Delivering a disease-modifying treatment for Huntington's disease

Drug Discovery Today  Volume 20, Number 1  January 2015

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Hunting diseases messages in the brain: emerging gene silencing approaches as novel therapeutic strategies for the treatment of Huntington’s disease.

Reviews  KEYNOTE REVIEW

Delivering a disease-modifying treatment for Huntington’s disease Bruno M.D.C. Godinho1,2, Meenakshi Malhotra1, Caitriona M. O’Driscoll1 and John F. Cryan1,2,3 1

Pharmacodelivery Group, School of Pharmacy, University College Cork, Cork, Ireland Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland 3 Laboratory of Neurogastroenterology, Alimentary Pharmabiotic Centre, Cork, Ireland 2

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 (muHTT) protein. Although no cure has yet been discovered, novel therapeutic strategies, such as RNA interference (RNAi), antisense oligonucleotides (ASOs), ribozymes, DNA enzymes, and genome-editing approaches, aimed at silencing or repairing the muHTT gene hold great promise. Indeed, several preclinical studies have demonstrated the utility of such strategies to improve HD neuropathology and symptoms. In this review, we critically summarise the main advances and limitations of each gene-silencing technology as an effective therapeutic tool for the treatment of HD.

Introduction HD is an autosomal dominant neurodegenerative disease caused by a CAG triplet mutation within the HTT gene and affects approximately 5–10 in 100 000 people in European, Australasian and American populations [1,2]. In general, symptoms strike during 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 and the 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 patients with HD. 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 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 Corresponding author: Cryan, J.F. ([email protected])

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Bruno M.D.C. Godinho is an invited lecturer in pharmacy at Escola Superior de Tecnologia da Sau´de de Lisboa, Instituto Polite´cnico de Lisboa, Portugal. He completed his PhD in pharmacy in a joint collaboration between the Pharmacodelivery Group/School of Pharmacy and the Department of Anatomy and Neuroscience, University College Cork. His PhD studies focussed on the development of modified cyclodextrins as novel nonviral vectors for neuronal siRNA delivery with a particular focus on neurodegenerative disorders, such as Huntington’s disease. Meenakshi Malhotra is a postdoctoral fellow in the laboratory of Caitriona M. O’Driscoll at the School of Pharmacy, University College Cork. Her work focusses on the design and development of novel surfacemodified cyclodextrin nanoparticles for siRNA delivery. She obtained her PhD from McGill University, Canada, where she studied cationic polymeric nanoparticles for siRNA delivery, targeting neurodegenerative diseases and cancer. Caitriona M. O’Driscoll completed her PhD in pharmaceutics at the University of Dublin, Trinity College. She held the post of senior lecturer in pharmaceutics at Trinity College until 2003. In 2003, she was appointed as the first professor of pharmaceutics at the School of Pharmacy, University College Cork and served as head of the school from 2003 to 2009 and 2010 to 2013. Her research interests are translational in nature and include formulation of nano-sized drug delivery constructs. Candidate drugs include biopharmaceuticals, peptide/proteins, nucleic acids and poorly soluble compounds. Delivery systems include lipid-based vehicles and nonviral gene delivery vectors using modified cyclodextrins, and targeted nanoparticles. John F. Cryan is professor and chair of the Department of Anatomy & Neuroscience, University College Cork. He received his PhD from the National University of Ireland, Galway. He was a visiting fellow at the Department of Psychiatry, University of Melbourne, which was followed by postdoctoral fellowships at the University of Pennsylvania, and The Scripps Research Institute. He spent 4 years at the Novartis Institutes for BioMedical Research, as lab head of behavioural pharmacology, before joining University College Cork (UCC) in 2005 as a senior lecturer in pharmacology. Currently, he is also a principal investigator in the Alimentary Pharmabiotic Centre, UCC.

1359-6446/06/$ - see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2014.09.011

Drug Discovery Today  Volume 20, Number 1  January 2015

REVIEWS

Environmental enrichment (d)

• Foetal striatal allografts

Huntington’s disease

• Human embryonic stem cells • Human-induced pluripotent stem cells

Reviews  KEYNOTE REVIEW

Interfering with muHTT

Cell replacement

• Genome-editing approaches (e.g., zinc finger proteins) • Post-transcriptional gene silencing (e.g., siRNA) • Post-translational targeting (e.g., scFV-C4 intrabody)

(a)

Counteracting pathological mechanisms

(c)

• Excitotoxicity (e.g,. Riluzole) • BDNF impairment (e.g., BDNF gene delivery) • Caspase activation (e.g., Minocycline) • Aggregate formation (e.g., Congo red) • Aggregate accumulation (e.g., Trehalose) • Mitochondrial dysfunction (e.g., Coenzyme Q10) • Transcriptional dysregulation (e.g., HDAC inhibitors)

(b) Drug Discovery Today

FIGURE 1

Novel emerging therapeutic approaches for Huntington’s disease (HD). Several strategies are being 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 mutant Huntingtin (muHTT) at the genomic level, post-transcriptionally or at post-translational level. Additionally, (d) environmental enrichment has also proven to be effective in delaying the progression of HD in animal models and could be used as a complementary approach to a pharmacological therapy in humans. Abbreviations: BDNF, brain-derived neurotrophic factor; HDAC, histone deacetylase; muHTT, mutant HTT; siRNA, short interfering RNA.

