CELL PENETRATING PEPTIDES: CHEMICAL MODIFICATION AND FORMULATION DEVELOPMENT

CELL PENETRATING PEPTIDES; CHEMICAL MODIFICATION AND FORMULATION DEVELOPMENT

Kariem Ezzat

Cell penetrating peptides; chemical modification and formulation

development

Licentiate thesis

Kariem Ezzat

©Kariem Ezzat, Stockholm 2011 ISBN 978-91-7447-216-5

Printed in Sweden by Universitetetsservice US-AB, Stockholm 2011 Distributor: Department of Neurochemistry, Stockholm University

To my family

List of pubilcations

This thesis is based on the following oublications referred to in text as paper I, paper II and paper III.

I. Lehto,T., Simonson,O.E., Mager,I., Ezzat,K., Sork,H., Copolovici,D.M., Viola,J.R., Zaghloul,E., Lundin,P., Moreno,P., Mäe,M., Oskolkov,N., Suhorutšenko,J., Smith,C.I.E. and

Adaloussi,S.E. (2011) A peptide-based vector for efficient gene transfer in vitro and in vivo. Mol. Ther., In press.

II. Andaloussi,S.E., Lehto,T., Mager,I., Rosenthal-Aizman,K., Oprea,J., Simonson,O.E., Sork,H., Ezzat,K., Copolovici,D.M., Kurrikoff,K., Viola,J.R., Zaghloul E.M., Sillard,R., Johansson,H., Hassane,F.S., Guterstam,P., Suhorutšenko,J., Moreno,M.D., Oskolov,N., Hälldin ,J., Tedebark,U., Metspalus,M., Lebleu,B., Lehtiö,J., Smith, C.I.E. and Langel, Ü. (2011) Design of a peptide- based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo. Nucleic Acids Res., In Press.

III. Ezzat,K., Andaloussi,S.E., Zaghloul,E., Lehto,T., Lindberg,S., More-

no,P., Viola,J.R., Magdy,T., Guterstam,P., Sillard,R., Hammond,S.M., Wood,M. J. A., Arzumanov,A., Gait,M.J., Smith,C.I.E, Hällbrink,M.

and Langel, Ü . PepFect 14, a novel cell-penetrating peptide for oligo-

Additional publications

Ezzat,K., El Andaloussi,S., Abdo,R. and Langel,U. (2010) Peptide-based

matrices as drug delivery vehicles. Curr. Pharm. Des., 16, 1167-1178. Re-

Lehto,T., Abes,R., Oskolkov,N., Suhorutsenko,J., Copolovici,D.M., Mag-

er,I., Viola,J.R., Simonson,O.E., Ezzat,K., Guterstam,P., et al. (2010) Deli-

very of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent

co-incubation strategy. J. Control. Release, 141, 42-51.

Abstract

Cell penetrating peptides (CPPs) have been extensively studied and exploited as drug delivery vectors for a wide variety of therapeu- tic cargos. However, several issues remain to be addressed regarding the enhancement of their efficiency and stability. In addition, to be available for patients, CPP-based therapeutics have to be formulated into suitable pharmaceutical forms that can be readily manufactured, transported, stored and conveniently used.

In this thesis, three chemically modified CPPs are developed having superior delivery properties for several nucleic acid-based the- rapeutic cargoes including: plasmids, small interfering RNA (siRNA) and splice switching oligonucleutides (SSOs), in different in-vitro and in-vivo models. In Paper I, we show that an N-terminally stearic acid- modified version of transportan-10 (TP10) can form stable nanopar- ticles with plasmids that efficiently transfect different cell types and can mediate efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, stearyl-TP10 is further modified with pH titratable trifluoromethylquinoline moieties to facilitate endosomal release. The new peptide, denoted PepFect 6 (PF6), elicited robust RNAi responses when complexed with siRNA in several cell models and promoted strong RNAi responses in differ- ent organs following systemic delivery in mice without any associated toxicity. In paper III , a new peptide with ornithine modification, PF14, is shown to efficiently deliver SSOs in different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne‟s muscular dystrophy (DMD). Additionally, we have developed a method for incorporating this delivery system into solid formulation that could be suitable for several therapeutic appli- cations. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocparticles in solution even when stored at elevated temperatures for several weeks.

Taken together, these results demonstrate that certain chemical

modifications could drastically enhance the activity and stability of

CPPs in-vitro and in-vivo. Moreover, we show that CPP-based thera-

peutics could be formulated into convenient and manufacturable do-

sage forms.

Contents

1. Introduction ... 1

1.1. Gene delivery ... 2

1.2. Silencing of disease-causing genes ... 2

1.2.1. Antisense ... 2

1.2.2. RNAi ... 3

1.3. Splice-switching therapeutics ... 3

1.4. Cell-penetrating peptides (CPPs) ... 5

1.4.1. History ... 5

1.4.2. Uptake mechanism ... 6

1.4.3. CPPs as drug delivery vehicles ... 7

1.4.3.1. Covalent linkage to cargo ... 7

1.4.3.2. Non-covalent complexation with cargo ... 8

1.4.4. Chemical modification ... 9

1.5. Pharmaceutical formulation ... 10

2. Aims of the study ... 11

3. Methodological considerations ... 12

3.1. Solid phase peptide synthesis ... 12

3.2. Cell cultures ... 13

3.3. Plasmid delivery (Paper I) ... 14

3.4. siRNA delivery (Paper II) ... 14

3.5. SSO delivery (Paper III) ... 15

3.6. Toxicity ... 15

3.7. Dynamic light scattering (DLS) and zeta-poteontial ... 15

3.8. Solid dispersion... 17

3.9. Animal Experiments ... 17

4. Results and discussion ... 19

4.1. Delivery of plasmids via stearyl-TP10 in-vitro and in-vivo (Paper I) ... 19

4.2. Delivery of siRNA via PF6 in-vitro and in-vivo (Paper II) ... 20

4.3. Delivery of SSOs via PF14 in-vitro and solid formulation development (Paper III) ... 22

5. Conclusions ... 25

6. Acknowledgements ... 26

7. References ... 27

Abbreviations

CPP CQ CR DLS DMD DNA GAPDH HPLC HPRT1 i.d i.m

MALDI-TOF MBHA MR mRNA ON PCR QN qPCR RNA RNAi RT-PCR siRNA SSOs SSPS TFA TIS

Cell-penetrating peptides Chloroquine

Charge ratio

Dynamic light scattering

Duchenne‟s muscular dystrophy Deoxyribonucleic acid

Glyceraldehyd 3-phosphate dehydrogenase High performance liquid chromatography

Hypoxanthine-guanine phosphoribosyl transferase Intradermal

Intramuscular

Matrix-assited laser desorption/ionization – Time of flight

p-methylbenzylhydralamine Molar ratio

Messenger RNA Oligonucleotide

Polymerase chain reaction

N-(2-aminoethyl)-N-methyl-N‟-[7-(trifl- oromethyl)-quinolin-4-yl]ethane-1,2-diamine

Quantitative real-time PCR Ribonucleic acid

RNA interference

Reverse transcriptase PCR Small interfering RNA

Splice-switching oligonucleotides Solid-phase peptide synthesis Trifluoroacetic acid

Triisopropylsilane

1. Introduction

Despite the tremendous success of basic biomedical research, new-drug output from pharmaceutical companies has been constant over the last decades and mostly based on small molecules (1). This gap between discoveries and therapeutics has recently led to intense interest in translational research; to transform biomedical discoveries into commercializable drug products (2). One of the major biomedical milestones in the last decade was the complete sequencing of the hu- man genome (3), which has significantly deepened our knowledge about the genetic causes of diseases. This led to the emergence of sev- eral methods to interfere with disease pathophysiology on the molecu- lar genetics level, a field that can collectively be called “gene thera- py”. Gene therapy approaches can be roughly divided into 3 types according to their therapeutic effect (figure 1):

 Restoration of lost gene function by gene delivery via viral vectors or plasmids.

