Cell-penetrating peptidesUptake mechanism and the role of receptors

Henrik Helmfors

Cover: Henrik Helmfors© Henrik Helmfors, Stockholm 2015

All papers were reprinted with permission

ISBN: 978-91-7649-259-8

Printed in Sweden by Holmbergs, Malmö 2015Distributor: Department of Neurochemistry

To Linda

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Abstract

Genes are the major regulators of biological processes in every living thing. Problems with gene regulation can cause serious problems for

the organism; for example, most cancers have some kind of genetic compo-nent. Regulation of biological processes using oligonucleotides can poten-tially be a therapy for any ailment, not just cancer. The problem so far has been that the targets for oligonucleotide-based therapies all reside on the in-side of cells, because the cellular plasma membrane is normally impermeable to large and charged molecules (such as oligonucleotides) a delivery method is needed. Cell-penetrating peptides are a class of carrier molecules that are able to induce the cellular membrane into taking them and their cargo mol-ecules into the cells. Understanding how and why cell-penetrating peptides work is one of the first and most important steps towards improving them to the point where they become useful as carriers for oligonucleotide-based therapies. This thesis is comprised of four scientific papers that are steps to-ward finding an uptake mechanism for cell-penetrating peptides that have been non-covalently complexed with oligonucleotides. In Paper I, we show that the scavenger receptors are responsible for uptake of the cell-penetrating peptide PepFect14 in complex with a short single-stranded oligonucleotide. Paper II expands upon this first finding and shows that the same receptors are key players in the uptake of several other cell-penetrating peptides that have been complexed with either, long double-stranded plasmid DNA or short double-stranded RNA. Paper III improves the luciferase-based assay for short oligonucleotide delivery by increasing the throughput 4-fold and reducing the cost by 95 %. The fourth manuscript uses the assay developed in paper III to investigate the effects on cell-penetrating peptide-mediated delivery by each of the constituents of a 264-member library of ligands for G-protein coupled receptors. We identify three ligands that dose-dependent-ly increase the luciferase expression compared to control cells. These three ligands are one positive-, one negative allosteric modulator of metabotropic glutamate receptor 5 and one antagonist of histamine receptor 3.

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List of publicationsThis thesis is based on tree papers and one manuscript, in the text referred to as Paper I, II, III and IV

I. Ezzat, K., Helmfors, H., Tudoran, O., Juks, C., Lindberg, S., Padari, K., EL Andaloussi, S., Pooga, M., Langel, Ü.: Scavenger receptor-mediated uptake of cell-penetrating peptide nanocomplexes with oligonucleotides. FASEB J. 26, 1172–1180 (2012)*.

II. Lindberg, S., Regberg, J., Eriksson, J., Helmfors, H., Muñoz-Alarcón, A., Srimanee, A., Figueroa, R.A., Hallberg, E., Ezzat, K., Langel, Ü.: A convergent uptake route for peptide- and polymer-based nucleotide delivery systems. J Control Release. 206, 58–66 (2015).

III. Helmfors, H., Eriksson, J., Langel, Ü.: Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides. Anal Biochem. 484, 136–142 (2015).

IV. Helmfors, H., Langel, Ü.: GPCR-ligands influence the short oligonucleotide transfection efficacy of the cell-penetrating peptide; Pepfect14, Manuscript.

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Additional publications

Publications not included in this thesis.

V. Eriksson, J., Helmfors, H., Langel, Ü.: A High-Throughput Kinetic Assay for RNA-Cleaving Deoxyribozymes. PLoS ONE. 10, e0135984 (2015).

VI. Helmfors, H., Lindberg, S., Langel, Ü.: SCARA Involvement in the Uptake of Nanoparticles Formed by Cell-Penetrating Peptides. In: Langel, Ü. (ed.) Cell-Penetrating Peptides. pp. 163–174. Springer New York, New York, NY (2015).

VII. Kim, T.K., Sul, J.-Y., Helmfors, H., Langel, Ü., Kim, J., Eberwine, J.: Dendritic glutamate receptor mRNAs show contingent local hotspot-dependent translational dynamics. CellReports. 5, 114–125 (2013).

VIII. Lindberg, S., Muñoz-Alarcón, A., Helmfors, H., Mosqueira, D., Gyllborg, D., Tudoran, O., Langel, Ü.: PepFect15, a novel endosomolytic cell-penetrating peptide for oligonucleotide delivery via scavenger receptors. Int J Pharm. 441, 242–247 (2013).

IX. Muñoz-Alarcón, A., Helmfors, H., Webling, K., Langel, Ü.: Cell-Penetrating Peptide Fusion Proteins. In: Fusion Proteins: Applications and Challenges. pp. 397–411. John Wiley & Sons, Inc., Hoboken, NJ, USA (2013).

X. Helmfors, H., Langel, Ü.: Recent developments in applications of cell-penetrating peptides Uptake mechanisms and oligonucleotide delivery. Chim Oggi. 30, 10–12 (2012).

XI. Andersson, O., Helmfors, H., Kanmert, D., Enander, K.: A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays. Biosens Bioelectron. 24, 2458–2464 (2009).

* Paper I in this thesis has previously been included in my licentiate thesis. ISBN 978-91-7447-782-5

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Contents1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Therapeut ic Ol igonucleot ides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.1.1 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.1.2 RNAi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.1.3 Ant isense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.1.4 Glybera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.2 Cel l-Penetrat ing Pept ides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.3 Ol igonucleot ide Del ivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.4 Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.4.1 Clathr in-Mediated Endocytosis . . . . . . . . . . . . . . . . . . . . . . 121.4.2 Caveolar Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.4.3 Other Endocytot ic Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.5 CPP entry and endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.6 Uptake Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.7 Scavenger Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.8 High-throughput Assay Development . . . . . . . . . . . . . . . . . . . . . . . . . 201.9 G-protein coupled receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.1 Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232.2 Paper I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.3 Paper I I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.4 Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.1 Sol id-Phase Pept ide Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.2 Cel l Culture and Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.3 Dynamic Light Scatter ing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.4 Luciferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.5 SCARA Inhibi tors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.6 Spl ice-Correct ion Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.7 SCO Del ivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.8 Plasmid Del ivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303.9 s iRNA Del ivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303.10 Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303.11 Toxici ty Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

4 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.1 Paper I & I I SCARAs and CPP Uptake . . . . . . . . . . . . . . . . . . . . . . . . 334.2 Paper I I I : CPP Del ivery Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354.3 Paper IV: GPCR Ligands Inf luence Uptake . . . . . . . . . . . . . . . . . 364.4 Summary and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

5 Future Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396 Populärvetenskaplig sammanfattning.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

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AbbreviationsAP2 adaptor protein 2ASMase acid sphingomyelinaseC. elegans Caenorhabditis elegansCAV1 caveolin 1CIE clathrin independent endocytosisCLIC clathrin- and dynamin-independent carrierCME clathrin-mediated endocytosisCPP cell-penetrating peptideCRISPR clustered regularly interspaced short palindromic repeatsD. melanogaster Drosophila melanogasterDAMPs danger associated molecular patternsDLS dynamic light scatteringDNA deoxyribonucleic aciddsRNA double-stranded RNAE. coli Escherichia coliFmoc 9-fluorenylmethyloxycarbonylGEEC CLIC-GPI-anchored protein-enriched early endosomal compartmentsGPI glycosylphosphatidylinositolHPLC high-performance liquid chromatography HS heparan sulphateLDL low-density lipoproteinLPLD lipoprotein lipase deficiencyLPS lipopolysaccharidesMALDI-TOF matrix-assisted laser desorption/ionization - time of flightmiRNA microRNAMQ Milli-Q ultrapure waterMR molar ratiomRNA messenger RNAMTS membrane translocating sequenceMTT 4-methyltritylNRP-1 neurophilin-1 receptornt nucleotidesON oligonucleotidePAMPs pathogen associated molecular patternspDNA plasmid DNAPI3K phosphatidylinositol 3-kinasePIP phosphaidyl inositol polyphosphatePNA peptide nucleic acidPoly C polycytidylic acid

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Poly I polyinosinic acidpri-miRNA primary miRNAPS-2’-OMe phosphorothioate 2’-O-methyl PTDs protein transduction domainsRhoA Ras homolog gene family, member ARISC RNA induced silencing complexRNA ribonucleic acidRNAi RNA interferenceRNase H ribonuclease HSCARA scavenger receptor class A SCO splice-correcting oligonucleotidesiRNA short interfering RNASPPS solid-phase peptide synthesisSR scavenger receptorSSO splice-switching oligonucleotideTEM transmission electron microscopyTFA trifluoroacetic acidTIS triisopropylsilaneuTR 3′-untranslated region

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1 Introduction

Humans have always used selective breeding to exert control over the genome of plants and animals. Then, ~70 years ago, science uncov-

ered that DNA contains the genetic information [1, 2]. With those discov-eries of the makeup and structure of the genome an entirely new scientific field emerged, it came to be known as molecular biology, a term likely coined by William Astbury in 1961. Ever since, our ability for exerting ever-finer control over the genome has grown. Already in the 1970s genes were trans-ferred between organisms [3] and eventually transgenic plants, bacteria and even mammals became commonplace. At the turn of the millennium the sequencing of the human genome [4] was completed. This was a monumen-tal effort, and it probably is the largest breakthrough in the biomedical sci-ences of the last half-century. We are finally beginning to reap the benefits of having cheaper and faster DNA sequencing. The control over the genome can now potentially be so fine that a single error in the genetic code can be corrected. In the coming decades genetic sequencing will be included in the panel of common lab tests. This in turn will bring even more data about dis-ease. The genetic contribution of each person’s illness will become especially interesting. This will bring truly personalized medicine. Mutations (genetic errors) within individuals suffering from hereditary disorders, mutations in individual tumors in cancer patients and other spontaneous mutations are all possible targets for gene-therapy.

Right now medicine is at the tipping point where gene therapy is just becoming possible, the first human treatment with gene therapy has already been approved in the European Union [5]. Alipogene tiparvovec treats adult patients with an adeno-associated virus-based gene therapy against familial

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lipoprotein lipase deficiency (LPLD). Now that the door has been opened, more therapies will become available, for many more conditions. These new therapies could work in four different ways. First, a gene could be restored like in the LPLD case above, to replace a lost or non-functional gene, as is common in hereditary diseases [6] via delivery of plasmid DNA (pDNA). Second, altering gene function by silencing a disease-causing gene, where a promising technique would be to use the RNA interference pathway [7], which in a therapeutic case would likely be mediated by short interfering RNAs (siRNA). Third, altering gene function by interfering with the splic-ing machinery either via anti-microRNAs or splice-switching oligonucle-otides (SSOs). In recent years a fourth way has received a lot of attention; the CRISPR/Cas system of editing genes [8, 9] has opened new possibilities of longer lasting genetic treatments; CRISPR/Cas also requires delivery of pDNA, the benefit of this technique is that it is simple and user friendly compared to the gene-editing techniques that preceded it.

