Note: Descriptions are shown in the official language in which they were submitted.
29/11 '0l 14:54 FAX ++49 89 92805444 BARDEftLE OFFICE -~ GOUDREAU 0]024
CA 02375854 2001-11-30
V36454PCCA
WO 00/74646 PCT/EP00/04678
Novel liposomal vector complexes and their use for gene therapy
The essential components of vectors for gene therapy have hitherto been
nucleic acid sequences which are complexed with a nonviral carrier (e.g.
cationic lipids or cationic polymers) or are inserted into a virus.
Previous experience with such vectors in the gene therapy of ail sorts of
diseases, but in particular of oncoses, shows that, in cell culture, nonviral
vector complexes particularly can only transduce a relatively low number of
cells (usually between 1 % and 30%); furthermore that after administration
of a gene therapy vector into the circulation of an organism these vectors
are eliminated from the circulation in a short time and are no longer
available for binding to the target cells and for the transfection of these
target cells (Ogris et a1. Gene Ther. 6: 595, 1999; Dash et al., Gene Ther.
t 5 6: 643, 1999; Li et al., Gene Ther. 5: 930, 1998; Liu et al. Gene Ther. 4:
517,1997) .
This elimination can take place due to degradation of the DNA or due to
rapid deposition of the vectors in the lung, the liver or the
'reticuloendothelial system' (RES) which is particularly developed in the
spleen and the lymph nodes (Zhu et al., Science 261: 209, 1993).
The causes of the rapid elimination are varied. They can be: an excessively
large negative or positive charge, an excessively large volume or an
opsoni2ation of the vector particles by blood proteins. In the case of viral
vectors, they can additionally be the binding of the virus coat proteins to
virus-specific receptors in the organs and/or alternatively antibodies or
immune cells specific for the viruses which bind to the vectors and thereby
eliminate these.
Previous experience additionally shows that the coupling or insertion of a
target cell-specific figand into the vector complex does not significantly
decrease its rapid elimination after administration into the blood
circulation.
In the knowledge of these problems, the urgent need exists for novel
preparations of vectors which transfects as many cells as possible in the
cell culture and which, after administration to a living organism, remain as
tong as possible in the circulation and are not prematurely eliminated from
the circulation. In order to decrease the elimination of cationic lipids or
29/11. '0l 14:55 FAX ++48 89 92805444 BARDEHLE OFFICE -~ GOUDREAU I~J025
CA 02375854 2001-11-30
2
cationic polymers as a complex with nucleic acid sequences from the blood
circulation, polyethylene glycol (Senior et al., Biochim. Biophys. Res. Acta
1062: 77, 1991; Mori et al., FEES Lett 284; 263, 1991; Ogris et al., Gene
Ther. 6: 595, 1999), vinyl polymers (Torchilin et al., Biochim. Biophys. Res.
Acta 1195: 181, 1994) or other amphipathic polymers (Woodle et al.,
Bioconjugat. Chem. 5: 493, i 994) were coupled to the cationic lipids or
cationic polymers or, with the aid of negatively charged lipids, anionic
liposomes were prepared in which the nucleic acid sequences were
included as a complex with cationic lipids or cationic polymers (US Patent
No. 4,946,787; US Patent No. 4,245,737; US Patent No. 5,480,463;
Heywood and Eanes, Calc. Tissue Int. 40: 149, 1992; Lee and Huang, J.
Biol. Chem. 271: 8481, 1996; Baiicki and Beutler, Blood 88: 3884, 1996;
Lucie et al., J. Lip. Res. 8: 57, 1998; Lakkaraju et al., J. Lip. Res. 8: 74,
1998; Turner et al., J. Lip. Res. 8: 114, 1998; Schoen et al., J. Lip. Res. 8:
485, i 998).
Modifications! of this type led, for example, to a stabilization of the vector
particle size, ,inhibited the aggregation of vectors with themselves or with
blood cells, reduced the opsonization of vectors by binding of
immunoglobulins, complement fractions, fibrinogen or fibronectin, protected
(adeno)viral vectors against elimination by antibodies (Chillon et al., Gene
Ther. 5: 995, 1998) and caused a prolongation of the blood residence time
of vectors, a markedly stronger concentration in tumors growing
subcutaneously and a transduction of the tumor cells (Ogris et al., Gene
Ther. 6: 595, 1999).
At the same time, however, it was also possible in the lung, spleen and
liver to detect va considerable concentration of the vectors and transduction
of the tissue cells in these organs (Ogris et al., Gene Ther. 8: 595, 1999),
so that it can gibe concluded that, for example, the coupling of PEG does
bring about arp improvement, but still no optimization of the distribution of
vectors.
General description of the invention
The invention relates to novel liposomal vector complexes for gene therapy
consisting of the following components:
a} a nucleic acid sequence of any desired length;
29/11 'O1 14:56 FAX ++49 89 92805444 BARDEHL.E OFFICE -~ GOiJDREAU C~J026
CA 02375854 2001-11-30
3
b) a cationic carrier which condenses component a) and is
lysosomolytic and/or lysosomotropic;
c} lipids and phospholipids which form a liposome;
d) optionally a ligand which has a binding site for a target cell;
e} optionally a fusogenic substance which can replace the
lysosomolytic and/or lysosomotropic function of component b);
where in the presence of a fusogenic substance{s) the cationic carrier (b)
must not be lysosomolytic and/or lysosornotropic.
