Cupid Peptides presentation to Clinical Innovation Partnership (CIP) : Proof of principle and basis of collaboration

1. William
Jonathan
Ryves
Cardiff,
U.K.

2.
Applica(ons
for
Cupid
Technology
• Cell
Marker
and
Tracking.
Real-­‐(me,
dye-­‐less
• Real-­‐(me
Protein
/
Pep(de
delivery
for
protein-­‐protein
interac(on
and
protein
func(on
mapping
• Drug
delivery
vehicle
for
cell-­‐impermeable
conjoined
API’s
• Regenera(ve
medicine
e.g
Safe
crea(on
of
Stem
Cells
ex-­‐vivo,
Cancer
diagnosis
/
therapy
in
vivo,
wound
/
burn
treatment
in
situ

3. Cell
Penetra(ng
Pep(des
classified
by
ac(on
Class
1
(e.g.
Synthe(c
Ca(onic
Pep(des)
• Short
strings
of
+vely
charged
amino
acids
e.g.
Polyarginine,
Polylysine
• Adhere
to
outer-­‐cell
membrane
through
charge
interac(on
• Endocytosed
into
vesicles
through
ac(on
of
membrane
recycling
machinery
Class
2
(e.g.
Viral
Pep(des)
• Short
strings
of
amino
acids
derived
from
viral
proteins
e.g.
TAT
from
HIV
• Adhere
to
outer-­‐cell
membrane
through
interac(on
with
receptor
protein
embedded
in
cell
membrane
• Endocytosed
into
vesicles
through
ac(on
of
receptor
recycling
machinery
Class
3
(Membrane-­‐Permeable
Pep(des)
• Short
strings
of
amphipathic
amino
acids
e.g.
Cupid
• Adhere
to
outer-­‐cell
membrane
through
charge
interac(on
• Pass
directly
through
lipid
bilayer
through
interac(on
with
both
hydrophilic
and
hydrophobic
parts

4. LIVE
FIXED
FIG. 2. Visualization of PTD–GFP fusion protein import in CHO cells. The recombinant proteins were added to the cells for 5 min at 37°C. The cells were washed
extensively with PBS and microscopy was performed either on live unfixed cells or after methanol fixation. FITC, fluorescein–isothiocyanate filter; PC, phase
contrast.
ARTICLEdoi:10.1016/S1525-0016(03)00135-7
FIG. 2. Visualization of PTD–GFP fusion protein import in CHO cells. The recombinant proteins were added to the cells for 5 min at 37°C. The cells were washed
extensively with PBS and microscopy was performed either on live unfixed cells or after methanol fixation. FITC, fluorescein–isothiocyanate filter; PC, phase
contrast.
ARTICLEdoi:10.1016/S1525-0016(03)00135-7
GFP
alone
VP-­‐22
GFP
TAT
GFP
K8
GFP
R8
GFP
FIG. 2. Visualization of PTD–GFP fusion protein import in CHO cells. The recombinant proteins were added to the cells for 5 min at 37°C. The cells were washed
extensively with PBS and microscopy was performed either on live unfixed cells or after methanol fixation. FITC, fluorescein–isothiocyanate filter; PC, phase
contrast.
FIG. 3. Adherence of PTD–GFP fusion proteins to prefixed CHO cells. The cells were fixed with methanol and rehydrated in PBS. Recombinant proteins were
added for 5 min at room temperature and the cells were washed extensively with PBS prior to microscopy. FITC, fluorescein–isothiocyanate filter; PC, phase
ARTICLEdoi:10.1016/S1525-0016(03)00135-7
BUT
Added
to
cells
AFTER
FIXATION
The
Lundberg
Revision:
How
different
condi(ons
can
result
in
misinterpreta(on
of
CPP
ac(on
Cell
surface
adherence
and
endocytosis
of
protein
transduc?on
domains.
Lundberg
M,
Wikström
S,
Johansson
M.
Mol
Ther.
2003
Jul;8(1):143-­‐50.
Problems
in
deploying
Class
1
and
2
CPP’s

