Note: Descriptions are shown in the official language in which they were submitted.
CA 02406654 2002-10-18
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METHODS OF ENHANCING SPLP-MEDIATED TRANSFECTION
USING ENDOSOMAL MEMBRANE DESTABILIZERS
CROSS-REFERENCES'TO RELATED APPLICATIONS
This patent application claims the benefit of U.S. Provisional Patent
Application No. 60/227,949, which was filed August 25, 2000, U.S. Patent
Application No.
09/553,639, which was filed April 20, 2000, and PCT Patent Application No. CA
00/00451,
which was filed April 20, 2000, the teachings of both of which axe
incorporated herein by
reference.
BACKGROUND OF THE INVENTION
An effective' and safe gene delivery system is required for gene therapy to be
clinically useful. Viral vectors are relatively efficient gene delivery
systems, but suffer from
a variety of limitations, such as the potential for reversion to the wild type
as well as immune
response concerns. As a result, nonviral gene delivery systems are receiving
increasing
attention (Worgall, et al., Human Gehe Therapy 8:37-44 (1997); Peeters, et
al., Human Gene
Therapy 7:1693-1699 (1996); Yei, et al., Gene Therapy 1:192-200 (1994); Hope,
et al.,
Molecular' Membrane Biology 15:1-14 (1998)). Plasmid DNA-cationic liposome
complexes
axe currently the most commonly employed nonviral gene delivery vehicles
(Felgner,
Scientific American 276:102-106 (I997); Chonn, et al., Cu~~ent Opinion in
Biotechnology
6:698-708 (1995)). However, complexes are large, poorly defined systems that
are not suited
for systemic applications and can elicit considerable toxic side effects
(Harrison, et al.,
Biotech~ciques 19:816-823 (1995); Huang, et al., Nature Biotechnology 15:620-
621 (1997);
Templeton, et al., Nature Biotechnology 15:647-652 (1997); Hofland, et al.,
Pharmaceutical
Research 14:742-749 (1997)).
Recent work has shown that plasmid DNA can be encapsulated in small
(~70 nm diameter) "stabilized plasmid-lipid particles" (SPLP) that consist of
a single plasmid
encapsulated within a bilayer lipid vesicle (Wheeler, et al., Gehe Therapy
6:271-281 (1999)).
These SPLPs typicaly contain the "fusogenic" lipid
dioleoylphosphatidylethanolamine
(DOPE), low levels of cationic lipid, and are stabilized in aqueous media by
the presence of a
polyethylene glycol) (PEG) coating. SPLP have systemic application as they
exhibit
extended circulation lifetimes following intravenous (i.v.) injection,
accumulate preferentially
at distal tumour sites due to the enhanced vascular permeability in such
regions, and can
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
mediate transgene expression at these tumour sites. The levels of transgene
expression
observed at the tumour site following i.v. injection of SPLP containing the
luciferase marker
gene are superior to the levels that can be achieved employing plasrnid DNA-
cationic
liposome complexes (lipoplexes) or naked DNA. Still, improved levels of
expression may be
required for optimal therapeutic benefit in some applications (see, e.g.,
Monck, et al., J. Drug
Targ. 7:439-452 (2000)).
Cationic polyethylene glycol) (PEG) lipids, or CPLs, have been designed for
insertion into lipid bilayers to impart a positive charge(see, Chen, et al.,
Bioconj. Chem.
11:433-437 (2000)). For example, CPL containing distearoyl-PE (DSPE) coupled
to PEG
containing one or more distal positive charges were synthesized, and shown to
promote
enhanced i~ vitro cellular binding and uptake of liposomes (Chen, et al.,
Biocohj. Chem.
11:433-7 (2000)).
Thus, there remains a strong need in the art for novel and more efficient
methods for introducing nucleic acids into cells. The present invention
addresses this and
other needs.
SUMMARY OF THE INVENTION
The present invention provides effective compositions, methods and uses for
delivering nucleic acids to cells. The inventive compositions and methods are
based upon the
surprising discovery that the presence of an endosomal membrane destabilizer
in a lipid
formulation leads to a dramatic increase in transfection efficiency. The
present compositions
and methods can be used in vitro or in vivo, and can be used to increase the
transfection
efficiency of any cell type, including mammalian cells (e.g., human).
As such, in one embodiment, the present invention provides a nucleic acid-
lipid particle composition for introducing a nucleic acid into a cell
comprising: a cationic
lipid, a conjugated lipid that inhibits aggregation of particles, a nucleic
acid and an
endosomal membrane destabilizer. In preferred aspects, the nucleic acid-lipid
particles are
"stabilized plasmid-lipid particles" (SPLP). Typically, SPLP are less than 150
nm in
diameter and comprise a single plasmid encapsulated within a bilayer lipid
vesicle. The
conjugated lipid that inhibits aggregation typically comprises a hydrophilic
polymer. In
preferred embodiments, the hydrophilic polymer is a PEG or polyamide (e.g.,
ATTA) having
a molecular weight of about 250 to about 7000 daltons. The endosomal membrane
destabilizer can be inside the particle, outside the particle, or both inside
and outside the
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CA 02406654 2002-10-18
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particle. Preferably, the endosomal membrane destabilizer is Cap ion. In
certain aspects, the
concentration of Ca ion is from about 0.1 mM to about 100 mM.
In certain embodiments, the conjugated lipid that inhibits aggregation is a
"cationic polymer lipid" (CPL). In preferred aspects, the CPL has the formula
A W Y
In Formula I, A is a lipid moiety, W is a hydrophilic polymer; and Y is a
polycationic moiety.
In certain preferred embodiments, Y is selected from lysine, arginine,
asparagine, glutamine,
and combinations thereof.
In another embodiment, the present invention provides a method for
introducing a nucleic acid into a cell, comprising contacting the cell with a
nucleic acid-lipid
particle composition, wherein the particle comprises a cationic lipid, a
conjugated lipid that
inhibits aggregation of particles, a nucleic acid; and an endosomal membrane
destabilizer.
The endosomal membrane destabilizer can be inside the particle, outside the
particle, or both
inside and outside the particle. In certain embodiments, the endosomal
membrane
destabilizer contacts the cell before the particle, after the particle,
simultaneously or
combinations thereof.
In still another embodiment, the present invention provides a method for
inducing HII phase structure in a lipid bilayer, comprising contacting the
lipid bilayer with an
endosomal membrane destabilizer, thereby inducing HB phase structure in the
lipid bilayer.
In certain aspects, the endosomal membrane destabilizer (e.g., Cap ion) acts
synergistically
or additively with low levels of the cationic lipid to trigger Ha phase
formation.
The present compositions, methods and uses offer numerous advantages. For
example, the presence of an endosomal membrane destabilizer leads to a
dramatic increase in
the transfection efficiency of nucleic acids. By increasing transfection
efficiency, the amount
of gene product within the cell is greatly increased. Moreover, the present
compositions and
methods can be used ih vitro or in vivo, and can be used to increase the
transfection efficiency
of any cell type, including human.
These and other advantages, objects and embodiments of the present
invention, will be described in more detail in conjunction with the following
figures and the
detailed description.
CA 02406654 2002-10-18
w0 ol/so9oo BEEF DESCRIPTION OF THE DRAWINGS P~T/CAOl/oosss
Figure 1. Effect and specificity of Ca2~ on SPLP transfection. Increasing
concentrations of CaCl2 ( ~ ), MgCl2 ( ~ ), or NaCI ( ~ ) (0 to 14 mM) were
titrated into SPLP
prior to their addition to cells. 0.5 ~,g of pCMVLuc plasmid encapsulated in
SPLP
(DODAC/DOPE/PEG-CerC20; 7:83:10 mol/mol/mol) vesicles was used to transfect
cells
plated at 1 x 104 cells/well of 96-well plates. Cells were incubated with SPLP
for 24 h, and
Luc activity was measured as described in Materials and Methods, Exampl I. All
experiments were performed in triplicate.
Figure 2. Effect of Ca2+ on the cellular uptake of SPLP. SPLP containing 0.5
mol% Rd-labeled DOPE (DODAC/DOPE/PEG-CerC20/Rh-DOPE; 7:82.5:I0:0.5
mol/mol/mol/mol) were employed to monitor cellular lipid uptake. 80 nmoles of
lipid
vesicles prepared in the presence of Ca2+ (0 to 14 mM) were incubated on cells
until the
appropriate time periods. Levels of lipid uptake were determined by measuring
Rd
fluorescence at 4 h ( ~ ), 8 h ( ~ ), or 24 h ( ~ ) as described in Materials
and Methods,
Example I. All experiments were performed in triplicate.
Figure 3. Fluorescence micrographs of the cellular localization of SPLP. 100
nmoles of 4 mol% Rh-DOPE labeled vesicles were incubated on cells (plated at
1x105 cells
per well of a 12-well plate) in the absence (A) or presence (B) of calcium (10
mM). At 8 h
time point, transfecting media was replaced with complete DMEM media and cells
were
examined using fluorescence microscopy. Fluorescence micrographs were talcen
on an
Axiovert 100 Zeiss Fluorescence microscope (Caxl Zeiss Jena GmbH) using a
rhodamine
filter from Omega Opticals (Brattleboro, VT) with the following
specifications, ~,eX 560 ~ 20
nm, 600 nm LP, and DC 590 nm.
Figure 4. Intracellular processing of plasmid DNA was affected by the
presence of Ca2+. SPLP containing 2.5 ~.g plasmid DNA was used to transfect
BHI~ cells in
the absence ( t ) or presence ( ~ ) of 8 mM Caa+ as described in Materials and
Methods,
Example I. At appropriate time points (2 h, 4 h, and 8 h), DNA was extracted
from the cells
and intracellular DNA was detected by hybridization to a specific 32P-labeled
plasmid DNA
probe. (A) Levels of plasmid DNA uptake determined by dot blot analysis as
described in
Materials and Methods. (B) Integrity of intracellular plasmid DNA determined
by Southern
blot analysis. Lanes 1 and 11: pCMVLuc control; lanes 2, 5, 8 and 12:
untransfected
control; lanes 3, 6, 9 and 13: cells transfected with SPLP; lanes 4, 7, I O
and 14: cells
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transtected with SPLP and 8 mM Ca ; and lane 15: cells transfected with ~YLY
and ~ mM
Mg2+. All experiments were performed in triplicate.
Figure 5. 31P NMR spectra of various model membrane systems in the
presence of CaCl2. (A) Ca2+ was titrated into the vesicles
(DOPE/DOPS/DOPC/Chol,
1:1:1:3) at Ca2+/DOPS ratios ranging from 0:1 to 0.5: I (molar ratios). (B)
Ca2+ was titrated
into the vesicles (DOPE/DOPS/DOPC/Ghol/DODAC, 1:1:1:3:0.25) at Ca2+/DOPS
ratios
ranging from 0:1 to 0.25:1 (molar ratios). Equilibration of the canons across
the lipid
bilayers was ensured by three cycles of freeze-thawing. Spectra have been
scaled to the same
peals height. Experiments were carried out as described in Materials and
Methods, Example
I.
Figure 6. Effect of Ca2+-containing SPLP on transfection. Ca2+ was loaded
employing A23187 in the presence of a pH gradient as described in Materials
and Methods.
Increasing concentrations of Ca2+ (0 to 14 mM) were added to both SPLP (~) and
Ca2+-
containing SPLP (~) prior to DMEM dilution. 0.5 ~.g of pCMVLuc plasmid
encapsulated in
SPLP were used to transfect cells plated at 1 x 104 cells/well of 96-well
plates. Luc activity
was measured as described in Materials and Methods, Example I. All experiments
were
performed in triplicate.
Figure 7. Effect of Ca2+ on improved SPLP systems. SPLP containing higher
DODAC content (14 mol%) or CPL (4 mol%) were used to transfection cells in the
presence
(dashed bars) or absence (open bars) of 8 mM Ca2+. 0.5 ~,g of pCMVLuc was used
in each
formulation in each transfection experiment. Cells were exposed to the
vesicles for 24 h
before assaying for Luc expression, as outlined in Materials and Methods,
Example I.
Experiments were performed in triplicate.
Figure 8. Production of SPLP-CPL4. A. Structure of dansylated CPL4. CPL4
possesses four positive charges at the end of a PEG34oo molecule attached to a
lipid achor,
DSPE. B. Protocol for insertion of CPL4 into preformed SPLP. The SPLP and CPL4
are
incubated together at 60°C for 3 h, and unincorporated CPL4 is removed
using Sepharose CL-
4B column chromatography. For further details see Materials and Methods,
Example II.
Figure 9. Time course for the insertion of CPL4 into SPLP at 60°C.
Dansylated CPL4 (0.3 pmol) was added to SPLP composed of 6 p,mol DOPE:PEG-
CerCao:DODAC:Rh-PE (83.5:10:6:0.5; mol%) containing 360 ~g pCMVLuc in a total
volume of 1.5 mL and incubated at 60°C. Aliquots (250 ~,L) of the
mixtuxe were taken at the
times indicated and unincorporated CPL4 was removed employing Sepharose CL-4B
column
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
chromatography. CPL4 incorporation was determined as described in Materials
and Methods,
Example II.
Figure 10. Effect of cation concentration on the de-aggregation of SPLP
following insertion of CPL4. SPLP were prepared and 4 mol% CPL4 was inserted
as
described in Materials and Methods, Example II. The mean diameter and standard
deviation
of the mean diameter of the SPLP-CPL4 in the presence of increasing
concentrations of Ca2+
(~) and Mg2+ (~) was determined by QELS. CaCl2 or MgCl2 from 500 mM stock
solutions
was added to SPLP-CPL4 (180 nmol in 400 ~.L). The addition of Ca2+ or Mg2+
results in a
more monodisperse preparation as indicated by a reduction in the standard
deviation of the
mean diameter at cation concentrations above 30 mM.
Figure 11. Freeze-fracture electron micrographs of (A) SPLP, (B) SPLP-
CPL4 and (C) SPLP-CPL4 in the presence of 40 mM CaCl2. The SPLP-CPL4 were
prepared
as described in Materials and Methods, Example II, and contained 4 mol% CPL4.
The bar in
plate A corresponds to 200 nm.
Figure 12. Serum stability of SPLP-CPL4 as assayed by Southern analysis of
encapsulated plasmid. SPLP were prepared as indicated in the legend to Figure
9 and 4
mol% of CPL4 inserted using the post-insertion protocol. SPLP-CPL4 containing
5 ~g
pCMVLuc were incubated in the presence of 50% mouse serum at 37°C for
the times
indicated, an aliquot of the mixture corresponding to 1 ~,g of plasmid DNA was
removed and
plasmid DNA was extracted and subjected to Southern analysis, as described in
the Materials
and Methods. Lanes 1-4 indicate the behaviour of naked plasmid DNA following
0, 1, 2, and
4 h incubation times respectively; lanes 5-8 indicate the behaviour of plasmid
extracted from
SPLP following 0, l, 2, and 4 h incubation times; and lanes 9-12 show the
behaviour of
plasmid DNA extracted from SPLP containing 4 mol% CPL4 following 0, 1, 2, and
4 h
incubation times.
Figure 13A. Influence of the amount of CPL4 incorporated into SPLP on the
uptake of SPLP-CPL4 into BHK cells. Uptake of SPLP containing 0 (~), 2 (~), 3
(1), or 4
(1) mol% CPL4 was investigated; the uptake of DOPE:DODAC lipoplexes (O) is
given for
comparison. The insertion of CPL4 into SPLP and the preparation of lipoplexes
was
performed as described in Materials and Methods, Example II. The SPLP-CPL4
media
contained 40 mM CaCl2 to prevent aggregation, addition to the BHK cells
resulted in dilution
of the CaCl2 concentration to 8 mM. The uptake protocol involved incubation of
SPLP-CPL4
(20 ~M total lipid) with 105 BHK cells in DMEM containing 10% FBS. Following
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
incubation, the cells were lysed and uptake of rhodamine-PE was measured as
described in
Materials and Methods, Example II. B. Fluorescence micrographs of BHK cells
following
uptake of SPLP (Panel I) and SPLP containing 4 mol% CPL4 (Panel II) following
a 4 h
incubation. The micrographs on the left were taken in the phase contrast mode
and those on
the right in the (rhodamine) fluorescence mode.
Figure 14. Luciferase expression in BHK cells following transfection by
SPLP containing various amounts of CPL4. SPLP containing 2, 3 and 4 mol% CPL4
were
prepared employing the post-insertion process. BHK cells (104) were
transfected with SPLP,
SPLP-CPL4 and DOPE:DODAC (1:l) lipoplexes containing 5.0 ~g/mL pCMVLuc using a
transfection time of 4 h and a complete incubation time of 24 h, as described
in Materials and
Methods, Example II. The CaCl2 concentration in the SPLP-CPL4-containing
systems
following dilution with media and addition to the BHK cells was 8 mM. After
transfection
the cells were lysed and the luciferase and BCA assays performed as described
in Materials
and Methods.
Figure 15. Influence of Ca2+ (~) and Mg2+ (~) on the transfection potency of
SPLP-CLP4. SPLP-CPL4 containing 4 mol% CPL4 were prepared by the post-
insertion
process as described in Materials and Methods, Example II. Increasing
concentrations of
CaCl2 or MgCl2 were added to the SPLP-CPL4 (5.0 pg pCMVLuc/mL), transferred to
BHK
cells and incubated for 48 h in DMEM containing 10% FBS. The cells were then
lysed and
the luciferase activity and protein content were measuxed as described in
Materials and
Methods, Example II.
Figure 16. Effect of Ca2+ (~) and Mg2+ (~) on the uptake of SPLP-CPL4 by
BHK cells. SPLP-CPL4 were prepared with increasing cation concentrations as
indicated for
Figure 8 and incubated with BHK cells (~80 ~,M lipid and ~5.0 ~g pCMVLuc/mL
per well)
for 4 h in DMEM containing 10% FBS. The cells were then lysed and the SPLP-
CPL4
content (as indicated by the Rh-PE lipid label) and cellular protein measured
as described in
Materials and Methods, Example II.
Figure 17. Luciferase expression in BHK cells as a function of transfection
time for SPLP, SPLP-CPL4 and lipoplexes. SPLP-CPL4 containing 4 rnol% CPL4
were
prepared by the post-insertion process. BHK cells in DMEM and 10% FBS were
incubated
with SPLP, SPLP-CPL4 and lipoplexes (5.0 ~g/mL pCMVLuc) employing transfection
times
of 4, 8 and 24 h and total incubation times of 24 h. The f nal CaCl2
concentration following
addition of media was 8 mM. The cells were then assayed for luciferase
activity and protein
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WO 01/80900 PCT/CA01/00555
content. Luciferase activity following transfection with SPLP-CPL4 (~), SPLP
(1),
DOPE:DODAC lipoplexes (~), and Lipofectin lipoplexes (1) is plotted as a
function of
transfection time. Lipoplexes were prepared at a charge ratio of 1.5:1.
Figure 18A. The transfection potency of SPLP-CPL4 (~) containing 4 mol%
S CPL4 and and Lipofectin lipoplexes (~) following extended transfection times
with BHK
cells. SPLP-CPL4 and lipoplexes were generated as indicated for Figure 10. BHK
cells were
transfected in DMEM containing 10% FBS for 24 and 48 h with SPLP-CPL4 and
Lipofectin
lipoplexes (charge ratio of 1.S:1) containing S.0 ~g/mL pCMVLuc. Following
transfection
the luciferase expression levels and cell protein Levels were determined in
the cell lysate. The
luciferase activity was normalized for protein content in the lysate and
plotted as a function
of transfection time. B. The toxicity of SPLP-CPL4 (~) containing 4 mol% CPL4
and and
Lipofectin lipoplexes (~) as a function of transfection time, as assayed by
cell survival based
on the protein concentration in the cell lysate.
Figure 19. Fluorescence and phase contrast micrographs of BHK cells
1 S transfected with SPLP-CPL4 and lipoplexes containing a plasmid coding for
GFP. Cells were
transfected with SPLP-CPL4 for 24 h (A1, A2) and 48 h (B1, B2) and with
lipofectin for 24 h
(C1, C2). SPLP and lipoplexes were prepared with pCMVGFP as described in
Materials and
Methods, Example II. SPLP-CPL4 containing 4 mo1% CPL was prepared by the post-
insertion process and contained CaCl2, resulting in an 8 mM CaCl2
concentration in the
transfection medium. BHK cells (105) were incubated with SPLP-CPL4 or
Lipofectin (S.0
~g/mL) in DMEM containing 10% FBS for the 24 and 48 h transfection times and
examined
immediately after the transfection period.
Figure 20. Mechanism for disruption of cellular membranes mediated by
cationic lipoplexes. Following binding (Step 1) and endocytosis (Step 2) into
a target cell,
2S cationic lipoplexes are transferred to late endosomal compartments (Step
3). Cationic lipids
induce destabilization of the endosomal membrane leading to fusion (Step 4) of
the lipoplex
with the endosomal membrane, or complete remodeling of the endosomal membrane
into a
nonbilayer phase (Step S).
Figure 21. A synthetic scheme for the preparation of cationic-PEG-lipid
conjugates having varying amount of charged head groups (a.) Et3N/CHC13; (b.)
TFA
/CHC13; c. Et3N / CHCI3 Na, Ns-di-t-Boc-L-Lysine N-hydroxysuccinide ester.
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DETAILED DESCRIPTION OF THE INVENTION
AND PREFERRED EMBODIMENTS
I. Introduction
The present invention provides novel and surprisingly effective compositions
and methods for delivering nucleic acids to Bells. These compositions and
methods are based
upon the discovery that the presence of an endosomal membrane destabilizes
(e.g., calcium)
leads to a dramatic increase in the transfection efficiency of nucleic acids
(e.g., plasmids)
formulated as SPLP or "stabilized nucleic acid (e.g., plasmid)-lipid
particles." Typically,
SPLP are less than about 150 nm in diameter (more preferably about 70 nm in
diameter) and
consist of a single plasmid encapsulated within a bilayer lipid vesicle.
As used herein, the term "endosomal membrane destabilizes" (EMD) refers to
an agents) that is believed to facilitate the disruption or destabilization of
the endosomal
membrane thereby enhancing the release of their contents. Endosomes are
typically distinct
intracellular compartments isolated from the rest of the cell by a selectively
permeable
membrane. Suitable EMDs include, but are not limited to, monovalent metal ions
such as
K+, Na~~, divalent metal ions such as Mg2+, Ca2+, Mn2+, Co2+, and combinations
of the metal
ions with cationic lipids. The most preferred EMD is Ca2+ ion wherein
approximately 106
times higher transfection efficiency is observed for SPLPs containing Ca2+
ions than SPLPs in
the absence of Ca2+ ions.
The present methods can be used in vitro or in vivo, and can be used to
increase the transfection efficiency of any cell type, including mammalian
cells. For
example, for in vitro transfection, an endosomal membrane destabilizes (e.g.,
calcium) can be
added to the transfection medium. For instance, any of a wide range of calcium
concentrations can be used, ranging, for example, from 0.1 mM to 100 mM.
Preferably, from
about 1 mM to about 20 mM is used, most preferably from about 8 to about 10
mM. In one
embodiment, the endosomal membrane destabilizes (e.g., calcium) is first added
to the SPLP
at a high concentration which will give rise to a desired final concentration
following the
dilution of the SPLP into the transfection medium. In other embodiments, the
endosomal
membrane destabilizes (e.g., calcium) is added to the SPLP at the time of
transfection into the
cells. The endosomal membrane destabilizes can be co-aehninistered with the
SPLP, it can be
administered prior to the administration of the SPLP or it can be administered
after the
administration of the SPLP.
Ih vivo, any method can be used that will result in a local increase of the
endosomal membrane destabilizes (e.g., calcium) concentration at the site of
transfection.
