Self-inactivating lentiviral vectors resist proviral methylation but do not confer position-independent expression in hematopoietic stem cells.
Oncoretroviral expression is transcriptionally silenced in embryonic and hematopoietic stem cells (HSCs). This is associated with methylation of viral and internal promoters. We determined whether self-inactivating (SIN) lentiviral vectors (LV) would circumvent proviral silencing in HSCs. We studied long-term expression, methylation, and position effects (PE) from two GFP-encoding SIN-LV containing erythroid enhancers and the human ankyrin-1 promoter (h-Ank-P) using the murine secondary bone marrow (BM) transplant assay. Proviral expression was detected in RBC 6-11 months following transplant only in 28 of 49 secondary mice, with 0.9 +/- 0.2 copy/cell and oligoclonally integrated provirus in BM, spleen, and thymus. Twenty-one of 49 secondary mice lacked integrated provirus. Secondary mice containing provirus also had GFP-expressing RBCs, although proviral copy number did not always correlate with expression, suggesting either proviral methylation or chromatin PE. The endogenous h-Ank-P was partially methylated in nonerythroid cell lines and unmethylated in erythroid cell lines. However, h-Ank-P in the provirus was unmethylated in erythroid and nonerythroid cells within secondary murine BM. Despite lack of methylation, GFP expression was variable in secondary BFU-e and in single-copy mouse erythroleukemia cell clones. Taken together, these data show that erythroid-specific SIN-LV express long term and resist methylation-associated proviral silencing, but may require additional elements to confer position-independent expression.
Division of Hematology-Oncology, Children's Hospital Los Angeles and University of Southern California Keck School of Medicine, 90027, USA.
Azim
A
Mohamedali
Francois
F
Moreau-Gaudry
Emmanuel
E
Richard
Ping
P
Xia
Jan
J
Nolta
Punam
P
Malik
D000818:Animals; D017487:Ankyrins / Q000235:genetics; D019175:DNA Methylation; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D011533:Proviruses / Q000235:genetics*; D014161:Transduction, Genetic
C110202:ANK1 protein, human; D017487:Ankyrins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins