Improvement of recombinant AD35 vector and related gene therapy

Abstract

Providing recombinant AD35 vectors and related gene therapy improvements. [Solution] This disclosure provides, among other things, a helper-dependent adenovirus serotype 35 (Ad35) vector. In various embodiments, the helper-dependent Ad35 vector can be used to deliver a therapeutic payload to a target that needs it. Examples of payloads may encode replacement proteins, antibodies, CARs, TCRs, small RNAs, and genome editing systems. In certain embodiments, the helper-dependent Ad35 vector is manipulated so that the payload is incorporated into the host cell genome. This disclosure further includes gene therapy methods comprising administering the helper-dependent Ad35 vector to a target that needs it.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 62 / 869,907 filed July 2, 2019, U.S. Provisional Application No. 62 / 935,507 filed November 14, 2019, and U.S. Provisional Application No. 63 / 009,385 filed April 13, 2020, the disclosures of each of these applications being incorporated herein by reference in their entirety.

[0002] Government support This invention was made with government support under grant numbers HL130040, HL141781, and CA204036 granted by the National Institutes of Health. The government has certain rights to this invention.

[0003] Description of sequence listings The sequence listing accompanying this application is provided in text format instead of as a paper copy and is incorporated herein by reference. The name of the text file containing this sequence listing is F053-0107PCT_ST25.txt. This text file (945KB) was created on July 2, 2020, and was filed electronically via EFS-Web. [Background technology]

[0004] Many medical conditions are caused by genetic mutations and / or are at least partially treatable by gene therapy. Such conditions include, for example, hemoglobin disorders, immunodeficiency, and cancer. Hereditary disorders known as hemoglobin disorders are one of the most common types of hereditary disorders globally, and their survival rates are significantly lower in patients born in developing countries. Examples of hemoglobin disorders include sickle cell anemia and thalassemia. Immunodeficiency can be primary or secondary. The number of primary immunodeficiency disorders recognized by the World Health Organization exceeds 80. Preventive and therapeutic interventions are needed for medical conditions caused by genetic mutations and / or that are at least partially treatable by gene therapy. [Overview of the project]

[0005] Gene therapy can treat many conditions involving genetic elements, including, but not limited to, hemoglobin disorders, immunodeficiency, and cancer. While molecular biology offers a variety of tools for genetic manipulation, applying these tools in the context of gene therapy (e.g., ex vivo and in vivo) presents new opportunities and challenges, at least in part, related to the development of genetic constructs for use in gene therapy vectors, as well as the development of such vectors themselves.

[0006] This disclosure includes, in particular, adenovirus vectors and adenovirus genomes (e.g., “recombinant” or “engineered” adenovirus vectors and adenovirus genomes) for expressing a base editor in target cells. This disclosure includes, in particular, adenovirus vectors and adenovirus genomes for expressing a CRISPR system in target cells, comprising a CRISPR enzyme and / or guide RNA (gRNA), which is a CRISPR-related RNA-guided endonuclease, wherein, optionally, the expression of at least one component of this CRISPR system is self-inactivated. This disclosure includes, in particular, adenovirus vectors and adenovirus genomes for expressing a base editing system in target cells, comprising a base editing system enzyme and / or guide RNA (gRNA), wherein, optionally, the expression of at least one component of this base editing system is self-inactivated. This disclosure includes, in particular, adenovirus vectors and adenovirus genomes containing regulatory sequences for expressing expression products (e.g., therapeutic expression products) in target cells, wherein the regulatory sequences include miRNA binding sites or β-globin locus regulatory regions (LCRs) (such as β-globin long chain LCRs). This disclosure includes, in particular, integrated adenovirus vectors and integrated adenovirus genomes for expressing multiple therapeutic expression products (e.g., therapeutic expression products) in target cells that jointly contribute to the treatment of a disease or condition. This disclosure includes, in particular, adenovirus vectors and adenovirus genomes for incorporating a payload containing β-globin long chain LCRs into a target cell genome. This disclosure includes, in particular, adenovirus vectors and adenovirus genomes with reduced immunogenicity compared to certain existing vectors (e.g., Ad5 vectors). This disclosure includes, in particular, an Ad35 adenovirus vector, an Ad35 adenovirus genome, an HDAd35 adenovirus vector, an HDAd35 adenovirus genome, a support vector, a support genome, an Ad35 helper vector, and an Ad35 helper genome, wherein the HDAd35 vector may have reduced immunogenicity compared to certain existing vectors (e.g., an Ad5 vector or an Ad5 / 35 vector).

[0007] This disclosure describes, in particular, CD46-targeted recombinant Ad35 vectors for in vivo gene editing of hematopoietic stem cells and related gene therapy improvements. In certain embodiments of the vector designs disclosed herein, all proteins are derived from serotype 35. In certain embodiments of the Ad35 vectors described herein, no viral genes remain in the vector. In certain embodiments, the ITR and packaging sequence are derived from Ad35. In certain embodiments, the Ad35 delivery vector has all genes encoding viral proteins removed and replaced with components relevant to the therapeutic application.

[0008] In certain embodiments, the Ad35 vector is helper-dependent, and this disclosure also provides a newly designed Ad35 helper vector. In certain embodiments, Ad35 is prepared by providing a helper-dependent transgene plasmid in an optimal ratio.

[0009] The gene therapy improvements described herein relate to one or more of the following: (i) novel Ad35 knob protein mutations that increase binding to CD46; (ii) vector features that enable positive selection of modified cells in vivo; (iii) microRNA regulatory systems that modulate the expression of therapeutic proteins within a clinically appropriate timeframe; (iv) use of homology arms to facilitate insertions targeting specific sites in the genome; (v) use of CRISPR to inactivate genomic suppressor regions to enable increased expression of endogenous genes; (vi) use of recruitment strategies to increase the delivery of Ad35 vectors to targeted CD46-expressing cells; (vii) use of small or long locus regulatory regions to increase gene expression; (viii) use of recombinase systems to increase the size of transposons that can be inserted using transposase systems; (ix) steroid delivery before vector delivery (e.g., glucocorticoids, dexamethasone); and (x) erythrocytes for generating and secreting therapeutic proteins. These related gene therapy improvements can be implemented using the Ad35 vectors described herein, as well as being usable with other viral vector delivery systems. For example, a mutant Ad35 knob protein with increased CD46 binding can be used with lentiviral or foam delivery systems.

[0010] The advances described herein include (i) an in vivo HSC transduction / selection technique for adding a transgene via SB100x using the HDAd5 / 35++ vector, (ii) increasing HbF reactivation by simultaneously targeting the erythrocyte bcl11a enhancer (thereby reducing BCL11A expression, for example) and the HBG1 / 2 promoter region (thereby increasing γ-globin expression), and (iii) in vivo genome genomics using CRISPR. This also relates to (iv) modification of thalassemia, (v) integration of γ gene addition and reactivation (SB100x system), (vi) CRISPR / Cas9 autoinactivation, (vii) targeted integration using HDAd as a donor vector containing an auto-releasing cassette, (viii) in vivo HSC gene therapy using erythrocytes as a factory for producing high levels of secretory therapeutic proteins, (ix) therapeutic methods for treating cancer (prophylactic and therapeutic), and (x) HDAd35++ vector.

[0011] One particular embodiment relates to a mutant knob protein capable of improving the targeting and specificity of therapeutic gene delivery by increasing binding to CD46.

[0012] Certain embodiments relate to the use of homology arms (which can be used to cause integration into chromosomes targeting a genome-safe harbor) to facilitate targeted insertion into the genome, where this targeted insertion is typically performed against open chromatin, which allows for increased expression levels of the transgene. As described herein, in certain embodiments, a 1.8b homology arm works well, with a lower limit of its size being 0.8. When the homology arm exceeds 1.8b, single nucleotide polymorphisms may begin to affect integration.

[0013] A particular embodiment relates to the use of a mobilization regimen to reduce the need for pre-transplant treatment.

[0014] In certain embodiments, (i) a low dose of O6 -MGMT, which can improve the therapeutic effect of short-term treatment with benzylguanine + bis-chloroethylnitrosourea. P140K (ii) an SB100X transposase-based integration mechanism, and (iii) an Ad35 in vivo gene therapy using a micro-LCR-promoting γ-globin gene are provided.

[0015] Specific embodiments include (i) a CRISPR / Cas9 cassette (which targets the BCL11A binding site in the HBG1 / 2 promoter to release the repression of an endogenous gene), and (ii) a γ-globin gene cassette (which is promoted by a 5kb β-globin mini LCR), and EF1α-MGMT. P140K The Ad35 adenovirus vector (HDAd-integrated) includes an expression cassette (which allows in vivo selection of transduced cells with the FRT site and the latter two cassettes adjacent to the transposon site).

[0016] In certain embodiments, a CRISPR / Cas9-mediated genome editing method is provided in adult CD34+ cells aimed at reactivating the expression of fetal gamma globin in erythrocytes. Because models in which CD34+ cells undergo erythrocyte differentiation have limitations in evaluating gamma globin reactivation, the repressor binding region in the gamma globin promoter was disrupted using a helper-dependent human CD46-targeted adenovirus vector (HDAd-HBG-CRISPR) expressing CRISPR / Cas9 in a human β-globin locus transgenic body.

[0017] In certain embodiments, a CD46-targeted embedded Ad35 vector system is provided, which includes (i) a β-globin locus regulatory region (LCR) that promotes the expression of the γ-globin gene, and (ii) an MGMT for positive selection of genetically modified HSCs in vivo. P140K The introduced gene includes EF1-α (a constitutive promoter) which promotes cassette expression.

[0018] In certain embodiments, a CD46-targeted embedded Ad35 vector system is provided, which includes (i) a 21.5kb (long-chain) human β-globin locus regulatory region (LCR(HS1~HS5)) that promotes the expression of the γ-globin gene and a β-globin promoter (1.6kb) (optionally including its 3'UTR), and (ii) an MGMT for positive selection of genetically modified HSCs in vivo. P140K The transgene includes EF1-α (a constitutive promoter) that promotes cassette expression. Some embodiments may further include 3'HS1 (human β-globin 3'HS1 (3kb) (e.g., 3'HS1 has the sequence at positions 5206867-5203839 on chromosome 11)). In various embodiments, 3'HS1 has the nucleic acid sequence shown in SEQ ID NO: 287 or a sequence with at least 80% sequence identity with SEQ ID NO: 287, for example, a sequence with 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 SEQ ID NO: 287. In such embodiments, a highly active transposase (e.g., SB100X) may be used in combination with a recombinase system (e.g., Flp / Frt;Cre / Lox). Therefore, in a particular embodiment, the Ad35 vector system may include, for example, a translocation transgene insert comprising a long-chain human β-globin locus regulatory region (21.5 kb), a human β-globin promoter (1.6 kb), a human γ-globin gene (including its 3'UTR (2.7 kb)), a human β-globin 3'UTR, and 3'HS1 (3 kb). The translocation transgene insert is, for example, MGMT. P140K The Ad35 vector system may further include EF1-α (a constitutive promoter) that promotes the expression of [the gene]. In various embodiments, the Ad35 vector system may include, for example, a 32.4 kb translocation transgene insert.

[0019] In certain embodiments, a miRNA regulatory system is provided that is activated only when an HSPC is recruited to a tumor to control the expression of a therapeutic transgene. These features of the disclosure have been demonstrated using anti-PDL1-γ1 as the transgene. Using such a system, the expression of a therapeutic transgene can be controlled under the conditions of the tumor microenvironment.

[0020] In various embodiments, a microRNA regulatory system may refer to a method or composition in which gene expression is controlled by the presence of a microRNA site (e.g., a nucleic acid sequence on which microRNA can interact), an example of which is shown in Example 5. In certain embodiments, gene expression was controlled by a microRNA regulatory system so that the gene is exclusively expressed in target cells (e.g., HSPCs (e.g., tumor-infiltrating HSPCs)). In some embodiments, a nucleic acid encoding a target protein or target nucleic acid (e.g., a therapeutic gene) comprises, is linked to, or functionally ligated to, a microRNA site, multiple identical microRNA sites, or multiple different microRNA sites. Means and techniques for linking a nucleic acid or a portion thereof having a sequence encoding a target gene to a microRNA site will be known to those skilled in the art, but certain specific methods are described herein. Non-limiting examples are given. For example, a target gene (e.g., a sequence encoding an αPD-L1γ1 antibody) may be located in nucleic acids such that its expression is regulated by the presence of one or more microRNA sites that suppress its expression in cells other than tumor-infiltrating leukocytes but not in tumor-infiltrating leukocytes. In a particular example, a target gene (e.g., a sequence encoding an αPD-L1γ1 antibody) may be located in nucleic acids such that its expression is regulated by the presence of one or more miR423-5p microRNA sites that suppress its expression in cells other than tumor-infiltrating leukocytes but not in tumor-infiltrating leukocytes.In various embodiments, the microRNA regulatory system may include a nucleic acid comprising one or more microRNA sites (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more microRNA sites), or a nucleic acid whose expression of a target protein or target nucleic acid is regulated by one or more microRNA sites (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more microRNA sites). In various embodiments, the microRNA regulatory system may include a nucleic acid containing one or more miR423-5p microRNA sites (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more miR423-5p microRNA sites), or a nucleic acid whose expression of a target protein or target nucleic acid is regulated by one or more miR423-5p microRNA sites (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more miR423-5p microRNA sites). In some specific embodiments, the microRNA regulatory system may include an αPD-L1γ1 antibody-coding nucleic acid containing one or more miR423-5p microRNA sites (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more miR423-5p microRNA sites (e.g., miR423-5p microRNA sites)), or an αPD-L1γ1 antibody-coding nucleic acid in which the expression of an αPD-L1γ1 antibody is regulated by one or more miR423-5p microRNA sites (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more miR423-5p microRNA sites (e.g., miR423-5p microRNA sites)).

[0021] This disclosure describes a CD46-targeted recombinant Ad35 vector for in vivo gene editing of hematopoietic stem cells and related gene therapy improvements. In certain embodiments, the Ad35 delivery vector is one from which all genes encoding viral proteins have been removed and replaced with components relevant to the therapeutic application. By removing all genes encoding viral proteins, a vector with a capacity of 30 kb is obtained, which is considerably larger than the space available on other viral vector delivery platforms. In certain embodiments, the Ad35 vector is helper-dependent, and this disclosure also provides a newly designed Ad35 helper vector. To avoid misunderstanding, the term “gene editing” as used herein includes, but is not limited to, the use of a vector or drug to modify a nucleic acid sequence.

[0022] This specification further provides vectors that encode nucleic acids provided herein (including, but not limited to, microRNA regulatory systems and other nucleic acids containing microRNA (also referred to herein as miRNA) sites (also referred to herein as target sites) disclosed herein) or that contain such nucleic acids and / or drugs disclosed herein (including, but not limited to, antibodies (such as αPD-L1 antibodies (e.g., αPD-L1γ1 antibodies))). In any of the various embodiments of this disclosure, the vector may be an Ad5 / 35 vector, and optionally, the Ad5 / 35 vector may be a helper-dependent Ad5 / 35 (HDAd5 / 35). In any of the various embodiments of this disclosure, the vector may be an Ad5 / 35 vector (e.g., an HDAd5 / 35 vector) containing variations (e.g., amino acid mutations) provided herein, and certain such vectors may be referred to as Ad5 / 35++ (e.g., HDAd5 / 35++). To avoid misunderstanding, it is intended that any embodiment using any of the vectors (including embodiments specifying vectors other than the Ad5 / 35 vector (e.g., vectors other than the Ad5 / 35++ vector or vectors other than the HDAd5 / 35++ vector)) will be clearly interpreted as disclosing vectors that are Ad5 / 35 vectors (e.g., including HDAd5 / 35 vectors, Ad5 / 35++ vectors, and HDAd5 / 35++ vectors) in addition to such vectors described in the related text, and that a person skilled in the art will understand from this disclosure.

[0023] In any of the various embodiments of this disclosure, the vector may be an Ad35 vector, and optionally, the Ad35 vector may be HDAd35. In any of the various embodiments of this disclosure, the vector may be an Ad35 vector (e.g., an HDAd35 vector) including variations (e.g., amino acid mutations) provided herein, and certain such vectors may be referred to as Ad35++ (e.g., HDAd35++). To avoid misunderstanding, it is intended that any embodiment using any of the vectors (including embodiments specifying vectors other than the Ad35 vector (e.g., vectors other than the Ad35++ vector or vectors other than the HDAd35++ vector)) will be clearly interpreted as disclosing vectors that are Ad35 vectors (e.g., HDAd35 vectors, Ad35++ vectors, and HDAd35++ vectors) in addition to such vectors described in the related text, and that those skilled in the art will understand from this disclosure.

[0024] As shown herein, the vectors described herein have many applications, including the treatment of sickle cell disease, addition and reactivation of the gamma globin gene, and targeting of multiple target sites for reactivating gamma globin. Furthermore, the disclosed method can be applied to other secreted proteins in addition to factor VIII (FVIII), and these other applications include, for example, (i) other coagulation factors (specifically FXI, FVII, von Willebrand factor (VWF), and trace coagulation factors (i.e., factor I, factor II, factor V, factor X, factor XI, or factor XIII)), (ii) enzymes currently used in enzyme replacement therapy (ERT) (utilizing cross-correction mechanisms) for lysosomal storage disorders such as Pompe disease, Gaucher disease, Fabry disease, and mucopolysaccharidosis type I (for the above lysosomal storage disorders, acid alpha(α)-glucosidase, glucocerebrosidase, α-galactosidase A, and α-L-iduronidase, respectively), and (iii) immunodeficiency (e.g., SCID-ADA (adenosine deaminase)). (iv) cardiovascular diseases (e.g., familial apolipoprotein E (ApoE) deficiency and atherosclerosis), (v) viral infection due to expression of viral decoy receptors (e.g., HIV-soluble CD4 or broad-spectrum neutralizing antibody (bNAb)) in HIV infection, chronic HCV infection, or HBV infection, (vi) cancer (e.g., expression control with monoclonal antibodies (e.g., trastuzumab) or checkpoint inhibitors (e.g., aPDL1), or protection of HSCs to allow therapeutic doses of chemotherapy), and (vii) the FANCA gene for Fanconi anemia, (viii) coagulation factor deficiencies selected at will from hemophilia A, hemophilia B, or von Willebrand disease, (ix) platelet disorders, (x) anemia, (xi) α1-antitrypsin deficiency, or (xii) immunodeficiency. Other additional uses are described in more detail elsewhere in this specification.

[0025] Therefore, in one embodiment, a CD46-targeted recombinant serotype 35 adenovirus (Ad35) vector is provided for in vivo gene editing of hematopoietic stem cells.

[0026] Another embodiment involves red blood cells genetically modified to express therapeutic proteins. For example, the therapeutic proteins may include coagulation factors or proteins that block or reduce viral infections. Optionally, the red blood cells secrete therapeutic proteins.

[0027] Uses of recombinant Ad35 vectors or erythrocytes as described herein are also provided. Such uses include: use to increase HbF reactivation by simultaneously targeting the erythrocyte bcl11a enhancer and HBG promoter region; use to integrate the addition of the γ-globin gene and the reactivation of the endogenous γ-globin gene; use for in vivo CRISPR genomic manipulation; use to provide therapeutic genes; (i) abnormal hemoglobin disorders; (ii) Fanconi anemia; (iii) coagulation factor deficiencies selected at will from hemophilia A, hemophilia B, or von Willebrand disease; (iv) blood Uses include (v) platelet disorders, (v) anemia, (vi) α1-antitrypsin deficiency, or (v) immunodeficiency; use to treat thalassemia; use to treat cancer, prevent or delay cancer recurrence, or prevent or delay cancer development in carriers of high-risk germline mutations (optionally, cancer is breast cancer or ovarian cancer); use to autoinactivate CRISPR / Cas9; and use for targeted incorporation using HDAd as a donor vector containing an auto-releasing cassette. Any of these uses may optionally include recruitment, for example, recruitment including administration of Gro-beta, GM-CSF, S-CSF, and / or AMD3100.

[0028] Another embodiment of use is the use of either the recombinant Ad35 vector or erythrocytes described herein, which includes administering a steroid (e.g., a glucocorticoid or dexamethasone), an IL-6 receptor antagonist, and / or an IL-1R receptor antagonist to a subject to whom the Ad35 vector and / or erythrocytes have been administered.

[0029] Embodiments of use using either the recombinant Ad35 vector or red blood cells described herein are also provided, which describe the use of an Ad35 vector and / or red blood cells administered to a subject. 6 The administration of BG and TMZ (temozolomide) or BCNU (carmustine) is included. Examples of such embodiments of use include the treatment of anaplastic astrocytoma, breast cancer, colorectal cancer, diffuse Yj endogenous brainstem glioma, Ewing's sarcoma, glioblastoma multiforme (GBM), malignant glioma, melanoma, metastatic malignant melanoma, nasopharyngeal cancer, or childhood cancer. 6 They are being administered BG and TMZ or BCNU.

[0030] Another embodiment is a recombinant adenovirus serotype 35 (Ad35) vector production system, which comprises a recombinant Ad35 helper genome comprising a nucleic acid sequence encoding an Ad35 fiber shaft, a nucleic acid sequence encoding an Ad35 fiber knob, and a recombinase DR adjacent to at least a portion of the Ad35 packaging sequence, and a recombinant helper-dependent Ad35 donor genome comprising a 5'Ad35 ITR, a 3'Ad35 ITR, an Ad35 packaging sequence, and a nucleic acid sequence encoding at least one heterologous expression product.

[0031] A recombinant Ad35 helper vector embodiment is also provided, comprising an adenovirus serotype 35 (Ad35) fiber shaft, an Ad35 fiber knob, and an Ad35 genome containing at least a portion of the Ad35 packaging sequence and an adjacent recombinase DR.

[0032] A recombinant Ad35 helper genome embodiment is also provided, comprising a nucleic acid sequence encoding an Ad35 fiber shaft, a nucleic acid sequence encoding an Ad35 fiber knob, and a recombinase DR adjacent to at least a portion of the Ad35 packaging sequence.

[0033] A recombinant helper-dependent Ad35 donor vector embodiment is also provided, comprising a nucleic acid sequence (this genome does not include a nucleic acid sequence encoding an Ad35 viral structural protein) including a 5'Ad35 ITR, a 3'Ad35 ITR, an Ad35 packaging sequence, and a nucleic acid sequence encoding at least one heterologous expression product, and an Ad35 fiber shaft and / or Ad35 fiber knob.

[0034] A recombinant helper-dependent Ad35 donor genome embodiment is also provided, comprising a 5'Ad35 ITR, a 3'Ad35 ITR, an Ad35 packaging sequence, and a nucleic acid sequence encoding at least one heterologous expression product, wherein the Ad35 donor genome does not include a nucleic acid sequence encoding an expression product encoded by the wild-type Ad35 genome.

[0035] Another embodiment is a method for producing a recombinant helper-dependent Ad35 donor vector, the method comprising isolating a recombinant helper-dependent Ad35 donor vector from a cell culture, the cell comprising a recombinant Ad35 helper genome comprising a nucleic acid sequence encoding an Ad35 fiber shaft, a nucleic acid sequence encoding an Ad35 fiber knob, and at least a portion of the Ad35 packaging sequence and adjacent recombinase DR, and 5'Ad35 The system includes a recombinant helper-dependent Ad35 donor genome comprising an ITR, a 3'Ad35 ITR, an Ad35 packaging sequence, and a nucleic acid sequence encoding at least one heterologous expression product.

[0036] Embodiments of recombinant Ad35 production systems are also provided, comprising a recombinant Ad35 helper genome comprising a nucleic acid sequence encoding an Ad35 fiber shaft, a nucleic acid sequence encoding an Ad35 fiber knob, and a recombinase DR located within 550 nucleotides from the 5' end of the Ad35 genome, which functionally disrupts the Ad35 packaging signal but does not functionally disrupt the 5'Ad35 ITR; and a recombinant Ad35 donor genome comprising a nucleic acid sequence encoding the 5'Ad35 ITR, the 3'Ad35 ITR, the Ad35 packaging sequence, and at least one heterologous expression product.

[0037] Another embodiment is a recombinant Ad35 helper vector comprising an Ad35 fiber shaft, an Ad35 fiber knob, and an Ad35 genome containing a recombinase DR located within 550 nucleotides from the 5' end of the Ad35 genome, which functionally disrupts the Ad35 packaging signal but does not functionally disrupt the 5'Ad35 ITR.

[0038] Another embodiment is a recombinant Ad35 helper genome, which comprises a nucleic acid sequence encoding an Ad35 fiber shaft, a nucleic acid sequence encoding an Ad35 fiber knob, and a DR located within 550 nucleotides from the 5' end of the Ad35 genome, which functionally disrupts the Ad35 packaging signal but does not functionally disrupt the 5'Ad35 ITR.

