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Theranostics 2017, Vol. 7, Issue 16
http://www.thno.org
3933
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2017; 7(16): 3933-3947. doi: 10.7150/thno.21529
Review
Nucleic Acid-Based Theranostics for Tackling
Alzheimer’s Disease
Madhuri Chakravarthy1, 2, Suxiang Chen1, 2, Peter R. Dodd3, Rakesh N. Veedu1, 2, 3
1. Centre for Comparative Genomics, Murdoch University, Murdoch, Perth, Australia 6150;
2. Perron Institute for Neurological and Translational Science, QEII Medical Centre, Nedlands, Perth, Australia 6005;
3. School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, Australia 4072.
Corresponding author: Rakesh N. Veedu, PhD. Centre for Comparative Genomics, Murdoch University, Building 390 Discovery Drive, Perth, Western
Australia, Australia 6150. Email: R.Veedu@murdoch.edu.au
© Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2017.06.20; Accepted: 2017.07.28; Published: 2017.09.05
Abstract
Nucleic acid-based technologies have received significant interest in recent years as novel
theranostic strategies for various diseases. The approval by the United States Food and Drug
Administration (FDA) of Nusinersen, an antisense oligonucleotide drug, for the treatment of spinal
muscular dystrophy highlights the potential of nucleic acids to treat neurological diseases, including
Alzheimer’s disease (AD). AD is a devastating neurodegenerative disease characterized by
progressive impairment of cognitive function and behavior. It is the most common form of
dementia; it affects more than 20% of people over 65 years of age and leads to death 7–15 years
after diagnosis. Intervention with novel agents addressing the underlying molecular causes is
critical. Here we provide a comprehensive review on recent developments in nucleic acid-based
theranostic strategies to diagnose and treat AD.
Key words: nucleic acids; Alzheimer’s disease; amyloid beta peptides; tau peptide; chemically modified
oligonucleotides; nucleic acid therapeutics.
Introduction
Nucleic acid-based technologies typically use
synthetic oligonucleotides ̴8–50 nucleotides in length,
most of which bind to RNA through Watson-Crick
base pairing to alter the expression of the targeted
RNA and protein. Novel chemical modifications and
conjugation strategies have been developed to
improve pharmacokinetics and tissue-specific
delivery. Vitravene, Kynamro, Nusinersen and
Eteplirsen are antisense oligonucleotides (AOs)
approved by the FDA to treat cytomegalovirus
retinitis, familial hypercholesterolemia, spinal
muscular atrophy, and Duchenne muscular
dystrophy respectively [1-3]. The nucleic acid aptamer
drug Macugen was approved for age-related macular
degeneration [4]. These successful clinical translations
demonstrate the potential of nucleic acid-based
technologies and provide scope for developing novel
therapeutics for AD. AD is the most common form of
dementia; it accounts for 70% of cases with that
diagnosis. Globally there are ~47 million current
cases; 7.7 million new cases are added each year [5].
AD is characterized by a progressive loss of memory
and cognitive function [6]. Patients eventually need
24-hour care that places emotional and economic
burdens on the community. There is no cure for AD,
nor any treatment that addresses its underlying
molecular cause [5]. Current treatments use
cholinesterase inhibitors [7] and N-methyl-D-aspartate
receptor (NMDA) antagonists [8] that improve
cognitive function and reduce symptoms temporarily
but do not stop the progression of the disease. The
current approach to diagnosis relies on a combination
of cognitive and clinical assessment, genetic profiling,
and magnetic resonance imaging to measure
anatomical changes in the brain [9], but confirmation
relies on post-mortem neuropathological assessment
Ivyspring
International Publisher
Theranostics 2017, Vol. 7, Issue 16
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and misdiagnosis is common [6]. Two hallmarks of
the disease are extracellular amyloid-β (Aβ) plaques
(mainly an agglomeration of Aβ peptides) and
intracellular neurofibrillary tangles (hyperpho-
sphorylated tau peptides). In this review we focus on
the potential of nucleic acid therapeutic, diagnostic,
and research strategies that target both Aβ and tau
pathologies to help diagnose and treat AD.
Amyloid β (Aβ) hypothesis
The Aβ hypothesis states that there is an
imbalance of toxic Aβ peptide production and
clearance [10-12]. The main Aβ species, Aβ
1-40 and
Aβ1-42, can aggregate to form fibrils and plaques
[10-12]. Aβ1-40 and Aβ1-42 are produced by the aberrant
splicing of amyloid precursor protein (APP) by β-site
APP cleaving enzyme 1 (BACE1) and γ-secretase
(Figure 1) [11-15]. Mutations in the APP and
Presenilin genes (PSEN1 codes for the catalytic
subunits of γ-secretase) increase Aβ1-42 levels [10-12,
14, 16-18] and lead to early-onset familial AD. Down
syndrome cases have an extra copy of chromosome
23, and hence of the APP gene, and develop Aβ
plaques early in adulthood [19]. Oligomers of Aβ
promote synaptic loss, neuronal dysfunction, and cell
death [20, 21]. Aβ1-42 inhibits the maintenance of
hippocampal long-term potentiation, resulting in
altered memory function [10, 22] and reduced
synaptic neurotransmission through NMDA
receptor-mediated signaling [10, 22, 23]. Aβ toxicity
has also been implicated in inflammation [11],
oxidative stress [11, 24], cholinergic transmission [23],
glucose metabolism [25, 26], and cholesterol
metabolism [27].
Tau hypothesis
Microtubule-associated protein tau (tau),
predominantly expressed in neuronal axons, is
involved in microtubule assembly and stability. Tau is
regulated by phosphorylation [28, 29].