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, several therapeutic approaches have been advanced (Fig. 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 (Box 1, Table 1). These novel therapeutic modalities include neuroprotective strategies targeting the underlying pathologic mechanisms of muHTT, and cell replacement therapies focussed 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 play a role in improving patients’ quality of life. However, and despite being potential alternatives to current pharmacotherapy, these strategies are aimed at downstream effects of muHTT and do not specifically target the root cause of the disease [7]. By contrast, 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 are genome-editing techniques and post-transcriptional gene silencing approaches using ribozymes and DNA enzymes, ASOs and RNAi, all of which enable a specific reduction of the synthesis of muHTT. In fact, these approaches target upstream processes of disease and might enable therapeutic intervention even before cellular damage arises [10]. Given their potential as therapeutic strategies, lately they have received

significant attention from the scientific community and the field has rapidly progressed. Therefore, here we aim to not only capture such significant development, but also identify limitations and hurdles that need to be overcome for these concepts to reach the clinical setting.

Post-transcriptional gene silencing: therapeutic potential for HD 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 been shown to modulate the translational efficiency through a process that involves cleavage, degradation, or translational suppression of the target messenger RNA (mRNA). Although they share the common concept of reducing muHTT mRNA (and, consequently impacting on muHTT protein load) to block or reverse HD neuropathology and symptoms, their mechanisms of action differ significantly (Fig. 2).

Catalytic nucleic acid approach Catalytic nucleic acids include ribozymes and DNA enzymes (DNAzymes), and are aimed at the elimination or repair of target mRNA transcripts.

Ribozymes: mechanism of action and advances towards a potential therapeutic approach for HD Ribozymes are naturally occurring RNA molecules with self-cleaving capabilities that comprise an effector catalytic core and two flanking sequences that enable specific binding to the mRNA www.drugdiscoverytoday.com

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BOX 1

Animal models of Huntington’s disease

Reviews  KEYNOTE REVIEW

To advance drug discovery, appropriate animal models of disease are required. These enable extensive testing of novel therapeutics before progression of such strategies to clinical trials in human subjects. A variety of animal models (rodents and nonhuman 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 categorised as nongenetic and genetic models [124]. The chemically induced, nongenetic HD models involve cell death by using excitotoxic agents, such as quinolinic acid and kainic acid [125,126] or mitochondrial disrupting agents, such as 3-nitropropionic acid and malonic acid [127]. However, these models do not mimic the actual pathological conditions of HD; for example, they lacked the production of muHTT. 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 is limited to studies that involve 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 into the genome of the animal, or a knock-in model that expresses the pathological trinucleotide (CAG) repeat specifically inserted 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 a 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 examples of knock-in mouse models 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 various roles, such as axonal guidance, cAMP signalling, calcium and glutamate signalling, and long-term potentiation and/or depression [137]. Table 1 in the main text summarises the most commonly used transgenic and knock-in mouse models for HD.

[11,12] (Fig. 2a). The catalytic core is able to cleave 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, their exploitation has been relatively limited to date [14]. That said, there are several in vitro and preclinical animal studies using this approach in HD. Specifically, hammerhead ribozymes, a class of ribozymes of 30–40 nucleotides (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 an approximately 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]. 52

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Drug Discovery Today  Volume 20, Number 1  January 2015

Interestingly, similar results were obtained when using short hairpin RNAs (shRNAs) 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 an approximately 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 has also been successfully demonstrated in a rat model of Parkinson’s disease (PD) [17]. AAV delivery of a ribozyme against a-synuclein, into the substantia nigra, reduced a-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) by up to 70%, and the total secretion level of amyloid-b peptides by 30% [18]. Therefore, these HDV ribozymes are been considered as potential therapeutics for Alzheimer’s disease (AD), but require further investigation in animal models. Additionally, other studies have used the transsplicing abilities of group I intron ribozymes to repair the expanded CUG repeat in the 30 untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA transcripts, defect that causes myotonic dystrophy type 1, which is an autosomal dominant neuromuscular disease [19]. In this proof-of-concept study, group I intron ribozymes were able to replace effectively the 30 end of the endogenous DMPK transcript in human fibroblasts for a new 30 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 that 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 knocked down the expression of a co-transfected muHTT construct by 85% in HEK293 cells [21]. Although DNAzymes have been widely investigated in vivo as a potential therapeutic approach for diseases such as HIV, hepatitis, and cancer, their application in vivo to neurodegenerative disorders, such as HD, requires further research [20].