[0039] Another embodiment is a method for producing a recombinant helper-dependent Ad35 donor vector, the method comprising isolating a recombinant helper-dependent Ad35 donor vector from a cell culture, the cell comprising a recombinant Ad35 helper genome comprising a nucleic acid sequence encoding an Ad35 fiber shaft, a nucleic acid sequence encoding an Ad35 fiber knob, and a recombinase DR located within 550 nucleotides from the 5' end of the Ad35 genome, which functionally disrupts the Ad35 packaging signal but does not functionally disrupt the 5'Ad35 ITR, and a recombinant Ad35 donor genome comprising a nucleic acid sequence encoding the 5'Ad35 ITR, the 3'Ad35 ITR, the Ad35 packaging sequence, and at least one heterologous expression product.

[0040] Another embodiment is a cell comprising the helper vector, helper genome, donor vector, or donor genome described herein, wherein the cell is optionally a HEK293 cell.

[0041] Another embodiment is a cell containing a donor genome from any one of the embodiments described herein, wherein the cell is optionally a erythrocyte, optionally a hematopoietic stem cell, a T cell, a B cell, or a myeloid cell, and optionally a cell that secretes an expression product.

[0042] A method for modifying cells is also provided, which involves contacting a cell with an Ad35 donor vector using one of the provided Ad35 donor vector embodiments.

[0043] A method for modifying target cells is also provided, which involves administering an Ad35 donor vector to the target using one of the Ad35 donor vector embodiments, and optionally, this method does not involve isolating cells from the target.

[0044] Another embodiment is a method of providing treatment to a subject in need of treatment for a disease or condition, the method comprising administering an Ad35 donor vector to a subject using one of the Ad35 donor vector embodiments provided herein, optionally, the administration being performed intravenously.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Affinity: As used herein, "affinity" refers to the strength of the sum of non-covalent interactions that occur between a particular 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 the target cell of the viral vector). 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 binding 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 binding rate constant (e.g., the binding 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.

[0050] Agent: As used herein, the term "agent" can refer to any chemical substance, and such chemical substances include, but are not limited to, any 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.

[0051] 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).

[0052] 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.

[0053] 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).

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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).

[0058] 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).

[0059] 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).

[0060] 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.

[0061] Effective dose: The "effective dose" is the amount of formulation necessary to produce the desired physiological change in a subject. Effective doses are often administered for research purposes.

[0062] Engineered: As used herein, the term “engineered” 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 engineered polynucleotide, then that polynucleotide is considered “engineered.” Those skilled in the art will understand that an “engineered” nucleic acid sequence or “engineered” amino acid sequence may be a recombinant nucleic acid sequence or recombinant amino acid sequence and may be referred to as “genetically engineered.” In some embodiments, the engineered polynucleotide includes coding and / or regulatory sequences that are found naturally in a functionally linked state with a first sequence but not naturally in a functionally linked state with a second sequence, and these coding and / or regulatory sequences are artificially linked to the second sequence in the engineered polynucleotide. In some embodiments, a cell or organism is considered “manipulated” or “genetically engineered” if it has been treated in such a way that its genetic information is altered (for example, if new genetic material that was not previously present is introduced (this introduction is carried out, for example, by transformation, crossing, somatic cell hybridization, transfection, transduction, or other mechanisms), or if existing genetic material is 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, a manipulated polynucleotide or offspring or copy (complete or incomplete) of a cell is typically still referred to as “manipulated,” even if the direct treatment was carried out on the original entity.

[0063] 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.

[0064] 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.

[0065] Adjacent: As used herein, a first element (e.g., a nucleic acid sequence or an amino acid sequence) present in a contiguous sequence containing a second and a third element is “adjacent” to the second and third elements if it is located between the second and third elements in the contiguous sequence. Thus, in such an arrangement, the second and third elements may be referred to as “adjacent” to the first element. Adjacent elements may be located immediately next to the adjacent element, or separated from the adjacent element by one or more related units. In various examples where the sequence is a nucleic acid sequence or an amino acid sequence, and the associated units are each a base or an amino acid residue, the number of units in the sequence between an adjacent element and a first adjacent element, and / or between the adjacent element and a second adjacent element, can independently be, for example, 50 units or less (e.g., 50 units or less, 45 units or less, 40 units or less, 35 units or less, 30 units or less, 25 units or less, 20 units or less, 15 units or less, 10 units or less, 5 units or less, 4 units or less, 3 units or less, 2 units or less, 1 unit or less, or 0 units).

[0066] 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, the polymer fragment contains or consists of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 25%, 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 96%, at least 97%, at least 98%, at least 99%, or more percent of monomer units (e.g., residues) found in the reference polymer. The 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 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 25%, 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 96%, at least 97%, at least 98%, at least 99%, or more. The fragment may or may not be produced by the physical fragmentation of the reference drug. In some cases, the fragment is produced by the physical fragmentation of the reference drug. In other cases, the fragment is not produced by the physical fragmentation of the reference drug, but instead may be produced by, for example, de novo synthesis or other means.

[0067] 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.

[0068] 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).

[0069] 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.

[0070] In various embodiments, host cells or target cells are identified by the presence, absence, or expression levels of various surface markers.

[0071] The statement that a cell or cell population is “positive” for a particular marker, or expresses a particular marker, means that the presence of the particular marker on or inside the cell is detectable. Where there is a reference to a surface marker, this term may mean that surface expression exists as detectable by flow cytometry, and this detection is performed, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, and this stain is detectable by flow cytometry at a level substantially higher than the stain detected by performing the same procedure under otherwise identical conditions using an isotype-matched control, and / or at a level substantially similar to that of cells known to be marker-positive, and / or at a level substantially higher than that of cells known to be marker-negative.

[0072] The statement that a cell or cell population is “negative” for a particular marker, or does not express the marker, means that the presence of the particular marker on or inside the cell is substantially undetectable. Where there is a reference to a surface marker, this term may mean that there is no surface expression as detectable by flow cytometry, and this detection is performed, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, and this stain is not detected by flow cytometry at a level substantially higher than the stain detected by performing the same procedure under otherwise identical conditions using an isotype-matched control, and / or at a level substantially lower than the level of cells known to be marker-positive, and / or at a level substantially similar to the level of cells known to be marker-negative.

[0073] 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). In the art, methods are known for calculating the percentage of identity between two sequences provided. The term “sequence identity %” refers to the relationship between two or more sequences determined by comparing those sequences. In the art, “identity” also means the degree of sequence relevance between such sequences, determined by the correspondence between protein sequences and the correspondence between nucleic acid sequences. “Identity” (often referred to as “similarity”) can be readily calculated by known methods, such as those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988), Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994), Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994), Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987), and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University This includes the method described in Press, NY (1992). Preferred identity determination methods are designed to maximize the agreement between the sequences being tested. Methods for determining identity and similarity are systematized in publicly available computer programs. For example, calculating the identity percentage of two nucleic acid sequences or polypeptide sequences can be done, for example, by aligning the two sequences (or complementary sequences of one or both sequences) for the purpose of optimal comparison (for example, gaps may be introduced in one or both of the first and second sequences to optimize the alignment, and non-identical sequences may be ignored for comparison purposes). A comparison of nucleotides or amino acids is then performed at 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, the number of gaps and the length of each gap may be considered, and such gaps may need to be introduced to optimize the alignment between the two sequences. Sequence comparison and identity percentage determination between two sequences can be achieved using computational algorithms (such as BLAST (Basic Local Alignment Search Tool)). Sequence alignment and identity percentage calculation can be performed using the Megalign program in the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple sequence alignment can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989)) with default parameters (gap penalty = 10, gap length penalty = 10).Related programs include the GCG program suite (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin), BLASTP, BLASTN, BLASTX (Altschul et al., J.Mol.Biol.215:403-410 (1990)), DNASTAR (DNASTAR, Inc., Madison, Wisconsin), and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput.Methods Genome Res., [Proc.Int.Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, NY). In the context of this disclosure, it will be understood that when sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the programs mentioned. "Default values" refer to any set of values ​​or parameters that are initially set by the software during the initial initialization.

[0074] "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.

[0075] 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 when it first occurred (whether in natural and / or experimental circumstances), and / or (2) artificially designed, generated, prepared, and / or manufactured. An isolated substance and / or entity may have been obtained by separating 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% of the other components that originally accompanied it. In some embodiments, the purity of the isolated drug is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99%. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may 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, for reasons of its origin or source, some or all of the components that accompany it in its natural state are absent; if, b) it is substantially free of 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 a) it is naturally associated with it and / or b) it is separated from other components that were associated with it when it was first produced.

[0076] Operafully linked: As used herein, “operably linked” or “operatively 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 sequence is linked to a nucleic acid coding sequence in such a way that the control sequence enables the expression control of the coding sequence, then the control sequence is “operably linked” to the coding sequence. In some embodiments, an “operably linked” control sequence is directly or indirectly covalently linked to the coding sequence (e.g., in a single nucleic acid). In some embodiments, the control sequence controls the expression of the coding sequence in trans, and it is not a requirement for operably linked that the control sequence be in the same nucleic acid as the coding sequence.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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 “functionally 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 the 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 contain such a DNA regulatory region. A promoter may be an expression regulatory sequence (such as an enhancer sequence and a repressor sequence), contain an expression regulatory sequence (such as an enhancer sequence and a repressor sequence), or be functionally linked to or functionally 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).

[0081] 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.

[0082] 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.).

[0083] 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.”

[0084] 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.

[0085] 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, this 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 the 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 actually require that the treatment be successfully 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).

[0086] 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. "Prophylactic treatment" includes treatments given to subjects who do not show signs or symptoms of a condition to be treated, or who show only early signs or symptoms of a condition to be treated, and such treatments are given with the aim of reducing, preventing, or decreasing the risk of developing such a condition. Thus, prophylactic treatment functions as a blocking treatment for a condition. "Therapeutic treatment" includes treatments given to subjects who show symptoms or signs of a condition, and such treatments are given to subjects with the aim of reducing the severity or progression of such a condition.

[0087] Unit Dose: As used herein, the term “unit dose” refers to a single dose of a pharmaceutical composition and / or an amount administered as a physically distinct unit. In many embodiments, a unit dose comprises a predetermined amount of the active agent and has, for example, 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, these may include an acceptable carrier (e.g., a pharmaceutically acceptable carrier), diluents, stabilizers, buffers, preservatives, etc. 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 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 used, the duration of treatment, any drugs and / or additional treatments used in combination or concurrently with the particular compound used, as well as similar factors well known in the medical field.

[0088] Many of the drawings submitted 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 subsequent proceedings. [Brief explanation of the drawing]

