WO2021097109A1 - Lentiviral vectors in hematopoietic stem cells to treat x-linked chronic granulomatous disease - Google Patents

Lentiviral vectors in hematopoietic stem cells to treat x-linked chronic granulomatous disease Download PDF

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WO2021097109A1
WO2021097109A1 PCT/US2020/060263 US2020060263W WO2021097109A1 WO 2021097109 A1 WO2021097109 A1 WO 2021097109A1 US 2020060263 W US2020060263 W US 2020060263W WO 2021097109 A1 WO2021097109 A1 WO 2021097109A1
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vector
nucleic acid
cybb
seq
fragment
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PCT/US2020/060263
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French (fr)
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Donald B. Kohn
Ryan L. WONG
Roger Paul Hollis
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The Regents Of The University Of California
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Priority to EP20886706.9A priority Critical patent/EP4058068A4/en
Priority to KR1020227019204A priority patent/KR20220097487A/en
Priority to CA3161175A priority patent/CA3161175A1/en
Priority to AU2020384286A priority patent/AU2020384286A1/en
Priority to US17/775,857 priority patent/US20220378937A1/en
Priority to JP2022527208A priority patent/JP2023502593A/en
Priority to CN202080086551.8A priority patent/CN114829614A/en
Publication of WO2021097109A1 publication Critical patent/WO2021097109A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/03—Oxidoreductases acting on NADH or NADPH (1.6) with oxygen as acceptor (1.6.3)
    • C12Y106/03001—NAD(P)H oxidase (1.6.3.1), i.e. NOX1

Definitions

  • P218P_ST25.txt created on November 11, 2019 and having a size of 46.4 kb.
  • the contents of the text file are incorporated by reference herein in their entirety.
  • X-linked chronic granulomatous disease is a primary immune deficiency caused by mutations in the CYBB gene which encodes for a vital subunit of the phagocyte NADPH Oxidase (PHOX) complex.
  • PHOX phagocyte NADPH Oxidase
  • Inflammation can occur in many different areas of the body in people with chronic granulomatous disease. Most commonly, granulomas occur in the gastrointestinal tract and the genitourinary tract. In many cases the intestinal wall is inflamed, causing a form of inflammatory bowel disease that varies in severity but can lead to stomach pain, diarrhea, bloody stool, nausea, and vomiting. Other common areas of inflammation in people with chronic granulomatous disease include the stomach, colon, and rectum, as well as the mouth, throat, and skin. Additionally, granulomas within the gastrointestinal tract can lead to tissue breakdown and pus production (abscesses). Inflammation in the stomach can prevent food from passing through to the intestines (gastric outlet obstruction), leading to an inability to digest food.
  • lymph nodes lymph nodes
  • bone marrow osteomyelitis
  • the PHOX complex is made of five different subunits encoded by five different genes. These are gp91 phox encoded by CYBB, p22 phox encoded by CYBA, p47 Phox encoded by NCF1, p67 phox encoded by NCF2, and p40 phox encoded by NCF4. Most common mutations are in the CYBB gene encoding for gp91 phox which accounts for ⁇ 56%-70% of all cases of CGD. The condition is X-linked and accordingly primarily affects males.
  • the disease was initially terms "fatal granulomatous disease of childhood" and without treatment patient rarely lived past their first decade of life.
  • Current standard of care utilizes routine prophylactic antibacterial and antifungal therapy and results in a mean age of survival around 30-40 years. These treatments do not provide a cure for the disease.
  • One potential curative therapy is an allogeneic hematopoietic stem cell transplantation from an HLA matched donor. However, this is not a viable option for many patients due to the unavailability of a suitable matched donor.
  • An alternative curative therapy is an autologous hematopoietic stem cell
  • HSC HSC transplantation with ex vivo gene therapy.
  • patients act as their own donor, eliminating the risk of immunological complications.
  • the patient's own blood HSCs are harvested and are genetically engineered ex- vivo to introduce a functional copy of gene of interest, and gene modified cells are reinfused
  • Previous viral-based therapies utilized a g-retroviral vector driven by the spleen focus-forming virus (SFFV) promoter. This provided a promising clinical benefit.
  • SFFV spleen focus-forming virus
  • 2/2 patients developed myelodysplasia due to insertional oncogenesis.
  • a current safer SIN lentiviral vector (pChim-CYBB; aka MSP-Gp91phox-WPRE) employs a chimeric “myeloid-specific promoter” (MSP) and initial results from current clinical trials indicate potential clinical benefits.
  • MSP myeloid-specific promoter
  • the pChim-CYBB construct fails to recapitulate wildtype levels of expression and regulation of Gp91 phox .
  • patient’s neutrophils post gene therapy under-express Gp91 phox compared to normal heathy donor cells.
  • LVs novel lentiviral vector(s)
  • the vectors described herein show better (higher) expression than the current lentiviral vector. Additionally, the vectors described herein possesses strict lineage and stage specific expression that mimics the expression pattern of the native CYBB gene.
  • Embodiment 1 A recombinant lentiviral vector (LV) for the treatment of chronic granulomatous disease, said vector comprising:
  • an expression cassette comprising a nucleic acid construct comprising:
  • CYBB promoter or promoter fragment CYBB promoter or promoter fragment.
  • Embodiment 2 The vector of embodiment 1, wherein said CYBB promoter or effective fragment thereof comprises a full-length endogenous CYBB promoter (SEQ ID NO:l).
  • Embodiment 3 The vector of embodiment 1, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
  • Embodiment 4 The vector of embodiment 3, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
  • Embodiment 5 The vector of embodiment 1, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
  • Embodiment 6 The vector of embodiment 5, wherein said CYBB promoter consists of an effective fragment of the CYBB promoter whose sequence consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
  • Embodiment 7 The vector according to any one of embodiments 1-6, wherein said expression cassette comprises an enhancer element 2 (SEQ ID NO:4) or an effective fragment thereof.
  • Embodiment 8 The vector of embodiment 7, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core (SEQ ID NO:5).
  • Embodiment 9 The vector of embodiment 8, wherein the sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 core (SEQ ID NO:5).
  • Embodiment 10 The vector of embodiment 7, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 ultra core (SEQ ID NO:6).
  • Embodiment 11 The vector of embodiment 10, wherein the sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 ultra core (SEQ ID NO:6).
  • Embodiment 12 The vector according to any one of embodiments 1-11, wherein said expression cassette further comprises a RELA TF binding site or an effective fragment thereof.
  • Embodiment 13 The vector of embodiment 12, wherein said RELA TF binding site comprises or consists of the nucleic acid sequence of SEQ ID NO:7).
  • Embodiment 14 The vector according to any one of embodiments 1-11, wherein said expression cassette comprises enhancer element 4 or an effective fragment thereof.
  • Embodiment 15 The vector of embodiment 14, wherein said expression cassette comprises an enhancer element 4R or an effective fragment thereof.
  • Embodiment 16 The vector of embodiment 15, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R core (SEQ ID NO: 10).
  • Embodiment 17 The vector of embodiment 15, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R ultra core (SEQ ID NO: 11).
  • Embodiment 18 The vector of embodiment 16, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment consists of the nucleic acid sequence of enhancer element 4R ultra core (SEQ ID NO: 11).
  • Embodiment 19 The vector according to any one of embodiments 1-18, wherein said expression cassette comprises an enhancer element 4L or an effective fragment thereof.
  • Embodiment 20 The vector of embodiment 19, wherein said expression cassette comprises an effective fragment of enhancer element 4L where said fragment comprises or consists of the sequence of 4L core sequence (SEQ ID NO: 13).
  • Embodiment 21 The vector according to any one of embodiments 1-20, wherein said expression cassette comprises an intron enhancer element 3 (SEQ ID NO: 14) or an effective fragment thereof.
  • said expression cassette comprises an intron enhancer element 3 (SEQ ID NO: 14) or an effective fragment thereof.
  • Embodiment 22 The vector of embodiment 21, wherein said expression cassette comprise an intron enhancer element 3 middle fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15.
  • Embodiment 23 The vector according to any one of embodiments 21-22, wherein said expression cassette comprises an intron enhancer element 3 right fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16.
  • Embodiment 24 The vector according to any one of embodiments 1- 23, wherein said nucleic acid that encodes a nucleic acid that encodes gp91 phox is a CYBB cDNA or a codon-optimized CYBB.
  • Embodiment 25 The vector of embodiment 24, wherein said nucleic acid that encodes gp91 phox is a CYBB cDNA (SEQ ID NO: 17).
  • Embodiment 26 The vector of embodiment 24, wherein said nucleic acid that encodes gp91 phox is a codon optimized CYBB.
  • Embodiment 27 The vector of embodiment 26, wherein the sequence of said nucleic acid that encodes gp91 phox is a codon optimized CYBB selected from the group consisting of jCAT codon optimized CYBB (SEQ ID NO: 18), GeneArt optimized CYBB (SEQ ID NO:20), IDT optimized CYBB SEQ ID NO:21), and previous clinical candidate (SEQ ID NO: 19).
  • Embodiment 28 The vector of embodiment 26, wherein the sequence of said nucleic acid that encodes gp91 phox is a jCAT codon optimized CYBB (SEQ ID NO: 18).
  • Embodiment 29 The vector according to any one of embodiments 1-28, wherein said vector comprises a y region vector genome packaging signal.
  • Embodiment 30 The vector according to any one of embodiments 1-29, wherein said vector comprise a 5' LTR comprising a CMV enhancer/promoter.
  • Embodiment 31 The vector according to any one of embodiments 1-30, wherein said vector comprises a Rev Responsive Element (RRE).
  • RRE Rev Responsive Element
  • Embodiment 32 The vector according to any one of embodiments 1-31, wherein said vector comprises a central polypurine tract.
  • Embodiment 33 The vector according to any one of embodiments 1-32, wherein said vector comprises a post-translational regulatory element.
  • Embodiment 34 The vector of embodiment 33, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE).
  • Embodiment 35 The vector according to any one of embodiments 1-34, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.
  • WPRE Woodchuck Post-transcriptional Regulatory Element
  • Embodiment 36 The vector of embodiment 1, wherein said vector comprises the features of full-sized 2-4R-Int3-pro-mCit-WPRE shown in Figure 19, where the mCit is replaced with a nucleic acid encoding Gp91 phox .
  • Embodiment 37 The vector of embodiment 1, wherein said vector comprises the features of UC 2-4R-Int3-pro-coGp91 phox -WRPE shown in Figure 20, panel A.
  • Embodiment 38 The vector of embodiment 37, wherein said vector comprise the features shown in the vector represented in Figure 20, panel B.
  • Embodiment 39 The vector of embodiment 38, wherein said vector comprises the nucleotide sequence of ultra core (UC) 2-4R-Int3-Pro-(GP91-jcat)-WPRE (SEQ ID NO: 22).
  • Embodiment 40 The vector according to any one of embodiments embodiment 1-39, wherein said vector shows high expression in CD33+ (bulk myeloid cells), high expression in CD19+ (B cells, high expression in CD66b+ CD15+ CDllb-i- CD16+ (mature neutrophils), and low or no expression in CD3+ T cells.
  • Embodiment 41 A host cell transduced with a vector according to any one of embodiments 1-40.
  • Embodiment 42 The host cell of embodiment 41, wherein the cell is a stem cell.
  • Embodiment 43 The host cell of embodiment 42, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.
  • Embodiment 44 The host cell of embodiment 41, wherein the cell is a human hematopoietic progenitor cell.
  • Embodiment 45 The host cell of embodiment 44, wherein the human hematopoietic progenitor cell is a CD34+ cell.
  • Embodiment 46 A method of treating a chronic granulomatous disease (X-
  • Embodiment 47 The method of embodiment 46, wherein the cell is a stem cell.
  • Embodiment 48 The host cell of embodiment 46, wherein said cell is a stem cell derived from bone marrow.
  • Embodiment 49 The method of embodiment 46, wherein the cell is a human hematopoietic stem and progenitor cell.
  • Embodiment 50 The method of embodiment 49, wherein the human hematopoietic progenitor cell is a CD34 + cell.
  • Embodiment 51 A recombinant nucleic acid encoding one or more of the following:
  • a CYBB promoter or an effective fragment thereof
  • a CYBB endogenous enhancer element 2 (CYBB B-cell enhancer), or an effective fragment thereof; and/or
  • a CYBB endogenous enhancer 4R CYBB endogenous myeloid enhancer
  • an effective fragment thereof CYBB endogenous myeloid enhancer
  • a CYBB endogenous enhancer 4L or an effective fragment thereof;
  • a CYBB endogenous myeloid Intron 3 enhancer or an effective fragment thereof;
  • Embodiment 52 The nucleic acid of embodiment 51, wherein said nucleic acid encodes a sequence comprising or consisting of a full-length endogenous CYBB promoter (SEQ ID NO:l).
  • Embodiment 53 The nucleic acid of embodiment 51, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
  • Embodiment 54 The nucleic acid of embodiment 53, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
  • Embodiment 55 The nucleic acid of embodiment 51, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
  • Embodiment 56 The nucleic acid of embodiment 55, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
  • Embodiment 57 The nucleic acid according to any one of embodiments 51-
  • nucleic acid encodes an effective fragment of a CYBB endogenous enhancer element 2 (CYBB B-cell enhancer).
  • Embodiment 58 The nucleic acid of embodiment 57, wherein the nucleic acid sequence of said a CYBB endogenous enhancer element 2 comprises or consists of the sequence of enhancer element 2 core (SEQ ID NO:5).
  • Embodiment 59 The nucleic acid of embodiment 57, wherein the nucleic acid sequence of said a CYBB endogenous enhancer element 2 comprises or consists of the sequence of enhancer element 2 ultra core (SEQ ID NO: 6).
  • Embodiment 60 The nucleic acid according to any one of embodiments 51-
  • nucleic acid comprises an effective fragment of a CYBB endogenous enhancer 4R (CYBB endogenous myeloid enhancer).
  • Embodiment 61 The nucleic acid of embodiment 60, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous enhancer 4R comprises or consists of the sequence of enhancer element 4R ultra core (SEQ ID NO: 10).
  • Embodiment 62 The nucleic acid according to any one of embodiments 51-
  • nucleic acid comprises an effective fragment of an enhancer element 4L.
  • Embodiment 63 The nucleic acid of embodiment 62, wherein said effective fragment of an enhancer element 4L comprises or consists of the sequence of the 4L core sequence (SEQ ID NO: 13).
  • Embodiment 64 The nucleic acid according to any one of embodiments 51-
  • nucleic acid comprises an effective fragment of a CYBB endogenous myeloid intron 3 enhancer.
  • Embodiment 65 The nucleic acid of embodiment 64, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous myeloid intron 3 enhancer comprises or consists of an element 3 middle fragment nucleic acid sequence (SEQ ID NO:15).
  • Embodiment 66 The nucleic acid according to any one of embodiments 64-
  • nucleic acid sequence of said effective fragment of a CYBB endogenous myeloid intron 3 enhancer comprises or consists of an intron enhancer element 3 right fragment (SEQ ID NO: 16).
  • Embodiment 67 The nucleic acid according to any one of embodiments 51-
  • nucleic acid comprises a jCAT codon optimized CYBB (SEQ ID NO: 18).
  • Embodiment 68 The nucleic acid according to any one of embodiments 51-
  • nucleic acid comprises an expression cassette.
  • Embodiment 69 The nucleic acid of embodiment 68, wherein said expression cassette is effective to express Gp91 phox in vivo.
  • Embodiment 70 The nucleic acid according to any one of embodiments 51-
  • nucleic acid comprises a lentiviral vector according to any one of embodiments 1-40.
  • a “promoter” refers to a regulatory sequence in a nucleic acid required to initiate transcription of a gene (e.g., a gene operably coupled to the promoter).
  • An “enhancer” refers to a regulatory DNA sequence that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene.
  • an "effective fragment” when used with respect to a promoter refers to a fragment of the full-length promoter that is sufficient to initiate transcription of a gene operably linked to that promoter.
  • an "effective fragment” when used with respect to an enhancer refers to a fragment of the full-length enhancer that is sufficient to provide regulate expression of an operably linked gene when bound by a transcription factor. In certain embodiments the regulation is comparable with respect to expression level and/or lineage offered by the full-length enhancer.
  • operably linked refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence.
  • a promoter is operably linked to a gene when that promoter is placed in a location that permits that promoter to initiate transcription of that gene.
  • An enhancer is operably linked to a gene when that enhancer, when bound by an appropriate transcription factor, is able to regulate (e.g., to upregulate) expression of that gene.
  • Recombinant is used consistently with its usage in the art to refer to a nucleic acid sequence that comprises portions that do not naturally occur together as part of a single sequence or that have been rearranged relative to a naturally occurring sequence.
  • a recombinant nucleic acid is created by a process that involves the hand of man and/or is generated from a nucleic acid that was created by hand of man (e.g., by one or more cycles of replication, amplification, transcription, etc.).
  • a recombinant vims is one that comprises a recombinant nucleic acid.
  • a recombinant cell is one that comprises a recombinant nucleic acid.
  • recombinant lentiviral vector or “recombinant LV) refers to an artificially created polynucleotide vector assembled from an LV and a plurality of additional segments as a result of human intervention and manipulation.
  • an effective amount is meant the amount of a required agent or composition comprising the agent to ameliorate or eliminate symptoms of a disease relative to an untreated patient.
  • the effective amount of composition(s) used to practice the methods described herein for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • Figure 1 illustrates the endogenous expression pattern of gp91 phox in human blood cells.
  • Figure 2 illustrates constructs used to probe enhancer activity.
  • Figure 5 shows expression of enhancer constructs in transduced RAMOs (B- cell line) D14 flow.
  • Figure 6 shows expression of enhancer constructs in transduced Jurkats (T- cell line) D16 flow.
  • Figure 7 shows expression of enhancer constructs in CB CD34+ differentiated
  • Figure 9 shows expression of enhancer constructs in transduced Jurkats (T- cell line) D16 flow.
  • Figure 10 shows expression of enhancer constructs in transduced RAMOs (B- cell line) D14 flow.
  • Figure 11 shows structure of E2-E4R-Int3-pro-mCit-WPRE vector (top) and the same vector where mCitrine is replaced with nucleic acid encoding Gp91 phox (bottom).
  • Figure 12 shows expression of the reduced size vectors in CB CD34+
  • Figure 13 shows expression of the reduced size vectors in CB CD34+
  • Figure 14 shows expression of the reduced size vectors in Jurkat Cells (T-Cell Line).
  • Figure 15 shows expression of the reduced size vectors in RAMOS Cells (B-
  • Figure 16 shows raw small scale titers of the "core”, the “ultra core”, the “extra core” and the “extra ultra core” constructs.
  • Figure 17 shows the expression levels produced by various codon optimizations of Gp91 phox in PLB-985 X-CGD' cells.
  • Figure 18 shows the raw titers of various codon optimizations of MSP-
  • Figure 19 illustrates one embodiment of a lenti viral vector for treatment of X-
  • the mCit reporter would be replaced with a nucleic acid sequence encoding a Gp91phox, e.g., as described herein.
  • FIG. 20 panels A-B, illustrate one embodiment of an optimized lentiviral vector for treatment of X-CGD.
  • Panel A schematically illustrates the elements of UC 2-4R- Int3-pro-coGp91 phox -WRPE.
  • Panel B shows a "map" of the vector.
  • Figure 21 illustrates improvement in titer (top panel) and infectivity (bottom panel) as the vector was optimized from the original 2-4R-Int3-pro-mCit-WPRE to the CORE variant and to the ULTRA CORE (UC) variant.
  • the UC variant MyeloVec is a lead candidate vector).
  • FIG. 22 shows that MyeloVec is able to recapitulate the endogenous expression pattern of the native CYBB gene in blood cells (panel A) and bone marrow cells (panel B) respectively.
  • Figure 23 shows that MyeloVec is able to recapitulate the temporal expression pattern of the native CYBB gene throughout neutrophil development. The expression gets higher as the neutrophils mature, mimicking the pattern of the native CYBB gene.
  • Figure 24 shows the restoration of Gp91 phox expression.
  • Figure 25 shows show the restoration of oxidase activity to wildtype levels.
  • Figure 26 shows restoration of Gp91 phox expression in neutrophils and monocytes in the peripheral blood.
  • Figure 27 shows restoration of oxidase activity near wildtype levels in the blood neutrophils and monocytes.
  • Figure 28 shows restoration of high levels of Gp91 phox expression in the bone marrow neutrophils and monocytes.
  • Figure 29 shows restoration of wildtype levels of oxidase activity.
  • Figure 30 shows the ability of MyeloVec to restore wildtype levels of
  • Figure 31 shows the ability of MyeloVec to restore wildtype levels of cellular oxidase activity in the human X-CGD neutrophils (DHR assay).
  • Figure 32 shows the ability of MyeloVec to restore wildtype levels of bulk oxidase activity in human X-CGD neutrophils at an average VCN of 1.63 (cytochrome C assay).
  • lentiviral vectors are provided for the treatment (or prophylaxis) of X-linked Chronic Granulomatous Disease (X-CGD) are provided.
  • X-CGD X-linked Chronic Granulomatous Disease
  • the vectors are optimized to reduce vector size, increase expression level and titer. Additionally, in various embodiments the vectors recapitulate the lineage specific expression pattern of the native CYBB gene, e.g., as described herein (see, e.g., Figure 1).
  • each putative enhancer element was cloned upstream of the endogenous CYBB promoter to drive expression of a reporter gene (mCitrine) (see, e.g., Figure 2).
  • mCitrine reporter gene
  • enhancer element 4 drives high levels of expression in mature neutrophils and in monocytes, with no expression in B-cells. It was also discovered that enhancer element 2 drives high levels of lineage specific expression in B-cells with no expression in neutrophils. None of the enhancer elements express in Jurkats (T-cells), suggesting lineage specific expression of each enhancer element.
  • enhancer element 4 is made of two distinct enhancer modules (4L and 4R) and these were evaluated to determine if one of these elements could be eliminated to decrease the size of the vector. [0142] Additionally, reduced variants of enhancer element 2, enhancer element 4, intron enhancer 3, and the CYBB endogenous promoter were made and evaluated. Codon optimizations of the nucleic acid encoding Gp91 phox were also evaluated.
  • a recombinant lentiviral vector (LV) for the treatment of chronic granulomatous disease comprising an expression cassette comprising a nucleic acid construct comprising a CYBB endogenous promoter or effective fragment thereof; and a nucleic acid that encodes gp91 phox operably linked to the CYBB promoter or promoter fragment.
  • the CYBB promoter or effective fragment thereof comprises a full-length endogenous CYBB promoter (see, e.g., Table 1, SEQ ID NO:l).
  • the CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (see, e.g., Table 1, SEQ ID NO:3). In certain embodiments the CYBB promoter consists of an effective fragment of the CYBB promoter whose sequence consists of the minimal CYBB promoter (see, e.g., Table 1, SEQ ID NO:3).
  • the expression cassette in the lentiviral vector comprises an enhancer element 2 (see, e.g., Table 1, SEQ ID NO: 4) or an effective fragment thereof.
  • the sequence of the effective fragment of enhancer element 2 comprises or consists of the sequence of enhancer element 2 "core” (see, e.g., Table 1, SEQ ID NO:5).
  • the sequence of the effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 core (see, e.g., Table 1, SEQ ID NO:5).
  • the sequence of the effective fragment of enhancer element 2 comprises or consists of the enhancer element 2 "ultra core” sequence (see, e.g., Table 1,
  • sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 ultra core (see, e.g., Table 1, SEQ ID NO:6).
  • the expression cassette comprising the lentiviral vector further comprises a RELA TF binding site or an effective fragment thereof.
  • the RELA TF binding site comprises or consists of the nucleic acid sequence of SEQ ID NO:7 in Table 1,
  • the expression cassette in the lentiviral vector comprises enhancer element 4 (see, e.g., Table 1, SEQ ID NO:8) or an effective fragment thereof.
  • the expression cassette comprises an enhancer element 4R (see, e.g., Table 1, SEQ ID NO: 9) or an effective fragment thereof.
  • the expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence comprises or consists of the nucleic acid sequence of enhancer element 4R core (see, e.g., Table 1, SEQ ID NO: 10).