 Silencing of disease causing genes by antisense, antigene or RNAi (RNA interference) approaches.

 Modification of gene function by interfering with the splicing ma- chinery via splice-switching oligonucleotides (SSOs)

However, all three approaches require the delivery of extreme-

ly large and charged nucleic-acid based molecules to their intracellular

targets across the plasma membrane, which is inherently imperemea-

ble to such molecules. Difficulties in delivering such new therapeutic

molecules to their target organs, tissues and subcellular compartments

led to the development of several drug delivery technologies in recent

years. In this thesis, the main gene therapy approaches will be briefly

illustrated, and the ability of newly developed chemically modified

CPPs to efficiently deliver plasmids, siRNA and SSOs will be hig-

hlighted.

1.1. Gene delivery

Loss of functional genes is the cause of several heritable diseases and cancers. Thus, the delivery of functional genes that could restore normal phenotype has been the holy grail of gene therapy for decades.

Viral vectors have been utilized for gene delivery, however, promising results have been tempered by the potential for insertional mutagene- sis that might lead to severe leukaemogenic side effects as has been reported recently (4, 5, 6). Also, humoral immunity directed against the viral vector particle is generally observed (7). Among the other safer alternatives for gene delivery is the delivery of plasmids carrying the desired gene via non-viral vectors. When the plasmid enters the cell, it is transcribed and translated by the cellular machinery without the need of genome integration. Cationic liposomes have been routine- ly used as delivery vectors for plasmids; however, toxicity remains a significant problem, according to several in-vivo findings (8).

1.2. Silencing of disease causing genes

Silencing of disease-causing genes can come in different ways including:

1.2.1. Antisense

There are two possible mechanisms for an antisense effect us-

ing antisense oligonucleotides (ONs). When the double-stranded DNA

or genes situated in the nucleus are targeted, the approach is called the

antigene strategy (9).The method that relies on targeting of the mRNA

is called the antisense strategy. Antisense activity can be achieved

either by blocking the binding sites for the 40S ribosomal subunit and

for other translation initiation signals or by the formation of a double-

stranded DNA/RNA complex that renders the RNA susceptible to

RNase H digestion (9) (figure 1). Natural DNA and RNA have been

used for antisense approaches together with several chemically mod-

ified analogues that offer better annealing with RNA targets and pos-

sess enhanced serum stability. Examples of chemically modified ONs

include: phosphothiate DNA, 2‟-O-methyl RNA (2‟-OMe), locked

nucleic acid (LNA), peptide nucleic acid (PNA) and phosphorodiami-

date morpholino oligo (PMO)(10).

1.2.2. RNAi

RNAi is a fundamental pathway in eukaryotic cells, where long pieces of double stranded RNA are cleaved by an enzyme called dicer into shorter fragments called small interfering RNA (siRNAs) that can cleave complementary mRNA sequences by the help of the RISC complex and argonaute 2 (figure 1) (11). The proof-of-principle study in 2001demonstrating that synthetic small interfering RNA (siRNA) could achieve sequence-specific gene knockdown in mam- malian cells marked the birth of siRNA therapeutics (12). What makes the siRNA approach more appealing is that it cleaves target mRNA in a catalytic manner, thus, lower doses are required to achieve gene knockdown compared to the conventional antisense approaches. That is why intensive research has been carried out in the last decade to develop delivery vectors for siRNA therapeutics (11).

1.3. Splice-switching therapeutics

Modification of gene function can be achieved by interfering with the splicing machinery; an approach termed splice-switching (13). Recent studies using high-throughput sequencing indicate that 95–100% of human pre-mRNAs have alternative splice forms (14).

Mutations that affect alternative pre-mRNA splicing have been linked to a variety of cancers and genetic diseases, and splice-switching oli- gonucleotides (SSOs) can be used to silence mutations that cause ab- errant splicing, thus restoring correct splicing and function of the de- fective gene (figure 1) (13, 15). SSOs are antisense ONs ranging from 15 to 25 bases in length that do not activate RNase H, which would destroy the pre-mRNA target before it could be spliced (13, 15). One example of genetic diseases amenable for SSO therapy that will be addressed in this thesis is Duchenne‟s muscular dystrophy (DMD).

DMD is a neuromuscular genetic disorder that affects 1 in 3500 young boys worldwide (16) . It is caused mainly by nonsense or frame-shift mutations in the dystrophin gene. SSOs are used to induce targeted

„exon skipping‟ and to correct the reading frame of mutated dystro- phin pre-mRNA such that shorter, partially-functional dystrophin forms are produced (17). SSOs targeting exon 51 are currently in hu- man clinical trials in various parts of Europe to treat DMD (18, 19).

However, translating the promising results of SSOs into products re-

quires optimization of many parameters ranging from enhancement of cellular uptake and biodistribution to pharmaceutical formulation and long term stability.

Figure.1. Different gene therapy approaches. a. Viral delivery and genome integration. b. Plasmid delivery. c. Antisense steric block of translation d. Antisense DNA/RNA hybrid and RNase H degradation.

e. Antigene. f. Splice-switching ONs. g. siRNA.

1.4.Cell-penetrating peptides (CPPs)

CPPs are polybasic and/or amphipathic peptides , usually less than 30 amino acids in length, that posses the ability to penetrate cells (Cell Penetrating Peptides) or transduce (Protein Transduction Domains) over cellular plasma membranes directly in a receptor independent manner (20, 21). CPPs have attracted much interest in recent years as promising vectors for the delivery of a wide variety of therapeutics ranging from small molecules up to nanoparticles.

1.4.1. History

Many peptides and proteins have desirable therapeutic effects, but being large and often charged molecules, they have always been thought incapable of bypassing the plasma membrane. This view was challenged in the year 1988, when two groups independently pub- lished results in the same issue of CELL showing that both the recom- binant and the chemically synthesized 86 amino acids long Tat protein were found to be rapidly taken up by cells in tissue culture (22, 23).

Few years later, the 60 amino acid homeodomain of the Antennapedia protein in Drosophila was also shown to penetrate cells (24). A very important advancement in the field came by showing that the cell pe- netration capability is imparted by relatively short peptide sequences.

The 16- mer peptide derived from the third helix of the homeodomain

of Antennapedia termed penetratin (25), the 11-mer peptide derived

from Tat protein (26), the 27-mer chimeric peptide termed transportan

(27) and even simple polyarginines (R8) (28) were all shown to tra-

verse the plasma membrane. These discoveries marked the birth of the

field of CPPs. Since then, many CPPs have been discovered and stu-

died as potential drug delivery vehicles, some of which are presented

in Table 1.

Table 1. Selection of CPPs and their sequences

CPP Sequence Ref

Penetratin RQIKIWFQNRRMKWKK

(25)

Tat (48-60) GRKKRRQRRRPPQ (26)

pVEC LLIILRRRIRKQAHAHSK-NH

(29)

bPrPp MVKSKIGSWILVLFVAMWSDVGLCKKRPKP-NH

(30) Transportan GWTLNSAGYLLGKINLKALAALAKKIL-NH

(27)

TP10 AGYLLGKINLKALAALAKKIL-NH

(31)

MAP KLALKLALKALKAALKLA-NH

(32)

Poly Arg (RRR)

c

(28)

Pep-1 KETWWETWWTEWSQPKKKRKV

(33)

MPG GALFLGWLGAAGSTMGAPKKKRKV

(34)

a

Peptides are C-terminal free acids unless stated otherwise.

Original- ly with a free acid C-terminally but later shown also to have CPP properties when amidated.

n equals 2-4.

C-terminal cysteamide group.

