U.S. patent application number 11/728134 was filed with the patent office on 2007-07-26 for lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use of gene transfer.
This patent application is currently assigned to The University of British Columbia. Invention is credited to Marcel B. Bally, Pieter R. Cullis, Michael Hope, Dorothy L. Reimer, Peter Scherrer, Jeffrey J. Wheeler, Yuan-Peng Zhang.
Application Number | 20070172950 11/728134 |
Document ID | / |
Family ID | 44123424 |
Filed Date | 2007-07-26 |
United States Patent
Application |
20070172950 |
Kind Code |
A1 |
Wheeler; Jeffrey J. ; et
al. |
July 26, 2007 |
Lipid-nucleic acid particles prepared via a hydrophobic
lipid-nucleic acid complex intermediate and use of gene
transfer
Abstract
Novel lipid-nucleic acid particulate complexes which are useful
for in vitro or in vivo gene transfer are described. The particles
can be formed using either detergent dialysis methods or methods
which utilize organic solvents. Upon removal of a solubilizing
component (i.e., detergent or an organic solvent) the lipid-nucleic
acid complexes form particles wherein the nucleic acid is
serum-stable and is protected from degradation. The particles thus
formed have access to extravascular sites and target cell
populations and are suitable for the therapeutic delivery of
nucleic acids.
Inventors: |
Wheeler; Jeffrey J.;
(Vancouver, CA) ; Bally; Marcel B.; (Bowen Island,
CA) ; Zhang; Yuan-Peng; (Vancouver, CA) ;
Reimer; Dorothy L.; (Vancouver, CA) ; Hope;
Michael; (Vancouver, CA) ; Cullis; Pieter R.;
(Vancouver, CA) ; Scherrer; Peter; (Vancouver,
CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 5400
SEATTLE
WA
98104
US
|
Assignee: |
The University of British
Columbia
Vancouver
CA
INEX Pharmaceuticals Corporation
Burnaby
CA
|
Family ID: |
44123424 |
Appl. No.: |
11/728134 |
Filed: |
March 23, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09431594 |
Nov 1, 1999 |
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11728134 |
Mar 23, 2007 |
|
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08660025 |
Jun 6, 1996 |
5976567 |
|
|
09431594 |
Nov 1, 1999 |
|
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08484282 |
Jun 7, 1995 |
5981501 |
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08660025 |
Jun 6, 1996 |
|
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08485458 |
Jun 7, 1995 |
5705385 |
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08660025 |
Jun 6, 1996 |
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Current U.S.
Class: |
435/458 ;
435/320.1 |
Current CPC
Class: |
A61K 47/6911 20170801;
Y10S 436/829 20130101; C12N 15/88 20130101; A61K 9/1272
20130101 |
Class at
Publication: |
435/458 ;
435/320.1 |
International
Class: |
C12N 15/88 20060101
C12N015/88 |
Claims
1. A hydrophobic lipid-nucleic acid complex consisting essentially
of cationic lipids and nucleic acids, said complex being
charge-neutralized and soluble in organic solvents.
2. A complex in accordance with claim 1, wherein said nucleic acid
is a plasmid.
3. A complex in accordance with claim 1, wherein said cationic
lipids are members selected from the group consisting of DODAC,
DDAB, DOTMA, DOSPA, DMRIE, DOGS and combinations thereof.
4. A method for the preparation of lipid-nucleic acid particles,
comprising: (a) contacting nucleic acids with a solution comprising
non-cationic lipids and a detergent to form a nucleic acid-lipid
mixture; (b) contacting cationic lipids with said nucleic
acid-lipid mixture to neutralize the negative charge of said
nucleic acids and form a charge-neutralized mixture comprising
detergent, nucleic acids and lipids; and (c) removing said
detergent from said charge-neutralized mixture to provide said
lipid-nucleic acid particles in which said nucleic acids are
protected from degradation.
5. A method in accordance with claim 4, wherein said solution of
step (a) further comprises an organic solvent.
6. A method in accordance with claim 4, wherein said cationic
lipids are members selected from the group consisting of DODAC,
DDAB, DOTMA, DOSPA, DMRIE, DOGS and combinations thereof.
7. A method in accordance with claim 4, wherein said non-cationic
lipids are selected from the group consisting of ESM, DOPE,
polyethylene glycol-based polymers and combinations thereof.
8. A method in accordance with claim 4, wherein said detergent is
octyl-.beta.-D-glucopyranoside, said cationic lipid is DODAC, said
non-cationic lipid is ESM, and said detergent is removed by
dialysis.
9. A method in accordance with claim 8, wherein said non-cationic
lipids are combinations of ESM and PEG-Ceramide.
10. A method for introducing a nucleic acid into a cell,
comprising; (a) preparing a lipid-nucleic acid particle according
to the method of claim 4; and (b) contacting said cell with said
lipid-nucleic acid particle for a period of time sufficient to
introduce said nucleic acid into said cell.
11. A method in accordance with claim 11, wherein said
lipid-nucleic acid particle comprises a plasmid, DODAC and ESM.
12. A method for the preparation of lipid-nucleic acid particles,
comprising: (a) contacting an amount of cationic lipids with
nucleic acids in a solution; said solution comprising of from about
15-35% water and about 65-85% organic solvent and said 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 said
hydrophobic lipid-nucleic acid complex in solution with
non-cationic lipids, to provide a lipid-nucleic acid mixture; and
(c) removing said organic solvents from said mixture to provide
said lipid-nucleic acid particles in which said nucleic acids are
protected from degradation.
13. A method in accordance with claim 12, wherein said cationic
lipids are members selected from the group consisting of DODAC,
DDAB, DOTMA, DOSPA, DMRIE, DOGS and combinations thereof.
14. A method in accordance with claim 12, wherein said non-cationic
lipids are members selected from the group consisting of ESM, DOPE,
polyethylene glycol-based polymers and combinations thereof.
15. A method in accordance with claim 12, wherein said organic
solvents are members selected from the group consisting of
methanol, chloroform, methylene chloride, ethanol, diethyl ether
and combinations thereof.
16. A method in accordance with claim 12, wherein said nucleic acid
is a plasmid, said cationic lipid is a member selected from the
group consisting of DODAC, DDAB, DOTMA, DOSPA, DMRIE, DOGS and
combinations thereof, said non-cationic lipid is a member selected
from the group consisting of ESM, DOPE, polyethylene glycol-based
polymers and combinations thereof, and said organic solvent is a
member selected from the group consisting of methanol, chloroform,
methylene chloride, ethanol, diethyl ether and combinations
thereof.
17. A method for introducing a nucleic acid into a cell,
comprising; (a) preparing a lipid-nucleic acid particle according
to the method of claim 13; and (b) contacting said cell with said
lipid-nucleic acid particle for a period of time sufficient to
introduce said nucleic acid into said cell.
18. A lipid-nucleic acid particle prepared according to claim
4.
19. A lipid-nucleic acid particle prepared according to claim
12.
20. A method for the preparation of serum-stable plasmid-lipid
particles, comprising: (a) combining a plasmid with cationic lipids
in a detergent solution to provide a coated plasmid-lipid complex;
(b) contacting non-cationic lipids with said coated plasmid-lipid
complex to provide a solution comprising detergent, a plasmid-lipid
complex and non-cationic lipids; and (c) removing said detergent
from said solution of step (b) to provide a solution of
serum-stable plasmid-lipid particles, wherein said plasmid is
encapsulated in a lipid bilayer and said particles are serum-stable
and have a size of from about 50 to about 150 nm.
