cell-penetrating peptides and bioactive cargoes. strategies and mechanisms.192279/... ·...

Cell-penetrating peptides and bioactive

cargoes. Strategies and mechanisms.

Kalle Kilk

Department of Neurochemistry and Neurotoxicology Arrhenius Laboratories for Natural Sciences

Stockholm University 2004

Doctoral dissertation 2004 Department of Neurochemistry and Neurotoxicology Arrhenius Laboratories for Natural Sciences Stockholm University, 10691 Stockholm Sweden

Abstract Cell membrane is an impermeable barrier for most macromolecules. Recently discovered cell-penetrating peptides (CPPs) have gained lot of attention because they can cross the membrane, and, even more, carry cargoes with them. How do CPPs enter the cells is still not clear, while the delivery of different cargoes has been convincingly shown. This thesis concentrates on evaluating CPPs as vectors for different biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by CPPs have been under particular interest and are explained in greater details. A new CPP, pIsl, was derived from Islet-1 transcription factor, and compared to archetypical CPPs penetratin and transportan. All three peptides resided in the headgroup region of lipid bilayers in model membranes. However, penetratin and pIsl did interact only with negatively charged membranes, while transportan did not distinguish negatively charged and neutral membranes. This suggests different translocation pathways for different CPPs. Biotinylated pIsl and penetratin were complexed with avidin, and uptake of avidin into human melanoma cell line Bowes was observed in both cases. This means that the protein is not unfolded during the translocation process, which is important in delivery of other, biologically active proteins. Transportan and its analogue TP10 were used for peptide nucleic acid (PNA) antisense oligonucleotide delivery. First, eight human galanin receptor type 1 targeting PNA oligomers were designed, conjugated to transportan and assayed for antisense efficiency. Unlikely to avidin-biotinylated peptide conjugate, a covalent bond between PNA oligomers and the transport peptide was necessary for cellular uptake of oligomers. A common problem in antisense technology is inactivity of antisense oligonucleotides due to the secondary structure of the target. Efficiencies of tested galanin receptor type 1 targeting PNA oligomers varied over two orders of magnitude. The most efficient oligomers were targeting coding sequence regions 24-38 and 27-38, and had EC50 values 70 and 80 nM, respectively. TP10-antisense PNA oligomer conjugates were targeted also to L-type voltage dependent Ca2+ channel subunits CaV1.2 and CaV1.3. Specific down-regulation of respective proteins was demonstrated by immunohistochemistry. Physiological response to the down-regulation of either of Ca2+ channels was studied by alteration of flexor reflex sensitisation. Rats treated with either of the antisense PNAs, but not with the scrambled PNA lost sensitisation ability. In conjunction with a variety of drugs, modulating the conductivity and excitability of neuronal membranes, a central role of L-type CaV channels in sensitisation was demonstrated. Nevertheless, also N-methyl-D-aspartate and glycine receptors were found be required. Finally, delivery of plasmids by TP10 was evaluated. In contrary to many similar CPPs, TP10 was incapable to translocate plasmids to cells. However, addition of TP10 or a TP10-PNA conjugate to polyethyleneimine-condensed plasmids increased the expression of reporter genes. In summary, different cargo types and coupling strategies were used to investigate carrier capacities of different CPPs. CPP-mediated antisense oligonucleotide delivery was used to identify accessible sites in human galanin receptor type 1 mRNA and to determine the role of L-type voltage dependent Ca2+ channels in axon potential windup.

© 2004 Kalle Kilk

List of publications

This thesis is based on work presented in the following original papers, referred to by Roman

numerals in the text:

I Kilk K, Magzoub M, Pooga M, Eriksson LE, Langel Ü, Gräslund A. Cellular

internalization of a cargo complex with a novel peptide derived from the third helix of the

islet-1 homeodomain. Comparison with the penetratin peptide.Bioconjug Chem. 2001 Nov-

Dec;12(6):911-6.

II Magzoub M, Kilk K, Eriksson LE, Langel Ü, Gräslund A. Interaction and structure

induction of cell-penetrating peptides in the presence of phospholipid vesicles. Biochim

Biophys Acta. 2001 May 2;1512(1):77-89.

III Kilk K, Elmquist A, Saar K, Pooga M, Land T, Bartfai T, Soomets S, Langel Ü.

Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA.

Neuropeptides Neuropeptides. 2004 Oct;38(5):316-24.

IV Fossat P, Dobremez E, Landry M, Kilk K, Sibon I, Benazzouz R, Le Masson G,

Langel Ü, and Nagy F. L-type calcium channels and NMDA receptors: a determinant duo for

short term nociceptive plasticity. Manuscript

V Kilk K, EL-Andaloussi S, Järver P, Valkna A, Bartfai T, Kogerman P, Metsis M,

Langel Ü. Evaluation of transportan 10 in PEI mediated plasmid delivery assay. Submitted to

Journal of Controlled Release

Additional publications by K. Kilk Mazarati A, Lu X, Kilk K, Langel Ü, Wasterlain C, Bartfai T.Galanin type 2 receptors regulate neuronal survival, susceptibility to seizures and seizure-induced neurogenesis in the dentate gyrus. Eur J Neurosci. 2004 Jun;19(12):3235-44. Hua XY, Hayes CS, Hofer A, Fitzsimmons B, Kilk K, Langel Ü, Bartfai T, Yaksh TL. Galanin Acts at GalR1 Receptors in Spinal Antinociception: Synergy with Morphine and AP-5.J Pharmacol Exp Ther. 2004 Feb;308(2):574-82. Epub 2003 Nov 10. Howitt SG, Kilk K, Wang Y, Smith DM, Langel Ü, Poyner DR.The role of the 8-18 helix of CGRP8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions.Br J Pharmacol. 2003 Jan;138(2):325-32. Conner AC, Hay DL, Howitt SG, Kilk K, Langel Ü, Wheatley M, Smith DM, Poyner DR.Interaction of calcitonin-gene-related peptide with its receptors.Biochem Soc Trans. 2002 Aug;30(4):451-5. Review. Saar K, Mazarati AM, Mahlapuu R, Hallnemo G, Soomets U, Kilk K, Hellberg S, Pooga M, Tolf BR, Shi TS, Hökfelt T, Wasterlain C, Bartfai T, Langel Ü.Anticonvulsant activity of a nonpeptide galanin receptor agonist.Proc Natl Acad Sci U S A. 2002 May 14; 99(10):7136-41. Oras A, Kilk K, Kunapuli S, Barnard EA, Järv J.Kinetic analysis of [35S]dATP alpha S interaction with P2y(1) nucleotide receptor.Neurochem Int. 2002 Apr; 40(5):381-6. Soomets,U., Kilk,K., Land,T., and Langel,Ü. Transportan, its analogues and applications. Proceedings of Solid Phase Peptide Synthesis, in : Innovation and Perspectives in Solid Phase Synthesis and Combinatorial Libraries 2000, Ed. R.Epton, Mayflower Worldwide Ltd., Kingswinford, England, 2001; pp. 131-136. Kilk, K and Langel Ü. Cellular Delivery of Peptide Nucleic Acids by Cell Penetrating Peptides. Peptide Synthesis and Applications. Ed. J. Howl, The Humana Press, Totowa, New Jersey, USA (in press)

Contents

1 INTRODUCTION................................................................................................. 1

1.1 Biological methods of passing cell membranes.......................................................... 1 1.1.1 Energy-independent carriers .................................................................................. 1 1.1.2 Energy-dependent carriers...................................................................................... 2 1.1.3 Endocytosis ............................................................................................................ 2

1.1.3.1 Membrane rafts .................................................................................................. 4 1.1.3.2 Phagocytosis....................................................................................................... 4 1.1.3.3 Pinocytosis ......................................................................................................... 4

1.1.3.3.1 Clathrin-mediated endocytosis ..................................................................... 4 1.1.3.3.2 Caveolar uptake............................................................................................ 5 1.1.3.3.3 Macropinocytosis ......................................................................................... 5

1.2 Cell-penetrating peptides............................................................................................. 6 1.2.1 Classes of CPPs...................................................................................................... 7

1.2.1.1 Penetratins .......................................................................................................... 7 1.2.1.2 Tat protein derived peptides............................................................................... 7 1.2.1.3 Transportans ....................................................................................................... 8 1.2.1.4 Other CPPs ......................................................................................................... 8

1.2.2 Cell-penetrating peptides in model membrane systems....................................... 10 1.2.3 Proposed internalisation mechanisms .................................................................. 11 1.2.4 Cargoes delivered by CPPs .................................................................................. 13 1.2.5 Cargo coupling methods....................................................................................... 18

1.3 Objectives for cargoes................................................................................................ 19 1.3.1 Antisense .............................................................................................................. 19

1.3.1.1 Antisense mechanisms ..................................................................................... 19 1.3.1.2 Design of antisense oligonucleotides ............................................................... 21 1.3.1.3 Side-effects and controls .................................................................................. 21 1.3.1.4 Peptide nucleic acids ........................................................................................ 23

1.3.2 Galanin and its receptors ...................................................................................... 24 1.3.3 Sensitisation ......................................................................................................... 25

1.3.3.1 Action potential windup ................................................................................... 25 1.3.3.2 N-methyl-D-aspartate receptors ....................................................................... 26 1.3.3.3 Ca2+ channels.................................................................................................... 26

1.3.4 Gene delivery ....................................................................................................... 27

2 AIMS: ................................................................................................................ 28

3 METHODOLOGICAL CONSIDERATION ......................................................... 29

3.1 Selection of CPPs........................................................................................................ 29

3.2 Selection of cargoes .................................................................................................... 29

3.3 Peptide synthesis......................................................................................................... 30

3.4 PNA synthesis ............................................................................................................. 31

3.5 Disulphide bridge formation ..................................................................................... 31

3.6 Interactions with model membranes ........................................................................ 33

3.7 mRNA structure prediction....................................................................................... 33

3.8 Cellular uptake studies .............................................................................................. 34

3.9 Galanin binding studies ............................................................................................. 34

3.10 Reverse-transcription PCR ....................................................................................... 35

3.11 Electrophysiological measurements.......................................................................... 35

4 RESULTS AND DISCUSSION.......................................................................... 36

4.1 Preparation of CPP-cargo conjugates ...................................................................... 36 4.1.1 Noncovalent complexes ....................................................................................... 36 4.1.2 Covalent complexes ............................................................................................. 37

4.2 CPP interactions with model membranes................................................................ 40

4.3 Cargo delivery ............................................................................................................ 41 4.3.1 Delivery of avidin................................................................................................. 41 4.3.2 Delivery of PNA................................................................................................... 42 4.3.3 Plasmid delivery................................................................................................... 44 4.3.4 Cargo uptake mechanisms.................................................................................... 45

4.4 Antisense effects of delivered oligonucleotides ........................................................ 47 4.4.1 Design of antisense PNA oligomers..................................................................... 47 4.4.2 Galanin receptor antisense ................................................................................... 48 4.4.3 Ca2+ L-channel antisense...................................................................................... 49 4.4.4 PNA antisense mechanism. .................................................................................. 50

5 CONCLUSIONS................................................................................................ 52

6 ACKNOWLEDGEMENTS ................................................................................. 53

7 REFERENCES.................................................................................................. 54

Abbrevations AcN acetonitrile asON antisense oligonucleotide ATP adenosine 5’- triphosphate CaV1.2 type 1.2 Ca2+ voltage dependent channel CaV1.3 type 1.3 Ca2+ voltage dependent channel CD circular dichroism cDNA DNA corresponding to mRNA sequence CPP cell penetrating peptide DCC dicyclohexylcarbodiimide DMF N,N-(dimethyl)formamide DMPC dimyristoylphosphatidylcholine DMPG dimyristoylphosphatidylglycerol DMSO dimethylsulfoxide DNA deoxyribonukleiinhape DOPG dioleoylphosphatidylglycerol EGFP enhanced green fluorescent protein EPR electron paramagnetic resonance FACS fluorescence activated cell sorter FITC fluoresceine isothiocyanate Fmoc 9-fluorenylmethoxy-carbonyl GABA γ-aminobutyric acid GalR1 galanin receptor type 1 GAPDH glyseraldehyde-3-phosphate dehydrogenase GFP green fluorescent protein GTP guanosine 5’- triphosphate hCT(9-32) human calcitonin fragment hGalR1 human galanin receptor type 1 HIV human immunodeficiency virus HOBt 1-hydroxybenzotriazole HPLC high pressure liquid chromatography LUV large unilabellar vesicle MALDI-ToF matrix-assisted laser desorption ionisation time of flight MAP model amphipatic peptide mRNA messenger RNA MW molar weight NLS nuclear localisation signal NMDA N-methyl-D aspartate NMR nuclear magnetic resonance Npys 3-Nitro-2-pyridinesulphenyl PCR polymerase chain reaction PEI polyethyleneimine pIsl Islet-1 derived cell penetrating peptide PNA peptide nucleic acid pTat HIV Tat protein derived cell penetarting peptide pVec vascular endothelial cadherin derived CPP. RISC RNA induced silencing complex

RT-PCR reverse transcription polymerase chain reaction SDS sodium dodecyl sulfate siRNA small interfering RNA SMCC succinimidyl trans-4-(maleimidylmethyl)cyclohexane- 1-carboxylate Tat HIV Transactivating regulatory protein t-Boc tert-butoxycarbonyl TBTU 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TFA trifluoroacetic acid TP10 transportan 10 TRITC tetramethylrhodamine isothiocyanate Three- and one-letter codes for amino acids: Ala A Alanine Arg R Arginine Asn N Asparagine Asp D Aspartic acid Cys C Cysteine Glu E Glutamic acid Gln Q Glutamine Gly G Glycine His H Histidine Ile I Isoleucine Leu L Leucine Lys K Lysine Met M Methionine Phe F Phenylalanine Pro P Proline Ser S Serine Thr T Threonine Trp W Tryptophan Tyr Y Tyrosine Val V Valine

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1 Introduction The majority of commercial drugs today target cell surface receptors. Compared to all

possible targets this is only a small fraction and accessing intracellular targets would open

many new possibilities. To gain this access, pathways designed by Mother Nature or artificial

methods can be employed. It is still unclear whether recently discovered cell-penetrating

peptides (CPPs) are artificial or exploit well-known natural processes for cargo delivery.

The present thesis concentrates on evaluating CPPs as vectors for different

biologically relevant cargoes. Proposed internalisation mechanisms are reviewed as well as

cargo coupling strategies. Biological activities of antisense oligonucleotides delivered by

CPPs have been under particular interest and are explained in greater details.

1.1 Biological methods of passing cell membranes Membranes are structures that separate cells or organelles from the external matrix. They

have hydrophobic nature and prevent free trafficking of energy and substances. It is highly

necessary for maintaining the cell functionality and to protect it from potential invaders. To

gain nutrients, a cell, however, needs traffic through membranes. Few molecules can pass

membranes by passive concentration gradient driven diffusion or osmotic pressure. It applies

to small (up to ca 500 daltons) lipid-soluble substances, water and gases. For uptake of large

and hydrophilic compounds cells have developed a variety of strategies (Fig. 1). They can be

divided into energy dependent and energy independent mechanisms, depending on the

translocation driving force.

