Note: Descriptions are shown in the official language in which they were submitted.
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MEMBRANE DISRUPTIVE PEPTIDES COVALENTLY OLIGOMERIZED
The present invention relates to a modified membrane disruptive peptide. The
present
invention also relates to a delivery complex comprising the modified membrane
disruptive
peptide and the use of the delivery complex.
There is a demand in the field of gene therapy for simple non-viral gene
delivery agents
that can be used to efficiently transfect cells. Cationic peptides condense
DNA by
electrostatic interaction and have been used to prepare simple and
reproducible
peptide:DNA formulations. Methods for their preparation and purification are
well
documented. Cationic peptides have also been used as chemical anchors for the
introduction of further peptidic or non-peptidic entities to assist in the
passage of a
packaged gene from an extracellular location to the nucleus of a desired
target cell.
Several groups have demonstrated that, when using cationic peptides as
delivery agents in
vitro, transfection is optimal only in the presence of exogenous agents that
are believed to
act by increasing the ability of genes undergoing transportation to escape
from intracellular
vesicles, which encapsulate them during or after cell uptake. Examples of such
exogenous
agents are chloroquine or membrane disruptive peptides. It is believed that
destabilisation
of biological membranes is central to the role of these agents. A breakdown in
the structure
of any membrane acting as a barrier to the passage of a delivery complex would
increase
its ability to access the nucleus. However, it is reasonable to assume that
for many in vivo
applications the use of exogenous agents such as the small organic molecule
chloroquine,
apart from complicating the formulation, would be hindered by the different in
vivo
clearance mechanisms and diffusion rates found between small molecules and
macromolecules. Fusogenic or membrane disruptive peptides such as those
described by
Wagner (Wagner et al., PNAS, 89, 7934-8, 1992) or Smith (International Patent
Application WO 97/35070) can be anchored either to condensing structures prior
to
complex formation or to pre-formed peptide:DNA complexes but this would also
complicate manufacture of the formulation. Moreover, such peptides have been
shown to
be less efficient than adenovirus particles at enhancing gene transfer
(Gottschalk et al.,
Gene Therapy, 3, 448-457, 1996; Wagner et al., PNAS, 89, 7934-8, 1992) and so
the
search continues for more effective peptides.
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A peptide that can both condense DNA and disrupt biological membranes would
simplify
the formulation and can lead to more efficient transfection.
Melittin is a well known membrane disruptive peptide; however, despite being
one of the
most extensively studied peptide sequences, there appear to be only two
instances in which
its use in gene delivery has been described. Firstly, in the form of dioleoyl
phosphatidylethanolamine-N-[3-(2-pyridyldithio) propionate] (a DOPE
derivative) linked
to melittin (Legendre et al., Bioconjugate Chem., 8, 57-63, 1997), and
secondly in US
patent US-A-5,547,932. In Legendre et al., the conjugation of a lipid is an
expensive and
complicating step and problems have been reported with the use of DOPE in
vivo. In US
patent US-A-5,547,932 formulations of an endosomolytic agent attached to a
nucleic acid
binding agent are described. The formulations described form relatively
inefficient
transfection formulations which are possibly also toxic to the cell being
transfected.
Indeed, the toxicity of melittin may explain why it has not been used as a
lone peptide in a
successful non-viral formulation.
The present invention provides a modified membrane disruptive peptide, wherein
the
membrane disruptive peptide has been modified to form a covalently linked
multimer.
Preferably, the modified membrane disruptive peptide is further modified to
form a
substantially continuous a helix.
By modifying the membrane disruptive peptide so that it forms a multimer and
preferably
so that it forms a substantially continuous a helix, it has been found that
the toxicity of the
peptide is reduced and that the peptide gives increased levels of transfection
when used to
deliver nucleic acids to cells.
Without being bound by any one theory, it is proposed that the membrane
disruptive
peptide must be in the form of a monomer in order to be able to insert into a
membrane
where it may aggregate to form a pore. The formation of the pore is believed
to be toxic to
the cell as it enhances passive ion permeability. The multimerisation of the
peptide
prevents the formation of the monomer and thereby prevents pore formation and
reduces
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cell toxicity. The multimers still retain some membrane disruptive properties
due to
predominantly non-polar amphiphilic a helices. By modifying the peptide so
that a
substantially continuous a helix is obtained, the membrane disruptive
properties of the
peptide are increased as the substantially continuous a helix is available to
interact with
cell membrane. The multimerisation of the peptide also has the advantage that
the peptide
is less likely to become dissociated from any bound nucleic acid.
The term "a membrane disruptive peptide" means a peptide that is capable of
promoting
membrane destabilisation and lowering the energy required for a molecule to
traverse the
membrane. Assays such as the erythrocyte lysis assay can be used to determine
if a
peptide is a membrane disruption peptide; however, different membrane
disruption
peptides have different cell specificities and may lyse a different cell type
to an
erythrocyte. Accordingly, other cell types should be used in lysis assays to
determine if a
peptide is a membrane disruption peptide. The membrane disruptive peptide is
preferably a
toxic membrane disruptive peptide. It is fiu-ther preferred that the membrane
disruptive
peptide is toxic by inserting itself into a membrane in the form of a monomer.
It is further
preferred that the membrane disruptive peptide is a toxic a helical membrane
disruptive
peptide such as melittin, cecropin A, cecropin P1, cecropin D, magainin 2,
bombolittins or
pardaxin (Saberwal et al., Biochimica et Biophysica Acta, 1197, 109-131,
1994). The
membrane disruptive peptide preferably forms an amphiphilic a helix with one
face rich in
cationic residues, which enable condensation of DNA, and a hydrophobic face
that is able
to interact with membrane lipids. It is also preferred that the membrane
disruptive peptide
comprises a a helix region and a basic region at the C-terminus of the
peptide, which can
condense a nucleic acid. Preferably the membrane disruptive peptide is not the
human
bactericidal/permeability-increasing protein (BPI) (Gray et al., J. Biol.