21. A method in accordance with claim 20, wherein said removing is
by dialysis.
22. A method in accordance with claim 20, wherein step (b) further
comprises adding a polyethylene glycol-lipid conjugate.
23. A method in accordance with claim 22, wherein said polyethylene
glycol-lipid conjugate is a PEG-ceramide conjugate.
24. A method in accordance with claim 20, further comprising; (d)
sizing said particles to achieve a uniform particle size.
25. A method in accordance with claim 20, wherein said cationic
lipids are selected from the group consisting of DODAC, DDAB,
DOTAP, DOTMA, DOSPA DOGS, DC-Chol and combinations thereof.
26. A method in accordance with claim 20, wherein said non-cationic
lipids are selected from the group consisting of DOPE, POPC, EPC
and combinations thereof.
27. A method in accordance with claim 20, wherein said detergent
solution comprises a detergent having a critical micelle
concentration of between about 20 mM and 50 mM.
28. A method in accordance with claim 8, wherein said detergent is
n-octyl-.beta.-D-glucopyranoside.
29. A method for the preparation of serum-stable plasmid-lipid
particles, comprising; (a) preparing a mixture comprising cationic
lipids and non-cationic lipids in an organic solvent; (b)
contacting an aqueous solution of plasmid with said mixture
prepared 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.
30. A method in accordance with claim 29, wherein said non-cationic
lipids comprise a polyethylene glycol-lipid conjugate.
31. A method in accordance with claim 30, wherein said polyethylene
glycol-lipid conjugate is a PEG-ceramide conjugate.
32. A method in accordance with claim 29, wherein said cationic
lipids are selected from the group consisting of DODAC, DDAB,
DOTAP, DOTMA, DOSPA, DOGS, DC-Chol and combinations thereof.
33. A method in accordance with claim 29, wherein said non-cationic
lipids are selected from the group consisting of DOPE, POPC, EPC
and combinations thereof.
34. A plasmid-lipid particle prepared according to claim 20.
35. A method for introducing a plasmid into a cell, comprising; (a)
preparing a plasmid-lipid particle according to the method of claim
20; and (b) contacting said cell with said plasmid-lipid particle
for a period of time sufficient to introduce said plasmid into said
cell.
36. A method in accordance with claim 35, wherein said
plasmid-lipid particle comprises a plasmid, DODAC, POPC and a
PEG-Ceramide selected from the group consisting of PEG-Cer-C.sub.20
and PEG-Cer-C.sub.14.
37. A method in accordance with claim 35, wherein said
plasmid-lipid particle comprises a plasmid, DODAC, DOPE and a
PEG-Ceramide selected from the group consisting of PEG-Cer-C.sub.20
and PEG-Cer-C.sub.14.
38. A plasmid-lipid particle prepared according to claim 29.
39. A method for introducing a plasmid into a cell, comprising; (a)
preparing a plasmid-lipid particle according to the method of claim
29; and (b) contacting said cell with said plasmid-lipid particle
for a period of time sufficient to introduce said plasmid into said
cell.
40. A method in accordance with claim 39, wherein said
plasmid-lipid particle comprises a plasmid, DODAC, POPC and a
PEG-Ceramide selected from the group consisting of PEG-Cer-C.sub.20
and PEG-Cer-C.sub.14.
41. A method in accordance with claim 39, wherein said
plasmid-lipid particle comprises a plasmid, DODAC, DOPE and a
PEG-Ceramide selected from the group consisting of PEG-Cer-C.sub.20
and PEG-Cer-C.sub.14.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/485,458 and of U.S. application Ser. No.
08/484,282, both filed on Jun. 7, 1995.
FIELD OF THE INVENTION
[0002] This invention relates to lipid-nucleic acid particles which
are useful for the introduction of nucleic acids into cells, and
methods of making and using them. The invention provides a
circulation-stable, characterizable delivery vehicle for the
introduction of plasmids or antisense compounds into cells. These
vehicles are safe, stable, and practical for clinical use.
BACKGROUND OF THE INVENTION
[0003] Gene transfer into genetically impaired host cells in order
to correct the genetic defects has vast potential for succesfully
treating a variety of thus far hitherto untreatable medical
conditions. There are currently six major non-viral methods by
which genes are introduced into host cells: (i) direct
microinjection, (ii) calcium phosphate precipitation, (ii)
DEAE-dextran complexes, (iv) electroporation, (v) cationic lipid
complexes and (vi) reconstituted viruses and virosomes (see Chang,
et al., Focus 10:88 (1988)).
[0004] Most reported examples of gene transfer have been performed
in vitro. In vivo gene transfer is complicated by serum
interactions, immune clearance, enzymatic degradation of the genes,
toxicity and biodistribution. In in vivo administration, selection
is not possible, and a reasonably high frequency of transformation
is necessary to achieve sufficient expression to compensate for a
defective endogenous gene.
[0005] The in vivo gene transfer methods under study in the clinic
consist almost entirely of viral vectors. Although viral vectors
have the inherent ability to transport nucleic acids across cell
membranes and some can integrate exogenous DNA into the
chromosomes, they can carry only limited amounts of DNA. In
addition, their use poses significant risks. One such risk is that
the viral vector may revert to a pathogenic genotype either through
mutation or genetic exchange with a wild type virus.
[0006] In view of these limitations and risks, alternative
non-viral-based gene transfer methods have been developed. These
methods use often plasmid vectors, which are small circular
sequences of DNA, as vectors for DNA delivery. However, most
plasmids do not possess the attributes required for intracellular
delivery and therefore sophisticated delivery systems are
required.
[0007] Cationic lipid complexes are presently the most effective
generally used means of introducing non-viral nucleic acids into
cells. A number of different formulations incorporating cationic
lipids are commercially available. These include:(i)
LIPOFECTIN.RTM. (which uses 1,2-dioleyloxy-3-
(N,N,N-trimethylamino)propane chloride, or DOTMA, see Eppstein, et
al., U.S. Pat. No. 4,897,355); LIPOFECTAMINE.RTM. (which uses
DOSPA, see Hawley-Nelson, et al., Focus 15(3):73 (1993)); and
LIPOFECTACE.RTM. (which uses N,N-distearyl-N,N-dimethyl-ammonium
bromide, or DDAB, see Rose, U.S. Pat. No. 5,279,833). Others have
reported alternative cationic lipids that work in essentially the
same manner but with different efficiencies, for example
1,2-dioleoyloxy-3-(N,N,N-trimethylamino) propane chloride, or DOTAP
(see Stomatatos, et al., Biochemistry 27: 3917-3925 (1988));
glycerol based lipids (see Leventis, et al., Biochem. Biophys. Acta
1023:124 (1990); lipopolyamines (see, Behr, et al., U.S. Pat. No.
5,171,678) and cholesterol based lipids (see Epand, et al., WO
93/05162, and U.S. Pat. No. 5,283,185). It has been reported that
DOTMA and related compounds are significantly more active in gene
transfer assays than their saturated analogues (see, Felgner, et
al., WO91/16024). However, both DOTMA and DOSPA based formulations,
despite their efficiency in effecting gene transfer, are
prohibitively expensive. DDAB on the other hand is inexpensive and
readily available from chemical suppliers but is less effective
than DOTMA in most cell lines. Another disadvantage of the current
lipid systems is that they are not appropriate for intravenous
injection.
