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C E L L - P E N E T R AT I N G P E P T I D E S A N D O L I G O N U C L E O T I D E S : D E S I G N , U P TA K E A N D T H E R A P E U T I C A P P L I C AT I O N S Andrés Muñoz-Alarcón Cell-penetrating peptides and oligonucleotides: Design, uptake and therapeutic applications Andrés Muñoz-Alarcón Cover: Andrés Muñoz-Alarcón, Cancún, Mexico ©Andrés Muñoz-Alarcón, Stockholm University 2015 ISBN 978-91-7649-085-3 Print: Holmbergs, Malmö 2015 Distributor: Department of Neurochemistry, Stockholm University To my family List of publications This thesis is based on the following papers, which are referred to in the text as paper I, paper II, paper III and paper IV: I Muñoz-Alarcón, A., Guterstam, P., Romero, C., Behlke, M.A., Lennox, K., Wengel, J., EL Andaloussi, S., and Langel, Ü. Modulating Anti-microRNA21 Activity and Specificity Using Oligonucleotide Derivatives and Length-Optimization. ISRN Pharmaceutics, 2012, vol. 2012, Article ID 407154, 7 pages. doi:10.5402/2012/407154 II Lindberg, S.*, Muñoz-Alarcón, A.*, Helmfors, H., Mosquiera, D., Gyllborg, D., Tudoran, O., and Langel, Ü. PepFect15, a Novel Endosomolytic Cell-Penetrating Peptide for Oligonucleotide Delivery via Scavenger Receptors. Int J Pharm 2013, 441,242247. doi:10.1016/j.ijpharm.2012.11.037. III Lindberg, S., Regberg, J., Eriksson, J., Helmfors, H., MuñozAlarcón, A., Srimanee, A., Figueroa, R., Hallberg, E., Ezzat, K., Langel, Ü. A Convergent Uptake Route for Peptide- and Polymer-Based Nucleotide Delivery Systems. J Control Release. 2015, 206:58-66. doi:10.1016/j.jconrel.2015.03.009. IV Muñoz-Alarcón, A., Eriksson, J., Langel, Ü. Novel Efficient Cell-Penetrating Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides. (Manuscript) *These authors contributed equally to this work. Additional publications 1. Muñoz-Alarcón, A., Pavlovic, M., Wismar, J., Schmitt, B. Eriksson, M., Kylsten, P., Dushay, MS. Characterization of Lamin Mutation Phenotypes in Drosophila and Comparison to Human Laminopathies. PLoS ONE, 2007, 2(6): e532. doi:10.1371/journal. pone.0000532. 2. Muñoz-Alarcón, A., Helmfors, H., Webling, K., and Langel,Ü. Cell-Penetrating Peptide Fusion Proteins in Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2013, pp. 397–411. 3. Yu, X. Muñoz-Alarcón, A., Ajayi, A., Webling, K., Steinhof, A., Langel, Ü. And Ström, A-L. Inhibition of Autophagy via p53Mediated Disruption of ULK-1 in a SCA7 Polyglutamine Disease Model. J Mol Neurosci, 2013, 50(3), 586-99. doi: 10.1007/s12031-013-0012-x. Abstract Regulation of biological processes through the use of genetic elements is a central part of biological research and also holds great promise for future therapeutic applications. Oligonucleotides comprise a class of versatile biomolecules capable of modulating gene regulation. Gene therapy, the concept of introducing genetic elements in order to treat disease, presents a promising therapeutic strategy based on such macromolecular agents. Applications involving charged macromolecules such as nucleic acids require the development of the active pharmaceutical ingredient as well as efficient means of intracellular delivery. Cell-penetrating peptides are a promising class of drug delivery vehicles, capable of translocation across the cell membrane together with molecules otherwise unable to permeate cells, which has gained significant attention. In order to increase the effectiveness of cell-penetrating peptide-mediated delivery, further understanding of the mechanisms of uptake is needed in addition to improved design to make the cell-penetrating peptides more stable and, in some cases, targeted. This thesis encompasses four scientific studies aimed at investigating cell-penetrating peptide and oligonucleotide designs amenable to therapeutic applications as well as elucidating the mechanisms underlying uptake of cell-penetrating peptide:oligonucleotide nanoparticles. It also includes an example of a therapeutic application of cell-penetrating peptide-mediated delivery of oligonucleotides. Paper I presents a study evaluating a range of chemically modified anti-miRNAs for use in the design of therapeutic oligonucleotides. All varieties of oligonucleotides used in the study target miRNA-21 and are evaluated using a dual luciferase reporter system. Paper II introduces a novel cell-penetrating peptide, PepFect15, aiming at combining the desirable properties of improved peptide stability and efficient cellular uptake with a propensity for endosomal escape, to produce a delivery vector well suited for delivery of oligonucleotides. The performance of this new cell-penetrating peptide was evaluated based on its delivery capabilities pertaining to splice-correcting oligonucleotides and anti-miRNAs. Paper III investigates the involvement of scavenger receptor class A in the uptake of various cell-penetrating peptides together with their oligonucleotide cargo. Finally, paper IV aims at using cell-penetrating peptide-mediated delivery to improve the efficiency of telomerase inhibition by antisense oligonucleotides targeting the telomerase enzyme ribonucleotide component. Contents List of publications ......................................................................................... vii Additional publications ..................................................................................viii Abstract .......................................................................................................... ix Contents .......................................................................................................... x 1. Introduction ................................................................................................. 1 1.1. Therapeutic oligonucleotides .................................................................................. 1 1.1.1. Antisense oligonucleotides............................................................................. 2 1.1.2. Splice-switching oligonucleotides ................................................................... 3 1.1.3. RNA interference ........................................................................................... 4 1.1.4. miRNA and anti-miRNA ................................................................................. 6 1.1.5. Antisense inhibition of ribonucleoproteins ...................................................... 7 1.2. Oligonucleotide modifications ................................................................................. 8 1.2.1. First-generation oligonucleotide modifications ............................................... 8 1.2.2. Second-generation oligonucleotide modifications ........................................ 10 1.2.3. Third-generation oligonucleotide modifications ............................................ 10 1.3. Delivery of oligonucleotides into cells ................................................................... 11 1.4. Cell-penetrating peptides ..................................................................................... 12 1.4.1. Uptake mechanisms .................................................................................... 13 1.4.2. Endosomal entrapment ................................................................................ 14 1.4.3. Scavenger receptors .................................................................................... 15 2. Aims of the study....................................................................................... 17 3. Methodological considerations .................................................................. 19 3.1. Solid-phase peptide synthesis (Paper II, III and IV) .............................................. 19 3.1.1. Peptides used in this thesis.......................................................................... 20 3.2. Dual luciferase reporter assay (Paper I and II) ..................................................... 20 3.3. Splice-correction assay (Paper II) ........................................................................ 20 3.4. Plasmid delivery (Paper III) .................................................................................. 22 3.5. siRNA delivery (Paper III) ..................................................................................... 22 3.6. Cell lines .............................................................................................................. 22 3.7. Cytotoxicity measurements (Paper II) .................................................................. 23 3.8. Dynamic light scattering and zeta-potential (Paper II, III and IV) .......................... 23 3.9. Scavenger receptor inhibitors (Paper II and III) .................................................... 24 3.10. Fluorescence microscopy (Paper III) .................................................................. 24 3.11. TRAPeze RT telomerase detection (Paper IV) ................................................... 24 3.12. Population doubling (Paper IV)........................................................................... 25 3.13. Cellular senescence assay (Paper IV) ............................................................... 25 4. Results and discussion ............................................................................. 26 4.1. Effects of chemical modifications and truncations of oligonucleotides on activity and specificity (Paper I) ............................................................................................... 26 4.2. Design of a novel cell-penetrating peptide with enhanced oligonucleotide delivery (Paper II) ..................................................................................................................... 28 4.3. Scavenger receptor-mediated uptake of CPP:ON complexes (Paper III) ............. 29 4.4. Cell-penetrating peptide-mediated delivery of telomerase inhibitor oligonucleotides enhances telomerase inhibition (Paper IV) .................................................................. 31 5. Conclusions ............................................................................................... 34 6. Concluding remarks and future perspectives ........................................... 35 6.1. Biological effects of antimiRs delivered by cell-penetrating peptides .................... 35 6.2. In vivo delivery of telomerase inhibitor oligonucleotides ....................................... 35 6.3. Functionalization of PepFects and further improvement of PepFect transfection technology .................................................................................................................. 36 7. Populärvetenskaplig sammanfattning på svenska.................................... 37 8. Acknowledgements ................................................................................... 40 9. References ................................................................................................ 