Delivering a disease-modifying treatment for Huntington's disease

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Delivering a Disease-Modifying Treatment for Huntington’s Disease

Bruno M.D.C. Godinho1,2

, Meenakshi Malhotra1,

Caitriona M. O’Driscoll1 and John F. Cryan

1,2,3*

1Pharmacodelivery group, School of Pharmacy, University College Cork, Cork, Ireland

2Dept. Anatomy and Neuroscience, University College Cork, Cork, Ireland

3Laboratory of Neurogastroenterology, Alimentary Pharmabiotic Centre, Cork, Ireland

Corresponding Author

*John F. Cryan , University College Cork, Western Gateway Building, Western Road, Cork, Ireland. Tel:

+353 214205426. Fax: +353427 3518. E-mail: [email protected]

Keywords

Ribozymes, Antisense oligonucletides, RNAinterference, Genome editing, neurodegenerative disease,

Huntington’s disease

This document is the unedited author's version of a Submitted Work that was subsequently accepted for

publication in Drug Discovery Today copyright © Elsevier after peer review. To access the final edited,

proof-corrected and published work, see doi: 10.1016/j.drudis.2014.09.011

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Abstract

Huntington’s disease (HD) is an incurable genetic neurodegenerative disorder that leads to motor and

cognitive decline. It is caused by an expanded polyglutamine tract within the Huntingtin (HTT) gene,

which translates into a toxic mutant HTT protein. Although no cure has yet been discovered, novel

therapeutic strategies, such as, RNA interference, antisense oligonucleotides, ribozymes, DNA enzymes

and genome editing approaches, aimed at silencing or repairing the mutant HTT gene hold great promise.

Indeed, several preclinical studies have demonstrated the utility of such strategies to improve HD

neuropathology and symptoms. This review critically summaries the main advances and limitations of

each gene silencing technology, as effective therapeutic tool for the treatment of HD.

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Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG triplet

mutation within the Huntingtin (HTT) gene and affects ~5-10 in 100,000 people in European,

Australasian and American populations [1,2]. In general, symptoms strike at middle-age and include

chorea (involuntary choreiform rapid movements), progressive motor and cognitive impairment,

depressive-like behaviour and mood alterations, usually leading to death 15-18 years after onset of

clinical manifestations [3,4]. Unfortunately, current pharmacotherapy is only able to provide temporary

symptomatic relief and fails to treat the underlying cause or progression of the disease [5]. Therefore, the

development of new therapeutic strategies to stop disease progression is crucial to improve the standard

of care for HD patients.

More than two decades have now passed since the identification of the causative mutation by The

Collaborative Huntington’s Research Group, and it is now well known that HD is caused by the

expression of a mutant HTT (muHTT) protein with an abnormally long polyglutamine (polyQ) tract (>40

Q) close to its N-terminus [6]. In addition, an increasing body of knowledge demonstrates that the disease

is caused by a toxic “gain of function” mechanism rather than merely by a loss of function of the wild-

type HTT (wtHTT) protein. Indeed, muHTT has been shown to interact with many intracellular targets

disrupting their normal function and consequently leading to neuronal dysfunction and loss in the

striatum, but also in other structures of the brain, such as the cortex [7]. Based on these understandings of

HD neuropathology, a number of therapeutic approaches have been advanced (see Figure 1). In addition,

a variety of animal models have been developed to evaluate the pathophysiological mechanisms of the

disease and the success of emerging therapeutic modalities (see Box 1, Table 1). These novel therapeutic

modalities include, neuroprotective strategies targeting the underlying pathologic mechanisms of muHTT,

and cell replacement therapies focused on counteracting neuronal loss in the brain [7]. Additionally,

preclinical evidence has also shown that environmental enrichment improves HD neuropathology in

transgenic rodent models of HD [8,9], which in turn suggests that this strategy may help in improving

patient’s quality of life. However, and despite being potential alternatives to current pharmacotherapy,

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these strategies are aimed at downstream effects of muHTT and do not specifically target the root cause

of the disease [7]. On the other hand, oligonucleotide therapeutic approaches that directly interfere with

muHTT by abrogating or reducing its expression have also been considered and presented encouraging

results [10]. Among such strategies there are genome editing techniques and post-transcriptional gene

silencing approaches using ribozymes and DNA enzymes, antisense oligonucleotides (ASOs) and RNA

interference (RNAi), all of which permit a specific reduction of the synthesis of muHTT. In fact, these

approaches target upstream processes of disease and may enable therapeutic intervention even before

cellular damage arises [10]. Due to their great potential as therapeutic strategies, lately they have received

significant attention from the scientific community and the field has rapidly progressed. Therefore, the

present review aims to capture such significant development, but also to identify limitations and hurdles

that need to be overcome in order for these concepts to reach the clinical setting.

Post-transcriptional gene silencing – therapeutic potentials for Huntington’s Disease

Post-transcriptional gene silencing approaches for HD have undergone considerable research and include

nucleic acids with catalytic capabilities (ribozymes and DNA enzymes), ASOs and RNAi [10,11]. These

nucleic acids have shown to modulate the translational efficiency through a process that involves

cleavage, degradation, or translational suppression of the targeted mRNA. Although they share the

common concept of reducing muHTT mRNA and consequently muHTT protein load in order to block or

reverse HD neuropathology and symptoms, their mechanisms of action differ significantly (Figure 2).

Catalytic nucleic acid approach

Catalytic nucleic acids, include ribozymes and DNA enzymes (DNAzymes), and are aimed at elimination

or repair of target mRNA transcripts.

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Ribozymes: Mechanism of action and advances towards a potential therapeutic approach for HD

Ribozymes are naturally occurring RNA molecules with self-cleaving capabilities consisting of an

effector catalytic core and two flanking sequences that allow for specific binding to the mRNA [11,12]

(Figure 2a). The catalytic core is able to cleavage substrates that contain a XUN motif, where X is any

nucleotide base and N is an unpaired A, C or U [12,13]. Although the discovery of such ribozymes

heralded much hope for novel gene silencing based therapeutics, there exploitation has been relatively

limited to date [14]. That said there are a number of in vitro and preclinical animal studies using this

approach in HD. Specifically, hammerhead ribozymes, a class of ribozymes of 30-40 nt in length, are

believed to cleave mRNAs at a preferred site with rapid degradation of mRNA fragments [11,12]. They

have been successfully used in in vitro models of HD [15]. In this study, specific ribozyme (HD6 or

HD7)-expressing adeno-associated virus-based (AAV) delivery systems enabled ~60% reduction in

muHTT mRNA expression when co-transfected with a specific plasmid (pCMV-R6/1), expressing human

exon1 with expanded CAG repeats into an artificial cell system (HEK293 cells) [15]. Interestingly,

similar results were obtained when using short hairpin RNA targeting the same regions in the muHTT

mRNA [16]. Furthermore, direct injection of HD7 ribozymes into the striatum of a mouse model of HD

(R6/1 HD mice) resulted in ~30% reduction of muHTT mRNA in the brain [15]. However, additional

preclinical studies are required to evaluate the potential improvements in HD behavioural deficits after

gene expression knockdown using hammerhead ribozymes. The potential application of hammerhead

ribozymes as a therapeutic approach for neurodegenerative diseases have also been successfully

demonstrated in a rat model of Parkinson’s disease [17]. AAV delivery of a ribozyme against α-synuclein,

(an aggregated protein that leads neuropathy in Parkinson’s disease) into the substantia nigra, reduced α-

synuclein protein levels and improved cell survival of tyrosine hydroxylase-positive neurons [17].