1.4.2. Uptake mechanism

The detailed structure activity relationship and the actual me-

chanism of cellular uptake for CPPs are not yet fully understood. Two

main pathways have been debated to be solely responsible for CPP

receptor-independent uptake; the direct translocation pathway and the

endocytic pathway. There are also debated sub-models within each

pathway. For the direct translocation, three different models have been

proposed; the carpet model, the pore formation model and the inverted

micelle-mediated model (35, 36) . Also, for the endocytic pathway,

three models have been proposed; classical clathrin mediated endocy-

tosis, caveolae-mediated endocytosis and macropinocytosis (37).

Recently, an increasing number of studies are emphasizing the role of endocytosis in their uptake (37, 38, 39, 40, 41, 42). However, it has become evident that different factors and experimental conditions af- fect the mechanism of uptake of CPPs and their cargos. Different CPPs, concentrations, incubation times and volumes, cell type, cargo type, cargo-coupling methodology, read-out assay, extent of toxicity and extent of degradation are all factors that affect the uptake mechan- ism. Moreover, endocytic entrapment has to be followed by physical membrane translocation if the peptide is to reach the cytoplasm (43).

So, both the interpretation of results and the design of new CPPs should be projected on to the detailed background including the che- mistry used, the type of cargo, the experimental conditions and the read-out assays.

1.4.3. CPPs as drug delivery vehicles

CPPs have attracted much interest as a promising alternative for intracellular delivery of therapeutic cargos, especially for proteins and nucleic acids. Most of the existing methodologies are either safe but not effective enough or very effective but suffer from safety con- cerns that limit their use. CPPs appear to combine both efficacy and safety being very potent for intracellular delivery of macromolecules and meanwhile easily biodegradable. Two main methods have been utilized to attach the CPP to its cargo, either via a covalent linkage or through the formation of a non-covalent complex.

1.4.3.1. Covalent linkage to cargo

CPPs have been linked to various cargos via covalent linkers.

Small molecules like antineoplastic agents and antibiotics have been

coupled to CPPs to enhance biodistribution and cellular uptake. For

example, SynB peptide was utilized for the delivery of benzylpenicil-

lin (B-Pc) to the brain and it was found that the brain uptake of

coupled B-Pc was increased eight-fold on average compared to free B-

Pc (44). An example of successful vectorization of chemostatics

comes from our group where two different CPPs, YTA2 and YTA4,

were utilized for the delivery of methotrexate (MTX) into MTX- resis-

tant breast cancer cells (45). CPPs have also been utilized to deliver a wide variety of proteins and peptides which are either coexpressed with CPPs or ligated with a disulphide bridge. For cancer treatment, a CPP derived from the fibroblast growth factor was conjugated to an anti-Akt single chain Fv antibody and administrated in vivo with sub- sequent reduction in tumor volume and neovascularization (46).

Another interesting example is using undeca-arginine (R11) expressed recombinantly with the four transcription factors, Oct4, Klf4, Sox2, and c-Myc, to generate induced pulripotent stem cells (iPS cells) (47).

This set-up was almost 10 times more efficient as compared to other approaches in generating iPS colonies without the risk of chromosom- al integration associated with viral vectors. For peptide delivery, sev- eral groups have used both Tat and penetratin (Table 1) to convey peptides derived from the tumor suppressor p53, or peptides that modulate p53 activity, in an attempt to reduce tumor growth and in- duce apoptosis (48, 49).

For gene therapy approaches, covalently coupling CPPs, which are positively charged, to negatively nucleic acids has been not very easy to achieve. That is why in most gene therapy approaches using the covalent coupling strategy, neutral PNA or PMO were used instead of normal nucleic acids. Also, they can be directly attached to the CPP utilizing the solid phase peptide synthesis chemistry. Successful deli- very of antisense ONs in-vivo using CPPs was for the first time dem- onstrated with an antisense PNA complementary to human galanin receptor 1 (GalR1) mRNA coupled to transportan or penetratin that specifically down-regulated these receptors in rat brains (50). A num- ber of endogenous proteins including dystrophin (51, 52), CD45, and the interleukin-2 (IL-2) receptor (53), have been targeted with PMOs using CPPs as well. However, the limitation of using only PNA or PMO has led the development of the other strategy of cargo linking to CPPs, which is the non-covalent complexation.

1.4.3.2. Non-covalent complexation with cargo

Some CPPs have been successfully exploited for plasmid and

ON delivery using a non-covalently complexation strategy (54). Hav-

ing net positive charge, such CPPs have been shown to form nano-

sized particles with negatively charged nucleic acids, which are effi-

ciently internalized by cells presumably via an endocytosis-dependant

mechanism (54, 54, 55, 56). This strategy has drastically expanded the range of therapeutic cargos that can be delivered via CPPs as it avoids laborious chemical conjugation and has almost no limitation on the size of the cargo. Moreover, it was found that lower concentrations of ONs are generally required to achieve a biological response utilizing this strategy (55, 57). Thus, it has been recently extensively utilized by our group and others for delivery of plasmids, siRNAs and SSOs (20).

1.4.4. Chemical modification

Several chemical modifications have been introduced to CPPs to enhance their membrane interaction, improve the nanoparticle forma- tion capability or to facilitate endosomal escape and release of cargo after internalization (55). C-terminal cysteamide modification was shown to be crucial for CPP-mediated siRNA delivery using the MPG and CADY peptides by increasing membrane association and stabiliz- ing particle formation by the formation of peptide dimers (58, 59, 60, 61). Also, addition of hydrophobic moieties to CPPs has been shown to be an efficient mean to increase the bioavailability of CPPs and associated cargo. Cholesteryl modification of some polyarginines was shown to enhance their activity for local delivery of siRNA in-vivo (62). Stearylation was also proven to be a successful method to intro- duce hydrophobicity and enhance delivery of CPPs in the case of plasmids (63). One major limitation of the non-covalent complexation strategy is that the formed nanoparticles are mainly internalized via endocytosis, and consequently much of the cargo is commonly re- tained in endosomes without reaching their target. Several approaches have been devised to enhance the endomal escape of CPPs and their cargos out of the endosomes to their target subcellular compartments.

One example is the development of a histidine-containing endosomo-

lytic α-helical penetratin analogue, EB1, which was able to form com-

plexes with siRNA and promote endosomal escape (64).Other me-

thods utilized fusogenic peptides , like HA2-peptide, conjugated to

CPPs in order to facilitate release from endosomes (65). One major

conclusion of the papers discussed in this thesis is that stearylation

does not only enhance nanoparticle formation and stability, but also

enhances the endosomal escape.

1.5. Pharmaceutical formulation

Pharmaceutical formulation is the process of transformation of the active drug substance into a final medicinal product, which is an in- dispensible process before the drug could reach the market and be used by patients. It has a great effect on enhancing the efficiency and stability of active pharmaceutical ingredients. Additionally, specific formulations have to be tailored for different therapeutic approaches.

Among the different pharmaceutical forms present, solid formulations

remain the most widely used. It is the form used in tablets, capsules,

powders for inhalation and even powders for injection. This is because

they are easy to handle and very stable upon storage and transporta-

tion. That is why in this thesis a method was developed to incorporate

CPP nanoparticles in a solid form that could be used in various thera-

peutic approaches.

2. Aims of the study

This thesis is primarily focusing on studying the effect of cer- tain chemical modifications on the activity of TP10 and its analogues for the delivery of plasmids, siRNA and SSOs utilizing a non-covalent complexation strategy. Furthermore, it describes a method for formu- lating such drug delivery systems into a solid dosage form that could be suitable for several therapeutic applications.

Paper I, studies the activity of stearic acid-modified TP10 to deliver plasmid DNA to various cell types in-vitro and in-vivo upon i.m (intramuscular) and i.d (intradermal) injection.

Paper II, studies the activity of a new chemically modified CPP named PF6, where endosomolytic trifluoromethylquinoline moieties are added to enhance endosomal escape, to deliver siRNA in various cell types and when administered systemically in-vivo.

