Constructs for multi-lineage expression of therapeutic agents

Abstract

The present disclosure provides nucleic acid constructs engineered to express therapeutic expression products from regulatory sequences that drive expression in hematopoietic cell populations. For example, the present disclosure includes nucleic acid constructs in which a first regulatory sequence drives expression of a first therapeutic expression product and a second regulatory sequence drives expression of a second therapeutic expression product. Nucleic acid constructs can be delivered to cells or subjects by viral vectors, including adenoviral vectors, e.g., for the treatment of cancer.

Description

ENO-00225 (38895-00225)CONSTRUCTS FOR MULTI-LINEAGE EXPRESSION OF THERAPEUTIC AGENTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims a right of priority from and the benefit of an earlier filing date of US Provisional Application No. 63 / 701,576, filed Septemeber 30, 2024, US Provisional Application No. 63 / 716,087, filed November 4, 2024, US Provisional Application No. 63 / 753,412, filed February 3, 2025, US Provisional Application No. 63 / 795,106, filed April 25, 2025, and US Provisional Application No. 63 / 804,637, filed May 13, 2025, each of which is hereby incorporated by reference in their entireites.SEQUENCE LISTING

[0002] A Sequence Listing in the form of an XML file (entitled “ENO-00225_SL.xml”, created on May 8, 2025, and having a size of 1,007,035 bytes) is hereby incorporated by reference in its entirety.BACKGROUND

[0003] Many medical conditions have the potential to be treated or ameliorated by gene therapy. Such conditions include, for example, genetic disorders, immune deficiencies, hemoglobinopathies, and cancers including solid tumors and hematological cancers. However, treatment of many such conditions has proven challenging.SUMMARY

[0004] The present disclosure provides compositions and methods related to the engineering of hematopoietic stem cells (HSCs) to express therapeutic products, including in vivo engineering of HSCs to express therapeutic products and / or other products useful in connection with the treatment of patients. The present disclosure includes the recognition that, in a subject, engineered HSCs differentiate into a variety of downstream lineages and cell populations. Cells of each lineage and type have distinct immunological capabilities and therapeutic utility. The present disclosure includes the recognition that expression in certain limited subsets of hematopoietic cell populations and / or lineage sometimes provides unsatisfactory therapeutic results, and / or results that can be improved by selective expression in multiple hematopoietic cell types and / or lineages. The present disclosure therefore provides, among other things, nucleic acid constructs, vectors including such constructs, andFH12901147.5ENO-00225 (38895-00225) use thereof for engineering of HSCs for expression of therapeutic agents in multiple cell populations and / or lineages, with advantages further disclosed herein.

[0005] In various embodiments, the present disclosure provides nucleic acid constructs in which a first regulatory sequence drives or causes expression of a first nucleic acid sequence encoding a therapeutic expression product and a second regulatory sequence drives or causes expression of a second nucleic acid sequence, where the first and second regulatory sequences drive or cause non-identical expression patterns in hematopoietic cell populations and / or HSC lineages, or subsets thereof. As one exemplary embodiment, in at least some examples, a first regulatory sequence can drive or cause expression in a first set of cell populations that include at least one lymphoid cell population, e.g., a cell population selected from T cells, NK cells, and B cells, while the second regulatory sequence can drive or cause expression in at least one myeloid cell (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells) population. In various embodiments, a first nucleic acid sequence encoding a first therapeutic expression product and / or a second nucleic acid sequence encoding a second therapeutic expression product can be operably linked with one or more HSC-related miRNA binding sites (e.g., two, three, or four HSC-related miRNA binding sites) that cause a decrease in expression in certain cell lineages as compared to a reference, but do not cause a decrease, or do not cause as great of a decrease, in one or more other cell lineages as compared to a reference. For the avoidance of doubt, while the first regulatory sequence and second regulatory sequence drive or cause non-identical expression patterns in hematopoietic cell populations and / or HSC lineages, or subsets thereof, there is not necessarily any limitation that the cell populations, HSC lineages, or subsets cannot overlap. Indeed, the expression driven or caused by a first regulatory sequence and second regulatory sequence may differ by the level expression in one or more cell populations or lineages, rather than by the absolute presence or absence of expression in any particular cell population or lineage. Moreover, it is not necessarily the case that any particular regulatory sequence limit expression to a single lineage, and still less to any single cell population.

[0006] Presently disclosed HSCs engineered to express therapeutic agents in one or more cell populations and / or HSC lineages are particularly useful in therapeutic methods in which undifferentiated engineered HSCs are present in a subject, e.g., by administration of engineered HSCs to a subject or as a result of in vivo engineering of HSCs. In many instances, a single type or lineage of hematopoietic cells such as T cells, NK cells, or B cells is engineered in vitro or ex vivo, and subsequently administered to a subject. In such cases, the range and amounts of engineered cells are necessarily limited by the engineered2FH12901147.5ENO-00225 (38895-00225) population. Such strategies cannot capture the advantages of achieving expression of one or more heterologous therapeutic agents in a plurality of HSC-derived cell populations and / or lineages. Moreover, insofar as each transgene encoding a therapeutic agent in an engineered cell is typically associated with a single particular regulatory sequence, the complexity of historically achieved expression patterns is limited to the performance of the single regulatory sequence. However, the present disclosure includes the recognition that expression of therapeutic agents in multiple engineered HSC cell populations and / or lineages as provided herein unlocks a previously unutilized degree of control and therapeutic utility, broadly empowering a class of therapeutics with complex expression of therapeutic agents. Indeed, in many systems, including systems in which only particular cell populations and / or lineages are targeted for engineering and / or in which nucleic acid construct limitations inhibit complex engineering, the advantages of the present disclosure cannot be achieved.

[0007] In some embodiments, payload vector comprises two or more payload cassettes in order to achieve two or more a desired effect in a host, e.g. expression of two payloads in two cell populations, expression of two transgenes, expression of a transgene and a gene editing system, expression of gene editing system to edit at least two different sites. Examples of gene editing systems, include but are not limited to, a CRISPR / Cas editing system, zinc finger nuclease (ZFN) editing system, transcription activator-like effector nucleases (TALEN) editing system, meganuclease editing system, base editing system, or prime editing system, epigenetic editing system, RNA editing system, and / or transposase-assisted targetsite integration system.

[0008] As one non-limiting, exemplary application of HSCs engineered for expression of one or more heterologous therapeutic agents in a plurality of cell populations and / or lineages, the present disclosure includes engineering of HSCs for the treatment of solid tumors. The present disclosure further includes the recognition that treatment of cancer, and in particular the treatment of solid tumors, using cells engineered to express CARs has proven challenging. One important reason for this challenge is that CAR therapy, when administered as CAR T cells, CAR NK cells, or CAR macrophage cells, are not able to penetrate, persist, and achieve sustained target cell killing in cancer compartments. The present disclosure includes a solution to improve the efficacy of CAR therapy against solid tumors by expressing CARs in multiple hematopoietic cell populations and / or HSC lineages, whereby each expressing cell population and / or HSC lineage contributes to enhancing treatment in accordance with its particular immunological and functional characteristics. More specifically, HSCs can be engineered, e.g., in vivo, to express a CAR in cell populations3FH12901147.5ENO-00225 (38895-00225) and / or HSC lineages including, without limitation, both T cells and / or NK cells, as well as myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells). In various such embodiments, without wishing to be bound by any particular scientific theory, CAR myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells)are able to infiltrate solid tumors (but do not necessarily mediate robust tumor cell killing), whereby activity of the CAR myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells)promote recruitment of CAR-T and / or CAR-NK cells that mediate robust target cell killing (but are not independently as effective for solid tumor infiltration). The combination of multi-lineage expression in lymphoid cells and myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells) thereby permits a more robust therapy for treatment of solid tumors. Accordingly, the present disclosure provides, among other things, compositions and methods related to the engineering of hematopoietic stem cells (HSCs) to express heterologous products for treatment of solid tumors.

[0009] In at least one aspect, the present disclosure provides a nucleic acid construct for expression of first and second expression products, including a nucleic acid sequence encoding a first therapeutic expression product operably linked with a first lineage-specific regulatory sequence, and a nucleic acid sequence encoding a second therapeutic expression product operably linked with a second lineage-specific regulatory sequence, where the first lineage-specific regulatory sequence causes expression of the first therapeutic expression product in at least a first cell population of a first HSC lineage, and where the second lineagespecific regulatory sequence is distinct from the first lineage-specific regulatory sequence and causes expression of the second therapeutic expression product in at least a second cell population of a second HSC lineage.

[0010] In some embodiments, the first lineage-specific regulatory sequence and / or the second lineage- specific regulatory sequence causes expression in a cell population of an HSC lineage selected from a lymphoid lineage or a myeloid lineage.

[0011] In some embodiments, the first lineage-specific regulatory sequence and / or the second lineage- specific regulatory sequence causes expression in one or more cell populations including at least one of T cells, NK cells, and / or myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

[0012] In some embodiments, the first cell population is distinct from the second cell population.

[0013] In some embodiments, the first HSC lineage is distinct from the second HSC lineage.4FH12901147.5ENO-00225 (38895-00225)

[0014] In some embodiments, the first lineage-specific regulatory sequence causes expression in at least one cell population and / or HSC lineage in which the second lineagespecific regulatory sequence does not cause expression, does not cause detectable expression, does not cause significant expression, and / or does not cause expression at a level that is comparable to that caused by the first lineage-specific regulatory sequence, optionally where expression is not comparable if expression is at least 100-fold less, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000-fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000- fold, or at least 100,000-fold less. In some embodiments, the second lineage- specific regulatory sequence causes expression in at least one cell population and / or HSC lineage in which the first lineage-specific regulatory sequence does not cause expression, does not cause detectable expression, does not cause significant expression, and / or does not cause expression at a level that is comparable to that caused by the first lineage-specific regulatory sequence, optionally where expression is not comparable if expression is at least 100-fold less, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000- fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, or at least 100,000-fold less.

[0015] In some embodiments, the first lineage-specific regulatory sequence causes expression in at least one cell type or cell population and / or HSC lineage in which the second lineage-specific regulatory sequence does not cause expression, does not cause detectable expression, and / or does not cause significant expression, if expression is at least 100-fold less, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000-fold, or at least 10000-fold less, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, or at least 100,000-fold less than expression from a ubiquitous promoter (e.g., a ubiquitous promoter provided herein).

[0016] In some embodiments, the first lineage-specific regulatory sequence causes expression in a cell population of a lymphoid lineage. In some embodiments, the first lineagespecific regulatory sequence causes expression in a plurality of cell populations that include the cell population of a lymphoid lineage. In some embodiments, the cell population of a lymphoid lineage is selected from T cells, NK cells, and / or B cells. In some embodiments, the first lineage-specific regulatory sequence causes expression in at least at least T cells and NK cells. In some embodiments, the first lineage-specific regulatory sequence causes expression in at least one cell population of a lymphoid lineage and at least one cell5FH12901147.5ENO-00225 (38895-00225) population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

[0017] In some embodiments, the second lineage-specific regulatory sequence causes expression in a cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells). In some embodiments, the second lineage-specific regulatory sequence causes expression in a plurality of cell populations that include the cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

[0018] In some embodiments, the cell population of a myeloid (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells) is selected from a monocyte, macrophage, myeloblast, granulocyte, neutrophil, eosinophil, basophil, megakaryocyte-erythroid progenitor cell, megakaryocyte, myeloid dendritic cell (mDC), monocyte-derived dendritic cell (MoDC), mast cell, platelet, and / or erythrocyte.

[0019] In some embodiments, the second lineage-specific regulatory sequence causes expression in at least one cell population of a lymphoid lineage and at least one cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells). In some embodiments, the first lineage-specific regulatory sequence causes expression in at least NK cells and a cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells), optionally where the first lineage-specific regulatory sequence causes expression in at least NK cells and macrophages. In some embodiments, T cells and / or cells of a lymphoid lineage include one or more of cytotoxic CD8+ T cells, and / or helper CD4+ T cells. In some embodiments, NK cells and / or cells of a lymphoid lineage include one or more of CD56brightNK cells, and / or CD56dimNK cells.

[0020] In some embodiments, myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells) and / or cells of a myeloid lineage include monocytes, macrophages, myeloblasts, granulocytes, neutrophils, eosinophils, basophils, megakaryocyte-erythroid progenitor cells, megakaryocytes, myeloid dendritic cells, monocyte-derived dendritic cells, mast cells, platelets, and / or erythrocytes.

[0021] In some embodiments, the first lineage-specific regulatory sequence causes expression of the first expression product in the first cell population that is at least 5-fold greater in the first cell population than in a reference cell population, optionally where expression of the first expression product in the first cell population is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least6FH12901147.5ENO-00225 (38895-00225)50,000-fold, or at least 100,000-fold greater in the first cell population than in the reference cell population, optionally where the reference cell population is a hematopoietic stem cell (HSC), an erythrocyte, platelet, LT-HSC (CD34+CD90+CD45RA- HSC), NK cell, T cell, B cell, or myeloid cell. In some embodiments, the second lineage-specific regulatory sequence causes expression of the second expression product in the second cell population that is at least 5-fold greater in the second cell population than in a reference cell population, optionally where expression of the second expression product in the second cell population is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100- fold, at least 1000-fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, or at least 100,000-fold greater in the second cell population than in the reference cell population, optionally where the reference cell population is a hematopoietic stem cell (HSC), an erythrocyte, platelet, LT-HSC (CD34+CD90+CD45RA- HSC), NK cell, T cell, B cell, or myeloid cell. In some embodiments, where, in erythroid cells, on the surface of erythroid cells, and / or in platelets, the first lineage-specific regulatory sequence does not cause expression, does not cause detectable expression, does not cause significant expression, and / or does not cause expression at a level that is comparable to that caused by the first lineage-specific regulatory sequence in the first cell population, optionally where expression is not comparable if expression is at least 100- fold less, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000-fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000- fold, or at least 100,000-fold less. In some embodiments, where, in erythroid cells, on the surface of erythroid cells, and / or in platelets, the second lineage-specific regulatory sequence does not cause expression, does not cause detectable expression, does not cause significant expression, and / or does not cause expression at a level that is comparable to that caused by the second lineage-specific regulatory sequence in the second cell population, optionally where expression is not comparable if expression is at least 100- fold less, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000-fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000- fold, or at least 100,000-fold less.

[0022] In some embodiments, the first lineage-specific regulatory sequence and / or the second lineage-specific regulatory sequence includes at least one enhancer, at least one promoter, at least one untranslated region (UTR), at least one intron, and / or at least one regulatory moiety binding site, optionally where the regulatory moiety includes a transcription factor. In some embodiments, the first lineage-specific regulatory sequence7FH12901147.5ENO-00225 (38895-00225) and / or the second lineage- specific regulatory sequence includes a promoter, or where the first lineage-specific regulatory sequence and / or the second lineage-specific regulatory sequence includes a promoter and a UTR.

[0023] In some embodiments, the first lineage-specific regulatory sequence or the second lineage-specific regulatory sequence includes a CD3d promoter (CD3dp), or a LCK promoter (LCKp), optionally where the first lineage-specific regulatory sequence includes a CD3d promoter (CD3dp), or a LCK promoter (LCKp). In some embodiments, the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence includes a promoter or regulatory sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 4-10, optionally where the first lineage-specific regulatory sequence includes a promoter having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 4-10.

[0024] In some embodiments, the first lineage-specific regulatory sequence or the second lineage-specific regulatory sequence includes, as a core promoter, a minimal CMV promoter (minCMV); other examples of core promoters can include, without limitation, YB_TATA, MLP, and minP. In some embodiments, the first lineage-specific regulatory sequence or the second lineage-specific regulatory sequence includes a minCMV promoter or a promoter derived therefrom having at least 80% sequence identity with SEQ ID NO: 45 or a portion thereof, optionally where the first lineage-specific regulatory sequence includes a promoter having at least 80% sequence identity with SEQ ID NO: 45 or a portion thereof. In some embodiments, the first lineage-specific regulatory sequence or the second lineage-specific regulatory sequence includes a minCMV promoter or a promoter derived therefrom having at least 90% sequence identity with SEQ ID NO: 45 or a portion thereof, optionally where the first lineage-specific regulatory sequence includes a promoter having at least 90% sequence identity with SEQ ID NO: 45 or a portion thereof.

[0025] In some embodiments, the first lineage-specific regulatory sequence or the second lineage-specific regulatory sequence includes an enhancer selected from PrF, PrE, TNK-C, TNK-A, PrG, PrA, or PrB, or present in another sequence provided herein, optionally where the first lineage-specific regulatory sequence includes an enhancer selected from PrF, PrE, TNK-C, TNK-A PrG, PrA, or PrB, or present in another sequence provided herein. In some embodiment, the first lineage-specific regulatory sequence or second lineage-specific regulatory sequence comprises the dLck-V2 promoter and an enhancer, wherein the enhancer is optionally CMVenh or SV40enh. In some embodiments, the first or second lineage-specific regulatory sequence is Smal-dLck or hCD3. In some embodiments, the first lineage-specific8FH12901147.5ENO-00225 (38895-00225) regulatory sequence or the second lineage-specific regulatory sequence includes an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs:65-76, optionally where the first lineage-specific regulatory sequence includes an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 65-76.

[0026] In some embodiments, the first lineage-specific regulatory sequence or the second lineage-specific regulatory sequence includes a CDl lb promoter, CD68 promoter, CX3CR1- P3 (-222) promoter, or CX3CR1-P3 (-498) promoter, optionally where the second lineagespecific regulatory sequence includes a CDl lb promoter, CD68 promoter, CX3CR1-P3 (- 222) promoter, or CX3CR1-P3 (-498) promoter. In some embodiments, the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence includes a promoter having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 20, 21, 24, and 25, optionally where the second lineage-specific regulatory sequence includes a promoter having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 20, 21, 24, and 25.

[0027] In some embodiments, the second lineage-specific regulatory sequence includes a CMV promoter or a promoter derived therefrom, optionally where the second lineagespecific regulatory sequence includes the CMV promoter or a promoter derived therefrom, optionally where the CMV promoter or promoter derived therefrom is a minimal CMV promoter (minCMV). In some embodiments, the second lineage- specific regulatory sequence includes a CMV promoter or a promoter derived therefrom having at least 80% sequence identity with SEQ ID NO: 45.

[0028] In some embodiments, the first lineage-specific regulatory sequence or the second lineage-specific regulatory sequence includes an enhancer selected from SV40, CMV, TNK- A, PrA, PrG, PrB, TNK-C, TNK-B, T-spe, PrF, a CMV enhancer, an SV40 enhancer, and WPRE, optionally where the second lineage-specific regulatory sequence includes an enhancer selected from SV40, CMV, TNK-A, PrA, PrG, PrB, TNK-C, TNK-B, T-spe, PrF, a CMV enhancer, an SV40 enhancer, and WPRE. In some embodiments, the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence includes an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 63 and 65-72, optionally where the second lineage-specific regulatory sequence includes an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 63 and 65-72.

[0029] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes an agent that, when expressed in a cell of a subject9FH12901147.5ENO-00225 (38895-00225) having a disease, disorder, or condition, directly or indirectly causes or contributes to the treatment of the disease, disorder, or condition, or of a symptom thereof.

[0030] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes a protein, peptide, nucleic acid aptamer, or ribonucleic acid (RNA), optionally where the RNA includes an inhibitory RNA.

[0031] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes an antigen-binding agent including an antigenbinding domain.

[0032] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes an antigen-binding agent including a Chimeric Antigen Receptor (CAR), T Cell Receptor (TCR), or antibody. In some embodiments, the first therapeutic expression product includes a CAR, TCR, or antibody. In some embodiments, the first therapeutic expression product includes a CAR. In some embodiments, the second therapeutic expression product includes a CAR, TCR, or antibody. In some embodiments, the second therapeutic expression product includes a CAR.

[0033] In some embodiments, the first therapeutic expression product includes an antigenbinding domain that binds an antigen that is not bound by the second therapeutic expression product. In some embodiments, the second therapeutic expression product includes an antigen-binding domain that binds an antigen that is not bound by the first therapeutic expression product. In some embodiments, the first therapeutic expression product includes an antigen-binding domain that is not comprised by the second therapeutic expression product. In some embodiments, the second therapeutic expression product includes an antigen-binding domain that is not comprised by the first therapeutic expression product. In some embodiments, the first therapeutic expression product includes a first antigen-binding domain and the second therapeutic expression product includes a second antigen-binding domain, where the first and second antigen-binding domains bind with the same antigen. In some embodiments, the first therapeutic expression product includes a first antigen-binding domain and the second therapeutic expression product includes a second antigen-binding domain, where the first and second antigen-binding domains have the same amino acid sequence.

[0034] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes an antigen-binding agent that binds a cancer-specific antigen or cancer-associated antigen, optionally where the cancer-specific antigen or cancer- associated antigen is characteristic of a solid tumor or of a liquid or hematological cancer. In10FH12901147.5ENO-00225 (38895-00225) some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes an antigen-binding agent that binds an antigen selected from HER2 (human epidermal growth factor receptor 2), CD19, CD20, CD22, CD 19 and CD 20, CD 19 and CD 22, AFP (alpha-fetoprotein), AXL (AXL receptor tyrosine kinase), BCMA, B7-H3, CD5, CD7, CD33, CD38, CD47, CD52, CD123, CD133, CD138, CD171, CD171, CD30, CD38 / CD123, CD80 / 86, CEA (carcinoembryonic antigen), Claudin 18.2, CLL-1, c- MET, DLL-3 (delta-like 3), DR5 (death receptor 5), EGFR (epidermal growth factor receptor), EGFR806, EGFRIII, EGFR VIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EpHA2 (EPH receptor A2), FAP, FLT3, FCRL5, FR-a (folate receptor alpha), GD2 (disganglioside molecule), Glypican-3, gplOO, GPC3 (glypican 3), GPRC5D, IL-13Ra2, Lewis Y, LMP1 (Epstein-Barr virus latent membrane protein 1), MAGE (melanoma antigen gene protein)-Al / 3 / 4, Mesothelin, Mesothelin, MUC1 (mucinl), MUC16 (mucinl6), Nectin4 / FAP (familial adenomatous polyposis), NKGD2 (natural killer group 2 member D), PD-L1, PMSA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), PSA, ROR1 (receptor tyrosine kinase-like orphan receptor 1), SIRPy, TPD52, VEGFRII (vascular endothelial growth factor receptor II), and / or VISTA. In some embodiments, an antibody or antigen-binding fragment thereof, or a therapeutic polypeptide comprising a binding domain, can bind to a cell surface protein or a secreted protein. In some further embodiment, the cell surface protein or secreted protein is a protein selected from the group consisting of AFP, ALPP, PD-1, PD-L1, LAG-3, TIM-3, BAFFR, B7-H3 (CD276), B7H4, CD4, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD34, CD37, CD38, CD44v6, CD52, CD56, CD70, CD73, CD79b, CD80, CD110, CD117, CD123, CD124, CD126,, CD133, CD138, CD147, CD171, CD269, CD276, C7R, Chlorotoxin, GLY, GUCY2C, IL1, IL2, IL6, a TCR specifically present on autoreactive T cells, IL4, IL10, IL12, IL13, IL13Ra2, ILIRa, IL1RAP, sILlRI, sILlRII, ILT3, TNF, ABCA3, ABCD1, ADA, AK2, APP, arginase, arylsulfatase A, AXL, A1AT, BCMA, CCCR, CD3D, CD3E, CD3G, CAIX, CD3Z, CEA, CFTR, CHD7, CIITA, CLDN, CLDN6, CLDN18.2, CLL1, CLN3, complement factor, CORO1A, CTLA, Cl inhibitor, C9ORF72, c-Met, DLL3, DCLRE1B, DCLRE1C, decoy receptors, DKC1, DRBl*1501 / DQB 1*0602, DR5, dystrophin, enzymes, EGFR, EGFRvIII, EpCam, EphA2, Factor VIII, FANC polypeptides (FancA, FancB, FancC, FancDl (BRCA2), FancD2, FancE, FancF, FancG, Fanci, FancJ (BRIP1), FancL, FancM, FancN (PALB2), FancO (RAD51C), FancP (SLX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)), FAP, Fas L,11FH12901147.5ENO-00225 (38895-00225)FLT3, FUS, GATA1, GFRalpha4, gplOO, globin polypeptides (i.e., y-globin), F8, GD2, glutaminase, GPC3, FRalpha, HBA1, HBA2, HBB, Herl, Her2, Her3, Her4, ICAM-1, IL7RA, JAK3, KLK2, LCK, LeY, LIG4, LMP1, LRRK2, MUC1, MUC16, MUC17, MSLN, Nectin4, NKG2D, NKG2DL, NKR2, NHEJ1, NLX2.1, NY-ESO-1, 0RAI1, PARK2, PARK7, phox, PINK1, PI3K, PNP, PRKDC, PSCA, PSEN1, PSEN2, PSMA, PTPN22, PTPRC, P53, pyruvate kinase, RAG1, RAG2, RFXANK, RFXAP, RFX5, RMRP, ribosomal proteins, RORE, R0R2, SLAMF7, SFTPB, SFTPC, SOD1, soluble CD40, STIM1, sTNFRI, sTNFRII, SLC46A1, SNCA, TDP43, TERT, TERC, TINF2, TM451F1, TnMUCl, TSLPR, TRBC1, TROP, ubiquilin 2, VEGFR2, WAS, WHN, ZAP70, yC, and other polypeptides described herein.

[0035] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes a HER2 CAR. In some embodiments, the HER2 CAR includes an antigen binding domain having at least 80% sequence identity with SEQ ID NO: 87. In some embodiments, the HER2 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 141. In some embodiments, the HER2 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 131. In some embodiments, the HER2 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 133. In some embodiments, the HER2 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 136. In some embodiments, the HER2 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 137. In some embodiments, the HER2 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 139.

[0036] In some embodiments, the anti- CD 19 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 163. In some embodiments, the anti- CD20 CAR includes a sequence having at least 80% sequence identity with SEQ ID NO: 169.In some embodiments, the first expression product and / or the second expression product include a synthetic receptor. In some embodiments, the synthetic receptor includes a synNotch receptor, optionally where the synNotch receptor is engineered such that proteolysis of membrane-bound synNotch receptor generates an intracellular transcription factor.

[0037] In some embodiments, the nucleic acid construct includes at least a first immunomodulatory domain. In some embodiments, the first immunomodulatory domain includes a pro-inflammatory cytokine and / or where the first immunomodulatory domain includes hGMCSF, hIL-18, hlFNa, truncated TGFbR II (dnTGFBr II), TGFbR2 / IFNgRl12FH12901147.5ENO-00225 (38895-00225) switch receptor, TGFbR2 / MyD88 / CD40 switch receptor, IL-2, IFNy, or GMCSF. In some embodiments, the first immunomodulatory domain includes an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 176, 178, 180, 182, 184, and 186. In some embodiments, the first immunomodulatory domain is operably linked with the first lineage-specific regulatory sequence. In some embodiments, a first immunomodulatory domain and the first therapeutic expression protein are encoded in a single open reading frame. In some embodiments, a first immunomodulatory domain and the first therapeutic expression protein are separated during or after translation of the single open reading frame.

[0038] In some embodiments, the single open reading frame encodes a cleavable linker or self-cleaving peptide positioned between the sequence encoding a first immunomodulatory peptide and the sequence encoding the first therapeutic expression product in the single open reading frame. In some embodiments, the first immunomodulatory domain is not operably linked with the first lineage-specific regulatory sequence and is operably linked with a third regulatory sequence. In some embodiments, the third regulatory sequence includes a lineagespecific regulatory sequence, or where the third regulatory sequence includes a ubiquitous regulatory sequence. In some embodiments, the nucleic acid construct includes at least a second immunomodulatory domain. In some embodiments, the second immunomodulatory domain includes a pro-inflammatory cytokine and / or where the second immunomodulatory domain includes hGMCSF, hIL-18, hlFNa, truncated TGFbR II (dnTGFBr II), TGFbR2 / IFNgRl switch receptor, TGFbR2 / MyD88 / CD40 switch receptor, IL-2, IFNy, or GMCSF. In some embodiments, the second immunomodulatory domain includes an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 176, 178, 180, 182, 184, and 186. In some embodiments, the second immunomodulatory domain is operably linked with the second lineage-specific regulatory sequence.

[0039] In some embodiments, the second immunomodulatory domain and the second therapeutic expression protein are encoded in a single open reading frame. In some embodiments, the second immunomodulatory domain and the second therapeutic expression protein are separated during or after translation of the single open reading frame.

[0040] In some embodiments, the single open reading frame encodes a cleavable linker or self-cleaving peptide positioned between the sequence encoding the second immunomodulatory peptide and the sequence encoding the second therapeutic expression product in the single open reading frame. In some embodiments, the second13FH12901147.5ENO-00225 (38895-00225) immunomodulatory domain is not operably linked with the second lineage-specific regulatory sequence and is operably linked with a fourth regulatory sequence. In some embodiments, the fourth regulatory sequence includes a lineage-specific regulatory sequence, or where the fourth regulatory sequence includes a ubiquitous regulatory sequence.

[0041] In some embodiments, where the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with one or more HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with at least 1-4 HSC-related miRNA binding sites. In some embodiments, each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with 2-4 HSC-related miRNA binding sites. In some embodiments, each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with 3 or 4 HSC-related miRNA binding sites.

[0042] In some embodiments, each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with a plurality of HSC-related miRNA binding sites, and where each binding site is the same. In some embodiments, each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with a plurality of HSC-related miRNA binding sites, and where the binding sites are different.

[0043] In some embodiments, the nucleic acid encoding the first therapeutic expression product is operably linked with one or more HSC-related miRNA binding sites. In some embodiments, nucleic acid encoding the first therapeutic expression product is operable linked with 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites. In some embodiments, nucleic acid encoding the first therapeutic expression product is operably liked with at least 1-4 HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the first therapeutic expression product is operably linked with 2-4 HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the first14FH12901147.5ENO-00225 (38895-00225) therapeutic expression product is operably linked with 3 or 4 HSC-related miRNA binding sites.

[0044] In some embodiments, the nucleic acid encoding the second therapeutic expression product is operably linked with one or more HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the second therapeutic expression product is operably linked with 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the second therapeutic expression product is operably linked with at least 1-4 HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the second therapeutic expression product is operably linked with 2-4 HSC-related miRNA binding sites. In some embodiments, the nucleic acid encoding the second therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with 3 or 4 HSC-related miRNA binding sites.

[0045] In some embodiments, the miRNA binding site is 3’ to the nucleic acid encoding the therapeutic expression product.

[0046] In some embodiments, each of the HSC-related miRNA binding sites associated with a nucleic acid encoding the therapeutic expression product is within 500 base pairs of the nucleic acid encoding the therapeutic expression product. In some embodiments, each of the HSC-related miRNA binding sites associated with a nucleic acid encoding the therapeutic expression product is within 250 base pairs of the nucleic acid encoding the therapeutic expression product. In some embodiments, each of the HSC-related miRNA binding sites associated with a nucleic acid encoding the therapeutic expression product is within 100 base pairs of the nucleic acid encoding the therapeutic expression product.

[0047] In some embodiments, HSC-related miRNA binding sites operably linked with a nucleic acid encoding a therapeutic expression product are positioned between (1) the terminal codon of the sequence encoding the therapeutic expression product, and (2) a polyadenylation signal.

[0048] In some embodiments, least 1 of the HSC-related miRNA binding sites is an miRNA binding site selected from a miR126 binding site, a miR218 binding site, a miR183 binding site, a miR223 binding site, a miR130a binding site, a miR486 binding site, or a miRlOa binding site. In some embodiments, at least 1 miRNA binding site miRNA binding site is a miR126 binding site.

[0049] In some embodiments, all HSC-related miRNA binding sites are a miR126 binding site, a miR218 binding site, a miR183 binding site, a miR223 binding site, a miR130a15FH12901147.5ENO-00225 (38895-00225) binding site, a miR486 binding site, or a miRlOa binding site. In some embodiments, all HSC-related miRNA binding sites are a miR126 binding site.

[0050] In some embodiments, at least 2 or more HSC-related miRNA binding sites are the same. In some embodiments, at least 2 or more HSC-related miRNA binding sites are different.

[0051] In some embodiments, the sequence of the miR126 binding site is set forth in SEQ ID NO: 381. In some embodiments, the sequence of the miR126 binding site includes at least 90% identity to the sequence set forth in SEQ ID NO: 381.

[0052] In some embodiments, the one or more HSC-related miRNA binding sites include a 4x miR126 binding site. In some embodiments, the sequence of the 4x miR126 binding site is set forth in SEQ ID NO: 388. In some embodiments, the sequence of the 4x miR126 binding site includes at least 90% identity to the sequence set forth in SEQ ID NO: 388.

[0053] In some embodiments, the sequence of the miR218 binding site is set forth in SEQ ID NO: 384. In some embodiments, the sequence of the miR218 binding site includes at least 90% identity to the sequence set forth in SEQ ID NO: 384.

[0054] In some embodiments, the sequence of the miR183 binding site is set forth in SEQ ID NO: 385. In some embodiments, the sequence of the miR183 binding site includes at least 90% identity to the sequence set forth in SEQ ID NO: 385.

[0055] In some embodiments, the sequence of the miR223 binding site is set forth in SEQ ID NO: 382. In some embodiments, the sequence of the miR223 binding site includes at least 90% identity to the sequence set forth in SEQ ID NO: 382.

[0056] In some embodiments, the sequence of the miR130a binding site is set forth in SEQ ID NO: 383. In some embodiments, the sequence of the miR130a binding site includes at least 90% identity to the sequence set forth in SEQ ID NO: 383.

[0057] In some embodiments, the one or more HSC-related miRNA binding sites cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked in one or more target cell types for suppression, as compared to a reference.

[0058] In some embodiments, one or more HSC-related miRNA binding sites cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked in one or more target cell types for suppression, as compared to a reference. In some embodiments, the decrease in expression is at least 50%. In some embodiments, the decrease in expression is at least 60%, at least 70%, at least 80%, at least 90%, or 100%. In some embodiments, the reference comprises a cell or population of cells of the one or more target cell types for suppression, in which the nucleic acid encoding the first therapeutic expression16FH12901147.5ENO-00225 (38895-00225) product and / or the nucleic acid encoding the second therapeutic expression is not operably linked with the one or more HSC-related miRNA binding sites. In some embodiments, the one or more target cell types for suppression comprise HSCs. In some embodiments, the one or more HSC-related miRNA binding sites: do not cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked in one or more non-target cell types, and / or do not cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked to as a great a degree in one or more non-target cell types as in the one or more target cell types for suppression. In some embodiments, the non-target cell type comprises a myeloid cell. In some embodiments, the non-target cell type comprises a T cell. In some embodiments, the non-target cell type comprises a NK cell. In some embodiments, the target cell type comprise an HSC. In some embodiments, the one or more non-target cell types comprise: A) a myeloid cell, optionally wherein the target cell type comprises an HSC; B) a T cell, optionally wherein the target cell type comprises an HSC; C) an NK cell, optionally wherein the target cell type comprises an HSC. In some embodiments, the one or more non-target cell types comprise a T cell, a NK cell, and a meyloid cell, optionally wherein the target cell type comprises an HSC. In some embodiments, the one or more non-target cell types comprise T cells, NK cells, and myeloid cells (e.g., monocytes and / or macrophages), optionally wherein the target cell type comprises an HSC. In some embodiments, the decrease in expression in the one or more target cell types for suppression as compared to a target reference is at least 50%, at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 50-fold, at least a 100-fold, or at least a 1,000-fold greater than the decrease in expression in the one or more non-target cell types as compared to a non-target reference. In some embodiments, the target reference comprises a cell or population of cells of the one or more target cell types for suppression, in which the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is not operably linked with the one or more HSC-related miRNA binding sites, and / or wherein the non-target reference comprises a cell or population of cells of the one or more non-target cell types, in which the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is not operably linked with the one or more HSC-related miRNA binding sites. In some embodiments, the reference includes a cell or population of cells of the one or more target cell types for suppression, in which the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is not operably linked with the one or more HSC-related miRNA binding sites.17FH12901147.5ENO-00225 (38895-00225)

[0059] In some embodiments, the one or more target cell types for suppression include HSCs.

