nihms304300.pdf

Gene Therapy in Mouse Models of Huntington Disease

Amber L. Southwell

and

Paul H. Patterson

Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC,

Canada

Division of Biology, California Institute of Technology, Pasadena, CA, USA

Abstract

Huntingtin, the protein that when mutated causes Huntington disease (HD), has many known

interactors and participates in diverse cellular functions. Mutant Htt (mHtt) engages in a variety of

aberrant interactions that lead to pathological gain of toxic functions as well as loss of normal

functions. The broad symptomatology of HD, including diminished voluntary motor control,

cognitive decline, and psychiatric disturbances, reflects the multifaceted neuropathology.

Although currently available therapies for HD focus on symptom management, the autosomal

dominant cause and the adult onset make this disease an ideal candidate for genetic intervention. A

variety of gene therapy approaches have been tested in mouse models of HD, ranging from those

aimed at ameliorating downstream pathology or replacing lost neuronal populations to more

upstream strategies to reduce mHtt levels. Here the authors review the results of these preclinical

trials.

Keywords

autophagy; intrabody; neurotrophin; RNAi; silencing; viral vector

Huntington disease (HD) is caused by the expansion of a polyglutamine (polyQ) tract in

exon 1 of the multifunctional protein Htt. Affected individuals have more than 36 glutamine

repeats, and longer polyQ tracts correlate with earlier onset and increased disease severity

(Huntington’s Disease Collaborative Research Group 1993). Expanded polyQ Htt induces

degeneration and eventual loss of susceptible neuronal populations in the striatum and

cortex. Chief among these are striatal medium spiny neurons (MSNs), whose population

undergoes an early and severe loss. The deficit in neurons and their processes results in a

dramatic atrophy of the putamen and caudate nucleus, as well as cortical thinning (Rosas

and others 2008). HD usually presents in midlife with chorea, dementia, and weight loss,

with death typically occurring 15 to 20 years after symptom onset (Walker 2007). Although

the appearance of gross motor deficits or dementia is commonly referred to as disease onset,

carriers of expanded polyQ in HD can display psychiatric symptoms such as anxiety,

depression and obsessive compulsive disorder, mild cognitive impairments of episodic

memory and executive function, and fine motor deficits years to decades before defined

clinical onset (Biglan and others 2009; Duff and others 2007). Elevated levels of

proinflammatory cytokines are also present in serum at least 16 years prior to motor

Corresponding Author:

Amber L. Southwell, Centre for Molecular Medicine and Therapeutics, University of British Columbia, 950

W 28th Ave, Hayden, Vancouver, BC, V5Z 4H4, Canada, asouthwell@cmmt.ubc.ca.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interests with respect to the authorship and/or publication of this article.

NIH Public Access

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Published in final edited form as:

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symptom onset, reflecting the role of inflammation in this disease (Bjorkqvist and others

2008).

The simple genetic nature and autosomal dominant transmission of HD should facilitate

therapy development. Unlike multicause, predominately idiopathic neurodegenerative

diseases such as Alzheimer and Parkinson diseases, identification of premanifest mutation

carriers and thus presymptomatic treatment initiation is possible for HD. This allows for

neuroprotective strategies rather than more complicated restorative methods or techniques

for coping with existing neuronal loss. Nonetheless, the therapies currently available to HD

patients, which include selective serotonin reuptake inhibitors (SSRIs) and atypical

antipsychotics for psychiatric disturbances and tetrabenazine for chorea, offer only moderate

symptom relief and have no effect on disease progression (De Marchi and others 2001;

Huntington Study Group 2006).

