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RATIONAL MODIFICATIONS OF CELLPENETRATING PEPTIDES FOR DRUG DELIVERY
Applications in tumor targeting and oligonucleotide delivery
Maarja Mäe
Rational modifications of cell-penetrating
peptides for drug delivery
Applications in tumor targeting and oligonucleotide delivery
Maarja Mäe
Cover picture: Stefanie Leuker
©Maarja Mäe, Stockholm 2009
ISBN 978-91-7155-792-6
Printed in Sweden by Universitetsservice, Stockholm 2009
Distributor: Department of Neurochemistry, Stockholm University
To my beloved mother
List of Publications
This thesis is based on the following publications, referred to in the text by
the corresponding Roman numerals:
I
Myrberg H, Zhang L, Mäe M and Langel Ü
Design of a tumor-homing cell-penetrating peptide
Bioconjugate chemistry, 2008; 19(1): 70-75.
II
Mäe M, Myrberg H, EL Andaloussi S and Langel Ü
Design of a tumor-homing cell-penetrating peptide for drug delivery
International Journal of Peptide Research and Therapeutics, 2009.
In press.
III
Mäe M, Enbäck J, Laakkonen P, Rosenthal Aizman K, Langel Ü
and Lindgren M
Target-activated cell-penetrating peptide
Manuscript.
IV
Mäe M, EL Andaloussi S, Lundin P, Johansson HJ, Guterstam P
and Langel Ü
A stearylated CPP for delivery of splice-correcting oligonucleotides using a non-covalent co-incubation strategy
Accepted in Journal of Controlled Release.
Additional publications
I
Mäe M,* Myrberg H,* Jiang Y, Paves H, Valkna A and Langel Ü
Internalisation of cell-penetrating peptides into tobacco protoplasts
Biochimica et Biophysica Acta, 2005; 1669(2), 101-107.
II
Mäe M and Langel Ü
Cell-penetrating peptides as vectors for peptide, protein and
oligonucleotide delivery
Current Opinion in Pharmacology, 2006; 6(5): 509-14.
III
Johansson HJ, EL Andaloussi S, Holm T, Mäe M, Jänes J,
Maimets T and Langel Ü
Characterization of a novel cytotoxic cell-penetrating peptide
derived from p14ARF protein
Molecular Therapy, 2008; 16(1): 115-23.
*These authors contributed equally to this work.
Abstract
High molecular weight biomolecules are becoming increasingly important in
the development of new therapeutics. However, the hydrophilic nature and
size of such molecules creates a major limitation for their application – poor
penetration through biological membranes. Over a decade ago, a new class
of peptides, cell-penetrating peptides (CPPs), with membrane translocating
ability, was discovered. They have shown remarkable capability to transport
various hydrophilic macromolecules inside the cells in a relatively non-toxic
fashion. However, there are at least two limiting factors for successful application of CPPs: the lack of cell-type specificity when applied in vivo and
restricted bioavailability resulting from endocytic uptake of CPPs and subsequent entrapment in endosomal compartments.
This thesis aims at designing delivery vehicles for therapeutic substances. In papers I-III, the CPPs have been rationally modified in order to
achieve in vivo selectivity towards cancer cells. The first two papers employ
tumor homing peptides as targeting moieties coupled to the N-termini of
CPPs and evaluated for cargo delivery. In the third paper, a CPP is Cterminally prolonged with a matrix metalloproteinase 2 (MMP-2) specific
cleavage site followed by an inactivating amino acid sequence. In tissues
overexpressing MMP-2, i. e. in proximity to cancer, the CPP is activated
after proteolytic removal of the inactivating sequence, thus the cargo can be
effectively transported inside the nearby cells. In paper IV, an amphipathic
CPP, transportan 10 (TP10), has been N-terminally prolonged with a stearic
acid tail and applied for the delivery of splice-correcting oligonucleotides.
We show that stearyl-TP10 is as effective in oligonucleotide cargo delivery
as commercial transfection agent Lipofectamine™ 2000. Moreover, stearylTP10 displays superior characteristics, preserved efficacy in serum and insignificant toxicity to cells, to the cationic lipid.
In conclusion, we believe that these results give new insights for improving drug delivery. More specifically, the rational modifications of CPPs
greatly potentiate their application in cargo delivery both in vitro and in vivo.
Contents
Introduction ..................................................................................................1
Cancer .........................................................................................................................1
Tumor angiogenesis..................................................................................................3
Matrix metalloproteinases........................................................................................4
MMPs in the hallmarks of cancer.......................................................................5
The function of MMP-2 in tumor development................................................6
Targeted tumor therapy...........................................................................................7
Tumor homing peptides ......................................................................................7
Target-activated prodrugs..................................................................................9
Antisense therapy......................................................................................................9
Alternative splicing ............................................................................................10
Aberrant splicing in diseases ...........................................................................11
Cell-penetrating peptides.......................................................................................13
Uptake mechanism of CPPs..............................................................................14
CPPs as delivery vectors ...................................................................................15
Aims of the study ......................................................................................20
Methodological considerations................................................................21
Design and choice of CPPs.....................................................................................21
Peptide and ON synthesis ......................................................................................22
Purification and characterization of synthesized peptides and ONs...............23
Coupling of cargos to the peptides (Paper I-IV)................................................23
Selection of cargos..................................................................................................24
Cell cultures..............................................................................................................24
Breast cancer cells (Paper I-III)......................................................................24
Endothelial cells (Paper III) .............................................................................25
HeLa cells (Paper IV).........................................................................................25
Evaluation of CPP uptake and cargo delivery.....................................................26
Fluorescence based methods...........................................................................26
Functional assays to evaluate CPPs as delivery vectors .............................27
Toxicity studies ........................................................................................................28
In vivo studies .........................................................................................................28
MDA-MB-435 tumor bearing mice (Paper I and II) .....................................28
MDA-MB-231 tumor bearing mice (Paper III) ..............................................28
Determination of MMP levels (Paper III).............................................................29
In vitro MMP-2 cleavage assay (Paper III).........................................................29
Serum stability.........................................................................................................29
Results and discussion .............................................................................30
Design and evaluation of tumor homing CPPs (Paper I and II)......................30
Target-activated CPP (Paper III)..........................................................................32
Stearylated CPPs for PS 2’-OMe RNA delivery using a non-covalent coincubation strategy (Paper IV)..............................................................................33
Conclusions .................................................................................................35
Populärvetenskaplig sammanfattning på svenska .............................36
Acknowledgements ...................................................................................38
References ..................................................................................................40
Abbreviations
2’-OMe RNA
2’-MOE RNA
AO
ATP
BMD
c
Cbl
CK2
CPP
Cys
DMD
EMMPRIN
Fmoc
i. v.
LDH
MALDI-TOF
MBHA
MMP
mRNA
MT
MTX
OA-Hy
OCT
ON
PMO
PNA
PTD
PS
RP-HPLC
siRNA
snRNP
SPPS
t-Boc
TFA
TIMP
2’-O-methyl RNA
2’-O-methoxy-ethyl RNA
Antisense oligonucleotide
Adenosine triphosphate
Becker muscular dystrophy
Cyclic
Chlorambucil
Casein kinase 2
Cell-penetrating peptide
Cysteine
Duchenne muscular dystrophy
Extracellular matrix metalloproteinase inducers
Fluorenylmethyloxycarbonyl
Intravenous
Lactate dehydrogenase
Matrix assisted laser desorption ionization time-of-flight
4-Methylbenzhydrylamine
Matrix metalloproteinase
Messenger RNA
Membrane type
Methotrexate
Hydroxamate derivative of oleic acid
Optimum cutting temperature compound
Oligonucleotide
Phosphorodiamidate morpholino oligomer
Peptide nucleic acid
Protein transduction domain
Phosphorothioate
Reversed phase high performance liquid chromatography
Short interfering RNA
Small nuclear ribonucleoprotein
Solid phase peptide synthesis
tert-Butyloxycarbonyl
Trifluoroacetic acid
Tissue inhibitor of metalloproteinase
TGF
TP10
WST-1
Transforming growth factor
Transportan 10
(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene disulfonate
Introduction
Drug delivery remains a challenge in modern medicine. A drug has to enter
the target cells in order to exert its biological activity. However, most bioactive molecules, like some anticancer drugs or antisense oligonucleotides,
have restricted access into the cell interior due to the highly selective membrane barrier. Therefore, the development of “carriers” for such therapeutics
capable of surmounting the greatest obstacle in drug delivery, the plasma
membrane, is highly desired.
There are several viral and non-viral vehicles developed for drug delivery. A class of peptides, cell-penetrating peptides (CPPs), has recently
gained growing attention as non-viral vectors. CPPs have the ability to translocate various cargos, ranging from small drug molecules and oligonucleotides (ONs) to large plasmids and proteins, through the plasma membrane in
a relatively non-toxic fashion. However, CPPs suffer from substantial entrapment in endosomal compartments as well as lack of cell-type specificity.
This thesis aims at improving CPPs as therapeutic vehicles using cancer as a model system. First, we concentrate on rational modifications of
already known CPPs with targeting moieties for application in tumor cellspecific drug delivery. The tumor microenvironment, more specifically angiogenesis and the overexpression of specific matrix metalloproteinases, is
taken into account when designing the peptides. Second, we modify wellknown CPPs with a hydrophobic moiety in order to improve the noncovalent cellular delivery of splice-correcting ONs.
Cancer
Cancer is one of the most frequent causes of death worldwide. It is easy to
neglect that cancer is not a single disease, but many diseases characterized
by uncontrolled cell proliferation. Tumors are classified into four major
groups according to their origin - carcinomas (epithelial), sarcomas (mesenchymal), hematopoietic cancers (blood and immune system) and neuroectodermal tumors (originate from nervous system). However, tumor tissue
comprises not only cancer cells, but also stromal cells including fibroblasts,
adipocytes, endothelial-, immune and inflammatory cells, which all have a
significant impact on tumor progression (Fig. 1) (Mueller and Fusenig
2004).
1
Six essential alterations have been proposed as the hallmarks of cancer, that together dictate the malignant growth of all tumors (Hanahan and
Weinberg 2000):
1.
2.
3.
4.
5.
6.
self-sufficiency in growth signals,
insensitivity to growth-inhibitory signals,
evasion of apoptosis,
limitless replicative potential,
sustained angiogenesis,
tissue invasion and metastasis.
Genomic instability, i. e. mutations in oncogenes and tumor-supressor genes
that control normal cell cycle processes result in the abovementioned six
alterations. The process when genetically unstable cancer cells escape from
the primary tumor and find their way through the blood or lymphatic stream
to a distant tissue and adapt to the tissue is called metastasis (Gupta and
Massagué 2006). The complex multistep nature of this process is the reason
why actually only a relatively small fraction of primary cancer cells are capable of developing metastasis. Cancer metastasis makes it especially complicated to treat this disease since it is difficult to identify cancer cells in
distant organs and moreover problematic to address with systemic therapies.
Figure 1. Tumor microenvironment. Tumor tissue comprises not only cancer cells,
but also stromal cells including fibroblasts, adipocytes, endothelial-, immune and
inflammatory cells. Different matrix metalloproteinases (MMPs) are synthesized by
both stromal and cancer cells. Adapted from Egeblad and Werb 2002.
