 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, calculation of the percent sequence identity of two nucleic acids or polypeptides can be performed, for example, by aligning the two sequences (or the complement of one or both sequences) for optimal comparison (e.g., gaps can be introduced in one or both of the first and second sequences to obtain 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 depends on 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 the percent identity between two sequences can be achieved using computational algorithms such as BLAST (Basic Local Alignment Search Tool). Sequence alignment and percent identity calculation can be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignment can also be performed using the Clustal alignment method (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, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, 1999). In the context of this disclosure, when sequence analysis software is used for the analysis, it will be understood that the results of the analysis will be based on the "default values" of the program being referred to. "Default values" refers to any set of values ​​or parameters that are originally loaded together when the software is first initialized.

[0046] "Improve," "Increase," "Inhibit," or "Decrease": As used herein, the terms "improve," "increase," "inhibit," and "decrease," and their grammatical equivalents, indicate a qualitative or quantitative difference from a reference.

[0047] Isolated: As used herein, "isolated" refers to a substance and / or entity that is (1) separated from at least some of the components with which it was associated when originally produced (whether in nature and / or in an experimental environment) and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. An isolated substance and / or entity 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 other components with which it was originally associated. In some embodiments, an isolated agent is greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 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 it has been 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 isolation rate or purity of the substance is calculated without including such carriers or excipients. By way of example, in some embodiments, a biological macromolecule, such as a naturally occurring polypeptide or polynucleotide, is considered to be "isolated" if it: a) is not associated with some or all of the components with which it is naturally associated in its native state because of its origin or source of origin; b) is substantially free of other polypeptides or nucleic acids of the same species from the species that naturally produces it; or c) is otherwise associated with components expressed by or derived from a cell or other expression system that is not the species that naturally produces it. Thus, for example, in some embodiments, a chemically synthesized polypeptide or a polypeptide synthesized in a cell system different from the cell system that 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 so long as 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.

[0048] Operably linked: As used herein, "operably linked" or "operably linked" refers to at least a first element and a second element in a relationship that allows the components to function as intended. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if the regulatory sequence and the coding sequence are related such that expression of the coding sequence can be controlled 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.

[0049] Pharmaceutically acceptable: As used herein, the term "pharmacologically acceptable" as applied to one or more or all of the ingredient(s) 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.

[0050] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulant, that facilitates the formulation of an agent (e.g., a pharmaceutical agent), modifies the bioavailability of an agent, or facilitates the transport of an agent from one organ or part of a subject to another. Some examples of materials which may function as pharma- ceutically 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 wax; 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; phosphate buffers; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0051] 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 pharma- ceutically acceptable carriers.

[0052] Promoter: As used herein, a "promoter" or "promoter sequence" may be a DNA regulatory region that is directly or indirectly involved (e.g., via a promoter-binding protein or substrate) in the initiation and / or processivity of transcription of a coding sequence. A promoter can initiate transcription of a coding sequence upon binding of one or more transcription factors and / or regulatory moieties to the promoter under suitable conditions. A promoter involved in initiating transcription of a coding sequence may be "operably linked" to a coding sequence. In certain cases, a promoter may be or include a DNA regulatory region that extends from the transcription initiation site (at its 3' end) to an upstream (5' direction) position, and such designated sequence includes one or both of a minimum number of bases or elements necessary to initiate a transcription event. A promoter may be, include, or be operably associated with or operably linked to expression control sequences such as enhancer and repressor sequences. In some embodiments, a promoter may be inducible. In some embodiments, a promoter may be a constitutive promoter. In some embodiments, a conditional (e.g., inducible) promoter may be unidirectional or bidirectional. Promoter may be or contain the same sequence as the sequence known to exist in the genome of a particular species. In some embodiments, promoter may be or contain hybrid promoter, where the sequence containing the transcriptional regulatory region can be obtained from one source, and the sequence containing the transcriptional initiation region can be obtained from a second source. Systems for linking control elements to coding sequences in transgenes are well known in the art (general molecular biology 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).

[0053] Reference: As used herein, "reference" refers to a standard or control to which comparison is made. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or a population thereof, or a representative measurement or characteristic thereof, is compared to a reference, agent, sample, sequence, subject, animal, or individual, or a population thereof, or a representative measurement or characteristic thereof. In some embodiments, the reference is a measurement. In some embodiments, the reference is an established standard or expected value. In some embodiments, the reference is a historical reference. The reference can be quantitative or qualitative. Typically, as understood by those of skill in the art, the reference and the value to which the reference is compared represent measurements under comparable conditions. Those of skill in the art will understand when there is sufficient similarity to justify reliance and / or comparison. In some embodiments, a suitable reference can be, for example, an agent, sample, sequence, subject, animal, or individual, or a population thereof, under conditions that the skilled artisan recognizes as comparable for the purpose of assessing one or more particular variables (e.g., the presence or absence of an agent or condition), or a representative measurement or characteristic thereof. Without wishing to be bound to any particular embodiment, in various embodiments, the reference sequence may be a sequence associated with the sequence accession numbers provided herein, some of which are provided in FIG.

[0054] Regulatory sequence: As used herein, a regulatory sequence is a nucleic acid sequence that, in the context of expression of a nucleic acid coding sequence, controls the expression of the coding sequence. In some embodiments, a regulatory sequence can control or affect one or more aspects of gene expression (e.g., cell type specific expression, inducible expression, etc.).

[0055] 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 afflicted with 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 afflicted with 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 has one or more characteristics characteristic of a susceptibility or risk for a disease, disorder, or condition. In some embodiments, the subject is one who has been tested for and / or administered therapy for a disease, disorder, or condition. In some cases, a human subject may be referred to interchangeably as a "patient" or an "individual."

[0056] 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 shows a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms or a human population. In some embodiments, the appropriate population may be defined by various criteria, such as a certain age group, sex, genetic background, pre-existing clinical conditions, 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 an agent that has been approved or requires approval by a government agency before being marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a prescription for administration to humans.

[0057] Therapeutically effective amount: As used herein, a "therapeutically effective amount" refers to an amount that produces a desired effect upon its administration. In some embodiments, the term refers to an amount that, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition according to a therapeutic administration regimen, is sufficient to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of a disease, disorder, and / or condition and / or delays the onset of one or more symptoms thereof. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not in fact require the achievement of successful treatment in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, produces a particular desired pharmacological response in a significant number of subjects. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount measured in one or more particular tissues (e.g., tissues affected by a disease, disorder, or condition) or in a bodily fluid (e.g., blood, saliva, serum, sweat, tears, urine, etc.). One of skill 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, e.g., as part of a dosing regimen.

[0058] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy that is performed with the intent of resulting in, or achieving, a partial or complete alleviation, amelioration, relief, inhibition, delay in onset, reduction in severity, and / or reduction in incidence of, one or more symptoms, characteristics, and / or causes of a particular disease, disorder, or condition. In some embodiments, such treatment may be to subjects who do not exhibit symptoms of the associated disease, disorder, or condition, and / or subjects who exhibit only early symptoms of the disease, disorder, or condition. Alternatively or additionally, such treatment may be to subjects who exhibit one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, treatment may be to subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In some embodiments, treatment may be to subjects who are known to have one or more susceptibility factors that are statistically correlated 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 being treated, or who exhibits only early signs or symptoms of the condition being treated, with the purpose of resulting in a reduction, prevention, or lowering of the risk of the occurrence of the condition. Thus, prophylactic treatment serves as a defensive treatment against the condition. "Therapeutic treatment" includes treatment administered to a subject who exhibits symptoms or symptoms of the condition, with the purpose of reducing the severity or progression of the condition.

[0059] Unit dose: As used herein, the term "unit dose" refers to an amount administered as a single dose and / or in a physically separate unit of pharmaceutical composition. In many embodiments, a unit dose contains a predetermined amount of active agent, e.g., a predetermined viral titer (number of viruses, virions, or viral particles in a given volume). In some embodiments, a unit dose includes all of the agent in a single dose. In some embodiments, more than one unit dose is administered to achieve the total amount of a single dose. In some embodiments, administration of multiple unit doses is necessary or expected to be necessary 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 solid therapeutic moieties, 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 in a formulation that includes any of a variety of components in addition to the therapeutic moiety(s). For example, acceptable carriers (e.g., pharma- ceutically acceptable carriers), diluents, stabilizers, buffers, preservatives, etc. may be included. In many embodiments, the appropriate total daily dosage of a particular therapeutic agent may be a portion or multiple of a unit dose, and may be determined, for example, by a medical practitioner within the scope of sound medical judgment, as will be understood by those skilled in the art.In some embodiments, the specific effective dosage level for any particular subject or organism may depend on a variety of factors, such as the disorder being treated and the severity of the disorder; the activity of the specific active compound employed; the age, weight, general health, sex, and diet of the subject of the specific composition employed; the administration time and excretion rate of the specific active compound employed; the duration of treatment; drugs and / or additional therapies used in combination or simultaneously with the specific compound(s) employed; and similar factors well known in the medical field. [Brief description of the drawings]

[0060] [Figure 1]1 is a chart 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 viral particles per cell or 2,000 viral particles per cell. For each serotype tested, the chart contains two replicates, with each replicate including analyses at 5,000 viral particles per cell and 2,000 viral particles per cell, in the order shown. Data represents infection efficiency. [Diagram 2] 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells infected with the indicated adenoviral serotypes. Cells were infected with 5,000 viral particles per cell or 2,000 viral particles per cell. For each serotype tested, the chart contains two replicates, with each replicate including the results of analysis at 5,000 viral particles per cell and 2,000 viral particles per cell, in the order shown. Data represents relative infection efficiency. [Diagram 3] FIG. 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 1 3 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Data represents the efficiency of infection. [Figure 4] FIG. 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 1 6 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Data represents the efficiency of infection. [Diagram 5] 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 1 3 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Data represents the efficiency of infection. [Figure 6]1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 1 6 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Data represents the efficiency of infection. [Figure 7] Figure 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 2 3 hours after infecting the cells with the indicated adenoviral serotype. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart includes data from two replicates. Data represent infection efficiencies. Two replicate preparations of serotype F35 were tested. GLN indicates that the indicated adenoviral vector contains an expression cassette encoding a GFP luminescent reporter. [Figure 8] FIG. 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 2 6 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Data represents the efficiency of infection. [Figure 9] 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 2 3 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Data represents the efficiency of infection. [Figure 10] 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 2 6 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Data represents the efficiency of infection. [Figure 11]FIG. 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 3 3 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Data represents the efficiency of infection. [Figure 12] FIG. 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 3 6 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart includes two or three replicates. Data represent infection efficiencies. [Figure 13] 1 is a chart showing the results of anti-hexon staining of CD34+ cells from donor 3 3 or 6 hours after infection of the cells with the indicated adenovirus serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains two replicates, with each replicate including analyses at 3 and 6 hours post-infection, in the order shown. Data represent infection efficiencies. [Figure 14] FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 1, 3 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 15] FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 1 6 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Data represent relative infection efficiencies. [Figure 16]FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 1 3 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 17] FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 1 6 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Data represent relative infection efficiencies. [Figure 18] FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 2, 3 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains two replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 19] FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 2 6 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 20] FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 2, 3 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 21]FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 2 6 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 22] FIG. 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 3, 3 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart contains three replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 23] FIG. 13 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 3 6 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 2,000 viral particles per cell. For each serotype tested, the chart includes two or three replicates. Error bars represent technical replicates. Data represent relative infection efficiencies. [Figure 24] 1 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 2 3 or 6 hours after infection of the cells with the indicated adenoviral serotypes. Cells were infected with 5,000 viral particles per cell. For each serotype tested, the chart contains two replicates, with each replicate including analyses at 3 and 6 hours post-infection in the order shown. Error bars represent technical replicates. Data represent relative infection efficiency. [Diagram 25]1 is an image of a gel showing digestion of the first generation adenovirus genome of serotype Ad11 obtained from an Ad preparation produced from HEK293 cells transfected with a plasmid containing the first generation adenovirus genome of serotype Ad11. As indicated in the table included in the figure, the purified adenovirus genome was digested with either BspHI (lane 3) or SmaI (lane 5), and for comparison, the parental plasmid was also digested (lanes 2 and 4, respectively). Also shown is a representation of the expected digestion fragments based on the sequences of the Ad genome and the plasmid. [Figure 26] 1 is an image of a gel showing digestion of the first generation adenovirus genome of serotype Ad34 obtained from an Ad preparation produced from HEK293 cells transfected with a plasmid containing the first generation adenovirus genome of serotype Ad34. Purified adenovirus genomes were digested with either SmaI (lane 2) or SspI (lane 3) as indicated in the table included in the figure. Also shown is a representation of the expected digestion fragments based on the sequence of the Ad genome. [Figure 27] This is an image of a gel showing digestion of the first generation Ad35++ genome obtained from an Ad preparation produced from HEK293 cells transfected with a plasmid containing the first generation Ad35++ genome. As indicated in the table included in the figure, the purified adenoviral genome was digested with BspHI (lane 2) and the parental plasmid was also digested for comparison (lane 3). Also shown is a representation of the expected digestion fragments based on the Ad genome and plasmid sequences. * indicates the lane of the replicate sample. [Figure 28]1 is an image of a gel showing digestion of the first generation Ad35++ genome from an Ad preparation produced from HEK293 cells transfected with a plasmid containing the first generation Ad35++ genome. The gel labeled Observation #1 was run for a longer time to separate the larger DNA fragments, and the gel labeled Observation #2 was run for a shorter time to separate the shorter DNA fragments. As indicated in the table included in the figure, the purified adenoviral genome was digested with SmaI (lane 2) and the parental plasmid was also digested for comparison (lane 3). Also shown is a representation of the expected digestion fragments based on the Ad genome and plasmid sequences. [Figure 29] FIG. 1 is a chart showing the results of GFP analysis of HEK293 cells 25 hours after infecting the cells with first generation adenoviral vectors of the indicated adenovirus serotypes. Cells were infected with 100, 200, 500, and 1,000 viral particles per cell. Data represent infection efficiency. NTC indicates no treatment control. [Diagram 30] 1 is a chart showing the results of GFP analysis of HEK293 cells 24 hours after infection of the cells with first generation adenoviral vectors of the indicated adenovirus serotypes. Cells were infected with 100, 200, 500, 1,000, and 2,000 viral particles per cell. Data represents the efficiency of infection. [Diagram 31] 1 is a chart showing the results of GFP analysis of K562 cells 24 hours after infection of the cells with first generation adenoviral vectors of the indicated adenovirus serotypes. Cells were infected with 100, 200, 500, 1,000, and 2,000 viral particles per cell. Data represents infection efficiency. [Diagram 32]1 is a chart showing the results of GFP analysis of CD34+ cells from donor 2 48 hours after infecting the cells with first generation adenoviral vectors of the indicated adenoviral serotypes. Cells were infected with 500, 2,000, and 5,000 viral particles per cell. For conditions using 2,000 and 5,000 viral particles per cell, the chart includes two replicates. Data represents infection efficiency. [Diagram 33] Figure 1 shows the gating used for CD34+ and CD34+ / CD90+ population analysis using flow cytometry. Purified CD34+ cells were stained with anti-CD34 and anti-CD90 antibodies, and transduction efficiency was measured in CD34+high / CD90+ cells. Boxes indicate the gates used to define cell populations. Arrows from one plot to another indicate that the population gated in the first plot is shown in the second plot. Percentages indicate the percent of cells included in each specified gate. Data shown in this figure correspond to CD34+ cells from donor 1 46 hours after infection of cells with first generation adenoviral vectors of serotype Ad34 at 5,000 viral particles per cell. [Diagram 34] FIG. 1 is a chart showing the results of GFP analysis of CD34+ and CD34+ / CD90+ cells from donor 1 46 hours after infection of the cells with first generation adenoviral vectors of the indicated adenoviral serotypes. The percentage of cells that are GFP positive is shown. Cells were infected with 2,000 and 5,000 viral particles per cell. Data represent infection efficiency. * indicates no data collected for the indicated condition. [Diagram 35] FIG. 1 is a chart showing the results of GFP analysis of CD34+ and CD34+ / CD90+ cells from donor 3 46 hours after infection of the cells with first generation adenoviral vectors of the indicated adenoviral serotypes. The percentage of cells that are GFP positive is shown. Cells were infected with 500, 2,000, and 5,000 viral particles per cell. Data represent the efficiency of infection. [Diagram 36]FIG. 1 is a chart showing the results of GFP analysis of CD34+ and CD34+ / CD90+ cells from donor 1 46 hours after infection of the cells with first generation adenoviral vectors of the indicated adenoviral serotypes. The GFP geometric mean fluorescence intensity (MFI) of GFP positive cells is shown. Cells were infected with 2,000 and 5,000 viral particles per cell. Data represent infection efficiency. * indicates no data collected for the indicated condition. [Figure 37] FIG. 1 is a chart showing the results of GFP analysis of CD34+ and CD34+ / CD90+ cells from donor 3 46 hours after infection of the cells with first generation adenoviral vectors of the indicated adenoviral serotypes. The GFP geometric mean fluorescence intensity (MFI) of GFP positive cells is shown. Cells were infected with 500, 2,000, and 5,000 viral particles per cell. Data represent infection efficiency. [Figure 38] 1 is a chart showing the results of qPCR analysis of adenoviral DNA in HEK293 cells 3 hours after infecting the cells with the indicated adenovirus serotypes. Cells were infected with 100 and 500 viral particles per cell. Data represents relative infection efficiency. [Figure 39] FIG. 13 is a chart showing the results of qPCR analysis of adenoviral DNA in CD34+ cells from donor 2, 3 hours after infecting the cells with the indicated adenoviral serotypes. Cells were infected with 500, 2,000, and 5,000 viral particles per cell. For conditions using 2,000 and 5,000 viral particles per cell, the chart includes two replicates. Data represent relative infection efficiencies. [Diagram 40] A list of nucleic acid and amino acid sequences corresponding to publicly available sequence accession numbers, some of which are included and / or utilized in whole and / or in part in the present disclosure, and / or some of which are incorporated herein by reference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present disclosure provides methods and compositions comprising adenoviral vectors advantageous for gene therapy targeted to HSCs. The methods and compositions of the present disclosure are based, at least in part, on the observation that adenoviral vectors of serotypes 3, 7, 11, 14, 16, 21, 34, 37, and 50 exhibit several advantageous properties for gene therapy targeted to HSCs, compared to at least one or more reference adenoviral vectors (e.g., Ad5 vectors or Ad5 / 35 vectors). Adenoviral (or interchangeably "adenoviral") vectors include viral particles characterized by one or more adenoviral protein sequences, and optionally include an adenoviral genome. The adenoviral genome includes a nucleic acid sequence that (a) supports packaging (including conditional packaging) of the nucleic acid sequence into the adenoviral vector, and (b) includes sufficient adenoviral sequences to express the coding sequence. The adenoviral genome can be a linear double-stranded DNA sequence and / or molecule. As will be appreciated by those of skill in the art, linear genomes such as the adenoviral genome can be present in circular plasmids, for example, for purposes of virus production. The length of the native adenoviral genome ranges from 26 kb to 45 kb depending on the serotype.

[0062] The present disclosure includes methods and compositions comprising engineered adenoviral vectors and adenoviral genomes. Adenoviral vectors include engineered adenoviral vectors that include engineered adenoviral proteins or engineered adenoviral genomes. Engineered adenoviral genomes can be engineered, for example, to add or remove adenoviral genome sequences compared to a reference sequence.

[0063] There are 57 known human serotypes of adenovirus. In particular, one of them, adenovirus serotype 5 (Ad5), has been widely used historically in gene therapy research and adenovirus vector construction. In one study, HDAd5 / 35 vectors were used that contain Ad5 capsid proteins, except that the fiber is chimeric in that it contains 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 to increase affinity for CD46 (e.g., Ad5 / 35++). In certain embodiments, the Ad5 / 35++ vector is a chimeric Ad5 / 35 vector that includes a mutated Ad35++ fiber knob (see, e.g., Wang et al. 2008 J. Virol. 82(21):10567-79; specifically incorporated herein by reference in its entirety with respect to the fiber knob mutation). In various embodiments, the Ad35++ mutated fiber knob is an Ad35 fiber knob that has been mutated to increase affinity for CD46, e.g., 25-fold, thereby increasing 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 for the fiber, which is chimeric in that it comprises an Ad5 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob (e.g., as described in Shayakhmetov 2000 J Virol. 74(6):2567-2583), where the Ad35 fiber knob is a mutated Ad35 fiber knob containing mutations D207G and T245A that result in increased affinity for CD46 (see, e.g., Wang 2008 J Virol. 82(21):10567-79), and optionally the genome encoding the Ad5 / 35 vector contains an E1 deletion.However, the majority of humans have neutralizing serum antibodies against the Ad5 capsid protein, which can block in vivo transduction with adenoviral vectors containing the Ad5 capsid, such as the HDAd5 / 35 vector. The presence of neutralizing serum antibodies against the Ad5 capsid protein does not negate the therapeutic value of adenoviral vectors containing the Ad5 capsid, but an adenoviral vector that does not contain the Ad5 capsid would provide additional benefits. At least one reason for this advantage is that Ad5 vectors may induce clinically significant immune responses in subjects with serum antibodies against the Ad5 capsid protein (see, e.g., Somanathan et al. 2020 Mol. Ther. 28(3):784-793), whereas serotypes that do not contain the Ad5 capsid protein are unlikely to induce such immune responses. At least a second reason is that neutralizing serum antibodies against the Ad5 capsid protein may reduce the therapeutic efficacy of Ad5 gene therapy vectors by inactivating vector particles, and serotypes that do not contain the Ad5 capsid protein are less likely to be inactivated.

[0064] The present disclosure, in various embodiments, includes adenovirus serotypes that exhibit infection of target HSCs, including increased infection of HSCs, compared to reference adenovirus serotypes, e.g., Ad5 and / or Ad5 / 35, and are therefore useful for producing adenoviral vectors for transduction into HSCs. The present disclosure includes adenoviral vectors of serotypes 3, 7, 11, 14, 16, 21, 34, 37, or 50.