Chem., 264, 9505,
1989 and US patent US-A-5,856,302). Preferably, the membrane disruptive
peptide is
melittin.
The term "peptide" as used herein refers to a polymer of amino acids having a
chain length of
between 10 and 150 amino acids. The term does not refer to or exclude
modifications of the
peptide, for example, glycosylations, acetylations and phosphorylations.
Included in the
definition are peptides containing one or more analogs of an amino acid
(including for
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example, unnatural amino acids), peptides with substituted linkages, as well
as other
modifications known in the art, both naturally occurring and synthesised.
The term "modified to form a multimer" means the membrane disruptive peptide
is
modified so that it is covalently linked to one or more other membrane
disruptive peptides.
Preferably the multimer is a dimer, trimer or a tetramer, more preferably a
dimer or a
tetramer and most preferably a dimer. The membrane disruptive peptides forming
the
multimer may be the same or different.
The membrane disruption peptides can be linked together at any point along the
length of
the peptides; however, it is preferred that the peptides are linked together
at the N-terminus
end and/or C-terminus end of each peptide and most preferred that the peptides
are linked
together at the N-terminus end of each peptide.
Preferably, the membrane disruptive peptide is modified to form a dimer by
replacing an
amino acid of the membrane disruptive peptide with an amino acid that forms a
covalent
bond directly via a disuphide bond or via linking group, with an amino acid of
another
modified membrane disruptive peptide. Alternatively, an amino acid that forms
a covalent
bond directly via a disulphide bond or via a linking group can be added to the
membrane
disruptive peptide. Preferably the amino acid added to or replacing an amino
acid of the
peptide is a cysteine amino acid, which can form a disulphide bond with a
cysteine residue
of another peptide. The membrane disruption peptides forming the dimer do not
have to be
identical; however, it is preferred that the peptides forming the dimer are
identical.
Preferably, the membrane disruptive peptide is modified to form a trimer or a
tetramer by
replacing two or more amino acids with two or more amino acids that form a
covalent
bond directly via a disulphide bond or via a linking group with an amino acid
of other
modified membrane disruptive peptides to form a trimer or a tetramer.
Alternatively, two
or more amino acids that form disulphide bonds directly or via a linking group
can be
added to the membrane disruptive peptide. The membrane disruption peptides
forming the
trimer or tetramer do not have to be identical; however, it is preferred that
the peptides
forming the trimer or tetramer are identical.
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The membrane disrupting peptides may be linked together via a linker such as
commercially available linkers including bismaleimide or bisvinylsulphone
linkers.
The term "a substantially continuous a helix" means the region of the membrane
disruptive
5 peptide that forms a a helix and does not comprise one or more amino acids
that disrupt a
helix formation. Preferably the substantially continuous helix is at least 10
amino acids in
length. Preferably the substantially continuous helix forms at least 5% of the
length of the
modified membrane disruption peptide, more preferably between 40% and 90% of
the
length of the modified membrane disruption peptide and most preferably about
80% of the
length of the modified membrane disruption peptide. The presence of a a helix
in a
peptide can easily be measured using standard circular dichroism analysis.
Amino acids that can disrupt a helix formation include proline, glycine,
tyrosine, threonine
and serine. However, as will be appreciated by one skilled in the art, the
ability of an
amino acid to disrupt a helix formation is dependent on the overall sequence
of the peptide
and other factors such as the pH of the solution in which the peptide is
folded.
Preferably the amino acid that can disrupt a helix formation is proline.
Preferably, the membrane disruptive peptide is modified to form a
uubstantially continuous
a helix by replacing an amino acid, which disrupts a helix formation with an
amino acid
that does not disrupt a helix formation.
In a preferred embodiment, the modified membrane disruptive peptide of the
present
invention has the amino acid sequence CIGAVLKVLTTGLAALISWIKRKRQQ. It is
further preferred that the modified membrane disruptive peptide forms a dimer
via a direct
disulphide linkage between the N-terminal cysteine amino acids.
The modified membrane disruption peptide of the present invention can be
further
modified by the addition of other functional groups such as lipids, targeting
groups such as
antibodies or antibody fragments which target the peptide to specific cell
types, antigenic
peptides, sugars and neutral hydrophilic polymers such as PEG and PVP.
Suitable
functional groups, which can be added to the modified membrane disruption
peptide of the
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present invention, are described in International Patent Application WO
96/41606 as well
as methods for attaching such groups to a peptide.
Preferably, the modified membrane disruption peptide of the present invention
is further
S modified by the addition of amino acids to the substantially continuous a
helix region of
the peptide, wherein the additional amino acids extend the length of the
substantially
continuous a helix region. Preferably, the substantially continuous a helix
region is
extended so that the a helix region is at least 10 and more preferably at
least 20 amino
acids in length.
Preferably, the modified membrane disruption peptide of the present invention
is further
modified by the addition of basic amino acids to the C-terminus region of the
peptide.
The C-terminus region of the modified membrane disruption peptide of the
present
invention is the region extending from the C-terminal amino acid to the region
forming the
substantially continuous a helix region.
Basic amino acids are well known to those skilled in the art and include
lysine, arginine
and histidine. Preferably, the C-terminus region is modified by the addition
of between 1
and SO basic amino acids, more preferably between 5 and 15 basic amino acids.
The present invention also provides a functional homolog of the modified
membrane
disruption peptide of the present invention.
Preferred functional homologs of the modified membrane disruption peptide of
the present
invention, are those that still retain their activity and preferably have a
homology of at least
80%, more preferably 90% and most preferably 95% to the peptide of the present
invention.
Preferably such functional homologs, which include fragments of the peptide of
the present
invention, differ by only 1 to 10 amino acids. It is further preferred that
the amino acid
changes are conservative. Conservative changes are those that replace one
amino acid with
one from the family of amino acids which are related in their side chains. For
example, it is
reasonable to expect that an isolated replacement of a leucine with an
isoleucine or valine, an
aspartate with a glutamate, a threonine with a serine, or a similar
conservative replacement of
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an amino acid with a structurally related amino acid will not have a major
effect on the
biological activity of the peptide.
