Anovasia is a Singapore-based company that has developed a platform technology called Molecular Painting (MP) to modify cell and virus surfaces. MP reagents can tag surfaces for research, diagnostics, vaccine development, and targeted drug delivery. MP allows non-specific surface modification without affecting native proteins. Anovasia supplies both stock and custom MP reagents and sees applications in basic research, diagnostics, vaccine development, and cell and gene therapy.

1. molecular painting
“just right on the face of it”

2. molecular painting
Technology Presentation (Nov. 2012)

3. Executive Summary
• Founded and based in Singapore, Anovasia is a solution provider for the life sciences
industry
• Anovasia has a unique platform technology called Molecular Painting (MP)
– MP is the surface modification of biomembranes
– MP reagents are based on natural products
• The main industrial application areas are:
– Basic and Applied Research
– Diagnostics, Biomarkers
– Vaccine Development
– Targeting and Delivery of Genes, Proteins and/or Drugs
– Cell and Gene Therapy
• Anovasia supplies products from its existing MP range as well as made-to-order,
designer reagents based on client’s individual needs
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4. Technology Overview
• Molecular Painting reagents uniquely allow non-specific biomembrane
surface modification
• Well documented for surface engineering of many cell types. Generally
CELL
considered to be possible with any cell type (except some forms of bacteria)
• Anovasia team was first to show for HIV lentivirus and MLV retrovirus (Metzner
et al., 2008)
VIRUS
• Anovasia team recently also shown for wild-type herpes and influenza viruses
(manuscript submitted)
EXOSOME • Other membrane entities such as membrane vesicles, lipsomes and exosomes
can be painted
• Anovasia team recently shown for exosomes (data unpublished, further work in
LIPOSOME
progress, patent application submitted in 2011)
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5. How does Molecular Painting Work?
- three easy steps
1. Prepare your sample
– example on next slide is a viral gene delivery vector
– other sample types: liposomal drug carrier, exosome, etc.
2. Chose the functionality of your MP reagent
– e.g. fluorescent tag (A)
B C D functional element
– e.g. immune-modulation (B) A
– e.g. magnetic particle (C) spacer region
– e.g. ligand-receptor targeting (D)
biomembrane prong
3. Mix together in a quick 10 min reaction
– see next slide
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6. Viral gene delivery vector modified with two MP reagents
MP reagent, e.g. ligand-receptor targeting
(pre-purification, targeting in vivo)
virus capsid
virus envelope
proteins
tissue/cell specific
promoter
therapeutic
gene
MP reagent, e.g. immune-modulation
(protection, longevity in vivo)
virus shell
(biomembrane)
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7. Key Advantages of the MP Technology
(compared to currently used / state-of-the-art technologies)
• Membrane modification is non-specific
– No prior information
– No antibodies required
– All entities can be tagged (liposomes, cells, viruses, exosomes etc.)
– No heavy lab equipment required for experiments
• Existing surface molecules NOT affected
– Viral vectors remain infectious
– Viral life cycle not influenced
– No need to change existing delivery vehicle (e.g. drug carrying liposome, gene vector)
• No genetic modification required
– Quick, simple and inexpensive
• Highly versatile
– Multiple MP molecules can be attached to the same target
– MP molecules can be dosed in varying amounts
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8. Technology Platform – Wide Fields of Application
• Research
– Tagging
– Isolation and purification
– Imaging
CELL – In vivo tracking
– Product development
• Diagnostics (infectious agents, biomarkers)
– Tagging
– Isolation and purification
– Imaging
VIRUS
• Vaccine Development
– Cell targeting
– In vivo vector protection
• Medical
EXOSOME – Immune evasion of transplant material
– Drug delivery (targeted liposomes)
• Cell and Gene Therapy
– Labelling of therapeutic stem cells
– Cell targeting
LIPOSOME – Protection from immune system
– Immuno-modulation
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9. I. Applications and Supportive Data
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10. Applications
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11. Nanoparticles for Linking to MP Reagents
Ni
Such complexes can be potentially used Iron, phospholipid micelle
for many applications such as purification,
Ni-NTA coated MNPs
isolation, concentration of vectors for
production or diagnostics used or even
for targeting in vivo.
Nickel based MNP Type 1 Nickel based MNP Type 2
Transmission electron microscopy 50 nm 5-100 nm
5-10 nm
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12. Biomarker / Diagnostics - Case Study
• Isolation of exosomes at physiological concentrations
– Use MP reagent to tag the surface of exosomes in a clinical sample and purify
– Use downstream method like quantitative real-time PCR to detect marker
2. Add tagged GPI protein 3. Add nickel or nickel-NTA
Fluorescence
coated magnetic particle
GPI-His
GPI-GFP Excitation
Ni
Viral Particlessample
1. Biological Visualisation/tracking of
4. Isolation from sample
virus particles
with magnet
5. Assay, e.g. EM, PCR, RT-PCR
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13. Cell Targeting – Transduction Targeting
F
B1 – B4
F
B
Producer cell R
Technology: any virus can be
quickly and easily surface
B Binding protein modified for targeting to any
F Fusogenic protein cell type in vivo
R Receptor Target cell
Application examples:
a) Antigen presenting cells can be targeted for more effective vaccination
b) Cancer cells can be targeted. The virus can be carrying marker genes for
imaging and/or suicide genes for specific cell killing.
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14. Multi-functional Vectors for Targeting and Delivery
allow the vector to
track down target
modify with MP
cell, deposit a
marker and/or
deliver genes or
drugs
e.g. liposome
carrying drug and/or gene
R
fluorescent MP reagent
targeting MP reagent
detection/ imaging
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15. Delivery of Protein Complexes
modify with MP
e.g. liposome Vector targets
based vector biomarker or
imaging target and
delivers substrate
for enhanced
reaction locally,
targeting MP reagent e.g. imagining,
biomarker
detection of cells
or other molecules
Linker MP reagent
protein complex payload
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16. Molecular Painting can be used to modulate the immune system (I)
Imagine a virus vector in the blood system...
…leading to its destruction.
viral vector in the blood
complement molecules
…it will be attacked by immunity factors…