1.1.1 Energy-independent carriers Energy-independent carriers are transmembrane or membrane-soluble proteins that either

form channels through the membrane or increase the lipophilicity of translocated substances.

Ion channels open by membrane potential or presence of specific ligands, and thereby

mediate ion flux down to the concentration gradient across the membrane (Fig. 1). Some

larger molecules internalise similarly, for example sugar moieties are passing through

maltoporin in E. coli outer membrane (Ferenci and Boos, 1980). Channels, in general, are

very specific towards substrates. Ion channels can discriminate ions like K+ and Na+, and

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allow only one of them pass, even if the excluded ion is sterically smaller and should

therefore pass easier.

Another pathway for energy independent delivery is capturing the substrate into a

lipophilic complex. After association, the substrate/carrier complex diffuses through the

membrane and dissociates on the other side. Also this mechanism is substrate specific and can

be saturated. The carriers are not necessarily membrane proteins. Ionomycin, an antibiotic

from Streptomyces conglobatus, works by this principle (Bennett et al., 1979).

1.1.2 Energy-dependent carriers A living organism is always at a higher energetic state than a dead organism. For functioning,

cells need to work against concentration gradient driven diffusion. Therefore they express

energy-dependent carriers that force specific substances to leave their energetically favourable

stages. In opposite to ion channels, energy-consuming pumps verify that vital unbalances, like

differences in Na+ and K+ extra- and intracellular concentrations are maintained. ATP or GTP

hydrolysis is the most common mechanism to gain the energy for translocation (Fig. 2).

Secondary active transporters do not use ATP hydrolysis, but the movement of a

solute down to concentration gradient to drive the translocation of the substrate. Na+/glucose

or amino acid transporters use the Na+ gradient for substance delivery against concentration

gradients. Di- or tripeptides as well as small peptidomimics are ligands for peptide

transporters PepT1 and PepT2, or peptide/histidine transporters PHT1 and PHT2 (reviewed

by Herrera-Ruiz and Knipp, 2003). These proteins are transmembrane channels utilising the

energy generated by a proton gradient.

All listed energy dependent and independent pathways are suitable for relatively small

substrates. The vast majority of macromolecules, including peptides, are taken up via other

mechanisms, collectively known as endocytosis.

1.1.3 Endocytosis

Endocytosis is a common term for mechanisms where substances are encapsulated into

membrane “bubbles” for their uptake. The “bubbles” can be formed in different ways and are

classified into clathrin-mediated and caveolae-mediated endocytoses, macropinocytosis and

phagocytosis (Fig. 1). Still unknown mechanisms, referred to as caveolae and clathrin

3

independent endocytosis, are likely to exist (Conner and Schmid, 2003). Phagocytosis (“cell

eating”) is the only known mechanism where no environmental liquid is taken up, all other

endocytotic pathways are superclassified into pinocytosis (“cell drinking”).

Figure 1. Trafficking through the outer cellular membrane has many different routes.

Early endocytotic vesicles are, after internalisation, merged with each other and sorted

inside the cell. Some are further targeted to lysosomes, where the content is degraded, others

are recycled without degradation or associate with the Golgi complex and endoplasmic

reticulum. Which route an endosome takes, depends on its size, and modifications by

associated proteins e.g. ubiquitinylation (reviewed by Maxfield and McGraw, 2004). Cell

surface receptors are often involved in endocytosis. It is a way to regulate receptor density on

cell surface and thereby also signal intensity. Still little is known, but evidences imply a

bidirectional regulation – change in either endocytosis rate or signal intensity echoes

somehow back in the other system. (Teis and Huber, 2003).

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1.1.3.1 Membrane rafts The cell membrane consists of a wide variety of lipids and proteins. The mixture is far from

an ideal solution and intermolecular interactions reduce the entrophy by forming specific

microdomains (reviewed by Mukherjee and Maxfield, 2004). The straight acyl chains of

saturated lipids do not fit well with unsaturated lipids. Cholesterol softens the borders by

penetrating into highly ordered saturated lipid chains and making them more “liquid”. These

cholesterol- and glycosphingolipid rich areas are called lipid rafts (reviewed by Pike, 2004).

Proteins, depending on their structure and counterparts, may be located in distinct regions, but

rafts are considered to be more protein rich than the rest of the membrane. Phospholipase C,

andenylate cyclase and many other central enzymes are located here. Due to its organised

structure, rafts are resistant to detergents and remain intact in detergent treatment. Depending

on detection method used, rafts are about ten to 300 nm in diameter. The identification of

signalling proteins in lipid rafts has led to the hypothesis that these domains are intimately

involved in signal transduction (reviewed by Simons and Toomre, 2000). Rafts are also

centres for endocytotic events (Nabi and Le, 2003) and cholesterol trafficking (Ikonen and

Parton, 2000).

1.1.3.2 Phagocytosis Phagocytosis is mainly conducted by cells specialized for clearing large-size pathogens like

bacteria and dead cells. When a bacteria interacts with membrane receptors of, for example, a

macrophage a signal cascade activates Rho-family of GTPases (Chimini and Chavrier, 2000).

The GTPases, especially dynamin, fuel actin reorganisation and this in turn drives membrane

zipping around the bacteria (Aderem and Underhill, 1999; Conner and Schmid, 2003).

Internalised phagosomes are fused with lysosomes and the content is degraded.

1.1.3.3 Pinocytosis 1.1.3.3.1 Clathrin-mediated endocytosis

At molecular level the clathrin-mediated pathway is the most well studied endocytosis

mechanism. It occurs in all cells and is important for nutrient uptake and signal transduction.

Receptor-mediated endocytotsis often utilises this pathway and the uptake of transferrin and

its receptors is a classical example (Hanover et al., 1984; van Dam and Stoorvogel, 2002). In

formation of endocytotic pits, a trimeric protein, clathrin, associates with the plasma

membrane and forms a polygonal coat on it (Fig. 1). Several adaptor proteins are additionally

5

needed for proper coating and membrane invagination (reviewed by Brodsky et al., 2001).

The accessory proteins also perform early sorting depending on the internalised cargo and

receptors involved (Warren et al., 1998). Clathrin-mediated endocytosis is powered by

dynamin (Song et al., 2004).

1.1.3.3.2 Caveolar uptake

Caveolae were first described by microscopic studies as smooth-surfaced flask-shaped pits of

typically 55–65 nm in diameter covering the surface of many mammalian cell types (Yamada,

1955). Caveolae are not found in lymphocytes and many neuronal cells, although they express

caveolin-1 (Galbiati et al., 1998; Hatanaka et al., 1998). Caveolin-1, a membrane bound

protein, seems to be the major (Fra et al., 1995; Rothberg et al., 1992) and sufficient

component for pit formation (Lipardi et al., 1998). The protein interacts with cholesterol and

lipid rafts (Sargiacomo et al., 1993; Murata et al., 1995) and caveolae is considered as a

special type of raft (Anderson, 1998; Kurzchalia and Parton, 1999; Bathori et al., 2004).

Similarly to clathrin mediated endocytosis, caveolar uptake is dynamin dependent. (Henley et

al., 1998; Oh et al., 1998). Heparan sulphate and other extracellular glycans that mediate

uptake of many substances, including growth factors and possibly CPPs, are proposed to

trigger caveolae formation and endocytosis (Belting, 2003).

1.1.3.3.3 Macropinocytosis

Macropinocytosis creates the largest pinocytotic vesicles with size in micrometer scale. It

starts similarly to phagocytosis with actin reorganization, but it does not “climb up” via the

surface of a membrane bound particle. Instead a ruffle rises from the cell membrane until it

eventually collapses back, locking a volume of extracellular milieu into a vesicle. (Conner and

Schmid, 2003). Macropinocytosis is mainly regulated by the small GTPase ARF6 but not by

dynamin (Radhakrishna and Donaldson, 1997; Radhakrishna et al., 1999; Brown et al., 2001).

Micropinosomes do not fuse with lysosomes according to Hewlett et al. (Hewlett et al., 1994)

and do fuse according to other studies (Racoosin and Swanson, 1993). Perhaps the fate

depends on the cell type (Swanson and Watts, 1995). Possible involvement of rafts in

macropinosome formation has been suggested (Liu et al., 2002; Watarai et al., 2002).

However, in contrary to caveosomes, micropinosomes are detergent sensitive.

6

1.2 Cell-penetrating peptides Cell-penetrating peptides (CPPs) are also known as membrane translocation sequences,

“Trojan” peptides or protein transduction domains. The last refers particularly to natural

protein derived peptides. CPPs are 7 to 30 amino acid long peptides capable of translocating

into cell without the help of a specific receptor. Since the internalisation mechanism is still a

hot debate, it is currently impossible to define them more precisely. Historically, CPPs were

envisaged to internalise in an endocytosis and energy-independent manner, but this has been

questioned in several recent studies (Richard et al., 2003; Lundberg and Johansson, 2001).

Important is that not only the peptides themselves translocate through plasma

membranes but for most of CPPs, cargo delivery has been demonstrated. Intriguingly, the

cargo size can exceed the vector size several fold. Therefore CPPs are not only interesting

because of their unknown internalisation mechanism, but their great potential as delivery

vectors.

One way to classify CPPs is to divide them into arginine rich peptides and

amphipathic peptides. Penetratin, pTat and polyarginine belong to the first class while

transportan and model amphipathic peptide are examples of the second family. Since the

internalisation mechanisms are not fully known yet, such classification may not be fully

correct, but several experiments support it. These classes differ in membrane interactions

(paper II, Magzoub et al., 2003), internalisation kinetics (Hällbrink et al., 2001), and

amphipathic peptides seem to be less influenced by endocytosis modulators (Simeoni et al.,

2003).

CPPs have been considered to translocate all cell lines with similar efficacy. This was

based on early observations that the peptides internalised all tested cell lines. On one hand, it

is positive, since the same delivery strategy can be used for all cell types. On the other hand,

lack of specificity can lead to waste of the material (Niesner et al., 2002). Resent reports

suggest that CPPs are not completely unspecific and uptake depends on used cell-line (Mai et

al., 2002; Violini et al., 2002; Ye et al., 2002). Extreme examples are tumour targeted

“homing” peptides, which are sometimes classified as CPPs (Essler and Ruoslahti, 2002;

Laakkonen et al., 2002). These peptides specifically internalise into cells carrying specific

membrane markers, some of which are already identified (Essler and Ruoslahti, 2002).

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1.2.1 Classes of CPPs 1.2.1.1 Penetratins Penetratin is the name of the Antennapedia homeodomain 43-58 peptide, which was the first

reported CPP. The parent protein is a drosophila transcription factor. It interacts with DNA

via a 60 amino acid long domain that consist of three alpha-helixes, third of which is

envisaged to be the most important DNA recognition site (Gehring et al., 1994). First it was

found that the 60 amino acid long polypeptide internalises to differentiated neurons in a

seemingly energy - independent manner (Joliot et al., 1991). To further investigate this

phenomenon several shorter peptides were synthesised (Derossi et al., 1994). Amino acids 43-

58 were found to be necessary and sufficient for temperature independent translocation. Later

studies have indicated that shorter fragments like amino acids 52-58 are also translocating into

cells (Fischer et al., 2000). Structure-activity studies of penetratin indicate that C-terminal

positive charges and tryptophan residues, particularly 48Trp, are important for internalisation

(Derossi et al., 1994; Fischer et al., 2000).

pIsl, studied in papers I and II belongs to the penetratin superfamily and is derived

from the homeodomain of the transcription factor Islet-1 (Isl-1) (Karlsson et al., 1990). Isl-1 is

known to bind and regulate the promoters of insulin, glucagon and somatostatin genes (Wang

and Drucker, 1995; Gay et al., 2000). The protein also possesses regulatory functions in

neuronal differentiation and regeneration (Thor and Thomas, 1997; Varela-Echavarria et al.,

1996; Hol et al., 1999).

1.2.1.2 Tat protein derived peptides pTat is the most widely used CPP sequence today and is in several meanings a pathfinder in

the CPP research. pTat is often used not as a separate peptide, but as a part of a recombinant

fusion protein. Energy-independent translocation of the HIV Tat protein was reported already

in 1988 (Frankel and Pabo, 1988; Green and Loewenstein, 1988). By the time penetratin was

first published, fusion proteins consisting of Tat protein fragments 37 – 72 or 37 – 62 and β-

galactosidase, horseradish peroxidase (Fawell et al., 1994) or a Fab antibody (Anderson et al.,

1993) were shown to internalise spontaneously to various cell-lines. However, Tat protein

derived short CPPs were published as late as 1997 by Vivés and coworkers (Vives et al.,

1997). Fragment 48-60 was the shortest reported peptide at this time found to translocate into

8

cells. Later structure-activity studies have given even shorter versions (Wender et al., 2000;

Futaki et al., 2001).

When, in the beginning of 2003, Richard and coworkers (Richard et al., 2003) rised

the question of artefacts in internalisation assays, it was again a work based on pTat and Arg9.

From FACS and live cell microscopy results, the authors concluded that the uptake of these

arginine rich peptides is endocytotic and energy dependent. This observation was in strict

contradiction with previous publications. Even mild fixation agents like paraformaldehyde

and glutaric aldehyde were found to cause artefacts similarly to methanol reported by

Lundberg and Johansson (Lundberg and Johansson, 2001).

1.2.1.3 Transportans In a search for new galanin receptor ligands, chimeric peptides consisting of galanin(1-13)

and other bioactive peptides were made. One of them, galanin(1-13)-mastoparan, was named

galparan (Langel et al., 1996). This peptide exerted unusual properties in receptor activation

studies. Apparently, the problem was that in contrary to other ligands, galparan was able to

internalise to cells in a receptor-independent manner and activate G-proteins (Zorko et al.,

1998). A peptide where 13Pro was replaced by Lys was called transportan (Pooga et al.,

1998b). Different cargoes coupled to the side-chain of 13Lys were shown to efficiently

internalise to cells (Pooga et al., 1998c; Pooga et al., 2001). Transportan, unfortunately, had

its own intracellular targets (i.e G-proteins) and its synthesis was problematic. Therefore

truncated and modified analogues were designed and tested in cellular penetration assays. One

of the analogues, “transportan 10” (TP10) or alternatively L262, retained CPP efficacy while

lacked GTPase activity (Soomets et al., 2000).