[0089] [Figure 1-1] Schematic diagrams of vector examples are shown. These schematic diagrams of vector examples illustrate possible component arrangements in embedded cassettes and transient expression cassettes useful in embodiments of the provided Ad35 vector. The embedded cassette includes transposons and other components between the frt sites. The HDAd vector may include expression products (Exp.Product) (e.g., γ-globin, GFP, mCherry, and hFVIII (ET3)), promoters (multiple possible) (e.g., EF1α promoter, PGK promoter, or β promoter), selection markers (multiple possible) (e.g., mgmtP140K), regulatory elements (Reg.Element) (e.g., promoter, poly-A tail), and / or insulators (e.g., cHS4). The transient expression cassette includes similar components, as well as DNA cleavage molecules (multiple possible) (e.g., spCas9) or base editors (multiple possible) and genome targeting guides (GTG; e.g., sgRNA). Transposase vectors include target-directed recombinases (e.g., FlpE) and transposases (e.g., SB100x). These vectors are shown in one orientation / direction, but may also be provided in the reverse orientation. [Figure 1-2]Schematic diagrams of vector examples are shown. These schematic diagrams of vector examples illustrate possible component arrangements in embedded cassettes and transient expression cassettes useful in embodiments of the provided Ad35 vector. The embedded cassette includes transposons and other components between the frt sites. The HDAd vector may include expression products (Exp.Product) (e.g., γ-globin, GFP, mCherry, and hFVIII (ET3)), promoters (multiple possible) (e.g., EF1α promoter, PGK promoter, or β promoter), selection markers (multiple possible) (e.g., mgmtP140K), regulatory elements (Reg.Element) (e.g., promoter, poly-A tail), and / or insulators (e.g., cHS4). The transient expression cassette includes similar components, as well as DNA cleavage molecules (multiple possible) (e.g., spCas9) or base editors (multiple possible) and genome targeting guides (GTG; e.g., sgRNA). Transposase vectors include target-directed recombinases (e.g., FlpE) and transposases (e.g., SB100x). These vectors are shown in one orientation / direction, but may also be provided in the reverse orientation. [Figure 1-3]Schematic diagrams of vector examples are shown. These schematic diagrams of vector examples illustrate possible component arrangements in embedded cassettes and transient expression cassettes useful in embodiments of the provided Ad35 vector. The embedded cassette includes transposons and other components between the frt sites. The HDAd vector may include expression products (Exp.Product) (e.g., γ-globin, GFP, mCherry, and hFVIII (ET3)), promoters (multiple possible) (e.g., EF1α promoter, PGK promoter, or β promoter), selection markers (multiple possible) (e.g., mgmtP140K), regulatory elements (Reg.Element) (e.g., promoter, poly-A tail), and / or insulators (e.g., cHS4). The transient expression cassette includes similar components, as well as DNA cleavage molecules (multiple possible) (e.g., spCas9) or base editors (multiple possible) and genome targeting guides (GTG; e.g., sgRNA). Transposase vectors include target-directed recombinases (e.g., FlpE) and transposases (e.g., SB100x). These vectors are shown in one orientation / direction, but may also be provided in the reverse orientation. [Figure 2A]This shows an embedded HDAd5 / 35++ vector for gene therapy of HSPC, a disorder of hemoglobinopathy. The vector structure is shown. In HDAd-γ-globin / mgmt, the reverse transposon repeat sequence (IR) and FRT site for embedding via the highly active Sleeping Beauty transposase (SB100X) derived from the HDAd-SB vector (right panel) are adjacent to an 11.8kb transposon. The γ-globin expression cassette contains a 4.3kb version of the β-globin LCR containing four DNase-sensitive (HS) regions and a 0.7kb β-globin promoter. The 76-Ile HBG1 gene, including the 3'-UTR (for stabilizing mRNA in red blood cells), was used. To avoid interference between the LCR / β-promoter and the EF1A promoter, a 1.2kb chicken HS4 chromatin insulator (Ins) was inserted between the cassettes. The HDAd-SB vector contains the gene for the enhanced SB100X transposase and the gene for Flpe recombinase (both under the control of ubiquitously active PGK and EF1A promoters, respectively). [Figure 2B] This paper describes an embedded HDAd5 / 35++ vector for gene therapy of HSPCs with abnormal hemoglobinopathy. It also shows in vivo transduction of recruited CD46tg mice. HSPCs were recruited by subcutaneous injection of human recombinant G-CSF for 4 days, followed by a single subcutaneous injection of AMD3100. 30 and 60 minutes after AMD3100 injection, a 1:1 mixture of HDAd-γ-globin / mgmt and HDAd-SB was intravenously injected into the animals (2 injections in total, 4 × 10¹⁰ virus particles each). For the next 4 weeks, mice were treated with immunosuppressants (IS) to avoid immune responses to human γ-globin and MGMTP140K. O6-BG / BCNU treatment was initiated in week 4 and repeated every 2 weeks for 3 cycles. The BCNU concentration was increased with each cycle from 5 mg / kg to 7.5 mg / kg to 10 mg / kg. Immunosuppression was restarted two weeks after the last O6-BG / BCNU injection. [Figure 2C]This shows an embedded HDAd5 / 35++ vector for gene therapy of HSPC (Hyperhemoglobinopathy). The percentage of human γ-globin + peripheral RBCs measured by flow cytometry is shown. Each symbol represents an individual animal. [Figure 2D] This shows an embedded HDAd5 / 35++ vector for gene therapy of HSPC (Hyperhemoglobinopathy). It shows the percentage of human γ-globin+ cells in peripheral blood mononuclear cells (MNCs), total cells, erythrocytes (Ter119+), and non-erythrocytes (Ter119-). Each symbol represents an individual animal. [Figure 2E] This shows an embedded HDAd5 / 35++ vector for gene therapy of HSPC (Hyperhemoglobinopathy). The percentage of human γ-globin protein relative to adult mouse globin chains (α, β-major, β-minor) in RBCs is shown, measured by HPLC at 18 weeks. Each symbol represents an individual animal. [Figure 2F] This shows an embedded HDAd5 / 35++ vector for gene therapy of HSPC (Hyperhemoglobinopathy). The percentage of human γ-globin mRNA relative to adult mouse β-major globin mRNA is shown, measured by RT-qPCR in whole peripheral blood cells at 18 weeks. Mice without any treatment were used as controls. Each symbol represents an individual animal. [Figure 3-1] Figure 3 shows the HPLC analysis of globin chains in RBCs obtained from hCD46tg control mice and representative CD46tg mice after in vivo transduction / selection. The numerical values ​​(volts) indicate peak intensity. A total of four mice from each group were analyzed, and similar results were obtained. A summary of this data is shown in Figure 2E. In Figure 3, the area under the curve (AUC) value is shifted to the left of the corresponding peak. [Figure 3-2]Figure 3 shows the HPLC analysis of globin chains in RBCs obtained from hCD46tg control mice and representative CD46tg mice after in vivo transduction / selection. The numerical values ​​(volts) indicate peak intensity. A total of four mice from each group were analyzed, and similar results were obtained. A summary of this data is shown in Figure 2E. In Figure 3, the area under the curve (AUC) value is shifted to the left of the corresponding peak. [Figure 4] This shows the analysis of mice transplanted with bone marrow Lin- cells ("secondary recipients") collected 18 weeks after in vivo transduction. (A) Engraftment is measured in blood samples at the indicated time point, based on the percentage of human CD46-positive cells in PBMCs. (B) Engraftment in bone marrow, spleen, and PBMCs at 20 weeks. (C) The ratio of human γ-globin protein to mouse α-globin protein in RBCs is measured by HPLC. Each symbol represents an individual animal. Statistical analysis was performed using the non-parametric Kruskal-Wallis test. [Figure 5A] This shows an analysis of transgene integration in bone marrow cells of secondary recipients at 20 weeks. The localization of integration sites on mouse chromosomes in bone marrow cells is shown. Representative mouse data is shown. Each line represents an integration site. The number of integration sites in this sample is 2,197. [Figure 5B] This report presents an analysis of transgene integration in bone marrow cells of secondary recipients at 20 weeks. The distribution of integrations within genomic regions is shown. Integration site data from five mice were pooled and used to create the graphs. [Figure 5C] This report presents an analysis of transgene integration in bone marrow cells of secondary recipients at week 20. The number and size of integrations overlapping with the continuous genome frame and with the randomized mouse genome frame were compared. Pooled data from Figure 5B were used. The Pearson χ² test p-value for similarity was 0.06381, suggesting that the integration pattern was nearly random. [Figure 5D]This report shows the analysis of transgene integration in bone marrow cells of secondary recipients at 20 weeks. The transgene copy number is indicated. Genomic DNA obtained from whole bone marrow cells derived from non-transduced control mice and secondary recipients at 20 weeks was subjected to qPCR using human γ-globin-specific primers. The copy number per cell is shown for each individual animal. Each symbol represents an individual animal. [Figure 5E] This paper presents an analysis of transgene integration in bone marrow cells of secondary recipients at 20 weeks. The transgene copy number in individual clonal progenitor cell colonies is shown. Bone marrow Lin- cells were seeded on methylcellulose, and individual colonies were picked after 15 days. qPCR was performed on genomic DNA. The qPCR signals in individual colonies are shown, normalized as the transgene copy number per cell (n=113). Each symbol represents the copy number in an individual colony derived from a single cell. [Figure 6] The VCN was measured by qPCR on single-cell-derived progenitor cell colonies (see Figure 7E). [Figure 7A] This shows hematological parameters after in vivo transduction / selection of HSPCs in CD46tg mice (18 weeks after HDAd injection). WBC counts are shown. Each symbol represents an individual animal: NE: neutrophil, LY: lymphocyte, MO: monocyte, BA: basophil. [Figure 7B] This shows hematological parameters after in vivo transduction / selection of HSPCs in CD46tg mice (18 weeks post-HDAd injection). Representative blood smears are shown from untreated mice and mice 18 weeks post-injection with HDAd-γ-globin / mgmt + HDAd-SB. Scale bar: 20 μm. WBC nuclei are stained purple. [Figure 7C]This report shows the hematological parameters of CD46tg mice (18 weeks after HDAd injection) after in vivo transduction / selection of HSPCs. Hematological parameters are shown below: Hb: Hemoglobin, HCT: Hematocrit, MCV: Mean Corpuscular Volume, MCH: Mean Corpuscular Hemoglobin, MCHC: Mean Corpuscular Hemoglobin Concentration, RDW: Red Blood Cell Distribution Width. n≧3, *P<0.05. Statistical analysis was performed using two-way ANOVA. Each symbol represents an individual animal: NE: Neutrophil, LY: Lymphocyte, MO: Monocyte, BA: Basophil. [Figure 7D] This shows hematological parameters after in vivo transduction / selection of HSPCs in CD46tg mice (18 weeks after HDAd injection). Bone marrow cell composition is shown for naive mice (control) and treated mice sacrificed at 18 weeks. Percentages of lineage marker-positive cells (Ter119+ cells, CD3+ cells, CD19+ cells, and Gr-1+ cells) and HSPCs (LSK cells) are shown. Each symbol represents an individual animal: NE: neutrophil, LY: lymphocyte, MO: monocyte, BA: basophil. [Figure 7E] This shows the hematological parameters of CD46tg mice (18 weeks after HDAd injection) after in vivo transduction / selection of HSPCs. It also shows the colony-forming ability of bone marrow lin-cells collected 18 weeks after in vivo transduction. The number of colonies formed after seeding 2,500 lin-cells is shown. Each symbol represents an individual animal: NE: neutrophil, LY: lymphocyte, MO: monocyte, BA: basophil. [Figure 8] This study demonstrates the generation of a CD46++ / Bhhth-3 thalassemia model. Female CD46tg mice were crossbred with male Hbbth-3 mice. The F1 hybrid mice were backcrossed with hCD46+ / + mice to obtain hCD46+ / + (homozygous) Hbbth-3 mice. [Figure 9A]This shows the phenotype of the CD46+ / + / Hbbth-3 mouse thalassemia model. Hematological parameters of CD46+ / + / Hbbth-3 mice (n=7) are compared to those of CD46tg (n=3) and Hbbth-3 mice (n=3). Each symbol represents an individual animal. *P≦0.05, **P≦0.0002, ***P≦0.00003. Statistical analysis was performed using two-way ANOVA. RET: Reticulocytes. [Figure 9B] This shows the phenotype of a CD46+ / + / Hbbth-3 mouse thalassemia model. A representative peripheral blood smear after May-Grünwald / Giemsa staining is shown. Scale bar: 20 μm. [Figure 9C] This shows the phenotype of the CD46+ / + / Hbbth-3 mouse thalassemia model. It compares extramedullary hematopoiesis (bottom left two panels) of liver and spleen sections of CD46+ / + / Hbbth-3 mice, as shown by H&E staining, with liver and spleen sections of CD46tg mice (top left two panels). Scale bar: 20 μm. The bottom left panel shows erythroblast clusters in the liver. In the bottom center panel, megakaryocytes in the spleen are marked with circles. The top right panel (CD46tg mouse) and bottom right panel (CD46+ / + / Hbbth-3 mouse) show iron deposition (bluish granular deposition) in the spleen, as shown by pearl Prussian blue staining. Scale bar: 25 μm. [Figure 10] This shows the analysis of leukocytes in thalassemia mice (Hbbth-3 and CD46+ / + / Hbbth-3) compared to those of "healthy" CD46tg mice. WBC: leukocytes, NEU: neutrophils, LY: lymphocytes, MONO: monocytes. *p≦0.05, **p≦0.0002, ***p≦0.00003. These are baseline levels in pre-treatment mice (CD46tg (n=8), Hbbth3 (n=4), CD46++ / Hbbth3 (n=20)). Each symbol represents an individual animal. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 11]This shows the recruitment of HSPCs in CD46+ / + / Hbbth-3 mice. The number of LSK (lineage- / Sca-1+ / c-Kit+ / ) cells recruited into peripheral blood 1 hour after the last AMD3100 injection is shown. There were 17 mice for recruitment treatment and 3 mice for untreated mice. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 12] This report describes in vivo transduction / selection in recruited CD46+ / + / Hbbth-3 mice. Transduction was performed in vivo in recruited CD46+ / + / Hbbth-3 mice. HSPCs were recruited by subcutaneous injection of human recombinant G-CSF for 6 days (days 1-6), followed by three subcutaneous injections of AMD3100 / prelixafor (days 5-7). A 1:1 mixture of HDAd-γ-globin / mgtm + HDAd-SB was intravenously injected into the animals 30 and 60 minutes after prelixafor injection (total of two injections, 4 × 10¹⁰ vp each). Immunosuppression was performed for 17 weeks after in vivo transduction to avoid immune responses to human γ-globin protein and MGMTP140K protein. At week 17, the treated mice were either used as donors for secondary transplantation or subjected to in vivo selection using O6-BG / BCNU. Secondary C57Bl / 6 recipients were sacrificed after 16 weeks of immunosuppression. Mice subjected to in vivo selection were treated with O6-BG / BCNU at escalating doses (5 mg / kg, 7.5 mg / kg, 10 mg / kg, 10 mg / kg) every week. Immunosuppression was restarted two weeks after the last O6-BG / BCNU dose. At week 29, the mice were sacrificed and their bone marrow was transplanted into C57Bl / 6 secondary recipients. [Figure 13A] This shows the analysis of in vivo transduced CD46+ / + / Hbbth-3 mice that were not treated with O6BG / BCNU. The percentage of human γ-globin in peripheral RBCs is shown, measured by flow cytometry. Three experiments were performed, and these are indicated by different symbolic shapes. [Figure 13B]This shows the analysis of in vivo transduced CD46+ / + / Hbbth-3 mice that were not treated with O6BG / BCNU. It shows the expression of γ-globin in erythrocytes (Ter119+) and non-erythrocytes (Ter119-). ***P ≤ 0.00003 (by one-way ANOVA). [Figure 13C] This section presents the analysis of in vivo transduced CD46+ / + / Hbbth-3 mice that were not treated with O6BG / BCNU. It shows RBC analysis of healthy (CD46tg) mice (n=3), CD46+ / + / Hbbth-3 mice before recruitment and in vivo transduction (n=14), and CD46+ / + / Hbbth-3 mice analyzed at 16 weeks after in vivo transduction (n=8). *P≦0.05. Statistical analysis was performed using two-way ANOVA. [Figure 13D] This shows the analysis of in vivo transduced CD46+ / + / Hbbth-3 mice that were not treated with O6BG / BCNU. Histological phenotypes are shown. Top: Blood smear. Center: Peripheral blood smear stained with brilliant cresil blue to detect reticulocytes. The percentage of positively stained reticulocytes in representative smears was 8%±0.8% for CD46tg, 39%±1.3% for CD46+ / + / Hbbth-3 before transduction, and 26%±0.45% for CD46+ / + / Hbbth-3 at 16 weeks post-transduction. Bottom: Extramedullary hematopoiesis. Scale bar: 20 μm. [Figure 13E] Analysis of in vivo transduced CD46+ / + / Hbbth-3 mice that were not treated with O6BG / BCNU is shown. Analysis of secondary recipients is also shown. Whole bone marrow obtained from 16-week-old in vivo transduced mice was transplanted into C57BL / 6 mice that had undergone sublethal pre-transplant treatment with busulfan. The mice were immunosuppressed during the observation period. Engraftment is shown based on the percentage of human CD46+ (hCD46+) PBMCs. (C57BL / 6 recipients do not express hCD46). Each symbol represents an individual animal. [Figure 13F]This shows the analysis of in vivo transduced CD46+ / + / Hbbth-3 mice that were not treated with O6BG / BCNU. The analysis of secondary recipients is also shown. Whole bone marrow obtained from 16-week-old in vivo transduced mice was transplanted into C57BL / 6 mice that had undergone sublethal pre-transplant treatment with busulfan. The mice were immunosuppressed during the observation period. The percentage of human γ-globin + RBCs is shown. Each symbol represents an individual animal. [Figure 14A] This figure shows the analysis of γ-globin expression in in vivo transduced CD46+ / + / Hbbth-3 mice after in vivo selection. The percentage of human γ-globin in peripheral RBCs is shown, measured by flow cytometry. Arrows indicate the time point of O6-BG / BCNU treatment. Different symbols represent three independent experiments. Data up to week 16 are identical to those in Figure 13A. [Figure 14B] This shows the analysis of γ-globin expression in in vivo transduced CD46+ / + / Hbbth-3 mice after in vivo selection. The percentage of γ-globin-expressing cells in hematopoietic tissue at sacrifice (week 29) is analyzed by flow cytometry. *P≦0.05, **P≦0.0002, ***P≦0.00003. [Figure 14C] This shows the analysis of γ-globin expression in in vivo transduced CD46+ / + / Hbbth-3 mice after in vivo selection. γ-globin expression in Ter119 cells purified by MACS is also shown. Ter119+ cells were selected immunomagnetically from bone marrow cells obtained from primary recipients at 29 weeks. γ-globin expression in Ter119+ and Ter119- cells was measured by flow cytometry. ***P≦0.0002. [Figure 14D]This shows the analysis of γ-globin expression in in vivo transduced CD46+ / + / Hbbth-3 mice after in vivo selection. It also compares the enrichment levels of γ-globin-positive erythrocytes (Ter119+) and γ-globin-positive non-erythrocytes (Ter119-) in peripheral blood, bone marrow, and spleen before and after in vivo selection (comparison between weeks 16 and 29). n=5, **P≦0.0002. [Figure 14E] This shows the analysis of γ-globin expression in in vivo transduced CD46+ / + / Hbbth-3 mice after in vivo selection. The percentage of human γ-globin protein relative to mouse α-globin protein in RBCs is shown, measured by HPLC. Statistical analysis was performed using the non-parametric Kruskal-Wallis test. [Figure 14F] This shows the analysis of γ-globin expression in in vivo transduced CD46+ / + / Hbbth-3 mice after in vivo selection. It also shows the levels of human γ-globin mRNA relative to adult mouse β-major globin mRNA in peripheral blood cells, measured by RT-qPCR. Untreated CD46+ / + / Hbbth-3 mice were used as a control. Each symbol represents an individual animal. [Figure 15A] The HPLC analysis of globin chains in RBCs is shown. (Figure 15A) A representative chromatogram showing the peaks of mouse globin in a control CD46tg mouse is shown. Peaks for adult mouse alpha (α) globin, beta (β)-minor globin, and β-major globin are labeled. (Figures 15B-15D) Chromatograms of RBCs obtained from CD46+ / + / Hbbth-3 mice (#71) are shown. Note that these mice are heterozygous for deletions of the β-minor and β-major genes. The presence of an additional peak around 29 min may be related to this. (Figure 15D) shows a peak specific to human γ-globin. The shown are representative chromatograms. The values ​​(volts) indicate peak intensity. In Figures 15C and 15D, the AUC values ​​are shifted to the left of the corresponding peaks. [Figure 15B] The HPLC analysis of globin chains in RBCs is shown. (Figure 15A) A representative chromatogram showing the peaks of mouse globin in a control CD46tg mouse is shown. Peaks for adult mouse alpha (α) globin, beta (β)-minor globin, and β-major globin are labeled. (Figures 15B-15D) Chromatograms of RBCs obtained from CD46+ / + / Hbbth-3 mice (#71) are shown. Note that these mice are heterozygous for deletions of the β-minor and β-major genes. The presence of an additional peak around 29 min may be related to this. (Figure 15D) shows a peak specific to human γ-globin. The shown are representative chromatograms. The values ​​(volts) indicate peak intensity. In Figures 15C and 15D, the AUC values ​​are shifted to the left of the corresponding peaks. [Figure 15C] The HPLC analysis of globin chains in RBCs is shown. (Figure 15A) A representative chromatogram showing the peaks of mouse globin in a control CD46tg mouse is shown. Peaks for adult mouse alpha (α) globin, beta (β)-minor globin, and β-major globin are labeled. (Figures 15B-15D) Chromatograms of RBCs obtained from CD46+ / + / Hbbth-3 mice (#71) are shown. Note that these mice are heterozygous for deletions of the β-minor and β-major genes. The presence of an additional peak around 29 min may be related to this. (Figure 15D) shows a peak specific to human γ-globin. The shown are representative chromatograms. The values ​​(volts) indicate peak intensity. In Figures 15C and 15D, the AUC values ​​are shifted to the left of the corresponding peaks. [Figure 15D]The HPLC analysis of globin chains in RBCs is shown. (Figure 15A) A representative chromatogram showing the peaks of mouse globin in a control CD46tg mouse is shown. Peaks for adult mouse alpha (α) globin, beta (β)-minor globin, and β-major globin are labeled. (Figures 15B-15D) Chromatograms of RBCs obtained from CD46+ / + / Hbbth-3 mice (#71) are shown. Note that these mice are heterozygous for deletions of the β-minor and β-major genes. The presence of an additional peak around 29 min may be related to this. (Figure 15D) shows a peak specific to human γ-globin. The shown are representative chromatograms. The values ​​(volts) indicate peak intensity. In Figures 15C and 15D, the AUC values ​​are shifted to the left of the corresponding peaks. [Figure 16] This shows DNA analysis of treated CD46++ / Hbbth-3 mice at 29 weeks. The number of transgene (γ-globin) copies per bone marrow cell is shown. Each symbol represents an individual animal. [Figure 17A] This study demonstrates phenotypic modification of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. RBC analysis (n=5) is shown for healthy (CD46tg) mice, CD46+ / + / Hbbth-3 mice before recruitment and in vivo transduction, and CD46+ / + / Hbbth-3 mice after in vivo transduction / selection (analyzed 29 weeks after HDAd injection). *P≦0.05, **P≦0.0002, ***P≦0.00003. Statistical analysis was performed using two-way ANOVA. [Figure 17B]Demonstrates the phenotypic correction of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. Shows peripheral blood smear specimens supravitally stained with brilliant cresyl blue to detect reticulocytes. Arrows indicate reticulocytes containing characteristic residual RNA and micro-organelles. The percentage of reticulocytes positively stained in representative smear specimens was 7% for CD46, 31% for untreated CD46+ / + / Hbbth-3, and 12% for treated CD46+ / + / Hbbth-3. Scale bar: 20 μm. [Figure 17C] Demonstrates the phenotypic correction of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. Top: Blood smear specimen. Scale bar: 20 μm. Center: Cytospin preparation of bone marrow. Arrows indicate erythroblasts at different maturation stages and show that the erythropoiesis dominated by proerythroblasts is normalized in treated mice. Scale bar: 25 μm. Bottom: Hemosiderin deposition in tissues by Perl's staining. Iron deposition is shown as cytoplasmic hemosiderin stained with blue pigment in spleen tissue sections. Images of blood smear specimens of control mice (CD46tg and CD46+ / + / Hbbth-3 before transduction) in Figures 17C and 18D were obtained from the same samples. [Figure 17D] Demonstrates the phenotypic correction of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. Shows macroscopic spleen images of one representative CD46tg mouse, one untreated CD46+ / + / Hbbth-3 mouse, and five treated CD46+ / + / Hbbth-3 mice. [Figure 17E] Demonstrates the phenotypic correction of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. Determined spleen size at sacrifice as the ratio of spleen weight to total body weight (mg / g). Each symbol represents an individual animal. Data are shown as mean ± SEM. *P≦0.05. Statistical analysis was performed using one-way ANOVA. [Figure 18A]This shows the analysis of secondary C57BL / 6 recipients transplanted with bone marrow cells obtained from treated CD46+ / + / Hbbth-3 mice. (Figure 18A) Engraftment rates are shown based on the percentage of human CD46+ (hCD46+) cells in PBMCs after pre-transplant treatment with busulfan or total body irradiation (TBI) (C57BL / 6 recipients do not express hCD46). (Figure 18B) Percentage of peripheral blood RBCs expressing human γ-globin. Immunosuppression was initiated at 4 weeks post-transplant in all mice. (Figure 18C) Percentage of γ-globin+ cells in hCD46+ (donor-derived) cells. (Figures 18C and 18D) γ-globin / CD46 expression in secondary C57BL / 6 recipients at 20 weeks post-transplant (pre-transplant treatment with busulfan). CD46+ cells were immunomagnetically isolated from the chimeric bone marrow of three representative secondary mice, and γ-globin expression was analyzed by flow cytometry. It is noteworthy that, unlike humans, huCD46tg mice express CD46 on RBCs. (Figure 18C) Shows the γ-globin / CD46 marking rates at sacrifice in primary and secondary recipients. (Figure 18D) Shows γ-globin expression in CD46+ selected cells from hematopoietic tissue of secondary recipients (20 weeks). Each symbol represents an individual animal. (Figure 18E) Shows γ-globin expression in secondary recipients (n=5) who underwent a new (second) HSPC recruitment / in vivo transduction. γ-globin and CD46 expression was analyzed in secondary recipients (pre-transplant treatment with busulfan) at 20 weeks post-transplant ("before in vivo transduction"). Next, these mice were recruited and transduced in vivo using HDAd-γ-globin vector + HDAd-SB vector. Four weeks after in vivo transduction, the mice were sacrificed and analyzed ("4 weeks after in vivo transduction"). ***P≦0.00003. Statistical analysis was performed using one-way ANOVA. [Figure 18B]This shows the analysis of secondary C57BL / 6 recipients transplanted with bone marrow cells obtained from treated CD46+ / + / Hbbth-3 mice. (Figure 18A) Engraftment rates are shown based on the percentage of human CD46+ (hCD46+) cells in PBMCs after pre-transplant treatment with busulfan or total body irradiation (TBI) (C57BL / 6 recipients do not express hCD46). (Figure 18B) Percentage of peripheral blood RBCs expressing human γ-globin. Immunosuppression was initiated at 4 weeks post-transplant in all mice. (Figure 18C) Percentage of γ-globin+ cells in hCD46+ (donor-derived) cells. (Figures 18C and 18D) γ-globin / CD46 expression in secondary C57BL / 6 recipients at 20 weeks post-transplant (pre-transplant treatment with busulfan). CD46+ cells were immunomagnetically isolated from the chimeric bone marrow of three representative secondary mice, and γ-globin expression was analyzed by flow cytometry. It is noteworthy that, unlike humans, huCD46tg mice express CD46 on RBCs. (Figure 18C) Shows the γ-globin / CD46 marking rates at sacrifice in primary and secondary recipients. (Figure 18D) Shows γ-globin expression in CD46+ selected cells from hematopoietic tissue of secondary recipients (20 weeks). Each symbol represents an individual animal. (Figure 18E) Shows γ-globin expression in secondary recipients (n=5) who underwent a new (second) HSPC recruitment / in vivo transduction. γ-globin and CD46 expression was analyzed in secondary