Paper III: In this paper, ornithine, a non-standard amino acid,

is used to chemically modify a TP-10 analogue together with N-

terminal stearylation. The new peptide named PF14 is tested in induc-

tion of splice-switching activity in several models and a method for

developing solid formulations utilizing PF14/SSO nanoparticles is

studied.

3. Methodological considerations

The methods used in this thesis are described in details in the con- tributing papers. This part will briefly discuss the main methods uti- lized with some theoretical aspects. The selections below are valid for all papers when nothing else is stated.

3.1. Solid phase peptide synthesis

All peptides in this thesis were synthesized by solid phase peptide synthesis (SPPS), which was pioneered by Bruce Merrifield in 1963 (66). SPPS is based on anchoring the growing peptide chain onto an insoluble solid matrix followed by adding an N- terminally protected amino acid then deprotection and adding a new amino acid in repeated cycles. This method enabled the synthesis of not only peptide chains but also other polymers including DNA and RNA revolutionizing en- tire fields of science.

All peptides used in this thesis were synthesized utilizing methyl- benzyl hydrylamine (MBHA) as an anchoring resin to produce C- terminal amidated peptides, and fmoc protection chemistry was used.

For PF6, four novel trifluoromethylquinoline-based derivatives (QN)

were introduced via a succinylated lysine tree. Peptides were thereaf-

ter cleaved from the resin using 95% trifluoroacetic acid (TFA), 2.5 %

triisopropylsilane (TIS) and 2.5 % H

O. Following cleavage and ex-

traction, peptides were subsequently freeze-dried. Crude peptides

products were purified using semi-preparative reversed-phase high

performance liquid chromatography (HPLC) and analyzed using ma-

trix-assisted laser desorption/ionization-time of flight (MALDI-TOF)

mass spectrometer. The main peptides used in this thesis are presented

in Table 2.

Table 2: The main peptides used in this thesis.

Name Sequence

Stearyl- TP10

Stearyl-AGYLLGKINLKALAALAKKIL-NH

PF6

PF14 Stearyl- AGYLLGKLLOOLAAAALOOLL-NH

3.2. Cell cultures

The first propagation of a human cell line in-vitro was the HeLa

cell line obtained from a cervical cancer patient named Henrietta

Lacks and propagated by George O. Gey in the 1950‟s. Since then, in-

vitro cell culture models are serving as indispensible tools in assessing

the activity of newly developed therapeutics before testing them on

animal and human subjects. Two types of cell cultures exist, primary

cultures that are obtained directly from the animal and can be kept for

a limited period of time, and permanent cultures, which are usually of

cancerous origin, and can be propagated indefinitely. More than 10

cell lines were used in this thesis including primary and permanent

cell lines. All cell lines were grown at 37°C, 5% CO2 in humidified

environment with the appropriate culture media supplemented with

nutrients and antibiotics.

3.3. Plasmid delivery (Paper I)

Non-covalent complexation strategy between the peptide and plasmids or ONs was exclusively utilized in this thesis. Different groups, including ourselves, have previously shown that CPPs can form nanoparticles with negatively charged ONs (including siRNAs) and plasmids with a diameter of 100–300 nm (67, 68) . These nano- particles are suggested to be taken up by the cells via an endocytosis- dependent process. In case of plasmids, pGL3 or pEGFP-C1 plasmid (4.7 kb), expressing luciferase or EGFP respectively were used. Plas- mids were mixed with CPPs at different peptide:plasmid charge ratios (CRs), which were calculated theoretically, taking into account the positive charges of the peptide and negative charges of the plasmid.

Complexes were formed for 1 h at room temperature then added to the cells. By measuring luciferase expression, the efficiency of delivery for different peptides and CRs can be assessed for different cell-lines.

3.4. siRNA delivery (Paper II)

In this paper, the nanoparticles were formed by mixing the peptide

and siRNA in water at different molar ratios (MRs). Reporter cell-

lines stably expressing luciferase were transfected with PF6 com-

plexed with luciferase siRNA, and the decrease in luciferase expres-

sion was used to quantify the transfection efficiency. In addition, we

investigated the targeting of an endogenous gene, hypoxanthine phos-

phoribosyl transferase 1 (HPRT1), in several other cell-lines by mea-

suring mRNA levels using qPCR (real time quantitative polymerase

chain reaction). HPRT1 was chosen due to the long cellular half-life

of the protein (around 48 h) and thereby minimal impact on the vitali-

ty of the transfected cell and limited off-target effects of the specific

HPRT1 siRNA sequence (69). In order to accurately determine the

degree of down-regulation, glyceraldehyde 3-phosphate dehydroge-

nase (GAPDH) was used as an internal standard for quantification.

3.5. SSOs delivery (Paper III)

Two cell lines were utilized in this study, Hela pLuc 705 and Mdx mouse myotubes. The HeLa pLuc705 cell-line is stably transfected with a luciferase-encoding gene interrupted by a mutated β-globin intron 2. This mutation causes aberrant splicing of luciferase pre- mRNA resulting in the synthesis of non-functional luciferase (70).

Masking the aberrant splice site with SSOs redirects splicing towards the correct mRNA and consequently restores luciferase activity. Mdx mouse myotubes were obtained from confluent H2K mdx cells, which is a myogenic cell-line derived from transgenic mice carrying a point mutation in exon 23 of the dystrophin gene. Thus this cell-line serves as the leading cell-model system for development of drugs and drug delivery systems for treatment of DMD. Masking the point mutation in exon 23 with SSOs leads to the production of a shorter, partially- functional dystrophin mRNA that can be quantified with reverse tran- scriptase PCR (RT-PCR). SSOs were non-covalently mixed with PF14 at different MRs in water. Splice-switching efficiency was cal- culated as the percentage of the corrected band (exon skipping %) to the sum of corrected and aberrant bands.

3.6. Toxicity

To exclude that the activity of the peptides in-vitro is associated with toxicity, WST-1 assay was used. This assay measures cell viabili- ty as a function of mitochondrial metabolic activity. Wst-1 measures the activity of the mitochondrial dehydrogenases to convert tetrazo- lium salts to formazan and cell viability and proliferation is directly correlated to the amount of formazan dye formed, which is quantified spectrophotometrically.

3.7. Dynamic light scattering (DLS) and zeta-poteontial

To investigate the physicochemical characteristics of the nanopar-

ticles formed upon complexation of the peptides and plasmids or ONs,

DLS and zeta-potential measurements were carried out. The nanopar-

ticles in solution after complexation can be regarded as a colloidal

dispersion system. Upon shining laser light on this system, the Brow- nian motion of the dispersed particles causes fluctuations in light- scattering intensity as a function of time, which can be correlated to the size of the scattering particles (71, 72). Each charged particle in a solution containing ions is surrounded by an electrical double layer of ions and counterions (figure 2) (73). The first layer of strongly bound counterions is commonly called Stern layer. Since it is strongly at- tached, this layer moves together with the particle in the medium. The second layer contains ions and counterions and is less strongly bound to the suspended particle, thus called “diffusion layer”. This layer is in direct contact with the bulk of the suspending liquid, and in contrast to the stern layer, ions of this layer do not move with the particle while moving in the medium. The potential difference across the double layer, i.e. between the edge of the Stern layer and the edge of the diffusion layer (bulk of suspending liquid) is called Zeta potential (figure 2) (72, 73).

Figure. 2. Different layers forming the zeta potential of the particle.

It is determined by electrophoresis of the sample and measuring the velocity of the particles using laser Doppler velocimetry (73). Zeta potential is highly sensitive to the type and amount of ions in the sus- pension and its pH.