[0008] Lipid-based vectors used in gene transfer have generally
been formulated in one of two ways. In one method, the nucleic acid
is introduced into preformed liposomes made of mixture of cationic
lipids and neutral lipids. The complexes thus formed have undefined
and complicated structures and the lipofection efficiency is
severely reduced by the presence of serum. A second method involves
the formation of DNA complexes with mono- or poly-cationic lipids
without the presence of a neutral lipid. These complexes are often
prepared in the presence of ethanol and are not stable in water.
Additionally, these complexes are adversely affected by serum (see,
Behr, Acc. Chem. Res. 26:274-78 (1993)).
[0009] An examination of the relationship between the chemical
structure of the carrier vehicle and its efficiency of gene
transfer has indicated that the characteristics which provide for
effective gene transfer would make a carrier unstable in
circulation (see, Ballas, et al., Biochim. Biophys. Acta 939:8-18
(1988)). Additionally, degradation either outside or inside the
target cell remains a problem (see, Duzghines, Subcellular
Biochemistry 11:195-286 (1985)). Others who have attempted to
encapsulate DNA in lipid-based formulations have not overcome these
problems (see, Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467
(1980); Deamer, U.S. Pat. No. 4,515,736, and Legendre, Pharm. Res.
9:1235-1242 (1992)).
[0010] Ideally, a delivery vehicle for a nucleic acid or plasmid
will have the following characteristics: a) ease of preparation, b)
capable of carrying a large amount of DNA per particle to enable
gene transfer of all sizes of genes and reduce the volume of
injection, c) homogenous, d) reproducible, e) is serum stable with
minimal serum interactions and shields DNA from extracellular
degradation, and f) is capable of transfecting target cells in such
a way that the DNA is not digested intracellularly.
[0011] The present invention provides such compositions and methods
for their preparation and use.
SUMMARY OF THE INVENTION
[0012] The present invention comprises novel, lipid-nucleic acid
particles. The invention also comprises methods of making and using
these particles.
[0013] In some embodiments, the particles are made by formation of
hydrophobic intermediate complexes in either detergent-based or
organic solvent-based systems, followed by removal of the detergent
or organic solvent. Preferred embodiments are
charge-neutralized.
[0014] In one embodiment, a plasmid is combined with cationic
lipids in a detergent solution to provide a coated plasmid-lipid
complex. The complex is then contacted with non-cationic lipids to
provide a solution of detergent, a plasmid-lipid complex and
non-cationic lipids, and the detergent is then removed to provide a
solution of serum-stable plasmid-lipid particles, in which the
plasmid is encapsulated in a lipid bilayer. The particles thus
formed have a size of about 50-150 nm.
[0015] In another embodiment, serum-stable plasmid-lipid particles
are formed by preparing a mixture of cationic lipids and
non-cationic lipids in an organic solvent; contacting an aqueous
solution of plasmid with the mixture of cationic and non-cationic
lipids to provide a clear single phase; and removing the organic
solvent to provide a suspension of plasmid-lipid particles, in
which the plasmid is encapsulated in a lipid bilayer, and the
particles are stable in serum and have a size of about 50-150
nm.
[0016] Another method of forming lipid-nucleic acid particles
involves:
[0017] (a) contacting nucleic acids with a solution of non-cationic
lipids and a detergent to form a nucleic acid-lipid mixture;
[0018] (b) contacting cationic lipids with the nucleic acid-lipid
mixture to neutralize the negative charge of said nucleic acids and
form a charge-neutralized mixture of nucleic acids and lipids:
and
[0019] (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.
[0020] Another method of forming lipid-nucleic acid particles
involves:
[0021] (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;
[0022] (b) contacting the hydrophobic, charge-neutralized
lipid-nucleic acid complex in solution with non-cationic lipids, to
provide a lipid-nucleic acid mixture; and
[0023] (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.
[0024] The lipid-nucleic acid particles of the present invention
are useful for the therapeutic delivery of nucleic acids. In one
embodiment, the particles are constructed via a hydrophobic
lipid-nucleic acid intermediate (or complex). Upon removal of a
solubilizing component (i.e., detergent or an organic solvent) the
nucleic acid becomes protected from degradation. The particles thus
formed are suitable for use in intravenous nucleic acid transfer as
they are stable in circulation, of a size required for
pharmacodynamic behavior resulting in access to extravascular sites
and target cell populations.
[0025] In particular, it is an object of this invention to provide
in vitro and in vivo methods for treatment of diseases which
involve the overproduction or underproduction of particular
proteins. In these methods, a nucleic acid encoding a desired
protein or blocking the production of an undesired protein, is
formulated into a lipid-nucleic acid particle, and the particles
are administered to patients requiring such treatment.
Alternatively, cells are removed from a patient, transfected with
the lipid-nucleic acid particles described herein, and reinjected
into the patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 illustrates a nucleic acid-lipid particle-mediated
gene transfer using "sandwich-type" complexes of DNA.
[0027] FIG. 2 illustrates an aggregation and precipitation which
commonly occurs during the entrapment of large nucleic acids in
lipid complexes.
[0028] FIG. 3 provides a schematic representation of the
preparation of plasmid-lipid particles according to certain
embodiments of the present invention.
[0029] FIG. 4 illustrates the recovery of .sup.3H-DNA from
encapsulated particles following the reverse-phase preparation of
the particles and extrusion through a 400 nm filter and a 200 nm
filter. Lipid composition is POPC:DODAC:PEG-Cer-C.sub.20.
PEG-CerC.sub.20 was held constant at 10 mole % and POPC and DODAC
were changed relative to each other. 20 mg lipid; 50 .mu.g plasmid
DNA (7.5 kbp).
[0030] FIG. 5 illustrates the recovery of 3H-DNA from particles
prepared using a reverse-phase procedure. The particles were
extruded through a 200 nm filter and eluted on a DEAE Sepharose
CL-6B anion exchange column. The percent recovery reported is based
on the amount recovered after filtration. Lipid composition is as
in FIG. 4.
[0031] FIG. 6 illustrates the recovery of .sup.14C-lipid from
encapsulated particles following the reverse-phase preparation of
the particles and extrusion through a 400 nm filter and a 200 nm
filter. Lipid composition is as in FIG. 4.
[0032] FIG. 7 illustrates the recovery of .sup.14C-lipid from
particles prepared using a reverse-phase procedure. The particles
were extruded through a 200 nm filter and eluted on a DEAE
Sepharose CL-6B anion exchange column. The percent recovery
reported is based on the amount recovered after filtration. Lipid
composition is as in FIG. 4.
[0033] FIG. 8 illustrates the effect of DODAC concentration on the
encapsulation of plasmid DNA. Encapsulation efficiency was measured
by anion exchange chromatography. Vesicles were composed of DOPE,
DODAC and 10 mole % PEG-Cer-C.sub.20 (symbol) or EPC, DODAC and 10
mole % PEG-Cer-C.sub.20 (symbol). Total lipid and DNA
concentrations were 10 mmole/ml and 50 .mu.g/ml, respectively.
[0034] FIGS. 9A and 9B illustrate the effect of serum nucleases on
free pCMVCAT DNA as assessed by column chromatography before (A)
and after (B) incubation in 80% mouse serum. Free .sup.3H-DNA
(pCMVCAT) was eluted on a Sepharose CL-4B column in HBS, pH
7.4.
[0035] FIG. 10 illustrates the effect of serum nucleases on
encapsulated pCMVCAT DNA (prepared by reverse-phase) as assessed by
column chromatography. Sepharose CL-4B column profile of
encapsulated pCMV plasmid incubated in 80% mouse serum for 30 min.
