Nanocarrier based co-delivery of siRNA against

BACE1 (ß-site APP cleavage enzyme 1) of amyloid

percussor protein (APP) as effective neurotherapy for
Alzheimer’s disease.

A project proposal submitted to

Department of Microbiology and Biotechnology Centre
The Maharaja Sayajirao University of Baroda.

As a part of
Master’s Degree in Microbiology
(2019-2021)
By
AYUSHI VAKIL
Department of Microbiology and Biotechnology Centre
Faculty of Science
The Maharaja Sayajirao University of Baroda
Vadodara- 390002 Gujarat, India.

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Content

Title Page no.

Abstract 3
Introduction 4-5
Origin of proposal 6
Rationale 7
Objectives 8
Work plan and methodologies 9-12
Schedule 13
Budget 14-15
References 16

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Abstract
The disorder of Alzheimer’s is marked by progressive pathophysiological
neurodegeneration. The amino acid peptides in the amyloid plaques found in the
brains of people with Alzheimer’s disease (AD) are known as amyloid-beta (Aβ).
Current treatments are not curative, and the effects associated with AD are
reduced. Improving treatment results involved the targeting of drugs at optimum
therapeutic concentration. Nanomedicine and nano delivery systems are a
relatively new but rapidly developing science where materials in the nanoscale
range are employed to serve as means of diagnostic tools or to deliver therapeutic
agents to specific targeted sites in a controlled manner. Nanotechnology offers
multiple benefits in treating chronic human diseases by site-specific, and target-
oriented delivery of precise medicines. Small interfering RNAs (siRNAs) show
great promise for AD therapy by specific silencing of BACE1.
However, lack of effective siRNA brain delivery approaches limits this strategy.
Here, I am intending to develop a glycosylated "triple-interaction" stabilized
polymeric siRNA nanomedicine (Gal-NP@siRNA) along with the dose of anti-
acetylcholinesterase to target BACE1 in APP/PS1 transgenic AD mouse model.
Gal-NP@siRNA would exhibits superior blood stability and can efficiently
penetrate the blood-brain barrier (BBB) via glycemia-controlled glucose
transporter-1 (Glutl)-mediated transport, thereby ensuring that siRNAs decrease
BACE1 expression and modify relative pathways. Gal-NP@siBACE1 when co-
delivered with the donepezil drug used in AD therapy, it is hypothesised that the
administration will restore the deterioration of cognitive capacity in AD mice
without notable side effects. This synergic strategy supports the utility of RNA
interference along with the neurotherapeutic drug in neurodegenerative diseases.

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Introduction
● Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder
characterized by memory, cognition and behavioural impairment
(dementia), and it eventually leads to mood fluctuation and fatal delirium.

● The hallmark histological abnormalities of AD comprise of the

extracellular aggregation of amyloid plaques and fibrillar aggregates of
the microtubule associated with tau protein. The deposition of amyloid
plaque, which is caused by the mounting production, accumulation, and
aggregation of the amyloid- ß(Aß), is the primary histopathological
characteristic of AD.

● The most widely accepted AD etiology is the amyloid cascade hypothesis,

the incorrect process of the Aß protein precursor (AßPP) by Ɣ-secretase,
which gives rise to the pathological 40–42 amino acid long cleaved peptide,
known as Aß, is fundamental to this hypothesis. The excess of Aß finally
results in the aggregated fibrils of plaques and neurotoxic oligomers.

● Conventional therapeutic approaches, including inhibitors of acetylcholine-

esterase, often fail due to insufficient solubility, decreased bioavailability
and a lack of obstacles to the brain and blood. This disease, one of the
biggest global healthcare problems, is the most common form of dementia.
It is a neurodegenerative disease causing progressive cognitive performance
and memory loss.

● Currently for AD, the early diagnosis could provide treatment opportunities
to high-risk groups, and the way to cure diseases under development seems
to be proven effective only at the very early stages of the initiated amyloid
deposition.