5. bind to negatively charged structures within the cells,
such as DNA, which become exposed upon membrane
disruption by cell fixation. The ability to of the proteins to
adhere to intracellular structures results in a redistribution
of protein during fixation, resulting in an apparent but
not true translocation across the cell membrane. The re-
vated caspase-3 [20], and Cre and Flp recombinases
[25,41–43]. A fixation artifact of protein import cannot
explain the results of these studies, since the biological
effects observed for the imported proteins require that the
cells are viable. Based on the data presented in the present
study, we suggest three possible mechanisms to explain
FIG. 5. Endocytosis of VP22-GFP. CHO cells were incubated 5 min, 1 h, or 24 h with VP22-GFP. Microscopy was performed on live unfixed cells.
ARTICLEdoi:10.1016/S1525-0016(03)00135-7
Class
2
CPP
stuck
on
cell
surface
Class
2
CPP
becomes
trapped
in
vesicles
Class
2
CPP
excluded
from
Parts
of
cell
e.g.
nucleus
With
live
imaging
the
class
2
CPPs
can
be
seen
to
be
trapped
in
vesicles
Cell
surface
adherence
and
endocytosis
of
protein
transduc?on
domains.
Lundberg
M,
Wikström
S,
Johansson
M.
Mol
Ther.
2003
Jul;8(1):143-­‐50.

6.
Problems
in
deploying
Class
1
and
2
CPP’s
CPPs
of
Class
1
and
2
have
a
problem
exi(ng
the
endosoma(c
pathway
to
meet
cellular
targets
=
Endocyto(c
vesicle
=
Degrada(on
pathway
CPP
1
CPP
2
Cell
?
Recycle

7. Early
work
with
CPP3
inhibi(ng
PKA
in
vivo
Free
living
Dictyostelium
amoeba
Starva?on:
Cells
release
when
they
begin
starving.
This
ac(vates
PKA
which
causes
them
to
Aggregate
within
24
hours
CPP3
alone
No
treatment
CPP3-­‐PKA
inhibitor
pep(de
Cells
aggregate
normally
Cells
fail
to
aggregate
PKA
inhibitor
pep(de
alone
Use
of
a
penetra?n-­‐linked
pep?de
in
Dictyostelium.
Ryves
WJ,
Harwood
AJ.
Mol
Biotechnol.
2006
Jun;33(2):123-­‐32.

8. • Success
in
Dictyostelium
–
PKA
inhibi(on
points
to
new
tools
to
inves(gate
protein
interac(ons
• Unlike
gene(cally
engineered
cells,
the
CPP3
based
research
is
fast
and
in
real
(me
• Unlike
CPP1
and
CPP2
related
work,
CPP3s
penetrate
cells
quickly
and
directly
without
using
receptors
or
vesicles.
Early
work
with
CPP3
inhibi(ng
PKA
in
vivo

9. CPP3 blockade of PTEN interaction with Drebrin.
A
CPP3-­‐linked
pep(de
inhibi(ng
PTEN
in
vivo
The interaction of PTEN with Drebrin was observed in vivo by co-expression of GFP-PTEN with
mCherry-Drebin in PC12 cells. Interaction was analyzed by measuring fluorescence resonance energy
transfer (FRET) using multiphoton fluorescence-lifetime imaging microscopy (FLIM) and demonstrated
these proteins bound together in a complex and this complex regulates the phosphorylation state of
Drebrin

10.
A
CPP3-­‐linked
pep(de
inhibi(ng
PTEN
in
vivo
Phosphorylation of the actin binding protein Drebrin at S647 is regulated by neuronal activity and PTEN.
Kreis P, Hendricusdottir R, Kay L, Papageorgiou IE, van Diepen M, Mack T, Ryves J, Harwood A, Leslie NR,
Kann O, Parsons M, Eickholt BJ. PLoS One. 2013 Aug 5;8(8):e71957. doi: 10.1371/journal.pone.0071957.
PTEN%
Drebrin%
Response%
B)%Depolarisa4on%s4mulus%decreases%Protein:Protein%interac4on%
Low$interac,on$
determined$by$
FRET$
Drebrin$in$Phosphorylated$state$
S4mulus%
Addition of a class 3 cell-permeable peptide containing
the D-Loop peptide, part of the PTEN protein, resulted
in separating these proteins by competing for PTEN D-
loop interactions.
PTEN%
Drebrin%
C)%Addi0on%of%CPP3%linked%to%‘D8Loop’%of%PTEN%pep0de%
Inhibits%Protein8Protein%interac0on%and%aAenuates%response%
Low$interac,on$
determined$by$
FRET$
Drebrin$in$Phosphorylated$state$
AAenuated%Response%
S0mulus%
CPP3%
+/-­‐