All these potential ON-based therapies have in common that they are based on the same kind of charged, water-soluble, macromolecules that all need to be on the inside of cells in order to exert their effect. However, the plasma membrane of the cell is exceptionally good at keeping exactly these kinds of molecules in place on either side of the membrane. By comparison conventional therapeutic drugs are small, hydrophobic molecules, these two properties alone are usually enough for them to be able to passively dif-fuse the plasma membrane. Because the cell membrane is not pearmable to hydrophilic and/or large molecules, a method for safely delivering siRNA, SSOs and pDNA across it is needed.

1.1 Therapeutic OligonucleotidesBefore the delivery problem can be addressed, the types of molecules that

need to be delivered have to be presented. Loss-of-function in a gene is not only associated with hereditary diseases such as hemophilia, cystic fibrosis and muscular dystrophy or the LPLD case mentioned above and described more in detail below, but it is also implicated in several cancers. A treat-ment that could possibly restore the correct phenotype is delivery of pDNA containing a functional gene. In the LPLD case, a successful method of delivering that gene is based on using a virus where the genomic material has been replaced with the gene of interest. The oligonucleotide-based therapy that is easiest to understand is based on delivery of a plasmid, where suc-cessful delivery could restore a protein function that has been lost due to a mutation or illness.

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1.1.1 Plasmids

A plasmid is a circular piece of DNA that is not part of the chromosomal DNA; it has the ability to replicate independently and can carry genes. The term plasmid was coined in 1952 by Joshua Lederberg [10], and defined as “a generic term for any extra-chromosomal hereditary determinant”. The defini-tion has since been narrowed, in order to exclude viruses, to genetic elements that can replicate autonomously and that exist outside of the chromosome. The first applications for plasmids in the treatment of human diseases were indirect. Almost 40 years ago plasmids were used to completely transform the production of insulin; from previously having to be harvested from ani-mals into being manufactured in Escherichia coli (E. coli) [11]. The next use of plasmids for the treatment of a human condition was to produce another protein, the human growth hormone. The methodology for production of both was the same, a plasmid was constructed and the protein was expressed in E. coli [12]. The protein was then harvested, purified and later injected in patients. Continuing with the insulin example, it would be much better to give the patients lacking the ability to produce insulin, the ability back rather than them having to inject insulin several times a day. Patients with type 1 diabetes have lost the insulin producing beta-cells, luckily it is possible to produce insulin in other cells, an early example, from 1983, are AtT20 (mouse pituitary corticotroph) cells [13]. Of course insulin secretion has to be tightly regulated [14], which is a different problem. The first attempts at using plasmids in vivo did not utilize any transfection vector, plasmid DNA was simply injected into the muscle [15, 16]. However, this method suffers from low efficacy, only about 1 % of the cells are transfected [17]. The low efficacy of plasmids when used alone indicates that there is large room for improvement of the transfection efficacy, most likely by delivery vectors.

1.1.2 RNAi

One of the most attractive potential therapies that has been introduced in the last couple of decades is the ability to activate the RNA interference path-way by introducing synthetic siRNA. The first step towards the discovery of this pathway was the realization in 1993 that the lin-4 gene, which is known to control the timing of Caenorhabditis elegans (C. elegans) larval develop-ment, did not code for a protein, but produced two small RNAs of 22 and 61 nucleotides (nt) respectively. These two RNAs regulate the translation of lin-14 gene [18]. In 1998, a previously hidden more general gene-regulation mechanism was uncovered in that nematode (C. elegans), RNA interference (RNAi). Andrew Fire and Craig Mello were awarded the 2006 Nobel Prize

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in medicine for their discovery. Their realization was that double-stranded RNA was significantly more potent in regulation of gene-expression than single-stranded antisense RNA [7]. The RNAi process is guided by small RNAs that include both siRNA and microRNA (miRNA). The two differ-ent RNAs have different effects and different origins, miRNA is expressed from the endogenous hairpin-shaped transcripts [19] into longer primary miRNA (pri-miRNA) [20]. The transcription of these genes occurs in a way analogous to that of messenger RNA (mRNA). The pri-miRNA is cleaved in the nucleus by an RNase called Drosha into a precursor miRNA of about 65-bp [21]. After cleavage the pre-miRNA is exported from the nucleus to the cytoplasm by Exportin5 [22]. The final step, that produces the short ~22-bp RNA duplex by cleaving the precursor, is carried out by another RNase III enzyme named Dicer [23, 24]. Dicer is highly conserved across almost all eukaryotes including yeast, plants and animals. Some organisms have multiple Dicer homologues, in Drosophila melanogaster (D. melanogaster) Dicer 1 is involved in miRNA biogenesis whereas Dicer 2 produces siRNA [25]. miRNAs act as guide molecules in post-transcriptional gene regula-tion by base-pairing with the target mRNAs, usually in the 3’ untranslated region (uTR). Binding of a miRNA to the target mRNA typically leads to translational repression [26]. Other types of regulation by miRNA, such as translational activation [27] and heterochromatin formation [28], have also been described. Currently the online repository for miRNAs, miRBase [29] contains 1881 human miRNAs, suggesting that the post-transcriptional regulatory mechanism mediated by small RNAs is even more general than previously appreciated. A simplified scheme of the process can be found in Figure 1.

The siRNA pathway is different because usually it is not transcribed from the chromosomal DNA but instead can begin when long double-stranded RNA (dsRNA) is processed into short siRNA duplexes; these fragments are 21 nt long [30, 31]. The source of the dsRNA can either be endogenous miRNA like above or typically exogenous dsRNA. They are both processed into suitable ssRNA by a ribonuclease III enzyme (Dicer) [32, 33]. Next the ssRNA duplexes are unwound and one strand, the so-called guide strand, is loaded into the RNA Induced Silencing Complex (RISC). This complex then searches the transcriptome for targets. Targets that are fully complimentary to the guide strand are then cleaved approximately 10-11 nucleotides from the 5’ end of the guide strand, by one of the components of the RISC com-plex, an argonaute protein. Humans have four argonaute proteins, Ago1-4. Only Ago2 cleaves bound target mRNA [34, 35]. The enzymatic cleavage of mRNA is what makes this process so efficient at repressing translation.

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Using this method it is potentially possible to target any protein and thereby any pathway, all that is necessary is to introduce a 21 bp dsRNA (same length as the Dicer cleavage fragments) with a specific target and that will suppress expression of any protein. It is therefore not surprising that siRNA has received some attention from the biotech/pharmaceuti-cal industry, a recent review lists over 50 clinical trials using 26 different siRNAs [36]. The targets for siRNA therapies are very diverse, from dry eye syndrome to Ebola infection.

One limitation of “naked” or “free” siRNA is that they are not taken into cells to any large extent and they are rapidly filtered via the kidneys [37]. Another problem with siRNA, as with all small oligonucleotides, is degrada-tion by serum nucleases. Delivery of RNAi therapeutics remains one of the most significant challenges to overcome [38]. siRNA can be delivered either as 21 bp oligonucleotides or as a plasmid expressing short hairpin RNA [39] that is then processed by Dicer the same way as above to form 21 bp siRNAs.

Figure 1. Simplified illustration of the RNAi process.

Drosha

Nucleuspri-miRNA

Exportin 5

pre-miRNA

DICERCytosol

dsRNA

miRNA siRNA

mRNA degradation

some miRNA

siRNA-RISCComplex

mRNA translational repression

miRNA-RISCComplex

RISC

AGO2 DICER

RISC

AGO2 DICER

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1.1.3 Ant isense

The idea behind using a complimentary oligonucleotide to suppress trans-lation has been known since at least 1978, when a short single-strand 13 nt oligonucleotide was used to inhibit viral production in chick embryo fibro-blasts [40]. This mechanism is similar to the RNAi pathway described above where the ON binds to the target mRNA and blocks translation or initiates degradation, except that the ON is not double-stranded and that Dicer and AGO enzymes are not recruited. The degradation of mRNA is performed by ribonuclease H (RNase H). A major drawback of this technique is that the process is not catalytic as the ON is also cleaved by RNase H. In order to recruit RNase H the ON has to be designed to span a RNase H cleavage site [41]. Antisense ONs can also block a cryptic splice site and restore proper splicing to an mRNA transcript [42].

1.1.4 Glybera

The only approved gene-delivery vector (in EU/USA) is based on the non-integrating and non-replicating adeno-associated virus (serotype 1), and it compensates for the rare (affecting 1 in 1 million people) LPLD; a disease where patients lack the ability to produce the enzyme necessary to break down fat from digested food and suffer from severe and/or multiple pancre-atitis attacks despite dietary restriction of fat. The therapy is administered as a one-time series of intramuscular injections in the legs. The virus carries the human gene LPL variant S447X, a mutation associated with several gain-of-function effects, for example; reduced triglyceride levels, increased high-density lipoprotein and a reduced risk of cardiovascular disease. The Worlds’ first approved gene therapy, in China over ten years ago [43] is also based on viral delivery, recombinant human serotype 5 adenovirus that delivers p53 [44]. A virus is of course one way that nature has always delivered functional ONs; unfortunately viral delivery has many drawbacks, not least being that our immune system has been trained against them for eons and that they can induce an immune response [45]. Another problem is the limited space available to load in the viral capsid, for example in adenovirus the limit is about 5kb [46].

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1.2 Cell-Penetrating PeptidesThe discovery of cell-penetrating peptides (CPPs) followed from the real-

ization that the HIV-TAT protein was able to enter cells and translocate into the nucleus [47, 48], and in 1991 the antennapedia homeodomain from D. melanogaster was also shown to spontaneously cross the plasma membrane [49]. The discovery of those two proteins capable of transducing the cell membrane eventually lead to the identification of the minimal part required for translocation. In the antennapedia homedomain, the minimal part was found to be located in the third helix, as that part of the protein alone was able to cross the cell membrane. This 16-mer peptide was named Penetratin [50]. A few years later the minimal cell-penetrating portion of the HIV-TAT peptide was also identified [51]. In the years since these initial discoveries many more CPPs have been identified, they have some common properties; they are poly-cationic like polyarginine [52] and/or amphipathic in nature (although, a poly-anionic CPP also exists [53]). Another characteristic prop-erty of CPPs is that they usually have less than 30 amino acids.

In 1998, our research group introduced the concept of CPPs when we demonstrated the ability of transportan [54], a chimeric peptide based on the N-terminal part of the neuropeptide galanin and the wasp venom pep-tide mastoparan, to cross the plasma membrane and bring covalently linked peptide nucleic acids (PNA) into the cells [55]. Ever since then the number of CPP publications has been growing each year. Figure 2. shows a timeline of important CPP discoveries.

There is some disagreement as to what these peptides should be called; there are those who insist on calling these molecules protein transduction domains (PTDs), presumably because they were first identified in protein domains. Later, peptides not derived from protein domains were found to

Figure 2. Timeline of CPP discoveries.