[0060] In some embodiments, the one or more HSC-related miRNA binding sites: i) do not cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked in one or more non-target cell types, and / or ii) do not cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked to as a great a degree in one or more non-target cell types compared to the one or more target cell types for suppression.

[0061] In some embodiments, wherein the one or more target cell types for suppression include HSCs. In some embodiments, the one or more target cell types for suppression or non-target cell types include myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells). In some embodiments, the non-target cell types include T cells. In some embodiments, the non-target cell types include NK cells. In some embodiments, the non-target cell types include B cells.

[0062] In some embodiments, the one or more non-target cell types include myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells), optionally wherein the one or more target cell types for suppression include HSCs . In some embodiments, the one or more non-target cell types include T cells, optionally wherein the one or more target cell types for suppression include HSCs. In some embodiments, the one or more non-target cell types include NK cells, optionally wherein the one or more target cell types for suppression include HSCs. In some embodiments, the one or more non-target cell types include T cells and NK cells, optionally where the one or more target cell types for suppression include HSCs In some embodiments, the one or more non-target cell types include T cells, NK cells, and myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells), optionally where the one or more target cell types for suppression include HSCs.

[0063] In some embodiments, the therapeutic expression product from the therapeutic expression cassette comprising the miRNA binding site(s) in a non-target cell types is a least 2-fold, at least a 5-fold, at least a 10-fold, at least a 50-fold, at least a 100-fold, or at least a 1 ,000-fold greater than the therapeutic expression product from the same therapeutic expression cassette in a target cell type.

[0064] In some embodiments, the nucleic acid construct includes one or more coding sequences each independently operably linked with one or more vaRNA binding sites, optionally where the vaRNA binding sites comprise a vaRNA 1 binding site and / or a vaRNA2 binding site, optionally where the one or more coding sequences each independently operably18FH12901147.5ENO-00225 (38895-00225) linked with one or more vaRNA binding sites include one or more of the nucleic acid sequence encoding the first therapeutic expression product, the nucleic acid sequence encoding the second therapeutic expression product, and / or the sequence encoding the enrichment marker.

[0065] In some embodiments, the nucleic acid construct includes a nucleic acid sequence encoding an enrichment marker. In some embodiments, the enrichment marker includes an inhibitor-resistant MGMT protein, optionally where the inhibitor-resistant MGMT protein is an MGMT protein that includes the mutation P140K (MGMTP14OK). In some embodiments, MGMTP140Kincludes an amino acid sequence having at least 80% identity with SEQ ID NO: 188. In some embodiments, the enrichment marker includes a signaling-enhanced EpoR protein. In some embodiments, the enrichment marker is operably linked with a regulatory sequence. In some embodiments, an enrichment marker is or includes an expression product that decreases expression of c-kit by a host cell, whereby contact with a selecting agent that selectively targets and / or kills cells expressing c-kit causes an enrichment of cells engineered to encode a nucleic acid construct of the present disclosure and express the product that decreases expression of c-kit. In some embodiments, an enrichment marker is or includes an expression product that decreases expression of CD33 by a host cell, whereby contact with a selecting agent that selectively targets and / or kills cells expressing CD33 causes an enrichment of cells engineered to encode a nucleic acid construct of the present disclosure and express the product that decreases expression of CD33.

[0066] In some embodiments, the enrichment marker is not operably linked with the first lineage-specific regulator sequence or the second lineage-specific regulatory sequence. In some embodiments, the enrichment marker is not operably linked with the third regulatory sequence and / or the fourth regulatory sequence. In some embodiments, the enrichment marker is operably linked with a fifth regulatory sequence.

[0067] In some embodiments, the enrichment marker is operably linked with a regulatory sequence including a ubiquitous promoter. In some embodiments, the ubiquitous promoter includes an EFla promoter, a UBC promoter, an SFFV promoter, a CAG promoter, a CMV promoter, or a CMV-derived promoter, optionally where the CMV-derived promoter is a minCMV.

[0068] In some embodiments, the nucleic acid construct includes a safety switch. In some embodiments, the safety switch includes tEGFR, tEGFR-RRR, and GMCSF. In some embodiments, the safety switch includes an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs:194, 196, and 198.19FH12901147.5ENO-00225 (38895-00225)

[0069] In some embodiments, the safety switch is operably linked with a regulatory sequence.In some embodiments, the safety switch is operably linked with the fifth regulatory sequence.

[0070] In some embodiments, the safety switch and the enrichment marker are encoded in a single open reading frame. In some embodiments, the where the safety switch and the enrichment marker are separated during or after translation of the single open reading frame. In some embodiments, the single open reading frame encodes a cleavable linker or selfcleaving peptide positioned between the sequence encoding the safety switch and the sequence encoding the enrichment marker in the single open reading frame.

[0071] In some embodiments, the safety switch is not operably linked with the fifth regulatory sequence and is operably linked with a sixth regulatory sequence. In some embodiments, the sixth regulatory sequence includes a lineage-specific regulatory sequence, or where the fourth regulatory sequence includes a ubiquitous regulatory sequence.

[0072] In some embodiments, the nucleic acid construct is engineered for integration into a target genome of a cell or subject by transposition.

[0073] In some embodiments, the nucleic acid construct is flanked by transposon inverted repeats (IRs), optionally where the transposon IRs are flanked by recombinase direct repeats (DRs).

[0074] In some embodiments, the transposon IRs are Sleeping Beauty (SB), piggyback, Mariner, frog prince, Tol2, TcBuster, or spinON IRs. In some embodiments, the transposon IRs are Sleeping Beauty (SB) IRs, optionally where the transposase is a Sleeping Beauty (SB) transposase, optionally where the transposase is Sleeping Beauty lOOx (SB100X) or Sleeping Beauty 150x (SB150X), optionally where the SB IRs are pT4 IRs. In some embodiments, the recombinase DRs (alternatively referred to as recombinase target sites) that flank the transposon IRs (alternatively referred to as transposon target sites) are FRT, loxP, rox, vox, AttB, or AttP sites. In some embodiments, the recombinase DRs that flank the transposon IRs are FRT sites. In some embodiments, the nucleic acid construct includes:A) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR); (ii) a lineage-specific regulatory sequence that causes expression in a myeloid cell, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage-20FH12901147.5ENO-00225 (38895-00225) specific miRNA binding sites; and (iii) a ubiquitous regulatory sequence operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence;B) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR); (ii) a lineage-specific promoter that causes expression in a myeloid cell, where the lineage-specific promoter is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage-specific miRNA binding sites; and (iii) a ubiquitous promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence;C) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), and further including a 5’ intron, where the lineagespecific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), and where the sequence encoding the therapeutic expression product is operably linked with four miR126 binding sites; (ii) a CD1 lb promoter operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage-specific miRNA binding sites; and (iii) an EFla promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence;D) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), and further including a 5’ intron, where the lineagespecific regulatory sequence is operably linked with a sequence encoding a HER2 CAR; (ii) a CD1 lb promoter operably linked with a sequence encoding a HER2 CAR, which sequence encoding a HER2 CAR is operably linked with four miR126 binding sites; and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence);21FH12901147.5ENO-00225 (38895-00225)E) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A) and futher including a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR having the structure [IgGk leader- (VL- whitlow linker- VH)-CD28 (Hinge-TM-ICD)-CD3z]; (ii) a CD 11b promoter operably linked with a sequence encoding a HER2 CAR having the structure [CD8 leader- VH-g4S-VL-CD8 (Hinge-TM)-CD3z], which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa); and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP140Kselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence);F) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A set forth in SEQ ID NO: 65 and further including a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 148; (ii) a CD1 lb promoter set forth in SEQ ID NO: 20 operably linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 146, which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa); and (iii) an EFla promoter set forth in SEQ ID NO: 43 operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch set forth in SEQ ID NO: 195, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence set forth in SEQ ID NO: 225);G) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage- specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), where a chicken beta actin 5’ UTR intron sequence is ;located 3’ of the lineage-specific regulatory sequence and 5’ to the coding sequence of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a lineage- specific regulatory sequence that causes expression in a myeloid cell, where the lineage-specific regulatory sequence is operably linked with a sequence22FH12901147.5ENO-00225 (38895-00225) encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage- specific miRNA binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (iii) a ubiquitous regulatory sequence operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence, optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);H) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), where a chicken beta actin 5’ UTR intron sequence is located 3’ to the lineage-specific regulatory sequence and 5’ to the coding sequence of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a lineage-specific promoter that causes expression in a myeloid cell, where the lineage-specific promoter is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineagespecific miRNA binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) a ubiquitous promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence, optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);I) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A)and further including a 5’ intron, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression23FH12901147.5ENO-00225 (38895-00225) product (e.g., a CAR, e.g., a HER2 CAR), and wherein the sequence encoding the therapeutic expression product is operably linked with four miR126 binding sites, where a chicken beta actin 5’ UTR intron is located 3’ to the lineage-specific regulatory sequence and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CD1 lb promoter operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage-specific miRNA binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence, optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);J) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), andfurther comprising a 5’ intron, where the lineagespecific regulatory sequence is operably linked with a sequence encoding a HER2 CAR, where a chicken beta actin 5’ UTR intron is located 3’ to the lineage-specific regulatory sequence and and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CD1 lb promoter operably linked with a sequence encoding a HER2 CAR, which sequence encoding a HER2 CAR is operably linked with four miR126 binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence), optionally where the sequence encoding the selectable marker and24FH12901147.5ENO-00225 (38895-00225) safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);K) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), and further including a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR having the structure [IgGk leader- (VL- whitlow linker- VH)-CD28 (Hinge-TM-ICD)-CD3z], where a chicken beta actin 5’ UTR intron is located 3’ to the lineage-specific regulatory sequence and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CDllb promoter operably linked with a sequence encoding a HER2 CAR having the structure [CD8 leader- VH-g4S-VL-CD8 (Hinge-TM)-CD3z], which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa), and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence), optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);L) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A set forth in SEQ ID NO: 65), and further including a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 148, where a chicken beta actin 5’ UTR intron sequence is 3’ to the promoter of the lineage-specific regulatory sequence and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CD1 lb promoter set forth in SEQ ID NO: 20 operably linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 146, which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which25FH12901147.5ENO-00225 (38895-00225) sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa), and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter set forth in SEQ ID NO: 43 operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR- RRR safety switch set forth in SEQ ID NO: 195, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence set forth in SEQ ID NO: 225), optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites).

[0075] In at least one aspect, the present disclosure provides a cell including the nucleic acid construct of any one of the foregoing embodiments.

[0076] In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a CD46+ cell. In some embodiments, the cell is a hematopoietic stem cell (HSC).

[0077] In some embodiments, the cell is a lymphoid lineage cell. In some embodiments, the cell is a T cell, NK cell, or B cell. In some embodiments, the cell is a T cell, optionally where the T cell is a CD8+ T cell, CD4+ T cell, or NKT cell. In some embodiments, the cell is an NK cell, optionally where the NK cell is a CD56brightNK cell, and / or CD56dimNK cell.

[0078] In some embodiments, the cell is a myeloid lineage cell. In some embodiments, the cell is a monocyte, macrophage, myeloblast, granulocyte, neutrophil, eosinophil, basophil, megakaryocyte-erythroid progenitor cell, megakaryocyte, myeloid dendritic cell (mDC), monocyte-derived dendritic cell (MoDC), mast cell, platelet, and / or erythrocyte.

[0079] In some embodiments, the cell expresses the first therapeutic expression product and / or the second therapeutic expression product. In some embodiments, the cell expresses the first therapeutic expression product and the second therapeutic expression product. In some embodiments, the cell expresses the first therapeutic expression product and does not express, does not detectably express, and / or does not significantly express the second therapeutic expression product. In some embodiments, the cell expresses the first therapeutic expression product at a level that is at least 10-fold, at least 20-fold, at least 30- fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 10,000- fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, or at least 100,000-fold than the expression level of the second therapeutic expression product. In some embodiments, the cell expresses the second therapeutic expression product and does not26FH12901147.5ENO-00225 (38895-00225) express, does not detectably express, and / or does not significantly express the first therapeutic expression product. In some embodiments, the cell expresses the second therapeutic expression product at a level that is at least 10-fold, at least 20-fold, at least 30- fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40, 000- fold, at least 50,000-fold, or at least 100,000-fold than the expression level of the first therapeutic expression product.

[0080] In some embodiments, the cell is derived from an HSC cell.

[0081] In some embodiments, the nucleic acid construct is integrated in the genome of the cell.

[0082] In at least one aspect, the present disclosure provides a helper-dependent adenoviral (HD Ad) genome including the nucleic acid construct of any of the foregoing embodiments.

[0083] In some embodiments, the HD Ad genome includes an adenoviral 5’ ITR positioned 5’ of the nucleic acid construct and an adenoviral 3’ ITR positioned 3’ of the nucleic acid construct. In some embodiments, the adenoviral 5’ ITR and adenoviral 3’ ITR are Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 ITRs. In some embodiments, the adenoviral 5’ ITR and adenoviral 3’ ITR are Ad5 or Ad6 ITRs.

[0084] In some embodiments, the HDAd genome includes an adenoviral packaging sequence. In some embodiments, the packaging sequence is an Ad3, Ad5, Ad6, Ad7, Adil, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 packaging sequence. In some embodiments, the packing sequence is an Ad5 or Ad6 packaging sequence.

[0085] In some embodiments, the HDAd genome does not include a nucleic acid sequence encoding a viral structural protein, optionally where the viral structural protein is selected from an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton. In some embodiments, the HDAd genome does not include nucleic acid sequences encoding any of an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton.

[0086] In at least one aspect, the present disclosure provides a helper-dependent adenoviral (HDAd) vector including the genome of any one of the foregoing embodiments. In some embodiments, the HDAd vector includes an adenoviral penton and an adenoviral hexon of the same adenoviral serotype, optionally where the adenoviral serotype of the hexon and penton is Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50, optionally where the adenoviral serotype of the hexon and penton is Ad5 or Ad6. In some embodiments, the HDAd vector includes a fiber that is heterologous in that it is wholly or27FH12901147.5ENO-00225 (38895-00225) partially derived from an adenoviral serotype that is distinct from the hexon and / or penton of the HD Ad vector. In some embodiments, the HD Ad vector includes a chimeric adenoviral fiber, where the adenoviral fiber includes an adenoviral fiber tail, fiber shaft, and fiber knob. In some embodiments, the heterologous fiber protein is a fiber derived from a particular adenoviral serotype (e.g., derived from a Group P adenoviral serotype, e.g., derived from Ad serotype 3, 7, 11, 14, 16, 21, 34, 35 50, or 55). In some embodiments, the heterologous fiber protein is a fiber is a chimeric fiber that includes at least a fiber knob (e.g., a fiber knob optionally together with a fiber shaft or portion thereof and / or fiber tail or portion thereof) derived from a particular adenoviral serotype (e.g., derived from a Group P adenoviral serotype, e.g., derived from Ad serotype 3, 7, 11, 14, 16, 21, 34, 35 (including 35++), 50, or 55), optionally with remaining portions derived from the same serotype as the hexon and / or penton (e.g., Ad5 or Ad6). Thus, in various embodiments, an HD Ad vector of the present disclosure can include a fiber knob sequence derived from of any one of the FH sequences of Ad serotypes 3, 7, 11, 14, 16, 21, 34, 35 (including 35++), 50, or 55. Accordingly, in various embodiments, an HDAd vector of the present disclosure can be, e.g., an HDAd5 / 3, HDAd5 / 7, HDAd5 / l l, HDAd5 / 14, HDAd5 / 16, HDAd5 / 21, HDAd5 / 34, HDAd5 / 35, HDAd5 / 35++, HDAd5 / 50, HDAd5 / 55, HDAd6 / 3, HDAd6 / 7, HDAd6 / ll, HDAd6 / 14, HDAd6 / 16, HDAd6 / 21, HDAd6 / 34, HDAd6 / 35, HDAd6 / 35++ vector, HDAd6 / 50, or HDAd6 / 55 HDAd vector.

[0087] In some embodiments, the chimeric adenoviral fiber includes an Ad5 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob. In some embodiments, the chimeric adenoviral fiber includes an Ad6 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob. In some embodiments, the chimeric adenoviral fiber includes an Ad5 fiber tail, an Ad5 fiber shaft, and an Ad35 fiber knob. In some embodiments, the chimeric adenoviral fiber includes an Ad6 fiber tail, an A35 fiber shaft, and an Ad35 fiber knob.

[0088] In some embodiments, the Ad35 fiber knob includes one or more mutations that increase affinity of the adenoviral fiber with CD46.

[0089] In some embodiments, the one or more mutations include at least one mutation selected from llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His. In some embodiments, the one or more mutations include each of llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His.

[0090] In some embodiments, the HDAd vector includes an Ad5 / 35++ adenoviral capsid.28FH12901147.5ENO-00225 (38895-00225)

[0091] In at least one aspect, the present disclosure provides an adenoviral vector system for delivery of the nucleic acid construct to a mammalian cell, including the HD Ad vector of any one of the foregoing embodiments and an integration vector, where the nucleic acid construct is flanked by the transposon inverted repeats (IRs), and where the transposon IRs are flanked by the recombinase direct repeats (DRs), where the integration vector is a helperdependent adenoviral (HD Ad) vector including a helper dependent adenoviral genome, where the integration vector genome encodes a transposase that mediates transposition of the nucleic acid construct flanked by the transposon IRs, and optionally where the integration vector genome encodes a recombinase for excision of the nucleic acid sequence flanked by the DRs. In some embodiments, the DRs include FRT, loxP, rox, vox, AttB, or AttP sites, and the recombinase includes a Flp, Cre, Dre, Vika, or PhiC31 recombinase. In some embodiments, the DRs include FRT sites, and the recombinase includes a Flp recombinase. In some embodiments, the nucleic acid sequence encoding the recombinase is operably linked with a regulatory sequence, optionally where the regulatory sequence operably linked with the recombinase includes an EFla promoter.

[0092] In some embodiments, the IRs include Sleeping Beauty (SB), piggyback, Mariner, frog prince, Tol2, TcBuster, or spinON IRs and the transposase includes a Sleeping Beauty (SB), piggyback, Mariner, frog prince, Tol2, TcBuster, or spinON transposase. In some embodiments, the IRs include Sleeping Beauty (SB) IRs and the transposase includes a Sleeping Beauty transposase, optionally where the IRs include pT4 IRs. In some embodiments, the transposase is SBIOOx or SB150X. In some embodiments, the nucleic acid sequence encoding the transposase is operably linked with a regulatory sequence, optionally where the regulatory sequence operably linked with the transposase includes a CAG, an EFla or a PGK promoter.

[0093] In some embodiments, the nucleic acid sequence encoding the recombinase and the transposase, and regulatory sequences operably linked thereto, is flanked by an adenoviral 5’ ITR and an adenoviral 3’ ITR positioned.

[0094] In some embodiments, the adenoviral 5’ ITR and adenoviral 3’ ITR of the integration vector genome are Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 ITRs.

[0095] In some embodiments, the adenoviral 5’ ITR and adenoviral 3’ ITR of the integration vector genome are Ad5 or Ad6 ITRs.

[0096] In some embodiments, the integration vector genome includes an adenoviral packaging sequence.29FH12901147.5ENO-00225 (38895-00225)

[0097] In some embodiments, the integration vector genome includes an Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 packaging sequence. In some embodiments, the integration vector genome includes an Ad5 or Ad6 packaging sequence. In some embodiments, the integration vector genome does not include a nucleic acid sequence encoding a viral structural protein, optionally where the viral structural protein is selected from an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton. In some embodiments, the integration vector genome does not include nucleic acid sequences encoding any of an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton.

[0098] In some embodiments, the integration vector includes an adenoviral penton and an adenoviral hexon of the same adenoviral serotype, optionally where the adenoviral serotype of the hexon and penton is Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50, optionally where the adenoviral serotype of the hexon and penton is Ad5 or Ad6. In some embodiments, the HD Ad v integration ector includes a fiber that is heterologous in that it is wholly or partially derived from an adenoviral serotype that is distinct from the hexon and / or penton of the integration vector. In some embodiments, the integration vector includes a chimeric adenoviral fiber, where the adenoviral fiber includes an adenoviral fiber tail, fiber shaft, and fiber knob. In some embodiments, the heterologous fiber protein is a fiber derived from a particular adenoviral serotype (e.g., derived from a Group P adenoviral serotype, e.g., derived from Ad serotype 3, 7, 11, 14, 16, 21, 34, 35 50, or 55). In some embodiments, the heterologous fiber protein is a fiber is a chimeric fiber that includes at least a fiber knob (e.g., a fiber knob optionally together with a fiber shaft or portion thereof and / or fiber tail or portion thereof) derived from a particular adenoviral serotype (e.g., derived from a Group P adenoviral serotype, e.g., derived from Ad serotype 3, 7, 11, 14, 16, 21, 34, 35 (including 35++), 50, or 55), optionally with remaining portions derived from the same serotype as the hexon and / or penton (e.g., Ad5 or Ad6). Thus, in various embodiments, an HD Ad vector of the present disclosure can include a fiber knob sequence derived from of any one of the FH sequences of Ad serotypes 3, 7, 11, 14, 16, 21, 34, 35 (including 35++), 50, or 55. Accordingly, in various embodiments, an HDAd vector of the present disclosure can be, e.g., an HDAd5 / 3, HDAd5 / 7, HDAd5 / l l, HDAd5 / 14, HDAd5 / 16, HDAd5 / 21, HDAd5 / 34, HDAd5 / 35, HDAd5 / 35++, HDAd5 / 50, HDAd5 / 55, HDAd6 / 3, HDAd6 / 7, HDAd6 / l l, HDAd6 / 14, HDAd6 / 16, HDAd6 / 21, HDAd6 / 34, HDAd6 / 35, HDAd6 / 35++ vector, HDAd6 / 50, or HDAd6 / 55 HDAd vector.

[0099] In some embodiments, the integration vector includes a chimeric adenoviral fiber including an Ad5 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob. In some30FH12901147.5ENO-00225 (38895-00225) embodiments, the integration vector includes a chimeric adenoviral fiber including an Ad6 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob. In some embodiments, the chimeric adenoviral fiber includes an Ad5 fiber tail, an Ad5 fiber shaft, and an Ad35 fiber knob. In some embodiments, the chimeric adenoviral fiber includes an Ad6 fiber tail, an A35 fiber shaft, and an Ad35 fiber knob.

[0100] In some embodiments, the Ad35 fiber knob of the integration vector fiber includes one or more mutations that increase affinity of the adenoviral fiber with CD46. In some embodiments, the Ad35 fiber knob of the integration vector fiber includes one or more mutations including at least one mutation selected from llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His. In some embodiments, the Ad35 fiber knob of the integration vector fiber includes each of llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His.

[0101] In some embodiments, the integration vector includes an Ad5 / 35++ adenoviral capsid.

[0102] In at least one aspect, the disclosure provides a kit or pharmaceutical composition including the adenoviral vector system of any one of the foregoing embodiments. In some embodiments, the pharmaceutical composition includes the HD Ad vector of any one of foregoing embodiments and the integration vector. In some embodiments, the pharmaceutical composition is a liquid, optionally where the liquid includes a buffer and / or where the pharmaceutical composition is formulated for intravenous administration.

[0103] In some embodiments, the kit includes a first pharmaceutical composition including the HD Ad vector of any one of the foregoing embodiments and a second pharmaceutical composition including the integration vector.

[0104] In some embodiments, the first pharmaceutical composition is a liquid, optionally where the liquid includes a buffer and / or where the pharmaceutical composition is formulated for intravenous administration, and / or where the second pharmaceutical composition is a liquid, optionally where the liquid includes a buffer and / or where the pharmaceutical composition is formulated for intravenous administration.

[0105] In some embodiments, the HD Ad vector of any one of the foregoing embodiments and the integration vector are present at a ratio of about 1-3:1 or 1:1-3 HD Ad vector:integration vector, optionally where the ratio is about 1:1. In some embodiments, the total dosage of all vector genomes present in the kit or pharmaceutical composition is about 2.5 x 1011GC / kg to about 1.25 x 1013GC / kg.31FH12901147.5ENO-00225 (38895-00225)

[0106] In at least one aspect, the present disclosure provides a method of modifying a hematopoietic stem cell including contacting an HSC with the adenoviral vector system of any one of the foregoing embodiments.

[0107] In at least one aspect, the present disclosure provides a method of modifying a hematopoietic stem cell of a mammalian subject including delivering to the subject the adenoviral vector system of any one of the foregoing embodiments.

[0108] In at least one aspect, the present disclosure provides a method of modifying a hematopoietic stem cell of a mammalian subject including administering to the subject the pharmaceutical composition, or first pharmaceutical composition and second pharmaceutical composition, of any one of the foregoing embodiments.

[0109] In at least one aspect, the present disclosure provides a method of treating a solid tumor in a subject in need thereof, the method including delivering to the subject the adenoviral vector system of any one of the foregoing embodiments.

[0110] In at least one aspect, the present disclosure provides a method of treating a solid tumor in a subject in need thereof, the method including administering to the subject the pharmaceutical composition, or first pharmaceutical composition and second pharmaceutical composition, of any one of the foregoing embodiments.

[0111] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product includes an antigen binding domain that binds a cancerspecific antigen or cancer- associated antigen, optionally where the cancer- specific antigen or cancer-associated antigen is characteristic of a solid tumor or of a liquid or hematological cancer.

[0112] In some embodiments, the first therapeutic expression product and / or the second therapeutic expression product bind an antigen selected from HER2 (human epidermal growth factor receptor 2), CD19, CD20, CD22, CD 19 and CD 20, CD 19 and CD 22, AFP (alpha-fetoprotein), AXL (AXL receptor tyrosine kinase), BCMA, B7-H3, CD5, CD7, CD33, CD38, CD47, CD52, CD123, CD133, CD138, CD171, CD171, CD30, CD38 / CD123, CD80 / 86, CEA (carcinoembryonic antigen), Claudin 18.2, CLL-1, c-MET, DLL-3 (delta-like 3), DR5 (death receptor 5), EGFR (epidermal growth factor receptor), EGFR806, EGFRIII, EGFRVIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EpHA2 (EPH receptor A2), FAP, FLT3, FCRL5, FR-a (folate receptor alpha), GD2 (disganglioside molecule), Glypican-3, gplOO, GPC3 (glypican 3), GPRC5D, IL- 13Ra2, Lewis Y, LMP1 (Epstein-Barr virus latent membrane protein 1), MAGE (melanoma antigen gene protein)-Al / 3 / 4, Mesothelin, Mesothelin, MUC1 (mucinl), MUC16 (mucinl6),32FH12901147.5ENO-00225 (38895-00225)Nectin4 / FAP (familial adenomatous polyposis), NKGD2 (natural killer group 2 member D), PD-L1, PMSA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), PSA, R0R1 (receptor tyrosine kinase-like orphan receptor 1), SIRPy, TPD52, VEGFRII (vascular endothelial growth factor receptor II), and / or VISTA, optionally where the first therapeutic expression product and / or the second therapeutic expression product includes a CAR. In some embodiments, the first therapeutic expression product and the second therapeutic expression product each include a CAR including an antigen-binding domain that binds an antigen selected from HER2 (human epidermal growth factor receptor 2), CD19, CD20, CD22, CD 19 and CD 20, CD 19 and CD 22, AFP (alpha-fetoprotein), AXL (AXL receptor tyrosine kinase), BCMA, B7-H3, CD5, CD7, CD33, CD38, CD47, CD52, CD123, CD133, CD138, CD171, CD171, CD30, CD38 / CD123, CD80 / 86, CEA (carcinoembryonic antigen), Claudin 18.2, CLL-1, c-MET, DLL-3 (delta-like 3), DR5 (death receptor 5), EGFR (epidermal growth factor receptor), EGFR806, EGFRIII, EGFRVIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EpHA2 (EPH receptor A2), FAP, FLT3, FCRL5, FR-a (folate receptor alpha), GD2 (disganglioside molecule), Glypican-3, gplOO, GPC3 (glypican 3), GPRC5D, IL-13Ra2, Lewis Y, LMP1 (Epstein-Barr virus latent membrane protein 1), MAGE (melanoma antigen gene protein)-Al / 3 / 4, Mesothelin, Mesothelin, MUC1 (mucinl), MUC16 (mucinl6), Nectin4 / FAP (familial adenomatous polyposis), NKGD2 (natural killer group 2 member D), PD-L1, PMSA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), PSA, ROR1 (receptor tyrosine kinase-like orphan receptor 1), SIRPy, TPD52, VEGFRII (vascular endothelial growth factor receptor II), and / or VISTA, optionally where the first therapeutic expression product and the second therapeutic expression product bind the same antigen, optionally where the antigen is HER2, CD19, or CD20, optionally where the antigen is HER2.

[0113] In some embodiments, an antibody or antigen-binding fragment thereof, or a therapeutic polypeptide comprising a binding domain, can bind to a cell surface protein or a secreted protein. In some further embodiment, the cell surface protein or secreted protein is a protein selected from the group consisting of AFP, ALPP, PD-1, PD-L1, LAG-3, TIM-3, BAFFR, B7-H3 (CD276), B7H4, CD4, CD5, CD7, CD19, CD20, CD22, CD30, CD33, CD34, CD37, CD38, CD44v6, CD52, CD56, CD70, CD73, CD79b, CD80, CD110, CD117, CD123, CD124, CD126,, CD133, CD138, CD147, CD171, CD269, CD276, C7R, Chlorotoxin, GLY, GUCY2C, IL1, IL2, IL6, a TCR specifically present on autoreactive T cells, IL4, IL10, IL12, IL13, IL13Ra2, ILIRa, IL1RAP, sILlRI, sILlRII, ILT3, TNF,33FH12901147.5ENO-00225 (38895-00225)ABCA3, ABCD1, ADA, AK2, APP, arginase, arylsulfatase A, AXL, A1AT, BCMA, CCCR, CD3D, CD3E, CD3G, CAIX, CD3Z, CEA, CFTR, CHD7, CIITA, CLDN, CLDN6, CLDN18.2, CLL1, CLN3, complement factor, C0R01A, CTLA, Cl inhibitor, C9ORF72, c- Met, DLL3, DCLRE1B, DCLRE1C, decoy receptors, DKC1, DRBl*1501 / DQBl*0602, DR5, dystrophin, enzymes, EGFR, EGFRvIII, EpCam, EphA2, Factor VIII, FANC polypeptides (FancA, FancB, FancC, FancDl (BRCA2), FancD2, FancE, FancF, FancG, Fanci, FancJ (BRIP1), FancE, FancM, FancN (PALB2), FancO (RAD51C), FancP (SEX4), FancQ (ERCC4), FancR (RAD51), FancS (BRCA1), FancT (UBE2T), FancU (XRCC2), FancV (MAD2L2), and FancW (RFWD3)), FAP, Fas L, FLT3, FUS, GATA1, GFRalpha4, gplOO, globin polypeptides (i.e., y-globin), F8, GD2, glutaminase, GPC3, FRalpha, HBA1, HBA2, HBB, Herl, Her2, Her3, Her4, ICAM-1, IL7RA, JAK3, KLK2, LCK, LeY, LIG4, LMP1, LRRK2, MUC1, MUC16, MUC17, MSEN, Nectin4, NKG2D, NKG2DL, NKR2, NHEJ1, NLX2.1, NY-ESO-1, 0RAI1, PARK2, PARK7, phox, PINK1, PI3K, PNP, PRKDC, PSCA, PSEN1, PSEN2, PSMA, PTPN22, PTPRC, P53, pyruvate kinase, RAG1, RAG2, RFXANK, RFXAP, RFX5, RMRP, ribosomal proteins, RORE, R0R2, SLAMF7, SFTPB, SFTPC, SOD1, soluble CD40, STIM1, sTNFRI, sTNFRII, SLC46A1, SNCA, TDP43, TERT, TERC, TINF2, TM451F1, TnMUCl, TSLPR, TRBC1, TROP, ubiquilin 2, VEGFR2, WAS, WHN, ZAP70, yC, and other polypeptides described herein.

[0114] In some embodiments, a cancer is a HER2 -positive cancer. In various embodiments, a “HER2-positive cancer” can refer to a cancer that has been determined to be Her2 positive according to a standard diagnostic method (e.g., immunohistochemistry (IHC) (score 3+) or fluorescence in situ hybridization (FISH)). In various embodiments, the HER-2 positive cancer can be selected from the group consisting of ovarian cancer, hepatocellular cancer, prostate cancer, cholangiocarcinoma (intrahepatic and / or extrahepatic), pancreatic adenocarcinoma, intestinal malignancy, lung cancer, head and neck carcinoma, central nervous system tumor, pediatric glioma, advanced solid tumor, colorectal cancer, uterine cancer, cervical cancer, testicular cancer, gastric adenocarcinoma, gallbladder cancer, breast cancer, esophageal cancer, esophagogastric junction cancer, bladder cancer, sarcoma, glioblastoma, biliary tract cancer, salivary gland cancer, and endometrial cancer.

[0115] In some embodiments, when the therapeutic expression product comprises an antibody protein, or fragment thereof, fused to a CAR (e.g., a CAR directed to the antigen HER2 (a HER2-CAR)), wherein the antibody binds HER2, and then the cancer to be treated is a HER2 positive cancer. In a further embodiment, then the therapeutic expression product is an a HER-CAR, then the cancer is a HER2 positive cancer that is selected from the group34FH12901147.5ENO-00225 (38895-00225) consisting of ovarian cancer, hepatocellular cancer, prostate cancer, cholangiocarcinoma (intrahepatic and / or extrahepatic), pancreatic adenocarcinoma, intestinal malignancy, lung cancer, head and neck carcinoma, central nervous system tumor, pediatric glioma, advanced solid tumor, colorectal cancer, uterine cancer, cervical cancer, testicular cancer, gastric adenocarcinoma, gallbladder cancer, breast cancer, esophageal cancer, esophagogastric junction cancer, bladder cancer, sarcoma, glioblastoma, biliary tract cancer, salivary gland cancer, and endometrial cancer.

