Naturally derived cell-penetrating
peptides and applications in gene
regulation
A study on internalization mechanisms and endosomal escape
Pontus Lundberg
Stockholm University
© Pontus Lundberg, Stockholm 2006
ISBN 91-7155-337-X
Typesetting: Intellecta Docusys
Printed in Sweden by Intellecta Docusys, Stockholm 2005
Distributor: Stockholm University Library
Though much is taken, much abides; and though
We are not now that strength which in old days
Moved earth and heaven; that which we are, we are;
One equal temper of heroic hearts,
Made weak by time and fate, but strong in will
To strive, to seek, to find, and not to yield
Tennyson (Ulysses)
List of publications
List of publications
This thesis is based on the following publications, referred to in the text by
the corresponding Roman numerals:
I
Lundberg P and Langel Ü
Uptake mechanism of novel cell-penetrating peptides derived
from the Alzheimer’s disease associated gamma-secretase
complex
Int. J. Pept. Res. Therap. (2006) 4(12): 105-114
II
Magzoub M1, Sandgren S1, Lundberg P1, Oglecka K, Lilja J,
Wittrup A, Göran Eriksson LE, Langel Ü, Belting M and
Gräslund A.
N-terminal peptides from unprocessed prion proteins enter
cells by macropinocytosis
Biochem. Biophys. Res. Commun. (2006) 348(2): 379-85.
III
Holm T, Johansson H, Lundberg P, Pooga M, Lindgren M and
Langel Ü
Studying the uptake of cell-penetrating peptides
Nat. Prot. (2006) 1(2): 1001 – 1005
IV
EL-Andaloussi S, Johansson H.J, Lundberg P and Langel Ü
Induction of splice correction by cell-penetrating peptide nucleic acids
J. Gene Med. (2006) 8(10): 1262-1273
V
Lundberg P, EL-Andaloussi S, Sütlü T, Johansson H and
Langel Ü
Delivery of short interfering RNA using endosomolytic cellpenetrating peptides
Faseb J. In press
1
(These authors contributed equally to this work)
Additional publications
Additional publications
Lundberg P, Magzoub M, Lindberg M, Hällbrink M, Jarvet J, Eriksson L,
Langel Ü and Gräslund, A
Cell membrane translocation of the N-terminal (1-28) part of the prion
protein
Biochem. Biophys. Res. Commun. (2002) 299(1): 85-90.
Lundberg P and Langel Ü
A brief introduction to Cell-penetrating peptides
J. Mol. Recognit. (2003) 16: 227-233
Ståhl A, Nilsson S, Lundberg P, Bhushan S, Moberg P, Morisset M, Vener
A, Mäler L, Langel Ü and Glaser E
Two novel targeting peptide degrading proteases (PrePs) in mitochondria and chloroplast: so similar and still different
J. Mol. Biol. (2005) 17;349(4): 847-60.
EL-Andaloussi S, Johansson H, Magnusdottir A, Järver P, Lundberg P and
Langel Ü
TP10, a delivery vector for decoy oligonucleotides targeting the Myc
protein
J. Control. Release (2005) 10;110(1): 189-201.
Hällbrink M, Kilk K, Elmquist A, Lundberg P, Lindgren M, Pooga M, Jiang
Y, Soomets U and Langel Ü
Prediction of cell-penetrating peptides
Int. J. Pept. Res. Therap. (2005) 11(04): 249-259
Lundberg P
Modifications of cell-penetrating peptide for enhanced delivery: Tissue
targeting and endosomal escape
Curr. Drug Del., In press
Lundberg P, Kilk K and Langel Ü.
Cell-penetrating peptide mediated delivery of peptide nucleic acid oligomers
Cold Spring Harbor, Methods in Gene Delivery, in press
EL-Andaloussi S, Johansson H.J, Lundberg P, and Langel, Ü.
Decoy and siRNA oligonucleotide as cargo in CPP deliveryHandbook of
Cell-Penetrating Peptides, 2nd Edition, CRC Press/Taylor & Francis, Ed.
Langel,Ü., Boca Raton, London, New York, (2006), pp. 375-386
Abstract
Abstract
Cell-penetrating peptides are a class of peptides which have achieved a lot of
recognition due to their vector abilities. Since their discovery over a decade
ago, there has been an uncertainty concerning the mechanism by which they
are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the
general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be
endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery.
The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious
particle in prion disorders. Furthermore, applications in gene regulation and
improvement of delivery efficacy by induction of endosomolysis were examined.
The results obtained confirm the uptake of cell-penetrating peptides to be
endocytic; however the internalization mechanism appears to be peptide
dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate
that the biological response can be increased manifold by the induction of
endosomolysis, either by using lysosomotropic agents or peptides able to
alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cellpenetrating peptides can be achieved by exploiting the intrinsic endocytic
mechanisms involved in the translocation process.
Table of contents
Table of contents
1. Introduction ...............................................................................................1
1.1 Alzheimer’s disease .................................................................................................. 1
1.1.1 The amyloid cascade hypothesis...................................................................... 2
1.1.2 Amyloid precursor protein processing .............................................................. 2
1.1.3 Treatment of Alzheimer’s disease .................................................................... 4
1.2 Prion disorders .......................................................................................................... 5
1.2.1 The prion protein and its structure .................................................................... 7
1.2.2 Prion infection mechanism................................................................................ 7
1.2.3 Prion pathogenesis ........................................................................................... 9
1.3 Endocytosis ............................................................................................................. 11
1.3.1 Endocytic trafficking and sorting ..................................................................... 11
1.3.2 Clathrin mediated endocytosis........................................................................ 12
1.3.3 Caveolae ......................................................................................................... 13
1.3.4 Macropinocytosis ............................................................................................ 14
1.3.5 Clathrin and caveolae independent endocytosis ............................................ 15
1.4 Gene regulation ....................................................................................................... 16
1.4.1 Short interfering RNA...................................................................................... 16
1.4.2 Alternative splicing and splice correction........................................................ 17
1.5 Cell-penetrating peptides ........................................................................................ 20
1.5.1 A brief history of cell-penetrating peptides ..................................................... 21
1.5.2 Mechanisms of CPP internalization ................................................................ 22
1.5.3 CPPs as cargo delivery vectors...................................................................... 23
1.5.4 Methods to improve CPP mediated delivery .................................................. 29
2. Aims of the study ....................................................................................32
3. Methodological considerations .............................................................33
3.1 Synthesis ................................................................................................................. 33
3.1.1 Synthesis and purification of peptides ............................................................ 33
3.1.2 PNA synthesis (Paper IV) ............................................................................... 34
3.1.3 Synthesis of peptide-PNA conjugates (Paper V)............................................ 34
3.1.4 Design of CPPs............................................................................................... 34
3.2 Complex formation (Paper V).................................................................................. 36
3.2.1 Gel shift assay ................................................................................................ 36
3.2.2 Ethidium bromide exclusion assay ................................................................. 37
3.3 Cell cultures............................................................................................................. 37
3.3.1 Chinese hamster ovary cells (Papers I and II)................................................ 37
3.3.2 HeLa cells (Papers III, IV and V) .................................................................... 37
3.4 Internalization and quantification of CPP uptake and delivery................................ 38
Table of contents
3.4.1 Quantitative uptake of fluorescently labeled CPPs (Papers I and III) ............ 38
3.4.2 Study of uptake and degradation of CPPs using HPLC (Paper III) ................ 39
3.4.3 Analysis of CPP uptake using HeLa pLuc 705 cells (Paper IV) ..................... 39
3.4.4 CPP mediated siRNA delivery (Paper V) ....................................................... 40
3.4.5 Confocal- and electron microscopy (Papers I, II, III and IV)........................... 41
3.4.6 Inhibition of γ-secretase activity using CPPs (Paper I)................................... 42
3.5 Methods to study CPP toxicity ................................................................................ 42
3.5.1 Membrane disturbance (Papers IV and V) ..................................................... 43
3.5.2 Examining viability using WST-1 (Papers IV and V)....................................... 44
4. Results and discussion ..........................................................................45
4.1 Uptake and mechanistic studies of CPPs derived from the γ-secretase (Paper I) . 45
4.2 Uptake mechanism of the N-terminal (1-30) part of the bovine prion protein (Paper
II).................................................................................................................................... 46
4.3 Assessing peptide uptake using various methods (Paper III)................................. 47
4.4 Evaluation of CPP mediated PNA delivery for splice correction (Paper IV) ........... 48
4.5 Promoting endosomal escape and siRNA delivery using endosomolytic CPPs
(Paper V) ....................................................................................................................... 49
5. Conclusions.............................................................................................53
Populärvetenskaplig sammanfattning på svenska........................................................ 54
6. Acknowledgements ................................................................................56
7. References...............................................................................................58
List of abbreviations
Abbreviations
Aβ
AChE
AD
AHNP
APH-1
APP
AS
ATP
bPrPp
CHO
CJD
CLIC
CME
CNS
COX
CPP
CT
CTB
DC
DGP
DMF
DMSO
DNA
dsRNA
EB1
ECV
FDC
FITC
flc
GAG
GPI
GVP
HA-2
HIV-1
HPLC
HS
IAP
LDH
LDL
amyloid beta
acetylcholinesterase
Alzheimer’s Disease
anti-HER-2/neu peptide mimetic
anterior pharynx defective
amyloid precursor protein
alternative splicing
adenosine triphosphate
bPrP (1-30)
Chinese hamster ovary
Creutzfeldt-Jakob disease
clathrin independent carriers
clathrin mediated endocytosis
central nervous system
cyclooxygenase
cell-penetrating peptide
cholera toxin
cholera toxin B subunit
dendritic cell
2-deoxy-D-glucose-6-phosphate
dimethylformamide
dimethyl sulfoxide
deoxyribonucleic acid
double stranded RNA
endosomolytic penetratin analogue
endosomal carrier vehicle
follicle dendrite cell
fluorescein isothiocyanate
fluorescein
glycosaminoglycan
glycosylphosphatidylinositol
Gal4 DNA-binding/VP16 transactivating domain
hemagglutinin N-terminal fusion domain
human immunodeficiency virus 1
high performance liquid chromatography
heparan sulfate
inhibitor of apoptosis proteins
lactate dehydrogenase
low-density lipoprotein
List of abbreviations
LTR
MBHA
miRNA
mPrPp
mRNA
MVBs
NAD
NCT
NLS
NMDA
Npys
NSAID
ON
PEG
PEN-2
PL1
PMO
PNA
PPT
Prnp
PrP
PrPC
PrPSc
PrPCtm
PrPNtm
PS1
PS2
RISC
RNAi
siRNA
ssRNA
SOD
t-Boc
TAT
Tf
TP
TP10
TSE
WST-1
long-terminal repeat
4-methylbenzylhydrylamine
micro RNA
mPrP (1-28)
messenger RNA
multivesicular bodies
nicotinamide adenine dinucleotide
nicastrin
nuclear localization signal
N-methyl-D-aspartate
3-nitro-2-pyridinesulfenyl
non-steroidal anti-inflammatory drugs
oligonucleotide
polyethylene glycol
presenilin enhancer-2
presenilin (151-162) [Ser158]
phosphorodiamidate morpholino oligomers
peptide nucleic acid
polypyrimidine tract
prion protein gene
prion protein
cellular prion protein
scrapie prion protein
C-terminal transmembrane isoform of PrP
N-terminal transmembrane isoform of PrP
presenilin 1
presenilin 2
RNA induced silencing complex
RNA interference
short interfering RNA
single stranded RNA
superoxide dismutase
tert-butuloxycarbonyl
transactivator of transcription
transferrin
transportan
transportan-10
transmissible spongiform encephalopathy
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2 H-5-tetrazolio]1, 3-benzene disulfonate sodium salt
1. Introduction
1. Introduction
The plasma membrane poses an impenetrable barrier for macromolecules not
having active transporters to aid in their delivery. Macromolecules are a
broad concept including a very diverse set of molecules, such as proteins,
carbohydrates, oligonucleotides (ONs) and fatty acids. As efficient delivery
of macromolecules is essential for researchers studying cellular functions or
in drug delivery, it is also a pivotal component in nature for survival and
development. Consequently, as the importance for developing delivery systems is crucial for life, including exclusion of unwanted and harmful compounds, the process is very efficiently and highly regulated. Despite this fact,
there are many infectious agents that are able to exploit the naturally developed system in order to infect its target cell, such as bacteria, viruses and
prions.
The purpose of this thesis is to elucidate the role of cell-penetrating peptides derived from naturally occurring proteins, methods to quantify their
internalization and what consequences this penetration property might have
in nature. Furthermore, using the knowledge obtained from these studies,
applications in the delivery of ONs for use in gene regulation is assessed. A
substantial portion of the peptides studied are derived from the γ-secretase
complex involved in Alzheimer’s disease, and the prion protein, thus these
two neurodegenerative disorders will be covered. Also, various means of
gene regulation where cell-penetrating peptides (CPPs) can be applied will
be discussed, as well as the general process of endocytosis which is responsible for the internalization of CPPs.
1.1 Alzheimer’s disease
Alzheimers’s disease (AD) is the most common cause of dementia among
elderly, and accounts for approximately 50% of the cases (Lobo et al. 2000),
with roughly 4.5 million suffering from the disease in the United States and
5 million in the European Union. With the present increase in life expectancy scientists estimate this figure to be doubled or tripled by the year 2050
(Cupers et al. 2006; Hodes 2006). The approximation of prevalence varies
between studies, although a rough estimate is that between 5.9% to 9.4% of
European citizens over 65 years of age have dementia, where AD represents
most of the cases (Hodes 2006). Increasing with age, as many as 20-40% of
the population above 80 may be affected (Small et al. 1997). Early symp1
1. Introduction
toms include short-term memory impairment and a general cognitive decline, which develops into deficits in spatial memory and motor abilities.
The duration is estimated to be 5-15 years; the cause of death usually originates from secondary illness such as pneumonia or other infections. The
most common form of AD is a late onset sporadic variant, which accounts
for approximately 90% of AD patients. Amongst the genetic causes for the
disease, carrying the ε4 allele of apolipoprotein E, a protein involved in lipoprotein metabolism, is considered the major risk factor.
Since its discovery in 1906 by Alois Alzheimer, the pathological hallmarks of AD include amyloid plaques and neurofibrillary tangles. Amyloid
plaques are extracellular deposits primarily consisting of the amyloid βpeptide (Aβ), which is found in two major forms harboring either 40 or 42
amino acids. Aβ originates from the amyloid precursor protein (APP), which
can be processed by three different protease complexes called α-, β-, and γsecretase.
1.1.1 The amyloid cascade hypothesis
In the early 1990’s it was discovered that mutations in the gene encoding
APP caused over production of Aβ42, with subsequent early onset of AD
(Citron et al. 1992; Cai et al. 1993; Suzuki et al. 1994). These early discoveries, supported by previous findings that individuals with Down’s syndrome
(trisomy 21, the chromosome containing the APP gene) develop AD at an
early age (Olson and Shaw 1969) led to the formulation of the amyloid cascade hypothesis (Selkoe 1991; Hardy and Higgins 1992). The theory, further
supported by the findings that mutations in the presenilin genes leads to
overproduction of Aβ42 (Borchelt et al. 1996; Duff et al. 1996; Citron et al.
1997), thus postulating a causative role of Aβ42 depositions in the pathogenesis of AD. Even though it is still recognized that amyloid deposits are
observed early on in AD, oligomeric forms of Aβ42, not the aggregates, are
thought to be the most neurotoxic species and recent findings suggests a
dodecameric species, designated Aβ*, to be responsible for causing the
memory deficits (Lesne et al. 2006).
1.1.2 Amyloid precursor protein processing
APP is a type 1 transmembrane protein differentially spliced into three diverse isoforms composed of 695, 751 and 770 amino acids, where the 695
isoform is the most abundant in neurons. It has been suggested to be involved in cell adhesion, signaling and gene regulation, where knock-out
mice are viable but displays age-related cognitive impairments and reduction
in synaptic density (Dawson et al. 1999).
2
1. Introduction
The main proteolytic processing of APP is performed by three different
proteases, α-, β-, and γ-secretase (Fig. 1.1). Processing of APP by αsecretase leads to a non-amyloidogenic pathway, whereas the amyloidogenic
processing occurs when APP is cleaved by β-secretase, a membrane bound
aspartyl protease (Vassar et al. 1999), followed by γ-secretase. Processing by
β- and γ-secretase also creates the APP intracellular domain (AICD). The
physiological role for this processing is not well characterized, although the
AICD has been implied to be transcriptionally active (Cao and Sudhof
2001), and to form complexes with Fe65 and Tip60, two proteins involved in
transcription regulation and signal transduction.