11. • The
PTEN
work
shows
efficacy
of
CPP3s
as
a
research
tool
but
scratches
the
surface
of
a
huge
opportunity
• Pep(des
can
be
engineered
to
inhibit
those
different
parts
of
proteins
which
play
key
roles
in
cell
propaga(on,
cell
func(oning
and
cell
death
• Each
of
those
pep(des
can
be
alached
to
a
CPP3
to
speed
up
in
vivo
research
• The
task
was
to
develop
a
superior
CPP3
and
then
to
produce
a
range
of
CPP3
linked
pep(des
as
an
ever
expanding
tool
kit
to
tackle
the
huge
research
task
ahead.
A
CPP3-­‐linked
pep(de
inhibi(ng
PTEN
in
vivo

12. Cupid
the
company
established
to:
• Patent
technology
• Produce
CPP3
linked
products
for
wider
research
and
commercial
use
• Develop
the
technology
to
aid
ease
of
produc(on,
ease
of
use
and
inves(gate
the
op(mal
environment
for
producing
and
using
Cupid
pep(des
Cupid
Pep(des
the
Company

13. A
few
Cupid-­‐linked
pep(de
products

14. nm
Ab
Spectrum
C.
Cupid-­‐GFP
Cupid
GFP
(2-­‐239
)
Tag
A.
N
42
31 32.3 kDMr
24
B.
Developing
Cupid-­‐GFP
Class
3
CPP
Column
elu(on
frac(ons
Mr

15. Time
15
mins
30
mins
45
mins
60
mins
Cupid
Class
3
CPP
is
able
to
directly
penetrate
cell
membranes
in
1
hour
0
Cupid-­‐GFP
5
uM
(NON-­‐Fluorescent)
LIVE
Mouse
heart
cell
culture
NO
wash
off
during
experiment
Dr
Chris
George,
Welsh
Na(onal
Heart
Ins(tute,
Cardiff
U.K.
1"
2"
3"
4"
0" 30" 60" 90" 120"
TOTALFluorescence
(mul%ple(of(baseline)(
Time(Minutes(
Cupid-­‐GFP
fluorescence
in
living
cells
increases
with
exposure
?me
Cupid-­‐GFP
refolds
to
fluorescence
within
1
hour

16. Cupid-­‐GFP
is
dispersed
throughout
cultured
Mouse
Heart
cells
Confocal
sec?ons
of
GFP
Fluorescence
Mouse
Cardiomyocytes
Top
of
cells
Base
of
Slide
Cupid-­‐GFP
fluorescence
is
distributed
throughout
the
interior
of
living
cells
Cupid-­‐GFP
5
uM
(NON-­‐Fluorescent)
LIVE
Mouse
heart
cell
culture
1
Hour
NO
wash
off
during
experiment
Cupid-­‐GFP
fluorescence
is
neither
trapped
in
vesicles
nor
excluded
from
structures
e.g.
nucleus

17. Cupid-­‐GFP
is
dispersed
throughout
cultured
Human
cells
Cupid-­‐GFP
1
uM
(NON-­‐Fluorescent)
LIVE
Human
HEC
cell
culture
1
Hour
NO
wash
off
during
experiment
Confocal
sec?ons
of
GFP
Fluorescence
Top
Base
Human
endometrioid
adenocarcinoma
images
courtesy
Dr
Lewis
Francis,
Swansea
University,
U.K.