[0116] In some embodiments, a cancer can be acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), agnogenic myeloid metaplasia, astrocytoma, atypical teratoid rhabdoid tumor, brain and central nervous system (CNS) cancer, breast cancer, carcinosarcoma, chondrosarcoma, chordoma, choroid plexus carcinoma, choroid plexus papilloma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), clear cell sarcoma of soft tissue, diffuse large B-cell lymphoma, ependymoma, epithelioid sarcoma, Ewing sarcoma, extragonadal germ cell tumor, extrarenal rhabdoid tumor, follicular lymphoma, gastrointestinal stromal tumor, glioblastoma, HBV-induced hepatocellular carcinoma, head and neck cancer, Hodgkin’s lymphoma, juvenile myelomonocytic leukemia, kidney cancer, lung cancer, lymphoma, malignant rhabdoid tumor, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloma, neuroglial tumor, nonHodgkin’s lymphoma, not otherwise specified (NOS) sarcoma, oligoastrocytoma, oligodendroglioma, osteosarcoma, ovarian cancer, ovarian clear cell adenocarcinoma, ovarian endometrioid adenocarcinoma, ovarian serous adenocarcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic endocrine tumor, pineoblastoma, prostate cancer, renal cell carcinoma, renal medullary carcinoma, rhabdomyosarcoma, sarcoma, schwannoma, skin squamous cell carcinoma, and / or stem cell cancer.

[0117] In some embodiments, a cancer can be lung cancer, osteosarcoma, glioblastoma, central nervous system tumor, pediatric glioma, advanced solid tumor, HER2 -positive cancer, BALL, B-CLL, leukemia, lymphoma, B-NHL / CLL, mantel cell leukemia / B-NHL, melanoma, Non-Hodgkins Lymphoma, B-Non Hodgkins Lymphoma, B-NHL, B-cell malignancy, B-ALL, D LBCL, B-ALL, B-NHL, hepatocellular carcinoma, liver, renal, hepatocellular carcinoma, neuroblastoma, neuroblastoma, lymphoma, B-cell malignancies, lung, breast cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, advanced solid tumor, breast, hepatocellular, lung cancer, hepatoma, lung cancer, liver cancer, stomach cancer, central nervous system tumor, pediatric glioma, glioblastoma and brain tumor, glioblastoma, colon, pancreatic, prostate, gastric, liver, glioma, malignant35FH12901147.5ENO-00225 (38895-00225) pleural mesothelioma, ovarian, brain cancer, neuroblastoma, osteosarcoma, liver, melanoma, lung squamous cell carcinoma, glioblastoma, advanced cancer, nasopharyngeal, lung, pancreatic cancer, ovarian, cervical, pancreatic, lung, advanced solid tumors, lung, ovarian, nectin4-positive advanced malignant solid tumor, leukemia, prostate cancer, lung, chronic lymphocytic leukemia, melanoma, brain cancer, and / or renal cancer.

[0118] In some embodiments, a cancer can be selected from cancers of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves and other parts of the CNS, such as glioblastomas or medulloblastomas); head and / or neck cancer, breast cancers, cancers of the circulatory system (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular cancers, and tumor-associated vascular tissue); cancers of the blood and lymphatic system (e.g., Hodgkin’s disease, Non-Hodgkin’s disease lymphoma, Burkitt’s lymphoma, AIDS-related lymphomas, malignant immunoproliferative diseases, multiple myeloma, and malignant plasma cell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell population, leukemia of unspecified cell population, unspecified malignant neoplasms of lymphoid, haematopoietic and related tissues, such as diffuse large cell lymphoma, T-cell lymphoma or cutaneous T-cell lymphoma); cancers of the excretory system (e.g., kidney, renal pelvis, ureter, bladder, and other urinary organs); cancers of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal); cancers involving the liver and intrahepatic bile ducts, gall bladder, and other parts of the biliary tract, pancreas, and other digestive organs; cancers of the oral cavity (e.g., lip, tongue, gum, floor of mouth, palate, parotid gland, salivary glands, tonsil, oropharynx, nasopharynx, puriform sinus, hypopharynx, and other sites of the oral cavity); cancers of the reproductive system (e.g., vulva, vagina, Cervix uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); cancers of the respiratory tract (e.g., nasal cavity, middle ear, accessory sinuses, larynx, trachea, bronchus and lung, such as small cell lung cancer and non-small cell lung cancer); cancers of the skeletal system (e.g., bone and articular cartilage of limbs, bone articular cartilage and other sites); cancers of the skin (e.g., malignant melanoma of the skin, nonmelanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi’s sarcoma); and cancers involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneoum and peritoneum, eye and adnexa, thyroid, adrenal gland, and other endocrine glands and related36FH12901147.5ENO-00225 (38895-00225) structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasms of other sites. In some embodiments a cancer is a solid tumor and the antigen is selected from GPC3, HER2, PSMA, B7-H3, CLDN18.2, DLL3, CEA, MSLN, CD70, TROP, and HER3.

[0119] In some embodiments, the method includes mobilizing hematopoietic stem cells of the subject prior to administering the adenoviral vector system. In some embodiments, mobilizing hematopoietic stem cells of the subject includes administering to the subject granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), an agent that inhibits binding of endogenous very late antigen 4 (VLA4) with its endogenous ligands (a VLA4 antagonist), AMD3100 (plerixafor), SCF, S-CSF, a CXCR4 antagonist, a CXCR2 agonist, Gro-Beta (GRO-P), truncated GRO- (tGRO-P), and / or motixifortide. In some embodiments, G-CSF and plerixafor are administered to the subject. In some embodiments, tGRO-beta and plerixafor are administered to the subject. In some embodiments, motixifortide and plerixafor are administered to the subject. In some embodiments, tGRO-beta and motixifortide are administered to the subject. In some embodiments, G-CSF is administered to the subject daily. In some embodiments, G-CSF is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, G-CSF is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of the adenoviral vector system. In some embodiments, granulocyte colony stimulating factor is administered on the day of administration of the adenoviral vector system. In some embodiments, plerixafor is administered to the subject daily. In some embodiments, plerixafor is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. In some embodiments, plerixafor is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of the adenoviral vector system. In some embodiments, plerixafor is administered to the subject on the day of administration of the adenoviral vector system.

[0120] In some embodiments, the method includes administering to the subject an immunosuppression regimen including one or more immunosuppression agents. In some embodiments, the immunosuppression regimen includes a corticosteroid, optionally where the corticosteroid includes or is a glucocorticoid, optionally where the glucocorticoid includes or is dexamethasone.

[0121] In some embodiments, the corticosteroid is administered to the subject daily. In some embodiments, the corticosteroid is administered to the subject for at least about 1, 2, 3,37FH12901147.5ENO-00225 (38895-00225)4, 5 or more days. In some embodiments, the corticosteroid is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the adenoviral vector system. In some embodiments, the corticosteroid is administered to the subject on the day of administration of the adenoviral vector system.

[0122] In some embodiments, the immunosuppression regimen includes an inflammatory signal inhibitor, optionally where the inflammatory signal inhibitor includes or is an interleukin- 1 signal inhibitor, optionally where the interleukin- 1 signal inhibitor includes or is an interleukin- 1 receptor antagonist, optionally where the interleukin- 1 receptor antagonist includes or is anakinra. In some embodiments, the inflammatory signal inhibitor is administered to the subject daily. In some embodiments, the inflammatory signal inhibitor is administered to the subject for at least about 1, 2, 3, 4, 5 or more days. In some embodiments, the inflammatory signal inhibitor is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the adenoviral vector system. In some embodiments, the inflammatory signal inhibitor is administered to the subject on the day of administration of the adenoviral vector system.

[0123] In some embodiments, the immunosuppression regimen includes an interleukin-6 receptor antagonist, optionally where the interleukin-6 receptor antagonist includes or is tocilizumab.

[0124] In some embodiments, the interleukin-6 receptor antagonist is administered to the subject daily. In some embodiments, the interleukin-6 receptor antagonist is administered to the subject for at least about 1, 2, 3, 4, 5 or more days. In some embodiments, the interleukin- 6 receptor antagonist is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the adenoviral vector system. In some embodiments, the interleukin-6 receptor antagonist is administered to the subject on the day of administration of the adenoviral vector system.

[0125] In some embodiments, the method includes administering to the subject one or more selecting agents. In some embodiments, the enrichment marker is MGMTP14OKand the one or more selecting agents include: (a) temozolomide (TMZ); and / or (b) O6-benzylguanine (O6BG).

[0126] In some embodiments, TMZ is administered to the subject orally. In some embodiments, TMZ is administered to the subject about once every 28 days or about monthly. In some embodiments, 1, 2, 3, 4, or more doses of TMZ are administered to the subject. In some embodiments, a first dose of TMZ is administered to the subject at about 4,5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of the adenoviral vector system. In38FH12901147.5ENO-00225 (38895-00225) some embodiments, temozolomide is administered to the subject at a dose of about 300-600 mg / m2.

[0127] In some embodiments, O6BG is administered to the subject intravenously. In some embodiments, O6BG is administered to the subject as an intravenous bolus and / or intravenous infusion. In some embodiments, the intravenous bolus is administered prior to the intravenous infusion. In some embodiments, O6BG is administered to the subject about once every 28 days or about monthly. In some embodiments, 1, 2, 3, 4, or more doses of O6BG are administered to the subject. In some embodiments, a first dose of O6BG is administered to the subject at about 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of the adenoviral vector system. In some embodiments, O6BG is administered to the subject at a dose of about 80-280 mg / m2.In some embodiments, a dose of O6BG includes an intravenous bolus dose of about 70-170 mg / m2O6BG and / or an intravenous infusion dose of about 10- 110 mg / m2.In some embodiments, both TMZ and O6BG are administered to the subject.

[0128] In some embodiments, the enrichment marker is CD117 comprising one or more mutations selected from the group consisting of F316S, M318V, I319K, V323I, I334V, E360K, P363V, E366D, E376Q, and H378R, wherein one or more selecting agents selected from the group consisting of an anti-CD117 antibody, antigen binding fragment thereof, or an anti-CD117 antibody drug conjugate. In some embodiments, the anti-CD117 antibody, antigen binding fragment thereof or anti-CD117 antibody drug conjugate selectively binds to CD117 that does not have one or more mutations selected from the group consisting of F316S, M318V, I319K, V323I, I334V, E360K, P363V, E366D, E376Q, H378R, T59A, L124S, D72G, W82R, D121K Y125H, E128G, I163T, Q190R, E191G, V195A, V214A, S215P, V216A, S217P, E227G, E228G, Y350H, and Y408H. In some embodiments, an antiCD 117 antibody can be selected from the group consisting of a protein comprising the CDRs or VH and VL of barzolvolimab, the CRs or VH and VL of briquilimab, or CDRs or VH and VL of CD79D (see WO2023159136, which is incorporated herein by reference in its entirety and with respect to anti-CD117 sequences).

[0129] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a patient. In some embodiments, the subject is an adult. In some embodiments, the subject is a child. In some embodiments, the subject is male. In some embodiments, the subject is a female.

[0130] In at least one aspect, the disclosure provides an HD Ad integration vector genome, where the integration vector genome includes a first ubiquitous promoter operably linked with a nucleic acid sequence encoding a transposase and a second ubiquitous promoter39FH12901147.5ENO-00225 (38895-00225) operably linked with a nucleic acid sequence encoding a recombinase, where the first ubiquitous promoter is an EFla promoter or CAG promoter, and / or where the second ubiquitous promoter is an EFla promoter or CAG promoter.

[0131] In at least one aspect, the disclosure provides an HD Ad integration vector genome, where the integration vector genome includes a first ubiquitous promoter operably linked with a nucleic acid sequence encoding a transposase and a second ubiquitous promoter operably linked with a nucleic acid sequence encoding a recombinase, where the transposase is Sleeping Beauty lOOx (SBIOOx), and where the nucleic acid sequence encoding the transposase is codon optimized for expression in a mammalian cell or subject, optionally where the nucleic acid sequence encoding the transposase is codon optimized for expression in a human cell or subject.

[0132] In at least one aspect, the disclosure provides an HD Ad integration vector genome, where the integration vector genome includes a first ubiquitous promoter operably linked with a nucleic acid sequence encoding a transposase and a second ubiquitous promoter operably linked with a nucleic acid sequence encoding a recombinase, where the nucleic acid sequence encoding the transposase and the nucleic acid sequence encoding the recombinase are expressed in the same orientation.

[0133] In some embodiments, the HD Ad integration vector genome includes a transposase, where the transposase is Sleeping Beauty lOOx (SBIOOx), and / or a recombinase, where the recombinase is a FLP recombinase.

[0134] In some embodiments, the HD Ad integration vector genome includes a first ubiquitous promoter, where the first ubiquitous promoter is an EFla promoter and / or a transposase, where the transposase is Sleeping Beauty lOOx (SBIOOx), a second ubiquitous promoter, where the second ubiquitous promoter is CAG, and a recombinase, where the recombinase is a FLP recombinase.

[0135] In some embodiments, the HD Ad integration vector genome a nucleic acid sequence, where the nucleic acid sequence encoding the transposase and the nucleic acid sequence encoding the recombinase are expressed in the same orientation.

[0136] In some embodiments, the HD Ad integration vector genome includes a nucleic acid sequence, where the nucleic acid sequence encoding the transposase is codon optimized for expression in a mammalian cell or subject, optionally wherein the nucleic acid sequence encoding the transposase is codon optimized for expression in a human cell or subject.

[0137] In some embodiments, the HD Ad integration vector genome includes a nucleic acid, where the nucleic acid sequences encoding the recombinase and the transposase, and40FH12901147.5ENO-00225 (38895-00225) regulatory sequences operably linked thereto, are flanked by an Ad5 5’ ITR and an Ad5 3’ ITR positioned.

[0138] In some embodiments, the HD Ad integration vector genome includes a first ubiquitous promoter, where the first ubiquitous promoter is an EFla promoter.

[0139] In some embodiments, the HD Ad integration vector genome includes a second ubiquitious promoter, where the second ubiquitous promoter is a CAG promoter.

[0140] In some embodiments, the HD Ad integration vector includes a first ubiquitous promoter and a second ubiquitious promoter where the first ubiquitous promoter is an EFla promoter and the second ubiquitous promoter is a CAG promoter.

[0141] In some embodiments, the HD Ad integration vector genome includes a sequence having at least 80% identity with SEQ ID NO: 227.

[0142] In some embodiments, the HD Ad integration vector genome includes a first ubiquitous promoter, where the first ubiquitous promoter is a CAG promoter.

[0143] In some embodiments, the HD Ad integration vector genome includes a second ubiquitous promoter, where the second ubiquitous promoter is an EFla promoter.

[0144] In some embodiments, the HD Ad integration vector includes a first ubiquitous promoter and a second ubiquitious promoter where the first ubiquitous promoter is CAG promoter and the second ubiquitous promoter is a an EFl a promoter.

[0145] In some embodiments, the HD Ad integration vector genome includes a sequence having at least 80% identity with SEQ ID NO: 228.DEFINITIONS

[0146] Lineage-Specific Regulatory Sequence: As used herein, a “lineage-specific regulatory sequence” refers to a regulatory sequence that controls (e.g., causes, drives, regulates, promotes, inhibits, modulates, and / or contributes to) expression of an operably linked coding sequence in at least one hematopoietic cell population and / or in at least one HSC lineage, but does not cause expression, does not cause detectable expression, does not cause significant expression, and / or causes significantly less expression in at least one other or reference hematopoietic cell population and / or HSC lineage, such as HSCs, erythroid cells, or platelets. In various embodiments, a lineage-specific regulatory sequence refers to a regulatory sequence that causes at least 100-fold greater (e.g., at least 500-fold, at least 1000- fold, at least 10,000-fold, at least 20, 000- fold, at least 30,000-fold, at least 40, 000- fold, at least 50,000-fold, or at least 100,000-fold greater) expression of an operably linked coding sequence in at least one hematopoietic cell population and / or lineage as compared to at least41FH12901147.5ENO-00225 (38895-00225) one other hematopoietic cell population and / or lineage (e.g., HSCs, erythroid cells, or platelets). In various embodiments, a lineage-specific regulatory sequence is not a ubiquitous promoter. In various embodiments, a lineage-specific regulatory sequence does not cause expression in a plurality of non-HSC cell lineages and / or non-HSC cell populations.

[0147] HSC-Related miRNA : As used herein, a “HSC-Related miRNA” refers to an miRNA that decreases expression of an operably linked coding sequence in a HSC or an HSC lineage cell population, but does not decrease expression, does not cause a detectable decrease in expression, does not cause a significant decrease in expression, and / or causes a significantly smaller decrease in expression in at least one other or reference hematopoietic cell lineage population and / or HSC lineage, such as T cells, NK cells, and / or myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells). In various embodiments, aHSC-Related miRNA refers to a miRNA that decreases expression of an operably linked coding sequence by at least 5-fold more (e.g., at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold in HSC or an HSC lineage cell population, compared to a reference hematopoietic cell population and / or HSC lineage, such as T cells, NK cells, and / or myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

[0148] HSC Lineage. As used herein, “HSC Lineage” refers collectively to a series of stages, cell populations, and cell states that occur between commitment of an HSC or its progeny to a particular pathway of differentiation toward (e.g., culminating in) a particular mature or differentiated hematopoietic cell population, or toward a plurality of mature or differentiated hematopoietic cell populations that share a common progenitor cell population. The lineage extends to and includes the mature or differentiated hematopoietic cell population or types. Cells or cell populations within a pathway of differentiation can be referred to as cells or cell populations of the HSC lineage. In various embodiments, an HSC lineage optionally does not include the HSC itself.

[0149] Operably linked'. As used herein, “operably linked” refers to the association of at least a first element and a second element such that the component elements are in a relationship permitting them to function in their intended manner. For example, a nucleic acid sequence or amino acid sequence is operably linked with another sequence if it modifies the expression, structure, or activity of the linked sequence, e.g., in an intended manner. In many cases, two nucleic acid sequences are operably linked if a first nucleic acid sequence includes a regulatory sequence and the second nucleic acid sequence includes a coding sequence (e.g., a coding sequence for a nucleic acid agent and / or a polypeptide), and the first nucleic acid sequence controls (e.g., causes, drives, regulates, promotes, inhibits, and / or modulates)42FH12901147.5ENO-00225 (38895-00225) expression of the coding sequence. Thus, for example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if the regulatory sequence and coding sequence are associated in a manner that permits control of expression of the coding sequence by the regulatory sequence. In some embodiments, an "operably linked" regulatory sequence is directly or indirectly covalently associated with a coding sequence (e.g., in a single nucleic acid). In some embodiments, a regulatory sequence controls expression of a coding sequence in trans and inclusion of the regulatory sequence in the same nucleic acid as the coding sequence is not a requirement of operable linkage. In many cases, two amino acid sequences are operably linked if they are expressed as a single polypeptide.

[0150] Promoter’. As used herein, a “promoter” or “promoter sequence” can be a DNA regulatory region that directly or indirectly (e.g., through promoter-bound proteins or substances) controls, causes, contributes to, and / or participates in initiation and / or processivity of transcription of a coding sequence. For example, a promoter may, under suitable conditions, initiate transcription of a coding sequence upon binding of one or more transcription factors and / or regulatory moieties with the promoter. A promoter that participates in initiation of transcription of a coding sequence can be “operably linked” to the coding sequence. In certain instances, a promoter can be or include a DNA regulatory region that extends from a transcription initiation site (at its 3’ terminus), or a site upstream or downstream from a transcription initiate site, to a position upstream (5’ direction) thereof, such that the sequence so designated includes one or both of a minimum number of bases or elements necessary to control, cause, contribute to, and / or participate in initiation and / or processivity of transcription (e.g., to initiate a transcription event). A promoter can be, include, or be operably associated with or operably linked to, expression control sequences such as a promoter, an enhancer, a transcription factor binding site, an untranslated region (UTR), a repressor, or a combination thereof.

[0151] Regulatory Sequence’. As used herein in the context of expression of a nucleic acid coding sequence, a regulatory sequence is a nucleic acid sequence that controls (e.g., causes, drives, regulates, promotes, inhibits, and / or modulates) expression of a coding sequence. In some embodiments, a regulatory sequence can control one or more aspects of gene expression (e.g., cell-type-specific expression, inducible expression, etc.). In various embodiments, a regulatory sequence can be or include, for example and without limitation, a promoter, an enhancer, a transcription factor binding site, an untranslated region (UTR), a repressor, an intronic sequence, or other sequence, or a combination thereof. In some embodiments, a regulatory sequence can include a plurality of elements that together control43FH12901147.5ENO-00225 (38895-00225) expression of a coding sequence, e.g., a promoter and a UTR, a promoter and an enhancer, or a promoter, an enhancer, and a UTR. In various embodiments a regulatory sequence can be a sequence upstream of a minimal promoter, which can be referred to herein as a cis-regulatory element (CRE).

[0152] About'. As used herein, the term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0153] Affinity: As used herein, “affinity” refers to the strength of the sum total of non- covalent interactions between a particular binding agent (e.g., a viral vector), and / or a binding moiety thereof, with a binding target (e.g., a cell). Unless indicated otherwise, as used herein, “binding affinity” refers to a 1: 1 interaction between a binding agent and a binding target thereof e.g., a viral vector with a target cell of the viral vector). Those of skill in the art appreciate that a change in affinity can be described by comparison to a reference (e.g., increased or decreased relative to a reference), or can be described numerically. Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD) and / or equilibrium association constant (KA). KD is the quotient of koff / kon, whereas KA is the quotient of kon / koff, where konrefers to the association rate constant of, e.g., viral vector with target cell, and koff refers to the dissociation of, e.g., viral vector from target cell. The konand koff can be determined by techniques known to those of skill in the art.

[0154] Administering'. As used herein, the term “administering” or “administration” typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be parenteral. In some embodiments, administration may be intravenous. In some embodiments, administration may be oral. In some embodiments, administration may be via injection. In some embodiments, administration may be systemic. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a44FH12901147.5ENO-00225 (38895-00225) plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., infusion, perfusion) for at least a selected period of time.

[0155] Agent: The term “agent” as used herein may refer to a compound or entity of any chemical class including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations thereof. As will be clear from context, in some embodiments, an agent can be or comprise a cell or organism, or a fraction, extract, or component thereof. In some embodiments, an agent is or comprises a natural product in that it is found in and / or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. Some particular embodiments of agents that may be utilized in accordance with the present disclosure include small molecules, antibodies, antibody fragments, aptamers, siRNAs, shRNAs, miRNAs, DNA / RNA hybrids, antisense oligonucleotides, ribozymes, peptides, peptide mimetics, small molecules, etc. In some embodiments, an agent is or comprises a polymer. In some embodiments, an agent is not a polymer and / or is substantially free of any polymer. In some embodiments, an agent contains at least one polymeric moiety. In some embodiments, an agent lacks or is substantially free of any polymeric moiety.

[0156] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In some embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically- engineered animal and / or a clone.

[0157] Antibody. As used herein, the term “antibody” refers to a polypeptide that includes one or more immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen (e.g., a heavy chain variable domain, a light chain variable domain, and / or one or more CDRs). As used herein, the term "antibody" includes an immunoglobulin molecule, or fragment thereof, that binds specifically to an epitope (e.g., of an antigen). Thus,45FH12901147.5ENO-00225 (38895-00225) the term antibody includes, without limitation, human antibodies, non-human antibodies, synthetic and / or engineered antibodies, fragments thereof, and agents including the same. Antibodies can be naturally occurring immunoglobulins (e.g., generated by an organism reacting to an antigen). Synthetic, non-naturally occurring, or engineered antibodies can be produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies known to those of skill in the art.

[0158] As is well known in the art, typical human immunoglobulins are approximately 150 kD tetrameric agents that include two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other to form a structure commonly referred to as a “Y-shaped” structure. Typically, each heavy chain includes a heavy chain variable domain (VH) and a heavy chain constant domain (CH). The heavy chain constant domain includes three CH domains: CHI, CH2 and CH3. A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the immunoglobulin. Each light chain includes a light chain variable domain (VL) and a light chain constant domain (CL), separated from one another by another “switch.” Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). In each VH and VL, the three CDRs and four FRs are arranged from aminoterminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of a heavy and / or a light chain are typically understood to provide a binding moiety that can interact with an antigen. Constant domains can mediate binding of an antibody to various immune system cells e.g., effector cells and / or cells that mediate cytotoxicity), receptors, and elements of the complement system. Heavy and light chains are linked to one another by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. When natural immunoglobulins fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure.

[0159] In some embodiments, an antibody is a polyclonal, monoclonal, monospecific, or multispecific antibody (e.g., a bispecific antibody). In some embodiments, an antibody includes at least one light chain monomer or dimer, at least one heavy chain monomer or46FH12901147.5ENO-00225 (38895-00225) dimer, at least one heavy chain-light chain dimer, or a tetramer that includes two heavy chain monomers and two light chain monomers. Moreover, the term “antibody” can include (unless otherwise stated or clear from context) any art-known constructs or formats utilizing antibody structural and / or functional features including without limitation intrabodies, domain antibodies, antibody mimetics, Zybodies®, Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, isolated CDRs or sets thereof, single chain antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFv), polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, camelized antibodies, masked antibodies (e.g., Probodies®), affybodies, anti-idiotypic (anti- Id) antibodies (including, e.g., anti-anti-Id antibodies), Small Modular ImmunoPharmaceuticals (“SMIPsTM”), single chain or Tandem diabodies (TandAb®), VHHs, Anticalins®, Nanobodies® minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Transbodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®s, CARs, engineered TCRs, and antigen-binding fragments of any of the above.

[0160] In various embodiments, an antibody includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR) or variable domain. In some embodiments, an antibody can be a covalently modified (“conjugated”) antibody (e.g., an antibody that includes a polypeptide including one or more immunoglobulin sequence elements sufficient to confer specific binding to a particular antigen, where the polypeptide is covalently linked with one or more of a therapeutic agent, a detectable moiety, another polypeptide, a glycan, or a polyethylene glycol molecule). In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art.

[0161] An antibody including a heavy chain constant domain can be, without limitation, an antibody of any known class, including but not limited to, IgA, secretory IgA, IgG, IgE and IgM, based on heavy chain constant domain amino acid sequence (e.g., alpha (a), delta (5), epsilon (a), gamma (y) and mu (p)). IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. “Isotype” refers to the Ab class or subclass e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. As used herein, a “light chain” can be of a distinct type, e.g., kappa (K) or lambda (I), based on the amino acid sequence of the light chain constant domain. In some embodiments, an antibody has constant region sequences that are characteristic of mouse,47FH12901147.5ENO-00225 (38895-00225) rabbit, primate, or human immunoglobulins. Naturally-produced immunoglobulins are glycosylated, typically on the CH2 domain. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation.

[0162] Antibody fragment : As used herein, an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof. An antibody fragment can be produced by any means. For example, in some embodiments, an antibody fragment can be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment can be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment can be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids.

[0163] Binding: As used herein, the term “binding” typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in one or more of a variety of contexts - for example, in some embodiments where interacting entities or moieties are studied in isolation, and / or in some embodiments where interacting entities or moieties are studied in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). Binding between two entities may be considered “specific” if, under the conditions assessed, the relevant entities are more likely to associate with one another than with other available binding partners. Those skilled in the art will be aware of circumstances where a particular degree of preference for one potential binding partner over another is required for binding to be deemed sufficiently “specific” for a particular purpose or situation.48FH12901147.5ENO-00225 (38895-00225)

[0164] Cancer: As used herein, the term “cancer” refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they display an abnormally elevated proliferation rate and / or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a cancer can include one or more tumors. In some embodiments, a cancer can be or include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a cancer can be or include a solid tumor. In some embodiments, a cancer can be or include a hematologic tumor.

[0165] CD46-binding adenoviral capsid'. As used herein, the term “CD46-binding adenoviral capsid” refers to a capsid that includes a fiber or a portion thereof, e.g., a fiber knob that binds CD46. In some embodiments, a fiber or a portion thereof, e.g., a fiber knob that binds CD46 is derived from a Group B adenovirus, e.g., from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55. In some embodiments, a CD46-binding adenoviral capsid includes a fiber knob that binds CD46, e.g., a fiber knob derived from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55. In some embodiments, a CD46-binding adenoviral capsid includes a hexon and / or penton derived from Ad5, 6, or 35. In some embodiments, an adenoviral vector includes a chimeric Ad5 / 3, Ad5 / 7, Ad5 / l l, Ad5 / 14, Ad5 / 16, Ad5 / 21, Ad5 / 34, Ad5 / 35, Ad5 / 50, Ad5 / 55 or Ad6 / 35 capsid, e.g., with a hexon and / or penton derived from Ad5 or Ad6 and a fiber, fiber tail and / or fiber knob derived from Ad3, 7, 11, 14, 16, 21, 34, 35, 50, or 55.

[0166] Chimeric antigen receptor’. As used herein, “Chimeric antigen receptor” or “CAR” refers to an engineered protein that includes (i) an extracellular domain that includes a moiety that binds a target antigen; (ii) a transmembrane domain; and (iii) an intracellular signaling domain that sends activating signals when the CAR is stimulated by binding of the extracellular binding moiety with a target antigen. A T cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR T cell. Thus, for example, when certain CARs are expressed by a T cell, binding of the CAR extracellular binding moiety with a target antigen can activate the T cell. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors.

[0167] Expression’. As used herein, “expression” refers individually and / or cumulatively to one or more biological process that result in production from a nucleic acid sequence of an encoded agent (i.e., an expression product), such as an RNA and / or a polypeptide. Expression specifically includes either or both of transcription and translation. A nucleic acid or cell that produces the encoded agent can be said to express the encoded agent.49FH12901147.5ENO-00225 (38895-00225)

[0168] Flank-. As used herein, a first element (e.g., a nucleic acid sequence or amino acid sequence) present in a contiguous sequence with a second element and a third element is “flanked” by the second element and third element if it is positioned in the contiguous sequence between the second element and the third element. Accordingly, in such arrangement, the second element and third element can be referred to as “flanking” the first element, and the first element can be referred to as “flanked” by the second element and third element. Flanking elements can be immediately adjacent to a flanked element or separated from the flanked element by one or more relevant units. In various examples, the contiguous sequence is a nucleic acid or amino acid sequence.

[0169] Heterologous: As used herein, a first nucleic acid sequence is “heterologous” to a second nucleic acid sequence if the first nucleic acid sequence is not operatively linked with the second nucleic acid sequence in nature. By extension, a polypeptide is “heterologous” to an expression control sequence if it is encoded by nucleic acid sequence heterologous the promoter.

[0170] Identity. As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., oligonucleotides, DNA, RNA, etc.) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When 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 the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity50FH12901147.5ENO-00225 (38895-00225) between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0171] Immunosuppressive agent: As used herein, the term “immunosuppressive agent,” in its broadest sense, refers to an agent, e.g., a therapeutic agent that suppresses or reduces the activation, activity, or efficacy of the immune system of a subject. Exemplary immunosuppressive agents include, but are not limited to, abatacept, abrocitinib, adalimumab, alemtuzumab, anakinra, atacicept, azathioprine, baricitinib, basiliximab, belatacept, belimumab, bortezomib, certolizumab, crovalimab, cyclophosphamide, cyclosporine, daclizumab, dexamethasone, eculizumab, efalizumab, epratuzumab, etanercept, everolimus, fingolimod, fluorouracil, golimumab, hydroxychloroquine, imlifidase, infliximab, leflunomide, mercaptopurine, methotrexate, methylprednisolone, mycophenolate mofetil, mycophenolate sodium, ocrelizumab, ofatumumab, pimecrolimus, prednisone, prednisolone, ridaforolimus, rilonacept, rituximab, ruxolitinib, secukinumab, sirolimus, tacrolimus, temsirolimus, tocilizumab, tofacitinib, upadacitinib, and veltuzumab. Additional immunosuppressive agents are known in the art.

[0172] Immunosuppressive regimen -. As used herein, the term “immunosuppressive regimen,” in its broadest sense, refers to a treatment regimen comprising one or more immunosuppressive agents.

[0173] Improve, increase, inhibit, decrease or reduce". As used herein, the terms “improve”, “increase”, “inhibit”, “decrease” and “reduce”, and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference.

[0174] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (e.g., animal, plant and / or microbe).

[0175] In vivo : As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant and / or microbe).

[0176] Level: As used herein with respect to a molecule such as a nucleic acid or polypeptide (e.g., a glycoform), “level” is used to refer to a measure indicative of an amount, concentration, ratio, or activity of the molecule, e.g., in a particular context such as a tissue,51FH12901147.5ENO-00225 (38895-00225) sample, organism, or a context representative thereof. An amount can be, for example, a mass or number of molecules. A concentration can be an amount relative to a context value, e.g., per a unit of mass or volume. A ratio can be a relationship between two values, such as an experimental value and a reference control value. Activity can be a measure of a function associated with a molecule, and can in various instances be measured relative to a context value, e.g., per a unit of mass or volume. Those of skill in the art will appreciate that the metric by which a level is expressed can vary depending, e.g., on the assay and purpose. Those of skill in the art will further appreciate that metrics such as amount, concentration, ratio, and activity are often interrelated and / or qualitatively or quantitatively informative of each other.

[0177] Linker: As used herein, “linker” is used to refer to that portion of a multi-element agent that connects different elements to one another. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide including a linker element has an overall structure of the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domains associated with one another by the linker. In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1 121-1123).

[0178] Nucleic acid'. The term “nucleic acid”, as used herein, includes any nucleotides and polymers thereof. The term “polynucleotide”, as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) or a combination thereof. These terms refer to the primary structure of the molecules and, thus, include double- and single-stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA comprising modified nucleotides and / or modified polynucleotides, such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligoribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA52FH12901147.5ENO-00225 (38895-00225) derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified internucleotidic linkages. The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified internucleotidic linkages. Examples include, and are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Unless otherwise specified, the prefix polyrefers to a nucleic acid containing 2 to about 10,000 nucleotide monomer units and wherein the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0179] One or more: As used herein, in some embodiments, “one or more” is 1-200, 1- 150, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. In some embodiments, “one or more” is one. In some embodiments, “one or more” is two. In some embodiments, “one or more” is three. In some embodiments, “one or more” is four. In some embodiments, “one or more” is five. In some embodiments, “one or more” is six. In some embodiments, “one or more” is seven. In some embodiments, “one or more” is eight. In some embodiments, “one or more” is nine. In some embodiments, “one or more” is ten. In some embodiments, “one or more” is at least one. In some embodiments, “one or more” is at least two. In some embodiments, “one or more” is at least three. In some embodiments, “one or more” is at least four. In some embodiments, “one or more” is at least five. In some embodiments, “one or more” is at least six. In some embodiments, “one or more” is at least seven. In some embodiments, “one or more” is at least eight. In some embodiments, “one or more” is at least nine. In some embodiments, “one or more” is at least ten.

