Improved enhancer and vector

Abstract

In some embodiments, improved enhancer elements, effective fragments thereof, and their use in vectors are described herein. In some embodiments, minimal backbone enhancer element structures and their use in vectors are described herein. In some embodiments, the expression of gene products (e.g., WAS protein) using such improved enhancer elements in vectors having, for example, the described minimal backbone enhancer element structures is described herein. In some embodiments, the vectors described herein are lentiviral vectors.

Description

Technical Field

[0001] Field The present disclosure relates to gene expression elements, specifically, enhancers and transgene delivery vehicles (e.g., vectors) containing the same. The present disclosure also relates to the treatment or prevention of disorders associated with expression defects of certain genes, such as the gene encoding the Wiskott-Aldrich syndrome (WAS) protein, using transgene delivery vehicles.

[0002] Sequence Listing This application includes the sequence listing of Table 1. The sequence listing has been submitted to EFS-Web and is hereby incorporated by reference in its entirety. The copy created on June 15, 2023, is named 21278.001_SL and has a size of 90,112 bytes.

Background Art

[0003] Background The WAS protein (WASp) expressed from hematopoietic cells is important for organizing the actin cytoskeleton, and the absence of functional WASp disrupts cell motility, endocytosis (and thus antigen recognition), cell-cell adhesion, and other cytokines.

[0004] WAS gene mutations can result in three different clinical manifestations: Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia (XLN). Mutations that completely inhibit WASp expression usually result in Wiskott-Aldrich syndrome (WAS). Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by one or more mutations or the absence of the WAS gene, affecting 1 to 10 males per million.

[0005] Gene mutations that result in the expression of defective WASp usually lead to X-linked thrombocytopenia (XLT). X-linked neutropenia can be caused by gain-of-function WASp mutations (constitutively activated WAS). See Albert et al., 2010, Blood 115(16):3231-3228.

[0006] Symptoms of WAS include thrombocytopenia, severe eczema, diarrhea with bleeding, and recurrent otitis media (in male infants). In addition to thrombocytopenia, WAS patients exhibit a reduction in platelet size (i.e., microthrombocytes). 30% of WAS patients exhibit an increase in eosinophil count (i.e., eosinophilia). WAS is characterized by an increased susceptibility to viral and bacterial infections, as well as an increased risk of autoimmune diseases and cancer (due to defects in adaptive and innate immune responses). Recurrent bacterial infections typically develop within the first 3 months of life, and children with WAS typically develop at least one autoimmune disorder. Up to one-third of WAS patients develop cancer (primarily lymphoma and leukemia). Patients with WAS exhibit altered immunoglobulin levels, where immunoglobulin G (IgG) levels may be normal, reduced, or elevated, IgM levels are typically reduced, and IgA and IgE levels are typically elevated.

[0007] If left untreated, WAS usually results in death in early childhood or adolescence.

[0008] Some patients with mutations in the WAS protein exhibit a milder phenotype called X-linked thrombocytopenia (XLT). XLT is a hereditary coagulation disorder that is a variant of WAS. XLT is also caused by mutations in the WAS gene. The symptoms of XLT are milder than those of WAS and can include thrombocytopenia, eczema, and infections.

[0009] Allogeneic stem cell transplantation is a common treatment for WAS and can be curative. However, this treatment requires the availability of a HLA-matched donor and may not be accessible to many patients due to the unavailability of a suitable (HLA-matched) donor.

[0010] An alternative to allogeneic stem cell transplantation is autologous hematopoietic stem cell (HSC) transplantation by in vivo or ex vivo gene therapy. Previous virus-based therapies include the CMMP-WAS γ-retroviral vector, however, the use of such vectors led to the development of acute leukemia (due to insertional carcinogenesis) in 7 out of 9 patients. See Braun, 2014, Sci Transl. Med. 6(227):227ra33. Current gene therapy trials utilize self-inactivating lentiviral vectors driven by a 1.6 kb promoter fragment of the endogenous WAS gene. Patients showed some clinical improvement after such gene therapy, however, variable modifications in platelet counts were observed and the majority of patients remained thrombocytopenic (likely due to low expression of the LV in the megakaryocytic lineage or low gene transfer into hematopoietic stem cells). See Magnani, 2022, Nature Medicine, 28, 71-80, doi: 10.1038 / s41591-021-01641-x, Ferrua, 2019, Lancet Haematol, 6(5):e239-e253, doi: 10.1016 / S2352-3026(19)30021-3, Abina, 2015, JAMA, 313(15):1550-63, doi: 10.1001 / jama.2015.3253. WO 2021 / 096887 describes a lentiviral vector (LV) for the treatment of WAS, in particular WASVec1.0, which is hereby incorporated by reference in its entirety. However, there is a need for further therapies for disorders associated with defective WAS protein expression, particularly gene therapies that can be used to avoid dependence on suitable donors. There is also a need for improved vectors that can be used in effective and safe gene therapies for WAS-related disorders.

Prior Art Documents

Patent Documents

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Patent Document 1

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Means for Solving the Problems

[0013] Summary This summary is provided to introduce, in simplified form, various concepts that will be further described in the following detailed description. This summary is not intended to identify key or essential features of the claimed subject matter nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following detailed description, including the aspects illustrated in the accompanying drawings and defined in the appended claims.

[0014] In some aspects, the invention describes an improved enhancer element, Element 14 (SEQ ID NO: 1), and its effective fragments, such as Element 14 core (SEQ ID NO: 2) and Element 14 ultra core (SEQ ID NO: 3). In other aspects, the disclosure provides additional regulatory sequences for improving gene expression, such as the uCore E2 element SEQ ID NO: 32. In certain aspects, the invention describes regulatory elements that assist in gene expression of a transgene in certain blood cell types (e.g., megakaryocytes and platelets). In certain aspects, the disclosure provides a vector for expressing the WAS protein in cells.

[0015] The use of such enhancer elements, minimal skeletal enhancer element constructs, and vectors for the treatment of diseases, such as diseases associated with WASp expression deficiency, such as Wiskott-Aldrich syndrome (WAS), is also described herein.

[0016] In some aspects, provided herein is a recombinant vector comprising a nucleic acid sequence of an enhancer, which comprises or consists of enhancer element 14 containing SEQ ID NO: 1 or an effective fragment thereof, a nucleic acid sequence of a promoter or an effective fragment thereof, and a nucleic acid encoding a gene product operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter.

[0017] In one aspect, provided herein is a recombinant vector comprising a nucleic acid sequence of an enhancer element 14 or a functional fragment thereof that comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, a nucleic acid sequence of a promoter or a functional fragment thereof, and a nucleic acid encoding a gene product operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter.

[0018] In some embodiments, the recombinant vector can be any transgene delivery vehicle. In some embodiments, the recombinant vector is a viral vector (e.g., a lentiviral vector). In some embodiments, the recombinant vector is a non-viral vector (e.g., a plasmid). In some embodiments, the nucleic acid encoding the gene product is a nucleic acid sequence encoding WASp (which can be cDNA and / or a codon-optimized sequence).

[0019] In some embodiments of the vectors provided herein, the nucleic acid sequence of enhancer element 14 or a functional fragment thereof is the nucleic acid sequence of the core fragment of element 14 that comprises or consists of SEQ ID NO: 2. In some embodiments of the vectors provided herein, the nucleic acid sequence of enhancer element 14 or a functional fragment thereof is the nucleic acid sequence of the core fragment of element 14 that comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2.

[0020] In some embodiments of the vectors provided herein, the nucleic acid sequence of enhancer element 14 or a functional fragment thereof is the nucleic acid sequence of the ultra-core fragment of element 14 that includes or consists of SEQ ID NO: 3. In some embodiments of the vectors provided herein, the nucleic acid sequence of enhancer element 14 or a functional fragment thereof is the nucleic acid sequence of the ultra-core fragment of element 14 that includes or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3.

[0021] In some embodiments of the vectors provided herein, the enhancer includes the first half of enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and / or enhancer element 2 core sub-element 5 of SEQ ID NO: 17. In some embodiments of the vectors provided herein, the enhancer includes the first half of enhancer element 2 core sub-element 1 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14, and / or enhancer element 2 core sub-element 5 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17.

[0022] In some embodiments of the vectors provided herein, the vector does not include the nucleic acid sequence of sub-sub-element 1 of element 2 of SEQ ID NO: 9 or a functional fragment thereof. In some embodiments of the vectors provided herein, the vector does not include the nucleic acid sequence of sub-sub-element 1 of element 2 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9.

[0023] In some embodiments of the vectors provided herein, the vector does not contain the nucleic acid sequence of sub - element 4 of enhancer element 2 of SEQ ID NO: 10 or a functional fragment thereof. In some embodiments of the vectors provided herein, the vector does not contain the nucleic acid sequence of sub - element 4 of enhancer element 2 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10.

[0024] In some embodiments of the vectors provided herein, the vector does not contain the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or a functional fragment thereof. In some embodiments of the vectors provided herein, the vector does not contain the nucleic acid sequence of enhancer element 9 slim of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7.

[0025] In some embodiments of the vectors provided herein, the vector does not contain the nucleic acid sequence of hypersensitive site 3 (HS3) core of SEQ ID NO: 8 or a functional fragment thereof. In some embodiments of the vectors provided herein, the vector does not contain the nucleic acid sequence of hypersensitive site 3 (HS3) core of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8.

[0026] In some embodiments of the vectors provided herein, the nucleic acid sequence of the enhancer consists of, or consists essentially of, (i) the nucleic acid sequence of enhancer element 14, or a functional fragment thereof, that includes or consists of SEQ ID NO: 1, and / or (ii) the nucleic acid sequence of the uCore E2 element of SEQ ID NO: 32. In some embodiments of the vectors provided herein, the nucleic acid sequence of the enhancer consists of, or consists essentially of, (i) the nucleic acid sequence of enhancer element 14, or a functional fragment thereof, that includes or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, and / or (ii) the nucleic acid sequence of the uCore E2 element of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32.

[0027] In one aspect, a recombinant vector comprising a nucleic acid sequence of an enhancer comprising or consisting of the first half of enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and enhancer element 2 core sub-element 5 of SEQ ID NO: 17, a nucleic acid sequence of a promoter or an effective fragment thereof, and a nucleic acid encoding a gene product operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, wherein optionally such a vector does not contain (i) the nucleic acid sequence of sub-sub-element 1 of element 2 of SEQ ID NO: 9 or an effective fragment thereof, (ii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of SEQ ID NO: 10 or an effective fragment thereof, (iii) the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or an effective fragment thereof, and / or (iv) the nucleic acid sequence of hypersensitive site 3 (HS3) core of SEQ ID NO: 8 or an effective fragment thereof, is provided herein. In one aspect, a recombinant vector comprising a nucleic acid sequence of an enhancer comprising or consisting of the first half of enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and enhancer element 2 core sub-element 5 of SEQ ID NO: 17, a nucleic acid sequence of a promoter or an effective fragment thereof, and a nucleic acid encoding a gene product operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, wherein such a vector does not contain (i) the nucleic acid sequence of sub-sub-element 1 of element 2 of SEQ ID NO: 9 or an effective fragment thereof, (ii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of SEQ ID NO: 10 or an effective fragment thereof, (iii) the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or an effective fragment thereof, and / or (iv) the nucleic acid sequence of hypersensitive site 3 (HS3) core of SEQ ID NO: 8 or an effective fragment thereof, is provided herein.

[0028] In one aspect, a recombinant vector comprising an enhancer nucleic acid sequence comprising or consisting of the first half of enhancer element 2 core sub-element 1 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14, and enhancer element 2 core sub-element 5 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17, a nucleic acid sequence of a promoter or an active fragment thereof, and a nucleic acid encoding a gene product operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, wherein optionally such a vector does not contain (i) the nucleic acid sequence of sub-sub-element 1 of element 2 of SEQ ID NO: 9 or an active fragment thereof (e.g., a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9), (ii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of SEQ ID NO: 10 or an active fragment thereof (e.g., a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10), (iii) the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or an active fragment thereof (e.g., a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7), and / or (iv) the nucleic acid sequence of hypersensitive site 3 (HS3) core of SEQ ID NO: 8 or an active fragment thereof (e.g., a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8) is provided herein.

[0029] In some embodiments, the recombinant vector can be any transgene delivery vehicle. In some embodiments, the recombinant vector is a viral vector (e.g., a lentiviral vector). In some embodiments, the recombinant vector is a non-viral vector (e.g., a plasmid or episome). In some embodiments, the nucleic acid encoding the gene product is a nucleic acid sequence encoding WASp (which can be cDNA and / or a codon-optimized sequence).

[0030] In some embodiments of the vectors provided herein, the vector comprises the nucleic acid sequence of enhancer element 14 or a functional fragment thereof, and the nucleic acid sequence of enhancer element 14 comprises or consists of SEQ ID NO: 1. In some embodiments of the vectors provided herein, the vector comprises the nucleic acid sequence of enhancer element 14 or a functional fragment thereof, and the nucleic acid sequence of enhancer element 14 comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0031] In some embodiments of the vectors provided herein, the vector comprises the nucleic acid sequence of the core fragment of element 14 that comprises or consists of SEQ ID NO: 2. In some embodiments of the vectors provided herein, the vector comprises the nucleic acid sequence of the core fragment of element 14 that comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2.

[0032] In some embodiments of the vectors provided herein, the vector comprises the nucleic acid sequence of the ultra-core fragment of element 14 that comprises or consists of SEQ ID NO: 3. In some embodiments of the vectors provided herein, the vector comprises the nucleic acid sequence of the ultra-core fragment of element 14 that comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3.

[0033] In some embodiments of the vectors provided herein, the nucleic acid sequence of the enhancer consists of, or consists essentially of, the nucleic acid sequence of the uCore E2 element of SEQ ID NO: 32. In some embodiments of the vectors provided herein, the nucleic acid sequence of the enhancer consists of, or consists essentially of, the nucleic acid sequence of the uCore E2 element of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32.

[0034] In some embodiments of the vectors provided herein, the nucleic acid sequence of the enhancer consists of, or consists essentially of, (i) the nucleic acid sequence of enhancer element 14 comprising or consisting of SEQ ID NO: 1 or a functional fragment thereof, and / or (ii) the nucleic acid sequence of the uCore E2 element of SEQ ID NO: 32. In some embodiments of the vectors provided herein, the nucleic acid sequence of the enhancer consists of, or consists essentially of, (i) the nucleic acid sequence of enhancer element 14 comprising or consisting of SEQ ID NO: 1 or a functional fragment thereof, and (ii) the nucleic acid sequence of the uCore E2 element of SEQ ID NO: 32.

[0035] In some embodiments of the vectors provided herein, the nucleic acid sequence of the enhancer consists of, or consists essentially of, (i) the nucleic acid sequence of enhancer element 14 comprising or consisting of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or a functional fragment thereof, and / or (ii) the nucleic acid sequence of the uCore E2 element of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32.

[0036] In some embodiments of the vectors provided herein, the gene product to be translated is the Wiskott-Aldrich syndrome protein (WASp).

[0037] In some embodiments of the vectors provided herein, the nucleic acid encoding WASp is a codon-optimized WAS nucleic acid sequence, and optionally, the codon-optimized WAS comprises or consists of SEQ ID NO: 21. In some embodiments of the vectors provided herein, the nucleic acid encoding WASp is a codon-optimized WAS nucleic acid sequence, and the codon-optimized WAS nucleic acid sequence comprises or consists of SEQ ID NO: 21. In some embodiments of the vectors provided herein, the nucleic acid encoding WASp is a codon-optimized WAS nucleic acid sequence, and the codon-optimized WAS nucleic acid sequence comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 21.

[0038] In some embodiments of the vectors provided herein, the codon-optimized WAS nucleic acid sequence is selected from the group consisting of a jCAT codon-optimized WAS nucleic acid sequence, a GeneArt-optimized WAS nucleic acid sequence, and an IDT-optimized WAS nucleic acid sequence. In some embodiments, the WAS nucleic acid sequence is a jCAT codon-optimized WAS nucleic acid sequence. In some embodiments, the WAS nucleic acid sequence is a GeneArt-optimized WAS nucleic acid sequence. In some embodiments, the WAS nucleic acid sequence is an IDT-optimized WAS nucleic acid sequence.

[0039] In some embodiments of the vectors provided herein, the promoter is a human promoter.

[0040] In some embodiments of the vectors provided herein, the promoter is the endogenous promoter of the WAS gene, for example, the endogenous human promoter of the WAS gene.

[0041] In some embodiments of the vectors provided herein, the promoter is the WAS gene promoter of SEQ ID NO: 11. In some embodiments of the vectors provided herein, the promoter is the WAS gene promoter of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11.

[0042] In some embodiments of the vectors provided herein, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene, having a maximum length of 600 bp and comprising the nucleic acid sequence of HS1pro of SEQ ID NO: 12. In some embodiments of the vectors provided herein, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene, having a maximum length of 600 bp and comprising the nucleic acid sequence of HS1pro of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12.

[0043] In some embodiments of the vectors provided herein, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS1pro (SEQ ID NO: 12). In some embodiments of the vectors provided herein, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene consisting of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12.

[0044] In some embodiments of the vectors provided herein, the vector is a recombinant lentiviral vector. In some embodiments, the vector comprises a Ψ packaging signal. In some embodiments, the vector comprises a Rev-responsive element (RRE). In some embodiments, the vector comprises a central polypurine tract. In some embodiments, the vector comprises a post-transcriptional regulatory element. In some embodiments, the post-transcriptional regulatory element is the woodchuck post-transcriptional regulatory element (WPRE).

[0045] In some embodiments, provided herein are vectors comprising or consisting of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 4. In some embodiments, provided herein are vectors comprising a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to any element or sequence in the vector of SEQ ID NO: 4.

[0046] In some embodiments, provided herein are vectors comprising or consisting of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 5. In some embodiments, provided herein are vectors comprising a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to any element or sequence in the vector of SEQ ID NO: 5.

[0047] In some embodiments, provided herein are vectors comprising or consisting of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 6. In some embodiments, provided herein are vectors comprising a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to any element or sequence in the vector of SEQ ID NO: 6.

[0048] In some embodiments of the vectors provided herein, the vector is capable of expressing a gene product (e.g., WASp) encoded by a transgene in a cell (e.g., a stem cell and / or a progenitor cell, e.g., a hematopoietic stem cell and / or a progenitor cell). In some of these embodiments, the vector is capable of expressing the gene product at its physiological level or at a near-physiological level. In some of these embodiments, the vector is capable of expressing the gene product at a high level (e.g., a level above the endogenous or physiological level of the corresponding native gene in a healthy subject). In some embodiments of the vectors provided herein, introduction of the vector into a cell results in expression of the gene product within about 60%, within 50%, within 40%, within 30%, or within 20% of the endogenous physiological level of expression of the corresponding native gene in a healthy subject. In some embodiments, the vector is effective to express WASp in a cell within about 60%, within 50%, within 40%, within 30%, or within 20% of the endogenous physiological level of WASp expression in a healthy subject.

[0049] In some aspects, the vectors provided herein are encapsulated within a viral particle, e.g., a viral capsid.

[0050] In some embodiments, a cell is transduced with any of the vectors or viral particles provided herein. In some embodiments, the cell is a stem cell or a progenitor cell. In some embodiments, the cell is a CD34+ hematopoietic stem cell and / or progenitor cell. In some embodiments, the cell is a cell derived from bone marrow, umbilical cord blood, and / or peripheral blood. In some embodiments, the cell is a dendritic cell, CD4 + T cell, or a peripheral blood B or T cell. In some embodiments, the cell is a human cell.

[0051] In some embodiments, a pharmaceutical composition is provided that includes any of the vectors provided herein, any of the viral particles provided herein, or any of the cells provided herein. In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0052] In some embodiments, a method is provided for treating or preventing a disease or disorder associated with a deficiency in the expression of a gene product in a subject in need thereof, the method including transducing a cell (e.g., a stem cell and / or a progenitor cell) with any of the vectors described herein or any of the viral particles described herein, and transplanting the cell into the subject (e.g., the cell or its derivatives express the gene product encoded by the vector or viral particle). In some embodiments, prior to transduction, the cell is derived from the subject (i.e., autologous to the subject to be treated). In some embodiments, the cell to be transduced is not derived from the subject to be treated.

[0053] In one aspect, provided herein is the use of any vector or viral particle described herein for treating or preventing a disease or disorder associated with a lack of expression of a gene product encoded by the vector or viral particle, in a subject in need thereof, comprising transducing a cell (e.g., a stem cell and / or a progenitor cell) with any vector described herein or any viral particle described herein, and transplanting the cell into the subject (e.g., the cell or its derivatives express the gene product encoded by the vector or viral particle). In some embodiments, prior to transduction, the cell is derived from the subject (i.e., autologous to the subject to be treated). In some embodiments, the cell to be transduced is not derived from the subject to be treated.

[0054] In some embodiments, the methods and uses provided herein are methods and uses for treating (rather than preventing) a disease or disorder.

[0055] In some embodiments, the syndrome, disease, or disorder is any syndrome, disease, or disorder associated with a lack of expression of WASp. In some embodiments, the syndrome, disease, or disorder is any disease or disorder associated with abnormal expression of WASp. In some embodiments, the syndrome, disease, or disorder is Wiskott-Aldrich syndrome (WAS). In some embodiments, the disease or disorder is X-linked thrombocytopenia (XLT) or X-linked congenital neutropenia (XLN). In some embodiments, the gene product expressed by the vector or viral particle is WASp. In some embodiments, the syndrome, disease, or disorder is any syndrome, disease, or disorder associated with a lack of expression of WASp, and the gene product expressed by the vector or viral particle is functional WASp.