1.2.1.4 Other CPPs Besides the three named peptide families many other CPPs exist. Table 1 gives an overview

of the most well known sequences. Many peptides have been derived from natural sequences

in attempts to study functions of parent proteins and their trafficking (Elmquist et al., 2001;

Lundberg et al., 2002). Others, like oligoarginine (Wender et al., 2000; Rothbard et al., 2000)

or model amphipathic peptide (Oehlke et al., 1998) are designed for simplification of

structure-activity studies and ‘as proofs of principle’. They show that arginines or

amphipathic structure are sufficient for translocation. The third group of CPPs is chimeraes,

like transportans. They consist of two parts, one usually hydrophilic (for example NLS

9

sequence form simian virus 40 large T antigen PKKKRKV) the other hydrophobic. Fourth,

recently the border-line between CPPs and antimicrobial peptides has lost coherence and

some antimicrobial peptides can apparently be classified as CPPs (Park et al., 1998; Sadler et

al., 2002; Takeshima et al., 2003).

Table 1. A selection of CPPs Origin Sequence Reference Protein derived pTat 48–60 HIV TAT GRKKRRQRRRPPQ (Vives et al.,

1997) Penetratin (pAntp)

Drosophila Antennapedia homeodomain

RQIKIWFQNRRMKWKK (Derossi et al., 1994)

pVec vascular endothelial cadherin

LLIILRRRIRKQAHAHSK-amide (Elmquist et al., 2001)

hCT(9-32) human calcitonin LGTYTQDFNKFHTFPQTAIGVGAP- amide (Schmidt et al., 1998)

VP22 herpes simplex virus envelope protein

DAATATRGRSAARPTERPRAPARSASRPRRPVE (Elliott and O'Hare, 1997)

PrP mouse prion protein

MANLGYWLLALFVTMWTDVGLCKKRPKP (Lundberg et al., 2002)

pIsl Rat transcription factor Islet-1

RVIRVWFQNKRCKDKK-amide I

Designed / chimaraes Transportan Galanin(1-12)-Lys-

Mastoparan, desigened as GalR ligand.

GWTLNSAGYLLGKINLKALAALAKKIL-amide (Pooga et al., 1998a)

TP10 truncation analogue of transportan

AGYLLGKINLKALAALAKKIL-amide (Soomets et al., 2000)

MAP model amphipathic peptide

KLALKLALKALKAALKLA-amide (Oehlke et al., 1998)

Pep-1 Cationic NLS + hydrophobic tail

KETWWETWWTEWSQPKKKRKV-cysteamide (Morris et al., 2001)

MPG Cationic NLS + Hydrophobic tail

GALFLGWLGAAGSTMGAPKKKRKV-cysteamide (Morris et al., 1997)

oligoArg ultimate arginine rich peptide

(R)n (Rothbard et al., 2000; Futaki et al., 2001)

Antibacterial peptides LL-37

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES-C-amide

(Sandgren et al., 2004)

Buforin 2 TRSSRAGLQFPVGRVHRLLRK (Park et al., 1998)

Magainin 2 GIGKFLHSAKKFGKAFVGEIMNS (Takeshima et al., 2003)

Bactenecin 7 RRIRPRPPRLPRPRPRPLPFPRPG (Sadler et al., 2002)

10

1.2.2 Cell-penetrating peptides in model membrane systems How CPPs access cell interior is an important question. Unfortunately, biological systems are

very complex, and because of an overwhelming amount of factors to consider, the crucial

steps for delivery cannot be studied in details in situ. Hence, detailed description of one or

another interaction requires simplified model systems. In case of CPPs, the crucial steps are

very likely their membrane interactions and following translocation process. Therefore,

artificial membranes, with well-characterised composition are necessary for detailed analysis.

For lipid layers several models can be used: monolayers, vesicles, micelles, bicelles and also

solvent mixtures (Gräslund and Eriksson, 2002). Which model to use, is mainly determined

by the detection principle. Large vesicles are considered better mimics of cells, but their

mobility and light scattering properties are not compatible with all data acquisition protocols.

Examples of biophysical methods that cannot be used for live cells include nuclear

magnetic resonance (NMR), several (tryptophan or tyrosine) fluorescence assays, Fourier

transform infrared spectroscopy and circular dicroism (CD). Additional methods have been

used as well (Brattwall et al., 2003, paper II). In order to study secondary structure of peptides

and positioning in membranes, the listed methods are most reliable.

Like most peptides, CPPs do not have a strict conformation in solution, and random

coil structure is dominant (Chaloin et al., 1997; Vidal et al., 1998). In the presence of

membrane mimics, α-helical or β-sheet conformations are adopted and peptides penetrate into

membranes disrupting their structure (Magzoub et al., 2003; Barany-Wallje et al., 2004;

Deshayes et al., 2004b). At the same time, presence of a (peptide) cargo does not trigger any

conformational change (Deshayes et al., 2004a).

Reports concerning penetratin structure are contradictory. While some studies have

shown α-helical structure (Drin et al., 2001a; Drin et al., 2001b), others support β-sheet as the

dominating structure (Bellet-Amalric et al., 2000; Binder and Lindblom, 2004). Magzoub et

al. systematically studied the interactions of penetratin with various membrane mimics and

confirmed the “chameleon” behaviour of penetratin (Magzoub et al., 2002). At high

lipid/peptide ratio and at low vesicle surface charge, the α-helical structure is dominating. As

soon as the lipid peptide ratio reduces or membrane charge increases α-helix transits to β-

sheet.

11

Model amphipathic peptides have also been investigated thoroughly. First, charge

interactions with highly negative membranes and the peptide helicity in contact with neutral

membranes were found important (Dathe et al., 1996). Later, amphipathicity was confirmed

to be the only essential characteristic (Scheller et al., 1999). Influence of hydrophobicity,

hydrophobic moment, and peptide position in the membrane was analysed and moderate

correlations were found between these parameters and hemolytic and antimicrobial activities,

but not with internalisation (Dathe et al., 2002). An interesting study by Van Mau et al.

concludes from surface tension analysis that, for amphipathic peptides lipid-peptide

interactions are stronger than either lipid-lipid or peptide-peptide interactions (Van Mau et al.,

1999).

Prof. Gräslund’s group has studied transportan, another amphipathic peptide, in

membrane models. NMR and CD spectras indicated that the peptide is α-helical while bound

to membranes and has a bend in 15Asn region. The C-terminal mastoparan part is buried into

the membrane, while the N-terminus and the hinge region lay on the surface. (Lindberg et al.,

2001; Barany-Wallje et al., 2004). When comparing transportan with penetratin and pIsl,

Magzoub et al. discovered two principal differences. Transportan, in contrary to the both

arginine rich peptides was able to interact with uncharged membrane and it caused much

higher membrane disruption (Magzoub et al., 2003).

An outstanding study has been performed by Boichot et al. (Boichot et al., 2004).

Authors have not studied only the hCT(9-32) derived CPP, but its conjugate (covalent and

uncovalent) with EGFP on model membranes. Atom force microscopy shows that the peptide

itself and its cargo complex aggregate on areas rich in unsaturated lipids. Addition of

cholesterol changes the pattern and peptide/cargo aggregates move to “lipid rafts”. This is in

good correlation with reports of raft mediated uptake (Fittipaldi et al., 2003; Wadia et al.,

2004), although the latter studies were performed with pTat peptides.

1.2.3 Proposed internalisation mechanisms How CPPs internalise is certainly the most challenging and hot debate today. Increasing

evidence suggests involvement of endocytosis, but proposed non-endocytotic pathways have

not been abandoned yet.

For penetratin the “inverted micelle” model has been proposed (Derossi et al., 1996).

According to this model, positively charged penetratin interacts with negatively charged

12

membrane and upon interaction the tryptophane residues penetrate into the membrane. The

latter act destabilises the membrane and provokes formation of an inverted micelle, which,

eventually, will collapse back to the planar bilayer. Depending on the direction of the

collapse, penetratin may be released to the intracellular milieu. This proposal does not have

any obvious faults for peptide internalisation, but considering cargoes in size of liposomes

and viruses, the universality of this mechanism is doubtful. Moreover, the impact of

tryptophans for penetratin translocation is controversial (Derossi et al., 1994; Fischer et al.,

2000; Thoren et al., 2003), and recent studies rather highlight Arg residues as the main

translocation motor (Thoren et al., 2003).

From the world of antimicrobial peptides more translocation proposals have risen

(reviewed by Leuschner and Hansel, 2004). Lytic peptides like melittin adopt amphipathic α-

helical conformation when bound to the membrane, a common feature for many CPPs. These

α-helices further associate into barrels and turn themselves perpendicularly to the membrane

surface. The hydrophobic sides of helices face fatty acid chains and hydrophilic sides form a

pore, through which small molecules and ions can freely diffuse. Again, pore formation does

not explain cargo delivery and the low lytic activity of CPPs satisfactorily.

Yet another non-endocytotic mechanism is the “carpet formation”. In this scenario

aggregates of peptides coat the cell surface and thereby disrupt its structure. Cracks in

disrupted membranes are the sites of internalisation for peptides and cargoes. Recent atom

force microscopy studies indicate that carpet-like mechanism may be involved in calcitonin

derived peptide hCT(9-32) and its cargo complex internalisation (Boichot et al., 2004).

As already mentioned, evidences for endocytotic uptake have risen. For pTat,

penetratin (Console et al., 2003; Richard et al., 2003; Wadia et al., 2004) and polyarginine

(Fuchs and Raines, 2004), little hope for non-endocytotic pathways is left. These studies also

suggest heparan sulphate as a “receptor” candidate for positively charged CPPs.

Proteoglycans act frequently as endocytosis accessories (reviewed by Belting, 2003). Such an

accessory molecule would also explain the identical uptake of L- and D-isomeric peptides,

because chirality does not matter for charge interactions. Doubt raises from evidences that

cellular proteoglycan levels are not always correlated with the uptake efficacy (Violini et al.,

2002). Earlier studies reported that Tat protein-GFP fusion, but not pTat, needs

glucoseaminoglycans (Silhol et al., 2002). However, this study used protocols that may be

associated with artefacts. Ross et al. used a clever methodology to study the involvement of

endocytosis (Ross et al., 2004). Since mitochondrion membranes lack vesicular transport

systems they studied translocation of pTat and penetratin (and their derivates) into

13

mitochondria. No internalisation was detected, and thus the authors concluded that there is no

non-endocytotic component involved in the translocation. For amphipathic peptides,

particularly MPG and Pep-1, a non-endocytotic mechanism is still supported (Deshayes et al.,

2004a) even in the case of cargo (siRNA) delivery (Simeoni et al., 2003).

An outstanding work by Wadia et al. demonstrated that pTat-Cre protein is

internalised via lipid raft mediated macropinocytosis because the internalisation was not

dynamin dependent but required cholesterol (Wadia et al., 2004). At the same time Ferrari et

al. visualised caveolae mediated uptake of pTat-EGFP fusion proteins in real time (Ferrari et

al., 2003; Fittipaldi et al., 2003). Caveolar uptake is also suggested for λ-phages (Eguchi et

al., 2001). Macropinocytosis and caveolar uptake are both raft mediated, making their

distinguishing difficult. Dynamin independence, on one hand (Wadia et al., 2004), and co-

localisation with caveolin on the other hand (Fittipaldi et al., 2003) are both strong arguments

for respective endocytic pathways. Possibly different routes can be taken and depending on

the peptide, cell type and presence of endocytosis inhibitors, their fraction varies (Thoren et

al., 2003).

To top the discrepancy of internalisation and its mechanisms, some reports claim no

uptake of CPPs at all. Falnes et al. report inability of pTat and VP22 recombinant conjugates

with diphtheria toxin A fragment to internalise (Falnes et al., 2001). Kelemen et al. could not

see any uptake of VP22 – peptide conjugate while pTat conjugate was internalised (Kelemen

et al., 2002). [99Tc]-Tat peptides are not internalised into cells in confluent monolayers

according to Violini et al. (Violini et al., 2002).

1.2.4 Cargoes delivered by CPPs Types of cargoes for CPPs do not seem to be limited by size. Starting from small ions and

fluorescence labels, covering proteins and oligonucleotides, cargo types end up with

nanoparticles, viruses and liposomes. Peptides, ions, polynucleotides, nanoparticles and

liposomes all have distinct chemical nature, which again is not a hindrance for CPP mediated

delivery, according to published data. An extensive review was published very recently

(Dietz and Bähr, 2004) and contains a detailed table with about 300 references to CPP

delivered cargoes until early 2004. The authors list the carrier peptides, cargoes, biological

activity and also whether the study was conducted in vivo or in vitro. Table 2 gives a brief

overview about delivered cargoes and their coupling to the carrier peptide.

14

Table 2. Cargoes for CPPs and their coupling methods. ND = not described. CPP cargo type cargo specification linkage CPP/

cargo ratio

reference

penetratin and a fibroblast growth factor derived CPP

low MW compounds

fluorophores covalent 1 (Fischer et al., 2002)

pTat low MW compounds

technecium, rhenium, gadolinium, dysprosium

chelate 1 (Polyakov et al., 2000; Bullok et al., 2002; Bhorade et al., 2000)

Arg7 low MW compounds

cyclosporin A a specific pH sensitive (covalent) linker

1 (Rothbard et al., 2000)

pTat, penetratin

peptide Mek1 N-terminus same chain synthesis

1 (Kelemen et al., 2002)

TP10 peptide PKC and cannabinoid receptor fragments

disulphide 1 (Howl et al., 2003)

pTat peptide PKC modulating peptides

disulphide 1 (Begley et al., 2004)

pTat, penetratin

peptide cyclin/cyclin dependent kinase inhibitors

same chain synthesis

1 (Chen et al., 1999)

pTat peptide MARCKS related protein fragments


1 (Corradin et al., 2002)

pTat peptide fragment of von Hippel-Lindau gene product

fusion 1 (Datta et al., 2001)

Arg11 and NLS peptide PKA inhibitor same chain synthesis

1 (Matsushita et al., 2001)

Arg7 peptide hemagglutinin epitope

disulphide 1 (Robbins et al., 2002)

pTat peptide anti-apoptotic Bcl-x(L) fragment

beta-alanine linker

1 (Shimizu et al., 2000)

pTat, penetratin

peptide p53 fragment same chain synthesis

1 (Snyder et al., 2004; Kanovsky et al., 2001)

pTat peptide protein kinase B substrates

disulphide and a photocleavable linker

1 (Soughayer et al., 2004)

pTat peptide antigenic peptide and controls


1 (Wang et al., 2002)

pTat peptide fragments of hypoxia-inducible factor-1

fusion 1 (Willam et al., 2002)

pTat peptide c-Jun kinase inhibitor same chain synthesis

1 (Borsello et al., 2003; Kaneto et al., 2004)

penetratin PNA-peptide chimerae

PNA-NLS disulphide 1 (Braun et al., 2002)

Pep-1 protein GFP, Pep-A noncovalent 6-8 for Pep-A 12-14 for

(Morris et al., 2001)

15

GFP pTat, Arg11 Arg9, Arg7

protein EGFP fusion 1 (Matsushita et al., 2001)

Pep-1 protein superoxide dismutase

fusion 1 (Sik Eum et al., 2004)