  • the expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R "ultra core” (see, e.g., Table 1, SEQ ID NO: 11). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment consists of the nucleic acid sequence of enhancer element 4R ultra core (see, e.g., Table 1, SEQ ID NO: 11).
  • the expression cassette in the lentiviral vector comprises an enhancer element 4L ((see, e.g., Table 1, SEQ ID NO: 12) or an effective fragment thereof.
  • the effective fragment of enhancer element 4L comprises or consists of the sequence of 4L core sequence (see, e.g., Table 1, SEQ ID NO: 13).
  • the effective fragment of enhancer element 4L consists of the sequence of 4L core sequence (see, e.g., Table 1, SEQ ID NO: 13).
  • the expression cassette in the lentiviral vector comprises an intron enhancer element 3 (see, e.g., Table 1, SEQ ID NO: 14) or an effective fragment thereof.
  • the expression cassette in the lentiviral vector comprises or consists of an intron enhancer element 3 middle fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15 in Table 1.
  • the expression cassette in the lentiviral vector consists of an intron enhancer element 3 middle fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15 in Table 1.
  • the expression cassette in the lentiviral vector comprises or consists of an intron enhancer element 3 right fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 in Table 1.
  • nucleic acid that encodes gp91 phox is a full CYBB gene, a CYBB cDNA, or a codon-optimized CYBB. In certain embodiments the nucleic acid that encodes gp91 phox is a CYBB cDNA (see, e.g., Table 1, SEQ ID NO:17).
  • the nucleic acid that encodes gp91 phox is a codon optimized CYBB (e.g., a jCAT codon optimized CYBB (see, e.g., Table 1, SEQ ID NO:18), a GeneArt optimized CYBB (see, e.g., Table 1, SEQ ID NO:20), an IDT optimized CYBB (see, e.g., Table 1, SEQ ID NO:21), and previous clinical candidate (see, e.g., Table 1, SEQ ID NO: 19)).
  • the sequence of said nucleic acid that encodes gp91 phox is a jCAT codon optimized CYBB (see, e.g., Table 1, SEQ ID NO: 18).
  • a recombinant nucleic acid comprising any one or more of the CYBB regulatory elements described herein is contemplated.
  • the recombinant nucleic acid comprises an expression cassette, e.g., an expression cassette effective to express Gp91 phox in vivo. It will be recognized that such an expression cassette can be used with other constructs, e.g., in conjunction with a CRISPR construct. Table 1. Nucleic acid sequences of various components of the lentiviral vectors for treatment of X-CGD as described herein.
  • the lentiviral vectors (LVs) described herein can have various "safety" features that can include, for example, the presence of an insulator (e.g., an FB insulator in the 3'LTR). Additionally, or alternatively, in certain embodiments, the HIV LTR has been substituted with an alternative promoter (e.g., a CMV) to yield a higher titer vector without the inclusion of the HIV TAT protein during packaging. Other strong promoters (e.g., RSV, and the like can also be used).
  • an alternative promoter e.g., a CMV
  • Other strong promoters e.g., RSV, and the like can also be used.
  • the lentiviral vectors described herein contain any one or more of the elements typically found in lentiviral vectors.
  • Such elements include, but need not be limited to a y region vector genome packaging signal, a Rev Responsive Element (RRE), a polypurine tract (e.g., a central polypurine tract, a 3' polypurine tract, etc.), a post- translational regulatory element (e.g., a modified Woodchuck Post-transcriptional Regulatory Element (WPRE)), an insulator, and the like, e.g., as described below.
  • the vector is a SIN vector substantially incapable of reconstituting a wild-type lentivirus through recombination.
  • the vector comprises the features of "ultra core” (UC)
  • the vector comprises the features shown in the vector represented in Figure 20, panel B.
  • the vector comprises the nucleotide sequence of ultra core (UC) 2-4R- Int3-Pro-(GP91-jcat)-WPRE (SEQ ID NO: 22).
  • the vector shows high expression in CD33+ (bulk myeloid cells), and/or high expression in CD19+ (B cells), high expression in CD66b+ CD15+ CDllb-i- CD16+ (mature neutrophils), and/or low or no expression in CD3+ (T cells).
  • the vector shows high expression in CD33+ (bulk myeloid cells), high expression in CD19+ (B cells, high expression in CD66b+ CD15+ CDllb-i- CD16+ (mature neutrophils), and low or no expression in CD3+ T cells.
  • Example 1 the vectors described herein are effective to transduce cells at high titer and to also provide high levels of expression of Gp91 phox .
  • LVs described herein e.g. , recombinant TAT-independent, SIN LVs that express a nucleic acid encoding a Gp91 phox can be used to effectively treat X-linked chronic granulomatous disease (X-CGD) in subjects (e.g. , human and non-human mammals). It is believed these vectors can be used for the modification of stem cells (e.g., hematopoietic stem and progenitor cells) that can be introduced into a subject in need thereof for the treatment of, e.g., subjects identified as having X-CGD.
  • stem cells e.g., hematopoietic stem and progenitor cells
  • the resulting cells will produce enough of the transgenic Gp91 phox protein to demonstrate significant improvement in subject health. It is also believed the vectors can be directly administered to a subject to achieve in vivo transduction of the target (e.g., hematopoietic stem or progenitor cells) and thereby also effect a treatment of subjects in need thereof.
  • the target e.g., hematopoietic stem or progenitor cells
  • the LVs described herein can comprise various safety features.
  • the HIV LTR has been substituted with a CMV promoter to yield higher titer vector without the inclusion of the HIV TAT protein during packaging.
  • an insulator e.g., the FB insulator
  • the LVs are also constructed to provide efficient transduction and high titer.
  • the lentiviral vector can comprise a
  • CYBB gene or cDNA the nucleic acid encoding Gp91phox is codon optimized.
  • Numerous methods of codon optimization are known to those of skill in the art.
  • One illustrative method is JCat (Java Codon Adaptation Tool).
  • the jCAT tool adapts gene codon usage to most sequenced prokaryotes and various eukaryotic gene expression hosts. In contrast to many tools, JCat does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators.
  • JCat The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the input sequence and the newly adapted sequence.
  • CAI Codon Adaptation Index
  • Still another codon optimization tool is IDT.
  • the IDT codon optimization tool was developed to optimize a DNA or protein sequence from one organism for expression in another by reassigning codon usage based on the frequencies of each codon’s usage in the new organism. For example, valine is encoded by 4 different codons (GUG, GUU, GUC, and GUA). In human cell lines, however, the GUG codon is preferentially used (46% use vs. 18, 24, and 12%, respectively).
  • the codon optimization tool takes this information into account and assigns valine codons with those same frequencies.
  • the tool algorithm eliminates codons with less than 10% frequency and re-normalizes the remaining frequencies to 100%.
  • the optimization tool reduces complexities that can interfere with manufacturing and downstream expression, such as repeats, hairpins, and extreme GC content.
  • the IDT optimization tool is available from IDT (Integrated DNA Technologies, Coralville, Iowa) and can be found at ww.idtdna.com/CodonOpt.
  • Other codon optimization tools include, but are not limited to CodonW an open source software program that can be found at codonw.sourceforge.net, and the OptimumGeneTM algorithm from GenScript.
  • the codon optimized Gp91 phox can be the sequence used in the current clinical candidate MSP-Gp91 phox -WPRE.
  • the lentiviral vectors described herein comprise a TAT-independent, self-inactivating (SIN) configuration.
  • SIN TAT-independent, self-inactivating
  • SIN vectors are ones in which the production of full-length vector RNA in transduced cells is greatly reduced or abolished altogether. This feature minimizes the risk that replication- competent recombinants (RCRs) will emerge. Furthermore, it reduces the risk that that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed.
  • SIN LVs can often permit full activity of the internal promoter.
  • the SIN design increases the biosafety of the LVs. The majority of the HIV
  • the LTR is comprised of the U3 sequences.
  • the U3 region contains the enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation.
  • Several of these promoter elements are essential for viral replication.
  • Some of the enhancer elements are highly conserved among viral isolates and have been implicated as critical virulence factors in viral pathogenesis. The enhancer elements may act to influence replication rates in the different cellular target of the vims
  • the retrovirus is self-inactivating (SIN) and those vectors are known as SIN transfer vectors.
  • self-inactivation is achieved through the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, /. ⁇ ? ., the DNA used to produce the vector RNA. During RT, this deletion is transferred to the 5' LTR of the proviral DNA.
  • this deletion is transferred to the 5' LTR of the proviral DNA.
  • the 5' end of the U3 region serves another essential function in vector transfer, being required for integration (terminal dinucleotide+att sequence).
  • the terminal dinucleotide and the att sequence may represent the 5' boundary of the U3 sequences which can be deleted.
  • some loosely defined regions may influence the activity of the downstream polyadenylation site in the R region. Excessive deletion of U3 sequence from the 3'LTR may decrease polyadenylation of vector transcripts with adverse consequences both on the titer of the vector in producer cells and the transgene expression in target cells.
  • the lentiviral sequences removed from the LTRs are replaced with comparable sequences from a non- lentiviral retrovirus, thereby forming hybrid LTRs.
  • the lentiviral R region within the LTR can be replaced in whole or in part by the R region from a non-lentiviral retrovirus.
  • the lentiviral TAR sequence a sequence which interacts with TAT protein to enhance viral replication, is removed, preferably in whole, from the R region.
  • the TAR sequence is then replaced with a comparable portion of the R region from a non- lentiviral retrovirus, thereby forming a hybrid R region.
  • the LTRs can be further modified to remove and/or replace with non-lentiviral sequences all or a portion of the lentiviral U3 and U5 regions.
  • the SIN configuration provides a retroviral LTR comprising a hybrid lentiviral R region that lacks all or a portion of its TAR sequence, thereby eliminating any possible activation by TAT, wherein the TAR sequence or portion thereof is replaced by a comparable portion of the R region from a non-lentiviral retrovirus, thereby forming a hybrid R region.
  • the retroviral LTR comprises a hybrid R region, wherein the hybrid R region comprises a portion of the HIV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 10 in US 2003/0039636) lacking the TAR sequence, and a portion of the MoMSV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9 in 2003/0039636) comparable to the TAR sequence lacking from the HIV R region.
  • the hybrid R region comprises a portion of the HIV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 10 in US 2003/0039636) lacking the TAR sequence, and a portion of the MoMSV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9 in 2003/0039636) comparable to the TAR sequence lacking from the HIV R region.
  • the entire hybrid R region comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11 in 2003/0039636.
  • Suitable lentiviruses from which the R region can be derived include, for example, HIV (HIV-1 and HIV-2), EIV, SIV and FIV.
  • Suitable retroviruses from which non- lentiviral sequences can be derived include, for example, MoMSV, MoMLV, Friend, MSCV, RSV and Spumaviruses.
  • the lentivirus is HIV and the non- lentiviral retrovirus is MoMSV.
  • the LTR comprising a hybrid R region is a left (5') LTR and further comprises a promoter sequence upstream from the hybrid R region.
  • Preferred promoters are non-lentiviral in origin and include, for example, the U3 region from a non-lentiviral retrovirus (e.g., the MoMSV U3 region).
  • the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 12 in US 2003/0039636.
  • the left (5') LTR further comprises a lentiviral U5 region downstream from the hybrid R region.
  • the U5 region is the HIV U5 region including the HIV att site necessary for genomic integration.
  • the U5 region comprises the nucleotide sequence shown in SEQ ID NO: 13 in US 2003/0039636.
  • the entire left (5') hybrid LTR comprises the nucleotide sequence shown in SEQ ID NO: 1 in US 2003/0039636.
  • the LTR comprising a hybrid R region is a right (3') LTR and further comprises a modified (e.g., truncated) lentiviral U3 region upstream from the hybrid R region.
  • the modified lentiviral U3 region can include the att sequence, but lack any sequences having promoter activity, thereby causing the vector to be SIN in that viral transcription cannot go beyond the first round of replication following chromosomal integration.
  • the modified lentiviral U3 region upstream from the hybrid R region consists of the 3' end of a lentiviral (e.g. , HIV) U3 region up to and including the lentiviral U3 att site.
  • the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15 in US 2003/0039636.
  • the right (3') LTR further comprises a polyadenylation sequence downstream from the hybrid R region.
  • the polyadenylation sequence comprises the nucleotide sequence shown in SEQ ID NO: 16 in US 2003/0039636.
  • the entire right (5') LTR comprises the nucleotide sequence shown in SEQ ID NO: 2 or 17 of US 2003/0039636.
  • the cassette expressing a nucleic acid encoding gp91 phox a SIN vector with the CMV enhancer/promoter substituted in the 5' LTR.
  • the CMV promoter typically provides a high level of non-tissue specific expression.
  • Other promoters with similar constitutive activity include, but are not limited to the RSV promoter, and the SV40 promoter.
  • Mammalian promoters such as the beta-actin promoter, ubiquitin C promoter, elongation factor lapromoter, tubulin promoter, etc. , may also be used.
  • the foregoing SIN configurations are illustrative and non-limiting. Numerous
  • LTR transcription is reduced by about 95% to about 99%.
  • LTR may be rendered at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% at least about 96%, at least about 97%, at least about 98%, or at least about 99% transcriptionally inactive.
  • insulators are inserted into the lentiviral vectors described herein.
  • Insulators are DNA sequence elements present throughout the genome. They bind proteins that modify chromatin and alter regional gene expression.
  • the placement of insulators in the vectors described herein offer various potential benefits including, inter alia : 1) Shielding of the vector from positional effect variegation of expression by flanking chromosomes (/. ⁇ ? ., barrier activity); and 2) Shielding flanking chromosomes from insertional trans- activation of gene expression by the vector (enhancer blocking).
  • insulators can help to preserve the independent function of genes or transcription units embedded in a genome or genetic context in which their expression may otherwise be influenced by regulatory signals within the genome or genetic context (see, e.g. , Burgess-Beusse el al. (2002) Proc. Natl. Acad. Sci. USA, 99: 16433; and Zhan el al. (2001) Hum. Genet., 109: 471).
  • insulators may contribute to protecting lenti virus -expressed sequences from integration site effects, which may be mediated by ex acting elements present in genomic DNA and lead to deregulated expression of transferred sequences.
  • LVs are provided in which an insulator sequence is inserted into one or both LTRs or elsewhere in the region of the vector that integrates into the cellular genome.
  • the first and best characterized vertebrate chromatin insulator is located within the chicken b-globin locus control region.
  • This element which contains a DNase-I hypersensitive site-4 (cHS4), appears to constitute the 5' boundary of the chicken b-globin locus (Prioleau el al. (1999) EMBO J. 18: 4035-4048).
  • cHS4 DNase-I hypersensitive site-4
  • a 1.2-kb fragment containing the cHS4 element displays classic insulator activities, including the ability to block the interaction of globin gene promoters and enhancers in cell lines (Chung et al. (1993) Cell, 74: 505-514), and the ability to protect expression cassettes in Drosophila (Id.), transformed cell lines (Pikaart el al.
  • One illustrative and suitable insulator is FB (FII/BEAD-A), a 77 bp insulator element, that contains the minimal CTCF binding site enhancer-blocking components of the chicken b-globin 5' HS4 insulators and a homologous region from the human T-cell receptor alpha/delta blocking element alpha/delta I (BEAD-I) insulator described by Ramezani et al. (2008) Stem Cell 26: 3257-3266.
  • the FB “synthetic” insulator has full enhancer blocking activity.
  • This insulator is illustrative and non-limiting.
  • Other suitable insulators may be used including, for example, the full-length chicken beta-globin HS4 or insulator sub-fragments thereof, the ankyrin gene insulator, and other synthetic insulator elements.
  • the vectors described herein further comprise a packaging signal.
  • a "packaging signal,” “packaging sequence,” or “PSI sequence” is any nucleic acid sequence sufficient to direct packaging of a nucleic acid whose sequence comprises the packaging signal into a retroviral particle. The term includes naturally occurring packaging sequences and also engineered variants thereof.
  • Packaging signals of a number of different retroviruses, including lentiviruses, are known in the art.
  • One illustrative, but non-limiting PSI is provided by SEQ ID NO:25.
  • the lenti viral vectors described herein comprise a Rev response element (RRE) to enhance nuclear export of unspliced RNA.
  • RREs are well known to those of skill in the art.
  • Illustrative RREs include, but are not limited to RREs such as that located at positions 7622-8459 in the HIV NL4-3 genome (Genbank accession number AF003887) as well as RREs from other strains of HIV or other retroviruses. Such sequences are readily available from Genbank or from the database with URL hiv- web.lanl.gov/content/index.
  • RRE Rev response element
  • the lentiviral vectors described herein further include a polypurine tract (e.g., central polypurine tract (cPPT), 3' poplypurine tract (3'PPT)). Insertion of a fragment containing the 3'PPT (see, e.g., SEQ ID NO:28) or the central polypurine tract (cPPT) in lentiviral (e.g., HIV-1) vector constructs is known to enhance transduction efficiency.
  • cPPT central polypurine tract
  • 3'PPT 3' poplypurine tract
  • the lentiviral vectors (LVs) described herein may comprise any of a variety of posttranscriptional regulatory elements (PREs) whose presence within a transcript increases expression of the heterologous nucleic acid (e.g., gp91 phox ) at the protein level.
  • PREs posttranscriptional regulatory elements
  • gp91 phox heterologous nucleic acid
  • PRE One type of PRE is an intron positioned within the expression cassette, which can stimulate gene expression.
  • introns can be spliced out during the life cycle events of a lentivirus.
  • introns are typically placed in an opposite orientation to the vector genomic transcript.
  • Posttranscriptional regulatory elements that do not rely on splicing events offer the advantage of not being removed during the viral life cycle.
  • Some examples are the posttranscriptional processing element of herpes simplex vims, the posttranscriptional regulatory element of the hepatitis B vims (HPRE) and the woodchuck hepatitis virus (WPRE). Of these the WPRE is typically preferred as it contains an additional cis-acting element not found in the HPRE.
  • This regulatory element is typically positioned within the vector so as to be included in the RNA transcript of the transgene, but outside of stop codon of the transgene translational unit.
  • the WPRE is characterized and described in U.S. Pat. No: 6,136,597.
  • the WPRE is an RNA export element that mediates efficient transport of RNA from the nucleus to the cytoplasm. It enhances the expression of transgenes by insertion of a cis- acting nucleic acid sequence, such that the element and the transgene are contained within a single transcript. Presence of the WPRE in the sense orientation was shown to increase transgene expression by up to 7- to 10-fold.
  • Retroviral vectors transfer sequences in the form of cDNAs instead of complete intron-containing genes as introns are generally spliced out during the sequence of events leading to the formation of the retroviral particle.
  • Introns mediate the interaction of primary transcripts with the splicing machinery. Because the processing of RNAs by the splicing machinery facilitates their cytoplasmic export, due to a coupling between the splicing and transport machineries, cDNAs are often inefficiently expressed. Thus, the inclusion of the WPRE (see, e.g., SEQ ID NO:27) in a vector results in enhanced expression of transgenes.
  • the recombinant lentiviral vectors (LV) and resulting virus described herein are capable of transferring a heterologous nucleic acid sequence (e.g. , a nucleic acid encoding a gp91 phox ) into a mammalian cell.
  • a heterologous nucleic acid sequence e.g. , a nucleic acid encoding a gp91 phox
  • vectors described herein are preferably used in conjunction with a suitable packaging cell line or co transfected into cells in vitro along with other vector plasmids containing the necessary retroviral genes (e.g., gag and pol) to form replication incompetent virions capable of packaging the vectors of the present invention and infecting cells.
  • the vectors are introduced via transfection into a packaging cell line.
  • the packaging cell line produces viral particles that contain the vector genome. Methods for transfection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art.
  • the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with or without a dominant selectable marker, such as neomycin, DHFR, Glutamine synthetase, followed by selection in the presence of the appropriate drug and isolation of clones.
  • the selectable marker gene can be linked physically to the packaging genes in the construct.
  • Stable cell lines wherein the packaging functions are configured to be expressed by a suitable packaging cell are known (see, e.g., U.S. Patent No. 5,686,279, which describes packaging cells).
  • a suitable packaging cell for the production of vims particles, one may employ any cell that is compatible with the expression of lentiviral Gag and Pol genes, or any cell that can be engineered to support such expression.
  • producer cells such as 293T cells and HT1080 cells may be used.
  • the packaging cells with a lentiviral vector incorporated therein form producer cells.
  • Producer cells are thus cells or cell-lines that can produce or release packaged infectious viral particles carrying the therapeutic gene of interest (e.g., a Gp91 phox ). These cells can further be anchorage dependent which means that these cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic.
  • Some examples of anchorage dependent cell lines used as lentiviral vector packaging cell lines when the vector is replication competent are HeLa or 293 cells and PERC.6 cells.
  • methods are provided of delivering a gene to a cell which is then integrated into the genome of the cell, comprising contacting the cell with a virion containing a lentiviral vector described herein.
  • the cell e.g., in the form of tissue or an organ
  • a subject e.g. , a mammal, animal or human
  • the gene e.g., a nucleic acid encoding gp91 phox
  • the cell can be autologous to the subject (/. ⁇ ?
  • the cells can be from a wide variety including, for example, bone marrow cells, mesenchymal stem cells (e.g., obtained from adipose tissue), and other primary cells derived from human and animal sources.
  • the virion can be directly administered in vivo to a subject or a localized area of a subject (e.g., bone marrow).
  • the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cells, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4 + T cell, a peripheral blood B or T lymphocyte cell, and the like.
  • particularly preferred targets are CD34 + hematopoietic stem and progenitor cells.
  • methods for transducing a human hematopoietic stem cell.
  • the methods involve contacting a population of human cells that include hematopoietic stem cells with one of the foregoing lentivectors under conditions to effect the transduction of a human hematopoietic progenitor cell in said population by the vector.
  • the stem cells may be transduced in vivo or in vitro, depending on the ultimate application. Even in the context of human gene therapy, such as gene therapy of human stem cells, one may transduce the stem cell in vivo or, alternatively, transduce in vitro followed by infusion of the transduced stem cell into a human subject.
  • the human stem cell can be removed from a human, e.g., an X-CGD patient, using methods well known to those of skill in the art and transduced as noted above.
  • the transduced stem cells are then reintroduced into the same or a different human.
  • Stem cell/progenitor cell gene therapy can be used to treat a human.
  • the lentivectors described herein are particularly useful for the transduction of human hematopoietic progenitor cells or hematopoietic stem cells (HSCs), obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4 + T cell, a peripheral blood B or T lymphocyte cell, and the like.
  • HSCs hematopoietic stem cells
  • particularly preferred targets are CD34 + hematopoietic stem and progenitor cells.
  • the vector particles are incubated with the cells using a dose generally in the order of between 1 to 50 multiplicities of infection (MOI) which also corresponds to 1 x 10 5 to 50 x 10 5 transducing units of the viral vector per 10 5 cells.
  • MOI multiplicities of infection
  • the amount of vector may be expressed in terms of HT-29 transducing units (TU).
  • cell-based therapies involve providing stem cells and/or hematopoietic precursors, transduce the cells with the lentivirus encoding, e.g., a Gp91 phox , and then introduce the transformed cells into a subject in need thereof (e.g., a subject with a mutation in the CYBB gene).
  • the lentivirus encoding e.g., a Gp91 phox
  • the methods involve isolating population of cells, e.g., stem cells from a subject, optionally expand the cells in tissue culture, and administer the lentiviral vector whose presence within a cell results in production of a Gp91 phox in the cells in vitro.
  • the cells are then returned to the subject, where, for example, they may provide a population of phagocytic cells that produce the Gp91 phox .
  • a population of cells which may be cells from a cell line or from an individual other than the subject, can be used.
  • Methods of isolating stem cells, immune system cells, etc., from a subject and returning them to the subject are well known in the art. Such methods are used, e.g., for bone marrow transplant, peripheral blood stem cell transplant, etc. , in patients undergoing chemotherapy.
  • stem cells are to be used, it will be recognized that such cells can be derived from a number of sources including bone marrow (BM), cord blood (CB), mobilized peripheral blood stem cells (mPBSC), and the like.
  • BM bone marrow
  • CB cord blood
  • mPBSC mobilized peripheral blood stem cells
  • IPCs induced pluripotent stem cells
  • HSCs hematopoietic stem cells
  • lentiviral vector described herein see, e.g., Figure
  • lentiviral compositions may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Commonly used routes of delivery include inhalation, parenteral, and transmucosal.
