Download - Cupid Peptides presentation wjr

William Jonathan Ryves

Cardiff, U.K.

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

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

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 washedextensively with PBS and microscopy was performed either on live unfixed cells or after methanol fixation. FITC, fluorescein–isothiocyanate filter; PC, phasecontrast.

FIG. 3. Adherence of PTD–GFP fusion proteins to prefixed CHO cells. The cells were fixed with methanol and rehydrated in PBS. Recombinant proteins wereadded for 5 min at room temperature and the cells were washed extensively with PBS prior to microscopy. FITC, fluorescein–isothiocyanate filter; PC, phasecontrast.

ARTICLEdoi:10.1016/S1525-0016(03)00135-7

145MOLECULAR THERAPY Vol. 8, No. 1, July 2003Copyright © The American Society of Gene Therapy



ARTICLEdoi:10.1016/S1525-0016(03)00135-7


GFP alone

VP-‐22 GFP

TAT GFP

K8 GFP

R8 GFP



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

bind to negatively charged structures within the cells,such as DNA, which become exposed upon membranedisruption by cell fixation. The ability to of the proteins toadhere to intracellular structures results in a redistributionof protein during fixation, resulting in an apparent butnot true translocation across the cell membrane. The re-location of PTD proteins during fixation explains whyprotein import into almost all cells in a cell population isdetected within only minutes of incubation as well aswhy the import process can occur at 37 and 4°C[5,18,26,27,29]. It also explains why many PTD sequencemutants, and even peptides with reversed amino acidsequences, retain the ability of protein import [18,27].The possibility of postfixation movement of proteins andpeptides thereby invalidates methods requiring fixationto study membrane translocation by PTDs. However,methods studying live cells such as flow cytometry alsorequire caution because they do not distinguish betweenprotein immobilized on the cell surface and protein thathas translocated across the cell membrane.

The binding of PTDs to the cell surface and artificialimport during fixation do not exclude that a smallamount of protein, undetectable by standard imagingtechniques, is imported into cells. The notion that PTDsin fact translocate across the cell membrane is supportedby several studies on biological effects mediated by PTDfusion proteins. These studies include the functional de-livery of p16INK4 [23], p27Kip1 [21], an HIV protease-acti-

vated caspase-3 [20], and Cre and Flp recombinases[25,41–43]. A fixation artifact of protein import cannotexplain the results of these studies, since the biologicaleffects observed for the imported proteins require that thecells are viable. Based on the data presented in the presentstudy, we suggest three possible mechanisms to explainthe biological effects observed: (i) PTD proteins exert ef-fects on the cell surface, (ii) the proteins exert their effectwithin endosomes, or (iii) the PTDs are released from theendosomes into the cytosol by endosomolysis. The firstmechanism implies that the PTD peptides and proteinsadhered to the cell surface may affect cell surface recep-tors, which results in a biological effect. The membrane-translocating property of TAT was first discovered whenrecombinant TAT was added to a cell line containing anHIV long terminal repeat promoter reporter construct [3].The exogenously added TAT was shown to activate thereporter gene, which was interpreted as import of TATinto the cell nucleus and TAT-mediated activation of thepromoter. However, subsequent studies suggested thatexogenously added TAT binds to the cell surface where itactivates cell surface receptors that in turn activate tran-scription factors and induce transcription of the promoter[44,45]. Accordingly, it is possible that some of the bio-logical effects of the PTD fusion proteins are mediated bycell surface receptor activation. The second possiblemechanism of PTD action suggested is that the proteinsexert a local effect within the endosomes and lysosomes.

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.

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

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.

•  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

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


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%

+/-‐

•  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.


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

A few Cupid-‐linked pep(de products

nm

Ab

Spectrum C. Cupid-‐GFP

Cupid GFP (2-‐239 ) Tag

A.

N

42

31 32.3 kD Mr

24

B.

Developing Cupid-‐GFP Class 3 CPP

Column elu(on frac(ons Mr

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"

TOTA

L Fl

uore

scen

ce

(mul%p

le(of(b

aseline)(

Time(Minutes(

Cupid-‐GFP fluorescence in living cells increases with exposure ?me Cupid-‐GFP refolds to fluorescence within 1 hour

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

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.

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)

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

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

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

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.

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

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:

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

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

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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

William Jonathan Ryves

Cardiff,

U.K.

www.cupidpep(des.com

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

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 : [email protected]