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also penetrate cells, hence the name, cell-penetrating peptides (CPPs). Just to add to the confusion, the name membrane translocating sequence (MTS) has also been used for these peptides [56]. The field has more or less settled on calling these molecules CPPs, although the other names are still used occasionally.

CPPs can be broadly divided into categories based on either their origin or chemical properties. The origins are perhaps interesting for historical purposes but classification by chemical properties like charge, hydropho-bicity and amphipathicity is more informative. The archetype for cationic CPPs would be poly-arginine, as it only contains cationic residues. Other examples of cationic CPPs are TAT [51] and (RxR)4 [57, 58]. CPPs with only hydrophobic residues are more rare but at least one (1) does exist [59]. Many CPPs can be categorized as amphipathic, examples include penetratin [50] and transportan [54].

In the end of the 1990s the group of Prof. Dowdy introduced the concept of CPP-fusion proteins, where a TAT-peptide was expressed as a chimera to-gether with another protein; the protein remained active and was transduced into cells in culture [60] and into cells of live animals [61].

However, covalent linking of peptides and cargo has many drawbacks, where the major one is laborious synthesis of the constructs. Since the pep-tides are cationic and ONs are anionic, the two should form electrostatic complexes in solution. It was the group of Prof. Divita that showed this for the first time. Oligoarginine was introduced in 2000 [62]. Just a year later in 2001 Futaki et al. showed that stearylation at N-terminal of the oligoar-ginine peptide improved its delivery efficacy for ON cargo about 100-fold. Since then more peptides that use this strategy have been developed [63-66].

We introduced the category of CPPs collectively known as PepFects, they are all derived from the transportan peptide, and they are all modified by a fatty acid at the N-terminal, but they incorporate different modifications like endosomolytic moieties and unnatural amino acids and fusions with targeting peptides, all of them are successful in delivering ON based cargoes [64, 65, 67-70].

Much of the most recent research has been aimed at elucidating the mechanism by which these peptides enter the cells, different groups have shown different mechanisms for the peptides that they are working with, there is currently nothing like a universal mechanism that applies to all CPPs. The lack of a universal and well-understood mechanism is further discussed below.

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Origin Peptide Delivery strategy Nucleic acid Application Reference

HIV-1 Transactivator Tat Covalent conjugation PNA In vitro,

in vivo [92, 93]

Tat-DRBD Mixing siRNA In vivo [94]

Antennapedia Penetratin Covalent conjugation PNA In vivo [95]

R6-Pen Covalent conjugation PNA In vitro [96]

EB1 Mixing siRNA In vitro [97]

HIV-1 gp41 + NLS of SV40 large T antigen

MPGNLS Mixing siRNA In vitro [98]

MPG-8/chol-MPG-8 Mixing siRNA In vivo [99]

Bee venom PEG/PLL/DMMAn-Melittin Covalent conjugation siRNA In vitro,

in vivo [100]

Galanin + mastoparan

Myristoryl-TP-LyP-1 Mixing siRNA In vivo [101, 102]

Stearyl-TP10 Mixing 2´-OMe ON In vitro [103]

PepFect6 Mixing siRNA In vivo [64]

Pepfect14 MixingsiRNA, 2’-OMe ON, pDNA

In vitro [65, 69, 104]

Pepfect15 MixingLNA, anti-miRNA, 2´-OMe ON

In vitro [68]

NickFects MixingsiRNA, 2’-OMe ON, pDNA

In vitro [63, 78]

Designed peptide Oligo Lys Covalent conjugation PNA In vivo [105-108]

Oligo Arg Mixing siRNA, pDNA In vitro [52, 109]

PE-R8 Liposome siRNA In vitro [110]

RVG-R9 Mixing siRNA In vivo [111]

DC3-R9 Mixing siRNA Ex vivo [112]

scFvCD7-R9 Mixing siRNA In vivo [113]

Pep-3, PEG-Pep-3 Mixing HypNA-pPNA In vivo [114]

MAP Mixing siRNA In vitro [109]

KALA Mixing siRNA In vitro [115, 116]

CADY Mixing siRNA In vitro [117-119]

(RX)8B, (RB)8B, (RXR)4, R9F2 Covalent conjugation PMO In vitro,

in vivo [120, 121]

(R-Ahx-R)4AhxB Covalent conjugation PMO Ex vivo [122]

Endo-porter Co-incubation siRNA In vitro [123]

ppTG20 Mixing pDNA In vitro,in vivo [124]

Table 1. List of peptides used for delivering nucleic acids.

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CPPs have been an active area of research for over 20 years and have been on the list of potentially exploitable vectors for gene-therapy almost the entire time. CPPs possess the ability to cross the cellular membrane and bring in cargo that would otherwise be unable to enter into the cytosol. The type of cargo carried by CPPs now includes almost every kind of biomacro-molecule from plasmids [69], siRNA [64] and other ONs [65, 68] to proteins [71], peptides [72] and peptide nucleic acids [66], for more ON examples see; Table 1. Many other therapeutic and potentially therapeutic substances and molecules [73-76], have also been delivered using CPPs. None of the types of cargo mentioned above, much like ONs, are normally taken up into cells in any significant quantities. Versatile carriers like CPPs are rare and interesting from a purely scientific point of view where understanding both the mechanism and perhaps also the reason they work can be a worthwhile pursuit in itself. Additionally, it is not difficult to imagine an application-centric view of CPPs where delivering a molecule in order to treat disease is the ultimate goal. In the years since the CPPs were discovered most of the research focus has been on the last part, developing new CPPs and develop-ing new applications for them, so much so that currently (September 2015) the online repository for CPPs lists over 800 members [77].

The use of cell-penetrating peptides as delivery vectors is attractive, not least because of the absorption, distribution, metabolism, and excretion (ADME) properties of peptides; they are cleared from the cells by the stan-dard metabolism of all cells, also they are relatively non-toxic, they don’t illicit an immune response in vivo and they successfully transfect entire populations of cells in vitro [63, 78]. That is not to say that there are not any toxic peptides, for example the conus venoms are a group of highly toxic peptides that target ion channels [79].

1.3 Oligonucleotide DeliverySince the molecules described above cannot efficiently enter cells by

themselves, a way to aid the entry has been the aim of many different re-search projects. The first chemical-based transfection method, using calcium phosphate, was discovered in 1973 [80]. This method precipitates the plas-mid together with the calcium phosphate onto the cells that are grown in a monolayer. The cells then take up some of the precipitate through what is believed to be an endocytotic mechanism [81]. The second method, based on poly-L-lysine emerged in the end of the following decade [82, 83]. Today lipid-based transfection systems are the most commonly used and could be regarded as the standard methods for transfection in molecular biology

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labs all over the world [84]. There are several commercial vendors of these systems. As each new manufacturer of oligonucleotide products comes to the market they seem to simultaneously introduce new “lipofection” reagents, some of the trade names are Lipofectamine™, Dharmafect™, HiPerFect™, ViaFect™, FuGENE™.

Non-chemical methods rely on using external forces to manipulate the cells into taking up the ON. The most direct method is to use microinjection, where a cell is injected with the foreign macromolecule by inserting a very small needle through the plasma membrane [85]; needless to say, this is an extremely time consuming way that requires a certain level of concentration and a very steady hand. Electroporation is the most well-known method, using a strong electrical field to increase the permeability of the cellular membrane [86]. Sonoporation is a similar method where ultrasound is used to generate pores in the membrane through the cavitation of gas bubbles on the surface, which in theory could allow for directed transfection but has side-effects like inducing cell-death and microvascular hemorrhage and disruption of tissue structure [87], which can be useful in vivo if cell-death is a desired outcome. An old technique that relies on generating transient pores is the optical transfection method where a highly focused laser “burns” a small transient hole in the plasma membrane [88], this could be useful for single-cell experiments. One of the more imaginative techniques from the 1980s is the so-called gene-gun. Here a particle of a heavy element like gold or tungsten is coated with DNA and then literarily shot from a 22. caliber nail gun at a cell; it has been mostly used in plants, and was originally used in an onion [89]. A recent application for this gene-gun technique was to label neurons, not with genetic material but with a dye [90].

A more recent idea is to use microfluidics to “squeeze” the cells to induce compression and shear forces that result in transient holes, which enable the diffusion of macromolecules into the cytosol [91]. This method could enable high-throughput transfection of all kinds of cells in combination with all kinds of macromolecules, as it is independent of transfection reagent and very efficient. However, because of the need to pass cells through a microflu-idic device, an in vivo application is difficult to imagine.

There are only limited successes when it comes to delivery of oligonucle-otides in humans, but they do exist and one of them is the above-mentioned Glybera.

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1.4 EndocytosisEndocytosis is the process by which all cells absorb molecules that are un-

able to passively diffuse the cellular membrane, for example water and large polar molecules. The name encompasses a variety of processes that each have different modes of action. Endocytosis has been divided into clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, phagocytosis, and flotillin-dependent endocytosis, and other even less defined pathways. A simpler endocytosis classification is either phagocytosis or pinocytosis [125]. The best understood and most researched method is the clathrin-mediated endocytosis [126-128]; it is the process by which eukaryotic cells take up and recycle receptors, internalize nutrients, antigens, growth factors and even pathogens [129, 130]. The different pathways are further described below.

1.4 .1 Clathr in-Mediated Endocy tosis

The introduction of glutaraldehyde fixation in the 1960s allowed for vi-sualization of uptake of material inside the cell. Using electron microscopy, images of vesicles with protein coats in different tissues [131] were generated. A decade later clathrin was identified as being the major protein component of the coat around those vesicles, described as “vesicles in a basket” [132, 133]. The pathway was named clathrin-mediated endocytosis (CME), after the major component. CME occurs constitutively in all mammalian cells, as it is involved in the uptake of nutrients. CME modulates signaling receptor levels [134], it can control the strength of synaptic transmission and CME is also involved in protein recycling from vesicles. The first step in CME is that the cellular membrane begins forming a pit, where cargo molecules cluster. The emerging pit is coated with the protein clathrin, and because clathrin does not interact directly with neither cargo nor cell membrane, adaptor proteins are necessary to mediate the interaction. The clathrin-coated vesicle is three-layered where adap-tor proteins attach to phosphatidyl-inositol-polyphosphate head groups on the inner membrane and then to clathrin [135]. The main such adaptor protein is thought to be adaptor protein 2 (AP2) [136], upon binding to a budding vesicle AP2 changes conformation, which in turn recruits the clathrin to bind [137]. AP2 is one of at least 5 adaptor proteins [138], out of the five only AP1 and AP2 bind to clathrin, the other adaptor proteins are also involved in trans-Golgi trafficking. When the invagination in the membrane is fully formed and the coat is fully assembled, vesicle scission is catalyzed by assembly of the GTPase dynamin at the neck of the pit. The size of the clathrin-coated vesicles differs between species and tissues. CME has been implicated in other processes like the uptake of micro-bubbles containing pDNA generated using sonoporation

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[139]. Many receptors like glutamate receptor (NMDR) [140] and g-protein coupled receptors (GPCRs) are known to be endocytosed via clathrin coated pits where the internalization is regulated via adaptor proteins [141].