  • compositions can include an LV in combination with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration ⁇ Supplementary active compounds can also be incorporated into the compositions.
  • active agents i.e., a lentiviral described herein and/or other agents to be administered together the vector
  • carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, poly glycolic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such compositions will be apparent to those skilled in the art. Suitable materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.
  • compositions are targeted to particular cell types or to cells that are infected by a vims.
  • compositions can be targeted using monoclonal antibodies to cell surface markers, e.g., endogenous markers or viral antigens expressed on the surface of infected cells.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit comprising a predetermined quantity of a LV calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.
  • Unit dose of the LV described herein may conveniently be described in terms of transducing units (T.U.) of lentivector, as defined by titering the vector on a cell line such as HeLa or 293.
  • unit doses can range from 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 s , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 T.U. and higher.
  • compositions can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to about 10 weeks; between about 2 to about 8 weeks; between about 3 to about 7 weeks; about 4 weeks; about 5 weeks; about 6 weeks, etc. It may be necessary to administer the therapeutic composition on an indefinite basis.
  • the skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • Treatment of a subject with a LV can include a single treatment or, in many cases, can include a series of treatments.
  • LV LV
  • appropriate doses of a LV may depend upon the particular recipient and the mode of administration ⁇ The appropriate dose level for any particular subject may depend upon a variety of factors including the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate: of excretion, other administered therapeutic agents, and the like.
  • lentiviral gene therapy vectors described herein can be delivered to a subject by, for example, intravenous injection, local administration, or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 3054).
  • vectors may be delivered orally or inhalationally and may be encapsulated or otherwise manipulated to protect them from degradation, enhance uptake into tissues or cells, etc.
  • Pharmaceutical preparations can include a LV in an acceptable diluent, or can comprise a slow release matrix in which a LV is imbedded.
  • a pharmaceutical preparation can include one or more cells which produce vectors.
  • Pharmaceutical compositions comprising a LV described herein can be included in a container, pack, or dispenser, optionally together with instructions for administration.
  • compositions, methods and uses are intended to be illustrative and not limiting. Using the teachings provided herein other variations on the compositions, methods and uses will be readily available to one of skill in the art.
  • This example describes the development of novel lentiviral vectors for the treatment of X-1 inked Chronic Granulomatous Disease (X-CGD).
  • X-CGD X-1 inked Chronic Granulomatous Disease
  • vector(s) that show higher expression levels than the current lentiviral vector undergoing clinical trials for X-CGD (pChim-CYBB, a.k.a. MSP-Gp91 phox -WPRE, see, e.g., Santilli el al.( 2011) Mol. Therapy., 19(1): 122-122).
  • This lentiviral vector uses a chimeric myeloid- specific promoter (MSP) and chronically under-expresses in the mature human neutrophil population and fails to recapitulate the lineage specific expression pattern of the native CYBB gene.
  • MSP myeloid-specific promoter
  • the vectors described in this example possesses strict lineage and stage specific expression that mimics the expression pattern of the native CYBB gene (see, e.g., Figure 1).
  • the native CYBB topologically associated domain comprises a 600kb window spanning lOOkb upstream to 500kb downstream of the CYBB gene. This CYBB TAD thus provides a 600,000 base pair window in the human genome to properly regulate the gene.
  • each putative enhancer element was cloned upstream of the endogenous CYBB promoter to drive expression of a reporter gene (mCitrine) (see, e.g., Figure 2).
  • mCitrine reporter gene
  • enhancer element 4 drives high levels of expression in mature neutrophils. Additionally, the expression level is significantly higher than that obtained using the current X-CGD vector undergoing clinical trials. Similarly, as shown in Figure 4 enhancer element 4 drives high levels of expression in monocytes as well, and again the expression levels are significantly higher than that obtained using the current X-CGD vector undergoing clinical trials.
  • Figure 5 shows that enhancer element 2 drives high levels of lineage specific expression in B-cells. None of the enhancer elements express in Jurkats (T-cells), suggesting lineage specific expression of each enhancer element (see, Figure 6). In contrast, the MSP- mCit-WPRE construct showed the highest level of off-target expression.
  • enhancer element 4 confers increased lineage specific expression in mature neutrophils and monocytes and shows 2 fold higher expression than the MSP-mCit-WPRE vector. No enhancer element 4 driven expression was observed in T-cells (Jurkats) or in B-cells (RAMOs). Enhancer element 2 appears to confer increased lineage specific expression in B-cells (RAMOs). No enhancer element 2 driven expression was observed in neutrophils, monocytes or T-cells.
  • enhancer element 4 is made of two distinct enhancer modules (4E and 4R) and these were evaluated to determine if one of these elements could be eliminated to decrease the size of the vector.
  • enhancer element 2 appears to increase expression in B cells (see, Figure 10).
  • Element 4 when combined with either of the myeloid enhancer elements 4, 4L or 4R remains a B-cell enhancer and is inert in the myeloid lineage.
  • the vector 2-4R-Int3-pro- mCit-WPRE expresses 1.6 fold higher than MSP-mCit-WPRE in CB CD34+ differentiated neutrophils and monocytes. However it has 50% of the expression of MSP-mCit-WPRE in RAMO cells (B-cell lineage), but this may be a sufficient amount of expression to be therapeutic.
  • MSP-mCit-WPRE in neutrophils and monocytes, respectively.
  • One X-CGD vector candidate of particular interest is 2-4R-Int3-pro-mCit-
  • WPRE in which mCit can be replaced with a nucleic acid encoding Gp91 phox see, e.g. ,
  • Another goal is to decrease the size of vector while maintaining expression.
  • designed deletions can make the ‘’core” and “ultra-core” variants. Modifications to make vectors of 7.6kb and 5.9kb respectively (w/ Gp91 phox in ORF).
  • a secondary goal is to shrink the vector while increasing expression. In certain embodiments this can involve adding the "extra 4L core” and/or "extra 2" to the core and ultra core variants.
  • different codon optimizations of Gp91 phox can be utilized to replace mCitrine in the open reading frame (ORF).
  • Full-length element 2 comprises 1092 base pairs.
  • a 200 bp deletion was made to generate the 892 bp "core” variant (see, e.g., Table 1, SEQ ID NO:5).
  • a 745 bp deletion was made to generate the 347 bp enhancer element 2 "ultra core” variant (see, e.g., Table 1, SEQ ID NO:6).
  • full length element 4R comprises 995 bp and a 496bp deletion was made to generate the 500bp enhancer element 4R "core” variant (see, e.g., Table 1, SEQ ID NO: 10).
  • a 741 bp deletion was made to generate the 254 bp element 4R enhancer "ultra core” variant (see, e.g., Table 1, SEQ ID NO: 11).
  • a 240bp deletion was made to the 507 bp full length CYBB endogenous promoter (see, e.g., Table 1, SEQ ID NO:l) to generate a 267 bp CYBB promoter "core” fragment (SEQ ID NO:2) and a 337 bp deletion was made to generate a minimal CYBB promoter " CYBB ultra core promoter" (see, e.g., Table 1, SEQ ID NO:3).
  • extra fragment are included.
  • the RELA TF binding site may increase B-cell expression.
  • RELA plays role in many cellular processes including inflammation and immunity.
  • the TF binding footprint can be included in the element 2 component (see, e.g., Table 1, SEQ ID NO: 7).
  • 4L "core” variant or a 4L “ultra core” variant can be included with the 4R component. Sizes of these "extra” fragment constructs are also shown in Table 2. [0236] The constructs shown in Table 3 were tested:
  • the ultra-core and extra ultracore variant vectors shows significantly higher expression in CB CD34+ differentiated neutrophils (CDllb+ CD66b+ CD15+ CD16+) ( Figure 12) and in CB CD34+ differentiated monocytes (CDllb+ CD15+) ( Figure 13) than the E2-E4R-Int3-pro-mCit-WPRE construct or the current clinical vector (MSP-Gp91 phox - WPRE).
  • one particularly suitable vector is the ultra-core variant of 2-4R-Int3-pro-mCit-WPRE (UC 2-4R-Int3-pro-mCit-WPRE).
  • the ORF of mCitrine can be replaced with the therapeutic transgene (a nucleic acid encoding Gp91 phox ) to provide a clinically relevant vector.
  • Gp91 phox -WPRE vector backbone the Int3-pro vector has high lineage specific expression and only expresses in mature neutrophils and did not express well in the PLB-985 CYBB' cell line (human promyeloblasts cell line).
  • PLB-985 X-CGD cell line human promyeloblasts cell line.
  • the lead codon optimized sequence can be transferred to the various X-CGD vectors described herein.
  • codon optimization is for optimization of expression within a specific species (possibly even cell type), however the ideal codon optimization should be independent of which promoter/vector it is expressed from.
  • jCAT is the optimal codon optimization of Gp91 phox .
  • This codon optimization increases expression over 2-fold higher than the native cDNA sequence and 1.2 fold higher than the current codon optimized sequence in the clinical MSP-Gp91 phox -WPRE vector.
  • This optimization also increases titer 1.2X higher than the native cDNA sequence (MSP-Gp91 phox -WPRE).
  • Example 1 describes the generation of an optimized lead candidate vector: UC 2-4R-Int3-pro-mCit-WPRE (aka MveloVec). This vector showed improved titer, improved infectivity, and improved expression.
  • Figure 21 demonstrates the improvement in titer (top panel) and infectivity
  • MyeloVec (expressing mCitrine) is able to recapitulate the endogenous expression pattern of the native CYBB gene.
  • HSCs healthy donor (HD) cord blood (CB) CD34+ hematopoietic stem and progenitor cells (HSPCs) and transplanted the cells into NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) mice.
  • the gene modified cells will give rise to all the different lineages of the hematopoietic system.
  • MyeloVec is able to recapitulate the endogenous expression pattern of the native CYBB gene — very high expression in neutrophils, high bulk myeloid expression, medium levels of B-cell expression and minimal expression in T-cells and HSPCs. This is shown in blood Figure 22, panel A, and bone marrow (Figure 22, panel B).
  • MyeloVec is also able to recapitulate the temporal expression pattern of the native CYBB gene throughout neutrophil development. The expression gets higher as the neutrophils mature, mimicking the pattern of the native CYBB gene (see, e.g., Figure 23).
  • the pattern of mCitrine expression from MyeloVec recapitulated the endogenous expression pattern of Gp91 phox across multiple lineages in the blood and bone marrow cells (see, e.g., Table 4).
  • Figures 24 and 25 demonstrate the ability of MyeloVec to functionally correct for the X-CGD phenotype in-vitro in mouse X-CGD HSPCs.
  • HSPCs murine X-CGD lineage negative (Lin-) Hematopoietic Stem and Progenitor Cells (HSPCs) and differentiate the cells to mature neutrophils to demonstrate the ability of our lead candidate vector (UC-2-4R-Int3-pro-Gp91 phox (jCAT)-WPRE) to restore expression of Gp91 phox (Figure 24) and oxidase activity Figure 25.
  • oxidase activity was assessed by the Dihydrorhodamine (DHR) assay.
  • DHR Dihydrorhodamine
  • MyeloVec can restore higher levels of Gp91 phox than the current clinical vector (MSP) in neutrophils differentiated from X-CGD mouse HPSCs. Additionally, MyeloVec is able to restore oxidase activity to WT levels in transduced murine X-CGD cells differentiation into mature neutrophils (see, e.g., Figure 25).
  • MSP current clinical vector
  • MyeloVec expresses Gp91phox 1.6 fold higher than MSP (current clinical vector) in murine CYBB Lin- in-vitro differentiated neutrophils and MyeloVec is able to restore oxidase activity to WT levels in murine CYBB Lin- in-vitro differentiated neutrophils.
  • Figures 26-29 demonstrate the ability of MyeloVec to correct the X-CGD phenotype in-vivo in the X-CGD mouse model. Briefly, HPSCs were isolated from X-CGD mice and transduced with MyeloVec. The gene modified cells were then transplanted into congenic B6.SJL-Ptprca Pepcb/BoyJ (Pepboy) mice. Mice were harvested 16 weeks post transplant for analysis of Gp91 phox expression and restoration of oxidase activity across the different hematopoietic lineages.
  • X-CGD cells in vitro we transduced human X-CGD HSPCs with MyeloVec and differentiated the cells to mature neutrophils in-vitro.
  • Figure 30 shows the ability of MyeloVec to restore wildtype levels of Gp91 phox expression in the human X-CGD neutrophils.
  • Figure 31 shows the ability of MyeloVec to restore wildtype levels of cellular oxidase activity in the human X-CGD neutrophils (DHR assay).
  • Figure 32 shows the ability of MyeloVec to restore wildtype levels of bulk oxidase activity in human X-CGD neutrophils at an average VCN of 1.63 (cytochrome C assay).
  • MyeloVec was able to restore oxidase activity to WT levels in bone marrow neutrophils and monocytes; [0268] MyeloVec achieved close to WT levels of oxidase activity in peripheral blood neutrophils and monocytes at a VCN of 1.74 and greater; and
  • SEP ID NO:2 _ Minimal CYBB promoter (core) tatctcttagttgtagaaattggtttcattttccactatgtttaattgtgactggatcatt atagacccttttttgtagttgttgaggtttaaagatttaagtttgttatggatgcaagct tttcagttgaccaatgattattagccaattttctgataaaagaaaaggaaaccgattgcccc agggctgtttcatttcctcattggaagaagaagcatagtatagaagaaaggcaaaca caacacattcaacctctgccacc
  • Enhancer element 4R cagagatcttcacctccatggactcccatagccacactctgaaccctgtcatctctcagaa gtgcactgcttctgaaatctgcatctcatacacccatcctctgactaccacctcctgttcc ctggcttcctaattcactcacacccaagatgactgtcctttgactctttgactttgctcccatcttgtgtgttcacttcttggcattctactcatct tagactcagttcacttctgccattttcttgcacaaatcctgaattctctcatgcagtgcccc tctgtaccacctgcaggcaaaaccaaccctgatcaactcaatttctctctgtaccacctgcaggcaaaaccaaccctgat
  • Enhancer element 4L core agccaataatgtgaggcaagtctttaaaagggtagcacaatcagtctgaggttacaccata gatatggttaaccatagtgtggtctccataacataggaagtcaagatccccttcactctt gaccagtcagattgcacctagaacatttttctcaattctgcataccacatttaaagaggaa gacaaaacccatgcgttgtgcagct

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Abstract

In certain embodiments a lentiviral vector for the treatment of X-linked chronic granulomatous disease (X-CGD) is provided. In certain embodiments the vector comprises an expression cassette comprising a nucleic acid construct comprising a CYBB promoter or effective fragment thereof; and a nucleic acid that encodes gp91phox operably linked to the CYBB promoter or promoter fragment.

Description

LENTIVIRAL VECTORS IN HEMATOPOIETIC STEM CELLS TO TREAT X-LINKED CHRONIC GRANULOMATOUS DISEASE
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claim priority to and benefit of USSN 62/934,352, filed on
11/12/2019, which is incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[ Not Applicable ]
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A
TEXT FILE
[0002] A Sequence Listing is provided herewith as a text file, “UCLA-
P218P_ST25.txt” created on November 11, 2019 and having a size of 46.4 kb. The contents of the text file are incorporated by reference herein in their entirety.
BACKGROUND
[0003] X-linked chronic granulomatous disease (X-CGD) is a primary immune deficiency caused by mutations in the CYBB gene which encodes for a vital subunit of the phagocyte NADPH Oxidase (PHOX) complex. A defective PH OX complex results in the inability of the phagocytic cells of the immune system to properly eliminate infections.
[0004] Patients are therefore highly susceptible and suffer from recurrent, life- threatening bacterial and fungal infections. In typical subjects, the immune system attempts to wall off the infection but is unable to eliminate it, leading to the characteristic formation of granulomas that can result in damage to those tissues. The features of chronic granulomatous disease usually first appear in childhood, although some individuals do not show symptoms until later in life.
[0005] People with chronic granulomatous disease typically have at least one serious bacterial or fungal infection every 3 to 4 years. The lungs are the most frequent area of infection and pneumonia is a common feature of this condition. Individuals with chronic granulomatous disease may develop a type of fungal pneumonia, called mulch pneumonitis, which causes fever and shortness of breath after exposure to decaying organic materials such as mulch, hay, or dead leaves. Exposure to these organic materials and the numerous fungi involved in their decomposition causes people with chronic granulomatous disease to develop fungal infections in their lungs. Other common areas of infection in people with chronic granulomatous disease include the skin, liver, and lymph nodes.
[0006] Inflammation can occur in many different areas of the body in people with chronic granulomatous disease. Most commonly, granulomas occur in the gastrointestinal tract and the genitourinary tract. In many cases the intestinal wall is inflamed, causing a form of inflammatory bowel disease that varies in severity but can lead to stomach pain, diarrhea, bloody stool, nausea, and vomiting. Other common areas of inflammation in people with chronic granulomatous disease include the stomach, colon, and rectum, as well as the mouth, throat, and skin. Additionally, granulomas within the gastrointestinal tract can lead to tissue breakdown and pus production (abscesses). Inflammation in the stomach can prevent food from passing through to the intestines (gastric outlet obstruction), leading to an inability to digest food. These digestive problems cause vomiting after eating and weight loss. In the genitourinary tract, inflammation can occur in the kidneys and bladder. Inflammation of the lymph nodes (lymphadenitis) and bone marrow (osteomyelitis), which both produce immune cells, can lead to further impairment of the immune system.
[0007] Rarely, people with chronic granulomatous disease develop autoimmune disorders, which occur when the immune system malfunctions and attacks the body's own tissues and organs.
[0008] Repeated episodes of infection and inflammation reduce the life expectancy of individuals with chronic granulomatous disease.
[0009] The PHOX complex is made of five different subunits encoded by five different genes. These are gp91phox encoded by CYBB, p22phox encoded by CYBA, p47Phox encoded by NCF1, p67phox encoded by NCF2, and p40phox encoded by NCF4. Most common mutations are in the CYBB gene encoding for gp91phox which accounts for ~56%-70% of all cases of CGD. The condition is X-linked and accordingly primarily affects males.
[0010] The disease was initially terms "fatal granulomatous disease of childhood" and without treatment patient rarely lived past their first decade of life. Current standard of care utilizes routine prophylactic antibacterial and antifungal therapy and results in a mean age of survival around 30-40 years. These treatments do not provide a cure for the disease.
[0011] One potential curative therapy is an allogeneic hematopoietic stem cell transplantation from an HLA matched donor. However, this is not a viable option for many patients due to the unavailability of a suitable matched donor. [0012] An alternative curative therapy is an autologous hematopoietic stem cell
(HSC) transplantation with ex vivo gene therapy. In this approach, patients act as their own donor, eliminating the risk of immunological complications. The patient's own blood HSCs are harvested and are genetically engineered ex- vivo to introduce a functional copy of gene of interest, and gene modified cells are reinfused
[0013] Previous viral-based therapies utilized a g-retroviral vector driven by the spleen focus-forming virus (SFFV) promoter. This provided a promising clinical benefit. However, 2/2 patients developed myelodysplasia due to insertional oncogenesis. A current safer SIN lentiviral vector (pChim-CYBB; aka MSP-Gp91phox-WPRE) employs a chimeric “myeloid-specific promoter” (MSP) and initial results from current clinical trials indicate potential clinical benefits. However, the pChim-CYBB construct fails to recapitulate wildtype levels of expression and regulation of Gp91phox. Thus, for example, patient’s neutrophils post gene therapy under-express Gp91phox compared to normal heathy donor cells.
SUMMARY
[0014] Described herein is the development of novel lentiviral vector(s) (LVs) for the treatment of X-CGD. The vectors described herein show better (higher) expression than the current lentiviral vector. Additionally, the vectors described herein possesses strict lineage and stage specific expression that mimics the expression pattern of the native CYBB gene.
This is in contrast to the MSP construct(s) that have off-target expression and fail to recapitulate the lineage specific expression pattern of the native CYBB gene.
[0015] Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following:
[0016] Embodiment 1 : A recombinant lentiviral vector (LV) for the treatment of chronic granulomatous disease, said vector comprising:
[0017] an expression cassette comprising a nucleic acid construct comprising:
[0018] a CYBB promoter or effective fragment thereof; and
[0019] a nucleic acid that encodes gp91phox operably linked to said
CYBB promoter or promoter fragment.
[0020] Embodiment 2: The vector of embodiment 1, wherein said CYBB promoter or effective fragment thereof comprises a full-length endogenous CYBB promoter (SEQ ID NO:l). [0021] Embodiment 3: The vector of embodiment 1, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
[0022] Embodiment 4: The vector of embodiment 3, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
[0023] Embodiment 5: The vector of embodiment 1, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
[0024] Embodiment 6: The vector of embodiment 5, wherein said CYBB promoter consists of an effective fragment of the CYBB promoter whose sequence consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
[0025] Embodiment 7: The vector according to any one of embodiments 1-6, wherein said expression cassette comprises an enhancer element 2 (SEQ ID NO:4) or an effective fragment thereof.
[0026] Embodiment 8: The vector of embodiment 7, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core (SEQ ID NO:5).
[0027] Embodiment 9: The vector of embodiment 8, wherein the sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 core (SEQ ID NO:5).
[0028] Embodiment 10: The vector of embodiment 7, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 ultra core (SEQ ID NO:6).
[0029] Embodiment 11 : The vector of embodiment 10, wherein the sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 ultra core (SEQ ID NO:6).
[0030] Embodiment 12: The vector according to any one of embodiments 1-11, wherein said expression cassette further comprises a RELA TF binding site or an effective fragment thereof. [0031] Embodiment 13: The vector of embodiment 12, wherein said RELA TF binding site comprises or consists of the nucleic acid sequence of SEQ ID NO:7).
[0032] Embodiment 14: The vector according to any one of embodiments 1-11, wherein said expression cassette comprises enhancer element 4 or an effective fragment thereof.
[0033] Embodiment 15: The vector of embodiment 14, wherein said expression cassette comprises an enhancer element 4R or an effective fragment thereof.
[0034] Embodiment 16: The vector of embodiment 15, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R core (SEQ ID NO: 10).
[0035] Embodiment 17: The vector of embodiment 15, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R ultra core (SEQ ID NO: 11).
[0036] Embodiment 18: The vector of embodiment 16, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment consists of the nucleic acid sequence of enhancer element 4R ultra core (SEQ ID NO: 11).
[0037] Embodiment 19: The vector according to any one of embodiments 1-18, wherein said expression cassette comprises an enhancer element 4L or an effective fragment thereof.
[0038] Embodiment 20: The vector of embodiment 19, wherein said expression cassette comprises an effective fragment of enhancer element 4L where said fragment comprises or consists of the sequence of 4L core sequence (SEQ ID NO: 13).
[0039] Embodiment 21: The vector according to any one of embodiments 1-20, wherein said expression cassette comprises an intron enhancer element 3 (SEQ ID NO: 14) or an effective fragment thereof.
[0040] Embodiment 22: The vector of embodiment 21, wherein said expression cassette comprise an intron enhancer element 3 middle fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15. [0041] Embodiment 23: The vector according to any one of embodiments 21-22, wherein said expression cassette comprises an intron enhancer element 3 right fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16.
[0042] Embodiment 24: The vector according to any one of embodiments 1- 23, wherein said nucleic acid that encodes a nucleic acid that encodes gp91phox is a CYBB cDNA or a codon-optimized CYBB.
[0043] Embodiment 25 : The vector of embodiment 24, wherein said nucleic acid that encodes gp91phox is a CYBB cDNA (SEQ ID NO: 17).
[0044] Embodiment 26: The vector of embodiment 24, wherein said nucleic acid that encodes gp91phox is a codon optimized CYBB.
[0045] Embodiment 27 : The vector of embodiment 26, wherein the sequence of said nucleic acid that encodes gp91phox is a codon optimized CYBB selected from the group consisting of jCAT codon optimized CYBB (SEQ ID NO: 18), GeneArt optimized CYBB (SEQ ID NO:20), IDT optimized CYBB SEQ ID NO:21), and previous clinical candidate (SEQ ID NO: 19).
[0046] Embodiment 28: The vector of embodiment 26, wherein the sequence of said nucleic acid that encodes gp91phox is a jCAT codon optimized CYBB (SEQ ID NO: 18).
