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![Page 1: Nicb Research Overview](https://reader031.fdocuments.us/reader031/viewer/2022030315/587e016a1a28abe11a8b4c0f/html5/thumbnails/1.jpg)
Nat ional I nst itute For Cellular B iotechnology
Research Overview
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NICB – Dublin City University
• Government-designated National Centre of Expertise in Basic
and Applied Molecular Cell Biotechnology since 1987
• New 3,200m2 building (opened 2006)
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Main Areas of Research
• Molecular basis for biopharmaceutical production by animal
cells
• Tissue Engineering/Stem Cell Therapy – ocular diseases,
diabetes
• Cells as research tools and models for disease research
• Cancer – drug resistance, invasion and biomarkers
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Cells as products
Stem cel l therapy/ Tissue engineering
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Tissue Engineering/
Stem Cell Therapy
• Twin focus – basic research and clinical application
• Corneal stem cell transplant – entering clinical phase
• Pancreatic islet transplant – currently technology
transfer stage (Partner: University of Oxford)
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• The Corneal surface consists of an
epithelial layer that, in the healthy eye,
is constantly renewed.
• Located at the limbus a narrow ring of
stem cells surrounding the cornea.
Stem Cell Treatments For
Eye Injur ies 1
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Stem Cell Treatments For
Eye Injur ies 2
• Patients with limbal deficiencies are unable to maintain a stable
cornea. e.g. Chemical and thermal burns, Stevens-Johnson syndrome
• Cornea transplantation in these patients typically fail due to
conjunctival invasion, vascularization, and persistent epithelial defect.
• The NICB in collaboration with its partners, proposes to introduce a
stem cell therapy for these patients.
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• Isolate and culture stem cells from the limbal ring.
These cultured cells are then transplanted on to the
surface of the injured eye, repopulating the
damaged cornea
• Research to refine techniques, find better
differentiation markers and investigate biology of
the process, e.g. using microarrays and proteomics
Stem Cell Treatments For Eye
Injur ies - 3
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Diabetes Research
at the NICB
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5-10% of these have severe
recurring hypoglycemia
23,000 adults in I re land
suffer f rom Type 1 Diabetes
(T1DM)
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Significant sustained decrease in
severe hypoglycaemic events and
improved quality of life.
Islet
transplantation
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Islets from the Donor’s pancreas are
infused into the recipient’s hepatic
portal vein and produce insulin
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Islet
Transplantation Islet Isolation &
Purification
Donated
Pancreas
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DRIVE – Horizon 2020
funded project
Diabetes
Reversing
Implants with enhanced
Viability and long-term
Efficacy
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Mitochondrial
Function
Oroboros oxygraph
Measures oxygen consumption
in cells and tissues in real time
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Cells as factories
Manufacture of recombinant
proteins
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Research Re la ted to B iopharmaceut ica l
Product ion
Our goal is to work closely with biopharmaceutical companies to improve basic
understanding of the molecular basis for recombinant protein production by
mammalian cells (including CHO and hybridomas) and
• to translate this understanding into improved biopharmaceutical
production processes
• Special focus on miRNA
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Why do we need improved b iopharmaceut ica l
product ion processes?
• Increasing number of products
• Current high unit cost
• Reduced production costs will contribute to
(a) Access for patients to treatments they need
(b) Long-term health of the biopharmaceutical industry
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Invited talks
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Cells as research tools
& as models for
disease research
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Cell culture
Access to l iving human material
- pr imar y cul tures
- ce l l l ines
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Cel l cul ture systems may not a lways
complete ly ref lect complex mult i -ce l l type
and spat ia l organisat ion in the body
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C o r e t e c h n o l o g i e s :
M o l e c u l a r p r o f i l i n g o f c e l l u l a r r e s p o n s e
e . g . t o d r u g s ; t o t e m p e r a t u r e c h a n g e : t o o x i d a t i v e s t r e s s
• mRNA (Affymetrix)
• miRNA (ABI)
• Proteins
• Importance of bioinformatics analysis
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Proteomics
• Proteomics Infrastructure at the NICB
• 2D DIGE (Difference Gel Electrophoresis)
• Mass Spectrometry for Protein
Identification/Characterisation, Including
phosphoproteomics
• Quantitative LC-MS approaches (i.e. gel-free)
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Mass Spectrometr y Suite
• LTQ Orbitrap XL (Thermo Fisher Scientific)
High mass accuracy
Interfaced with Dionex Ultimate 3000
1D and 2D LC capabilities
• LTQ XL with ETD
Electron Transfer Dissociation (ETD)
PTM analysis (e.g. phosphorylation)
• MALDI TOF-TOF 4800 (Applied Biosystems)
High throughput MALDI MS/MS
LC-MALDI
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Using proteomic profi l ing to identify
new cancer markers in blood
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Cancer Biomarkers
for What?
