Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 128

agents for HD by reducing abnormal mHtt load in striatal neurons.
An alternative strategy is to remove mHtt at the genetic level through
RNA interference, which we will review in the next section.

1.3 RNA Interference
Gene Silencing

RNA interference (RNAi) has received much attention as a power-
ful gene silencing tool in therapy development for neurological
disorders. Naturally, HD is a prime candidate for RNAi-based ther-
apeutics due to its dominant inheritance of a single causative gene.
If executed successfully, the suppression of mutant HTT gene
expression is an appealing treatment strategy because it would
lessen the burden of misfolded protein aggregates by halting dis-
ease progression at the most upstream point of the pathogenic cas-
cade and may be comparatively more effective than trophic
factor-based therapies. RNAi technology exploits an evolutionarily
conserved gene silencing pathway found in most eukaryotic cells
including mammalian cells [ 65 ]. In this pathway, posttranscrip-
tional gene silencing is driven by short non-coding RNA sequences
(~22 nucleotides) called microRNAs (miRNAs). When RNA-
induced silencing complex (RISC) is recruited by mature miRNA,
the mode of silencing is determined by sequence complementation
between the miRNA and its target mRNA. Partial base pairing
usually results in translational repression and does not alter mRNA
levels, while complete base pairing promotes degradation of mRNA
transcripts [ 66 ]. Nowadays, artificial inhibitory RNAs which mimic
the action of endogenously expressed miRNAs can be designed to
suppress specific genes of interest. Therapeutic RNAi primarily
involves three types of small RNA molecules: short interfering
RNA (siRNA), short hairpin RNA (shRNA), and artificial
miRNA. These RNAi agents enter the natural RNAi pathway at
different stages and undergo differential processing. The therapeu-
tic promise of downregulating mHtt expression was first estab-
lished in a conditional mouse model of HD where abolishing mHtt
transgene expression led to symptomatic improvement and reduc-
tion in nuclear inclusions, suggesting that HD pathogenesis might
be reversible [ 67 ]. RNAi-assisted downregulation of mHtt has led
to considerable phenotypic improvements in animal models of HD
when treatments were initiated presymptomatically or after the
development of symptoms, demonstrating the potential for RNAi
to be a preventive or remedial intervention in HD. Although most
HD patients are heterozygous for HTT, nonallele-specific RNAi
may be a viable treatment option if long-term reduction of wild-
type Htt expression can be safely tolerated in humans.

The beneficial effects of RNAi-mediated mHtt suppression in vivo
were first demonstrated with shRNAs produced in target cells after
plasmid- or viral vector-mediated gene transfer. Structurally similar
to pre-miRNAs, shRNAs undergo cytoplasmic processing by Dicer
into double-stranded siRNAs that are incorporated into RISC to

1.3.1 Short Hairpin RNAs