An in vivo characterization of chromatin state and its relationship to the expression of foreign, non-integrating transgenic DNA in mouse liver
Abstract/Contents
- Abstract
- The pursuit of gene therapy to treat a wide-spectrum of diseases holds great promise, but its application in the clinic is still blocked by several barriers. Gene therapy approaches that rely on a virus to carry genetic material are plagued by problems with a host immune response and difficulty reaching therapeutic doses. Nonviral approaches have the advantage that delivering naked DNA does not illicit the same significant immune response and therapeutic levels are often initially easily attained. But, one prominent barrier to nonviral gene therapy is the lack of in vivo sustained expression from a foreign transgene. In the early stages after delivery, robust expression can be achieved from transgenic DNA, but this expression is very quickly and efficiently silenced, with loss of the desired effect of gene replacement. Our work took a two-step approach to further our understanding of how to create a better expression vector, both for gene therapy purposes as well as basic scientific goals of sustaining expression from delivered transgenic DNA. First, we studied whether we could manipulate and control the chromatin structure that a delivered gene adopts in vivo via nucleosome positioning signals on plasmid DNA in mouse liver. We developed a selective hybridization assay that allowed us to use a high-throughput sequencing approach to more rapidly screen the nucleosome occupancy and positioning of numerous constructs with varied DNA elements. We were able to transiently control the initial chromatin structure, but other forces in the cell soon overcame the thermodynamic preferences of nucleosome formation. We now believe that the use of nucleosome eviction elements may be a more promising approach: it will likely be more feasible to exclude nucleosome formation using rigid DNA sequences instead of permanently positioning a nucleosome using DNA sequence alone. Secondly, we gained a more fundamental understanding of the mechanism(s) responsible for the silencing of episomal (non-integrating) DNA constructs in mouse liver. We studied the differences between plasmid DNA and minicircle DNA, which was derived from a parental plasmid but lacked bacterial backbone sequences. At 6 weeks after delivery, plasmid DNA was effectively silenced but there was still active expression from the minicircle. We used high-throughput sequencing approaches to quantitate the levels of transcription and the enrichment of various histone modifications and RNA polymerase II (Pol II) on plasmid and minicircle. We found that Pol II appeared to stall at the transcription start site on the plasmid but was present at the 5' and 3' ends of the gene on the minicircle. This observation may explain the 28-fold higher level of transcript produced by the minicircle versus the plasmid. Though we saw enrichment of activating and silencing histone modifications on both plasmid and minicircle (likely due to our analysis of a population of cells which at any given point could have a construct that is actively expressed or silenced) we observed a striking abundance of a well-characterized histone modification associated with silencing on the plasmid and not on the minicircle. These results represent a contribution to the fields of gene therapy and chromatin biology that will help us move in the direction of achieving safe, therapeutic, and sustained expression of transgenic DNA.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Gracey, Lia |
|---|---|
| Associated with | Stanford University, Department of Genetics |
| Primary advisor | Fire, Andrew Zachary |
| Primary advisor | Kay, Mark Allan |
| Thesis advisor | Fire, Andrew Zachary |
| Thesis advisor | Kay, Mark Allan |
| Thesis advisor | Baker, Julie, (Professor of genetics) |
| Thesis advisor | Ford, James M. (James Matthew) |
| Thesis advisor | Wysocka, Joanna, Ph. D |
| Advisor | Baker, Julie, (Professor of genetics) |
| Advisor | Ford, James M. (James Matthew) |
| Advisor | Wysocka, Joanna, Ph. D |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Lia Eun Hee Gracey. |
|---|---|
| Note | Submitted to the Department of Genetics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Lia Gracey
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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