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WO 01/80900 PCT/CA01/00555
For example, particles can be formulated to incorporate the endosomal membrane
destabilizes, particles can be soaked in a solution containing a high
endosomal membrane
destabilizes (e.g., calcium) concentration prior to administration, or the
particles can be
administered in a buffer or formulation containing a high endosomal membrane
destabilizes
(e.g., calcium) concentration. Such methods are especially useful for the
local delivery of
particles, e.g., intratumoral injection, where the co-administration of, e.g.,
calcium ions, can
produce a locally high calcium concentration, thereby leading to enhanced
transfection of the
particles into cells at or near the site of delivery. Again, the endosomal
membrane
destabilizes can be co-administered with the SPLP, it can be administered
prior to the
administration of the SPLP or it can be administered after the administration
of the SPLP.
In certain ih vivo or i~ vitro embodiments, the SPLP are formulated to include
on their surface chelating molecules for chelating the endosomal membrane
destabilizes, e.g.,
lipids derivatized with a endosomal membrane destabilizes chelator, thereby
allowing the
generation of a locally high endosomal membrane destabilizes concentration
even following
systemic delivery of the particles. For instance, in certain ih vivo or i~c
vitro embodiments,
particles are formulated to include calcium chelating molecules on the
surface, e.g., lipids
derivatized with a calcium chelator, thereby allowing the generation of a
locally high calcium
concentration even following systemic delivery of the particles.
Any SPLP particle can be used to practice the present invention. For example,
SPLP comprising any of a broad range of concentrations of cationic and other
lipids can be
used. Similarly, the SPLP can comprise any of a wide variety of cationic and
other lipids.
The SPLP can be prepared with any plasmid, from any source and comprising any
polynucleotide sequence, and can be prepared using any of a large number of
methods.
The present invention also provides SPLP containing cationic PEG lipids,
called SPLP-CPL. In a preferred embodiment, SPLP-CPL4 is used, comprising a
PEG lipid
having four positive charges. SPLP and SPLP-CPL can be derivatized to include
any of a
number of functional groups, including, but not limited to, calcium chelators,
cell or tissue-
specific targeting molecules, labels, and others.
Suitable SPLP and SPLP-CPL for use in the present invention, and methods of
malting and using SPLP and SPLP-CPL, are taught, e.g., in U.S. Application
Nos. 60/130,151
and 09/553,639, as well as in PCT International Application PCT/CA00/00451,
the teachings
of each of which is incorporated herein in its entirety by reference.
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II. ~etinitions
The term "lipid" refers to a group of organic compounds that include, but are
not limited to, esters of fatty acids and are characterized by being insoluble
in water, but
soluble in many organic solvents. They are usually divided into at least three
classes: (1)
"simple lipids' which include fats and oils as well as waxes; (2) "compound
lipids" which
include phospholipids and glycolipids; (3) "derived lipids" such as steroids.
The term "vesicle-forming lipid" is intended to include any amphipathic lipid
having a hydrophobic moiety and a polar head group, and which by itself can
form
spontaneously into bilayer vesicles in water, as exemplified by most
phospholipids.
The term "vesicle-adopting lipid" is intended to include any amphipathic lipid
which is stably incorporated into lipid bilayers in combination with other
amphipathic lipids,
with its hydrophobic moiety in contact with the interior, hydrophobic region
of the bilayer
membrane, and its polar head group moiety oriented toward the exterior, polar
surface of the
membrane. Vesicle-adopting lipids include lipids that on their own tend to
adopt a
nonlamellar phase, yet which are capable of assuming a bilayer structure in
the presence of a
bilayer-stabilizing component. A typical example is DOPE
(dioleoylphosphatidylethanolamine). Bilayer stabilizing components include,
but are not
limited to, conjugated lipids that inhibit aggregation of the SPLPs, polyamide
oligomers (e.g.,
ATTA-lipid derivatives), peptides, proteins, detergents, lipid-derivatives,
PEG-lipid
derivatives such as PEG coupled to phosphatidylethanolamines, and PEG
conjugated to
ceramides (see, U.S. Application Serial No. 08/485,608, now U.S. Patent No.
5,885,613,
which is incorporated herein by reference).
The teen "amphipathic lipid" refers, in part, to any suitable material wherein
the hydrophobic portion of the lipid material orients into a hydrophobic
phase, while the
hydrophilic portion orients toward the aqueous phase. Amphipathic lipids are
usually the
major component of a lipid vesicle. Hydrophilic characteristics derive from
the presence of
polar or charged groups such as carbohydrates, phosphato, carboxylic, sulfato,
amino,
sulfhydryl, nitro, hydroxy and other like groups. Hydrophobicity can be
conferred by the
inclusion of apolar groups that include, but are not limited to, long chain
saturated and
unsaturated aliphatic hydrocarbon groups and such groups substituted by one or
more
aromatic, cycloaliphatic or heterocyclic group(s). Examples of amphipathic
compounds
include, but axe not limited to, phospholipids, aminolipids and sphingolipids.
Representative
examples of phospholipids include, but are not limited to,
phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid,
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CA 02406654 2002-10-18
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palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine,
disteaxoylphosphatidylcholine or dilinoleoylphosphatidylcholine. Other
compounds lacking
in phosphorus, such as sphingolipid, glycosphingolipid families,
diacylglycerols and (3-
acyloxyacids, are also within the group designated as amphipathic lipids.
Additionally, the
amphipathic lipid described above can be mixed with other lipids including
triglycerides and
sterols.
The term "neutral lipid" refers to any of a number of lipid species that exist
either in an uncharged or neutral zwitterionic form at a selected pH. At
physiological pH,
such lipids include, for example, diacylphosphatidylcholine,
diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and
diacylglycerols.
The term "hydrophopic lipid" refers to compounds having apolar groups that
include, but are not limited to, long chain saturated and unsaturated
aliphatic hydrocarbon
groups and such groups optionally substituted by one or more aromatic,
cycloaliphatic or
heterocyclic group(s). Suitable examples include, but are not limited to,
diacylglycerol,
dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane and 1,2-
dialkyl-3-
aminopropane.
The term "diacylglycerolyl" denotes 2-fatty acyl chains, Rl and R2 having
independently between 2 and 30 caxbons bonded to the 1- and 2-position of
glycerol by ester
linkages. The acyl groups can be saturated or have varying degrees of
unsaturation.
The term "dialkylglycerolyl" denotes two C1-C3o alkyl chains bonded to the l-
and 2-position of glycerol by ether linkages.
The term "N-N-dialkylamino" denotes
~Ci Cso a~'1
N
\Ci C3o a~'1
The term "1,2-diacyloxy-3-aminopropane" denotes 2-fatty acyl chains C1-C3o
bonded to the 1- and 2-position of propane by an ester linkage. The acyl
groups can be
saturated or have varying degrees of unsaturation. The 3-position of the
propane molecule
has a -NH- group attached. 1,2-diacyloxy-3-aminopropanes have the following
general
formula:
12
CA 02406654 2002-10-18
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CH20 \R1
O
CH-O~ RZ
CHzN-
The term "1,2-dialkyl-3-aminopropane" denotes 2-alkyl chains (C1-C3o)
bonded to the 1- and 2-position of propane by an ether linkage. The 3-position
of the propane
molecule has a -NH- group attached. 1,2-dialkyl-3-aminopropanes have the
following
general formula:
CHZO Cl-Cso-A~'1
CH-O Cl-C3o-Allcyl
CHzN-
The term "noncationic lipid" refers to any neutral lipid as described above as
well as anionic lipids. Examples of anionic lipids include, but are not
limited to,
phosphatidylglycerol, caxdiolipin, diacylphosphatidylserine,
diacylphosphatidic acid, N-
dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-
glutarylphosphatidylethanolamines, lysophosphatidylglycerols, and other
anionic modifying
groups joined to neutral lipids.
The term "cationic lipid" refers to any of a number of lipid species that
carry a
net positive charge at a selected pH, such as physiological pH. Such lipids
include, but are
not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC"); N-(2,3-
dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ("DOTMA"); N,N-distearyl-
N,N-
dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N-
trimethylammonium chloride ("DOTAP"); 3 -(N-(N',N'-dimethylaminoethane)-
carbamoyl)cholesterol ("DC-Chol") and N-(1,2-dimyristyloxyprop-3-yl)-N,N-
dimethyl-N-
hydroxyethyl ammonium bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids are available which can be used in the present
invention.
These include, for example, LIPOFECTIN~ (commercially available cationic
liposomes
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compmsmg I~OTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine ("DOPE"), from
GIBCO/BRL, Grand Island, New York, USA); LIPOFECTAMINE~ (commercially
available cationic liposomes comprising N-(1-(2,3-dioleyloxy)propyl)-N-(2-
(sperminecaxboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA")
and("DOPE"), from GIBCO/BRL); and TRANSFECTAM~ (commercially available
cationic
lipids comprising dioctadecylamidoglycyl carboxyspermine ("DOGS") in ethanol
from
Promega Corp., Madison, Wisconsin, USA). The following lipids are cationic and
have a
positive charge at below physiological pH: DODAP, DODMA, DMDMA and the like.
The term "fusogenic" refers to the ability of a liposome, an SPLP or other
drug delivery system to fuse with membranes of a cell. The membranes can be
either the
plasma membrane or membranes surrounding organelles, e.g., endosome, nucleus,
etc.
Fusogenesis is the fusion of a liposome to such a membrane.
The term "dendrimer" includes reference to branched polymers that possess
multiple generations. In dendrimers, each generation creates multiple branch
points.
The teen "ligand" includes any molecule, compound or device with a reactive
functional group and includes lipids, amphipathic lipids, carrier compounds,
chelating
moities, bioaffinity compounds, biomaterials, biopolymers, biomedical devices,
analytically
detectable compounds, therapeutically active compounds, enzymes, peptides,
proteins,
antibodies, immune stimulators, radiolabels, fluorogens, biotin, drugs,
haptens, DNA, RNA,
polysaccharides, liposomes, virosomes, micelles, immunoglobulins, functional
groups,
targeting agents, or toxins. The foregoing list is illustrative and not
intended to be
exhaustive.
The term "ATTA" or "polyamide" refers to, but is not limited to, compounds
disclosed in U.S. Patent Application No. 09/218,988, filed December 22, 1998.
These
compounds include a compound having the formula
R1 O R2
R N (CH2CH20)m (CH2)p C-(NH-H C)q R3
O n
wherein: R is a member selected from the group consisting of hydrogen, alkyl
and acyl; R1 is
a member selected from the group consisting of hydrogen and alkyl; or
optionally, R and Rl
and the nitrogen to which they axe bound form an azido moiety; R2 is a member
of the group
selected from hydrogen, optionally substituted alkyl, optionally substituted
aryl and a side
chain of an amino acid; R3 is a member selected from the group consisting of
hydrogen,
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halogen, hydroxy, allcoxy, mercapto, hydrazino, amino and NR4R5, wherein 1Z
and RJ are
independently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4; and q
is 0 or 1. It will
be apparent to those of skill in the art that other polyamides can be used in
the compounds of
the present invention.
As used herein, the term "alkyl" denotes branched or unbranched hydrocarbon
chains, such as, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-
butyl, tertbutyl,
octa-decyl and 2-methylpentyl. These groups can be optionally substituted with
one or more
functional groups which are attached commonly to such chains, such as,
hydroxyl, bromo,
fluoro, chloro, iodo, mercapto or thio, cyano, alkylthio, heterocyclyl, aryl,
heteroaryl,
carboxyl, carbalkoyl, alkyl, alkenyl, vitro, amino, alkoxyl, amido, and the
like to form alkyl
groups such as trifluoromethyl, 3- hydroxyhexyl, 2-carboxypropyl, 2-
fluoroethyl,
carboxymethyl, cyanobutyl and the like.
The term "allcylene" refers to a divalent alkyl as defined above, such as
methylene (-CHZ-), propylene (-CH2CH2CH2-), chloroethylene (-CHCICH2-), 2-
thiobutene (-
CHZCH(SH)CH2CH2-), 1-bromo-3-hydroxyl-4-methylpentene
(-CHBrCH2CH(OH)CH(CH3)CHZ-), and the like.
The term "alkenyl" denotes branched or unbranched hydrocarbon chains
containing one or more carbon-carbon double bonds.
The term "alkynyl" refers to branched or unbranched hydrocarbon chains
containing one or more carbon-carbon triple bonds.
The term "aryl" denotes a chain of carbon atoms which form at least one
aromatic ring having preferably between about 6-14 carbon atoms, such as
phenyl, naphthyl,
indenyl, and the like, and which may be substituted with one or more
functional groups
which are attached commonly to such chains, such as hydroxyl, bromo, fluoro,
chloro, iodo,
mercapto or thio, cyano, cyanoamido, alkylthio, heterocycle, aryl, heteroaryl,
carboxyl,
carbalkoyl, alkyl, alkenyl, vitro, amino, alkoxyl, amido, and the like to form
aryl groups such
as biphenyl, iodobiphenyl, methoxybiphenyl, anthryl, bromophenyl, iodophenyl,
chlorophenyl, hydroxyphenyl, methoxyphenyl, formylphenyl, acetylphenyl,
trifluoromethylthiophenyl, trifluoromethoxyphenyl, alkylthiophenyl,
trialkylammoniumphenyl, amidophenyl, thiazolylphenyl, oxazolylphenyl,
imidazolylphenyl,
imidazolylmethylphenyl, and the like.
The term "acyl' denotes the -C(O)R group, wherein R is alkyl or aryl as
defined above, such as formyl, acetyl, propionyl, or butyryl.
The term "alkoxy" denotes -OR-, wherein R is alkyl.
CA 02406654 2002-10-18
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The term "amido"denotes an amide linkage: -C(O)NR- (wherein K is
hydrogen or all~yl).
The term "amino" denotes an amine linkage: -NR-, wherein R is hydrogen or
alkyl or a terminal NH2,
S The term "carboxyl" denotes the group -C(O)O-, and the term "carbonyl"
denotes the group -C(O)-.
The term "carbonate" indicates the group -OC(O)O-.
The term "carbamate" denotes the group -NHC(O)O-, and the term "urea"
denotes the group -NHC(O)NH-.
The term "phosphoro" denotes the group -OP(O)(OH)O-.
The term "basic amino acid" refers to naturally-occurring amino acids as well
as synthetic amino acids and/or or amino acid mimetics having a net positive
charge at a
selected pH, such as physiological pH. This group includes, but is not limited
to, lysine,
arginine, asparagine, glutamine, histidine and the like.
1 S The term "phosphorylethanolamino" denotes the group
-OP(O)(OH)OCH2CH2NH-.
The term "phosphorylethanolamido" denotes the group
-OP(O)(OH)OCH2CH2NHC(O)-.
The term "phospho" denotes a pentavalent phosphorous moiety -P(O)(OH)O-.
The term "phosphoethanolamino" denotes the group
-P(O)(OH)OCH2CH2NH-.
The term "phosphoethanolamido" denotes the group
-P(O)(OH)OCH2CH2NHC(O)-.
The term "ethylene oxide unit" denotes the group -OCH2CH2-.
2S The term "CPL" refers to a cationic-polymer-lipid, e.g., cationic-PEG-
lipid.
Preferred CPLs are compounds of Formulae I and II. Such CPLs are disclosed in
U.S. Patent
Application No. 09/SS3,639, which was filed April 20, 2000, and PCT Patent
Application
No. CA 00/00451, which was filed April 20, 2000 and which published as WO
00/62813 on
October 26, 2000.
The term "d-DSPE-CPL-M" is encompassed by the term "CPL1" which refers
to a DSPE-CPL having one positive charge. The "d-" in d-DSPE-CPL-M indicates
that the
CPL contains a fluorescent dansyl group. It will be apparent to those of skill
in the art that a
CPL can be synthesized without the dansyl moiety, and thus the term "DSPE-CPL-
M" is
encompassed by in the term "CPL1" as defined above.
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The term "d-DSPE-CPL-D" is encompassed by the term "CPL2" which refers
to DSPE-CPL having two positive charges.
The term "d-DSPE-CPL-Tl" is encompassed by the term "CPL3" which
refers to DSPE-CPL having three positive charges.
The term "d-DSPE-CPL-Ql" is encompassed by the term "CPL4a" which
refers to DSPE-CPL having four positive charges.
The term "d-DSPE-CPL-Q5," or, alternatively, DSPE-PEGQuadS, or,
alternatively, DSPE-CPL-4, are all encompassed by the term "CPL4 (or CPL4b)"
which refer
to a DSPE-CPL having four positive charges. By modifying the headgroup region,
CPLs
were synthesized which contained 1 (mono, or M), 2 (di, or D), 3 (tri, or T),
and 4 (quad, or
Q) positive charges. Various Quad CPLs were synthesized, hence these are
numbered Q1
through Q5.
The abbreviations "HBS" refers to Hepes-buffered saline, "Rho-PE" refers to
rhodamine-phosphatidylethanolamine, and "LUVs" refers to "large unilamellar
vesicles."
II. Nucleic Acid-Lipid Particles (SPLPs and Properties Thereof
The nulceic acid-lipid particles or, alternatively, SPLPs typically comprise
cationic lipid and nucleic acids. Such SPLPs also preferably comprise
noncationic lipid and a
bilayer stabilizing component or, more preferably, a conjugated lipid that
inhibits aggregation
of the SPLPs. The SPLPs of the present invention have a mean diameter of less
than about
150 nm and are substantially nontoxic. In addition, the nucleic acids when
present in the
SPLPs of the present invention are resistant to aqueous solution to
degradation with a
nuclease. Such SPLPs are disclosed in great detail in U.S. Patent No.
5,976,567 and PCT
Patent Publication No. WO 96/40964, the teachings of both of which are
incoporated herein
by reference.
A. SPLP Components
Various suitable cationic lipids may be used in the present invention, either
alone or in combination with one or more other cationic lipid species or
neutral lipid species.
Cationic lipids wluch are useful in the present invention can be any of a
number of lipid species which carry a net positive charge at physiological pH,
for example:
DODAC, DOTMA, DDAB, DOTAP, DOSPA, DOGS, DC-Chol and DMRIE, or
combinations thereof. A number of these lipids and related analogs, which are
also useful in
the present invention, have been described in co-pending USSN 08/316,399; U.S.
Patent Nos.
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
S,ZU~,U36, 5,264,618, 5,279,833 and 5,283,185, the disclosures of which are
incorporated
herein by reference. Additionally, a number of commercial preparations of
cationic lipids are
available and can be used in the present invention. These include, for
example,
LIPOFECTIN° (commercially available cationic liposomes comprising DOTMA
and DOPE,
from GIBCO/BRL, Grand Island, New York, USA); LIPOFECTAMINE°
(commercially
available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and
TR.ANSFECTAM~ (commercially available cationic liposomes comprising DOGS from
Promega Corp., Madison, Wisconsin, USA).
The noncationic lipids used in the present invention can be any of a variety
of
neutral uncharged, zwitterionic or anionic lipids capable of producing a
stable complex.
They are preferably neutral, although they can alternatively be positively or
negatively
charged. Examples of noncationic lipids useful in the present invention
include:
phospholipid-related materials, such as lecithin, phosphatidylethanolamine,
lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
sphingomyelin,
cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate,
dioleoylphosphatidylcholine (DOPC), dipalmitoyl-phosphatidylcholine (DPPC),
dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-
phosphatidylethanolamine (DOPE), palmitoyloleoy-lphosphatidylcholine (POPC),
palmitoyloleoyl- phosphatidylethanolamine (POPE) and dioleoyl-
phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal). Additional
nonphosphorous containing lipids are, e.g., stearylamine, dodecylamine,
hexadecylamine,
acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl
myristate, amphoteric
acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate
polyethyloxylated fatty
acid amides, dioctadecyldimethyl ammonium bromide and the like,
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,
sphingomyelin,
cephalin, and cerebrosides. Other lipids such as lysophosphatidylcholine and
lysophosphatidylethanolamine may be present. Noncationic lipids also include
polyethylene
glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene glycol
conjugated to
phospholipids or to ceramides (referred to as PEG-Cer), as described in co-
pending USSN
08/316,429, incorporated herein by reference.
In preferred embodiments, the noncationic lipids are
diacylphosphatidylcholine (e.g., dioleoylphosphatidylcholine,
dipalmitoylphosphatidylcholine and dilinoleoylphosphatidylcholine),
diacylphosphatidylethanolamine (e.g., dioleoylphosphatidylethanolamine and
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palmitoyloleoylphosphatidylethanolamine), ceramide or sphingomyelin. ~he acyl
groups m
these lipids are preferably acyl groups derived from fatty acids having Clo-
Cz4 carbon chains.
More preferably the acyl groups are lauroyl, myristoyl, palmitoyl, stearoyl or
oleoyl. In
particularly preferred embodiments, the noncationic lipid will be 1,2-sn-
dioleoylphosphatidylethanolamine, or egg sphingomyelin (ESM).
In one embodiment, the SPLP further comprises a bilayer stabilizing
component (BSC). Suitable BSCs include, but are not limited to, polyamide
oligomers,
peptides, proteins, detergents, lipid-derivatives, PEG-lipids such as PEG
coupled to
phosphatidylethanolamine, and PEG conjugated to ceramides (see, U. S. Patent
No.
5,885,613, which is incorporated herein by reference). Preferably, the bilayer
stabilizing
component is a PEG-lipid, or an ATTA-lipid. In a presently preferred
embodiment, the BSC
is a conjugated lipid that inhibits aggregation of the SPLPs. Suitable
conjugated lipids
include, but are not limited to PEG-lipid conjugates, ATTA-lipid conjugates,
cationic-
polymer-lipid conjugates (CPLs) or mixtures thereof. In a presently preferred
embodiment,
the SPLPs comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate
together with a
CPL.
The CPLs used in the present invention have the following architectural
features: (1) a lipid anchor, such as a hydrophobic lipid, for incorporating
the CPLs into the
lipid bilayer; (2) a hydrophilic spacer, such as a polyethylene glycol, for
linking the lipid
anchor to a cationic head group; and (3) a polycationic moiety, such as a
naturally occurring
amino acid, to produce a protonizable cationic head group. As such, the
present invention
provides a compound of Formula I:
A W Y I
wherein A, W and Y are as follows.
With reference to Formula I, "A" is a lipid moiety such as an amphipathic
lipid, a neutral lipid or a hydrophobic lipid that acts as a lipid anchor.
Suitable lipid examples
include vesicle-forming lipids or vesicle adopting lipids and include, but are
not limited to,
diacylglycerolyls, dialkylglycerolyls, N-N-dialkylaminos, 1,2-diacyloxy-3-
aminopropanes
and 1,2-dialkyl-3-aminopropanes.
"W" is a polymer or an oligomer, such as a hydrophilic polymer or oligomer.
Preferably, the hydrophilic polymer is a biocompatable polymer that is
nonimmunogenic or
possesses low inherent immunogenicity. Alternatively, the hydrophilic polymer
can be
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CA 02406654 2002-10-18
WO 01/80900 , PCT/CA01/00555
weamy antigenic if used with appropriate adjuvants. Suitable nonimmunogemc
polymers
include, but are not limited to, PEG, polyamides, polylactic acid,
polyglycolic acid, polylactic
acid/polyglycolic acid copolymers and combinations thereof. In a preferred
embodiment, the
polymer has a molecular weight of about 250 to about 7000 daltons.
"Y" is a polycationic moiety. The term polycationic moiety refers to a
compound, derivative, or functional group having a positive charge, preferably
at least 2
positive charges at a selected pH, preferably physiological pH. Suitable
polycationic
moieties include basic amino acids and their derivatives such as arginine,
asparagine,
glutamine, lysine and histidine; spermine; spennidine; cationic dendrimers;
polyamines;
polyamine sugars; and amino polysaccharides. The polycationic moieties can be
linear, such
as linear tetralysine, branched or dendrimeric in structure. Polycationic
moieties have
between about 2 to about 15 positive charges, preferably between about 2 to
about 12 positive
charges, and more preferably between about 2 to about 8 positive charges at
selected pH
values. The selection of which polycationic moiety to employ may be determined
by the type
of liposome application which is desired.