In striking contrast to the simplicity of HD genetics, the convoluted disease mechanisms in

this disorder have hindered development of disease-modifying therapies. The Htt protein is a

large, promiscuous protein that is known to associate with many molecular partners,

including proteins, DNA, and mRNA, as well as cellular components that include the

endoplasmic reticulum (ER), mitochondria, microtubules, the plasma membrane, and

endocytic, synaptic, and autophagic vesicles (Imarisio and others 2008). Wild-type Htt

(wtHtt) is a jack of all trades, playing roles in numerous, seemingly unrelated processes that

include transcriptional regulation, apoptosis suppression, ER stress signaling, calcium

homeostasis, axonal transport, endocytosis, and synaptic transmission (Zuccato and others

2010). In HD, mHtt fails to adequately perform these essential roles and also acquires toxic

functions related to accumulation, aggregation, and sequestration of essential proteins, as

well as aberrant interactions. This combination of loss and gain of function creates a

network of cellular dysfunction that leads to impaired proteolysis, transcriptional regulation,

intracellular trafficking, mitochondrial metabolism, calcium signaling, and synaptic activity

(Imarisio and others 2008; Ramaswamy, Shannon, and others 2007). Consequently, there is

a wide variety of potential HD therapeutic targets—from the mHtt protein itself to myriad

downstream factors, many of which have been evaluated for gene therapy efficacy in mouse

models of HD.

Downstream Targets

Neurotrophic Factor Delivery

Neurotrophic factors are secreted growth factors that can enhance differentiation, function,

and survival of neurons. Ectopic expression of neurotrophic factors mediated either by viral

vectors or the transplantation of genetically modified cells has shown some promise in the

treatment of mouse models of HD.

Nerve growth factor (NGF) is the prototypical neurotrophin that not only promotes neuronal

survival but can also down-regulate Htt expression in cultured striatal neurons (Haque and

Isacson 2000). NGF delivery by transplantation of transfected neural stem cells (NSCs) is

neuroprotective when administered prior to injection of quinolinic acid (QA), an

-methyl-

-aspartate (NMDA) receptor agonist that induces excitotoxicity and neuropathology similar

to that found in HD (Martinez-Serrano and Bjorklund 1996). Transplantation of

mesenchymal stem cells (MSCs) genetically engineered to overexpress NGF into the

striatum of four-month-old YAC128 mice (a transgenic model expressing full-length mHtt)

results in moderate improvements in rotarod and clasping behavior but no increase in striatal

neuron number or expression of dopamine and cyclic-AMP-regulated phosphoprotein

(DARPP-32), a marker of healthy MSNs (Dey and others 2010).

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Brain-derived neurotrophic factor (BDNF), which is required for differentiation and survival

of striatal MSNs, is reduced in the human HD striatum. Expression of BDNF is activated by

wtHtt, a function that is lost with polyQ expansion (Zuccato and Cattaneo 2007). Viral

delivery of BDNF to the striatum of rats prior to injection of QA is neuroprotective,

resulting in reduced cell loss and lesion volume (Bemelmans and others 1999; Kells and

others 2004). Astrocytes cultured from transgenic mice expressing BDNF driven by the glial

fibrillary acid protein (GFAP), astrocyte-specific promoter, increase their BDNF production

in response to inflammatory insults. When grafted into the striatum of nude mice one week

or one month before QA injection, these cells produce excess BDNF, which reduces lesion

volume and prevents neuronal death, as measured by NeuN, a marker of neuronal nuclei.

This neuroprotection is selective, resulting in increased DARPP-32-positive MSNs and

parvalbumin-positive but not choline acetyltransferase–positive interneurons. In addition,

BDNF-expressing astrocyte grafts in unilaterally lesioned animals decrease apomorphine-

induced rotation, indicating that function is preserved in the protected neurons (Giralt and

others 2010). The beneficial effects of ectopic BDNF expression have also been

demonstrated in the YAC128 transgenic model of HD. In four-month-old mice, striatal

injection of MSCs harvested from mouse femurs and transfected to produce BDNF results in

improved clasping and rotarod performance. At 13 months of age, BDNF-MSC-grafted mice

have displayed more NeuN- and DARPP-32-positive striatal neurons than unmodified MSC-

grafted or vehicle-injected mice, illustrating the long-term benefit of this treatment (Dey and

others 2010).