(A) External DNA was removed by ion exchange chromatography prior
to incubation in serum. (B) External DNA was not removed prior to
incubation in serum. Lipid composition was
POPC:DODAC:PEG-Cer-C.sub.20. Total lipid and plasmid concentrations
were 20 .mu.mole/ml and 50 .mu.g/ml prior to anion exchange
chromatography.
[0036] FIGS. 11A and 11B illustrate the effect of serum nucleases
on encapsulated pCMVCAT DNA (prepared by detergent dialysis) as
assessed by column chromatography. Sepharose CL-4B column profile
of encapsulated pCMV plasmid incubated in 80% mouse serum for 30
min. (A) External DNA was removed by ion exchange chromatography
prior to incubation in serum. (B) External DNA was not removed
prior to incubation in serum. The lipid composition was
DOPE:DODAC:PEG-Cer-C.sub.20 (84:6:10). Total lipid and plasmid
concentrations were 10 .mu.mole/ml and 400 .mu.g/ml prior to anion
exchange chromatography.
[0037] FIGS. 12A and 12B illustrate the resistance of plasmid
complexed to preformed liposomes composed of DOPE:DODAC(50:50) (A)
and plasmid encapsulated within DOPE:DODAC:PEG-Cer-C.sub.14
particles (B) to digestion by DNAse I. Plasmid DNA was extracted
and subjected to PCR (polymerized chain reaction) to amplify for
visualization on a gel. Free plasmid was used as a control. Lane
1:1 kb DNA marker; Lane 2: PCR negative control (no DNA); Lane 3:
free plasmid alone; Lane 4: free plasmid in 0.05% detergent (Triton
X-100); Lane 5: free plasmid incubated with DNAse I in the absence
of detergent; Lane 6: free plasmid incubated with DNAse I in the
presence of detergent: Lane 7: complexed (A) or encapsulated (B)
plasmid alone; Lane 8: complexed (A) or encapsulated (B) plasmid in
0.05% detergent; Lane 9: complexed (A) or encapsulated (B) plasmid
incubated in DNAse I in the absence of detergent; Lane 10:
complexed (A) or encapsulated (B) plasmid incubated in DNAse I in
the presence of detergent.
[0038] FIG. 13 illustrates the effect of plasmid DNA concentration
on encapsulation efficiency (detergent dialysis). Vesicles were
composed of DOPE:DODAC:PEG-Cer (84:6:10) at a lipid concentration
of 10 .mu.mole/ml.
[0039] FIG. 14 illustrates the effect of NaCl concentration on the
optimal DODAC concentration for plasmid entrapment. Lipid
composition was DOPE:DODAC:PEG-Cer-C.sub.14 (or PEG-Cer-C.sub.20).
PEG-Cer was held constant at 10 mole %. Total lipid concentration
was 10 .mu.mole/ml. Plasmid concentration was 50 .mu.g/ml.
[0040] FIG. 15 illustrates the size distribution of plasmid:lipid
particles prepared by the detergent dialysis procedure (Volume
weighted analysis). Lipid composition was
DOPE:DODAC:PEG-Cer-C.sub.20) (84:6:10).
[0041] FIG. 16 illustrates the size distribution of plasmid:lipid
particles prepared by the detergent dialysis procedure (Number
weighted analysis). Lipid composition was
DOPE:DODAC:PEG-Cer-C.sub.20 (84:6: 10).
[0042] FIGS. 17A and 17B provide electron micrographs of liposomes
composed of DOPE:DODAC:PEO-Cer-C.sub.20 without encapsulated
plasmid (A) and the plasmid:lipid particles (B). The small arrows
denote empty liposomes approximately 100 nm in diameter. These are
compared to electron-dense particles surrounded by a membrane
bilayer (large arrows). Scale bar=100 nm.
[0043] FIG. 18 shows the clearance of .sup.3H-DNA and
.sup.14C-lipid from particles (prepared by reverse-phase methods)
after injection into ICR mice. The figure includes free .sup.3H-DNA
after injection as a comparison. Lipid composition is
POPC:DODAC:PEG-Cer-C.sub.20.
[0044] FIGS. 19A and 19B show the clearance .sup.3H-DNA and
.sup.14C-lipid from particles (prepared by detergent dialysis
methods) after injection into ICR mice. Lipid compositions were (A)
DOPE:DODAC-PEG-Cer-C.sub.20 (84:6:10) and (B)
DOPE:DODAC-PEG-Cer-C.sub.14 (84:6:10).
[0045] FIG. 20 shows the results of in vivo gene transfer which
occurs in the lungs of mice. Lipid composition is
DOPE-DODAC-PEG-Cer-C.sub.20 or DOPE:DODAC:PEG-Cer-C.sub.14
(84:6:10).
[0046] FIG. 21 shows the results of in vivo gene transfer which
occurs in the liver mice. Lipid composition is
DOPE-DODAC-PEG-Cer-C.sub.20 or DOPE:DODAC:PEG-Cer-C.sub.14
(84:6:10).
[0047] FIG. 22 shows the results of in vivo gene transfer which
occurs in the spleen of mice. Lipid composition is
DOPE-DODAC-PEG-Cer-C.sub.20 or DOPE:DODAC:PEG-Cer-C.sub.14
(84:6:10).
[0048] FIG. 23 shows the effect of increasing amounts of
LIPOFECTIN.RTM. (DOTMA/DOPE; 50:50 mol ratio) on the recovery of
.beta. gal plasmid DNA in the aqueous phase following Bligh and
Dyer extraction of the lipid-nucleic acid complexes.
[0049] FIGS. 24A and 24B show the effect of increasing amounts of
cationic lipid on the recovery of plasmid DNA in the aqueous (A)
and organic (B) phase following Bligh and Dyer extraction of the
lipid-nucleic acid complexes.
[0050] FIGS. 25A, 25B, 25C and 25D show the recovery of plasmid DNA
from aqueous (A and C) and organic (B and D) fractions following
Bligh and Dyer extraction and expressed as a function of charge
ratio (+/-).
[0051] FIGS. 26A and 26B illustrate the DNA condensation by
poly-L-lysine and DODAC assayed by TO-PRO-1 dye intercalation.
Condensation state was assessed in a Bligh and Dyer monophase (A)
and in 100 mM OGP (B).
[0052] FIG. 27 illustrates the effects of increasing amounts of OGP
on the recovery of plasmid DNA from the aqueous and organic phases
following Bligh and Dyer extraction of lipid-nucleic acid complexes
(plasmid/DODAC).
[0053] FIG. 28 shows the effects of increasing amounts of NaCl on
the recovery of plasmid DNA from the aqueous phase following Bligh
and Dyer extraction of lipid-nucleic acid complexes.
[0054] FIGS. 29A and 29B show the effect of poly-L-lysine and DODAC
on the electrophoretic mobility of plasmid DNA.
[0055] FIG. 30 illustrates a protocol for preparing lipid-nucleic
acid particles using detergent dialysis.
[0056] FIGS. 31A and B are bar graphs which illustrates the QELS
results of a typical lipid-nucleic acid complex mixture prepared
from .beta.-gal plasmid/DODAC/ESM.
[0057] FIG. 32 is a bar graph which illustrates the fluorescence
spectroscopic evaluation of DNA condensation in the lipid-nucleic
acid complexes using TO-PRO-1 dye intercalation. The results show
that .beta.-gal plasmid in DODAC/ESM is condensed and protected
against dye intercalation by the lipid, and that OGP can uncondense
the particle.