Hyperphosphorylation decreases the ability of tau to
bind to microtubules, leading to reduced trafficking,
destabilization of microtubules, and synaptic loss [29,
30] (Figure 2). Abnormal tau can aggregate into paired
helical filaments to form neurofibrillary tangles [31] in
the cytosol and sequester normal tau to inhibit
microtubule assembly [29]. Alternatively, tau
aggregation may be a protective mechanism to stop
hyperphosphorylated tau sequestering normal tau
and inhibit microtubule assembly [29]. Tau
hyperphosphorylation is detrimental in various
neurodegenerative diseases termed “tauopathies” [28,
32]. Hyperphosphorylation of tau correlates with
neurodegeneration and cognitive decline [29, 32].
Other post-translational modifications of tau,
including abnormal glycosylation and reduced
β-linked acylation of N-acetylglucosamine, increase
hyperphosphorylation [29, 33]. Inhibition of the
ubiquitin-proteasome system may also increase the
aggregation of hyperphosphorylated tau [31].
Other hypothesis of AD
Drugs currently approved by the FDA for the
treatment of AD are Donepezil, Rivastigmine,
Galantamine and Memantine (Table 1) [34-37]. These
agents enhance cholinergic and glutamatergic
neurotransmission and improve cognitive function
temporarily. However, they do not slow the
progression of the disease. Oxidative stress [38],
inflammation [39], insulin impairment [40, 41] and
abnormal cholesterol metabolism [27] may also play
roles (Table 1), but will not be considered in depth
here.
Figure 1. Non-amyloidogenic and amyloidogenic pathways in AD neurons. In the amyloidogenic pathway the APP is aberrantly spliced by BACE1 and γ-secretase to
overproduce toxic Aβ species.
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Figure 2. The roles of tau in normal neurons and of hyperphosphorylation in AD neurons that lead to neuronal toxicity.
Table 1. Therapeutic molecules in clinical trials, their targets, and trial outcomes.
Drug molecule
Role/ Target
Trial stage
Results
References
Donepezil (Pfizer)
Cholinesterase inhibitor
FDA approved- Although they improve the symptoms temporarily these
drugs do not stop the progression of the disease.
[34-37]
Rivastigmine (Novartis)
Cholinesterase inhibitor
Galantamine (Jansen-Cilag)
Cholinesterase inhibitor
Memantine (Lundbeck)
NMDA receptor antagonist
Tramiprosate
Aβ aggregation inhibitor
Phase III
No significant benefit. May promote abnormal tau
aggregation
[47-49]
Colostrinin
Aβ aggregation inhibitor
Phase III
Modest improvements not sustained
[50-52]
Scyllo-inositol
Stabilizes Aβ aggregates and
inhibits toxicity
Phase II
No statistically significant effect. Reduced Aβ in
cerebrospinal fluid
[53]
Aβ vaccination
Aβ aggregation inhibitor
Phase II
Halted because patients developed
meningo-encephalitis
[54]
Bapineuzumab
Aβ aggregation inhibitor
Phase III
End points not significantly different
[55]
Solanezumab
Aβ aggregation inhibitor
Phase III
End points not significantly improved
[56]
Anti-amyloid Ab
Aβ aggregation inhibitor
Phase III
No positive primary outcome
[57]
Other mAbs
Aβ aggregation inhibitor
Various
No positive outcome
[42, 58, 59]
Tarenflurbil
γ-secretase inhibitor
Phase III
No significant improvement
[60-62]
LY450139 (Eli Lilly)
γ-secretase inhibitor
Phase III
Discontinued: no Aβ40/42 reduction
[63]
BMS-708163 (B-M Squibb)
γ-secretase inhibitor
Phase II
Terminated due to lack of favorable pharmacodynamics
[42, 64]
Verubecestat
BACE1 inhibitor
Phase III
Currently running
[65]
Rogiglitazone
BACE1 inhibitor and Type 2
diabetes drug
Phase III
No positive outcome
[66]
Pioglitaozone
BACE1 inhibitor and Type 2
diabetes drug
Phase III
No positive outcome
[66]
Methyl thionium chloride
Tau aggregation inhibitor
Phase II
Significantly improved cognitive function
[67, 68]
Tideglusib
GSK3β
Phase IIb
No positive outcome
[68, 69]
Davunetide
Microtubule stabilizer
Phase III
No significant improvement
[30]
Antioxidants
ROS
Phase III
No positive outcome
[42, 70]
Anti-inflammatories
Inflammation
Phase III
No significant improvement
[25, 39, 59, 71-73]
Intranasal insulin
Insulin impairment
Pilot
Improvement in patients without APOE-ε4 allele
[40, 74]
Other anti-diabetics
Insulin impairment
Phase III
Currently running
Statins
Cholesterol metabolism
Phase III
Preliminary results positive; mechanism unknown.
[27, 75]
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Current therapeutic molecules and
clinical trials for the treatment of AD
Many disease-modifying therapeutics show
positive outcomes in animal models but
disappointing results in clinical trials (for drug
candidates and ongoing trials see Table 1). Current
strategies have been comprehensively reviewed [42].
Poor outcomes might have arisen because each agent
is targeting a single pathway, whereas AD is a
complex disease and it may be important to aim at
multiple targets [43, 44]. Success in developing a
suitable therapeutic approach is challenging because
the pathogenesis of AD is unknown [45]. Trials might
be affected by factors such as genetics, metabolism,
and diet [46], but there is clearly a need to develop
novel therapeutics for this disease.
Nucleic-acid based molecules for tackling
AD
Unlike conventional small-molecule drugs,
nucleic acid-based therapeutic agents such as AOs,
small-interfering RNAs (siRNAs), microRNA
moieties that target oligonucleotides (antimiRs and
miRNA mimics), and DNAzymes/ribozymes can
regulate the expression of key proteins by selectively
targeting their mRNAs. The outcome is mRNA
cleavage, repair, or steric blockade (Figure 3). The
class of modified nucleic acids called aptamers can
target proteins and inhibit their function (Figure 3).
Nucleic acid-based strategies could be an effective
alternative to drug development for AD because they
can target a range of pathological features.
