Note: Descriptions are shown in the official language in which they were submitted.
f
CA 02377211 2001-12-24
2
The invention relates to the field of gene transfer,
particular to non-viral vectors.
The availability of stable, efficient gene vectors is a
prerequisite for the clinical viability of gene-therapy
strategies. Certainly, most known gene transfer
carriers in systemic use with the objective of somatic
gene therapy still have problems attached to them.
For efficient gene transfer in vi vo basically the
following two transport problems have to be solved:
1) transfer of the agent which is to be transferred
(e.g. plasmid-DNA, oligonucleotides) from the site of
administration in the body to the target cell (the
extracellular aspect) and 2) transfer of the agent
which is to be transferred from the cell surface into
the cytoplasm or the cell nucleus (the cellular
aspect). An essential prerequisite for receptor-
mediated gene transfer is the compacting of DNA to
virus-sized particles and the release of DNA from
internal vesicles after endocytotic uptake into the
cells. This prerequisite is satisfied by compacting DNA
with specific cationic polymers the chemical nature of
which ensures that DNA complexes are liberated from
internal vesicles (endosomes, lysosomes) after
endocytotic uptake into cells (Boussif et al., 1995;
Ferrari et al., 1997; Haensler & Szoka, 1993; Tang et
al., 1996). Such an effect is also achieved by
incorporating pH-dependent membrane-destroying peptides
into DNA complexes (Plank et al., 1994; WO 93/07283).
With DNA complexes of suitable composition, a specific
uptake and efficient gene transfer into cells can be
CA 02377211 2001-12-24
n
3
achieved by receptor-ligand interaction (Kircheis et
al., 1997, Zanta et al., 1997). Complexes of DNA with
cationic peptides are also particularly suitable for
receptor-mediated gene transfer (Gottschalk et al.,
1996; Wadhwa et al., 1997; Plank et al., 1999).
The clinical implementation of the promising research
results obtained in gene transfer with nonviral vectors
is made more difficult inter alia by the fact that the
extracellular aspect of the transport problem is not
completely solved. One of the causes of this problem
is the chemical/physical nature of the nonviral gene
transfer vectors, on account of which they engage in
powerful interactions with-blood and tissue components
when used systemically (e.g. by opsonisation, the
attachment of serum protein), thereby restricting, in
particular, the receptor-mediated gene transfer
directed to specific target cells. It was shown that
the surface modification of DNA complexes with
poly(ethyleneglycol) significantly reduces their blood
protein-binding properties (Plank et al., 1996; Ogris,
1998; WO 98/59064). A further restriction to the use of
nonviral vectors is the insufficient solubility (or
stability) of DNA complexes in vivo. Using known
methods it has not hitherto been possible to complex
DNA with a polycation in sufficiently high
concentrations for intravenous use (e. g. in the region
of 1 mg/ml) as the DNA complexes aggregate at
physiological salt concentrations and are precipitated
from the solution.
CA 02377211 2001-12-24
4
Similar problems arise when low molecular chemical
compounds are used. In the field of "conventional"
drugs biodegradable synthetic polymers are used to
package pharmaceuticals in a form which guarantees a
longer retention time in the body and leads to the
desired bioavailability in the target organ (controlled
release). For this, the surface modification of
colloidal particles with polyethyleneglycol is designed
so that the unwanted opsonisation is suppressed. There
is extensive literature on the synthesis and
characterisation of biodegradable polymers for use in a
variety of medical applications (Coombes et al., 1997).
Depending on the substance and application the chemical
bonds in the polymer backbone are varied. By suitable
positioning of ester, amide, peptide or urethane bonds
the desired instability in a physiological medium can
be achieved, while the sensitivity to attack by enzymes
is varied in controlled manner. For rapid and efficient
synthesis of biologically active substances,
combinatory methods of synthesis have proved useful
(Balkenhohl et al., 1996). By systematically varying a
few parameters a large number of compounds can be
obtained having the desired basic structure (Brocchini
et al., 1997). With a suitable meaningful biological
selection system it is possible to search a pool of
compounds for those which have the desired properties.
US Patent No. 5,455,027 describes polymers consisting
of alternate units of a polyalkylene oxide and a
functionalised alkane, with a pharmacologically active
substance covalently coupled to the functional side
group of the alkane.
CA 02377211 2001-12-24
In recent years the following essential findings have
crystallised out with regard to the use of nonviral
gene transfer systems:
5
a) Complexes of plasmid-DNA and cationic polymers are
suitable for gene transfer in vitro and in vivo, while
complexes with polymers with secondary and tertiary
amino groups can also be credited with an inherent
endosomolytic activity which leads to efficient gene
transfer (Boussif et al., 1995; Tang et al., 1996).
b) Branched cationic peptides are suitable, upwards of
a certain chain length of the cationic part, for
efficiently binding to DNA and forming particular DNA
complexes (Plank et al., 1999).
c) Polycation-DNA complexes engage in powerful
interactions with blood components and activate the
complement system (Plank et al., 1996).
d) Powerful interactions of particular structures with
blood components can be reduced or prevent by
modification with polyethyleneglycol; this is also true
0~ polycation-DNA complexes (Plank et al., 1996; Ogris
et al., 1999) .
The aim of the present invention was to provide a new
improved nonviral gene transfer system based on nucleic
acid-polycation complexes.
1
CA 02377211 2001-12-24
6
The solution to the underlying problem of the present
invention started from the idea of surrounding the
nucleic acid or nucleic acid complexes with a charged
polymer which physically stabilises the complexes and
protects them from opsonisation.
In a first aspect the present invention relates to a
charged copolymer of general formula I
R X
p
En 1
wherein R is amphiphilic polymer or a homo- or hetero-
bifunctional derivative thereof,
and wherein X
i) denotes an amino acid or an amino acid derivative, a
peptide or a peptide derivative or a spermine or
spermidine derivative; or
ii) wherein X is
a
d C b
c
where
CA 02377211 2001-12-24
7
a denotes H or optionally halo- or dialkylamino-
substituted C1-C6-alkyl;
and where
b, c and d denotes identical or different, optionally
halo- or dialkylamino-substituted C1-C6-alkylene; or
iii) wherein X is
a
c~N~b
to
while
a denotes H or optionally halo- or dialkylamino-
substituted C1-C6-alkyl, and
b and c, which may be identical or different, denote
optionally halo- or dialkylamino-substituted C1-C6-
alkylene; or
iv) wherein X is
a substituted aromatic compound with three functional
groupings W1Y1Z1, where W, Y and Z have the meanings
given below;
while
W, Y or Z are identical or different groups CO, NH, O
or S or a linker group capable of reacting with SH, OH,
NH or NH2;
CA 02377211 2001-12-24
8
and the effector molecule E denotes
a cationic or anionic peptide or peptide derivative or
a spermine or spermidine derivative or a
glycosaminoglycan or a non-peptidic oligo/polycation or
polyanion; wherein
m and n independently of one another are 0, 1 or 2;
wherein
p is preferably 3 to 20; and wherein
1 i s 1 to 5, preferably 1.
If 1 is >1, the unit X-Zm En may be identical or
different.
Aromatic compound for the purposes of the invention
denotes a monocyclic or bicyclic aromatic hydrocarbon
group with 6 to 10 ring atoms which may optionally be
independently substituted - apart from the substituents
mentioned hereinbefore - with one or more other
substituents, preferably with one, two or three
substituents selected from among C1-C6-alkyl, -0-(C1-C6-
alkyl), halogen - preferably fluorine, chlorine or
bromine - cyano, nitro, amino, mono-(C1-C6-alkyl)amino,
di-(C1-C6-alkyl)amino. The phenyl group is preferred.
Aromatic compound for the purposes of the invention may
also denote a heteroaryl group, i.e. a monocyclic or
bicyclic aromatic hydrocarbon group with 5 to 10 ring
atoms which contains, independently of one another,
CA 02377211 2001-12-24
9
one, two or three ring atoms selected from among N, O
or S, the remaining ring atoms being C.
Alkylamino or dialkylamino, unless otherwise stated,
denote an amino group which is substituted by one or
two C1-C6-alkyl groups, while if two alkyl groups
are present the two alkyl groups may also form a
ring.
C1-C6-alkyl, unless otherwise stated, generally
denotes a branched or unbranched hydrocarbon group
with 1 to 6 carbon atom(s), which may optionally be
substituted by one or more halogen atoms) -
preferably fluorine - which may be identical to or
different from each other. The following hydrocarbon
groups are mentioned by way of example:
methyl, ethyl, propyl, 1-methylethyl (isopropyl),
n-butyl, 1-methylpropyl, 2-methylpropyl,
1,1-dimethylethyl, pentyl, 1-methylbutyl,
2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl,
1,2-dimethylpropyl, 2,2-dimethylpropyl,
1-ethylpropyl, hexyl, 1-methylpentyl,
2-methylpentyl, 3-methylpentyl, 4-methylpentyl,
1,1-dimethylbutyl, 1,2-dimethylbutyl,
. 1.t3-dimethylbutyl, 2,2,-dimethylbutyl,
2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl,
2-ethylbutyl, 1,1,2-trimethylpropyl,
1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl and
1-ethyl-2-methylpropyl.
Unless otherwise stated, lower alkyl groups with 1
to 4 carbon atoms, such as methyl, ethyl, propyl,
CA 02377211 2001-12-24
iso-propyl, n-butyl, 1-methylpropyl, 2-methylpropyl
or 1,1-dimethylethyl, are preferred.
Accordingly, alkylene denotes a branched or
unbranched double-bonded hydrocarbon bridge with 1
5 to 6 carbon atoms which may optionally be
substituted by one or more halogen atoms) -
preferably fluorine - which may be identical to or
different from each other.
The amphiphilic polymer R is preferably a
10 polyalkylene oxide, polyvinylpyrrolidone,
polyacrylamide, polyvinylalcohol, or a copolymer of
these polymers. _
Examples of suitable polyalkylene oxides are
polyethyleneglycols (PEG), polypropyleneglycols,
polyisopropyleneglycols and polylbutyleneglycols.
Polyalkylene oxides, particularly PEG, are preferred
within the scope of the present invention.
The polyalkylene oxide may be present in the copolymer
as is it or in the form of a thio, carboxy or amino
derivative.
The polymer R preferably has a molecular weight of 500-
10,000, preferably 1000 - 10,000.
In case i), wherein X is an amino acid, an amino acid
with three functional groups may be used to synthesise
the copolymer, two of these groups being capable of
CA 02377211 2001-12-24
11
copolymerisation with the polymer and one being capable
of coupling to the effector molecule E; in this case Z
is not needed. The natural amino acids glutamic acid,
aspartic acid, lysine, ornithine and tyrosine are
preferred. Theoretically, synthetic amino acids (e. g.
corresponding spermine and spermidine derivatives) may
also be used instead of the natural amino acids.
In case i), the synthesis may be carried out using an
amino acid derivative which contains two functional
groups for copolymerisation with the polymer and which
has been obtained by modification of an amino acid
(glutamic acid, aspartic acid, lysine or ornithine)
with a linker grouping for coupling of the effector
molecule, with Z being omitted (m = 0); examples of
linker groupings are pyridylthiomercaptoalkyl
carboxylates (cf. Fig. 1) or maleimidoalkane
carboxylates.
In case i) X may also be a peptide (derivative). In the
case of an uncharged peptide or peptide derivative E is
coupled directly thereto or via Z.