In order to study CME, many different methods of inhibiting CME have been employed, the two most common are treatment with chlorpromazine [142] and potassium depletion [143]. Chlorpromazine has been used to study CPP-uptake [144-151]. However, chlorpromazine may have effects on the bio-genesis of large intracellular vesicles such as phagosomes, macropinosomes, and granules [152], because of this chlorpromazine should be complemented with other, more specific, tools that do not affect other cellular processes. Other points that are important when considering CME is that clathrin function is not limited to only endocytosis, but it is crucial in many other processes like mitosis and protein secretion from the trans-Golgi network. It has been suggested that when looking to target CME with RNAi, it is more specific towards endocytosis to knock down the α or µ2-subunit of AP2 than to target clathrin itself [153].

1.4 .2 Caveolar Endocy tosis

Caveolae were first identified by electron microscopy in the 1950s [154] and are now defined as characteristically regular, flask-shaped or spherical pits, without any obvious coat on the plasma membrane [155]. The size of the pits is approximately 60–80 nm diameter [156]. The main protein component of caveolae, Caveolin 1 (CAV1) is a more recent discovery, from 1992 [157], than that of clathrin in CME, which is from 1976 [132]. The clathrin coat can be seen from electron micrographs whereas caveolin cannot. Up until the early 1990s there was no consensus as to whether clathrin-independent endocytosis (CIE) existed at all [158]. The linking of caveolae to CIE finally resolved this issue with the finding that dynamin is recruited to caveolae, as well as clathrin-coated pits, which suggest that the caveolae can bud off the membrane [159]. The role of the caveolin proteins for specific endocytosis of cargo remains to some extent unresolved. SV40 virus and CAV1 clearly co-localize when the virus is internalized in cultured cells indicating that CAV1 is involved in the uptake of SV40 [160]. In CAV1 knockout mice the rate of SV40 internalization is increased, suggesting that CAV1 inhibits the uptake of SV40 [161]. Further confounding the role of CAV1 in endocyto-sis, there are seemingly conflicting reports involving the endocytosis and transcytosis of albumin, where in one case CAV1 is required for albumin to be transported [162], and in the other case for CAV1 knockout mice show normal levels of albumin, indicating that transport occurs normally also in

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the absence of CAV1 [163]. What is known is that Caveolin proteins play crucial roles in many processes, CAV1 [163] and CAV3 [164] are involved in the formation of caveolae and CAV2 seems to not have any effect on the formation of caveolae [165]. Additionally CAV1 seems to be involved in lipid regulation, expression of CAV1 facilitates the uptake of fatty acids into cells and increases the levels of free cholesterol in cells and rate of cholesterol export from cells [166-168]. More recently caveolin has been found in other cellular organelles like mitochondria, nuclei and endoplasmic reticulum [169]. Caveolin proteins interact with many signaling and transmembrane proteins, like GPCRs, Src kinases, endothelial nitric oxide synthethase, ad-enylyl cyclases, protein kinase A, mitogen activated kinases and ion channels [170]. Like CAV1 and CAV3 the cavin family of proteins (cavin 1-4) are also involved in the formation of caveolae (cavin1), plays a role in the membrane invagination (cavin2), is responsible for trafficking and budding of vesicles (cavin3). Cavin4 is specific for muscle cells [170-173]. Caveolae in smooth muscle cells do not take part in endocytosis, a reason could be that these caveolae lack tyrosine resides that can be phosphorylated [174], but it does not fully explain why muscle cells are not using caveolae for endocytosis.

Caveolin-1 and CDC42 (a small GTPase), play an important role in the endocytosis of silica coated iron oxide nanoparticles and of superparamag-netic iron oxide nanoparticles with negative surface charge and a diameter around 17 to 30 nm in HeLa cells in vitro [175].

1.4 .3 Other Endocy tot ic Pathways

Clathrin- and caveolar endocytosis recruit proteins that coat the vesicles, but there are endocytotic pathways that are independent of these two mecha-nisms. Glycosylphosphatidylinositol (GPI) anchored proteins were one of the first cargo proteins to be identified as markers for a pathway independent of both clathrin and caveolin. That pathway is now termed clathrin- and dynamin-independent carrier (CLIC)-GPI-anchored protein-enriched early endosomal compartments (GEEC), CLIC-GEEC. The internalization via this pathway is unaffected by inhibition of clathrin or dynamin but it is sensitive to inhibition of the small GTP-binding protein CDC42 [176]. The pathway is most active on the leading edge of migrating cells [177]. The car-riers then fuse with early endosomes through a mechanism that is dependent on phosphatidylinositol 3-kinase (PI3K) activity [178].

Other forms of clathrin independent endocytosis include a pathway as-sociated with Arf6 [179-181], one that is dependent on dynamin and Rho [182], and others that depend on flotillin [183].

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Macropinocytosis originates in ruffled regions on the plasma membrane, an invagination of the cell membrane forms a pocket that pinches off into the cell to form a vesicle (0.5–5 µm in diameter). The vesicle is filled with a comparatively large volume of extracellular fluid and molecules (roughly about 100 clathrin-coated vesicles). The filling of the macropinosomes is non-specific [184]. The pathway is likely to involve most of the molecular players that are common to all the pathways described above, except caveolae [185]. Simultaneously it is clear that the macropinosomes are substantially larger than all the other pathways pointing towards a mechanistic difference.

From the discussion above it is obvious that the pathways to some extent overlap, for example dynamin is involved in multiple pathways, endocytosis of GPCRs seems to occur both through caveloae and clathrin-coated pits, and there is significant crosstalk between the different pathways cavin1 and cavin3 are potent inhibitors of the CLIC/GEEC pathway [186]. For an il-lustration of some of the overlapping proteins in the different endocytotic pathways see Figure 3. Even the “well-defined” clathrin pathway may turn out to be more than one pathway, because there is evidence of cargo sort-ing at all stages of the internalization process [187], it logically follows that the protein constitution of the vesicles must be non-identical. The clathrin-independent pathways may in turn be revealed as red herrings as there is some doubt as to if these pathways contribute to endocytosis in mammalian cells at all [188].

Phagocytosis

CME

CLIC/GEEC

Arf6

FlotillinMacropinocytosis

Caveolae

Rab5

Dyna

min

Arf6

Cdc42

RhoA

Arf6

Rac1

Cdc42actin

actin actinsrcPKC

src

Other implicated protein

Small G-protein depencence

Figure 3. Illustration of the overlap between the different endocytotic pathways; the ovals represent a defined pathway, the diamonds a protein, the cylinders represent Small G-proteins, note especially that dynamin is implicated in 4 out of the 7 pathways, and actin in all but 2 pathways.

Adapted from Table 1. in Doherty, G.J., McMahon, H.T.: Mechanisms of Endocytosis. Annu Rev Biochem. 78, 857–902 (2009). [254]

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1.5 CPP entry and endocytosisThe endocytotic uptake route of CPPs is not yet fully understood, for

example; siRNA knockdown of Caveolin-1, and flotillin-1 showed no effect on the uptake of the cationic peptides TAT and R8 [145]. There are reports that TAT uses CME to enter cells [189] although this result relies mostly on inhibition of CME using chlorpromazine which as discussed earlier may have side effects. For TAT and larger cargo like TAT-fusion proteins the ca-veolar endocytotic route is implicated because of co-localization with CAV1 [190]. While TAT-fusion peptide uptake in cells expressing a dominant negative mutant of dynamin, was not inhibited suggesting that neither the caveolar nor the clathrin pathway is necessary for uptake [191]. Other CPP-cargo complexes are taken up via all of the previously mentioned endocytic pathways, with the exception of phagocytosis [192].

A method that could provide insight also for CPP-based delivery was described recently in a paper that presents a way to visualize and detect the release of siRNA from endosomes and correlating it with knockdown of a GFP-reporter for lipid based delivery vectors. The escape occurs early in the uptake process and only a small portion of siRNA escapes, it will be interesting to se if this is similar for CPPs [193].

1.6 Uptake Mechanisms For many CPP-based delivery systems, including PepFects, stearyl-

polyarginine, and TAT-DRBD, endocytosis has been suggested as the main mechanism behind the uptake into cells [64, 65, 94, 194, 195]. For other systems, like the CADY-peptide, the group of Prof. Divita has shown that the translocation of CADY:siRNA complexes is independent of both endo-cytosis and/or surface proteoglycans and is probably energy-independent, suggesting that the uptake mechanism must be direct translocation [119]. However, one of the papers in this thesis partly question direct translocation by demonstrating that the CADY-peptide when in complex with siRNA is also taken up through scavenger receptors. It is, of course, entirely possible that CPPs take different routes into the cells when in complex with cargo and when alone.

The arginine-rich peptide TAT has been shown to interact with the membrane of giant unilamellar vesicles, it induces a specific conformational change causing a membrane curvature that leads to the formation of pores that allows the peptide to enter [196]. When the TAT-peptide is coupled

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to cargo that is too large for these pores, the peptide was proposed to in-teract with actin inside of the vesicle and induce conformational changes, reminiscent of membrane the blebbing associated with macropinocytosis [196]. Verdurmen et al. showed that the endocytosis independent entry of poly-arginines depends on a CPP-induced translocation of acid sphingomy-elinase (ASMase) to the outer leaflet of the plasma membrane and ceramide formation via hydrolysis of sphingomylin. This changes the composition of the plasma membrane and allows for entry into the cytosol, the mechanism of enzyme activation is not yet clear. The threshold for direct CPP translo-cation could be lowered through addition of ASMase and it enhances the translocation of poly-arginine coupled to low-molecular weight cargos, but not high-molecular weight cargos [197].

When studying the uptake mechanism of PepFects, the peptides and ONs are complexed prior to treatment. The complexation is done with the peptide in excess, in a molar-ratio (MR) ranging from just a few to tens of times more peptide than ON [64, 65]. It could be expected that these complexes would have an excess of positive charge and that this charge is important for at least the initial interaction with the negatively charged cell mem-brane if not the entire translocation. However, the particles formed by the CPP PepFect14, and splice-correcting oligonucleotides (SCOs) form com-plexes with a negative surface charge in bio-relevant media, as determined by ζ-potential measurements [65, 198]. A particle with net negative charge should not interact with the negative plasma membrane or glycosaminogly-cans on the cell-surface. This contradiction is further explored later in the thesis.