Component a} can be a nonmodified or modified DNA sequence or a
nonmodified or modified RNA sequence. The nucleotide sequence can
exert an anti-DNA {triplex) or anti-RNA {antisense; ribozyme) function or
can code for an active RNA sequence of this type or for a protein. The
nucleotide sequences and their modification can be such that the
nucleotide sequence is largely resistant to degradation by DNAses or
RNAses. Examples of nucleotide sequences of this type and their
modifications are shown in Breaker, Nature Biotechnol. 15: 427, 1997;
Gerwik, Critical Reviews in Oncogenesis 8: 93, 1997; Mukhopadhyay et al.,
Crit. Rev. Oncogen. 7: 151, 1996; Mercola et al., Cancer Gene Ther. 2: 47,
1995; Frank-Kamenetski, Annu. Rev. Biochem. 64: 65, 1995 and Fraser et
al., Exp. Opin. Invest. Drugs 4: 637, 1995. The DNA sequence can be
linear or circular, for example in the form of a plasmid.
Component a) can additionally be a virus, preferably a virus in which a
nucleic acid sequence foreign to the virus has been inserted using the
methods known to the person skiNed in the art. Examples of viruses of this
type are RTV, AAV and lentiviruses: Examples of this type and further
examples have been described by Vile, Nature Biotechnol. 15: 840; 1997;
McKeon et al., Human Gene Ther. 7: 1615, 1996; Flotte et al., Gene Ther.
2: 357; 1995; Jolly, Cancer Gene Ther. 1: 51, 1994; Dubensky et al., J.
Virol. 70: 508, 1996.
Component b} is a cationic carrier which condenses component a) and at
the same time has lysosomolyticaily and/or lysosomotropically and/or
iysosomotropic properties.
According to this invention, in a particular embodiment component b) is a
cationic polymer, for example described by Boussif et al., Proc. Natl. Acad.
Sci. USA 92: 7297, 1995; Kaneda et al., Science 243: 375, 1989; Keown et
29/1.1 'O1 14:56 FAX ++49 89 92805444 BARDEALE OFFICE -. GOLTDREAiJ f~/027
CA 02375854 2001-11-30
4
al., Methods in Encymology 185: 527, 1990; Baker et al., Gene Ther. 4:
773, 1997; Fritz et al., Human Gene Ther. 7: 1395, 1996; Wolfert et ai.,
Human Gene Ther. 7: 2123, 1996 and Solodin et al., Biochem. 34: 13537,
1995. The polyethyleneimine (PEI} described by Boussif, when used as a
vector for gene therapy according to the method described by the same
author, finally leads to a swelling and bursting of the lysosomes, i.e. PEI
acts lysosomolytically.
In a further particular embodiment of this invention, component b) is a
polyethyleneimine (PEI), in a further particular embodiment of this invention
the polyethyleneimine has a molecular weight in a range of 500-25,000 Da
and in a further embodiment a molecular weight of 5000-10,000 Da in a
further embodiment of the invention a molecular weight of on average
approximately 2000 Da and was prepared as described in the patent
application EP-A 0 905 254. High-branched-chain PEI (LupasolC, BASF,
Ludwigshafen, Germany) and a low-branched-chain PEI derivative, which
was prepared according to Fischer et al. (Pharm. Res. 16, 1273-1279,
1999}, are used in a particular embodiment of the invention.
Component c) is any desired liposome having any desired composition
known to the person skilled in the art. In a particular embodiment, this
liposome has an anionic charge. In a further particular embodiment the lipid
and phospholipid composition of the anionic liposome is similar to the
composition of a virus coat. The preparation of liposornes having anionic
charge has already been widely described, for example from in US patents
Nos. US 4,946,787, US 4,245,737, US 5,480,463, and also in Heywood
and Eanes, Calc. Tissue Int. 40: 149, 1992; Lee and Huang, J. Biol. Chem.
271: 8481, 1996; Balicki and Beutler, Blood 88: 3884, 1996; Lucie et al., J.
Lip. Res. 8: 57, 1998; Lakkaraju et al., J. Lip. Res. 8: 74, 1998; Turner et
al., J. Lip. Res. 8: 114, 1998; Schoen et al., J. Lip. Res. 8: 485, 1998.
The preparation of liposomes which are similar to virus coats was
described, for example, in the US patents Nos. US 5,252,348; US
5,753,258; US 5,766,625 and EP-A 0 555 333.
The invention additionally relates to the completion of the liposornal vector
complexes according to the invention by addition of a component d).
29/11 'O1 14:57 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU I~J028
CA 02375854 2001-11-30
This component d) is a ligand which has a binding site for the target cell
and is conjugated to a lipid. The target cell specificity of the ligand can be
arbi#rary.
5 Preferred target cell specificities of ligands are selected from a group
described in detail in
polyfunctional ligand systems for the target cell-specific transfer of
nucleotide sequences EP-A 0 846 772
- single-chain, double antigen-binding molecules (DE 198161417, still
unpublished)
- specific cell membrane-penetrating molecules (DE 19850987.1, still
unpublished) or
- target cell-specific, multivalent proteins (DE 19910419.0, still
unpublished).
The type of lipid can be arbitrary, but naturally occurring lipids, such as
described, far example, in US patent Nos. US 5,252,348; US 5,753,258;
US 5,766,625 and EP-A 0 555 333 are preferred.
The conjugation of lipids to the target cell-specific tigand is carried out
using one of the methods known to the person skilled in the art, for
example as described in US patent No. US 5,662,930.
The insertion of component d) into the liposome according to the invention
(component c) is carried out using the method known to the person skilled
in the art, for example described in US patents Nos. 5,252,348 and US
5,753,258).