Figure 3. Nucleic acid-based therapeutic strategies. mRNA: messenger RNA; RNase H: ribonuclease H; siRNA: small interfering RNA; RISC: RNA inducing silencing
complex; AO: antisense oligonucleotide; antimiR: anti-microRNA; miRNA mimic: microRNA mimic
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Figure 4. Examples of chemically-modified nucleotide analogues. 2’-OMe: 2’-O-methyl; 2’-MOE:2’-O-methoxyethyl; 2’-F: 2’-fluoro; 2’-NH2: 2’-amino; FANA:
fluoroarabinonucleotide; LNA: locked nucleic acid; TNA: threose nucleic acid; PNA: peptide nucleic acid; PMO: phosphorodiamidate morpholino oligomer; MNA:
morpholino nucleic acid; HNA: hexitol nucleic acid; CeNA: cyclohexenyl nucleic acid; ANA: anhydrohexitol nucleic acid
Improving the stability and efficacy of nucleic
acid-based therapeutics
Therapeutic oligonucleotides composed of
naturally occurring nucleotides are rapidly degraded
in vivo, which makes them unsuitable for drug
development. To improve their pharmacokinetic
properties, chemically modified nucleotide analogues
with high resistance to nucleases are normally used. A
number of analogues have been developed by
modifying the base or sugar moieties, or the
inter-nucleotide linkages (see Figure 4) [76-78].
Phosphorothioate DNA [79], 2'-O-methyl (2’-OMe)
RNA [80], 2'-fluoro (2’-F) RNA [81],
2'-O-methoxyethyl (2’-MOE) RNA [82], and
phosphorodiamidate morpholino (PMO) [83]
analogues have been successfully utilized in
FDA-approved oligonucleotide drugs. Analogues
such as locked nucleic acids (LNA) [84, 85], peptide
nucleic acids (PNA) [86], tricyclo-DNA (tcDNA) [87],
and cyclohexenyl nucleic acids (CeNA) [88] also show
excellent biophysical properties and offer further
scope for novel oligonucleotide development. These
chemistries can be used to construct fully modified or
mixmer oligonucleotides. Aptamers can be modified
during the selection or post-selection stages to
improve their affinity and bioavailability [77].
Another challenge in the clinical utilization of
unmodified oligonucleotides is rapid renal clearance
from the blood due to their small size that falls under
the renal filtration threshold. To increase their
bioavailability, oligonucleotides can be conjugated
with polyethylene glycol (PEG) to increase their size,
which could also improve their resistance to nucleases
[89]. Several PEGylated drugs have been approved by
the FDA for clinical use [89, 90]. Other strategies
include conjugating the oligonucleotides to albumin,
which has a size of around 7 nm and shows reduced
renal clearance and can therefore increase the
circulation half-life of the oligonucleotides.
Phosphorothioate modified oligonucleotides also
showed reduced renal clearance by binding to plasma
proteins like albumin to avoid glomerular filtration
[91]. Another strategy is the synthesis of neutral
siRNA (masking the negative charge on the
phosphate backbone). Neutral siRNA showed
reduced renal clearance [92].
Recent progress in modified nucleic acids
for AD
Antisense oligonucleotides
A classical nucleic acid approach to controlling
the expression of proteins is to use AOs, short
single-stranded synthetic oligonucleotides, which can
precisely target an mRNA transcript to regulate the
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expression of the protein it codes for. Antisense
mechanisms include RNase H recruitment and
cleavage of mRNA, modulation of splicing in
pre-mRNA, and steric blockade of either mature or
pre-mRNAs (Figure 3). RNase H-mediated cleavage
involves designing a short DNA oligonucleotide that
binds to the target mRNA to form a RNA-DNA
duplex [93]. The duplex is recognized and cleaved by
endogenous RNase H. AOs that modulate pre-mRNA
splicing can be used to repair defective RNA and
eliminate disease-associated splice variants [94].
Many pre-mRNA transcripts are alternatively spliced
to produce different mRNA, and hence protein,
variants [94].
APP
Many groups have designed AOs that target
APP to reduce APP expression. An early study by
Allinquant et al. [95] developed AOs that successfully
blocked rat APP synthesis. Administration of the AOs
showed that APP played a role in axonal and
dendritic growth, and thus in neuronal differentiation
[95]. ISIS Pharmaceuticals (now Ionis Pharma) have
patented (US 2003/0232435 A1) 78 gapmer AOs with
2'-MOE wings and central DNA region. The AOs
target various regions of APP mRNA and inhibit
39–82% of APP protein expression [96].
Kumar and colleagues [97] developed
phosphorothioated DNA AOs against sequences that
correspond to the Aβ region of APP (17-42 amino
acids). Administration of the AOs led to improved
cognitive function in senescence-accelerated
mouse-prone 8 (SAMP8) mice. SAMP8 mice have a
natural mutation that leads to APP over-expression,
impaired Aβ removal, and loss of memory with
increasing age. The AOs that target the mid-Aβ region
reduced APP levels by 43–68% in the amygdala,
septum and hippocampus [97]. The mice showed
improvement in acquisition and retention in the
footshock avoidance paradigm, which reversed their
deficits in learning and memory [97]. AOs that target
the sequences that correspond to the region of APP
coding for the first 17–30 amino acids of Aβ were the
subject of intellectual property protection [98]. Banks
and colleagues [99] showed that a radioactively
tagged phosphorothioate DNA AO targeting the Aβ
region of APP could transit the blood-brain barrier
(BBB) of mice to enter the cerebrospinal fluid. When a
100-fold higher dose of the AO was injected into the
brain by intracerebroventricular injection it reversed
the learning and memory deficits in SAMP8 mice,
possibly through reduced oxidative stress. Poon et al.
[100] used proteomics to show that lower Aβ levels
result in reduced oxidative stress in brain.