Fig 1.1. Secretase processing of APP. APP processed by α-secretase leads to the non amyloidogenic pathway as the Aβ sequence is interrupted. If APP is processed by β- and γ-secretase
Aβ is formed, which can oligomerize and aggregate, probably causing the memory deficits
and neurotoxicity observed in AD.
The elusive intramembrane γ-secretase cleavage is performed by a high
molecular weight protein complex consisting of presenilin-1 (PS1), nicastrin
(NCT), anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN2) (Fig. 1.2). As previously mentioned, PS1 and presenilin-2 were first identified for their linkage with early onset familial AD (FAD). PS1 is a multispanning transmembrane protein containing eight (Vassar, et al. 1999) or
nine (Laudon et al. 2005) transmembrane regions, and is subjected to endoproteolysis generating a 30 kDa N-terminal fragment and a 20 kDa Cterminal fragment (Thinakaran et al. 1996), which can form highly stable
complexes with the other γ-secretase constituents. It is thought that PS1 is
activated by this endoproteolysis, being a crucial step in γ-secretase matura-
3
1. Introduction
tion, and that the full length PS1 is an inactive precursor (Ratovitski et al.
1997).
NCT was first discovered through partial purification of PS1 interacting
proteins, and subsequently shown to modulate γ-secretase processing of APP
(Yu et al. 2000). It is a type 1 transmembrane glycoprotein and has an expected molecular weight of 80 kDa, but in its mature form being approximately 150 kDa due to glycosylations and sialylations within the secretory
pathway. APH-1 and PEN-2 were both identified in genetic screens for mutations affecting γ-secretase activity in C. elegans (Francis et al. 2002). It
was subsequently shown by Takasugi and colleagues that the role of APH-1
in the γ-secretase is to stabilize the complex, whereas PEN-2 is required for
endoproteolytic processing of PS1 yielding the γ-secretase activity (Takasugi
et al. 2003).
Fig 1.2. Membrane topology of the constituents. The exact protein-protein interactions between the involved proteins are unknown. PS1 is thought to be activated through an endoproteolytic cleavage, and is here shown in its 8-transmembrane topology, which is the most
commonly accepted model. The other γ-secretase constituents are modulating the γ-secretase
activity although the exact mechanisms are unknown.
1.1.3 Treatment of Alzheimer’s disease
There are currently two classes of drugs approved for the treatment of AD,
acetylcholinesterase (AChE) inhibitors and N-methyl-D-aspartate (NMDA)
receptor antagonists. AChE inhibitors prolong the action of acetylcholine in
the synapse by reducing the breakdown, a strategy resulting in improvements
of cognition, mood and behavior. NMDA receptor antagonists are thought to
mediate their effect by regulating the levels of glutamate. Even though both
of these approaches for treatment of AD show to some extent a positive effect on cognition, neither of them are able to effectively influence the disease
4
1. Introduction
progression. Also, approximately 30-40% of the patients given AChE inhibitors do not respond to the treatment (Mount and Downton 2006).
1.2 Prion disorders
Transmissible spongiform encephalopathies (TSEs), often called prion disorders, are neurodegenerative disorders affecting both humans and animals
(Table 1.1). The neuropathological characteristics within the central nervous
system (CNS) include spongiform alterations, neuronal loss, glial activation
and accumulation of amyloidal aggregates. The most common form of TSE
in humans is Creutzfeldt-Jakob disease (CJD), which encompass the sporadic (sCJD), familial (fCJD), variant (vCJD) and iatrogenic (iCJD) forms.
Even though the sporadic form of CJD is the most common, the annual incidence is only 0.6-1.2 × 10-6 (Ladogana et al. 2005). The etiology of sCJD is
unknown, where no endogenous or exogenous cause have been identified so
far. The familial forms of CJD are transmitted as autosomal dominant traits
which always correlate with the Prnp gene encoding the prion protein, although some experimental results suggest that additional factors could contribute to the disease progression. (Stephenson et al. 2000; Lloyd et al. 2001;
Manolakou et al. 2001; Moreno et al. 2003). Iatrogenic prion dsorder is usually caused by the transplantation of tissues or pituitary hormones from deceased patients suffering from an unrecognized TSE. The vCJD is the prion
disorder that has gained most attention the last decade due to an extreme rise
of cases in the UK during the late 1990’s, where an epidemic was feared.
The origin of the disease was cattle ingesting TSE contaminated meat and
bonemeal, which was further transferred to humans eating BSE infected
meat. However, since then the numbers of reported cases have been stabilized, and even reduced (Valleron et al. 2001). However, as a result of the
long incubation time of CJD, varying between 1.5 to more than 40 years, the
threat is not over yet.
Prion stems from a word jumble of proteinaceous infectious particle
(Prusiner 1982). PrPC (cellular prion protein) is expressed in a wide variety
of cells (Moudjou et al. 2001), and its endogenous function is unknown,
although there are some experimental evidence suggesting that PrPC might
be a superoxide dismutase (SOD) (Brown et al. 1997; Brown et al. 1999).
The first prion disease described was scrapie in sheep, as early as in the mid
18th century, but the first cases of human CJD were described in the early
1920s (Creutzfeldt 1920; Jakob 1921). The first successful experimental
transmission was performed in 1939 (Cuille and Chelle 1939), and approximately 20 years later kuru was described among the Fore people of Papua
New Guinea (Gajdusek and Zigas 1957), followed by the discovery of the
resemblance between kuru and scrapie (Hadlow 1959). Experiments aiming
5
1. Introduction
to inactivate the infectious particle, following the protocols used for bacteria
and viruses, failed, which led to the first statement of the protein-only hypothesis (Griffith 1967), stating that a protein was the infectious particle.
More than a decade later, Prusiner and colleagues purified a hydrophobic
protease resistant protein from brains of scrapie infected hamsters (Prusiner
et al. 1980), and a few years afterwards PrPC was cloned for the first time
(Chesebro et al. 1985; Oesch et al. 1985) and it was established that the PrPC
and the infectious scrapie form (PrPSc) isoforms were encoded by the same
host gene (Basler et al. 1986). These discoveries led to the conclusion that it
was a proteinaceous particle, the prion, which was responsible for the transmission of the diseases reported. The discovery that Prnp 0/0 (double
knockout of the gene encoding for PrPC) mice were resistant to scrapie disease (Büeler et al. 1993), and that there were structural differences between
PrPC and PrPSc (Pan et al. 1993), were the final proofs needed for to understand that the cause of the disease was the lethal conversion of endogenous
PrPC to PrPSc (Büeler, et al. 1993).
Table 1.1. Selection of prion disorders affecting humans and animals.
Natural
host species
Transmission route
or disease induction
mechanism
Creutzfeld-Jacob disease
Humans
Sporadic, familial or
iatrogenic
Variant CreutzfeldJacob disease
Humans
Ingestion of BSE
contaminated meat
Kuru
Humans
Ingestion
Fatal familial insomnia
Humans
Familial
(Gajdusek and Zigas
1957; Gajdusek et al.
1967)
(Tateishi et al. 1995)
Gerstmanm-SträusslerSheinker syndrome
Humans
Familial
(Tateishi et al. 1984)
Scrapie
Sheep and
goats
Horizontal
(Gibbs et al. 1980;
Bruce et al. 2002)
Bovine spongiform
encephalopathy
Cattle
Ingestion of contaminated feed
(Lasmezas et al. 1996;
Bruce, et al. 2002)
Disease
6
Reference
(Gibbs and Gajdusek
1969; Taguchi et al.
2003)
(Taguchi, et al. 2003)
1. Introduction
1.2.1 The prion protein and its structure
The human PrPC consists of an unstructured N-terminal region (residues 23124) and an ordered C-terminal region (125-228) containing three α-helices
and two short β-strands flanking the first α-helix (Zahn et al. 2000). The
hypothesis is that the prion protein itself is the infectious particle responsible
for prion diseases, and upon infection, PrPC is converted by PrPSc into the
protease resistant scrapie form of the protein. The difference in structural
elements is drastic, where PrPC encompasses approximately 3% β-sheet
structure compared to 45% for PrPSc (Prusiner 1998). However, the intrinsic
property of PrPSc to form large aggregates, hypothesized to act as a seed for
the conversion of PrPC to PrPSc, have precluded experiments trying to solve a
high resolution structure for the protein. Given that the theory postulating
PrPSc to act as a seed for the conversion of PrPC to PrPSc proves correct, it is
unlikely that a monomeric form of PrPSc exists. During the last couple of
years there has emerged much evidence indicating that prion propagation has
the characteristics of a nucleated polymerization reaction, both in fungi and
mammalian cells. Even though the data generated from studies in fungi
strongly support this statement, there is some discrepancy regarding mammalian cells. Studies generating fibrils from recombinantly produced PrP
have drawn the conclusion that the species barrier in prion infection is due to
intrinsic properties of PrP which will be translated into the amyloid fibre
structure (Baskakov 2004; Jones and Surewicz 2005; Leffers et al. 2005).
These conformational differences are often referred to as strains. The different strains have characteristic features such as variances in incubation time
preceding the disease, severity of the neuropathological lesions, neuroanatomic distribution and protease resistance. The differences also affect the
species barrier. An example of the species barrier is that human kuru or CJD
brain tissue are unable to transmit the disease onward to goats or ferrets, but
if transmitted to cats or primates, their brain tissue will further transmit to
goats or ferrets.
1.2.2 Prion infection mechanism
How transmission of a prion disease occurs is often unclear, although PrPC
expression is a necessity to be susceptible to the disease (Büeler, et al. 1993).
Considering the cases found in the UK, being infected with BSE contaminated meat, and the ones in Papua New Guinea, where ritualistic cannibals
were infected after consumption of diseased brains (Gajdusek and Zigas
1957), it is clear that infection can occur by oral ingestion of PrPSc contaminated meat. However, the pathway of infection after the oral exposure is
somewhat unclear. The first area to be exposed to the prion agents are the
lymphoid tissues, where accumulation occurs, such as in the spleen and
7
1. Introduction
lymph nodes. After this initial step, the pathogen can be further spread to the
nervous system. If the infectious agent still lingers in the lymphoid tissue is
debatable, since none has been detected in natural BSE cases in cattle (Terry
et al. 2003), whereas it is abundant in vCJD cases in humans, although with
a big variation in the measured levels (Wadsworth et al. 2001). Possibly, this
variation is connected to the different strains reported of the prion agent, as a
variation of infected tissue can be observed within a host species depending
on which type of prion disease that was acquired (Glatzel et al. 2003). Interestingly, the initial accumulation in lymphoid tissue, usually in follical dendritic cells (FDCs), is essential for effective neuroinvasion, and depletion of
FDCs might prove to be one of the few ways to treat prion diseases (Mabbott
et al. 2000; Montrasio et al. 2000), although this approach would only be
efficient before neuroinvasion occurs. The accumulation in FDCs and other
lymphoid structures can also provide insight into the extreme variation in
incubation time observed between species and individuals. One hypothesis is
that the levels of expression of PrPC in the affected lymphoid tissue could
determine the rate by which early accumulation develops into neuroinvasion,
where FDCs express high levels of PrPC and are therefore the primary source
of prion replication (Brown et al. 1999).
However, before the FDCs are reached, the infectious agent needs to
cross the gut epithelium. The mechanism by which the prion crosses this
barrier is largely unknown, although some recent findings have hypothesized
that M cells and dendritic cells (DCs) might be responsible. M cells are
epithelial cells specialized in the trans-epithelial transport of macromolecules (Neutra et al. 1996). They are exploited by bacteria and viruses in transcytotic transport (Neutra et al. 1996), and have furthermore been suggested
to be responsible for prion transport across the gut epithelium (Heppner et al.
2001), although in vivo confirmation has so far not been reported. The reports concerning transepithelial prion transport by DCs are not altogether
conclusive. Degradation of PrPSc occurs in DCs by cysteine proteases (Luhr
et al. 2004), however, they can also contain and transport non-degraded
PrPSc (Huang et al. 2002), showing optimal features for being a prion transporter.
Emerging from the gut epithelium to the lymphoid tissues, the next step
of transport for the prions is to their final destination, the brain. The spread
of prions from the gut epithelium is mediated by the enteric nerve system
(Haik et al. 2003), which is a component of the autonomic nerve system
regulating intestinal stimuli from sympathetic- and parasympathetic nerves.
The prion infection is thought to spread through both of these systems
(Beekes et al. 1998; Glatzel et al. 2001), where the parasympathetic nerves
allow direct integration of the prions in the CNS without passing through the
spinal cord (Beekes, et al. 1998). However, how the initial spread from the
FDCs to the peripheral nervous system occurs is unknown.
8
1. Introduction
1.2.3 Prion pathogenesis
The damage that occurs in prion disorders is mainly manifested in the CNS.
Much research on prion toxicity has been made on the PrP (106-126), which
has been shown to be toxic to cultured neuronal cells (Forloni et al. 1993). It
is, however, hard to establish proofs that the oligomeric forms of PrP (106126) will be able to mimic the complex aggregates that are formed in vivo by
PrPSc. Furthermore, it has been commonly assumed that these aggregates
formed by PrPSc are the primary source for neurodegeneration in prion diseases, however, like in AD, there are reports that the neurotoxicity observed
has little correlation with PrPSc aggregation. Also, massive PrPSc aggregation
can occur without any obvious toxicity. As in AD, an intermediate between
PrPC and PrPSc designated PrP* has been proposed to be the neurotoxic species (Cohen et al. 1994), however so far there have been no experimental
evidence supporting this theory.
Recent experiments have indicated that an unusual transmembrane form
of PrPC may play a pathogenic role. When the prion protein is synthesized, it
is, after transport to the plasma membrane, attached to the outer leaflet of the
membrane via a glycosylphosphatidylinositol (GPI) anchor. However, two
other forms can be observed, termed PrPCtm and PrPNtm (Hegde et al. 1998),
which have alternate topology, PrPCtm being attached to the plasma membrane via a GPI anchor but with a transmembrane topology and retaining the
signal sequence (1-22), whereas the PrPNtm form is situated in the membrane
as a type 1 transmembrane protein lacking N-glycosylations (Fig. 1.3). Both
of these proteins span the membrane with a highly conserved hydrophobic
part of the prion protein (amino acids 111-134). Although these alternative
forms are expressed in comparatively small amounts (<10% of the total PrP),
mutations in close proximity to, or within the hydrophobic region such as the
A117V mutation, can increase the relative proportion of PrPCtm to as much as
20-30% of the total PrP (Stewart et al. 2001; Stewart and Harris 2001).
Transgenic mice expressing mutated PrP with a propensity for the PrPCtm
form spontaneously develop scrapie-like symptoms, without any detectable
levels of PrPSc (Hegde, et al. 1998). Furthermore, the toxicity level incurred
by PrPSc depends on the predilection of the host cell to produce PrP in the
PrPCtm form (Hegde et al. 1999). Also, PrPCtm generation closely follows the
time course of PrPSc accumulation. Thus, the levels of PrPCtm can be altered
either by mutations in PrP, leading to an increased ratio of PrPCtm as compared to PrPC, or by accumulation of PrPSc; where a combination of these
events would definitely push the PrPCtm levels above the critical threshold
needed for developing the disease. This gives PrPCtm a critical role both in
the infectious pathway of prion diseases, as well as in genetic variants.
9
1. Introduction
Fig 1.3. The proposed topologies for PrPC, PrPCtm and PrPNtm, where the two latter forms are
transmembrane. PrPCtm has a retained signal sequence N-terminally, and is suggested to have
a crucial role in prion toxicity.
The exact mechanism leading to neurodegeneration in prion disorders is
unknown, although apoptosis has been observed in scrapie infected mice
(Giese et al. 1995). The molecular cause for triggering apoptosis is unknown. Isolated microglia treated with PrP (106-126) yields increased levels
of reactive oxygen species and cytokines which ultimately could lead to
apoptosis (Brown et al. 1996). Also, if the hypothesis that PrPC is a SOD, the
loss of SOD function upon conversion to PrPSc could in itself be a neurotoxic
event. Another mechanism thought to be involved in prion toxicity is the
retention of PrPCtm in the ER. The retained signal sequence in PrPCtm is
probably caused by an inability to enter the lumen of the ER, where a signal
peptidase normally cleaves off the signal peptide. A proposed mechanism for
the toxicity of PrPCtm is that the observed in vitro accumulation of this form
in the ER would trigger stress-induced signals that are activated by an accumulation of misfolded proteins in the ER. Another theory is that accumulation of PrPCtm, possibly in combination with PrPSc, will overload the proteasome system, triggering stress responses resulting in apoptosis. Surprisingly,
PrPCtm was recently shown to accumulate in the Golgi apparatus in vivo
(Stewart and Harris 2005), and maybe a revised theory of the neurotoxic
mechanism is needed.
