Integration of large adenovirus payloads

Abstract

The present disclosure provides recombinant adenoviral vectors and adenoviral genomes that can accommodate or contain large transposon payloads, for example, transposon payloads up to 40 kb. The adenoviral vectors and genomes can deliver large transposon payloads to target genomes, for example, for gene therapy. The present disclosure includes, inter alia, the adenoviral vectors and adenoviral genomes of the present disclosure, systems comprising two or more adenoviral vectors and / or adenoviral genomes, and uses of such adenoviral vectors, adenoviral genomes, and systems.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority and benefits therefrom based on the earlier filing date of U.S. Provisional Application No. 63 / 009,298, filed April 13, 2020, which is incorporated herein by reference in its entirety.

[0002] Description of research or development supported by the federal government. This invention was made with government support under grant numbers HL128288 and HL136135 granted by the National Institutes of Health. The government has certain rights to this invention.

[0003] Areas of disclosure This disclosure provides, in particular, recombinant adenovirus vectors and adenovirus genomes capable of accommodating or containing large transposon payloads, such as transposon payloads up to 40 kb. Some of the adenovirus vectors and genomes can deliver large transposon payloads to a target genome, for example, for gene therapy. [Background technology]

[0004] Background of Disclosure Gene therapy presents numerous challenges. Viral vectors are one means of gene therapy. Various challenges in the development of viral vectors for gene therapy include, in some cases, vector payload capacity, efficiency of transgene integration into the target cell genome, cell type specificity of transgene expression, levels of transgene expression, and site effects of integration. Various methods of gene therapy using viral vectors require resource-intensive steps, such as extracting cells from the target and manipulating and / or amplifying them ex vivo before administration to the target. For these reasons, and especially considering the increasing number of therapies utilizing viral vectors, there is a great need for improved viral vector design.

[0005] Hemoglobin disorders are among the most common genetic disorders worldwide, and their survival rates are significantly lower, especially in patients born in developing countries. Examples of hemoglobin disorders include sickle cell disease and thalassemia. Patient-specific hematopoietic stem / progenitor cell (HSPC) gene therapy offers a promising treatment for hemoglobin disorders.

[0006] The World Health Organization recognizes more than 80 primary immunodeficiency disorders. These disorders are characterized by an endogenous defect in the immune system (in some cases, the body is unable to produce any or sufficient antibodies against an infection). In other cases, the cellular defenses that fight infection do not function properly. Typically, primary immunodeficiency is a hereditary disorder.

[0007] Secondary immunodeficiency, or acquired immunodeficiency, is not the result of inherited genetic abnormalities, but rather occurs when the immune system is impaired by factors outside the immune system in an individual. Examples include trauma, viruses, chemotherapy, toxins, and contamination. Acquired immunodeficiency syndrome (AIDS) is an example of secondary immunodeficiency caused by a virus (human immunodeficiency virus (HIV)), in which T lymphocytes are depleted, making it impossible for the body to fight infection.

[0008] X-linked severe combined immunodeficiency (SCID-X1) is a condition in which both cellular and humoral immunity are depleted due to a mutation in the common gamma chain gene (γC). As a result of this mutation, T lymphocytes and natural killer (NK) lymphocytes are absent, and non-functional B lymphocytes are present. SCID-X1 is fatal by the age of two unless the immune system is reconstituted (e.g., through bone marrow transplantation (BMT) or gene therapy).

[0009] Since the majority of individuals lack a suitable donor for BMT or non-autologous gene therapy, haplotype-matched parental bone marrow depleted of mature T cells is often used; however, complications include graft-versus-host disease (GVHD), inability to produce adequate antibodies and therefore requiring long-term immunoglobulin supplementation, late T cell loss due to inability to engraft hematopoietic stem and progenitor cells (HSPCs), chronic warts, and lymphocyte dysregulation.

[0010] Fanconi anemia (FA) is a hereditary blood disorder that causes bone marrow failure. FA is partially characterized by a deficiency in DNA repair mechanisms. At least 20% of FA patients develop cancer (acute myeloid leukemia, as well as cancers of the skin, liver, gastrointestinal tract, and gynecological systems). Skin and gastrointestinal tumors 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.

[0011] Treatments using in vivo gene therapy, including direct delivery of viral vectors to patients, are being explored. In vivo gene therapy is a simple and attractive approach because it does not require any genotoxic pre-transplant treatment (or may require less genotoxic pre-transplant treatment), nor does it require ex vivo cell processing, and therefore can be administered by injection, similar to what has already been done worldwide for vaccine delivery, making it potentially adoptable in many facilities around the world, including those in developing countries.

[0012] Adenoviruses are particularly well-suited for use as gene transfer vectors due to their medium-sized genome, ease of manipulation, high titer, broad target cell range, and high infectivity. Both ends of the viral genome contain 100-200 base pair reverse repeat sequences (ITRs), which are cis-elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units, separated by the timing of viral DNA replication initiation. The E1 region (E1A and E1B) encodes proteins responsible for regulating viral genome transcription and a small number of cellular genes. When the E2 region (E2A and E2B) is expressed, proteins for viral DNA replication are synthesized. These proteins are involved in DNA replication, late gene expression, and host cell blockade. The late gene products (including most of the viral capsid protein) are expressed only after a single primary transcript, generated by the major late promoter (MLP), has been extensively processed. MLPs are particularly efficient during the later stages of infection, and all mRNA generated from this promoter has a three-part leader (TPL) sequence at the 5' end, which makes such mRNA more suitable for translation.

[0013] For successful gene therapy without site-specific effects and transcriptional silencing, the introduced gene must be expressed at high levels in the desired tissue or cell. Locus regulatory regions (LCRs) are particularly well-suited for this task because they are characterized by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy-number-dependent manner at ectopic chromatin sites. The components of LCRs generally co-localize to DNAse I-highly sensitive (HS) sites in the chromatin of expressing cells. The central determinants at individual HSs consist of a series of multiple ubiquitous and lineage-specific transcription factor binding sites. [Overview of the project] [Means for solving the problem]

[0014] overview This disclosure includes, in particular, adenovirus vectors and adenovirus genomes of this disclosure, systems comprising two or more adenovirus vectors and / or adenovirus genomes, and the use of such adenovirus vectors, adenovirus genomes, and systems. In certain embodiments of this disclosure, the present invention includes, for example, adenovirus vectors and / or adenovirus genomes comprising a transposon payload of 1 kb to 40 kb. In certain embodiments of this disclosure, the transposase can cause the incorporation of, for example, a transposon payload of up to 40 kb into the genome of a target cell. Accordingly, this disclosure includes, in particular, vectors, genomes, and systems that enable the incorporation of a payload of up to 40 kb present in an adenovirus donor vector into the genome of a target cell. As those skilled in the art will understand, in at least part, the incorporation capacity limits the length and / or complexity of the therapeutic payload, and therefore, vector incorporation capacity is in itself one of the critically important features in gene therapy systems.

[0015] Certain examples of long and / or complex nucleic acid payloads recognized in this disclosure include payloads containing long locus regulatory regions. Due to their length, long locus regulatory regions have historically been unsuitable for inclusion in adenovirus payloads; however, long and / or complex nucleic acid payloads containing long locus regulatory regions, without limitation, can be incorporated into target cell genomes according to the vectors, genomes, and systems disclosed herein.

[0016] Therefore, in one embodiment, an adenovirus donor vector is provided, comprising (a) an adenovirus capsid and (b) a linear double-stranded DNA genome including (i) a transposon payload of at least 10 kb, (ii) a transposon reverse repeat sequence (IR) adjacent to the transposon payload, and (iii) a recombinase co-direction repeat sequence (DR) adjacent to the transposon reverse repeat sequence.

[0017] Another embodiment is an adenovirus donor genome comprising (a) a transposon payload of at least 10 kb, (b) a transposon reverse repeat sequence (IR) adjacent to the transposon payload, and (c) a recombinase same-direction repeat sequence (DR) adjacent to the transposon reverse repeat sequence.

[0018] (a) an adenovirus donor vector as described herein, and (b) an adenovirus support vector comprising an adenovirus support genome comprising (i) an adenovirus capsid and (ii) a transposase-encoding nucleic acid sequence are also provided.

[0019] Another embodiment is an adenovirus transposition system comprising (a) an adenovirus donor genome described herein, and (b) an adenovirus support genome comprising a nucleic acid sequence encoding a transposase.

[0020] Furthermore, an adenovirus production system is provided, comprising (a) a nucleic acid comprising an adenovirus donor genome described herein, and (b) a nucleic acid comprising an adenovirus helper genome comprising a conditional packaging element.

[0021] Further embodiments include cells (e.g., hematopoietic stem cells) comprising a vector, genome, or system relating to any one of the various embodiments described herein.

[0022] Also described are cells (one or more) (e.g., hematopoietic stem cells) whose genome contains a transposon payload of any embodiment described herein, wherein the transposon payload present in the cell's genome is flanked by a transposon-reverse repeat sequence.

[0023] Another embodiment is an adenovirus-producing cell comprising an adenovirus-producing system according to any one of the embodiments described herein, which is optionally HEK293 cells.

[0024] A method for modifying cells, comprising contacting the cells with a vector, genome, or system relating to any one of the embodiments described herein.

[0025] Another embodiment is a method for modifying cells of interest, comprising administering to the subject a vector, genome, or system relating to any one of the embodiments described herein.

[0026] Another embodiment is a method for modifying cells of a subject without isolating the cells from the subject, comprising administering to the subject a vector, genome, or system relating to any one of the embodiments described herein.

[0027] Also provided are methods for treating a disease or condition in a subject requiring treatment of the disease or condition, the method comprising administering to the subject a vector, genome, or system relating to any one of the embodiments described herein.

[0028] In at least one aspect, the disclosure provides an adenovirus donor vector comprising (a) an adenovirus capsid and (b) a linear double-stranded DNA genome comprising (i) a transposon payload of at least 10 kb, (ii) a transposon reverse repeat sequence (IR) adjacent to the transposon payload, and (iii) a recombinase co-direction repeat sequence (DR) adjacent to the transposon reverse repeat sequence.

[0029] In at least one aspect, the disclosure provides an adenovirus donor genome comprising (a) a transposon payload of at least 10 kb, (b) a transposon reverse repeat sequence (IR) adjacent to the transposon payload, and (c) a recombinase co-direction repeat sequence (DR) adjacent to the transposon reverse repeat sequence.

[0030] In at least one aspect, the disclosure provides an adenovirus transposition system comprising (a) an adenovirus donor vector as described in Embodiment 1, and (b) an adenovirus support vector comprising an adenovirus support genome comprising a nucleic acid sequence encoding (i) an adenovirus capsid and (ii) a transposase.

[0031] In at least one aspect, the disclosure provides an adenovirus transposition system comprising (a) an adenovirus donor genome as described in Embodiment 2, and (b) an adenovirus support genome comprising a nucleic acid sequence encoding a transposase.

[0032] In at least one aspect, the disclosure provides an adenovirus production system comprising (a) a nucleic acid comprising an adenovirus donor genome as described in Embodiment 2, and (b) a nucleic acid comprising an adenovirus helper genome comprising a conditional packaging element.

[0033] In various embodiments, the transposon payload includes long-chain LCRs, which, if necessary, are β-globin long-chain LCRs including β-globin LCRs HS1-HS5. In various embodiments, the long-chain LCRs have a length of at least 27 kb. In various embodiments, the transposon payload includes LCRs shown in Table 1. In various embodiments, the transposon payload has a length of at least 15 kb, at least 16 kb, at least 17 kb, at least 18 kb, at least 19 kb, at least 20 kb, at least 21 kb, at least 22 kb, at least 23 kb, at least 24 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 38 kb, or at least 40 kb. In various embodiments, the transposon payload has a length of 10kb to 35kb, 10kb to 30kb, 15kb to 35kb, 15kb to 30kb, 20kb to 35kb, or 20kb to 30kb. In various embodiments, the transposon payload has a length of 10kb to 32.4kb, 15kb to 32.4kb, or 20kb to 32.4kb.

[0034] In various embodiments, the transposon payload comprises a nucleic acid sequence encoding a protein, and optionally, the protein is a therapeutic protein. In various embodiments, the protein is selected from the group consisting of β-globin replacement proteins and γ-globin replacement proteins. In various embodiments, the protein is a factor VIII replacement protein. In various embodiments, the nucleic acid sequence encoding the protein is operably linked to a promoter, and optionally, the promoter is a β-globin promoter.

[0035] In various embodiments, the transposon reverse repeat sequence is a Sleeping Beauty (SB) reverse repeat sequence, and optionally the SB reverse repeat sequence is a pT4 reverse repeat sequence. In various embodiments, the transposase is a Sleeping Beauty (SB) transposase, and optionally the transposase is a Sleeping Beauty 100x (SB100x). In various embodiments, the recombinase co-recombination repeat sequence is an FRT site. In various embodiments, the adenovirus support genome includes a nucleic acid encoding the recombinase. In various embodiments, the recombinase is an FLP recombinase. In various embodiments, the transposon payload includes a β-globin long chain LCR, and the transposon payload includes a nucleic acid sequence encoding β-globin operably linked to a β-globin promoter, the reverse repeat sequence is an SB reverse repeat sequence, and the recombinase co-recombination repeat sequence is an FRT site.

[0036] In various embodiments, the transposon payload includes a selection cassette, and if necessary, the selection cassette is mgmt P140K It contains a nucleic acid sequence that codes for [something].

[0037] In various embodiments, the adenovirus capsid is modified to increase its affinity for CD46, and, if necessary, the adenovirus capsid is an Ad35++ capsid.

[0038] In various embodiments, the conditional packaging element of the adenovirus helper genome includes a packaging sequence flanked by a recombinase co-directed repeat sequence.

[0039] In various embodiments, the packaging sequence of the conditional packaging element and the adjacent recombinase co-directional repeat sequence are LoxP sites.

[0040] In various embodiments, the Disclosure provides cells comprising a vector, genome, or system relating to the Disclosure.

[0041] In various embodiments, the Disclosure provides a cell whose genome contains a transposon payload according to the Disclosure, wherein the transposon payload present in the cell's genome is adjacent to a transposon-reverse repeat sequence.

[0042] In various embodiments, the cells are hematopoietic stem cells.

[0043] In various embodiments, the Disclosure provides adenovirus-producing cells comprising an adenovirus-producing system relating to the Disclosure, which may optionally be HEK293 cells.

[0044] In various embodiments, the Disclosure provides a method for modifying cells, which includes contacting the cells with a vector, genome, or system relating to the Disclosure.

[0045] In various embodiments, the Disclosure provides a method for modifying cells of interest, comprising administering to the subject a vector, genome, or system relating to the Disclosure.

[0046] In various embodiments, the Disclosure provides a method for modifying cells of a subject without isolating the cells from the subject, the method comprising administering a vector, genome, or system relating to the Disclosure to the subject.

[0047] In various embodiments, the Disclosure provides a method for treating a disease or condition in a subject requiring treatment of the disease or condition, the method comprising administering to the subject a vector, genome, or system relating to the Disclosure.

[0048] In various embodiments, the adenovirus donor vector is administered intravenously to the subject.

[0049] In various embodiments, the method comprises administering a mobilizer to a subject, the mobilizer optionally comprising one or more of granulocyte colony-stimulating factor (G-CSF), a CXCR4 antagonist, and a CXCR2 agonist. In various embodiments, the CXCR4 antagonist is AMD3100. In various embodiments, the CXCR2 agonist is GRO-β.

[0050] In various embodiments, the transposon payload comprises a selection cassette, and the method comprises administering a selection agent to a subject. In various embodiments, the selection cassette comprises mgmt P140K The code is O 6 It is BG / BCNU.

[0051] In various embodiments, the method causes the incorporation and / or expression of at least one copy of the transposon payload in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of cells expressing CD46. In various embodiments, the method causes hematopoietic stem cells and / or erythrocyte lineage Ter119 +The method causes the incorporation and / or expression of at least one copy of the transposon payload in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of cells. In various embodiments, the method causes the incorporation of an average of at least two copies of the transposon payload in the genome of cells containing at least one copy of the transposon payload. In various embodiments, the method causes the incorporation of an average of at least 2.5 copies of the transposon payload in the genome of cells containing at least one copy of the transposon payload. In various embodiments, the method causes the expression of the protein encoded by the transposon payload at a level that is at least about 20% of the reference level, where the reference is, if necessary, the expression of an endogenous reference protein in the subject or reference population. In various embodiments, the method causes the expression of the protein encoded by the transposon payload at a level that is at least about 25% of the reference level, where the reference is, if necessary, the expression of an endogenous reference protein in the subject or reference population.

[0052] In various embodiments, the subject is a subject suffering from intermediate thalassemia, and the transposase payload includes a long-chain β-globin LCR containing β-globin LCR HS1-HS5, and nucleic acid sequences encoding β-globin replacement protein and / or γ-globin replacement protein operably linked to the β-globin promoter. In various embodiments, the subject is a subject suffering from hemophilia, and the transposase payload includes a long-chain β-globin LCR containing β-globin LCR HS1-HS5, and nucleic acid sequences encoding factor VIII replacement protein operably linked to the β-globin promoter. In various embodiments, protein expression in the subject reduces at least one symptom of intermediate thalassemia and / or treats intermediate thalassemia.

[0053] definition A, An, The: As used herein, “a,” “an,” and “the” refer to one or more (i.e., at least one) grammatical objects of such articles. For example, “an element” discloses embodiments of strictly one element and embodiments comprising multiple elements.

[0054] Approximately: As used herein, the term “approximately” refers, when used in relation to a value, to a value that is similar to the value being referred to. Generally, those skilled in the art will understand, in addition to the context, the appropriate degree of variation encompassed by “approximately” in that context. For example, in some embodiments, the term “approximately” may encompass a range of variation in a value that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than that of the value being referred to.

[0055] Administration: As used herein, the term “administration” typically refers to administering a composition to a subject or system in order to achieve delivery of a drug that is a composition or a drug contained in a composition.

[0056] Adoptive cell therapy: As used herein, “adoptive cell therapy” or “ACT” involves transferring therapeutically active cells into a subject (e.g., a subject requiring treatment for a condition, disorder, or disease). In some embodiments, ACT involves transferring cells into a subject after ex vivo and / or in vitro cell manipulation and / or proliferation.

[0057] Affinity: As used herein, "affinity" refers to the strength of the sum of non-covalent interactions that occur between a specific binding agent (e.g., a viral vector) and / or its binding moiety and a binding target (e.g., a cell). Unless otherwise specified, "binding affinity" as used herein refers to a 1:1 interaction between a binding agent and its binding target (e.g., a 1:1 interaction between a viral vector and its target cell). Those skilled in the art will understand that changes in affinity can be described by comparison to a reference (e.g., an increase or decrease relative to the reference) or numerically. Affinity can be measured and / or expressed in many ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and / or the equilibrium association constant (K A ). K D is the quotient of k off / k on , and K A is the quotient of k on / k off , where k on refers to the association rate constant (e.g., the association rate constant between a viral vector and a target cell), and k off refers to the dissociation rate constant (e.g., the dissociation rate constant of a viral vector from a target cell). k on and k off can be determined by techniques known to those skilled in the art.

[0058] Agent: As used herein, the term "agent" can refer to any chemical substance, including but not limited to one or more of atoms, molecules, compounds, amino acids, polypeptides, nucleotides, nucleic acids, proteins, protein complexes, liquids, solutions, sugars, polysaccharides, lipids, or combinations or complexes thereof.

[0059] Allogeneic: As used herein, the term "allogeneic" refers to the situation where any material is obtained from one subject and then introduced into another subject (e.g., allogeneic T cell transplantation).

[0060] Between or from: As used herein, the term “between” means that the contextual content falls between the indicated upper and lower limits, or between (including the limits) a first boundary and a second boundary. Similarly, when used in the context of a range of values, the term “from” means that the contextual content contained within such a range falls between the indicated upper and lower limits, or between (including the limits) a first boundary and a second boundary.

[0061] Binding: As used herein, the term “binding” refers to a non-covalent bond between two or more drugs. “Direct” binding involves physical contact between drugs, while indirect binding involves a physical interaction resulting from physical contact with one or more intermediate drugs. Binding between two or more drugs can occur and / or be evaluated in any of a variety of situations, including when the interacting drugs are tested in isolation, or when the interacting drugs are tested in a more complex system setting (e.g., covalently or otherwise bound to a carrier drug and / or present in a biological system or cell).

[0062] Cancer: As used herein, the term “cancer” refers to a condition, disorder, or disease in which cells exhibit relatively abnormal, uncontrolled and / or autonomous growth, resulting in such cells exhibiting abnormally increased growth rates and / or abnormal growth phenotypes characterized by a marked loss of control over cell growth. In some embodiments, cancer may comprise one or more tumors. In some embodiments, cancer may comprise cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic. In some embodiments, cancer may comprise solid tumors. In some embodiments, cancer may comprise hematological malignancies.

[0063] Chimeric Antigen Receptor: As used herein, “chimeric antigen receptor” or “CAR” refers to an engineered protein comprising (i) an extracellular domain containing a portion that binds to a target antigen, (ii) a transmembrane domain, and (iii) an intracellular signaling domain that emits an activation signal when the CAR is stimulated by the binding of the extracellular binding portion to the target antigen. T cells that have been genetically engineered to express a chimeric antigen receptor may be called CAR T cells. Thus, for example, if a T cell expresses a particular CAR, the T cell may be activated when the CAR’s extracellular binding portion binds to a target antigen. CARs are also known as chimeric T cell receptors or chimeric immune receptors.

[0064] Combination Therapy: As used herein, the term “combination therapy” refers to the administration of two or more drugs or regimens to a subject so that the condition, disorder, or disease of interest is treated together by two or more drugs or regimens. In some embodiments, two or more therapeutic agents or regimens may be administered simultaneously, sequentially, or in overlapping drug regimens. Combination therapy includes, but is not necessarily, the administration of two drugs or regimens together in a single composition or simultaneously, as will be understood by those skilled in the art.

[0065] Control of Expression or Activity: As used herein, if the expression or activity of a second element (e.g., a protein, or a nucleic acid encoding a drug (e.g., a protein)) is entirely or partially dependent on the status (e.g., presence, absence, conformation, chemical modification, interaction, or other activity) of a first element (e.g., a protein (e.g., a transcription factor) or a nucleic acid sequence (e.g., a promoter)) under at least one set of conditions, then the first element "controls" or "promotes" the expression or activity of the second element. Control of expression or activity can be substantial control of expression or activity in that, for example, under at least one set of conditions, a change in the status of the first element can result in a change in the expression or activity of the second element compared to a reference control by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, 100%, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 20x, at least 30x, at least 40x, at least 50x, at least 100x).

[0066] Corresponding to: As used herein, the term “corresponding to” may be used to specify the position / identity of a structural element in a compound or composition through comparison with a suitable reference compound or reference composition. For example, in some embodiments, monomeric residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) may be identified as “corresponding to” residues in a suitable reference polymer. For example, a person skilled in the art will understand that residues in a provided polypeptide sequence or polynucleotide sequence are often designated (e.g., numbered or labeled) according to the scheme of the related reference sequence (even if, for example, such designation does not reflect the numbering in the literature of the provided sequence). As an example, 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 positions in the second related sequence may be said to “correspond to” positions 100-110 of the reference sequence. Corresponding positions can be easily identified (for example, by aligning the sequences), and those skilled in the art will understand that such alignments are generally achieved by any of the various known tools, strategies, and / or algorithms. Such tools, strategies, and / or algorithms include, but are not limited to, software programs (e.g., 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).

[0067] Dosage regimen: As used herein, the term “dosage regimen” may refer to the administration of one or more identical or different sets of unit doses to a target, such administration typically includes the administration of multiple unit doses, each dose separated from other doses by duration. In various embodiments, one or more or all of the unit doses in a dosage regimen may be the same or different (e.g., they may be increased or decreased over time, or adjusted according to the target and / or physician’s decision). In various embodiments, one or more or all of the periods between each dose may be the same or different (e.g., they may be prolonged or shortened over time, or adjusted according to the target and / or physician’s decision). In some embodiments, a given therapeutic agent has a recommended dosage regimen, such a recommended dosage regimen may include one or more doses. Typically, a commercially available drug has at least one recommended dosage regimen known to those skilled in the art. In some embodiments, the medication regimen, when administered across the relevant population, correlates with a desired or beneficial outcome (i.e., it is a therapeutic medication regimen).

[0068] Downstream and Upstream: As used herein, the term “downstream” means that the first DNA region is closer to the C-terminus of the nucleic acid containing the first and second DNA regions than the second DNA region. As used herein, the term “upstream” means that the first DNA region is closer to the N-terminus of the nucleic acid containing the first and second DNA regions than the second DNA region.

[0069] Manipulated: As used herein, the term “manipulated” refers to an aspect of being artificially processed. For example, if two or more sequences that are not naturally linked together in that order are artificially linked to each other directly in the manipulated polynucleotide, then that polynucleotide is considered “manipulated.” Those skilled in the art will understand that a “manipulated” nucleic acid sequence or “manipulated” amino acid sequence may be a recombinant nucleic acid sequence or a recombinant amino acid sequence. In some embodiments, the manipulated polynucleotide includes coding and / or regulatory sequences that are found naturally in an operably linked state with a first sequence but not naturally in an operably linked state with a second sequence, and these coding and / or regulatory sequences are present in the manipulated polynucleotide and artificially operably linked to the second sequence. In some embodiments, a cell or organism is considered “engineered” if it has been processed in such a way that its genetic information is altered (for example, if new genetic material that was not previously present has been introduced (this introduction is carried out, for example, by transformation, crossing, somatic hybridization, transfection, transduction, or other mechanisms), or if genetic material that was already present has been modified or removed (this modification or removal is carried out, for example, by substitution, deletion, or crossing)). As is commonly practiced and understood by those skilled in the art, an engineered polynucleotide or offspring or copy (complete or incomplete) of a cell is typically still referred to as “engineered,” even if the direct processing was carried out on the original entity.

[0070] Additives: As used herein, “additives” refers to non-therapeutic agents that may be included in a pharmaceutical composition, by which such non-therapeutic agents may be included in the pharmaceutical composition to obtain, or assist in obtaining, a desired consistency or stabilizing effect, for example. In some embodiments, suitable pharmaceutical additives may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, fat milk powder, glycerol, propylene, glycol, water, ethanol, or similar.

[0071] Expression: As used herein, “expression” refers individually and / or cumulatively to one or more biological processes resulting from the nucleic acid sequence of an encoded drug (such as a protein). Expression specifically includes one or both of transcription and translation.

[0072] Fragment: As used herein, “fragment” refers to a structure that includes and / or consists of an isolated portion of a reference drug (sometimes referred to as the “parent” drug). In some embodiments, the fragment does not contain one or more portions found in the reference drug. In some embodiments, the fragment includes or consists of one or more portions found in the reference drug. In some embodiments, the reference drug is a polymer (such as a polynucleotide or polypeptide). In some embodiments, the polymer fragment includes or consists of monomeric units (e.g., residues) of the reference polymer, the number of such monomeric units being at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, or a number greater than that.In some embodiments, a polymer fragment contains or consists of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 25%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more percent of monomer units (e.g., residues) found in the reference polymer. A reference polymer fragment is not necessarily identical to the corresponding portion of the reference polymer. For example, a fragment of a reference polymer may be a polymer having a sequence of residues in which the percentage of identity with the reference polymer is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 25%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more. The fragment may or may not be produced by the physical fragmentation of the reference agent. In some cases, the fragment is produced by the physical fragmentation of the reference agent. In other cases, the fragment is not produced by the physical fragmentation of the reference agent, but instead may be produced by, for example, de novo synthesis or other means.

