Lentiviral vectors containing an enhancer-less ubiquitously acting chromatin opening element (UCOE) provide highly reproducible and stable transgene expression in hematopoietic cells.
Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy, we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters, the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters, suggesting a relative resistance to silencing. Furthermore, an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector, largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile.
Zhang
Fang
F
Centre for Immunodeficiency, Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom.
Thornhill
Susannah I
SI
Howe
Steven J
SJ
Ulaganathan
Meera
M
Schambach
Axel
A
Sinclair
Joanna
J
Kinnon
Christine
C
Gaspar
H Bobby
HB
Antoniou
Michael
M
Thrasher
Adrian J
AJ
D000818:Animals; D002843:Chromatin / Q000235:genetics*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D004195:Disease Models, Animal; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D020868:Gene Silencing; D015316:Genetic Therapy; D005822:Genetic Vectors; D016679:Genome, Viral / Q000235:genetics; D006367:HeLa Cells; D006412:Hematopoietic Stem Cells; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D011401:Promoter Regions, Genetic / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013155:Spleen Focus-Forming Viruses / Q000235:genetics; D014161:Transduction, Genetic; D019076:Transgenes / Q000502:physiology; D016662:Virus Integration / Q000235:genetics; D053632:X-Linked Combined Immunodeficiency Diseases / Q000235:genetics / Q000628:therapy*
C518294:CBX3 protein, human; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D007376:Interleukin-2; C439768:hnRNP A2