therapeutic potential of small interfering rna for brain …therapeutic potential of small...

Therapeutic Potential of Small InterferingRNA for Brain Diseases

Amy E. Lovett-Racke

Contents

1 RNA Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

2 Spinocerebellar Ataxia and Other Polyglutamine-Associated Ataxias . . . . . . . . . . . . . . . . . . . . 279

3 Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

4 Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

5 Amyotrophic Lateral Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284

6 Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

7 Brain Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291

Abstract RNA interference (RNAi) is an evolutionarily conserved mechanismfound in all eukaryotes to degrade messenger RNA (mRNA) that has sequence

homology to short double-stranded RNA (dsRNA). It is believed that the usual

biological function of RNAi is to safeguard the host cells from RNA viruses and

transposons, which can endanger the genome. Expression of dsRNA in the host cell

signals a cascade of intracellular events that generate small interfering RNA

(siRNA) segments that mediate the degradation of complementary mRNA and

subsequent failure to express protein. Recently, engineered siRNA has been used

to study the normal biological function and significance of a vast number of genes

in a variety of cell types, as well as the role of specific genes in disease patho-

genesis. Thus, siRNA has therapeutic potential to specifically suppress the expres-

sion of proteins associated with disease processes. The exquisite specificity of

siRNA-mediated gene suppression provides an opportunity to target genes with

single point mutations found in hereditary disorders, genes unique to pathogenic

tumor cells in malignancies, and genes that are critical for mediating the pathology

of many diseases. The fact that RNAi functions in the cell at the transcription level

A.E. Lovett‐RackeDepartment of Molecular Virology, Immunology and Medical Genetics, Ohio State University,

43065, Columbus, OH, USA

e-mail: [email protected]

V.A. Erdmann et al. (eds.), Therapeutic Ribonucleic Acids in Brain Tumors, 275DOI: 10.1007/978‐3‐642‐00475‐9_13, # Springer‐Verlag Berlin Heidelberg 2009

means that virtually any gene can be targeted for suppression, providing a unique

strategy to develop therapeutics for many neurological diseases, many of which

have no effective therapies.

1 RNA Interference

RNA interference (RNAi) was initially discovered in plants, when RNA for

pigment-producing genes was introduced into petunias to enhance color (Napoli

et al. 1990; Van der Krol et al. 1990). Surprisingly, these petunias expressed very

little color, and this paradoxical effect was termed cosuppression. A similar loss of

gene function was observed in fungi transformed with albino transgene that

silenced the endogenous albino gene and was referred to as quelling (Romano

and Macino 1992). Like many other genetic discoveries, the recognition that these

specific and potent suppressive effects were provoked by double-stranded RNA

(dsRNA) was made in Caenorhabditis elegans when dsRNA homologous to unc-22mRNA was injected into C. elegans, producing a twitching phenotype associatedwith unc-22 loss of function (Fire et al. 1998). The clear association of dsDNA with

suppression of a complementary gene was termed RNAi and led to Drs. Fire and

Mello receiving the Nobel Prize in Physiology or Medicine in 2006. The observa-

tion that 22–25 nucleotide RNA fragments in plants undergoing posttranscriptional

gene silencing led to the realization that small interfering RNA (siRNA) were

ultimately responsible for mediating the silencing effect of dsRNA observed in

these diverse organisms (Hamilton and Baulcombe 1999).

Since these original observations, RNAi, as a posttranscriptional gene-silencing

mechanism, has been well defined. It is thought that RNAi evolved as a means to

reduce the effect of potentially dangerous dsRNA, not usually expressed in eukar-

yotes, but commonly by viruses. Dicer, a cytoplasmic ribonuclease III-like enzyme,

recognizes dsRNA and cleaves it into 21–23 nucleotide segments that contain 2–3

nucleotide overhangs (Fig. 1). These 21–23 nucleotide fragments, termed siRNA,

bind to a multiprotein complex called the RNA-induced silencing complex (RISC).

The RISC causes linearization of the siRNA and separation of the strands. The

single-stranded RNA within the RISC, known as the guide strand, hybridizes to

complementary messenger RNA (mRNA). An enzyme within the RISC, argo-

naute 2, cleaves the mRNA into fragments so that translation of the transcript is

no longer possible. The guide strand within the RISC can repeatedly bind comple-

mentary mRNA, continuing the degradation of the target mRNA, allowing pro-

longed suppression of the target gene.

Scientists have since used the RNAi pathway to target self-genes as a mecha-

nism to understand and define the normal biological function of gene products. This

has been accomplished by delivering to cells plasmid or viral vectors containing

specific RNA sequences that are processed into short hairpin DNA that contain a

21-base dsRNA sequence that can be cleaved by Dicer into siRNA. Alternatively,

synthetic siRNA have been shown to readily transfect cells and were shown to be

276 A.E. Lovett-Racke

capable of directly activating the RISC, degrading the target mRNA. Similarly,

RNAi has been used to suppress genes known to be associated with diseases, as well

as discover genes critical to pathogenic processes. Because siRNA usually only

recognize mRNA that are completely homologous, siRNA have been developed

Long ds RNA

shRNA expressed from plasmid or viral vectors

Dicer

Dicer

siRNA

Synthetic siRNA

siRNA

5’

5’

5’3’

5’3’

5’

5’

RNA induced silencing complex (RISC)

Argonaute 2

5’AAAmRNA

5’

AAA

5’

NUCLEUS

Plasmid or viral vector containing

siRNAsequence

CYTOPLASM

RNA degradation

5’

mRNAcomplementary

to siRNA

Virus with dsRNAgenome

Plasmids can carry siRNAsequences

Viruses can be engineered to contain siRNA

sequences

5’

RISC with guide strand can continue to bind mRNA

Guide strand of siRNA

Synthetic siRNA

5’