[0073] Genes, Transgenes: As used herein, the term “gene” means either a DNA sequence that is a coding sequence (i.e., a DNA sequence that codes for an expression product (such as an RNA product and / or polypeptide product)) or a DNA sequence that contains a coding sequence, such coding sequence together with some or all of the regulatory sequences that control the expression of such coding sequence. In some embodiments, a gene includes non-coding sequences (such as introns, but not limited to them). In some embodiments, a gene may include both coding sequences (e.g., exon sequences) and non-coding sequences (e.g., intron sequences). In some embodiments, a gene includes a regulatory sequence that is a promoter. In some embodiments, a gene includes either or both of (i) DNA nucleotides extending upstream of the coding sequence by a predetermined number of nucleotides in a reference environment (such as an origin genome), and (ii) DNA nucleotides extending downstream of the coding sequence by a predetermined number of nucleotides in a reference environment (such as an origin genome). In various embodiments, the given number of nucleotides may 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 present in the reference environment or is placed in the reference environment by manipulation, but is not inherent in the reference environment or is not originally present in the reference environment.

[0074] Gene product or expression product: As used herein, the terms “gene product” or “expression product” generally refer to RNA transcribed from a gene (before and / or after processing) or polypeptides encoded by RNA transcribed from a gene (before and / or after modification).

[0075] Host Cells, Target Cells: As used herein, “host cells” refers to cells into which exogenous DNA (recombinant or otherwise) (such as a transgene) has been introduced. Those skilled in the art will understand that “host cells” may be the cell into which the exogenous DNA was first introduced and / or its complete or incomplete offspring or copies. In some embodiments, the host cells contain one or more viral genes or transgenes. In some embodiments, the intended or potential host cells may be referred to as target cells.

[0076] Identity: As used herein, the term “identity” refers to the overall relationship between polymer molecules (e.g., between nucleic acid molecules (e.g., between DNA molecules and / or RNA molecules) and / or between polypeptide molecules). Methods for calculating the percentage of identity between two sequences provided are known in the art. The calculation of the percentage of identity between two nucleic acid sequences or polypeptide sequences can be carried out, for example, by aligning the two sequences (or complementary sequences of one or both sequences) for the purpose of optimal comparison (e.g., gaps may be introduced in one or both of the first and second sequences so that the alignment is optimized, and non-identical sequences may be ignored for comparison purposes). A comparison of nucleotides or amino acids is then made at the corresponding positions. 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, then these molecules are identical at that position. The identity percentage between two sequences is a function of the number of identical positions shared by those sequences, and optionally, considering the number of gaps and the length of each gap, these gaps may need to be introduced so that the alignment between the two sequences is optimized. Sequence comparison and determination of the identity percentage between two sequences can be achieved using computational algorithms (such as BLAST (Basic Local Alignment Search Tool)).

[0077] "Improve," "Increase," "Inhibit," or "Reduce": The terms "improve," "increase," "inhibit," and "reduce" as used herein, as well as their grammatical equivalents, are intended to produce qualitative or quantitative differences from the references.

[0078] Isolated: As used herein, “isolated” means that a substance and / or entity has been (1) separated from at least some of the components that were originally associated with it (whether in natural and / or experimental circumstances), and / or (2) artificially designed, produced, prepared, and / or manufactured. An isolated substance and / or entity may be one from which about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components that originally accompanied it. In some embodiments, the purity of the isolated drug is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%. As used herein, a substance is “pure” if it substantially contains no other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure” after being combined with certain other components (e.g., one or more carriers or additives (e.g., buffers, solvents, water, etc.)). In such embodiments, the isolation percentage or purity of the substance is calculated without including such carriers or additives. To give just one example, in some embodiments, a naturally occurring biological polymer (such as a polypeptide or polynucleotide) is considered “isolated” if (a) for reasons of its origin or source, some or all of the components that are associated with it in its natural state are not present with it; (b) it substantially does not contain other polypeptides or nucleic acids of the same species that naturally produces it; or (c) it is expressed by cells or other expression systems that are not of the species that naturally produces it, or components derived from such cells or other expression systems are otherwise associated with it.Therefore, for example, in some embodiments, a polypeptide is considered an “isolated” polypeptide if it is chemically synthesized or synthesized in a cell system different from the one that naturally produces it. Alternatively or additionally, in some embodiments, a polypeptide subjected to one or more purification methods may be considered an “isolated” polypeptide insofar as it is (a) naturally associated with it and / or (b) separated from other components that were associated with it when it was first produced.

[0079] Operatably linked: As used herein, “operably linked” means that at least a first element and a second element are linked so that these constituent elements are in a relationship that enables them to function in their intended manner. For example, if a nucleic acid control element is linked to a nucleic acid coding element in such a way that the control element enables the control of the expression of the coding element, then the control element is “operably linked” to the coding element. In some embodiments, an “operably linked” control element is directly or indirectly covalently linked to the coding element (e.g., in a single nucleic acid). In some embodiments, the control element controls the expression of the coding element in trans, and it is not a requirement for operably linked elements that the control element is contained in the same nucleic acid as the coding element.

[0080] Medicinally acceptable: As used herein, the term “medically acceptable” means that, where applied to one or more components of a formulation of a composition disclosed herein, each component must be compatible with the other components of the composition and harmless to its recipient.

[0081] pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable substance, composition, or medium (such as a liquid or solid extender, diluent, additive, or solvent encapsulant) that facilitates the formulation of a drug (e.g., a pharmaceutical substance), modifies the bioavailability of a drug, or facilitates the transport of a drug from one organ or part of a target to another. Some examples of substances that can act as pharmaceutically acceptable carriers include sugars (such as lactose, glucose, and sucrose), starches (such as corn starch and potato starch), cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate), tragacanth powder, malt, gelatin, talc, additives (such as cocoa butter and suppository waxes), oils (such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycols (such as propylene glycol), polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol), esters (such as ethyl oleate and ethyl laurate), agar, buffers (such as magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, pH buffers, polyesters, polycarbonates, and / or polyacid anhydrides, as well as other non-toxic suitable substances used in pharmaceutical formulations.

[0082] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.

[0083] Promoter: As used herein, “promoter” or “promoter sequence” may be a DNA regulatory region that is directly or indirectly involved (e.g., via a protein or substance that binds to the promoter) in initiating and / or processing the transcription of a coding sequence. A promoter may initiate the transcription of a coding sequence under appropriate conditions when one or more transcription factors and / or regulatory moieties bind to the promoter. A promoter involved in initiating the transcription of a coding sequence may be “operably linked” to the coding sequence. In certain cases, a promoter may be a DNA regulatory region (this DNA regulatory region extends upstream (5' direction) from a transcription start site (located at its 3' end), and as a result, the sequence so called contains one or both of the minimum number of bases or elements required to initiate a transcription event) or may include such a DNA regulatory region. A promoter may be an expression regulatory sequence (such as an enhancer sequence and a repressor sequence), or may be functionally linked to or operably linked to an expression regulatory sequence (such as an enhancer sequence and a repressor sequence). In some embodiments, the promoter may be inductive. In some embodiments, the promoter may be a constitutive promoter. In some embodiments, a conditional (e.g., inductive) promoter may be unidirectional or bidirectional. The promoter may be identical to, or contain, a sequence known to occur in the genome of a particular species. In some embodiments, the promoter may be a hybrid promoter or include a hybrid promoter in which the sequence containing the transcriptional regulatory region may be derived from a first origin, and the sequence containing the transcriptional initiation region may be derived from a second origin.Systems for linking regulatory elements to coding sequences within a transgene are well known in the field (general molecular biological and recombinant DNA techniques are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0084] Reference: As used herein, “reference” refers to the standard or control on which the comparison is made. For example, in some embodiments, a drug, sample, sequence, subject, animal, or individual, or population thereof, or its measure or characteristic representative, is compared to the reference drug, sample, sequence, subject, animal, or individual, or population thereof, or its measure or characteristic representative. In some embodiments, the reference is a measured value. In some embodiments, the reference is an established standard or predicted value. In some embodiments, the reference is a historical reference. The reference may be quantitative or qualitative. Typically, as a person skilled in the art would understand, the reference and its comparison value are measures under equivalent conditions. A person skilled in the art would understand when there is sufficient similarity to justify the reliability and / or comparison. In some embodiments, a suitable reference may be a drug, sample, sequence, subject, animal, or individual, or population thereof ((for example, under conditions that a person skilled in the art would recognize as equivalent for the purpose of evaluating one or more specific variables (e.g., the presence or absence of a drug or condition)) or its measure or characteristic representative.

[0085] Regulatory Sequence: As used herein in the context of nucleic acid coding sequence expression, a regulatory sequence is a nucleic acid sequence that controls the expression of a coding sequence. In some embodiments, a regulatory sequence may control or influence one or more aspects of gene expression (e.g., cell type-specific expression, inducible expression, etc.).

[0086] Subject: As used herein, the term “subject” refers to an organism, typically a mammal (e.g., human, rat, or mouse). In some embodiments, the subject suffers from a disease, disorder, or condition. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not suffer from a disease, disorder, or condition. In some embodiments, the subject exhibits none of the symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject has one or more characteristics characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the subject is a subject that has been examined and / or treated for a disease, disorder, or condition. In some cases, a human subject may be interchangeably referred to as “patient” or “individual.”

[0087] Therapeutic Agent: As used herein, the term “therapeutic agent” refers to any agent that, when administered to a subject, induces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, the suitable population may be a model organism population or a human population. In some embodiments, the suitable population may be defined by various criteria, such as a particular age group, sex, genetic background, or pre-existing conditions. 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 or needs to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent that requires a medical prescription for administration to humans.

[0088] Therapeutic effective dose: As used herein, “therapeutic effective dose” refers to the amount that produces the desired effect for which the administration is intended. In some embodiments, the term refers to an amount sufficient to treat a disease, disorder, and / or condition when administered according to a therapeutic drug regimen to an affected or susceptible population of the disease, disorder, and / or condition. In some embodiments, a therapeutic effective dose is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, and / or delays their onset. Those skilled in the art will understand that the term “therapeutic effective dose” does not require that successful treatment is actually achieved in a particular individual. Rather, a therapeutic effective dose may be an amount that, when administered to patients requiring such treatment, produces a particular desired pharmacological response in a significant number of subjects. In some embodiments, a reference to a therapeutic effective dose may refer to an amount measured in one or more specific tissues (e.g., affected tissue of the disease, disorder, or condition) or body fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a therapeutically effective amount of a particular drug or treatment may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective drug may be formulated and / or administered in multiple doses (e.g., as part of a medication regimen).

[0089] Treatment: As used herein, the term “treatment” (also referred to as “treat” or “treating”) means administering treatment to partially or completely reduce, alleviate, mitigate, suppress, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition, or administering treatment aimed at achieving any such result. In some embodiments, such treatment may be for subjects who do not show signs of the disease, disorder, and / or condition in question, and / or for subjects who show only initial signs of the disease, disorder, or condition. Alternatively or additionally, such treatment may be for subjects who show one or more established signs of the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who have been diagnosed with the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who have been found to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, or condition.

[0090] Unit Dose: As used herein, the term “unit dose” refers to a single dose and / or an amount administered as a physically distinct unit of a pharmaceutical composition. In many embodiments, a unit dose comprises a predetermined amount of the 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 comprises the total single dose of the agent. In some embodiments, multiple unit doses are administered to achieve a total single dose. In some embodiments, administration of multiple unit doses is required or expected to be required to achieve the desired effect. A unit dose may be, for example, a fixed volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic parts, a solid form containing a predetermined amount of one or more therapeutic parts, or a sustained-release formulation or drug delivery device containing a predetermined amount of one or more therapeutic parts. It will be understood that formulations in which a unit dose may exist may optionally include various components in addition to the therapeutic part(s). For example, they may include an acceptable carrier (e.g., a pharmaceutically acceptable carrier), diluents, stabilizers, buffers, preservatives, and the like. Those skilled in the art will understand that in many embodiments, the appropriate total daily dose of a particular therapeutic agent may consist of some or more unit doses and may be determined, for example, by the attending physician within the bounds of sound medical judgment. In some embodiments, a particular effective dose level for any particular subject or organism may depend on a variety of factors, including the disorder being treated and its severity, the activity of the particular active compound(s) used, the particular composition used, the subject's age, weight, overall health, sex, and diet, the timing and rate of administration and excretion of the particular active compound(s) used, the duration of treatment, any drugs and / or additional treatments used in combination with or concurrently with the particular compound(s) used, and similar factors well known in the medical field.

[0091] One or more of the submitted drawings in this specification are easier to understand in color. The applicant will consider color versions of the drawings as part of the initial submission and reserves the right to present color images of the drawings in later proceedings. [Brief explanation of the drawing]

[0092] [Figure 1A-B] Figures 1A-1D. Ex vivo transduction of HSPCs using HDAd-long LCR. (Figure 1A) Vector structure. A 21.5kb β-globin LCR, a 1.6kb β-globin promoter, and a γ-globin gene under the control of the 3'HS1 region, which is also derived from the β-globin locus. The β-globin gene UTR was ligated to the 3' end of the γ-globin gene to stabilize the RNA in erythrocytes. The vector also includes an mgmtP140K expression cassette, which enables in vivo selection of transduced HSPCs and HSPC offspring. The γ-globin expression cassette and mgmt expression cassette are separated by a chicken globin HS4 insulator. The 32.4kb LCR-γ-globin / mgtm transposon has a reverse repeat sequence (IR) (recognized by SB100x) and an ftr site (which enables circularization of the transposon by Flpe recombinase) adjacent to each other. (Figure 1B) Experimental regimen. Transduction of bone marrow Lin- cells obtained from CD46 transgenic mice was performed using HDAd-long chain LCR and HDAd-SB at a total MOI of 500 vp / cell. After 1 day of culture, transduced cells were transplanted into lethal irradiated C57Bl / 6 mice at a rate of 1 × 10⁶ cells / mouse. O6BG / BCNU treatment was started at week 4 and repeated every 2 weeks for a total of 4 cycles. With each cycle, the BCNU concentration was increased from 5 mg / kg → 7.5 mg / kg → 10 mg / kg (twice). Mice were sacrificed at week 20. (Figure 1C) Percentage of human γ-globin-positive peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 1D) Representative flow cytometry data showing human γ-globin expression in erythrocytes (Ter119+) bone marrow cells (lower panel) at 20 weeks post-transplantation. The top panel shows mice transplanted with cells transduced using a mock-up. [Figure 1C-D]Figures 1A-1D. Ex vivo transduction of HSPCs using HDAd-long LCR. (Figure 1A) Vector structure. A 21.5kb β-globin LCR, a 1.6kb β-globin promoter, and a γ-globin gene under the control of the 3'HS1 region, which is also derived from the β-globin locus. The β-globin gene UTR was ligated to the 3' end of the γ-globin gene to stabilize the RNA in erythrocytes. The vector also includes an mgmtP140K expression cassette, which enables in vivo selection of transduced HSPCs and HSPC offspring. The γ-globin expression cassette and mgmt expression cassette are separated by a chicken globin HS4 insulator. The 32.4kb LCR-γ-globin / mgtm transposon has a reverse repeat sequence (IR) (recognized by SB100x) and an ftr site (which enables circularization of the transposon by Flpe recombinase) adjacent to each other. (Figure 1B) Experimental regimen. Transduction of bone marrow Lin- cells obtained from CD46 transgenic mice was performed using HDAd-long chain LCR and HDAd-SB at a total MOI of 500 vp / cell. After 1 day of culture, transduced cells were transplanted into lethal irradiated C57Bl / 6 mice at a rate of 1 × 10⁶ cells / mouse. O6BG / BCNU treatment was started at week 4 and repeated every 2 weeks for a total of 4 cycles. With each cycle, the BCNU concentration was increased from 5 mg / kg → 7.5 mg / kg → 10 mg / kg (twice). Mice were sacrificed at week 20. (Figure 1C) Percentage of human γ-globin-positive peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 1D) Representative flow cytometry data showing human γ-globin expression in erythrocytes (Ter119+) bone marrow cells (lower panel) at 20 weeks post-transplantation. The top panel shows mice transplanted with cells transduced using a mock-up.

[0093] [Figure 2A-B]Figures 2A-2C. iPCR analysis of vector / chromosome ligatures in bone marrow cells derived from animals 20 weeks post-transplant. (Figure 2A) Schematic diagram of iPCR analysis. 5 micrograms of genomic DNA were digested with SacI, re-ligated, and subjected to nested inverse PCR using the primers shown (see Materials and Methods). (Figure 2B) Agarose gel electrophoresis of cloned plasmids containing the integration ligatures. The bands shown were excised and sequenced. Chromosome integration sites are shown below the gel. (Figure 2C) Examples of ligation sequences: 5' end vector sequence, Sleeping beautyIR / DR sequence, integration ligament (chr15, 6805206) SEQ ID NO: 1; 5' end vector sequence, Sleeping beautyIR / DR sequence, integration ligament (chrX, 16897322) SEQ ID NO: 2; 3' end vector sequence, Sleeping beautyIR / DR sequence, integration ligament (chr4, 10207667) SEQ ID NO: 3. Vector sequences and IR / DR sequences are shown in plain text and underlined, respectively. Chromosome sequences are shown in bold text. TA dinucleotides located at the junction of the IR and chromosomal DNA (used by SB100x) are shown in square brackets. [Figure 2C]Figures 2A-2C. iPCR analysis of vector / chromosome ligatures in bone marrow cells derived from animals 20 weeks post-transplant. (Figure 2A) Schematic diagram of iPCR analysis. 5 micrograms of genomic DNA were digested with SacI, re-ligated, and subjected to nested inverse PCR using the primers shown (see Materials and Methods). (Figure 2B) Agarose gel electrophoresis of cloned plasmids containing the integration ligatures. The bands shown were excised and sequenced. Chromosome integration sites are shown below the gel. (Figure 2C) Examples of ligation sequences: 5' end vector sequence, Sleeping beautyIR / DR sequence, integration ligament (chr15, 6805206) SEQ ID NO: 1; 5' end vector sequence, Sleeping beautyIR / DR sequence, integration ligament (chrX, 16897322) SEQ ID NO: 2; 3' end vector sequence, Sleeping beautyIR / DR sequence, integration ligament (chr4, 10207667) SEQ ID NO: 3. Vector sequences and IR / DR sequences are shown in plain text and underlined, respectively. Chromosome sequences are shown in bold text. TA dinucleotides located at the junction of the IR and chromosomal DNA (used by SB100x) are shown in square brackets.

[0094] [Figure 3A-B]Figures 3A-3E. In vivo transduction of HSPC using HDAd-long chain LCR containing a 32.4kb transposon, and in vivo transduction of HSPC using HDAd-short chain LCR containing an 11.8kb transposon. (Figure 3A) Instead of the 21.5kb HS1-HS5 LCR and 3'HS1 (Figure 1A HDAd-short chain LCR), this vector contains a 4.3kb miniature LCR containing the core region of DNase-sensitive sites (HS) 1-4. (Figure 3B) Treatment regimen. hCD46tg mice were recruited and IV injected with either HDAd-short chain LCR + HDAd-SB or HDAd-long chain LCR + HDAd-SB (two injections of a 1:1 mixture of both viruses (4 × 10¹⁰ vp each)). After 5 weeks, O6BG / BCNU treatment was initiated. The BCNU concentration was increased in each cycle from 2.5 mg / kg to 7.5 mg / kg to 10 mg / kg. The O6BG concentration was 30 mg / kg in all three treatments. Mice were observed for 20 weeks, at which point the animals were sacrificed for analysis and Lin-cell transplantation to secondary recipients. The secondary recipients were then observed for 16 weeks. Immunosuppressants (IS) were administered in vivo to animals transduced with HSPC to inhibit the immune response to human γ-globin and mgtm protein. (Figure 3C) Percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. In mice transduced with mocks, γ-globin-positive cells were less than 0.1%. (Figure 3D) γ-globin protein chain levels in RBCs at 20 weeks after transduction with HSPC in vivo, measured by HPLC. The percentage of human γ-globin protein chain relative to mouse α-globin protein chain is shown. (Figure 3E) mRNA levels of γ-globin in whole blood 20 weeks after transduction of HSPC in vivo, measured by qRT-PCR. The percentage of human γ-globin mRNA relative to mouse α-globin mRNA is shown. [Figure 3C-E]Figures 3A-3E. In vivo transduction of HSPC using HDAd-long chain LCR containing a 32.4kb transposon, and in vivo transduction of HSPC using HDAd-short chain LCR containing an 11.8kb transposon. (Figure 3A) Instead of the 21.5kb HS1-HS5 LCR and 3'HS1 (Figure 1A HDAd-short chain LCR), this vector contains a 4.3kb miniature LCR containing the core region of DNase-sensitive sites (HS) 1-4. (Figure 3B) Treatment regimen. hCD46tg mice were recruited and IV injected with either HDAd-short chain LCR + HDAd-SB or HDAd-long chain LCR + HDAd-SB (two injections of a 1:1 mixture of both viruses (4 × 10¹⁰ vp each)). After 5 weeks, O6BG / BCNU treatment was initiated. The BCNU concentration was increased in each cycle from 2.5 mg / kg to 7.5 mg / kg to 10 mg / kg. The O6BG concentration was 30 mg / kg in all three treatments. Mice were observed for 20 weeks, at which point the animals were sacrificed for analysis and Lin-cell transplantation to secondary recipients. The secondary recipients were then observed for 16 weeks. Immunosuppressants (IS) were administered in vivo to animals transduced with HSPC to inhibit the immune response to human γ-globin and mgtm protein. (Figure 3C) Percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. In mice transduced with mocks, γ-globin-positive cells were less than 0.1%. (Figure 3D) γ-globin protein chain levels in RBCs at 20 weeks after transduction with HSPC in vivo, measured by HPLC. The percentage of human γ-globin protein chain relative to mouse α-globin protein chain is shown. (Figure 3E) mRNA levels of γ-globin in whole blood 20 weeks after transduction of HSPC in vivo, measured by qRT-PCR. The percentage of human γ-globin mRNA relative to mouse α-globin mRNA is shown.

[0095] [Figure 4]Vector copy count per cell in bone marrow MNCs collected 20 weeks after transduction of HSPC in vivo. The difference between the two groups is not statistically significant.

[0096] [Figure 5A-B] Figures 5A-5D. Hematological parameters at 20 weeks after in vivo transduction of HSPC. (Figure 5A) Leukocytes (WBC), neutrophils (NE), leukocytes (LY), monocytes (MO), eosinophils (EO), and basophils (BA). (Figure 5B) Erythropoiesis parameters. RBC: erythrocytes, Hb: hemoglobin, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: erythrocyte distribution width. The differences between the three groups were not significant. (Figure 5C) Bone marrow cell composition. (Figure 5D) Colony-forming ability of bone marrow Lin-cells. In Figures 5A-5D, the differences between groups were not significant. The data in the panel of Figure 5 show that in vivo transduction of HSPC using HDAd-short chain LCR and / or HDAd-long chain LCR vectors does not affect hematopoiesis and cell distribution in the bone marrow. [Figure 5C-D] Figures 5A-5D. Hematological parameters at 20 weeks after in vivo transduction of HSPC. (Figure 5A) Leukocytes (WBC), neutrophils (NE), leukocytes (LY), monocytes (MO), eosinophils (EO), and basophils (BA). (Figure 5B) Erythropoiesis parameters. RBC: erythrocytes, Hb: hemoglobin, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: erythrocyte distribution width. The differences between the three groups were not significant. (Figure 5C) Bone marrow cell composition. (Figure 5D) Colony-forming ability of bone marrow Lin-cells. In Figures 5A-5D, the differences between groups were not significant. The data in the panel of Figure 5 show that in vivo transduction of HSPC using HDAd-short chain LCR and / or HDAd-long chain LCR vectors does not affect hematopoiesis and cell distribution in the bone marrow.

[0097] [Figure 6]The localization of the NheI and KpnI sites in the HDAd-globin vector is shown in relation to the Sleeping Beauty reverse repeat sequence (IR). The cleavage sites of these enzymes are close to but outside the SB IR / DR, and using these enzymes reduces background noise from unintegrated vectors. The remaining genomic DNA derived from bone marrow Lin- cells was digested with NheI and KpnI, and after thermal inactivation, further digested with NlaIII. NlaIII is a 4-base recognition enzyme, which creates short DNA fragments. Next, the digested DNA was ligated with double-stranded oligonucleotides with known sequences and ends compatible with the digested NlaIII fragments. After thermal inactivation and purification, the ligation product with the linker was used for linear amplification, which created a population of single-stranded (ss) DNA starting from the left arm of the SB. Since the primers were biotinylated, ssDNA could be collected with streptavidin beads. After thorough washing, ssDNA was eluted from the beads and subjected to further amplification by two nested PCR cycles. The PCR amplification products were purified on a gel, cloned, sequenced, and mapped to the mouse genome sequence to mark integration sites.