[0047] Embodiment 29: The vector according to any one of embodiments 1-28, wherein said vector comprises a y region vector genome packaging signal.
[0048] Embodiment 30: The vector according to any one of embodiments 1-29, wherein said vector comprise a 5' LTR comprising a CMV enhancer/promoter.
[0049] Embodiment 31 : The vector according to any one of embodiments 1-30, wherein said vector comprises a Rev Responsive Element (RRE).
[0050] Embodiment 32: The vector according to any one of embodiments 1-31, wherein said vector comprises a central polypurine tract.
[0051] Embodiment 33: The vector according to any one of embodiments 1-32, wherein said vector comprises a post-translational regulatory element.
[0052] Embodiment 34: The vector of embodiment 33, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE). [0053] Embodiment 35: The vector according to any one of embodiments 1-34, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.
[0054] Embodiment 36: The vector of embodiment 1, wherein said vector comprises the features of full-sized 2-4R-Int3-pro-mCit-WPRE shown in Figure 19, where the mCit is replaced with a nucleic acid encoding Gp91phox.
[0055] Embodiment 37: The vector of embodiment 1, wherein said vector comprises the features of UC 2-4R-Int3-pro-coGp91phox-WRPE shown in Figure 20, panel A.
[0056] Embodiment 38: The vector of embodiment 37, wherein said vector comprise the features shown in the vector represented in Figure 20, panel B.
[0057] Embodiment 39: The vector of embodiment 38, wherein said vector comprises the nucleotide sequence of ultra core (UC) 2-4R-Int3-Pro-(GP91-jcat)-WPRE (SEQ ID NO: 22).
[0058] Embodiment 40: The vector according to any one of embodiments embodiment 1-39, wherein said vector shows high expression in CD33+ (bulk myeloid cells), high expression in CD19+ (B cells, high expression in CD66b+ CD15+ CDllb-i- CD16+ (mature neutrophils), and low or no expression in CD3+ T cells.
[0059] Embodiment 41 : A host cell transduced with a vector according to any one of embodiments 1-40.
[0060] Embodiment 42: The host cell of embodiment 41, wherein the cell is a stem cell.
[0061] Embodiment 43: The host cell of embodiment 42, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.
[0062] Embodiment 44: The host cell of embodiment 41, wherein the cell is a human hematopoietic progenitor cell.
[0063] Embodiment 45 : The host cell of embodiment 44, wherein the human hematopoietic progenitor cell is a CD34+ cell.
[0064] Embodiment 46: A method of treating a chronic granulomatous disease (X-
CGD), in a subject, said method comprising:
[0065] transducing a stem cell and/or progenitor cell from said subject with a vector according to any one of embodiments 1-40; and
[0066] transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express said Gp91phox.
[0067] Embodiment 47 : The method of embodiment 46, wherein the cell is a stem cell.
[0068] Embodiment 48: The host cell of embodiment 46, wherein said cell is a stem cell derived from bone marrow.
[0069] Embodiment 49: The method of embodiment 46, wherein the cell is a human hematopoietic stem and progenitor cell.
[0070] Embodiment 50: The method of embodiment 49, wherein the human hematopoietic progenitor cell is a CD34+ cell.
[0071] Embodiment 51: A recombinant nucleic acid encoding one or more of the following:
[0072] a CYBB promoter, or an effective fragment thereof; and/or
[0073] a CYBB endogenous enhancer element 2 (CYBB B-cell enhancer), or an effective fragment thereof; and/or
[0074] a CYBB endogenous enhancer 4R (CYBB endogenous myeloid enhancer), or an effective fragment thereof; and/or
[0075] a CYBB endogenous enhancer 4L, or an effective fragment thereof; and/or
[0076] a CYBB endogenous myeloid Intron 3 enhancer, or an effective fragment thereof; and/or
[0077] a codon optimized nucleic acid encoding Gp91phox.
[0078] Embodiment 52: The nucleic acid of embodiment 51, wherein said nucleic acid encodes a sequence comprising or consisting of a full-length endogenous CYBB promoter (SEQ ID NO:l).
[0079] Embodiment 53: The nucleic acid of embodiment 51, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
[0080] Embodiment 54: The nucleic acid of embodiment 53, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (core) (SEQ ID NO: 2). [0081] Embodiment 55: The nucleic acid of embodiment 51, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
[0082] Embodiment 56: The nucleic acid of embodiment 55, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
[0083] Embodiment 57: The nucleic acid according to any one of embodiments 51-
56, wherein said nucleic acid encodes an effective fragment of a CYBB endogenous enhancer element 2 (CYBB B-cell enhancer).
[0084] Embodiment 58: The nucleic acid of embodiment 57, wherein the nucleic acid sequence of said a CYBB endogenous enhancer element 2 comprises or consists of the sequence of enhancer element 2 core (SEQ ID NO:5).
[0085] Embodiment 59: The nucleic acid of embodiment 57, wherein the nucleic acid sequence of said a CYBB endogenous enhancer element 2 comprises or consists of the sequence of enhancer element 2 ultra core (SEQ ID NO: 6).
[0086] Embodiment 60: The nucleic acid according to any one of embodiments 51-
59, wherein said nucleic acid comprises an effective fragment of a CYBB endogenous enhancer 4R (CYBB endogenous myeloid enhancer).
[0087] Embodiment 61: The nucleic acid of embodiment 60, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous enhancer 4R comprises or consists of the sequence of enhancer element 4R ultra core (SEQ ID NO: 10).
[0088] Embodiment 62: The nucleic acid according to any one of embodiments 51-
61, wherein said nucleic acid comprises an effective fragment of an enhancer element 4L.
[0089] Embodiment 63 : The nucleic acid of embodiment 62, wherein said effective fragment of an enhancer element 4L comprises or consists of the sequence of the 4L core sequence (SEQ ID NO: 13).
[0090] Embodiment 64: The nucleic acid according to any one of embodiments 51-
63, wherein said nucleic acid comprises an effective fragment of a CYBB endogenous myeloid intron 3 enhancer.
[0091] Embodiment 65 : The nucleic acid of embodiment 64, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous myeloid intron 3 enhancer comprises or consists of an element 3 middle fragment nucleic acid sequence (SEQ ID NO:15).
[0092] Embodiment 66: The nucleic acid according to any one of embodiments 64-
65, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous myeloid intron 3 enhancer comprises or consists of an intron enhancer element 3 right fragment (SEQ ID NO: 16).
[0093] Embodiment 67: The nucleic acid according to any one of embodiments 51-
66, wherein said nucleic acid comprises a jCAT codon optimized CYBB (SEQ ID NO: 18).
[0094] Embodiment 68: The nucleic acid according to any one of embodiments 51-
67, wherein said nucleic acid comprises an expression cassette.
[0095] Embodiment 69: The nucleic acid of embodiment 68, wherein said expression cassette is effective to express Gp91phox in vivo.
[0096] Embodiment 70: The nucleic acid according to any one of embodiments 51-
69, wherein said nucleic acid comprises a lentiviral vector according to any one of embodiments 1-40.
Definitions.
[0097] A "promoter" refers to a regulatory sequence in a nucleic acid required to initiate transcription of a gene (e.g., a gene operably coupled to the promoter).
[0098] An "enhancer" refers to a regulatory DNA sequence that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene.
[0099] An "effective fragment" when used with respect to a promoter (e.g. , an effective fragment of a CYBB promoter) refers to a fragment of the full-length promoter that is sufficient to initiate transcription of a gene operably linked to that promoter.
[0100] An "effective fragment" when used with respect to an enhancer (e.g., an effective fragment of a CYBB enhancer) refers to a fragment of the full-length enhancer that is sufficient to provide regulate expression of an operably linked gene when bound by a transcription factor. In certain embodiments the regulation is comparable with respect to expression level and/or lineage offered by the full-length enhancer.
[0101] The term "operably linked" refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to a gene when that promoter is placed in a location that permits that promoter to initiate transcription of that gene. An enhancer is operably linked to a gene when that enhancer, when bound by an appropriate transcription factor, is able to regulate (e.g., to upregulate) expression of that gene.
[0102] "Recombinant" is used consistently with its usage in the art to refer to a nucleic acid sequence that comprises portions that do not naturally occur together as part of a single sequence or that have been rearranged relative to a naturally occurring sequence. A recombinant nucleic acid is created by a process that involves the hand of man and/or is generated from a nucleic acid that was created by hand of man (e.g., by one or more cycles of replication, amplification, transcription, etc.). A recombinant vims is one that comprises a recombinant nucleic acid. A recombinant cell is one that comprises a recombinant nucleic acid.
[0103] As used herein, the term "recombinant lentiviral vector" or "recombinant LV) refers to an artificially created polynucleotide vector assembled from an LV and a plurality of additional segments as a result of human intervention and manipulation.
[0104] By "an effective amount" is meant the amount of a required agent or composition comprising the agent to ameliorate or eliminate symptoms of a disease relative to an untreated patient. The effective amount of composition(s) used to practice the methods described herein for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
BRIEF DESCRIPTION OF THE DRAWINGS 0105] Figure 1 illustrates the endogenous expression pattern of gp91phox in human blood cells.
[0106] Figure 2 illustrates constructs used to probe enhancer activity.
[0107] Figure 3 expression of enhancer constructs in CB CD34+ differentiated neutrophils day 16 (CDllb+ CD66b+ CD15+ CD16+).
[0108] Figure 4 expression of enhancer constructs in CB CD34+ differentiated monocytes day 16 (CDllb+ CD15+ ).
[0109] Figure 5 shows expression of enhancer constructs in transduced RAMOs (B- cell line) D14 flow. [0110] Figure 6 shows expression of enhancer constructs in transduced Jurkats (T- cell line) D16 flow.
[0111] Figure 7 shows expression of enhancer constructs in CB CD34+ differentiated
Neutrophils Day 16 (CDllb+ CD66b+ CD15+ CD16+). [0112] Figure 8 shows expression of enhancer constructs in CB CD34+ differentiated
Monocytes Day 16 (CDllb+ CD15+ ).
[0113] Figure 9 shows expression of enhancer constructs in transduced Jurkats (T- cell line) D16 flow.
[0114] Figure 10 shows expression of enhancer constructs in transduced RAMOs (B- cell line) D14 flow.
[0115] Figure 11 shows structure of E2-E4R-Int3-pro-mCit-WPRE vector (top) and the same vector where mCitrine is replaced with nucleic acid encoding Gp91phox (bottom).
[0116] Figure 12 shows expression of the reduced size vectors in CB CD34+
Differentiated Neutrophils Day 16. (CDllb+ CD66b+ CD 15+ CD 16+)
[0117] Figure 13 shows expression of the reduced size vectors in CB CD34+
Differentiated Monocytes Day 16
(CDllbf CD15+ )
[0118] Figure 14 shows expression of the reduced size vectors in Jurkat Cells (T-Cell Line).
[0119] Figure 15 shows expression of the reduced size vectors in RAMOS Cells (B-
Cell Line).
[0120] Figure 16 shows raw small scale titers of the "core", the "ultra core", the "extra core" and the "extra ultra core" constructs. [0121] Figure 17 shows the expression levels produced by various codon optimizations of Gp91phox in PLB-985 X-CGD' cells.
[0122] Figure 18 shows the raw titers of various codon optimizations of MSP-
Gp91 phox- WPRE. [0123] Figure 19 illustrates one embodiment of a lenti viral vector for treatment of X-
CGD. For use in a treatment the mCit reporter would be replaced with a nucleic acid sequence encoding a Gp91phox, e.g., as described herein.
[0124] Figure 20 panels A-B, illustrate one embodiment of an optimized lentiviral vector for treatment of X-CGD. Panel A schematically illustrates the elements of UC 2-4R- Int3-pro-coGp91phox-WRPE. Panel B shows a "map" of the vector.
[0125] Figure 21 illustrates improvement in titer (top panel) and infectivity (bottom panel) as the vector was optimized from the original 2-4R-Int3-pro-mCit-WPRE to the CORE variant and to the ULTRA CORE (UC) variant. The UC variant (MyeloVec is a lead candidate vector).
[0126] Figure 22, panels A-B, shows that MyeloVec is able to recapitulate the endogenous expression pattern of the native CYBB gene in blood cells (panel A) and bone marrow cells (panel B) respectively.
[0127] Figure 23 shows that MyeloVec is able to recapitulate the temporal expression pattern of the native CYBB gene throughout neutrophil development. The expression gets higher as the neutrophils mature, mimicking the pattern of the native CYBB gene.
[0128] Figure 24 shows the restoration of Gp91phox expression.
[0129] Figure 25 shows show the restoration of oxidase activity to wildtype levels.
[0130] Figure 26 shows restoration of Gp91phox expression in neutrophils and monocytes in the peripheral blood.
[0131] Figure 27 shows restoration of oxidase activity near wildtype levels in the blood neutrophils and monocytes.
[0132] Figure 28 shows restoration of high levels of Gp91phox expression in the bone marrow neutrophils and monocytes. [0133] Figure 29 shows restoration of wildtype levels of oxidase activity.
[0134] Figure 30 shows the ability of MyeloVec to restore wildtype levels of
Gp91phox expression in the human X-CGD neutrophils.
[0135] Figure 31 shows the ability of MyeloVec to restore wildtype levels of cellular oxidase activity in the human X-CGD neutrophils (DHR assay). [0136] Figure 32 shows the ability of MyeloVec to restore wildtype levels of bulk oxidase activity in human X-CGD neutrophils at an average VCN of 1.63 (cytochrome C assay).
DETAILED DESCRIPTION
[0137] In various embodiments, lentiviral vectors are provided for the treatment (or prophylaxis) of X-linked Chronic Granulomatous Disease (X-CGD) are provided. In certain embodiments the vectors are optimized to reduce vector size, increase expression level and titer. Additionally, In various embodiments the vectors recapitulate the lineage specific expression pattern of the native CYBB gene, e.g., as described herein (see, e.g., Figure 1).
[0138] As described herein (see, e.g., Example 1), analysis of bioinformatics information about the CYBB gene, which produces the gp91phox component of the phagocytic cell anti-microbial oxidase system, identified several putative transcriptional regulatory domains, based on histone marks, DNAse hypersensitivity sites and sequence motifs for binding transcriptional factor.
[0139] Fifteen putative endogenous enhancer elements were identified within the native CYBB topologically associated domain (TAD). In order to experimentally identify the critical enhancer elements that regulate the CYBB gene, each putative enhancer element was cloned upstream of the endogenous CYBB promoter to drive expression of a reporter gene (mCitrine) (see, e.g., Figure 2). To elucidate the function of each putative enhancer element, we assayed the activity of each of the vectors in cord blood (CB) CD34+ differentiated mature neutrophils and monocytes as well as RAMOS cells (B -lymphocyte cell line) which are 3 on-target cell lineages.
[0140] It was discovered that enhancer element 4 drives high levels of expression in mature neutrophils and in monocytes, with no expression in B-cells. It was also discovered that enhancer element 2 drives high levels of lineage specific expression in B-cells with no expression in neutrophils. None of the enhancer elements express in Jurkats (T-cells), suggesting lineage specific expression of each enhancer element.
[0141] It was also discovered that enhancer element 4 is made of two distinct enhancer modules (4L and 4R) and these were evaluated to determine if one of these elements could be eliminated to decrease the size of the vector. [0142] Additionally, reduced variants of enhancer element 2, enhancer element 4, intron enhancer 3, and the CYBB endogenous promoter were made and evaluated. Codon optimizations of the nucleic acid encoding Gp91phox were also evaluated.
[0143] In view of these discoveries, in various embodiments, a recombinant lentiviral vector (LV) for the treatment of chronic granulomatous disease is provided where the vector comprises an expression cassette comprising a nucleic acid construct comprising a CYBB endogenous promoter or effective fragment thereof; and a nucleic acid that encodes gp91phox operably linked to the CYBB promoter or promoter fragment. In certain embodiments the CYBB promoter or effective fragment thereof comprises a full-length endogenous CYBB promoter (see, e.g., Table 1, SEQ ID NO:l). In certain embodiments the CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (see, e.g., Table 1, SEQ ID NO:3). In certain embodiments the CYBB promoter consists of an effective fragment of the CYBB promoter whose sequence consists of the minimal CYBB promoter (see, e.g., Table 1, SEQ ID NO:3).
[0144] In certain embodiments the expression cassette in the lentiviral vector comprises an enhancer element 2 (see, e.g., Table 1, SEQ ID NO: 4) or an effective fragment thereof. In certain embodiments the sequence of the effective fragment of enhancer element 2 comprises or consists of the sequence of enhancer element 2 "core" (see, e.g., Table 1, SEQ ID NO:5). In certain embodiments the sequence of the effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 core (see, e.g., Table 1, SEQ ID NO:5). In certain embodiments the sequence of the effective fragment of enhancer element 2 comprises or consists of the enhancer element 2 "ultra core" sequence (see, e.g., Table 1,
SEQ ID NO:6). In certain embodiments the sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 ultra core (see, e.g., Table 1, SEQ ID NO:6).
[0145] In certain embodiments the expression cassette comprising the lentiviral vector further comprises a RELA TF binding site or an effective fragment thereof. In certain embodiments the RELA TF binding site comprises or consists of the nucleic acid sequence of SEQ ID NO:7 in Table 1,
[0146] In certain embodiments the expression cassette in the lentiviral vector comprises enhancer element 4 (see, e.g., Table 1, SEQ ID NO:8) or an effective fragment thereof. In certain embodiments the expression cassette comprises an enhancer element 4R (see, e.g., Table 1, SEQ ID NO: 9) or an effective fragment thereof. In certain embodiments the expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence comprises or consists of the nucleic acid sequence of enhancer element 4R core (see, e.g., Table 1, SEQ ID NO: 10). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R "ultra core" (see, e.g., Table 1, SEQ ID NO: 11). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment consists of the nucleic acid sequence of enhancer element 4R ultra core (see, e.g., Table 1, SEQ ID NO: 11).
[0147] In certain embodiments the expression cassette in the lentiviral vector comprises an enhancer element 4L ((see, e.g., Table 1, SEQ ID NO: 12) or an effective fragment thereof. In certain embodiments the effective fragment of enhancer element 4L comprises or consists of the sequence of 4L core sequence (see, e.g., Table 1, SEQ ID NO: 13). In certain embodiments the effective fragment of enhancer element 4L consists of the sequence of 4L core sequence (see, e.g., Table 1, SEQ ID NO: 13).
[0148] In certain embodiments the expression cassette in the lentiviral vector comprises an intron enhancer element 3 (see, e.g., Table 1, SEQ ID NO: 14) or an effective fragment thereof. In certain embodiments the expression cassette in the lentiviral vector comprises or consists of an intron enhancer element 3 middle fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15 in Table 1. In certain embodiments the expression cassette in the lentiviral vector consists of an intron enhancer element 3 middle fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15 in Table 1. In certain embodiments the expression cassette in the lentiviral vector comprises or consists of an intron enhancer element 3 right fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 in Table 1.
[0149] In certain embodiments the nucleic acid that encodes gp91phox is a full CYBB gene, a CYBB cDNA, or a codon-optimized CYBB. In certain embodiments the nucleic acid that encodes gp91phox is a CYBB cDNA (see, e.g., Table 1, SEQ ID NO:17). In certain embodiments the nucleic acid that encodes gp91phox is a codon optimized CYBB (e.g., a jCAT codon optimized CYBB (see, e.g., Table 1, SEQ ID NO:18), a GeneArt optimized CYBB (see, e.g., Table 1, SEQ ID NO:20), an IDT optimized CYBB (see, e.g., Table 1, SEQ ID NO:21), and previous clinical candidate (see, e.g., Table 1, SEQ ID NO: 19)). In certain embodiments the sequence of said nucleic acid that encodes gp91phox is a jCAT codon optimized CYBB (see, e.g., Table 1, SEQ ID NO: 18).
[0150] It will be recognized that the expression cassettes described herein in the context of lentiviral vectors need not be limited to this context. Accordingly, in certain embodiments, a recombinant nucleic acid comprising any one or more of the CYBB regulatory elements described herein is contemplated. In certain embodiments the recombinant nucleic acid comprises an expression cassette, e.g., an expression cassette effective to express Gp91phox in vivo. It will be recognized that such an expression cassette can be used with other constructs, e.g., in conjunction with a CRISPR construct. Table 1. Nucleic acid sequences of various components of the lentiviral vectors for treatment of X-CGD as described herein.
Figure imgf000018_0001
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
[0151] In various embodiments, the lentiviral vectors (LVs) described herein can have various "safety" features that can include, for example, the presence of an insulator (e.g., an FB insulator in the 3'LTR). Additionally, or alternatively, in certain embodiments, the HIV LTR has been substituted with an alternative promoter (e.g., a CMV) to yield a higher titer vector without the inclusion of the HIV TAT protein during packaging. Other strong promoters (e.g., RSV, and the like can also be used).
[0152] In various embodiments the lentiviral vectors described herein contain any one or more of the elements typically found in lentiviral vectors. Such elements include, but need not be limited to a y region vector genome packaging signal, a Rev Responsive Element (RRE), a polypurine tract (e.g., a central polypurine tract, a 3' polypurine tract, etc.), a post- translational regulatory element (e.g., a modified Woodchuck Post-transcriptional Regulatory Element (WPRE)), an insulator, and the like, e.g., as described below. [0153] In various embodiments the vector is a SIN vector substantially incapable of reconstituting a wild-type lentivirus through recombination.
[0154] In certain embodiments the vector comprises the features of "ultra core" (UC)
2-4R-Int3-Pro-(GP91-jcat)-WPRE shown in Figure 20, panel A. In certain embodiments the vector comprises the features shown in the vector represented in Figure 20, panel B. In certain embodiments the vector comprises the nucleotide sequence of ultra core (UC) 2-4R- Int3-Pro-(GP91-jcat)-WPRE (SEQ ID NO: 22).
[0155] In various embodiments the vector shows high expression in CD33+ (bulk myeloid cells), and/or high expression in CD19+ (B cells), high expression in CD66b+ CD15+ CDllb-i- CD16+ (mature neutrophils), and/or low or no expression in CD3+ (T cells). In various embodiments the vector shows high expression in CD33+ (bulk myeloid cells), high expression in CD19+ (B cells, high expression in CD66b+ CD15+ CDllb-i- CD16+ (mature neutrophils), and low or no expression in CD3+ T cells.
[0156] As shown above, in Example 1 , the vectors described herein are effective to transduce cells at high titer and to also provide high levels of expression of Gp91phox.
[0157] In view of these results, it is believed that LVs described herein, e.g. , recombinant TAT-independent, SIN LVs that express a nucleic acid encoding a Gp91phox can be used to effectively treat X-linked chronic granulomatous disease (X-CGD) in subjects (e.g. , human and non-human mammals). It is believed these vectors can be used for the modification of stem cells (e.g., hematopoietic stem and progenitor cells) that can be introduced into a subject in need thereof for the treatment of, e.g., subjects identified as having X-CGD. Moreover, it is believed that the resulting cells will produce enough of the transgenic Gp91phox protein to demonstrate significant improvement in subject health. It is also believed the vectors can be directly administered to a subject to achieve in vivo transduction of the target (e.g., hematopoietic stem or progenitor cells) and thereby also effect a treatment of subjects in need thereof.
[0158] As noted above, in various embodiments the LVs described herein can comprise various safety features. For example, the HIV LTR has been substituted with a CMV promoter to yield higher titer vector without the inclusion of the HIV TAT protein during packaging. In certain embodiments an insulator (e.g., the FB insulator) can be introduced into the 3'LTR for safety. The LVs are also constructed to provide efficient transduction and high titer. [0159] It will be appreciated that the foregoing elements are illustrative and need not be limiting. In view of the teachings provided herein, suitable substitutions for these elements will be recognized by one of skill in the art and are contemplated within the scope of the teachings provided herein.
Gp91phox codon optimization.
[0160] As noted above, in various embodiments the lentiviral vector can comprise a
CYBB gene or cDNA. However, in certain embodiments the nucleic acid encoding Gp91phox is codon optimized. Numerous methods of codon optimization are known to those of skill in the art. One illustrative method is JCat (Java Codon Adaptation Tool). The jCAT tool adapts gene codon usage to most sequenced prokaryotes and various eukaryotic gene expression hosts. In contrast to many tools, JCat does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators. The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the input sequence and the newly adapted sequence. JCat optimization is described by Grote et al. (2005) Nucleic Acids Res. 33(suppl 2): W526-W531) and a JCat tool is available online at www.jcat.de.