VP22 protein proapoptotic BH3 domain family member Bak

fusion 1 (Brewis et al., 2003)

pTat protein Cre recombinase fusion 1 (Caron et al., 2004; Peitz et al., 2002)

pTat protein Rho GTPase fusion 1 (Chellaiah et al., 2000)

pTat and penetratin

protein avidin biotin-avidin 1(4) (Console et al., 2003)

VP22 protein GFP fusion 1 (Fang et al., 1998) (Boenicke et al., 2003)

Arg8 protein His-tagged pro-apoptotic peptide and EGFP

Ni2+ - His (noncovalent)

1 (Futaki et al., 2004b)

pTat protein dominant negative Ras

fusion 1 (Myou et al., 2002)

NLS(various) protein β-galactosidase, lysozyme

chemical crosslinking

2 (Ragin et al., 2002)

proline rich Bac7 analogues

protein Avidin biotin-avidin (noncovalent)

1 (4) (Sadler et al., 2002)

pTat protein clostridium botulinum C3 exoenzyme

fusion 1 (Sauzeau et al., 2001)

pTat protein p27 fusion 1 (Snyder et al., 2003)

pTat protein heat shock protein HSP20

fusion 1 (Tessier et al., 2004)

tat protein caspase-3 fusion 1 (Vocero-Akbani et al., 1999)

pTat, Arg8 protein RNase S CPP-RNse S (1-20) covalent chimera, which binds RNase S (21-124) noncovalently (KD = 10-5)

1 (Futaki et al., 2004a)

NLS(various) oligonucleotide K-ras oncogene 10mer

disulphide 1 (Ragin et al., 2002)

penetratin oligonucleotide nonsense PNA peptide bond 1 (Simmons et al., 1997)

penetratin oligonucleotide superoxide dismutase antisense oligodeoxynucleotide

disulphide 1 (Troy et al., 1996)

penetratin oligonucleotide PNA targeting telomerase RNA component

disulphide 1 (Villa et al., 2000)

60 amino acid antennapedia homeodomain

oligonucleotide amyloid precursor peptide asON

disulphide 1 (Allinquant et al., 1995)

penetratin oligonucleotide prepro-oxytocin antisense PNA

beta-alanine linker

1 (Aldrian-Herrada et al., 1998)

16

transportan oligonucleotide X-chromosome targeting PNAs

disulphide 1 (Beletskii et al., 2001)

NLS based CPPs

oligonucleotide Ca2+ L channel β subunit phospho-thioate asON

disulphide 1 (Chaloin et al., 1998)

pVec oligonucleotide PNA 6mer same chain cyntesis

1 (Elmquist et al., 2001)

MPG oligonucleotide 18 and 36 mer fragments of HIV natural primer binding site

charge interaction

total 20, in complex 3-8

(Morris et al., 1997)

penetratin transportan

oligonucleotide GalR1 antisense PNA

disulphide 1 (Pooga et al., 1998c)

transportan oligonucleotide anti HIV transactivation response element PNA

disulphide 1 (Kaushik et al., 2002)

Transportan, TP10

oligonucleotide NFκB binding site decoy

disulphide to PNA, and PNA hybridisation to oligonucleotides

1 Fisher et al., 2004

pTat polyanions DNA, heparan sulphate

charge interaction

≥ 7 (Sandgren et al., 2002)

pTat polymer N-(2-hydroxypropyl) metharcyl-amide MW~26 kDa


ca 1 (Nori et al., 2003)

pep-1 plasmid luciferase, GFP charge interaction

> 104 (Morris et al., 1999)

branched tat plasmid luciferase charge interaction

ca 1500

(Tung et al., 2002)

LL-37 plasmid luciferase charge interaction

ca 1500

(Sandgren et al., 2004)

pTat plasmid β-galactosidase and apolipoprotein A-I

noncovalent > 1500 (Ignatovich et al., 2003)

pTat nanoparticles superparamagnetic iron-oxide

disulphide 6.7 (Josephson et al., 1999)

pTat nanoparticles cross-linked superparamagnetic iron oxide


5 - 15 (Zhao et al., 2002)

pTat nanoparticles superparamagnetic iron-oxide particles (total size about 45 nm)


4.1 and 9.7

(Lewin et al., 2000) (Wunderbaldinger et al., 2002)

transportan colloidal gold colloidal gold – avidin covalent complex

biotin avidin 1 (4) (Pooga et al., 2001)

pTat, long verison of NLS

virus lambda phage expression on phage surface

ND (Eguchi et al., 2001; Akuta et al., 2002)

penetratin virus adenovirus free peptide (noncovalent)

> 105 (Gratton et al., 2003)

pTat and penetratin

liposome chemical crosslinking

ND ≥ 5

(Console et al., 2003) (Tseng et al., 2002)

pTat liposome chemical crosslinking

ca 500 (Torchilin et al., 2001; Torchilin et al., 2003)

17

Fluorophores, green fluorescent protein and radionuclide conjugates are good for

visualising internalisation of CPPs. They, as well as magnetic nanoparticles can also be used

for in vivo imaging of tissues (reviewed by Franc et al., 2003), without affecting cell

metabolism. Other proteins, peptides and oligonucleotides are designed to have a specific

effect on the level of cell metabolism. Examples of biological activities of peptide cargoes are

inhibition of c-Jun kinase (Borsello et al., 2003; Kaneto et al., 2004), inhibition of calcineurin

and NFAT (nuclear factor of activated T cells) interactions (Noguchi et al., 2004), inhibition

of IκB and NFkB essential modulator interactions (May et al., 2000), and secretion of β-

hexosaminidase (Howl et al., 2003). The list is long and consists mainly of peptides that

mimic certain protein-protein or protein-nucleotide interaction sites and thereby compete with

the parent protein. Since these inhibitory peptides themselves are not CPPs, for reaching

targets, they must be conjugated to a CPP. Samples of cargo proteins with specific effects are:

caspase-3, which kills HIV infected cells (Vocero-akbani 1999), cyclin E and D3, which

restore proliferation in defected cells (Hsia et al., 2002), p53, which induces apoptosis in

target cells (Phelan et al., 1998; Takenobu et al., 2002). Again, the list is significantly longer

(Table 2; Dietz and Bähr, 2004). It must be noted that fusion proteins with arginine rich CPP

moieties are in vast majority and less work has been carried out with amphipathic peptides.

First attempts in gene delivery with CPPs have been reported (Table 2).

Expectationally, for preliminary studies, the delivered genes are mainly expressing

biologically neutral reporter proteins (GFP, luciferase, β-galactosidase). Penetratin delivered

adenovirus carrying a vascular endothelial growth factor gene has been shown to promote

angiogenesis (Gratton et al., 2003). Additionally, a plasmid, delivered by MPG, and encoding

the cell-cycle regulatory protein cdc25C targeting antisense, has been shown to be active and

arrest the cell cycle (Morris et al., 1999).

Short oligonucleotides have relatively few distinct biological functions. They either

exert antisense effects by nucleotide-nucleotide interactions (discussed in chapter 1.3.1) or

compete with endogenous nucleotide-protein interactions (reviewed by Gambari, 2004).

Antisense technology has suffered from two problems: delivery and design of

oligonucleotides (Stein, 1999). The decade that has passed from the first CPP delivered

antisense oligonucleotides (Allinquant et al., 1995) has demonstrated CPP suitability for

antisense oligonucleotide delivery. Desired effects of have been obtained in correction of

aberrant luciferase gene splicing (Astriab-Fisher et al., 2002), superoxide dismutase antisense

(Troy et al., 1996) and inhibition of HIV production by blocking the transactivation response

18

element (Kaushik et al., 2002) (see also Table 2. and Dietz and Bähr, 2004). As mentioned,

also protein-nucleotide interactions can be inhibited by exogenous oligonucleotides. Short

double stranded decoy nucleotides bearing a transcription factor recognition site efficiently

compete with genomic DNA as demonstrated for NFκB (Fisher et al., 2004).

1.2.5 Cargo coupling methods How to couple a cargo to the carrier peptide depends on the cargo type. Cargoes like peptides,

proteins and peptide nucleic acids can be linked directly to the same polypeptide chain in

synthesis or recombinant expression (does not apply to peptide nucleic acids). For other

cargoes this is not possible. In fact, also for peptides and proteins, direct linkage is not always

advised. The cargo may alter the CPP translocation properties, and the CPP may alter the

cargo’s biological functions. It can be the case in some studies reporting no uptake or

biological activity. A less stable bond, like a disulphide bridge, or entirely noncovalent

interactions might be preferred in some cases.

Noncovalent, charge-charge interactions have, within the last 5 years, seemingly made

a breakthrough in CPP-cargo coupling. Simple mixing of CPP and proteins (Morris et al.,

2001), siRNAs (Simeoni et al., 2003), plasmids (Morris et al., 1999) and viruses (Gratton et

al., 2003) has been shown to increase the uptake of the listed cargoes. Regarding noncovalent

conjugation, biotin-(strept)avidin complexation and metal chelates must be mentioned.

Although noncovalent, the stability of these complexes is high. Similarly other specific non-

covalent interactions can be used. Futaki et al. recently demonstrated delivery of RNase S in a

noncovalent but specific peptide-protein complex (Futaki et al., 2001). Yet another specific

non-covalent linkage has been used. Oligonucleotides (Fisher et al., 2004) and plasmids (IV,

Brandén et al., 1999) have been attached to CPPs via sequence specific hybridisation to a

PNA-CPP conjugate, where PNA acts as the “glue”. The complex stability can be easily

regulated by PNA/DNA lengths and mismatches and is well characterised.

Bifunctional crosslinkers provide another type of covalent conjugation method. For

several biochemical purposes reagents with two specific reaction centres have been

developed. While one reacts to, for example, amino groups of a protein the other remains

stabile and can later be specifically reacted to, for example, thiol groups of a peptide. This is

essential for coupling cargoes that cannot be synthesised in the same chain with the peptide

(like liposomes), or if the same chain synthesis alters CPP or cargo activity.

19

One aspect to consider, but not often discussed, is the ratio of CPPs per cargo. In other

words CPP/cargo ratio is not always strictly 1, but higher and can be hundreds of CPPs per

cargo (Table. 2). Particularly it is the case of non-covalent complexes. Judging from

published data, it can be concluded that from a certain cargo size (plasmids, nanoparticles) a

single CPP is obsolete, while several CPP molecules still perform the delivery. Work of Zhao

et al. demonstrates that by increasing the number of pTat molecules per nanoparticle from 1

to 15, uptake increases exponentially (Zhao et al., 2002). About 5 pTat peptides per cargo was

the threshold above which intracellular signal differentiated significantly from the

background. Independently, Tseng et al. analysed liposome uptake and concluded that for

uptake of small unilamellar vesicles (<100 nm) at least 5 to 10 penetratin molecules must be

attached to it (Tseng et al., 2002). Five pTat per cargo, lowest tested ratio, yielded uptake in

all tested cell lines, therefore it was impossible to determine the minimum loading

requirements. In nanoparticle delivery, again 4 peptides per particle were used (Lewin et al.,

2000). For smaller covalently linked cargoes the conjugate structure is known making it

certain that one vector corresponds to one cargo. In recombinant expression of CPP-protein

fusion, of course, the ratio is also strictly 1. Cargoes in size of recombinant proteins or smaller

are generally delivered efficiently by a single CPP.

1.3 Objectives for cargoes

1.3.1 Antisense Antisense technology exploits sequence specific nucleotide-nucleobase interactions for

manipulation of gene expression. Traditionally, it means application of a short oligonucleotide

(antisense oligonucleotide (asON)), which leads to degradation or inactivation of the target

mRNA upon hybridisation to a complementary sequence. If the asON interferes with genes, it

is also referred to as antigene effect and antigene agent (Helene and Toulme, 1990; Praseuth

et al., 1999).

1.3.1.1 Antisense mechanisms In general antisense can work in one of the two principles: either cause degradation or

sterically block its target (Crooke, 2000). The first can be obtained via intracellular enzymes

that recognise asON-target complex or by chemical activity of the asON itself. The first

generation antisense molecules were DNA oligomers and oligophosphothioates, where one of

20

the non-bridging oxygens in the phosphate is replaced by sulphur (De Clercq et al., 1969).

When such oligomers bind to RNA, a nuclease, RNase H, recognises the duplex and cleaves

the RNA. Different RNases recognise different complexes or structural moieties that allow

variable strategies for RNA cleavage (reviewed by Baker and Monia, 1999). Noteworthy is,

that also small interfering RNAs (siRNA), which have recently been found to be an

endogenous regulatory system applies the degradation principle. First the RNase III Dicer cuts

a 21-23 nucleotides long fragment of double stranded RNAs. The short RNA oligomers

activate the RNA-induced silencing complex (RISC), which in turn associates and cleaves

mRNAs complementary to the short RNA fragment. As shown recently RISC also tolerates

chemically modified nucleotides (Chiu and Rana, 2003).

Another approach is to incorporate the cleaving moiety into the asON. Ribozymes,

RNA based enzymes that occur naturally and can be engineered artificially, interact with

complementary targets and at the same time function as an RNase. Secondly, the cleaving

moiety may be a simpler chemical compound or heavy metal ion attached to the asON (He et

al., 2004). Heavy metal ions form complexes with the DNA, and thereby inactivate or degrade

it.

Steric hindrance is a relatively new strategy, although analogous endogenous

processes may exist (Morris et al., 2004). The first generation of asONs (i.e. phosphothioates)

were recognised by cells as natural oligonucleotides. This was necessary for RNase H

activation. On the other hand, also DNases recognised these oligomers and degraded them.

Second and third generation DNA analogues with improved nuclease resistance and longer

half-life failed also RNase H activation. However, if they are not disassociated when a

ribosome attaches to or moves along the mRNA, down-regulation in respective protein level

still occurs. To name some, peptide nucleic acids (PNAs), morpholino oligos and locked

nucleic acids (LNA) are capable of sterical hindrance. Either by a yet uncharacterised

interaction with RNases or by disruption of important mRNA stabilisation motifs, degradation

of a fraction of the target mRNA cannot be excluded. Steric hindrance antisense principle can

also be utilised for increasing the expression of a gene, since it can be used for redirecting

aberrant splicing Astriab-Fisher et al., 2002.

The level at which asONs work involves all steps in the DNA and RNA processing:

transcription start and elongation, pre-mRNA capping, polyadenylation, splicing and delivery

to cytoplasm, mature mRNA maintenance and finally translation start and elongation

(reviewed by Baker and Monia, 1999). Degradation makes sense in any listed step, and there

are always protein/RNA interactions that can be sterically hindered.

21

1.3.1.2 Design of antisense oligonucleotides Some restrictions are set by the chosen strategy, like PNAs suffer from ineffective uptake in

most cells and ribozymes are likely to bind intracellular proteins. Design of oligonucleotide

sequences is, however, the greatest challenge and cannot always be overcome by changing the

type of asON or delivery vector.

Secondary and tertiary structures of targets always influence the antisense efficacy.

Targeting a structurally hindered region is envisaged as the most common failure in antisense.