However, it is sometimes desirable to alter amino acids in order to alter the
biological activity
of the peptide. For example, mutations which abolish or enhance one or more of
the functions
of the peptide can be particularly useful. Such mutations can generally be
made by altering
any conserved sequences of the peptide. It is preferred that such homologs
have a homology
of at least 80%, more preferably 90% and most preferably 95% to the protein or
a fragment
thereof of the present invention. Preferably such altered proteins or
fragments thereof differ
by only 1 to 10 amino acids.
The present invention also provides the use of the modified membrane
disruption peptide
of the present invention in a delivery complex to deliver a nucleic acid to a
cell.
The modified membrane disruption peptide of the present invention can be used
to deliver a
negatively charged polymer, preferably a nucleic acid, to any cell type.
Preferred cell types
include prokaryotic cell types such as E. coli and eukaryotic cell types such
as mammalian
cells, including ex vivo primary cells, such as, HCTVEC and DC cells mammalian
cell lines
including HeLa, HepG2, CHO and myeloma cell lines, and lower eukaryotic cell
types such
as yeasts. Preferably, the modified membrane disruption peptide of the present
invention is
used to deliver a nucleic acid to a mammalian cell.
The present invention also provides a delivery complex comprising the modified
membrane disruption peptide of the present invention and a nucleic acid.
Preferably, the delivery complex consists of a nucleic acid to be delivered
and the modified
membrane disruption peptide of the present invention.
The delivery complex of the present invention may be any delivery complex
which comprises
a nucleic acid to be delivered and the modified membrane disruption peptide of
the present
invention.
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Numerous delivery complexes for delivering a nucleic acid to a cell are well
known to those
skilled in the art. Peptides derived from the amino acid sequences of viral
envelope proteins
have been used in gene transfer when co-administered with polylysine DNA
complexes (Plant
et al, 1994, J. Biol. Chem.. 269: 12918-24); Trubetskoy et al, 1992.
Bioconju~ate Chem.. 3:
323-327; Mack et al, 1994, Am. J. Med. Sci.. 307: 138-143) suggest that
cocondensation of
polylysine conjugates with cationic lipids can lead to improvement in gene
transfer efficiency.
WO 95/02698 discloses the use of viral components to attempt to increase the
efficiency of
cationic lipid gene transfer.
In a preferred embodiment, the delivery complex of the present invention
comprises the
modified membrane disruption peptide and the nucleic acid encapsulated within
nano- or a
micro-particles such as polylactide glycolide particles and liposomes etc.
In a fiu-ther preferred embodiment the delivery complex of the present
invention comprises a
nucleic acid, a nucleic acid condensing peptide and the modified membrane
disruption
peptide of the present invention.
The nucleic acid condensing peptide can be any peptide that condenses nucleic
acids
including polylysine and histone derived peptides. Preferred nucleic acid
condensing peptides
are described in International Patent Applications WO 96/41606 and WO
98/35984.
Preferably the delivery complex is formed by condensing the nucleic acid with
the nucleic
acid condensing peptide to form a condensed nucleic acid complex. The
condensed
nucleic acid complex is then coated with the modified membrane disruptive
peptide of the
present invention.
The present invention provides a method for forming a delivery complex
according to the
present invention comprising:
1. condensing a nucleic acid with a nucleic acid condensing peptide to form a
condensed nucleic acid complex; and
2. coating the condensed nucleic acid complex with a modified membrane
disruptive peptide according to the present invention.
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It has been found that the presence of serum during transfection increases the
level of
transfection. Preferably the serum is foetal calf serum. Other suitable serums
that could be
used are well known to those skilled in the art and include normal human serum
and
normal mouse serum.
The modified membrane disruptive peptide of the present invention can be used
to deliver
therapeutic nucleic acids to cells in vivo, in vitro and for ex vivo
treatments. The
therapeutic uses of nucleic acids in a variety of diseases is well known to
those skilled in
the art.
The therapeutic nucleic acid to be delivered to cells can be any form of DNA
or RNA vector,
including plasmids, linear nucleic acid molecules, ribozymes and
deoxyribozymes and
episomal vectors. Expression of heterologous genes has been observed after
injection of
plasmid DNA into muscle (Wolff J. A. et al., 1990, Science, 247: 1465-1468;
Carson D.A. et
al., US Patent No. 5,580,859), thyroid (Sykes et al., 1994, Human Gene Ther.,
5: 837-844),
melanoma (Vile et al., 1993, Cancer Res., 53: 962-967), skin (Hengge et al.,
1995, Nature
Genet., 10: 161-166), liver (Hickman et al., 1994, Human Gene Therany, S: 1477-
1483) and
after exposure of airway epithelium (Meyer et al., 1995, Gene Theranv, 2: 450-
460).
Useful therapeutic nucleic acid sequences include those encoding recelators,
enzymes, ligands,
regulatory factors, and structural proteins. Therapeutic nucleic acid
sequences also include
sequences encoding nuclear proteins, cytoplasmic proteins, mitochondria)
proteins, secreted
proteins, plasmalemma-associated proteins, serum proteins, viral antigens,
bacterial antigens,
protozoa) antigens and parasitic antigens. Therapeutic nucleic acid sequences
useful
according to the invention also include sequences encoding proteins,
lipoproteins,
glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such as ribozymes
or antisense
nucleic acids). Proteins or polypeptides encoded by the nucleic acid include
hormones,
growth factors, neurotransmitters, enzymes, clotting factors, apolipoproteins,
receptors, drugs,
oncogenes, tumor antigens, tumor suppressors, structural proteins, viral
antigens, parasitic
antigens and bacterial antigens. Specific examples of these compounds include
proinsulin,
growth hormone, dystrophin, androgen receptors, insulin-like growth factor I,
insulin-like
growth factor II, insulin-like growth factor binding proteins, epidermal
growth factor TGF-a,
TGF-Vii, PDGF, angiogenesis factors (acidic fibroblast growth factor, basic
fibroblast growth
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factor and angiogenin), matrix proteins (Type IV collagen, Type V11 collagen,
laminin),
phenylalanine hydroxylase, tyrosine hydroxylase, oncogenes (ras, fos, myc,
erb, src, sis, jun),
E6 or E7 transforming sequence, p53 protein, Rb gene product, cytokines (e.g.