recipients (pre-transplant treatment with busulfan) at 20 weeks post-transplant ("before in vivo transduction"). Next, these mice were recruited and transduced in vivo using HDAd-γ-globin vector + HDAd-SB vector. Four weeks after in vivo transduction, the mice were sacrificed and analyzed ("4 weeks after in vivo transduction"). ***P≦0.00003. Statistical analysis was performed using one-way ANOVA. [Figure 18C]This shows the analysis of secondary C57BL / 6 recipients transplanted with bone marrow cells obtained from treated CD46+ / + / Hbbth-3 mice. (Figure 18A) Engraftment rates are shown based on the percentage of human CD46+ (hCD46+) cells in PBMCs after pre-transplant treatment with busulfan or total body irradiation (TBI) (C57BL / 6 recipients do not express hCD46). (Figure 18B) Percentage of peripheral blood RBCs expressing human γ-globin. Immunosuppression was initiated at 4 weeks post-transplant in all mice. (Figure 18C) Percentage of γ-globin+ cells in hCD46+ (donor-derived) cells. (Figures 18C and 18D) γ-globin / CD46 expression in secondary C57BL / 6 recipients at 20 weeks post-transplant (pre-transplant treatment with busulfan). CD46+ cells were immunomagnetically isolated from the chimeric bone marrow of three representative secondary mice, and γ-globin expression was analyzed by flow cytometry. It is noteworthy that, unlike humans, huCD46tg mice express CD46 on RBCs. (Figure 18C) Shows the γ-globin / CD46 marking rates at sacrifice in primary and secondary recipients. (Figure 18D) Shows γ-globin expression in CD46+ selected cells from hematopoietic tissue of secondary recipients (20 weeks). Each symbol represents an individual animal. (Figure 18E) Shows γ-globin expression in secondary recipients (n=5) who underwent a new (second) HSPC recruitment / in vivo transduction. γ-globin and CD46 expression was analyzed in secondary recipients (pre-transplant treatment with busulfan) at 20 weeks post-transplant ("before in vivo transduction"). Next, these mice were recruited and transduced in vivo using HDAd-γ-globin vector + HDAd-SB vector. Four weeks after in vivo transduction, the mice were sacrificed and analyzed ("4 weeks after in vivo transduction"). ***P≦0.00003. Statistical analysis was performed using one-way ANOVA. [Figure 18D]Analysis of secondary C57BL / 6 recipients transplanted with bone marrow cells obtained from treated CD46+ / + / Hbbth-3 mice is shown. (FIG. 18A) Engraftment rates measured in the periphery based on the percentage of human CD46+ (hCD46+) cells in PBMC after pre-treatment with busulfan or total body irradiation (TBI) (C57BL / 6 recipients do not express hCD46). (FIG. 18B) Percentage of human γ-globin-expressing peripheral blood RBCs is shown. Immunosuppression was initiated at 4 weeks post-transplantation in all mice. (FIG. 18C) Percentage of γ-globin+ cells in hCD46+ (donor-derived) cells is shown. (FIGS. 18C and 18D) Expression of γ-globin / CD46 in secondary C57BL / 6 recipients at 20 weeks post-transplantation (pre-treatment with busulfan). CD46+ cells were immunomagnetically separated from the chimeric bone marrow of 3 representative secondary mice and analyzed for γ-globin expression by flow cytometry. Notably, unlike humans, huCD46tg mice express CD46 on RBCs. (FIG. 18C) γ-globin / CD46 marking rates at sacrifice in primary and secondary recipients are shown. (FIG. 18D) γ-globin expression in cells selected for CD46+ from the hematopoietic tissue of secondary recipients (20 weeks). Each symbol represents an individual animal. (FIG. 18E) γ-globin expression in secondary recipients (n = 5) that underwent a new (second) HSPC mobilization / in vivo transduction is shown. Secondary recipients (pre-treated with busulfan) were analyzed for γ-globin and CD46 expression at 20 weeks post-transplantation ("before in vivo transduction"). Next, these mice were mobilized and transduced in vivo using an HDAd-γ-globin vector + HDAd-SB vector. Four weeks after in vivo transduction, the mice were sacrificed and analyzed ("4 weeks after in vivo transduction"). ***P ≦ 0.00003. Statistical analysis was performed using one-way ANOVA. [Figure 18E]This shows the analysis of secondary C57BL / 6 recipients transplanted with bone marrow cells obtained from treated CD46+ / + / Hbbth-3 mice. (Figure 18A) Engraftment rates are shown based on the percentage of human CD46+ (hCD46+) cells in PBMCs after pre-transplant treatment with busulfan or total body irradiation (TBI) (C57BL / 6 recipients do not express hCD46). (Figure 18B) Percentage of peripheral blood RBCs expressing human γ-globin. Immunosuppression was initiated at 4 weeks post-transplant in all mice. (Figure 18C) Percentage of γ-globin+ cells in hCD46+ (donor-derived) cells. (Figures 18C and 18D) γ-globin / CD46 expression in secondary C57BL / 6 recipients at 20 weeks post-transplant (pre-transplant treatment with busulfan). CD46+ cells were immunomagnetically isolated from the chimeric bone marrow of three representative secondary mice, and γ-globin expression was analyzed by flow cytometry. It is noteworthy that, unlike humans, huCD46tg mice express CD46 on RBCs. (Figure 18C) Shows the γ-globin / CD46 marking rates at sacrifice in primary and secondary recipients. (Figure 18D) Shows γ-globin expression in CD46+ selected cells from hematopoietic tissue of secondary recipients (20 weeks). Each symbol represents an individual animal. (Figure 18E) Shows γ-globin expression in secondary recipients (n=5) who underwent a new (second) HSPC recruitment / in vivo transduction. γ-globin and CD46 expression was analyzed in secondary recipients (pre-transplant treatment with busulfan) at 20 weeks post-transplant ("before in vivo transduction"). Next, these mice were recruited and transduced in vivo using HDAd-γ-globin vector + HDAd-SB vector. Four weeks after in vivo transduction, the mice were sacrificed and analyzed ("4 weeks after in vivo transduction"). ***P≦0.00003. Statistical analysis was performed using one-way ANOVA. [Figure 19A]This shows the safety of in vivo transduction / selection in the CD46+ / + / Hbbth-3 mouse model. (Figure 19A) Shows WBC and platelet (PLT) counts during and after in vivo selection. O6BG / BCNU treatment is indicated by an asterisk. n≧3. (Figure 19B) Shows the absolute number of circulating WBC subpopulations. n≧3. (Figure 19C) Shows the bone marrow cell composition of control mice and treated mice sacrificed at 29 weeks. Percentages of lineage marker-positive cells (Ter119+ cells, CD3+ cells, CD19+ cells, and Gr-1+ cells) and HSPC (LSK cells) are shown. (Figure 19D) Shows the colony-forming ability of bone marrow cells collected at 29 weeks. Each symbol represents an individual animal. *P≦0.05, **P≦0.0002, ***P≦0.00003. Statistical analysis was performed using two-way ANOVA. NEU: Neutrophil, LY: Lymphocyte, MO: Monocyte [Figure 19B] This shows the safety of in vivo transduction / selection in the CD46+ / + / Hbbth-3 mouse model. (Figure 19A) Shows WBC and platelet (PLT) counts during and after in vivo selection. O6BG / BCNU treatment is indicated by an asterisk. n≧3. (Figure 19B) Shows the absolute number of circulating WBC subpopulations. n≧3. (Figure 19C) Shows the bone marrow cell composition of control mice and treated mice sacrificed at 29 weeks. Percentages of lineage marker-positive cells (Ter119+ cells, CD3+ cells, CD19+ cells, and Gr-1+ cells) and HSPC (LSK cells) are shown. (Figure 19D) Shows the colony-forming ability of bone marrow cells collected at 29 weeks. Each symbol represents an individual animal. *P≦0.05, **P≦0.0002, ***P≦0.00003. Statistical analysis was performed using two-way ANOVA. NEU: Neutrophil, LY: Lymphocyte, MO: Monocyte [Figure 19C]This shows the safety of in vivo transduction / selection in the CD46+ / + / Hbbth-3 mouse model. (Figure 19A) Shows WBC and platelet (PLT) counts during and after in vivo selection. O6BG / BCNU treatment is indicated by an asterisk. n≧3. (Figure 19B) Shows the absolute number of circulating WBC subpopulations. n≧3. (Figure 19C) Shows the bone marrow cell composition of control mice and treated mice sacrificed at 29 weeks. Percentages of lineage marker-positive cells (Ter119+ cells, CD3+ cells, CD19+ cells, and Gr-1+ cells) and HSPC (LSK cells) are shown. (Figure 19D) Shows the colony-forming ability of bone marrow cells collected at 29 weeks. Each symbol represents an individual animal. *P≦0.05, **P≦0.0002, ***P≦0.00003. Statistical analysis was performed using two-way ANOVA. NEU: Neutrophil, LY: Lymphocyte, MO: Monocyte [Figure 19D] This shows the safety of in vivo transduction / selection in the CD46+ / + / Hbbth-3 mouse model. (Figure 19A) Shows WBC and platelet (PLT) counts during and after in vivo selection. O6BG / BCNU treatment is indicated by an asterisk. n≧3. (Figure 19B) Shows the absolute number of circulating WBC subpopulations. n≧3. (Figure 19C) Shows the bone marrow cell composition of control mice and treated mice sacrificed at 29 weeks. Percentages of lineage marker-positive cells (Ter119+ cells, CD3+ cells, CD19+ cells, and Gr-1+ cells) and HSPC (LSK cells) are shown. (Figure 19D) Shows the colony-forming ability of bone marrow cells collected at 29 weeks. Each symbol represents an individual animal. *P≦0.05, **P≦0.0002, ***P≦0.00003. Statistical analysis was performed using two-way ANOVA. NEU: Neutrophil, LY: Lymphocyte, MO: Monocyte [Figure 20A]The effect of anti-HDAd5 / 35++ antibody on the second transduction is shown. (Figure 20A) CD46tg mice were recruited and injected with HDAd-mgmt / GFP+HDAd-SB. Serum samples were collected as shown. (Figures 20B, 20C) Flow cytometry analysis of PBMCs at 4 days and 4 weeks after recruitment / transduction is shown. (Figure 20D) GFP analysis after the second recruitment / transduction at 4 weeks is shown. (Figure 20E) The titer of anti-HDAd5 / 35++ antibody based on OD450 is shown. An OD450 titer of 0.2 is considered to indicate neutralization. (Figure 20F) Percentage of GFP-positive PBMCs measured in different cohorts (see Figures 20B-D) is shown. The control was untreated CD46tg mice. Each symbol in (Figure 20E) and (Figure 20F) represents an individual animal. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 20B] The effect of anti-HDAd5 / 35++ antibody on the second transduction is shown. (Figure 20A) CD46tg mice were recruited and injected with HDAd-mgmt / GFP+HDAd-SB. Serum samples were collected as shown. (Figures 20B, 20C) Flow cytometry analysis of PBMCs at 4 days and 4 weeks after recruitment / transduction is shown. (Figure 20D) GFP analysis after the second recruitment / transduction at 4 weeks is shown. (Figure 20E) The titer of anti-HDAd5 / 35++ antibody based on OD450 is shown. An OD450 titer of 0.2 is considered to indicate neutralization. (Figure 20F) Percentage of GFP-positive PBMCs measured in different cohorts (see Figures 20B-D) is shown. The control was untreated CD46tg mice. Each symbol in (Figure 20E) and (Figure 20F) represents an individual animal. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 20C]The effect of anti-HDAd5 / 35++ antibody on the second transduction is shown. (Figure 20A) CD46tg mice were recruited and injected with HDAd-mgmt / GFP+HDAd-SB. Serum samples were collected as shown. (Figures 20B, 20C) Flow cytometry analysis of PBMCs at 4 days and 4 weeks after recruitment / transduction is shown. (Figure 20D) GFP analysis after the second recruitment / transduction at 4 weeks is shown. (Figure 20E) The titer of anti-HDAd5 / 35++ antibody based on OD450 is shown. An OD450 titer of 0.2 is considered to indicate neutralization. (Figure 20F) Percentage of GFP-positive PBMCs measured in different cohorts (see Figures 20B-D) is shown. The control was untreated CD46tg mice. Each symbol in (Figure 20E) and (Figure 20F) represents an individual animal. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 20D] The effect of anti-HDAd5 / 35++ antibody on the second transduction is shown. (Figure 20A) CD46tg mice were recruited and injected with HDAd-mgmt / GFP+HDAd-SB. Serum samples were collected as shown. (Figures 20B, 20C) Flow cytometry analysis of PBMCs at 4 days and 4 weeks after recruitment / transduction is shown. (Figure 20D) GFP analysis after the second recruitment / transduction at 4 weeks is shown. (Figure 20E) The titer of anti-HDAd5 / 35++ antibody based on OD450 is shown. An OD450 titer of 0.2 is considered to indicate neutralization. (Figure 20F) Percentage of GFP-positive PBMCs measured in different cohorts (see Figures 20B-D) is shown. The control was untreated CD46tg mice. Each symbol in (Figure 20E) and (Figure 20F) represents an individual animal. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 20E]The effect of anti-HDAd5 / 35++ antibody on the second transduction is shown. (Figure 20A) CD46tg mice were recruited and injected with HDAd-mgmt / GFP+HDAd-SB. Serum samples were collected as shown. (Figures 20B, 20C) Flow cytometry analysis of PBMCs at 4 days and 4 weeks after recruitment / transduction is shown. (Figure 20D) GFP analysis after the second recruitment / transduction at 4 weeks is shown. (Figure 20E) The titer of anti-HDAd5 / 35++ antibody based on OD450 is shown. An OD450 titer of 0.2 is considered to indicate neutralization. (Figure 20F) Percentage of GFP-positive PBMCs measured in different cohorts (see Figures 20B-D) is shown. The control was untreated CD46tg mice. Each symbol in (Figure 20E) and (Figure 20F) represents an individual animal. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 20F] The effect of anti-HDAd5 / 35++ antibody on the second transduction is shown. (Figure 20A) CD46tg mice were recruited and injected with HDAd-mgmt / GFP+HDAd-SB. Serum samples were collected as shown. (Figures 20B, 20C) Flow cytometry analysis of PBMCs at 4 days and 4 weeks after recruitment / transduction is shown. (Figure 20D) GFP analysis after the second recruitment / transduction at 4 weeks is shown. (Figure 20E) The titer of anti-HDAd5 / 35++ antibody based on OD450 is shown. An OD450 titer of 0.2 is considered to indicate neutralization. (Figure 20F) Percentage of GFP-positive PBMCs measured in different cohorts (see Figures 20B-D) is shown. The control was untreated CD46tg mice. Each symbol in (Figure 20E) and (Figure 20F) represents an individual animal. Statistical analysis was performed using the nonparametric Kruskal-Wallis test. [Figure 21A]This shows the in vivo distribution of vector DNA 18 weeks after HDAd injection (after 10 weeks of in vivo selection). The primer design is also shown. The light gray primer is specific to the transgene cassette and detects both the integrated vector DNA and episomal vector DNA. The dark gray primer detects vector stuffer DNA derived from plasmid pHCA. When SB100x-mediated integration occurs, the target region corresponding to the dark gray primer disappears. Therefore, the dark gray primer is used for measuring episomal vector copies. [Figure 21B] This shows the in vivo distribution of vector DNA 18 weeks after HDAd injection (after 10 weeks of in vivo selection). The standard curve for the copy number of the incorporated transgene is also shown. [Figure 21C] This shows the in vivo distribution of vector DNA 18 weeks after HDAd injection (after 10 weeks of in vivo selection). The standard curve for HCA (episome vector) copy number is also shown. [Figure 21D] This graph shows the in vivo distribution of vector DNA 18 weeks after HDAd injection (following 10 weeks of in vivo selection). It indicates the number of integrated transgene copies per cell. The total vector copy number (light gray primer) is subtracted from the episomal vector copy number (dark gray primer). Vector-specific signals were normalized using GAPDH. Each symbol represents an individual animal. [Figure 22A] The mutagenicity of O6BG / BCNU treatment was evaluated using an in vitro assay. After restoring cells overnight from cryopreservation, CD34+ cells were transduced using HDAd-mgmt / GFP or HDAd control at an MOI of 3000 vp / cell. Transduction at this MOI resulted in GFP expression in 50% of cells after 2 days. Next, the cells were treated with 10 mM O6BG, followed by treatment with 25 mM BCNU (or DMSO solvent) for 2 hours. After washing, the cells were seeded on methylcellulose for the CFU assay (3000 cells / 35 mm dish). Colonies and pooled cells were counted after 14 days, and genomic DNA was subjected to whole exome sequencing. [Figure 22B]This shows the results of an in vitro assay evaluating the mutagenicity of O6BG / BCNU treatment. The number of pooled cells per plate is shown. Each symbol represents the number of cells in an individual 35mm dish. Statistical analysis was performed using the non-parametric Kruskal-Wallis test. [Figure 22C] This shows the results of an in vitro assay evaluating the mutagenicity of O6BG / BCNU treatment. Representative colonies obtained from the HDAd-mgmt / GFP+O6BG / BCNU group are shown. This demonstrates that GFP expression is reduced around the colony due to the loss of the episomal viral genome, but GFP expression is present in the majority of the cell. The scale bar is 1 mm. [Figure 23]The vector structures are shown. HDAd-short LCR: This vector contains a 4.3kb small LCR consisting of a core region of DNase-sensitive sites (HS) 1-4 and a 0.66kb β-globin promoter. The transposon length is 11.8kb. HDAd-long LCR: The γ-globin gene is regulated by a 21.5kb β-globin LCR (chr11:5292319-5270789), a 1.6kb β-globin promoter (e.g., chr11:5228631-5227023 or chr11:5228631-5227018), and a 3'HS1 region (chr11:5206867-5203839) (also derived from the β-globin locus). To stabilize RNA in erythrocytes, the γ-globin gene UTR was ligated to the 3' end of the γ-globin gene. The vector also contains an mgmtP140K expression cassette, enabling in vivo selection of transduced HSPCs and HSPC offspring. The γ-globin expression cassette and mgmt expression cassette are separated by chicken globin HS4 insulator (cHS4). The 32.4kb LCR-γ-globin / mgmt transposon has a reverse repeat sequence (IR) (recognized by SB100x) and an ftr site (enabling transposon cyclization by Flpe recombinase) adjacent to each other. HDAd-SB: A second vector required for integration, this second vector contains an expression cassette for the enhanced Sleeping Beauty SB100x transposase and an expression cassette for Flpe recombinase. [Figure 24A]This study demonstrates that a 32.4kb transposon is incorporated via SB100x after ex vivo HSPC transduction using HDAd-long chain LCR. 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 500vp / 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 initiated at week 4 and repeated every 2 weeks for a total of 4 cycles. The BCNU concentration was increased with each cycle from 5 mg / kg to 7.5 mg / kg to 10 mg / kg (twice). Mice were sacrificed at week 20. [Figure 24B] This shows that a 32.4kb transposon is incorporated via SB100x after ex vivo HSPC transduction using HDAd-long chain LCR. The percentage of human γ-globin-positive peripheral red blood cells (RBCs) measured by flow cytometry is also shown. Each symbol represents an individual animal. [Figure 24C] This study demonstrates that a 32.4kb transposon is incorporated via SB100x after ex vivo HSPC transduction using HDAd-long LCR. Representative flow cytometry data showing human γ-globin expression in erythrocytes (Ter119+) bone marrow cells (lower panel) 20 weeks post-transplantation are shown. The upper panel shows mice transplanted with cells transduced using a mock-up. [Figure 24D] This study demonstrates that a 32.4kb transposon is incorporated via SB100x after ex vivo HSPC transduction using HDAd-long LCR. Schematic diagram of iPCR analysis: 5 micrograms of genomic DNA were digested with SacI, re-ligated, and subjected to nested inverse PCR using the indicated primers (see Materials and Methods). [Figure 24E]This study demonstrates that a 32.4kb transposon is incorporated via SB100x after ex vivo HSPC transduction using HDAd-long LCR. The agarose gel electrophoresis of the cloned plasmid containing the integration ligation site is shown. The indicated bands were excised and sequenced. The localization of the integration site on the chromosome is shown below the gel. [Figure 24F] This shows that a 32.4kb transposon is incorporated via SB100x after ex vivo HSPC transduction using HDAd-long LCR. Examples of ligation site sequences: 5' end vector sequence, Sleeping beautyIR / DR sequence, incorporated ligament (chr15, 6805206) SEQ ID NO: 1; 5' end vector sequence, Sleeping beautyIR / DR sequence, incorporated ligament (chrX, 16897322) SEQ ID NO: 2; 3' end vector sequence, Sleeping beautyIR / DR sequence, incorporated 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 ligation site between IR and chromosomal DNA (used by SB100x) are shown in square brackets. [Figure 25A] This report describes 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. 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 to 5 mg / kg → 7.5 mg / kg → 10 mg / kg with each cycle. The O6BG concentration was 30 mg / kg in all four treatments. Mice were followed up until week 20, when the animals were sacrificed for analysis. Bone marrow lin- cells were used for transplantation into secondary recipients. Next, secondary recipients were followed up for 16 weeks. [Figure 25B] This paper shows in vivo transduction of HSPC using a DAd-long chain LCR containing a 32.4kb transposon, and in vivo transduction of HSPC using an HDAd-short chain LCR containing an 11.8kb transposon. The percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs), measured by flow cytometry, is also shown. Each symbol represents an individual animal. In mice transduced using mocks, the percentage of γ-globin-positive cells was less than 0.1%. [Figure 25C] This document describes in vivo transduction of HSPC using DAd-long chain LCRs containing a 32.4kb transposon, and in vivo transduction of HSPC using HDAd-short chain LCRs containing an 11.8kb transposon. It also shows the γ-globin protein chain levels in RBCs measured by HPLC 20 weeks after in vivo HSPC transduction. The percentage of human γ-globin protein chain relative to mouse α-globin protein chain is shown. [Figure 25D] This document describes in vivo transduction of HSPC using a DAd-long LCR containing a 32.4kb transposon, and in vivo transduction of HSPC using an HDAd-short LCR containing an 11.8kb transposon. It also shows the mRNA levels of gamma globin in whole blood measured by qRT-PCR 20 weeks after in vivo HSPC transduction. The percentage of human gamma globin mRNA relative to mouse α-globin mRNA is shown. [Figure 25E] This paper describes in vivo transduction of HSPC using a DAd-long chain LCR containing a 32.4kb transposon, and in vivo transduction of HSPC using an HDAd-short chain LCR containing an 11.8kb transposon. It also shows the vector copy count per cell of bone marrow mononuclear cells collected 20 weeks after in vivo HSPC transduction. The difference between the two groups is not significant. Statistical analysis was performed using two-way ANOVA. [Figure 26A]Shows the hematological parameters at the 20th week after in vivo transduction of HSPC. (Figure 26A) Shows white blood cells (WBC), neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO), and basophils (BA). (Figure 26B) Shows erythropoietic 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 26C) Shows the bone marrow cell composition. (Figure 26D) Shows the colony-forming ability of bone marrow Lin- cells. In Figures 26A - D, the differences between groups were not significant. [Figure 26B] Shows the hematological parameters at the 20th week after in vivo transduction of HSPC. (Figure 26A) Shows white blood cells (WBC), neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO), and basophils (BA). (Figure 26B) Shows erythropoietic 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 26C) Shows the bone marrow cell composition. (Figure 26D) Shows the colony-forming ability of bone marrow Lin- cells. In Figures 26A - D, the differences between groups were not significant. [Figure 26C] Shows the hematological parameters at the 20th week after in vivo transduction of HSPC. (Figure 26A) Shows white blood cells (WBC), neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO), and basophils (BA). (Figure 26B) Shows erythropoietic 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 26C) Shows the bone marrow cell composition. (Figure 26D) Shows the colony-forming ability of bone marrow Lin- cells. In Figures 26A - D, the differences between groups were not significant. [Figure 26D]Figure 26A shows hematological parameters at 20 weeks after transduction of HSPC in vivo. It shows white blood cells (WBC), neutrophils (NE), white blood cells (LY), monocytes (MO), eosinophils (EO), and basophils (BA). Figure 26B shows erythropoiesis parameters: RBC: red blood cells, 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 26C shows bone marrow cell composition. Figure 26D shows the colony-forming ability of bone marrow Lin-cells. In Figures 26A-D, the differences between groups were not significant. [Figure 27] A schematic diagram of insertion site analysis is shown. The localization of the NheI and KpnI sites in the HDAd-long LCR 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 the background of the unintegrated vector. Genomic DNA obtained from bone marrow Lin- cells was digested with NheI and KpnI, thermally inactivated, and then further digested with NlaIII. NlaIII is a 4-base recognition enzyme, which creates short DNA fragments. Next, the digested DNA was ligated with a double-stranded oligonucleotide with a known sequence and a terminal that matches the digested NlaIII fragment. 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 are biotinylated, it is possible to collect ssDNA 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. [Figure 28A]This shows the analysis of vector integration sites in HSPCs using LAM-PCR / NGS. Genomic DNA was isolated from bone marrow cells collected 20 weeks after in vivo transduction using HDAd-long LCR + HDAd-SB. The distribution of integration sites on the chromosomes is shown. Integration sites are marked with vertical lines. [Figure 28B] This paper shows the analysis of vector integration sites in HSPCs using LAM-PCR / NGS. Genomic DNA was isolated from bone marrow cells collected 20 weeks after in vivo transduction using HDAd-long LCR + HDAd-SB. 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. Chromosome 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 28C] This report shows the analysis of vector integration sites in HSPCs using LAM-PCR / NGS. Genomic DNA was isolated from bone marrow cells collected 20 weeks after in vivo transduction using HDAd-long LCR + HDAd-SB. The integration sites were mapped to the mouse genome, and their locations were analyzed in relation to genes. The percentage of integration events occurring is shown as follows: 1kb upstream of the transcription start site (TSS) (0.0%), 5'UTR of the exon (0.0%), protein coding sequence (0.0%), intron (17.0%), 3'UTR (0.0%), 1kb downstream of the 3'UTR (0.0%), and between genes (83.0%). [Figure 28D]This report describes the analysis of vector integration sites in HSPCs using LAM-PCR / NGS. Genomic DNA was isolated from bone marrow cells collected 20 weeks after in vivo transduction using HDAd-long chain LCR + HDAd-SB. The integration patterns in mouse genome frames are shown. 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 integrations with 1 per frame was higher. [Figure 29A] The analysis of secondary recipients is presented. Bone marrow lin cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. Engraftment rates based on the percentage of CD46-positive PBMCs at 4, 8, 12, and 16 weeks post-transplant are shown. There was no significant difference between the two groups. [Figure 29B] The analysis of secondary recipients is shown. Bone marrow lin cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. The percentage of peripheral blood RBCs expressing γ-globin was measured by flow cytometry and is shown. The difference between the two groups was not statistically significant. [Figure 29C] The analysis of secondary recipients is shown. Bone marrow Lin- cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. The vector copy number per cell in bone marrow MNCs collected at 20 weeks after in vivo transduction of HSPC is shown. The difference between the two groups is not significant. [Figure 29D]The analysis of secondary recipients is shown. Bone marrow lin cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were observed for 16 weeks. The analysis of human γ-globin chains in the RBCs of secondary recipients by HPLC is shown. The percentage of human γ-globin relative to adult mouse α-globin is shown. ***p<0.0001. [Figure 29E] The analysis of secondary recipients is shown. Bone marrow lin cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were observed for 16 weeks. The levels of γ-globin mRNA in all blood cells are shown compared to those of mouse α-globin mRNA. [Figure 29F] The analysis of secondary recipients is presented. Bone marrow lin- cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. The percentage of γ-globin-expressing erythrocytes (Ter119+ cells) in the total bone marrow MNC is shown. Statistical analysis was performed using two-way ANOVA. [Figure 29G] The analysis of secondary recipients is shown. Bone marrow lin cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. The levels of γ-globin mRNA in bone marrow MNCs at 16 weeks post-transduction are shown. The percentage of human γ-globin mRNA relative to mouse α-globin mRNA and β-major globin mRNA is shown. [Figure 29H] The analysis of secondary recipients is shown. Bone marrow lin- cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. Erythrocyte specificity is shown. The percentage of γ-globin+ cells in erythrocytes (Ter119+) and non-erythrocytes (Ter119-) is shown. [Figure 29I]The analysis of secondary recipients is shown. Bone marrow Lin- cells collected at 20 weeks from in vivo transduced CD46tg mice were transplanted into lethally irradiated C57Bl / 6 mice. Secondary recipients were followed for 16 weeks. The vector copy number (VCN) per cell in bone marrow MNCs collected at 20 weeks after in vivo transduction of HSPC is shown. The difference between the two groups is not significant. [Figure 30] The following shows hematological parameters in secondary recipients at 16 weeks post-transplant. (A) White blood cell count. (B) Erythrocyte production parameters. RBC: red blood cells, Hb: hemoglobin, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: erythrocyte distribution width. (C) Bone marrow cell composition. (D) Bone marrow Lin-cell colony formation ability. There were no significant differences between groups in A-D. Statistical analysis was performed using two-way ANOVA. [Figure 31A] This shows an in vitro study using human CD34+ cells. (Figure 31A) 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 started 5 days after ED. Cells were analyzed on day 18 by flow cytometry (Figure 31B) and HPLC (Figure 31C). (Figure 31D) Vector copy number on day 18 is shown. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 31B] This shows an in vitro study using human CD34+ cells. (Figure 31A) 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 started 5 days after ED. Cells were analyzed on day 18 by flow cytometry (Figure 31B) and HPLC (Figure 31C). (Figure 31D) Vector copy number on day 18 is shown. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 31C] This shows an in vitro study using human CD34+ cells. (Figure 31A) 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 started 5 days after ED. Cells were analyzed on day 18 by flow cytometry (Figure 31B) and HPLC (Figure 31C). (Figure 31D) Vector copy number on day 18 is shown. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 31D] This shows an in vitro study using human CD34+ cells. (Figure 31A) 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 started 5 days after ED. Cells were analyzed on day 18 by flow cytometry (Figure 31B) and HPLC (Figure 31C). (Figure 31D) Vector copy number on day 18 is shown. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 32A]Figure 32A shows the 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. The treatment regimens are shown. In contrast to Figures 25A-25E, Figures 32A-32D show the results in thalassemia Hbbth3 / CD46 mice. (Figure 32B) Shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 32C) Shows the level of γ-globin protein chains in RBCs measured by HPLC 18 weeks after transduction of HSPC in vivo. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. (Figure 32D) Representative chromatograms of untreated Hbbth3 / CD46 mice (left panel) and mice 21 weeks after treatment are shown. This indicates that human γ-globin was added in addition to the mouse α-chain and β-chain. [Figure 32B] Figure 32A shows the 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. The treatment regimens are shown. In contrast to Figures 25A-25E, Figures 32A-32D show the results in thalassemia Hbbth3 / CD46 mice. (Figure 32B) Shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 32C) Shows the level of γ-globin protein chains in RBCs measured by HPLC 18 weeks after transduction of HSPC in vivo. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. (Figure 32D) Representative chromatograms of untreated Hbbth3 / CD46 mice (left panel) and mice 21 weeks after treatment are shown. This indicates that human γ-globin was added in addition to the mouse α-chain and β-chain. [Figure 32C]Figure 32A shows the 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. The treatment regimens are shown. In contrast to Figures 25A-25E, Figures 32A-32D show the results in thalassemia Hbbth3 / CD46 mice. (Figure 32B) Shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 32C) Shows the level of γ-globin protein chains in RBCs measured by HPLC 18 weeks after transduction of HSPC in vivo. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. (Figure 32D) Representative chromatograms of untreated Hbbth3 / CD46 mice (left panel) and mice 21 weeks after treatment are shown. This indicates that human γ-globin was added in addition to the mouse α-chain and β-chain. [Figure 32D] Figure 32A shows the 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. The treatment regimens are shown. In contrast to Figures 25A-25E, Figures 32A-32D show the results in thalassemia Hbbth3 / CD46 mice. (Figure 32B) Shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. (Figure 32C) Shows the level of γ-globin protein chains in RBCs measured by HPLC 18 weeks after transduction of HSPC in vivo. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. (Figure 32D) Representative chromatograms of untreated Hbbth3 / CD46 mice (left panel) and mice 21 weeks after treatment are shown. This indicates that human γ-globin was added in addition to the mouse α-chain and β-chain. [Figure 32E]Figure 32E shows the expression of human γ-globin after in vivo HSPC gene therapy in Hbbth3 / CD46+ / + mice using HDAd-short chain LCR and HDAd-long chain LCR. Treatment regimen: In contrast to the study shown in Figure 25, this study was performed using thalassemia Hbbth3 / CD46 mice. Figure 32F shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. Figure 32G shows the level of γ-globin protein chains in RBCs measured by HPLC 10-16 weeks after in vivo HSPC transduction. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. Figure 32H shows representative chromatograms of untreated Hbbth3 / CD46+ / + mice (left panel) and mice 16 weeks after treatment. The study demonstrates the addition of human γ-globin to the mouse α- and β-chains. It is noteworthy that two independent studies were conducted using Hbbth3 / CD46+ / + mice. Primary study: N=6 for HD-long chain LCR, N=2 for HDAd-short chain LCR, 21 weeks of follow-up. Secondary study: N=4 for HD-long chain LCR, N=5 for HDAd-short chain LCR, 16 weeks of follow-up. Figure 32F shows the integrated data up to week 21. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 32F]Figure 32E shows the expression of human γ-globin after in vivo HSPC gene therapy in Hbbth3 / CD46+ / + mice using HDAd-short chain LCR and HDAd-long chain LCR. Treatment regimen: In contrast to the study shown in Figure 25, this study was performed using thalassemia Hbbth3 / CD46 mice. Figure 32F shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. Figure 32G shows the level of γ-globin protein chains in RBCs measured by HPLC 10-16 weeks after in vivo HSPC transduction. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. Figure 32H shows representative chromatograms of untreated Hbbth3 / CD46+ / + mice (left panel) and mice 16 weeks after treatment. The study demonstrates the addition of human γ-globin to the mouse α- and β-chains. It is noteworthy that two independent studies were conducted using Hbbth3 / CD46+ / + mice. Primary study: N=6 for HD-long chain LCR, N=2 for HDAd-short chain LCR, 21 weeks of follow-up. Secondary study: N=4 for HD-long chain LCR, N=5 for HDAd-short chain LCR, 16 weeks of follow-up. Figure 32F shows the integrated data up to week 21. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 32G]Figure 32E shows the expression of human γ-globin after in vivo HSPC gene therapy in Hbbth3 / CD46+ / + mice using HDAd-short chain LCR and HDAd-long chain LCR. Treatment regimen: In contrast to the study shown in Figure 25, this study was performed using thalassemia Hbbth3 / CD46 mice. Figure 32F shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. Figure 32G shows the level of γ-globin protein chains in RBCs measured by HPLC 10-16 weeks after in vivo HSPC transduction. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. Figure 32H shows representative chromatograms of untreated Hbbth3 / CD46+ / + mice (left panel) and mice 16 weeks after treatment. The study demonstrates the addition of human γ-globin to the mouse α- and β-chains. It is noteworthy that two independent studies were conducted using Hbbth3 / CD46+ / + mice. Primary study: N=6 for HD-long chain LCR, N=2 for HDAd-short chain LCR, 21 weeks of follow-up. Secondary study: N=4 for HD-long chain LCR, N=5 for HDAd-short chain LCR, 16 weeks of follow-up. Figure 32F shows the integrated data up to week 21. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 32H]Figure 32E shows the expression of human γ-globin after in vivo HSPC gene therapy in Hbbth3 / CD46+ / + mice using HDAd-short chain LCR and HDAd-long chain LCR. Treatment regimen: In contrast to the study shown in Figure 25, this study was performed using thalassemia Hbbth3 / CD46 mice. Figure 32F shows the percentage of human γ-globin-positive cells in peripheral erythrocytes (RBCs) measured by flow cytometry. Each symbol represents an individual animal. Figure 32G shows the level of γ-globin protein chains in RBCs measured by HPLC 10-16 weeks after in vivo HSPC transduction. The percentage of human γ-globin protein chains relative to mouse α-globin protein chains is shown. Figure 32H shows representative chromatograms of untreated Hbbth3 / CD46+ / + mice (left panel) and mice 16 weeks after treatment. The study demonstrates the addition of human γ-globin to the mouse α- and β-chains. It is noteworthy that two independent studies were conducted using Hbbth3 / CD46+ / + mice. Primary study: N=6 for HD-long chain LCR, N=2 for HDAd-short chain LCR, 21 weeks of follow-up. Secondary study: N=4 for HD-long chain LCR, N=5 for HDAd-short chain LCR, 16 weeks of follow-up. Figure 32F shows the integrated data up to week 21. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 33] Analysis of bone marrow at the time of sacrifice is shown. Bone marrow was collected 16 weeks after in vivo transduction of HSPC in Hbbth3 / CD46+ / + mice. (A) Vector copy number per cell in bone marrow MNCs is shown. The difference between the two groups is not significant. (B) Mean fluorescence intensity (MFI) of gamma globin in erythrocyte (Ter119+) cells is shown. Statistical analysis was performed using two-way ANOVA. [Figure 34] 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) are shown. [Figure 35]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) are shown. [Figure 36A] The phenotypic modifications are shown. (Figures 36A and 36B) Blood cell morphology is shown; the left panel shows blood smears stained with Giemsa, and the right panel shows blood smears stained with May-Grünwald. Nuclear and cytoplasmic residues in reticulocytes are stained purple. (Figure 36A) A comparison is shown between before treatment and 14 weeks after treatment. (Figure 36B) A comparison of Giemsa staining and reticulocytes is shown for Hbbth3 / CD46 mice before treatment with CD46tg and 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 36C) Cytospin preparations of bone marrow are shown. It can be seen that erythropogenesis, which is predominantly proerythroblasts, has been normalized in the treated mice. The scale bar is 20 μm. [Figure 36B] The phenotypic modifications are shown. (Figures 36A and 36B) Blood cell morphology is shown; the left panel shows blood smears stained with Giemsa, and the right panel shows blood smears stained with May-Grünwald. Nuclear and cytoplasmic residues in reticulocytes are stained purple. (Figure 36A) A comparison is shown between before treatment and 14 weeks after treatment. (Figure 36B) A comparison of Giemsa staining and reticulocytes is shown for Hbbth3 / CD46 mice before treatment with CD46tg and 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 36C) Cytospin preparations of bone marrow are shown. It can be seen that erythropogenesis, which is predominantly proerythroblasts, has been normalized in the treated mice. The scale bar is 20 μm. [Figure 36C]The phenotypic modifications are shown. (Figures 36A and 36B) Blood cell morphology is shown; the left panel shows blood smears stained with Giemsa, and the right panel shows blood smears stained with May-Grünwald. Nuclear and cytoplasmic residues in reticulocytes are stained purple. (Figure 36A) A comparison is shown between before treatment and 14 weeks after treatment. (Figure 36B) A comparison of Giemsa staining and reticulocytes is shown for Hbbth3 / CD46 mice before treatment with CD46tg and 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 36C) Cytospin preparations of bone marrow are shown. It can be seen that erythropogenesis, which is predominantly proerythroblasts, has been normalized in the treated mice. The scale bar is 20 μm. [Figure 37A] Phenotypic correction (week 16) is shown. (Figure 37A) Left panel: Blood smear stained with Giemsa / May-Grünwald stain (5 min). Right panel: Blood smear stained with Brilliant Cresil Blue for reticulocytes. Nuclear and cytoplasmic residues in reticulocytes appear stained purple. (Figure 37B) Cytospin preparations of bone marrow stained with Giemsa / May-Grünwald stain (15 min) are shown. (Figures 37A and 37B) Top panel: Bone marrow cells are normally distributed - the erythrocyte lineage consists of all stages of erythrocyte differentiation. Middle panel: Erythrocyte lineage is dominant over leukocyte lineage - the erythrocyte lineage consists mainly of proerythroblasts and basophilic erythroblasts. Bottom panel: Bone marrow cells are normally distributed - the erythrocyte lineage consists mainly of maturing polychromatic erythroblasts and orthochromatic erythroblasts. The scale bar is 25 μm. [Figure 37B]Phenotypic correction (week 16) is shown. (Figure 37A) Left panel: Blood smear stained with Giemsa / May-Grünwald stain (5 min). Right panel: Blood smear stained with Brilliant Cresil Blue for reticulocytes. Nuclear and cytoplasmic residues in reticulocytes appear stained purple. (Figure 37B) Cytospin preparations of bone marrow stained with Giemsa / May-Grünwald stain (15 min) are shown. (Figures 37A and 37B) Top panel: Bone marrow cells are normally distributed - the erythrocyte lineage consists of all stages of erythrocyte differentiation. Middle panel: Erythrocyte lineage is dominant over leukocyte lineage - the erythrocyte lineage consists mainly of proerythroblasts and basophilic erythroblasts. Bottom panel: Bone marrow cells are normally distributed - the erythrocyte lineage consists mainly of maturing polychromatic erythroblasts and orthochromatic erythroblasts. The scale bar is 25 μm. [Figure 38] The graphs show the normalization of red blood cell parameters at week 1 (upper panel) and week 10 (lower panel) for long-chain LCR vectors, short-chain LCR vectors, and control CD46tg. [Figure 39A] Figure 39A shows the hematological parameters of Hbbth3 / CD46+ / + mice before and after (16 weeks) in vivo HSPC gene therapy. Figure 39B shows the hematological parameters. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 39B-1] Figure 39A shows the hematological parameters of Hbbth3 / CD46+ / + mice before and after (16 weeks) in vivo HSPC gene therapy. Figure 39B shows the hematological parameters. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 39B-2] Figure 39A shows the hematological parameters of Hbbth3 / CD46+ / + mice before and after (16 weeks) in vivo HSPC gene therapy. Figure 39B shows the hematological parameters. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 39B-3]Figure 39A shows the hematological parameters of Hbbth3 / CD46+ / + mice before and after (16 weeks) in vivo HSPC gene therapy. Figure 39B shows the hematological parameters. Statistical analysis was performed using two-way ANOVA. *p<0.05, **p<0.0001. [Figure 40A] This shows phenotypic modifications of extramedullary hematopoiesis in the spleen and liver. Spleen size at slaughter (16 weeks) is shown. Left panel: Representative spleen image. Right panel: Summary. Each symbol represents an individual animal. Statistical analysis was performed using one-way ANOVA. **p<0.0001. The difference between the two vectors is not significant. [Figure 40B] This shows phenotypic modification of extramedullary hematopoiesis in the spleen and liver. Extramedullary hematopoiesis is shown 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. The scale bar is 20 μm. The images shown are representative. [Figure 41] This image shows phenotypic correction of hemosiderin deposition in the spleen and liver (week 16). Iron deposition is indicated by Pearl staining, where cytoplasmic hemosiderin in spleen and liver sections is stained blue. The scale bar is 20 μm. Representative sections are shown. (Exposure time: 2.24 msec, Gain: 4.1×, Saturation: 1.50, Gamma: 0.60). [Figure 42A] This shows the analysis of bone marrow at the time of sacrifice (week 21). Bone marrow was collected at week 21 after in vivo HSC transduction in Hbbth3 / CD46tg mice. (Figure 42A) Shows the number of vector copies per cell in bone marrow MNCs. (Figures 42B, 42C) Shows the erythrocyte specificity of γ-globin expression. (Figure 42B) Shows the percentage of γ-globin-expressing erythrocytes (Ter119+) and non-erythrocytes (Ter119-). *p<0.05. Statistical analysis was performed using two-way ANOVA. [Figure 42B]This shows the analysis of bone marrow at the time of sacrifice (week 21). Bone marrow was collected at week 21 after in vivo HSC transduction in Hbbth3 / CD46tg mice. (Figure 42A) Shows the number of vector copies per cell in bone marrow MNCs. (Figures 42B, 42C) Shows the erythrocyte specificity of γ-globin expression. (Figure 42B) Shows the percentage of γ-globin-expressing erythrocytes (Ter119+) and non-erythrocytes (Ter119-). *p<0.05. Statistical analysis was performed using two-way ANOVA. [Figure 42C] This shows the analysis of bone marrow at the time of sacrifice (week 21). Bone marrow was collected at week 21 after in vivo HSC transduction in Hbbth3 / CD46tg mice. (Figure 42A) Shows the number of vector copies per cell in bone marrow MNCs. (Figures 42B, 42C) Shows the erythrocyte specificity of γ-globin expression. (Figure 42B) Shows the percentage of γ-globin-expressing erythrocytes (Ter119+) and non-erythrocytes (Ter119-). *p<0.05. Statistical analysis was performed using two-way ANOVA. [Figure 43] This shows extramedullary hematopoiesis obtained 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 is shown by Pearl staining, where cytoplasmic hemosiderin in the spleen is stained with blue dye. [Figure 44A] This study demonstrates phenotypic modification of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. RBC analysis (n=5) is shown for healthy (CD46tg) mice, CD46+ / + / Hbbth-3 mice before recruitment and in vivo transduction, and CD46+ / + / Hbbth-3 mice after in vivo transduction / selection (analyzed 29 weeks after HDAd injection). *P≦0.05, **P≦0.0002, ***P≦0.00003. Statistical analysis was performed using two-way ANOVA. [Figure 44B]This shows phenotypic modification of CD46+ / + / Hbbth-3 mice by transduction / selection of HSPC in vivo. Peripheral blood smears stained with brilliant cresyl blue for the detection of reticulocytes are shown. Arrows indicate reticulocytes containing characteristic residual RNA and microorganelles. In representative smears, the percentage of positively stained reticulocytes was 7% for CD46, 31% for untreated CD46+ / + / Hbbth-3, and 12% for treated CD46+ / + / Hbbth-3. Scale bar: 20 μm. [Figure 44C] This shows phenotypic modification of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. Top: Blood smear. Scale bar: 20 μm. Center: Bone marrow cytospin preparation. Arrows indicate erythroblasts at different stages of maturation and show that erythropoiesis, which was predominantly proerythroblasts, is normalized in the treated mice. Scale bar: 25 μm. Bottom: Hemosiderin deposition in tissue by Pearl staining. Iron deposition is shown as cytoplasmic hemosiderin stained blue in spleen tissue sections. The blood smear images of control mice (CD46tg and CD46+ / + / Hbbth-3 before transduction) in C and Figure 5D were obtained from the same sample. [Figure 44D] This paper demonstrates phenotypic modification of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. Macroscopic spleen images are shown from one representative CD46tg mouse, one untreated CD46+ / + / Hbbth-3 mouse, and five treated CD46+ / + / Hbbth-3 mice. [Figure 44E] This report demonstrates phenotypic modification of CD46+ / + / Hbbth-3 mice by in vivo transduction / selection of HSPC. Spleen size at sacrifice was determined as the ratio of spleen weight to total body weight (mg / g). Each symbol represents an individual animal. Data are presented as mean Å}SEM. *P≦0.05. Statistical analysis was performed using one-way ANOVA. [Figure 45]The bone marrow cell composition of CD46 mice and treated Hbbth3 / CD46 mice 16 weeks after in vivo transduction is shown. There were no significant differences between the groups. Statistical analysis was performed using two-way ANOVA. [Figure 46] This document outlines the gating strategy for human gamma globin. Red blood cell cells (RBCs) obtained from CD46 / Hbbth3 mice were immobilized and permeabilized, and then subjected to staining for the erythrocyte marker Ter-119 and intracellular gamma globin. [Figure 47A-1] This study demonstrates the effect of SB100x-mediated incorporation on the transcriptome of CD34+ cells. A schematic diagram of the experiment is shown. CD34+ cells were infected with HDAd5 / 35++ vector containing a GFP / mgmt cassette, either alone or in combination with HDAd-SB, under the control of the EF1α promoter. Transduced cells were grown in erythrocyte differentiation medium for 16 days. GFP-positive cells with transposon incorporation were enriched by two O6BG / BCNU selections (50 μM O6BG + 35 μM BCNU). GFP-positive cells were selected by FACS on day 16 (sample #6). CD34+ cells transduced with mgmt / GFP vector alone and then selected were used as a comparison (sample #5). These control cells did not express SB100x and were therefore GFP-negative due to the loss of the episomal mgmt / GFP vector. Total RNA obtained from both samples was subjected to RNA-Seq (performed by Omega Bioservices). [Figure 47A-2]This study demonstrates the effect of SB100x-mediated incorporation on the transcriptome of CD34+ cells. A schematic diagram of the experiment is shown. CD34+ cells were infected with HDAd5 / 35++ vector containing a GFP / mgmt cassette, either alone or in combination with HDAd-SB, under the control of the EF1α promoter. Transduced cells were grown in erythrocyte differentiation medium for 16 days. GFP-positive cells with transposon incorporation were enriched by two O6BG / BCNU selections (50 μM O6BG + 35 μM BCNU). GFP-positive cells were selected by FACS on day 16 (sample #6). CD34+ cells transduced with mgmt / GFP vector alone and then selected were used as a comparison (sample #5). These control cells did not express SB100x and were therefore GFP-negative due to the loss of the episomal mgmt / GFP vector. Total RNA obtained from both samples was subjected to RNA-Seq (performed by Omega Bioservices). [Figure 47B] This shows the effect of SB100x-mediated integration on the transcriptome of CD34+ cells. Genes whose mRNA expression changed (log2 ratio change) are ranked based on their p-values. [Figure 48] This figure shows the expression levels of mgmt mRNA in bone marrow MNCs 16 weeks after in vivo transduction. Human mgmtP140K levels and mouse mRPL10 levels in whole bone marrow MNCs were measured by qRT-PCR (mRPL10 is a mouse housekeeping gene). Relative levels were further divided by VCN (see Figure 33). Statistical analysis was performed using two-way ANOVA. [Figure 49]This report compares in vivo HSC transduction in hCD46tg mice using "long-chain" LCR vectors and "short-chain" LCR vectors. In vivo transduction of Hbbth3 / CD46 mice was performed using the vectors. Group 1 shows the results of in vivo transduction of 7 mice using HDAd-long-chain LCR-γ-globin / mgmt + HDAd-SB / Flpe. Group 2 shows the results of in vivo transduction of 3 mice using HDAd-short-chain LCR-γ-globin / mgmt + HDAd-SB / Flpe. Only 3 selection cycles were required for selection using O6BG and BCNU. [Figure 50-1] The Thbb mouse test (week 6) is shown. The graph results show 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. [Figure 50-2] The Thbb mouse test (week 6) is shown. The graph results show 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. [Figure 51-1] The results of the Thbb mouse test (week 8) are shown. 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. [Figure 51-2] The results of the Thbb mouse test (week 8) are shown. 