In the early days of CPP research the uptake mechanisms of CPPs were thought to be mostly independent of any endocytosis pathway and rely on direct penetration of the cellular membrane. However, in 2003 a re-evalua-tion of the methodology used to determine the uptake mechanism dispelled much of the previously held beliefs. The earlier results were likely caused by artifacts related to the fixation of cells [199]. Because of this, imaging and fluorescence activated cell sorting (FACS) is now done on live cells that have been heparin washed and trypsinized. In the following decade, reports indicating one or another kind of endocytotic mechanism as responsible for the uptake of CPPs has steadily increased in number.

It had been known since before the artifacts due to cell fixation came to light that internalization of the most famous and well-studied CPPs, the cationic TAT-peptide requires cell-surface heparan sulphate (HS) proteogly-cans [200], this is also true for other arginine-rich peptides [201]. This is the first step of interaction with the cell membrane that then stimulates uptake.

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The picture is, however, not as clear as that because there are reports that state the opposite i.e. HS-interaction inhibits the uptake of cationic CPPs [202]. On the other hand, when it comes to the other major class of CPPs, the amphipathic ones, cell-surface clustering of HS-proteoglycans neither contributes to, nor inhibits the uptake [203]. The endocytotic mechanism by which the TAT-peptide is then taken up after the initial interaction with cell-surface proteoglycans has been reported to be either; macropinocytotis [201], lipid raft caveolar endocytosis [190], CME [189], or all three mecha-nisms simultaneously [144]. More recently a report detailing the ways in which the TAT-peptide can interact with, and be internalized by, cells, added some clarity to this apparent lack of agreement. The TAT-peptide can bind to HS-proteoglycans and become internalized by endocytosis; it can also interact with the membrane and generate saddle-splay curvature and in this way induce pore-like structures in the membrane. If the cargo is sufficiently small the peptide can be internalized directly, larger cargo is anchored to the membrane via the peptide and a strong interaction between TAT and actin can remodel the cytoskeleton and promote other pathways like macropinocytosis [196].

Direct translocation for arginine-rich CPPs is likely related to the ability of the guanidinium moieties on arginines to form hydrogen bonds with mem-brane lipids [204]. Each cationic guanidinium head group has a rigid planar array of hydrogen bond donors that allows for highly effective bidentate hydrogen bond formation with negatively charged carboxylates, phosphates, and sulfates [205]. Direct penetration is also supported by the ability of the arginine-rich TAT-peptide to induce pores in artificial membranes [206].

In 2012 Tanaka et al [207] reported that chemokine receptor type 4 (CXCR4) acts to stimulate the uptake of a dodecamer of arginine. Stimulation of CXCR4 with the intrinsic ligand SDF-1α induces macropi-nocytosis. Additionally they showed that siRNA knockdown of the receptor prevents the formation of lamellipodia, further indicating that for oligo-arginine macropinocytosis is one uptake route.

Peptides that bind to prostate cancer cells may bind to the neurophilin-1 receptor (NRP-1) and then be quickly internalized. These peptides have a C-terminal consensus sequence of R/KXXR/K which is similar to sequences found in many CPPs [208]. This is the same consensus sequence that is found in the C-terminal end of VEGF-A165 and some semaphorins that are known to bind to NRP-1.

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We have reported that our most recent generation of CPPs, peptides that have been modified using both a N-terminal stearylation, phosphorylated tyrosine or a side-chain extension of the peptide chain were also are taken up through differing endocytotic routes. Inhibitors for the different path-ways revealed that NickFect1 is endocytosed via both the CME pathway and through macropinocytosis, whereas NickFect51 is internalized mainly through macropinocytosis [209].

1.7 Scavenger ReceptorsAfter their discovery of the low density lipoprotein receptors (LDL recep-

tor) [210, 211], the Nobel laureates Brown and Goldstein went on to discover the scavenger receptors (SR) in patients who lack LDL receptors [212]. SRs are a family of cell-surface glycoproteins that where first recognized to bind modified lipoproteins such as acetylated and oxidized LDLs. Initially these receptors were thought to only scavenge acetylated LDL into macrophages [213]. This first definition has since grown to encompass receptors that can bind a diverse set of ligands, like modified endogenous proteins and lipo-proteins; since these modified molecules are potential hazards to the cells, they are called “danger-associated molecular patterns” (DAMPs). In addition to DAMPs, the receptors promiscuously bind several different exogenous molecules like bacterial lipopolysaccharides (LPS) [214], microorganisms like Staphylococcus aureus bacteria [215], PrP106–126 prion protein [216], and viral RNA [217], HCV virus [218], together these have been called “pathogen associated molecular patterns” (PAMPs) [219]. Other foreign particles that are not necessarily PAMPs like silver-nanoparticles [220] are also recognized by scavenger receptors. Also, they recognize several sulfated polysaccharides (dextran sulfate and fucoidan but not chondroitin sulfate) and polyribonucleotides (polyinosinic (poly I) and polyguanilic (poly G) acid but not polyadenilic (poly A) or polycytidylic acid (poly C)) [221]. SR class A (SCARA) was found to recognize the exchangeable apolipo-proteins A-I and E in both lipid-free and lipid-associated form, suggesting the shared amphipathic alpha helix as a potential recognition motif [222].

Since SRs recognize so many different PAMPs, it is not surprising that they are highly expressed on macrophages, where they were also initially identified. Later, SRs were found in many different cell types, where sub-types were identified in cells such as smooth muscle cells, epithelial and endothelial cells, splenic dendritic cells, and fibroblasts. The number of cell-surface receptors classified as scavenger receptors is continually grow-ing from the first suggestion of classes A-F in 1997 [223] to now include

20

19 receptors that are divided into classes A through I [219]. The receptors share the ability to bind poly-anionic ligands with low specificity. The term scavenger receptor used to be defined as: an extracellular glycoprotein (either soluble or membrane-bound) involved in the recognition and/or endocytosis of negatively charged molecules [224]. Today many more functions for the receptors have been reported and they are generally considered a sub-class of the membrane-bound pattern recognition receptors [219]. An emerging role for SRs is cellular adhesion where SCARAs were found to contribute to the majority of the macrophage adhesion to the extracellular matrix both in the presence and absence of serum and almost all of the adhesion when divalent ions were chelated [225]. The class A scavenger receptors are multi-meric receptors with a cytoplasmic domain, a trans-membrane domain and collagenous domain and some have an additional cysteine-rich domain or a c-type lectin domain [226].

The SRs ability to take up viral DNA and other negatively charged mol-ecules are the properties that made us suspect that SRs could be involved in the uptake of CPP:ON complexes. We have shown, in several papers, that SCARAs are involved in the uptake of CPP:ON complexes using both a number of pharmacological inhibitors and by knocking down the receptor using siRNA [68, 69, 198, 209, 227], two of those publications are included in this thesis.

1.8 High-throughput Assay DevelopmentThe invention and standardization of the micro-plate has been a paradigm

shift in the life sciences. The subsequent technological developments have brought various plate-readers, which now enable “everyone” to do some form of higher throughput experiments. For example, even a simple experiment like determination of protein concentration in all 96 wells of a standard cell-culture plate using the much cited Lowry protein assay [228], increases throughput almost 100-fold compared to using cuvettes. The increasing number of wells in the standard format micro-plates enables ever-higher throughput; a state-of-the-art RNAi HTS project that uses 1536-well plates will be able to go through all the known human genes in less than 15 plates. However, using plates with such small wells necessarily requires that no hu-man be involved in any of the liquid handling steps, robots and high levels of automation are necessary for repeatable experiments. The more difficult problem is the evaluation of data, it is not trivial to determine what is a “hit” when there are thousands of data points. The first question to answer is whether the assay is robust; this is done by evaluation of the so-called

21

Z’-score [229], which compares the averages and standard deviations of the positive and negative controls see Equation 1. A score > 0.5 is considered an excellent assay, whereas 0 < Z’ < 0.5 is considered a marginal assay and score below zero means that the variation between the negative and positive controls overlap and that screening is essentially impossible. If the normally accepted value of p < 0.05 was used, over a thousand hits would be identified just by chance in a whole human genome siRNA scan, or over a dozen in the 264-member library used here. To put it differently; almost five hits by chance for each 96-well plate. From this it is obvious that a threshold set too low will give many false positives, while a threshold set too high will exclude many potential hits. In this work a hit was further investigated if it deviated more than 3 standard deviations from the mean of the positive controls in at least three consecutive experiments. This approach still leaves an acceptable one in one-thousand chance of a false positive.

1.9 G-protein coupled receptorsG-protein coupled receptors (GPCRs) are the largest family of cell-surface

receptors, they perform many diverse functions that include for example vision in the eye, taste on the tongue, they regulate mood and alertness. The ligands that bind to the receptors are diverse and include small molecules and peptides. The receptors have in common that they span the membrane seven times and on the inside of cells they associate with G-proteins. The 2012 Nobel Prize in chemistry was awarded to Brian Kobilka and Robert Lefkowitz for their work “crucial for understanding how G-protein-coupled receptors function”. The receptors can become internalized upon binding a ligand and there is mounting evidence that the signaling function of the receptors continues even after internalization [230].

Equation 1. Z’-score.Mean (µ) and standard deviation (δ) of the positive and negative controls.

Z '=1−3δ + +3δ −

µ+ − µ−

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2 Aim

The primary aim of our research group is to use CPPs to improve the delivery of therapeutic ONs; to this end this thesis includes a couple

of different projects aimed at identifying the uptake mechanism for peptide-based delivery vectors and methodologies for evaluation and comparison of as many peptides as possible.

The over-arching aims are:

• DevelopmentofnewCPPvectorsforgenedeliveryapplications.

• ApplicationofCPPsin vitro and eventually in vivo.

The aims for each paper are detailed below.

2.1 Paper IOur finding that surface charge was negative for CPP:ON complexes

in cell-media was perplexing and counter to the hypothesis that cellular uptake of CPPs was initiated via the opposite charges of the CPPs and cell membrane attracting each other. The aim here was to identify a receptor or other mediator of an interaction between a negatively charged particle and the negatively charged cell-membrane.

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2.2 Paper IIThe findings of paper I suggested that SCARAs were responsible for the

uptake of PepFect CPP:ON complexes, the main aim of the study was to determine if this uptake mechanism is also responsible for the uptake of other CPP:ON and cationic-polymer:ON complexes.

2.3 Paper IIIThe aim of this paper was to miniaturize our standard 24-well plate assay

for delivery of short ONs, i.e. the assay used in paper I and II, and simulta-neously reduce cost by developing a homemade luciferase assay buffer. And reduce complexity and decreased time required for experiments, in order to make the assay amiable to high-throughput screening. Additionally this development was necessary in order to perform the experiments required for paper IV.

2.4 Paper IVThe aim of this study was to use a library of GPCR ligands to stimulate

cell-surface receptors and look for changes that affect the uptake of CPP:ON complexes. The secondary aim was to perhaps identify a novel mechanism either for uptake or endosomal escape.