The invention additionally relates to the completion of the liposomal vector
complexes according to the invention by addition of a component e). This
component e) is the functional sequence of a fusion peptide, preferably
from subunit HA-2 of the hemagglutinin from the influenza virus (Wagner et
al., Proc. Natl. Acad. Sci. USA 89{17), 7934-7938) ligands, which facilitate
the release of the liposome contents from the endosome.
The preparation of the vector complex according to the invention consisting
of the components a), b) and c} or a), b, c) and d) or a}, b), c}, d) and e)
is
29/11 '0l 14:57 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU 0]029
CA 02375854 2001-11-30
6
carried out using methods known to the person skilled in the art, for
example in such a way that
- in the 1s~ step component a) is mixed with component b}, the mixing
ratio being adjusted such that the net charge of the resulting total
complex is preferably either cationic or anionic and subsequently
- in the 2"d step the complex resulting from step (1} is inserted into the
component c}, which can already contain component d), the mixing
ratio of all components being adjusted such that the net charge of the
resulting overall complex is preferably either anionic or cationic;
- in a 3~d step component d) can also optionally be inserted into
component c) following step 2; and
- in a 4t" step component e) is optionally inserted into the complex
resulting from step 2 or 3, or, alternatively, component e} is added to
component c) before step 2.
The liposomal vector complexes resulting from these preparation steps
have a diameter of 100 - 600 nm and a cationic or anionic ctlarge,
preferably a diameter of 100 - 300 nm and an anionic charge.
The liposomes according to the invention are enriched, for example, in the
tumor vascular bed (Unezaki et al., Int. J. Pharmac. 174:11, 1996; Sadzuka
et al., Cancer Lett. 127:99, 1998; Wunder et al., Int. J. Oncol. 11:497,
1997). In addition, the liposomes according to the invention bind via their
component d) to the target cell and transfects this such that the nucleic acid
sequence in the vector complex according to the invention is released in
the cell.
This nucleic acid sequence can display its action, depending on
composition, in the target cell, i.e. for example inhibits the transcription
or
translation of a certain gene or of a certain RNA, or transduce the cell for
the expression of the RNA ar of the protein encoded by this nucleic acid
sequence.
The iransduction rate by the liposomal vector complexes according to the
invention is considerably improved in comparison to the existing technique
29/11 'O1 14:58 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU f~ 030
CA 02375854 2001-11-30
7
known to the person skilled in the art and is, for example in the cell
culture,
over 80% of the cells which carry a receptor for component d) and are
brought into contact with the liposomal vector complexes according to the
invention.
The vector complexes according to the invention are thus preferably
suitable for in vitro transduction of cells and for in vivo administration
with
the aim of prophylaxis or therapy of diseases.
The invention relates to a liposomai vector complex comprising the
following components:
f) a nucleic acid sequence of any desired length;
g) a cationic carrier which condenses component a) and is lyosomolytic
and/or lysosomotropic;
h) lipids and phospholipids which form a liposome;
t) optionally a ligand which has a binding site for a target cell;
j) optionally a fusogenic substance which can replace the
lysosomolytic and/or lysasomotropic function of component b);
where in the presence of a fusogenic substances) the cationic carrier {b)
must not be lysosomolytic and/or lysosomotropic; and in which component
a) is preferably a polynucleic acid, component b) a cationic protein, a
cationic polymer or a combination of both.
In a further embodiment of the invention, the cationic carrier is protamine
sulfate.
In a further embodiment of the invention, component b) is a cationic
polymer, in particular polyethyleneimine (PEI), particularly preferably PEI
having a molecular weight of on average 2,000 - t 0,000 Da, very
particularly preferably a high-branched-chain or low-branched-chain PEI.
In a further embodiment of the invention, component c) is constructed of
phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine,
anchor lipid, and cholesterol, a particularly preferred anchor lipid is an N-
carboxyphosphatidylethanolamine, e.g. an N-gtutarylphosphatidylethanol-
amine; and component d) is preferably conjugated to one of the
components a} - c) without an anchor, via an anchor or via an anchor lipid.
29/11 'O1 14:58 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOiTDREAU C~J031
CA 02375854 2001-11-30
A further embodiment of the invention is a liposomal vector complex in
which component d) is embedded noncovalently in the liposome surface.
A further embodiment of the invention is a liposomal vector complex whose
target cell is a tissue cell, an epithelial cell, an endothelial cell, a blood
cell,
a leukemia cell or a tumor cell.
A further embodiment of the invention is a liposomal vector complex whose
component e) is the functional sequence from the subunit HA-2 of the
hemagglutin of the influenza virus or a synthetic derivative thereof.
A further embodiment of the invention is a liposomal vector complex for the
transduction and transfection of cells in vitro or in vivo, serum preferably
being used in vitro.
A further embodiment of the invention is the use of a liposomal vector
complex for the production of a diagnostic for use in vitro and in vivo and/or
for the production of a therapeutic for the prophylaxis or therapy of a
disease in vivo and ex vivo, administration preferably taking place on the
skin, on a mucus membrane, in the lung, on the eye, in a body cavity, in the
connective tissue, in the muscle, in an organ or in the blood circulation.
A further embodiment of the invention is a process for the preparation of a
liposomal vector complex, where
{1 ) component a) is mixed with component b),
(2) the complex resulting from step (1 ) is introduced into component
c), the mixing ratio of all components being adjusted such that
the net charge of the resulting overall complex is preferably
either cationic or anionic;
(3) component d) is optionally inserted into component c) before or
after complex formation;
(4) component e) is optionally inserted into the complex resulting
from steps (2) and (3) or into component c) before complex
formation;
the resulting product preferably being lyophilized or aerosolized.