[0180] In some embodiments, a name that refers to a nucleic acid (e.g., a gene or coding sequence) can be used to designate a class of nucleic acids and / or sequences that encode a relevant expression product and / or share a relevant level of sequence identity. For such classes, the present specification provides, and / or those skilled in the art will be aware of, exemplary nucleic acids within the class. In some embodiments, a unifying characteristic of a class can be defined in relation to a reference nucleic acid (e.g., by percent identity with a reference nucleic acid). In some embodiments, a nucleic acid has at least 80%, 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 a reference nucleic acid sequence or designated53FH12901147.5ENO-00225 (38895-00225) portion thereof (e.g., coding sequence(s) thereof). In some embodiments, a nucleic acid differs from a reference nucleic acid sequence or designated portion thereof (e.g., coding sequence(s) thereof) by no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 nucleic acid sequence differences, where each nucleic acid sequence difference can be independently selected from an insertion, deletion, or substitution (e.g., a substitution that changes an encoded amino acid or that does not change an encoded amino acid) of a nucleic acid residue.

[0181] Patient'. As used herein, the term “patient” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more conditions, diseases, or disorders. In some embodiments, a patient displays one or more symptoms of a condition, disease, or disorder. In some embodiments, a patient has been diagnosed with one or more conditions, diseases, or disorders. In some embodiments, the patient is receiving or has received certain therapy to diagnose and / or to treat a condition, disease, or disorder.

[0182] Pharmaceutically acceptable'. As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0183] Pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive54FH12901147.5ENO-00225 (38895-00225) 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; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or poly anhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0184] Pharmaceutical composition-. As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, an active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0185] Polypeptide: The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class,55FH12901147.5ENO-00225 (38895-00225) is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0186] Protein : As used herein, the term “protein” refers to a polypeptide (z.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins can include moieties other than amino acids e.g., can be glycoproteins, proteoglycans, etc.) and / or can be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides can contain L-amino acids, D-amino acids, or both and can contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can include natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0187] Reference: As used herein, “reference” refers to a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or population thereof, or a measure or characteristic representative thereof, is compared with a reference agent, sample, sequence, subject, animal, or individual, or population thereof, or measure or characteristic representative thereof. In some embodiments, a reference is a measured value. In some embodiments, a reference is an established standard or expected value. In some embodiments, a reference is a historical reference. A reference can be quantitative of qualitative. Typically, as would be understood by those of skill in the art, a reference and the value to which it is compared represent assessments under comparable conditions. Those of skill in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison. In some56FH12901147.5ENO-00225 (38895-00225) embodiments, an appropriate reference may be an agent, sample, sequence, subject, animal, or individual, or population thereof, under conditions those of skill in the art will recognize as comparable, e.g., for the purpose of assessing one or more particular variables (e.g., presence or absence of an agent or condition), or a measure or characteristic representative thereof.

[0188] Solid Tumor. As used herein, the term “solid tumor” refers to an abnormal mass of tissue including cancer cells. In various embodiments, a solid tumor is or includes an abnormal mass of tissue that does not contain cysts or liquid areas. In some embodiments, a solid tumor can be benign; in some embodiments, a solid tumor can be malignant. Examples of solid tumors include, but are not limited to, carcinomas, lymphomas, and sarcomas. In some embodiments, solid tumors can be or include adrenal, bile duct, bladder, bone, brain, breast, cervix, colon, endometrium, esophagum, eye, gall bladder, gastrointestinal tract, head & neck, kidney, larynx, liver, lung, nasal cavity, nasopharynx, oral cavity, ovary, pancreas, penis, pituitary, prostate, retina, salivary gland, skin, small intestine, stomach, testis, thymus, thyroid, uterine, vaginal, and / or vulval tumors.

[0189] Subject-. As used herein, the term “subject” or “test subject” refers to any organism to which a compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject is a human. In some embodiments, a subject may be suffering from and / or susceptible to a disease, disorder and / or condition.

[0190] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0191] Susceptible to : An individual who is “susceptible to” a disease, disorder and / or condition is one who has a higher risk of developing the disease, disorder and / or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition is predisposed to have that disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not have been diagnosed with the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may exhibit symptoms of the disease, disorder and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition may not exhibit symptoms of the disease, disorder and / or condition. In some embodiments,57FH12901147.5ENO-00225 (38895-00225) an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0192] Therapeutic agent: As used herein, the term “therapeutic agent” refers to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms or a human population. In some embodiments, an appropriate population can be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used for treatment of a disease, disorder, or condition. In some embodiments, a therapeutic agent is an agent that has been or is required 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 for which a medical prescription is required for administration to humans.

[0193] Therapeutically effective amount'. As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0194] Treat-. As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does58FH12901147.5ENO-00225 (38895-00225) not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0195] Tumor: As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor can include cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments, a tumor can be a disperse tumor or a liquid tumor. In some embodiments, a tumor can be a solid tumor.

[0196] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into a viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In some embodiments, the term “vector” refers to an agent capable of transporting a nucleic acid, wherein the agent comprises the nucleic acid. In some embodiments, a vector comprises or is an agent capable of transporting a nucleic acid.

[0197] Wild-type: As used herein, the term “wild-type” has its art-understood meaning that refers to an entity having a structure and / or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).BRIEF DESCRIPTION OF THE DRAWINGS

[0198] FIG. 1 is a schematic timeline of in vivo helper-dependent adenovirus (HD Adj- mediated hematopoietic stem cell (HSC) transduction and enrichment in mobilized human CD46 transgenic (hCD46tg) mice.59FH12901147.5ENO-00225 (38895-00225)

[0199] FIG. 2 is a graph showing frequency of HDAd nucleic acid construct expression (expression product expressed on the cell surface) in peripheral blood CD45+ immune cells on Day 5 post-HDAd administration. Data represent mean + / - SEM. Mock n = 10, GFP n = 16, HER2 CAR n = 8.

[0200] FIGs. 3A-3C are graphs showing frequency of HDAd nucleic acid construct expression in HSC-derived peripheral blood CD45+ immune cells over time in all treatment groups (FIG. 3A), GFP-expressing mice (FIG. 3B), and HER2 CAR-expressing mice (FIG. 3C). Mice received O6-benzylguanine / Carmustine (O6BG / BCNU) chemotherapy in Weeks4, 6, 8, and 10 post-HDAd administration. Data represent mean + / - SEM. Mock n = GFP n =5, HER2 CAR n = 6.

[0201] FIG. 4 is a graph showing frequency of peripheral blood immune cell populations at Week 18 post-HDAd administration. Data represent mean + / - SEM. Mock n = 10; GFP n = 9; HER2 CAR n = 5.

[0202] FIG. 5 is a graph showing body weights of on- study mice surviving to study endpoint. Data represent mean + / - SEM. Mock n = 10; GFP n = 9; HER2 CAR n = 6. GFP- expressing and HER2 CAR-expressing mice experience a suppression of weight resulting from O6BG / BCNU administration.

[0203] FIG. 6 is a graph showing complete blood counts of on-study mice surviving to study endpoint. GFP and HER2 CAR HD Ad-treated mice experience a drop in cbc during O6BG / BCNU chemotherapy administered at Weeks 4, 6, 8, and 10. Data represent mean + / - SEM. Mock n = 10; GFP n = 9; HER2 CAR n = 6.

[0204] FIGs. 7A-7C are graphs showing frequency of HDAd nucleic acid construct expression in peripheral blood immune cells (FIG. 7A), spleen cells (FIG.7B), and bone marrow immune cells (FIG. 7C) at Week 20 post HDAd administration. FIG. 7A data represent mean + / - SEM. Mock n = 10, GFP n = 6; HER2 CAR n = 8. FIG.7B data represent mean + / - SEM. Mock n = 10, GFP n = 6; HER2 CAR n = 8. FIG. 7C data represent mean + / - SEM. Mock n = 10 , GFP n = 6; HER2 CAR n = 8.

[0205] FIG. 8 is a graph showing frequency of HDAd nucleic acid construct expression peripheral blood immune cells on Day 3 after HDAd administration in mobilized hCD46tg mice. Data represent Mean + / - SEM. Mock n = 5; GFP n = 9; HER2 CAR n = 10.

[0206] FIG. 9 is a graph showing HER2 CD3z CAR-M mediate dose-dependent target cell killing ex vivo. Negative specific killing values in control groups were plotted as zero for data presentation. Data represent Mean + / - SEM of 6 to 9 technical replicates.FH12901147.5ENO-00225 (38895-00225)

[0207] FIG. 10 is a graph showing HER2 CD3z CAR-T mediate dose-dependent target cell killing ex vivo. Data represent Mean + / - SEM of 3 to 9 technical replicates.

[0208] FIG. 11 is a schematic timeline showing secondary transplant following in vivo HSC transduction.

[0209] FIG. 12 is a graph showing frequency of hCD46+ peripheral blood immune cells in wild-type C57BL / 6 mice at Week 18 after transplant with hCD46+ HSPCs originating from mock or HD Ad-treated mice. Data represent mean + / - SEM. Mock n = 13; GFP n = 5; HER2 CAR n = 16.

[0210] FIG. 13 is a graph showing frequency of gene marking in peripheral blood immune cells at Week 18 post-transplant. Data represent mean + / - SEM. Mock n = 13; GFP n = 5; HER2 CAR n = 16.

[0211] FIGs. 14A-14C are graphs showing EO771 / huHER2-Fluc tumor burden expressed as total flux (photos per second) measured by bioluminescent imaging (BLI) in all treatment groups (FIG. 14), GFP-expressing mice (FIG. 14B), and HER2 CAR-expressing mice (FIG. 14C). Data represent Mean + / - SD. GFP n = 7, HER2 CAR n = 3.

[0212] FIG. 15 is a graph showing TNF-a quantified in the serum of tumor-bearing mice on Day 3 post-tumor implant. Data represent Mean + / - SEM,* p value = 0.0244 by One-way ANOVA. Mock n = 5; GFP n - 7, HER2 CAR n = 3.

[0213] FIG. 16 is a graph showing gene marked cells. EO771 / huHER2 tumors were harvested on Day 5 post-implant to assess immune infiltrates by flow cytometry. The frequency of infiltrating gene marked cells was equivalent in GFP and HER2 CAR gene marked mice. N = 5 per group. Data represent Mean + / - SEM

[0214] FIGs. 17A-17B are graphs showing monocyte activation. Mock, GFP, and HER2 CAR gene marked mice were implanted with EO771 / huHER2-Fluc tumors and harvested on Day 5 post-implant. Tumors were digested to single cell suspension and assayed by flow cytometry to assess tumor-infiltrating immune cells. HER2 CAR-expressing mice show an increase in the frequency of MHC Class II-expressing myeloid cells when measured by Ly6C, indicative of myeloid cell activation (FIG. 17A). Further gating on gene marked (GFP+ or CAR+) myeloid cells show a similar increase in MHC Class Il-positive cells when measured by Ey6C (FIG. 17B). FIG. 17A data represent Mean + / - SEM. * p-value = 0.0128, *** p-value = 0.0005 by One-way ANOVA for multiple comparisons N = 5 per group. FIG. 17B data represent Mean + / - SEM. ** p-value = 0.0054 by Paired t-test N = 5 per group.

[0215] FIGs. 18A-18D are graphs showing MC38 / huHER2-Fluc tumor burden expressed as total flux (photos per second) measured by bioluminescent imaging (BLI) in all treatment61FH12901147.5ENO-00225 (38895-00225) groups (FIG. 18A), mock mice (FIG. 18B), GFP-expressing mice (FIG. 18C), and HER2 CAR-expressing mice (FIG. 18D). Data represent Mean + / - SD. * p = 0.0248 by two-way ANOVA with Tukey’s test for multiple comparisons. Mock n = 5, GFP n = 4, HER2 CAR n = 4.

[0216] FIG. 19 is a schematic timeline of ex vivo hCD46tg HSPC transduction and transplant to irradiated wild-type (WT) recipients.

[0217] FIG. 20 is a graph showing white blood cell count (WBC) over time following hCD46tg HSPC transplant. O6BG / TMZ chemotherapy was administered at Week 5 and 9. Data represent mean + / - SEM. GFP n = 8, CAG-HER2 CAR n = 7, CD1 lb-HER2 CAR n = 7.

[0218] FIG. 21 is a graph showing frequency of GFP+ or HER2 CAR+ immune cells in peripheral blood at indicated time points after hCD46tg HSPC transplant. Data represent mean + / - SEM. GFP n = 8, CAG-HER2 CAR n = 7, CDllb-HER2 CAR n = 7.

[0219] FIG. 22 is a graph showing frequency of GFP+ or HER2 CAR+ specified peripheral blood immune cell lineages at Week 12 after hCD46tg HSPC transplant. Data represent mean + / - SEM. GFP n = 8, CAG-HER2 CAR n = 7, CD1 lb-HER2 CAR n = 7.

[0220] FIG. 23 is a graph showing per-cell surface level of HER2 CAR expression measured by geometric mean fluorescence intensity (gMFI). Data represent mean + / - SD. CAG-HER2 CAR n = 7, CDllb-HER2 CAR n = 7.

[0221] FIG. 24 is a graph showing MC38 / huHER2-Fluc tumor burden measured by bioluminescence imaging (BLI) and expressed as Total Flux (photons / second). GFP vs CAG *** p value = 0.0002; GFP vs CDl lb *** p value = 0.0007 by Mixed-effects analysis with multiple comparisons, ns on Day 3. Data represent mean + / - SD. CAG-HER2 CAR n = 7, CDl lb-HER2 CAR n = 7

[0222] FIGs. 25A-25C is a graph showing frequency of GFP+ or HER2 CAR+ specified peripheral blood immune cell lineages (FIG. 25A), spleen immune cell lineages (FIG.25B), and bone marrow immune cell lineages (FIG. 25C) at Week 13 after hCD46tg HSPC transplant. FIG. 25A data represent mean + / - SEM. GFP n = 5, CAG-HER2 CAR n = 6, CD1 lb-HER2 CAR n = 7. FIG. 25B data represent mean + / - SEM. GFP n = 5, CAG-HER2 CAR n = 6, CD1 lb-HER2 CAR n = 7. FIG. 25C data represent mean + / - SEM. GFP n = 5, CAG-HER2 CAR n = 6, CDllb-HER2 CAR n = 7.

[0223] FIGs. 26A-26B is a graph showing frequency of HER2 CAR and MGMT expression in bone marrow LSK HSCs (FIG. 26A) and LT-HSCs (FIG. 26B) at Week 13 after hCD46tg HSPC transplant. FIG. 26A data represent mean + / - SEM. GFP n = 5, CAG-62FH12901147.5ENO-00225 (38895-00225)HER2 CAR n = 6, CD1 lb-HER2 CAR n = 7. FIG. 26B data represent mean + / - SEM. GFP n = 5, CAG-HER2 CAR n = 6, CD1 lb-HER2 CAR n = 7.

[0224] FIG. 27 is a graph showing HER2 CD3z driven by the CD1 lb promoter induces on-target CAR-M cytotoxicity ex vivo. Negative specific killing values in control groups were plotted as zero for data presentation. Data represent Mean + / - SEM of 6 to 9 technical replicates.

[0225] FIG. 28 provides a graph showing percentages of transduced primary T-cells expressing reporters from the lentivirus screening construct. mVenus expressing populations are gated on cells that are expressing mKate.

[0226] FIGs 29A-29B provide histograms of mVenus (FIG. 29 A) and mKate (FIG. 29B) expression for primary T-cells transduced with CRE screening construct. Gated on live, mKate-expressing, single cells. The x-axis denotes gMFI for mVenus and mKate, respectively, and the y-axis denotes number of cells. Dotted line corresponds to median histogram value for the “untreated” sample.

[0227] FIG. 30 provides a graph showing normalized expression of CRE constructs in primary T and Nalm6 cells. The gMFI of mVenus was divided by the gMFI of mKate for each construct. The “no treatment” control was set to 1, and all samples with expression greater than 1 have increased expression in that cell population.

[0228] FIG. 31 provides a schematic of hematopoietic cell differentiation that includes hematopoietic stem cells, progenitor cells, and terminally differentiated cells.

[0229] FIG. 32 provides a schematic showing an HD Ad vector system including a payload vector genome and an integration vector genome. The payload vector genome includes a nucleic acid construct in which a first lineage-specific promoter operably linked with a first sequence of interest (SOI), a second lineage-specific promoter operably linked with a second sequence of interest (SOI), and a ubiquitous promoter operably linked with a nucleic acid sequence encoding an enrichment marker. The nucleic acid construct is flanked, in turn, by pT4 transposase IRs, FRT recombinase sites, and Ad5 ITRs. The integration vector includes a first ubiquitous promoter operably linked with a nucleic acid sequence encoding a sleeping beauty transposase, and a second ubiquitous promoter operably linked with a nucleic acid sequence encoding an FLP recombinase. The integration construct is flanked by Ad5 ITRs.

[0230] FIG. 33 are graphs showing Sleeping beauty protein expression at D2 in human T cells transduced with PGK-Sleeping Beauty; EFla-FLP, EFla-Sleeping Beauty ;CAG-FLP63FH12901147.5ENO-00225 (38895-00225) and CAG-Sleeping Beauty; EFla-FLP. Data are representative of three independent experiments.

[0231] FIG. 34 is a graph showing gene marking frequency at D 19 in human T cells transduced with PGK-Sleeping Beauty; EFla-FLP, EFla-Sleeping Beauty;CAG-FLP and CAG-Sleeping Beauty; EFla-FLP paring with the indicated payloads. Data are representative of two independent experiments.

[0232] FIG. 35 is a timeline of in vivo HD Ad-mediated PBMC transduction to assess integration in human T cells in IL-15tg NSG mice.

[0233] FIG. 36 is a graph showing CD 19 CAR expression in human T cells over time in the peripheral blood in IL15tg NSG mice. PGK-Sleeping Beauty; EFla-FLP n =5, EFla- Sleeping Beauty;CAG-FLP n =6. CAG-Sleeping Beauty; EFla-FLP n = 6.

[0234] FIG. 37 is a timeline of HD Ad mediated HSPCs transduction and neutrophil differentiation assay to assess integration in mouse HSPCs in vitro. Neutrophil differentiation is initiated at Day 3.

[0235] FIG. 38 is a graph showing GFP+ cells frequency in live cells at day 3 and day 10 post transduction.

[0236] FIG. 39 is a timeline of in vivo HD Ad-mediated HSC transduction and single cell CFU assay to assess integration in HSPCs.

[0237] FIG. 40 is a graph showing frequency of GFP + cells in LSK derived from bone marrow of CD46 mice at day 7 after injection with different DPs. Data represent mean + / - SEM. DPs = 5n, Mock =3n

[0238] FIG. 41 is a graph showing integration in in vivo transduced mouse HSPCs measured by frequency of GFP+ CFUs in total number of CFUs.

[0239] FIG. 42 is a graph showing integration in human CD34+ cells measured by frequency of GFP+ CFUs in total number of CFUs. EFla-Sleeping Beauty ;CAG-FLP 3n, PGK-Sleeping Beauty; EFla-FLP 3n, Payload only = 2n, UT (untreated) 2n

[0240] FIG. 43 is a timeline of in vivo HD Ad-mediated HSC transduction and enrichment in mobilized human CD46 transgenic (hCD46tg) mice.

[0241] FIG. 44 is a graph showing frequency of Hdad payload expression in CD45+ cells in peripheral blood (PB). PB was collected 8 weeks after virus and 4 weeks after 06BG / TMZ administration. UT (untreated) = 5n, PGK-Sleeping Beauty; EFla-FLP = 5n, EFla-Sleeping Beauty;CAG-FLP = 5n, PGK-Sleeping Beauty; EFla-FLP + O6BG + TMZ = 12n, EFla- Sleeping Beauty;CAG-FLP + O6BG + TMZ = 22n64FH12901147.5ENO-00225 (38895-00225)

[0242] FIG. 45 is a graph showing frequency of Hdad payload expression in peripheral blood immune cells. PB was collected 8 weeks after virus and 4 weeks after O6BG / TMZ administration PGK-Sleeping Beauty; EFla-FLP + O6BG + TMZ=12n, EFla-Sleeping Beauty;CAG-FLP +O6BG + TMZ = 22n

[0243] FIGs. 46A-46D. VLPs efficiently deliver Her2-CAR to immune cells in vivo. A) is a schematic of a vector encoding an anti-HER2 CAR to mediate tumor targeting and an MGMTP14OKcassette to enable chemotherapy-based enrichment of integrated cells. B) is an experimental timeline of in vivo VLP dosing in HSC-mobilized human CD46 transgenic (hCD46tg) immune competent mice. C) is a graph showing the frequency of HER2 CAR- expressing myeloid, NK and CD8+T cells in the blood on Day 3 post- VLP dosing. D) is a graph showing the absolute number of HER2 CAR-expressing myeloid, NK and CD8+T cells in the blood on Day 3 post- VLP dosing.

[0244] FIGs. 47A-47D. In vivo CAR Engineered HSCs give rise to multi-lineage CAR- expressing immune cells. A) is an experimental timeline to assess in vivo VLP HSC transduction, enrichment, and CAR expression in mature immune cell lineages. B) is a graph showing HER2 CAR expression in peripheral blood CD45+immune cells over time indicating long-term, stable integration in HSCs and derived lineages. C) is a graph showing CD45+cell gene marking in blood and lymphoid tissues is similar at Week 20 post- VLP dose. D) is a graph showing HER2 CAR is detected in HSC-derived immune cells.

[0245] FIGs. 48A-48C. In vivo engineered HSCs provide a durable, self-renewing source of CAR+ immune effectors. A) is a schematic of transplantation of gene marked HSPCs into irradiated wild-type BL / 6 mice to assess long-term engineered HSC function. B) is a graph showing 100% reconstitution of hCD46+immune cells in BL / 6 recipients at Week 18 posttransplant. C) is a graph showing HER2 CAR+expression in immune cells at Week 18 posttransplant is similar to that of primary VLP-dosed mice, demonstrating long-term function of stably-integrated HSCs.

[0246] FIGs. 49A-49C. Lineage-specific promoter drives restricted CAR expression and suppresses tumor growth in vivo. A) is a graph showing HER2 CAR driven by a native myeloid-specific promoter is active in human CD 14+ monocytes but not Jurkat T cells in vitro. B) is a graph showing gene marking of HSPC-derived immune cells lineages at Week 12 post-transplant. C) is a graph showing suppression of MC38 / huHER2-Fluc tumor growth in mice expressing HER2 CAR driven by a myeloid-specific promoter.

[0247] FIGs. 50A-50B. Dose-dependent anti-tumor activity of in vzvo-generated HER2 CAR immune cells. Tumor burden, quantified as total flux of MC38 / huHER2-FLuc cells,65FH12901147.5ENO-00225 (38895-00225) over time in mice expressing varying percentages of HER2 CAR+ immune effector cells. Mice with fewer than 2% of circulating immune cells expressing HER2 CAR demonstrate robust tumor regression compared to mock control mice.

[0248] FIGs. 51A-51C. Composite and individual mouse growth curves demonstrate HER2 CAR-mediated EO771 / muHER2TZBgrowth suppression in the presence of HER2 CAR-expressing immune cells. Tumor control is maintained over 3 weeks post-transplant of whole bone marrow cells. Pre-transplant gene marking: The immune cell profile and frequency of gene marked cells was assessed by flow cytometry at the time of transplant. Tumor control: In vivo-generated HER2 CAR immune cells mediate durable tumor control.

[0249] Figs. 52A-52D depict HER2 CAR expression in mouse and human macrophages in vitro. (A) is a graph of percentage and (B) is a graph of geometric mean fluorescence intensity (gMFI) of HER2 CAR positive cells in hCD46tg murine macrophages. (C) is a graph of percentage and (D) is a graph of gMFI of HER2 CAR positive cells in human PBMC-derived macrophages. Data represented as mean+ / - SDEV (n=3 technical replicates).

[0250] FIGs. 53A-53D depicts CD1 lb promoter-driven HER2 CAR expression with or without miRNA126 binding arrays. Cell surface HER2 CAR expression was assessed by flow cytometry. All groups except ‘No HD Ad’ were treated with O6BG and TMZ at 4-weeks post-transplant (A) is a graph of HER2 CAR expression in peripheral blood monocytes as a percent of all monocytes or (B) geometric mean fluorescence intensity (gMFI). Data represent Mean + / - SEM, n = 3-5 mice per group at each time point. (C) is a graph of HER2 CAR expression in Ein-neg Scal+, cKit-i- cells (LSKs) and (D) is a graph of long-term hematopoietic stem cells (LT-HSCs) at 6 weeks post-transplant; = 1 mouse per group.

[0251] FIG. 54 depicts HER2 CAR and / or MGMT expression in peripheral blood immune cells. Expression of HER2 CAR and MGMT(P140K) (top) or HER2 CAR alone (bottom) in peripheral blood immune cell types (monocytes, NK and T cells) is shown. Cell surface HER2 CAR and intracellular MGMT expression was assessed by flow cytometry. All groups except ‘No HD Ad’ and ‘No integration HD Ad’ were treated with O6BG and TMZ at 5- and 9-weeks post HD Ad dosing. N = 4-6 per group, Data represent Mean + / - SEM.

[0252] FIG. 55 depicts HER2 CAR expression in peripheral blood immune cells with alternative enrichment regimen. Expression of HER2 CAR in peripheral blood immune cell types (monocytes, NK and T cells) is shown. Cell surface HER2 CAR was assessed by flow cytometry. All groups except ‘No HD Ad’ and ‘No enrichment’ were treated with O6BG and TMZ at 1-, 3-, and 5-weeks post HD Ad dosing. N = 4-6 per group, Data represent Mean + / - SEM.66FH12901147.5ENO-00225 (38895-00225)

[0253] FIG. 56 is a graph depicting HER2 CAR expression in peripheral blood immune cells. Flow cytometry was used to characterize HER2 CAR expression in myeloid, NK, T, and B cells at Week 16 post- transplant. CAR positive percentage of parent population is shown. N = 4 per group, Data represent Mean + / - SEM.

[0254] FIGs. 57A-57B depict an ex vivo T cell cytotoxicity assay. (A) T cells harvested from spleens of gene marked mice were stained for HER2 CAR expression. (B) Isolated T cells were co-cultured with MC38 / huHER2 target cells at the indicated effector to target (E:T) ratios for 78 hours. For HER2 CAR-expressing groups, E:T was normalized for % CAR+. T cells expressing HER2 CAR driven by CAG or TNK-A-promoter show specific killing of target cells. Data represent Mean + / - SDEV, n = 5 technical replicates

[0255] FIG. 58 depicts gene marked immune cell reconstitution during tumor challenge. Irradiated BL / 6 mice were implanted with IxlO6EO771 / muHER2Tzbtumor cells followed by a transplant of gene marked whole bone marrow. Mice were bled three weeks post-transplant to assess gene marking (GFP or CAR expression) in peripheral blood immune cells by flow cytometry Data represent Mean + / -SEM, n = 10 mice per group

[0256] FIGs. 59A-59C depict EO771 / muHER2TZBtumor burden. Irradiated BL / 6 mice were implanted with IxlO6EO771 / muHER2Tzbtumor cells followed by a transplant of gene marked whole bone marrow. Tumor burden was measured twice weekly. (A) shows composite data of tumor growth over time. Data represent Mean + / -SEM, n = 10 mice per group (B) depicts growth curves of individual mice over time for each treatment group. (C) depicts tumor volume of each animal on Day 17 post- transplant.

[0257] FIGs. 60A-60E depict an assessment of tumor-infiltrating myeloid cells. EO771 tumors were harvested on Day 25 post-transplant and processed to single cells for flow cytometric analysis (A) depicts frequency of total myeloid cell populations in EO771 tumors (B) depicts frequency of macrophages co-expressing Ml activation markers MHC Class II and CD80. Frequency (C) and absolute count (D) of macrophages expressing HER2 CAR are depicted. (E) Geometric mean fluorescence intensity (gMFI) of CD80 (left) and CD86 (right) on non-gene marked and gene-marked macrophages in the tumor are shown. Data represent Mean + / - SEM, n = 4 mice per group, * p-value < 0.05 by One-way ANOVA for multiple comparisons.

[0258] FIGs. 61A-61C depicts an Assessment of tumor-infiltrating gene marked T cells. EO771 tumors were harvested on Day 25 post-transplant and processed to single cells for flow cytometric analysis (A) depicts absolute count of GFP+or CAR+T cells in EO771 tumors. (B) depicts absolute count of CD8+GFP+or CAR+T cells. (C) depicts absolute count67FH12901147.5ENO-00225 (38895-00225) of CD25+GFP+or CAR+T cells. Data represent Mean + / - SEM, n = 4 mice per group, * p- value < 0.05; ** p-value < 0.01; *** p value < 0.001, **** p-value < 0.0001 by One-way ANOVA for multiple comparisons.

[0259] FIG. 62 depicts macrophages (left) or T cells (right) isolated from spleen or bone marrow, respectively, of gene marked mice. VLPs encoding anti-HER2 CAR(s) driven by Myel-Pr and / or T / NK-Pr, and an EFla driven MGMTP140K cassette. Co-cultures were setup with HER2+ tumor cells and CAR effector cells (CAR M or CAR T) at normalized ratios.

[0260] FIGs. 63A-63E depict multiplexed CAR-M / NK / T cells mediate anti-tumor activity and remodel the tumor microenvironment in vivo. (A) depicts an experimental timeline to assess tumor control mediated by in vivo VLP HSC transduction, enrichment, and CAR expression in mature immune cell lineages. Bone marrow transplanted into HER2+ tumor bearing and irradiated C57BL / 6 mice to assess tumor control. (B) depicts composite data of tumor growth over time. Tumors were harvested on Day 25 post-transplant and processed to single cells for flow cytometric analysis. (C) depicts geometric mean fluorescence intensity (gMFI) of CD80 (left) and CD86 (right) on non-gene marked and gene-marked macrophages in tumor. (D) depicts an absolute count of GFP+ or CAR+ T cells in tumors. (E) depicts absolute counts of CD25+ GFP+ or CAR+ T cells.

[0261] FIG. 64 depicts miR binding arrays for CD34+ repression knocks down transgene expression in CD34+ cells. Lentivirus expression vectors were tested in CD34+ cells to assess expression of copGFP regulated by various miR binding arrays. Cells transduced with lentivirus express mKate2, and the frequency and MFI of copGFP was assessed in this population to assess knockdown by miR binding sites. copGFP MFIs are plotted in histogram form where the strongest copGFP expression is delineated by the dotted line on the right, and the MFI of the untreated population is noted by the dotted line on the right. For all constructs bearing miR binding sites, notable knockdown of copGFP is observed relative to the no miR binding site control.

[0262] FIGs. 65A-65C depict the frequency of HER2 CAR expression in peripheral blood immune cell types (A) and bone marrow cells (B) at 10- and 6.5-weeks post-HSPC transplant, respectively. Bone marrow cells include Eineage-, Scal+, c-Kit-i- (LSK) and longterm hematopoietic stem cell (LT-HSCs). (C) depicts HER2 CAR frequency (left) geometric mean fluorescence intensity (gMFI; right) in human CD 14+ monocytes treated with VEP and differentiated to macrophages. VLP vector encoding anti-HER2 CAR driven by Myel-Pr with or without binding sites for candidate miRNA, anti-HER2 CAR driven by T / NK-Pr, and an EFla driven MGMTP140K cassette.68FH12901147.5ENO-00225 (38895-00225)

[0263] FIG. 66 depicts that HDAd5 / 35++ transduction can be affected by level of expression of CD46 on red blood cells (RBCs). In brief, whole blood from human, CD46tg mice, C57BL / 6 mice, rhesus monkey, and cynomolgus monkey was washed twice with PBS. Resulting washed whole blood was then added to HEK293 cells and followed by addition of HDAd5 / 35++ vector containing a GFP reporter sequence at a MOI of 1, 10, 100, 500, or 1000. After incubation for 1 hour, cells were washed twice with PBS and incubated for an additional 48 hours. Cells were then assessed for expression of GFP using flow cytometry. Exemplary data are shown in graph. The X-axis displays the MOI tested; the Y-axis depicts percentage of GFP+ cells. For each MOI (no virus, 1, 10, 100, 500, 1000), the columns represent from left to right: No Blood, C57BL / 6, Human, CD46tg, Rhesus, Cynomolgus.DETAILED DESCRIPTION

[0264] Genetic engineering is a promising avenue for treatment of many conditions, including without limitation genetic disorders, immune deficiencies, hemoglobinopathies, and cancers including solid tumors and hematological cancers. Engineering of target cells can occur in vitro, ex vivo, or in vivo. Various challenges have arisen in the application of genetic engineering to therapeutic purposes. One of those challenges is that expression in particular hematopoietic cell populations and / or HSC cell lineages can be important for efficacy and outcomes of some therapies, including without limitation CAR therapies, e.g., for the treatment of cancers including solid tumors and hematological cancers. However, many hematopoietic cell engineering approaches are limited in their ability to regulate the hematopoietic cell populations and / or HSC cell lineages in which encoded therapeutic products are expressed. For example, when NK cells, T cells, and B cells are engineered in vitro or ex vivo, and administered to a subject, the numbers and types of engineered cells present in the subject is limited by the engineered population. Moreover, when expression products are operably linked with a single particular regulatory sequence in hematopoietic cells, expression patterns are limited by the behavior of the single regulatory sequence. In many instances, further means of controlling expression of one or more therapeutic products in a plurality of hematopoietic cell populations and / or HSC cell lineages provides a broadly applicable advantage in refining the expression of encoded therapeutic products.

[0265] In some existing therapeutic strategies, cells including T cells and NK cells can be engineered to target therapeutic antigens, e.g., by expression of a chimeric antigen receptor (CAR). One common strategy is to engineer cells in a laboratory environment in vitro or ex vivo) and administer the engineered cells to a subject. Upon administration to the subject, the69FH12901147.5ENO-00225 (38895-00225) in vitro or ex vivo engineered cells (e.g., T cells or NK cells) can direct immune activity against bound targets. Such strategies do not involve modification of other endogenous cells, and the administered cells do not proliferate to establish immune cell compartments. By contrast, engineering of hematopoietic stem cells (HSCs), and in particular in vivo engineering of HSCs, presents a distinct scenario. HSCs can divide and differentiate to produce numerous cell lineages, and in vivo engineering of HSCs can produce lasting changes in the subject’s immune system, populating numerous downstream lineages, cell populations, and compartments with engineered cells (e.g., engineered NK cells, T cells, and myeloid cells).

[0266] The present disclosure provides nucleic acid constructs in which two or more distinct regulatory sequences control expression of operably linked sequences encoding therapeutic products in a plurality of hematopoietic cell populations and / or HSC lineages. The present disclosure includes, among other things, in vivo engineering of HSCs whereby, in cells differentiated from engineered HSCs or progeny thereof, the regulatory sequences can cause expression of operably linked, encoded therapeutic products in target cell populations and / or HSC lineages, e.g., for treatment of a disease, disorder, or condition.