[0056] In some embodiments, the stem cells and / or progenitor cells are human hematopoietic stem cells and / or progenitor cells. In some embodiments, the stem cells and / or progenitor cells are human hematopoietic stem cells and / or progenitor cells derived from bone marrow. In some embodiments, the cells are CD34+ cells. In some embodiments, the cells are megakaryocytes. In some embodiments, the cells are derived from mPBSC.

[0057] In some aspects, provided herein is a method of treating or preventing a disease or disorder associated with a deficiency in the expression of a gene product in a subject in need thereof, the method comprising administering to the subject any vector described herein, any viral particle described herein, any cell transduced with a vector or viral particle described herein, or any pharmaceutical composition comprising a vector, viral particle, or cell transduced therewith described herein.

[0058] In some aspects, provided herein is the use of any vector, viral particle, cell, or pharmaceutical composition described herein for treating or preventing a disease or disorder associated with a deficiency in the expression of a gene product in a subject in need thereof, the use comprising administering to the subject such a vector, viral particle, cell, or pharmaceutical composition described herein.

[0059] In some embodiments, the methods and uses provided herein are methods and uses for treating (not preventing) a disease or disorder.

[0060] In some embodiments, the subject to be treated using any method or nucleic acid (e.g., viral vector) described herein is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is male. In some embodiments, the subject to be treated using any method or use described herein is less than 21 years old, less than 18 years old, less than 16 years old, less than 14 years old, less than 12 years old, less than 10 years old, less than 8 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, less than 2 years old, or less than 1 year old. In some embodiments, the subject is an infant. In some embodiments, the subject is a toddler.

[0061] In some embodiments, the treatment comprises a single administration of a vector, viral particle, transduced cell, or pharmaceutical composition described herein.

[0062] In some embodiments, the treatment comprises parenteral (e.g., intravenous) administration of a vector, viral particle, transduced cell, or pharmaceutical composition described herein. In some embodiments, the treatment is by intravenous infusion. In some embodiments, the treatment comprises topical administration. In some embodiments, the treatment comprises intramuscular administration.

[0063] In some embodiments, the vector or viral particle described herein is administered at a dose in the range of about 1×10 5 TU / ml to about 1×10 8 TU / ml. In some cases, the vector is administered at a dose not exceeding 1×10 5 TU / ml. In some cases, the vector is 1×10 1 ~1×10 12 TU / m, 1×10 1 ~1×10 11 TU / mL, 1×10 1 ~1×10 10 TU / mL, 1×10 1 ~1×10 9 TU / mL, 1×10 1 ~1×10 8 TU / mL, 1×10 1 ~1×107 TU / mL, 1×10 1 ~1×10 6 TU / mL, 1×10 1 ~1×10 5 TU / mL, or 1×10 1 ~1×10 4 is administered at a dose of TU / mL.

[0064] In some embodiments, provided herein are recombinant nucleic acids or expression cassettes that contain a nucleic acid sequence of enhancer element 14 that comprises or consists of SEQ ID NO: 1 or a valid fragment thereof. In some embodiments, provided herein are recombinant nucleic acids or expression cassettes that contain a nucleic acid sequence of enhancer element 14 or a valid fragment thereof that comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1.

[0065] In some embodiments, the recombinant nucleic acid contains a nucleic acid sequence of the core fragment of element 14 that comprises or consists of SEQ ID NO: 2. In some embodiments, the recombinant nucleic acid contains a nucleic acid sequence of the core fragment of element 14 or a valid fragment thereof that comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2.

[0066] In some embodiments, the recombinant nucleic acid contains a nucleic acid sequence of the ultra-core fragment of element 14 that comprises or consists of SEQ ID NO: 3. In some embodiments, the recombinant nucleic acid contains a nucleic acid sequence of the ultra-core fragment of element 14 or a valid fragment thereof that comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3.

[0067] In some embodiments, the recombinant nucleic acid comprises the nucleic acid sequence of the first half of enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and / or enhancer element 2 core sub-element 5 of SEQ ID NO: 17. In some embodiments, the recombinant nucleic acid comprises the nucleic acid sequence of the first half of enhancer element 2 core sub-element 1 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14 and / or the nucleic acid sequence of enhancer element 2 core sub-element 5 of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17.

[0068] In some embodiments, the recombinant nucleic acid comprises or consists of the nucleic acid sequence of the uCore E2 element of SEQ ID NO: 32. In some embodiments, the recombinant nucleic acid comprises or consists of the nucleic acid sequence of the uCore E2 element of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32.

[0069] In some embodiments, the recombinant nucleic acid comprises, or consists of, a nucleic acid sequence of enhancer element 14, or a functional fragment thereof, that (i) comprises, or consists of, SEQ ID NO: 1 and optionally comprises, or consists of, SEQ ID NO: 2 or SEQ ID NO: 3, and / or (ii) a combination with the nucleic acid sequence of the uCore E2 element of SEQ ID NO: 32, or consists of the combination. In some embodiments, the recombinant nucleic acid comprises, or consists of, a combination of (i) a nucleic acid sequence of enhancer element 14, or a functional fragment thereof, that comprises, or consists of, a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 and optionally comprises, or consists of, SEQ ID NO: 2 or SEQ ID NO: 3, and (ii) a nucleic acid sequence of the uCore E2 element of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32, or consists of the combination.

[0070] In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence of any human promoter or a functional fragment thereof.

[0071] In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence of the endogenous promoter of the WAS gene. In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence of the WAS gene promoter that comprises, or consists of, SEQ ID NO: 11. In some embodiments, the recombinant nucleic acid comprises a nucleic acid sequence of the WAS gene promoter that comprises, or consists of, a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11.

[0072] In some embodiments, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene, having a maximum length of 600 bp and comprising the nucleic acid sequence of HS1pro of SEQ ID NO: 12. In some embodiments, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene, having a maximum length of 600 bp and comprising a nucleic acid sequence of HS1pro having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene consisting of the sequence of SEQ ID NO: 12. In some embodiments, the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene consisting of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12.

[0073] In some embodiments, the recombinant nucleic acid does not include (i) the nucleic acid sequence of sub-sub-element 1 of element 2 of SEQ ID NO: 9 or an effective fragment thereof, (ii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of SEQ ID NO: 10 or an effective fragment thereof, (iii) the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or an effective fragment thereof, and / or (v) the nucleic acid sequence of hypersensitive site 3 (HS3) of SEQ ID NO: 8 or an effective fragment thereof.

[0074] In some embodiments, the recombinant nucleic acid does not include (i) a nucleic acid sequence of sub-sub-element 1 of element 2 or a functional fragment thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9, (ii) a nucleic acid sequence of sub-element 4 of enhancer element 2 or a functional fragment thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10, (iii) a nucleic acid sequence of enhancer element 9 slim or a functional fragment thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7, and / or (v) a nucleic acid sequence of hypersensitive site 3 (HS3) or a functional fragment thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8.

[0075] In some embodiments, the recombinant nucleic acid comprises a transgene. In some embodiments of the nucleic acids described herein, the recombinant nucleic acid comprises a transgene operably linked to any enhancer element described herein (e.g., enhancer element 14 or a functional fragment thereof) and / or any promoter described herein. In some embodiments, the recombinant nucleic acid comprises a transgene encoding WASp. In some embodiments, the recombinant nucleic acid is contained within an expression cassette. In some embodiments, the expression cassette can express a gene product (e.g., WASp) encoded by the transgene in a cell (e.g., a stem cell and / or a progenitor cell, e.g., a hematopoietic stem cell and / or a progenitor cell). In some embodiments, the expression cassette drives expression of the gene product at or near physiological levels in cells of a healthy subject. In some embodiments, the expression cassette drives expression of the gene product at a high level (e.g., a level that exceeds the endogenous or physiological level of the corresponding native gene). In some embodiments, the expression cassette is effective to express WASp at physiological levels or at a high level in a healthy subject when transduced into a cell (e.g., a megakaryocyte). In some embodiments, the expression cassette is effective to express WASp within about 60%, 50%, 40%, 30%, or 20% of the endogenous physiological level of WASp in a healthy subject.

[0076] The term As used herein, the terms "about" and "approximately," when used to modify a numerical value, indicate that a deviation of up to 10% above or below that numerical value is included in the intended meaning of the recited value.

[0077] "Promoter" refers to a nucleic acid sequence capable of initiating transcription of a gene (e.g., a gene operably linked to the promoter). The term "promoter," as used herein, has the meaning generally known in the art.

[0078] An "enhancer" generally refers to a nucleic acid sequence that, when bound by one or more specific proteins, generally referred to as transcription factors, regulates the transcription of an operably linked gene (e.g., enhances it by increasing its rate or likelihood). This specification describes novel enhancers not generally known in the art. Generally, an enhancer can act by increasing the activity of a promoter operably linked to the same gene. An enhancer may be located away from the gene, either upstream or downstream from the start site, e.g., up to 1,000,000 bp away from the gene. In some cases, this application describes new enhancer elements and their properties. In some embodiments, the invention provides a new enhancer element, e.g., Element 14. The invention also describes Element 14 Core and Element 14 Ultra Core, which are functional fragments of Element 14. The examples presented herein show that gene transfer and virus titer in viral vectors were improved by the use of such enhancer elements.

[0079] As used herein, the term "effective fragment" when used in connection with a promoter (e.g., an effective fragment of the WAS promoter) refers to a fragment of the full-length promoter that is sufficient for promoter activity, i.e., capable of initiating transcription of a gene operably linked to that promoter. In some embodiments, the effective fragment provides the same, substantially the same, or similar expression level and / or pattern of the operably linked gene as compared to the full-length promoter. In some embodiments, the effective fragment provides a better expression level of the operably linked gene as compared to the full-length promoter.

[0080] As used herein, the term "effective fragment" when used in connection with an enhancer (e.g., an effective fragment of the WAS enhancer) refers to a fragment of the full-length enhancer that is sufficient for enhancer activity, i.e., capable of enhancing the transcription of an operably linked gene when bound by a transcription factor. In some embodiments, the effective fragment provides the same, substantially the same, or a similar expression level and / or pattern of the operably linked gene as compared to the full-length enhancer. In some embodiments, the effective fragment provides a better expression level of the operably linked gene as compared to the full-length enhancer.

[0081] The term "operably linked" refers to a nucleic acid sequence placed in a functional relationship with another nucleic acid sequence. The term "operably linked" as used herein has the meaning generally known in the art. For example, a promoter is operably linked to a gene if the promoter is positioned at a location that enables it to initiate transcription of the gene. An enhancer is operably linked to a gene if the enhancer can regulate (e.g., enhance) the expression of the gene when the enhancer is bound by an appropriate transcription factor.

[0082] "Recombinant" is used in accordance with its usage in the art to refer to a nucleic acid sequence that is not naturally occurring, e.g., contains portions that do not naturally occur together as part of a single sequence or is rearranged compared to a naturally occurring sequence. Recombinant nucleic acids are made by a process that includes human intervention and manipulation and / or are generated from nucleic acids made in such a manner. Recombinant viruses are those that contain recombinant nucleic acids. Recombinant cells are those that contain recombinant nucleic acids.

[0083] As used herein, the term "recombinant vector" refers to an artificially produced polynucleotide vector.

[0084] As used herein, the term "percent sequence identity" with respect to a reference nucleic acid or amino acid sequence is the percentage of nucleic acid bases or amino acid residues within a candidate sequence that are identical to the nucleic acid bases or amino acid residues in the reference sequence, after aligning the sequences and introducing gaps as needed to achieve the maximum percent sequence identity. Methods of sequence alignment are well known in the art. Optimal alignment of sequences can be performed by the methods described in Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, Pearson and Lipman, 1988, PNAS 85:2444, or by computer implementations of these algorithms. Alignment can be performed using publicly available computer software such as BLASTp, BLASTn, BLAST-2, ALIGN, or MegAlign Pro (DNASTAR) software.

[0085] As used herein, the term "effective amount" refers to the amount of an agent or a composition containing such agent required to bring about a particular physiological effect, e.g., to ameliorate or eliminate the symptoms of a disease as compared to a non-treated patient. The effective amount of a particular agent can be expressed in various ways, based on the nature of the agent, e.g., mass / volume, number of cells / volume, particles / volume, (mass of agent) / (mass of subject), number of cells / (mass of subject), or particles / (mass of subject). The effective amount of an agent or composition described herein for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such amount is referred to as an "effective" amount.

[0086] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of exemplary embodiments, in conjunction with the accompanying drawings.

Brief Description of the Drawings

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Best Mode for Carrying Out the Invention

[0135] It should be understood that the drawings are not necessarily to scale (e.g., schematic diagrams), and that like reference numerals indicate like characteristics.

[0136] Detailed Description In some embodiments, the present invention is based on the elucidation of a new enhancer element, Element 14, and its effective fragments, Element 14 Core and Element 14 Ultra Core. The examples presented herein show that improved gene transfer and viral titers were obtained by the use of such enhancer elements in a WAS expression vector.

[0137] In some embodiments, the present invention is based on the elucidation of a minimal enhancer element that can be placed in a vector backbone to assist gene expression in a particular cell type, e.g., a particular blood cell type. Such enhancers can be optimized to minimize vector size (e.g., the enhancer element backbone in WASVec2.0 V3). The examples presented herein show that improved gene transfer and viral titers were obtained by the use of such minimal enhancer element vector backbones in a WAS expression vector.

[0138] In some embodiments, vectors are provided herein that contain Element 14 or an effective fragment thereof (e.g., Element 14 Core and Element 14 Ultra Core). The vectors described herein may further contain a minimal enhancer element vector backbone (e.g., that of WASVec2.0 V3). The examples presented herein demonstrate that vectors having any one of such enhancer elements achieve excellent hematopoietic stem and progenitor cell (HPSC) gene transfer and improved viral titers. The examples also demonstrate that such vectors can maintain the ability to restore physiological levels of WASp expression in WAS− / − cells.

[0139] The examples presented in this specification also show that the enhancer elements identified by the inventors, when used in a WASp expression vector in vivo, resulted in improved WASp expression in all affected hematopoietic lineages and restoration of platelet counts and platelet function to levels in healthy subjects.

[0140] Accordingly, in some embodiments, enhancer element 14 having the nucleic acid sequence of SEQ ID NO: 1, or a functional fragment thereof, is provided herein. In some embodiments, an enhancer element having at least 70% or more, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 1, or a functional fragment thereof, is provided herein. In some embodiments, a vector comprising any such enhancer, e.g., enhancer element 14 of SEQ ID NO: 1, an enhancer element having at least 70% or more (or any of the identity percentages described above) nucleic acid sequence identity to SEQ ID NO: 1, or a functional fragment thereof, is provided herein. In some embodiments, the vector is a viral vector, e.g., a lentiviral vector. In some embodiments, the vector is for the expression of the WAS protein (i.e., the vector drives the expression of the gene encoding WASp).

[0141] In some embodiments, an enhancer element 14 core having the nucleic acid sequence of SEQ ID NO: 2, or a functional fragment thereof, is provided herein. In some embodiments, an enhancer element having at least 70% or more, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 2, or a functional fragment thereof, is provided herein. In some embodiments, an enhancer comprising, or consisting of, a nucleic acid sequence of an enhancer derived from WASVec2.0 V1 (SEQ ID NO: 4), or a nucleic acid sequence having at least 70% or more (or any of the above identity percentages) nucleic acid sequence identity to the enhancer derived from SEQ ID NO: 4, or a functional fragment thereof, is provided herein. In some embodiments, a vector comprising any such enhancer, e.g., the enhancer element 14 core of SEQ ID NO: 2, an enhancer element having at least 70% or more nucleic acid sequence identity (or any of the above identity percentages) to SEQ ID NO: 2, or a functional fragment thereof, is provided herein. In some embodiments, the vector is a viral vector, e.g., a lentiviral vector. In some embodiments, the vector is for the expression of the WAS protein (i.e., the vector drives the expression of the gene encoding WASp). In some embodiments, the vector WASVec2.0 V1 (SEQ ID NO: 4), or a vector substantially identical to WASVec2.0 V1 (SEQ ID NO: 4), is provided herein. In some embodiments, a vector having at least 50% or more, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 4 is provided herein.

[0142] In some embodiments, an enhancer element 14 ultra core having the nucleic acid sequence of SEQ ID NO: 3, or a functional fragment thereof, is provided herein. In some embodiments, an enhancer element having at least 70% or more, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 3, or a functional fragment thereof, is provided herein. In some embodiments, an enhancer comprising, or consisting of, a nucleic acid sequence of an enhancer derived from WASVec2.0 V2 (SEQ ID NO: 5), or a nucleic acid sequence having at least 70% or more (or any of the above identity percentages) nucleic acid sequence identity to an enhancer derived from SEQ ID NO: 5, or a functional fragment thereof, is provided herein. In some embodiments, a vector comprising any such enhancer, e.g., the enhancer element 14 ultra core of SEQ ID NO: 3, an enhancer element having at least 70% or more nucleic acid sequence identity (or any of the above identity percentages) to SEQ ID NO: 3, or a functional fragment thereof, is provided herein. In some embodiments, the vector is a viral vector, e.g., a lentiviral vector. In some embodiments, the vector is for the expression of the WAS protein (i.e., the vector drives the expression of the gene encoding WASp). In some embodiments, a vector WASVec2.0 V2 (SEQ ID NO: 5), or a vector substantially identical to WASVec2.0 V2 (SEQ ID NO: 5) is provided herein. In some embodiments, a vector having at least 50% or more, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 5 is provided herein.

[0143] In some embodiments that refer to an array having a percent identity to a given array, the array is effective to perform the function of the given array. In some embodiments that refer to an array having a percent identity to a given enhancer array, the array is effective to perform the enhancer function of the given array.

[0144] In some embodiments, enhancer element 14 or a functional fragment thereof (e.g., enhancer element 14 core or enhancer element 14 ultra-core) is used in combination with one or more additional enhancer elements. In some embodiments, the one or more additional enhancer elements comprise (i) the first half of enhancer element 2 core sub-element 1 (or a functional fragment thereof) comprising the nucleic acid sequence of SEQ ID NO: 14, and / or (ii) enhancer element 2 core sub-element 5 (or a functional fragment thereof) comprising the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the one or more additional enhancer elements comprise, or consist of, a uCore E2 element consisting of the first half of enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and enhancer element 2 core sub-element 5 of SEQ ID NO: 17. In some embodiments, the one or more additional enhancer elements comprise, or consist of, a uCore E2 element of SEQ ID NO: 32. In some embodiments, the additional enhancer element comprises a sequence having at least 50% or more, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 14, 17, or 32. In some embodiments, the additional enhancer element comprises a sequence having at least 50% or more, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 14, 17, or 32 and is effective to perform the function of SEQ ID NO: 14, 17, or 32.

[0145] In some embodiments, the vectors provided herein comprise enhancer element 14 or a functional fragment thereof (e.g., enhancer element 14 core or enhancer element 14 ultra-core).

[0146] In some embodiments, the vectors provided herein include enhancer element 14 or a functional fragment thereof (e.g., enhancer element 14 core or enhancer element 14 ultra-core), the first half of enhancer element 2 core sub-element 1 comprising the nucleic acid sequence of SEQ ID NO: 14 (or a functional fragment thereof), and enhancer element 2 core sub-element 5 comprising the nucleic acid sequence of SEQ ID NO: 17 (or a functional fragment thereof). In some embodiments, the vectors provided herein include enhancer element 14 or a functional fragment thereof (e.g., enhancer element 14 core or enhancer element 14 ultra-core), and the uCore E2 element consisting of the first half of enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and enhancer element 2 core sub-element 5 of SEQ ID NO: 17. In some embodiments, the vectors provided herein include an enhancer element, and the enhancer element comprises or consists essentially of enhancer element 14 or a functional fragment thereof (e.g., enhancer element 14 core or enhancer element 14 ultra-core) and the uCore E2 element of SEQ ID NO: 32.

[0147] WASVec1.0 is described in International Publication No. WO2021096887A (referred to as Figure 20, SEQ ID NO: 17 in International Publication No. WO2021096887A), and the entire disclosure thereof, particularly the disclosure regarding the enhancer, promoter, WAS, and vector elements described therein, for example, the disclosure regarding WASVec1.0 is hereby incorporated by reference in its entirety. The enhancer element used in WASVec1.0 is described in International Publication No. WO2021096887A.

[0148] In some embodiments, the enhancer provided herein is reduced to a minimal backbone compared to the enhancer in WASVec1.0. In some embodiments, the minimal backbone enhancer provided herein lacks (i) "element 9 slim" (E9s) having the nucleic acid sequence of SEQ ID NO: 7, a valid fragment thereof, or an element substantially identical thereto, (ii) "hypersensitive site 3 slim" (HS3s) having the nucleic acid sequence of SEQ ID NO: 8, a valid fragment thereof, or an element substantially identical thereto, (iii) sub-sub-element 1 of element 2 having the nucleic acid sequence of SEQ ID NO: 9, a valid fragment thereof, or an element substantially identical thereto, and / or (iv) sub-element 4 of E2 having the nucleic acid sequence of SEQ ID NO: 10, a valid fragment thereof, or an element substantially identical thereto. In some embodiments, the minimal backbone enhancer provided herein lacks "element 9 slim" (E9s) having the nucleic acid sequence of SEQ ID NO: 7 and / or a valid fragment thereof. In some embodiments, the minimal backbone enhancer provided herein lacks "hypersensitive site 3 slim" (HS3s) having the nucleic acid sequence of SEQ ID NO: 8 and / or a valid fragment thereof. In some embodiments, the minimal backbone enhancer provided herein lacks sub-sub-element 1 of element 2 having the nucleic acid sequence of SEQ ID NO: 9 and / or a valid fragment thereof. In some embodiments, the minimal backbone enhancer provided herein lacks sub-element 4 of E2 having the nucleic acid sequence of SEQ ID NO: 10 and / or a valid fragment thereof.