5’

5’

5’AAA

5’

Fig. 1 Mechanism of RNA interference. Double-stranded RNA (dsRNA) is cleaved by theenzyme Dicer into 21–23 nucleotide fragments, termed small interfering RNA (siRNA). The

dsRNA can come from viruses with dsRNA genomes, or from plasmids or viral vectors carrying

sequences for shRNA. The siRNA enter a multiprotein complex called the RNA-induced silencing

complex (RISC) that linearizes and separates the siRNA. The guide strand binds complementary

mRNA and initiates degradation of the mRNA, preventing translation and protein production

Therapeutic Potential of Small Interfering RNA for Brain Diseases 277

that efficiently distinguish between wild-type DNA sequences and point mutations

in autosomal dominant genes. For neurological diseases, the question arose as to

whether axons had the machinery for RNAi. Developing axons and growth cones

have local mRNA that are translated in response to specific extracellular signaling

molecules which are critical for axon development and neurotransmitter produc-

tion. These same signaling pathways would be necessary in axonal repair following

injury. The molecular mechanisms that mediate the translation of mRNA in axons

and growth cones in not well defined and thus mechanisms that regulate mRNA in

cell bodies may not be the same. However, developing axons express argonaute and

Dicer, and can assemble functional RISC when distal axons are transfected with

siRNA (Hengst et al. 2006).

One of the most significant obstacles to RNAi in both research and its advance-

ment to therapeutics is delivery of the siRNA to the target cells, especially in the

central nervous system (CNS) (Begley 2004). In particular, cells in the CNS are

protected by the blood–brain barrier (BBB) which significantly limits access to

these tenuous cells. Nonspecific delivery systems, such as liposomes and cholester-

ol, have been beneficial in enhancing siRNA uptake and reducing degradation.

Viral delivery of shRNA that can be cleaved by Dicer into siRNA has been used to

deliver siRNA and can provide some cell specificity based on the type of virus and

cell surface molecules that mediate viral entry. In addition, viruses add the benefit

of prolonged expression of the siRNA which should result in extended suppression

of the target gene. Synthetic siRNA have been shown to be effective at suppressing

target genes when administered intravenously if the target is in highly vascularized

tissue, such as the liver and spleen (Lovett-Racke et al. 2004; Thakker et al. 2004).

This hydrodynamic approach is limited because siRNA delivered in large volumes

of saline intravenously, which enhances tissue perfusion, can cause heart damage

and may be inappropriate for humans. Chemical modifications of synthetic siRNA

that reduce digestion of siRNA may improve bioavailability. Substitutions with

locked nucleic acids, 20F, 20O-Me, and 20H residues can stabilize siRNA andenhanced benefits have been observed in vivo in mouse models using these chemi-

cal modifications (Elman et al. 2005; Morrissey et al. 2005). Conjugation of siRNA

to carriers may also enhance cell uptake and specificity of delivery. Peptides,

aptamers or monoclonal antibodies that recognize specific cell surface receptors

can be conjugated to siRNA, allowing entry into cells with the required receptor

(Muratovska and Ecles 2004; Juliano 2005; Chu et al. 2006; Kumar et al. 2008).

Although intracerebral and intrathecal administration of siRNA has been used in

experimental models of CNS disease and is possible in humans, it is not the optimal

strategy in humans given the potential risks. Systemic administration of siRNA

does not efficiently cross the BBB, providing a serious obstacle to the development

of siRNA-based therapies for human neurological disease. However, strategies to

facilitate transport across the BBB are being developed. Trojan horse liposomes

have been developed that may overcome many of the obstacles with siRNA

delivery to the CNS (Boado 2007). Nucleic acids are encapsulated in a liposome

that protects the nucleic acid from nuclease-mediated degradation (Fig. 2).

The liposome is constructed of polyethyleneglycol that stabilizes the liposomes.


Monoclonal antibodies specific for receptors on the BBB are engineered into the

polyethyleneglycol which allow transcytosis across the BBB by the liposomes. The

Trojan horse liposomes can contain siRNA or plasmids that generate shRNA. In the

case of plasmids, they can be engineered such that the promoter is specific for a

particular cell type so that the shRNA is only expressed in the appropriate CNS cell

type. Alternatively, the liposomes can contain two types of monoclonal antibodies,

one that recognizes a BBB to allow transport across the BBB and a second

monoclonal antibody that binds to a cell type-specific receptor to promote uptake

of the nucleic acid by the appropriate cell. This strategy would be feasible with both

plasmids and siRNA. Although delivery has been a major challenge in all forms of

gene therapy, technology is advancing to the point that this will soon be overcome.

2 Spinocerebellar Ataxia and Other Polyglutamine-AssociatedAtaxias

Polyglutamine-associated ataxias are a group of autosomal dominant neurodegen-

erative disorders. As such, one allele of the affected gene contains a mutation that

results in expanded CAG repeats, while the other allele is normal. The mutant

proteins cause intranuclear inclusions that are toxic to neurons. Since RNAi targets

Blood Brain Barrier CNS Target CellsTrojan Horse Liposome

MAb to BBB

MAb to Cell

siRNAplasmid

Fig. 2 Delivering siRNA to cells in the CNS. Trojan horse liposomes are composed of poly-ethyleneglycol bound to monoclonal antibodies that recognize proteins unique to the blood-brain

barrier to allow transport into the CNS and monoclonal antibodies to a cell type-specific receptor

to allow transport of the siRNA into the target cell


and degrades complementary mRNA sequences, it was theoretically possible that

siRNA containing the mutation could be used to specifically target the mutant

mRNA, allowing the nonmutant mRNA to translate into normal protein. The

potential of siRNA as a therapeutic option for CNS diseases was first observed in

a mouse model for spinocerebellar ataxia type 1 (SCA1), which is a dominantly

inherited, progressive neurodegenerative disease caused by an expanded polyglu-

tamine (CAG) sequence in the ataxin 1 protein. Initially, plasmids containing short

hairpin RNA (shRNA) sequences within the human ataxin 1 complementary DNA

sequence to target the ataxin 1 protein were tested in neuronal cell lines (Xia et al.