Cilliary neurotrophic factor (CNTF), a member of the interleukin (IL)–6 or neuropoietic

cytokine family, promotes the survival of a broad range of neuronal types, including those

most susceptible to mHtt-induced toxicity (Ip and Yancopoulos 1996). Although

neuroprotective in QA rat models of HD (De Almeida and others 2001; Emerich and others

1996; Régulier and others 2002), CNTF has shown no benefit and, in some cases, a

detriment in HD transgenic mice. Lentiviral delivery of CNTF into YAC72 mice results in

excellent striatal expression for up to one year following surgery. Although no exacerbation

of behavioral deficits is found in treated animals, reduced DARPP-32 expression and NeuN-

positive cells as well as increased GFAP expression are seen in the striatum of both wild-

type and YAC72-treated mice (Zala and others 2004). Moreover, adeno-associated virus

(AAV) delivery of CNTF into the striatum of 10-week-old R6/1 mice results in earlier onset

of rotarod deficits. In addition, this treatment increases body weight loss and reduces

expression of striatally enriched genes such as DARPP-32 and preproenkephalin in both

transgenic and wild-type mice (Denovan-Wright and others 2008). These studies indicate

that long-term overexpression of CNTF is not well tolerated, and therefore this neurotrophic

factor is not a good candidate for gene therapy in HD.

Glial cell line–derived neurotrophic factor (GDNF) is produced in the developing striatum

and is expressed by adult striatal projection neurons as well as interneurons (Pérez-Navarro

and others 2000). Viral- and cell-mediated delivery of GDNF prior to QA injection is

neuroprotective in rats (Kells and others 2004; Pérez-Navarro and others 1999; Pérez-

Navarro and others 1996). Transplantation of NSCs transfected to produce GDNF protects

against QA toxicity in nude mice, resulting in moderately reduced loss of NeuN-positive

striatal neurons and reduced amphetamine-induced rotation (Pineda and others 2006). When

given after symptom onset, lentiviral delivery of GDNF to four- to five-week-old R6/2 mice

has no effect on rotarod, clasping, or open field behavior. Intranuclear inclusions, BrdU

staining, and neuronal cross-sectional area are also unaffected in these animals (Popovic and

others 2005). However, presymptomatic AAV-GDNF delivery to five-week-old N171-82Q

transgenic mice significantly delays onset of clasping and rotarod deficits. At 16 weeks of

age, compared with untreated mice, the striata of treated mice have decreased inclusions and

increased neuronal number with larger cross-sectional areas, although striatal volume loss is

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not improved (McBride and others 2006). Moreover, transplantation of GDNF-secreting

mouse neural progenitor cells (NPCs) into 60-day-old N171-82Q mice following symptom

onset results in delayed body weight loss and dramatic improvement in rotarod performance.

GDNF-NPC transplantation also results in reduced inclusions and increased NeuN-positive

cells in the striatum but no improvement in cortical thinning. Although some loss of tyrosine

hydroxylase is seen in areas proximal to the graft, no loss of dopaminergic neurons is

observed (Ebert and others 2010). Thus, GDNF can be a restorative as well as a preventative

treatment.

Neuturin (Ntn), a member of the GDNF family, promotes survival of projection neurons that

mediate hypo-kinetic movement, making it a logical candidate for the treatment of HD, a

disease characterized by hyperkinetic movement (Alberch and others 2002). Cell-mediated

delivery of Ntn protects projection neurons but not interneurons in QA-lesioned rat striata,

which is the opposite effect seen with GDNF overexpression (Pérez-Navarro and others

2000). This indicates that although these neurotrophins share expression patterns and

receptor family members, they have differential mechanisms of action. As MSNs, the most

susceptible neurons in HD, are projection neurons, Ntn may be more suitable for HD gene

therapy than GDNF. Viral-mediated delivery of Ntn into rats treated with the mitochondrial

toxin 3-NP is neuroprotective and ameliorates motor deficits (Ramaswamy, McBride, and

others 2007). AAV delivery of Ntn to the striatum of six-week-old N171-82Q mice results

in increased stride length and delayed rotarod deficits and clasping compared to AAV-GFP-

or vehicle-treated transgenic mice. Treatment with AAV-Ntn dramatically increases striatal

neuron as well as layer 5 and 6 cortical neuron numbers. This therapy, however, has no

effect on neuronal cross-sectional area, inclusion deposition, or mouse survival

(Ramaswamy and others 2008). Interestingly, inclusion deposition and neuronal cross-

sectional area are measures that are improved by GDNF treatment, indicating that there may

be increased benefit of combining these very promising neurotrophic factor gene therapies.

