Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.
The Locus Control Region (LCR) requires intronic elements within beta-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 beta-globin 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 Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR 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.
Buzina
Alla
A
Developmental and Stem Cell Biology Program, SickKids, Toronto, Ontario, Canada.
Lo
Mandy Y M
MY
Moffett
Angela
A
Hotta
Akitsu
A
Fussner
Eden
E
Bharadwaj
Rikki R
RR
Pasceri
Peter
P
Garcia-Martinez
J Victor
JV
Bazett-Jones
David P
DP
Ellis
James
J
D000755:Anemia, Sickle Cell / Q000235:genetics / Q000628:therapy; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic; D005260:Female; D015870:Gene Expression; D005816:Genetic Complementation Test; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics*; D007438:Introns; D019901:Locus Control Region; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D050811:Octamer Transcription Factor-1 / Q000378:metabolism; D011247:Pregnancy; D011994:Recombinant Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D050811:Octamer Transcription Factor-1; C494166:Pou2f1 protein, mouse; D011994:Recombinant Proteins; D005914:Globins