[0149] In some embodiments, the enhancer provided herein that is reduced to a minimal backbone compared to the enhancer in WASVec1.0 lacks the elements described in the preceding paragraph (e.g., the "Element 9 Slim" (E9s), "Hypersensitive Site 3 Slim" (HS3s), sub-element 4 of E2, and sub-sub-element 1 of Element 2 of the sequences mentioned above, or their effective fragments), but includes the first half of the enhancer element 2 core sub-element 1 containing the nucleic acid sequence of SEQ ID NO: 14 and the enhancer element 2 core sub-element 5 containing the nucleic acid sequence of SEQ ID NO: 17 (e.g., that in WASVec2.0 V3 provided herein).

[0150] In some embodiments, the enhancer provided herein that is reduced to a minimal backbone compared to the enhancer in WASVec1.0 lacks the elements described in the preceding paragraph (e.g., the "Element 9 Slim" (E9s), "Hypersensitive Site 3 Slim" (HS3s), sub-element 4 of E2, and sub-sub-element 1 of Element 2 of the sequences mentioned above, or their effective fragments), but includes the uCore E2 element consisting of the first half of the enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and the enhancer element 2 core sub-element 5 of SEQ ID NO: 17 (e.g., that in WASVec2.0 V3 provided herein). In some embodiments, the enhancer provided herein that is reduced to a minimal backbone compared to the enhancer in WASVec1.0 lacks the elements described in the preceding paragraph (e.g., the "Element 9 Slim" (E9s), "Hypersensitive Site 3 Slim" (HS3s), sub-element 4 of E2, and sub-sub-element 1 of Element 2 of the sequences mentioned above, or their effective fragments), but includes the uCore E2 element of SEQ ID NO: 32. In some embodiments, the enhancer provided herein includes or consists essentially of the uCore E2 element of SEQ ID NO: 32. In some embodiments, the enhancer provided herein includes or consists essentially of the combination of SEQ ID NO: 14 and SEQ ID NO: 17.

[0151] In some embodiments, the vectors provided herein include or consist of an enhancer that includes the nucleic acid sequence of the enhancer in WASVec2.0 V3. In some embodiments, the vectors provided herein include or consist of an enhancer that includes the nucleic acid sequence of the enhancer in WASVec2.0 V2.

[0152] In some embodiments, the enhancer provided herein includes the first half of enhancer element 2 core sub-element 1, including the nucleic acid sequence of SEQ ID NO: 14, a valid fragment thereof, or a sequence substantially identical thereto. In some embodiments, the enhancer provided herein includes an enhancer element having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 14, or a valid fragment thereof. Vectors containing such enhancers are also contemplated.

[0153] In some embodiments, the enhancer provided herein includes enhancer element 2 core sub-element 5, including the nucleic acid sequence of SEQ ID NO: 17, a valid fragment thereof, or a sequence substantially identical thereto. In some embodiments, the enhancer provided herein includes an enhancer element having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 17, or a valid fragment thereof. Vectors containing such enhancers are also contemplated.

[0154] In some embodiments, the enhancer provided herein comprises a uCoreE2 element comprising the nucleic acid sequence of SEQ ID NO: 32, a valid fragment thereof, or a sequence substantially identical thereto. In some embodiments, the enhancer provided herein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 32, and comprises an enhancer element or a valid fragment thereof. Vectors containing such enhancers are also contemplated.

[0155] In some embodiments, an expression construct (e.g., a vector) comprising a nucleic acid sequence of an enhancer consisting of or comprising the first half of the enhancer element 2 core sub-element 1 of SEQ ID NO: 14 and the enhancer element 2 core sub-element 5 of SEQ ID NO: 17, a nucleic acid sequence of a promoter or a valid fragment thereof, and a nucleic acid encoding the Wiskott-Aldrich syndrome protein (WASp) operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, wherein the expression construct (e.g., a vector) does not contain (i) the nucleic acid sequence of enhancer element 2 of SEQ ID NO: 13, (ii) the nucleic acid sequence of sub-sub-element 1 of element 2 of SEQ ID NO: 9 or a valid fragment thereof, (iii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of SEQ ID NO: 10 or a valid fragment thereof, (iv) the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or a valid fragment thereof, and / or (v) the nucleic acid sequence of hypersensitive site 3 (HS3) of SEQ ID NO: 8 or a valid fragment thereof, is provided herein. In some embodiments, the vector is a lentiviral vector.

[0156] In some embodiments, an expression construct (e.g., a vector) comprising or consisting of a nucleic acid sequence of an enhancer comprising the uCore E2 element of SEQ ID NO: 32, a nucleic acid sequence of a promoter or a functional fragment thereof, and a nucleic acid encoding Wiskott - Aldrich syndrome protein (WASp) operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, wherein the expression construct (e.g., a vector) does not include (i) the nucleic acid sequence of enhancer element 2 of SEQ ID NO: 13, (ii) the nucleic acid sequence of sub - sub - element 1 of element 2 of SEQ ID NO: 9 or a functional fragment thereof, (iii) the nucleic acid sequence of sub - element 4 of enhancer element 2 of SEQ ID NO: 10 or a functional fragment thereof, (iv) the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or a functional fragment thereof, and / or (v) the nucleic acid sequence of hypersensitive site 3 (HS3) of SEQ ID NO: 8 or a functional fragment thereof, is provided herein. In some embodiments, the vector is a lentiviral vector.

[0157] In some embodiments, an expression construct (e.g., a vector) comprising a nucleic acid sequence of an enhancer comprising enhancer element 14 of SEQ ID NO: 1, enhancer element 14 core of SEQ ID NO: 2, or enhancer element 14 ultra - core of SEQ ID NO: 3, a nucleic acid sequence of a promoter or a functional fragment thereof, and a nucleic acid encoding Wiskott - Aldrich syndrome protein (WASp) operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, is provided herein. In some embodiments, the vector is a lentiviral vector.

[0158] In some embodiments, an expression construct (e.g., a vector) comprising a nucleic acid sequence of an enhancer comprising enhancer element 14 of SEQ ID NO: 1, enhancer element 14 core of SEQ ID NO: 2, or enhancer element 14 ultra core of SEQ ID NO: 3, a nucleic acid sequence of a promoter or a functional fragment thereof, and a nucleic acid encoding Wiskott-Aldrich syndrome protein (WASp) operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, wherein the expression construct (e.g., a vector) does not include (i) the nucleic acid sequence of enhancer element 2 of SEQ ID NO: 13, (ii) the nucleic acid sequence of sub-sub-element 1 of element 2 of SEQ ID NO: 9 or a functional fragment thereof, (iii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of SEQ ID NO: 10 or a functional fragment thereof, (iv) the nucleic acid sequence of enhancer element 9 slim of SEQ ID NO: 7 or a functional fragment thereof, and / or (v) the nucleic acid sequence of hypersensitive site 3 (HS3) of SEQ ID NO: 8 or a functional fragment thereof, is provided herein. In some embodiments, the vector is a lentiviral vector.

[0159] In some embodiments of the expression constructs (e.g., vectors) described herein, the promoter is any human promoter (e.g., any human promoter known in the art). In some embodiments of the expression constructs (e.g., vectors) described herein, the promoter is the endogenous promoter of the WAS gene, e.g., the human endogenous WAS gene promoter. In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 11, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 11. In some embodiments, the promoter has a maximum length of 600 bp and comprises the sequence of HS1pro (SEQ ID NO: 12). In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 12, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 12. In some embodiments, the promoter is a functional fragment of the endogenous promoter of the WAS gene consisting of or substantially consisting of the sequence of HS1pro (SEQ ID NO: 12).

[0160] In some embodiments referring to a sequence having a percent identity to a given promoter sequence, the sequence is effective to perform the promoter function of the given sequence.

[0161] In some embodiments, the vectors described herein comprise a transgene operably linked to any one of the enhancers and / or promoter elements described herein. In some embodiments, the transgene encodes a protein (e.g., WASp).

[0162] In some embodiments, the vectors described herein comprise any or all of the vector characteristics shown in Figure 2.

[0163] In some embodiments, the vectors described herein include any or all of the characteristics of the vectors shown in FIG. 5A.

[0164] In some embodiments, the vectors described herein include any or all of the characteristics of the vectors shown in FIG. 5B.

[0165] In some embodiments, vectors comprising the nucleic acid sequence of SEQ ID NO: 4 are described herein. In some embodiments, vectors comprising nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 4 are described herein.

[0166] In some embodiments, vectors comprising the nucleic acid sequence of SEQ ID NO: 5 are described herein. In some embodiments, vectors comprising nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 5 are described herein.

[0167] In some embodiments, vectors comprising the nucleic acid sequence of SEQ ID NO: 6 are described herein. In some embodiments, vectors comprising nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 6 are described herein.

[0168] In some embodiments, the vectors provided herein increase the expression level and titer of a transgene, such as WAS. In some embodiments, the vectors provided herein can express a transgene, such as WAS, in a cell at a physiological level (e.g., at or near the expression level of the native gene corresponding to the transgene). In some embodiments, the vectors provided herein can express a transgene, such as WAS, in a cell at a level of at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the expression level of the native gene corresponding to the transgene. In some embodiments, the vectors provided herein can express a transgene, such as WAS, in a cell at a high level (e.g., higher than the expression level of the native gene corresponding to the transgene). In some embodiments, the vector is a lentiviral vector.

[0169] In some embodiments, the vectors provided herein are optimized to reduce vector size. In some embodiments, the vectors provided herein are less than 6 kb in size. In some embodiments, the vectors provided herein are about 5.9 kb or less in size. In some embodiments, the vectors provided herein are about 5.8 kb or less in size. In some embodiments, the vectors provided herein are about 5.7 kb or less in size. In some embodiments, the vectors provided herein are about 5.6 kb or less in size. In some embodiments, the vectors provided herein are about 5.5 kb or less in size. In some embodiments, the vectors provided herein are about 5.4 kb in size. In some of these embodiments, the vector is a lentiviral vector. In some of these embodiments, the vector is a lentiviral vector and the transgene encodes WASp.

[0170] In some embodiments, the vectors provided herein are for the treatment of any disorder associated with a deficiency in the expression of a protein encoded by a transgene. In some embodiments, the vectors provided herein are for the treatment of any disorder associated with a deficiency in the expression of the WAS protein. In some embodiments, the vectors provided herein are for the treatment of Wiskott-Aldrich syndrome (WAS). In some embodiments, the vectors provided herein are for the treatment of XLT.

[0171] When reference is made herein to the expression of the WAS transgene and / or WASp, the use of other transgenes and / or the expression of other gene products (e.g., those described herein) is also contemplated.

[0172] When reference is made herein to a vector, the use of other transgene delivery vehicles (e.g., those described herein) is also contemplated.

[0173] Additional Explanation Regarding Enhancer Element Through the bioinformatics analysis and enhancer screening described in the examples of this application, a sequence of approximately 2173 bp, designated as Element 14, which increases WAS expression 1.7-fold more than the endogenous WAS promoter alone in human HSPC-derived megakaryocytes, was identified. This disclosure contemplates that Element 14 and its functional fragments can be used to drive the expression of various other genes in certain blood cell types when driving expression from at least the WAS promoter, the WAS minimal promoter, or similar promoters. In some embodiments, the 2173 bp nucleic acid sequence of SEQ ID NO: 1, herein referred to as "Element 14 (E14)", is described herein.

[0174] A functional 555 bp region within E14 was identified by the inventors. In some embodiments, the 555 bp nucleic acid sequence of SEQ ID NO: 2, herein referred to as "Element 14 Core", is described herein.

[0175] A functional 234 bp region within the Element 14 Core was further identified by the inventors. In some embodiments, the 234 bp nucleic acid sequence of SEQ ID NO: 3, herein referred to as "Element 14 Ultra Core", is described herein.

[0176] In some embodiments, the Element 14 Core fragment is incorporated into WASVec2.0 V3 to generate WASVec2.0 V1. In some embodiments, the Element 14 Ultra Core fragment is incorporated into WASVec2.0 V3 to generate WASVec2.0 V2.

[0177] In some embodiments, WASVec2.0 V1, WASVec2.0 V2, and WASVec2.0 V3 exhibit increased viral titers compared to WASVec1.0 after transfection into human cells.

[0178] In some embodiments, WASVec2.0 V1, WASVec2.0 V2, and WASVec2.0 V3 exhibit increased gene transfer compared to WASVec1.0 after transfection into human cells.

[0179] In some embodiments, the expression cassette comprises a Slim enhancer element 2 (SEQ ID NO: 13) or a functional fragment thereof. In some embodiments, the expression cassette comprises a functional fragment of enhancer element 2, the fragment comprising enhancer element 2 core sub-element 1 (SEQ ID NO: 14 + SEQ ID NO: 9). In some embodiments, the expression cassette comprises a functional fragment of enhancer element 2, the fragment comprising enhancer element 2 core sub-element 4 (SEQ ID NO: 10). In some embodiments, the expression cassette comprises enhancer element 2 core sub-element 5 (SEQ ID NO: 17). In some embodiments, the expression cassette comprises a functional fragment of enhancer element 2, the fragment comprising enhancer element 2 core sub-element 1 (SEQ ID NO: 14 + SEQ ID NO: 9), enhancer element 2 core sub-element 4 (SEQ ID NO: 10), and enhancer element 2 core sub-element 5 (SEQ ID NO: 17).

[0180] In some embodiments, the expression cassette does not comprise Slim enhancer element 2 (SEQ ID NO: 13). In some embodiments, the expression cassette does not comprise (SEQ ID NO: 9). In some embodiments, the expression cassette does not comprise enhancer element 2 core sub-element 4 (SEQ ID NO: 10).

[0181] In some embodiments, the expression cassette comprises a functional fragment of enhancer element 2, the fragment comprising the first half of enhancer element 2 core sub-element 1 (SEQ ID NO: 14) and enhancer element 2 core sub-element 5 (SEQ ID NO: 17). In some embodiments, the expression cassette comprises a functional fragment of enhancer element 2, the fragment consisting of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO: 14) and enhancer element 2 core sub-element 5 (SEQ ID NO: 17).

[0182] In some embodiments, the expression cassette comprises an enhancer element E9 core sequence (SEQ ID NO: 7). In some embodiments, the expression cassette comprises an enhancer element HS3 core sequence (SEQ ID NO: 8).

[0183] In some embodiments, the expression cassette does not comprise an enhancer element E9 core sequence (SEQ ID NO: 7). In some embodiments, the expression cassette does not comprise an enhancer element HS3 core sequence (SEQ ID NO: 8).

[0184] In some embodiments, the expression cassette comprises an enhancer element E9 core sequence (SEQ ID NO: 7), an enhancer element HS3 core sequence (SEQ ID NO: 8), and a Slim enhancer element 2 sequence (SEQ ID NO: 13).

[0185] In some embodiments, the expression cassette does not comprise a combination of an enhancer element E9 core sequence (SEQ ID NO: 7), an enhancer element HS3 core sequence (SEQ ID NO: 8), and a Slim enhancer element 2 sequence (SEQ ID NO: 13).

[0186] In some embodiments, the expression cassette does not comprise the second half of the core sub-element 1 of enhancer element 2 (SEQ ID NO: 9). In some embodiments, the expression cassette does not comprise the core sub-element 4 of enhancer element 2 (SEQ ID NO: 10).

[0187] In some embodiments, the expression cassette does not comprise a combination of an enhancer element E9 core sequence (SEQ ID NO: 7), an enhancer element HS3 core sequence (SEQ ID NO: 8), the second half of the core sub-element 1 of enhancer element 2 (SEQ ID NO: 9), and the core sub-element 4 of enhancer element 2 (SEQ ID NO: 10).

[0188] In some embodiments, the expression cassette comprises an enhancer element E9 core sequence (SEQ ID NO: 7), an enhancer element HS3 core sequence (SEQ ID NO: 8), an enhancer element 2 core sub-element 1 (SEQ ID NO: 14 + SEQ ID NO: 9), an enhancer element 2 core sub-element 4 (SEQ ID NO: 10), and an enhancer element 2 core sub-element 5 (SEQ ID NO: 17).

[0189] In some embodiments, the expression cassette does not comprise a combination of an enhancer element E9 core sequence (SEQ ID NO: 7), an enhancer element HS3 core sequence (SEQ ID NO: 8), an enhancer element 2 core sub-element 1 (SEQ ID NO: 14 + SEQ ID NO: 9), an enhancer element 2 core sub-element 4 (SEQ ID NO: 10), and an enhancer element 2 core sub-element 5 (SEQ ID NO: 17).

[0190] In some embodiments, the expression cassette comprises an enhancer element E9 core sequence (SEQ ID NO: 7), an enhancer element HS3 core sequence (SEQ ID NO: 8), the first half of enhancer element 2 core sub-element 1 (SEQ ID NO: 14), and an enhancer element 2 core sub-element 5 (SEQ ID NO: 17).

[0191] In some embodiments, the expression cassette does not comprise a combination of an enhancer element E9 core sequence (SEQ ID NO: 7), an enhancer element HS3 core sequence (SEQ ID NO: 8), the first half of enhancer element 2 core sub-element 1 (SEQ ID NO: 14), and an enhancer element 2 core sub-element 5 (SEQ ID NO: 17).

[0192] In some embodiments, the expression cassette comprises an enhancer that comprises or consists essentially of the uCore E2 element of SEQ ID NO: 32.

[0193] In some embodiments, the expression cassette comprises or consists essentially of a combination of element 14 or a functional fragment thereof (e.g., element 14 core or element 14 ultra core) and the uCore E2 element of SEQ ID NO: 32, and includes an enhancer.

[0194] In some embodiments, the expression cassette includes an enhancer that comprises or consists essentially of a combination of (i) SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and / or (ii) SEQ ID NO: 32.

[0195] In some embodiments, the enhancer can operably link a transgene to be expressed (e.g., in megakaryocytes) at a level of at least 25% or more, at least 30% or more, at least 35% or more, at least 40% or more, at least 45% or more, at least 50% or more, at least 55% or more, at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more, at least 90% or more, at least 95%, or at least 100% or more of the wild-type expression of the gene or gene expression in a healthy subject. In some embodiments, the transgene encodes WASp.

[0196] In some embodiments, the enhancer can cause an operably linked transgene to be expressed at a physiological or endogenous level (the normal level of expression of the corresponding wild-type gene) or near that level in cells, such as megakaryocytes. Near-normal levels of expression can be within 5%, 10%, 15%, 20%, 25%, or 30% of the wild-type expression level of the gene (e.g., 70% - 100% of the wild-type level, 80 - 100% of the wild-type level, 90 - 100% of the wild-type level, 100% - 130% of the wild-type level, or 75% - 125% of the wild-type level, or any range therebetween). In some embodiments, the transgene encodes WASp.

[0197] In some embodiments, the enhancer can cause an operably linked transgene to be expressed at a physiological level in all affected cell lineages. In some embodiments, the enhancer can cause an operably linked transgene to be expressed at a physiological level in megakaryocytes. In some embodiments, the transgene encodes WASp. In some embodiments, the enhancer can cause WASp to be expressed at a level that increases platelet count. In some embodiments, the enhancer can cause WASp to be expressed at a level that restores platelet count to healthy subject levels in vivo. In some embodiments, the enhancer can cause WASp to be expressed at a level that increases platelet engraftment and / or improves or restores platelet function to healthy subject levels in vivo.

[0198] In some embodiments, the enhancer can cause an operably linked transgene to be expressed at a high level (i.e., overexpressed) in cells, such as megakaryocytes, platelets.

[0199] The enhancers described herein can be used with any of the promoters described herein.

[0200] The enhancers described herein can be used in any of the vectors described herein. In some embodiments, the enhancers described herein are for use in viral vectors (e.g., LV). In some embodiments, the enhancers described herein are for use in non-viral vectors (e.g., plasmids or transposons).

[0201] In some embodiments, the enhancers described herein can be used in single-stranded oligonucleotides and / or double-stranded DNA homologous recombination repair templates for CRISPR gene editing.

[0202] Promoter In some embodiments, the promoters described herein are any promoter (e.g., any human promoter).

[0203] In some embodiments, the promoter is the minimal CMV promoter. In some embodiments, the promoter is a constitutively active promoter, such as the elongation factor alpha short (EFS) promoter or the phosphoglycerate kinase (PGK) promoter.

[0204] Systematic analysis of the biochemical compatibility of 1000 enhancer sequences and 1000 promoter sequences in recent years has revealed extensive compatibility between human enhancer and promoter elements. See Bergman et al., 2022; Nature, doi: 10.1038 / s41586-022-04877-w. Thus, in some embodiments, any expression cassette (e.g., vector) described herein may include any human promoter (e.g., the endogenous promoter of any human gene). In some embodiments, the expression cassette (e.g., vector) described herein includes the endogenous promoter of any human gene. In some embodiments, the promoter is a human CMV promoter, a human phosphoglycerate kinase gene promoter, a human elongation factor 1 alpha (EF1-alpha) promoter, a human U6 promoter, a human ubiquitin promoter (e.g., human ubiquitin C promoter).

[0205] In some embodiments, the promoter described herein is the endogenous promoter (e.g., human promoter) of a transgene (e.g., human gene) or a functional fragment thereof.