The charges on the polycationic moieties can be either distributed around the
entire liposome moiety, or alternatively, they can be a discrete concentration
of charge
density in one particular area of the liposome moiety e.g., a charge spike. If
the charge
density is distributed on the liposome, the charge density can be equally
distributed or
unequally distributed. All variations of charge distribution of the
polycationic moiety are
encompassed by the present invention.
The lipid "A," and the nonimmunogenic polymer "W," can be attached by
various methods and preferably, by covalent attachment. Methods known to those
of skill in
the art can be used for the covalent attachment of "A" and "W." Suitable
linkages include,
but are not limited to, amide, amine, carboxyl, carbonate, carbamate, ester
and hydrazone
linkages. It will be apparent to those skilled in the art that "A" and "W"
must have
complementary functional groups to effectuate the linlcage. The reaction of
these two groups,
one on the lipid and the other on the polymer, will provide the desired
linkage. For example,
when the lipid is a diacylglycerol and the terminal hydroxyl is activated, for
instance with
NHS and DCC, to form an active ester, and is then reacted with a polymer which
contains an
amino group, such as with a polyamide (see, U.S. patent Application No.
09/218,988, filed
December 22, 1998), an amide bond will form between the two groups.
In certain embodiments, "W" is bound, preferably covalently bound, to "Y".
As with "A" and "W", a covalent attachment of "W" to "Y" can be generated by
CA 02406654 2002-10-18
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complementary reactivity of functional groups, one on the polymer and the
other on the
polycationic moiety. For example, an amine functional group on "W" can be
reacted with an
activated carboxyl group, such as an acyl chloride or NHS ester, to form an
amide. By
suitable choice of reactive groups, the desired coupling can be obtained.
Other activated
carboxyl groups include, but are not limited to, a carboxylic acid, a
carboxylate ester, a
carboxylic acid halide and other activated forms of carboxylic acids, such as
a reactive
anhydride. Reactive acid halides include for example, acid chlorides, acid
bromides, and acid
fluorides.
In certain instances, the polycationic moiety can have a ligand attached, such
as a targeting ligand or a chelating moiety for complexing calcium.
Preferably, after the
ligand is attached, the cationic moiety maintains a positive charge. In
certain instances, the
ligand that is attached has a positive charge. Suitable ligands include, but
are not limited to,
a compound or device with a reactive functional group and include lipids,
amphipathic lipids,
carrier compounds, bioaffinity compounds, biomaterials, biopolymers,
biomedical devices,
analytically detectable compounds, therapeutically active compounds, enzymes,
peptides,
proteins, antibodies, immune stimulators, radiolabels, fluorogens, biotin,
drugs, haptens,
DNA, RNA, polysaccharides, liposomes, virosomes, micelles, immunoglobulins,
functional
groups, other targeting moieties, or toxins. Suitable chelating moieties for
chelating or
complexing the endosomal membrane destabilizer are described below.
In certain preferred embodiments, other moieties are incorporated into the
compounds of Formula I to form the compounds of Formula II:
A X (CH2 CH2 O)" Z Y
II
In Formula II, "A" is a lipid moiety such as, an amphipathic lipid, a neutral
lipid or a
hydrophobic lipid moiety. Suitable lipid examples include, but are not limited
to,
diacylglycerolyl, dialkylglycerolyl, N-N-dialkylamino, ~1,2-diacyloxy-3-
aminopropane and
1,2-dialkyl-3-aminopropane.
In Formula II, "X" is a single bond or a functional group that covalently
attaches the lipid to at least one ethylene oxide unit. Suitable functional
groups include, but
are not limited to, phosphatidylethanolamino, phosphatidylethanolamido,
phosphoro,
phospho, phosphoethanolamino, phosphoethanolamido, carbonyl, carbamate,
carboxyl,
carbonate, amido, thioamido, oxygen, NR wherein R is a hydrogen or alkyl group
and sulfur.
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In certain instances, the lipid "A" is directly attached to the ethylene oxide
unit by a single
bond. The number of ethylene oxide units can range from about 1 to about 160
and
preferably from about 6 to about 50.
In Formula II, "Z" is a single bound or a functional group that covalently
attaches the ethylene oxide unit to the polycationic moiety. Suitable
functional groups
include, but are not limited to, phospho, phosphoethanolamino,
phosphoethanolamido,
carbonyl, carbamate, carboxyl, amido, thioamido, NR wherein R is a member
selected from
the group consisting of hydrogen atom ox alkyl group. In certain embodiments,
the terminal
ethylene oxide unit is directly attached to the polycationic moiety.
In Formula II, "Y" is a polycationic moiety as described above in connection
with Formula I. In Formula II, the index "n" is an integer ranging in value
from about 6 to
about 160.
In an illustrative embodiment, compounds of Formula II can be synthesized
using a generalized procedure as outlined in Figure 21. Figure 21 illustrates
one particular
embodiment of the present invention and thus, is merely an example that should
not limit the
scope of the claims herein. Clearly, one of ordinary skill in the art will
recognize many other
variations, alternatives, and modifications that can be made to the reaction
scheme illustrated
in Figure 21. With reference to Figure 21, a solution of a lipid, such as
DSPE, and a base,
such as triethylamine in a chloroform solution is added to (t-Boc-NH-PEG34oo-
C~2NHS), and
the solution is stirred at ambient temperature. The solution is then
concentrated under a
nitrogen stream to dryness. The residue is then purified by repeated
precipitation of the
chloroform mixture solution with diethyl ether until disappearance of the
lipid using
chromatography. The purified CPL conjugate is dissolved in a solvent, followed
by addition
of TFA, and the solution is stirred at room temperature. The solution can
again be
concentrated under a nitrogen stream. The residue is then purified by repeated
precipitation
of the mixture with diethyl ether to offer a lipid-PEG-NH2, such as a DSPE-PEG-
NH2 or,
alternatively, DSPE-CPL-1 with one protonizable cationic head group. The ratio
of the
phosphoryl-lipid anchor and the distal primary amine can then be measured by
phosphate and
flourescamine assays as described herein.
In this illustrative embodiment, the number of protonizable amino groups can
be increased to create a polycationic moiety. By incrementally adding
stoichiometric
amounts of, for example, a Noc,Ns-di-t-Boc-L-Lysine N-hydroxysuccinide ester,
the
polycationic moiety can be increase from about 2 to about 16 positive charges.
As describe
22
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
previously, the positive charges can be incorporated using any number of
suitable
polycationic moieties such as lysine, arginine, asparagine, glutamine,
histidine, polyamines
and derivatives or combinations thereof. Using the synthesis methods of the
present
invention, the number of cationic groups, such as amino groups, can be readily
controlled
during the CPL synthesis.
In addition, as explained above, the endosomal membrane destabilizer can be
incorporated into the nucleic acid-Iipid particle. In such embodiments. the
endosomal
membrane destabilizer can be loaded into the nucleic-acid lipid particle using
any of a
number of different loading techniques (see, Examples I and II). Exemplar
loading methods
are disclosed, for example, in U.S. Patent No. 4,885,172, U.S. Patent No.
5,059,421, and U.S.
Patent No. 5,171,578, the teachings of which are incorporated herein by
reference. In
addition, a particularly preferred ionophore-mediated loading process is
disclosed and
claimed in U.S. Patent No. 5,837,282, the teachings of which are incorporated
herein by
reference.
Moreover, as explained above, a chelating moiety suitable for chelating the
endosomal membrane destabilizer can be attached, linked or coupled to any of
the lipid
components of the SPLP, such as the CPL. In a presently preferred embodiment,
the
chelating moiety is a metal chelator. Metal chelators, such as
ethylenediaminetetxaacetic acid
(EDTA), diethylenetriaxninepentaacetic acid (DTPA),
ethylenebis(oxyethylenenitrilo)-
tetraacetic acid (EGTA), I,4,7,10-tetraazacyclododecane-N,N',N",N"'-
tetraacetic acid, trans-
1,2-cyclohexylenediamine-N,N,N',N'-tetraacetic acid, N6-carboxymethyl- N3, N9 -
[2,3-
dihydroxy-N-methylpropylcarbamoylmethyl]-3,6,9-triazaundecanedioic acid, N6-
carboxymethyl- N3, N9 -bis (methylcarbamoylmethyl)-3,6,9-triazaundecanedioic
acid, N3,
N6-bis (carboxymethyl)- N9-3-oxapentamethylene-carbamoylmethyl-3,6,9-
triazaundecanedioic acid or N3, N6-bis (carboxymethyl)- N9[3,3-bis
(dihydroxyphosphoryl)-
3-hydroxypropyl-carbamoylmethyl]-3,6,9-triazaundecan edioic acid, metal ion
transporters,
metal ion transport proteins, metal sequesters, metal chelate ligands, and the
like can be used
to chelate the endosomal membrane destabilizer. In addition, the metal
chelators disclosed
in U.S. Patent No. 5,876,695, which is incorporated herein by reference, can
also be used.
Other chelators suitable for use in the compositions and_methods of the
present invention
witll be known to those of skill in the art.
2. Nucleic Acid Component
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
While the invention is described in the examples with reference to the use of
plasmids, one of skill in the art will understand that the methods described
herein are equally
applicable to other larger nucleic acids or oligonucleotides. As such,
suitable nucleic acids
include, but are not limited to, plasmids, antisense oligonucleotides,
ribozymes as well as
other poly- and oligo-nucleotides.
The nucleic acids which are useful in the present invention (including both
the
complexes and particles) are typically nucleotide polymers having from 10 to
100,000
nucleotide residues. Typically, the nucleic acids are to be administered to a
subject for the
purpose of repairing or enhancing the expression of a cellular protein.
Additionally, the
nucleic acid can carry a label (e.g., radioactive label, fluorescent label or
colorimetric label)
for the purpose of providing clinical diagnosis relating to the presence or
absence of
complementary nucleic acids. Accordingly, the nucleic acids, or nucleotide
polymers, can be
polymers of nucleic acids including genomic DNA, cDNA, mRNA or
oligonucleotides
containing nucleic acid analogs, for example, the antisense derivatives
described in a review
by Stein, et al., Sciev~ce 261:1004-l0I 1 (1993) and in U.S. Patent Nos.
5,264,423 and
5,276,019, the disclosures of which are incorporated herein by reference.
Still further, the
nucleic acids may encode transcriptional and translational regulatory
sequences including
promoter sequences and enhancer sequences.
The nucleotide polymers can be single-stranded DNA or RNA, or double-
stranded DNA or DNA-RNA hybrids. Examples of double-stranded DNA include
structural
genes, genes including control and termination regions, and self replicating
systems such as
plasmid DNA.
Single-stranded nucleic acids include antisense oligonucleotides
(complementary to DNA and RNA), ribozymes and triplex-forming
oligonucleotides. In
order to increase stability, some single-stranded nucleic acids will
preferably have some or all
of the nucleotide linlcages substituted with stable, nonphosphodiester
linkages, including, for
example, phosphorothioate, phosphorodithioate, phosphoroselenate, or O-alkyl
phosphotriester linkages.
The nucleic acids used in the present invention will also include those
nucleic
acids in which modifications have been made in one or more sugar moieties
and/or in one or
more of the pyrimidine or purine bases. Examples of sugar modifications
include
replacement of one or more hydroxyl groups with halogens, alkyl groups,
amines, azido
groups or functionalized as ethers or esters. Additionally, the entire sugar
may be replaced
with sterically and electronically similar structures, including aza-sugars
and carbocyclic
24
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
sugar analogs. Modifications in the purine or pyrimidine base moiety include,
for example,
allcylated purines and pyrimidines, acylated purines or pyrimidines, or other
heterocyclic
substitutes known to those of skill in the art.
Multiple genetic sequences can be also be used in the present methods. Thus,
the sequences for different proteins may be located on one strand or plasmid.
Nonencoding
sequences may be also be present, to the extent that they are necessary to
achieve appropriate
expression.
The nucleic acids used in the present method can be isolated from natural
sources, obtained from such sources as ATCC or GenBanlc libraries or prepared
by synthetic
methods. Synthetic nucleic acids can be prepared by a variety of solution or
solid phase
methods. Generally, solid phase synthesis is preferred. Detailed descriptions
of the
procedures for solid phase synthesis of nucleic acids by phosphite-triester,
phosphotriester,
and H-phosphonate chemistries are widely available. See, for example, Itakura,
U.S. Patent
No. 4,401,796; Caruthers, et al., U.S. Patent Nos. 4,458,066 and 4,500,707;
Beaucage, et al.,
Tetrahedron Lett., 22:1859-1862 (1981); Matteucci, et al., J. Am. Chem.
Soc.,103:3185-
3191 (1981); Caruthers, et al., Genetic Ercgir~eering, 4:1-17 (1982); Jones,
chapter 2,
Atkinson, et al., chapter 3, and Sproat, et al., chapter 4, in
Oligorcucleotide Synthesis: A
Practical Approach, Gait (ed.), IRL Press, Washington D.C. (1984); Froehler,
et al.,
Tetrahedron Lett., 27:469-472 (1986); Froehler, et al., Nucleic Acids Res.,
14:5399-5407
(1986); Sinha, et al. Tetrahedrov~ Lett., 24:5843-5846 (1983); and Sinha, et
al., Nucl. Acids
Res., 12:4539-4557 (1984) which are incorporated herein by reference.
a. Vectors for introduction and expression of genes in cells
An important aspect of this invention is the use of the lipid-nucleic acid
particles provided herein to introduce selected genes into cells ih vitro and
ih vivo, followed
by expression of the selected gene in the host cell. Thus, the nucleic acids
in the particles
specificlly encompass vectors that are capable of being expressed in a host
cell. Promoter,
enhancer, stress or chemically-regulated promoters, antibiotic-sensitive or
nutrient-sensitive
regions, as well as therapeutic protein encoding sequences, may be included as
required.
In brief summary, the expression of natural or synthetic nucleic acids is
typically achieved by operably linking a nucleic acid of interest to a
promoter (which is either
constitutive or inducible), incorporating the construct into an expression
vector, and
introducing the vector into a suitable host cell. Typical vectors contain
transcription and
translation terminators, transcription and translation initiation sequences,
and promoters
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
useful for regulation of the expression of the particular nucleic acid. The
vectors optionally
comprise generic expression cassettes containing at least one independent
terminator
sequence, sequences permitting replication of the cassette in eukaryotes, or
prokaryotes, or
both, (e.g., shuttle vectors) and selection markers for both prokaryotic and
eukaryotic
systems. Vectors are suitable for replication and integration in prokaryotes,
eukaryotes, or
preferably both. See, Giliman and Smith (1979), Gene, 8: 81-97; Roberts et al.
(1987),
Nature, 328: 731-734; Berger and I~immel, Guide to Molecular Clohiug
Techniques,
Methods ih Enzymology, volume 152, Academic Press, Inc., San Diego, CA
(Berger);
Sambrook et al. (1989), MOLECULAR CLONING - A LABORATORY MANUAL (2nd ed.) Vol.
1-
3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y., (Sambrook);
and F.M.
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current
Protocols, a
joint venture between Greene Publishing Associates, Inc, and John Wiley &
Sons, Inc., (1994
Supplement) (Ausubel). Product information from manufacturers of biological
reagents and
experimental equipment also provide information useful in known biological
methods. Such
manufacturers include the SIGMA chemical company (Saint Louis, MO), R&D
systems
(Minneapolis, MN), Pharmacia LKB Biotechnology (Piscataway, NJ), CLONTECH
Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.
(Gaithersberg,
MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
and
Applied Biosystems (Foster City, CA), as well as many other commercial sources
known to
one of skill.
Vectors to which foreign nucleic acids are operably linked may be used to
introduce these nucleic acids into host cells and mediate their replication
and/or expression.
"Cloning vectors" are useful for replicating and amplifying the foreign
nucleic acids and
obtaining clones of specific foreign nucleic acid-containing vectors.
"Expression vectors"
mediate the expression of the foreign nucleic acid. Some vectors are both
cloning and
expression vectors.
In general, the particular vector used to transport a foreign gene into the
cell is
not particularly critical. Any of the conventional vectors used for expression
in the chosen
host cell may be used.
An expression vector typically comprises a eukaryotic transcription unit or
"expression cassette" that contains all the elements required for the
expression of exogenous
genes in eukaryotic cells. A typical expression cassette contains a promoter
operably linked
26
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
to the DNA sequence encoding a desired protein and signals required for
efficient
polyadenylation of the transcript.
Eukaryotic promoters typically contain two types of recognition sequences,
the TATA box and upstream promoter elements. The TATA box, located 25-30 base
pairs
upstream of the transcription initiation site, is thought to be involved in
directing RNA
polymerase to begin RNA synthesis. The other upstream promoter elements
determine the
rate at which transcription is initiated.
Enhancer elements can stimulate transcription up to 1,000 fold from linked
homologous or heterologous promoters. Enhancers are active when placed
downstream or
upstream from the transcription initiation site. Many enhancer elements
derived from viruses
have a broad host range and are active in a variety of tissues. For example,
the SV40 early
gene enhancer is suitable for many cell types. Other enhancer/promoter
combinations that
are suitable for the present invention include those derived from polyoma
virus, human or
marine cytomegalovirus, the long term repeat from various retroviruses such as
marine
leukemia virus, marine or Rous sarcoma virus and HIV. See, E~chahce~s ahd
Eukaryotic
Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is
incorporated
herein by reference.
In addition to a promoter sequence, the expression cassette should also
contain
a transcription termination region downstream of the structural gene to
provide for efficient
termination. The termination region may be obtained from the same source as
the promoter
sequence or may be obtained from a different source.
If the mRNA encoded by the selected structural gene is to be efficiently
translated, polyadenylation sequences are also commonly added to the vector
construct. Two
distinct sequence elements are required for accurate and efficient
polyadenylation: GU or U
rich sequences located downstream from the polyadenylation site and a highly
conserved
sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
Termination
and polyadenylation signals that are suitable for the present invention
include those derived
from SV40, or a partial genomic copy of a gene already resident on the
expression vector.
In addition to the elements already described, the expression vector of the
present invention may typically contain other specialized elements intended to
increase the
level of expression of cloned nucleic acids or to facilitate the
identification of cells that carry
the transduced DNA. For instance, a number of animal viruses contain DNA
sequences that
promote the extra chromosomal replication of the viral genome in permissive
cell types.
Plasmids bearing these viral replicons are replicated episomally as long as
the appropriate
27
CA 02406654 2002-10-18
faWo s ~e provided by genes either carried on the plasmid or with the genome~o
the host
cell.
The expression vectors of the present invention will typically contain both
prokaryotic sequences that facilitate the cloning of the vector in bacteria as
well as one or
more eukaryotic transcription units that are expressed only in eukaryotic
cells, such as
mammalian cells. The prolcaryotic sequences are preferably chosen such that
they do not
interfere with the replication of the DNA in eukaryotic cells.
Selected genes are normally be expressed when the DNA sequence is
functionally inserted into a vector. "Functionally inserted" means that it is
inserted in proper
reading frame and orientation and operably linked to proper regulatory
elements. Typically,
a gene will be inserted downstream from a promoter and will be followed by a
stop codon,
although production as a hybrid protein followed by cleavage may be used, if
desired.
Expression vectors containing regulatory elements from eukaryotic viruses
such as retroviruses are typically used. SV40 vectors include pSVT7 and pMT2.
Vectors
derived from bovine papilloma virus include pBV-1MTHA, and vectors derived
from Epstein
Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG,
pAV009/A+,
pMTOlO/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing
expression of
proteins under the direction of the SV-40 early promoter, SV-40 later
promoter,
metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma
virus
promoter, polyhedrin promoter, or other promoters shown effective for
expression in
eukaryotic cells.
While a variety of vectors may be used, it should be noted that viral vectors
such as retroviral vectors are useful for modifying eukaryotic cells because
of the high
efficiency with which the retroviral vectors transfect target cells and
integrate into the target
cell genome. Additionally, the retroviruses harboring the retroviral vector
are capable of
infecting cells from a wide variety of tissues.
In addition to the retroviral vectors mentioned above, cells may be lipofected
with adeno-associated viral vectors. See, e.g., Methods in Enzymology, Vol.
185, Academic
Press, Inc., San Diego, CA (D.V. Goeddel, ed.) (1990) or M. I~rieger (1990),
Gene T~ansfe~
and Exp~essiovc -- A Laboratory Manual, Stockton Press, New York, NY, and the
references
cited therein. Adeno associated viruses (AAVs) require helper viruses such as
adenovirus or
herpes virus to achieve productive infection. In the absence of helper virus
functions, AAV
integrates (site-specifically) into a host cell's genome, but the integrated
AAV genome has no
pathogenic effect. The integration step allows the AAV genome to remain
genetically intact
28
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
until the host is exposed to the appropriate environmental conditions (e.g., a
lytic helper
virus), whereupon it re-enters the lytic life-cycle. Samulski (1993), Cu~~e~ct
Opinion in
Genetic and Development, 3: 74-80, and the references cited therein provides
an overview of
the AAV life cycle. See also West et al. (1987), hi~ology, 160: 38-47; Carter
et al. (1989),
U.S. Patent No. 4,797,368; Carter et al. (1993), WO 93/24641; Kotin (1994),
Human Gehe
The~~apy, 5: 793-801; Muzyczka (1994), J. Clin. Invest., 94: 1351 and
Samulski, supra, for an
overview of AAV vectors.
Plasmids designed for producing recombinant vaccinia, such as pGS62,
(Langford, C. L. et al. (1986), Mol. Cell. Biol., 6:3191-3199) may also be
used. This plasmid
consists of a cloning site for insertion of foreign nucleic acids, the P7.5
promoter of vaccinia
to direct synthesis of the inserted nucleic acid, and the vaccinia TK gene
flanking both ends
of the foreign nucleic acid.
Whatever the vector is used, generally the vector is genetically engineered to
contain, in expressible form, a gene of interest that encodes a gene product
of interest.
Suitable classes of gene products include, but are not limited to,
cytotoxic/suicide genes,
immunomodulators, cell receptor ligands, tumor suppressors, and anti-
angiogenic genes. The
particular gene selected will depend on the intended purpose or treatment.
Examples of such
genes of interest are described below and throughout the specification.
Cytotoxic/suicide
genes are those genes that are capable of killing cells, causing apoptosis, or
arresting cells in
the cell cycle. Such genes include, but are not limited to, genes for
immunotoxins, thymidine
kinase, a cytochrome P450 2B1, a deoxycytidine kinase, or a cytosine
deaminase. Agents
such as acyclovir and ganciclovir (for thymidine kinase), cyclophosphoamide
(for
cytochrome P450 2B1), 5-fluorocytosine (for cytosine deaminase), are typically
administered
systemically in conjunction (e.g., simulatenously or nonsimulatenously) with
the lipid-nucleic
compositions of the present invention to achieve the desired cytotoxic or
cytostatic effect.
Immunomodulator genes are genes that modulate one or more immune responses.
Examples
of immunomodulator genes include cytokines such as growth factors (e.g., TGF-
a., TGF- p,
EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2,
IL-12, IL-
15, IL-20, etc.), interferons (e.g., IFN-6, IFN-p, IFN-y, etc.) and TNF. Cell
receptor Iigands
include ligands that are able to bind to cell surface receptors (e.g., insulin
receptor, EPO
receptor, G-protein coupled receptors, receptors with tyrosine kinase
activity, cytokine
receptors, growth factor receptors, etc.), to modulate (e.g,. inhibit,
activate, etc.) the
physiological pathway that the receptor is involved in (e.g., glucose level
modulation, blood
cell development, mitogenesis, etc. ). Examples of cell receptor ligands
include cytokines,
29
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
growth factors, interleukins, interferons, erythropoietin (EPO), insulin,
glucagon, Ci-protein
coupled receptor ligands, etc.). Tumor suppressor genes are genes that are
able to inhibit the
growth of a cell, particularly tumor cells. Thus, delivery of these genes to
tumor cells is
useful in the treatment of cancers. Tumor suppressor genes include, but are
not limited to,
p53 (Lamb et al., Mol. Cell. Biol. 6:1379-1385 (1986), Ewen et al., Science
255:85-87
(1992), Ewen et al. (1991) Cell 66:1155-1164, and Hu et al., EMBO J. 9:1147-
1155 (1990)),
RBl (Toguchida et al. (1993) Genomics 17:535-543), WTl (Hastie, N. D., Cu~~.
