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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
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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
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