If X is a positively or negatively charged peptide or
peptide derivative or a spermine or spermidine
derivative, X itself represents the effector molecule
(Z and E are omitted, m=n=0). In the simplest case the
peptide in this case consists of a linear sequence of
two or more identical or different natural or synthetic
amino acids, these amino acids being selected so that
the peptide as a whole is either positively or
negatively charged. Alternatively, the peptide may be
branched. In these cases the peptide as such is the
CA 02377211 2001-12-24
12
effector, Z and E are omitted (m = n = 0). Examples of
one type of suitable branched cationic peptides have
been described by Plank et al., 1999.
Suitable anionic peptide derivatives X have the general
formula (peptide)n-B-spacer-(Xaa). The peptide is a
sequence of amino acids or amino acid derivatives with
an overall negative charge. Preferably, the peptide
consists of three to 30 amino acids, and preferably it
consists exclusively of glutamic acid and/or aspartic
acid residues. n denotes the number of branches
depending on the functional groups contained in B. B is
a branching molecule, preferably lysine or a molecule
of type X, in cases ii) to iv). The spacer is a peptide
consisting of 2 to 10 amino acids or an organic
aminocarboxylic acid with 3 to 9 carbon atoms in the
carboxylic acid skeleton, e.g. 6-aminohexanoic acid.
The spacer serves to separate the charged effector
molecule spatially from the polymer skeleton. Xaa is
preferably a trifunctional amino acid, particularly
glutamic acid or aspartic acid, and may generally be a
compound of type X, cases i) - iv).
Alternatively, in case i), X may be a peptide
derivative, the modification of the peptide consisting
of a charged grouping other than an amino acid;
Examples of such groupings are sulphonic acid groupings
or charged carbohydrate groups such as neuramino acids,
or sulphated glycosaminoglycans. The modification of
the peptide may be carried out using standard methods,
either directly in the course of the peptide synthesis
or afterwards, on the finished peptide.
CA 02377211 2001-12-24
13
The effector molecule E may, like X in case i), be a
polycationic or polyanionic peptide or peptide
derivative or a spermine or spermidine derivative. In
the simplest case the peptide in this case too
constitutes a linear sequence of two or more identical
or different natural or synthetic amino acids, the
amino acids being selected so that the peptide as a
whole is either positively or negatively charged.
Alternatively, the peptide may be branched. Examples of
suitable branched cationic peptides are described by
Plank et al., 1999. Suitable anionic molecules E have
the general formula (peptide)n-B-spacer-(Xbb), where Xbb
is preferably an amino acid with a reactive group which
can be coupled to X directly or via Z.
The coupling of the effector peptide E to Z or directly
to X is effected via a reactive group already present
in the peptide or subsequently introduced into the
peptide, e.g. a thiol group (in a cysteine or by
introduction of a mercaptoalkanoic acid group).
Alternatively, depending on Z, the coupling may also
take place via amino or carboxylic acid groups which
are already present or introduced afterwards.
Alternatively, E, like X in case i), may be a peptide
derivative, the modification of the peptide consisting
of a charged grouping other than an amino acid;
examples of such groupings are sulphonic acid groupings
or charged carbohydrate groups, such as neuramino
acids, or sulphated carbohydrate groups. In this case,
too, the coupling to X is effected directly or via Z.
The copolymer of general formula I
CA 02377211 2001-12-24
14
The copolymer of general formula I
R X Y
P
E~ 1
is preferably synthesised as a strictly alternating
block copolymer.
The copolymer may optionally be modified with a
cellular ligand for the target cell (receptor ligand
L). In this case the majority of the linker positions Z
are occupied by E, with a cellular ligand coupled in
between to individual positions of the linker Z instead
of the cationic or anionic effector E. Alternatively,
the ligand is present coupled to individual positions
of the effector molecule E. The ratio E:L is preferably
about 10:1 to 4:1. The receptor ligand may be of
biological origin (e. g. transferrin, antibody,
carbohydrate groups) or may be synthetic (e. g. RGD-
peptides, synthetic peptides, derivatives of synthetic
peptides); examples of suitable ligands are given in
W~ 93/07283.
The copolymers according to the invention may be
prepared by the following method:
The copolymerisation partner X or X-Zm-En , if it is a
peptide or peptide analogue, is synthesised by standard
methods, e.g. on the solid phase (solid phase peptide
CA 02377211 2001-12-24
synthesis, SPPS) by the Fmoc method (Fields et al.,
1990). The activation of the amino acid derivatives is
carried out with TBTU/HOBt or with HBTU/HOBt (Fields et
al., 1991). The following derivatives in their N-
5 terminally Fmoc-protected form are used for the ionic
amino acid positions:
(a) cationic side chains:
R(Pbf), K(Boc,Trt), ornithine(Boc), carboxyspermine or
-spermidine (Boc).
10 (b) anionic side chains:
D(O-tert.Bu), E(O-tert.Bu).
Fmoc-K(Fmoc)-OH is used for the branching site B of the
molecule (peptide)n-B-spacer-(Xaa) or (peptide)n-B-
15 spacer-(Xbb). The peptides are cleaved from the resin
with TFA/DCM.
If the polymerisation partner X is a peptide of general
structure (peptide)n-B-spacer-(Xaa) in the subsequent
copolymerisation, glutamic acid or aspartic acid which
has a benzyl protecting group at a carboxyl position is
used at position Xaa. This is selectively removed by
hydrogenolysis (Felix et al., 1978). The N-terminally
situated amino acid positions of the peptide chain are
occupied by Boc-protected amino acids in order that the
cleaving of the protecting group can be carried out in
one step after copolymerisation of the peptide with
PEG .
CA 02377211 2001-12-24
16
3-mercaptopropionic acid, 6-aminohexanoic acid), this
may be obtained in liquid phase by conventional methods
of peptide chemistry. Mercaptopropionic acid is reacted
with 2,2'-dithiodipyridine and purified by
chromatography. The reaction product is reacted with
carboxyl-protected glutamic acid (0-t. butyl),
activating with HOBt/EDC (cf. Fig. 1). 6-Fmoc
aminohexanoic acid is reacted analogously. The carboxyl
protecting groups are eliminated in TFA/DCM, the
resulting glutamic acid derivative is purified by
chromatographic methods.
The copolymers may be prepared by the following
principles; their preparati-on is illustrated by means
of a PEG-peptide copolymer:
(1) poly(PEG -O- OC-) matrix ("polyester"):
The copolymerisation of the ionic, partially side
chain-protected peptide-dicarboxylic acids or glutamic
or aspartic acid derivatives with PEG-macromonomers in
defined molecular mass ranges (MW 400-20000 obtainable
commercially, e.g. Fluka) yields a matrix based on PEG-
ester. This is a system which is susceptible to
hydrolysis in a physiological environment (Ulbrich et
al., 1985) .
The p(PEG-peptide)-copolymers are synthesised by
established methods, e.g. with
dicyclohexylcarbodiimide/DMAP, preferably in strictly
alternating sequence (Zalipsky et al., 1984; Nathan,
A., 1992). DCC / DMAP is added to the PEG macromonomer,
together with a side chain-protected peptide or
glutamic or aspartic acid derivative in dichloromethane
solution. After the urea derivative formed has been
CA 02377211 2001-12-24
17
separated off, the polymer can be obtained by
precipitation with cold ether. The remaining side chain
protecting groups are cleaved with TFA in
dichloromethane (under these conditions the PEG ester
bond is stable (Zalipsky et al., 1984)). The ionic
polymer is obtained by precipitation and finally
chromatography. Control of the reaction makes it
possible to monitor the degree of polymerisation and
the proportion of charge per PEG unit in the polymer.
(2) poly(PEG -HN- OC-) matrix ("polyamide")
As an alternative to the polyester, an amide polymer
matrix may be synthesised if excessively rapid
hydrolysis and consequently too much instability are to
be expected when a copolymer-DNA complex is used
systemically in gene therapy. In this case diamino-PEG-
derivatives are used instead of the PEG macromonomers
which are copolymerised with the ionic peptides or
glutamic or aspartic acid derivatives analogously to
the method of synthesis described above. In this
synthesis, a hydrolysis-stable amide structure is
obtained. Diamino-modified polyethyleneglycols are
commercially obtainable as basic materials in defined
molecular mass ranges of between 500 and 20000 (e. g.
Fluka). The remaining acid-unstable side chain
protecting groups of the peptide components are cleaved
e.g. with TFA/DCM, and the polymers are purified by
chromatographic methods.
Copolymers of glutamic or aspartic acid derivatives are
reacted in a further step with anionic or cationic
peptides which contain a suitable reactive group. For
example, copolymers of 3-(2'-thin-pyridyl)-
CA 02377211 2001-12-24
18
example, copolymers of 3-(2'-thio-pyridyl)-
mercaptopropionyl-glutamic acid are reacted with
peptides which contain a free cysteine-thiol group. The
Fmoc protecting group is removed under basic conditions
from copolymers which have been obtained from 6-Fmoc-
aminohexanoyl-glutamic acid. The product is reacted
with a carboxyl-activated, protected peptide. The
peptide protecting groups (t-Boc or O-t.butyl) are
eliminated in DCM/TFA and the end product is purified
by chromatography. Alternatively the amino group of Ahx
(6-aminohexanoic acid) may be derivatised with
bifunctional linkers and then reacted with a peptide.
The ligand L may be coupled directly by activation of
carboxyl groups on the effector E (preferably in
anionic copolymers) or on the ligand or via
bifunctional linkers such as succinimidyl-pyridyl-
dithiopropionate (SPDP; Pierce or Sigma) and similar
compounds. The reaction product may be purified by gel
filtration and ion exchange chromatography.
This copolymerisation mixture may also be reacted in
accordance with combinatory principles. The selectable
variables are primarily the type and the molecular
weight (degree of polymerisation) of the polymer R, the
identity of the polymerisation partner X-Zm-En or of the
effector molecule E (e. g. a series of anionic peptides
with an increasing number of glurtamic acids) and the
overall degree of polymerisation p.
By varying the molecular masses of the PEG
macromonomers, the nature of the ionic species used and
CA 02377211 2001-12-24
19
the amount thereof in the copolymer and the degree of
polymerisation of the polymer matrix, a multiparameter
system is created which allows the rapid parallel
construction of a homologous series of different
copolymers and consequently, after complexing with
nucleic acid, of various nonviral vectors. The
synthesis concept is converted to the scale of a cell
culture plate (e. g. 96 wells per plate). To do this,
the chemical synthesis is adapted to the desired micro
scale (reaction volumes in the region of 500 u1). This
allows the polymers synthesised in parallel to be
transferred directly into the biological assay and thus
contributes to the rapid screening of a number of
systems and fast finding of suitable compounds. In
order to carry out the biological selection process
with regard to the preferred use of the copolymers
according to the invention for gene transfer, the
copolymers are combined with DNA complexes, for
example, and then subjected to tests which enable the
qualities of the polymers to be evaluated in terms of
their intended use (e. g. gene transfer). The same
selection methods may be used for copolymer-coated
nanoparticles. Screening and selection methods of this
kind may be, for example, complement activation tests
in the 96-well plate format (Plank et al., 1996),
turbidimetric measurements of aggregation induced by
serum albumin or salt in the same format or in vitro
gene transfer studies in the same format (Plank et al.,
1999) or optical fluorescence processes in the same
format.
CA 02377211 2001-12-24
Such investigations provide information e.g. as to
which copolymers from a combinatory synthetic batch are
suitable for modifying the surface of DNA complexes so
that their solubility is sufficient for gene transfer
5 applications in vivo in which the interaction with
blood and tissue components is restricted, so that
their retention time and duration of effect in the
bloodstream is increased sufficiently to allow
receptor-mediated gene transfer in target cells.
The copolymers according to the invention are
preferably used for transporting nucleic acids into
higher eukaryotic cells.
In another aspect the present invention thus relates to
complexes containing one or more nucleic acid molecules
and one or more charged copolymers of general formula
I.