ASO approach Mechanism of action For more than 20 years, ASOs have been suggested as an ideal strategy to silence aberrant gene expression in various disease states [22]. ASO technology involves the use of single-stranded DNA molecules, typically 20–25 base pairs (bp) long, which have complementary sequence to the target mRNA [23,24]. ASOs hybridise with the pre-mRNA in the nucleus and cause inhibition of 50 cap formation, inhibition of splicing, and/or activation of

Drug Discovery Today  Volume 20, Number 1  January 2015

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TABLE 1

Summary of the most widely used mouse models of HD and their key featuresa

Promoter PolyQ repeat HTT protein expression versus endogenous Hdh HD-related neuropathology Mutant HTT aggregates Gliosis Dark neurons (striatum) Striatal cell loss Striatal volume Cortical volume Brain weight HD behavioural phenotypes Cognitive deficits Reversal learning Morris water maze Anxiety-like behaviour Motor deficits Wheel running Rotarod Grip strength Open field Stride Length Rear and climbing Survival Body weight

Transgenic

Knock-in

R6/2

N171-82Q

YAC128

BACHD

Hdh111

CAG140

Hdh(CAG)150

Human HTT 150 CAG 75%

Murine prion 82 CAG 20%

Human HTT 128 CAG 75%

Human HTT 97 CAA/CAG 150%

Murine Hdh 111 CAG 50%/100%

Murine Hdh 140 CAG 50%/100%

Murine Hdh 150 CAG 100%

"""

""" "" "

""

"

""

""

""

" 10–15%/52 wk

" 3.5%/104 wk

"

" # # #

### 20%/12 wk

## ## "" ###/4.5 wk ###/10 wk ###/10 wk ###/8 wk ##/12 wk ###/4.5 wk ###/12–13 wk. #/7 wk

18%/52 wk 15%/52 wk 7%/52 wk 10%/52 wk

" 40%/100 wk # 40%/100 wk

# 28%/52 wk # 32%/52 wk # 14%/52 wk

##/8 wk ""

##/24 wk

###/24 wk

##/8 wk

##/8 wk

# 100 wk

#/104 wk

#/12 wk

#/8 wk

##/8 wk

"/8 wk

"/8 wk

##/4 wk #/52 wk

##/8 wk #/100 wk

#/70 wk

a

Abbreviations: (") Increase; (#) reduction; BACHD, Bacterial artificial chromosome Huntington’s disease; HD, Huntington’s disease; HTT, Huntingtin; PolyQ, Polyglutamine; wk, week; YAC, Yeast artificial chromosome.

RNase H degradation [23]. However, when hybridisation occurs in the cytoplasm, translation is inhibited by steric hindrance or by RNase H degradation of the mRNA transcript, and is limited to the inhibition of one mRNA copy [25] (Fig. 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 was speculated to be because of 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 reduce successfully muHTT expression, improve HD-like neuropathology, and ameliorate symptoms of disease [29,30] (Table 2). Indeed, a recent study demonstrated a dose-dependent reduction in muHTT mRNA level (approximately 38%) with chemically (20 -O-methoxyethyl) modified ASOs in the BACHD mouse model of HD [30]. In this study was 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]. Moreover, these animals had improved motor activity and/or coordination, and displayed alleviated anxiety and delayed formation of polyQ aggregates for an extended 3 months of post-treatment termination [30]. In a follow-up study in nonhuman 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 allelespecific targeting of the human muHTT gene with ASOs by selective identification of single nucleotide polymorphism (SNP) targets in the human muHTT gene [29]. Results indicated allele-specific knockdown of human muHTT by approximately 52% in transgenic BACHD mice containing the elongated CAG tract, whereas no effect was observed in the YAC18 transgenic mice containing the nonpathological CAG tract [29]. Other studies have followed and also focused on the allele-selective downregulation of muHTT gene expression, showing significant reduction in muHTT mRNA in fibroblasts derived from patients with HD and a humanised mouse model of HD [31]. In addition, confirming the clinical utility of the ASO approach in neurological disorders, ASOs have recently undergone clinical trials for amyolateral sclerosis, further advocating their 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].