Opih.
Genet. Dev. 3:408-4I3 (1993)), NFI (Trofatter et al., Cell 72:791-800 (1993),
Cawthon et al.,
Cell 62:193-201 (1990)), VHL (Latif et al., Science 260:1317-1320 (1993)) and
APC
(Gorden et al., Cell 66:589-600 (1991)). Anti-angiogenic genes are able to
inhibit
angiogenesis. These genes are particularly useful for treating those cancers
in which
angiogenesis plays a role in the pathological development of the disease.
Examples of anti-
angiogenic genes include, but are not limited to, endostatin (see e.g., U.S.
Patent No.
6,174,861) and augiostatin (see, e.g., U.S. Patent No. 5,639,725).
The vectors further usually comprise selectable markers which result in
nucleic acid amplification such as the sodium, potassium ATPase, thyrnidine
kinase,
aminoglycoside phosphotransferase, hygromycin B phosphotransferase, xanthine-
guanine
phosphoribosyl transferase, CAD (carbamyl phosphate synthetase, aspartate
transcarbamylase, and dihydroorotase), adenosine deaminase, dihydrofolate
reductase, and
asparagine synthetase and ouabain selection. Alternatively, high yield
expression systems
not involving nucleic acid amplification are also suitable, such as using a
baculovirus vector
in insect cells, with the encoding sequence under the direction of the
polyhedrin promoter or
other strong baculovirus promoters.
When nucleic acids other than plasmids are used the nucleic acids can contain
nucleic acid analogs, for example, the antisense derivatives described in a
review by Stein, et
al., Science 261:1004-1011 (1993) and in U.S. Patent Nos. 5,264,423 and
5,276,019, the
disclosures of which are incorporated herein by reference.
Unlike viral-based gene therapy vectors which can only incorporate a
relatively small nonviral nucleic acid sequence into the viral genome because
of size
limitations for packaging virion particles, the lipid-nucleic acid complexes
of the prtesent
invention may be used to transfer large (e.g., 50-5,000 kilobase) exogenous
nucleic acids into
cells. This aspect of lipofection is particularly advantageous since many
genes which may be
targets for gene therapy span over 100 kilobases (e.g., amyloid precursor
protein (APP) gene,
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
Huntmgton's chorea gene) and large homologous targeting constructs or
transgenes may be
required for therapy.
Cells can be lipofected with an exogenous nucleic acid at high efficiency and
with cell type specificity by contacting the cells with a receptor-recognition
transfection
complex comprising: (1) an exogenous nucleic acid, (2) a receptor-ligand
protein ("rlp")
which is covalently linked to a polycation, and (3) a cationic or neutral
lipid. It has been
found that a combination of a polycation-linked receptor-recognition protein
and a suitable
cationic (or neutral) lipid can be used to transfect nucleic acids, and that
the combination
retains cell type targeting specificity conferred by the receptor-recognition
protein and also
exhibits high efficiency transfection conferred, in part, by the inclusion of
a cationic lipid,
neutral lipid, or lipopolyamine.
The exogenous nucleic acid is typically dsDNA, ssDNA, ssRNA, dsRNA;
most typically the exogenous nucleic acid is dsDNA such as a cloned DNA
sequence in a
cloning vector such as a plasmid or viral genome. Multiple species of
exogenous nucleic acid
may be combined in a transfection complex, such as for co-transfection of
unlinked nucleic
acid sequences or to accomplish in vivo homologous recombination shuffling.
Frequently,
the exogenous nucleic acids) are not capable of autonomous replication in
cells which
incorporate the transfection complex, and are either transiently expressed or
are stably
integrated into a host cell chromosome by homologous recombination or
nonhomologous
integration. Often at least one selectable marker (e.g., a ~ceoR expression
cassette) is included
in the exogenous nucleic acids) to facilitate selection of cells which have
incorporated the
exogenous nucleic acid(s). Typically, an exogenous nucleic acid comprises a
structural gene
encoding a polypeptide to be expressed in a target cell which has incorporated
the exogenous
nucleic acid, and the structural gene usually is operably linked to
appropriate cis-acting
regulatory elements (e.g., promoter, enhancer, polyadenylation site). Although
gene therapy
may be performed in a variety of ways, a typical receptor-recognition
lipofection complex
comprises a nucleic acid which comprises at least one transcriptional unit.
The lipid nucleic acid particles of the invention can be designed to contain,
in
addition to the species of nucleic acid, a receptor-recognition molecule
(rlm), such as a
protein. The rlm can be covalently bound to lipids that comprise the nucleic
acid-lipid
particle. Its presence on the particle increases the efficiency aand
specificity with the particle
contacts and enters target cells. For example, a suitable rlm is a
nonimmunoglobulin protein
that binds to a cell surface receptor of a target cell which mediates
internalization of a
transfection complex comprising the rlm-polycation conjugate by, for example,
the process of
31
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
endocytosis and/or membrane fusion. Additional suitable rlm species typically
are naturally-
occurring physiological ligands which comprise a polypeptide portion (e.g.,
adhesion
molecules such as ICAM-1, ICAM-2, ELAM-l, VCAM-1). Viral proteins (e.g., spike
glycoproteins) which bind to viral receptors on eukaryotic cells and mediate
virus
internalization may also be used as rlm species for forming rlm-polycation
conjugates.
Examples also include viral glycoproteins which attach to cell surface
receptors and lead to
internalization and/or membrane fusion include the gB, gC, gD, gE, gH, and gI
virion
glycoproteins of HSV-1, and gp120 of HIV-1.
Fragments and analogs of naturally-occurring proteins may be used as well as
full-length mature proteins as rlm species in forming transfection complexes
of the invention.
For example, fragments, analogs, and fusion proteins comprising a portion of
an adhesion
molecule or virion attachment protein which mediates attachment to a target
cell may be used
as rlm species without other portions of the naturally-occurring full-length
protein that are not
essential for cell attachment and/or membrane fusion. Thus, for example, a
cytoplasmic tail
peptide portion of a virion glycoprotein usually may be omitted and the
resultant protein may
still serve as a suitable rlm.
The rlm selected will vary with the particular target cell type. For specific
targeting to hepatocytes, asialoglycoproteins (galactose-terminal) are
preferred as rlm
species. Examples of asialoglycoproteins include asialoorosomucoid,
asialofetuin, and
desialylated vesicular stomatitis virus virion proteins. These can be formed
by chemical or
enzymatic desialylation of those glycoproteins that possess terminal sialic
acid and
penultimate galactose residues. Alternatively, rlm species suitable for
forming lipofection
complexes that selectively target hepatocytes may be created by coupling
lactose or other
galactose-terminal carbohydrates (e.g., arabinogalactan) to nongalactose-
bearing proteins by
reductive lactosamination. Other useful galactose-terminal carbohydrates for
hepatocyte
targeting include carbohydrate trees obtained from natural glycoproteins,
especially tri- and
tetra-antennaxy structures that contain either terminal galactose residues or
that can be
enzymatically treated to expose terminal galactose residues. For targeting
macrophages,
endothelial cells, or lymphocytes, rlm species comprising mannose or mannose-6-
phosphate,
or complex carbohydrates comprising these terminal carbohydrate structures may
be used.
Since a variety of different cell surface receptors exist on the surfaces of
mammalian cells, cell-specific targeting of nucleic acids to nonhepatic cells
can involve
lipofection complexes that comprise various rlm species. For example,
transferrin can be
used as a suitable rlm for forming receptor-recognition transfection complexes
to cells
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expressW g transferrin receptors. Other receptor ligands such as polypeptide
hormones (e.g.,
growth hormone, PDGF, FGF, EGF, insulin, IL-2, IL-4, etc.) may be used to
localize
receptor-recognition transfection complexes to cells expressing the cognate
receptor.
The nucleic acid-lipid particles may comprise multiple rlm species.
Frequently, an agent having membrane fusion activity (e. g. , influenza virus
hemagglutinin,
HSV-1 gB and gD) is used as an rlm for forming rlm-polycation complexes,
either alone or in
combination with other rlm species, typically with those which lack membrane
fusion
activity.
These transfection methods generally comprise the steps of: (1) forming a
nucleic acid-lipid-rlm particle consisting essentially of an exogenous nucleic
acid, a
polycation conjugate consisting essentially of a polycation linked to a
nonimmunoglobulin
receptor-recognition molecule that binds to a predetermined cell surface
receptor, and a lipid
component consisting essentially of a neutral or cationic lipid (optionally
including a
quaternary ammonium detergent and/or a lipopolyamine), and (2) contacting
cells expressing
the predetermined cell surface receptor with a composition comprising the
receptor-
recognition transfection complex under physiological transfection conditions
which permit
uptake of the exogenous nucleic acid into said cells. In alternative
embodiments, the rlm is
attached to the polycation by covalent linkage, frequently by covalent linkage
through a
crosslinking agent or by peptide linkage.
III. Prebaration of SPLPs and SPLP-CPLs and Sizing
In one embodiment, the present invention provides lipid-nucleic acid particles
produced via hydrophobic nucleic acid-lipid intermediate complexes. The
complexes are
preferably charge-neutralized. Manipulation of these complexes in either
detergent-based or
organic solvent-based systems can lead to particle formation in which the
nucleic acid is
protected.
The present invention provides a method of preparing serum-stable plasmid-
lipid particles in which the plasmid or other nucleic acid is encapsulated in
a lipid bilayer and
is protected from degradation. Additionally, the particles formed in the
present invention are
preferably neutral or negatively-charged at physiological pH. For in vivo
applications,
neutral particles are advantageous, while for in vitro applications the
particles are more
preferably negatively charged. This provides the further advantage of reduced
aggregation
over the positively-charged liposome formulations in which a nucleic acid can
be
encapsulated in cationic lipids.
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The particles made by the methods of this invention have a size of about 50 to
about 150 nm, with a majority of the particles being about 65 to 85 nm. The
particles can be
formed by either a detergent dialysis method or by a modification of a reverse-
phase method
which utilizes organic solvents to provide a single phase during mixing of the
components.
Without intending to be bound by any particular mechanism of formation, a
plasmid or other
nucleic acid is contacted with a detergent solution of cationic lipids to form
a coated plasmid
complex. These coated plasmids can aggregate and precipitate. However, the
presence of a
detergent reduces this aggregation and allows the coated plasmids to react
with excess lipids
(typically, noncationic lipids) to form particles in which the plasmid or
other nucleic acid is
encapsulated in a lipid bilayer. The methods described below for the formation
of
plasmid-lipid particles using organic solvents follow a similar scheme.
In some embodiments, the particles are formed using detergent dialysis. Thus,
the present invention provides a method for the preparation of serum-stable
plasmid-lipid
particles, comprising:
(a) combining a plasmid with cationic lipids in a detergent solution to form a
coated plasmid-lipid complex;
(b) contacting noncationic lipids with the coated plasmid-lipid complex to
form a
detergent solution comprising a plasmid-lipid complex and noncationic lipids;
and
(c) dialyzing the detergent solution of step (b) to provide a solution of
serum-
stable plasmid-lipid particles, wherein the plasmid is encapsulated in a lipid
bilayer and the particles are serum-stable and have a size of from about 50 to
about 150 nm.
An initial solution of coated plasmid-lipid complexes is formed by combining
the plasmid
with the cationic lipids in a detergent solution.
In these embodiments, the detergent solution is preferably an aqueous solution
of a neutral detergent having a critical micelle concentration of 15-300 mM,
more preferably
20-50 mM. Examples of suitable detergents include, for example, N,N'-
((octanoylimino)-bis-
(trimethylene))-bis-(D-gluconamide) (BIGCHAP); BRIJ 35; Deoxy-BIGCHAP;
dodecylpoly(ethylene glycol) ether; Tween 20; Tween 40; Tween 60; Tween 80;
Tween 85;
Mega 8; Mega 9; Zwittergent° 3-08; Zwittergent° 3-10; Triton X-
405; hexyl-, heptyl-, octyl-
and nonyl-[3-D-glucopyranoside; and heptylthioglucopyranoside; with octyl [3-D-
glucopyranoside and Tween-20 being the most preferred. The concentration of
detergent in
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CA 02406654 2002-10-18
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the detergent solution is typically about 100 mM to about 2 M, preferably from
about zUU
mM to about 1.5 M.
The cationic lipids and plasmid will typically be combined to produce a charge
ratio (+/-) of about 1:1 to about 20:1, preferably in a ratio of about 1:1 to
about 12:1, and
more preferably in a ratio of about 2:1 to about 6:1. Additionally, the
overall concentration
of plasmid in solution will typically be from about 25 ~,g/mL to about 1
mg/mL, preferably
from about 25 ~,g/mL to about 200 ~,g/mL, and more preferably from about 50
~,g/mL to
about 100 ~,g/mL. The combination of plasmids and cationic lipids in detergent
solution is
kept, typically at room temperature, for a period of time which is sufficient
for the coated
complexes to form. Alternatively, the plasmids and cationic lipids can be
combined in the
detergent solution and warmed to temperatures of up to about 37°C. For
plasmids which are
particularly sensitive to temperature, the coated complexes can be formed at
lower
temperatures, typically down to about 4°C.
In a preferred embodiment, the nucleic acid to lipid ratios (mass/mass ratios)
in a formed SPLP will range from about 0.01 to about 0.08. The ratio of the
starting
materials also falls within this range because the purification step typically
removes the
unencapsulated nucleic acid as well as the empty liposomes. In another
preferred
embodiment, the SPLP preparation uses about 400 ~g nucleic acid per 10 mg
total lipid or a
nucleic acid to lipid ratio of about 0.01 to about 0.08 and, more prederably,
about 0.04, which
corresponds to 1.25 mg of total lipid per 50 ~g of nucleic acid.
The detergent solution of the coated plasmid-lipid complexes is then contacted
with noncationic lipids to provide a detergent solution of plasmid-lipid
complexes and
noncationic lipids. The noncationic lipids which are useful in this step
include,
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceraxnide,
sphingomyelin,
cephalin, cardiolipin, and cerebrosides. In preferred embodiments, the
noncationic lipids are
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide or
sphingomyelin.
The acyl groups in these lipids are preferably acyl groups derived from fatty
acids having
Clo-Caa carbon chains. More preferably the acyl groups are lauroyl, myristoyl,
palmitoyl,
stearoyl or oleoyl. In particularly preferred embodiments, the noncationic
lipid will be 1,2-
sn-dioleoylphosphatidylethanolaxnine (DOPE), palmitoyl oleoyl
phosphatidylcholine (POPC)
or egg phosphatidylcholine (EPC). In the most preferred embodiments, the
plasmid-lipid
particles will be fusogenic particles with enhanced properties ih vivo and the
noncationic lipid
will be DOPE. In other preferred embodiments, the noncationic lipids will
further comprise
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polyethylene glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene
glycol
conjugated to ceramides, as described in co-pending USSN 08/316,429,
incorporated herein
by reference.
The amount of noncationic lipid which is used in the present methods is
typically about 2 to about 20 mg of total lipids to 50 pg of plasmid.
Preferably the amount of
total lipid is from about 5 to about 10 mg per 50 ~,g of plasmid.
Following formation of the detergent solution of plasmid-lipid complexes and
noncationic lipids, the detergent is removed, preferably by dialysis. The
removal of the
detergent results in the formation of a lipid-bilayer which surrounds the
plasmid providing
serum-stable plasmid-lipid particles which have a size of from about 50 nm to
about 150 nm.
The particles thus formed do not aggregate and are optionally sized to achieve
a uniform
particle size.
The serum-stable plasmid-lipid particles can be sized by any of the methods
available for sizing liposomes. The sizing may be conducted in order to
achieve a desired
size range and relatively narrow distribution of particle sizes.
Several techniques are available for sizing the particles to a desired size.
One
sizing method, used for liposomes and equally applicable to the present
particles is described
in U.S. Patent No. 4,737,323, incorporated herein by reference. Sonicating a
particle
suspension either by bath or probe sonication produces a progressive size
reduction down to
particles of less than about 50 nm in size. Homogenization is another method
which relies on
shearing energy to fragment larger particles into smaller ones. In a typical
homogenization
procedure, particles are recirculated through a standard emulsion homogenizer
until selected
particle sizes, typically between about 60 and 80 nm, are observed. In both
methods, the
particle size distribution can be monitored by conventional laser-beam
particle size
discrimination, or QELS.
Extrusion of the particles through a small-pore polycarbonate membrane or an
asymmetric ceramic membrane is also an effective method for reducing particle
sizes to a
relatively well-defined size distribution. Typically, the suspension is cycled
through the
membrane one or more times until the desired particle size distribution is
achieved. The
particles may be extruded through successively smaller-pore membranes, to
achieve a gradual
reduction in size.
In another group of embodiments, the present invention provides a method for
the preparation of serum-stable plasmid-lipid particles, comprising;
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(a) preparing a mixture comprising cationic lipids and noncatiomc lipids m an
organic solvent;
(b) contacting an aqueous solution of nucleic acid with said mixture in step
(a) to
provide a clear single phase; and
(c) removing said organic solvent to provide a suspension of plasmid-lipid
particles, wherein said plasmid is encapsulated in a lipid bilayer, and said
particles are stable in serum and have a size of from about 50 to about 150
nm.
The plasmids (or nucleic acids), cationic lipids and noncationic lipids which
are useful in this group of embodiments are as described for the detergent
dialysis methods
above.
The selection of an organic solvent will typically involve consideration of
solvent polarity and the ease with which the solvent can be removed at the
later stages of
particle formation. The organic solvent, which is also used as a solubilizing
agent, is in an
amount sufficient to provide a clear single phase mixture of plasmid and
lipids. Suitable
solvents include chloroform, dichloromethane, diethylether, cyclohexane,
cyclopentane,
benzene, toluene, methanol, or other aliphatic alcohols such as propanol,
isopropanol,
butanol, tert-butanol, iso-butanol, pentanol and hexanol. Combinations of two
or more
solvents may also be used in the present invention.
Contacting the plasmid with the organic solution of cationic and noncationic
lipids is accomplished by mixing together a first solution of plasmid, which
is typically an
aqueous solution and a second organic solution of the lipids. One of skill in
the art will
understand that this mixing can take place by any number of methods, for
example by
mechanical means such as by using vortex mixers.
After the plasmid has been contacted with the organic solution of lipids, the
organic solvent is removed, thus forming an aqueous suspension of serum-stable
plasmid-
lipid particles. The methods used to remove the organic solvent will typically
involve
evaporation at reduced pressures or blowing a stream of inert gas (e.g.,
nitrogen or argon)
across the mixture.
The serum-stable plasmid-lipid particles thus formed will typically be sized
from about SO nm to 150 nm. To achieve further size reduction or homogeneity
of size in the
particles, sizing can be conducted as described above.
In other embodiments, the methods will further comprise adding nonlipid
polycations which are useful to effect the transformation of cells using the
present
compositions. Examples of suitable nonlipid polycations include,
hexadimethrine bromide
37
CA 02406654 2002-10-18
(so d uorhde tile brandname POLYBRENE~, from Aldrich Chemical Co., M
lwaukee~oosss
Wisconsin, USA) or other salts of heaxadimethrine. Other suitable polycations
include, for
example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-
lysine,
polyallylamine and polyethyleneimine.
In other embodiments, the polyoxyethylene conjugates which are used in the
plasmid-lipid particles of the present invention can be prepared by combining
the conjugating
group (i. e. phosphatidic acid or phosphatidylethanolamine) with an
appropriately
functionalized polyoxyethylene derivative. For example,
phosphatidylethanolamine can be
combined with polyoxyethylene bis(p-toluenesulfonate) to provide a
phosphatidylethanolamine-polyoxyethylene conjugate. See, Woodle, et al.,
Biochim.
Biophys. Acta 1105:193-200 (1992), incorporated herein by reference.
In certain embodiments, the formation of the lipid-nucleic acid complexes can
be carried out either in a monophase system (e.g., a Bligh and Dyer monophase
or similar
mixture of aqueous and organic solvents) or in a two phase system with
suitable mixing.
When formation of the complexes is carried out in a monophase system, the
cationic lipids and nucleic acids are each dissolved in a volume of the
monophase mixture.
Combination of the two solutions provides a single mixture in which the
complexes form.
Alternatively, the complexes can form in two-phase mixtures in which the
cationic lipids bind
to the nucleic acid (which is present in the aqueous phase), and "pull" it in
to the organic
phase.
In another embodiment, the present invention provides a method for the
preparation of lipid-nucleic acid particles, comprising:
(a) contacting nucleic acids with a solution comprising noncationic lipids and
a
detergent to form a nucleic acid-lipid mixture;
(b) contacting cationic lipids with the nucleic acid-lipid mixture to
neutralize a
portion of the negative charge of the nucleic acids and form a chaxge-
neutralized mixture of
nucleic acids and lipids; and
(c) removing the detergent from the charge-neutralized mixture to provide the
lipid-nucleic acid particles in which the nucleic acids are protected from
degradation.
In one group of embodiments, the solution of noncationic lipids and detergent
is an aqueous solution. Contacting the nucleic acids with the solution of
noncationic lipids
and detergent is typically accomplished by mixing together a first solution of
nucleic acids
and a second solution of the lipids and detergent. One of skill in the art
will understand that
this mixing can take place by any number of methods, for example by mechanical
means
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CA 02406654 2002-10-18
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such as by using vortex mixers. Preferably, the nucleic acid solution is also
a detergent
solution. The amount of noncationic lipid which is used in the present method
is typically
determined based on the amount of cationic lipid used, and is typically of
from about 0.2 to 5
times the amount of cationic lipid, preferably about 0.5 to 2 times the amount
of cationic lipid
used.
The nucleic acid-lipid mixture thus formed is contacted with cationic lipids
to
neutralize a portion of the negative charge which is associated with the
nucleic acids (or other
polyanionic materials) present. The amount of cationic lipids used will
typically be sufficient
to neutralize at least 50% of the negative charge of the nucleic acid.
Preferably, the negative
charge will be at least 70% neutralized, more preferably at least 90%
neutralized. Cationic
lipids which are useful in the present invention, include, for example, DODAC,
DOTMA,
DDAB, DOTAP, DC-Chol and DMRIE. These lipids and related analogs have been
described in co-pending USSN 08/316,399; U.S. Patent Nos. 5,208,036,
5,264,618, 5,279,833
and 5,283,185, the disclosures of which are incorporated herein by reference.
Additionally, a
number of commercial preparations of cationic lipids are available and can be
used in the
present invention. These include, for example, LIPOFECTIN~ (commercially
available
cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island,
New
York, USA); LIPOFECTAMINE~ (commercially available cationic liposomes
comprising
DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAMC~ (commercially available
cationic lipids comprising DOGS in ethanol from Promega Corp., Madison,
Wisconsin,
USA).
Contacting the cationic lipids with the nucleic acid-lipid mixture can be
accomplished by any of a number of techniques, preferably by mixing together a
solution of
the cationic lipid and a solution containing the nucleic acid-lipid mixture.
Upon mixing the
two solutions (or contacting in any other manner), a portion of the negative
charge associated
with the nucleic acid is neutralized. Nevertheless, the nucleic acid remains
in an
uncondensed state and acquires hydrophilic characteristics.
After the cationic lipids have been contacted with the nucleic acid-lipid
mixture, the detergent (or combination of detergent and organic solvent) is
removed, thus
forming the lipid-nucleic acid particles. The methods used to remove the
detergent will
typically involve dialysis. When organic solvents are present, removal is
typically
accomplished by evaporation at reduced pressures or by blowing a stream of
inert gas (e.g.,
nitrogen or argon) across the mixture.