10
1. Introduction
1.3 Endocytosis
Endocytosis is an essential process for cells in order to regulate nutrient uptake and signal transduction. Depending on the type of stimuli, various forms
of endocytosis can occur. Although being a highly regulated process, endocytosis can also be exploited for the internalization of pathogens, such as
viruses, bacteria and parasites. Endocytosis integrates extracellular stimuli
and intertwines them with intracellular signaling pathways, hence being one
of the most important parts in cellular organization. There are several types
of endocytosis, such as clathrin mediated endocytosis (CME), which includes most receptor mediated endocytosis, and clathrin independent endocytosis, consisting of e.g. caveolae and macropinocytosis (Fig 1.4.). To establish which endocytic pathway that is used for a certain particle, endocytosis inhibitors, markers and antibodies can be used. After internalization, the
endocytic vesicle is “tagged” to denote what the final destination is. Subsequent to docking and fusion to the target membrane, the endocytic vesicle
and, when called for, receptors, are recycled to the plasma membrane, if not
the fate of the internalized material is degradation.
Fig 1.4. Schematic representation of the different endocytic processes taking place in mammalian cells (modified from (Conner and Schmid 2003)). Macropinocytosis is a protrusion of
actin filaments, whereas the other endocytic mechanisms are initially seen as membrane
invaginations.
1.3.1 Endocytic trafficking and sorting
The trafficking of endocytic vesicles largely depends on the internalization
route, where receptors necessary for the maintenance of the cell are constitutively endocytosed, independent of ligand occupancy, whereas the internalization level of other receptors and endocytic pathways are dependant on
ligand occupancy and other stimuli. Internalized receptors are either recycled
11
1. Introduction
to the plasma membrane or retained in the cell with concomitant degradation
in the proteosomal or lysosomal pathways (Kurten 2003). Hence, for exploiting endocytic pathways, it is of great importance to understand how to trigger endocytosis, and direct the uptake so that the molecule of interest is not
degraded.
In clathrin CME (described below), recycling receptors are rapidly
(t1/2~2.5 min) detached from the ligand and recycled back to the plasma
membrane. The sorting of receptors is probably depending on certain amino
acid sorting motifs, although so far this characterization is inconclusive
(Gruenberg 2001). If recycling does not occur, as in the case for e.g. downregulated receptors, the cargo will be transported to the late endosomes, and
further on to the lysosomes for degradation. Transport between the early and
late endosomes are ascribed an entity called multivesicular bodies (MVBs)
or endosomal carrier vehicle (ECV) (Gruenberg and Stenmark 2004). Upon
maturation to late endosomes, the vesicles are again sorted, some directed
towards Golgi and others to the lysosomal compartements. The lysosomes
are the final destination, where the proteins targeted for down-regulation will
be degraded by numerous proteases, and its constituents reused.
1.3.2 Clathrin mediated endocytosis
Clathrin mediated uptake, often referred to as the “classical” endocytosis, is
the best characterized of the endocytic pathways. CME was previously often
referred to as receptor based endocytosis, but it is nowadays well established
that most pinocytic pathways also involve a specific receptor-ligand interaction to induce membrane ruffling with concomitant uptake. CME is a process constantly taking place in mammalian cells for continuous uptake of
essential nutrients, such as the low-density lipoprotein (LDL) and transferrin
(Tf) for internalization of cholesterol and iron, respectively. CME has a central role in regulating cellular responses by controlling the levels of cellsurface receptors as well as mediating the rapid clearance and downregulation of already activated receptors. Furthermore, it is essential for neuronal activity, being responsible for internalization of voltage-gated calcium
channels in order to control the strength of the synaptic responses, as well as
recycling membrane proteins after neurotransmission (De Camilli and Takei
1996). However, CME is also the endocytosis route most preferred for cellular entry of viruses. It transports viruses along with receptors into early and
late endosomes, and is a rapid and efficient way for viral entry. The internalized viruses are often exposed to an acidic milieu within minutes, leading to
the structure conversion of necessary proteins with subsequent penetration of
the endosomal membrane. Depending on the pH threshold for such processes, the endosomal escape would either be promoted at the early endosomes (pH~5.5-6.5) or late endosomes (pH 5-6). In certain cases, pH
12
1. Introduction
structure conversion of certain proteins per se is not sufficient for endosomal
fusion, e.g. the ebola- and SARS viruses need an acid-dependent proteolytic
cleavage in specific viral proteins in order to become “activated” and enter
the penetration competent state (Chandran et al. 2005; Simmons et al. 2005).
CME is initiated through the binding of a ligand to its receptor, upon
which a lateral movement of the receptor-ligand complex occurs, concentrating them in coated pits, endocytic “hot spots”, on the plasma membrane.
These pits are formed by the assembly of cytosolic coat proteins, the main
part constituted by clathrin together with adaptor proteins. These coated pits
eventually pinch off from the plasma membrane, a process regulated by the
GTPase dynamin, which is a master regulator of membrane trafficking also
involved in phagocytosis, CME, caveolae-mediated endocytosis and CME
and caveolae independent endocytosis (Conner and Schmid 2003). The role
of dynamin is essential, although its exact role is not clear and two different
hypotheses exist (Sweitzer and Hinshaw 1998; Stowell et al. 1999). After the
endocytic vesicle is pinched off, coat proteins such as clathrin are recycled
back to the plasma membrane and reused, whereas the endocytic vesicle is
targeted to the early endosome for sorting.
As described below, PrPC has been suggested to be internalized via caveolae mediated endocytosis, however, PrPC is also suggested to be able to leave
the lipid raft and be endocytosed by CME, a process ascribed to be dependent on the short cationic N-terminal (amino acids 23-28) motif (NH2KKRPKP) (Sunyach et al. 2003). Furthermore, extracellular stimuli, such as
treatment with Cu+2 seem to trigger PrPC to be endocytosed via CME
(Sunyach, et al. 2003).
1.3.3 Caveolae
Caveolae are small membrane invaginations, ~60 nM in diameter, whose
role in endocytosis have been highly debated. Caveolae have been reported
to be responsible for the internalization of various pathogens, such as cholera
toxin (CT) and the SV40 virus. It has also been implicated that caveolae are
responsible for the internalization of GPI-anchored proteins, as a high concentration of these can be found in caveolae structures. However, recent
studies using fluorescence recovery after photobleaching (FRAP) techniques
have shown that caveolae structures are highly immobile, and have a very
slow turn-over at the plasma membrane (Thomsen et al. 2002).
Caveolae were discovered over 50 years ago using electron microscope
techniques, when it was observed that certain cells were covered with thousands of small pits, which more than a decade later were shown to be responsible for transendothelial transport (Bruns and Palade 1968). Since then,
various toxins and viruses have been reported to be internalized via caveolae. The caveolar pits are covered with the protein caveolin (Rothberg et al.
13
1. Introduction
1992), which was the first molecular marker for caveolae. Depending on cell
type studied, either caveolin-1, caveolin-2 or caveolin-3 can be found, where
caveolin-1 and 2 often colocalize throughout the body (Scherer et al. 1997)
while caveolin-3 is preferably localized in skeletal and cardiac muscles
(Way and Parton 1995). Caveolin-1 null mice lack caveolae in all cell types,
except in differentiated striated muscle cells where caveolin-3 is situated
(Drab et al. 2001; Park et al. 2002). Being ubiquitously expressed, it is likely
that caveolin have a crucial role in bodily functions such as signaling and
transport. Surprisingly, knock out mice lacking caveolin-1 show no phenotypic changes and appear healthy. However, in depth analysis of these mice
showed that they had pulmonary defects and abnormities in vasoconstriction
and dilation (Drab, et al. 2001). Also, changes in albumin levels were observed, supporting the role of caveolae as a vesicular transporter.
As mentioned earlier, caveolae have been reported to be responsible for
the internalization of CT, which also have been used as a molecular marker
for caveolar endocytosis. However, recent reports claim the uptake of CT not
to be exclusively mediated by caveolae endocytosis, but also via clathrin
coated pits. Even more confusing; expression of a dominant-negative dynamin mutant, thus inhibiting both clathrin- and caveolae-mediated endocytosis, only causes a 50-70% inhibition of CT uptake, indicating that there
could be an additional mechanism responsible for the uptake (Torgersen et
al. 2001), or that there is a compensation mechanism for the loss of these
pathways of endocytosis. Also, reports claiming the uptake of SV40 viruses
not to be exclusively mediated by caveolae- or clathrin-mediated endocytosis
show that CT might not be an isolated case (Damm et al. 2005). In addition,
the original statement that caveolae were responsible for the internalization
of GPI-anchored proteins has also proven at least partly faulty, as uptake of
the GPI-anchored diphtheria toxin receptor is via a non-caveolin mediated
endocytosis (Skretting et al. 1999). However, another GPI-anchored protein,
the prion protein, has been shown to be, at least partly, internalized via
caveolae. In this study, immunogold labeled PrPC was monitored in several
cell lines, and found to be enriched in both caveolae and lamellae structures
(Peters et al. 2003). Furthermore, the PrPC containing endosomes did not
intersect with classical early endocytic sorting, as clathrin mediated endocytosis, and was separate from other GPI-anchored proteins, a result which is
in concurrence with the study described above by Skretting and colleagues.
1.3.4 Macropinocytosis
Macropinocytosis is an actin dependent endocytosis where the membrane is
ruffled as a response to some stimuli, followed by the internalization of huge
(>1µm) endosomes created by the fusion of the protruded actin “arm” with
the plasma membrane. There is so far no evidence that macropinocytosis, in
14
1. Introduction
contrary to CME, concentrate receptors prior to endocytosis. It has, similarly
with CME and caveolae, been implied to be responsible for the uptake of
certain pathogens, such as bacteria and viruses, e.g. Mycobacterium tuberculosis (Garcia-Perez et al. 2003) and adenoviruses (Meier et al. 2002), but is
normally induced as a response to certain growth factors or other signals.
The membrane ruffles creating macropinosomes have been attributed to colocalize with lipid rafts, and alike caveolae, macropinocytosis is inhibited by
the depletion of cholesterol (Grimmer et al. 2002). Although the exact
mechanism of macropinocytosis is undefined, inhibition of phosphoinositide
3 (PI3)-kinase activity diminishes the vesicle formation, interestingly without affecting the membrane ruffling (Araki et al. 1996; Araki et al. 2003).
Also, cytochalasins blocking actin polymerization and amiloride which inhibits the Na+/H+ exchange pump are potent inhibitors of macropinocytosis.
Experimental data suggest that there are two distinct forms of micropinocytosis, where the recycled variant is induced by growth factors, whereas processive macropinocytosis is responsible for the uptake of particles and bacteria; a mechanism mediated by a diverse group of receptors such as the mannose receptor and CD14 (Meier and Greber 2004).
1.3.5 Clathrin and caveolae independent endocytosis
As previously discussed, many so called specific markers of a perticular
endocytic pathways have been shown not to be exclusively internalized by
the pathway intended, and in many cases reported to be internalized via an
uncharacterized endocytic pathway. These novel endocytosis variants,
termed CLICs (clathrin independent carriers), have at least two separate
endocytic pathways, one being dynamin dependent, and one independent
(Kirkham and Parton 2005). The dynamin independent mechanism has been
shown to be responsible for the internalization of GPI-anchored proteins, the
uptake directed to the recycling endosomal compartment but not to Golgi,
and being dependent on the small GTPase cdc42 (Sabharanjak et al. 2002).
Furthermore, the same pathway seems to be responsible for the internalization of the CT B subunit (CTB) in both caveolin-1 containing and caveolin-1
null cells. Further characterization showed the vesicle uptake of CTB to colocalize with fluid phase markers such as dextran and HRP, but not with Tf
(Kirkham et al. 2005). In a very interesting study, a macropinocytosis variant, possibly CLIC, was shown to promote the uptake of heparan sulfate
proteoglycans (syndecan-4) with a bound growth factor, FGF-2 (Tkachenko
et al. 2004). As syndecan-4 is associated with PKCα, it indicates that syndecan-4 might work as a receptor for certain ligands, without containing any
intrinsic catalytic activities. A recent report has shown that the protein flotillin-1 strongly co-localize with CTB, especially in cell expressing a dominant-negative mutant of dynamin-2 (Glebov et al. 2006), suggesting that
15
1. Introduction
flotillin-1 is a separate type of region in the plasma membrane, distinct from
those of clathrin and caveolin-1. This data indicate that flotillin-1 could be a
selective marker for the CLIC endocytic pathway.
The other CLIC pathway, called CLIC-d for its dynamin dependence, was
shown, by using Eps15 dominant negative mutants, thus blocking CME, to
internalize the interleukin-2 receptor (Lamaze et al. 2001). The same endocytic process was further shown to be responsible for endocytic uptake of the
common γc cytokine receptor (Sauvonnet et al. 2005).
All these results together show that CLIC and CLIC-d might be important
endocytic components responsible for the uptake of GPI-anchored proteins,
growth factor receptors and possibly ligands interacting with glycosaminoglycans (GAGs).
1.4 Gene regulation
Regulating gene expression in order to e.g. target cancer growth or characterize protein function can be achieved in a variety of ways; either directly
through down-regulation of the gene of interest via transcriptional blocking,
or modification of expression by interfering with pre-mRNA processing and
translation. Depending on the target and the preferred longevity of the effect,
a range of nucleotides and analogues can be used.
1.4.1 Short interfering RNA
The strategy for gene regulation receiving the most attention the last years is
RNA interference (RNAi) using short interfering RNA (siRNA). It is a very
appealing strategy to use for down-regulation of target mRNA due to its
specificity and high gene-silencing effect (Davidson and Paulson 2004).
RNAi was initially described in plants and C. elegans when it was observed
that double stranded RNA (dsRNA) was much more efficient in gene silencing than either the sense or anti-sense strand RNA (Fire et al. 1998). RNAi is
a process mainly used for silencing endogenous gene expression, e.g. in embryonic development, but it is also used as a protection mechanism against
viruses in plants and lower vertebrates.
Processing of double stranded RNA by Dicer and Drosha
The RNAi mechanism is divided into two separate steps. First, dsRNA is
processed into a 21-23 nucleotide long siRNA by two dsRNA specific
RNase-III-type endonucleases, Dicer and Drosha. Drosha is required for the
processing of micro RNA (miRNA) precursors, which are endogenous RNAi
16
1. Introduction
molecules first synthesized as complementary strands connected via a hairpin loop, which can be processed by Drosha into dsRNA (Lee et al. 2003).
After this process, which takes place in the nucleus, the processed miRNA is
translocated into the cytosol (Lund et al. 2004), where it is further processed
by Dicer (Grishok et al. 2001). There are two forms of Dicer, called 1 and 2,
where Dicer-1 preferentially processes miRNA and Dicer-2 long dsRNA
(Lee et al. 2004). This process yields the bioactive siRNA and miRNA duplexes which can be incorporated into the RNA-induced silencing complex
(RISC) to direct gene silencing.
Cleavage of mRNA by RISC
The second step of RNAi is the assembly of RISC and incorporation of a
single stranded RNA (ssRNA) into the complex for concomitant mRNA
cleavage. The incorporation of RNA into RISC is an energy dependent process, most probably because of the required unwinding of dsRNA. Interestingly, the sequence composition of dsRNA seems to affect the ratio of sense
(same sequence as target gene) and antisense (complementary to the target
gene) incorporation into RISC, where thermodynamic stability of the 3’ and
5’ ends directs the ratio of sense/antisense incorporation (Khvorova et al.
2003; Schwarz et al. 2003). Once incorporated into RISC, the ssRNA is very
tightly bound to the enzyme complex. The incorporated ssRNA guides the
RISC dependent mRNA degradation in a sequence specific manner, targeting the complementary sequence of the RISC-bound RNA. RISC induced
cleavage of mRNA occurs in the middle of the mRNA complementary sequence, a mechanism which is not energy dependent; however presence of
ATP can yield a more efficient cleavage progress (Hutvagner and Zamore
2002). The exact protein responsible for this hydrolysis is unclear; some
theories ascribe the process to be dependent on Dicer, although the small
molecular mass of the minimal RISC argues against this (Martinez et al.
2002). However, recent studies have shown that the protein Argonaute 2 is a
critical component of RISC, thought to be the smallest component required
for RISC activity thus mediating mRNA cleavage (Lingel et al. 2003;
Meister et al. 2004; Rivas et al. 2005).