RNAi approach Mechanism of action Over the past two decades, RNAi technology has emerged with great promise in areas of gene therapy development. RNAi is an endogenous cellular pathway that enables post-transcriptional regulation of gene expression, and was first identified in petunia plants [34], later on in the nematode Caenorhabditis elegans [35] www.drugdiscoverytoday.com

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Mouse model

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Drug Discovery Today  Volume 20, Number 1  January 2015

(a) Ribozymes

Degraded mRNA

Ribozymes

Reviews  KEYNOTE REVIEW

mRNA

(b) ASOs

DNA

ASO ASO Degraded mRNA

DNA

RNAse H Pre-mRNA

No protein

Cy

mRNA

Mature mRNA

to

pl

A

RN

(c) RNAi

Ribosome

as

A

DN

eus Nucl

d

de

m

a gr

m

e g D

dsRNA

Dicer

siRNA + RISC

AS-siRNA mRNA + RISC

in bind

Drug Discovery Today

FIGURE 2

Mechanism of action of RNA therapeutics: (a) ribozymes hybridise, sequence specifically, with the mature mRNA in the cytoplasm and induce catalytic cleavage of the target mRNA. (b) Antisense oligonucleotides (ASOs) can act in both the nucleus and the cytoplasm and lead to RNAse H-induced mRNA cleavage. However, inside the nucleus, ASOs can also hybridise with pre-mRNA and inhibit and/or interfere with any of the following processes: 50 capping, polyadenylation, or intronexon 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 (RNAi): RNAse III-like enzyme (Dicer) cleaves the double-stranded (ds)RNA into short interfering RNAs (siRNAs). The siRNA is incorporated into a multiprotein RNA-induced silencing complex (RISC) that comprises an Argonaute (Ago) as one of the main protein components. The retained antisense strand in the RISC complex guides the RISC to the complementary mRNA to induce endonucleolytic cleavage of the mRNA. Abbreviation: AS-siRNA; antisense short interfering RNA.

and finally in mammalian cells [36] (Figs 1,2c). This intracellular pathway enables cells to autoregulate 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) that 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 can contain several stem–loop structures, into small interfering RNAs (siRNAs) with 21–25 oligonucleotides (nt) [39]. siRNAs are loaded on an RNA-induced silencing complex (RISC), which is activated upon unwinding of the siRNA and thermodynamic selection of the guide and/or antisense strand. Activated RISC searches the transcriptome for specific complementary mRNAs, targeting them for degradation, therein inhibiting translation of the protein product [40,41] (Fig. 2c). Activated RISCs are able to catalyse multiple turnover reactions, enhancing the potency of this gene-silencing approach when compared with ASOs. Additionally, not only because of the sequence mismatch and partial complementarity, but also through the ability of binding to several UTR sequences, miRNAs have been shown to silence several mRNA targets [38]. This RNAi machinery can be artificially hijacked to induce specific gene expression knockdown, which is commonly 54

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performed using synthetic siRNAs, not only in combination with nonviral delivery systems, but also through shRNA, usually utilising 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, not only 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 polyQinduced 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, were administered into the striatum of HD transgenic mice (N171-82Q) [54]. Significant

ASO in vivo studies for HD in mammalian modelsa Animal model

Disease stage: age of intervention

Delivery system

Route of administration

HTT gene expression knockdown

Protein knockdown

Improvement in HD pathology

Behavioural outcomes

Refs

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 brain

YAC128: # muHTT protein by 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: U elevated plus maze U Ambient motor activity U Lifespan

[30]

Hu97/18 mice C57B16 mouse and Sprague–Dawley rat

Not reported

ASO 30 in PBS

# muHTT protein

Not reported

Not reported

[31]

BACHD and YAC18 mice

Not reported

Allele nonspecific ASO in PBS Human allele- specific ASO in PBS

Intracerebroventricular and intrathecal injections Intraparenchymal bolus injections

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% # muHTT mRNA

Not reported

Nonallele-specific ASO: YAC18 and BACHD: # muHTT protein by 80.77% and 82.56%, respectively Human allele-specific ASO: YAC18 and BACHD: # muHTT protein by 3.31% and 52.56%, respectively

Not reported

Not reported

[29]

Not reported

MkHuASO

# muHTT mRNA: frontal cortex 47%, occipital cortex 63%, striatum 25%, spinal cord 46%

Not reported

Not reported

Not reported

[30]

Rodents BACHD mice, YAC128 mice, and R6/2 mice

Nonhuman primates Rhesus monkey

Intrathecal infusion

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TABLE 2

Abbreviations: (") Increase; (#) reduction; (U) Improvement; () No improvement; ASOs, Antisense oligonucleotides; BACHD, Bacterial artificial chromosome Huntington’s disease; HTT, Huntingtin; HD, Huntington’s disease; HTT, Huntingtin; Hu, human; muHTT, mutant HTT; PBS, phosphate buffered saline; YAC, Yeast artificial chromosome.