● RNA interference (RNAi) is emerging as a powerful approach in cancer

treatment. siRNA is an important RNAi tool composed short length (18—
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 25 nucleotides) double strand RNAs that either inhibit translation initiation
in ribosomes or destruct target mRNAs by cellular ribonucleases. Due to
sequence specific targeting of siRNA and its potential to inhibit protein
translation; siRNA is considered as a potential drug for gene therapy.
However, there are two major obstacles for siRNA treatment are; first quick
degradation and elimination of siRNA from blood stream and body fluids,
and second less cellular uptake of siRNA by target cells.

● The use of nanotechnology to develop nano delivery systems can overcome

these limitations. Nanocarriers are colloidal drug carrier systems having
submicron particle size typically~500nm.The characteristic features of
nanocarrier are high surface area to volume ratio, ability to alter basic
properties and bioactivity of drugs, improved pharmacokinetics and
biodistribution, decreased toxicities, improved solubility and stability,
controlled release and site-specific delivery. Therefore, the use of
nanocarriers to deliver siRNA, prevents both renal clearance and RNase
degradation by protecting siRNA chains, increasing their half-life in blood
and through active targeting mechanism the uptake of siRNA by target cell
can also be increased.

● Applying nanotechnology in drug delivery systems has improve the

bioavailability and kinetic profile of drugs in biological systems. Advances
in nanotechnology aid in targeting drugs to specific molecular targets and
safely delivering drugs to specific sites of action. The sustained release of
nano-drug delivery systems enhances the controlled release profile of
loaded drugs, thereby minimizing the dosage-regimen. Thus, the overall
effectiveness of drug candidates can be enhanced by adapting nano-drug
delivery systems along with combinational drugs for the symptomatic
treatment in AD therapeutics.

Origin of Proposal
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Alzheimer’s disease (AD) is the most common age-related neurodegenerative
disorder, characterized by progressive deterioration of cognitive capacity.
Currently, clinical therapy using acetylcholinesterase inhibitors or N-methyl-D-
aspartate receptor antagonists are palliative treatment options, which only
moderately improve cognition and behaviour in Alzheimer’s patients but do not
slow disease progression. Hence, it is imperative to develop therapeutics
targeting pathological mechanisms in AD.

The precise pathological mechanisms leading to AD are not fully understood.

However, plaques composed of aggregated amyloid-ß peptide (Aß),
neurofibrillary tangles containing hyperphosphorylated tau protein, and
neuroinflammation are pathological hallmarks. Among these, the aberrant
accumulation of Aß resulting from the sequential cleavage of the amyloid
precursor protein (APP) by BACE1 (ß-site APP cleavage enzyme 1) and Ɣ-
secretase activity is believed to be a key pathogenic event in AD, strategies that
reduce BACE1 activity, and thereby Ap levels, have been considered as potential
therapeutics for AD.

BACE1 can become an ideal drug target since this aspartyl protease is involved
in the abnormal production of ß amyloid plaques (Aß), Thus evidence suggests
that there is a strong connection between AD and BACE1; BACE1 appears to be
a prime target to prevent Aß generation in AD. Silencing of BACE1 expression
by treatment with siRNA along with nanocarrier in combination with the anti-
acetylcholinesterase drug may lead to therapeutic efficiency of the drug and
prevent the later stage amyloid plaque formation as well as increases the level of
acetylcholine in the neuro receptors and thus causes improvement in the
cognitive capacity and memory loss. Moreover, for targeted delivery and
prevention of premature siRNA degradation in-vivo nano encapsulation of
siRNA and antimitotic drug with specific surface ligand can be performed. This
approach, if successful, can lead to rapid treatment of the diseased neuron with
minimal damage to the nearby healthy cells.

Rationale
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Nanocarrier-based co-delivery systems using siRNA/anti- acetylcholinesterase
drug combinations show a new ray of hope for novel treatment strategies for
Alzheimer’s disease.