[0267] The present disclosure includes combinations of regulatory sequences that together achieve expression of operably linked, encoded therapeutic products in a plurality of target hematopoietic cell populations and / or HSC lineages. For the avoidance of doubt, the present disclosure encompasses use of first and second regulatory sequences that are not identical but cause expression in overlapping (e.g., incompletely overlapping) sets or subsets of hematopoietic cells, cell populations, and / or cell lineages. Indeed, first and second regulatory sequences of use in the present disclosure can cause expression in one or more of the same cell populations (e.g., among others) and may be differentiated by (and in some instances only differentiated by) the level of expression of operably linked therapeutic products in relevant cells, cell populations, and / or cell lineages.

[0268] As disclosed herein, nucleic acid constructs of the present disclosure can encode, e.g., a plurality of therapeutic products operably linked with respective linage-specific regulatory sequences, where each therapeutic product can be independently selected from a therapeutic protein or therapeutic nucleic acid, for example, a proteinaceous binding agent, a gene editing enzyme or system, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a nucleic acid aptamer, or a functional ribonucleic acid (RNA) such as a small and / or inhibitory RNA. In some embodiments, a plurality of therapeutic products (e.g., first and / or second therapeutic products) can be independently selected from70FH12901147.5ENO-00225 (38895-00225) binding agents such as an antibody, a chimeric antigen receptor (CAR), or a T cell receptor (TCR). In various embodiments, therapeutic products can be selected to target cancer antigens and / or for the treatment of cancer (e.g., a solid tumor a hematological cancer).

[0269] The present disclosure provides vectors encoding nucleic acid constructs provided herein, and use thereof. In various embodiments, a nucleic acid construct of the present disclosure is present in a vector genome, e.g., an adenoviral vector genome. In various embodiments, a nucleic acid construct of the present disclosure is present in the genome of an adenoviral vector, optionally wherein the adenoviral vector is of a particular serotype (e.g., Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50) and / or can be a pseudotyped adenoviral vector and / or an adenoviral engineered for HSC tropism (e.g., an Ad5 / 35++ adenoviral vector or an Ad6 / 35++ adenoviral vector). In various embodiments, a nucleic acid construct of the present disclosure is present in a helper-dependent adenoviral (HD Ad) genome and / or vector.

[0270] Adenoviral genomes and vectors provided herein can be particularly useful for in vivo gene therapy and / or in vivo engineering of HSCs. The present disclosure recognizes that in vivo gene therapy and / or engineering of HSCs provides numerous advantages over in vitro and / or ex vivo approaches. For example, certain such methods have a variety of drawbacks, including, without limitation, the cost and technical complexity of therapies based on laboratory-engineered cells, including the need for sophisticated facilities to isolate, modify, expand, and reformulate engineered cells. Additional challenges of certain such therapies can include difficulty achieving engraftment in recipients (sometimes requiring, e.g., high-dose chemotherapy). For at least these reasons, there is a need for in vivo gene therapy strategies and protocols.I. Hematopoietic Cells

[0271] Hematopoiesis refers to the process by which various types of blood cells are produced from HSCs (see, e.g., FIG.31). Without wishing to be bound by any particular scientific theory, HSCs are understood to be self-renewing and multipotent, differentiating into progenitors that further differentiate to produce mature and / or terminally differentiated hematopoietic cell populations. A pathway of differentiation entails increasing differentiation relative to an HSC or other temporally prior state and / or further change away from an HSC, toward a mature or terminally differentiated cell population.

[0272] According to some estimates, an adult human can include tens of thousands of HSCs, giving rise to hundreds of millions of progenitor cells that differentiate into precursor71FH12901147.5ENO-00225 (38895-00225) cells and eventually mature effector cells. Thus, a population of multipotent self-renewing HSCs generates large numbers of differentiated progeny by amplification and progressive lineage restriction. As referred to herein, hematopoietic cell populations refer to any and all types of cells that are, or are derived from, hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs), including without limitation particular hematopoietic cell populations and HSC lineages disclosed herein.

[0273] Hematopoietic cell populations (e.g., target hematopoietic cell populations) of the present disclosure include hematopoietic cells of all lineages and stages of hematopoietic cell differentiation. Target cell populations of the present disclosure include, without limitation, HSCs (e.g., CD34+ long-term (LT)-HSCs and / or CD34+ short-term (ST)-HSCs), common lymphoid progenitors (CLPs), T cells, NK cells, colony forming unit (CFU)-pre B cells, B cells, common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), CFU-M cells, monoblasts, monocytes, macrophages, CFU-G cells, myeloblasts, granulocytes, neutrophils, eosinophils, basophils, megakaryocyte-erythrocyte progenitors (MEPs), BFU-E cells, CFU-E cells, erythroblasts, erythrocytes, CFU-Mk cells, megakaryocytes, and / or platelets. Hematopoietic cell populations (e.g., target hematopoietic cell populations) of the present disclosure include CD34+ hematopoietic cells.

[0274] Without wishing to be bound by any particular scientific theory, HSCs can be divided into two subpopulations according to their CD34 expression: CD34+ long-term (LT)- HSCs and CD34+ short-term (ST)-HSCs. LT-HSCs differentiate into ST-HSCs, and subsequently, ST-HSCs differentiate into multipotent progenitors (MPPs). In various embodiments, a hematopoietic cell population is or includes CD34+ hematopoietic cells. In various embodiments, HSCs or subsets thereof may be detected by CD46 receptor expression.

[0275] In some embodiments, an HSC is a cell characterized by expression of CD34 (CD34+). HSCs can generally be identified as CD34+ cells. CD34 is expressed by ST-HSCs and LT-HSCs. In some embodiments, an HSC is a cell characterized by expression of CD90 (CD90+). In some embodiments, an HSC is a cell characterized by expression of CD34 and CD90 (CD34+CD90+). In some embodiments, an HSC is a cell characterized by expression of CD46 (CD46+). In some embodiments, an HSC is a cell characterized by greater expression of CD46 than one or more mature immune cell populations, e.g., T cells, NK cells, monocytes, neutrophils, granulocytes, and / or B cells. In some embodiments, an HSC is a cell characterized by expression of CD34 and CD46 (CD34+CD46+). In some72FH12901147.5ENO-00225 (38895-00225) embodiments, an HSC is a cell characterized by expression of CD34, CD46, and CD90 (CD34+CD46+CD90+).

[0276] HSCs or subsets thereof can also be identified by any of the following marker profiles: CD34+; Lin- / CD34+ / CD38- / CD45RA- / CD90+ / CD49f+ (HSC1); CD34+ / CD38- / CD45RA- / CD90- / CD49f+ / (HSC2). In various embodiments, human HSC1 can be identified by any of the following profiles: CD34+ / CD38- / CD45RA- / CD90+ or CD34+ / CD45RA- / CD90+ and mouse LT-HSC can be identified by Lin-Scal+ckit+CD150+CD48-Flt3-CD34- (where Lin represents the absence of expression of any marker of mature cells including CD3, CD4, CD8, CDl lb, CDl lc, NK1.1, Grl, and TERI 19). In particular embodiments, HSC are identified by a CD 164+ profile. In particular embodiments, HSC are identified by a CD34+ / CD164+ profile. HSCs can be targeted for in vivo genetic modification by binding CD46.

[0277] Without wishing to be bound by theory, progenitors are understood to lack the capacity for self-renewal and are characterized by restricted differentiation, in that they can only yield cells of a particular lineage. Progenitors can be myeloid lineage progenitors or lymphoid lineage progenitors (referred to respectively as common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs)).

[0278] CMPs can differentiate into granulocyte-macrophage progenitors (GMPs) and megakaryocyte-erythrocyte progenitors (MEPs). GMPs can differentiate into granulocytes (e.g., neutrophils, eosinophils, and basophils), and monocytes (which can differentiate into to macrophages). MEPs can differentiate into megakaryocytes / platelets and erythrocytes. CLPs can differentiate into T, NK, and B cells.

[0279] Hematopoiesis further includes cell populations that are referred to by names that are based on their identification in colony forming unit assays. Cells that form hematopoietic colonies (so-called CPUs or CFCs) can represent steps or stages of hematopoietic differentiation between HSCs and more terminally differentiated cells. CPUs can be identified by culturing hematopoietic cells in a semisolid media (typically methylcellulose or agar) supplemented with cytokines that promote the localized expansion and differentiation of hematopoietic cells in discrete colonies. CPUs can be identified by factors including, without limitation, the number of cells in a colony, the time required to produce the colony, and / or the types of cells in the colony. In general, without wishing to be bound by any particular scientific theory, progenitor cells can produce colonies that include, e.g., at least 30,000 cells including cell populations of multiple lineages, e.g., by day 15-18 of culture. In various embodiments, culturing can produce colonies that generate erythroid bursts (e.g., of73FH12901147.5ENO-00225 (38895-00225)5,000 cells), referred to as burst-forming unit erythroid (BFU-E). Other colony types can include granulomonocytic colonies (colony forming unit, granulomonocytic (CFU-GM)) and colonies of, e.g., 50-200 cells that are erythroid cells (colony-forming unit, erythroid (CFU- E)), granulocytic cells (CFU-G), or monocytic cells (CFU-M). These descriptions of colonies are solely for general illustration, and methods and techniques for colony analysis and identification are known in the art.

[0280] CEPs can also be referred to as CFU-L cells. In various embodiments, CFU-L cells can differentiate into CFU-B cells that differentiate into Pre-B Eymphocytes that can differentiate into B Lymphoblasts and subsequently into B Lymphocytes. In various embodiments, CFU-L cells can differentiate into CFU-T cells that differentiate into Pre-T Lymphocytes that can differentiate into T Lymphoblasts and subsequently into T Lymphocytes.

[0281] CMPs can also be referred to as CFU-GEMM cells. GMPs can also be referred to as CFU-GM cells. In various embodiments, CFU-GM cells can differentiate into CFU-M cells that differentiate into monoblasts and CFU-G cells that differentiate into neutrophils (e.g., via myeloblasts and neutrophilic myelocytes). MEPs can differentiate into BFU-E cells that can differentiate into CFU-E cells that can differentiate into erythroblasts (e.g., via rubriblasts, rubricytes, and metarubricytes). MEPs can differentiate into CFU-Mk cells that differentiate into megakaryocytes. CFU-Gemm can also differentiate into CFU-Eo cells that differentiate into eosinophils (e.g., via myeloblasts and eosinophilic myelocytes) and CFU- Baso cells that differentiate into basophils (e.g., via myeloblasts and basophilic myelocytes). Megakaryocyte lineage progenitors can include BFU-MK cells that differentiate into more mature progenitor cells referred to as CFU-MK cells.

[0282] Several different subsets of T-cells have been discovered, each with a distinct function. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of several proteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCRP) genes and are called a- and P-TCR chains.

[0283] y5 T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface. In y5 T-cells, the TCR is made up of one y-chain and one 5-chain. This group of T-cells is much less common (2% of total T-cells) than the aP T-cells.

[0284] CD3 is expressed on all mature T cells. Activated T-cells express 4-1BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.74FH12901147.5ENO-00225 (38895-00225)

[0285] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T- cells (CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface. Helper T-cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0286] Cytotoxic T-cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.

[0287] In particular embodiments, CARs are genetically modified to be expressed in cytotoxic T-cells.

[0288] ‘ ‘Central memory” T-cells (or “TCM”) as used herein refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof, and does not express or has decreased expression of CD45RA as compared to naive cells. In particular embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.

[0289] ‘ ‘Effector memory” T-cell (or “TEM”) as used herein refers to an antigen experienced T-cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell. In particular embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA. Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.

[0290] ‘ ‘Naive” T-cells as used herein refers to a non-antigen experienced T cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells. In particular embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.75FH12901147.5ENO-00225 (38895-00225)

[0291] B cells are mediators of the humoral response and are responsible for production and release of antibodies specific to an antigen. Several types of B cells exist which can be characterized by key markers. In general, immature B cells express CD19, CD20, CD34, CD38, and CD45R, and as they mature the key expressed markers are CD19 and IgM.

[0292] NK Cells can be defined as CD3-CD56+ cells that can also be CD7+CD127- NKp46+T-bet+Eomes+. Different subtypes of human NK cells have been identified that are either CD3-CD56dimCD16+ or CD3-CD56brightCD16-. The CD56dimCD16+ subset of NK cells is predominantly found in the blood and is highly cytotoxic, while the CD56brightCD16- subset is the main subtype found in the lymph nodes and has only weak cytotoxic potential. Some studies have suggested that CD56brightCD16- subset are precursors of mature CD56dimCD16+ NK cells.

[0293] In addition, NK cells can express cell surface receptors that regulate their activation. These can include the human killer immunoglobulin-like receptors (KIRs), CD94- NKG2 heterodimeric receptors, NKG2D, natural cytotoxicity receptors (NCRs). In some embodiments, an NK cell is a mature NK cell in peripheral blood, where the NK cell is characterized as CD56dim.

[0294] Various hematopoietic cell populations contribute to native responses to cancer. For example, tumor infiltrating lymphocytes are some of the first cells to infiltrate tumors during native antitumor responses. Tumor infiltrating lymphocytes, including native natural killer (NK) cells, are non-antigen specific lymphocytes that express receptors and signaling pathways which are highly responsive to microenvironmental cues. NK cells further have cell-killing effector materials such as granzyme and perforin to induce apoptosis of target cells. The presence of these cells also contributes to an antitumor immune response by secreting cytokines such as IFN-y to enhance the immune reaction. Because NK cells do not express antigen-specific receptors, they cannot detect specific tumor cells (e.g., those expressing HER2). The ability to target NK cells to a tumor {e.g., a solid tumor) would bolster the body’s innate ability to respond to the tumor. In additional to ILCs, cytotoxic lymphocytes, such as CD 8+ and CD4+ T cells play a significant role in the anti-tumor response of the immune system. T cells are activated when they encounter tumor antigens and can effectively eliminate target cancer cells. Another major component of the anti-tumor response is the activity of myeloid cells e.g., macrophages, dendritic cells, and monocytes). Myeloid cells can exert anti-tumor functions by activating NK cells and T cells. Discovery of methods which utilize the various cells of the immune response are essential to develop targeted and effective cancer therapies.76FH12901147.5ENO-00225 (38895-00225)II. Regulatory Sequences

[0295] The present disclosure includes a variety of regulatory sequences and regulatory sequence elements useful to control expression of operably linked, encoded products in a variety of contexts. In some embodiments, a regulatory sequence is useful to cause expression of an operably linked, encoded product in at least a target hematopoietic cell population and / or HSC lineage. In some embodiments, a regulatory sequence is useful to cause expression of an operably linked, encoded product in a constitutive and / or ubiquitous manner.

[0296] The present disclosure includes the recognition that in some embodiments a regulatory sequence can be or include a promoter. In various embodiments, a promoter, e.g., a promoter of the present disclosure, can be sufficient to cause lineage-specific expression of an operably linked coding sequence that would not otherwise (e.g., with a different promoter, e.g., with a ubiquitous promoter) be expressed in a lineage-specific manner. The present disclosure further recognizes that, in various embodiments, a regulatory sequence can include a promoter and one or more additional regulatory elements (e.g., one or more of a UTR, enhancer, and / or intronic sequence) that together cause lineage-specific expression. In some embodiments, a combination of a promoter and one or more additional regulatory elements can cause an expression pattern that would not be caused by the promoter in the absence of the one or more additional regulatory elements. In some embodiments, a combination of a promoter and one or more additional regulatory elements can cause a lineage-specific expression pattern that would not be caused by the promoter in the absence of the one or more additional regulatory elements, optionally wherein the expression pattern that would be caused by the promoter in the absence of the one or more additional regulatory elements would not be a lineage-specific expression pattern.

[0297] In some embodiments, a lineage-specific regulatory sequence causes expression of an operably linked coding sequence in a first hematopoietic cell population (e.g., a first cell population of a first HSC lineage) or first HSC lineage, but does not cause expression in at least one other or reference hematopoietic cell population and / or HSC lineage. In some embodiments, a lineage-specific regulatory sequence causes expression of an operably linked coding sequence in a first hematopoietic cell population (e.g., a first cell population of a first HSC lineage) or first HSC lineage, but does not cause detectable expression in at least one other or reference hematopoietic cell population and / or HSC lineage. In some embodiments, a lineage-specific regulatory sequence causes expression of an operably linked coding77FH12901147.5ENO-00225 (38895-00225) sequence in a first hematopoietic cell population (e.g., a first cell population of a first HSC lineage) or first HSC lineage, but does not cause significant expression in at least one other or reference hematopoietic cell population and / or HSC lineage. In some embodiments, a lineage-specific regulatory sequence causes expression of an operably linked coding sequence in a first hematopoietic cell population (e.g., a first cell population of a first HSC lineage) or first HSC lineage, causes significantly less expression in at least one other or reference hematopoietic cell population and / or HSC lineage.

[0298] In various embodiments, the other or reference hematopoietic cell population and / or HSC lineage is an HSC. In various embodiments, the other or reference hematopoietic cell population and / or HSC lineage is an erythroid cell. In various embodiments, the other or reference hematopoietic cell population and / or HSC lineage is an HSC. In various embodiments, the other or reference hematopoietic cell population and / or HSC lineage is a platelet.

[0299] In various embodiments, a regulatory sequence can be referred to as not causing expression, or as not causing detectable expression, in the other or reference hematopoietic cell population and / or HSC lineage if, under conditions in which a nucleic acid construct that includes the regulatory sequence operably linked with the coding sequence causes expression of the coding sequence in the first cell population, the same nucleic acid construct does not cause expression, essentially does not cause expression does not cause detectable expression, and / or essentially does not cause detectable expression of the coding sequence in the other or reference cell population or lineage. In various embodiments, a regulatory sequence can be referred to as not causing significant expression in the other or reference hematopoietic cell population and / or HSC lineage if, under conditions in which a nucleic acid construct that includes the regulatory sequence operably linked with the coding sequence cause expression of the coding sequence in a first cell population, the same nucleic acid construct does not cause sufficient expression of the coding sequence in the other or reference cell population or lineage to produce a detectable biological phenotype as determined by an assay relevant to the encoded product.

[0300] In various embodiments, a regulatory sequence can be referred to as causing significantly less expression in the other or reference hematopoietic cell population and / or HSC lineage if, under conditions in which a nucleic acid construct that includes the regulatory sequence operably linked with the coding sequence cause expression of the coding sequence in a first cell population, the same nucleic acid construct causes expression in the other or reference cell population or lineage that is at least 50-fold lower than in the first cell78FH12901147.5ENO-00225 (38895-00225) population. In some embodiments, expression in the other or reference cell population or lineage is at least 100-fold lower than in the first cell population. In some embodiments, expression in the other or reference cell population or lineage is at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5000-fold, at least lOOOO-fold, at least 20000-fold, at least 30000-fold, at least 40000-fold, at least 50000-fold, or at least lOOOOO-fold lower than in the first cell population.

[0301] In various embodiments in which expression is compared, e.g., expression of a coding sequence operably linked with a lineage-specific regulatory sequence compared between a first cell population and a reference cell population, there are a variety of approaches known in the art to determine expression. For example, those of skill in the art will appreciate that expression can be determined in a laboratory test in relevant cell populations under controlled conditions. Expression can be determined in cells of a cell culture. Expression can be determined in cells that are derived from a laboratory cell line, cells isolated from a subject, or cells present in a subject. In various embodiments, expression can be determined by measurement of the amount or concentration of mRNA having a sequence transcribed from a coding sequence of a construct of interest. In various embodiments, expression can be determined by measurement of the amount or concentration of a protein having a sequence translated from a coding sequence of a construct of interest. In various embodiments, expression can be determined by measurement of a reporter construct operably linked with a regulatory sequence. For example, a reporter can be a protein that produces a visible phenotype, such as a fluorescent protein, whereby those of skill in the art will appreciate that the measured fluorescence is indicative of the level of expression. For example, a reporter can be an enzyme that participates in a detectable reaction, whereby those of skill in the art will appreciate that the measured activity of the enzyme based on the completion of the reaction is indicative of the level of expression. Means of determining mRNA levels, protein levels, and fluorescence are well known to those of skill in the art.

[0302] The present disclosure includes, among other things, lineage-specific regulatory sequences and regulatory sequence elements that can be used to express operably linked, encoded therapeutic products in one or more hematopoietic cell populations and / or HSC lineages selected from NK cells, T cells, B cells, and / or myeloid cells. In various embodiments, a nucleic acid construct of the present disclosure can include two or more lineage-specific regulatory sequences each controlling expression of at least one distinct therapeutic expression product (e.g., antibody, CAR, or TCR) in distinct hematopoietic cell79FH12901147.5ENO-00225 (38895-00225) populations and / or distinct HSC lineages, in distinct sets or subsets thereof, and / or at distinct expression levels.

[0303] In some embodiments, a lineage-specific regulatory sequence can include a promoter or regulatory element that causes expression, exclusively or non-exclusively, in at least one lymphoid cell population and / or lymphoid lineage. In some embodiments, a lineagespecific regulatory sequence can include a promoter or regulatory element that causes expression, exclusively or non-exclusively, in at least one myeloid cell population and / or myeloid lineage. Because a lineage-specific regulatory sequence that causes expression in at least one lymphoid cell population and / or lymphoid lineage can also, in various embodiments, also cause expression of the same operably linked, encoded product in at least one myeloid cell population and / or myeloid lineage, the present disclosure recognizes that many lineage-specific promoters cannot be unilaterally or absolutely described as specific to any single cell population, or to any single lineage. Rather, the present disclosure recognizes and appreciates that various regulatory sequences and regulatory sequence elements provided herein can provide significant utility, individually and / or in combination, to achieve diverse expression patterns of therapeutic utility. Therapeutic utility is not necessarily dependent on absolute isolation of expression to any single particular cell population or lineage, nor to any set or subset thereof. The power and utility of various embodiments disclosed herein is derived at least in part from the ability to achieve and tune broader and different expression than can be achieved, e.g., from a single regulatory sequence and / or by the engineering of any single hematopoietic cell population.

[0304] Moreover, non-lineage-specific regulatory sequences are also used in certain contexts of the present disclosure, e.g., for expression of agents other than therapeutic expression products. As found throughout the present disclosure, not all expression products require and / or benefit from lineage-specific expression and do not require operable linkage with a lineage-specific regulatory sequence. For example, presently disclosed enrichment markers, safety switches, transposases, and / or recombinases do not necessarily require and / or benefit from lineage- specific expression and do not require operable linkage with a lineagespecific regulatory sequence.

[0305] For the avoidance of doubt, those of skill will appreciate that an “h” as the first letter of a name used to designate a sequence, e.g., a promoter sequence, particularly when followed by a capital letter is a common syntax used to indicate that sequence is derived from a human genome sequence. Similarly, those of skill will appreciate that an “m” as the first letter of a name used to designate a sequence, e.g., a promoter sequence, particularly when80FH12901147.5ENO-00225 (38895-00225) followed by a capital letter is a common syntax used to indicate that sequence is derived from a mouse genome sequence. Those of skill in the art will further appreciate that such terminology refers solely to the source of a sequence or portion thereof, rather than to its potential utility; sequences derived from non-mammalian or non-human organisms are commonly found to have utility for expression constructs in other organisms, including without limitation mammals such as humans.11(A). Promoter Sequences

[0306] In various embodiments, a lineage-specific regulatory sequence of the present disclosure can include a promoter. In some embodiments, a promoter included in a lineagespecific regulatory sequence is itself a sequence that can cause lineage-specific expression of an operably linked coding sequence. In other embodiments, a promoter included in a lineagespecific regulatory sequence causes (e.g., only causes) lineage-specific expression of an operably linked coding sequence when used in combination with one or more other regulatory elements present in the lineage-specific regulatory sequence.

[0307] In various embodiments, a promoter of the present disclosure can be capable of causing and / or contributing to, when used independently and / or when included in a lineagespecific regulatory sequence, expression of an operably linked coding sequence in one or more hematopoietic cell populations and / or HSC lineages, optionally selected from one or more of T cells, NK cells, B cells, and / or myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells). In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is an NK cell. In some embodiments, the cell is a cytotoxic CD8+ T cell. In some embodiments, the cell is a helper CD4+ T cell. In some embodiments, the cell is an NKT cell. In some embodiments, the cell is a CD56brightNK cell. In some embodiments, the cell is a CD56dimNK cell.

[0308] In various embodiments, a promoter included in a lineage-specific regulatory sequence of the present disclosure can be or include a nucleic acid sequence derived from a mammalian genome (e.g., a human, non-human primate, mouse, rat, pig, goat, cow, or horse genome). In various embodiments, a promoter included in a lineage-specific regulatory sequence of the present disclosure can be or include a nucleic acid sequence derived from a human genome. In various embodiments, a promoter included in a lineage-specific regulatory sequence of the present disclosure can be or include a nucleic acid sequence derived from a bacterial genome, such as a CMV sequence (e.g., a CMV or CMV-derived promoter or81FH12901147.5ENO-00225 (38895-00225) enhancer). In various embodiments, a promoter included in a lineage-specific regulatory sequence of the present disclosure can be or include a synthetic sequence (e.g., YB_TATA).II(A)(i). Exemplary Promoter Sequences for use in Regulatory Sequences that cause Expression in Lymphoid Cells

[0309] In some embodiments, a promoter of the present disclosure that causes, or can be included in a regulatory sequence that causes, expression in a lymphoid hematopoietic cell population and / or in a lymphoid HSC lineage. In various embodiments, a regulatory sequence or regulatory sequence element of the present section can include one or more regulatory elements together with a promoter, optionally including an enhancer and / or a UTR, e.g., as illustrative embodiments of regulatory sequences including a promoter of the present disclosure. In some embodiments, a promoter, and / or a regulatory sequence including a promoter, and / or a regulatory sequence including a promoter, of the present disclosure that causes, or can be included in a regulatory sequence that causes, expression in a lymphoid hematopoietic cell population and / or in a lymphoid HSC lineage can be a hB29, mB29, hCD3A, mCD3A, CD19, or dLCK promoter.

[0310] In some embodiments, a promoter or regulatory sequence can be or include an hB29 promoter or regulatory sequence. In various embodiments, an hB29 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 4 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 4). In various embodiments, an hB29 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 4.

[0311] In some embodiments, a promoter or regulatory sequence can be or include an mB29 promoter or regulatory sequence. In various embodiments, an mB29 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 5 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 5). In various embodiments, an mB29 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 5.

[0312] In some embodiments, a promoter or regulatory sequence can be or include an hCD3d promoter or regulatory sequence. In various embodiments, an hCD3d promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 6 (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at82FH12901147.5ENO-00225 (38895-00225) least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 6). In various embodiments, an hCD3d promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 6.

[0313] In some embodiments, a promoter or regulatory sequence can be or include an mCD3D promoter or regulatory sequence. In various embodiments, an mCD3D promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 7 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 7). In various embodiments, an mCD3D promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 7.

[0314] In some embodiments, a promoter or regulatory sequence can be or include a CD 19 promoter or regulatory sequence. In various embodiments, a CD 19 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 8 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 8). In various embodiments, a CD19 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 8.

[0315] In some embodiments, a promoter or regulatory sequence can be or include a dLCK promoter or regulatory sequence. In various embodiments, a dLCK promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 9 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 9). In various embodiments, a dLCK promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 9.The present disclosure further specifically includes the recognition that a promoter or regulatory sequence, such as a dLCK promoter or regulatory sequence, can be included in a nucleic acid construct of the present disclosure as a sequence set forth herein (e.g., in SEQ ID NO: 9), optionally in the context of a larger sequence corresponding to a reference genome (e.g., in a larger fragment from the reference sequence, e.g., a Smal-dLCK fragment that includes a dLCK promoter or regulatory sequence, e.g., according to SEQ ID NO: 10) and / or in combination with an enhancer. In some embodiments, a promoter or regulatory sequence can be or include a dLCK promoter or regulatory sequence present in a Smal-dLCK sequence. In various embodiments, a Smal-dLCK sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 10 (e.g., at least 80%, at least 85%,83FH12901147.5ENO-00225 (38895-00225) at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 10). In various embodiments, a Smal-dLCK sequence can have the nucleic acid sequence set forth in SEQ ID NO: 10.Expression in Myeloid Cells

[0316] In some embodiments, a promoter of the present disclosure that causes, or can be included in a regulatory sequence that causes, expression in a myeloid hematopoietic cell population and / or in a myeloid HSC lineage. In various embodiments, a regulatory sequence or regulatory sequence element of the present section can include one or more regulatory elements together with a promoter, optionally including an enhancer and / or a UTR, e.g., as illustrative embodiments of regulatory sequences including a promoter of the present disclosure. In some embodiments, a promoter, and / or a regulatory sequence including a promoter, of the present disclosure that causes, or can be included in a regulatory sequence that causes, expression in a myeloid hematopoietic cell population and / or in a myeloid HSC lineage can be a CDl lb, CD68, CX3CR1, CX3CR1 Pl, CX3CR1 P2, CX3CR1 P3, CX3CR1 P3[-222], CX3CR1 P3[-498], CTSG / FES, coGP91, GP91 intron 1, CD43, CD14, HBBlongLCR, HBBpLCR, CDl lb+UTR, CD68+UTR, WASP, or CYBB promoter.

[0317] In various embodiments, promoters that can be associated with causing expression in B lymphocytes include hB29, mB29, and CD19 promoters. In various embodiments, promoters that can be associated with causing expression in macrophages include mCD68 and hCD68 promoters. In various embodiments, promoters that can be associated with causing expression in monocytes include CDllb and CD14 promoters. In various embodiments, promoters that can be associated with causing expression in leukocytes and platelets include hCD43 and SV40en-hCD43 promoters. In various embodiments, regulatory sequences and / or promoters that can be associated with causing expression in T lymphocytes include hCD3d, mCD3d, and dLCK promoters. In various embodiments, promoters that can be associated with causing expression in neutrophils include CTSG-FES promoters. In various embodiments, promoters that can be associated with causing expression in erythrocytes include HBBlongLCR and HBBpLCR promoters. In various embodiments, promoters that can be associated with causing expression in megakaryocytes include WASP promoters.

[0318] In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1+UTR (-222) promoter or regulatory sequence. In various embodiments, a84FH12901147.5ENO-00225 (38895-00225)CX3CR1+UTR (-222) promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 14 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 14). In various embodiments, a CX3CR1+UTR (-222) promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 14.

[0319] In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1+UTR (-498) promoter or regulatory sequence. In various embodiments, a CX3CR1+UTR (-498) promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 15 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 15). In various embodiments, a CX3CR1+UTR (-498) promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 15.

[0320] In some embodiments, a promoter or regulatory sequence can be or include a CD1 lb promoter or regulatory sequence. In various embodiments, a CD1 lb promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 20 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 20). In various embodiments, a CDl lb promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 20.

[0321] In some embodiments, a promoter or regulatory sequence can be or include an hCD68 promoter or regulatory sequence. In various embodiments, an hCD68 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 22 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 22). In various embodiments, an hCD68 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 22.

[0322] In some embodiments, a promoter or regulatory sequence can be or include an mCD68 promoter or regulatory sequence. In various embodiments, an mCD68 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 23 (e.g., 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%, at least 99%, or85FH12901147.5ENO-00225 (38895-00225)100% identity with SEQ ID NO: 23). In various embodiments, an mCD68 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 23.

[0323] In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1 promoter or regulatory sequence. In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1 Pl promoter or regulatory sequence. In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1 P2 promoter or regulatory sequence. In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1 P3 promoter or regulatory sequence.

[0324] In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1 P3[-222] promoter or regulatory sequence. In various embodiments, a CX3CR1 P3[-222] promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 24 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 24). In various embodiments, a CX3CR1 P3[-222] promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 24.

[0325] In some embodiments, a promoter or regulatory sequence can be or include a CX3CR1 P3[-498] promoter or regulatory sequence. In various embodiments, a CX3CR1 P3[-498] promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 25 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 25). In various embodiments, a CX3CR1 P3 [-498] promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 25.

[0326] In some embodiments, a promoter or regulatory sequence can be or include a CTSG / FES promoter or regulatory sequence. In various embodiments, a CTSG / FES promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 26 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 26). In various embodiments, a CTSG / FES promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 26.

[0327] In some embodiments, a promoter or regulatory sequence can be or include a coGP91 promoter or regulatory sequence. In various embodiments, a coGP91 promoter or86FH12901147.5ENO-00225 (38895-00225) regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 27 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 27). In various embodiments, a coGP91 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 27.

[0328] In some embodiments, a promoter or regulatory sequence can be or include a GP91 Intron #1 promoter or regulatory sequence. In various embodiments, a GP91 Intron #1 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 28 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 28). In various embodiments, a GP91 Intron #1 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 28.

[0329] In some embodiments, a promoter or regulatory sequence can be or include a CYBB promoter or regulatory sequence. In various embodiments, a CYBB promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 29 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 29). In various embodiments, a CYBB promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 29.

[0330] In some embodiments, a promoter or regulatory sequence can be or include an hCD43 promoter or regulatory sequence. In various embodiments, an hCD43 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 30 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 30). In various embodiments, an hCD43 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 30.

[0331] In some embodiments, a promoter or regulatory sequence can be or include an SV40en-hCD43 promoter or regulatory sequence. In various embodiments, an SV40en- hCD43 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 31 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 31). In various embodiments, an87FH12901147.5ENO-00225 (38895-00225)SV40en-hCD43 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 31.

[0332] In some embodiments, a promoter or regulatory sequence can be or include a CD 14 promoter or regulatory sequence. In various embodiments, a CD 14 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 32 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 32). In various embodiments, a CD14 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 32.

[0333] In some embodiments, a promoter or regulatory sequence can be or include an HBBlongLCR promoter or regulatory sequence. In various embodiments, an HBBlongLCR promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 33 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 33). In various embodiments, an HBBlongLCR promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 33.

[0334] In some embodiments, a promoter or regulatory sequence can be or include an HBBpLCR promoter or regulatory sequence. In various embodiments, an HBBpLCR promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 34 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 34). In various embodiments, an HBBpLCR promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 34.

[0335] In some embodiments, a promoter or regulatory sequence can be or include a CD1 Ib+UTR promoter or regulatory sequence. In various embodiments, a CD1 Ib+UTR promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 12 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 12). In various embodiments, a CD1 Ib+UTR promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 12.88FH12901147.5ENO-00225 (38895-00225)

[0336] In some embodiments, a promoter or regulatory sequence can be or include a CD68+UTR promoter or regulatory sequence. In various embodiments, a CD68+UTR promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 13 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 13). In various embodiments, a CD68+UTR promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 13.

[0337] In some embodiments, a promoter or regulatory sequence can be or include a WASP promoter or regulatory sequence. In various embodiments, a WASP promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 3 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 3). In various embodiments, a WASP promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 3.II(A)(iii). Minimal, Ubiquitous, Hematopoietic, and / or Constitutive Promoters

[0338] In some embodiments, a promoter of the present disclosure can be a minimal promoter, ubiquitous promoter, hematopoietic promoter, and / or constitutive promoter. In various embodiments, a nucleic acid construct of the present disclosure can include, for example, a minimal, ubiquitous, hematopoietic, and / or constitutive promoter. In various embodiments, minimal, ubiquitous, hematopoietic, and / or constitutive can be used, e.g., to express coding sequences that encode, e.g., enrichment markers, safety switches, transposases, and / or recombinases. Moreover, in various embodiments, minimal and / or ubiquitous promoters can be used in combination with additional regulatory sequences in a lineage-specific regulatory sequences that causes expression of an operably linked coding sequence in one or more hematopoietic cell populations and / or HSC lineages.