[0206] In some embodiments, the promoter described herein is the endogenous promoter of the WAS gene. In some embodiments, the promoter described herein is the endogenous promoter of the human WAS gene. In some embodiments, the promoter described herein is a genomic fragment of about 1.6 kb derived from the human WAS promoter. In some embodiments, the promoter described herein is a 1.6 kb (or about 1.6 kb) genomic fragment derived from the human WAS promoter disclosed in Dupre et al., 2005; Mol. Ther, 10(5):903-15, doi: 10.1016 / j.ymthe.2004.08.008, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the promoter described herein comprises the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the promoter described herein comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 11, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 11.

[0207] In some embodiments, the expression cassettes (e.g., vectors) described herein include a WAS promoter (e.g., the endogenous promoter of the human WAS gene). In some embodiments, the expression cassettes (e.g., vectors) described herein include a promoter that includes a genomic fragment of about 1.6 kb derived from the human WAS promoter (e.g., the 1.6 kb WAS promoter disclosed in Dupre et al., 2005; Mol. Ther, 10(5):903-15, doi: 10.1016 / j.ymthe.2004.08.008). In some embodiments, the expression cassettes (e.g., vectors) described herein include a promoter that comprises or consists of a nucleic acid sequence of SEQ ID NO: 11, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 11.

[0208] In some embodiments, the promoter described herein is an effective fragment of the endogenous promoter of the WAS gene. In some embodiments, the promoter described herein is an effective fragment of the endogenous promoter of the human WAS gene. In some embodiments, the promoter described herein is the minimal effective fragment of the endogenous promoter of the WAS gene (e.g., the human WAS gene). In some embodiments, the promoter described herein is the minimal effective fragment of the endogenous promoter of the WAS gene, and the promoter has a maximum length of 600 bp and comprises the sequence of HS1pro (e.g., as described in International Publication No. WO2021 / 096887A1, the disclosure of which related to the promoter is hereby incorporated by reference in its entirety). In some embodiments, the promoter described herein is the minimal effective fragment of the endogenous promoter of the WAS gene, and the promoter comprises the sequence of HS1pro (e.g., as described in International Publication No. WO2021 / 096887A1). In some embodiments, the promoter described herein is HS1pro (e.g., as described in International Publication No. WO2021 / 096887A1), and consists of, or consists essentially of, for example, the sequence of HS1pro. In some embodiments, the promoter described herein comprises, or consists of, SEQ ID NO: 12. In some embodiments, the promoter described herein comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 12, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 12.

[0209] In some embodiments, the expression cassette (e.g., vector) described herein comprises a functional fragment of the endogenous promoter of the WAS gene. In some embodiments, the expression cassette (e.g., vector) described herein comprises the endogenous promoter of the human WAS gene. In some embodiments, the expression cassette (e.g., vector) described herein comprises the minimal functional fragment of the endogenous promoter of the WAS gene (e.g., human WAS gene). In some embodiments, the expression cassette (e.g., vector) described herein comprises the minimal functional fragment of the endogenous promoter of the WAS gene, said promoter having a maximum length of 600 bp and comprising the sequence of HS1pro (e.g., as described in International Publication No. WO2021 / 096887A1, the disclosure of which related to the promoter is incorporated herein by reference in its entirety). In some embodiments, the expression cassette (e.g., vector) described herein comprises the minimal functional fragment of the endogenous promoter of the WAS gene, said minimal functional fragment comprising the sequence of HS1pro (e.g., as described in International Publication No. WO2021 / 096887A1). In some embodiments, the expression cassette (e.g., vector) described herein comprises a promoter, the promoter being HS1pro (e.g., as described in International Publication No. WO2021 / 096887A1), for example, consisting of or substantially consisting of the sequence of HS1pro. In some embodiments, the expression cassette (e.g., vector) described herein comprises a promoter comprising or consisting of SEQ ID NO: 12. In some embodiments, the expression cassette (e.g., vector) described herein comprises a promoter comprising the nucleic acid sequence of SEQ ID NO: 12, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 12.

[0210] The promoters described herein can be used with any enhancer element described herein. The promoters described herein can be used in any vector described herein. In some embodiments, the promoters described herein are for use in viral vectors (e.g., LV). In some embodiments, the promoters described herein are for use in non-viral vectors (e.g., plasmids or transposons).

[0211] Transgene In some embodiments, any vector, expression construct, enhancer, and / or promoter described herein can be used for the expression of any transgene. In some embodiments, the transgene encodes a polypeptide, e.g., a polypeptide having a therapeutic benefit. In some embodiments, the expression of the polypeptide encoded by the transgene (using any vector, expression construct, enhancer, and / or promoter described herein) compensates for the deficiency or absence of the expression of the endogenous polypeptide in the cell. One of ordinary skill in the art knows or can determine the suitability of any particular transgene for use with the vectors, expression constructs, enhancers, and / or promoters described herein.

[0212] In some embodiments, the transgene encodes a therapeutic peptide or protein. In some embodiments, the transgene encodes a chimeric antigen receptor. In some embodiments, the transgene encodes a coagulation factor.

[0213] In some embodiments, the transgene encodes a WAS protein. In some embodiments, the transgene encodes a human WAS protein. In some embodiments, the transgene comprises a nucleic acid, e.g., DNA of the WAS gene (e.g., DNA of human WAS). In some embodiments, the transgene comprises a WAS cDNA (e.g., cDNA of the human WAS gene). In some embodiments, the transgene is a WAS cDNA having the nucleic acid sequence of SEQ ID NO: 20 or comprises the same. In some embodiments, the transgene is a WAS cDNA having a nucleic acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 20 or comprises the same. In some embodiments, the transgene comprises a codon-optimized WAS cDNA (e.g., codon-optimized cDNA of the human WAS gene). In some embodiments, the transgene is a codon-optimized WAS cDNA having the nucleic acid sequence of SEQ ID NO: 21 or comprises the same. In some embodiments, the transgene is a WAS cDNA having a nucleic acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity to SEQ ID NO: 21 or comprises the same.

[0214] Polynucleotide or nucleic acid In some embodiments, provided herein is a polynucleotide or nucleic acid comprising any enhancer described herein, any promoter described herein, and any transgene described herein. In some embodiments, provided herein is a polynucleotide or nucleic acid comprising any enhancer described herein and any promoter described herein operably linked to any transgene described herein.

[0215] In some embodiments, the polynucleotides or nucleic acids described herein can express a gene product encoded by a transgene.

[0216] In some embodiments, the polynucleotides or nucleic acids described herein are codon-optimized (e.g., with respect to human codon usage).

[0217] In some embodiments, the polynucleotides or nucleic acids described herein further comprise untranslated regions (UTRs), signal sequences, Kozak sequences, transcription start sites, polyadenylation sequences, stop codons, and / or transcription termination signals.

[0218] In some embodiments, the polynucleotide or nucleic acid is a recombinant polynucleotide or nucleic acid.

[0219] Vector In some embodiments, delivery of nucleic acids using any of the expression cassettes, enhancers, and / or promoters described herein is by use of a vector. The vector can be a vector known in the art or any viral or non-viral vector described herein. For example, viral and non-viral vectors and delivery systems are described in Sung & Kim 2019, Biomaterials Research 23:8, doi: 10.1186 / s40824-019-0156-z, Mali, 2013, Indian Journal of Human Genetics, 19(1):3-8, Hardee et al., 2017, Genes 8:65, Bulcha et al., 2020, Signal Transduction and Targeted Therapy, Ghosh et al., 2020, Applied Biosafety: Journal of ABSA International 25(1):7-18, the disclosures of each of which are incorporated herein by reference in their entirety.

[0220] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector (LV), a retroviral vector (RV), an adenoviral vector (AV), an adeno-associated viral vector (AAV), a herpes simplex viral vector (HSV), or a poxviral vector.

[0221] In some embodiments, an LV (e.g., a recombinant LV) comprising any of the expression cassettes, enhancers, and / or promoters described herein is provided herein.

[0222] In some embodiments, an RV comprising any of the expression cassettes, enhancers, and / or promoters described herein is provided herein.

[0223] In some embodiments, a gamma-retroviral vector comprising any of the expression cassettes, enhancers, and / or promoters described herein is provided herein.

[0224] In some embodiments, an AV comprising any of the expression cassettes, enhancers, and / or promoters described herein is provided herein.

[0225] In some embodiments, an AAV comprising any of the expression cassettes, enhancers, and / or promoters described herein is provided herein.

[0226] In some embodiments, an HSV comprising any of the expression cassettes, enhancers, and / or promoters described herein is provided herein.

[0227] In some embodiments, a poxvirus-based vector comprising any of the expression cassettes, enhancers, and / or promoters described herein is provided herein.

[0228] In some embodiments, the viral vectors described herein are engineered to be safe by rendering them unable to replicate. In some embodiments, the viral vectors described herein are replication - incompetent.

[0229] In some embodiments, the viral vectors described herein are replication - competent.

[0230] In some embodiments, the viral vectors described herein have no or low toxicity (i.e., have no effect on the physiology of normal host cells).

[0231] In some embodiments, the viral vectors described herein are stable (e.g., genomic rearrangements do not occur).

[0232] In some embodiments, the vector is a non - viral vector. In some embodiments, the non - viral vector is naked DNA (e.g., a DNA plasmid). In some embodiments, the non - viral vector is a plasmid. In some embodiments, the non - viral vector is delivered in a lipid composition, in a chromosome, with a cationic polymer, or as a conjugate complex. In some embodiments, the non - viral vector is a liposome or lipid vector comprising plasmid DNA and a lipid solution. In some embodiments, the non - viral vector is a transposon vector.

[0233] The non - viral vector or plasmid DNA can be transfected into cells, for example, by chemical or physical transfection. Chemical transfection can be achieved by calcium phosphate, lipids, or protein complexes. Physical transfection can be achieved by electroporation or microinjection.

[0234] In some embodiments, the vectors described herein exhibit high expression in MEG-01 cells (megakaryocyte cell line) and / or Jurkat cells (T cell line) and / or RAMO cells (B cell line). In some embodiments, the vectors described herein exhibit high expression in CB CD34+ differentiated megakaryocytes including "pro-megakaryocytes", megakaryocytes, and platelets.

[0235] The expression cassettes, enhancers, and / or promoters described herein in connection with lentiviral vectors need not be limited to their use in lentiviral vectors and may be incorporated into essentially any other construct in which expression of a transgene (e.g., WASp) is desired. Thus, in some embodiments, nucleic acid constructs comprising any of the expression cassette components (e.g., enhancers, promoters, and / or combinations thereof) described herein are contemplated.

[0236] In some embodiments, the vectors described herein can be delivered to both dividing and non-dividing cells. In some embodiments, the vectors described herein can be delivered to non-dividing cells. In some embodiments, the vectors described herein can be delivered to dividing cells.

[0237] In some embodiments, any of the vectors described herein are recombinant vectors.

[0238] Any of the vectors described herein can be transduced or introduced into cells.

[0239] Lentiviral vector In some embodiments, the vectors described herein are lentiviral vectors (LV).

[0240] In some embodiments, the LV described herein includes any one or more of the elements typically found in lentiviral vectors. Such elements can include, but are not necessarily limited to, a vector genome packaging signal, a Rev-responsive element (RRE), a polypurine tract (e.g., a central polypurine tract, a 3' polypurine tract, etc.), a post-translational regulatory element (e.g., a modified woodchuck post-transcriptional regulatory element (WPRE)), an insulator, such as any one or more of those described below.

[0241] In some embodiments, the LV described herein can include various safety features. In some embodiments, the LV described herein is self-inactivating (SIN). In some embodiments, the LV described herein is TAT-independent. Various "safety" features can include, for example, the presence of an insulator (e.g., a 3' LTR, a PB insulator in the terminal repeat). In some embodiments, an insulator (e.g., a PB insulator) is introduced into the 3' LTR for safety. In some embodiments, the HIV LTR is replaced with an alternative promoter (e.g., CMV) so that high-titer vectors can be obtained without the inclusion of the HIV TAT protein during packaging.

[0242] Methods for constructing self-inactivating, replication-deficient, and / or TAT-independent LVs are known in the art.

[0243] In some embodiments, the LV provided herein is constructed to provide efficient transduction and high titers. Methods for constructing LVs that achieve efficient transduction and / or high titers are known in the art.

[0244] In some embodiments, the lentiviral vectors described herein include self-inactivating (SIN) and TAT-independent configurations. This self-inactivating ability serves as a biosafety feature. In SIN vectors, the production of full-length vector RNA in transduced cells is significantly reduced or completely halted. This property reduces the chance of generating replication-competent recombinants (RCR). Furthermore, the chance of abnormal expression of cellular coding sequences located adjacent to the vector integration site is reduced.

[0245] In some embodiments, an LTR region having reduced promoter activity compared to the wild-type LTR is utilized in the LVs described herein.

[0246] In some embodiments, the LV is a SIN vector that is substantially incapable of reconstituting wild-type lentivirus through recombination.

[0247] In some embodiments, the SIN design reduces the potential for interference between the LTR and the promoter driving the expression of the transgene.

[0248] In some embodiments, self-inactivation is achieved through a deletion in the U3 region of the 3' LTR of the LV DNA, i.e., the DNA used to produce the vector RNA. During RT, this deletion is transferred to the 5' LTR of the proviral DNA.

[0249] In some embodiments, it is desirable to eliminate as many transcriptionally important motifs as possible from the LTR while preserving the polyadenylation determinant.

[0250] In some embodiments, the LV described herein includes a Rev response element (RRE) to enhance the nuclear export of unspliced RNA. Exemplary RREs include, but are not limited to, the RRE located at positions 7622-8459 of the HIV NL4-3 genome (Genbank accession number AF003887), as well as RREs derived from other strains of HIV or other retroviruses. Such sequences are readily available from Genbank or from the database at the URL hiv-web.lanl.gov / content / index. One exemplary but non-limiting RRE is shown in SEQ ID NO: 25. In some embodiments, the LV described herein includes SEQ ID NO: 25.

[0251] In some embodiments, the LV described herein includes a polypurine tract (e.g., a central polypurine tract (cPPT) or a 3' polypurine tract (3'PPT)). One exemplary but non-limiting 3'PPT is shown in SEQ ID NO: 27. In some embodiments, the LV described herein includes SEQ ID NO: 27.

[0252] In some embodiments, it is known that the insertion of a fragment containing a 3'PPT (e.g., see SEQ ID NO: 27) or a central polypurine tract (cPPT) in a lentivirus (e.g., HIV-1) vector construct enhances transduction efficiency.

[0253] Post-transcriptional regulatory element (PRE) In some embodiments, the vector described herein includes a post-transcriptional regulatory element (PRE). In some embodiments, the vector described herein includes one or more post-transcriptional regulatory elements (PREs) that increase the expression of a heterologous nucleic acid (e.g., a nucleic acid encoding WASp) at the protein level.

[0254] In some embodiments, the LV described herein includes one or more post-transcriptional regulatory elements (PREs) that increase the expression of a heterologous nucleic acid (e.g., a nucleic acid encoding WASp) at the protein level. In some embodiments, the PRE may be particularly useful in a lentiviral construct having a moderate promoter.

[0255] Post-transcriptional regulatory elements that are independent of splicing events are not excised during the viral life cycle. Some examples include the post-transcriptional processing element of herpes simplex virus, the post-transcriptional regulatory element of hepatitis B virus (HPRE), and the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE). WPRE contains additional cis-acting elements not found in HPRE. In some embodiments, the post-transcriptional regulatory element is WPRE.

[0256] WPRE is characterized and described in U.S. Patent No. 6,136,597, which is hereby incorporated by reference in its entirety and particularly with respect to its description of WPRE. As described herein, WPRE is an RNA transport element. WPRE promotes the transport of RNA from the nucleus to the cytoplasm. It inserts a cis-acting nucleic acid sequence such that the element and the transgene are contained within a single transcript and enhance the expression of the transgene. The presence of WPRE in the sense orientation has been shown to increase the expression of the transgene by up to 7 - 10-fold.

[0257] In some embodiments, inclusion of WPRE in a vector results in enhanced expression of the transgene.

[0258] One exemplary but non-limiting WPRE is provided by SEQ ID NO: 26. In some embodiments, the vectors described herein include SEQ ID NO: 26. In some embodiments, the LV described herein includes SEQ ID NO: 26.

[0259] Packaging signal In some embodiments, the vectors described herein include a packaging signal. A "packaging signal," "packaging array," or "PSI sequence" is any nucleic acid sequence sufficient to direct the packaging of a nucleic acid (whose sequence includes the packaging signal) into retroviral particles. This term includes naturally occurring packaging sequences and engineered variants thereof. Packaging signals for several different retroviruses, including lentiviruses, are known in the art. One exemplary but non-limiting PSI is provided by SEQ ID NO: 24. In some embodiments, the vectors described herein include SEQ ID NO: 24. In some embodiments, the LVs described herein include SEQ ID NO: 24.

[0260] Packaging cell In some embodiments, the vectors described herein do not encode a particular virion protein and require a suitable packaging cell line to package the viral vector genome into virions. In some embodiments, the vectors described herein are used with a suitable packaging cell line or co-transfected into cells in vitro with another vector plasmid containing the necessary genes (e.g., necessary retroviral genes such as gag and pol) to form replication-incompetent virions that can package and infect cells with the vectors described herein.

[0261] In some embodiments, the vector is transfected into a packaging cell line that produces virus particles containing the vector genome. The recombinant virus is recovered from the cell culture medium by standard methods after co-transfection of the packaging vector and the transfer vector into the packaging cell line and can be titrated. Methods for the production and transfection of virions (e.g., replication-incompetent virions) are well known in the art.

[0262] In some embodiments, the packaging construct is introduced into a mammalian (e.g., human) cell line by calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging construct is introduced into a mammalian (e.g., human) cell line together with a dominant selectable marker, such as neomycin, DHFR, kanamycin, or glutamine synthetase, and subsequent selection is performed in the presence of an appropriate drug to isolate clones that express the marker. In some embodiments, the selectable marker gene is physically linked to the packaging gene within the construct.

[0263] Stable cell lines are known that are configured such that the packaging function is expressed by a suitable packaging cell (see, e.g., U.S. Patent No. 5,686,279, which describes packaging cells and which is hereby incorporated by reference in its entirety, and the disclosure thereof related to stable packaging cell lines is hereby specifically incorporated by reference).

[0264] To produce lentiviral particles, any cell that is compatible with the expression of the lentiviral Gag and Pol genes, or any cell that can be engineered to assist such expression, can be utilized. For example, producer cells such as 293T cells and HT1080 cells may be used.

[0265] Any suitable cell can be used to produce viral particles. For example, producer cells such as HEK293, 293T cells, or HT1080 cells may be used.

[0266] To produce lentiviral particles, any cell that is compatible with the expression of the lentiviral Gag and Pol genes, or any cell that can be engineered to assist such expression, can be used. For example, producer cells such as 293T cells or HT1080 cells may be used.

[0267] Ex vivo cell transfection and gene therapy In some embodiments, methods for transfecting cells (e.g., human cells) are provided.

[0268] Using the vectors and other delivery vehicles described herein, heterologous nucleic acid sequences (e.g., nucleic acids encoding WASp) can be introduced into mammalian cells (e.g., human cells).

[0269] In some embodiments, the methods provided herein include contacting a cell population with any one of the viral vectors and other delivery vehicles (e.g., LV) described herein under conditions suitable to affect cell transfection.

[0270] In some embodiments, methods are provided for delivering a transgene to a cell, which is then integrated into the genome of the cell, the method including contacting the cell with a viral vector or another delivery vehicle described herein.

[0271] In some embodiments, the cells are stem cells and / or progenitor cells (e.g., human stem cells and / or progenitor cells). In some embodiments, the cells are hematopoietic stem cells and / or progenitor cells (e.g., human hematopoietic stem cells and / or progenitor cells). In some embodiments, the cells are hematopoietic stem cells (e.g., human hematopoietic stem cells). In some embodiments, the cells are hematopoietic progenitor cells (e.g., human hematopoietic progenitor cells).

[0272] In some embodiments, the cells to be transfected are human CD4+ T cells. In some embodiments, the cells to be transfected are peripheral blood B or T lymphocyte cells. In some embodiments, the cells to be transfected are CD34+ cells. In some embodiments, the cells to be transfected are CD34+ hematopoietic stem cells and / or progenitor cells (e.g., human cells).

[0273] In some embodiments, the cells are induced pluripotent stem cells (iPSCs).

[0274] In some embodiments, the cells are transduced in vitro or ex vivo. Methods for introducing the vectors or nucleic acids described herein into cells are known in the art. In some embodiments, the vector or nucleic acid is introduced by, for example, but not limited to, viral or bacteriophage infection, transfection, conjugation, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, microinjection, nanoparticle-mediated nucleic acid delivery, or any other method known in the art. Suitable methods depend on the specific delivery vehicle being used, as is known in the art.

[0275] In some embodiments, the cells are transduced with a vector at a dose in the range of about 1×10 5 TU / ml to about 1×10 8 TU / ml. In some embodiments, the cells are transduced using a vector dose in the range of about 1×10 7 TU / ml to about 1×10 8 TU / ml. In some embodiments, the cells are transduced using a vector dose in the range of about 1×10 6 TU / ml to about 1×10 7 TU / ml. In some embodiments, the cells are transduced using a vector dose in the range of about 1×10 5 TU / ml to about 1×10 6 TU / ml. In some embodiments, the cells are transduced using a vector dose in the range of about 1×10 5 TU / ml to about 1×10 7 TU / ml. In some embodiments, the cells are transduced using a vector dose of 1×10 8 TU / ml or less, 1×10 7 TU / ml or less, 1×10 6Transduce using a vector dose of TU / ml or less, or 1×10 5 TU / ml or less.