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3 Methods

3.1 Solid-Phase Peptide Synthesis

Solid-phase peptide synthesis (SPPS) is the most common method for producing peptides for both research and therapeutic purposes, it is a

technique that earned its inventor, Robert Bruce Merrifield [231] the Nobel Prize in chemistry in 1984. The peptide is attached via its C-terminus to a solid phase, which allows for filtration to be used to remove spent reagents. The 9-fluorenylmethyloxycarbonyl (Fmoc) scheme was used in this work. The process is a stepwise, iterative cycle of addition followed by de-protection of each amino acid. Addition occurs via the carboxylic acid to a free amine on the anchored peptide chain, using the Fmoc method the peptide is at-tached to the solid support via an acid labile linker. In order to make the reaction specific, a protection group is used on the α-amine of the amino acid being attached; it is protected with the Fmoc group. This group is easily removed (or de-protected) using a base like for example piperidine or pipera-zine. The amino acid side-chains are also protected with acid labile groups. After addition of each amino acid the Fmoc group is removed and then the next amino acid is coupled. When the peptide is ready the solid support and side chains are removed using acid, yielding an unprotected peptide.

In the event that one of the side chains has to be functionalized further, an orthogonal protecting group can be used. The peptide is first synthesized to completion and the side chain is then de-protected in conditions that only affect that one specific site. In this work all side-chain modifications have

26

been performed at a lysine. In this case the lysine is protected with the acid labile 4-methyltrityl (MTT)-group that can be removed without cleaving the resin, using weak acid.

All peptides were synthesized using one of two automated peptide synthe-sis machines (Syro II, Multisyntech GmBH or Alstra+, Biotage AB, Uppsala, Sweden). Stearic acid modification was carried out either in a manual step after automated synthesis or coupled under microwave heating conditions in the Alstra+; 5,6 carboxy-fluorescein was coupled manually after removal of the acid-labile side-chain protecting group from lysine 7 on PepFect 14. All peptides were synthesized using a H-Rink-Amide-ChemMatrix resin (PCAS Biomatrix, St-Jean-sur-Richelieu (province of Quebec), Canada), this kind of resin produces peptides with amidated C-termini. The peptides were cleaved from the resin, using 95 % tri-fluoroacetic acid (TFA), 2.5 % triisopropylsi-lane (TIS) and 2.5 % H2O, precipitated in ether and lyophilized. Crude pep-tides were purified using semi-preparative reversed-phase high performance liquid chromatography (HPLC) and analyzed using matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF) mass spectrometry. After purification the peptides where lyophilized again and reconstituted in ultra-pure water (Milli Q, Merck Millipore) before use. All the peptides that were used in this thesis were synthesized in this manner, except for the CADY peptide, which was a kind gift provid-ed by Dr. Gilles Divita and Dr. Sebastien Deshayes. Table 2 lists the peptides used in this thesis. Figure 4 shows the structure of PF14, it was used in all papers included in the thesis.

A-G-Y-L-L-G-K-L-L-O-O-L-A-A-A-A-L-O-O-L-L NH2

O

8

Figure 4. The structure of PepFect14.

CPP Sequence

PF6 Stearyl-AGYLLGKaINLKALAALAKKIL-NH2

PF14 Stearyl-AGYLLGKbLLOOLAAAALOOLL-NH2

dPF14 Stearyl-agyllgklloolaaaalooll-NH2

S-(RxR)4 Stearyl-RxRRxRRxRRxR-NH2

CADY Ac-GLWRALWRLLRSLWRLLWRA-Cya

Table 2. aFour chloroquine-analogs coupled via a lysine tree. b 5,6 Carboxy-Flourescein (only for labeled PF14) * small caps = d-amino acid, x= amino hexanoic acid, O=Ornithine, Ac= acetylation, Cya = Cysteamine

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3.2 Cell Culture and TreatmentThe first cell line, HeLa, that was propagated indefinitely in vitro came

from a cervical cancer patient named Henrietta Lacks. The man who first generated it in the 1950s, George O. Gay, named the cell line HeLa after the patient. Mrs. Lack’s cells have been an essential tool in the life sciences ever since. More information and a popularized story of Mrs. Lacks and her cells can be found in the book “The Immortal Life of Henrietta Lacks” by Rebecca Skloot.

HeLa pLuc705 cells are a gift from Prof. Ryszard Kole (University of North Carolina, Chapel Hill, NC, USA). The HeLa pLuc705 cell line is stably transfected with a luciferase-encoding gene interrupted by a mutated β-globin intron 2. These cells were used in all the papers included in this work and are further described below.

The cells used for siRNA knockdown in paper II were U2OS-SAMP1-YFP cells, these cells stably express a yellow fluorescent protein (YFP) fusion of the inner nuclear membrane protein SAMP1 [232].

The cells used in the siRNA knockdown experiments in paper III (U87 MG Luc cells were provided to us from Dr. Kaido Kurrikoff (University of Tartu, Tartu, Estonia); these cells stably express the luciferase gene in high quantity.

Cells were grown at 37°C, 5 % CO2, in Dulbecco’s modified Eagle’s me-dium with glutamax supplemented with 0.1 mM non-essential amino acids, 10 % fetal bovine serum, 200 U/ml penicillin, and 200 µg/ml streptomycin (Invitrogen, Stockholm, Sweden).

HeLa pLuc705 cells (7.5×104) were seeded 24 h prior to experiments into 24-well plates. PF14 was mixed with PS-2’-OMe SCOs at molar ratio 5 in MQ-water in 10 % of the final treatment volume (i.e., 10 µl). The final concentrations were 200 nM SCO and 1 µM PF14/well. Complexes were allowed to form for 30–45 min at room temperature.

3.3 Dynamic Light ScatteringIn order to further characterize the physiochemical properties of the

particles that formed when mixing the peptides with ONs, dynamic light scattering (DLS) was used. DLS is also known as photon correlation spec-troscopy and as quasi-elastic light scattering [233]. This is a method that

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allows for the determination of the size distribution of small particles in so-lution. It is built on measuring the intensity of light scattering of particles in Brownian motion [234] where time-dependent fluctuations are correlated to size. If particles are small compared to the wavelength of the laser used then the light scatters in all directions (Rayleigh scattering [235]). In a way, it is amazing that the initial explanation of scattering from 1871 was derived at a time when everyone believed in the luminiferous ether. Lord Rayleigh went on to use the preferential scattering of blue light (shorter wavelength) by the atmosphere to explain why the sky is blue and the sunsets are red [236]. Due to the Brownian motion the distance between the particles is constantly changing, therefore scattered light intensity fluctuates. The time-scale of the fluctuations is directly related to the translational diffusion coefficient of the scattering particles, which in turn is related to their size [237]. From this an autocorrelation function that relates the fluctuations in intensity to the size can be used for size determination. One drawback of this method is that intensity peaks have to be separated by a factor of at least two for the method to be able to distinguish between them, if they are closer they will yield a single broader peak [238]. Another problem is that the method is more sensitive to larger species as they give higher intensity.

The ζ-potential is the difference in potential between the stationary layer of fluid attached to a dispersed particle and that of the dispersion medium. From an instrumental aspect it is fortunate that the electrophoretic velocity is proportional to the measurable electrophoretic mobility. This allows for the use of the above-described method of DLS to be adapted to electro-phoretic light scattering. Here the Doppler effect on particles undergoing electrophoresis shifts the frequency of the light scattered from the particles. In this work an instrument that can do both types of measurements was used; Zetasizer Nano ZS (Malvern Instruments, Malvert, UK).

PF14-SCO nano-complexes were prepared as described above and diluted in MQ-water, 150 mM NaCl solution, Opti-MEM (Invitrogen), or Opti-MEM with 10 % serum into a final volume of 1 ml. Samples were assessed in disposable low-volume cuvettes. Data was converted to relative intensity plots, from which the mean hydrodynamic diameter was derived.

3.4 LuciferaseLuciferase from firefly is an enzyme that catalyzes a reaction that emits

light; it is what gives the flies their characteristic glow. The gene that encodes it can be used as a reporter gene in many different types of delivery assays. Because the reaction produces light, is a convenient and easy way to measure

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differences in gene expression. All that is needed is addition of the substrate for the luciferase enzyme to the lysed sample, For example delivering a lucif-erase encoding plasmid to a cell that does not normally produce the enzyme gives a measurable increase light output that is proportional to the amount of luciferase present. Conversely, delivering siRNA to a cell that already expresses luciferase will result in a decrease in the amount of enzyme and therefore a reduction in light output. The assay described below (Section 3.6) is another way to use luciferase as reporter gene in an ON assay.

3.5 SCARA InhibitorsScavenger receptor-specific “inhibitors” were used in paper I and II in order

to elucidate the effect of this family of receptors on the uptake of CPP:ON complexes; the inhibitors used are fucoidan, dextran sulfate and Polyinosinic acid (Poly I). Chemically similar molecules were used as controls. Fucoidan is a sulfated polysaccaride found in brown algae, dextran sulfate is a sulfated analog of dextran and Poly I is a polymer of the inosinic monophosphate. The controls used where galactose for fucoidan and chondroitin sulfate for dextran sulfate, and polycytidylic acid (Poly C) was used as control for the Poly I. These molecules have been used previously in studies of SRs [221, 239-241].

3.6 Splice-Correction AssayThe splice-correction assay that was developed by Kole et al. [42] provides

an elegant quantitative assessment of cellular delivery efficiency of SCOs. The assay uses HeLa cells that have been stably transfected with pLuc705, a plasmid containing a luciferase-encoding gene interrupted by a mutated intron from a β-thalassemic globin gene. The intronic mutations activate a cryptic splice site that produces non-functional luciferase. Masking the mutated site with an antisense ON re-orients the splicing machinery to pro-duce functional luciferase. Subsequent luminescence measurement allows for quantification of uptake using an instrument as simple as a luminometer.

3.7 SCO DeliveryIn papers I, III and IV SCOs were delivered, using non-covalent complex-

ation with CPPs, to cells that stably express an incorrectly spliced, and non-functional, luciferase. The successful delivery results in functional luciferase.

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In the first two papers a 24-well assay was used and in Papers III and IV a 96-well assay was used. In the 96-well assay the CPP:SCO complexes were already present in the micro-plate wells when cells were seeded. In paper IV a library of GPCR receptors was also present in the micro-plate wells prior to seeding the cells. The cells and the assay are described above.

3.8 Plasmid DeliveryCPPs and two cationic polymers were used to deliver the pGL3 plasmid,

which expresses the luciferase gene. Plasmid delivery was performed again by non-covalent complexation with the selected CPPs and polymers. In this case the cells were pre-treated with SCARA inhibitors (and their controls) to assess the effects of removal of the SCARAs from the uptake process of CPP:pGL3 and polymer:pGL3 complexes. Additionally, plasmids expressing the SCARA-3 and -5 genes were used to over-express the receptors and study the effect on the uptake. In the over-expression experiments, a commercial transfection reagent (LipoFectamie 2000) was used to transfect HeLa cells with the SCARA genes before addition of the CPP/Polymer:pGL3 complexes.