[0058] FIG. 33 shows the results of electrophoresis of DNA
extracted from lipid-nucleic acid complexes following digestion
with DNase I. DNA within the complex is protected from DNase I
degradation whereas uncomplexed DNA is not protected.
[0059] FIG. 34 provides the results of CHO cell lipofection using
.beta.-gal plasmid/DODAC/ESM as assayed by .beta.-gal enzyme
activity.
[0060] FIGS. 35A and B show changes in sample turbidity measured by
90.degree. light scattering at 600 nm during the preparation of
nucleic acid-lipid particles in the presence of 100 mM (A) or 20 mM
(B) n-octyl .beta.-D-glucopyranoside (OGP).
[0061] FIG. 36 shows solubilization of preformed DODAC
(.circle-solid.) and SM (.box-solid.) vesicles in OGP as measured
by 90.degree. light scattering. The concentrations of lipids used
were 200 .mu.M (solid lines) and 800 .mu.M (broken lines).
[0062] FIGS. 37A, 37B and 37C show volume-weighing particle size
distribution determined by QELS operating in solid particle
analysis mode for a nucleic acid-lipid particle formulation
composed of pCMV.beta./DODAC/SM (charge ratio of 2:1, DODAC/SM mole
ratio of 1:1) and prepared using 20 mM OGP before (.circle-solid.)
and after (.box-solid.) dialysis (A). The same nucleic acid-lipid
particle formulation after dialysis was also examined by electron
microscopy (B, negative stain and C, freeze-fracture). Bar=100
nm.
[0063] FIGS. 38A and 37B depict the agarose gel electrophoresis of
DNA isolated from formulations prepared in 100 mM and 20 mM OGP
(charge ratio of 2.1 and SM/DODAC ratio of 1:1) and tested for
DNase I sensitivity in the absence (A) and presence (B) of OGP.
Panel A: molecular weight standards (lane 1), pCMV.beta. in the
absence of added lipid or DNase I (lane 2), pCMV.beta. following
incubation with DNase I (lane 3), DNA isolated from a dialyzed
nucleic acid-lipid particle formulation prepared using 100 mM OGP
following incubations in the absence (lane 4) and presence (lane 5)
of DNase I, and DNA isolated from particles prepared using 20 mM
OGP and dialyzed following incubations in the absence (lane 6) and
presence (lane 7) of DNase 1. The first 3 lanes in panel B are
identical to those in panel A except that pCMV.beta. was incubated
in 20 mM OGP in the absence (lane 2) and presence (lane 3) of DNase
1. DNA isolated from a formulation prepared in 20 mM OGP (prior to
detergent removal) was incubated in the absence (lane 4) and
presence (lane 5) of DNase I in 20 mM OGP. Arrow indicates degraded
DNA.
[0064] FIGS. 39A, 39B and 39C show in vitro Chinese Hamster Ovary
(CHO) cell lipofection using nucleic acid-lipid particle
formulations composed of pCMV.beta./SM/DODAC (SM/DODAC mole ratio
of 1:1 and charge ratio of 1:1 to 8:1) prepared using 100 mM OGP
followed by dialysis. (A) Influence of charge ratio on
.beta.-galactosidase lipofection. (B) Particle induced toxicity as
measured by reduced .beta.-galactosidase activity per well for
formulations prepared using a charge ratio of 4:1. (C)
.beta.-galactosidase lipofection achieved with nucleic acid-lipid
particles prepared using SM (solid bar) or DOPE (hatched bar) as
the neutral lipid (charge ratio of 4:1 and DODAC to neutral lipid
mole ratio of 1:1).
[0065] FIG. 40 is a model describing the intermediates that may be
involved in the generation of a novel lipid-DNA particle.
[0066] FIGS. 41A, 41B and 41C illustrate the encapsulation of
plasmid DNA in a lipid vesicles by the detergent dialysis method
using different cationic lipids.
[0067] FIGS. 42A, 42B and 42C demonstrate the stability of plasmid
containing vesicles prepared with different cationic lipids.
[0068] FIG. 43 demonstrates the encapsulation of plasmid DNA with
the ionizable lipid AL-1 (pK.sub.a=6.6) by the dialysis method
[0069] FIGS. 44 and 45 show the stability of the plasmid containing
vesicles formed with AL-1 at pH 4.8 and the protection of the
entrapped DNA from degradation by serum nucleases at pH 7.5.
[0070] FIG. 46 demonstrates the effect of the PEG-ceramide
concentration on the encapsulation efficiency by the dialysis
method with 7.5% DODAC and DOPE.
DETAILED DESCRIPTION OF THE INVENTION
Contents
[0071] I. Glossary [0072] II. General [0073] III. Embodiments of
the invention [0074] A. Lipid-Nucleic Acid Particles, and
Properties Thereof [0075] B. Methods of Formulating Lipid-Nucleic
Acid Particles [0076] C. Pharmaceutical Preparations [0077] D.
Administration of Lipid-Nucleic Acid Particle Formulations for Gene
Transfer [0078] IV. Examples [0079] V. Conclusion
I. Glossary
[0080] The following abbreviations are used herein: CHO, Chinese
hamster ovary cell line; B16, murine melanoma cell line; DC-Chol,
3.beta.-(N-(N',N'-dimethylaminoethane)carbamoyl) cholesterol (see,
Gao, et al., Biochem. Biophys. Res. Comm. 179:280-285 (1991));
DDAB, N,N-distearyl-N,N-dimethylammonium bromide; DMRIE,
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide; DODAC, N,N-dioleyl-N,N-dimethylammonium chloride; DOGS,
diheptadecylamidoglycyl spermidine; DOPE,
1,2-sn-dioleoylphoshatidylethanolamine; DOSPA,
N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethy-
lammonium trifluoroacetate; DOTAP,
N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride;
DOTMA, N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride; EPC, egg phosphatidylcholine; ESM, egg sphingomyelin; RT,
room temperature; TBE, Tris-Borate-EDTA (89 mM in Tris-borate and 2
mM in EDTA); HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; HBS, HEPES buffered saline (150 mM NaCl and 20 mM HEPES);
PEG-Cer-C.sub.20, 1-O-(2'-(.omega.-methoxypolyethyleneglycol)
succinoyl)-2-N-arachidoyl-sphingosine; PEG-Cer-C.sub.14,
1-O-(2'-(.omega.-methoxypolyethyleneglycol)succinoyl)-2-N-myristoyl-sphin-
gosine; PBS, phosphate-buffered saline; EGTA,
ethylenebis(oxyethylenenitrilo)-tetraacetic acid; OGP, n-octyl
.beta.-D-glycopyranoside (Sigma Chemical Co., St. Louis, Mo.);
POPC, palmitoyl oleoyl phosphatidylcholine (Northern Lipids,
Vancouver, BC); QELS, quasielastic light scattering; TBE, 89 mM
Tris-borate with 2 mM EDTA; and EDTA, Ethylenediaminetetraacetic
acid (Fisher Scientific, Fair Lawn, N.J.).
[0081] The term "acyl" refers to a radical produced from an organic
acid by removal of the hydroxyl group. Examples of acyl radicals
include acetyl, pentanoyl, palmitoyl, stearoyl, myristoyl, caproyl
and oleoyl.