Moreover, Hepatitis Delta Virus (HDV) ribozymes, a new generation of ribozymes, have been

successfully used in SH-SY5Y neuroblastoma cell cultures to reduce the expression of amyloid protein

precursor (APP) up to 70%, and the total secretion level of amyloid-β peptides by 30% [18]. Therefore

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these HDV ribozymes are been considered as potential therapeutics for Alzheimer’s disease, but require

further investigation in animal models. Additionally, other studies have used the trans-splicing abilities of

group I intron ribozymes to repair the expanded CUG repeat in the 3’ UTR of the Dystrophia myotonica

protein kinase (DMPK) mRNA transcripts, defect that causes Myotonic Dystrophy Type 1 – an autosomal

dominant neuromuscular disease [19]. In this proof-of-concept study, group I intron ribozymes were able

to effectively replace the 3’ end of the endogenous DMPK transcript in human fibroblasts with a new 3’

region with a smaller repeat length [19]. However, the application of such strategy to repair mutant

DMPK transcripts in vivo, or even to other mutated trinucleotide-containing disease transcripts, still

warrants further investigation.

DNAzymes: Mechanism of action and advances towards a potential therapeutic approach for HD

DNAzymes are another class of single stranded catalytic nucleic acids which are also able to bind

complementary mRNAs transcripts through substrate-binding arms, similar to ribozymes [20]. These

catalytic DNA molecules often have cation-dependent catalytic core and are not naturally occurring,

being derived and selected in vitro. In the context of HD, DNAzymes have specifically knockdown the

expression of a co-transfected muHTT construct by 85% in HEK293 cells [21]. In spite of the fact that

DNAzymes have been widely investigated in vivo as a potential therapeutic approach for diseases such as,

HIV, hepatitis and cancer, its application in vivo to neurodegenerative disorders, such as HD, requires

further research [20].

Antisense oligonucleotide approach

Mechanism of action

For more than 20 years ASOs have been proposed to be an ideal strategy to silence aberrant gene

expression in disease states [22]. ASO technology involves the use of single stranded DNA molecules,

typically ~20-25 bp long, which have complementary sequence to the target mRNA [23,24]. ASOs

hybridize with the pre-mRNA in the nucleus and cause inhibition of 5’ cap formation, inhibition of

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splicing and/or activation of RNase H degradation [23]. However, when hybridisation takes place in the

cytoplasm translation is inhibited by steric hindrance or by RNase H degradation of the mRNA transcript.

ASOs are limited to the inhibition of one mRNA copy [25] (Figure 2b).

Advances towards a potential therapeutic approach for HD

In early in vivo studies, although ASOs successfully penetrated neurons with no remarkable toxicity, no

significant reduction in muHTT was observed [26]. The lack of efficacy in these studies were speculated

to be due to high susceptibility to nuclease degradation [25]. In contrast, modified ASOs have recently

been shown to successfully reduce the expression of HTT in human fibroblasts [27,28]. Furthermore, it

was recently demonstrated in several rodent models of HD that modified ASOs are able to successfully

reduce muHTT expression, improve HD-like neuropathology and ameliorate symptoms of disease [29,30]

(Table 2). A recent study demonstrated a dose dependent reduction in muHTT mRNA level (~38%) with

chemically (2’-O-methoxyethyl) modified ASOs in the BACHD mouse model of HD [30]. They further

reported that older mice from two different rodent models of HD (YAC128 and BACHD) showed

sustained reduction of the muHTT mRNA by 42% and protein by 43% [30]. These animals improved

motor activity/coordination and displayed alleviated anxiety, and delayed formation of polyQ aggregates,

for an extended three months of post-treatment termination [30]. In a follow-up study in non-human

primates researchers performed intrathecal infusion of ASOs for 21 days and reported a sustained

reduction (4 weeks) of muHTT mRNA levels in the frontal cortex (53%), the occipital cortex (68%) and

the spinal cord (46%) [30]. In another approach, Haydon and colleagues demonstrated allele-specific

targeting of the human muHTT gene with ASOs by selective identification of single nucleotide

polymorphism (SNP) targets in the human HD gene [29]. Results indicated allele-specific knockdown of

human muHTT by ~52% in transgenic BACHD mice containing the elongated CAG tract, while no effect

was observed in the YAC18 transgenic mice containing non-pathological CAG tract [29]. Other studies

have followed and also focused on the allele-selective downregulation of muHTT gene expression,

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showing significant reduction in muHTT mRNA in HD-patient derived fibroblasts and a humanized

mouse model of HD [31].

In addition, confirming the clinical utility of the ASO approach in neurology ASOs have very recently

undergone clinical trials as the potential therapy for amyolateral sclerosis, further advocating its promise

for other neurodegenerative diseases [32]. Despite their therapeutic potential, ribozymes, DNAzymes and

ASOs, have been largely superseded by the enhanced gene silencing efficiency and longer effects of

RNAi approaches [25,33].

RNA interference approach

Mechanism of action

In the past two decades, RNAi technology has emerged with great promise in areas of gene therapy

development. RNAi is an endogenous cellular pathway that allows post-transcriptional regulation of gene

expression, and was first identified in petunia plants [34], later on in nematode C.Elegans [35] and finally

in mammalian cells [36] (Figure 1 and 2c). This intracellular pathway enables cells to auto-regulate gene

expression through micro RNAs (miRNAs) and has been shown to have a crucial role during

development [37,38]. Briefly, the pathway initiates in the nucleus with a long double-stranded RNA

(dsRNA) which is then processed by an endoribonuclease enzyme (Drosha) and transported to the

cytoplasm by the nuclear exportin-5 [38]. Another cytoplasmic endoribonuclease enzyme (Dicer) cleaves

these miRNAs, which may contain several stem-loop structures, into a small interfering RNAs (siRNAs)

with ~21-25 oligonucleotides (nt) [39]. siRNA are loaded to an RNA-induced silencing complex (RISC),

which is activated upon unwinding of the siRNA and thermodynamic selection of the guide/antisense

strand. Activated RISC searches the transcriptome for specific complementary mRNAs, targeting them

for degradation, therein, inhibiting translation to the protein product [40,41] (Figure 2c). Activated RISCs

are able to catalyze multiple turnover reactions enhancing the potency of this gene silencing approach

when compared to ASOs and catalytic nucleic acids. Additionally, not only due to sequence mismatch

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and partial complementarity, but also through the ability of binding to several UTR sequences, miRNAs

have been shown to be able to silence several mRNA targets [38].