2004). Two sequences that flanked the CAG repeat region coding for the poly-

glutamine sequence suppressed ataxin 1 levels. These shRNA sequences were

subsequently inserted into recombinant adeno-associated virus (AAV) vectors to

determine if ataxin 1-specific siRNA had a biological effect in the transgenic mouse

model for SCA1. Intracerebellar injection of the AAV-siRNA resulted in enhanced

motor function, improved neuropathologic findings, and total loss of ataxin 1

inclusions in transfected Purkinje cells, demonstrating a clear therapeutic benefit.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease,

is also an autosomal dominant neurodegenerative disease that results from an

expanded polyglutamine repeat within the ataxin 3 gene. Initial attempts to sup-

press ataxin 3 by RNAi targeting various sequences within the ataxin 3 gene

resulted in efficient, but not allele-specific, suppression of the mutant and normal

alleles (Miller et al. 2003). To enhance the probability that the mutant allele would

be preferentially targeted, siRNA were developed that were specific for a single

nucleotide polymorphism in the ataxin 3 gene which is in linkage disequilibrium

with the expanded CAG repeat, and thus typically segregates with the disease allele.

Development of synthetic siRNA that had the single mutation failed to differentiate

between the mutant and normal alleles. However, siRNA that added a mutation

within the siRNA sequence, so that it contained two mismatched bases with the

normal sequence and a single mismatch to the mutant allele, demonstrated signifi-

cant suppression of the mutant allele while maintaining expression of the normal

allele. Subsequently, ataxin3-specific siRNA was developed that placed the mutant

base in the center of the siRNA sequence, which demonstrated allele-specific

suppression. Recent studies have developed strategies for designing siRNA that

specifically distinguish between alleles that differ by a single nucleotide (Schwarz

et al. 2006). The ataxin3 siRNA sequences with single base changes were subse-

quently inserted in plasmid and AAV vectors as shRNA and demonstrated similar

inhibition of the mutant ataxin3 in vitro, showing that efficacy is maintained when

the siRNA is produced by different modes.

Huntington disease is also a dominantly inherited neurodegenerative disease

which causes dementia and motor deficits, ultimately resulting in death. Expansive

CAG repeats, typically greater than 35, within the huntingtin gene create a protein

that is prone to aggregation and toxic to neurons, predominately in the cerebral

cortex and striatum. Although the huntingtin gene is expressed in all cells, its

function remains unknown. Mice deficient in the huntingtin gene fail to produce

viable offsprings, indicating that the huntingtin gene product is essential for normal


development (Duyao et al. 1995; Nasir et al. 1995; Zeitlin et al. 1995). Initially,

a novel strategy was used to target the huntingtin gene by inserting shRNA into a

transposon system, which inserts efficiently into almost any DNA sequence, pro-

moting continuous suppression of the target gene (Chen et al. 2005). Using this

system, expression of the normal huntingtin gene was suppressed in various cell

lines. To repress the mutant huntingtin gene in vivo, AAV was used to deliver of

mutant-specific huntingtin shRNA to the brain in mouse models of Huntington

Disease. This resulted in improved motor function, and decreased mutant hunt-

ingtin gene expression (Harper et al. 2005; Machida et al. 2006). Since shRNA can

integrate into the genome randomly, off-target effects can be observed and were

noted in a model of Huntington Disease (Denovan-Wright et al. 2008). Therefore,

synthetic siRNA may provide a safer alternative for therapeutic use of RNAi to

suppress specific genes. To enhance cell uptake in vivo of synthetic siRNA,

cholesterol conjugated-synthetic siRNA specific for the mutant huntingtin gene

were developed. Mice were injected with cholesterol conjugated-siRNA in the

striata following administration of AAV vectors containing huntingtin cDNA

encoding amino acids 1–400 with expanded (100 CAG) or typical (18 CAG)

number of CAG repeats (DiFiglia et al. 2007). Mice receiving the mutant huntingtin

cDNA with expanded CAG repeats developed motor deficits and neuropathology

similar to Huntington Disease, while the mice that received the huntingtin cDNA

with the wild-type number of CAG repeats remained healthy. Mice that received

both the expanded CAG huntingtin cDNA and the mutant-specific siRNA had

prolonged survival of striatal neurons, fewer neutrophil aggregates, smaller inclu-

sion size, and improved motor function, indicating that siRNA had similar

efficacy as the shRNA. Currently, therapeutic intervention in Huntington Disease

is ineffective. Since Huntington Disease is a dominant familial disorder, families

live with the burden that many will be stricken with this devastating disease.

Genetic therapies, such as siRNA, provide the best hope of preventing the expres-

sion of the mutant gene and suppressing the disease phenotype.