Breast cancer is by far the most frequent cancer in women worldwide, comprising 23% of all cancers, with an estimated 1.15 million new cases per year
2
(Parkin, et al. 2005). Not the primary tumors, but distant metastasis are the
cause of mortality in breast cancer (Weigelt, et al. 2005). Most common
metastatic sites of this malignancy are lung, bone, liver and pleural cavity
(Weigelt, et al. 2005).
There are countless of molecules and a number of processes that results in genomic instability required for tumor development and progression;
however in the next sections only an overview of angiogenesis and matrix
metalloproteinases (MMPs) as well as their role in cancer progression will
be discussed in more detail.
Tumor angiogenesis
Angiogenesis, the formation of new blood vessels from pre-existing ones, is
essential during growth and development, wound healing, female reproductive cycle and pregnancy, but is otherwise quiescent in adult organisms. Furthermore, angiogenesis plays an important role also in many pathological
processes such as cancer (Bergers and Benjamin 2003), stroke, Alzheimer’s
disease, and in eye morbidity and blindness.
The hypothesis that tumor growth is angiogenesis-dependent was first
postulated by Judah Folkman in 1971 (Folkman 1971) and afterwards has
been confirmed by a large number of research groups (reviewed in Bergers
and Benjamin 2003). It is now established that tumors cannot grow beyond a
size of 1-2 mm3 without blood supply. Angiogenesis is not merely a prerequisite for tumor growth but also for the development and progression into
metastasis. Malignant tumor cells recruit blood as well as lymphatic vessels
to spread to distant organs.
Tumor blood vessels are morphologically different from their normal
counterparts: they are tortuous, irregularly shaped, leaky, dilated and often
have blind ends (Dewhirst, et al. 1989). Additionally, blood flows irregularly
and more slowly (McDonald and Choyke 2003). This all results in hypoxic
(oxygen deficit) tumor tissue, which in turn induces the sprouting of new
blood vessels, so called “angiogenic switch” (Folkman and Hanahan 1991,
Yancopoulos, et al. 2000). The onset of angiogenesis is determined by the
type of tumor as well as its microenvironment. Tumors express various proangiogenic factors that induce the formation of new blood vessels, for instance vascular endothelial growth factor A, B, C (Ferrara 2002), fibroblast
growth factor, platelet derived growth factor B, angiopoietin-1
(Yancopoulos, et al. 2000), placental growth factor (Carmeliet, et al. 2001),
as well as various MMPs (Genís, et al. 2006, Kim and Kim 1999, van Hinsbergh and Koolwijk 2008).
Moreover, certain proteins, i. e. tumor vascular markers, are expressed
on the tumor endothelial cells but not on the vasculature of normal tissues.
Some of these well-characterized proteins are roundabout-4, endothelial3
specific protein disulphide isomerase, fibronectin, DELTA4 and tumor endothelial markers (reviewed in Neri and Bicknell 2005). Such tumor-specific
molecules, already known and yet to be discovered, create an excellent possibility for tumor targeting.
Matrix metalloproteinases
MMPs are proteolytic enzymes capable of degrading various proteins. They
play an important role not only in angiogenesis, bone remodeling, and normal wound healing, but also in pathological processes like arthritis, atherosclerosis and cancer. The primary function of MMPs in normal physiological
processes is the maintenance of extracellular matrix.
MMPs are zinc-dependent endopeptidases first discovered by Gross
and Lapiere in 1962 when they demonstrated that cultivating bullfrog tadpole tissues on collagen gels resulted in degradation of the substrate into
collagen peptides (Gross and Lapiere 1962). To date more than 20 different
MMPs are recognized and they are commonly divided into 2 main groups,
secreted or membrane-bound MMPs. These groups are in turn divided into
six subgroups according to their structure and putative substrate specificity:
collagenases, gelatinases, stromeolysins, matrilysins, membrane type (MT)
MMPs and other types that do not fit into the abovementioned classification
(Vihinen and Kähäri 2002). Most of the MMPs are not constitutively expressed in the adult body; instead their expression is rapidly induced when
needed by external stimuli like cytokines, growth factors, hormones, cellmatrix and cell-cell interactions (Westermarck and Kähäri 1999). Additionally, the activity of already secreted MMPs is tightly controlled by endogenous inhibitors like tissue inhibitors of MMPs (TIMPs) (reviewed in Baker,
et al. 2002), α2-macroglobulin (Sottrup-Jensen 1989) and thrombospondin-2
(Yang, et al. 2001). In cancer, for instance, tumor cells produce factors, extracellular MMP inducers (EMMPRINs) that enhance the production of
MMPs from stromal fibroblasts (Biswas, et al. 1995). All MMPs are synthesized as latent proenzymes that can be activated by other MMPs or various
other proteins (reviewed in Chakraborti, et al. 2003). Latency of these enzymes is maintained through the so called “cysteine switch” mechanism
proposed by Springman, et al. 1990. The pro-domain of MMPs contains a
conserved cysteine (Cys) that interacts with an active site zinc atom (bound
to three histidines) and maintains the enzyme inactive (Fig. 3). Hence the
dissociation of zinc atom from the Cys residue leads to activation of the enzyme, and thus this process is called “cysteine switch”.
4
MMPs in the hallmarks of cancer
Initially, MMPs were thought to be exclusively important in tumor invasion
and metastasis, however, later studies have shown that MMPs are involved
in various steps of cancer development (Hanahan and Weinberg 2000).
MMPs have shown to play a crucial role in both cancer-promoting and
cancer-inhibiting events (Fig. 2). MMPs are able to promote tumor cell
growth by releasing the precursors of growth factors bound to the cell membrane, for example transforming growth factor α (TGF-α) (Peschon, et al.
1998). On the other hand, activation of TGF-β by MMPs can negatively
regulate cancer-cell growth (Derynck, et al. 2001). Additionally, MMPs are
capable of controlling whether a tumor cell undergoes apoptosis or not, possibly by indirectly influencing the integrin signaling; and moreover they
have shown to be able to induce angiogenesis, that is highly essential for
tumor growth beyond the microscopic size (Egeblad and Werb 2002). One
interesting aspect is the functional role that MMPs have in the immune response to cancer. Indeed, MMPs may be involved in cancer cell escape from
the immune surveillance (Coussens and Werb 2002).
However, the most well-characterized function of MMPs is their role
in tumor invasion and metastasis. Overexpression of certain MMPs promote
tumor progression while TIMPs have an inhibitory effect (thoroughly reviewed in Egeblad and Werb 2002). More specifically, MMP-2 is widely
reported to play a pivotal role in tumor associated events.
Figure 2. The role of MMPs in cancer development.
5
The function of MMP-2 in tumor development
The MMPs are a large and diverse group of enzymes, therefore only MMP-2
and related MT1-MMP (MMP-14) that play important roles in this thesis
will be depicted in more detail.
MMP-2 is a 72 kDa gelatinase A (and type IV collagenase) particularly important in tumor development and progression (Fig. 3). Since MMP2 and MMP-9 degrade type IV collagen, the core component of the basement
membrane and the main barrier separating in situ and invasive tumor, it is
believed to play a crucial role in tumor metastasis. There have been studies
suggesting that MMP-2 overexpression is involved in breast cancer metastasis for example to brain (Mendes, et al. 2007) and lungs (Gupta, et al. 2007).
Moreover, it has been shown that active MMP-2 is detected more frequently
in malignant than benign breast carcinomas (Hanemaaijer, et al. 2000).
Figure 3. Schematic diagram of MMP-2 (a) and MT1-MMP (b). Signal, N-terminal
signal sequence; Pro, pro-peptide; Cys, cysteine thiol group interacting with zinc;
Catalytic; catalytic domain; Fi, gelatin- or collagen-binding type II repeats of fibronectin; Zn, zinc binding site; H, hinge; Hemopexin; determines the substrate
specificity and has interaction site for tissue inhibitors of MMPs; Fu, recognition site
for intracellular furin-like serin proteases; TM, single span transmembrane domain;
Cy, short cytoplasmic domain.
As mentioned above, like all MMPs, MMP-2 is expressed as a zymogen and
its activation is a well-studied event where MT1-MMP (Fig. 3) and TIMP-2
play important roles (Kinoshita, et al. 1998, Strongin, et al. 1995, Butler, et
al. 1998, Sato, et al. 2005, Zucker, et al. 1998). First, active MT1-MMP
binds to the N-terminus of TIMP-2 and attaches soluble TIMP-2 to the cell
surface. This MT1-MMP–TIMP-2 complex acts as a receptor for pro-MMP2, in such that pro-MMP-2 binds to the C-terminus of TIMP-2. Consequently, the prodomain of latent MMP-2 is cleaved by an adjacent MT1MMP (TIMP-2 free) creating an activated intermediate which is fully activated through intermolecular autocleavage (Atkinson, et al. 1995). In case
TIMP-2 is expressed in excess to MT1-MMP, the first step of MMP-2 acti-
6
vation process is blocked and no MMP-2 activation occurs (Kinoshita, et al.
1996, Sato, et al. 1996, Strongin, et al. 1995).
In tumors, cancer cell interaction with fibroblasts, endothelial cells
and other tumor stromal cells determine the expression of different MMPs.
In breast cancer, both MMP-2 and MT1-MMP have been shown to be expressed by vascular endothelial cells (Chun, et al. 2004, Jia, et al. 2000) and
MT1-MMP additionally by myofibroblasts adjacent to breast cancer cells
(Bisson, et al. 2003). In conclusion, it is difficult to discriminate which cells
in the tumor tissue that are expressing specific type of MMPs, but it is sure
that cellular interplay determines the level of particular MMP expression.
Targeted tumor therapy
Although there are number of treatments available today to fight cancer, the
need for improved therapeutics is still desired (Clarke, et al. 2005a, Clarke,
et al. 2005b). The development of new compounds for the systemic treatment of cancer is a challenging task mainly due to the heterogeneic nature of
tumors. Moreover, a successful therapeutic must first be delivered to the
target site and then enter the target cell to exert its biological effect while
having little or no effect on non-targeted cells for minimal the side-effects.
In order to fulfill those criteria, a drug has to first overcome the physiological barriers like rapid degradation in the blood stream, renal clearance, and
the immune system, and then cellular barriers like cellular membranes, entrapment in endosomes, and intracellular degradative enzymes. This simplified description gives an idea of how complicated it is to develop promising
cancer therapeutics.
Novel strategies are required to improve the targeting of anticancer
agents to tumor tissue and to minimize the adverse effects on healthy organs.
Next, two approaches, that take advantage of tumor vasculature and the
molecules expressed on the tumor blood vessels as well as tumor stroma, are
described as possible vehicles for targeted tumor therapies.
Tumor homing peptides
Tumor homing peptides are a class of peptides that bind to tumor vasculature
after intravenous (i. v.) injection. These peptides are discovered by screening
of phage display libraries first introduced by Smith in 1985 (Smith 1985).
Phage libraries are created by incorporating random DNA sequences into the
phage genome; in turn each phage expresses only one type of peptide on its
surface. The phage libraries can then be applied for in vivo screening by
injecting to the tail vein of mice and allowing them to distribute throughout
the body (Ruoslahti 2002). Phages that display a tumor-specific peptide on
their surface become concentrated in the tumor tissue, tumor is isolated,
7
phages extracted and amplified and injected for repeated rounds until one
type of phage is in majority. Finally, the DNA that encodes the expressed
peptide is sequenced and this peptide can be used for in vivo tumor targeting.
Table 1. Selection of tumor homing peptides applied in cargo delivery.
