| Intracellular Transport of Synthetic Gene Vectors (Experimental) Nishit Doshi Gene therapy shows great potential as a treatment method for a variety of diseases. However the challenge of safely delivering the transgene to the nucleus of targeted cells has proved to be the nemesis of most gene therapy trials. Synthetic vectors, which seek to mimic viruses, have been conceived and designed to surmount the safety issues associated with viral vectors, but they have been found to have much lower efficiencies than their natural counterparts. However, these synthetic vectors can be designed better if their interactions with the cell machinery are understood. The vector (viral or synthetic) which is to deliver the DNA to the host cell has to cross a number of biological barriers along the way. Assuming that it reaches the location of the host cell, it must enter the cell by endocytosis, after which it is still contained in a membranous vesicle called endosome. The vector is then carried towards the nucleus, through the viscous cell cytoplasm on the cell "subway" called microtubules. The vector then needs to escape from the endosome, protect the DNA cargo from the hostile degradative environment of the cytoplasm and finally deliver the DNA to the nucleus, where it can be transcribed by the host’s machinery. Viruses are smart and do this job effortlessly, but not synthetic vectors. Our work is focused on investigating this pathway systematically and quantitatively. We use Fluorescence Video Microscopy to image the intracellular vectors and then use Image Processing and Multiple Particle Tracking to quantify their distribution and trafficking. The experimental findings will then be integrated with mathematical models and simulations being developed in our own laboratory by Tuan Dinh. Thus, we hope to build an understanding of the basic principles that govern this fascinating phenomenon, which will help in designing better vectors to make gene therapy a clinical reality.
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