Opazo et al. [101] transfected the AOs described
by Kumar and colleagues [97] into the CTb cell line, a
neuronal line from mice that overexpresses APP, and
the CNh cell line from normal mice. The AOs resulted
in APP knockdown in CTb cells by 36%, 40% and 50%
compared with normal CNh cells after 24 h, 48 h and
72 h respectively [101]. By 72 h after AO transfection,
choline uptake was similar to that in CNh cells and
there was increased choline release in response to
glutamate, nicotine and KCl depolarization, which
reached similar levels to those observed in CNh cells.
The CTb cells come from a Down syndrome mouse
model, which show some learning deficits and
cholinergic dysfunction that are similar to those found
in AD [102]. Similarly, Rojas et al. [103] showed that
APP overexpression reduced the expression and
retrograde transport of nerve growth factor. This
reduced nicotine-induced stimulation of α3β2 nicotinic
acetylcholine receptor and in consequence lowered
intracellular Ca2+ responses in CTb cells. The effects of
APP overexpression were restored close to normal by
treatment with AOs targeting APP expression.
Chauhan and colleagues [104] designed gapmer
AOs with 2'-OMe and DNA nucleotides on a
phosphorothioate backbone that target the β-secretase
cleavage site of APP and found that they reduced
brain Aβ40 and Aβ42 levels in a mouse model of AD.
The AOs were delivered intracerebroventricularly
and showed rapid uptake and retention for 30
minutes. They efficiently crossed cell membranes into
the nuclear and cytoplasmic compartments of
neuronal and non-neuronal cells. Chauhan and Siegel
[105] designed two additional AOs targeting the β-
and γ-secretase site of APP in the Tg2576 mouse
model that expresses APP. The AO targeting the
mutated β-secretase site increased soluble APPα by
43% and decreased soluble Aβ40 and Aβ42 levels by
39%, whereas the AO targeting the γ-secretase site
had no effect. The AO targeting β-secretase also
inhibited acetylcholinesterase activity, increasing
acetylcholine by five-fold in cortex compared with
controls.
Erickson et al. [106] peripherally administered an
APP AO to SAMP8 mice. This resulted in a 30%
increase in APP levels but no change in soluble Aβ
levels. The treated mice showed improved memory.
They also showed [107] that AO-mediated APP
knockdown in Tg2576 mouse brains reduced cytokine
expression and improved learning and memory.
Attenuating APP overexpression may improve
learning and memory by reducing inflammation (also
implicated in AD pathology).
BACE1
Yan et al. [108] developed two AOs that target
β-secretase aspartyl protease and found that they
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reduced the release of Aβ40 and Aβ42 by 50–80%.
Vassar and colleagues [109] also used AOs that target
β-secretase to reduce Aβ40 and Aβ42 production by
around 30%. These studies showed that β-secretase is
important for the production of Aβ40 and Aβ42 and
highlighted BACE1 as an important target for AD.
Wolfe et al. [110] designed splice-modulating AOs to
target BACE1, since alternatively spliced transcript
variants at exons 2 and 3 do not show β-secretase
activity. The AOs reduced Aβ production
significantly in cells without altering total BACE1
mRNA.
Presenilin 1 (PSEN1)
Refolo et al. [111] found that AOs targeting
PSEN1 in a human cell line reduced PSEN1
holoprotein by 80% 12 days after treatment and by
90% after 14 days. This was correlated with a two-fold
increase in Aβ42 levels. Grilli et al. [112] found that
hippocampal primary neurons overexpressing
mutant PSEN1 were vulnerable to excitotoxic and
hypoxia-hypoglycemic damage and increased cell
death. They designed two phosphorothioate AOs
targeting PSEN1 in wild type mice. In contrast to
Refolo et al. [111] they found that lower PSEN1
expression reduced cell death and provided
neuroprotection [112]. Fiorini et al. [113] administered
AOs targeting PSEN1 to aged SAMP8 mice and found
they reduced brain oxidative stress biomarkers. In the
T-maze foot shock avoidance and novel object
recognition tests the mice showed a reversal of
learning and memory deficits.
Tau
Caceres et al. [114] showed that an AO targeting
the 5' end of the tau gene, in the region before the start
codon, showed strong inhibition of neurite elongation
in primary rat neurons. Immunoblotting revealed that
the tau protein level was reduced in AO-treated mice
but not in control mice. The effect of AO treatment on
cognition needs to be assessed. DeVos et al. [115]
screened 80 AOs targeting tau and selected the three
that showed the best knockdown of tau to test in vivo.
The latter reduced tau mRNA levels by more than
75%. The best AO was selected for further testing in
mice; it lowered brain tau mRNA and protein
significantly in a dose-dependent manner. Behavioral
impacts and neurotoxicity were not measured.
Kalbfuss et al. [116] developed splice-modulating AOs
modified with 2'-OMe nucleotides to target the tau
exon 10 splice junctions to reduce exon 10 inclusion.
Exclusion of exon 10 increases the ratio of tau proteins
lacking the microtubule-binding domain. In
consequence, the microtubule cytoskeleton becomes
destabilized as observed in frontotemporal dementia
and parkinsonism.
Peacey et al. [117] designed bipartite AOs that
bound to the hairpin structure at the boundary
between exon 10 and intron 10 of tau to inhibit exon
10 splicing, reversing the effect of disease-causing
mutations in cells. Liu et al. [118] developed a
small-molecule (mitoxantrone) conjugated to a
bipartite AO that binds to the tau RNA hairpin
structure. The conjugate also inhibited exon 10
splicing in cell-free conditions more effectively than
mitoxantrone or the bipartite AO alone, but induced
cytotoxicity. The same group used a PNA-modified
bipartite AO conjugated to mitoxantrone that
inhibited tau splicing but was also cytotoxic [110].
