Development of murine leukemia virus-based retroviral vectors with a minimum possibility of cis-activation
| Autor: | Sunyoung Kim, Jiwon Jang, Jung Young Kim, Joonyeop Lee, Keejung Yoon, Kyeong Seok Lee |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Ribosomal Proteins
Ribosomal Protein L10 Genetic Vectors Cell Line Viral vector Insertional mutagenesis Mice Transduction (genetics) Transduction Genetic Murine leukemia virus Genetics Animals Humans Promoter Regions Genetic Enhancer Molecular Biology Gene DNA Primers Gammaretrovirus biology Terminal Repeat Sequences Genetic Therapy biology.organism_classification Virology Leukemia Virus Murine Molecular Medicine Expression cassette |
| Zdroj: | Gene Therapy. 18:240-249 |
| ISSN: | 1476-5462 0969-7128 |
| Popis: | The possibility of insertional mutagenesis in retroviral gene therapy can be reduced by using a vector lacking the enhancer sequence present in the U3 of the long-terminal repeats. However, such vectors suffer from many pitfalls. We attempted to improve a murine leukemia virus-based retroviral vector containing the enhancer-free U3, first by making it easier to construct a producer line and then by introducing the cellular RPL10 promoter as an internal promoter. The reverse orientation of the expression cassette of the transgene was found to give higher transducing titer and higher-level gene expression. The deletion analysis revealed that the 54-bp-long sequence of U3 (34 and 20 bp present at 5' and 3' extreme ends, respectively) was sufficient for the functions of retroviral vectors. The data from the in vitro cell culture assay indicated that the final construct, ROK, containing all these features, had little cis-activation activity, even if it was placed right upstream from the RNA start site of the neighboring gene. Our data suggested that the newly developed vector might provide increased safety, while still producing high viral titer from a stable producer line and high-level gene expression in various target cells including human CD34(+) stem cells. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|