RNA structures are complex and therefore hard to predict. The first and second generations of

asONs were not capable of invading double stranded structures. Members of the third

generation of asONs like PNAs, have put double strand invasion and thereby the entire

antisense strategy into a different light (Peffer et al., 1993). Unfortunately, problems in target

structure predictions have hampered data collection in this aspect. Hence up to date, scanning

an mRNA with different antisense agents is the best method to find a good asON. Scanning

can be done by random “shots” or by systematic arrays of nucleotides (reviewed by Sohail

and Southern, 2000). If the mRNA is available in vitro (i.e. purified form), random libraries

and RNase H or other RNA cleaving enzymes can be used to digest end-labelled mRNA and

map all the accessible sites (reviewed by Sohail and Southern, 2000).

Comupter prediction of structures is an alternative to screening. They are less

laborious, but not always correct (Milner et al., 1997; Sohail et al., 1999). New types of

asONs and continuous improvements of existing algorithms (Mathews et al., 2004), however,

call for additional tests and evaluation of new strategies.

1.3.1.3 Side-effects and controls There is always a risk that a drug designed for a specific target has side-effects in complex

biological systems. Some of them can be predicted, others not. Results from Matson and

Krieg indicating that the first generation asONs alter the intracellular nucleotide pool and

thereby DNA metabolism is, for example, predictable (Matson and Krieg, 1992). Ribozymes

are natural aptamers, i.e. nucleotides with very conserved secondary structures that bind

proteins specifically. Therefore protein–ribozyme interactions are always a valid side-effect in

ribozyme utilisation. Also very short or long oligomers have logically high probability to

22

cause side-effects. Short asONs (< 10 nucleotides) have, likely, more than one fully

complementary target. Long oligomers can form stable complexes with only partly

complementary sequences. It has been demonstrated that an internal complementary 5-mer is

enough for inducing RNase H activation and target cleavage (Monia et al., 1993).

Twelve to fifteen nucleotides are calculated to be the minimum length for a specific

sequence, assuming entirely random distribution of the bases within the 3.2 billion basepairs

of the human genome (Woolf et al., 1992). Since the genome contains many repeats these

calculated values cannot be taken as a rule of thumb, but experiments, some of which

covering enormous database of asON sequences, indicate the rightfulness of this theory

(Lloyd et al., 2001). Interestingly, longer sequences are not necessarily more specific. In

contrary, longer sequences may contain more probable sub-sequences that are complementary

to unspecific targets.

As our knowledge of genomes and splicing products as well as search-engines

improve, finding cross-targets becomes easier and easier, though the final goal is not reached

yet. Unspecific interactions with proteins are more difficult to predict since protein-nucleotide

interactions are not well characterised. asONs, like oligophosphothioathes are polyanions and

may electrostatically interact with positively charged proteins (Cazenave et al., 1989). An

extreme example is the discovery of a tyrosine kinase p210 inhibitor from antisense

screening, whereas the inhibition occurred at nucleotide-protein, and not at antisense level

(Bergan et al., 1994).

To minimise the risk of side effects, controls should always be used. Firstly, different

asONs should be applied and, secondly, more than one outcome must be checked (cell

viability, housekeeping proteins vs desired effect). For a long time, mismatch, scrambled and

sense controls were suggested for asONs (Stein and Krieg, 1994). Sense control was

suggested because it maintains the structural features of the antisense sequence. This is true

only partly, since guanidine, but not its counterpart cystine, can form quatruplexes (Panyutin

et al., 1990). Hence, the sense sequence may adopt a different structure than the antisense

oligomer. Moreover, in case of antigene, where the target has both strands, sense control may

show specific effects. Confirmed by editors of the Oligonucleotides journal (formerly

Antisense and Nucleic Acid Drug Development), sense is not the best control and mismatched

or scrambled asONs are preferred (Stein, 1999). Moreover, G rich oligomers (particularly

with 4 or more adjacent G) should be avoided (Stein, 1999). The mismatch control has a few

(often only one) nucleotides replaced, and should demonstrate specificity by not giving the

same effect as the wild-type asON. Although a negative effect (no down regulation) from the

23

mismatch oligomer is an excellent proof of specificity, positive results do not prove much.

There is no direct evidence of side effects, because the target discrimination threshold may

not be exceeded. Due to minimal differences from original sequence the mismatch may still

interact with the original target sequence. Scramble oligonucleotides or non-related sequences

are more universal controls. A positive result from them is a clear evidence of side effects,

since the target should not recognise these oligomers under any circumstances. Scramble

oligonucleotides with switched nucleobase composition have identical total pyrimidine/purine

ratio with original asON. At least in the case of PNA mediated antisense, it may be important

(Wittung et al., 1997).

1.3.1.4 Peptide nucleic acids Peptide nucleic acids were designed by Nielsen et al. in 1991 (Nielsen et al., 1991). The

phosphoribose backbone of RNA was replaced by a polyamide without altering the positions

of nucleobases. Several different polyamides have been tested for backbone replacement, but

the most common so far is N-(2-aminoethyl)glycine (Fig. 2A). Due to the neutral backbone of

PNA, complementary RNA and DNA complexes with PNA are more stable than double

stranded DNA (Egholm et al., 1993; Giesen et al., 1998). Mismatch discrimination is strongly

pronounced in PNA/DNA interactions. Additionally, PNA/DNA duplexes are less sensitive to

ionic strength of the environment than DNA duplexes (Tomac et al., 1996) and PNA is highly

resistant to enzymes (Demidov et al., 1994). A major drawback of PNA is poor cellular

uptake, except for neuronal cells (Tyler et al., 1998). PNAs are also capable to form triplexes

using Hoogsteen hydrogen bonding besides Watson-Crick bonds (Fig. 2B; Egholm et al.,

1993). Depending on the pyrimidine (C and T nucleobases) content of the PNA oligomer,

PNA2/DNA, DNA2/PNA or PNA/DNA complexes can form (Wittung et al., 1997).

PNA asONs have been shown to function at the levels of: reverse transcription of HIV

RNA (Boulme et al., 1998) mitocondrial DNA replication (Muratovska et al., 2001), mRNA

splicing (Karras et al., 2001; Sazani et al., 2002), and transcription initiation and prolongation

(Dias et al., 1999; Dryselius et al., 2003). Besides antisense, PNAs offer improved methods

for PCR (Orum et al., 1993; Demers et al., 1995), investigations of tRNA functions

(Ninomiya et al., 2001) and also gene up-regulation (Mollegaard et al., 1994; Wang and Xu,

2004). Excellent reviews about PNA use are available in literature (Nielsen, 2000; Nielsen,

2002).

24

Figure 2 A) DNA and PNA structures. B) Watson-Crick and Hoogsteen hydrogen bonding between nucleobases.

1.3.2 Galanin and its receptors Galanin is a 29 (30 in humans) amino acid long neuropeptide. It is widely distributed in both

the central and peripheral nervous system. It is co-released with other neurotransmitters, e.g.

acetylcholine, and modulates their activity. Today the galaninergic system is known to be

involved in pain transmission and hyperalgesia (Kerr et al., 2000; Sun et al., 2004), recovery

of neurons after injury (Holmes et al., 2000; Hwang et al., 2004), epilepsy (Mazarati et al.,

1998; Mazarati et al., 2004), feeding (Kyrkouli et al., 1986; Moron et al., 2004), learning

(Ögren et al., 1999; Kinney et al., 2003), sexual behaviour (Poggioli et al., 1992; Bloch et al.,

1996) and Alzheimer’s disease (reviewed by Counts et al., 2003). Its N-terminal 13 amino

acids long sequence is conserved throughout all species and is the interaction region with

galanin receptors. Three galanin receptor subtypes have been cloned (Habert-Ortoli et al.,

1994; Bloomquist et al., 1998; Borowsky et al., 1998; Wang et al., 1997). Galanin receptor 1

(GalR1) messenger RNA has been detected exclusively in the central and peripheral nervous

system with highest expression in hypothalamus, amygdala, spinal cord and dorsal root

ganglia. Galanin receptor 2 messenger RNA is highly expressed in hypothalamus, dorsal root

ganglia, and kidney with moderate expression in several other tissues. Galanin receptor 3

25

messenger RNA is widely distributed at low to moderate levels in many central and peripheral

tissues (Waters and Krause, 2000). Little is known about functions of different subtypes, due

to lack of subtype specific ligands for long time. 2001 Liu et al (Liu et al., 2001) reported that

Gal(2-11) has a 500-fold preference to rat GalR2 over human GalR1. Furthermore, constant

failure in creation of subtype specific antibodies has hampered the galanin research. One way

to study the function of each receptor subtype specifically is to down-regulate them by asONs.

However, for reliable results efficient asONs must be designed.

1.3.3 Sensitisation Pain is defined as “perception of an aversive or unpleasant sensation that originates from a

specific region of the body” (Kandel et al., 1991). Nociception is a closely related term and

refers to sensory signals conveying information about potential tissue damage. If the

nociceptive signal is repeated sufficiently, sensitisation of spinal cord neurons can occur and

pain is felt upon less intense signal than normal. Some molecular aspects of sensitisation are

closely related to learning and memory (Ji et al., 2003).

1.3.3.1 Action potential windup Regardless of being physiological, inflammatory or neuropathic pain, sensitisation is a result

of neuronal plasticity – the capacity of neurons to change their function, chemical profile, or

structure (Woolf and Salter, 2000). One of these mechanisms is action potential windup. The

principle of the windup is an increase of the neuronal excitability upon certain stimulation.

Dorsal horn neurons wind up if the C-fiber fires with a frequency of 0.2 – 20 Hz and the

maximum level of windup is reached after 8 – 70 stimuli (Herrero et al., 2000). The effect has

been known for almost 40 years (Mendell, 1966), but its mechanism is still obscure. It is

known that windup is not the only way of sensitisation (Laird et al., 1995) and it does not

happen in all types of neurons (Schouenborg and Sjolund, 1983). On the molecular level the

involvement of N-methyl-D-aspartate (NMDA) receptors, tachykinin receptors and Ca2+

channels has been shown (Davies and Lodge, 1987; Russo and Hounsgaard, 1994; Barbieri

and Nistri, 2001), but the exact function of these needs to be clarified (Morisset and Nagy,

2000).

26

1.3.3.2 N-methyl-D-aspartate receptors The most important excitatory neurotransmitter in nervous systems is glutamate. NMDA is a

specific ligand for a set of ionotropic glutamate receptors, which are therefore called NMDA

receptors. They consist of four subunits. The most common arrangement contains two, so

called, NR1 and two NR2 subunits. In the middle of the subunits is the pore, which has

especially high permeability for Ca2+. Gating of the pore is regulated at many stages. The

highest conductance requires glycine and glutamate as co-agonists and a slightly depolarised

membrane.

NMDA receptors are not involved in acute pain to the same degree as in long term

processes, including the action potential windup. NMDA antagonists (Medvedev et al., 2004)

and siRNA mediated NMDA receptor down-regulation (Shimoyama et al., 2004; Tan et al.,

2004) suppress pain like behaviour of rats in chronic pain models.

1.3.3.3 Ca2+ channels Ca2+ is an important regulator of cell signalling. Ca2+- currents are divided into L, T, N, P, Q

and R types. Channels mediating these currents are composed of four different subunits α1,

α2δ (heterodimer), β and γ, where α1 is the actual pore. The channel type is primarily defined

by the α1 subunit and at least 10 versions of α1 are known. Their historical names are α1A to

α1I and α1S and newly recommended systematic names are CaVX.Y, where numerical X and

Y refer to super and subclasses (Ertel et al., 2000). For example α1C is synonymous to

CaV1.2 and α1D to CaV1.3. Furthermore, each of these subclasses consists of several

individual proteins. The CaV1.3 gene, for example, encodes for at least 16 different splicing

variants (Safa et al., 2001).

CaV1.2 expression is restricted to the soma, axons and basal dendrites, while CaV1.3

can be found more widely on dendrites and is more concentrated in synaptic sites

(Westenbroek et al., 1998; Simon et al., 2003). Additionally, CaV1.3 activates at about 20 mV

more hyperpolarised membrane potentials than CaV1.2 (Xu and Lipscombe, 2001). This all

suggest separate functions for both L-channel subtypes, which, however, is problematic to

verify experimentally. Knock-out mice die in early embryonic stage and neither is it possible

to relay on specificity of ligands. Like in the case of galanin, antisense offers a plausible

strategy to distinguish the channels in vivo.

27

1.3.4 Gene delivery Adding genes to a living organism during its lifetime holds enormous potential for gene

therapy. Identification of genes and entire genomes of many organisms as well as

development of recombinant DNA technologies opens novel research and medical care

possibilities. Unfortunately, gene technology is strongly hampered by difficulties in delivering

genes to desired locations. All existing methods to introduce new genes to a living organism

have drawbacks. The most efficient vectors today are viruses. Viral DNA is partially replaced,

but infection of host cells occurs in the ways that viruses have developed during the evolution.

Although such particles are efficient vectors they are still recognised by host immunosystem,

which causes problems in in vivo applications (reviewed by Robbins and Ghivizzani, 1998.

In non-viral gene delivery the gene is usually incorporated into a plasmid. Plasmids

are circular DNAs from bacteria capable to replicate and transcribe products independently

from host chromosomes. Their size is in range of megadaltons. Electrical and mechanical

delivery of plasmids, like electroporation or DNA particle bombardment use a “brutal force”

principle and damage the organism. Chemical delivery vectors are more versatile, because

they can use different routes that can be optimised for safety. Liposomes and polycationes are

the main types of chemical carriers (reviewed by Luo and Saltzman, 2000). The former

encapsulates DNA into a hydrophobic vesicle and thereby increases solubility in membranes.

This works well in cell cultures, if cells are not sensitive to foreign lipids. In vivo, such lipid

micelles are not as effective, mainly because of low stability (reviewed by Lee and Huang,

1997).

Polycations interact with DNA through charge interactions and neutralise the charge

of DNA and, at the same time, condense it into smaller particles. Upon adsorptive binding to

the cell membrane DNA/polycation complexes (polyplexes) are internalised via endocytosis.

Also polycations are toxic in vivo. The general statement of all delivery methods is that higher

delivery efficiency causes higher toxicity (Luo and Saltzman, 2000).

Polyplexes and also liposomes can be modified with surface coats, for example

polyethylene glycol, and functional entities (Kircheis et al., 2001). While former reduces

unwanted bioactivities of the particle the latter allows targeting and enhanced delivery. pTat

modified liposomes are demonstrated to be efficient and relatively non-toxic both in vitro and

in vivo (Torchilin et al., 2003). Targeting can be obtained via specific receptor ligands like

transferrin (Ogris et al., 2001b).

28

2 Aims:

Papers I and II: To compare a novel CPP, pIsl, to penetratin in the means of membrane

interactions and internalisation with and without a protein cargo.

Paper III: In vitro delivery of antisense PNA oligomers and optimisation of hGalR1 targeting

antisense.