Peptide
Cargo
a
a
NGR , RGD Dox
F3
b
2’OMe/PS
RNA
Fl/−
LyP-1a
RGDa
AdLuc
b
Cancer
Analyses/Effect
Reference
Breast
Inhibited tumor growth
Arap, et al. 1998
Breast
Inhibited tumor growth and
metastasis
Breast
Tumor imaging/Inhibited
tumor growth
Breast/Brain Tumor imaging
Henke, et al. 2008
Laakkonen, et al.
2004
Niu, et al. 2007
CREKA
Nanoparticle Breast
Self-amplifying homing
GX1a
99
Gastric
Tumor imaging
Lipo-Dox
Lung, oral
Inhibited tumor growth
Simberg, et al.
2007
Zhi, et al. 2004,
Hui, et al. 2008
Lee, et al. 2007
Fl
Metastasis
Metastasis imaging
Yang, et al. 2008
SP5-52b
TMTP1
b
TcmO4-
−, no cargo; acyclic; blinear; 2’OMe, 2’-O-methyl RNA; AdLuc, adenovirus carrying firefly
luciferase reporter gene; Dox, doxorubicin; Fl, fluorescein; Lipo-Dox, liposome carrying
doxorubicin; PS, phosphorpthioate; 99Tc, technetium 99.
A number of tumor homing peptides targeting different tumors have been
identified by screening such aforementioned libraries. Many of these peptides have been successfully applied in tumor imaging and treatment (Table
1). One excellent study published recently by Henke et al. utilized the tumor
homing peptide F3 (Porkka, et al. 2002) which is believed to bind to cell
surface nucleolin (Christian, et al. 2003). They utilized F3 for antisense ON
delivery to breast tumor xenografts (Henke, et al. 2008). F3-conjugated to
2’OMe/PS RNA antisense was applied to downregulate Id1, a transcription
factor required for tumor invasiveness, metastasis and angiogenesis which
led to significant inhibition of tumor growth and metastasis.
The majority of tumor homing peptides bind to markers on tumor
blood vessels, however there are also peptides homing to tumor lymphatics.
Laakkonen et al. discovered a cyclic breast tumor lymphatic’s specific peptide, LyP-1, which is not only homing to primary tumors but also to their
metastasis (Laakkonen, et al. 2004). Recently, a mitochondrial/cell-surface
protein p32 was shown to be a receptor for LyP-1 (Fogal, et al. 2008).
Moreover, there is no need for conjugating LyP-1 to an anticancer drug because the systemic treatment with only the peptide inhibited tumor growth.
All the given reports in Table 1 illustrate the vast potential of tumor homing
peptides in cancer treatment and imaging.
8
Target-activated prodrugs
The second strategy for tumor targeting involves so called prodrugs, designed to be non-toxic until cleaved by enzymes that are overexpressed in
tumor environment. There are several proteases shown to be abundantly
expressed in proximity to tumors but not in normal tissues. For example,
several cancers have reported to have increased levels of MMP-2, -9, -14
(Duffy, et al. 2000, Fingleton 2006, Garzetti, et al. 1996, Yamamura, et al.
2002), plasmin (Dano, et al. 2005), cathepsin B (Jedeszko and Sloane 2004),
urokinase-type plasminogen activator (Dano, et al. 2005) and prostatespecific antigen (Partin, et al. 2002).
The prodrug idea has been employed to improve the anticancer drugs’
therapeutic index, i. e. the ratio between the toxic dose and the therapeutic
dose of a drug. For instance, Albright et al. utilized a MMP-selective prodrug where doxorubicin was conjugated to a heptapeptide, which prevented
the prodrug from entering cells. Upon MMP cleavage doxorubicin is “activated” and taken up by nearby cells. Moreover, they showed that this prodrug cured 8 out of 10 mice with HT1080 xenografts at doses below the
maximum tolerated dose, while doxorubicin alone cured only 2 out of 20
mice at its maximum tolerated dose (Albright, et al. 2005). Additionally, two
other groups developed anticancer drug-peptide derivatives that have the
ability to bind to circulating albumin (Mansour, et al. 2003, Warnecke, et al.
2007). Interestingly, the albumin-bound drugs were found to be specifically
and efficiently cleaved by target enzymes. Moreover, such prodrugs demonstrated superior antitumor efficacy in vivo compared to free drug. Table 2
provides an overview of several studies that have taken advantage of various
tumor-specific enzymes in prodrug design and applied in tumor targeting.
Table 2. Selection of target-activated prodrugs applied in tumor treatment.
Carrier
Enzyme specificity Cancer
Cargo
Reference
Mansour, et al. 2003
Albumin
MMP-2
Melanoma
Dox
Avidin
MMP-2
Melanoma
Biotinyl-melittin Holle, et al. 2003
Tripeptide
Plasmin
Breast
Dox
Heptapeptide MMP-2, -9, -14
Fibrosrcoma Dox
Devy, et al. 2004
Albright, et al. 2005
Hexapeptide
MMP-2, -9
Fibrosrcoma MTX
Chau, et al. 2006
Albumin
Cathepsin B
Ovarian
Warnecke, et al. 2007
MTX
Dox, doxorubicin; MTX, methotrexate.
Antisense therapy
Antisense technology holds a great potential as a therapeutic tool in gene
therapy. In 1978 Zamecnik and Stephenson reported, what is now considered
9
as a proof-of-principle for antisense technology, that an ON complementary
to the 3’ end of Rous sarcoma virus could block viral replication in chicken
fibroblasts (Zamecnik and Stephenson 1978). Antisense technology exploits
15-22 nucleotide long oligonucleotide (AO) analogues designed to be complementary to a selected gene (transcriptional level) or mRNA (posttranscriptional level) and have the ability to bind to target sequence through
Watson-Crick hybridization (Crooke 2004). These interactions can lead to
inhibition of transcription of chromosomal DNA (Cutrona, et al. 2000, Janowski, et al. 2005); or translational arrest of mRNA, through RNase H
cleavage of mRNA-ON duplex or through steric hindrance of the ribosomal
assembly on mRNA (Gleave and Monia 2005).
There is a growing repertoire of antisense mechanisms that can be exploited for post-transcriptional gene silencing. The most recognized are
RNA interference first published by Fire et al. where the double stranded
short interfering RNA (siRNA) duplexes suppressed gene activity in
Caenorhabditis elegans (Fire, et al. 1998); micro RNAs, naturally occurring
post-transcriptional gene regulatory elements (Ambros 2001), and splicecorrecting AOs, selectively manipulating alternative pre-mRNA splicing.
The latter will be discussed below in more detail.
Alternative splicing
In constitutive splicing, introns (non-coding sequences) are removed from
newly transcribed pre-mRNAs and all exons (coding sequences) are joined
together in the order of which they occur in the pre-mRNAs. In alternative
splicing, different mRNAs are produced from the same gene (for example
through exon skipping); it is a key post-transcriptional modification that
contributes to protein diversity through highly regulated splicing events.
Pre-mRNA splicing takes place in the cell nucleolus and is carried out
by a splicing machinery called the spliceosome. The spliceosome consists of
small nuclear ribonucleoproteins (snRNP) and a large number of nonsnRNPs. The spliceosome recognizes intron/exon boundaries, the so-called
5’ and 3’ splice sites (invariant GU and AG intronic nucleotides, respectively), branch point and polypyrimidin tract and catalyses accurate cleavage
and rejoining of exons (Srebrow and Kornblihtt 2006). Additionally, lessconserved consensus motifs (intronic and exonic splicing enhancers and
silencers) participate in the splicing process.
Mutations in cis- and trans-acting elements of splicing might result in
disruption of normal splicing which can lead to various serious diseases. In
the following section the role of aberrant splicing in disease, as a direct
cause or contribution for disease development like cancer, will be described.
10
Aberrant splicing in diseases
As already mentioned above, the disruption of normal alternative splicing
patterns can lead to various diseases, such as cystic fibrosis, muscular dystrophies, β-thalassaemia, and several neurological disorders (Garcia-Blanco,
et al. 2004, Pajares, et al. 2007). For example, mutations in the dystrophin
gene can lead to Duchenne muscular dystrophy (DMD) which is a severe
disease characterized by progressive degenerative myopathy or to its milder
allelic disorder called Becker muscular dystrophy (BMD) (Koenig, et al.
1989). Most nonsense mutations within this gene lead to a shift in the translational reading frame and thus complete absence of functional dystrophin
protein resulting in development of DMD. In BMD, the nonsense mutations
occur in exonic splicing enhancer sequences in the dystrophin gene, which
leads to partial in-frame skipping of an exon and production of partly functional dystrophin protein.
Aberrations in alternative splicing have also been observed in many
cancer-related genes (thoroughly reviewed in (Pajares, et al. 2007, Srebrow
and Kornblihtt 2006). Cis-acting mutations that affect the splicing of oncogenes and tumor supressors can have contributory roles in cancer development. However, most-cancer associated splicing abnormalities are not
caused by mutations in the affected gene suggesting there are mutations in
trans-acting splicing elements (Wang and Cooper 2007).
Application of ONs for splice correction
As already mentioned, several types of AOs have been utilized to downregulate gene expression through RNase-H mediated degradation of mRNA
or by steric blockage of gene promoter elements. However, growing knowledge about the importance of aberrant splicing in human diseases has resulted in development of various strategies for abrogating splicing defects.
For instance, small molecule inhibitors are attractive in targeting different
protein isoforms (Garcia-Blanco, et al. 2004) or trans-splicing approach
repairing genetic defects in the mature mRNA (reviewed in Wood, et al.
2007).
Additionally, splice-correcting AOs can be employed for altering aberrant splicing patterns on the pre-mRNA level. Since unmodified RNA or
DNA have poor stability in biological systems, several ON analogues have
been developed to improve their stability and even specificity and affinity
for target sequences. Most commonly used RNA analogues include phosphorothioate (PS) (Campbell, et al. 1990), 2’-O-methyl (2’OMe) (Morvan, et
al. 1993), 2’-O-methoxy-ethyl (MOE) (Baker, et al. 1997) modified RNAs,
as well as phosphorodiamidate morpholino oligomers (PMO) (Summerton
1999) and peptide nucleic acids (PNAs) (Egholm, et al. 1993) (Fig. 4).
These AOs are reported to block cryptic splice sites; induce exon skipping or
inclusion to alter expression and; induce exon removal to eliminate a non11
sense mutation to restore the reading frame around a genomic deletion.
Some examples of such manipulations are given in Table 3.
Additionally, it is possible to direct natural alternative splicing towards a desired protein isoform. For example, the bcl-x gene is alternatively
spliced to two proteins with antagonistic functions, the pro-apoptotic Bcl-xS
and the anti-apoptotic Bcl-xL. The latter has been shown to be overexpressed in various cancers (Kirkin, et al. 2004). By blocking the alternative
5’ splice site in the bcl-x intron 2 with AOs, the splicing can be shifted from
the bcl-xL to the bcl-xS isoform and thereby make cells susceptible to apoptotic stimuli (Mercatante, et al. 2001, Taylor, et al. 1999).
Figure 4. A selection of widely utilized ON analogues. 1st generation: PS-RNA
(phosphorothioate RNA) has an improved stability in human serum. 2nd generation:
2’-OMe RNA (2’-O-methyl RNA) and 2’-MOE (2’-O-methoxy-ethyl) have reduced
toxicity and slightly increased affinity for RNA and is not recognized by RNase H.