1.4.2 Alternative splicing and splice correction
One of the most surprising discoveries originating from the Human Genome
Project was that the number of protein encoding genes was much lower than
originally thought, where the previously predicted 100.000 protein-encoding
genes have been gradually revised down, and now it is clear that it is approximately 20.000-25.000 genes (1.5 %) of the total genome (Consortium
2004). This observation led to the conclusion that there must be another uni17
1. Introduction
versal mechanism responsible for the diversification of the genome, enabling
the synthesis of existing proteins. Alternative splicing (AS) is a process taking place in the nucleolus, where exons of the primary transcript (premRNA) is ordered into different arrangements, resulting in structurally and
functionally distinct mRNA and proteins from the same pre-mRNA. It is
now believed that AS is one of the most important processes in eukaryotes
for achieving macromolecular and cellular complexity. The splicing mechanism is very intricate, an example of this being the Dscam gene of Drosophila which has the potential to encode as many as 38.000 different splice
variants (Graveley 2005), and it is thought that in humans, approximately
60-80% of all genes are alternatively spliced. Seemingly, the amount of AS
is correlated with the complexity of the organism (Modrek and Lee 2002).
Splicing mechanism and disease related mutations
The process of splicing takes place after capping and editing of the premRNA, during which introns are removed resulting in a spliced mRNA and
an intron lariat. Splicing requires a 3’ and 5’ splice site, a branchpoint and a
variable stretch of pyrimidines termed polypyrimidine tract (PPT). Splicing
involves the assembly of small ribonucleoprotein particles (snRNPs), which,
together with a large number of non snRNP proteins, form the spliceosome
(Jurica and Moore 2003). Intron excision occurs in two steps, where first the
5’ splice site is cleaved, with concomitant intron lariat formation, after which
the 3’ splice site is cleaved and exon ligation can take place. When splicing
has successfully occurred, the constituents of the spliceosome disassemble to
be able to create new spliceosomes.
In higher eukaryotes, AS is one of the main reasons for genomic diversity, however, there is also a downside to AS. It has been estimated that 50%
of disease causing mutations lead to aberrant splicing (Lopez-Bigas et al.
2005), either by damaging or modifying splice sites, branch points, PPTs or
enhancer elements. Mutations can lead to gain of splice function, where
cryptic splicing sites are created which affect exonic/intronic splicing inhibitors/enhancers. An example of this is the disease β-thalassemia where a 3’
cryptic splice site is activated in the β-globin gene leading to severe anemia
and in certain cases death. Loss of splice function mutations inactivate cisacting splice elements, which has been observed in e.g. hemophilia B where
aberrant splicing of factor IX occurs (Rees et al. 1985). Also, mutations in
splicing factors required for constitutive splicing could lead to severe diseases, as is the case in some cancers and other diseases. Some mutations
causing aberrant splicing directly affects the pre-mRNA in such a way that
the created protein either has a loss of function or gain of function, and in
some cases protein isoform ratios are altered leading to a disease.
18
1. Introduction
Treatment strategies for alternative splicing disorders
There are several approaches that can be used in order to treat splicing disorders. Targeting protein isoforms using small molecules is an attractive strategy used today, which is based on the fact that diverse protein isoforms have
unique properties which can be exploited. An example of targeting protein
isoforms is the nonsteroidal anti-inflammatory drugs (NSAIDs) like ibuprofen. NSAIDs target the cyclooxygenase (COX) enzymes responsible for the
conversion of arachidonic acid to prostaglandin, and recently NSAIDs able
to target the specific isoform COX-2 were developed, which decreases gastrointenstinal complications, as COX-1 expressed in these areas is not affected. Although COX-1 and 2 stem from two different genes, it is a proof of
concept that selectively targeting protein isoforms is possible. Recent discoveries showing that there are two COX-1 splice variants, COX-3 and
COX-1a (Simmons 2003), which are differentially expressed in e.g. dog
tissues, imply that tissue specificity can be achieved by targeting different
splice isoforms. Small molecules can also be used for targeting expression of
different splice isoforms by interacting either directly with the gene expression, or by modulating splice patterns by targeting splicing factors.
Another approach for gene-specific therapy is mediated by ONs and ON
analogues, which can be used in a variety of ways to modify gene expression. ONs have mainly been used as antisense agents to silence gene expression, either through steric hindrance of the translation process, or by RNAse
H activation. For targeting splicing disorders, ONs can be used in a variety
of ways. The most obvious approach is exon specific targeting, which would
lead to silencing of a specific protein or isoform. Another strategy which can
be employed is to direct splicing towards a certain isoform by blocking
splice sites, or correction of aberrant splicing by masking cryptic splice sites
which otherwise would lead to non-functional transcripts. An excellent example were ONs were used to alter the AS of a transcript is Bcl2l1. This
gene encodes for one pro-apoptotic protein, Bcl-xS and one anti-apoptotic
protein, Bcl-xL. The anti-apoptotic Bcl-xL is over expressed in various cancers, and by shifting the splicing towards the pro-apoptotic Bcl-xS, apoptosis
was induced (Mercatante et al. 2001), indicating that this could in future be a
means to treat diseases where AS is involved, such as some cancers. As previously mentioned, ONs can also be used to block cryptic splice sites, where
a mutation is altering the splicing pattern, leading to non-functional transcripts. β-thalassemia is in many cases caused by a mutation in the β-globin
gene leading to a cryptic splice site. It has been shown that by blocking this
site using ONs, splice correction is induced, leading to the synthesis of normal hemoglobin A (Dominski and Kole 1993; Lacerra et al. 2000). An ON
suitable for splice correction is peptide nucleic acid (PNA), which is a DNA
analogue with a peptide backbone (Fig 1.5). The increased flexibility and
lack of negative charges allow PNA to bind more tightly to complementary
19
1. Introduction
DNA or mRNA as compared to ordinary oligonucleotides, and it does not
activate RNase H (Egholm et al. 1993; Giesen et al. 1998).
Fig 1.5. Comparison of the DNA and PNA structure. As PNA has a peptide backbone it is has
no negative charges, thus being able to bind more strongly to RNA and DNA than its DNA
counterpart. Furthermore this modification also renders PNA stable against nuclease degradation.
1.5 Cell-penetrating peptides
CPPs are a class of peptides able to transport cargos such as ONs, proteins
and plasmids across the plasma membrane. It has been debated whether the
internalization of CPPs can be attributed a direct penetration mechanism or
receptor independent endocytosis. A series of recent articles have ascribed
the uptake to be endocytic, where macropinocytosis is the most probable
candidate. CPPs are a very heterologous class, where some peptides are
mainly cationic and derived from naturally occurring proteins, such as Tat,
others are more hydrophobic and synthetically derived (Table 1.2). The features connecting various CPPs are the positive net charge of the peptide and
their vector abilities.
20
1. Introduction
Table 1.2. Selection of CPPs for comparison of the length and primary structure differences.
Name
Sequence
Origin
Reference
Penetratin
RQIKIWFQNRRMKWKK1
Protein derived
(Derossi et al. 1994)
Tat
GRKKRRQRRRPPQ2
Protein derived
(Vivès et al. 1997)
bPrPp
MVKSKIGSWILVLFVAM
WSDVGLCKKRPKP-amide
Protein
derived
(Magzoub et al. 2006)
Transportan
GWTLNSAGYLLGKINLKA
LAALAKKIL-amide
Chimeric
(Pooga et al. 1998)
MPG ΔNLS
GALFLGFLGAAGSTMGA
WSQPKSKRKV3
Chimeric
(Simeoni et al. 2003)
Pep-1
KETWWETWWTEWSQPK
KKRKV3
Synthetic
(Morris et al. 2001)
1
Originally synthesized as a C-terminal free acid
Originally synthesized with a C- and N-terminal cysteine
3
Synthesized as a C-terminal cysteamide
2
1.5.1 A brief history of cell-penetrating peptides
The first indication that proteins could contain sequences responsible for
their translocation across cellular membranes originated from the observation that living cells internalized an 86 amino acid long fragment from the
HIV-1 TAT protein, activating HIV-LTR driven transcription (Frankel and
Pabo 1988; Green and Loewenstein 1988). In one of these reports it was
proven that the sequence that today corresponds to the CPP Tat was necessary for trans-activation and that replacing three of the arginine residues with
alanine residues greatly reduced the activity (Green and Loewenstein 1988).
Some years later it was discovered that the 60 amino acid homeodomain of
the Antennapedia protein of Drosophila also was able to translocate across
cell membranes (Joliot et al. 1991), proving that translocation of the TAT
protein was not an isolated case. In order to understand the driving force for
the internalization, the homeodomain was modified by site-directed
mutagenesis, leading to the discovery that its third helix was necessary and
sufficient for membrane translocation, resulting in the development of the 16
amino acid long CPP penetratin (Derossi, et al. 1994), nowadays sometimes
referred to as pAntp.
21
1. Introduction
1.5.2 Mechanisms of CPP internalization
Much of the research during the last decade has been directed towards understanding the interactions necessary for CPP internalization, and how
modifications of known CPPs can increase their uptake. For arginine rich
peptides, uptake seems to be dependent on charge interactions, and possibly
hydrogen bonding of the guanidinium group of arginine (Futaki 2005), as
replacement of arginine residues with other amino acids as well as constructing a spacer between the backbone and the guanidinium head group significantly change uptake kinetics and distribution pattern. These changes probably alter the interactions between the CPP and negatively charged GAGs on
the cell surface, an interaction reported to be crucial for uptake of arginine
rich peptides (Tyagi et al. 2001; Suzuki et al. 2002). The initial interaction
with GAGs seems to trigger a series of events leading to the internalization
of the peptide by endocytosis. The exact endocytic pathway responsible for
CPP internalization has been highly debated, and could be dependent on the
cargo size. Richard and colleagues reported that Tat colocalize with Tf
(Richard et al. 2003), thus being internalized via clathrin mediated endocytosis, whereas Fittipaldi et al. reported no co-localization between Tat-EGFP
and Tf, claiming the uptake to be caveolar (Fittipaldi et al. 2003). A couple
of more recent studies imply the uptake of Tat and polyarginines to be mediated by macropinocytosis, both with Tat (Lundberg and Langel 2006) and
octaarginine (Nakase et al. 2004), as well as with a Tat-Cre fusion protein
(Wadia et al. 2004). A recent mechanistic study of Tat reveals the uptake to
be cargo dependent, where internalization of cargo shorter than 50 amino
acids was dependent on membrane potential, whereas uptake of proteins was
observed in large cytoplasmic vesicles (Tunnemann et al. 2006). Interestingly, co-incubation of the endosomolytic fusion peptide Tat-HA2 did not
significantly alter the uptake pattern of Tat-Cre, which still was retained in
endosomes, even though co-localization of the two Tat conjugates was substantial.
For non-arginine rich peptides, such as transportan (TP) and MPG, few
studies regarding the internalization mechanisms have been reported. Protein
conjugated TP, and its shorter analogue TP10, in accordance with arginine
rich peptides, seem to be internalized via a lipid raft dependent endocytosis
which probably can be ascribed macropinocytosis (Säälik et al. 2004; Padari
et al. 2005). MPG on the other hand, which is able to deliver both plasmids
and siRNA, has been reported to be internalized independently of endocytosis (Simeoni, et al. 2003). In a recent study, using a splice correction system
which can quantify ON uptake developed by R. Kole’s group (Kang et al.
1998), it was confirmed that the uptake of penetratin, Tat and TP is endocytic, where both uptake and splice correction are reduced with decreasing
temperature, and significantly increased by addition of the lysosomotropic
agent chloroquine (EL-Andaloussi et al. 2006). Co-localization of CPP-PNA
22
1. Introduction
conjugates with the fluid phase marker dextran using confocal microscopy,
shows a higher degree of TP internalized by fluid-phase endocytosis as compared to penetratin and Tat, who demonstrate co-localization with dextran to
a lesser extent. As previously mentioned in the endocytosis section of the
thesis, the read out of mechanistic studies using co-localization markers and
endocytosis inhibitors is not fully reliable and, at least in some cases, the
CPP internalization might well be attributed the novel uptake mechanism
described above that is neither depending on caveolae or clathrin nor mediated by macropinocytosis.
1.5.3 CPPs as cargo delivery vectors
For CPPs to increase the delivery of cargos such as proteins and ONs, the
cargo should either be connected to the CPP via a non-cleavable covalent
bond, cleavable covalent bond or in the form of a nanoparticle with CPPs
surrounding the cargo (Table 1.3). The selected strategy depends on laboratory facilities, the CPP used and what cargo to transport. CPPs can also be
used to modify liposomes in order to increase ON delivery, both in vitro and
in vivo, a method which is not covered in this thesis.
Delivery of oligonucleotides
The most attractive and used approach for delivery of ONs and analogues
using CPPs is conjugation via a cleavable disulphide bond. The rational is to
have a bond which is stable in a non-reductive environment, thus when approaching the target cell, but once internalized the disulphide bond will be
reduced and the cargo will be free to exert its biological effect. There are
several advantages of this method: first, it allows conjugation between peptides and e.g. siRNA and DNA modified to contain a thiol group. Second, if
conjugated to PNA it will facilitate the synthesis, as synthesis without using
a linker between the CPP and PNA will create long one-chain sequences
resulting in low yields. Finally, the fact that the peptide and cargo are detached after entering the cell, contrary to using a stable bond, suggests that
the ON more easily can perform its biological role, as no sterically hindering
peptide affects its interactions. The only drawback of this conjugation strategy is the possible unwanted reduction of the labile bond between the CPP
and ON before the target cell is reached, especially in in vivo settings, resulting in a reduced biological effect. Two recent articles have used the splice
correction system, and compared how different conjugation strategies between the CPP and ON influence the biological response. Moulton et al.
focused on how the number of arginines in the CPP, choice of cross-linker,
peptide conjugation position, and length of the phosphorodiamidate mor23
1. Introduction
pholino oligomer (PMO) affected both the uptake and biological response of
the construct (Moulton et al. 2003). PMOs are attractive DNA analogues
where the negatively charged phosphodiester intranucleoside DNA linkage
is replaced by a neutral phosphorodiamidate bond, and the five–membered
ring of deoxyribose in DNA is replaced by a six-membered morpholino ring,
making PMOs more resistant to enzymatic degradation. It was observed that
antisense effect (splice correction) was dependent on spacer length between
the CPP and PMO moiety, site of conjugation and also PMO length. Interestingly, even though the same antisense effect was observed using a stable
thiomaleimide linker and labile disulphide linker, the uptake pattern and
intracellular concentrations of the PMO varied dramatically. A similar approach was used by Bendifallah and colleagues, using the same splice correction system, with the difference that the delivered nucleotide analogue in
this case was PNA (Bendifallah et al. 2006), and the CPPs evaluated were
not only arginine rich peptides, but also TP and (KFF)3K. In concurrence
with Moulton and colleagues they observed the importance of the linker
between the CPP and ON (PNA), and molecular site of conjugation. No direct correlation between length of the linker and antisense effect could be
concluded, but rather that labile bonds, such as biologically cleavable ester
and disulphide bonds, increased the biological effect. Two recent reports
have used the disulphide conjugation strategy for the delivery of siRNA.
Muratovska and Eccles used both penetratin and TP to transport siRNA targeting the luciferase- and GFP protein, claiming the CPP-siRNA construct to
be as effective, or more effective, than lipofectamine 2000 (Muratovska and
Eccles 2004). In an interesting study, Davidson and colleagues showed the
effect of a penetratin-siRNA disulphide conjugate on primary hippocampal
and sympathetic neurons, targeting the proteins SOD1 and members of the
caspase family. Remarkably, a decrease in protein level could be observed as
early as 6 hours after addition of the penetratin-siRNA conjugate, whereas a
decrease in mRNA levels was observed after 24-hours, indicating an antisense effect of siRNA on a translational level, prior to mRNA degradation
(Davidson et al. 2004).
Another means for ON delivery is using the co-incubation strategy. Using
this approach, the CPP is incubated with the ON in a molar excess, typically
10-50 times. The positive amino acids within the CPP will interact with
negative charges on the phosphate groups, and a semi-stable nanoparticle
will be formed. Contrary to using a disulphide bond, there is no need for
additional conjugation steps, as only mixing of the ON and CPP is sufficient,
making this approach practical for laboratories without synthesis facilities. It
has been reported that the formed nanoparticle is stable in serum media and
can deliver plasmid DNA and siRNA effectively (Simeoni, et al. 2003),
making it a very attractive approach for in vivo applications.
24
1. Introduction
The drawback of the co-incubation strategy, as compared to conjugation via
a disulphide bond, is the need for higher peptide concentrations to deliver the
same amount of ON, which could possibly lead to toxic side effects and also
making the method less cost effective.