Activation of Endogenous Neural Stem Cells

One approach to coping with neuronal loss is the activation of endogenous NSCs in the adult

brain. One site of NSCs throughout life is within the subventricular zone (Taupin and Gage

2002). Delivery of adenoviral-BDNF (AdB) into the subventricular zone results in increased

NSC proliferation and migration of newly formed cells into the striatum, although the

majority of these cells differentiate into glia. Coexpression of noggin, an inhibitor of

gliogenesis, pushes these newly generated cells toward a neuronal fate. Intraventricular

delivery of BDNF and noggin (AdB/N) into R6/2 mice increases neurogenesis, leading to

recruitment of new striatal MSNs, which project axons to their targets. AdB/N gene therapy

also results in improved rotarod performance and increased spontaneous activity as well as a

17% increase in survival time (Cho and others 2007). It is remarkable that the new neurons

“know where to go and what to do when they get there.” Because BDNF can stimulate the

generation of several other types of neurons, it seems that the local environment in the

damaged striatum contains the cues necessary for MSN differentiation.

Normalization of Calcium Signaling and Mitochondrial Function

Mitochondrial dysfunction in HD, which includes altered respiration, membrane potential,

and metabolism, leads to disruption of calcium homeostasis, excess production of reactive

oxygen species, and excitotoxicity (Damiano and others 2010). One of the ways that the

mHtt protein induces energy dysregulation is by repressing transcription of PGC-1

, a

regulator of mitochondrial metabolism. Lentiviral delivery of PGC-1

into the striatum of

R6/2 mice completely prevents loss of individual neuronal volumes, supporting

mitochondrial involvement in HD-related neurodegeneration (Cui and others 2006).

Mitochondria from HD patient–derived lymphocytes display reduced calcium-buffering

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capacity, leading to excess cytosolic calcium (Panov and others 2002). This causes

excitotoxicity and aberrant calcium signaling. Calmodulin (CaM), a regulatory protein that

associates with mHtt, activates a number of enzymes upon calcium binding. Among these is

transglutaminase, an enzyme that catalyzes covalent cross-linking of glutamine residues and

possibly contributes to mHtt-induced toxicity. Intrastriatal, AAV-mediated delivery of a

CaM fragment to R6/2 mice disrupts mHtt-CaM interaction, leading to a reduction in the

level of transglutaminase-modified mHtt. AAV-CaM gene therapy also improves rotarod

performance, gait, and hypoactivity, as well as deposition of inclusions and loss of neuronal

size and body weight, although mouse survival is unaffected (Dai and others 2009). It will

be of interest to determine the effects of this form of gene therapy in full-length models of

HD.

Targeting mHtt

Increased Clearance

Considering the multiplicity of pathogenic mechanisms of HD, a more comprehensive,

upstream therapeutic approach is to reduce mHtt protein levels by increasing its degradation.

Removal of mHtt is less likely to have deleterious off-target effects and more likely to

ameliorate all aspects of the HD phenotype than are therapies directed at downstream

dysfunctions. Interventions that selectively reduce levels of mHtt while sparing the wt

protein are preferable because long-term reduction of wtHtt may not be well tolerated. As

mentioned at the outset, wtHtt protein is important for neuronal health throughout life, and

mice with wtHtt conditionally knocked out in adulthood develop progressive motor

impairment and neuropathology (Dragatsis and others 2000). Small-molecule-mediated

increases in mHtt clearance have been unsuccessful thus far, ostensibly because of the

elaborate and somewhat mysterious mechanisms of endogenous mHtt degradation. On the

other hand, gene therapy approaches to selectively increase mHtt clearance have shown

considerable promise.