18.
Cupid-­‐GFP
treated
cells
exhibit
normal
viability
Viability
test
using
MTT
assay
Cupid-­‐GFP
5
uM
(NON-­‐Fluorescent)
Human
HEC-­‐50
cell
culture
0
20
40
60
80
100
120
140
0
4
8
24
Viability
%
of
Control
Time
(Hours)

19. Applica(ons
of
Cupid
Technology
Induced
pluripotent
stem
cells
(iPSCs)
Adult
cells
that
have
been
gene(cally
reprogrammed
to
an
embryonic
stem
cell–like
state
by
factors
important
for
maintaining
the
defining
proper(es
of
embryonic
stem
cells
•
iPSCs
were
first
generated
by
Shinya
Yamanaka
at
Kyoto
University,
Japan
in
2006.
•
Yamanaka
used
genes
that
had
been
iden(fied
as
par(cularly
important
in
embryonic
stem
cells
(ESCs),
and
used
retroviruses
to
transduce
mouse
fibroblasts
with
a
selec(on
of
those
genes.
•
Eventually,
four
key
pluripotency
genes
essen(al
for
the
produc(on
of
pluripotent
stem
cells
were
isolated;
Oct-­‐3/4,
SOX2,
c-­‐Myc,
and
Klf4

20.
Why
are
iPSCs
important?
iPS
cell
research
allows
−
both
wild-­‐type
and
disease-­‐specific
pluripotent
cells
to
be
derived
from
accessible
sources
iPS
cells
will
help
researchers
−
create
gene(c
models
for
disease
−
understand
molecular
controls
influencing
cell
development
iPS
cells
hold
the
promise
of
−
reducing
drug
development
(mes
−
improving
drug
safety
−
bringing
us
closer
to
Personalized
Medicine
and
targeted
therapies

21. Genera(on
of
iPSC
cells
with
Reprogramming
Factors
(RFs)
Soma(c
cells
(e.g.
Fibroblasts)
Add
genes
for
reprogramming
factors
e.g.
Oct-­‐3/4,
SOX2,
c-­‐Myc,
and
Klf4
Select
and
expand
iPSC
colonies

22. iPSC
Problems
-­‐ Protocols
do
not
exist
to
harmonize
results
from
research
laboratories
u(lizing
iPS
cell
lines
necessary
to
validate
findings.
-­‐ Current
iPSC
genera(on
protocols
use
gene(c
delivery
systems
of
Reprogramming
Proteins
(RP’s),
risking
integra(on
with
iPSC
DNA
and
gene(c
problems
downstream.
-­‐
Furthermore
Cupid
RP
factors
proteins
themselves
have
oncogenic
poten(al
•
Oncogenesis
Problem
•
Gene?c
Instability
Problem:
•
Valida?on
Criteria
Problem:
-­‐
iPS
cells
have
demonstrated
significant
gene(c
variability
upon
reprogramming
and
subsequent
culture.

23. Genera(ng
iPSC
cells
with
CPP3
linked
Reprogramming
Factors
(RFs)
Soma(c
cells
(e.g.
Fibroblasts)
Add
the
reprogramming
factors
(e.g.
Oct-­‐3/4,
SOX2,
c-­‐Myc,
and
Klf4)
as
CPP3-­‐Proteins
Select
and
expand
iPSC
colonies

24.
Cupid
Solu(ons
to
iPSC
problems
-­‐ Cupid
RFs
are
applied
to
the
media
and
therefore
dosing
regimes
are
very
controllable.
This
will
allows
result
evalua(on
and
protocol
harmoniza(on.
-­‐
Unlike
retroviral
or
other
gene(c
delivery
systems,
Cupid-­‐linked
reprogramming
factors
(Cupid
RFs)
are
proteins
and
will
not
integrate
with
iPSC
DNA,
avoiding
the
poten(al
to
cause
gene(c
problems
downstream.
-­‐
Furthermore
Cupid
RFs
will
be
recycled
(‘turned
over’)
just
like
other
proteins
and
therefore
will
be
removed
following
simple
media
exchange.
-­‐ Variability
caused
by
differences
in
modes
of
gene(c
delivery
systems
or
uneven
delivery
between
individual
iPS
cells
could
poten(ally
be
controlled
or
negated
with
a
Cupid
RF
delivery
system.
•
Oncogenesis
Solu?on
•
Gene?c
Instability
Solu?on:
•
Valida?on
Criteria
Solu?on:

25. Prototype
Cupid-­‐GFP-­‐KLF4
Cupid
GFP
(2-­‐239
)
Tag
Cupid-­‐GFP-­‐KLF4
N
KLF4
(2-­‐479
)
Total
Amino
acids:
759
98
62
49
38
28
Cell
Penetra(on
of
Cupid-­‐GFP-­‐83kD
in
living
cells
(5
uM,
1
Hour).
Star(ng
product
is
Non-­‐Fluorescent
Mr:
83
kD

26. Cupid-­‐GFP
sa(sfies
the
characteris(cs
required
from
CPP
technology
• Pure,
water-­‐soluble,
stable
in
storage
• Capable
of
carrying
large
cargo
• Non-­‐toxic
at
applied
concentra(ons
• Able
to
directly
access
cytosol
to
allow
refolding
and
subsequent
target
interac(on
• Ini(ally
non-­‐fluorescent,
regaining
fluorescence
within
cells.
-­‐
Eliminates
need
for
washing
of
cells
-­‐
Allows
tracking
of
CPP
throughout
experiment
✔
✔
✔
✔
✔

27. Summary
Cupid
technology
has
reached
the
stage
where
it
can
be
applied
to
a
range
of
bioscience
applica?ons:
•
Cell
Tracking,
protein-­‐protein
interac(on
and
protein
func(on
mapping
•
Regenera(ve
medicine:
Crea(on
of
Stem
Cells,
cell
treatment
ex-­‐vivo,
•
Drug
delivery
vehicle
Cupid
manufactures
Cell-­‐Penetra?ng
proteins
linked
to
our
proprietary
molecule
Cupid
-­‐ Cupid
products
are
added
to
the
cell
medium
and
directly
accesses
the
interior
of
cells
-­‐ Penetra(on
and
dispersal
are
monitored
by
imaging
the
refolding
of
GFP
within
living
cells
-­‐ Rapid
Cell
penetra(on
is
observed
in
real
(me

28. William
Jonathan
Ryves
Cardiff,
U.K.
www.cupidpep(des.com

29. Collabora(on
with
Cardiff
and
Vale
UHB
and
Cardiff
University
Goal:
By
working
together
Cupid
and
CVUHB
/
CU
could
establish
Cardiff
as
the
global
centre
for
Cell
Penetra(ng
Pep(de
(CPP)
technology
Benefits:
a) Inward
commercial
investment
in
Wales
b) Enhance
biotechnology
profile
of
CVUHB
/
CU
c) Increase
employment
Provides
Cupid
CVUHB
/
CU
1. Access
to
CPP
patented
technology
2. Leadership
in
CPP
experiments
3. Novel
CPP
products
Receives
1. Access
1st
class
labs
and
equipment
2. Personnel
to
conduct
experiments
3. Know-­‐how
in
specific
scien(fic
areas
1. Enhanced
recogni(on
of
Cupid
2. Accelera(on
of
Cupid
development
3. Improved
access
to
R
&
D
funding
1. Access
to
leading
CPP
products
2. Development
of
CPP
skill
set
3. Opportunity
to
publish
in
and
open
up
new
research
areas
4. Improve
access
to
grant
funding

30. Exis(ng
Development
Program
Cardiff:
a) Adrian
Harwood
b) Trevor
Dale
c) Rachel
Errington
Swansea:
Lewis
Francis,
Nano
&
Micro
technologies
for
Healthcare
(NMH)
(Exploring
penetra(on
mechanism
with
Atomic
Force
Microscopy)
Commercial
Development:
Contacts
with
firms
interested
in
using
Cupid
as
a
drug
delivery
mechanism
a) European
Cancer
Stem
Cell
Research
Ins(tute
(ECSCR
-­‐
Cardiff)
b) Neuroscience
and
Mental
Health
Research
Ins(tute
(NMHRI
-­‐
Cardiff)
c) Research
and
development
funding
sources
d) Suppliers
to
CU
/
CVUHB
to
make
use
of
Cupid-­‐GFP
tracking
technology
Poten(al
addi(onal
contact
areas
Contact
details:
W
J
Ryves
:
jonnyryves@cupidpep(des.com
A
W
Speirs
:
andspeirs@gmail.com