[0065] 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. The native adenoviral capsid contains three proteins: fiber, penton, and hexon. The hexon occupies the majority of the viral capsid and forms 20 triangular faces. A penton base is located at each of the 12 vertices of the capsid, and a fiber (also called knobbed fiber) projects from each penton base. The pentons and fibers, especially the fiber knobs, are particularly important for receptor binding and internalization, as they facilitate the attachment of the capsid to the host cell.

[0066] The adenoviral genome comprises adenoviral DNA flanked by serotype-specific terminal inverted 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 100-200 base pairs (e.g., about 160 base pairs) in length and are most highly conserved at the nucleotide position (e.g., about 50 base pairs) closest to the end of the adenoviral genome. 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 sequences are located in the left portion of the genome.

[0067] The native adenoviral genome encodes several proteins, including the early transcription units E1, E2, E3, and E4, as well as the late transcription unit that encodes the structural protein components of the adenoviral vector. Early (E) and late (L) transcription are separated by the initiation of replication of the viral genome. The E1 region (E1A and E1B) encodes proteins involved in the regulation of transcription of the viral genome. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for the replication of the viral genome. 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 the major processing of a single primary transcript generated by the major late promoter (MLP). The MLP is particularly efficient at the later stages of infection. The mRNA transcribed using this promoter may contain a 5'-triple leader (TPL) sequence that enhances translation.

[0068] 1(B).Ad3, 7, 11, 14, 16, 21, 34, 37, and 50 gene therapy vectors The present disclosure includes the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genomes. In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome is a single- or double-stranded DNA sequence that includes the ITRs of an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector (e.g., a 5' ITR according to SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, or 129 and a 3' ITR according to SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, or 130), 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, 7, 11, 14, 16, 21, 34, 37, or 50 genome is a single- or double-stranded DNA sequence that includes a packaging sequence of an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector (e.g., a packaging sequence according to SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, or 131), or a packaging sequence having 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, 7, 11, 14, 16, 21, 34, 37, or 50 genome is a single-stranded or double-stranded DNA sequence that includes a sequence having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to all, a portion, or a contiguous or non-contiguous corresponding portion of a reference Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome (e.g., SEQ ID NOs: 145, 146, 147, 148, 149, 150, 151, 152, or 153).

[0069] In various embodiments, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome is any nucleotide sequence that includes at least the ITRs of an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector (e.g., a 5' ITR according to SEQ ID NO: 1, 17, 33, 49, 65, 81, 97, 113, or 129 and a 3' ITR according to SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, or 130), 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, 7, 11, 14, 16, 21, 34, 37, or 50 genome is an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome that has one or more nucleotides, coding sequences, and / or genes completely or partially deleted compared to a reference sequence. For example, in some embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome can be a genome that does not include one or more of E1, E2, E3, and E4. In certain embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome is a genome that does not contain any coding sequences of the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome (e.g., a "gutless" vector that includes ITRs with at least 75% sequence identity to the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome ITRs, but does not contain any of the coding sequences present in the reference Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome).

[0070] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome includes, does not include, or includes a deletion of all or a portion of the E1 sequence according to SEQ ID NO: 4, 20, 36, 52, 68, 84, 100, 116, or 132, 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).

[0071] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome includes, does not include, or includes a deletion of all or a portion of the E2 sequence according to SEQ ID NO: 5, 21, 37, 53, 69, 85, 101, 117, or 133, 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).

[0072] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome includes, does not include, or includes a deletion of all or a portion of the E3 sequence according to SEQ ID NO: 46, 22, 38, 54, 70, 86, 102, 118, or 134, 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).

[0073] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome includes or does not include a fiber-encoding sequence, where 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, 23, 39, 55, 71, 87, 103, 119, or 135.

[0074] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome includes or does not include a sequence encoding a fiber shaft, where 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, 24, 40, 56, 72, 88, 104, 120, or 136.

[0075] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome includes or does not include a sequence encoding a fiber knob, where 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, 25, 41, 57, 73, 89, 105, 121, or 137.

[0076] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome may or may not include a sequence encoding a fiber tail, where 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 the fiber tail of SEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, or 135 (e.g., to a portion of the fiber sequence including all nucleotides 5' to the sequence encoding the fiber shaft and / or including all nucleotides encoding a portion of the fiber at the N-terminus of the fiber shaft).

[0077] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome includes or does not include a sequence encoding a penton, where 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, 26, 42, 58, 74, 90, 106, 122, or 138.

[0078] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome may or may not include a sequence encoding a hexon, where 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, 27, 43, 59, 75, 91, 107, 123, or 139.

[0079] The disclosure includes Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors comprising 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 an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 fiber (e.g., a fiber according to SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, or 140).

[0080] The disclosure includes Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors comprising a fiber tail 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber tail (e.g., a fiber tail of a fiber according to SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, or 140, e.g., the fiber tail is a portion of the fiber that includes all the amino acids at the N-terminus of the fiber shaft).

[0081] The disclosure includes Ad3, 7, 11, 14, 16, 21, 34, 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber shaft (e.g., a fiber shaft according to SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 125, or 141).

[0082] The disclosure includes Ad3, 7, 11, 14, 16, 21, 34, 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 an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 fiber knob (e.g., a fiber knob according to SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, or 142).

[0083] The disclosure includes Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors comprising 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, 7, 11, 14, 16, 21, 34, 37, or 50 penton (e.g., a penton according to SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, or 143).

[0084] The disclosure includes Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors comprising 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, 7, 11, 14, 16, 21, 34, 37, or 50 hexon (e.g., a hexon according to SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, or 144).

[0085] In various embodiments, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector is any adenoviral vector that includes 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber (e.g., a fiber according to SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, or 140).

[0086] In various embodiments, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector is any adenoviral vector that includes 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber tail (e.g., the fiber tail of a fiber according to SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, or 140, e.g., the fiber tail is a portion of the fiber that includes all the amino acids at the N-terminus of the fiber shaft).

[0087] In various embodiments, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector is any adenoviral vector that includes 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber shaft (e.g., a fiber shaft according to SEQ ID NO: 13, 29, 45, 61, 77, 93, 109, 125, or 141).

[0088] In various embodiments, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector is any adenoviral vector that includes 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber knob (e.g., a fiber knob according to SEQ ID NO: 14, 30, 46, 62, 78, 94, 110, 126, or 142).

[0089] In various embodiments, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector is any adenoviral 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 an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 penton (e.g., a penton according to SEQ ID NO: 15, 31, 47, 63, 79, 95, 111, 127, or 143).

[0090] In various embodiments, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector is any adenoviral vector that includes 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, 7, 11, 14, 16, 21, 34, 37, or 50 hexon (e.g., a hexon according to SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144).

[0091] Thus, an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector can be a chimeric adenovirus 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber knob, and at least one protein or portion thereof (such as a 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.

[0092] The Ad3, 7, 11, 14, 16, 21, 34, 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber shaft and at least one protein or portion thereof (such as a 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.

[0093] The Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector can be a chimeric adenovirus vector comprising 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, 7, 11, 14, 16, 21, 34, 37, or 50 fiber tail and at least one protein or portion thereof (such as a 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.

[0094] An Ad3, 7, 11, 14, 16, 21, 34, 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, 7, 11, 14, 16, 21, 34, 37, or 50 penton and at least one protein or portion thereof (such as a 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.

[0095] The Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector can be a chimeric adenovirus vector comprising 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, 7, 11, 14, 16, 21, 34, 37, or 50 hexon and at least one protein or portion thereof (such as a 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.

[0096] Exemplary sequences for each component (e.g., ITRs, packaging sequences, genes, and proteins) of Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 are provided in the table below. Viral polypeptides include proteins and portions or fragments thereof that are components of the viral vector, such as fiber, fiber knob, fiber shaft, fiber tail, penton, or hexon.

[0097] For example, various sequences by accession number disclosed herein, including the accession sequences designated SEQ ID NOs: 145, 146, 147, 148, 149, 150, 151, 152, and / or 153 shown in Tables 1-18 herein, are provided herein in Figure 40. One of skill in the art will understand that such sequences, including the sequences disclosed in Figure 40, 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]

[0098] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector or genome comprises a modification that reduces and / or eliminates viral replication in the recipient. Broadly speaking, there are three known "generations" of adenoviral vectors and genomes that have been engineered to reduce and / or eliminate viral replication in the recipient. The adenoviral vectors of the present disclosure can include vectors from any of these three generations.

[0099] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 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 interest serotype) in that at least the regulatory E1 genes (E1a and E1b) have been deleted from the Ad genome (a "first generation" vector modification). E1a and E1b are the first transcriptional regulators produced during the adenovirus replication cycle. E1 deletion reduces or eliminates the expression of certain viral genes controlled by E1, and thus the E1 deleted helper virus is replication defective. Thus, the first generation Ad vector is replication defective in the recipient. In some embodiments, the first generation adenovirus vector is engineered to delete the E1 and E3 genes. The retained portion of the reference genome may be sequence identical to the reference genome, or may have less than 100% identity with 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 the expression of a particular viral gene. By way of example, if an E1-deleted Ad5 vector encodes Ad5 E4orf6, the helper vector can be propagated in a cell line that expresses Ad5 E1. As an exemplary cell type for adenoviral vector production, HEK293 cells express Ad5 E1b55k, which is known to form a complex with Ad5 E4 protein ORF6.

[0100] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome differs from the 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 are deleted ("second generation" modification). Second generation Ad have a larger payload capacity than first generation Ad and are less replicative than first generation viruses. In some embodiments, second generation adenoviral vectors are engineered to delete nonstructural genes E2 and E4 in addition to E1 / E3 deletion, 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, for example, at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity.

[0101] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome differs from the reference Ad sequence in that at least all viral coding sequences have been removed from the Ad genome and engineered to retain only the genomic ITRs and genomic packaging sequences or functional fragments thereof (a "third generation" modification). Third generation adenoviral vectors can also be referred to as gutless high-capacity adenoviral vectors or helper-dependent adenoviral vectors (HdAd). The retained portion of the reference genome can be sequence identical to the reference genome or can have less than 100% identity with the reference genome, for example, at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% identity.

[0102] Third generation Ad genomes are helper-dependent because they do not code for proteins necessary for viral production. Helper-dependent genomes can only be packaged into vectors when present in cells that contain nucleic acid sequences that provide viral proteins in trans. These helper-dependent vectors are also characterized by a larger capacity and lower immunogenicity than first and second generation vectors. HDAd vectors do not express viral genes when used as vectors, reducing the risk of cytotoxicity or interferon responses in recipients.

[0103] Helper-dependent adenoviral vectors (HDAd), engineered to lack all viral coding sequences, can efficiently transduce a wide variety of cell types and mediate long-term transgene expression with little chronic toxicity. By deleting viral coding sequences and leaving only the cis-acting elements required for genome replication (ITRs) and packaging (ψ), the cellular immune response to the Ad vector is reduced. HDAd vectors have a large cloning capacity and are capable of delivering large payloads. These payloads can include large therapeutic genes as well as multiple transgenes and large regulatory elements to enhance, extend, and regulate transgene expression. It has also been observed that certain HDAd vector genomes can be packaged most efficiently when the genome is at least a minimum full length, e.g., a minimum to at least 20 kb full length (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 kb), which may include, for example, a therapeutic payload and / or a "stuffer" sequence. If the payload does not utilize a number of nucleotides that results in an adenoviral genome having at least the desired length, a stuffer sequence can be used to achieve or exceed the desired length. The present disclosure includes that beneficial use of the vectors provided herein does not require a minimum length for efficient packaging, and meeting any desired length may be advantageous, but is not necessary for use of the compositions and methods provided herein. As with other adenoviral vectors, a typical HDAd genome generally remains episomal and is not incorporated into the host genome.

[0104] Because HDAd vectors do not encode viral proteins necessary for the production of viral particles, viral proteins must be provided in trans, e.g., expressed in and / or by the cells in which the HDAd genome resides. In some HDAd vector systems, one viral genome (the helper genome) encodes all of the proteins necessary for replication (e.g., all of the structural viral proteins), but has a conditional deletion in the packaging sequence that renders it less likely to be packaged into a vector under certain vector production conditions (e.g., in the presence of an agent that reduces the function of the conditionally deleted packaging sequence). Thus, the HDAd donor viral genome comprises (e.g., comprises 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), such that the HDAd donor viral genome can be selectively packaged into HDAd viral vectors produced from structural elements expressed from the helper vector genome. In other words, Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper vectors can be used to produce Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vectors. Production of HD Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors can involve co-transfection of a plasmid containing the HDAd vector genome with a packaging-defective helper virus that provides structural and non-structural viral proteins. The helper virus genome can aid in the amplification of the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector, and the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector can 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.

[0105] 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 can contain a packaging sequence or a functional fragment thereof (e.g., a fragment of a packaging sequence that is sufficient for packaging, necessary for packaging, or necessary for efficient packaging of the Ad genome into a capsid) flanked by recombinase (e.g., loxP) sites, such that upon contact with a corresponding recombinase (e.g., Cre recombinase), the packaging sequence or a functional fragment thereof is excised from the helper genome by recombinase-mediated (e.g., Cre-mediated) site-specific recombination between the recombinase sites (e.g., loxP sites). The disclosure includes, inter alia, Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper vectors and genomes that contain two recombination sites flanking a packaging sequence or a functional fragment thereof, where the two recombination sites correspond to (i.e., act on or by) the same recombinase.

[0106] In various embodiments, the helper genome can contain a deletion of E1, e.g., where the helper genome contains all of the viral genes except E1, 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 genome present in the producer cell and the HDAd donor genome, a "stuffer" sequence can be inserted into the E3 region, rendering the recombinant too large to package and / or too inefficiently packaged.

[0107] For production of HDAd vectors, the HDAd donor genome can be delivered to a cell expressing a recombinase that excises the conditional packaging sequence of the helper vector (e.g., 293 cells expressing Cre recombinase (HEK293)), optionally delivered to the cell in a non-viral vector form, such as a bacterial plasmid form (e.g., the HDAd donor genome is present in a bacterial plasmid (pHDAd) and / or released by restriction enzyme digestion). The helper genome can be introduced into the cell, comprising the packaging sequence or a functional fragment thereof flanked by recombinase sites (e.g., loxP sites). In this manner, a producer cell can be transfected with an HDAd donor genome and a helper genome having a packaging sequence or a functional fragment thereof flanked by recombinase sites (e.g., loxP sites) can be introduced, where the cell expresses a recombinase (e.g., Cre) corresponding to the recombinase sites, thereby excising the packaging sequence or a functional fragment thereof and rendering the helper virus genome packaging-deficient (e.g., unable to be packaged), but still providing all of the trans-acting factors necessary for production of an HDAd donor vector containing the HDAd donor genome.

[0108] Similarly, HDAd production systems have been developed that use FLP (e.g., FLPe) / frt site-specific recombination, in which packaging of the helper genome is reduced or abolished in producer cells expressing FLP by FLP-mediated recombination between frt sites that flank the packaging sequence of the helper genome or a functional fragment thereof.

[0109] The HDAd vector, including the payload-containing donor vector genome, can be isolated from the producer cell. The HDAd donor vector can be further purified from the helper vector by physical means. In general, some degree of contamination of the helper vector and / or helper genome in HDAd viral vectors and HDAd viral vector preparations can occur, but is tolerable.

[0110] HDAd3, 7, 11, 14, 16, 21, 34, 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, 7, 11, 14, 16, 21, 34, 37, or 50 vectors or genomes are helper-dependent Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors or genomes. Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper vectors are vectors that contain a helper genome that contains a packaging sequence or a fragment thereof that is conditionally expressed (e.g., flanked by frt or loxP sites) and encodes all of the trans-acting factors required for the production of Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 virions that can package the donor genome.

[0111] The disclosure further includes an HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector production system comprising a cell comprising an HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor genome and an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper genome. In such particular cells, viral proteins encoded and expressed by the helper genome can be utilized to produce an HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector into which the HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor genome is packaged. Thus, the disclosure includes methods of producing HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vectors by culturing cells containing an HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor genome and an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper genome. In some embodiments, the cells encode and express a recombinase that corresponds to the recombinase orthogonal repeats that flank the packaging sequence of the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper vector. In some embodiments, the flanked packaging sequences of the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper genome are excised.

[0112] In some embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper genome encodes all of the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 coding sequences. In some embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 helper genome encodes and / or expresses all of the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 coding sequences except for one or more of the E1 and / or E3 coding sequences and / or E4 coding sequences. In various embodiments, the helper genome that does not encode and / or express the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 E1 gene does not encode and / or express the Ad3, 7, 11, 14, 16, 21, 34, 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 for producing HDAd donor vectors can be cells that express the E1 expression product.

[0113] The disclosure includes, inter alia, HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vectors and genomes that contain an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 ITR (a 5'Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 ITR and a 3'ITR of the same serotype), e.g., two Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 ITRs flanking the packaging sequence and payload. The disclosure includes, inter alia, HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vectors and genomes that have a deletion of E1 or a fragment thereof. The disclosure includes, inter alia, HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors and genomes in which E3 or fragments thereof have been deleted.

[0114] In various embodiments, excision of the packaging sequence or a functional fragment thereof from the Ad3, 7, 11, 14, 16, 21, 34, 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., at least 20%, 30%, 40%, 50%, 60%, 70%, and at least ... %, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100% reduction in vector growth, where optionally percent growth is measured as the number of viral particles produced by growth of an excised vector (a vector in which the sequences flanked by the recombinase sites have not been excised) compared to an intact vector (a vector in which the sequences flanked by the recombinase sites have been excised) or compared to a wild-type Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector under comparable conditions.

[0115] A further optional operational consideration may be to engineer the helper genome to have a size that allows the helper vector to be separated from the HDAd3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector by centrifugation, e.g., by CsCl ultracentrifugation. One means of achieving this result is to increase the size of the helper genome compared to a typical Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome. In particular, the adenoviral genome can be engineered to be increased to at least 104% of the length of the wild type. Certain helper vectors of the present disclosure can accommodate payload and / or stuffer sequences.

[0116] The disclosure includes that in various embodiments, the vectors or genomes of the disclosure can include a selection of elements selected from or having at least 75% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the corresponding sequences of a single particular serotype. To provide an illustrative example, all elements can correspond to Ad34 (e.g., can have at least 75% sequence identity to the sequences), except for sequences (e.g., payloads, e.g., heterologous payloads) that are otherwise indicated.

[0117] I(C).Ad3, 7, 11, 14, 16, 21, 34, 37, and 50 gene therapy vector payloads The Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vectors and genomes of the present disclosure may comprise a variety of heterologous nucleic acid payloads, which may 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 protein of therapeutic interest, or expression of a gene editing system, e.g., a CRISPR / Cas system or a base editing system, that results in a sequence modification of therapeutic interest.

[0118] In some embodiments, the payload may include a gene. A gene may 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, insulators, etc. A gene may include introns and other DNA sequences spliced ​​from the expressed mRNA transcript, as well as variants resulting from alternative splice sites. A coding sequence may also include alternative synonymous codon usage compared to a reference sequence, for example, altered codon usage compared to the reference according to the codon preferences of a specific organism or type of target cell.

[0119] A payload may comprise a single gene or multiple genes. A payload may comprise a single coding sequence or multiple coding sequences. A payload may comprise a single regulatory sequence or multiple regulatory sequences. A payload may comprise multiple coding sequences, where the individual expression products of the coding sequences function together, for example, as in the case of an endonuclease and a guide RNA, or function independently, for example, 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 element (e.g., expression product or regulatory sequence encoded by the payload) that is not encoded by the reference wild-type Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 genome may be referred to herein as a heterologous expression product.

[0120] 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% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, 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.

[0121] I(C)(i).Payload expression product The payload of the adenoviral donor vector or adenoviral donor genome of the present disclosure may 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 a disease or condition characterized by low expression or activity of a biologically active protein compared to a reference level. 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, it is not necessary for all or a portion of the donor vector payload to be integrated into the host cell genome in order to deliver the donor vector or genome to a target cell and produce the intended or desired effect, for example, in certain cases where the intended or desired 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 in order to deliver the donor vector or genome to the target cell and produce an intended or desired effect, e.g., if expression of the payload-encoded expression product is desired in a progeny cell of the introduced target cell. In various embodiments, the payload may include a nucleic acid sequence engineered to integrate, e.g., by recombination or transposition, into the host cell genome (an "integration element").

[0122] Genetic sequences encoding one or more therapeutic proteins can be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences. In certain embodiments, the genetic sequences encoding any of these sequences may also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence to provide for easy excision of the genetic sequence encoding the sequence and replacement with another genetic sequence encoding a different sequence. In certain embodiments, the genetic sequence encoding the sequence may be codon-optimized for expression in mammalian cells.

[0123] 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), Fan cP(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 on autoreactive T cells, IL4, IL10, IL12, IL13, IL1Ra, sIL1RI, sIL1RII, sTNFRI, sTNFRII, globin family genes, WAS, phox, dystrophin, pyruvate, quinine Examples of genes that may be involved in the expression of the present invention 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 agents and / or expression products described herein.

[0124] The therapeutic gene can be selected to provide a therapeutically effective response to a disease related to red blood cells and blood clotting. In a particular embodiment, the disease is a hemoglobinopathy, such as thalassemia or sickle cell disease / proneness. The therapeutic gene can be, for example, a gene that induces or increases the production of hemoglobin, induces or increases the production of β-globin, γ-globin, or α-globin, or increases oxygen availability to cells in the body. The therapeutic gene can be, for example, HBB or CYB5R3. Exemplary effective treatments can, for example, increase blood cell counts, improve blood cell function, or increase cellular oxygenation in a patient. In another particular embodiment, the disease is hemophilia. The therapeutic gene may be, for example, a gene that increases the production of blood clotting / clotting factor VIII or blood clotting / clotting factor IX, resulting in the production of normal forms of blood clotting factor VIII or blood clotting factor IX, a gene that decreases the production of antibodies against blood clotting / clotting factor VIII or blood clotting / clotting factor IX, or a gene that results in the proper formation of blood clots. Exemplary therapeutic genes include F8 and F9. Exemplary effective treatments may, for example, increase or induce the production of blood clotting / clotting factor VIII and factor IX, improve the function of blood clotting / clotting factor VIII and factor IX, or reduce blood clotting in a subject.