[0276] In some embodiments, the cells transduced ex vivo are then administered to a subject (e.g., a human subject). In some embodiments, the cells are transduced ex vivo and then the transduced cells are infused into a human subject.

[0277] In some embodiments, the cells are autologous to the subject (derived from the subject to be treated).

[0278] In some embodiments, the cells are non - autologous (i.e., allogeneic or xenogeneic) to the subject (the subject to be treated).

[0279] In some embodiments, cells (e.g., human hematopoietic stem cells and / or progenitor cells) are removed from a human using methods known in the art and can be transduced as described. In some embodiments, the transduced cells are then re - introduced into the same or a different human. In some embodiments, the human is a human in which the expression of a gene product is deficient or absent, and the transgene delivered by the vector encodes that gene product. In some embodiments, the human is a human in which the expression of the WAS protein is deficient or absent, and the transgene delivered by the vector comprises the human WAS gene.

[0280] When attempting to use stem cells, it will be recognized that such cells can be derived from several sources, including bone marrow (BM), cord blood (CB), mobilized peripheral blood stem cells (mPBSC), and the like. In some embodiments, the cells are derived from BM. In some embodiments, the cells are derived from CB. In some embodiments, the cells are derived from mPBSC. Methods for isolating any such cells, transducing such cells, and introducing them into a mammalian subject are well known in the art. For example, methods commonly used for bone marrow transplantation, peripheral blood stem cell transplantation (e.g., in patients undergoing chemotherapy) can be used in this context. In some embodiments, cells derived from a cell line or from an individual other than the subject can be used.

[0281] In some embodiments, the vectors described herein are introduced into bone marrow cells, mesenchymal stem cells (e.g., obtained from adipose tissue), or other primary cells derived from a mammalian (e.g., human) source.

[0282] In some embodiments, the cells to be transduced are human hematopoietic stem cells and / or human hematopoietic progenitor cells obtained from bone marrow, peripheral blood, or cord blood.

[0283] In some embodiments, a cell-based therapy includes providing stem cells and / or progenitor cells (e.g., human hematopoietic stem cells and / or hematopoietic progenitor cells), transducing the cells with a vector (e.g., an LV) containing a transgene encoding a gene product, and subsequently introducing the transduced cells into a subject in need thereof (e.g., a subject having a mutation in a gene product that results in its loss of expression).

[0284] In some embodiments, a cell-based therapy involves providing stem cells and / or progenitor cells (e.g., human hematopoietic stem cells and / or hematopoietic progenitor cells), transducing the cells with a vector (e.g., LV) comprising a nucleic acid encoding WASp, and subsequently introducing the transduced cells into a subject in need thereof (e.g., a subject having a mutation in the WAS gene that results in a deficiency in WASp expression).

[0285] In some embodiments, administration of the vectors (e.g., LV) described herein to cells results in the production of a normal copy of the transgene (e.g., normal WASp) in the cells in vitro or ex vivo. In some embodiments, administration of the vectors described herein to cells in vitro results in the production of the transgene at endogenous or wild-type levels (e.g., wild-type levels of WASp) in cells that are deficient in transgene expression (e.g., having a loss-of-function mutation or deletion in the WAS gene). In some embodiments, the cells are first expanded in tissue culture before the vector (e.g., LV) is administered. After administration of the vector (e.g., LV), the cells are then returned to the subject, where they can provide a population of cells (e.g., red blood cells) that produce the gene product (e.g., WASp).

[0286] In some embodiments, the LV described herein is used in gene therapy (using stem cells and / or progenitor cells) for WAS (or another disease associated with a deficiency in WASp expression) by introducing a nucleic acid encoding WASp into cells of a patient having WAS and subsequently performing autologous transplantation.

[0287] In some embodiments, the transduced cells described herein are administered to a subject parenterally (e.g., by intravenous infusion). In some embodiments, the transduced cells are administered to a local region of the subject (e.g., bone marrow).

[0288] In some embodiments, the transduced cells described herein are administered to a subject in a therapeutically effective amount.

[0289] A therapeutically effective amount is an amount capable of achieving a therapeutic effect. In some embodiments, the therapeutic effect includes, without limitation, an increase in the expression level of a protein encoded by a transgene in a subject, or a restoration of its normal (wild-type or physiological) expression level, treatment or prevention of a disorder caused by a lack of expression of a transgene, improvement or amelioration of any symptoms of a disorder caused by a lack of expression of a transgene, or improvement in the survival or lifespan of the subject being treated.

[0290] In some embodiments, the cells are administered to a subject at a dose in the range of about 1×10 5 ~ about 1×10 7 cells per kg of body weight. In some embodiments, the cells are administered to a subject at a dose in the range of about 1×10 6 ~ about 50×10 6 cells per kg of body weight. In some embodiments, the cells are administered to a subject at a dose in the range of about 1×10 6 ~ about 20×10 6 cells per kg of body weight. In some embodiments, the cells are administered to a subject at a dose equal to or less than 50×10 6 cells per kg of body weight, equal to or less than 30×10 6 cells per kg of body weight, equal to or less than 20×10 6 cells per kg of body weight, equal to or less than 10×10 6 cells per kg of body weight, or equal to or less than 5×10 6 cells per kg of body weight.

[0291] In some embodiments, administration of the transduced cells described herein to a subject achieves a therapeutic effect. In some embodiments, administration of the transduced cells described herein to a subject increases the expression level of the protein encoded by the transgene in the subject or restores its normal or wild-type expression level. In some embodiments, administration of the transduced cells described herein to a subject is effective to treat or ameliorate the symptoms of a disorder caused by a deficiency in transgene expression. In some embodiments, administration of the transduced cells described herein to a subject is effective to prevent a disorder caused by a deficiency in transgene expression. In some embodiments, the transgene comprises a nucleic acid of the WAS gene and the protein encoded by the transgene is WASp. In some embodiments, the disorder to be treated is WAS. In some embodiments, the disorder to be treated is XLT.

[0292] In some embodiments, administration of the transduced cells described herein to a subject results in physiological or near-physiological levels of transgene expression in all cell lineages affected. In some embodiments, administration of the transduced cells described herein to a subject results in physiological or near-physiological levels of transgene expression in megakaryocytes. In some embodiments, the transgene encodes WASp. In some embodiments, administration of the transduced cells described herein to a subject when the transgene encodes WASp results in increased platelet counts in the subject. In some embodiments, administration of the transduced cells described herein to a subject when the transgene encodes WASp results in rescue of platelet counts in the subject to levels in healthy subjects. In some embodiments, administration of the transduced cells described herein to a subject when the transgene encodes WASp results in improved platelet engraftment and / or improvement or restoration of platelet function to levels in healthy subjects.

[0293] The transduced cells described herein can be administered to a subject once or, if desired, several times at various intervals over different periods of time.

[0294] In some embodiments, the transduced cells described herein are administered to a subject once in a single administration. In some embodiments, a single administration achieves a therapeutic effect. In some embodiments, a single administration significantly increases the expression level of the protein encoded by the transgene in the subject or significantly restores its normal or wild-type expression level. In some embodiments, a single administration is effective in treating or ameliorating the symptoms of a disorder caused by a deficiency in transgene expression. In some embodiments, a single administration is effective in preventing a disorder caused by a deficiency in transgene expression. In some embodiments, the transgene comprises a nucleic acid of the WAS gene and the protein encoded by the transgene is WASp. In some embodiments, the disorder to be treated is WAS. In some embodiments, the disorder to be treated is XLT. In some embodiments, the disorder to be treated is XLN.

[0295] In some embodiments, the transduced cells are administered to a subject once every 10 years, once every 5 years, once every 3 years, once every 1 year, once every 6 months, once every 3 months, or once a month. In some embodiments, the transduced cells are administered to the subject for an appropriate period, for example, at least 1 year or less, at least 2 years or less, at least 3 years or less, at least 5 years or less, at least 10 years or less, or at least 20 years or less, or as needed.

[0296] In some embodiments, the transduced cells are administered to the subject the number of times required to achieve the desired effect.

[0297] In some embodiments, treatment of a subject with LV can include a single treatment. In some embodiments, treatment of a subject with LV can include a series of treatments.

[0298] As is known in the art, certain factors can affect the dosage and timing required to effectively treat a subject, including, but not limited to, the general health and / or age of the subject, the severity of the disease or disorder, previous treatments, and other diseases present.

[0299] In vivo cell transfection and gene therapy In some embodiments, the cells are transfected in vivo. In some embodiments, the subject is treated by direct in vivo introduction of the vectors, virus particles, or virions described herein. In some embodiments, the vectors, virus particles, or virions described herein are administered directly to the subject. In some embodiments, the vectors, virus particles, or virions described herein are administered directly to a local region of the subject (e.g., bone marrow).

[0300] In some embodiments, the vectors, virus particles, or virions described herein are administered to the subject in a therapeutically effective amount. A therapeutically effective amount is an amount capable of achieving a therapeutic effect. In some embodiments, therapeutic effects include, without limitation, an increase in the expression level of a protein encoded by the transgene in the subject, or a restoration of its normal (wild-type or physiological) expression level, treatment or prevention of a disorder caused by a lack of expression of the transgene, improvement or amelioration of any symptoms of a disorder caused by a lack of expression of the transgene, and improvement in the survival or lifespan of the treated subject.

[0301] In some embodiments, the vectors, virus particles, or virions described herein are administered to the subject at a dosage in the range of about 1×10 5 TU / ml to about 1×10 8 TU / ml. In some embodiments, the vectors, virus particles, or virions described herein are about 1×10 7 TU / ml to about 1×108 It is administered to a subject at a dose in the range of TU / ml. In some embodiments, the vectors, virus particles, or virions described herein are about 1×10 6 TU / ml to about 1×10 7 It is administered to a subject at a dose in the range of TU / ml. In some embodiments, the vectors, virus particles, or virions described herein are about 1×10 5 TU / ml to about 1×10 6 It is administered to a subject at a dose in the range of TU / ml. In some embodiments, the vectors, virus particles, or virions described herein are about 1×10 5 TU / ml to about 1×10 7 It is administered to a subject at a dose in the range of TU / ml. In some embodiments, the vectors, virus particles, or virions described herein are 1×10 8 TU / ml or less, 1×10 7 TU / ml or less, 1×10 6 TU / ml or less, or 1×10 5 It is administered to a subject at a dose of TU / ml or less, or 1×10 8 TU / ml or less, or about 1×10 7 It is administered to a subject at a dose of TU / ml.

[0302] In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein achieves a therapeutic effect. In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein increases the expression level of the protein encoded by the transgene in a subject or restores its normal or wild-type expression level. In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein is effective to treat or ameliorate the symptoms of a disorder caused by a deficiency in transgene expression. In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein is effective to prevent a disorder caused by a deficiency in transgene expression. In some embodiments, the transgene comprises the nucleic acid of the WAS gene, and the protein encoded by the transgene is WASp. In some embodiments, the disorder to be treated is WAS. In some embodiments, the disorder to be treated is XLT.

[0303] In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein results in physiological or near-physiological levels of transgene expression in cells (e.g., all cell lineages). In some embodiments, the transgene encodes WASp. In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein results in increased platelet counts in a subject. In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein results in a rescue of platelet counts in a subject to healthy subject levels. In some embodiments, in vivo administration of the vectors, virions, or pharmaceutical compositions described herein results in improved platelet engraftment and / or improves or restores platelet function to healthy subject levels.

[0304] The vectors, virions, and pharmaceutical compositions described herein can be administered once or, if desired, several times over different periods at various intervals.

[0305] In some embodiments, the vectors, virions, and pharmaceutical compositions described herein are administered to a subject once by a single administration. In some embodiments, the single administration achieves a therapeutic effect. In some embodiments, the single administration significantly increases the expression level of the protein encoded by the transgene in the subject or significantly restores its normal or wild-type expression level. In some embodiments, the single administration is effective in treating or ameliorating the symptoms of a disorder caused by a deficiency in the expression of a transgene. In some embodiments, the single administration is effective in preventing a disorder caused by a deficiency in the expression of a transgene.

[0306] In some embodiments, the vectors, virions, and pharmaceutical compositions described herein are administered to a subject once every 10 years, once every 5 years, once every 3 years, once every 1 year, once every 6 months, once every 3 months, or once a month. In some embodiments, the vectors, virions, and pharmaceutical compositions described herein are administered to a subject for an appropriate period, e.g., at least 1 year or less, at least 2 years or less, at least 3 years or less, at least 5 years or less, at least 10 years or less, or at least 20 years or less, or as needed.

[0307] In some embodiments, the vectors, virions, and pharmaceutical compositions described herein are administered to a subject the number of times required to achieve the desired effect.

[0308] In some embodiments, the treatment of a subject with LV can include a single treatment. In some embodiments, the treatment of a subject with LV can include a series of treatments.

[0309] As is known in the art, certain factors can affect the dosage and timing required to effectively treat a subject, which includes, but is not limited to, the general health and / or age of the subject, the severity of the disease or disorder, previous treatments, and other existing diseases.

[0310] Pharmaceutical Compositions and Methods of Treatment / Prevention The cells, vectors, virus particles, and virions (as well as other delivery vehicles) described herein can be formulated into pharmaceutically acceptable compositions (e.g., any composition suitable for human administration known in the art). In some embodiments, the pharmaceutical composition comprises a cell, vector, virus particle, or virion (e.g., LV) in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers suitable for the delivery of cells, vectors, virus particles, or virions (e.g., LV) are known in the art. Suitable pharmaceutically acceptable carriers are determined by the specific composition being administered and the specific method of administration being used.

[0311] In some embodiments, the pharmaceutical composition comprises one or more cells transduced with the vectors described herein.

[0312] In some embodiments, the pharmaceutical composition comprises the vectors described herein.

[0313] In some embodiments, the pharmaceutical composition comprises virus particles or virions capable of infecting cells, and the infected cells express the transgene as described herein.

[0314] The cells, vectors, virus particles, and virions described herein, and the pharmaceutically acceptable compositions containing them, can be formulated for delivery by any available route including, but not limited to, parenteral (e.g., intravenous), intramuscular, intradermal, subcutaneous, transdermal (topical), transmucosal, vaginal, and rectal. In some embodiments, the cells, vectors, virus particles, and virions described herein, and the pharmaceutically acceptable compositions containing them, are administered parenterally (e.g., intravenously by infusion, such as continuous infusion). In some embodiments, the cells, vectors, virus particles, and virions described herein, and the pharmaceutically acceptable compositions containing them, are administered intravenously, intraarterially, or intraperitoneally. In some embodiments, the cells, vectors, virus particles, and virions described herein, and the pharmaceutically acceptable compositions containing them, are administered locally to a tissue or organ.

[0315] In some embodiments, the LV gene therapy vectors described herein can be delivered to a subject, for example, by intravenous injection, local administration, or stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 3054). The pharmaceutical preparation can contain the LV in an acceptable diluent or can contain a slow release matrix in which the LV is embedded.

[0316] Pharmaceutical compositions for parenteral delivery can include, for example, sterile isotonic injection solutions that contain, for example, buffers and / or antioxidants. Pharmaceutical compositions for parenteral delivery can include, for example, sterile suspensions that contain, for example, suspending agents, thickening agents, solubilizing agents, stabilizing agents, and / or preservatives.

[0317] In some embodiments, liposomes are used as pharmaceutically acceptable carriers. These can be prepared according to methods known in the art, for example, as described in U.S. Patent No. 4,522,811, which is hereby incorporated by reference in its entirety.

[0318] In some embodiments, the cells, vectors, virus particles, and virions described herein, and pharmaceutical compositions containing them, can be encapsulated or otherwise manipulated to protect them from degradation and rapid elimination from the body and to enhance their uptake into tissues or cells, etc.

[0319] In some embodiments, the pharmaceutical compositions described herein include, for example, microencapsulated delivery systems with biodegradable and / or biocompatible polymers (such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polylactic acid, and polyorthoesters).

[0320] The pharmaceutical composition can be in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unit dosages for the subject to be treated, each unit containing a predetermined quantity of cells, vectors, or virions (such as LV) calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0321] The pharmaceutical compositions described herein can be contained in unit dose containers, such as ampoules or vials, or multiple unit dose containers or packs, together with instructions for administration if necessary.

[0322] The unit dose can be for continuous infusion over a set period. The unit dose of a vector or virion (such as LV) described herein can be described in terms of transduction units (T.U.) defined by titration of the vector in cell lines such as HeLa or HEK293. In some embodiments, the unit dose can be in the range of 10 4 ~10 10 T.U. In some embodiments, the unit dose can be in the range of about 10 5 ~ about 10 9 T.U. In some embodiments, the unit dose can be in the range of about 10 5~ about 10 8 may be in the range of T.U. In some embodiments, the unit dose is about 10 6 ~ about 10 8 may be in the range of T.U. In some embodiments, the unit dose is about 10 7 ~ about 10 8 may be in the range of T.U.

[0323] In some embodiments, the pharmaceutical composition is targeted to a particular cell type. In some embodiments, the composition is targeted to a particular cell type using a monoclonal antibody against a cell surface marker, such as an endogenous marker or a viral antigen expressed on the surface of an infected cell.

[0324] In some embodiments, the pharmaceutical compositions described herein are administered to a subject in a therapeutically effective amount. A pharmaceutically acceptable composition can be used in a method of treating a subject having a deficiency in the expression of a gene product encoded by a transgene, treating a disorder caused by the deficiency in expression, or preventing a disorder caused by the deficiency in expression. Treating can include, for example, (i) obtaining a desired biological result (e.g., increased expression of a gene product), (ii) obtaining a desired clinical result (e.g., reduction of symptoms known to be caused by or associated with a deficiency in the expression of a gene product), (iii) causing a reduction or regression of the onset of a disease or disorder known to be caused by or associated with a deficiency in the expression of a gene product. Preventing a disorder or disease can include, for example, preventing the clinical symptoms of a disease or disorder (such as those resulting from a deficiency in the expression of a gene product) from developing in a subject that may be predisposed or at risk for the disease or disorder. In some embodiments, the gene product is WASp. In some embodiments, the disease or disorder is WAS. In some embodiments, the disease or disorder is XLT.

[0325] A therapeutically effective amount is an amount capable of achieving a therapeutic effect. In some embodiments, the therapeutic effect can include, without limitation, an increase in the expression level of a protein encoded by a transgene in a subject or a restoration of its normal (wild-type or physiological, or substantially wild-type / physiological) expression level, treatment or prevention of a disorder caused by a deficiency in transgene expression, amelioration or improvement of any symptoms of a disorder caused by a deficiency in transgene expression, or improvement of the survival or lifespan of the subject being treated.

[0326] In some embodiments, when the transgene is WAS encoding WASp, the therapeutic effects include (i) an increase or restoration of physiological WASp expression (or a restoration of WASp expression within 60%, 50%, 40%, 30%, 20%, 20%, or 10% above or below its physiological or wild-type level), and (ii) alleviation of one, two, three, or more symptoms of WASp-related disorders such as thrombocytopenia, microthrombocytopenia, eczema, and / or autoimmune symptoms, but are not limited thereto. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein improves or eliminates thrombocytopenia in a subject. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein increases the platelet count of the treated subject by at least 20%, or more than that, at least 30%, or more than that, at least 40%, or more than that, at least 50%, or more than that, at least 60%, or more than that, at least 70%, or more than that, at least 80%, or more than that, at least 90%, or more than that, or at least 100%, or more than that compared to the platelet count in the subject before treatment. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein increases the platelet count of the treated subject by at least 2-fold, or more than that, at least 3-fold, or more than that, at least 4-fold, or more than that, at least 5-fold, or more than that, at least 6-fold, or more than that, at least 7-fold, or more than that, at least 8-fold, or more than that, at least 9-fold, or more than that, at least 10-fold, or more than that, at least 15-fold, or more than that, or at least 20-fold, or more than that compared to the platelet count in the subject before treatment.In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein increases the platelet count of the treated subject to within a range of at least 60%, or more than 60%, at least 50%, or more than 50%, at least 40%, or more than 40%, at least 30%, or more than 30%, at least 20%, or more than 20%, at least 15%, or more than 15%, at least 10%, or more than 10%, or at least 5%, or more than 5% of the platelet count in a healthy subject or a subject having wild-type WASp expression. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein restores the platelet count of the treated subject to a physiological or near-physiological level (e.g., the level in a healthy subject or a subject having wild-type WASp expression). In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein results in high or improved platelet engraftment and / or improves or restores platelet function to healthy subject levels. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein improves or eliminates thrombocytopenia in the subject. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein prevents the onset of thrombocytopenia in the subject. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein improves or eliminates microthrombocytopenia in the subject. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein prevents the onset of microthrombocytopenia in the subject. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein improves or eliminates eczema in the subject. In some embodiments, when the transgene is WAS encoding WASp, treatment by the methods described herein prevents the onset of eczema in the subject.In some embodiments, when the transgene is WAS that encodes WASp, treatment by the methods described herein improves or eliminates the symptoms of an autoimmune disease in a subject. In some embodiments, when the transgene is WAS that encodes WASp, treatment by the methods described herein prevents the onset of symptoms of an autoimmune disease in a subject. In some embodiments, when the transgene is WAS that encodes WASp, treatment by the methods described herein prevents the onset of cancer thrombocytopenia in a subject, e.g., the subject does not develop cancer within 5 years, 10 years, 15 years, 20 years, 25 years, or 30 years after administration.