42 Abbreviations 2’OMe RNA AMO AntimiR ASMase BNA CPP CQ DLS DNA EGFP FACS Fmoc GAG HA2 HeLa HSPG HPLC hTERT hTR LF2000 LNA MALDI-TOF MBHA miRNA MO MPO MR mRNA ON ORF QN qPCR PEI PF 2’-O-methyl RNA Anti-miRNA oligonucleotide Anti-miRNA Acid sphingomyelinase Bridged nucleic acids Cell-penetrating peptide Chloroquine Dynamic light scattering Deoxyribonucleic acid Enhanced green fluorescent protein Fluorescence-activated cell-sorting 9-Fluorenylmethyloxycarbonyl Glycosaminoglycan Hemaglutinin 2 Henrietta Lacks Heparan sulfate proteoglycan High-performance liquid chromatography Human telomerase reverse transcriptase Human telomerase RNA template Lipofectamine 2000 Locked nucleic acid Matrix-assisted laser desorption/ionization–time of flight 4-Methylbenzhydrylamine Micro RNA Morpholino oligonucleotides Methylphosphonate oligonucleotide Molar ratio (peptide:oligonucleotide) Messenger RNA Oligonucleotide Open reading frame N-(2-aminoethyl)-N-methyl-N’-[7(trifluoromethyl)-quinoline-4-yl]ethane-1,2-diamine Quantitative real-time polymerase chain reaction Polyethylenimine PepFect PF14-Fl pDNA piRNA PLO PN PNA PO PolyC PolyI PS RISC RNA RNAi RNP SA-β–Gal SCARA SCO siRNA SPPS SR SSO SCO s-RxR4 UNA WST-1 YFP 5,6-carboxy-fluorescein-labeled PepFect14 Plasmid DNA Piwi-interacting RNA Poly-L-Ornithine Phosphoramidite Peptide nucleic acid Phosphodiester Polycytidylic acid Polyinosinic acid Phosphorothioate RNA-induced silencing complex Ribonucleic acid RNA interference Ribonucleoprotein Senescence-associated β–galactosidase Scavenger receptor class A Splice-correcting oligonucleotide Small interfering RNA Solid-phase peptide synthesis Scavenger receptor Splice-switching oligonucleotide Splice-correcting oligonucleotide Stearyl-RxR4 Unlocked nucleic acid 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5tetrazolio]-1,3-benzene disulfonate Yellow fluorescent protein 1. Introduction The genome is a vast repository of genetic information that provides the information required by our cells to produce all the essential proteins needed to sustain life. Our DNA not only contains the blueprints for the proteins produced by our cells but also regulatory elements that direct and adjust the levels and nature of the myriad of biomolecules present. Aberrantly regulated or damaged genes can cause pathological states due to disruption of various biological processes [1,2]. In some instances, these genetic aberrations are inherited insofar as they are present in the germline cells but in other instances external influences from pathogens can cause disease for example via production of foreign proteins or, through harmful alterations of the regulations of the biological processes in our cells. As a result of our improved understanding of the information contained within the genome, new venues of both research and clinical applications have presented themselves. Therein lies a tremendous potential to treat many pathological conditions both inherited as well as acquired by use of molecules capable of repairing or adjusting the regulation of our biological processes. The obstacles to a full realization of the promise of gene therapy and other molecular genetic-based therapeutics present a tripartite challenge. First, treatment of any pathological disorder requires a solid understanding of the molecular mechanisms of pathogenesis and appropriate therapeutic targets. Secondly, non-toxic, stable molecules with the capacity to interact with and modulate genetic elements with an appropriate efficiency and specificity are required. The third and final challenge is to enable the efficient delivery of the therapeutic molecules. 1.1. Therapeutic oligonucleotides Oligonucleotides (ONs) comprise a class of versatile biomolecules with tremendous pharmacological potential capable of interaction with, and modulation of the activity of, nucleic acids. The members of this group of molecules are short strands of nucleic acids commonly chemically modified to enhance their biological properties. They are able to affect biological processes on several levels. They can be designed to interfere with transcription, splicing and translation either directly by targeting complementary sequences of 1 DNA, mRNA or indirectly by targeting other regulatory RNA molecules [36]. In some instances they may also be used to target complementary sequences within ribonucleoproteins (RNPs). As modulators of gene regulation they are indispensible for gene therapeutic application and research. 1.1.1. Antisense oligonucleotides Antisense ONs have been used for many years to modify the expression of specific genes both in vivo and in vitro [7]. Antisense ONs are short singlestranded nucleic acids consisting of either natural DNA, RNA or chemically modified nucleic acid analogues. By virtue of mainly Watson-Crick base pair hybridization, the ONs target complementary sequences of DNA or RNA and suppress expression. Two basic approaches exist when using antisense ONs (Figure 1). Antigene strategies employ the use of antisense ONs targeting regions of genomic DNA [3]. In the antigene approach, transcription of the targeted genomic regions is blocked by the annealing of the antisense ON. The second strategy, the one that is most commonly just referred to as antisense strategy, DNA (A) Transcription RNase H 40S mRNA (B) (C) 60S Translation Figure 1. Antisense strategies. Antigene ONs blocks transcription of targeted regions of the genomic DNA (A). Regular antisense ON inhibits translation of targeted mRNA molecules via activation of RNase H mediated degradation (B), or by blocking interaction with the ribosomes (C). 2 targets RNA. The targeted RNA is generally mRNA. Antisense ONs targeting mRNAs block translation either by blocking the mRNA interaction with the ribosomal translation machinery, or by eliciting RNase H-mediated degradation. The RNase H–endonuclease exclusively cleaves RNA when it is hybridized as a heteroduplex with DNA. Previous studies have indicated that antisense ON capable of eliciting RNase H-mediated degradation of target RNA sequences are much more potent than those that are incapable of recruiting RNase H [8,9]. Many synthetic nucleic acid analogues are unable to recruit RNase H and thus design of potent ONs often requires synthesis of ONs of mixed chemical composition. However, enzymatic degradation of targeted RNA molecules may not always be appropriate depending on the application and its desired outcome. For example, antisense ONs design to correct aberrant splicing by masking cryptic splice sites derived from mutation should not activate RNase H since intact mRNAs are required for splicing and subsequent translation of the “corrected” mRNA transcript. 1.1.2. Splice-switching oligonucleotides In addition to suppression of gene expression, antisense technology can be used in order to manipulate the cellular splicing machinery to circumvent loss- or gain-of-function mutations that can otherwise cause pathological states (Figure 2). Cryptic splice sites, generated by mutations, which lead to aberrant splicing, can be targeted by ONs complementary to the undesired splice site. The ON anneals to the target sequence and masks the splice site, re-directing the splicing machinery to the proper splice site. In other instances, where mutation causes deletions rendering parts of gene product defective, ONs can be used to mask splice sites to “skip” affected exons yielding a Spliceosome Pre-mRNA Alternative splicing Figure 2. Splice-switching oligonucleotides. Oligonucleotides with complementary sequences (red) can mask splice sites to redirect the spliceosomes to alternative splice sites. 3 slightly shorter but more functional protein. ONs that re-direct splicing are commonly referred to as splice-switching ONs (SSOs), splice-correcting ONs (SCOs) and exon-skipping ONs. These types of ONs are the focus for several studies aiming to find therapeutic strategies to treat genetic disorders such as β-thalassemia, Becker and Duchenne muscular dystrophy [10,11]. 1.1.3. RNA interference The term RNA interference (RNAi) refers to an important regulatory pathway fundamental in eukaryotic cells. Investigated thoroughly in nematodes by Fire et al. who were awarded the Nobel Prize in Medicine 2006 for their pioneering work, RNAi encompasses a range of post-transcriptional mechanisms that use small non-coding RNA molecules to direct gene silencing of specific genes. RNAi can be mediated through a variety of effectors including small interfering RNA (siRNA), microRNA (miRNA), short hairpin RA (shRNA), piwi-interacting RNA (piRNA). Among all the known RNAi effector molecules, siRNAs and miRNAs are currently the best studied (Figure 3). Both siRNA and miRNA are short fragments of RNA, derived from longer double-stranded RNA that is enzymatically processed via interaction with the RNase III-like endoribouclease Dicer. Dicer, digests the double-stranded precursor RNA into short (20-25 nucleotides) with 3’ overhang [16]. The resulting regulatory RNA molecules, whether synthetic or endogenous, then integrates into a multiprotein complex referred to the RNA-induced silencing complex (RISC) [17]. Subsequently, RISC targets mRNA sequences with full or partial complementarity to its integrated siRNA or miRNA. RISC can either block translation or cleave the target RNA sequences. Members of the Argonaute (AGO) protein family constitute a core active component of RISC that catalyzes RNA cleavage [18,19]. Which inhibitory mechanism is employed is decided by the level of complementarity between the guiding strand of the integrated RNAi effector inside RISC and its target. Perfect complementarity between the target sequence and the RNAi effector sequence promotes degradation, whereas partial complementarity induce translational suppression [20]. A common difference between siRNAs and miRNAs is that while, siRNAs usually have perfect complementarity to target sequences, miRNAs usually only exhibit partial complementary via the seed region, 6-8 nucleotides at the 5’ end of the miRNA strand [21]. The imprecise matching between miRNAs and their target mRNAs enable an individual miRNA sequence to potentially regulate a large repertoire of mRNAs. Gene silencing resulting from introduction of exogenous siRNA is often transient due to RNA degradation and dilution of effector molecule concentration as a natural consequence of cell proliferation. Expression vectors producing shRNAs capable of maturing into functional siRNA after under4 going processing by endogenous Dicer provides an efficient strategy to induce RNAi effects over longer periods of time. PiRNAs comprise yet another class of RNAi effector molecules. Longer than siRNAs and miRNAs, piRNAs are usually 25-33 nucleotides in length and depend on different nucleases for their biosynthesis. The piRNAs act to protect germline cells from mobile genetic elements in both vertebrates and invertebrates. These RNA molecules interact with PIWI-domain proteins, members of the AGO protein family and silence genetic elements such as transposons or act as components of RISC [15,22]. The wide range of various RNAi effector molecules can be used experimRNA (A) RISC mRNA degradation mRNA (B) RISC mRNA degradation or translational block mRNA (C) RISC Translation Figure 3. Sample RNAi effector mechanisms. RISC guided by siRNA complementary to target mRNA mediates degradation (A). RISC guided by miRNA with perfect or partial complementarity to target mRNA mediates either degradation or translational blocking respectively (B). AntimiRNA (red) interferes with the miRNA component of RISC, preventing degradation or translational block (C). 5 mentally for laboratory research and for the development of novel therapies for disease treatment. 1.1.4. miRNA and anti-miRNA A miRNA is a short (generally around 22 nucleotides long), endogenous RNA that directs posttranscriptional regulation of gene expression vital for many developmental and cellular functions. MiRNAs interact with one or several RNA transcripts possessing partial or full complementarity and inhibit translation by blocking target mRNA or by invoking a RISC-mediated degradation of the mRNA. As a consequence of their widespread regulatory function, many of which involve tumor suppression mechanisms, miRNA dysregulation has been implicated in the pathogenesis of a multitude of cancers in addition to human developmental disorders and diseases [23-25]. The miRNAs are potentially very interesting both as targets and as bioactive agents in future therapeutic applications. Previous studies have shown miRNAs to possess the ability to regulate multiple functionally related mRNAs, such as sets of metabolic genes, a powerful feature that may enable miRNA-based therapeutics that circumvent redundant mechanisms that might otherwise bypass single inhibited targets. Manipulation of miRNAs utilizing synthetic ONs presents an approach to both elucidate mechanisms underlying miRNA regulation and discover novel therapies for many pathological conditions [26-29]. Commonly referred to as anti-miRNAs (antimiRs) or anti-miRNA ONs (AMOs), these ONs have been synthesized with various chemical modifications aimed at improving potency, specificity and stability. 6 1.1.5. Antisense inhibition of ribonucleoproteins Antisense can be used to inhibit or alter the functions of RNPs. This is achieved by specifically targeting the RNA component of the RNP. An interesting example is telomerase. Telomerase is the enzyme responsible for maintaining the telomeres in nearly all eukaryotic cells [30]. It is a RNP that utilizes a short sequence within its RNA subunit as the template for reverse transcription, synthesizing d(TG)-rich repeats [31] (Figure 4). The enzyme can be inhibited using ONs directed against the human telomerase RNA template (hTR) component. The hTR contains a ribonucleotide component Figure 4. Mechanism of telomerase (adapted from Sinauer Associates). Telomeres shorten progressively during each replication (A). Telomerase utilizes its RNA component as a template for DNA and adds telomeric repeats to the chromosomal 3’ termini (B). The original length of the chromosomal DNA is restored (C). Telomerase inhibitor ONs disables telomere restoration by blocking the RNA component of telomerase. 