[0339] Minimal promoters can be relatively short sequences that permit formation of a transcription initiation complex, e.g., at a transcription start site. In various embodiments, a minimal promoter can be a ubiquitous, hematopoietic, and / or constitutive promoter. For the avoidance of doubt, a minimal promoter, ubiquitous promoter, hematopoietic promoter, and / or constitutive promoter can be used for expression according to its independent expression function (e.g., for ubiquitous express) or can be used in combination with one or89FH12901147.5ENO-00225 (38895-00225) more other elements (e.g., an enhancer) that further specify a resulting expression pattern, e.g., to achieve lineage- specific expression of an operably linked coding sequence.

[0340] In some embodiments, a minimal promoter of the present disclosure can be a minCMV, YB TATA, miniTK, MLP, or minP promoter. Exemplary minCMV, YB TATA, miniTK, MLP, or minP promoter. Exemplary minCMV, YB TATA, miniTK, MLP, and minP promoters are provided in Table 1.

[0341] In some embodiments, a promoter of the present disclosure can be a ubiquitous and / or constitutive promoter. In some embodiments, a ubiquitous and / or constitutive promoter of the present disclosure can be a CAG, EFla, mini EFl, EFS, CMV, PGK, hPGK, mPGK, SFFV, SV40, GAPDH, ACTB, UBC, UBB, hROSA, mROSA, KIN, heIF4Al, meIF4Al, hB29, mB29, LSE beta-glucuronidase (GUSB), ubiquitous chromatin opening element (UCOE), chicken beta actin promoter, or A2-SFFV promoter. In some embodiments, a promoter can be a CMV promoter. In some embodiments, a promoter can be a CAG promoter. Exemplary sequences of CAG, EFla, mini EFl, EFS, CMV, PGK, hPGK, mPGK, SFFV, SV40, GAPDH, ACTB, UBC, UBB, hROSA, mROSA, KIN, heIF4Al, meIF4Al, hB29, mB29, LSE beta-glucuronidase (GUSB), ubiquitous chromatin opening element (UCOE), chicken beta actin promoter, and A2-SFFV promoters are provided in Table 2.

[0342] In some embodiments, a hematopoietic promoter of the present disclosure can be an hCD45 or SV40en-hCD45 promoter. Exemplary sequences of hCD45 or SV40en- hCD45promoters are provided in Table 3.

[0343] In some embodiments, a promoter or regulatory sequence can be or include a YB_TATA promoter or regulatory sequence. In various embodiments, a YB_TATA promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 2 (e.g., at least 80%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 2). In various embodiments, a YB_TATA promoter or regulatory sequence can include a nucleic acid sequence having no more than five sequence differences from SEQ ID NO: 2 (e.g., no more than 5, not more than 4, no more than 3, no more than 2, or no more than sequence differences from SEQ ID NO: 2), where a sequence difference can be an insertion, deletion, or substitution. In various embodiments, a YB_TATA promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, the minimal promoter or regulatory sequence is a YB_TATA promoter or regulatory sequence. The YB_TATA promoter or regulatory sequence is a synthetic sequence developed by Hansen et al. (Hansen J., Transplantation of prokaryotic two-componentFH12901147.5ENO-00225 (38895-00225) signaling pathways into mammalian cells. Proc. Natl. Acad. Sci. USA. 2014; 111:15705- 15710).

[0344] In various embodiments, a minCMV promoter or regulatory sequence promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 35 (e.g., at least 80%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 35). In various embodiments, a minCMV promoter or regulatory sequence can include a nucleic acid sequence having no more than five sequence differences from SEQ ID NO: 35 (e.g., no more than 5, not more than 4, no more than 3, no more than 2, or no more than sequence differences from SEQ ID NO: 35), where a sequence difference can be an insertion, deletion, or substitution. In various embodiments, a minCMV promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 35.

[0345] In various embodiments, a minCMV promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 38 (e.g., at least 80%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 38). In various embodiments, a minCMV promoter or regulatory sequence can include a nucleic acid sequence having no more than five sequence differences from SEQ ID NO: 38 (e.g., no more than 5, not more than 4, no more than 3, no more than 2, or no more than sequence differences from SEQ ID NO: 38), where a sequence difference can be an insertion, deletion, or substitution. In various embodiments, a minCMV promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 38.

[0346] In some embodiments, a promoter or regulatory sequence can be or include a minP promoter or regulatory sequence. In various embodiments, a minP promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 36 (e.g., at least 80%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 36). In various embodiments, a minP promoter or regulatory sequence can include a nucleic acid sequence having no more than five sequence differences from SEQ ID NO: 36 (e.g., no more than 5, not more than 4, no more than 3, no more than 2, or no more than sequence differences from SEQ ID NO: 36), where a sequence difference can be an insertion, deletion, or substitution. In various embodiments, a minP promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 36.

[0347] In some embodiments, a promoter or regulatory sequence can be or include a herpes simplex thymidine kinase promoter or regulatory sequence (mini TK) promoter or regulatory sequence. In various embodiments, a mini TK promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 37 (e.g., at91FH12901147.5ENO-00225 (38895-00225) least 80%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 37). In various embodiments, a mini TK promoter or regulatory sequence can include a nucleic acid sequence having no more than five sequence differences from SEQ ID NO: 37 (e.g., no more than 5, not more than 4, no more than 3, no more than 2, or no more than sequence differences from SEQ ID NO: 37), where a sequence difference can be an insertion, deletion, or substitution. In various embodiments, a mini TK promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 37.

[0348] In some embodiments, a promoter or regulatory sequence can be or include a YB_TATA promoter or regulatory sequence. In various embodiments, a YB_TATA promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 39 (e.g., at least 80%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 39). In various embodiments, a YB_TATA promoter or regulatory sequence can include a nucleic acid sequence having no more than five sequence differences from SEQ ID NO: 39 (e.g., no more than 5, not more than 4, no more than 3, no more than 2, or no more than sequence differences from SEQ ID NO: 39), where a sequence difference can be an insertion, deletion, or substitution. In various embodiments, a YB_TATA promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 39.

[0349] In some embodiments, a promoter or regulatory sequence can be or include an MLP promoter or regulatory sequence. In various embodiments, an MLP promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 40 (e.g., at least 80%, at least 90%, at least 95%, or 100% identity with SEQ ID NO: 40). In various embodiments, an MLP promoter or regulatory sequence can include a nucleic acid sequence having no more than five sequence differences from SEQ ID NO: 40 (e.g., no more than 5, not more than 4, no more than 3, no more than 2, or no more than sequence differences from SEQ ID NO: 40), where a sequence difference can be an insertion, deletion, or substitution. In various embodiments, an MLP promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 40.

[0350] In some embodiments, a promoter or regulatory sequence can be or include a CAG promoter or regulatory sequence. In various embodiments, a CAG promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 42 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 42). In various embodiments, a CAG promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 42.92FH12901147.5ENO-00225 (38895-00225)

[0351] In some embodiments, a promoter or regulatory sequence can be or include an EFla promoter or regulatory sequence. In various embodiments, an EFla promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 43 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 43). In various embodiments, an EFla promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 43.

[0352] In some embodiments, a promoter or regulatory sequence can be or include an EFla promoter or regulatory sequence. In various embodiments, an EFla promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 401 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 401). In various embodiments, an EFla promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 401.

[0353] In some embodiments, a promoter or regulatory sequence can be or include an EFS promoter or regulatory sequence. In various embodiments, an EFS promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO:44 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 44). In various embodiments, an EFS promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 44.

[0354] In some embodiments, a promoter or regulatory sequence can be or include a CMV promoter or regulatory sequence. In various embodiments, a CMV promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO:45 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 45). In various embodiments, a CMV promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 45.

[0355] In some embodiments, a promoter or regulatory sequence can be or include an hPGK promoter or regulatory sequence. In various embodiments, an hPGK promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 46 (e.g., 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%, at least 99%, or93FH12901147.5ENO-00225 (38895-00225)100% identity with SEQ ID NO: 46). In various embodiments, an hPGK promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 46.

[0356] In some embodiments, a promoter or regulatory sequence can be or include an mPGK promoter or regulatory sequence. In various embodiments, an mPGK promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 47 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 47). In various embodiments, an mPGK promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 47.

[0357] In some embodiments, a promoter or regulatory sequence can be or include an SFFV promoter or regulatory sequence. In various embodiments, an SFFV promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 48 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 48). In various embodiments, an SFFV promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 48.

[0358] In some embodiments, a promoter or regulatory sequence can be or include an SV40 promoter or regulatory sequence. In various embodiments, an SV40 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 49 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 49). In various embodiments, an SV40 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 49.

[0359] In some embodiments, a promoter or regulatory sequence can be or include a GAPDH promoter or regulatory sequence. In various embodiments, a GAPDH promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 50 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 50). In various embodiments, a GAPDH promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 50.

[0360] In some embodiments, a promoter or regulatory sequence can be or include an ACTB promoter or regulatory sequence. In various embodiments, an ACTB promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 51 (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at94FH12901147.5ENO-00225 (38895-00225) least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 51). In various embodiments, an ACTB promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 51.

[0361] In some embodiments, a promoter or regulatory sequence can be or include a UBC promoter or regulatory sequence. In various embodiments, a UBC promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO:52 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 52). In various embodiments, a UBC promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 52.

[0362] In some embodiments, a promoter or regulatory sequence can be or include a UBB promoter or regulatory sequence. In various embodiments, a UBB promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO:53 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 53). In various embodiments, a UBB promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 53.

[0363] In some embodiments, a promoter or regulatory sequence can be or include a HROSA promoter or regulatory sequence. In various embodiments, a HROSA promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 54 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 54). In various embodiments, a HROSA promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 54.

[0364] In some embodiments, a promoter or regulatory sequence can be or include an MROSA promoter or regulatory sequence. In various embodiments, an MROSA promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 55 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 55). In various embodiments, an MROSA promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 55.

[0365] In some embodiments, a promoter or regulatory sequence can be or include a KIN promoter or regulatory sequence. In various embodiments, a KIN promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO:95FH12901147.5ENO-00225 (38895-00225)56 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 56). In various embodiments, a KIN promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 56.

[0366] In some embodiments, a promoter or regulatory sequence can be or include an HEIF4A1 promoter or regulatory sequence. In various embodiments, an HEIF4A1 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 57 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 57). In various embodiments, an HEIF4A1 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 57.

[0367] In some embodiments, a promoter or regulatory sequence can be or include an MEIF4A1 promoter or regulatory sequence. In various embodiments, an MEIF4A1 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 58 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 58). In various embodiments, an MEIF4A1 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 58.

[0368] In some embodiments, a promoter or regulatory sequence can be or include an A2- SFFV promoter or regulatory sequence. In various embodiments, an A2-SFFV promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 59 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 59). In various embodiments, an A2-SFFV promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 59.

[0369] In some embodiments, a promoter or regulatory sequence can be or include an hCD45 promoter or regulatory sequence. In various embodiments, an hCD45 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 1 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 1). In various embodiments, an hCD45 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 1.

[0370] In some embodiments, a promoter or regulatory sequence can be or include an SV40en-hCD45 promoter or regulatory sequence. In various embodiments, an SV40en-96FH12901147.5ENO-00225 (38895-00225) hCD45 promoter or regulatory sequence can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 2 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 2). In various embodiments, an SV40en- hCD45 promoter or regulatory sequence can have the nucleic acid sequence set forth in SEQ ID NO: 2.Table 1. Minimal Promoter SequencesTable 2. Ubiquitous Promoters97FH12901147.5ENO-00225 (38895-00225)98FH12901147.5ENO-00225 (38895-00225)99FH12901147.5ENO-00225 (38895-00225)100FH12901147.5ENO-00225 (38895-00225)101FH12901147.5ENO-00225 (38895-00225)102FH12901147.5ENO-00225 (38895-00225)103FH12901147.5ENO-00225 (38895-00225)Table 3. Hematopoietic Promoters104FH12901147.5ENO-00225 (38895-00225)

[0371] In some embodiments, the present specification provides promoters for use in regulatory sequences that cause expression in myeloid cells. Non-limiting examples of promoters for use in regulatory sequences that cause expression in myeloid cells include the following SEQ ID NOs: 14 (CX3CR1+UTR (-222)), 15 (CX3CR1+UTR (-498)), 20 (CDl lb), 21 (CD68 ), 22 (hCD68), 23 (mCD68), 24 (CX3CR1 P3[-222]), 25 (CX3CR1 P3[-105FH12901147.5ENO-00225 (38895-00225)498]), 26 (CTSG / FES promoter ), 27 (coGP91), 28 (GP91 Intron #1), 29 (CYBB), 30 (hCD43), 31 (SV40-en-hCD43), 32 (CD 14), 33 (HBBlongLCR), 34 (HBBpLCR), 3 (WASP), 12 (CDl lb+UTR), and 13 (CD68+UTR).11(B). UTR, Intronic Sequences, and Enhancers

[0372] In some embodiments, a regulatory sequence of the present disclosure, e.g., a lineage-specific regulatory sequence, can include one or more regulatory elements including, without limitation, a UTR, intronic sequence, and / or an enhancer. In various embodiments, a UTR or intronic sequence can be included in a regulatory sequence to promote or increase mRNA stability and / or expression of an encoded expression product. In various embodiments, an enhancer can be included to increase transcription and / or translation of an operably linked coding sequence. Enhancers can be used to enhance the efficiency of mRNA translation in cells for example, to provide efficient processing and polyadenylation to boost protein expression levels. In some embodiments, an enhancer can be sequence that causes lineage-specific expression of an operably-linked coding sequence. In some embodiments, an enhancer can be sequence that does not causes lineage-specific expression, or is not included to cause lineage-specific expression, but which provides a lineage-nonspecific increase in expression (e.g., transcription) of an operably- linked coding sequence. Exemplary UTR sequences, intronic sequences, and / or enhancer sequences are provided in Table 6.

[0373] In various embodiments, UTR sequences, intronic sequences, and / or enhancer sequences, when included in a regulatory sequence, e.g., in combination with a promoter, can cause, control, modify, or direct expression of an operably linked coding sequence. For example, certain enhancers, when present a regulatory sequence with a promoter, can cause, control, modify, or direct expression of an operably linked coding sequence in one or more hematopoietic cell populations and / or HSC lineages. In some embodiments, UTR sequences, intronic sequences, and / or enhancer sequences utilized in a regulatory sequence with a promoter of the present disclosure can cause lineage-specific expression. For example, enhancers that can control (e.g., cause or contribute to) lineage-specific expression of an operably linked coding sequence in lymphoid cells (e.g., T cells, NK cells, and / or B cells) can include SV40, CMV, TNK-A, PrA, PrG, PrB, TNK-C, TNK-B, T-spe, PrF, a CMV enhancer, an SV40 enhancer, and WPRE. For example, enhancers that can provide a lineage- nonspecific increase in expression (e.g., transcription) of an operably-linked coding sequence include a CMV enhancer and an SV40 enhancer. For the avoidance of doubt, indication of a reference chromosome (e.g., in the names used to designate enhancers such as TNK-A, TNK-106FH12901147.5ENO-00225 (38895-00225)C, and TNK-B are solely used to distinguish provided sequences, and are not intended to, and do not, provide information relating to the location or context of the indicated element in any given nucleic acid construct.

[0374] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes an intronic sequence (e.g., a chimeric intronic sequence). In various embodiments, an intronic sequence (e.g., a chimeric intronic sequence) can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 60 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 60). In various embodiments, an intronic sequence (e.g., a chimeric intronic sequence) can have the nucleic acid sequence set forth in SEQ ID NO: 60.

[0375] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a UTR. In various embodiments, a UTR can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 61 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 61). In various embodiments, a UTR can have the nucleic acid sequence set forth in SEQ ID NO: 61.

[0376] In various embodiments, a UTR can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 62 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 62). In various embodiments, a UTR can have the nucleic acid sequence set forth in SEQ ID NO: 62.

[0377] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes an enhancer. Exemplary enhancers of the present disclosure can include a WPRE enhancer, an HPRE enhancer, a CTE enhancer, or a derivative or hybrid of any of one or more of the foregoing.

[0378] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a WPRE enhancer. In various embodiments, a WPRE enhancer can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 63 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 63). In various embodiments, a WPRE enhancer can have the nucleic acid sequence set forth in SEQ ID NO: 63.107FH12901147.5ENO-00225 (38895-00225)

[0379] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a polyadenylation (poly A) signal, optionally wherein the polyadenylation (poly A) signal can be a bovine growth hormone polyadenylation signal (bGH poly A), a small polyA signal (SPA), a human growth hormone polyadenylation signal (hGH poly A), a SV40 polyA signal, a SV40 late polyA signal, or a derivative or hybrid of any of one or more of the foregoing.

[0380] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a bGH polyA In various embodiments, a bGH polyA can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 64 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 64). In various embodiments, a bGH polyA can have the nucleic acid sequence set forth in SEQ ID NO: 64.

[0381] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a TNK-A. In various embodiments, a TNK-A can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 65 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 65). In various embodiments, a TNK-A can have the nucleic acid sequence set forth in SEQ ID NO: 65.

[0382] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a PrA. In various embodiments, a PrA can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 66 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 66). In various embodiments, a PrA can have the nucleic acid sequence set forth in SEQ ID NO: 66.

[0383] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a PrG. In various embodiments, a PrA can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 67 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 67). In various embodiments, a PrG can have the nucleic acid sequence set forth in SEQ ID NO: 67.

[0384] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a PrB. In various embodiments, a PrB can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 68 (e.g., at least 80%, at least 85%, at least108FH12901147.5ENO-00225 (38895-00225)90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 68). In various embodiments, a PrB can have the nucleic acid sequence set forth in SEQ ID NO: 68.

[0385] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a TNK-C. In various embodiments, a TNK-C can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 69 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 69). In various embodiments, a TNK-C can have the nucleic acid sequence set forth in SEQ ID NO: 69.

[0386] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a TNK-B. In various embodiments, a TNK-B can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 70 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 70). In various embodiments, a TNK-B can have the nucleic acid sequence set forth in SEQ ID NO: 70.

[0387] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a T-spe. In various embodiments, a T-spe can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 71 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 71). In various embodiments, a T-spe can have the nucleic acid sequence set forth in SEQ ID NO: 71.

[0388] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a PrF. In various embodiments, a PrF can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 72 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 72). In various embodiments, a PrF can have the nucleic acid sequence set forth in SEQ ID NO: 72.

[0389] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a CMV enhancer. In various embodiments, a CMV enhancer can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 73 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 73). In various embodiments, a CMV enhancer can have the nucleic acid sequence set forth in SEQ ID NO: 73.109FH12901147.5ENO-00225 (38895-00225)

[0390] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a SV40 enhancer. In various embodiments, a SV40 enhancer can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 74 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 74). In various embodiments, a SV40 enhancer can have the nucleic acid sequence set forth in SEQ ID NO: 74.

[0391] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes UTR. In various embodiments, a UTR can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 408 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 408). In various embodiments, a UTR can have the nucleic acid sequence set forth in SEQ ID NO: 408.

[0392] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes UTR. In various embodiments, a UTR can include a nucleic acid sequence having at least 80% identity with SEQ ID NO: 409 (e.g., 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%, at least 99%, or 100% identity with SEQ ID NO: 409). In various embodiments, a UTR can have the nucleic acid sequence set forth in SEQ ID NO: 409.

[0393] In some embodiments, a regulatory sequence (e.g., a lineage-specific regulatory sequence) includes a Kozak sequence.Table 4. Regulatory Elements110FH12901147.5ENO-00225 (38895-00225)111FH12901147.5ENO-00225 (38895-00225)112FH12901147.5ENO-00225 (38895-00225)11(C). Lineage-specific miRNA Binding Sites

[0394] In various embodiments, a regulatory sequence of the present disclosure can include one or more HSC-related miRNA binding sites operably linked with a nucleic acid sequence, e.g., a nucleic acid sequence encoding a therapeutic expression product.MicroRNAs (miRNA) are inhibitory RNA capable of regulating gene expression. Organisms113FH12901147.5ENO-00225 (38895-00225) naturally produce miRNA which complexes with proteins to form an RNA-induced silencing complex (RISC). The miRNA guides the RISC complex to an miRNA binding site on mRNA resulting in degradation of the mRNA. Inclusion of miRNA binding sites of endogenously expressed miRNAs in engineered nucleic acid constructs can cause regulation of encoded sequences based on expression patterns of the endogenous miRNA.

[0395] As used herein, a “HSC-related miRNA binding site” refers to a sequence that is complementary to at least a portion of an miRNA and / or that hybridizes with an miRNA (e.g., an miRNA endogenously expressed by a subject or cell type), whereby binding of the site by the miRNA decreases expression of a coding sequence operably linked with the miRNA binding site in at least one hematopoietic cell population and / or in at least one HSC lineage, but does not decrease expression, does not detectably decrease expression, does not significantly decrease expression, and / or decreases expression to a lesser degree in at least one other or reference hematopoietic cell population and / or HSC lineage. In various embodiments, a decrease in expression is measured by comparison to a reference, e.g., where expression of an expression product is measured in a cell type or population that includes a nucleic acid sequence in which one or more miRNA binding sites are operably linked with the nucleic acid encoding the expression product, and the measured expression is compared to expression of the same expression product in a reference cell type or population comprising a nucleic acid sequence that is the same or comparable except that it lacks one or more, or all, of the miRNA binding sites.

[0396] Examples of HSC-related miRNA binding sites can be found in WO 2010 / 125471 (which is incorporated by reference in its entirety and with respect to miRNA and miRNA binding site sequences). In various embodiments, a nucleic acid sequence encoding a first therapeutic expression product is operably linked with one or more HSC-related miRNA binding sites, wherein the miRNA binding sites are targeted by miRNA expressed in, e.g., HSCs. In various embodiments, a nucleic acid sequence encoding a second therapeutic expression product is operably linked with one or more HSC-related miRNA binding sites, wherein the miRNA binding sites are targeted by miRNA expressed in, e.g., HSCs. In various embodiments, a nucleic acid sequence encoding a first therapeutic expression product and a nucleic acid sequence encoding a second therapeutic expression product are each operably linked with one or more HSC-related miRNA binding sites, wherein the miRNA binding sites are targeted by an miRNA in HSCs.

[0397] In some embodiments, the targeting of the HSC-related miRNA binding sites by miRNA decreases expression in a target cell as compared to a non-target cell. In some114FH12901147.5ENO-00225 (38895-00225) embodiments, non-target cells can include, but are not limited to, B cells, myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells), T cells, NK cells, or a cell in which the provided nucleic acid constructs are manufactured. In some embodiments, a nontarget cell is a B cell. In some embodiments, a non-target cell is a myeloid cell. In some embodiments, a non-target cell is a T cell. In some embodiments, a non-target cell is a NK cell. In some embodiments, a non-target cell is a cell in which the provided nucleic acid constructs are manufactured.

[0398] In some embodiments, a HSC-related miRNA binding site is useful to suppress expression of an operably linked, encoded product in CD34+ CD90+ CD45RA- cells.

[0399] In some embodiments, one or more operably linked HSC-related miRNA binding sites cause a decrease in expression of a therapeutic expression product as compared to a reference that does not comprise the HSC-related miRNA binding sites. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause at least a 50% decrease in expression of a therapeutic expression product as compared to a reference. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause at least a 60% decrease in expression of a therapeutic expression product as compared to a reference. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause at least a 70% decrease in expression of a therapeutic expression product as compared to a reference. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause at least an 80% decrease in expression of a therapeutic expression product as compared to a reference. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause at least a 90% decrease in expression of a therapeutic expression product as compared to a reference. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause a 100% decrease in expression of a therapeutic expression product as compared to a reference.

[0400] In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 2-fold greater than the decrease observed in a non-target cell (expression in each cell type respectively measured by comparison to a reference). In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 5-fold greater than the decrease observed in a non-target cell. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 10-fold greater than the decrease observed in a non-target cell. In some embodiments, one or115FH12901147.5ENO-00225 (38895-00225) more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 20-fold greater than the decrease observed in a non-target cell. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 50-fold greater than the decrease observed in a non-target cell. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 100-fold greater than the decrease observed in a non-target cell. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 250-fold greater than the decrease observed in a non-target cell. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 500-fold greater than the decrease observed in a non-target cell. In some embodiments, one or more operably linked HSC-related miRNA binding sites cause expression of a therapeutic expression product to be decreased by at least 1000-fold greater than the decrease observed in a non-target cell.

[0401] In some embodiments, a HSC-related miRNA binding site operably linked to a nucleic acid encoding a therapeutic expression product does not cause a decrease, or does not cause as great of a decrease, in one or more non-target cell types (e.g., one or more target HSC lineages) as in one or more target cell types for suppression (e.g., one or more other, non-target HSC lineages). In some embodiments, a HSC-related miRNA binding site operably linked to a nucleic acid encoding a therapeutic expression product does not cause a decrease, or does not cause as great of a decrease, in myeloid, T, or NK cells. In some embodiments, a HSC-related miRNA binding site operably linked to a nucleic acid encoding a therapeutic expression product does not cause a decrease, or does not cause as great of a decrease, in myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells). In some embodiments, a HSC-related miRNA binding site operably linked to a nucleic acid encoding a therapeutic expression product does not cause a decrease, or does not cause as great of a decrease, in T cells. In some embodiments, a HSC-related miRNA binding site operably linked to a nucleic acid encoding a therapeutic expression product does not cause a decrease, or does not cause as great of a decrease, in NK cells.

[0402] In some embodiments, a nucleic acid sequence encoding a first therapeutic expression product is operably linked with one or more HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapeutic expression product is operably linked with 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more HSC-related116FH12901147.5ENO-00225 (38895-00225) miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapeutic expression product is operably linked with at least 1-4 HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapeutic expression product is operably linked with 2-5 HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapeutic expression product is operably linked with 3 or 4 HSC-related miRNA binding sites.

[0403] In some embodiments, a nucleic acid sequence encoding a second therapeutic expression product is operably linked with one or more HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a second therapeutic expression product is operably linked with 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a second therapeutic expression product is operably linked with at least 1-4 HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a second therapeutic expression product is operably linked with 2-5 HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a second therapeutic expression product is operably linked with 3 or 4 HSC-related miRNA binding sites.

[0404] In some embodiments, a nucleic acid sequence encoding a first therapaeutic expression product and a nucleic acid sequence encoding a second therapeutic expression product are each operably linked with one or more HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapaeutic expression product and a nucleic acid sequence encoding a second therapeutic expression product are each operably linked with 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapaeutic expression product and a nucleic acid sequence encoding a second therapeutic expression product are each operably linked with at least 1-4 HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapaeutic expression product and a nucleic acid sequence encoding a second therapeutic expression product are each operably linked with 2-5 HSC-related miRNA binding sites. In some embodiments, a nucleic acid sequence encoding a first therapaeutic expression product and a nucleic acid sequence encoding a second therapeutic expression product are each operably linked with 3 or 4 HSC-related miRNA binding sites.

[0405] In some embodiments, at least 2 HSC-related miRNA binding sites operably linked with a particular nucleic acid sequence encoding a therapeutic expression product are the same. In some embodiments, all miRNA binding sites operably linked with a particular117FH12901147.5ENO-00225 (38895-00225) nucleic acid sequence encoding a therapeutic expression product are the same. In some embodiments, at least 2 miRNA binding sites operably linked with a particular nucleic acid sequ...

Claims

1. ENO-00225 (38895-00225)CLAIMS1. A nucleic acid construct for expression of first and second expression products, comprising a nucleic acid sequence encoding a first therapeutic expression product operably linked with a first lineage-specific regulatory sequence, and a nucleic acid sequence encoding a second therapeutic expression product operably linked with a second lineage- specific regulatory sequence, wherein the first lineage- specific regulatory sequence causes expression of the first therapeutic expression product in at least a first cell population of a first HSC lineage, and wherein the second lineage-specific regulatory sequence is distinct from the first lineage-specific regulatory sequence and causes expression of the second therapeutic expression product in at least a second cell population of a second HSC lineage.

2. The nucleic acid construct of claim 1, wherein the first lineage-specific regulatory sequence and / or the second lineage-specific regulatory sequence causes expression in a cell population of an HSC lineage selected from a lymphoid lineage or a myeloid lineage.

3. The nucleic acid construct of claim 1 or 2, wherein the first lineage-specific regulatory sequence and / or the second lineage-specific regulatory sequence causes expression in one or more cell populations including at least one of T cells, NK cells, and / or myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

4. The nucleic acid construct of any one of claims 1-3, wherein the first cell population is distinct from the second cell population.

5. The nucleic acid construct of any one of claims 1-4, wherein the first HSC lineage is distinct from the second HSC lineage.

6. The nucleic acid construct of any one of claims 1-5, wherein the first lineage- specific regulatory sequence and / or second lineagespecific regulatory sequence do not cause expression, do not cause detectable expression, do not cause significant expression in HSC cells, and / or do not cause expression in HSCs at a level that is comparable to that caused by the first lineage-specific regulatory sequence and / or395FH12901147.5ENO-00225 (38895-00225) the second lineage-specific regulatory sequence in the first cell population or the second cell population, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000-fold, at least 10, 000- fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, or at least 100,000-fold less.

7. The nucleic acid construct of any one of claims 1-7, wherein the first lineagespecific regulatory sequence causes substantial expression in a cell population of a lymphoid lineage.

8. The nucleic acid construct of any one of claims 1-7, wherein the second lineagespecific regulatory sequence causes substantial expression in a cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

9. The nucleic acid construct of any one of claims 1-8, wherein the first lineagespecific regulatory sequence causes expression in a plurality of cell populations that include the a cell population of a lymphoid lineage.

10. The nucleic acid construct of any one of claims 1-9, wherein the cell population of a lymphoid lineage comprises T cells, NK cells, and / or B cells, or a combination thereof.

11. The nucleic acid construct of any one of claims 1-10, wherein the first lineagespecific regulatory sequence causes expression in at least at least T cells and NK cells.

12. The nucleic acid construct of any one of claims 1-11, wherein the first lineagespecific regulatory sequence causes expression in at least one cell population of a lymphoid lineage and at least one cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

13. The nucleic acid construct of any one of claims 1-12, wherein the second lineagespecific regulatory sequence causes expression in a cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).396FH12901147.5ENO-00225 (38895-00225)14. The nucleic acid construct of any one of claims 1-13, wherein the second lineagespecific regulatory sequence causes expression in a plurality of cell populations that include the cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

15. The nucleic acid construct of any one of claims 2-14, wherein the cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells) is selected from a monocyte, macrophage, myeloblast, granulocyte, neutrophil, eosinophil, basophil, megakaryocyte-erythroid progenitor cell, megakaryocyte, myeloid dendritic cell (mDC), monocyte-derived dendritic cell (MoDC), mast cell, platelet, and / or erythrocyte.

16. The nucleic acid construct of any one of claims 1-15, wherein the second lineagespecific regulatory sequence causes expression in at least one cell population of a lymphoid lineage and at least one cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells).

17. The nucleic acid construct of any one of claims 1-16, wherein the first lineagespecific regulatory sequence causes expression in at least NK cells and a cell population of a myeloid lineage (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells), optionally wherein the first lineage-specific regulatory sequence causes expression in at least NK cells and macrophages.

18. The nucleic acid construct of any one of claims 2-17, wherein the T cells and / or cells of a lymphoid lineage comprise one or more of cytotoxic CD8+ T cells, and / or helper CD4+ T cells.

19. The nucleic acid construct of any one of claims 2-18, wherein the NK cells and / or cells of a lymphoid lineage comprise one or more of CD56brightNK cells, and / or CD56dimNK cells.

20. The nucleic acid construct of any one of claims 2-19, wherein the myeloid cells (e.g., macrophages, monocytes, neutrophils, and / or dendritic cells) and / or cells of a myeloid lineage comprise monocytes, macrophages, myeloblasts, granulocytes, neutrophils, eosinophils, basophils, megakaryocyte-erythroid progenitor cells, megakaryocytes, myeloid397FH12901147.5ENO-00225 (38895-00225) dendritic cells (mDCs), monocyte-derived dendritic cells (MoDCs), mast cells, platelets, and / or erythrocytes.

21. The nucleic acid construct of any one of claims 1-20, wherein the first lineagespecific regulatory sequence causes expression of the first expression product in the first cell population that is at least 5 -fold greater in the first cell population than in a reference cell population, optionally wherein expression of the first expression product in the first cell population is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50- fold, at least 100-fold, at least 500-fold, at least 1000-fold, or at least 10000-fold greater in the first cell population than in the reference cell population, optionally wherein the reference cell population is a hematopoietic stem cell (HSC), an erythrocyte, platelet, LT-HSC (CD34+CD90+CD45RA- HSC), NK cell, T cell, B cell, or myeloid cell.

22. The nucleic acid construct of any one of claims 1-21, wherein the second lineagespecific regulatory sequence causes expression of the second expression product in the second cell population that is at least 5-fold greater in the second cell population than in a reference cell population, optionally wherein expression of the second expression product in the second cell population is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or at least 10000-fold greater in the second cell population than in the reference cell population, optionally wherein the reference cell population is a hematopoietic stem cell (HSC), erythrocyte, platelet, LT- HSC (CD34+CD90+CD45RA- HSC), NK cell, T cell, B cell, or myeloid cell. .

23. The nucleic acid construct of any one of claims 1-22, wherein, in erythroid cells, on the surface of erythroid cells, and / or in platelets, the first lineage- specific regulatory sequence does not cause expression, does not cause detectable expression, does not cause significant expression, and / or does not cause expression at a level that is comparable to that caused by the first lineage-specific regulatory sequence in the first cell population, optionally wherein expression is not comparable if expression is at least 100-fold less, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000-fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40, 000- fold, at least 50,000-fold, or at least 100, 000- fold less.398FH12901147.5ENO-00225 (38895-00225)24. The nucleic acid construct of any one of claims 1-23, wherein, in erythroid cells, on the surface of erythroid cells, and / or in platelets, the second lineage- specific regulatory sequence does not cause expression, does not cause detectable expression, does not cause significant expression, and / or does not cause expression at a level that is comparable to that caused by the second lineage-specific regulatory sequence in the second cell population, optionally wherein expression is not comparable if expression is at least 100-fold less, optionally wherein expression is not comparable if expression is at least 500-fold, at least 1000- fold, at least 10,000-fold, at least 20,000-fold, at least 30,000-fold, at least 40,000-fold, at least 50,000-fold, or at least 100,000-fold less.