3.8. Solid dispersion

In paper III, to test the feasibility of formulating PF14/SSO nano- particles into solid formulation, solid dispersion technique was uti- lized. Although initially invented to increase the solubility of poorly water-soluble drugs by dispersing them over hydrophilic solid matric- es (74), it is not used for the same purpose in the present work. This is because PF14/SSO nanoparticles are already water-soluble. However, the technique was adopted to enable us obtain uniform distribution of the nanocomplexes over water-soluble excipients by solvent evapora- tion. The suspension of nanoparticles was first mixed with different excipient solutions at different concentrations. For solvent evapora- tion (in this case water), we chose speed drying under elevated tem- perature (55 -60 ⁰C) and reduced pressure. Despite being relatively stressful drying method, it is more relevant to the widely used, cost- effective, heat-based pharmaceutical drying techniques.

3.9. Animal Experiments

In-vivo animal experiments were carried out in paper I and paper II. All animal experiments were approved by The Swedish Local Board for Laboratory Animals, and were designed to minimize the suffering and pain of the animals.

In paper I, stearyl-TP10/plasmid nanoparticles were injected i.d. or

intramuscularly i.m. into M. tibialis anterior. Gene expression was

assessed by imaging of the reporter gene (firefly luciferase) expres-

sion. In paper II, in luciferase experiments, mice were first injected by

high dose of a luciferase expressing plasmid via hydrodynamic injec-

tion to stably express luciferase gene in the liver. After 3 weeks, the

animals were systemically injected (tail vein injection) with PF6/luc-

siRNA nanoparticles, and the downregulation was quantified by imag-

ing of luciferase expression in the liver. In HPRT1 experiments, ani-

mals were systemically injected with PF6/HPRT1-siRNA nanopar-

ticles and downregulation of the HPRT1 mRNA was measured in dif-

ferent organs by qPCR. In addition, blood samples were collected in

heparinized tubes to assess acute toxicity by measuring the levels of

liver transaminases (ALT/AST), creatinine, white blood cells and C-

reactive protein.

4. Results and discussion

The three papers in this thesis cover three newly developed chemi- cally modified CPPs used for the delivery of different nucleic-acid based therapeutics in-vitro and in-vivo.

4.1. Delivery of plasmids via stearyl-TP10 in-vitro and in- vivo (Paper I)

Here, we demonstrate that stearylation of the TP10 peptide has a significant impact on plasmid delivery in different cell-lines including Chinese hamster ovary (CHO), Human Embryonic Kidney (HEK293), human glioblastoma (U87), human osteosarcoma (U2OS) and the hard-to transfect primary mouse embryonal fibroblasts (MEF).

Stearyl-TP10 (Table 2) transfection efficiency was in parity with the

commercial lipid-based transfection reagent Lipofectamine-2000™ ,

with less toxicity. In addition, stearyl-TP10 was able to transfect entire

cell population in a ubiquitous manner. However, the transfection ef-

ficiency was dependent on the charge ratio (CR) between the peptide

and the plasmid. DLS studies showed that the nanoparticles are in the

range of 120-150 nm. Compared to the unstearylated version, stearyl-

TP10 resulted in around 4 log increases in luciferase expression levels

as compared to plasmid-treated cells. Clearly, stearic acid renders the

peptide more hydrophobic and presumably hydrophobicity plays an

important role in particle formation, as it enables more pronounced

plasmid DNA condensation and the formation of small, stable par-

ticles. This protects plasmid DNA, making it more stable against the

degrading capacity of serum enzymes. The drastic increase in activity

of stearyl-TP10 compared to TP10 could also be a result of increased

endosomal escape. To corroborate this, the uptake levels of fluoresce-

inyl-labelled plasmid complexed with TP10 and stearyl-TP10 were

assessed in serum free conditions, as TP10 nanoparticles are very la-

bile in the presence of serum. Minor differences were observed in the levels of uptake between the two peptides, which indicated that the higher activity of stearyl –TP10 might be due to better endosomal es- cape.

These stearyl-TP10/plasmid nanoparticles facilitated efficient gene delivery in muscle and skin, after i.m. or i.d. injections, respec- tively. In both cases, luciferase activity was increased around 1 log as compared to the background levels and these effects were shown to be dose-dependent. We also confirmed that the gene delivery did not trigger any immune response in- vivo and that these treatments were not associated with any systemic toxicity. Interestingly, these effects were only seen with the nanoparticles formed at CR1, while at other CRs, no effect on luciferase activity was seen. The critical dependency on certain CR possibly emanates from the avidity of stearyl-TP10 to- wards the plasmid and, therefore, the stability of these nanoparticles.

Probably, at higher CRs release of the plasmid from the complexes is perturbed and the affinity of stearyl-TP10 towards it is too great for in-vivo applicability. Therefore, the plasmid cannot escape from the nanoparticle complex and, consequently, does not reach the cytoplasm throughout the tissue with higher efficacy than naked plasmid. These differences between the avidity at different CRs were confirmed by other assays in the paper.

4.2. Delivery of siRNA via PF6 in-vitro and in-vivo (Paper II)

siRNA has attracted of lot of attention in recent years as it was re- garded as the ideal therapeutic molecule having the properties of spe- cificity, potency, tolerability, and universality, except for being im- permeable through the cell membrane due to its size and charge (75).

In this paper, we tried to further enhance the endosomal escape prop-

erties of stearyl-TP10 and utilize it for siRNA delivery in-vitro and in-

vivo. We hypothesized that incorporation of chloroquine (CQ) analo-

gues into CPP backbone would mimic the pH buffering and osmotic

endosome swelling effects of CQ (76). Hence, we introduced four

novel trifluoromethylquinoline-based derivatives (QN) via a succiny-

lated lysine tree into the stearyl-Tp10 sequence to improve endosomal

release of siRNA (Table 2). The mean diameter of PF6/siRNA nano-

particles, formed at various molar ratios (MRs) was below 200 nm.

The endosomolytic design rendered PF6 highly efficient in promoting siRNA-mediated RNAi in various cells, targeting either reporter genes (EGFP and luciferase), or endogenous genes (HPRT1 and Oct-4). In contrast to lipofection, PF6 promoted siRNA-mediated gene silencing in entire cell populations and, thus, dose-dependently exceeded the activity of the commercial lipofection reagents in practically all tested cells. Most importantly, the activity was well preserved in serum. Fur- thermore, while primary cells and suspension cells remain relatively refractory to most transfection strategies, PF6 efficiently promoted siRNA-mediated, dose-dependent gene silencing in different refrac- tory cells such as Huvec, Jurkat, C17.2 neuronal stem cells and em- bryonic stem cells with IC

values ranging from 10–50 nM. These results collectively show that PF6 has the potential to transfect differ- ent cell types without affecting the phenotype of cells, which opens new possibilities for large-scale RNAi screens in disease relevant pri- mary cells.

After confirming the potency of PF6 in cell culture, we next as- sessed the suitability of the vector for systemic delivery of siRNA in mice. Using the HPRT1 house-keeping gene as a target, we observed pronounced knockdown in liver, kidney and lung, following systemic delivery of PF6/HPRT1-siRNA, possibly reflecting their high blood supply. The majority of previous other successful RNAi reports are indeed based on results achieved from the liver, whereas efficient gene knockdown in other organs requires much more complicated strategies and multicomponent delivery vectors(77, 78). Interestingly, we ob- served over 60% gene silencing also in kidneys and lung, by using PF6 as a single-component vehicle without additional targeting motifs and without associated toxicity.

Based on the encouraging results of strong RNAi response in liver we utilized another animal model to analyze gene silencing in liver only. In this model, mice with luciferase expressed in the liver were treated with PF6/luc-siRNA nanoparticles. By using doses up to 1 mg/kg, substantial luciferase knockdown was observed, which lasted for more than a week. Hydrodynamic injection is frequently consi- dered the golden standard technique for achieving siRNA-mediated RNAi responses in liver (79). Intriguingly, RNAi responses with PF6/siRNA in liver were at least in line with that standard technique.