3.9 siRNA DeliveryIn Paper II, siRNA knockdown of SCARA-3 and -5 was performed to

study the effects of the SCARAs on uptake. In the experiments, U2OS-SAMP1-YFP cells were transfected with siRNA targeting SCARA-3 and -5, in this case by using a commercial transfection reagent, (Lipofectamine RNAiMAX). After knockdown of the SCARA receptors, the cells were treated with CPP:ON complexes. In paper II, siRNA against green fluores-cent protein (which also targets yellow fluorescent protein (YFP)) was deliv-ered using both CADY and s-RxR4. Subsequent analysis of YFP knockdown was performed using FACS.

3.10 Fluorescence MicroscopyOne of the experiments in paper II uses fluorescence microscopy. A tech-

nique that is now an essential tool in life sciences because it allows for visual-ization and identification of cells, sub-microscopic cellular components and fluorescently labeled molecules, today the method is no longer limited by diffraction, as super-resolution microscopy [242] is becoming more common, although that is not used in this thesis it will become a tool in CPP research.

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Fluorescence microscopy enables the study of single molecules as well as allowing for the identification of several target molecules simultaneously. In this thesis, the technique has been applied to study the cellular uptake of labeled CPPs and ON-cargo and to investigate the role played by SCARA in the uptake of PF14-Fluorescein alone or PF14-Fluorescein in complex with Alexa568-labelled siRNA.

3.11 Toxicity AssaysTo exclude that the cell-penetrating and delivering activity of the peptides

in vitro is caused by toxicity, the WST-1 assay was used. The same assay was also used for the fourth paper to exclude that the effects of the added ligands (SCARA inhibitors and GPCR library) on splice-correction were due to toxicity. This assay measures cell viability as a function of mitochon-drial metabolic activity. The activity of the mitochondrial dehydrogenases is measured by their conversion of tetrazolium salts to formazan, cell-viability and cell-proliferation is directly correlated to the amount of formazan dye formed, more cells results in more formazan dye. The dye can be elegantly quantified spectrophotometrically, using UV-absorption at 420 nm, more in-tense staining means more active mitochondrial dehydrogenase. The WST-1 reagent and the formazan product are both water-soluble, which means that measurements can be performed 1-4 h after addition, making it a very rapid and convenient assay compared to the commonly used MTT assay, which requires solubilization of the formazan salt.

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4 Results and Discussion

4.1 Paper I & II SCARAs and CPP Uptake

In Paper I: We show involvement of the scavenger receptor class A for the uptake of PepFect14:splice-correcting oligonucleotide complexes. In

Paper II: We show that the findings from paper I, namely the involvement of the scavenger receptors in uptake, can be expanded to peptides and poly-mers and ON cargo that are different from PepFect14. When we first started characterizing the physiochemical properties of the CPP:ON complexes we found, much to our surprise, that even though the peptide was always in molar excess (for example, at MR 5:1 for PF14, there would be 30 positive charges from the five peptides to the 18 negative charges from the one SCO) there was still a net negative surface charge (ζ-potential) in most dispersion media. We also found that the surface charge was highly dependent on the dispersion medium. In pure water the surface charge was entirely dependent on the ratio of charges between the peptide and the oligonucleotide, being negative at MR3, neutral at MR5 and positive at MR10. However, when the dispersion medium was changed to better mimic the conditions that the complexes would encounter in a cell (like isotonic NaCl or serum-free/serum-containing medium) the ζ-potential became negative for all MRs. As this change in surface charge happens even in the absence of serum, it would be unlikely that the serum proteins are responsible for the change, but that salt concentration and pH is. The net negative surface charge should cause the CPP:ON complexes to be repelled by the negatively charged cell mem-brane and the negatively charged GAGs on the cell-surface.

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We believed that something must have been mediating this interaction, that there was something between the cell-surface and the complexes. Literature searches gave us the SCARAs as potential candidates; because they are known to bind with low specificity to polyanionic ligands (section 1 SCARAs). The simplest conceivable experiment at the time was to use the splice-correction assay and pre-treat the cells with some ligands that com-petitively bind to the receptors. Even at low concentrations of the SCARA ligands, fuciodan, dextran sulfate and poly i, the splice-switching effect of PF14:SCOs was completely abolished in a dose-dependent manner. The next question was of course which, if any, of the SCARA receptors are expressed in the HeLa pLuc705 cells, we performed real-time PCR and found that SCARA-5 and SCARA-3 (with its two splice variants) are expressed whereas some of the others could not be detected.

Once the receptors had been identified the next step was to try to knock them down with siRNA. Knockdown of SCARA-3 and SCARA-5 resulted in an inhibition of the luminescence signal from luciferase by more than 50 %. In order to try to gather more evidence immunofluorescence and trans-mission electron microscopy (TEM) were performed in order to directly vi-sualize changes in membrane association and localization at the cell-surface. The TEM images clearly show the effects of the absence of SCARAs, when the receptors are not available, because of pretreatment with dextran sulfate, CPP:ON complexes are not associated with the cell membrane.

This is the first time that a specific receptor is demonstrated to be in-volved in the uptake of cell-penetrating peptides; in Paper I we demonstrated SCARA involvement using both a number of pharmacological inhibitors and by knocking down the receptor using siRNA and thus abolishing uptake of PF14/SCO complexes. We later identified SCARAs as a receptor for the Pepfect15 and the NickFect class of CPPs.

In Paper II, we went further with this investigation into the SCARA receptors and their impact on the uptake of CPP:ON complexes. The initial results from the first paper cover only one of the several hundred CPPs that have been reported. The whole ensemble of CPPs is, as can be seen from (Section 1.2), a heterogeneous collection of peptides with the common abil-ity to transport cargo into cells. We did not at the time know if the effects we had seen were valid only for some peptides that all derive from the trans-portan peptide, or if what we had discovered was a more general mechanism. We also included two cationic polymers to broaden the scope even further.

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The discovery that SCARAs are involved in the uptake of CPP:ON complexes offers new and interesting information that can be used both in scavenger receptor research as well as in the development of CPP technology. Original research on these receptors identified them in macrophages, later they were identified in many different cell types, where different subtypes were identified in different cells such as smooth muscle cells, epithelial and endothelial cells, splenic dendritic cells, and fibroblasts.

The SCARAs are clearly very promiscuous and bind to all manner of ligands, without any discernible preference for one thing over the other, except maybe for dextran sulfate. At first it could seem as if linking one of these ligands to the CPP, in a manner analogous to the endosomal-escape moieties in PF6 [64] and PF15 [68], would be a possible way to enhance uptake. However, such a modification could diminish the ability of the pep-tides to form complexes with ONs. Because of the opposite charge of CPPs and dextran sulphate, the CPP would lose some of its ability to complex ONs via charge interactions, as some of the charges on the CPP would be neutralized.

4.2 Paper III: CPP Delivery AssayIn this paper we develop an assay that is cheaper, faster and higher

throughput than what we previously used, going from commercially sourced reagents and 24-well plates to homemade reagents and 96-well plates while reducing cost by 95 %. The assay that was developed in this work was born out of necessity. The original intention was to use the library in Paper IV, to determine if stimulation of other cell-surface receptors may have effects other than the inhibitory effect on uptake that saturation of SCARAs has. Discovering any effects would be interesting and discovering a ligand that increased the uptake could eventually lead to an improvement of delivery efficacy. The assumption was that simply scaling the assay based on the treatment volume differences between 24- and 96-well plates would yield a satisfactory experiment. That assumption turned out to be completely incor-rect. The difference in volume is 5-fold and the difference in surface area is approximately 6-fold, whereas there are 4 times more wells in a 96-well than in a 24-well plate. The problem is then what, if any, scaling factor should be used. In the initial attempts to run the library, consisting of 264 ligands, in the 96-well format we used the difference in treatment volume as the scaling factor. That did not yield any usable data. The major problem was that, all of a sudden, there was much more variance within one experiment and ex-periments performed independently yielded sometimes opposite results. This

36

more or less forced the development and optimization of the assay. Initially, the experiments were performed in way that was analogous to that for the 24-well plates. The cells were seeded on the first day, treated on the second day and finally the luciferase measurement was performed on the third day. Two things were quickly realized; first, the commercially sourced reagent is not ideal when using a luminometer without reagent injectors, because the luciferase signal changes by approximately 20 % during the time it takes to read one plate. Second, this was not the only source of variance. The first problem led to the development of a homemade luciferase reagent, which is more stable during measurement. Fortunately this homemade reagent is sig-nificantly less costly; representing a discount of more than 95 % compared to the commercial reagent used previously.

With a more stable luciferase reagent, it became clear what at least one of the additional sources of variance was. Seeding ten-thousand cells on the first day, treating the cells on the next day and measuring 24 hours later caused large row and column effects. These effects are currently not explained, even though attempts at discovering the source of the row effects were made. However these problems are avoidable by seeding fewer cells and treating the cells on the day of seeding. Overall, this assay is successful at yielding experiments with low variation and a good separation between positive and negative controls as evidenced by the excellent Z’ scores.

4.3 Paper IV: GPCR Ligands Influence UptakeWe use the above assay and a library of 264 GPCR ligands to identify

receptor ligands that increase the observed luminescence from luciferase in the splice-correction assay. We also determined that the inhibition of luciferase activity, initially observed in a library screen, was most likely due to toxicity. Three ligands (or about 1 % of the library) have a stimulatory effect on the luminescence from luciferase in the splice-correction assay, two of them target the metabotropic glutamate receptor 5 (mGluR5) and one targets histamine H3. We also investigate 5 ligands that seem to have an inhibitory effect, all of them target estrogen receptors. The observed in-crease in luminescence from luciferase is fairly large and repeatable across multiple experiments. The decrease we observed is disappointingly difficult to separate from toxicity. It is not surprising that high toxicity results in a decrease of luciferase activity, if not for any other reason than that there are fewer viable cells in the treated wells. The over 3-fold increase we observed in luminescence is interesting, not least for the potential to improve the delivery efficacy of CPPs. However, that the increase of luminescence is due

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to the ON being delivered more efficiently is yet to be confirmed. The result can be an unspecific effect of something other than delivery. Stimulating the receptor could trigger a signaling event, which in turn may cause anything from increased transcription to reorganization at the cell-membrane, for example it has been shown that GPCRs can signal from endosomes [243, 244]. The observed effect could be related to the stability of the endosomes. The least exciting possibility is that the identified ligands may simply act to disrupt the endosomes like in the case for other lysosomotropic amines [245].

