1. No category

Improving the Diagnosis and Treatment of Alzheimer's Disease
Neurodegenerative Disorder
Affects memory, thinking and behavior.
Characterized by beta-amyloid plaques and
a specific miRNA profile.
Affects 5 million Americans. Kills more than
breast and prostate cancer combined.
Current diagnoses revolve around cognitive and
psychological evaluations.
-Amyloid Detection:
B-Cell Receptor
A: LilrB2 and PirB are natural, transmembrane receptors that selectively
bind beta-amyloid oligomers (Kim et
al. Science 2012). Once bound (1), they
recruit the cofactor cofilin (2), which is
fused to a protease. Upon recruitment
to the receptor, the protease cleaves
(3), releasing a transcription factor,
which can activate a treatment
response circuit (4).
C
B
E
HEK293 cells transfected with BCR B
DNA and assayed for beta-amyloid
binding using biotinylated betaamyloid oligomers and AlexaFluor
594-conjugated streptavidin. Flow
cytometry shows no correlation
between transfection and bound
beta-amyloid. B: Cells with no BCR
DNA (control). C: Cells with BCR DNA.
C
A
B
D
C
E
104
103
102
0
104
103
102
0
0
102
103
104
eBFP
105
0
102
103
104
105
D: Flow cytometry of HEK293 cells
transfected with L7ae repressible
output shows close to full repression
of output.
eBFP
E
F
HEK293 cells transfected with receptor DNA
and assayed for beta-amyloid binding
using biotinylated beta-amyloid oligomers
and AlexaFluor 594-conjugated
streptavidin. D: Confocal microscopy. E:
LilrB2 flow cytometry shows increase in red
fluorescence. PirB shows simlar
results.
Treatment
A
Delivery
Potential delivery mechanisms were
used to inform decisions regarding
our project. Two were
explored:
F: Acitvation of LilrB2 with beta
amyloid results in lower levels of
output (mKate) relative to when
no beta-amyloid is present. PirB
shows similar results. Sterics of
interaction between receptors
and cofilin may be responsible
for unexpected results.
D: HEK293 cells transfected with BCR
DNA
and
immunostained
with
AlexaFluor 488-conjugated antibodies
confirm BCR expression and membrane
localization.
Neurons in Alzheimer's express a
different miRNA profile than healthy
neurons. Sensors were built to detect
low levels of miRNA (A) and high
levels of miRNA (B). Truth table (C)
shows overall logic.
105
105
D
F
miRNA Detection
A: Synthetic B-cell receptor engineered to
bind beta-amyloid oligomers (Ostrowitzki
et al, Arch Neurol, 2011). Upon binding
(1), BCR is activated, recruiting the
cofactor Syk (2). Syk is fused to a
protease, cleaving upon recruitment to the
receptor (3). Cleavage releases a
transcription factor, which can activate a
treatment response circuit (4).
Alexa Fluor 594
HEK293 cell transfected with
receptor DNA and immunostained with AlexaFluor
488-conjugated antibodies
confirm LilrB2 expression. A:
Confocal microscopy. B: Flow
cytometry. PirB shows similar
results.
D
A
Current Treatments Inneffective
Alexa Fluor 594
A
-Amyloid Detection:
Native Receptors
6th Leading Cause of Death
E: Acitvation of BCR results in lower levels of
output (mKate) relative to inactive conditions.
Sterics of interaction between receptors and
Syk may be responsible for unexpected
results.
G
E: Modeling shows that different
repression mechanisms result in
different orders of magnitude of
repression.
H
HEK293 cells were transfected with high sensor for miR-125b. F: Cells
transfected with high sensor and no siRNA (control). G: Cells trasnfected with
high sensor and siRNA show increased output (GFP) indicating that high sensor
works as expected. H: Flow cytometry shows higher output (GFP) in cells that
were also transfected with siRNA corresponding to miR-125b.
C
B
D
A. BACE1 is involved in production of betaamyloid. BACE2 has been shown to degrade
beta-amyloid (Abdul-Hay, et al Molecular
Neurodegeneration, 2012).
The treatment
module down-regualtes BACE1 and upregulates BACE2, thereby reducing overall
levels of beta-amyloid.
B. BACE2 activity assay shows that
BACE2 is not properly induced.
HEK293 cells were tranfected with miRNA generating vectors.
mKate was used as a proxy for correct miRNA processing. C: Flow
cytometry showing proper miRNA processing (+ve control). D:
Little red fluorescence indicates incorrect processing of miRNA.
Ex-Vivo Delivery
In-Vivo Delivery
Involves extracting cells and engineering
them before introducing them back into the
patient.
No risk of random genome integration of
circuit.
No access to chemical pathways within
diseased cells.
No FDA approved treatments; several
clinical trials in progress (Wang, Discovery
Medicine, 2014).
Involves using a delivery vector (virus or
liposome) to deliver therapeutic DNA
directly to patient cells.
Risk of random genome integration of the
circuit.
Requires risky procedures to deliver.
Easy access to diseased cells.
One approved therapy and several in
clinical trials (Wang, Discovery Medicine,
2014).
Experience with Alzheimer's
and Receptiveness to Gene Thereapy
James Anderson, Lyla Atta, Kathryn Brink, Gary Burnett, Andrew Wei Chen, Erik Ersland, Alexa
Garcia, Alex Leffell, Jing Wei "Raymond" Liu, Raashed Raziuddin, Alex Leffell, Christian
Richardson, Shinjini Saha, Alexander Smith, Abigail Weiss, Jiaqi Xie
Advisors: Ron Weiss, Brian Teague, Jonathan Babb
E. BACE1 activity assay shows that miRNA generators
successfuly inhibit BACE1 activity
Hypothesis: People with personal experience with
Alzheimer's disease are more willing to undergo
experimental treatments than people who do not have
personal experinece with the disease.
Neither experience with Alzheimer's nor knowledge
of gene therapy affects respondent's willingness to
receive genetic therapeutics.
Respondents prefer less invasive delivery methods.
Survey results helped inform our decision to use
neuron-specific detection and treatment methods.