In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 240

backbone. There is no reason why artifi cial or native miRNAs
cannot be used instead of shRNAs for the purpose of gene silencing
in an experimental or therapeutic setting. In fact, such choice may
offer distinct advantages. Recent research demonstrates that mis-
matches in the stem resulting in imperfect siRNA increase the
functionality and may improve selectivity of shRNAs [ 56 ]. Similarly
to dsRNAs, shRNAs have been shown to trigger interferon-
mediated immunological response in cultured cells [ 57 , 58 ].
Furthermore, shRNAs that effectively knockdown mutant hun-
tingtin in the mouse model of Huntington's disease also caused
toxicity in the brain, whereas the same sequences in the miRNA
context were just as effective without toxicity [ 59 ]. Today, miRNA
as well as shRNA vectors, design programs, and predesigned and
validated sequences are available commercially, although shRNA
are still used more frequently.

1.3 Delivery of RNAs
by Viral Vectors

For gene silencing in mammalian cultured cells, the expression of
artifi cial shRNAs or miRNAs encoded by mammalian expression
vector can be achieved by cell transfection. Obviously, this is not an
option if the goal is gene silencing in tissues and organs of living
organisms. To transfer these sequences into target cells in living
animals, viral vectors are required. Lentiviral vectors are by far the
most commonly used to direct the expression of foreign DNA in
nervous cells. They are the ones offered by most companies for the
miRNA expression in cells. The major advantage of these vectors
for neurobiology is their ability to effectively infect neurons but
not most glial cells. Additionally, lentiviruses do not, as a rule,
induce a strong immunological reaction in the host organism,
which is important in functional studies. Furthermore, lentiviruses
incorporate into the host genome, thus driving a long-term, if not
permanent, ectopic expression of the gene of interest, which might
be important in long-term studies.
Among other viral vectors, adenoviral vectors for miRNA clon-
ing are also commercially available (Invitrogen and Cell Biolabs)
although they are less suitable for the knockdown in the brain due
to limited ability of adenoviruses to transduce neurons. Adeno-
associated viruses (AAV) are popular in neurobiology for gene
delivery, because they effectively infect many types of neurons.
AAVs have been used successfully to express miRNA in the brain in
vivo [ 60 , 61 ], although no commercial system is yet available for
that purpose. A word of caution is in order, however. AAVs are
known to drive a very high level of transgene expression. The high
AAV-mediated concentration of shRNAs can cause toxicity or even
death due to saturation of the miRNA-processing pathway and
resulting decrease in endogenous miRNAs [ 59 , 62 , 63 ]. shRNAs
are more prone to such effects than miRNAs [ 59 ], but probably
even miRNAs can be overexpressed to the point of interfering with
the endogenous miRNA equilibrium.