[0082] As used herein, the term "pharmaceutically acceptable anion"
refers to anions of organic and inorganic acids which provide
non-toxic salts in pharmaceutical preparations. Examples of such
anions include chloride, bromide, sulfate, phosphate, acetate,
benzoate, citrate, glutamate, and lactate. The preparation of
pharmaceutically acceptable salts is described in Berge, et al., J.
Pharm. Sci. 66:1-19 (1977), incorporated herein by reference.
[0083] The term "lipid" refers to any fatty acid derivative which
is capable of forming a bilayer such that a hydrophobic portion of
the lipid material orients toward the bilayer while a hydrophilic
portion orients toward the aqueous phase. Amphipathic lipids are
necessary as the primary lipid vesicle structural element.
Hydrophilic characteristics derive from the presence of phosphato,
carboxylic, sulfato, amino, sulfhydryl, nitro, and other like
groups. Hydrophobicity could be conferred by the inclusion of
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). Preferred lipids are phosphoglycerides and sphingolipids,
representative examples of which include phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
phosphatidic acid, palmitoyloleoyl phosphatidylcholine,
lysophosphatidylcholine, lysophosphatidylethanolamine,
dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,
distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine
could be used. Other compounds lacking in phosphorus, such as
sphingolipid and glycosphingolipid families are also within the
group designated as lipid. Additionally, the amphipathic lipids
described above may be mixed with other lipids including
triglycerides and sterols.
[0084] The term "neutral" refers to any of a number of lipid
species which exist either in an uncharged form, a neutral
zwitterionic form. Such lipids include, for example
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, and cerebrosides.
[0085] The term "non-cationic lipid" refers to any neutral lipid as
described above as well as anionic lipids. Examples of anionic
lipids include cardiolipin, diacylphosphatidylserine and
diacylphosphatidic acid.
[0086] The term "cationic lipid" refers to any of a number of lipid
species which carry a net positive charge at physiological pH. Such
lipids include, but are not limited to, DODAC, DOTMA, DDAB, DOTAP,
DC-Chol and 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.RTM.
(commercially available cationic liposomes comprising DOTMA and
DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE.RTM.
(commercially available cationic liposomes comprising DOSPA and
DOPE, from GIBCO/BRL); and TRANSFECTAM.RTM. (commercially available
cationic lipids comprising DOGS from Promega Corp., Madison, Wis.,
USA).
[0087] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form.
Unless otherwise specified, the term nucleic acid is used
interchangeably with gene, DNA, cDNA, RNA, and mRNA. The term
specifically encompasses ribozymes; nucleic acid cloning and/or
expression vectors such as plasmids; genetically engineered viral
genomes, expression cassettes, and chromosomes from mammalian
(especially human) sources.
[0088] The terms "gene transfer", "transfection", and
"transformation" are used herein interchangeably, and refer to the
introduction of polyanionic materials, particularly nucleic acids,
into cells. The term "lipofection" refers to the introduction of
such materials using lipid-based complexes. The polyanionic
materials can be in the form of DNA or RNA which is linked to
expression vectors to facilitate gene expression after entry into
the cell. Thus the polyanionic material used in the present
invention is meant to include DNA having coding sequences for
structural proteins, receptors and hormones, as well as
transcriptional and translational regulatory elements (i.e.,
promoters, enhancers, terminators and signal sequences) and
vectors. Methods of incorporating particular nucleic acids into
expression vectors are well known to those of skill in the art, but
are described in detail in, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring
Harbor Laboratory, (1989) or Current Protocols in Molecular
Biology, F. Ausubel et al., ed. Greene Publishing and
Wiley-Interscience, New York (1987), both of which are incorporated
herein by reference. "Expression vectors", "cloning vectors", or
"vectors" are nucleic acid molecules (such as plasmids) that are
able to replicate in a chosen host cell. Expression vectors may
replicate autonomously, or they may replicate by being inserted
into the genome of the host cell, by methods well known in the art.
Vectors that replicate autonomously will have an origin of
replication or autonomous replicating sequence (ARS) that is
functional in the chosen host cell(s). Often, it is desirable for a
vector to be usable in more than one host cell, e.g., in E. coli
for cloning and construction, and in a mammalian cell for
expression.
[0089] The term "hydrophobic" as applied to DNA and DNA complexes,
refers to complexes which are substantially more soluble in organic
solvents than in aqueous solutions. More particularly, hydrophobic
DNA and DNA complexes are those which are at least 50% soluble in
organic solvents such as chloroform/methanol mixtures, and
preferably more than 70% soluble, more preferably more than 90%
soluble in such organic solvents.
II. General
[0090] Gene transfer techniques that involve the use of liposomes
have been described previously in the art (U.S. Pat. Nos.
5,049,386; 4,946,787; and 4,897,355). General lipofection protocols
are also described in the following references: Behr et al. (1989)
Proc. Natl. Acad. Sci. (U.S.A.) 86: 6982; Demeneix et al. (1991)
Int. J. Dev. Biol. 35: 481; Loeffler et al. (1990) J. Neurochem.
54; 1812; Bennett et al. (1992) Mol. Pharmacol. 41: 1023; Bertling
et al. (1991) Biotechnol. Appl. Biochem. 13: 390; Felgner et al.
(1987) Proc. Natl. Acad. Sci. (U.S.A.) 84: 7413; Felgner and
Ringold (1989) Nature 337: 387; Gareis et al. (1991) Cell. Mol.
Biol. 37: 191; Jarnagin et al. (1992) Nucleic Acids Res. 20: 4205;
Jiao et al. (1992) Exp. Neurol. 115: 400; Lim et al. (1991)
Circulation 83: 2007; Malone et al. (1989) Proc. Natl. Acad. Sci.
(U.S.A.) 86: 6077; Powell et al. (1992) Eur. J. Vasc. Surg. 6: 130,
Strauss and Jaenisch (1992) EMBO J. 11: 417; and Leventis and
Silvius (1990) Biochim. Biophys. Acta 1023: 124. Lipofection
reagents are sold commercially (e.g., "Transfectam" and
"Lipofectin"). Cationic and neutral lipids that are reportedly
suitable for efficient lipofection of nucleic acids include those
of Felgner (WO91/17424; WO91/16024). In addition, a combination of
neutral and cationic lipid has been shown to be highly efficient at
lipofection of animal cells and showed a broad spectrum of
effectiveness in a variety of cell lines (Rose et al. (1991)
BioTechniques 10: 520. The above lipofection protocols may be
adapted for use in the present invention, and the preceding
references are therefore incorporated in their entirety.
III. EMBODIMENTS OF THE INVENTION
A. Lipid-Nucleic Acid Particles, and Properties Thereof
[0091] In one aspect, the present invention provides novel,
lipid-nucleic acid complexes consisting essentially of cationic
lipids and nucleic acids.
[0092] 1. Lipid Components
[0093] 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.
[0094] Cationic lipids which 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 U.S. Ser. No.
08/316,399, U.S. Pat. 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.RTM.
(commercially available cationic liposomes comprising DOTMA and
DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE.RTM.
(commercially available cationic liposomes comprising DOSPA and
DOPE, from GIBCO/BRL); and TRANSFECTAM.RTM. (commercially available
cationic liposomes comprising DOGS from Promega Corp., Madison,
Wis., USA).
[0095] The non-cationic 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 non-cationic 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 non-phosphorous 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. Non-cationic 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 U.S. Ser. No. 08/316,429,
incorporated herein by reference.