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. [Figure 52] The graph shows the percentage of human gamma-globin-expressing RBCs in mice. This graph demonstrates that 100% marking occurs after only three in vivo selection cycles. [Figure 53]This graph shows the relative values ​​of human gamma-globin to mouse HBA (at week 10) based on HPLC. This graph demonstrates that significantly higher gamma-globin levels are obtained with long-chain LCR compared to short-chain LCR. [Figure 54] The graph shows an example of HPLC analysis of blood obtained at 10 weeks from mouse #57 containing a long-chain LCR vector. [Figure 55A] This report characterizes AAVS1-specific CRISPR / Cas9 vectors and donor vectors for HDR-mediated integration. The HDAd-CRISPR vector structure: AAVS1-specific sgRNA is transcribed from the U6 promoter by PolIII, and the spCas9 gene is under the control of the EF1α promoter. Cas9 expression is regulated by miR-183-5p and miR-218-5p, which repress Cas9 expression in HDAd-producing 116 cells but do not negatively affect Cas9 expression in CD34+ cells (Sayadaminova et al., Mol Ther Methods Clin Dev, 1, 14057, 2015). The corresponding microRNA target site (miR-T) was embedded in the 3' untranslated region (3'UTR) of the β-globin gene. [Figure 55B] This document describes the characterization of AAVS1-specific CRISPR / Cas9 vectors and donor vectors for HDR-mediated integration. It shows the frequency of target site cleavage in human CD34+ cells measured by the T7E1 assay 3 days after transduction with HDAd-CRISPR at an MOI of 2000 vp / cell. The 474 bp and 294 bp segments are products of specific cleavage. Cleavage efficiency is shown below the gel. [Figure 55C]This document characterizes AAVS1-specific CRISPR / Cas9 vectors and donor vectors for HDR-mediated integration. The top 13 most frequently observed indels in HDAd-CRISPR-transduced CD34+ cells (sequences 6-18, from top to bottom) are shown. Sequences highlighted in light gray indicate guide RNA targets, and TAM sequences are highlighted in medium gray. CRISPR / Cas9 cleavage sites are indicated by vertical arrows. Green indicates insertions induced by NHEJ. [Figure 55D] This document characterizes the AAVS1-specific CRISPR / Cas9 vector and donor vector for HDR-mediated integration. The structure of the donor vector (HDAd-GFP-donor) for integration into the AAVS1 site is shown. The mgmtP140K gene is linked to the GFP gene via a self-cleaved picornavirus 2A peptide. These genes are under the control of the EF1α promoter. PA: polyadenylation signal. The transgene cassette is adjacent to a 0.8kb region homologous to the AAVS1 locus, similar to those in previously published studies (Lombardo et al., Nat Methods 8, 861-869, 2011). Upstream and downstream of these homologous regions are AAVS1-specific CRISPR / Cas9 recognition sites for releasing the donor cassette. [Figure 55E] This document characterizes AAVS1-specific CRISPR / Cas9 vectors and donor vectors for HDR-mediated integration. The release of the donor cassette is shown. CD34+ cells were infected using HDAd-GFP-donor (used at an MOI of 1000 vp / cell or 2000 vp / cell) alone or in combination with HDAd-CRISPR (MOI 1000 vp / cell). Genomic DNA was subjected to Southern blotting using a GFP-specific probe after 3 days. The electrophoretic position of the (linear) full-length HDAd-donor-GFP genome is 36 kb. The electrophoretic position of the free cassette is 4.7 kb. The cleavage frequency is shown below the gel. [Figure 56A]This paper compares targeted integration and SB100x-mediated integration in HUDEP-2 cells. The experimental scheme is shown below. HUDEP-2 cells were transduced using the HDAd vector shown, with each virus's MOI set at 1000 vp / cell. After 21 days of growth, GFP-positive cells were selected and seeded into 96-well plates. Single-cell clones were obtained by further growth for two weeks. GFP expression was measured in the cell population on day 2 and day 21 after transduction, and in the cell clones on day 35 after transduction. [Figure 56B] This shows a comparison of target-directed integration and SB100x-mediated integration in HUDEP-2 cells. The GFP levels in cells treated with the donor vector alone, or cells treated with the donor vector along with a vector possessing either the target-directed integration mechanism or the SB100x integration mechanism, were analyzed by flow cytometry on days 2 and 21. [Figure 56C] This shows a comparison of target-directed incorporation and SB100x-mediated incorporation in HUDEP-2 cells. The average fluorescence intensity of GFP in all GFP+ cells is compared between cells with target-directed incorporation and those with SB100x-mediated incorporation (day 21). The data shown (mean ± SD) represent three independent experiments. [Figure 56D] This paper compares target-directed integration and SB100x-mediated integration in HUDEP-2 cells. The average fluorescence intensity of GFP in a single clone is shown. Each symbol represents one cell clone. The data shown (mean ± SD) are representative of two independent experiments. [Figure 56E] This shows a comparison of target-directed incorporation and SB100x-mediated incorporation in HUDEP-2 cells. Flow cytometry is shown indicating GFP expression in representative cell clones that underwent target-directed incorporation or SB100x-mediated incorporation. [Figure 56F]This shows a comparison of target-directed integration and SB100x-mediated integration in HUDEP-2 cells. The vector copy number in cell clones, measured by qPCR using GFP primers, is also shown. [Figure 57A] This shows the analysis of integration in HUDEP-2 clones transduced with a target-directed integration vector. (Figure 57A) This shows the analysis of integration sites by inverse PCR. The upper figure shows the location of the NcoI site used and the primers (single arrow. Dark gray: EF1α primer for the 5' junction, light gray: pA primer for the 3' junction). The expected amplification product size is shown for each side of the target-directed integration. The gel photograph below shows the results of iPCR. Each lane represents one cell clone. A 1kb ladder obtained from New England Biolabs was used. Because an Ef1α primer was used, an extra endogenous Ef1α band was detected. For clone #20, the amplification product size was different from what was expected, but cloning and sequencing revealed that it was a clone in which targeted integration occurred. (Figure 57B) This shows the in-out PCR analysis. The upper figure shows the position of the primers. Expected product sizes for various integration patterns are listed. The gel image below demonstrates that targeted incorporation into one allele occurred in most clones (Figure 57A). In relation to the results obtained from (Figure 57A), the unexpected amplification product sizes obtained from clones #17, #20, and #36 may be due to chain-like incorporation. [Figure 57B]This shows the analysis of integration in HUDEP-2 clones transduced with a target-directed integration vector. (Figure 57A) This shows the analysis of integration sites by inverse PCR. The upper figure shows the location of the NcoI site used and the primers (single arrow. Dark gray: EF1α primer for the 5' junction, light gray: pA primer for the 3' junction). The expected amplification product size is shown for each side of the target-directed integration. The gel photograph below shows the results of iPCR. Each lane represents one cell clone. A 1kb ladder obtained from New England Biolabs was used. Because an Ef1α primer was used, an extra endogenous Ef1α band was detected. For clone #20, the amplification product size was different from what was expected, but cloning and sequencing revealed that it was a clone in which targeted integration occurred. (Figure 57B) This shows the in-out PCR analysis. The upper figure shows the position of the primers. Expected product sizes for various integration patterns are listed. The gel image below demonstrates that targeted incorporation into one allele occurred in most clones. In relation to the results obtained from (Figure 57A), the unexpected amplification product sizes obtained from clones #17, #20, and #36 may be due to chain-like incorporation. [Figure 58A] This shows cleavage of the AAVS1 target site in AAVS1 / CD46tg mice. In vitro analysis is also shown. The frequency of target site cleavage in myeloid-negative cells obtained from AAVS1 / CD46tg mice was measured 3 days after in vitro transduction by HDAd-CRISPR at the indicated MOI. [Figure 58B] This shows the cleavage of the AAVS1 target site in AAVS1 / CD46tg mice. The percentage of total AAVS1 indels obtained by deep sequencing of DNA from whole bone marrow mononuclear cells 14 weeks post-transplantation is shown. Each symbol represents an individual animal. [Figure 58C]This shows the cleavage of AAVS1 target sites in AAVS1 / CD46tg mice. The top 29 most frequently observed indels in the mice are shown (from top to bottom: SEQ ID NOs. 19-23, SEQ ID NOs. 21, SEQ ID NOs. 21, SEQ ID NOs. 26-30, SEQ ID NOs. 27, SEQ ID NOs. 32, SEQ ID NOs. 28, SEQ ID NOs. 34-47). The data shown are representative. Yellow sequences indicate guide RNA targets, and TAM sequences are marked in blue. CRISPR / Cas9 cleavage sites are marked with vertical arrows. [Figure 59A] This diagram shows the results of ex vivo transduction of AAVS1 / CD46 Lin- cells using HDAd-AAVS1 and HDAd-GFP donors, followed by transplantation of these AAVS1 / CD46 Lin- cells into lethal irradiation recipients. Schematic diagram of the experiment: Bone marrow was collected from AAVS1 / CD46tg mice, and lineage-negative cells (Lin-) were isolated by MACS. Lin- cells were transduced using HDAd-CRISPR and HDAd-GFP donors, either individually or in combination (total MOI 500vp / cell). After 1 day of culture, 1 × 10⁶ transduced cells / mouse were transplanted into lethal irradiation C57Bl / 6 mice. O6BG / BCNU treatment was started at week 4 and repeated every 2 weeks for a total of 3 cycles. The BCNU concentration was increased from 5 mg / kg → 7.5 mg / kg → 10 mg / kg with each cycle. Mice were sacrificed at 14 weeks, and bone marrow Lin- cells were used for transplantation into lethally irradiated secondary C57Bl / 6 recipients. These secondary C57Bl / 6 recipients were then observed for 16 weeks. [Figure 59B]This shows the results of ex vivo transduction of AAVS1 / CD46Lin- cells using HDAd-AAVS1 and HDAd-GFP- donors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The percentage of GFP-positive cells in peripheral blood mononuclear cells (PBMCs) is shown, measured by flow cytometry. The results show groups transplanted with Lin- cells transduction using HDAd-CRISPR alone, Lin- cells transduction using HDAd-GFP- donors alone, and Lin- cells transduction using HDAd-CRISPR + HDAd-GFP- donors. Each symbol represents an individual animal. [Figure 59C] This shows the results of ex vivo transduction of AAVS1 / CD46Lin- cells using HDAd-AAVS1 and HDAd-GFP- donors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The percentage of GFP+ cells in PBMCs obtained from representative mice transplanted with Lin- cells is shown. Data obtained from week 4 (before selection) and week 12 (after selection) are presented. [Figure 59D] This shows the results of ex vivo transduction of AAVS1 / CD46Lin- cells using HDAd-AAVS1 and HDAd-GFP- donors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The percentage of GFP+ cells in lineage-positive cells (CD3+ (T cells), CD19+ (B cells), Gr-1+ (myeloid cells)) and HSCs (LSK cells) is shown. [Figure 60A] This shows the engraftment of ex vivo transduced Lin- cells. Engraftment of transplanted cells is shown based on human CD46 expression on PBMCs measured by flow cytometry. Each symbol represents an individual animal. It is noteworthy that the transduced donor cells expressed CD46, while the recipient C57Bl / 6 mice did not. [Figure 60B]This shows the engraftment analysis of ex vivo transduced Lin- cells. The percentage of CD46-positive cells in PBMCs (blood), spleen, and bone marrow at week 14 is shown. [Figure 60C] This shows the engraftment analysis of ex vivo transduced Lin- cells. The percentage of GFP-positive cells in PBMCs, spleen, and bone marrow at week 14 is shown. [Figure 60D] This shows the engraftment analysis of Lin- cells transduced ex vivo. The percentages of LSK cells and lineage-positive cells under different transduction conditions are shown. The differences between the three groups are not statistically significant. [Figure 60E] This panel shows the analysis of engraftment of ex vivo transduced Lin- cells. Analysis of GFP+ colonies is also shown. Whole bone marrow Lin- cells obtained from mice were seeded at 14 weeks, and GFP expression in the colonies was analyzed at 12 days. Each symbol represents the average number of GFP+ colonies in each individual mouse (left panel). Cells obtained from all colonies were pooled and analyzed by flow cytometry (right panel). [Figure 61A] This section describes the analysis of GFP marking in secondary recipients. Bone marrow cells derived from responder mice transplanted with Lin- cells transduced using HDAd-GFP-donors or HDAd-CRISPR+HDAd-GFP-donors were collected 14 weeks post-transplantation, subjected to depletion of lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. GFP flow cytometry of PBMCs in four recipient mice is shown. The right panel shows a typical analysis. The vertical axis shows hCD46 staining, and the horizontal axis shows GFP staining. [Figure 61B] This section describes the analysis of GFP marking in secondary recipients. Bone marrow cells derived from responder mice transplanted with Lin- cells transduced using HDAd-GFP-donors or HDAd-CRISPR+HDAd-GFP-donors were collected at 14 weeks post-transplantation, subjected to depletion of lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. The percentage of GFP-positive cells in PBMCs, spleen, and bone marrow at 16 weeks is shown. [Figure 61C]This section describes the analysis of GFP marking in secondary recipients. Bone marrow cells derived from responder mice transplanted with Lin- cells transduced using HDAd-GFP-donors or HDAd-CRISPR+HDAd-GFP-donors were collected at 14 weeks post-transplantation, subjected to treatment to remove lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. The GFP flow analysis of lineage-positive and lineage-negative cells in recipients at 16 weeks post-transplantation is shown. [Figure 61D] This section describes the analysis of GFP marking in secondary recipients. Bone marrow cells derived from responder mice transplanted with Lin- cells transduced using HDAd-GFP-donors or HDAd-CRISPR+HDAd-GFP-donors were collected at 14 weeks post-transplantation, subjected to treatment to remove lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. The analysis of GFP+ colonies is shown. Whole bone marrow Lin- cells obtained from mice were seeded at 16 weeks, and GFP expression in the colonies was analyzed at 12 days. Each symbol represents the average number of GFP+ colonies in an individual mouse (left panel). Cells obtained from all colonies were pooled and analyzed by flow cytometry (right panel). [Figure 61E] This section describes the analysis of GFP marking in secondary recipients. Bone marrow cells derived from responder mice transplanted with Lin- cells transduced using HDAd-GFP-donors or HDAd-CRISPR+HDAd-GFP-donors were collected 14 weeks post-transplantation, subjected to treatment to remove lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. Engraftment of transplanted cells is shown based on human CD46 expression on PBMCs measured by flow cytometry. [Figure 61F] This report presents an analysis of GFP marking in secondary recipients. Bone marrow cells derived from responder mice transplanted with Lin- cells transduced using HDAd-GFP-donors or HDAd-CRISPR+HDAd-GFP-donors were collected 14 weeks post-transplantation, subjected to treatment to remove lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. The percentages of lineage-positive and lineage-negative cells under different transduction conditions are shown. The difference between the two groups is not statistically significant. [Figure 62A] This report describes in vivo transduction of AAVS1 / hCD46tg mice using HDAd-AAVS1-CRISPR+HDAd-GFP-donor. The treatment regimen is as follows: AAVS1 / hCD46tg mice were recruited and IV-injected with HDAd-CRISPR+HDAd-GFP-donor (two injections of a 1:1 mixture of both viruses (4 × 10¹⁰ vp each)). After 4 weeks, O6BG / BCNU treatment was initiated. The BCNU concentration was increased to 2.5 mg / kg → 7.5 mg / kg → 10 mg / kg with each cycle. The O6BG concentration was 30 mg / kg for all three treatments. Mice were observed until week 12, 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. [Figure 62B] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-AAVS1-CRISPR+HDAd-GFP-donors. The percentage of GFP-positive cells in peripheral blood mononuclear cells (PBMCs) is shown as measured by flow cytometry. [Figure 62C] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-AAVS1-CRISPR+HDAd-GFP-donors. The percentage of GFP-positive cells in PBMCs, spleen, and bone marrow at 14 weeks is also shown. [Figure 62D] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-AAVS1-CRISPR+HDAd-GFP-donors. It also shows the percentage of GFP+ cells in lineage-positive cells (CD3+ (T cells), CD19+ (B cells), Gr-1+ (myeloid cells)) and HSCs (LSK cells). [Figure 62E]This report describes the in vivo transduction of AAVS1 / CD46tg mice using HDAd-AAVS1-CRISPR+HDAd-GFP-donors. Analysis of GFP+ colonies is also shown. Whole bone marrow Lin- cells obtained from mice were seeded at 14 weeks, and GFP expression in the colonies was analyzed at 12 days. Each symbol represents the average number of GFP+ colonies in each individual mouse (left panel). Cells obtained from all colonies were pooled and analyzed by flow cytometry (right panel). [Figure 62F] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-AAVS1-CRISPR+HDAd-GFP-donors. The percentages of lineage-positive and lineage-negative cells at 14 weeks are shown. [Figure 63A] Figures 59A-59D show the analysis of secondary recipients. At week 14, bone marrow Lin- cells obtained from in vivo transduced AAVS1 / hCD46tg mice were transplanted into lethal irradiated C57Bl / 6 recipients. GFP flow cytometry of PBMCs in six recipient mice is shown. [Figure 63B] Figures 59A-59D show the analysis of secondary recipients. At week 14, bone marrow Lin- cells obtained from in vivo transduced AAVS1 / hCD46tg mice were transplanted into lethal irradiated C57Bl / 6 recipients. GFP expression in mononuclear cells in blood, spleen, and bone marrow is shown. [Figure 63C] Figures 59A-59D show the analysis of secondary recipients. At week 14, bone marrow Lin- cells obtained from in vivo transduced AAVS1 / hCD46tg mice were transplanted into lethal irradiated C57Bl / 6 recipients. GFP flow analysis of lineage-positive and lineage-negative cells in the recipients 16 weeks after transplantation is shown. [Figure 63D] Figures 59A-59D show the analysis of secondary recipients. At week 14, bone marrow Lin- cells obtained from in vivo transduced AAVS1 / hCD46tg mice were transplanted into lethal irradiated C57Bl / 6 recipients. Engraftment of transplanted cells is shown based on human CD46 expression on PBMCs measured by flow cytometry. [Figure 63E] Figures 59A-59D show the analysis of secondary recipients. At week 14, bone marrow Lin- cells obtained from in vivo transduced AAVS1 / hCD46tg mice were transplanted into lethal irradiated C57Bl / 6 recipients. The percentages of lineage-positive and lineage-negative cells at week 16 are shown. [Figure 64A] This shows AAVS1 / CD46Lin- cells transduced ex vivo using the HDAd-AAVS1 vector and the HDAd-donor-γ-globin vector, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The donor structure is shown. The overall structure is the same as that of the HDAds-GFP-donor vector (see Figure 55D). In the novel HDAd-globin-donor vector, the homology region is elongated (comparison of 1.8kb and 0.8kb). The γ-globin expression cassette contains a 4.3kb version of γ-globin LCR containing four DNAse-high sensitivity (HS) regions and a γ-globin promoter (Lisowski et al., Blood. 110, 4175-4178, 1996). Full-length γ-globin cDNA including the 3'UTR (for stabilizing mRNA in red blood cells) was used. The mgmtP140K gene is under the control of a ubiquitously active EF1α promoter. Bidirectional SV40 polyadenylation signaling was used to terminate transcription. To avoid interference between the LCR / β-promoter and the EF1α promoter, a 1.2kb chicken HS4 chromatin insulator (Emery et al., Proc Natl Acad Sci USA, 97, 9150-9155, 2000) was inserted between the cassettes. [Figure 64B] This figure shows the results of transduction of AAVS1 / CD46Lin- cells ex vivo using HDAd-AAVS1 vectors and HDAd-donor-γ-globin vectors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The treatment regimen is the same as that shown in Figure 57A. [Figure 64C]This image shows AAVS1 / CD46Lin- cells transduced ex vivo using HDAd-AAVS1 vectors and HDAd-donor-γ-globin vectors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The percentage of human γ-globin-positive cells in peripheral red blood cells (RBCs) is shown, measured by flow cytometry. [Figure 64D] This shows the results of transduction of AAVS1 / CD46Lin- cells ex vivo using HDAd-AAVS1 vectors and HDAd-donor-γ-globin vectors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The percentage of human γ-globin-positive cells in erythrocytes (Ter119+) and non-erythrocytes (Ter119-) in blood and bone marrow 16 weeks after in vivo transduction is shown. *p<0.05. [Figure 64E] This shows the results of transduction of AAVS1 / CD46Lin- cells ex vivo using HDAd-AAVS1 vectors and HDAd-donor-γ-globin vectors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The mean fluorescence intensity of human γ-globin-positive cells in erythrocytes (Ter119+) and non-erythrocytes (Ter119-) in blood and bone marrow 16 weeks after in vivo transduction is shown. *p<0.05. [Figure 64F] This image shows AAVS1 / CD46Lin- cells transduced ex vivo using HDAd-AAVS1 vectors and HDAd-donor-γ-globin vectors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The percentage of γ-globin chains relative to mouse β-major chains in RBCs was measured by HPLC at week 16. [Figure 64G]This image shows AAVS1 / CD46Lin- cells transduced ex vivo using HDAd-AAVS1 vectors and HDAd-donor-γ-globin vectors, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The percentage of γ-globin mRNA relative to mouse β-major RNA in RBCs is measured by qRT-PCR at week 16. [Figure 64H] This shows the results of transduction of AAVS1 / CD46Lin- cells ex vivo using HDAd-AAVS1 vector and HDAd-donor-γ-globin vector, followed by transplantation of these AAVS1 / CD46Lin- cells into lethal irradiation recipients. The vector copy number per cell in the colonies generated from Lin- cells is shown. Each symbol represents one colony. Differences between animals are not statistically significant. [Figure 65] This shows the engraftment of AAVS1 / CD46Lin- cells transduced with HDAd-CRISPR vectors and HDAd-globin-donor vectors. (A) Engraftment of transplanted cells based on human CD46 expression on PBMCs measured by flow cytometry. (B) Percentage of CD46-positive cells in lineage-positive PBMCs (blood), spleen, and bone marrow cells, as well as bone marrow LSK cells, at 16 weeks. [Figure 66A] Figures 64A-64H show the analysis of secondary recipients. Bone marrow cells derived from mice transplanted with Lin- cells transduced using HDAd-CRISPR+HDAd-globin-donors were collected 16 weeks post-transplantation, subjected to treatment to remove lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. γ-globin flow cytometry of RBCs in five recipient mice is shown. [Figure 66B] Figures 64A-64H show the analysis of secondary recipients. Bone marrow cells derived from mice transplanted with Lin- cells transduced with HDAd-CRISPR+HDAd-globin-donors were collected 16 weeks post-transplantation, subjected to treatment to remove lineage-positive cells, and then transplanted into lethal-irradiated C57Bl / 6 mice. The percentage of CD46-positive cells in lineage-positive PBMCs is shown. [Figure 66C] Figures 64A-64H show the analysis of secondary recipients. Bone marrow cells derived from mice transplanted with Lin- cells transduced using HDAd-CRISPR+HDAd-globin-donors were collected 16 weeks post-transplantation, subjected to treatment to remove lineage-positive cells, and then transplanted into lethally irradiated C57Bl / 6 mice. The bone marrow composition of secondary recipients 16 weeks post-transplantation is shown. [Figure 67A] This document describes the in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. The treatment regimen is shown. [Figure 67B] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. The percentage of γ-globin-positive RBCs is shown. [Figure 67C] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. A representative dot plot is shown showing the percentage of γ-globin expression in peripheral RBCs obtained from non-transduced control mice or mice 16 weeks post-transduction. [Figure 67D] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. The mean fluorescence intensity of γ-globin in erythrocytes (Ter119+) and non-erythrocytes (Ter119-) in blood and bone marrow is shown. *p<0.05. [Figure 67E] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. The percentage of γ-globin chains relative to mouse β-major chains in RBCs was measured by HPLC at week 16. *p<0.05. [Figure 67F] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. The percentage of γ-globin mRNA relative to mouse β-major RNA in RBCs was measured by qRT-PCR at week 16. *p<0.05. [Figure 67G] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. The vector copy count per cell in colonies derived from Lin- cells obtained from four responder mice is shown. Each symbol represents one colony. Differences between animals are not statistically significant. [Figure 67H] This shows in vivo transduction of AAVS1 / CD46tg mice using HDAd-CRISPR + HDAd-globin donor. It also shows the composition of lineage-positive cells in blood, spleen, and bone marrow, as well as in LSK cells in the bone marrow, 16 weeks after in vivo transduction. [Figure 68A] Figures 67A-67H show the analysis of secondary recipients. Engraftment of transplanted cells is shown based on the expression of human CD46 on PBMCs measured by flow cytometry. [Figure 68B] Figures 67A-67H show the analysis of secondary recipients, illustrating the expression of γ-globin in RBCs. [Figure 68C] The analysis of secondary recipients obtained from Figures 67A to 67H is shown. The percentage of γ-globin chains relative to mouse β-major chains in the RBCs of secondary recipients is shown, measured by HPLC at week 16. [Figure 68D] Figures 67A-67H show the analysis of secondary recipients. They show the composition of lineage-positive cells in blood, spleen, and bone marrow 16 weeks after in vivo transduction. [Figure 69A] This image shows the localization and structure of the AAVS1 locus in AAVS1 / CD46 transgenic mice. TLA data showing a mismatch on chromosome 14 is also shown. AAVS1-specific primer pairs were used. The right panel shows an enlarged section of chromosome 14, revealing an 18kb gap. This gap corresponds to the added human AAVS1 locus. [Figure 69B] This shows the localization and structure of the AAVS1 gene locus in AAVS1 / CD46 transgenic mice. [Figure 70]The detailed structure of the AAVS1 locus, which indicates genomic localization, is shown. The shaded AAVS1 region was confirmed by Sanger sequencing. Blank areas were estimated from restriction enzyme analysis and genetic background information of AAVS1 tg mice obtained from Jackson Laboratory. CRISPR / Cas9 cleavage sites are indicated by scissors. Repeat sequences #2 to #5 are complete 8.2kb human AAVS1 EcoRI fragments, while repeat sequences #1 and #5 contain only a portion of this EcoRI fragment. Notably, repeat sequence #5 completely lacks a 5' homology arm. The results obtained depend on the CRISPR / Cas9 cleavage of the multi-copy AAVS1 locus present in AAVS1 tg mice. The rules regarding cleavage locations are as follows: a) One cleavage in repeat sequences #1 to #4: occurs preferentially. b) One cleavage in repeat sequence #5: its priority is lower due to the incomplete left homology arm. c) Two cleavages in two reverse-oriented repeat sequences (e.g., #1 and #4): HDR-mediated target-directed integration does not occur due to the absence of a right homology arm. d) Two cleavages in two repeat sequences oriented in the same direction (e.g., #1 and #2): This occurs preferentially. e) More than two cleavages (only those located near the mouse gDNA sequence on each side are conceivable): Therefore, rule c) or rule d) applies. f) Cleavages occur in repeat sequences #1 and #5, resulting in deletion of the central region. Furthermore, if HDR-mediated target-directed integration occurs in repeat sequences #2-#4, and adjacent repeat sequences (e.g., #1 and #5) are successively cleaved by CRISPR, the already integrated transgene may be lost. [Figure 71A] This image shows the integration sites of genomic DNA isolated at 16 weeks after transduction of HSCs ex vivo or in vivo using HDAd-CRISPR+HDAd-GFP donors, analyzed by Southern blotting. Hybridization with AAVS1-specific probes is shown. The upper panel shows the expected EcoRI fragment size and probe localization. The lower panel shows the analysis of individual mice obtained from ex vivo and in vivo transduction status. Larger bands represent AAVS1 locus repeat sequences that were not targeted. [Figure 71B] This shows the integration sites of genomic DNA isolated at 16 weeks after transduction of HSCs ex vivo or in vivo using HDAd-CRISPR+HDAd-GFP donors, analyzed by Southern blotting. Hybridization with a GFP-specific probe on DNA digested with BlpI is also shown. The band pattern will be discussed elsewhere. [Figure 72A] This figure shows the results of inverse PCR (iPCR) analysis of the integration sites of genomic DNA isolated at 16 weeks after transduction of HSCs ex vivo or in vivo using HDAd-CRISPR+HDAd-GFP-donors. The figure shows the location of the NcoI site and the primers (single arrow: EF1a primer for the 5' junction, light gray: pA primer for the 3' junction). The expected amplification product sizes for each side of targeted integration to repeat sequence #5 are shown. [Figure 72B] This shows the results of inverse PCR (iPCR) analysis of the integration sites of genomic DNA isolated 16 weeks after transduction of HSCs ex vivo or in vivo using HDAd-CRISPR+HDAd-GFP-donors. The results of iPCR using genomic DNA obtained from whole bone marrow cells are shown. Each lane represents one mouse. #009, #023, #943, #944, and #946 are mice transduction of HSCs ex vivo. #147, #304, and #467 are animals transduced in vivo. [Figure 72C]This shows the results of inverse PCR (iPCR) analysis of genomic DNA isolated at 16 weeks after transduction of HSCs ex vivo or in vivo using HDAd-CRISPR+HDAd-GFP-donors, analyzing the integration sites. The iPCR analysis of GFP-positive colonies is shown. Bone marrow Lin- cells obtained from mice were seeded at 14 weeks, and genomic DNA was isolated from GFP+ colonies at 20 days and used for iPCR. Mice #943 and #946 were analyzed. Each lane represents one colony. Light gray arrows: targeted integration, dark gray arrows: off-target integration, medium gray arrows: whole HDAd virus genome integration. [Figure 73] This figure shows the results of inverse PCR (iPCR) analysis of integration sites in genomic DNA isolated at 16 weeks after transduction of HSCs ex vivo or in vivo using HDAd-CRISPR + HDAd-globin donor. (A) The figure shows the location of the NcoI site and primers (single arrow; black: EF1a primer for the 5' junction, gray: pA primer for the 3' junction). The expected amplification product size on each side of targeted integration to repeat sequence #5 is shown. (B) The results of iPCR using genomic DNA obtained from whole bone marrow cells are shown. Each lane represents one mouse. #321, #322, #856, #857, #858, and #945 are mice transduced ex vivo. #504, #816, #869, and #898 are animals transduced in vivo. White arrowheads indicate targeted integration. Gray dotted arrowhead: Off-target integration; White full arrow: Integration of the entire HDAd viral genome. [Figure 74A]The HDAd5 / 35++ vector for in vivo transduction of HSPCs is shown. In HDAd-GFP / mgmt, the transposon is adjacent to the reverse transposon repeat sequence (IR) and frt site for integration via the highly active Sleeping Beauty transposase (SB100X) supplied from the HDAd-SB vector. The transgene cassette contains a PGK-promoter-promoting GFP gene linked to the β-globin 3'UTR, as well as an EF1α-promoter-promoting mgmtP140K cassette. Both cassettes are isolated by a chicken globin HS4 insulator. HSPCs were recruited in neu / CD46 transgenic mice by subcutaneous injection of human recombinant G-CSF (5 μg / mouse / day, 4 days), followed by subcutaneous injection of AMD3100 (5 mg / kg) 18 hours after the last G-CSF injection. One hour after AMD3100 injection, HDAd-GFP / mgmt + HDAd-SB (total viral particle count 8 × 10¹⁰) was administered intravenously. To