Preferably, the nucleic acid molecule is condensed with
an organic polycation or a cationic lipid.
In another aspect the present invention thus relates to
complexes of nucleic acid and an organic polycation or
a-cationic lipid, which are characterises in tnat they
have a charged copolymer of general formula I bound to
their surface by ionic interaction.
The nucleic acids to be transported into the cell may be
DNAs or RNAs, with no restrictions as to the nucleotide
sequence and size. The nucleic acid contained in the
complexes according to the invention is defined
CA 02377211 2001-12-24
21
primarily by the biological effect to be achieved in the
cell, e.g. when used in gene therapy by the gene or gene
portion which is to be expressed, or by the intended
substitution or repair of a defective gene or any
desired target sequence (Yoon et al., 1996; Kren et al.
1998), or by the target sequence of a gene which is to
be inhibited (e. g. when using antisense
oligoribonucleotides or ribozymes). Preferably, the
nucleic acid to be transported into the cell is plasmid-
DNA, which contains a sequence coding for a
therapeutically effective protein. For use in cancer
therapy, the sequence codes e.g. for one or more
cytokines such as interleukin-2, IFN-a, IFN-y,
TNF-a, or for a suicide gene which is used in
conjunction with the substrate. For use in so-called
genetic tumour vaccination the complexes contain DNA,
coding for one or more tumour antigens or fragments
thereof, optionally combined with DNA, coding for one or
more cytokines. Uther examples of therapeutically active
nucleic acids are recited in WO 93/07283.
The copolymer according to the invention has the
property of sterically stabilising the nucleic acid-
polycation complex and reducing or suppressing its
unwanted interaction with components of body fluids
(e. g. with serum proteins).
Suitable organic polycations for complexing nucleic acid
for transporting into eukaryotic cells are known; on the
basis of their interaction with the negatively charged
nucleic acid the latter is compacted and brought into a
CA 02377211 2001-12-24
22
form which is suitable for uptake in the cells. Examples
are polycations which are used for receptor-mediated
gene transfer (EP 0388 758; WO 93/07283), such as
homologous linear cationic polyamino acids (such as
polylysine, polyarginine, polyornithine) or heterologous
linear mixed cationic-neutral polyamino acids
(consisting of two or more cationic and neutral amino
acids), branched and linear cationic peptides (Plank et
al., 1999; Wadhwa et al. 1997), non-peptidic polycations
(such as linear or branched polyethyleneimines,
polypropyleneimines), dendrimers (spheroid polycations
which can be synthesised with a well defined diameter
and an exact number of terminal amino groups; (Haensler
and Szoka, 1993; Tang et al., 1996; WO 95/02397),
cationic carbohydrates, e.g. chitosan (Erbacher et al.
1998). The polycations may also be modified with lipids
(Zhou et al. 1994; WO 97/25070).
Other suitable cations are cationic lipids (Lee et al.
1997), some of which are commercially obtainable (e. g.
Lipofectamine, Transfectam).
Unless otherwise stated, the term "polycation"
hereinafter represents both polycations and cationic
lipids.
Preferred polycations within the scope of the present
invention are polyethyleneimines, polylysine and
dendrimers, e.g. polyamidoamine dendrimers ("PAMAM"
dendrimers).
The size or charge of the polycations may vary over a
wide range; it is selected so that the complex formed
with nucleic acid does not dissociate at physiological
CA 02377211 2001-12-24
23
saline concentration, as may easily be determined by the
Ethidium Bromide Displacement Assay (Plank et al, 1999).
In a further step, a defined amount of nucleic acid is
incubated with increasing amounts of the selected
polycation, the complex formed is applied to the cells
which are to be transfected and the gene expression is
measured by standard methods (generally using a reporter
gene construct, e.g. luciferase).
The nucleic acid complexes are synthesised by
electrostatic interactions. The DNA may be present in an
excess, in relation to the polycation, so that these
complexes have a negative surface charge; conversely, if
the polycation condensing the nucleic acid is present in
excess, the complexes have a positive surface charge.
Within the scope of the present invention the polycation
is preferably present in an excess.
Preferably, the ratio of polycation:nucleic acid in the
case of a positive charge excess is adjusted so that the
zeta potential is about +20 to +50 mV, while if certain
polycations, e.g. polylysine, are used, it may be
higher.
In the case of a negative charge excess the zeta
p9tential is about -50 to -20mV.
The zeta potential is measured by established standard
methods as described e.g. by Erbacher et al. 1998.
The polycation is optionally conjugated with a cellular
ligand or antibody; suitable ligands are described in
WO 93/07283. For target cell-directed gene transfer
within the scope of tumour therapy, ligands or
CA 02377211 2001-12-24
24
antibodies for tumour cell-associated receptors (e. g.
CD87; uPA-R) which are capable of increasing gene
transfer into tumour cells are preferred.
In the preparation of the complexes the nucleic acid,
generally plasmid-DNA, is incubated with the polycation
(optionally derivatised with a receptor ligand) which is
present in a charge excess. Particles are then formed
which can be incorporated into cells by receptor-
mediated endocytosis. Then the complexes are incubated
with a negatively charged copolymer according to the
invention, preferably polyethyleneglycol copolymer. The
effector E in the copolymer is preferably a polyanionic
peptide. Alternatively, the copolymer is first mixed
with nucleic acid and then incubated with polycation,
or, as a third alternative, first mixed with polycation
and then incubated with nucleic acid.
Alternatively the nucleic acid is incubated with a
polycation which is present in a charge deficit and then
a cationic copolymer is added. Here again, the sequence
of the mixing steps can be varied as described above for
anionic copolymers. The relative proportions of the
individual components are selected so that the resulting
DNA complex has a slightly positive, neutral or slightly
negative zeta potential (+10 mV to -10 mV).
30
When positively charged copolymers are used they may be
used as sole nucleic acid-binding and -condensing
polycationic molecules; the polycation or cationic lipid
ingredient can thus be omitted. The relative proportions
of the individual components are also selected in this
case so that the resulting DNA complex has a slightly
CA 02377211 2001-12-24
case so that the resulting DNA complex has a slightly
positive, neutral or slightly negative zeta potential
(+10 mV to -10 mV).
5 In the complexes, polycation and/or copolymer is/are
optionally modified with identical or different cellular
ligands.
The nucleic acid complexes according to the invention
10 which are stabilised in size by the electrostatically
bound copolymer of general formula I and are thus
protected from aggregation have the advantage of being
capable of being stored in solution for fairly lengthy
periods (weeks). In addition, they have the advantage
15 of interacting less or not at all with components of
body fluids (e.g. with serum proteins) because of the
protective effect of the bound copolymer.
In another aspect, the invention relates to a
20 pharmaceutical composition containing a therapeutically
active nucleic acid, the copolymer according to the
invention and optionally an organic polycation or
cationic lipid.
25 The pharmaceutical composition according to the
invention is preferably in lyophilised form, optionally
with the addition of sugar such as sucrose or dextrose,
in a quantity which yields a physiological concentration
in the finished solution. The composition may also be in
the form of a cryoconcentrate.
CA 02377211 2001-12-24
26
The composition according to the invention may also be
deep-frozen (cryopreserved) or it may take the form of a
cooled solution.
In another aspect, the positively or negatively charged
copolymers according to the invention serve to
sterically stabilise colloidal particles
("nanoparticles"), as developed for the application of
conventional drugs, and to reduce or suppress their
unwanted interaction with components of body fluids
(e. g. with serum proteins). Moreover, the copolymers
modified with receptor ligands according to the
invention may be used to provide nanoparticles of this
kind with receptor ligands an their surface in order to
transfer drugs to target cells with increased
specificity ("drug targeting").
Summary of the Figures
Fig. 1: Preparation of the copolymer structures from
3-(2'-thio-pyridyl)-mercaptopropionyl-glutamic
acid and O,O'-bis(2-aminoethyl)-
poly(ethyleneglycol) 6000 or O,O'-bis(2-
- aminoethyl)poly(ethyleneglycol) 3400
Fig. 2: Coupling of charged peptides to the copolymer
structure
Fig. 3: Preparation of the copolymer structure from
the protected peptide E4EPROT and 0, O' -bis ( 2-
aminoethyl)poly(ethyleneglycol) 6000
CA 02377211 2001-12-24
27
Fig. 4: Complement activation tests
Fig. 5: Erythrocyte lysis test
Fig. 6: Electron microscope photographs of PEI-DNA
complexes (N/P = 8) in the presence of the
copolymers P3YE5C
Fig. 7: Zeta potential of PEI- and DOTAP/cholesterol-
DNA complexes depending on the quantity of
copolymer P3YE5C or P6YE5C added
Fig. 8: Preparation of DNA/polycation/copolymer
complexes
Fig. 9: Gene transfer into K562 cells with PEI(25 kD)-
DNA complexes in the presence and absence of
the copolymer P3YE5C
Fig. 10: Transfection of the breast cancer cell line
MDA-MB435S with polylysine-DNA complexes in
the presence and absence of the coat polymer
P3INF7
Fig. 11: Lipofection into NIH3T3-cells in the presence
and absence of copolymers P3YE5C
Fig. 12: Transfection of HepG2-cells with
DOTAP/cholesterol-DNA and PEI-DNA in the
presence and absence of P6YE5C
Fig.l3: Transfection of HepG2-cells with PEI-DNA
complexes in the presence of different
amounts of copolymer under different charging
conditions
CA 02377211 2001-12-24
28
Fig. 14: Dependency of the transfection efficiency on
the amount of copolymer, the purification of
the complex and the presence of salt
Fig. 15: Intravenous gene transfer in vivo using
DNA/polycation complexes with a copolymer
shell
Fig. 16: Gene transfer with copolymer-protected PEI-
DNA-vectors in vivo. P3YE5C-PEI-DNA in a
T-cell lymphoma model in DBA2 mice
Fig. 17: Gene transfer with copolymer-protected PEI-
DNA-vectors in vivo - PYESC-PEI-DNA in DBA2
mice -
Fig.l8: P6YE5C increases the transportation of DNA-PEI
complexes into the tumour (tumour targeting)
Fig. 19: Interaction of PEI-DNA complexes and
constituents of human serum
Example 1
Preparation of charged copolymers of general formula I
R X Y
I p
CA 02377211 2001-12-24
29
1.1. Preparation of the copolymer structures from 3-(2'-
thio-pyridyl)-mercaptopropionyl-glutamic acid and
O,O'-bis(2-aminoethyl)poly(ethyleneglycol) 6000 or
0,O'-bis(2-aminoethyl)poly(ethyleneglycol)
3400 (diamino-PEG-3400; Fluka)
In this case, in general formula I:
W=Y=NH;
X=3-mercaptopropionyl-glutamic acid, i.e. an amino acid
derivative according to case i), which was obtained by
coupling the linker grouping 3-(2'-thiopyridyl)-
mercaptopropionic acid to glutamic acid;
therefore there is no Z(m=0).
a) reaction of 3-mercaptopropionic acid with
2,2'-dithiodipyridine (1):
1 g of DTDP (Fluka) was dissolved in 4 ml of absolute
ethanol (Merck). After the addition of 100 u1 of
triethylamine (Aldrich) 87 u1 (lmmol) of 3-
m~rcaptopropionic acid were added. After 1 h the
reaction mixture was purified in aliquots using RP-
HPLC: preparative C18 column (Vydac, 218TP1022), flow
rate 25 ml/min, 0.1 % trifluoroacetic acid, 0-40 %
acetonitrile in 24 min, 40-100 % acetonitrile in 5 min,
100 % acetonitrile in 5 min. The product peak eluted at
about 20 % acetonitrile. The product fractions were
combined and freeze-dried.
