Lentiviral Vectors Useful for Treating Disease

Abstract

The present disclosure relates generally to lentiviral vectors useful for the treatment of a disease or disorder, such as Wiskott-Aldrich Syndrome (WAS) or Sickle Cell Disease (SCD).

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 179,993, entitled "Lentiviral vectors useful for the treatment of Wiskott Aldrich Syndrome," filed April 26, 2021, and U.S. Provisional Application No. 63 / 180,001, entitled "Lentiviral vectors useful for the treatment of Sickle Cell Disease," filed April 26, 2021, the contents of which are incorporated by reference in their entireties herein.

[0002] FIELD OF THEINVENTION The present disclosure relates generally to lentiviral vectors useful for the treatment of a disease or condition, such as Wiskott-Aldrich Syndrome (WAS) or Sickle Cell Disease (SCD). [Background technology]

[0003] Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency (PID) disorder characterized by recurrent infections, small platelets, microthrombocytopenia, eczema, and increased risk of autoimmune manifestations and tumors. Wiskott-Aldrich syndrome involves mutations in the Wiskott-Aldrich syndrome protein (WASP) gene. The gene encoding the WAS protein is located on the short arm of the X chromosome (XP11.22-11.23), is approximately 9 kb containing 12 exons, and encodes 502 amino acids. To date, mutations in WASP, including missense / nonsense, splicing, small deletions, small insertions, total deletions, and total insertions, have been identified in patients with Wiskott-Aldrich syndrome.

[0004] Wiskott-Aldrich syndrome protein is a proline-rich, hematopoietic lineage-specific, intracellular signal transduction molecule that is expressed exclusively in hematopoietic cell lineages. Wiskott-Aldrich syndrome protein is thought to be a key regulator of the actin cytoskeleton that has been found to be expressed in all leukocytes. It is thought to be involved in dynamic cytoskeletal changes essential for numerous cellular functions such as adhesion, migration, phagocytosis, immune synapse formation, and receptor-mediated cellular activation processes (e.g., antigen receptors on B and T cells). As a result, both innate and cytoadaptive immunity are affected in patients with Wiskott-Aldrich syndrome, making these patients highly susceptible to infections.

[0005] In general, mutations in the WAS gene that cause the absence of expression of the protein result in "classical Wiskott-Aldrich syndrome." Reduced expression of the Wiskott-Aldrich syndrome protein results in X-linked thrombocytopenia. Activating gain-of-function mutations in the Wiskott-Aldrich syndrome protein result in X-linked neutropenia. Depending on the mutation in the WAS gene product, there is a wide variety of clinical disease. In one study of 154 Wiskott-Aldrich syndrome patients, only 30% had the classic presentation with thrombocytopenia, small platelets, eczema, and immunodeficiency, 84% had clinical signs and symptoms of thrombocytopenia, 80% had eczema, 20% had only hematological abnormalities, and 5% had only signs of infection (see Sullivan (1994), J Pediatr., 125(6 Pt 1):876-85). Autoimmune disease is common, occurring in up to 40-70% of patients. There also appears to be a significantly increased risk (10-20%) of lymphoreticular malignancies, including lymphoma, leukemia, and myelodysplasia. Another review of 55 patients with Wiskott-Aldrich syndrome over a 20-year period in a single French hospital found that 70% of patients had an autoimmune or inflammatory condition, most commonly autoimmune hemolytic anemia.

[0006] Wiskott-Aldrich syndrome was one of the first conditions to be successfully treated by allogeneic hematopoietic stem cell transplantation (HSCT) approximately 40 years ago (Galy, Roncarolo et al. (2008), Expert Opinion on Biological Therapy, Vol. 8(2): pp. 181-190; Candotti (2018), Journal of Clinical Immunology, 33: pp. 13-27). Reports of gene therapy approaches for the treatment of WAS also continue, including, for example, Aiuti et al. (2013), Science, 341, p. 1233151; Hacein-Bey Abina, et al. (2015), JAMA, 313, pp. 1550-1563; Koldej et al. (2013), Human Gene Therapy Clinical Development, Vol 24, pp. 77-85; Wielgosz et al. (2015), Molecular Therapy: Methods & Clinical Development Vol 2, pp. 14063, and Singh et al. (2017), Molecular Therapy: Methods & Clinical Development Vol. 4 pp. 1-16.

[0007] Bone marrow transplantation remains considered the only proven cure for the disease, and outcomes are largely good for patients treated with HLA-matched donors (available in less than 20% of patients). Hematopoietic stem cell gene therapy (HSC-GT) offers a novel potentially curative option for patients who do not have a matched donor. Gene therapy offers several potential advantages over allogeneic HSCT: it would theoretically be available to all patients, would reduce the risk of graft rejection, and would possibly avoid the risks associated with graft-versus-host disease (GvHD).

[0008] Clinical trials of HSC-GT using integrative viral vectors, such as lentiviral vectors, for the treatment of WAS have shown that this approach can be therapeutically effective, but patients in clinical trials using gammaretroviral vectors developed leukemia due to integration events (see, e.g., Braun et al. (2014), Sci Transl Med. 6(227):227ra33). This highlights the continuing need to develop lentiviral vectors with improved safety profiles. Summary of the Invention [Problem to be solved by the invention]

[0009] Cryptic splice sites in lentiviral vectors (and indeed other viral vectors) can result in alternative splicing of transgene RNA leading to the production of potentially non-therapeutic truncated transcripts and proteins, as well as alternative splicing of lentiviral genomic RNA leading to truncated viral RNA and potentially non-viable viruses. Additionally, cryptic splice sites in lentiviral vectors can result in alternative splicing of transcripts from genes into which the vector genome is integrated. Alternative splicing of transcripts of genes such as HMGA2, into which lentiviral vectors are known to integrate, can result in the proliferation of cells with clonal growth advantages and those cells expressing alternatively spliced ​​transcripts. This is likely due, at least in part, to the absence in these truncated or fused transcripts of one or more of the let-7 binding sites present in the complete HMGA2 transcript, which normally binds to the let-7 family of tumor suppressor microRNAs to negatively regulate their expression. HMGA2 is not considered an oncogene, and the clonal proliferation resulting from the overexpression of the result of truncated or fused transcripts is generally considered benign, but when the patient is a child, as in the case of the target population for the treatment of WAS, even the benign cell proliferation resulting from the administration of therapeutic lentiviral vectors is less tolerable.For other genetic diseases, it is also desirable to treat patients as early as possible to reduce the impact of the disease and improve quality of life from an early stage, and therefore, all gene therapies must reduce alternative splicing. [Means for solving the problem]

[0010] The present disclosure is based, at least in part, on the identification of cryptic splice acceptor sites in cHS4-derived insulators, including the HS4-650 insulator or the HS4-400 insulator, present in therapeutic lentiviral vectors useful for treating diseases or conditions, including Wiskott-Aldrich syndrome (WAS) or sickle cell disease (SCD). These cryptic splice acceptor sites are located in the reverse complement (i.e., on the negative or reverse strand) of the HS4-650 insulator and the HS4-400 insulator. For the HS4-650 insulator, the first cryptic splice acceptor site is designated splice acceptor site 1 (SA1) and is located at nucleotides 385-386 of SEQ ID NO:2 (i.e., splicing occurs between nucleotides 385 and 386), where SEQ ID NO:2 is the reverse complement of the unmodified HS4-650 insulator set forth in SEQ ID NO:1. The second cryptic splice acceptor site is designated splice acceptor 2 (SA2) and is located at nucleotides 446-447 of SEQ ID NO:2 (i.e., splicing occurs between nucleotides 446 and 447), and the third cryptic splice acceptor site is designated splice acceptor 3 (SA3) and is located at nucleotides 456-457 of SEQ ID NO:2 (i.e., splicing occurs between nucleotides 456 and 457). For the HS4-400 insulator, SA2 is located at nucleotides 190-191 of SEQ ID NO:90, where SEQ ID NO:90 is the reverse complement of the unmodified HS4-400 insulator set forth in SEQ ID NO:89. SA3 is located at nucleotides 200-201 of SEQ ID NO:90.

[0011] The 1.2 kb fragment containing hypersensitive site 4 (cHS4) from chicken β-globin locus is a well-characterized insulator with barrier and enhancer blocking functions. Thus, lentiviral vectors are provided herein that contain modified cHS4-derived insulators, such as modified HS4-650 or modified HS4-400 insulators. In particular, lentiviral vectors are provided herein that contain modified HS4-650 insulators in which one or more of SA1, SA2, and SA3 are inactivated, and lentiviral vectors are provided herein that contain modified HS4-400 insulators in which one or both of SA2 and SA3 are inactivated. Thus, the resulting lentiviral vectors may have reduced risk of alternative splicing associated therewith when introduced into cells, such as hematopoietic stem cells. The modified HS4-650 insulators may have mutations that inactivate SA1, SA2, or SA3 relative to the "wild-type" or unmodified HS4-650 insulator. Alternatively, the modified HS4-650 insulator may be oriented within the lentiviral vector and / or relative to the transgene (e.g., the WAS transgene) in such a manner that SA1, SA2, and / or SA3 are effectively inactivated, e.g., SA1, SA2, and SA3 are not on the positive or forward strand of the viral RNA and / or transcript (e.g., the WAS transcript). Similarly, the modified HS4-400 insulator may have a mutation relative to the wild-type or unmodified HS4-400 insulator that inactivates one or both of SA2 and SA3. Alternatively, the modified HS4-400 may be oriented within the lentiviral vector and / or relative to the transgene (e.g., the globin transgene) in such a manner that SA2 and / or SA3 are effectively inactivated, e.g., SA2 and SA3 are not on the positive or forward strand of the viral RNA and / or transcript (e.g., the globin transcript).

[0012] That is, in one embodiment, a first promoter operably linked to a first nucleic acid sequence encoding a Wiskott-Aldrich syndrome protein; A lentiviral vector is provided that contains a modified HS4-650 insulator, When present in a vector, the modified HS4-650 insulator contains an inactivated splice acceptor site 1 (SA1) relative to the unmodified HS4-650 insulator; SA1 is located at nucleotide positions 385-386 in the unmodified HS4-650 insulator, as numbered relative to SEQ ID NO:2, which is the reverse complement of the unmodified HS4-650 insulator shown in SEQ ID NO:1; and / or SA1 has the sequence TTGCATCCAG ∧ ACACCATCAA (SEQ ID NO: 60), ∧ indicates the splice position.

[0013] In some embodiments, the modified HS4-650 insulator comprises a mutation that inactivates SA1 relative to the unmodified HS4-650 insulator. In one example, the mutation is an A mutation at position 384 (e.g., an A to T mutation) and / or a G mutation at position 385, numbered relative to SEQ ID NO:2. In certain embodiments, the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:3, 12, 21, 30, 39, and 48.

[0014] In some examples, the modified HS4-650 insulator further comprises a mutation that inactivates splice acceptor site 2 (SA2) relative to the unmodified HS4-650 insulator, where SA2 is present in the unmodified HS4-650 insulator at nucleotide positions 446-447, numbered relative to SEQ ID NO:2. For example, the mutation can be an A mutation at position 445 (e.g., an A to T mutation) and / or a G mutation at position 446, numbered relative to SEQ ID NO:2. In certain embodiments, the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:4, 13, 22, 31, 40, and 49.

[0015] In a further example, the modified HS4-650 insulator also includes a mutation that inactivates splice acceptor site 3 (SA3) relative to the unmodified HS4-650 insulator, e.g., an A mutation at position 455 (e.g., an A to T mutation) and / or a G mutation at position 456, numbered relative to SEQ ID NO:2, where SA3 is located at nucleotide positions 456-457 in the unmodified HS4-650 insulator, numbered relative to SEQ ID NO:2. In particular examples, the modified HS4-650 insulator includes the sequences set forth in SEQ ID NOs:5, 6, 14, 15, 23, 24, 32, 33, 41, 42, 50, and 51.

[0016] In some embodiments, the modified HS4-650 insulator is in the opposite orientation relative to the first nucleic acid sequence, hi certain embodiments, the first nucleic acid is in a forward orientation and the modified HS4-650 insulator is in a reverse orientation in the lentiviral vector.

[0017] In alternative embodiments, the modified HS4-650 insulator is in the same orientation as the first nucleic acid sequence, thereby inactivating SA1. In a particular example, the first nucleic acid and the modified HS4-650 insulator are in a forward orientation within the lentiviral vector.

[0018] In another embodiment, the present invention provides a method for the detection of a Wiskott-Aldrich syndrome protein comprising the steps of: A lentiviral vector is provided that contains a modified HS4-650 insulator, When present in a vector, the modified HS4-650 insulator contains an inactivated splice acceptor site 2 (SA2) relative to the unmodified HS4-650 insulator; SA2 is located at nucleotide positions 446-447 in the unmodified HS4-650 insulator, as numbered relative to SEQ ID NO:2, which is the reverse complement of the unmodified HS4-650 insulator shown in SEQ ID NO:1; and / or SA2 has the sequence ATCCCCCCAG ∧ GTGTCTGCAG (SEQ ID NO: 61), ∧ indicates the splice position.

[0019] In some embodiments, the modified HS4-650 insulator comprises a mutation relative to the unmodified HS4-650 insulator that inactivates SA2, such as an A mutation at position 445 (e.g., an A to T mutation) and / or a G mutation at position 446, numbered relative to SEQ ID NO:2. In some examples, the reverse complement sequence of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:7, 16, 25, 34, 43, and 52.

[0020] The modified HS4-650 insulator may also comprise a mutation that inactivates splice acceptor site 1 (SA1) relative to the unmodified HS4-650 insulator, where SA1 is present in the unmodified HS4-650 insulator at nucleotide positions 385-386, numbered relative to SEQ ID NO:2. In some examples, the mutation is an A mutation at position 384 (e.g., an A to T mutation) and / or a G mutation at position 385, numbered relative to SEQ ID NO:2. In certain embodiments, the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:4, 13, 22, 31, 40, and 49.

[0021] The modified HS4-650 insulator may further comprise a mutation that inactivates splice acceptor site 3 (SA3) relative to the unmodified HS4-650 insulator, e.g., an A mutation (e.g., an A to T mutation) at position 455 and / or a G mutation at position 456, numbered relative to SEQ ID NO:2, where SA3 is at nucleotide positions 456-457 in the unmodified HS4-650 insulator, numbered relative to SEQ ID NO:2. In some examples, the reverse complement sequence of the modified HS4-650 insulator comprises the sequences set forth in SEQ ID NOs:5, 6, 14, 15, 23, 24, 32, 33, 41, 42, 50, and 51.

[0022] In some embodiments of this aspect, the modified HS4-650 insulator is in the opposite orientation relative to the first nucleic acid sequence. In one example, the first nucleic acid is in a forward orientation and the modified HS4-650 insulator is in a reverse orientation in the lentiviral vector.

[0023] In other embodiments of this aspect, the modified HS4-650 insulator is in the same orientation as the first nucleic acid sequence, thereby inactivating SA2. In one example, the first nucleic acid and the modified HS4-650 insulator are in a forward orientation within the lentiviral vector.

[0024] In a further aspect, the present invention provides a method for the production of a gene encoding a Wiskott-Aldrich syndrome protein, comprising: A lentiviral vector is provided that contains a modified HS4-650 insulator, When present in a vector, the modified HS4-650 insulator contains an inactivated splice acceptor site 3 (SA3) relative to the unmodified HS4-650 insulator; SA3 is located at nucleotide positions 456-457 in the unmodified HS4-650 insulator, as numbered relative to SEQ ID NO:2, which is the reverse complement of the unmodified HS4-650 insulator shown in SEQ ID NO:1; and / or SA3 has the sequence GTGTCTGCAG ∧ GCTCAAAGAG (SEQ ID NO: 62), ∧ indicates the splice position.

[0025] In one example, the modified HS4-650 insulator comprises a mutation that inactivates SA3 relative to the unmodified HS4-650 insulator. In some embodiments, the mutation is an A mutation at position 455 (e.g., an A to T mutation) and / or a G mutation at position 456, numbered relative to SEQ ID NO:2. In one example, the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:9, 18, 27, 36, 45, and 54.

[0026] The modified HS4-650 insulator may also comprise a mutation that inactivates splice acceptor site 1 (SA1) relative to the unmodified HS4-650 insulator, where SA1 is present in the unmodified HS4-650 insulator at nucleotide positions 385-386, numbered relative to SEQ ID NO:2. In one example, the mutation is an A mutation at position 384 (e.g., an A to T mutation) and / or a G mutation at position 385, numbered relative to SEQ ID NO:2. In some embodiments, the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs: 14, 23, 32, 41, and 50.

[0027] The modified HS4-650 insulator may further comprise a mutation that inactivates splice acceptor site 2 (SA2) relative to the unmodified HS4-650 insulator, where SA2 is present in the unmodified HS4-650 insulator at nucleotide positions 446-447, as numbered relative to SEQ ID NO:2, such as an A mutation (e.g., an A to T mutation) at position 445 and / or a G mutation at position 446, as numbered relative to SEQ ID NO:2. In some examples, the reverse complement sequence of the modified HS4-650 insulator comprises the sequences set forth in SEQ ID NOs:6, 8, 15, 17, 24, 26, 33, 35, 42, 44, 51, and 53.

[0028] In certain embodiments of this aspect, the modified HS4-650 insulator is in the opposite orientation relative to the first nucleic acid sequence. In some examples, the first nucleic acid is in a forward orientation and the modified HS4-650 insulator is in a reverse orientation in the lentiviral vector.

[0029] In other embodiments of this aspect, the modified HS4-650 insulator is in the same orientation as the first nucleic acid sequence, thereby inactivating SA3. In some examples, the first nucleic acid and the modified HS4-650 insulator are in a forward orientation within the lentiviral vector.

[0030] In some embodiments of the above aspects, the modified HS4-650 insulator is downstream of the first nucleic acid sequence. In further embodiments, the Wiskott-Aldrich syndrome protein comprises an amino acid sequence set forth in SEQ ID NO: 76, or a sequence having at least 95% sequence identity thereto. In particular examples, the first nucleic acid sequence comprises a sequence set forth in any one of SEQ ID NOs: 73-75, or a sequence having at least 90% sequence identity thereto.

[0031] The lentiviral vector may further include a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) between the first nucleic acid sequence and the modified HS4-650 insulator, for example a WPRE comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 77-78 or a sequence having at least 95% sequence identity thereto. In some embodiments, the lentiviral vector comprises a sequence selected from the group consisting of the sequence set forth as nucleotides 3098-6006 of SEQ ID NO:57, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; the sequence set forth as nucleotides 3098-6009 of SEQ ID NO:58, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and the sequence set forth as nucleotides 3098-6006 of SEQ ID NO:59, or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0032] In some examples, the first promoter is an MND promoter, such as an MND promoter comprising a nucleic acid sequence set forth in any one of SEQ ID NO: 72, or a sequence having at least 90% sequence identity thereto. In particular examples, the vector comprises a sequence selected from the group consisting of the sequence set forth as nucleotides 2710-6006 of SEQ ID NO:57 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, the sequence set forth as nucleotides 2710-6009 of SEQ ID NO:58 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence set forth as nucleotides 2710-6006 of SEQ ID NO:59 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0033] The lentiviral vector may further comprise a second promoter operably linked to a second nucleic acid sequence encoding a nucleic acid that inhibits expression of HPRT. In some examples, the nucleic acid that inhibits expression of HPRT is an shRNA, such as an shRNA comprising a hairpin loop sequence as set forth in SEQ ID NO: 66, and / or a nucleic acid sequence as set forth in any one of SEQ ID NOs: 67-68, or a sequence having at least 95% sequence identity thereto. In some examples, the second promoter comprises a Pol III promoter or a Pol II promoter, such as a promoter comprising 7sk (e.g., a promoter comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs: 69-71, or a sequence having at least 95% sequence identity thereto). In some examples, the second promoter and the operably linked second nucleic acid sequence are in a reverse orientation and are upstream of the first promoter and the operably linked first nucleic acid. In some embodiments, the vector comprises a sequence selected from the group consisting of the sequence set forth as nucleotides 2402-6006 of SEQ ID NO:57, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, the sequence set forth as nucleotides 2402-6009 of SEQ ID NO:58, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence set forth as nucleotides 2402-6006 of SEQ ID NO:59, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0034] The lentiviral vector may further comprise a polyadenylation signal downstream of the first nucleic acid and the modified HS4-650 insulator.

[0035] In some instances, the vector is a plasmid. In other instances, the vector is a viral particle.

[0036] Also provided are host cells comprising or transduced by the lentiviral vectors of the present disclosure. In some examples, the host cells are hematopoietic stem cells (HSCs), such as allogeneic or autologous HSCs.

[0037] Also provided is a method of treating a subject having Wiskott-Aldrich syndrome, comprising administering to the subject the host cells described above and herein. In certain embodiments, the method comprises administering to the subject the host cells, and then administering to the subject a purine analog (e.g., 6-thioguanine ("6TG"), 6-mercaptopurine ("6MP"), or azathioprine ("AZA") to increase engraftment of the host cells. In further embodiments, the method comprises preconditioning the subject with the purine analog prior to administration of the host cells. Use of the host cells of the present disclosure for the preparation of a medicament for the treatment of Wiskott-Aldrich syndrome is also provided.

[0038] Embodiments of the present disclosure will now be described, by way of non-limiting example only, with reference to the following drawings: [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 is an alignment of the reverse complement sequences of the HS4-650 insulator. [Diagram 2] FIG. 2 is a schematic diagram of pBRNGTR47. [Diagram 3] FIG. 3 is a schematic diagram of pBRNGTR47 showing the cryptic splice acceptor sites SA1, SA2, and SA3 in the HS4-650 insulator (650 bp Ins). [Figure 4] FIG. 4 is a schematic diagram of pBRNGTR84. [Diagram 5] FIG. 5 is a schematic diagram of pBRNGTR88. [Figure 6] FIG. 6 is a schematic diagram of pBRNGTR92. [Figure 7] FIG. 7 is a schematic diagram of pBRNGTR120. [Figure 8] Figure 8 shows the ratio of HMGA2 exon 2-3 / exon 4-5 transcripts assessed by ddPCR on day 7 (solid bars, left) and day 14 (right). "650" refers to the construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to the construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to the construct containing the HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to the control construct containing the original unmodified insulator), and "mock" refers to the control. [Figure 9] Figure 9 shows the frequency of edited cells in culture from day 7 (solid bars) to day 26 (hashed bars), indicating the reduction or elimination of the selective cell growth advantage over time in culture for constructs containing modified insulators in KG1 cells. "650" refers to a construct containing an unmodified HS4-650 insulator with an unmodified splice site, "2xSA" refers to a construct containing an HS4-650 insulator with two corrected cryptic splice acceptor sites, "3xSA" refers to a construct containing an HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing an HS4-650 insulator in a forward orientation relative to the transgene (or in a reverse orientation relative to a control construct containing the original unmodified insulator), and "mock" or "MND-GFP" refers to controls. [Figure 10]FIG. 10 shows the frequency of edited cells in culture from day 5 (solid bars, left) to day 26 (hashed bars, right), indicating a reduction or elimination of the selective cell growth advantage over time in culture for constructs containing modified insulators in CD34+ cells. "650" refers to a construct containing an unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to a construct containing an HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing an HS4-650 insulator in a forward orientation relative to the transgene (or in a reverse orientation relative to a control construct containing the original unmodified insulator), and "mock" or "GFP" refers to controls. [Figure 11] FIG. 11 is a schematic diagram of the mapping of custom baits for enrichment of exons 1, 2, and 3 of HMGA2. [Figure 12] FIG. 12 shows the expression levels of HMGA2 and AAV fusion transcripts in KG1 cells. (A) Measurement of total HMGA2 expression transcripts compared to untreated cells. (B) Measurement of the levels of fusion transcripts expressed in cells normalized to 3xSA. "650" refers to a construct containing an unmodified HS4-650 insulator with unmodified splice sites, "2xSA" refers to a construct containing an HS4-650 insulator with two corrected cryptic splice acceptor sites, "3xSA" refers to a construct containing a 650bp cHS4 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing an HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to the control construct containing the original unmodified insulator), and "mock" or "fwd_LTRrev" refers to the control. [Figure 13]Figure 13 shows the expression levels of HMGA2 and AAV fusion transcripts in CD34+ cells. (A) Measurement of total HMGA2 expression transcripts compared to untreated cells. (B) Measurement of the levels of fusion transcripts expressed in cells normalized to 3xSA. "650" refers to a construct containing an unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to a construct containing an HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing an HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to a control construct containing the original unmodified insulator), and "mock" refers to a control. [Figure 14] Figure 14 shows the percentage of exon 3-LVV splice junctions mapped from HMGA2 transcript assays in CD34+ cells. "650" refers to a construct containing the unmodified HS4-650 insulator with an unmodified splice site, "3xSA" refers to a construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing the HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to a control construct containing the original unmodified insulator), and "mock", "AAV_only", or "MND_GFP" refer to controls. [Figure 15]Figure 15 shows the percentage of HMGA2 exon 3-exon 4 splice junctions mapped from HMGA2 transcript assays in CD34+ cells. "650" refers to a construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to a construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing the HS4-650 insulator in forward orientation relative to the transgene (or reverse orientation relative to a control construct containing the original unmodified insulator), and "mock", "AAV_only", or "MND_GFP" refer to controls. [Figure 16] Figure 16 shows the ratio of LVV fusion transcripts to HMGA2 isoform 1 mapped from HMGA2 transcript assays in CD34+ cells. "650" refers to a construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to a construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing the HS4-650 insulator in forward orientation relative to the transgene (or reverse orientation relative to a control construct containing the original unmodified insulator), and "mock", "AAV_only", or "MND_GFP" refer to controls. [Figure 17]Figure 17 shows the percentage of exon 3 LVV splice junctions mapped from HMGA2 transcript assays in KG1 cells. "650" refers to a construct containing the unmodified HS4-650 insulator with an unmodified splice site, "3xSA" refers to a construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing the HS4-650 insulator in forward orientation relative to the transgene (or reverse orientation relative to a control construct containing the original unmodified insulator), and "mock", "AAV_only", or "MND_GFP" refer to controls. [Figure 18] Figure 18 shows the percentage of HMGA2 exon 3-exon 4 splice junctions mapped from an HMGA2 transcript assay in KG1 cells. "650" refers to a construct containing the unmodified HS4-650 insulator with an unmodified splice site, "3xSA" refers to a construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing the HS4-650 insulator in forward orientation relative to the transgene (or reverse orientation relative to a control construct containing the original unmodified insulator), and "mock", "AAV_only", or "MND_GFP" refer to controls. [Figure 19]Figure 19 shows the ratio of LVV fusion transcripts to HMGA2 isoform 1 mapped from HMGA2 transcript assays in KG1 cells. "650" refers to a construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to a construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing the HS4-650 insulator in forward orientation relative to the transgene (or reverse orientation relative to a control construct containing the original unmodified insulator), and "mock", "AAV_only", or "MND_GFP" refer to controls. [Figure 20] FIG. 20 shows the results of a LIM domain only 2 (LMO2) activation assay in a single cell assay. LMO2 mRNA levels in mScarlet+ cells normalized to PPIA (%, y-axis) relative to a control construct without insulator. "650" refers to a construct containing an unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to a construct containing an HS4-650 insulator with three corrected cryptic splice acceptor sites, and "fwd" refers to a construct containing an HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to the control construct containing the original unmodified insulator). "No promoter" refers to a control construct lacking a promoter, and "No insulator" refers to a control construct lacking an insulator. [Figure 21]FIG. 21 shows the results of the LMO2 activation assay in bulk cell assay. (A) LMO2 mRNA levels (%, y-axis) of mScarlet+ cells normalized to PPIA versus the insulator-free control construct in bulk cell assay. (B) Expanded plot extracted from (A), i.e., LMO2 mRNA levels (%, y-axis) of mScarlet+ cells normalized to PPIA versus the insulator-free control construct. "650" refers to the construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3xSA" refers to the construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to the construct containing the HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to the control construct containing the original unmodified insulator). "No-Ins" refers to the control construct lacking the insulator. NOTE: The average data from three independent duplicate experiments are represented. [Figure 22] Figure 22 shows the AAV / HMGA2 ratios in exemplary constructs containing modified or unmodified insulators in (A) KG1 cells and (B) CD34+ cells, calculated as the ratio of AAV reads to HMGA2 downstream exon reads. "650" refers to a construct containing an unmodified HS4-650 insulator with an unmodified splice site, "2xSA" refers to a construct containing an HS4-650 insulator with two corrected cryptic splice acceptor sites, "3xSA" refers to a construct containing an HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing an HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to a control construct containing the original unmodified insulator), "mock" refers to a control, and "(r1)"..."(r2)" refer to the duplicates of the samples. [Diagram 23]Figure 23 shows expression of WAS in mouse lineage negative (Linneg) WAS KO cells transduced with selected WAS-LVV. Transgene expression is shown as MFI (y-axis) in cells transduced at multiplicity of infection (MOI) 1-10 (as indicated). "650" refers to the construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3SA" refers to the construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to the construct containing the HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to the control construct containing the original unmodified insulator), "KO" refers to untransduced WAS KO cells, and "WT" refers to wild type cells (Linneg cells) as negative and positive controls. [Figure 24] Figure 24 shows expression of WAS in human U937 WAS KO cells transduced with selected WAS LVVs. Transgene expression is shown as MFI (y-axis) in cells transduced at MOIs of 1-10 (as indicated). "650" refers to the construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3SA" refers to the construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to the construct containing the HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to the control construct containing the original unmodified insulator), "KO" refers to untransduced WAS KO cells, and "WT" refers to wild-type cells (U937 cells) as negative and positive controls. [Diagram 25]Figure 25 shows a dose-dependent increase in vector copy integration (VCN) in mouse Linneg WAS KO cells transduced with selected WAS LVVs at MOIs of 1, 2, 10, and 20 (as indicated). "650" refers to a construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3SA" refers to a construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to a construct containing the HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to a control construct containing the original unmodified insulator), "KO" refers to untransduced WAS KO cells, and "WT" refers to wild-type cells (Linneg cells) as negative and positive controls. [Figure 26] Figure 26 shows a dose-dependent increase in VCN in human U937 WAS KO cells transduced with selected WAS LVVs at MOI 1, 2, 10, and 20 (as indicated). "650" refers to the construct containing the unmodified HS4-650 insulator with unmodified splice sites, "3SA" refers to the construct containing the HS4-650 insulator with three corrected cryptic splice acceptor sites, "fwd" refers to the construct containing the HS4-650 insulator in forward orientation relative to the transgene (or in reverse orientation relative to the control construct containing the original unmodified insulator), "KO" refers to untransduced WAS KO cells, and "WT" refers to wild type cells (U937 cells) as negative and positive controls. [Figure 27] Figure 27 shows the arrangement of genes and elements in pCalH10. (A) High level overview schematic of pCalH10. (B) Detailed schematic of pCalH10. [Figure 28] Figure 28 shows the results of Southern blot analysis of HeLa cells transduced with virions produced from pCalH10. (A) Southern blot showing the sizes of fragments observed in the cells. (B) Quantitation of the contribution of each fragment to the population. [Figure 29] FIG. 29 is a schematic diagram of pCalH10 showing the locations of the splice donor site (SD1) and splice acceptor site 2 (SA2) and the fusions produced following alternative splicing at these sites. [Diagram 30] FIG. 30 is a schematic diagram of pCalH10 showing the locations of splice donor site 1 (SD1), splice acceptor site 2 (SA2), and splice acceptor site 3 (SA3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0041] The articles "a" and "an" are used to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0042] As used herein, "and / or" means and includes every possible combination of one or more of the associated listed items, as well as the absence (or) of a combination when indicated in the alternative.