This RNAi machinery can be artificially hijacked to induce specific gene expression knockdown, which is

commonly performed using synthetic siRNAs – in combination with non-viral delivery systems, but also

through short hairpin RNAs (shRNA) – usually utilizing viral delivery systems [42-44]. After delivery,

shRNAs are expressed within the nucleus and need to be transported by exportin-5 to the cytoplasm,

whereas synthetic siRNAs bypass this nuclear step. Indeed, RNAi has been widely exploited as a research

tool for target validation, providing greater understanding of gene and protein functions [39,45], but also

as a mean of generating in vivo models of disease [46-48]. Finally, harnessing the RNAi pathway to

induce specific gene silencing effects has also shown great potential as a therapeutic strategy for incurable

diseases of the central nervous system (CNS), ranging from brain cancers to neurodegenerative diseases,

such as HD [49-51].

Advances towards a potential therapeutic approach for HD

Initial studies using RNAi as a therapeutic strategy to treat polyQ-induced neurodegenerative disorders

successfully improved cell survival in in vitro models of spinobulbar muscular atrophy [52], and later

enabled the reduction of both HTT mRNA and protein in in vitro models of HD [53]. Almost

simultaneously, Davidson and colleagues conducted the first RNAi-based in vivo preclinical trial for HD,

whereby single bilateral injections of an AAV delivery system, coding anti-HTT shRNA, into the

striatum of HD transgenic mice (N171-82Q) [54]. Significant reductions in muHTT mRNA levels (~55%)

and in the number of HTT inclusions were observed. Moreover, behavioural improvements in stride

length and in rotarod deficits were also reported [54]. However, in this study there were no improvements

in weight profiles, and this was attributed to the systemic nature of the disease or to muHTT-mediated

hypothalamic dysfunction [54].

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Since Davidson’s pioneering study over 15 other preclinical studies have been conducted in various in

vivo models of HD, from rodents to nonhuman primates, using different RNAi delivery systems (Table

3). Indeed, another study also reported a reduction in HTT mRNA levels (~75%), decreased number of

HTT inclusions (25-38%) and improvement of hindlimb clasping behaviour in the R6/1 transgenic mouse

model, upon delivery of specific shRNAs (shHUNT1 and shHUNT2) using AAV delivery systems [16].

In addition, muHTT suppression in the striatum increased expression of dopamine- and cAMP-regulated

neuronal phosphoprotein (DARPP-32) and preproenkephalin (ppENK) when compared to untreated R6/1.

Nevertheless, in this study RNAi treatment failed to improve weight gain and performance in the rotarod

task of R6/1 mice [16]. Also in 2005, the first pre-clinical study using non-viral technologies to deliver

siRNAs emerged [55]. In this study, Wang and co-workers used a lipid-formulated siRNAs (siRNA-

HDExon1), targeting a sequence upstream of the CAG repeats of the human muHTT transcript [55].

Nanoparticles (entities typically 1-100 nm but not >500 nm in diameter which can be used as a delivery

vehicle to transport therapeutic molecules into the cells, see Box 2) were successfully delivered into the

intracerebral ventriculum (i.c.v.) of postnatal day 2, R6/2 mice, yielding a significant reduction in muHTT

mRNA levels (~70%), coupled with sustained effects up to 7 days [55]. Furthermore, this suppression of

muHTT resulted in a reduced number of nuclear aggregates and reduced general brain atrophy, which is

characteristic in the R6/2 transgenic mouse model [55]. Additionally, RNAi treatment had unexpected

long lasting effects on R6/2 behavioural deficits, delaying the onset of the clasping behaviour, improving

spontaneous locomotor activity in the open field and improving rotarod motor deficits. Furthermore, less

severe weight loss and increased survival when compared to untreated R6/2 mice were also reported [55].

We have recently shown using a different nanoparticle-based siRNA delivery approach the ability of

locally delivered, amphiphilic cyclodextrins-siRNA nanoparticles to successfully knockdown the muHTT

gene (85%) in the striatum of R6/2 mouse model of HD and showed improved phenotypic effects on

repeated brain injections [56]. In another approach, the use of cholesterol-conjugated siRNAs (cc-

siRNA-HTT) for RNAi in HD was demonstrated in AAV-based mouse model of HD (AAV-HTT100Q)

[57]. Co-administration of AAV-HTT100Q and cc-siRNA-HTT into adult mouse striatum resulted in

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approximately 66% knockdown of the HTT transcript and a reduction of HTT aggregates in the striatum

[57]. Results also showed increased neuronal survival and significant behavioural improvements in beam

walking and clasping behaviour [57].

Interestingly, in all of the above mentioned in vivo studies RNAi treatment was initiated when animals

were still pre-symptomatic, and therefore, limited conclusions about reversal of neuropathology and HD

symptoms could be drawn. However, following studies were carried out in symptomatic rodents and have

shown that RNAi treatment is also able to reverse, at least partially, the number of HTT inclusions and

improve striatal dysfunction (Table 3) [58,59]. Despite these encouraging results, further investigations

are now warranted to assess if such improvements in HD neuropathology also result in reversal of HD

behavioural deficits. Similarly, additional studies are needed in order to elucidate which are the most

efficient RNAi delivery systems, but also to identify any safety issues regarding long-term use of RNAi

technologies for HD.

Limitations of gene silencing technologies for Huntington’s Disease therapeutics

The translation of gene silencing technologies for the treatment of CNS disorders, such as HD, to the

clinic setting faces three major setbacks: the lack of effective and non-toxic strategies for the delivery of

such technologies to the brain; the so-called “off-target effects”; and the saturation of endogenous

pathways.

Delivery issues

One of the primary obstacles to the progress of CNS gene silencing technologies to the clinic is the lack

of effective, non-toxic and safe delivery systems able to adequately overcome the different CNS barriers

[60]. In fact, nucleic acids (shRNA and ribozyme expressing vectors and ASOs – DNA molecules – and

synthetic siRNAs – RNA molecules) are highly hydrophilic macromolecules with poor cell penetrating

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properties, normally requiring adequate systems for neuronal delivery and effective protection from

nuclease degradation [60]. AAV and lentiviral vectors have great tropism over a wide range cell types and

are by far the most widely used delivery systems for gene and shRNA delivery to the CNS [61]. However,

recent fatal reports have raised awareness to possible adverse reactions to viral vectors due to toxicity and

activation of immune responses [62]. Alternatively, several lipid- or polymer-based non-viral vectors

have been engineered and are now commercially available as transfection reagents for nucleic acids (e.g.