CA 02377211 2001-12-24
In a modified version of this synthesis, before the
purification by RP-HPLC, excess DTDP was precipitated
by the slow addition of water with stirring. The
precipitate is twice taken up in ethanol and again
5 precipitated by adding water. The combined aqueous
phases are purified by RP-HPLC as above.
b) Synthesis of 3-(2'-thio-pyridyl)-
mercaptopropionyl-glutamic acid (2b):
10 Product 1 obtained in a) (cf. Fig.l; 0.5 mmol) was
dissolved in 25 ml dichloromethane. While stirring and
cooling with ice, one mmol mach of di-t-butyl glutamate
(Glu(OtBu)OtBu, Bachem), 1-hydroxybenzotriazole
(Aldrich), N-ethyl-N'-(dimethylaminopropyl)-
15 carbodiimide (Aldrich) and diisopropylethylamine
(Aldrich) were added successively to a 50 ml
polypropylene test tube (Messrs. Peske). After 48 h
reaction the mixture was evaporated down in the rotary
evaporator to an oily residue which was taken up in 20
20 ml of ethyl acetate. This solution was extracted twice
with 0.5 M hydrochloric acid, saturated sodium hydrogen
carbonate solution and saturated sodium chloride
s9lution. The organic phase was evaporated down in the
rotary evaporator to an oily residue and dried
25 overnight in a high vacuum (product 2a; cf. Fig. 1).
Product 2a was taken up in 30 ml of
dichloromethane:trifluoroacetic acid (2:1) without any
further purification to eliminate the t-butyl
protecting groups and stirred for two hours at ambient
30 temperature. It was evaporated down in the rotary
CA 02377211 2001-12-24
31
evaporator to an oily residue which was washed with
ice-cold ether. After drying in a high vacuum the
product was dissolved in 100 mM Hepes pH 7.4 and
purified in aliquots using RP-HPLC (same conditions as
for product 1). The product fractions were combined.
Product 2b (cf. Fig.l) was obtained in a yield of
270 umol (27 ~ over all stages). Theoretical molecular
weight: 344.05.
Found: 345.0 (MH+).
c1) Copolymerisation of pyridyl-(2-dithiopropionyl)-
glutamic acid (2b) with O,O'-bis(2-
aminoethyl)poly(ethyleneglycol) 6000 (diamino-
PEG-6000; Fluka):
Product 2b was dissolved in 3 ml dimethylformamide
(Fluka) and made up to 20 ml with dichloromethane.
506 mg of diamino-PEG-6000 (84 umol, corresponding to
1.25 equivalents; Fluka), 30 mg of
dicyclohexylcarbodiimide (135 umol, 2 equivalents,
135 u1 of a 1 M solution in DMF) and 2 mg of
dimethylaminopyridine (0.25 equivalents, 1 M solution
~ in DMF) were added successively to 5 ml of this
s9lution (67.5 umol). After 2 h, 10 u1 were removed for
a ninhydrin test which showed only faint blue
coloration. Crude product 3 (cf. Fig. 1) was obtained
after cooling to -20°C by precipitation from the
reaction mixture with t-butyl-methylether, with
stirring. The product was dried in vacuo. Aliquots were
taken up in water, and after elimination of the
insoluble residue by filtration (Ultra-Free MC,
CA 02377211 2001-12-24
32
Millipore) it was purified by gel filtration. For this
an XK 16/40 column (Pharmacia) was filled with Superdex
75 (Pharmacia) in accordance with the manufacturer's
instructions. Aliquots of 20 mg of crude product 3 were
purified at a flow rate of 1 ml/min with 20 mM Hepes pH
7.3 as eluant. The main fraction eluted at an apparent
molecular weight of 40,000 Da after higher-molecular,
clearly distinct preliminary fractions, which were
collected separately.
c2) Copolymerisation of pyridyl-(2-dithiopropionyl)-
glutamic acid (2b) with O,O'-bis(2-
aminoethyl)poly(ethyleneglycol) 3400 (diamino-
PEG-3400; Fluka) (product 4; cf. Fig. l):
Product 4 was obtained by the same method and with the
same purification as product 3. The main fraction (54 ~
of the product fractions) obtained after gel filtration
was a product which eluted at an apparent molecular
weight of 22.800 Da (secondary fractions are a product
having 64 kD, 14 s of the total amount, and a product
having 46 kD, 32 8 of the total amount).
The reaction diagram of synthesis steps a) to c) which
yield the copolymer structure is shown in Fig. 1:
3=mercaptopropionic acid is reacted with 2,2'-
dithiodipyridine, product (1) is coupled to carboxyl-
protected glutamic acid (product 2a). After the
t-butyl-protecting groups have been cleaved, 3-(2'-
thio-pyridyl)-mercaptopropionyl-glutamic acid (2b) is
obtained which is copolymerised with O,O'-bis(2-
aminoethyl)poly(ethyleneglycol) 6000 or O,O'-bis(2-
' CA 02377211 2001-12-24
33
aminoethyl)poly(ethyleneglycol) 3400, activating with
DCC. Products 3 or 4 are obtained.
1.2. Peptide synthesis
The peptides were prepared by the FastMoc~ method in an
Applied Biosystems 431A peptide synthesizer.
i) Peptide YESC (sequence [Ac-YEEEEE]2-ahx-C) was
prepared using 330 mg of cysteine-charged chlorotrityl
resin (0.5 mmol/g; Bachem) with the protecting groups
trityl (Cys), di-Fmoc (Lys) and O-t-butyl (Glu). 1 mmol
of the protected amino acids was used in each case.
After the branching point (Lys) double couplings were
made throughout. The acetyl-ation of the N-termini was
carried out on the peptide resin with 2 mmol of acetic
anhydride in 2 ml of N-methylpyrrolidone in the
presence of 2 mmol of diisopropylethylamine. The
peptide was obtained as a crude product after cleaving
from the resin (500 u1 of water, 500 u1 of thioanisol,
250 u1 of ethanedithiol in 10 ml of trifluoroacetic
acid) and precipitation with diethylether. The crude
product was dissolved in 100 mM of HEPES pH 7.9 and
purified by perfusion chromatography (Poros 20 HQ,
Boehringer Mannheim, packed into a 9 x 100 mm PEEK
column. 0 - 0.5 M NaCl in 8 min, flow rate 10 ml/min).
The extinction coefficient of the peptide in 50 mM of
sodium phosphate buffer in 6 M guanidinium
hydrochloride is 2560 M-lcm 1 at 280 nm (Gill and von
Hippel 1989).
ii) Peptide INF7 (sequence GLFEAIEGFIENGWEGMIDGWYGC)
was synthesised by the same procedure on 500 mg of
chlorotrityl resin (0.5 mmol/g), cleaved from the resin
CA 02377211 2001-12-24
34
as described for YESC and precipitated with
diethylether. The crude product was dried in vacuo. 20
mg aliquots were dissolved in 500 u1 of 1 M
triethylammonium hydrogen carbonate buffer pH 8 and
purified by gel filtration (Sephadex G-10 made by
Pharmacia, packed into an HR 10/30 column made by
Pharmacia. Flow rate 1 ml/min. The eluant used was 20
mM HEPES pH 7.3 / 150 mM NaCl or 100 mM TEAB or 100 mM
ammonium hydrogen carbonate). Extinction coefficients:
278 nm 12600; 279 nm 12665 280 nm 12660 M-lcm 1.
iii) Peptide SF029-ahx (sequence K2K-ahx-C) was
synthesised analogously (500 mg Fmoc-Cys(Trt)-
chlorotrityl resin made by Bachem; 0.5 mmol/g) and
purified by standard methods (Sephadex G10 with 0.1
TFA as eluant; Reverse phase HPLC, 0.1 s TFA -
acetonitrile gradient). The lysine at the branching
point was alpha, epsilon-di-Fmoc-L-lysine, the following
lysines were alpha-Fmoc-epsilon-Boc-L-lysine.
iv) Peptide E4E (sequence [EEEE]2KGGE) was synthesised
analogously. Batch size 0.25 mmol Fmoc-Glu(OBzl)-
chlorotrityl resin. The peptide was deposited on the
resin by suspending corresponding amounts of
O-chlorotritylchloride resin (Alexis) in absolute
dichloromethane and adding 2 eq. each of
FmocGlu(OBzl)OH and diisopropylethylamine. After
several hours' agitation it was filtered off and washed
several times with dimethylformamide, methanol,
isopropanol, dichloromethane and diethylether. A
modified Fmoc method was used. The N-terminal amino
acid carries a Boc protecting group, in order to obtain
a fully protected base-stable peptide derivative of the
CA 02377211 2001-12-24
sequence (E (Boc) [E (tBu) ] 3) 2KGGE (OBzl) OH (E4EPR°T) from
the solid phase synthesis.
Cleaving from the resin was carried out with
dichloromethane / acetic acid / trifluoroethanol 8:1:1
5 at ambient temperature. The benzyl ester protecting
group of the C-terminal glutamic acid was selectively
cleaved with H2 / palladium on active charcoal by
standard methods.
The peptide masses were determined by Electrospray Mass
10 Spectroscopy and this confirmed the identity of the
peptides.
1.3. Coupling of the peptides to the copolymer
structure (4) or (5)
15 The solutions of 1.2 equivalents of C-terminal
cysteine-containing peptide and copolymer structure,
obtained in 1.1 (based on the thiopyridyl groups on the
polymer), in 20 mM Hepes, pH 7.4 are combined and
stirred or shaken for 15 hours at ambient temperature.
20 In order to determine the equivalents to be used, the
available thiopyridyl binding sites are detected by
combining a dilute solution of the polymer with
2-mercaptoethanol and then measuring the extinction of
the 2-thiopyridone released at a wavelength of 342 nm,
25 and the concentration of the free thiol functions of
the cysteine-containing peptides is determined with
Ellman's reagent at a wavelength of 412 nm according to
Lambert-Beer.
CA 02377211 2001-12-24
36
After the end of the reaction, the completeness of
which was determined by the absorption of the
thiopyridone released at 342 nm, the reaction mixture
was evaporated down and the product was fractionated by
gel filtration (Superdex 75-Material, Pharmacia).
1.3.1 Preparation of the copolymer P3YE5C
The branched peptide YESC, sequence (YEEEEE)ZK(ahx)C,
was used, which is connected via a disulphide bridge of
the cysteine-thiol to the 3-mercaptopropionyl-glutamic
acid grouping.
a) Copolymer P3YE5C was prepared from fraction 3
(22,800 Da) of the product (4) and purified peptide.
The product obtained was a compound with an apparent
molecular weight of 35,000 Da. Taking into account the
molecular weight of the peptide and the starting
polymer this denotes a degree of polymerisation p = 6
(6 repeating units).
b) Copolymer P6YE5C was prepared from fraction 3
(40,200 Da) of the product (3) and purified peptide.
The product obtained was a compound with an apparent
molecular weight of 55,800 Da. The degree of
polymerisation is about 7.
1.3.2 Preparation of the copolymer P3INF7
The endosomolytic peptide INF7 was used, which is
linked to the 3-mercapto-propionyl-glutamic acid
grouping via a disulphide bridge of the cysteine-thiol.
' CA 02377211 2001-12-24
37
a) Copolymer P3INF7 was prepared from fraction 3
(22,800 Da) of the product (4) and purified influenza
peptide.
b) Copolymer P6INF7 was prepared from fraction 3
(40,200 Da) of the product (3) and purified influenza
peptide INF7.
1.3.3 Preparation of a receptor ligand-modified
("lactosylated") copolymer
Part of the lactosylated peptide SF029-ahx and 9 parts
of the branched peptide YESC, which are linked to the
3-mercaptopropionyl-glutamic acid grouping via a
disulphide bridge of the cysteine-thiol, are used.