Thus, developing a nanocarrier siRNA-based gene therapy with anti-

acetylcholinesterase might help in:
1. Directly blocking causative gene expression
2. High target specificity
3. Low effective dose
4. Simple drug development process
5. Reduction of side effects caused by neurotherapeutic drugs

Objectives
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 ⮚ Objective 1- Formation of glycosylated triple-interaction stabilized siRNA
nanomedicine (Gal-NP@siRNA) enclosing si-BACE1 and anti-
acetylcholinesterase drug (Donepezil) as an active target against AD.

⮚ Objective 2- To evaluate the uptake of the targeted NPs into the affected
Neuro-2a; APP/PS1 transgenic mice with a PBS Gal-NP@siRNA as
control.

⮚ Objective 3- To test behavioural evaluation and the reduction in amyloid

plaque of AD compromised neurons of double transgenic mouse by the
synergistic effect of siRNA and anti-acetylcholinesterase drugs.

⮚ Objective 4- To evaluate the bioactivity of NPs in the increased level of

acetylcholine in the neurons.

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 Work plan and Methodologies
OBJECTIVE 1- Formation of glycosylated triple-interaction
stabilized siRNA nanomedicine (Gal-NP@siRNA) enclosing si-
BACE1 and anti-acetylcholinesterase drug (Donepezil) as an active
target against AD.

o A glycosylated nano-delivery system is designed using glycemia-

controlled Glutl (glucose transporter-1) to facilitate nanomedicine
penetration through BBB for more effective AD therapy.

o To improve biophysiological protection of the encapsulated siRNA,

a ―triple-interaction‖ stabilization method is introduced, in which the
anti-acetylcholinesterase drug- ―Donepezil‖ alongside with guanidium
phosphate (Gu+/PO3) would forms a salt bridge to provide a stabilizing
electrostatic and hydrogen bond interaction in addition to hydrophobic
interaction formed during addition of siRNA in the carrier.

o siRNA concept is introduced in the nanomedicine by complexation

between siRNA and Gal-PEG-b-P(GUF) by reversible addition
fragmentation chain transfer and co-polymerization of hydrophobic
monomer and siRNA.

o The characterization of these nanocarrier medicines is done using

dynamic light scattering (DES) and transmission electron microscopy
(TEM), which will show the morphology and size of the nanomedicine.

Expected outcome- Galactose-modified siRNA along with the anti-

acetylcholinesterase drug, nanomedicines/nanocarrier should bind to
Glutl to efficiently penetrate the BBB by glycemia-controlled Glutl -
mediated transport.

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OBJECTIVE 2- To evaluate the uptake of the targeted NPs into the
affected Neuro-2a; APP/PS1 transgenic mice with a PBS Gal-
NP@siRNA as control.

o Flow cytometry analysis and confocal imaging will show whether the
glycosylated and non-glycosylated (CONTROL)siRNA nanomedicine is
taken up by the Neuro-2a cells of the APP/PS1 double transgenic mouse
model along with a PBS-GalNp@siRNA as control.

o To quantify the efficiency of siRNA silencing of the target gene, BACE1

mRNA and protein expression will be examined in a Neuro-2a cell sample
using gel retardation assay of GAL-NPsiRNA.

Expected outcome- BACE1 genes will be sufficiently silenced in Neuro-2a cells

when treated with Gal-NP@siRNA (siBACE1) and its mRNA and protein will
show down-regulation expecting no obvious toxicity in multiple neuron related
cells. While in contrast to that the control system will fail to reduce BACE1
mRNA and proteins level, corroborating the sequence specific gene silencing
activity of siBACE1

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OBJECTIVE 3- To test behavioural evaluation and the reduction in
amyloid plaque of AD compromised neurons of double transgenic
mouse by synergistic effect of siRNA and anti-acetylcholinesterase
drugs.

The affected/ observed APP/Ps1 double transgenic mouse model would be assessed for
behavioural tests of learning and memory impairment
o Behavioural tests will be conducted and it will include the Novel
Recognition test (NOR) and Morris Water Maze (MWM)to examine spatial
learning and memory. Nest building test will also be performed to accesses
the general health and hippocampal function.