[0125] In various embodiments of the present disclosure, the donor vector encodes a globin gene, where the globin protein encoded by the globin gene is selected from gamma-globin, beta-globin, and / or alpha-globin. The globin gene of the present disclosure may include one or more regulatory sequences, such as, for example, a promoter, operably linked to a nucleic acid sequence encoding the globin protein. As will be appreciated by those skilled in the art, gamma-globin, beta-globin, and / or alpha-globin are each components of fetal and / or adult hemoglobin and are therefore useful in the various vectors disclosed herein.

[0126] In various embodiments, increased 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 that can be identified as γ-globin (or alternatively β-globin or α-globin) by one of skill in the art or as described herein), regardless of the sequence of each protein; and / or (iii) expressing a heterologous globin protein in a cell or system, e.g., a globin protein that was not encoded by the host cell prior to gene therapy.

[0127] The following references provide 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 (beta and gamma globin sequences), which sequences are incorporated herein by reference: (1) GenBank Accession No. Z84721 (Mar. 19, 1997); (2) GenBank Accession No. NM_000517 (Oct. 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 in Augusta, Ga. (available online at globin.cse.psu.edu); (5) GenBank Accession No. NM_000517 (Oct. 31, 2000); No.J00179(Aug.26,1993) or U01317.1;(6)Tagle et al.,Genomics(1992)13(3):741-760;(7)Grovsfeld et al.,Cell(1987)51(6):975-985;(8)Li et al. al.,Blood(1999)93(7):2208-2216;(9)Gorman et al.,J.Biol.Chem.(2000)275(46):35914-35919;(10)Slightom et al.,Cell(1980)21(3):627-638;(11)Fritsch et al. al.,Cell(1980)19(4):959-972;(12)Marotta et al. 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.

[0128] An exemplary amino acid sequence of hemoglobin subunit β is provided, for example, in NCBI Accession No. P68871. An exemplary amino acid sequence of β-globin is provided, for example, in NCBI Accession No. NP_000509.

[0129] 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.

[0130] As another example, the therapeutic gene can be selected to provide a therapeutically effective response to a lysosomal storage disorder. In certain embodiments, the lysosomal storage disorder is mucopolysaccharidosis (MPS) type I; MPS type II or Hunter syndrome; MPS type III or Sanfilippo syndrome; MPS type IV or Morquio syndrome; MPS type V; MPS type VI or Maroteaux-Lamy syndrome; MPS type 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 that codes for an enzyme or induces the production of oxygen, or otherwise causes 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 disorders may, for example, encode or induce the production of enzymes involved in the breakdown of various substances in lysosomes, reduce, eliminate, prevent, or delay swelling in various organs including the head (especially macrocephaly), liver, spleen, tongue, or vocal cords, reduce fluid in the brain, reduce heart valve abnormalities, prevent or widen narrowing of airways, prevent infections and associated upper respiratory tract conditions such as sleep apnea, reduce, eliminate, prevent, or delay destruction of neurons and / or associated symptoms.

[0131] As another example, a therapeutic gene 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 that encodes an enzyme, a gene that encodes an antibody, or a gene that encodes a hormone. Exemplary therapeutic genes and gene products (in addition to those otherwise enumerated herein) include 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, CNTF, 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-1 1, ING1, interferon α, interferon β, interferon γ, IRF-1, JUN, KRAS, LUCA-1 (HYAL1), LUCA-2 (HYAL2), LYN, MADH4, MADR2, These include MCC, mda7, MDM2, 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, resulting in a reduction in the number of cancer cells, a decrease in tumor size, a slowing or elimination of tumor growth, or an alleviation of symptoms caused by the tumor.

[0132] As another example, therapeutic genes can be selected to provide a therapeutically effective response to an infectious disease. In a particular embodiment, the infectious disease is human immunodeficiency virus (HIV). The therapeutic genes can be, for example, genes that render immune cells resistant to HIV infection or enable immune cells to effectively neutralize the virus through immune reconstitution, polymorphisms in genes that code for proteins expressed by immune cells, genes that are not expressed in the patient and that are advantageous in fighting infection, genes that code for infectious agents, receptors or co-receptors, genes that code for ligands for receptors or co-receptors, viral and cellular genes essential for viral replication, such as genes that code for ribozymes, antisense RNAs, small interfering RNAs (siRNAs) or decoy RNAs to block the action of certain transcription factors, genes that code for dominant-negative viral proteins, intracellular antibodies, intrakines and suicide genes. 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.A therapeutically effective amount for treating HIV may, for example, increase the subject's immunity to HIV, ameliorate symptoms associated with AIDS or HIV, or induce a subject's innate or adaptive immune response to HIV.An immune response to HIV may include antibody production, provide prevention of AIDS, and / or ameliorate symptoms of AIDS or HIV infection in a subject, or reduce or eliminate HIV infectivity and / or virulence.

[0133] 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. The sequences encoding the binding domains can encode, for example, antibodies, chimeric antigen receptors, TCRs, or other binding polypeptides.

[0134] Antibodies and antibody fragments are exemplary of binding domains. The term "antibody" may refer to a polypeptide that includes one or more standard immunoglobulin sequence elements (e.g., heavy chain variable domain, light chain variable domain, and / or one or more CDRs) sufficient to confer specific binding to a particular antigen. Thus, the term antibody includes, but is not limited to, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents that contain them. An antibody may be a naturally occurring immunoglobulin (e.g., generated by an organism in response to an antigen). Synthetic, non-naturally occurring, or engineered antibodies may be generated by recombinant engineering, chemical synthesis, or other artificial systems or methods known to those skilled in the art.

[0135] 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 about 50 kD) and two identical light (L) chain polypeptides (each about 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 a "switch" connects 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 known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four "framework" regions (FR1, FR2, FR3, and FR4) that are somewhat invariant. In each VH and VL, the three CDRs and four FRs are arranged from the amino terminus to the 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 a binding moiety that can interact with an antigen. The constant domains can mediate the binding of the antibody to various immune system cells (e.g., effector cells and / or cells that mediate cytotoxicity), receptors, and elements of the complement system. The heavy and light chains are linked together by one disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions together, thereby linking the dimers together to form a tetramer. When a native immunoglobulin folds, the FR regions form β-sheets that confer a structural framework to the domain, and the CDR loop regions of both the heavy and light chains come together in three dimensional space to form a single hypervariable antigen-binding site located at the tip of the Y-shaped structure.

[0136] In some embodiments, the antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (including 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" may include any construct or format known in the art that utilizes the structural and / or functional features of antibodies (unless otherwise indicated or apparent from the context), including, but not limited to, 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 IgNAR or fragments thereof), camelid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affibodies, anti-idiotypic (anti-Id) antibodies (e.g., Examples of antibodies that may be used include, for example, anti-anti-Id antibodies), small modular immunopharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAb®), 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.

[0137] In various embodiments, the antibody comprises one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or a variable domain. In some embodiments, the 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 linked 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.

[0138] An antibody 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 of skill in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. As used herein, a "light chain" can be of distinct types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the light chain constant domain. In some embodiments, an antibody has a constant region sequence characteristic of a mouse, rabbit, primate, or human immunoglobulin. Naturally produced immunoglobulins are typically glycosylated at the CH2 domain. As is known in the art, the affinity and / or other binding attributes of an Fc region for an Fc receptor can be modulated via glycosylation or other modifications. In some embodiments, an antibody may lack covalent modifications (e.g., attachment of glycans) that it would have if produced naturally. In some embodiments, an antibody produced and / or utilized in accordance with the present invention comprises a glycosylated Fc domain, e.g., an Fc domain that has been modified or engineered, such as by glycosylation.

[0139] The term "antibody fragment" may refer to a portion of an antibody or antibody agent described herein, typically a portion including an antigen-binding portion or variable region thereof. Antibody fragments may be produced by any means. For example, in some embodiments, antibody fragments may be produced enzymatically or chemically by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, antibody fragments may be produced recombinantly (i.e., by expression of an engineered nucleic acid sequence). In some embodiments, antibody fragments may be produced synthetically, in whole or in part. In some embodiments, antibody fragments (particularly antigen-binding antibody fragments) may 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.

[0140] In some cases, it is beneficial for the binding domain to be derived from the same species as the species 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. Antibodies of human origin or humanized antibodies have less or no immunogenicity in humans and a smaller number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and engineered fragments thereof are generally selected to have reduced levels or no antigenicity in human subjects.

[0141] 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).

[0142] The present disclosure provides antibodies and other binding domains that bind to CD4, CD5, CD7, CD52, etc.; antibodies; 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); CI ITA; CLN3; complement factors, CORO1A; CTLA; C1 inhibitor; C9ORF72; DCLRE1B; DCLRE1C; 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), F ancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)); FasL; FUS; GATA1; globin family genes (i.e., γ-globin); F8; glutaminase; HBA1; HBA2; HBB; IL7RA; JAK3; LCK; LIG4; LRRK2; NHEJ1; NLX2.1; neutralizing antibodies; ORAI1; PARK2; PARK7; ph ox; 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; γC; as well as other therapeutic genes described herein.

[0143] HSCs 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 extracellular and intracellular components.

[0144] The extracellular CAR element may contain a binding domain that specifically binds to a marker that is frequently present on the surface of unwanted cells. When the binding domain binds to such a marker, the intracellular element induces the cell to destroy the cancer cell to which it is bound. The binding domain is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but may also be based on other formats that include an antibody-like antigen-binding site.

[0145] Intracellular CAR elements provide activation signals based on the incorporation of effector domains. 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) in the intracellular effector domain, whereas third generation CARs utilize CD3ζ in combination with CD28 and 401BB.

[0146] The intracellular or other cytoplasmic signaling elements of a CAR are responsible for the activation of cells expressing the CAR. Thus, the term "intracellular signaling element" or "intracellular element" is meant to include any portion of the intracellular domain sufficient to transmit an activation signal. The intracellular element of an expressed CAR may include an effector domain. An effector domain is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response in a 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 elicits a signal from the effector domain. An effector domain may 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, an effector domain indirectly promotes a cellular response by associating with one or more other proteins, such as a costimulatory domain, that directly promotes a cellular response.

[0147] 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. The 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 element that includes a T cell receptor and a costimulatory domain that can include a cytoplasmic sequence derived from a co-receptor or costimulatory molecule.

[0148] The effector domain may comprise one, two, three or more receptor signaling domains, intracellular signaling elements (e.g., cytoplasmic signaling sequences), costimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from 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 include 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, I and signaling and costimulatory domains selected from TGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, 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.

[0149] The stimulatory intracellular signaling element sequence may comprise an iTAM. Examples of iTAMs that comprise primary cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, and common FcRγ (FCER1G), FcγRlla, FcRβ (FcεRib), DAP10, and DAP12. In certain embodiments, variants of CD3ζ retain at least one, two, three, or all of the ITAM regions.

[0150] In certain embodiments, the effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, where the cytoplasmic signaling protein is a lymphocyte receptor or a signaling domain thereof, a protein containing multiple ITAMs, a costimulatory domain, or any combination thereof.

[0151] Further examples of intracellular signaling elements include cytoplasmic sequences of the CD3 zeta chain and / or co-receptors that act in concert to initiate signal transduction following engagement of the binding domains.

[0152] A costimulatory domain is a domain whose activation may be required for efficient lymphocyte response to cell marker binding. Several molecules are interchangeable as intracellular signaling elements or costimulatory domains. Examples of costimulatory domains include 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 ligands that specifically bind to CD83. For example, costimulation of CD27 has been shown to improve the expansion, effector function, and survival of human CART cells in vitro, and to increase the persistence and anticancer activity of human T cells in vivo (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, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDllb, ITGAX, CDllc, ITGBl, CD2 9, 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, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, and CD19a.

[0153] In certain embodiments, the amino acid sequence of the intracellular signaling element comprises a variant of CD3ζ and a portion of the 4-1BB intracellular signaling element.

[0154] In certain embodiments, the intracellular signaling element 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.

[0155] Intracellular elements may also include those 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 protein may include one or more of the following proteins: a G-protein coupled receptor, GPCR (Frizzled or Smoothened); a serine / threonine kinase receptor (BMPR or TGFR); or a cytokine receptor (IL1R, IL2R, IL7R, or IL15R).

[0156] CARs also generally contain one or more linker sequences that are used for various purposes within the molecule. For example, a transmembrane domain can be used to link the extracellular elements of the CAR to the intracellular elements. A flexible linker sequence that is membrane-proximal to the binding domain is often referred to as a spacer region and can be used to create additional distance between the binding domain and the cell membrane. This can be beneficial to reduce steric hindrance to binding due to proximity to the membrane. A common spacer region used for this purpose is the IgG4 linker. Depending on the cell marker to be targeted, a more compact spacer or a longer spacer can be used. Other possible CAR subelements are detailed elsewhere herein. Here, the following elements of CAR are described in more detail: (a) binding domain, (b) intracellular signaling element, (c) linker, (d) transmembrane domain, (e) connecting amino acid, and (f) regulatory function including tag cassette.

[0157] The transmembrane domain in the CAR molecule often functions to connect extracellular and intracellular elements through the cell membrane, and can anchor the expressed molecule to the membrane of the engineered cell.

[0158] The transmembrane domain may be derived from either natural and / or synthetic origin. If the origin is natural, the transmembrane domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may include at least the transmembrane region(s) of the α, β or ζ chain of the T cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In certain embodiments, the transmembrane domain comprises at least one of the following: e.g., 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, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CD may include the transmembrane region(s) of IIa, ITGAM, CDIIb, ITGAX, CDIIc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C. In certain embodiments, various human hinges may also be employed, including human Ig (immunoglobulin) hinges (e.g., IgG4 hinges, IgD hinges), GS linkers (e.g., GS linkers described herein), KIR2DS2 hinges, or CD8a hinges.

[0159] 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 an element of a CAR. For example, a CAR / TCR hybrid can have a naturally occurring TCR binding domain and an effector domain with which the TCR binding domain is not naturally associated. A CAR / TCR hybrid can have a TCR binding domain that includes a mutation and an ITAM signaling domain. A CAR / TCR hybrid can have a naturally occurring TCR and an inserted spacer region or transmembrane domain that is not naturally occurring.

[0160] I(C)(i)(b). Gene editing systems and components In various embodiments, the payload of the present disclosure encodes at least one or all of the elements of a gene editing system. The gene editing system of the present disclosure includes a CRISPR system, a base editing system, and a prime editing system. In general, the gene editing system may include multiple elements, 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 may include either (i) a CRISPR enzyme that is a CRISPR-associated RNA-guided endonuclease and at least one guide RNA (gRNA) in the case of a CRISPR system, (ii) a base editing enzyme and at least one gRNA in the case of a base editing system, or (iii) at least one prime editing gRNA in the case of a prime editing system. The nucleotide sequence encoding the gene editing system disclosed herein is typically too large to be included in most volume-limited vector systems, but the large volume of adenoviral vectors allows such sequences to be included in the adenoviral vectors and genomes of the present disclosure. A further advantage of the adenoviral vectors and genomes comprising payloads encoding the gene editing systems or elements of the present disclosure is that the adenoviral genome does not naturally integrate into the host cell genome, thereby facilitating transient expression of the gene editing systems and elements, which may be desirable, e.g., to avoid immunogenicity and / or genotoxicity.

[0161] In other embodiments, the gene editing system may include engineered Zing Finger Nucleases (ZFNs). For example, ZFNs are artificial endonucleases consisting of engineered zinc finger proteins (ZFPs) 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 engineering, ZFNs are targeted to cleave selected genomic sequences. ZFN-induced cleavage events trigger cellular repair processes that mediate efficient modification of the target locus. If ZFN-induced cleavage events are resolved by non-homologous end joining, small deletions or insertions can occur, resulting in effective gene knockdown. If the cleavage is resolved by homologous processes in the presence of a researcher-provided donor, small changes or entire transgenes can be introduced into the chromosome, often without selection. This is referred to as "gene correction" and "gene addition," respectively.

[0162] In some embodiments, a gene editing system (e.g., a CRISPR system, a base editing system, or a prime editing system) manipulates a nucleic acid sequence encoding γ-globin, for example, to increase the expression of γ-globin. Hemoglobin F (HbF), the main fetal form of hemoglobin, is formed by pairing of γ-globin and α-globin polypeptide subunits. Human fetal γ-globin genes (HBG1 and HBG2; two highly homologous genes produced by evolutionary duplication) are normally silenced around the time of birth, and the expression of adult β-globin gene expression (HBB and HBD) increases. Mutations that cause or allow the continued expression of fetal γ-globin throughout life can improve the phenotype of β-globin deficiency. Thus, reactivating fetal γ-globin genes can be therapeutically beneficial, especially in subjects with β-globin deficiency. A variety of mutations that increase γ-globin expression are known in the art (see, e.g., Wienert, Trends in Genetics 34(12):927-940, 2018; the entirety of which and the contents relating to mutations that increase γ-globin expression are incorporated herein by reference). Some of these mutations are found in the HBG1 or HBG2 promoters.

[0163] 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 encoded γ-globin 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 expression of γ-globin by modifying and / or inactivating an erythroid bcl11a enhancer that reduces expression of the BCL11A repressor protein in erythrocytes. In various embodiments, the gene editing system designed to increase γ-globin expression comprises a gRNA targeted to create a loss-of-function mutation in the gene encoding BCL11A.

[0164] I(C)(i)(b)(1). CRISPR payload expression product The CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated proteins) nuclease system is an engineered nuclease system used in bacterial-based genetic engineering. It is based in part on the adaptive immunity of many bacteria and archaea. When a virus or plasmid invades a bacterium, the invader's DNA segment 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, and guides the Cas nuclease to a region of homology to the crRNA in the target DNA, called the "protospacer." The Cas nuclease cleaves the DNA to generate a blunt end at the double-stranded break at the site specified by a 20-nucleotide complementary sequence contained within the crRNA transcript. In some cases, the Cas nuclease requires both the crRNA and tracrRNA for site-specific DNA recognition and cleavage.

[0165] A guide RNA (gRNA) is an example of a targeting element. In its simplest form, a gRNA provides a sequence that targets a site in a genome based on complementarity (e.g., crRNA). However, as described below, a gRNA can also include additional elements. For example, in certain embodiments, a gRNA can include a targeting sequence (e.g., crRNA) and an element that links the targeting sequence to a cleavage element. This linking element 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). The gRNA can also be linked to a cleavage element by other mechanisms, such as nanoparticles or expression or construction of dual or multi-purpose molecules. One of skill in the art will understand that gRNAs or other targeting elements to effect correction or modification of a selected nucleic acid sequence, for example, in a host cell of an adenoviral donor vector or genome of the present disclosure, can be readily designed and implemented, for example, based on available sequence information.

[0166] In certain embodiments, a targeting element (e.g., gRNA) can include one or more modifications (e.g., base modifications, backbone modifications) that provide new or improved characteristics (e.g., improved stability) to the nucleic acid. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not retain a phosphorus atom in the backbone. Suitable modified backbones containing a phosphorus atom can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates, with normal 3'-5' linkages, as well as those with inverted polarity where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages. Suitable targeting elements with inverted polarity may contain a single 3'-3' linkage (i.e., a single inverted nucleoside residue in which a nucleobase is missing or has a hydroxyl group in its place) at the 3'-most internucleotide linkage. Various salts (e.g., potassium chloride or sodium chloride), mixed salts, and free acid forms may be included.

[0167] Examples of cleavage elements include nucleases. CRISPR-Cas loci have more than 50 gene families, and there are no strictly universal genes, indicating that loci are rapidly evolving and highly diverse in structure. Exemplary Cas nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cpfl, C2c3, C2c2 and C2clCsyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, These include Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4.

[0168] There are three main types of Cas nucleases (type I, type II, and type III) and 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 Casl, Cas2, Csn2, and Cas9. These Cas nucleases are well known to those of skill in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth in NCBI reference sequence number NP269215, and the amino acid sequence of the Streptococcus thermophilus wild-type Cas9 polypeptide is set forth in NCBI reference sequence number WP_011681470.

[0169] In certain embodiments, Cas9 refers to an RNA-guided double-stranded DNA binding nuclease protein 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 activated. In some embodiments, the Cas9 enzyme comprises one or more catalytic domains of Cas9 proteins from bacteria such as Corynebacter, Sutterella, Legionella, Treponema, Filiforme, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. In some embodiments, Cas9 is a fusion protein, e.g., the two catalytic domains are derived from different bacterial species.

[0170] In some embodiments, the crRNA and tracrRNA can be combined into one molecule called single gRNA (sgRNA). In this engineering 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 generate a double-stranded break at a desired target in the cell's genome and utilize the cell's endogenous machinery to repair the induced break by HDR or NHEJ. Certain embodiments described herein utilize homology arms to facilitate HDR at a defined integration site.

[0171] In various embodiments, the variant of Cas9 nuclease contains a single inactive catalytic domain, such as RuvC "or HNH" enzyme or nickase. Cas9 nickase has only one active functional domain, and in some embodiments, it creates a single-strand break or nick by cleaving only one strand of the target DNA. In some embodiments, the mutant Cas9 nuclease with at least D10A mutation is Cas9 nickase. In other embodiments, the mutant Cas9 nuclease with at least H840A mutation is Cas9 nickase. Other examples of mutations present in Cas9 nickase include N854A and N863A. Cas9 nickase is used to introduce double-strand breaks, using at least two DNA-targeting RNAs that target opposite DNA strands. The double-nicked 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 depending on the target cell or organism.