Table 1.3. Selection of recent ON delivery studies using CPPs
Delivery
strategy
Target/ Biological effect
Reference
bPrPp and EB1
Co-incubation
Luciferase expression
(Lundberg et al. 2006)
MPG ΔNLS
Co-incubation
Luciferase expression
(Simeoni, et al. 2003)
Penetratin
Disulphide
SOD1 and caspase proteins
(Davidson, et al. 2004)
Penetratin, TP
Disulphide
Luciferase/GFP
expression
(Muratovska and Eccles
2004)
Disulphide and
stable linkers
Inhibition of
TAT-dependent
transactivation
(Turner et al. 2005)
Disulphide
Splice correction
(EL-Andaloussi, et al.
2006)
Disulphide, onechain synthesis
and SMCC
Splice correction
(Bendifallah, et al. 2006)
CPP
siRNA delivery:
PNA delivery:
(R)9-F2, (R)6Penetratin, SV40
NLS-(K)8, Tat, TP
and TP10
Penetratin, Tat and
TP
NLS, Penetratin,
Polyarginines,
SynB3, (KFF)3K, Tat
and TP
Delivery of proteins
Contrary to ONs, where lipid vectors can be used efficiently, at least in vitro,
no such tools are available for the delivery of biologically active proteins.
Therefore, protein delivery using CPPs is an attractive means for characterization of protein function and also in therapeutic applications. Protein delivery using CPPs was initially described using a TAT protein domain (37-72),
which was able to deliver beta-galactosidase into cells (Fawell et al. 1994).
Preceding studies have used shorter versions of Tat, which is without comparison the most widely used CPP for protein delivery (Table 1.4). A recent
CPP application for targeting various diseases is to create transduction com25
1. Introduction
petent antibodies. In an in vitro study, the CPP Tat, fused to an anti-TAT
single chain antibody, retained both antibody specificity and transduction
ability, resulting in an inhibition of TAT-dependent transcription of a HIV-1
reporter gene by >80% (Theisen et al. 2006).
Table 1.4. Selection of CPP delivery of bioactive proteins
CPP
Delivery
strategy
Tat
Cargo
Readout
Reference
Fusion protein
β-galactosidase
Tissue distributiona
(Schwarze et al. 1999)
Tat
Fusion protein
Bcl-xL
Apoptosisa
(Cao et al. 2002)
Tat
Fusion protein
Apoptin
Apoptosisb
(Guelen et al. 2004)
Tat
Fusion protein
β-galactosidase
Tissue distributiona
(Schwarze, et al. 1999)
Tat
Fusion protein
Anti-TAT
scFv antibody
Transcription
suppressionb
(Theisen, et al. 2006)
MTS
Fusion protein
scFv antibody
Tumor volume
reductiona
(Shin et al. 2005)
Penetratin
Coadministration
scFv anibody
Tumor targeting and antibody retentiona
(Jain et al. 2005)
Pep-1
Co-incubation
GFP/ βgalactosidase
Protein expressionb
(Morris, et al. 2001)
a
b
In vivo
In vitro
Also in the case of protein delivery, co-incubation strategies have been successfully used for the delivery of biologically active proteins (Morris, et al.
2001). This principle has also been used for the delivery of single chain antibodies in vivo using Tat and penetratin, where co-addition of peptide, most
significantly penetratin, increased tumor retention of a single chain antibody
24 h after administration (Jain, et al. 2005). Increasing the concentration of
penetratin as compared to antibody yielded higher tumor specific delivery up
to a certain point, after which high unspecific delivery was observed, imply26
1. Introduction
ing the importance of titrating peptide/protein ratio for optimal delivery and
specificity.
Delivery of peptides
An attractive approach for mimicking or inhibiting protein interactions is the
synthesis of short peptide fragment derived from the wild type protein. Ideally, the peptide in itself should be a CPP, or in the case where this is not
feasible, it could be conjugated to a CPP either by a disulphide bond, or synthesized in one chain, the CPP N- or C-terminally. Some excellent studies
have been made where short peptides are used to direct malignant cell into
apoptosis (Table 1.5). The lack of specificity for current cancer treatments
and intrinsic resistance of many tumors for established cancer treatments,
emphasize the need for improved therapeutics.
Table 1.5. Selection of bioactive peptide delivery using CPPs
CPP
Cargo
Readout
Reference
Tat
P53 C’ peptide
Tumor growth, survivala
(Snyder et al. 2004)
Tat
Mdm-2 blocking peptide
Proliferationb
(Jarajapu et al. 2005)
Tat
CK2 blocking peptide
Tumor growtha
(Perea et al. 2004)
Tat
Smac peptide
Tumor sensitizationa
(Fulda et al. 2002)
Tat and
penetratin
Shepherdin
Tumor growtha
(Plescia et al. 2005)
Penetratin
P53 C’ peptide
Apoptosisb
(Li et al. 2005)
Penetratin
P53 C’ peptide
Apoptosisa
(Senatus et al. 2006)
Penetratin
T lymphosite peptide
T-cell responseb
(Pouniotis et al. 2006)
a
b
In vivo
In vitro
The optimal solution for achieving cancer-specific targeting is to either use a
peptide taken up by all cells, but only affecting cancer cells, or cancer-cellspecific delivery. Fulda and colleagues developed a cell-penetrating Smac(second mitochondria-derived activator of caspase) Tat fusion peptide consisting of the seven N-terminal residues of Smac linked to Tat. Smac is a
mitochondrial protein shown to promote cytochrome c-dependent caspase
activation by eliminating IAP (inihibtion of apoptosis proteins) activation
27
1. Introduction
(Du et al. 2000; Verhagen et al. 2000). Treatment of the Smac-Tat fusion
peptide strongly enhanced the anti-tumor activity of TRAIL (Apo-2L/tumor
necrosis factor-related apoptosis-inducing ligand) in an intracranial xenograft model, and also sensitized cells to treatment with anti-CD95 or cytotoxic drugs (Fulda, et al. 2002). The usefulness of this approach was further
established by an independent group using a mouse lung cancer xenograft
model, showing that a Smac-octaarginine sensitized the cancer cells for cisplatin therapy (Yang et al. 2003).
Another impressive study exploited the p53 signaling pathway, thought to
be involved in most cancers (Vousden and Lu 2002; Trigiante and Lu 2006),
using a fusion peptide between Tat and a peptide derived from the Cterminus of p53 (termed p53C’). This C-terminal p53 moiety has previously
been shown by Selivanova et al. to restore the function of certain p53 mutants in cancer cells (Selivanova et al. 1997; Selivanova et al. 1999). To increase the stability of the Tat-p53C’ peptide, it was synthesized using Damino acids, and to minimize the changes in surface topology it was synthesized in a retro-inverso form, yielding a stable peptide with essentially retained secondary structure (Snyder, et al. 2004). This peptide preserved the
p53-activating function of its parental peptide, and achieved significant effect in three different human cancer models. Analogous studies using penetratin linked to a C-terminal p53 peptide have also proved effective (Li, et al.
2005; Senatus, et al. 2006). A similar approach also exploiting the p53 signaling pathway was successful using a peptide blocking the MDM-2 binding
of the ARF protein, P14ARF, which induced apoptosis after attaching it to
either Tat or penetratin (Jarajapu, et al. 2005).
A good example of rational peptide design for disturbing protein-protein
interaction in an anticancer setup was performed by Plescia et al. In order to
decrease cancer proliferation, they targeted the interaction between heat
shock protein 90 (Hsp90) and the antiapoptotic and mitotic regulator survivin, which they had previously shown to be possible using a 12 amino acid
long peptide (Fortugno et al. 2003). A nine amino acid truncated version of
this peptide called shepherdin, which specifically abolished the Hsp90survivin interaction, was further conjugated to Tat and penetratin to achieve
cell-penetrating properties (Plescia, et al. 2005). Even though very high peptide concentrations were needed to achieve a biological response on cell
cultures (IC50 ~25-75 µM, ≥100 µM shepherdin-penetratin was used for
most studies), a cell-permeable retro-inverso D-analogue of shepherdin was
able to inhibit prostate cancer xenograft growth in vivo. The fact that shepherdin seems to have no effect on non-cancer cells suggests that this strategy
could be a base for developing future cancer therapeutics.
28
1. Introduction
1.5.4 Methods to improve CPP mediated delivery
Enhancement of endosomal escape
The mechanistic studies using lysosomotropic agents to increase biological
response, suggest that entrapment of CPP-cargo molecules in endocytic
vesicles after internalization appears to be the major caveat in CPP delivery,
and a rough estimate is that at best only a few percentages of the internalized
CPP-cargo molecules are able to penetrate the endosomal membrane and
exert their biological effect. Thus, increasing the endosomal escape properties of CPPs could increase cargo delivery several-fold. As viruses also are
taken up by endocytosis they have developed special peptides, so called fusion peptides, which enable them to escape from endosomes. This naturally
selected way to increase endosomal escape of viruses has been applied to the
CPP-field with some success. The best characterized of these fusion peptides
is the 20 amino acid NH2-terminal part of the influenza protein hemagglutinin (HA2). Upon acidification, at a pH of approximately five, the peptide
will change conformation, with a concomitant insertion into the endosomal
membrane resulting in endosomolysis and escape of the viral particles into
the cytosol. This approach has lately been applied to increase CPP mediated
cargo delivery with promising results, especially when applied to protein
transduction (Wadia, et al. 2004; Michiue et al. 2005), but also in ON
delivery (EL-Andaloussi, et al. 2006; Lundberg, et al. 2006).
Another means to increase the endosomal escape is also based on the
acidification which occurs in early-late endosomes. Using peptides or polymers with histidyl modifications, which can be protonated in acidic endosomes, have yielded interesting results. Histidyl modifications of peptides
and polymers have been pioneered by the group of P. Midoux, whose group
have shown that histidyl modifications of the HA-2 peptide (H5WYG) enhances membrane permeabilization at acidic pH (Midoux et al. 1998). The
probably most promising applications of peptides which are activated at a
low pH are in cancer therapy. These peptides are inert at neutral pH, thus
unable to enter cells, but upon protonation in hypoxic tumor areas the peptide will be activated and able to enter the cancer cells specifically. Also,
histidyl modified polyplexes show increased gene transfer ability, both for
ONs and plasmids, implying the imidazole group of histidine to have membrane destabilizing abilities (Midoux and Monsigny 1999; Benns et al. 2000;
Pichon et al. 2000).
29
1. Introduction
Tissue targeting
To achieve satisfactory biological activity, while minimizing dangerous
toxic side effects, either the peptide need to be activated only in the target
cell, or tissue specific delivery is needed. Also, one must take into consideration that the administration pathway could influence the in vivo distribution
(Cai et al. 2006).
An attractive means to achieve tissue specific delivery is to exploit that
several human malignancies over-express certain proteases on the cell surface, such as the prostate specific antigen (PSA) in prostate cancer, or matrix
metalloproteases (MMP) as a more general cancer marker. The rational is to
exploit the necessity for cationic interactions needed for CPP internalization,
and by neutralizing them before the target cell is reached, no uptake in nontarget tissue will occur. R. Tsien’s group showed that by adding acidic
amino acid residues to nonaarginine, using a linker with an MMP cleavage
site separating the cationic and anionic parts, the cargo uptake was 18-fold
less when having the anionic inactivating sequence on, as compared to when
an equimolar mixture of the cationic and anionic moieties were added to HT1080 cells (Jiang et al. 2004). Making several analogues with different
lengths of the cationic, anionic and cleavage sequence, the highest observed
uptake increase between inactivated and activated peptide was >100 fold.
The in vivo data achieved using a PEGylated version of the peptide, to prevent excretion, was not as impressive as the in vitro data, but also here an
increase in tumor fluorescence could be observed as compared to a noncleavable all D analogue. One might speculate that the PEG moiety could
interact with the peptide in an uncontrollable way, decreasing the cleavage
of the inactivator sequence, and, also, that a better in vivo result could be
achieved if the PEG part was bigger, as e.g. glomerular filtration will excrete
compounds with a molecular weight less than approximately 70 kDa (also
depending on charge and secondary structure (Brown 2005)) and free PEGs
under 30 kDa (Harris and Chess 2003).
A similar study by Gounand colleagues used a PSA linker between the CPP
and inactivating sequence. This article addressed the importance of using a
suitable inactivator sequence, by synthesizing a library of different constructs with different length and composition of the inactivating sequence
(Goun et al. 2006). They found that inactivation using aspartate residues
spaced by glycine residues (DGGDGGDGGD) provided the highest inhibition of non-cleaved constructs, yielding uptake similar to that of pentaarginine. Also, once the construct is cleaved, the octaarginine moiety showed
uptake ratios comparable to that of octaarginine, indicating that the inactivating sequence dissociates from the CPP, a characteristic necessary for efficient delivery.
30
1. Introduction
Targeting over-expressed cell surface proteins
Over-expressed proteins on the cell surface provide an excellent means for
selective tumor targeting. The idea is to create a peptide with the ability to
interact with the protein being over-expressed, and after initial interaction
the peptide should be internalized and, using some sort of warhead, selectively kill the tumor tissue. Problems to be addressed are: (1) Sufficient difference between tumor cell and non-tumor cell in production of the target
protein for minimal uptake in non-target tissue and (2) Constructing a peptide that is not internalized in all tissues because it contains a too “strong”
CPP sequence, thus not needing the interaction with the target protein before
internalization. Even though achieving these two parameters might seem far
fetched, one recent publication showed that it is not impossible. In an article
by Tan and colleagues a member of the epidermal growth factor family,
ErbB2 (or HER-2/neu), was targeted, a protein being over-expressed in approximately 30% of breast cancers. For targeting and efficient internalization
in tumor tissue, a chimerical peptide consisting of a truncated version of Tat
conjugated to the anti-HER-2/neu peptide mimetic (AHNP) was constructed
(Tan et al. 2006). The reason for truncating the Tat peptide was to reduce
unspecific delivery, as previous reports regarding polyarginines state that a
certain length is required for internalization (Mitchell et al. 2000). Unfortunately, no uptake comparison between wild type Tat and truncated Tat (P3)
was made when conjugated to AHNP which, considering that cargo and
peptide length influence uptake, could possibly decrease the specificity observed using the shorter Tat analogue. Adding the AHNP moiety to P3 increased the uptake in 435.eB cells, an MDA-MB-435 cell line constitutively
expressing active ErbB2, whereas uptake in wt MDA-MB-435 cells was
unchanged. This is probably a result of increased initial binding sites of the
peptide, as P3 alone can only interact with heparan sulfates whereas the P3AHNP can interact with both heparan sulfate and ErbB2, resulting in an
approximately two-fold increase in uptake.
Further elongating this peptide with a STAT3-blocking peptide, which
should affect ErbB2-induced cell transformation and tumor progression but
still retaining its specificity for ErbB2 expressing cells, the authors successfully targeted ErbB2 over-expressing breast cancer cells in vivo. Using
xenografts expressing both 435.eB cells and MDA-MB-435 cells, they concluded that the effect seen was specific for ErbB2 over-expressing cells, as
no effect on tumor growth could be observed on the MDA-MB-435 xenografts.
31
2. Aims of the study
2. Aims of the study
In papers I and II, we were looking at naturally occurring peptide motifs
from proteins involved in neurodegenerative disorders, namely γ-secretase
involved in Alzheimer’s disease and the prion protein responsible for prion
diseases. In paper I we wanted to examine if various peptides derived from
the γ-secretase were able to interact with APP processing in such a way that
the production of Aβ would be reduced or even abolished, and the fashion by
which they are internalized. In paper II we hypothesized that CPPs could be
involved in the infectivity of prions, a paper which is a follow up study of
Lundberg and colleagues from 2002 (Lundberg et al. 2002). Paper III is a
summary of methods that can be used in order to quantify peptide uptake,
mechanisms of internalization and qualitative uptake using confocal microscopy. In paper IV and V we were studying ON delivery using two different
methods and read outs, elucidating mechanisms of internalization and various ways by which ON delivery could be enhanced. The main goals are presented below.
• Develop and optimize methods for quantification of CPP internalization
(Paper I and III)
• Examine the role, biological activity and translocation efficacy of CPP
motifs observed in naturally occurring proteins (Paper I and II)
• Elucidate the mechanisms responsible for CPP internalization (Paper I, II,
III and IV)
• Study CPP applications in gene regulation (Paper IV and V)
• Investigate various ways by which CPP delivery and gene regulation can
be enhanced (Paper IV and V)
• Evaluate toxicity and membrane disturbance induced by CPPs when applied in biological systems (Paper IV and V)
32
3. Methodological considerations
3. Methodological considerations
The methods used in this thesis are thoroughly described in each paper,
hence here some theoretical and practical aspects of the methods will be
discussed.
