β-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy

Public Deposited
Analytics

Downloadable Content

Download PDF
Request Version for Screen Reader
Creator
  • Buzina, Alla
    • Other Affiliation: SickKids
  • Lo, Mandy Y. M.
    • Other Affiliation: SickKids
  • Moffett, Angela
    • Other Affiliation: SickKids
  • Hotta, Akitsu
    • Other Affiliation: SickKids
  • Fussner, Eden
    • Other Affiliation: SickKids
  • Bharadwaj, Rikki R.
    • Other Affiliation: SickKids
  • Pasceri, Peter
    • Other Affiliation: SickKids
  • Garcia-Martinez, J. Victor
    • Affiliation: School of Medicine, Center for AIDS Research
    • Other Affiliation: University of Texas Southwestern Medical Center
  • Bazett-Jones, David P.
    • Other Affiliation: SickKids
  • Ellis, James
    • Other Affiliation: SickKids
Abstract
  • The Locus Control Region (LCR) requires intronic elements within b-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional bglobin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igm 39MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.
Date of publication
Keyword
DOI
Identifier
Resource type
  • Article
Rights statement
  • In Copyright
License
  • Attribution 3.0 United States
Journal title
  • PLoS Genetics
Journal volume
  • 4
Journal issue
  • 4
Language
  • English
ISSN
  • 1553-7390

Relations

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details journal.pgen.1000051.PDF 2020-06-15 Public Download