Sud et al. [119] developed PMO AOs to modulate
the splicing of tau and tau expression. The AOs were
designed to target sequences at the donor and
acceptor splice sites, the splicing branch points, and
splicing enhancers and inhibitors to induce exon
skipping. Exons 0, 1, 4, 5, 7, 9, and 10 were targeted.
Exons 1, 4, 5, 7, and 9 are found in all 6 isoforms of tau
while exon 10 is present in only three of the six
isoforms. Of the 31 AOs tested, AO E1.4 targeting the
splice donor site at the exon 1 – intron 1 junction
reduced tau mRNA expression by 50%. The other
AOs effective in this region were a combination of
AOs that targeted the splice donor and acceptor sites
and the start codon. AO E5.3 targeted the splice donor
site at the exon 5 –intron 5 junction and reduced total
tau mRNA expression by 29–46%. It also reduced tau
protein level by 58–62%. The resulting transcript was
missing exons 4 to exon 7 using the normal splice
sites. AO E7.7 targeted the splice donor site on exon 7;
it reduced tau mRNA expression by 30% and tau
protein levels by 67%. E5.3 injected into mice in vivo
produced lower tau mRNA levels than in non-injected
regions.
GSK-β
Farr et al. [120] showed that a phosphorothioated
AO that targets GSK-3β decreased GSK-3β protein
levels in the cortex of SAMP8 mice. There were
improvements in learning and memory, reduced
oxidative stress, increased levels of the antioxidant
transcription factor nuclear factor erythroid-2 related
factor 2, and decreased tau phosphorylation.
Acetylcholinesterase (AChE)
Fu et al. [121] found that AOs against human
AChE mRNA reduced AChE activity in an AD mouse
model after 8 h; the effect lasted till 42 h. Lower
enzymic activity was accompanied by improvement
on behavioral tasks, which showed increased memory
retention and improved water maze performance
(shorter swimming time).
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Apolipoprotein E receptor 2 (ApoER2)
ApoER2 may be a primary risk factor for
late-onset AD [122, 123]. Dysregulation of ApoER2
splicing may result in impaired synaptic homeostasis.
Cerebral injection of mice with AOs targeting the
adjacent introns enhanced exon 19 inclusion, an effect
that persisted for up to 6 months [123]. The mice
showed improvement in Aβ-induced cognitive
defects. It was postulated that the AOs bind to the
splicing factor SRF1 to reduce its expression and
increase the inclusion of exon 19, thereby increasing
the level of the active form of ApoER2 to enhance
NMDA receptor phosphorylation.
siRNA
These are short synthetic double stranded RNA
oligonucleotides that target complementary mRNA
and silence gene expression through the assembly of
the RNA-induced silencing complex (RISC) (Figure 3)
[93, 124]. Chemical modifications can be introduced
into the siRNA to increase its stability against
nucleases and increase its selectivity for the target
(Figure 4).
APP
Miller and colleagues [125] found that siRNAs
against the Swedish mutant in APP that causes a
familial form of AD silenced the expression of mutant
alleles. The siRNAs were designed to ensure that they
bound specifically to the mutant alleles and not the
wild-type. The mutation was placed in the central
region of the siRNA duplex to achieve high silencing
efficiency.
BACE1
McSwiggen and colleagues [126] patented 325
siRNAs that target BACE (NCBI ID: NM_012104). The
patent covers sequences with various chemically
modified siRNAs that include 2'-deoxy, 2'-F and
2'-OMe pyrimidine and purine nucleotides,
phosphorothioate internucleotide linkages and
inverted deoxyabasic caps. Four of the siRNAs
reduced BACE expression by 40–90% at 25 nM
concentration, but there was no data on whether this
altered Aβ40 and Aβ42 expression. Basi et al. [127]
made a siRNA that reduced the BACE1 mRNA level
by 50% and BACE protein level by more than 90%. It
decreased the secretion of Aβ peptide without
affecting BACE2 expression, indicating specificity for
BACE1. Kao et al. [128] also designed siRNAs, where
two of the siRNAs reduced BACE1 mRNA by more
than 90% and Aβ production by 36–41%. Pretreatment
of neurons with the siRNA increased neuroprotection
against hydrogen peroxide-induced oxidative stress.
Modarresi et al. [129] injected LNA-modified siRNAs
targeting BACE1 antisense transcripts into the third
ventricle of Tg-19959 mice to downregulate BACE1
and BACE1 antisense transcripts, which led to lower
BACE1 protein levels and less Aβ production and
aggregation in the brain. Notably, Cai et al. [130]
showed that siRNAs targeting BACE1 inhibited it in
mice and increased choroidal neovascularization:
BACE1 is also expressed in the neural retina and in in
vitro and in vivo angiogenesis. Although BACE1
inhibition may be therapeutically beneficial in AD, it
may contribute to retinal pathologies and exacerbate
conditions such as age-related macular degeneration.
Heterogeneous nuclear ribonucleoprotein H
A G-rich region in exon 3 of BACE1 may form a
G-quadruplex structure and recruit a splicing
regulator, heterogeneous nuclear ribonucleoprotein
H, that regulates splicing to increase generation of the
BACE1 501 isoform. Fisette et al. [131] reported that
siRNA and short hairpin RNA candidates that target
heterogeneous nuclear ribonucleoprotein H reduced
its expression and thereby decreased BACE1 501
isoform levels and Aβ production.
AntimiRs and miRNA mimics
miRNAs are short non-coding RNAs that
regulate protein expression post-transcriptionally.
miRNA mimics can modulate RNA and protein
expression by acting like their endogenous miRNA
counterparts. AntimiRs can modulate RNA and
protein expression by inhibiting endogenous miRNA
similar to AOs (Figure 3). miRNAs silence gene
expression by translational repression and/or mRNA
degradation [132, 133]. miRNAs are first transcribed
by RNA polymerases II or III to form long primary
miRNA with a 5’ CAP and a poly(A) tail [133-135].