[0043] The terms "active agent" and "therapeutic agent" are used interchangeably herein and refer to an agent that prevents, reduces, or ameliorates at least one symptom of a disease or disorder.

[0044] The terms "concurrent administration" or "administering simultaneously" or "co-administering" and the like refer to administration of a single composition containing two or more agents, or administration of each agent delivered by separate routes either simultaneously or sequentially as separate compositions and / or within a sufficiently short period of time that the effective results are comparable to those obtained when all such agents are administered as a single composition. "Concurrently" means that the agents are administered at substantially the same time, preferably together in the same formulation. "Concurrently" means that the agents are administered close in time, for example, one agent is administered within about one minute to about one day before or after another agent. Any concurrent time is useful. However, when not administered simultaneously, the agents are often administered within about one minute to about eight hours, suitably within about one hour to less than about four hours. When administered concurrently, the agents are suitably administered at the same site relative to the subject. The term "same site" includes the exact location, but may be within about 0.5 to about 15 centimeters, preferably within about 0.5 to about 5 centimeters. The term "separately" as used herein means that the agents are administered at time intervals, for example, from about one day to several weeks or months. The agents may be administered in any order. The term "sequentially" as used herein means that the agents are administered one after the other, for example, at intervals of minutes, hours, days, or weeks. Where appropriate, the agents may be administered in a regular repeating cycle.

[0045] Throughout this specification, unless the context requires otherwise, it will be understood that the word "comprise" implies the inclusion of a recited step or element or group of steps or elements, but does not imply the exclusion of any other step or element or group of steps or elements. That is, the use of the term "comprise" or the like indicates that the recited elements are necessary or required, while other elements are optional and may or may not be present. "Consisting of" includes and is limited to everything before the phrase "consisting of". That is, the phrase "consisting of" indicates that the recited elements are necessary or required, while other elements may or may not be present. "Consisting essentially of" means including any element recited before the phrase, and is limited to other elements that do not intervene or contribute to the activity or action specified in this disclosure for the recited element. That is, the phrase "consisting essentially of" indicates that the recited elements are necessary or required, while other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited element.

[0046] As used herein, "corresponding nucleotides," "corresponding amino acid residues," or "corresponding positions" refer to nucleotides, amino acids, or positions occurring at aligned loci. Related or variant polynucleotide or polypeptide sequences are aligned by any method known to one of skill in the art. Such methods typically maximize matches (e.g., identical nucleotides or amino acids at a position) and include methods using manual alignments and the like and methods using the many alignment programs available (e.g., BLASTN, BLASTP, ClustlW, ClustlW2, EMBOSS, LALIGN, Kalign, and others) and others known to one of skill in the art. By aligning the sequences of polynucleotides, one of skill in the art can identify corresponding nucleotides. For example, by aligning the HS4-650 insulator set forth in SEQ ID NO:2 with other HS4-650 insulators (e.g., as shown in FIG. 1), one of skill in the art can identify regions or nucleotides in the other insulators that correspond to various regions or nucleotides in the insulator set forth in SEQ ID NO:2. For example, the A at position 384 of SEQ ID NO:2 is or corresponds to the corresponding nucleotide of the A at position 375 of SEQ ID NO:11. In another example, the SA1 site at nucleotides 385-386 of SEQ ID NO:2 corresponds to the SA1 site at nucleotides 375-376 of SEQ ID NO:20. That is, when a nucleotide or position is referred to herein with respect to a particular sequence (e.g., an HS4 650 insulator sequence), it is understood that a reference is also made to the corresponding nucleotide or position in another sequence (e.g., another HS4 650 insulator sequence), where appropriate. For example, a reference to an SA1 in an HS4-650 insulator "at nucleotide positions 385-386, numbered relative to SEQ ID NO:2" means an SA1 at positions 385-386 of the HS4-650 insulator set forth in SEQ ID NO:2 and an SA1 in other HS4-650 insulators, where the SA1 is at a position corresponding to 385-386 of the HS4-650 insulator set forth in SEQ ID NO:2.In another example, a reference to an HS4-650 insulator comprising an A mutation at position 384, numbered relative to SEQ ID NO:2, encompasses not only HS4-650 insulators shown in SEQ ID NO:2 having an A mutation at position 384, but also other HS4-650 insulators having an A mutation at the position corresponding to position 384 of SEQ ID NO:2.

[0047] An "effective amount" in the context of treating a disease or condition means an amount of an agent or composition administered to an individual in need of such treatment or prevention, either as a single dose or as part of a series, that is effective for preventing the worsening of symptoms of the condition, suppressing such symptoms, and / or treating existing symptoms. The effective amount varies according to the age, health, and physical condition of the individual to be treated, whether the disease state is evident, the taxonomic group of the individual to be treated, the formulation of the composition, the evaluation of the medical condition, and other relevant factors. The optimal dosing schedule can be calculated from the measurement of the accumulation of the drug in the subject's body. The optimal dosage varies according to the relative capacity of the individual subject, and can generally be estimated based on the EC50 values ​​found to be effective in in vitro and in vivo animal models. Those skilled in the art can easily determine the optimal dosage, method of administration, and number of repetitions. It is expected that the amount will fall within a relatively broad range that can be determined by routine testing.

[0048] The terms "subject," "patient," and "individual," as used interchangeably herein, refer to any subject, particularly a vertebrate subject, and more particularly a mammalian subject (e.g., a human). In some embodiments, the term "subject" refers to a mammalian subject (e.g., a human) with WAS. In other embodiments, the term "subject" refers to a mammalian subject (e.g., a human) with SCD.

[0049] As used herein, the term "expression cassette" refers to one or more gene sequences in a vector that can subsequently express RNA and, in some embodiments, proteins. An expression cassette comprises at least one promoter and at least one gene of interest. In some embodiments, an expression cassette comprises at least one promoter, at least one gene of interest, and at least one additional nucleic acid sequence that encodes a molecule for expression (e.g., a transgene or RNAi). In some embodiments, the expression cassette is positionally and sequentially oriented in the vector, so that the nucleic acid in the cassette can be transcribed into RNA, translated into a protein or polypeptide if necessary, subjected to appropriate post-translational modifications required for activity in transformed cells (e.g., transduced stem cells), and translocated to the appropriate compartment for biological activity by targeting to the appropriate intracellular compartment or secretion into an extracellular compartment. In some embodiments, the cassette has its 3' and 5' ends adapted for ready insertion into a vector, e.g., it has a restriction endonuclease site at each end.

[0050] As used herein, the term "host cell" refers to a cell to be modified using the method of the present disclosure. In some embodiments, the host cell is a mammalian cell into which a lentiviral vector can be introduced. Suitable mammalian host cells include, but are not limited to, human cells, mouse cells, non-human primate cells (e.g., rhesus monkey cells), human progenitor or stem cells, 293 cells, HeLa cells, D17 cells, MDCK cells, BHK cells, and Cf2Th cells. In certain embodiments, the host cell comprising the expression vector of the present disclosure is a hematopoietic cell, such as a hematopoietic progenitor / stem cell (e.g., CD34-positive hematopoietic progenitor / stem cell), monocyte, macrophage, peripheral blood mononuclear cell, CD4+ T lymphocyte, CD8+ T lymphocyte, or dendritic cell. The hematopoietic cell (e.g., CD4+ T lymphocyte, CD8+ T lymphocyte, and / or monocyte / macrophage) transduced with the expression vector of the present disclosure can be from an allogeneic, autologous, or matched sibling. The hematopoietic cells, in some embodiments, are CD34 positive and can be isolated from the patient's bone marrow or peripheral blood. The isolated CD34 positive hematopoietic cells (and / or other hematopoietic cells described herein) are, in some embodiments, transduced with an expression vector described herein.

[0051] As used herein, the term "hematopoietic stem cell" or "HSC" refers to a pluripotent cell that can differentiate into all cell types of the hematopoietic system, including but not limited to granulocytes, monocytes, erythrocytes, megakaryocytes, lymphocytes, and dendritic cells, and has self-renewal activity, i.e., the ability to divide and generate at least one daughter cell that has the same (e.g., self-renewal) characteristics as the parent cell.

[0052] As used herein, HPRT is an enzyme involved in purine metabolism, encoded by the HPRT1 gene. HPRT1 is located on the X chromosome and is therefore present in a single copy in males. HPRT1 encodes a transferase that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate by transferring the 5-phosphoribosyl group from 5-phosphoribosyl 1-pyrophosphate to a purine. The enzyme functions primarily to salvage purines from degraded DNA for use in renewed purine synthesis.

[0053] As used herein, the term "lentivirus" refers to a genus of retroviruses that can infect dividing and non-dividing cells.Some examples of lentiviruses include HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), the causative agent of human acquired immunodeficiency syndrome (AIDS), visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, caprine arthritis-encephalitis virus, which causes immunodeficiency, arthritis, and encephalitis in goats, equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalitis in horses, feline immunodeficiency virus (FIV), which causes immunodeficiency in cats, bovine immunodeficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle, and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalitis in subhuman primates.

[0054] As used herein, the term "lentiviral vector" is used to refer to any form of nucleic acid derived from lentivirus and used to transfer genetic material into cells via transduction.This term includes lentiviral vector nucleic acids such as DNA and RNA, encapsulated forms of these nucleic acids, and viral particles in which viral vector nucleic acids are packaged.

[0055] As used herein, the term "mutated" refers to a change in a sequence, such as a sequence of nucleotides or amino acids, from the native, standard or reference version of the respective sequence, i.e., the non-mutated sequence.

[0056] As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, an enhancer, or an array of transcription factor binding sites) and a second nucleic acid sequence, where the expression control sequence affects the transcription and / or translation of the nucleic acid corresponding to the second sequence when an appropriate molecule (e.g., a transcriptional activator protein) is bound to the expression control sequence.

[0057] As used herein, the term "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In some embodiments, a promoter operable in a mammalian cell contains an AT-rich region located about 25-30 bases upstream from the site where transcription is initiated and / or another sequence found about 70-80 bases upstream from the start of transcription, e.g., a CNCAAT region, where N can be any nucleotide.

[0058] As used herein, the term "small hairpin RNA" or "shRNA" refers to an RNA molecule that comprises an antisense region, a loop portion, and a sense region, where the sense region has complementary nucleotides that base-pair with the antisense region to form a double-stranded stem.Following post-transcriptional processing, the small hairpin RNA is converted into small interfering RNA by a cleavage event mediated by the enzyme DICER, which is a member of the RNase III family.As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription, for example, mediated by the enzyme DICER and / or Drosha.

[0059] As used herein, the term "transduce" or "transduction" refers to the delivery of a gene using a viral or retroviral vector by means of infection rather than transfection. For example, an anti-HPRT gene carried by a retroviral vector (a modified retrovirus used as a vector to introduce nucleic acid into cells) can be transduced into a cell by infection and proviral integration. That is, a "transduced gene" is a gene that has been introduced into a cell by infection and proviral integration with a lentivirus or vector. The viral vector (e.g., a transduction vector) transduces a gene into a "target cell" or host cell.

[0060] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect in a subject in need of treatment, i.e., a subject having a disease or disorder. "Treatment" refers to improving or preventing one or more symptoms or effects (e.g., consequences) of a disease or disorder. Reference to "treatment", "treat", or "treating" does not necessarily mean reversing or preventing every symptom or effect of a disease or disorder. For example, although a subject may ultimately suffer from one or more symptoms or effects, the number and / or severity of the symptoms or effects are reduced and / or the quality of life is improved compared to before treatment.

[0061] Each embodiment described herein applies mutatis mutandis to all embodiments unless specifically stated otherwise.

[0062] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0063] 2. Lentiviral Vectors The present disclosure provides lentiviral vectors useful for gene therapy applications to treat diseases or conditions, including, for example, WAS or SCD. In one embodiment, the lentiviral vector comprises a first promoter operably linked to a transgene (i.e., operably linked to a first nucleic acid sequence encoding a protein or polynucleotide, such as a therapeutic protein or polynucleotide) and a modified HS4-650 insulator. The modified HS4-650 insulator comprises inactivation of one or more cryptic splice acceptor sites present in the unmodified HS4-650 insulator when the insulator is present in the vector. In another embodiment, the lentiviral vector comprises a first promoter operably linked to a transgene (i.e., operably linked to a first nucleic acid sequence encoding a protein or polynucleotide, such as a therapeutic protein or polynucleotide) and a modified HS4-400 insulator. The modified HS4-400 insulator comprises inactivation of one or more cryptic splice acceptor sites present in the unmodified HS4-400 insulator when the insulator is present in the vector.

[0064] Thus, the lentiviral vectors of the present disclosure, when integrated into the genome of a cell, as described herein, can be associated with reduced alternative splicing (e.g., of transcripts of genes into which the lentiviral vector has been integrated in a cell, of lentiviral vector RNA, and / or of transcripts of transgenes encoded by the lentiviral vector) compared to lentiviral vectors comprising an unmodified HS4-650 insulator or an unmodified HS4-400 insulator. In some embodiments, the level of alternative splicing is reduced by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0065] For the purposes of this disclosure, a lentiviral vector is a vector that contains a nucleic acid that contains at least one component part that can be derived from a lentivirus. This component part can be involved in the biological mechanism by which the vector infects cells, expresses genes, or replicates. That is, a lentiviral vector includes a nucleic acid molecule such as a plasmid and a viral particle.

[0066] The basic structure of the genomes of retroviruses and lentiviruses share many common features, such as the 5'LTR and 3'LTR, between or within which are located the packaging signal that allows the genome to be packaged, the primer binding site, the integration site that allows integration into the genome of the host cell, and the gag, pol, and env genes that code for packaging components, which are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as the rev and rev response element (RRE) sequences, which allow efficient export of the RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cell. In the provirus, the viral genes are flanked on both sides by regions called long terminal repeats (LTRs). The LTRs are involved in the integration and transcription of the provirus. The LTRs can also act as enhancer-promoter sequences and control the expression of the viral genes. The LTRs themselves are identical sequences that can be divided into three elements called "U3", "R", and "U5". U3 is derived from a sequence unique to the 3' end of the RNA, R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably between different viruses.

[0067] In one embodiment, at least a portion of one or more protein coding regions essential for replication may be removed from the vector, thereby rendering the vector replication-defective. A portion of the viral genome may be replaced with nucleic acid to generate a vector that contains nucleic acid capable of transducing target non-dividing host cells and / or integrating its genome into the host genome. In one embodiment, the lentiviral vector is a non-integrating vector, as described in US Patent Application No. 12 / 138,993 (herein incorporated by reference).

[0068] Lentiviral vectors may have genomes engineered to remove non-essential elements and retain essential elements to provide the functions necessary to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell (see, for example, U.S. Patent No. 6,669,936, incorporated by reference). In some embodiments, the genome is limited to sufficient lentiviral genetic information to allow packaging of an RNA genome into a viral particle capable of infecting a target cell in the presence of packaging components. Infection of a target cell may include reverse transcription and integration into the target cell genome. In some embodiments, the vector is incapable of independent replication to produce infectious lentiviral particles in the final target cell. In some embodiments, the lentiviral vector lacks a functional gag-pol and / or env gene and / or other genes essential for replication.

[0069] In some examples, the lentiviral vector is a self-inactivating vector. A self-inactivating vector can be constructed by deleting the transcription enhancer or enhancer and promoter in the U3 region of the 3'LTR. After rounds of vector reverse transcription and integration, these changes are copied into both the 5' and 3' LTRs, producing a transcriptionally inactive provirus (Yu et al. (1986), Proceedings Nat'l Acad. Sci. USA, 83:3194-98; Dougherty and Temin et al. (1987), Proceedings Nat'l Acad. Sci. USA, 84:1197-01; Hawley (1987), Proceedings Nat'l Acad. Sci. USA, 84:2406-10; Yee et al. (1994), Proceedings Nat'l Acad. Sci. USA, 91:9564-68). However, any promoters internal to the LTR in such vectors may still be transcriptionally active. This strategy was adopted to eliminate the influence on transcription of enhancers and promoters in the viral LTR from internally placed genes. Such effects include increased transcription (Jolly et al. (1983), Nucleic Acids Research, 11:1855-72) or repression of transcription (Emerman and Temin (1984), Cell, 39:449-67). This strategy can also be used to eliminate transcription downstream from the 3'LTR into genomic DNA (Herman & Coffin (1987), Science, 236:845-48).

[0070] The plasmid vector used to produce the viral genome in the host cell / packaging cell will also contain transcriptional regulatory control sequences operably linked to the lentiviral genome that direct the transcription of the genome in the host cell / packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed lentiviral sequence, i.e., the 5'U3 region, or they may be another viral promoter, such as a heterologous or modified promoter, such as the CMV promoter or the 7tetO promoter / operator. Some lentiviral genomes require additional sequences for efficient virus production. For example, in the case of HIV-based lentiviral vectors, rev and RRE sequences are preferably included. However, the need for rev and RRE may be reduced or eliminated by codon optimization (see U.S. Patent Application No. 12 / 587,236, incorporated by reference). Alternative sequences that perform the same function as the rev / RRE system are also known. For example, a functional analogue of the rev / RRE system is found in the Mason-Pfizer monkey virus, which contains an RRE-type sequence in its genome known as a constitutive transport element (CTE) that is believed to interact with factors in infected cells. The factor in the cell can be considered as a rev analogue. That is, CTE can be used as a substitute for the rev / RRE system. Any other functional equivalents known or available can also be associated with the vector of the present disclosure. For example, the Rex protein of HTLV-1 can functionally replace the Rev protein of HIV-1. Rev and Rex are also known to have similar effects on IRE-BP.

[0071] In some embodiments, the expression vector comprises sequences from the 5' and 3' long terminal repeats (LTRs) of lentivirus. In some embodiments, the vector comprises the R and U5 sequences from the 5'LTR of lentivirus and the inactivated or self-inactivating 3'LTR from lentivirus. In some embodiments, the LTR sequences are HIV LTR sequences.

[0072] In some embodiments, the lentiviral vectors contemplated herein may be integrative or non-integrative (also referred to as integration-defective lentiviruses). As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus with an integrase that lacks the ability to integrate the viral genome into the genome of a host cell. In some applications, the use of an integrative lentiviral vector can avoid the possibility of insertional mutagenesis induced by an integrative lentivirus. An integration-defective lentiviral vector is typically generated by mutating the lentiviral integrase gene or modifying the attachment sequence of the LTR (see, for example, Sarkis et al. (2008), Curr. Gene. Ther., 6: 430-437). Lentiviral integrase is encoded by the HIV-1 Pol region, which cannot be omitted since this region encodes other important activities including reverse transcription, nuclear import, and assembly of viral particles. Mutations in pol that alter the integrase protein fall into one of two classes: those that selectively affect only integrase activity (Class I), or those that have pleiotropic effects (Class II). Mutations across the N- and C-termini and catalytic core regions of the integrase protein generate Class II mutations that affect multiple functions, including particle formation and reverse transcription. Class I mutations limit their effects to catalytic activity, DNA binding, linear episome processing, and integrase multimerization. The most common Class I mutation site is a triad of residues in the catalytic core of integrase that includes D64, D116, and E152. Each mutation has been shown to efficiently inhibit integration, with integration frequencies up to 4 logs lower than that of normal integrating vectors, while maintaining transgene expression of NILV. Another alternative method to block integration is to introduce mutations in the DNA binding site of integrase (LTR att site) within a 12 base pair region of the U3 region or within an 11 base pair region of the U5 region at the 5' and 3' ends of the LTR, respectively.These sequences contain conserved terminal CA dinucleotides that are exposed following integrase-mediated end processing. Single or double mutations in the conserved CA / TG dinucleotides result in up to a 3-4 log decrease in integration frequency, while retaining all other functions required for efficient viral transduction.

[0073] 2.1 Transgene A transgene can be any gene that encodes a therapeutic expression product (e.g., a protein or polynucleotide) capable of correcting a defect in a target cell (e.g., HSC). Transgenes can include genomic sequences, cDNA sequences, and small engineered gene segments that express, or can be adapted to express, proteins, polypeptides, domains, fusion proteins, and mutants that maintain some or all of the therapeutic function of the full-length polypeptide encoded by the transgene.

[0074] In certain embodiments, the transgene encodes a Wiskott-Aldrich syndrome (WAS) protein (WASP). That is, in some embodiments, the lentiviral vector of the present disclosure comprises a first nucleic acid sequence encoding a WASP. Exemplary WASPs include a WASP comprising an amino acid sequence set forth in SEQ ID NO:76, and a WASP having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% sequence identity to the WASP set forth in SEQ ID NO:76. In some embodiments, the nucleic acid sequence encoding WASP comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% sequence identity to a nucleic acid sequence set forth in any one of SEQ ID NOs: 73-75. In other embodiments, the transgene is a globin transgene, such as a γ-globin transgene.

[0075] In certain embodiments, the transgene encodes a globin transgene. That is, in some embodiments, the lentiviral vector of the present disclosure comprises a first nucleic acid sequence encoding a globin transgene. Exemplary globin transgenes include transgenes comprising the amino acid sequence set forth in SEQ ID NO: 103, and transgenes having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% sequence identity with the protein set forth in SEQ ID NO: 103. In some embodiments, the nucleic acid sequence encoding the globin protein comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% sequence identity to a nucleic acid sequence set forth in any one of SEQ ID NOs:101-102.

[0076] 2.2 HS4-650 insulator Some lentiviral vectors of the present disclosure comprise a modified HS4-650 insulator that has one or more inactivated or disrupted splice acceptor sites relative to the unmodified HS4-650 insulator.

[0077] Insulator elements have two important activities: "enhancer-blocking activity," which prevents the interaction of enhancers with promoters, and "barrier activity," which prevents silencing of transgenes by chromatin condensation. While the barrier activity can effectively increase transgene expression, the enhancer-blocking activity can prevent enhancers in a vector from acting on normally inactive oncogenic gene promoters when integrated nearby.

[0078] The best-characterized insulator with barrier and enhancer-blocking functions is a 1.2 kb fragment containing hypersensitive site 4 (cHS4) from the chicken β-globin locus. This insulator is effective in increasing transgene expression and reducing undesired promoter activity, but it has been shown to reduce viral titers, thereby limiting large-scale virus production for clinical use. An alternative form of the 650bp cHS4 insulator, containing HS4-Core (250bp) and HS4-Ext (400bp), termed HS4-650 (or cHS4-650), retains the barrier and enhancer blocking functions but does not affect virus production as well as the 1.2kb fragment (see, for example, Arumugam et al. (2009), PLoS ONE, 4(9):e6995; Wielgosz et al. (2015), Molecular Therapy -Methods & Clinical Development, 2, 14063). Another alternative form of the 400bp cHS4 insulator, containing HS4-Ext (400bp), termed HS4-400 (or cHS4 400), retains the barrier and enhancer blocking functions but does not affect virus production as well as the 1.2kb fragment.

[0079] As determined herein, insulators derived from cHS4, including the HS4-650 and HS4-400 insulators, may contain cryptic splice acceptor sites when present in a viral vector. These splice acceptor sites were identified in the HS4-650 insulator, shown in SEQ ID NO:1, when the insulator was present in a lentiviral vector in a reverse orientation, whereby the splice acceptor site was in the positive strand of the vector. That is, the splice acceptor site was in the reverse complement of SEQ ID NO:1. This reverse complement is shown as SEQ ID NO:2. The splice acceptor sites include splice acceptor site 1 (SA1), splice acceptor site 2 (SA2), and splice acceptor site 3 (SA3). Table 2 sets forth the sequences and locations of these splice sites in the HS4-650 insulator, shown in SEQ ID NO:2.

[0080] [Table 2]

[0081] 2.2.1 SA1 As shown in Table 2, SA1 is present at positions 385-386 of SEQ ID NO:2 (i.e., splicing occurs between G at position 385 and A at position 386) and at the corresponding positions of other reverse complement HS4-650 insulator sequences, including those shown in SEQ ID NOs:11, 20, 29, 38, and 47 (see FIG. 1). SA1 has the sequence TTGCATCCAG ∧ It may also be defined as comprising ACACCATCAA (SEQ ID NO: 60), ∧ indicates the splice position.

[0082] The lentiviral vectors of the present disclosure comprise a modified HS4-650 insulator that, when present in the lentiviral vector, comprises an inactivated SA1 (relative to the unmodified HS4-650 insulator when present in the lentiviral vector). That is, the modified HS4-650 insulator, when present in the vector, comprises a modification to the unmodified HS4-650 insulator, where the modification results in inactivation of SA1. That is, the lentiviral vector comprising the modified HS4-650 insulator, when transduced into a cell, exhibits reduced splicing at positions 385-386, numbered relative to SEQ ID NO:2, compared to the splicing that occurs at positions 385-386 in a lentiviral vector comprising the unmodified HS4-650 insulator. In some examples, splicing is reduced by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the modification is or includes a mutation in the sequence of the modified HS4-650 insulator relative to the unmodified HS4-650 insulator. In other examples, the modification is a change in the orientation of the modified HS4-650 insulator in a vector relative to the orientation of the unmodified HS4-650 insulator when in the vector. As will be appreciated, if the modification is a change in the orientation of the insulator, there may be no modification of the sequence of the modified HS4-650 insulator compared to the unmodified HS4-650 insulator.