Il-1, IL-6, IL-
8) or their receptors, viral capsid protein, and proteins from viral,
bacterial and parasitic
5 organisms which can be used to induce an immunologic response, and other
proteins of usefi~l
significance in the body. The compounds which can be incorporated are only
limited by the
availability of the nucleic acid sequence for the protein or polypeptide to be
incorporated.
One skilled in the art will readily recognize that as more proteins and
polypeptides become
identified they can be integrated into the delivery complex of choice,
transfected using the
10 modified membrane disruption peptide of the present invention and
expressed.
Nucleic acids delivered using the modified membrane disruption peptide of the
present
invention include those that encode proteins for which a patient might be
deficient or that
might be clinically effective in higher-than-normal concentration as well as
those that are
designed to eliminate the translation of unwanted proteins. Nucleic acids of
use for the
elimination of deleterious proteins are antisense RNA and ribozymes, as well
as DNA
expression constructs that encode them.
Ribozymes of the hammerhead class are the smallest known ribozymes, and lend
themselves
both to in vitro synthesis and delivery to cells (summarized by Sullivan,
1994, J. Invest.
Dermatol., 103: 85S-98S; Usman et al., 1996, Curr. O_pin. Struct. Biol., 6:
527-533).
The present invention also provides the modified membrane disruptive peptide
or the
delivery complex of the present invention for use in therapy.
The present invention further provides the use of the modified membrane
disruptive
peptide or delivery complex of the present invention in the manufacture of a
composition
for the treatment of a genetic disorder.
A genetic disorder is defined as a disorder that can be treated at the genetic
level i.e. by
delivering a nucleic acid to the patient in need of such treatment. Genetic
disorders include,
but are not limited to, enzymatic deficiencies (e.g. those of the liver,
digestive system, skin
and nervous system), endocrine deficiencies (e.g. deficiencies of growth
hormone,
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reproductive hormones, vasoactive and hydrostatic hormones), exocrine
deficiencies (such as
deficiencies of pancreatic hormone secretion), neurodegenerative disorders
(such as
Alzheimer's Disease, amyotrophic lateral sclerosis, Huntington's disease, Tay
Sachs' disease,
etc.), cancer, muscular dystrophy and albinism.
The present invention also provides a method of treating a genetic disorder
comprising
administering to a patient in need of such treatment an effective dose of a
delivery complex
comprising a therapeutic nucleic acid and the modified membrane disruptive
peptide of the
present invention.
The present invention is now illustrated in the appended examples with
reference to the
following figures.
Figure 1 shows the erythrocyte lysis activity of CP36 dimer (CP36D), CP36
monomer
(CP36M) and melittin (CP1).
Figure 2 shows the erythrocyte lysis activity of CP36 and CP48.
Figure 3 shows the transfection of HepG2 cells when transfected with CP1 2,
18, 36, 39, 41,
42, 43, 44, 45, and 48 complexed at various charge ratios in the presence or
absence of DTT
and in the presence of 10% foetal calf serum.
Figure 4 shows the protein levels of HepG2 cells when transfected with CP1 2,
18, 36, 39, 41,
42, 43, 44, 45, and 48 complexed at various charge ratios in the presence or
absence of DTT
and in the presence of 10% foetal calf serum. Reduced protein levels are
indicative of a toxic
effect on the cells.
Figure 5 shows the transfection of HepG2 cells with pCMV~i complexed to CP36/1
(first
batch), CP36/1.2 (second batch) in the presence or absence of DTT and in the
presence or
absence of 10% foetal calf serum. The nomenclature Red means in reduced form
i.e. in the
presence of SmM DTT; and NR means in non-reduced form i.e. without added DTT.
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Figure 6 shows data from an experiment comparing luciferase expression from I-
iLTVEC
transfected with CP36 complexed-DNA from different batches of CP36 peptide, at
four
charge ratios. Batches of peptide refer to different syntheses and were termed
CP36/l, /3, /4,
/5, /6.
Figure 7 shows a compilation from a number of experiments (no. of experiments
shown in
brackets) comparing average luciferase expression from HCTVEC transfected with
CP36
complexed-DNA and PEI complexed-DNA at various charge or N:P ratios, PEI used
was
22kDa linear PEI (Exgen-500).
Figure 8 shows a compilation from a number of experiments (no. of experiments
shown in
brackets) comparing average % cells expressing GFP from HLJVEC transfected
with CP36
complexed-DNA and PEI complexed-DNA at various charge or N:P ratios. PEI used
was
22kDa linear PEI (Exgen-500).
Figure 9 shows luciferase expression in vivo after dosing with 75~.g DNA using
various
peptides in a number of tissues.
Figure 10 shows the average luciferase expression from HLJVEC transfected with
NBC28:DNA particles coated with CP36 dimer.
EXAMPLES
Materials and Methods
Peptide synthesis and purification
Method l: Multiple Peptide Synthesiser (MPSI
The peptides were synthesised on a P.E. Biosystems Pioneer peptide synthesiser
running
version 1.7 of the instrument software. The instrument was equipped with a
single MPS
unit attached to column position 2, controlled by a workstation running
software version
1.3.
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Each synthesis was carned out on a O.OSmmol scale using Fmoc-PAL-PEG-PS resin
(P.E.
Biosystems). For the synthesis the extended, slow activation and coupling
cycles were used
which were provided with the instrument. Deprotection was carried out using a
solution of
20% Piperidine in DMF. The following amino acid derivatives were used as
appropriate
for the peptide; Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Cys(Trt)-OH, Fmoc-L-
Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH,
Fmoc-Nle-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-
Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Lys(Fmoc)-OH. Activation of the amino acids
was achieved using TBTU and DIPEA. In the final synthesis step the Fmoc group
was
removed followed by solvent exchange into dichloromethane and drying of the
resin under
a stream of dry nitrogen. The columns containing the resin were then taken and
dried
further under vacuum for 2-3 hours at room temperature.
