Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 131

mHtt transcripts levels occurred 4 days after a single intraventricular
injection of lipid-encapsulated siRNA-HDExon1, but the transient
silencing effect subsided 1 week later. At 8 weeks, the brains
showed profound reduction in the number and size of neuronal
intranuclear inclusions, the extent of striatal atrophy and ventricu-
lar enlargement. Despite the short period of gene suppression,
long-lasting improvements in motor function were reported in the
rotarod and open-field spontaneous activity tests as well as feet-
clasping behavior. Moreover, the treatment reduced weight loss
and increased the survival of rats. Although liposome-related cyto-
toxicity can be minimized with appropriate choices of transfection
reagents and dosage, it is still a hindrance to the safe application of
siRNAs. Another study utilized cholesterol-conjugated siRNA
(cc-siRNA) as an alternative delivery system [ 82 ]. Co-injections
of cc-siRNA against Htt and AAV vector expressing an expanded
Htt fragment were made unilaterally into the mouse striatum, and
suppression of exogenous mHtt protein expression was evident
within 3 days. Neuropil aggregation, inclusion formation, and
neuronal loss were reduced compared to the contralateral striatum.
In addition, balance beam deficits and clasping behavior were ame-
liorated in HD mice 7-14 days after injection, respectively. Chronic
or repeated administration of siRNA-based treatments would be
essential to maintain long-term gene silencing and may be achieved
by the use of AAV vectors. A recent study reported widespread trans-
duction in the striatum of YAC128 mice following AAV-siRNA
delivery which lasted up to 5 months and suppressed wild-type and
mHtt mRNA levels by ~45 % with no overt inflammation or neu-
rotoxicity [ 83 ]. Treated animals showed behavioral improvements
in the rotarod and forced swim tests. The treatment also reduced
striatal mHtt aggregates and partially corrected the altered tran-
scriptional profile of YAC128 mice.

Due to the limitations of shRNAs and siRNAs, recent studies have
begun to use artificial miRNA shuttles as alternative RNAi-
triggering molecules. Like shRNAs, artificial miRNAs are typically
expressed in target cells following gene delivery by plasmid DNA
or viral vectors. They are structurally analogous to primary miRNA
transcripts and usually have endogenous miRNA backbones [ 84 ].
The mature miRNA duplex in the central stem is replaced by
sequences specifically designed for target transcripts, but the native
flanking sequences which direct cleavage by Drosha and Dicer are
preserved. Retention of natural cleavage sites promotes the correct
and efficient processing of precursor RNA molecules to form pre-
dictable miRNA final products, thereby ensuring robust gene
silencing activity [ 85 ]. Furthermore, artificial miRNAs can be pro-
duced from RNA polymerase II promoters which allow the use of
tissue-specific promoters and inducible expression systems, or alter-
natively, high-level expression may be driven by RNA polymerase

1.3.3 microRNAs