[0327] Patient population Patients or subjects treated by the methods described herein include, but are not limited to, humans and non-human mammals. In some embodiments, the subject to be treated is a primate. In some embodiments, the subject to be treated is a human. In certain embodiments, the subject to be treated is a domestic animal (e.g., cattle, sheep, horse, goat, cow, pig, etc.) or a companion animal (e.g., dog or cat). In some embodiments, the subject to be treated is a laboratory animal (e.g., used in research), e.g., a rodent, rabbit, or primate.

[0328] In some embodiments, the subject is male. In some embodiments, the subject is female.

[0329] In some embodiments, the subject is an infant or a young child. In some embodiments, the subject is less than 1 year old, less than 2 years old, less than 3 years old, less than 4 years old, or less than 5 years old. In some embodiments, the subject is less than 6 years old, less than 7 years old, less than 8 years old, less than 9 years old, or less than 10 years old. In some embodiments, the subject is less than 16 years old or less than 18 years old.

[0330] In some embodiments, the subject to be treated by the methods described herein has a deficiency in the expression of the gene product encoded by the transgene. In some embodiments, the subject to be treated by the methods described herein has a mutation or deletion thereof in the gene to be replaced using the transgene. In some embodiments, the subject to be treated by the methods described herein has a loss-of-function mutation in the gene to be replaced using the transgene.

[0331] In some embodiments, the subject to be treated by the methods described herein has a deficiency in the expression of the WAS protein. In some embodiments, the subject to be treated by the methods described herein has a mutation or deletion of the WAS gene. In some embodiments, the subject to be treated by the methods described herein has a loss-of-function mutation in the WAS gene.

[0332] In some embodiments, the subject to be treated by the methods described herein has WAS (e.g., is diagnosed with WAS). In some embodiments, the subject to be treated by the methods described herein has XLT (e.g., is diagnosed with XLT).

[0333] In some embodiments, the subject to be treated has not been treated with allogeneic stem cell transplantation.

Example

[0334] (Example 1) Identification of the Minimal Enhancer Element of a Vector / Plasmid Expressing WASp This example demonstrates the identification of the minimal enhancer element for WASp (Wiskott-Aldrich syndrome protein) expression that, upon its use, results in excellent hematopoietic stem and progenitor cell (HPSC) gene transfer and improved viral titers while maintaining the ability to restore physiological levels of WASp expression in WAS- / - cells.

[0335] The redesign of the lentiviral vector WASVec1.0 described in International Publication No. WO 2021 / 096887 (referred to as Figure 20, SEQ ID NO: 17 in International Publication No. WO2021096887A) was performed using an approach based on improved and refined bioinformatics. The goal of the redesign was to achieve excellent hematopoietic stem and progenitor cell (HPSC) gene transfer and improved viral titers while maintaining the ability to restore physiological levels of WASp expression (e.g., at a VCN of approximately 1) in WAS− / − cells.

[0336] In this study, new endogenous elements of the WAS locus were identified and utilized, and the minimal functional boundaries of important enhancer elements were redefined to reduce the proviral length (for increasing gene transfer and titer).

[0337] To design a new lead candidate LV, WASVec1.0 was systematically dissected to retain only the important functional elements. The following elements: "Element 9 Slim" (E9s), "Hypersensitive Site 3 Slim" (HS3s), "Sub-Sub-Element 1 of Element 2", and "Sub-Element 4 of Element 2" were removed, and a total of 1.1 kb of sequence was removed from WASVec1.0 to generate "WASVec2.0 V3", which served as the minimal backbone for the redesign.

[0338] Figure 1 shows the expression of mCitrine driven from a series of LVs. A series of lentiviral LVs containing either the original fragment or the “slim” fragment of important enhancer constructs were cloned to define the minimal functionality boundary. For example, the “slim” version of the element 2 enhancer (E2) was cloned upstream of the endogenous minimal WAS promoter (HS1) that drives the expression of the mCitrine reporter gene (mCit) (fifth from the left) and compared with the LV containing the original E2. The important element (seventh from the left) within WASVec1.0 was also removed or “slimmed” to define the minimal functionality boundary of the important element within WASVec1.0 and to identify which enhancer elements could be removed without significantly decreasing expression. The sub-element 4 of element 2, third from the right, was removed.

[0339] These constructs were packaged into LV particles in 293T cells as previously described in Cooper, 2011, J Virol Methods, 177(1):1-9 (doi: 10.1016 / j.jviromet.2011.06.019, https: / / pubmed.ncbi.nlm.nih.gov / 21784103 / ). The packaged constructs were used to transduce fetal liver CD34+ hematopoietic stem and progenitor cells (HSPCs) cultured in X-VIVO15 medium supplemented with 50 ng / mL of hSCF, hTPO, and hFL3L (previously described in Masiuk, 2019, Cell Stem Cell, 24(2):309-317.e7 (doi: 10.1016 / j.stem.2018.12.003, https: / / pubmed.ncbi.nlm.nih.gov / 30639036 / ), which were subsequently differentiated into megakaryocytes through 12 days of culture in StemSpan medium + 50 ng / mL of hTPO (previously described by Perdomo et al., 2017, J Vis Exp (130), e56420, doi:10.3791 / 56420). After 12 days of differentiation, the expression of mCitrine in CD41+ CD42+ megakaryocytes was evaluated.

[0340] Data indicated that "Element 9 Slim" (E9s), "Hypersensitive Site 3 Slim" (HS3s), sub-element 4 of E2, and sub-sub-element 1 of Element 2 were not major drivers of expression. Therefore, these four elements were removed from WASVec1.0 to generate WASVec2.0 V3, which served as the minimal backbone for vector redesign. Figure 2 shows a schematic of WASVec2.0 V3.

[0341] (Example 2) Development of improved expression vectors for hematopoietic stem cells, progenitor cells, and certain blood lineages This example shows the identification of new regulatory enhancer elements that regulate gene expression in hematopoietic cells, such as the expression of WAS.

[0342] After defining the minimal LV backbone, WASVec 2.0 V3, the WAS locus was re-analyzed using a refined bioinformatics approach to identify new endogenous regulatory elements that regulate WAS gene expression. Figure 3 shows a schematic diagram of a vector construct generated as part of an LV library for screening to identify a new enhancer of the endogenous WAS gene.

[0343] To elucidate the function of each of the putative regulatory elements, a series of lentiviral vectors were generated that each had a single putative enhancer element cloned upstream of the endogenous WAS promoter that drives the expression of mCitrine. Since the main goal was to identify the endogenous megakaryocyte enhancer responsible for controlling the expression of the WAS gene in the human genome, the LV library was transduced into human HSPCs, which were subsequently differentiated into megakaryocytes in vitro to identify a strong megakaryocyte enhancer.

[0344] Specifically, a series of LVs were cloned that each had a single putative regulatory / enhancer element cloned upstream of the minimal WAS promoter driving expression of the mCitrine reporter gene. As a comparator, an LV (WAS1.6) currently being evaluated in clinical trials and WASVec1.0 (labeled WASVEC in Figure 4) were also tested. WASVec1.6 is currently in clinical trials and is driven by a 1.6 kb promoter fragment of the WAS gene (Abina, 2015, JAMA, 313(15):1550-63, doi: 10.1001 / jama.2015.3253). These LV constructs were packaged into LV particles (previously described by Cooper et al., 2011, J Virol Methods, 177(1):1-9, doi: 10.1016 / j.jviromet.2011.06.019) and used to transduce fetal liver CD34+ hematopoietic stem and progenitor cells (HSPCs) cultured in X-VIVO15 medium supplemented with 50 ng / mL of hSCF, hTPO, and hFL3L (previously described), followed by differentiation into megakaryocytes through 12 days of culture in StemSpan medium + 50 ng / mL of hTPO (previously described by Perdomo et al. (2017; J Vis Exp, (130), e56420, doi:10.3791 / 56420)). After 12 days of differentiation, mCitrine expression in CD41+ CD42+ megakaryocytes was evaluated.

[0345] Figure 4 shows the results of screening of this refined single element for WAS where 26 new putative regulatory elements were identified. The newly identified endogenous elements were located within a 1100 kb topologically associated domain (TAD) spanning from 261 kb upstream to 839 kb downstream of the WAS gene.

[0346] Screening of this enhancer identified Element 14, a 2173 bp sequence, which increased expression 1.7-fold over the endogenous WAS promoter alone in megakaryocytes derived from human HSPCs. The identified Element 14 (WAS 14) drove high levels of expression in the megakaryocyte lineage, expressing nearly two-fold higher than the minimal WAS promoter (HS1-pro) alone.

[0347] Enhancer screening revealed that Elements 2 and 10 contributed to driving expression, but it was also found that Element 10 contributed less to expression when all elements were combined. To reduce the proviral size, Element 10 was removed from a particular vector being evaluated.

[0348] Using additional bioinformatics data, such as DNaseI hypersensitivity, transcription factor binding by ChIP-seq, and sequence conservation, the core and ultra-core fragments of E14 (the next two slides) were identified. Specifically, additional bioinformatics data were used to identify a 555 bp "core" fragment of Element 14 and a 235 bp "ultra-core" fragment of Element 14.

[0349] The identified "core" (E14 core) and "ultra-core" (E14 ultra-core) fragments of Element 14 were incorporated into WASVec2.0 V3 (i.e., the minimal backbone vector shown in Figure 2) to generate WASVec2.0 V1 and WASVec2.0 V2, respectively, which are shown in Figures 5A and 5B, respectively. Figure 5C shows the key elements of WASVec2.0 V2. The vector expresses a codon-optimized variant of WASp. The goal of this redesign was to reduce the proviral length in an attempt to increase titer and gene transfer while maintaining expression.

[0350] Describe the design of previously known vectors and newly designed vectors:

[0351] WASVec 1.0 is 6.4 kb (contains WASp in the ORF): E9 Slim - HS3 - Slim - (E2 Slim 1, 4, 5) - HS1 - WASp (JCAT co-op) -WRPE.

[0352] WASVec2.0 V1 is 5.9 kb (increased maximum expression, smaller provirus size): E14 Core - (first half and 5 of E2 Slim 1) - HS1 - WASp (JCAT co-op) -WRPE.

[0353] WASVec2.0 V2 is 5.6 kb (increased / maintained expression, even smaller provirus size): E14 Ultra Core - (first half and 5 of E2 Slim 1) - HS1 - WASp (JCAT co-op) -WRPE.

[0354] WASVec2.0 V3 is 5.3 kb (slight decrease in expression, smallest provirus size): (first half and 5 of E2 Slim 1) - HS1 - WASp (JCAT co-op) -WRPE.

[0355] (Example 3) Viral titers and dose responses in human cells transfected with the new WAS vectors This example shows that the new vectors WASVec2.0 V1, V2, and V3 exhibit increased viral titers and gene transfer compared to the previously known WASVec1.0.

[0356] WASVec1.0 described in International Publication No. WO2021 / 096887 has a proviral genome length of 6.4 kb, resulting in low HPSC gene transfer and viral titers. There is a negative correlation between proviral length and both HSPC gene transfer and viral titers (see Morgan, 2020, 28(1):328 - 340. doi: 10.1016 / j.ymthe.2019.09.020).

[0357] The newly designed LV, WASVec2.0 V1, V2, and V3, have proviral lengths of 5.9 kb, 5.6 kb, and 5.4 kb, respectively. When these LVs were packaged in HT-29 cells and titrated one by one (previously described in Cooper, 2011, J Virol Methods, 177(1):1-9, doi: 10.1016 / j.jviromet.2011.06.019, https: / / pubmed.ncbi.nlm.nih.gov / 21784103 / ), a stepwise increase in titer was observed as the proviral length decreased. Specifically, WASVec2.0 V1, V2, and V3 showed 1.097-fold, 1.36-fold, and 1.6-fold increases in titer, respectively, compared to WASVec1.0. See Figure 6.

[0358] To evaluate the improvement in gene transfer, human CD34+ mobilized peripheral blood stem cells (PBSC) were transduced with increasing doses of WASVec1.0, or WASVec2.0 V1, V2, or V3, along with WAS1.6 and WASVec1.0, and cultured for 14 days to measure the stable vector copy number.

[0359] Specifically, as previously described (Masiuk, 2019, Cell Stem Cell, 24(2):309-317.e7. doi: 10.1016 / j.stem.2018.12.003, https: / / pubmed.ncbi.nlm.nih.gov / 30639036 / ), human CD34+ PBSCs were pre-stimulated for 24 hours in X-VIVO15 medium supplemented with 50 ng / mL of hSCF, hTPO, and hFLT3L, and then LV supernatant was added for an additional 24 hours. The cells were then cultured for an additional 14 days in bone marrow basal medium (BBMM) containing IMDM supplemented with 20% FBS, 0.52% BSA, 5 ng / mL of hIL3, 10 ng / mL of IL6, and 25 ng / mL of hSCF. After culturing for 14 days, genomic DNA (gDNA) was extracted from the cells, and the vector copy number was evaluated by ddPCR as previously described (Masiuk, 2019, Cell Stem Cell, 24(2):309-317.e7. doi: 10.1016 / j.stem.2018.12.003).

[0360] At an equal vector dose of 4.8×10^7 TU / mL, WASVec2.0 V1, V2, and V3 were found to have 2.9-fold, 4.2-fold, and 3.0-fold improvements in gene transfer, respectively, compared to WASVec1.0. See Figure 7. WASVec2.0 V2 had the highest gene transfer into HSPCs and had a 4.2-fold increase in gene transfer compared to WASVec1.0 at an equal vector dose of 1×10^7 TU / mL.

[0361] (Example 4) The new vector restores WAS protein to endogenous levels in WAS- / - cells This example shows that a new vector with a newly designed enhancer element can restore wild-type levels of WASp per transduced cell.

[0362] To evaluate expression from each LV construct, human WAS− / − HSPCs generated by CRISPR knockout were transduced with WASVec2.0 V1, V2, V3, WASVec1.0, or WAS1.6 at a range of vector doses and differentiated into megakaryocytes in vitro. Figure 8A is a schematic diagram detailing the strategy used to evaluate rescue of WAS expression by the new vectors in megakaryocytes differentiated from FL CD34+ WAS KO cells.

[0363] Specifically, to evaluate expression from each construct, healthy donor (HD) fetal liver (FL) CD34+ HSPCs were transduced with the above LV constructs as previously described (Masiuk, 2019, Cell Stem Cell, 24(2):309 - 317.e7. doi: 10.1016 / j.stem.2018.12.003), and 24 hours later, CRISPR - Cas9 targeting the endogenous WAS gene (using the conditions specified in Figure 8A) was electroporated to knockout the endogenous WAS gene. Cells were maintained in megakaryocyte differentiation medium for 11 days and then cultured for 12 days in StemSpan medium + 50 ng / mL hTPO (previously described in Perdomo, 2017, J Vis Exp, (130), e56420, doi:10.3791 / 56420).

[0364] Recovery of WASp expression was measured on day 14 in the CD41+ CD42+ megakaryocyte population. See Figures 8B - 8E.

[0365] Figure 8B shows the restoration of WASp (Wiskott-Aldrich syndrome protein) in WAS knockout megakaryocytes (derived from 3 pooled CD34+ donors) that were transduced to achieve various VCNs for comparison of the expression of each LV. WASVec1.0, WASVec2.0 v1, v2, and v3 were all found to be able to restore WASp to HD levels. WASVec2.0 V2 expressed WASp similar to WASVec1.0 (despite the much smaller proviral length of WASVec2.0), and both restored WASp to HD levels. In the WAS KO control arm without LV transduction, no WASp expression was detected.

[0366] Figure 8C shows the second iteration of the experiment shown in Figure 8B using cells derived from 2 pooled CD34+ donors. Again, WASVec1.0, WASVec2.0 v1, v2, and v3 were all found to be able to restore WASp to HD levels. WASVec2.0 V2 expressed WASp similar to WASVec1.0 (despite the much smaller proviral length of WASVec2.0), and both restored WASp to HD levels. In the WAS KO control arm without LV transduction, no WASp expression was detected.

[0367] Figure 8D shows the third iteration of the experiment shown in Figure 8B using cells derived from 2 pooled CD34+ donors. At equivalent VCN, WASVec2.0 V2 was found to express WASp better (more amount) than WAS1.6.

[0368] Figure 8E shows the results obtained in the third iteration of the above experiment. WASVec2.0 V2 was able to restore WASp to HD levels at a VCN of 1.29, while WAS1.6 required a VCN of 2.61 to restore HD WASp in WAS knockout megakaryocytes.

[0369] Overall, at a VCN of 1.29, WASVec2.0 V2 was able to restore WASp expression to wild-type levels in megakaryocytes derived from WAS- / - HSPCs, while WAS1.6 was found to be able to restore WASp expression to only 64% of wild-type levels. It was also found that both WASVec2.0 V2 and WASVec1.0 were able to restore wild-type levels of WASp in WAS- / - megakaryocytes.

[0370] To evaluate expression in T cells, human WAS- / - T cells generated by CRISPR knockout were transduced with WASVec2.0 V2 or WAS1.6 at a range of vector doses. Figure 9A is a schematic diagram detailing the strategy used to evaluate the rescue of WAS expression by WASVec2.0 V2 compared to WAS1.6 in WAS gene knockout (KO) T cells.

[0371] Specifically, to evaluate the ability of WASVec2.0 to restore WASp expression in WAS- / - T cells, HD T cells were electroporated with CRISPR to knockout the endogenous WAS gene and subsequently transduced with either WASVec2.0 V2 or WAS1.6, both of which express WASp. The T cells were further cultured for 11 days in X-VIVO15 medium supplemented with 5% human serum and 100 U / mL of hIL-2. After 14 days, WASp recovery and VCN were measured.

[0372] It was found that WASVec2.0 V2 was able to restore wild-type levels of WASp per cell transduced in WAS KO T cells.

[0373] Specifically, WASVec2.0 V2 is a previously known vector, WASVec1.0 (e.g., 4.8×10 7For the vector dose of TU / mL, an improvement of 1.37-fold in virus titer and 4.2-fold in gene transfer was found in HSPC (human peripheral blood stem cells). Notably, WASVec2.0 V2 was also found to restore physiological levels of WASp expression in transduced WAS− / − cells. See Figure 9B. Thus, WASVec2.0 V2 was found to maintain physiological expression per integrated copy.

[0374] To evaluate WASp expression from WASVec1.6 and WASVec2.0 V2, CD34+-mobilized peripheral blood stem cells (PBSCs) were transduced with either WASVec2.0 or WAS1.6 (previously described in Masiuk, 2019, Cell Stem Cell, 24(2):309-317.e7, doi: 10.1016 / j.stem.2018.12.003). After 24 hours, to enable measurement of the ability of each LV to restore WAS levels, cells were electroporated with CRISPR-Cas9 targeting the endogenous WAS gene (using the conditions from Figure 8A) to knockout the endogenous WAS gene. Cells were incubated for 13 days in megakaryocyte differentiation medium StemSpan medium + 50 ng / mL of hTPO (previously described in Perdomo, 2017, J Vis Exp, (130), e56420, doi:10.3791 / 56420).

[0375] Figures 10A and 10B show the expression levels of intracellular WASp measured by flow cytometry. The CRISPR-Cas9 targeting construct was able to knockout WAS with high efficiency. Furthermore, at an average VCN within 1.1, WASVec2.0 V2 was able to restore WASp to healthy donor levels in gene-corrected cells.

[0376] (Example 5) Restoration of WASp Levels in vivo This example demonstrates that a new vector having the novel enhancer element described herein can restore WASp expression across different hematopoietic lineages (including megakaryocytes) in vivo. Further, this example shows that the WASp expression levels achieved using such vectors resulted in restoration to healthy donor levels of platelet number and platelet function.

[0377] To evaluate the ability of WASVec2.0 V2 to restore WAS expression across different hematopoietic lineages in vivo, the protocol was optimized to generate WAS− / − knockout HSPCs for transplantation into NBSWG mice. Figure 11 shows a schematic of the optimized protocol.

[0378] Healthy donor (HD) male fetal liver (FL) CD34 HPSCs were thawed and pre-stimulated for 24 hours in X-VIVO15 medium containing 100 ng / mL of hSCF, 100 ng / mL of hTPO, and 100 ng / mL of Flt3L, and then transduced with WASVec2.0 V2 at low or high vector doses of 5e5 TU / mL or 5e6 TU / L, respectively, for an additional 24 hours. The next day, using a CRISPR-based method, cells were electroporated with a ribonucleoprotein complex (RNP) composed of 100 uM of sgRNA targeting the endogenous WAS gene, 60 uM of HifiCas9 protein, and 100 uM of IDT electroporation enhancer to knockout the endogenous WAS gene in the transduced cells. Two hours after electroporation, WAS- / - knockout cells transduced with WASVec2.0 V2 were transplanted into busulfan-conditioned NBSWG mice by intrahepatic injection. The WASVec2.0 V2 construct was codon-optimized and designed not to be recognized by the sgRNA targeting the endogenous WAS gene. The NBSWG model was selected because this model has previously been shown to assist human platelet reconstitution better than irradiated NSG mice. As a positive control, unmodified HD FL HSPCs were transplanted, and as a negative control, WAS- / - knockout non-transduced FL HSPCs were transplanted. When the two cell products introduced into the mice were maintained in culture for 14 days, VCNs of 0.69 and 3.21 were measured for the low and high doses, respectively.