These results are comparable to recently published results on the use

of lipid nanoparticles (LNPs) for siRNA, targeting luciferase in liver of mice(80). Using LNPs with optimized composition (right amount of cationic liposomes, polyethylene glycol and cholesterol), the au- thors reported on significant RNAi responses for 10 days using doses of 3 mg/kg siRNA. The strong RNAi responses observed in liver could relate to the negative zeta-potential observed for PF6/siRNA in serum media since it has been previously reported that various lipid- based systems with negative surface charge efficiently target hepatic cells in-vivo(81, 82).

4.3. Delivery of SSOs via PF14 in-vitro and solid formulation development (Paper III)

In this paper we present a new chemically modified CPP, PF14. Starting from stearyl-TP10, we utilized ornithines as the main source of positive charges instead of lysines. The design of this pep- tide was based on earlier reports showing that poly-L-ornithine dem- onstrated superior transfection efficiency (up to x 10-fold) compared to equivalent poly-L-lysine-based systems (83). The superior efficien- cy of poly-L-ornithines was related to the higher affinity for DNA and the ability to make more stable complexes at lower charge ratios (83).

Furthermore, we hypothesized that ornithine, being a nonstandard amino acid, would be less prone to serum proteases, and thus could retain the activity in serum conditions.

PF14 was tested in HeLa 705 and mdx mouse myotubes cell-

lines utilizing different MRs. Robust splice-switching was observed in

both cell lines in a dose dependent manner. This was confirmed on the

protein and mRNA level by measuring the amount of functional luci-

ferase enzyme produced in the HeLa 705 cell-line and by measuring

the intensity of corrected mRNA bands via RT-PCR in both cell lines

respectively. The activity was not significantly affected by serum con-

ditions and at certain MRs in HeLa 705 cell-line PF14 significantly

exceeded the activity of Lipofectamine2000

without associated tox-

icity. In addition, PF14 elicited a fast onset of its splice-switching ac-

tivity as early as 8 hours and was able to transfect entire cell popula-

tion without being very sensitive to increasing cell densities. Co-

localization with endocytosis markers demonstrated that endocytosis

is responsible for the uptake of PF14/SSO nanoparticles and this was

further corroborated by the enhanced splice-switching activity upon addition of CQ.

Finally, we wanted to take our delivery system a step further by developing a suitable formulation for administration. Although CPPs have been extensively exploited in recent years for the delivery of various therapeutics (56), to our knowledge, formulation of CPPs into different pharmaceutical forms has never been thoroughly stu- died. Therefore, to expand the range of therapeutic applications of CPP-based therapeutics, there is a need for studies exploring the pos- sibility of incorporating CPPs in different pharmaceutical forms; espe- cially the solid form which is widely used in several pharmaceuticals.

We applied the solid dispersion technique by mixing the naoparticles in suspension with excipient solutions then drying at relatively high temperatures ( 55- 60 ⁰C) under vacuum. Mannitol, lactose and PEG 6000 were used as excipients at different concentrations. It was clear that different excipients and concentrations thereof had a huge impact on the activity of the formulation, with lactose at a concentration of 3.33 % demonstrating splice-switching activity nearly identical to the freshly prepared nanoparticles in solution. On the contrary, Lipofec- tamine2000

almost lost its entire activity upon application of this drying and dispersion procedure. In order to assess that the presence of intact nanoparticles after the formulation procedure, DLS studies were performed comparing the freshly prepared nanoparticles with the solid formulations. We found that the particles size and particle-size distribution is highly affected by the type of excipient used and the formulation which mediated the highest splice-switching activity, also had DLS profile most similar to the freshly prepared nanocomplexes.

Upon measuring the zeta potential of the nanoparticles, we found that they are negatively charged either freshly prepared or in formulation similar to the results obtained for PF6/siRNA nanoparticles.

Next, we subjected the best performing formulation (lactose at

3.33%) to stressful stability testing conditions at elevated temperatures

for 2 months. Storage at high temperatures increases the rate of degra-

dation reactions that could take years to occur (84). PF14 formulations

demonstrated an excellent stability profile under such conditions

where no statistically significant loss in transfection efficiencies at any

time-point except for the 60 ⁰C-8-weeks point where the transfection

efficiency decreased to 70% of the initial value. Compared to lyophi-

lized lipoplexes (85, 86), PF14 formulations demonstrated an excel-

lent stability profile without further additives or special storage condi-

tions.

5. Conclusions

This thesis described three new chemically modified CPPs that were used to deliver nucleic-acid based molecules in various gene therapy approaches. We clearly demonstrate that these peptides form stable nanoparticles with the negatively charged cargos and are effi- ciently internalized in several cell-lines in-vitro and in-vivo. The na- noparticles were also active in solid formulation that can be used for different routes of drug administration.

Paper I: Introduces a new CPP, stearyl-TP10, for delivery of plasmids in-vitro in various cell-lines and in-vivo after i.m and i.d in- jection.

Paper II: Introduces a new CPP, PF6, for delivery of siRNA in- vitro and after systemic administration in-vivo.

Paper III: Describes a new CPP, PF14, for delivery of SSOs in different cell models including mdx mouse cell-line, an in-vitro model for Duchenne‟s muscular dystrophy. A method to formulate

PF14/SSO nanoparticles into a solid form was also developed.

6. Acknowledgements

- First of any acknowledgements goes to God (Allah) for His in- numerable blessings and His guidance for me to the right path.

- To my sincere mother and honorable father who have sacri- ficed everything for me to be successful and happy. Also, my brother who is my intimate companion and friend.

- To my beloved wife, whom I couldn‟t have achieved anything here without her support and tenderness. Dear Sarah, thank you and I love you.

- To Ülo of course, who has taken the risk of hiring a Binladen in his lab without being afraid that I would use the CPP tech- nology to invade the world. For his patience on my crazy, of- ten non-working ideas until something finally worked out. And for his being so kind and helpful to me, on the contrary to what he looks like in seminars.

- To Samir EL-Andaloussi, although stressful to a great extent, I learned basically everything from him: lab work, paper writ- ing, initiating a project, making collaborations, pushing people to work for you… everything. Thanks a lot.

- To Johan Runesson, the kindest and most helpful person I have ever see, who is wishing to help you in your often very silly tasks with a charming smile always on his face. Thank you.

- To all Ülo‟s group members that were here or still here includ- ing: Peter Guterstam, Staffan Lindberg, Andres Munoz and all the others in the boys room, which is always filled with mer- riment and laughter.

- To all of my collaborators and all of the people working in the

department of neurochemistry for being so helpful. Thanks.

7. References

1. Munos,B. (2009) Lessons from 60 years of pharmaceutical innova- tion. Nat. Rev. Drug Discov., 8, 959-968.

2. Butler,D. (2008) Translational research: Crossing the valley of death. Nature, 453, 840-842.

3. Lander,E.S., Linton,L.M., Birren,B., Nusbaum,C., Zody,M.C., Baldwin,J., Devon,K., Dewar,K., Doyle,M., FitzHugh,W., et al.

(2001) Initial sequencing and analysis of the human genome. Nature, 409, 860-921.

4. Cavazzana-Calvo,M. (2009) Basic research tries to decrease the risks of translational medicine. Gene Ther., 16, 309-310.

5. Hacein-Bey-Abina,S., Garrigue,A., Wang,G.P., Soulier,J., Lim,A., Morillon,E., Clappier,E., Caccavelli,L., Delabesse,E., Beldjord,K., et al. (2008) Insertional oncogenesis in 4 patients after retrovirus-

mediated gene therapy of SCID-X1. J. Clin. Invest., 118, 3132-3142.