3.1 Synthesis
3.1.1 Synthesis and purification of peptides
The peptides examined in this thesis (Table 3.1) were synthesized using
solid phase peptide synthesis, a method introduced in 1963 by B. Merrifield
(Merrifield 1963). More specifically, amino acids protected with the t-Boc
group were coupled to a solid phase (resin). After addition of each preactivated amino acid in excess, the resin with attached amino acids was
washed, deprotected from the t-Boc protecting group, whereupon a new
amino acid was added. This cycle was repeated for each amino acid that was
to be coupled, the process being fast and resulting in high yields as compared to classic peptide synthesis performed in solution. After the last amino
acid was coupled, the peptide was cleaved off from the resin using a mixture
of hydrofluoric acid, which is an extremely strong acid necessary for cleaving the bond between the peptide and resin. Scavengers, p-cresol, and if the
peptide contains sulphur, p-thiocresol, will scavenge the reactive carbocations formed in the cleavage process. Following cleavage, the peptide was
filtered in order to remove resin fragments, and freeze-dried to remove solvents. Depending on the peptide synthesized, the purity of the crude product
varies, but a rule of thumb is that the shorter the synthesized peptide is, the
higher the yield will be. Independently of the purity of crude peptide, it has
to be further processed to remove impurities; a procedure usually performed
using reversed-phase high performance liquid chromatography (HPLC).
Depending on resin used in the synthesis procedure, the peptide will be
either amidated at the C-terminus, or a free carboxylic acid. In the studies
performed, C-terminally amidated peptides have been used, since they show
improved stability as compared to the non-amidated counterparts, hence a 4methylbenzhydrylamine (MBHA) resin was used.
33
3. Methodological considerations
In order to quantify peptide uptake, 5,6-carboxyfluorescein was coupled Nterminally, and to assess protein delivery, biotin was coupled N-terminally.
Biotin binds strongly and practically irreversibly to avidin and streptavidin,
where both proteins have four binding pockets for biotin, enabling monitoring of fluorescently labeled protein bound to peptides. Where peptide-PNA
conjugates were synthesized, a cysteine with a 3-nitro-2-pyridinesulfenyl
(Npys) containing group was attached N-terminally on the peptide, with the
exception of TP where it was added orthogonally on Lys13.
3.1.2 PNA synthesis (Paper IV)
PNA synthesis was performed in essentially the same fashion as peptide
synthesis using t-Boc chemistry. As long PNA chains tend to aggregate and
terminate chain growth, the resin used for PNA synthesis was downloaded to
approximately 0.10 mmol/g substitution, as compared to peptide synthesis
where 0.72-1.36 mmol/g was used. In order to increase the PNA solubility,
two lysine residues were attached both N- and C-terminally, and to enable
further conjugation with a peptide moiety the PNA was synthesized with an
N-terminal cysteine.
3.1.3 Synthesis of peptide-PNA conjugates (Paper IV)
One way to increase the bioavailability of PNA is to conjugate it to a CPP
using a disulphide bond. To improve the kinetics and specificity of said conjugation, cysteine activated with the Npys group was attached to the peptide.
Previous studies have used DMSO/DMF in combination with different buffers for the conjugation reaction, where the organic solvents DMSO and
DMF were used to increase the solubility of PNA. In our study the disulphide formation was performed overnight in 20% acetonitrile/water containing 0.1% TFA, with subsequent separation using reversed phase HPLC. We
were able to use this acetonitrile/water solution for the disulphide formation
reaction since the PNA had four lysine residues attached to it, two N- and
two C-terminally, thereby increasing the solubility.
3.1.4 Design of CPPs
The selection of potential CPP sequences in paper I was made by choosing
peptides which contained at least three cationic residues, equal to or longer
than 12 amino acids in length, not containing any negatively charged amino
acids and total sum of at least three positive charges. Also, sequences containing as many bulky amino acids as possible, such as phenylalanine, tyro34
3. Methodological considerations
sine and tryptophan, were preferred. The reason for this selection is that
many protein-protein interactions are formed between bulky amino acids,
which are inserted in hydrophobic pockets. As the original aim of paper I
was to find peptides able to break up protein-protein interactions, having
bulky hydrophobic amino acids while at the same time being CPPs, thus
containing several positive charges, were ideal characteristics.
Table 3.1 List of synthesized peptides used in the thesis and their original reference. All peptides used in the thesis were amidated at their C-terminus, the exception being MPG ΔNLS
which was synthesized as a C-terminal cysteamide.
Peptide
Sequence
Reference
APH-1 (85-98)
VFRFAYYKLLKKA
(Lundberg and Langel 2006)
bPrPp
MVKSKIGSWILVLFVAMWSD
VGLCKKRPKP
(Magzoub et al. 2006)
EB1
LIRLWSHLIHIWFQNRRLK
WKKK
(Lundberg, et al. 2006)
HA2-penetratin
GLFGAIAGFIENGWEGMIDG
RQIKIWFQNRRMKWKK
(EL-Andaloussi, et al. 2006)
NCT (38-53)
RKIYIPLNKTAPCVR
(Lundberg and Langel 2006)
Penetratin
RQIKIWFQNRRMKWKK
(Derossi, et al. 1994)
PS1 (151-162)
VVLYKYRCYKVI
(Lundberg and Langel 2006)
PL1
VVLYKYRSYKVI
(Lundberg and Langel 2006)
pVEC
LLIILRRRIRKQAHAHSK
(Elmquist et al. 2001)
Tat (48-60)
GRKKRRQRRRPPQ
(Vivès, et al. 1997)
(KFF)3K
KFFKFFKFFK
(Vaara and Porro 1996)
MPG ΔNLS
GALFLGFLGAAGSTMGAWS
QPKSKRKV
(Simeoni, et al. 2003)
TP
GWTLNSAGYLLGKINLKALA
ALAKKIL
(Pooga, et al. 1998)
TP10
AGYLLGKINLKALAALA
KKIL
(Soomets et al. 2000)
Protein derived
Synthetic/Chimeric
35
3. Methodological considerations
Paper II is a follow-up study of a preceding report on the mouse prion
peptide mPrP (1-28) (Lundberg, et al. 2002), which consists of a signal sequence, amino acids (1-22) and an NLS-like motif, amino acids (23-28). The
study on mPrP (1-28) was based on its similarity with a previously discovered CPP consisting of a hydrophobic N-terminus and a cationic NLS Cterminally (Chaloin et al. 1997), and bPrPp in paper II is its bovine counterpart (Table 3.1).
The idea for the histidine analogue of penetratin in paper V was based on
the viral HA2 peptide, which is able to change conformation during acidification of the endosomes (Midoux, et al. 1998; Michiue, et al. 2005). The
rational was to replace certain amino acids in the penetratin sequence with
histidines, yielding a peptide which potentially would be transformed into an
α-helix upon protonation, but being inert in its non-protonated form (Table
3.1). The necessity for such modification was based on observations that the
uptake of penetratin was endocytic, which made us hypothesize that a peptide able to induce endosomolysis would increase the biological effect of a
delivered cargo several-fold.
3.2 Complex formation (Paper V)
There are various ways to conjugate a CPP to its cargo. The mainly used
methods are via a disulphide bond to an ON, or via recombinant techniques
to a protein. However, for the delivery of these cargoes, co-incubation between the CPP and cargo can also be applied. In paper V we applied the coincubation strategy for the delivery of siRNA, where two different approaches were used to quantify the optimal CPP:siRNA ratios for delivery.
3.2.1 Gel shift assay
The most basic method for examination of complex formation ability is to
incubate the CPP of interest with siRNA at various ratios, and to see if retardation can be observed using polyacrylamide electrophoresis. The method
allows fast screening for determination of the optimal peptide/siRNA ratio
needed for complete complex formation. However, being somewhat crude in
the read out as no exact numbers of the optimal ratio can be concluded, in
some cases a more precise method to use is the ethiduim bromide exclusion
assay.
36
3. Methodological considerations
3.2.2 Ethidium bromide exclusion assay
In order to more precisely determine and compare the complex formation
ability between two peptides and siRNA in paper V, an ethidium bromide
exclusion assay was performed. SiRNA was incubated with increasing
amount of CPP, and after 30 min incubation, ethidium bromide was added to
the solution. The fluorescence measured gives a direct value of the complex
formation amount, as the peptides bound to the siRNA complicate the
ethidium bromide binding, whereupon a loss in fluorescence takes place.
3.3 Cell cultures
Different cell lines have several characteristics that distinguish them, including protein expression and proliferation rate. Therefore, the choice of cell
line is of outmost importance when performing studies measuring peptide
uptake and biological effects.
3.3.1 Chinese hamster ovary cells (Papers I and II)
Chinese hamster ovary (CHO)-K1 cells is a widely used cell line for various
studies. CHO cells were used in these studies as they have two mutant cell
line derivatives: pgsA-745 which are deficient in the enzyme UDP-Dxylose:serine beta-1, 3-D-xylolyltransferase leading to a defective GAG
synthesis and pgsD-677 cells which are deficient in the polymerization of
heparin sulfate (HS) disaccharide chains because of a mutation affecting
both N-acetylglucosaminyltransferase and glucuronosyl-transferase activities
(Esko et al. 1985). Since GAGs, and especially HS, have been implied to be
of great importance for CPP internalization (Console et al. 2003), these cells
provide an excellent toolbox to study the early events of CPP binding to its
target cell.
3.3.2 HeLa cells (Papers III, IV and V)
HeLa cells are an immortalized cell line derived from a cervical cancer obtained from Henrietta Lacks in 1951. The HeLa cells in paper V were used
mainly because most early studies on siRNA were performed using this cell
line and it is considered to be easily transfected with plasmids, thus providing a good model system for our experiments where we look at downregulation of luciferase expression using transiently transfected cells. In paper IV, a HeLa Tet-Off cell line stably transfected with the recombinant
37
3. Methodological considerations
plasmid (pLuc/705) was used (Kang, et al. 1998). This plasmid carries the
luciferase gene, interrupted by a mutated human beta-globin intron 2. This
intron mutation causes aberrant splicing of the luciferase pre-mRNA, thus
resulting in a non-functional mRNA not able to translate into the luciferase
enzyme. However, upon treatment of the cells with an ON targeted to the
mutation site this aberrant splicing can be corrected, leading to accurate premRNA splicing and a functional luciferase enzyme (Fig 3.1). These cells,
referred to as HeLa pLuc 705 cells, provide an excellent means to quantify
ON delivery into the nucleolus.
3.4 Internalization and quantification of CPP
uptake and delivery
To compare various CPPs and examine how different modifications of the
peptide primary structure affect their characteristics, CPP uptake and delivery can be quantified and monitored qualitatively. There are many different
assays which can be applied for characterization, which all have their advantages and pitfalls.
3.4.1 Quantitative uptake of fluorescently labeled CPPs
(Papers I and III)
The most basic and applied method to study and quantify CPP uptake is by
labeling the peptide with a fluoresceinyl moiety, typically N-terminally using standard peptide chemistry. There are several advantages and disadvantages using quantification of fluoresceinyl-CPP (CPP-flc). The main reason
for using this method is that it is very fast, as the CPP-flc is added to the
cells, left to incubate for the desired time period, after which the cells are
lysed and fluorescence measured. This allows fast screening of different CPP
sequences and for determination of how various modifications of the peptide
primary structure affects its uptake. Also, normalization of uptake to total
protein content of the lysate partially corrects for errors caused by variances
in cell seeding density. There are however some pitfalls when using this
method. The main problem is that it is hard to distinguish which peptides
that are internalized into the cells and which remain bound to the plasma
membrane or extracellular structures. One way used to avoid the problem of
membrane associated peptides is trypsinizing the cells after the incubation
process, thus in theory degrading the non-internalized peptides, after which a
more correct fluorescence reading can be acquired. In practice however, this
method has got its flaws. The main predicament is that a large fraction of the
38
3. Methodological considerations
extracellular peptides are either buried in the plasma membrane, or bound to
extracellular structures such as HS, making the peptides hard to process by
the bulky trypsin enzyme. Also, some of the CPPs used have a propensity to
aggregate on the cell surface, resulting in non-degradable aggregates giving
false readings as internalized peptides since they cannot be degraded by
trypsin. Another drawback of measuring CPP-flc added to the cells is that
much of the readings will come from peptides retained in the endosomes,
thus not able to deliver a cargo which is to perform its biological effect. One
last consideration of using CPP-flc is the importance of which fluorophore
that is used, where it is conjugated to the peptide and how this might influence the uptake rate and localization of the CPP; thus for a more correct
view a set a fluorophores and sites of conjugation should be used to assess
uptake (Fischer et al. 2002).
3.4.2 Study of uptake and degradation of CPPs using HPLC
(Paper III)
A more recent method used to quantify CPP uptake is based on analysis of
cell lysate using HPLC. In this method, after incubation of the CPP-flc, the
cells are treated with diazotized 2-nitroaniline, which modifies peptides residing extracellularly, thus enabling separation of internalized and membrane
bound peptides (Oehlke et al. 1998). An advantage of this method is that the
diazotized- 2-nitroaniline is a small molecule able to reach and modify peptides which cannot be processed by trypsin, thus less false signal from extracellular peptide will be measured. Also, upon analysis of the peptides
using HPLC, one can perform a peak analysis to study the degradation pattern of the peptide in order to determine the amount of intact peptide, which
in combination with mass spectrometry can show the metabolites that are
formed. This is a good way to measure the half-life of the peptide, at the
same time as it gives an indication of which residues that are most prone to
enzymatic degradation hence can be modified to increase peptide stability.
If the interest lies in the use of CPPs for delivery of ONs, there are other
more reliable, although more cumbersome, ways to evaluate CPP internalization by measuring biological response.
3.4.3 Analysis of CPP uptake using HeLa pLuc 705 cells
(Paper IV)
As previously mentioned, the stably transfected HeLa pLuc 705 splice correction cells provide an excellent tool for the quantification of CPP mediated
delivery. The advantage of this method is that it relies on measuring a bio39
3. Methodological considerations
logical response inside the cell; correction of splicing leading to luciferase
enzyme translation, rather than indirect observations using fluorophores (Fig
3.1). Observing biological response of the CPP-cargo, or in some cases the
CPP itself, minimizes the possibilities of false signals from non-internalized
CPPs. Also, in this assay the quantification method for monitoring cargo
delivery gives a positive readout, increased luciferase activity, which is preferable to assays using negative readout, being more sensitive to toxic side
effects. Another great benefit of the splice correction system is that it enables
uptake quantification of fluorescently labeled cargo, in combination with
biological response, thus direct correlation between uptake and biological
effect of the cargo can be concluded. Since there are many variables which
are important to achieve activity in the splice correction system; uptake,
endosomal escape, ability to translocate to the nucleoli and binding affinity
to the pre-mRNA, there are numerous factors to consider when optimizing
the delivery efficacy. Consequently, the observed effect in splice correction
can be attributed to several characteristics of the CPP studied, for instance
the ability of the CPP to transport the cargo into the nucleoli. Therefore, the
results from this system cannot be applied directly to methods where the
means of quantification resides in another compartment, such as the cytosol.
Figure 3.1. Pre-mRNA of the modified luciferase gene, where the human β-globin intron 2
carrying a T-to-G mutation at nucleotide 705 is inserted. (Adapted from Kang et al. 1998)
3.4.4 CPP mediated siRNA delivery (Paper V)
For quantification of CPP mediated siRNA delivery, measuring mRNA and
protein levels can be performed. A previous study on CPP-mediated siRNA
delivery observed that effects on protein levels can be observed as soon as 6
hours after addition of the CPP-siRNA, and precedes the decrease in mRNA
levels, which is evident after 24 hours (Davidson, et al. 2004). This effect is
of course dependent on protein turnover, protein levels and mRNA stability.
We have studied siRNA gene-silencing effect on protein levels measuring
luciferase activity in cells transiently transfected with a luciferase plasmid.
40
3. Methodological considerations
Briefly, HeLa cells were transfected and the following day seeded out in 24well plates. The subsequent day, the transfected cells were treated with various concentrations of CPP/siRNA complexes, which were formed by coincubating CPP with siRNA in DMEM at different molar ratios 30 min before addition to the cells. The lowest siRNA concentration assessed for genesilencing was 10 nM and the lowest peptide:siRNA ratio was 10:1. The
knock-down of protein levels was examined either 24 or 48 h after addition
of the CPP/siRNA complexes by measuring enzyme activity, which was
normalized to the total protein content of the cells. This is a comparatively
simple setup, although with certain flaws. The main problem when using
transiently transfected cell is that the transfection levels vary between experiments, which would not be the case had stably transfected cells been
used. Therefore, the stochiometry of siRNA versus mRNA levels might differ between experiments thus increasing the chance for experimental errors.
However, as siRNA degradation of its target mRNA is an active process,
unlike sterically hindering ONs such as PNA, the stochiometry is not as crucial in these experiments as it had been using antisense PNA. Another disadvantage as compared to using stably transfected cells is that the transiently
transfected cells will lose their gene expression over time, thus complicating
studies examining long term siRNA effects.