3 Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by demen-

tia that affects up to 30 million people worldwide and accounts for the majority of

cognitive impairment in elderly patients inWestern Europe and North America. AD

is characterized by two major hallmarks, extracellular senile plaques containing

b-amyloid fragments cleaved from the amyloid precursor protein (APP) and intra-cellular neurofibrillary tangles composed of hyperphosphorylated tau protein. The

most critical element for the use of siRNA as a therapeutic agent in the treatment of

diseases is the identification of relevant and specific target genes. The molecular

characterization of plaques and neurofibrillary tangles, and the discovery of genes

involved in the pathogenesis of both familial and sporadic AD, have identified

several potential targets for RNAi. APP was the first gene directly associated with


AD (Mullan et al. 1992). Autosomal dominant mutations in APP result in an early-

onset, aggressive form of the disease (Goate et al. 1991). Approximately 20 APP

missense mutations near the cleavage site of secretases have been found, suggesting

that altered cleavage of APP may negatively affect b-amyloid deposition (Fig. 3).Given the relative large number of APP mutations, identification of APP mutations

associated with pathology is a critical first step before targeting a specific mutant

allele with siRNA. Plasmids expressing shRNA, containing siRNA sequences,

specific for the Swedish APP allele were developed, since this is the most common

mutation in AD (Miller et al. 2004). The Swedish APP allele contains two adjacent

point mutations which were targeted with siRNA that altered the position of the

mutations within the siRNA sequence. The mutant APP allele could be suppressed

to varying degrees in vitro based on the location of the mutations within the siRNA

sequence, while maintaining the normal APP expression. Using a novel lentiviral-

based mouse model for b-amyloid overexpression, herpes simplex viral vectorsexpressing a shRNA specific for the Swedish APP allele were found to significantly

g-secretase

a-secretase

g-secretase

b-secretase

AmyloidPrecursor

Protein

membrane

a-APP

P3

P7

b-APP

Ab39-43

P7

NormalAPP

Processing

PathogenicAPP

Processing

APPCleavageProducts

APP PointMutations

APHnic PEN2

PS1 PS2

APHnic PEN2

PS1 PS2

Insoluble Ab peptides

Fig. 3 Multiple proteins in the processing amyloid precursor protein (APP) may be targets forsiRNA-based therapy in Alzheimer Disease. APP is normally processed into a-APP, P3, and P7 bya-secretase and g-secretase. However, mutations in APP and g-secretase are associated withabnomal APP cleavage, generating b-APP, Ab39–43 peptides and P7. Aggregation of Ab39–43peptides results in neurodegeneration


reduce b-amyloid accumulation, suggesting that in vivo suppression of mutant APPis achievable via RNAi (Hong et al. 2006).

AD, as well as the inherited frontotemporal dementia with Parkinsonism linked to

chromosome 17, progressive supranuclear palsy, and corticobasal ganglionic degen-

eration (Houlden et al. 2001), are collectively called tauopathies due to mutations in

the tau gene that cause an altered sequence or aberrant splicing (Lewis et al. 2001;

Oddo et al. 2003). A mutant allele of the tau protein was successfully targeted in vitro

using shRNA plasmids, demonstrating that a single nucleotide mutation could be

used to suppress mutant tau by siRNA (Miller et al. 2004). As observed in the APP

siRNA studies, the position of the mutation within the siRNA sequence was critical

for selective inhibition of the mutant allele (Miller et al. 2003, 2004).

Additional targets for AD include the apolipoprotein E e4 allele (ApoE4), thesecretases, and the presenilin genes. In addition to increasing age, ApoE4 is

considered the most significant risk factor for familial and nonfamilial AD (Corder

et al. 1993). Homozygosity for ApoE4 was shown to be sufficient to cause AD in

most individuals over age 80. Suppression of ApoE4 with siRNA could potentially

reduce the probability of developing AD or delay its onset, at least in heterozygotes.

Because ApoE4 plays a critical role in cholesterol and triglyceride transport, it is

necessary for normal metabolism, and, thus, the risk-benefit ratio of gene targeting

in ApoE4 homozygotes is less clear.

More than 150 mutations have been identified in the presenilin (PS) genes, which

result in an early-onset aggressive form of AD. Presenilins (PS1/PS2) are the

catalytic subunit of g-secretase, which also includes nicastrin, APH-1a, and PEN2.Mutations in PS1/PS2 enhance production of the highly self-aggregating Ab42peptide. AAP cleavage occurs via two pathways. Typically, APP is cleaved by

a- and g-secretases into nontoxic, soluble a-APP protein and two smaller proteinsP3 and P7 (Nawrot 2004). In healthy individuals, these proteins protect neuronal

cells from oxidative stress and play a role in wound repair. In AD patients, APP is

cleaved at the N-terminus of the Ab region by b-secretase and at the C-terminus byg-secretase, generating b-APP protein, P7 and 39–43 amino acid long b-amyloidpeptides (Fig. 3). PS1 control and missense sequences were cloned into pSilencer

plasmids that generate siRNA and then transfected into PS1/APP-expressing cell

lines, resulting in reduced PS1 and Ab42 peptide (Luo et al. 2004). Given the largenumber of mutations found in the PS genes, therapeutic siRNA would need to be

designed for individual patients or AD families based on the specific pathogenic PS

mutation in that patient or family. Since b-secretase-deficient mice have no knowndeficits, this particular enzyme may also be a good therapeutic target (Luo et al.

2001). Suppression of b-secretase, also known as beta-site APP-cleaving protein(BACE1), with siRNA inhibited Ab production, as well as the neurotoxicityassociated with oxidative stress, in neuronal cultures from normal and Swedish

APP mutant mice (Kao et al. 2004). Using lentivirus vectors expressing siRNA that

target BACE1, amyloid production, behavioral deficits and neurodegeneration were

significantly reduced in an APP transgenic model of AD, indicating that reducing

BACE1 reduced APP cleavage and the associated pathology (Singer et al. 2005).

The relatively large number of targets for therapeutic intervention in AD and our


enhanced understanding of designing mutant or allele-specific siRNA make AD a

good candidate for developing therapeutic siRNA.

