Cell-penetrating peptides:

news for dated cellular Trojan horses

Assunta Senatore

August 5th 2014

Technical Journal Club

Overview

Cell-penetrating peptides: classes and mechanisms of cell entry

Overview of the described applications

Papers:

• A new cell-penetrating peptide with endosomolytic properties

• Rapid and reversible knockdown of endogenous proteins by peptide-

directed lysosomal degradation

CPPs are short amphipathic or purely cationic peptides of less than 30 amino acids

which possess a positive net charge, are able to penetrate biological membranes

and transfer covalently or non-covalently attached bioactive cargoes into cells

Cell-penetrating peptides (CPPs)

Intracellular pathways of cell entry for cell-penetrating peptides (CPPs)

Applications of Cell-penetrating peptides as molecular delivery vehicles for a

variety of drugs, nucleic acids, proteins, therapeutics, and imaging agents

Brain-specific SOD1 silencing by intravenous

injection of RVG-9R/ siRNA complex Intravenous treatment with antiviral siRNA/RVG-9R

complex protects mice against JEV encephalitis

RVG R R R R R R R R R

acfTAT nrfTAT

Dimeric-fluorescent TAT (dfTAT)

dfTAT

Cytosolic delivery of dfTAT in live cells is efficient

tris(2-carboxyethyl)phosphine

dfTAT displays higher endosomolytic activity when compared to acfTAT

dfTAT penetrates the cytosol by escaping from the endocytic pathway

dfTAT-mediated delivery does not substantially affect cell proliferation and transcription

dfTAT-mediated endosomal escape can be repeated

Red Blue Green

Bafilomycin

Delivery of intact and functional proteins using co-incubation with dfTAT

dfTAT EGFP

EGFP Stop

Cre dfTAT

dfTAT

EGFP

EGFP

EGFP TMR

Delivery of EGFP by dfTAT does not require physical interaction

FRET assay

16

17

dfTAT-mediated delivery improves the delivery and transcriptional output of

transcription factor HOXB4

HOXB4 inducible

Luciferase

NIH 3T3 cell

HOXB4

HOXB4-TAT

dfTAT

dfTAT is efficient at delivery proteins and peptide into the cytosol of cells by endosomal

escape

Further studies to assess the roles of the two copies of TAT and fluorescent moiety in

the endosomolytic activity

Minimal cell responses associated with the efficient endosomal leakage (1h incubation).

Delivery of cell-impermeable molecules of different structure and properties :

Cre,

HOXB4

DEAC-K9

EGFP: no physical interaction is required

delivery of different cargos simultaneously or in successive steps.

In vivo application?

Advantages and limitation

speed: days to weeks

specificity: all post-translational modified versions of targeted proteins

viral delivery in vivo, limiting their potential for clinical use:

(“retrovirus, adenovirus (types 2 and 5), adeno-associated virus, herpes virus, pox virus, human

foamy virus (HFV), and lentivirus. All viral vector genomes have been modified by deleting some

areas of their genomes so that their replication becomes deranged and it makes them more safe,

but the system has some problems, such as their marked immunogenicity that causes induction of

inflammatory system leading to degeneration of transducted tissue; and toxin production, including

mortality, the insertional mutagenesis; and their limitation in transgenic capacity size”)

DNA and RNA-based protein manipulation

Nouri Nayerossadat et al., Adv Biomed Res. 2012, 1:27

http://www.ncbi.nlm.nih.gov/pubmed/?term=Nayerossadat N[auth]

1: cell entry 2: to target the POI

3: to direct the POI

for degradation

The strategy of CTM-directed protein degradation

Step1:

Chaperone-targeting motiv (CTM)-directed protein degradation

Lysosome proteasome

Step2: a model for a protein binding domain

DAPK1 targeting peptide knocks down active DAPK1 in HEK cells.

Weihong Tu et al., Cell, 2010

Step 3: cell-penetrating peptide delivery in primary neurons

DAPK1-targeting peptide specifically degrades activated endogenous DAPK1 in neuronal culture.