Paper IV: Identification of roles of NMDA receptors and L-type Ca2+ channels in axon

potential windup. Discrimination of CaV1.2 and CaV1.3 L-channels by in vivo down-

regulation either of them with a specific CPP - antisense PNA conjugate.

Paper V: Evaluation of TP10 as an independent gene delivery vector and as a component of

PEI transfection protocols.

29

3 Methodological consideration 3.1 Selection of CPPs pIsl was designed and characterised in papers I and II with the penetratin structure-activity

relationship in mind. Although pIsl is a naturally occurring sequence, it is, at the same time,

an analogue of penetratin. For penetratin internalisation, positive charges and tryptophan

residues have been suggested to be important (Derossi et al., 1994). Therefore, the

replacement of hydrophobic 14Trp of penetratin to negative Glu in the highly positively

charged C-terminus is of particular interest. Furthermore, peptide position in lipid layers is

commonly identified from Trp fluorescence. Penetratin, unlike pIsl, has two Trp residues,

which complicates data interpretations.

In paper III, transportan was used because the study itself was a sequel to the first

demonstration of antisense efficacy of GalR1 targeting PNAs (Pooga et al., 1998c). It was

started right after obtaining the first results with GalR1 antisense PNA and before transportan

structure-activity studies were performed. Later TP10, a transportan analogue with fewer side

effects, was developed (Soomets et al., 2000) and thus papers IV and V are based on TP10.

3.2 Selection of cargoes We have delivered three types of cargoes – a protein, PNA oligonucleotides and plasmids.

The avidin protein was not expected to have any specific biological activity, rather it was

chosen due to its capability to form strong complexes with biotin. Using this approach we

could elucidate whether a protein loses its functional structure during the internalisation

process. Since no avidin would be taken up if the protein unfolds, biotin-avidin complexation

was a plausible model system for that.

In paper V, we evaluated TP10 as a gene delivery vector. Peptides have been shown to

enhance plasmid uptake before (Brandén et al., 1999; Morris et al., 1999; Sandgren et al.,

2004), and our aim was to evaluate TP10 in similar assays. To characterise TP10 as a

transfection agent, well-characterised reporter genes encoding enhanced green fluorescence

protein (EGFP) and luciferase were studied. Expression of these genes is a reliable measure of

delivery efficacy.

30

Evaluation of the bioactivity of the cargo was the main goal in papers III and IV. CPP

mediated uptake and PNA antisense had been performed earlier (reviewed by Gait, 2003) and

shown great promise. We used CPP-PNA conjugates to target hGalR1 and L-type Ca2+

channel subunit CaV1.2 and CaV1.3. The first protein has been one of the main research

subjects of our group and the latter of our co-workers in Prof. Nagy’s group.

3.3 Peptide synthesis 1963 Bruce Merrifield introduced the principle of solid phase synthesis. Its essence lies in a

solid support (resin), which serves as an anchor for growing peptide chains. Compared to

traditional solution phase synthesis, the protection schedule and washing procedures were

significantly simplified by the cost of an additional step in the final cleavage of peptide from

the resin. Today solid phase synthesis is routinely used in manual and automated synthesis.

Two alternative chemistries exist – tert-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxy-

carbonyl (Fmoc). Both strategies are named after the protection group on α-amino groups of

amino acids. Both are widely used and give high-yield products. Traditions of our lab favour

t-Boc chemistry therefore all peptides were assembled from t-Boc protected amino acids in an

Applied Biosystems 413A peptide synthesiser (Applied Biosystems, Forster City, CA). For

efficient peptide bond formation, carboxyl group of amino acids must be activated. In

automated synthesis we used dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole

(HOBt) activation.

In case of transportan and TP10 t-Boc-Lys(Fmoc)-OH was used as the building block

in positions 13 or 7, respectively. The Fmoc group can be specifically removed after

completion of main peptide chain synthesis by 20% piperidine in dimethylformamide (DMF)

treatment and the ε-amino group of Lys becomes available for cargo coupling. As disulphide

bridges were of special interest, t-Boc-Cys(Npys)-OH was coupled to the 13Lys or 7Lys with

2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU)/HOBt

activation. For pIsl used in papers I and II cargo was linked to the N-terminus.

Anhydrous HF on ice for 45 min was used for cleaving the peptides from 4-

methylbenzhydrylamine (MBHA) resins resulting in C-terminally amidated peptides. In the

cleavage step, p-cresol or a p-cresol/p-thiocresol mixture was used as scavenger for reactive

carbocations. A p-thiocresol containing mixture was used for peptides with free, but not Npys

labelled, thiol groups.

31

The cleaved peptides were purified on preparative reverse phase HPLC (Gynkotek,

Munich, Germany) with C18 column and a H2O 0.1% TFA/ acetonitrile 0.1% TFA gradient.

Molecular weights of the peptides were analysed on Bioion 20 plasma desorption mass

spectrometry (Uppsala, Sweden) (papers I and II) or Voyager STR-DE MALDI-ToF (Applied

Biosystems, Foster City, CA) (papers III - V).

3.4 PNA synthesis PNA synthesis was carried out on an Applied Biosystems 433A peptide synthesiser (Applied

Biosystems, Foster City, CA) according to the protocol of Koch (Koch et al., 1997). In

principle the synthesis was similar to the peptide synthesis described above, except that N-O-

(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) esters

were used instead of DCC/HOBt or TBTU/HOBt for monomer activation. Additionally, after

acetylation steps a 10% piperidine wash was performed. Piperidine wash is recommended for

removal of acetic anhydride traces that can cause unwanted termination of growing PNA

chains during coupling steps (Koch et al., 1997). MBHA resin loading was not 1.0 mmol/g as

for peptides but 0.15 mmol/g. Downloading was achieved through assembly of t-Boc-

Lys(ClZ)-OH to resin and subsequent acetylation. At higher loadings growing PNA chains

start to aggregate and thereby fail to grow to desired length. Loading 1 mmol/g spontaneously

terminated the chain growth after 9th to 12th monomer.

3.5 Disulphide bridge formation In intracellular delivery assays disulphide bridges behave vastly differently from other

covalent bonds. The reducing milieu of the cytoplasm cleaves disulphide bonds through the

action of glutathione and thereby separates the cargo from the vehicle. This may be important

for the activity of the cargo, because even noncovalent, but high-affinity binding carriers

create a potential barrier for cargo activity (Schaffer et al., 2000).

Heterodimers of PNA and CPP were formed in an exchange reaction of Npys-labelled

disulphide and free thiols. The reaction is schematically envisaged in Fig. 3.

32

Figure 3. Reaction between 3-Nitro-2-pyridinesulphenyl (Npys) labelled peptides and cysteine containing PNAs.

Different conditions were evaluated to achieve best yields. First, the protocol of

Bernatowicz (Bernatowicz et al., 1986) was tested. Peptide and 1.2-fold molar excess of PNA

were dissolved in 0.1 mM KH2PO4 pH 4.5, stirred for 2 h and separated by a semipreparative

C18 HPLC column, with a gradient of 20 to 100% of acetonitrile (0.1% TFA) in H2O (0.1%

TFA) for 45 min. In fact, the solution remained usually cloudy due to PNA insolubility. The

outcome was in best cases 15% of the theoretically calculated value (estimated from weight).

To increase the yield organic solvents – DMSO and DMF were added to the mixture, which

slightly increased the outcome, but often still failed to dissolve PNAs. Finally the following

protocol was established: PNA and CPP in 1:1 to 1:2 ratio was weighted up in the same test

tube and 200 µl DMSO, 200 µl DMF and 100µl acetic buffer were added. If an insoluble

pellet remained after 5 min of vortexing and warming, about 20 µl TFA was added to the

pellet. The reaction was protected from light and gently stirred overnight (8-16 h). Products

were separated according to protocols described above.

PNA nucleobases absorb strongly at the wavelength of 260 nm, whereas peptide bonds

of PNA and CPPs absorb around 215 nm. Acquiring both wavelengths and considering

hydrophobicities of the PNA, the peptide and the conjugate, we were able to identify the

conjugate from HPLC profiles. MALDI-ToF mass spectrometry was used to ensure the

correctness of our conjugates. A more detailed protocol can be found in literature (Kilk and

Langel, 2005)

33

3.6 Interactions with model membranes A label (fluorescein or biotin) is, in principle, a cargo and may alter CPP membrane

interactions. Aromatic amino acids, especially Trp are also fluorescent in the UV range,

allowing detection of the peptide. Moreover, the fluorescence, as an outcome of electron

configuration, is influenced by the proximal environment of the fluorophore. Hence, by

identifying Trp emission peak wavelength (excitated at 280 nm and emission scanned from

300 to 500 nm) an estimation of penetration deepness into lipid layer can be done.

Unfortunately live cells have very high UV fluorescence background and Trp of the peptide

cannot be specifically followed. Thus, large unilamellar vesicles (LUV) were used in paper I

and II as model membranes.

Electron paramagnetic resonance (EPR) spectroscopy requires a paramagnetic spin

label. Typically it is a stable free (nitroxyl) radical conjugated to a cysteine side chain

(Berliner, 1998) An EPR spectrum consists of three peaks, which shape depends on the

immediate environment and, most interestingly, freedom of motion of the beacon radical. For

CPPs this method had not been used so far, therefore we included it in paper II.

3.7 mRNA structure prediction Prediction of secondary structure of mRNAs was performed using the Mfold algorithm

(Jaeger et al., 1989; Zuker, 1989) based RNAStructure 3.5 and 3.7 software

(http://128.151.176.70/RNAstructure.html). Parameters were set to 10% of maximal energy

difference between the most optimal and possible suboptimal structures, with a maximum of

20 different structures. Meaning that 20 or less structures are predicted and the formation

energy of any particular structure is not more than 10% different from the most stable

structure. Prediction of more than a single structure increases the probability to hit the correct,

naturally occurring structure. Folded proteins and mRNAs can “breathe”, since folding is a

reversible process composing of many interactions. All individual interactions do not have to

be “structured” all the time and therefore minor local changes usually do not change the

overall structure of the biomolecule. In our prediction, structures with minimal differences

were considered as a single structure with the parameters of the most stable form.

Used hGalR1 mRNA sequence has the NCBI reference number XM_050937 and

GeneBank Accession number gi:17457665. CaV1.2 and CaV1.3 references are M67516,

gi:206575 and MN 017289, gi:8393029, respectively.

34

For analysis of energetical parameters of mRNA and asON interactions, the OligoWalk

function of the RNAStructure program was applied. The local structure around the target site,

but not the entire RNA was recalculated for each oligomer. Designed PNA oligomer

sequences were screened by standard nucleotide-nucleotide BLAST

(http://www.ncbi.nlm.nih.gov/ BLAST/) for possible cross-reactivity to unrelated genes and

RNAs.

3.8 Cellular uptake studies In papers I and III, cellular uptake experiments were carried out on methanol or

paraformaldehyde fixed cells. At this time we were not aware of potential fixation artefacts

(Lundberg and Johansson, 2001; Richard et al., 2003) and indirect staining allowed the use of

biotinylated peptides/PNA oligomers instead of fluoresceinylated ones. Biotin was a more

cost-efficient label and known to be compatible with t-Boc chemistry. Later fluoresceinyl pIsl

and PNA oligomers were synthesised according to Fischer et al. (Fischer et al., 2003) and

their uptake was measured in live cells (unpublished results, see 4.3.1).

3.9 Galanin binding studies Western blot could not be used for GalR1 quantification (paper IV) due to lack of antibodies.

Quantification was achieved through radioligand, 125I-galanin, binding to receptors. Bowes

cells are known to express only type 1 receptors (Heuillet et al., 1994). Human, rat and

porcine galanins have identical affinities for this receptor (Hulting et al., 1993) and therefore 125I labelled porcine galanin (NEN, Boston, MA) was used as tracer. Only porcine galanin has

a Tyr in position 26 that can be iodinated by chloramine T method without alteration of

affinity (Fisone et al., 1989). Nonspecific binding was determined by addition of 1 µM human

galanin and specific binding was defined as the difference between total and nonspecific

bindings. In order to decrease galanin degradation during incubation and absorption to assay

equipment, the protease inhibitor bacitracin was added to membrane preparations and

equipments were silanized (Land et al., 1991).

35

3.10 Reverse-transcription PCR Intracellular GalR1 mRNA levels were analysed by semi-quantitative reverse-transcription

PCR. Primers were targeted to the 3’ end of the mRNA, bases 1700-1719 and 1873-1892

from starting codon, respectively. Primers covering the PNA target region -110 to -89 and -

207 to -229 were also tested, but due to technical problems they were not chosen for main

experiments. The GalR1 mRNA level is very low compared to mRNAs of housekeeping

proteins. In random primer based cDNA transcription from total RNA, no GalR1 cDNA

suitable for PCR amplification could be obtained. Since total RNA consists mainly of

ribosomal RNA, probably not enough mRNAs were reverse-transcribed. In the case of oligo

dT primer, which is specific to only mRNAs, 5’ end of GalR1 mRNA was reverse-transcribed

only seldomly in irreproducible experiments. Apparently the reverse transcriptase falls off

before reaching the other end of the 2 kb long mRNA. Thus, PCR primers targeted to the 3’

end and not to the PNA target site were used for highest reproducibility.

3.11 Electrophysiological measurements Sensitisation was studied in 16-21 days old wistar rats. All drugs, including asON-CPP

conjugates were injected intrathecally to fourth and fifth lumbar vertebrae area. Direct

recording on spinal cord is technically more problematic and less reproducible than

measurements on motoneurons (Herrero et al., 2000). Therefore windup recordings were

made on the hind paw biceps femoris muscle motoneuron. For studying the windup, precise

control over the C-fiber firing frequency is essential. This was achieved by stimulating the C-

fiber with electric impulses from electrodes placed under the paw skin in the territory of the

sural nerve. All experimental procedures on animals were approved by the local ethic

committee.

36

4 Results and discussion 4.1 Preparation of CPP-cargo conjugates

4.1.1 Noncovalent complexes In paper I we used noncovalent biotin-avidin complexation for protein delivery. For that

biotinylated peptides (4 eq) were incubated with avidin (1 eq) solution for 5 min. Biotin and

avidin have an affinity constant of about 10–15 M, which is orders of magnitudes higher than

common antibody-antigen affinity (10-7 to 10-11 M). Hence the complexation is specific, and

judging from the results processed as expected. The same strategy has been used for cargo

delivery elsewhere (Pooga et al., 2001; Säälik et al., 2004; Sadler et al., 2002; Console et al.,

2003).

Figure 4. pEGFP-N1 mobility on 1.8% agarose gel after 30 min incubation with PEI

or TP10 at listed charge ratios.

Since it is technically problematic to link peptides covalently to plasmids without

abolishing plasmid’s functionality, in paper V non-covalent interactions were used. Co-

incubation of TP10 and short oligonucleotides prior transfection, enhances DNA uptake

37

comparably to a commercial vector OligofectamineTM (data not shown). In case of plasmids,

complexes were formed but not with a particularly high affinity. Even a 4-fold excess of

positive charges (all originate from TP10) was not enough to force the peptide/DNA complex

to migrate towards the cathode in agarose gel electrophoresis (Fig. 4). Complexation with PEI

was found to be much stronger and reverse the plasmid migration direction already at charge

ratio one.

