Cell-penetrating peptides: news for dated cellular Trojan horses

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1 Technical Journal Club Cell-penetrating peptides: news for dated cellular Trojan horses Assunta Senatore August 5 th 2014

2 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 peptidedirected lysosomal degradation

3 Cell-penetrating peptides (CPPs) 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

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

5 Applications of Cell-penetrating peptides as molecular delivery vehicles for a variety of drugs, nucleic acids, proteins, therapeutics, and imaging agents

6 RVG R R R R R R R R R 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

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8 Dimeric-fluorescent TAT (dftat) acftat dftat nrftat

9 Cytosolic delivery of dftat in live cells is efficient tris(2-carboxyethyl)phosphine

10 dftat displays higher endosomolytic activity when compared to acftat

11 dftat penetrates the cytosol by escaping from the endocytic pathway

12 dftat-mediated delivery does not substantially affect cell proliferation and transcription

13 dftat-mediated endosomal escape can be repeated Red Blue Green Bafilomycin

14 Delivery of intact and functional proteins using co-incubation with dftat EGFP dftat Stop EGFP Cre dftat

15 Delivery of EGFP by dftat does not require physical interaction EGFP EGFP TMR EGFP dftat FRET assay

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17 dftat-mediated delivery improves the delivery and transcriptional output of transcription factor HOXB4 HOXB4 dftat HOXB4 inducible HOXB4-TAT Luciferase NIH 3T3 cell 17

18 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 Advantages and limitation delivery of different cargos simultaneously or in successive steps. In vivo application?

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20 DNA and RNA-based protein manipulation 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 ) Nouri Nayerossadat et al., Adv Biomed Res. 2012, 1:27

21 The strategy of CTM-directed protein degradation 1: cell entry 2: to target the POI 3: to direct the POI for degradation

22 Step1: Chaperone-targeting motiv (CTM)-directed protein degradation proteasome Lysosome

23 Step2: a model for a protein binding domain DAPK1 targeting peptide knocks down active DAPK1 in HEK cells. Weihong Tu et al., Cell, 2010

24 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 200 um, 60 min before and 30 min during NMDA time-dependence prolonged effect by multiple application

25 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

26 sirna-directed knockdown of Lamp2a reduces TAT-GluN2BCTM induced DAPK1 degradation

27 Pep1-mediated intracellular delivery of short synthetic GluN2B-CTM peptides specifically knocks down active native DAPK1 in cultured neurons. Pep Cysteamine Ac - KETWWETWWTEWSQPKKKRKV - cysteamine Non covalent binding 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, (1 December 2001)

28 Target peptide mediated respective degradation of α-synuclein and PSD-95 in cultured neurons alpha-syn beta-syn Shaltiel-Karyo R et al., PLoS One PSD-95 NR2B Aarts M et al., Science 2002 no toxicity

29 Target peptide mediated degradation of mutant α-synuclein

30 Step 4: can targeting-peptide mediated knockdown have a pathophysiological relevant phenotype? TAT-GluN2Bct-CTM knocks down H 2 O 2 - activated DAPK1, protecting neurons against H 2 O 2 - induced neurotoxicity in neuronal cultures

31 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

32 Neuroprotective effect of TAT-GluN2BCTM mediated knocks down of DAPK1 in ischemic brain areas in the MCAo model of focal ischemia in rats.

33 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

34 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 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 , November 2012

35 AAV2/8 long term expression dividing and non dividing cells safe inefficient in different cell types

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

37 Cell penetrating peptides enhance viral transduction in vivo Cell-permeable peptides (CPPs) facilitate gene delivery of adeno-associated virus type 2 (AAV2) in mouse muscles. No cytotoxicity of cell-permeable peptides (CPPs) was detected in vivo.

38 Brain-penetrating inputs generating ideas Thank you for your attention!

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