1 S The peptides with no cysteine residues were cleaved from the resin using
trifluoroacetic
acid (TFA)/Water/Triisopropylsilane(TIS) 95:2.5:2.5 and those containing
cysteine
residues were cleaved using trifluoroacetic acid (TFA)/ Water/
Triisopropylsilane (TIS)/
ethanedithiol (EDT) 92.5:2.5:2.5:2.5. An empty Sml syringe was attached to one
end of
each column and a 2.Sm1 syringe containing the cleavage mixture was attached
to the other
end. Cleavage mixture (lml) was injected onto each column and tljey were left
to stand for
mins, then a further lml was injected and after 30 mins the last O.SmI. After
standing
for 30 rains the empty 2.Sml syringe was removed and the remaining cleavage
mixture
drawn into the 5ml syringe. The column was removed from the Sml syringe,
inverted and
replaced, then a fresh 2.Sm1 syringe containing cleavage mixture attached to
the other end
25 and the cleavage procedure repeated. The contents of the Sml syringe were
then expelled
into a SOml centrifuge tube containing diethylether (45m1). The resulting
precipitate was
allowed to settle for 1-2 hours at room temperature and the supernatant was
poured off.
The remaining diethylether was blown of under a stream of dry nitrogen gas and
the pellet
dried further under vacuum for 2-3h.
Method 2: Continuous flow smthesis of the Peptides.
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14
The peptides were synthesised on a P.E. Biosystems Pioneer peptide synthesiser
running
version 1.7 of the instrument software which was controlled by a workstation
running
software version 1.3.
Each synthesis was carried out on a 0.2mmol scale using Fmoc-PAL-PEG-PS resin
(P.E.
Biosystems). For the coupling of all the amino acids except for cysteine,
extended coupling
cycles were used which were provided with the instrument. For the coupling of
cysteine a
special extended (1 hour coupling time) solvent exchange cycle was used so
that coupling
took place in DMF/DCM 1:1. Deprotection was carried out using a solution of
20%
Piperidine in DMF. The following amino acid derivatives were used as
appropriate for the
peptide: Fmoc-L-Ala-OH, Fmoc-L-Arg(Pbf)-OH; Fmoc-L-Cys(Trt)-OH, Fmoc-L-
Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH,
Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH,
Fmoc-L-Trp(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Lys(Fmoc)-OH. Activation of the
amino acids except cysteine was achieved using TBTU and DIPEA in DMF.
Activation of
cysteine was achieved using TBTU in DMF and Sym-Collidine in DCM. In the final
synthesis step the Fmoc group was removed followed by solvent exchange into
Dichloromethane and drying of the resin under a stream of dry nitrogen. The
resin was
washed out of the column into a filter funnel allowed to air dry for a few
minutes the
transferred to a pre-weighed flask and dried further under vacuum until a
constant weight
was achieved.
The peptides with no cysteine residues were cleaved from the resin using
trifluoroacetic
acid (TFA)/Water/Triisopropylsilane(TIS) 95:2.5:2.5 (20m1) for l.Sh at room
temperature
and those containing cysteine residues were cleaved using trifluoroacetic acid
(TFA)/
Water/ Triisopropylsilane (TIS)/ ethanedithiol (EDT) 92.5:2.5:2.5:2.5 (20m1)
for l.Sh at
room temperature. In each case the resin was then filtered off, washed with
neat TFA
(3xSm1) and the combined filtrate and washes evaporated down to a volume of
ca. 3m1.
The residue was transferred to a SOmI centrifuge tube containing
diethylether(45m1). The
resulting precipitate was allowed to settle for 1-2 hours at room temperature
and the
supernatant was poured off. The remaining diethylether was blown of under a
stream of
dry nitrogen gas and the pellet dried further under vacuum for 2-3h.
CA 02370284 2001-10-25
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Purification of the peptides
The peptides were purified by reverse phase h.p.l.c. The more polar peptides
were purified
on either a Shandon Hypersil SAS column (120, lOp,, 150x21.2mm) using a
gradient of
S water(0.1 % TFA) Acetonitrile (0.1 % TFA) typically 10-65% Acetonitrile in
water over 20
minutes with a flow rate of 24mllmin or a Phenomenex Jupiter C4 column (120.x,
5~,
250x10 mm) using a gradient of water(0.1% TFA) Acetonitrile (0.1% TFA)
typically 30-
100% Acetonitrile in water over 30 minutes with a flow rate of 6 ml/min.
10 The fractions containing the peptide of interest, as measured by matrix
assisted lazer
desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) and HPLC
analysis were pooled and lyophilised.
Peptide Multimer Formation
For complete dimerisation or oligomerisation via a disulphide or cystine
linkage, pure
peptide was dissolved in fresh 20mM ammonium bicarbonate and left at room
temperature
for 16 hours. The dimerisation was confirmed by analytical gel filtration
chromatography
using a Pharmacia Superdex Peptide HR 10/30 column run in 20% acetonitrile in
water
containing 0.1 % TFA, with a flow rate of between 0.8 and 1.Om1/min. The
decrease in
elution time of the dimerised peptide was confirmed by monitoring absorbance
of eluent at
214nm.
For the synthesis of bisvinylsulphone linked conjugates (CP48), 2.Omg Biolink
6
(Molecular Biosciences, Colerodo) was dissolved in 100p.1 acetonitrile and
made up to a
l.Om1 volume using 25mM HEPES, pH7.2. 200p1 of this solution was then added to
15
mg of CP36 monomer (5.2 p,mol) dissolved in 800p.1 buffer. The reaction was
monitored
by MALDI and 1ZP-HPLC and judged complete after 1 hour at 24°C. The
final product
was isolated by purification of Phenomenex Jupiter C4 semipreparative column
using a 30
to 100% gradient of increasing acetonitrile in water containing 0.1% TFA over
30 minutes.