One such approach uses viral-mediated delivery of intrabodies (iAbs), which are

intracellularly expressed Ab fragments that recognize and bind Htt in living cells. Several

anti-Htt iAbs have been developed as potential therapeutics, and the most promising of these

reduce mHtt-induced toxicity by promoting its degradation. EM48 is an iAb that recognizes

an epitope in the C-terminus of HDx1. Although this epitope is common to mutant and

wtHtt, EM48 preferentially binds mHtt on Western blots, possibly because of polyQ

expansion–induced conformational changes. Observations in a cellular model of HD reveal

that EM48 binding increases the ubiquitination and subsequent degradation of mHtt.

Intrastriatal Ad delivery of this iAb into R6/2 and N171-82Q mouse models results in

increased Htt cleavage and reduced neuropil aggregates, demonstrating in vivo efficacy of

EM48-enhanced mHtt degradation. EM48-treated N171-82Q mice also display improved

gait and rotarod performance. However, EM48 gene therapy does not prevent loss of body

weight or enhance the survival of these animals (Wang C-E and others 2008).

Another iAb, Happ1, binds the proline-rich region of Htt adjacent to the polyQ tract. Despite

its recognition of wtHtt under denaturing conditions, Happ1 binding selectively increases

the turnover of mutant but not wtHtt in cellular models of HD (Southwell and others 2008).

This iAb-induced turnover involves enhanced calpain cleavage of mHtt (Southwell and

others 2011). The target epitope of Happ1 is the site of multiple endogenous protein-protein

interactions, which are enhanced by polyQ expansion, suggesting increased epitope

availability in the context of mHtt (Passani and others 2000; Qin and others 2004; Steffan

and others 2000). This is likely to be responsible for the selectivity of Happ1 action under

native conditions. Intrastriatal AAV delivery of Happ1 is beneficial in five different mouse

models of HD. In a unilateral, acute lentiviral-mHtt mouse model, Happ1 treatment prevents

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the amphetamine-induced rotation phenotype and reduces striatal lesion size and intensity as

well as mHtt aggregation. In genetic HD models, Happ1 treatment improves rotarod and

beam-crossing performance of R6/2, N171-82Q, YAC128, and BACHD mice and

spontaneous climbing in YAC128 and BACHD mice. Happ1 treatment also normalizes

open-field exploration in YAC128 and BACHD mice and object learning in YAC128 mice,

demonstrating benefit in the psychiatric and cognitive dimensions of the HD-like phenotype.

An object learning deficit in BACHD mice is unchanged by Happ1 treatment, however.

Happ1 treatment also improves neuropathological measures, including ventricular

enlargement in R6/2, YAC128, and BACHD mice and inclusion load in R6/2. Importantly,

Happ1 gene therapy increases body weight and increases life span by 30% in the N171-82Q

model (Fig. 1), although there is no benefit of Happ1 treatment in these outcomes in the

more severe and earlier onset R6/2 model (Southwell and others 2009).

The ability of anti-Htt iAbs to increase clearance of mHtt and subsequent therapeutic benefit

can be improved by fusion to targeting sequences, which direct the degradation of the bound

Htt-iAb complex. The iAb C4, which recognizes the N-terminus of HDx-1, reduces mHtt-

induced toxicity and aggregation in cell culture, brain slice, and fly models of HD (Lecerf

and others 2001; Murphy and Messer 2004; Wolfgang and others 2005), but AAV-mediated

C4 gene therapy in R6/1 HD model mice confers only modest phenotypic benefit (Snyder-

Keller, McLear and others 2010). A fusion protein of this iAb and a PEST signal sequence, a

peptide rich in proline, glutamic acid, serine, and threonine, efficiently targets mHtt for

proteasomal degradation. C4-PEST significantly reduces levels of both soluble and insoluble

mHtt in cotransfected cultured striatal cells (Snyder-Keller, Butler and others 2010). It will

be interesting to see how this modification affects C4 gene therapy efficacy in HD mice.

