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Direct Nuclear Transport of Aptamer-RNA Chimeras to Specific Cell Types
Jonathan W Kotula, Jeffrey J Kovacs, Robert J Lefkowitz, and Bruce A Sullenger,
Duke University Medical Center, Durham, NC 27710, USA
Introduction- Aptamers are an emerging class
of therapeutics rivaling antibodies in binding
specificity and affinity at a fraction of their size
and with minimal immunogenic activity.
Furthermore, aptamer-chimeras (ACs) are
developing into potent cell-type specific vectors
for delivering RNA or DNA cargoes. However,
current methods have limited efficacy due to the
cell-types that the ACs can target and their
method of entering the cell, receptor-mediated
endocytosis. We have shown that a DNA
aptamer against the protein nucleolin binds to the
protein at the cell surface and is transported
directly to the nucleus by an as yet unidentified
mechanism. We hypothesize that this method of
cell targeting and internalization will have a
wider range of cancer cell targets than current
ACs and a wider range of cargo molecules.
Materials and Methods- Cells were monitored
for AC internalization by either fluorescent or
radio-labeling. The ability of the cells to process
the delivered cargo was assayed by northern
analysis, and the effects of the cargo were
assayed by western analysis, flow cytometric
analysis of fluorescent proteins, and apoptotic
assays.
Results- The ACs underwent direct nuclear
transport in every cancer cell type tested,
including; renal, mammary, various prostate
cancer, leukemia, lung, and melanoma cancer
cell lines, as well as HeLa cells. Moreover, the
ACs did not internalize to the non-cancer cells
tested, including primary renal epithelial cells,
and primary mammary epithelial cells. Using
these ACs, we are presenting multiple novel
strategies for controlling cell fate including
transcript knockdown, and protein inhibition by
generating ACs that differ in their effector
molecule. We have shown the ability knockdown
GFP levels in cells by delivering engineered
miRNAs targeting GFP. We have also begun
experiments and have had success inducing cells
towards apoptosis by delivering engineered
miRNAs that target Plk1, a tumor suppressor
protein, in cancerous cell lines compared to noncancerous renal epithelial cells. The knockdown
of Plk1 by the AC led to higher levels of
activated caspases 3 and 7, which are signals of
apoptosis, in the cancerous over the non-
cancerous cells. By delivering an aptamer that
inhibits ß-arrestin with the nucleolin-targeting
aptamer, we have been able to inhibit ß-arrestin
from activating downstream pathways by
delivering an aptamer with the nucleolintargeting aptamer.
Discussion and Conclusions- Based on this
evidence, we believe that we have identified
potential avenues of therapy that can specifically
target and affect specific cancer cell types.
However, these techniques have great potential
to impact the field of tissue engineering and
regenerative medicine, by allowing you to block
specific signals throughout tissue development
or more safely generate iPS cells without a viral
vector as a few examples.