3rd generation: PMO (phosphorodiamidate morpholino oligomer) and PNA (peptide nucleic acid), uncharged ON analogues with improved target specificity.
All examples given in Table 3 illustrate the potency of such AOs to be applied in clinical trials. However, the efficacy of these RNA-based AOs still
remains poor due to delivery-related issues. Commonly used transfection
systems including cationic liposomes, cationic polymers, viruses, micro in12
jection and electroporation are all facing several drawbacks. For example,
cationic liposomes are restricted to in vitro applications due to their sensitivity to serum proteins (Liu, et al. 2003) and are generally toxic. Polyethylene
imine (Boussif, et al. 1995), the most commonly used cationic polymer, is
highly toxic probably due to its non-degradable nature. Viruses are limited to
plasmid delivery and are possibly immunogenic in vivo and moreover carry a
risk for viral recombination.
All the listed problems with current transfection methods for ONs
clearly indicate that new better agents are needed for successful ON delivery. A relatively new class of peptides, cell-penetrating peptides (CPPs), is
especially promising for this application.
Table 3. Modulation of aberrant splicing in various diseases.
Disease
β-thalassemiaa
β-thalassemiab
β-thalassemiac
Hutchinson-Gilford
progeria syndromea
Cystic fibrosisa
Ataxia-telangiectasiaa
Duchenne muscular
dystrophyc
Duchenne muscular
dystrophyc
Frontotemporal dementia
and parkinsonisma
Target gene
AO
Blockage of cryptic splice site
β-globin
2’OMe RNA
β-globin
PMO
β-globin
MOE
Lamin
PMO
CFTR
ATM
2’OMe RNA
PMO
Exon skipping
Dystrophin
2’OMe RNA
Dystrophin
PMO
Tau
2’OMe RNA
Reference
Sierakowska, et al. 1996
Lacerra, et al. 2000
Sazani, et al. 2002
Scaffidi and Misteli
2005
Friedman, et al. 1999
Du, et al. 2007
Lu, et al. 2005
Alter, et al. 2006,
Fletcher, et al. 2006
Kalbfuss, et al. 2001
Exon inclusion
Spinal muscular atrophya
SMN
MOE
Hua, et al. 2007
Glioblastomaa
FGFR-1
PMO
Bruno, et al. 2004
a
in vitro; bex vivo; cin vivo; ATM, ataxia-telangiectasia mutated; CFTR, cystic fibrosis
transmembrane conductance regulator; FGFR, fibroblast growth factor receptor; SMN,
survival motor neuron.
Cell-penetrating peptides
The cellular plasma membrane possesses an effective barrier for most hydrophilic macromolecules used for therapeutic purposes. The need to deliver
biologically active agents into cells has encouraged researchers to develop
various delivery vehicles. Unfortunately, most delivery systems used today
suffer from different limitations that need to be overcome to be applicable in
vivo.
14 years ago a new class of peptides, CPPs, also referred to as protein
transduction domains (PTDs), with membrane translocation ability was dis13
covered. The first presented CPP, penetratin (also named Antp), was derived
from the third helix of the Drosophila melanogaster antennapedia transcription factor homeodomain (amino acids 43-58) (Derossi, et al. 1994). Penetratin, pVEC (Elmquist, et al. 2001) as well as Tat (48-60) (Vivés, et al.
1997) peptides represent CPPs derived from naturally occurring proteins.
There are also chimeric CPPs like transportan, which has 12 amino acids
derived from the neuropeptide galanin fused together with 14 amino acids
derived from the wasp venom mastoparan (Pooga, et al. 1998). Additionally,
two widely used chimeric CPPs are MPG (Morris, et al. 1997) and the deletion analogue of transportan, TP10 (Soomets, et al. 2000). A third group of
CPPs is the synthetic peptide family, where polyarginines are the best studied members (Wender, et al. 2000).
Generally, CPPs are relatively short (5-40 amino acids), cationic
and/or amphipathic peptides that are capable of carrying diverse biological
cargos across cellular membranes and possess relatively low cellular toxicity.
Uptake mechanism of CPPs
Although the uptake mechanism of CPPs is still debated, the new findings in
this research field during the past few years have contributed to the understanding of how these short peptides make their way into the cell interior. It
is important to gain information about their uptake mechanism in order to be
able to rationally design new CPPs or to modify the “old” ones for cargo
transport into specific cellular compartments. Based on a number of investigations, four main events for CPP uptake have been suggested: first, interactions with cell surface; second, translocation through cell membrane either
via different types of endocytosis or direct translocation through cell membrane; third, destination to different intracellular compartments; and fourth,
in the case of endocytic uptake, the endosomal release of the CPPs.
Independent of the subsequent cellular uptake route, it is believed that
internalization of CPPs or CPP-cargo complexes begins with interactions
with components on the surface of the plasma membrane. It has been shown
that this occurs through binding to cell surface proteoglycans, mostly
through heparane sulfate proteoglycans (Foerg, et al. 2007, Gerbal-Chaloin,
et al. 2007, Poon and Gariepy 2007, Pujals, et al. 2006, Lundin, et al. 2008,
Richard, et al. 2005). This binding is followed by activation of small Rho
GTPases which in turn results in remodeling of the actin network and thus
contributes to the cell membrane fluidity and following translocation processes. Additionally, hydrophobic interactions between CPP sequences and
cell membrane lipids have been proposed to be important in creating the first
contact (Pujals, et al. 2006).
14
As mentioned above after CPP-cell surface interactions either direct
translocation or endocytosis can occur (Table 4). Energy-dependent fluid
phase endocytosis comprises at least macropinocytosis, clathrin-mediated
endocytosis, caveolae-mediated endocytosis and clathrin- and caveolaeindependent endocytosis (thoroughly reviewed in Conner and Schmid 2003,
Futaki, et al. 2007, Henriques, et al. 2007, Herce and Garcia 2007). There is
evidence that CPPs can utilize several different endocytosis pathways simultaneously, thus when blocking one pathway the other one/ones become active (Duchardt, et al. 2007, Padari, et al. 2005, Lundin, et al. 2008). Moreover, the choice of endocytic pathway seems to be dependent on the size and
nature of attached cargo. Additionally, energy independent direct translocation through cell membrane has also been supported by several studies
(Duchardt, et al. 2007, Palm-Apergi, et al. 2008).
Table 4. Defined uptake mechanisms for Tat, transportan, TP10, penetratin and
polyarginine.
Cargo
Mechanism
Fluorescein
Tat
Clathrin-mediated endocytosis
Macropinocytosis
EGFP
Phage
Cre-recombinase
Avidin-gold
PNA
PNA
Fluorescein
Fluorescein/biotin
Direct translocation
Caveolae-mediated endocytosis
Caveolae-mediated endocytosis
Macropinocytosis
Transportan
Mainly macropinocytosis
Clathrin-mediated endocytosis
TP10
Clathrin-mediated endocytosis
Polyarginine
Clathrin-mediated endocytosis
Macropinocytosis
Penetratin
Direct translocation
Fluorescein
Macropinocytosis
EGFP, enhanced green fluorescent protein.
Reference
Richard, et al. 2005,
Kawamura, et al. 2006
Nakase, et al. 2004,
Nakase, et al. 2007
Tünnemann, et al. 2006
Fittipaldi, et al. 2003
Eguchi, et al. 2001
Wadia, et al. 2004
Padari, et al. 2005
EL Andaloussi, et al. 2006
Lundin, et al. 2008
Kawamura, et al. 2006
Nakase, et al. 2004,
Nakase, et al. 2007
Dupont, et al. 2007,
Christiaens, et al. 2004
Sugita, et al. 2008
The intracellular fate of CPPs is determined by the internalization pathway.
For example, if the uptake occurs through clathrin-mediated endocytosis, the
peptides are destined to the degradative route. On the other hand micropinocytosis has been proposed to bypass the lysosomal pathway (Hewlett, et al.
1994).
15
The results from the plethora of experiments are not fully conclusive,
as it seems that the translocation mechanism utilized is dependent on the
nature of cargo, cell-type, CPP and the concentration of CPP. Recent studies
have shown that at higher concentrations, uptake occurs via direct translocations, and at lower concentrations via the endocytosis pathway (Duchardt, et
al. 2007, Fretz, et al. 2007). Suggested uptake mechanisms for some most
utilized CPPs – Tat, transportan, TP10, penetratin and polyarginine - are
given in Table 4.
CPPs as delivery vectors
CPPs are versatile tools and their ability to enter a wide variety of cell types
in a non-discriminating manner forms the favorable basis for their practical
use. Cargos that have been transported by CPPs are peptides, proteins, drugs,
nanoparticles, lipsomes, ONs, phages, plasmids and imaging agents (thoroughly described in recent reviews: Dietz and Bähr 2004, EL Andaloussi, et
al. 2005, Stewart, et al. 2008). However, the application of CPPs in systemic
tumor targeting and for splice correction in vitro will be discussed below in
more detail.
CPPs in tumor targeting
In the case of systemic delivery for treatment of cancer, the nondiscriminating mode of cell translocation is an undesirable characteristic.
Therefore, strategies to regulate the selective uptake of CPPs are highly required for in vivo applications, such as in cancer treatment. In order to
achieve selectivity of CPPs in vivo, there are two main possibilities; coupling
of a targeting moiety to the CPP (Table 5) or by modifying the CPP in such
that it becomes inactivated and can be activated locally, e. g. in proximity to
tumors, by enzymes expressed at the site of action (Table 6).
Table 5. Selection of studies describing the application of CPPs in systemic tumor
targeting in vivo.
CPP
Tat
Penetratin
Cargo
P15 peptide
p53 peptide
TLM
Ovalbumin
Tat
Penetratin
β-galactosidase
scFv antibody
Penetratin,
SynB
Doxorubicin
Cellular response
Reduction of tumor mass
Induction of apoptosis in glioma
cells
Improvement of protein
vaccination
Monitoring of tissue distribution
Delivery and prolonged retention
of scFv antibody into solid tumors
Accumulation of doxorubicin into
mouse brain parenchyma
Reference
Perera, et al. 2008
Senatus, et al. 2006
Bleifuss, et al. 2006
Cai, et al. 2006
Jain, et al. 2005
Rousselle, et al.
2000
16
Perea et al. screened a random phage display library and identified a cyclic 9
amino acids long peptide, P15, which abrogated casein kinase 2 (CK2)
phosphorylation (Perera, et al. 2008). CK2, a serine-threonine kinase, is
frequently dysregulated in many human tumors. By fusing Tat to P15 and
injecting it into TC-1 tumor bearing mice, the tumor mass was substantially
decreased. Recently, two interesting studies were carried out by the same
group with a p53 derived C-terminal peptide linked to the N-terminus of
penetratin (p53p-Ant) (Li, et al. 2005, Senatus, et al. 2006). In the first
study, Li et al. showed that p53p-Ant induced selective cell death only in
pre-malignant or malignant cells, without affecting normal cells or null p53
cells (Li, et al. 2005). In the second study, Senatus et al. showed that the
same chimeric peptide induced apoptosis in human and rat glioma cells both
in vitro and in vivo (Senatus, et al. 2006).