[0083] Unmodified HS4-650 insulators include insulators that, when present in a lentiviral vector, contain an active SA1, i.e., an insulator that contains a sequence and orientation within the lentiviral vector that can facilitate splicing at SA1. Exemplary unmodified HS4-650 insulators include sequences set forth in SEQ ID NOs: 1, 10, 19, 28, 37, and 46 (with reverse complements set forth in SEQ ID NOs: 2, 11, 20, 29, 38, and 47) and sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (provided that the SA1 site is still present, e.g., that the reverse complement of the HS4-650 insulator contains the sequence TTGCATCCAGACACCATCAA (SEQ ID NO: 60)). In some examples, the unmodified HS4-650 insulator is an insulator in a reverse orientation in the lentiviral vector, whereby SA1 is on the positive strand. In further examples, the unmodified HS4-650 insulator is an insulator in a reverse orientation relative to the transgene, whereby SA1 is on the positive strand of the transgene transcript.

[0084] In certain examples, the modified HS4-650 insulator comprises a mutation (e.g., a deletion, insertion, or substitution of a nucleotide) relative to the unmodified HS4-650 insulator, which inactivates the SA1 present in the unmodified HS4-650 insulator (or reduces splicing at positions 385-386 of the reverse complement of the modified HS4-650 insulator compared to splicing occurring at positions 385-386 of the reverse complement of the unmodified HS4-650 insulator, numbered relative to SEQ ID NO:2). The mutation may be any mutation that inactivates or disrupts SA1. In some examples, the mutation is a deletion or substitution of any nucleotide in the SA1 sequence, or an insertion of a nucleotide (e.g., the sequence TTGCATCCAGACACCATCAA (SEQ ID NO:60)) into the SA1 sequence. In particular examples, the mutation is a mutation (e.g., a deletion or substitution) of A at position 384, G at position 385, A at position 386, and / or C at position 387, numbered relative to SEQ ID NO:2. For example, the modified HS4-650 insulator may include an A to T, A to C, or A to G mutation at position 384, a G to C, G to A, or G to T mutation at position 385, an A to T, A to C, or A to G mutation at position 386, and / or a C to G, C to T, or C to A mutation at position 387, numbered relative to SEQ ID NO:2. In other examples, the mutation includes an insertion of a nucleotide after position 384, 385, or 386. In some examples, the modified HS4-650 insulator includes two or more such mutations.

[0085] In one example, the modified HS4-650 insulator comprises an A to T mutation at position 384 in the reverse complement sequence (i.e., in the complementary strand) numbered relative to SEQ ID NO: 2. In certain embodiments, the reverse complement sequence of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs: 3, 12, 21, 30, 39, and 48, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of a T at position 384 numbered relative to SEQ ID NO: 2).

[0086] In some examples, the modified HS4-650 insulator described herein having a mutation that inactivates SA1 is in the opposite orientation to the transgene (i.e., the opposite orientation to the first nucleic acid sequence). In particular examples, the first nucleic acid is in a forward orientation and the modified HS4-650 insulator is in a reverse orientation in the lentiviral vector.

[0087] In a further example, the modified HS4-650 insulator is in the opposite orientation in the lentiviral vector to the unmodified HS4-650 insulator when in the lentiviral vector, i.e. the orientation of the modified HS4-650 insulator is inverted relative to the unmodified HS4-650 insulator, thereby inactivating SA1. In a particular example, the modified HS4-650 insulator is in the forward orientation in the vector. That is, a lentiviral vector is also provided, comprising a first promoter operably linked to a first nucleic acid sequence encoding WASP and the HS4-650 insulator in the forward orientation in the vector. In some examples, the first nucleic acid sequence is also in the forward orientation in the vector. In some examples, the HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:1, 10, 19, 28, 37, and 46, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0088] 2.2.2 SA2 SA2 is located at positions 446-447 of SEQ ID NO:2 (i.e., splicing occurs between G at position 446 and G at position 447) and the corresponding position in other reverse complement HS4-650 insulator sequences, including those shown in SEQ ID NOs:11, 20, 29, 38, and 47 (see FIG. 1). SA2 is located at the sequence ATCCCCCCAG ∧ GTGTCTGCAG (SEQ ID NO: 61), ∧ indicates the splice position.

[0089] The lentiviral vectors of the present disclosure comprise a modified HS4-650 insulator that, when present in the lentiviral vector, comprises an inactivated SA2 (relative to the unmodified HS4-650 insulator when present in the lentiviral vector). That is, the modified HS4-650 insulator, when present in the vector, comprises a modification to the unmodified HS4-650 insulator, where the modification results in inactivation of SA2. That is, the lentiviral vector comprising the modified HS4-650 insulator, when transduced into a cell, exhibits reduced splicing at positions 446-447, numbered relative to SEQ ID NO:2, compared to the splicing that occurs at positions 446-447 in a lentiviral vector comprising the unmodified HS4-650 insulator. In some examples, splicing is reduced by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the modification is or includes a mutation in the sequence of the modified HS4-650 insulator relative to the unmodified HS4-650 insulator. In other examples, the modification is a change in the orientation of the modified HS4-650 insulator in a vector relative to the orientation of the unmodified HS4-650 insulator when in the vector. As will be appreciated, if the modification is a change in the orientation of the insulator, there may be no modification of the sequence of the modified HS4-650 insulator compared to the unmodified HS4-650 insulator.

[0090] Unmodified HS4-650 insulators include insulators that, when present in a lentiviral vector, contain an active SA2, i.e., an insulator that contains a sequence and orientation within the lentiviral vector that can facilitate splicing at SA2. Exemplary unmodified HS4-650 insulators include sequences set forth in SEQ ID NOs: 1, 10, 19, 28, 37, and 46 (with reverse complements set forth in SEQ ID NOs: 2, 11, 20, 29, 38, and 47) and sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (provided that the SA2 site is still present, e.g., that the reverse complement of the HS4-650 insulator contains the sequence ATCCCCCCAGGTGTCTGCAG (SEQ ID NO: 61)). In some examples, the unmodified HS4-650 insulator is an insulator in a reverse orientation in the lentiviral vector, whereby SA2 is on the positive strand. In further examples, the unmodified HS4-650 insulator is an insulator in a reverse orientation relative to the transgene, whereby SA2 is on the positive strand of the transgene transcript.

[0091] In certain examples, the modified HS4-650 insulator comprises a mutation (e.g., a deletion, insertion, or substitution of a nucleotide) relative to the unmodified HS4-650 insulator, which inactivates the SA2 present in the unmodified HS4-650 insulator (or reduces splicing at positions 446-447 of the reverse complement of the modified HS4-650 insulator compared to the splicing occurring at positions 446-447 of the reverse complement of the unmodified HS4-650 insulator, numbered relative to SEQ ID NO:2). The mutation may be any mutation that inactivates or destroys SA2. In some examples, the mutation is a deletion or substitution of any nucleotide in the SA2 sequence, or an insertion of a nucleotide (e.g., the sequence ATCCCCCCAGGTGTCTGCAG (SEQ ID NO:61)) into the SA2 sequence. In particular examples, the mutation is a mutation (e.g., a deletion or substitution) of A at position 445, G at position 446, G at position 447, and / or T at position 448, numbered relative to SEQ ID NO:2. For example, the modified HS4-650 insulator may include an A to T, A to C, or A to G mutation at position 445, a G to C, G to A, or G to T mutation at position 446, a G to C, G to T, or G to A mutation at position 447, and / or a T to A, T to C, or T to G mutation at position 448, numbered relative to SEQ ID NO:2. In other examples, the mutation includes an insertion of a nucleotide after position 445, 446, or 447. In some examples, the modified HS4-650 insulator includes two or more such mutations.

[0092] In one example, the modified HS4-650 insulator comprises an A to T mutation at position 445 in the reverse complement sequence, numbered relative to SEQ ID NO: 2. In certain embodiments, the reverse complement sequence of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs: 7, 16, 25, 34, 43, and 52, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of a T at position 445 in the numbering relative to SEQ ID NO: 2).

[0093] In some examples, the modified HS4-650 insulator described herein having a mutation that inactivates SA2 is in the opposite orientation to the transgene (i.e., the opposite orientation to the first nucleic acid sequence). In particular examples, the first nucleic acid is in a forward orientation and the modified HS4-650 insulator is in a reverse orientation in the lentiviral vector.

[0094] In a further example, the modified HS4-650 insulator is in the opposite orientation in the lentiviral vector relative to the unmodified HS4-650 insulator when in the lentiviral vector, i.e., the orientation of the modified HS4-650 insulator is reversed relative to the unmodified HS4-650 insulator, thereby inactivating SA2. In a particular example, the modified HS4-650 insulator is in the forward orientation in the vector.

[0095] 2.2.3 SA3 SA3 is located at positions 456-457 of SEQ ID NO:2 (i.e., splicing occurs between G at position 456 and G at position 457) and at the corresponding position in other reverse complement HS4-650 insulator sequences, including those shown in SEQ ID NOs:11, 20, 29, 38, and 47 (see FIG. 1). SA3 has the sequence GTGTCTGCAG ∧ GCTCAAAGAG (SEQ ID NO: 62), ∧ indicates the splice position.

[0096] The lentiviral vectors of the present disclosure comprise a modified HS4-650 insulator that, when present in the lentiviral vector, comprises an inactivated SA3 (relative to the unmodified HS4-650 insulator when present in the lentiviral vector). That is, the modified HS4-650 insulator, when present in the vector, comprises a modification to the unmodified HS4-650 insulator, where the modification results in inactivation of SA3. That is, the lentiviral vector comprising the modified HS4-650 insulator, when transduced into a cell, exhibits reduced splicing at positions 456-457, numbered relative to SEQ ID NO:2, compared to the splicing that occurs at positions 456-457 in a lentiviral vector comprising the unmodified HS4-650 insulator. In some examples, splicing is reduced by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the modification is or includes a mutation in the sequence of the modified HS4-650 insulator relative to the unmodified HS4-650 insulator. In other examples, the modification is a change in the orientation of the modified HS4-650 insulator in a vector relative to the orientation of the unmodified HS4-650 insulator when in the vector. As will be appreciated, if the modification is a change in the orientation of the insulator, there may be no modification of the sequence of the modified HS4-650 insulator compared to the unmodified HS4-650 insulator.

[0097] Unmodified HS4-650 insulators include insulators that, when present in a lentiviral vector, contain an active SA3, i.e., an insulator that contains a sequence and orientation within the lentiviral vector that can facilitate splicing at SA3. Exemplary unmodified HS4-650 insulators include sequences set forth in SEQ ID NOs: 1, 10, 19, 28, 37, and 46 (with reverse complements set forth in SEQ ID NOs: 2, 11, 20, 29, 38, and 47) and sequences having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (provided that a SA3 site is still present, e.g., that the reverse complement of the HS4-650 insulator contains the sequence GTGTCTGCAGGCTCAAAGAG (SEQ ID NO: 62)). In some examples, the unmodified HS4-650 insulator is an insulator in a reverse orientation in the lentiviral vector, whereby SA3 is on the positive strand. In further examples, the unmodified HS4-650 insulator is an insulator in a reverse orientation relative to the transgene, whereby SA3 is on the positive strand of the transgene transcript.

[0098] In certain examples, the modified HS4-650 insulator comprises a mutation (e.g., a deletion, insertion, or substitution of a nucleotide) relative to the unmodified HS4-650 insulator, which inactivates the SA3 present in the unmodified HS4-650 insulator (or reduces splicing at positions 456-457 of the reverse complement of the modified HS4-650 insulator compared to the splicing occurring at positions 456-457 of the reverse complement of the unmodified HS4-650 insulator, numbered relative to SEQ ID NO:2). The mutation may be any mutation that inactivates or destroys the SA3. In some examples, the mutation is a deletion or substitution of any nucleotide in the SA3 sequence, or an insertion of a nucleotide (e.g., the sequence GTGTCTGCAGGCTCAAAGAG (SEQ ID NO:62)) into the SA3 sequence. In particular examples, the mutation is a mutation (e.g., a deletion or substitution) of A at position 455, G at position 456, G at position 457, and / or C at position 458, numbered relative to SEQ ID NO:2. For example, the modified HS4-650 insulator may include an A to T, A to C, or A to G mutation at position 455, a G to C, G to A, or G to T mutation at position 456, a G to C, G to T, or G to A mutation at position 457, and / or a C to A, C to G, or C to T mutation at position 458, numbered relative to SEQ ID NO:2. In other examples, the mutation includes an insertion of a nucleotide after position 455, 456, or 457. In some examples, the modified HS4-650 insulator includes two or more such mutations.

[0099] In one example, the modified HS4-650 insulator comprises an A to T mutation at position 455 in the reverse complement sequence, numbered relative to SEQ ID NO: 2. In certain embodiments, the reverse complement sequence of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs: 9, 18, 27, 36, 45, and 54, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of a T at position 455 in the numbering relative to SEQ ID NO: 2).

[0100] In some examples, the modified HS4-650 insulator described herein having a mutation that inactivates SA3 is in the opposite orientation to the transgene (i.e., the opposite orientation to the first nucleic acid sequence). In particular examples, the first nucleic acid is in a forward orientation and the modified HS4-650 insulator is in a reverse orientation in the lentiviral vector.

[0101] In a further example, the modified HS4-650 insulator is in the opposite orientation in the lentiviral vector relative to the unmodified HS4-650 insulator when in the lentiviral vector, i.e., the orientation of the modified HS4-650 insulator is reversed relative to the unmodified HS4-650 insulator, thereby inactivating SA3. In a particular example, the modified HS4-650 insulator is in the forward orientation in the vector.

[0102] 2.2.4 Combinatorial mutations The modified HS4-650 insulator may contain two or more mutations that inactivate two or more of SA1, SA2, or SA3 relative to the unmodified HS4-650 insulator. Any of the above mutations to inactivate SA1, SA2, and / or SA3 can be combined in the modified HS4-650 insulator.

[0103] In one example, the modified HS4-650 insulator comprises a mutation that inactivates SA1 and a mutation that inactivates SA2. For example, the modified HS4-650 insulator may comprise an A to T mutation at position 384 in the reverse complement sequence numbered relative to SEQ ID NO:2, and an A to T mutation at position 445 in the reverse complement sequence numbered relative to SEQ ID NO:2. In certain embodiments, the reverse complement sequence of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:4, 13, 22, 31, 40, and 49, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of a T at position 384 and a T at position 445 in the numbering relative to SEQ ID NO:2).

[0104] In another example, the modified HS4-650 insulator comprises a mutation that inactivates SA1 and a mutation that inactivates SA3. For example, the modified HS4-650 insulator may comprise an A to T mutation at position 384 in the reverse complement sequence numbered relative to SEQ ID NO:2 and an A to T mutation at position 455 in the reverse complement sequence numbered relative to SEQ ID NO:2. In certain embodiments, the reverse complement sequence of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:5, 14, 23, 32, 41, and 50, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of a T at position 384 and a T at position 455 in the numbering relative to SEQ ID NO:2).

[0105] The modified HS4-650 insulator may comprise an SA2 inactivating mutation and an SA3 inactivating mutation. For example, the modified HS4-650 insulator may comprise an A to T mutation at position 445 in the reverse complement sequence numbered relative to SEQ ID NO:2 and an A to T mutation at position 455 in the reverse complement sequence numbered relative to SEQ ID NO:2. In certain embodiments, the reverse complement sequence of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs:8, 17, 26, 35, 44, and 43, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of a T at position 445 and a T at position 455 in the numbering relative to SEQ ID NO:2).

[0106] In another example, the modified HS4-650 insulator comprises an SA1 inactivating mutation, an SA2 inactivating mutation, and an SA3 inactivating mutation. For example, in some embodiments, the modified HS4-650 insulator may comprise an A to T mutation at position 384 in the reverse complement sequence, an A to T mutation at position 445 in the reverse complement sequence, and an A to T mutation at position 455 in the reverse complement sequence, numbered relative to SEQ ID NO:2. In certain embodiments, the reverse complement of the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs: 6, 15, 24, 33, 42, and 51, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (subject to the presence of a T at position 384, a T at position 445, and a T at position 455, numbered relative to SEQ ID NO: 2).

[0107] 2.3 HS4-400 insulator The lentiviral vector of the present disclosure may comprise an HS4-400 insulator. In certain embodiments, the HS4-400 insulator is a modified HS4-400 insulator that has one or more inactivated or destroyed splice acceptor sites relative to the unmodified HS4-400 insulator. An exemplary HS4-400 insulator is an insulator that comprises a sequence as set forth in SEQ ID NO:89 or a sequence having at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% sequence identity thereto.

[0108] As determined herein, the HS4-400 insulator may contain cryptic splice acceptor sites when present in a viral vector. These splice acceptor sites were identified in the HS4-400 insulator, shown in SEQ ID NO:89, when the insulator was present in a lentiviral vector in a reverse orientation, whereby the splice acceptor site was in the positive strand in the vector. That is, the splice acceptor site was in the reverse complement of SEQ ID NO:89. This reverse complement is shown as SEQ ID NO:90. The splice acceptor sites include splice acceptor site 2 (SA2) and splice acceptor site 3 (SA3). Table 3 sets forth the sequences and locations of these splice sites in the HS4-400 insulator sequence, shown in SEQ ID NO:90.

[0109] [Table 3]

[0110] 2.3.1 SA2 SA2 is located at positions 190-191 of SEQ ID NO:90 (i.e., splicing occurs between G at position 190 and G at position 191) and at the corresponding positions in other reverse complement HS4-400 insulator sequences. SA2 is located at the sequence ATCCCCCCAG ∧GTGTCTGCAG (SEQ ID NO: 61) ∧ represents a splice position), or may be defined as comprising the sequence of nucleotides 181 to 200 of the complementary strand of the HS4-400 insulator, numbered relative to SEQ ID NO:90.

[0111] The lentiviral vectors of the present disclosure comprise a modified HS4-400 insulator that, when present in the lentiviral vector, comprises an inactivated SA2 (relative to the unmodified HS4-400 insulator when present in the lentiviral vector). That is, the modified HS4-400 insulator, when present in the vector, comprises a modification to the unmodified HS4-400 insulator, where the modification results in inactivation of SA2. That is, the lentiviral vector comprising the modified HS4-400 insulator, when transduced into a cell, exhibits reduced splicing at positions 190-191, numbered relative to SEQ ID NO: 90, compared to the splicing that occurs at positions 190-191 in a lentiviral vector comprising the unmodified HS4-400 insulator. In some examples, splicing is reduced by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the modification is or includes a mutation in the sequence of the modified HS4-400 insulator relative to the unmodified HS4-400 insulator. In other examples, the modification is a change in the orientation of the modified HS4-400 insulator in a vector relative to the orientation of the unmodified HS4-400 insulator when in the vector. As will be appreciated, if the modification is a change in the orientation of the insulator, there may be no modification of the sequence of the modified HS4-400 insulator compared to the unmodified HS4-400 insulator.

[0112] Unmodified HS4-400 insulators include insulators that contain active SA2 when present in a lentiviral vector, i.e., insulators that contain a sequence and orientation within the lentiviral vector that can facilitate splicing at SA2. Exemplary unmodified HS4-400 insulators include the sequence shown in SEQ ID NO:89 (with the reverse complement shown in SEQ ID NO:90) and sequences with at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (provided that the SA2 site is still present, e.g., the reverse complement of the HS4-400 insulator contains the sequence ATCCCCCCAGGTGTCTGCAG (SEQ ID NO:61)). In some examples, unmodified HS4-400 insulators are insulators in a reverse orientation in a lentiviral vector, whereby SA2 is present on the positive strand.

[0113] In certain examples, the modified HS4-400 insulator comprises a mutation (e.g., a deletion, insertion, or substitution of a nucleotide) relative to the unmodified HS4-400 insulator, which inactivates the SA2 present in the unmodified HS4-400 insulator (or reduces splicing at positions 190-191 of the reverse complement of the modified HS4-400 insulator compared to the splicing occurring at positions 190-191 of the reverse complement of the unmodified HS4-400 insulator, numbered relative to SEQ ID NO: 90). The mutation may be any mutation that inactivates or destroys SA2. In some examples, the mutation is a deletion or substitution of any nucleotide in the SA2 sequence, or an insertion of a nucleotide (e.g., the sequence ATCCCCCCAGGTGTCTGCAG (SEQ ID NO: 61)) into the SA2 sequence. In particular examples, the mutation is a mutation (e.g., a deletion or substitution) of A at position 189, G at position 190, G at position 191, and / or T at position 192, numbered relative to SEQ ID NO: 90. For example, the modified HS4-400 insulator may include an A to T, A to C, or A to G mutation at position 189, a G to C, G to A, or G to T mutation at position 190, a G to C, G to T, or G to A mutation at position 191, and / or a T to A, T to C, or T to G mutation at position 192, numbered relative to SEQ ID NO: 90. In other examples, the mutation includes an insertion of a nucleotide after position 189, 190, or 191. In some examples, the modified HS4-400 insulator includes two or more such mutations.

[0114] In one example, the modified HS4-400 insulator comprises an A to T mutation at position 189 in the reverse complement sequence, numbered relative to SEQ ID NO: 90. In certain embodiments, the reverse complement sequence of the modified HS4-400 insulator comprises the sequence set forth in SEQ ID NO: 93 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of a T at position 189, numbered relative to SEQ ID NO: 90).

[0115] In some instances, the modified HS4-400 insulator described herein having a mutation that inactivates SA2 is in a reverse orientation within the lentiviral vector.

[0116] In a further example, the modified HS4-400 insulator is in the opposite orientation in the lentiviral vector to the unmodified HS4-400 insulator when in the lentiviral vector, i.e. the orientation of the modified HS4-400 insulator is inverted relative to the unmodified HS4-400 insulator, thereby inactivating SA2. In a particular example, the modified HS4-400 insulator is in the forward orientation in the vector. That is, a lentiviral vector is also provided that includes a first promoter operably linked to a first nucleic acid sequence that includes a modified γ-globin transgene that includes HBB intron 2, and the HS4-400 insulator in the forward orientation in the vector. In some examples, the first nucleic acid sequence is in the reverse orientation in the vector. In some examples, the HS4-400 insulator comprises a sequence set forth in SEQ ID NO:90 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0117] 2.3.2 SA3 SA3 is present at positions 200-201 of SEQ ID NO:90 (i.e., splicing occurs between G at position 200 and G at position 201) and at corresponding positions in other reverse complement HS4-400 insulator sequences. SA3 has the sequence GTGTCTGCAG ∧ GCTCAAAGAG (SEQ ID NO: 62) (wherein ∧ represents a splice position), or may be defined as comprising the sequence of nucleotides 191 to 210 of the complementary strand of the HS4-400 insulator, numbered relative to SEQ ID NO:90.

[0118] The lentiviral vectors of the present disclosure comprise a modified HS4-400 insulator that, when present in the lentiviral vector, comprises an inactivated SA3 (relative to the unmodified HS4-400 insulator when present in the lentiviral vector). That is, the modified HS4-400 insulator, when present in the vector, comprises a modification to the unmodified HS4-400 insulator, where the modification results in inactivation of SA3. That is, the lentiviral vector comprising the modified HS4-400 insulator, when transduced into a cell, exhibits reduced splicing at positions 200-201, numbered relative to SEQ ID NO: 90, compared to the splicing that occurs at positions 200-201 in a lentiviral vector comprising the unmodified HS4-400 insulator. In some examples, splicing is reduced by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the modification is or includes a mutation in the sequence of the modified HS4-400 insulator relative to the unmodified HS4-400 insulator. In other examples, the modification is a change in the orientation of the modified HS4-400 insulator in a vector relative to the orientation of the unmodified HS4-400 insulator when in the vector. As will be appreciated, if the modification is a change in the orientation of the insulator, there may be no modification of the sequence of the modified HS4-400 insulator compared to the unmodified HS4-400 insulator.

[0119] Unmodified HS4-400 insulators include insulators that contain active SA3 when present in a lentiviral vector, i.e., insulators that contain sequences and orientations in lentiviral vectors that can facilitate splicing at SA3. Exemplary unmodified HS4-400 insulators include the sequence shown in SEQ ID NO:90 (with the reverse complement shown in SEQ ID NO:89) and sequences with at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (provided that the SA3 site is still present, e.g., that the reverse complement of the HS4-400 insulator contains the sequence GTGTCTGCAGGCTCAAAGAG (SEQ ID NO:62)). In some examples, unmodified HS4-400 insulators are insulators in reverse orientation in lentiviral vectors, whereby SA3 is present on the positive strand.

[0120] In certain examples, the modified HS4-400 insulator comprises a mutation (e.g., a deletion, insertion, or substitution of a nucleotide) relative to the unmodified HS4-400 insulator, which inactivates the SA3 present in the unmodified HS4-400 insulator (or reduces splicing at positions 200-201 of the reverse complement of the modified HS4-400 insulator compared to the splicing occurring at positions 200-201 of the reverse complement of the unmodified HS4-400 insulator, numbered relative to SEQ ID NO: 90). The mutation may be any mutation that inactivates or destroys the SA3. In some examples, the mutation is a deletion or substitution of any nucleotide in the SA3 sequence, or an insertion of a nucleotide (e.g., the sequence GTGTCTGCAGGCTCAAAGAG (SEQ ID NO: 62)) into the SA3 sequence. In particular examples, the mutation is a mutation (e.g., a deletion or substitution) of A at position 199, G at position 200, G at position 201, and / or C at position 202, numbered relative to SEQ ID NO: 90. For example, the modified HS4-400 insulator may include an A to T, A to C, or A to G mutation at position 199, a G to C, G to A, or G to T mutation at position 200, a G to C, G to T, or G to A mutation at position 201, and / or a C to A, C to G, or C to T mutation at position 202, numbered relative to SEQ ID NO: 90. In other examples, the mutation includes an insertion of a nucleotide after position 199, position 200, or position 201. In some examples, the modified HS4-400 insulator includes two or more such mutations.

[0121] In one example, the modified HS4-400 insulator comprises an A to T mutation at position 199 in the reverse complement sequence, numbered relative to SEQ ID NO: 90. In certain embodiments, the reverse complement sequence of the modified HS4-400 insulator comprises a sequence set forth in any one of SEQ ID NOs: 95 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming there is an A to T mutation at position 199, numbered relative to SEQ ID NO: 90).

[0122] In some instances, the modified HS4-400 insulator described herein having an SA3-inactivating mutation is in a reverse orientation within the lentiviral vector.

[0123] In a further example, the modified HS4-400 insulator is in the opposite orientation relative to the unmodified HS4-400 insulator when in the lentiviral vector, i.e., the orientation of the modified HS4-400 insulator is reversed relative to the unmodified HS4-400 insulator, thereby inactivating SA3. In a particular example, the modified HS4-400 insulator is in the forward orientation in the vector.

[0124] 2.3.3 Combinatorial mutations The modified HS4-400 insulator may contain two or more mutations relative to the unmodified HS4-400 insulator that inactivate both SA2 and SA3. Any of the above mutations for inactivating SA2 or SA3 can be combined in the modified HS4-400 insulator.

[0125] In one example, the modified HS4-400 insulator comprises an A to T mutation at position 189 in the reverse complement sequence, numbered relative to SEQ ID NO: 90 (i.e., includes a T at position 189), and an A to T mutation at position 199 in the reverse complement sequence, numbered relative to SEQ ID NO: 90 (i.e., includes a T at position 199). In certain embodiments, the reverse complement sequence of the modified HS4-400 insulator comprises the sequence set forth in SEQ ID NO: 94 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of the T mutation at position 189 and the T mutation at position 199, numbered relative to SEQ ID NO: 90).

[0126] 2.4 Components that inhibit HPRT gene expression In some embodiments, the lentiviral vector of the present disclosure comprises a nucleic acid sequence that encodes an agent that inhibits the expression of HPRT.That is, in some examples, the lentiviral vector comprises a second promoter that is operably linked to a second nucleic acid sequence that encodes a nucleic acid that inhibits the expression of HPRT.In some embodiments, the RNAi agent is shRNA, microRNA, or a hybrid thereof.

[0127] 2.4.1 RNAi In some embodiments, the expression vector comprises a second nucleic acid sequence encoding RNAi. RNA interference is an approach for post-transcriptional silencing of gene expression by triggering the degradation of homologous transcripts through a complex multi-step enzymatic process, for example, a process involving sequence-specific double-stranded small interfering RNA (siRNA). A simplified model of the RNAi pathway is based on two steps, each involving a ribonuclease enzyme. In the first step, the trigger RNA (dsRNA or miRNA primary transcript) is processed into short interfering RNA (siRNA) by the RNase II enzymes DICER and Drosha. In the second step, the siRNA is loaded into the effector complex, the RNA-induced silencing complex (RISC). The siRNA is unwound during RISC assembly, and the single-stranded RNA hybridizes to the mRNA target. Gene silencing is believed to be the result of nucleolytic degradation of the target mRNA by the RNase H enzyme Argonaute (Slicer). If the siRNA / mRNA duplex contains a mismatch, the mRNA is not cleaved. Rather, gene silencing is the result of translational inhibition.