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The particles thus formed will typically be sized from about 1 UU nm to
several
microns. To achieve further size reduction or homogeneity of size in the
particles, the lipid-
nucleic acid particles can be sonicated, filtered or subjected to other sizing
techniques which
are used in liposomal formulations and are known to those of skill in the art.
In other embodiments, the methods will further comprise adding nonlipid
polycations which are useful to effect the lipofection of cells using the
present compositions.
Examples of suitable nonlipid polycations include, hexadimethrine bromide
(sold under the
brandname POLYBRENE~, from Aldrich Chemical Co., Milwaukee, Wisconsin, USA) or
other salts of hexadimethrine. Other suitable polycations include, for
example, salts of poly-
L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine and
polyethyleneimine. Addition of these salts is preferably after the particles
have been formed.
In another aspect, the present invention provides methods for the preparation
of lipid-nucleic acid particles, comprising:
(a) contacting an amount of cationic lipids with nucleic acids in a solution;
the
solution comprising of from about 15-35% water and about 65-85% organic
solvent and the
amount of cationic lipids being sufficient to produce a +/- charge ratio of
from about 0.85 to
about 2.0, to provide a hydrophobic, charge-neutralized lipid-nucleic acid
complex;
(b) contacting the hydrophobic, charge-neutralized lipid-nucleic acid complex
in
solution with noncationic lipids, to provide a lipid-nucleic acid mixture; and
(c) removing the organic solvents from the lipid-nucleic acid mixture to
provide
lipid-nucleic acid particles in which the nucleic acids are protected from
degradation.
The nucleic acids, noncationic lipids, cationic lipids and organic solvents
which are useful in this aspect of the invention are the same as those
described for the
methods above which used detergents. In one group of embodiments, the solution
of step (a)
is a monophase. In another group of embodiments, the solution of step (a) is
two-phase.
In preferred embodiments, the cationic lipids are DODAC, DDAB, DOTMA,
DOSPA, DMRIE, DOGS or combinations thereof. In other preferred embodiments,
the
noncationic lipids axe ESM, DOPE, polyethylene glycol-based polymers (e.g.,
PEG 2000,
PEG 5000, PEG-modified phospholipids or PEG-modified ceramides) or
combinations
thereof. In still other preferred embodiments, the organic solvents axe
methanol, chloroform,
methylene chloride, ethanol, diethyl ether or combinations thereof.
In a particularly preferred embodiment, the nucleic acid is a plasmid; the
cationic lipid is DODAC, DDAB, DOTMA, DOSPA, DMRIE, DOGS or combinations
thereof; the noncationic lipid is ESM, DOPE, polyethylene glycol-based
polymers or
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combinations thereof; and the organic solvent is methanol, chloroform,
methylene chloride,
ethanol, diethyl ether or combinations thereof
As above, contacting the nucleic acids with the cationic lipids is typically
accomplished by mixing together a first solution of nucleic acids and a second
solution of the
lipids, preferably by mechanical means such as by using vortex mixers. The
resulting
mixture contains complexes as described for one aspect of the invention above.
These
complexes are then converted to particles by the addition of noncationic
lipids and the
removal of the organic solvent. The addition of the noncationic lipids is
typically
accomplished by simply adding a solution of the noncationic lipids to the
mixture containing
the complexes. A reverse addition can also be used. Subsequent removal of
organic solvents
can be accomplished by methods known to those of skill in the art and also
described above.
The amount of noncationic lipids which is used in this aspect of the invention
is typically an amount of from about 0.2 to 5 times the amount (on a mole
basis) of cationic
lipids which was used to provide the charge-neutralized lipid-nucleic acid
complex.
Preferably, the amount is from 0.5 to 2 times the amount of cationic lipids
used.
In yet another aspect, the present invention provides lipid-nucleic acid
particles which are prepared by the methods described above. In these
embodiments, the
lipid-nucleic acid particles are either net charge neutral or carry an overall
charge which
provides the particles with greater gene lipofection activity. Preferably, the
nucleic acid
component of the particles is a nucleic acid which encodes a desired protein
or blocks the
production of an undesired protein. In particularly preferred embodiments, the
nucleic acid is
a plasmid, the noncationic lipid is egg sphingomyelin and the cationic lipid
is DODAC.
A variety of general methods for malting SPLP-CPLs (CPL-containing
SPLPs) axe discussed herein. Two general techniques include "post-insertion"
technique, that
is, insertion of a CPL into for example, a pre-formed SPLP, and the "standard"
technique,
wherein the CPL is included in the lipid mixture during for example, the SPLP
formation
steps. The post-insertion technique results in SPLPs having CPLs mainly in the
external face
of the SPLP bilayer membrane, whereas standard techniques provide SPLPs having
CPLs on
both internal and external faces.
In particular, "post-insertion" involves forming SPLPs (by any method), and
incubating the pre-formed SPLPs in the presence of CPL under appropriate
conditions
(preferably 2-3 hours at 60°C). Between 60-80% of the CPL can be
inserted into the external
leaflet of the recipient vesicle, giving final concentrations up to about 5 to
10 mol % (relative
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CA 02406654 2002-10-18
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to total lipid). The method is especially useful for vesicles made from
phosphohpids (which
can contain cholesterol) and also for vesicles containing PEG-lipids (such as
PEG-Ceramide).
In an example of a "standard" technique, the CPL-SPLPs of the present
invention can be formed by extrusion. In this embodiment, all of the lipids
including the
CPL, are co-dissolved in chloroform, which is then removed under nitrogen
followed by high
vacuum. The lipid mixture is hydrated in an appropriate buffer, and extruded
through two
polycarbonate filters with a pore size of 100 nm. The resulting SPLPs contain
CPL on both
of the internal and external faces. In yet another standard technique, the
formation of CPL-
SPLPs can be accomplished using a detergent dialysis or ethanol dialysis
method, for
example, as discussed in U.S. Patent Nos. 5,976,567 and 5,981,501, both of
which are
incorporated herein by reference.
IV. Pharmaceutical Preparations
The nucleic acid-lipid particles of the present invention can be administered
either alone or in mixture with a physiologically-acceptable carrier (such as
physiological
saline or phosphate buffer) selected in accordance with the route of
administration and
standard pharmaceutical practice. Generally, normal saline will be employed as
the
pharmaceutically acceptable carrier. Other suitable carriers include, e.g.,
water, buffered
water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for
enhanced stability,
such as albumin, lipoprotein, globulin, etc.
The pharmaceutical carrier is generally added following particle formation.
Thus, after the particle is formed, the particle can be diluted into
pharmaceutically acceptable
carriers such as normal saline.
The concentration of particles in the pharmaceutical formulations can vary
widely, i.e., from less than about 0.05%, usually at or at least about 2-5% to
as much as 10 to
30% by weight and will be selected primarily by fluid volumes, viscosities,
etc., in
accordance with the particular mode of administration selected. For example,
the
concentration may be increased to lower the fluid load associated with
treatment. This may
be particularly desirable in patients having atherosclerosis-associated
congestive heart failure
or severe hypertension. Alternatively, particles composed of irritating lipids
may be diluted
to low concentrations to lessen inflammation at the site of administration.
As described above, it is often desirable to include PEG-lipid conjugates,
such
as PEG-ceramides or PEG-PE, ganglioside GMl-modified lipids or ATTA-lipids to
the
particles. Addition of such components prevents particle aggregation and
provides a means
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for increasing circulation lifetime and increasing the delivery of the lipid-
nucleic acid
particles to the target tissues. Typically, the concentration of the component
in the particle
will be about 1-20 % and, more preferably from about 3-10 %.
The pharmaceutical compositions may be sterilized by conventional, well
known sterilization techniques. Aqueous solutions can be packaged for use or
filtered under
aseptic conditions and lyophilized, the lyophilized preparation being combined
with a sterile
aqueous solution prior to administration. The compositions can contain
pharmaceutically
acceptable auxiliary substances as required to approximate physiological
conditions, such as
pH adjusting and buffering agents, tonicity adjusting agents and the like, for
example, sodium
acetate, sodium lactate, sodium chloride, potassium chloride, and calcium
chloride.
Additionally, the particle suspension may include lipid-protective agents
which protect lipids
against free-radical and lipid-peroxidative damages on storage. Lipophilic
free-radical
quenchers, such as alphatocopherol and water-soluble iron-specific chelators,
such as
ferrioxamine, are suitable.
In another example of their use, lipid-nucleic acid particles can be
incorporated into a broad range of topical dosage forms including but not
limited to gels, oils,
emulsions and the like. For instance, the suspension containing the nucleic
acid-lipid
particles can be formulated and administered as topical creams, pastes,
ointments, gels,
lotions and the like.
The present invention also provides lipid-nucleic acid particles in kit form.
The lcit will typically be comprised of a container which is compartmentalized
for holding the
various elements of the lipid-nucleic acid particles and the endosomal
membrane destabilizer
(e.g., calcium ions). The kit will contain the compositions of the present
inventions,
preferably in dehydrated form, with instructions for their rehydration and
administration. In
still other embodiments, the particles and/or compositions comprising the
particles will have
a targeting moiety attached to the surface of the particle. Methods of
attaching targeting
moieties (e.g., antibodies, proteins) to lipids (such as those used in the
present particles) are
known to those of skill in the art.
V. Administration of Lipid-Nucleic Acid Particle Formulations
The serum-stable nucleic acid-lipid particles of the present invention are
useful for the introduction of nucleic acids into cells. Accordingly, the
present invention also
provides methods for introducing a nucleic acids (e.g., a plasmid) into a
cell. The methods
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CA 02406654 2002-10-18
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are carried out ivc vitro or in vivo by first forming the particles as
described above, then
contacting the particles with the cells for a period of time sufficient for
transfection to occur.
The nucleic acid-lipid particles of the present invention can be adsorbed to
almost any cell type with which they are mixed or contacted. Once adsorbed,
the particles
can either be endocytosed by a portion of the cells, exchange lipids with cell
membranes, or
fuse with the cells. Transfer or incorporation of the nucleic acid portion of
the particle can
take place via any one of these pathways. In particular, when fusion takes
place, the particle
membrane is integrated into 'the cell membrane and the contents of the
particle combine with
the intracellular fluid.
1. In vitro gene transfer
For in vitro applications, the delivery of nucleic acids can be to any cell
grown
in culture, whether of plant or animal origin, vertebrate or invertebrate, and
of any tissue or
type. In preferred embodiments, the cells will be animal cells, more
preferably mammalian
cells, and most preferably htunan cells.
Contact between the cells and the lipid-nucleic acid particles, when carried
out
in vitro, takes place in a biologically compatible medium. The concentration
of particles
varies widely depending on the particular application, but is generally
between about 1 ~,mol
and about 10 mmol. Treatment of the cells with the nucleic acid-lipid
particles is generally
carried out at physiological temperatures (about 37° C) for periods of
time of from about 1 to
48 hours, preferably of from about 2 to 4 hours.
In one group of preferred embodiments, a lipid-nucleic acid particle
suspension is added to 60-80% confluent plated cells having a cell density of
from about 103
to about 105 cells/mL, more preferably about 2 x 104 cells/mL. The
concentration of the
suspension added to the cells is preferably of from about 0.01 to 0.2 ~g/mL,
more preferably
about 0.1 ~g/mL.
2. In vivo gene transfer
Alternatively, the compositions of the present invention can also be used for
the in vivo gene transfer, using methods which are known to those of skill in
the art. In
particular, Zhu, et al., Science 261:209-211 (1993), incorporated herein by
reference,
describes the intravenous delivery of cytomegalovirus (CMV)-chloramphenicol
acetyltransferase (CAT) expression plasmid using DOTMA-DOPE complexes. Hyde,
et al.,
Nature 362:250-256 (1993), incorporated herein by reference, describes the
delivery of the
44
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
cystic fibrosis transmembrane conductance regulator (CFTR) gene to epithelia
of the airway
and to alveoli in the lung of mice, using liposomes. Brigham, et al., Am. J.
Med. Sci.
298:278-281 (1989), incorporated herein by reference, describes the in vivo
transfection of
lungs of mice with a functioning prokaryotic gene encoding the intracellular
enzyme
chloramphenicol acetyltransferase (CAT).
For ih vivo administration, the pharmaceutical compositions are preferably
administered parenterally, i.e., intraaxticularly, intravenously,
intraperitoneally,
subcutaneously, or intramuscularly. More preferably, the pharmaceutical
compositions are
administered intravenously or intraperitoneally by a bolus injection. For
example, see
Stadler, et al., U.S. Patent No. 5,286,634, which is incorporated herein by
reference.
Intracellular nucleic acid delivery has also been discussed in Straubringer,
et al., METHODS
IN ENZYMOLOGY, Academic Press, New York. 101:512-527 (1983); Maxmino, et al.,
Biotechniques 6:682-690 (1988); Nicolau, et al., Cr~it. Rev. Ther. Drug
Ca~~ier Syst.
6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993). Still other
methods of
administering lipid-based therapeutics are described in, for example, Ralunan
et al., U.S.
Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410; Papahadjopoulos et
al., U.S. Patent
No. 4,235,871; Schneider, U.S. Patent No. 4,224,179; Lenk et al., U.S. Patent
No. 4,522,803;
and Fountain et al., U.S. Patent No. 4,588,578.
In certain embodiments, the pharmaceutical preparations may be contacted
with the target tissue by direct application of the preparation to the tissue.
The application
may be made by topical, "open" or "closed" procedures. By "topical", it is
meant the direct
application of the pharmaceutical preparation to a tissue exposed to the
environment, such as
the skin, orophaxynx, external auditory canal, and the like. "Open" procedures
are those
procedures which include incising the skin of a patient and directly
visualizing the underlying
tissue to which the pharmaceutical preparations are applied. This is generally
accomplished
by a surgical procedure, such as a thoracotomy to access the lungs, abdominal
laparotomy to
access abdominal viscera, or other direct surgical approach to the target
tissue. "Closed"
procedures are invasive procedures in which the internal target tissues are
not directly
visualized, but accessed via inserting instruments through small wounds in the
skin. For
example, the preparations may be administered to the peritoneum by needle
lavage.
Likewise, the pharmaceutical preparations may be administered to the meninges
or spinal
cord by infusion during a lumbar puncture followed by appropriate positioning
of the patient
as commonly practiced for spinal anesthesia or metrazamide imaging of the
spinal cord.
Alternatively, the preparations may be administered through endoscopic
devices.
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
The lipid-nucleic acid particles can also be administered in an aerosol
inhaled
into the lungs (see, Brigham, et al., Am. J. Sci. 298(4):278-281 (1989)) or by
direct injection
at the site of disease (Culver, HUMAN G$NE THERAPY, MaryAnn Liebert, Inc.,
Publishers,
New York. pp.70-71 (1994)).
The methods of the present invention may be practiced in a variety of hosts.
Preferred hosts include mammalian species, such as humans, nonhuman primates,
dogs, cats,
cattle, horses, sheep, and the like.
The amount of particles administered will depend upon the the ratio of nucleic
acid to lipid; the particular nucleic acid used, the disease state being
diagnosed; the age,
weight, and condition of the patient and the judgement of the clinician; but
will generally be
between about 0.01 and about 50 mg per kilogram of body weight; preferably
between about
0.1 and about 5 mg/kg of body weight or about 108-101° particles per
injection.
3. Insertion of Functional Copy of a Gene
Solve methods of gene therapy serve to compensate for a defect in an
endogenous gene by integrating a functional copy of the gene into the host
chromosome. The
inserted gene replicates with the host DNA and is expressed at a level to
compensate for the
defective gene. Diseases amendable to treatment by this approach are often
characterized by
recessive mutations. That is, both copies of an endogenous gene must be
defective for
symptoms to appear. Such diseases include cystic fibrosis, sickle cell anemia,
(3-thalassemia,
phenylketonuria, galactosemia, Wilson's disease, hemochromatosis, severe
combined
immunodeflciency disease, alpha-1-antitrypsin deficiency, albinism,
alkaptonuria, lysosomal
storage diseases, Ehlers-Danlos syndrome, hemophilia, glucose-6-phosphate
dehydrogenase
deficiency, agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome,
muscular
dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, fragile X-syndrome, and
the like.
Other recessive mutations are known in the art, and the use of the methods of
the present
invention to treat them is contemplated herein.
There are several methods for introducing an exogenous functional gene to
compensate for the above genetic defects. In one approach, cells axe removed
from a patient
suffering from the disease and contacted with a lipid-vector complex ih vitro.
Cells should be
removed from a tissue type in which disease symptoms are manifested. If the
cells are
capable of replication, and the vector used includes a selective marker, cells
having
internalized and expressed the marker can be selected. Particularly if
selection is not
46
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
performed, it is important that the frequency of gene transfer into cells be
high, for example,
at least about 1, S, 10, 2S or SO% of cells.
After integration of the vector into the cellular genome, and optionally,
selection, cells are reintroduced into the patient. In this application, and
others discussed
S below (except site-specific recombination to correct dominant mutations), it
is not necessary
that the gene supplied by the lipid-nucleic acid particle be delivered to the
same site as is
occupied by the defective gene for which it is compensating.
Alternatively, the lipid-vector complex can be introduced directly into a
patient as a pharmaceutical composition. The complex is delivered to the
tissues) affected
by the genetic disorder being treated in a therapeutically effective dose. In
this and other
methods, a therapeutically effective dose is an amount sufficient to cure, or
at least partially
arrest, the symptoms of the disease and its complications. Effective doses of
the
compositions of the present invention, for the treatment of the above
described conditions
will vary depending upon many different factors, including means of
administration, target
1 S site, physiological state of the patient, and other medicants
administered. Thus, treatment
dosages will need to be titrated to optimize safety and efficacy. Doses
ranging from about 10
ng to 1 g, 100 ng to 100 mg, 1 ~,g to 10 mg, or 30-300 ~,g DNA per patient are
typical.
Routes of administration include oral, nasal, gastric, intravenous,
intradennal and
intramuscular.
The nucleic acid-lipid complexes can also be used to transfect embryonic stem
cells or zygotes to achieve germline alterations.' See Jaenisch, Science,
240:1468-1474
(1988); Gordon et al., Methods Enzymol. 101, 414 (1984); Hogan et al.,
Manipulation of the
Mouse Embryo: A Laboratory Manual, C.S.H.L. N.Y. (1986); and Hammer et al.,
Nature
315:680 (1985); Gandolfi et al., J. Reprod. Fert. 81:23-28 (1987); Rexroad et
al., J. Ahim.
2S Sci. 66:947-9S3 (1988) and Eyestone et al., J. Rep~od. Fert. 85:71 S-720
(1989); Camous et
al., J. Rep~od. Feet. 72:779-785 (1984); Heyman et al., The~iogehology 27:5968
(1987).
However, these methods are presently more suitable for veterinary applications
that human
treatment due to ethical and regulatory constraints in manipulating human
embryos.
As an example, cystic fibrosis (CF) is a usually fatal recessive genetic
disease,
having a high incidence in Caucasian populations. The gene responsible for
this disease was
isolated by Riordan et al, Science 24S:1OS9-1065 (1989). It encodes a protein
called the
cystic fibrosis transmembrane conductance regulator (CFTR) which is involved
in the
transfer of chloride ions (C1) through epithelial cell membranes. Mutations in
the gene cause
defects of Cl- secretion in epithelial cells leading to the various clinical
manifestations.
47
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
Although CF has a number of symptoms including thickened exocrine gland
secretions,
pancreatic deficiency, intestinal blockage and malabsorption of fat, the most
serious factor
affecting mortality is chronic lung disease. Accordingly, to treat a CF
patient, a vector
containing a coding sequence for a functional CFTR gene product can be
complexed with
lipid, and optionally, a pharmaceutical excipient and introduced into the
patient via nasal
administration so that the vector-lipid composition reaches the lungs. The
dose of vector-
lipid complex is preferably about 108-101° particles.
As another example, defects in the a or 'y globin genes (see McDonagh &
Nienhuis in Hematology ofhcfancy and Childhood (eds. Nathan & Oski, Saunders,
PA, 1992)
at pp. 783-879) can be compensated for by ex vivo treatment of hemopoietic
stem cells with
an nucleic acid-lipid complex containing a functional copy of the gene. The
gene integrates
into the stem cells which are then reintroduced into the patient. Defects in
the gene
responsible for Fanconi Anemia Complement Crroup C can be treated by an
analogous
strategy (see Walsh et al., J. Clih. Invest. 94:1440-1448 (1994)).
Other applications include the introduction of a functional copy of a tumor
suppressor gene into cancerous cell or cells at risk of becoming cancerous.
Individuals
having defects in one or both copies of an endogenous tumor suppressor gene
are particularly
at risk of developing cancers. For example, Li-Fraumeni syndrome is a
hereditary condition
in which individuals receive mutant p53 alleles, resulting in the early onset
of various cancers
(Harris, Science 262:1980-1981 (1993) Frebourg et al., PNAS 89:6413-6417
(1992); Malkin
et al., Science 250:1233 (1990)). Expression of a tumor suppressor gene in a
cancerous cell
or a cell at risk of becoming cancerous is effective to prevent, arrest and/or
reverse cellular
proliferation and other manifestations of the cancerous state. Suitable tumor
suppressor
genes for use in the invention include p53 (Buchman et al., Gene 70:245-252
(1988)), APC,
DCC, Rb, WT1, and NF1 (Marx, Science 260:751-752 (1993); Marshall, Cell 64:313-
326
(1991)). Lipid-nucleic acid complexes bearing a functional copy of a tumor
suppressor gene
are usually administered in vivo by the route most proximal to the intended
site of action. For
example, skin cancers can be treated by topical administration and leukemia by
intravenous
administration.
4. Suppression of Gene Expression
Methods of gene therapy using the nucleic acid-lipid complexes of the
invention can also be used for prophylactic or therapeutic treatment of
patients or cells,
infected with or at risk of being infected with, a pathogenic microorganism,
such as HIV.
48
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
The effectiveness of antisense molecules in blocking target gene functions of
impeding virus
replication has been demonstrated in a number of different systems (Friedman
et al., Nature
335:452-54 (1988), Malim et al., Cell 58:205-14 (1989) & Trono at al., Cell
59:113-20
(1989)). The vector used includes a DNA segment encoding an antisense
transcript, which is
complementary to a segment of the genome from the pathogenic microorganism.
The
segment should preferably play an essential role in the lifecycle of the
microorganism, and
should also be unique to the microorganism (or at least absent from the genome
of the natural
genome of a patient undergoing therapy). For example, suitable sites for
inhibition on the
HIV virus includes TAR, REV or nef (Chatterjee et al., Science 258:1485-1488
(1992)). Rev
is a regulatory RNA binding protein that facilitates the export of unspliced
HIV pre mRNA
from the nucleus. Malim et al., Nature 338:254 (1989). Tat is thought to be a
transcriptional
activator that functions by binding a recognition sequence in 5' flanking
mRNA. Karn &
Graeble, Trends Genet. 8:365 (1992). The nucleic acid-lipid complex is
introduced into
leukocytes or hemopoietic stem cells, either ex vivo or by intravenous
injection in a
therapeutically effective dose. The treatment can be administered
prophylactically to HIV-
persons, or to persons already infected with HIV.
Analogous methods are used for suppressing expression of endogenous
recipient cell genes encoding adhesion proteins. Suppression of adhesion
protein expression
in useful in aborting undesirable inflammatory responses. Adhesion proteins
that can be
suppressed by antisense segments present in seelcted vectors include
integrins, selectins, and
immunoglobulin (Ig) superfamily members (see Springer, Nature 346:425-433
(1990).