Lipofectamine2000, Lipofecatmine RNAi Max, Exgen500, Superfect, INTERFERin). Indeed, these

nanosystems have been widely used to transfect ASOs [27,63,64] and siRNAs [55,56,65] in both in vitro

and in vivo models of HD, with various degrees of efficiency. Despite been considered relatively inert

materials with negligible or no toxic effects, and having considerable advantages over viral counterparts

regarding their toxicology [60], non-viral vectors are now known to cause several biological, genomic and

inflammatory disturbances [66,67]. Indeed, some of these cationic transfection reagents are somewhat

limited in their application, as they show non-specific targeting, cellular toxicity and activation of the

immune response, in vivo [65]. Several studies have reported differential toxicological and inflammatory

responses to lipid- and polymer-based non-viral vectors, further suggesting that certain biomaterials be

more likely to enhance adverse effects in CNS than others (see Box 2) [65,68]. In addition, it is worth

noting that muHTT renders striatal neurons more sensitive to toxic stimulus [69], and, therefore, the

selection of appropriate delivery systems to enable therapeutic gene silencing in HD is crucial.

Off-target effects

Off-target effects are another common obstacle for the therapeutic application of gene silencing strategies

and these arise from interference with non-target mRNA transcripts or activation of components of the

immune system [70]. In the specific context of HD, anti-HTT ribozymes, have been reported to alter

mouse transcriptional activity which affected other mRNAs including DARPP32 (downregulation) and

NGFI-A (upregulation) [15]. Despite the fact that such off-target effects were suggested to be sequence-

dependent and were also seen with shRNAs (siHUNT2) [16], no other human mRNA sequence presented

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significant similarity to the targeted regions. Therefore, although these unwanted effects may be restricted

to the context of the mouse transcriptome, further studies are now required to evaluate possible “off-target

effects” in humans.

Synthetic siRNAs and shRNAs can cause the miRNA-like off-target effect whereby they down-regulate

untargeted mRNAs by partial complementarity [71]. Furthermore, shRNA and siRNA have been shown

to activate RNA helicases (PKR and RIG-1) leading to a protein synthesis arrest through phosphorylation

of eIF2α and upregulation of a subset of genes from the IFN pathway [72]. In addition, siRNA have also

been shown to activate endosomal pattern-recognition toll-like receptors (TLR) 3, 7 and 8, thereby,

increasing the expression of pro-inflammatory cytokines [73-75]. Both of these unwanted effects,

miRNA-like off target and immunogenic effects, have been found to occur in a dose-dependent manner

and, therefore, the use of a minimal dose is critical [76]. Moreover, the use of rational algorithmic design

tools is essential to guarantee correct loading of the antisense strand to the RISC (G/C content influences

thermodynamic selection of strands), but also to generate highly complementary siRNA/shRNA to their

target mRNAs ensuring low potential for cross-hybridization with untargeted mRNA transcripts [77,78].

Chemical modifications can also be introduced in siRNA sequence to enhance its stability against serum

nucleases and reduce immunological activation [70,79,80].

Saturation of the endogenous RNAi pathway

Overload of the RNAi endogenous pathway can also lead to toxicity and be detrimental. Indeed, dose-

dependent saturation of the endogenous RNAi pathway leading to liver toxicity and increased morbidity

has been reported in mice upon intravenous injection of a shRNA-AAV expressing vector [81,82]. In

these studies, shRNAs prevented endogenous miRNA maturation by overloading nuclear exportin-5,

leading to a global shutdown of the miRNA pathway [83,84]. Selection of adequate promoters for modest

expression of shRNAs or co-expression of recombinant exportin-5 are the main approaches being

evaluated to reduce shRNA-mediated toxicity [81,85]. In addition, others have suggested using an

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artificial miRNA-based expression system, which is well tolerated in vitro and in vivo [86,87]. In contrast,

synthetic siRNAs bypass nuclear processing and do not overload nuclear transport, thereby circumventing

this issue.

Allele-specificity: The holy grail for silencing therapies?

In dominantly inherited neurodegenerative diseases, such as HD, most of the affected individuals are

heterozygous, carrying one copy of the normal allele and another mutated form of the allele [88].

Although muHTT proteins are causative of disease, wtHTT proteins have essential functions in

embryonic development and several other cellular processes, such as, vesicle trafficking, protection

against apoptosis and transcription regulation [7]. In fact, a study conducted in a zebrafish model of HD

revealed a 5-fold increase in caspase 3 activity and a decrease in BDNF levels leading to neuronal cell

death, following ASO-mediated knockdown of wtHTT [89]. These results highlight the detrimental

effects of non-specific silencing of the wild-type alleles, especially if chronic administration is needed,

and emphasize that allele-specific targeting of mutant genes might be an alternative to circumvent this

issue [89,90]. Indeed, this has been successfully achieved by exploiting the nucleotide differences

between mutated and wild-type genes and the specificity of gene silencing mechanisms. As an example,

rational design of siRNA (or short hairpin RNAs (shRNAs) targeting the site of the mutations has enabled

allele-specific silencing of the mutant forms of superoxide dismutase (SOD)-1 in vitro and in vivo [91],

and of tau and amyloid precursor protein (APP) in vitro [92]. However, targeting the mutant CAG

expansion using siRNAs in polyQ disorders, such as HD and spinocerebellar ataxia (SCA), has led to an

unintended suppression of the wild-type allele and also of other genes normally containing CAG repeats

[28,92]. On the other hand, ASOs, peptide nucleic acids (PNA) and locked nucleic acids (LNA) seem to

be a better allele-selective alternative when targeting these elongated CAGs, since these approaches are

able to discriminate CAG repeat lengths based on energetically different structures, rather than solely in

base differences [27,28,64]. Chemically modified ASOs have enhanced RNAse H nucleotide

discrimination above 100-fold, sparing the downregulation of non-targeted HTT mRNA transcript. In the

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specific case of siRNAs, allele-specificity might only be achievable by targeting disease-linked

polymorphisms.

Approximately 60 single nucleotide polymorphisms (SNPs) have been identified in the coding and in the

3’ untranslated regions (UTR) of the human HTT gene [93]. From those, a selection of 25 SNPs and a

GAG deletion (in exon 58) were recently found to have enough heterozygosity among a cohort of HD

Caucasian Europeans, with 86% of patients being heterozygous for at least one of these polymorphisms

[94]. Initial studies showed specific silencing of the muHTT allele in artificial HeLa cell systems

containing plasmids with the nucleotide sequence of the SNP incorporated [95]. Further studies revealed

that allele-specific silencing is also possible by targeting HTT polymorphisms in human HD fibroblasts,

naturally harbouring the muHTT [94,96]. Additionally, rationally designed siRNAs against a subset of 3-

7 SNPs have achieved allele-specific knockdown of the muHTT in vitro and may be suitable to treat at

least three quarters of the US and European HD populations [96,97]. It is also important to note that

allele-specific silencing has also been achieved using ASO technology in several in vitro and in vivo

studies, further supporting the feasibility of this approach [29,31]. However, this approach would require

genotyping of all SNP sites of interest, selection of the SNP to be targeted and the design of the allele-

specific siRNAs accordingly, all of which may lead to an increased cost for implementation of such

strategy.