The possibility to deliver proteins inside cells in order to elucidate the
protein function in complex biological processes has been challenged by
difficulties in traditional delivery approaches. Plasmid-based genetic recombination techniques have several disadvantages such as limited transfection
efficiency and cellular toxicity. Recently, several studies have been published utilizing CPPs for delivery of biologically active full-length proteins
into animals. In study by Jain et al. penetratin was shown to improve tumor
retention of single-chain Fv antibodies (Jain, et al. 2005). Monoclonal antibody-based radiopharmaceuticals have two major impediments, long circulation times and extremely poor penetration. Thus, they showed that coadministration of penetratin and the scFv antibody resulted in improved retention and distribution of single-chain Fvs.
A novel prodrug design of tumor targeting CPPs based on enzyme activation of precursor peptides has recently been developed by Jiang et al.
2004. They showed that the uptake of nona-arginine was effectively blocked
when fused to polyanionic domain. They introduced MMP-2 and MMP-9
cleavable linkers between the CPP and the inactivating sequence, hence in
the presence of these enzymes the CPP could be activated and cargo could
be transported into nearby cells. This selective targeting strategy was reported to be applicable as a tumor imaging approach in mice bearing human
fibrosarcoma xenografts (Jiang, et al. 2004). At least two other studies with
similar setup have been reported, however, both these studies were carried
out only in vitro (Table 6).
Table 6. Target-activated CPPs.
CPP
Inactivating
Cargo
Target enzyme
sequence
All D-Arg9
Glu6
Fl, Cy5
MMP-2/-9
All L-Penetratin NLS
Fl
Cathepsin B
All-D-Arg8
Poly-Asp
Fl
PSA
Asp, aspartic acid; Fl, fluorescein; PSA, prostate specific antigen.
Reference
Jiang, et al. 2004
Pipkorn, et al. 2006
Goun, et al. 2006
17
Chemical modification of CPPs
One main consideration when utilizing CPPs for cargo delivery is their cellular fate and, hence, the effects on the bioactivity of the cargo. As mentioned
in the previous section, CPPs are taken up primarily via endocytic pathways,
and consequently the cargo is commonly retained in endosomal compartments without reaching either the cytoplasm or other cellular compartments
such as the nucleus and mitochondria. In vitro this obstacle can be overcome
with the use of different lysosomotropic agents, e. g. chloroquine, but this
approach is not optimal for in vivo applications.
In order to promote endosomal escape and increase the transfection
efficiency of CPPs, a number of different strategies have been employed. For
example the fusion of the Tat peptide with the influenza virus HA2 domain
(Wadia, et al. 2004) and the development of a histidine-containing endosomolytic alpha-helical penetratin analogue (Lundberg, et al. 2007). Stearylation of CPPs has proven to be another successful methodology to increase
the transfection efficiency of both plasmids (Futaki, et al. 2001) and siRNA
(Nakamura, et al. 2007, Tönges, et al. 2006), through a non-covalent approach resulting in the formation of nanoparticle complexes. In addition to
N-terminal stearylation, the presence of a C-terminal cysteamide appears to
be crucial for CPP-mediated siRNA delivery using the MPG peptide
(Simeoni, et al. 2003). This modification has been believed to increase
membrane association (Moulton, et al. 2003, Weller, et al. 2005) and stabilize complex formation (Gros, et al. 2006).
CPPs in splice-correcting ON delivery
Splice-correcting ONs, negatively charged or uncharged ON-analogues,
have shown promising therapeutic potential in redirecting splicing in various
diseases. However, the commonly used delivery methods for such ONs suffer, as already mentioned, from number of drawbacks. Therefore, as CPPs
are increasingly utilized for transport of a variety of cargos, they have also
been applied for the delivery of splice-correcting ON analogues such as
PNAs, PMOs and 2’-OMe RNAs (Abes, et al. 2007, EL Andaloussi, et al.
2006, Moulton, et al. 2007).
Kole and co-workers have developed a functional splice correction assay for evaluating the cellular delivery efficiency of antisense oligonucleotides (Kang, et al. 1998). This assay is based on the HeLa pLuc 705 cell-line
that is stably transfected with a luciferase coding gene interrupted by a mutated β-globin intron 2. This mutation causes aberrant splicing of luciferase
pre-mRNA resulting in synthesis of non-functional luciferase. Masking the
aberrant splicing site with an antisense ON redirects splicing towards the
correct mRNA and consequently restores luciferase activity. This positive
read-out assay has been extensively used for evaluating the delivery efficacy
of various CPPs (Table 7). Early studies utilized mainly classical CPPs like
Tat and penetratin (Astriab-Fisher, et al. 2002, Moulton, et al. 2003), while
18
later several other CPPs like M918 (EL Andaloussi, et al. 2007) and (RXR)4
(McClorey, et al. 2006) have been found to be more potent for these applications.
Table 7. Application of CPPs in delivery of splice-correcting oligonucleotides.
CPP
Tat, Pen
Tat
Tat, TP, Pen
M918
Tat
Cargo
2’-OMe RNA
PMO
PNA
PNA
PNA
Targeted pre-mRNA
Luciferase
Luciferase
Luciferase
Luciferase
Luciferase
Reference
Astriab-Fisher, et al. 2002
Moulton, et al. 2003
EL Andaloussi, et al. 2006
EL Andaloussi, et al. 2007
Moulton, et al. 2007,
Shiraishi and Nielsen 2006
MAP
PNA
Luciferase
Wolf, et al. 2006
(RXR)4
PMO
Dystrophin
Moulton, et al. 2007
McClorey, et al. 2006
Pip2
PNA
Luciferase, dystrophin
Ivanova, et al. 2008
B peptide, P007
PMO
Dystrophin
Wu, et al. 2008, Yin, et al.
2008a, Yin, et al. 2008b
R6-penetratin
PNA
Luciferase
Abes, et al. 2007
MAP, model amphipathic peptide; Pen, penetratin; PMO, phosphorodiamidate morpholino
oligomers; PNA, peptide nucleic acid; TP, transportan.
Moreover, a number of research groups have successfully applied CPPconjugated antisense ONs to restore dystrophin expression in vivo (Table 7)
(McClorey, et al. 2006, Wu, et al. 2008, Yin, et al. 2008a, Yin, et al. 2008b).
They utilized murine models with muscular dystrophy and demonstrated,
after i. v. injection of CPP-morpholino conjugates, widespread correction of
dystrophin expression in skeletal as well as in cardiac muscle.
Despite great progress with CPPs in delivery of splice-correcting ONs,
it is important to mention that all the studies so far have been carried out
with CPPs covalently conjugated to splice-correcting ONs. However, the
covalent conjugation of ON cargo is considerably more laborious, thus a
simple co-incubation approach would be highly desirable.
19
Aims of the study
The main goal of this thesis was to improve CPPs as cargo delivery vehicles
for tumor targeting in vivo and ON delivery in vitro. Specific aims for each
paper are presented below:
Paper I & II
Design of tumor homing CPPs by conjugating pVEC to two different homing peptides and evaluation of these chimeric peptides in cargo delivery.
Paper III
Rational design of MMP-2 activatable CPP, NoPe, and application in tumor
targeting.
Paper IV
Evaluation of stearylated CPPs in delivery of splice-correcting PS 2’-OMe
RNA utilizing a simple non-covalent co-incubation strategy.
20
Methodological considerations
The materials and methods used in the thesis are described in detail in each
paper. Therefore, mainly theoretical and practical aspects of selecting methods will be discussed here and the protocols will only be briefly explained.
Design and choice of CPPs
Several CPPs have been exploited in this thesis for modification and hence
facilitated targeted intracellular delivery of various cargos (Table 8).
In paper I, the cyclic homing sequence PEGA was selected for its
ability to accumulate in breast tumors (Essler and Ruoslahti 2002). In order
to make PEGA a cell-penetrating peptide it was conjugated to the Nterminus of pVEC (Elmquist, et al. 2001). The resulting PEGA-pVEC was
studied regarding homing and cell-penetrating capacity.
Paper II is a follow-up study to paper I. Here, the breast tumor homing peptide CREKA (Simberg, et al. 2007) was selected to be coupled to
pVEC. The CREKA peptide contains only 5 amino acids and is not cyclic,
thus, making it easier to synthesize than the PEGA peptide. CREKA was
conjugated to the N-terminus of pVEC and the conjugate was assessed for
homing ability and cell-penetrating capacity. CREKA conjugated to scrambled pVEC was used as a negative control.
In paper III, a previously reported CPP, YTA4 (Lindgren, et al. 2006)
was selected to be modified in order to achieve selectivity in vivo. The novel
peptide, NoPe, was designed with the help of a CPP prediction algorithm
published by Hällbrink et al. (Hällbrink, et al. 2005). The YTA4 peptide was
prolonged C-terminally with a sequence consisting of mainly negatively
charged amino acids spaced with glycines. In addition, an MMP-2 specific
cleavage sequence (Kratz, et al. 2001, Mansour, et al. 2003) was introduced
between the CPP and the negatively charged inactivating sequences. The
applicability of NoPe in tumor targeting was assessed in a pre-clinical model
for breast cancer.
In paper IV, several well-known CPPs were evaluated as vectors for
splice-correcting PS 2’-OMe RNA delivery. Since CPPs are known to be
primarily taken up by endocytic pathway, the bioactive cargo will most
likely be entrapped in the endosomes. In order to facilitate the release of the
21
cargo inside the cells we introduced two modifications to the peptides − Nterminal stearylation or C-terminal cysteamidation. Modified as well as unmodified peptides were utilized for transport of splice-correcting ONs inside
the nucleolus and the transfection efficiencies were compared with Lipofectamine™ 2000.
Table 8. Peptides utilized in the thesis.
Peptide
Sequence
Reference
PEGA
a,b
pVEC
a,b
Essler and Ruoslahti
2002
Elmquist, et al. 2001
PEGA-pVEC
a,b
CREKA
a,b
Simberg, et al. 2007
CREKA-pVEC
a,b
Mäe, et al. 2008
Scr-pVEC
a,b
YTA4
a,c
NoPe
a,c
Penetratin
d
TP10
d
AGYLLGKINLKALAALAKKILf-NH2
Arg9
d
RRRRRRRRR-NH2
MPG
e
cCPGPEGAGC-NH2
LLIILRRRIRKQAHAHSK-NH2
cCPGPEGAGCLLIILRRRIRKQAHAHSK-NH2 Myrberg, et al. 2008
CREKA-NH2
CREKALLIILRRRIRKQAHAHSK-NH2
AARIKLRSRQHIKLRHL-NH2
Elmquist and Langel
2003
Lindgren, et al. 2006
IAWVKAFIRKLRKGPLG-NH2
IAWVKAFIRKLRKGPLG-IAGEDGDEGF-NH2 Paper III
RQIKIWFQNRRMKWKK-NH2
Derossi, et al. 1994
GALFLGFLGAAGSTMGAWSQPKKKRKV
Soomets, et al. 2000
Futaki, et al. 2001
f
Morris, et al. 1997
Peptides were N-terminally labeled with fluoresceinyl carboxylic acida, chlorambucilb, methotrexatec or had either N-terminal stearyl-d or acetyl-e modification or C-terminal cysteamide
modificationf. c, cyclilized between the cysteines; Scr, scrambled.