Osborn, Cell 62:3 (1990); Hynes, Cell 69:11 (1992)). Integrins are
heterodimeric
transmembrane glycoproteins consisting of an a chain (120-180 kDa) and a (3
chain (90-110
kDa), generally having short cytoplasmic domains. The three known integrins,
LFA-l, Mac-
l and P 15 0,95, have different alpha subunits, designated CD 11 a, CD 11 b
and CD 11 c, and a
common beta subunit designated CD18. LFA-1 (aL(32) is expressed on
lymphocytes,
granulocyte and monocytes, and binds predominantly to an Ig-family member
counter-
receptor termed ICAM-1 (and perhaps to a lesser extent ICAM-2). ICAM-1 is
expressed on
many cells, including leukocytes and endothelial cells, and is up-regulated on
vascular
endothelium by cytokines such as TNF and IL-1. Mac-1 (aM(32) is distributed on
neutrophils
and monocytes, and also binds to ICAM-1 (and possibly ICAM-2). The third (32
integrin,
P150,95 (ax(32), is also found on neutrophils and monocytes. The selectins
consist of L-
selectin, E-selectin and P-selectin.
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
5. Cells to be transformed
The compositions and methods of the present invention are used to treat a
wide variety of cell types, in vivo and in vitro. Among those most often
targeted for gene
therapy are hematopoietic precursor (stem) cells. Other cells include those of
which a
proportion of the targeted cells are nondividing or slow dividing. These
include, for example,
fibroblasts, keratinocytes, endothelial cells, skeletal and smooth muscle
cells, osteoblasts,
neurons, quiescent lymphocytes, terminally differentiated cells, slow or
noncycling primary
cells, parenchyma) cells, lymphoid cells, epithelial cells, bone cells, etc.
The methods and
compositions can be employed with cells of a wide variety of vertebrates,
including
mammals, and especially those of veterinary importance, e.g, canine, feline,
equine, bovine,
ovine, caprine, rodent, lagomorph, swine, etc., in addition to human cell
populations.
To the extent that tissue culture of cells may be required, it is well known
in
the art. Freshney (1994) (Culture ofAnimal Cells, a Manual of Basic Technique,
third
edition Wiley-Liss, New York), Kuchler et al. (1977) Biochemical Methods in
Cell Culture
and Virology, Kuchler, R.J., Dowden, Hutchinson and Ross, Inc., and the
references cited
therein provides a general guide to the culture of cells. Cultured cell
systems often will be in
the form of monolayers of cells, although cell suspensions are also used.
Gene therapy relies on the efficient delivery of therapeutic genes to target
cells. Most of the somatic cells that have been targeted for gene therapy,
e.g., bematopoietic
cells, skin fibroblasts and keratinocytes, hepatocytes, endothelial cells,
muscle cells and
lymphocytes, are normally nondividing. Retroviral vectors, which are the most
widely used
vectors for gene therapy, unfortunately require cell division for effective
transduction (Miller
et al., Mol. Cell. Biol. 10:4239-4242 (1990)). This is also true with other
gene therapy
vectors such as the adeno-associated vectors (Russell et al., Proc. Nat).
Acad. Sci. USA
91:8915-8919 (1994); Alexander et al., J. Virol. 68:8282-8287 (1994);
Srivastrava, Blood
Cells 20:531-538 (1994)). Recently, HIV-based vectors has been reported to
transfect
nondividing cells. Nonetheless, the majority of stem cells, a preferred target
for many gene
therapy treatments, are normally not proliferating. Thus, the efficiency of
transduction is
often relatively low, and the gene product may not be expressed in
therapeutically or
prophylactically effective amounts. This has led investigators to develop
techniques such as
stimulating the stem cells to proliferate print to or during gene transfer
(e.g., by treatment
with growth factors) pretreatment with 5-fluorouracil, infection in the
presence of cytokines,
and extending the vector infection period to increase the likelihood that stem
cells are
dividing during infection, but these have met with limited success.
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
6. Detection of foreign nucleic acids
After a given cell is transduced with a nucleic acid construct that encodes a
gene of interest, it is important to detect which cells or cell lines express
the gene product and
to assess the level of expression of the gene product in engineered cells.
This requires the
detection of nucleic acids that encode the gene products.
Nucleic acids and proteins are detected and quantified herein by any of a
number of means well known to those of skill in the art. These include
analytic biochemical
methods such as spectrophotometry, radiography, electrophoresis, capillary
electrophoresis,
high performance liquid chromatography (HPLC), thin layer chromatography
(TLC),
hyperdiffusion chromatography, and the like, and various immunological methods
such as
fluid or gel precipitin reactions, immunodiffusion (single or double),
immunoelectrophoresis,
radioimmunoassays (RIAs), enzyme-linked inununosorbent assays (ELISAs),
immunofluorescent assays, and the like. The detection of nucleic acids
proceeds by well
known methods such as Southern analysis, northern analysis, gel
electrophoresis, PCR,
radiolabeling, scintillation counting, and affinity chromatography.
The selection of a nucleic acid hybridization format is not critical. A
variety
of nucleic acid hybridization formats are known to those skilled in the art.
For example,
common formats include sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in "Nucleic Acid
Hybridization, A Practical
Approach," Ed. Hames, B.D. and Higgins, S.J., IRL Press, 1985.
The sensitivity of the hybridization assays may be enhanced through use of a
nucleic acid amplification system which multiplies the target nucleic acid
being detected. In
vitro amplification techniques suitable for amplifying sequences for use as
molecular probes
or for generating nucleic acid fragments for subsequent subcloning are known.
Examples of
techniques sufficient to direct persons of skill through such in vitro
amplification methods,
including the polymerase chain reaction (PCR) the ligase chain reaction (LCR),
Q(3-replicase
amplification and other RNA polymerase mediated techniques (e.g., NASBA) are
found in
Berger, Sambrook, and Ausubel, as well as Mullis et al. (1987), U.S. Patent
No. 4,683,202;
PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic
Press Inc.
San Diego, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990), C&EN 36-
47; The
Journal OfNIHResea~ch, 3:81-94 (1991); (Kwoh et al., Proc. Natl. Acad. Sci.
USA, 86:1173
(1989); Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell et
al., J. Clin.
Chem., 35:1826 (1989); Landegren et al., Science, 241:1077-1080 (1988); Van
Brunt,
Biotechnology, 8:291-294 (1990); Wu and Wallace, Gene, 4:560 (1989); Barringer
et al.,
51
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
Gehe, ~~:117 (1990), and Sooknanan and Malek, Biotechnology, 13:563-564
(1NN5).
Improved methods of cloning ih vitro amplified nucleic acids are described in
Wallace et al.,
U.S. Pat. No. 5,426,039. Other methods recently described in the art are the
nucleic acid
sequence based amplification (NASBATM, Cangene, Mississauga, Ontario) and Q
Beta
Replicase systems. These systems can be used to directly identify mutants
where the PCR or
LCR primers are designed to be extended or ligated only when a select sequence
is present.
Alternatively, the select sequences can be generally amplified using, for
example, nonspecific
PCR primers and the amplified target xegion later probed for a specific
sequence indicative of
a mutation.
Oligonucleotides for use as probes, e.g., in in vitro amplification methods,
for
use as gene probes, or as inhibitor components are typically synthesized
chemically
according to the solid phase phosphoramidite triester method described by
Beaucage and
Caruthers, Tetrahed~°on Letts., 22(20):1859-1862 (1981), e.g., using an
automated
synthesizer, as described in Needham-VanDevanter et al., Nucleic Acids Res.,
12:6159-6168
(1984). Purification of oligonucleotides, where necessary, is typically
performed by either
native acrylamide gel electrophoresis or by anion-exchange HPLC as described
in Pearson
anal Regnier, J. Ch~om., 255:137-149 (1983). The sequence of the synthetic
oligonucleotides
can be verified using the chemical degradation method of Maxam and Gilbert
(1980) in
Grossman and Moldave (eds.) Academic Press, New York, Methods in Enzymology,
65:499-560.
An alternative means for determining the level of expression of the gene is i~
situ hybridization. Ih situ hybridization assays are well known and are
generally described in
Angerer et al., Methods Enzymol., 152:649-660 (1987). In an ih situ
hybridization assay cells
are fixed to a solid support, typically a glass slide. If DNA is to be probed,
the cells are
denatured with heat or alkali. The cells are then contacted with a
hybridization solution at a
moderate temperature to permit annealing of specific probes that are labelled.
The probes are
preferably labelled with radioisotopes or fluorescent reporters.
7. Detection of foreign gene products
The expression of the gene of interest to produce a product may be detected or
quantified by a variety of methods. Preferred methods involve the use of
specific antibodies.
Methods of producing polyclonal and monoclonal antibodies are known to
those of skill in the art. See, e.g., Coligan (1991), CURRENT PROTOCOLS IN
IMMUNOLOGY,
WileylGreene, NY; and Harlow and Lane (1989), ANTIBODIES: A LABORATORY MANUAL,
52
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
Cold ~prmg Harbor Press, NY; Stites et al. (eds.) BASIC AND CLINICAL
IMMUNOLOGY (4th
ed.) Lange Medical Publications, Los Altos, CA, and references cited therein;
Goding (1986),
MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New
York,
NY; and Kohler and Milstein, Nature, 256:495-497 (1975). Such techniques
include
antibody preparation by selection of antibodies from libraries of recombinant
antibodies in
phage or similar vectors. See, Huse et al., Sciehee, 246:1275-1281 (1989); and
Ward et al.,
Nature, 341:544-546 (1989). Specific monoclonal and polyclonal antibodies and
antisera
will usually bind with a KD of at least about .1 mM, more usually at least
about 1 ~.M,
preferably at least about .1 ~,M or better, and most typically and preferably,
.O1 ~,M or better.
The presence of a desired polypeptide (including peptide, transcript, or
enzymatic digestion product) in a sample may be detected and quantified using
Western blot
analysis. The technique generally comprises separating sample products by gel
electrophoresis on the basis of molecular weight, transferring the separated
proteins to a
suitable solid support, (such as a nitrocellulose filter, a nylon filter, or
derivatized nylon
filter), and incubating the sample with labeling antibodies that specifically
bind to the analyte
protein. The labeling antibodies specifically bind to analyte on the solid
support. These
antibodies are directly labeled, or alternatively are subsequently detected
using labeling
agents such as antibodies (e.g., labeled sheep anti-mouse antibodies where the
antibody to an
analyte is a marine antibody) that specifically bind to the labeling antibody.
V. Examples
Example l: The Effect of Calcium oh the Ti~ahsfectioh Potency of SPLP
A. Materials and Methods
1. Materials. N,N-dioleyl-N,N-dimethylammonium chloride (DODAC)
was obtained from Dr. S. Ansell and 1-O-(2-(w-methoxyethyleneglycol)succinoyl)-
2-N-
arachidoylsphingosine (PEG-CerC2o) was synthesized by Dr. Z. Wang at Inex
Pharmaceuticals Corporation (Burnaby, BC). 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)
were
obtained from Northern Lipids (Vancouver, BC). 1,2-dioleoyl-sn-glycero-3-
(phospho-L-
serine) (DOPS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(Lissamine
Rhodamine B Sulfonyl) (Rh-DOPE) were purchased from Avanti Polar Lipids
(Alabaster,
AL). Cholesterol (Chol), octylglucopyranoside (OGP), HEPES, MgCl2, and NaCI
were
53
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
obtained from Sigma Chemical Co. (St. Louis, MO). DEAF Sepharose CL-bti amomc-
exchange column and Sepharose CL-4B sizing column materials were obtained from
Sigma
Chemical Co. (St. Louis, MO). The luciferase assay kit was purchased from
Promega Corp.
(Madison, WI). Picogreen dsDNA detection reagent was obtained from Molecular
Probes
(Eugene, OR). Plasmid DNA (pCMVLuc) coding for the luciferase reporter gene
under the
control of the human CMV immediate early promoter-enhancer element was
obtained from
Inex Pharmaceuticals Corporation (Burnaby, BC). Bovine hamster kidney (BHK)
cells were
obtained from the American Tissue Culture Collection (ATCC CCL-10, Rockville,
MD) and
cultured in Dulbecco modified Eagle medium (DMEM) supplement with 10% fetal
bovine
serum (FBS), 100 U/ml of penicillin and 100 ~.g/ml of streptomycin. BHK cells
were
maintained as a monolayer at 37°C in a htunidified atmosphere
containing 5.0% C02.
2. Preparation of SPLP. SPLP were prepared as described by Wheeler,
et al., Gene Therapy 6:271-281 (1999)) with some modifications. Briefly, a
total of 10
.moles of DODAC, DOPE, PEG-CerC2o (7:83:10; mol/mol/mol) were dissolved in
chloroform and dried under a stream of nitrogen gas. Residual solvent was
removed under
high vacuum for 2 h. The resulting lipid film was hydrated in 1 mI of HBS
buffer (20 mM
HEPES and 150 mM NaCI, pH 7.5) containing 0.2 M OGP with continuous vortexing.
Plasmid DNA (400 ~,g/ml) was added to the hydrated lipids and the mixtures
were dialysed
against HBS buffer for 36 to 48 h with 2 buffer changes. Nonencapsulated
plasmid was
removed by DEAE anion exchange chromatography and empty lipid vesicles were
removed
by employing a sucrose density gradient as previously described (Mok, et al.,
Biochimica et
BiophysicaActa 1419:137-150 (1999)). For the high DODAC content formulation
(DODAC/DOPE/PEG-CerCZO, 14:76:10, mol/mol/mol), SPLP were initially prepared
in HBS
buffer containing 30 mM sodium citrate as described previously (Zhang, et al.,
Gehe Therapy
6:1438-1447 (1999)). SPLP were characterized with respect to plasmid
entrapment using a
previously described Picogreen assay (Zhang, et al., Gene Therapy 6:1438-1447
(1999)) and
sized using quasielastic light scattering.
3. Transfection in the presence of Ca2~. Prior to transfection, BHK
cells were plated at a density of 1 x 104 cells per well in a 96-well plate
overnight. 200 mM
CaCl2 stock solution was prepared in dH20 and sterilized by filtering. 0.5 ~.g
plasmid DNA
encapsulated in SPLP was used per well of transfection. SPLP were first added
to
appropriate concentrations of Ca2+ as required by the experiment, after which
culture media
54
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
was added to the mixtures to obtain the final transfection volume of 100
~Cl/well. Ca'T
concentration was calculated with respect to the final volume of the
transfection medium
applied to cells. The final volume contained 20% vol Ca2+ and SPLP mixtures
and 80% vol
culture media. Cells were incubated with the transfection complexes for the
appropriate time
periods before assaying for gene expression as described previously (Wheeler,
et al., Gene
Therapy 6:271-281 (1999)). Relative luciferase activity was normalized against
total cellular
protein determined by using the Micro BCA protein assay reagent kit (Pierce,
Illinois).
4. Determination of cellular lipid uptake. BHK cells were plated at 1 x
105 cells per well of 12-well plates the day prior to the experiment. SPLP
were prepared with
0.5 mol% Rh-DOPE incorporated into the lipid formulations. SPLP mixed with
increasing
concentrations of Ca2+ (0 to 14 mM) were added to cells at a lipid dose of 80
nmoles in
complete media (1 ml final volume). After incubation at 37°C for 4, 8,
and 24 h, cells were
washed with PBS and lysed by the addition of buffer containing 0.1% TX-100 in
250 mM
phosphate buffer (pH 8.0). Rhodamine fluorescence of the lysate was measured
on a Perkin
Elmer Luminescence Spectrophotometer using ~,eX of 560 nm and ~,e,T, of 590 nm
with slit
widths of 10 and 10 nm, respectively. Lipid uptake was determined by comparing
lysate
fluorescence to that of a lipid standard normalizing it to the total cellular
protein. To
determine the intracellular SPLP localization, fluorescence microscopy was
employed. Cells
were transfected with vesicles labeled with 4 mol% Rh-DOPE. The transfection
media was
replaced with complete media prior to analysis under the fluorescence
microscope.
Fluorescence micrographs were taken on an Axiovert 100 Zeiss Fluorescent
microscope (Carl
Zeiss Jena GrnbH) using a rhodamine filter from Omega Opticals (Brattleboro,
VT) with the
following specifications, 7~eX 560 ~ 20 nm, 600 nm LP, and DC 590 nm.
5. 31P NMR spectroscopy. Solid-state 31P NMR spectra were recorded
with broad-band decoupling at 81.02 MHz on a Bruker MSL 200 spectrometer,
using a 3.8-
~s 60° pulse and a 1.5-s repeat time. The free induction decay (FID)
was accumulated over
2500-3000 scans and was Fourier transformed with 50-Hz line broadening.
Phospholipid
mixtures (25 ~.mol of total phospholipid) were dispersed by vortex mixing in 2
ml of buffer
(20 mM HEPES buffer, pH 7.4). Increasing concentrations of Ca2+ were titrated
into the
vesicles by adding aliquots of 200 mM CaCl2 stock. Caa+equilibration was
ensured by
performing three cycles of freeze-thawing. The temperature was maintained at
25 °C with a
CA 02406654 2002-10-18
Brwu k wagro able temperature unit. A mixture of phosphoric acid/D20 was sed a
tlieoosss
reference for chemical shifts in all 31P NMR spectra.
6. Intracellular processing of plasmid DNA. BHK cells were plated at
3 x 105 cells per well of 6-well plates the day prior to the experiment. 2.5
~g plasmid DNA
encapsulated in SPLP were incubated with cells for 2, 4, and 8 h, in the
absence or presence
(8 mM) of Caa+. At the appropriate time points, cells were washed with PBS and
external
SPLP were removed by trypsinization. Trypsinized cells were pelleted by
centrifugation and
cells were resuspended and washed with isotonic buffer (250 mM sucrose, 3 mM
MgCl2, 50
mM HEPES, pH 7.2). Subsequently, pelleted cells were lysed by incubating with
250 ~,1 of
lysis buffer (10 mM Tris, pH 7.5, 0.5% SDS, 1 mM EDTA) containing Pronase E at
1 mg/ml
(Sigma) overnight at 37°C. DNA (genomic DNA and delivered plasmid DNA)
were
extracted as described previously (Sambrook, et al., In Molecular Cloning: A
Laboratory
Manual I, 1.21-1.52 (1989). Cold Spring Harbor, New York, C. Nolan, editor.
Cold Spring
Harbor Laboratory). DNA recovery was determined by measuring the absorbaizce
at 260 nrn.
6 ~g of total DNA from each sample was either dot blotted onto a nylon
transfer membrane
(Amersham) with a set of pCMVLuc standards (0 to 5 pg) or loaded into a 1%
agarose gel
and size fractionated at 60 V for 2 h for the Southern analysis. Both blots
were hybridized
overnight at 68°C to a 32P-labeled plasmid DNA probe, which was
prepared with PstI cut-
pCMVLuc plasmid using the T~QuickPrimeTM Kit (Pharmacia Biotech). Blots were
washed 3
times with 2x SSC containing 0.1% SDS, and were then exposed on a
PhosphoImager screen
which was subsequently scanned (Molecular Dynamics - PhosphoImagerTMSI).
7. Entrapment of Ca2+ inside SPLP. SPLP (DODAC/DOPE/PEG-
CerC20/Rd-DOPE, 10:79.5:10:0.5, mol/mol/mol) were initially prepared in
citrate buffer
(150 mM sodium citrate and 150 mM citric acid) at pH 4. Nonencapsulated
plasmid was
removed by DEAF anion exchange chromatography equilibrated in HBS buffer (pH
7.5) and
empty lipid vesicles were removed by employing a sucrose density gradient as
previously
described (Mok, et al., Biochimica et Biophysica Acta 1419:137-150 (1999)).
Ca2+ loading
was performed by incubation of the DNA-loaded vesicles with 2.5 mM CaCl2 and
the
ionophores A23187 (O.l~,g/pmole lipids) for 30 min at room temperature.
Unloaded Ca2+
and ionophores were removed by dialysis in HBS buffer with 2 buffer changes.
Internal Ca2+
concentrations were determined in the absence and presence of TX-I00 (0.2%) by
employing
the membrane nonpermeant absorbant indicator Asenazo III (0.1 mM in 10 mM
HEPES
56
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
buffer, pH 7), against a CaCl2 standard curves (0 to 50 nmoles). Absorbances
at 650 nm were
measured as an indicator of Ca2+ presence. SPLP were characterized with
respect to plasmid
entrapment using a previously described Picogreen assay (Zhang, et al., Gene
Therapy
6:1438-1447 (1999)) and sized using quasielastic light scattering. Internal
concentrations of
Ca2+ were found to be 175 tnM.
8. Insertion of CPL. Prior to CPL insertion, SPLP were prepared as
described in the previous section with some modification. SPLP containing
total of 10
p,moles of DODAC, DOPE, PEG-CerC2o, and Rd-DOPE (7:82.5:10:0.5;
mol/mol/mol/mol)
were hydrated in 1 ml of HBS buffer (20 mM HEPES and 150 mM NaCI, pH 7.5)
containing
0.2 M OGP with continuous vortexing. Plasmid DNA (400 ~,g/ml) was added to the
hydrated
lipids and the mixtures were dialysed against HBS buffer for 36 to 48 h with 2
buffer
changes. Nonencapsulated plasmid was removed by DEAE anion exchange
chromatography.
CPL stocks in methanol labeled with a dansyl fluorescence marker were added to
the SPLP to
give the desired molar ratio (up to 4 mol% CPL relative to vesicle lipid). CPL
and SPLP
were incubated for up to 3 h at 60°C, and cooled on ice to room
temperature. Both empty
lipid vesicles and noninserted CPL were removed by employing a sucrose density
gradient.
The insertion levels of CPL were quantitated by using the Perkin Elmer
Luminescence
Spectrophotometer. Briefly, initial dansyl/rhodamine (D/R;) fluorescence ratio
prior to
sucrose density gradient and the final D/R (D/Rf) ratio of the isolated CPL-
SPLP were
measured. Rhodamine flouresence was assayed at ~,eX= 560 nm and 7~e", = 590
nm, wlule
dansyl fluorescence was assayed at ~,eX= 340 nm and ~,e", = 510 nm, with slit
widths of 10
and 10 nm. The%-insertion was calculated as follows:
-insertion = ([D/R] f)* 100/(D/R);
CPL-SPLP were further characterized with respect to plasmid entrapment
using a previously described Picogreen assay (Zhang et al., 1999) and sized
using
quasielastic light scattering.
B. Results
1. The transfection potencies of SPLP are dramatically enhanced in
the presence of Ca2+. Previous work has shown that SPLP, particularly SPLP
stabilized by
PEG-CerC2o, can exhibit lower levels of transfection irc vitro (Wheeler, et
al., Gehe They~apy
6:271-281 (1999); Mok et al., Biochimica et Biophysica Acta, 1419:137-150
(1999)). Here,
57
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
the effects of La2+ on the transfection potency of SPLP were examined. SPLY
prepared i~om
DOPE/DODAC/PEG-CerC2o (84:6:10; mol:mol:mol) lipid mixture and pCMVLuc
employing the detergent dialysis method were purified from empty vesicles and
unencapsulated plasmid as described in the above Materials and Methods.
Appropriate
amounts of CaCl2 were then added to the SPLP preparation to give rise to the
desired Ca2+
concentrations following dilution into the media before applying to the BHI~
cells. The BHK
cells and the SPLP were incubated together for 24 h, after which the
transfected cells were
assayed for luciferase expression.
As shown in Figure 1, the presence of Ca2+ resulted in dramatic enhancements
in luciferase expression levels, with a 600-fold increase in SPLP transfection
potency
observed at the optimal Ca2+ concentrations. This Ca2+-mediated increase in
transfection is
significantly greater for the SPLP system than previously observed for plasmid
DNA-cationic
lipid complexes. The optimal concentrations of Ca2+ required for stimulating
SPLP
transfection potencies were in the range of 8 to 10 mM, somewhat lower than
that required
(5-25 mM) for optimal stimulation of the transfection potencies of plasmid DNA-
cationic
lipid complexes. Further, the ability of Ca2+ to stimulate the transfection
potency of SPLP
was highly specific. As shown in Figure 1, if MgCl2 or NaCI was substituted
for CaCl2 no
enhancement in transfection potency was observed.