6. Howe,S.J., Mansour,M.R., Schwarzwaelder,K., Bartholomae,C., Hubank,M., Kempski,H., Brugman,M.H., Pike-Overzet,K., Chat- ters,S.J., de Ridder,D., et al. (2008) Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J. Clin. Invest., 118, 3143-3150.

7. Kay,M.A., Glorioso,J.C. and Naldini,L. (2001) Viral vectors for gene therapy: The art of turning infectious agents into vehicles of the- rapeutics. Nat. Med., 7, 33-40.

8. Dass,C.R. (2004) Lipoplex-mediated delivery of nucleic acids: Fac-

tors affecting in vivo transfection. J. Mol. Med., 82, 579-591.

9. Rakoczy,P.E. (2000) Antisense DNA technology. Methods Mol Med., 47, 89-104.

10. Bell,N.M. and Micklefield,J. (2009) Chemical modification of oligonucleotides for therapeutic, bioanalytical and other applications.

Chembiochem, 10, 2691-2703.

11. Whitehead,K.A., Langer,R. and Anderson,D.G. (2009) Knocking down barriers: Advances in siRNA delivery. Nat. Rev. Drug Discov., 8, 129-138.

12. Elbashir,S.M., Harborth,J., Lendeckel,W., Yalcin,A., Weber,K.

and Tuschl,T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494-498.

13. Bauman,J., Jearawiriyapaisarn,N. and Kole,R. (2009) Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides, 19, 1-13.

14. Nilsen,T.W. and Graveley,B.R. (2010) Expansion of the eukaryo- tic proteome by alternative splicing. Nature, 463, 457-463.

15. Sazani,P. and Kole,R. (2003) Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J. Clin. Invest., 112, 481-486.

16. Wood,M.J. (2010) Toward an oligonucleotide therapy for du- chenne muscular dystrophy: A complex development challenge Sci.

Transl. Med., 2, 25ps15.

17. Ivanova,G.D., Arzumanov,A., Abes,R., Yin,H., Wood,M.J., Leb- leu,B. and Gait,M.J. (2008) Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res., 36, 6418-6428.

18. van Deutekom,J.C., Janson,A.A., Ginjaar,I.B., Frankhuizen,W.S.,

Aartsma-Rus,A., Bremmer-Bout,M., den Dunnen,J.T., Koop,K., van

der Kooi,A.J., Goemans,N.M., et al. (2007) Local dystrophin restora-

tion with antisense oligonucleotide PRO051. N. Engl. J. Med., 357,

2677-2686.

19. Kinali,M., Arechavala-Gomeza,V., Feng,L., Cirak,S., Hunt,D., Adkin,C., Guglieri,M., Ashton,E., Abbs,S., Nihoyannopoulos,P., et al.

(2009) Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in duchenne muscular dystrophy: A single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol., 8, 918-928.

20. Ezzat,K., El Andaloussi,S., Abdo,R. and Langel,U. (2010) Pep- tide-based matrices as drug delivery vehicles. Curr. Pharm. Des., 16, 1167-1178.

21. Verdurmen,W.P. and Brock,R. (2010) Biological responses to- wards cationic peptides and drug carriers. Trends Pharmacol. Sci., . 22. Green,M. and Loewenstein,P.M. (1988) Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell, 55, 1179-1188.

23. Frankel,A.D. and Pabo,C.O. (1988) Cellular uptake of the tat pro- tein from human immunodeficiency virus. Cell, 55, 1189-1193.

24. Joliot,A.H., Triller,A., Volovitch,M., Pernelle,C. and Pro- chiantz,A. (1991) Alpha-2,8-polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide. New Biol., 3, 1121-1134.

25. Derossi,D., Joliot,A.H., Chassaing,G. and Prochiantz,A. (1994) The third helix of the antennapedia homeodomain translocates through biological membranes. J. Biol. Chem., 269, 10444-10450.

26. Vives,E., Brodin,P. and Lebleu,B. (1997) A truncated HIV-1 tat protein basic domain rapidly translocates through the plasma mem- brane and accumulates in the cell nucleus. J. Biol. Chem., 272, 16010- 16017.

27. Pooga,M., Hallbrink,M., Zorko,M. and Langel,U. (1998) Cell pe- netration by transportan. FASEB J., 12, 67-77.

28. Futaki,S., Suzuki,T., Ohashi,W., Yagami,T., Tanaka,S., Ueda,K.

and Sugiura,Y. (2001) Arginine-rich peptides. an abundant source of

membrane-permeable peptides having potential as carriers for intracel-

lular protein delivery. J. Biol. Chem., 276, 5836-5840.

29. Elmquist,A., Lindgren,M., Bartfai,T. and Langel,U. (2001) VE- cadherin-derived cell-penetrating peptide, pVEC, with carrier func- tions. Exp. Cell Res., 269, 237-244.

30. Magzoub,M., Sandgren,S., Lundberg,P., Oglecka,K., Lilja,J., Wit- trup,A., Goran Eriksson,L.E., Langel,U., Belting,M. and Graslund,A.

(2006) N-terminal peptides from unprocessed prion proteins enter cells by macropinocytosis. Biochem. Biophys. Res. Commun., 348, 379-385.

31. Soomets,U., Lindgren,M., Gallet,X., Hallbrink,M., Elmquist,A., Balaspiri,L., Zorko,M., Pooga,M., Brasseur,R. and Langel,U. (2000) Deletion analogues of transportan. Biochim. Biophys. Acta, 1467, 165- 176.

32. Oehlke,J., Scheller,A., Wiesner,B., Krause,E., Beyermann,M., Klauschenz,E., Melzig,M. and Bienert,M. (1998) Cellular uptake of an alpha-helical amphipathic model peptide with the potential to de- liver polar compounds into the cell interior non-endocytically. Bio- chim. Biophys. Acta, 1414, 127-139.

33. Morris,M.C., Depollier,J., Mery,J., Heitz,F. and Divita,G. (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol., 19, 1173-1176.

34. Morris,M.C., Vidal,P., Chaloin,L., Heitz,F. and Divita,G. (1997) A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Res., 25, 2730-2736.

35. Oren,Z. and Shai,Y. (1998) Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers, 47, 451-463.

36. Derossi,D., Calvet,S., Trembleau,A., Brunissen,A., Chassaing,G.

and Prochiantz,A. (1996) Cell internalization of the third helix of the antennapedia homeodomain is receptor-independent. J. Biol. Chem., 271, 18188-18193.

37. Lundin,P., Johansson,H., Guterstam,P., Holm,T., Hansen,M., Lan-

gel,U. and EL Andaloussi,S. (2008) Distinct uptake routes of cell-

penetrating peptide conjugates. Bioconjug. Chem., 19, 2535-2542.

38. Letoha,T., Gaal,S., Somlai,C., Venkei,Z., Glavinas,H., Kusz,E., Duda,E., Czajlik,A., Petak,F. and Penke,B. (2005) Investigation of penetratin peptides. part 2. in vitro uptake of penetratin and two of its derivatives. J. Pept. Sci., 11, 805-811.

39. Richard,J.P., Melikov,K., Vives,E., Ramos,C., Verbeure,B., Gait,M.J., Chernomordik,L.V. and Lebleu,B. (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J. Biol.

Chem., 278, 585-590.

40. Drin,G., Cottin,S., Blanc,E., Rees,A.R. and Temsamani,J. (2003) Studies on the internalization mechanism of cationic cell-penetrating peptides. J. Biol. Chem., 278, 31192-31201.

41. Fuchs,S.M. and Raines,R.T. (2004) Pathway for polyarginine en- try into mammalian cells. Biochemistry, 43, 2438-2444.

42. Nakase,I., Niwa,M., Takeuchi,T., Sonomura,K., Kawabata,N., Koike,Y., Takehashi,M., Tanaka,S., Ueda,K., Simpson,J.C., et al.

(2004) Cellular uptake of arginine-rich peptides: Roles for macropino- cytosis and actin rearrangement. Mol. Ther., 10, 1011-1022.

