Boundary sequences stabilize transgene expression from subtle position effects in retroviral vectors.
Transgene expression shut-down, attenuation and/or variability from integrated retroviral vectors pose a major obstacle to gene therapy trials involving hematopoietic cells. We have undertaken a systematic assessment of the behavior of different configurations containing IFN-beta SAR and/or 5'HS4 beta-globin insulator sequences within a gammaretroviral vector optimized for high-level expression, focusing on the long-term achievement of stable, homogeneous transgene expression in the successfully transduced cells. Introduction of these cis regulatory elements did not perturb virus production and stability. Conversely, the SAR/5'HS4 insulator combination appeared to increase the homogeneity of EGFP expression in mass cultures. Furthermore, a clonal analysis of the dispersion of EGFP expression revealed that the IFN-SAR/5'HS4 insulator dyad was particularly effective in reducing the variability of transgene expression when both sequences were placed in opposite orientations within the retroviral backbone. These results may prove useful for the design of more stable retroviral expression cassettes able to counteract chromosomal position effects.
Moreno
Rafael
R
Medical and Molecular Genetics Center, Institut d'Investigació Biomèdica de Bellvitge, Hospital Duran i Reynals, Barcelona, Spain.
Martinez
Itziar
I
Petriz
Jordi
J
González
Juan Ramón
JR
Gratacós
Eduard
E
Aran
Josep M
JM
D045744:Cell Line, Tumor; D055012:Chromosomal Position Effects; D019204:GTP-Binding Proteins / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D038101:Insulator Elements; D016899:Interferon-beta / Q000235:genetics; D012190:Retroviridae; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics*; D055544:beta-Globins / Q000378:metabolism*
D055544:beta-Globins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016899:Interferon-beta; D019204:GTP-Binding Proteins; C505672:SAR protein, human