[0128] In some embodiments, the RNAi agent is an inhibitory or silencing nucleic acid. As used herein, "silencing nucleic acid" refers to any polynucleotide that can interact with a specific sequence to inhibit gene expression. Examples of silencing nucleic acids include RNA duplexes (e.g., siRNA, shRNA), locked nucleic acids ("LNA"), antisense RNA, DNA polynucleotides that code the sense and / or antisense sequence of siRNA or shRNA, DNAzymes, or ribozymes. Those skilled in the art will recognize that inhibition of gene expression does not necessarily have to be gene expression from the specific sequence listed, but can be gene expression from the sequence controlled by the specific sequence, for example.

[0129] Methods for constructing interfering RNA are known in the art. For example, interfering RNA can be assembled from two separate oligonucleotides, one strand being a sense strand and the other being an antisense strand, and the antisense strand and the sense strand are self-complementary (i.e., each strand comprises a nucleotide sequence that is complementary to the nucleotide sequence in the other strand, e.g., the antisense strand and the sense strand form a duplex or double-stranded structure). The antisense strand comprises a nucleotide sequence that is complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof (i.e., an undesirable gene), and the sense strand comprises a nucleotide sequence that corresponds to the target nucleic acid sequence or a portion thereof. Alternatively, interfering RNA can be assembled from a single oligonucleotide, and the self-complementary sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker. Interfering RNA can be a polynucleotide with double stranded, asymmetric double stranded, hairpin or asymmetric hairpin secondary structure with self-complementary sense and antisense regions, antisense region comprises nucleotide sequence complementary to nucleotide sequence in individual target nucleic acid molecule or part thereof, and sense region has nucleotide sequence corresponding to target nucleic acid sequence or part thereof.Interfering RNA can be a circular single stranded polynucleotide with stem comprising two or more loop structures and self-complementary sense and antisense regions, antisense region comprises nucleotide sequence complementary to nucleotide sequence in target nucleic acid molecule or part thereof, and sense region has nucleotide sequence corresponding to target nucleic acid sequence or part thereof, and circular polynucleotide can be processed in vivo or in vitro to generate active siRNA molecule that can mediate RNA interference.

[0130] In some embodiments, the region encoding the interfering RNA encodes a self-complementary RNA molecule having a sense region, an antisense region, and a loop region. When expressed, such an RNA molecule desirably forms a "hairpin" structure and is referred to herein as an "shRNA." In some embodiments, the loop region is generally about 2 to about 10 nucleotides in length. In other embodiments, the loop region is about 6 to about 9 nucleotides in length. In some embodiments, the sense and antisense regions are about 15 to about 30 nucleotides in length. After post-transcriptional processing, the small hairpin RNA is converted to an siRNA by a cleavage event mediated by the enzyme DICER, a member of the RNase III family. The siRNA can then inhibit expression of genes with which it shares homology. Further details are provided in Brummelkamp et al. (2002), Science, 296:550-553,; Lee et al. (2002), Nature Biotechnol., 20, 500-505; Miyagishi and Taira (2002), Nature Biotechnol., 20:497-500; Paddison et al. (2002), Genes & Dev., 16:948-958; Paul (2002), Nature Biotechnol., 20, 505-508; Sui (2002), Proceedings Nat'l Acad. Sci. USA, 99(6), 5515-5520; and Yu et al. (2002), Proceedings Nat'l Acad. Sci. USA 99:6047-6052. These disclosures are incorporated herein by reference in their entireties.

[0131] 2.4.2 shRNA In some embodiments, the second nucleic acid sequence encodes an shRNA that inhibits HPRT. In certain embodiments, the shRNA is sh734, such as an shRNA comprising a sequence as set forth in SEQ ID NO:66 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto. In another embodiment, sh734 comprises multiple t-termination sequences required for Pol III promoters such as 7SK. That is, in some embodiments, sh734 comprises a sequence as set forth in SEQ ID NO:67 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto. In a further embodiment, sh734 comprises a single t-stop sequence and thus comprises, for example, the sequence set forth in SEQ ID NO:68 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto.

[0132] 2.4.3 MicroRNAs MicroRNAs (miRs) are a group of non-coding RNAs that post-transcriptionally regulate the expression of their target genes. These single-stranded molecules are thought to form miRNA-mediated silencing complexes (miRISCs) with other proteins that bind to the 3' untranslated regions (UTRs) of their target mRNAs, thereby preventing their translation in the cytoplasm.

[0133] In some embodiments, the shRNA sequence is embedded in a microRNA secondary structure ("microRNA-based shRNA"). In some embodiments, the shRNA nucleic acid sequence targeting HPRT is embedded in a microRNA secondary structure. In some embodiments, the microRNA-based shRNA targets a coding sequence in HPRT to achieve knockdown of HPRT expression. This is considered equivalent to using an shRNA targeting HPRT without the associated pathway saturation and cytotoxic or off-target effects. In some embodiments, the microRNA-based shRNA is a de novo artificial microRNA shRNA. The production of such de novo microRNA-based shRNAs is described in Fang, W. & Bartel, David P. The Menu of Features that Define Primary MicroRNAs and Enable De Novo Design of MicroRNA Genes. Molecular Cell 60, 131-145, the disclosure of which is incorporated herein by reference in its entirety.

[0134] Exemplary miRNAs are provided in International Patent Publication No. WO2020139796.

[0135] 2.4.4 Alternatives to RNAi As an alternative to incorporating RNAi, in some embodiments, the vector may include a nucleic acid sequence encoding an antisense oligonucleotide that binds to a site of messenger RNA (mRNA). The antisense oligonucleotides of the present disclosure specifically hybridize with a nucleic acid encoding a protein and interfere with the transcription or translation of the protein. In some embodiments, the antisense oligonucleotides target DNA and interfere with its replication and / or transcription. In other embodiments, the antisense oligonucleotides specifically hybridize with RNA, including pre-mRNA (i.e., precursor mRNA, which is an immature single strand of mRNA) and mRNA. Such antisense oligonucleotides may affect, for example, the translocation of RNA to the site of protein translation, the translation of RNA into protein, the splicing of RNA to obtain one or more mRNA species, and catalytic activities that may involve or be facilitated by RNA. The overall effect of such interference will be to modulate, reduce, or inhibit the expression of the target protein.

[0136] 2.5 Other Elements Other elements that may be present in the lentiviral vectors of the present disclosure include, for example, promoters, operators, termination signals, polyadenylation signals, etc. One of skill in the art can readily identify appropriate elements present in the vector for proper processing, transcription, and / or translation of the nucleic acid encoded by the vector.

[0137] In one example, the vector comprises a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE). In certain embodiments, the WPRE is downstream of the first nucleic acid sequence and upstream of the modified HS4-650 insulator (i.e., between the first nucleic acid sequence and the modified HS4-650 insulator). In some embodiments, the WPRE is a WPRE mut6 comprising a sequence as set forth in SEQ ID NO: 77 or a WPRE mut7 comprising a sequence as set forth in SEQ ID NO: 78, or comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity to the sequence as set forth in SEQ ID NO: 77 or 78.

[0138] In some examples, the promoter is an MND promoter, such as a promoter comprising a sequence set forth in SEQ ID NO: 72, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 77 or 78. In one embodiment, the first promoter is an MND promoter and is operably linked to a first nucleic acid comprising a transgene.

[0139] In some examples, the promoter is a 7SK RNA promoter, such as a promoter set forth in any one of SEQ ID NOs: 69-71, or a promoter comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity to a sequence set forth in SEQ ID NOs: 69-71. In one embodiment, the second promoter is a 7SK RNA promoter and is operably linked to a second nucleic acid encoding a nucleic acid that inhibits expression of HPRT.

[0140] In some examples, the lentiviral vector comprises a 7tetO promoter / operator, such as a promoter / operator comprising a sequence set forth in SEQ ID NO:79 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity to the sequence set forth in SEQ ID NO:79.

[0141] In a further example, the lentiviral vector comprises a β-globin poly(A) signal, such as a signal comprising a sequence set forth in SEQ ID NO:80 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity to the sequence set forth in SEQ ID NO:80.

[0142] 2.6 Vector production The lentiviral vectors of the present disclosure can be produced using any method, such methods being well known to one of skill in the art. In some embodiments, a first promoter and modified HS4-650 insulator (and any other expression cassettes or elements described herein, as appropriate) operably linked to a first nucleic acid sequence encoding a therapeutic protein, such as a Wiskott-Aldrich syndrome protein, are expressed in a plasmid, such as pTL20c, pTL20d, FG, pRRL, pCL20, pLKO.1 puro, pLKO.1, pLKO.3G, Tet-pLKO-puro, pSico, pLJM1-EGFP, FUGW, pLVTHM, pLVUT-tTR-KRAB, pLL3.7, pLB, pWPXL, pWPI, EF.CMV.RFP, pLenti CMV Puro The plasmid is inserted into a lentiviral vector, which is a plasmid selected from the group consisting of DEST, pLenti-puro, pLOVE, pULTRA, pLJM1-EGFP, pLX301, pInducer20, pHIV-EGFP, Tet-pLKO-neo, pLV-mCherry, pCW57.1, pLionII, pSLIK-Hygro, and pInducer10-mir-RUP-PheS. In other embodiments, the lentiviral vector into which the first promoter, the first nucleic acid sequence, and the modified HS4-650 insulator are inserted is selected from the group consisting of AnkT9W vector, T9Ank2W vector, TNS9 vector, Lentiglobin HPV569 vector, Lentiglobin BB305 vector, BG-1 vector, BGM-1 vector, GLOBE vector, G-GLOBE vector, V5 vector, V5m3 vector, V5m3-400 vector, G9 vector, and BCL11A shmir vector. In certain embodiments, the lentiviral expression vector is pTL20c.

[0143] In one example, an expression cassette having a first promoter and a modified HS4-650 insulator operably linked to a first nucleic acid sequence may be inserted into a pTL20c vector according to the methods described in U.S. Patent Publication No. 20180112233 and International Patent Publication No. WO2020139796.

[0144] In some examples, an expression cassette having a first promoter operably linked to a first nucleic acid sequence and an appropriately modified HS4-400 insulator may be inserted into a pTL20c vector according to the methods described in U.S. Patent Publication No. 20180112233 and International Patent Publication No. WO2020139796.

[0145] Lentiviral particles or virions (or recombinant lentivirus) can be produced using standard methods known in the art. In one example, a stable producer cell line derived from one of the GPR, GPRG, GPRT, GPRGT, or GPRT-G packing cell lines is utilized to generate the virus. In some embodiments, the stable producer cell line is derived from the GPRT-G cell line. In some embodiments, the stable producer cell line is generated by (a) synthesizing a vector by cloning a nucleic acid sequence encoding an anti-HPRT shRNA and WASP into a recombinant plasmid (i.e., the synthesized vector can be any one of the vectors described herein encoding an anti-HPRT shRNA and WASP), (b) generating DNA fragments from the synthesized vector, (c) forming a concatameric array from (i) the DNA fragments generated from the synthesized vector and (ii) the DNA fragments derived from an antibiotic resistance cassette plasmid, (d) transfecting the formed concatameric array into one of the packaging cell lines, and (e) isolating a stable producer cell line. Further methods of generating stable producer cell lines are described in U.S. Patent Publication No. 20180112233.

[0146] 2.7 Exemplary Vectors Exemplary lentiviral vectors of the present disclosure include a 7sk promoter (e.g., any one of SEQ ID NOs: 69-72) operably linked downstream from 5' to 3' to a central polypurine tract (cPPT), a REV response element (RRE), a nucleic acid encoding sh734 (e.g., a nucleic acid encoding sh734 comprising a sequence set forth in any one of SEQ ID NOs: 66-68 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto). a 7sk-sh734 expression cassette comprising a promoter comprising a sequence as set forth in any one of SEQ ID NOs:73 to 75 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto), a transgene encoding WASP (e.g., a transgene comprising a sequence as set forth in any one of SEQ ID NOs:73 to 75 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto), operably linked MND promoter (e.g., a promoter comprising a sequence set forth in SEQ ID NO: 72 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto), a WASP expression cassette comprising a WPRE (e.g., a promoter comprising a sequence set forth in SEQ ID NO: 77 or 78 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto); a 5'LTR (comprising a 7tetO promoter / operator, R, and U5 (e.g., as shown diagrammatically in Figures 2-7)) that is a WPRE comprising a 7tetO promoter / operator, R, and U5 (e.g., as shown diagrammatically in Figures 2-7) and an HS4-650 insulator (e.g., an unmodified HS4-650 insulator as set forth in any one of SEQ ID NOs: 1, 10, 19, 28, 37, and 46, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, in a forward orientation;or a modified HS4-650 insulator as described herein having an inactivated SA1, SA2, and / or SA3, comprising a complement comprising the modified HS4-650 insulator in a reverse orientation, e.g., a sequence as set forth in any one of SEQ ID NOs: 3, 12, 21, 30, 39, and 48, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, wherein the sequence comprises a T at position 384 in the numbering relative to SEQ ID NO: 2. a modified HS4-650 insulator comprising a sequence as set forth in any one of SEQ ID NOs: 7, 16, 25, 34, 43, and 52, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, and a complementary strand comprising a sequence as set forth in any one of SEQ ID NOs: 7, 16, 25, 34, 43, and 52, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, and a modified HS4-650 insulator comprising a T at position 445 in the numbering relative to SEQ ID NO: 2, a sequence as set forth in any one of SEQ ID NOs: 9, 18, 27, 36, 45, and 54, or a sequence having at least 85% sequence identity thereto; a modified HS4-650 insulator comprising a T at position 455 in the numbering relative to SEQ ID NO:2; a sequence as set forth in any one of SEQ ID NOs:4, 13, 22, 31, 40, and 49, or a complementary strand comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% sequence identity thereto; a modified HS4-650 insulator comprising a sequence as set forth in any one of SEQ ID NOs: 5, 14, 23, 32, 41, and 50, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, and comprising a T at position 384 and a T at position 455, as set forth in SEQ ID NO: 8,a modified HS4-650 insulator comprising a complementary strand comprising a sequence as set forth in any one of SEQ ID NOs: 17, 26, 35, 44, and 43, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, the sequence comprising a T at position 445 and a T at position 455, as numbered relative to SEQ ID NO: 2; or a sequence as set forth in any one of SEQ ID NOs: 6, 15, 24, 33, 42, and 51, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto; or a complementary strand comprising a sequence having 99% sequence identity to SEQ ID NO:2, wherein the sequence comprises a modified HS4-650 insulator comprising a T at position 384, a T at position 445, and a T at position 455, as numbered relative to SEQ ID NO:2), U3, R, and a 3'LTR comprising a β-globin poly(A) signal (e.g., a signal comprising the sequence set forth in SEQ ID NO:31 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto). In these vectors, typically the 7sk-sh734 expression cassette is in a reverse orientation and the WASP expression cassette is in a forward orientation.

[0147] In some embodiments, the lentiviral vector comprises a sequence set forth as nucleotides 3098-6006 of SEQ ID NO:57, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the sequence comprises an inactivation of SA1 and SA2, e.g., a T at position 384 and a T at position 445, numbered relative to SEQ ID NO:2), a sequence set forth as nucleotides 3098-6009 of SEQ ID NO:58, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the sequence comprises an inactivation of SA1 and SA2, e.g., a T at position 384 and a T at position 445, numbered relative to SEQ ID NO:2), 59, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the polynucleotide comprises inactivations of SA1, SA2, and SA3, and the sequence comprises inactivations of SA1, SA2, and SA3, e.g., a T at position 384, a T at position 445, and a T at position 455, numbered relative to SEQ ID NO:2).

[0148] In some embodiments, the lentiviral vector comprises a sequence set forth as nucleotides 2710-6006 of SEQ ID NO:57, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the sequence comprises an inactivation of SA1 and SA2, e.g., a T at position 384 and a T at position 445, numbered relative to SEQ ID NO:2), a sequence set forth as nucleotides 2710-6009 of SEQ ID NO:58, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the sequence comprises an inactivation of SA1 and SA2, e.g., a T at position 384 and a T at position 445, numbered relative to SEQ ID NO:2), 59, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the sequence comprises inactivation of SA1, SA2, and SA3, e.g., a T at position 384, a T at position 445, and a T at position 455, numbered relative to SEQ ID NO:2).

[0149] In some embodiments, the lentiviral vector comprises a sequence set forth as nucleotides 2402-6006 of SEQ ID NO:57, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the sequence comprises an inactivation of SA1 and SA2, e.g., a T at position 384 and a T at position 445, numbered relative to SEQ ID NO:2), a sequence set forth as nucleotides 2402-6009 of SEQ ID NO:58, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the sequence comprises an inactivation of SA1 and SA2, e.g., a T at position 384 and a T at position 445, numbered relative to SEQ ID NO:2), 59, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (wherein the polynucleotide comprises inactivations of SA1, SA2, and SA3, and the sequence comprises inactivations of SA1, SA2, and SA3, e.g., a T at position 384, a T at position 445, and a T at position 455, numbered relative to SEQ ID NO:2).

[0150] In one embodiment, a lentiviral vector of the disclosure comprises a sequence set forth in SEQ ID NO:57 or 82, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, wherein the sequence comprises inactivation of SA1 and SA2, e.g., a T at position 384 and a T at position 445, numbered relative to SEQ ID NO:2.

[0151] In another embodiment, a lentiviral vector of the present disclosure comprises a sequence set forth in SEQ ID NO: 58 or 83, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0152] In another embodiment, a lentiviral vector of the disclosure comprises a sequence set forth in SEQ ID NO:59 or 84, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, wherein the sequence comprises inactivation of SA1, SA2, and SA3, e.g., a T at position 384, a T at position 445, and a T at position 455, numbered relative to SEQ ID NO:2.

[0153] 3.Host cells The present disclosure also provides a host cell that contains, is transformed with, or is transduced with the lentiviral vector of the present disclosure. "Host cell" or "target cell" refers to a cell that is transformed or transduced using the method and vector of the present disclosure. In some embodiments, the host cell is a mammalian cell that can express the vector therein. Suitable mammalian host cells include, but are not limited to, human cells, mouse cells, non-human primate cells (e.g., rhesus monkey cells), human progenitor or stem cells, 293 cells, HeLa cells, D17 cells, MDCK cells, BHK cells, and Cf2Th cells. In certain embodiments, the host cell that contains the expression vector of the present disclosure is a hematopoietic cell, such as a hematopoietic progenitor / stem cell (e.g., CD34-positive hematopoietic progenitor / stem cell), monocyte, macrophage, peripheral blood mononuclear cell, CD4+ T lymphocyte, CD8+ T lymphocyte, or dendritic cell.

[0154] Hematopoietic stem cells (e.g., CD4+ T lymphocytes, CD8+ T lymphocytes, and / or monocytes / macrophages) to be transduced with the vectors of the present disclosure may be allogeneic, autologous, or of matched sibling origin. HSCs, in some embodiments, are CD34 positive and can be isolated from the patient's bone marrow or peripheral blood. The isolated CD34 positive HSCs (and / or other hematopoietic cells described herein) are, in some embodiments, transduced with vectors as described herein.

[0155] In some embodiments, the host cells or transduced host cells are combined with a pharma- ceutically acceptable carrier. In some embodiments, the host cells or transduced host cells are formulated with PLASMA-LYTE A (e.g., a sterile, pyrogen-free, isotonic solution for intravenous administration, where 1 liter of PLASMA-LYTE A has ionic concentrations of 140 mEq sodium, 5 mEq potassium, 3 mEq magnesium, 98 mEq chloride, 27 mEq acetate, and 23 mEq gluconate). In other embodiments, the host cells or transduced host cells are formulated in a solution of PLASMA-LYTE A, i.e., a solution comprising about 8% to about 10% dimethyl sulfoxide (DMSO). In some embodiments, about 2×10 β-lactams are administered per mL of formulation comprising PLASMA-LYTE A and DMSO. 7 There are less than 10 host cells / transduced host cell.

[0156] In some embodiments, the host cell is substantially HPRT-deficient following transduction with a vector of the present disclosure. In some embodiments, the level of HPRT gene expression is reduced by at least 50%. In some embodiments, the level of HPRT gene expression is reduced by at least 55%. In some embodiments, the level of HPRT gene expression is reduced by at least 60%. In some embodiments, the level of HPRT gene expression is reduced by at least 65%. In some embodiments, the level of HPRT gene expression is reduced by at least 70%. In some embodiments, the level of HPRT gene expression is reduced by at least 75%. In some embodiments, the level of HPRT gene expression is reduced by at least 80%. In some embodiments, the level of HPRT gene expression is reduced by at least 85%. In some embodiments, the level of HPRT gene expression is reduced by at least 90%. In some embodiments, the level of HPRT gene expression is reduced by at least 95%. Cells with 20% or less residual HPRT gene expression are considered sensitive to purine analogs, such as 6TG, and can be selected for by purine analogs.

[0157] In some embodiments, the transduction of host cells may be increased by contacting the host cells in vitro, ex vivo, or in vivo with an expression vector of the present disclosure and one or more compounds that increase the efficiency of transduction.For example, in some embodiments, the one or more compounds that increase the efficiency of transduction are compounds that stimulate the prostaglandin EP receptor signaling pathway, i.e., one or more compounds that increase the downstream cell signaling activity of the prostaglandin EP receptor in cells contacted with the one or more compounds, compared to the downstream cell signaling activity of the prostaglandin EP receptor in the absence of the one or more compounds.In some embodiments, the one or more compounds that increase the efficiency of transduction are prostaglandin EP receptor ligands, including but not limited to prostaglandin E2 (PGE2) or its analogs or derivatives. In other embodiments, the one or more compounds that increase the efficiency of transduction include, but are not limited to, RetroNectin (a 63 kD fragment of recombinant human fibronectin fragment, available from Takara), Lentiboost (a membrane-sealing poloxamer, available from Sirion Biotech), protamine sulfate, cyclosporine H, and rapamycin.

[0158] 4. Pharmaceutical Compositions The present disclosure also provides compositions, including pharmaceutical compositions, that include one or more vectors and / or non-viral delivery vehicles (e.g., nanocapsules) disclosed herein. In some embodiments, the pharmaceutical composition comprises an effective amount of at least one of the vectors and / or non-viral delivery vehicles described herein and a pharma- ceutically acceptable carrier. For example, in certain embodiments, the pharmaceutical composition comprises an effective amount of the vector and a pharma- ceutically acceptable carrier. The effective amount can be easily determined by those skilled in the art based on factors such as the subject's size, weight, age, health, sex, ethnicity, and viral titer.

[0159] The phrase "pharmacologically acceptable" or "pharmacologically acceptable" refers to a molecular entity or composition that does not produce adverse, allergic, or other untoward reactions when administered to an animal or human. For example, an expression vector may be formulated with a pharma- ceutically acceptable carrier. As used herein, "pharma-ceutically acceptable carrier" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents acceptable for use in pharmaceutical formulations, and the like, suitable for administration to humans, for example. Methods for formulating compounds with pharmaceutical carriers are known in the art and are described, for example, in Remington's Pharmaceutical Science, (17th ed. Mack Publishing Company, Easton, Pa. 1985); and Goodman & Gillman's: The Pharmacological Basis of Therapeutics (11th Edition, McGraw-Hill Professional, 2005). The disclosures of each of which are incorporated herein by reference in their entirety.

[0160] In some embodiments, the pharmaceutical composition may comprise any of the vectors, nanocapsules, or compositions disclosed herein in any concentration that allows the administered silencing nucleic acid to achieve a concentration ranging from about 0.1 mg / kg to about 1 mg / kg. In some embodiments, the pharmaceutical composition may comprise the expression vector in an amount of about 0.1% to about 99.9% by weight. Suitable pharma- ceutically acceptable carriers for inclusion in any pharmaceutical composition include water, buffered water, saline solutions, such as normal saline or balanced salt solutions, such as Hanks or Earle's balanced solution, glycine, hyaluronic acid, and others. The pharmaceutical composition may be formulated for parenteral administration, such as intravenous, intramuscular, or subcutaneous administration. Pharmaceutical compositions for parenteral administration include pharma- ceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, solvents, diluents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and mixtures thereof, vegetable oils (e.g., olive oil), injectable organic esters (e.g., ethyl oleate).

[0161] Pharmaceutical compositions may be formulated for oral administration.Solid dosage forms for oral administration may include, for example, tablets, dragees, capsules, pills, and granules.In such solid dosage forms, the composition may include at least one pharma-ceutically acceptable carrier, such as sodium citrate and / or dicalcium phosphate, and / or fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, moisturizing agents, such as glycerol, disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, silicates, and sodium carbonate, wetting agents, such as acetyl alcohol, glycerol monostearate, absorbents, such as kaolin and bentonite clay, and / or wetting agents, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. Liquid dosage forms for oral administration may include, for example, pharma- ceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.Liquid dosage forms may include inert diluents, such as water or other solvents, solubilizers and / or emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g. cottonseed oil, corn oil, germ oil, castor oil, olive oil, sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0162] The pharmaceutical composition may contain a penetration enhancer to facilitate its delivery. The penetration enhancer may include fatty acids such as oleic acid, lauric acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, ricinate, monoolein, dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate, mono- and di-glycerides and physiologically acceptable salts thereof. The composition may further include a chelating agent such as ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g. sodium salicylate, 5-methoxysalicylate, homovanillate).

[0163] The pharmaceutical composition may include any of the vectors disclosed herein in encapsulated form. For example, the vector may be encapsulated in a nanocapsule, e.g., a nanocapsule comprising one or more biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). In some embodiments, the vector is encapsulated in a polymeric nanocapsule. In other embodiments, the vector is encapsulated in a biodegradable and / or erodible polymeric nanocapsule. In some embodiments, the polymeric nanocapsule is comprised of two different positively charged monomers, at least one neutral monomer, and a crosslinker. In some embodiments, the nanocapsule further comprises at least one targeting moiety. In some embodiments, the nanocapsule comprises 2-6 targeting moieties. In some embodiments, the targeting moiety is an antibody. In some embodiments, the targeting moiety targets any one of CD117, CD10, CD34, CD38, CD45, CD123, CD127, CD135, CD44, CD47, CD96, CD2, CD4, CD3, and CD9 markers. In some embodiments, the targeting moiety targets any one of the human mesenchymal stem cell CD markers including CD29, CD44, CD90, CD49a-f, CD51, CD73 (SH3), CD105 (SH2), CD106, CD166, and Stro-1 markers. In some embodiments, the targeting moiety targets any one of the human hematopoietic stem cell CD markers including CD34, CD38, CD45RA, CD90, and CD49.

[0164] 5. Treatment Method As an example, a lentiviral vector described herein that includes a nucleic acid sequence encoding WASP may be administered to genetically correct Wiskott-Aldrich syndrome or to alleviate pathology associated with Wiskott-Aldrich syndrome. In some embodiments, a population of host cells transduced with the vector may be administered to correct Wiskott-Aldrich syndrome or to alleviate pathology associated with Wiskott-Aldrich syndrome. This method is believed to be advantageous over currently available therapies because it is available to all patients, especially those who do not have a matched sibling donor. It is further believed that this method has the potential to be applied as a one-time treatment that provides lifelong correction. This method is advantageously free of any immune side effects, and any side effects that do occur may be alleviated by administering a dihydrofolate reductase inhibitor (e.g., MTX or MPA) as noted herein. It is further believed that an effective gene therapy approach will revolutionize the way Wiskott-Aldrich syndrome is treated, ultimately improving patient outcomes.

[0165] In some embodiments, treatment with the vectors or transduced host cells described herein genetically corrects or alleviates one or more of the symptoms associated with Wiskott-Aldrich syndrome, such as those outlined below. In some embodiments, symptoms that can be genetically corrected or alleviated by administering an expression vector or transduced host cells to a patient include, but are not limited to, microthrombocytopenia, eczema, autoimmune disease, and recurrent infections. Eczema skin eruptions are common in patients with classical WAS. In young children, eczema can occur on the face or scalp and resemble "cradle cap". It can have the appearance of severe diaper rash and can be more generalized to involve the arms and legs. In older boys, eczema is often localized to the skin creases near the front of the elbows or behind the knees, behind the ears, or near the wrists. Eczema is so itchy that patients often scratch themselves until they bleed, even while sleeping. These areas where the skin barrier is disrupted become entry points for bacteria that can cause infections of the skin and bloodstream.