A different strategy for reducing mHtt levels involves induction of autophagy. Autophagy is

a lysosomal degradation process for long-lived and misfolded cytoplasmic proteins that can

selectively degrade mHtt (Sarkar and Rubinsztein 2008). Chemical induction of autophagy

results in accelerated clearance of mHtt and subsequent therapeutic benefit in fly and mouse

HD models (Ravikumar and others 2004; Rose and others 2010; Sarkar and others 2008).

Drug-mediated global induction of autophagy, however, is not specific to Htt and could

potentially have off-target effects, particularly over the extremely long treatment duration

required for HD therapy. The lack of understanding of the regulation of endogenous,

autophagy-mediated mHtt degradation hampers selective drug development, and therefore

there is interest in gene therapy approaches. Specific targeting of mHtt for autophagic

degradation has been demonstrated in the R6/2 mouse model by intrastriatal AAV-mediated

delivery of an expanded polyQ binding peptide (QBP1), which binds mutant but not wtHtt.

This peptide is fused to a synthetic HSC70 chaperone-binding motif (HSC70bm), which

targets bound proteins to lysosomes for degradation. Artificial, chaperone-mediated,

autophagic degradation of mHtt in these animals results in reduced levels of both aggregated

and soluble mHtt protein in brain lysates and inclusions in transduced striata. QBP1-

HSC70bm gene therapy improves clasping and rotarod performance and reduces striatal

atrophy. This promising therapy also improves body weight and dramatically increases the

survival of this very severe model (Bauer and others 2010). One would like to see this

approach tested in a full-length mHtt model.

Silencing

The most upstream and direct strategy for the treatment of HD is silencing expression of

mHtt mRNA, thus preventing generation of the toxic protein and all subsequent pathology.

At least 10 RNAi gene therapy efficacy preclinical trials have been performed since 2005,

using a variety of rodent models of HD. These studies, recently reviewed by Harper (2009),

are briefly summarized here.

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Intrastriatal AAV delivery of a short hairpin RNA (shRNA), HDsh2.1, in N171-82Q mice

results in reduced inclusions and improved rotarod performance and gait but no change in

loss of body weight (Harper and others 2005). Another shRNA, siHUNT-1, which targets

the 5

′

UTR of Htt, delivered by AAV into the striatum of R6/1 mice, reduces inclusions and

moderately improves clasping behavior and transcriptional dysregulation but not loss of

body weight (Rodriguez-Lebron and others 2005). Postsymptomatic onset, intrastriatal AAV

delivery of an shRNA in the HD190QG mouse model causes reduction of inclusion load and

a modest restoration of DARPP-32 expression (Machida and others 2006). In an AAV-

generated mouse model of HD, intrastriatal co-injection of mHtt plus cholesterol-conjugated

small interfering RNA (siRNA), cc-siRNA-Htt, reduces inclusions, improves clasping and

beam-crossing performance, and ameliorates neuronal loss but not neuronal shrinkage

(DiFiglia and others 2007). Adenoviral delivery of an shRNA targeting HDx-1 in the

striatum of R6/2 mice as well as an Ad-HDx-1 model of HD reduces transgene expression

and inclusions (Huang and others 2007). Co-delivery of an shRNA, AAV-shHD2, along

with AAV-HDx-1, ameliorates loss of NeuN- and calbindin-positive cells in the striatum of

rats and normalizes asymmetric forepaw use in unilaterally injected animals (Franich and

others 2008). The most positive results to date involve intraventricular delivery of a

cholesterol-conjugated siRNA targeting HDx-1 in neonatal R6/2 mice, resulting in reduced

mHDx-1 expression. Compared to control-treated mice, those receiving the HDx-1-siRNA

display reduced inclusions, delayed clasping and loss of body weight, improved rotarod

performance and spontaneous activity, reduced striatal atrophy, and an increase in longevity

of 14% (Wang Y-L and others 2005).