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a

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56 TABLE 3

RNAi in vivo studies for HD in mammalian modelsa Animal model

Delivery system

Route of administration

Presymptomatic; 4weeks old

AAV1 shRNA (shHD2.1)

Intrastriatal injection # 51–55% in the (bilateral). striatum Intracerebellar injection.

R6/1 mice

Presymptomatic; 6weeks old

AAV5 shRNA (siHUNT-1 and -2)

Intrastriatal injection (bilateral)

R6/2 mice

Presymptomatic; postnatal day 2

Lipofectamine2000 siRNA-HDExon1

i.c.v. injection

HD190Q EGFP mice

Symptomatic; 12weeks old

AAV2/AAV5 shRNA (shEGFP)

Intrastriatal injection (unilateral)

AAV1/8-based mouse model overexpressing HTT100Q

Presymptomatic

cc-siRNA-HTT Intrastriatal injection (co-injection with the AAV1/8 HTT100Q)

Adenoviral-based mouse model overexpressing HTTN171Q128 R6/2 mice CAG140 heterozygous knock in mice AAV1/2-based rat model

R6/2 symptomatic; 5- Ad shRNA (shHTT) Intrastriatal injection weeks old (co-injection with Ad (bilateral) HTTN171Q12)

Rodents HD-N171-82Q mice

Lentiviral-based rat model overexpressing HTT171-82Q HD-N171-82Q mice

5-weeks old Presymptomatic

Symptomatic 2 months after expression started Presymptomatic 7-weeks old

AAV2/1 shRNA and miRNA AAV2/1 shRNA (shHD2)

Intrastriatal injection (bilateral) Intrastriatal injection (bilateral)

DOX regulated Intrastriatal injection lentiviral shRNA sihtt1.1system AAV2/1 shRNA Intrastriatal injection (sh2.4) and miRNA (bilateral) (mi2.4) (also targeted endogenous HTT homologue)

HTT gene expression knockdown b

Protein knockdownb

Improvement in HD pathologyb

# HTT inclusions (striatum and cerebellum)

# HTT inclusions (striatum and cerebellum)

Behavioural outcomes

URotarod deficits UGait deficits (front and rear stride length) Weight loss # 75% in the # 25–38% in the # HTT nuclear inclusions UClasping behaviour striatum striatum " 24% ppENK, " 16% DARPP-32 mRNA Weight loss Rotarod deficits # General brain atrophy USurvival # 70% in the striatum # HTT nuclear # HTT nuclear UWeight loss aggregates in the striatum aggregates in the URotarod deficits striatum UClasping behaviour USpontaneous locomotor activity Not reported # 82% human HTT- " ppENK and " DARPP- No improvement in positive aggregates, 32 mRNA behaviour and # 65.9% ubiquitin survival (due to aggregates unilateral injection) Not reported # 66% human HTT # Size of nuclear U Clasping inclusions behaviour # Neurophil aggregates U Beam walking " Survival of striatal neurons (Nissl-stain) Not reported # HTT aggregates in # HTT aggregates in Not reported transduced areas transduced areas

50–60% in transduced areas # 80–90% in the striatum

Refs

[54]

[16]

[55]

[58]

[57]

[141]

Not reported

Not reported

Not reported

[87]

# 50% HTT in the striatum

" Neuronal survival # Number of degenerating neurons " DARPP-32 mRNA, # Ubiquitin inclusions

U Spontaneous exploratory forepaw use Not reported

[142]

Not reported

U Rotarod deficits U Trend to improved survival  Weight loss

Not reported for muHTT

# HTT inclusions

# 60–75% in the striatum

Not reported

[59]

[100]

Drug Discovery Today  Volume 20, Number 1  January 2015

Disease stage/age of intervention

Animal model

Disease stage/age of intervention

Delivery system

Route of administration

HTT gene expression knockdown b

Protein knockdownb

Improvement in HD pathologyb

Behavioural outcomes

Refs

BACHD mice

Not reported

Intrastriatal injections

# 60% in the striatum

Not reported

Not reported

Not reported

[101]

Wistar rats

N/A

AAV2/1 miRNA (miHDS1) (also targeted endogenous HTT homologue) cc-siRNA-HTT (targeting endogenous HTT homologue)

MRIgFUS combined with i.v. injection

# 35% in the striatum

Not reported

N/A

N/A

[143]

Nonhuman primates Adult rhesus monkeys (males)

N/A

Intrastriatal injections (3 # 45% in mid and injections per caudal putamen hemisphere)

Not reported

N/A

N/A

[101]

Intrastriatal injections (5 # 30% injections per hemisphere)

