Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells.
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.
Santilli
Giorgia
G
Molecular Immunology Unit, Institute of Child Health, London, UK.
Almarza
Elena
E
Brendel
Christian
C
Choi
Uimook
U
Beilin
Chiara
C
Blundell
Michael P
MP
Haria
Sneha
S
Parsley
Kathryn L
KL
Kinnon
Christine
C
Malech
Harry L
HL
Bueren
Juan A
JA
Grez
Manuel
M
Thrasher
Adrian J
AJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism; D056649:Cathepsin G / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D056915:DNA Copy Number Variations; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D050172:Genes, X-Linked; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D022423:Myeloid Cells / Q000378:metabolism / Q000502:physiology*; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051578:Proto-Oncogene Proteins c-fes / Q000235:genetics*; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D013155:Spleen Focus-Forming Viruses / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism; D020079:Terminal Repeat Sequences; D015534:Trans-Activators / Q000378:metabolism; D019076:Transgenes; D000099232:Proto-Oncogene Protein Spi-1
D022762:CCAAT-Enhancer-Binding Proteins; D056649:Cathepsin G; D019255:NADPH Oxidases; D011518:Proto-Oncogene Proteins; D051578:Proto-Oncogene Proteins c-fes; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000099232:Proto-Oncogene Protein Spi-1; C105949:Pirb protein, mouse