Importin‑4 Regulates Gene Delivery by Enhancing
Nuclear Retention and Chromatin Deposition by Polyplexes
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Abstract
For successful gene delivery, plasmid
DNA must be able to access
the nucleus in order to be transcribed. Numerous studies have shown
that gene delivery occurs more readily in dividing cells, which is
attributed to increased nuclear access when the nuclear envelope disassembles
during mitosis; however, nonviral carriers continue to have low transfection
efficiencies and require large quantities of DNA per cell to achieve
reasonable gene transfer, even in dividing cells. Therefore, we hypothesized
that using histone-derived nuclear localization sequences (NLS)s to
target polyplexes might enhance nuclear delivery by facilitating interactions
with histone effectors that mediate nuclear partitioning and retention
during mitosis. We discovered a novel interaction between polyplexes
linked to histone 3 (H3) N-terminal tail peptides and the histone
nuclear import protein importin-4, as evidenced by strong spatial
colocalization as well as significantly decreased transfection when
importin-4 expression was reduced. A fraction of the histone-targeted
polyplexes was also found to colocalize with the retrotranslocon of
the endoplasmic reticulum, Sec61. Super resolution microscopy demonstrated
a high level of polyplex binding to chromatin postmitosis, and there
also was a significant decrease in the amount of chromatin binding
following importin-4 knockdown. These results provide evidence that
natural histone effectors mediate both nuclear entry and deposition
on chromatin by histone-targeted polyplexes, and a translocation event
from the endoplasmic reticulum into the cytosol may occur before mitosis
to enable the polyplexes to interact with these essential cytoplasmic
proteins