# 45% # HTT immunostaining

N/A

N/A

[102]

# 32% in the putamen # HTT immunostaining with decreasing distance from the catheter

N/A

N/A

[103]

Adult rhesus monkeys (females) N/A

Adult rhesus monkeys (females) N/A

AAV2/1 miRNA (miHDS1) (targeting endogenous HTT homologue) AAV2 shRNA (shHD5) (targeting endogenous HTT homologue) 14 C-siRNA (siHTT) (targeting endogenous HTT homologue)

CED in the striatum for 28 days

# 44% in the putamen

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TABLE 3 (Continued )

a Abbreviations: (") Increase; (#) reduction; (U) Improvement; () No improvement; AAV, Adeno-associated virus; BACHD, Bacterial artificial chromosome Huntington’s disease; cc-siRNA, cholesterol-conjugated siRNA; CED, convection enhanced delivery; DARPP-32, dopamine and cAMP-responsive phosphoprotein 32 kDa; DOX, doxycycline; EGFP, enhanced green fluorescent protein; HD, Huntington’s disease; HTT, Huntingtin; i.v., intravenous injection; miRNA, micro RNA; MRIgFUS, magnetic resonance imaging guided-focused ultrasound; N/A, not applicable; ppENK, preproenkephalin; shRNA, short hairpin RNA. b Versus diseased control or sham treated.

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reductions in muHTT mRNA levels (approximately 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 either the systemic nature of the disease or to muHTT-mediated hypothalamic dysfunction [54]. 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 (approximately 75%), decreased number of HTT inclusions (25–38%) and improvement of hind-limb 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 with 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 preclinical study using nonviral 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 that can be used as a delivery vehicle to transport therapeutic molecules into the cells; 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 (approximately 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 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 with untreated R6/2 mice were also reported [55]. Using a different nanoparticlebased siRNA delivery approach, locally delivered, amphiphilic cyclodextrins-siRNA nanoparticles have shown to knockdown the muHTT gene successfully (85%) in the striatum of the R6/2 mouse model of HD, also selectively improving phenotypic deficits upon repeated brain injections [56]. In another approach, the utility of cholesterol-conjugated siRNAs (cc-siRNA-HTT) for HD was demonstrated in an AAV-based mouse model of HD (AAVHTT100Q) [57]. Co-administration of AAV-HTT100Q and ccsiRNA-HTT into adult mouse striatum resulted in 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 the above-mentioned in vivo studies, RNAi treatment was initiated when animals were still pre-symptomatic and, therefore, limited conclusions about the reversal of neuropathology and HD symptoms can be drawn. However, following studies were carried out in symptomatic rodents and have shown 58

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BOX 2

Key features of an ideal nonviral nanosystem for therapeutic delivery to the CNS The design and synthesis of a nonviral nanosystem has a crucial role in the successful delivery of a therapeutic macromolecules, such as siRNAs and ASOs, to the targeted site. Several extracellular and intracellular biological barriers exist and must be overcome 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 nonviral vector to deliver effectively to the brain [60]. In this regard, Kostarelos and Miller suggested a practical and meaningful paradigm for optimisation of nonviral vectors based on the self-assembly ‘ABCD’ nanoparticles concept [138]. Based on this concept, if nonviral vectors are to be administered through localised intraparenchymal administrations to a specific target site within the brain, the simpler ‘AB’ formulations might suffice, where ‘A’ is the therapeutic payload and ‘B’ is the polymer that encapsulates/complexes the ‘A’ [138]. However, to achieve successful gene silencing, this ‘AB’ delivery systems should still (i) protect siRNA from enzymatic degradation; (ii) be able to transfect relevant target cell types within the CNS; and (iii) escape endosomal degradation releasing the siRNAs to the cytoplasm [60,139]. In addition, neurons are difficult to transfect, most likely because of their postmitotic nature and, therefore, pose great challenges to nonviral delivery systems [140]. Alternatively, and given that the systemic route is preferred, cationic nonviral vectors might need to be further stabilised by forming ‘ABC’-type nanoparticles, where ‘C’ refers to a stealth layer. This can be achieved by incorporation of a polyethylene glycol polymer (PEG), which improves the 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 Blood-Brain Barrier (BBB) is likely to be required to access the brain [60]. For this, the incorporation of targeting moieties is essential, which relates to layer ‘D’ in the ABCD nanoformulation 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: (i) scalable, reproducible, and cost effective; (ii) synthesised from a biodegradable material; (iii) nontoxic and nonimmunogenic (iv) 100–200 nm in size with preferably neutral or negative charge; (v) allows surface modification; (vi) stable in blood with a prolonged circulation time; and (vii) avoid uptake by reticuloendothelial system. 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 whether such improvements in HD neuropathology also result in the reversal of HD behavioural deficits. Similarly, additional studies are needed not only to elucidate which are the most efficient RNAi delivery systems, but also to identify any safety issues regarding the long-term use of RNAi technologies for HD.