• Early detection
• Monitoring of tumour burden (response to
therapy)
• Detection of relapse
• Predicting response to specific therapy
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Advanced Lung
Cancer Haptoglobin ELISA
0
1000
2000
3000
4000
5000
6000
ug
/mL
Control
Squamous
Adenocarcinoma
Small cell carcinoma
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Ultimate aims of our
Cancer Research Programmes
Pa i red d iagnos t i c and therapeut i c approaches to
t rea tment of ind iv idua l cancer pa t ients
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Toxicity Testing Drug Resistance in Cancer Mechanisms of Cancer Cel l Invasion
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CHEMOTHERAPY WORKS,
BUT ONLY SOMETIMES:
WHY?
One reason is drug resistance
(acquired or intrinsic)
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Adr iamyc in D is t r ibut ion in Res i s tant Cancer Ce l l s
DLKP-A resistant cancer cells
after exposure to Adriamycin.
Fluorescent view
DLKP sensitive cancer cells after
exposure to Adriamycin
Fluorescent view
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Immunodetection of MRP-1
MRP-1 Negative Breast Carcinoma MRP-1 Positive Breast Carcinoma
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Cancer invasion & Metastasis
• Cell models, including clonal variants
• In vitro properties and investigation of relevance in human cancer
• Molecular profiling
• New antigens/monoclonal antibodies
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Therapeutic Targeting Using ADCs
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Ident i f i ca t ion and Inves t iga t ion of nove l
membrane prote in ta rgets w i th h igh cancer
spec i f i c express ion for potent ia l the rapeut i c
ta rget ing us ing Ant ibody Drug Con jugates (ADCs )
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Colon Cancer
The 9E1 target antigen is highly
expressed in colon cancer and show
limited expression in normal colon
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Pancreatic Cancer
Novel membrane target is highly
expressed in Pancreatic Cancer
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Breast Cancer
Novel Membrane Target is highly
expressed in triple negative breast
cancer (TNBC) and shows very
limited expression in normal breast
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Development of funct ional ant i - invas ive
MAbs to ident i f y potent ia l drug targets
associated with cancer invas ion/metastas is .
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MAb 7B7 target ing the Ku70/80 heterodimer
s igni f icant ly b locks the invas ion of
MiaPaCa-2 pancreat ic cancer ce l ls
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Ef fec t of LY6G6F s iRNA on 2D co lony format ion
of Mia Paca-2 ce l l s
Cell number seeded:
100 200 400
Control
Negative
siRNA
LY6G6F
siRNA #2
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Uveal Melanoma
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Proteomic Analysis of uveal melanoma
to understand metastatic disease
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Enucleation & Plaque
Radiotherapy
Treatments for
Primary Tumor
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metastasis in
50% of cases
Primarily to the liver with
average survival of
5-8 months
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Loss of chromosome 3
Class I v Class 2 gene signature
Loss of BAP1
GNAQ/GNA11 mutation
Current
Biomarkers
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A i m s o f t h e S t u d y :
I d e n t i f y d i f f e re n t i a l l y e x p re s s e d p ro t e i n s i n p r i m a r y
u v e a l m e l a n o m a t i s s u e s o f pa t i e n t s w h o d e v e l o p e d
m e t a s t a t i c d i s e a s e v e r s u s t h o s e w h o d i d n o t .
U n d e r s t a n d t h e b i o l o g y o f t h e d i s e a s e
I d e n t i f y n e w t h e r a p e u t i c t a rg e t s
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Cancer Biotherapeutics
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Background
• NSABP-B31 (Paik et al. 2007) and N9831 (Perez et al.
2010)
• Could a strong immune response to trastuzumab in the
adjuvant setting be responsible for the response in HER2
low patients?
• Trastuzumab has two mechanisms of action 1) it inhibits
HER2 signaling and 2) engages the immune system
through ADCC.
