Design: Epigenetic features
Key objectives
Genetic and epigenetic factors regulate gene function in mammalian cells. While the genetic information can easily be manipulated during vector design we must understand how alteration of DNA sequence impacts on gene function through epigenetic criteria. Episomal vectors that are used as gene expression systems must inevitably use the host cell machinery for DNA and RNA synthesis. The rational design of episomal vectors for any long-term application requires a detailed understanding of the role epigenetic features that influence:
- The establishment and maintenance of chromatin states defined by the histone code that are permissive for sustained gene expression.
- The dynamic properties of the episomal DNA that will contribute to the ability of the associated gene expression cassettes to engage the host cell functional compartments.
- Mechanisms such as binding the nuclear matrix and functional nuclear compartments that regulate chromatin functions such as gene expression, DNA replication and episome segregation during mitosis.
Deliverables
Prototype vectors and second generation vectors will be assessed to establish the efficiency with which they perform DNA and RNA synthesis as a function of their ability to engage the relevant host cell nuclear compartment. A detailed analysis of the chromatin organization of the vectors will be performed and evaluated in the context of the dynamic properties of vector sequences within nuclei in living cells. This will reveal the mechanisms that regulate nuclear interactions that are essential to maintain the active status of episomal genes. The behavior of different constructs will be assessed and compared. This information will allow us to make predictions about design parameters that will be used to inform the principles of rational vector design for specific applications. An iterative approach will be used to refine the performance of vectors produced within EPI-vector.