Examples for illustration of the concept of the invention
29/11. '0l 14:59 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOLTDREAU I~j032
CA 02375854 2001-11-30
9
The following examples are intended to illustrate how the present invention
could be carried out.
Example 1:
Modified peptide having an RGD sequence
For the improvement of the targeting of the integrin receptors, a cyclic
peptide was synthesized. It is a CDCRGDCFC peptide (Arap W.,
Pasqualini, R. and Ruoslahti, E. (1998) Science, 279: 377-380; Pasqualini,
R. Koivunen, E. Ruoslathi E., Nature Biotech. (1997) 15:542-546) having
an additional arginine at the N-terminal end (FW 1163.35). The terminal
amino acid reduces the extent of the coupling of the peptide to the active
center, the RGD sequence.
The cyclization takes place by means of oxidation of the thiol group to
disulfide bridges. Successful cyclization is checked by means of HPLC
analysis.
After the HPLC purification, the peptide is lyophilized, stored at
4°C and
dissolved in buffer (250 ~g/150 ~I of tris buffer 10 mM pH 7.4 or PBS buffer
pH 7.4) before use.
Preparation of the liposomes
Material
Substance Manufacturer Batch Parts FW
DAPS Avanti 181 PS-P36a 3 810
Sodium salt
DLPE S ena 0998 3 579.76
CholesterolCalbiochem 228111 3 386.7
N-glut-PE in-house synthesis16118 1 805.97
or Avanti
29/11 'O1 14:59 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~J033
CA 02375854 2001-11-30
. 10
Stock solutions
Substance ConcentrationBatch Vol. Amount of
[p,mol/ml] employed substance
[~I] employed
[~mol]
DOPS 16.13 15128 i 86 3
DLPE 33.47 09039 89.7 3
Cholesterol24.95 09039 120.2 3
N- It-PE 2 16118 500 1
The liposomes are prepared by the film/hydration method. The lipids
dissolved in chloroform and the lipid anchors are pipetted into a 100 ml
round-bottomed flask and the chloroform is stripped off for 15 min. The
flask dips during the course of this into a temperature-controlled water bath
having a temperature which is above the phase-transition temperature of
the lipids, in this specific case 30°C. For the complete removal of the
solvent, the film is dried in a high vacuum for 15 min.
Hydration of the film with buffer follows (tris 10 mM, pH 7.4 or PBS pH 7.4,
other buffers and pHs are likewise possible). The buffer is added to the
flask together with a few small glass beads and the batch is rotated for 45
min with N2 aeration, the flask also dips into the warm water bath at
30°C
here. For the swelling of the lipid film and for the production of
multilamellar liposomes (MLV), the batch is allowed to stand at room
temperature for 2 hours [1, p.38]. The MLV suspension is transferred to a
special sonicator glass vessel and the batch is sonicated for 15 sec by
means of a probe sonicator (here Soniprep 150 (preferably adjusted
amplitude in microns 8-12)). The suspension dips into an ice bath in the
course of this. After the sonication, a pause of 30 sec is inserted for the
cooling of the suspension. This procedure (sonication - pause) is repeated
10 times. The size measurement of the SUV obtained affords a size of 120
to 300 nm. After subsequent extrusion [1, pp.52-56][2] by means of
LiposoFast through a polycarbonate filter, pore size 50 nm, the size in this
example is reduced to a value between 107.5-128 nm. The liposomes thus
prepared are stable for at least 2 months and do not measureably change
their size in this time. The bottling of the liposomes for further processing
is
carried out under an air hood in a sterile Eppendorf cap.
29/7.1 'O1 15:00 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU f~034
CA 02375854 2001-11-30
17
References
[1 ) Roger R.C. New, "Preparation of liposomes", chapter 2, Liposomes a
practical approach (1989)
[2] Olson et al. (1980} Biochim. Biophys. Acta, 394, 483
Coupling of the peptide to lipid anchors (Weissig, V., Lasch, J., Klibanov,
A.L. Torchilin, V.P., A new hydrophobic anchor for the attachment of
proteins to liposomal membranes. FEES Lett. 202, 1986, 86-90; Bogdanov
et al., "Protein immobilization on the surface of liposomes via carbodiimide
activation in the presence of N-hydroxysulfosuccinimide", FEBS Lett. 231,
1988, 381-384; Weissig, Qualifying thesis for university Lecturers
"Methoden zur Darstellung funktionalisierter Liposomen mit Adjuvanseffekt"
[Methods for the preparation of functionalized liposomes having an
adjuvant effect] MLU Halle-Wittenberg (1992); Thesis Ragna Schmidt,
Halle University (1997). [3] Martin et al. "Covalent attachment of protein to
liposomes", chapter 4, Roger R.C. New, Liposomes a practical approach
(1989})
Material
Substance Conc. Batch Solvent Amount Amount
of
mark employed substance
em to ed
Liposomes 10 pmoi/ml12039 Iris 400 w1 4 ~cmol
buffer
H 7.4
EDAC pure 25H0993, solid 3.5 mg 18.26
substanceSi ma pmol
RGD 250 fig/ 22049 tris 150 fc( 0.215
buffer =
solution 150 ~1 H 7.4 250 ~c umol
Firstly, the carboxyl group of the giutaric acid radical of the lipid anchor
is
activated by the addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
[3, p. 170]. For this, 400 ~l of the liposome suspension (4 pmol of total
lipid,
0.4 ~mol of N-glut-PE, medium tris buffer pH 8, 10 mM, other buffers and
pHs are likewise possible) are vortexed and 3.5 mg of EDC are weighed in.