The increase and decrease in transfection efficacy can also be related to the endocytotic process of caveolae, there are indications that both SCARA [246] and mGluR5 [247] are localized within caveolae on the cell membrane, as are scavenger receptors class B [248], and many other GPCRs [249], as well as the Na+/K+ ATPase [250]. Effects on caveolae that are involved in cell-membrane lipid organization and stability, receptor signaling regulation, and even mechanosensing of the cell membrane [251] could be a lead into what is happening that causes the uptake to change. This study leaves many unanswered questions, about the uptake of CPPs and about the complexities of the workings of transport at the cellular membrane. An illustration of the discussion above can be found in Figure 5.

Figure 5. GPCR internalization is mostly associated with clathrin-coated pits, CPPs can be taken up via multiple endocytotic mechanisms and SRs are also associated with clathrin and caveolae, what role if any the receptors play in endosomal escape has not yet been explored.

ClathrinCaveolae

GPCR

SCARA? ?

CPP:ON

SCARA

Endosome

Biological effect

Cytosol

?

Signaling

Signaling

Endosomal escape

?

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4.4 Summary and ConclusionThis thesis presents three findings; first scavenger receptors are involved

in, if solely not responsible for, the uptake of many types of ONs in complex with a number of different cationic molecules; we have shown this for the peptides developed by us and by others as well as for polymers.

Second, this thesis contains an optimized assay for the delivery of short ONs that will allow for the use of cell-penetrating peptides in high-through-put screening of for example siRNA libraries. Additionally the methods used for evaluation of transfection efficacy and optimization of transfection conditions for CPPs will be much higher throughput using this assay. This method is faster, cheaper and more efficient that previously used methods.

Third, the assay above was used to screen a library of GPCR-ligands, look-ing for changes in uptake of CPPs. From the library, we identified ligands that increase the uptake efficacy as measured by the luciferase-splicing assay.

The long-term goal of this scientific endeavor is to use CPPs to safely de-liver ON-based molecules as therapies. The contribution of this work towards that goal is to try to understand the uptake mechanism of cell-penetrating peptides. Identifying first the SCARAs negative modulators of uptake and then the mGlu5R and Histamine H3 receptors as positive modulators could induce new directions to take. Understanding the uptake should reasonably provide insight into how CPPs can be improved from where they stand today.

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5 Future Outlook

The aim of this work was to clarify the uptake mechanism further, and the first two papers seem to do that, as the involvement of the

SCARAs is quite clear and independent of which peptide or in fact what delivery system is used. Recent studies suggest that it is the nano-particulate form, not the composition of the particles that is important for the recogni-tion and uptake by SCARA receptors [252]. We see similar results in Paper II; the uptake of fluorescently labeled PF14 alone is also inhibited by the SCARA ligands, even when no ONs are present. It is likely that PF14 forms complexes with itself even in the absence of ONs.

The last manuscript suggests the possibility that the mechanism is more convoluted and perhaps affected by other cellular processes. Currently we don’t have a working hypothesis of the complete mechanism. Looking at the normal course of events when a GPCR is activated can provide some suggestions at what to investigate next; one possibility is to look for acti-vation of protein kinases, which in turn activate other cellular responses. Effects on the recruitment of caveolin and trafficking of caveolae should be further investigated. Perhaps the effect we observe using the mGluR5 and Histamine H3 ligands is due of the release of the CPP:ON complex from the endosomes, this could perhaps be visualized using the endosome release assay mentioned earlier [193].

Another aspect that is not yet fully investigated by us, is which endocy-totic pathway PF:ON complexes is using. Studies using different pharmaco-logical inhibitors and smaller cargo than ONs [149, 253] indicate that both the clathrin and caveolin pathways are involved. The investigation using the NickFect family of peptides revealed, using the pharmacological inhibitors

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chlorpromazine and nystatin, that NF1 and NF51 were utilizing different pathways [209]. Because of the overlap between the different pathways (Section 1.4) and because of the overlap of the effects of chlorpromazine [153] it will be difficult to answer definitely which pathway(s) are involved. We knocked down Caveolin and Clathrin light- or heavy-chain using siRNA without finding conditions from which useful information could be drawn. The most recent suggestions are to use siRNA to target the adaptor pro-teins, the cavin proteins and Arf6. Super-resolution microscopy is another possibility to further explore the content of the vesicles that contain the endocytosed CPPs. This in combination with other new techniques like the endosome escape assay, will eventually uncover the hidden machinery that surrounds the uptake of CPPs and when all is revealed it will be easier to design new peptides that take advantage of the new insights to become even better transfection vectors.

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6 Populärvetenskaplig sammanfattning

DNA innehåller all biologisk information som behövs för att åstad-komma alla proteiner i en cell. Olika celler i kroppen utför olika

uppgifter; ögonen ser, lungorna tar upp syre från inandningsluften och hjär-tat pumpar blodet ut till kroppens alla delar. Allt det här arbetet utförs av olika specialiserade proteiner vars utseende, uppbyggnad och funktion be-stäms av våra gener, av vårt DNA. Kostnaden för DNA-sekvensering sjunker snabbare än det någonsin gjort för en tidigare teknologi, jämförelsen som ligger närmast är beräkningskraft i datorer (Moores lag). En dag kommer sekvensering bli lika vanligt som ett blodprov. Med all kunskap om hur generna påverkar sjukdom som redan finns, och som ytterligare utökas när fler sjukdomstillstånd kartläggs, är det naturligt att vilja påverka dem, till exempel genom att stänga av en gen som inte borde vara aktiv, eller tvärtom. För att kunna göra det behöver man föra in oligonukleotid-baserade moleky-ler i cellen, oligonukleotider är uppbyggda av DNA eller RNA. Det största problemet är att cellen är mycket bra på att hålla sådana molekyler på en sida av cellmembranet. För att göra det möjligt för dem att komma in i cel-len behövs någon form av bärare eller transportör. Vi arbetar med en klass av peptider som fungerar just som sådana transportörer. Cell-penetrerande peptider (CPPer) har till skillnad från oligonukleotider positivt elektrisk laddning och kan därmed skapa elektrostatiska komplex tillsammans med dem. Ett sådant komplex kan sedan tas upp av cellen. Innan det här arbetet var det känt att cellerna kunde ta upp komplexen och att det mest troligen skedde genom en process som kallas endocytos. Den processen är det natur-liga sättet för celler att ta upp näring och kommunicera med andra celler. Cellmembranet har negativ laddning och man trodde att den första kontak-ten som inledde upptaget av CPPer var en elektrostatisk interaktion mellan

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dem. Till vår förvåning upptäckte vi i Artikel I att komplexen mellan CPPer och oligonuleotider inte hade positiv laddning. Det i sin tur ledde oss till att tro att det borde finnas någonting i cell-membranet som fungerar som en länk mellan en negativ partikel och det negativa cellmembranet. I Artikel I identifierar vi Scavenger receptor class A som den länken, i alla fall när det gäller en viss peptid och en bestämd typ av oligonukleotid. I Artikel II vi-sar vi att mekanismen som medieras genom Scavenger receptorerna är mer generell och gäller många olika typer av oligonuleotider och flera olika cell-penetrerande peptider samt ett par andra positivt laddade polymerer som även de används för att få in oligonukleotider i celler.

För att göra det enklare, snabbare och billigare att utvärdera CPPers ef-fektivitet som oligonukleotidbärare har vi i Artikel III utvecklat vår metod, till att ha hälften så många steg, ta en dag mindre i anspråk och spara 95 % av kostnaderna, samtidigt som den låter oss mäta 4 gånger så många prov samtidigt.

Denna metod användes sedan i Artikel IV till att undersöka om stimu-lans av den största familjen av receptorer på cellytan, G-proteinkopplade receptorer, kunde påverka upptaget av CPP:oligo komplexen. Ur ett bibliotek med 264 olika substanser identifierade vi tre stycken som var och en ökade upptaget med upp till tre gånger.

Sammanfattningsvis innehåller den här avhandligen en metod som är optimerad för att kunna göra experiment med CPPer snabbare och billigare och samtidigt i större skala. En beskrivning av hur SCARA receptorer är in-blandade i upptag av CPP:oligo komplex samt en indikation på att upptaget ytterligare kan förbättras genom att ta andra receptorer i beaktande.

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7 Acknowledgments

This is the section where I admit that none of the pages in this book would have been possible without Linda my wife, who with her pa-

tience as I commuted back and forth the first four years and always with her love and support made sure that I could finish this, these words are not enough to express the gratitude and love that I feel.

Ülo, my supervisor who can motivate and inspire in ways I would have thought were impossible. Thank you for letting me do this, and thank you for you generosity.

My parents who must not have noticed that I stayed in school for what must have seemed an indefinitely long time instead of getting a “real job”. Thank you for everything.

Markus and Sara thank you for a place to stay on the days where the Swedish railway system refused to cooperate, and thank you for your con-vincing interest in this work.

Staffan. It would have been a lot worse without you, thank you for the company in the lab, the office and the conference bars.

Andrés who can talk more sense and more nonsense than anyone I know. You have been great discussion partner irregardless of the subject. Scientific, non-scientific or just entertaining subjects have all been memorable. Never stop talking.

Jakob who knows all the detailed minutiae of the inner workings of the academic bureaucracy. And without whom we all would have incapable of distinguishing between an excellent meal and a merely good one. Thank you for knowing the difference. And thank you for reading and correcting my thesis.

44

Jonas who is the most ambitious and smartest scientist that I know, he read this thesis and corrected the errors, saw the inconsistent parts found the wrongly used words and made it better, thank you.

Tõnis really put an impressive amount of time and effort into reading this thesis, the input I got made it a lot better and more readable, thank you.

Kristin we started together in the same group, I was lucky enough to get the CPP project while you were lucky enough to get a real neurochemistry project, thank you for always being so kind.

Carmine you are the nicest person I know, we have the same idea about what a synthesis-lab fume hood should look like, but because you are so nice and kind I worry that you won’t be as ruthless as may be necessary towards the unlabeled tubes.

Ying you seem to always be in a hurry, but I am happy that you decided to stay in our group your are good addition to it.

Daniel thank you for all the discussions, stay on the surfboard.

Kariem you started the SCARA project, it is your idea, thank you for letting me be a part of it.

Johan lured me into becoming the computer responsible person, thank you for that.

Moataz thank you for all the discussions.

Marie-Louise whom I consider one of the few “adults” at the department, thank you for knowing everything and making sure that all the inmates at this asylum are well taken care of, we would not know what to do without you.

The master-thesis students that I have supervised, Aimee, Diogo, Chetan, Sini, Maxime, Maria you all taught me what to expect, In most cases what I thought beforehand was not enough, because all of you exceeded my ex-pectations you were all better smarter and in less need of me then I though you would be.

Thanks to all of my co-authors without whom the publications would not exist.

Lastly it is more than likely that I have forgotten some friends, co-workers students and colleagues. Doing a PhD is crazy, fun, interesting and full of freedom in a way that won’t come again, but it is not the work itself but the people that you work with that make it a worthwhile pursuit, so thank you all.

45

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