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Antibody-Dependent Cell-
mediated Cytotoxicity
ADCC
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NK cells and peripheral blood
mononuclear cells (PBMCs)
Immune cells from
healthy Volunteers
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Immunoblotting and high
content analysis.
HER2 Levels
quantified
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Examine the effects of TKIs on
HER2 expression in a HER2-
positive cell line (SKBR3)
Laser scanning
Confocal
Microscopy
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Determining the effect of PD-1/PD-L1
inhibition on response to
trastuzumab in preclinical HER2-
expressing breast cancer models.
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Assessing the expression of
immunomodulatory proteins in
HER2-targeted therapy-resistant
tumour cells.
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Pancreatic Cancer Programme
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• St Lukes Radiation Oncology
Network, Rathgar
• University of Buffalo
Collaboration
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Generation of primary pancreatic
cancer and stromal cell lines
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With pancreatic cancer
cells and how they impact
treatment success
Stromal Cancer
Cell interactions
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L e v e l o f c o l o n y f o r m a t i o n i n P a n c - 1 c e l l s p o s t c o - c u l t u r e
w i t h p a n c r e a t i c p a t i e n t - d e r i v e d f i b r o b l a s t s .
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Medicinal Inorganic Chemistry
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artificial
metallonuceases
• First reported “self-active” oxidative system
capable of inducing single-stranded DNA scission
in the absence of exogenous reductant or oxidant.
• Copper based complex, di-nuclear structure.
• Displays excellent in vitro chemotherapeutic
activity toward cisplatin-resistant ovarian cancer.
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• Detection and quantification DNA
oxidation by synthetic artificial
metallonucleases using capillary
electrophoresis.
• Analysis completed using Agilent
Bioanalyzer 2100 located within the
molecular biological laboratory at NICB.
Chemical nuclease detection
using microfluid ics
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• Potent non-covalent DNA binding agents where nucleic
acid recognition is achieved through use of the
“phosphate clamp”.
• Phosphate clamp-DNA interactions result in
condensation of superhelical and B-DNA.
• Triplatin-DNA binding inhibits endonuclease activity by
type II restriction enzymes.
• High chemotherapeutic potential for human cancer.
Novel chemotherapeutic
platinum( I I ) complexes
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• Detection of apoptosis induced mitochondrial
depolarization through exposure to ROS active
copper developmental therapeutics.
• Flow cytometry and confocal imaging analyses.
• Broad spectrum of activity identified using the
National Cancer Institute (NCI) 60-human cancer
cell panel.
• Unique mechanism compared to marketed
therapeutics.
Copper chemotherapeutic
drug development
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• Individual morphine molecules and derivatives
thereof lack nucleic acid recognition properties.
• In the triplet drug form unique properties
emerge with this alkaloid substructure interacting
with dsDNA and condensing superhelical DNA.
Tripodal opio ids as
unique DNA interacting
agents
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Phenazine based
biomaterials
• Enhanced high affinity DNA binding
• Distinctive nucleotide binding
specificity.
• High intercalative capacity
• In vitro chemotherapeutic potential
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• Metal catalyzed reactive oxygen species (ROS)
in biological systems can cause a wide variety of
pathological conditions including cancer.
• The extent of DNA damage owing to these
radicals can be quantified through 8-oxo-2’-
deoxyguanosine (8-oxo-dG) lesion detection
using both ELISA or LC-MS/MS analysis.
Detection of free radical
DNA damage
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Metallodrug
topoisomerase
inhibitors
• Development of intercalating metal complexes to
unwind and relax negatively supercoiled DNA.
• Applications as unique topoisomerase inhibitors.
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Collaboration with Industry
• Cells as factories
• Cells as products
• Cells as Research Tools
• Cells for toxicity assay and bioassay
• Making monoclonal antibodies
• Whole genome mRNA and miRNA profiling
• Proteomic profiling
• Pharmacokinetics/drug analysis
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Some past & present industr y col laborators
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Quest ions?
N a t i o n a l I n s t i t u t e f o r C e l l u l a r
B i o t e c h n o l o g y
D u b l i n C i t y U n i v e r s i t y ,
G l a s n e v i n , D u b l i n 9 , I r e l a n d .
+ 3 5 3 1 7 0 0 5 7 0 0
w w w. n i c b . i e
n i c b @ d c u . i e