[0172] I(C)(i)(b)(2). Base editor payload expression products The present disclosure includes, inter alia, base editors and nucleic acids encoding same, for example, the base editors or nucleic acids encoding same are present in an adenoviral vector or genome. The base editing system may include, as its components, a base editing enzyme and / or at least one gRNA. In some specific embodiments, the base editors and / or base editing systems of the present disclosure are present in an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 adenoviral vector.

[0173] Base editing refers to the selective modification of a nucleic acid sequence by converting a base or base pair in genomic DNA or cellular RNA to a different base or base pair (Rees & Liu, Nature Reviews Genetics, 19:770-788, 2018). There are two general classes of DNA base editors: (i) cytosine base editors (CBEs), which convert a guanine-cytosine base pair to a thymine-adenine base pair, and (ii) adenine base editors (ABEs), which convert an adenine-thymine base pair to a guanine-cytosine base pair. In certain embodiments, elements of the CRISPR system are combined with other enzymes or biologically active fragments thereof to directly introduce, cause, or generate a mutation, such as a point mutation, in a nucleic acid, e.g., DNA or RNA, without, for example, creating, causing, or generating one or more double-stranded breaks in the mutated nucleic acid. Certain combinations of such elements are known as base editors.

[0174] DNA base editors may contain noncatalytic nucleases fused to nucleobase deaminase enzymes and, in some cases, DNA glycosylase inhibitors. RNA base editors use RNA base-modifying elements to achieve similar changes.

[0175] When binding to the target locus in DNA, base pairing occurs between guide RNA and target DNA strand, and a small segment of single-stranded DNA is displaced.The DNA base in this single-stranded DNA bubble can be modified by deaminase enzyme.In certain embodiments, to improve efficiency in eukaryotic cells, the non-catalytic nuclease also generates nicks in the non-edited DNA strand, and uses the edited strand as a template to induce cells to repair the non-edited strand.

[0176] In the case of CBE, CRISPR-based editors can be created by linking cytosine deaminase with Cas nickase, e.g., Cas9 nickase (nCas9). To provide an example, nCas9 creates a nick in the target DNA by cutting a single strand, which can reduce the possibility of harmful indel formation compared to methods that require double-strand breaks. After binding to DNA, CBE deaminates the target cytosine (C) to a uracil (U) base. The resulting UG pair is then either repaired by the cellular mismatch repair machinery to convert the original CG pair to TA, or reverted to the original CG by base excision repair mediated by uracil glycosylase. In various embodiments, expression of a uracil glycosylase inhibitor (UGI), e.g., a UGI present in the payload, reduces the occurrence of the second outcome and increases the generation of TA base pairing.

[0177] In the case of adenosine base editors (ABEs), exemplary adenosine deaminases that can act on DNA for adenine base editing include mutant TadA adenosine deaminase (TadA*), which accepts DNA as a substrate. E. coli TadA typically acts as a homodimer and deaminates adenosines 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 inosine excision repair in cells is not as active as uracil excision, ABEs do not require any additional inhibitor proteins, such as UGI in CBEs. In some embodiments, a typical ABE may include three elements, including a wild-type E. coli tRNA-specific adenosine deaminase (TadA) monomer that can play a structural role during base editing, a TadA* mutant TadA monomer that catalyzes the deamination of deoxyadenosine, and a Cas nickase, such as Cas9 (D10A). In certain embodiments, a linker is placed between TadA and TadA*, and in certain embodiments, a linker is placed between TadA* and the Cas nickase. In various embodiments, one or both linkers include at least 6 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 a sequence according to (SGGS)2-XTEN-(SGGS)2, or a sequence otherwise known to one of skill in the art.

[0178] Base editors can directly convert one base or base pair to another, allowing efficient introduction of point mutations in non-dividing cells without producing excessive amounts of undesirable editing by-products such as insertions and deletions (indels). For example, base editors can result 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% indels.

[0179] DNA base editors can insert such point mutations into non-dividing cells without generating double-strand breaks. Because there are no double-strand breaks, base editors do not result in excessive amounts of undesirable editing by-products, such as insertions and deletions (indels). For example, base editors can produce 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% indels compared to techniques that rely on double-strand breaks.

[0180] The components of most base editing systems include (1) a targeted DNA-binding protein, (2) a nucleobase deaminase enzyme, and (3) a DNA glycosylase inhibitor.

[0181] Any nuclease in the CRISPR system can be inactivated and used in the base editing system. Exemplary Cas nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cpfl, C2c3, C2c2 and C2clCsyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5 , Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4 and mutants thereof.

[0182] Certain embodiments utilize nuclease-inactive Cas9 (dCas9) as a catalytically inactive nuclease. However, any nuclease of the CRISPR system (many of which are described above) can be inactivated and used within the base editing system. In certain embodiments, a high-fidelity Cas9 domain is selected that exhibits reduced electrostatic interactions between the Cas9 domain and the sugar phosphate backbone of DNA compared to the wild-type Cas9 domain. In some embodiments, the Cas9 domain (e.g., the wild-type Cas9 domain) comprises 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, et al., Nature 529, 490-495, 2016; and Slaymaker et al., Science 351, 84-88, 2015.

[0183] Nucleases from other gene editing systems can also be used. For example, base editing systems can utilize zinc finger nucleases (ZFNs) (Urnov et al., Nat Rev Genet., 11(9):636-46, 2010) and transcription activator-like effector nucleases (TALENs) (Joung et al., Nat Rev Mol Cell Biol. 14(1):49-55, 2013). For more information on DNA-binding nucleases, see US2018 / 0312825A1.

[0184] In certain embodiments, the nucleobase deaminase enzyme comprises a cytidine deaminase domain or an adenine deaminase domain.

[0185] Certain embodiments utilize a cytidine deaminase domain as the nucleobase deaminase enzyme. Certain embodiments utilize an adenine deaminase domain as the nucleobase deaminase enzyme. Additionally, certain embodiments utilize a uracil glycosylase inhibitor (UGI) as the glycosylase inhibitor. For example, in certain embodiments, a dCas9 or aCas9 nickase can be fused to a cytidine deaminase domain. A dCas9 or aCas9 nickase fused to a cytidine deaminase domain can be fused to one or more UGI domains. Base editors that include more than one UGI domain can deaminate target nucleic acids more efficiently with fewer indels.

[0186] In certain embodiments, the deaminase domain (cytidine and / or adenine) is fused to the N-terminus of the non-catalytic nuclease. This is because a cytidine deaminase domain fused to the N-terminus of Cas9 may improve base editing efficiency compared to other configurations. In these embodiments, a glycosylase inhibitor (e.g., a UGI domain) may be fused to the C-terminus of the non-catalytic nuclease. When multiple glycosylase inhibitors are used, each of them may be fused to the C-terminus of the non-catalytic nuclease.

[0187] In certain embodiments, CBEs utilizing a cytidine deaminase domain convert guanine-cytosine base pairs to thymine-adenine base pairs by deaminating the exocyclic amine of cytosine to generate uracil. Examples of cytosine deaminase enzymes include APOBEC1, APOBEC3A, APOBEC3G, CDA1, and AID. APOBEC1 in particular accepts single-stranded (ss) DNA as a substrate but cannot act on double-stranded (ds) DNA.

[0188] Most base editing systems may include a DNA glycosylase inhibitor, which functions to disable natural DNA repair mechanisms that might otherwise repair the intended base edit. In certain embodiments, the DNA glycosylase inhibitor includes a uracil glycosylase inhibitor, such as the uracil DNA glycosylase inhibitor protein (UGI) described in Wang et al. (Gene 99,31-37,1991).

[0189] The base editor elements can be fused directly (e.g., by direct covalent bonding) or via a linker. For example, a non-catalytic 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 one of skill in the art, linkers can be used to link any peptide or portion thereof.

[0190] 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.

[0191] The linker may also include a functionalized moiety that facilitates 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.

[0192] In certain embodiments, the linker ranges from 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.

[0193] A number of base editing (BE) systems formed by linking targeted DNA-binding proteins with cytidine deaminase enzymes and DNA glycosylase inhibitors (e.g., UGI) have been described. These complexes include, for example, BE1 ([APOBEC1-16 amino acid (aa) linker-Sp dCas9(D10A,H840A)] Komer et al., Nature, 533, 420-424, 2016), BE2 ([APOBEC1-16aa linker-Sp dCas9(D10A,H840A)-4aa linker-UGI] Komer et al., 2016 (supra)), BE3 ([APOBEC1-16aa linker-Sp nCas9(D10A)-4aa linker-UGI] Komer et al. (supra)), HF-BE3 ([APOBEC1-16aa linker-HF nCas9(D10A)-4aa linker-UGI] Rees et al., Nature, 533, 420-424, 2016), and HF-BE4 ([APOBEC1-16aa linker-HF nCas9(D10A)-4aa linker-UGI] Komer et al., Nature, 533, 420-424, 2016). al., Nat. Commun. 8, 15790, 2017), BE4, BE4max ([APOBEC1-32aa linker-Sp nCas9(D10A)-9aa linker-UGI-9aa linker-UGI] Koblan et al., Nat. Biotechnol 10.1038 / nbt.4172, 2018; Komer et al., Sci. Adv., 3, eaao4774, 2017), BE4-GAM ([Gam-16aa linker-APOBEC1-32aa linker-Sp nCas9(D10A)-9aa linker-UGI-9aa linker-UGI] Komer et al. al., 2017 (supra)), YE1-BE3 ([APOBEC1 (W90Y, R126E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI] Kim et al., Nat. Biotechnol. 35, 475-480, 2017), EE-BE3 ([APOBEC1 (R126E, R132E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI] Kim et al., 2017 (supra)), YE2-BE3 ([APOBEC1 (W90Y, R132E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI] Kim et al.,2017(above)), YEE-BE3 ([APOBEC1(W90Y,R126E,R132E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI] Kim et al.,2017(above)), VQR-BE3 ([APOBEC1-16aa linker-Sp VQR nCas9(D10A)-4aa linker-UGI] Kim et al.,2017(above)), VRER-BE3 ([APOBEC1-16aa linker-Sp VRER nCas9(D10A)-4aa linker-UGI] Kim et al., Nat. Biotechnol. 35, 475-480,2017), Sa-BE3 ([APOBEC1-16aa linker-Sa nCas9(D10A)-4aa linker-UGI] Kim et al., 2017 (supra)), SA-BE4 ([APOBEC1-32aa linker-Sa nCas9(D10A)-9aa linker-UGI-9aa linker-UGI] Komer et al., 2017 (supra)), SaBE4-Gam ([Gam-16aa linker-APOBEC1-32aa linker-Sa nCas9(D10A)-9aa linker-UGI-9aa linker-UGI] Komer et al., 2017 (supra)), SaKKH-BE3 ([APOBEC1-16aa linker-Sa KKH nCas9(D10A)-4aa linker-UGI] Kim et al. al., 2017 (supra), Cas12a-BE ([APOBEC1-16aa linker-dCas12a-14aa linker-UGI] Li et al., Nat. Biotechnol. 36, 324-327, 2018), Target-AID ([Sp nCas9(D10A)-100aa linker-CDA1-9aa linker-UGI] Nishida et al., Science, 353, 10.1126 / science.aaf8729, 2016), Target-AID-NG ([Sp nCas9(D10A)-NG-100aa linker-CDA1-9aa linker-UGI] Nishimasu et al. al.,Science,361(6408):1259-1262,2018), xBE3 ([APOBEC1-16aa linker-xCas9(D10A)-4aa linker-UGI]Hu et al.,Nature,556,57-63,2018), eA3A-BE3([APOBEC3A(N37G)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI]Gerkhe et al., Nat.Biotechnol.,10.1038 / nbt.4199,2018), A3A-BE3([hAPOBEC3A-16aa linker-Sp nCas9(D10A)-4aa linker-UGI]Wang et al., Nat.Biotechnol.10.1038 / nbt.4198,2018), and BE-PLUS([10X GCN4-Sp nCas9(D10A) / ScFv-rAPOBEC1-UGI] Jiang et al., Cell. Res, 10.1038 / s41422-018-0052-4, 2018). For further examples of BE complexes that contain adenine deaminase base editors, see Rees & Liu Nat. Rev Genet. 19(12):770-788, 2018.

[0194] For more information regarding base editors, see US2018 / 0312825A1, WO2018 / 165629A, Urnov et al., Nat Rev Genet. 11(9):636-46, 2010; Joung et al., Nat Rev Mol Cell Biol. 14(1):49-55, 2013; Charpentier et al., Nature.; 495(7439):50-1, 2013; Seo & Kim, Nature Medicine, 24, 1493-1495, 2018, and Rees & Liu, Nature Reviews Genetics, 19, 770-78, 2018, each of which is incorporated by reference in its entirety and for specific content regarding base editors. Certain base editor constructs that can be used in various embodiments of the disclosure are described in Zafra et al., Nat Biotech, 36(9):888-893, 2018, and Koblan et al., Nat Biotech 36(9):843-846, 2018, each of which is incorporated by reference in its entirety and for specific content relating to base editor constructs.

[0195] I(C)(i)(b)(3). Prime editor payload expression product Prime editing can introduce any type of point mutation, small insertion, and small deletion in a precise and targeted manner. Prime editors are fusion proteins that contain a Cas9 nickase domain (e.g., inactivated HNH nuclease) and an engineered reverse transcriptase domain. The prime editor enzyme is targeted to the editing site by an engineered prime editing gRNA (pegRNA), which not only specifies the target site in its spacer sequence, but also encodes the desired edit in an extension, typically at the 3' end of the pegRNA.

[0196] At least three prime editor systems have been characterized. PE1 contains a fusion of Cas9 nickase and wild-type Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT). PE2 is similar to PE1 but contains an engineered pentamutant M-MLV RT, which increases editing efficiency by approximately 3-fold. PE3 combines the PE2 fusion protein and pegRNA with an additional sgRNA that targets nicking of the non-edited strand. A variant of the PE3 system, called PE3b, contains a nicking sgRNA that targets only the edited sequence, thereby reducing the level of indel products by preventing nicking of the non-edited DNA strand until the other strand is converted to the edited sequence.

[0197] I(C)(i)(c). Small RNA payload expression products 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.

[0198] 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 the reduced translation of the target mRNA, a process called 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 through fragments cleaved from free double-stranded RNA (dsRNA) and directs the decomposition mechanism to other similar RNA sequences. Alternatively, RNAi can be manufactured, for example, to suppress the expression of a target gene. Exemplary RNAi molecules include small hairpin RNA (shRNA, also called short hairpin RNA) and small interfering RNA (siRNA).

[0199] Without limiting the disclosure or being bound by theory, natural RNA interference and / or some embodiments are typically a two-step process. In the first, initiation step, the 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 by a transgene or virus) in an ATP-dependent manner. Successive cleavage events degrade the RNA into 19-21 base pair (bp) duplexes (siRNAs), each of which has a 2-nucleotide 3' overhang.

[0200] 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.

[0201] The remarkable efficacy of RNAi suggests an amplification step in the RNAi pathway.Amplification can occur by copying input dsRNA, which can generate more siRNA, or by replicating the siRNA formed.Alternatively or additionally, amplification can occur by multiple turnover events of RISC.

[0202] 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, so that the first sequence and the second sequence form a double-stranded region, the ends of which are connected by a linking sequence to form a hairpin loop structure. The first sequence may be capable of hybridizing to any part of a polynucleotide encoding a transgene. The double-stranded stem domain of the shRNA may include a restriction endonuclease site.

[0203] Transcription of shRNAs is thought to initiate at the polymerase III (PolIII) 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 then processed to convert the shRNAs into 21- to 23-nucleotide siRNA-like molecules.

[0204] The stem-loop structure of the shRNA may have optional nucleotide overhangs, such as 2 bp overhangs, e.g., 3'UU overhangs. Although variations are possible, the stem typically ranges from 15-49, 15-35, 19-35, 21-31 bp, or 21-29 bp, and the loop ranges from 4-30 bp, e.g., 4-23 bp. In certain embodiments, the shRNA sequence comprises 45-65 bp; 50-60 bp; or 51, 52, 53, 54, 55, 56, 57, 58, or 59 bp. In certain embodiments, the shRNA sequence comprises 52 bp or 55 bp. In certain embodiments, the siRNA has 15-25 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 with a length of less than 16 nucleotides or more than 24 nucleotides can also function to mediate RNAi. Longer RNAi agents have been shown to cause interferon or protein kinase R (PKR) response in certain mammalian cells, which may be undesirable.Preferably, RNAi agents do not cause PKR response (i.e., are sufficiently short in length).However, longer RNAi agents may be useful, for example, in situations where PKR response is downregulated or attenuated by alternative means.

[0205] In certain exemplary embodiments, the disclosure includes an adenoviral vector payload encoding an shRNA targeted to the gene encoding BCL11A, where the shRNA results in decreased translation of BCL11A.

[0206] I(C)(ii). Payload regulatory sequence I(C)(ii)(a) Promoter regulatory sequences A promoter is a non-coding genomic DNA sequence, usually upstream (5') of an associated coding sequence, to which RNA polymerase binds to initiate transcription. This binding aligns the RNA polymerase so that transcription begins at a specific transcription initiation site. The nucleotide sequence of the promoter determines the nature of the enzyme and other associated protein factors that bind to it, as well as the rate of RNA synthesis. RNA is processed into messenger RNA (mRNA), which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5' non-translated leader sequence is a region of the mRNA upstream of the coding region that may be involved in the initiation and translation of the mRNA. The 3' transcription termination / polyadenylation signal is a non-translated region downstream of the coding region that functions in plant cells to terminate RNA synthesis and to effect the addition of polyadenylate nucleotides to the 3' end.

[0207] The promoter may include a general promoter, a tissue-specific promoter, a cell-specific promoter, and / or a cytoplasm-specific promoter. The promoter may include a strong promoter, a weak promoter, a constitutive expression promoter, and / or an inducible (conditional) promoter. An inducible promoter induces or controls expression in response to a certain condition, signal, or cellular event. For example, the promoter may be an inducible promoter that requires a specific ligand, small molecule, transcription factor, hormone, or hormone protein to cause transcription from the promoter.Specific examples of promoters include AFP (alpha-fetoprotein) promoter, amylase 1C promoter, aquaporin-5 (AP5) promoter, alphal-antitrypsin promoter, beta-act promoter, beta-globin promoter, beta-Kin promoter, B29 promoter, CCKAR promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, CEA promoter, c-erbB2 promoter, COX-2 promoter, CXCR4 promoter, desmin promoter, E2F-1 promoter, human elongation factor 1 alpha promoter (EF1α), CMV (cytomegalovirus) promoter, minCMV promoter, SV40 (simian virus 40) immediate early promoter, EGR1 promoter, eIF4A1 promoter, elastase-1 promoter, endoglin promoter, FerH promoter, FerL promoter, fibronectin promoter, Flt-1 promoter, GAPDH promoter, GFAP promoter, and GPIIb promoter. -, GRP78 promoter, GRP94 promoter, HE4 promoter, hGR1 / 1 promoter, hNIS promoter, Hsp68 promoter, Hsp68 minimal promoter (proHSP68), HSP70 promoter, HSV-1 viral TK gene promoter, hTERT promoter, ICAM-2 promoter, kallikrein promoter, LP promoter, major late promoter (MLP), Mb promoter, Rho promoter, MT (metallothionein) promoter, MUC1 promoter, NphsI promoter, OG-2 promoter, PGK (phosphoglycerate kinase) promoter, PGK-1 promoter, polymerase III (PolIII) promoter, PSA promoter, ROSA promoter, SP-B promoter, Survivn promoter, SYN1 promoter, SYT8 gene promoter, TRP1 promoter, Tyr promoter, ubiquitin B promoter, WASP promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter.

[0208] The promoter can be obtained as a natural promoter or a composite promoter. A natural promoter or a minimal promoter refers to a promoter that comprises a nucleotide sequence derived from the 5' region of a given gene. A natural promoter comprises a core promoter and its natural 5'UTR. In certain embodiments, the 5'UTR comprises an intron. A composite promoter refers to a promoter that is obtained by combining promoter elements of different origins, or by combining a minimal promoter of the same or different origins with a distal enhancer.

[0209] In certain embodiments, the promoter comprises a wild-type promoter sequence and a sequence with optional changes (including insertions, point mutations or deletions) at specific positions relative to the wild-type promoter. In certain embodiments, the promoter differs from the naturally occurring promoter 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 native sequence is modified by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. The length of the promoter can vary from a 50 nucleotide LTR sequence to a 100, 200, 250, or 350 nucleotide LTR sequence, with or without other viral sequences.

[0210] Some promoters are tissue or cell specific and some promoters are tissue or cell non-specific. Each gene in a mammalian cell has its own promoter and some promoters may only be active in certain cell types. Non-specific promoters, or ubiquitous promoters, help initiate transcription of a gene or nucleotide sequence operably linked to the promoter sequence in a wide range of cells, tissues and cell cycles. In certain embodiments, the promoter is a non-specific promoter. In certain embodiments, the non-specific promoter includes a CMV promoter, a RSV promoter, a SV40 promoter, a mammalian elongation factor 1 alpha (EF1 alpha) promoter, a β-act promoter, an EGR1 promoter, an eIF4A1 promoter, a FerH promoter, a FerL promoter, a GAPDH promoter, a GRP78 promoter, a GRP94 promoter, an HSP70 promoter, a β-Kin promoter, a PGK-1 promoter, a ROSA promoter, and / or a ubiquitin B promoter. A specific promoter aids in the cell-specific expression of a nucleotide sequence operably linked to the promoter sequence.

[0211] I(C)(ii)(b) MicroRNA site regulatory sequence In various embodiments, a microRNA (or miRNA) regulatory system may refer to a method or composition in which expression of a gene is regulated by the presence of a microRNA site (e.g., a nucleic acid sequence with which a microRNA may interact). In various embodiments, the present disclosure includes an adenoviral donor vector comprising a payload, wherein a nucleic acid sequence encoding an expression product in the payload is operably linked to a miRNA target site such that expression of the expression product is controlled by the presence, level, activity, and / or contact of the corresponding miRNA. For the avoidance of doubt, the present disclosure contemplates that, for example, a nucleic acid sequence operably linked to a miRNA site disclosed herein may be a nucleic acid sequence encoding any of one or more expression products provided herein.