The batch is again vortexed and shaken for 5 hours protected against light
(IKA Vibrax VXR}. The activated intermediate, the O-acyl intermediate, is
formed, to which the peptide having a free amino function binds with
formation of an amide. 250 ~g of RGD dissolved in PBS buffer pH 7.4 or
29/11 '01 15:01 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~J035
CA 02375854 2001-11-30
12
tris buffer 10 mM pH 7.4 (250 ~eg/150 p1, other buffers and pHs are likewise
possible) are added, and the batch is vortexed and shaken overnight, After
coupling has taken place, the unbound peptide is separated from' the
liposomes by gel chromatography. Size exclusion chromatography is
carried out using a Sephadex G 50 column, the eluent is tris buffer 10 mM,
pH 7.4 (other buffers and pHs are likewise possible).
The coupling yield is carried out by means of a simultaneously carried out
batch containing fluorescent dye {5-DTAF)-labeled RGD {Product
information sheet 5-DTAF, Molecular Probes (MP 00143 08/27/95)
"Conjugation with Amine-Reactive Probes") and is at least 6 ~g of RGD/
1 Imo! of PL (calculated for actual amounts of lipid using cholesterol). The
determination of the coupling efficiency can also alternatively be carried out
by means of HPLC (Gyongyossy-Issa et al. "The Covalent Coupling of Arg-
Gly-Asp-Containing Peptides to Liposomes: Purification and Biochemical
Function of the Lipopeptide" Archives of Biochemistry and Biophysics, Vol.
353, No. 1, May 1 (1998)). The size of the liposomes coupled using RGD is
between 100-150 nm. The coupling can be modified according to
Bogdanov in order to increase the coupling efficiency. The coupling can
also be carried out on its own using an anchor lipid according to the
process described by Weissig. The resulting lipid anchor-peptide construct
can be employed, like the lipid, in the preparation of liposomes.
Example 2: Preparation of a liposomal vector complex according to the
invention in the sequence plasmid, liposome, protamine sulfate and PEI
The plasmid was condensed with PEI, which had been prepared according
to the method described by Fischer et al. {Pharm. Res. 16, 7 273-1279,
1999), or using Lupasol (BASF, Ludwigshafen, Germany).
Complex formation is firstly carried out by mixing together the negatively
charged constituents plasmid DNA (pGl3, Clontech, Heidelberg, Germany)
and liposomes (DLPE, D(JPS, cholesterol, N-glutaryl-PE 3:3:3:1 ): In this
process, the dilution of the solutions is to be taken into account in order to
prevent irreversible precipitate formation. The final volume of all
constituents mentioned is 100 ~I. Firstly, the buffer (iris 10 mM, pH 7.4,
other buffers and pHs are likewise possible) is introduced and 10 ~g of
plasmid (10 ~cg/60 p1) and also 40 ~cg of Jiposomes (variable, between 1 and
6 pg/pl) are mixed together by simple pipetting. The mixture is vortexed.
29/11 'O1 15:01 FAX ++49 89 92805444 BARDEHLE OFFICE -. GOUDREAU C~03B
CA 02375854 2001-11-30
13
The condensation of the DNA is then carried out by addition of the cationic
agent, firstly 19.96 ~g of protamine sulfate is added (charge ratio +I-
3.3:1).
For this, the protamine sulfate is added rapidly thereto using an Eppendorf
pipette and the batch is mixed by pipetting to and fro 10 times and a
coating of the complex with the lipids is achieved. 29.7 beg (amounts up to
16.2 ~g are likewise possible) of PEI (NIP ratio 20.7, reduction to 7.5
possible), a further cationic agent, are then added. For this, 33 p.1 of PEI
solution 0.9 mg/ml are diluted with 250 ~,l of high-purity water and added to
the batch in substeps. The addition is carried out in 2 x 100 ~! and 1 x 85 ~I
steps, pipetting the batch to and fro 10 times each after the addition and
waiting for 15 min. A complex of this type has a size of 180-300 nm 1 h
after the preparation and is used immediately after the preparation for the
cell culture experiments. The complexes are stable in the cell culture
medium M199 + 10% FCS used for the transfection (size 360-500 nm).
Example 3: Preparation of a liposomal vector complex according to the
invention in the sequence plasmid, liposome, and PEI
The batch as described in Example 2 can also be prepared without
protamine sulfate. The preparation steps are identical, with the exception of
the addition of 19.96 ug of protamine sulfate. Transfection experiments
show almost identical efficiency.
example 4: Preparation of a liposomal vector complex according to the
irhvention in the sequence plasmid, liposome, fusion peptide, and PEI
T'he preparation is carried out according to the method and sequence as
described in Example 3. In addition, a "fusion peptide", hemagglutinin (HA)
originating from the membrane protein of the influenza virus {Wagner et al.,
Proc. Natl. Acad. Sci. USA 89, 7934-7938, 1992; Smoes & Slepushkin
Gene Therapy 5, 955-964, 1998), is added to the liposomes at the start of
complex formation and the mixture is then used like the pure iiposomes
according to Example 3. The concentration of the WA peptide can be
between 0.1 and 1 nmol (1 - 10 fig) per batch.
Example 5: Preparation of a liposomal vector complex according to the
invention in the sequence plasmid, PEI, liposome, fusion peptide.
29/11 ' O1. 15: 02 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAIT f~ 037
CA 02375854 2001-11-30
- 14
Complex formation can likewise be carried out by condensation of the DNA
by PEI. For this, firstly both substances are mixed together (single addition
or in portions, see above) wait for 15 min. The liposomes are then pipetted
in and the mixture is pipetted to and fro several times {preferably 10 x).