[0098] [Figure 7A-1]Figures 7A-7D. Analysis of vector integration sites in HSPC. Genomic DNA was isolated from bone marrow Lin- cells collected 20 weeks after in vivo transduction using HDAd-long LCR + HDAd-SB. (Figure 7A, spanning two pages) Distribution of integration sites on the chromosome. Sleeping Beauty integration throughout the genome. Integration sites are marked with vertical lines. (Figure 7B) Examples of junction sequences: Sleeping Beauty IR / DR sequence, integration junction (chr7, 79796094) SEQ ID NO: 4; Sleeping Beauty IR / DR sequence, integration junction (repetitive sequence region) SEQ ID NO: 5. IR / DR sequences are shown in underlined and bold text. Chromosomal sequences are shown in plain text. TA dinucleotides located at the junction between IR and chromosomal DNA (used by SB100x) are shown in bold. (Figure 7C) Sleeping Beauty integration throughout the genome, associated with RefSeq annotation. Integration sites were mapped to the mouse genome, and their locations were analyzed in association with genes. The percentage of integration events occurring between the 1kb upstream of the transcription start site, the 3'UTR of exons, protein coding sequences, introns, the 3'UTR, the 1kb downstream of the 3'UTR, and genes is shown (Figure 7D). Sleeping Beauty integration patterns compared to randomized controls. Integration patterns in mouse genome frames. The number and size of integrations overlapping with continuous genome frames and with randomized mouse genome frames were compared. This indicates that the integration patterns are similar in continuous and randomized frames. In any given frame, the maximum number of integrations never exceeded 3, and the incidence of 1 integration per frame was higher. Values ​​represent mean ± sd. The data in the panel of Figure 7 show near-random integration patterns without gene selectivity. [Figure 7A-2]Figures 7A-7D. Analysis of vector integration sites in HSPC. Genomic DNA was isolated from bone marrow Lin- cells collected 20 weeks after in vivo transduction using HDAd-long LCR + HDAd-SB. (Figure 7A, spanning two pages) Distribution of integration sites on the chromosome. Sleeping Beauty integration throughout the genome. Integration sites are marked with vertical lines. (Figure 7B) Examples of junction sequences: Sleeping Beauty IR / DR sequence, integration junction (chr7, 79796094) SEQ ID NO: 4; Sleeping Beauty IR / DR sequence, integration junction (repetitive sequence region) SEQ ID NO: 5. IR / DR sequences are shown in underlined and bold text. Chromosomal sequences are shown in plain text. TA dinucleotides located at the junction between IR and chromosomal DNA (used by SB100x) are shown in bold. (Figure 7C) Sleeping Beauty integration throughout the genome, associated with RefSeq annotation. Integration sites were mapped to the mouse genome, and their locations were analyzed in association with genes. The percentage of integration events occurring between the 1kb upstream of the transcription start site, the 3'UTR of exons, protein coding sequences, introns, the 3'UTR, the 1kb downstream of the 3'UTR, and genes is shown (Figure 7D). Sleeping Beauty integration patterns compared to randomized controls. Integration patterns in mouse genome frames. The number and size of integrations overlapping with continuous genome frames and with randomized mouse genome frames were compared. This indicates that the integration patterns are similar in continuous and randomized frames. In any given frame, the maximum number of integrations never exceeded 3, and the incidence of 1 integration per frame was higher. Values ​​represent mean ± sd. The data in the panel of Figure 7 show near-random integration patterns without gene selectivity. [Figure 7B-C]Figures 7A-7D. Analysis of vector integration sites in HSPC. Genomic DNA was isolated from bone marrow Lin- cells collected 20 weeks after in vivo transduction using HDAd-long LCR + HDAd-SB. (Figure 7A, spanning two pages) Distribution of integration sites on the chromosome. Sleeping Beauty integration throughout the genome. Integration sites are marked with vertical lines. (Figure 7B) Examples of junction sequences: Sleeping Beauty IR / DR sequence, integration junction (chr7, 79796094) SEQ ID NO: 4; Sleeping Beauty IR / DR sequence, integration junction (repetitive sequence region) SEQ ID NO: 5. IR / DR sequences are shown in underlined and bold text. Chromosomal sequences are shown in plain text. TA dinucleotides located at the junction between IR and chromosomal DNA (used by SB100x) are shown in bold. (Figure 7C) Sleeping Beauty integration throughout the genome, associated with RefSeq annotation. Integration sites were mapped to the mouse genome, and their locations were analyzed in association with genes. The percentage of integration events occurring between the 1kb upstream of the transcription start site, the 3'UTR of exons, protein coding sequences, introns, the 3'UTR, the 1kb downstream of the 3'UTR, and genes is shown (Figure 7D). Sleeping Beauty integration patterns compared to randomized controls. Integration patterns in mouse genome frames. The number and size of integrations overlapping with continuous genome frames and with randomized mouse genome frames were compared. This indicates that the integration patterns are similar in continuous and randomized frames. In any given frame, the maximum number of integrations never exceeded 3, and the incidence of 1 integration per frame was higher. Values ​​represent mean ± sd. The data in the panel of Figure 7 show near-random integration patterns without gene selectivity. [Figure 7D]Figures 7A-7D. Analysis of vector integration sites in HSPC. Genomic DNA was isolated from bone marrow Lin- cells collected 20 weeks after in vivo transduction using HDAd-long LCR + HDAd-SB. (Figure 7A, spanning two pages) Distribution of integration sites on the chromosome. Sleeping Beauty integration throughout the genome. Integration sites are marked with vertical lines. (Figure 7B) Examples of junction sequences: Sleeping Beauty IR / DR sequence, integration junction (chr7, 79796094) SEQ ID NO: 4; Sleeping Beauty IR / DR sequence, integration junction (repetitive sequence region) SEQ ID NO: 5. IR / DR sequences are shown in underlined and bold text. Chromosomal sequences are shown in plain text. TA dinucleotides located at the junction between IR and chromosomal DNA (used by SB100x) are shown in bold. (Figure 7C) Sleeping Beauty integration throughout the genome, associated with RefSeq annotation. Integration sites were mapped to the mouse genome, and their locations were analyzed in association with genes. The percentage of integration events occurring between the 1kb upstream of the transcription start site, the 3'UTR of exons, protein coding sequences, introns, the 3'UTR, the 1kb downstream of the 3'UTR, and genes is shown (Figure 7D). Sleeping Beauty integration patterns compared to randomized controls. Integration patterns in mouse genome frames. The number and size of integrations overlapping with continuous genome frames and with randomized mouse genome frames were compared. This indicates that the integration patterns are similar in continuous and randomized frames. In any given frame, the maximum number of integrations never exceeded 3, and the incidence of 1 integration per frame was higher. Values ​​represent mean ± sd. The data in the panel of Figure 7 show near-random integration patterns without gene selectivity.

[0099] [Figure 8A-D]Figures 8A-8E. Analysis of secondary recipients. Bone marrow lin- cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethal irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. (Figure 8A) Engraftment rate based on the percentage of CD46-positive PBMCs. The difference between the two groups was not significant. (Figure 8B) Percentage of γ-globin-expressing peripheral blood RBCs measured by flow cytometry. The difference between the two groups was not significant. (Figure 8C) Analysis of human γ-globin chains in RBCs of secondary recipients by HPLC. The percentage of human γ-globin relative to adult mouse α-globin at 4, 8, 12, and 16 weeks after transplantation is shown. *p<0.0001. Statistical analysis was performed using two-way ANOVA. (Figure 8D) Levels of γ-globin mRNA in all blood cells. The percentage of human γ-globin mRNA relative to mouse α-globin mRNA and β-major globin mRNA is shown. (Figure 8E) Levels of γ-globin mRNA in bone marrow MNCs at 16 weeks post-transduction. The percentage of human γ-globin mRNA relative to mouse α-globin mRNA and β-major globin mRNA is shown. The panels of Figures 8 and 9 show that the incorporation of the "32.4" kb transposon occurred in long-term regrowing cells, that the level of γ-globin expression from vectors with long LCRs increased over time compared to vectors with short LCRs, and that vectors with long LCRs produced more precise erythrocyte specificity of γ-globin expression. [Figure 8E]Figures 8A-8E. Analysis of secondary recipients. Bone marrow lin- cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethal irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. (Figure 8A) Engraftment rate based on the percentage of CD46-positive PBMCs. The difference between the two groups was not significant. (Figure 8B) Percentage of γ-globin-expressing peripheral blood RBCs measured by flow cytometry. The difference between the two groups was not significant. (Figure 8C) Analysis of human γ-globin chains in RBCs of secondary recipients by HPLC. The percentage of human γ-globin relative to adult mouse α-globin at 4, 8, 12, and 16 weeks after transplantation is shown. *p<0.0001. Statistical analysis was performed using two-way ANOVA. (Figure 8D) Levels of γ-globin mRNA in all blood cells. The percentage of human γ-globin mRNA relative to mouse α-globin mRNA and β-major globin mRNA is shown. (Figure 8E) Levels of γ-globin mRNA in bone marrow MNCs at 16 weeks post-transduction. The percentage of human γ-globin mRNA relative to mouse α-globin mRNA and β-major globin mRNA is shown. The panels of Figures 8 and 9 show that the incorporation of the "32.4" kb transposon occurred in long-term regrowing cells, that the level of γ-globin expression from vectors with long LCRs increased over time compared to vectors with short LCRs, and that vectors with long LCRs produced more precise erythrocyte specificity of γ-globin expression.

[0100] [Figure 9A] Figures 9A-9C. Erythrocyte specificity of γ-globin expression in the bone marrow of secondary recipients (16 weeks post-transplant). (Figure 9A) Percentage of γ-globin-expressing erythrocytes (Ter119+ cells) in whole bone marrow MNCs. (Figure 9B) Erythrocyte specificity. Percentage of γ-globin+ cells in erythrocytes (Ter119+) and non-erythrocytes (Ter119-). (Figure 9C) Vector copy number per cell (VCN) in bone marrow MNCs collected 20 weeks after transduction of HSPC in vivo. The difference between the two groups is not significant. [Figure 9B-C] Figures 9A-9C. Erythrocyte specificity of γ-globin expression in the bone marrow of secondary recipients (16 weeks post-transplant). (Figure 9A) Percentage of γ-globin-expressing erythrocytes (Ter119+ cells) in whole bone marrow MNCs. (Figure 9B) Erythrocyte specificity. Percentage of γ-globin+ cells in erythrocytes (Ter119+) and non-erythrocytes (Ter119-). (Figure 9C) Vector copy number per cell (VCN) in bone marrow MNCs collected 20 weeks after transduction of HSPC in vivo. The difference between the two groups is not significant.

[0101] [Figure 10A-D] Figures 10A-11D. Hematological parameters in secondary recipients at 16 weeks post-transplant. (Figure 10A) Leukocytes. (Figure 10B) Erythropoiesis parameters. RBC: Red blood cells, Hb: Hemoglobin, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, RDW: Red blood cell distribution width. The differences between the three groups were not significant. (Figure 10C) Bone marrow cell composition. (Figure 10D) Colony-forming ability of bone marrow Lin-cells.

[0102] [Figure 11A-C] Figures 11A-11C. In vitro studies using human CD34+ cells. (Figure 11A) Schematic diagram of the experiment. CD34+ cells were transduced using HDAd-long chain LCR+HD-SB or HDAd-short chain LCR+HDAd-SB, and these CD34+ cells were subjected to erythrocyte differentiation (ED). In vitro selection using O6BG-BCNU was initiated 5 days after ED. Cells were analyzed on day 18 by flow cytometry (Figure 11B) and HPLC (Figure 11C). The panel in Figure 11 shows that in human cell lines, the HDAd-long chain LCR vector results in higher γ-globin expression after erythrocyte differentiation of transduced human HSC / CD34+ cells.

[0103] [Figure 12A-B]Figures 12A-12B. In vivo HSC transduction in hCD46tg mice using "long-chain" LCR vectors and "short-chain" LCR vectors. (Figure 12A) HDAd-long-chain LCR-γ-globin / mgmt vector and HDAd-short-chain LCR-γ-globin / mgmt vector. (Figure 12B) In vivo transduction of Hbbth3 / CD46 mice using vectors. Group 1 shows in vivo transduction of 7 mice using HDAd-long-chain LCR-γ-globin / mgmt + HDAd-SB / Flpe. Group 2 shows in vivo transduction of 3 mice using HDAd-short-chain LCRγ-globin / mgmt + HDAd-SB / Flpe. Only 3 selection cycles were required for selection using O6BG and BCNU.

[0104] [Figure 13-1] Figure 13. Thbb mouse test (week 6). The graph shows no difference between mice transduced with long-chain LCR vectors and mice transduced with short-chain LCR vectors, indicating that human γ-globin expression is almost absent in these mice. (Spanning two pages.) [Figure 13-2] Figure 13. Thbb mouse test (week 6). The graph shows no difference between mice transduced with long-chain LCR vectors and mice transduced with short-chain LCR vectors, indicating that human γ-globin expression is almost absent in these mice. (Spanning two pages.)

[0105] [Figure 14-1] Figure 14. Thbb mouse test (week 8). The graph shows a difference between mice transduced with long-chain LCR vectors and mice transduced with short-chain LCR vectors, but it is unclear whether the short-chain LCR virus was killed in the mice. (Spans 2 pages.) [Figure 14-2]Figure 14. Thbb mouse test (week 8). The graph shows a difference between mice transduced with long-chain LCR vectors and mice transduced with short-chain LCR vectors, but it is unclear whether the short-chain LCR virus was killed in the mice. (Spans 2 pages.)

[0106] [Figure 15] A graph showing the percentage of human gamma-globin-expressing RBCs in mice. This graph demonstrates that 100% marking occurs after only three in vivo selection cycles.

[0107] [Figure 16] HPLC graph showing the relative values ​​of human gamma-globin to mouse HBA (at week 10). This graph indicates that significantly higher gamma-globin levels are obtained with long-chain LCR compared to short-chain LCR.

[0108] [Figure 17] The graph shows an example of HPLC analysis of blood obtained at 10 weeks from mouse #57 containing a long-chain LCR vector.

[0109] [Figure 18A-B]Figures 18A-18D. Expression of human γ-globin after in vivo HSC gene therapy in Hbbth3 / CD46 mice using HDAd-short chain LCR and HDAd-long chain LCR. (Figure 18A) Treatment regimen. In contrast to Figures 3A-3E, Figures 18A-18D show results within thalassemia Hbbth3 / CD46 mice. (Figure 18B) Percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 18C) Levels of γ-globin protein chains in RBCs 18 weeks after transduction of HSPCs in vivo, measured by HPLC. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. (Figure 18D) Representative chromatograms of untreated Hbbth3 / CD46 mice (left panel) and mice 21 weeks after treatment. This shows the addition of human γ-globin in addition to mouse α- and β-chains. The panel data in Figure 18 demonstrate that 100% GRP marking can be achieved with the long-chain LCR-HDAd vector at low in vivo selectivity and / or fewer rounds and / or lower doses. γ-globin expression levels are within the expected range for effective treatment (20% or greater). [Figure 18C-D]Figures 18A-18D. Expression of human γ-globin after in vivo HSC gene therapy in Hbbth3 / CD46 mice using HDAd-short chain LCR and HDAd-long chain LCR. (Figure 18A) Treatment regimen. In contrast to Figures 3A-3E, Figures 18A-18D show results within thalassemia Hbbth3 / CD46 mice. (Figure 18B) Percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 18C) Levels of γ-globin protein chains in RBCs 18 weeks after transduction of HSPCs in vivo, measured by HPLC. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. (Figure 18D) Representative chromatograms of untreated Hbbth3 / CD46 mice (left panel) and mice 21 weeks after treatment. This shows the addition of human γ-globin in addition to mouse α- and β-chains. The panel data in Figure 18 demonstrate that 100% GRP marking can be achieved with the long-chain LCR-HDAd vector at low in vivo selectivity and / or fewer rounds and / or lower doses. γ-globin expression levels are within the expected range for effective treatment (20% or greater).

[0110] [Figure 19] Microscopic images showing the normalization of erythrocyte morphology in C57BL6 (normal mice) and Townes SCA mice (before treatment and 10 weeks after long-chain LCR treatment).

[0111] [Figure 20] Microscopic images showing the normalization of erythrocyte production (reticulocyte count) in Townes mice (before treatment) and Townes mice (10 weeks after long-chain LCR treatment).

[0112] [Figure 21A]Figures 21A-21C. Phenotypic modification. (Figures 21A, 21B) Show blood cell morphology; the left panel shows blood smears stained with Giemsa, and the right panel shows blood smears stained with May-Grünwald. Nuclei and cytoplasmic residues in reticulocytes are stained purple. (Figure 21A) Comparison before treatment and 14 weeks after treatment. (Figure 21B) Comparison of Giemsa staining and reticulocytes in Hbbth3 / CD46 mice before treatment with CD46tg, HDAd-long chain LCR, Hbbth3 / CD46 mice 18 weeks after treatment with HDAd-long chain LCR, and Hbbth3 / CD46 mice 21 weeks after treatment with HDAd-long chain LCR. (Figure 21C) Cytospin preparation of bone marrow. It can be seen that erythrocyte production, which is predominantly proerythroblasts, is normalized in the treated mice. The scale bar is 20 μm. The data in the panel of Figure 21 show that blood cell morphology normalized after in vivo HSC gene therapy with HDAd-long chain LCR vector. [Figure 21B] Figures 21A-21C. Phenotypic modification. (Figures 21A, 21B) Show blood cell morphology; the left panel shows blood smears stained with Giemsa, and the right panel shows blood smears stained with May-Grünwald. Nuclei and cytoplasmic residues in reticulocytes are stained purple. (Figure 21A) Comparison before treatment and 14 weeks after treatment. (Figure 21B) Comparison of Giemsa staining and reticulocytes in Hbbth3 / CD46 mice before treatment with CD46tg, HDAd-long chain LCR, Hbbth3 / CD46 mice 18 weeks after treatment with HDAd-long chain LCR, and Hbbth3 / CD46 mice 21 weeks after treatment with HDAd-long chain LCR. (Figure 21C) Cytospin preparation of bone marrow. It can be seen that erythrocyte production, which is predominantly proerythroblasts, is normalized in the treated mice. The scale bar is 20 μm. The data in the panel of Figure 21 show that blood cell morphology normalized after in vivo HSC gene therapy with HDAd-long chain LCR vector. [Figure 21C]Figures 21A-21C. Phenotypic modification. (Figures 21A, 21B) Show blood cell morphology; the left panel shows blood smears stained with Giemsa, and the right panel shows blood smears stained with May-Grünwald. Nuclei and cytoplasmic residues in reticulocytes are stained purple. (Figure 21A) Comparison before treatment and 14 weeks after treatment. (Figure 21B) Comparison of Giemsa staining and reticulocytes in Hbbth3 / CD46 mice before treatment with CD46tg, HDAd-long chain LCR, Hbbth3 / CD46 mice 18 weeks after treatment with HDAd-long chain LCR, and Hbbth3 / CD46 mice 21 weeks after treatment with HDAd-long chain LCR. (Figure 21C) Cytospin preparation of bone marrow. It can be seen that erythrocyte production, which is predominantly proerythroblasts, is normalized in the treated mice. The scale bar is 20 μm. The data in the panel of Figure 21 show that blood cell morphology normalized after in vivo HSC gene therapy with HDAd-long chain LCR vector.

[0113] [Figure 22] Hematological parameters of Hbbth3 / CD46+ mice before and after in vivo HSC gene therapy. Hbbth3 / CD46+ mice exhibit an intermediate thalassemia phenotype. Mice were treated with adenovirus donor vectors containing a γ-globin nucleic acid sequence operably linked to either long-chain or short-chain LCRs. Mice were sampled at 1 and 10 weeks post-treatment. Figure 22 shows graphs of normalized erythrocyte parameters (WBC, RBC, Hb, HCT, MCV, MCH, MCHC, and RDW) at 1 week (upper panel) and 10 weeks (lower panel) for mice treated with long-chain LCR vectors, mice treated with short-chain LCR vectors, and samples from control CD46tg.

[0114] [Figure 23A]Figure 23. Hematological parameters of Hbbth3 / CD46+ mice before and after in vivo HSC gene therapy. Hbbth3 / CD46+ mice exhibit an intermediate thalassemia phenotype. Mice were treated with an adenovirus donor vector containing a γ-globin nucleic acid sequence operably linked to either a long-chain or short-chain LCR, among other things. Mice were sacrificed and sampled 18 weeks post-treatment. The percentage of reticulocytes was counted on blood smears (Figure 23A; reticulocyte count). The hematological parameters at 18 weeks post-transduction in vivo were indistinguishable from their control CD46tg-corresponding hematological parameters, suggesting complete phenotypic modification, including normalization of leukocyte and erythrocyte counts, as well as erythrocyte characteristics (Hb, HCT, MHCH, and RDW) (Figure 23B; hematological parameters). [Figure 23B-1] Figure 23. Hematological parameters of Hbbth3 / CD46+ mice before and after in vivo HSC gene therapy. Hbbth3 / CD46+ mice exhibit an intermediate thalassemia phenotype. Mice were treated with an adenovirus donor vector containing a γ-globin nucleic acid sequence operably linked to either a long-chain or short-chain LCR, among other things. Mice were sacrificed and sampled 18 weeks post-treatment. The percentage of reticulocytes was counted on blood smears (Figure 23A; reticulocyte count). The hematological parameters at 18 weeks post-transduction in vivo were indistinguishable from their control CD46tg-corresponding hematological parameters, suggesting complete phenotypic modification, including normalization of leukocyte and erythrocyte counts, as well as erythrocyte characteristics (Hb, HCT, MHCH, and RDW) (Figure 23B; hematological parameters). [Figure 23B-2]Figure 23. Hematological parameters of Hbbth3 / CD46+ mice before and after in vivo HSC gene therapy. Hbbth3 / CD46+ mice exhibit an intermediate thalassemia phenotype. Mice were treated with an adenovirus donor vector containing a γ-globin nucleic acid sequence operably linked to either a long-chain or short-chain LCR, among other things. Mice were sacrificed and sampled 18 weeks post-treatment. The percentage of reticulocytes was counted on blood smears (Figure 23A; reticulocyte count). The hematological parameters at 18 weeks post-transduction in vivo were indistinguishable from their control CD46tg-corresponding hematological parameters, suggesting complete phenotypic modification, including normalization of leukocyte and erythrocyte counts, as well as erythrocyte characteristics (Hb, HCT, MHCH, and RDW) (Figure 23B; hematological parameters). [Figure 23B-3] Figure 23. Hematological parameters of Hbbth3 / CD46+ mice before and after in vivo HSC gene therapy. Hbbth3 / CD46+ mice exhibit an intermediate thalassemia phenotype. Mice were treated with an adenovirus donor vector containing a γ-globin nucleic acid sequence operably linked to either a long-chain or short-chain LCR, among other things. Mice were sacrificed and sampled 18 weeks post-treatment. The percentage of reticulocytes was counted on blood smears (Figure 23A; reticulocyte count). The hematological parameters at 18 weeks post-transduction in vivo were indistinguishable from their control CD46tg-corresponding hematological parameters, suggesting complete phenotypic modification, including normalization of leukocyte and erythrocyte counts, as well as erythrocyte characteristics (Hb, HCT, MHCH, and RDW) (Figure 23B; hematological parameters).

[0115] [Figure 24A]Figure 24. Phenotypic correction of extramedullary hematopoiesis in the spleen and liver. (Figure 24A) Spleen size at sacrifice (21 weeks). The top two panels show representative spleen images. The bottom panel is a dot plot summarizing the results. Each symbol represents an individual animal. Data are shown as mean ± standard error of the mean (SEM). *p ≤ 0.05. Statistical analysis was performed using one-way ANOVA. (Figure 24B) Extramedullary hematopoiesis by hematoxylin / eosin staining of liver and spleen sections. Erythroblast clusters in the liver and megakaryocyte clusters in the spleen of Hbbth3 / CD46 mice are indicated by black arrows. Scale bar is 20 μm. [Figure 24B] Figure 24. Phenotypic correction of extramedullary hematopoiesis in the spleen and liver. (Figure 24A) Spleen size at sacrifice (21 weeks). The top two panels show representative spleen images. The bottom panel is a dot plot summarizing the results. Each symbol represents an individual animal. Data are shown as mean ± standard error of the mean (SEM). *p ≤ 0.05. Statistical analysis was performed using one-way ANOVA. (Figure 24B) Extramedullary hematopoiesis by hematoxylin / eosin staining of liver and spleen sections. Erythroblast clusters in the liver and megakaryocyte clusters in the spleen of Hbbth3 / CD46 mice are indicated by black arrows. Scale bar is 20 μm.

[0116] [Figure 25] Phenotypic correction of hemosiderin deposition in the spleen and liver. Iron deposition is shown by Pearl staining as cytoplasmic hemosiderin staining with blue dye in spleen and liver sections. Scale bar is 20 μm. (Exposure time: 2.24 ms, Gain: 4.1 ×, Saturation: 1.50, Gamma: 0.60).

[0117] [Figure 26A-B]Figures 26A-26C. Analysis of bone marrow at sacrifice (week 21). Bone marrow was collected at week 21 after in vivo HSC transduction in Hbbth3 / CD46tg mice. (Figure 26A) Vector copy number per cell in bone marrow MNCs. The difference between the two groups is not significant, but it may become significant if analyzed with a larger sample size. (Figures 26B, 26C) Erythrocyte specificity of γ-globin expression. (Figure 26B) Percentage of γ-globin-expressing erythrocytes (Ter119+) and non-erythrocytes (Ter119-). *p<0.05. Statistical analysis was performed using two-way ANOVA. [Figure 26C] Figures 26A-26C. Analysis of bone marrow at sacrifice (week 21). Bone marrow was collected at week 21 after in vivo HSC transduction in Hbbth3 / CD46tg mice. (Figure 26A) Vector copy number per cell in bone marrow MNCs. The difference between the two groups is not significant, but it may become significant if analyzed with a larger sample size. (Figures 26B, 26C) Erythrocyte specificity of γ-globin expression. (Figure 26B) Percentage of γ-globin-expressing erythrocytes (Ter119+) and non-erythrocytes (Ter119-). *p<0.05. Statistical analysis was performed using two-way ANOVA.

[0118] [Figure 27] Extramedullary hematopoiesis was shown by hematoxylin / eosin staining of liver and spleen sections obtained from CD46tg mice and CD46+ / + / Hbbth-3 mice (before administration of adenovirus donor vector). Iron deposition was shown by Pearl staining, where cytoplasmic hemosiderin in the spleen was stained blue.

[0119] [Figure 28]Schematic diagram of the experimental design for comparing the integration efficiency of SB100x transposase using different reverse repeat sequences (IRs). Three plasmids were used in which the mgmt / GFP transposon payloads were adjacent to (i) pT0ITR; (ii) pT2ITR; or (iii) pT4ITR, and the plasmids were otherwise identical. 293 cells were transfected with the three plasmids containing the mgmt / GFP transposon payload, with or without the support plasmid encoding pSB100x. The cells were cultured for 17 days with or without selection. Culture samples were removed on days 3, 12, and 17 for unselected cells, and on day 17 for selected cells with a single addition of 50 μM O6BG / BCNU on day 3.

[0120] [Figure 29] Percentage of GFP expression in 293 cells cultured with or without the SB100x plasmid for T0, T2, and T4 plasmids, respectively, at days 12 and 17 of culture.

[0121] [Figure 30] Percentage of GFP expression in 293 cells at 17 days of culture under selection with O6BG / BCNU in cells cultured with or without the SB100x plasmid for T0, T2, and T4 plasmids, respectively.

[0122] [Figure 31]Schematic diagram (integrated cassette) of nucleic acid (pWEAd5-PT4-LCR-globin-mgmt) containing a 31.776kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to the transposon IR (specifically, Sleeping Beauty IR), which is then adjacent to the recombinase co-directed repeat sequence (DR, specifically, FRT DR). The transposon includes (i) a beta promoter, a long LCR containing HS1-HS5, and a gamma-globin coding sequence operably ligated to 3'HS1, and (ii) an MGMTP140K selective cassette in which the MGMTP140K coding sequence is operably ligated to the Ef1a promoter.

[0123] [Figure 32] Schematic diagram (embedded cassette) of nucleic acid (HDAd5-PT4-long-chain LCR globin-rhMGMT) containing a 31.772kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to the transposon IR (specifically, Sleeping Beauty IR), which is then adjacent to the recombinase DR (specifically, FRT DR). The transposon includes (i) a beta promoter, a long-chain LCR containing HS1-HS5, and a gamma-globin coding sequence operably ligated to 3'HS1, and (ii) an MGMTP140K selective cassette in which the MGMTP140K coding sequence is operably ligated to the Ef1a promoter.

[0124] [Figure 33] Schematic diagram (embedded cassette) of nucleic acid (HDAd-Ad5-PT4-LCR-hACE2 / mgmt) containing a 13.173kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to the transposon IR (specifically, Sleeping Beauty IR), which is then adjacent to the recombinase DR (specifically, FRT DR). The transposon includes (i) a beta promoter and a recombinant human ACE2 coding sequence operably ligated to a long LCR containing HS1-HS4, and (ii) an MGMTP140K coding sequence operably ligated to the Ef1a promoter, in an MGMTP140K selective cassette.

[0125] [Figure 34] Schematic diagram (integrated cassette) of nucleic acid (pWEHCB-microLCR-globin / mgmt) containing a 12.169kb transposon payload. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is flanked by the transposon IR (specifically, Sleeping Beauty IR), which is then flanked by the recombinase DR (specifically, FRT DR). The transposon comprises (i) a beta promoter and a gamma globin coding sequence operably ligated to a long LCR containing HS1-HS4, and (ii) an MGMTP140K selective cassette in which the MGMTP140K coding sequence is operably ligated to the Ef1a promoter.

[0126] [Figure 35] Schematic diagram (embedded cassette) of nucleic acid (pWEHCA-Faconi-GFP) containing a 9.382 kb transposon payload. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is flanked by the transposon IR (specifically, Sleeping Beauty IR), which is then flanked by the recombinase DR (specifically, FRT DR). The transposon contains (i) a FancA coding sequence operably ligated to the pgk promoter, and (ii) a GFP coding sequence operably ligated to the Ef1a promoter.

[0127] [Figure 36] Schematic diagram (integrated cassette) of nucleic acid (pHCA-T4-rhMGMT-GFP) containing a 5.490kb transposon payload. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is flanked by a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then flanked by a recombinase co-direction repeat sequence (DR, specifically FRT DR). The transposon comprises (i) a GFP coding sequence operably ligated to a PGK promoter, and (ii) an MGMTP140K selective cassette in which an MGMTP140K coding sequence operably ligated to an EF1a promoter.