In each case 3.32 umol of copolymer (4) or (5) (based
on the thiopyridyl groups present) dissolved in 1 ml of
20 mM HEPES pH 7.4 were combined with a mixture of
500 nmol of lactosylated SF029-ahx and 4.48 ~unol of
peptide YESC in 1.1 ml of HEPES buffer. This
corresponds to a 1.5-fold excess of free thiol groups
of the peptides over the available thiopyridyl groups,
and the proportion of the lactosylated peptide in the
total peptide is 10 0. The reaction proceeded overnight
to completion. The products were purified by gel
filtration (Superdex 75) as described.
The reaction diagram for coupling the charged peptides
to the copolymer structure according to 1.3 is shown in
Fig. 2: peptides with free thiol groups, e.g. the
peptide INF7 (on the left) or the peptide YESC are
coupled to product 3 or 4. The products P3INF7 (from
O,0'-bis(2-aminoethyl)poly(ethyleneglycol) 3400),
CA 02377211 2001-12-24
38
P6INF7 (from O,O'-bis(2-aminoethyl)poly(ethyleneglycol)
6000) and analogously P3YE5C and P6YE5C are obtained.
Example 2
Preparation of the copolymer structure from Fmoc-6-
aminohexanoyl-glutamic acid and
O,O'-bis(2-aminoethyl)poly(ethyleneglycol) 6000
(diamino-PEG-6000; Fluka) or
O,O'-bis(2-aminoethyl)poly(ethyleneglycol) 3400
(diamino-PEG-3400; Fluka).
In this case, for general formula I:
W=Y=NH; X=Fmoc-6-aminohexanoyl-glutamic acid.
This means that X according to i) is an amino acid
derivative obtained by coupling Fmoc-6-aminohexanoic
acid to glutamic acid. For coupling the effector
molecule E, Z is not required or may be a bifunctional
linker such as SPDP or EMCS.
A suitable effector E for coupling to this polymer
structure may be a peptide of type E4EpROT (Z is not
present) or of type YESC, which reacts via the
c~isteine-thiol with the linker molecule Z (e.g. SPDP or
EMCS) .
a) Synthesis of the dipeptide Fmoc-6-aminohexanoic
acid-GluOH (6)
' CA 02377211 2001-12-24
39
1 g of Fmoc-protected 6-aminohexanoic acid (2.82 mmol),
1.2 eq. Glu(OtBu)OtBu and 1.2 eq. 1-
hydroxybenzotriazole are dissolved in 200 ml
dichloromethane. After cooling to 0°C the mixture was
combined with 1.2 eq. of N-ethyl-N'-
(dimethylaminopropyl)-carbodiimide and 1.7 ml of
diisopropylethylamine (pH = 8). After one hour at O°C
the mixture was stirred for another 18 hours at ambient
temperature. The solvent was then totally distilled
off, the residue was taken up with ethyl acetate and
extracted with 0.5 N hydrochloric acid, saturated
sodium hydrogen carbonate solution and saturated sodium
chloride solution. After removal of the solvent, Fmoc-
6-aminohexanoic acid-Glu(OtBu)OtBu (5) was obtained
after freeze-drying.
Di-t-butyl-protected derivative (5) was dissolved in 30
ml dichloromethane/trifluoroacetic acid 2:1 and stirred
for one hour at ambient temperature. After the end of
the reaction (reaction monitored by reversed phase
HPLC) the solvent was reduced to about 5~ of the
starting volume and the product (6) was obtained by
precipitation from diethylether. The final purification
was by RP-HPLC with an acetonitrile/ water/ O.1~TFA
gradient.
b) Copolymerisation of Fmoc-6-aminohexanoic acid-GluOH
(6) with O,O'-bis(2-aminoethyl)poly(ethyleneglycol)
3400' (diamino-PEG-3400, Fluka), product (7)
10 mg (6), 1.5 eq. of O,O'-bis(2-
aminoethyl)poly(ethyleneglycol) 3400', 2 eq. of
dicyclohexylcarbodiimide and 0.25 eq. of 4-
~
CA 02377211 2001-12-24
(dimethylamino)-pyridine are dissolved in 5 ml of
dichloromethane. After 30 minutes' stirring at ambient
temperature the solution was concentrated, then
filtered, and then the solvent was totally distilled
5 off. The residue was suspended in 500 u1 water and
freeze-dried.
After cleaving the Fmoc protecting group (20$
piperidine in dimethylformamide or dichloromethane)
from the polymer the copolymer may be conjugated with
10 any desired peptides having a free C-terminus using
current peptide coupling chemistry.
Example 3
Preparation of the copolymer structures from the
15 protected peptide E4EpROT and O, O' -bis (2-
aminoethyl)poly(ethyleneglycol) 6000 (diamino-PEG-6000;
Fluka or O,O'-bis(2-aminoethyl)poly(ethyleneglycol)
3400 (diamino-PEG-3400; Fluka)
In this case in general formula I
20 W=Y=NH; X = the branched peptide E4EPROT_
I~ this Example a polyanionic peptide X according to i)
itself constitutes the effector; therefore Z and E are
not present (m = n = 0)
Copolymerisation of E4EPROT with O,O'-bis(2-aminoethyl)-
25 poly-(ethyleneglycol) 6000 (diamino-PEG-6000,
Fluka) (8);
' CA 02377211 2001-12-24
41
50 umol E4EPROT, 1.5 eq. of O,O'-bis(2-aminoethyl)-
poly(ethyleneglycol) 6000', 2 eq. of
dicyclohexylcarbodiimide and 0.25 eq. of 4-
(dimethylamino-)pyridine were dissolved in 10 ml
dichloromethane. After four hours' stirring at 4°C the
solution was concentrated and filtered and the solvent
was distilled off completely. The residue was suspended
in 500 u1 water and freeze-dried.
In order to cleave the remaining acid-unstable side
chain protecting groups, trifluoroacetic acid was added
as described in the literature, with the addition of up
to 5~ of scavenger (preferably ethanedithiol,
triethylsilan, thioanisol) _and stirred for two hours at
ambient temperature. The crude product was isolated by
precipitation from diethylether. The final purification
was carried out as described above by gel filtration
(Superdex75, Pharmacia).
Fig. 3: shows the reaction diagram: The benzyl
protecting group at carboxylate 1 of the C-terminal
glutamic acid of the fully protected peptide E4EPROT is
selectively cleaved with H2/palladium on active
charcoal. The product is copolymerised with 0,0'-bis(2-
aminoethyl)poly(ethyleneglycol) 6000 or with O,O'-
bis(2-aminoethyl)poly(ethyleneglycol) 3400, activating
with DCC. In the final step the protecting groups of
the N-terminally situated glutamic acids are cleaved
with TFA in DCM.
~
CA 02377211 2001-12-24
42
Example 4
Complement activation studies
The test was carried out essentially as described in
Plank et al., 1996.
a) Polylysine-DNA complexes with and without
copolymer P6INF7
Polylysine (average chain length 170; Sigma) - DNA was
prepared as a stock solution by adding 64 ug of pCMVLuc
(corresponds to pCMVL, described in WO 93/07283) in 800
u1 of HBS to 256 ug of pL in 800 u1 HBS and mixing them
by pipetting; this corresponds to a calculated charging
ratio of 6.3. Of this suspension (the DNA-polycation
complexes are hereinafter also referred to as
"polyplexes"), 50 u1 batches were added as positive
controls to columns 1 A-F of a 96-well plate and
combined with 100 u1 of GVB2+ buffer. All the other
wells contained 50 u1 of GVBZ+ buffer. 100 u1 were
transferred from column 1 to column 2, and mixed etc.
as described in Plank et al. 1996.
In addition, 350 u1 of the polylysine-DNA stock
solution were combined with 35, 70 and 105 nmol (based
o~ fraction INF7) of the polymer P6INF7 and after 15
min incubation made up to 1050 u1 with GVB2+ buffer. 150
u1 aliquots of the resulting suspension were poured
onto rows A to F of column 1 of a 96-well plate. A 1.5
fold dilution series in GVBZ+ buffer and the further
complement activation test were carried out as above
and in Plank et al. 1996.
CA 02377211 2001-12-24
43
The final concentrations of the components in column 1
are 2/3 ug for DNA, 8/3 ug for pL and 0, 5, 10, 15 nmol
for the polymer (based on INF7) per 200 u1 of total
volume.
b) Complement activation by PEI-DNA complexes with and
without a copolymer shell
The test was carried out as described.
PEI (25 kD, Aldrich) - DNA complexes were prepared by
combining equal volumes of a DNA solution (80 ug/ml in
20 mM HEPES pH 7.4) and a PEI solution (83.4 ug/ml in
mM HEPES pH 7.4). The DNA complexes were centrifuged
three times for 15 min at 350 x g in Centricon-100-
Filter Tube (Millipore) to eliminate excess unbound
PEI, topping up to the original volume between
15 centrifugations with 20 mM HEPES pH 7.4. After the last
centrifugation step a stock solution of DNA complex was
obtained, corresponding to a DNA concentration of 300
ug/ml. 182 u1 of this solution were diluted with 20 mM
HEPES pH 7.4 to 2520 u1. 610 u1 aliquots of this
20 solution (corresponding to 13.2 ug of DNA) were
pipetted into solutions of P6YE5C in 277.6 u1 of 20 mM
HEPES pH 7.4. The resulting solutions were adjusted
with 5 M NaCl to a salt concentration of 150 mM. 150 u1
aliquots of the resulting solutions were transferred
into column 1, A to F, of a 96-well plate. The dilution
series in GVBZ+ buffer was carried out as described
(Plank et al. 1996).
Similarly, 610 u1 aliquots of PEI-DNA complex of a
higher concentration (86 ng DNA per u1) were incubated
with 277.6 u1 of solutions of the polymer P3YE5C. The
CA 02377211 2001-12-24
44
solutions contained 0, 1, 2 ,3 charging equivalents of
peptide YESC with regard to the DNA used. After 15 min
were 27.45 u1 aliquots of 5 M NaCl were added
(resulting total volume 915 u1). 150 u1 aliquots of the
resulting solution were transferred into column 1, rows
A to F of a 96-well plate (this corresponds to 8.6 ug
of DNA and 9 ug PEI). The dilution series in GVB2+ and
the remainder of the test were carried out as described
above for pL-DNA.
The result of the complement activation test is shown
in Fig. 4:
A) Complement activation by polylysine-DNA complexes in
the presence and absence of the copolymer P6INF7. The
CH50-Value denotes a specific serum dilution which
leads to the lysis of 50 0 of the sheep erythrocytes in
this test structure. The value CH50maX denotes the CH50
value which is obtained with untreated human serum. In
the test described, human serum was incubated with gene
vectors. The CH50 values which are obtained with serum
treated in this way are lower than CH50maX. if the
vectors activate the complement cascade. The data are
shown as a percentage of CH50maX. The potent complement
activation observed with polylysine-DNA complexes can
be totally prevented by coat polymer P6INF7.
B) The peptide INF7 itself, in free form or polymer-
bound, is a weak complement activator. When
incorporated in a polylysine-DNA complex this
complement activation disappears.
C) Complement activation by PEI-DNA complexes (N/P = 8)
in the presence of the copolymer P3YE5C. The
~
CA 02377211 2001-12-24
unprotected DNA complex is a powerful activator of the
complement system. The copolymer P3YE5C reduces the
complement activation depending on the amount of
copolymer added, but does not lead to total protection
5 in the range under investigation.
D) On the other hand, copolymer P6YE5C gives total
protection from complement activation even when added
in small amounts.