The mass of the final product was confirmed by MALDI-TOF MS.
Erythrocyte Lysis Assay
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16
To 9.0 ml blood, 1.0 ml 110 mM citrate, pHS, was added to stop coagulation.
The blood
was spun at 2000 rpm for 5 min. The plasma supernatant was aspirated and the
pellet
washed with HBS at least six times, by serial mixing, centrifugation and
aspiration. The
clear supernatant was aspirated and the pellet washed twice with the
appropriate buffer:
either HBS (pH7.4) or lSmM sodium acetate, pHS, 150mM NaCI. The appropriate
buffer
was added to the blood pellet to make a total volume of 6 rnl and lml taken
from this stock
preparation and diluted 15 times with buffer to give the final working
solution. The
peptides were tested as serial dilutions in a 96-well plate in triplicate by
adding 75m1 of
blood solution, in appropriate buffer, to 100m1 of peptide solution in
corresponding buffer,
and mixing. The blood was incubated with the peptide for 1 h at 37°C.
At this stage 1%
Triton X-100 was added to blood solution containing no peptide to act as a
control for
100% lysis. The cells were spun down at 2500rpm for 5 min and 80m1 of
supernatant taken
for spectrophotometric analysis at 450nm.
Preparation of Complexes
Plasmid DNA at 40p.g/ml in HBS (HEPES buffered saline) pH 7.4 was rapidly
mixed with
an equal volume of an appropriate concentration of transfection agent in HBS
and allowed
to incubate for up to 1 hour at room temperature. The concentration of
transfection agent
was determined for peptides by the final desired charge ratio, or for PEI
(Exgen-500,
Euromedex, France) by the final desired ratio of nitrogen (from PEI):
phosphate (from
DNA). The charge ratio was calculated according to the definition by Felgner
et al (1997)
(Nomenclature for synthetic gene delivery systems. Gene Therapy, (1997) 8:511-
512).
CA 02370284 2001-10-25
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17
HepG2 Transfection Assay
1.13-Gal Transfection Protocol
HepG2s were plated the day before transfection at 5 x 104 cells/well in a 96-
well plate in
DMEM + 10% FCS (with antibiotics) and incubated at 37°C. The next day
the cells were
washed with 100 ~1/well PBS. 901 HEPES buffered RPMI containing 10% FCS and
antibiotics was added to the cells followed by 101 transfection complex,
prepared as
described above comprising plasmid pCMV(3 reporter plasmid (plasmid encoding
for (3-
galactosidase). The complexes were formulated in the presence or absence of
SmM DTT to
reduce all peptide disulphide bonds. Using a relevant control, it was
confirmed that the
presence of DTT in the formulation itself had no observable effect on
transfection. Cells
were transfected in triplicate with each complex. The cells were centrifuged
at 1100 rpm,
then incubated under humid conditions in a non-gassed incubator at 37°C
for 5 h. After 5 h
the transfection medium was removed and the cells washed with 100~1/well PBS
before
incubation in DMEM/10% FCS (with antibiotics) medium for 19-20 h in a C02-
gassed
incubator. Finally, the cells were washed twice with PBS, lysed with 30m1 0.1%
Triton,
250mM Tris, pHB, and frozen at -20°C before (3-gal assay.
2. f3-Gal Assav
The frozen lysed cells were thawed at room temperature and 10~~ v:om each well
removed
for protein assay. The remaining cell lysate was assayed for 13-gal reporter
using a
Galacton-Sstar !3-gal assay (TROPIX) and luminescence was measured using a 96
well
TopCount scintillation counter running in SPC mode. A DC protein assay kit
(BioRad) was
used to assay lysate for total protein content. The transfection counts were
reported as pg
13-gal/ ng total protein, using a 13-gal standard curve prepared in cell
lysate from
untransfected cells on the same 96-well plate.
HUVEC Transfection Assays
Luciferase Transfection Assays
primary HI7VECs (Promocell, Germany) were plated at 1x104 cells/well the day
before
transfection in a 0.1 % gelatin coated 96-well plate in Endothelial Growth
Medium with
Supplements (EGMS) (Promocell, Germany) and incubated at 37° in a COZ-
gassed
CA 02370284 2001-10-25
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18
incubator. The next day cells were washed with PBS. 901 per well medium M199
(Sigma) containing 10% FCS and antibiotics was~,added to the cells followed by
101 per
well transfection complex, prepared with pCMuc reporter plasmid (plasmid
encoding
for firefly luciferase). Cells were transfected in triplicate for each
complex. The cells
were centrifuged at 1100 rpm for 5 minutes, theri;incubated for 1 hour at
37°C in a COZ-
gassed incubator. After 1 hour the transfection medium was removed and the
cells washed
with100~1/well PBS before incubation in 100~1/v~ell EGMS for 20-24 hours at
37°C in a
C02-gassed incubator.
Luciferase Assav
After 20-24 hours Luciferase expression was determined using LucScreen assay
(TROPIX)
with luminescence measured using a 96-well TopCount scintillation counter
(Packard)
running in SPC mode
GFP Transfection Protocol
Primary HLTVECs were seeded at 1.5x105 cells/well the day before transfection
in 2 ml of
EGMS, in 6 well plates. The next day cells were washed twice with 2 ml per
well PBS,
and 1 ml per well of transfection solution was added, consisting of 1001
transfection
complex prepared with pCMVEGFP reporter plasmid (plasmid encoding for green
fluorescent protein) mixed with 900p1 M199 containing 10% FCS and antibiotics.
Cells
were transfected in duplicate for each complex. dells were incubated for 2
hours at 37°C
in a C02-gassed incubator, after which the medium was changed back to 2ml per
well
EGMS. The cells were incubated for a further 24'~ours at 37°C in a C02-
gassed incubator.
GFP Assav
After 24 hours, cells were washed twice with 2 ml per well PBS, trypsinised
and
resuspended with M199 media + 10%FCS. The % transfected cells was determined
by
FACS analysis.