[0128] [Figure 37]Schematic diagram (embedded cassette) of nucleic acid containing a 3.797kb transposon payload. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is flanked by a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then flanked by a recombinase same-direction repeat sequence (DR, specifically FRT DR). The transposon contains (i) a GFP coding sequence and (ii) an MGMTP140K coding sequence operably ligated to the EF1a promoter.

[0129] [Figure 38] Schematic diagram (embedded cassette) of nucleic acid (pBHCA-PT0-EF1a-mgmt / GFP) containing a 3.709kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then adjacent to a recombinase same-direction repeat sequence (DR, specifically FRT DR). The transposon contains (i) an eGFP coding sequence and (ii) an MGMTP140K coding sequence operably ligated to the EF1a promoter.

[0130] [Figure 39]Schematic diagram (embedded cassette) of nucleic acid (pHCA(Ad35)-PT4-EF1a-mgmt / GFP) containing a 3.547kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then adjacent to a recombinase same-direction repeat sequence (DR, specifically FRT DR). The transposon contains (i) a GFP coding sequence and (ii) an MGMTP140K coding sequence operably ligated to the EF1a promoter.

[0131] [Figure 40] Schematic diagram (embedded cassette) of nucleic acid (pHCA-Ad5-PT4-Ef1a-mgmt / GFP) containing a 3.543kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then adjacent to a recombinase same-direction repeat sequence (DR, specifically FRT DR). The transposon contains (i) a GFP coding sequence and (ii) an MGMTP140K coding sequence operably ligated to the EF1a promoter.

[0132] [Figure 41]Schematic diagram (embedded cassette) of nucleic acid (pHCA(Ad35)-PT4-EF1a-mgmt) containing a 2.781kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then adjacent to a recombinase same-direction repeat sequence (DR, specifically FRT DR). The transposon contains an MGMTP140K selective cassette in which the MGMTP140K coding sequence is operably ligated to the EF1a promoter.

[0133] [Figure 42] Schematic diagram (integrated cassette) of nucleic acid (pHCA-T4-Ef1a-rhMGMT) containing a 2.777kb transposon payload. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is flanked by a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then flanked by a recombinase same-direction repeat sequence (DR, specifically FRT DR). The transposon includes an MGMTP140K selective cassette in which the MGMTP140K coding sequence is operably ligated to the EF1a promoter.

[0134] [Figure 43]A schematic diagram (embedded cassette) of a nucleic acid (pHCA-Ad5-PT4-Ef1a-mgmt) containing a 2.751kb transposon payload. The schematic diagram is divided into two overlapping parts for ease of display, the relationship between the parts will be apparent to those skilled in the art. The schematic diagram provides the transposon payload in the context of a circular plasmid. Those skilled in the art will understand that in other contexts, for example in a viral vector genome, the transposon payload can be readily utilized using molecular biological techniques. The transposon payload is adjacent to a transposon reverse repeat sequence (IR, specifically Sleeping Beauty IR), which is then adjacent to a recombinase same-direction repeat sequence (DR, specifically FRT DR). The transposon contains an MGMTP140K selective cassette in which the MGMTP140K coding sequence is operably ligated to the EF1a promoter. [Modes for carrying out the invention]

[0135] Detailed explanation This disclosure includes, in particular, adenovirus vectors, adenovirus vector genomes, and combinations and uses thereof. The adenovirus vectors and adenovirus vector genomes of this disclosure can contain transposon payloads of up to, for example, 20, 25, 30, or even greater than 30 kb, and furthermore, in various embodiments, successfully integrate such large transposon payloads into the genome of host cells. As those skilled in the art will understand, since integration capacity limits the length and / or complexity of the therapeutic payload, at least in part, vector integration capacity is itself one of the critically important features in gene therapy systems. Accordingly, the methods and compositions provided herein provide, in particular, a platform for effective gene therapy using adenovirus vectors that enables transmissible integration of nucleic acid payloads of, for example, 20, 25, 30, or even greater than 30 kb into the genome of host cells. As those skilled in the art will understand from this disclosure, and as illustrated by the various embodiments herein, such integration capacity enables the manipulation of therapeutic payloads with greater complexity and diversity than is possible with various prior systems.

[0136] The methods and compositions disclosed herein overcome certain previously understood limitations in incorporation capacity. Some such limitations relate to the viral vector type. For example, the lentiviral vector payload capacity is approximately 9 kb, the retroviral payload capacity is approximately 8 kb, and the adeno-associated virus (AAV) payload capacity is approximately 5 kb. Other such limitations were previously understood to be inherent to transposition. For example, studies have shown that transposon incorporation is length-dependent—as length increases, the ability to transpose rapidly declines, a phenomenon sometimes referred to in the art as "length-dependent." Given these existing assumptions, the discovery that the compositions and methods disclosed herein break the previously understood limits of adenovirus transposable incorporation capacity is a surprising result revealed by the disclosure and the examples provided herein. To the best of our knowledge, this study represents the first demonstration that the methods and compositions provided herein can incorporate various transposon payloads of certain sizes disclosed herein. This finding is illustrated, for example, by the incorporation of transposon payloads containing large regulatory regions (locus regulatory regions or "LCRs") for improved transgene expression. However, to avoid any doubt, those skilled in the art will understand that such examples represent the more general finding of high transmissibility incorporation capacity of adenovirus compositions and methods provided herein, and their importance in various fields, particularly in the field of gene therapy.

[0137] Next, aspects of this disclosure will be described in further detail for the purpose of further enhancement, as follows: (I) Incorporation of a viral vector payload into a target cell genome; (II) Types of large payloads; (III) Long LCRs; (IV) Coding sequences operably linked to long LCRs; (V) Transposases; (VI) Regulatory elements; (VII) Vectors; (VIII) Formulations; (IX) Applications; (X) Exemplary embodiments; (XI) Experimental examples (one or more); and (XII) Conclusions.

[0138] (I) Integration of viral vector payload into target cell genome

[0139] Gene therapy often requires the integration of a desired nucleic acid payload into the genome of target cells. Given the diversity of conditions that can be treated by various gene therapies, many strategies have been considered for designing nucleic acid payloads. However, in practice, the delivery of therapeutic payloads is limited in many contexts by the difficulty of integrating large payloads into the target cell genome. For example, the payload capacity of lentiviral vectors is approximately 9 kb, retroviral payloads are approximately 8 kb, and adeno-associated virus (AAV) payloads are approximately 5 kb. These represent substantial limitations given the current interest in expressing large genes, utilizing large human regulatory sequences, and / or payloads capable of expressing multiple genes. Furthermore, as is well understood by those skilled in the art, each viral platform is related to a diversity of different characteristics that make each one uniquely suitable to some extent for a variety of uses, and these factors may include, but are not limited to, recipient immune responses (e.g., inflammation and / or interactions with existing antibodies), difficulties in vector production, effectiveness of cell transduction, effectiveness of payload integration, transgene expression characteristics, target-oriented cell types, risk of genotoxicity (e.g., carcinogenicity), and others, any or all of which can be weighed independently by researchers and physicians in various contexts. The Disclosure acknowledges that the efficiency of transposon payload incorporation using certain known compositions and methods in one or more systems depends on one or more of the target cell type, plasmid backbone, and / or transposon length, and that certain such dependencies are reduced or eliminated in at least certain compositions and methods of the Disclosure, e.g., compositions and methods comprising an adenovirus genome containing a transposon payload flanked by an SB reverse repeat sequence (e.g., for transposition by SB100x transposase or another SB transposase in human target cells, e.g., hematopoietic stem cells, and / or in vivo therapy).

[0140] Adenovirus vectors are one of the most commonly used gene therapy vectors. For example, according to at least several reports, adenovirus vectors are the most commonly used vectors for cancer gene therapy. In fact, more than 400 gene therapy clinical trials have been initiated and / or completed using human Ad vectors, for example, for vaccine use, therapeutic gene delivery, and / or cancer treatment. The various advantages of adenovirus vectors that contribute to and / or at least partly cause their widespread use in gene therapy are well known in the art. Nevertheless, even with commonly used vectors, gene therapy remains a challenging task, at least in some cases, because long-term phenotypic correction requires sufficiently efficient and stable integration and expression of the therapeutic gene.

[0141] While some adenovirus vectors are known to have high cloning capacities of up to approximately 36–37 kb, the ability to physically generate vectors with large payloads does not reflect the vector's ability to efficiently mediate the integration of the payload into the target cell genome. In fact, adenovirus vector genomes, which are typically 26–45 kb (e.g., approximately 36 kb for Ad5) linear double-stranded DNA genomes, are typically not naturally integrated into host cell genomes. On the contrary, adenovirus vectors are characterized by episomal maintenance of the viral genome in host cells. Episomal maintenance minimizes the risk of insertion effects, but among several difficulties known to those skilled in the art, episomal genomes are often poorly retained by target cells and their progeny. For these reasons, at least, attempts have been made to produce adenovirus vectors engineered for integration into host cell genomes, unlike their natural counterparts. Such approaches, too, have not been without challenges. For example, one of the problems associated with certain embedded adenovirus vectors was site preference characterized by genotoxic effects.

[0142] One method for manipulating adenovirus vectors to incorporate a payload into a host cell genome is to obtain embedded viral hybrid vectors. In embedded viral hybrid vectors, the genetic elements of a vector that efficiently induces transduction of target cells are integrated with the genetic elements of a vector that stably incorporates its own vector payload. Desired embedded elements (e.g., for use in combination with adenovirus vectors) include those of bacteriophage integrase PHiC31, retrotransposons, retroviruses (e.g., those mediated by LTRs or retroviral embedding), zinc finger nucleases, DNA-binding domain-retroviral integrase fusion proteins, AAVs (e.g., those mediated by AAV-ITRs or AAV-Rep proteins), and Sleeping Beauty (SB) transposases.

[0143] Like the vector itself, the integration system of an embedded viral hybrid vector suffers its own unique advantages and disadvantages, including characteristic positional integration patterns and payload capacity. Studies have shown, for example, that transposon integration is length-dependent; as length increases, the ability to displace decreases rapidly, a phenomenon sometimes referred to in the art as "length-dependent." In the case of SB transposases, studies have shown that SB transposon effectiveness decreased by 30% for every 1kb of transposon (payload) length added, and was completely lost above approximately 9kb. While some studies have indicated that a small fraction of SB transposon integration was retained up to at least approximately 10kb, evidence has demonstrated that larger SB transposons are not integrated as efficiently as their smaller counterparts. Certain transposon-binding systems modified to enhance integration effectiveness also suffered from significant length-dependent effects, albeit at substantially reduced levels of transposon integration (Turchiano et al., PLOS One, 9: e112712, 2014).

[0144] This disclosure provides, in particular, our remarkable discovery that transposon payloads of at least about 30 kb to about 35 kb can be incorporated into host cell genomes with sufficient efficacy for therapeutic use. In various embodiments, this disclosure provides vectors, genomes, and systems for the incorporation of large payloads (e.g., at least about 30 kb to about 35 kb), comprising an adenovirus genome containing a transposon payload flanked by SB reverse repeat sequences flanked by FRT recombination sites, such that the genome or portion containing the transposon payload is circularized in the presence of a recombinase, and the recombinase discovered by the inventors enables the incorporation of the large transposon payload into the target cell genome in the presence of an SB transposase. This disclosure further provides that such compositions are sufficiently efficient for incorporation and transgene expression, for example, to achieve in vivo therapy. These remarkable findings, sharply contrasting with previous concepts of length dependence and incorporation efficacy, open the door to therapeutic and research uses of adenovirus vectors that were previously considered unattainable.

[0145] (II) Types of large payloads

[0146] In certain embodiments, the invention disclosed herein facilitates the delivery and incorporation of large transposon payloads. Large payloads include, for example, code sequences linked to long chain LCRs, including long chain LCRs as described herein. In certain embodiments, the payload is at least 10kb. In certain embodiments, the payload is at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 35kb, at least 40kb, or longer. In certain embodiments, the payload has a length of 10kb to 35kb, 10kb to 30kb, 15kb to 35kb, 15kb to 30kb, 20kb to 35kb, or 20kb to 30kb. In certain embodiments, the payload has a length of 10kb to 32.4kb, 15kb to 32.4kb, or 20kb to 32.4kb. In certain embodiments, the payload encodes a single long (large) protein. In certain embodiments, the payload encodes multiple proteins; for example, two or more proteins, e.g., two, three, four, or five or more proteins. In embodiments where the payload encodes multiple proteins, it is not necessary to consider any individual protein thus encoded independently as "large" or "long"; rather, it is understood that the entire payload held by the adenovirus vector is "large," even if it contains sequences encoding a number of smaller individual proteins. In certain embodiments, the payload includes a long chain LCR.

[0147] (III) Long-chain LCR

[0148] The ability to integrate large payloads into host cell genomes opens the door to the integration of constructs previously considered too large for effective therapeutic use. Beyond the immediately apparent general utility of being able to integrate large payloads, one category of large payloads includes payloads containing long locus regulatory regions (or long LCRs). In some cases, regulatory regions larger than those accommodated by at least certain currently existing vector systems for gene therapy, such as lentivirals and AAV systems, are useful for achieving therapeutically effective transgene expression from payloads, and / or increasing the level of expression (e.g., the number or frequency of production of mRNA encoding the transgene expression product and / or the transgene expression product encoded by the transgene) and / or the specificity of expression (e.g., the timing of expression and / or cell or tissue specificity).

[0149] Without wishing to be constrained by any particular scientific theory, the human genome is an organized three-dimensional structure that includes long-range direct and / or indirect interactions between regulatory regions (such as transcription factor binding sites) and coding regions whose expression it modulates, for example, via loop formation. In many instances, such long-range interactions occur in the context of topologically related domains (TADs). TADs are thought to be functional units of chromosomal organization that can facilitate interactions between enhancers and other regulatory regions for regulating transcription. TADs are thought to be demarcated by boundaries that limit the search space for enhancers and promoters, preventing the formation of undesirable regulatory contacts. TAD boundaries on both sides of such domains are conserved among different mammalian cell types, and even between species.

[0150] Due to its crucial role in the genome, particularly in the organization of nucleic acid sequences and proteins that contribute to gene and transgene expression, TADs can be used to increase the safety and / or efficacy of gene therapy. TADs themselves are too large to be contained in any currently available viral vectors. The median size of a TAD is 880 kb. However, certain functional elements present within TADs have been identified that capture some or all of the gene or transgene expression effects of the TAD, and these are of a size suitable for inclusion in the adenovirus vectors disclosed herein, although in many instances they are still too large to be contained in certain other vectors, such as lentiviruses and AAV vectors. In some cases, a control sequence containing one or more nucleic acid sequences of a TAD may be referred to as a LCR. LCRs are engineered to have varying lengths, for example, in some cases to be relatively short in order to be contained in vectors with relatively small payload capacities, such as lentiviruses or AAV vectors. However, without wishing to be constrained by any particular theory, those skilled in the art will understand that longer sequences, having a greater capacity, impart a favorable expression effect to the endogenous sequence from which they originate or on which they are based, either entirely or partially, to the relevant gene or transgene. Therefore, some LCRs have been engineered to have relatively short lengths, for example, less than 5kb, less than 6kb, less than 7kb, less than 8kb, or less than 9kb. In contrast, this disclosure recognizes that long LCRs (e.g., regulatory sequences of 9kb or more, 10kb or more, 11kb or more, 12kb or more, 13kb or more, 14kb or more, 15kb or more, 20kb or more, 25kb or more, or 30kb or more) can be incorporated into the host cell genome using the vectors, genomes, and methods provided herein.In various embodiments, the long-chain LCR includes a control sequence having a length range having a lower limit selected from any of 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, 15kb, 16kb, 17kb, 18kb, 19kb, 20kb, 21kb, 22kb, 23kb, 24kb, 25kb, 26kb, 27kb, 28kb, 29kb, and 30kb, and an upper limit selected from any of 30kb, 31kb, 32kb, 33kb, 34kb, 35kb, 36kb, 37kb, 38kb, 39kb, and 40kb. The long-chain LCR may also have any length of any of the LCRs provided herein, and such lengths may be considered as the lower or upper limit in various embodiments.

[0151] Examples of LCRs are shown in Table 1. Unless otherwise indicated or obvious to those skilled in the art, the reference genome is a GRCh38 reference genome such as GRCH38 / hg38 or GRCh38.p13.

[0152] [Table 1]

[0153] β-globin LCR is an example of at least some LCRs in at least some respects. For example, like many other LCRs, β-globin LCRs produce increased expression of operably linked genes or transgenes (e.g., increased transcription, increased translation, and / or increased cell-specific or tissue-specificity), and include DNAse-sensitive (HS) regions, which are understood by those skilled in the art to mediate the LCR's expressive action. Furthermore, like many other LCRs, β-globin LCRs can be used in whole or in part, for example, as a nucleic acid containing a β-globin LCR sequence including all of the β-globin LCR HS regions (HS1-HS5), or as a nucleic acid containing a subset of the β-globin LCR HS regions (e.g., HS1-HS4).

[0154] An example of the nucleic acid sequence of the Homo sapiens β-globin region located on chromosome 11 is provided under GenBank acceptance number NG_000007. The β-globin long chain recurrence region (LCR) may, in some cases, be a sequence located at 6-22 kb on the 5' end of the first (embryonic) globin gene at the locus, or may contain such a sequence. The β-globin long chain LCR may contain five DNAseI-sensitive sites (5'HS1-5). Li et al., Blood, 100(9):3077-3086, 2002. NG_000007 provides the locations of restriction enzyme recognition sites representing the boundaries of DNAseI-sensitive sites HS1, HS2, HS3, and HS4 within the regulatory region of the gene locus (e.g., the SnaBI and BstXI restriction enzyme recognition sites of HS2, the HindIII and BamHI restriction enzyme recognition sites of HS3, and the BamHI and BanII restriction enzyme recognition sites of HS4), and NG_000007, in whole and in particular the portion relating to the locations of the sensitive sites, is incorporated herein by reference. The sequence and location of HS1 are described, for example, by Pasteri et al., Ann NY Acad.Sci. 1998;850:377-381, Pasteri et al., Blood. 92:653-663, 1998, and Milot et al., Cell. 87:105-114, 1996. In certain embodiments, the HS2 region extends to positions 16,671–17,058 in the regulatory locus. The SnaBI restriction enzyme recognition site and the BstXI restriction enzyme recognition site of HS2 are located at positions 17,093 and 16,240, respectively. The HS3 region extends to positions 12,459–13,097 in the regulatory locus. The BamHI restriction enzyme recognition site and the HindIII restriction enzyme recognition site of HS3 are located at positions 12,065 and 13,360, respectively. The HS4 region extends to positions 9,048–9,713 in the regulatory locus. The BamHI restriction enzyme recognition site and the BanII restriction enzyme recognition site of HS4 are located at positions 8,496 and 9,576, respectively.

[0155] In certain embodiments disclosed herein, a small portion of a β-globin LCR is utilized. The small portion contains fewer than five HS regions (e.g., HS1, HS2, HS3, HS4, and / or HS5), provided that the LCR does not contain all five segments of the β-globin LCR. The 4.3kb HS1-HS4 LCR used in Example 1 of this disclosure is an example of a small LCR. Other small LCRs may include, for example, HS1, HS2, and HS3; HS2, HS3, and HS4; HS3, HS4, and HS5; HS1, HS3, and HS5; HS1, HS2, and HS5; and HS1, HS4, and HS5. For examples of additional small LCRs, see Sadelain et al., Proc.Nat.Acad.Sci.(USA)92:6728-6732,1995, and Lebouich et al., EMBO J.13:3065-3076,1994. In certain embodiments, small β-globin LCRs may be used in combination with a β-globin promoter. In certain embodiments, this combination yields a 5.9 kb LCR-promoter integrated. With respect to LCRs, the terms "small" and "micro" are used interchangeably herein.

[0156] In certain embodiments disclosed herein, the long-chain portion of a locus regulatory region (LCR) is utilized. The long-chain β-globin LCR may include HS1, HS2, HS3, HS4, and HS5. In certain embodiments, the long-chain LCR includes a sequence of approximately 21.5 kb containing HS1, HS2, HS3, HS4, and HS5 of the β-globin LCR. By combining the long-chain β-globin LCR with a β-globin promoter, the protein can be expressed at high levels.

[0157] Certain embodiments may include the long-chain β-globin LCR position 5292319~5270789 (21,531 bp) (SEQ ID NO: 6) on human chromosome 11, as shown in GRCH38 / hg38. In various embodiments, the total length of the long-chain LCR may be 18kb or greater, 18.5kb or greater, 19kb or greater, 19.5kb or greater, 20kb or greater, 20.5kb or greater, 21kb or greater, 21.5kb or greater, or 21.531kb or greater. In various embodiments, the total length of the long-chain LCR may be 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater of the length of the sequence SEQ ID NO: 6. In various embodiments, the long LCR may comprise at least 18kb, at least 18.5kb, at least 19kb, at least 19.5kb, at least 20kb, at least 20.5kb, at least 21kb, or at least 21.5kb of SEQ ID NO: 6. In any of the various embodiments provided herein, the long LCR may comprise or comprise a nucleic acid having an identity percentage of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the corresponding contiguous portion of the sequence of SEQ ID NO: 6. In various embodiments, the long LCR may differ from the natural genome sequence in that it contains one or more restriction sites, such as an XhoI restriction site (see, for example, SEQ ID NO: 98, where an exemplary XhoI site (italicized) is provided at positions 10655–10661). In any of the various embodiments provided herein, the long LCR may include HS1, HS2, HS3, HS4, and HS5.

[0158] In various embodiments, the Ad35 vector system may include a transposing transgene insert containing, for example, the position 5228631-5227018 (1614 bp) of human chromosome 11 (SEQ ID NO: 7), as a β-globin promoter. In various embodiments, the full length of the β-globin promoter may be, for example, 1.0 kb or more, 1.1 kb or more, 1.2 kb or more, 1.3 kb or more, 1.4 kb or more, 1.5 kb or more, 1.6 kb or more, or 1.609 kb or more. In various embodiments, the β-globin promoter may contain at least 1.0 kb, at least 1.1 kb, at least 1.2 kb, at least 1.3 kb, at least 1.4 kb, at least 1.5 kb, at least 1.6 kb, or at least 1.609 kb of the sequence of SEQ ID NO: 7. In various embodiments, the full length of the β-globin promoter may include, for example, 100 bp or more, 200 bp or more, 300 bp or more, 400 bp or more, 500 bp or more, 1 kb or more, 1.5 kb or more, 2 kb or more, 2.5 kb or more, 3 kb or more, 4 kb or more, or 5 kb or more of nucleic acid sequences located upstream of a gene whose expression is controlled by the β-globin LCR (e.g., immediately upstream of the first coding nucleotide of such a gene), and such genes include, but are not limited to, the epsilon (HBE1) globin gene, the G-gamma (HBG2) globin gene, the A-gamma (HBG1) globin gene, the delta (HBD) globin gene, and the beta (HBB) globin gene, and / or one or more genes located at the hemoglobin β locus (11:5,225,463~5,227,070, complementary strand). In various embodiments, the full length of the β-globin promoter may include, for example, 100 bp or more, 200 bp or more, 300 bp or more, 400 bp or more, 500 bp or more, 1 kb or more, 1.5 kb or more, 2 kb or more, 2.5 kb or more, 3 kb or more, 4 kb or more, or 5 kb or more of a nucleic acid sequence located upstream (e.g., immediately upstream) of position 5227021 at chromosome 11 NC_000011.10.In various embodiments, the full length of the β-globin promoter may be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the length of the sequence of SEQ ID NO: 7. In any of the various embodiments provided herein, the β-globin promoter may be a nucleic acid having a sequence whose identity percentage with respect to the corresponding contiguous portion of the β-globin promoter sequence present in the reference genome is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and optionally the β-globin promoter may include the sequence of SEQ ID NO: 7.

[0159] In various embodiments, β-globin LCR (such as long-chain β-globin LCR) expresses an operably linked coding sequence in red blood cells. In various embodiments, the operably linked coding sequence is also operably linked to a β-globin promoter as shown herein or otherwise known in the art.

[0160] Immunoglobulin heavy chain locus B cell LCRs are examples of LCRs that produce enhanced expression of operably linked coding sequences (e.g., increased transcription, increased translation, and / or increased cell-specific or tissue-specificity). Enhanced coding sequence expression can be achieved when operably linked to an immunoglobulin heavy chain locus B cell LCR containing a complete immunoglobulin heavy chain locus B cell LCR sequence and / or its expression regulatory fragments. It is understood by those skilled in the art that immunoglobulin heavy chain locus B cell LCRs contain DNase I hypersensitive sites (HSs), and such HSs mediate at least some of the expression-enhancing effects of immunoglobulin heavy chain locus B cell LCRs. An immunoglobulin heavy chain locus B cell LCR contains four DNase I hypersensitive sites (HS1, HS2, HS3, and HS4) in the 3'Cα region of the immunoglobulin heavy chain (IgH) locus and functions as an enhancer locus regulatory region (LCR). Therefore, the immunoglobulin heavy chain locus B cell LCR can be a complete immunoglobulin heavy chain locus B cell LCR containing all HS1-HS4, or it can be its expression regulatory fragment containing a subset of the highly sensitive HS1-HS4 sites. These HS sites map to approximately 10-30 kb of the IgH C gene and can act as a lymphoid cell-specific and developmentally regulated enhancer element in transient transfection assays. This nucleic acid sequence has been observed to lead to similar expression patterns when ligated with the c-myc gene in Burkitt lymphoma and plasmacytoma cell lines. In Burkitt lymphoma and plasmacytoma, c-myc regulation by the B cell LCR occurs, and this regulation is due to a characteristic chromosomal translocation in which the c-myc gene is juxtaposed with the IgH sequence, resulting in abnormal c-myc transcription.For further explanations regarding the addition of B-cell LCRs, see, for example, Madisen et al., Mol Cell Biol. 18(11):6281-92, 1998; Giannini et al., J.Immunol. 150:1772-1780, 1993; Madisen & Groudine, Genes Dev. 8:2212-2226, 1994; and Michaelson et al., Nucleic Acids Res. 23:975-981, 1995.

[0161] Certain embodiments may include the immunoglobulin heavy chain locus B cell LCR location, chromosome 14 - NC_000014.9 (105586437~106879844, complement) (1,293,408 bp) or its expression regulatory fragment. In various embodiments, the immunoglobulin heavy chain locus B cell LCR may be equal to or greater than 70%, equal to or greater than 75%, equal to or greater than 80%, equal to or greater than 85%, equal to or greater than 90%, equal to or greater than 91%, equal to or greater than 92%, equal to or greater than 93%, equal to or greater than 94%, equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98%, equal to or greater than 99%, or have 100% of the full length of the immunoglobulin heavy chain locus B cell LCR position 105586437-106879844. In various embodiments, the immunoglobulin heavy chain locus B cell LCR may include at least 10kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, or at least 30kb of immunoglobulin heavy chain locus B cell LCR locations 105586437 to 106879844. In any of the various embodiments provided herein, the long chain LCR may be a nucleic acid having an identity percentage of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the corresponding contiguous portion of the immunoglobulin heavy chain locus B cell LCR positions 105586437-106879844.

[0162] In various embodiments, the Ad35 vector may include, for example, an immunoglobulin heavy chain locus B cell LCR provided herein in a payload that includes, for example, an immunoglobulin heavy chain locus B cell LCR and, optionally, a promoter of a gene typically operably linked to the immunoglobulin heavy chain locus B cell LCR in the human genome. In various embodiments, the gene operably linked to the immunoglobulin heavy chain locus B cell LCR is an immunoglobulin heavy chain gene. In various embodiments, the immunoglobulin heavy chain gene promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the immunoglobulin heavy chain gene promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, or at least 5.0 kb upstream of the first coding nucleotide of the immunoglobulin heavy chain gene in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of the coding sequence of the gene that is typically operably linked to the immunoglobulin heavy chain locus B cell LCR in the human genome is the first coding nucleotide of the immunoglobulin heavy chain gene.