[0212] I(C)(iii). Selection sequence In certain embodiments, the vector comprises a selection element comprising 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 terminating transcription of the independent transcription element.

[0213] The selection cassette can encode one or more proteins that (a) confer resistance to antibiotics or other toxins, (b) compensate for an auxotrophic deficiency, or (c) provide an essential nutrient that cannot be obtained from complex media, for example, a gene encoding Bacilli D-alanine racemase. Any number of selection systems can be used to recover transformed cell lines. In certain embodiments, the positive selection cassette comprises a resistance gene for neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In certain embodiments, the positive selection cassette comprises a DHFR (dihydrofolate reductase) gene that confers resistance to methotrexate, an O (olanin) gene, or a β-lactamase gene that confers resistance to methotrexate. 6 MGMT is involved in resistance to BG / BCNU P140K The selection agents include the genes for the enzyme phospholipase (HPRT) involved in the conversion of specific bases present in the HAT selection medium (aminopterin, hypoxanthine, thymidine), and other genes for detoxification against several drugs. In a particular embodiment, the selection agents are neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, viomycin, ampicillin, O 6 These include BG / BCNU, methotrexate, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gln synthetase, or ADA.

[0214] In a particular embodiment, the negative selection cassette contains genes for converting a substrate present in the culture medium into a substance toxic to the cells expressing that gene. These molecules include the detoxification gene for diphtheria toxin (DTA) (Yagi et al., Anal Biochem. 214(1):77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221, 1999), the herpesvirus kinase thymidine gene (HSV TK) that is sensitive to the presence of ganciclovir or FIAU. The HPRT gene can also be used as a negative selection by adding 6-thioguanine (6TG) to the medium. Also, for all positive and negative selections, polyA transcription termination sequences from different sources are most classically derived from SV40 polyA, or from eukaryotic gene polyA (bovine growth hormone, rabbit β-globin, etc.).

[0215] In certain embodiments, the selection cassette comprises the MGMT P140K In certain embodiments, the selective agent is O 6 Includes BG / BCNU.

[0216] The drug resistance gene MGMT, which encodes human alkylguanine transferase (hAGT), is 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 nitrosourea toxicity and is coadministered with TMZ to potentiate the cytotoxic effects of the agent. Some mutant forms of MGMT, which encode variants of AGT, are highly resistant to inactivation by 6-BG while retaining the ability to repair DNA damage (Maze et al., J. Pharmacol. Exp. Ther. 290:1467-1474, 1999). MGMT P140K)-based drug resistance gene therapy 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).

[0217] In certain embodiments, the combination with an in vivo selection cassette may be an important factor for diseases where the genetically corrected cells have no selective advantage. For example, in SCID and some other immunodeficiencies and FA, the corrected cells are advantageous and it is sufficient to introduce the therapeutic gene into a "small number" of HSPCs to obtain a therapeutic effect. In other diseases where the therapeutically modified cells do not show a competitive advantage, such as hemoglobinopathies (i.e., sickle cell disease and thalassemia), the therapeutically modified cells may show no competitive advantage, for example, MGMT P140K In vivo selection of modified cells by expression of an in vivo selection cassette such as , allows a small number of transduced HSPCs to be selected and the gene-corrected cells expanded to achieve a therapeutic effect. This approach can also be applied to HIV, where HSPCs can be made HIV-resistant in vivo rather than genetically modified ex vivo.

[0218] 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 size of the genome closer 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.

[0219] Stuffer sequences are used to achieve efficient packaging and stability of the vector. In certain embodiments, stuffer sequences are used to reduce the genome size to 70%-110% of the size of the wild-type virus.

[0220] The stuffer sequence can be any DNA, preferably 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.

[0221] Stuffer sequences, when used to keep the size of the vector at a certain size, can be any non-coding sequence or sequence that can keep the genome 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 parts of centromeres and / or telomeres.

[0222] 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. A variety of such systems can include one or more of specific payload sequence characteristics and a support vector and a support genome (support genome).

[0223] One way to make an adenoviral vector that integrates a payload into the host cell genome is to generate an integrative virus hybrid vector.An integrative virus hybrid vector combines a vector genetic element that efficiently introduces into target cells with a vector genetic element that stably integrates the vector payload.For example, the integrative elements that can be used in combination with an adenoviral vector 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.

[0224] The Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors described herein may optionally include transposase and transposon transposase elements. Transposase includes retrotransposon-derived or retroviral origin integrases, as well as enzymes that are elements of functional nucleic acid protein complexes that allow transposition and mediate transposition. The transposition reaction includes a transposon and a transposase or integrase enzyme. In certain embodiments, the use of such transposable elements may improve the integration efficiency, 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. A transposon includes 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.

[0225] Several transposases have been described in the art that facilitate the insertion of nucleic acids into the genome of vertebrates, including humans. Examples of such transposases include sleeping beauty ("SB", e.g., from the genome of salmonids); piggyback (e.g., from lepidopteran cells and / or Myotis lucifugus); mariner (e.g., from Drosophila); frog prince (e.g., from Rana pipiens); Tol1; Tol2 (e.g., from medaka); TcBuster (e.g., from the red flour beetle Tribolium castaneum), Helraiser, Himar1, Passport, Minos, Ac / Ds, PIF, Harbinger, Harbinger3-DR, HSmar1, and spinON.

[0226] PiggyBac (PB) transposase is a compact functional transposase protein and is 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.

[0227] Further information regarding DNA transposons can be found, for example, in Munoz-Lopez & Garcia Perez, Curr Genomics, 11(2):115-128, 2010.

[0228] 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 the Hyperactive Sleeping Beauty SB100x transposase enzyme. SB transposons transpose most efficiently when present in circular nucleic acid molecules (Yant et al., Nature Biotechnology, 20:999-1005, 2002).

[0229] Systematic mutagenesis studies have been carried out to enhance the activity of SB transposase. For example, Yant et al. systematically changed the N-terminal 95AA of SB transposase to alanine (Mol. Cell Biol. 24: 9239-9247, 2004). Ten of these substitutions resulted in 200-400% increased 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. Further hyperactive SB variants are described by Zayed et al. (Molecular Therapy 9(2): 292-304, 2004) and US 9,840,696.

[0230] SB transposase transfers 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, and contains 32bp long imperfect orthogonal repeats that function as transposase recognition signals.

[0231] In various embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector or genome comprises a payload comprising SB100x transposon inverted repeats flanking integration elements comprising at least one coding sequence encoding a β-globin or γ-globin expression product.

[0232] In various embodiments, the adenoviral transfer system comprises an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector or genome comprising an integration element flanked by transposon inverted repeats, and may further comprise an adenoviral support vector or genome. In various embodiments, the support vector comprises (i) an adenoviral capsid and (ii) an adenoviral support genome comprising a nucleic acid sequence encoding a transposase corresponding to the inverted repeats flanking the integration element. Thus, in various embodiments, at least one function of the support vector or genome may be to encode, express, and / or deliver to a target cell a transposase for transposing an integration element present in the donor vector administered to the target cell. For example, in some embodiments, the Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 donor vector or genome comprises SB100x transposon inverted repeats flanking an integration element 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 element is flanked by recombinase direct repeats, e.g., the integration element is flanked by transposon inverted repeats, which 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 recombining recombinase sites present in the donor vector administered to the target cell. In various embodiments, the support vector or supporting genome can encode, express, and / or deliver to the target cell a recombinase for recombining recombinase sites present in a donor vector administered to the target cell, and can also encode, express, and / or deliver to the target cell a transposase for transposing integration elements present in a donor vector administered to the target cell.

[0233] 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 an inverted repeat sequence that recognizes the transposase also contains at least one recombinase recognition site. Thus, in certain embodiments, the present disclosure also provides a method of integrating a therapeutic gene into a genome, comprising administering (a) a transposon containing a therapeutic gene, where the therapeutic gene is flanked by (i) an inverted repeat sequence recognized by the transposase and (ii) a recombinase recognition site, and b) a transposase and a recombinase that function 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 a nucleic acid encoding the protein(s). In some embodiments, the transposon and the nucleic acid encoding the protein(s) of (b) are present on separate vectors. In some embodiments, the nucleic acid encoding the transposon and 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 region encoding the transposase and / or recombinase is located outside the region flanked by the inverted repeats and / or recombinase recognition sites. In the above method, the transposase protein recognizes the inverted repeats flanking the insert nucleic acid, such as the nucleic acid to be inserted into the target cell genome. The use of recombinases and recombinase recognition sites can further increase the size of the transposon that can be integrated into the genome.

[0234] 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) that catalyzes DNA recombination at Frt recognition sites. Variants of the Flp protein include GenBank: ABD57356.1) and GenBank: ANW61888.1.

[0235] 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 recombines the 34 bp loxP DNA sequence by binding to a 13 base pair inverted repeat sequence and catalyzing strand cleavage and religation within the spacer region. The staggered DNA cleavage generated by Cre in the spacer region is separated by 6 base pairs, providing an overlap region and acting as a homology sensor to ensure that only recombination sites with the same overlap region are recombined. Further variants of the recognition site that can be used include lox2272; lox511; lox66; lox71; loxM2; and lox5171. The VCre / VloxP recombinase system was isolated from Vibrio plasmid p0908. The sCre / SloxP system is described in WO2010 / 143606. The Dre / rox system is described in US 7,422,889 and US 7,915,037B2. Generally, it comprises the Dre recombinase isolated from the Enterobacteria phage D6 and a rox recognition site. The Vika / vox system is described in US 10,253,332. Furthermore, the PhiC31 recombinase recognizes the AttB / AttP binding site.

[0236] The amount of vector nucleic acid comprising a transposon (including an inverted repeat and / or a recombinase recognition site), and in various embodiments, the amount of vector nucleic acid encoding a transposase and / or a recombinase, introduced into a cell is sufficient to effect the desired excision and insertion of the transposon nucleic acid into the target cell genome. Thus, the amount of vector nucleic acid introduced should provide a sufficient amount of transposase activity and / or recombinase activity as well as a sufficient copy number of the transposon desired to be inserted into the target cell genome. Particular embodiments include a 1:1, 1:2, or 1:3 ratio of transposon to transposase / recombinase.

[0237] The subject methods result in stable integration of the nucleic acid into the genome of the target cell, meaning that the nucleic acid continues to be present in the genome of the target cell for more than a transient period of time and that a portion of the chromosomal genetic material is passed on to progeny of the target cell.

[0238] As shown above, certain embodiments utilize homology arms to facilitate targeted insertion of gene constructs using homology-directed repair. Homology arms can be of any length with sufficient homology to the genomic sequence of the cleavage site, for example, 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequence that flanks or directly flanks the cleavage site, for example, within 50 bases, for example, within 30 bases, within 15 bases, within 10 bases, within 5 bases, of the cleavage site, to support HDR between the homologous genomic sequence. Homology arms are generally identical to the genomic sequence, for example, the genomic region where the double-strand break (DSB) occurs. However, as shown, absolute identity is not necessary.

[0239] Certain embodiments may utilize homology arms having 25, 50, 100, or 200 nucleotides (nt), or greater than 200 nt, of sequence homology between the homology recombination repair template and the target genomic sequence (or any integer value between 10 and 200 nucleotides or greater). 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 may also be symmetric or asymmetric.

[0240] Certain embodiments may utilize first and / or second homology arms, each of which comprises 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 nucleotides or more and have 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, with 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, with a length of 40-1,000 nucleotides, 500-2,500 nucleotides, 700-2,000 nucleotides, or 800-1800 nucleotides, or at least 800 nucleotides or at least 850 nucleotides. The first and second homology arms can have the same, similar, or different lengths.

[0241] For more information regarding homology arms, see Richardson et al., Nat Biotechnol. 34(3):339-44, 2016.

[0242] In certain embodiments, the genetic construct (e.g., a gene that expresses 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 obtain 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 (measured by DNA cleavage by natural or engineered nucleases), (iv) a location outside of a gene transcription unit, and (v) a location outside of ultraconserved regions (UCRs), microRNAs or long non-coding RNAs in the genome.

[0243] In certain embodiments, to meet the criteria for genomic safe harbor, the chromatin site must be more than 150 kb away from a known cancer gene, more than 30 kb away from a known transcription start site, and have no overlap with coding mRNAs. In certain embodiments, to meet the criteria for genomic safe harbor, the chromatin site must be more than 200 kb away from a known cancer gene, more than 40 kb away from a known transcription start site, and have no overlap with coding mRNAs. In certain embodiments, to meet the criteria for genomic safe harbor, the chromatin site must be more than 300 kb away from a known cancer gene, more than 50 kb away from a known transcription start site, and have no overlap with coding mRNAs. In certain embodiments, the genomic safe harbor meets the aforementioned criteria (more than 150 kb, 200 kb or 300 kb away from a known transcription start site and has no overlap with any coding mRNA, more than 40 kb or 50 kb away from a known transcription start site and has no overlap with any coding mRNA) and further has 100% homology between the animal and human genomes of the relevant animal model, allowing for rapid clinical translation of relevant results.

[0244] In certain embodiments, a genomic safe harbor meets the criteria described herein and also demonstrates a 1:1 forward:reverse ratio of lentiviral integration and further demonstrates that the locus does not affect the surrounding genetic material.

[0245] Specific genomic safe harbor sites include CCR5, HPRT, AAVS1, Rosa and albumin. For further 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.

[0246] Various techniques known in the art can be used to directly integrate integrating elements into specific genomic loci, such as genomic safe harbors. For example, AAV-mediated gene targeting, 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 into predetermined genomic loci, such as genomic safe harbors.

[0247] In certain embodiments, integration of an integration element at a particular genomic locus, such as a genomic safe harbor, may include integration by homologous recombination using CRISPR enzyme-mediated cleavage of the target genome. A CRISPR enzyme (e.g., Cas9) cleaves double-stranded DNA at a site specified by a guide RNA (gRNA). The double-stranded break may be repaired by homology-directed repair (HDR) if a donor template (such as an Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 payload integration element that contains left and right homology arms) is present. 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 is several orders of magnitude more efficient than natural recombination of a DNA template, indicating that CRISPR-mediated gene insertion may be an effective tool for genome editing. Exemplary methods for integrating a nucleic acid sequence into a specific genomic locus via homologous recombination are known in the art, e.g., Richardson et al. (Nat Biotechnol. 34(3):339-44, 2016).

[0248] II. Target Cell Population In various embodiments, the donor vectors and genomes of the present disclosure can be introduced into hematopoietic stem cells (HSCs). HSCs can be targeted for genetic modification in vivo by binding to CD46. HSCs or subsets thereof can also be identified 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 identified by either of the following profiles: CD34+ / CD38- / CD45RA- / CD90+ or ​​CD34+ / CD45RA- / CD90+, and mouse LT-HSC can be identified by Lin- / Sca1+ / ckit+ / CD150+ / CD48- / Flt3- / CD34- (wherein Lin indicates the absence of expression of any marker 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.

[0249] For the avoidance of doubt, in various embodiments, the donor vectors and genomes of the present disclosure can infect and / or be introduced into CD34+ hematopoietic cells. In various embodiments, the donor vectors and genomes of the present disclosure can infect and / or be introduced into CD34+ / CD90+ cells. In various embodiments, CD34+ cells and / or CD34+ phenotype can refer to cells that are found to express CD34+, e.g., based on binding of the cells to a labeled anti-CD34 antibody, e.g., as shown in Example 6 and / or FIG. 33. In various embodiments, CD90+ cells and / or aCD90+ phenotype can refer to cells that are found to express CD90+, e.g., based on binding of the cells to a labeled anti-CD90 antibody, e.g., as shown in Example 6 and / or FIG. 33. In various embodiments, CD34+ cells and / or CD34+ phenotype can refer to cells in a sample or population that are most robustly labeled with a label for CD34+ (e.g., most robustly labeled with a labeled anti-CD34 antibody). For example, in various embodiments where a sample or population comprises cells labeled with a label for CD34+, CD34+ cells and / or CD34+ phenotype may refer to (i) all cells labeled with a label for CD34%, or (ii) 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells most robustly labeled with a label for CD34, where the CD34+ cells may optionally be referred to as CD34+ high cells. In various embodiments, the label and / or robustness of the label may be determined by any of a variety of methods known in the art, including, but not limited to, the relative presence of the label, such as the fluorescence of a fluorescent label. In various embodiments, the labeling and / or labeling robustness can be measured by techniques including methods such as fluorescence activated cell sorting (FACS).Thus, in various embodiments, CD34+ / CD90+ cells may refer to a population of cells that are (i) CD34+ cells and / or cells determined to have a CD34+ phenotype, and (ii) CD90+ cells and / or cells determined to have a CD90+ phenotype. In various embodiments, CD34+ / CD90+ cells may refer to a population of CD34+high / CD90+ cells that are (i) CD34+high cells and / or cells determined to have a CD34+high phenotype, and (ii) CD90+ cells and / or cells determined to have a CD90+ phenotype. In various such embodiments, the cells may be hematopoietic cells. In various embodiments, the cells may be CD45RA-. In various embodiments, the cells may be CD45RA+.

[0250] Without wishing to be bound by any particular scientific theory, the present disclosure includes that, for example, in hematopoietic cells, expression of CD34+ (e.g., CD34 labeling and / or intensity of labeling) can correlate with expression of CD46 and / or susceptibility to infection and / or transduction. Without wishing to be bound by any particular scientific theory, the present disclosure includes that the vectors disclosed herein are particularly advantageous for infection and / or transduction of CD34+ cells, CD34+high cells, CD34+ / CD90+ cells, and / or CD34+high / CD90+ cells, for example, when the cells are hematopoietic cells (e.g., can selectively infect and / or transduce CD34+ cells, CD34+high cells, CD34+ / CD90+ cells, and / or CD34+high / CD90+ cells).

[0251] HSCs can beneficially 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).

[0252] 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 vector described herein can be formulated for administration to a subject. The formulation comprises an adenoviral vector that codes for a therapeutic agent and one or more pharmaceutically acceptable carriers.

[0253] 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.

[0254] The selection or use of any 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. Accordingly, the vector may be formulated for administration by parenteral modes (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intranasal, intraocular, pulmonary, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural, intracerebral, intracranial, intracarotid, and intracisternal injection and infusion. Parenteral administration routes may be by injection, nasal, pulmonary, or transdermal administration. Administration can be systemic or local, by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

[0255] In various embodiments, the vectors of the present invention may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for stable storage at high concentrations. Sterile solutions for injection can be prepared by incorporating the compositions described herein in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as needed, followed by filtered sterilization. In general, dispersions are prepared by incorporating the compositions described herein into a sterile vehicle, which contains a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for preparing sterile solutions for injection, preparation methods include vacuum drying and freeze-drying, which obtain the compositions described herein and any desired additional ingredients (see below) from a previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition can be brought about by including an agent that delays absorption, for example, monostearate salts and gelatin, in the composition.

[0256] The vector can be administered parenterally in the form of an injection, including a sterilized solution or suspension in water or another pharma- ceutically acceptable liquid. For example, the vector can be formulated by suitably combining the therapeutic molecule with a pharma- ceutically acceptable vehicle or medium, such as sterile water and saline, vegetable oil, emulsifiers, suspending agents, surfactants, stabilizers, flavoring excipients, diluents, vehicles, preservatives, binders, and the like, and then mixing in a unit dosage form required by generally accepted pharmaceutical practice. The amount of vector included in the pharmaceutical preparation is such that a suitable dose within the specified range is provided. Non-limiting examples of oily liquids include sesame oil and soybean oil, which may be mixed 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 injection can be packaged in suitable ampoules.

[0257] In various embodiments, subcutaneous administration may be accomplished by a device such as a syringe, a prefilled syringe, an auto-injector (e.g., disposable or reusable), a pen injector, a patch injector, a wearable injector, an outpatient syringe infusion pump with a subcutaneous infusion set, or other subcutaneous injection device.

[0258] 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" refers to delivery that does not depend on the vector or the transport of the vector to its intended target tissue or site via the vascular system.For example, the vector can be delivered by injection or implantation of the composition or agent, or by injection or implantation of a device containing the composition or agent.In certain embodiments, following local administration in the vicinity of the target tissue or site, the composition or agent, or one or more elements thereof, can diffuse to the intended target tissue or site that is not the administration site.

[0259] In some embodiments, the compositions provided herein are in unit dosage form, and unit dosage form can be suitable for self-administration.Such unit dosage form can be provided in a container, typically in, for example, vial, cartridge, pre-filled syringe or disposable pen.Dosing device, such as the dosing device described in US6,302,855, can also be used with the injection system described herein, for example.

[0260] The pharmaceutical form of the vector preparation suitable for injection may include a sterile aqueous solution or dispersion. The preparation may be sterile and must be fluid to allow suitable entry and exit into a syringe. The preparation may also be stable under the conditions of manufacture and storage. The carrier may be a solvent or dispersion medium, including, for example, water and physiological saline or buffered aqueous solution. Preferably, an isotonic agent, such as sugar or sodium chloride, may be used in the preparation.

[0261] The suitable dose of the vectors described herein may depend on a variety of factors, including, for example, the age, sex, and weight of the subject being treated, the condition or disease being treated, and the particular vector being used. Other factors that affect the dose administered to the subject include, for example, the type or severity of the condition or disease. Other factors may include, for example, other medical disorders that have affected the subject simultaneously or previously, the general health condition of the subject, the genetic predisposition of the subject, diet, time of administration, excretion rate, drug combinations, and any other additional treatments administered to the subject. The suitable means of administration of the vector may be selected based on the condition or disease being treated and the age and condition of the subject. The dose and method of administration may vary depending on the patient's weight, age, condition, etc., and can be suitably selected by those skilled in the art as necessary. The specific dosage and treatment regimen for any particular subject may be adjusted based on the judgment of a medical professional.