25. The nucleic acid construct of any one of claims 1-24, wherein the first lineagespecific regulatory sequence and / or the second lineage-specific regulatory sequence comprises at least one enhancer, at least one promoter, at least one untranslated region (UTR), at least one intron, and / or at least one regulatory moiety binding site, optionally wherein the regulatory moiety comprises a transcription factor.

26. The nucleic acid construct of any one of claims 1-25, wherein the first lineagespecific regulatory sequence and / or the second lineage-specific regulatory sequence comprises a promoter, or wherein the first lineage-specific regulatory sequence and / or the second lineage-specific regulatory sequence comprises a promoter and a UTR.

27. The nucleic acid construct of any one of claims 1-26, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises a CD3d promoter (CD3dp), or a LCK promoter (LCKp), optionally wherein the first lineage-specific regulatory sequence comprises a CD3d promoter (CD3dp), or a LCK promoter (LCKp).

28. The nucleic acid construct of any one of claims 1-27, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises a promoter or regulatory sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 4-10, optionally wherein the first lineage-specific regulatory sequence comprises a promoter or regulatory sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 4-10.399FH12901147.5ENO-00225 (38895-00225)29. The nucleic acid construct of any one of claims 1-28, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises a minimal CMV promoter (minCMV).

30. The nucleic acid construct of any one of claims 1-29, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises a CMV promoter or a promoter derived therefrom having at least 80% sequence identity with SEQ ID NO: 45 or a portion thereof, optionally wherein the first lineage-specific regulatory sequence comprises a promoter having at least 80% sequence identity with SEQ ID NO: 45 or a portion thereof.

31. The nucleic acid construct of any one of claims 1-30, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises an enhancer selected from PrF, PrE, TNK-C, TNK-A, PrG, PrA, or PrB, optionally wherein the first lineage-specific regulatory sequence comprises an enhancer selected from PrF, PrE, TNK-C, TNK-A, PrG, PrA, or PrB.

32. The nucleic acid construct of any one of claims 1-31, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 65-76, optionally wherein the first lineage-specific regulatory sequence comprises an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 65-76.

33. The nucleic acid construct of any one of claims 1-32, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises a CDl lb promoter, CD68 promoter, CX3CR1-P3 (-222) promoter, or CX3CR1-P3 (-498) promoter, optionally wherein the second lineage-specific regulatory sequence comprises a CDl lb promoter, CD68 promoter, CX3CR1-P3 (-222) promoter, or CX3CR1-P3 (-498) promoter.400FH12901147.5ENO-00225 (38895-00225)34. The nucleic acid construct of any one of claims 1-33, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises a promoter having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 20, 21, 24, and 25, optionally wherein the second lineage-specific regulatory sequence comprises a promoter having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 20, 21, 24, and 25.

35. The nucleic acid construct of any one of claims 1-34, wherein the second lineagespecific regulatory sequence comprises a CMV promoter or a promoter derived therefrom, optionally wherein the second lineage-specific regulatory sequence comprises theCMV promoter or a promoter derived therefrom, optionally wherein the CMV promoter or promoter derived therefrom is a minimal CMV promoter (minCMV).

36. The nucleic acid construct of any one of claims 1-35, wherein the second lineagespecific regulatory sequence comprises a CMV promoter or a promoter derived therefrom having at least 80% sequence identity with SEQ ID NO: 45.

37. The nucleic acid construct of any one of claims 1-36, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises an enhancer selected from SV40, CMV, TNK-A, PrA, PrG, PrB, TNK-C, TNK-B, T-spe, PrF, a CMV enhancer, an SV40 enhancer, and WPRE, optionally wherein the second lineage-specific regulatory sequence comprises an enhancer selected from SV40, CMV, TNK-A, PrA, PrG, PrB, TNK-C, TNK-B, T-spe, PrF, a CMV enhancer, an SV40 enhancer, and WPRE.

38. The nucleic acid construct of any one of claims 1-37, wherein the first lineagespecific regulatory sequence or the second lineage-specific regulatory sequence comprises an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 63 and 65-72, optionally wherein the second lineage-specific regulatory sequence comprises an enhancer having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 63 and 65-72.401FH12901147.5ENO-00225 (38895-00225)39. The nucleic acid construct of any one of claims 1-38, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises an agent that, when expressed in a cell of a subject having a disease, disorder, or condition, directly or indirectly causes or contributes to the treatment of the disease, disorder, or condition, or of a symptom thereof.

40. The nucleic acid construct of any one of claims 1-39, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises a protein, peptide, nucleic acid aptamer, or ribonucleic acid (RNA), optionally wherein the RNA comprises an inhibitory RNA.

41. The nucleic acid construct of any one of claims 1-40, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises an antigenbinding agent comprising an antigen-binding domain.

42. The nucleic acid construct of any one of claims 1-41, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises an antigenbinding agent comprising a Chimeric Antigen Receptor (CAR), T Cell Receptor (TCR), or antibody.

43. The nucleic acid construct of any one of claims 1-42, wherein the first therapeutic expression product comprises a CAR, TCR, or antibody.

44. The nucleic acid construct of any one of claims 1-43, wherein the first therapeutic expression product comprises a CAR.

45. The nucleic acid construct of any one of claims 1-44, wherein the second therapeutic expression product comprises a CAR, TCR, or antibody.

46. The nucleic acid construct of any one of claims 1-45, wherein the second therapeutic expression product comprises a CAR.402FH12901147.5ENO-00225 (38895-00225)47. The nucleic acid construct of any one of claims 1-46, wherein the first therapeutic expression product comprises an antigen-binding domain that binds an antigen that is not bound by the second therapeutic expression product.

48. The nucleic acid construct of any one of claims 1-47, wherein the second therapeutic expression product comprises an antigen-binding domain that binds an antigen that is not bound by the first therapeutic expression product.

49. The nucleic acid construct of any one of claims 1-48, wherein the first therapeutic expression product comprises an antigen-binding domain that is not comprised by the second therapeutic expression product.

50. The nucleic acid construct of any one of claims 1-49, wherein the second therapeutic expression product comprises an antigen-binding domain that is not comprised by the first therapeutic expression product.

51. The nucleic acid construct of any one of claims 1-46, wherein the first therapeutic expression product comprises a first antigen-binding domain and the second therapeutic expression product comprises a second antigen-binding domain, wherein the first and second antigen-binding domains bind with the same antigen.

52. The nucleic acid construct of any one of claims 1-46, wherein the first therapeutic expression product comprises a first antigen-binding domain and the second therapeutic expression product comprises a second antigen-binding domain, wherein the first and second antigen-binding domains have the same amino acid sequence.

53. The nucleic acid construct of any one of claims 1-52, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises an antigenbinding agent that binds a cancer-specific antigen or cancer-associated antigen, optionally wherein the cancer-specific antigen or cancer-associated antigen is characteristic of a solid tumor or of a liquid or hematological cancer.

54. The nucleic acid construct of any one of claims 1-53, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises an antigen-403FH12901147.5ENO-00225 (38895-00225) binding agent that binds an antigen selected from HER2 (human epidermal growth factor receptor 2), CD19, CD20, CD22, CD 19 and CD 20, CD 19 and CD 22, AFP (alphafetoprotein), AXL (AXL receptor tyrosine kinase), BCMA, B7-H3, CD5, CD7, CD33, CD38, CD47, CD52, CD123, CD133, CD138, CD171, CD171, CD30, CD38 / CD123, CD80 / 86, CEA (carcinoembryonic antigen), Claudin 18.2, CLL-1, c-MET, DLL-3 (delta-like 3), DR5 (death receptor 5), EGFR (epidermal growth factor receptor), EGFR806, EGFRIII, EGFRVIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EpHA2 (EPH receptor A2), FAP, FLT3, FCRL5, FR-a (folate receptor alpha), GD2 (disganglioside molecule), Glypican-3, gplOO, GPC3 (glypican 3), GPRC5D, IL- 13Ra2, Lewis Y, LMP1 (Epstein-Barr virus latent membrane protein 1), MAGE (melanoma antigen gene protein)-Al / 3 / 4, Mesothelin, Mesothelin, MUC1 (mucinl), MUC16 (mucinl6), Nectin4 / FAP (familial adenomatous polyposis), NKGD2 (natural killer group 2 member D), PD-L1, PMSA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), PSA, ROR1 (receptor tyrosine kinase-like orphan receptor 1), SIRPy, TPD52, VEGFRII (vascular endothelial growth factor receptor II), and / or VISTA.

55. The nucleic acid construct of any one of claims 1-54, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises a HER2 CAR.

56. The nucleic acid construct of claim 55, wherein the HER2 CAR comprises an antigen binding domain having at least 80% sequence identity with SEQ ID NO: 87.

57. The nucleic acid construct of claim 55, wherein the HER2 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 141.

58. The nucleic acid construct of claim 55, wherein the HER2 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 131.

59. The nucleic acid construct of claim 55, wherein the HER2 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 133.404FH12901147.5ENO-00225 (38895-00225)60. The nucleic acid construct of claim 55, wherein the HER2 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 135.

61. The nucleic acid construct of claim 55, wherein the HER2 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 137.

62. The nucleic acid construct of claim 55, wherein the HER2 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 139.

63. The nucleic acid construct of any one of claims 1-54, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises an anti-CD19 CAR.

64. The nucleic acid construct of claim 63, wherein the anti- CD 19 CAR comprises an antigen binding domain having at least 80% sequence identity with SEQ ID NO: 163.

65. The nucleic acid construct of claim 63, wherein the anti- CD 19 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 163.

66. The nucleic acid construct of any one of claims 1-54, wherein the first therapeutic expression product and / or the second therapeutic expression product comprises an anti-CD20 CAR.

67. The nucleic acid construct of claim 66, wherein the anti- CD20 CAR comprises an antigen binding domain having at least 80% sequence identity with SEQ ID NO: 169.

68. The nucleic acid construct of claim 66, wherein the anti- CD20 CAR comprises a sequence having at least 80% sequence identity with SEQ ID NO: 169.

69. The nucleic acid construct of any one of claims 1-68, wherein the first expression product and / or the second expression product comprise a synthetic receptor.

70. The nucleic acid construct of claim 69, wherein the synthetic receptor comprises a synNotch receptor, optionally wherein the synNotch receptor is engineered such that405FH12901147.5ENO-00225 (38895-00225) proteolysis of membrane-bound synNotch receptor generates an intracellular transcription factor.

71. The nucleic acid construct of any one of claims 1-70, wherein the nucleic acid construct comprises at least a first immunomodulatory domain.

72. The nucleic acid construct of claim 71, wherein the first immunomodulatory domain comprises a pro-inflammatory cytokine and / or wherein the first immunomodulatory domain comprises hGMCSF, hIL-18, hlFNa, truncated TGFbR II (dnTGFBr II), TGFbR2 / IFNgRl switch receptor, TGFbR2 / MyD88 / CD40 switch receptor, IL-2, IFNy, or GMCSF.

73. The nucleic acid construct of claim 71 or 72, wherein the first immunomodulatory domain comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 176, 178, 180, 182, 184, and 186.

74. The nucleic acid construct of any one of claims 71-73, wherein the first immunomodulatory domain is operably linked with the first lineage-specific regulatory sequence.

75. The nucleic acid construct of any one of claims 71-74, wherein the first immunomodulatory domain and the first therapeutic expression product are encoded in a single open reading frame.

76. The nucleic acid construct of claim 75, wherein the first immunomodulatory domain and the first therapeutic expression product are separated during or after translation of the single open reading frame.

77. The nucleic acid construct of claim 75 or 76, wherein the single open reading frame encodes a cleavable linker or self-cleaving peptide positioned between the sequence encoding a first immunomodulatory peptide and the sequence encoding the first expression product in the single open reading frame.406FH12901147.5ENO-00225 (38895-00225)78. The nucleic acid construct of any one of claims 71-73, wherein the first immunomodulatory domain is not operably linked with the first lineage-specific regulatory sequence and is operably linked with a third regulatory sequence.

79. The nucleic acid construct of claim 78, wherein the third regulatory sequence comprises a lineage-specific regulatory sequence, or wherein the third regulatory sequence comprises a ubiquitous regulatory sequence.

80. The nucleic acid construct of any one of claims 1-79, wherein the nucleic acid construct comprises at least a second immunomodulatory domain.

81. The nucleic acid construct of claim 80, wherein a second immunomodulatory domain comprises a pro-inflammatory cytokine and / or wherein the second immunomodulatory domain comprises hGMCSF, hIL-18, hlFNa, truncated TGFbR II (dnTGFBr II), TGFbR2 / IFNgRl switch receptor, TGFbR2 / MyD88 / CD40 switch receptor, IL-2, IFNy, or GMCSF.

82. The nucleic acid construct of claim 80 or 81, wherein a second immunomodulatory domain comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 176, 178, 180, 182, 184, and 186.

83. The nucleic acid construct of any one of claims 80-82, wherein a second immunomodulatory domain is operably linked with the second lineage-specific regulatory sequence.

84. The nucleic acid construct of any one of claims 80-83, wherein the second immunomodulatory domain and the second therapeutic expression protein are encoded in a single open reading frame.

85. The nucleic acid construct of claim 84, wherein the second immunomodulatory domain and the second therapeutic expression protein are separated during or after translation of the single open reading frame.407FH12901147.5ENO-00225 (38895-00225)86. The nucleic acid construct of claim 84 or 85, wherein the single open reading frame encodes a cleavable linker or self-cleaving peptide positioned between the sequence encoding the second immunomodulatory peptide and the sequence encoding the second therapeutic expression product in the single open reading frame.

87. The nucleic acid construct of any one of claims 80-82, wherein the second immunomodulatory domain is not operably linked with the second lineage-specific regulatory sequence and is operably linked with a fourth regulatory sequence.

88. The nucleic acid construct of claim 87, wherein the fourth regulatory sequence comprises a lineage-specific regulatory sequence, or wherein the fourth regulatory sequence comprises a ubiquitous regulatory sequence.

89. The nucleic acid construct of any one of claims 1-88, wherein the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with one or more HSC-related miRNA binding sites.

90. The nucleic acid of claim 89, wherein each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites.

91. The nucleic acid construct of claim 89 or claim 90, wherein each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with at least 1-4 HSC-related miRNA binding sites.

92. The nucleic acid construct of any one of claims 89-91, wherein each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with 2-4 HSC-related miRNA binding sites.

93. The nucleic acid construct of any one of claims 89-92, wherein each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the408FH12901147.5ENO-00225 (38895-00225) second therapeutic expression is operably linked with 3 or 4 HSC-related miRNA binding sites.

94. The nucleic acid construct of any one of claims 89-93, wherein each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with a plurality of HSC-related miRNA binding sites, and wherein each binding site is the same.

95. The nucleic acid construct of any one of claims 89-93, wherein each of the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with a plurality of HSC-related miRNA binding sites, and wherein the binding sites are different.

96. The nucleic acid construct of any one of claims 1-88, wherein the nucleic acid encoding the first therapeutic expression product is operably linked with one or more HSC- related miRNA binding sites.

97. The nucleic acid construct of any one of claims 1-88 or 96, wherein the nucleic acid encoding the first therapeutic expression product is operable linked with 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites.

98. The nucleic acid construct of claim 96 or claim 97, wherein the nucleic acid encoding the first therapeutic expression product is operably liked with at least 1-4 HSC- related miRNA binding sites.

99. The nucleic acid construct of any one of claims 95-98, wherein the nucleic acid encoding the first therapeutic expression product is operably linked with 2-4 HSC-related miRNA binding sites.

100. The nucleic acid construct of any one of claims 95-99, wherein the nucleic acid encoding the first therapeutic expression product is operably linked with 3 or 4 HSC-related miRNA binding sites.409FH12901147.5ENO-00225 (38895-00225)101. The nucleic acid construct of any one of claims 1-100, wherein the nucleic acid encoding the second therapeutic expression product is operably linked with one or more HSC-related miRNA binding sites.

102. The nucleic acid construct of claim 101, wherein the nucleic acid encoding the second therapeutic expression product is operably linked with 2 or more, 3 or more, 4 or more, or 5 or more HSC-related miRNA binding sites.

103. The nucleic acid construct of claim 101 or 102, wherein the nucleic acid encoding the second therapeutic expression product is operably linked with at least 1-4 HSC-related miRNA binding sites.

104. The nucleic acid construct of any one of claims 101-103, wherein the nucleic acid encoding the second therapeutic expression product is operably linked with 2-4 HSC-related miRNA binding sites.

105. The nucleic acid construct of any one of claims 101-104, wherein the nucleic acid encoding the second therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is operably linked with 3 or 4 HSC-related miRNA binding sites.

106. The nucleic acid construct of any one of claims 89-105, wherein the miRNA binding site is 3’ to the nucleic acid encoding the therapeutic expression product and 5’ to the poly adenylation site.

107. The nucleic acid construct of any one of claims 89-112, wherein at least 1 of the HSC-related miRNA binding sites is an miRNA binding site selected from a miR126 binding site, a miR218 binding site, a miR183 binding site, a miR223 binding site, a miR130a binding site, a miR486 binding site, or a miRlOa binding site.

108. The nucleic acid construct of claim 107, wherein at least one of the HSC-related miRNA binding sites is a miR126 binding site.410FH12901147.5ENO-00225 (38895-00225)109. The nucleic acid construct of claim 107, wherein at least one of the HSC-related miRNA binding sites is a miR218 binding site.

110. The nucleic acid construct of claim 107, wherein at least one of the HSC-related miRNA binding sites is a miR183 binding site.

111. The nucleic acid construct of claim 107, wherein at least one of the HSC-related miRNA binding sites is a miR223 binding site.

112. The nucleic acid construct of claim 107, wherein at least one of the HSC-related miRNA binding sites is a miR130a binding site.

113. The nucleic acid construct of claim 107, wherein at least one of the HSC-related miRNA binding sites is a miR486 binding site.

114. The nucleic acid construct of claim 107, wherein at least one of the HSC-related miRNA binding sites is a miRlOa binding site.

115. The nucleic acid construct of any one of claims 89-114, wherein all HSC-related miRNA binding sites are a miR126 binding site, a miR218 binding site, a miR183 binding site, a miR223 binding site, a miR130a binding site, a miR486 binding site, or a miRlOa binding site.

116. The nucleic acid construct of any one of claims 89-115, wherein all HSC-related miRNA binding sites are a miR126 binding site.

117. The nucleic acid construct of any one of claims 89-116, wherein at least 2 or more HSC-related miRNA binding sites are the same.

118. The nucleic acid construct of any one of claims 89-115, wherein at least 2 or more HSC-related miRNA binding sites are different.

119. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR126 binding site is set forth in SEQ ID NO: 381.411FH12901147.5ENO-00225 (38895-00225)120. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR126 binding site comprises at least 90% identity to the sequence set forth in SEQ ID NO: 381.

121. The nucleic acid construct of any one of claims 89-120, wherein the one or more HSC-related miRNA binding sites comprise a 4x miR126 binding site.

122. The nucleic acid construct of claim 121, wherein the sequence of the 4x miR126 binding site is set forth in SEQ ID NO: 388.

123. The nucleic acid construct of claim 121, wherein the sequence of the 4x miR126 binding site comprises at least 90% identity to the sequence set forth in SEQ ID NO: 388.

124. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR218 binding site is set forth in SEQ ID NO: 384.

125. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR218 binding site comprises at least 90% identity to the sequence set forth in SEQ ID NO: 384.

126. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR183 binding site is set forth in SEQ ID NO: 385.

127. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR183 binding site comprises at least 90% identity to the sequence set forth in SEQ ID NO: 385.

128. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR223 binding site is set forth in SEQ ID NO: 382.

129. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR223 binding site comprises at least 90% identity to the sequence set forth in SEQ ID NO: 382.412FH12901147.5ENO-00225 (38895-00225)130. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR130a binding site is set forth in SEQ ID NO: 383.

131. The nucleic acid construct of any one of claims 89-118, wherein the sequence of the miR130a binding site comprises at least 90% identity to the sequence set forth in SEQ ID NO: 383.

132. The nucleic acid construct of any one of claims 89-131, wherein the one or more HSC-related miRNA binding sites cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked in one or more target cell types for suppression, as compared to a reference.

133. The nucleic acid construct of claim 132, wherein the decrease in expression is at least 50%.

134. The nucleic acid construct of claim 132 or claim 133, wherein the decrease in expression isat least 60%, at least 70%, at least 80%, at least 90%, or 100%.

135. The nucleic acid construct of any one of claims 132-134, wherein the reference comprises a cell or population of cells of the one or more target cell types for suppression, in which the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is not operably linked with the one or more HSC-related miRNA binding sites.

136. The nucleic acid construct of any one of claims 132-135, wherein the one or more target cell types for suppression comprise HSCs.

137. The nucleic acid construct of any one of claims 89-136, wherein the one or more HSC-related miRNA binding sites do not cause a decrease in the expression of the nucleic acid sequence(s) with which they are operably linked in one or more non-target cell types.

138. The nucleic acid construct of any one of claims 89-137, wherein the one or more HSC-related miRNA binding sites do not cause a decrease in the expression of the nucleic413FH12901147.5ENO-00225 (38895-00225) acid sequence(s) with which they are operably linked to as a great a degree in one or more non-target cell types as in the one or more target cell types for suppression.

139. The nucleic acid construct of claim 138, wherein the one or more target cell types for suppression comprise HSCs.

140. The nucleic acid construct of claim 138 or 139, wherein the non-target cell type comprises a myeloid cell.

141. The nucleic acid construct of any one of claims 138-140, wherein the non-target cell type comprises a T cell.

142. The nucleic acid construct of any one of claims 138-141, wherein the non-target cell type comprises a NK cell.

143. The nucleic acid construct of any one of claims 138-142, wherein the target cell types for suppression comprise an HSC.

144. The nucleic acid construct of any one of claims 138-143, wherein the one or more non-target cell types comprise: a myeloid cell, optionally wherein the target cell types for suppression comprises an HSC.

145. The nucleic acid construct of any one of claims 138-143, wherein the one or more non-target cell types comprise: a T cell, optionally wherein the target cell types for suppression comprises an HSC.

146. The nucleic acid construct of any one of claims 138-143, wherein the one or more non-target cell types comprise: an NK cell, optionally wherein the target cell types for suppression comprises an HSC.

147. The nucleic acid construct of any one of claims 138-146, wherein the one or more non-target cell types comprise a T cell, a NK cell, and a meyloid cell.414FH12901147.5ENO-00225 (38895-00225)148. The nucleic acid construct of any one of claims 138-147, wherein the target cell types for suppression comprises an HSCs.

149. The nucleic acid construct of any one of claims 138-148, wherein the one or more non-target cell types comprise a T cell, a NK cell, and a meyloid cell.

150. The nucleic acid construct of any one of claims 138-149, wherein the target cell types for suppression comprises an HSCs.

151. The nucleic acid construct of any one of claims 132-150, wherein the decrease in expression in the one or more target cell types for suppression as compared to a target reference is at least 50% greater than the decrease in expression in the one or more non-target cell types as compared to a non-target reference.

152. The nucleic acid construct of any one of claims 132-151, wherein the decrease in expression in the one or more target cell types for suppression as compared to a target reference is at least 50%, at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 50- fold, at least a 100-fold, or at least a 1,000-fold greater than the decrease in expression in the one or more non-target cell types as compared to a non-target reference.

153. The nucleic acid construct of claim 152, wherein the target reference comprises a cell or population of cells of the one or more target cell types for suppression, in which the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is not operably linked with the one or more HSC-related miRNA binding sites, and / or wherein the non-target reference comprises a cell or population of cells of the one or more non-target cell types, in which the nucleic acid encoding the first therapeutic expression product and / or the nucleic acid encoding the second therapeutic expression is not operably linked with the one or more HSC-related miRNA binding sites.

154. The nucleic acid construct of any one of claims 1-153, wherein the nucleic acid construct comprises one or more coding sequences each independently operably linked with one or more vaRNA binding sites, optionally wherein the vaRNA binding sites comprise a vaRNAl binding site and / or a vaRNA2 binding site,415FH12901147.5ENO-00225 (38895-00225) optionally wherein the one or more coding sequences each independently operably linked with one or more vaRNA binding sites comprise one or more of the nucleic acid sequence encoding the first therapeutic expression product, the nucleic acid sequence encoding the second therapeutic expression product, and / or the sequence encoding the enrichment marker.

155. The nucleic acid construct of any one of claims 1-153, wherein the nucleic acid construct comprises one or more coding sequences each independently operably linked with a chicken beta actin 5’ UTR intron sequence, optionally wherein the one or more coding sequences each independently operably linked with the chicken beta actin 5’ UTR intron sequence comprise one or more of the nucleic acid sequence encoding the first therapeutic expression product, the nucleic acid sequence encoding the second therapeutic expression product, and / or the sequence encoding the enrichment marker.

156. The nucleic acid construct of any one of claims 1-155, wherein the nucleic acid construct comprises a nucleic acid sequence encoding an enrichment marker, optionally wherein the enrichment marker is an inhibitor-resistant MGMT protein, an expression product that decreases expression of c-kit, or an expression product that decreases expression of CD33.

157. The nucleic acid construct of claim 156, wherein the enrichment marker comprises an inhibitor-resistant MGMT protein, optionally wherein the inhibitor-resistant MGMT protein is an MGMT protein that includes the mutation P140K (MGMTP14OK).

158. The nucleic acid construct of claim 156, wherein MGMTP14OKcomprises an amino acid sequence having at least 80% identity with SEQ ID NO: 188.

159. The nucleic acid construct of claim 156, wherein the enrichment marker comprises a signaling-enhanced EpoR protein.

160. The nucleic acid construct of any one of claims 156-159, wherein the enrichment marker is operably linked with a regulatory sequence.416FH12901147.5ENO-00225 (38895-00225)161. The nucleic acid construct of any one of claims 156-160, wherein the enrichment marker is not operably linked with the first lineage-specific regulator sequence or the second lineage-specific regulatory sequence.

162. The nucleic acid construct of any one of claims 156-160, wherein the enrichment marker is not operably linked with the third regulatory sequence and / or the fourth regulatory sequence.

163. The nucleic acid construct of any one of claims 156-160, wherein the enrichment marker is operably linked with a fifth regulatory sequence.

164. The nucleic acid construct of any one of claims 156-163, wherein the enrichment marker is operably linked with a regulatory sequence comprising a ubiquitous promoter.

165. The nucleic acid construct of claim 164, wherein the ubiquitous promoter comprises an EFla promoter, a UBC promoter, an SFFV promoter, a CAG promoter, a CMV promoter, or a CMV-derived promoter, optionally wherein the CMV-derived promoter is a minCMV.

166. The nucleic acid construct of any one of claims 1-165, wherein the nucleic acid construct comprises a safety switch.

167. The nucleic acid construct of claim 166, wherein the safety switch comprises tEGFR EGFR, and GMCSF.

168. The nucleic acid construct of claim 166 or 167, wherein the safety switch comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NOs:194, 196, and 198.

169. The nucleic acid construct of any one of claims 166-168, wherein the safety switch is operably linked with a regulatory sequence.

170. The nucleic acid construct of any one of claims 166-169, wherein the safety switch is operably linked with the fifth regulatory sequence.417FH12901147.5ENO-00225 (38895-00225)171. The nucleic acid construct of any one of claims 166-170, wherein the safety switch and the enrichment marker are encoded in a single open reading frame.

172. The nucleic acid construct of claim 171, wherein the wherein the safety switch and the enrichment marker are separated during or after translation of the single open reading frame.

173. The nucleic acid construct of claim 171 or 172, wherein the single open reading frame encodes a cleavable linker or self-cleaving peptide positioned between the sequence encoding the safety switch and the sequence encoding the enrichment marker in the single open reading frame.

174. The nucleic acid construct of any one of claims 166-168, wherein the safety switch is not operably linked with the fifth regulatory sequence and is operably linked with a sixth regulatory sequence.

175. The nucleic acid construct of claim 174, wherein the sixth regulatory sequence comprises a lineage-specific regulatory sequence, or wherein the fourth regulatory sequence comprises a ubiquitous regulatory sequence.

176. The nucleic acid construct of any one of claims 1-175, wherein the nucleic acid construct is engineered for integration into a target genome of a cell or subject by transposition.

177. The nucleic acid construct of claim 176, wherein the nucleic acid construct is flanked by transposon inverted repeats (IRs), optionally wherein the transposon IRs are flanked by recombinase direct repeats (DRs).

178. The nucleic acid construct of claim 177, wherein the transposon IRs are Sleeping Beauty (SB), piggyback, Mariner, frog prince, Tol2, TcBuster, or spinON IRs179. The nucleic acid construct of claim 178, wherein the transposon IRs are Sleeping Beauty (SB) IRs, optionally wherein the transposase is a Sleeping Beauty (SB) transposase,418FH12901147.5ENO-00225 (38895-00225) optionally wherein the transposase is Sleeping Beauty lOOx (SB100X) or Sleeping Beauty 150x (SB150X).

180. The nucleic acid construct of claim 178 or 179, wherein the SB IRs are pT4 IRs.

181. The nucleic acid construct of any one of claims 177-180, wherein the recombinase DRs that flank the transposon IRs are FRT, loxP, rox, vox, AttB, or AttP sites, optionally the recombinase DRs that flank the transposon IRs are FRT sites.

182. The nucleic acid construct of any one of claims 1-181, wherein the nucleic acid construct comprises:A) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR); (ii) a lineage-specific regulatory sequence that causes expression in a myeloid cell, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineagespecific miRNA binding sites; and (iii) a ubiquitous regulatory sequence operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence;B) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR); (ii) a lineage-specific promoter that causes expression in a myeloid cell, where the lineage-specific promoter is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage-specific miRNA binding sites; and (iii) a ubiquitous promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence;C) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), and further comprising a 5’ intron, where the419FH12901147.5ENO-00225 (38895-00225) lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), and wherein the sequence encoding the therapeutic expression product is operably linked with four miR126 binding sites; (ii) a CD1 lb promoter operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage-specific miRNA binding sites; and (iii) an EFla promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence;D) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), and further comprising a 5’ intron, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR; (ii) a CD 11b promoter operably linked with a sequence encoding a HER2 CAR, which sequence encoding a HER2 CAR is operably linked with four miR126 binding sites; and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence);E) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A) and futher comprising a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR having the structure [IgGk leader- (VL- whitlow linker- VH)-CD28 (Hinge-TM-ICD)-CD3z]; (ii) a CD 11b promoter operably linked with a sequence encoding a HER2 CAR having the structure [CD8 leader- VH-g4S-VL-CD8 (Hinge-TM)-CD3z], which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa); and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP140Kselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence);F) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A set forth in SEQ ID NO: 65 and further comprising a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably420FH12901147.5ENO-00225 (38895-00225) linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 148; (ii) a CD1 lb promoter set forth in SEQ ID NO: 20 operably linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 146, which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa); and (iii) an EFla promoter set forth in SEQ ID NO: 43 operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch set forth in SEQ ID NO: 195, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence set forth in SEQ ID NO: 225);G) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage- specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), where a chicken beta actin 5’ UTR intron sequence is ;located 3’ of the lineage-specific regulatory sequence and 5’ to the coding sequence of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a lineage- specific regulatory sequence that causes expression in a myeloid cell, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage- specific miRNA binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (iii) a ubiquitous regulatory sequence operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence, optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);H) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells, where the lineage-specific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), where a chicken beta actin 5’ UTR intron sequence is ;located 3’ to the lineage-specific regulatory421FH12901147.5ENO-00225 (38895-00225) sequence and 5’ to the coding sequence of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a lineage-specific promoter that causes expression in a myeloid cell, where the lineage-specific promoter is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineagespecific miRNA binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) a ubiquitous promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence, optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);I) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A)and further comprising a 5’ intron, where the lineagespecific regulatory sequence is operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), and wherein the sequence encoding the therapeutic expression product is operably linked with four miR126 binding sites, where a chicken beta actin 5’ UTR intron is located 3’ to the lineage-specific regulatory sequence and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CD1 lb promoter operably linked with a sequence encoding a therapeutic expression product (e.g., a CAR, e.g., a HER2 CAR), which sequence encoding a therapeutic expression product can optionally be operably linked with one or more lineage-specific miRNA binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter operably linked with a sequence encoding a selectable marker and a safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence, optionally where the sequence encoding the422FH12901147.5ENO-00225 (38895-00225) selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);J) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), andfurther comprising a 5’ intron, where the lineagespecific regulatory sequence is operably linked with a sequence encoding a HER2 CAR, where a chicken beta actin 5’ UTR intron is located 3’ to the lineage-specific regulatory sequence and and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CD1 lb promoter operably linked with a sequence encoding a HER2 CAR, which sequence encoding a HER2 CAR is operably linked with four miR126 binding sites, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence), optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);K) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A), and further comprising a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR having the structure [IgGk leader- (VL- whitlow linker- VH)-CD28 (Hinge-TM-ICD)-CD3z], where a chicken beta actin 5’ UTR intron is located 3’ to the lineage-specific regulatory sequence and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CDllb promoter operably linked with a sequence encoding a HER2 CAR having the structure [CD8 leader- VH-g4S-VL-CD8 (Hinge-TM)-CD3z], which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa), and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites423FH12901147.5ENO-00225 (38895-00225)(e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR-RRR safety switch, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence), optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites);L) (i) a lineage-specific regulatory sequence that causes expression in T cells and / or NK cells (e.g. including TNK-A set forth in SEQ ID NO: 65), and further comprising a 5’ intron (e.g., a 5’ UTR intron), where the lineage-specific regulatory sequence is operably linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 148, where a chicken beta actin 5’ UTR intron sequence is 3’ to the promoter of the lineage-specific regulatory sequence and 5’ to the coding region of the therapeutic expression product, and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); (ii) a CD1 lb promoter set forth in SEQ ID NO: 20 operably linked with a sequence encoding a HER2 CAR set forth in SEQ ID NO: 146, which sequence encoding the HER2 CAR is operably linked with four miR126 binding sites, and which sequence encoding the HER2 CAR is followed by a polyadenylation signal (e.g., SV40pa), and optionally where the sequence encoding the therapeutic expression product is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites); and (iii) an EFla promoter set forth in SEQ ID NO: 43 operably linked with a sequence encoding an MGMTP14OKselectable marker and an EGFR- RRR safety switch set forth in SEQ ID NO: 195, where the selectable marker and safety switch are present in a single open reading frame and, when expressed as a protein, are separated by a cleavable sequence (e.g., a T2A cleavable sequence set forth in SEQ ID NO: 225), optionally where the sequence encoding the selectable marker and safety switch is operably linked with one or more vaRNA binding sites (e.g., one or more vaRNAl binding sites and / or one or more vaRNA2 binding sites).

183. A cell comprising the nucleic acid construct of any one of claims 1-182.

184. The cell of claim 183, wherein the cell is a CD34+ cell.424FH12901147.5ENO-00225 (38895-00225)185. The cell of claim 183 or 184, wherein the cell is a CD46+ cell.