3.4.5 Confocal- and electron microscopy (Papers I, II, III and
IV)
Although the methods to quantify delivery of CPP and attached cargo give
valuable information concerning the characteristics and mechanisms of internalization, there are occasions when the sub-cellular localization is of
interest. The predominantly used method for studying CPP localization is by
live confocal microscopy. Early studies on CPPs were often performed on
fixed cells, either using methanol or paraformaldehyde, but a series of studies indicating that the fixation step could influence CPP uptake pattern, giving rise to artifacts, stressed the importance of using unfixed cells (Lundberg
and Johansson 2001; Richard, et al. 2003). In previous studies on fixed cells
a strong nuclear staining of the peptides was often observed, something
which is very rarely detected using live microscopy. Using live confocal
microscopy, it is clear that endocytosis is responsible for most, if not all, of
the CPP uptake. However, one caveat when using this method is that the
accumulation of peptide in the endosomes provides such a strong signal that
it is practically impossible to distinguish peptide which have been taken up
by an alternative route, or successfully escaped from the endosomes. This
was observed in paper IV, where a strong luminescence signal from corrected splicing was achieved, but no fluorescently labeled PNA could be
41
3. Methodological considerations
observed other than in endosomes residing in the cytosol or on the cell surface (EL-Andaloussi, et al. 2006).
Even though there are some shortcomings using microscopy, it is a valuable tool for studying CPP internalization mechanisms using endocytosis
markers. In this thesis Tf, for studying clathrin mediated endocytosis, and
dextran, for monitoring fluid phase endocytosis, have been used. As with all
microscopy studies, co-localization is very subjective and by increasing the
levels of either the CPP or marker, it is easy to change something which
looks like modest co-localization to an almost perfect match. Hence, much
care must be taken, and several experiments performed, in order to optimize
the signal strength and exposure time to get a picture which resembles reality
as closely as possible.
To study CPP internalization at a higher resolution, electron microscopy
can be applied. In this thesis, intracellular monitoring of CPP-DNA complexes using streptavidin-gold conjugates was performed. As the resolution
of electron microscopy is very high (~0.2-0.4 nm) individual endosomes
containing gold particles can be visualized.
3.4.6 Inhibition of γ-secretase activity using CPPs (Paper I)
To screen if the γ-secretase derived peptides were able to destabilize the γsecretase protein-protein interactions, thus modifying APP processing, a
luciferase reporter system was used. This assay utilizes a modified APP
which is already processed by β-secretase, thus being a substrate for γsecretase cleavage. Further modification include a Gal4 DNA-binding/VP16
transactivation (GVP) domain, which allows translocation to the nucleus
following γ-secretase cleavage, resulting in the expression of luciferase
(Karlström et al. 2002). Examining the peptides listed in paper I, none of the
peptides, even at high concentrations (50 µM), were able to alter γ-secretase
activity. The most probable reason for this is the incomplete tertiary structure of the γ-secretase complex, only allowing educated guesses as to what
parts of the proteins in the complex that interacts with the other proteins.
However, the method in itself proved to be efficient, as addition of an already established γ-secretase inhibitor, S-2188 (Sigma Aldrich), significantly
reduced APP processing.
3.5 Methods to study CPP toxicity
When treating cells with synthetic peptides, it is of outmost importance to
take into account certain side effects that can arise from the peptides intrinsic
properties. Between different CPPs the toxic effects vary dramatically,
42
3. Methodological considerations
where some peptides can induce membrane perturbance at as low concentrations as 1 µM, others are practically inert at 100 µM. When studying peptide
toxicity there are several methods that can be applied, where the short term
toxicity assays often measure membrane disturbance and long term toxicity
is assessed using viability assays.
3.5.1 Membrane disturbance (Papers IV and V)
Membrane disturbance effect, as previously mentioned, differs vastly between CPPs, where short cationic peptides often barely have any effect on
membrane integrity, longer amphipathic or hydrophobic peptides can have
detrimental consequences on membrane stability. A frequently applied
method to study membrane disturbance is to monitor lactate dehydrogenase
(LDH) leakage from the cells. LDH is an enzyme responsible for the interconversion of lactate and pyruvate with concomitant exchange of NAD+ and
NADH. Released LDH is measured in the supernatant using an enzymatic
assay where reazurin is converted into the end product resorufin, which can
be quantified by measuring fluorescence at 560Ex/590Em.
LDH
NAD
+
+ lactate
→ pyruvate
+ NADH
Diaphorase
NADH
+ reazurin
→ NAD
+
+ resorufin
Fig 3.2. Release of LDH from damaged cells is measured by addition of lactate, NAD+ and
reazurin as substrates in the presence of diaphorase.
The LDH leakage assay is a rapid way to evaluate membrane toxicity,
where different concentrations and endpoints can establish which CPP concentrations that are safe to use without having toxic side effects. Although,
as LDH is a fairly large protein (MW~140 kDa), there is a possibility that
small pores can be created in the plasma membrane which will not be detectable using this assay, as the LDH is too large to leak out. In these cases a
membrane integrity study where the read out is based on smaller molecules
must be performed.
To study whether smaller holes in the membrane are present, 2-deoxy-Dglucose-6-phosphate (DGP) leakage is a good choice. In this assay, cells are
43
3. Methodological considerations
loaded with a glucose analogue, 2-deoxy-D-glucose, which is taken up by
glucose carriers and phosphorylated by hexokinase. The product, DGP, is
unable to leave the cell provided that the plasma membrane is intact (Walum
and Peterson 1982). Upon addition of the CPP of interest, the efflux of DGP
into the supernatant is a direct measurement of the peptide induced plasma
membrane disturbance.
3.5.2 Examining viability using WST-1 (Papers IV and V)
Even when no CPP induced membrane toxicity can be observed, long term
toxic effect by unspecific protein binding or induction of receptor internalization can occur. WST-1 is an assay for examining long term toxic effects
on cell viability by measuring dehydrogenase activity of the mitochondria,
which can be directly correlated with the amount of viable cells in the culture. WST-1 is incubated with the cells until a satisfactory signal is achieved,
usually 30 min to 1 h, during which time the WST-1 is converted to a quantifiable formazan dye by viable cells. As the absorbance at 420 nm is directly
correlated with cell number, comparing treated cells with untreated and control cells gives an estimate of how much, if any, the CPP treatment has affected viability.
44
4. Results and discussion
4. Results and discussion
The articles providing the framework of this thesis are divided in two intertwining parts. The first three articles are analyzing uptake of fluoresceinyl
labeled CPPs, and different ways by which internalization efficacy and
mechanistic studies can be performed. The last two articles are using the
knowledge base achieved from the first studies, while investigating applications which can be used in gene regulation. Below the main findings are
presented and discussed.
4.1 Uptake and mechanistic studies of CPPs
derived from the γ-secretase (Paper I)
In paper I we wanted to asses whether there were specific motifs in any of
the γ-secretase constituents APH-1, NCT, PEN-2 and PS1, which had cellpenetrating properties. Out of eight synthesized peptides, three yielded uptake levels equivalent or higher than that of Tat, one from each γ-secretase
protein with the exception of PEN-2 where no sequences able to fulfill the
selection criteria were found. One of the CPPs, PL1, was a peptide derived
from the wild type PS1 (151-162), where the cysteine is substituted for a
serine in order to decrease aggregation, however, also the wild type peptide
was able to enter cells as determined by confocal microscopy. To characterize the uptake, we wanted to elucidate what interactions and endocytic
mechanism that were responsible for the uptake of the CPPs found in the
first screening, and for comparison penetratin and Tat were used. Assessment of internalization in CHO cells deficient in GAG and HS synthesis, we
concluded that for all CPPs studied the absence of these sugars molecules
dramatically decreased the uptake. This was in concordance with previous
studies on Tat and penetratin (Console, et al. 2003). To further elucidate the
endocytic process responsible for the uptake after the initial interaction with
GAGs, we used the PI3-kinase inhibitor wortmannin, which is an inhibitor of
macropinocytosis (Araki, et al. 1996). Following treatment with wortmannin, a decreased cellular uptake could be observed for all peptides studied,
with the exception of PL1. Also, incubating the cells at 4°C, thus efficiently
inhibiting endocytosis, decreased the uptake of all peptides, however not
significantly for PL1 and Tat.
To confirm that the internalization was mediated via macropinocytosis,
co-localization studies using confocal microscopy was performed. In this
45
4. Results and discussion
study the fluid phase marker dextran was used, and after 30 min incubation
together with the different CPPs, co-localization could be observed; penetratin having the highest level, followed by APH-1 (85-98), NCT (38-53)
and PL1 having the least co-localization. This is in agreement with the endocytosis inhibitor study, where the strongest decrease was observed with
penetratin and APH-1 (85-98) whereas the decrease in NCT (38-53) was not
significant, and no reduction in uptake could be observed with PL1.
These results prompted us to investigate whether the observed difference
in uptake mechanism could alter the ability to transport a protein cargo. Coincubation of biotinyl CPPs in a four times molar excess with FITC labeled
streptavidin, with concomitant addition to the cells following standard uptake protocols, showed that penetratin had by far the best vector abilities,
followed by APH-1 (85-98) whereas neither NCT (38-53) nor PL1 were able
to increase the uptake. We believe this can be ascribed the fact that different
uptake mechanisms are responsible for the uptake of the CPPs examined in
this study, and, seemingly, the only universal characteristic for the observed
CPP translocation is the initial binding to GAGs before internalization is
initiated.
4.2 Uptake mechanism of the N-terminal (1-30) part
of the bovine prion protein (Paper II)
The infectivity of prion proteins remains a mystery, and in a previous study
from our group (Lundberg, et al. 2002) we hypothesized that the infectivity
could be ascribed a posttranslational error, where the N-terminal signal peptide was not cleaved off, leading to a retained cell-penetrating motif which
could aid the translocation of the entire protein. That study was based on the
sequence of the mouse prion protein, and, considering the cases suffering
from mad cow disease, we were compelled to further proceed with examining its bovine counterpart. Preceding studies on bPrP (1-30) (bPrPp) had
shown that it, alike the mouse variant, had a predilection for forming βstructures and induce calcein leakage due to pore formation, a study performed on model membranes (Magzoub et al. 2005). It has also got the interesting ability to escape from artificial endosomes (Magzoub et al. 2005).
To be able to make use of the endosomal escape abilities, the initial uptake
must be mediated via endocytosis, whereupon internalization, endsomolysis
is induced, a process which is probably concentration dependent and could
be reliant on a structure conversion of the peptide when reaching acidic compartments.
Using a fluoresceinyl labeled peptide derivative, we could clearly see that
bPrPp was able to translocate into CHO cells, where it seemed to localize in
46
4. Results and discussion
the cytosol. Even though bPrPp has an NLS-like motif, KKRPKP, no fluorescence was observed in the nucleus. To elucidate the mechanism responsible for the uptake, we evaluated the uptake in GAG deficient cells, and examined if we could observe any co-localization using the endosomal markers
dextran and Tf in wild type CHO cells. In the GAG deficient CHO cells, no
substantial internalization could be observed, indicating the uptake to be
dependent on this initial interaction. Using the endosomal markers, an almost perfect co-localization with dextran could be observed, whereas none
could be detected with Tf, concluding that the peptide is taken up via fluid
phase endocytosis, which probably can be attributed macropinocytosis. To
investigate whether the peptide is able to promote endosomal escape, we
incubated the peptide with a luciferase plasmid, and added the mixture to
CHO cells. After 48 h of treatment, a concentration dependent luciferase
signal could be observed, concluding that the peptide is able to induce endocytosis and endosomal escape of the plasmid. The fact that the peptide was
taken up by macropinocytosis was further confirmed using electron microscopy, where gold-labeled DNA in complex with peptides were localized in
endocytic vesicles approximately 0.4 µm in diameter, a size which can only
be ascribed macropinocytotic vesicles.
4.3 Assessing peptide uptake using various
methods (Paper III)
This study was performed to unify the different methods used for CPP internalization in order to facilitate comparison of studies made by different
groups where protocols often vary. The protocol used for quantitative uptake
is the same as described above (Lundberg and Langel 2006), and here we
also wanted to assess the effect of the trypsination step used in the above
mentioned protocol. The effect of the trypsin treatment was dramatic, both
for TP10 and pVEC, where a decrease between approximately 3 and 4 times
could be observed, respectively. This indicates that a significant fraction of
the peptide bound to the cell surface was degraded and subsequently washed
away during the trypsin treatment. However, it is not certain that all peptide
was washed away, but as a step enhancing the reliability of a high throughput screening protocol the results were satisfactory. We also wanted to promote the idea of using confocal microscopy for determination of peptide
intracellular localization and internalization mechanisms. In this this case the
CPP was TP10, and to track endocytosis, co-localization with dextran was
assessed. We can clearly see that TP10 is internalized into HeLa cells, where
an extensive fluorescent signal can be observed perinuclearly, possibly coinciding with Golgi. The co-localization study showed that the main pathway
47
4. Results and discussion
for internalization of TP10 is through fluid phase endocytosis, as a massive
co-localization with dextran could be observed. The last method described
uses HPLC as the quantification method, where both uptake and degradation
can be assessed. Using this method we could clearly observe that the CPP
(KFF)3K was internalized into S. cerevisiae and that, within the hour, a substantial part of the peptide was degraded.
4.4 Evaluation of CPP mediated PNA delivery for
splice correction (Paper IV)
Correction of aberrant splicing using ONs holds a great promise for future
therapeutics, and the splice correction system used in paper IV is an excellent tool for comparison of CPP mediated PNA delivery, as well as mechanistic studies using a biological response as read out. The three peptides
studied, penetratin, Tat and TP are well known CPPs which have been used
for delivery both in vitro and in vivo. The PNA was connected via a disulphide bridge to the CPP, so that once entering the cells the labile bond can
be reduced and the PNA will be able to exert its biological role.
Our results showed that the splice correction efficacy of the CPP-PNA
conjugates was dose dependent, TP having the strongest effect, followed by
penetratin and Tat. Interestingly, a significant decrease in splice correction
was observed when the conjugate was added in serum media as compared to
serum free media. In serum-containing media TP had the best effect, where a
significant splice correction could be observed at 2 µM, as compared to
penetratin and Tat, where 5 µM and 10 µM were needed for a significant
effect, respectively. The decrease in splice correction in serum containing
media might either be attributed to an additional degradation of the CPP
extracellularly, or most probably to unspecific binding of the conjugate to
serum proteins.
Besides being an easy and efficient system to study, another advantage of
the splice correction system is that correlation between quantitative uptake
and splice correction can be evaluated in the same cell lysate using fluoresceinyl-PNA. We could observe a relationship between internalization
efficacy and splice correction for both penetratin and Tat, whereas TP at
lower concentrations had little relation between splice correction and the
observed quantitative uptake. Also, observing that the splice correction was
significantly decreased when inhibiting endocytosis, we wanted to assess
whether increased endosomolysis could enhance splice correction of the
conjugates. In order to evaluate this, we used the lysosomotropic agent
chloroquine, and the viral fusion peptide HA2 conjugated to penetratin. Addition of chloroquine increased the splice correction 2-4 times, the strongest
48
4. Results and discussion
effect observed with TP. The same trend could be observed when adding
HA2-penetratin together with the conjugates, although in this case the increase was not significant. Interestingly, upon adding agents to promote endosomolysis, especially chloroquine, the quantitative uptake of penetratin
and TP conjugates decreased dramatically, an effect which can probably be
ascribed a slow-down of the endocytosis machinery following treatment.
Taken together, these data showed that the uptake of all CPP-PNA conjugates was mediated by endocytosis, and by enhancing the propensity for the
conjugates to escape from the endosomes an increased effect in splice correction could be observed. We could clearly observe that all peptides to
some extent co-localized with dextran, where penetratin and Tat both
showed partial co-localization and TP an almost complete co-localization.
These results are clearly supporting the notion that the uptake of CPPs is
endocytic, and that to achieve an enhanced biological effect, modifications
in order to increase endosomal escape is possible. Also, even though the
uptake of all three CPPs was endocytic, TP was the only peptide showing a
complete co-localization with dextran, whereas the other two peptides, even
though showing a somewhat strong co-localization with dextran, were taken
up by at least two mechanisms where fluid phase endocytosis was one.
4.5 Promoting endosomal escape and siRNA
delivery using endosomolytic CPPs (Paper V)
RNA interference has achieved increasing attention the last few years as a
result of its high efficacy and specificity in gene silencing. Thereof we
wanted to assess whether effective internalization of siRNA could take place
using the well characterized CPPs TP10, penetratin, MPG ΔNLS, bPrPp, and a
penetratin analogue with certain amino acids replaced by histidines in order
to theoretically achieve an α-helix upon protonation. The last peptide, EB1,
was also elongated with sufficient amino acids to be able to span the plasma
membrane, the rational being that upon protonation the α-helix would be
inserted into the endosomal membrane, with a concomitant endosomolysis
(Fig 4.1).