[0262] In various cases, the vector may be formulated to include a pharma- ceutically acceptable carrier or excipient. Examples of pharma-ceutically acceptable carriers include, but are not limited to, any physiologically compatible solvent, dispersion medium, coating agent, antibacterial and antifungal agent, isotonicity agent, and absorption delaying agent, etc. The composition of the present invention may include a pharma-ceutically acceptable salt, such as an acid addition salt or a base addition salt.

[0263] Exemplary pharma- ceutically acceptable carriers in common use include any absorption retarders, antioxidants, binders, buffers, fillers or bulking agents, chelating agents, coatings, disintegrants, dispersions, gels, isotonicity agents, lubricants, preservatives, salts, solvents or cosolvents, stabilizers, surfactants, and / or delivery vehicles.

[0264] In various embodiments, the composition comprising the vector described herein, for example, the sterile preparation for injection, can be formulated according to conventional pharmaceutical practice, using distilled water for injection as vehicle.For example, saline or isotonic solution containing glucose and other additives such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be used as the aqueous solution for injection, and can be optionally combined with suitable solubilizers, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and non-ionic surfactants such as polysorbate 80 (trademark), HCO-50, etc.

[0265] The formulations disclosed herein can be formulated, for example, for administration by injection. For injection, the formulations can be formulated as aqueous solutions, such as Hanks' solution, Ringer's solution, or buffered solutions including saline, or in culture media such as Iscove's modified Dulbecco's medium (IMDM). Aqueous solutions can contain formulation agents, such as suspending agents, stabilizing agents, and / or dispersing agents. Alternatively, the formulations can be in lyophilized and / or powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0266] Any formulation disclosed herein may advantageously include any other pharma- ceutically acceptable carrier, including those that do not produce significant adverse reactions, allergic reactions, or other side effects that outweigh the benefits of administration. Exemplary pharma-ceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, the formulations may be prepared to meet the sterility, pyrogenicity, general safety, and purity standards required by the US FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0267] 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 another example, the dose may range from 5×10 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, 10×10 8 In certain embodiments, a therapeutically effective amount of an adenoviral vector associated with a therapeutic gene may comprise 4×10 IU or more. 8 IU. In 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 recruitment factors.

[0268] In various embodiments of the present disclosure, the in vivo gene therapy comprises administration to a subject of at least one viral gene therapy vector in combination with at least one immunosuppressive regimen. In an in vivo gene therapy comprising more than one vector species, e.g., a first vector that is a supporting viral gene therapy vector in combination with a second vector that is a supporting vector, the first vector and the second vector 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 at the same time or at different times, e.g., during the same hour or during non-overlapping hours. In various embodiments, the first and second vectors can be administered at the same time 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 in the same dosage or at different dosages, e.g., dosages measured as total number of viral particles or number of viral particles per kilogram of subject. In various embodiments, the first and second vectors can be administered in a predefined ratio. In various embodiments, the ratio ranges from 2:1 to 1:2, for example, a 1:1 ratio.

[0269] In various embodiments, the vector is administered to the subject in a single total dose in one day. In various embodiments, the vector is administered in two, three, four or more unit doses, which together constitute a total dose. In various embodiments, the subject is administered one unit dose of the vector per day on each of one, two, three, four or more consecutive days. In various embodiments, the subject is administered two unit doses of the vector per day on each of one, two, three, four or more consecutive days. Thus, in various embodiments, a daily dose can refer to the dose of the vector administered to the subject during one day. In various embodiments, the term day refers to a 24-hour period, such as the 24 hours from midnight on a first calendar day to midnight on the next calendar day.

[0270] 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 dose of a viral gene therapy vector and a support vector combined, 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 dose of a viral gene therapy vector and a support vector combined, can be 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.

[0271] 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, and optionally the viral gene therapy vector unit dose, daily dose, or total dose is at least 1E10, 5E 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 5E10vp / kg and an upper limit selected from 1E9, 5E9, 1E10, 5E10, 1E11, and 5E11vp / kg.

[0272] In various embodiments, the support 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 supporting viral gene therapy 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, and optionally the unit dose, daily dose, or total dose of the supporting vector is at least 1E10, 5E10, 1E 11, 5E11, 1E12, and 5E12, vp / kg and an upper limit selected from 1E11, 5E11, 1E12, 5E12, 1E13, 5E13, 1E14, and 1E15vp / kg, and / or the unit dose, daily dose, or total dose of the supporting viral gene therapy vector is within a range having a lower limit selected from 1E8, 5E8, 1E9, 5E9, 1E10, and 5E10vp / kg and an upper limit selected from 1E9, 5E9, 1E10, 5E10, 1E11, and 5E11vp / kg. In various embodiments, the supporting viral gene therapy vector and the supporting vector are administered at a predefined ratio. In various embodiments, the ratio is in the range of 2:1 to 1:2, for example, a ratio of 1:1.

[0273] IV.Applications The methods and compositions provided herein are disclosed, at least in part, for use in in vivo gene therapy. However, for the avoidance of doubt, the present disclosure expressly includes the use of the compositions and methods provided herein for ex vivo manipulation of cells and / or tissues, as well as in vitro use, including the manipulation of cells and / or tissues for research purposes. Gene therapy includes the use of the vectors, genomes, or systems of the present disclosure in methods for introducing exogenous DNA into a host cell (such as a target cell) and / or a nucleic acid (such as a target nucleic acid, e.g., a target genome, e.g., the genome of a target cell). The present disclosure includes descriptions and illustrations of compositions and methods relating to in vivo, in vitro, and ex vivo therapy, and one of skill in the art will appreciate that the various methods and compositions provided herein are generally applicable to the introduction of a nucleic acid payload into a subject, e.g., a host or target cell. Such compositions and methods are useful for general utility, e.g., in gene therapy, and are therefore useful both as tools in gene therapy in general and in a variety of specific conditions, including those provided herein.

[0274] IV(A). In vivo gene therapy Treatment using in vivo gene therapy, including direct delivery of viral vectors to patients, is being explored. In vivo gene therapy is an attractive approach because it does not require any genotoxic conditioning (or can require less genotoxic conditioning), does not require ex vivo cell processing, and can be administered by injection, similar to what is already being done worldwide as vaccine delivery, and can be adopted in many facilities around the world, including developing countries. In various embodiment methods, the method of in vivo gene therapy using the adenoviral vector of the present disclosure can include one or more steps of (i) target cell recruitment, (ii) immunosuppression, (iii) administration of the vector, genome, system or formulation provided herein, and / or (iv) selection of transduced cells and / or cells that have integrated the integration elements of the payload of the adenoviral vector or genome.

[0275] The adenoviral vector formulations disclosed herein can be used to treat subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cows, goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice, fish, etc.). Treating a subject includes delivering a therapeutically effective amount of one or more vectors, genomes, or systems of the present disclosure. A therapeutically effective amount includes those that provide an effective amount, prophylactic treatment, and / or therapeutic treatment.

[0276] The vectors described herein can be administered with mobilization factors. In certain embodiments, the adenoviral vector formulations described herein can be administered in conjunction with HSPC mobilization. In certain embodiments, administration of the adenoviral donor vector is performed simultaneously with administration of one or more mobilization factors. In certain embodiments, administration of the adenoviral donor vector is performed after administration of one or more mobilization factors. In certain embodiments, administration of the adenoviral donor vector is performed after administration of a first one or more mobilization factors and simultaneously with administration of a second one or more mobilization factors. Agents for HSPC mobilization include, for example, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), AMD3100, SCF, S-CSF, CXCR4 antagonist, CXCR2 agonist, and Gro-Beta (GRO-β). In various embodiments, the CXCR4 antagonist is AMD3100 and / or the CXCR2 agonist is GRO-β.

[0277] G-CSF is a cytokine whose functions in HSPC mobilization may include promoting the expansion of granulocytes and attenuating both protease-dependent and -independent adhesion molecules and disrupting the SDF-1 / CXCR4 axis. In certain embodiments, any commercially available form of G-CSF known to those of skill in the art may be used in the methods and formulations disclosed herein, such as Filgrastim (Neupogen®, Amgen Inc., Thousand Oaks, CA) and PEGylated Filgrastim (Pegfilgrastim, NEULASTA®, Amgen Inc., Thousand Oaks, CA).

[0278] GM-CSF, also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein that functions as a cytokine and is naturally secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. In certain embodiments, any commercially available form of GM-CSF known to those of skill in the art can be used in the methods and formulations disclosed herein, such as Sargramostim (Leukine, Bayer Healthcare Pharmaceuticals, Seattle, WA) and molgramostim (Schering-Plough, Kenilworth, NJ).

[0279] AMD3100 (MOZOBIL™, PLERIXAFOR™; Sanofi-Aventis, Paris, France), a synthetic organic molecule of the bicyclam class, is a chemokine receptor antagonist that reversibly inhibits SDF-1 binding to CXCR4 and promotes HSPC mobilization. AMD3100 has been approved in combination with G-CSF for HSPC mobilization in patients with myeloma and lymphoma. The structure of AMD3100 is: [ka]

[0280] SCF, also known as KIT ligand, KL, or steel factor, is a cytokine that binds to the c-kit receptor (CD117). SCF can exist as both a transmembrane protein and a soluble protein. This cytokine plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. In certain embodiments, any commercially available form of SCF known to those of skill in the art can be used in the methods and formulations disclosed herein, for example, recombinant human SCF (Ancestim, STEMGEN®, Amgen Inc., Thousand Oaks, Calif.).

[0281] Chemotherapy used in intensive myelosuppressive treatment also mobilizes HSPCs into the peripheral blood as a result of compensatory neutrophil production after chemotherapy-induced hypoplasia. In certain embodiments, chemotherapeutic agents that can be used to mobilize HSPCs include cyclophosphamide, etoposide, ifosfamide, cisplatin, and cytarabine.

[0282] Further agents that can be used for cell recruitment include CXCL12 / CXCR4 modulators (e.g., CXCR4 antagonists: POL6326 (Polyphor, Allschwil, Switzerland), a synthetic cyclic peptide that reversibly inhibits CXCR4; BKT-140 (4F-benzoyl-TN14003; Biokine Therapeutics, Rehovit, Israel); TG-0054 (Taigen Biotechnology, Taipei, Taiwan); the CXCL12 neutralizer NOX-A12 (NOXXON Pharma, Berlin, Germany), which binds to SDF-1 and inhibits its binding to CXCR4); sphingosine-1-phosphate (S1P) agonists (e.g., SEW2871, Juarez et al. Blood 119:707-716,2012); vascular cell adhesion molecule-1 (VCAM) or very late antigen 4 (VLA-4) inhibitors (e.g., Natalizumab, a recombinant humanized monoclonal antibody against the α4 subunit of VLA-4 (Zohren et al. Blood 111:3893-3895,2008); BIO5192, a small molecule inhibitor of VLA-4 (Ramirez et al. Blood 114:1340-1343,2009)); parathyroid hormone (Brunner et al. Exp Hematol. 36:1157-1166,2008); proteasome inhibitors (e.g., Bortezomib, Ghobadi et al. ASH Annual Meeting Abstracts.p.583,2012); Groβ, a member of the CXC chemokine family that stimulates neutrophil chemotaxis and activation by binding to the CXCR2 receptor (e.g., SB-251353, King et al. Blood 97:1534-1542,2001); stabilization of hypoxia-inducible factor (HIF) (e.g., FG-4497, Forristal et al. ASH Annual Meeting Abstracts.p.216,2012); Firategrast, an α4β1 and α4β7 integrin inhibitor (α4β1 / 7) (Kim et al.Blood 128:2457-2461,2016); Vedolizumab, a humanized monoclonal antibody against α4β7 integrin (Rosario et al. Clin Drug Investig 36:913-923,2016); and BOP (N-(benzenesulfonyl)-L-prolyl-LO-(1-pyrrolidinylcarbonyl)tyrosine) which targets integrin α9β1 / α4β1 (Cao et al. Nat Commun 7:11007,2016). Further agents that can be used for HSPC mobilization are described, for example, in Richter R et al. Transfus Med Hemother 44:151-164, 2017, Bendall & Bradstock, Cytokine & Growth Factor Reviews 25:355-367, 2014, WO2003043651, WO2005017160, WO2011069336, US5,637,323, US7,288,521, US9,782,429, US2002 / 0142462, and US2010 / 02268.

[0283] In certain embodiments, the therapeutically effective amount of G-CSF comprises 0.1 μg / kg to 100 μg / kg. In certain embodiments, the therapeutically effective amount of G-CSF comprises 0.5 μg / kg to 50 μg / kg. In certain embodiments, the therapeutically effective amount of G-CSF comprises 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 11 μg / kg, 12 μg / kg, 13 μg / kg, 14 μg / kg, 15 μg / kg, 16 μg / kg, 17 μg / kg, 18 μg / kg, 19 μg / kg, 20 μg / kg or more. In certain embodiments, the therapeutically effective amount of G-CSF comprises 5 μg / kg. In certain embodiments, G-CSF can be administered subcutaneously or intravenously. In certain embodiments, G-CSF can be administered for 1 day, 2 consecutive days, 3 consecutive days, 4 consecutive days, 5 consecutive days or more. In certain embodiments, G-CSF can be administered for 4 consecutive days. In certain embodiments, G-CSF can be administered for 5 consecutive days. In certain embodiments, G-CSF can be used subcutaneously daily as a single agent at a dose of 10 μg / kg, starting 3, 4, 5, 6, 7, or 8 days prior to adenoviral delivery. In certain embodiments, G-CSF can be administered as a single agent and then co-administered with another mobilizing factor. In certain embodiments, G-CSF can be administered as a single agent and then co-administered with AMD3100. In certain embodiments, the treatment protocol includes 5 days of treatment, where G-CSF may be administered on days 1, 2, 3, and 4, and on day 5, G-CSF and AMD3100 are administered 6-8 hours prior to adenovirus administration.

[0284] The therapeutically effective amount of GM-CSF administered may include, for example, a dose in the range of 0.1-50 μg / kg or 0.5-30 μg / kg. In certain embodiments, doses at which GM-CSF may be administered include 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 11 μg / kg, 12 μg / kg, 13 μg / kg, 14 μg / kg, 15 μg / kg, 16 μg / kg, 17 μg / kg, 18 μg / kg, 19 μg / kg, 20 μg / kg. In certain embodiments, GM-CSF may be administered subcutaneously for 1 day, 2 consecutive days, 3 consecutive days, 4 consecutive days, 5 consecutive days, or more. In certain embodiments, GM-CSF can be administered subcutaneously or intravenously. In certain embodiments, GM-CSF can be administered subcutaneously daily at a dose of 10 μg / kg, beginning 3, 4, 5, 6, 7, or 8 days prior to adenoviral delivery. In certain embodiments, GM-CSF can be administered as a single agent followed by co-administration with another mobilizing factor. In certain embodiments, GM-CSF can be administered as a single agent followed by co-administration with AMD3100. In certain embodiments, the treatment protocol includes a 5-day treatment, where GM-CSF can be administered on days 1, 2, 3, and 4, and on day 5, GM-CSF and AMD3100 are administered 6-8 hours prior to adenoviral administration. The dosing regimen for Sargramostim is 200 μg / m 2 , 210 μg / m 2 , 220 μg / m 2 , 230 μg / m 2 , 240 μg / m 2 , 250 μg / m 2 , 260 μg / m 2 , 270 μg / m 2 , 280 μg / m 2 , 290 μg / m 2 , 300 μg / m 2, or more. In certain embodiments, Sargramostim can be administered for 1 day, 2 consecutive days, 3 consecutive days, 4 consecutive days, 5 consecutive days or more. In certain embodiments, Sargramostim can be administered subcutaneously or intravenously. In certain embodiments, the dosing regimen for Sargramostim can include 250 μg / m2 intravenously or subcutaneously. 2 / day and can continue until target cell mass is reached in peripheral blood, or can continue for 5 days. In certain embodiments, Sargramostim can be administered as a single agent followed by co-administration with another mobilizing factor. In certain embodiments, Sargramostim can be administered as a single agent followed by co-administration with AMD3100. In certain embodiments, the treatment protocol includes 5 days of treatment, where Sargramostim can be administered on days 1, 2, 3, and 4, and on day 5, Sargramostim and AMD3100 are administered 6-8 hours prior to adenovirus administration.

[0285] In certain embodiments, the therapeutically effective amount of AMD3100 comprises 0.1 mg / kg to 100 mg / kg. In certain embodiments, the therapeutically effective amount of AMD3100 comprises 0.5 mg / kg to 50 mg / kg. In certain embodiments, the therapeutically effective amount of AMD3100 comprises 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg or more. In certain embodiments, the therapeutically effective amount of AMD3100 comprises 4 mg / kg. In certain embodiments, the therapeutically effective amount of AMD3100 comprises 5 mg / kg. In certain embodiments, a therapeutically effective amount of AMD3100 includes 10 μg / kg to 500 μg / kg or 50 μg / kg to 400 μg / kg. In certain embodiments, a therapeutically effective amount of AMD3100 includes 100 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 350 μg / kg or more. In certain embodiments, AMD3100 can be administered subcutaneously or intravenously. In certain embodiments, AMD3100 can be administered subcutaneously at 160 to 240 μg / kg 6 to 11 hours prior to adenoviral delivery. In certain embodiments, a therapeutically effective amount of AMD3100 can be administered simultaneously with another recruitment factor. In certain embodiments, a therapeutically effective amount of AMD3100 can be administered following administration of another recruitment factor. In certain embodiments, a therapeutically effective amount of AMD3100 can be administered following administration of G-CSF. In certain embodiments, the treatment protocol includes 5 days of treatment, with G-CSF administered on days 1, 2, 3, and 4, and on day 5, G-CSF and AMD3100 administered 6-8 hours prior to adenovirus injection.

[0286] The therapeutically effective amount of SCF administered may include, for example, a dose ranging from 0.1 to 100 μg / kg / day or 0.5 to 50 μg / kg / day. In certain embodiments, SCF may be administered at doses of 0.5 μg / kg / day, 1 μg / kg / day, 2 μg / kg / day, 3 μg / kg / day, 4 μg / kg / day, 5 μg / kg / day, 6 μg / kg / day, 7 μg / kg / day, 8 μg / kg / day, 9 μg / kg / day, 10 μg / kg / day, 11 μg / kg / day, 12 μg / kg / day, 13 μg / kg / day, 14 μg / kg / day, 15 μg / kg / day, 16 μg / kg / day, 17 μg / kg / day, 18 μg / kg / day, 19 μg / kg / day, 20 μg / kg / day, 21 μg / kg / day, 22 μg / kg / day, 23 μg / kg / day, 24 μg / kg / day, 25 μg / kg / day, 26 μg / kg / day, 27 μg / kg / day, 28 μg / kg / day, 29 μg / kg / day, 30 μg / kg / day, 31 μg / kg / day, 32 μg / kg / day, 33 μg / kg / day, 34 μg / kg / day, 35 μg / kg / day, 36 μg / kg / day, 37 μg / kg / day, 38 μg / kg / day, 39 μg / kg / day, 40 μg / kg / day, 41 μg / kg / day, 42 μg / kg / day, 43 μg / kg / day, 44 μg / kg / day, 45 μg / In certain embodiments, SCF can be administered for 1 day, 2 consecutive days, 3 consecutive days, 4 consecutive days, 5 consecutive days, or more. In certain embodiments, SCF can be administered subcutaneously or intravenously. In certain embodiments, SCF can be injected subcutaneously at 20 μg / kg / day. In certain embodiments, SCF can be administered as a single agent followed by co-administration with another mobilizing factor. In certain embodiments, SCF can be administered as a single agent followed by co-administration with AMD3100. In certain embodiments, the treatment protocol involves 5 days of treatment, where SCF can be administered on days 1, 2, 3, and 4, with SCF and AMD3100 administered 6-8 hours prior to adenovirus administration on day 5.

[0287] In certain embodiments, the growth factors GM-CSF and G-CSF can be administered to mobilize HSPCs in the bone marrow niche into the peripheral circulation and increase the proportion of HSPCs circulating in the blood. In certain embodiments, mobilization can be achieved by administration of G-CSF / Filgrastim (Amgen) and / or AMD3100 (Sigma). In certain embodiments, mobilization can be achieved by administration of GM-CSF / Sargramostim (Amgen) and / or AMD3100 (Sigma). In certain embodiments, mobilization can be achieved by administration of SCF / Ancestim (Amgen) and / or AMD3100 (Sigma). In certain embodiments, administration of G-CSF / Filgrastim precedes administration of AMD3100. In certain embodiments, administration of G-CSF / Filgrastim is simultaneous with administration of AMD3100. In certain embodiments, administration of G-CSF / Filgrastim precedes administration of AMD3100, followed by co-administration of G-CSF / Filgrastim and AMD3100. US20140193376 describes a mobilization protocol using a CXCR4 antagonist in combination with an S1P receptor 1 (S1PR1) modulator. US20110044997 describes a mobilization protocol using a CXCR4 antagonist in combination with a vascular endothelial growth factor receptor (VEGFR) agonist.

[0288] Adenoviral vectors (e.g., Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors) are examples of vectors that can be administered in conjunction with HSPC mobilization. In certain embodiments, administration of the adenoviral vector is performed simultaneously with administration of one or more mobilization factors. In certain embodiments, administration of the adenoviral vector is performed after administration of one or more mobilization factors. In certain embodiments, administration of the adenoviral vector is performed after administration of a first one or more mobilization factors and simultaneously with administration of a second one or more mobilization factors.

[0289] In certain embodiments, HSC expansion agents such as CD19 immunotoxin or 5-FU can be administered to expand HSPCs. CD19 immunotoxin can be used to deplete all CD19 lineage cells, which account for 30% of bone marrow cells. Depletion promotes mobilization from bone marrow. Proliferation of HSPCs (e.g., via CD19 immunotoxin 5-FU) stimulates HSPC differentiation and mobilization from bone marrow, increasing transgene marking in peripheral blood cells.