Alternatively, non-allele specific targeting has been recently suggested as a valid approach that would

circumvent the economic cost of individualized therapy. Complete (or almost complete) ablation of the

mouse homologue Hdh gene has led to complications in embryogenesis and progressive degeneration in

the adult brain [98,99]. However, recent in vivo preclinical studies have shown that partial reduction of

the wtHTT protein might be tolerable [59,100,101]. In these studies, partial silencing of the endogenous

HTT homologue did not exacerbate HD pathology or cause detrimental effects in neuronal survival, and

has been found to be well tolerated for several months [59,100]. Furthermore, studies carried out in non-

human primates have also shown that the reduction of endogenous HTT homologue by ~45% does not

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induce neuronal degeneration, astrogliosis or even motor deficits [101-103]. Thus, the residual levels of

wtHTT protein may be sufficient to maintain cellular needs. However, silencing the endogenous wtHTT

has led to transcriptomic changes in other genes related to the functions of HTT and, therefore, a clear

assessment of the impact of these is needed before progressing to the clinic [59,100]. Finally, despite the

favourable outcomes, most of these in vivo studies were conducted only up to 6-9 months and, therefore,

the effect of long term gene expression knockdown of wtHTT still needs further investigations.

A novel combinatory therapy has been reported as a useful strategy to target diseases where mutations

have a high level of heterogeneity, such as dominant retinitis pigmentosa [104]. The method consists of

utilising RNAi for non-allele specific gene suppression of the mutated gene and gene therapy for

supplementing an RNAi-resistant wild-type gene. Thus, this approach can be an alternative, if in the long

run, weighing up issues related to both the cost and efficiency of the treatment, RNAi approaches

targeting both wtHTT and muHTT alleles are to be used, one may consider gene replacement as a strategy

to maintain the adequate levels of expression of the wtHTT protein. Such an approach has been

successfully used in vivo for α-1 antitrypsin (AAT) deficiency, preventing liver pathology and increasing

blood levels of AAT, and for dominant retinitis pigmentosa, improving retinal structures and function

[104,105]. The applicability to neurodegenerative diseases has also been explored with initial studies in

SOD1 and SCA showing effective knockdown and replacement; however no functional effects were

reported [106,107]. The suppression and replacement approach could also be possibly used for HD,

however, this has not yet been investigated.

Genome editing approaches for Huntington’s Disease Therapeutics

Specific gene targeting/silencing can also be achieved at the transcriptional level through engineered

nucleases, such as meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector

nucleases (TALEN) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas systems

[108]. These chimeric nucleases are able to bind to specific DNA sequences, repressing gene transcription

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and/or inducing DNA double strand breaks and enabling correction of mutated genes [108]. Some of

these approaches have been successfully applied in X-linked severe combined immune deficiency (X-

SCID) [109], hemophilia B [110], sickle-cell disease [111], Parkinson’s Disease [112], Retinitis

Pigmentosa [113] and HIV [114]. In fact, ZFNs that interfere with the C-C chemokine receptor type 5,

which in turn confers HIV resistance, are now undergoing clinical testing (ClinicalTrial.gov Identifiers:

NCT01252641, NCT00842634 and NCT01044654), and promising safety results have just recently been

published [115]. Furthermore, ZFN-based strategies have also reached the clinical phase for the treatment

of glioblastoma targeting the glucocorticoid receptor gene as part of a T cell-based cancer immunotherapy

(NTC01082926). In the specific case of HD, it has recently been demonstrated that zinc finger proteins

(ZFP) are able to effectively silence the muHTT, without affecting the expression of wtHTT, in vitro and

in the R6/2 mouse brain (~40% reduction in muHTT) [116]. In this study, ZFP repressors were delivered

intraparenchymally using an AAV delivery system, resulting in significant improvements, in HD-related

neuropathology and motor deficits [116]. Although these technologies are still at their infancy, they hold

great promise not only for HD but other monogenic disorders.

Conclusions and Future Perspectives

Although the function of HTT is not fully understood yet, it is now known that HD is caused by the

expression of a muHTT protein. Thus, lowering muHTT levels is likely to be beneficial for the treatment

of HD, and this strategy is now being considered for delaying or even blocking disease progression. In

this regard, genome editing and post-transcriptional gene silencing approaches, such as RNAi, ASOs,

DNAzymes and ribozymes appear to promise a new intervention, reducing or even eliminating the

production of pathogenic mutant protein.

Genome editing approaches are gradually shaping into a powerful therapeutic strategy for monogenic

disorders, such as HD, due to the possibility of replacing/repairing native mutated genes. In fact, the use

of such strategy in HD would enable modification and correction of the native mutant loci, with the

18

advantage that this change will persist for the lifetime of the cell and its progeny, therefore, reducing the

need for continuous exposure to therapeutics. However, several issues still need to be addressed, mainly

regarding the specificity and safety of engineered nucleases, before progression of genome editing to the

clinic as a viable approach for HD. The development of highly specific and effective nucleases is highly

costly and dependent on the fine combination of zinc fingers in ZFN and of the right amino acid repeat in

TALENs. On the other hand, the development of more efficient methods to detect and control for off-

target cleavages and insertions is crucial, since these undesired effects may lead to a reduced efficiency

but more importantly to cytotoxicity. Therefore, despite its great promise as a potential therapeutic

approach for HD, more work is needed in different rodent models and even non-human primate models of

HD to support the growing evidence of its applicability in human disease.

Post-transcriptional gene silencing approaches have also shown very promising results in many in vitro

and in vivo models of HD, enabling knockdown of the muHTT mRNA transcript and reducing muHTT

protein levels, along with the improvement in HD neuropathology at molecular and phenotypic levels. In

this regard, the two strategies that have been most extensively tested are the RNAi approach and the ASO

approach. Although the RNAi approach allows for strong gene silencing effect and follows a targeted

approach, most in vivo studies carried out so far targeted sequences with shared homology to both human

wtHTT and muHTT mRNA transcripts. Furthermore, and despite that partial suppression of wtHTT has

been well tolerated in preclinical studies, the effects of chronic suppression of wtHTT has not been

assessed in humans. Thus, application of such approaches to human therapy may need further

investigation and refinement, such as implementing an allele-specific strategy by targeting disease-linked

SNPs. However, one should bear in mind that SNP-based strategies may present additional challenges at

the clinical stage, since multiple clinical trials may be required to establish the efficacy of each of the

different SNP-targeted molecules. Alternatively, to date ASOs may be considered to have an edge over

siRNA-mediated gene silencing in terms of enabling allele-specific targeting of the muHTT through the

pathological CAG tract, thus sparing the wtHTT protein for its essential cellular processes.