In the next approach TP10 was coupled to plasmids via a PNA oligomer, characterised

by Zhang et al. (Zhang et al., 2000). The sequence AGGATCTAGGTGAA-amide was shown

to interact with a specific site within the pUC bacterial replication origin of plasmids. The

binding site should have little or no influence on plasmid expression in mammalian cells. To

increase the freedom of the peptide movement, a biglycinyl linker was used additionally to the

cysteine linker between the PNA and the peptide (Table 3). Results of Zhang et al. (Zhang et

al., 2000) were further confirmed by us. PNA and TP10-PNA shifted complementary DNA

oligomer but not mismatched oligomer (5’ TCCTAGATTCACTT 3’, where the mismatch is

shown in bold) bands on acrylamide gel (Fig. 5).

Finally, after demonstrating the necessity of plasmid condensation with high MW

polycations, TP10 was covalently linked to PEI and thereafter complexed with DNA. DNA

condensation by polycations has been described elsewhere (Tang and Szoka, 1997; Ramsay et

al., 2000).

4.1.2 Covalent complexes In paper V a bifunctional crosslinker, succinimidyl trans-4-(maleimidylmethyl)cyclohexane-

1-carboxylatelinked (SMCC), was first reacted with amino groups of PEI and then with thiol

groups of cysteinylated TP10. The molar extinction coefficient of TP10 was measured from a

set of standard solutions and was found to equal EC220 = 9490 M-1cm-1. This value was used

to estimate the success of TP10/PEI crosslinking reactions. The outcome of five separate

conjugation reactions were found to equal 1.5, 5, 7.5, 13, and 24 TP10 molecules per PEI. In

other words, TP10/plasmid (CPP/cargo) ratio in transfection assays was between 40–2500.

38

Figure 5. Interaction of the PNA used in paper V with complementary and mismatched (mmDNA) sequence.

For use in papers III, IV and V, disulphide bridges between cargoes and CPPs were

created. Several different solvents for this reaction were tested as described in the

methodological considerations section. The conjugation yield varied from 0 to 60% of

theoretical values (calculated from weight). No or very little conjugate was achieved when

PNA remained in suspension. In addition to PNA solubility the quality of both compounds

seemed to be important. Not quality in means of overall purity, but rather presence of thiol

containing by-products, including the ratio of oxidised and reduced forms of Npys-TP10. As

said in methods, conjugates were identified by high absorbencies at 220 and 260 nm and

verified by MALDI-ToF mass spectrometry. A semipreparative and an analytical HPLC

profile of the TP10-scrambled CaV1.2 PNA conjugate are shown on Fig. 6 A and B. α-cyano-

4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid, the most recommended matrixes for

peptide/PNA analysis in MALDI-ToF mass spectrometry (Butler et al., 1996), unfortunately,

fail to fully protect disulphide bridges (Crimmins et al., 1995). In analysis of Npys labelled

peptides (it contains a disulphide bridge), peaks with and without the label were observed, and

their ratio depended on laser intensity and acquisition duration. Identically, in PNA-peptide

conjugate analysis, both components were seen in parallel with the conjugate (Fig. 6 C).

39

Figure 6. Conjugation of 0.4 µmol scrambled antiC-aV1.2 PNA and 1.0 µmol Npys-TP10 in 200 µl DMSO, 20 µl TFA, 300 µl acetic buffer pH 5.5. A) Semi-preparative HPLC profile 10 h after reaction start, main peaks from left to right: DMSO, PNA, conjugate and TP10. B) analytical profile of conjugate from (A) showing 85% purity at λ = 215 nm and 99.9% purity at λ = 260 nm. C) MALDI-ToF mass spectra of the conjugate.

40

4.2 CPP interactions with model membranes In papers I and II, we have studied membrane interactions of penetratin, pIsl and transportan.

All peptides contain a tryptophane, allowing fluoroscopic studies without conjugation with

additional fluorophores.

Penetratin and pIsl did not bind to neutral dimyristoylphosphatidylcholine (DMPC)

vesicles (I Fig. 3 and II Fig. 2) judged from identical Trp fluorescence spectrum in the

presence and absence of lipids. In case of negatively charged sodium dodecyl sulfate (SDS) (I

Fig. 3) or dimyristoylphosphatidylglycerol (DMPG) and dioleoylphosphatidylglycerol

(DOPG) (II Fig. 2) vesicles, fluorescence emission peak shifted from 354 to 340 indicating

partial shielding of Trp by lipids. Important is the linearity of the penetratin spectra, meaning

an identical state of both Trp. The more hydrophobic transportan peptide was found to interact

with any kind of lipids and not to prefer negatively charged ones to neutral lipids. Trp was

shielded to the same degree as in penetratin and pIsl, indicating same deep internalisation into

lipid bilayers. Electron paramagnetic resonance spectroscopy confirmed the results obtained

for penetratin. Additionally, the N-terminal paramagnetic probe showed after penetratin

insertion into vesicle membranes significantly higher mobility than the vesicle itself. Hence

the peptide, or at least its N-terminus, has considerable local mobility even in a membrane-

bound state.

CD spectroscopy was used for peptide conformation analysis. All peptides in buffer

were mainly random coils, but additionally to that about 33-42% of homeopeptides were in β-

sheet and 30% of transportan in α-helical structure. Presence of negatively charged vesicles

(diameter 100 nm) raised the β-sheet content of penetratin and pIsl to 66 and 56%,

respectively. SDS micelles, almost 100 times smaller than vesicles and consisting of a single

lipid layer, forced homeopeptides into α-helical conformation. DMPG vesicles are, due to

their size and structure, certainly a better mimic of biological membranes than SDS micelles,

hence results from them are more relevant. Later the switching between α-helical and β-sheet

structures of penetratin was found to be dependent on peptide/lipid ratio and surface charge

density (Magzoub et al., 2002; see 1.2.2). Transportan adopts 45-60% α-helical conformation

in presence of any membrane mimics, and the rate depended more on lipid/peptide ratio than

on lipid type. Recent works indicate that the lipid peptide ratio is in good correlation with the

41

uptake of amphipathic Pep-1 (Henriques and Castanho, 2004) and MAP (Hällbrink, Oehlke

unpublished).

Conclusions from biophysical studies are as follows: peptides are not fixed in the

membranes and retain local mobility. Arginine-rich penetratin and pIsl interact with

membranes on distinct principles from transportan. Secondary structures support the diversity

or, on the other hand, suggest that in the case of a uniform internalisation mechanism, it must

be independent of peptide secondary structure. Incorporation of prolines, imino acids that

disrupt α-helical and β-sheet structures does not to alter penetratin uptake (Derossi et al.,

1996; Lindgren et al., 2000) but abolishes transportan internalisation (Lindgren et al., 2000).

It could be that the structure is unimportant for Arg-rich, highly charged CPPs, but is decisive

for peptides with amphipathicity. For the antimicrobial peptide buforin 2, the internal proline-

bend has been demonstrated to be crucial for non-lytic internalisation (Kobayashi et al.,

2000). Also transportan, in opposite to the non-penetrating mastoparan part, is known to be

bent (Lindberg et al., 2001). Perhaps the activity of some CPPs relies on small, still

undefined, secondary structure features.

4.3 Cargo delivery

4.3.1 Delivery of avidin In paper I we have delivered TRITC-avidin with biotinylated pIsl, a newly characterised CPP,

and penetratin. Confocal microscopy (paper I Fig. 2) shows strong staining in the cytoplasm

and nucleus. Small punctual structures can be seen, indicating either cargo aggregation or

accumulation in small intracellular compartments e.g. endocytotic vesicles. These studies

were performed before fixation artefacts were reported and should therefore be taken

cautiously. To investigate the effect of fixation procedure, pIsl uptake has later been

conformed quantitatively in non-fixed cells. For that cells were incubated with 5 µM

fluoresceinylated peptide for 1 h at 37 oC, washed trice, trypsinated for 5 min and washed

again. For quantification water-soluble intracellular fractions were separated from membranes

by ultracentrifugation of cell lysis. pIsl and penetratin both are taken up significantly greater

extent than dextrane, a general pinocytosis marker (Fig. 7). In regards of avidin delivery into

non-fixed cells, very recently Säälik et al. have made a scrupulous study covering penetratin,

pTat, transportan and pVec (Säälik et al., 2004). Using FACS analysis on live cells and

spectrofluorimetry on cell lysates uptake of FITC-avidin was detected with all carriers.

42

Hyperosmolar medium (0.45 M sucrose), cellular energy depletion (NaN3 and 2-

deoxyglucose) and cholesterol removal (methyl-β-cyclodextrin) reduced uptake, suggesting

involvement of endocytosis. Moreover, in the same study confocal pictures of transportan-

avidin complexes prior and after fixation are presented and no differences could be seen. One

year before, Console et al. (Console et al., 2003) studied streptavidin delivery by biotinylated

pTat and penetratin quantitatively, and no differences in fixed and unfixed cells were

observed. Additionally Console et al. found that the uptake is abolished if peptides are

preincubated with soluble heparan sulphates, suggesting that extracellular proteoglycans

mediate the uptake.

Figure 7. Quantitative uptake of fluoresceinylated pIsl in comparison to TP10 and dextran. Shown as picomole peptide or dextran per total protein.

4.3.2 Delivery of PNA Except neuronal tissues, cells do not take up PNA oligomers. The simplest way to overcome it

could be the addition of Lys residues in one of the PNA terminus (Sazani et al., 2002). This

methodology has, unfortunately, been used in only a few studies. Therefore, it is currently

impossible to estimate its efficacy under different conditions of various assays, as well as how

much the Lys residues reduce the target specificity due to charge interactions with mRNAs.

For targeted delivery, receptor ligands can be employed (Pardridge et al., 1995). In cases

when tissue/cell specific targeting is of minor importance, CPPs are a plausible choice. In

43

paper III indirect fluorescence microscopy confirmed uptake of all PNA-transportan

conjugates but naked PNAs, into human melanoma cell line Bowes. Although presented

pictures were taken again on fixed cells, delivery of TP10-PNA(18-38) (EC50 = 1.8 µM) and

TP10-PNA(18-32) (EC50 > 10 µM) has been successfully repeated in live cells (Fig. 8). The

shorter PNA showed slightly higher uptake than the longer one. When disulphide bridges

were reduced in both complexes, the longer PNA failed to internalise. The shorter oligomer

was still taken up, but internalisation was three folds lower compared to TP10 conjugates.

Thus, for analysis of asON efficiency a carrier is obligatory, otherwise insufficient delivery

adds a new level of complexity.

In paper IV we targeted spinal cord neurons. Although a carrier is not highly necessary

and intrathecally injected naked GalR1 PNA has shown specific antisense effect (Rezaei et

al., 2001) we used TP10-PNA conjugate to ensure maximal delivery. Aldrian-Herrada et al. in

1998 (Aldrian-Herrada et al., 1998) demonstrated that penetratin conjugated PNA is taken up

more rapidly than naked PNA. Three micromolar concentration of the conjugate reached its

peak intracellular concentration in 10 min of incubation, while PNA itself required 2 h.

In our case, conjugates injected intrathecally into lumbar L4/L5 vertebrae knocked

down target proteins in local area, but did not diffuse to upper thoracic (T6) regions (paper

IV, Fig. 3).

0%

10%

20%

30%

40%

50%

60%

dextrane TP10-PNA(18-32)

TP10-PNA(18-38)

TP10-PNA(18-32) +

DTT

TP10-PNA(18-38) +

DTT

Figure 8. Quantitative uptake of dextrane, TP10-PNA(18-32), TP10-PNA(18-38) and both conjugates pretreated with dithiothreitol (DTT) into Bowes cells. The fluorescence label is on the N-terminus of PNA oligomer and internalisation is shown as percentage of total added fluorescence.

44

4.3.3 Plasmid delivery Several groups have reported plasmid delivery with CPPs or antimicrobial peptides (Morris et

al., 1999; Ogris et al., 2001a; Sandgren et al., 2004). In paper IV, we have evaluated TP10 in

this aspect.

Bioplex is a strategy originally used by Branden et al. in a work where they

demonstrated improved nuclear uptake of NLS-PNA hybridised plasmids (Brandén et al.,

1999). In this and later similar studies, a polycation, PEI, has been required to mediate

plasmid uptake into the cytoplasm. CPP properties of NLS are not well established, therefore

more archetypical CPPs should be tested in respective means. Based on our results also TP10

is incapable to deliver native plasmids. PEI condensed TP10-PNA/plasmid, on the other hand,

showed improved reporter gene expression. Interestingly, 24 h post transfection, in fact, fewer

cells expressed the reporter gene, but at a significantly higher level than the control, PEI

transfected cells. After 48 additional hours also the population of transfected cells was larger

in TP10-PNA than in the standard protocol. Seemingly it took more time for plasmids to start

expression, but when started, expression level was higher. Since the phenomenon was not

observed with TP10 or PNA alone, the conjugate must be required. We speculate that the

bifunctional (PNA and peptide) conjugate interacts to plasmids with both ends, and leads to

crosslinking of two plasmids. Large complexes of two or more plasmids require more time for

intracellular trafficking and unpacking, but higher copy number of genes leads to higher

protein levels.

Secondly we tried to form charge-charge complex of TP10 and plasmids. This is the

method used for the antimicrobial peptide LL-37 (Sandgren et al., 2004), MPG (Morris et al.,

1999), hCT(9-32) (Krauss et al., 2004) and pTat (Ignatovich et al., 2003). In contrary to

named peptides, at charge ratios up to 1:20, TP10 failed to promote transfection. This is

surprising since MPG and LL-37 are not significantly more charged than TP10, although

charges in MPG are more localised. Agarose gel shift assay indicated that TP10 forms

complexes with the plasmid, although the affinity is low compared to PEI (Fig. 4). Hence,

TP10 interacts with DNA, but for the uptake promotion TP10 concentrations above 10 µM

must be required. This could cause cytotoxicity. PEI transfection can be improved by coating

it with functional peptides, as shown with melittin (Ogris et al., 2001a). We successfully

linked TP10 to PEI via SMCC crosslinker. Unconjugated TP10 and PEI were included for

transfection experiments as a control. Surprisingly both protocols had similar efficacies,

hinting obsoleteness of the covalent linkage.

45

In all cases the TP10 effect was more notable at low PEI concentrations. Interestingly,

the same tendency has been observed in transferrin-PNA + PEI mediated transfection (Liang

et al., 2000). However, it does not necessarily mean an identical mechanism for TP10 and

transferrin. Transferrin was demonstrated to improve delivery via receptor mediated

endocytosis, but no receptors are known for TP10. We speculate that TP10 and PEI functions

overlap and at high PEI concentrations it eclipses the effect of TP10. Improving transfection

at low PEI concentrations, has a great impact in development of systemic in vivo gene transfer

vectors. As systematically analysed by Prokop et al., low positive net charge of the vector is a

key factor for in vivo activity (Prokop et al., 2002).