[0379] At 8 weeks post-transplantation, mouse peripheral blood was analyzed to evaluate the recovery of WASp across different affected hematopoietic lineages. Figure 12 shows that the knockout (KO) strategy had high efficiency, resulting in complete absence or very low levels of WASp across reconstituted T cells, B cells, and myeloid cells in engrafted mice compared to mice transplanted with unmodified HD HSPC. Even at a low vector dose of 5e5 TU / mL, WASVec2.0 V2 restored WASp to physiological levels in the WASp+ population across different hematopoietic lineages. As shown in Figure 13, surprisingly, high levels of platelet engraftment were observed in transplanted mice with the presence of the CD41+ CD45- population, thereby enabling the evaluation of the expression of WASVec2.0 V2 in the platelet compartment. Mice transplanted with CRISPR WAS KO cells had very low levels of WASp expression in peripheral blood (PB) platelets compared to mice transplanted with unmodified HD HSPC. Mice transplanted with WAS KO HPSC transduced with WASVec2.0 V2 were able to restore WASp expression to HD levels in the WASp+ platelet population.

[0380] Figure 14 shows a clear increase in WASp+ cells among different cell lineages of mice transplanted with cells transduced with low (5e5 TU / mL) or high (5e6 TU / mL) doses of WASVec2.0 V2. Figures 14 and 15 show that at a high vector dose of 5e6 TU / mL, mice transplanted with cells transduced with WASVec2.0 V2 had 87% HD levels of WASp+ platelets compared to 92% seen in mice transplanted with WT HSPC, and thus a clear dose response was observed. This suggests that WAS levels correlate with platelet reconstitution in this model. Confirming expression across different lineages, WASVec2.0 V2 was able to restore WASp expression to physiological levels.

[0381] Since robust platelet engraftment was observed at 8 weeks post-transplantation, we evaluated whether mice transplanted with WAS KO HSPCs exhibited thrombocytopenia compared to mice transplanted with WT HPSCs and whether WASVec2.0 V2 could rescue this platelet defect. Figure 16 shows that mice transplanted with WAS KO HPSCs showed much lower levels of circulating platelets compared to mice transplanted with unmodified HD HPSCs. Transduction of WAS KO HPSCs with WASVec2.0 V2 at a vector dose of 5e6 TU / mL corrected the platelet count to healthy donor levels. Partial correction of the platelet count was observed in mice transplanted with WAS KO cells transduced with WASVecV2 2.0 at a low dose of 5e5 TU / mL, suggesting that correction of the platelet count depends on the expression of WASp and the number of genetically modified cells. Next, to evaluate the function of these platelets, the responsiveness of platelets to thrombin stimulation and CD62p expression after stimulation were measured.

[0382] The results shown in Figure 17 indicate that platelets derived from WT platelets and HPSCs transduced with WASVec2.0 V2 at higher vector doses had similar responsiveness to activation, while WAS KO platelets had a blunted response.

[0383] (Example 6) The uCore E14 and uCore E2 regulatory elements assist in excellent expression of the gene encoding WASp protein from the minimal WAS promoter in megakaryocytes and restore the platelet count to healthy donor levels in vivo An incoming plasmid (Figure 18) containing novel megakaryocyte enhancers (uCore E14 and uCore E2) was cloned upstream of the minimal WAS promoter to drive the expression of a codon-optimized version of WASp. To successfully correct platelet numbers and function, the vector was evaluated for its ability to restore WASp expression at healthy donor levels in the MK / platelet lineage. This example demonstrates the claimed vector's ability to restore WASp expression at healthy donor levels in WASp knockout (KO) MK and platelets and to restore platelet numbers in a preclinical model.

[0384] Development and research design of WAS animal models

[0385] IMVC-003 (Figure 18) was evaluated in a humanized mouse model to assess in vivo WASp expression and correction of thrombocytopenia and platelet function. NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl / ThomJ (NBSGW) mice assist in multi-lineage human HSPC engraftment and human platelet generation, providing a suitable model for testing platelet numbers. As a surrogate for WAS patient HSPC, WASp KO fetal liver CD34 + HSPCs were generated by electroporation of CRISPR / Cas9 ribonucleoprotein (RNP) with a guide RNA targeting exon 1 of the WAS gene. Mock electroporation was performed on WT cells. WASp KO CD34 - cells were transduced with WAS1.6 and IMVC-003 at equivalent vector doses and transplanted into 21-day-old NBSGW mice by IV injection (Figure 19A).

[0386] Figure 19A shows NOD.Cg-Kit to restore WASp expression at healthy donor levels in the MK / platelet lineage to successfully correct platelet numbers and function. W-41J Tyr + Prkdc scid Il2rgtm1Wjl Schematic of the experimental setup used to verify the activity of a novel enhancer in the / ThomJ(NBSGW) humanized mouse model. In the WT arm, mock electroporation was performed. In the KO arm, electroporation and editing were performed using CRISPR / Cas9 with a guide RNA targeting the WAS locus. In the WAS1.6 and WASVec2.0 V2 (referred to as WasVec in the figure) arms, the IMVC-003 and Was1.6 lentiviral vectors were transduced and edited to KO endogenous WASp. 500,000 fetal liver CD34 + cells per mouse were injected into 21-day-old NBSGW adult mice by retro-orbital injection via the IV route.

[0387] At 20 weeks post-transplantation, the mean stable vector copy number (VCN) in the bone marrow (BM) was 3.6 in the IMVC-003 arm and 1.35 in the WAS1.6 arm, reflecting excellent gene transfer from IMVC-003. All mice had stable engraftment, and the mean engraftment for all arms exceeded 50% (Figure 19B). Figure 19B shows human chimerism calculated for all experimental arms using flow cytometry, showing the ratio of total CD45 + mice to CD45 + human cells. Successful knockout of WASp in vivo was confirmed by sequencing the WAS gene from gDNA derived from the BM of all mice, and the frequency of frameshift insertions / deletions was on average 94% and higher than 84% for all samples (Figure 19C). Figure 19C shows the results of the analysis of genomic DNA extracted from the BM of all mice, with PCR performed to amplify the edited region of the WAS locus, and the WAS KO frequency calculated by measuring the indel frequency compared to unedited WT DNA using the ICE tool (Synthego). The abbreviations used in Figures 19B and 19C are as follows: BM: bone marrow, indel: insertion-deletion, IV: intravenous, KO: knockout, NBSGW: NOD.Cg-Kit W-41JTyr + Prkdc scid Il2rg tm1Wjl / Thom J, PCR: Polymerase Chain Reaction, WAS: Wiskott-Aldrich Syndrome gene, WASp: Wiskott-Aldrich Syndrome protein, WASVec: IMVC-003 also known as WASVec2.0 V2, WT: wild type.

[0388] (Example 7) IMVC-003 corrects the megakaryocyte and platelet lineages in the WAS animal model The abbreviations used in Figures 20A - 20F are as follows: BM: bone marrow, KO: knockout, MFI: mean fluorescence intensity, WASp: Wiskott-Aldrich Syndrome protein, WASVec: IMVC-003 also known as WASVec2.0 V2, WT: wild type.

[0389] Briefly, at 20 weeks post-transplantation, BM, spleen, and blood were harvested from all mice and immune cells were isolated from each tissue. The immune cells were analyzed for WASp expression using flow cytometry by intracellular staining. (20A - 20C) WASp expression was measured by (upper) MFI and (lower) % WASp cells in platelets and megakaryocytes. (20D - 20F) Histograms are shown for each individual mouse, and WASp MFI is measured on the x-axis. The vertical lines represent the mean MFI of WT cells for each cell type. Data represent pooled data from 3 different donors (n = 4 - 7 mice / arm). One-way ANOVA with Tukey's multiple comparisons.

[0390] Platelets are formed by MKs present in the BM and released into the peripheral blood (PB). To evaluate the modification of the MK / platelet lineage, WASp expression was evaluated in platelets in blood and spleen, as well as in the MK lineage in the BM. IMVC-003 platelets had WASp expression equivalent to WT platelets and on average two-fold higher levels than WAS1.6 platelets as measured by mean fluorescence intensity (MFI) (Figures 20A - 20C, upper panel). Notably, almost all IMVC-003 platelets were WASp+ (on average 90%) equivalent to WT platelets, while only 50% of WAS1.6 platelets were WASp+ on average (Figures 20A - 20C, lower panel).

[0391] MKs in the BM of IMVC-003 also had WT levels of WASp expression as measured by MFI, and almost all MKs in the IMVC-003 arm were WASp+, with no significant difference compared to WT (Figure 20B). When WASp expression was tested using histograms measuring MFI, platelets and MKs of IMVC-003 had WT levels of WASp expression. In addition, WASp expression in WAS1.6 was highly variable and consistently lower than WT levels (Figures 20D - 20F).

[0392] Methods: At 20 weeks post-transplantation, BM, spleen, and blood were collected from all mice, and immune cells were isolated from each tissue. Immune cells were analyzed for WASp expression using flow cytometry by intracellular staining. (20A - 20C) WASp expression was measured by (upper) MFI and (lower) % WASp cells in platelets and megakaryocytes. (20D - 20F) Histograms are shown for each individual mouse, and WASp MFI is measured on the x-axis. Vertical lines represent the mean MFI of WT cells for each cell type. Data represent pooled data from 3 different donors (n = 4 - 7 mice / arm). One-way ANOVA with Tukey's multiple comparisons.

[0393] (Example 8) IMVC-003 produces functional platelets at healthy donor levels The abbreviations used in FIGS. 21A - 21B are as follows: BM: bone marrow, KO: knockout, PB: peripheral blood, PLT: platelet, WASp: Wiskott - Aldrich syndrome protein, WASVec2.0V2: IMVC-003, WT: wild type.

[0394] Briefly, the absolute platelet count in peripheral blood was evaluated by flow cytometry and normalized by engraftment. IMVC-003 restored the platelet count to WT levels, while the WAS1.6 platelet count remained at one - fifteenth of the WT and IMVC-003 platelet counts (FIG. 21A). There was an increase in the WAS1.6 platelet count compared to KO, but the difference was not significant (FIG. 21A). In addition to thrombocytopenia, WAS patients have impaired platelet function, which can be quantified by a decrease in the expression of CD62p (a platelet activation marker) when activated with adenosine diphosphate (Sereni et al. 2019, Rai et al. 2020). The MFI ratio of CD62p / CD61 (a platelet lineage marker) was tested for all arms compared to unstimulated platelets, and IMVC-003 platelet activation was found to be equivalent to WT platelets. Platelets from both WAS1.6 and IMVC-003 were significantly more activated compared to KO platelets. The inventors also tested the relationship between WASp expression and the recovery of platelet count in the MK / platelet lineage. The inventors found that the platelet count in PB was positively correlated with WASp expression in both PB platelets and BM MK (FIG. 21B).

[0395] Methods: PB was collected from the retro-orbital vein, and PLT was quantified using flow cytometry. (A) The bar graph represents the number of platelets per 1 μL of blood. (B) PB PLT was activated with adenosine diphosphate (10 μM) for 10 minutes and immediately stained for flow cytometry analysis. The CD62p / CD61 ratio was calculated using the mean fluorescence intensity values of the respective markers. Data represent pooled data from 3 different donors (n = 4 - 7 per arm). Unpaired t-tests with Welch's correction were performed.

[0396] (Example 9) WASp expression is restored in multiple cell lineages in vivo by IMVC-003 We also considered the restoration of WASp expression in multiple lineages. IMVC-003 demonstrated superior WASp expression compared to WAS1.6 in preclinical studies. Briefly, at 20 weeks post-transplantation, BM and spleen were harvested from all mice, and immune cells were isolated from each tissue. Immune cells were analyzed for WASp expression using flow cytometry by intracellular staining. WASp expression was measured by (upper) MFI and (lower) % WASp cells in CD34+ HSPC, CD33+ myeloid cells, and CD19+ B cells. Data represent pooled data from 3 different donors (n = 4 - 7 per arm). One-way ANOVA with Tukey's multiple comparisons. Testing BM CD34+ cells, CD33+ myeloid cells, and spleen CD19+ B cells, the IMVC-003 arm showed higher WASp MFI (Figures 22A - 22C, upper) and higher % WASp+ cells (Figures 22A - C, lower) compared to WAS1.6. Manipulation of IMVC-003 with an additional MK-specific enhancer targeting increased MK expression explains the slightly lower expression compared to MK in other cell lineages.

[0397] (Example 10) IMVC-003 restores WASp expression and IL-2 production in WASp KO T cells in vitro In T cells, WASp sets the threshold of activation driven by the T cell receptor (TCR) by regulating the dynamics of lipid raft membrane microdomains during immunological synapse formation, and WASp - / - T cells show impaired responses to TCR stimulation, including defective cytokine production. A contribution to clinical immunodeficiency has been observed in WAS patients.

[0398] To test the activity of IMVC-003 in T cells, WASp KO T cells were generated from healthy donor CD4+ cells by electroporation of CRISPR / Cas9 RNP with a guide RNA targeting exon 1 of the WAS gene (see schematic in Figure 23A). Briefly, resting healthy donor CD4 T cells from three independent donors were edited with CRISPR / Cas9 RNP with a guide RNA targeting the endogenous WAS gene. Control (WT) T cells were mock electroporated without RNP. Cells were rested overnight and activated the next day with CD3 / CD28 magnetic beads (Dynabeads). WASP KO cells were transduced with lentiviral vectors Was1.6 and IMVC-003. Cells were expanded and analyzed for WASp expression by flow cytometry 5 days after transduction. Cells were reactivated on day 9 with CD3 / CD28 Dynabeads and the supernatant was evaluated for IL2 production by enzyme-linked immunosorbent assay 72 hours after reactivation. Genomic DNA was extracted from cells on day 14 and analyzed for vector copy number and endogenous WAS gene KO.

[0399] The average KO efficiency measured by sequencing of the WAS gene ranged from 73 - 80% across all experimental arms (Figure 23B). WASp KO cells were transduced with Was1.6 and IMVC-003, and the average VCN measured 14 days after transduction was 2.5 and 4.2 for Was1.6 and IMVC-003, respectively, which reflected excellent gene transfer of IMVC-003 (Figure 23C).

[0400] Compared to the WT (mock electroporated) control, both Was1.6 and IMVC-003 restored WASp expression. The percentage of WASp+ cells was 79% for Was1.6 and 88% for IMVC-003 (Figure 24A). WASp expression in total CD4 cells measured by mean fluorescence intensity (MFI) of whole cells was 53% of the WT level for Was1.6 and 77% of the WT level for IMVC-003 (Figure 24B). WASp expression per transduced cell measured by MFI within the WASP+ gate was 77% of the WT level for Was1.6 and 95% of WT for IMVC-003 (Figure 24C). Collectively, these results indicate that IMVC-003 achieved restoration of WASp protein in 88% of T cells, and the corrected T cells contained 95% of the WT level of WASp protein per cell.

[0401] T cell functionality was also tested by the ability of cells to produce IL-2 when reactivated with CD3 / CD28 beads. Was1.6 and IMVC-003 restored 60% and 63%, respectively, of the WT level of IL-2 production. These data confirm that IMVC-003 restores functionality to WASp KO T cells. Figure 24D represents the WASp MFI of WASP + cells and represents the average level of WASp per transduced (WASp + ) cell. Figure 24E shows IL2 production measured by enzyme-linked immunosorbent assay after activation with CD3 / CD28 Dynabeads. To normalize for donor variability, experimental arms were normalized as a percentage of the WT arm for each donor. Data represent pooled data from three different donors using two replicates per donor (n = 6 / arm). One-way ANOVA with Tukey's multiple comparisons.

[0402] In summary, gene therapy with WAS1.6 has been shown to successfully correct T cell function, infectious complications, and autoimmunity in WAS patients (Hacein-Bey Abina et al. 2015, Labrosse et al. 2019, Magnani et al. 2022). IMVC-003 achieves a level of T cell correction equal to or better than that of WAS1.6, suggesting that the level of correction achieved by IMVC-003 will similarly achieve a clinical solution for infectious complications and autoimmunity in WAS patients.

[0403] (Example 11) Dose-response study in murine lin-cells A forward LV dose-response study was performed in wild-type murine lineage-negative cells to select the LV dose for transplantation. 100,000 WT murine lineage-negative cells were transduced with w1.6W LV or WasVec2.0 v2 (referred to as WASVec in the figure) LV at a range of doses and cultured in BBMM cultures for 14 days, followed by genomic DNA extraction and VCN analysis. Based on the dose-response results, an LV dose of 6e6 TU / mL (MOI 6.6) was selected for the transplantation study. This LV dose resulted in a VCN of 0.8 for w1.6W and 2.7 for WasVec in vitro.

[0404] Figure 25 shows the results of an experiment evaluating the effect of vector dose-response in murine lin-cells. Briefly, an in vitro LV dose escalation was performed in murine lin-cells to select the optimal LV dose for comparison of WasVec2.0 v2 (referred to as WasVec in the figure) and w1.6W LV. The graph in Figure 25 represents the administered LV dose (TU / mL) and the resulting VCN after 14 days of expansion in murine myeloid differentiation medium.

[0405] (Example 12) In vivo transplantation Ten WAS recipient mice per arm (five males and five females) were prospectively identified for transplantation, assigned a study number by ear punch for identification, and irradiated on day - 1 (24 hours prior to transplantation). During isolation of mouse lin - cells, the male mouse lin - cells obtained were only sufficient in amount to transplant into three out of five female mice in the WT arm. Therefore, two female mice (mouse 9, WT; mouse 17, WT) that received ear punches and irradiation were not transplanted, euthanized, and excluded from the study.

[0406] Six study-related deaths (1×WT, 3×WAS NTD, and 2×w1.6W) occurred within the first 30 days post-transplant, the predictive time frame for engraftment failure after BMT. At the time points of +32 to +42 days post-transplant, all mice in the study developed ulcerative dermatitis (UD), a known side effect of radiation exposure. The dermatitis was characterized by ulcerative pruritic lesions spanning the dorsal neck. Topical treatment with GenOne (gentamicin / betamethasone) spray was initiated 5 days per week, and nail trimming was performed weekly. In two cases (mouse 14 WAS NTD and mouse 19 w1.6W), the lesions became severe, and on day +53, euthanasia was performed by a veterinarian from the UCLA Division of Laboratory Medicine, blinded to the study and treatment groups. Three additional mice with UD (mouse 32 WasVec2.0 v2 (referred to as WASVec in the figure), mouse 34 WAS NTD, and mouse 35 w1.6W) died spontaneously between days +40 and +69. UD resolved by day +84 in all other mice with treatment. The overall attrition due to dermatitis in the cohort (5 / 38, 13%) was within the typical estimated range for mice on a C57Bl / 6J background (4 - 20%23,24). All mice that died prior to 12 weeks were excluded from the analysis. One mouse (mouse 13, WT) was analyzed for platelet WASP expression at 12 weeks and had a CBC analysis at 15 weeks and was found dead at the time point of +122 days (17 weeks). This mouse was excluded from all terminal (20-week) analyses.

[0407] Analysis of peripheral blood platelets: WASP expression

[0408] At 12 weeks post-transplant, peripheral blood was collected and platelets were stained for WASP expression by flow cytometry (Figure 26A). Briefly, peripheral blood was stained for CD41a (a platelet marker) and then fixed and permeabilized for intracellular staining of human WASP protein. FACS plots were FSC low SSC low CD41a+ Shows WASP expression in platelets. Figure 26B shows the percentage of WASP+ platelets in each transplantation arm. In mice that received cells transduced with WasVec2.0 v2 (referred to as WASVec in the figure) (IMVC-003), platelets were >97% WASP+ (Figure 26B). In contrast, mice that received cells transduced with w1.6W showed lower and more variable levels of WASP+ platelets (mean = 41%, range 1-88%). Furthermore, the level of WASP protein per platelet, as measured by MFI, was 7-fold higher in mice treated with WasVec2.0 v2 (referred to as WASVec in the figure) compared to mice treated with w1.6W (Figure 26C). As predicted, platelets expressing human WASP were not detected in mice that received WT and non-transduced WAS cells. The monoclonal antibody EP2541Y used for WASP detection detects only the human WASP transgene and does not cross-react with the mouse WASP protein.

[0409] CBC analysis: Platelet count

[0410] Figure 27 shows the results of platelet counts from CBC analysis of peripheral blood at 15 weeks post-transplantation. At 15 weeks post-transplantation, peripheral blood was collected for CBC analysis. Here, the inventors found that mice that received non-transduced WAS cells had significantly lower platelet counts (about half) compared to mice that received WT cells. Treatment with cells transduced with WasVec2.0 v2 (referred to as WASVec in the figure) (IMVC-003) significantly increased platelet counts, and 6 out of 7 mice achieved platelet counts within the normal reference range. In contrast, only 2 out of 6 treated mice that received cells transduced with w1.6W achieved normal platelet counts. Statistical analysis: *=p<0.05 vs WT, #=p<0.05 vs WAS NTD, one-way ANOVA with Tukey's multiple comparisons.

[0411] CBC analysis: Hematopoietic lineages in peripheral blood

[0412] Figures 28A - 28H show the results of CBC analysis of peripheral blood at 15 weeks post - transplantation. The mean CBC values of mice in all four study arms were within the normal reference range, with the following exceptions: Mice in all four study arms had elevated eosinophil counts, but there was no significant difference between study arms. Elevated absolute neutrophil counts were observed in mice receiving non - transduced WAS cells, but were within the normal range in mice receiving cells transduced with w1.6W and WasVec2.0 v2 (referred to as WASVec in the figure). Statistical analysis: *= p < 0.05 vs WT, # = p < 0.05 vs WAS NTD, one - way ANOVA with Tukey's multiple comparisons.