These are then processed in the nucleus into short
70-nucleotide hairpin structures called precursor
miRNAs (pre-miRNA) by the microprocessor
complex [133-135]. The pre-miRNAs are exported to
the cytoplasm by Exportin 5 and processed by Dicer
into double-stranded miRNA duplexes, which are
approximately 22 nucleotides long [133-135].
BACE1
An endogenous non-coding BACE1 antisense
transcript stabilizes the BACE1 transcript and may
upregulate BACE1 in AD cases. BACE1 antisense
binds to BACE1 at the miR-485-5p binding site and
suppresses BACE1 expression. Faghihi et al. [136]
found that LNA-antimiRs that target miR-485-5p
decreased miRNA-induced suppression of BACE1
and increased BACE1 antisense expression. Hebért et
al. [137] showed that miR-29a/b-1 cluster was
significantly reduced in sporadic AD patients and
correlated with increased BACE1 expression and Aβ
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generation, and therefore may be potential targets for
miRNA mimics as a therapeutic strategy for AD.
Tau
Mi34a reduces endogenous tau expression at
both the mRNA and protein level in M17D cells by
binding to the 3' UTR region of tau [110], whereas
miR-34c levels are elevated in the hippocampus of AD
patients and mouse AD models [138]. Wolfe et al. [110]
used LNA antimiRs to inhibit miR-34a,
-34b and -34c and found increased tau expression.
Zovolis et al. [138] found that an antimiR that targets
miR-34c rescued learning in mouse models.
Acetyl-CoA acyl transferase
Acetyl-CoA acyl transferase has a role in lipid
metabolism that has been implicated in the
pathogenesis of AD. Murphy et al. [139] inhibited
Acetyl-CoA acyl transferase using an artificial miRNA
to reduce Aβ plaque burden and improve cognition in
a mouse model of AD. The miRNA also reduced
full-length human APP levels.
Brain-derived neurotropic factor
Brain-derived neurotropic factor regulates
synaptic plasticity and memory and is decreased in
AD brains [140-142], while miR-206 suppresses
brain-derived neurotropic factor levels and memory
function in AD mice [143]. Lee et al. [143] injected an
anti-miR candidate AM-206 that targets miR-206 into
the third ventricle of Tg2576 mice. It increased brain
levels of brain-derived neurotropic factor, enhanced
hippocampal synaptic density, neurogenesis, and
memory. Intranasally administered AM-206 also
reached the brain and had similar effects to the
injected AM-206.
DNAzymes/Ribozymes as therapeutic
candidates for AD
A target RNA can be cleaved to reduce its
expression using catalytic oligonucleotides such as
DNAzymes and ribozymes [144]. The arms of these
enzymes hybridise with the target RNA and cleave
their targets through the catalytic loop in the middle.
DNAzymes and ribozymes cleave the phosphodiester
bond at the purine-pyrimidine or purine-purine
junction (Figure 3).
BACE1
Nawrot et al. [145] designed RNA-cleaving
hammerhead ribozymes that downregulated BACE1
mRNA expression by more than 90% in HEK293 and
SH-SY5Y cells and reduced Aβ40 and Aβ42 production
by more than 80%. They also showed that a
DNAzyme with the 10–23 catalytic loop reduced
BACE mRNA expression by 70%. However, whether
the reduced BACE mRNA expression leads to
reduced Aβ production is unknown and requires
validation.
Nucleic acid aptamers
Aptamers are short single stranded RNA or
DNA oligonucleotides with unique three-dimensional
structure that bind to targets with high affinity and
specificity. Aptamers can be developed against a
variety of targets ranging from small molecules to
complex proteins over whole cells. Aptamers can be
used for therapeutic, diagnostic (biosensors and
molecular imaging), and targeted drug delivery
applications. They are typically selected from large
DNA and RNA oligonucleotide libraries through a
process called Systematic Evolution of Ligands by
EXponential enrichment (SELEX) [146, 147].
Aβ
Ylera et al. [148] were the first to report novel
RNA aptamers that bound to Aβ1-40 fibrils with high
affinity (29–48 nM). Bunka et al. [149] made aptamers
against amyloid-like fibrils from β2-microglobulin.
They bound to the target with high affinity, but also
bound to other amyloid fibrils including, but not
confined to, those found in dialysis-related
amyloidosis patients. Rahimi et al. [150] also
developed RNA aptamers against Aβ fibrils, but these
also interacted with other amyloidogenic proteins by
binding to a common β-sheet motif. They bound to
fibrils with ≥15-fold higher sensitivity than
thioflavin-T, suggesting that aptamers might be
diagnostic tools for AD. Takahashi et al. [151] isolated
two RNA aptamers, N2 and E2, that bound to
monomeric Aβ40 with dissociation constants of 21.6
and 10.9 µM respectively. Though the affinities were
quite low for clinical use, enzyme-linked
immunosorbent assay (ELISA) showed that they
could inhibit Aβ aggregation efficiently. When
conjugated to AuNP gold nanoparticles, N2 and E2
bound to both Aβ monomers and oligomers. Mathew
et al. [152] showed that the N2 aptamer conjugated to
curcumin-polymer nanoparticles enhanced binding
to, and disaggregated, amyloid plaques, which were
then cleared by phagocytosis. The study targeted
peripheral amyloid as peripheral organs may also
generate amyloid proteins, which have also been
implicated in AD. Targeting peripheral amyloid is
easier due to the challenges in the brain delivery of
aptamers.
Farrar et al. [153] developed a fluorescently
tagged aptamer that bound to Aβ oligomers in both
AD and transgenic mouse brain tissue. The aptamer
may be useful for Aβ imaging, which has diagnostic
implications. Similarly, Babu and colleagues [154]
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developed an aptamer complexed with ruthenium
that binds to, and inhibits the formation of, Aβ
oligomers. The aptamer-ruthenium interaction
increases luminescence intensity, which is reduced
when the aptamer binds to Aβ monomer or
oligomers.