7 that serves as a template for telomere replication via the catalytically active human telomerase reverse transcriptase (hTERT) domain. Antisense ON inhibition of telomerase results in a progressive shortening of the telomeres that in some cases induces apoptosis. Telomerase is believed to enable limitless replicative potential and the fact that most forms of cancer display telomerase activity [32], whereas most normal cells don’t, makes telomerase an attractive target for anticancer therapy. Currently, a lipid-conjugated thiophosphoramidate ON is undergoing clinical Phase II trials. The ON referred to as GRN163L [33], developed by Geron Corporation will be part of the focus in paper IV. 1.2. Oligonucleotide modifications Therapeutic applications of ONs face several major obstacles. The limitations of natural unmodified nucleic acids have spurred the development and synthesis of novel synthetic ON analogues. Currently, a plethora of distinct chemical modifications that, in addition to overcoming issues with stability, impart additional desirable properties of enhanced activity, specificity and membrane-permeability [34-37]. Modified synthetic nucleotide monomers can be incorporated selectively in limited amounts to produce “gamers”, ONs of mixed composition, or used exclusively to create a uniformly modified synthetic ON. A selection of synthetic nucleotide analogues is presented in Figure 5. 1.2.1. First-generation oligonucleotide modifications The first generation of modified ONs is represented mainly by nucleic acid backbone modifications such as methylphosphonate ONs (MPOs) and phosphorothioate (PS) ONs, the latter often referred to as s-oligos. MPOs, non-charged nucleic acid analogues, in which a non-bridging oxygen atom is substituted by a methyl group at each phosphorous in the ON strand, were among the first chemically synthesized modified ON [38]. Although displaying excellent stability, MPOs suffered from significantly lower affinity to target DNA or RNA sequences compared to natural DNA [39]. Furthermore, the lack of charges reduced the solubility as well as cellular uptake. Additionally, MPOs did not activate RNase H, which severely restricted their use especially in antisense ON applications [40-44]. 8 Figure 5. A selection of synthetic nucleic acid analogues. 9 PS-ONs are charged nucleic acid analogues, in which a sulphur atom replaces a non-bridging oxygen atom at each phosphorous in the ON chain. Their excellent nuclease stability and relative ease of synthesis makes them one of the most widely studied type of ON. Additional modifications of the pentose ring can be combined with PS-linkages to enhance cellular uptake in addition to further improving stability [45,46]. In contrast to MPOs, PS-ONs are highly soluble and possess the capacity to trigger RNase H activity, rendering them highly efficient for antisense applications. PS-ONs do have several disadvantageous properties. Similarly to MPO, PS linkages have a duplex de-stabilizing property (although minor compared to that of MPOs). Perhaps more importantly, PS ONs exhibit sequence-independent effects deriving from a length-dependent high affinity for various cellular proteins [47-50]. However, PS-modifications have been used and continue to be highly used in the design of ONs in clinical trial settings. 1.2.2. Second-generation oligonucleotide modifications Second-generation ONs attempt to address various problems inherent to the first generation of nucleic acid analogues. The members of this new generation of ONs are nuclease-resistant and hybridize to their target DNA or RNA with high specificity and affinities higher than natural phosphodiester (PO) ONs and first-generation ONs such as MPOs and PS-ONs. Secondgeneration ON modifications commonly involve substitution of the hydrogen of the 2’-position of ribose by an O-alkyl group. Two examples are 2’O-methyl (2’OMe) RNA and 2′-O-(2-methoxy)ethyl ONs. The 2’-OMe ribonucleotides are modified with a methyl group at their 2’-OH residue of the ribose molecule [51]. The 2’-O-alkyl modifications provide improved resistance to hydrolysis and nuclease degradation, as well as enhanced thermostability of duplexes formed with complementary strands [52]. ONs with 2’O-alkyl insertions are not readily recognized by many enzymes, which is both seen as an advantage in form of nuclease resistance, and a disadvantage, in the sense of a slightly limited utility. Nevertheless, even though their inability to induce RNase H activity, these ONs have still been shown to be potent antisense ONs [53]. Overall, the second-generation ONs have been reported to display higher affinity, increased nuclease stability, improved half-life, better uptake properties and lesser toxicity as compared to first-generation ONs [54]. 1.2.3. Third-generation oligonucleotide modifications Continuing the development of nucleic acid analogues, third generation ON include but are not limited to bridged nucleic acids (BNAs), peptide nucleic acids (PNAs), morpholino ONs (MOs), Unlocked nucleic acid (UNA) and phosphoramidites (PNs) (Fig. 1). Many, if not most third-generation ONs do 10 not possess natural phosphate-ribose backbones. PNAs are nucleic acid analogues that for very stable duplexes or triplexes with single- or doublestranded DNA or RNA [55,56]. Structurally, they possess an uncharged polyamide backbone with nucleobases attached via methylene carbonyl linkers [55,57]. The PN-ONs, in which an amine group substitutes the oxygen at the 3’ position on ribose, are able form very stable duplexes with complementary RNA and DNA [58,59]. PN-ONs have been used in several studies due to their amenity to antisense applications, exhibiting high specificity and specific antisense activity both in vitro and vivo. Although they do not induce any RNase H activation, the involvement of another uncharacterized nucleolytic enzyme has been suggested [60,61]. MOs possess several properties considered desirable for antisense purposes. The modification consists of a replacement of the deoxyribose moiety by a morpholine ring, and the charged phosphodiester linkage is replaced by an uncharged phosphorodiamidate linkage [62]. MOs display a very high stability in biological systems and efficiency in antisense [63-65] though MOs do not elicit any RNase H activation. Furthermore, due to the absence of charge, MOs do not form complexes with cationic delivery reagents. BNAs consist of a group of ribonucleic acid analogues with constrained structure. BNA monomers contain a bridged structure synthetically incorporated at the 2’, 4’-position of the ribose. The first and widely known BNA is locked nucleic acid (LNA). LNA monomers, like other BNAs, are essentially nuclease-resistant and significantly enhance the hybridization properties of ONs [34,66,67]. BNA and other third generation ON technology has successfully been implemented in several studies, including use in antimiR strategies (in addition to antisense applications) allowing as short as 8 nucleotide long seed region LNA antimiRs to effectively target and regulate and range of miRNAs [68-70]. UNA, also known as 2’,3’-seco-RNA, is an acyclic RNA mimic that enables modulation of ON affinity and specificity [71,72]. In stark contrast to the structural rigidity of BNAs, UNAs acyclic structure grants the molecule enhanced flexibility. Insertion of UNA monomers into an ON sequence greatly reduces the hybridization properties of the ON in an additive manner allowing for gradual decrease of thermostability by introduction into DNA or RNA ONs [73]. 1.3. Delivery of oligonucleotides into cells The therapeutic potential of ONs and other macromolecules are largely hampered by their inability to cross the lipid bilayers of cellular membranes. Consequently, with or without the aid of delivery, in order to exert any biological effect, any genetic material administered must be able to efficiently 11 translocate across the plasma membrane. Absent any delivery method, simple addition of naked ONs or plasmid DNA (pDNA) to cells is a very inefficient way to introduce genetic material into cells as the stability is usually poor and the cellular uptake very low. ONs alone enter cells mainly by endocytosis [44,74,75]. Various methods have been developed and employed in order to efficiently transfer genetic material into cells in vitro and in vivo. Physical methods include rapid injection of nucleic acids dissolved in appropriate buffer solution, electroporation, and ballistic gene delivery (a.k.a. “gene-gun delivery”) [76-78]. Other methods, involve the formation of nanoparticles consisting of the genetic material associated with a delivery vector. The carrier molecules can be inorganic, such as nucleic-acid-coated gold nanoparticles and calcium phosphate that condenses nucleic acids into particles more readily taken up by cells, or organic delivery molecules. Popular organic transfection reagents include cationic lipids, such as the commercial transfection reagent Lipofectamine 2000 (LF2000), as well as viral-based delivery vehicles and cellpenetrating peptides (CPPs). Cationic liposome formulations are commonly used for in vitro delivery of ONs. The liposome encapsulate the ON and protect them from nuclease degradation in addition to facilitating their cellular entry. However, their utility in vivo is severely limited due to their toxicity and a marked sensitivity to the presence of serum [79]. Viral delivery vectors are often favored for their efficiency yet suffer from persistent drawbacks related to potential immunogenicity, insertional mutagenesis, and reversion into pathogenic viruses [80-83]. Additionally, biosynthesis of virions is generally expensive, viral tropism varying and the size of delivered nucleic acids limited by the size of the viral capsid [84]. 1.4. Cell-penetrating peptides Since their initial discovery over two decades ago cell-penetrating peptides (CPPs) have attracted significant attention as molecular delivery vectors with great therapeutic potential [85-87]. The term CPP encompasses a heterogeneous class of short and most commonly either polybasic and/or amphipathic peptides [88-90]. One of their defining attributes is their ability to translocate across cellular membranes. Additionally, they are able to co-translocate associated molecules otherwise unable to enter cells. There is no unified classification of CPPs. They are commonly divided into groups on the basis of origin (e.g. natural, synthetic or chimeric), linkage to cargo (covalent and non-covalent), charge or amphipathicity. Naturally derived peptides, include peptides originating from naturally occurring proteins such as the HIV-1 Tat protein and the antennapedia homeobox protein [85,86]. Synthetic CPPs on the other hand include completely artificial peptides such as poly-arginines [91]. Chimeric CPPs include peptides that 12 derive part of their amino acid sequence from two or more proteins or peptides [92]. Classification based on amphipathicity divides CPP into primary amphipathic, secondary amphipathic and non-amphipathic peptides [93]. Primary amphipathic CPPs display amphipathicity in their primary structure, whereas the amphipathic properties in the secondary amphipathic CPPs are revealed in the secondary structure. An assortment of CPPs is presented in Table 1. To this date, a variety of cargos have been delivered into cells ranging from antigenic peptides, PNAs and ONs to full-length proteins, nanoparticles and liposomes. CPP Amino acid sequence Penetratin RQIKIWFQNRRMKWKK Tat (48-60) GRKKRRQRRRPPQ Transportan GWTLNSAGYLLGKINLKALAALAKKIL-NH2 TP10 AGYLLGKINLKALAALAKKIL-NH2 PF3 Stearyl-AGYLLGKINLKALAALAKKIL-NH2 Poly-arginine Rn SAP(E) (VELPPP)3 RxR4 RxRRxRRxRRxR-NH2 Table 1. An assortment of CPPs. Ref. [85] [86] [92] [94] [95] [96] [89] [97] 1.4.1. Uptake mechanisms The cellular uptake mechanisms of CPPs and their associated biomolecular cargo has been a matter of controversy. Initially, the mechanisms