2. SPLP are stable in the presence of Ca2+. SPLP with PEG-CerC2o are
highly stable systems that exhibit extended circulation times in vivo, protect
encapsulated
plasmid from external nucleases, and do not interact readily with cells
(Wheeler, et al., Gehe
Therapy 6:271-281 (1999); Mok, et al., Biochimica etBiophysicaActa 1419:137-
150 (1999);
Monck, et al., Journal of Drug Targeting 7:439-452 (2000)). It was therefore
important to
demonstrate that the enhanced transfection properties of SPLP in the presence
of Ca2+ was
not due to destabilization or aggregation of the SPLP leading to enhanced cell
uptake. The
stability of the SPLP in the presence of Ca2+ was examined employing quasi-
elastic light
scattering (QELS) to detect changes in size and the Picogreen fluorophore
assay to detect
DNA leakage. For the QELS experiments, CaCl2 was added to the SPLP suspension
to
achieve concentrations as high as 50 mM. No change in the SPLP size or size
distribution
was observed. For the plasmid release experiments, SPLP were incubated at
37°C in HBS
buffer containing 10% FBS in the presence or absence of 8 mM Ca2+. Plasmid
release was
assayed over 24 h employing the Picogreen assay. No plasmid release was
observed.
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CA 02406654 2002-10-18
WO 01/80900 Z+ CT/CA01/00555
3. Ca does not influence the cellular uptake of SPY. l;he ability of
Ca2+ to enhance the transfection activity of plasmid DNA-cationic lipid
complexes has been
attributed to an increase in the uptake of the complexes into cells in the
presence of Ca2+
(Lam, et al., Biochim Biophys Acta 1463:279-290 (2000)). In this regard, the
low
transfection potencies of SPLP as compared to complexes arise, at least in
part, from very
low levels of cellular uptake of SPLP (Mok, et al., Biochimica et Biophysics
Acta 1419:137-
150 (1999)). It was therefore of interest to determine whether Ca2+ stimulated
SPLP
transfection potencies by increasing SPLP uptake into cells. SPLP containing
0.5 mol% Rh-
DOPE were employed to determine SPLP uptake into BHK cells in the presence of
up to 12
mM Ca2+ as described in the above Materials and Methods. The SPLP were
incubated with
cells for 4, 8, and 24 h and the levels of intracellular lipid determined.
Lipid uptake at each
time-point was normalized against total cell protein in order to account for
cell growth. As
shown in Figure 2, the results indicate that Ca2+ did not significantly
increase the cellular
uptalee of SPLP even though the transfection potencies of the SPLP varied by
several
hundred-fold over the range of Ca2+ concentrations tested.
4. Fluorescence studies indicate enhanced endosomal destabilization
following SPLP uptake in the presence of Ca2+. The fact that uptake of SPLP is
not
stimulated by addition of Ca2+ suggests that the Ca2+-dependent enhancement of
transfection
must arise from more efficient utilization of SPLP that are accumulated. One
possibility is
that Ca2+ somehow facilitates destabilization of endosomes following uptake of
SPLP, thus
enhancing intracellular delivery of plasmid. Previous work has shown that
endosornal
destabilization following uptake of vesicles containing fluorescently-labeled
lipids can be
detected by fluorescence microscopy as a diffuse intracellular fluorescence,
whereas uptake
into stable endosomes gives rise to a localized "punctate" appearance
(Felgner, et al., Proc.
Natl. Acad. Sci. USA 84:7413-7417 (1987)). In order to be able to visualize
the cellular
distribution of SPLP, a higher level of Rh-DOPE (4 mol%) was incorporated with
the vesicle
formulation. Such Rh-labeled SPLP were incubated on BHK cells in the presence
and
absence of 10 mM Ca2+ and the cell morphology was examined at 8 h by
fluorescence
microscopy. Similar levels of rhodamine fluorescence were detected in the
absence or
presence of Ca2+, in agreement with the quantitative measurements of SPLP
uptake noted in
the previous section. However, as shown in Figure 3, the appearance of the
cells as detected
by fluorescence microscopy was quite different in the presence or absence of
Ca2+. Although
some punctate structures are observed, BHK cells containing the fluorescently-
labeled SPLP
59
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
exhibited a more diffuse pattern when Ca2+was included. In the absence of Ca'
, the
fluorescence pattern was largely punctate, consistent with SPLP retention in
the endosomal
compartments.
5. Intracellular processing of plasmid DNA. The preceding fluorescent
microscopy results suggest that Ca2~ enhances transfection by destabilizing
the endosomal
compartments, thus enhancing cytoplasmic delivery of the SPLP-associated
plasmid. If
SPLP plasmid can escape from the endosome more readily in the presence of
Ca2~, it will
avoid breakdown in the lysosomal pathway and more intact intracellular plasmid
DNA
should be present. A dot blot assay was employed to measure intracellular
delivery of
plasmid DNA, and the integrity of the plasmid was examined by using the
Southern blot
analysis. Cells were incubated with SPLP in the absence or presence of 8 mM
Ca2+ for 2, 4,
and 8 h. The levels of intact, intracellular plasmid DNA for the different
systems were
compared after isolation of DNA from the cells as described in the above
Materials and
Methods, and the results are shown in Figure 4. As shown in Figure 4A, when
cells were
transfected with the SPLP in the presence of Ca2+, the amount of intact
plasmid in the BHK
cells was increased by approximately 10-fold after an 8 h incubation period.
This is also
reflected by a Southern analysis which showed that more intact plasmid DNA was
present in
cells transfected with SPLP prepared in the presence of Ca2+ (Figure 4B). Such
enhanced
levels of intact plasmid DNA were not observed when Mg2+ was substituted for
Ca2~,
demonstrating the specificity of Ca2+ (Figure 4B).
6. Ca2~ destabilizes bilayer lipid structures in a manner consistent
with an ability to destabilize endosomal membranes. Recent work suggests that
cationic
lipids stimulate intracellular delivery of macromolecules such as plasmid DNA
by combining
with anionic lipids and forming ion pairs that destabilize bilayer membranes
by inducing
nonbilayer (HII phase) structure. In this regard, it is well known that Ca2+
can destabilize
lipid bilayers containing acidic lipids such as phosphatidylserine (PS) in
combination with
unsaturated PEs by inducing the nonbilayer hexagonal Hn phase structure (Hope,
et al., FEBS
Letters 107:323-326 (1979); Tilcock, et al., Biochimica et Biophysics Acta
641:189-201
(1981)). It has also been shown that Ca2+ can induce HB phase structure in
related systems
containing phosphatidylcholine (PC) and cholesterol. For example, addition of
Ca ~ to
mixtures of DOPC/DOPE/DOPS/Cholesterol (1:1:1:3; molar ratios) also triggers
bilayer to
hexagonal HII phase transitions (Tilcock, et al., Biochemistry 23:2696-2703
(1984)). It is
CA 02406654 2002-10-18
WO 01/80900 Z+ PCT/CA01/00555
thus possible that Ca stimulates SPLP transfection by acting in concert mth
the cationic
lipid in the SPLP to destabilize the lipid bilayer of endosomal membranes.
In order to investigate this possibility, the Ca2+-dependent polymorphism of
MLV composed of DOPC/DOPE/DOPS/Chol (1:1:1:3; molar ratios) was investigated
in the
absence and presence of small amounts of DODAC employing 31P NMR. Considerable
previous work has shown that phospholipids in the bilayer organization give
rise to
asymmetric 31P NMR line shapes with a low field shoulder and high field peak,
whereas
phospholipids in the hexagonal HII phase give rise to a line shape with
reversed asymmetry
that is a factor of two narrower (Cullis, et al., Biochimica et Biophysics
Acta 559:399-420
(1979)). As shown in Figure 5A, in the absence of DODAC, Ca2+ is able to
stimulate a
transition from the bilayer to the hexagonal HII phase as reported by 31P NMR
at the Ca2+-to-
DOPS ratio of 0.5:1. Alternatively, in MLV containing small amounts of DODAC
(DOPC/DOPE/DOPS/Cholesterol/DODAC; 1:1:1:3:0.25; molar ratios), Ca2+-to-DOPS
ratios
of only 0.25:1 are required to induce predominantly Hn phase structure (Figure
SB). The
narrow central peak may arise from small lamellar structures or lipid in
nonbilayer structures
such the as cubic phase in which component phospholipids experience isotropic
motional
averaging.
7. External Ca2+ is required to enhance SPLP transfection potency.
A final set of experiments was conducted to determine whether Ca2+
encapsulated within the
SPLP could stimulate transgene expression. As detailed elsewhere (Felgner, et
al., Journal of
Biological Chemistry 269:2550-2561 (1994)), Ca2+ can be loaded into large
unilamellar
vesicles (LUV) in response to a pH gradient (inside acidic) when the Ca2+
ionophore A23187
is present. Internal Ca2+ concentrations as high as 200 mM ca,n be achieved.
As described in
the above Materials and Methods, SPLP could be readily prepared at pH 4 in the
presence of
a citrate buffer and then the external pH could be raised to pH 7.5 following
the detergent
dialysis procedure. Addition of external CaCl2 and ionophore then resulted in
loading of Ca2+
into the SPLP to achieve internal concentrations of 175 mM. As shown in Figure
6,
although the presence of encapsulated Ca2+ result in enhancement of SPLP
transfection
potency, it appears that external levels of Ca2+ play the dominant roles in
stimulating the
transfection process.
8. Effect of Ca2+ on improved SPLP systems. One limitation of SPLP
is that the system is not optimally taken by cells as a result of limited
cationic lipid and
61
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
presence of Y~G on the vesicles (Mok, et al., Biochimica et Biophysica Acta
141:137-150
(1999)). One straightforward way to increase the positive charges is by
increasing the
cationic content (Zhang, et al., Gehe Therapy 6:1438-1447 (1999)). However,
transfection
e~ciencies increased with SPLP increased DODAC concentrations, and such
systems are
thus compromised with lower DNA encapsulation. Recently, a new class of
cationic lipid
known as cationic polyethylene glycol) lipid conjugates (CPL) has been
synthesized (Chen,
et al., Bioconjugate Chem. 11:433-437 (2000)). A typical CPL employed in this
study
contains a hydrophobic ceramide anchor, which is attached to a hydrophilic PEG
spacer that
is linked to a cationic headgroup made of four lysine residues. It has been
shown that SPLP
w/ CPL inserted onto its surface show enhanced interaction between the
liposomes and cell
plasma membrane (Chen, et al., Bioconjugate Chem. 11:433-437 (2000)).
Experiments were carried out to determine the influence of Ca2+ on the CPL-
SPLP system. SPLP containing higher DODAC content (14 mol%) was also included.
For
the CPL-SPLP preparation, plasmid DNA was loaded into liposomes using the
previously
described detergent dialysis method and CPL were inserted into the preformed
SPLP using a
characterized insertion method. CPL were inserted to obtain a final 4 moI%
insertion
effciency, as this level has been shown to provide optimal cellular binding
and uptake. Ca2+
at 8 mM was added to the SPLP and CPL-SPLP preparations, diluted into the
media before
applying to the BHI~ cells. Gene expression was determined by assaying for
luciferase, 24 h
after incubation of the BHI~ cells together with the transfecting Iiposomes.
As shown in
Figure 7, a 2000-fold increase and a 105-fold increase in transfection were
detected for the
SPLP containing either higher DODAC content or CPL, respectively.
C. Discussion
This example demonstrates that Ca2+ gives rise to a large enhancement of
SPLP transfection potency in vitro.
The mechanism whereby Ca2+ stimulates the transfection potency of SPLP
must account for several observations. First, the enhanced transfection
appears to result from
higher intracellular levels of intact plasmid in the presence of Caz+; these
higher levels of
plasmid do not appear to arise from increased uptake of SPLP into cells,
however. Second,
the process is associated with a reduction in the "punctate" appearance of
cells following
uptake of fluorescently labeled SPLP. Finally, the effect is Ca2+ specific.
The first two
observations are clearly consistent with enhanced endosomal destabilization of
the BHK cells
following endocytosis of SPLP. The question thus remaining is how Ca2+ could
promote this
62
CA 02406654 2002-10-18
WO 01/80900 , PCT/CA01/00555
destabilization m a specific manner. In this regard, there is presently no
consensus as to how
endosomes can be destabilized to enhance release of their contents, however a
number of
leading observations have been made. Chief amongst these is the observation
that cationic
lipids can dramatically enhance the intracellular delivery of macromolecules
such as plasmids
and antisense oligonucleotides (Bennett, et al., Mol. Pharmacol. 41:1023-1033
(1992);
Baryon, et al., Gene Ther. 6:1179-1183 (1999)) and that this process appears
to rely on an
ability of cationic lipids to destabilize endosomal membranes, thus
facilitating intracellular
release of endosomal contents (Wattiaux, et al., FEBS Letters 417:199-202
(1997); Xu, et al.,
Biochemistry 35:5616-5623 (1996)). Recent work has shown that cationic lipids
exhibit as a
general property the ability to combine with anionic lipids to form nonbilayer
hexagonal Hn
phase structure, leading to the proposal that the mechanism whereby cationic
lipids
destabilize endosomes relies on an ability to disrupt the bilayer organization
of the endosomal
membrane. In the same vein, if Ca2+ could disrupt bilayer organization and
induce Hn phase
structure similar enhancements in intracellular delivery would be expected.
There is considerable evidence that Ca2+ can induce Hu phase structure iri
previously bilayer lipid systems containing anionic lipids, and that this
effect is Ca2+-specific,
as other cations such as Mg2+ either cannot induce Ha structure or require
higher
concentrations to produce similar effects (Tilcock, et al., Biochemistry,
23:2696-2703
(1984)). As shown in the present example, Ca2+ can induce Hu phase structure
in bilayers
composed ofDOPC:DOPE:DOPS:Chol and can act in synergy with low levels of the
cationic
lipid DODAC to trigger HII phase formation. While it is difficult to directly
relate the model
membrane behaviour to the behaviour inside the endosome, it is known that the
anionic lipid
content of endosomes increases as they move from "early" to "late" stages due
to formation
of a novel acidic lipid (lysobisphosphatidic acid; LBPA) and that mixtuxes of
LBPA with
cationic lipids such as DODAC adopt the HII phase. These results therefore
support the
theory that Ca2+ enhances transfection by promoting endosomal destabilization
in synergy
with cationic lipid. Such a proposal is also in agreement with the observation
that the
addition of Ca2+ to LBPA results in forniation of the Hu phase. Other workers
have
suggested that Ca2+ plays a role in mediating endosomal release during calcium
phosphate
(CaP;) mediated transfection (Loyter, et al., Proc. Natl. Acad. Sci. USA
79:422-426 (1982);
Orrantia, et al., Experimental Cell Research 190:170-174 (1990)), as well as
in polycation-
mediated gene transfer (Bottger, et al., Biochimica et Biophysics Acta I39S:78-
87 (1998);
Haberland, et al., Biochimica et Biophysics Acta 1445:2I-30 (1999)).
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
A surprising aspect of the present study concerns the discrepancy between the
influence of Ca2+ on the transfection properties of plasmid DNA-cationic lipid
complexes
previously reported (Lam, et al., Bioehim. Biophys. Acta 1463:279-290 (2000))
and the
results reported here for SPLP. In particular, the previous work demonstrated
that Ca2+ could
enhance the transfection potency of complexes by up to 20-fold and that this
could be
attributed to enhanced uptake of the complexes into the cells, rather than
enhanced
endosomal release. The surprising aspect is that the increased transfection
potency of SPLP
in the presence of Ca2+ could not be related to increased uptake of SPLP by
the cells, whereas
Ca2+ caused at least a 2-fold increase in uptake of complexes as evidenced by
uptake of both
I 0 lipid and plasmid (Lam, et al., Biochim Biophys Acta 1463 :279-290
(2000)). It is likely that
this discrepancy is related to the much different physical properties of SPLP
as compared to
complexes. Complexes are large, positively charged systems containing high
(equimolar)
levels of cationic lipid, whereas SPLP are small, stable, essentially neutral
vesicles with a
PEG coating that contain low levels of cationic lipid. The low levels of
cationic lipid in
I S SPLP as compared to complexes may be directly related to enhanced
sensitivity to Ca 2+, as
the cationic lipid present in the SPLP may be insufficient to combine with all
available
anionic lipid in the endosome, thus requiring the additional presence of
Ca2+to achieve
maximum destabilization.
The final topic of discussion concerns extension of the results presented here
20 to generate SPLP that exhibit enhanced transfection potencies in vivo. As
emphasized
elsewhere (Wheeler, et al., Gene Therapy 6:271-281 (1999); Zhang, et al., Gene
Therapy
6:1438-1447 (1999)), a preferred method of SPLP delivery is by systemic
application, where
long circulation lifetimes and accumulation at disease sites such as tumour
sites is required.
The present results suggest that Ca2+ is preferably outside the SPLP in order
to give rise to
25 enhanced transfection. To achieve this, strategies aimed at increasing
surface Ca2+
concentrations by attachment of Ca2+-chelating agents to SPLP should give rise
to enhanced
i~ vivo transfection. In addition, a local increase in calcium concentration
can be produced at
the site of transfection, e.g., by local (e.g., intratumoral) delivery of the
SPLP along with a
high concentration of calcium, or by systemic delivery of the SPLP combined
with local
30 delivery of calcium to the desired site of transfection.
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CA 02406654 2002-10-18
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Example 11: Stabilized Plasmid-Lipid Particles Containing Cationic P~c_i LWids
~;xlvbit
Enhanced Transfection Potencies
A. Materials and Methods
1. Materials. DOPE was obtained from Northern Lipids Inc. (Vancouver,
BC). Rh-PE, and PicoGreen were obtained from Molecular Probes (Eugene, OR).
DODAC
was synthesized and supplied by Dr. S. Ansell of Inex Pharmaceuticals
(Vancouver, BC).
PEG-CerC2o was synthesized as indicated elsewhere (Webb, et al., Biochim.
Biophys. Acta
1372:272-282 (1998)) and was supplied by Dr. Z. Wang of Inex Pharmaceuticals
(Vancouver, BC). The pCMVLuc plasmid encodes the Photihus pyralis luciferase
gene
I O under the control of the human CMV early promoter and was supplied by Dr.
P. Tam of Inex
Pharmaceuticals (Vancouver, BC). The pCMVGFP plasmid contains the gene for the
green
fluorescent protein from Aequorea victoria and was supplied by Dr. P. Tam of
Inex
Pharmaceuticals (Vancouver, BC). DEAF-Sepharose CL-6B, Sepharose CL-4B, octyl-
(3-D-
galactoside, and HEPES were obtained from Sigma-Aldrich (Oakville, ON).
Lipofectin was
obtained from Gibco BRL (Burlington, ON). BHK cells were obtained from Dr. R.
MacGillivray of the Department of Biochemistry and Molecular Biology, UBC.
2. Preparation of SPLP-CPL4. SPLP composed of
DOPE:DODAC:PEG-CerC2o (84:6:10) and containing the plasmid pCMVLuc (or
pCMVGFP) were prepared according to the method of Wheeler, et al. (Gene
Therapy 6:271-
281 (1999)) using purification by anion exchange (DEAF-Sepharose CL-6B)
chromatography and sucrose density gradient centrifugation to remove
unencapsulated
plasmid and empty vesicles, respectively. SPLP containing Rh-PE were prepared
by
dissolving Rh-PE with other component lipids in CHC13 at a molar ratio of
83.5:10:6:0.5
(DOPE:DODAC:PEG-CerC2o:Rh-PE) prior to forming the lipid film.
CPL4 was inserted into preformed SPLP by incubating SPLP (500 nrnol lipid)
with CPL4 (12.5, 19, and 30 nmol) at 60°C for 2 to 3 h in Hepes
buffered saline (HBS),
pH 7.5, unless otherwise indicated. Unincorporated CPL4 was removed by gel
filtration
chromatography on a Sepharose CL-4B column equilibrated in HBS. Fractions (1
ml) were
collected and assayed for CPL4, phospholipid and DNA content. Fractions
containing all
three components were pooled and concentrated. CPL4 content was determined by
the
fluorescence of the dansyl labeled CPL at ~,em= 510 nm following excitation at
~,eX = 340 nm
employing a Perkin Elmer LS52 Luminescence spectrophotometer with excitation
and
CA 02406654 2002-10-18
WO 01/80900 , PCT/CA01/00555
emission slit widths of 10 and 20 nm, respectively. A standard curve was
derived lTOm a
stoclc solution of dansylated CPL in HBS. For SPLP containing Rh-PE the
phospholipid
content was determined from the fluorescence of the Rh label measured at 7~em
590 nm
following excitation at ~,eX=560 nm, using excitation and emission slit widths
of 10 and 20
nm, respectively. For SPLP that did not contain the Rh label, phospholipid was
determined
using the method of Fislce-Subbarow (J. Biol. Chem. 66:375-400 (1925))
following lipid
extraction according to Bligh and Dyer (Cah. J. Biochem. Physiol. 37:911-917
(1959)).
Plasmid DNA was determined using the PicoGreen Assay kit (Molecular Probes,
Eugene,
Oregon) as previously described (Mok, et al., Biochim. Biophys. Acta 1419:137-
150 (1999)).
For the Rh-PE containing systems, the incorporation of CPL4 was determined
by dividing the dansyl to rhodamine ratio before the Sepharose column by that
after the
column multiplied by 100%. For the other systems, incorporation was determined
by
dividing the CPL4 content by the total lipid content and multiplying by 100%.
Lipoplexes were prepared at a charge ratio of 1.5:1 (positive-to-negative) by
adding 25 ~,L of 88 ~g/mL plasmid DNA (pCMVLuc or pCMVGFP) with 25 ~,L of
DOPE:DODAC (0.8 mM) while vortexing followed by incubation at room temperature
for
30 min prior to addition to cells. Lipofectin lipoplexes were similarly
prepared.
Quasi-elastic light scattering (QELS) studies were conducted employing a
Nicomp Model 270 Submicron Particle Sizer operating in the vesicle mode.
Freeze-fracture
electron microscopy studies were performed as described by Wheeler et al.,
supra.
DNA for Southern analysis was extracted using a phenol:chloroform
extraction following incubation of SPLP systems with 50% mouse serum. The
resulting
DNA was then subjected to electrophoresis through a 1% agarose gel,
transferred to a nylon
membrane (Amersham) and subjected to Southern analysis. The membrane was
exposed to
random-primed 32P-labelled PvuII restriction fragment from the luciferase gene
according to
current protocols. Hybridization intensities were quantified using a
PhosphorimagerTM SI
from Molecular Dynamics. The data were converted to give amounts of intact DNA
relative
to undigested DNA.
Levels of PEG-CerC2o and DOPE were determined by HPLC analyses
performed by Northern Lipids, Inc, Vancouver, B.C.
3. Uptake and transfection studies. A transformed BHK cell line (tk-)
was used for all uptake and transfection studies. To determine the cellular
uptake of SPLP,
1x105 BHK cells were seeded in each well of a 12-well plate and incubated
overnight in 2 ml
66
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555,
of complete media (DMEM containing 10% FBS) at 37°C in 5% C02. SPLY,
~YLY-C;YL4 m
media containing 40 mM CaCl2, or DOPE:DODAC lipoplexes in a volume of 200 ~,L
were
mixed with 800 ~,L of complete media at a final lipid dose of 20 ~.M and was
added to the
cells. Plasmid DNA concentrations corresponded to 1.4 ~.g/mL and 2.2 ~.glmL
for the SPLP
systems and the lipoplexes, respectively. Cells were incubated at 37°C
for indicated periods,
washed twice with PBS and lysed with 600 ~,L of lysis buffer (0.1% Triton X-
100 in PBS).