[0161] Another codon optimization tool is provided by GeneArt (from ThermoFisher
Scientific®.
[0162] Still another codon optimization tool is IDT. The IDT codon optimization tool was developed to optimize a DNA or protein sequence from one organism for expression in another by reassigning codon usage based on the frequencies of each codon’s usage in the new organism. For example, valine is encoded by 4 different codons (GUG, GUU, GUC, and GUA). In human cell lines, however, the GUG codon is preferentially used (46% use vs. 18, 24, and 12%, respectively). The codon optimization tool takes this information into account and assigns valine codons with those same frequencies. In addition, the tool algorithm eliminates codons with less than 10% frequency and re-normalizes the remaining frequencies to 100%. Moreover, the optimization tool reduces complexities that can interfere with manufacturing and downstream expression, such as repeats, hairpins, and extreme GC content. The IDT optimization tool is available from IDT (Integrated DNA Technologies, Coralville, Iowa) and can be found at ww.idtdna.com/CodonOpt. [0163] Other codon optimization tools include, but are not limited to CodonW an open source software program that can be found at codonw.sourceforge.net, and the OptimumGene™ algorithm from GenScript.
[0164] In one embodiment, illustrated in Example 1, the codon optimized Gp91phox, can be the sequence used in the current clinical candidate MSP-Gp91phox-WPRE.
[0165] These codon optimizations are illustrative and non-limiting. Using the teaching provided here and in Example 1, the Gp91phox codon usage can readily be optimized for particular applications.
TAT-Independent and Self inactivating lentiviral vectors.
[0166] To further improve safety, in various embodiments, the lentiviral vectors described herein comprise a TAT-independent, self-inactivating (SIN) configuration. Thus, in various embodiments it is desirable to employ in the LVs described herein an LTR region that has reduced promoter activity relative to wild-type LTR. Such constructs can be provided that are effectively "self-inactivating" (SIN) which provides a biosafety feature.
SIN vectors are ones in which the production of full-length vector RNA in transduced cells is greatly reduced or abolished altogether. This feature minimizes the risk that replication- competent recombinants (RCRs) will emerge. Furthermore, it reduces the risk that that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed.
[0167] Furthermore, a SIN design reduces the possibility of interference between the
LTR and the promoter that is driving the expression of the transgene. SIN LVs can often permit full activity of the internal promoter.
[0168] The SIN design increases the biosafety of the LVs. The majority of the HIV
LTR is comprised of the U3 sequences. The U3 region contains the enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation. Several of these promoter elements are essential for viral replication. Some of the enhancer elements are highly conserved among viral isolates and have been implicated as critical virulence factors in viral pathogenesis. The enhancer elements may act to influence replication rates in the different cellular target of the vims
[0169] As viral transcription starts at the 3' end of the U3 region of the 5' LTR, those sequences are not part of the viral mRNA and a copy thereof from the 3' LTR acts as template for the generation of both LTR's in the integrated provirus. If the 3' copy of the U3 region is altered in a retroviral vector construct, the vector RNA is still produced from the intact 5'
LTR in producer cells, but cannot be regenerated in target cells. Transduction of such a vector results in the inactivation of both LTR's in the progeny virus. Thus, the retrovirus is self-inactivating (SIN) and those vectors are known as SIN transfer vectors.
[0170] In certain embodiments self-inactivation is achieved through the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, /.<?., the DNA used to produce the vector RNA. During RT, this deletion is transferred to the 5' LTR of the proviral DNA. Typically, it is desirable to eliminate enough of the U3 sequence to greatly diminish or abolish altogether the transcriptional activity of the LTR, thereby greatly diminishing or abolishing the production of full-length vector RNA in transduced cells. However, it is generally desirable to retain those elements of the LTR that are involved in polyadenylation of the viral RNA, a function typically spread out over U3, R and U5. Accordingly, in certain embodiments, it is desirable to eliminate as many of the transcriptionally important motifs from the LTR as possible while sparing the polyadenylation determinants.
[0171] The SIN design is described in detail in Zufferey et al. (1998) J Virol. 72(12):
9873-9880, and in U.S. Patent No: 5,994,136. As described therein, there are, however, limits to the extent of the deletion at the 3' LTR. First, the 5' end of the U3 region serves another essential function in vector transfer, being required for integration (terminal dinucleotide+att sequence). Thus, the terminal dinucleotide and the att sequence may represent the 5' boundary of the U3 sequences which can be deleted. In addition, some loosely defined regions may influence the activity of the downstream polyadenylation site in the R region. Excessive deletion of U3 sequence from the 3'LTR may decrease polyadenylation of vector transcripts with adverse consequences both on the titer of the vector in producer cells and the transgene expression in target cells.
[0172] Additional SIN designs are described in U.S. Patent Publication No:
2003/0039636. As described therein, in certain embodiments, the lentiviral sequences removed from the LTRs are replaced with comparable sequences from a non- lentiviral retrovirus, thereby forming hybrid LTRs. In particular, the lentiviral R region within the LTR can be replaced in whole or in part by the R region from a non-lentiviral retrovirus. In certain embodiments, the lentiviral TAR sequence, a sequence which interacts with TAT protein to enhance viral replication, is removed, preferably in whole, from the R region. The TAR sequence is then replaced with a comparable portion of the R region from a non- lentiviral retrovirus, thereby forming a hybrid R region. The LTRs can be further modified to remove and/or replace with non-lentiviral sequences all or a portion of the lentiviral U3 and U5 regions.
[0173] Accordingly, in certain embodiments, the SIN configuration provides a retroviral LTR comprising a hybrid lentiviral R region that lacks all or a portion of its TAR sequence, thereby eliminating any possible activation by TAT, wherein the TAR sequence or portion thereof is replaced by a comparable portion of the R region from a non-lentiviral retrovirus, thereby forming a hybrid R region. In a particular embodiment, the retroviral LTR comprises a hybrid R region, wherein the hybrid R region comprises a portion of the HIV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 10 in US 2003/0039636) lacking the TAR sequence, and a portion of the MoMSV R region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 9 in 2003/0039636) comparable to the TAR sequence lacking from the HIV R region.
In another particular embodiment, the entire hybrid R region comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11 in 2003/0039636.
[0174] Suitable lentiviruses from which the R region can be derived include, for example, HIV (HIV-1 and HIV-2), EIV, SIV and FIV. Suitable retroviruses from which non- lentiviral sequences can be derived include, for example, MoMSV, MoMLV, Friend, MSCV, RSV and Spumaviruses. In one illustrative embodiment, the lentivirus is HIV and the non- lentiviral retrovirus is MoMSV.
[0175] In another embodiment described in US 2003/0039636, the LTR comprising a hybrid R region is a left (5') LTR and further comprises a promoter sequence upstream from the hybrid R region. Preferred promoters are non-lentiviral in origin and include, for example, the U3 region from a non-lentiviral retrovirus (e.g., the MoMSV U3 region). In one particular embodiment, the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 12 in US 2003/0039636. In another embodiment, the left (5') LTR further comprises a lentiviral U5 region downstream from the hybrid R region. In one embodiment, the U5 region is the HIV U5 region including the HIV att site necessary for genomic integration. In another embodiment, the U5 region comprises the nucleotide sequence shown in SEQ ID NO: 13 in US 2003/0039636. In yet another embodiment, the entire left (5') hybrid LTR comprises the nucleotide sequence shown in SEQ ID NO: 1 in US 2003/0039636.
[0176] In another illustrative embodiment, the LTR comprising a hybrid R region is a right (3') LTR and further comprises a modified (e.g., truncated) lentiviral U3 region upstream from the hybrid R region. The modified lentiviral U3 region can include the att sequence, but lack any sequences having promoter activity, thereby causing the vector to be SIN in that viral transcription cannot go beyond the first round of replication following chromosomal integration. In a particular embodiment, the modified lentiviral U3 region upstream from the hybrid R region consists of the 3' end of a lentiviral (e.g. , HIV) U3 region up to and including the lentiviral U3 att site. In one embodiment, the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15 in US 2003/0039636. In another embodiment, the right (3') LTR further comprises a polyadenylation sequence downstream from the hybrid R region. In another embodiment, the polyadenylation sequence comprises the nucleotide sequence shown in SEQ ID NO: 16 in US 2003/0039636. In yet another embodiment, the entire right (5') LTR comprises the nucleotide sequence shown in SEQ ID NO: 2 or 17 of US 2003/0039636.
[0177] Thus, in the case of HIV based LV, it has been discovered that such vectors tolerate significant U3 deletions, including the removal of the LTR TATA box (e.g. , deletions from -418 to -18), without significant reductions in vector titers. These deletions render the LTR region substantially transcriptionally inactive in that the transcriptional ability of the LTR in reduced to about 90% or lower.
[0178] It has also been demonstrated that the trans- acting function of Tat becomes dispensable if part of the upstream LTR in the transfer vector construct is replaced by constitutively active promoter sequences (see, e.g., Dull et al. (1998) J Virol. 72(11): 8463- 8471. Lurthermore, we show that the expression of rev in trans allows the production of high-titer HIV-derived vector stocks from a packaging construct which contains only gag and pol. This design makes the expression of the packaging functions conditional on complementation available only in producer cells. The resulting gene delivery system, conserves only three of the nine genes of HIV-1 and relies on four separate transcriptional units for the production of transducing particles.
[0179] In one embodiments illustrated in Example 1, the cassette expressing a nucleic acid encoding gp91phox a SIN vector with the CMV enhancer/promoter substituted in the 5' LTR.
[0180] It will be recognized that the CMV promoter typically provides a high level of non-tissue specific expression. Other promoters with similar constitutive activity include, but are not limited to the RSV promoter, and the SV40 promoter. Mammalian promoters such as the beta-actin promoter, ubiquitin C promoter, elongation factor lapromoter, tubulin promoter, etc. , may also be used. [0181] The foregoing SIN configurations are illustrative and non-limiting. Numerous
SIN configurations are known to those of skill in the art. As indicated above, in certain embodiments, the LTR transcription is reduced by about 95% to about 99%. In certain embodiments LTR may be rendered at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% at least about 96%, at least about 97%, at least about 98%, or at least about 99% transcriptionally inactive.
Insulator element
[0182] In certain embodiments, to further enhance biosafety, insulators are inserted into the lentiviral vectors described herein. Insulators are DNA sequence elements present throughout the genome. They bind proteins that modify chromatin and alter regional gene expression. The placement of insulators in the vectors described herein offer various potential benefits including, inter alia : 1) Shielding of the vector from positional effect variegation of expression by flanking chromosomes (/.<?., barrier activity); and 2) Shielding flanking chromosomes from insertional trans- activation of gene expression by the vector (enhancer blocking). Thus, insulators can help to preserve the independent function of genes or transcription units embedded in a genome or genetic context in which their expression may otherwise be influenced by regulatory signals within the genome or genetic context (see, e.g. , Burgess-Beusse el al. (2002) Proc. Natl. Acad. Sci. USA, 99: 16433; and Zhan el al. (2001) Hum. Genet., 109: 471). In the present context insulators may contribute to protecting lenti virus -expressed sequences from integration site effects, which may be mediated by ex acting elements present in genomic DNA and lead to deregulated expression of transferred sequences. In various embodiments LVs are provided in which an insulator sequence is inserted into one or both LTRs or elsewhere in the region of the vector that integrates into the cellular genome.
[0183] The first and best characterized vertebrate chromatin insulator is located within the chicken b-globin locus control region. This element, which contains a DNase-I hypersensitive site-4 (cHS4), appears to constitute the 5' boundary of the chicken b-globin locus (Prioleau el al. (1999) EMBO J. 18: 4035-4048). A 1.2-kb fragment containing the cHS4 element displays classic insulator activities, including the ability to block the interaction of globin gene promoters and enhancers in cell lines (Chung et al. (1993) Cell, 74: 505-514), and the ability to protect expression cassettes in Drosophila (Id.), transformed cell lines (Pikaart el al. (1998) Genes Dev. 12: 2852-2862), and transgenic mammals (Wang et al. (1997) Nat. Biotechnol., 15: 239-243; Taboit-Dameron et al. (1999) Transgenic Res., 8: 223- 235) from position effects. Much of this activity is contained in a 250-bp fragment. Within this stretch is a 49-bp cHS4 core (Chung el al. (1997) Proc. Natl. Acad. Sci., USA, 94: 575- 580) that interacts with the zinc finger DNA binding protein CTCF implicated in enhancer blocking assays (Bell et al. (1999) Cell, 98: 387-396).
[0184] One illustrative and suitable insulator is FB (FII/BEAD-A), a 77 bp insulator element, that contains the minimal CTCF binding site enhancer-blocking components of the chicken b-globin 5' HS4 insulators and a homologous region from the human T-cell receptor alpha/delta blocking element alpha/delta I (BEAD-I) insulator described by Ramezani et al. (2008) Stem Cell 26: 3257-3266. The FB “synthetic” insulator has full enhancer blocking activity. This insulator is illustrative and non-limiting. Other suitable insulators may be used including, for example, the full-length chicken beta-globin HS4 or insulator sub-fragments thereof, the ankyrin gene insulator, and other synthetic insulator elements.
Packaging signal.
[0185] In various embodiments the vectors described herein further comprise a packaging signal. A "packaging signal," "packaging sequence," or "PSI sequence" is any nucleic acid sequence sufficient to direct packaging of a nucleic acid whose sequence comprises the packaging signal into a retroviral particle. The term includes naturally occurring packaging sequences and also engineered variants thereof. Packaging signals of a number of different retroviruses, including lentiviruses, are known in the art. One illustrative, but non-limiting PSI is provided by SEQ ID NO:25.
Rev Responsive Element (RRE).
[0186] In certain embodiments the lenti viral vectors described herein comprise a Rev response element (RRE) to enhance nuclear export of unspliced RNA. RREs are well known to those of skill in the art. Illustrative RREs include, but are not limited to RREs such as that located at positions 7622-8459 in the HIV NL4-3 genome (Genbank accession number AF003887) as well as RREs from other strains of HIV or other retroviruses. Such sequences are readily available from Genbank or from the database with URL hiv- web.lanl.gov/content/index. One illustrative, but non-limiting RRE is shown in SEQ ID NO:26).
PolyPurine Tract (cPPT, 3'PPT).
[0187] In various embodiments the lentiviral vectors described herein further include a polypurine tract (e.g., central polypurine tract (cPPT), 3' poplypurine tract (3'PPT)). Insertion of a fragment containing the 3'PPT (see, e.g., SEQ ID NO:28) or the central polypurine tract (cPPT) in lentiviral (e.g., HIV-1) vector constructs is known to enhance transduction efficiency.
Expression-Stimulating Posttranscriptional Regulatory Element (PRE)
[0188] In certain embodiments the lentiviral vectors (LVs) described herein may comprise any of a variety of posttranscriptional regulatory elements (PREs) whose presence within a transcript increases expression of the heterologous nucleic acid (e.g., gp91phox) at the protein level. PREs may be particularly useful in certain embodiments, especially those that involve lentiviral constructs with modest promoters.
[0189] One type of PRE is an intron positioned within the expression cassette, which can stimulate gene expression. However, introns can be spliced out during the life cycle events of a lentivirus. Hence, if introns are used as PRE's they are typically placed in an opposite orientation to the vector genomic transcript.
[0190] Posttranscriptional regulatory elements that do not rely on splicing events offer the advantage of not being removed during the viral life cycle. Some examples are the posttranscriptional processing element of herpes simplex vims, the posttranscriptional regulatory element of the hepatitis B vims (HPRE) and the woodchuck hepatitis virus (WPRE). Of these the WPRE is typically preferred as it contains an additional cis-acting element not found in the HPRE. This regulatory element is typically positioned within the vector so as to be included in the RNA transcript of the transgene, but outside of stop codon of the transgene translational unit.
[0191] The WPRE is characterized and described in U.S. Pat. No: 6,136,597. As described therein, the WPRE is an RNA export element that mediates efficient transport of RNA from the nucleus to the cytoplasm. It enhances the expression of transgenes by insertion of a cis- acting nucleic acid sequence, such that the element and the transgene are contained within a single transcript. Presence of the WPRE in the sense orientation was shown to increase transgene expression by up to 7- to 10-fold. Retroviral vectors transfer sequences in the form of cDNAs instead of complete intron-containing genes as introns are generally spliced out during the sequence of events leading to the formation of the retroviral particle. Introns mediate the interaction of primary transcripts with the splicing machinery. Because the processing of RNAs by the splicing machinery facilitates their cytoplasmic export, due to a coupling between the splicing and transport machineries, cDNAs are often inefficiently expressed. Thus, the inclusion of the WPRE (see, e.g., SEQ ID NO:27) in a vector results in enhanced expression of transgenes.
Transduced Host Cells and Methods of cell transduction.
[0192] The recombinant lentiviral vectors (LV) and resulting virus described herein are capable of transferring a heterologous nucleic acid sequence (e.g. , a nucleic acid encoding a gp91phox) into a mammalian cell. In various embodiments, for delivery to cells, vectors described herein are preferably used in conjunction with a suitable packaging cell line or co transfected into cells in vitro along with other vector plasmids containing the necessary retroviral genes (e.g., gag and pol) to form replication incompetent virions capable of packaging the vectors of the present invention and infecting cells.
[0193] In certain embodiments the vectors are introduced via transfection into a packaging cell line. The packaging cell line produces viral particles that contain the vector genome. Methods for transfection are well known by those of skill in the art. After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art. Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with or without a dominant selectable marker, such as neomycin, DHFR, Glutamine synthetase, followed by selection in the presence of the appropriate drug and isolation of clones. In certain embodiments the selectable marker gene can be linked physically to the packaging genes in the construct.
[0194] Stable cell lines wherein the packaging functions are configured to be expressed by a suitable packaging cell are known (see, e.g., U.S. Patent No. 5,686,279, which describes packaging cells). In general, for the production of vims particles, one may employ any cell that is compatible with the expression of lentiviral Gag and Pol genes, or any cell that can be engineered to support such expression. For example, producer cells such as 293T cells and HT1080 cells may be used.
[0195] The packaging cells with a lentiviral vector incorporated therein form producer cells. Producer cells are thus cells or cell-lines that can produce or release packaged infectious viral particles carrying the therapeutic gene of interest (e.g., a Gp91phox). These cells can further be anchorage dependent which means that these cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic. Some examples of anchorage dependent cell lines used as lentiviral vector packaging cell lines when the vector is replication competent are HeLa or 293 cells and PERC.6 cells.
[0196] Accordingly, in certain embodiments, methods are provided of delivering a gene to a cell which is then integrated into the genome of the cell, comprising contacting the cell with a virion containing a lentiviral vector described herein. The cell (e.g., in the form of tissue or an organ) can be contacted (e.g. , infected) with the virion ex vivo and then delivered to a subject (e.g. , a mammal, animal or human) in which the gene (e.g., a nucleic acid encoding gp91phox) will be expressed. In various embodiments the cell can be autologous to the subject (/.<?., from the subject) or it can be non-autologous (/.<?., allogeneic or xenogenic) to the subject. Moreover, because the vectors described herein are capable of being delivered to both dividing and non-dividing cells, the cells can be from a wide variety including, for example, bone marrow cells, mesenchymal stem cells (e.g., obtained from adipose tissue), and other primary cells derived from human and animal sources. Alternatively, the virion can be directly administered in vivo to a subject or a localized area of a subject (e.g., bone marrow).
[0197] In certain embodiments, the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cells, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ hematopoietic stem and progenitor cells.
Gene therapy.
[0198] In still other embodiments, methods are provided for transducing a human hematopoietic stem cell. In certain embodiments the methods involve contacting a population of human cells that include hematopoietic stem cells with one of the foregoing lentivectors under conditions to effect the transduction of a human hematopoietic progenitor cell in said population by the vector. The stem cells may be transduced in vivo or in vitro, depending on the ultimate application. Even in the context of human gene therapy, such as gene therapy of human stem cells, one may transduce the stem cell in vivo or, alternatively, transduce in vitro followed by infusion of the transduced stem cell into a human subject. In one aspect of this embodiment, the human stem cell can be removed from a human, e.g., an X-CGD patient, using methods well known to those of skill in the art and transduced as noted above. The transduced stem cells are then reintroduced into the same or a different human. Stem cell/progenitor cell gene therapy.
[0199] In various embodiments the lentivectors described herein are particularly useful for the transduction of human hematopoietic progenitor cells or hematopoietic stem cells (HSCs), obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ hematopoietic stem and progenitor cells.
[0200] When cells, for instance CD34+ cells, dendritic cells, peripheral blood cells or tumor cells are transduced ex vivo, the vector particles are incubated with the cells using a dose generally in the order of between 1 to 50 multiplicities of infection (MOI) which also corresponds to 1 x 105 to 50 x 105 transducing units of the viral vector per 105 cells. This can include amounts of vector corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 MOI. Typically, the amount of vector may be expressed in terms of HT-29 transducing units (TU).
[0201] In certain embodiments cell-based therapies involve providing stem cells and/or hematopoietic precursors, transduce the cells with the lentivirus encoding, e.g., a Gp91phox, and then introduce the transformed cells into a subject in need thereof (e.g., a subject with a mutation in the CYBB gene).
[0202] In certain embodiments the methods involve isolating population of cells, e.g., stem cells from a subject, optionally expand the cells in tissue culture, and administer the lentiviral vector whose presence within a cell results in production of a Gp91phox in the cells in vitro. The cells are then returned to the subject, where, for example, they may provide a population of phagocytic cells that produce the Gp91phox.
[0203] In some illustrative, but non-limiting, embodiments, a population of cells, which may be cells from a cell line or from an individual other than the subject, can be used. Methods of isolating stem cells, immune system cells, etc., from a subject and returning them to the subject are well known in the art. Such methods are used, e.g., for bone marrow transplant, peripheral blood stem cell transplant, etc. , in patients undergoing chemotherapy.
[0204] Where stem cells are to be used, it will be recognized that such cells can be derived from a number of sources including bone marrow (BM), cord blood (CB), mobilized peripheral blood stem cells (mPBSC), and the like. In certain embodiments the use of induced pluripotent stem cells (IPSCs) is contemplated. Methods of isolating hematopoietic stem cells (HSCs), transducing such cells and introducing them into a mammalian subject are well known to those of skill in the art.
[0205] In certain embodiments a lentiviral vector described herein (see, e.g., Figure
19) is used in stem cell gene therapy for X-CDG by introducing a nucleic acid that encodes Gp91phox the into the bone marrow stem cells of patients with X-CGD followed by autologous transplantation.
Direct introduction of vector.
[0206] In certain embodiments direct treatment of a subject by direct introduction of the vector(s) described herein is contemplated. The lentiviral compositions may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Commonly used routes of delivery include inhalation, parenteral, and transmucosal.
[0207] In various embodiments pharmaceutical compositions can include an LV in combination with a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration· Supplementary active compounds can also be incorporated into the compositions.
[0208] In some embodiments, active agents, i.e., a lentiviral described herein and/or other agents to be administered together the vector, are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, poly glycolic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such compositions will be apparent to those skilled in the art. Suitable materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.
4,522,811. In some embodiments the composition is targeted to particular cell types or to cells that are infected by a vims. For example, compositions can be targeted using monoclonal antibodies to cell surface markers, e.g., endogenous markers or viral antigens expressed on the surface of infected cells. [0209] It is advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit comprising a predetermined quantity of a LV calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.
[0210] A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the LV described herein may conveniently be described in terms of transducing units (T.U.) of lentivector, as defined by titering the vector on a cell line such as HeLa or 293. In certain embodiments unit doses can range from 103, 104, 105, 106, 107, 10s, 109, 1010, 1011, 1012, 1013 T.U. and higher.
[0211] Pharmaceutical compositions can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to about 10 weeks; between about 2 to about 8 weeks; between about 3 to about 7 weeks; about 4 weeks; about 5 weeks; about 6 weeks, etc. It may be necessary to administer the therapeutic composition on an indefinite basis. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Treatment of a subject with a LV can include a single treatment or, in many cases, can include a series of treatments.