Limitations of post-transcriptional gene-silencing technologies for HD 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 nontoxic 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, nontoxic and safe delivery systems able to overcome adequately 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 properties, normally requiring adequate systems for neuronal delivery and effective protection from nuclease degradation [60]. AAV and lentiviral vectors have great tropism over a 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 of possible adverse reactions to viral vectors because of toxicity and activation of immune responses [62]. Alternatively, several lipid- or polymerbased nonviral vectors have been engineered and are now commercially available as transfection reagents for nucleic acids (e.g., Lipofectamine2000, Lipofecatmine RNAi Max, Exgen500, Superfect, and 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], nonviral 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, because they show nonspecific 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 nonviral vectors, further suggesting that certain biomaterials are more likely to enhance adverse effects in CNS than others (Box 2) [65,68]. In addition, 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 nontarget mRNA transcripts or activation of components of the immune system [70]. In the specific context of HD, antiHTT ribozymes have been reported to alter mouse transcriptional activity that affected other mRNAs including DARPP-32 (downregulation) and nerve growth factor inducible A (NGFI-A) binding protein (upregulation) [15]. Despite the fact that such offtarget effects were suggested to be sequence dependent and were also seen with shRNAs (siHUNT2) [16], no other human mRNA sequence presented significant similarity to the targeted regions. Therefore, although these unwanted effects might 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 offtarget effect whereby they downregulate untargeted mRNAs by partial complementarity [71]. Furthermore, shRNA and siRNA

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have been shown to activate RNA helicases (Protein kinase R (PKR) and retinoic acid-inducible gene 1 (RIG-1)) leading to protein synthesis arrest through phosphorylation of eukaryotic initiation factor 2 (eIF2a) and upregulation of a subset of genes from the interferon (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 miRNA-like off-target and immunogenic effects have been found to occur in a dosedependent manner and, therefore, the use of a minimal dose is crucial [76]. Moreover, the use of rational algorithmic design tools is essential not only 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-hybridisation with untargeted mRNA transcripts [77,78]. Chemical modifications can also be introduced on siRNA sequences to enhance their 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 artificial miRNA-based expression system, which is well tolerated in vitro and in vivo [86,87]. By 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 a copy of the 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 fivefold increase in caspase 3 activity and a decrease in brainderived neurotrophic factor (BDNF) levels leading to neuronal cell death, following ASO-mediated knockdown of wtHTT [89]. These results highlight the detrimental effects of nonspecific silencing of the wild-type alleles, especially if chronic administration is needed, and emphasise 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 shRNAs) targeting the site of the mutations has enabled allele-specific silencing of the mutant forms of www.drugdiscoverytoday.com

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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]. By contrast, ASOs, peptide nucleic acids (PNA), and locked nucleic acids (LNA) seem to be a better alleleselective alternative when targeting these elongated CAGs, because these approaches are able to discriminate CAG repeat lengths based on energetically different structures, rather than solely on base differences [27,28,64]. Chemically modified ASOs have enhanced RNAse H nucleotide discrimination more than 100-fold, sparing the downregulation of nontargeted HTT mRNA transcripts. In the specific case of siRNAs, allele specificity might only be achievable by targeting disease-linked polymorphisms. Approximately 60 SNPs have been identified in the coding and the 30 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 three to seven SNPs have achieved allele-specific knockdown of the muHTT in vitro and might 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, all of which could lead to an increased cost for implementation of such strategy. Alternatively, nonallele-specific targeting has been recently suggested as a valid approach that would circumvent the economic cost of individualised 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 nonhuman primates have also shown that the reduction of endogenous HTT homologue by approximately 45% does not induce neuronal degeneration, astrogliosis, or even motor deficits [101–103]. Thus, the residual levels of wtHTT protein might 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 60

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only up to 6–9 months and, therefore, the effect of long-term gene expression knockdown of wtHTT still needs further investigation. 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 comprises utilising RNAi for nonallele-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 might 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 a-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 gene expression knockdown and effective gene replacement; however, no functional effects were reported [106,107]. The suppression and replacement approach could also represent a possibility for HD therapeutics; however, this has not yet been investigated.

Genome-editing approaches for HD therapeutics Specific gene targeting and/or 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 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], haemophilia B [110], sickle-cell disease [111], PD [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 (ClinicalTrial.gov Identifier: NCT01082926). In the specific case of HD, it has recently been demonstrated that zinc finger proteins (ZFP) are able to silence effectively muHTT, without affecting the expression of wtHTT, in vitro and in the R6/2 mouse brain (approximately 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 in their infancy, they hold great promise not only for HD, but also other monogenic disorders.