This complex is likewise allowed to stand for 15 min before use. The
amount of liposomal formulation of plasmid, cationic agent and liposomes
needed for the transfection can be reduced to 5 pg of plasmid/3 cm dish. In
addition, the volume of the formulation can be decreased.
Complex formation is firstly carried out by mixing together the constituents
plasmid DNA and PEI. In this process, the dilution of the solutions is to be
taken into account in order to prevent irreversible precipitate formation. The
final volume of the two constituents mentioned is 245.93 ~.1. Firstly, the
buffer (tris 10 mM, pH 7.8, other buffers and pHs are likewise possible) is
introduced and 15 ug of plasmid (15 ~.g/90 ~!) are added thereto. The
condensation of the DNA is then carried out by addition of the cationic
agent, of 44.55 ~g of PEI (N!P ratio 20.7, larger amounts and a reduction to
9.79 ug are likewise possible). For this, 49.5 p1 of PEI solution 0.9 mg/ml
are diluted with 106.43 y1 of high-purity water and added to the batch in
substeps. The addition is carried out in 1 x 100 ~I and 1 x 55.93 u1 steps,
the batch is pipetted to and fro 5 x after the addition and finally pipetted
to
and fro 5 x with a 100 u1 volume, wait for 15 min. 60 ~g of liposomes
(variable, between 1 and 6 ~cg/~I) are mixed with 15 ~g of HA fusion peptide
(reduction of the amount to 0.1 ~cg possible) and added to the plasmidIPEI
complex by simple pipetting. For this, the batch is pipetted to and fro 10 x
with a 100 ~I volume after the addition. The mixture is vortexed. Complexes
of similar activity are also obtained by simple mixing of all components
indicated. After preparation, a complex of this type has a size of 180-250
nm and is used immediately after preparation for the cell culture
experiments. The complexes are stable in the cell culture medium M 199 +
10% FCS used for the transfection (size 300-400 nm).
Example 6: Preparation of a liposomal vector complex according to the
invention in the sequence plasmid, PEf, liposome
The liposomal formulation can be prepared without the HA fusion peptide.
For this, the abovementioned preparation procedure is used and only the
addition of the HA peptide is omitted.
29/11 '01. 15:02 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU f~038
CA 02375854 2001-11-30
_ 15
Example 7: Preparation of a liposomal vector complex according to the
invention in the sequence plasmid, PEI, liposome, with and without HA
peptide, with variable volumes.
Complex formation is firstly carried out by mixing together the constituents
plasmid DNA and PEI. In this process, the dilution of the solutions is to be
taken into account in order to prevent irreversible precipitate formation. The
fins! volume of the two constituents mentioned is, depending on the batch
type: = a) 465 ~I, b) 245.93 ~I, c) 196.4 ~I, d} 150 p1. Firstly, the buffer
(tris
10 mM, pH 7.8, other buffers and pHs are likewise possible) is introduced
and 15 ~g of plasmid (15 ~g/90 ~l or 75 u1) added thereto. The
condensation of the DNA is then carried out by addition of the cationic
agent, of 44.55 beg of PEI (N/P ratio 20.7, larger amounts and a reduction to
9.79 fxg are likewise possible). For this, 49.5 u! of PEI solution 0.9 mg/ml
are diluted with high-purity water (325.5, 106.43, 5fi.9, 25.5 ~I) and added
to the batch in substeps. The addition is carried out in 100 ;~I and/or
variable (55.9, 75, 106 u1) steps, after the addition the batch is pipetted to
and fro 5 x each and finally pipetted to and fro 5 x with a 100 ~l volume,
wait for 15 min. so ug of liposomes (variable, between 1 and 6 ~gipl) are
mixed with 15 pg of HA fusion peptide (reduction of the amount to 0.1 ~g
possible). This addition of the HA fusion peptide is optional, it can be
omitted without a relatively large decrease in transfection. The liposomes
are added to the plasmid/PEI complex by simple pipetting. For this, the
batch is pipetted to and fro 10 x after the addition with a 100 u1 volume.
The mixture is vortexed. Complexes of similar activity are also obtained by
simple mixing of all components indicated. After preparation, a complex of
this type has a size of 180-250 nm and is used immediately after
preparation for the cell culture experiments. The complexes are stable in
the cell culture medium M199 + 10% FCS used for the transfection (size
300-400 nm).
Example 8: Preparation of a Iiposomal vector complex according to the
invention in the sequence plasmid, protamine sulfate, ~PEl~and liposome.
Complex formation is firstly carried out by mixing together the constituents
plasmid DNA and protamine sulfate. In this process, the dilution of the
solutions is to be taken into account in order to prevent irreversible
precipitate formation. The final volume of the two constituents mentioned is,
depending on the batch type: = a) 369 p1, b) 320 ~I. Firstly, the buffer (tris
29/1.1 'O1 15:03 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU ~ 039
CA 02375854 2001-11-30
16
mM, pH 7.8, other buffers and pHs likewise possible) is introduced and
p.g of plasmid (15 ug/90 ~I) added thereto. The condensation of the DNA
is then carried out by addition of the protamine sulfate {+/- ratio 3.3,
larger
or smaller amounts are likewise possible). Protamine sulfate is diluted with
5 high-purity water {29.94 pg/105 ~I) and added to the DNA, the batch being
pipetted to and fro 10 x. The complex is allowed to stand for 15 min for
maturation. 49.5 -~I of PEI solution 0.9 mg/ml are then diluted with high-
purity water (106.43, 56.9 1u1) and added to the batch in substeps. The
addition is carried out in 700 p! and/or 55.9 p1 steps, after the addition the
10 batch is pipetted to and fro 5 x each and finally pipetted to and fro 5 x
with
a 100 p1 volume, wait for 15 min. 60 pg of liposomes (variable, between 1
and 6 ~g/~I) are mixed with 15 mg of HA fusion peptide {reduction of the
amount to 0.1 pg possible). This addition of the HA fusion peptides is
optional, it can be omitted without a relatively large increase in
transfection.