BACE1
Rentmeister et al. [155] made an RNA aptamer
that binds to the short cytoplasmic tail of BACE1. It is
a good research tool to investigate the biological
function of the cytoplasmic tail without interfering
with BACE1 transport and localization. Liang et al.
[156] developed two DNA aptamers, A1 and A4, that
bind to the extracellular domain of BACE1 with high
affinity (Kd 15–69 nM) and specificity. They have
similar affinities to the anti-BACE1 antibody. In vitro,
APP Swedish mutant cells treated with A1 showed
lower Aβ40 and Aβ42 levels than control cells. sAPPβ
expression decreased with A1 treatment compared
with untreated controls.
Tau
Kim et al. [157] used recombinant his-tagged
tau40 to select aptamers from an RNA library through
SELEX. 12 rounds of selection produced a tau-1
aptamer, which represented ~76% of identified
aptamers, that reduced the levels of oligomeric tau (by
~94%) in vitro in a dose-dependent manner. However,
it could not de-oligomerize pre-existing tau oligomers
and had no effect on tau degradation. The aptamer
bound to tau protein and inhibited its
oligomerization, unlike control aptamers. Primary
neurons treated with tau-1 aptamer showed less
cytotoxicity than controls but no difference in
membrane integrity or viability; there was little effect
on normal tau function. Primary rat cortical neurons
administered tau oligomers and treated with tau-1
aptamers showed significantly less oligomeric tau
phosphorylation at Ser199/202 but there was no effect
on monomeric tau. Extracellular tau oligomers also
stress neighboring neurons. Administration of tau
oligomers leads to severe neurotoxicity, which was
reduced by tau-1 aptamer treatment. Tau-1 aptamers
can prevent or reverse cytotoxicity mediated by tau
oligomerization both in a non-neuronal cell line and
in primary rat cortical neurons. Unfortunately, the
tau-1 aptamers isolated by Kim et al. bound only to
one of the six isoforms of tau. Therefore, the effects of
tau-1 aptamers observed in mice may not translate
clinically, because six isoforms are prone to
aggregation and implicated in neurodegeneration. To
be successful clinically, the aptamers must be able to
cross the BBB and the neuronal cell membrane, and
disaggregate the neurofibrillary tangles after binding
[158]. Kim et al. [159] reported a DNA
aptamer-antibody sandwiched to the tau-381 isoform
that detected tau in human plasma at femtomolar
concentrations by surface plasmon resonance.
The ubiquitin-proteasome system
Lee et al. [160] developed an aptamer against
USP14, an enzyme that delays protein degradation by
the ubiquitin-proteasome system. Recombinant
USP14 was incubated with a random RNA library for
SELEX. Three aptamers, USP14-1, USP14-2 and
USP-14-3, were identified, all of which bound to
USP14 with high affinity. USP14-3 showed the
strongest inhibition of deubiquitination, which may
be due to its ability to bind both USP14 and UCH37.
UCH37 is a protein that also slows protein
degradation in the proteasome. The aptamers have
yet to be tested in mice for their effect on tau
oligomerization and degeneration.
Prion protein
Mashima et al. [161] isolated aptamers against
bovine prion protein by SELEX that may have
therapeutic potential in prion diseases and AD. Aβ
oligomers bind to the prion protein to block long-term
potentiation. Thus, prion protein may mediate Aβ
oligomer-induced synaptic dysfunction.
Brain delivery of nucleic acid molecules
Receptor-mediated endocytosis or nanoparticle
conjugation strategies
The barrier for any successful drug to treat
neurological diseases is its failure to cross the BBB.
Various approaches have been made to overcome this
issue, and in many instances the drug can be
conjugated with other molecules to improve brain
delivery. Lipophilic molecules under 500 Da can cross
the BBB by simple diffusion. Therapeutic nucleic acids
are typically too large to cross the BBB, although
nanotechnology is now starting to overcome this
problem. This was the subject of a comprehensive
review by Kanwar et al. [162]. Nucleic acids can be
transported through the BBB by receptor-mediate
endocytosis when conjugated to molecules such as
transferrin, insulin, leptin, and insulin-like growth
factor 1: these bind to their receptors on the BBB,
which allows them to cross the BBB. Many studies
have used nucleic acids conjugated to molecules that
target the transferrin receptor. Transferrin-conjugated
nanoparticles, or nanoparticles conjugated to
transferrin receptor antibodies, can transport drugs
across the BBB [163, 164]. An aptamer that targeted
the mouse transferrin receptor allowed a lysosomal
enzyme to enter cells via endocytosis; this can be
applied to drug transport [165]. Two aptamers that
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3943
bound to epithelial cell adhesion molecule and
transferrin receptor were fused together; the product
could bind to cancer cells expressing the epithelial cell
adhesion molecule after crossing the BBB by
transferrin-receptor targeting [166].
Cell-penetrating peptide-based delivery systems
The use of cell-penetrating peptide-based
delivery systems is another approach for transporting
nucleic acids across the BBB. These systems generally
contain between 8 and 30 amino acids. Fluorescein
isothiocyanate-labeled cell-penetrating peptides were
conjugated to a morpholino AO targeting the mutant
ataxia telangiectasia gene and were able to cross the
BBB [167]. Heitz et al. [168] reviewed the development
of cell-penetrating peptides.
Intracerebroventricular infusion
Nucleic acids can also be introduced directly into
the cerebrospinal fluid by intracerebroventricular
infusion. The advantage of this over the use of
targeting molecules is that the drugs are delivered at
therapeutic concentrations quickly. However,
intracerebroventricular infusions are highly invasive
and rely on diffusion of the drugs throughout the
ventricular system. The drugs can then enter the
blood stream, because the cerebrospinal fluid turns
over every 4–5 hours [169]. Pardridge et al. [169]
described the advantages and disadvantages of
different drug delivery methods to the brain.