[0096] In preferred embodiments, the non-cationic lipids are
diacylphosphatidylcholine (e.g., dioleoylphosphatidylcholine,
dipalmitoylphosphatidylcholine and dilinoleoylphosphatidylcholine),
diacylphosphatidylethanolamine (e.g.,
dioleoylphosphatidylethanolamine and
palmitoyloleoylphosphatidylethanolamine), ceramide or
sphingomyelin. The acyl groups in these lipids are preferably acyl
groups derived from fatty acids having C.sub.10-C.sub.24 carbon
chains. More preferably the acyl groups are lauroyl, myristoyl,
palmitoyl, stearoyl or oleoyl. In particularly preferred
embodiments, the non-cationic lipid will be
1,2-sn-dioleoylphosphatidylethanolamine, or egg sphingomyelin
(ESM).
[0097] 2. Nucleic Acid Components
[0098] 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.
[0099] 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., Science
261:1004-1011 (1993) and in U.S. Pat. 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.
[0100] 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.
[0101] 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 linkages substituted with stable,
non-phosphodiester linkages, including, for example,
phosphorothioate, phosphorodithioate, phosphoroselenate, or O-alkyl
phosphotriester linkages.
[0102] 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 sugar analogs. Modifications in the purine or
pyrimidine base moiety include, for example, alkylated purines and
pyrimidines, acylated purines or pyrimidines, or other heterocyclic
substitutes known to those of skill in the art.
[0103] 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. Non-encoding sequences may be
also be present, to the extent that they are necessary to achieve
appropriate expression.
[0104] The nucleic acids used in the present method can be isolated
from natural sources, obtained from such sources as ATCC or GenBank
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.
Pat. No. 4,401,796; Caruthers, et al., U.S. Pat. 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 Engineering, 4:1-17 (1982); Jones,
chapter 2, Atkinson, et al., chapter 3, and Sproat, et al., chapter
4, in Oligonucleotide 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. Tetrahedron Lett., 24:5843-5846
(1983); and Sinha, et al., Nucl. Acids Res., 12:4539-4557 (1984)
which are incorporated herein by reference.
[0105] a. Vectors for Introduction and Expression of Genes in
Cells
[0106] An important aspect of this invention is the use of the
lipid-nucleic acid particles provided herein to introduce selected
genes into cells in vitro and in vivo, followed by expression of
the selected gene in the host cell. Thus, the nucleic acids in the
particles specifically 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.
[0107] 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
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 Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology,
volume 152, Academic Press, Inc., San Diego, Calif. (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, Minn.), Pharmacia
LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc.
(Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie AG, Buchs, Switzerland), and Applied
Biosystems (Foster City, Calif.), as well as many other commercial
sources known to one of skill.
[0108] 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.
[0109] 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.
[0110] 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 to the DNA sequence encoding a desired protein and
signals required for efficient polyadenylation of the
transcript.
[0111] 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.
[0112] 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 murine cytomegalovirus, the long term
repeat from various retroviruses such as murine leukemia virus,
murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic
Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
1983, which is incorporated herein by reference.
[0113] 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.
[0114] 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.
[0115] 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 factors are
provided by genes either carried on the plasmid or with the genome
of the host cell.
[0116] 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 prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells.
[0117] 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.
[0118] 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.sup.+, pMTO10/A.sup.+, 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.
[0119] 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.
[0120] 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,
Calif. (D. V. Goeddel, ed.) (1990) or M. Krieger (1990), Gene
Transfer and Expression--A Laboratory Manual, Stockton Press, New
York, N.Y., 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 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), Current 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), Virology, 160: 38-47; Carter et al. (1989), U.S. Pat. No.
4,797,368; Carter et al. (1993), WO 93/24641; Kotin (1994), Human
Gene Therapy, 5: 793-801; Muzyczka (1994), J. Clin. Invest., 94:
1351 and Samulski, supra, for an overview of AAV vectors.
[0121] 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.
[0122] Whatever the vector is used, generally the vector is
genetically engineered to contain, in expressible form, a gene of
interest. The particular gene selected will depend on the intended
treatment. Examples of such genes of interest are described below
at Section D.3. Insertion of Functional Copy of a Gene, and
throughout the specification.
[0123] The vectors further usually comprise selectable markers
which result in nucleic acid amplification such as the sodium,
potassium ATPase, thymidine 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 bacculovirus vector in insect
cells, with the encoding sequence under the direction of the
polyhedrin promoter or other strong baculovirus promoters.
[0124] 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. Pat. Nos. 5,264,423 and 5,276,019,
the disclosures of which are incorporated herein by reference.
B. Methods of Making the Particles
[0125] In one embodiment, the present invention provides
lipid-nucleic acid particles produced via novel, 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.
[0126] Lipid-nucleic acid formulations can be formed by combining
the nucleic acid with a preformed cationic liposome (see, U.S. Pat.
Nos. 4,897,355, 5,264,618, 5,279,833 and 5,283,185). In such
methods, the nucleic acid is attracted to the cationic surface
charge of the liposome and the resulting complexes are thought to
be of the liposome-covered "sandwich-type." As a result, a portion
of the nucleic acid or plasmid remains exposed in serum and can be
degraded by enzymes such as DNAse I. Others have attempted to
incorporate the nucleic acid or plasmid into the interior of a
liposome during formation. These methods typically result in the
aggregation in solution of the cationic lipid-nucleic acid
complexes (see FIG. 2). Passive loading of a plasmid into a
preformed liposome has also not proven successful. Finally, the
liposome-plasmid complexes which have been formed are typically 200
to 400 nm in size and are therefore cleared more rapidly from
circulation than smaller sized complexes or particles.
[0127] The present invention provides a method of preparing
serum-stable plasmid-lipid particles in which the plasmid 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.
[0128] 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, FIG. 3 depicts a detergent
dialysis approach to the formation of the plasmid-lipid particles.
With reference to FIG. 3, a plasmid or other large 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, non-cationic lipids) to form particles in which
the plasmid is encapsulated in a lipid bilayer. As noted above,
these particles differ from the more classical liposomes both in
size (liposomes being typically 200-400 nm) in that there is little
or no aqueous medium encapsulated by the particle's lipid bilayer.
The methods described below for the formation of plasmid-lipid
particles using organic solvents follow a similar scheme.
[0129] 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: [0130] (a) combining a plasmid with cationic lipids in
a detergent solution to form a coated plasmid-lipid complex; [0131]
(b) contacting non-cationic lipids with the coated plasmid-lipid
complex to form a detergent solution comprising a plasmid-lipid
complex and non-cationic lipids; and [0132] (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.
[0133] 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.RTM. 3-08; Zwittergent.RTM. 3-10; Triton X-405;
hexyl-, heptyl-, octyl- and nonyl-.beta.-D-glucopyranoside; and
heptylthioglucopyranoside; with octyl .beta.-D-glucopyranoside and
Tween-20 being the most preferred. The concentration of detergent
in the detergent solution is typically about 100 mM to about 2 M,
preferably from about 200 mM to about 1.5 M.
[0134] 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 .mu.g/mL to about 1 mg/mL, preferably from about 25
.mu.g/mL to about 200 .mu.g/mL, and more preferably from about 50
.mu.g/mL to about 100 .mu.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.degree. C. For plasmids which are
particularly sensitive to temperature, the coated complexes can be
formed at lower temperatures, typically down to about 4.degree.
C.
[0135] The detergent solution of the coated plasmid-lipid complexes
is then contacted with non-cationic lipids to provide a detergent
solution of plasmid-lipid complexes and non-cationic lipids. The
non-cationic lipids which are useful in this step include,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cardiolipin, and cerebrosides.