10 Example 5
Erythrocyte lysis test
The test carried out serves to investigate the ability
of peptides to lyse natural membranes in pH-dependent
manner.
15 The erythrocytes used in this Example were obtained as
follows: 10 ml of fresh blood was taken from volunteers
and immediately taken up in 10 ml of Alsevers solution
(Whaley 1985; Plank et al., 1996). 3 ml aliquots were
washed 3 times with the corresponding buffer (40 ml
20 citrate or HBS) (after the addition of buffer and
agitation, centrifugation at 2500 x g and discarding of
the supernatant). The concentration of the erythrocytes
was determined with an "extinction coefficient" of
2.394 x 10-8 ml/cells at 541 nm. In order to obtain the
25 extinction coefficient the cell number was determined
in one aliquot with a Neubauerkammer and then the
extinction of this solution was determined after the
addition of 1 u1 of 1 ~ Triton X-100 at 541 nm.
CA 02377211 2001-12-24
46
In a 96-well plate, aliquots of INF7 or copolymer-
coupled INF7 (P3INF7) in 150 u1 10 mM sodium citrate pH
/ 150 mM NaCl or in HBS buffer were placed in column
1 (usually 45 umol peptide). 50 u1 aliquots of buffer
5 (citrate or HBS) were placed in all the other wells.
100 u1 were transferred with a multichannel pipette
from column 1 to column 2, mixed by pipetting, 100 u1
were transferred from column 2 to column 3 etc. The
other 100 u1 from column 11 were discarded, while
column 12 contained only buffer. The resulting 1.5-fold
dilution series was topped up to 100 u1 with 50 u1
buffer (citrate or HBS). Then 3 x 106 human erythrocytes
were added, the plates were covered with Parafilm and
agitated at 400 rpm in a shaking incubator (Series 25
Incubator Shaker; New Brunswick Scientific Co.; NJ,
USA) at 37°C for 1 h. The plates were then centrifuged
at 2500 x g, 150 u1 aliquots of supernatant were
transferred into a flat-bottomed 96-well plate and
released haemoglobin was measured at 410 nm with an
ELISA Plate Reader. 100 ~ lysis was determined by
adding 1 u1 of 1 ~ Triton X-100 to individual wells
from column 12 (before transferring into the flat-
bottomed plate), 0 ~ lysis was determined from
untreated samples from column 12.
The result of the erythrocyte lysis test is shown in
Fig. 5: peptide INF7 showed high pH-specific activity.
Four fractions (decreasing in molecular weight from 1
to 4) were obtained from the synthesis of the copolymer
P3INF7 after chromatographic resolution (Superdex 75,
Pharmacia). Of these, fractions 2 and 3 showed a higher
lysis activity than the free peptide INF7. In every
CA 02377211 2001-12-24
47
case the activity was strictly pH-dependent, i.e. no
lysis at neutral pH (not shown).
Example 6
Determining the size of DNA complexes using dynamic
light scattering and electron microscopy
Preparation of PEI-DNA polyplexes and application of
the polymer shell: 40 ug of DNA (pCMVLuc) in 333 u1 of
20 mM HEPES pH 7.4 were pipetted into 41.7 ug of PEI
(25 kD, Aldrich) in 333 u1 HEPES pH 7.4 and mixed.
After 10 - 15 min incubation, 0, 0.5, 1, 1.5, 2 or
3 charging equivalents (with respect to the charging of
the DNA used) of polymers P3YE5C or P6YE5C in 333 u1 of
HEPES were added (or 0, 1, 2, 3 and 5 equivalents in a
second experiment). This corresponds to an amount,
based on the peptide YESC, of 0, 152, 303, 455, 606 or
909 pmol of polymer per ug of DNA. DOTAP/cholesterol-
DNA complexes were prepared from DOTAP/cholesterol (1:1
mol/mol) liposomes in 330 u1 of 20 mM HEPES pH 7.4 and
DNA in the same volume at a charging ratio of 5. The
lipoplexes were incubated with 0, 1, 2, 3 and 5
equivalents of the copolymer P3YE5C in 330 u1 of
buffer. The final DNA concentration of the complex was
10 ug/ml.
The size of the DNA complexes was determined on the one
hand by dynamic light scattering (Zetamaster 3000,
Malvern Instruments) immediately after the addition of
polymer and then at various times over several hours,
CA 02377211 2001-12-24
48
on the other hand by electron microscopy as described
in Erbacher et al., 1998, and Erbacher et al., 1999.
Fig. 6 shows the electron-microscope photographs of
PEI-DNA complexes (N/P = 8) in the presence of the
copolymer P3YE5C.
A) In the presence of one charging equivalent of the
copolymer (with respect to the charges of the DNA
used). The particle size is 20 to 30 nm.
B) In the presence of two charging equivalents of the
copolymer. The majority of the particles are around 20
nm in size. These are monomolecular DNA complexes, i.e.
one plasmid molecule packed into one particle.
C) In the presence of 1.5 charging equivalents of the
copolymer after the addition of BSA to give a final
concentration of 1 mg/ml and incubation overnight.
Copolymer-protected DNA complexes remain stable and do
not aggregate, unlike unprotected PEI-DNA complexes
which precipitate immediately under these conditions
(not shown).
Example 7
Determining the zeta potential of DNA complexes
The zeta potentials were determined on the same samples
as in Example 6 with the Malvern apparatus, adjusting
the parameters of refractive index, viscosity and
' CA 02377211 2001-12-24
49
dielectric constant to the values of deionised water,
which can only be an approximation.
Fig. 7A shows the zeta potential of PEI- and
DOTAP/cholesterol-DNA complexes depending on the amount
of added copolymer P3YE5C. The zeta potential as a
measurement of the surface charge of the complexes
decreases from strongly positive through neutral to
slightly negative with increasing amounts of the
copolymer added. This shows that the copolymer binds to
the DNA complexes and equalises or screens their
electrostatic charge.
Fig. 7B shows the zeta potential of purified PEI-DNA
complexes. Excess PEI was eliminated as described in
Example 4B. From the PEI-DNA stock solution which
resulted from purification, aliquots were taken which
corresponded to a DNA amount of 40 ug, and made up to
666 u1 with 20 mM HEPES pH 7.4. The resulting PEI-DNA
suspensions were combined with solutions of P3YE5C in
333u1 of 20 mM HEPES pH 7.4 and mixed by pipetting. The
solutions of P3YE5C contained amounts of polymer
corresponding to 1, 2, 3 and 5 charging equivalents
relative to the amount of DNA used. The zeta potentials
were determined as described in Example 7A.
Example 8
Preparation of DNA complexes and transfections
Unless otherwise stated, the following materials and
methods were used for the cell culture and transfection
experiments carried out in the following Examples:
CA 02377211 2001-12-24
a) Gene transfer in cell culture in a 96-well plate
Adherent cells are seeded, on the day before the
transfection, in flat-bottomed plates in amounts of
20,000 - 30,000 cells per well (depending on the rate
5 of division of the cells. During transfection the
confluence should be about 70 - 80 0).
Before the transfection medium is sucked out. For the
transfection 150 u1 of medium are added to the cells
and then 50 u1 of DNA complexes are added.
10 b) Composition of the DNA complexes: preferably 1 ug
of DNA/well final concentration; calculation for
1.2-fold amount; 20 u1 volume per component (DNA, PEI,
polymer). Finally, 50 u1 of DNA complex are used for
the transfection. Buffer: 20 mM HEPES pH 7.4/ 150 mM
15 NaCl = HB5. The volumes of buffer used remain the same.
If DNA complexes are required for 96 individual tests,
the calculation is appropriately carried out as if 100
individual tests were being carried out: e.g.
DNA: 1 ug x 100 x 1.2 = 120 pg in 20 x 100 u1 HBS =
20 2 ml total volume.
polyethyleneimine (PEI): To achieve an N/P ratio of 8
it is calculated according to the formula
Nl P = gP~~ X 330
43 (~gDNA~
g - gPEI X 330
43 (120
' CA 02377211 2001-12-24
51
that 125.09 ug of PEI are required, specifically in
20 x 100 u1 = 2 ml HBS.
Shell polymer: If for example shell polymer is to be
used in an amount of 2 charging equivalents (with
regard to DNA) for the given amount of DNA and PEI, the
required volume of shell polymer is calculated
according to the formula below where 'Hiillpol.' is
'shell polymer' and 'Ladungsaqu' is 'charging
equivalent'
gDNA Ladungsaqu.
l0 p,1 (Hiillpol.) =1000 x 330 " c (Hullpol. [~mol/ml])
in a concentration c of 11.1 u.mol / ml as
p,1 (Hiillpol.) =1000 x 120 x 2 = 65.5 p1 ,
330 11.1
which is also topped up to 2 ml with HB5.
This is an example of 100 tests each using 1 ug of DNA.
Normally about 5 tests are carried out and e.g. N/P-
ratios of 4, 5, 6, 7, 8 with 0, l, 2, 3 charging
equivalents of shell polymer are investigated.
c~ Mixing of the DNA complexes:
After preparation of the desired preliminary dilutions,
DNA is added to PEI with vortexing. After 15 min shell
polymer is again added to the pre-prepared PEI-DNA
complex with vortexing. After another 30 min, 50 u1 of
DNA complexes are added to the cells, which are in 150
u1 of medium.
' CA 02377211 2001-12-24
52
The containers used will depend on the total volume
calculated. In the above case PEI is appropriately
placed in a 14 ml polypropylene test tube (e. g. Falcon
2059), the other two components are placed in 6 ml test
tubes (e.g. Falcon 2063). For individual tests in the
96-well plate the components may also be mixed in a 96-
well plate. If the final total volume of the DNA
complexes is 1 - 1.5 ml, Eppendorf tubes are
appropriate. For mixing, the components may be pipetted
up and down with a micropipette instead of vortexing.
Conversion to 3 cm dishes (6-well plate):
For 3 cm dishes (6-well plate) amounts of 2 to 5 ug of
DNA are preferably used, vo-lame per component about 100
u1 of each, calculated as above. In the 12-well plate
amounts of about 1 ug of DNA per test are appropriately
used.
Fig. 8 diagrammatically shows the formulation of DNA
complexes: Preferably, first polycation is incubated
with plasmid-DNA, leading to a positively charged DNA
complex (e. g. PEI, N/P = 8). Then negatively charged
copolymer is added which binds electrostatically to the
pre-formed complex. The copolymer may be modified with
receptor ligands, symbolised by 5terne (on the right).
d) Luciferin substrate buffer
The luciferin substrate buffer used was a mixture of
60 mM dithiothreitol, 10 mM magnesium sulphate, 1 mM
ATP, 30 uM D (-)-luciferin, in 25 mM glycyl-glycine
buffer pH 7.8.
CA 02377211 2001-12-24
53
e) Measurement of protein in cell lysates
The protein content of the lysates was determined by
the Bio-Rad protein assay (Messrs. Bio-Ra): 150 ~,1 (or
155 ~,1) dist. water and 40 ~,1 Bio-Rad protein assay dye
concentrate were added to 10 ~,1 (or 5 ~.1) of the lysate
in a well of a transparent 96-well plate (flat bottom
type, Messrs. Nunc, Denmark) and the absorption was
measured at 630 nm with the "Biolumin 690" Absorbance
Reader and the "Xperiment" computer program (both made
by Messrs. Molecular Dynamics, USA). Concentrations of
50, 33.3, 22.3, 15, 9.9, 6.6, 4.4, 2.9, 2.0, 1.3, 0.9
and 0 ng of BSA / ~,1 were measured as the calibration
curve. Bovine Serum Albumin (BSA) was bought as Bio-Rad
Protein Assay Standard II. Thus it was possible to give
the data in pg of luciferase per mg of protein.