Taken together, the RNAi results appear very promising. An important caveat of these

studies is, however, that they have all employed species-specific RNAi reagents that only

target human Htt. As a result, endogenous mouse Htt is not silenced, although it would be in

comparable human HD applications. Some studies have found that nonspecific Htt silencing

is relatively nontoxic in mice, resulting in gene expression changes but no overt behavioral

phenotype (Boudreau and others 2009; Drouet and others 2009; McBride and others 2008).

Nonspecific silencing has in fact been shown to confer therapeutic benefit in a transgenic

mouse model of HD. AAV-mediated delivery of miRNA2.4 into the striatum of seven-

week-old N171-82Q mice results in a 75% reduction of both human transgene and mouse

endogenous Htt mRNAs. Compared to AAV-GFP- and vehicle-treated mice, miRNA-

treated mice display improved rotarod performance and a trend toward increased survival

but no change in loss of body weight (Boudreau and others 2009). This suggests that

nonspecific Htt silencing would be a viable therapeutic option for human HD. The

prolonged treatment duration required in humans and the general resistance to Htt-associated

neurodegeneration in the mouse brain should be considered, however. If sensitivity to Htt-

associated neuropathology were similar between mice and humans, heterozygous knock-in

HD mice with polyQ lengths equivalent to those seen in patients would model human HD

fairly closely. In actuality, these mice have no clear HD-related phenotype (White and others

1997), and to model human HD in mice, polyQ expansion lengths well above those seen in

humans are used, often in the context of a highly toxic N-terminal fragment. For these

reasons, allele-specific mHtt gene silencing that does not disrupt wtHtt expression will be

preferable for therapeutic applications.

As the HD-causing mutation is an expansion of a normally occurring sequence, development

of allele-specific silencing reagents has required ingenuity. Peptide and locked nucleic acid

oligonucleotides targeting expanded CAG have shown modest success in selective mHtt

silencing in cellular models of HD, although selectivity of these reagents declines in the

absence of a large repeat length difference between the normal and mutant allele (Hu and

others 2009). Nonetheless, the extremely focused therapeutic mechanism and the potential

benefit to not only those afflicted with HD but to those with other CAG expansion diseases

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warrants further development and optimization of expanded CAG-targeting

oligonucleotides. Allele-specific mHtt silencing could also be accomplished by targeting

single-nucleotide polymorphisms (SNPs). Many SNPs that are enriched in expanded polyQ

human Htt genes have been identified, and silencing reagents targeting a small number of

these disease-associated SNPs could provide a therapeutic option for the majority of HD

patients (Lombardi and others 2009; Pfister and others 2009; Warby and others 2009).

Development of silencing reagents targeting these SNPs is currently under way, and gene

therapy efficacy trials in rodent models of HD are eagerly expected in the near future.

Perspectives

It is apparent that a wide variety of gene therapy approaches display promise for prevention

as well as treatment of HD symptoms. Several of these approaches also have potential

application in other polyQ diseases, as well as in other neurodegenerative diseases that

involve protein misfolding, such as Parkinson and Alzheimer diseases and the prionoses.

What are the next steps toward human trials? In some cases, the gene therapies must be

tested in full-length HD models. In other cases, the reagents must be optimized for use in

humans. There is some debate as to the steps after that. One possibility is to test the reagents

in animals with larger brains so as to optimize delivery of the vectors (Ciron and others

2009; Fiandaca and others 2009; Varenika and others 2009). Another possibility is to test the

therapies for efficacy in nonhuman primates. This could be done using lentiviral delivery of

mHtt (Palfi and others 2007). There are also genetic HD models being developed using

monkeys, pigs, and sheep (Jacobsen and others 2010; Yang D and others 2010; Yang S-H

and others 2008). It will also be extremely important to develop inducible viral vectors so

that a transgene that turns out to be ineffective or harmful can be turned off.