prevent the release of pro-inflammatory cytokines after HDAd injection, dexamethasone (10 mg / kg) was administered intraperitoneally to the animals 16 hours and 2 hours prior to viral injection. Six weeks later, O6BG / BCNU (intraperitoneal) was applied three times (30 mg / kg O6BG + 5 mg / kg, 7.5 mg / kg, and 10 mg / kg BCNU) to activate the release of transduced HSPCs into peripheral blood circulation. Seventeen weeks after in vivo transduction, 1 × 10⁶ MMC cells were transplanted into the mammary fat body. Five weeks later, tumor and other tissues were collected and analyzed for GFP expression. [Figure 74B] Left panel: Percentage of GFP expression in PBMCs at different time points after in vivo transduction. Each symbol represents an individual animal. Right panel: Percentage of GFP+ cells in panleukocyte marker CD45-stained cells present in bone marrow, spleen, blood, and tumors digested with collagenase / dispase. [Figure 74C] The image shows tumor sections stained with antibodies against GFP and laminin (an extracellular matrix protein). The scale bar represents 50 μm. [Figure 74D]This shows the immunophenotyping of GFP+ PBMCs in the blood and GFP+ cells in tumors. [Figure 75] This image shows rat Neu expression in MMC cells. Cells were stained with Neu-specific monoclonal antibody 7.16.4, followed by anti-mouse Ig-FITC. Representative confocal microscopy images of cultured MMC cells are shown. Neu-specific signals appear as whiter hues. The scale bar is 20 μm. [Figure 76-1] This document outlines a gate strategy for determining immunophenotypes. [Figure 76-2] This document outlines a gate strategy for determining immunophenotypes. [Figure 77] This shows the immunophenotyping of GFP+ cells in bone marrow and spleen (MMC model). See Figure 74D for details. [Figure 78A] This shows GFP expression in tumor-infiltrating leukocytes after transduction of HSPC in vivo (TC-1 model). A schematic diagram of the experiment is shown. HSPCs were recruited in CD46tg transgenic mice by subcutaneous injection of human recombinant G-CSF (5 mg / mouse / day, 4 days), followed by subcutaneous injection of AMD3100 (5 mg / kg) 18 hours after the last G-CSF injection. One hour after the AMD3100 injection, HDAd-GFP / mgmt + HDAd-SB (total viral particle count 8 × 10¹⁰) was administered intravenously. To prevent the release of pro-inflammatory cytokines after HDAd injection, dexamethasone (10 mg / kg) was administered intraperitoneally to the animals 16 hours and 2 hours before viral injection. Six weeks later, three intraperitoneal administrations of O6BG / BCNU (30 mg / kg O6BG + 5 mg / kg, 7.5 mg / kg, and 10 mg / kg BCNU) were performed to activate the release of transduced HSPCs into peripheral blood circulation. Seventeen weeks after in vivo transduction, 5 × 10⁴ TC-1 cells were transplanted into the mammary fat body. Five weeks later, tumor and other tissues were collected and analyzed for GFP expression. [Figure 78B]This graph shows GFP expression in tumor-infiltrating leukocytes after transduction of HSPCs in vivo (TC-1 model). It also shows the percentage of GFP expression in PBMCs at different time points after transduction in vivo. Each symbol represents an individual animal. [Figure 78C] This shows GFP expression in tumor-infiltrating leukocytes after transduction of HSPC in vivo (TC-1 model). It also shows the percentage of GFP+ cells in panleukocyte marker CD45-stained cells present in bone marrow, spleen, blood, and tumors digested with collagenase / dispase. [Figure 78D] This shows GFP expression in tumor-infiltrating leukocytes after transduction of HSPC in vivo (TC-1 model). Representative flow cytometry data for GFP+ cells and GFP+-positive leukocytes in all (malignant + tumor-infiltrating) cells are shown. [Figure 78E] This image shows GFP expression in tumor-infiltrating leukocytes after transduction of HSPC in vivo (TC-1 model). Representative tumor sections are shown. Left panel: GFP fluorescence. Right panel: Staining with antibody against GFP (white) and antibody against extracellular matrix protein laminin (gray). Scale bar is 50 mm. [Figure 78F] This study demonstrates GFP expression in tumor-infiltrating leukocytes after transduction of HSPC in vivo (TC-1 model). It also shows immunophenotyping of GFP+ cells in tumors and circulating PBMCs. Lymphocyte flow cytometry panel 8c (CD45, CD3, CD4, CD8, CD25, CD19) and myeloid panel 9c (CD45, CD11c, F4 / 80, MHCII, SiglecF-PecCP, Ly6C, CD11b, Ly6G) obtained from BD Biosciences were used. [Figure 79A]This study demonstrates the selection of miRNAs to induce repression in cells other than tumor-infiltrating leukocytes. It also shows that the tissue specificity of transgene expression is regulated based on miRNAs. miRNAs function as guide molecules by forming base pairs with target sequences, known as miRNA target sites (miR-Ts), and are typically located in the 3' untranslated region (3'UTR) of native mRNA. This interaction recruits effector complexes, mediating mRNA cleavage or translational repression. If the transgene mRNA contains a miR-T for a miRNA highly expressed in a given cell type, transgene expression will be inhibited in that cell type. Conversely, in cell types that do not express this specific miRNA, the transgene will be expressed (Brown et al., Nat Med. 2006;12:585-591). [Figure 79B] This study demonstrates the selection of miRNAs to induce suppression in cells other than tumor-infiltrating leukocytes. MicroRNA-Seq was performed on pooled RNA from five mice (neu / CD46tg-MMC model, 17 days post-tumor inoculation). The normalized microRNA read counts (read count per 1 million mapped microRNA reads + 1) identified by small molecule RNA sequencing of the spleen, bone marrow, and blood are shown compared to those of 13 GFP+ tumor samples. MicroRNAs not present in the tumor (including miR-423) are listed on the left of the scatter plot with a pseudo-number of 1 added. miR-423-5p is shown in the blot. [Figure 79C] This study demonstrates the selection of miRNAs that induce suppression in cells other than tumor-infiltrating leukocytes. MicroRNA-Seq was performed on pooled RNA from five mice (CD46tg / TC-1 model, day 17). The top 10 miRNAs with relative expression levels compared to tumor levels (set to 1) are shown. [Figure 80A]This shows the effect of overexpression of the miR-423-5p target site on HSPC. The vector structure is shown. HDAd-GFP-miR-423 contains four miR-423-5p target sites in the 3'UTR linked to the GFP gene. [Figure 80B] This study demonstrates the effects of overexpression of the miR-423-5p target site on HSPCs. Mouse HSPCs (M) (Lin- cells obtained from the bone marrow of CD46 transgenic mice) and human HSPCs (Hu) (CD34+ cells) were infected with either HDAd-GFP or HDAd-GFP-miR423 at MOIs of 500 vp / cell or 3000 vp / cell, respectively. After 3 days, CDKN1A in the cell lysates was analyzed by Western blotting. The blots were re-searched with an anti-β-actin antibody to adjust for load differences. The right panel shows the quantified CDKN1A signal normalized by the β-actin signal. The signals obtained from the corresponding mouse HDAd-GFP / mgmt and human HDAd-GFP / mgmt samples were set to 100%. [Figure 80C] This study demonstrates the effect of overexpression of the miR-423-5p target site on HSPCs. It also shows the effect on colony formation of progenitor cells. One day after HDAd infection, mouse Lin- cells (2.5 × 10³ cells / 35 mm dish) or human CD34+ cells (3 × 10³ cells / dish) were seeded for colony assay. Colonies were counted after 12 days. N=3. *p<0.05. Statistical significance was calculated using a two-sided Student's t-test (Microsoft Excel) (consistent with previous studies (Li et al., Mol Ther Methods Clin Dev. 2018;9:390-401, Li et al., Mol Ther Methods Clin Dev. 9:142-152, 2018), where relatively high MOI during HSPC infection slightly reduced HSPC colony formation ability). [Figure 81]This report demonstrates the validation of miR-423-5p expression by Northern blotting. Total RNA (2 μg) obtained from myeloid-negative cells, spleen, whole blood cells, and MMC- / TC-1-tumor-infiltrating leukocytes was isolated on a 15% denatured polyacrylamide gel. The blots were subjected to hybridization with a probe specific to muRNA-423-5p, followed by hybridization with a probe for U6 RNA (load control). Mir-423 has a precursor of 70 bp in length, and its mature miRNA is 23 bp long. A miR-423-5p specific signal was observed in blood, bone marrow, and spleen, but was absent in tumor-infiltrating cells of both tumor models. [Figure 82A] This shows the expression of miRNA423-5p in humans. The levels of miRNA423-5p are shown as published in Ludwig et al., Nucleic Acids Res. 2016;44:3865-3877. The y-axis labels, from left to right, include adipocytes, arteries, colon, dura mater, kidneys, livers, lungs, muscles, cardiac muscle, skin, spleen, stomach, testes, thyroid gland, small intestine and duodenum, small intestine and jejunum, pancreas, adrenal gland, renal cortex, renal medulla, esophagus, prostate, bone marrow, veins, lymph nodes, pleura, pituitary gland, spinal cord, thalamus, white matter, caudate nucleus, gray matter, temporal lobe of the cerebral cortex, frontal lobe of the cerebral cortex, occipital lobe of the cerebral cortex, and cerebellum. [Figure 82B] This shows the expression of miRNA423-5p in humans. A plot of miRNA-Seq data obtained from two pooled ovarian cancer patients is shown. CD45+ cells were isolated from biopsy specimens of high-grade serous ovaries. RNA was isolated from tumor-infiltrating leukocytes and comparative PBMCs and subjected to miRNA-Seq by LCSciences, LLC. miRNA-423-5p is shown. [Figure 83A] This image shows in vivo HSPC αPD-L1-γ1 immune checkpoint inhibitor treatment in a neu / MMC model. PDL1 expression in MMC tumor cells (white) is shown. The scale bar is 20 μm. [Figure 83B]This describes in vivo HSPC αPD-L1-γ1 immune checkpoint inhibitor therapy in the neu / MMC model. The overall structure of the therapeutic vector is the same as that shown in Figure 74A. This vector contains an HA tag and secretory signaling (LS) located at the 5' end, a human IgG1 hinge-CH2-CH3 domain located at the 3' end, and a myc tag, along with an expression cassette of scFv anti-mouse PD-L1 ligated to it. The miR423-5p target site was inserted into the 3'UTR to limit αPD-L1-γ1 expression to tumor-infiltrating cells through regulation by miR423-5p. The vector also contains an expression cassette of mgtmP140K. [Figure 83C] This shows in vivo treatment of HSPCs with αPD-L1-γ1 immune checkpoint inhibitors in a neu / MMC model. The tumor volume is shown after inoculation (day 0) of MMC cells into mice with HSPCs transduced in vivo with HDAd-GFP / mgmt and mice with HSPCs transduced in vivo with HDAd-αPD-L1-γ1. The HDAd-αPD-L1-γ1 group mice were reloaded with 1 × 10⁵ MMC cells by subcutaneous injection 80 days after the first tumor cell injection. Each curve represents an individual animal. [Figure 83D] This report describes in vivo HSPC αPD-L1-γ1 immune checkpoint inhibitor treatment in a neu / MMC model. Flow cytometry analysis of T cell responses is also presented. Splenocytes from naive neu transgenic mice and HDAd-αPD-L1-γ1 treated mice (day 100) were analyzed by flow cytometry for CD4 and CD8 staining, as well as intracellular IFNγ or Neu tetramer staining. N=3. *p<0.05. [Figure 83E]This study demonstrates in vivo HSPC αPD-L1-γ1 immune checkpoint inhibitor treatment in a neu / MMC model. It shows the IFNγ response to stimulation with Neu+ and Neu cells. Splenocytes obtained from naive neu transgenic mice and HDAd-αPDL1-γ1 treated mice (day 100) were exposed to arrested MMC cells (Neu+) or spleen cells from neu transgenic mice (Neu-), or treated with PMA / ionomycin ("noAg"). IFNγ concentrations in the culture supernatant are shown. N=3. *p<0.005. [Figure 84A] This shows the kinetics of αPD-L1-γ1 expression. A Western blot of αPD-L1-γ1 using an anti-HA tagged antibody is shown. Three animals were sacrificed on day 17, and their tissues were analyzed for αPD-L1-γ1 expression by Western blotting. The αPD-L1-γ1 protein was not completely reduced, resulting in a complete αPD-L1-γ1 (130kDa) residue with two scFv chains (see the right panel for the structure of αPD-L1-γ1). β-actin-stained tissue was used as a load control. Representative samples are shown. Quantified Western blot signals are also shown. N=5 mice. [Figure 84B] This study demonstrates the kinetics of αPD-L1-γ1 expression. It shows αPD-L1-γ1 mRNA expression in tumor-infiltrating leukocytes, PBMCs, myeloid cells, and splenocytes. Mouse PPIA mRNA was used as an internal control. Results were calculated according to the 2(-ΔΔCt) method and are expressed as relative expression percentages, with the cDNA level of the corresponding tumor sample set to 100%. [Figure 84C] This shows the kinetics of αPD-L1-γ1 expression. The levels of αPD-L1-γ1 secreted into the serum are shown, measured by ELISA. Recombinant mouse PD-L1 was used for capture, and an anti-HA antibody-HRP conjugate was used for detection. Each symbol represents an individual animal. *p<0.05. Statistical significance was calculated using a two-tailed Student's t-test (Microsoft Excel). [Figure 85A]This report describes an immunopreventive study in an ID8-p53- / -brca2- / - ovarian cancer model. It also shows the analysis of ID8-p53- / -brca2- / - tumors. ID8-p53- / -brca2- / - cells (total cell count 2 × 10⁶) were injected intraperitoneally into CD46 transgenic mice. Ascites / cachexia developed after 6–8 weeks. The tumors were then removed and digested with dispase / collagenase for flow cytometry. Cell fractions were selected for tumor-associated macrophages (TAMs), neutrophils (TANs), and T cells (TILs) for Northern blot analysis (see Figure 76). [Figure 85B] This shows an immunopreventive study in an ID8-p53- / -brca2- / - ovarian cancer model. It also shows the immunophenotyping of tumor-associated leukocytes. [Figure 85C] This report shows an immunoprevention study in an ID8-p53- / -brca2- / - ovarian cancer model. Northern blots for miR-423-5p are shown. A total of 1 μg of RNA was loaded per lane. The upper panel shows the signal after detection with a 32P-labeled miR-423-5p probe. This blot was subjected to probe removal and re-detected with a U6 RNA-specific probe (lower panel). The right lane was run with a 32P-labeled Decade marker obtained from Ambion. [Figure 85D] This report describes an immunoprevention study in an ID8-p53- / -brca2- / - ovarian cancer model. The experimental scheme is as follows: CD46 transgenic mice were recruited and injected with HDAd-αPDL1γ1miR423+HDAd-SB, HDAd-GFP-miR423+HDAd-SB, or a mock. Four in vivo selections were performed using O6BG / BCNU. Two weeks after the last O6BG / BCNU treatment, ID8-p53- / -brca2- / - cells were injected intraperitoneally. Serum αPDL1γ1 levels were analyzed two, six, and eleven weeks after tumor cell injection. The development of ascites or pathological condition / cachexia was used as the endpoint. [Figure 85E] This shows an immunopreventive trial in an ID8-p53- / -brca2- / - ovarian cancer model. A Kaplan-Meier survival plot is shown. N=7 [Figure 85F] This shows immunoprevention studies in an ID8-p53- / -brca2- / - ovarian cancer model. Serum αPDL1γ1 levels are measured by ELISA. Each symbol represents an individual animal. *p<0.05. Statistical significance was calculated using a two-tailed Student's t-test (Microsoft Excel). [Figure 86A] This report describes immunotherapy trials in an ID8-p53- / -brca2- / - ovarian cancer model. It also describes the clinical context for preventing cancer recurrence. In vivo transduction of HSCs would be initiated after the majority of the tumor has been surgically removed, or in conjunction with chemotherapy if surgery is not an option. In vivo selection with O6BG / BCNU may be used in combination with chemotherapy. As a result of in vivo transduction / selection of HSPCs, armed HSPCs would remain dormant until cancer recurs, triggering differentiation of HSPCs and activation of effector gene expression. [Figure 86B] This report describes an immunotherapy trial in an ID8-p53- / -brca2- / - ovarian cancer model. The experimental scheme is as follows: 1 × 10⁶ ID8-p53- / -brca2- / - tumor cells were injected intraperitoneally into CD46 transgenic mice. Once the tumor was established, in vivo transduction and selection of HSPCs were performed. Activation of a miR-423-based expression system was monitored based on serum αPDL1γ1 levels. [Figure 86C] This report shows immunotherapy trials in an ID8-p53- / -brca2- / - ovarian cancer model. Kaplan-Meier survival plots are shown. In the control group, HDAd-GFP-miR423 was injected. N=9. [Figure 86D] This report presents immunotherapy trials in an ID8-p53- / -brca2- / - ovarian cancer model. Serum αPDL1γ1 levels are shown, measured by ELISA. Each symbol represents an individual animal. *p<0.05. Statistical significance was calculated using a two-tailed Student's t-test (Microsoft Excel). [Figure 87]This shows the autoimmune response in animals sacrificed on day 17 (before reversal from tumor growth), when αPD-L1-γ1 levels peaked. (A) The treated animals (right panel) show hair discoloration compared to untreated animals (left panel). (B) Histological analysis of organs obtained from treated animals is shown. Sections were stained with H&E. Representative regions are shown. The scale bar is 20 mm. Note the infiltration of mononuclear cells. [Figure 88A] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effect of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (four times every four days). Individual mouse tumor volumes are shown. [Figure 88B] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effect of in vivo HSC transduction on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). A Kaplan-Meier survival plot is shown, indicating that anti-PD-L1 treatment prolongs survival. The endpoint was defined as a tumor volume of 1000 mm³. The difference between the two groups was not statistically significant. [Figure 88C] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu transgenic mice with MMC tumors, and the effect of in vivo HSC transduction on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). Figure 85D shows the blood cell count of hCD46 transgenic mice two weeks after in vivo HSCPC transduction. [Figure 88D]This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu transgenic mice with MMC tumors, and the effect of in vivo HSC transduction on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). Hematological parameters are shown: RBC: red blood cells, Hb: hemoglobin, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: erythrocyte distribution width. Statistical analysis was performed using two-way ANOVA. Differences between the three groups were not significant. [Figure 88E-1] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effect of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). NiRNA-Seq data from the GFP+ cell fraction are shown. [Figure 88E-2] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effect of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). NiRNA-Seq data from the GFP+ cell fraction are shown. [Figure 88F-1]This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effects of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (four times every four days). The kinetics of αPDL1 expression are shown by Western blotting, qRT-PCR, and serum ELISA. [Figure 88F-2] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effects of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (four times every four days). The kinetics of αPDL1 expression are shown by Western blotting, qRT-PCR, and serum ELISA. [Figure 88G-1] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effects of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (four times every four days). The study showed miRNA-regulated gene expression. [Figure 88G-2] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effects of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (four times every four days). The study showed miRNA-regulated gene expression. [Figure 88H-1]This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effect of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). A schematic diagram summarizing the immunoprevention and cancer recurrence prevention is shown. [Figure 88H-2] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effect of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). A schematic diagram summarizing the immunoprevention and cancer recurrence prevention is shown. [Figure 88H-3] This study demonstrates the efficacy of anti-PD-L1 monoclonal antibody therapy in neu-transgenic mice with MMC tumors, and the effect of in vivo transduction of HSCs on hematopoiesis. When the tumor volume reached 100 mm³, mice were intraperitoneally injected with either the anti-mouse PD1-L1 monoclonal antibody muDX400* or an isotype control antibody (5 mg / kg intraperitoneally) (4 injections every 4 days). A schematic diagram summarizing the immunoprevention and cancer recurrence prevention is shown. [Figure 89A] This shows data related to GFP expression from red blood cells. [Figure 89B] This shows data related to GFP expression from red blood cells. [Figure 89C] This shows data related to GFP expression from red blood cells. [Figure 89D] This shows data related to GFP expression from red blood cells. [Figure 89E] This shows data related to GFP expression from red blood cells. [Figure 89F] This shows data related to GFP expression from red blood cells. [Figure 89G]This shows data related to GFP expression from red blood cells. [Figure 89H] This shows data related to GFP expression from red blood cells. [Figure 90A] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90B] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90C] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90D] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90E] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90F] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90G] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90H-1] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90H-2] This shows data related to the expression of human factor VIII from red blood cells. [Figure 90I] This shows data related to the expression of human factor VIII from red blood cells. [Figure 91A] This indicates that no hematological abnormalities were observed. [Figure 91B] This indicates that no hematological abnormalities were observed. [Figure 91C] This indicates that no hematological abnormalities were observed. [Figure 91D] This indicates that no hematological abnormalities were observed. [Figure 92A] This study demonstrates that the phenotype of hemophilia A can be modified even if inhibitory antibodies are produced. [Figure 92B] This study demonstrates that the phenotype of hemophilia A can be modified even if inhibitory antibodies are produced. [Figure 92C] This study demonstrates that the phenotype of hemophilia A can be modified even if inhibitory antibodies are produced. [Figure 92D] This study demonstrates that the phenotype of hemophilia A can be modified even if inhibitory antibodies are produced. [Figure 92E] This study demonstrates that the phenotype of hemophilia A can be modified even if inhibitory antibodies are produced. [Figure 92F] This study demonstrates that the phenotype of hemophilia A can be modified even if inhibitory antibodies are produced. [Figure 92G] This study demonstrates that the phenotype of hemophilia A can be modified even if inhibitory antibodies are produced. [Figure 93A] This document demonstrates in vivo transduction of traits into macaques (M. fascicularis). The experimental timeline is shown. [Figure 93B] This shows in vivo transduction into macaques (M. fascicularis). GFP marking of CD34+ cells recruited into peripheral blood is also shown. [Figure 93C] This shows in vivo transduction into macaques (M. fascicularis). GFP marking of CD34+ cells recruited into peripheral blood is also shown. [Figure 93D] This shows in vivo transduction into macaques (M. fascicularis). GFP marking of CD34+ cells recruited into peripheral blood is also shown. [Figure 93E] This shows in vivo transduction of macaques (M. fascicularis). The bone marrow (day 3) is shown. [Figure 94A] This demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K confers a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). The MGMTp140k vector is shown. [Figure 94B]This study demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K confers a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). The experimental design showing the timeline and injection dose is presented. [Figure 94C] This demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K confers a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). Data showing the percentage of GFP+ cells in PBMCs are presented. [Figure 94D] This demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K confers a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). Data showing the percentage of GFP+ cells in bone marrow at 26 weeks are presented. [Figure 94E] This demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K provides a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). The Ad5 / 35-GFP vector is shown. [Figure 94F]This study demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K confers a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). An experimental protocol is presented showing that pig-tailed monkeys are mobilized for 4 days, followed by an Ad5 / 35 injection. [Figure 94G] This study demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K confers a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). Animal IDs, as well as doses of G-CSF, SCF, AMD3100, and Ad5 / 35-GFP, are shown. [Figure 94H] This study demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K provides a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). Using AMD3100, total CD34+ stem cell levels were increased 3-fold compared to G-CSF / SCF alone, and 65-fold compared to baseline. The left panel shows the percentage of CD34+ stem cells in peripheral blood. The right panel shows the number of CD34+ cells. [Figure 94I]This study demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K provides a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). The study shows that cells recruited after AD5 / 35 injection form healthy colonies without lineage bias. The left panel shows numerical data indicating the frequency and number of colonies 0–6 hours after Ad5 / 35 injection. The right panel shows a visual examination of the morphology of CD34+ cells. [Figure 94J] This demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K provides a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). The upper panel shows flow cytometry data of Ad5 / 35-GFP cells 0–6 hours after injection. The lower panel shows numerical data of the number of Ad5 / 35-GFP-containing colonies 0, 2, and 6 hours after injection. [Figure 94K] This study demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K provides a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). It shows that more than 3% of peripheral CD34+ cells express GFP after injection of Ad5 / 35. The upper panel shows CD34+ cells extracted from the mononuclear cell (MNC) layer 0–8 days after Ad5 / 35 injection. The lower panel shows the mean GFP+ expression levels 2 and 6 hours after injection. [Figure 94L]This study demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K provides a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). The study shows confirmation of successful transduction of circulating cells after recruitment and Ad5 / 35 injection using multiple methods. The left panel shows Taqman detection of vector DNA. The right panel shows flow cytometry data of GFP expression. [Figure 94M] This demonstrates integrated transduction selection of HSCs in vivo. mgmtP140K provides a mechanism for genetically modified cells to possess drug resistance and selectively proliferate. The P140K variant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance to the MGMT inhibitor O(6)-(4-bromotenyl)guanine (O6BG) (also known as benzylguanine). This shows that the modified cells return to the bone marrow. The left panel shows flow cytometry data showing changes in CD34+ and GFP+ cells at 3, 7, and 73 days after injection of Ad5 / 35. The right panel shows the percentage of GFP+CD34+ cells at baseline, 3, 7, and 73 days after injection of Ad5 / 35. [Figure 95] The following describes the characteristics of representative Ad35 helper viruses and vectors as described herein. The five-pointed star indicates the following text: Integration of SB100x and target-directed (addition and reactivation), multiple sgRNAs for CRISPR or BE, miRNA (miR187 / 218) regulatory Cas9 expression, and Cas9 autoinactivation. [Figure 96] A schematic diagram of the HDAd-TI integrated vector is shown. The CRISPR system targets two different sites (HBG promoter and erythrocyte bcl11a enhancer), thereby increasing gamma reactivation. [Figure 97A]When HDAd-SB and HDAd-integration are simultaneously introduced, Flpe is expressed, releasing transposons adjacent to the IR, which are then incorporated