4 Parkinson’s Disease

Parkinson’s disease (PD) is a prevalent neurodegenerative disease, second only to

AD. Both familial and idiopathic forms of PD exist with characteristic symptoms

including resting tremor, rigidity, bradykinesia, and a unique freezing phenomenon

that typically appears on one side of the body. The symptoms result from a loss of the

dopaminergic neurons of the substantia nigra pars compacta and progressive exhaus-

tion of striatal dopamine. Pathologically, intracytoplasmic inclusions, called Lewy

Bodies, are present in the neurons. Since the pathology appears to be limited to

dopaminergic neurons in the substantia nigra, finding proteins unique to these

neurons is critical to identifying therapeutic targets for siRNA intervention. Current

pharmacotherapy is replacement of levodopa, but side-effects such as dyskinesias can

offset therapeutic benefit over time. Thus, the need for an effective therapy is great.

The first gene linked to PD was PARK1, later identified as a-synuclein whichcontained single base mutations associated with familial disease (Polymeropoulos

et al. 1997). Duplicate and triplicate gene copies of a-synuclein have also beenidentified in some families with PD, indicating that excess native a-synuclein isneurotoxic (Singleton et al. 2003). Idiopathic PD has been associated with genetic

variability in the promoter of the a-synuclein gene (Pals et al. 2004). Lewy bodies arecomposed primarily of a-synuclein which appears to be misfolded and aggregateddue to the mutation or overexpression. RNAi of normal a-synuclein in dopaminergicneuroblastoma cells protected the cells from N-methyl-4-phenylpyridinium toxicity

and reduced dopamine transport, illustrating a role for a-synuclein dopamine homeo-stasis (Fountaine and Wade-Martins 2007). An allele-specific siRNA was developed

for the A53T mutation in human a-synuclein that specifically suppressed the targetgene in vitro (Sapru et al. 2006). Using a lentivirus expression system, shRNA

specific for a-synuclein suppressed human a-synuclein in vivo in rat brains thatwere transgenic for human a-synuclein. Unfortunately, there are currently no goodanimal models of PD to evaluate the therapeutic potential of the a-synuclein siRNAthat specifically targets mutant alleles. Other proteins implicated in PD, such as

PTEN, sirtuin 2, and LRRK-2, have been inhibited with siRNA in vitro, and

preliminary data suggest that they may be potential targets for siRNA therapy in

PD (Outeiro et al. 2007; Zhu et al. 2007; Habig et al. 2008).

5 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive, usually fatal, neurodegenera-

tive disease caused by motor neuron deterioration due to oxidative stress. Only

5–10% of ALS is associated with a specific genetic or familial factor. The most


common genetic defect in ALS is a point mutation in copper/zinc superoxide

dismutase (SOD1), an enzyme responsible for scavenging free radicals. Since

SOD1 performs critical functions in cells, silencing of the mutant SOD1 must be

specific, so that the normal allele is unaffected. This was accomplished in 2004

when SOD1 mutant-specific siRNA were introduced into neuroblastoma cell lines

that expressed mutant SOD1 (Maxwell et al. 2004; Yokota et al. 2004). Mutant-

specific siRNA for SOD1 protected the neuroblastoma cells from cyclosporin

A-induced cell death. In addition, siRNA was found to be more efficient at

suppressing mutant SOD1 than ribozyme or DNA enzyme.

Since more than 100 SOD1 mutations have been found, siRNA would need to be

developed for each specific mutation found in an individual ALS patient or ALS

family. However, this may not be possible because not all nucleic acid sequences

are able to elicit a silencing effect, probably due to the ability of a sequence to bind

the RISC or the ability of enzymes within the RISC to linearize and separate the

siRNA strands. Therefore, a siRNA-based strategy was developed that would

target all SOD1 sequence and replace the wild-type SOD1 (Xia et al. 2005). Two

constructs were developed that contained shRNA sequences specific for both

mutant and wild-type SOD1. However, these constructs also contained an RNAi-

resistant SOD1 transgene that were generated by inserting silent point mutations in

the wild-type SOD1 sequence that were not recognized by the shRNA. Transfection

of these constructs into HEK293 cells demonstrated that endogenous and

mutant SOD1 expression were suppressed, but that the transfected cells were not

susceptible to hydrogen peroxide toxicity because the shRNA-resistant SOD1 gene

was able to reverse the affects of the hydrogen peroxide. This study demonstrated

the feasibility of using siRNA to target all mutations within the SOD1 gene by

targeting all endogenous SOD1 and replacing the SOD1 gene with a wild-type

functioning gene that is resistant to RNAi. Extending this strategy to animal models

of ALS will determine if this will be feasible in patients.

Allele-specific siRNA for SOD1 have been evaluated in mouse models of ALS.

Intramuscular and intraspinal injection of lentivirus expressing mutant human

SOD1 substantially retarded disease onset and progression in mice that overexpress

a mutant form of SOD1 (Raoul et al. 2005; Ralph et al. 2005; Xia et al. 2006).

Motor neuron survival in the brainstem and spinal cord was significantly improved

and nearly 80% of mice had a normal life span, demonstrating that siRNA can be

used in vivo to specifically suppress the mutant human allele. Intramuscular

injection with AAV constructs carrying SOD1 siRNA and a green fluorescent

protein gene demonstrated that retrograde transport of the AAV occurs, allowing

expression of the siRNA in the spinal cord (Miller et al. 2005). To determine if

siRNA can be used to actually prevent dominantly inherited diseases such as ALS,

transgenic mice that express a shRNA specific for mutant SOD1 that was ubiqui-

tously expressed throughout the brain were developed (Saito et al. 2005). These

mice were crossed to the human mutant SOD1 transgenic mouse that spontaneously

develops an ALS-like disease. However, the double transgenic mice failed to

develop disease when monitored for 300 days, twice the time it takes until the

mice transgenic for mutant SOD1 to die. In addition, these mice had no CNS


pathology suggesting that the transgenic siRNA had completely prevented the

expression of mutant SOD1.