responsible for CPP internalization was considered to be receptor-independent and mediated via electrostatic interactions between cationic charges of CPPs and the negatively charged plasma membrane [85]. This lead to a common acceptance that endocytosis was not involved. However, later studies revealed critical flaws in the experiments that used high concentrations of fluorophore-labeled CPPs and cell fixation procedures. This led to the consensus regarding CPP uptake to be challenged. Perhaps most notably, one study demonstrated that cell fixation could generate artifactual uptake of CPPs [98]. Currently, two distinct mechanistic pathways predominate [98-100] the direct translocation pathway and the endocytic pathway, the latter being the main entry route for several distinct type of CPP-associated nanoparticles [101-105]. Several models for the direct translocation of peptides have been proposed such as the pore-formation model and the inverted micelle model [106-109]. Endocytic uptake of CPPs and CPP-nanoparticles can occur via several different pathways such as clathrin-mediated endocytosis, caveolaemediated endocytosis, macropinocytosis and combinations thereof [110113]. Recent studies have suggested that uptake of CPPs is affected by inter13 action with extracellular components such as heparin sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) [114], though other studies implies that while these components may facilitate the initial adherence of CPPs to cells, they are not essential for the actual uptake [115-119]. For direct translocation of cationic peptides, investigations by Verdurmen et al. suggests direct penetration requires acid sphingomyelinase (ASMase)induced formation of ceramide [120]. In that study, ASMase was shown to translocate to the outer leaflet of the plasma membrane upon interaction between CPP and the cell membrane. Ultimately, the specific uptake mechanisms used by a cell can vary depending on cell type, CPP, concentration of peptide and cargo, as well as the type of intramolecular linkage used between CPP and cargo. It is also possible that more than one mechanism occurs [113]. 1.4.2. Endosomal entrapment ONs and CPP:ON nanoparticles taken up by cells via endocytosis tend to accumulate in the endosomal/lysosomal compartments. There most of the particles are either degraded or expelled via exocytosis. Thus, the effective concentration of biomolecules able to exert any biological effect on the biological processes of targeted cells is drastically lower than the administered dose. This delivery problem presents a significant bottle-neck as only a small fraction manages to escape endosomal entrapment. Several strategies have been used in order to facilitate endosomal escape. Strategies that mimic those used by viruses, certain plants and bacterial toxins [121-123] have been employed with some success. For example, adding the hemaglutinin 2 (HA2) domain derived from the influenza virus to peptide delivery vectors is one approach that has been tested. HA2 and other pH-sensitive fusogenic peptides undergo conformational changes during the acidification processes of the endosomes and facilitate endosomal escape through interactions with the endosomal membrane. Other strategies make use of endosomolytic reagents such as chloroquine (CQ) and CQ-like analogues that are believed to mediate osmotic swelling and subsequent rupturing of endosomes [124]. EL Andaloussi et al. successfully designed a CPP, PepFect6 (PF6), with endosomolytic properties facilitating endosomal escape of its associated cargo via covalent linkage of CQ-like analogues to the peptide backbone via a lysine tree [101]. Overcoming the challenge presented by undesired accumulation of CPPs and ONs in endosomal compartments will significantly enhance the effectiveness and therapeutic potential of nonviral delivery vector strategies. 14 1.4.3. Scavenger receptors Recent physicochemical studies on CPP:ON nanoparticles showed that they have a net negative charge. This goes against the expectations for cationic CPP complexes that can directly interact with the negatively charged plasma membrane. More specifically, the study by Ezzat et al. suggested that the uptake mechanism of such nanoparticles be receptor-mediated [125]. Scavenger receptors (SRs) are members of a group of promiscuous membrane-spanning receptors that recognize and mediate uptake of extracellular modified low-density lipoproteins and anionic macromolecules [126-129]. Uptake occurs via induction of endocytosis, resulting in the internalization of the receptor-ligand complex. SRs are broadly divided into 8 classes (A-H) based on structural similarities and conserved domains. The study conducted by Ezzat et al. suggests class A scavenger receptors (SCARA), more specifically subtypes 3 and 5, play a major role in the uptake of CPP:ON nanocomplexes. SCARAs are trimeric type II transmembrane glycoproteins with several structural features being shared within the group as well as with members of other classes of SRs [130] (Figure 6). Figure 6. Schematic representation of five SCARA subtypes. Adapted from Canton et al. [131]. 15 Previously, SCARAs have been shown to take up nucleic acids [132-136] and ON-functionalized gold-particles [137]. Initially discovered in macrophages, they have since been detected in many other cell types such as endothelial cells, fibroblasts, epithelial cells and smooth muscle cells [136,138]. SCARAs with their ability to recognize and mediate entry of nucleic acids are important to this thesis as several of the studies included pertain to elucidating the relevance of SCARAs in the uptake of CPP:ON nanoparticles. The ability of SRs to discriminate between certain sulfated polysaccharides (fucoidan and dextran sulfate vs. chondroitin sulfate) as well as between polyribonucleotides such as polyinosinic acid (polyI) and polycytidylic acid (polyC) is utilized to set up SR inhibitor and inhibitor control experiments. 16 2. Aims of the study This thesis focuses primarily on ways to improve upon therapeutic ON methodology and the design and synthesis of novel peptide delivery vectors. It includes the use of ONs designed against various targets including miRNAs, mRNAs as well as ribonucleoproteins. General aims:  To develop more efficient CPPs for delivery of ONs.  To evaluate various nucleotide modifications amenable to CPPmediated delivery in both therapeutic and research applications.  To gain an improved understanding of mechanisms underlying uptake of CPP:ON nanoparticles.  Explore possible therapeutic applications for ONs delivered by CPPs. Specific aims: Paper I: In this study we evaluated various modifications of an antimiR targeting miRNA-21 on the basis of their effects on ON activity and mismatch discrimination in vitro. The main objective was to screen for ON modifications that allowed for simultaneously high potency and specificity for potential delivery with CPP. Paper II: In this paper, we aimed at enhancing cellular delivery of ONs, more specifically SCOs and antimiRs, through rational modification of CPP vector PepFect 14 (PF14). The study aimed to assess whether the addition of an endosomolytic modification would significantly improve the delivery of ONs and whether or not the modification would alter the SCARA-dependent uptake mechanism of the CPP:ON nanocomplexes. Paper III: In light of previous studies suggesting a SCARA-dependent mechanism for uptake of PF CPP:ON complexes, one of the main aims of this study was to assess whether SCARA was also involved in the uptake of other types of CPPs in complex with ONs. Additionally, we investigated 17 whether SCARA could interact with, and mediate uptake of PF14 nanoparticles without either ONs or serum proteins being required. To broaden the scope of the study, we also included two cationic polymers used for transfection, PLO and PEI. Paper IV: The main aim of this study was to evaluate the potential for therapeutic applications of PF CPPs, combining the previously designed PepFect15 (PF15) (paper II) with the lessons learned regarding ON modifications. More specifically, we aimed at enhancing the efficiency of a class of ONs used in clinical trials to treat cancer by inhibiting telomerase. 18 3. Methodological considerations The methods used in this thesis are described in each contributed paper; consequently, the theoretical and methodological aspects will not be depicted in detail here. The selections below are valid for all papers if no special emphasis is mentioned. 3.1. Solid-phase peptide synthesis (Paper II, III and IV) The peptides used in this thesis were synthesized through solid-phase peptide synthesis (SPPS). SPPS was introduced by Bruce Merrifield in 1963 [139] and resulted in a paradigm shift in the peptide synthesis community, replacing liquid-phase peptide synthesis as the mainstream technique for peptide synthesis. SPPS is based on a principle of iterative cycles of amino acid coupling and de-protection. The synthesis starts at the C-terminus, which is covalently anchored to a resin, an insoluble polymer acting as support during the synthesis procedure. The desired peptide is then built sequentially through successive addition of amino acids from C-terminus to Nterminus in a step-wise manner. As the growing peptide chain is linked to an insoluble support, excess reagents and by-products can easily be removed by repetitive washing steps with the appropriate solvents. Upon completion of the peptide sequence the desired peptide is cleaved from the resin. All peptides synthesized for use in the experiments described in this thesis were produced utilizing 9-fluorenylmethyloxycarbonyl (Fmoc) protection of the α-amino groups. 4-methylbenzylhydrylamine (MBHA) and H-RinkAmide-ChemMatrix resins were used as insoluble supports, producing amidated C-termini as result. For PF15, four trifluoromethylquinoline-based derivated (QN) were introduced via a succinylated lysine tree on a lysine in the CPP backbone. The peptides were cleaved from the resin using 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS) and 2.5% H20. The crude peptide products were then freeze-dried and then purified using semipreparative reverse-phase high performance liquid chromatography (HPLC). Purified product was subsequently subjected to analysis using matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer. 19 The peptides used in this thesis are listed in below. The PF CPPs are based on a stearylated form of TP10. N-terminal stearic acid modifications of CPPs were first utilized by Futaki et al. [140] to enhance the transfection efficiency of arginine-rich peptides. CADY was kindly provided by Dr. Gilles Divita and Dr. Sebastien Deshayes. 3.1.1. Peptides used in this thesis CPP PF6 PF14 PF15 dPF14b PF14-Fl s-RxR4(d) CADY(e) Sequence Stearyl-AGYLLG(Ka)INLKALAALAKKIL-NH2 Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2 Stearyl-AGYLLG(Ka)LLOOLAAAALOOLL-NH2 Stearyl-agyllgklloolaaaalooll-NH2 Stearyl-AGYLLG(Kc)LLOOLAAAALOOLL-NH2 Stearyl-RxRRxRRxRRxR-NH2 Ac-GLWRALWRLLRSLWRLLWRA-Cya Ref. [101] [104] [141] [142] [143] (a) Four trifluoromethyl-quinolinyl moieties coupled via a lysine tree. (b) Small letters denoted D-amino acids. (c) 5,6 carboxyfluorescein. (d) x denotes 6-aminohexanoic acid. (e) Ac indicates an acetyl moiety. Cya denotes a cysteamide residue. 3.2. Dual luciferase reporter assay (Paper I and II) The dual-luciferase assay utilizes a psiCHECK-2 plasmid (Figure 7). The psiCHECK-2 plasmid contains both a Firefly luciferase open reading frame (ORF) and an ORF encoding Renville luciferase. The plasmid used in paper I is modified with a miR21-binding site inserted into the 3’ UTR of the Renilla luciferase gene. Endogenous miR21 effectively blocks Renilla expression through binding to the 3’ UTR of the gene [144]. The presence of excess anti-miR21 ON inhibits miR21, causing a de-repression of Renilla luciferase. Consequently, the luminescence generated by expression of Renilla correlates with the activity of the anti-miR ON. This method utilizes the constitutively active Firefly luciferase as an internal standard. 