19

Delivery of functional ribozymes and DNAzymes, ASOs and siRNAs/shRNAs, is one of the major

hurdles when developing such post-transcriptional silencing approaches for human therapy. Indeed, these

approaches have their own advantages and limitations, including synthesis, stability and delivery. Various

chemical modification techniques have been employed to improve performance of siRNAs and ASOs in

vivo, especially in terms of their stability against serum proteins and nucleases. In addition, numerous

studies are being conducted to identify safe and efficient CNS delivery vectors which enable specific

delivery of oligonucleotides. A wide variety of delivery vectors are under investigation for site specific

delivery of the oligonucleotides for the treatment of neurodegenerative diseases. In fact, the design and

synthesis of a delivery carrier is another class of study to achieve translational success. This includes the

ability to safely complex/encapsulate the therapeutic molecule and deliver it to the targeted site in

relevantly sufficient quantities and be safe to CNS tissues by being non-immunogenic/non-

toxic/biodegradable. Furthermore, to avoid invasive brain surgery in HD patients, a targeted delivery

system that can cross the blood-brain barrier (BBB) via transcytosis and specifically deliver the cargo to

affected structures would be ideally required. To this end, several cell penetrating and cell targeting

ligands/peptides have been investigated to enhance transport across the BBB or to increase delivery to

neurons [117,118]. Among cell targeting peptides, the 29 amino acid fragment derived from the rabies

virus glycoprotein (RVG) has lately received much attention [119]. This targeting ligand interacts with

acetylcholine (Ach) receptors expressed in the BBB and neuronal cells, leading to translocation across the

BBB by receptor-mediated endocytosis. Another, recently discovered peptide is TGN, which identified by

phage display library and was reported to specifically target BBB [120]. Transferrin [121] lactoferrin

[122] and angiopep [123] have also been widely investigated for CNS delivery, however, the receptors for

this proteins/peptides are widely expressed in other tissues and may not confer specificity for the CNS.

This demonstrates that further improvements of current nano-formulations for specific targeting to

neurons will be required, and will eventually enable delivery to specific sub-populations of neurons.

Thus, gene silencing and gene delivery are two approaches that go hand-in-hand for the actualization of

gene therapy. Successful outcomes in this regard may lead to clinical testing of oligonucleotide-based

20

therapies, not only for HD but for other neurodegenerative disorders, potentially delaying the progression

of the disease and improving quality of life.

Acknowledgements

Authors wish to acknowledge research funding provided by Science Foundation Ireland (Grant no.

07/SRC/B1154) and the Irish Drug Delivery Network.

21

Box 1: Animal Models of Huntington’s Disease

In order to advance drug discovery appropriate animal models of disease are required. These enable

extensive testing of novel therapeutics prior to progression of such strategies to clinical trials in human

subjects. A variety of animal models (rodents and non-human primates) have been developed that

specifically exhibit HD pathology and have been extensively used in research to study gene therapy. The

animal models are usually categorized in non-genetic and genetic models [124]. The chemically induced,

non-genetic HD models involve cell death by using excitotoxic agents (Quinolinic acid and Kainic acid)

[125,126] or mitochondrial disrupting agents (3-nitropropionic acid and malonic acid) [127]. These

models, however, did not mimic the actual pathologic conditions of HD, for example, they lacked the

production of mutant HTT. Moreover, HD is a hereditary disease and leads to progressive cell-death over-

time, whereas, in chemically induced models, the cell death is immediate. Thus, their use was limited to

studies that involved neurorestorative and neuroprotective therapies. The genetic animal models

developed for HD, closely mimic the HD pathology and its progression over-time [128]. These include

either a traditional transgenic animal model expressing a truncated or a full-length form of the human

muHTT gene randomly inserted in the animal’s genome or a knock-in model which expresses the

pathological trinucleotide (CAG) repeat inserted specifically in the within the endogenous HTT gene of

the animal [128]. Some of the examples of transgenic HD rodent models are R6/1 (114 CAG), R6/2 (150

CAG) [129], N171-82Q (82 CAG) [130], yeast artificial chromosome (YAC) (72 or 128 CAG) [131,132]

and bacterial artificial chromosome (BAC) (97 CAG/CAA) [133]. The knock-in models are considered

the most faithful reproduction of HD from the genetic standpoint given that the expression of muHTT is

regulated by the endogenous promoter, and therefore protein synthesis is spatially and temporally

accurate [124]. Some of the examples of knock-in mouse model include HdhQ111 and HdhQ92 [134],

CAG140 [135] and CAG150 [136]. The development of these models has enabled the investigation of

allele-specific targeting of the mutant allele using post-transcriptional gene silencing approaches. Specific

targeting of mutant allele preserves the expression of its wild-type counterpart that is usually involved in

22

various roles such as axonal guidance, cAMP signalling, calcium and glutamate signalling and long-term

potentiation/depression [137]. Table 1 summarises the most commonly used transgenic and knock-in

mouse models for HD.

23

Box 2: Key features of an ideal non-viral nanosystem for therapeutic delivery to CNS

The design and synthesis of a non-viral nanosystem plays a crucial role for successful delivery of a

therapeutic macromolecule, such as siRNAs and ASOs, at the targeted site. Several extracellular and

intracellular biological barriers exist and must be overcome in order to achieve the required silencing

effect at a specific target site [60]. Based on the knowledge about these different barriers and according to

the selected route of administration, it is possible to foresee the “ideal” characteristics required by the

non-viral vector to effectively deliver to the brain [60]. In this regard, Kostarelos and Miller have

suggested a practical and meaningful paradigm for optimisation of non-viral vectors based on the self-

assembly “ABCD” nanoparticles concept [138]. Based on this concept, if non-viral vectors are to be

administered through localised intraparenchymal administrations to a specific target site within the brain,

the simpler “AB” formulations may suffice, where “A” is the therapeutic payload and “B” is the polymer

that encapsulates/complexes the “A” [138]. However, in order to achieve successful gene silencing, this

“AB” delivery systems should still: protect siRNA from enzymatic degradation; be able to transfect

relevant target cell-types within the CNS; and, escape endosomal degradation releasing the siRNAs to the

cytoplasm [60,139]. In addition, it is worth noting that neurons are notoriously difficult to transfect, most

likely due to their post-mitotic nature, and, therefore, pose great challenges to non-viral delivery systems

[140]. Alternatively, and given that the systemic route is preferred, cationic non-viral vectors may need to

be further stabilised by forming “ABC”-type nanoparticles, where “C” refers to an addition of a stealth

layer such as, incorporation of a polyethylene glycol polymer (PEG) to improve stability of the

nanosystem under physiological conditions (reducing salt- and serum-induced aggregation) [138]. Finally,

once stable siRNA nanoparticles have been formulated, targeting across the BBB is likely to be required

to access the brain [60]. For which, the incorporation of targeting moieties is essential, which relates to

layer “D” in the ABCD nano-formulation concept. Addition of targeting ligands can enhance the transport

of the delivery vehicle across the BBB, mediating ease of access to the targeted cells. Thus, in brief the

key features include: a nanoparticle formulation that is, 1) scalable, reproducible and cost effective, 2)

24

synthesized from a biodegradable material, 3) non-toxic and non-immunogenic, 4) 100-200 nm in size

with preferably neutral or negative charge, 5) allows surface-modification, 6) stable in blood with

prolonged circulation time, and 7) avoid uptake by reticuloendothelial system.

