Long-Term Episomal Transgene Expression from Mitotically Stable Integration-Deficient Lentiviral Vectors
| Autor: | Frank A. Giordano, Angela F. Davies, Hanna Kymalainen, George Dickson, Sherif G. Ahmed, Rafael J. Yáñez-Muñoz, Stephanie Laufs, Juergen Bode, Manfred G. Schmidt, Caroline Mackie Ogilvie, Uwe Appelt |
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| Rok vydání: | 2014 |
| Předmět: |
Time Factors
Virus Integration Transgene Genetic Vectors Green Fluorescent Proteins Gene Expression Mitosis Replication Origin CHO Cells Biology Gene delivery Minicircle Polymerase Chain Reaction Viral vector Transduction (genetics) Cricetulus Plasmid Transduction Genetic Cricetinae Genetics Animals Transgenes Scaffold/matrix attachment region Molecular Biology Research Articles Cell Nucleus Chinese hamster ovary cell Lentivirus High-Throughput Nucleotide Sequencing Cell Cycle Checkpoints Dependovirus Matrix Attachment Regions Molecular biology Clone Cells Molecular Medicine DNA Circular Plasmids |
| Zdroj: | Human Gene Therapy. 25:428-442 |
| ISSN: | 1557-7422 1043-0342 |
| Popis: | Nonintegrating gene delivery vectors have an improved safety profile compared with integrating vectors, but transgene retention is problematic as nonreplicating episomes are progressively and rapidly diluted out through cell division. We have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression. We found that a transient cell cycle arrest at the time of transduction with IDLVs resulted in 13–45% of Chinese hamster ovary (CHO) cells expressing the transgene for over 100 cell generations in the absence of selection. The use of a scaffold/matrix attachment region did not result in improved episomal retention in this system, and episomes did not form after transduction with adeno-associated viral or minicircle vectors under the same conditions. Investigations into the episomal status of the vector genome using (1) linear amplification-mediated polymerase chain reaction followed by deep sequencing of vector–genome junctions, (2) Southern blotting, and (3) fluorescent in situ hybridization strongly suggest that the vector is not integrated in the vast majority of cells. In conclusion, we have developed an IDLV procedure generating mitotically stable episomes capable of long-term transgene expression. The application of this approach to stem cell populations could significantly improve the safety profile of a range of stem and progenitor cell gene therapies. |
| Databáze: | OpenAIRE |
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