Apart from the experimental hurdles that must be overcome, there is a psychological barrier

unique to gene therapy in the brain that needs to be confronted. Many researchers believe

that inserting genes into the brain is too dangerous and so only small-molecule approaches

should be developed. This view reflects a widespread lack of awareness of the many gene

therapy trials that are already ongoing or have been concluded in humans. These include

trials of potential treatments for Parkinson and Alzheimer diseases as well as others

(Christine and others 2009; Kaplitt and others 2007; Mandel 2010; Marks and others 2008).

In general, such trials have shown that the safety of the surgery and vector delivery is not a

cause for great concern, although new methods are still under development. There is also the

issue of side effects; direct delivery of vectors into the brain avoids peripheral side effects,

and gene therapies with high target specificity (as with iAbs, for instance) may avoid

potential side effects of delivering small-molecule reagents to the periphery.

A daunting reality is that most of the therapies developed in mouse models of cancer, for

instance, fail in human trials. For that reason alone, it is important that the wide variety of

gene therapy approaches summarized here proceed in parallel toward human testing.

Acknowledgments

Financial Disclosure/Funding

The author(s) disclosed receipt of the following financial support for the research and/or authorship of this article:

research from our laboratory that is cited here was funded by the National Institute of Neurological Disease and

Stroke and the Hereditary Disease Foundation.

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Figure 1.

Happ1 gene therapy improves body weight of N171-82Q Huntington disease (HD) model

mice. The green fluorescent protein (GFP)–treated N171-82Q mouse (

left

) displays reduced

body weight, hunched posture, and piloerection (ruffled coat), whereas the Happ1-treated

N171-82Q mouse (

right

) displays normal body weight and appearance. The mice shown are

male littermates.

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Southwell and Patterson

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Table 1

Summary of the Most Promising Gene Therapy Trials in Mice to Date

Study

Therapeutic

Reagent

Mechanism of

Action

Delivery

HD Model(s)

Motor

Performance

Cognitive/

Psychiatric

Performance

Neuropathology

Body

Weight

Survival

Ebert and

others 2010

GDNF

Trophic support

NPC transplant

N171-82Q

Inclusions, striatal neuron

count, no change cortical

thinning

Transient increase

Ramaswamy

and others

2008

Neurturin

Trophic support

IS-AAV

N171-82Q

RR, gait, clasping

Striatal and cortical

neuron count, no change

inclusions or neuronal

size

No change

Cho and

others 2007

BDNF and noggin

Activates

endogenous neural

stem cells

ICV-Ad

R6/2

RR, SA

New striatal MSNs

Transient decrease

17% increase

Dai and

others 2009

CaM fragment

Disrupts aberrant

mHtt-CaM

interaction

IS-AAV

R6/2

RR, gait, hypoactivity

Inclusions, neuronal size

Increase

No change

Southwell

and others

2009

Happ1 iAb

Selectively

increases mHtt

degradation

IS-AAV

Lentiviral

AIR

Inclusions

No deficit

R6/2

RR, BC

Inclusions, VE

No change

N171-82Q

RR, BC, clasping

Increase

30% increase

YAC128

RR, BC, climbing

OF, OL

No change

BACHD

RR, BC, climbing

OF, no

change OL

No change

Bauer and

others 2010

QBP1-HSC70bm

Targets mHtt for

chaperonemediated

autophagy

IS-AAV

R6/2

Clasping, RR

Inclusions, striatal atrophy

Increase

30% increase

Wang Y-L

and others

2005

Cholesterol conjugated siRNA

Nonselectively

silences Htt gene

expression

ICV

R6/2

Clasping, RR, SA

Inclusions, striatal atrophy

Transient increase

14% increase

AAV = adeno-associated virus; AIR = amphetamine-induced rotation; BC = beam crossing; BDNF = brain-derived neurotrophic factor; GDNF = glial cell line–derived neurotrophic factor; HD =

Huntington disease; ICV = intracerebroventricular; IS = intrastriatal; MSN = medium spiny neuron; ND = not determined; NPC = neural progenitor cell; OF = open-field exploration; OL = object learning;

RR = rotarod; SA = spontaneous activity; siRNA = small interfering RNA; VE = ventricular enlargement.

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