Peptide and ON synthesis
All peptides were synthesized using solid phase peptide synthesis (SPPS),
first introduced by Bruce Merrifield in 1963 (Merrifield 1963). The principle
of SPPS is that the amino acids are anchored to an insoluble support, resin,
which is stable to the chemical reactions carried out during the synthesis. An
excess of pre-activated amino acids with either tert-Butyloxycarbonyl (tBoc) or 9-fluorenylmethyloxycarbonyl (Fmoc) protected Nα-amino groups
are assembled in a step-wise manner on a resin forming a peptide bond between the amino group and the Nα-protected amino acid. After each coupling, excess of coupling reagents are easily washed away and temporary
Nα-protecting group is removed in order to be able to couple the next amino
acid. t-Boc is removed with trifluoroacetic acid (TFA) and Fmoc with
piperidine. Before coupling a new amino acid, the resin is washed once
again. This cycle is repeated until the last amino acid has been coupled.
In order to generate C-terminally amidated peptides, t-Boc chemistry
with 4-methylbenzhydrylamine (MBHA) resin or Fmoc chemistry with Rink
22
amide resin was applied. The choice of chemistry was determined by the
compatibility of the cargo with final cleavage conditions. The final cleavage
from the solid support was carried out with hydrofluoric acid (in t-Boc
chemistry) or TFA (in Fmoc chemistry). In order to get C-terminally cysteamide modified peptides cysteamine-2 chlorotrityl resin was used (Fmoc
chemistry).
Synthesis of PS 2’-OMe RNA was performed using disposable columns packed with polystyrene-based solid support, functionalized for synthesis of ON sequences with 2’-OMe RNA monomers at the 3’-end. 5’labelling was carried out using Cy5 amidite.
Purification and characterization of synthesized
peptides and ONs
The synthesized peptides were purified on preparative or semi-preparative
reversed phase high performance liquid chromatography (RP-HPLC) on a
C18 column using acetonitrile/water gradient containing 0.1% trifluoracetic
acid. The identity of the purified products was verified by analytical RPHPLC and by matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass-spectrometry. The mass-spectra were acquired in positive ion reflector mode using α-cyano-4-hydroxycinnamic acid as a matrix.
Crude 2’-OMe RNAs were purified by anion exchange chromatography, desalted, and freeze dried.
Coupling of cargos to the peptides (Paper I-IV)
In general, two main strategies have been employed to couple CPPs to different cargos: covalent coupling, where the cargo and the CPP are conjugated via a covalent bond, or non-covalent complex formation, where the
cargo is simply co-incubated with the CPP.
The advantage of using the first strategy is that a defined molecule is
obtained, which is desirable in therapeutic applications. Also, the amount of
peptide needed in this setting can be less as compared to the complex formation strategy, which could be beneficial as some CPPs display toxicity at
higher concentrations. However, this strategy is rather laborious and expensive, requiring pre-activation of peptides as well as extensive purification
both before and after conjugation.
The advantage of using the co-incubation strategy is the simplicity; as
only mixing of CPP and cargo is necessary, followed by addition of the mixture to cells and, thus, complicated chemical conjugation is not required.
23
Additionally, these complexes are highly stable to serum degradation, an
attractive feature for in vivo applications.
Selection of cargos
5(6)-carboxyfluorescein (further referred to as fluorescein), chlorambucil
(Cbl) and methotrexate (MTX) were covalently coupled to the N-terminus of
the peptides and phosphorothioate 2’-O-methyl RNA (PS 2’-OMe RNA)
was non-covalently complexed with CPPs. Peptides used for MTX coupling
were synthesized by Fmoc chemistry since MTX is not compatible with
hydrogen fluoride treatment.
Fluorescein is a commonly used fluorophore with an excitation wavelength of 494 nm and an emission wavelength of 518 nm. Fluorescein is pHsensitive and for optimal results when quantifying fluorescence, the pH
should be kept between 6 and 10.
The nitrogen mustard analog, Cbl, is a well characterized cytotoxic
agent, which acts by alkylating and crosslinking the DNA, resulting in inhibition of further cell proliferation. The effect is mediated through alkylation
of guanine N7 in the major groove of DNA and subsequent interstrand
crosslinking (Faguet 1994, Mattes, et al. 1986). Moreover, this interstrand
crosslink may induce apoptosis (Mattes, et al. 1986, Whiteside, et al. 2003).
MTX is a folate antagonist clinically used in anti-cancer therapy
(McGuire 2003). Briefly, MTX inhibits the metabolism of folates which are
important in synthesis of nucleotides (especially thymine), thus inhibiting
DNA replication and concomitantly decreasing cell proliferation (Genestier,
et al. 2000).
Cell cultures
Breast cancer cells (Paper I-III)
All breast cancer cell-lines used in this thesis are derived from tumor metastasis pleural effusions. MCF-7 is an invasive human ductal carcinoma and it
is the most commonly used breast cancer cell-line in cancer research (Soule,
et al. 1973). MDA-MB-231, a human breast adenocarcinoma cell-line, was
originally obtained from a patient who had acquired MTX resistance during
chemotherapy (Worm, et al. 2001). This cell-line was selected because they
lack the reduced folate carrier, i. e. the MTX transport protein. MDA-MB435 is likewise one of the most commonly used breast cancer cell-lines and
is, similar to MCF-7, derived from invasive ductal carcinoma (Cailleau, et
al. 1978).
24
Endothelial cells (Paper III)
The EA.hy 926 cell-line is a hybrid cell-line derived by fusing human umbilical vein endothelial cells with permanent human lung carcinoma cells
A549 (Edgell, et al. 1983). They have been shown to express and secrete a
number of molecules characteristic to endothelial cells (Edgell, et al. 1983,
Emeis and Edgell 1988, Nelimarkka, et al. 1997, Nelimarkka, et al. 1998),
among others MMPs and their tissue inhibitors (Nelimarkka, et al. 1998).
These cells were chosen in paper III to represent the vascular endothelial
cells in a co-culture assay with MDA-MB-231 to mimic tumor environment
in vitro.
HeLa cells (Paper IV)
HeLa cells are an immortalized cell line derived from cervical cancer cells
taken from Henrietta Lacks after her death in 1951. Since then, this has been
the most widely used cell line within various research areas and, in particular, in the field of cancer. The main beneficial characteristics with these cells
are that they grow rapidly and are very robust.
In paper IV, a stably transfected HeLa cell line, HeLa pLuc 705 cells
(Fig. 5), was used (Kang, et al. 1998). Here, a plasmid carrying the
luciferase coding sequence interrupted by an insertion of intron 2 from βglobin pre-mRNA carrying a cryptic splice site is stably transfected into
cells. Unless the aberrant splice site is masked by antisense ONs, the premRNA of luciferase will be improperly processed. Thus, by using these
cells, various vectors can be evaluated by measuring luciferase activity.
Figure 5. HeLa pLuc 705 reporter system. Luciferase pre-mRNA was modified with
insertion of β-globin intron 2 carrying a point mutation at nucleotide 705. Blockage
of this site with antisense ON redirects splicing towards the fuctional mRNA.
25
Evaluation of CPP uptake and cargo delivery
In this thesis, the internalization efficiency and cargo delivery by CPPs have
been studied with two different methods; quantification of fluoresceinyllabeled CPPs by spectrofluorometry and confocal microscopy. One general
problem with peptides is that they are rapidly degraded both intracellularly
and extracellularly (Palm, et al. 2007, Tréhin, et al. 2004). The risk for
measuring fluorescent degradation products should therefore always be considered. Each method has advantages as well as disadvantages and to understand the cell-penetrating ability of peptides several different methods to
study the translocation over the plasma membrane should be combined.
Fluorescence based methods
Quantitative uptake
The quantified CPP is usually N-terminally labeled with fluorescein. The
quantification method is described in detail by Holm, et al. 2006. In short,
fluoresceinyl-CPP was incubated with the cells. After incubation, cells were
washed and peptides bound outside of the cell membrane were removed by
trypsination. The cells were lysed in sodium hydroxide solution, thus the
fluorescence was not measured from intact cells, but from cell lysates. Before measuring the fluorescence of internalized peptide or peptide-cargo
complexes by spectrofluorometry, the lysed cells were centrifuged in order
to eliminate the risk of measuring peptides on the cell membrane. The
amount of internalized cargo was normalized against the total protein content in the cell lysate. This normalization helps to correct for small variances
in the amount of cells from different experiment.
Even though the method is straightforward and gives a quantitative
number of the amount of internalized cargo, one should always consider the
possible false interpretations of the results. For instance, it is impossible to
differ between cytosolic, nuclear, or endosomal localization of the peptide.
Confocal microscopy (Paper I-III)
Microscopy is a useful method for studying localization of fluorophorelabeled peptide inside the cell or in tissue sections. Early microscopy studies
of CPPs were performed on fixed cells, using methanol or paraformaldehyde
fixation. However, the fixation step appeared to cause artifacts on the CPP
uptake pattern and a reevaluation of the uptake mechanism led the field to
use unfixed, live cells. In paper I-IV confocal microscopy was used were
only light from a specific focal plane is detected. By scanning a focal plane
crossing the nucleus of a cell, there will be reduced risk of detecting fluoresceinyl-labeled peptide bound to the outside of the cell membrane.
26
Functional assays to evaluate CPPs as delivery vectors
As a result of the previously mentioned flaws associated with using fluorescence-based methods to assess CPP internalization, introduction of functional assays are highly desirable. In this thesis two of those have been employed: splice correction and cell proliferation.
Splice correction assay (paper IV)
The splice correction assay developed by Kole and co-workers 1998, was
previously described in the thesis (Kang, et al. 1998). In addition to obtaining information of the delivery efficacy, it also provides information concerning the sub-cellular localization of conjugates. In contrary to siRNA
assays that targets mRNA for degradation and other methods that exert their
effect by down regulation, this assay generates a positive read-out. Therefore, this assay was used to evaluate uptake of CPPs complexed with 2´OMe RNA targeting the aberrant splice site. Briefly, cells were treated with
2´-OMe RNA complexed with CPP for 4 h, after which media was replaced
and cells were allowed to grow for additionally 20 h. After cell lysis,
luciferase activity was measured and normalized to protein content.
Cell proliferation assays
In order to evaluate whether CPPs are successful at transporting drug molecules inside cells, two cell viability assays were used. To make sure that the
toxic effects were mediated by the cargo and not from the CPP, peptides
without cargos were used as controls.
In paper I the CellTiter-Glo™ Luminescent Cell Viability Assay from
Promega was used. This assay measures the amount of viable cells based on
the quantity of ATP that is produced by metabolically active cells. The
luciferase from the assay produces oxyluciferin in a reaction together with
luciferin, oxygen and ATP. Oxyluciferin releases light and the amount of
light produced is proportional to the number of viable cells.
In papers II-IV cell proliferation reagent WST-1 from Roche Molecular Biochemicals was utilized. This method has been used for quantification
of cell proliferation and cell viability by monitoring the mitochondrial activity in cells. Mitochondrial dehydrogenases in viable cells cleaves the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene disulfonate) to formazan. The produced formazan can be quantified by measuring absorbance at 420 nm. The amount of formazan formed is
proportional to the number of metabolically active, viable cells.
27
Toxicity studies
The lactate dehydrogenase (LDH) leakage assay is a simple and commonly
applied in vitro assay to evaluate the integrity of the plasma membrane. LDH
is a cytosolic enzyme that catalyzes the conversion of lactate to pyruvate
with concomitant exchange of NAD+ to NADH. In the CytoTox-ONE™
assay used in this thesis, released LDH is measured via an enzymatic reaction in which nicotinamide adenine dinucleotide (NADH) reacts with resazurin that is converted to the end product resorufin which can be quantified by
measuring fluorescence at 560/590 (ex/em) nm. The main advantage with
this assay is that it is fast. However, LDH is quite large molecule, with a
molecular weight of 132 kDa, thus it should be kept in mind that small pores
or early membrane disruption may not be detected with this method. On then
other hand, Johansson et al. demonstrated that LDH assay correlated quite
well with a substantially smaller molecule, deoxyglucose, leakage
(Johansson, et al. 2008).