[0166] Thrombocytopenia (decreased platelet count) appears to be a common feature in patients with Wiskott-Aldrich syndrome. In addition to the decreased numbers, the platelets themselves are small and dysfunctional, less than half the size of normal platelets. As a result, patients with Wiskott-Aldrich syndrome bleed easily even without trauma. In some embodiments, bleeding into the skin can produce a bluish-red spot called a petechiae the size of a pinhead, or can be larger and resemble a bruise.

[0167] The immune deficiency associated with Wiskott-Aldrich syndrome is thought to result in significant abnormalities in the function of B and T lymphocytes. As a result, infections are common in the classical form of Wiskott-Aldrich syndrome and can involve all kinds of microorganisms. In some embodiments, these infections can include upper and lower respiratory tract infections, such as ear infections, sinus infections, and pneumonia. More severe infections, such as sepsis (bloodstream infection or "toxemia"), meningitis, and severe viral infections, occur less frequently but can occur. Sometimes patients with the classical form of Wiskott-Aldrich syndrome can develop pneumonia caused by a fungus (pneumocystis jiroveci carinii). In some embodiments, the skin can become infected with bacteria, such as Staphylococcus, in areas where the patient has scratched the rash. In some embodiments, a viral skin infection called molluscum contagiosum is also common in Wiskott-Aldrich syndrome. Vaccination to prevent infection is likely to be ineffective in Wiskott-Aldrich syndrome because patients do not mount the normal protective antibody response to the vaccine.

[0168] In some embodiments, the recurrent infection includes, but is not limited to, otitis media, skin abscess, pneumonia, enterocolitis, meningitis, sepsis, and urinary tract infection. In some embodiments, the recurrent infection is a skin infection. In some embodiments, the eczema experienced by patients diagnosed with Wiskott-Aldrich syndrome is classified as refractory eczema.

[0169] By way of example, autoimmune diseases frequently experienced by patients with Wiskott-Aldrich syndrome include hemolytic anemia, vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA nephropathy, Henoch-Schönlein-like purpura, dermatomyositis, recurrent angioedema, and uveitis. In some embodiments, the recurrent infections may be caused by either bacterial, viral, or fungal infections. In some embodiments, treatment with the vectors or transduced host cells described herein genetically corrects or reduces multiple pathologies associated with Wiskott-Aldrich syndrome, such as those outlined below.

[0170] As noted herein, in addition to therapeutic genes, the expression vectors of the present disclosure contain agents (e.g., shRNA against HRPT) designed to inhibit or knock down the expression of HPRT, thus providing an in vivo chemoselection strategy that exploits the essential role that HPRT plays in the metabolism of purine analogs, such as 6TG, into myelotoxic drugs. Since HPRT deficiency does not impair hematopoietic cell development or function, it can be removed from hematopoietic cells used for transplantation. Purine analog conditioning and chemoselection are discussed further herein.

[0171] With respect to treating or alleviating pathologies associated with Wiskott-Aldrich syndrome, treating a subject includes identifying a subject in need of such treatment, transducing HSCs (e.g., autologous HSCs, allogeneic HSCs, sibling matched HSCs) with a lentiviral vector of the present disclosure, and transplanting or administering the transduced HSCs to the subject. In some embodiments, the subject in need of such treatment is a subject afflicted with pathologies associated with Wiskott-Aldrich syndrome.

[0172] In some embodiments, the method further comprises a step of myeloablative conditioning (e.g., using a purine analog, chemotherapy, radiation therapy, treatment with one or more internalizing immunotoxins or antibody-drug conjugates, or any combination thereof) prior to administration of the transduced HSCs. In some embodiments, the method further comprises a step of preconditioning or in vivo chemoselection utilizing a purine analog (e.g., 6TG) following administration of the transduced HSCs. In some embodiments, the method further comprises a step of negative selection utilizing a dihydrofolate reductase inhibitor (e.g., MTX or MPA) in case of adverse reactions (e.g., GvHD).

[0173] 5.1 Conditioning and chemoselection with purine analogues In some embodiments, the method of treatment includes an additional step of (i) conditioning prior to transplantation of the HSCs, and / or (ii) in vivo chemical selection. Either or both steps may utilize a purine analog. In some embodiments, the purine analog is selected from the group consisting of 6-thioguanine ("6TG"), 6-mercaptopurine ("6MP"), or azathioprine ("AZA"). It is believed that the grafted Wiskott-Aldrich syndrome protein-containing HSCs that are deficient in HPRT activity are highly resistant to the cytotoxic effects of the introduced purine analogs. A combined conditioning and chemical selection strategy can achieve efficient and high engraftment of Wiskott-Aldrich syndrome protein-containing HSCs that lack HPRT with low overall toxicity. It is believed that the resulting expression of Wiskott-Aldrich syndrome protein, combined with enhanced engraftment and chemical selection of the genetically modified HSCs, can result in the production of sufficient protein to alleviate the pathology associated with Wiskott-Aldrich syndrome.

[0174] 6TG is a purine analogue with both anticancer and immunosuppressive activity. Thioguanine competes with hypoxanthine and guanine for the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) and is itself converted to 6-thioguanylic acid (TGMP). This nucleotide reaches high intracellular concentrations at therapeutic doses. TGMP interferes in several ways with the synthesis of guanine nucleotides. It inhibits de novo purine biosynthesis by pseudofeedback inhibition of glutamine-5-phosphoribosylpyrophosphate amidotransferase, the first enzyme specific to the de novo pathway for purine ribonucleotides. TGMP also inhibits the conversion of inosinic acid (IMP) to xanthylic acid (XMP) by competing for the enzyme IMP dehydrogenase. TGMP was once thought to be a key inhibitor of ATP:GMP phosphotransferase (guanylate kinase), but recent results show otherwise. Thioguanylate is further converted to the di- and tri-phosphates, thioguanosine diphosphate (TGDP) and thioguanosine triphosphate (TGTP) (and their deoxyribosyl analogs), by the same enzymes that metabolize guanine nucleotides.

[0175] As will be appreciated by those skilled in the art, by considering the inclusion of an agent designed to inhibit the expression of HPRT in the vector of the present disclosure, for example an RNAi agent that knocks down HPRT, the resulting transduced HSCs are HPRT-deficient or substantially HPRT-deficient (e.g., have 20% or less residual HPRT gene expression). Thus, HSCs expressing HPRT, i.e., HPRT wild-type cells, can be selectively depleted by administering one or more doses of 6TG. In some embodiments, 6TG may be administered for myeloablative conditioning of HPRT wild-type recipients and for the in vivo chemoselection process of donor cells. Thus, it is believed that this strategy allows for in vivo selection of genetically modified cells, i.e., in vivo selection of genetically modified cells containing Wiskott-Aldrich syndrome protein.

[0176] In some embodiments, following collection of HSCs from a donor, the HSCs are transduced with a vector according to the present disclosure. The resulting HSCs are HPRT-deficient and express the WAS gene. In parallel, the patient receiving the HSCs is first treated with a myeloablative conditioning step. Following conditioning, the transduced HSCs are transplanted or administered to the patient. The HSCs containing the WAS gene may then be selected in vivo using 6TG as discussed herein.

[0177] Myeloablative conditioning may be achieved using high dose conditioning radiation, chemotherapy, and / or treatment with a purine analog (e.g., 6TG). In some embodiments, the HSCs are administered about 24 to about 96 hours after treatment with the conditioning regimen. In other embodiments, the patient is treated with the HSC graft about 24 to about 72 hours after treatment with the conditioning regimen. In yet other embodiments, the patient is treated with the HSC graft about 24 to about 48 hours after treatment with the conditioning regimen. In some embodiments, the HSC graft is administered in an amount of about 2×10 6 cells / kg ~ approx. 15×10 6 cells / kg of patient weight. In some embodiments, the HSC graft comprises 6×10 6 Aim for greater than 2 x 10 cells / kg, with a minimum of 2 x 10 6cells / kg. In some embodiments, at least 10% of the administered cells are transduced by a lentiviral vector described herein. In some embodiments, at least 20% of the administered cells are transduced by a lentiviral vector described herein. In some embodiments, at least 30% of the administered cells are transduced by a lentiviral vector described herein. In some embodiments, at least 40% of the administered cells are transduced by a lentiviral vector described herein. In some embodiments, at least 50% of the administered cells are transduced by a lentiviral vector described herein.

[0178] In some embodiments, the transgene-containing HPRT-deficient HSCs are selected for in vivo using a low-dose schedule of a purine analog, such as 6TG, which is believed to have minimal adverse effects outside of hematopoietic tissues. In some embodiments, a dosage of a purine analog, such as 6TG, for in vivo chemoselection ranging from about 0.2 mg / kg / day to about 0.6 mg / kg / day is provided to the patient following introduction of the HSCs into the patient. In some embodiments, the dosage ranges from about 0.3 mg / kg / day to about 1 mg / kg / day. In some embodiments, the dosage is up to about 2 mg / kg / day.

[0179] In some embodiments, the amount of 6TG administered per dose is based on the determination of the patient's HPRT enzyme activity. Those skilled in the art will recognize that a dose that exhibits a high level of HPRT enzyme activity may be provided with a dose that has a small amount of purine analogs such as 6TG. The higher the level of HPRT, the greater the conversion of purine analogs such as 6TG to toxic metabolites. Therefore, to achieve the same goal, a lower dose must be administered.

[0180] Prior to formulating 6TG conditioning, TPMT genotype and / or TPMT enzyme activity may be measured to identify individuals with low or no TPMT enzyme activity. Thus, in other embodiments, the amount of 6TG administered is based on thiopurine S-methyltransferase (TPMT) levels or TPMT genotype.

[0181] In some embodiments, a dosage of a purine analog such as 6TG for in vivo chemoselection is administered to a patient 1-3 times per week on a cyclic schedule selected from the group consisting of: (i) weekly; (ii) every other week; (iii) 1 week on treatment followed by 2, 3, or 4 weeks off; (iv) 2 weeks on treatment followed by 1, 2, 3, or 4 weeks off; (v) 3 weeks on treatment followed by 1, 2, 3, 4, or 5 weeks off; (vi) 4 weeks on treatment followed by 1, 2, 3, 4, or 5 weeks off; (vii) 5 weeks on treatment followed by 1, 2, 3, 4, or 5 weeks off; and (viii) monthly.

[0182] In some embodiments, about 3 to about 10 dosages of a purine analog such as 6TG are administered to a patient over a dosing period ranging from 1 week to about 4 weeks, in some embodiments, 4 or 5 dosages of 6TG are administered to a patient over a period of 14 days.

[0183] 5.2 Negative selection with dihydrofolate reductase inhibitors Furthermore, HPRT-deficient cells can be negatively selected by using dihydrofolate reductase inhibitors (e.g., MTX), which inhibit the enzyme dihydrofolate reductase (DHFR) in the de novo synthesis pathway of purines. This was developed as a safety procedure to remove genetically modified HSCs if unexpected adverse effects are observed. That is, patients can be treated with dihydrofolate reductase inhibitors (e.g., MTX or MPA) if any adverse side reactions occur. Adverse side reactions include, for example, abnormal blood counts / clonal growth or cytokine storms indicating insertion mutations in certain clones of cells.

[0184] Dihydrofolate reductase inhibitors (e.g., MTX or MPA) are believed to competitively inhibit dihydrofolate reductase (DHFR), an enzyme that contributes to the synthesis of tetrahydrofolate (THF). DHFR catalyzes the conversion of dihydrofolate to active tetrahydrofolate. Folic acid is required for the de novo synthesis of thymidine, a nucleoside required for DNA synthesis. Folate is also essential for the biosynthesis of purine and pyrimidine bases, and therefore synthesis is inhibited. Thus, dihydrofolate reductase inhibitors (e.g., MTX or MPA) inhibit the synthesis of DNA, RNA, thymidylates, and proteins. MTX or MPA blocks the de novo pathway by inhibiting DHFR. HPRT- / - cells do not have a functional salvage or de novo pathway, which results in no purine synthesis and therefore cell death. However, HPRT wild-type cells have a functional salvage pathway, which results in purine synthesis and cell survival.

[0185] In view of the sensitivity of the modified HSCs produced according to the present disclosure, a dihydrofolate reductase inhibitor (e.g., MTX or MPA) may be used to selectively eliminate HPRT-deficient cells. In some embodiments, the dihydrofolate reductase inhibitor (e.g., MTX or MPA) is administered as a single dose. In some embodiments, multiple doses of the dihydrofolate reductase inhibitor are administered.

[0186] In some embodiments, the amount of MTX administered is about 2 mg / m 2 / injection~about 100mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 90mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 80mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 70mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 60mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 50mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 40mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 30mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 20mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m 2 / injection~about 10mg / m 2 In some embodiments, the amount of MTX administered ranges from about 2 mg / m2 / injection~approx.8mg / m 2 In another embodiment, the amount of MTX administered is about 2.5 mg / m 2 / injection~about 7.5mg / m 2 In yet other embodiments, the amount of MTX administered ranges from about 5 mg / m 2 In a further embodiment, the amount of MTX administered is about 7.5 mg / m 2 / Injection.

[0187] In some embodiments, 2-6 infusions are administered, each of which may include the same dosage or a different dosage (e.g., increasing dosages, decreasing dosages, etc.) In some embodiments, administration may be on a weekly basis or on a bimonthly basis.

[0188] In some embodiments, MPA is administered in an amount of about 500 mg to about 1500 mg per day. In some embodiments, the dose of MPA is administered in a single bolus. In some embodiments, the dose of MPA is divided into multiple individual doses totaling about 500 mg to about 1500 mg per day.

[0189] In some embodiments, analogs or derivatives of MTX or MPA may be substituted for MTX or MPA. Derivatives of MTX are described in U.S. Patent No. 5,958,928 and PCT Publication No. WO / 2007 / 098089, the disclosures of which are incorporated herein by reference in their entireties. In some embodiments, the antifolates may be selected from the group consisting of ribavarin (an inhibitor of IMPDH), VX-497 (an inhibitor of IMPDH) (see Jain J, VX-497: a novel, selective IMPDH inhibitor and immunosuppressive agent, (2001), J Pharm Sci., 90(5):625-37)), lometrexol (DDATHF, LY249543) (an inhibitor of GAR and / or AICAR), thiophene analogs (LY254155) (an inhibitor of GAR and / or AICAR), furan analogs (LY222306) (an inhibitor of GAR and / or AICAR) (Habeck et al., A Novel Class of Monoglutamated Antifolates Exhibits Tight-binding Inhibition of Human Glycinamide Ribonucleotide Formyltransferase and Potent Activity against Solid Tumors, (1994), Cancer Research, 54, 1021-2026), DACTHF (an inhibitor of GAR and / or AICAR) (Cheng et. al. Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway, (2005) Bioorg Med Chem., 13(10):3577-85), AG2034 (an inhibitor of GAR and / or AICAR) (Boritzki et. al.AG2034: a novel inhibitor of glycinamide ribonucleotide formyltransferase, (1996), Invest New Drugs., 14(3):295-303), LY309887 (inhibitor of GAR and / or AICAR) ((2S)-2-[[5-[2-[(6R)-2-amino-4-oxo-5,6,7,8-tetrahydro-1H-pyrido[2,3-d]pyrimidin-6-yl]ethyl]thiophene-2-carbonyl]amino]pentanedioic acid), Alimta (LY231514) (inhibitor of GAR and AICAR) (Shih et. al. LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes, (1997) Cancer Research, 57(6):1116-23), dmAMT (an inhibitor of GAR and / or AICAR), AG2009 (an inhibitor of GAR and / or AICAR), forodesine (Immucillin H, BCX-1777, trade names Mundesine and Fodosine) (an inhibitor of purine nucleoside phosphorylase [PNP]) (see Kicska et. al., Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes, (2001) Proceedings Nat'l Acad. Sci. USA, 98 (8) 4593-4598), and Immucillin-G (an inhibitor of purine nucleoside phosphorylase [PNP]) may be used in place of MTX or MPA.

[0190] 6. Combination Therapy Another aspect of the present disclosure is a combination therapy in which antibacterial, antifungal, and / or antiviral active pharmaceutical ingredients (depending of course on the particular infection presented) are administered before, during, or after administration or transplantation of transduced HSCs (above) to a patient in need of such treatment to treat Wiskott-Aldrich syndrome. In some embodiments, patients suffering from Wiskott-Aldrich syndrome and having severe thrombocytopenia may be treated with high doses of intravenous immunoglobulin (2 gm / kg / day) and / or corticosteroids (2 mg / kg / day) before, during, or after administration or transplantation of transduced HSCs (above) to a patient in need of such treatment. Alternatively, allogeneic transplantation of stem cells from healthy donors may be performed before or after treatment with the expression vector or transduced stem cells of the present disclosure.

[0191] 7. Lentiviral Vectors Useful for the Treatment of Sickle Cell Disease β-Hemoglobinopathies, including beta-thalassemia and sickle cell disease (SCD), are a heterogeneous group of generally inherited disorders that affect the function or levels of hemoglobin. SCD and β-thalassemia major are the most common monogenic disorders affecting approximately 400,000 newborns worldwide each year. Clinical signs typically appear in the first few months of life during the switch from fetal hemoglobin (HbF) to adult β-globin (HbA) and can be severe with significant morbidity and mortality. Allogeneic bone marrow transplantation is curative, but only in patients with a suitably matched donor. Autologous gene therapy, utilizing the patient's own cells, is an attractive treatment option.

[0192] β-thalassemia is an inherited blood disorder characterized by reduced levels of functional hemoglobin. β-thalassemia is caused by mutations in the hemoglobin subunit beta (hereinafter "HBB gene"), which is believed to be inherited in an autosomal recessive manner. β-thalassemia major, defined as clinically transfusion-dependent, is caused by reduced or absent synthesis of the beta chain of hemoglobin. The severity of the disease depends on the nature of the mutation, with outcomes ranging from severe anemia to clinically asymptomatic individuals.

[0193] Hundreds of different mutations have been described that affect the levels of beta globin through effects on a wide range of processes including transcription, mRNA splicing / processing, RNA stability, translation, and globin peptide stability. It is believed that low beta globin content allows excess alpha globin chains to precipitate in erythroid precursor cells. It is further believed that alpha globin aggregates damage cell membranes, leading to premature death of erythroid precursor cells. The resulting reduced effectiveness of erythropoiesis in patients can be severe enough to require frequent blood transfusions.

[0194] Sickle cell anemia (SCA) results from a single point mutation in exon 1 of the beta globin gene that results in the replacement of glutamic acid at position 6 with valine in hemoglobin S (HbS), a mutated sickle form of hemoglobin. In addition to homozygous hemoglobin S ("HbSS"), there are other genetic types that can result in SCD. Classical SCA is often defined as homozygous HbSS, but also includes homozygous hemoglobin C ("HbSC") and HbS / β 0 are common genotypes with essentially the same disease manifestations. HbS polymerizes upon deoxygenation resulting in sickle-shaped red blood cells ("RBCs"), which occlude the microvasculature. SCD is clinically characterized by variable degrees of anemia and episodic vaso-occlusive crises, which lead to multi-organ damage and premature death. In addition to sickling, there is a state of excessive hemolysis and chronic inflammation.

[0195] Patients with SCD are responsible for approximately 75,000 hospitalizations per year in the United States, resulting in an estimated annual cost of $475 million. Globally, SCD is the second most prevalent monogenic disorder after thalassemia, with over 200,000 children born with the disease each year in Africa. Currently available medical management options for SCD include supportive management of vaso-occlusive crises, chronic transfusions to avoid or prevent recurrence of serious complications of SCD such as stroke or acute chest syndrome, and induction of fetal hemoglobin (HbF) with hydroxyurea. Transplantation of matched allogeneic hematopoietic stem cells (HSCs) is considered curative but is limited by the availability of matched related donors and has the potential for serious complications. During fetal life, the gamma globin gene (alpha, which results in HbF) is expressed in the alpha-gamma globin gene (alpha, which results in HbF) and is expressed in the alpha-gamma globin gene (alpha, which results in HbF). 2 gamma 2 ) is the major gene expressed by the beta globin locus, and beta globin gene expression is repressed. However, after birth, expression of the fetal gamma globin gene decreases to negligible levels, with a concomitant increase in beta globin expression. During adulthood, the fetal gamma globin transcript is highly silenced; that is, gene expression is controlled to prevent or reduce the expression of gamma globin. This change in expression is accompanied by a decrease in HbF and a corresponding increase in HbA (alpha 2 beta 2 Gamma globin is known to have anti-sickling properties, therefore addition of this gene is being considered for gene therapy.

[0196] Hemoglobinopathies, particularly SCD, are prime targets for gene therapy for a variety of reasons. The high prevalence of SCD, significant morbidity and mortality, and the resulting high costs of lifelong palliative medical care predict that a curative therapy could greatly improve patient outcomes and significantly reduce associated medical costs. Autologous CD34 +Gene therapy for β-hemoglobinopathies by ex vivo lentiviral transfer of therapeutic β-globin genes into hematopoietic stem / progenitor cells (HSPCs) was evaluated in human clinical trials. Myeloablative therapy-based autologous HSC transplantation resulted in transfusion independence or transfusion reduction in β-thalassemia patients for more than 12 months after gene therapy.

[0197] Although clinical trials of gene therapy using viral vectors, such as lentiviral vectors, for the treatment of SCD and other β-hemoglobinopathies have demonstrated that this approach can be therapeutically effective, there is a continuing need for the development of vectors with improved efficacy and improved safety profiles.

[0198] Thus, in another aspect, lentiviral vectors are provided herein that contain a modified globin transgene with inactivation of SD1. As will be appreciated, such globin transgenes contain intron 2 from β-globin. In certain embodiments, lentiviral vectors are provided that contain a modified γ-globin transgene with inactivation of SD1. Also provided herein are lentiviral vectors that contain a modified HS4-400 insulator with inactivation of one or both of SA2 and SA3. Also provided are vectors that do not contain the HS4-400 insulator. Thus, the lentiviral vectors of the present disclosure may have reduced risk of alternative splicing associated therewith when introduced into cells, such as hematopoietic stem cells.

[0199] This aspect of the disclosure is based, at least in part, on the identification of a cryptic splice donor site in intron 2 (derived from β-globin) in the γ-globin transgene present in a therapeutic lentiviral vector. This cryptic splice donor site, also referred to as SDI, is in the positive strand of the vector in β-globin intron 2 in the γ-globin transgene. Because the γ-globin transgene is present in the vector in the reverse orientation, SD1 is in the complementary strand of the γ-globin transgene, i.e., in the reverse complement of the γ-globin transgene. SD1 is located at nucleotides 933-934 of SEQ ID NO:122 (where SEQ ID NO:122 is the reverse complement of the γ-globin transgene shown in SEQ ID NO:121, i.e. splicing can occur between G at position 933 and G at position 934) and at nucleotides 150-151 of SEQ ID NO:119 (where SEQ ID NO:119 is the reverse complement of the γ-globin transgene shown in SEQ ID NO:118, i.e. splicing can occur between G at position 150 and G at position 151). When referring to β-globin intron 2, the cryptic splice donor site is located at nucleotides 20-21 of SEQ ID NO:88, where SEQ ID NO:88 is the reverse complement of β-globin intron 2 shown in SEQ ID NO:87, i.e. splicing can occur between G at position 20 and G at position 21 of SEQ ID NO:88.

[0200] That is, in one aspect, 1. A lentiviral vector comprising a first promoter operably linked to a first nucleic acid sequence comprising a modified γ-globin transgene containing β-globin intron 2, the modified γ-globin transgene comprises a mutation relative to the unmodified γ-globin transgene, the mutation inactivating splice donor site 1 (SD1) present in the unmodified γ-globin transgene; SD1 is present in the unmodified γ-globin transgene at nucleotide positions 933-934, numbered relative to SEQ ID NO:122, where SEQ ID NO:122 is the reverse complement of the unmodified γ-globin transgene shown in SEQ ID NO:121; SD1 is present in the unmodified γ-globin transgene at nucleotide positions 150-151, numbered relative to SEQ ID NO: 119, where SEQ ID NO: 119 is the reverse complement of the unmodified γ-globin transgene shown in SEQ ID NO: 118; and / or SD1 has the sequence AAGATAAGA ∧ GTATGAACAT (SEQ ID NO:96), ∧ represents a splice site.

[0201] In some embodiments, the mutation is an A at position 932, a G at position 933, a G at position 934, and / or a T at position 935, numbered relative to SEQ ID NO: 122. In particular examples, the mutation is a nucleotide substitution, e.g., a G to A mutation at position 934, numbered relative to SEQ ID NO: 122. In some examples, the modified γ-globin transgene comprises the sequence set forth in SEQ ID NO:91.

[0202] In some embodiments, the lentiviral vector further comprises a modified HS4-400 insulator.

[0203] In one example, the modified HS4-400 insulator, when present in a vector, comprises a splice acceptor site 2 (SA2) that is inactivated relative to the unmodified HS4-400 insulator, wherein SA2 is present in the unmodified HS4-400 insulator at nucleotide positions 190-191, numbered relative to SEQ ID NO: 90, where SEQ ID NO: 90 is the reverse complement of the unmodified HS4-400 insulator set forth in SEQ ID NO: 89, and / or SA2 is represented by the sequence ATCCCCCCAG ∧ GTGTCTGCAG (SEQ ID NO: 61), ∧represents a splice position. In some examples, the modified HS4-400 insulator comprises a mutation that inactivates SA2 relative to the unmodified HS4-400 insulator, such as an A at position 189 (e.g., an A to T mutation), a G at position 190, a G at position 191, and / or a T at position 192, numbered relative to SEQ ID NO:90. In some examples, the reverse complement sequence of the modified HS4-400 insulator comprises the sequence set forth in SEQ ID NO:93.

[0204] In another example, the modified HS4-400 insulator, when present in a lentiviral vector, comprises a mutation that inactivates splice acceptor site 3 (SA3) relative to the unmodified HS4-400 insulator, wherein SA3 is present in the unmodified HS4-400 insulator at nucleotide positions 200-201, numbered relative to SEQ ID NO: 90, and / or SA3 is represented by the sequence GTGTCTGCAG ∧ GCTCAAAGAG (SEQ ID NO: 62), ∧ represents a splice position. In some examples, the mutation is an A at position 199 (e.g., an A to T mutation), a G at position 200, a G at position 201, and / or a C at position 202, numbered relative to SEQ ID NO: 90. In certain embodiments, the reverse complement sequence of the modified HS4-400 insulator comprises a sequence set forth in any one of SEQ ID NOs: 94-95.

[0205] In some examples, the modified HS4-400 insulator is in a reverse orientation within the lentiviral vector. In certain embodiments, the first nucleic acid is in a reverse orientation and the modified HS4-400 insulator is in a reverse orientation within the lentiviral vector.

[0206] In another example, the modified HS4-400 is in a forward orientation within the lentiviral vector.

[0207] In another embodiment, a lentiviral vector is provided comprising a first promoter operably linked to a first nucleic acid sequence comprising a modified γ-globin transgene comprising a β-globin intron 2, and a modified HS4-400 insulator, the modified HS4-400 insulator, when present in the vector, contains an inactivated splice acceptor site 2 (SA2) relative to the unmodified HS4-400 insulator; SA2 is present at nucleotide positions 190-191 in the unmodified HS4-400 insulator, as numbered relative to SEQ ID NO: 90, and SEQ ID NO: 90 is the reverse complement of the unmodified HS4-400 insulator set forth in SEQ ID NO: 89; and / or SA2 has the sequence ATCCCCCCAG ∧ GTGTCTGCAG (SEQ ID NO: 61), ∧ represents a splice site.

[0208] In one embodiment, the modified HS4-400 insulator comprises a mutation relative to the unmodified HS4-400 insulator that inactivates SA2, such as an A at position 189 (e.g., an A to T mutation), a G at position 190, a G at position 191, and / or a T at position 192, numbered relative to SEQ ID NO:90. In a particular example, the reverse complement sequence of the modified HS4-400 insulator comprises the sequence set forth in SEQ ID NO:93.