3.3. Splice-correction assay (Paper II) The splice correction assay originally developed by Kole et al. [145] provides a useful quantitative assessment of cellular delivery efficiency of antisense ONs (Figure 8). The assay utilizes HeLa cells stably transfected with pLuc705, a plasmid containing a luciferase-encoding gene interrupted by a 20 SV40 early enhancer/promoter T7 hRluc Ampr psiCHECK-2 627 3 bp Multiple cloning region Synthetic poly(A) SV40 Late poly(A) HSV-TK promoter hluc+ Figure 7. PsiCHECK-2 vector map (Vector NTI). The ORFs for Renilla and Firefly luciferase are denoted hRluc and hluc respectively. L Figure 8. Splice-correction assay. A plasmid carrying a luciferase ORF with an insertion of intron 2 from β-globin pre-mRNA containing an aberrant splice-site that, unless masked by a SCO generates an aberrant luciferase mRNA. Adapted from Kang et al. [145]. 21 mutated intron from a β-thalassemic globin gene. The intronic mutations activate a cryptic splice site, which produces non-functional luciferase. Masking the mutated site with an antisense ON re-directs the splicing machinery to produce functional luciferase. Subsequent luminescence allows for quantification using a luminometer. 3.4. Plasmid delivery (Paper III) A range of CPPs and two cationic polymers were used to deliver the pGL3 plasmid, which contains a luciferase ORF. Plasmid delivery was performed by non-covalent complexation with the selected CPPs and polymers. Cells were pre-treated with or without SCARA inhibitors and their respective controls to assess the role of SCARA played in the uptake of CPPs and polymers with their associated pGL3 plasmid cargo. In addition to pGL3, plasmids containing SCARA subtype 3 and 5 ORFs were used for overexpression experiments to study the effects on uptake. In those experiments, commercial transfection reagent LF2000 was used to transfect HeLa cells prior to addition of CPP- or polymer-pGL3 complexes. 3.5. siRNA delivery (Paper III) Paper III includes experiments where siRNA was used to study the potential effects on uptake caused by knockdown of SCARA 3 and 5. In these studies, U2OS-SAMP1-YFP cells were transfected with siRNA targeting SCARA 3 and 5, using commercial transfection reagent Lipofectamine RNAiMAX, prior to treatment with CPP:ON complexes. In paper III, siRNA against EGFP (which also targets YFP) was delivered using both CADY and sRxR4. Subsequent analysis of YFP knockdown was performed using fluorescence activated cell-sorting (FACS). 3.6. Cell lines Cells derived from the HeLa cell line were used throughout all the experiments in this thesis. The HeLa cell line originate from immortalized cervical cancer cells appropriated from a woman named Henrietta Lacks in the early 1950s. The cells are very robust, proliferate rapidly and are easily transfected. HeLa pLuc705, carries the pLuc705 plasmid that contains a luciferaseencoding gene interrupted by a mutated intron from a β-thalassemic globin gene. Without intervention, the intronic mutations activate a cryptic splice site, producing non-functional luciferase. HeLa pLuc705 cells are amenable for use in evaluating ON delivery as mentioned in the description of the 22 splice-correction assay above. Additionally, in paper III we make use of U2OS-SAMP1-YFP cells. All cells were cultivated with appropriate culture medium supplemented with nutrients and antibiotics in a humidified environment at 37⁰C and 5% CO2. 3.7. Cytotoxicity measurements (Paper II) Toxicity assessments of CPP:ON complexes were performed using a standard WST-1 assay [146]. It is a rapid and convenient colorimetric assay that utilizes a modified tetrazolium salt that reacts with metabolic enzymes to form formazan dye. The amount of formazan dye directly correlates to the amount of metabolically active cells in the culture, requires no solubilization since the formazan formed is water-soluble, and can be quantified spectrofotometrically to assess the amount of viable cells. 3.8. Dynamic light scattering and zeta-potential (Paper II, III and IV) DLS is a method that was used to determine the size of the CPP:ON nanoparticles at different molar ratios (MRs) in paper II. While in solution, small particles undergo Brownian motion. Dynamic light scattering (DLS) utilizes a laser to measure particle size correlated to time-dependent fluctuations in light-scattering intensity caused by these motions [147,148]. Charged particles in solution become surrounded by an electrical double layer comprised of ions and counter-ions [149]. The inner layer follows the movement of the particle in the medium, consists of strongly bound counterions and is commonly referred to as the Stern layer. The outer layer is more diffuse and contains ions and counter-ions less strongly bound to the particle. The electrical potential between across these two layers is termed zetapotential. The zeta-potential can be determined by applying an electric field to the particle solution. Particles with a zeta-potential will move towards the electrode of opposite charge. The velocity of this electrophoretic movement is proportional to the magnitude of the potential and can be measured using laser Doppler anemometry [149]. It is important to note that the zetapotential can vary drastically depending on the medium in which the measurements are conducted due to for example significant differences in ionic concentrations. 23 3.9. Scavenger receptor inhibitors (Paper II and III) In addition to RNAi, some of the experiments included in this thesis utilize pharmacological inhibitors to repress SR function. These inhibitors, dextran sulfate, fucoidan and polyI, are ligands that bind specifically to SRs and has been used previously in studies relating to SRs and their uptake of various ligands [128,129,135,150]. These ligands bind competitively to the SCARA, preventing interaction with CPP:ON and SCARA as a result. Chemically related substances; chondroitin sulfate, galactose and polyC, do not bind SCARA and are used as inhibitor controls in the experiments where SCARA-mediated uptake is assessed. Cells were pre-treated with inhibitors and their controls respectively, one hour prior to addition of CPP:ON nanocomplexes. 3.10. Fluorescence microscopy (Paper III) One experiment in paper III makes use of fluorescence microscopy. The technique of fluorescence microscopy has become an essential tool in life sciences as it allows for visualization and identification of cells, submicroscopic cellular components and fluorescently labeled molecules with a high degree of specificity. Through the application of fluorophores, fluorescence microscopy enables the study of single molecules as well as allowing for the identification of several target molecules simultaneously. In the context of this thesis, the technique has been applied to study the cellular uptake of labeled CPPs and ON cargo. We use fluorescence microscopy to investigate the role played by SCARA in the uptake of PF14-Fl alone or PF14-Fl in complex with Alexa568-labelled siRNA. Initially, HeLa cells were treated with SCARA inhibitors and their respective controls one hour prior to treatment with PF14-Fl or PF14-Fl:siRNA complexes. Following one hour incubation, cells were washed and analyzed by fluorescence microscopy. 3.11. TRAPeze RT telomerase detection (Paper IV) The TRAPeze RT telomerase detection assay is a highly sensitive and robust PCR-based method for fluorometric detection and real time quantitation of telomerase activity. Based on the original method, telomerase repeat amplification protocol (TRAP) [30,151], the TRAPeze RT telomerase detection assay has the ability to quantitate telomerase activity using fluorescence energy transfer primers. The resulting fluorescently labeled TRAP products permit non-isotopic, quantitative analysis of telomerase activity. The primers only fluoresce upon incorporation into the trap products. During the first part of the reaction, telomerase enzyme adds a number of telomeric repeats 24 (GGTTAG) onto the 3’ end of a substrate ON. In the following step the extended products are amplified by a Taq polymerase using PCR with the substrate ON and fluorescein-labeled primer. The fluorescence emission produced is directly proportional to the amount of TRAP products generated. 3.12. Population doubling (Paper IV) In order to assess effects on cellular proliferation and senescence we study the growth of treated cells in the presence or absence of telomerase inhibitor. A fixed number of cells are seeded and treated. The cells are then left to proliferate for a period of three days after which they are counted, the population doubling calculated, re-seeded in the same amount as at the start of the experiment, treated and allowed to once more proliferate. Cells are monitored and population doubling calculated thus over a longer period of time. 3.13. Cellular senescence assay (Paper IV) Previous studies have shown that several human cells express a histochemically detectable β-galactosidase (SA-β–Gal) upon senescence in cell culture. This expression was observed in senescent but not pre-senescent cells and it was absent from terminally differentiated cells as well as immortal cells. However, it was induced in the latter cells by genetic manipulation that reversed immortality [152]. In paper IV, cells in the cellular senescence experiments are seeded at a fixed density (1,5x104 cells) on petri dishes. Subsequently, the cells were fixed and subjected to histochemical staining for human SA-β–Gal activity. Fixation and staining was done as previously described [152]. Both cells positively stained for SA-β–Gal activity (blue) and negative cells (unstained) are counted using a standard light microscopy without the use of phase contrast filter. The percentage of blue cells was calculated by combining data from ten separate fields, totaling at least 200 counted cells. 25 4. Results and discussion The scientific reports included in this thesis aim at investigating CPPs and ON designs suitable for therapeutic applications as well as elucidating the role of scavenger receptor-mediated endocytosis in the uptake of CPP:ON nanoparticles. In paper I we present a study evaluating the potency and specificity of a range of chemically modified antimiRs of varying lengths for use in the design of therapeutic ONs. Paper II introduces a novel CPP with enhanced endosomolytic properties, PF15, and studies its ON delivery capabilities and uptake route. Paper III investigates the involvement of SCARAmediated endocytosis in the uptake of several structurally unrelated CPPs of different origin and their associated ON cargo. Paper IV presents a possible therapeutic application of CPP-mediated ON delivery in a study aiming to enhance the efficiency of telomerase inhibitor ONs. 4.1. Effects of chemical modifications and truncations of oligonucleotides on activity and specificity (Paper I) This report encompasses an evaluation of modified synthetic antimiRs based on their potency and specificity. The study involved twenty 2’OMe antimiRs of different lengths, modified chemically with various nucleotide analogs. The ONs were evaluated for their ability to bind the miRNA-21 sequence and interfere with its binding to the target. Dysregulation of miRNA-21 has previously been strongly implicated in the development of several types of cancers as well as other diseases [153-156]. Overexpression of miRNA-21 causes the excessive down-regulation of its target genes, many of which are tumor suppressors. Consequently, the approach presented in this work aimed at optimizing the inhibitory effect of the antimiR ONs, suppressing the activity of miRNA-21. Advancing our understanding of the effects that ON length and various chemical modifications have on antimiR potency and specificity allows for improved rational design and development of therapeutic ONs. Additionally, the same principles can be utilized for design of ONs used as molecular tools in research to obtain a greater understanding of miRNA functions and mechanisms. The ONs used in this study were chosen in order to determine which chemical modifications are more suitable for use in the design of antimiRs. 