[0290] A therapeutically effective amount of an HSC mobilizing factor and / or HSC expanding agent can be administered by any suitable route of administration, such as by injection, infusion, perfusion, more specifically by one or more of bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal injection, infusion, or perfusion.

[0291] In certain embodiments, the methods of the disclosure may include selecting cells that have been modified to express a selectable marker (e.g., a mutant form of MGMT that is resistant to inactivation by 6-BG while retaining the ability to repair DNA damage). For example, specific embodiments include selecting cells that express a mutant form of MGMT P140K In the case of an adenoviral vector comprising a selection marker, the combination regimen includes mobilization (e.g., the mobilization protocol described herein) and administration of the adenoviral vector described herein in combination with administration of BCNU or benzylguanine and temozolomide. In certain embodiments, the in vivo selection marker is MGMT as described in Olszko et al., Gene Therapy 22:591-595, 2015. P140K Therefore, MGMT P140K Selecting cells expressing the gene can aid in the selection of transduced cells and / or contribute to the therapeutic effect.

[0292] The adenoviral vector can be administered simultaneously with, or following, the administration of one or more immunosuppressive agents or regimens.

[0293] IV(B). In vitro and ex vivo gene therapy In vitro gene therapy includes the use of the vectors, genomes, or systems of the present disclosure in a method of introducing exogenous DNA into a host cell (such as a target cell) and / or a nucleic acid (such as a target nucleic acid, e.g., a target genome), where the host cell or nucleic acid is not present in a multicellular organism (e.g., in a laboratory). In some embodiments, the target cell or nucleic acid is derived from a multicellular organism, such as a mammal (e.g., a mouse, a rat, a human, or a non-human primate). In vitro manipulation of cells from a multicellular organism may also be referred to as ex vivo manipulation and can be used in ex vivo therapy. In various embodiments, the methods and compositions of the present disclosure are utilized to modify a target cell or nucleic acid derived from a first multicellular organism, e.g., as disclosed herein, and then administer the engineered target cell or nucleic acid to a second multicellular organism, such as a mammal (e.g., a mouse, a rat, a human, or a non-human primate), e.g., in a method of adoptive cell therapy. In some cases, the first and second organisms are the same single subject organism. Returning an in vitro manipulated material to the subject from which it originated can be an autologous therapy. In some cases, the first and second organisms are different organisms (e.g., two organisms of the same species, e.g., two mice, two rats, two humans, or two non-human primates of the same species). Transplantation of engineered material from a first subject into a second, different subject can be an allogeneic therapy.

[0294] Ex vivo cell therapy may involve isolating stem cells, progenitor cells or differentiated cells from a patient or normal donor, expanding the isolated cells ex vivo (with or without genetic manipulation), and administering the cells to a subject to establish a transient or stable graft of the injected cells and / or their progeny. Such ex vivo approaches can be used, for example, to treat genetic, infectious or neoplastic diseases, regenerate tissues, or deliver therapeutic agents to disease sites. In various ex vivo therapies, the subject is not directly exposed to the gene transfer vector, and the cells targeted for transduction can be selected, expanded and / or differentiated before or after any genetic manipulation to improve efficacy and safety.

[0295] Ex vivo therapies include hematopoietic stem cell (HSC) transplantation (HCT). Autologous HSC gene therapy is a treatment option for several monogenic and storage disorders of the blood and immune system and may be the first-line treatment option for certain disease states.

[0296] Applications of ex vivo therapy include reconstituting dysfunctional cell lineages. In the case of genetic diseases characterized by defective or absent cell lineages, the lineages can be regenerated by functional progenitor cells derived from either normal donors or autologous cells that have been subjected to ex vivo gene transfer to correct the defect. One example is SCID, in which a defect in any one of several genes inhibits the development of mature lymphoid cells. Transplantation of unmanipulated normal donor HSCs allows the generation of functional hematopoietic cells of various lineages from the donor in the host, making it a treatment option not only for SCID but also for many other diseases affecting the blood and immune system. Autologous HSC gene therapy can replenish functional copies of defective genes in transplanted hematopoietic stem / progenitor cells (HSPCs) and, similar to HCT, can provide a stable supply of functional progeny, which may have several advantages, such as reduced risk of graft-versus-host disease (GvHD), reduced risk of graft rejection, and reduced need for post-transplant immunosuppression.

[0297] Applications of ex vivo therapy include increasing the dosage of therapeutic genes. In some applications, HSC gene therapy can enhance the therapeutic efficacy of allogeneic HCT. The dosage of therapeutic genes can be manipulated to exceed the normal level of transplanted cells.

[0298] Applications of ex vivo therapy include the introduction of new functions and targeted gene therapy. Ex vivo gene therapy can confer new functions to HSCs or their progeny, for example, by expressing RNA-based agents (e.g., ribozymes, RNA decoys, antisense RNA, RNA aptamers and small interfering RNA) and protein-based agents (e.g., dominant negative mutant viral proteins, fusion inhibitors and engineered nucleases that target the genome of pathogens), establishing drug resistance that allows the administration of high-dose antitumor chemotherapy regimes, or establishing resistance to previously established infections by viruses such as HIV or other pathogens.

[0299] IV(C). Conditions treatable by gene therapy At least in part, because the adenoviral vectors of the present disclosure (e.g., Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vectors) can be used in vivo, in vitro, or ex vivo to modify host and / or target cells, and further because the adenoviral vectors can contain payloads encoding a wide variety of expression products, it will be apparent from this specification that the various techniques provided herein have broad applicability and can be used to treat a wide variety of conditions. Examples of conditions treatable by administration of the adenoviral vectors, genomes, or systems of the present disclosure include, but are not limited to, hemoglobinopathies, immunodeficiencies, point mutation conditions, cancers, protein deficiencies, infectious diseases, and inflammatory conditions.

[0300] In certain embodiments, the vectors, genomes, systems, and formulations disclosed herein can be used to treat subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cows, goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice, fish, etc.). Treating a subject includes delivery of a therapeutically effective amount. A therapeutically effective amount includes one that provides an effective amount, prophylactic treatment, and / or therapeutic treatment.

[0301] In certain embodiments, the methods and formulations disclosed herein can be used to treat hematological disorders, hi certain embodiments, the formulations are administered to a subject to treat hemophilia, β-thalassemia major, Diamond-Blackfan anemia (DBA), paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia (PRCA), refractory anemia, severe aplastic anemia, and / or hematological cancers, such as leukemia, lymphoma, and myeloma.

[0302] Hemoglobinopathies are a global health burden with disproportionate outcomes. Defects in the expression of hemoglobin proteins or globin genes can result in diseases called hemoglobinopathies. Hemoglobinopathies are among the most common genetic disorders worldwide.

[0303] Each year, 1.1 million newborns worldwide are at risk of hemoglobinopathies, affecting 25 out of every 1,000 live births in areas where falciparum malaria is endemic, due to natural resistance to malaria infection conferred by hemoglobin (Hb) gene mutations. In developed regions, patients are at risk of iron overload from long-term transfusions. In less developed regions, survival is extremely low. For example, in Africa, childhood mortality in patients with hemoglobinopathies is 40%, compared to an overall childhood mortality rate of 16%.

[0304] Mutations in globin genes can result in an imbalance of globin chains in cells, resulting in the production of abnormal forms of hemoglobin or reduced production of alpha or beta polypeptides, as in sickle cell disease (SCD) and hemoglobin C, D, and E diseases. The latter conditions are called alpha or beta-thalassemia, depending on which globin chain is impaired. Five percent of the world's population carries a significant hemoglobin variant, with the sickle cell mutation (conversion of glutamic acid to valine; historically E6V, modern E7V) in the b-globin (HBB) gene being the most common (40% of carriers). The high prevalence and severity of hemoglobin disorders represents a significant burden, affecting not only the lives of affected individuals, but also the health care system, as lifelong care of patients is costly.

[0305] Hemoglobin exists in two forms: fetal (HbF), which contains two alpha (α) and two gamma (γ) chains, and adult (HbA), which contains two alpha and two beta (β) chains. The natural replacement of HbF with HbA occurs soon after birth and is regulated by transcriptional repression of the γ-globin gene by factors including the master regulator bcl11a. Importantly, various clinical observations have shown that the severity of β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, can be improved by increasing the production of HbF.

[0306] In certain embodiments, a therapeutically effective treatment is one that induces or increases expression of HbF, induces or increases production of hemoglobin, and / or induces or increases production of β-globin. In certain embodiments, a therapeutically effective treatment improves blood cell function and / or increases cellular oxygenation.

[0307] In various embodiments, the present disclosure includes the treatment of blood disorders using the adenoviral donor vector of the present disclosure, which comprises a coding nucleic acid sequence that encodes a protein or agent for the treatment of blood disorders.In various embodiments, the blood disorder is thalassemia, and the protein is a β-globin or γ-globin protein, or a protein that partially or completely functionally replaces β-globin or γ-globin otherwise.In various embodiments, the blood disorder is hemophilia, and the protein is ET3, or a protein that partially or completely functionally replaces factor VIII otherwise.In various embodiments, the blood disorder is a point mutation disease, such as sickle cell anemia, and the agent is a gene editing protein.

[0308]

[0309] The β-globin may have or include the following amino acid sequence: SEQ ID NO: 155. In various embodiments, the β-globin replacement protein may have an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 155 (MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH).

[0310] Gamma-globin may have or include the following amino acid sequence: SEQ ID NO: 156. In various embodiments, a gamma-globin replacement protein may have an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 156 (MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDATKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYH).

[0311] Over 80 primary immunodeficiencies are recognized by the World Health Organization. These diseases are characterized by an essential defect in the immune system, and in some cases, the body does not produce enough or no antibodies to fight infection. In other cases, the cellular defenses that fight infection do not work properly. Typically, primary immunodeficiencies are genetic disorders.

[0312] Secondary or acquired immunodeficiencies occur in individuals whose immune system is compromised by factors other than the immune system, rather than as a result of a congenital genetic abnormality. Examples include trauma, viruses, chemotherapy, toxins, and pollution. Acquired immune deficiency syndrome (AIDS) is an example of a secondary immunodeficiency caused by the human immunodeficiency virus (HIV), which depletes T lymphocytes and renders the body unable to fight infection.

[0313] X-linked severe combined immunodeficiency (SCID-X1) is a depletion of both cellular and humoral immunity caused by mutations in the common gamma chain gene (γC), resulting in a lack of T and natural killer (NK) lymphocytes and nonfunctional B lymphocytes. SCID-X1 is fatal within the first 2 years of life unless the immune system is reconstituted, for example, by bone marrow transplantation (BMT) or gene therapy.

[0314] As most individuals do not have a suitable donor for BMT or non-autologous gene therapy, haploidentical parental bone marrow depleted of mature T cells is often used, although complications include graft-versus-host disease (GVHD), inability to produce sufficient antibodies resulting in prolonged immunoglobulin supplementation, late loss of T cells due to failure of hematopoietic stem and progenitor cells (HSPC) engraftment, chronic vegetations, and lymphocyte dysregulation.

[0315] Fanconi anemia (FA) is a congenital blood disorder that results in bone marrow failure. It is characterized, in part, by a deficiency in DNA repair mechanisms. At least 20% of FA patients develop cancers, including acute myeloid leukemia, as well as cancers of the skin, liver, gastrointestinal tract, and gynecological system. Tumors of the skin and gastrointestinal tract are usually squamous cell carcinomas. The average age of patients developing cancer is 15 years for leukemia, 16 years for liver tumors, and 23 years for other tumors.

[0316] The therapeutic gene can be selected to provide a therapeutically effective response to a condition that, in certain embodiments, is congenital. In certain embodiments, the condition can be Graves' disease, rheumatoid arthritis, pernicious anemia, multiple sclerosis (MS), inflammatory bowel disease, systemic lupus erythematosus (SLE), adenosine deaminase deficiency (ADA-SCID) or severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), Fanconi anemia (FA), Batten disease, adrenoleukodystrophy (ALD) or metachromatic leukodystrophy (MLD), muscular dystrophy, pulmonary alveolar proteinosis (PAP), pyruvate kinase deficiency, Shwachman-Diamond-Blackfan anemia, dyskeratosis congenita, cystic fibrosis, Parkinson's disease, Alzheimer's disease, or amyotrophic lateral sclerosis (Lou Gehrig's disease). In certain embodiments, the therapeutic gene may be a gene encoding a protein and / or a gene whose function is inhibited, depending on the condition.

[0317] In certain embodiments, the methods and formulations disclosed herein can be used to treat cancer. In certain embodiments, the formulations are administered to a subject to treat acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma, juvenile myelomonocytic leukemia, multiple myeloma, myelodysplasia, and / or non-Hodgkin's lymphoma.

[0318] Additional exemplary cancers that may be treated include astrocytoma, atypical teratoid rhabdoid tumor, brain and central nervous system (CNS) cancer, breast cancer, carcinosarcoma, chondrosarcoma, chordoma, choroid plexus carcinoma, choroid plexus papilloma, clear cell sarcoma, diffuse large B-cell lymphoma, ependymoma, epithelial sarcoma, extragonadal germ cell tumor, extrarenal rhabdoid tumor, Ewing's sarcoma, gastrointestinal stromal tumor, glioblastoma, HBV-induced hepatocellular carcinoma, head and neck cancer, kidney cancer, and the like. Cancer includes lung cancer, malignant rhabdoid tumor, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, glioma, sarcoma not otherwise specified (NOS), oligoastrocytoma, oligodendroglioma, osteosarcoma, ovarian cancer, ovarian clear cell adenocarcinoma, ovarian endometrioid adenocarcinoma, ovarian serous adenocarcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic endocrine tumor, pineoblastoma, prostate cancer, renal cell carcinoma, renal medullary carcinoma, rhabdomyosarcoma, sarcoma, schwannoma, cutaneous squamous cell carcinoma, and stem cell carcinoma. In various specific embodiments, the cancer is ovarian cancer. In various specific embodiments, the cancer is breast cancer. In certain embodiments, the formulation is administered to a subject to prevent or delay the recurrence of cancer, or to prevent or delay the onset of cancer in carriers of high-risk germline mutations.

[0319] In the context of cancer, a therapeutically effective amount may result in a reduction in the number of tumor cells, a reduction in the number of metastases, a reduction in tumor volume, an increase in life expectancy, an induction of apoptosis in cancer cells, an induction of cancer cell death, an induction of chemotherapy or radiation sensitivity in cancer cells, an inhibition of angiogenesis near cancer cells, an inhibition of cancer cell proliferation, an inhibition of tumor growth, prevention of metastasis, an increase in the survival of a subject, a reduction in cancer-associated pain, a reduction in the number of metastases, and / or a reduction in the recurrence or relapse of cancer following treatment.

[0320] In certain embodiments, the methods and formulations disclosed herein can be used to treat point mutation conditions. In certain embodiments, the formulations are administered to a subject to treat sickle cell disease, cystic fibrosis, Tay-Sachs disease, and / or phenylketonuria. In various embodiments, the transposon payload of the present disclosure encodes a CRISPR-Cas that provides correct editing of the nucleic acid lesion. In various embodiments, the transposon payload of the present disclosure encodes a base editor that provides correct editing of the nucleic acid lesion. In various embodiments, the transposon payload of the present disclosure encodes a prime editor that provides correct editing of the nucleic acid lesion.

[0321] In certain embodiments, the methods and formulations disclosed herein can be used to treat certain enzyme deficiencies. In certain embodiments, the formulations are administered to a subject to treat Hurler syndrome, selective IgA deficiency, hyper IgM, IgG subclass deficiency, Niemann-Pick disease, Tay-Sachs disease, Gaucher disease, Fabry disease, Krabbe disease, glucosmia, maple syrup urine disease, phenylketonuria, glycogen storage disease, Friedreich's ataxia, Zellweger syndrome, adrenoleukodystrophy, complement disorders, and / or mucopolysaccharidoses.

[0322] A therapeutically effective amount may provide function to immune cells and other blood cells and / or microglial cells, or, depending on the condition being treated, may inhibit lymphocyte activation, induce apoptosis of lymphocytes, eliminate various subsets of lymphocytes, inhibit T cell activation, eliminate or inhibit autoreactive T cells, inhibit Th-2 or Th-1 lymphocyte activity, antagonize IL-1 or TNF, reduce inflammation, induce selective tolerance to inducing agents, reduce or eliminate immune-mediated conditions, and / or reduce or eliminate symptoms of immune-mediated conditions. A therapeutically effective amount may result in functional DNA repair mechanisms; expression of surfactant proteins; maintenance of telomeres; lysosomal function; degradation of lipids or other proteins such as amyloid; enabling ribosomal function; and / or generating otherwise undeveloped mature blood cell lineages such as macrophages, other white blood cell types.

[0323] In certain embodiments, the methods of the present disclosure can restore a T cell-mediated immune response in a subject in need thereof, including restoration of thymic output and / or restoration of normal T lymphocyte development.

[0324] In certain embodiments, restoring thymic output can include restoring the proportion of CD3+ T cells expressing CD45RA in peripheral blood to a level comparable to a reference level obtained from a control population. In certain embodiments, restoring thymic output can include restoring the number of T cell receptor rearrangements (TRECs) per 106 mature T cells to a level comparable to a reference level obtained from a control population. The number of TRECs per 106 mature T cells can be determined as described in Kennedy et al., Vet Immunol Immunopathol 142:36-48, 2011.

[0325] In certain embodiments, restoring normal T lymphocyte development includes restoring the ratio of CD4+ cells:CD8+ cells to 2. In certain embodiments, restoring normal T lymphocyte development includes detecting the presence of αβ TCR in circulating T lymphocytes. The presence of αβ TCR in circulating T lymphocytes can be detected, for example, by flow cytometry using antibodies that bind to the α and / or β chains of the TCR. In certain embodiments, restoring normal T lymphocyte development includes detecting the presence of a diverse TCR repertoire that is comparable to a reference level obtained from a control population. TCR diversity can be assessed by TCR Vβ spectratype, which analyzes genetic rearrangements in the variable regions of the TCR β gene. A robust normal spectratype profile can be characterized by a Gaussian distribution of fragment sizes across 17 families of TCR Vβ segments. In certain embodiments, restoring normal T lymphocyte development includes restoration of T cell specific signaling pathways. Restoration of T cell specific signaling pathways can be assessed by lymphocyte proliferation following exposure to the T cell mitogen phytohemagglutinin (PHA). In certain embodiments, restoration of normal T lymphocyte development includes restoration of white blood cell counts, neutrophil cell counts, monocyte cell counts, lymphocyte cell counts, and / or platelet cell counts to levels comparable to reference levels obtained from a control population.

[0326] In certain embodiments, the methods of the present disclosure can improve lymphocyte reconstitution kinetics and / or clonal diversity in a subject in need thereof. In certain embodiments, improving lymphocyte reconstitution kinetics can include increasing the number of circulating T lymphocytes to within a range of reference levels obtained from a control population. In certain embodiments, improving lymphocyte reconstitution kinetics can include increasing the absolute CD3+ lymphocyte count to within a range of reference levels obtained from a control population. The range can be the range of values ​​observed or exhibited in normal (i.e., non-immunocompromised) subjects for a given parameter. In certain embodiments, improving lymphocyte reconstitution kinetics can include shortening the time required to reach a normal lymphocyte count compared to a subject in need thereof who has not been administered a therapy described herein. In certain embodiments, improving lymphocyte reconstitution kinetics can include increasing the abundance of gene-corrected lymphocytes compared to a subject in need thereof who has not been administered a therapy described herein. In certain embodiments, improving lymphocyte reconstitution kinetics can include increasing the diversity of the clonal repertoire of gene-corrected lymphocytes in a subject compared to a subject in need thereof who has not been administered a gene therapy described herein. Increasing the diversity of the clonal repertoire of gene-corrected lymphocytes can include increasing the number of unique RIS clones as measured by retroviral insertion site (RIS) analysis.

[0327] In certain embodiments, the methods of the present disclosure can restore bone marrow function in a subject in need thereof. In certain embodiments, restoring bone marrow function can include improving bone marrow repopulation with gene-corrected cells compared to a subject in need thereof not administered the therapy described herein. Improving bone marrow repopulation with gene-corrected cells can include increasing the percentage of gene-corrected cells. In certain embodiments, the cells are selected from white blood cells and bone marrow-derived cells. In certain embodiments, the percentage of gene-corrected cells can be measured using an assay selected from quantitative real-time PCR and flow cytometry.

[0328] In certain embodiments, the methods of the present disclosure can normalize primary and secondary antibody responses to immunization in a subject in need thereof. Normalizing primary and secondary antibody responses to immunization can include restoring B cell and / or T cell cytokine signaling programs that function in class switching and memory responses to antigens. Normalizing primary and secondary antibody responses to immunization can be measured by bacteriophage immunoassays. In certain embodiments, restoration of B cell and / or T cell cytokine signaling programs can be assayed after immunization with the T cell-dependent neoantigen bacteriophage ΨX174. In certain embodiments, normalizing primary and secondary antibody responses to immunization can include increasing IgA, IgM, and / or IgG levels in a subject in need thereof to levels comparable to reference levels obtained from a control population. In certain embodiments, normalizing primary and secondary antibody responses to immunization can include increasing the levels of IgA, IgM, and / or IgG in a subject in need thereof to a level greater than that of a subject in need thereof who has not been administered the gene therapy described herein. The levels of IgA, IgM, and / or IgG can be measured, for example, by immunoglobulin testing. In certain embodiments, the immunoglobulin testing includes antibodies that bind to IgG, IgA, IgM, kappa light chains, lambda light chains, and / or heavy chains. In certain embodiments, the immunoglobulin testing includes serum protein electrophoresis, immunoelectrophoresis, radial immunodiffusion, nephelometry, and turbidimetry. Commercially available immunoglobulin testing kits include MININEPH™ (Binding site, Birmingham, UK), and immunoglobulin testing systems from Dako (Denmark) and Dade Behring (Marburg, Germany). In certain embodiments, samples that can be used to measure immunoglobulin levels include blood samples, plasma samples, cerebrospinal fluid samples, and urine samples.