Concluding and future perspectives Although the function of HTT is not fully understood, 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 the pathogenic mutant protein. Genome-editing approaches are gradually shaping into a powerful therapeutic strategy for monogenic disorders, such as HD, because of the possibility of replacing and/or repairing native mutated genes. In fact, the use of such a strategy in HD would enable modification and correction of the native mutant loci, with the 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 costly and dependent on the fine combination of zinc fingers in ZFN and of the right amino acid repeat in TALENs. By contrast, the development of more efficient methods to detect and control for off-target cleavages and insertions is crucial, because these undesired effects can lead not only to a reduced efficiency, but more importantly, also to cytotoxicity. Therefore, despite its great promise as a potential therapeutic approach for HD, more work is needed in different rodent models and even nonhuman primate models of HD to support the growing evidence of its applicability in human disease. Post-transcriptional gene-silencing approaches have also shown 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 and ASO approaches. Although the RNAi approach allows for a strong gene-silencing effect and follows a targeted approach, most in vivo studies carried out so far targeted sequences with shared homology with both human wtHTT and muHTT mRNA transcripts. Furthermore, and although partial suppression of wtHTT has been well tolerated in preclinical studies, the effects of chronic suppression of wtHTT have not been assessed in humans. Thus, application of such approaches to human therapy 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 might present additional challenges at the clinical stage, because multiple clinical trials might be required to establish the efficacy of each of the different SNP-targeted molecules. Alternatively, to date, ASOs can be considered to have an edge over siRNA-mediated gene silencing in terms of enabling allele-specific targeting of the

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muHTT through the pathological CAG tract, thus sparing the wtHTT protein for its essential cellular processes. 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 used 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 that 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 complex and/or encapsulate safely the therapeutic molecule and deliver it to the targeted site in relevantly sufficient quantities and be safe to CNS tissues by being nonimmunogenic, nontoxic, and biodegradable. Furthermore, to avoid invasive brain surgery in patients with HD, a targeted delivery system that can cross the BBB via transcytosis and specifically deliver the cargo to affected structures would be required. To this end, several cell-penetrating and cell-targeting ligands and/or peptides have been investigated to enhance transport across the BBB or to increase delivery to neurons [117,118]. Among the 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 was identified by using a phage display library and was reported to target BBB specifically [120]. Transferrin [121], lactoferrin [122], and angiopep [123] have also been widely investigated for CNS delivery; however, the receptors for these proteins and/or peptides are widely expressed in other tissues and might not confer specificity for the CNS. This demonstrates that further improvements of current nanoformulations for specific targeting to neurons will be required, and will eventually enable delivery to specific subpopulations of neurons. Thus, gene silencing and gene delivery are two approaches that go hand-in-hand for the actualisation of gene therapy. Successful outcomes in this regard could lead to clinical testing of oligonucleotide-based therapies, not only for HD, but also for other neurodegenerative disorders, potentially delaying the progression of the disease and improving quality of life.

Acknowledgements The authors wish to acknowledge research funding provided by Science Foundation Ireland (Grant no. 07/SRC/B1154) and the Irish Drug Delivery Network.

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136 Lin, C-H. et al. (2001) Neurological abnormalities in a knock-in mouse model of Huntington’s disease. Human Mol. Genet. 10, 137–144 137 Cattaneo, E. et al. (2005) Normal huntingtin function: an alternative approach to Huntington’s disease. Nat. Rev. Neurosci. 6, 919–930 138 Kostarelos, K. and Miller, A.D. (2005) Synthetic, self-assembly ABCD nanoparticles; a structural paradigm for viable synthetic non-viral vectors. Chem. Soc. Rev. 34, 970–994 139 Guo, J. et al. (2010) Therapeutic targeting in the silent era: advances in non-viral siRNA delivery. Mol. Biosyst. 6, 1143–1161 140 Krichevsky, A.M. and Kosik, K.S. (2002) RNAi functions in cultured mammalian neurons. Proc. Natl. Acad. Sci. U. S. A. 99, 11926–11929 141 Huang, B. et al. (2007) High-capacity adenoviral vector-mediated reduction of huntingtin aggregate load in vitro and in vivo. Human Gene Ther. 18, 303–311 142 Franich, N.R. et al. (2008) AAV vector-mediated RNAi of mutant huntingtin expression is neuroprotective in a novel genetic rat model of Huntington’s disease. Mol. Ther. 16, 947–956 143 Burgess, A. et al. (2012) Focused ultrasound for targeted delivery of siRNA and efficient knockdown of Htt expression. J. Control. Release 163, 125–129