Later the mice will be sacrificed and brain tissues will be collected for analysis of BACE1
suppression and its impact on Aß and tau pathological accumulation.
o To further assess the biocompatibility and systemic response to the
nanomedicine, routine blood parameters and chemistry would be accessed
by measuring plasma alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP), plasma urea (BUN),
uric acid (UA), creatinine (CR), as well as blood platelet (PLT), red blood
cells (RBCs), and white blood cells (WBCs).

o Confocal microscopy would be performed on the Neuro-2a cells which

would be cultured on microscope slides in 24-well plates (1 x 105 cells per
well) and incubated with Nanomedicine.

Expected outcome- Gal-NP@siBACEl nanomedicine mediates highly effective

siRNA brain delivery to significantly improve cognitive performance in
APP/PS1 mice.

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OBJECTIVE 4- To evaluate the bioactivity of NPs in the increased level of
acetylcholine in the neurons.

o The techniques such as micro dialysis will be employed on regular time

interval to check the level of acetylcholine in the brain of the affected
mouse.

Expected outcome- with increasing dose of Nanomedicines and their effects the
level of acetylcholine is expected to rise with time, as the drug directed in the
nanocarrier will work in favour of inhibiting the anti-acetylcholinesterase and
increasing the amount of acetylcholine in the brain.

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 Schedule
Objective Time
required
Formation of glycosylated triple-interaction stabilized
siRNA nanomedicine (Gal-NP@siRNA) enclosing si-
BACE1 and anti-acetylcholinesterase drug (Donepezil) as an 4 months
active target against AD.

To evaluate the uptake of the targeted NPs into the affected

Neuro-2a; APP/PS1 transgenic mice with a PBS Gal-
NP@siRNA as control. 4 months

To test behavioural evaluation and the reduction in amyloid

plaque of AD compromised neurons of double transgenic
mouse the by synergistic effect of siRNA and anti- 6 months
acetylcholinesterase drugs.

To evaluate the bioactivity of NPs in the increased level of

acetylcholine in the neurons 1 month

Total 15 months

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 Budget
Non- Recurring cost
Name of the item Quantity Price
(INR)
Mouse neuroblastoma cell line 1 34,930
(N2a[TRb1])
Double transgenic mutant mouse 6 19,31,580
Double-stranded siRNA oligomers 1 48000
Lipofectamine RNAiMax (Invitrogen) 1 36000
Coulter DNA Prep Reagents kit 1 600000
(Beckman-Coulter)
LSRII flow cytometer (Becton- 1 1520000
Dickinson)
Vi-CELL Viability Analyzer (Beckman 1 1098000
Coulter)
Ultracentrifuge 1 900000
UV spectrophotometer 1 18000
Zeiss Confocal Microscope 1 100,00,000
system (Zeiss 880)
Zetasizer advance-DLS 1 6,50,000
Zeiss TEM 1 6,650,000
Molecular Devices, microplate reader 1 1,00,000
Molecular 1 17,2000
Imager FX (Bio-Rad, Hercules, CA)
Near-infrared fluorescence imaging 1 2500000
system (Lumina,
IVIS III
RT-PCR 1 15,00000
standard individual ventilation cages 6 42,000
animal experimental system
radioimmunoprecipitation assay lysis 5 19,250
buffer with a proteinase and
phosphorylase inhibitor cocktail
(Thermo Fisher Scientific)
BACE1 polyclonal antibody 1 38,871
Total Non-Recurring Cost- 27,216,631
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Recurring cost
1 Chemicals and consumables 2,00,000
2 Rat maintenance 1,00,000
3 JRF (25,000 per month + 20%HRA) 5,70,000
4 Overhead expenditure 1,00,000
Total recurring cost: - 9,70,000

TOTAL COST OF THE PROJECT- 28,186,631

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