[0212] Illustrative, but non-limiting, doses for administration of gene therapy vectors and methods for determining suitable doses are known in the art. It is furthermore understood that appropriate doses of a LV may depend upon the particular recipient and the mode of administration· The appropriate dose level for any particular subject may depend upon a variety of factors including the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate: of excretion, other administered therapeutic agents, and the like.
[0213] In certain embodiments lentiviral gene therapy vectors described herein can be delivered to a subject by, for example, intravenous injection, local administration, or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 3054). In certain embodiments vectors may be delivered orally or inhalationally and may be encapsulated or otherwise manipulated to protect them from degradation, enhance uptake into tissues or cells, etc. Pharmaceutical preparations can include a LV in an acceptable diluent, or can comprise a slow release matrix in which a LV is imbedded. Alternatively or additionally, where a vector can be produced intact from recombinant cells, as is the case for retroviral or lentiviral vectors as described herein, a pharmaceutical preparation can include one or more cells which produce vectors. Pharmaceutical compositions comprising a LV described herein can be included in a container, pack, or dispenser, optionally together with instructions for administration.
[0214] The foregoing compositions, methods and uses are intended to be illustrative and not limiting. Using the teachings provided herein other variations on the compositions, methods and uses will be readily available to one of skill in the art.
Example 1
Development of Lentiviral Vectors for Treatment of X-CGD [0215] This example describes the development of novel lentiviral vectors for the treatment of X-1 inked Chronic Granulomatous Disease (X-CGD). In particular, we described the development of vector(s) that show higher expression levels than the current lentiviral vector undergoing clinical trials for X-CGD (pChim-CYBB, a.k.a. MSP-Gp91phox-WPRE, see, e.g., Santilli el al.( 2011) Mol. Therapy., 19(1): 122-122). This lentiviral vector uses a chimeric myeloid- specific promoter (MSP) and chronically under-expresses in the mature human neutrophil population and fails to recapitulate the lineage specific expression pattern of the native CYBB gene. In contrast, the vectors described in this example possesses strict lineage and stage specific expression that mimics the expression pattern of the native CYBB gene (see, e.g., Figure 1).
[0216] We have implemented a bioinformatics approach to elucidate the elements which regulate the endogenous CYBB gene in the human genome. The native CYBB topologically associated domain (TAD) comprises a 600kb window spanning lOOkb upstream to 500kb downstream of the CYBB gene. This CYBB TAD thus provides a 600,000 base pair window in the human genome to properly regulate the gene.
[0217] We attempted to elucidate the functional enhancer elements within the
600,000 base pair window and package the vital elements in a lentiviral vector of less than 9,000 base pairs. Using a bioinformatics approach, fifteen putative endogenous elements were identified within the native CYBB topologically associated domain (TAD).
[0218] In order to experimentally identify the critical enhancer elements that regulate the CYBB gene, each putative enhancer element was cloned upstream of the endogenous CYBB promoter to drive expression of a reporter gene (mCitrine) (see, e.g., Figure 2). In order to elucidate the function of each putative enhancer element, we assayed the activity of each of the vectors in cord blood (CB) CD34+ differentiated mature neutrophils and monocytes as well as RAMOS cells (B -lymphocyte cell line) which are 3 on-target cell lineages. All of the vectors were compared to the MSP-mCit-WPRE construct.
[0219] As shown in Figure 3, enhancer element 4 drives high levels of expression in mature neutrophils. Additionally, the expression level is significantly higher than that obtained using the current X-CGD vector undergoing clinical trials. Similarly, as shown in Figure 4 enhancer element 4 drives high levels of expression in monocytes as well, and again the expression levels are significantly higher than that obtained using the current X-CGD vector undergoing clinical trials.
[0220] Figure 5 shows that enhancer element 2 drives high levels of lineage specific expression in B-cells. None of the enhancer elements express in Jurkats (T-cells), suggesting lineage specific expression of each enhancer element (see, Figure 6). In contrast, the MSP- mCit-WPRE construct showed the highest level of off-target expression.
[0221] Thus, it appears that enhancer element 4 confers increased lineage specific expression in mature neutrophils and monocytes and shows 2 fold higher expression than the MSP-mCit-WPRE vector. No enhancer element 4 driven expression was observed in T-cells (Jurkats) or in B-cells (RAMOs). Enhancer element 2 appears to confer increased lineage specific expression in B-cells (RAMOs). No enhancer element 2 driven expression was observed in neutrophils, monocytes or T-cells.
[0222] It was thus determined to incorporate enhancer elements 2 and 4 into a lentiviral vector to design a vector possessing on-target lineage specific expression in neutrophils, monocytes and B-cells. We note that enhancer element 4 is made of two distinct enhancer modules (4E and 4R) and these were evaluated to determine if one of these elements could be eliminated to decrease the size of the vector.
[0223] Accordingly, five new vectors were produced for evaluation. These were 4E -
Int3 -pro-mCit-WPRE, 4R - Int3-pro-mCit-WPRE, 2 + 4E - Int3-pro-mCit-WPRE, 2 + 4R - Int3-pro-mCit-WPRE, and 2+ 4 - Int3 -pro-mCit-WPRE. These new vectors were evaluated in in CB CD34+ differentiated neutrophils and monocytes and in RAMOs and Jurkats.
[0224] As shown in Figure 7, the two fragments of enhancer element 4, 4F and 4R, act synergistically in neutrophils. However, element 4R alone still has higher expression than the MSP vector (current vector undergoing clinical trials). In monocytes, the 4R fragment seems to express at a similar level to the entire element 4 (see, Figure 8). Fineage specificity was maintained (see, Figure 9). Unlike MSP-mCit-WPRE (current vector undergoing clinical trials), all candidate vectors provided no off-target expression in T-cells.
Additionally, incorporation of enhancer element 2 appears to increase expression in B cells (see, Figure 10).
[0225] In view of these results, we conclude that the right half of element 4 (4R) seems to be the key contributor to lineage specific enhancer activity in neutrophils and monocytes. However, 4L and 4R seem to have a synergistic increase in expression when combined together in neutrophils and an additive effect when combined together in monocytes. Element 2 when combined with either of the myeloid enhancer elements 4, 4L or 4R remains a B-cell enhancer and is inert in the myeloid lineage. The vector 2-4R-Int3-pro- mCit-WPRE expresses 1.6 fold higher than MSP-mCit-WPRE in CB CD34+ differentiated neutrophils and monocytes. However it has 50% of the expression of MSP-mCit-WPRE in RAMO cells (B-cell lineage), but this may be a sufficient amount of expression to be therapeutic.
[0226] The 2-4Full-Int3-pro-mCit-WPRE expresses 2 fold and 1.6 fold higher than
MSP-mCit-WPRE in neutrophils and monocytes, respectively.
[0227] One X-CGD vector candidate of particular interest is 2-4R-Int3-pro-mCit-
WPRE in which mCit can be replaced with a nucleic acid encoding Gp91phox (see, e.g. ,
Figure 11) and which achieves the goal of possessing lineage specific expression, recapitulating the expression pattern of the native CYBB gene, and also expressing higher than the MSP-mCit-WPRE in mature neutrophils and monocytes.
[0228] Another goal is to decrease the size of vector while maintaining expression. In certain embodiments designed deletions can make the ‘’core” and “ultra-core” variants. Modifications to make vectors of 7.6kb and 5.9kb respectively (w/ Gp91phox in ORF). A secondary goal is to shrink the vector while increasing expression. In certain embodiments this can involve adding the "extra 4L core" and/or "extra 2" to the core and ultra core variants. Additionally, different codon optimizations of Gp91phox can be utilized to replace mCitrine in the open reading frame (ORF).
[0229] Full-length element 2 comprises 1092 base pairs. A 200 bp deletion was made to generate the 892 bp "core" variant (see, e.g., Table 1, SEQ ID NO:5). A 745 bp deletion was made to generate the 347 bp enhancer element 2 "ultra core" variant (see, e.g., Table 1, SEQ ID NO:6). [0230] Similarly, full length element 4R comprises 995 bp and a 496bp deletion was made to generate the 500bp enhancer element 4R "core" variant (see, e.g., Table 1, SEQ ID NO: 10). A 741 bp deletion was made to generate the 254 bp element 4R enhancer "ultra core" variant (see, e.g., Table 1, SEQ ID NO: 11).
[0231] Similarly, a 242 bp deletion was made to the intron 3 enhancer (1778 bp) to generate a 1536 bp intron 3 enhancer "core" variant and a 1058bp deletion was made to generate the 720bp intron 3 enhancer "ultra core" fragment which comprises a middle fragment (see, e.g., Table 1, SEQ ID NO:15) and a right fragment (see, e.g., Table 1, SEQ ID NO:16).
[0232] A 240bp deletion was made to the 507 bp full length CYBB endogenous promoter (see, e.g., Table 1, SEQ ID NO:l) to generate a 267 bp CYBB promoter "core" fragment (SEQ ID NO:2) and a 337 bp deletion was made to generate a minimal CYBB promoter " CYBB ultra core promoter" (see, e.g., Table 1, SEQ ID NO:3).
[0233] By making the “core” and “ultra-core” deletions, the vector size decreases by
1182bp and 2882bp, respectively as shown in Table 2.
Table 2. Size of "core" and "ultra core" vector variants.
Figure imgf000044_0001
[0234] Additionally, in certain embodiments "extra" fragment are included. Thus for example we hypothesized that the RELA TF binding site may increase B-cell expression. RELA plays role in many cellular processes including inflammation and immunity. Moreover, there is a B-cell lineage specific DNAsel hypersensitivity at the RELA binding size. Accordingly, in certain embodiments, the TF binding footprint can be included in the element 2 component (see, e.g., Table 1, SEQ ID NO: 7).
[0235] Additionally, in certain embodiments 4L "core" variant or a 4L "ultra core" variant can be included with the 4R component. Sizes of these "extra" fragment constructs are also shown in Table 2. [0236] The constructs shown in Table 3 were tested:
Table 3. Lenti viral constructs tested.
Figure imgf000045_0001
Figure imgf000046_0001
[0237] The ultra-core and extra ultracore variant vectors shows significantly higher expression in CB CD34+ differentiated neutrophils (CDllb+ CD66b+ CD15+ CD16+) (Figure 12) and in CB CD34+ differentiated monocytes (CDllb+ CD15+) (Figure 13) than the E2-E4R-Int3-pro-mCit-WPRE construct or the current clinical vector (MSP-Gp91phox- WPRE).
[0238] All of the ultra-core and extra ultra core variant vectors showed low expression (lower than the current clinical vector) in Jurkat Cells (Figure 14).
[0239] As show in Figure 16 ultra core vector and extra ultra core vectors showed higher titers than the core and extra core variants. [0240] In view of this we conclude that by making 2.9kb of deletions to our lead vector, we have increased expression as follows:
• 180% increase in neutrophils (3.4X higher than MSP)
• 150% increase in monocytes (2.2X higher than MSP)
• 129% increase in RAMOs (B-cell line) (1.16X higher than MSP) The vector also retains specificity with no T-cell expression (no change). Additionally, by making 1.2kb of deletions, we have decreased expression as follows:
• 15% reduction in neutrophils
• 33% reduction in monocytes
• 6% reduction in RAMOS (B-cell line) [0241] In certain embodiments one particularly suitable vector is the ultra-core variant of 2-4R-Int3-pro-mCit-WPRE (UC 2-4R-Int3-pro-mCit-WPRE). The ORF of mCitrine can be replaced with the therapeutic transgene (a nucleic acid encoding Gp91phox) to provide a clinically relevant vector.
[0242] Moreover, to maximize expression and titer a number of different codon optimizations were evaluated. These include jCAT, GeneArt, IDT, the codon optimized sequence in the current clinical vector (MSP-Gp91phox-WPRE) and a Gp91phox cDNA.
[0243] We originally screened the different codon optimization in the Int3-pro-
Gp91phox-WPRE vector backbone. However, the Int3-pro vector has high lineage specific expression and only expresses in mature neutrophils and did not express well in the PLB-985 CYBB' cell line (human promyeloblasts cell line). In order to use the PLB-985 X-CGD cell line, we decided to screen the different codon optimizations of Gp91phox with the MSP- Gp91phox-WPRE vector backbone.
[0244] The lead codon optimized sequence can be transferred to the various X-CGD vectors described herein. We note that codon optimization is for optimization of expression within a specific species (possibly even cell type), however the ideal codon optimization should be independent of which promoter/vector it is expressed from.
[0245] As shown in Figure 17, the jCAT optimization of gp91phox produced the highest expression level of the optimizations tested. The raw titers of the various optimizations are shown in (MSP-Gp91phox-WPRE) optimization and the jCAT optimization did not significantly differ (Figure 18).
[0246] In view of the foregoing, we conclude that jCAT is the optimal codon optimization of Gp91phox. This codon optimization increases expression over 2-fold higher than the native cDNA sequence and 1.2 fold higher than the current codon optimized sequence in the clinical MSP-Gp91phox-WPRE vector. This optimization also increases titer 1.2X higher than the native cDNA sequence (MSP-Gp91phox-WPRE).
[0247] By implementing a bioinformatics guided approach, we have rationally designed a novel X-CGD lentiviral vector possessing strict lineage and stage specific expression which mimics the expression pattern of the native CYBB gene. One lead candidate vector is the ultra core: UC 2-4R-Int3-pro-Gp91phox(jCAT)-WPRE vector, e.g., as illustrated in Figure 20, panel A. A map of this vector is shown in Figure 20, panel B, and the sequence is shown in Table 1, (SEQ ID NO:22). Example 2
Lineage-Specific Expression of MeloVec
[0248] Example 1 , above describes the generation of an optimized lead candidate vector: UC 2-4R-Int3-pro-mCit-WPRE (aka MveloVec). This vector showed improved titer, improved infectivity, and improved expression.
[0249] A number of different codon optimizations were screened and it was decided to replace the open reading frame of mCtrine with a jCAT codon optimization Gp91phox to express the actual therapeutic transgene.
[0250] As described herein in-vitro lineage specific expression of MyeloVec
(expressing mCitrine) was demonstrated by transplanting transduced human healthy donor (HD) cord blood (CB) CD34+ cells into NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) mice. Additionallyh the the ability of MyeloVec (expressing codon optimized Gp91phox) to functionally correct for the X-CGD phenotype was demonstrated by:
[0251] 1) Transducing murine X-CGD lineage negative (Lin-) hematopoietic stem and progenitor cells (HSPCs) and in-vitro differentiating to neutrophils to evaluate restoration of Gp91phox expression and restoration of functional oxidase activity;
[0252] 2) Transducing murine X-CGD Lin- HSPCs and transplanting cells into congenic B6.SJL-Ptprca Pepcb/BoyJ (Pepboy) mice to demonstrate in-vivo functional correct of the disease; and
[0253] 3) Transducing human X-CGD patient cells and in-vitro differentiating to neutrophils to demonstrate restoration of Gp91phox expression and functional oxidase activity.
[0254] Figure 21 demonstrates the improvement in titer (top panel) and infectivity
(bottom panel) as we optimized our vector from the original 2-4R-Int3-pro-mCit-WPRE to the CORE variant and to the ULTRA CORE (UC) variant. We improved titer and infectivity of as we decreased size of our vector from the original 2-4R-Int3-pro-mCit-WPRE to the CORE variant and to the Ultra Core (UC) variant.
[0255] As shown in Figure 22 MyeloVec (expressing mCitrine) is able to recapitulate the endogenous expression pattern of the native CYBB gene. In this experiment, we transduced healthy donor (HD) cord blood (CB) CD34+ hematopoietic stem and progenitor cells (HSPCs) and transplanted the cells into NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) mice. The gene modified cells will give rise to all the different lineages of the hematopoietic system. By evaluating which lineages are expressing mCitrine and to what degree, we can determine the lineage specific expression pattern of our vector and see if it mimics the endogenous expression pattern of the native CYBB gene.
[0256] MyeloVec is able to recapitulate the endogenous expression pattern of the native CYBB gene — very high expression in neutrophils, high bulk myeloid expression, medium levels of B-cell expression and minimal expression in T-cells and HSPCs. This is shown in blood Figure 22, panel A, and bone marrow (Figure 22, panel B).
[0257] MyeloVec is also able to recapitulate the temporal expression pattern of the native CYBB gene throughout neutrophil development. The expression gets higher as the neutrophils mature, mimicking the pattern of the native CYBB gene (see, e.g., Figure 23). [0258] Thus, in transduced human cord blood CD34+ cells transplanted into NSG mice, the pattern of mCitrine expression from MyeloVec recapitulated the endogenous expression pattern of Gp91phox across multiple lineages in the blood and bone marrow cells (see, e.g., Table 4).
Table 4. Expression pattern of MyeloVec.
Figure imgf000049_0001
[0259] We then replaced the open reading frame containing the mCitrine reporter gene with a jCAT codon optimized version of Gp91phox to express the therapeutic transgene for our functional studies.
[0260] Figures 24 and 25 demonstrate the ability of MyeloVec to functionally correct for the X-CGD phenotype in-vitro in mouse X-CGD HSPCs. For this experiment, we transduced murine X-CGD lineage negative (Lin-) Hematopoietic Stem and Progenitor Cells (HSPCs) and differentiate the cells to mature neutrophils to demonstrate the ability of our lead candidate vector (UC-2-4R-Int3-pro-Gp91phox(jCAT)-WPRE) to restore expression of Gp91phox (Figure 24) and oxidase activity Figure 25. In these experiments, oxidase activity was assessed by the Dihydrorhodamine (DHR) assay.
[0261] As shown in Figure 24, it is believed that MyeloVec can restore higher levels of Gp91phox than the current clinical vector (MSP) in neutrophils differentiated from X-CGD mouse HPSCs. Additionally, MyeloVec is able to restore oxidase activity to WT levels in transduced murine X-CGD cells differentiation into mature neutrophils (see, e.g., Figure 25). Thus, it appears that MyeloVec expresses Gp91phox 1.6 fold higher than MSP (current clinical vector) in murine CYBB Lin- in-vitro differentiated neutrophils and MyeloVec is able to restore oxidase activity to WT levels in murine CYBB Lin- in-vitro differentiated neutrophils.
[0262] Figures 26-29 demonstrate the the ability of MyeloVec to correct the X-CGD phenotype in-vivo in the X-CGD mouse model. Briefly, HPSCs were isolated from X-CGD mice and transduced with MyeloVec. The gene modified cells were then transplanted into congenic B6.SJL-Ptprca Pepcb/BoyJ (Pepboy) mice. Mice were harvested 16 weeks post transplant for analysis of Gp91phox expression and restoration of oxidase activity across the different hematopoietic lineages.
[0263] High levels of Gp91phox expression was restored in neutrophils and monocytes in the peripheral blood (see, e.g., Figure 26). This led to a restoration of oxidase activity near wildtype levels in the blood neutrophils and monocytes (see, e.g., Figure 27). High levels of Gp91phox expression was also restored in the bone marrow neutrophils and monocytes (see, e.g., Figure 28) which led to restoration of wildtype levels of oxidase activity (see, e.g.,
Figure 26). Thus, it appears that we have corrected the X-CGD mouse model in-vivo.
[0264] To demonstrate the ability of MyeloVec to functionally correct human patient
X-CGD cells in vitro, we transduced human X-CGD HSPCs with MyeloVec and differentiated the cells to mature neutrophils in-vitro. We then measured restoration of Gp91phox expression and restoration of oxidase activity by the DHR assay and the cytochrome C assay. Figure 30 shows the ability of MyeloVec to restore wildtype levels of Gp91phox expression in the human X-CGD neutrophils. Figure 31 shows the ability of MyeloVec to restore wildtype levels of cellular oxidase activity in the human X-CGD neutrophils (DHR assay). Figure 32shows the ability of MyeloVec to restore wildtype levels of bulk oxidase activity in human X-CGD neutrophils at an average VCN of 1.63 (cytochrome C assay). [0265] Thus, by implementing a bioinformatic-based design approach we developed our lead candidate X-CGD vector UC-2-4R-Int3-pro-coGp91phox-WPRE (MyeloVec) (see, e.g., Figure : 20, panels A and B, and SEQ ID NO: 22).