[0163] In various embodiments, an immunoglobulin heavy chain locus B cell LCR, such as a long-chain immunoglobulin heavy chain locus B cell LCR, induces the expression of an operably ligated coding sequence in B cells. In various embodiments, the operably ligated coding sequence is also operably ligated to an immunoglobulin heavy chain gene promoter, either as shown herein or otherwise known in the art.

[0164] Another exemplary LCR is the T cell LCR at the T cell receptor alpha / delta locus, which enhances the expression of an operably ligated coding sequence. At the T cell receptor (TCR) alpha / delta locus, the LCR can control differential tissue and developmental expression, as well as the rearrangement of the TCR alpha and delta genes. The expression of the coding sequence can be enhanced when operably ligated with the T cell LCR of the T cell receptor alpha / delta locus LCR, which includes the complete T cell LCR of the T cell receptor alpha / delta locus LCR sequence and / or its expression control fragments. The T cell LCR of the T cell receptor alpha / delta locus LCR includes a DNAse hypersensitive site (HS), which is understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the T cell LCR of the T cell receptor alpha / delta locus LCR. The T cell LCR was identified as a region located at 3' of the TCR alpha / delta locus, containing eight T cell-specific nuclease hypersensitive domains (HS-1 to HS-8). Therefore, the T cell LCR of the T cell receptor alpha / delta locus LCR may be either the complete T cell LCR of the T cell receptor alpha / delta locus LCR, including all HS1-HS8, or its expression regulatory fragment, including a subset of the highly sensitive sites HS1-HS8. In transgenic mice, the TCR alpha gene linked to this region was observed to be expressed at high levels, regardless of the site of integration, and correlated with gene copy number. This transgene was expressed in alpha-beta cells but not in the gamma-delta T cell subset, and was activated at the correct time during development. LCR function requires at least HS-2-HS-6. Additional descriptions of B cell LCRs can be found, for example, in Diaz et al., Immunity 1(3):207-17, 1994.

[0165] In various embodiments, the Ad35 vector may contain, for example, the T cell LCR of the T cell receptor alpha / delta locus LCR provided herein in a payload that includes, for example, the T cell LCR of the T cell receptor alpha / delta locus LCR and, optionally, a promoter of a gene typically operably ligated to the T cell LCR of the T cell receptor alpha / delta locus LCR in the human genome. In various embodiments, the gene operably ligated to the T cell LCR of the T cell receptor alpha / delta locus LCR is TCR alpha on chromosome 14, NC_000014.9(21621904..22552132) or TCR delta locus on chromosome 14, NC_000014.9(22422546..22466577). In various embodiments, the TCR alpha or TCR delta promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb.In various embodiments, the TCR alpha or TCR delta promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upstream of the first coding nucleotide of the TCR alpha or TCR delta in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upstream of the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably ligated to the T cell LCR at the T cell receptor alpha / delta locus LCR in the human genome is the first coding nucleotide of TCR alpha or TCR delta.

[0166] In various embodiments, T cell LCRs of the T cell receptor alpha / delta locus, such as long T cell LCRs of the T cell receptor alpha / delta locus LCR, induce the expression of an operably ligated coding sequence in T cells. In various embodiments, the operably ligated coding sequence is also operably ligated to a TCR alpha or TCR delta promoter, as shown herein or otherwise known in the art.

[0167] Adenosine deaminase LCRs are exemplary LCRs that enhance the expression of operably ligated coding sequences. The expression of coding sequences can be enhanced when operably ligated with an adenosine deaminase LCR, which includes a complete adenosine deaminase LCR sequence and / or an expression control fragment thereof. Adenosine deaminase LCRs include DNAse hypersensitive sites (HSs) that are understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the adenosine deaminase LCR. Adenosine deaminase LCRs include hypersensitive sites 1-6. Thus, an adenosine deaminase LCR may be a complete adenosine deaminase LCR containing all of HS1-HS6, or an expression control fragment thereof containing a subset of hypersensitive sites HS1-HS6.

[0168] Certain embodiments may include the adenosine deaminase LCR position NC_000020.11 44629004~44651567 (22,564 bp) on human chromosome 20 or a regulatory fragment thereof. In various embodiments, the adenosine deaminase LCR may have a total length equal to or greater than 70%, equal to or greater than 75%, equal to or greater than 80%, equal to or greater than 85%, equal to or greater than 90%, equal to or greater than 91%, equal to or greater than 92%, equal to or greater than 93%, equal to or greater than 94%, equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98%, equal to or greater than 99%, or 100% of the total length of the adenosine deaminase LCR at positions 44629004 to 44651567. In various embodiments, the adenosine deaminase LCR may include at least 10kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, or at least 22kb of adenosine deaminase LCR positions 44629004 to 44651567. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having an identity percentage of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the corresponding contiguous portion of adenosine deaminase LCR positions 44629004 to 44651567, or may include such nucleic acid.

[0169] In various embodiments, the Ad35 vector may include, for example, the adenosine deaminase LCR provided herein in a payload that includes a promoter of a gene typically operably ligated to the adenosine deaminase LCR in the human genome, and optionally the adenosine deaminase LCR. In various embodiments, the gene operably ligated to the adenosine deaminase LCR is adenosine deaminase (20:44,619,518~44,651,757, complement). In various embodiments, the adenosine deaminase promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the adenosine deaminase promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upstream of the first coding nucleotide of adenosine deaminase in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upstream of the corresponding nucleic acid sequence that is immediately upstream.In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably linked to adenosine deaminase LCR in the human genome is the first coding nucleotide of adenosine deaminase on chromosome 20 - NC_000020.11 44651607.

[0170] In various embodiments, adenosine deaminase LCRs, such as long-chain adenosine deaminase LCRs, induce the expression of operably linked coding sequences in one or more of the blood, intestinal, and lymphoid tissues. In various embodiments, the operably linked coding sequences are also operably linked to adenosine deaminase promoters as shown herein or otherwise known in the art.

[0171] Apolipoprotein E / C LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with an apolipoprotein E / C LCR, which includes a complete apolipoprotein E / C LCR sequence and / or its expression control fragments. An apolipoprotein E / C LCR includes DNAse hypersensitive sites (HSs) that are understood by those skilled in the art to mediate at least a portion of the apolipoprotein E / C LCR's expression-enhancing effect. An apolipoprotein E / C LCR includes hypersensitive sites 1-6. Thus, an apolipoprotein E / C LCR may be a complete apolipoprotein E / C LCR containing all of HS1-HS6, or its expression control fragments containing a subset of hypersensitive sites HS1-HS6.

[0172] In various embodiments, the Ad35 vector may include the apolipoprotein E / C LCR provided herein in a payload that includes, for example, the apolipoprotein E / C LCR and, optionally, a promoter of a gene typically operably ligated to the apolipoprotein E / C LCR in the human genome. In various embodiments, the gene operably ligated to the apolipoprotein E / C LCR is apolipoprotein E (19:44,905,795~44,909,394). In various embodiments, the apolipoprotein E promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the apolipoprotein E promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream.In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably linked to apolipoprotein E / C LCR in the human genome is the first coding nucleotide of apolipoprotein E on chromosome 19 - NC_000019.10(44906625).

[0173] In various embodiments, apolipoprotein E / C LCRs, such as long-chain apolipoprotein E / C LCRs, induce the expression of operably ligated coding sequences in erythrocytes. In various embodiments, the operably ligated coding sequences are also operably ligated to apolipoprotein E / C promoters as shown herein or otherwise known in the art.

[0174] A Th2 cytokine LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with a Th2 cytokine LCR, which includes a complete Th2 cytokine LCR sequence and / or an expression control fragment thereof. A Th2 cytokine LCR includes DNAse hypersensitive sites (HS) that are understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the Th2 cytokine LCR. A Th2 cytokine LCR includes hypersensitive sites RHS5–RHS7. Thus, a Th2 cytokine LCR may be a complete Th2 cytokine LCR including all of RHS5–RHS7, or an expression control fragment thereof including a subset of the hypersensitive sites RHS5–RHS7.

[0175] Certain embodiments may include the Th2 cytokine LCR position NC_000005.10 (132629263~132642195) (12,933 bp) on human chromosome 5 or a regulatory fragment thereof. In various embodiments, the Th2 cytokine LCR may have a total length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the Th2 cytokine LCR position 132629263~132642195. In various embodiments, the Th2 cytokine LCR may include at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 11 kb, or at least 12 kb of the Th2 cytokine LCR positions 132629263 to 132642195. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding contiguous portion of the Th2 cytokine LCR positions 132629263 to 132642195.

[0176] In various embodiments, the Ad35 vector may contain the Th2 cytokine LCR provided herein in a payload comprising, for example, the Th2 cytokine LCR and, optionally, a promoter of a gene typically operably ligated to the Th2 cytokine LCR in the human genome. In various embodiments, the gene operably ligated to the Th2 cytokine LCR is a Th2 cytokine, for example, IL-4, IL-13, or IL-5. In various embodiments, the Th2 cytokine promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the Th2 cytokine promoter includes, for example, 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb upstream of the first coding nucleotide of the Th2 cytokine in the reference genome, for example, 100 bp, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the corresponding nucleic acid sequence that is immediately upstream.

[0177] In various embodiments, Th2 cytokine LCRs, such as long-chain Th2 cytokine LCRs, induce the expression of operably linked coding sequences in T cells. In various embodiments, the operably linked coding sequences are also operably linked to Th2 cytokine promoters shown herein or otherwise known in the art.

[0178] A CD2 LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with a CD2 LCR, which includes a complete CD2 LCR sequence and / or an expression control fragment thereof. A CD2 LCR includes DNAse hypersensitive sites (HS) which are understood by those skilled in the art to mediate at least a portion of the CD2 LCR's expression-enhancing effect. A CD2 LCR includes hypersensitive sites 1-3. Thus, a CD2 LCR may be a complete CD2 LCR containing all of HS1-HS3, or an expression control fragment thereof containing a subset of hypersensitive sites HS1-HS3.

[0179] Certain embodiments may include the CD2 LCR position NC_000001.11 116769217~116774826 (5,610 bp) of human chromosome 1 or an expression control fragment thereof. In various embodiments, the CD2 LCR may have a length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the entire length of the CD2 LCR position 116769217~116774826. In various embodiments, the CD2 LCR may include at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, or at least 5 kb of CD2 LCR positions 116769217 to 116774826. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having an identity percentage of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the corresponding contiguous portion of CD2 LCR positions 116769217 to 116774826.

[0180] In various embodiments, the Ad35 vector may include the CD2 LCR provided herein in a payload that includes, for example, the CD2 LCR and, optionally, a promoter of a gene typically operably ligated to the CD2 LCR in the human genome. In various embodiments, the gene operably ligated to the CD2 LCR is CD2(1:116,754,429~116,769,228). In various embodiments, the CD2 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the CD2 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upstream of the first coding nucleotide of CD2 in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the first coding nucleotide of a gene coding sequence typically operably linked to the CD2 LCR in the human genome is the first coding nucleotide of CD2 on chromosome 1 - NC_000001.11(116754493).

[0181] In various embodiments, CD2 LCRs, such as long-chain CD2 LCRs, induce the expression of operably ligated coding sequences in T cells. In various embodiments, the operably ligated coding sequences are also operably ligated to CD2 promoters as shown herein or otherwise known in the art.

[0182] S100β LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with an S100β LCR, which includes a complete S100β LCR sequence and / or an expression control fragment thereof. The S100β LCR includes a DNAse-sensitive site (HS) which is understood by those skilled in the art to mediate at least a portion of the S100β LCR's expression-enhancing effect.

[0183] In various embodiments, the Ad35 vector may include the S100β LCR provided herein in a payload that includes, for example, the S100β LCR and, optionally, a promoter of a gene typically operably ligated to the S100β LCR in the human genome. In various embodiments, the gene operably ligated to the S100β LCR is S100β(21:46,598,603~46,605,242, complement). In various embodiments, the S100β promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the S100β promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably ligated to the S100β LCR in the human genome is the first coding nucleotide of S100β (chromosome 21 - NC_000021.9(46602415)).

[0184] In various embodiments, S100β LCRs, such as long-chain S100β LCRs, induce the expression of operably ligated coding sequences in brain astrocytes. In various embodiments, the operably ligated coding sequences are also operably ligated to S100β promoters as shown herein or otherwise known in the art.

[0185] Growth hormone LCRs are exemplary LCRs that enhance the expression of operably linked coding sequences. The expression of coding sequences can be enhanced when operably linked with growth hormone LCRs, which include a complete growth hormone LCR sequence and / or its expression control fragments. Growth hormone LCRs include DNAse hypersensitive sites (HS) that are understood by those skilled in the art to mediate at least a portion of the growth hormone LCR's expression-enhancing effect. Growth hormone LCRs include hypersensitive sites 1-5. Thus, a growth hormone LCR may be a complete growth hormone LCR containing all of HS1-HS5, or its expression control fragments containing a subset of hypersensitive sites HS1-HS5.

[0186] Certain embodiments may include the growth hormone LCR position NC_000017.11 (63917193~63958852) (41,660 bp) of human chromosome 17 or a regulatory fragment thereof. In various embodiments, the growth hormone LCR may have a total length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total length of the growth hormone LCR position 63917193~63958852. In various embodiments, the growth hormone LCR may include at least 10kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, or at least 30kb of the growth hormone LCR position 63917193 to 63958852. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 7%, at least 98%, or at least 99% identity with the corresponding contiguous portion of growth hormone LCR positions 63917193 to 63958852, or comprising such nucleic acid.

[0187] In various embodiments, the Ad35 vector may contain the growth hormone LCR provided herein in a payload that includes, for example, the growth hormone LCR and, optionally, a promoter of a gene typically operably linked to the growth hormone LCR in the human genome. In various embodiments, the gene operably linked to the growth hormone LCR may be GH1 (growth hormone 1), CSHL1 (chorionic somatomammotropin hormone-like 1), CSH1 (chorionic somatomammotropin hormone 1 (placental lactogen)), GH2 (growth hormone 2), or CSH2 (chorionic somatomammotropin hormone 2). In various embodiments, the GH1, CSHL1, CSH1, GH2, or CSH2 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the GH1, CSHL1, CSH1, GH2, or CSH2 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upstream of the first coding nucleotide of GH1, CSHL1, CSH1, GH2, or CSH2 in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% upstream of the corresponding nucleic acid sequence that is immediately upstream.In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably linked to the growth hormone LCR in the human genome is the first coding nucleotide of the growth hormone (17:63,917,202~63,918,838, complement) at position NC_000017.11 (63918776).

[0188] In various embodiments, growth hormone LCRs, such as long-chain growth hormone LCR, induce the expression of operably coupled coding sequences in the pituitary gland. In various embodiments, the operably coupled coding sequences are also operably coupled to GH1, CSHL1, CSH1, GH2, or CSH2 promoters as shown herein or otherwise known in the art.

[0189] Apolipoprotein B LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with an apolipoprotein B LCR, which includes a complete apolipoprotein B LCR sequence and / or an expression regulatory fragment thereof. The apolipoprotein B LCR includes a DNAse-sensitive site (HS) which is understood by those skilled in the art to mediate at least a portion of the apolipoprotein B LCR's expression-enhancing effect.

[0190] In various embodiments, the Ad35 vector may include the apolipoprotein B LCR provided herein in a payload that includes, for example, the apolipoprotein B LCR and, optionally, a promoter of a gene typically operably ligated to the apolipoprotein B LCR in the human genome. In various embodiments, the gene operably ligated to the apolipoprotein B LCR is APOB(2:21,001,428~21,044,072, complement). In various embodiments, the APOB promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the APOB promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb upstream of the first coding nucleotide of APOB in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably linked to apolipoprotein B LCR in the human genome is the first coding nucleotide of APOB at position 2 - NC_000002.12(21043945).

[0191] In various embodiments, apolipoprotein B LCRs, such as long-chain apolipoprotein B LCRs, induce the expression of operably ligated coding sequences in the intestine and / or liver. In various embodiments, the operably ligated coding sequences are also operably ligated to APOB promoters, as shown herein or otherwise known in the art.

[0192] A β-myosin heavy chain (LCR) is an exemplary LCR that enhances the expression of an operably linked coding sequence. The expression of a coding sequence can be enhanced when operably linked with a β-myosin heavy chain (LCR) containing a complete β-myosin heavy chain (LCR) sequence and / or an expression control fragment thereof. A β-myosin heavy chain (LCR) contains DNAse hypersensitive sites (HS) which are understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the β-myosin heavy chain (LCR). A β-myosin heavy chain (LCR) contains hypersensitive sites 1 and 2. Thus, a β-myosin heavy chain (LCR) may be a complete β-myosin heavy chain (LCR) containing both HS1 and HS2, or an expression control fragment thereof containing a subset of the hypersensitive sites (HS1 or HS2).

[0193] In various embodiments, the Ad35 vector may include the β-myosin heavy chain LCR provided herein in a payload that includes, for example, a β-myosin heavy chain LCR and, optionally, a promoter of a gene typically operably ligated to the β-myosin heavy chain LCR in the human genome. In various embodiments, the gene operably ligated to the β-myosin heavy chain LCR is the β-myosin heavy chain (14:23,412,739~23,435,676, complement). In various embodiments, the β-myosin heavy chain promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the β-myosin heavy chain promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of a gene coding sequence typically operably linked to the β-myosin heavy chain LCR in the human genome is the first coding nucleotide of the β-myosin heavy chain on chromosome 14 - NC_000014.9(23433732).

[0194] In various embodiments, β - myosin heavy chain LCRs, such as the long - chain β - myosin heavy chain LCR, cause the expression of an operably linked coding sequence in cardiac muscle and / or skeletal muscle. In various embodiments, the operably linked coding sequence is also operably linked to a β - myosin heavy chain promoter shown herein or otherwise known in the art.

[0195] The MHC class I HLA - B7 LCR is an exemplary LCR that enhances the expression of an operably linked coding sequence. The expression of the coding sequence can be enhanced when operably linked to the MHC class I HLA - B7 LCR, including the complete MHC class I HLA - B7 LCR sequence and / or its expression - control fragments. The MHC class I HLA - B7 LCR contains DNAse hypersensitive sites (HS) that are understood by those skilled in the art to mediate at least part of the expression - enhancing effect of the MHC class I HLA - B7 LCR.

[0196] In various embodiments, the Ad35 vector may include the MHC class I HLA-B7 LCR provided herein in a payload that includes, for example, an MHC class I HLA-B7 LCR and, optionally, a promoter of a gene typically operably ligated to the MHC class I HLA-B7 LCR in the human genome. In various embodiments, the gene operably ligated to the MHC class I HLA-B7 LCR is MHC class I HLA-B7. In various embodiments, the MHC class I HLA-B7 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the MHC class I HLA-B7 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, or at least 5.0 kb, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, where the identity percentage with the corresponding nucleic acid sequence that is immediately upstream of the first coding nucleotide of MHC class I HLA-B7 in the reference genome is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0197] In various embodiments, MHC class I HLA-B7 LCRs, such as the long-chain MHC class I HLA-B7 LCR, cause the expression of operably linked coding sequences in or ubiquitous in many cell types. In various embodiments, the operably linked coding sequences are also operably linked to an MHC class I HLA-B7 promoter shown herein or otherwise known in the art.

[0198] The MHC class I HLA-G LCR is an exemplary LCR that enhances the expression of operably linked coding sequences. The expression of the coding sequences can be enhanced when operably linked to an MHC class I HLA-G LCR, including the full MHC class I HLA-G LCR sequence and / or including its expression control fragments. The MHC class I HLA-G LCR contains DNase hypersensitive sites (HS) that are understood by those skilled in the art to mediate at least part of the expression enhancing effect of the MHC class I HLA-G LCR.

[0199] In various embodiments, the Ad35 vector may include, for example, an MHC class I HLA-G LCR provided herein in a payload that includes a promoter of a gene typically operably ligated to the MHC class I HLA-G LCR in the human genome, and optionally the MHC class I HLA-G LCR. In various embodiments, the gene operably ligated to the MHC class I HLA-G LCR is MHC class I HLA-G. In various embodiments, the MHC class I HLA-G promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the MHC class I HLA-G promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb upstream of the first coding nucleotide of MHC class I HLA-G in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream.

[0200] In various embodiments, MHC class I HLA-G LCRs, such as long-chain MHC class I HLA-G LCRs, induce the expression of operably ligated coding sequences in many cell types or ubiquitously. In various embodiments, the operably ligated coding sequences are also operably ligated to MHC class I HLA-G promoters as shown herein or otherwise known in the art.

[0201] Keratin 18 LCRs are exemplary LCRs that enhance the expression of operably ligated coding sequences. The expression of coding sequences can be enhanced when operably ligated with keratin 18 LCRs, including a complete keratin 18 LCR sequence and / or its expression control fragments. Keratin 18 LCRs include DNAse hypersensitive sites (HSs), which are understood by those skilled in the art to mediate at least a portion of the keratin 18 LCR's expression-enhancing effect. Keratin 18 LCRs include hypersensitive sites 1-4. Therefore, a keratin 18 LCR may be a complete keratin 18 LCR containing all of HS1-HS4, or its expression control fragments containing a subset of the hypersensitive sites HS1-HS4.

[0202] Certain embodiments may include keratin 18 LCR position NC_000012.12 (52948039~52956706) (8,668 bp) of human chromosome 12 or an expression control fragment thereof. In various embodiments, the keratin 18 LCR may have a total length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total length of keratin 18 LCR position 52948039~52956706. In various embodiments, the keratin 18 LCR may include at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, or at least 8 kb of keratin 18 LCR positions 52948039 to 52956706. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding contiguous portion of keratin 18 LCR positions 52948039 to 52956706, or may include such nucleic acid.

[0203] In various embodiments, the Ad35 vector may include the keratin 18 LCR provided herein in a payload that includes, for example, the keratin 18 LCR and, optionally, a promoter of a gene typically operably ligated to the keratin 18 LCR in the human genome. In various embodiments, the gene operably ligated to the keratin 18 LCR is keratin 18 (12:52,948,870~52,952,905). In various embodiments, the keratin 18 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the keratin 18 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of a gene coding sequence typically operably linked to the keratin 18 LCR in the human genome is the first coding nucleotide of keratin 18 on chromosome 12 - NC_000012.12(52949174).

[0204] In various embodiments, keratin 18 LCRs, such as long-chain keratin 18 LCRs, induce the expression of operably linked coding sequences in epithelial cells. In various embodiments, the operably linked coding sequences are also operably linked to keratin 18 promoters as shown herein or otherwise known in the art.

[0205] The complement component C4A / B LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with a complement component C4A / B LCR, which includes a complete complement component C4A / B LCR sequence and / or its expression control fragment. The complement component C4A / B LCR includes DNAse-sensitive sites (HS) that are understood by those skilled in the art to mediate at least a portion of the complement component C4A / B LCR's expression-enhancing effect.

[0206] In various embodiments, the Ad35 vector may include the complement component C4A / B LCR provided herein in a payload that includes, for example, the complement component C4A / B LCR and, optionally, a promoter of a gene typically operably ligated to the complement component C4A / B LCR in the human genome. In various embodiments, the gene operably ligated to the complement component C4A / B LCR is C4A(6:31,982,056~32,002,680). In various embodiments, the C4A promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the C4A promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of a gene coding sequence typically operably ligated with complement component C4A / B LCR in the human genome is the first coding nucleotide of C4A on chromosome 6 - NC_000006.12(31982108).

[0207] In various embodiments, complement components C4A / B LCRs, such as long-chain complement component C4A / B LCRs, induce the expression of operably ligated coding sequences in the liver. In various embodiments, the operably ligated coding sequences are also operably ligated to C4A promoters as shown herein or otherwise known in the art.

[0208] Red-green opsin (LCR) LCRs are exemplary LCRs that enhance the expression of operably ligated coding sequences. The expression of coding sequences can be enhanced when operably ligated with red-green opsin (LCR) LCRs, which include a complete red-green opsin (LCR) sequence and / or its expression control fragments. Red-green opsin (LCR) LCRs include DNAse hypersensitive sites (HS) which are understood by those skilled in the art to mediate at least a portion of the red-green opsin (LCR) expression-enhancing effect. Red-green opsin (LCR) LCRs include hypersensitive sites 1-3. Thus, a red-green opsin (LCR) LCR may be a complete red-green opsin (LCR) containing all of HS1-HS3, or its expression control fragments containing a subset of hypersensitive sites HS1-HS3.

[0209] Certain embodiments may include the human X chromosome red-green opsin (LCR) position NC_000023.11 (154137727~154144286) (6,560 bp) or a regulatory fragment of its expression. In various embodiments, the red-green opsin (LCR) can have a total length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total length of the red-green opsin (LCR) LCR positions 154137727 to 154144286. In various embodiments, the red-green opsin LCR may include at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, or at least 6 kb of the red-green opsin LCR positions 154137727 to 154144286. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding contiguous portion of the red-green opsin LCR positions 154137727 to 154144286, or may include such nucleic acid.

[0210] In various embodiments, the Ad35 vector may include, for example, the red-green opsin (LCR) provided herein in a payload that includes the LCR and, optionally, a promoter of a gene typically operably linked to the LCR in the human genome. In various embodiments, the gene operably linked to the LCR is opsin 1 (X:154,144,242~154,159,031), long-wavelength sensitive (OPN1LW), opsin 1, medium-wavelength sensitive (OPN1MW), OPN1MW2, or OPN1MW3. In various embodiments, the OPN1LW, OPN1MW, OPN1MW2, or OPN1MW3 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the OPN1LW, OPN1MW, OPN1MW2, or OPN1MW3 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb upstream of the first coding nucleotide of OPN1LW, OPN1MW, OPN1MW2, or OPN1MW3 in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream.In some embodiments, the first coding nucleotide of a gene coding sequence typically operably linked to the red-green opsin (LCR) in the human genome is either the first coding nucleotide of OPN1LW on the X chromosome - NC_000023.11(154144284) or the first coding nucleotide of OPN1MW on the X chromosome - NC_000023.11(154182678).

[0211] In various embodiments, red-green opsin (opsin) LCRs, such as long-chain red-green opsin (LCRs), induce the expression of operably linked coding sequences in cone photoreceptors. In various embodiments, the operably linked coding sequences are also operably linked to the OPN1LW, OPN1MW, OPN1MW2, or OPN1MW3 promoters shown herein or otherwise known in the art.

[0212] α-globin LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with an α-globin LCR, which includes a complete α-globin LCR sequence and / or an expression control fragment thereof. The α-globin LCR includes DNAse hypersensitive sites (HS) which are understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the α-globin LCR. The α-globin LCR includes hypersensitive sites MCS-R1 to MCS-R4. Thus, the α-globin LCR may be a complete α-globin LCR including all of MCS-R1 to MCS-R4, or an expression control fragment thereof including a subset of the hypersensitive sites MCS-R1 to MCS-R4.

[0213] Certain embodiments may include the α-globin LCR position NC_000016.10 (87808~152854) (65,047 bp) of human chromosome 16 or an expression control fragment thereof. In various embodiments, the α-globin LCR may have a total length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total length of the α-globin LCR position 87808~152854. In various embodiments, the α-globin LCR may include at least 10kb, at least 15kb, at least 16kb, at least 17kb, at least 18kb, at least 19kb, at least 20kb, at least 21kb, at least 22kb, at least 23kb, at least 24kb, at least 25kb, at least 26kb, at least 27kb, at least 28kb, at least 29kb, or at least 30kb of the α-globin LCR at positions 87808 to 152854. In any of the various embodiments provided herein, the long-chain LCR is a nucleic acid having an identity percentage of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the corresponding contiguous portion of α-globin LCR positions 87808 to 152854, or may include such nucleic acid.