Intranasal methods are non-invasive and can deliver
nucleic acids directly to the brain. They have been
used successfully to deliver insulin to AD patients
[170]. Various molecules delivered intranasally have
improved cognitive function in a mouse model of AD
and in clinical trials, as summarized in the review by
Hanson et al. [171]. Many reviews describe how
nose-to-brain delivery occurs, the different drugs that
have been successfully delivered this way, and its
potential for treating neurodegenerative diseases such
as AD [172, 173].
Conclusions and Future Perspectives
Nucleic acid-based approaches offer great
promise for developing novel therapeutics for AD, a
complex neurodegenerative disease with several
pathological features. Confounds include genetic
factors, metabolic disorders including high cholesterol
levels, insulin resistance due to impaired glucose
metabolism, and dysfunction in various molecular
pathways. Existing therapies only treat AD
symptoms, not the underlying molecular causes.
Although many drug molecules have shown success
in cell and animal models, this effect often cannot be
replicated in human trials. There is an unmet need for
better theranostic strategies. The drug Nusinersen,
recently approved by the FDA for spinal muscular
atrophy, shows that nucleic acids have potential for
the treatment of neurological diseases, including AD.
Their efficacy in targeting several pathways that
underlie AD highlights their potential to be
developed as novel therapeutics for AD.
Abbreviations
FDA: Food and Drug Administration; AD:
Alzheimer’s disease; AO: antisense oligonucleotides;
NMDA: N-methyl-D- aspartate receptor; Aβ: amyloid
β; APP: amyloid precursor protein; BACE1: β-site
amyloid precursor protein cleaving enzyme 1; PSEN:
Presenilin; tau: microtubule associated protein;
siRNA: small interfering RNA; antimiR:
anti-microRNA; 2’-OMe: 2’-O-methyl; 2’-MOE:
2’-O-methoxyethyl; 2’-F: 2’-fluoro; LNA: locked
nucleic acids; PNA: peptide nucleic acids; PMO:
phosphorodiamidate morpholino; tcDNA:
tricyclo-DNA; CeNA: cyclohexenyl nucleic acid; PEG:
polyethylene glycol; mRNA: messenger RNA;
SAMP8: senescence- accelerated mouse-prone 8; BBB:
blood brain barrier; AChE: acetylcholinesterase;
ApoER2: Apolipoprotein E receptor 2; RISC: RNA
induced silencing complex; SELEX: systemic
evolution of ligands by exponential enrichment.
Acknowledgements
RNV thanks the funding from the McCusker
Charitable Foundation and Perron Institute for
Neurological Diseases and Translational Science.
RNV and MC thank the funding support from Greg
and Dale Higham.
Competing Interests
The authors have declared that no competing
interest exists.
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Author Biography
Madhuri Chakravarthy
obtained her Bachelor’s (2011)
in Biomedical Science and
Master’s (2014) in Biomedical
Science from the University of
Auckland, New Zealand. She
is currently a PhD student
under the supervision of Dr.
Rakesh N. Veedu at the
Murdoch University, Western Australia. Her research
is centered on developing novel nucleic acid
technologies to tackle neurological diseases.
Suxiang Chen received
his Bachelor degree of
Agriculture in 2010 in South
China Agricultural University.
He then obtained his Master
degree in Biotechnology
(Advanced) and a second
Master degree in Technology
and Innovation Management from the University of
Queensland in 2013 and 2014 respectively. Later, he
worked in Darui Biotechonology Co. Ltd and Gene
Denovo Biotechnology Co. Ltd in Guangzhou, China
as a core technician and technical support team
Theranostics 2017, Vol. 7, Issue 16
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3947
member respectively. Since 2016, he has been
pursuing a PhD degree at Murdoch University under
the supervision of Dr. Rakesh N. Veedu. His current
research focuses include the development of novel
RNA targeting therapies for tackling Duchenne
muscular dystrophy and Type-2 Diabetes.
A/Prof. Peter R. Dodd
obtained his PhD from
Imperial College, University
of London, and is currently
the Director of Queensland
Brain Bank at the School of
Chemistry and Molecular
Biosciences, The University of Queensland, Brisbane,
Australia. His lab uses human autopsy tissues to
study amino acid neurotransmission, and has
developed protocols to prepare good-quality mRNA,
miRNA, and proteins for mapping and quantification.
His lab studied phenotype-genotype interactions in
alcoholics and dementia cases, partitioned by sex and
comorbid disease, with key findings including altered
expression of GABA receptors in alcoholics without
comorbid disease and of glutamate receptors in
cirrhotic alcoholics, and altered expression of
glutamate transporters and receptors in Alzheimer
disease. Prof. Dodd’s group was the first to use
microarrays to study the human brain transcriptome.
Overall, his lab researches the pathogenesis of human
neurological diseases.
Rakesh N. Veedu is
currently leading the precision
nucleic acid theranostics group
at Murdoch University and
Perron Institute for Neuro-
logical and Translational
Science. He obtained his PhD
in synthetic organic chemistry in 2006 from The
University of Queensland, Australia under the
supervision of Prof. Curt Wentrup after completing
his MSc from Griffith University, Australia. He then
continued his postdoctoral career under the
supervision of Prof. Jesper Wengel at the Nucleic Acid
Center, University of Southern Denmark in the field
of nucleic acid chemical biology. Later in 2009, he was
appointed as a Research Associate Professor within
the Nucleic Acid Center. He then returned to The
University of Queensland in mid-2010 and established
his independent research career in Functional Nucleic
Acid Theranostics Development. His current research
is focused on developing novel precision nucleic acid
theranostics for tackling solid cancers, neurological
diseases including Alzheimer’s disease and infectious
diseases using nucleic acid aptamers, antisense
oligonucleotides, siRNA, antimiRs, molecular beacons
and DNAzymes.
