Lentiviral vector-mediated gene transfer in vitro and in vivo

Zhang, Bing. (2004). Lentiviral vector-mediated gene transfer in vitro and in vivo PhD Thesis, School of Medicine, The University of Queensland.

       
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Author Zhang, Bing.
Thesis Title Lentiviral vector-mediated gene transfer in vitro and in vivo
Formatted title

Lentiviral vector-mediated gene transfer in vitro and in vivo


School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2004
Thesis type PhD Thesis
Supervisor Dr. Ming Wei
Prof Geoff Cleghom
Prof Malcolm West
Total pages 147
Collection year 2004
Language eng
Subjects L
321011 Medical Genetics
730107 Inherited diseases (incl. gene therapy)
Formatted abstract

At this early stage of gene therapy research, the development of efficient vectors for gene transfer and the means of their mass production under aseptic conditions are crucial elements in moving the technology forward.     

In the thesis presented, a new method for harvesting high titre lentiviral vector stocks from large volumes of cell supernatant was devised and the results showed that it is possible to obtain high yields of vector (up to 75% of viral vector particles were recovered) in a relatively short time as compared to the methods currently in use. The new method of harvesting lentiviral vector involves the treatment of viral vector harvests with poly-L-Lysine (PLL) followed by a low speed centrifugation. Optimal conditions have been established when 0.005% of PLL is added to vector supernatant harvests, followed by incubation for 30 min and centrifugation at 10,000 g for 2 hour at 4 °C. Direct comparison with the current method of ultracentrifugation of untreated viral particles demonstrated that the new method consistently produces large volumes (6ml) of high-titre viral vector at 1 x 10 transduction unit (TU/ml) from about 3 litres of supernatant in one round of concentration. Electron microscope analysis showed that PLL/viral vector forms complexes, which facilitate easy precipitation at low speed (10000 g), a speed, which does not usually precipitate viral particles efficiently. Transfection of several cell lines in vitro and transduction in vivo in the liver with the lentivector/PLL complexes demonstrated efficient gene transfer without any significant signs of vector damage or toxicity by the PLL. These results are presented in Chapter 3.  

 

For successful gene therapy, there is a prerequisite of a reliable method for determining the true number of active vector particles. This is a central issue in developing an appropriate protocol for use in the treatment of a human disease. A number of cell lines have been compared with regard to their efficiency of gene transfer with a view to optimising the yield and hence the economy of lentiviral vector production. On the basis of their susceptibility to the transduction of a marker gene, the commonly used cell lines 293, 293T or NIH 3T3 have been compared with a new liver embryonic cell line FRL 19. It was shown that FRL 19 is 1.9 fold more susceptible than line 293T, 3.6 fold than in the case of NIH 3T3 and 5.6 fold more susceptible than 293 to lentivector-mediated gene transfer. The marker gene used was green fluorescent protein. It can be noted that the use of the common cell lines 293 etc. leads to a marked underestimate of active lentiviral vector particles which could be of significance in human gene therapy. A further advantage in the use of FRL 19 is that it is a less demanding in its nutritional requirements than many other cell lines which can lead to major economies for lentiviral culture. These results and their discussion are found in Chapter 4 

 

The third-generation packaging unit of HIV-1-based vectors that contain only three of the nine genes present in the genome of the parental vims: gag, pol, and rev has been reported. Based on the third generation of packaging unit, a new lentiviral vector with a new marker gene, the Renilla luciferase, was generated in this study.    

 

The practical application of gene transfer requires confidence of the means by which the gene is delivered into the animal. For this study, there is an investigation into whether polyethyleneimine (PEI) is a suitable vehicle to facilitate lentiviral vector   mediated gene transfer. In the course of these experiments, PEI and PLL were compared as DNA transfection reagents in vitro and PEI was compared as an adjuvant for delivery of DNA and of lentivirus in vivo. There are theoretical considerations which suggest that PEI efficiently increased transgene expression in vivo. The results from these experiments confirmed that PEI efficiently increased plasmid DNA transfection efficiency in vitro, but in vivo experiments results showed that PEI/lentivector failed to lead to significantly higher transduction efficiency as compared to the lentivector alone. The main challenge for successful gene transfer is the development of safe and effective gene transfer techniques that allow long-term, tissue-specific, and regulated expression of the desired protein. A method of efficiently delivering the lentivector into animal liver was evaluated in the last part of this study. A repeated portal vein infusion of PLL/lentivector shows 40-80% of rat liver cells were GFP positive 2 weeks after gene delivery as measured by confocal microscopy. The third-generation lentivector with or without PEI were administered into rat liver by tail vein injection, and luciferase activity measured. The results show that relatively efficient transgene expression is achieved in both groups through the time course from day 2 to week 8. There were no toxicity effects in either liver or kidney by both delivery methods. The results and discussion are found in chapter 5.

Keyword Gene therapy

Document type: Thesis
Collection: UQ Theses (RHD) - UQ staff and students only
 
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Created: Fri, 24 Aug 2007, 18:32:23 EST