Biomedical Engineering Reference
In-Depth Information
interaction for retardation in a column, is a common method
for purification of CPP-fusion proteins and has been utilized
several times recently [40,45,84].
Using the immunsystem is also one possible therapeutic
approach to cancer treatment. Dendric cells present antigens
on their surface for T cells in the immune system and by
fusing a T-cell peptide or protein to dendritic cells were
reported to specifically enhance the immune response 3- to
10-fold when compared to free peptide/protein [74,92].
The possibility of a novel cancer treatment that can
circumvent the difficulties with pharmaceutical resistance
among certain cancers is coadministration of chemotherapy
and immunotherapy, the anticancer drug temozolomide and
TAT-survivin-pulsed dendritic cells gave an increased sur-
vival rate in animal models at lower concentrations than
temozolomide and TAT-survivin-pulsed dendritic cells alone
[93]. One well known approach in coadministration is the
herpes simplex virus type I thymidine kinase, HSV-TK, that
enhances the sensitivity of tumor cells for the drug ganci-
clovir but the clinical relevance is limited due to low
transduction efficiency [94]. The fusion of TAT to HSV-
TK showed an increased cytotoxicity in 3 out of 12 human
tumor cell lines [94].
In autoimmune diseases, such as arthritis and diabetes,
the immune system targets substances and tissues normally
present in the body. In arthritis, interleukin 1 (IL-1) plays a
key role by activating the immune response but also by
controlling the production of reactive oxygen species
(ROS) that mediate degradation of collagen [26]. The
cellular defense against ROS is, among others, superoxide
dismutase (SOD), a protein that regulates the redox state in
the cell and are known to cause inflammatory response
[26,95,96]. Human cartilage contains extracellular SOD
and patients with osteoarthritis have as much as a fourfold
less SOD [97]. A topical formulation of TAT-SOD fusion
protein has been administrated into murine ears in an
inflammation model [95]. The TAT-SOD fusion protein
increased the concentration of SOD and reduced the
proinflammatory response created by administrating 12-
O-tetradecanoylphorbol-13-acetate, TPA, both in vivo and
in vitro [95]. This is promising for a possible new thera-
peutic treatment for arthritis.
NF-
k
B is an important mediator in inflammation by
regulating proinflammatory cytokines and its activity is
regulated by I
k
B
a
[47,98]. A nondegradable mutant of
I
k
B
a
, named srI
k
B
a
, fused to TAT efficiently transduced
and inhibited NF-
k
B activation [98]. This could be of
interest for treatment of different inflammatory diseases.
Essential for immunology self-tolerance are a specific
type of T cells called regulatory T cells or T
reg
cells [16].
These T
reg
cells are dependent of the transcription factor
Foxp3, which have been identified as a possible therapeutic
target for autoimmune diseases, such as diabetes and arthri-
tis, and also asthma. Clinical applications have been limited
due to several problems in isolating T
reg
cells and the fact
that the use of virus-mediated gene delivery gave very low
transfection efficacy [16]. A human-derived CPP fused to
26.3.9 Solid Phase Peptide Synthesis and High
Performance Liquid Chromatography
Solid-phase peptide synthesis (SPPS), first described by
Merrifield [80], is the standard method for synthesizing
CPPs that are not expressed recombinantly together with
their fusion partner protein in bacteria. High performance
liquid chromatography (HPLC) is the standard method for
purification of peptides synthesized using SPPS [1].
26.4 SUCCESSES AND FAILURES IN
PRECLINICAL AND CLINICAL RESEARCH
There are roughly four major therapeutic areas that have
generated a great deal of research, namely cancer, neuro-
protection, cardioprotection, and inflammation/infection.
26.4.1 Preclinical Research
Currently used anticancer therapeutics are unspecific and
affect normal cells as well as cancer cells, which give side
effects. In order to circumvent this, much effort is put into
enhancing the specificity toward cancer cells in different ways.
The use of macromolecules has been of interest since tumor
cells often accumulate them due to leaky vasculature; tumor
cells also have an increased temperature compared to normal
cells. The use of an elastin-like polypeptide (ELP) fused to
TAT were modified to aggregate in cells that had an elevated
temperature and tested in four different cancer cell lines with
encouraging result in specificity [85]. A specific inhibition of
cell migration and proliferation of cancer cells was also seen
in animal models of ovarian cancer [86]. Recently, the CPP
Bac-7 was fused to ELP and a cell-cycle inhibitor, p21, to
produce Bac-ELP1-p21, which was reported to be more
specific and specifically accumulated in ovarian cancer cell
lines with a temperature above 39
C and aggregated. No toxic
effects were seen in cells at physiological temperature [87].
A different therapeutic approach to specifically target
cancer cells is by activating the tumor suppressor protein,
p53, which have attracted attention since it ismutated in a very
high percentage of several different types of cancers
[53,55,88]. Previous studies have used adenovirus-mediated
transduction of p53 into cancer cells resulted in cell-cycle
arrest and apoptosis and clinical studies have been performed
[55,89]. TheVP22-p53 fusion proteinwas shown to transduce
between cells and induce apoptosis in a p53 negative cancer
cell line in 1998 [53] but since then, the therapeutic utility
of the VP22-p53 fusion protein have been under discussion
since the intercellular trafficking properties of VP22 seems
to vary with different cargoes [88,90,91].
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