We first wanted to elucidate the ability of the peptides TP10, penetratin,
bPrPp and MPG ΔNLS to bind to siRNA at different molar ratios. Using a gel
shift retardation assay, we could see that at a 10 times molar excess of penetratin, the siRNA was not able to migrate, thus indicating that complete
complex formation had occurred.
49
4. Results and discussion
Fig 4.1 Proposed mechanism of CPP induced endosomolysis. After the initial interaction with
GAGs on the cell surface, the CPP-cargo is internalized via endocytosis. Upon acidification in
the endosomes, the peptide is able to change secondary structure, whereupon endosomolysis
can be induced.
50
4. Results and discussion
For TP10 and MPG ΔNLS the same results were observed at a 20 times molar
excess, whereas for bPrPp a full complex formation was not achieved even
at a 20 times molar excess.
Knowing that the CPPs were able to form complexes with siRNA, we
wanted to establish whether this interaction was enough to promote complex
internalization. The lowest molar ratios used were where a full complex
formation could be observed in the gel shift assay, and, surprisingly, at this
ratio only a small amount of internalization could be observed, with the exception for bPrPp where a massive internalization was seen. However, at
increasing peptide concentration, the other peptides were also able to promote siRNA uptake. However, the uptake of TP10 and MPG ΔNLS at the
highest ratio assessed, 100:1, was 1/3 of the uptake achieved with penetratin
and bPrPp at the same molar ratio. In comparison with Lipofectamine 2000,
the penetratin and bPrPp mediated siRNA delivery was approximately five
times higher.
As quantitative uptake not necessarily correlates well with biological activity, we wanted to confirm that the internalized complexes had gene silencing effect. To measure down-regulation, a previously published siRNA targeting the luciferase mRNA was used (Ui-Tei et al. 2004), where end point
measurement of luciferase activity was used as read out for quantification of
gene silencing effect. At the highest concentration, all peptides except penetratin and TP10 had significant gene silencing activity, thus able to transport
biologically active siRNA across the plasma membrane. It is surprising that
penetratin and TP10 were unable to induce gene silencing as they have previously been used to transport ONs, where TP10 has been shown to work
also using the co-incubation strategy (Davidson, et al. 2004; El-Andaloussi
et al. 2005). Considering the high quantitative siRNA uptake achieved using
penetratin, we concluded that the penetratin/siRNA complexes were retained
in endosomes, thus not able to exert their biological effect. Of the other peptides, bPrPp, MPG ΔNLS and EB1, a significant reduction of luciferase activity could be observed at both 50 and 100 nM siRNA concentration; however
at 10 nM only MPG ΔNLS and EB1 showed significant gene silencing effect.
The molar ratios of peptide:siRNA used in these studies were 50:1 for all
peptides, with the exception of MPG ΔNLS and EB1 were a 25:1 molar ratio
was used.
Observing that the modifications of penetratin led to increased gene silencing, we performed a side by side study comparing penetratin and EB1 to
establish why EB1 and not penetratin was able to induce gene silencing. The
ability of the two peptides to form complexes with siRNA was determined
by an ethidium bromide exclusion assay. Being more reliable in quantifying
complex formation ability than the previously used gel shift assay, we could
observe that EB1 much more readily formed complexes with siRNA as
compared to penetratin, where a 2-5 times higher molar ratio was needed for
penetratin in order to form complexes to the same extent as EB1. This was
51
4. Results and discussion
also translated into the quantitative uptake experiments, where at a 10:1 molar ratio EB1 yielded a ten times higher uptake than penetratin, the effect
being somewhat slighter at 25:1 where the increase was approximately two
times. Although the penetratin modifications we made affected the complex
formation ability, the main reason for constructing the EB1 peptide was to
induce endosomal escape. To confirm that the lack of gene silencing ability
observed with penetratin was due to its inability to escape from endosomes,
we used the fusion peptide HA2-penetratin to induce endosomolysis and
compared the effect with the one using only EB1. When adding HA2penetratin to the cells simultaneously with penetratin/siRNA complexes, a
significant gene silencing could be observed, concluding that penetratin on
its own is unable to promote endosomal escape, but when endosomolysis is
induced the complexes can promote efficient gene silencing. Although an
improvement of penetratin mediated siRNA gene silencing was observed
with the addition of HA2-penetratin, the effect was not as significant as that
of EB1/siRNA induced down-regulation. Thus, we conclude that designing
peptides whith improved endosomal escape properties can be made for enhancement of cargo delivery using CPPs.
52
5. Conclusions
5. Conclusions
Research in the field of CPPs have recent years progressed rapidly, both in
elucidating internalization mechanisms and in applications of the CPP technology. Only 3-4 years ago the main mechanism responsible for CPP translocation was thought to be energy independent, whereas today most research
point towards an internalization process driven by endocytosis. We have
studied uptake mechanisms using different methods, where quantitative uptake, live confocal microscopy and assays measuring biological effect of
cargos have been the major tools. In these studies, the uptake mechanisms
have been assessed using endocytosis inhibitors, co-localization markers and
lysosomotropic agents. All studies we have performed demonstrate the uptake of CPPs to be, at least mainly, mediated by endocytosis, most probably
macropinocytosis. However, none of the endocytosis inhibitors have been
able to fully reduce the uptake process, and most of the microscopy studies
using endosomal markers have yielded substantial co-localization, but not
total. Therefore we cannot exclude additional mechanisms responsible for
CPP uptake besides macropinocytosis, a process which is likely to be ascribed one of the recently identified endocytic pathways CLIC and CLIC-d,
or internalized via membrane penetration.
Besides studying uptake mechanisms, we have also used the CPPs as a
vector for ON delivery, both in splice correction and RNAi. From these studies it is clear that the CPP technology proves a great tool for ON delivery.
The applications of both splice correction and RNAi in disease prevention
and control are endless, although to reach such results, further optimization
is necessary to achieve efficient delivery without serious side effects. One
approach we have used to improve siRNA delivery is by developing a peptide which is designed to be able to shift secondary structure upon protonation following acidification in the endosomes. Consequently, the topology of
the peptide will be changed, resulting in endosomal escape of the endocytosed material. This strategy for increasing CPP mediated delivery could prove
to be an effective and necessary tool for future CPP studies, both in vitro and
in vivo.
53
Populärvetenskaplig sammanfattning på svenska
Populärvetenskaplig sammanfattning på svenska
Peptider är likt proteiner uppbyggda av aminosyror men är per definition
kortare i längd. Temat för denna avhandling är cellpenetrerande peptider
(CPP), alltså peptider som har förmågan att ta sig in i levande celler. Dessa
peptider upptäcktes för ungefär 10 år sedan, och sedan upptäckten har det
varit debatterat huruvida de tar sig in i cellerna genom en process som är
aktiverad av cellen, s.k endocytos, eller om de kan penetrera cellen direkt
utan dess hjälp. Tills för några år sedan var den allmänt vedertagna teorin att
CPP kunde penetrera cellväggen direkt, men de senaste årens forskning har
visat att så inte är fallet, utan att de till stor del tas upp av endocytos.
Förutom att det i sig är viktigt att veta att det är endocytos som står för
upptaget i cellerna så är det också av stor vikt att karakterisera vilken typ av
endocytotisk process som ansvarar för upptaget. En av delarna i denna
avhandling handlar om att bestämma vilken endocytosisk process som är
ansvarig för upptaget av CPP, samt vilka metoder som bäst används för att
avgöra detta. Förutom att dessa peptider har förmågan att korsa plasma
membranet på cellerna, så kan de också ta med sig en last, t.ex. ett protein
eller nukleinsyror som kan användas för genreglering. I de tre första
artiklarna i denna avhandling har vi bestämt hur dessa peptider utan last tas
upp.
Den bredaste applikationen av CPP är för transport av en last, och i
artiklarna fyra och fem har vi undersökt två olika sätt att mediera upptag av
nukleinsyror m.h.a. CPP. I artikel fyra har vi konjugerat CPP direkt med
lasten genom en kemisk bindning som kan bli klyvd när cellmembranet har
korsats. Detta är ett enkelt och effektivt sätt för att transportera nukleobaser
in i celler, och en metod som även kan appliceras i levande organismer och
som framtida läkemedel. I artikel fem har vi försökt att leverera nukleobaser
genom att blanda ett överskott av CPP med dessa. Eftersom nukleobaserna
har flera negativa laddningar, och CPP flera positiva, så kommer dessa att
attrahera varandra, och de kan tillsammans bilda ett komplex. En fördel med
denna metod jämfört med den förra är att det är snabbare och enklare att
konstruera dessa komplex, samt att den s.k. nanopartikeln som skapas är
tämligen stabil och inte bryts ned lika snabbt som konjugatet om man
använder den första metoden. Nackdelen är att det behövs mer peptid,
eftersom överskottet av peptid i jämförelse med nukleobasen är ca. 50
gånger.
Den sista delen i artikel fem behandlar hur man kan modifiera CPP för att
öka effektiviteten av dess last inne i cellen. När CPP tas upp av endocytos så
är detta endast det första steget i vad som behövs för att transportera en last
in i cellen. En endocytotisk vesikel är som en vätskefylld boll innehållandes
54
Populärvetenskaplig sammanfattning på svenska
extracellulärt material, och om inte detta kan komma ut ur vesikeln kommer
det att transporteras vidare in i cellen för att slutligen degraderas av olika
enzym, och dess beståndsdelar återanvändas för att t.ex. bygga upp nya
proteiner eller DNA. För att detta inte ska ske har vi i artikel fem konstruerat
en CPP som har både egenskaperna att tas upp av celler, samt att efter
upptaget i endosomerna kunna påverka det endocytotiska membranet på ett
sådant sätt att det går sönder, och lasten som transporterats med CPP kan
komma ut ur vesikeln. Genom dessa modifikationer har vi lyckats att öka
den biologiska effekten av vår last. Denna förändring för att öka
effektiviteten av CPP är så pass generell att den kan appliceras inom all slags
CPP medierad transport, vilket kan vara till nytta för framtida forskning.
55
6. Acknowledgements
6. Acknowledgements
Det absolut sista jag skriver i denna avhandling läser du nu. Efter att ha haft några sköna
veckor på landet sitter jag nu i skrivandets stund på den plats som varit min i nästan fem år.
Jag minns fortfarande när jag första gången träffade min handledare, Ülo, som tyckte det var
meningslöst att jag skulle läsa fler kurser och som när jag träffade på en torsdagseftermiddag
sa att jag skulle börja på ”måndag morgon klockan åtta”. Det vore en lögn att säga att det inte
funnits tidpunkter i mitt liv när jag kanske skulle önskat att mitt val varit annorlunda, men
dessa är förvånandsvärt få. Jag har under dessa år fått lära mig mycket om både vetenskap och
människor runtomkring mig, och dessutom fått möjligheten att resa till roliga ställen för att
medverka på konferenser.
Mitt största tack går av naturliga skäl till min boss och handledare, Ülo, först och främst
för att vi under åren haft många roliga stunder, främst i dina yngre dar, men också för att du
antog mig som doktorand. Som handledare liknar du ingen annan jag träffat, och jag har
sällan skådat någon med sådan uthållighet att se resultat på ett så positivt sätt som möjligt,
som tycker om att chansa och på något outgrundligt vis får saker att fungera. När jag ändå är i
farten vill jag också skicka ett tack till alla lärare och administrativ personal som jag under
åren träffat. Anders för att du är rolig att diskutera med när du är i farten och alltid har full
koll. Tiit för att du alltid tar dig tid och har ett oslagbart lugn. Kerstin och Anna för trevliga
pratstunder, framför allt på fester, och Mattias för att du är en kul snubbe att dricka öl och
snacka med. Jag vill också tacka Coco för att du under den tid du var här verkligen bidrog
med mycket kunskap och engagemang, och jag tycker att det alltid är trevligt att träffa dig.
Birgitta för att du alltid vet var saker finns, hur de ska göras och att du alltid hjälper till med
allt. Jag förstår inte hur institutionen ska klara sig utan dig. Siv och Ulla för alla trevliga
pratstunder på morgonen inmundigandes frukost; jag kommer att sakna er.
Ett tack till alla doktorander som jag träffat under åren. Schlokrummet bestående av
Jonzo, Pecke, Samme, Henke, Florén och nytillskottet sweet-Pete. En och annan stund har jag
tillbringat i detta rum, samt joggandes och diskuterandes med Jonzo, pumpandes med Samme
(tack för alla samarbeten) och Henke och festandes med Pecke.
Alzheimer’s tjejerna Linda A, Linda F, Veronica, Malin för allt roligt under åren, och
Sofia för trevligt ressällskap på Jamaica! Järnladies bestående av Katarina och Sandra och
giftdamerna Johanna, Helena G för skönt festsällskap. Tidigare och nuvarande synteskungar i
Undéns grupp, Ove, Katri, Micke och Karolina (hoppas datoransvaret inte tar kål på dig).
Mattias mastodontgrupp bestående av Caroline, som jag har haft mycket kul med under de 9
år vi har känt varandra.
Ülos grupp som var vuxit under åren, Helena, Ulla S, Emelia, Maria och Yang (I
especially remember the first time Yang joined us for champagne...) The Estonian possy,
Margus, Külliki, Meeri, Maarja, Kalle, Mats and Pille.
56
6. Acknowledgements
Ett stort tack till alla i rum 409 under åren för underbart sällskap och för att ni smittat av
lite av er seriositet på mig, det behövs... Daniel, Anna (för att du hjälpte mig enormt mycket i
början), Vickan för att du är en underbar person (och har roliga kids) och att du hjälp mig
genom åren samt varit en otrolig fadder, Tina för att du lyckas se på de mest tråkiga saker på
ett fantastiskt positivt sätt, och för att du uppskattar allt jag avskyr ;) Another former 409,
Ursel, for all the fun moments we have had during these years, for supervising me in the
beginning and for always being incredibly happy!
Alla mina samarbetspartners under åren, framför allt Astrid Gräslund för att du är så snäll
och tror på alla vilda idéer.
Ni som jag pluggade med på södertörns högskola, främst Johan, Tobban (tro inte du kan
borsta bort vår vänskap likt mjäll från dina axlar) samt Lotta (vad ska jag säga, all tid vi
spenderat ihop, sena festnätter med följande föreläsningar, Gullholmen och allt annat, hoppas
vi kommer ses mer framöver...)
Mina kära vänner från gymnasiet, Ullis och Sabina samt hela danskgänget, framför allt
Nils och Sören, för all tid vi hängt och underbara stunder på Lindholmen. Mange och Skyla
för att vi alltid fann tid utanför och under skoltiden för att dricka folkbärs. Monkan, Solis och
Sandra (min gamla sambo, hoppas du flyttar hem från bratwurstätarna så vi kan ses mer).
Hanna R (som jag känt från mellanstadiet) och Hanna L (vet inte var jag ska börja, det finns
få jag känner så bra som dig och jag hoppas verkligen att jag alltid kommer att vara så).
Mina ”main men”, Johnsson, Klang och Roos (som jag känt i all evighet), Milbo, Okko
och Macke glaskäken B ( I love you guys!) och Davve koma Samuelsson för att du är en
underbar och lost vän. Tjörngänget, Emma go o glad, terriern Carro för att du irriterar mig så
enormt ibland, men är så smart och härlig, Maria B och Ingo (för ni är underbara!).
De norrländska spexen, i form av Elin med tillhörande göteborgsspex-Patrik (underbara
segelturer) och Jenps för att du är så omtänksam.
Tack till min norrländska familj, Thore, Britta, Robban och Helena, för att jag får hänga
med er i Hemavan och ha helt underbara stunder där.
Min extra familj tvärs över gatan, Lena, Göran och Karin, ni har alltid funnits där för mig
sedan vi flyttade in bredvid er för 25 år sedan.
Linda för allt under åren sedan vi först träffades här på institutionen. Vi har haft några
underbara år och jag hoppas det blir många fler.
Farmor Anna-Lisa för att jag har fått bo hos dig och för att du är så rolig. En tanke går
också till mormor Siri, morfar John och farfar Torsten som tyvärr inte är med oss längre.
Min underbara familj, Morsan, Farsan och Fritte, som jag älskar över allt annat. För allt ni
gjort för mig under åren och all stöd och kärlek ni gett mig; hur ni stog ut när jag var ett
otroligt jobbigt energiknippe är beundrantsvärt. Fritte för att du är världens bästa syster.
57
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