Application of endocytosis modulators, indicated that the uptake mechanism is still

endocytosis. Enhancement in reporter gene expression by TP10 can originate from increased

endosmal escape of DNA or by increased number of endosomes. General cytotoxicity of

endocytosis modulators makes it difficult to judge the cause directly from experiments.

4.3.4 Cargo uptake mechanisms In paper I we demonstrated TRITC labelled avidin delivery by biotinylated pIsl and

penetratin. Fixed cells showed strong intracellular staining after 2 h. This demonstrates that a

covalent link between CPP and cargo is not necessary if the carrier and the cargo can interact

in other ways. Transportan and PNA do not interact per se, therefore a disulphide bridge is

obligatory, as demonstrated in fig. 8. Furthermore, during the avidin uptake process, the

protein does not unfold, because that would weaken interactions with biotin. Nevertheless,

conformation may change in a minor extent, maybe even to a molten globule state, as

speculated by Schwarze et al. (Schwarze et al., 1999). They found that peak enzymatic

activity of β-galactosidase is reached about 2 h later than the peak intracellular concentration

of the protein. Considering the size of an unfolded protein, vesicular (endocytotic, inverted

micelle) uptake appears most reasonable. Creating a pore or crack with needed extent must be

energetically costly and, likely, higher toxicity should be observed. Considering also the

results of Säälik et al. (Säälik et al., 2004) we can be certain that pIsl and penetratin mediate

protein uptake via endocytosis.

Questioning whether enhancement in reporter gene expression in paper V was because

of TP10 mediated delivery, we used various endocytosis modificators. Swelling and

disrupting endosomes by chloroquine increased reporter gene expression even more.

46

Disruption of the cytosceleton or energy depletion with NaN3 and 2-deoxyglucose attenuated

the expression. Hence, even if TP10 mediates the cargo uptake, it must be internalised via

endocytosis. PEI itself is taken up via endocytosis (Godbey et al., 1999; Remy-Kristensen et

al., 2001). The vesicles are approximately 200 nm in size suggesting clathrin mediated

endocytosis or macropinocytosis (Remy-Kristensen et al., 2001). Analysing PEI co-

localisation with the endocytosis marker FM4-64 authors speculate that the mechanism of

uptake is macropinocytotic. LL-37 mediated transfection is shown to be cholesterol and

proteoglycan dependent (Sandgren et al., 2004), and pTat mediated transfection shows

punctual distribution and is temperature and Ca2+ dependent (Ignatovich et al., 2003). The

first report proposes caveolar uptake, the second supports caveolae, but only for some cell-

lines. PEI/DNA complexes formed in high ionic strength buffers have size >1000 nm. As in

our protocol low ionic strength in DNA/PEI complexation was used, the forming particles are

about 40 nm in size according to Ogris et al. (Ogris et al., 1998). Therefore caveolae size (ca

90 nm) would not be a limiting factor. In conclusion different PEI and TP10 based vectors for

plasmids are likely to internalise via either macropinocytosis or caveolae based endocytosis.

In regards of CPP ratio to cargo, tetrameric composition of avidin should not be

forgotten. Each cargo has 4 peptides attached to it, each directing differently from the surface

of the complex. It is reasonable to ask whether they co-operate or not in uptake process.

Association with a planar membrane can sterically not involve more than two peptides. The

co-operation might be necessary in cargo encapsulation into a vesicle. Also cargo-membrane

interactions might be necessary, although chemical variability of delivered cargos makes it

doubtful. Yet more probable is that in cases where multiple peptides per cargo are required,

the initial step is not association with a planar membrane. Instead, the initial association

requires more than one interaction with either the membrane (possible in membrane

invaginations) or accessory molecules e.g. heparan sulphate. Only if there are enough

interaction sites, a necessary change in the membrane stability can be induced and uptake

process completed.

In plasmid delivery, range 1 to 2.5 × 104 of TP10/plasmid ratio was used. Without

PEI, none of complexes was internalised, meaning that CPPs have their limits even in co-

operational mode. With PEI, on the other hand, all TP10/ plasmid ratios showed improved

delivery.

47

4.4 Antisense effects of delivered oligonucleotides

4.4.1 Design of antisense PNA oligomers Pooga et al. (Pooga et al., 1998c) targeted sequences 1-21 and 18-38 of the hGalR1 and found

the latter to be a more efficient antisense agent. EC50 values were 0.6 and 0.2 µM,

respectively. We “dissected” the better asON into shorter fragments (Table 3) in order to

investigate structural elements that determine the high efficacy. Furthermore, we computed

the structure of the target mRNA and found it to be conserved among all predicted

possibilities.

Table 3. PNA sequences used in papers III-V.

target PNA sequence hGalR1 18-38 Cys-GCGTTGCCCTCGCTGAGGTTC-Lys-amide

hGalR1 18-26 Cys-CTGAGGTTC-Lys-amide

hGalR1 18-29 Cys-TCGCTGAGGTTC-Lys-amide

hGalR1 18-32 Cys-CCCTCGCTGAGGTTC-Lys-amide

hGalR1 18-35 Cys-TTGCCCTCGCTGAGGTTC-Lys-amide

hGalR1 21-38 Cys-GCGTTGCCCTCGCTGAGG-Lys-amide

hGalR1 24-38 Cys-GCGTTGCCCTCGCTG-Lys-amide

hGalR1 27-38 Cys-GCGTTGCCCTCG-Lys-amide

hGalR1 18-38 scr Cys-GGCATGGCTGCTCTCCGTCGT-Lys-amide

Anti-CaV1.2 Cys-CGTGAGATTGTAATG-amide

Anti-CaV1.3 Cys-GTTACTGATAGGTAG-amide

Scrambled Cys-CGTGAATTAGTTAGG-amide

pUC Cys-Gly-Gly-AGGATCTAGGTGAA-Lys-amide

mRNA structure prediction for L-channel subunits was performed analogously to

GalR1 mRNA prediction. However, we did not choose to target the translation initiation

region but rather the 5’ end: nucleobases -233 to -219 of CaV1.2 and -94 to -80 of CaV1.3

from the starting codon. The targeted region of CaV1.3 mRNA was stable among all predicted

possibilities, and consisted entirely of an unpaired region (Fig. 9C). The target sequence in

CaV1.2 had different conformations in predicted structures, indicating lack of nearby

structurally conserved motifs and high flexibility. All structures for CaV1.2 target regions had

percentages of unpaired nucleotides similar to GalR1(18-38) (Fig. 9A and B). Intriguingly all

three sequences, including the scrambled, share the same nucleobase composition, meaning

that CaV1.2 antisense was designed to be a CaV1.3 scrambled sequence and vice versa (Table

3).

48

Figure 9. Predicted structures of A) hGalR1 B) CaV1.2, C) CaV1.3 mRNAs. PNA target sites are highlighted with a solid line parallel to the mRNA.

4.4.2 Galanin receptor antisense hGalR1 (18-38) antisense PNA conjugates with transportan or penetratin have been found to

internalise Bowes melanoma cells in culture and specifically affect pain transmission in rats

(Pooga et al., 1998c). Additional studies have been carried out with this particular PNA,

showing galanin synergy with morphine and reduction of nonciceptive transmission in the

49

spinal cord (Rezaei et al., 2001; Hua et al., 2004), GalR1 role in antinociception in the arcuate

nucleus (Sun et al., mauscript in preparation), in learning and feeding (Mclaughin et al.,

manuscript in preparation) and also in epilepsy (Saar et al., 2002). In paper II our aim was to

analyse what determines the efficiency of this PNA oligomer on the level of PNA/RNA

interactions.

A 12- (positions 24-38) and a 15-mer (positions 27-38) appeared to have the highest

GalR1 knock-down potency with EC50 of 80 ± 30 and 70 ± 30 nM, respectively. At the same

time other 12- and 15-mer, had the lowest activity (EC50 > 10 µM). The rest of the asONs had

moderate efficiencies with EC50 ranging from 0.18 to 1.8 µM. No straight correlation between

PNA structural characteristics (length, pyrimidine content) and efficiency was found. Neither

correlated calculated thermodynamical parameters or combinations thereof with the

efficacies.

By comparing the effects of different oligomers it was possible to evaluate the

importance of nucleotide triplets within 18-38. The greatest impact was assigned for

nucleotides 33-35, which according to computer prediction is the longest unpaired region.

This indicates that despite of duplex invasion capabilities of PNA, single strands are still

preferable targets. On the other hand, unpaired nucleotides: 27 and 20-21 did not contribute to

antisense efficiency.

4.4.3 Ca2+ L-channel antisense Paper III questioned the roles of Ca2+-modulated membrane conductivity and NMDA

receptors in sensitisation. To address these aims, the effect of Ca2+ channel and NMDA as

well as α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and γ-amino-

glutamic acid (GABA) receptor ligands on neuronal activity was measured. First we

confirmed that our experimental setup was appropriate for windup tests. Rat hindpaw

motoneurons sensitised at frequencies above 0.3 Hz and was saturated after 8-10 stimuli as

characteristic for motoneuron windups. The L-channel antagonist nifedipine and the agonist

Bay K 8644 were tested, and as expected, inhibition and enhancement of windup,

respectively, were observed. Fluphenamic acid, a blocker of all Ca2+ activated cationic

currents, again, blocked the windup.

Expanding our methods we included antisense PNA oligomers that were delivered to

targets by TP10. Immunohistolochemical staining of rat spinal cord slices indicated specific

50

down-regulation of CaV1.2 or CaV1.3 protein (paper IV Fig. 3). Suppression occurred locally

in lumbar (lower third of the spinal cord) regions and the TP10-PNA conjugate did not diffuse

elsewhere. Only respective PNAs altered the immunostaining of respective proteins.

Scrambled or the irrespective PNA (which, as described in 4.4.1, can be considered as a

scrambled) were inactive.

The electrophysiological response to either CaV1.2 or CaV1.3 downregulation was a

complete abolishment of flexor reflex sensitisation in rats, meaning a central role of both L-

channels in axon potential windup. At the same time no change in mechanical or thermal

sensitivity was determined. In contrary, animals injected with controls (scrambled PNA or

buffer) were slightly more sensitive than intact animals. The injection procedure might have

caused unwanted sensitisation, which would explain the change in control animals and its lack

in antisense treated rats.

Next we applied the NMDA receptor blocker AP5, and verified its ability to suppress

the windup. Strychnine, a blocker of inhibitory glycine receptors, enhanced the sensitisation,

meaning that NMDA and glycine receptors are involved in windup regulation. This is not

surprising since they are regulators in many neuronal processes at the spinal cord level.

Additionally, we found that the windup was restored if NMDA receptors and glycine

receptors were blocked simultaneously. Hence, in the absence of inhibitory glycinergic

signals, NMDA receptors are not essential for the windup. On the other hand, simultaneous

blockage of glycine receptors and Ca2+ stimulated cationic currents (either selectively via L-

channels or the total current) did not restore windup.

In conclusion we show that windup onset is primarily a result of membrane

conductance and particularly dependent on L-type Ca2+ currents. NMDA receptors do not

directly mediate sensitisation, but maintain the balance of excitatory and inhibitory synapses,

which in turn is needed for activation of CaV channels.

4.4.4 PNA antisense mechanism. Identification of more potent hGalR1 antisense oligomers and attempts to find the principles

of antisense design, were not the only goals of article II. Reports about PNA antisense

mechanism are contradictional – sometimes mRNA downregulation is reported (Rapozzi et

al., 2002; van Rossenberg et al., 2003), sometimes not. By performing semi-quantitative

reverse-transcription PCR we showed that no hGalR1 mRNA downregulation occured after

51

treatment with the most the effective conjugate, TP-PNA(24-38), or respective PNA or carrier

peptide alone.

Figure 10. GalR1 mRNA level during antisense experiment. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as housekeeping mRNA. The numbers between lines show the quantification results (FujiFilm, ScienceLab, ImageGauge 3.45) for this particular gel. Shown as ratio of band intensities compared to intensities of untreated control [(tIGalR1/tIGAPDH) / (0IGalR1/0IGAPDH)]

Besides positive results, paper III is also an example how misprints can pass the careful

evaluation of scientific publications. Protein concentration was measured 36 h after conjugate

treatment as stated previously (Pooga et al., 1998c) and in Fig. 3 (paper III) legend, and not 12

h as stated in the text throughout paper III. Accordingly also reverse-transcription PCR was

carried out at longer time points (Fig. 10). Despite an erroneous time schedule, the results are

still fully valid. In summary of 2–5 independent experiments, no statistically significant

change in GalR1 mRNA concentration was observed.

52

5 Conclusions CPPs are interesting in two meanings. First their internalisation mechanism, which was first

indicated to be energy independent and is now under re-investigation. Second, their ability to

deliver cargoes is a promising methodology for research and medical applications.

Work presented in this thesis, has demonstrated the following:

Penetratin and a novel CPP, pIsl, share all characteristics of their translocation and

protein delivery. Both peptides are able to deliver a protein in its folded form across the cell

membrane and there are no differences in interactions with model membrane systems. The

main force driving these peptides to, and perhaps also through, the membrane is electrostatic

interaction between the peptide and negatively charged membranes. In (model) membranes

peptides reside partly buried into lipid head group layer and retain their local mobility.

Transportan, a more amphipathic peptide, relies on hydrophobic interactions in respect

to insertion into membranes. This confirms that arginine rich and amphipathic peptides are

distinct classes of CPPs.

PNA oligomers can efficiently be delivered both in vitro and in vivo, when conjugated

to transportan or TP10. Conjugates with PNA asONs targeting hGalR1, CaV1.2 and CaV1.3

exhibited desired activity and suppressed expression of respective proteins. Some correlation

between observed antisense efficiency and computer predicted target mRNA structure was

found. Judging from these results, despite triplex forming and double strand invasion

capabilities of PNA oligomers, unpaired sequences are preferred targets for PNA asONs. In

the case of Ca2+ channel antisense, a central role of both L-type Ca2+ channels in flexor reflex

sensitisation was demonstrated.

TP10 alone even in a co-operational mode, thousand of peptides per plasmid, failed to

deliver reporter genes. A high molar weight polycation was needed for efficient transfection,

and TP10 could be used to enhance the transfection mediated by the polycation (PEI). This

means that the cargo delivery capacity of CPPs is limited. For each cargo, the carrier peptide

and its conjugation method should be evaluated.

53

6 Acknowledgements

54

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Aldrian-Herrada, G., Desarmenien, M. G., Orcel, H., Boissin-Agasse, L., Mery, J., Brugidou, J. and Rabie, A. 1998. A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons. Nucleic Acids Res 26(21): 4910-6.

Allinquant, B., Hantraye, P., Mailleux, P., Moya, K., Bouillot, C. and Prochiantz, A. 1995. Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro. J Cell Biol 128(5): 919-27.

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