Development of a CENH3-based and non-viral vector system

New vectors for successful transgene delivery in patients are more
than needed. Current gene therapeutic vectors are mostly based on
virus genomes. A main reason is the very efficient administration
of the vector into the target cells. The viral background of these
systems also has severe side effects like e.g. immunological
reactions of the patient or the activation of oncogenes due to
insertional mutagenesis, which can result in cancer development. A
possibility to overcome these problems is the establishment of
extrachromosomally maintained, autonomously replicating and
non-viral vectors. The goal of this work was to establish such a
novel system with the help of the CENH3 protein. The
centromere-specific histone 3 variant CENH3 (H.sapiens:
CENH3CENP-A, D.melanogaster: CENH3CID) is sufficient for centromere
formation. It confers a stable and epigenetically heritable mark at
the centromeric region and does this also on plasmids. With the
help of CENH3 I changed the passive piggyback segregation mechanism
of Epstein-Barr virus-derived vectors into an active segregation
mechanism for plasmid DNA (pDNA) vectors. I demonstrated that the
new and active pDNA vector segregation mechanism prolongs pDNA
vector retention in cells. The information gained from basic
research might have great impact in the field of gene-therapeutic
research, as this mechanism might be a helpful tool in future gene
therapeutic vectors.