[0214] In various embodiments, the Ad35 vector may include, for example, the α-globin LCR provided herein in a payload comprising the α-globin LCR and, optionally, a promoter of a gene typically operably linked to the α-globin LCR in the human genome. In various embodiments, the gene operably linked to the α-globin LCR is HBZ (hemoglobin, zeta), HBA2 (hemoglobin, alpha 2), HBA1 (hemoglobin, alpha 1), or HBQ1 (hemoglobin, theta 1) within the alpha-globin gene cluster (major α-globin loci: 16:172,875-173,709). In various embodiments, the HBZ (hemoglobin, zeta), HBA2 (hemoglobin, alpha-2), HBA1 (hemoglobin, alpha-1), or HBQ1 (hemoglobin, theta-1) promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb.In various embodiments, the HBZ (hemoglobin, zeta), HBA2 (hemoglobin, alpha-2), HBA1 (hemoglobin, alpha-1), or HBQ1 (hemoglobin, theta-1) promoter has, for example, a 70%, 75%, or less identity percentage with the corresponding nucleic acid sequence that is immediately upstream of the first coding nucleotide of HBZ (hemoglobin, zeta), HBA2 (hemoglobin, alpha-2), HBA1 (hemoglobin, alpha-1), or HBQ1 (hemoglobin, theta-1) in the reference genome. At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the data consists of at least 100bp, at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 1.0kb, at least 1.5kb, at least 2.0kb, at least 2.5kb, at least 3.0kb, at least 4.0kb, or at least 5.0kb. In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably linked to α-globin LCR in the human genome is HBA1, the first coding nucleotide of chromosome 16 - NC_000016.10(176717), HBA2, the first coding nucleotide of chromosome 16 - NC_000016.10(172913), HBZ, the first coding nucleotide of chromosome 16 - NC_000016.10(152910), or HBQ1, the first coding nucleotide of chromosome 16 - NC_000016.10(180487).

[0215] In various embodiments, α-globin LCRs, such as long-chain α-globin LCRs, induce the expression of operably linked coding sequences in erythrocytes. In various embodiments, the operably linked coding sequences are also operably linked to promoters shown herein or otherwise known in the art.

[0216] Desmin LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with a desmin LCR, which includes a complete desmin LCR sequence and / or an expression control fragment thereof. The desmin LCR includes DNAse hypersensitive sites (HS) which are understood by those skilled in the art to mediate at least a portion of the desmin LCR's expression-enhancing effect. The desmin LCR includes hypersensitive sites 1-5. Thus, the desmin LCR may be a complete desmin LCR containing all of HS1-HS5, or an expression control fragment thereof containing a subset of hypersensitive sites HS1-HS5.

[0217] Certain embodiments may include the desmin LCR position NC_000002.12 (219399709~219418452) (18,743 bp) of human chromosome 2 or its expression control fragment. In various embodiments, the desmin LCR may have a length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total length of the desmin LCR position 219399709~219418452. In various embodiments, the desmin LCR may include at least 10kb, at least 15kb, at least 16kb, at least 17kb, or at least 18kb of the desmin LCR positions 219399709 to 219418452. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding contiguous portion of the desmin LCR positions 219399709 to 219418452, or may include such nucleic acid.

[0218] In various embodiments, the Ad35 vector can include, for example, the desmin LCR, and, optionally, a payload comprising a promoter of a gene typically operably linked to the desmin LCR in the human genome, including the desmin LCR provided herein. In various embodiments, the gene operably linked to the desmin LCR is desmin (2:219,418,376 - 219,426,733). In various embodiments, the desmin promoter can have a full length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the desmin promoter has, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a corresponding nucleic acid sequence that is upstream of the first coding nucleotide of desmin in a reference genome, for example, immediately upstream, and includes at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb. In some embodiments, the first coding nucleotide of the coding sequence of the gene typically operably linked to the desmin LCR in the human genome is the first coding nucleotide of desmin on chromosome 2 - NC_000002.12(21941863).

[0219] In various embodiments, desmin LCRs, such as long-chain desmin LCRs, induce the expression of operably coupled coding sequences in cardiac muscle, skeletal muscle, and / or smooth muscle. In various embodiments, the operably coupled coding sequences are also operably coupled to desmin promoters as shown herein or otherwise known in the art.

[0220] The nuclear factor, erythroid 2-like 1 (NFE2L1) LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with an NFE2L1 LCR containing a complete NFE2L1 LCR sequence and / or its expression regulatory fragment. The NFE2L1 LCR contains a DNAse-sensitive site (HS) which is understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the NFE2L1 LCR.

[0221] Certain embodiments may include the NFE2L1 LCR position NC_000017.11 (48048359~48061545) (13,186 bp) of human chromosome 17 or an expression control fragment thereof. In various embodiments, the NFE2L1 LCR may have a total length equal to or greater than 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the total length of the NFE2L1 LCR position 48048359~48061545. In various embodiments, the NFE2L1 LCR may include at least 10kb, at least 11kb, at least 12kb, or at least 13kb of the NFE2L1 LCR positions 48048359 to 48061545. In any of the various embodiments provided herein, the long-chain LCR may be a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding contiguous portion of the NFE2L1 LCR positions 48048359 to 48061545.

[0222] In various embodiments, the Ad35 vector may include the NFE2L1 LCR provided herein in a payload that includes, for example, the NFE2L1 LCR and, optionally, a promoter of a gene typically operably ligated to the NFE2L1 LCR in the human genome. In various embodiments, the gene operably ligated to the NFE2L1 LCR is NFE2L1(17:48,048,358~48,061,544). In various embodiments, the NFE2L1 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the NFE2L1 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably ligated with the NFE2L1 LCR in the human genome is the first coding nucleotide of NFE2L1 on chromosome 17 - NC_000017.11(48051119).

[0223] In various embodiments, NFE2L1 LCRs, such as long-chain NFE2L1 LCRs, induce the expression of operably ligated coding sequences in erythrocytes. In various embodiments, the operably ligated coding sequences are also operably ligated to NFE2L1 promoters as shown herein or otherwise known in the art.

[0224] A CD4 LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with a CD4 LCR, which includes a complete CD4 LCR sequence and / or an expression control fragment thereof. A CD4 LCR includes DNAse hypersensitive sites (HSs) that are understood by those skilled in the art to mediate at least a portion of the CD4 LCR's expression-enhancing effect. A CD4 LCR contains up to 17 hypersensitive sites DH1–DH17. Thus, a CD4 LCR may be a complete CD4 LCR containing all of DH1–DH17, or an expression control fragment thereof containing a subset of the hypersensitive sites DH1–DH17.

[0225] In various embodiments, the Ad35 vector may include the CD4 LCR provided herein in a payload that includes, for example, the CD4 LCR and, optionally, a promoter of a gene typically operably ligated to the CD4 LCR in the human genome. In various embodiments, the gene operably ligated to the CD4 LCR is CD4(12:6,789,527~6,820,809). In various embodiments, the CD4 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the CD4 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of a gene coding sequence typically operably linked to the CD4 LCR in the human genome is the first coding nucleotide of CD4 on chromosome 12 - NC_000012.12(6800139).

[0226] In various embodiments, CD4 LCRs, such as long-chain CD4 LCRs, induce the expression of operably ligated coding sequences in CD4+ T cells. In various embodiments, the operably ligated coding sequences are also operably ligated to CD4 promoters as shown herein or otherwise known in the art.

[0227] α-Lactalbumin LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with an α-Lactalbumin LCR comprising a complete α-Lactalbumin LCR sequence and / or an expression regulatory fragment thereof. The α-Lactalbumin LCR comprises a DNAse-sensitive site (HS) which is understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the α-Lactalbumin LCR.

[0228] In various embodiments, the Ad35 vector may include, for example, the α-lactalbumin LCR provided herein in a payload that includes the α-lactalbumin LCR and, optionally, a promoter of a gene typically operably ligated to the α-lactalbumin LCR in the human genome. In various embodiments, the gene operably ligated to the α-lactalbumin LCR is α-lactalbumin (12:48,567,683~48,571,882). In various embodiments, the α-lactalbumin promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the α-lactalbumin promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream.In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably linked to α-lactalbumin LCR in the human genome is the first coding nucleotide of α-lactalbumin on chromosome 12 - NC_000012.12(48570020).

[0229] In various embodiments, α-lactalbumin LCRs, such as long-chain α-lactalbumin LCRs, induce the expression of operably linked coding sequences in the mammary gland. In various embodiments, the operably linked coding sequences are also operably linked to α-lactalbumin promoters, either those shown herein or otherwise known in the art.

[0230] The CYP19 / aromatase LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of a coding sequence can be enhanced when operably ligated with a CYP19 / aromatase LCR comprising a complete CYP19 / aromatase LCR sequence and / or an expression control fragment thereof. The CYP19 / aromatase LCR comprises a DNAse-sensitive site (HS) which is understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the CYP19 / aromatase LCR.

[0231] In various embodiments, the Ad35 vector may include the CYP19 / aromatase LCR provided herein in a payload that includes, for example, the CYP19 / aromatase LCR and, optionally, a promoter of a gene typically operably ligated to the CYP19 / aromatase LCR in the human genome. In various embodiments, the gene operably ligated to the CYP19 / aromatase LCR is CYP19A1 (15:51,208,056~51,338,595). In various embodiments, the CYP19A1 promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the CYP19A1 promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream.In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably ligated with CYP19 / aromatase LCR in the human genome is the first coding nucleotide of CYP19A1 on chromosome 15 - NC_000015.10(51242912).

[0232] In various embodiments, CYP19 / aromatase LCRs, such as long-chain CYP19 / aromatase LCRs, induce the expression of operably linked coding sequences in multiple different tissues. In various embodiments, the operably linked coding sequences are also operably linked to CYP19A1 promoters as shown herein or otherwise known in the art.

[0233] The C-fes proto-oncogene LCR is an exemplary LCR that enhances the expression of an operably ligated coding sequence. The expression of the coding sequence can be enhanced when operably ligated with a C-fes proto-oncogene LCR that includes a complete C-fes proto-oncogene LCR sequence and / or its expression regulatory fragment. The C-fes proto-oncogene LCR includes DNAse-sensitive sites (HS) that are understood by those skilled in the art to mediate at least a portion of the expression-enhancing effect of the C-fes proto-oncogene LCR.

[0234] In various embodiments, the Ad35 vector may include the C-fes proto-oncogene LCR provided herein in a payload that includes, for example, the C-fes proto-oncogene LCR and, optionally, a promoter of a gene typically operably ligated to the C-fes proto-oncogene LCR in the human genome. In various embodiments, the gene operably ligated to the C-fes proto-oncogene LCR is FES(15:90,884,420~90,895,775). In various embodiments, the FES promoter may have a total length equal to or greater than 100 bp, equal to or greater than 200 bp, equal to or greater than 300 bp, equal to or greater than 400 bp, equal to or greater than 500 bp, equal to or greater than 1.0 kb, equal to or greater than 1.5 kb, equal to or greater than 2.0 kb, equal to or greater than 2.5 kb, equal to or greater than 3.0 kb, equal to or greater than 4.0 kb, or equal to or greater than 5.0 kb. In various embodiments, the FES promoter includes, for example, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 1.0 kb, at least 1.5 kb, at least 2.0 kb, at least 2.5 kb, at least 3.0 kb, at least 4.0 kb, or at least 5.0 kb upstream of the first coding nucleotide of the FES in the reference genome, for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the corresponding nucleic acid sequence that is immediately upstream. In some embodiments, the first coding nucleotide of the coding sequence of a gene typically operably ligated with the C-fes proto-oncogene LCR in the human genome is the first coding nucleotide of FES on chromosome 15 - NC_000015.10(90885046).

[0235] In various embodiments, C-fes oncogenes, such as long-chain C-fes proto-oncogenes (LCRs), induce the expression of operably ligated coding sequences in myeloid cells, including macrophages and neutrophils. In various embodiments, the operably ligated coding sequences are also operably ligated to FES promoters, either those shown herein or otherwise known in the art.

[0236] (IV) Code sequences operably coupled with long chain LCRs

[0237] (IV-b) Protein therapy, e.g., protein / enzyme replacement therapy

[0238] In certain embodiments, the coding sequence operably linked to the long LCR includes a transgene encoding a therapeutic protein. The coding sequence refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) encoding one or more therapeutic proteins described herein. This definition includes various sequence polymorphisms, mutations, and / or sequence variants, such modifications not substantially affecting the function of the encoded one or more therapeutic proteins. The coding sequence or “gene” may include not only the coding sequence but also regulatory regions such as promoters, enhancers, and termination regions. The term may further include introns and all other DNA sequences spliced ​​from mRNA transcripts, along with variants arising from alternative splice sites. A gene sequence encoding a molecule may be DNA or RNA that directs the expression of one or more therapeutic proteins. These nucleic acid sequences may be DNA strand sequences transcribed to RNA, or RNA sequences translated to proteins. Nucleic acid sequences include both full-length nucleic acid sequences and incomplete-length sequences derived from full-length proteins. The sequence may also include degenerate codons of native sequences (one or more) that can be introduced to result in codon preference in specific cell types.

[0239] Gene sequences encoding one or more therapeutic proteins can be readily prepared from relevant amino acid sequences by synthetic or recombination methods. In certain embodiments, the gene sequences encoding any of these sequences may have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence to allow for easy excision of the sequence-encoding gene sequence and substitution with another gene sequence encoding a different sequence. In certain embodiments, the sequence-encoding gene sequence may have codons optimized for expression in mammalian cells. The coding sequences of therapeutic proteins are referred to herein as therapeutic genes.

[0240] Therapeutic genes may be selected to produce a therapeutically effective response to a condition (in certain embodiments, a hereditary condition). In certain embodiments, such conditions may 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), Wiscott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), Fanconi anemia (FA), Batten disease, adrenoleukodystrophy (ALD) or metachromatic leukodystrophy (MLD), muscular dystrophy, alveolar proteinosis (PAP), pyruvate kinase deficiency, Schwachmann-Diamond-Blackfan anemia, congenital keratosis, cystic fibrosis, Parkinson's disease, Alzheimer's disease, or amyotrophic lateral sclerosis (Lou Gehrig's disease). In certain embodiments, depending on the state, the therapeutic gene may be a gene encoding a protein whose function is impaired and / or a gene whose function is impaired.

[0241] Examples of therapeutic genes and gene products include antibodies against CD4, CD5, CD7, and CD52; antibodies against IL1, IL2, and IL6; antibodies against TCRs specifically present on autoreactive T cells; IL4; IL10; IL12; IL13; IL1Ra; sIL1RI; sIL1RII; antibodies against TNF; ABCA3; ABCD1; ADA; AK2; APP; arginase; arylsulfatase A; A1AT; CD3D; CD3E; CD3G; CD3Z; CFTR; CHD7; chimeric antigen receptor (CAR); CIITA; CLN3; complement factor, 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), FancP (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; HB A1;HBA2;HBB;IL7RA;JAK3;LCK;LIG4;LRRK2;NHEJ1;NLX2.1;neutralizing antibody;ORAI1;PARK2;PARK7;phox;PINK1;PNP;PRKDC;PSEN1;PSEN2;PTPN22;PTPRC;P53;pyruvate kinase;RAG1;RAG2;RFXANK;RFXAP;RFX5;RMRP;ribosomal protein gene;SFTPB;SFTPC;SOD1;soluble CD40;STIM1;sTNFRI;sTNFRII;SLC46A1;SNCA;TDP43;TERT;TERC;TINF2;ubiquilin2;WAS;WHN;ZAP70;γC;and other therapeutic genes described herein.

[0242] A therapeutically effective dose may confer function to immune cells and other blood cells and / or microglial cells, or, alternatively, depending on the condition being treated, suppress lymphocyte activation, induce lymphocyte apoptosis, eliminate various lymphocyte subsets, suppress T cell activation, eliminate or suppress autoreactive T cells, suppress Th-2 or Th-1 lymphocyte activity, antagonistize IL-1 or TNF, reduce inflammation, induce selective tolerance to provocatives, reduce or eliminate immune-mediated conditions, and / or reduce or eliminate the symptoms of immune-mediated conditions. A therapeutically effective dose may confer functional DNA repair mechanisms, express surfactant proteins, maintain telomeres, confer lysosomal function, cause degradation of lipids or other proteins (such as amyloid), enable ribosome function, and / or enable the development of mature blood cell lineages (such as macrophages and other leukocyte types) that would not otherwise occur without a therapeutically effective dose.

[0243] As another example, therapeutic genes may be selected to produce a therapeutically effective response to diseases related to red blood cells and coagulation. In a particular embodiment, the disease is a hemoglobin disorder such as thalassemia or sickle cell anemia / phenotype. The therapeutic gene may be, for example, a gene that induces or increases hemoglobin production, a gene that induces or increases the production of β-globin, γ-globin, or α-globin, or a gene that improves oxygen availability to cells in the body. The therapeutic gene may be, for example, HBB or CYB5R3. Examples of effective treatments may be, for example, those that increase the number of blood cells, improve blood cell function, or increase cellular oxygenation in the patient. In another particular embodiment, the disease is hemophilia. Therapeutic genes may include, for example, genes that increase the production of coagulation factor VIII or coagulation factor IX, genes that induce the production of normal versions of coagulation factor VIII or coagulation factor IX, genes that reduce the production of antibodies against coagulation factor VIII or coagulation factor IX, or genes that induce proper blood clot formation. Examples of therapeutic genes include F8 and F9. Examples of effective treatments may include, for example, those that increase or induce the production of coagulation factor VIII and coagulation factor IX in the subject, those that improve the function of coagulation factor VIII and coagulation factor IX, or those that reduce clotting time.

[0244] The following references provide specific exemplary sequences of functional globin genes. References 1-4 concern α-globin sequences, and references 4-12 concern β-globin sequences (including β and γ-globin sequences): (1) GenBank access number Z84721 (March 19, 1997); (2) GenBank access number NM_000517 (October 31, 2000); (3) Hardison et al., J. Mol. Biol. 222(2):233-249, 1991; (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 access number J00179 (August 26, 1993); (6) Tagle et al., Genomics 13(3):741-760, 1992;(7)Grovsfeld et al., Cell 51(6):975-985, 1987;(8)Li et al., Blood 93(7):2208-2216, 1999;(9)Gorman et al., J. Biol. Chem .275(46):35914-35919, 2000;(10)Slightom et al., Cell 21(3):627-638, 1980;(11)Fritsch et al., Cell 19(4): 959-972, 1980;(12)Marotta et al., J. Biol. Chem. 252(14):5040-5053, 1977. For additional information on the coding and non-coding regions of genes encoding globin, see, for example, 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.

[0245] An example of the amino acid sequence of the hemoglobin β-subunit is provided, for example, under NCBI access number P68871. An example of the amino acid sequence of β-globin is provided, for example, under NCBI access number NP_000509.

[0246] As another example, therapeutic genes may be selected to produce a therapeutically effective response to lysosomal storage disorders. In certain embodiments, lysosomal storage disorders include mucopolysaccharidosis (MPS) type I, MPS type II (i.e., Hunter syndrome), MPS type III (i.e., Sanfilippo syndrome), MPS type IV (i.e., Morquio syndrome), MPS type V, MPS type VI (i.e., Maloto-Lamy syndrome), MPS type VII (i.e., Sly syndrome), α-mannosidosis, β-mannosidosis, glycogen storage disease type I (GSDI, also known as von Gierke disease or Tay-Sachs disease), Pompe disease, Gaucher disease, and Fabry disease. Therapeutic genes may be, for example, genes that encode or induce the production of enzymes or otherwise cause the degradation of mucopolysaccharides in lysosomes. Examples of therapeutic genes include IDUA (i.e., idulonidase), IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, and HYAL1. Examples of gene therapies effective for lysosomal storage disorders may include those that encode or induce the production of enzymes responsible for the degradation of various substances in lysosomes; those that reduce, eliminate, inhibit, or delay swelling of various organs (including the head (macrocephaly), liver, spleen, tongue, or vocal cords); those that reduce fluid in the brain; those that reduce heart valve abnormalities; those that block or dilate airway stenosis and prevent related upper airway conditions such as infections and sleep apnea; and those that reduce, eliminate, inhibit, or delay neuronal destruction and / or related symptoms.

[0247] As another example, therapeutic genes may be selected to produce a therapeutically effective response to a hyperproliferative disorder. In certain embodiments, the hyperproliferative disorder is cancer. The therapeutic gene may be, for example, a tumor suppressor gene, an apoptosis-inducing gene, an enzyme-coding gene, an antibody-coding gene, or a hormone-coding gene. Examples of therapeutic genes and gene products (in addition to those described elsewhere in this specification) 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, Examples 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, thrombospongin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, and zac1.Effective genetic therapies may include those that suppress or eliminate tumors, reduce the number of cancer cells, reduce tumor size, slow or eliminate tumor growth, or alleviate symptoms caused by the tumor.

[0248] As another example, therapeutic genes may be selected to produce a therapeutically effective response to an infectious disease. In a particular embodiment, the infectious disease is human immunodeficiency virus (HIV). Therapeutic genes may be, for example, genes that make immune cells resistant to HIV infection or genes that enable immune cells to efficiently neutralize the virus through immune reconstitution, polymorphisms of genes encoding proteins expressed by immune cells, genes that are not expressed in the patient but are advantageous in fighting infection, infectious pathogens, genes encoding receptors or co-receptors, genes encoding ligands for receptors or co-receptors, viral and cellular genes essential for viral replication (including genes encoding ribozymes, antisense RNA, small interfering RNA (siRNA), or decoy RNA to block the action of certain transcription factors), genes encoding dominant-negative viral proteins, genes encoding intracellular antibodies, genes encoding intrakines, and genes encoding suicide genes. Examples of therapeutic genes and therapeutic 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 receptors. A therapeutically effective dose for HIV treatment may be, for example, one that enhances the target's immunity to HIV, alleviates symptoms of AIDS or HIV-related symptoms, or induces an innate or adaptive immune response to HIV in the target. An immune response to HIV may include antibody production, which may block AIDS and / or alleviate the symptoms of an AIDS or HIV infection in a subject, or reduce or eliminate the infectivity and / or pathogenicity of HIV.

[0249] (IV-c) antibody, CAR and TCR

[0250] In addition to therapeutic genes and / or gene products, coding sequences may also encode therapeutic molecules such as antibodies, chimeric antigen receptor molecules specific to one or more cancer antigens, and / or T cell receptors specific to one or more cancer antigens.

[0251] Significant progress has been made in genetically engineering immune system T cells to target and kill undesirable cell types (such as cancer cells). Many of these T cells are genetically engineered to express chimeric antigen receptor (CAR) constructs. CARs are proteins containing several different dependent components that enable genetically modified T cells to recognize and kill cancer cells. These dependent components include at least extracellular and intracellular elements.

[0252] The extracellular element contains a binding domain that specifically binds to markers selectively present on the surface of undesirable cells. When the binding domain binds to such a marker, the intracellular element directs T cells to destroy the bound cancer cells. The binding domain is typically a single-stranded variable fragment (scFv) derived from a monoclonal antibody (mAb), but can also be based on other forms including antibody-like antigen-binding sites.

[0253] Intracellular elements provide activation signals based on the presence of effector domains. In first-generation CARs, the cytoplasm of CD3ζ was used as the effector domain. In second-generation CARs, CD3ζ was used in combination with surface antigen classification 28 (CD28) or 4-1BB (CD137), and in third-generation CARs, CD3ζ was used in combination with CD28 and 4-1BB within the intracellular effector domain.

[0254] CARs generally also contain one or more linker sequences within the molecule, which are used for various purposes. For example, a transmembrane domain may be used to link the extracellular element of a CAR to an intracellular element. The mobile linker sequence is often referred to as a spacer region, which is located closer to the membrane than the binding domain and can be used to create further distance between the binding domain and the cell membrane. This distance creation may be beneficial in reducing steric hindrance to binding due to proximity to the membrane. Depending on the target cell marker, a more compact or longer spacer may be used. Other potential CAR dependent elements are described in more detail elsewhere in this specification. The components of a CAR are described in further detail below: binding domain; intracellular signaling element; linker; transmembrane domain; junctional amino acids; and control portion including the tag cassette. The description of the binding domain is also relevant to antibodies as therapeutic molecules.

[0255] Binding domain. A binding domain contains any substance that binds to a cell marker and forms a complex. The choice of binding domain may depend on the type and number of cell markers that define the surface of the target cell. Examples of binding domains include cell marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (e.g., T cell receptors), chimeric antigen receptors (CARs), or combinations and manipulated fragments or forms thereof.

[0256] Antibodies are examples of binding domains that specifically bind to cell markers, and include whole antibodies or antibody-binding fragments, such as Fv, Fab, Fab', F(ab')2, and single-chain (sc) forms, as well as fragments thereof. Antibodies or antigen-binding fragments may include all or part of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant antibodies, chimeric antibodies, bispecific antibodies, minibodies, and linear antibodies.

[0257] Antibodies are produced from two genes: a heavy chain gene and a light chain gene. Generally, an antibody contains two identical copies of the heavy chain and two identical copies of the light chain. Within the variable heavy chain and variable light chain, segments called complementarity-determining regions (CDRs) determine epitope binding. Each heavy chain has three CDRs (i.e., CDRH1, CDRH2, and CDRH3), and each light chain has three CDRs (i.e., CDRL1, CDRL2, and CDRL3). The CDR regions are adjacent by framework residues (FRs).

[0258] In some cases, it is beneficial to derive the binding domain from the same species in which it will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain to contain a human antibody, a humanized antibody, or a fragment or engineered form thereof. Antibodies of human origin or humanized antibodies have reduced or no immunogenicity in humans and a smaller number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and their engineered fragments are generally selected to have low or no antigenicity in human subjects.

[0259] In certain embodiments, the binding domain comprises a humanized antibody or an engineered fragment thereof. In certain embodiments, a non-human antibody is humanized, and one or more amino acid residues of such antibody are modified to increase similarity to antibodies or fragments naturally produced in humans. These non-human amino acid residues are often referred to as “implant” residues, and these “implant” residues are typically obtained from “implant” variable domains. As provided herein, a humanized antibody or humanized antibody fragment comprises a framework region in addition to one or more CDRs derived from a non-human immunoglobulin molecule, wherein the amino acid residues comprising such framework are entirely or mostly derived from the human germline. In one embodiment, the antigen-binding domain is humanized.Humanized antibodies can be produced using a variety of techniques known in the art, including CDR transplantation (see, for example, European Patent No. EP239,400, WO91 / 09967, and US5,225,539, US5,530,101, and US5,585,089), veneering, or resurfacing (see, for example, EP592,106 and EP519,596, Padlan, Molecular Immunology, 28(4 / 5):489-498, 1991; Studnicka et al., Protein Engineering, 7(6):805-814, 19944, and Roguska et al.) See, for example, al., PNAS, 91:969-973, 1994), chain shuffling (see, for example, U.S. Patent No. 5,565,332), and, for example, U.S. Patent Application Publication No. 2005 / 0042664, U.S. Patent Application Publication No. 2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, WO9317105, Tan et al., J.Immunol., 169:1119-25, 2002, Caldas et al., Protein Eng., 13(5):353-60, 2000, Morea et al., Methods, 20(3):267-79, 2000, Baca et The techniques disclosed are included in al., J. Biol. Chem., 272(16):10678-84, 1997; Roguska et al., Protein Eng., 9(10):895-904, 1996; Couto et al., Cancer Res., 55(23 Supp):5973s-5977s, 1995; Couto et al., Cancer Res., 55(8):1717-22, 1995; Sandhu, Gene, 150(2):409-10, 1994; and Pedersen et al., J. Mol. Biol., 235(3):959-73, 1994. In many cases, by substituting framework residues in the framework region with corresponding residues derived from CDR donor antibodies, for example, cell marker binding is modified (e.g., improved).These framework substitutions are identified by methods well known in the art, such as modeling the interaction between CDR residues and framework residues to identify framework residues important for cell marker binding, and by performing sequence comparisons to identify framework residues that are not normally present at specific positions (see, for example, U.S. Patent No. 5,585,089 and Riechmann et al., Nature, 332:323, 1988).