25

Figure 1. Novel emerging therapeutic approaches for HD. Several strategies are been considered as

therapeutic alternatives to current symptom management: (A) Cell replacement approaches, which aim to

compensate for neuronal loss that occurs mainly in the Striatum; (B) Strategies that counteract the

underlying pathological mechanisms of HD, which try to avoid neuronal dysfunction and loss; and (C)

directly interfering with the cause of the disease by targeting the muHTT at the genomic level, post-

transcriptionally or at post-translational level. Additionally, (D) environmental enrichment has also

proven to be effective delaying the progression of HD in animal models and may be used as a

complementary approach to a pharmacological therapy in humans. Abbreviations: BDNF, brain-derived

neurotrophic factor; HDAC, histone deacetylase; muHTT, mutant Huntingtin; siRNA, short interfering

RNA.

26

Figure 2. Mechanism of action of RNA therapeutics. (A) Ribozymes: Ribozymes hybridize, sequence

specifically, with the mature mRNA in the cytoplasm and induce catalytic cleavage of the target mRNA.

(B) Antisense oligonucleotides: ASOs can act both in the nucleus and in the cytoplasm and lead to

RNAse H induced mRNA cleavage. However, inside the nucleus, ASOs can also hybridize with pre-

mRNA and inhibit/interfere with any of the following processes: 5’ capping, polyadenylation, intron-exon

splicing. In the cytoplasm, ASOs usually bind with the mature mRNA and recruit RNAse H to induce

mRNA cleavage or inhibit ribosomal binding to the mRNA, leading to protein inhibition. (C) RNA

interference: RNAse III like enzyme (Dicer) cleaves the dsRNA into siRNAs. The siRNA is incorporated

into a multiprotein RNA induced silencing complex (RISC), that consist of an Argonaute (Ago) as one of

the main protein components that cleaves and discards the sense strand of the siRNA. The retained

antisense strand in the RISC complex, guides the RISC to the complementary mRNA to induce

endonucleolytic cleavage of the mRNA. Abbreviations: mRNA, messenger RNA; siRNA, short

interfering RNA; ASO, antisense oligonucleotides; dsRNA, double-stranded RNA; AS-siRNA; antisense

short interfering RNA; RISC, RNA induced silencing complex; RNAi, RNA interference.

27

Table 1. Summary of most widely used mouse models of HD and their features.

Transgenic Knockin

Mouse model R6/2 N171-82Q YAC128 BACHD Hdh111 CAG140 Hdh(CAG)150

Promoter Human HTT Murine

prion Human HTT Human HTT

Murine

Hdh

Murine

Hdh Murine Hdh

PolyQ repeat 150 CAG 82 CAG 128 CAG 97 CAA/CAG 111 CAG 140 CAG 150 CAG

HTT Protein

expression vs.

Endogenous Hdh

75% 20% 75% 150% 50% /

100%

50% /

100% 100%

HD-related Neuropathology

Mutant HTT

aggregates ↑↑↑ ↑↑↑ ↑↑ ↑ ↑↑ ↑↑ ↑↑

Gliosis

↑↑

Dark neurons

(Striatum) ↑ ↑

↑ 10–15% / 52

wk

↑ 3.5% /

104 wk

Striatal cell loss

↑ 18% / 52 wk

↑ 40% / 100

wk

Striatal volume ↓↓↓

↓ 15% / 52 wk ↓ 28% / 52 wk

↓ 40% / 100

wk

Cortical volume

↓ 7% / 52 wk ↓ 32% / 52 wk

Brain weight 20% / 12 w

↓ 10% / 52 wk ↓ 14% / 52 wk

HD Behavioural Phenotypes

Cognitive Deficits

Reversal learning ↓↓

↓↓ / 8 wk

Morris water maze ↓↓

Anxiety-like behav. ↑↑

↑↑

Motor Deficits

Wheel running ↓↓↓ / 4.5 wk

Rotarod ↓↓↓ / 10 wk

↓↓ / 24 wk ↓↓↓ / 24 wk

↓ 100 wk

Grip strength ↓↓↓ / 10 wk

Open field ↓↓↓ / 8 wk

↓↓ / 8 wk ↓↓ / 8 wk

↓↓ / 4 wk ↓↓ / 8 wk

Stride Length ↓↓ / 12 wk

↓ / 104 wk ↓ / 52 wk ↓ / 100 wk

Rear & Climbing ↓↓↓ / 4.5 wk

↓ / 8 wk ↓↓ / 8 wk

Survival ↓↓↓ / 12-13

wk.

Body weight ↓ / 7 wk ↓ / 12 wk ↑ / 8 wk ↑ / 8 wk

↓ / 70 wk

Abbreviations: (↑) Increase; (↓) reduction; wk, week; HD, Huntington’s disease; HTT, Huntingtin; ASO, Antisense

oligonucleotide; PBS, Phosphate Buffered Saline; mRNA, messenger RNA; shRNA, short hairpin RNA; siRNA, short interfering

RNA; Hu, Human

28

Table 2. ASO in vivo studies for Huntington’s Disease in mammalian models

Category Animal model

Disease

stage/Age of

intervention

Delivery

system

Route of

administration

HTT gene expression

knockdown* Protein knockdown*

Improvement

in HD

pathology*

Behavioura

l outcomes Ref.

Rodents

BACHD mice,

YAC128 mice

and R6/2 mice

BACHD – Not

reported

YAC128 – 3

months and 6

months

(therapeutic

test)

R6/2 – 8 weeks

BACHD and

YAC128: Dose

of HuASO in

Saline via Alzet

osmotic pumps

R6/2 mice:

HuASOEx1 in

Saline via Alzet

osmotic pumps

Surgical

implantation of

the osmotic

pump/catheter

into the brain

BACHD: ↓ Human HTT

mRNA. Ipsilateral: cortex

28%, Striatum 19%;

Contralateral: cortex 36%,

striatum 39%; Caudal

region: Thalamus – 25%,

Midbrain - 53%,

Brainstem – 54%.

YAC128: ↓muHTT

mRNA – 42%

R6/2: ↓Human HTT

mRNA – 44%

YAC128: ↓mHTT

protein – 44%

R6/2 mice: no effect on

protein aggregates

BACHD and

YAC128: ↓

HTT aggregates

R6/2 mice : ↓

Astrocytosis

and microgliosis

in

R6/2 mice : ↓

loss of brain

mass

BACHD

and

YAC128:

Elevated

plus maze

Ambient

motor

activity

Lifespan

[30]

Hu97/18 mice

C57B16 mouse

and Sprague–

Dawley rat

Not reported

ASO 30 - PBS

Intracerebrovent

ricular and

intrathecal

injections

↓mHTT mRNA ↓mHTT Protein Not reported Not reported [31]

BACHD and

YAC18 mice Not reported

Allele non-

specific ASO

in PBS

Human Allele-

specific ASO

in PBS

Intraparenchym

al bolus

injections

Not reported

Non-allele specific

ASO: YAC18 and

BACHD: ↓mHTT

protein – 80.77%

and 82.56%,

respectively

Human-allele specific

ASO: YAC18 and

BACHD: ↓mHTT

protein – 3.31% and

52.56%, respectively

Not reported Not reported [29]

29

Non-human

primates Rhesus Monkey Not reported MkHuASO

Intrathecal

infusion

↓mHTT mRNA – Frontal

cortex: 47%, Occipital

cortex: 63%, Striatum:

25%, spinal cord: 46%

Not reported Not reported Not reported [30]

Abbreviations: (↑) Increase; (↓) reduction; () Improvement; () No improvement; HTT, Huntingtin; ASO, Antisense oligonucleotide; PBS, Phosphate Buffered Saline; mRNA,

messenger RNA; shRNA, short hairpin RNA; siRNA, short interfering RNA; Hu, Human.