[0329] In certain embodiments, the method of the present disclosure can be used to treat SCID-X1. In certain embodiments, the method of the present disclosure can be used to treat SCID (e.g., JAK3 kinase deficiency SCID, purine nucleoside phosphorylase (PNP) deficiency SCID, adenosine deaminase (ADA) deficiency SCID, MHC class II deficiency or recombinase activating gene (RAG) deficiency SCID). In certain embodiments, the therapeutic effect can be observed by improving lymphocyte reconstitution, clonal diversity and thymopoiesis, reducing infections, and / or improving patient outcomes. The therapeutic effect can also be observed by one or more of weight gain and growth, improved gastrointestinal function (e.g., reducing diarrhea), reducing upper respiratory tract symptoms, reducing fungal infections of the oral cavity (thrush), reducing the incidence and severity of pneumonia, reducing meningitis and bloodstream infections, and reducing ear infections. In certain embodiments, the treatment of SCIDX-1 by the method of the present disclosure includes restoring the function of γC-dependent signaling pathway. The functionality of the γC-dependent signaling pathway can be assayed by measuring the tyrosine phosphorylation of the effector molecules STAT3 and / or STAT5 after in vitro stimulation with IL-21 and / or IL-2, respectively, which can be measured by intracellular antibody staining.

[0330] In certain embodiments, the methods of the present disclosure can be used to treat FA. In certain embodiments, therapeutic benefit can be observed by improved lymphocyte reconstitution, clonal diversity and thymopoiesis, reduced infections, and / or improved patient outcomes. Therapeutic benefit can also be observed by one or more of weight gain and growth, improved gastrointestinal function (e.g., reduced diarrhea), reduced upper respiratory tract symptoms, reduced fungal infections of the oral cavity (thrush), reduced incidence and severity of pneumonia, reduced meningitis and bloodstream infections, and reduced ear infections. In certain embodiments, treating FA with the methods of the present disclosure includes increasing the resistance of bone marrow-derived cells to mitomycin C (MMC). In certain embodiments, resistance of bone marrow-derived cells to MMC can be measured by cell survival assay in methylcellulose and MMC.

[0331] In certain embodiments, the disclosed method can be used to treat hypogammaglobulinemia. Hypogammaglobulinemia is caused by a lack of B-lymphocytes and is characterized by low antibody levels in the blood. Hypogammaglobulinemia can occur as a result of both leukemia-related immune dysfunction and treatment-related immune suppression in patients with chronic lymphocytic leukemia (CLL), multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), and other related malignancies. Patients with acquired hypogammaglobulinemia secondary to such hematological malignancies, and patients after HSPC transplantation, are susceptible to bacterial infections. Lack of humoral immunity is a major contributor to the increased risk of infection-related morbidity and mortality in these patients, especially from encapsulated microorganisms. For example, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus, as well as Legionella and Nocardia spp., are frequent pathogens causing pneumonia in CLL patients. Opportunistic infections, such as Pneumocystis carinii, fungi, viruses, and mycobacteria, have also been observed, and the number and severity of infections in these patients is greatly reduced by administration of immune globulin (Griffiths et al. Blood 73:366-368, 1989; Chapel et al. Lancet 343:1059-1063, 1994).

[0332] In certain embodiments, the formulation is for use in the treatment of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenoleukodystrophy, primary myelofibrosis, amegakaryocytic thrombocytopenia / congenital thrombocytopenia, ataxia telangiectasia, β-thalassemia, chronic granulomatous disease, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, common variable immunodeficiency (CVID), complement disorders, congenital agammaglobulinemia, Diamond-Blackfan anemia (DBA), diffuse large B-cell lymphoma, familial hemophagocytic lymphohistiocytosis, leukemia ... In some embodiments, the therapeutic agent is administered to a subject to treat an immune deficiency disorder, such as idiopathic leukemia, myelodysplastic syndrome, myeloma, leukemia, myelodysplastic syndrome, leukemia, myeloma, non-Hodgkin's lymphoma, paroxysmal nocturnal hemoglobinuria (PNH), primary immunodeficiency, pure red cell aplasia, refractory anemia, Schwachman-Diamond syndrome, selective IgA deficiency, severe aplastic anemia, sickle cell disease, specific antibody deficiency, Wiskott-Aldrich syndrome, and / or X-linked agammaglobulinemia (XLA).

[0333] Certain embodiments include the treatment of secondary or acquired immune deficiencies, such as immune deficiencies caused by trauma, viruses, chemotherapy, toxins, pollution, and the like. As indicated above, Acquired Immune Deficiency Syndrome (AIDS) is an example of a secondary immune deficiency caused by a virus, Human Immunodeficiency Virus (HIV), which depletes T lymphocytes and renders the body unable to fight infection. Thus, as another example, genes can be selected to provide a therapeutically effective response to an infectious disease. In certain embodiments, the infectious disease is Human Immunodeficiency Virus (HIV). Therapeutic genes can be, for example, genes that render immune cells resistant to HIV infection or enable immune cells to effectively neutralize the virus through immune reconstitution, polymorphisms in genes that code for proteins expressed by immune cells, genes that are not expressed in the patient and that are advantageous in fighting infection, genes that code for infectious agents, receptors or co-receptors, genes that code for ligands for receptors or co-receptors, viral and cellular genes essential for viral replication, for example genes that code for ribozymes, antisense RNA, small interfering RNA (siRNA) or decoy RNA to block the action of certain transcription factors, genes that code for dominant negative viral proteins, intracellular antibodies, intrakines and suicide genes. 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.A therapeutically effective amount for treating HIV may, for example, increase a subject's immunity to HIV, ameliorate symptoms associated with AIDS or HIV, or induce a subject's innate or adaptive immune response to HIV.An immune response against HIV includes antibody production and may result in the prevention of AIDS and / or ameliorate the symptoms of AIDS or HIV infection in a subject, or reduce or eliminate HIV infectivity and / or virulence.

[0334] Patients with MGMT-expressing tumors P140K It may be beneficial to administer an adenoviral vector (e.g., Ad3, 7, 11, 14, 16, 21, 34, 37, or 50 vector) that contains a therapeutic payload (such as CAR, TCR, or checkpoint inhibitor) combined with an in vivo selection cassette. The ex vivo approach shows the applicability of this approach. In certain embodiments, therapeutic amounts of TMZ and benzylguanine or BCNU are administered to reduce tumor burden or volume.

[0335] In certain embodiments, a therapeutically effective amount may provide a function to immune cells and other blood cells, reduce or eliminate an immune-mediated condition, and / or reduce or eliminate the symptoms of an immune-mediated condition.

[0336] The exemplary embodiment and example(s) provided herein are included to illustrate specific embodiments of the present disclosure. Those skilled in the art will, in light of the present disclosure, recognize that many changes can be made to the specific embodiments disclosed herein and still obtain equivalent or similar results without departing from the spirit and scope of the present disclosure. EXAMPLES

[0337] This example shows that certain adenovirus serotypes are particularly effective at infecting CD34+ cells, such as HSCs. Since HSCs are a therapeutically important target for gene therapy, the identification of vectors that are effective at transducing CD34+ cells is of great clinical importance. Several adenovirus serotypes tested were as effective or more effective at infecting CD34+ cells than those commonly associated with gene therapy testing and research, such as Ad5 and Ad5 / 35++.

[0338] Example 1: Analysis of adenoviral vector infection of CD34+ cells by anti-hexon staining This example uses anti-hexon staining to measure infection of CD34+ cells by various adenoviral vectors. Serotypes used in this experiment included Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad26, Ad34, Ad35, Ad37, Ad48, Ad50, and Ad52, as well as Ad5 / 35++ vectors containing an E1 deletion ("F35"). Vectors were wild-type human adenoviral vectors unless otherwise noted.

[0339] Human CD34+ cells (REF: 4Y-101C, LOT: 3038009, donor ID: 15846) were infected with wild-type human adenovirus at 5,000 or 2,000 viral particles per cell (vp / c) (identified by Ad type number). After 3 hours of incubation, cells were first washed with phosphate-buffered saline (PBS), quickly trypsinized to remove all extracellular viral particles, and washed with PBS. The washed cells were then split into two aliquots and utilized for intracellular adenovirus particle analysis by anti-hexon staining in this example and adenovirus DNA internalization analysis by qPCR in Example 2, respectively. Additionally, replicates were performed in which CD34+ cells were infected with 2,000, 10,000, and 20,000 viral particles per cell (vp / c).

[0340] In this example, cells were first fixed with fixation medium (Thermofisher) for 15 min at room temperature. After a PBS washing step, cells were suspended in permeabilization medium (Thermofisher). Anti-adenovirus hexon antibody (clone 20 / 11, MAB8052, Sigma) was added to the permeabilization medium and incubated overnight at 4°C. On the second day, cells were washed twice with PBS and stained with Alexa Fluor 488-labeled secondary antibody (catalog number A-21121, Thermofisher) in permeabilization medium. Staining was stopped by two PBS washing steps and cells were analyzed on a Beckman Coulter Gallios Flow Cytometer. Background signals were obtained by analyzing isotype controls, which refer to staining using Mouse IgG1 Isotype Control antibody (Sigma, REF: M5284-.1MG, clone: ​​MOPC21). The percentage of FITC-positive cells is shown in Figure 1. For each virus, two samples are shown per virus dose.

[0341] The results of anti-hexon staining are shown in Figure 1. As shown in Figure 1, the reference serotypes in this example include Ad5 and Ad5 / 35++ (F35) serotypes, which are frequently used, for example, in gene therapy studies or adenoviral vector constructs. Unexpectedly, several adenoviral vector serotypes consistently outperformed these reference serotypes for internalization into CD34+ cells. These included Ad3, 7, 11, 14, 16, 21, 34, 35, and 50. In contrast, serotypes Ad26, Ad37, Ad48, and Ad52 did not consistently outperform the reference serotypes for internalization into CD34+ cells. These data indicate that Ad3, 7, 11, 14, 16, 21, 34, 35, and 50 are particularly unexpectedly useful for engineering vectors for transduction into CD34+ cells, such as HSCs.

[0342] Example 2: Analysis of adenovirus particle internalization into CD34+ cells by qPCR This example uses qPCR to measure the internalization of adenoviral particles into CD34+ cells by various adenovirus serotypes. The serotypes used in the experiments of this example included Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad26, Ad34, Ad37, Ad35, Ad48, Ad50, and Ad52, as well as the Ad5 / 35++ vector ("F35") that contains an E1 deletion. The viruses used were purified wild-type human adenoviruses, unless otherwise stated. Cells were prepared as described in Example 1.

[0343] In this example, total genomic DNA was isolated using Monarch® Genomic DNA Purification Kit (NEB). For qPCR analysis, samples were divided into two experiments. The first experiment was Ad3, 7, 11, 14, 16, 21, 34, 35, and 50, and the second experiment was Ad26, Ad37, Ad48, Ad52, Ad5, and F35. In the first experiment, primers and probes targeted to DNA polymerase were used for amplification, and purified plasmids containing the Ad35 genome (pAd35) were used for generating standard curves. In the second experiment, primers and probes targeted to hexon were used for amplification, and purified plasmids containing the Ad5 genome (pAd5) were used for generating standard curves. For normalization, primers amplifying the gene hB2M were applied.

[0344] The results of the qPCR analysis of this example are shown in Figure 2. In general, the highest viral copy numbers per cell were obtained using Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad50, and F35. Viral copies per cell were also detected for Ad3, Ad37, Ad48, Ad52, and Ad5. The lowest viral copy numbers per cell were obtained for Ad26.

[0345] Example 3: Analysis of adenoviral vector infection of CD34+ cells by anti-hexon staining This example uses anti-hexon staining to measure infection of CD34+ cells by various adenoviral vectors. Serotypes used in this experiment included Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad26, Ad34, Ad35, Ad37, Ad48, Ad50, and Ad52, as well as Ad5 / 35++ vectors containing an E1 deletion ("F35"). Vectors were wild-type human adenoviral vectors unless otherwise noted.

[0346] Human CD34+ cells from three donors were infected with wild-type human adenovirus at 5,000 or 2,000 viral particles per cell (vp / c) (identified by Ad type number). Donor 1 cells (Lonza, REF: 4Y-101C, LOT: 3038009, donor ID: 15846) and donor 2 cells (Lonza, REF: 4Y-101E, LOT: 3046829, donor ID: 14538) were derived from a donor subjected to hematopoietic stem cell (HSC) mobilization with G-CSF, and donor 3 cells (Hemacare, REF: M34C-MOZ-1, LOT: 20063998) were derived from a donor subjected to HSC mobilization with plerixafor. After 3 or 6 hours of incubation, the cells were first washed with phosphate-buffered saline (PBS), quickly trypsinized to remove any extracellular viral particles, and washed with PBS. The washed cells were then divided into two aliquots, which were used in this example for intracellular adenovirus particle analysis by anti-hexon staining (Example 1) and adenovirus DNA internalization analysis by qPCR (Example 4), respectively.

[0347] In this example, cells were first fixed with Fixation Medium (Thermofisher) for 15 min at room temperature. After a PBS washing step, cells were resuspended in Permeabilization Medium (Thermofisher). Anti-adenovirus hexon antibody (clone 20 / 11, MAB8052, Sigma) was added to the permeabilization medium and incubated overnight at 4 °C. On the second day, cells were washed twice with PBS and stained with Alexa Fluor 488-labeled secondary antibody (catalog number A-21121, Thermofisher) in permeabilization medium. Staining was stopped by two PBS washing steps and cells were analyzed on a Beckman Coulter Gallios Flow Cytometer. Background signals were obtained by analyzing negative controls, referring to uninfected cells stained with the same antibody as the samples and / or isotype controls, referring to staining using a Mouse IgG1 Isotype Control antibody (Sigma, REF: M5284-.1MG, clone: ​​MOPC21). The percentage of FITC-positive cells is shown in Figures 3-13. For each virus, two or three samples are shown per viral dose.

[0348] The results of anti-hexon staining are shown in Figures 3-13. As shown in Figures 3-13, the reference serotypes in this example include Ad5 and Ad5 / 35++ (F35) serotypes, which are frequently used, for example, in gene therapy studies or adenoviral vector constructs. Unexpectedly, some adenovirus serotypes consistently outperformed the reference Ad5 serotype, and in some cases, the reference F35 serotype, for internalization into CD34+ cells. These included Ad3, 7, 11, 14, 16, 21, 34, 35, 37, and 50. Serotype Ad37 outperformed the reference serotype Ad5 for internalization into CD34+ cells from donors 2 and 3, but not donor 1. In contrast, serotypes Ad26, Ad48, and Ad52 did not consistently outperform the reference serotypes for internalization into CD34+ cells. These data indicate that Ad3, 7, 11, 14, 16, 21, 34, 35, 37, and 50 are particularly and unexpectedly useful for engineering vectors for transduction into CD34+ cells, such as HSCs.

[0349] Example 4: Analysis of adenovirus particle internalization into CD34+ cells by qPCR This example uses qPCR to measure the internalization of adenoviral particles into CD34+ cells by various adenovirus serotypes. The serotypes used in the experiments of this example included Ad3, Ad5, Ad7, Ad11, Ad14, Ad16, Ad21, Ad26, Ad34, Ad37, Ad35, Ad48, Ad50, and Ad52, as well as the Ad5 / 35++ vector ("F35") that contains an E1 deletion. The viruses used were purified wild-type human adenoviruses, unless otherwise stated. Cells were prepared as described in Example 3.

[0350] In this example, total genomic DNA was isolated using Monarch® Genomic DNA Purification Kit (NEB). For qPCR analysis, samples were divided into two experiments. The first experiment was Ad3, 7, 11, 14, 16, 21, 34, 35, and 50, and the second experiment was Ad26, Ad37, Ad48, Ad52, Ad5, and F35. In the first experiment, primers and probes targeted to DNA polymerase were used for amplification, and purified plasmids containing the Ad35 genome (pAd35) were used for generating standard curves. In the second experiment, primers and probes targeted to hexon were used for amplification, and purified plasmids containing the Ad5 genome (pAd5) were used for generating standard curves. For normalization, primers amplifying the gene hB2M were applied. During testing, background signals were obtained by analyzing negative controls, which refer to genomic DNA isolated from non-infected cells, and / or water (HO) controls, which refer to the use of water instead of genomic DNA in the qPCR reaction.

[0351] The results of the qPCR analysis of this example are shown in Figures 14-24. In general, the highest viral copy numbers per cell were obtained using Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad37, Ad50, and F35. Viral copies per c...

Claims

1. A pharmaceutical composition for use in a method of in vivo gene therapy in a mammalian subject, comprising an adenoviral vector, said method comprising administering to said subject said pharmaceutical composition, said adenoviral vector comprising: (a) a capsid comprising one or more viral polypeptides of the Ad34, Ad11, Ad3, Ad7, Ad14, Ad16, 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) hexon and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload; 13. A pharmaceutical composition comprising:

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

3. The pharmaceutical composition of claim 1 or 2, wherein the method comprises mobilization of the subject's hematopoietic stem cells prior to administration of the adenoviral vector.

4. The heterologous nucleic acid payload comprises a selection marker, optionally wherein the selection marker is MGMT. Ｐ１４０Ｋ 4. The pharmaceutical composition of claim 1, wherein optionally the method comprises administering to the subject a selection agent, optionally the selection agent comprises O 6 BG and / or BCNU.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the method comprises administering to the subject one or more immunosuppressive agents, optionally prior to administration of the adenoviral vector.

6. 1. An adenoviral donor vector, (a) a capsid comprising one or more viral polypeptides of the Ad34, Ad11, Ad3, Ad7, Ad14, Ad16, 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) hexon and (b) a double-stranded DNA genome comprising a heterologous nucleic acid payload; The adenovirus donor vector comprising:

7. The genome comprises: (a) a 3' ITR and a 5' ITR, each of which is of a viral polypeptide serotype; (b) a packaging sequence, the packaging sequence being of the viral polypeptide serotype; The vector of claim 6, further comprising:

8. The heterologous nucleic acid payload comprises a selection marker, optionally wherein the selection marker is MGMT. Ｐ１４０Ｋ The vector according to claim 6 or 7,

9. 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 (s) Penton and Hexon The pharmaceutical composition or vector according to any one of claims 1 to 8, comprising:

10. (a) the fiber knob has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, and 142; (b) the fiber shaft has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 125, and 141; (c) the fiber tail has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 157, 158, 159, 160, 161, 162, 163, 164, and 165; (d) the penton has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 15, 31, 47, 63, 79, 95, 111, 127, and 143; and / or (e) the hexon has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, and 144; A pharmaceutical composition or vector according to any one of claims 1 to 9.

11. The pharmaceutical composition or vector according to any one of claims 1 to 10, wherein the adenoviral vector comprises a fiber of a serotype of the viral peptide.

12. 12. The pharmaceutical composition or vector of any one of claims 1 to 11, wherein the fiber has a sequence having at least 80% identity to a sequence selected from SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, and 140.

13. The pharmaceutical composition or vector of any one of claims 1 to 12, wherein 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.

14. The pharmaceutical composition or vector according to any one of claims 1 to 13, wherein the adenoviral vector is a helper-dependent vector.

15. 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 Ad34, Ad11, Ad3, Ad7, Ad14, Ad16, Ad21, Ad37, or Ad50 serotypes, respectively; (b) a packaging sequence, the packaging sequence being of the ITR serotype; and (c) a heterologous nucleic acid payload; The adenovirus donor vector genome comprising:

16. The heterologous nucleic acid payload comprises a selection marker, optionally wherein the selection marker is MGMT. Ｐ１４０Ｋ The genome of claim 15.

17. (a) the heterologous nucleic acid payload encodes a protein; (b) the heterologous nucleic acid payload encodes a chimeric antigen receptor (CAR), a T cell receptor (TCR), or a small RNA, optionally wherein the small RNA is an shRNA; or (c) 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; A pharmaceutical composition, vector or genome according to any one of claims 1 to 16.

18. the heterologous nucleic acid payload comprising: (a) glioblastoma, hemoglobinopathies, platelet disorders, Fanconi anemia, alpha-1 antitrypsin deficiency, sickle cell anemia, thalassemia, thalassemia intermedia, von Willebrand's disease, hemophilia A, hemophilia B, factor V deficiency, factor VII deficiency, factor X deficiency, factor XI deficiency, factor XII deficiency, factor XIII deficiency, Bernard-Soulier syndrome, gray platelet syndrome, mucopolysaccharidoses, cystic fibrosis, Tay-Sachs disease, chronic granulomatous disease, Wiskott-Aldrich syndrome, and phenylketonuria; or (b) Graves' disease, rheumatoid arthritis, pernicious anemia, multiple sclerosis (MS), inflammatory bowel disease, systemic lupus erythematosus (SLE), adenosine deaminase deficiency (ADA-SCID) or severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), Fanconi anemia (FA), Batten disease, adrenoleukodystrophy (ALD) or metachromatic leukodystrophy (MLD), muscular dystrophy, pulmonary alveolar proteinosis (PAP), pyruvate kinase deficiency, Shwachman-Diamond-Blackfan anemia, dyskeratosis congenita, cystic fibrosis, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (Lou Gehrig's disease).

18. A pharmaceutical composition, vector or genome according to any one of claims 1 to 17, encoding an agent for the treatment of a condition selected from:

19. The pharmaceutical composition, vector or genome according to any one of claims 1 to 18, wherein the serotype of the viral polypeptide is Ad34.

20. A pharmaceutical composition comprising the adenoviral vector of any one of claims 6 to 19, the pharmaceutical composition being formulated for injection into a subject in need thereof.

21. 21. The pharmaceutical composition, vector or genome of any one of claims 1 to 20, wherein the adenoviral vector is infecting and / or transducing into CD34+ cells, CD34+high cells, CD34+ / CD90+ cells, and / or CD34+high / CD90+ cells, optionally wherein the cells are hematopoietic cells.

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