[0209] In a further embodiment, the modified HS4-400 insulator further comprises a mutation that inactivates splice acceptor site 3 (SA3) relative to the unmodified HS4-400 insulator, where SA3 is present in the unmodified HS4-400 insulator at nucleotide positions 200-201, numbered relative to SEQ ID NO: 90, and / or SA3 is the sequence GTGTCTGCAG ∧ GCTCAAAGAG (SEQ ID NO: 62), ∧represents a splice position. In some examples, the mutation is an A at position 199 (e.g., an A to T mutation), a G at position 200, a G at position 201, and / or a C at position 202, numbered relative to SEQ ID NO:90. In certain embodiments, the reverse complement sequence of the modified HS4-400 insulator comprises the sequence set forth in SEQ ID NO:94.

[0210] In one example of this embodiment, the modified HS4-400 insulator is in a reverse orientation within the lentiviral vector, hi another example, the modified HS4-400 insulator is in a forward orientation within the lentiviral vector, thereby inactivating SA2.

[0211] In another embodiment, a lentiviral vector is provided comprising a first promoter operably linked to a first nucleic acid sequence comprising a modified γ-globin transgene comprising a β-globin intron 2, and a modified HS4-400 insulator, the modified HS4-400 insulator, when present in the vector, contains an inactivated splice acceptor site 3 (SA3) relative to the unmodified HS4-400 insulator; SA3 is present at nucleotide positions 200-201 in the unmodified HS4-400 insulator, as numbered relative to SEQ ID NO: 90, and SEQ ID NO: 90 is the reverse complement of the unmodified HS4-400 insulator set forth in SEQ ID NO: 89; and / or SA3 has the sequence GTGTCTGCAG ∧ GCTCAAAGAG (SEQ ID NO: 62), ∧ represents a splice site.

[0212] In one embodiment, the modified HS4-400 insulator comprises a mutation relative to the unmodified HS4-400 insulator that inactivates SA3, such as an A at position 199 (e.g., an A to T mutation), a G at position 200, a G at position 201, and / or a C at position 202, numbered relative to SEQ ID NO:90. In some embodiments, the modified HS4-400 insulator comprises the sequence set forth in SEQ ID NO:95.

[0213] In some examples, the modified HS4-400 insulator further comprises a mutation that inactivates splice acceptor site 2 (SA2) relative to the unmodified HS4-400 insulator, where SA2 is present in the unmodified HS4-400 insulator at nucleotide positions 190-191 as numbered relative to SEQ ID NO:90, which is the reverse complement of the unmodified HS4-400 insulator set forth in SEQ ID NO:89, and / or SA2 is present at the sequence ATCCCCCCAG ∧ GTGTCTGCAG (SEQ ID NO: 61), ∧ represents a splice position. In some examples, the mutation is an A at position 189 (e.g., an A to T mutation), a G at position 190, a G at position 191, and / or a T at position 192, numbered relative to SEQ ID NO:90. In certain embodiments, the modified HS4-400 insulator comprises the sequence set forth in SEQ ID NO:94.

[0214] In some examples of this embodiment, the modified HS4-400 insulator is in a reverse orientation within the lentiviral vector, hi other examples, the modified HS4-400 insulator is in a forward orientation within the lentiviral vector, thereby inactivating SA3.

[0215] In some embodiments, the unmodified γ-globin transgene comprises a sequence set forth in SEQ ID NO: 85 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In one example, the modified γ-globin transgene encodes a γ-globin comprising an amino acid sequence set forth in SEQ ID NO: 103 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0216] In some examples, the transgene only contains the γ-globin coding sequence (e.g., as set forth in SEQ ID NO: 101 or 102). In particular examples, the γ-globin transgene contains exons and introns and is associated with other non-coding elements. In one example, the γ-globin transgene contains γ-globin exon 1 (or HBG exon 1, e.g., as set forth in SEQ ID NO: 98), γ-globin exon 2 (or HBG exon 2, e.g., as set forth in SEQ ID NO: 99), and γ-globin exon 3 (or HBG exon 3, e.g., as set forth in SEQ ID NO: 100). The introns may contain γ-globin intron 1 (or HBG intron 1), and β-globin intron 2 (HBB intron 2, e.g., the truncated HBB intron 2, as set forth in SEQ ID NO: 87). In certain embodiments, the transgene comprises the sequence set forth in SEQ ID NO: 118 (i.e., HBG exon 1, HBG intron 1, HBG exon 2, HBB truncated intron 2, and HBG exon 3), or SEQ ID NO: 121 (i.e., HBG exon 1, HBG intron 1, HBG exon 2, HBB truncated intron 2, HBG exon 3, and the 3'UTR / polyA signal). The transgene may be associated with other non-coding elements, such as the β-globin locus control region (LCR) (e.g., set forth in SEQ ID NO: 105).

[0217] As determined herein, a γ-globin transgene containing β-globin intron 2 may have a cryptic splice donor site (SD1) when in a lentiviral vector. This splice donor site was identified in the γ-globin transgene shown in SEQ ID NOs: 118 and 121 when the transgene was in the reverse orientation in the lentiviral vector, whereby SD1 was in β-globin intron 2 in the positive strand of the vector. That is, SD1 was in the reverse complement of SEQ ID NOs: 118 and 121. These reverse complements are shown as SEQ ID NOs: 120 and 122.

[0218] SD1 is present at positions 933-934 of SEQ ID NO:121 (i.e., splicing can occur between G at position 933 and G at position 934) and at positions 150-151 of SEQ ID NO:119 (i.e., splicing can occur between G at position 150 and G at position 151), and at corresponding positions in other γ-globin transgenes that contain β-globin intron 2. SD1 has the sequence AAGATAAGA ∧ GTATGAACAT (SEQ ID NO: 96) ∧ represents a splice position), or as comprising the sequence of nucleotides at positions 924 to 943 of the complementary strand of a γ-globin transgene containing a β-globin intron 2 in numbering relative to SEQ ID NO: 121, or as comprising the sequence of nucleotides at positions 141 to 160 of the complementary strand of a γ-globin transgene containing a β-globin intron 2 in numbering relative to SEQ ID NO: 119. When considering the β-globin intron 2, SD1 is located at positions 20 to 21 of SEQ ID NO: 88 (i.e. splicing occurs between G at position 20 and G at position 21) and corresponding positions of other β-globin introns 2. Thus, SD1 can also be defined as comprising the sequence of nucleotides at positions 11 to 30 of the complementary strand of a β-globin intron 2 in numbering relative to SEQ ID NO: 88.

[0219] That is, in some embodiments, a lentiviral vector of the present disclosure comprises a first promoter operably linked to a first nucleic acid sequence comprising a modified γ-globin transgene comprising β-globin intron 2, the modified γ-globin transgene comprising a mutation relative to an unmodified γ-globin transgene, the mutation inactivating SD1. The lentiviral vector comprising the modified γ-globin transgene, when transduced into a cell, may exhibit reduced splicing at positions 924-943 or 150-151, numbered relative to SEQ ID NO: 122 or 119, respectively, compared to the splicing occurring at positions 924-943 or 150-151 by a lentiviral vector comprising an unmodified γ-globin transgene. In some examples, splicing is reduced by at least or about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0220] Unmodified γ-globin transgenes include transgenes that contain an active SD1 when present in a lentiviral vector, i.e., transgenes that contain a sequence and orientation within the lentiviral vector that can facilitate splicing at SD1. Exemplary unmodified γ-globin transgenes include transgenes that encode γ-globin and contain the sequences set forth in SEQ ID NOs: 118 and 121 (with the reverse complements set forth in SEQ ID NOs: 119 and 122, respectively) and sequences having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (provided that the SD1 site is still present, e.g., the reverse complement of the γ-globin transgene contains the sequence AAGATAAGAGGGTATGAACAT (SEQ ID NO: 96)).

[0221] In certain examples, the modified γ-globin transgene comprises a mutation (e.g., a deletion, insertion, or substitution of a nucleotide) relative to the unmodified γ-globin transgene, which inactivates SD1 present in the unmodified γ-globin transgene (or reduces splicing at positions 924-943 of the reverse complement of the modified γ-globin transgene compared to the splicing occurring at positions 924-943 of the reverse complement of the unmodified γ-globin transgene, numbered relative to SEQ ID NO: 122). The mutation may be any mutation that inactivates or destroys SD1. In some examples, the mutation is a deletion or substitution of any nucleotide in the SD1 sequence, or an insertion of a nucleotide (e.g., the sequence AAGATAAGAGGGTATGAACAT (SEQ ID NO: 96)) into the SD1 sequence. In particular examples, the mutation is a mutation (e.g., a deletion or substitution) of A at position 932, G at position 933, G at position 934, and / or T at position 935, numbered relative to SEQ ID NO:122 (e.g., a mutation of A at position 149, G at position 150, G at position 151, and / or T at position 152, numbered relative to SEQ ID NO:119). For example, a modified γ-globin transgene may comprise an A to T, A to C, or A to G mutation at position 932, a G to C, G to A, or G to T mutation at position 933, a G to C, a G to T or G to A mutation at position 934, and / or a T to A, T to C, or T to G mutation at position 935, numbered relative to SEQ ID NO:122 (i.e., an A to T, A to C, or A to G mutation at position 149, a G to C, G to A, or G to T mutation at position 150, a G to C, G to T, or G to A mutation at position 151, and / or a T to A, T to C, or T to G mutation at position 152, numbered relative to SEQ ID NO:119). In other examples, the mutation includes an insertion of nucleotides after positions 932, 933, and / or 934, numbered relative to SEQ ID NO:122 (i.e., an insertion of nucleotides after positions 149, 150, and / or 151, numbered relative to SEQ ID NO:119).In some instances, the modified γ-globin transgene contains two or more such mutations.

[0222] In one example, the modified γ-globin transgene comprises a G to A mutation at position 934 in the reverse complement sequence as numbered relative to SEQ ID NO: 122 (i.e., an A at position 934 in the numbering relative to SEQ ID NO: 122). That is, in some examples, the modified γ-globin transgene comprises a G to A mutation at position 151 in the reverse complement sequence as numbered relative to SEQ ID NO: 119 (i.e., an A at position 151 in the numbering relative to SEQ ID NO: 119). In certain embodiments, the reverse complement sequence of the γ-globin transgene comprises the sequence set forth in SEQ ID NO: 123 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of an A at position 934 in the numbering relative to SEQ ID NO: 122). In other embodiments, the reverse complement of the gamma-globin transgene comprises the sequence set forth in SEQ ID NO:120 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (assuming the presence of an A at position 151 in the numbering relative to SEQ ID NO:119).

[0223] In some examples, the modified γ-globin transgene described herein having an inactivating mutation in SD1 is in a reverse orientation within the lentiviral vector.

[0224] In a further embodiment, the first promoter is a β-globin promoter, for example a promoter comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 115-117, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0225] In one embodiment, the first promoter is a β-globin promoter and is operably linked to a first nucleic acid comprising a γ-globin transgene.

[0226] In some embodiments, the lentiviral vector further comprises a second promoter operably linked to a second nucleic acid sequence encoding a nucleic acid that inhibits expression of HPRT. In some examples, the nucleic acid that inhibits expression of HPRT is an shRNA, such as an shRNA comprising a hairpin loop sequence as set forth in SEQ ID NO: 66 and / or comprising a sequence as set forth in any one of SEQ ID NOs: 67-68, or comprising at least 95% sequence identity thereto. In one example, the second promoter comprises a Pol III promoter or a Pol II promoter, such as a promoter comprising 7sk (e.g., a promoter comprising a nucleic acid sequence as set forth in any one of SEQ ID NOs: 69-71, or a sequence having at least 95% sequence identity thereto). In certain embodiments, the second promoter and the operably linked second nucleic acid sequence are in a forward orientation and downstream of the first promoter and the operably linked first nucleic acid, which are in a reverse orientation.

[0227] In some examples, the lentiviral vector further comprises a polyadenylation signal in the 3'LTR of the vector. The polyadenylation signal may be, for example, a rabbit β-globin polyadenylation signal comprising the nucleic acid sequence set forth in SEQ ID NO: 103 or a nucleic acid sequence having at least 95% sequence identity thereto.

[0228] In another aspect of the disclosure, downstream thereof from 5' to 3', a central polypurine tract (cPPT), a REV response element (RRE) (e.g., a sequence shown in SEQ ID NO: 106 or an element comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto), a γ-globin transgene (e.g., the modified γ-globin transgene described herein having inactivated SD1, e.g., a transgene comprising a sequence shown in SEQ ID NO: 123 or a complementary strand comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, the sequence of which comprises an A at position 934 numbered relative to SEQ ID NO: 122, or a transgene comprising a sequence shown in SEQ ID NO: 120 or a complementary strand comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, the sequence of which comprises an A at position 151 numbered relative to SEQ ID NO: 119) operably linked to a β-globin promoter (e.g., a promoter shown by any one of SEQ ID NOs: 115 - 117 or a promoter comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto), a γ-globin expression cassette comprising a β-globin LCR (e.g., a LCR shown in SEQ ID NO: 105 or a LCR comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto), a 7sk-sh734 expression cassette comprising a 7sk promoter operably linked to a nucleic acid encoding sh734, a 5' LTR (comprising a 7tetO promoter / operator, R, and U5 (e.g., schematically shown in FIG. 27)), and an HS4-400 insulator (e.g., the modified HS4-400 insulator described herein having inactivated SA2 and / or SA3,For example, an insulator comprising a complementary strand comprising a sequence as set forth in SEQ ID NO:93 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, wherein the sequence comprises a T at position 189 in the numbering relative to SEQ ID NO:93; an insulator comprising a complementary strand comprising a sequence as set forth in SEQ ID NO:95 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, wherein the sequence comprises a T at position 199 in the numbering relative to SEQ ID NO:95; Provided is a lentiviral vector comprising a nucleic acid vector (e.g., a plasmid) comprising a 3' LTR comprising an insulator comprising a complementary strand comprising a sequence having 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, wherein the sequence comprises a T at position 189 and a T at position 199, numbered relative to SEQ ID NO:94), R, and a β-globin poly(A) signal (e.g., comprising a sequence as set forth in SEQ ID NO:104 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto) and a lentiviral virion (or viral particle). In some examples, the γ-globin expression cassette is an expression cassette in which the complementary strand comprises a sequence set forth in SEQ ID NO:91 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, and the sequence comprises an A at position 934 numbered relative to SEQ ID NO:86. In these vectors, typically the γ-globin expression cassette is in a reverse orientation and the 7sk-sh734 expression cassette is in a forward orientation.

[0229] In some embodiments, the lentiviral vector is a plasmid. In other embodiments, the lentiviral vector is a viral particle.

[0230] In one embodiment, the vector is a plasmid and comprises a sequence as set forth in SEQ ID NO: 109 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, provided that the vector comprises an A at position 934 of the gamma-globin expression cassette as numbered relative to SEQ ID NO: 91.

[0231] In another embodiment, the vector is a plasmid and comprises a sequence as set forth in SEQ ID NO:110, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, provided that the vector comprises a T at position 189 and a T at position 199 of the HS4-400 insulator, numbered relative to SEQ ID NO:94.

[0232] In another embodiment, the vector is a plasmid and comprises a sequence as set forth in SEQ ID NO:111 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, provided that the vector comprises an A at position 934 of the γ-globin expression cassette when numbered relative to SEQ ID NO:91, and provided that the vector comprises a T at position 189 and a T at position 199 of the HS4-400 insulator when numbered relative to SEQ ID NO:94.

[0233] In one embodiment, the vector is a plasmid and comprises a sequence as set forth in SEQ ID NO:112 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, provided that the vector comprises an A at position 934 of the gamma-globin expression cassette as numbered relative to SEQ ID NO:91.

[0234] In another embodiment, the vector is a plasmid and comprises a sequence as set forth in SEQ ID NO:113, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, provided that the vector comprises a T at position 189 of the HS4-400 insulator, numbered relative to SEQ ID NO:93.

[0235] In another embodiment, the vector is a plasmid and comprises a sequence as set forth in SEQ ID NO:114 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity thereto, provided that the vector comprises an A at position 934 of the γ-globin expression cassette as numbered relative to SEQ ID NO:91 and provided that the vector comprises a T at position 189 of the HS4-400 insulator as numbered relative to SEQ ID NO:93.

[0236] Also provided is a host cell that comprises or is transduced by the lentiviral vector of the present disclosure.In some examples, the host cell is a hematopoietic stem cell (HSC) (e.g., allogeneic or autologous HSC).In some examples, the host cell is HPRT-deficient.

[0237] In a further aspect, a method of treating a subject having sickle cell disease or β-thalassemia is provided, comprising administering to the subject a host cell as described above and herein. In one embodiment, the method comprises administering to the subject a host cell, and then administering to the subject a purine analog (e.g., 6-thioguanine ("6TG"), 6-mercaptopurine ("6MP"), or azathioprine ("AZA")) to increase engraftment of the host cell. In some examples, the method further comprises preconditioning the subject with the purine analog prior to administration of the host cell. Use of the host cell for the preparation of a medicament for the treatment of sickle cell disease or β-thalassemia is also provided.

[0238] Genetic correction of HSCs with a vector encoding the gamma globin gene would result in continuous (i.e. permanent) production of anti-sickling HbF, thereby preventing or reducing sickling of RBCs throughout the subject's life. This method would have advantages over currently available therapies, including its applicability to all patients, particularly those who do not have a matched sibling donor, and being a one-time treatment that results in lifelong correction. Advantageously, this method would also be free of immune system side effects. It is further believed that an effective gene therapy approach would revolutionize the way SCD is treated and improve outcomes for patients with this devastating disorder.

[0239] As noted herein, in addition to the γ-globin transgene, the vectors of the present disclosure may contain agents (e.g., shRNA) designed to inhibit or knock down expression of HPRT, thus providing an in vivo chemoselection strategy that exploits the important role that HPRT plays in metabolizing purine analogs, such as 6TG, into myelotoxic drugs. Because HPRT deficiency does not impair hematopoietic cell development or function, it can be removed from hematopoietic cells used for transplantation. Purine analog conditioning and chemoselection are discussed further herein.

[0240] With respect to treating sickle cell anemia or β-thalassemia, treating a subject includes the steps of identifying a subject in need of the treatment, transfecting hematopoietic stem cells (HSCs) (e.g., autologous HSCs) with a vector (e.g., a lentiviral vector) of the present disclosure (i.e., a vector containing a mutated human gamma globin gene and an shRNA to HPRT), and transplanting the transfected HSCs into the subject.

[0241] In some embodiments, the method for treating hemoglobinopathies comprises (i) transducing HSCs with a vector comprising at least two nucleic acid sequences, a nucleic acid sequence encoding an shRNA to the HPRT gene and a nucleic acid sequence encoding a gamma globin gene, and (ii) administering the transduced HSCs to a mammalian subject. In some embodiments, the method further comprises a step of myeloablative conditioning prior to administration of the transduced HSCs. In some embodiments, the method further comprises a step of in vivo chemical selection using a purine analog (e.g., 6TG) following administration of the transduced HSCs. In some embodiments, the method further comprises a step of negative selection using MTX or MTA.

[0242] In some embodiments, fetal hemoglobin after transplantation is greater than at least 20%, and F cells constitute at least two-thirds of the circulating red blood cells. Fetal hemoglobin per F cell constitutes at least one-third of the total hemoglobin in sickle red blood cells, and at least 20% of the genetically modified HSCs repopulate the bone marrow of the subject. In some embodiments, fetal hemoglobin after transplantation is greater than 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, fetal hemoglobin after transplantation is greater than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, F cells constitute at least 70%, 75%, 80%, 85%, 90%, 95% or more of the circulating red blood cells. In some embodiments, fetal hemoglobin per F cell constitutes at least one-third of the total hemoglobin in sickle red blood cells. In some embodiments, fetal hemoglobin per F cell accounts for at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total hemoglobin in the sickle cell. In some embodiments, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the genetically modified HSCs repopulate the bone marrow of the subject.

[0243] Another embodiment of the disclosure is a method of treating an immune deficiency, a genetic disease, a blood disease (e.g., hemophilia, a hemoglobin disorder), a lysosomal storage disease, a neurological disease, a vascular disorder, or cancer, comprising administering to a mammalian subject an effective amount of a vector comprising at least two nucleic acid sequences: a nucleic acid sequence encoding an RNAi against the HPRT gene and a nucleic acid sequence encoding a therapeutic gene.

[0244] Another aspect of the present disclosure is a method of treating hemoglobinopathies, comprising administering to a mammalian subject an effective amount of a vector comprising at least two nucleic acid sequences, namely, a nucleic acid sequence encoding an RNAi that knocks out or otherwise reduces expression of the HPRT gene and a nucleic acid sequence encoding the gamma globin gene. In some embodiments, the method comprises administering to the patient an effective amount of a pharmaceutical composition comprising (i) a vector comprising at least two nucleic acid sequences, namely, a nucleic acid sequence encoding an shRNA against the HPRT gene and a nucleic acid sequence encoding the gamma globin gene, and (ii) a pharmaceutically acceptable carrier. In some embodiments, the method further comprises a step of myeloablative conditioning prior to administration of the transduced HSCs. In some embodiments, the method further comprises a step of in vivo chemoselection utilizing 6TG following administration of the transduced HSCs. In some embodiments, the method further comprises a step of negative selection utilizing MTX.

[0245] In order that the present invention may be readily understood and put to practical use, certain preferred embodiments will now be described by way of the following non-limiting examples. EXAMPLES

[0246] [Example 1] Identification of cryptic splice sites in WAS LVV The lentiviral vector containing the WAS cDNA was evaluated for cryptic splice sites. This lentiviral vector is the plasmid pBRNGTR47_pTL20c_SK734rev_MND_WAS_650 (or pBRNGTR47) with the sequence shown in SEQ ID NO: 55. As can be seen in FIG. 1, pBRNGTR47 contains a first expression cassette in forward orientation containing the WAS cDNA under the control of the MND promoter. Downstream of the WAS cDNA is the Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE), followed by the HS4-650 insulator (in reverse orientation) and the β-globin poly(A) signal. A second expression cassette, upstream of the first expression cassette and in reverse orientation, contains a nucleic acid encoding shRNA 734 under the control of the 7sk promoter. The lentiviral DNA (including the viral genes and LTR elements) is under the control of the 7tetO promoter / operator (see FIG. 1). The location and orientation of each of these elements within the vector is provided in Table 4 below.

[0247] [Table 4]

[0248] Bioinformatics splice site prediction analysis (Netgene2) was used to identify possible splice sites in pBRNGTR47. Three important splice acceptor sites (splice acceptor site 1 (SA1), splice acceptor site 2 (SA2), and splice acceptor site 3 (SA3)) were identified on the positive strand of the vector, with confidence levels ranging from 0.30 to 0.82, the HS4-650 insulator (see Figure 2). Since the HS4-650 insulator is in the reverse orientation in pBRNGTR47, the splice acceptor sites are in the reverse complement of the HS4-650 insulator. These three sites were considered to be particularly susceptible to the induction of aberrant transcripts. Table 5 below describes the details of SA1, SA2, and SA3.

[0249] [Table 5]

[0250] [Example 2] Generation of vectors with inactivated splice acceptor sites A series of modified vectors were generated to inactivate SA1, SA2, and / or SA3. These vectors contain a mutation in the splice acceptor site or an inversion of the HS4-650 insulator so that it is present in the vector in a forward orientation (as in the WAS cDNA) whereby the splice acceptor site sequence is on the reverse strand. For vectors containing a mutation in the HS4-650 insulator that inactivates the splice acceptor site, the mutation was an A to T, as shown in Table 6 below.

[0251] [Table 6]

[0252] Table 7 summarizes the vectors that were generated. Some vectors (pBRNGTR83, pBRNGTR87, pBRNGTR91, and pBRNGTR119) lack the second expression cassette (i.e., the p7sk-shRNA 734 expression cassette). All vectors contain a WPRE downstream of the WAS cDNA, but the sequence varies, with the WPRE in pBRNGTR47 containing seven mutations (WPRE mut7) compared to the wild-type sequence, and the newly generated vectors that utilize the WPRE containing six mutations (mut6) compared to the wild-type sequence (also referred to in the literature as WPRE mut6). All newly generated vectors also contain an additional 2 bp in the U3 sequence upstream of the insulator. It is deleted in pBRNGTR47 but reintroduced in pBRNGTR83, pBRNGTR84, pBRNGTR87, pBRNGTR88, pBRNGTR91, pBRNGTR92, pBRNGTR119, and pBRNGTR120. Vectors with two point mutations that inactivate SA1 and SA2 include pBRNGTR87 and pBRNGTR88, and vectors with three point mutations that inactivate SA1, SA2, and SA3 include pBRNGTR119 and pBRNGTR120. Vectors with an inversion of the HS4-650 insulator that inactivates the splice site include pBRNGTR91 and pBRNGTR92.

[0253] [Table 7]

[0254] The location and orientation of the various elements in pBRNGTR84, pBRNGTR88, pBRNGTR92, and pBRNGTR120 are provided below in Tables 8-11 and illustrated in Figures 3-6.

[0255] [Table 8]

[0256] [Table 9]

[0257] [Table 10]

[0258] [Table 11]

[0259] [Example 3] Assessment of aberrant splicing by HMGA2 fusion transcript assay A new HDR-based gene editing assay was developed to directly assess LV vector fusion transcripts in the HMG2A locus following integration into intron 3 of the HMGA2 gene. This approach was utilized because LV integration through this intron was identified in LV studies (De Ravin et al. (2016), Science Translational Medicine, Vol. 8, pp. 335ra57).

[0260] Briefly, we designed sgRNAs that target multiple sites within HMGA2 intron 3 and showed high cleavage efficiency (NHEJ rate 70-90%) in cell lines. We designed and produced a series of AAV homology-directed repair (HDR) donors with 0.6 kb homology arms. Each donor contained homology arm flanking sequences derived from the LVV LTR-containing insulator element with an engineered insulator. The AAV donors were designed to be used for co-delivery with the sgRNA.

[0261] Following generation of recombinant AAV vector stocks, the AAV donor and sgRNA (delivered as RNPs) were transfected into KG-1 cell line or primary human CD34+ In addition to the LTR / insulator donor construct, a control AAV HDR donor was generated that contained the same homology arms designed to deliver the MND.GFP.polyadenylation cassette. This control provides a rapid means to access the targeted integration rate by flow cytometry. Using this control construct, an HDR rate of approximately 40% was observed in KG-1 cells. Following co-delivery of the RNP and AAV LTR donor, HDR rates and fusion transcripts were measured in genomic DNA and RNA isolated from edited cells >1 week post-editing.

[0262] Preliminary experiments in KG-1 cells demonstrated efficient targeting by the AAV donor with an average HDR rate of 31% (range 23-37%). Fusion transcripts using SA sites were detected in cells edited with the cHS4 element. The sequences of the fusion constructs were confirmed by DNA sequencing and were consistent with the predicted use of the highest scoring SA sites.

[0263] Initial studies suggest that modifications of both SA1 and SA2 inactivate or eliminate splicing at these sites. However, fusion transcripts resulting from splicing at unmodified SA3 were observed in these constructs. Fusion transcripts were not detected in constructs containing the inverted HS4-650 insulator, i.e., in the forward orientation relative to the WAS transgene, suggesting that inversion of the orientation eliminates the splice site. Constructs containing combinations of mutations in SA1, SA2, and SA3 were evaluated.

[0264] Assessment of the HMGA2 exon 2-3 / exon 4-5 transcript ratio by ddPCR showed reduced fusion transcripts resulting from aberrant splicing in the constructs containing the modified insulator ("3xSA" and "fwd") compared to the construct containing the unmodified 650 bp insulator with unmodified splice sites ("650"). See Figure 8.

[0265] Furthermore, assessment of the frequency of edited cells in culture over time indicated that reduction of HMGA2 fusion transcripts (i.e., constructs containing modified insulators "fwd" and "3xSA") correlated with a reduced or eliminated advantage in selective cell proliferation compared to that observed for a construct containing the unmodified 650 bp insulator with an unmodified splice site ("650"). Results from KG1 cells are shown in Figure 9 and results from CD34+ cells are shown in Figure 10.

[0266] [Example 4] Enrichment-based RNA-SEQ method for assessment of aberrant splicing Sequencing of total RNA in a sample (RNA-Seq) is a useful next-generation sequencing method to directly assess the signal of gene expression in a sample. However, RNA-Seq samples are often highly complex and require deep sequencing to fully resolve the signal of typically relatively rare transcripts of interest. RNA-Seq hybridization capture kits may be used to enrich targets from complex samples prior to RNA-Seq. To this end, we designed a custom RNA bait to target HMGA2 and enrich for HMGA2 mRNA transcripts.

