Lentivirus vectors incorporating the immunoglobulin heavy chain enhancer and matrix attachment regions provide position-independent expression in B lymphocytes.
In the present studies we developed lentivirus vectors with regulated, consistent transgene expression in B lymphocytes by incorporating the immunoglobulin heavy chain enhancer (E micro ) with and without associated matrix attachment regions (E micro MAR) into lentivirus vectors. Incorporation of these fragments upstream of phosphoglycerate kinase (PGK) or cytomegalovirus promoters resulted in a two- to threefold increase in enhanced green fluorescent protein (EGFP) mean fluorescence intensity (MFI) in B-lymphoid but not T-lymphoid, myeloid, fibroblast, or carcinoma cell lines. A 1-log increase in EGFP expression was observed in B-lymphoid cells (but not myeloid cells) differentiated from human CD34(+) progenitors in vitro transduced with E micro - and E micro MAR-containing lentivectors. Lastly, we evaluated the expression from the E micro MAR element in mice 2 to 24 weeks posttransplant with transduced hematopoietic stem cells. In mice receiving vectors with the E micro and E micro MAR elements upstream of the PGK promoter, there was a 2- to 10-fold increase in EGFP expression in B cells (but not other cell types). Evaluation of the coefficient of variation of expression among different cell types demonstrated that consistent, position-independent transgene expression was observed exclusively in B cells transduced with the E micro MAR-containing vector and not other cells types or vectors. Proviral genomes with the E micro MAR element had increased chromatin accessibility, which likely contributed to the position independence of expression in B lymphocytes. In summary, incorporation of the E micro MAR element in lentivirus vectors resulted in enhanced, position-independent expression in primary B lymphocytes. These vectors provide a useful tool for the study of B-lymphocyte biology and the development of gene therapy for disorders affecting B lymphocytes, such as immune deficiencies.
Lutzko
Carolyn
C
Division of Research Immunology and Bone Marrow Transplantation at Childrens Hospital Los Angeles, California 90027, USA.
Senadheera
Dinithi
D
Skelton
Dianne
D
Petersen
Denise
D
Kohn
Donald B
DB
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D004742:Enhancer Elements, Genetic; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D036961:Matrix Attachment Region Binding Proteins / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic; D019076:Transgenes
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D008164:Luminescent Proteins; D036961:Matrix Attachment Region Binding Proteins; D049452:Green Fluorescent Proteins