Another issue that must be addressed with diseases such as ALS is whether

siRNA have therapeutic benefit after the onset of symptoms. To generate stable

siRNA that could potentially be used in ALS patients, allele-specific mutant SOD1

synthetic siRNA were developed with chemical modification to enhance stability

and cellular uptake (Wang et al. 2008). Stability of the siRNA was enhanced

by 20fluoro modifications at the 20-hydroxyl position which should maintain theA-helical structure of the siRNA required for initiating the RNAi cascade. Phos-

porothioate modifications were also made to enhance uptake of the siRNA that was

then infused into the spinal cord using an osmotic pump. Long-term infusion

resulted in widespread distribution of the siRNA, decreased levels of mutant

SOD1, slowed disease progression, and increased survival. This approach to patient

treatment may initially be the safest option since infusion dose and rate of synthetic

siRNA can be closely monitored and stopped if adverse effects are observed.

Since mutations in the SOD1 gene only account for 2–3% of ALS cases, siRNA

targets that are common to all forms of ALS may be more widely applicable. Motor

neuron death in ALS is hypothesized to be mediated by Fas–Fas ligand apoptosis.

To determine if motor neurons might be preserved if this apoptotic pathway is

suppressed, Fas-specific siRNA was infused into the CNS of mutant SOD1 trans-

genic mice (Locatelli et al. 2007). The number of Fas positive cells in the spinal

cord decreased by nearly 50% and other molecules in the Fas pathway were

similarly reduced. Motor deficits were delayed and survival prolonged in the treated

mice, but not to the extent observed with mutant SOD1-specific siRNA. Suppres-

sing genes such as Fas which are ubiquitously expressed also poses the problem that

unforeseen side-effects are more likely than when targeting mutant alleles or genes

unique to the target cell population. Expression of Fas in the CNS is believed to play

a critical role in protecting neurons and resident CNS cells from potentially

damaging affects of inflammatory cells that express FasL, resulting in apoptosis

of the inflammatory cells. Thus, silencing Fas in the CNS may be associated with

increased inflammation which could be problematic.

6 Multiple Sclerosis

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of unknown

etiology. The neurologic deficits appear to be mediated by demyelination and

axonal transaction mediated by inflammatory cells. Thus, targeting components

of the immune and nervous system may be necessary for optimal therapeutic

intervention in MS. Diseases such as MS may be more easily modulated with

siRNA than other CNS diseases because systemic administration of siRNA can

readily target immune cells and potentially the site of active lesions due to BBB

breakdown. Several inflammatory mediators appear to play a critical etiological

role in MS. Specifically, it was demonstrated that systemic administration of


interferon gamma causes disease exacerbations (Panitch et al. 1987). Also, immu-

nomodulation with agents that promote an anti-inflammatory cytokine profile are

approved and currently being used for MS therapy (Hartung et al. 2004). In

experimental autoimmune encephalomyelitis (EAE), a murine model of MS, the

disease is mediated by myelin-specific CD4þ T cells that produce IFN-g, andsuppression of IFN-g in myelin-specific CD4þ T cells reduces the ability of thesecells to transfer disease to naı̈ve recipient mice (Olsson 1992; Racke et al. 1994,

1995). Surprisingly, mice genetically deficient in IFN-g develop severe EAE,indicating that systemic loss of IFN-g results in immune dysregulation that mayactually enhance EAE (Ferber et al. 1996; Willenborg et al. 1996). Since IFN-g isexpressed by different types of immune cells that may be critical for normal

immune regulation, siRNA specific for a transcription factor, T-bet, was developed

to specifically inhibit IFN-g in CD4þ T cells since it appears that T-bet specificallyregulates IFN-g gene transcription in CD4þ T cells. Transfection of myelin-specificT cells in vitro with T-bet-specific siRNA prevented these cells from inducing EAE

following transfer of these siRNA-transfected cells into naive mice (Lovett-Racke

et al. 2004). More important, systemic administration of T-bet-specific siRNA at

the time of EAE induction prevented the onset of disease, indicating that inhibition

of a transcription factor critical to the development of the pathogenic T cells was

sufficient to prevent disease. Since the cause of MS is unknown, potential therapies

must be able to treat established disease. To address this issue, the T-bet-specific

siRNA was administered to mice with established EAE and significantly amelio-

rated disease, suggesting that T-bet was a viable target in ongoing disease patho-

genisis (Gocke et al. 2007). More recently, it has been postulated that another

population of CD4þ T cells that express IL-17 may be pathogenic in MS and EAE(Langrish et al. 2005). Interestingly, these IL-17-producing T cells have been

shown to be suppressed with administration of T-bet siRNA in EAE, suggesting

that both known populations of potentially pathogenic T cells may be targeted by

the same siRNA (Gocke et al. 2007). Another transcription factor, NR4A2 (an

orphan nuclear receptor), was found by microarray to be upregulated in peripheral

blood T cells fromMS patients (Doi et al. 2008). Further analysis demonstrated that

IFNg and IL-17 expression was enhanced by NR4A2. Suppression of NR4A2 withsiRNA in vitro suppressed IFNg and IL-17 production. Similar to T-bet siRNA,NR4A2 siRNA reduced the ability of encephalitogenic T cells to transfer EAE into

recipient mice, suggesting that NR4A2 is critical for the pathogenic features of

myelin-specific T cells (Doi et al. 2008). Other immune cell markers that are readily

targeted in the periphery, such as IL-23 (Cua et al. 2003) and osteopontin (Chabas

et al. 2001), which appear to play an essential role in the function of pathogenic T

cells in immune-mediated demyelinating disease and whose deficiency does not

appear to compromise the immune system, may also be potential targets for siRNA

therapy in MS.

