In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 239

Ago-2 is guided by mature miRNA to its mRNA targets. The details
of the miRNA processing pathways are still being actively eluci-
dated. Recent reviews provide more detailed information on the
miRNA processing in vertebrate and in invertebrate animals, on the
sources of miRNAs and siRNA in vivo, and on recently discovered
noncanonical miRNA pathways [ 38 , 42 , 43 ].

In most cases, shRNAs are used to achieve long-term gene knock-
down. Initially, before the details of the miRNA pathways were elu-
cidated, shRNA vectors contained just the hairpin sequence
transcribed under the control of RNA polymerase III (Pol III)
[ 44 - 47 ]. The shRNA hairpin is processed by Dicer to produce siR-
NAs (Fig. 1 ). Importantly, since, at least in human cells, processing
of pre-miRNA and formation of RISC are functionally coupled,
and the cleavage of the target mRNA is tenfold more effective when
RISC is initially loaded with pre-miRNA as compared to siRNA
duplex [ 34 ]. This discovery has clear implications for the design of
shRNA. Consequently, later generations of shRNA incorporate
additional structural elements borrowed from miRNAs to allow for
effi cient entry into the miRNA-processing pathway. One such mod-
ifi cation consists of incorporating the backbone from the human
miRNA-30 into shRNAs, where the hairpin is replaced by an artifi -
cial shRNA construct [ 48 - 51 ]. Such shRNAs are transcribed simi-
larly to the miRNA primary transcripts, then are effectively processed
and mediate the target knockdown. One advantage of these modi-
fi ed shRNAs is that they are usually transcribed under control of
RNA polymerase II (Pol II) as endogenous miRNAs [ 52 ], and not
from a Pol III promoter used in earlier shRNA constructs. Pol III
promoters specialize in driving the transcription of highly expressed
housekeeping small noncoding RNAs, such as tRNAs and 5S rRNA
[ 53 ] (although it might transcribe some miRNAs, for review see
[ 54 ]), which made them suitable to control shRNA expression. The
use of Pol II promoters to transcribe such miRNA-adapted shRNA
opens the possibility of tissue-specifi c and regulated expression, as
many tissue-specifi c Pol II promoters are available.
The evidence of the asymmetry in the strand loading into
RISC suggested strategies to improve shRNA design by modifying
the sense strand to increase the chances of the antisense strand to
enter the RISC [ 39 , 40 ]. Such modifi cation may improve the
knockdown effi cacy as well as selectivity, since the sense strand can
direct the off-target silencing [ 55 ]. Therefore, newer generation of
shRNA vectors have become more “miRNA-like.” The distinction
between the two constructs is somewhat blurred today. The
construct with the perfect hairpin stem is referred to as “shRNA”
regardless of the structure of the fl anking sequences, whereas the
construct with mismatches in the stem, whether native or engi-
neered, is usually called “miRNA.” In some publications, terms like
“miRNA-based shRNA” or “miRNA-adapted shRNA” are used
indicating a perfectly matched shRNA with the miRNA-derived

1.2 Short Hairpin
RNAs and MicroRNAs