In vivo studies
Mice bearing two commonly used tumor types were utilized as models for in
vivo studies.
MDA-MB-435 tumor bearing mice (Paper I and II)
To produce tumors, nude BALB/c mice were orthotopically injected with
MDA-MB-435 tumor cells in the mammary fat pad. The tumor bearing mice
were used for homing analysis of peptides when the tumor diameter reached
about 10 mm. Tissue distribution of fluoresceinyl-labeled peptides was studied by i. v. injecting the peptides into the tumor-bearing mice. The injected
peptides were allowed to circulate wherafter the mice were perfused with 4%
parafolmaldehyde through the left ventricle of the heart. Tumors and control
tissues were dissected and frozen in optimum cutting temperature compound
(OCT) embedding medium after which the frozen sections were prepared for
immunohistological analysis and observed under a confocal microscope to
determine the accumulation of peptides in vivo.
MDA-MB-231 tumor bearing mice (Paper III)
Athymic, 4-6-weeks old female BALB/c nu/nu mice from Taconic Europe
were maintained under specific pathogen free-conditions in a temperature
and humidity controlled environment. Mice under anesthesia (Ketamin 90
mg/kg, Xylazin 10 mg/kg) were injected subcutaneously with 2.5 x 106
MDA-MB-231 human tumor cells to induce tumors. Animals were used for
28
3 independent in vivo biodistribution experiments at 8-12 weeks after tumor
implantation. All animal studies were conducted according to the guidelines
of the Provincial Governement of Southern Finland and the protocol was
approved by the Experimental Animal Committee at University of Helsinki,
Finland.
Determination of MMP levels (Paper III)
The Amersham Biotrak Activity Assay System (GE Healthcare, Uppsala,
Sweden) was utilized to determine the levels of MMP-2 and MMP-14 in the
cell-lines according to the manufacture’s description. Briefly, 150,000
cells/well were seeded in complete medium two days prior the experiment
on a 12-well plate. One day prior the measurement of protein levels, cell
medium was changed to serum free complete medium in order to exclude
MMPs found in serum.
In vitro MMP-2 cleavage assay (Paper III)
In order to study the MMP-2-specific cleavage of NoPe, the fluoresceinylpeptide was incubated with active human MMP-2 enzyme for 30 min atroom
temperature. The cleavage was monitored at 494/518 (ex/em) nm with spectrofluorometry. The increase in fluorescence by MMP-2 cleavage was inhibited by the MMP-2 selective inhibitor hydroxamate derivative of oleic acid
(OA-Hy) (Emonard, et al. 1999). Additionally, the cleavage of MTX-NoPe
with recombinant MMP-2 was confirmed with analytical RP-HPLC. The
identity of the cleavage products was verified by MALDI-TOF massspectrometry.
Serum stability
MTX-NoPe was incubated in 100% fetal bovine serum and frozen after 5,
10, 15, 30 and 60 min to stop the activity of serum enzymes. Thereafter the
degradation products were separated from the serum proteins using Millipore’C18 ZipTips and analyzed by MALDI-TOF mass-spectrometer.
29
Results and discussion
The articles included in this thesis describe the possibilities to modify CPPs
in order to be applied in tumor targeting in vivo and improving ON delivery
in vitro. In paper I and II, a well-known CPP pVEC has been coupled to two
different cancer homing peptides to obtain tumor targeting and cellular uptake in vivo. In paper III, tumor cell specificity has been achieved through
inactivation of a CPP, YTA4, with mainly negatively charged amino acids.
An MMP-2 specific amino acid sequence was placed between the CPP and
the inactivation sequence, so that the peptide could be activated in the presence of MMP-2, i. e. in close proximity to tumors. In paper IV, several CPPs
have been N-terminally stearylated in order to improve the delivery of
splice-correcting 2’-OMe RNA and compared with commercially available
transfection agent Lipofectamine™ 2000.
Design and evaluation of tumor homing CPPs
(Paper I and II)
Chemotherapy used for treatment of cancer has severe side effects, which
are mainly caused by the toxicity of the drugs to normal healthy cells and
additionally, in many cases tumor cells have developed resistance to the anticancer drugs. Clearly tumor cell-specific delivery vehicles are urgently required to overcome these impediments. As CPPs have gained extensive attention as promising vehicles for therapeutics we designed two tumor homing CPPs by conjugating homing peptide to the pVEC peptide.
In paper I, we conjugated a cyclic homing peptide PEGA to the Nterminus of pVEC. PEGA has been isolated by screening in vivo phage display library and it has been shown to target normal breast vasculature as well
as premalignant breast tissue and primary breast tumors in vivo (Essler and
Ruoslahti 2002). However, the PEGA-peptide is not able to translocate the
plasma membrane. Therefore, in order to overcome this barrier PEGA was
coupled to pVEC (Elmquist, et al. 2001).
The cell-penetrating ability of fluoresceinyl-PEGA-pVEC was first
demonstrated in vitro and compared with fluoresceinyl-PEGA and -pVEC.
Both, confocal microscopy and quantitative uptake experiments showed that
fluoresceinyl-PEGA-pVEC was able to translocate into MCF-7, MDA-MB231 and MDA-MB-435 (only with microscopy) cell lines. The uptake pat30
tern was similar in both MCF-7 and MDA-MB-231 cells. PEGA alone did
not display any cell-penetrating properties, whereas when coupled to pVEC,
the uptake was greatly improved, although not reaching the same levels as
for pVEC itself. There are two possible explanations for decreased uptake of
PEGA-pVEC as compared to pVEC. As mentioned in the introduction, CPPs
are rich in positively charged amino acids, thus when coupling PEGA peptide, that has one glutamic acid in the sequence, the net positive charge is
reduced, most likely resulting in reduced uptake. Another explanation is that
the hydrophobic amino acids in the N-terminus of pVEC have been shown
previously to be important for its uptake (Elmquist, et al. 2006). Coupling
the homing peptide to the N-terminus of CPP can restrict the full interaction
of the peptide with plasma membrane and cause reduced uptake.
Second, we wanted to demonstrate the drug delivery ability of PEGApVEC in vitro. The DNA alkylating agent Cbl was chosen to exert its anticancer properties. Cbl is believed to enter cells by passive diffusion (Adair
and McElnay 1986), which is an extremely inefficient process. In order to
improve the uptake of Cbl, we coupled it to the N-terminus of PEGA-pVEC
via an amide bond leaving the alkylating moiety of Cbl available for DNA
cross-linking. After 48 h exposure of Cbl or Cbl-peptide conjugates the cytotoxicity of the drug was improved with the factor of 4 when conjugated to
PEGA-pVEC.
Third, the tumor homing properties of the fluorescein-labeled chimeric peptide were evaluated. Fluorecseinyl-peptides were injected into the
tail vein of mice bearing MDA-MB-435 tumors, allowed to circulate for 2 h
whereafter the mice were sacrificed, tumor and control organs were isolated,
and the accumulation of fluorescence was assessed with fluorescence microscopy. Fluoresceinyl-PEGA, -pVEC and -PEGA-pVEC were all found in
tumor tissue where they partially co-localized with the blood vessel marker
MECA-32. The co-localization was particularly prominent with fluoresceinyl-PEGA-pVEC. In some areas, fluoresceinyl-PEGA-pVEC appeared
to be also in tumor cells, although it is not fully accurate to draw such conclusion from that experiment, since the tissue sections are fixed in paraformaldehyde and it is know that even mild fixation can lead to artifactual redistribution of CPPs (Richard, et al. 2003). Apparently, the accumulation of
fluoresceinyl-PEGA-pVEC in control organs was substantially lower than
that of control peptides.
In paper II, another linear homing peptide CREKA was chosen to be
coupled to pVEC. CREKA, a 5 amino acid long peptide has been previously
shown to accumulate in various types of tumors in vivo (Simberg, et al.
2007). CREKA selectively homes to tumor blood vessels and stroma where
it recognizes clotted plasma proteins. We showed that CREKA alone is not
taken up by cells, but when coupled to pVEC it becomes a CPP. The advantages of CREKA-pVEC over PEGA-pVEC are the shorter length, easier to
synthesize and does not include an extra cyclization step. Moreover, Cbl
alone did not have any effect on MDA-MB-231 cell proliferation at concen31
trations studied, while Cbl-pVEC and Cbl-CREKA-pVEC reduced the percentage of proliferating cells from 100% to approximately 40% at 10 µM
and 20 µM concentration, respectively. In conclusion, the resultant homing
cell-penetrating peptides demonstrated the conserved desirable properties of
the parent peptides, making them promising drug delivery vectors.
Target-activated CPP (Paper III)
Bearing in mind the abovementioned drawbacks associated with CPPs as
well as with small molecule chemotherapeutics, we designed an inactivated
CPP, activatable in the vicinity of tumors overexpressing MMPs. This peptide was named NoPe as in no non-specific cellular penetration. We chose
the previously published CPP, YTA4 (Lindgren, et al. 2006), and prolonged
it C-terminally with mainly negatively charged amino acids spaced with
glycines and exploited its drug (MTX) delivery properties.
Figure 6. Schematic diagram of NoPe.
In order to mimic tumor environment in vitro, we set up a co-culture experiment comprising of breast cancer and endothelial cells overexpressing
MMP-2 and MMP-14, respectively. We first tested the uptake of fluoresceinyl-NoPe and -YTA4 in both these cell-lines separately and could observe diminished uptake of fluoresceinyl-NoPe as compared to YTA4.
Thereafter we tested whether the NoPe peptide could be taken up by cells in
the co-culture assay. Indeed, fluoresceinyl-NoPe was detected inside the
cells and moreover this uptake could be significantly decreased when MMP2 specific inhibitor was added prior to treatment.
Additionally, the correct cleavage of NoPe was demonstrated using
recombinant active MMP-2. The cleavage was monitored spectrofluorometrically and with analytical HPLC and the expected cleavage products were determined with mass-spectrometry. Moreover, the MTX-NoPe
was found intact after 1 h incubation in 100% fetal bovine serum, which
could be compared to the suggested half-life of penetratin, 5 min (Palm, et
al. 2007).
32
Next, to validate the applicability of our target activated CPP in vivo,
we injected fluoresceinyl-NoPe i. v. to MDA-MB-231 tumor-bearing mice.
After 1 h circulation the peptide was mainly found in tumor tissue and substantially lower amounts were also detected in the spleen and liver. In contrast to NoPe, fluoresceinyl-YTA4, displayed negligible levels in tumor tissue while high levels were found in control organs.
MDA-MB-231 cells are known to be resistant to MTX treatment
(Ref), thus our goal was not only to target MTX specifically to tumor tissue,
but upon activation also transport the drug inside cancer cells. We first tested
MTX-NoPe in vitro in a co-culture assay, but we could not detect any significant effect of the conjugate on cell proliferation. This indicates that in
cell culture the level of MMPs is too low to activate enough of the MTXpeptide to have an effect on cell proliferation. Therefore, further in vivo experiments are required for evaluating the potency of this prodrug.