Example 9
Gene transfer in K562-cells with PEI(25 kD)-DNA
complexes in the presence and absence of the copolymer
P3YE5C
K562 cells (ATCC CC1, 243) were cultivated in RPMI-1640
medium with the addition of 10 % FCS, 100 units/ml
penicillin, 100 ug/ml streptomycin and 2 mM glutamine
at 37°C in an atmosphere of 5 % C02. The evening before
the transfection, the cells were combined with
desferoxamine to give a final concentration of 10 uM.
Immediately before the transfection the medium was
changed. 50,000 cells in 160 }11 of medium were placed
in the wells of a 96-well plate.
' CA 02377211 2001-12-24
54
Transferrin-PEI (hTf-PEI 25 kD) was prepared essentially
as described by Kircheis et al. (Kircheis et al., 1997)
by reductive amination. A product was obtained which has
on average 1.7 transferrin molecules coupled per PEI
molecule.
A preliminary test detected a composition of hTf-PEI-
polyplexes which yields high transfection and wherein a
significant influence of the receptor ligand can be
determined.
32.4 ug of hTf-PEI (amount based on hTf) in 600 u1 of
HBS were combined with 36 ug of PEI (25kD) in 600 u1 of
HBS. 40 ug of DNA (pCMVLuc) in 600 u1 HBS were pipetted
into this mixture and mixed. 270 u1 of the resulting
solution were added after 15 min to 90 u1 of solutions
of the polymer P3YE5C in HBS or HBS on its own. These
solutions contained amounts of polymer which contained
0/0.5/1/1.5/2/3 charging equivalents based on the
charging of the DNA used. Similarly, DNA complexes
without hTf were prepared with the equivalent amount of
PEI (40 ug of DNA + 42 pg PEI + shell polymer). 60 u1 of
the resulting mixtures (corresponding to an amount of 1
ug of DNA / well) were placed in 5 wells of a round-
bottomed 96-well plate and 50,000 K562 in 160 u1 of RPMI
medium were added. After 24 h the cells were sedimented
by centrifugation, the supernatant was removed by
aspiration and 100 u1 of lysing buffer (250 mM Tris pH
7.8; 0.1 s Triton X-100) were added. After 15 min
incubation it was mixed by pipetting and 10 u1 of sample
were transferred into a black plate (Costar) for the
luciferase test in the 96-well plate format and combined
with 100 u1 of luciferin substrate buffer. The resulting
CA 02377211 2001-12-24
4
light emission was measured using a "Top Count"
Microplate Scintillation & Luminescence Counter (Messrs.
Canberra-Packard, Dreieich). The measuring time was 12
seconds, the measuring delay was 10 min. and background
5 values were subtracted automatically. As the standard,
100, 50, 25, 12.6, 6.25, 3.13, 1.57, 0.78, 0.39, 0.2,
0.1, 0.05, 0.025, 0.013, 0.007 and 0 ng of luciferase
(Boehringer Mannheim) were measured under the same
conditions in 10 ~1 of lysing buffer (= 2-fold dilution
10 series) and a calibration curve was plotted.
Fig. 9 shows the results of the gene transfer
experiments with PEI-DNA complexes (N/P = 8) in K562-
cells in the presence and absence of transferrin as
receptor ligand with the addition of the copolymer
15 P3YE5C. The copolymer does not interfere with gene
transfer and even increases it if a receptor ligand is
present in the DNA complex. The expression of the
reporter gene luciferase based on the amount of total
protein in the cell extract is shown (average values
20 and standard deviations from triplicates).
Example 10
a~ Transfection of the breast cancer cell line MDA-
MB435S with polylysine-DNA complexes in the presence
25 and absence of the shell polymer P3INF7
MDA-MB435S cells (ATCC 45526; human breast cancer cell
line) were cultivated in DMEM medium with the addition
of 10 ~ FCS, 100 units/ml of penicillin, 100 ug/ml of
streptomycin and 2 mM of glutamine at 37°C in an
CA 02377211 2001-12-24
56
atmosphere of 5 ~ C02. On the evening before the
transfection the cells were placed in 96-well plates
(flat-bottomed) in amounts of 20,000 cells per well.
The DNA complexes were prepared as follows:
Calculation for 1 well: The amount to be achieved is 1
ug of DNA pro well, 4 ug of pL170 in a total volume of
60 u1 of HB5. These amounts were multiplied by 1.2. The
DNA complexes were mixed as shown in the following
Table, first adding DNA to polylysine and then in turn,
after 15 minutes, adding this to polymer P3INF7 or to
the buffer. The tests were carried out in triplicates.
60 u1 of DNA complexes were added to cells, covered
with 150 u1 of medium. After 4 h the medium was
changed, after 24 h, after washing with PBS and the
addition of 100 u1 lysing buffer, the luciferase test
and the protein test were carried out as described in
Example 9.
No. P3INF7 HBS pL170 HBS 7.2ug of
DNA in
u1 u1 = ug HBS(ul)
1 144.6 71.4 28.8 79.2 108
(5 nmol)
2 289.2 - 28.8 42.6 71.4
(10 nmol)
3 - - 28.8 187.2 216
Fig. 10A shows the results of the gene transfer
experiments with polylysine-DNA complexes into the
human breast cancer cell line MDA-MB435S in the
' CA 02377211 2001-12-24
57
presence and absence of the copolymer P3INF7. In the
absence of the copolymer there is no measurable
reporter gene expression. The pH-dependent membrane-
destroying and thus endosomolytic activity of the
copolymer brings about efficient gene transfer. 5 nmol
or 10 nmol of P3INF7 relate to the amount of the
polymer-bound peptide INF7 used.
b) Transfection of the breast cancer cell line MDA-
MB435S with polylysine-DNA complexes in the presence
and absence of the shell polymer P3INF7
In another test series with MDA-MB435S cells the
efficiency of the transfection was investigated using
DNA-polylysine complexes in the presence and absence of
the shell polymer P3INF7 at different charging ratios
of pL/DNA. The results of these tests are shown in Fig.
10B.
Example 11
Lipofection in the presence of shell polymers (Fig. 11)
NIH3T3-cells (ATCC CRL 1658) were cultivated in DMEM
medium with the addition of 10 o FCS, 100 units/ml
penicillin, 100 ug/ml streptomycin and 2 mM glutamine at
37°C in an atmosphere of 5 % CO2.
On the evening before the transfection, transferred into
in 6-well plates in a density of 500,000 cells per well.
Preparation of the DNA complexes:
16 ug of DNA in 240 u1 of 20 mM HEPES pH 7.4 were
combined with a solution of 242 nmol DOTAP/cholesterol
liposomes in 240 u1 of the same buffer. This produces a
CA 02377211 2001-12-24
58
charging ratio (+/_) of 5. 210 u1 of the resulting
solution were pipetted into 105 u1 of a solution which
contained 6.36 nmol of the polymer P3YE5C (based on the
peptide content YESC; this corresponds to 3 DNA-
charging equivalents). For the control, 210 u1 of
DOTAP/cholesterol-DNA were pipetted into 105 u1 of 20
mM HEPES pH 7.4. 90 u1 aliquots of the resulting DNA
complexes were added to the cells which were in 800 u1
of fresh medium; this corresponds to 2 ug of DNA per
well. The tests were carried out in triplicate.
Similarly the test was carried out with Lipofectamine'n'
instead of DOTAP/cholesterol. In this case, an amount
of Lipofectamine (DOSPA) which leads to a charging
ratio of 7 (~/_) was used.
30 min after the addition of the DNA complexes, 1 ml of
fresh medium were added to the cells, followed by
another 2 ml after 3 h. The medium was not changed. 22
h after the addition of the complex the cells were
washed with PBS and lysed in 500 u1 of lysing buffer.
Aliquots of the cell lysate were used in the luciferase
test and used for protein measurement.
Fig. 11 shows the results of lipofection in NIH3T3
cells in the presence and absence of the copolymer
P3YE5C. Neither the transfection with
DOTAP/cholesterol-DNA nor that with Lipofectamine is
significantly lowered (3 charging equivalents of the
copolymer; DOTAP/cholesterol-DNA has a neutral zeta
potential in this composition, cf. Fig. 7A).
' CA 02377211 2001-12-24
59
Example 12
a) Transfection of HepG2 cells with DOTAP/cholesterol-
DNA and PEI-DNA in the presence and absence of P6YE5C
HepG2-cells (ATCC HB 8065) were cultivated in DMEM
medium with the addition of 10 o FCS, 100 units/ml of
penicillin, 100 ug/ml of streptomycin and 2 mM
glutamine at 37°C in an atmosphere of 5 ~ CO2.
Two days before the transfection the cells were placed
in 6-well plates in a density of 500,000 cells per
well. The transfection with DOTAP/cholesterol was
carried out exactly as described above for NIH3T3
cells, this time using polymer P6YE5C. Moreover, 7 ug
of DNA in 105 u1 HEPES buffer was pipetted into 7.3 ug
of PEI 25 kD dissolved in the same volume. After 15 min
incubation this solution was pipetted into 105 u1 of a
solution of the polymer P6YE5C which contained 3 DNA-
charging equivalents PYESC. 90 u1 of this solution were
added to the cells. The tests were carried out in
triplicate.
Fig. 12 shows the gene transfer into HepG2 cells in the
presence and absence of the copolymer P6YE5C (labelled
as P6 in the Fig.). Transfection by DOTAP/cholesterol-
D~iA is not significantly inhibited. The transfection by
PEI-DNA complexes is reduced (3 charging equivalents of
the copolymer).
Example 13
' CA 02377211 2001-12-24
Transfection of HepG2-cells with PEI-DNA complexes in
the presence of different amounts of P3YE5C or P6YE5C
in different charging ratios
The complexes were prepared in 20mM HEPES pH 7.4, as
5 described in Example 14, using unpurified complexes and
adjusting them to a concentration of 5 ~ glucose before
addition to the cells. The tests the results of which
are shown in Fig. 13 were carried out three times, the
error bars show the standard deviation. It was found
10 that the shell copolymers with a higher charging ratio
increase the transfection efficiency.
Example 14
Transfections of K-562, HepG2 and NIH3T3 cells
15 The DNA-PEI complexe were prepared as follows:
The polyplexes (N/P=8) were prepared as stock solutions
in 20 mM HEPES pH 7.4, with each component (DNA,
polycation, copolymer) being dispersed in the same
volume, so as to obtain a final concentration of 1 ug
20 of DNA per 50 u1, corresponding to the amount applied
to the cells in each well (in the case of the purified
complexes the same amounts of DNA complex in the same
volume were also added as well as buffer). First of
all, the DNA was added to the polycation with gently
25 vortexing or mixing with a pipette, and after 15 min
the copolymers P3YE5C or P6YE5C were added; after
another 30 min or more the complexes were added to the
cells. When using purified complexes the purification
was carried out as follows: stock solutions of PEI-DNA
' CA 02377211 2001-12-24
61
complexes (N/P=8; 100 ug of DNA per ml in 20 mM HEPES
pH 7,4) were centrifuged using Centricon-100 membranes
(Millipore) 4 x 15 min at 500 g on a Beckman JA-20
Rotor with a fixed angle. Between the centrifugations
the solutions were diluted again to the original
concentrations. Care was taken to ensure that a DNA
concentration of 500 ug per ml was not exceeded. The
final centrifugation yielded complexes of 300 - 500 ug
of DNA per ml. The DNA concentration was determined on
the basis of the absorption at 260 nm (1 OD26o = 0.45 ug
per ml for dsDNA in a PEI-DNA complex, determined using
a standard dilution of a DNA complex with a known DNA
content). The amount of free, non-DNA-associated PEI
was determined in the combined filtrates using
trinitrobenzosulphonic acid assay (Snyder and
Sobocinski, 1975).