43. Henriques,S.T., Melo,M.N. and Castanho,M.A. (2006) Cell- penetrating peptides and antimicrobial peptides: How different are they? Biochem. J., 399, 1-7.

44. Rousselle,C., Clair,P., Temsamani,J. and Scherrmann,J.M. (2002) Improved brain delivery of benzylpenicillin with a peptide-vector- mediated strategy. J. Drug Target., 10, 309-315.

45. Lindgren,M., Rosenthal-Aizman,K., Saar,K., Eiriksdottir,E., Jiang,Y., Sassian,M., Ostlund,P., Hallbrink,M. and Langel,U. (2006) Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide. Biochem. Pharmacol., 71, 416-425.

46. Shin,I., Edl,J., Biswas,S., Lin,P.C., Mernaugh,R. and Arteaga,C.L.

(2005) Proapoptotic activity of cell-permeable anti-akt single-chain

antibodies. Cancer Res., 65, 2815-2824.

47. Zhou,H., Wu,S., Joo,J.Y., Zhu,S., Han,D.W., Lin,T., Trauger,S., Bien,G., Yao,S., Zhu,Y., et al. (2009) Generation of induced pluripo- tent stem cells using recombinant proteins. Cell. Stem Cell., 4, 381- 384.

48. Senatus,P.B., Li,Y., Mandigo,C., Nichols,G., Moise,G., Mao,Y., Brown,M.D., Anderson,R.C., Parsa,A.T., Brandt-Rauf,P.W., et al.

(2006) Restoration of p53 function for selective fas-mediated apopto- sis in human and rat glioma cells in vitro and in vivo by a p53 COOH- terminal peptide. Mol. Cancer. Ther., 5, 20-28.

49. Snyder,E.L., Meade,B.R., Saenz,C.C. and Dowdy,S.F. (2004) Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide. PLoS Biol., 2, E36.

50. Pooga,M., Soomets,U., Hallbrink,M., Valkna,A., Saar,K., Re- zaei,K., Kahl,U., Hao,J.X., Xu,X.J., Wiesenfeld-Hallin,Z., et al.

(1998) Cell penetrating PNA constructs regulate galanin receptor le- vels and modify pain transmission in vivo. Nat. Biotechnol., 16, 857- 861.

51. Fletcher,S., Honeyman,K., Fall,A.M., Harding,P.L., Johnsen,R.D., Steinhaus,J.P., Moulton,H.M., Iversen,P.L. and Wilton,S.D. (2007) Morpholino oligomer-mediated exon skipping averts the onset of dy- strophic pathology in the mdx mouse. Mol. Ther., 15, 1587-1592.

52. McClorey,G., Moulton,H.M., Iversen,P.L., Fletcher,S. and Wil- ton,S.D. (2006) Antisense oligonucleotide-induced exon skipping re- stores dystrophin expression in vitro in a canine model of DMD. Gene Ther., 13, 1373-1381.

53. Marshall,N.B., Oda,S.K., London,C.A., Moulton,H.M., Iver- sen,P.L., Kerkvliet,N.I. and Mourich,D.V. (2007) Arginine-rich cell- penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing. J. Immunol. Me- thods, 325, 114-126.

54. Deshayes,S., Morris,M., Heitz,F. and Divita,G. (2008) Delivery of

proteins and nucleic acids using a non-covalent peptide-based strate-

gy. Adv. Drug Deliv. Rev., 60, 537-547.

55. Mae,M., Andaloussi,S.E., Lehto,T. and Langel,U. (2009) Chemi- cally modified cell-penetrating peptides for the delivery of nucleic acids. Expert Opin. Drug Deliv., 6, 1195-1205.

56. Heitz,F., Morris,M.C. and Divita,G. (2009) Twenty years of cell- penetrating peptides: From molecular mechanisms to therapeutics. Br.

J. Pharmacol., 157, 195-206.

57. Lehto,T., Abes,R., Oskolkov,N., Suhorutsenko,J., Copolovi- ci,D.M., Mager,I., Viola,J.R., Simonson,O.E., Ezzat,K., Guterstam,P., et al. (2010) Delivery of nucleic acids with a stearylated (RxR)4 pep- tide using a non-covalent co-incubation strategy. J. Control. Release, 141, 42-51.

58. Simeoni,F., Morris,M.C., Heitz,F. and Divita,G. (2003) Insight into the mechanism of the peptide-based gene delivery system MPG:

Implications for delivery of siRNA into mammalian cells. Nucleic Acids Res., 31, 2717-2724.

59. Weller,K., Lauber,S., Lerch,M., Renaud,A., Merkle,H.P. and Zerbe,O. (2005) Biophysical and biological studies of end-group- modified derivatives of pep-1. Biochemistry, 44, 15799-15811.

60. Gros,E., Deshayes,S., Morris,M.C., Aldrian-Herrada,G., Depol- lier,J., Heitz,F. and Divita,G. (2006) A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction. Biochim.

Biophys. Acta, 1758, 384-393.

61. Crombez,L., Aldrian-Herrada,G., Konate,K., Nguyen,Q.N., McMaster,G.K., Brasseur,R., Heitz,F. and Divita,G. (2009) A new potent secondary amphipathic cell-penetrating peptide for siRNA de- livery into mammalian cells. Mol. Ther., 17, 95-103.

62. Kim,W.J., Christensen,L.V., Jo,S., Yockman,J.W., Jeong,J.H., Kim,Y.H. and Kim,S.W. (2006) Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma. Mol. Ther., 14, 343-350.

63. Futaki,S., Ohashi,W., Suzuki,T., Niwa,M., Tanaka,S., Ueda,K.,

Harashima,H. and Sugiura,Y. (2001) Stearylated arginine-rich pep-

tides: A new class of transfection systems. Bioconjug. Chem., 12, 1005-1011.

64. Lundberg,P., El-Andaloussi,S., Sutlu,T., Johansson,H. and Lan- gel,U. (2007) Delivery of short interfering RNA using endosomolytic cell-penetrating peptides. FASEB J., 21, 2664-2671.

65. El-Sayed,A., Masuda,T., Khalil,I., Akita,H. and Harashima,H.

(2009) Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape. J. Control.

Release, 138, 160-167.

66. Merrifield,R.B. (1963) Solid phase peptide synthesis. I. the syn- thesis of a tetrapeptide. 85, 2154.

67. Mae,M., El Andaloussi,S., Lundin,P., Oskolkov,N., Johans- son,H.J., Guterstam,P. and Langel,U. (2009) A stearylated CPP for delivery of splice correcting oligonucleotides using a non-covalent co- incubation strategy. J. Control. Release, 134, 221-227.

68. Morris,M.C., Chaloin,L., Mery,J., Heitz,F. and Divita,G. (1999) A novel potent strategy for gene delivery using a single peptide vector as a carrier. Nucleic Acids Res., 27, 3510-3517.

69. Collingwood,M.A., Rose,S.D., Huang,L., Hillier,C., Amarz- guioui,M., Wiiger,M.T., Soifer,H.S., Rossi,J.J. and Behlke,M.A.

(2008) Chemical modification patterns compatible with high potency dicer-substrate small interfering RNAs. Oligonucleotides, 18, 187- 200.

70. Kang,S.H., Cho,M.J. and Kole,R. (1998) Up-regulation of lucife- rase gene expression with antisense oligonucleotides: Implications and applications in functional assay development. Biochemistry, 37, 6235- 6239.

71. Philo,J.S. (2006) Is any measurement method optimal for all ag- gregate sizes and types? AAPS J., 8, E564-71.

72. Domingues,M.M., Santiago,P.S., Castanho,M.A. and Santos,N.C.

(2008) What can light scattering spectroscopy do for membrane-active

peptide studies? J. Pept. Sci., 14, 394-400.