Testing a non-viral vector for its long term stability and replication ability
Başlık çevirisi mevcut değil.
- Tez No: 403141
- Danışmanlar: DR. PHILIP LAIPIS
- Tez Türü: Yüksek Lisans
- Konular: Genetik, Tıbbi Biyoloji, Mikrobiyoloji, Genetics, Medical Biology, Microbiology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2011
- Dil: İngilizce
- Üniversite: Unıversıty Of Florıda
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: Belirtilmemiş.
Özet
Özet yok.
Özet (Çeviri)
An ideal non-viral gene therapy vector should provide long term transgene expression in dividing and non-dividing cells without being dependent on integration since it was shown that integration can cause potential mutations. Stable expression without integration can be achieved by using vectors which persist episomally and replicate upon cell division. Thus far, Scaffold/Matrix Attachment Region (SMAR)-based vectors have attracted attention for their replication abilities in the presence of an initial selection. Here, we tested the long-term maintenance of SMAR vectors in 293 cells in terms of reporter Green Fluorescent Protein (GFP) expression without applying an initial selection pressure. The rate of GFP expression loss over time was similar for SMAR and non-SMAR control vectors suggesting that initial selection pressure is necessary for long-term SMAR-based vector replication. We also designed and tested a novel vector which combines a SMAR element with the minimal replicator (Latency Associated Nuclear Antigen and Terminal Repeats) of Kaposi Sarcoma-Associated Herpesvirus (KSHV). This vector was designed with a goal of increasing episomal maintenance and partition efficiency. The novel vector was also unable to provide long term GFP expression and GFP positive cells displayed a dying cell phenotype. The underlying reason for rapid loss of GFP positive cells was hypothesized to be the competition between the two different episomal maintenance factors, SMAR and LANA-TR, for binding to different nuclear locations. To address that possibility, a fluorescence-tagged KSHV replicator protein was utilized to identify the presence of disordered nuclear structures in the SMAR/LANA-TR vector transfected cells. It was found that the novel vector causes multi-nucleation after several cell divisions. In summary, it was found that the SMAR element is insufficient for long term maintenance without initial selection and the initial attempts of constructing a LANASMAR combination vector did not yield a vector suitable for gene therapy purposes. Vectors derived from episomally stable viruses are still a promising area of inquiry, but further development will be necessary.
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