Stearylated CPPs for PS 2’-OMe RNA delivery
using a non-covalent co-incubation strategy
(Paper IV)
Utilizing CPPs to facilitate cellular uptake and to improve the general
bioavailability of various types of ONs holds great promise for future therapeutic purposes. CPPs have mainly been covalently attached to ONs; however this adds additional synthesis steps that can be, on occasion, somewhat
laborious. Therefore, many groups have recently employed a simple coincubation approach, where peptides and the negatively charged ONs are
mixed at certain molar ratios in order to create nanoparticle complexes that
are simply added to the cells (Meade and Dowdy 2008). Previously, two
chemical modifications, N-terminal stearylation and C-terminal cysteamidation, have been shown to be critical to improve the non-covalent cellular
delivery of ONs. Therefore in paper IV, we modified three peptides- TP10,
penetratin and nona-arginine N-terminally with stearyl moiety and compared
their transfection efficiency with the commercially available transfection
agent Lipofectamine™ 2000 and previously published MPG peptide.
First, the transfection efficiency of Cy5-labelled PS 2’-OMe RNA
with unmodified CPPs TP10, penetratin, and nonaarginine were evaluated.
In spite of considerable quantitative uptake of ONs no significant splice correction was detected. This contradiction can be explained with the fact that
the complexes were probably entrapped inside endosomes. This was further
confirmed in the case of TP10 and Pen; when the lysosomotrophic agent,
chloroquine was added to the cells simultaneously with complexes, the
splice correction was significantly increased.
33
Due to the low transfection efficiency observed for unmodified peptides, we stearylated all three peptides N-terminally and evaluated their
transfection efficiency. No significant difference between the peptides in
quantitative uptake could be detected, however stearyl-TP10 proved to be
exceptionally effective for the delivery of splice-correcting ONs. Furthermore, it is worth mentioning that correctly spliced luciferase intensity
reached levels comparable with results achieved with Lipofectamine™ 2000.
However, no significant increase in splice correction for stearyl-Pen and
-Arg9 in complex with 2’-OMe ON was detected, thus, apparently the
stearyl moiety does not potentiate all CPPs.
Since the cationic lipids are associated with quite severe cellular toxicity we evaluated the effect of stearylated TP10 on cell proliferation.
Stearyl-TP10 displayed no long-term toxicity even at the highest CPP:ON
ratios, on the other hand Lipofectamine™ 2000 induced significant impairment of metabolic activity at the concentration suggested by the manufacturer.
In conclusion, we have shown that stearylation of TP10 results in a
non-toxic and highly efficient ON delivery vector reaching transfection efficiencies compareable with commercial transfection agent Lipofectamine™
2000. Moreover, the splice correction mediated by stearyl-TP10 was not
affected by the presence of serum. Taken together, these results are particularly encouraging for future in vivo applications of TP10.
34
Conclusions
The major findings of this thesis are listed and described below:
Paper I and II
Conjugation of tumor homing peptides to CPPs results in chimeric peptides
with both homing and cell-penetrating properties. Moreover, by coupling
Cbl to the N-terminus of these peptides and the drug, efficacy is improved
several-fold in vitro. These results are encouraging for future applications in
targeted drug delivery.
Paper III
Rationally designed MMP-2 activated CPP, fluoresceinyl-NoPe, prove to be
applicable in tumor imaging, since it selectively accumulates in breast tumor
xenografts in vivo.
Paper IV
Introduction of an N-terminal stearyl-moiety to an amphipathic CPP, TP10,
significantly improves the non-covalent cellular delivery of splice-correcting
oligonucleotides. Futhermore, the efficacy is preserved in serum and the
complex do not display any significant cytotoxicity − characteristics being of
great importance for future in vivo applications.
This thesis has proved that the rational modification of CPPs greatly potentiate their application in cargo delivery both in vitro and in vivo.
35
Populärvetenskaplig sammanfattning på
svenska
Cancer är ett tillstånd där celler delar sig okontrollerat och förstör
omkringliggande vävnad, något som idag är en av västvärldens vanligaste
dödsorsaker. Behandlingen av cancer har utvecklats över tid och idag
existerar en rad olika behandlingsmetoder. Strålbehandling, kirurgi,
immunterapi och cytostatikabehandling är exempel på metoder som används.
Vilken behandlingsmetod som är effektiv är avhängig på typen av cancer
och tumörens stadium.
Cytostatikabehandling innefattar användandet av läkemedel vars syfte
är att döda cancercellerna eller stoppa tumörers tillväxt. Huvudproblemet
med många av dagens cytostatikabehandlingar är att de är toxiska. Detta
innebär att även normala celler påverkas av behandlingen, en effekt som
resulterar i biverkningar såsom till exempel håravfall och magbesvär. En
särskild problematik uppstår då mängden läkemedel måste begränsas för att
inte orsaka alltför svåra bieffekter trots att tumören egentligen kräver en
högre dos. Forskningen för att göra cytostatikabehandlingen mer effektiv är
utbredd och fokus ligger på att utveckla metoder som gör att läkemedlet
endast angriper cancercellerna.
En metod att transportera olika ämnen och läkemedel in i en cell är
med hjälp av cell-penetrerande peptider. En peptid, som består av
aminosyror, är en beståndsdel i ett protein. Vanligtvis upptas cellpenetrerande peptider av alla typer av celler, men genom att modifiera dessa
peptider är det möjligt göra upptagningen specifik för cancerceller. Genom
att använda dessa modifierade cell-penetrerande peptider och koppla
cytotoxiska läkemedel till dem, skapas cytostatika som bara angriper
cancercellerna och inte normala celler. I förlängningen medför detta en mer
effektiv cancerterapi, samtidigt som biverkningarna minskar.
I den första och andra artikeln har en cell-penetrerande peptid
kopplats till målsökande peptider och en cancerdrog. Den målsökande
peptiden urskiljer cancercellerna och med hjälp av den cell-penetrerande
peptiden ökar cancerdrogens effektivitet genom en bättre upptagning i
cellen.
Den tredje artikeln behandlar en cell-pentrerande peptid med en
inaktiverande aminosyrasekvens, vilken aktiveras vid förekomsten av ett
specifikt enzym, vilket frigör den cell-penetrerade peptiden. Även här är
36
peptiden kopplad till en cancerdrog. Enzymet förekommer rikligt i närheten
av tumörer.
I den fjärde och sista artikeln modifieras en cell-penetrerande peptid
med en fettmolekyl som bidrar till att upptagningen av splicing korrigerande
oligonukleotider förbättras. Dessa oligonukleotider kan korrigera en muterad
gen så att ett funktionellt protein bildas. Ett flertal genetiska sjukdomar och
olika former av cancer är i många fall ett resultat av mutationer som gör att
proteiner överuttrycks eller att inte korrekta proteiner bildas. Genom att
använda ovannämnda oligonukleotider med cell-penetrerande peptider som
transportörer skulle flera av dessa sjukdomar möjligen kunna botas.
Sammantaget påvisar modifieringarna av de cell-penetrerande
peptider som beskrivs i avhandlingen, en förbättrad upptagning av
oligonukleotider och droger i cancerceller.
37
Acknowledgements
Finally, this long journey is coming to an end. There are so many mixed
feelings when I am looking back at the past four years. There have been
tough times, but there have also been good times and I must say, these last
years have probably been the best time of my life. First and foremost, I appreciate all the amazing people I have met during my time here.
First I would like to thank my supervisor Ülo, for giving me the opportunity to do my PhD in your group. Thank you for always having time for
me when I wanted to discuss my projects or when I had something on my
mind, even when I was so called “unstable”. Now I actually appreciate the
way you have pushed me even though I never imagined saying this out
loud...
I would also like to thank other teachers from the department, Anders, for your interesting lectures in neurochemistry; Mattias, for your constructive comments after each seminar; Anna, Kerstin, and Tiit for everything you have done at the department. And of course Birgitta, MarieLouise, Siv and Ulla for all your administrative help which made my life a
lot easier.
Then I would like to thank all my collaborators and colleagues, especially the ones from my group: Helena, for being such a good friend and for
all the support in the lab, and most of all I have really missed our daily “confessions” since you defended your thesis; Maria, for being always so positive in life and in research, and at all times listening to my ideas as well as
my complaints; Katri, for your help with the peptide synthesis and for sharing your expert knowledge in chemistry with me; Per, it was a pleasure to
work with you on the fourth paper, you are an impressive person and I am
really glad I met you; and last but definitely not least Samir, for being such
an inspiring person, I have learned so much from you. But most of all, thank
you for being the best killkompis, we have had so much fun! I would also
like to thank all the other people in Ülo’s group: Emelia, for being a great
friend and for teaching me to understand chemistry and irony (now I can
laugh at all your jokes); Ulla, I am so happy that you moved into our office,
I hope I can support you as much when you are writing your thesis; Tina,
your energy is making me jealous and I have really missed your company in
the lab; Henrik, for always helping me when I needed it and for being so
calm and reasonable, what would we have done without you in Australia?!
Peter G, for synthesizing valuable oligonucleotides. Indrek and Imre, for
38
being so positive and fun guys. All the “old” members in Ülo’s group, Mats,
I could not have managed without your computer knowledge! Linda,
Pontus, Peter J, Yang, Kalle, Külliki, Riina and Ursel you all have a special place in my heart.
I would also like to thank all the other PhD students at the department
for creating a great atmosphere. Especially, Johanna, for being who you
are! We have come a long way together and our friendship means a lot to
me.Thank you for our great conversations and parties, and for influencing
me to become more tough and not to be afraid to say what I think; Karolina,
for being such a wonderful person and for helping me with confusing chemistry questions; Jessica, for being so thoughtful, always trying to make people glad; Caroline, good luck in States, you have really worked hard for it!
Thank you again Helena, Maria, Per and Samir for proof-reading my
thesis; Farid and Imre for helping me with the popular scientific overviews.
All my foreign friends: Anne B, Agatha, Ganesh and Nomchit. You
have thought me so much about other cultures and I have enjoyed our delicious special-food dinners and talks.
I would also like to thank all my estonian friends outside the lab. Anneli, for being such a good friend, who is there for me whenever I need it;
Tõnu, for always making me laugh; Jaana, for wonderful parties and I hope
you will continue visiting me in Sweden; Anne M, I am so glad that we have
been keeping in touch; Kairi, I have really missed you here in Stockholm;
Erki, for your never ending interest in my “blossoming” proteins; Margret,
Ave, Peeter and Raul, for our great dinners and get-togethers, Leili, Ago,
Martin and Madis for being my extra family; and of course Evelin, no one
will ever understand the unique bond between us, thank you for everything!!! Among other things I really enjoy your ability to make everything
sound so glamorous and interesting!
Thank you Mimmi, Gerd and Dan for always making me feel like
home in Visserfjärda.
My beloved family – without your support I would never have been
where I am now in the first place. Heli and Andres and your wonderful sons
Franck and Patrick, thank you for making me feel so welcomed and loved
every single time I come home. My little brother and sisters, thanks for
always making me smile. Piret, thank you for your never-ending kindness
and for making my father’s life so wonderful! My dear father, you have
been the biggest and most important support through all my study years!!!
Thank you for inspiring me and providing me everything I have ever needed.
Farid, no words are enough to describe how much you mean to me!!!
Thank you for your never-ending support and for making me believe in myself. I cannot imagine my life without you anymore! I love you so much!
39
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