The β-interferon scaffold attachment region confers high-level transgene expression and avoids extinction by epigenetic modifications of integrated provirus in adipose tissue-derived human mesenchymal stem cells.
Because of their abundance and ease of isolation, multilineage differentiation, and paracrine and immunoregulatory capabilities, genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) might combine cell- and gene therapy-based strategies for efficacious tissue repair/regeneration. In this report, we aimed to analyze and influence the long-term dynamics of transgene expression in ASCs transduced with different gammaretroviral vector configurations incorporating the human β-interferon scaffold attachment region (IFN-SAR) and/or chicken 5'HS4 β-globin insulator sequences. In our undifferentiated ASC culture model, naked retroviral vectors experienced EGFP transgene extinction correlating with increases in both H3 histone deacetylation and CpG dinucleotide methylation within the 5' long terminal repeat-primer-binding site proviral region. Retroviral configurations incorporating the referred boundary elements alone or combined were able to prevent the development of the above epigenetic events and to reduce transgene extinction to different degrees. Particularly, the IFN-SAR sustained the highest levels of H3 histone acetylation and transgene expression throughout the study. Analogously, ASCs differentiating to adipocytes or osteocytes experienced a gradual decline of EGFP expression using naked retroviral vectors. In contrast, only retroviral configurations including the IFN-SAR alone were able to overcome the epigenetic pressure, yielding high-level, uniform transgene expression throughout both lineage differentiation processes. Thus, embedding the IFN-SAR in retroviral vectors should have positive implications in gene repair strategies using ASCs.
Moreno
Rafael
R
Medical and Molecular Genetics Center, Institut d'Investigació Biomèdica de Bellvitge (IDIBELL), Barcelona, Spain.
Martínez
Itziar
I
Petriz
Jordi
J
Nadal
Marga
M
Tintoré
Xavier
X
Gonzalez
Juan Ramón
JR
Gratacós
Eduard
E
Aran
Josep M
JM
D000107:Acetylation; D017667:Adipocytes / Q000166:cytology; D000273:Adipose Tissue / Q000166:cytology; D000328:Adult; D000818:Animals; D002454:Cell Differentiation; D002645:Chickens; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D038101:Insulator Elements / Q000235:genetics; D016899:Interferon-beta / Q000235:genetics*; D045170:Matrix Attachment Regions / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000821:virology*; D010011:Osteocytes / Q000166:cytology; D011533:Proviruses / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics*; D016662:Virus Integration / Q000235:genetics*
D006657:Histones; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016899:Interferon-beta