[0267] It has been recognized that HMGA2 has five known transcript variants, each of which results in the expression of a distinct protein isoform. All isoforms share exons 1, 2, and 3. An HMGA2 target enrichment kit was designed with baits targeting exons 1, 2, and 3 of HMGA2. Baits for three housekeeping genes (B2M, PPIA, GAPDH) were also designed as controls for normalization (Figure 11). To allow for the assessment of aberrant splice events by sequencing and quantification of the abundance of downstream HMGA2 exons compared to the downstream lentiviral sequence, the following protocol was used to enrich for mRNAs containing HMGA2 exons 1-3 from complex RNA-Seq samples. a) Hybridization - First, barcoded NGS cDNA libraries were heat denatured and hybridized over the course of several hours with a complex mixture of complementary biotinylated RNA baits. Adapter-specific blocking oligos were used to prevent random annealing of library molecules at the common adapter sites. b) Washing - After hybridization is complete, the biotin present on each bait is bound to streptavidin-coated magnetic beads. The washing step helps remove off-target or poorly hybridized library molecules. c) Amplification - The remaining library molecules bound to their complementary baits were denatured by heat and amplified with universal library primers. This "enriched" library was sequenced and evaluated.

[0268] Samples from KG1 cells (35 days after editing with AAV donor and RNP) and CD34+ cells (13 days after editing with AAV donor and RNP) were evaluated, respectively. All reads mapping to either the human genome or the AAV construct were extracted. Reads mapping to i) PPIA, ii) GAPDH, iii) B2M, iv) HMGA2 or AAV (combination) were counted. Reads mapping to HMGA2 or AAV were further divided into i) HMGA2 upstream of the AAV insertion site, ii) HMGA2 downstream of the AAV insertion site, and iii) AAV sequence.

[0269] The ratio of AAV reads to HMGA2 downstream reads can be used as a measure of splicing activity for LVVs containing modified insulator sequences. Expression levels of AAV fusion transcripts were calculated and normalized to the selected housekeeping genes (controls) above. HMGA2 upstream reads were calculated and normalized to the housekeeping genes to assess total HMGA2 expression transcripts.

[0270] In KG1 cells, a 5.5-fold decrease in the AAV / HMGA2 ratio was observed with constructs containing the 3xSA 650bp corrected insulator, calculated as the ratio of AAV reads to HMGA2 downstream exon reads (Figure 22A). In CD34+ cells, a 3.6-fold decrease in the AAV / HMGA2 ratio was observed with constructs containing the 3xSA 650bp corrected insulator (Figure 22B). A similar decrease in the AAV / HMGA2 ratio was observed with constructs containing the HS4-650 insulator in forward orientation relative to the transgene (reverse orientation relative to the control construct containing the original unmodified insulator) (Figures 22A and 22B, "fwd").

[0271] Figure 12 and Figure 13 show the expression levels of HMGA2 transcripts and AAV fusion transcripts in KG1 and CD34+ cells, respectively. Specifically, Figure 12A and Figure 13A show the levels of total HMGA2 expression transcripts compared to untreated cells. Figure 12B and Figure 13B show the levels of fusion transcripts expressed in cells normalized to 3xSA modified insulator constructs. "650" refers to the construct containing unmodified HS4-650 insulator with unmodified splice sites. "2xSA" refers to the construct containing HS4-650 insulator with two corrected cryptic splice acceptor sites. "3xSA" refers to the construct containing HS4-650 insulator with three corrected cryptic splice acceptor sites. "fwd" denotes constructs containing the HS4-650 insulator in forward orientation relative to the transgene (or reverse orientation relative to the control construct containing the original unmodified insulator). "Mock" or "fwd_LTRrev" denote controls. In both KG1 and CD34+ cells, constructs containing modified insulators ("3xSA" and "fwd") exhibited reduced expression levels of AAV fusion transcripts compared to the construct containing the unmodified insulator ("650").

[0272] To quantify the detected splicing events between HMGA2 exon 3 and exon 4 (major isoform) or LVV sequences (AAV insert), reads containing HMGA2 exon 3 sequences were extracted and spliced ​​to the next exon downstream. This analysis was performed on CD34+ cells (Figures 14-16) and KG1 cells (Figures 17-19). In both cell types, the percentage of HMGA2 exon 3-LVV splicing was reduced in both constructs containing the modified insulator compared to the construct containing the unmodified HS4-650 insulator with the unmodified splice site (Figures 14 and 17).

[0273] [Example 5] Assessment of insulator activity To evaluate the enhancer-blocking activity of the modified HS4-650 insulator, the LIM domain only 2 (LMO2) activation assay can be used to verify the function of the modified insulator. A similar assay is described in Ryu et al., 2008, Blood, Vol. 111, pp. 1866 and Goodman et al. 2018, Journal of Virology, Vol. 92 pp. e01639-17.

[0274] Briefly, vector constructs are evaluated using Jurkat cell lines with targeted integration of provirus into the promoter or first intron of the LMO2 gene (Ryu et al., 2008; Zhou et al., 2010). When the modified insulator sequence retains its insulator enhancer blocking function, LMO2 expression similar to that of the unmodified insulator sequence is observed, corresponding to a clear reduction in LMO2 expression compared to the uninsulated provirus. When the insulator function is reduced or destroyed by the modification of the insulator sequence, higher LMO2 expression is observed than that of the unmodified insulator sequence.

[0275] RT-qPCR was used as a measure of LMO2 expression and thus enhancer blocking activity of exemplary insulators. LVV proviral constructs containing the MND promoter driving the mScarlet-I reporter transgene and with various insulator sequences in the LTR were used for this assay. MoMLV and LVV proviral constructs containing the MND promoter driving the mScarlet-I reporter transgene but lacking the insulator were used as positive controls for LMO2 activation. Proviral constructs without promoter or with the EF1 alpha promoter were used as negative controls for LMO2 activation. Both single clone assays (Figure 20) and bulk cell assays (Figures 21A and 21B) were completed. Exemplary constructs containing modified insulators ("fwd" and "3xSA") showed comparable LMO2 expression to the unmodified HS4-650 insulator ("650") with unmodified splice sites, indicating that the modified insulators retained the desired insulator-enhancer blocking function. Positive and negative controls included constructs lacking a promoter ("no promoter") and an insulator ("no insulator" or "no-Ins").

[0276] [Example 6] In vitro immortalization assay (IVIM assay) The in vitro immortalization assay (IVIM assay) is used to evaluate vector-mediated genotoxic events following gene therapy with lentiviral vectors. IVIM is a rapid mutagenesis assay that uses a simple cell culture model to quantify the risk of transformation of hematopoietic cells. The IVIM assay allows quantification of the incidence of genotoxic mutants based on the initial number of transduced cells and the clonal characterization of mutants that show robust replating after limiting dilution. It also allows characterization of transformation of common insertion sites (CIS).

[0277] The IVIM assay has been described (Modlich et al. (2006), Blood, Vol. 108 pp. 2545; and Modlich et al. (2009), Molecular Therapy, Vol. 17 pp.1919-1928). Briefly, lineage-negative (Lin-) bone marrow cells are isolated from complete bone marrow. Prestimulated Lin- bone marrow cells are transduced with LVV vectors by spinoculation (retronectin-coated suspension culture dishes). After two rounds of transduction, cells are harvested for flow cytometry (FACS) and DNA samples for real-time PCR analysis (copy number).

[0278] After transduction, the cells are expanded as a large-scale culture for about 2 weeks. After the large-scale culture expansion, the cells are seeded into 96-well plates. After about 2 weeks, the positive wells are counted and the frequency of the re-seeded cells is calculated. Selected clones may be expanded for further characterization.

[0279] [Example 7] Transduction and expression of WASP Expression of WAS from an exemplary lentiviral vector construct was demonstrated in two different cell types: mouse lineage negative (Lin neg ) cells and (human) U937 cells. WAS KO and WT cells in both cell types were used as positive controls for the assay.

[0280] MouseLin neg Cells: Lineage-negative cells from bone marrow of WAS KO mice were depleted by magnetic labeling using the Direct Lineage Cell Depletion Kit (Miltenyi). After lineage depletion, 200,000 Lin neg WASp KO cells were mixed with 150 μL of medium containing transduction enhancer (TE; 1× LentiBoost and 10 μM dmPGE2) in a 96-well plate and incubated at 37 °C, 5% CO 2 Various WASP LVs were added to the cells at MOI 1–10 in a final volume of 200 μL / well and incubated at 37°C, 5% CO 2 The cells were incubated in 0.1% CO for 12–16 h. Each transduction was performed in triplicate wells. After the indicated incubation times, the cells were washed with medium to remove the viral supernatant and cultured (in 24-well plates) for 7 days.

[0281] U937 cells: U937 is a promonocytic human myeloid leukemia cell line known to express high levels of WASP. For characterization of WASP LV, U937 WASP KO cells (clone 19B) were used. WASP KO clones were generated by CRISPR / Cas9 targeting of exon 7 of the WASP locus. neg Similarly, WAS KO U937 cells were transduced with various WAS LVs and incubated at 37°C, 5% CO 2 The MOIs used for U937 cells were 0.5, 1, and 10. neg As with cells, each transduction was performed in triplicate and cells were cultured (in 24-well plates) for 21 days after transduction.

[0282] Expression of WAS protein was determined by negU937 cells were analyzed 7 days after transduction and U937 cells 21 days after transduction. Briefly, cells were harvested and permeabilized to allow staining of intracellular WAS protein. WAS protein was stained with Alexa-Fluor 647-labeled WAS antibody (5A5, BD Biosciences, labeled in-house). Untransduced WAS KO cells and WT cells (Lin neg and U937 cells) were used as negative and positive controls, respectively, and the expression of WAS is expressed as median fluorescence intensity (MFI). neg For cells, comparable levels of WASP expression were observed in all LVVs, including those containing modified insulators. In U937 cells, WASP expression was above WT controls (WT:KO 1-2 and 1-3), indicating that insulator modifications to address aberrant splicing do not reduce or disrupt transgene expression (Lin et al., 2013). neg (see Figure 23 for U937 cells and Figure 25 for U937 cells).

[0283] In addition to WAS protein expression, total DNA from cells was extracted and the number of vector copy integrations (VCN) was analyzed by q-PCR and expressed as VCN / cell. A dose-dependent increase in VCN was observed in both cell types (Lin et al., 2013). neg See Figure 24 for U937 cells and Figure 26 for U937 cells).

[0284] [Example 8] Transplantation of LVV-modified cells into WAS KO mice In vivo WAS rescue is evaluated in the WAS KO mouse model. neg Cells are modified with the exemplary WAS LVV with corrected insulators using the same protocol described in Example 6. After LV modification, cells are washed and transplanted into preconditioned (lethally irradiated) WAS KO mice (approximately 2×10 6Cells from donor and recipient mice are distinguishable based on the cognate alleles of CD45.1 or CD45.2. WT to WAS KO and WAS KO to WAS KO groups are used as positive and negative controls, respectively. Peripheral blood from mice is collected at regular intervals to monitor the engraftment and development of different immune cell lineages and the expression of WASP in each cell type. To evaluate the function of WASP rescue, T cells from the spleens of transplanted mice (at the time of sacrifice) are analyzed for their response to stimulation.

[0285] [Example 9] Identification of cryptic splice sites in SCD LVV A lentiviral vector containing a human γ-globin transgene was evaluated for cryptic splice sites. The lentiviral vector is the plasmid pCalH10_TL20c_rGbGM_7SKsh734 ("pCalH10") having the sequence shown in SEQ ID NO: 109. As can be seen in Figure 27, pCalH10 is a human γ-globin transgene containing a 3.2 kb β-globin locus control region (LCR) consisting of human γ-globin (HBG) exons (with a G16D point mutation), β-globin (HBB) non-coding sequences, β-globin promoter, and hypersensitive sites (HS2, HS3, and HS4 elements) cloned in the viral backbone in the reverse orientation to the viral RNA transcript. G16D Contains an expression cassette. The β-globin non-coding region contains a truncated HBB intron 2. Human γ-globin G16D The second expression cassette, downstream in forward orientation, contains the 7sk promoter (sh734) operably linked to a nucleic acid encoding shRNA734. Downstream of this in the LTR is the HS4-400 insulator in reverse orientation. Transcription of the lentiviral DNA is driven by the 7tetO promoter / operator (see FIG. 27).

[0286] We used bioinformatics splice site prediction analysis (Netgene2) to identify possible splice sites in pCalH10, which revealed approximately 100 possible splice sites, suggesting that there may be aberrant splicing associated with this vector.

[0287] The stability of the pCalH10 vector insert was then analyzed. Briefly, virions produced from pCalH10 were transduced into HeLa cells with the same insert as described above and shown in Figure 27. After 6 days, cell DNA was extracted and the DNA was subjected to restriction enzyme digestion and Southern blot analysis to compare with non-transduced HeLa cells (negative control) and pCalH10 (positive control). AflIII and NotI restriction enzymes were used on the test samples to release approximately 6.7 kb of vector insert sequence, and MfeI and XbaI were used to generate a probe fragment from pCalH10. As shown in Figure 28, a fragment of approximately 7 kb was observed as the major species (87.5%), and fragments of approximately 4 kb and 2.5 kb were observed as minor species (3% and 9.5%, respectively).

[0288] Next generation sequencing of DNA from transduced cells was also performed to identify the donor and acceptor sites that contributed to the generation of the truncated 2.5 kb fragment, which accounted for 9.5% of the population. The splice donor site was determined to be the splice donor site (SD1) on the positive strand of the vector in the truncated β-globin intron 2 in the γ-globin transgene. Since the γ-globin transgene is in the reverse orientation in the vector, SD1 is in the complementary strand of the γ-globin transgene, i.e., in the reverse complement of the γ-globin transgene. The splice acceptor site (SA2) is on the positive strand of the vector in the HS4-400 insulator. Since the HS4-400 insulator is in the reverse orientation in pCalH10, SA2 is on the complementary strand of the HS4-400 insulator, i.e., in the reverse complement. Aberrant splicing at these sites results in a truncated lentiviral fragment in which portions of the γ-globin expression construct, namely the β-globin LCR and the 7sk-sh734 expression construct, are deleted (Figure 29). Table 12 below details SD1 and SA2, as well as an additional splice acceptor site, SA3, that was deemed to be of interest (see Figure 30).

[0289] [Table 12]

[0290] [Example 10] Generation of vectors with inactivated splice sites A series of modified vectors were generated to inactivate SD1, SA2, and / or SA3. Most of these vectors are based on pCalH10 and contain mutations in one or more sequences at the splice sites that inactivate them. As shown in Table 13 below, a G to A mutation was made for those vectors containing a mutation in the γ-globin transgene that inactivates SD1, and an A to T mutation was made for those vectors containing a mutation in the HS4-400 insulator that inactivates SA2 and / or SA3. In two vectors, the HS4-400 insulator was simply deleted. Table 14 summarizes the vectors that were generated.

[0291] [Table 13]

[0292] [Table 14]

[0293] [Example 11] Assessment of insulator activity To evaluate the activity of the modified HS4-400 insulator, the LIM domain only 2 (LMO2) activation assay may be used to verify the function of the modified insulator. A similar assay is described in Ryu et al. (2008), Blood, Vol. 111, pp. 1866 and Goodman et al. (2018), Journal of Virology, Vol. 92 pp. e01639-17.

[0294] Briefly, vector constructs are evaluated using Jurkat cell lines with targeted integration sites in the promoter or first intron of the LMO2 gene (Ryu et al. (2008); Zhou et al. (2010), Blood, Vol. 116(6), pp. 900-908). If the modified insulator sequence retains its insulator function, reduced LMO2 expression is observed. If the insulator function is reduced or destroyed by modification of the insulator sequence, little or no reduction in LMO2 expression is observed.

[0295] [Example 12] In vitro immortalization assay (IVIM assay) The in vitro immortalization assay (IVIM assay) can be used to evaluate vector-mediated genotoxic events following gene therapy with lentiviral vectors. IVIM is a rapid mutagenesis assay that uses a simple cell culture model to quantify the risk of transformation of hematopoietic cells. The IVIM assay allows quantification of the incidence of genotoxic mutants based on the initial number of transduced cells and the clonal characterization of mutants that show robust replating after limiting dilution. It also allows characterization of transformation of common insertion sites (CIS).

[0296] The IVIM assay has been described (Modlich et al. (2006), Blood, Vol. 108 pp. 2545; and Modlich et al. (2009), Molecular Therapy, Vol. 17 pp.1919-1928). Briefly, lineage-negative (Lin-) bone marrow cells are isolated from complete bone marrow. Prestimulated Lin- bone marrow cells are transduced with LVV vectors by spinoculation (retronectin-coated suspension culture dishes). After two rounds of transduction, cells are harvested for flow cytometry (FACS) and DNA samples for real-time PCR analysis (copy number).

[0297] After transduction, the cells are expanded as a large-scale culture for about 2 weeks. After the large-scale culture expansion, the cells are seeded into 96-well plates. After about 2 weeks, the positive wells are counted and the frequency of the re-seeded cells is calculated. Selected clones may be expanded for further characterization.

[0298] array Unmodified HS4-650 insulator (SEQ ID NO:1) GAGCTCACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGC ACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGATACGGGGAAAAAGCTTGATATCATGTGTCTGAGCCTGCATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAGCTGGG TCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCATACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCCAAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCACAGTTCTTCAT CACCTCCAGGGACGGTGACCCCCCACCTCCGTGGGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAATCCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGAGTCAGTGAGGATGGGGCT Reverse complement (rc) of the unmodified HS4-650 insulator (SEQ ID NO:2) AGCCCCATCCTCACTGACTCCGTCCTGGAGTTGGATGAGATAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATTTAGCAAGATTTACCTTCTCCAAAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGGGGGGTCACCGTCCCTGGAGGT GATGAAGAACTGTGGGGATGTGGCACTGAGGGACATGGCCAGTGGGCACGGTGGGTGGGTTGGGGTTGGTCTTGGGGATCTTGGAGGGCTTTTCCAGCCTTCATGATTTGACGATTGTATGAACATCTACATGGCAATTCTCCAGCTGCCTGTCCCAGTCCTACTG ACCCAGCTGTATCTCTCCAGGCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATCAAACATGCAGGCTCAGACACATGATATCAAGCTTTTTCCCCGTATCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCG TGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAGCTC A to T mutation in engineered HS4-650 insulator (rc)-SA1 (bold and underlined mutation) (SEQ ID NO:3) TIFF2024519524000019.tif48160 Modified HS4-650 insulator (rc)-A to T mutations in SA1 and SA2 (bold and underlined mutations) (SEQ ID NO: 4) TIFF2024519524000020.tif48160 Modified HS4-650 insulator (rc)-A to T mutations in SA1 and SA3 (bold and underlined mutations) (SEQ ID NO:5) TIFF2024519524000021.tif48160 Modified HS4-650 insulator (rc)-A to T mutations in SA1, SA2, and SA3 (bold and underlined mutations) (SEQ ID NO:6) TIFF2024519524000022.tif48160 A to T mutation in modified HS4-650 insulator (rc)-SA2 (bold and underlined mutation) (SEQ ID NO: 7) TIFF2024519524000023.tif48160 Modified HS4-650 insulator (rc)-A to T mutations in SA2 and SA3 (bold and underlined mutations) (SEQ ID NO: 8) TIFF2024519524000024.tif48160 A to T mutation in modified HS4-650 insulator (rc)-SA3 (bold and underlined mutation) (SEQ ID NO: 9) TIFF2024519524000025.tif48160 Unmodified HS4-650 insulator (Genbank Acc. No. JN000001) (SEQ ID NO: 10) ACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAAGCTTTAGGCTTGTGTCTGAGCCTGCATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAGCTGGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCATACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCCAAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCACAGTTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTGGGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAATCCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGAGTCAGTGAG HS4-650 insulator with unchanged reverse complement (r-c) (Genbank Acc. No. JN000001) (SEQ ID NO: 11) CTCACTGACTCCGTCCTGGAGTTGGATGAGAGATAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATTTAGCAAGATTTACCTTCTCCAAAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGGGGGGTCACCGTCCCTGGAGGTGATGA AGAACTGTGGGGATGTTGGCACTGAGGGACATGGCCAGTGGCACGGTGGGTGGGTTGGGGTTGGTCTTGGGGATCTTGGAGGGCTTTTCCAGCCTTCATGATTTGACGATTGTATGAACATCTACATGGCAATTCTCCAGCTGCCTGTCCCAGTCCTACTGA CCCAGCTGTATCTCTCCAGGCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATCAAACATGCAGGCTCAGACACAAGCCTAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCC CGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGT Modified HS4-650 insulator (Genbank Acc. No. JN000001) (rc)-A to T mutation in SA1 (bold and underlined mutation) (SEQ ID NO: 12) TIFF2024519524000026.tif48160 Modified HS4-650 insulator (Genbank Acc. No. JN000001) (rc)-A to T mutations in SA1 and SA2 (bold and underlined mutations) (SEQ ID NO: 13) TIFF2024519524000027.tif48160 Modified HS4-650 insulator (Genbank Acc. No. JN000001) (rc)-A to T mutations in SA1 and SA3 (bold and underlined mutations) (SEQ ID NO: 14) TIFF2024519524000028.tif48160 Modified HS4-650 insulator (Genbank Acc. No. JN000001) (rc)-A to T mutations in SA1, SA2, and SA3 (bold and underlined mutations) (SEQ ID NO: 15) TIFF2024519524000029.tif48160 A to T mutation in modified HS4-650 insulator (Genbank Acc. No. JN000001) (rc)-SA2 (bold and underlined mutation) (SEQ ID NO: 16) TIFF2024519524000030.tif48160 Modified HS4-650 insulator (Genbank Acc. No. JN000001) (rc)-A to T mutations in SA2 and SA3 (bold and underlined mutations) (SEQ ID NO: 17) TIFF2024519524000031.tif48160 Modified HS4-650 insulator (Genbank Acc. No. JN000001) (rc)-A to T mutation in SA3 (bold and underlined mutation) (SEQ ID NO: 18) TIFF2024519524000032.tif48160 Unmodified HS4-650 insulator (US2016003218) (SEQ ID NO: 19) AAGATCTGCTCACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAATGTGTCTGAGCCTGCATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAGCTGGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCATACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCCAAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCACAGTTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTGGGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAATCCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGAGTCAGTGAGAATATT Reverse complement of the unmodified HS4-650 insulator (US2016003218) (SEQ ID NO: 20) AATATTCTCACTGACTCCGTCCTGGAGTTGGATGAGATAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATTTAGCAAGATTTACCTTCTCCAAAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGGGGGGTCACCGTCCCTGGAGGT GATGAAGAACTGTGGGGATGTGGCACTGAGGGACATGGCCAGTGGGCACGGTGGGTGGGTTGGGGTTGGTCTTGGGGATCTTGGAGGGCTTTTCCAGCCTTCATGATTTGACGATTGTATGAACATCTACATGGCAATTCTCCAGCTGCCTGTCCCAGTCCTAC TGACCCAGCTGTATCTCTCCAGGCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATCAAACATGCAGGCTCAGACACATTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCT TCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTGAGCAGATCTT A to T mutation in the engineered HS4-650 insulator (US2016003218)(rc)-SA1 (bold and underlined mutation) (SEQ ID NO:21) TIFF2024519524000033.tif48160 Modified HS4-650 insulator (US2016003218) (rc)-A to T mutations in SA1 and SA2 (bold and underlined mutations) (SEQ ID NO:22) TIFF2024519524000034.tif48160 Modified HS4-650 insulator (US2016003218) (rc)-A to T mutations in SA1 and SA3 (bold and underlined mutations) (SEQ ID NO:23) TIFF2024519524000035.tif48160 Modified HS4-650 insulator (US2016003218) (rc)-A to T mutations in SA1, SA2, and SA3 (bold and underlined mutations) (SEQ ID NO:24) TIFF2024519524000036.tif48160 A to T mutation in modified HS4-650 insulator (US2016003218)(rc)-SA2 (bold and underlined mutation) (SEQ ID NO:25) TIFF2024519524000037.tif48160 Modified HS4-650 insulator (US2016003218) (rc)-A to T mutations in SA2 and SA3 (bold and underlined mutations) (SEQ ID NO:26) TIFF2024519524000038.tif48160 A to T mutation in modified HS4-650 insulator (US2016003218)(rc)-SA3 (bold and underlined mutation) (SEQ ID NO:27) TIFF2024519524000039.tif48160 Unmodified HS4-650 insulator (Genbank Acc. No. MN044710.1) (SEQ ID NO: 28) CCTGTACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAATGTGTCTGAGCCTGCATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAGCTGGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCATACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCCAAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCACAGTTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTGGGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAATCCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGAGTCAGTGAGGATGGGGCT Reverse complement of the unmodified HS4-650 insulator (Genbank Acc.No.MN044710.1) (SEQ ID NO: 29) AGCCCCATCCTCACTGACTCCGTCCTGGAGTTGGATGAGATAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATTTAGCAAGATTTACCTTCTCCAAAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGGGGGGTCACCGTCCCTGG AGGTGATGAAGAACTGTGGGGATTGGCACTGAGGGACATGGCCAGTGGGGCACGGTGGGTGGGTTGGGGTTGGTCTTGGGGATCTTGGAGGGCTTTTCCAGCCTTCATGATTTGACGATTGTATGAACATCTACATGGCAATTCTCCAGCTGCCTGTCCCAGT CCTACTGACCCAGCTGTATCTCTCCAGGCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATCAAACATGCAGGCTCAGACACATTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGC CACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTACAGG Modified HS4-650 insulator (Genbank Acc. No. MN044710.1) (rc)-A to T mutation in SA1 (bold and underlined mutation) (SEQ ID NO: 30) TIFF2024519524000040.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044710.1) (rc)-A to T mutations in SA1 and SA2 (bold and underlined mutations) (SEQ ID NO: 31) TIFF2024519524000041.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044710.1) (rc)-A to T mutations in SA1 and SA3 (bold and underlined mutations) (SEQ ID NO: 32) TIFF2024519524000042.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044710.1) (rc)-A to T mutations in SA1, SA2, and SA3 (bold and underlined mutations) (SEQ ID NO: 33) TIFF2024519524000043.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044710.1) A to T mutation in (rc)-SA2 (bold and underlined mutation) (SEQ ID NO: 34) TIFF2024519524000044.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044710.1) (rc)-A to T mutations in SA2 and SA3 (bold and underlined mutations) (SEQ ID NO: 35) TIFF2024519524000045.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044710.1) A to T mutation in (rc)-SA3 (bold and underlined mutation) (SEQ ID NO: 36) TIFF2024519524000046.tif48160 Unmodified HS4-650 insulator (Genbank Acc. No. MN044709) (SEQ ID NO: 37) CCTGTACGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAATGTGTCTGAGCCTGCATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAGCTGGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCATACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCCAAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCACAGTTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTGGGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAATCCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGAGTCAGTGAGGATGGGGCT Reverse complement of the unmodified HS4-650 insulator (Genbank Acc.No.MN044709) (SEQ ID NO:38) AGCCCCATCCTCACTGACTCCGTCCTGGAGTTGGATGAGAGATAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATTTAGCAAGATTTACCTTCTCCAAAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGGGGGGTCACCGTCCCTG GAGGTGATGAAGAACTGTGGGGATTGGCACTGAGGGACATGGCCAGTGGCACGGTGGGTGGGTTGGGGTTGGTCTTGGGATCTTGGAGGGCTTTTCCAGCCTTCATGATTTGACGATTGTATGAACATCTACATGGCAATTCTCCAGCTGCCTGTCCCA GTCCTACTGACCCAGCTGTATCTCTCCAGGCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATCAAACATGCAGGCTCAGACACATTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCG TGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCGTG Modified HS4-650 insulator (Genbank Acc. No. MN044709) (rc)-A to T mutation in SA1 (bold and underlined mutation) (SEQ ID NO: 39) TIFF2024519524000047.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044709) (rc)-A to T mutations in SA1 and SA2 (bold and underlined mutations) (SEQ ID NO: 40) TIFF2024519524000048.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044709) (rc)-A to T mutations in SA1 and SA3 (bold and underlined mutations) (SEQ ID NO: 41) TIFF2024519524000049.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044709) (rc)-A to T mutations in SA1, SA2, and SA3 (bold and underlined mutations) (SEQ ID NO: 42) TIFF2024519524000050.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044709) A to T mutation in (rc)-SA2 (bold and underlined mutation) (SEQ ID NO: 43) TIFF2024519524000051.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044709) (rc)-A to T mutations in SA2 and SA3 (bold and underlined mutations) (SEQ ID NO: 44) TIFF2024519524000052.tif48160 Modified HS4-650 insulator (Genbank Acc. No. MN044709) (rc)-A to T mutation in SA3 (bold and underlined mutation) (SEQ ID NO: 45) TIFF2024519524000053.tif48160 Unmodified HS4-650 insulator (Genbank Acc. No. KF569217) (SEQ ID NO: 46) CGGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAATGTGTCTGAGCCTGCATGTTTGATGGTGTCTGGATGCAAGCAGAAGGGGTGGAAGAGCTTGCCTGGAGAGATACAGCTGGGTCAGTAGGACTGGGACAGGCAGCTGGAGAATTGCCATGTAGATGTTCATACAATCGTCAAATCATGAAGGCTGGAAAAGCCCTCCAAGATCCCCAAGACCAACCCCAACCCACCCACCGTGCCCACTGGCCATGTCCCTCAGTGCCACATCCCCACAGTTCTTCATCACCTCCAGGGACGGTGACCCCCCCACCTCCGTGGGCAGCTGTGCCACTGCAGCACCGCTCTTTGGAGAAGGTAAATCTTGCTAAATCCAGCCCGACCCTCCCCTGGCACAACGTAAGGCCATTATCTCTCATCCAACTCCAGGACGGAGTCAGTGAGAATA Reverse complement of the unmodified HS4-650 insulator (Genbank Acc.No.KF569217) (SEQ ID NO:47) TATTCTCACTGACTCCGTCCTGGAGTTGGATGAGAGATAATGGCCTTACGTTGTGCCAGGGGAGGGTCGGGCTGGATTTAGCAAGATTTACCTTCTCCAAAGAGCGGTGCTGCAGTGGCACAGCTGCCCACGGAGGTGGGGGGGTCACCGTCCCTGGAGG TGATGAAGAACTGTGGGGATTGGCACTGAGGGACATGGCCAGTGGGCACGGTGGGTGGGTTGGGGTTGGTCTTGGGGATCTTGGAGGGCTTTTCCAGCCTTCATGATTTGACGATTGTATGAACATCTACATGGCAATTCTCCAGCTGCCTGTCCCAGT CCTACTGACCCAGCTGTATCTCTCCAGGCAAGCTCTTCCACCCCTTCTGCTTGCATCCAGACACCATCAAACATGCAGGCTCAGACACATTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCC GTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCCG Modified HS4-650 insulator (Genbank Acc. No. KF569217) (rc)-A to T mutation in SA1 (bold and underlined mutation) (SEQ ID NO: 48) TIFF2024519524000054.tif48160 Modified HS4-650 insulator (Genbank Acc. No. KF569217) (rc)-A to T mutations in SA1 and SA2 (bold and underlined mutations) (SEQ ID NO: 49) TIFF2024519524000055.tif48160 Modified HS4-650 insulator (Genbank Acc. No. KF569217) (rc)-A to T mutations in SA1 and SA3 (bold and underlined mutations) (SEQ ID NO: 50) TIFF2024519524000056.tif48160 Modified HS4-650 insulator (Genbank Acc. No. KF569217) (rc)-A to T mutations in SA1, SA2, and SA3 (bold and underlined mutations) (SEQ ID NO:51) TIFF2024519524000057.tif48160 Modified HS4-650 insulator (Genbank Acc. No. KF569217) (rc)-A to T mutation in SA2 (bold and underlined mutation) (SEQ ID NO: 52) TIFF2024519524000058.tif48160 Modified HS4-650 insulator (Genbank Acc. No. KF569217) (rc)-A to T mutations in SA2 and SA3 (bold and underlined mutations) (SEQ ID NO: 53) TIFF2024519524000059.tif48160 Modified HS4-650 insulator (Genbank Acc. No. KF569217) (rc)-A to T mutation in SA3 (bold and underlined mutation) (SEQ ID NO: 54) TIFF2024519524000060.tif48160pBRNGTR47_pTL20c_SK734rev_MND_WAS_650 (SEQ ID NO: 55) pBRNGTR84_pTL20c_SK734rev_MND_WAS_650 (SEQ ID NO: 56) pBRNGTR88_pTL20c_SK734rev_MND_WAS_650_SAmut (SEQ ID NO: 57) pBRNGTR92_pTL20c_SK734rev_MND_WAS_650fwd (SEQ ID NO: 58) pBRNGTR120_pTL20c_SK734rev_MND_WAS_650_3xSAmut (SEQ ID NO: 59) SA1 sequence (SEQ ID NO:60) TTGCATCCAGACACCATCAA SA2 sequence (SEQ ID NO:61) ATCCCCCCAGGTGTCTGCAG SA3 sequence (SEQ ID NO:62) GTGTCTGCAGGCTCAAAGAG Inactivated SA1 sequence (SEQ ID NO:63) TTGCATCC T GACACCATCAA Inactivated SA2 sequence (SEQ ID NO:64) ATCCCCCC T GGTGTCTGCAG Inactivated SA3 sequence (SEQ ID NO:65) GTGTCTGC T GGCTCAAAGAG sh734 (SEQ ID NO:66) AGGATATGCCCTTGACTATTTGTCCGACATAGTCAAGGGCATATCC sh734 (SEQ ID NO:67) with multiple t-terminator sequences AGGATATGCCCTTGACTATTTGTCCGACATAGTCAAGGGCATATCCTTTTTT shRNA734 single t-terminator sequence (SEQ ID NO:68) AGGATATGCCCTTGACTATTTGTCCGACATAGTCAAGGGCATATCCT 7SK RNA promoter (SEQ ID NO:69) ATCGACGTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTACCTC 7SK RNA promoter (SEQ ID NO: 70) ATCGACGTGCAGTCGGGCTACTGCCCCACCCATAGTACCGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGTAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTACCTC 7SK RNA promoter (SEQ ID NO: 71) CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCCGGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATGCTGGTTAAATTAGATTTTAGTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGATTTCCTTCAGGTTTATATAGCTTGTGCGCCGCCTGGGTACCTC MND promoter (SEQ ID NO: 72) GAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC WASWT cDNA (wild type ORF) (SEQ ID NO: 73) WASWT cDNA (Genbank accession no. AB590224.1) (SEQ ID NO: 74) WASWT cDNA-codon optimized (SEQ ID NO:75) WASP (SEQ ID NO:76) MSGGPMGGRPGGRGAPAVQQNIPSTLLQDHENQRLFEMLGRKCLTLATAVVQLYLALPPGAEHWTKEHCGAVCFVKDNPQKSYFIRLYGLQAGRLLWEQELYSQLVYSTPTPFFHTFAGDDCQAG LNFADEDEAQAFRALVQEKIQKRNQRQSGDRRQLPPPPTPANEERRGGLPPLPLHPGGDQGGPPVGPLSLGLATVDIQNPDITSSRYRGLPAPGPSPADKKRSGKKKISKADIGAPSGFKHVSHVG WDPQNGFDVNNLDPDLRSLFSRAGISEAQLTDAETSKLIYDFIEDQGGLEAVRQEMRRQEPLPPPPPPSRGGNQLPRPPIVGGNKGRSGPLPPVPLGIAPPPPTPRGPPPPGRGGPPPPPPPATG RSGPLPPPPPGAGGPPMPPPPPPPPPPPSSGNGPAPPPLPPALVPAGGLAPGGGRGALLDQIRQGIQLNKTPGAPESSALQPPPQSSEGLVGALMHVMQKRSRAIHSSDEGEDQAGDEDEDDEWDD WPRE mut6 (SEQ ID NO: 77) AATCAACCTCTGGATTACAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCTTGTATAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCTGTGTGTCACTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATGGCCACCACTGTCAGCTCTTCCGGACTTTCGCTTTCCCC TCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGTGTTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTCCGCTCTTCGCCTTCCCTCCAGACGAGTCGGATCTCCCTTGGGCCGCCTCCCGCA. WPRE Mut7 AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGTTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCA 7tet operator (SEQ ID NO: 79) TTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCAGTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAA β-globin poly(A) signal (SEQ ID NO: 80) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC pBRNGTR83_pTL20c_MND_WAS_650 (SEQ ID NO: 81) pBRNGTR87_pTL20c_MND_WAS_650_SAmut (SEQ ID NO: 82) pBRNGTR91_pTL20c_MND_WAS_650fwd (SEQ ID NO: 83) pBRNGTR119_pTL20c_MND_WAS_650_3xSAmut (SEQ ID NO: 84) Unmodified gamma-globin expression cassette (capital letters are exons) (SEQ ID NO:85) Unmodified Gamma-Globin Expression Cassette - Reverse Complement (Capital Letters are Exons) (SEQ ID NO:86) Unmodified truncated HBB intron 3 (SEQ ID NO: 87) Gtgagtctatgggacccttgatgttttctttccccttcttttctatggttaagttcatgtcataggaaggggagaagtaacagggtacacatattgaccaaatcagggtaattttgcat ttgtaattttaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttccctaatctctttctttcagggcaataatgatacaatgtatcatgcctctttg caccattctaaagaataacagtgataatttctgggttaaggcaatagcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagc agctacaatccagctaccattctgcttttattttatggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcatgttcatacctcttatcttcctcccacag Unmodified truncated HBB intron 3-reverse complement (SEQ ID NO: 88) Ctgtgggaggaagataagaggtatgaacatgattagcaaaagggcctagcttggactcagaataatccagccttatcccaaccataaaataaaagcagaatggtagctggattgtagctgctattagcaatatgaaacctcttacatcagttacaatttatatgcagaaatatttatatgcagaaatattgctattgccttaacccagaaattatcactgttattctttagaatggtgcaaagaggcatgatacattgtatcattattgccctgaaagaaagagattagggaaagtattagaaataagataaacaaaaaagtatattaaaagaagaaagcattttttaaaattacaaatgcaaaattaccctgatttggtcaatatgtgtaccctgttacttctccccttcctatgacatgaacttaaccatagaaaagaaggggaaagaaaacatcaagggtcccatagactcac Unmodified HS4-400 insulator (SEQ ID NO: 89) Ggggagctcacggggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggcctgggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcccagagccacatccagcctggccttgaatgcctgcagggat Unmodified HS4-400 - reverse complement (SEQ ID NO: 90) Atccctgcaggcattcaaggccaggctggatgtggctctgggcagcctgggctgctggttgatgaccctgcacatagcaggggggttggatctggatgagcact gtgctcctttgcaacccaggccgttctatgattctgtcattctaaatctctctttcagcctaaagctttttccccgtatccccccaggtgtctgcaggctcaa agagcagcgagaagcgttcagaggaaagcgatcccgtgccaccttccccgtgcccgggctgtccccgcacgctgccggctcggggatgcggggggagcgccgg accggagcggagccccgggcggctcgctgctgccccctagcgggggagggacgtaattacatccctgggggctttggggggggggctgtccccgtgagctcccc Modified γ-globin transgene - reverse complement (bold and underlined mutations) (SEQ ID NO:91) TIFF2024519524000061.tif144160 Modified truncated HBB intron 3-reverse complement (bold and underlined mutations) (SEQ ID NO:92) TIFF2024519524000062.tif32160 A to T mutation in modified HS4-400-reverse complement-SA2 (bold and underlined mutations) (SEQ ID NO:93) TIFF2024519524000063.tif32160 Modified HS4-400-Reverse complement-A to T mutations in SA2 and SA3 (bold and underlined mutations) (SEQ ID NO:94) TIFF2024519524000064.tif32160 A to T mutation in modified HS4-400-reverse complement-SA3 (bold and underlined mutations) (SEQ ID NO:95) TIFF2024519524000065.tif32160 Splice donor site 1 (SD1) (SEQ ID NO: 96) AAGATAAGAGGTATGAACAT Inactivated SD1 (underlined mutation) (SEQ ID NO: 97) AAGATAAGAG A TATGAACAT Gamma-globin exon 1 (SEQ ID NO: 98) CCTTCCCAGGGTTTCTCCTCCAGCATCTTCCACATTCACCTTGTCCCACAGGCTTGTGATAGTAGCCTTGTCCTCCTCTGTGAAATGACCCAT Gamma-globin exon 2 (SEQ ID NO: 99) CTTGAAGTTCTCAGGATCCACATGCAGCTTGTCACAGTGCAGTTCACTCAGCTGGGCAAAGGTGCCCTTGAGATCATCCAGGTGCTTTATGGCATCTCCCAAGGAAGTCAGCACCTTCTTGCCATGTGCCTTGACTTTGGGTTGCCCATGATGCAGAGGCAGAGGACAGGTTGCCAAAGCTGTCAAAGAACCTCTGGGTCCATGGGTAGACAACCAGGAGC Gamma-globin exon 3 (SEQ ID NO: 100) GTGATCTCTCAGCAGAATAGATTTATTATTTGTATTGCTTGCAGAATAAAGCCTATCCTTGAAAGCTCTGAATCATGGGCAAGAGGCTCAGTGGTATCTGGAGGACAGGGCACTGGCCACTGCAGTCACCATCTTCTGCCAGGAAGCCTGCACCTCAGGGGTGAATTCTTTGCCAAAGTGAATGGCCAGCACGGTGACCAGCACGTTGCCCAGGAG γ-globin coding sequence (SEQ ID NO:101) CAGACACCATGGGTCATTTCACAGAGGAGGACAAGGCTACTATCACAAGCCTGTGGGACAAGGTGAATGTGGAAGATGCTGGAGGAGAAACCCTGGGAAGGCTCCTGGTTGTCTACCCATGGACCCAGAGGTTCTTTGACAGCTTTGGCAACCTGTCCTCTGCCTCTGCCATCATGGGCAACCCCAAAGTCAAGGCACATGGCAAGAAGGTGCTGACTTCCTTGGGAGATGCCATAAAGCACCTGGATGATCTCAAGGGCACCTTTGCCCAGCTGAGTGAACTGCACTGTGACAAGCTGCATGTGGATCCTGAGAACTTCAAGCTCCTGGGCAACGTGCTGGTCACCGTGCTGGCCATTCACTTTGGCAAAGAATTCACCCCTGAGGTGCAGGCTTCCTGGCAGAAGATGGTGACTGCAGTGGCCAGTGCCCTGTCCTCCAGATACCACTGAGCCTCTTGCCCATGATTCAGAGCTTTCAAGGATAGGCTTTATTCTGCAAGCAATACAAATAATAAATCTATTCTGCTGAGAGATCAC γ-globin coding sequence (SEQ ID NO: 102) ATGGGTCATTTCACAGAGGAGGACAAGGCTACTATCACAAGCCTGTGGGACAAGGTGAATGTGGAAGATGCTGGAGGAGAAACCCTGGGAAGGCTCCTGGTTGTCTACCCATGGACCCAGAGGTTCTTTGACAGCTTTGGCAACCTGTCCTCTGCCTCTGCCATCATGGGCAACCCCAAAGTCAAGGCACATGGCAAGAAGGTGCTGACTTCCTTGGGAGATGCCATAAAGCACCTGGATGATCTCAAGGGCACCTTTGCCCAGCTGAGTGAACTGCACTGTGACAAGCTGCATGTGGATCCTGAGAACTTCAAGCTCCTGGGCAACGTGCTGGTCACCGTGCTGGCCATTCACTTTGGCAAAGAATTCACCCCTGAGGTGCAGGCTTCCTGGCAGAAGATGGTGACTGCAGTGGCCAGTGCCCTGTCCTCCAGATACCACTGA γ-globin G16D (GbGMG16D) protein (SEQ ID NO: 103) MGHFTEEDKATITSLWDKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDAIKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYH β-globin poly(A) signal (SEQ ID NO: 104) GATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC β-globin locus control region (LCR) (SEQ ID NO: 105) REV response element (RRE) (SEQ ID NO: 106) AGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCG pCalH10_TL20c_rGbGM_7SKsh734 (SEQ ID NO: 107) pCalH20_TL20d_rGbGM_7SKsh734 (SEQ ID NO: 108) pCalH21_TL20d_rGbGM_G3320A_7SKsh734 (SEQ ID NO: 109) A pCalH32_TL20c_rGbGM_7SKsh734_400_2AT (SEQ ID NO: 110) T GGTGTCTGC T pCalH13_TL20c_rGbGM_G3320A_7SKsh734_400_2AT (SEQ ID NO: 111) AT GGTGTCTGC T pCalH11_TL20c_rGbGM_G3320A_7SKsh734 (SEQ ID NO: 112) A pCalH31_TL20c_rGbGM_7SKsh734_400_1AT (SEQ ID NO: 113) T pCalH12_TL20c_rGbGM_G3320A_7SKsh734_400_1AT (SEQ ID NO: 114) AT β-globin promoter (SEQ ID NO:115) GGTGTCTGTTTGAGGTTGCTAGTGAACACAGTTGTGTCAGAAGCAAATGTAAGCAATAGATGGCTCTGCCCTGACTTTTATGCC β-globin promoter (SEQ ID NO:116) AAGCATAGATGGCTCTGCCCTGACTTTTATGCCCAGCCCTGGCTCCTGCCCTCCCTGCTCCTGGGAGTAGATTGGCCAACCCTAGGGTGTGGCTCCACAGGGTGAGGTCTAAGTGATGACAGCCGTACCTG TCCTTGGCTCTTCTGGCACTGGCTTAGGAGTTGGACTTCAAACCCTCAGCCCTCCCTCTAAGATATATCTCTTGGCCCCATACCATCAGTACAAATTGCTACTAAAAAACATCCTCCTTTGCAAGTGTATTTAC β-globin promoter (SEQ ID NO:117) GGTGTCTGTTTGAGGTTGCTAGTGAACACAGTTGTGTCAGAAGCAAATGTAAGCAAGATGGCTCTGCCCTGACTTTTATGCCCAGCCCTGGCTCCTGCCCTCCCTGCTCCTGGGAGTAGATTGGCCAACCCTAGGGTGTGGCTCCACAGGGTGAGG TCTAAGTGATGACAGCCGTACCTGTCCTTGGCTCTTCTGGCACTGGCTTAGGAGTTGGACTTCAAACCCTCAGCCCTCCCTCTAAGATATATCTCTTGGCCCCATACCATCAGTACAAATTGCTACTAAAACATCCTCCTTTGCAAGTGTATTTACGA Unmodified γ-globin transgene (SEQ ID NO: 118) Unmodified gamma-globin transgene-reverse complement (capital letters are exons) (SEQ ID NO: 119) Unmodified gamma-globin transgene-reverse complement-G to A mutation in SD1 (bold and underlined mutation) (SEQ ID NO: 120) TIFF2024519524000066.tif75160 Unmodified γ-globin transgene + polyA signal (SEQ ID NO: 121) Unmodified γ-globin transgene + polyA signal - reverse complement (SEQ ID NO: 122) Unmodified gamma-globin transgene + polyA signal - reverse complement - G to A mutation in SD1 (bold and underlined mutation) (SEQ ID NO: 123) TIFF2024519524000067.tif123160

[0299] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0300] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the present application.

[0301] Throughout this specification, the objective is to describe preferred embodiments of the invention without limiting the invention to any embodiment or particular collection of features. Thus, those skilled in the art will recognize in light of this disclosure that various modifications and changes can be made in the specific embodiments exemplified without departing from the scope of the invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

1. a first promoter operably linked to a first nucleic acid sequence encoding a Wiskott-Aldrich syndrome protein; and Modified HS4-650 insulator wherein, when present in the vector, the modified HS4-650 insulator comprises an inactivated splice acceptor site 1 (SA1) relative to the unmodified HS4-650 insulator, said SA1 being present at nucleotide positions 385-386 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:2, which is the reverse complement of the unmodified HS4-650 insulator set forth in SEQ ID NO:1, and / or said SA1 has the sequence TTGCATCCAG ∧ ACACCATCAA (SEQ ID NO:60), ∧ represents the splice location, Lentiviral vectors.

2. The lentiviral vector of claim 1, wherein the modified HS4-650 insulator comprises a mutation that inactivates SA1 relative to the unmodified HS4-650 insulator.

3. 3. The lentiviral vector of claim 2, wherein the mutation is an A mutation at position 384 and / or a G mutation at position 385, numbered relative to SEQ ID NO:

2.

4. The lentiviral vector of claim 3 , wherein the A mutation at position 384 is an A to T mutation.

5. 2. The lentiviral vector of claim 1, wherein the modified HS4-650 insulator a) further comprises a mutation that inactivates splice acceptor site 2 (SA2) relative to the unmodified HS4-650 insulator, where SA2 is located at nucleotide positions 446-447 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:2, and / or b) further comprises a mutation that inactivates splice acceptor site 3 (SA3) relative to the unmodified HS4-650 insulator, where SA3 is located at nucleotide positions 456-457 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:

2.

6. The lentiviral vector of claim 5, wherein the mutation is a) an A mutation at position 445 and / or a G mutation at position 446, numbered relative to SEQ ID NO:2, and / or b) an A mutation at position 455 and / or a G mutation at position 456, numbered relative to SEQ ID NO:

2.

7. 7. The lentiviral vector of claim 6, wherein the A mutation at position 445 is an A to T mutation and / or the A mutation at position 455 is an A to T mutation.

8. The lentiviral vector of claim 1, wherein the reverse complement sequence of the modified HS4-650 insulator comprises any one of the sequences shown in SEQ ID NOs: 3-6, 12-15, 21-24, 30-33, 39-42 and 48-51.

9. a first promoter operably linked to a first nucleic acid sequence encoding a Wiskott-Aldrich syndrome protein; and Modified HS4-650 insulator wherein, when present in the vector, the modified HS4-650 insulator comprises a splice acceptor site 2 (SA2) that is inactivated relative to the unmodified HS4-650 insulator, said SA2 being present at nucleotide positions 446-447 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:2, SEQ ID NO:2 being the reverse complement of the unmodified HS4-650 insulator set forth in SEQ ID NO:1, and / or said SA2 has the sequence ATCCCCCCAG ∧ GTGTCTGCAG (SEQ ID NO:61), ∧ represents the splice location, Lentiviral vectors.

10. The lentiviral vector of claim 9, wherein the modified HS4-650 insulator comprises a mutation that inactivates SA2 relative to the unmodified HS4-650 insulator.

11. 11. The lentiviral vector of claim 10, wherein the mutation is an A mutation at position 445 and / or a G mutation at position 446, numbered relative to SEQ ID NO:

2.

12. The lentiviral vector of claim 11, wherein the mutation at position 445 is an A to T mutation.

13. 10. The lentiviral vector of claim 9, wherein the modified HS4-650 insulator a) further comprises a mutation that inactivates splice acceptor site 1 (SA1) relative to the unmodified HS4-650 insulator, where SA1 is located at nucleotide positions 385-386 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:2, and / or b) further comprises a mutation that inactivates splice acceptor site 3 (SA3) relative to the unmodified HS4-650 insulator, where SA3 is located at nucleotide positions 456-457 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:

2.

14. The lentiviral vector of claim 13, wherein the mutation is a) an A mutation at position 384 and / or a G mutation at position 385, numbered relative to SEQ ID NO:2, and / or b) an A mutation at position 455 and / or a G mutation at position 456, numbered relative to SEQ ID NO:

2.

15. 15. The lentiviral vector of claim 14, wherein the A mutation at position 384 is an A to T mutation and / or the A mutation at position 455 is an A to T mutation.

16. The lentiviral vector of claim 9, wherein the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs: 4, 6, 7, 8, 13, 15, 16, 17, 22, 24, 25, 26, 31, 33, 34, 35, 40, 42, 43, 44, 49, 51, 52 and 53.

17. a first promoter operably linked to a first nucleic acid sequence encoding a Wiskott-Aldrich syndrome protein; and Modified HS4-650 insulator wherein, when present in the vector, the modified HS4-650 insulator comprises an inactivated splice acceptor site 3 (SA3) relative to the unmodified HS4-650 insulator, said SA3 being present at nucleotide positions 456-457 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:2, which is the reverse complement of the unmodified HS4-650 insulator set forth in SEQ ID NO:1, and / or said SA3 is the sequence GTGTCTGCAGG ... ∧ GCTCAAAGAG (SEQ ID NO:62), ∧ represents the splice location, Lentiviral vectors.

18. The lentiviral vector of claim 17, wherein the modified HS4-650 insulator comprises a mutation that inactivates SA3 relative to the unmodified HS4-650 insulator.

19. 19. The lentiviral vector of claim 18, wherein the mutation is an A mutation at position 455 and / or a G mutation at position 456, numbered relative to SEQ ID NO:

2.

20. 20. The lentiviral vector of claim 19, wherein the A mutation at position 455 is an A to T mutation.

21. 18. The lentiviral vector of claim 17, wherein the modified HS4-650 insulator a) further comprises a mutation that inactivates splice acceptor site 1 (SA1) relative to the unmodified HS4-650 insulator, where SA1 is located at nucleotide positions 385-386 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:2, and / or b) further comprises a mutation that inactivates splice acceptor site 2 (SA2) relative to the unmodified HS4-650 insulator, where SA2 is located at nucleotide positions 446-447 in the unmodified HS4-650 insulator as numbered relative to SEQ ID NO:

2.

22. The lentiviral vector of claim 21, wherein the mutation is a) an A mutation at position 384 and / or a G mutation at position 385, numbered relative to SEQ ID NO:2, and / or b) an A mutation at position 445 and / or a G mutation at position 446, numbered relative to SEQ ID NO:

2.

23. 23. The lentiviral vector of claim 22, wherein the A mutation at position 384 is an A to T mutation and / or the A mutation at position 445 is an A to T mutation.

24. The lentiviral vector of claim 17, wherein the modified HS4-650 insulator comprises a sequence set forth in any one of SEQ ID NOs: 5, 6, 8, 9, 14, 15, 17, 18, 23, 24, 26, 27, 32, 33, 35, 36, 41, 42, 44, 45, 50, 51, 53 and 54.

25. The lentiviral vector of any one of claims 1, 9 and 17, wherein the modified HS4-650 insulator is in the opposite orientation relative to the first nucleic acid sequence.

26. 20. The lentiviral vector of any one of claims 1, 9 and 17, wherein the first nucleic acid is in a forward orientation and the modified HS4-650 insulator is in a reverse orientation within the lentiviral vector.

27. 27. The lentiviral vector of claim 26, wherein the modified HS4-650 insulator is in the same orientation as the first nucleic acid sequence, thereby inactivating SA1, SA2 or SA3.

28. The lentiviral vector of claim 27, wherein the first nucleic acid and the modified HS4-650 insulator are in a forward orientation within the lentiviral vector.

29. The lentiviral vector of any one of claims 1, 9 and 17, wherein a modified HS4-650 insulator is downstream of the first nucleic acid sequence.

30. The lentiviral vector of any one of claims 1, 9 and 17, having one or more of the following features: (a) the Wiskott-Aldrich syndrome protein comprises the amino acid sequence set forth in SEQ ID NO:76, or a sequence having at least 95% sequence identity thereto; (b) the first nucleic acid sequence comprises a sequence set forth in any one of SEQ ID NOs:73-75, or a sequence having at least 95% sequence identity thereto; (c) further comprising a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) between the first nucleic acid sequence and the modified HS4-650 insulator; (d) further comprising a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) between the first nucleic acid sequence and the modified HS4-650 insulator, wherein the WPRE comprises a nucleic acid sequence set forth in any one of SEQ ID NOs:77-78, or a sequence having at least 95% sequence identity thereto; (e) comprising a sequence selected from the group consisting of the sequence set forth as nucleotides 3098-6006 of SEQ ID NO:57, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, the sequence set forth as nucleotides 3098-6009 of SEQ ID NO:58, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence set forth as nucleotides 3098-6006 of SEQ ID NO:59, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (f) the first promoter is an MND promoter; (g) the first promoter is an MND promoter, wherein the MND promoter comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 72, or a sequence having at least 95% sequence identity thereto; (h) comprising a sequence selected from the group consisting of the sequence set forth as nucleotides 2710-6006 of SEQ ID NO:57, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, the sequence set forth as nucleotides 2710-6009 of SEQ ID NO:58, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence set forth as nucleotides 2710-6006 of SEQ ID NO:59, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (i) further comprising a polyadenylation signal downstream of the first nucleic acid and the modified HS4-650 insulator; and (j) comprises a sequence as set forth in SEQ ID NO: 84 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

31. 20. The lentiviral vector of claim 1, 9 or 17, which is a plasmid or a viral particle.

32. A host cell comprising or transduced with a lentiviral vector according to any one of claims 1, 9 and 17.

33. A pharmaceutical composition comprising the host cell of claim 32 for the treatment of Wiskott-Aldrich syndrome.

Images