In Yivo Delivery of pCMVLuc Using Peptides
pCMVLuc at SOOIxg/ml in HBS was mixed with an equal volume (typically 500,1)
of
peptide or polylysine (127mer) (Sigma) at the appropriate concentration to
give the desired
CA 02370284 2001-10-25
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19
charge ratio. Charge ratio was as defined by Felgner et al (Hum. Gene Ther., 8
S , 511-2,
1997). Peptide or polylysine was added to DNA over 3-4 seconds whilst mixing
on a
vortex mixer at 800rpm. Complexes were incubated for 1 hour at room
temperature. 300p1
of complex was injected into the tail vein of CD-1 mice. 20 hours later mice
were
sacrificed and 80-200mg of each organ was removed, briefly blotted to remove
excess
fluid, and frozen in liquid nitrogen and stored at -80°C. Frozen tissue
was weighed and
then thawed in lysis buffer (lOmM sodium phosphate, containing 1mM EDTA, 1%
Triton
X-100, 15% glycerol, 8mM MgCl2, O.SmM PMSF, 1mM DTT), and homogenised for 0.3-
2 minutes using a Mini bead beater-8 (Stratech Ltd) and lmm glass beads. The
homogenate was removed and the glass beads washed with lysis buffer and the
washings
combined with the homogenate. Particulates were removed by centrifugation for
5 minutes
at 13000rpm, and 801 assayed for luciferase activity in a Berthold LB593
luminometer,
using O.ImM luciferin, 0.44mM ATP, and a 4 second acquisition time. Results
are
expressed as RLU corrected to mg weight of each tissue.
The following peptides were prepared as monomers, or where possible as dimers
or other
multimers using the methods described above:
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CP 1 GIGAVLKVLTTGLPALISWIKRKRQQ-CONHZ
CP2 CIGAVLKVLTTGLPALIS WIKRKRQQ-CONH2
CP 18 GIGAVLKVLTTGLAALIS WIKRKRQQ-CONHZ
CP36 CIGAVLKVLTTGLAALISWIKRKRQQ-CONH2
CP37 GIGAVLEVLTTGLAALIS WLERERQQC-CONH2
CP39 lVle-IGAVLKVLTTGLAALIS WIKRKRQQ-CONH2
CP41 CIGAVLKVLTTGLAALISWLKRKRQQ-CONH2
CP42 CIGAVLKVLTTGLAALLS WLKRKRQQ-CONH2
CP43 CIGAVLKVLTTGLWALISWLKRKRQQ-CONHZ
CP44 CIGAVLKVLTTGLAWLIS WLKRKRQQ-CONHZ
CP45 CIGAVLKVLTWGLAALISWLKRKRQQ-CONH2
CP48 CIGAVLKVLTTGLAALISWIKRI~2QQ-NH2
o=s=o
o=s=o
s
CIGAVLKVLTTGLAALISWIKRKRQQ-NH2
CP-46 NH2-LLQSLLSLLQSLLSLLLQWLKRKRQQ-CONH2
CP-47 NH2-CLLQSLLSLLQSLLSLLLQWLKRKRQQ-CONH2
CP-49 NH2-CIGAVLKVLTTGLAALISWIKRKItQQC-CONH2
CP-50 NH2-GIGAVLKVLTTGLAALISWIKRKRQQC-CONH2
CP-51 NH2-CIGAVLEVLTTGLAALISWLERERQQ-CONHZ
CP-52 NH2-CIGAVLKVLTTGLAALISWIKRKRQQK-CONH2
NH2-CIGAVLKVLTTGLAALISWIKRKRQQ-CONH2
CP-53 NH2-GIGAVLKVLTTGLAALISWIKRKRQQKC-CONH2
NH2-GIGAVLKVLTTGLAALISWIKRKRQQ-CONH2
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21
CP-54 NH2-CIGAVLKVLTTGLAALISWLAALISWIKRKRQQ-CONH2
CP-55 NH2-CIGAVLKVLTTGLAALISWIKRKRQQ-CONH2
S
S
NH2-GIGAVLEVLTTGLAALIS WLERERQQC-CONH2
CP56 NH2-CIGAVLKVLTTGLAALISWIKRKRQQ-CONH2
S
S
NH2-CIGAVLKVLTTGLAALIS WIKRKRQQK-CONH2
NH2-CIGAVLKVLTTGLAALIS WIKRKRQQ-CONH2
S
NH2-CIGAVLKVLTTGLAALIS WIKRKRQQ-CONH2
CP-57 NH2-GIGAVLKVLTTGLPALISWIKRCRQQ-CONH2
CP-5 8 NH2-CIGAVLKVLTTGLAALIS WIKRKRQQ-CONH2
\
S
S
NH2-GIGAVLKVLTTGLAALIS WIKRKRQQC-CONH2
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22
CP-59 NH2-GIGAVLEVLTTGLAALISWLERERQQC-CONIi2
S
S
NH2'-CIGAVLKVLTTGLAALIS WIKRKRQQK-CONH2
NH2-CIGAVLKVLTTGLAALIS WIKRKRQQ-CONH2
S
S
NH2-GIGAVLEVLTTGLAALISWLERERQQC-CONH2
CP-60 NH2-GIGAVLKVLTTGLAALISWIKRKRQQ-CONH2
NH2-GIGAVLKVLTTGLAALIS WIKRKRQQKC-CONH2
S
S
NH2-GIGAVLKVLTTGLAALIS WIKRKRQQKC-CONH2
NH2-GIGAVLKVLTTGLAALISWIKRKRQQ-CONH2
CP1 is melittin, the main toxic component of bee venom.
CP2 is CPl(Gl to C). This peptide was designed to form N-terminal cysteine
linked dimers
of CP 1.
CP18 is CP1(P14 to A). This peptide was modified to form a substantially
continuous helix.
CP36 is CPl(Gl to C; P14 to A). This peptide modified so that it forms a dimer
and has a
substantially continuous helix.
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23
CP39 is CP36(Cl to Norleucine). The modification was introduced to increase
the
hydrophobicity at the N-terminus.