Fig. 14 shows the dependency of the transfection
efficiency on the amount of P3YE5C and P6YE5C, on the
complex purification by ultrafiltration (unpurified:
solid bars; purified: shaded bars) and on the presence
of salt during or after (for the purified complexes)
complex formation (black bars: 150 mM NaCl; grey bars:
no salt). (A): K562 cells; (B): HepG2 cells; (C):
NIH-3T3 cells.
Example 15
Intravenous gene transfer in vivo
a) Control (PEI-DNA, N/P = 8):
~
CA 02377211 2001-12-24
62
150 ug of DNA (pCLuc) in 337.5 u1 of 20 mM HEPES pH 7.4
were pipetted into 156.4 u1 PEI (25 kD, Aldrich) in the
same volume of HEPES buffer. After 15 min 75 u1 of 50 %
glucose were added. Of this solution 100 u1 aliquots
were injected into the caudal vein of mice
(corresponding to a dose of 20 ug of DNA per animal).
b) Control (DOTAP/cholesterol-DNA; charging ratio +/_
- 5)
DOTAP-cholesterol liposomes were prepared by standard
methods (Barron et al., 1998). In this case liposomes
were prepared with a molar ratio of DOTAP to
cholesterol of 1:l and a final concentration of DOTAP
of 5 mM in 5 % Glucose. 134 ug of DNA in 91.1 u1 of 20
mM HEPES pH 7.4 were added to 393.5 u1 of liposome
suspension. After 15 min 65 u1 of 50 % glucose were
added. Of this solution 100 u1 aliquots were injected
in the caudal vein of mice (corresponding to a dose of
ug of DNA per animal).
c) PEI-DNA (N/P = 8) with copolymer shell:
20 150 ug of DNA in 2475 u1 were added to 156.4 ug of PEI
(25 kD) an equal volume, with vortexing. After 15 min 3
charging equivalents (with respect to the charges of
tie amount of DNA used) of polymer P3YE5C in 2475 u1
HEPES buffer were added with vortexing. After a further
30 min the DNA complexes were concentrated by
centrifugation in Centricon 30 test tubes to a DNA
concentration of 454 ug/ml. This solution was then
adjusted to a final concentration of 200 ug of DNA per
ml and 5 % glucose with the addition of 50 % glucose
and 20 mM HEPES pH 7.4. Of this solution 100 u1
~
CA 02377211 2001-12-24
63
aliquots were injected in the caudal vein of mice
(corresponding to a dose of 20 ug of DNA per animal).
d) DOTAP/cholesterol-DNA (5:1) with a copolymer
shell: 393.9 u1 of liposome suspension were pipetted
directly into a solution of 130 ug of DNA in 65.3 u1
water. After 15 min, 3 charging equivalents of P3YE5C
in 216.9 u1 HEPES buffer were added and after another
30 min 75 u1 of 5 o glucose. Of this solution 115.5 u1
aliquots were injected in the caudal vein of mice
(corresponding to a dose of 20 ug of DNA per animal).
Fig. 15 shows the results of the gene transfer
experiments in vivo: PEI-DNA or DOTAP/cholesterol DNA
complexes with or without bound copolymer P3YE5C (3
charging equivalents) were injected into the caudal
vein of mice (n = 6). The animals were sacrificed 24 h
after the injection, and the reporter gene expression
in organs was determined. The maximum activity was
measured at the injection site, with PEI-DNA copolymer
significant reporter gene expression occurred in the
lung and heart, while gene transfer into the lung by
DOTAP/cholesterol-DNA was inhibited when the copolymer
was used.
Example 16
Steric stabilisation of PEI-DNA complexes
PEI-DNA complexes were prepared exactly as described in
Example 6 (PEI-DNA, N/P = 8, 0/1,5/3 charging
equivalents of copolymer P3YE5C or P6YE5C). The size of
the complexes was determined by dynamic light
CA 02377211 2001-12-24
64
scattering and was 20 to 30 nm. Then 5 M NaCl was added
to give a final concentration of 150 mM. PEI-DNA
without copolymer aggregated immediately (after 5 min
there was a population of particles >500 nm to be
measured; after 15 min the majority of the particles
were >1000 nm; overnight the complexes were
precipitated from the solution). In the presence of
P3YE5C or P6YE5C (1,5 or 3 charging equivalents) the
particle size remained stable over at least 3 days.
Similarly, the addition of BSA to give a final
concentration of 1 mg/ml led to immediate precipitation
of PEI-DNA. In the presence of P3YE5C or P6YE5C (1.5
charging equivalents or more) the particle size remains
constant over at least 24 h (cf. also Fig. 6).
Example 17
Gene transfer studies with copolymer-protected PEI-DNA-
vectors in vivo
a) P3YE5C-PEI-DNA in a T-cell lymphoma model in DBA2
mice
200,000 tumour cells were injected intradermally into
ti'ie mice (5 weeks old) on day 0, as described (Kruger
et al. 1994). On day 21, 200 u1 of DNA complex
containing 50 ug of DNA (p55pCMV-IVS-luc+, provided by
Dr. Andrew Baker of Bayer Corp., USA.) was injected
into the animals through the caudal vein.
DNA complexes: DNA (250 ug in 700 u1 of 20 mM HEPES
pH7.4) was mixed with PEI (260.6 ug in 700 u1 of the
~
CA 02377211 2001-12-24
same buffer) by pipetting. After 15 min incubation the
DNA complexes were pipetted into 400 p1 of shell
polymer solution which contained 5 charging equivalents
of P3YE5C. After another 15 min incubation 200 u1 of
5 50~ glucose (in water) are added. The complexes were
purified of excess PEI by ultrafiltration as described
in Example 4b. The vector suspension was finally made
up to a volume of 1 ml with 20 mM HEPES. 200 u1 was
injected into each test animal through the caudal vein.
10 After 24 hours the animals were sacrificed and the
reporter gene expression in the organs was determined:
The animals were fully anaesthetised using an
intraperitoneal injection of 100 mg of ketamine and 5
mg of xylazine per kg of body weight. Then the
15 abdominal cavity was opened up and perfused with 20 ml
of isotonic sodium chloride solution through the Vena
cava caudalis. The organs were transferred into 2 ml
screw-topped test tubes containing 500 u1 of lysing
buffer (0.1 ~ Triton X-100 in 250 mM Tris-HC1 pH 7.8)
20 and zirconium beads (2.5 mm in diameter; Biospec
Products, Bartlesville, USA). The tissue samples were
homogenised 2 x 20 sec with a Mini-Bead-Beater (Biospec
Products). 50 u1 aliquots of homogenised tissue were
used in the luciferase test (Promega Luciferase Assay
25 Kit); the results are shown in Fig. 16.
b) P6YE5C-PEI-DNA in DBA2 mice:
DNA complexes: 350 ug of DNA in 420 u1 of 20 mM HEPES
pH 7.4 were mixed with 364 ug of PEI in the same volume
of the same buffer. After 5 min. incubation either 420
30 u1 of 20 mM HEPES or 3 charging equivalents of P6YE5C
in 420 u1 of 20 mM HEPES pH 7.4 were added and mixed.
CA 02377211 2001-12-24
66
Finally 140 u1 of 50 ~ glucose in water were added. 200
u1 aliquots (corresponding to 50 ug DNA) were injected
into the animals (5 weeks old) through the caudal vein.
The tests were evaluated as described above, except
that this time 750 u1 of lysing buffer from the Promega
Luciferase Assay Kit were used to homogenise the organs
and that 20 u1 of homogenised tissue were used for the
luciferase test. The results are shown in Fig. 17.
Example 18
P6YE5C increases the transfer of DNA-PEI complexes into
the tumour (tumour targeting)
First, tumours were set by placing 1x106 murine melanoma
M3 cells (ATCC No. CCL 53.1) in 100 u1 Ringer's
solution subcutaneously in DBA/2 mice.
To prepare the polyplexes 50ug of pCMVLuc DNA were
complexed with PEI(25) (N/P =6) in 0.5 x HBS. The
complexes were then incubated at ambient temperature
for 20 min. When using shell copolymer 3 equivalents of
P6YE5C were added, in accordance with the formula:
amount of P6YE5C (u1) - [1000*(ug of DNA)*charging
equivalent of P6YE5C]/330*19.3; the charging equivalent
being (3) as defined above and 19.3 being the
concentration in mM of the solution of P6YE5C used.
Before the complexes were administered 2.5~ glucose was
added in order to prepare an isotonic solution for the
inj ection in vivo.
CA 02377211 2001-12-24
67
20 days after the setting of the tumours - by this time
the tumours measured 15mm x 15mm on average - the mice
were treated. 250u1 aliquots of the polyplexes were
injected per mouse strictly by i.v. route (into the
lateral caudal vein). Two groups were treated
consisting of 3 mice, the first group receiving
unprotected DNA-PEI complexes and the second group
being given complexes coated with P6YE5C.
After 24 hours the animals were euthanased and the
organs (liver, spleen, kidneys, heart and lungs) and
the tumour were removed. The further treatment of the
organs as well as the measurement of the luciferase
activity were carried out as described in the foregoing
Examples; the results of the tests are shown in
Fig. 18.
Example 19
Biosensor studies of the interaction of PEI-DNA
complexes and constituents of human serum
To investigate the interaction of DNA complexes and
constituents of human serum, surface plasmon resonance
measurements were carried out (BIAcore, Uppsala,
Sweden). Human complement serum (Sigma, Deisenhofen,
Germany) was dissolved according to the manufacturer's
instructions and covalently coupled to a "Research
Grade" Sensor-Chip (CM5), following the standard
procedure (Johnsson et a1. 1991). After N-(3-
dimethylaminopropyl)-N'-ethyl-carbodiimide / N-
hydroxysuccinimide activation of the carboxylate groups
CA 02377211 2001-12-24
68
on the sensor chip, serum dilution (100 ug/ml in 10 mM
sodium acetate pH 4) was automatically applied to the
chip. Unreacted N-hydroxysuccinimide esters were
inactivated with 1 M ethanolamine, pH 8.5 in water. The
immobilisation process and the binding studies were
carried out at 25°C with 10 mM HEPES pH 7.4 / 150 mM
sodium chloride / 3.4 mM EDTA / 0.05 Surfactant P20 as
a continuous eluting buffer at a flow rate of 5 ul/min.
DNA complexes were prepared as described in 20 mM HEPES
pH 7.4 with a final DNA concentration of 10 ug/ml.
Immediately before the injection onto the sensor chip
the DNA complex suspension was automatically applied
directly to 150 mM sodium chloride in the apparatus by
the addition of a 5 molar sodium chloride solution.
Fig. 19 shows the biospecific interaction analysis
(BIAcore) of PEI polyplexes in the presence of
copolymers with serum ingredients: PEI-DNA complexes
(N/P = 8), incubated with the described amounts of
copolymer, were injected onto the sensor chip twice
over 4 minutes (arrows), followed by washing steps
until the measuring signal stabilised (flow rate 5
ul/min). Binding to the serum-charged sensor chip is
demonstrated by an increasing measuring signal
(Resonance Units, RU, y-axis). The time axis over the
whole experiment (x-axis) was 1200 seconds. On the left
P3YE5C-protected complexes, on the right P6YE5C-
protected complexes (individual sensograms
superimposed). A reduction in the interaction with the
serum-charged chip as the amount of copolymer increases
is obvious. The charging signal of "naked" PEI-DNA
complexes ("0 equiv.") does not reach a plateau because
' CA 02377211 2001-12-24
69
of the continuing aggregation on the sensor chip during
the injection in salt-containing buffer.
~
CA 02377211 2001-12-24
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