17. Molecular Painting can be used to modulate the immune system (II)
However, a vector that is painted…
…with a cell targeting MP reagent…
…as well as a immune-protective MP reagent…
e.g. cancer or antigen
…will be protected from immune attack… presenting cell
complement molecules
…allowing unhindered, targeted gene
transfer or immune stimulation.

18. Tracking therapeutic stem cells
Challenge:
• The fate of many therapeutic stem cells in vivo remains an
open question
• Scientists, companies, authorities and regulators alike require
better control and understanding of this
Solution:
• Molecular Painting allows tagging of stem cells for tracking,
imaging and potential removal
• The unique characteristics of the stem cells are not changed
because MP does not interfere with cell surface proteins or
cause signal transduction
– The MP reagent is simply inserted into inert areas of the outer-
membrane, where they go undetected by the cell
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19. Summary of MP features for which Anovasia
has Experimental Proof-of-Concept
• Labeling for tagging, tracking, imaging and/or isolation from dilute samples
• Link to nano- and micro-particles
• Deliver genetic material
and/or
• Deliver a functional protein load
• Protect from active serum
• Possible with any protein (produced/tested so far)
• Proteins are always functional (produced/tested so far)
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20. II. Technical Overview of Molecular Painting
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21. The Key to MP – the GPI Anchor
o Glycosylphosphatidylinositol
anchor
o Post-translational modification
o Conserved backbone structure
o External face of cell membrane
o GPI signalling sequence
o Recombinant anchoring
o Releasing enzymes
o Hypermobility
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22. Engineering Methods for Viral Vectors
Metzner C et al., Virology 2008
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23. Detecting Virus Surface Modification
Viral Particles
Purification Analysis
CD59his
Painting
RV c
CD59 kD
170
130
MLV CA 95
- + - + - + 72
CD59his 5%
ME PC VP 56
43
34
LV CD59 26
17
HIV p24
100 %
10 %
Before
After
CD59his 10% - + - + - + Marker
ME PC VP
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24. Publications
• Metzner, C., Mostegl, M.M., Günzburg, W.H., Salmons, B. and Dangerfield, J.A. (2008) Association of
glycosylphosphatidylinositol-anchored protein with retroviral particles. FASEB Journal 22(8): 2734-2739.
• Metzner, C., Salmons, B., Günzburg, W.H. and Dangerfield, J.A. (2008) Rafts, anchors and viruses – A role for
glycosylphosphatidylinositol-anchored proteins in the modification of viruses and viral vectors. Virology 382(2): 125-31.
• Lear, M.J., Salmons, B., Günzburg, W.H. and Dangerfield, J.A. (2009) Singapore R&D and globetrotting.
Biotechnology Journal 4: 179-85.
• Dangerfield, J.A. and Metzner, C. Main and co-editor for the E-Book “GPI Membrane Anchors - The Much Needed
link” (2010) Bentham Science Publishers. eISBN: 978-1-60805-123-6.
• Metzner, C., Legler, D.F. and Dangerfield, J.A. (2010) Chapter 6: Surface engineering of biomembranes with GPI-
anchored proteins and its applications, p. 83-97. In J. A. Dangerfield, Metzner C. (ed.), GPI Membrane Anchors - The
Much Needed Link, vol. 1. Bentham Science Publishers. E-Book, eISBN: 978-1-60805-123-6.
• Metzner, C. and Dangerfield, J.A. (2011) Chapter 3: Surface modification of retroviral vectors for gene therapy, p. 41-
72, in K. Xu (ed.), Viral Gene Therapy, vol. 1. InTech Open Access Publishers. E-Book, eISBN: 978-953-307-539-6.
• Metzner, C., Kochan, F. and Dangerfield, J.A. (2012) Fluorescence molecular painting of enveloped viruses.
Molecular Biotechnology, Oct 28, DOI: 10.1007/s12033-012-9616-6.
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25. Current Products and Pipeline
please see www.anovasia.com for more details
• Off-the-shelf MP Reagents
Ano-P001: Green-Glow*
Ano-P002: Immuno-Sheath*
Ano-P003: Targo-Tag*
• Made-to-Order MP Reagents
Based on customer requirements
• MP Reagents in Development
AnoR&D4: Immuno-Stim
AnoR&D5: Strepto-Multilink
AnoR&D6: Tomato-Tag
*contains linker(s)
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26. Contact
John A. Dangerfield
Founder and Managing Director
Anovasia Pte Ltd
20 Biopolis Way
#05-518 Centros
Singapore 138668
ACRA Bus. Reg. No. 201101692G
Tel: +65 9339 4927
Fax: +65 6774 5569
john@anovasia.com
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