Peptides CP41 to CP45 were designed to study the effects of conserved amino
acid changes
on CP36 activity.
CP37 is CP18(I20 to L) and all lysines are replaced with glutamates.
CP41 is CP36(I20 to L).
CP42 is CP36(I17 to L; I20 to L).
CP43 is CP36(A14 to W; I20 to L).
CP44 is CP36(A15 to W; I20 to L).
CP45 is CP36(T11 to W; I20 to L).
CP46 is a functional homolog of melittin containing conserved amino acid
changes and is
based on the sequence described by DeGrado et al., (1981), J. Am. Chem. Soc.,
103, 679-681.
CP47 is CP46 with an added N-terniinal cysteine for dimer formation.
CP49 is CP36 with an added C-terminus cysteine for multimer formation.
CP50 is CP18 with an added C-terminus cysteine for multimer formation.
CP51 is CP41 with all the lysine residues substituted by glutamates.
CP52 is a dimer of CP36 formed by bis-amine modification of an additional C-
terminus
lysine.
CP53 is a dimer of CP36 with an added C-terminus lysine and a cysteine residue
linked to
CP36
CP54 is CP36 with LAALISW inserted at position 20 to increase the length of
the a helix
CP55 is a heterodimer of CP36 and CP51.
CP56 is a tetramer consisting of CP52 and 2 CP36 peptides.
CP57 is CPl(K23 to C).
CP58 is a heterodimer of CP36 and CP50.
CP59 is a tetramer consisting of a dimer of CP52 linked to two CP37 peptides.
CP60 is a tetramer consisting of a dimer of CP53.
CP61 is a dimer consisting of CP36 and CP51, wherein a disulphide bond is
formed between
the N- terminal cysteines.
It was observed that although melittin (CP1) could effectively bind to DNA, as
determined
by plasmid retardation on an agarose gel, melittin:DNA complexes conveyed
little or no
transfection of HepG2 cells and that melittin was toxic to cells. After
incubation of cells
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24
with such complexes, the total protein content per cell decreased with
increasing ratios of
melittin to DNA (figure 4). This implied a toxic effect caused by the ability
of melittin to
form numerous structures that are perturbing to biological membranes.
Literature on the
mechanism of melittin suggests that although the peptide exists as a tetramer
in solution at
high concentrations and/or high ionic strength, it must be able to dissociate
into a
monomeric form in order to be able to insert into a membrane, in which it can
aggregate
into a pore-forming tetramer. The pore forming tetramer of melittin is
believed to enhance
passive ion permeability and is considered to be toxic to cells. It was
proposed that the
dimerisation or tetramisation of melittin should result in a construct that
would be less
toxic to cells, the dimerisation or tertramisation effectively preventing
initiation of pore-
forming tetramer formation. Melittin dimers or tetramers would nevertheless
possess
membrane disruptive properties due to predominantly non-polar amphiphilic
helices. It
was decided to optimise the interaction between amphiliphilic helices of
melittin and
membrane surfaces by replacing Pro residues at position 14 of melittin, which
causes a
kink in helical structure, with a residue that would allow continuation of the
helix.
As indicated above, dimers were constructed and their membrane disruptive
activity
measured by an erythrocyte lysis assay. Surprisingly, although the lytic
activity of dimers
was greater than melittin at pH 7 and 5 (see Figures 1 and 2), the dimers
appeared less
toxic to mammalian cell lines (see Figure 4). It is therefore proposed that
pore formation is
toxic but other membrane disruption/destabilisation mechanisms are not toxic.
The dimerisation of melittin together with helix elongation, as described,
resulted in
constructs which could bind DNA; the resulting DNA complexes were
significantly less
harmful to HepG2 cells, as determined by protein content determination (see
Figure 4), and
conveyed efficient transfection of number of cell types in the absence of
exogenous agents
(see Figures 3 and 5). It was also found that the presence of foetal calf
serum during
transfection gave increased levels of transfection (see figure 5).
CA 02370284 2001-10-25
WO 00/64929 PCT/GB00/01588
Figure 9 shows that peptides CP36 and CP61 of the present invention are
effective at
increasing transfection of DNA to a number of tissues in vivo.
5 Delivery Complex Coated with CP36 Dimer
The delivery complex was prepared as follows.
1. Preparation NBC28:DNA complexes at charge ratio ~4 and DNA concentration of
25~g:
Solutions of NBC28 at 92.6p,g/ml and of pCMVluc DNA at SO~g/ml were prepared
in
l OmM HEPES pH7.4. The peptide solution was added to the DNA solution in a 1:1
(v/v)
10 ratio and left to stand for 1 hour at RT. NBC28 is a nucleic acid
condensing peptide
having the amino acid sequence: T YCG.
2. Coating of the complexes:
The coating peptide was added to the complexes followed by some l OmM HEPES
buffer
15 to make the volume up to 97% of the final volume required. The resulting
complexes were
vortexed for l Osec. then stored at 4°C overnight.
3. Salt spike:
Next morning (ca. 18h. later) SM salt (3% of the final volume to give 150mM
salt) was
20 added to the complexes; wfiic~were then vortexed for 10 sec., and left to
stand for a --
further hour at RT.
4. Reconstitution of the complexes:
The complexes, in l.Sml Eppendorfs, were spun down for 30min. at 13000rpm in
an MSE
25 Microcentaur centrifuge. Supernatant (75%) was removed and an equivalent
volume of
fresh HBS was added with slow vortexing and sucking up and down with the
Gilson
pipette for 30 sec.
5. Sonication of the complexes
The complexes, in l.Sml Eppendorf tubes, were sonicated for 30sec. in a
sonication bath
prior to transfection.
CA 02370284 2001-10-25
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26
The complexes were then transfected into HLTVEC cells in accordance with the
method
described above.
Figure 10 shows that the complexes coated with CP36 dimer give good
transfection of
HWEC cells. In particular, the level of transfection is greater than that
obtained with
CP36 or NBC28 alone.
