Biology Reference
In-Depth Information
4.3.3
Transport Through the Cytoplasm
Different types of RNAi triggers require delivery to different intracellular compartments.
Mechanism of action of pri-miRNA, pre-miRNA, and shRNA demands delivery to
the nucleus. Even siRNA may benefit from controlled localization within the cyto-
plasm, as evidenced by higher activity of siRNA delivered to the perinuclear region
of the cell. The cytoplasmic environment is composed of densely packed soluble
proteins and cytoskeleton protein structures such as actin filaments and microtu-
bules. This dense packing creates a substantial barrier for trafficking of large deliv-
ery vectors because their passive diffusion is severely limited. The trafficking of the
delivery vectors, and endosomes containing the vectors, mainly relies on microtu-
bules and kinesin/dynein motor proteins [ 80, 81 ]. Disrupting the microtubule net-
work using nocodazole to depolymerize tubulin significantly decreases the
cytoplasmic transport of the delivery vectors. In contrast, applying cyclic mechani-
cal stretch to the cells to reorganize and stabilize the microtubules leads to tubulin
acetylation and enhanced endosomal trafficking leading to improved transfection
ef fi ciency [ 82 ] .
4.3.4
Nuclear Entry
Early research from the 1980s to 1990s showed that microinjection of exogenous
genes into the nucleus produced strong gene expression, while injection into the
cytoplasm resulted in poor expression [ 83- 85 ] . These fi ndings clearly demonstrated
that the nuclear envelope is a crucial barrier for delivery of nucleic acids to the
nucleus. Successful delivery of shRNA and pri- and pre-miRNA vectors requires
efficient entry to the nucleus, and the challenges thus mirror those encountered in
gene delivery. This problem is somewhat minimized in rapidly dividing cells because
delivery vectors can gain access to the nucleus during the process of mitosis when
the nuclear envelope breaks down and becomes permeable to the material present in
the cytoplasm [ 86 ]. However, in non-dividing cells, the nuclear envelope is a major
barrier for nonviral delivery vectors. Significant effort has been made in improving
nuclear delivery of DNA in the past, but not until recently have similar approaches
been applied to delivery of RNAi triggers.
First discovered in the SV40 large T-antigen [ 87 ] , nuclear localization signals
(NLS) have become a convenient tool for targeting delivery systems to the nucleus.
The NLS sequence when conjugated to the delivery vector provides the ability to
guide through the nuclear pore complex into the nucleus. Rahbek et al. have designed
delivery vectors based on reducible copolypeptides of endosomolytic peptide and
an NLS peptide to deliver siRNA and pri-miRNA. The inclusion of NLS promoted
nuclear localization of the RNAi molecules, allowing for transcriptional silencing
of EF1A and Drosha- and Dicer-dependent expression of mature miRNA.
The authors observed an NLS-dependent effect on the production of mature miRNA.
An optimized content of NLS was necessary to achieve maximum levels of mature
miRNA when delivering pri-miRNA using the described delivery vectors [ 11 ] .
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