[0413] Engraftment and vector copy number

[0414] Briefly, at 20 weeks post - transplantation, bone marrow, thymus, and spleen were harvested and analyzed for vector copy number (Figures 29A - 29C) and engraftment (Figures 30A - 30C) by digital PCR. The mean VCN of bone marrow was 1 for w1.6W and 4 for WasVec2.0 v2 (referred to as WASVec in the figure), reflecting improved gene transfer with WasVec2.0 v2 (referred to as WASVec in the figure) at equivalent TU / mL and MOI. The mean engraftment exceeded 90% in all transplant arms in all tissues analyzed. Figures 29A - 29C show the results of vector copy number (VCN) analysis in bone marrow (A), thymus (B), and spleen (C) at 20 weeks post - transplantation. Figures 30A - 30C show the results of engraftment measured by ddPCR to determine the percentage of donor cells in bone marrow (A), thymus (B), and spleen (C) at 20 weeks post - transplantation.

[0415] Lineage distribution in bone marrow, spleen, and thymus by lineage

[0416] At 20 weeks post-transplantation, bone marrow, thymus, and spleen were harvested and analyzed for lineage distribution by flow cytometry, as shown in Figures 31-33, respectively. Lineage distribution across all arms was as predicted overall, with the following exceptions: compared to the WT arm, mice receiving non-transduced WAS cells had a slight increase in the percentage of monocytes (bone marrow and spleen) and neutrophils (spleen), as well as a decrease in the percentage of B cells (spleen) and CD8 T cells (spleen), all of which were normalized in mice receiving cells transduced with WasVec2.0 v2. Mice receiving non-transduced cells, cells transduced with w1.6W, and cells transduced with WasVec2.0 v2 all had a slight but statistically significant increase in the percentage of CD4 T cells. No differences were seen in thymic lineage distribution among the groups.

[0417] As shown in Figures 31-33, the bone marrow, spleen, and thymus lineage distribution graphs show the percentage of each defined hematopoietic lineage in either the bone marrow, spleen, or thymus. Percentages were calculated as the percentage of cells within the defined lineage relative to the total viable mCD45 + cells. LSK: lineage negative, Sca1+, c-kit+. NK cells: natural killer cells. Statistical analysis: *=p<0.05 vs WT, #=p<0.05 vs WAS NTD, one-way ANOVA with Tukey's multiple comparisons.

[0418] In conclusion, WAS mice receiving IMVC-003 showed sustained (20-week) multi-lineage engraftment (100%) in the bone marrow, spleen, and thymus. The average in vivo copy number was 4.5, 4.2, and 4.0 in the BM, spleen, and thymus, respectively. Mice engrafted with IMVC-003 showed multi-lineage expression of the human WASP transgene in hematopoietic cells in the range of 40-80% and in platelets above 97%.

[0419] Importantly, IMVC-003 completely dissipated thrombocytopenia, and 8 out of 9 treated mice achieved platelet counts within the normal reference range, with no significant difference between mice receiving IMVC-003 or WT cells.

[0420] (Example 13) Expression of the IMVC-003 vector dissipated thrombocytopenia by restoring platelet expression. Bone marrow, thymus, and spleen were further analyzed for intracellular human WASP (hWASP) expression by flow cytometry. Mice receiving IMVC-003 had an average of 40 - 80% hWASP+ cells in all lineages analyzed. Lineage negative, Sca1+, c-kit+ (LSK) cells indicating engrafted HSCs were 60% hWASP+, indicating sustained modification and engraftment of long-term HSCs. In all lineages, the percentage of hWASP+ cells and the MFI of hWASP in each lineage were significantly higher in mice receiving WasVec2.0 v2 (referred to as WASVec in the figure) / IMVC-003 compared to mice receiving cells transduced with w1.6WLV. As predicted, cells expressing human WASP were not detected in mice receiving WT and non-transduced WAS cells. The monoclonal antibody EP2541Y used for WASP detection detected only the human WASP transgene and did not cross-react with the mouse WASP protein. Figure 34A shows the percentage of hWASP+ cells within each defined hematopoietic lineage in the bone marrow. Figure 34B shows the mean fluorescence intensity (MFI) of hWASP in each defined lineage in the bone marrow. Similarly, Figures 35A and 36A show the percentage of hWASP+ cells within each defined hematopoietic lineage in the spleen (35A) and thymus (36A), respectively. Similarly, Figures 35B and 36B show the mean fluorescence intensity (MFI) of hWASP in each defined lineage in the spleen (35B) and thymus (36B), respectively.

[0421] (Example 14) Identification of the Minimal Enhancer Element for WASp (Wiskott-Aldrich Syndrome Protein) Expression This example demonstrates the identification of the minimal enhancer element for WASp (Wiskott-Aldrich Syndrome protein) expression that, upon its use, results in excellent hematopoietic stem and progenitor cell (HPSC) gene transfer and improved viral titers while maintaining the ability to restore physiological levels of WASp expression in WAS− / − cells.

[0422] Briefly, total serum IgE was measured by ELISA at 20 weeks post-transplantation (Figure 37). WT C57Bl / 6J mice typically have total serum IgE levels of less than 100 ng / mL. Titration with WasVec2.0v2 / IMVC-003 significantly reduced the mean IgE levels, with only 1 out of 9 mice treated with WasVec2.0v2 having an IgE level above 500 ng / mL (Figure 37). *=p < 0.05 vs. WT, #=p < 0.05 vs. WAS NTD, and one-way ANOVA with Tukey's multiple comparisons.

[0423] Furthermore, sera were collected at 20 weeks and analyzed by ELISA for the presence of anti-dsDNA antibodies. In total, 5 mice within the study cohort had positive levels of anti-dsDNA antibodies (above 2.5-fold the ELISA detection limit). As shown in Figure 38A, the concentration of serum anti-dsDNA IgG was calculated by ELISA. Anti-dsDNA antibodies were found in 0% of WAS mice receiving WT cells, and 40% of WAS mice receiving non-transduced WAS gave rise to anti-dsDNA antibodies. In contrast, only 11% of mice receiving WasVec2.0 v2 (referred to as WASVec in the figure) / IMVC-003 gave rise to positive anti-dsDNA antibodies, indicating a decrease in autoantibody production. Figure 38B shows the percentage of mice in each arm that were positive for dsDNA antibodies.

[0424] (Example 15) Response to Vaccination with PneumoVax23 The B cell antibody response to type II T-independent antigens, such as carbohydrate-based pneumococcal vaccines, is impaired in WAS mice. To evaluate the efficacy of IMVC-003 in restoring B cell function, mice were immunized with 2 μg of Pneumovax23 at 16 weeks post-transplantation. Serum was collected at 20 weeks post-transplantation for the evaluation of anti-pneumococcal IgM. Treatment with WasVec2.0 v2 (referred to as WASVec in the figure) / IMVC-003 significantly increased anti-pneumococcal IgM, but the levels were significantly lower than those in mice that received WT cells. In contrast, no significant increase in antibody response was seen in mice that received w1.6W LV.

[0425] Figure 39 shows the results of the experiment. More specifically, mice were immunized with PneumoVax23 (2 μg / mouse, intraperitoneal injection) at 16 weeks post-transplantation. Serum was collected at 20 weeks post-transplantation (28 days after immunization) and analyzed for the presence of anti-pneumococcal antibodies by ELISA. The graph shows the ELISA O.D. values for anti-pneumococcal IgM. Statistical analysis: *=p<0.05 vs WT, #=p<0.05 vs WAS NTD, one-way ANOVA with Tukey's multiple comparisons.

[0426] B cell dysfunction is a major feature of WAS, accompanied by impaired antibody responses to carbohydrate-based vaccines and dysregulation of immunoglobulin production. Treatment with IMVC-003 resulted in the expression of the WAS transgene in approximately 60% of splenic B cells. IMVC-003 led to an improvement in the antibody response to the PneumoVax23 vaccine. WAS mice treated with IMVC-003 also showed normalization of elevated anti-dsDNA antibodies and elevated IgE levels. Thus, the use of IMVC-003 represents an effective treatment option for the treatment of WAS patients, contributing to the improvement of immunodeficiency, autoimmunity, and thrombocytopenia in this patient group, and thus potentially contributing to a better quality of life. The present invention is satisfied by embodiments in many different forms, which will be described in detail in conjunction with the preferred embodiments of the present invention. It should be understood that the present disclosure is considered as an exemplification of the principles of the present invention and is not intended to limit the present invention to the specific embodiments illustrated and described herein. Numerous variations can be made by those skilled in the art without departing from the spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents. The abstract and the title are not intended to limit the scope of the present invention as they are intended to enable appropriate authorities and the general public to quickly grasp the general nature of the present invention. In the following claims, unless the term "means" is used, none of the recited characteristics or elements are to be construed as a means-plus-function limitation under 35 U.S.C. §112, ¶6. Array: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10]

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Claims

1. The enhancer nucleic acid sequence includes an enhancer element 14 containing or consisting of sequence number 1, or a nucleic acid sequence of an effective fragment thereof, The nucleic acid sequence of the promoter or an effective fragment thereof, The nucleic acid encoding a gene product operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, Recombinant vectors containing [specific vectors].

2. The vector according to claim 1, wherein the nucleic acid sequence of the enhancer element 14 or an effective fragment thereof is the nucleic acid sequence of a core fragment of element 14 comprising or consisting of sequence number 2.

3. The vector according to claim 1, wherein the nucleic acid sequence of the enhancer element 14 or an effective fragment thereof is the nucleic acid sequence of an ultracore fragment of the element 14 comprising or consisting of sequence number 3.

4. The vector according to any one of claims 1 to 3, wherein the enhancer includes the first half of the enhancer element 2 core sub-element 1 of sequence number 14 and / or the enhancer element 2 core sub-element 5 of sequence number 17.

5. The vector according to claim 1, which does not include the nucleic acid sequence of sub-sub-element 1 of element 2 of sequence number 9 or an effective fragment thereof.

6. The vector according to claim 1, which does not include the nucleic acid sequence of sub-element 4 or an effective fragment of enhancer element 2 of sequence number 10.

7. The vector according to claim 1, which does not include the nucleic acid sequence of enhancer element 9 slim or an effective fragment of sequence number 7.

8. The vector according to claim 1, which does not include the nucleic acid sequence of the supersensitive site 3 (HS3) core or an effective fragment of SEQ ID NO:

8.

9. The vector according to claim 1, wherein the nucleic acid sequence of the enhancer comprises (i) a nucleic acid sequence of an enhancer element 14 or an effective fragment thereof comprising or comprising SEQ ID NO: 1, and (ii) a nucleic acid sequence of the uCore E2 element of SEQ ID NO:

32.

10. A nucleic acid sequence of an enhancer, comprising or consisting of the first half of enhancer element 2 core sub-element 1 of sequence number 14 and enhancer element 2 core sub-element 5 of sequence number 17, The nucleic acid sequence of the promoter or an effective fragment thereof, The nucleic acid encoding a gene product operably linked to the nucleic acid sequence of the enhancer and the nucleic acid sequence of the promoter, A recombinant vector containing, A recombinant vector that does not include (i) the nucleic acid sequence of sub-sub-element 1 of element 2 of sequence number 9 or an effective fragment thereof, (ii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of sequence number 10 or an effective fragment thereof, (iii) the nucleic acid sequence of enhancer element 9 slim of sequence number 7 or an effective fragment thereof, and / or (iv) the nucleic acid sequence of supersensitive site 3 (HS3) core or an effective fragment thereof of sequence number 8.

11. The vector according to claim 10, comprising a nucleic acid sequence of an enhancer element 14 or an effective fragment thereof, wherein the nucleic acid sequence of the enhancer element 14 includes or consists of Sequence ID No.

1.

12. The vector according to claim 10, comprising a nucleic acid sequence of a core fragment of element 14 containing or consisting of sequence number 2.

13. The vector according to claim 10, comprising a nucleic acid sequence of an ultracore fragment of element 14 containing or consisting of sequence number 3.

14. The vector according to claim 10, wherein the nucleic acid sequence of the enhancer comprises (i) a nucleic acid sequence of an enhancer element 14 or an effective fragment thereof comprising or comprising SEQ ID NO: 1, and (ii) a nucleic acid sequence of the uCore E2 element of SEQ ID NO: 32, or substantially comprising therefrom.

15. The vector according to claim 1 or 10, wherein the gene product is Wiscott-Aldrich syndrome protein (WASp).

16. The vector according to claim 15, wherein the nucleic acid encoding WASp is WAS cDNA, and optionally the WAS cDNA includes or consists of SEQ ID NO:

20.

17. The vector according to claim 15, wherein the nucleic acid encoding the WASp is a codon-optimized WAS, and optionally the codon-optimized WAS includes or consists of SEQ ID NO:

21.

18. The vector according to claim 17, wherein the codon-optimized WAS is selected from the group consisting of jCAT codon-optimized WAS, GeneArt-optimized WAS, and IDT-optimized WAS.

19. The vector according to claim 1 or claim 10, wherein the promoter is a human promoter.

20. The vector according to claim 1 or claim 10, wherein the promoter is the endogenous promoter of the WAS gene.

21. The vector according to claim 1 or claim 10, wherein the promoter is the WAS gene promoter of Sequence ID No.

11.

22. The vector according to claim 1 or 10, wherein the promoter or the effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene having a maximum length of 600 bp and containing the nucleic acid sequence of SEQ ID NO: 12 HS1pro.

23. The vector according to claim 1 or 10, wherein the promoter or the effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene, comprising the sequence HS1pro (SEQ ID NO: 12).

24. The vector according to claim 1 or claim 10, which is a recombinant lentiviral vector.

25. The vector according to claim 24, wherein wild-type lentivirus cannot be reconstituted through recombination.

26. A vector according to claim 1 or claim 10, comprising a Ψ packaging signal.

27. A vector according to claim 1 or claim 10, comprising a Rev-responsive element (RRE).

28. A vector according to claim 1 or claim 10, comprising a central polyprint lactate.

29. A vector according to claim 1 or claim 10, comprising a post-transfer regulatory element.

30. The vector according to claim 29, wherein the post-transfer adjustment element is a woodchuck post-transfer adjustment element (WPRE).

31. A vector comprising or consisting of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity with respect to Sequence ID No.

4.

32. A vector comprising or consisting of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity with respect to Sequence ID No.

5.

33. A vector comprising or consisting of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% nucleic acid sequence identity with respect to Sequence ID No.

6.

34. The vector according to any one of claims 1, 10, and 31-33, which, when introduced into cells, or megakaryocytes, results in the expression of the gene product, or, if necessary, physiological expression or high expression of the gene product.

35. A viral particle comprising the vector according to any one of claims 1, 10, and 31 to 33.

36. A cell transduced with a vector according to any one of claims 1, 10, and 31-33, or a viral particle containing the vector according to any one of claims 1, 10, and 31-33.

37. The cell according to claim 36, which is a stem cell or progenitor cell.

38. The cell according to claim 36, which is a CD34+ hematopoietic stem cell and / or progenitor cell.

39. The cells according to claim 36, which are cells derived from bone marrow, umbilical cord blood, and / or peripheral blood.

40. Dendritic cells, CD4 ＋ The cell according to claim 36, which is a T cell, or a peripheral blood B or T cell.

41. The cell according to claim 36, which is a human cell.

42. A pharmaceutical composition comprising a vector according to any one of claims 1, 10 and 31 to 33, a viral particle containing the vector according to any one of claims 1, 10 and 31 to 33, or a cell into which the vector or the viral particle has been transduced, further comprising a pharmaceutically acceptable carrier.

43. A composition for use in a method of treating a disease or disorder associated with a deficiency in the expression of a gene product in a subject in need thereof, comprising a vector according to any one of claims 1, 10 and 31 to 33, wherein the method comprises the steps of transducing the composition into stem cells and / or progenitor cells derived from the subject, and transplanting the transduced stem cells and / or progenitor cells into the subject, wherein the cells or their derivatives express the gene product.

44. A composition for use in a method of treating Wiscott-Aldrich syndrome (WAS) in a subject requiring such treatment, comprising a vector according to any one of claims 1, 10 and 31-33, wherein the method comprises the steps of transducing the composition into stem cells and / or progenitor cells derived from the subject, wherein the gene product expressed by the vector is WASp; and transplanting the transduced stem cells and / or progenitor cells into the subject, wherein the cells or their derivatives express WASp.

45. The composition according to claim 43, wherein the stem cells and / or progenitor cells are human hematopoietic stem cells and / or progenitor cells.

46. The composition according to claim 45, wherein the human hematopoietic stem cells and / or progenitor cells are derived from bone marrow and / or CD34+ cells.

47. A vector according to any one of claims 1, 10, and 31-33, a viral particle containing the vector according to any one of claims 1, 10, and 31-33, or a composition comprising a cell transduced with the vector or the viral particle, or a pharmaceutical composition comprising the vector, the viral particle, or the cell and a pharmaceutically acceptable carrier, for treating or preventing a disease or disorder associated with a gene product expression deficiency in a subject requiring such treatment or prevention.

48. A composition comprising a vector according to any one of claims 1, 10 and 31-33, a viral particle containing the vector according to any one of claims 1, 10 and 31-33, or a cell transduced with the vector or the viral particle, or a pharmaceutical composition comprising the vector, the viral particle, or the cell and a pharmaceutically acceptable carrier, for treating or preventing WAS in a subject requiring such treatment.

49. The composition according to claim 43, wherein the subject is a human.

50. The composition according to claim 43, wherein the vector is a lentiviral vector.

51. The composition is approximately 1 × 10 ５ TU / ml ~ approx. 1×10 ８ The composition according to claim 43, characterized in that it is administered in a dose of the vector in the range of TU / ml.

52. Recombinant nucleic acid comprising enhancer element 14 containing or consisting of sequence number 1, or a nucleic acid sequence of an effective fragment thereof.

53. The nucleic acid according to claim 52, comprising the nucleic acid sequence of a core fragment of element 14 which includes or consists of sequence number 2.

54. The nucleic acid according to claim 52, comprising the nucleic acid sequence of an ultracore fragment of element 14 containing or consisting of sequence number 3.

55. The nucleic acid according to claim 52, comprising the first half of the enhancer element 2 core sub-element 1 of sequence number 14 and / or the nucleic acid sequence of the enhancer element 2 core sub-element 5 of sequence number 17.

56. The nucleic acid according to claim 52, comprising (i) a nucleic acid sequence of an enhancer element 14 or an effective fragment thereof, including or comprising SEQ ID NO: 1, and optionally including or comprising SEQ ID NO: 2 or SEQ ID NO: 3, and (ii) a combination of the nucleic acid sequences of the uCore E2 element of SEQ ID NO:

32.

57. The nucleic acid according to claim 52, comprising a nucleic acid sequence of any human promoter or an effective fragment thereof.

58. The nucleic acid according to claim 52, comprising the nucleic acid sequence of the endogenous promoter of the WAS gene.

59. The nucleic acid according to claim 52, comprising a nucleic acid sequence of a WAS gene promoter including or consisting of SEQ ID NO:

11.

60. The nucleic acid according to claim 52, wherein the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene, having a maximum length of 600 bp and containing the nucleic acid sequence of HS1pro of SEQ ID NO:

12.

61. The nucleic acid according to claim 52, wherein the promoter or an effective fragment thereof is an effective fragment of the endogenous promoter of the WAS gene, comprising the sequence HS1pro (SEQ ID NO: 12).

62. The nucleic acid according to claim 52, which does not include (i) the nucleic acid sequence of subsub-element 1 of element 2 of sequence number 9 or an effective fragment thereof, (ii) the nucleic acid sequence of sub-element 4 of enhancer element 2 of sequence number 10 or an effective fragment thereof, (iii) the nucleic acid sequence of enhancer element 9 slim of sequence number 7 or an effective fragment thereof, and / or (v) the nucleic acid sequence of supersensitive site 3 (HS3) of sequence number 8 or an effective fragment thereof.

63. The nucleic acid according to claim 52, comprising a transgene.

64. The nucleic acid according to claim 63, wherein the introduced gene encodes WASp.

65. A nucleic acid according to any one of claims 52 to 64, contained within an expression cassette.

66. The nucleic acid according to claim 65, wherein when the expression cassette is transduced into a cell, it can express a gene product encoded by a transgene operably linked to the nucleic acid sequence of the enhancer element 14, and, if necessary, can express the gene product in the cell at a physiological expression level or a high expression level.

67. The nucleic acid according to claim 65, wherein the introduced gene encodes WASp, and the expression cassette can express WASp when transduced into a cell, and can express WASp at a physiological level or a high level in the cell as needed.

68. The composition according to claim 44, wherein the stem cells and / or progenitor cells are human hematopoietic stem cells and / or progenitor cells.

69. The composition according to claim 68, wherein the human hematopoietic stem cells and / or progenitor cells are derived from bone marrow and / or CD34+ cells.

70. The composition according to claim 44, wherein the subject is a human.

71. The composition according to claim 44, wherein the vector is a lentiviral vector.

72. The composition according to claim 44, characterized in that the composition is administered in a dose of the vector in the range of about 1 × 10⁵ TU / ml to about 1 × 10⁸ TU / ml.

73. The composition according to claim 47, wherein the subject is a human.

74. The composition according to claim 47, wherein the vector is a lentiviral vector.

75. The composition according to claim 47, characterized in that the composition is administered in a dose of the vector in the range of about 1 × 10⁵ TU / ml to about 1 × 10⁸ TU / ml.

76. The composition according to claim 48, wherein the subject is a human.

77. The composition according to claim 48, wherein the vector is a lentiviral vector.

78. The composition according to claim 48, characterized in that the composition is administered in a dose of the vector in the range of about 1 × 10⁵ TU / ml to about 1 × 10⁸ TU / ml.

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