Conclusions [0266] The experiments described above, demonstrate the ability to correct the X-
CGD phenotype in-vivo in the murine X-CGD mouse model. In transduced murine X-CGD Lin- cells transplanted into lethally irradiated PepBoy mice:
[0267] MyeloVec was able to restore oxidase activity to WT levels in bone marrow neutrophils and monocytes; [0268] MyeloVec achieved close to WT levels of oxidase activity in peripheral blood neutrophils and monocytes at a VCN of 1.74 and greater; and
[0269] In-vitro differentiated neutrophils from human X-CGD patient CD34+
HSPCs transduced with MyeloVec restored Gp91phox expression and functional oxidase activity to healthy donor levels at an average VCN of 1.63. [0270] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
SEPUENCE LISTING
SEP ID NO:l CYBB promoter tagcacataaaattggcacatattaagcattttgtaaatatcaaccattacaattgttacta cttttctcagcaaggctatgaatgctgttccagcctgtcaaaatcacacctgtttaatgtgt tttacccagcacgaagtcatgtctagttgagtggcttaaaaattgtgatcaaatagctggtt agttaaaaagttatttcactgtgtaaaatacatcccttaaaatgcactgttatttatctctt agttgtagaaattggtttcattttccactatgtttaattgtgactggatcattatagaccct ttttttgtagttgttgaggtttaaagatttaagtttgttatggatgcaagcttttcagttga ccaatgattattagccaatttctgataaaagaaaaggaaaccgattgccccagggctgctgt tttcatttcctcattggaagaagaagcatagtatagaagaaaggcaaacacaacacattcaa cctctgccacc
SEP ID NO:2 _ Minimal CYBB promoter (core) tatctcttagttgtagaaattggtttcattttccactatgtttaattgtgactggatcatt atagaccctttttttgtagttgttgaggtttaaagatttaagtttgttatggatgcaagct tttcagttgaccaatgattattagccaatttctgataaaagaaaaggaaaccgattgcccc agggctgctgttttcatttcctcattggaagaagaagcatagtatagaagaaaggcaaaca caacacattcaacctctgccacc
SEP ID NP:3 _ Minimal CYBB promoter (ultra-core) tttaagtttgttatggatgcaagcttttcagttgaccaatgattattagccaatttctgat aaaagaaaaggaaaccgattgccccagggctgctgttttcatttcctcattggaagaagaa gcatagtatagaagaaaggcaaacacaacacattcaacctctgccacc
SEP ID NP:4 _ Enhancer element 2 gcttagtcatgttggtcccaaagtcatagttgatgagaagtagcaagttaagagagaaaga cttctagagataggtacatacacaatgataacaagtgacatcagagaacctaaggaagggc aaagaaagaaacactgcaaagcagactcaaacacttaaaagcatagcagcttggggccagt tagtgtaagagaaaaggagctccatatgcctcaatagaacctaagagcatcattgtactgc atttattcattcattcacttcacatgtttattcaacaaatgctatgtatactgagattttt ctctggtcattgtactggctagaacctaaaggagtgagactattaattagagtttacaatc tggcaatgatattaacagtctattcacaaaagggttaactcaagttaagccggcctaaatg tttatgcaaaataggatttttgcctaagtctaaagggtatcagaaaagtgtagccattgag aatgactcatttcatggtgttctcggatggcttaagtattattaatatgtctccatttcta gtgcaggaacctccacgttttagaggaaaggaggaaagaatttgtgaagactgtgcctaaa aaaggtagaaatttgtttacaatttatttaaagataaaagtaaagaactaggttgctttaa aaaagggagggaaagaaaatcaaaatacatcttatttgaggcattaaaacttttttaagaa aataaaatttaaaataaagttgtattcttctaaaaataattttttaaaccagctgaaaatg aaaaatgcagattatactaagaagcaactgttttacattctgctttctgaatggtatttaa aaactcagttattttcagaaatgaggaagtcttgatctgctagatgaaggtcggctgcagg tggtgtttattgctttatgatggcaacaaaccgtaaacccatcactcagtaaatattaaac tggctgaatgaatccaaagcatgtctaacatacaggaaaaacacagccctgttaagcagtc ttgaaacccacaagctacatggaaaacacagattcaactacatcataaaaattca
SEP ID NP:5 _ Enhancer element 2 core gagctccatatgcctcaatagaacctaagagcatcattgtactgcatttattcattcattc acttcacatgtttattcaacaaatgctatgtatactgagatttttctctggtcattgtact ggctagaacctaaaggagtgagactattaattagagtttacaatctggcaatgatattaac agtctattcacaaaagggttaactcaagttaagccggcctaaatgtttatgcaaaatagga tttttgcctaagtctaaagggtatcagaaaagtgtagccattgagaatgactcatttcatg gtgttctcggatggcttaagtattattaatatgtctccatttctagtgcaggaacctccac gttttagaggaaaggaggaaagaatttgtgaagactgtgcctaaaaaaggtagaaatttgt ttacaatttatttaaagataaaagtaaagaactaggttgctttaaaaaagggagggaaaga aaatcaaaatacatcttatttgaggcattaaaacttttttaagaaaataaaatttaaaata aagttgtattcttctaaaaataattttttaaaccagctgaaaatgaaaaatgcagattata ctaagaagcaactgttttacattctgctttctgaatggtatttaaaaactcagttattttc agaaatgaggaagtcttgatctgctagatgaaggtcggctgcaggtggtgtttattgcttt atgatggcaacaaaccgtaaacccatcactcagtaaatattaaactggctgaatgaatcca aagcatgtctaacatacaggaaaaacacagccctgttaagcagtcttgaaacccacaagct acatggaaaacacagattcaactacatcataaaaattc
SEQ ID NO:6 _ Enhancer element 2 ultra core Aaatcaaaatacatcttatttgaggcattaaaacttttttaagaaaataaaatttaaaata aagttgtattcttctaaaaataattttttaaaccagctgaaaatgaaaaatgcagattata ctaagaagcaactgttttacattctgctttctgaatggtatttaaaaactcagttattttc agaaatgaggaagtcttgatctgctagatgaaggtcggctgcaggtggtgtttattgcttt atgatggcaacaaaccgtaaacccatcactcagtaaatattaaactggctgaatgaatcca aagcatgtctaacatacaggaaaaacacagccctgttaagca
SEQ ID NO : 7 _ Element 2 RELA TF binding site aactgcccaggccatccacagatgactgtagatacatgtgtaagttcagttcacatcctca gaaccacccagatgtcctgtagatgcatgagaaatgttaaatgcttgttgttttaagccac taacttcagagtagtttgttatataacaaaaccgctgatgcaaatggcatcaaaaattgtt gaaagagagatgggggttcagggtgagagctgtaggtgattgtatctgtgctaataccaca tagcccttttttggggattgccatgaataatatattagctttgctatgagtaaaatactat atcctctgaattgtcatgaattacgtggagtcatacgtgttttggaagtgtgaaagtccct gggctcagataaaaggtgttgccatctggaaagtacaggtagtttatttcaattctgctcc aataactagcacgtcattccattcatgtagaaataagctactggctatctcactatctgaa atagaagtatgaactgtgggtaagtgggtgaggacaatgtctgagcaaccaaaaaggagct caaatcc
SEQ ID NO:8 _ Enhancer element 4 aaactaatatgaccttataagaggaggaagttggggcacaggcatgtacacacagaggaaa gaccatacagaggaaagaccatattaagataaaggaagaggatgaccatctacaagccaag caaaggggccccagaaggaaaccaaacatgctgaaaccttgatcttgaatttgtagcttct aaaactgtgagaaaataaatttctgttgtttaaaacatccaggctgaggtactttgttatg gaagccctgtcaaactaatgcaacaacatttcctcccattagatttcttaattcgtgtata gctggcctgataatgtcttatcagctaccccaactcaattgctgcaaatacatttttaaaa gttctggtggttgtagttgattgcacacttctgtatgagccaataatgtgaggcaagtctt taaaagggtagcacaatcagtctgaggttacaccatagatatggttaaccatagtgtggtc tccataacataggaagtcaagatcccccttcactcttgaccagtcagattgcacctagaac atttttctcaattctgcataccacatttaaagaggaagacaaaacccatgcgttgtgcagc taccacatgtcgagcatcagactatgtgcactgtgtacacttagtcctcccaccaacccaa tgaagatggtattaatacccacctcccattgtacagatgaggagactggggctaaatgagg tcaaataggttgctcaacagagatcttcacctccatggactcccatagccacactctgaac cctgtcatctctcagaagtgcactgcttctgaaatctgcatctcatacacccatcctctga ctaccacctcctgttccctggcttcctaattcactcacacccaagatgactgtccttcaac ctcatcaaactttgagttctttttgactctttgactttgctcccatcttgtgttcacttct tggcattctactcatcttagactcagttcacttctgccattttcttgcacaaatcctgaat tctctcatgcagtgcccttctgtaccacctgcaggcaaaaaccaaccctgatcaactcaat tgtcctctatacttgctcgtgggtgggtaagaaaagctagaaaagctacccacagactcct accattactgatttatgagctccaggctcaactgggcccttatctgggcctggaaatcatt ttgcatttctacagtcaagtctcctttctgaacaaaagatacaacattgaaaactgtcttc tgtttcctgaaatgtctactcactacctcactttcaacagataaccttgccctctctttca caaaggaaatggaaaccacaaagaggaagtccctcaccctgctgtccccagccctacaaat cctcctgcatctgcactctgctccttccctctttttacagagaggaggcccctcctgtcta aagcaaattccatttccttcctgccttgggctcagaaatctcaccccatccaaaatcttcc atggttagcctgtccctttgttgcgactctttctcaatatttacaagctcctatatttttt aaaataataaaactaggtcctcctggtgttcacatgttttcccaattgtagccaagtcctc tcattcttatcacagcctcagacattttgaggtgtctcactacctcacctcaacccacaac atctggcttccctcattgttttccagtaggcccctt
SEQ ID NO:9 Enhancer element 4R cagagatcttcacctccatggactcccatagccacactctgaaccctgtcatctctcagaa gtgcactgcttctgaaatctgcatctcatacacccatcctctgactaccacctcctgttcc ctggcttcctaattcactcacacccaagatgactgtccttcaacctcatcaaactttgagt tctttttgactctttgactttgctcccatcttgtgttcacttcttggcattctactcatct tagactcagttcacttctgccattttcttgcacaaatcctgaattctctcatgcagtgccc ttctgtaccacctgcaggcaaaaaccaaccctgatcaactcaattgtcctctatacttgct cgtgggtgggtaagaaaagctagaaaagctacccacagactcctaccattactgatttatg agctccaggctcaactgggcccttatctgggcctggaaatcattttgcatttctacagtca agtctcctttctgaacaaaagatacaacattgaaaactgtcttctgtttcctgaaatgtct actcactacctcactttcaacagataaccttgccctctctttcacaaaggaaatggaaacc acaaagaggaagtccctcaccctgctgtccccagccctacaaatcctcctgcatctgcact ctgctccttccctctttttacagagaggaggcccctcctgtctaaagcaaattccatttcc ttcctgccttgggctcagaaatctcaccccatccaaaatcttccatggttagcctgtccct ttgttgcgactctttctcaatatttacaagctcctatattttttaaaataataaaactagg tcctcctggtgttcacatgttttcccaattgtagccaagtcctctcattcttatcacagcc tcagacattttgaggtgtctcactacctcacctcaacccacaacatctggcttccctcatt gttttccagtaggcccctt
SEO ID NO:10 Enhancer element 4R Core catgcagtgcccttctgtaccacctgcaggcaaaaaccaaccctgatcaactcaattgtcc tctatacttgctcgtgggtgggtaagaaaagctagaaaagctacccacagactcctaccat tactgatttatgagctccaggctcaactgggcccttatctgggcctggaaatcattttgca tttctacagtcaagtctcctttctgaacaaaagatacaacattgaaaactgtcttctgttt cctgaaatgtctactcactacctcactttcaacagataaccttgccctctctttcacaaag gaaatggaaaccacaaagaggaagtccctcaccctgctgtccccagccctacaaatcctcc tgcatctgcactctgctccttccctctttttacagagaggaggcccctcctgtctaaagca aattccatttccttcctgccttgggctcagaaatctcaccccatccaaaatcttccatggt tagcctgtccct
SEO ID NO:ll Enhancer element 4R ultra core gcccttatctgggcctggaaatcattttgcatttctacagtcaagtctcctttctgaacaa aagatacaacattgaaaactgtcttctgtttcctgaaatgtctactcactacctcactttc aacagataaccttgccctctctttcacaaaggaaatggaaaccacaaagaggaagtccctc accctgctgtccccagccctacaaatcctcctgcatctgcactctgctccttccctctttt tacagagagg
SEO ID NO:12 Enhancer element 4L aaactaatatgaccttataagaggaggaagttggggcacaggcatgtacacacagaggaaa gaccatacagaggaaagaccatattaagataaaggaagaggatgaccatctacaagccaag caaaggggccccagaaggaaaccaaacatgctgaaaccttgatcttgaatttgtagcttct aaaactgtgagaaaataaatttctgttgtttaaaacatccaggctgaggtactttgttatg gaagccctgtcaaactaatgcaacaacatttcctcccattagatttcttaattcgtgtata gctggcctgataatgtcttatcagctaccccaactcaattgctgcaaatacatttttaaaa gttctggtggttgtagttgattgcacacttctgtatgagccaataatgtgaggcaagtctt taaaagggtagcacaatcagtctgaggttacaccatagatatggttaaccatagtgtggtc tccataacataggaagtcaagatcccccttcactcttgaccagtcagattgcacctagaac atttttctcaattctgcataccacatttaaagaggaagacaaaacccatgcgttgtgcagc taccacatgtcgagcatcagactatgtgcactgtgtacacttagtcctcccaccaacccaa tgaagatggtattaatacccacctcccattgtacagatgaggagactggggctaaatgagg tcaaataggttgctcaa
SEO ID NO:13 Enhancer element 4L core agccaataatgtgaggcaagtctttaaaagggtagcacaatcagtctgaggttacaccata gatatggttaaccatagtgtggtctccataacataggaagtcaagatcccccttcactctt gaccagtcagattgcacctagaacatttttctcaattctgcataccacatttaaagaggaa gacaaaacccatgcgttgtgcagct
SEO ID NO:14 Full Intron 3 Enhancer gatcatccctccttgacttccatacatgtggggattacaggcatgagtcacctgcctggcg agttccttgtttctaaggagacacaattcatttttattctccctacccccattagaatagt ttctatttagaggaagtaaagcctgagaaacaggcaatgttttcaccaagatggcctgtta agaaatcttggttagtctacaagtccaaatttcactgccggtgagcaccatgtcccatgag cagcacatgttgtaatgccagctagaggtctcaatcattgaaactttgctttgtaatcctt ctggttacctagagaaagaaagccccagggttgcccaccccaccactccaggaaaggtagg ggtaaaggctctcagactgctttgttgagaaaaatggagaatgggtgaagctcagcacaca aaaatctctgaggaagccttaaaaacccccaacttgccatgcagaaactaatttctgtctg gatggcagtcctagtcttaagatcagaaagaaacaggaaggtgagagggtgaggttttatc tgttaccttatatagtctgggagtcagaggcactcagtgtgcctctatctttaatcacgtg gtctagcactagtctcttgggctttctgtctcatagtttttttttttagttgaaaaacagg tcaactaacacaaatgtaagaaggcatatgttggtctaaaagtatattaattgtttaagtc tgtcaattagtgagttgtcagtcaataaatatttgttgagtgccatttatgtgctaagcac tggggacatgtggtaagtaaagattaagttatagataggccatgagcttaaggagcttaga gtgttaacaggagagacagagaataaatatggaacttccaaattataaacagtgctatgca aataaggtagtgttattcatatttatcagatattctactgccagcaggtgtggatattact gtcaacttacttgcctgagttctgtagattcaaagttggattttgtaatttctcccagttg cgtataaatatctaaatcagatacattgatggtgcgtgtggtgagatcaagtgtacaaaaa gtagagcttttgagtttctgtaaagtgttacaccccataaaatatgtacttctttttagtt ccacttcccattttcttgaaatatttttttcttactcagtttcaatagagcatagaaatct gctgaagtgactcaataatctcccttgcattagaatggtagtttattgaaatcgggcaagg cttccggtgacagtaacagagaaacttccctttagaagtcaatggcagaaagtaaagtaag ttagtaaggaagctatggggcatgatggcaacgtggataattgggaagtggctggcaataa tttagaagtaactcaaagcatataaatgcaatctgcctgatgatggggaacaaaaaattat gggcagtcacagacagtaaagtccttccttcctatgccaccaaccggttgtctcgcctcct tttttaaggaagtggtgaggagatggtattcttaaaagcccagtatcagcatgacttgtgg cttctttttggatttgtttgccattcctgtccacaccaaagagggtaggtgggaaaaatta gggatttgtgccctgatggttggacccactccactgatecattagttactagtaatctcac tttttcctttcaatataatatatgtgttttacattaactagctttttaaaaattacctatt aagatgaaa
SEQ ID NO : 15 Middle fragment INT3 enhancer ultra core cttaaaaacccccaacttgccatgcagaaactaatttctgtctggatggcagtcctagtct taagatcagaaagaaacaggaaggtgagagggtgaggttttatctgttaccttatatagtc tgggagtcagaggcactcagtgtgcctctatctttaatcacgtggtctagcactagtctct tgggctttctgtctcatagtttttttttttagttgaaaaacaggtcaactaacacaaatgt aagaaggcatatgttggtctaaaagtatatta
SEQ ID NO:16 INT3 enhancer right fragment ultra core
Agcttttgagtttctgtaaagtgttacaccccataaaatatgtacttctttttagttccac ttcccattttcttgaaatatttttttcttactcagtttcaatagagcatagaaatctgctg aagtgactcaataatctcccttgcattagaatggtagtttattgaaatcgggcaaggcttc cggtgacagtaacagagaaacttccctttagaagtcaatggcagaaagtaaagtaagttag taaggaagctatggggcatgatggcaacgtggataattgggaagtggctggcaataattta gaagtaactcaaagcatataaatgcaatctgcctgatgatggggaacaaaaaattatgggc agtcacagacagtaaagtccttccttcctatgccaccaaccggttgtctcgcctccttttt taaggaagtggtgagga
SEQ ID NO: 17 Gp91Phox cDNA atggggaactgggctgtgaatgaggggctctccatttttgtcattctggtttggctggggt tgaacgtcttcctctttgtctggtattaccgggtttatgatattccacctaagttctttta cacaagaaaacttcttgggtcagcactggcactggccagggcccctgcagcctgcctgaat ttcaactgcatgctgattctcttgccagtctgtcgaaatctgctgtccttcctcaggggtt ccagtgcgtgctgctcaacaagagttcgaagacaactggacaggaatctcacctttcataa aatggtggcatggatgattgcacttcactctgcgattcacaccattgcacatctatttaat gtggaatggtgtgtgaatgcccgagtcaataattctgatccttattcagtagcactctctg aacttggagacaggcaaaatgaaagttatctcaattttgctcgaaagagaataaagaaccc tgaaggaggcctgtacctggctgtgaccctgttggcaggcatcactggagttgtcatcacg ctgtgcctcatattaattatcacttcctccaccaaaaccatccggaggtcttactttgaag tcttttggtacacacatcatctctttgtgatcttcttcattggccttgccatccatggagc tgaacgaattgtacgtgggcagaccgcagagagtttggctgtgcataatataacagtttgt gaacaaaaaatctcagaatggggaaaaataaaggaatgcccaatccctcagtttgctggaa accctcctatgacttggaaatggatagtgggtcccatgtttctgtatctctgtgagaggtt ggtgcggttttggcgatctcaacagaaggtggtcatcaccaaggtggtcactcaccctttc aaaaccatcgagctacagatgaagaagaaggggttcaaaatggaagtgggacaatacattt ttgtcaagtgcccaaaggtgtccaagctggagtggcacccttttacactgacatccgcccc tgaggaagacttctttagtatccatatccgcatcgttggggactggacagaggggctgttc aatgcttgtggctgtgataagcaggagtttcaagatgcgtggaaactacctaagatagcgg ttgatgggccctttggcactgccagtgaagatgtgttcagctatgaggtggtgatgttagt gggagcagggattggggtcacacccttcgcatccattctcaagtcagtctggtacaaatat tgcaataacgccaccaatctgaagctcaaaaagatctacttctactggctgtgccgggaca cacatgcctttgagtggtttgcagatctgctgcaactgctggagagccagatgcaggaaag gaacaatgccggcttcctcagctacaacatctacctcactggctgggatgagtctcaggcc aatcactttgctgtgcaccatgatgaggagaaagatgtgatcacaggcctgaaacaaaaga ctttgtatggacggcccaactgggataatgaattcaagacaattgcaagtcaacaccctaa taccagaataggagttttcctctgtggacctgaagccttggctgaaaccctgagtaaacaa agcatctccaactctgagtctggccctcggggagtgcatttcattttcaacaaggaaaact tctaa
SEO ID NO:18 iCAT Codon optimized Gp91phox atgggcaactgggccgtgaacgagggcctgagcatcttcgtgatcctggtgtggctgggcc tgaacgtgttcctgttcgtgtggtactaccgcgtgtacgacatcccccccaagttcttcta cacccgcaagctgctgggcagcgccctggccctggcccgcgcccccgccgcctgcctgaac ttcaactgcatgctgatcctgctgcccgtgtgccgcaacctgctgagcttcctgcgcggca gcagcgcctgctgcagcacccgcgtgcgccgccagctggaccgcaacctgaccttccacaa gatggtggcctggatgatcgccctgcacagcgccatccacaccatcgcccacctgttcaac gtggagtggtgcgtgaacgcccgcgtgaacaacagcgacccctacagcgtggccctgagcg agctgggcgaccgccagaacgagagctacctgaacttcgcccgcaagcgcatcaagaaccc cgagggcggcctgtacctggccgtgaccctgctggccggcatcaccggcgtggtgatcacc ctgtgcctgatcctgatcatcaccagcagcaccaa gaccatccgccgcagctacttcgaggtgttctggtacacccaccacctgt tcgtgatcttcttcatcggcctggccatccacggcgccgagcgcatcgtgcgcggccagac cgccgagagcctggccgtgcacaacatcaccgtgtgcgagcagaagatcagcgagtggggc aagatcaaggagtgccccatcccccagttcgccggcaacccccccatgacctggaagtgga tcgtgggccccatgttcctgtacctgtgcgagcgcctggtgcgcttctggcgcagccagca gaaggtggtgatcaccaaggtggtgacccaccccttcaagaccatcgagctgcagatgaag aagaagggcttcaagatggaggtgggccagtacatcttcgtgaagtgccccaaggtgagca agctggagtggcaccccttcaccctgaccagcgcccccgaggaggacttcttcagcatcca catccgcatcgtgggcgactggaccgagggcctgttcaacgcctgcggctgcgacaagcag gagttccaggacgcctggaagctgcccaagatcgccgtggacggccccttcggcaccgcca gcgaggacgtgttcagctacgaggtggtgatgctggtgggcgccggcatcggcgtgacccc cttcgccagcatcctgaagagcgtgtggtacaagtactgcaacaacgccaccaacctgaag ctgaagaagatctacttctactggctgtgccgcgacacccacgccttcgagtggttcgccg acctgctgcagctgctggagagccagatgcaggagcgcaacaacgccggcttcctgagcta caacatctacctgaccggctgggacgagagccaggccaaccacttcgccgtgcaccacgac gaggagaaggacgtgatcaccggcctgaagcagaagaccctgtacggccgccccaactggg acaacgagttcaagaccatcgccagccagcaccccaacacccgcatcggcgtgttcctgtg cggccccgaggccctggccgagaccctgagcaagcagagcatcagcaacagcgagagcggc ccccgcggcgtgcacttcatcttcaacaaggagaacttctaa
SEO ID NO:19 Clinical co-op Gp91Phox atgggcaactgggccgtgaacgagggcctgagcatcttcgtgatcctggtgtggctgggcc tgaacgtgttcctgttcgtgtggtactaccgggtgtacgacatcccccccaagttcttcta cacccggaagctgctgggcagcgccctggccctggccagagcccctgccgcctgcctgaac ttcaactgcatgctgatcctgctgcccgtgtgccggaacctgctgtccttcctgcggggca gcagcgcctgctgcagcaccagagtgcggcggcagctggaccggaacctgaccttccacaa gatggtggcctggatgatcgccctgcacagcgccatccacaccatcgcccacctgttcaac gtggagtggtgcgtgaacgcccgggtgaacaacagcgacccctacagcgtggccctgagcg agctgggcgaccggcagaacgagagctacctgaacttcgcccggaagcggatcaagaaccc cgagggcggcctgtacctggccgtgaccctgctggccggcatcaccggcgtggtgatcacc ctgtgcctgatcctgatcatcaccagcagcaccaagaccatccggcggagctacttcgagg tgttctggtacacccaccacctgttcgtgatctttttcatcggcctggccatccacggcgc cgagcggatcgtgaggggccagaccgccgagagcctggccgtgcacaacatcaccgtgtgc gagcagaaaatcagcgagtggggcaagatcaaagagtgccccatcccccagttcgccggca acccccccatgacctggaagtggatcgtgggccccatgttcctgtacctgtgcgagcggct ggtgcggttctggcggagccagcagaaagtggtgattaccaaggtggtgacccaccccttc aagaccatcgagctgcagatgaagaaaaagggcttcaagatggaagtgggccagtacatct ttgtgaagtgccccaaggtgtccaagctggaatggcaccccttcaccctgaccagcgcccc tgaagaggacttcttcagcatccacatcagaatcgtgggcgactggaccgagggcctgttc aatgcctgcggctgcgacaagcaggaattccaggacgcctggaagctgcccaagatcgccg tggacggcccctttggcaccgccagcgaggacgtgttcagctacgaggtggtgatgctggt cggagccggcatcggcgtgacccccttcgccagcatcctgaagagcgtgtggtacaagtac tgcaacaacgccaccaacctgaagctgaagaagatctacttctactggctgtgccgggaca cccacgccttcgagtggttcgccgatctgctgcagctgctggaaagccagatgcaggaacg gaacaacgccggcttcctgagctacaacatctacctgaccggctgggacgagagccaggcc aaccacttcgccgtgcaccacgacgaggaaaaggacgtgatcaccggcctgaagcagaaaa ccctgtacggcaggcccaactgggacaacgagtttaagaccatcgccagccagcaccccaa cacccggatcggcgtgtttctgtgcggccctgaggccctggccgagacactgagcaagcag agcatcagcaacagcgagagcggccccaggggcgtgcacttcatcttcaacaaagaaaact tctga
SEQ ID NO:20 GeneArt optimized Gp91phox atgggaaactgggccgtgaatgagggcctgagcatcttcgtgatcctcgtgtggctgggcc tgaacgtgttcctgttcgtgtggtactaccgggtgtacgacatccctcctaagttcttcta cacccggaagctgctgggctctgctctggctcttgctagagcaccagccgcctgcctgaac ttcaactgcatgctgatcctgctgcctgtgtgccggaacctgctgagctttctgagaggca gcagcgcctgctgtagcaccagagttagacggcagctggacagaaacctgaccttccacaa gatggtggcctggatgatcgccctgcacagcgccattcacacaatcgcccacctgttcaac gtcgagtggtgcgtgaacgccagagtgaacaacagcgacccttacagcgtggccctgagcg agctgggcgatagacagaatgagagctacctgaatttcgcccggaagcggatcaagaaccc tgaaggcggactgtacctggccgtgacactgctggctggaatcacaggcgtggtcatcacc ctgtgcctgatcctgatcatcaccagcagcaccaagaccatccggcggagctacttcgagg tgttctggtacacccaccacctgtttgtgatctttttcatcggcctggccatccacggcgc cgagagaatcgttagaggacagacagccgagtctctggccgtgcacaatatcaccgtgtgc gagcagaaaatcagcgagtggggcaagatcaaagagtgccccattcctcagttcgccggca atcctcctatgacctggaagtggatcgtgggccccatgttcctgtacctgtgcgaaagact cgtgcggttctggcggagccagcagaaggtggtcattaccaaggtcgtgacacaccccttt aagaccatcgagctgcagatgaagaaaaagggcttcaagatggaagtgggccagtacatct ttgtgaagtgccccaaggtgtccaagctggaatggcaccccttcacactgacaagcgcccc tgaagaggacttcttcagcatccacatccggatcgtcggcgattggaccgagggcctgttt aatgcctgcggctgcgacaagcaagagttccaggatgcttggaagctgcccaagatcgccg tggacggaccttttggaacagccagcgaggacgtgttcagctacgaggtcgtgatgctcgt tggagccggcatcggcgtgacaccttttgccagcatcctgaagtctgtgtggtacaagtac tgcaacaacgccaccaacctgaagctcaagaagatctacttctactggctgtgccgggaca cccacgcctttgagtggttcgctgatctcctgcagctgctggaaagccagatgcaagagag aaacaacgccggcttcctgagctacaacatctacctgaccggctgggatgagagccaggcc aatcactttgccgtgcaccacgacgaagagaaggacgtgatcaccggcctgaagcagaaaa ccctgtacggcagacccaactgggacaacgagttcaagacaatcgcctctcagcaccccaa taccagaatcggagtgtttctgtgcggccctgaggctctggccgaaacactgagcaagcag agcatcagcaacagcgagtctggccctagaggcgtgcacttcatcttcaacaaagagaact tctga
SEQ ID NO:21 IDT optimized Gp91Phox atgggtaactgggcagtgaacgaggggctttctatctttgtcatactcgtgtggcttggcc tcaacgtgttcttgttcgtctggtactaccgagtgtacgacattcctcctaaattctttta cacacgcaaactccttgggtctgctttggcgctcgctcgggcacctgcagcgtgcctgaat tttaactgtatgctgatcctccttcctgtgtgccgaaaccttctttcattcctgcgaggta gttccgcttgctgctcaactcgggtgcgcaggcagcttgaccgcaacctgacgttccataa gatggtagcatggatgattgcgttgcattccgcgatccacactatcgcgcacctctttaac gtggaatggtgtgtaaacgcgagagtaaataacagcgacccatactctgtagcactttccg aacttggagaccggcagaacgaatcttaccttaacttcgctaggaagagaattaaaaaccc agaaggtggcctttatctcgcggttacgctgcttgctggcattaccggcgttgtcataact ctctgtttgatacttataattacaagctccaccaagactataagacgatcctactttgaag tcttctggtacacgcaccacctgttcgtaattttctttataggactggctattcacggtgc ggaaaggattgtacgaggtcagacagctgaatccctcgcggtgcacaacattacggtatgc gagcagaagataagtgagtggggaaaaattaaagagtgccccataccacagttcgccggca atccaccaatgacatggaagtggatcgtgggcccaatgttcctctacctgtgtgagcgcct tgtaaggttttggcgaagccaacagaaagtagtgataacgaaagtagttacacacccgttc aagacaatagagctccagatgaaaaaaaaaggcttcaagatggaagtcggtcaatacatat tcgtgaagtgcccgaaagtctcaaagttggaatggcacccattcactctcacatcagcgcc tgaagaagactttttctccattcatattcgcattgtgggcgattggacggaagggctcttt aacgcttgcgggtgtgataaacaagagtttcaagacgcatggaaattgcctaagatagcag ttgatggcccgttcggaaccgccagcgaagatgttttcagttacgaggtcgtcatgctcgt tggtgctggaatcggagttactccgtttgcttccatacttaagagcgtctggtacaaatat tgtaataatgccaccaatttgaaactcaagaagatttacttttattggttgtgtagggata ctcacgctttcgaatggttcgcagaccttctccagctccttgaaagccaaatgcaggaacg aaataacgcaggatttttgagctacaatatataccttacgggttgggacgaatctcaggct aatcatttcgcggtacaccatgatgaagaaaaggatgttataacgggtttgaaacaaaaaa cactctatggacgacctaactgggataatgaatttaaaacaatcgccagccaacatcctaa cacccggattggagttttcctgtgcgggccagaggcactcgcggagacgctgagtaaacaa tcaattagcaactctgagtccgggccacgcggggtgcattttatttttaacaaagagaact tctag
SEO ID NO:22 full vector