[0260] Antibodies having binding domains that specifically bind to cell markers can be prepared using methods for obtaining monoclonal antibodies, phage display methods, methods for generating human antibodies or humanized antibodies, or methods using transgenic animals or plants engineered to produce antibodies, such methods are known to those skilled in the art (see, e.g., US6,291,161 and US6,291,158). For partially or fully synthetic antibodies, phage display libraries are available, and antibodies or fragments capable of binding to cell markers can be screened in these phage display libraries. For example, binding domains can be identified by screening Fab fragments that specifically bind to cell markers in Fab phage libraries (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies are also available. Furthermore, the binding domain can also be developed using conventional hybridoma development strategies that utilize cell markers as immunogens in simpler systems (e.g., mice, HuMAb mice® (GenPharm Int'l. Inc., Mountain View, CA), TC mice® (Kirin Pharma Co. Ltd., Tokyo, JP), KM- mice® (Medarex, Inc., Princeton, NJ), llamas, chickens, rats, hamsters, rabbits, etc.). Once identified, the amino acid sequence of the antibody and the gene sequence encoding the antibody can be isolated and / or determined.

[0261] In some cases, scFv can be prepared according to methods known in the art (see, for example, Bird et al., Science 242:423-426 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). scFv molecules can be produced by linking the VH and VL regions of an antibody together using a mobile polypeptide linker. When a short polypeptide linker is used (e.g., between 5 and 10 amino acids), intrachain folding is prevented. Interchain folding is also required to unite the two variable regions and form a functional epitope binding site. For examples of linker orientation and size, see, for example, Hollinger et al., Proc Natl Acad. Sci. USA 90:6444-6448, 1993, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and WO2006 / 020258 and WO2007 / 024715. More specifically, the linker sequences used to connect the VL and VH of scFv are generally 5 to 35 amino acids long. In certain embodiments, the number of amino acids contained in the VL-VH linker is 5 to 35, 10 to 30, or 15 to 25. Varying the linker length can maintain or enhance activity and may result in superior efficacy in activity testing. scFv is commonly used as the CAR binding domain.

[0262] Additional examples of antibody-based binding domain formats include scFv-based grababody and soluble VH domain antibodies. In these antibodies, the binding domain is formed using only the heavy chain variable region. See, for example, Jespers et al., Nat. Biotechnol. 22:1161, 2004, Cortez-Retamozo et al., Cancer Res. 64:2853, 2004, Baral et al., Nature Med. 12:580, 2006, and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0263] In certain embodiments, the VL region in the binding domain of the Disclosure is derived from or based on the VL of a known monoclonal antibody and contains, compared to the VL of the known monoclonal antibody, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above modifications. The insertions, deletions, or substitutions may be located at any point in the VL region, including the amino or carboxyl terminus or both termini, provided that each CDR contains no modifications, or at most one, at most two, or at most three, and that the binding domain containing the modified VL region is still able to bind specifically to its target with the same affinity as the wild-type binding domain.

[0264] In certain embodiments, the binding domain VH region of the Disclosure may be derived from or based on the VH of a known monoclonal antibody and may contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or a combination of the above modifications compared to the VH of the known monoclonal antibody. The insertions, deletions, or substitutions may be located at any point in the VH region, including the amino or carboxyl terminus or both terminus, provided that each CDR contains no modifications, or at most one, at most two, or at most three, and that the binding domain containing the modified VH region can still specifically bind to its target with the same affinity as the wild-type binding domain.

[0265] In certain embodiments, the binding domain comprises or is a sequence having an amino acid sequence identity percentage of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% with the light chain variable region (VL) or the heavy chain variable region (VH) or both, and each CDR contains no modifications from a monoclonal antibody or a fragment or derivative thereof that specifically binds to the cell marker of interest, or at most one, at most two, or at most three such modifications.

[0266] Alternative sources for the binding domain include sequences encoding random peptide libraries or sequences encoding variously manipulated amino acids in the loop region of alternative non-antibody backbones. These alternative non-antibody backbones include single-chain (sc) T cell receptors (scTCRs) (see, e.g., Lake et al., Int.Immunol.11:745,1999; Maynard et al., J.Immunol.Methods 306:51,2005; US8,361,794), fibrinogen domains (see, e.g., Weisel et al., Science 230:1388,1985), Knitz domains (see, e.g., US6,423,498), and engineered ankyrin repeat proteins (DARPin; Binz et al., J.Mol.Biol.332:489,2003 and Binz et al. al., Nat. Biotechnol. 22:575, 2004), fibronectin-binding domain (adnectin or monobody; Richards et al., J. Mol. Biol. 326:1475, 2003, Parker et al., Protein Eng. Des. Selec. 18:435, 2005, and Hackel et al., J. Mol. Biol. 381:1238-1252, 2008), cystine knot miniprotein (Vita et al., Proc. Nat'l. Acad. Sci. (USA) 92:6404-6408, 1995; Martin et al., Nat. Biotechnol. 21:71, 2002, and Huang et al., Structure 13:755, 2005), tetratricopeptide repeat domain (Main (e.g., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008), leucine-rich repeat domain (Stumpp et al., J. Mol. Biol. 332:471, 2003), lipocalin domain (e.g., WO2006 / 095164, Beste et al., Proc. Nat'l. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et al., Proc. Nat'l.See Acad.Sci.(USA)106:8198,2009), V-like domains (see, for example, US2007 / 0065431), C-type lectin domains (Zelensky & Gready, FEBS J.272:6179,2005, Beavil et al., Proc.Nat'l.Acad.Sci.(USA)89:753,1992, and Sato et al., Proc.Nat'l.Acad.Sci.(USA)100:7779,2003), Fc region with mAb2 or antigen-binding domain (Fcab(trademark)) (F-Star Biotechnology, Cambridge UK, see, for example, WO2007 / 098934 and WO2006 / 072620), armadillo repeat proteins (see, for example, Madhurantakam et al.) See also al., Protein Sci. 21:1015, 2012, WO2009 / 040338), affilin (Ebersbach et al., J. Mol. Biol. 372:172, 2007), affibody, avimer, notchin, fynomer, atrimer, cytotoxic T lymphocyte-associated protein-4 (Weidle et al., Cancer Gen. Proteo. 10:155, 2013), or similar (Nord et al., Protein Eng. 8:601, 1995, Nord et al., Nat. Biotechnol. 15:772, 1997, Nord et al., Euro. J. Biochem. 268:4269, 2001, Binz et al. Examples include al., Nat. Biotechnol. 23:1257, 2005; and Boersma & Pluckthun, Curr. Opin. Biotechnol. 22:849, 2011.

[0267] Peptide aptamers include peptide loops (specific to cell markers) bound to a protein backbone at both ends. This dual structural constraint elevates the binding affinity of peptide aptamers to levels comparable to antibodies. The amino acid length of the variable loop is typically 8–20, and the backbone can be any stable, soluble, small, and non-toxic protein. Peptide aptamer selection can be carried out using various systems (such as yeast two-hybrid systems (e.g., Gal4 yeast two-hybrid systems) or LexA interaction capture systems).

[0268] In certain embodiments, the binding domain is an scT cell receptor (scTCR) containing Vα / β chains and Cα / β chains (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ), or an scTCR containing Vα-Cα, Vβ-Cβ, and Vα-Vβ pairs specific to a cell marker peptide-MHC complex.

[0269] In certain embodiments, the manipulated binding domain comprises a Vα, Vβ, Cα, or Cβ region derived from or based on Vα, Vβ, Cα, or Cβ, and includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or a combination of the above modifications, compared to the referenced Vα, Vβ, Cα, or Cβ. The insertions, deletions, or substitutions include the amino or carboxyl terminus or both of the terminus of the region. L , V H The modified CDR may be present in any of the Vα, Vβ, Cα, or Cβ regions, provided that each CDR contains no changes, or at most one, at most two, or at most three, and that the target-binding domain containing the modified Vα, Vβ, Cα, or Cβ region can still specifically bind to its target with the same affinity and action as the wild type.

[0270] In certain embodiments, the manipulated binding domain comprises a sequence having an amino acid sequence identity percentage of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% with a known or identified binding domain, wherein each CDR contains no modifications from known or identified binding domains or fragments or derivatives thereof that specifically bind to a target cell marker, or contains at most one, at most two, or at most three.

[0271] The precise amino acid sequence boundaries of a given CDR or FR can be easily determined using one of many well-known schemes, including the one described by Kabat et al. (1991) “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md (Kabat numbering scheme), the one described by Al-Lazikani et al., J Mol Biol 273:927-948, 1997 (Chothia numbering scheme), the one described by Maccallum et al., J Mol Biol 262:732-745, 1996 (Contact numbering scheme), the one described by Martin et al., Proc. Natl. Acad. Sci., 86:9268-9272, 1989 (AbM numbering scheme), and the one described by Lefranc et al., Dev Comp Immunol This includes the IMGT numbering scheme described in 27(1):55-77,2003 and the "Aho" numbering scheme described in Honegger & Pluckthun, J Mol Biol 309(3):657-670,2001. The boundaries of a given CDR or FR may differ depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. For both the Kabat and Chothia schemes, numbering is based on the most common antibody region sequence length, insertions are considered by insertion letters (e.g., "30a"), and deletions are observed depending on the antibody. The locations of certain insertions and deletions ("indels") determined by these two schemes differ, resulting in differences in numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. In certain embodiments, the antibody CDR sequences disclosed herein are numbered by Kabat.

[0272] CARs are engineered receptors designed to bind to a specific target and elicit a response. When expressed on a cell, a CAR contains several distinct dependent elements that enable a genetically modified cell to recognize and kill unwanted cells, such as cancer cells or virus-infected cells. The dependent elements include at least an extracellular element and an intracellular element. The extracellular element contains a binding domain that specifically binds to a marker selectively present on the surface of the unwanted cell. Once the binding domain binds to such a marker, the intracellular element activates the genetically modified cell to destroy the bound cell. In addition, a CAR contains a transmembrane domain that links the extracellular element to the intracellular element, and other dependent elements that can enhance the function of the CAR. For example, the inclusion of one or more linker sequences, such as a spacer region, can allow the CAR to have additional structural mobility, often increasing the ability of the binding domain to bind to target cell markers.

[0273] The extracellular domain of a CAR includes a binding domain. The binding domain has been previously described and can contain antibodies, scFvs, ligands, peptides, peptide aptamers, or receptors.

[0274] In certain embodiments, the manipulated CAR comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% amino acid sequence identity with a known or identified TCR Vα, TCR Vβ, TCR Cα, or TCR Cβ, wherein each CDR contains no changes from the TCR or its fragments or derivatives that specifically bind to a target cell marker, or contains at most one, at most two, or at most three such changes.

[0275] In certain embodiments, the manipulated CAR includes a Vα, Vβ, Cα, or Cβ region derived from or based on the Vα, Vβ, Cα, or Cβ of a known or identified TCR (e.g., a high-affinity TCR), and includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) insertions, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) deletions, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or a combination of the above changes compared to the Vα, Vβ, Cα, or Cβ of such known or identified TCR. Insertions, deletions, or substitutions may occur anywhere within the Vα, Vβ, Cα, or Cβ regions (including the amino-terminus, carboxy-terminus, or both), provided that the number of changes in each CDR is zero, at most one, at most two, or at most three, and that the target-binding domain containing the modified Vα, Vβ, Cα, or Cβ region can still specifically bind to its target with the same affinity and action as the wild type.

[0276] In certain embodiments, the binding domain of the CAR contains or is a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity with the amino acid sequence of the light chain variable region (VL) or the heavy chain variable region (VH) or both, and each CDR contains no modifications from a monoclonal antibody or a fragment or derivative thereof that specifically binds to a target cell marker, or at most one, at most two, or at most three such modifications.

[0277] In certain embodiments, the VL region in the CAR of the Disclosure is derived from or based on the VL of a known monoclonal antibody and includes, compared to the VL of such known monoclonal antibody, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) insertions, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) deletions, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above changes. Insertions, deletions, or substitutions may occur anywhere within the VL region (including the amino-terminus, carboxy-terminus, or both), provided that the number of changes in each CDR is zero, at most one, at most two, or at most three, and that the binding domain containing the modified VL region can still specifically bind to its target with the same affinity as the wild-type binding domain.

[0278] In certain embodiments, the binding domain VH region of the CAR of the Disclosure may be derived from or based on the VH of a known monoclonal antibody and may include, compared to the VH of a known monoclonal antibody, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) insertions, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) deletions, one or more (e.g., two, three, four, five, six, seven, eight, nine, ten) amino acid substitutions (e.g., conserved or non-conserved amino acid substitutions), or a combination of the above changes. Insertions, deletions, or substitutions may occur anywhere within the VH region (including the amino-terminus, carboxyl-terminus, or both), provided that the number of changes in each CDR is zero, at most one, at most two, or at most three, and that the binding domain containing the modified VH region can still specifically bind to its target with the same affinity as the wild-type binding domain.

[0279] Specific cellular markers associated with prostate cancer include PSMA, WT1, prostate stem cell antigen (PSCA), and SV40 T. Specific cellular markers associated with breast cancer include HER2 and ERBB2. Specific cellular markers associated with ovarian cancer include L1-CAM, the extracellular domain of MUC16 (MUC-CD), folate-binding protein (folate receptor), Lewis Y, mesothelin, and WT-1. Specific cellular markers associated with pancreatic cancer include mesothelin, CEA, and CD24. Specific cellular markers associated with multiple myeloma include BCMA, GPRC5D, CD38, and CS-1. Specific markers associated with leukemia and / or lymphoma include CLL-1, CD123, CD33, and PD-L1.

[0280] In certain embodiments, the CAR binding domain binds to the cell marker Her2. In certain embodiments, the HER2-binding domain is derived from trastuzumab (Herceptin). In certain embodiments, the binding domain includes a variable light chain comprising a CDRL1 sequence containing SEQ ID NO: 8, a CDRL2 sequence containing SEQ ID NO: 9, and a CDRL3 sequence containing SEQ ID NO: 10, as well as a variable heavy chain comprising a CDRH1 sequence containing SEQ ID NO: 11, a CDRH2 sequence containing SEQ ID NO: 12, and a CDRH3 sequence containing SEQ ID NO: 13.

[0281] In certain embodiments, the CAR binding domain binds to the cell marker PD-L1. In certain embodiments, the binding domain that binds to PD-L1 is derived from at least one of pembrolizumab or FAZ053 (Novartis). In certain embodiments, the binding domain includes a variable light chain comprising a CDRL1 sequence containing SEQ ID NO: 14, a CDRL2 sequence containing SEQ ID NO: 15, and a CDRL3 sequence containing SEQ ID NO: 16, and a variable heavy chain comprising a CDRH1 sequence containing SEQ ID NO: 17, a CDRH2 sequence containing SEQ ID NO: 18, and a CDRH3 sequence containing SEQ ID NO: 19.

[0282] Exemplary binding domains for PD-L1 may include or be derived from avelumab or atezolizumab. In certain embodiments, the variable heavy chain of avelumab includes SEQ ID NO: 20.

[0283] In certain embodiments, the variable light chain of avelumab includes SEQ ID NO: 21.

[0284] In certain embodiments, the CDR region of avelumab includes CDRH1 (sequence number 22); CDRH2 (sequence number 23); CDRH3 (sequence number 24); CDRL1 (sequence number 25); CDRL2 (sequence number 26); and CDRL3 (sequence number 27).

[0285] In certain embodiments, the variable heavy chain of atezolizumab includes SEQ ID NO: 28. In certain embodiments, the variable light chain of atezolizumab includes SEQ ID NO: 29.

[0286] In certain embodiments, the CDR region of atezolizumab includes CDRH1 (SEQ ID NO: 30); CDRH2 (SEQ ID NO: 31); CDRH3 (SEQ ID NO: 32); CDRL1 (SEQ ID NO: 33); CDRL2 (SEQ ID NO: 34); and CDRL3 (SEQ ID NO: 35).

[0287] In certain embodiments, the CAR binding domain binds to the cell marker PSMA. In certain embodiments, the binding domain includes a variable light chain comprising a CDRL1 sequence containing SEQ ID NO: 36, a CDRL2 sequence containing SEQ ID NO: 37, and a CDRL3 sequence containing SEQ ID NO: 38. In certain embodiments, the binding domain includes a variable heavy chain comprising a CDRH1 sequence containing SEQ ID NO: 39, a CDRH2 sequence containing SEQ ID NO: 40, and a CDRH3 sequence containing SEQ ID NO: 41.

[0288] In certain embodiments, the CAR binding domain binds to the cell marker MUC16. In certain embodiments, the binding domain is human or humanized and includes a variable light chain containing a CDRL1 sequence including SEQ ID NO: 42, a CDRL2 sequence including GAS, and a CDRL3 sequence including SEQ ID NO: 43. In certain embodiments, the binding domain is human or humanized and includes a variable heavy chain containing a CDRH1 sequence including SEQ ID NO: 44, a CDRH2 sequence including SEQ ID NO: 45, and a CDRH3 sequence including SEQ ID NO: 46.

[0289] In certain embodiments, the CAR binding domain binds to the cell marker FOLR. In certain embodiments, the binding domain that binds to FOLR is derived from farletuzumab. In certain embodiments, the binding domain includes a variable light chain comprising a CDRL1 sequence containing SEQ ID NO: 47, a CDRL2 sequence containing SEQ ID NO: 48, and a CDRL3 sequence containing SEQ ID NO: 49, and a variable heavy chain comprising a CDRH1 sequence containing SEQ ID NO: 50, a CDRH2 sequence containing SEQ ID NO: 51, and a CDRH3 sequence containing SEQ ID NO: 52.

[0290] An exemplary binding domain for mesothelin may include or be derived from amatsuximab.

[0291] In certain embodiments, the variable heavy chain of amatsuximab includes SEQ ID NO: 53. In certain embodiments, the variable light chain of amatsuximab includes SEQ ID NO: 54.

[0292] In certain embodiments, the CDR region of amatsuximab includes CDRH1 (sequence number 55); CDRH2 (sequence number 56); CDRH3 (sequence number 57); CDRL1 (sequence number 58); CDRL2 (sequence number 59); and CDRL3 (sequence number 60).

[0293] Binding domains specific to infectious disease pathogens are also intended (for example, by binding to infectious pathogen antigens). These include, for example, viral antigens or other viral markers (e.g., those expressed by virus-infected cells). Examples of viruses include adenoviruses, arenaviruses, bunyaviruses, coronaviruses, flaviviruses, hantaviruses, hepadnaviruses, herpesviruses, papillomaviruses, paramyxoviruses, parvoviruses, picornaviruses, poxviruses, orthomyxoviruses, retroviruses, reoviruses, rhabdoviruses, rotaviruses, spongiform encephalopathy viruses, or togaviruses. In additional embodiments, the viral antigen marker may include peptides expressed by CMV, common cold virus, Epstein-Barr virus, influenza virus, hepatitis A virus, hepatitis B virus, and hepatitis C virus, herpes simplex virus, HIV, influenza virus, Japanese encephalitis virus, measles virus, poliovirus, rabies virus, polynuclear respiratory virus, rubella virus, smallpox virus, varicella-zoster virus, or West Nile virus.

[0294] Other specific examples include cytomegalovirus antigens, which include envelope glycoprotein B and CMV pp65; Epstein-Barr virus antigens, which include EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens, which include HBV S, M, and L proteins, HBV pre-S antigen, HBCAG delta, HBV HBE, hepatitis C virus RNA, HCV NS3, and HCV NS4; herpes simplex virus antigens, which include pre-initial protein and glycoprotein D; and HIV antigens, which include the gene products of the gag gene, pol gene, and env gene (such as HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, Nef protein, and reverse transcriptase). Influenza antigens contain hemagglutinin and neuraminidase. Japanese encephalitis virus antigens contain E protein, ME protein, ME-NS1 protein, NS1 protein, NS1-NS2A protein, and 80% E protein. Measles antigens contain measles virus fusion protein. Rabies antigens contain rabies glycoprotein and rabies nucleoprotein. Respiratory polynuclear virus antigens contain RSV fusion protein and M2 protein. Rotavirus antigens contain VP7sc. Rubella antigens contain E1 protein and E2 protein. Varicella-zoster virus antigens contain gpI and gpII. Examples of additional specific viral antigen sequences include Nef(66~97)(SEQ ID NO: 61), Nef(116~145)(SEQ ID NO: 62), Gag p17(17~35)(SEQ ID NO: 63), Gag p17~p24(253~284)(SEQ ID NO: 64), and Pol325~355(RT158~188)(SEQ ID NO: 65). For more examples of additional viral antigens, see Fundamental Virology, Second Edition, eds. Fields, B.N. and Knipe, DM (Raven Press, New York, 1991).

[0295] Intracellular signaling elements. The intracellular or cytoplasmic signaling elements of a CAR are responsible for activating the cell expressing the CAR. Therefore, the terms “intracellular signaling element” or “intracellular element” are intended to include any portion of the intracellular domain sufficient for transmitting the activation signal. The intracellular elements of an expressed CAR may include the effector domain. The 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 reception of the appropriate signal. In certain embodiments, the effector domain is a portion of a protein or protein complex that receives a signal after binding, or the protein or protein complex directly binds to a target molecule (which generates the signal from the effector domain). If the effector domain contains one or more signaling domains or signaling motifs (such as an immune receptor tyrosine activation motif (ITAM)), it may directly promote a cellular response. In other embodiments, the effector domain indirectly promotes a cellular response by being linked to one or more other proteins (such as a costimulatory domain) that directly promote a cellular response.

[0296] The effector domain may activate at least one function of the modified cell when it binds to a cellular marker expressed by cancer cells. Activation of the modified cell may include one or more of the following: differentiation, proliferation, and / or activation, or other effector functions. In certain embodiments, the effector domain may include an intracellular signaling element comprising a T cell receptor and a costimulatory domain which may include a cytoplasmic sequence derived from a coreceptor or costimulatory molecule.

[0297] An effector domain may contain one, two, three, or more receptor signaling domains, intracellular signaling elements (e.g., cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Examples of 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, examples of effector domains include ligands that specifically bind to CD86, FcγRIIa, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, CDDS, 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, and ITGAM. Examples include signal transduction domains and co-stimulatory domains selected from 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.

[0298] Intracellular signaling element sequences that function in a stimulus-like manner may include iTAMs. Examples of iTAMs containing primary cytoplasmic signaling sequences include those derived from CD3γ, CD3δ, CD3ε, CD3ζ, CD5, CD22, CD66d, CD79a, CD79b, as well as those derived from the common FcRγ(FCER1G), FcγRlla, FcRβ(FcεRib), DAP10, and DAP12. In certain embodiments, variants of CD3ζ may retain at least one, at least two, at least three, or all of the ITAM regions.

[0299] In certain embodiments, the effector domain comprises a cytoplasmic portion bound to a cytoplasmic signaling protein, the cytoplasmic signaling protein being a lymphocyte receptor or its signaling domain, a protein containing multiple ITAMs, a co-stimulatory domain, or any combination thereof.

[0300] Examples of additional intracellular signaling elements include coreceptors, where the cytoplasmic sequence and / or binding domain of the CD3ζ chain work synergistically to initiate signal transduction after binding.

[0301] Co-stimulatory domains are domains that may need to be activated to efficiently produce a lymphocyte response to binding to cellular markers. Depending on the molecule, they can be interchangeable as either intracellular signaling elements or co-stimulatory domains. Examples of co-stimulatory 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 (those that specifically bind to CD83). For example, CD27 co-stimulation has been shown to enhance the proliferation, effector function, and survival of human CAR T cells in vitro, and to enhance the persistence and anticancer activity of human T cells in vivo (Song et al. Blood. 2012;119(3):696-706). Other examples of such co-stimulatory domain molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB, and CD2 Examples include 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.

[0302] In certain embodiments, the amino acid sequence of the intracellular signaling element includes a variant of CD3ζ and a portion of the 4-1BB intracellular signaling element.

[0303] In certain embodiments, the intracellular signaling element includes (i) all or part of the signaling domain of CD3ζ, (ii) all or part of the signaling domain of 4-1BB, or (iii) all or part of the signaling domains of CD3ζ and 4-1BB.

[0304] Intracellular components include proteins of the Wnt signaling pathway (e.g., LRP, Ryk, or ROR2), proteins of the NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, or NOTCH4), proteins of 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, tropomyosin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinases) This also includes one or more of the following: the LTK (long-term kinase) receptor family, the tyrosine kinase 1 (TIE) receptor family with immunoglobulin-like and EGF-like domains, the receptor tyrosine kinase-like orphan (ROR) receptor family, the discoidin domain (DDR) receptor family, the reorganization during transfection (RET) receptor family, the tyrosine-protein kinase-like (PTK7) receptor family, the receptor tyrosine kinase-related (RYK) receptor family, or the muscle-specific kinase (MuSK) receptor family, G protein-coupled receptors (GPCRs (Frizzled or Smoothened)), serine / threonine kinase receptors (BMPR or TGFR), or cytokine receptors (IL1R, IL2R, IL7R, or IL15R). It is visible.

[0305] Linkers. As used herein, linkers can be any part of a CAR molecule that serves to link two other dependent elements of that molecule. While many linkers serve additional purposes, some linkers serve no purpose other than linking other elements. Linkers in the context of linking the binding domain of scFv (derived from the VL and VH of the antibody) are described above. Linkers may also include spacer regions and linking amino acids.

[0306] A spacer region is a type of linker region used to create appropriate distance and / or mobility from other linking elements. In certain embodiments, the length of the spacer region may be customized for each individual cell marker on the unwanted cell to optimize the recognition and destruction of the unwanted cell. The spacer may be of a length that improves the responsiveness of the cell after antigen binding compared to the absence of a spacer. In certain embodiments, the length of the spacer region may be selected based on the location of the cell marker epitope, the affinity of the binding domain to the epitope, and / or the ability of modified cells expressing the molecule to proliferate in vitro and / or in vivo in response to recognition of the cell marker. The spacer region may also allow for increased expression levels in the modified cell.

[0307] In certain embodiments, the spacer region includes a hinge region containing a type II / type C lectin interdomain (stalk) region or a surface antigen classification (CD) molecule stalk region. As used herein, “wild-type immunoglobulin hinge region” refers to naturally occurring upper hinge amino acid sequences and intermediate hinge amino acid sequences, which are found in the heavy chain of antibodies and interposed between the CH1 and CH2 domains (for IgG, IgA, and IgD) to link them, or interposed between the CH1 and CH3 domains (for IgE and IgM) to link them.