26 Effects on specificity and potency caused by introduction of LNA, UNA and PS modifications as well as truncations of the antimiR sequence were studied. The activity of these ONs on the target was evaluated in HeLa cells using a dual luciferase reporter assay. The reporter system consisted of a modified psiCHECK-2 vector containing the miRNA-21 binding site in the 3’ UTR of the plasmids Renilla luciferase ORF whereas a second constitutively active ORF, Firefly luciferase, acted as an internal control. In contrast, the expression of Renilla luciferase was dependent on the amount of free endogenous miRNA-21. Without any antimiR present, the miRNA-21 binds to the miRNA-21 binding site in the 3’ UTR and effectively blocks Renilla expression. The greater the interaction between antimiRs present in the cells and endogenous miRNA-21, the greater the Renilla expression as the miRNA21-dependent repression is reduced. The potency of the tested antimiRs were assessed on the basis of the elicited light signal produced as a result of Renilla luciferase expression. In contrast, specificity was measured by comparing the difference in signal strength between antimiRs of given lengths and chemical composition identical except for two mismatched bases. Our results show that LNA modification significantly increased the potency yet at also reduced mismatch discrimination. Although a high drug activity is certainly a desired characteristic of a potential therapeutic agent, high specificity is also a desired characteristic in order to avoid unwanted off-target effects. In addition to nucleotide modifications, modifying the linkages between nucleotides provides further possibilities to enhance ON activity. AntimiRs with PS backbone modifications evaluated in this report displayed a significantly improved activity compared to ONs with a phosphodiester backbone. Furthermore, the PS modifications appeared to ameliorate the impairing effects on target specificity by LNA as PS-LNA antimiRs displayed mismatch discrimination superior to their PO-LNA counterparts. Additionally, moderate truncations of the antimiRs used in this study also counteracted the negative effect on mismatch discrimination caused by LNA. Reducing the length of the 22 nucleotide full-length antimiR by as little as four nucleotides improved the specificity while retaining an activity equal to that of the full-length ON. Modifications that greatly enhances the affinity of an ON to its complementary target sequence can potentially compromise the specificity given that the affinity increases to the point where it compensates for otherwise reduced affinity for mismatched sequences. In contrast, modifications that reduce binding affinity such as PS linkages [46] and moderate truncations may improve specificity. This may also provides a scientific context for why short LNAs have been shown to be highly specific [68]. UNA has been reported to have duplex-destabilizing properties [71-73] and were introduced to ONs in order to assess whether those properties could modulate the negative impact of LNA on mismatch discrimination. However, initially the UNA modifications resulted in an all but completely abolished activity most likely deriving from an unbalanced LNA to UNA 27 ratio and, consequently, an excessive duplex de-stabilization. A considerable reduction of the amount of UNA monomers in the ON sequence resulted in low but measurable activity with significant mismatch discrimination. In conclusion, although limited to a small number of constructs, the report offers insights in how to modulate both the activity and specificity of antimiRs utilizing modifications of ON length and chemical composition. 4.2. Design of a novel cell-penetrating peptide with enhanced oligonucleotide delivery (Paper II) The therapeutic potential of biologically active ONs is limited not only by the ability of the delivery vector to promote cellular uptake but also by the final localization of the ON once it has entered the cell. Various endocytosis mechanisms exist and mediate a large part of cellular uptake. If endocytosis ultimately leads to the majority of the bioactive molecules to become trapped inside endosomes the risk is great that the molecules will not be able to exert their biological effect properly. Thus, avoiding or overcoming endosomal entrapment is of paramount importance to ensure the bioavailability of delivered ONs. In this study we aimed at facilitating endosomal escape of ONs delivered by CPP through the design of a novel endosomolytic CPP named PF15. PF15, based on its predecessor peptide PF14, utilizes CQ analogs covalently linked to a lysine-tree situated on a lysine residue located in the CPP backbone. The rationale behind this modification was to impart the protonbuffering and endosomolytical effects of CQ [124,157]. Previously, our group has successfully utilized the same modification in the design of another CPP named PF6 [158]. CQ and its analogs, lysosomotropic agents that preferentially accumulate in the acidic lysosome, which regularly fuse with late endosomes, are frequently used in vitro to enhance the effects of delivery. Initially, PF15 was used to deliver SCOs at different MRs into HeLa pLuc705 cells in order to establish optimal conditions for transfection. PF15 performed significantly better when delivering SCO compared to its parent peptide PF14. The difference in performance could be explained both by an increased uptake as well as an improved endosomal escape. In order to differentiate between the two possible explanations we performed two control experiments. First, we performed an uptake study delivering ONs labeled with Cy5. The results showed no difference in the uptake between PF14 and PF15. Secondly, in order to assess whether an improved capacity for endosomal escape could be the cause of the improved performance of PF15 we performed an experiment where free CQ was added at high concentration. This experiment showed no significant effect on the read-out for SCO delivered by PF15 in the presence of CQ whereas SCOs delivered by PF14 in the 28 presence of CQ elicited a significantly higher read-out than without addition of CQ. Taken together, the results from the two experiments strongly indicated that the difference in performance between PF15:SCO and PF14:SCO nanoparticles could largely be attributed to differences in endosomal escape. In addition to SCO delivery, we utilized our new CPP to mediate delivery of an antimiR targeting miRNA-21, a miRNA involved in several forms of cancer [153-156]. The antimiR used in this study was a 2’OMe/LNA POmodified mixmer ON designed in a previously conducted evaluation screen of ON modifications [159]. The antimiR was delivered into HeLa cells transiently transfected with a modified psiCHECK-2 vector containing a dual luciferase read-out system used to evaluate delivery efficiency. Both PF15 and PF14 successfully delivered the antimiR although PF15 did so at a much greater efficiency as compared to both its parent peptide and commercial transfection reagent Lipofectamine®2000 (Invitrogen). Previously, SCARA was revealed as a mediator of endocytosis of PF14:SCO nanoparticles [125]. In light of those findings, we performed a number of experiments to assess whether SCARA was also responsible for the uptake of PF15:SCO complexes. We set up an experiment using a range of pharmacological inhibitors of SCARA [134,135,137,150,160]. The results show that addition of SCARA inhibitors drastically reduces the read-out of the SCO luciferase reporter system. This strongly suggest that SCARAmediated endocytosis is the main route of entry for our CPP:ON nanocomplexes and that the QN moieties of PF15 effectively enhances ON delivery without altering the uptake mechanism. In summary, we designed a novel member of the PepFect CPP family to produce a CPP with improved ability to deliver ONs by facilitating endosomal escape of its delivered cargo. 4.3. Scavenger receptor-mediated uptake of CPP:ON complexes (Paper III) PF CPP:ON nanoparticles have previously been shown be taken up largely via SCARA-mediated endocytosis [125]. SCARA are known to promiscuously bind to and mediate uptake of negatively charged macromolecules [135,137,160]. PF:ON nanoparticles have been shown to possess a negative zeta-potential in bio-relevant medium, which resonates well with the findings that their uptake is mediated by SCARA. However, there are many CPPs structurally unrelated to and with completely different origins than the PF CPPs. In this study, we wanted to assess the whether SCARAs involvement in uptake of CPPs and their associated ON cargo also included CPPs other than PFs. We conduct a series of experiments using different CPPs and cargos. Initially, we measured the zeta-potential of all the CPP:ON com29 plexes included in this study. The results from the zeta-potential measurements revealed all tested complexes to be negatively charged although sRxR4 in complex with pGL3 plasmid were near neutral. We used PF6 and PF14 to deliver pGL3 plasmid into HeLa cells. Delivery efficiency was measured on the basis of luciferase activity yielded as a result of successful transfection of luciferase-encoding pGL3. Addition of SCARA inhibitors drastically reduced the luciferase activity whereas inhibitor controls did not. We then performed an experiment where SCARA expression was knocked down using siRNA targeting SCARA3 and SCARA5, the two subtypes of SCARA previously shown to be present in HeLa cells [125], prior to delivery of pGL3 in complex with PF14, dPF14 and s-RxR4 respectively. For all three CPPs, cells pre-treated with siRNA targeting SCARA 3 and 5 showed significantly reduced luciferase activity compared to cells pre-treated with control siRNA or without any siRNA treatment. We conducted an experiment where SCARA 3 and 5 was up-regulated to study the effects on CPP:ON uptake. The results revealed that overexpressing SCARA increased the luciferase activity measured 4 to 6–fold for cells where pGL3 was delivered by either PF14, dPF14 or s-RxR4. In order to eliminate the possibility that neither knockdown nor up-regulation of SCARA indiscriminately affected all types of uptake, we performed a control experiment in which commercial lipid-based transfection reagent LF2000 was included to deliver pGL3 into HeLa cells. An unrelated control plasmid was also used. The results showed no statistically significant effects on the luciferase activity from cells where pGL3 had been delivered by LF2000. Furthermore, a decomplexation assay was performed to exclude the possibility that effects seen in experiments where SCARA inhibitors were used were due to a disruption of the CPP:pGL3 complexes. The results indicated no decomplexation in the presence of SCARA inhibitors. Next, U2OS-SAMP1-YFP cells were assessed for subtypes of SCARA expressed by use of RT-PCR. The results show that both SCARA 3 and 5 are present in the cells. Next, cells were pre-treated with siRNA targeting SCARA 3 and 5. Subsequently PF6 or CADY in complex with siRNA targeting EGFP was added to the cells in an attempt to assess if knockdown of the SCARA receptors affected the CPP:siRNA-mediated down-regulation of YFP expression in the U2OS-SAMP1-YFP cells. The results showed that YFP expression did not diminish significantly by addition of CPP:siRNA, indicating that knockdown of SCARA 3 and 5 affects the uptake of both PF6:ON as well as CADY:ON nanoparticles. Judging from the results it is easy to come to the conclusion that the CPP does not matter in the context and that it is the ON that conveys the interaction with SCARA due to the negative charge. The primary sequence of the tested CPPs seems inconsequential for whether SCARA mediates uptake or not. However, serumprotein opsonization could also be a likely mediator of SCARA interaction. In order to elucidate the matter we conducted a fluorescence microscopy 30 experiment in which fluorescein-labeled PF14 (PF14-Fl) and Alexa568labeled siRNA were used. The experiment was setup in both medium with and without serum. The results show that uptake of both PF-Fl:siRNA in complex and PF-Fl alone was either abolished completely or significantly reduced in the presence of SCARA inhibitors. This suggests that the ON is not needed for recognition by SCARA. In addition, comparable cellular internalization of both PF14-Fl:siRNA and PF14-Fl alone strongly suggests that opsonization by serum-proteins is not required for SCARA interaction. Two cationic polymers, PLO and PEI were included in the paper to broaden the scope of the study with transfection reagents other than CPPs. pGL3 in complex with either PLO or PEI was added to HeLa cells pretreated with either SCARA inhibitors or inhibitor controls. Cells pre-treated with SCARA inhibitors displayed a drastically reduced luciferase activity whereas the luminescence of cells pre-treated with the inhibitor controls was not adversely affected. Once more, a de-complexation assay was performed to exclude the possibility that the effects seen were due to a disruption of the polymer:pGL3 complexes. Once again, the results indicated no decomplexation caused by the presence of SCARA inhibitors. Conclusively, the results indicate SCARA as a major mediator of cellular uptake not only of CPPs but also of the two polymers PLO and PEI with their associated cargo. In summary, this paper shows that SCARA-mediated endocytosis constitutes an important uptake route for several structurally disparate CPPs and two cationic polymers. For CPPs, L- or D-configuration does not appear to have any impact on the uptake via SCARA. 