Although MS typically begins as a relapsing-remitting disease, most patients

ultimately develop permanent functional deficits and advance to secondary pro-

gressive MS. Permanent neurologic deficits in MS patients appear to result from

axonal damage mediated by repeated inflammation, and currently no therapeutic


agents promote axonal repair. Nogo-A is a potent inhibitor of neurite outgrowth and

probably plays a critical role in negatively regulating regeneration and plasticity in

the adult nervous system (Buffo et al. 2000; Prinjha et al. 2000). Studies inhibiting

Nogo-A or its receptor with monoclonal antibodies or peptide inhibitors in experi-

mental models of acute spinal cord injury have shown enhanced functional recov-

ery, apparently mediated by axonal sprouting (GrandPre et al. 2000, 2002). The

Nogo receptor associates with a transmembrane receptor, p75NTR, which initiate

inhibitory signals that cause growth cone collapse and cessation of axonal growth.

Suppression of p75NTR with siRNA in rat retinal ganglion cells decreased Rho-A

activation and increase neurite sprouting, supporting the hypothesis that siRNA can

be used to target inhibitory proteins to promote axonal repair (Ahmed et al. 2006).

Suppression of axonal growth inhibitors, such as Nogo-A or the Nogo receptor,

with siRNA may be beneficial in the promoting axonal repair in MS. Since there is

episodic BBB breakdown in EAE and MS, systemic administration of siRNA may

be sufficient to target the areas of the lesions, while areas of the CNS that are not

affected by lesions, and potentially adversely affected by suppressing these pro-

teins, would not be exposed to the siRNA. The glial reaction to injury results in the

production of many other inhibitory and potentially toxic molecules that damage

axons and prevent axonal repair which are also potential therapeutic targets for MS

siRNA therapies (Fawcett and Asher 1999). Other CNS proteins that contribute to

demyelination and wallerian degeneration may also be targets for RNAi in MS and

other CNS diseases with similar mediators of pathology and repair.

7 Brain Tumors

Primary brain tumors remain one of the most difficult tumors to treat. Glioblastoma

multiforme (GBM) is the most malignant form of human astrocytomas, accounting

for almost 22% of all brain tumors. The worldwide incidence of brain tumors is

approximately 3 per 100,000. Five-year survival for GBM is less than 3%, empha-

sizing the critical need for better therapies for CNS malignancies. In addition,

peripheral malignancies that metastasize to the CNS are often as difficult to treat

as primary brain tumors. Although new therapies such as Herceptin, which inhibits

growth of Her-2 positive cancer cells, has been shown to be very effective in breast

cancer, this agent is not effective against breast cancer cells that have metastasized

to the CNS (Bendell et al. 2003). Treatment of brain malignancies is hindered by

the inability of most therapeutic agents to cross the BBB, as well as poor respon-

siveness of brain tumors to current therapeutic agents. Molecular characterization

of brain tumors has defined several molecules that appear to be essential for tumor

survival, proliferation, metastasis and angiogenesis. Thus, siRNA technology tar-

geted against specific molecules critical for tumors may be more effective therapies

for brain tumors.

One molecule that appears to be a potential target for the treatment of many

cancers is epidermal growth factor receptor (EGFR). Proliferation of 90% of GBM


is driven by EFGR (Kaun et al. 2001; Kleihues and Ohgaki 1999), indicating that

minimizing EFGR-mediated signaling may be an effective therapeutic option for

GBM. In addition, EGFR plays an oncogenic role in nearly 70% of peripheral solid

tumors that metastasize to the CNS (Nicholson et al. 2001). In tumors, the EGFR

gene is frequently amplified which enhances the proliferative capacity of the tumor

cells, or the EGFR gene contains mutations that alter signaling. The most common

EGFRmutation is an in-frame deletion of exons 2–7 which generates the EGFR vIII

protein that is not very responsive to its natural ligands, but signals continuously

promoting the proliferation of cancer cells (Kaun et al. 2001). In an attempt to target

the EGFR vIII protein which is observed in 40% of GBM, siRNA were developed

that targeted the exon1–exon 8 junction sequence of mutant EGFR (Fan and Weiss

2005). In human glioblastoma cell lines, this siRNA reduced phosphorylated Akt,

enhanced apoptosis, and caused partial arrest of the cell cycle at the G2M phase. To

determine if amplified wild-type EGFR could be targeted in glioma cells, plasmids

that expressed shRNA targeting native EGFR were transfected into human glio-

blastoma cell lines (Kang et al. 2005; Saydam et al. 2005). The transfected cells had

a diminished capacity to proliferate and enhanced apoptosis. Transwell studies

demonstrated that invasiveness was also inhibited. More importantly, suppression

of EGFR with shRNA inhibited the growth of human glioblastoma cells in athymic

mice, indicating that targeting EGFR reduced the capacity of malignant brain

tumors to thrive in vivo.

Since the ultimate goal in the treatment of brain tumors is to develop therapeu-

tics that are specific and effective, combining targeted delivery with RNAi may

provide optimal benefit in treating CNS tumors. Plasmids expressing shRNA

specific for wild-type EGFR were encapsulated in monoclonal antibody-coated

pegylated liposomes, often referred to as Trojan horse liposomes (Zhang et al.

2004). One monoclonal antibody recognizes the mouse transferrin receptor that can

mediate transport of the liposomes across the BBB, and another monoclonal

antibody recognized the human insulin receptor that is expressed on human glio-

blastoma cells (Fig. 2). Receptor-mediated transport would allow the liposomes to

cross the BBB and specifically deliver the plasmids containing EGFR shRNA to the

glioma cells. Human glioblastoma cells were injected into the brain of scid mice

and the antibody-coated liposomes carrying the plasmids carrying shRNA specific

for EGFR were infused intravenously once per week. EGFR expression on tumor

cells was significantly decreased and survival increased almost 90% in this in vivo

model of human glioblastoma. This study demonstrated that two of the greatest

hurdles in brain tumor therapies, BBB and efficacy of the therapeutic agent, can be

overcome with targeted delivery of siRNA.