186. The cell of claim 184, wherein the cell is a hematopoietic stem cell (HSC).

187. The cell of claim 183, wherein the cell is a lymphoid lineage cell.

188. The cell of claim 187, wherein the cell is a T cell, NK cell, or B cell.

189. The cell of claim 188, wherein the cell is a T cell, optionally wherein the T cell is a CD8+ T cell or CD4+ T cell.

190. The cell of claim 188, wherein the cell is an NK cell, optionally wherein the NK cell is a CD56brightNK cell, and / or CD56dimNK cell.

191. The cell of claim 183, wherein the cell is a myeloid lineage cell.

192. The cell of claim 191, wherein the cell is a monocyte, macrophage, myeloblast, granulocyte, neutrophil, eosinophil, basophil, megakaryocyte-erythroid progenitor cell, megakaryocyte, myeloid dendritic cell (mDC), monocyte-derived dendritic cell (MoDC), mast cell, platelet, and / or erythrocyte.

193. The cell of any one of claims 187-192, wherein the cell expresses the first therapeutic expression product and / or the second therapeutic expression product.

194. The cell of claim 193, wherein the cell expresses the first therapeutic expression product and the second therapeutic expression product.

195. The cell of claim 193, wherein the cell expresses the first therapeutic expression product and does not express, does not detectably express, and / or does not significantly express the second therapeutic expression product.

196. The cell of claim 193, wherein the cell expresses the first therapeutic expression product at a level that is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at425FH12901147.5ENO-00225 (38895-00225) least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or at least lOOOO-fold greater than the expression level of the second therapeutic expression product.

197. The cell of claim 193, wherein the cell expresses the second therapeutic expression product and does not express, does not detectably express, and / or does not significantly express the first therapeutic expression product.

198. The cell of claim 193, wherein the cell expresses the second therapeutic expression product at a level that is at least 10-fold, at least 20-fold, at least 30- fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or at least 10000- fold greater than the expression level of the first therapeutic expression product.

199. The cell of any one of claims 187-191, wherein the cell is derived from an HSC cell.

200. The cell of any one of claims 183-199, wherein the nucleic acid construct is integrated in the genome of the cell.

201. A helper-dependent adenoviral (HD Ad) genome comprising the nucleic acid construct of any one of claims 1-182.

202. The HD Ad genome of claim 201, wherein the HD Ad genome comprises an adenoviral 5’ ITR positioned 5’ of the nucleic acid construct and an adenoviral 3’ ITR positioned 3’ of the nucleic acid construct.

203. The HDAd genome of claim 202, wherein the adenoviral 5’ ITR and adenoviral 3’ ITR are Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 ITRs.

204. The HDAd genome of claim 202 or 203, wherein the adenoviral 5’ ITR and adenoviral 3’ ITR are Ad5 or Ad6 ITRs.

205. The HDAd genome of any one of claims 201-204, wherein the HDAd genome comprises an adenoviral packaging sequence.426FH12901147.5ENO-00225 (38895-00225)206. The HDAd genome of claim 205, wherein the packaging sequence is an Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 packaging sequence.

207. The HDAd genome of claim 205 or 206, wherein the packing sequence is an Ad5 or Ad6 packaging sequence.

208. The HDAd genome of anyone of claims 201-207, wherein the HDAd genome does not comprise a nucleic acid sequence encoding a viral structural protein, optionally wherein the viral structural protein is selected from an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton.

209. The HDAd genome of claim 208, wherein the HDAd genome does not comprise nucleic acid sequences encoding any of an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton.

210. A pharmaceutical composition comprising the HDAd genome of any one of claims 201-209.

211. A pharmaceutical composition comprising the HDAd genome of any one of claims 201-209 and a pharmaceutically acceptable carrier.

212. A helper-dependent adenoviral (HDAd) vector comprising the genome of any one of claims 201-209.

213. The HDAd vector of claim 212, wherein the HDAd vector comprises an adenoviral penton and an adenoviral hexon of the same adenoviral serotype, optionally wherein the adenoviral serotype of the hexon and penton is Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50, optionally wherein the adenoviral serotype of the hexon and penton is Ad5 or Ad6.

214. The HDAd vector of claim 212 or 213, wherein the HDAd vector comprises a chimeric adenoviral fiber, wherein the adenoviral fiber comprises an adenoviral fiber tail, fiber shaft, and fiber knob.427FH12901147.5ENO-00225 (38895-00225)215. The HD Ad vector of claim 214, wherein the chimeric adenoviral fiber comprises an Ad5 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob.

216. The HDAd vector of claim 214, wherein the chimeric adenoviral fiber comprises an Ad6 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob.

217. The HDAd vector of claim 215 or 216, wherein the Ad35 fiber knob comprises one or more mutations that increase affinity of the adenoviral fiber with CD46.

218. The HDAd vector of claim 217, wherein the one or more mutations comprise at least one mutation selected from llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His.

219. The HDAd vector of claim 217, wherein the one or more mutations comprise each of llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His.

220. The HDAd vector of any one of claims 212-219, wherein the HDAd vector comprises an Ad5 / 35++ adenoviral capsid.

221. A pharmaceutical composition comprising the HDAd vector of any one of claims 212-220.

222. A pharmaceutical composition comprising the HDAd vector of any one of claims 212-220 and a pharmaceutically acceptable carrier.

223. An adenoviral vector system for delivery of the nucleic acid construct to a mammalian cell, comprising the HDAd vector of any one of claims 212-219 and an integration vector, wherein the nucleic acid construct is flanked by the transposon inverted repeats (IRs), and wherein the transposon IRs are flanked by the recombinase direct repeats (DRs), wherein the integration vector is a helper-dependent adenoviral (HDAd) vector comprising a helper dependent adenoviral genome,428FH12901147.5ENO-00225 (38895-00225) wherein the integration vector genome encodes a transposase that mediates transposition of the nucleic acid construct flanked by the transposon IRs; and optionally wherein the integration vector genome encodes a recombinase for excision of the nucleic acid sequence flanked by the DRs.

224. The adenoviral vector system of claim 223, wherein the DRs comprise FRT, loxP, rox, vox, AttB, or AttP sites, and the recombinase comprises a Flp, Cre, Dre, Vika, or PhiC31 recombinase.

225. The adenoviral vector system of claim 223 or 224, wherein the DRs comprise FRT sites, and the recombinase comprises a Flp recombinase.

226. The adenoviral vector system of any one of claims 223-225, wherein the nucleic acid sequence encoding the recombinase is operably linked with a regulatory sequence, optionally wherein the regulatory sequence operably linked with the recombinase comprises an EFla promoter.

227. The adenoviral vector system of any one of claims 223-226, wherein the IRs comprise Sleeping Beauty (SB), piggyback, Mariner, frog prince, Tol2, TcBuster, or spinON IRs and the transposase comprises a Sleeping Beauty (SB), piggyback, Mariner, frog prince, Tol2, TcBuster, or spinON transposase.

228. The adenoviral vector system of any one of claims 223-227, wherein the IRs comprise Sleeping Beauty (SB) IRs and the transposase comprises a Sleeping Beauty transposase, optionally wherein the IRs comprise pT4 IRs.

229. The adenoviral vector system of any one of claims 223-228, wherein the transposase is SBIOOx or SB150X.

230. The adenoviral vector system of any one of claims 223-229, wherein the nucleic acid sequence encoding the transposase is operably linked with a regulatory sequence, optionally wherein the regulatory sequence operably linked with the transposase comprises an Ela promoter, a UBC promoter, an SFFV promoter, a CAG promoter, or a PGK promoter.429FH12901147.5ENO-00225 (38895-00225)231. The adenoviral vector system of claim of any one of claims 223-230, wherein the nucleic acid sequence encoding the recombinase and the transposase, and regulatory sequences operably linked thereto, is flanked by an adenoviral 5’ ITR and an adenoviral 3’ ITR positioned.

232. The adenoviral vector system of claim 231, wherein the adenoviral 5’ ITR and adenoviral 3’ ITR of the integration vector genome are Ad3, Ad5, Ad6, Ad7, Adi l, Ad 14, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 ITRs.

233. The adenoviral vector system of claim 231 or 232, wherein the adenoviral 5’ ITR and adenoviral 3’ ITR of the integration vector genome are Ad5 or Ad6 ITRs.

234. The adenoviral vector system of any one of claims 223-233, wherein the integration vector genome comprises an adenoviral packaging sequence.

235. The adenoviral vector system of claim 234, wherein the integration vector genome comprises an Ad3, Ad5, Ad6, Ad7, Adil, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50 packaging sequence.

236. The adenoviral vector system of claim 234 or 235, wherein the integration vector genome comprises an Ad5 or Ad6 packaging sequence.

237. The adenoviral vector system of any one of claims 223-236, wherein the integration vector genome does not comprise a nucleic acid sequence encoding a viral structural protein, optionally wherein the viral structural protein is selected from an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton.

238. The adenoviral vector system of any one of claims 223-270, wherein the integration vector genome does not comprise nucleic acid sequences encoding any of an adenoviral fiber, adenoviral hexon, and / or an adenoviral penton.

239. The adenoviral vector system of any one of claims 223-271, wherein the integration vector comprises an adenoviral penton and an adenoviral hexon of the same adenoviral serotype, optionally wherein the adenoviral serotype of the hexon and penton is430FH12901147.5ENO-00225 (38895-00225)Ad3, Ad5, Ad6, Ad7, Adi l, Adl4, Adl6, Ad21, Ad34, Ad35, Ad37, or Ad50, optionally wherein the adenoviral serotype of the hexon and penton is Ad5 or Ad6.

240. The adenoviral vector system of any one of claims 223-239, wherein the integration vector comprises a chimeric adenoviral fiber, wherein the adenoviral fiber comprises an adenoviral fiber tail, fiber shaft, and fiber knob.

241. The adenoviral vector system of any one of claims 223-240, wherein the integration vector comprises a chimeric adenoviral fiber comprising an Ad5 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob.

242. The adenoviral vector system of any one of claims 223-240, wherein the integration vector comprises a chimeric adenoviral fiber comprising an Ad6 fiber tail, an Ad35 fiber shaft, and an Ad35 fiber knob.

243. The adenoviral vector system of any one of claims 223-242, wherein the Ad35 fiber knob of the integration vector fiber comprises one or more mutations that increase affinity of the adenoviral fiber with CD46.

244. The adenoviral vector system of claim 243, wherein the Ad35 fiber knob of the integration vector fiber comprises one or more mutations comprising at least one mutation selected from llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His.

245. The adenoviral vector system of claim 244, wherein the Ad35 fiber knob of the integration vector fiber comprises each of llel92Val, Asp207Gly or Glu207Gly, Asn217Asp, Thr226Ala, Thr245Ala, Thr254Pro, lle256Leu, lle256Val, Arg259Cys, and Arg279His.

246. The adenoviral vector system of any one of claims 223-245, wherein the integration vector comprises an Ad5 / 35++ adenoviral capsid.

247. The adenoviral vector system of any one of claims 223-246, wherein the integration vector genome comprises a first ubiquitous promoter operably linked with a431FH12901147.5ENO-00225 (38895-00225) nucleic acid sequence encoding a transposase and a second ubiquitous promoter operably linked with a nucleic acid sequence encoding a recombinase, wherein the first ubiquitous promoter is an EFla promoter, wherein the transposase is Sleeping Beauty lOOx (SBIOOx), wherein the second ubiquitous promoter is CAG, wherein the recombinase is a FLP recombinase, and wherein the wherein the nucleic acid sequences encoding the recombinase and the transposase, and regulatory sequences operably linked thereto, are flanked by an Ad5 5’ ITR and an Ad5 3’ ITR positioned.

248. The adenoviral vector system of any one of claims 223-247, wherein the nucleic acid sequence encoding the transposase and the nucleic acid sequence encoding the recombinase are expressed in the same orientation.

249. The adenoviral vector system of any one of claims 223-248, wherein the nucleic acid sequence encoding the transposase is codon optimized for expression in a mammalian cell or subject, optionally wherein the nucleic acid sequence encoding the transposase is codon optimized for expression in a human cell or subject.

250. The adenoviral vector system of any one of claims 223-249, wherein the integration vector genome comprises a sequence having at least 80% identity with SEQ ID NO: 227.

251. A kit or pharmaceutical composition comprising the adenoviral vector system of any one of claims 223-246.

252. The kit or pharmaceutical composition of claim 251, wherein the pharmaceutical composition comprises the HDAd vector of any one of claims 212-219 and the integration vector.

253. The kit or pharmaceutical composition of claim 252, wherein the pharmaceutical composition is a liquid, optionally wherein the liquid comprises a buffer and / or wherein the pharmaceutical composition is formulated for intravenous administration.432FH12901147.5ENO-00225 (38895-00225)254. The kit or pharmaceutical composition of claim 251 , wherein the kit comprises a first pharmaceutical composition comprising the HD Ad vector of any one of claims 212-219 and a second pharmaceutical composition comprising the integration vector.

255. The kit or pharmaceutical composition of claim 254, wherein the first pharmaceutical composition is a liquid, optionally wherein the liquid comprises a buffer and / or wherein the pharmaceutical composition is formulated for intravenous administration, and / or wherein the second pharmaceutical composition is a liquid, optionally wherein the liquid comprises a buffer and / or wherein the pharmaceutical composition is formulated for intravenous administration.

256. The kit or composition of any one of claims 251-255, wherein the HD Ad vector of any one of claims 212-219 and the integration vector are present at a ratio of about 1-3:1 or 1:1-3 HD Ad vector:integration vector, optionally wherein the ratio is about 1:1.

257. The kit or pharmaceutical composition of any one of claims 251-256, wherein the total dosage of all vectors genomes present in the kit or pharmaceutical composition is about 2.5 x 1011GC / kg to about 1.25 x 1013GC / kg.

258. A method of modifying a hematopoietic stem cell comprising contacting an HSC with the adenoviral vector system of any one of claims 223-246.

259. A method of modifying a hematopoietic stem cell of a mammalian subject comprising delivering to the subject the adenoviral vector system of any one of claims 222- 246.

260. A method of modifying a hematopoietic stem cell of a mammalian subject comprising administering to the subject the pharmaceutical composition, or first pharmaceutical composition and second pharmaceutical composition, of any one of claims 251-257.433FH12901147.5ENO-00225 (38895-00225)261. A method of treating a solid tumor in a subject in need thereof, the method comprising delivering to the subject the adenoviral vector system of any one of claims 223- 246.

262. A method of treating a solid tumor in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition, or first pharmaceutical composition and second pharmaceutical composition, of any one of claims 251-257.

263. The method of claim 261 or 262, wherein first therapeutic expression product and / or the second therapeutic expression product comprises an antigen binding domain that binds a cancer-specific antigen or cancer-associated antigen, optionally wherein the cancerspecific antigen or cancer-associated antigen is characteristic of a solid tumor or of a liquid or hematological cancer.

264. The method of any one of claims 261-263, wherein the first therapeutic expression product and / or the second therapeutic expression product bind an antigen selected from HER2 (human epidermal growth factor receptor 2), CD19, CD20, CD22, CD 19 and CD 20, CD 19 and CD 22, AFP (alpha-fetoprotein), AXL (AXL receptor tyrosine kinase), BCMA, B7-H3, CD5, CD7, CD33, CD38, CD47, CD52, CD123, CD133, CD138, CD171, CD171, CD30, CD38 / CD123, CD80 / 86, CEA (carcinoembryonic antigen), Claudin 18.2, CLL-1, c- MET, DLL-3 (delta-like 3), DR5 (death receptor 5), EGFR (epidermal growth factor receptor), EGFR806, EGFRIII, EGFR VIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EpHA2 (EPH receptor A2), FAP, FLT3, FCRL5, FR-a (folate receptor alpha), GD2 (disganglioside molecule), Glypican-3, gplOO, GPC3 (glypican 3), GPRC5D, IL-13Ra2, Lewis Y, LMP1 (Epstein-Barr virus latent membrane protein 1), MAGE (melanoma antigen gene protein)-Al / 3 / 4, Mesothelin, Mesothelin, MUC1 (mucinl), MUC16 (mucinl6), Nectin4 / FAP (familial adenomatous polyposis), NKGD2 (natural killer group 2 member D), PD-L1, PMSA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), PSA, ROR1 (receptor tyrosine kinase-like orphan receptor 1), SIRPy, TPD52, VEGFRII (vascular endothelial growth factor receptor II), and / or VISTA, optionally wherein the first therapeutic expression product and / or the second therapeutic expression product comprises a CAR.434FH12901147.5ENO-00225 (38895-00225)265. The method of any one of claims 261-264, wherein the first therapeutic expression product and the second therapeutic expression product each comprise a CAR comprising an antigen-binding domain that binds an antigen selected from HER2 (human epidermal growth factor receptor 2), CD19, CD20, CD22, CD 19 and CD 20, CD 19 and CD 22, AFP (alphafetoprotein), AXL (AXL receptor tyrosine kinase), BCMA, B7-H3, CD5, CD7, CD33, CD38, CD47, CD52, CD123, CD133, CD138, CD171, CD171, CD30, CD38 / CD123, CD80 / 86, CEA (carcinoembryonic antigen), Claudin 18.2, CLL-1, c-MET, DLL-3 (delta-like 3), DR5 (death receptor 5), EGFR (epidermal growth factor receptor), EGFR806, EGFRIII, EGFRVIII (epidermal growth factor receptor variant III), EpCAM (epithelial cell adhesion molecule), EpHA2 (EPH receptor A2), FAP, FLT3, FCRL5, FR-a (folate receptor alpha), GD2 (disganglioside molecule), Glypican-3, gplOO, GPC3 (glypican 3), GPRC5D, IL- 13Ra2, Lewis Y, LMP1 (Epstein-Barr virus latent membrane protein 1), MAGE (melanoma antigen gene protein)-Al / 3 / 4, Mesothelin, Mesothelin, MUC1 (mucinl), MUC16 (mucinl6), Nectin4 / FAP (familial adenomatous polyposis), NKGD2 (natural killer group 2 member D), PD-L1, PMSA (prostate-specific membrane antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), PSA, ROR1 (receptor tyrosine kinase-like orphan receptor 1), SIRPy, TPD52, VEGFRII (vascular endothelial growth factor receptor II), and / or VISTA, optionally wherein the first therapeutic expression product and the second therapeutic expression product bind the same antigen, optionally wherein the antigen is HER2, CD19, or CD20, optionally wherein the antigen is HER2.

266. The method of any one of claims 261-265, wherein the cancer is a solid tumor.

267. The method of any one of claims 261-265, wherein the cancer is a liquid or hematological cancer.

268. The method of claim 261, wherein the cancer is selected from:(i) a HER2 positive cancer, optionally where the cancer is selected from ovarian cancer, glioblastoma, hepatocellular cancer, prostate cancer, cholangiocarcinoma (intrahepatic and / or extrahepatic), pancreatic adenocarcinoma, intestinal malignancy, lung cancer, head and neck carcinoma, osteosarcoma, central nervous system tumor, pediatric glioma, advanced solid tumor, colorectal cancer, uterine cancer, cervical cancer, testicular cancer, gastric adenocarcinoma, gallbladder cancer, breast cancer, esophageal cancer,435FH12901147.5ENO-00225 (38895-00225) esophagogastric junction cancer, bladder cancer, sarcoma, glioblastoma, biliary tract cancer, salivary gland cancer, and endometrial cancer;(ii) a cancer selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), agnogenic myeloid metaplasia, astrocytoma, atypical teratoid rhabdoid tumor, brain and central nervous system (CNS) cancer, breast cancer, carcinosarcoma, chondrosarcoma, chordoma, choroid plexus carcinoma, choroid plexus papilloma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), clear cell sarcoma of soft tissue, diffuse large B-cell lymphoma, ependymoma, epithelioid sarcoma, Ewing sarcoma, extragonadal germ cell tumor, extrarenal rhabdoid tumor, follicular lymphoma, gastrointestinal stromal tumor, glioblastoma, HBV-induced hepatocellular carcinoma, head and neck cancer, Hodgkin’s lymphoma, juvenile myelomonocytic leukemia, kidney cancer, lung cancer, lymphoma, malignant rhabdoid tumor, medulloblastoma, melanoma, meningioma, mesothelioma, multiple myeloma, myeloma, neuroglial tumor, nonHodgkin’s lymphoma, not otherwise specified (NOS) sarcoma, oligoastrocytoma, oligodendroglioma, osteosarcoma, ovarian cancer, ovarian clear cell adenocarcinoma, ovarian endometrioid adenocarcinoma, ovarian serous adenocarcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic endocrine tumor, pineoblastoma, prostate cancer, renal cell carcinoma, renal medullary carcinoma, rhabdomyosarcoma, sarcoma, schwannoma, skin squamous cell carcinoma, and / or stem cell cancer;(iii) a cancer selected from lung cancer, osteosarcoma, glioblastoma, central nervous system tumor, pediatric glioma, advanced solid tumor, HER2 -positive cancer, BALL, B-CLL, leukemia, lymphoma, B-NHL / CLL, mantel cell leukemia / B-NHL, melanoma, Non-Hodgkins Lymphoma, B-Non Hodgkins Lymphoma, B-NHL, B-cell malignancy, B-ALL, D LBCL, B-ALL, B-NHL, hepatocellular carcinoma, liver, renal, hepatocellular carcinoma, neuroblastoma, neuroblastoma, lymphoma, B-cell malignancies, lung, breast cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, advanced solid tumor, breast, hepatocellular, lung cancer, hepatoma, lung cancer, liver cancer, stomach cancer, central nervous system tumor, pediatric glioma, glioblastoma and brain tumor, glioblastoma, colon, pancreatic, prostate, gastric, liver, glioma, malignant pleural mesothelioma, ovarian, brain cancer, neuroblastoma, osteosarcoma, liver, melanoma, lung squamous cell carcinoma, glioblastoma, advanced cancer, nasopharyngeal, lung, pancreatic cancer, ovarian, cervical, pancreatic, lung, advanced solid tumors, lung, ovarian, nectin4-positive advanced malignant solid tumor, leukemia, prostate cancer, lung, chronic lymphocytic leukemia, melanoma, brain cancer, and / or renal cancer; and / or436FH12901147.5ENO-00225 (38895-00225)(iv) a cancer can be selected from cancers of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves and other parts of the CNS, such as glioblastomas or medulloblastomas); head and / or neck cancer, breast cancers, cancers of the circulatory system (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular cancers, and tumor-associated vascular tissue); cancers of the blood and lymphatic system (e.g., Hodgkin’s disease, Non-Hodgkin’s disease lymphoma, Burkitt’s lymphoma, AIDS-related lymphomas, malignant immunoproliferative diseases, multiple myeloma, and malignant plasma cell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell population, leukemia of unspecified cell population, unspecified malignant neoplasms of lymphoid, haematopoietic and related tissues, such as diffuse large cell lymphoma, T-cell lymphoma or cutaneous T-cell lymphoma); cancers of the excretory system (e.g., kidney, renal pelvis, ureter, bladder, and other urinary organs); cancers of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal); cancers involving the liver and intrahepatic bile ducts, gall bladder, and other parts of the biliary tract, pancreas, and other digestive organs; cancers of the oral cavity (e.g., lip, tongue, gum, floor of mouth, palate, parotid gland, salivary glands, tonsil, oropharynx, nasopharynx, puriform sinus, hypopharynx, and other sites of the oral cavity); cancers of the reproductive system (e.g., vulva, vagina, Cervix uteri, uterus, ovary, and other sites associated with female genital organs, placenta, penis, prostate, testis, and other sites associated with male genital organs); cancers of the respiratory tract (e.g., nasal cavity, middle ear, accessory sinuses, larynx, trachea, bronchus and lung, such as small cell lung cancer and non-small cell lung cancer); cancers of the skeletal system (e.g., bone and articular cartilage of limbs, bone articular cartilage and other sites); cancers of the skin (e.g., malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma, Kaposi’s sarcoma); and cancers involving other tissues including peripheral nerves and autonomic nervous system, connective and soft tissue, retroperitoneoum and peritoneum, eye and adnexa, thyroid, adrenal gland, and other endocrine glands and related structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasms of other sites.437FH12901147.5ENO-00225 (38895-00225)269. The method of any one of claims 258-268, wherein the method comprises mobilizing hematopoietic stem cells of the subject prior to administering the adenoviral vector system.

270. The method of claim 269, wherein mobilizing hematopoietic stem cells of the subject comprises administering to the subject granulocyte colony stimulating factor (G- CSF), granulocyte macrophage colony stimulating factor (GM-CSF), an agent that inhibits binding of endogenous very late antigen 4 (VLA4) with its endogenous ligands (a VLA4 antagonist), AMD3100 (plerixafor), SCF, S-CSF, a CXCR4 antagonist, a CXCR2 agonist, Gro-Beta (GRO-P), truncated GRO- (tGRO-P), and / or motixifortide.

271. The method of claim 270, wherein G-CSF and plerixafor are administered to the subject.

272. The method of claim 270, wherein tGRO-beta and plerixafor are administered to the subject.

273. The method of claim 270, wherein motixifortide and plerixafor are administered to the subject.

274. The method of claim 270, wherein tGRO-beta and motixifortide are administered to the subject.

275. The method of any one of claims 270-274, wherein G-CSF is administered to the subject daily.

276. The method of any one of claims 270-275, wherein G-CSF is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

277. The method of any one of claims 269-276, wherein G-CSF is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of the adenoviral vector system.438FH12901147.5ENO-00225 (38895-00225)278. The method of any one of claims 269-277, wherein granulocyte colony stimulating factor is administered on the day of administration of the adenoviral vector system.

279. The method of any one of claims 269-278, wherein plerixafor is administered to the subject daily.

280. The method of any one of claims 269-279, wherein plerixafor is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

281. The method of any one of claims 269-280, wherein plerixafor is administered to the subject for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days prior to administration of the adenoviral vector system.

282. The method of any one of claims 269-281, wherein plerixafor is administered to the subject on the day of administration of the adenoviral vector system.

283. The method of any one of claims 258-282, wherein the method comprises administering to the subject an immunosuppression regimen comprising one or more immunosuppression agents.

284. The method of claim 283, wherein the immunosuppression regimen comprises a corticosteroid, optionally wherein the corticosteroid comprises or is a glucocorticoid, optionally wherein the glucocorticoid comprises or is dexamethasone.

285. The method of claim 284, wherein the corticosteroid is administered to the subject daily.

286. The method of claim 284 or 285, wherein the corticosteroid is administered to the subject for at least about 1, 2, 3, 4, 5 or more days.

287. The method of any one of claims 284-286, wherein the corticosteroid is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the adenoviral vector system.439FH12901147.5ENO-00225 (38895-00225)288. The method of any one of claims 284-287, wherein the corticosteroid is administered to the subject on the day of administration of the adenoviral vector system.

289. The method of any one of claims 283-288, wherein the immunosuppression regimen comprises an inflammatory signal inhibitor, optionally wherein the inflammatory signal inhibitor comprises or is an interleukin- 1 signal inhibitor, optionally wherein the interleukin- 1 signal inhibitor comprises or is an interleukin- 1 receptor antagonist, optionally wherein the interleukin- 1 receptor antagonist comprises or is anakinra.

290. The method of claim 289, wherein the inflammatory signal inhibitor is administered to the subject daily.

291. The method of claims 289 or 290, wherein the inflammatory signal inhibitor is administered to the subject for at least about 1, 2, 3, 4, 5 or more days.

292. The method of any one of claims 289-291, wherein the inflammatory signal inhibitor is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the adenoviral vector system.

293. The method of any one of claims 289-292, wherein the inflammatory signal inhibitor is administered to the subject on the day of administration of the adenoviral vector system.

294. The method of any one of claims 283-293, wherein the immunosuppression regimen comprises an interleukin-6 receptor antagonist, optionally wherein the interleukin-6 receptor antagonist comprises or is tocilizumab.

295. The method of claim 294, wherein the interleukin- 6 receptor antagonist is administered to the subject daily.

296. The method of claim 294 or 295, wherein the interleukin-6 receptor antagonist is administered to the subject for at least about 1, 2, 3, 4, 5 or more days.440FH12901147.5ENO-00225 (38895-00225)297. The method of any one of claims 294-296, wherein the interleukin-6 receptor antagonist is administered to the subject for at least about 1, 2, 3, 4, 5 or more days prior to administration of the adenoviral vector system.

298. The method of any one of claims 294-297, wherein the interleukin-6 receptor antagonist is administered to the subject on the day of administration of the adenoviral vector system.

299. The method of any one of claims 258-298, wherein the method comprises administering to the subject one or more selecting agents.

300. The method of claim 299, wherein the enrichment marker is MGMTP14OKand the one or more selecting agents comprise:(a) temozolomide (TMZ);(b) O6-benzylguanine (O6BG); and / or(c) BCNU.

301. The method of claim 300, wherein TMZ is administered to the subject orally.

302. The method of claim 300 or 301, wherein TMZ is administered to the subject about once every 28 days or about monthly.

303. The method of any one of claims 300-302, wherein 1, 2, 3, 4, or more doses of TMZ are administered to the subject.

304. The method of any one of claims 300-303, wherein a first dose of TMZ is administered to the subject at about 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of the adenoviral vector system.

305. The method of any one of claims 300-304, wherein temozolomide is administered to the subject at a dose of about 300-600 mg / m2.

306. The method of any one of claims 300-305, wherein O6BG is administered to the subject intravenously.441FH12901147.5ENO-00225 (38895-00225)307. The method of any one of claims claim 300-306, wherein O6BG is administered to the subject as an intravenous bolus and / or intravenous infusion.

308. The method of claim 307, wherein the intravenous bolus is administered prior to the intravenous infusion.

309. The method of any one of claims 300-308, wherein O6BG is administered to the subject about once every 28 days or about monthly.

310. The method of any one of claims 300-309, wherein 1, 2, 3, 4, or more doses of O6BG are administered to the subject.

311. The method of any one of claims 300-310, wherein a first dose of O6BG is administered to the subject at about 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks following administration of the adenoviral vector system.

312. The method of any one of claims 300-311, wherein O6BG is administered to the subject at a dose of about 80-280 mg / m2.

313. The method of any one of claims 300-312, wherein a dose of O6BG comprises an intravenous bolus dose of about 70-170 mg / m2O6BG and / or an intravenous infusion dose of about 10-110 mg / m2.

314. The method of any one of claims 300-313, wherein both TMZ and O6BG are administered to the subject.

315. The method of claim 299, wherein the enrichment marker is CD117 comprising one or more mutations selected from the group consisting of F316S, M318V, I319K, V323I, I334V, E360K, P363V, E366D, E376Q, and H378R and the one or more selecting agents comprise an anti-CDl 17 antibody or antigen binding fragment thereof.

316. The method of claim 316, wherein the anti-CDl 17 antibody or antigen binding fragment thereof selectively binds to CD 117 not comprising the one or more mutations442FH12901147.5ENO-00225 (38895-00225) selected from the group consisting of F316S, M318V, I319K, V323I, I334V, E360K, P363V, E366D, E376Q, and H378R.

317. The method of any one of claims 258-316, wherein the subject is a mammal.

318. The method of any one of claims 258-317, wherein the subject is a human.

319. The method of any one of claims 258-318, wherein the subject is a patient.

320. The method of any one of claims 258-319, wherein the subject is an adult.

321. The method of any one of claims 258-320, wherein the subject is a child.

322. The method of any one of claims 258-321, wherein the subject is male.

323. The method of any one of claims 258-322, wherein the subject is a female.

324. An HD Ad integration vector genome, wherein the integration vector genome comprises a first ubiquitous promoter operably linked with a nucleic acid sequence encoding a transposase and a second ubiquitous promoter operably linked with a nucleic acid sequence encoding a recombinase, wherein the first ubiquitous promoter is an EFla promoter or CAG promoter, and / or wherein the second ubiquitous promoter is an EFla promoter or CAG promoter.

325. An HDAd integration vector genome, wherein the integration vector genome comprises a first ubiquitous promoter operably linked with a nucleic acid sequence encoding a transposase and a second ubiquitous promoter operably linked with a nucleic acid sequence encoding a recombinase, wherein the transposase is Sleeping Beauty lOOx (SBIOOx), and wherein the nucleic acid sequence encoding the transposase is codon optimized for expression in a mammalian cell or subject, optionally wherein the nucleic acid sequence encoding the transposase is codon optimized for expression in a human cell or subject.443FH12901147.5ENO-00225 (38895-00225)326. An HD Ad integration vector genome, wherein the integration vector genome comprises a first ubiquitous promoter operably linked with a nucleic acid sequence encoding a transposase and a second ubiquitous promoter operably linked with a nucleic acid sequence encoding a recombinase, wherein the nucleic acid sequence encoding the transposase and the nucleic acid sequence encoding the recombinase are expressed in the same orientation.

327. The HD Ad integration vector genome of any one of claims 324-326, wherein: wherein the transposase is Sleeping Beauty lOOx (SBIOOx), and / or wherein the recombinase is a FLP recombinase.

328. The HDAd integration vector genome of any one of claims 324-327, wherein: wherein the first ubiquitous promoter is an EFla promoter and / or wherein the transposase is Sleeping Beauty lOOx (SBIOOx), wherein the second ubiquitous promoter is CAG, and wherein the recombinase is a FLP recombinase.

329. The HDAd integration vector genome of any one of claims 324-328, wherein the nucleic acid sequence encoding the transposase and the nucleic acid sequence encoding the recombinase are expressed in the same orientation.

330. The HDAd integration vector genome of any one of claims 324-329, wherein the nucleic acid sequence encoding the transposase is codon optimized for expression in a mammalian cell or subject, optionally wherein the nucleic acid sequence encoding the transposase is codon optimized for expression in a human cell or subject.

331. The HDAd integration vector genome of any one of claims 324-330, wherein the wherein the nucleic acid sequences encoding the recombinase and the transposase, and regulatory sequences operably linked thereto, are flanked by an Ad5 5’ ITR and an Ad5 3’ ITR positioned.

332. The HDAd integration vector genome of any one of claims 324-331, wherein the first ubiquitous promoter is an EFla promoter.444FH12901147.5ENO-00225 (38895-00225)333. The HDAd integration vector genome of any one of claims 324-332, wherein the second ubiquitous promoter is a CAG promoter.

334. The HDAd integration vector genome of any one of claims 324-333, wherein the first ubiquitous promoter is an EFla promoter and the second ubiquitous promoter is a CAG promoter.

335. The HDAd integration vector genome of any one of claims 324-334 wherein the integration vector genome comprises a sequence having at least 80% identity with SEQ ID NO: 227.

336. The HDAd integration vector genome of any one of claims 324-330, wherein the first ubiquitous promoter is a CAG promoter.

337. The HDAd integration vector genome of any one of claims 324-330, wherein the second ubiquitous promoter is an EFla promoter.

338. The HDAd integration vector genome of any one of claims 324-330, 336, or 337, wherein the first ubiquitous promoter is a CAG promoter and the second ubiquitous promoter is an EFla promoter.

339. The HDAd integration vector genome of any one of claims 324-330, or 336-338, wherein the integration vector genome comprises a sequence having at least 80% identity with SEQ ID NO: 228.445FH12901147.5

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