4.4. Cell-penetrating peptide-mediated delivery of telomerase inhibitor oligonucleotides enhances telomerase inhibition (Paper IV) Every time a cell divides, the ends of its chromosomes, the telomeres, shorten. This is due to the inability of the conventional DNA replication machinery to duplicate the ends of linear chromosomes. Once the telomeres are reduced to a critical length, cells may undergo replicative senescence, losing their ability to proliferate, and commonly experiencing chromosomal instability – a phenomenon referred to as cell crisis. During crisis, most cells undergo apoptotic cell death [161-163]. Telomerase is the enzyme responsible for maintaining the telomeres in nearly all eukaryotic cells. It is a RNP that utilizes a short RNA template for reverse transcription [164]. In human tissues, telomerase activity is present in germline and stem cells but is absent in most somatic or differentiated cells [165]. 31 Acquisition of telomerase activity enables tumor cells to circumvent telomerase-dependent mechanisms of cell mortality and achieve limitless proliferative potential [166-168]. Targeting telomerase presents several key advantages compared to most other cancer targets. The enzyme expressed in the majority of tumors from all cancer types and studies indicate that cancer stem cells are also telomerase-positive [161,169,170]. Furthermore, most normal cells do not use telomerase and those that do exhibit low or transient levels of enzyme expression, which reduces potential toxicity stemming from telomerase-based therapies. Finally, maintenance of the telomeres and replicative immortality appear to be crucial for tumor progression and telomerase is critical in these biological mechanisms. Consequently, tumor cells are considered less likely to develop resistance to telomerase-based therapies than to other therapies targeting more redundant cancer targets such as growth factor receptors [169]. Currently there are several therapeutic approaches for targeting telomerase in tumors [169,171]. One in particular, is based on chemically modified antisense ON targeting the RNA template within the hTERT domain. Geron Corporation has produced several telomerase inhibitor ONs, most importantly GRN163 and a 5’palmitoylated form of GRN163 named GRN163L (also referred to as Imetelstat). Although GRN163 displays a significantly lower IC50 value than GRN163L in cell-free evaluation systems (1.4 nM vs 7.8 nM) [33] , poor cellular uptake (IC50 > 20 μM compared to 150nM for GRN163L) makes GRN163L superior for use in therapeutic applications [172,173]. GRN163L is currently undergoing clinical phase II studies. Previous studies using CPPs to deliver telomerase antagonists have been limited to PNA covalently conjugated with classical CPPs such as Tat, polyarginine and poly-lysine [174,175]. The CPP-PNA conjugates in those studies were less efficient than the GRN163L ON, the interaction between PNA and the telomerase RNA template likely suffering from a sterical interference of the conjugated CPP. Furthermore, several advances in the field of rational design of CPPs have given rise to new and more efficient CPPs capable of transfecting a broader range of cell types [101,104,141]. The high efficiency of our CPPs combined with the lack of need for covalent modification of the cargo and versatility in the kind of ONs amenable to delivery suggests the possibility to enhance uptake of therapeutic ONs such as the GRN163 without the need of a fatty acid conjugation, which probably cause steric hindrance [172]. We haven’t seen any signs of acute toxicity in the in vivo experiments we’ve performed with our peptides in the past [101]. Histopathological examination hasn’t revealed any signs of toxicity in liver, lung or kidneys, and several other tested clinical or hematological parameters remain unaffected. Furthermore, in vivo studies show no evidence of enhanced IL-1 or TNF-1 32 cytokine production after 24 or 48h treatment with a range of our peptides [176]. In this study we aim at determining whether the efficiency of telomerase inhibitors can be enhanced via CPP-mediated delivery in vitro and in vivo. We evaluate our CPP delivery strategies and formulation protocols with telomerase antagonists using a quantitative real-time PCR-based (qPCR) TRAP assay to quantify telomerase activity. The ON used in this study are PS-modified LNAs. HeLa cells were used, as they are simple to cultivate and have been reported to express telomerase [177,178]. We determined the size and zeta-potential of the CPP:ON complexes and found that the complexes are approximately 400 nm in diameter and possess a negative zeta-potential, which is in accordance with previous CPP:ON formulations that have involved a SCARA-mediated uptake route. By addition of SCARA inhibitors we show that inhibition of SCARA significantly reduces the telomerase inhibition mediated by the ONs. However, we do see some reduction of telomerase activity even when SCARA inhibitors are added. This could be due to several reasons. First, there is still room for optimizing the inhibitor concentrations further, but it is also possible that it’s not possible to totally block all uptake of the CPP:ON complexes via SCARA, or that enough quantities of ONs enter via an uptake route unaffected by SCARA inhibitors. Our results suggests IC50 values for our telomerase inhibitor ON significantly lower than that of GRN163L in HeLa cells; ~8,4 nM (paper IV) compared to ~200 nM [179]. We conducted a population doubling experiment spanning over 27 days and a subsequent histochemical staining for SA-β–Gal. A previous study performed such an experiment over a period of 60 days, but due to time constraints we were unable to run the experiment longer. Unfortunately, telomerase inhibitor treatment did not yield any tangible effects on HeLa cell proliferation rates nor senescence (paper IV). Telomerase inhibition is not a treatment expected to give fast results as it takes time for the telomeres to shorten with each cell division and cancer cells with impaired telomerase activity have been shown to prioritize maintaining the shortest telomeres in order to prolong their survival [180]. Despite of the significantly lower IC50 value of our tested CPP:ON formulations, obtaining a strong biological effect on cancer cell proliferation and senescence will likely require a longer treatment than 27 days. Given the liver accumulation of PS-ONs, lipid-conjugated ONs and the reported hepatotoxicity of GRN163L it would be interesting to conduct experiments studying GRN163L and our CPP:ON formulations side by side, while also investigating the possibility to block SCARA heavily expressed in Kupffer cells that might be contributing significantly to the harmful accumulation [181]. In summary, our in vitro results suggest a significant potential to enhance telomerase-based ON therapies through delivery by CPPs. 33 5. Conclusions The major findings of this thesis are listed and described below: Paper I: We describe a variety of ways with which to improve the efficiency and modulate the specificity of antimiRs using a range of different chemical modifications and moderate truncations of ONs. Paper II: We have expanded on existing PepFect technology and produced a novel endosomolytic CPP, PF15, with enhanced delivery efficiency compared to both its parent peptide PF14 as well as the commercial transfection reagent LF2000. Furthermore, we show that PF15 forms nanoparticles together with the associated ON cargo and that cellular internalization of those nanoparticles is mediated via a scavenger receptors-mediated uptake route. Paper III: We show that scavenger receptor-mediated endocytosis is not exclusive to PepFect CPP:ON nanoparticles but that this uptake route is also involved in the uptake of several structurally and chemically different CPPs as well as two cationic polymers, in complex with ONs. Paper IV: Delivering telomerase inhibitors into telomerase positive HeLa cells, we show that CPPs are able to enhance the enzymatic inhibition compared to naked ON. This is also the first known case of a non-covalent CPP:ON targeting applied to a RNP. 34 6. Concluding remarks and future perspectives 6.1. Biological effects of antimiRs delivered by cellpenetrating peptides In paper I we present an array of various ON modifications that can be utilized to tune both the activity and specificity of selected antimiRs targeting miRNA-21. However, no comprehensive evaluation of their compatibility with CPP-mediated delivery has yet been conducted. In paper II, the designed CPP PF15, is shown to successfully deliver an LNA-modified POON, eliciting a signal via the dual luciferase reporter assay. However, preliminary studies suggest that, depending on application, all ON modifications are not amenable to delivery by CPPs. PS-ON antimiRs performed poorly compared to their PO-ON counterparts. In contrast, for splicecorrection, PO-ONs performs inadequately while PS-ONs performs well (data not shown). These disparities should be of great interest to further investigate to determine what the underlying mechanisms are and if they can be extrapolated to delivery by other CPPs than PFs. Furthermore, the dual luciferase reporter assay used in paper I only reveals if the selected antimiRs are able to sequester enough endogenous miRNA-21 to de-repress Renilla luciferase expression. It is not known whether or not the activity of the tested antimiRs, or that the concentrations used, would be sufficient to elicit a proper biological response in the form of for example reduced tumor cell viability or proliferation. 6.2. In vivo delivery oligonucleotides of telomerase inhibitor In paper IV we have initiated an evaluation of CPP-mediated delivery of antisense ONs targeting the ribonucleotide component of hTERT. Using PF15 we achieve a significantly improved IC50 value compared to that based on previous studies in HeLa cells utilizing clinical trial Phase II drug candidate Imetelstat (GRN163L). However, regardless of how promising initial investigations appear, further in vitro experimentation as well as in vivo studies are needed in order to properly evaluate the use of CPPs in this type of cancer therapy. Hepatocellular carcinomas and lung cancers could prove 35 interesting targets, as those tissues are known to express high levels of SRs that may facilitate delivery. Furthermore, more investigations conducted on additional cell lines and studies of effects over longer time-periods should be performed. Ideally, we would also like to compare the effects of our constructs side-by-side with Imetelstat, alone and together with our PepFect delivery vectors. 6.3. Functionalization of PepFects and further improvement of PepFect transfection technology The field of CPP and CPP-mediated delivery continues to grow and as such it is of great importance to continue to further improve our understanding of their uptake mechanisms as well as how they interact to form particles with associated cargo. The PF family of CPPs has been tested against a wide range of cells, which have been successfully transfected yet some remain untested. Considering the importance and impact of cancer stem cells in our current understanding of tumorigenesis and recurrence of cancer, this subclass of tumor cells would be of great interest to investigate for their susceptibility to CPP-mediated delivery. Further developing the targeting-capabilities of CPPs would be an interesting area of research. Functionalizing CPPs with short, target specific ligands (e.g. RGD-motifs) to direct delivery to specific tissues and cells provides a potential route to obtaining more specialized peptide delivery vectors. Making CPPs more specialized yet relatively simple to synthesize presents a significant challenge that most likely needs addressing in order to translate CPPs into functional drug-delivery vehicles appealing to the pharmaceutical industry. Discovering ways to tune the particle size of CPP:ON nanocomplexes is also an important area of research [182]. Currently our particles size lie in the range of between 70-700 nm depending on the type and concentrations of CPP and cargo as well as the type of media used [101,104]. 