30

Table 3. RNAi in vivo studies for Huntington’s Disease in mammalian models

Category Animal

model

Disease

stage/Age of

intervention

Delivery

system

Route of

administration

HTT gene

expression

knockdown

*

Protein

knockdown

*

Improvement in

HD pathology*

Behavioural

outcomes Ref.

Rodents

HD-N171-

82Q mice

Pre-

symptomatic

4-week old

AAV1 shRNA

(shHD2.1)

Intrastriatal

injection

(bilateral).

Intracerebellar

injection.

↓ 51-55% in

the striatum

↓ HTT

inclusions

(striatum

and

cerebellum)

↓ HTT inclusions

(striatum and

cerebellum)

Rotarod deficits

Gait deficits (Front

and rear stride length)

Weight loss

[54]

R6/1 mice

Pre-

symptomatic

6-week old

AAV5 shRNA

(siHUNT-1 and

-2)

Intrastriatal

injection

(bilateral)

↓~75% in

the striatum

↓ 25-38% in

the striatum

↓HTT nuclear

inclusions

↑ 24% ppENK, ↑

16% DARPP-32

mRNA

Clasping behaviour

Weight loss

Rotarod deficits

[16]

R6/2 mice

Pre-

symptomatic

Post natal day

2

Lipofectamine2

000 siRNA-

HDExon1

i.c.v. injection ↓70% in the

striatum

↓ HTT

nuclear

aggregates

in the

striatum

↓General brain

atrophy

↓ HTT nuclear

aggregates in the

striatum

Survival

Weight loss

Rotarod deficits

Clasping behaviour

Spontaneous

locomotor activity

[55]

HD190Q

EGFP mice

Symptomatic

12-week old

AAV2/AAV5

shRNA

(shEGFP)

Intrastriatal

injection

(unilateral)

Not reported

↓ ~82%

human

HTT-

positive

aggregates,

↓ ~65.9%

ubiquitin

aggregates

↑ ppENK and

↑DARPP-32

mRNA

No improvement in

behaviour and

survival (due to

unilateral injection)

[58]

AAV1/8-

based mouse

model

overexpressi

ng

HTT100Q

Pre-

symptomatic

cc-siRNA-HTT

(co-injection

with the

AAV1/8

HTT100Q)

Intrastriatal

injection

Not reported ↓~66%

human HTT

↓Size of nuclear

inclusions

↓Neurophil

aggregates

↑Survival of

striatal neurons

(Nissl-stain)

Clasping behaviour

Beam walking [57]

31

Adenoviral-

based mouse

model

overexpressi

ng

HTTN171Q

128

R6/2 mice

R6/2

symptomatic.

5 week-old

Ad shRNA

(shHTT)

(co-injection

with Ad

HTTN171Q12)

Intrastriatal

injection

(bilateral)

Not reported

↓ HTT

aggregates

in

transduced

areas

↓ HTT

aggregates in

transduced areas

Not reported [141]

CAG140

heterozygou

s knock in

mice

5 week-old

AAV2/1

shRNA and

miRNA

Intrastriatal

injection

(bilateral)

~50-60% in

transduced

areas

Not reported Not reported Not reported [87]

AAV1/2-

based rat

model

Pre-

symptomatic

AAV2/1

shRNA

(shHD2)

Intrastriatal

injection

(bilateral)

↓~80-90%

in the

striatum

↓~50% HTT

in the

striatum

↑ Neuronal

survival

↓Number of

degenerating

neurons

Spontaneous

exploratory forepaw

use

[142]

Lentiviral-

based rat

model

overexpressi

ng HTT171-

82Q

Symptomatic

2 months after

expression

started

DOX regulated

lentiviral

shRNA

sihtt1.1system

Intrastriatal

injection

Not reported

for muHTT

↓ HTT

inclusions

↑DARPP-32

mRNA,

↓ubiquitin

inclusions

Not reported [59]

HD-N171-

82Q mice

Pre-

symptomatic

7 week-old

AAV2/1

shRNA (sh2.4)

and miRNA

(mi2.4)

(also targeted

endogenous

HTT

homologue)

Intrastriatal

injection

(bilateral)

↓ ~60-75%

in the

striatum

Not reported Not reported

Rotarod deficits

Trend to improved

survival

Weight loss

[100]

BACHD

mice Not reported

AAV2/1

miRNA

(miHDS1)

(also targeted

endogenous

HTT

homologue)

Intrastriatal

injections

↓ ~60% in

the striatum Not reported Not reported Not reported [101]

Wistar rats N/A

cc-siRNA-HTT

(targeting

endogenous

HTT

homologue)

MRIgFUS

combined with

i.v. injection

↓ ~35% in

the striatum Not reported N/A N/A [143]

32

Nonhuman

primates

Adult rhesus

monkeys

(males)

N/A

AAV2/1

miRNA

(miHDS1)

(targeting

endogenous

HTT

homologue)

Intrastriatal

injections (3

injections per

hemisphere)

↓ ~45% in

mid and

caudal

putamen

Not reported N/A N/A [101]

Adult rhesus

monkeys

(females)

N/A

AAV2 shRNA

(shHD5)

(targeting

endogenous

HTT

homologue)

Intrastriatal

injections (5

injections per

hemisphere)

↓~30%

↓~45%

↓ HTT

immunostain

-ing

N/A N/A [102]

Adult rhesus

monkeys

(females)

N/A

14C-siRNA

(siHTT)

(targeting

endogenous

HTT

homologue)

CED in the

striatum for 28

days

↓ ~44% in

the putamen

↓ ~32% in

the putamen

↓HTT

immunostain

-ing with

decreasing

distance

from the

catheter

N/A

N/A [103]

* (vs diseased control or sham treated)

Abbreviations: (↑) Increase; (↓) reduction; () Improvement; () No improvement; AAV, adeno-associated virus; cc-siRNA, cholesterol-conjugated siRNA; CED, convection

enhanced delivery; DARPP-32, dopamine and cAMP-responsive phophoprotein 32 kDa; DOX, doxycycline; EGFP, enhanced green fluorescent protein; HTT, Huntingtin; i.v.,

intravenous injection; ppENK, preproenkephalin; MRIgFUS, magnetic resonance imaging guided focused ultrasound; mRNA, messenger RNA; N/A, not applicable; shRNA, short

hairpin RNA; siRNA, short interfering RNA.

33

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