Rhodamine fluorescence was determined using a ~,eX of 560 nm and a 7~e", of
600 nrn with slit
widths of 10 and 20 nm, respectively. An emission filter of 530 nm was also
used. Lipid
uptake was determined by comparison of the fluorescence in the lysate to that
of a lipid
standard and normalized to the cell number as determined by the BCA protein
assay (Pierce,
Rockford, IL). Where indicated, fluorescence micrographs were obtained using
an Axiovert
100 Zeiss Fluorescent microscope (Carl Zeiss Jena GmbH) using a rhodamine
filter from
Omega Opticals (Brattleboro, VT) with the following specif canons: excitation
560~20/dichroic filter 590/long pass emission 600.
The effect of Ca2+ and Mg2+ on lipid uptake was determined as described
above with the following exceptions. BHI~ cells (5x104 per well) were seeded
in a 24-well
plate in 1 mL of complete media and incubated overnight at 37°C. SPLP-
CPL4 (40 nmol)
were mixed with CaCl2 or MgCl2 in a total volume of 100 ~,L. Complete media
(400 ~L) was
added to the SPLP-CPL4 resulting in final cation concentrations of 4 to 14 mM.
This mixture
was then added to the cells and incubated for 4 h at 37°C. Cells were
then washed twice with
PBS and lysed in 600 ~.L of lysis buffer (0.1 % Triton X-100 in PBS).
Unless otherwise indicated, transfection studies were performed employing
1 x 104 BHK cells plated in each well of a 96-well plate in 150 p,L complete
media prior to
overnight incubation at 37°C in 5% C02. SPLP and SPLP-CPL4
corresponding to 0.5 ~,g of
pCMVLuc in 20 ~L HBS (SPLP), or HBS containing 40 mM CaCl2 (SPLP-CPL4) were
added to 80 ~.L of complete media for a plasmid concentration of 5.0 ~,g/mL. A
transfection
time of 4 h with a total incubation time of 24 h was used routinely. The
transfection time is
defined as the time the cells are incubated with the plasmid-containing
particles whereas the
total incubation time is the transfection time (after which the transfection
media is replaced)
plus the subsequent time the cells are incubated for prior to assaying for
transgene
expression. After 24 h, the cells were lysed with 100 ~,L of lysis buffer, and
40 p,L of the
lysate was transferred to a 96-well luminescence plate. Luciferase activity
was determined
using a Luciferase reaction kit (Promega, Madison, WI), a luciferase standard
(Boehringer-
67
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
Manheim), and a ML3200 microtiter plate luminometer from Molecular Dynamics
(Chantilly, VA). Activity was normalized to the number of cells as determined
by the BCA
protein assay (Pierce, Rockford, IL).
The transfection time course study included SPLP, SPLP-CPL, and Lipofectin
(Gibco BRL, Burlington, ON) and DOPE/DODAC lipoplexes containing pCMVLuc. The
lipoplexes were prepared as described earlier. After transfection times of 4,
8, and 24 h the
transfection media was removed and in the case of the 4 and 8 h transfections,
was replaced
with complete media for a total incubation time of 24 h. At 24 h, all cells
were lysed and
assayed for luciferase activity and protein content (BCA assay), as described
above.
SPLP-CPL4, DOPE:DODAC lipoplexes and Lipofectin lipoplexes containing
pCMVGFP were prepared as described for pCMVLuc. The transfections were
performed as
described above at a plasmid DNA dose of 5.0 ~,g/mL. Following incubation of
the samples
for 24 and 48 h, the transfection media was removed, the cells were washed,
and fresh media
was added to the cells. The cells were then viewed under a Zeiss fluorescence
microscope.
The number of cells expressing GFP were counted using a fluorescein filter
(Omega
Opticals) with the following specifications: excitation 475~20/dichroic filter
500/emission
535~22.5. The transfection efficiency was expressed as percentage of cells
expressing GFP.
B. Results
1. Cationic PEG lipids can be inserted into preformed SPLP.
Previous work has shown that SPLP exhibit lower uptake into cells and lower
transfection
potencies than lipoplexes (Mok, et al., Biochim. Biophys. Acta 1419:137-150
(1999)). It has
also been shown that surface-associated cationic PEG lipids (CPL),
particularly those
containing four charges at the end of the PEG molecule (CPL4; for structure
see Figure 8A),
can dramatically enhance the uptake of LUV into cells. Further, CPL can be
inserted into
preformed LUV with lipid compositions similar to SPLP employing a
straightforward
incubation protocol. It was thus examined. whether a similax procedure could
be developed to
insert CPL4 into SPLP. SPLP containing pCMVLuc were prepared by the detergent
dialysis
procedure of Wheeler et al. (Gene Therapy 6:271-281 (1999)) from a lipid
mixture
containing 6 mol% of the cationic lipid N,N-dioleoyl-N,N-dimethyl ammonium
chloride
(DODAC), 84 mol% of the "fusogenic" helper lipid dioleoyl
phosphatidylethanolamine
(DOPE) and 10 mol% of a stabilizing lipid consisting of PEG2ooo attached to a
ceramide
(Cer) anchor (PEG-Cer). The ceramide anchor of the PEG-Cer contained a C2o
acyl chain
(PEG-CerC2o) that does not readily exchange out of the vesicle, thus
contributing to a highly
68
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
stable SPLP system (Wheeler, et al., Gene Therapy 6:271-28,1 (1999)). The
detergent
dialysis procedure results in the formation of a mixture of SPLP containing
one plasmid per
vesicle, free plasmid, and empty vesicles. SPLP were purified by removing free
plasmid and
empty vesicles by DEAE column chromatography and density centrifugation,
respectively, as
described elsewhere (Wheeler, et al., Gene Therapy 6:271-281 (1999)).
The procedure for post-insertion of CPL4 into the preformed SPLP is
illustrated in Figure 8B. Purified SPLP were incubated with CPL4 (~5 mol%) at
60°C for up
to 3 h and then separated from nonincorporated CPL4 by column chromatography.
As shown
in Figure 9, this resulted in association of up to 80% of the available CPL4
with the SPLP,
corresponding to 4 mol% of the total lipid in the SPLP-CPL4 system.
2. SPLP-CPL4 aggregate following insertion of CPL4 and de-
aggregate following addition of divalent rations. Previous work has shown that
LUV
containing CPL tend to aggregate and that this aggregation can be inhibited by
increasing the
ionic strength of the medium. It was found that SPLP-CPL4 were also
susceptible to
aggregation, and that this aggregation could be reversed by adding NaCI, CaCl2
or MgCl2 to
the SPLP-CFL4 formulation. This effect is illustrated in Figure 10 which shows
the effect of
the addition of CaCl2 and MgCl2 on aggregation of SPLP-CPL4 as monitored by
the change
in the standard deviation of the mean diameter of the particles measured by
quasi-elastic light
scattering (QELS). Fox both rations the standard deviation decreased with
increasing ration
concentration with optimal de-aggregation occurring above 30 mM. This
behaviour could
also be visualized by freeze-fracture electron microscopy. As shown in Figure
11A, freeze-
fracture micrographs of SPLP reveal small monodisperse particles, whereas SPLP-
CPL4
prepared in the absence of CaCl2 are highly aggregated (Figure 11B). As shown
in Figure
11C, the addition of 40 mM CaCl2 reverses this aggregation to produce
monodisperse
particles similar to the SPLP preparation.
The sizes of SPLP and SPLP-CPL4 in the presence of CaCl2 were compared
using QELS and freeze-fracture electron microscopy. QELS studies revealed the
mean
diameter of SPLP and SPLP-CPL4 to be 80 ~ 19 nm and 76 ~ 15 nm, respectively,
whereas
the freeze-fracture studies indicated diameters of 68 ~ 11 nm and 64 ~ 14 nm.
These values
fox SPLP diameters are in close agreement with previous studies (Wheeler, et
al., Gene
Therapy 6:271-281 (1999)).
69
CA 02406654 2002-10-18
w0 o1/so9oo3. pEG-CerC2o content and stability of SPLP-CPL4. The obse/rv tion
that CPL4 can be inserted to achieve levels as high as 4 mol% of the total
SPLP lipid
indicates that the level of CPL4 in the outer monolayer of the SPLP-CPL4 is 8
mol%. Given
that the initial concentration of PEG-CerC2o is 10 mol%, this suggests that
the total levels of
PEG-lipids in the outer monolayer of the SPLP-CPL4 can approach 18 mol%. These
levels
are higher than the levels of PEG-lipids that can usually be incorporated into
lipid vesicles
(Woodle, et al., Biochim. Biophys. Acta 1113:171-199 (1992)) leading to the
possibility that
some of the PEG-CerC2o in the outer monolayer exchanged out as CPL4 was
inserted. This
was examined by measuring the ratio of PEG-CerC2o-to-DOPE for the SPLP before
and after
insertion of CPL4 employing HPLC. CPL4 was inserted into SPLP as described
previously.
Analysis following removal of nonincorporated material determined that 4 mol%
CPL4
(normalized to the total SPLP lipid) was inserted into the SPLP. Prior to
insertion of the
CPL4 the PEG-CerC2o-to-DOPE ratio was 0.091, corresponding to a PEG-CerC2o
content of
7.6 mol%, assuming that the DOPE constituted 84 mol% of the lipid content.
Following
insertion of the CPL4 the PEG-CerC2o-to-DOPE ratio was found to be 0.072,
indicating a
PEG-CerC2o content of 6.0 mol%. Assuming that all of the PEG-CerC2o lost from
the SPLP
during insertion of the CPL4 is lost from the outer monolayer, this indicates
that the PEG-
CerC2o content of the outer monolayer decreases from 7.6 mol% to 4.4 mol%
during the
insertion process. The total PEG-lipid content in the outer monolayer of the
SPLP-CPL4 can
then be estimated to be 12.4 mol% of the outer monolayer lipid.
The stability of SPLP and SPLP-CPL4 following incubation in 50% mouse
serum for up to 4 h is illustrated in Figure 12. In all cases, the
encapsulated plasmid DNA
was fully protected from serum degradation. In contrast, essentially complete
degradation of
the plasmid in lipoplexes was observed within 30 min of incubation in serum.
4. SPLP-CPL4 exhibit enhanced uptake into BHK cells and
dramatically enhanced transfection potency. The next set of experiments was
aimed at
determining the influence of incorporated CPL4 on the uptake of SPLP into BHK
cells and
the resulting transfection potency of the SPLP-CPL4 system. SPLP containing up
to 4 mol%
CPL4 were prepared in the presence of 40 mM CaCla and were added to BHI~ cells
(final
CaCl2 concentration 8 mM) and incubated for varying times. The cells were then
assayed for
associated SPLP-CPL4 as indicated in the above Materials and Methods. As shown
in Figure
13A, while uptake of SPLP that contain no CPL4 is minimal even after 8 h of
incubation,
uptake is dramatically improved for SPLP containing 3 mol% or higher levels of
CPL4. For
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
example, SPLP containing 4 mol% CPL4 exhibit accumulation levels at 8 h that
are
approximately SO-fold higher than achieved for SPLP in the absence of CPL.
This enhanced
uptake is visually illustrated in Figure 13B, which shows fluorescence
micrographs of BHK
cells following incubation with rhodamine-labeled SPLP and SPLP-CPL4 for 4 h.
The transfection properties of SPLP, SPLP-CPL4 and plasmid DNA-cationic
liposome lipoplexes (DODAC/DOPE; 1:1) were examined using the transfection
protocol
described in the above Materials and Methods. This protocol involves
incubation of BHK
cells with SPLP or lipoplexes for 4 h (the transfection time) followed by
replacement of
media and further incubation to maximize transgene expression. The total
incubation time
(transfection time plus time of incubation following the media change) was
kept constant at
24 h. As shown in Figure 14, the presence of increasing amounts of CPL4
resulted in
dramatic increases in the transfection potency for the SPLP system. SPLP-CPL4
containing 4
mol% CPL4 exhibited luciferase expression levels some 3x103 higher than
achieved with
SPLP.
5. Ca~'+ is required for transfection activity of SPLP-CPL4.
Example I, supra, demonstrates that the transfection potency of SPLP is highly
sensitive to
the presence of Ca2+, where the presence of ~10 mM Ca2+ enhances transfection
potency
several hundred-fold. It was therefore of interest to determine the influence
of Caz+ on the
transfection activity of SPLP-CPL4. SPLP containing 4 mol% CPL4 were incubated
with
BHK cells for 48 h in the presence of varying amounts of MgCl2 and CaCl2, and
the
luciferase activities were determined. As shown in Figure 15, the transfection
activity was
primarily dependent on the presence of Ca2+ in the transfection medium. At the
optimum
CaCl2 concentration of 10 mM, SPLP-CPL4 exhibited transfection potencies that
were more
than 1 OS times higher than if the corresponding amount of MgCl2 was present.
a In order to determine whether the different transfection properties of SPLP-
CPL4 in the presence of Ca2+ or Mga+ could be accounted for by differences in
uptake into
cells, the accumulation of SPLP-CPL4 into BHK cells was monitoxed following a
4 h
incubation in the presence of MgCl2 or CaCI2. As shown in Figure 16, uptake of
SPLP-CPL4
into BHK cells is the same for both Ca2+ and Mg2+. It may be noted that SPLP-
CPL4 uptake
decreases slightly as the concentration of divalent cations increases, likely
due to the
shielding of the negatively charged CPL4 binding sites on the surface of BHK
cells. These
results are consistent with a previous study indicating that Ca2~ has little
effect on the cellular
uptake of SPLP.
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
SPLP-CPL4 exhibit transfection potencies ih vitro that are comparable to or
greater than the transfection potencies of lipoplexes. The data presented in
Figure 14
indicate that DOPE/DODAC lipoplexes yield 100-fold higher levels of gene
expression than
SPLP-CPL4 when applied to BHK cells for a period of 4 h. Given that SPLP-CPL4
are stable
systems, uptake can conceivably continue over extended time periods. The
transfection
levels achieved when SPLP-CPL4 or the lipoplexes were applied to BHK cells for
transfection times of 4, 8 and 24 h were thus examined. Two types of
lipoplexes were used,
namely DOPE:DODAC (1:l) lipoplexes (charge ratio 1.5) and lipoplexes generated
using the
transfection reagent Lipofectin, consisting of DOPE/DOTMA (1:1) lipoplexes at
a charge
ratio of 1.5. As shown in Figure 17, the potency of SPLP-CPL4 increases
markedly with
increased transfection times, suggesting that the rate of uptake of the SPLP-
CPL4 system may
be a limiting factor for transfection. For the 24 h transfection time, where
the cells are
assayed for luciferase expression immediately after the transfection period,
transfection levels
are comparable to those achieved by Lipofectin or the DOPE/DODAC lipoplexes.
Further experiments were conducted to determine transfection levels after
transfection times of 24 and 48 h with SPLP-CPL4 and lipoplexes where
luciferase activities
were assayed immediately following the transfection period. As shown in Figure
18A the
activity of Lipofectin (DOPE:DOTMA) lipoplexes leveled off at 2000 ng
luciferase per mg
of cell protein after 24 h. Similar results were obtained for the DOPE:DODAC
lipoplexes.
In contrast, the activity of the SPLP-CPL4 formulation continued to increase
as the incubation
time was increased, achieving luciferase expression levels corresponding to
4000 ng per mg
of cell protein at 48 h. This activity is approximately 106 times higher than
observed for
SPLP (in the absence of Ca2~ and almost double the levels that can be achieved
by
Lipofectin Iipoplexes.
6. SPLP-CPL4 are nontoxic and efficient transfection agents. It is
well known that lipoplexes can be toxic to cells. The SPLP-CPL4 contain low
levels of
cationic lipid and are potentially less toxic than lipoplexes. The toxicities
of SPLP-CPL4 and
lipoplexes were assayed by determining cell viability following a 24 h and 48
h exposure to
levels of SPLP-CPL4 and lipoplexes corresponding to 5.0 ~.g/mL plasmid,
corresponding to
total lipid doses of approximately 80 pM and 45 pM for SPLP-CPL4 and
lipoplexes,
respectively. As shown in Figure 18B, SPLP-CPL4 exhibited little toxicity,
whereas
lipoplexes were highly toxic. Cell survival was only 30% after a 48 h
incubation with
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
Lipofectin Iipoplexes, whereas ~95% of the cells were viable following a 48 h
incubation
with SPLP-CPL4.
Studies were also conducted to determine the efficiency of transfection as
indicated by the proportion of cells transfected by SPLP-CPL4. The proportion
of transfected
cells was determined by employing plasmid containing the green fluorescent
protein (GFP)
gene. GFP expression was detected by fluorescence microscopy. As shown in
Figures 19A
and 19B, approximately 35% of the cells at 24 h and 50% at 48 h were
transfected by SPLP-
CPL4, with no apparent cell death. In contrast, Lipofectin lipoplexes exhibit
maximum
transfection efficiencies of less than 35% and only ~50% cell survival after
the 24 h
transfection period (Figure 19C). Similar low transfection efficiencies and
high toxicities
were also seen with DOPE:DODAC lipoplexes.
C. Discussion
These results demonstrate that the incorporation of CPL4 into SPLP results in
improved uptake into BHK cells and dramatically enhanced transfection
potencies of SPLP
when Ca2+ is present. There are three points of interest. The first concerns
the chemical
composition and structure of the SPLP-CPL4 system and the generality of the
post-insertion
procedure for modifying the trophism and transfection potency of SPLP. The
second
concerns the relation between enhanced uptake of SPLP, the presence of Ca2+
and the
transfection activities observed. Finally, it is of interest to compare the
properties of the
SPLP-CPL4 system with lipoplexes. Each of these areas is addressed below in
turn.
The results presented here demonstrate that the cationic PEG lipid CPL4 can
be inserted into preforr~ed SPLP employing a simple process involving
incubation at 60°C.
The ability to insert CPL4 to levels corresponding, for example, to about 8
mol% of the total
lipid in the SPLP outer monolayer is consistent with results of other workers
demonstrating
that PEG-PE can be inserted into preformed LUV employing a similar incubation
protocol,
resulting in systems exhibiting extended circulation lifetimes (Uster, et al.,
FEBS Lett.
386:243-246 (1996)). It is also consistent with previous results showing that
CPL4 can be
inserted into preformed LUV with a lipid composition similar to the SPLP
system. The total
levels of PEG-lipids achieved in the outer monolayer (12.4 mol%) are high
given that
maximum levels of incorporation of PEG-lipids into LUV are commonly 7-10 mol%
(Woodle, et al., Biochim. Biophys. Acta 1113:171-199 (1992)). However, a
number of
authors have reported that PEG-lipids can be incorporated into LUV to levels
as high as 15
mol% before Iytic effects are observed (Edwards, et al., Biophys. .J. 73:258-
266 (1997);
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
Kenworthy, et al., Biophys. J. 68:1903-1920 (1995); Hristova, et al.,
Macromolecules
28:7693-7699 (1995)). These include cryo-electron microscopy studies which
indicate that
structural changes (from spheres to discs) are only observed for
distearoylphosphatidylcholine (DSPC) liposomes at PEG-PE levels above I2 moI%,
with
lytic effects observed above 15 mol% (Edwards, et al., Biophys. J. 73:258-266
(1997)).
Similarly, X-ray studies indicate that nonbilayer micellar structures are only
observed for
PEG-lipid levels above 1 S mol% (Kenworthy, et al., Biophys. J. 68:1903-1920
(1995);
Hristova, et al., Macromolecules 28:7693-7699 (1995)).
The tendency for the SPLP-CPL4 system to aggregate following insertion of
the CPL4 is consistent with previous observations that LUV containing CPL4
also aggregate.
The reason for this aggregation is not currently understood, although two
general points can
be made. First, the interaction is likely due to electrostatic interactions
between vesicles
given the inhibition of aggregation at higher ionic strengths. Second, the
aggregation is not a
consequence of the post-insertion process itself as such aggregation is also
observed for LUV
containing CPL4, where the CPL4 was present in the lipid mixture from which
the LUV were
formed. It is possible that the cationic headgroup interacts with opposed
membranes at the
level of the phospholipid phosphate group. Alternatively, the aggregation
phenomenon may
be related to the ability of PEG coatings to adopt a conformation that is able
to bind proteins
such as streptavidin (Sheth, et al., Proc. Natl. Acad. Sci. USA 94:8399-8404
(1997)).
The second point of discussion concerns the mechanism whereby CPL4
increases the transfection potency of the SPLP system. A number of studies
have indicated
that the cationic lipids contained in lipoplex systems play a direct role in
stimulating uptake
into cells (Miller, et al., Biochemistry 37:12875-12883 (1998)) and that this
uptake arises due
to the positive charge on the lipoplexes (van der Woude, et al., Biochim
Biophys Acta
2S 1240:34-40 (1995)). It has been suggested that heparin sulfonated
proteoglycans on the cell
surface play a primary role in this process (Mislick, et al., Proc. Natl.
Acad. Sci. USA
93:12349-12354 (1996); Mounkes, et al., J. Biol. Chem. 273:26164-26170
(1998)).
Enhanced uptake of SPLP following addition of the CPL4 could be due to similar
mechanisms, although the increase in transfection potency is largely dependent
on the
additional presence of Ca2+. Example I shows that the presence of Ca2~ results
in an increase
in SPLP transfection potency of 600 fold, and that this increase results from
an ability of
Caz+ to assist in destabilizing the endosomal membrane following uptake,
rather than from an
increase in uptake. It is therefore possible that the observed improvements in
transfection
potency for SPLP-CPL4 over SPLP result from improvements in uptake mediated by
the
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CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
CPL4 coupled with enhanced abilities to destabilize the endosomal membrane due
to the
presence of Ca2+. In this regard, the transfection potency of SPLP-CPL4 (in
the presence of
Ca2+) is increased by a factor of 104 (Figure 14) in comparison to the
transfection potency
of SPLP in the absence of Ca2+. This could be accounted for by an increase in
uptake of
SPLP into BHK cells by approximately 50-fold due to the presence of 4 mo1%
CPL4 (Figure
13A, 4 h incubation) multiplied by a factor of --600 due to the presence of
Ca2+.
The final area of discussion concerns the advantages of the SPLP-CPL4
system over other nonviral vectors, which include the well-defined modular
nature of the
SPLP-CPL4 system as well as toxicity and potency issues. First, SPLP-CPL4 are
small,
Z O homogeneous, stable systems containing one plasmid per particle (Wheeler,
et al., Gene
Therapy 6:271-281 (1999)), in contrast with other nonviral systems such as
lipoplexes, which
are large, heterogeneous, unstable systems containing ill-defined numbers of
plasmids per
particle. An important point is that SPLP are basic components of more
sophisticated
systems, such as SPLP-CPL4, that can be constructed in a modular fashion. For
example,
post-insertion of PEG-lipids containing specif c targeting ligands in place of
the cationic
groups of CPL should result in SPLP that are specifically targeted to
particular cells and
tissues. With regard to toxicity, SPLP-CPL4 are markedly less toxic to BHK
cells in tissue
culture than are lipoplexes. This is presumably related to the low proportions
of cationic lipid
contained in SPLP as compared to lipoplexes. Finally, SPLP without CPL4 or
Ca2+ exhibit
transfection properties in vivo following systemic administration that are
already superior to
the transfection properties of plasmid DNA-cationic lipid complexes or naked
plasmid DNA.
The results presented here suggest that further significant gains can be
expected through the
use of ligands that encourage SPLP uptake into cells and methods leading to
local increases
in Ca2~ concentrations.
In summary, the results presented here demonstrate that a cationic PEG lipid
can be post-inserted into SPLP, resulting in well-defined SPLP-CPL4 systems
that exhibit
improved uptake into BHK cells in vitro. In the presence of Ca2+ SPLP- CPL4
systems give
rise to transfection potencies that are increased by up to 106-fold as
compared to SPLP in the
absence of Ca2+. These results indicate that the SPLP system is a nontoxic,
highly
transfection potent entity following uptake into cells and indicates that SPLP
targeted to cell-
surface ligands that undergo endocytosis should lead to significant
enhancement of
transfection potency in vivo.
CA 02406654 2002-10-18
WO 01/80900 PCT/CA01/00555
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims. All publications, patents,
and patent
applications cited herein are hereby incorporated by reference in their
entirety for all
purposes.
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