into the genome by the SB100x transposase. Simultaneously, HBG1 CRISPR and bcl11a-E CRISPR are expressed, generating DNA indels, which reactivate γ-globin. When transposons are released via Flp, the CRISPR cassette is degraded, thereby avoiding toxicity. The CRISPR system targets two different sites (HBG promoter and erythrocyte bcl11a enhancer), which increases γ-globin reactivation. [Figure 97B] We will outline a target-oriented strategy. [Figure 97C] It exhibits red blood cell-specific BCL11A enhancer. [Figure 97D] The BCL11A binding site of the HBG promoter (SEQ ID NO: 48) is shown. Schematic diagrams of HDAd-SB and HdAd-integrated-SB can be seen in Figure 102. [Figure 98A] This document demonstrates dual CRISPR vectors and γ-globin reactivation. It also shows HDAd-Bcl11ae-CRISPR, HDad-HBG-CRISPR, HDAd-dual-CRISPR, and HDAd-scrambled vector designs. [Figure 98B] This shows a dual CRISPR vector and γ-globin reactivation. It also shows the HD-Ad5 / 35++ CRISPR vector as a dual gRNA vector. [Figure 98C] This shows dual CRISPR vectors and γ-globin reactivation. Transduction of a human erythrocyte progenitor cell line (HUDEP-2) with HD-Ad5 / 35++CRISPR is shown along with images of HUDEP-2 before and after differentiation. A timeline is shown below the HUDEP-2 cell images. [Figure 98D]This study demonstrates dual CRISPR vectors and γ-globin reactivation. It shows that the HD-AD5 / 35++ "dual" gRNA vector does not negatively affect cell viability compared to untreated (UNTR), BCL11A vectors, or HBG vectors. [Figure 98E] This study demonstrates dual CRISPR vector and γ-globin reactivation. It shows that the HD-AD5 / 35++ "dual" gRNA vector does not negatively affect proliferation compared to UNTR, BCL11A vector, or HBG vector. [Figure 98F] This study demonstrates dual CRISPR vectors and γ-globin reactivation. It shows that the dual vector achieves a similar level of editing to that observed with a single gRNA vector at the target locus (Bcl11a enhancer). [Figure 98G] This study demonstrates dual CRISPR vectors and γ-globin reactivation. It shows that the dual vector achieves a similar level of editing to that observed with a single gRNA vector at the target locus (HBG promoter). [Figure 98H] This demonstrates dual CRISPR vectors and γ-globin reactivation. It shows that the HD-AD5 / 35++ "dual" gRNA vector achieves target locus editing levels similar to those observed with single gRNA vectors. [Figure 98I] This shows dual CRISPR vectors and γ-globin reactivation. HUDEP-2 cells transduced with the HD-Ad5 / 35 "dual" gRNA vector showed a significantly higher percentage of HbF+ cells observed by flow cytometry compared to cells transduced with single gRNA vectors. A bar graph summarizing the flow cytometry data is shown below the flow cytometry data. [Figure 98J] This demonstrates dual CRISPR vectors and γ-globin reactivation. In samples subjected to dual targeting, total gamma-globin expression (measured by HPLC) was significantly higher. [Figure 98K]The double CRISPR vector and γ-globin reactivation are observed. The expression level of fetal globin observed in the double knockout clone is significantly higher compared to the single knockout clone, suggesting that the two mutations may have a synergistic effect, thereby increasing gamma expression / cells. [Figure 98L] This diagram shows the dual CRISPR vector and γ-globin reactivation. A schematic diagram illustrates the transduction of CD34+ cells recruited into peripheral blood using the HDAd5 / 35++ CRISPR vector. To minimize CRISPR / Cas9 cytotoxicity, cell transduction was continued using the HDAd5 / 35++ vector expressing an anti-Cas9 peptide. The cells were transplanted into sublethal irradiated NSG mice and analyzed. [Figure 98M] This study demonstrates dual CRISPR vectors and γ-globin reactivation. At 10 weeks post-transplantation, cells transduced with the HD-Ad5 / 35 "dual" gRNA vector showed similar engraftment to cells transduced with a single gRNA vector. Lineage composition was similar across all groups. [Figure 98N] This study demonstrates the use of a dual CRISPR vector and γ-globin reactivation. It shows that CD34+ cells transduced and edited using a dual gRNA vector efficiently engrafted in NSG mice. Furthermore, despite relatively low editing levels, the engrafted dual-targeted cells expressed gammaglobin at higher control-counter levels after erythrocyte differentiation compared to single-targeted cells. [Figure 99A]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99B]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99C]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99D]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99E]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99F]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99G]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99H]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99I]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99J]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99K]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99L]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99M]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99N]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99O]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99P]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99Q]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99R]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99S]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99T]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 99U]Figure 99A shows the experimental design. Figure 99B shows HBF expression and Figure 99C shows MFI in colonies of normal CD34+ cells at day 15. * indicates p=0.034. Figure 99D shows flow cytometry data illustrating HBF expression in colonies of normal CD34+ cells at day 15. Figure 99E shows HBF expression and Figure 99F shows MFI after erythrocyte differentiation (ED) of normal CD34+ cells. * indicates p=0.01. Figure 99G shows TE71 at the HBG site and Figure 99H shows TE71 at the BCL11A site 48 hours after transduction (txd) into normal CD34+ cells. Figure 99I shows flow cytometry data illustrating HBF expression and erythrocyte differentiation in EC. (Figures 99J-99U) Show Thalassemia CD34+ cells. (Figure 99J) Shows the immunophenotypes of cells, untransduced cells, and CRISPR-double transduced cells on day 0. (Figure 99K) Shows the growth curves comparing untransduced cells and CRISPR-double transduced cells over 11 days. (Figure 99L) Shows HBF expression and (Figure 99M) MFI in colonies on day 15. ** indicates p=0.0046. (Figure 99N) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double transduced and untransduced cells. (Figure 99O) Shows a comparison of HBF expression in the erythrocyte and myeloid compartments between CRISPR-double A transduced cells, CRISPR-double B transduced cells, and untransduced cells. (Figure 99P) Shows HBF expression in EC, and (Figure 99Q) shows MFI. *** indicates p=0.0003, and **** indicates p=0.00003. (Figure 99R) Shows flow cytometry data explaining HBF expression at P04 and P18. (Figures 99S, 99T) Shows TE71 at the HBG site at (Figure 99S) p04 and (Figure 99T) P18 during erythrocyte differentiation. (Figure 99U) Shows TE71 at the BCL11A site 48 hours after transduction. [Figure 100]A summary diagram illustrating the integration of γ-globin gene addition and endogenous γ-globin reactivation is shown. [Figure 101] The HDAd5 / 35++ vector used herein is shown. Addition of the γ-globin gene is achieved via the SB100x transposase system, which consists of a transposon vector (see HDAd-integration and HDAd-SB-addition) having an expression cassette and adjacent IR and frt sites, and a second vector (HDAd-SB) supplying SB100x and Flpe recombinase in trans. The transposon cassette for random incorporation consists of a small β-globin LCR / promoter for erythrocyte-specific expression of human γ-globin. The 3'UTR helps stabilize mRNA in erythrocytes. The γ-globin expression unit and the cassette for expressing mgmtP140K from a ubiquitously active PGK promoter are separated by a chicken globin HS4 insulator. The CRISPR / Cas9 cassette in the HDAd-CRISPR vector and the HDAd-integrated vector contains a U6 promoter-promoting sgRNA specific to the BCL11A binding site in the HBG1 / 2 promoter, and SpCas9 under the control of the EF1α promoter. Cas9 expression in HDAd-producing cells is suppressed by the miRNA regulatory system (Saydaminova et al., Mol Ther Methods Clin Dev. 2015, 1:14057, 2015). In HDAd-integration, the CRISPR / Cas9 cassette is located outside the transposon, and as a result, the CRISPR / Cas9 cassette disappears when Flpe / SB100x-mediated integration occurs (see Figure 102). [Figure 102]A schematic diagram of Cas9 expression regulation is shown. In HDAd integration, when Flpe recombinase interacts with the frt site, the transposon becomes cyclized, leaving behind a linear fragment of the vector containing the CRISPR cassette. Previous studies using the SB100x / Flpe system have demonstrated that these vector portions rapidly disappear while the cyclized transposon is integrated into the host genome by SB100x (Yant et al., Nat Biotechnol., 20:999-1005, 2002). [Figure 103A] This document presents in vitro studies analyzing Cas9 and γ-globin expression using HUDEP-2 cells. Western blotting analysis of Cas9 expression is shown. HUDEP-2 cells were transduced using HDAd-integration alone or in combination with HDAd-SB (i.e., a vector supplying Flpe and SB100x in trans). In vitro erythrocyte differentiation was initiated 4 days after transduction and continued for 8 days (erythrocyte differentiation enables γ-globin expression). Right panel: Representative Western blots using Cas9 antibody and β-actin antibody as probes. Left panel: Summary of Cas9 signaling. The bars compare Cas9 levels with and without co-infection with HDAd-SB, indicating a decrease in Cas9 due to the Flpe / SB100x mechanism. [Figure 103B] This document presents in vitro studies analyzing Cas9 and γ-globin expression using HUDEP-2 cells. Western blotting analysis of Cas9 expression is shown. HUDEP-2 cells were transduced using HDAd-integration alone or in combination with HDAd-SB (i.e., a vector supplying Flpe and SB100x in trans). In vitro erythrocyte differentiation was initiated 4 days after transduction and continued for 8 days (erythrocyte differentiation enables γ-globin expression). Right panel: Representative Western blots using Cas9 antibody and β-actin antibody as probes. Left panel: Summary of Cas9 signaling. The bars compare Cas9 levels with and without co-infection with HDAd-SB, indicating a decrease in Cas9 due to the Flpe / SB100x mechanism. [Figure 103C]This document presents in vitro studies analyzing Cas9 and γ-globin expression using HUDEP-2 cells. It also shows the results of γ-globin expression analysis by flow cytometry. HUDEP-2 cells were transduced using HDAd-CRISPR ("cleavage"), HDAd-SB-addition ("addition") + HDAd-SB, or HDAd-integration ("integration") + HDAd-SB, and these HUDEP-2 cells were analyzed at the indicated time points. [Figure 103D] This shows an in vitro study analyzing Cas9 and γ-globin expression using HUDEP-2 cells. γ-globin mRNA levels determined by qRT-PCR are shown. dpt: days after transduction. Diff: differentiation. *p<0.05. [Figure 104A] This report describes the in vivo transduction of γ-globin expression in CD46 / β-YAC mice. A schematic diagram of the experiment is shown. HSPCs were recruited by subcutaneous injection of human recombinant G-CSF for 4 days, followed by a single subcutaneous injection of AMD3100. 30 and 60 minutes after the AMD3100 injection, a 1:1 mixture of the following HDAd vectors was intravenously injected into the animals (2 injections in total, 4 × 10¹⁰ vp each): HDAd-integration + HDAd-SB, HDAd-SB-addition + HDAd-SB, and HDAd-cleavage. For the next 4 weeks, mice were treated with immunosuppressants (IS) to avoid immune responses to human γ-globin and MGMT. In week 4, O6-BG / BCNU treatment was initiated and repeated every 2 weeks for a total of 3 cycles. With each cycle, the BCNU concentration was increased from 5 mg / kg to 7.5 mg / kg to 10 mg / kg. At 18 weeks, the animals were sacrificed for tissue sample analysis, and their bone marrow Lin- cells were collected and secondarily transplanted into lethally irradiated C57Bl / 6 mice. These mice were then observed for a further 16 weeks. [Figure 104B] This shows the gamma globin expression test after transduction in CD46 / β-YAC mice in vivo. The gamma globin expression in peripheral erythrocytes of the "integrated" and "cleaved" groups was detected by flow cytometry. [Figure 104C]This shows the in vivo transduction-followed γ-globin expression test of CD46 / β-YAC mice. γ-globin protein levels measured by HPLC are shown. Right panel: Chromatogram of RBC lysate (week 18) (human β-globin, reactivated human Aγ, and added γ-globin chains are marked). Left panel: Summary of HPLC data. The percentage of total γ-globin relative to human β-globin in CD46 / β-YAC mice treated with "cleavage," "addition," and "integration" vectors is shown. *: p<0.05, ns. [Figure 104D] This shows the γ-globin expression test after transduction in CD46 / β-YAC mice in vivo. It shows the expression of γ-globin mRNA (measured by qRT-PCR) relative to the expression of mouse β-major mRNA. [Figure 104E] This shows the γ-globin expression test after in vivo transduction of CD46 / β-YAC mice. The percentage of target site cleavage by CRISPR / Cas9 is shown. Genomic DNA obtained from PBMCs and bone marrow MNCs collected at 18 weeks from mice transduced in vivo using "cleavage" and "integration" was subjected to the T7EI assay. A summary of the data obtained from Figure 105 is shown. *p<0.05. [Figure 104F] This shows the γ-globin expression test after in vivo transduction in CD46 / β-YAC mice. The number of integrated vector copies in the bone marrow HSPC was measured 18 weeks after transduction using "additional vector" and "integrated" vectors. The difference between groups was not statistically significant. [Figure 104G] This shows the γ-globin expression test after transduction in CD46 / β-YAC mice in vivo. The VCN spectra in individual CFUs obtained from mice treated with the "integrated" vector are shown. Bone marrow Lin- cells were seeded for a progenitor cell assay, and VCN in individual colonies was measured by qPCR. Data from four different mice are shown. [Figure 104H]This shows the γ-globin expression test after transduction in CD46 / β-YAC mice in vivo. The results of HPLC analysis of human γ / human β-globin proteins are also shown. [Figure 104I] This shows the gamma globin expression test after transduction in CD46 / β-YAC mice in vivo. The percentage of human gamma globin mRNA expression relative to mouse β-major mRNA expression is shown. [Figure 105A] The chromatograms of RBC lysates are shown, with peaks for human β-globin and γ-globin marked. The top panel shows β-YAC mice before treatment. The middle panel shows the mice 18 weeks after transduction by HDAd-CRISPR ("cleavage"). The left panel shows the reactivation of both Gγ and Aγ. The bottom panel shows the mice 18 weeks after transduction by HDAd-CRISPR ("cleavage"). [Figure 105B] The chromatograms of RBC lysates are shown, with the peaks for human β-globin and γ-globin marked. In the last lower panel, the peaks are labeled. Each chromatogram is from an individual animal. Note that human β-globin decreases with increasing γ-globin (reverse globin switching). [Figure 106] This shows T7EI assay data obtained from MNCs (microclavic cells) acquired from blood, spleen, and bone marrow 16 weeks after transduction with "cleavage" and "integration" vectors. Specific CRISPR / Cas9 cleavage fragments (255 bp and 110 bp) are marked with arrows. Below each lane, the cleavage percentage is shown based on quantification of the band signal. [Figure 107A] This shows the analysis of secondary recipients of Lin- cells obtained from CD46 / β-YAC transduced mice. The percentage of human γ-globin-expressing peripheral blood RBCs at the indicated time point is shown. Immunosuppression was initiated in all mice from 4 weeks post-transplantation. [Figure 107B] This shows the analysis of secondary recipients of Lin- cells obtained from CD46 / β-YAC transduced mice. The levels of γ-globin protein relative to human β-globin at 16 weeks post-transplantation are shown. [Figure 107C] This shows the analysis of secondary recipients of Lin- cells obtained from CD46 / β-YAC transduced mice. It also shows the levels of γ-globin protein relative to mouse β-major-globin and human β-globin. [Figure 107D] This shows the analysis of secondary recipients of Lin- cells obtained from CD46 / β-YAC transduced mice. It also shows the levels of γ-globin protein relative to mouse β-major-globin and human β-globin. [Figure 107E] This shows the analysis of secondary recipients of Lin- cells obtained from CD46 / β-YAC transducible mice. It also shows the lineage-positive cell composition in blood, spleen, and bone marrow MNCs 16 weeks after transduction with the "integrated" vector, compared to non-transduced control mice. [Figure 107F] This shows the analysis of secondary recipients of Lin- cells obtained from CD46 / β-YAC transduced mice. It also shows the vector copy number per cell in total leukocytes obtained from the HDAd-integrated group, measured by qPCR using γ-globin primers. [Figure 108A] This paper describes the generation and characterization of triple transgenic CD46 / Townes mice as a model for SCD. It also describes the mating and breeding of CD46 / Townes mice. Townes mice (hα / hα::βS / βS) were mated with CD46 transgenic mice three times. Homozygous animals for CD46, HbS, and HBA were used in in vivo transduction studies. [Figure 108B] This document describes the generation and characterization of triple-transgenic CD46 / Townes mice as a model for SCD. Peripheral blood smears from CD46 / Townes mice exhibiting typical features of the human disease, including deformed erythrocytes, polychromatic cells (black arrows), sickle cells, and fragmented cells (starred black arrows). Scale bar is 15 μm. [Figure 108C]This paper describes the generation and characterization of triple transgenic CD46 / Townes mice as a model for SCD. Peripheral blood samples obtained from CD46 / Townes mice were hematologically analyzed and compared to "healthy" parental CD46 transgenic mice. Ret: reticulocytes, RBC: erythrocytes, Hb: hemoglobin, HCT: hematocrit, WBC: leukocytes. All differences are statistically significant (p<0.05). [Figure 108D] This paper describes the generation and characterization of triple transgenic CD46 / Townes mice as a model for SCD. It shows splenomegaly in CD46 / Townes mice. The ratio of spleen to body weight in CD46tg mice and CD46 / Townes mice is shown. N=3. [Figure 109A] This shows γ-globin expression after in vivo transduction of HSPC into CD46 / Townes mice. Mice were recruited, injected with HDAd-integrated + HDAd-SB, and treated with O6BG / BCNU as described for Figure 104. γ-globin marking in peripheral RBCs was measured by flow cytometry. White squares indicate marking in RBCs of untreated CD46 / Townes mice. Vertical arrows indicate the in vivo selection cycle. [Figure 109B]This shows the expression of γ-globin after in vivo transduction of HSPC into CD46 / Townes mice. Mice were recruited, injected with HDAd-integration + HDAd-SB, and treated with O6BG / BCNU as described for Figure 104. The γ-globin levels in RBCs were measured by HPLC at week 13. Left panel: Summary of total γ-globin levels relative to human α-globin and β-s-globin chains in individual mice. White squares indicate RBC levels in untreated CD46 / Townes mice. Right panel: Representative chromatogram of CD46 / Townes mice before treatment (upper panel), and representative chromatogram of CD46 / Townes mice at week 13 after in vivo transduction of HSPC with HDAd-integration + HDAd-SB. Peaks for human β-globin, β-s-globin, reactivated Aγ, and added γ-globin are shown. [Figure 109C] This shows the expression of γ-globin after in vivo transduction of HSPC into CD46 / Townes mice. Mice were recruited, injected with HDAd-integrated + HDAd-SB, and treated with O6BG / BCNU as described for Figure 104. The percentage of reactivated Aγ based on HPLC is shown. [Figure 109D] This shows the expression of γ-globin after in vivo transduction of HSPC into CD46 / Townes mice. Mice were recruited, injected with HDAd-integrated + HDAd-SB, and treated with O6BG / BCNU as described for Figure 104. The percentage of total γ-globin mRNA relative to human α-globin and β-s-globin mRNA in each mouse is shown. [Figure 109E] This shows the expression of gamma globin after in vivo transduction of HSPCs in CD46 / Townes mice. Mice were recruited, injected with HDAd-integrated + HDAd-SB, and treated with O6BG / BCNU as described for Figure 104. The number of embedded vector copies in bone marrow HSPCs at 163 weeks after transduction with HDAd-integrated is shown. [Figure 109F]This shows γ-globin expression after in vivo transduction of HSPC into CD46 / Townes mice. Mice were recruited, injected with HDAd-integration + HDAd-SB, and treated with O6BG / BCNU as described for Figure 104. This shows cleavage of HBG1 / 2 target sites in all bone marrow nuclei, Lin- cells, PBMCs, and splenic cells of CD46 / Townes mice 13 weeks post-injection with HDAd-integration. Specific CRISPR / Cas9 cleavage fragments (255 bp and 110 bp) are marked with arrows. Below each lane, the cleavage percentage is shown based on quantification of band signaling. [Figure 110] This shows the analysis of secondary recipients transplanted with Lin- cells obtained from transduced CD46 / Townes mice. (A) Percentage of peripheral blood RBCs expressing human γ-globin. (B) Levels of γ-globin protein relative to human α-globin and β-S globin at 16 weeks post-transplantation. [Figure 111A] This shows phenotypic modifications in blood. A blood smear with reticulocytes stained with brilliant cresyl blue is shown. This dye stains residues in the nuclear and cytoplasmic compartments (quantified results can be seen in Figure 109C (first group of bars)). The scale bar is 20 μm. [Figure 111B] This shows phenotypic modification in the blood. It presents a blood smear demonstrating normocytic erythrocyte morphology after gene therapy with HDAd-integration. [Figure 111C] The results show phenotypic modifications in the blood. Hematological analysis of peripheral blood is also shown. The difference between "CD46" and "CD46 / Townes at 13 weeks after treatment with integrated vector" is not significant. [Figure 112A]Phenotypic modifications in the spleen and liver are shown. Histological analyses are also shown. Top panel: Iron deposition in the spleen. Hemosiderin was detected in spleen sections by pearl Prussian blue staining. Scale bar is 20 μm. Middle and bottom panels: Hematoxylin / eosin staining of extramedullary hematopoiesis in spleen and liver sections. Erythroblast clusters in the liver and megakaryocyte clusters in the spleen of CD46 / Townes mice are indicated by white arrows. Scale bar is 20 μm. The images shown are representative. [Figure 112B] Phenotypic modifications are observed in the spleen and liver. Spleen size (a measurable compensatory hematopoietic feature) in treated CD46 / Townes mice is comparable to that of paternal CD46 mice. [Figure 112C] This shows phenotypic modifications in the spleen and liver. Figure 112A shows a 4x magnified view of a liver section image. In CD46 / Townes mice before treatment, sickle cell red blood cells (RBCs) are trapped in the hepatic sinusoids (left panel), while after treatment, sickle cell red blood cells are absent in the hepatic sinusoids (right panel). [Figure 113]The left end of the Ad5 / 35 helper virus genome is shown. Dark gray shaded sequences correspond to the native Ad5 sequence; i.e., sequences without a shadow or highlighted in light gray are artificially introduced. The light gray highlighted sequences are two copies of the (tandem repeat) loxP sequence. In the presence of the "cre recombinase" protein, the nucleotide sequence between the two loxP sequences is deleted (leaving one loxP copy). The Ad5 sequence between the loxP sites is essential for packaging adenovirus DNA into the capsid (in the nucleus of producing cells), so its deletion makes packaging of helper adenovirus genomic DNA impossible. Consequently, the efficiency of the deletion process directly affects the level of helper genomic DNA packaging (undesirable helper virus "contamination"). From the above perspective, in order to apply the same scheme to adenovirus serotypes other than Ad5, it is desirable to achieve the following: 1. Identify sequences essential for packaging, place such sequences adjacent to loxP sequence insertions, and ensure they can be deleted in the presence of cre recombinase. Identifying such sequences is not easy if there is little similarity between the sequences. 2. Determine the location in the native DNA sequence where the impact of loxP sequence insertions on helper virus replication and packaging (in the absence of cre recombinase) will be minimized. 3. Determine the spacing between loxP sequences that will allow the packaging sequences to be efficiently deleted during the production of helper-dependent adenovirus (i.e., production in cre recombinase-expressing cell lines (e.g., cell line 116)) and minimize the packaging of the helper virus. [Figure 114]This shows the alignment of the Ad5 packaging signal (SEQ ID NO: 49) and the Ad35 packaging signal (SEQ ID NO: 50). Aligning the left-terminal sequences of Ad5 and Ad35 is useful for identifying packaging signals. Important Ad5 sequence motifs for packaging (AI~AV) are indicated by boxes (see Figure 1B in Schmid et al., J Virol., 71(5):3375-4, 1997). The location of loxP insertion sites is indicated by black arrows. These insertions are adjacent to AI~AIV and disrupt AV. Note that further packaging signals AVI and AVII (as shown in Schmid et al.) are deleted from this vector as part of the E1 deletion of the Ad5 helper virus. [Figure 115] A schematic diagram of pAd35GLN-5E4 is shown. This is a first-generation (E1 / E3 deletion) Ad35 vector obtained from an Ad35 genome (Holden strain obtained from ATCC) (PMID: 28538186) vectorized using a recombinant technique. Next, this vector plasmid was used for loxP site insertion. [Figure 116]Information regarding plasmid packaging signals is provided. Packaging site (PS)1: The LoxP insertion site is located after nucleotides 178 and 344. This results in the removal of AI~AIV. The remaining packaging signal, including AVI and AVII (located after 344), is deleted (deleted as part of the E1 deletion (345~3113)). PS2: The LoxP insertion site is located after nucleotides 178 and 481. Furthermore, nucleotides 179~365 are deleted, so AI~AV is absent. The remaining packaging motifs (AVI and AVII) can be removed by cre recombinase during HDAd production. 482~3113 are deleted in the E1 deletion. PS3: The LoxP insertion site is located after nucleotides 154 and 481. These three manipulated vectors are rescueable. The percentage of viral genomes with rearranged loxP sites was 50%, 20%, and 60% for PS1, PS2, and PS3, respectively. Rearrangement occurs when the loxP site critically affects viral replication and gene expression. Vectors with rearranged loxP sites may be packaged and contaminate HDAd preparations. Sequence IDs 286, 51, and 52 are examples of vectors, which are illustrated as PS1, PS2, and PS3, respectively. [Figure 117]This report compares the next-generation HDAd35 platform with the current HDAd5 / 35 platform. Both vectors contain a CMV-GFP cassette. This Ad35 vector does not contain the immunogenic Ad5 capsid protein. The transduction efficiency of CD34+ cells in vitro shown by these is equivalent. Bridging tests demonstrate equivalent transduction efficiency of CD34+ cells in vitro. Transduction of human HSCs (peripheral CD34+ cells) obtained from G-CSF-recruited donors was performed at MOIs of 500 vp / cell, 1000 vp / cell, and 2000 vp / cell using HDAd35 (produced with Ad35 helper P-2) or a chimeric vector containing an Ad5 capsid with Ad35-derived fibers. The percentage of GFP-positive cells was measured 48 hours after virus addition in three independent experiments. Notably, at 48 hours, infection with HDAd35 induced a cytopathic effect due to helper virus contamination. [Figure 118] The PS2 helper vector was reconstructed to focus on monkey studies. The following operations were performed: deletion of the E1 region, adjacent placement of the mutant packaging signal and Loxp, use of the mutant packaging sequence, deletion of the E3 region (27435→30540), substitution at Ad5 E4orf6, insertion of the copGFP cassette and adjacent stuffer DNA, and creation of Ad35K++ by introducing mutations to the knob. [Figure 119] The provided mutant packaging signal sequence is shown. Residues 1-137 are Ad35 ITR. Bold text indicates SwaI sites, Loxp sites ...

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[Claim 1] The invention described in the specification.

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