Growth factors, such as vascular endothelial growth factor (VEGF) and pleio-

trophin (PTN), have also been shown to play a role in glioblastoma pathogenesis,

and siRNA has been used to target these growth factors with some success

(Grzelinski et al. 2006; Niola et al. 2006). Growth factor receptors, such as

EGFR, VEGFR, platelet-derived growth factor receptor (PDGFR), and insulin

growth factor receptor (IGFR), are all frequently overexpressed in malignant

gliomas and share a common signaling pathway. Therefore, signaling molecules


in this pathway, such as PI3 kinase, may be more effective targets for siRNA

therapy since all of them can be suppressed simultaneously. The catalytic subunit

of PI3 kinase, p110 beta, was target with plasmid-expressed siRNA in human

glioma cell lines, resulting in decreased proliferation, arrest of cell cycle, decreased

invasiveness, and increased apoptosis (Pu et al. 2006). Growth of human glioma

cells in nude mice was also significantly impaired, illustrating the potential of PI3

kinase as a therapeutic siRNA target.

Identification of molecules critical in tumor survival, proliferation, and invasive-

ness remains a critical issue in developing siRNA-based therapeutics given the vast

heterogeneity of brain tumors. Numerous genes have been shown to be critical in

different types of human brain cancers and attempts to target them with siRNA have

shown promise. Downregulation of Notch-1 and its ligands, which are overex-

pressed in primary human gliomas, inhibit proliferation, and induce apoptosis

(Purow et al. 2005). In addition, transfection of glioma cells with siRNA

specific for Notch-1 prior to injection into mice enhances survival. However,

Notch signaling is critical in developmental cell fate and adult nervous system

plasticity, which may limit Notch as a viable therapeutic target. Proteases that

enhance extracellular matrix degradation promote tumor invasion and progression.

Cathepsin B, urokinase-type plasminogen activator receptor, and matrix metallo-

proteinase have been inhibited with siRNA both in vitro and in vivo in several brain

tumor models (Gondi et al. 2004, 2007; Lakka et al. 2005; Tummalapalli et al.

2007; Kargiotis et al. 2008). Both tumor invasion and angiogenesis were reduced,

and established intracranial tumors in mice demonstrated significant regression

following intracranial injection of plasmids expressing these siRNAs. Similar to

Notch-1, these proteases are abundantly expressed and play central roles in normal

biological functions such as immune surveillance. Therefore, these targets should

be studied cautiously and evaluated carefully to determine if the therapeutic benefit

is greater than potential side-effects in fatal illness such as brain tumors.

However, proteins like tenascin-C which is typically expressed in adults under

pathological conditions, including human brain tumors, may be better therapeutic

targets. Although the role of tenascin-C in brain tumors in unclear, its expression is

associated with tumor grade. To determine if tenascin-C could be targeted in

patients, ten high-grade and low-grade glioma patients with poor prognosis were

chosen for siRNA therapy following surgical transection (Zukiel et al. 2006).

Plasmids carrying shRNA specific for tenascin-C were injected directly into neo-

plastic brain tissue that was inaccessible to surgical removal. Postsurgical imaging

showed reduced tumor burden in the areas of injection of the shRNA, but some

patients had new tumors in remote areas, suggesting that the efficacy of the RNAi

therapy was limited to the injection site.

RNAi technology has also been used to enhance the efficacy of other therapeutic

modalities in models of human glioblastomas. Suppression of galectin 1, a hypoxia-

regulated proangiogenic factor that promotes glioblastoma cell migration, enhances

the therapeutic effect of the proautophagic agent temozolomide in vivo (Le Mercier

et al. 2008). The anti-apoptotic molecule Bcl-2 has also been targeted with siRNA,

enhancing apoptosis of glioma cell lines, but the apoptotic effect was significantly


Joseph GeorgeRectangle

increased with the chemotherapeutic agent taxol (George et al. 2008). A VEGF

siRNA, shown to significantly reduce vascularization of glioblastoma cells in nude

mice, did not appear to alter tumor growth in vivo (Niola et al. 2006). However,

using a retroviral vector that expressed VEGF siRNA and an IL-4 transgene, tumor

growth was totally abolished, illustrating the synergistic effect between a siRNA

and transgene. Since combination therapy has proved to be the most beneficial

therapeutic option for most types of cancers, the use of siRNA with other thera-

peutic modalities may prove to be the future for brain tumors.

8 Conclusions

Most pharmacotherapies that are currently being used for treatment of neurodegen-

erative diseases, inflammatory CNS disorders, and brain tumors target molecules

that are localized downstream in the pathogenic cascade. As a consequence, their

effects are often not specific and their efficacy is moderate with regard to disease

modulation. Furthermore, some of these therapeutics, such as chemotherapeutic

agents for brain tumors and MS, are often associated with adverse effects that limit

their use. RNAi technology has two major advantages over conventional therapeu-

tic strategies. They can be used diagnostically to identify gene mutations associated

with a clinical phenotype, and they can directly inhibit the gene product involved in

the pathology of a disease. With our ever-accumulating knowledge of pathways that

mediate pathogenic processes, it will soon be possible to design RNAi-based

therapies that are specific for certain pathologic conditions and have relatively

few systemic adverse effects.

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References1 RNA Interference2 Spinocerebellar Ataxia and Other Polyglutamine-Associated Ataxias3 Alzheimer's Disease4 Parkinson's Disease5 Amyotrophic Lateral Sclerosis6 Multiple Sclerosis7 Brain Tumors8 ConclusionsReferences

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