15 The liposomes are added to the plasmid/PS/PEI complex by simple
pipetting. For this, the batch is pipetted to and fro 10 x after the addition
with a 100 u1 volume. The mixture is vortexed. Complexes of similar activity
are also obtained by simple mixing of ail components indicated. After
preparation, a complex of this type has a size of 180-300 nm and is used
immediately after preparation for the cell culture experiments. The
complexes are stable in the cell culture medium M199 + 10% FCS used for
the transfection (size 300-500 nm). These complexes can be prepared with
other charge ratios, it being possible to vary both the proportion of the PS
and of the PEI. !n this case, larger or smaller volumes can likewise be
chosen.
Example 9: Preparation of a liposomai vector complex according to the
invention in the sequence plasmid, protamine sulfate, PEI and liposome
having a reduced PEI content.
Complex formation is firstly carried out by mixing together the constituents
plasmid DNA and protamine sulfate. In this process, the dilution of the
solutions is to be taken into account in order to prevent irreversible
precipitate formation. The final volume of the two constituents mentioned is,
depending on the batch type: = 319 p1. Firstly, the buffer (tris 10 mM, pH
7.8, olher buffers and pHs likewise possible) is introduced and 15 ~,g of
plasmid {15 pgI90 ~I) are added. The condensation of the DNA is then
carried out by addition of the protamine sulfate (+I- ratio 3.3, larger or
smaller amounts are likewise possible). Protamine sulfate is diluted with
29/1.1 ' Ol 1.5 : 04 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOiIDREAI1 l~J
040
CA 02375854 2001-11-30
_ '! 7
high-purity water (29.94 pg/105 ~I) and added to the DNA, the batch being
pipetted to and fro 10 x. The complex is allowed to stand for t 5 min for
maturation. 30 ~! (NIP ratio 12.5) or 18 ~I (NIP ratio 7.5) of PEI solution
0.9 mg/ml are then diluted to 106 ~I with high-purity water (76 or 88 pf) and
added to the batch in substeps. The addition is carried out in 106 p1 steps,
after the addition pipette the batch to and fro 10 x each, wait for 15 min. 60
pg of liposomes (variable, between 1 and 6 pgl~l) are mixed with 15 ~g of
HA fusion peptide (reduction of the amount to 0.1 ~.g possible). This
addition of the HA fusion peptides is optional, it can be omitted without a
relatively large decrease in transfection. The liposomes are added to the
plasmid/PSIPEI complex by simple pipetting. For this, the batch is pipetted
to and fro 10 x with a 100 p1 volume after addition. The mixture is vortexed.
Complexes of similar activity are also obtained by simple mixing of all
components indicated.
Example 10: Preparation of a liposomal vector complex according to the
invention in the sequence plasmid, PEI, protamine sulfate and liposome.
Complex formation is firstly carried out by mixing togefher the constituents
plasmid DNA and PEI. !n this process, the dilution of the solutions is to be
taken into account in order to prevent irreversible precipitate formation. The
final volume of the two constituents mentioned is, depending on the batch
type: = 246 Vii. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and
pHs
likewise possible) is introduced and 15 ~g of plasmid (15 pg/90 ~~I) are
added thereto. The condensation of the DNA is Then carried out by addition
of 44.55 pg of PEI (NIP ratio 20.7, larger amounts and a reduction are
likewise possible). For this, 49.5 p1 of PEI solution 0.9 mg/ml are diluted
with high-purity water (to 155.93 p1) and added to the batch in substeps.
The addition is carried out in 100 ~I and 55.9 ,~l steps, after the addition
the
batch is pipetted to and fro 5 x each and finally pipetted to and fro 5 x with
a 100 p1 volume, wait for 15 min. The protamine sulfate (+/- ratio 3.3, larger
or smaller amounts are likewise possible) is then added. Protamine sulfate
is diluted with high-purity water (29.94 ~g/105 ~I) and added to the DNA,
the batch being pipetted to and fro 10 x. The complex is allowed to stand
for 15 min for maturation. 60 ug of liposomes (variable, between 1 and 6
~,g/~I) are mixed with 15 ~g of HA fusion peptide (reduction of the amount
to 0.1 ~g possible). This addition of the HA fusion peptides is optional, it
can be omitted without a relatively large decrease in transfection. The
liposomes are added to the plasmid/PEI/PS complex by simple pipetting.
29/11 'O1 15:05 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOUDREAU C~j041
CA 02375854 2001-11-30
18
For this, the batch is pipetted to and fro i0 x after the addition with a 100
~I
volume. The mixture is vortexed. Complexes of similar activity are also
obtained by simple mixing of all components indicated.
Example 11: Preparation of a iiposomal vector complex according to the
invention in the sequence plasmid, PE1, protamine sulfate and liposome
having a reduced PEI content.