[0308] The "Stork region" of a type II C-type lectin or CD molecule refers to the portion of the extracellular domain of a type II C-type lectin or CD molecule located between the C-type lectin-like domain (CTLD (for example, similar to the CTLD of the natural killer cell receptor)) and the hydrophobic portion (transmembrane domain). For example, the extracellular domain of human CD94 (GenBank acceptance number AAC50291.1) corresponds to amino acid residues 34-179, while the CTLD corresponds to amino acid residues 61-176. Therefore, the stalk region of the human CD94 molecule includes amino acid residues 34-60, which are located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:15, 1999. For other descriptions of the stalk region, also see Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992 and Figdor et al., Nat. Rev. Immunol. 2:11, 2002). Such type II C-type lectins or CD molecules may also have linking amino acids (described below) between the stalk region and the transmembrane domain or CTLD. In another example, the 233-amino acid human NKG2A protein (GenBank acceptance number P26715.1) has a hydrophobic region (transmembrane domain) in the range of amino acids 71–93 and an extracellular domain in the range of amino acids 94–233. The CTLD contains amino acids 119–231, and the stalk region contains amino acids 99–116, which may be adjacent to additional linkage amino acids. Other type II C-type lectins or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs, are known in the art (for example, see GenBank acceptance numbers NP001993.2, AAH07037.1, NP001773.1, AAL65234.1, and CAA04925.1, respectively, for the sequences and descriptions of human CD23, CD69, CD72, NKG2A, and NKG2D).

[0309] To further elaborate on the spacer region, the extracellular elements of a fusion protein may optionally include an extracellular non-signaling spacer region or an extracellular non-signaling linker region, which may be capable of optimizing intercellular contact, antigen binding, and activation, for example, by positioning the binding domain away from the host cell (e.g., T cell) surface (Patel et al., Gene Therapy 6:412-419, 1999). As shown, the extracellular spacer region of a fusion-binding protein is generally located between the hydrophobic portion or transmembrane domain and the extracellular binding domain, and the length of the spacer region may be modified to maximize antigen recognition (e.g., tumor recognition) based on the size and affinity of the selective target molecule, selective binding epitope, or antigen-binding domain (see, e.g., Guest et al., J.Immunother.28:203-11, 2005, PCT Publication WO2014 / 031687). In certain embodiments, the spacer region includes an immunoglobulin hinge region. The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is a human immunoglobulin hinge region. The immunoglobulin hinge region may be a hinge region of IgG, IgA, IgD, IgE, or IgM. The IgG hinge region may be a hinge region of IgG1, IgG2, IgG3, or IgG4. Other examples of hinge regions used in the fusion-binding proteins described herein include hinge regions (which may be wild-type or variants thereof) located in the extracellular domains of type 1 membrane proteins (such as CD8α, CD4, CD28, and CD7).

[0310] In certain embodiments, the extracellular spacer region comprises all or part of an Fc domain selected from the CH1 domain, CH2 domain, CH3 domain, CH4 domain, or any combination thereof. The Fc domain or part thereof may be wild-type or modified (for example, to reduce antibody effector function). In certain embodiments, the extracellular element comprises an immunoglobulin hinge region, a CH2 domain, a CH3 domain, or any combination thereof, positioned between the binding domain and the hydrophobic portion.

[0311] Linking amino acids can be linkers used to ligate the sequences of CAR domains when the distance obtained by a spacer is unnecessary and / or undesirable. Linking amino acids are short amino acid sequences that can be used to ligate costimulatory intracellular signaling elements. In certain embodiments, the number of amino acids in the linking amino acids is 9 or less.

[0312] The linking amino acids may be short-chain oligolinkers or short-chain protein linkers, preferably with amino acid lengths of 2 to 9 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) that form such linkers. In certain embodiments, a glycine-serine doublet may be used as a suitable linking amino acid linker. In certain embodiments, a single amino acid (e.g., alanine, glycine) may be used as a suitable linking amino acid.

[0313] Transmembrane domain. As shown, the transmembrane domain within a CAR molecule often functions to link extracellular and intracellular elements across the cell membrane. The transmembrane domain can fix expression molecules to the membrane of modified cells.

[0314] The transmembrane domain may originate from natural and / or synthetic sources. If the origin is natural, the transmembrane domain may originate from any membrane-bound or transmembrane protein. The transmembrane domain may include at least one or more transmembrane regions 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 may include at least one or more transmembrane regions, such as 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, I L2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11 b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT These are AM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, or NKG2C.

[0315] In certain embodiments, the transmembrane domain has a thermodynamically stable three-dimensional structure within the cell membrane, and generally has an amino acid length in the range of 15 to 30. The structure of the transmembrane domain may include α-helices, β-barrels, β-sheets, β-helices, or any combination thereof.

[0316] The transmembrane domain may contain one or more additional amino acids adjacent to the transmembrane region (e.g., one or more amino acids within the extracellular region of the CAR (such amino acids may be, for example, up to 15 of the amino acids in the extracellular region) and / or one or more additional amino acids within the intracellular region of the CAR (such additional amino acids may be, for example, up to 15 of the amino acids in the intracellular element)). In one embodiment, the transmembrane domain originates from the same protein from which the signaling domain, costimulatory domain, or hinge domain originates. In another embodiment, the transmembrane domain does not originate from the same protein from which any of the other domains of the CAR originate. In some cases, interaction with other unintended members of the receptor complex may be minimized by selecting or modifying the transmembrane domain with amino acids so that such domain does not bind to the transmembrane domain of the same or different surface membrane protein. In one embodiment, the transmembrane domain has the ability to homodimerize with another CAR on the cell surface of a CAR-expressing cell. In a different embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell. In certain embodiments, the transmembrane domain includes the amino acid sequence of the CD28 transmembrane domain.

[0317] The transduction marker may be selected from at least one of the following: cleaved CD19 (tCD19; see Budde et al., Blood 122:1660, 2013), cleaved human EGFR (tEGFR; see Wang et al., Blood 118:1255, 2011), the extracellular domain of human CD34, and / or RQR8, which is a combination of a target epitope derived from the CD34 antigen (see Fehse et al., Mol. Therapy 1(5 Pt 1):448-456, 2000) and a target epitope derived from the CD20 antigen (see Philip et al., Blood 124:1277-1278, 2014).

[0318] In certain embodiments, a polynucleotide encoding the i-caspase 9 construct (iCasp9) can be inserted into the CAR nucleotide construct as a suicide switch.

[0319] Multiple control units may exist as multiple copies within the CAR, or they may be expressed as different molecules using skipping elements. In certain embodiments, the transduction marker includes tEGFR. Examples of transduction markers and corresponding pairs are described in U.S. Patent No. 8,802,374.

[0320] One advantage of including at least one control unit in a CAR is that the CAR-expressing cells administered to a target can be depleted by using a corresponding binding molecule to the control unit or by using a second CAR-expressing modified cell with specificity to the control unit. Removal of the modified cell can be achieved using a depleting agent specific to the control unit.

[0321] In certain embodiments, modified cells expressing a chimeric molecule can be detected or tracked in vivo by using an antibody that specifically binds to the control molecule or another corresponding binding molecule that specifically binds to the control molecule, and such binding partners to the control molecule are conjugated with fluorescent dyes, radiotracers, iron oxide nanoparticles, or other contrast agents (as known in the art for detection by X-ray, CT scans, MRI scans, PET scans, ultrasound, flow cytometry, near-infrared contrast systems, or other imaging diagnostic methods) (see, for example, Yu, et al., Theranostics 2:3, 2012).

[0322] Therefore, modified cells expressing at least one control group along with the CAR may be easier to identify, isolate, sort, induce proliferation, track, and / or remove compared to modified cells that lack a tag cassette, for example.

[0323] T cell receptors (TCRs) are molecules found on the surface of T cells that enable T cell recognition of peptides bound to the major histocompatibility complex (MHC).

[0324] TCR refers to a naturally occurring T cell receptor. HSCs can be modified in vivo to express a selective TCR. CAR / TCR hybrids refer to proteins that have elements of both a TCR and a CAR. For example, a CAR / TCR hybrid may have a naturally occurring TCR-binding domain, which may have an effector domain added that is not naturally bound to such a TCR-binding domain. CAR / TCR hybrids may have a mutant TCR-binding domain and an ITAM signaling domain. CAR / TCR hybrids may have a naturally occurring TCR with an inserted non-natural spacer region or transmembrane domain.

[0325] Certain CAR / TCR hybrids include the TRuC® (T cell receptor fusion construct) hybrid (TCR2 Therapeutics, Cambridge, MA). For example, the generation of TCR fusion proteins is described in international patent publications WO2018 / 026953 and WO2018 / 067993, and patent application publication US2017 / 0166622.

[0326] In certain embodiments, the CAR / TCR hybrid includes a “T cell receptor (TCR) fusion protein,” or “TFP.” The TFP comprises recombinant polypeptides derived from various polypeptides, which generally include TCRs that i) bind to surface antigens on target cells and ii) interact with other polypeptide elements of the intact TCR complex (typically when co-localized within or on the surface of T cells).

[0327] (IV-d)CRISPR

[0328] The CRISPR (Crystal-Repeatable Short Repeat Palindromic Sequence) / Cas (CRISPR-Related Protein) nuclease system is a bacterial-based, engineered nuclease system used in genetic engineering. The CRISPR / Cas nuclease system is partly based on the adaptive immune responses of many bacteria and archaea. When a virus or plasmid invades a bacterium, the bacterial "immune" response converts segments of the invader's DNA into CRISPR RNA (crRNA). The crRNA then binds to another type of RNA (called tracrRNA) via a partially complementary region, leading the Cas nuclease to a target DNA region homologous to the crRNA (called a "protospacer"). When the Cas nuclease cleaves the DNA at a site identified by a 20-nucleotide complementary strand sequence in the crRNA transcript, a double-strand break occurs, resulting in a blunt end. In some cases, the Cas nuclease requires both crRNA and tracrRNA for site-specific recognition and cleavage of DNA.

[0329] Guide RNA (gRNA) is an example of a target-directed element. In its simplest form, gRNA provides a sequence (e.g., crRNA) that targets a genomic site based on complementarity. On the other hand, as described below, gRNA can also contain additional components. For example, in certain embodiments, gRNA may include a target-directed sequence (e.g., crRNA) and an element for linking this target-directed sequence to a cleavage element. This linking element may be tracrRNA. In certain embodiments, as described below, gRNA containing crRNA and tracrRNA may be expressed as a single molecule called a single gRNA (sgRNA). gRNA can also be linked to cleavage elements through other mechanisms (such as via nanoparticles or by expressing or constructing molecules with two or more purposes).

[0330] In certain embodiments, nucleic acids with novel or enhanced features (e.g., improved stability) can be obtained by incorporating one or more modifications (e.g., base modifications, skeleton modifications) into a target-directed element (e.g., gRNA). Modified skeletons may include those that retain phosphorus atoms in the skeleton and those that do not. Suitable phosphorus atom-containing modified skeletons may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates and other alkylphosphonates (such as 3'-alkylene phosphonates and 5'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), phosphorodiamidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, selenophosphates, and boranophosphates (those with the usual 3'-5' linkage, 2'-5' linkage analogues, and those with inverted polarity (where one or more internucleotide bonds are 3'-3', 5'-5', or 2'-2')). A suitable inverted polarity-containing target-directed element may contain one 3'-3' bond at the furthest 3' internucleotide bond position (i.e., one inverted nucleoside residue that either lacks a nucleic acid base or has a hydroxyl group at that position). Various salt forms (e.g., potassium chloride or sodium chloride), mixed salt forms, and free acid forms may also be included.

[0331] The target-directed element may include one or more phosphorothioate nucleoside bonds and / or heteroatom nucleoside bonds, specifically -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (i.e., methylene (methylimino) or MMI skeleton), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- (the natural phosphodiester nucleotide bond is represented as -OP(=O)(OH)-O-CH2-).

[0332] In certain embodiments, the target-directing element may include a morpholino skeletal structure. For example, the target-directing element may include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, the phosphodiester bond is replaced by a phosphorodiamidate nucleoside bond or other non-phosphodiester nucleoside bond.

[0333] In certain embodiments, the target-directing element may comprise one or more substituted sugar moieties. Suitable polynucleotides may comprise sugar substituents selected from OH, F, O-alkyl, S-alkyl, or N-alkyl, O-alkenyl, S-alkenyl, or N-alkenyl, O-alkynyl, S-alkynyl, or N-alkynyl, or O-alkyl-O-alkyl (wherein such alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and C2-C10 alkynyl). Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2 (wherein n and m are independently 1 to 10).

[0334] Examples of cleavage elements include nucleases. The CRISPR-Cas locus has more than 50 gene families, and there are no strictly universal genes, which indicates that the locus structure has evolved rapidly and is extremely diverse. Examples of Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cpf1, C2c3, C2c2, and C2c1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, C Examples include sa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

[0335] There are three main types of Cas nucleases (type I, type II, and type III) and ten subtypes (including five type I proteins, three type II proteins, and two type III proteins) (see, for example, Hochstrasser and Doudna, Trends Biochem Sci, 40(l):58-66, 2015). Type II Cas nucleases include Cas1, Cas2, Csn2, and Cas9. These Cas nucleases are known in the art. For example, the amino acid sequence of the wild-type Cas9 polypeptide of Streptococcus pyogenes is shown, for example, in NBCI reference sequence number NP269215, and the amino acid sequence of the wild-type Cas9 polypeptide of Streptococcus thermophilus is shown, for example, in NBCI reference sequence number WP_011681470.

[0336] In certain embodiments, Cas9 refers to an RNA-guided double-stranded DNA-binding nuclease protein or double-stranded DNA-binding nickase protein. The wild-type Cas9 nuclease has two functional domains (e.g., RuvC and HNH) that cleave different DNA strands. Cas9 can introduce double-strand breaks into genomic DNA (target DNA) when both functional domains are active. In some embodiments, the Cas9 enzyme comprises one or more catalytic domains of a Cas9 protein derived from bacteria (such as Corynebacter, Sutterella, Legionella, Treponema, Filif actor, 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, for example, in which the two catalytic domains originate from different bacterial species.

[0337] As previously demonstrated, the CRISPR / Cas system can be manipulated so that, in certain cases, crRNA and tracrRNA can be integrated into a single molecule (called a single gRNA (sgRNA)). In this manipulation technique, Cas is guided by the sgRNA to target an arbitrary 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 manipulated to create double-strand breaks at desired targets in the cell's genome and to utilize the cell's endogenous mechanisms to repair the introduced breaks by HDR or NHEJ. In the specific embodiments described herein, homology arms are used to facilitate HDR at a defined integration site.

[0338] Useful variants of Cas9 nuclease include enzymes in which one of the catalytic domains is inactive (RuvC ” Enzymes or HNH ” This includes enzymes (such as nickase). Cas9 nickase has only one active functional domain and, in some embodiments, cleaves only one strand of target DNA, thereby creating a single-strand break, or nick. In some embodiments, a mutant Cas9 nuclease having at least the D10A mutation is a Cas9 nickase. In other embodiments, a mutant Cas9 nuclease having at least the H840A mutation is a Cas9 nickase. Other examples of mutations present in Cas9 nickase include N854A and N863A. When at least two DNA targeting RNAs targeting opposing DNA strands are used, a double-strand break is introduced using Cas9 nickase. The double-strand break with doubly introduced nicks is repaired by HDR or NHEJ. This gene editing strategy is generally favorable to HDR and reduces the frequency of indel mutations occurring at off-target DNA sites. In some embodiments, Cas9 nuclease or Cas9 nickase is used for codon optimization for target cells or target organisms.

[0339] In certain embodiments, Staphylococcus aureus Cas9 (SaCas9) may be used. In certain embodiments, SaCas9 having mutations in one or more of the following positions may be used: E782, N968, and / or R1015. In certain embodiments, SaCas9 having mutations in one or more of the following positions may be used: E735, E782, K929, N968, A1021, K1044, and / or R1015. In some embodiments, the variant SaCas9 protein contains one or more of the following mutations: R1015Q, R1015H, E782K, N968K, E735K, K929R, A1021T, and / or K1044N. In some embodiments, the variant SaCas9 protein includes mutations D10A, D556A, H557A, and N580A, for example, D10A / H557A and / or D10A / D556A / H557A / N580A. In some embodiments, the variant SaCas9 protein includes mutations at one or more positions selected from E735, E782, K929, N968, R1015, A1021, and / or K1044. In some embodiments, the SaCas9 variant may include one of the following sets of mutations: E782K / N968K / R1015H (KKH variant), E782K / K929R / R1015H (KRH variant), or 782K / K929R / N968K / R1015H (KRKH variant).

[0340] Class II and V CRISPR-Cas classes (Cpf1 being an example) have been identified (Zetsche et al., Cell 163(3):759-771, 2015). In particular, the Cpf1 nuclease offers flexibility in target site selection due to its short three-base pair recognition sequence (TTN) (known as the protospacer adjacent motif (PAM)). The cleavage site of Cpf1 is at least 18 bp away from the PAM sequence. Furthermore, alternating double-sequence bridges (DSBs) with protruding ends allow for orientation-specific insertion of the donor template, which is advantageous in non-dividing cells.

[0341] In certain embodiments, manipulated Cpf1 may be used. For example, US2018 / 0030425 describes Cpf1 nucleases derived from Lachnospiraceae bacterium ND2006 and a species of the genus Acidaminococcus BV3L6 that have been manipulated to alter and improve target specificity. Certain variants include those from Lachnospiraceae bacterium ND2006, which have mutations in at least amino acids 19–1246 (i.e., native amino acids are replaced by different amino acids (e.g., alanine, glycine, or serine)), and such mutations are located in one or more of the following positions: S202, N274, N278, K290, K367, K532, K609, K915, Q962, K963, K966, K1002, and / or S1003. Certain Cpf1 variants may include Cpf1 (AsCpf1) of Acidaminococcus species BV3L6, which is mutated (i.e., the native amino acid is replaced by a different amino acid (e.g., alanine, glycine, or serine) (except when the native amino acid is serine)), and such mutations are located at one or more of the following positions: N178, S186, N278, N282, R301, T315, S376, N515, K523, K524, K603, K965, Q1013, Q1014, and / or K1054.

[0342] Other Cpf1 variants include the Cpf1 homologs and orthologs of the Cpf1 polypeptide disclosed in Zetsche et al., Cell 163:759-771, 2015, and the Cpf1 polypeptide disclosed in U.S. Patent Application Publication No. 2016 / 0208243. Other manipulated Cpf1 variants are known to those skilled in the art and are included in the scope of this disclosure (see, for example, WO / 2017 / 184768).

[0343] As previously shown, in embodiments, homology arms are utilized to facilitate the insertion of a genetic construct into a target using homologous recombination repair. The homology arm may support HDR between the homology arm and the genome sequence to which the homology arm is homologous by having a sufficiently large length of homology to the genome sequence at the cleavage site (for example, the homology percentage between the cleavage site and the adjacent nucleotide sequence may be 70%, 80%, 85%, 90%, 95%, or 100%, and these adjacent nucleotide sequences may be located, for example, within 50 bases of the cleavage site (e.g., within 30 bases, within 15 bases, within 10 bases, within 5 bases, or immediately adjacent to the cleavage site)). The homology arm is generally identical to the genome sequence (e.g., the genome region where the double-strand break (DSB) occurs). However, as shown, absolute identity is not required.

[0344] Certain embodiments may utilize homology arms having 25, 50, 100, 200, or greater than 200 nucleotides having sequence homology between the homologous recombination repair template and the target genome sequence (or any integer between 10 and 200 or greater). In certain embodiments, the length of the homology arm is between 40 nucleotides (nt) and 1000 nt. In certain embodiments, the number of base pairs in the homology arm is between 500 and 2500, 700 and 2000, or 800 and 1800. In certain embodiments, the homology arm contains at least 800 base pairs or at least 850 base pairs. The length of the homology arm may be symmetric or asymmetric. For additional information regarding homology arms, see Richardson et al., Nat Biotechnol. 34(3):339-44, 2016.

[0345] Additional information regarding the CRISPR-Cas system and its components can be found in US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233, and US8999641. , and related applications, as well as WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, WO2014 / 093661, WO2014 / 093694, WO2014 / 093701, WO2014 / 093709, WO2014 / 093712, WO201 4 / 093718, WO2014 / 145599, WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, W O2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 08935 4, as described in WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016 / 205711, WO2017 / 106657, WO2017 / 127807, and related applications.

[0346] (IV-e) Base editing system

[0347] Base editing refers to the selective modification of nucleic acid sequences by converting bases or base pairs in genomic DNA or cellular RNA to different bases or base pairs (Rees & Liu, Nature Reviews Genetics, 19:770-788, 2018). There are two common classes of DNA base editors: (i) cytosine base editors (CBEs) that convert guanine-cytosine base pairs to thymine-adenine base pairs, and (ii) adenine base editors (ABEs) that convert adenine-thymine base pairs to guanine-cytosine base pairs.

[0348] DNA base editors can insert such point mutations into non-dividing cells without generating double-strand breaks. Because no double-strand breaks occur, base editors do not produce excessive amounts of undesirable editing byproducts (such as insertions and deletions (indels)). For example, the rate of indel generation by base editors may be less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1% compared to techniques that rely on double-strand breaks.

[0349] Most base editing systems consist of (1) a target-directed DNA-binding protein, (2) a nucleic acid base deaminase enzyme, and (3) a DNA glycosylase inhibitor.

[0350] Any nuclease in the CRISPR system can be inactivated and used in base editing systems. Examples of Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cpf1, C2c3, C2c2, and C2c1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, and Csa5. Examples include Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and their variants.

[0351] Nucleases derived from other gene editing systems may also be used. For example, in base editing systems, 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) may be used. For additional information on DNA-binding nucleases, see US2018 / 0312825A1.

[0352] In certain embodiments, the nucleic acid base deaminase enzyme comprises a cytidine deaminase domain or an adenine deaminase domain.

[0353] In certain embodiments, CBEs utilizing the cytidine deaminase domain convert guanine-cytosine base pairs to thymine-adenine base pairs by deaminating the extracyclic amine of cytosine to produce 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 does not have the ability to act on double-stranded (ds) DNA. 【035...

Claims

1. A helper-dependent adenovirus (HDAd) donor vector, (a) Adenovirus capsids containing Ad5 / 35++ fibers, and (b) (i) A transposon payload of at least 25 kb, wherein the transposon payload is (1) A β-globin long chain gene locus regulatory region (LCR), wherein the β-globin long chain LCR includes β-globin HS1 to HS5 and is at least 15 kb, (2) β-globin promoter, (3) A nucleic acid sequence encoding a γ-globin protein, which is operably linked to the β-globin promoter, and (4) 3'HS1 region Transposon payloads, including (ii) Sleeping Beauty (SB) reverse repeat sequence (IR) adjacent to the transposon payload, and (iii) The FRT region adjacent to the SB reverse repeating sequence. Linear double-stranded DNA genome containing HDAd donor vectors, including...

2. A helper-dependent adenovirus (HDAd) donor genome, (a) A transposon payload of at least 25 kb, wherein the transposon payload is (1) A β-globin long chain gene locus regulatory region (LCR), wherein the β-globin long chain LCR includes β-globin HS1 to HS5 and is at least 15 kb, (2) β-globin promoter, (3) A nucleic acid sequence encoding a γ-globin protein, which is operably linked to the β-globin promoter, and (4) 3'HS1 region Transposon payloads, including (b) Sleeping Beauty (SB) reverse repeat sequence (IR) adjacent to the transposon payload, and (c) FRT region adjacent to the SB reverse repeating sequence HDAd donor genome, including.

3. Adenovirus translocation system, (a) The HDAd donor vector according to claim 1, and (b) (i) Adenovirus capsid, and (ii) Adenovirus support genome containing nucleic acid sequences encoding Sleeping Beauty (SB) transposase and FLP recombinase Adenovirus support vectors containing Adenovirus translocation systems, including those mentioned above.

4. The system according to claim 3, wherein the SB transposase is Sleeping Beauty 100x (SB100x).

5. Adenovirus production system, (a) a nucleic acid comprising the HDAd donor genome described in claim 2, and (b) Nucleic acids containing an adenovirus helper genome with a conditional packaging element Adenovirus production systems, including those mentioned above.

6. The vector, genome, or system according to any one of claims 1 to 5, wherein the transposon payload has a length of at least 30 kb.

7. The vector, genome, or system according to any one of claims 1 to 6, wherein the SB reverse repeat sequence is a pT4 reverse repeat sequence.

8. The vector, genome, or system according to any one of claims 1 to 7, wherein the transposon payload includes a selection cassette.

9. The aforementioned selection cassette is mgmt Ｐ１４０Ｋ The vector, genome, or system according to claim 8, comprising a nucleic acid sequence encoding a

10. The adenovirus production system according to claim 5, wherein the conditional packaging element of the adenovirus helper genome includes a packaging sequence adjacent by a recombinase co-directed repeat sequence.

11. The adenovirus production system according to claim 10, wherein the recombinase co-direction repeat sequence adjacent to the packaging sequence of the conditional packaging element is a LoxP site.

12. A cell comprising the vector, genome, or system according to any one of claims 1 to 11.

13. The cell according to claim 12, which is a hematopoietic stem cell.

14. Adenovirus-producing cells comprising the adenovirus-producing system according to any one of claims 5 to 11.

15. The cell according to claim 14, which is a HEK293 cell.

16. An in vitro method for modifying cells, comprising contacting the cells with a vector or adenovirus translocation system described in any one of claims 1 to 4 and 6 to 9.

17. A composition for use in a method for modifying target cells, The composition comprises the vector or adenovirus translocation system described in any one of claims 1 to 4 and 6 to 9. A composition comprising the method of administering the composition to the subject.

18. A composition for use in a method for modifying cells of a subject without isolating the cells from the subject, The composition comprises the vector or adenovirus translocation system described in any one of claims 1 to 4 and 6 to 9. A composition comprising the method of administering the composition to the subject.

19. A composition for use in a method of treating a disease or condition in an object requiring treatment of a disease or condition, The composition comprises the vector or adenovirus translocation system described in any one of claims 1 to 4 and 6 to 9. The aforementioned disease or condition is abnormal hemoglobinosis, thalassemia, or sickle cell disease. A composition comprising the method of administering the composition to the subject.

20. The composition for use according to any one of claims 17 to 19, wherein the composition is administered intravenously to the subject.

21. The composition for use according to any one of claims 17 to 20, wherein the method further comprises administering a mobilizing agent to the subject.

22. The composition for use according to claim 21, wherein the mobilizer comprises one or more of granulocyte colony-stimulating factor (G-CSF), a CXCR4 antagonist, and a CXCR2 agonist.

23. The composition for use according to claim 22, wherein the CXCR4 antagonist is AMD3100.

24. The composition for use according to claim 22 or 23, wherein the CXCR2 agonist is GRO-β.

25. The composition for use according to any one of claims 17 to 24, wherein the transposon payload comprises a selection cassette, and the method comprises administering a selectant to the subject.

26. The aforementioned selection cassette is mgmt Ｐ１４０Ｋ The code is, and the selector is O ６ A composition for use according to claim 25, wherein BG / BCNU.

27. The composition for use according to any one of claims 17 to 26, wherein the method causes the incorporation and / or expression of at least one copy of the transposon payload in at least 20% of the cells expressing CD46.

28. Hematopoietic stem cells and / or erythrocytes Ter119 ＋ A composition for use according to any one of claims 17 to 26, which causes the incorporation and / or expression of at least one copy of the transposon payload in at least 20% of the cells.

29. A composition for use according to any one of claims 17 to 28, which causes the incorporation of an average of at least two copies of the transposon payload into the genome of a cell containing at least one copy of the transposon payload.

30. A composition for use according to any one of claims 17 to 29, which causes the incorporation of an average of at least 2.5 copies of the transposon payload into the genome of a cell containing at least one copy of the transposon payload.

31. The composition for use according to any one of claims 17 to 30, wherein the method causes the expression of the γ-globin protein encoded by the transposon payload at a level of at least 20% of the reference level.

32. The composition for use according to any one of claims 17 to 31, wherein the method causes the expression of the γ-globin protein encoded by the transposon payload at a level of at least 25% of the reference level.

33. The composition for use according to claim 31 or 32, wherein the reference is the expression of an endogenous reference protein in the subject or reference population.

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