Lysososme-dependent

degradation dose-dependence

time-dependence prolonged effect by

multiple application

200 uM , 60 min before

and 30 min during NMDA

TAT-GluN2BCTM synthetic peptide–induced degradation of DAPK1 in various

subcellular compartments in neuron cultures

Shorter

synthetic

peptides 25 uM , 60 min before and

30 min during NMDA

siRNA-directed knockdown of Lamp2a reduces TAT-GluN2BCTM

induced DAPK1 degradation

Pep1-mediated intracellular delivery of short synthetic GluN2B-CTM peptides

specifically knocks down active native DAPK1 in cultured neurons.

Pep - 1 - Cysteamine

Ac - KETWWETWWTEWSQPKKKRKV - cysteamine

Pep-1 “This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically

active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents

several advantages for protein therapy, including stability in physiological buffer, lack of toxicity, and lack of sensitivity to serum”.

May C. Morris et al., Nature Biotechnology 19, 1173–1176 (1 December 2001)

Non covalent

binding

Target peptide–mediated respective degradation of α-synuclein and PSD-95 in cultured neurons

beta-syn

alpha-syn

Shaltiel-Karyo R et al.,

PLoS One. 2010

NR2B

PSD-95

Aarts M et al.,

Science 2002

no toxicity

Target peptide–mediated degradation of mutant α-synuclein

TAT-GluN2Bct-CTM knocks down H2O2- activated DAPK1, protecting neurons against H2O2-

induced neurotoxicity in neuronal cultures

Step 4: can targeting-peptide mediated knockdown have a pathophysiological relevant phenotype?

TAT-GluN2BCTM specifically knocks down DAPK1 in ischemic brain areas and reduces

neuronal damage in the MCAo model of focal ischemia in rats.

tetrazolium chloride staining

Neuroprotective effect of TAT-GluN2BCTM mediated knocks down of DAPK1

in ischemic brain areas in the MCAo model of focal ischemia in rats.

Cell membrane-permeant targeting peptide-based system to

knockdown endogenous protein by lysosomal degradation

Simple

Rapid (hours)

Versatile: 19 kDa alpha-synuclein and its pathological variant, 160 kDa DAPK1, synaptic

scaffolding protein PSD-95,

Reversible: dose and time dependent

peptide generated either as recombinant protein or synthesized

non-virally mediated

native neuronal protein knockdown

in vitro: primary neuronal cultures

in vivo: brain of animals when the targeting peptides was administered systemically

(penetration of the BBB)

ready potential for clinical translation

Neuroprotection with NA-1 (Tat-NR2B9c), an inhibitor of postsynaptic density-95 protein, was

assessed in case of ischemic brain damage in human beings

(ruptured or unruptured intracranial aneurysm amenable to endovascular repair)

Patients received an intravenous infusion of either NA-1 or saline control at the end of their

endovascular procedure

The primary outcome was safety and primary clinical outcomes were the number and volume of

new ischemic strokes defined by MRI at 12–95 h after infusion.

No difference between groups in the volume of lesions by MRI.

Patients in the NA-1 group sustained fewer ischemic infarcts than did patients in the placebo

group

The Lancet Neurology, Volume 11, Issue 11, Pages 942 - 950, November 2012

http://www.thelancet.com/journals/laneur/issue/vol11no11/PIIS1474-4422(12)X7024-8

AAV2/8

• long term expression

• dividing and non dividing cells

• safe

• inefficient in different cell types

Cell –penetrating peptides enhance viral transduction in primary cells and tissue

Cell –penetrating peptides enhance viral transduction in vivo

No cytotoxicity of cell-permeable peptides

(CPPs) was detected in vivo. Cell-permeable peptides (CPPs) facilitate gene

delivery of adeno-associated virus type 2

(AAV2) in mouse muscles.

Brain-penetrating inputs generating ideas

Thank you for your attention!