Complex formation is firstly carried out by mixing together the constituents
plasmid DNA and PEI. In this process, the dilution of the solutions is to be
taken into account in order to prevent irreversible precipitate formation. The
final volume of the two constituents mentioned is, depending on the batch
type: = 196 u1. Firstly, the buffer (tris 10 mM, pH 7.8, other buffers and pHs
likewise possible} is introduced and 15 pg of plasmid (15 ug/90 yi) are
added. The condensation of the DNA is then carried out by addition of
9.75 fig, 16.2 ~g or 27 ~g of PEI (N/P ratio 4.5, 7.5, 12.5, larger amounts
and a reduction are likewise possible, e.g. N/P ratio 1.8). For this , 10.86
~I
18 ~I or 30 ~l of PEI solution 0.9 mg/ml are diluted with high-purity water
(to
106 pI) and added to the batch while pipetting to and fro 10 x. The
protamine sulfate is then added (+/- ratio 3.3, larger or smaller amounts are
likewise possible}. Protamine sulfate is diluted with high-purity water
(29.94 ~g1105 ~.I) and added to the DNA/PEI complex, the batch being
pipetted to and fro 10 x. The complex is allowed to stand for 15 min for
maturation. 60 pg of liposomes (variable, between 1 and 6 ~g/~,I) are mixed
with 15 ~g of HA fusion peptide (reduction of the amount to O.i ~g
possible). This addition of the HA fusion peptides is optional, it can be
omitted without a relatively large decrease in transfection. The liposomes
are added to the plasmid/PEI/PS complex by simple pipetting. For this,
after the addition the batch is pipetted to and fro 10 x with a 100 ~l volume.
The mixture is vortexed. Complexes of similar activity are also obtained by
simple mixing of all components indicated.
Example 12: Preparation of a liposomal vector complex according to the
invention in the sequence plasmid, PEI and liposome having an increased
and reduced lipid content
Complex formation is firstly carried out by mixing together the constituents
plasmid DNA and PEI. In this process, the dilution of the solutions is to be
taken into account in order to prevent irreversible precipitate formation. The
29/11 'O1 15:08 FAX ++49 89 92805444 BARDEHLE OFFICE ~ GOLTDREALT f~j042
CA 02375854 2001-11-30
i9
final volume of the two constituents mentioned is 245.93 ~l. Firstly, the
buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is
introduced and 15 pg of plasmid (15 pgI90 p1) are added thereto. The
condensation of the DNA is then carried out by addition of the cationic
agent, 44.55 pg of PEl (NIP ratio 20.7, larger amounts and a reduction are
likewise possible). For this, 49.5 p1 of PEI solution 0.9 mglml are diluted
with 106.43 ~l of high-purity water and added to the batch in subsieps. The
addition is carried out in 1 x 100 fd and 1 x 55.93 ~l steps, after the
addition
the batch is pipetted to and fro 5 x each and finally pipetted to and fro 5 x
with a 100 ~d volume, wait for 15 min. 75, 45 and 30 ~g of liposomes
(between 1 and 6 pglpl) are added to the plasmidIPEI complex by simple
pipetting. For this, the batch is pipetted to and fro 10 x after the addition
with a 100 p1 volume. The content of the liposomes used can moreover be
markedly increased to 10 x the amount.
Example 13: Biological testing of the liposomal vector complexes according
to the invention in various cell cultures
Complex formation is firstly carried out by mixing together the constituents
plasmid DNA and PEI. In this process, the dilution of the solutions is to be
taken into account in order to prevent irreversible precipitate formation. The
final volume of the two constituents mentioned is 245.93 p1. Firstly, the
buffer (tris 10 mM, pH 7.8, other buffers and pHs likewise possible) is
introduced and 15 ~g of plasmid (15 ug/90 ~l) are added thereto. The
condensation of the DNA is then carried out by addition of the cationic
agent, 44.55 pg PEl (NIP ratio 20.7, larger amounts and a reduction are
likewise possible). For this, 49.5 ~1 of PEI solution 0.9 mg/rnf are diluted
with 106.43 w1 of high-purity water and added to the batch in substeps. The
addition is carried out in 1 x 100 p! and 1 x 55.93 ~I steps, after the
addition
the batch is pipetted to and fro 5 x and finally pipetted to and fro 5 x with
a
100 u1 volume, wait for 15 min. 60 ~g of liposomes (between 1 and 6 pg/~l)
with and without coupled RGD targeter (RGD binds to the a"~ receptor)
are added to the plasmid/PEI complex by simple pipetting. For this, the
batch is pipetted to and fro 10 x after the addition with a 100 p1 volume.
The batch is divided into 3 aliquots and one aliquot each is added to a 3 cm
dish. The triplicate batch was added to a 10 cm dish for FRCS analysi .
The liposomal vector complexes, prepared as in Examples 1-11, were
added to the cells (in 10 cm dishes for FACS analysis; 3 cm dishes for
luciferase and GFP microscopy) and incubated at 37°C for 1-6 hours.
29/11 'O1 ---15:06 FAX ++49 89 92805444 BARDEHLE OFFICE -~ GOUDREAU ~ 043
CA 02375854 2001-11-30
Subsequently, the cells were washed and incubated for a further 24-48
hours in fresh cell culture medium. The successful absorption of the
complexes into the cells, the transcription, and the expression of the
reporter gene in the plasmid by the defection of the GFP autofluorescence,
5 luciferase assay, and FACS analysis were then measured. Results of the
FACS analysis are compiled in the following table.
f Cells + RGD - RGD
HUVEC rima endothelial cells 73 29
MeWo melanoma cells 66 5
MSM melanoma cells 37 12
HMB-2 melanoma cells 30 7
B254 melanoma cells 10 4
DX-3 melanoma cells 11 3
Saos-2 osteosarcoma cells 15 28
DU-145 rostate carcinoma cells 1 1
pC3 rostate carcinoma cells 95 21
HeLa cervical carcinoma cells 4 33
LoVo colon carcinoma cells 8 1
A549 lun carcinoma cells 23 11
MCF-7 breast cancer cells ~3 34
'
JEG-3 chorionic carcinoma cells ~4