36 7. Populärvetenskaplig sammanfattning på svenska I vårt DNA, som utgör vår så kallade arvsmassa, finns kopiösa mängder biologisk information lagrad. En av DNAs huvudsakliga uppgifter är att via våra gener tillhandahålla information till cellerna hur alla de för kroppen livsnödvändiga proteiner ska produceras, i vilka mängder samt hur de ska processas för att fungera korrekt. I takt med att vår förståelse för den genetiska informationen ökar så ökar även vår insikt i hur skadliga förändringar i våra gener kan resultera i att olika biologiska processer i kroppen rubbas vilket kan ge upphov till olika sjukdomar. I många fall är sådana sjukdomstillstånd ärftliga då de skadliga förändringarna nedärvs från föräldrar till barnen men ibland kan även yttre påverkan från t.ex. virus eller mikroorganismer orsaka problem genom exempelvis produktion av främmande proteiner och/eller oönskade förändringar i regleringen av våra cellers biologiska processer. Som resultat av vår ökade förståelse har man upptäckt möjligheten att behandla många av dessa sjukdomstillstånd med hjälp av molekyler som förmår reparera skador i eller justera regleringen av våra egna cellers genetiska information. Därutöver kan liknande molekyler angripa eller kontrollera arvsmassan hos skadliga mikroorganismer för att på så vis neutralisera deras förmåga att orsaka problem i våra kroppar. Dessa molekyler tillhör en klass av genetiska element som kallas oligonukleotider och de består rent kemiskt av korta sekvenser av DNA eller RNA och förmår interagera med andra specifika sekvenser av DNA och RNA, helt beroende på hur deras egen sekvens ser ut. Olyckligtvis är denna klass av molekyler till naturen relativt stora och därtill elektriskt laddade vilket gör dem näst intill helt oförmögna att ta sig genom cellernas plasmamembran. Plasmamembranet består nästan uteslutande av fetter och proteiner och utgör ett selektivt och skyddande filter kring cellen som reglerar vad som kan färdas in och ut ur cellen. På grund av oligonukleotidernas storlek och laddning släpps de inte igenom plasmamembranet utan assistans. Därtill bryts de snabbt ner av olika enzymer inuti cellerna vars uppgift bl.a. är att skydda kroppen från främmande DNA och RNA. En möjlig lösning på dessa problem är att kemiskt modifiera oligonukleotider. Sådana modifikationer kan göra dem mer resistenta mot nedbrytning och underlätta passage genom cellens membran. Exempelvis 37 kan man göra dem mer fettlösliga med en tillkopplad fettmolekyl vilket gör det lättare för de annars vattenlösliga molekylerna att passera det fettrika plasmamembranet. Modifikationer av dessa slag har dock sina begränsningar. Dels så är cellens upptag av oligonukleotiderna fortfarande inte särskilt stort, dels så kan kemiska modifikationer i vissa fall påverka oligonukleotidens önskade biologiska effekt negativt genom att blockera interaktionen med målet inuti cellen. Alternativt kan modifikationer påverka oligonukleotidernas aktivitet, deras förmåga att binda till sina mål, så att specificiteten blir lidande vilket kan ge upphov till oönskade sidoeffekter. En annan lösning oligonukleotiders oförmåga att på egen hand få tillträde till är att använda sig av så kallade vektorer, bärarmolekyler, för att transportera ämnen in i cellen. Cell-penetrerande peptider (CPPer) utgör en sådan klass av lovande leveransmolekyler. Denna avhandling med dess fyra artiklar fokuserar på i huvudsak tre områden. Det första området är oligonukleotider. Vi är intresserade av hur olika modifikationer av dessa kan nyttjas för att förbättra deras terapeutiska potential. Det andra området är CPPer. CPPer utgör ett intressant, effektivt och icke-toxiskt alternativ att föra in ämnen som normalt sett har svårt att på egen hand ta sig in i celler. Vi intresserar oss för hur CPPer kan designas till att bli effektivare på att leverera molekyler såsom oligonukleotider samt öka vår förståelse för hur dessa tas upp av cellerna. Slutligen intresserar vi oss för olika praktiska tillämpningsområden. Vi vill identifiera olika biologiska processer och/eller sjukdomstillstånd som lämpar sig väl för användning av CPPer och oligonukleotider. I den första artikeln, utvärderar vi effekten av olika modifikationer på anti-miRNA oligonukleotider. Målet var att studera hur kemiska modifikationer samt olika längd på oligonukleotiderna kunde påverka dels deras förmåga att binda till sina mål, men även deras förmåga att särskilja mellan specifika och ospecifika mål. Slutsatserna var att kemiska modifikationer med starkt positiv effekt på oligonukleotidernas förmåga att interagera effektivt med sina mål kunde ha negativa effekter på deras specificitet och vice versa. Andra modifikationer upptäcktes ha positiva effekter på såväl aktiviteten som specificiteten hos de testade oligonukleotiderna. Det visade sig även vara möjligt att förkorta längden på oligonukleotiderna utan att förlora någon nämnvärd aktivitet emedan specificiteten tycktes öka något. Resultaten från denna artikel visar på riskerna med att konstruera oligonukleotider med mycket hög aktivitet då specificiteten riskerar bli lidande, men pekar även på möjligheten att kombinera olika modifikationer för att uppnå en balans mellan aktivitet och specificitet. I den andra artikeln designar vi en ny cell-penetrerande peptid med avsikten att möjliggöra en förbättrad leverans av olika typer av oligonukleotider in i cellen. Den nya peptiden, benämnd PepFect15, är kapabel att leverera sin last in i cellen och på ett effektivt sätt hjälper oligonukleotiderna att undgå 38 att fångas inuti endosomer, membranblåsor avknoppade från plasmamembranet inuti vilka inträdande partiklar normalt sett transporteras, inuti cellen. Tidigare studier har visat att oligonukleotider i komplex med CPPer designeade i vårt labb tas upp via membranreceptorer benämnda Scavengerreceptorer. Då CPPer kan se ut på många olika sätt och ha mycket olika kemiska egenskaper var det inte säkert att denna upptagsmekanism även ansvarade för upptag av andra CPPer i komplex med oligonukleotider. I den tredje artikeln studerar vi en rad olika CPPer och undersöker Scavengerreceptorernas roll i cellers upptag av CPPer av olika slag tillsammans med oligonukleotider. Tidigare har den generella uppfattningen varit att cellens upptag av CPP tillsammans med oligonukleotider initierats av CPP. Resultaten från våra studier antyder dock att Scavenger-receptorerna spelar en viktig roll i upptaget av en bredare urval av CPP och oligonukleotider. Denna upptäckt är mycket intressant dels för förståelsen för hur CPPer och oligonukleotider tas upp i celler, men även för ny design och praktiska tillämpningar av CPPer. I den fjärde och sista artikeln tillämpar vi våra tidigare resultat på cancerceller. Med hjälp av PepFect15 levererar vi oligonukleotider designade för att blockera telomeras, ett enzym essentiellt för majoriteten av alla idag kända cancerformer. Cancercellerna utnyttjar ett oreglerat telomerasenzym för att kringgå begränsningarna för celldelning vilket ger dem en gränslös förmåga att dela sig. Våra resultat tyder på att vi lyckas uppnå en mycket hög blockering av telomerasenzymet, jämförbar eller bättre än cancerläkemedel som riktar sig mot telomeras i pågående kliniska prövningar. Vår förhoppning är att resultaten från vår studie dels kan öka chanserna för att en ny klass av effektiva cancerläkemedel utvecklas, samt att förespråka CPPer som lovande läkemedelstransportörer. 39 8. Acknowledgements After many years, many more than I had first anticipated, I am now finally at the end of a very big chapter of my life. During all these years, a lot of different people have had great influence on me, helping me to find my way and overcoming all the challenges. First of all I would like to thank my supervisor, Ülo, for giving me the opportunity to join his group and for believing in me. Thank you so much for all the fun and challenging experiences. A big thank you to all the great people of our group, thank you for the wonderful environment you provide and all the scientific discussions and good laughs. You’re all fantastic people. Special thanks to Henrik for your great sense of humor, your patience and all the technical help and to Kristin, our own paragon of cheerfulness, for the fun times teaching together. Jakob, our groups own food-critic, I’m happy you decided to join our group, thanks for all the great times during our joint conference trips. Rania and Moataz, I enjoyed all our talks, ranging from Arabic lessons, scientific discussions and whatnot. Jonas, incredibly smart, friendly and very serious. A huge thank you for your invaluable help proof-reading this work! Ying, I’m glad you joined the group; you are so helpful and friendly. Staffan, thanks for all the great times we had, the trips, the talks, you’re a BöMäzin’ guy. Daniel you’re a really funny and friendly guy. I’m impressed that you surf during winter in Sweden! Carmine, our resident Italian, so social and friendly! Mattias, thank you for your feedback during presentations and our conversations, you’re a very bright man with a great smile and sense of humor! Tõnis, you’re a funny guy and I really like the õ in your name  I also want to thank some people who aren’t in our group anymore: A big thanks to my old friend Samir for helping me find my way back to science (I miss the good old time we had together as students at Södertörn!), to Peter Guterstam for the great albeit short time we worked together. Thank you Kariem for your friendship and all the nice discussions we had during your time at the department. Anders Florén, I learnt a lot of good things when you supervised me during my undergraduate project at the department. I’d like to thank the Estonian part of “Team Langel”, Taavi, Imre, Indrek, Piret, Julia, Kaido, Ly and all the others for the great times, science-related and other. Kent (and Merlin), thank for all the fun in Tartu, it was awesome! To all my collegues and friends at the department; thank you for making the department of Neurochemistry a tremendously fun and interesting place 40 to work in. Marie-Louise, Staffan wrote it in his thesis and I wouldn’t be surprised if others have and will do the same: You are invaluable to the department as a whole, an absolute gem! A huge thank you! Linda Tracy, I will never forget the time we shared office and all the conversations we had. Sylvia (as well as her “predecessors” Ulla and Siv), thank you for all your kind assistance. Your help and support has been amazing. Anna-Lena, it was always a pleasure to teach in your course and great to collaborate scientifically. Xin, you are a really nice and friendly person whom I have enjoyed talking and working with. Thank you Per Kylsten for memorable experiences both fun and trying during my time at Södertörn that made me all the wiser. A heartfelt thank you to Catarina Ludwig for helping me regain my footing when I needed it and a lot of love to my old friends and colleagues at Södertörn where I started my scientific journey, especially Fergal, Lina and Maja. Tack till alla mina vänner utanför institutionen. Särskilt vill jag tacka mina vänner Khidir, Harry, (Carl)-Daniel, Sebastian Weil och Sebastian “Ranne” Ranhem. Tack för att ni är så awesome!  Děkuji Jessačku, för vår tid ihop, för din kärlek och ditt stöd. Sist men definitivt inte minst vill jag tacka min underbara familj. Ert ändlösa stöd och er kärlek betyder allt. In case I’ve forgotten someone: a big thank you to you too! 41 9. References [1] T.P. O'Connor, R.G. Crystal, Genetic medicines: treatment strategies for hereditary disorders, Nat. Rev. Genet. 7 (2006) 261-276. [2] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell. 144 (2011) 646-674. [3] P.E. Rakoczy, Antisense DNA technology, Methods Mol. Med. 47 (2001) 89-104. [4] K.A. 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