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA GGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGC CTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAG TGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC TTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcccgaacagggacttgaaagcgaaaggg aaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcga ggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatg ggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggtta aggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctaga acgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggac agctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagca accctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagat agaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaat tgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagag cagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgca gcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaa caatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatca agcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctgggg atttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggag taataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaatta acaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaat gaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatag tttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcag acccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggaga gagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaactttta aaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaaca gacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgatcacga gactagcctcgagAAATCAAAATACATCTTATTTGAGGCATTAAAACTTTTTTAAGAAAATA AAATTTAAAATAAAGTTGTATTCTTCTAAAAATAATTTTTTAAACCAGCTGAAAATGAAAAA TGCAGATTATACTAAGAAGCAACTGTTTTACATTCTGCTTTCTGAATGGTATTTAAAAACTC AGTTATTTTCAGAAATGAGGAAGTCTTGATCTGCTAGATGAAGGTCGGCTGCAGGTGGTGTT TATTGCTTTATGATGGCAACAAACCGTAAACCCATCACTCAGTAAATATTAAACTGGCTGAA TGAATCCAAAGCATGTCTAACATACAGGAAAAACACAGCCCTGTTAAGCAGCCCTTATCTGG GCCTGGAAATCATTTTGCATTTCTACAGTCAAGTCTCCTTTCTGAACAAAAGATACAACATT GAAAACTGTCTTCTGTTTCCTGAAATGTCTACTCACTACCTCACTTTCAACAGATAACCTTG CCCTCTCTTTCACAAAGGAAATGGAAACCACAAAGAGGAAGTCCCTCACCCTGCTGTCCCCA GCCCTACAAATCCTCCTGCATCTGCACTCTGCTCCTTCCCTCTTTTTACAGAGAGGCTTAAA AACCCCCAACTTGCCATGCAGAAACTAATTTCTGTCTGGATGGCAGTCCTAGTCTTAAGATC AGAAAGAAACAGGAAGGTGAGAGGGTGAGGTTTTATCTGTTACCTTATATAGTCTGGGAGTC AGAGGCACTCAGTGTGCCTCTATCTTTAATCACGTGGTCTAGCACTAGTCTCTTGGGCTTTC TGTCTCATAGTTTTTTTTTTTAGTTGAAAAACAGGTCAACTAACACAAATGTAAGAAGGCAT ATGTTGGTCTAAAAGTATATTAAGCTTTTGAGTTTCTGTAAAGTGTTACACCCCATAAAATA TGTACTTCTTTTTAGTTCCACTTCCCATTTTCTTGAAATATTTTTTTCTTACTCAGTTTCAA TAGAGCATAGAAATCTGCTGAAGTGACTCAATAATCTCCCTTGCATTAGAATGGTAGTTTAT TGAAATCGGGCAAGGCTTCCGGTGACAGTAACAGAGAAACTTCCCTTTAGAAGTCAATGGCA GAAAGTAAAGTAAGTTAGTAAGGAAGCTATGGGGCATGATGGCAACGTGGATAATTGGGAAG TGGCTGGCAATAATTTAGAAGTAACTCAAAGCATATAAATGCAATCTGCCTGATGATGGGGA ACAAAAAATTATGGGCAGTCACAGACAGTAAAGTCCTTCCTTCCTATGCCACCAACCGGTTG TCTCGCCTCCTTTTTTAAGGAAGTGGTGAGGATTTAAGTTTGTTATGgatgcaagcttttca gttgaccaatgattattagccaatttctgataaaagaaaaggaaaccgattgccccagggct gctgttttcatttcctcattggaAGAAGAAGCATAGTATAGAAGAAAGGCAAACACAACACA TTCAACCTCTGCCACCATGGGCAACTGGGCCGTGAACGAGGGCCTGAGCATCTTCGTGATCC TGGTGTGGCTGGGCCTGAACGTGTTCCTGTTCGTGTGGTACTACCGCGTGTACGACATCCCC CCCAAGTTCTTCTACACCCGCAAGCTGCTGGGCAGCGCCCTGGCCCTGGCCCGCGCCCCCGC CGCCTGCCTGAACTTCAACTGCATGCTGATCCTGCTGCCCGTGTGCCGCAACCTGCTGAGCT TCCTGCGCGGCAGCAGCGCCTGCTGCAGCACCCGCGTGCGCCGCCAGCTGGACCGCAACCTG ACCTTCCACAAGATGGTGGCCTGGATGATCGCCCTGCACAGCGCCATCCACACCATCGCCCA CCTGTTCAACGTGGAGTGGTGCGTGAACGCCCGCGTGAACAACAGCGACCCCTACAGCGTGG CCCTGAGCGAGCTGGGCGACCGCCAGAACGAGAGCTACCTGAACTTCGCCCGCAAGCGCATC AAGAACCCCGAGGGCGGCCTGTACCTGGCCGTGACCCTGCTGGCCGGCATCACCGGCGTGGT GATCACCCTGTGCCTGATCCTGATCATCACCAGCAGCACCAAGACCATCCGCCGCAGCTACT TCGAGGTGTTCTGGTACACCCACCACCTGTTCGTGATCTTCTTCATCGGCCTGGCCATCCAC GGCGCCGAGCGCATCGTGCGCGGCCAGACCGCCGAGAGCCTGGCCGTGCACAACATCACCGT GTGCGAGCAGAAGATCAGCGAGTGGGGCAAGATCAAGGAGTGCCCCATCCCCCAGTTCGCCG GCAACCCCCCCATGACCTGGAAGTGGATCGTGGGCCCCATGTTCCTGTACCTGTGCGAGCGC CTGGTGCGCTTCTGGCGCAGCCAGCAGAAGGTGGTGATCACCAAGGTGGTGACCCACCCCTT
CAAGACCATCGAGCTGCAGATGAAGAAGAAGGGCTTCAAGATGGAGGTGGGCCAGTACATCT TCGTGAAGTGCCCCAAGGTGAGCAAGCTGGAGTGGCACCCCTTCACCCTGACCAGCGCCCCC
GAGGAGGACTTCTTCAGCATCCACATCCGCATCGTGGGCGACTGGACCGAGGGCCTGTTCAA
CGCCTGCGGCTGCGACAAGCAGGAGTTCCAGGACGCCTGGAAGCTGCCCAAGATCGCCGTGG ACGGCCCCTTCGGCACCGCCAGCGAGGACGTGTTCAGCTACGAGGTGGTGATGCTGGTGGGC GCCGGCATCGGCGTGACCCCCTTCGCCAGCATCCTGAAGAGCGTGTGGTACAAGTACTGCAA CAACGCCACCAACCTGAAGCTGAAGAAGATCTACTTCTACTGGCTGTGCCGCGACACCCACG CCTTCGAGTGGTTCGCCGACCTGCTGCAGCTGCTGGAGAGCCAGATGCAGGAGCGCAACAAC GCCGGCTTCCTGAGCTACAACATCTACCTGACCGGCTGGGACGAGAGCCAGGCCAACCACTT CGCCGTGCACCACGACGAGGAGAAGGACGTGATCACCGGCCTGAAGCAGAAGACCCTGTACG GCCGCCCCAACTGGGACAACGAGTTCAAGACCATCGCCAGCCAGCACCCCAACACCCGCATC GGCGTGTTCCTGTGCGGCCCCGAGGCCCTGGCCGAGACCCTGAGCAAGCAGAGCATCAGCAA CAGCGAGAGCGGCCCCCGCGGCGTGCACTTCATCTTCAACAAGGAGAACTTCTAActgcagg aattcgagcatcttaccgccatttattcccatatttgttctgtttttcttgatttgggtata catttaaatgttaataaaacaaaatggtggggcaatcatttacatttttagggatatgtaat tactagttcaggtgtattgccacaagacaaacatgttaagaaactttcccgttatttacgct ctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaac tatgttgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgc ttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgtctctttatgagg agttgtggcccgttgtccgtcaacgtggcgtggtgtgctctgtgtttgctgacgcaaccccc actggctggggcattgccaccacctgtcaactcctttctgggactttcgctttccccctccc gatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgc tgggcactgataattccgtggtgttgtcggggaagggcctgctgccggctctgcggcctctt ccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctgg aattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactt tttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgcttt ttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaacta gggaacctactgcttaagcctcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCG TCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCT CTAGCagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaata tcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcat cacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactca tcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccc taactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgca gaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggagg cctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgt tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatc cccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttg cgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggt ggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttct tcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccct ttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatgg ttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgt tctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattct tttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaaca aaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcgggg aaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctca tgagacaataaccctgataaatgettcaataatagcacctagatcaagagacaggatgagga tcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagag gctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggc tgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaa ctgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgt gctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcagg atctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcgg cggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcga gcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatc aggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggat ctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttc tggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggcta cccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggt atcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaat tattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcac accgcatcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttcta aatacattcaaatatgtatccgctcatgaccaaaatcccttaacgtgagttttcgttccact gagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgta atctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaaga gctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttc ttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggtt ggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgca cacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatga gaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgg aacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcg ggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagccta tggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctca catgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgag ctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaa gagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggca cgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctca ctcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtg agcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaa ccctcactaaagggaacaaaagct ggagctgcaagcttggccattgcatacgttgtatccatatcataatatgtacatttatattg gctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatca attacggggtcattagttcatagcccatatatgg
SEO ID NO:23 CMV:
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA GGTCTATATAAGCAGAGCTCGTTTAGTGAACCG
SEO ID NO:24 3’R/U5:
GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT
GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG
ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC SEO ID NO:25 PSI:
Tcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtga gtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagt attaagcgggggag
SEO ID NO:26 RRE:
Tccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggta caggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattga ggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacct
SEO ID NO:27 WPRE:
Cccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatgg tggggcaatcatttacatttttagggatatgtaattactagttcaggtgtattgccacaaga caaacatgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggatt acaaaatttgtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtgga tatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctc cttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtg gcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgt caactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgc ctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgt cggggaagggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacg agtcggatctccctttgggccgcctccccgcctgga
SEO ID NO:28 3’ PPT: tttttaaaagaaaaggggggac
SEO ID NO:29 3’ delta U3/R/U5 tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctc tggttagaccagatctgagcctgggagctctctggctaactagggaacctactgcttaagcc tcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA
ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC
SEO ID NO:30 SV40 ori:
Atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttt tatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggct tttttggaggcctagg
SEO ID NO:31 KANr:
Attgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggcta tgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcagg ggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgag gcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgt cactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcat ctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacg cttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtac tcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgc cagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacc catggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggatteatega ctgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattg ctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctccc gattcgcagcgcatcgccttctatcgccttcttgacgagttcttctga SEP ID NO:32 _ COLEI: agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaa aaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttag gccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca gtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttacc ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaa cgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaa gggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggga gcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg

Claims

CLAIMS What is claimed is:
1. A recombinant lenti viral vector (LV) for the treatment of chronic granulomatous disease, said vector comprising: an expression cassette comprising a nucleic acid construct comprising: a CYBB promoter or effective fragment thereof; and a nucleic acid that encodes gp91phox operably linked to said CYBB promoter or promoter fragment.
2. The vector of claim 1, wherein said CYBB promoter or effective fragment thereof comprises a full-length endogenous CYBB promoter (SEQ ID NO:l).
3. The vector of claim 1, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
4. The vector of claim 3, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
5. The vector of claim 1, wherein said CYBB promoter comprises an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
6. The vector of claim 5, wherein said CYBB promoter consists of an effective fragment of the CYBB promoter whose sequence consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
7. The vector according to any one of claims 1-6, wherein said expression cassette comprises an enhancer element 2 (SEQ ID NO:4) or an effective fragment thereof.
8. The vector of claim 7, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core (SEQ ID NO:5).
9. The vector of claim 8, wherein the sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 core (SEQ ID NO:5).
10. The vector of claim 7, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 ultra core (SEQ ID NO:6).
11. The vector of claim 10, wherein the sequence of said effective fragment of enhancer element 2 consists of the sequence of enhancer element 2 ultra core (SEQ ID NO:6).
12. The vector according to any one of claims 1-11, wherein said expression cassette further comprises a RELA TF binding site or an effective fragment thereof.
13. The vector of claim 12, wherein said RELA TF binding site comprises or consists of the nucleic acid sequence of SEQ ID NO:7).
14. The vector according to any one of claims 1-11, wherein said expression cassette comprises enhancer element 4 or an effective fragment thereof.
15. The vector of claim 14, wherein said expression cassette comprises an enhancer element 4R or an effective fragment thereof.
16. The vector of claim 15, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R core (SEQ ID NO:10).
17. The vector of claim 15, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment comprises or consists of the nucleic acid sequence of enhancer element 4R ultra core (SEQ ID NO:ll).
18. The vector of claim 16, wherein said expression cassette comprises an effective fragment of enhancer element 4R where the nucleic acid sequence of said fragment consists of the nucleic acid sequence of enhancer element 4R ultra core (SEQ ID NO: 11).
19. The vector according to any one of claims 1-18, wherein said expression cassette comprises an enhancer element 4L or an effective fragment thereof.
20. The vector of claim 19, wherein said expression cassette comprises an effective fragment of enhancer element 4L where said fragment comprises or consists of the sequence of 4L core sequence (SEQ ID NO: 13).
21. The vector according to any one of claims 1-20, wherein said expression cassette comprises an intron enhancer element 3 (SEQ ID NO: 14) or an effective fragment thereof.
22. The vector of claim 21, wherein said expression cassette comprise an intron enhancer element 3 middle fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15.
23. The vector according to any one of claims 21-22, wherein said expression cassette comprises an intron enhancer element 3 right fragment comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16.
24. The vector according to any one of claims 1- 23, wherein said nucleic acid that encodes a nucleic acid that encodes gp91phox is a CYBB cDNA or a codon-optimized
CYBB.
25. The vector of claim 24, wherein said nucleic acid that encodes gp91phox is a CYBB cDNA (SEQ ID NO: 17).
26. The vector of claim 24, wherein said nucleic acid that encodes gp91phox is a codon optimized CYBB.
27. The vector of claim 26, wherein the sequence of said nucleic acid that encodes gp91phox is a codon optimized CYBB selected from the group consisting of jCAT codon optimized CYBB (SEQ ID NO: 18), GeneArt optimized CYBB (SEQ ID NO:20), IDT optimized CYBB SEQ ID NO:21), and previous clinical candidate (SEQ ID NO: 19).
28. The vector of claim 26, wherein the sequence of said nucleic acid that encodes gp91phox is a jCAT codon optimized CYBB (SEQ ID NO: 18).
29. The vector according to any one of claims 1-28, wherein said vector comprises a y region vector genome packaging signal.
30. The vector according to any one of claims 1-29, wherein said vector comprise a 5' LTR comprising a CMV enhancer/promoter.
31. The vector according to any one of claims 1-30, wherein said vector comprises a Rev Responsive Element (RRE).
32. The vector according to any one of claims 1-31, wherein said vector comprises a central polypurine tract.
33. The vector according to any one of claims 1-32, wherein said vector comprises a post-translational regulatory element.
34. The vector of claim 33, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE).
35. The vector according to any one of claims 1-34, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.
36. The vector of claim 1, wherein said vector comprises the features of full-sized 2-4R-Int3-pro-mCit-WPRE shown in Figure 19, where the mCit is replaced with a nucleic acid encoding Gp91phox.
37. The vector of claim 1, wherein said vector comprises the features of UC 2-4R-Int3-pro-coGp91phox-WRPE shown in Figure 20, panel A.
38. The vector of claim 37, wherein said vector comprise the features shown in the vector represented in Figure 20, panel B.
39. The vector of claim 38, wherein said vector comprises the nucleotide sequence of ultra core (UC) 2-4R-Int3-Pro-(GP91-jcat)-WPRE (SEQ ID NO: 22).
40. The vector according to any one of claims claim 1-39, wherein said vector shows high expression in CD33+ (bulk myeloid cells), high expression in CD19+ (B cells, high expression in CD66b+ CD15+ CDllb-i- CD16+ (mature neutrophils), and low or no expression in CD3+ T cells.
41. A host cell transduced with a vector according to any one of claims 1- 40.
42. The host cell of claim 41, wherein the cell is a stem cell.
43. The host cell of claim 42, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.
44. The host cell of claim 41, wherein the cell is a human hematopoietic progenitor cell.
45. The host cell of claim 44, wherein the human hematopoietic progenitor cell is a CD34+ cell.
46. A method of treating a chronic granulomatous disease (X-CGD), in a subject, said method comprising: transducing a stem cell and/or progenitor cell from said subject with a vector according to any one of claims 1-40; and transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express said Gp91phox.
47. The method of claim 46, wherein the cell is a stem cell.
48. The host cell of claim 46, wherein said cell is a stem cell derived from bone marrow.
49. The method of claim 46, wherein the cell is a human hematopoietic stem and progenitor cell.
50. The method of claim 49, wherein the human hematopoietic progenitor cell is a CD34+ cell.
51. A recombinant nucleic acid encoding one or more of the following: a CYBB promoter, or an effective fragment thereof; and/or a CYBB endogenous enhancer element 2 (CYBB B-cell enhancer), or an effective fragment thereof; and/or a CYBB endogenous enhancer 4R (CYBB endogenous myeloid enhancer), or an effective fragment thereof; and/or a CYBB endogenous enhancer 4L, or an effective fragment thereof; and/or a CYBB endogenous myeloid Intron 3 enhancer, or an effective fragment thereof; and/or a codon optimized nucleic acid encoding Gp91phox.
52. The nucleic acid of claim 51, wherein said nucleic acid encodes a sequence comprising or consisting of a full-length endogenous CYBB promoter (SEQ ID NO:l).
53. The nucleic acid of claim 51, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
54. The nucleic acid of claim 53, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (core) (SEQ ID NO: 2).
55. The nucleic acid of claim 51, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment comprises or consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
56. The nucleic acid of claim 55, wherein said nucleic acid encodes a sequence comprising an effective fragment of a CYBB promoter where said fragment consists of the minimal CYBB promoter (ultra core) (SEQ ID NO:3).
57. The nucleic acid according to any one of claims 51-56, wherein said nucleic acid encodes an effective fragment of a CYBB endogenous enhancer element 2 (CYBB B-cell enhancer).
58. The nucleic acid of claim 57, wherein the nucleic acid sequence of said a CYBB endogenous enhancer element 2 comprises or consists of the sequence of enhancer element 2 core (SEQ ID NO:5).
59. The nucleic acid of claim 57, wherein the nucleic acid sequence of said a CYBB endogenous enhancer element 2 comprises or consists of the sequence of enhancer element 2 ultra core (SEQ ID NO: 6).
60. The nucleic acid according to any one of claims 51-59, wherein said nucleic acid comprises an effective fragment of a CYBB endogenous enhancer 4R (CYBB endogenous myeloid enhancer).
61. The nucleic acid of claim 60, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous enhancer 4R comprises or consists of the sequence of enhancer element 4R ultra core (SEQ ID NO: 10).
62. The nucleic acid according to any one of claims 51-61, wherein said nucleic acid comprises an effective fragment of an enhancer element 4L.
63. The nucleic acid of claim 62, wherein said effective fragment of an enhancer element 4L comprises or consists of the sequence of the 4L core sequence (SEQ ID NO:13).
64. The nucleic acid according to any one of claims 51-63, wherein said nucleic acid comprises an effective fragment of a CYBB endogenous myeloid intron 3 enhancer.
65. The nucleic acid of claim 64, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous myeloid intron 3 enhancer comprises or consists of an element 3 middle fragment nucleic acid sequence (SEQ ID NO: 15).
66. The nucleic acid according to any one of claims 64-65, wherein the nucleic acid sequence of said effective fragment of a CYBB endogenous myeloid intron 3 enhancer comprises or consists of an intron enhancer element 3 right fragment (SEQ ID NO: 16).
67. The nucleic acid according to any one of claims 51-66, wherein said nucleic acid comprises a jCAT codon optimized CYBB (SEQ ID NO: 18).
68. The nucleic acid according to any one of claims 51-67, wherein said nucleic acid comprises an expression cassette.
69. The nucleic acid of claim 68, wherein said expression cassette is effective to express Gp91phox in vivo.
70. The nucleic acid according to any one of claims 51-69, wherein said nucleic acid comprises a lenti viral vector according to any one of claims 1-40.
PCT/US2020/060263 2019-11-12 2020-11-12 Lentiviral vectors in hematopoietic stem cells to treat x-linked chronic granulomatous disease WO2021097109A1 (en)

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