In preferred embodiments, the non-cationic lipids are
diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide
or sphingomyelin. The acyl groups in these lipids are preferably
acyl groups derived from fatty acids having C.sub.10-C.sub.24
carbon chains. More preferably the acyl groups are lauroyl,
myristoyl, palmitoyl, stearoyl or oleoyl. In particularly preferred
embodiments, the non-cationic lipid will be
1,2-sn-dioleoylphosphatidylethanolamine (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 in vivo and the
non-cationic lipid will be DOPE. In other preferred embodiments,
the non-cationic lipids will further comprise polyethylene
glycol-based polymers such as PEG 2000, PEG 5000 and polyethylene
glycol conjugated to ceramides, as described in co-pending U.S.
Ser. No. 08/316,429, incorporated herein by reference.
[0136] The amount of non-cationic lipid which is used in the
present methods is typically about 2 to about 20 mg of total lipids
to 50 .mu.g of plasmid. Preferably the amount of total lipid is
from about 5 to about 10 mg per 50 .mu.g of plasmid.
[0137] Following formation of the detergent solution of
plasmid-lipid complexes and non-cationic 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.
[0138] 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.
[0139] 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. Pat. 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.
[0140] 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.
[0141] In another group of embodiments, the present invention
provides a method for the preparation of serum-stable plasmid-lipid
particles, comprising; [0142] (a) preparing a mixture comprising
cationic lipids and non-cationic lipids in an organic solvent;
[0143] (b) contacting an aqueous solution of nucleic acid with said
mixture in step (a) to provide a clear single phase; and [0144] (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.
[0145] The plasmids (or nucleic acids), cationic lipids and
non-cationic lipids which are useful in this group of embodiments
are as described for the detergent dialysis methods above.
[0146] 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.
[0147] Contacting the plasmid with the organic solution of cationic
and non-cationic 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.
[0148] 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.
[0149] The serum-stable plasmid-lipid particles thus formed will
typically be sized from about 50 nm to 150 nm. To achieve further
size reduction or homogeneity of size in the particles, sizing can
be conducted as described above.
[0150] 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 norilipid polycations include, hexadimethrine bromide
(sold under the brandname POLYBRENE.RTM., from Aldrich Chemical
Co., Milwaukee, Wis., 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] Without intending to be bound by any particular theory of
formation, FIG. 1 provides a model for the binding of monocationic
lipids to DNA which results in the formation of a hydrophobic
(organic-soluble) lipid-nucleic acid complex. In this figure,
cationic lipids first bind to the DNA to form a complex in which
the DNA is uncondensed. This complex is soluble in the organic
phase or in a monophase and the DNA remains uncondensed. Upon the
addition of other lipids and removal of solvent, and hydration, the
complexes form particles (described in more detail below).
[0155] In another embodiment, the present invention provides a
method for the preparation of lipid-nucleic acid particles,
comprising: [0156] (a) contacting nucleic acids with a solution
comprising non-cationic lipids and a detergent to form a nucleic
acid-lipid mixture; [0157] (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 charge-neutralized mixture
of nucleic acids and lipids; and [0158] (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.
[0159] Without intending to be limited by any particular aspect of
the illustration, FIG. 30 provides a depiction of one method of
forming the particles using detergent dialysis. In this figure, DNA
in an aqueous detergent solution (OGP) is combined with
non-cationic lipids (ESM) in an aqueous detergent solution and
allowed to anneal for about 30 min. A previously sonicated mixture
of cationic lipid (DODAC) in detergent is added and the resulting
mixture is dialyzed for 3 days to remove detergent and thereby form
lipid-nucleic acid particles. One of skill in the art will
understand that for the kinetic formation of such particles, the
order of addition of cationic lipids and non-cationic lipids could
be reversed, or the lipids could be added simultaneously. In
addition, it is possible to cover the nucleic acid with multivalent
cations, such that it now binds anions.
[0160] In one group of embodiments, the solution of non-cationic
lipids and detergent is an aqueous solution. Contacting the nucleic
acids with the solution of non-cationic 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
such as by using vortex mixers. Preferably, the nucleic acid
solution is also a detergent solution. The amount of non-cationic
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.
[0161] 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 U.S.
Ser. No. 08/316,399; U.S. Pat. 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.RTM.
(commercially available cationic liposomes comprising DOTMA and
DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE.RTM.
(commercially available cationic liposomes comprising DOSPA and
DOPE, from GIBCO/BRL); and TRANSFECTAM.RTM. (commercially available
cationic lipids comprising DOGS in ethanol from Promega Corp.,
Madison, Wis., USA).
[0162] 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.
[0163] 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.
[0164] The particles thus formed will typically be sized from about
100 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.
[0165] 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.RTM., from Aldrich Chemical Co.,
Milwaukee, Wis., 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.
[0166] In another aspect, the present invention provides methods
for the preparation of lipid-nucleic acid particles, comprising:
[0167] (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; [0168] (b) contacting the hydrophobic,
charge-neutralized lipid-nucleic acid complex in solution with
non-cationic lipids, to provide a lipid-nucleic acid mixture; and
[0169] (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.
[0170] The nucleic acids, non-cationic 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.
[0171] In preferred embodiments, the cationic lipids are DODAC,
DDAB, DOTMA, DOSPA, DMRIE, DOGS or combinations thereof. In other
preferred embodiments, the non-cationic lipids are 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 are methanol, chloroform, methylene chloride,
ethanol, diethyl ether or combinations thereof.
[0172] 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 non-cationic lipid is ESM, DOPE,
polyethylene glycol-based polymers or combinations thereof; and the
organic solvent is methanol, chloroform, methylene chloride,
ethanol, diethyl ether or combinations thereof.
[0173] 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 non-cationic lipids and the removal of
the organic solvent. The addition of the non-cationic lipids is
typically accomplished by simply adding a solution of the
non-cationic 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.
[0174] The amount of non-cationic 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.
[0175] 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 non-cationic lipid
is egg sphingomyelin and the cationic lipid is DODAC.
[0176] As noted above, the lipid-nucleic acid particles are useful
for the lipofection of cells, either in vitro or in vivo.
Accordingly, the present invention provides, in yet another aspect,
a method for introducing a nucleic acid into a cell, comprising;
[0177] (a) preparing a lipid-nucleic acid particle according to the
methods above; and [0178] (b) contacting the cell with the
lipid-nucleic acid particle for a period of time sufficient to
introduce the nucleic acid into the cell.
[0179] Although discussed in more detail below, preferred
embodiments are those in which the lipid-nucleic acid particle
comprises a plasmid, DODAC and ESM.
[0180] Unlike viral-based gene therapy vectors which can only
incorporate a relatively small non-viral nucleic acid sequence into
the viral genome because of size limitations for packaging virion
particles, the lipid-nucleic acid complexes of the present
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, Huntington's chorea gene) and large homologous
targeting constructs or transgenes may be required for therapy.
[0181] 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.
[0182] 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 acid(s)
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 neo.sup.R expression cassette)
is included in the exogenous nucleic acid(s) 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.
[0183] 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 (rim), such as a protein. The rim can
be covalently bound to lipids that comprise the nucleic acid-lipid
particle. Its presence on the particle increases the efficiency and
specificity with the particle contacts and enters target cells. For
example, a suitable rim is a non-immunoglobulin 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
endocytosis and/or membrane fusion. Additional suitable rim species
typically are naturally-occurring physiological ligands which
comprise a polypeptide portion (e.g., adhesion molecules such as
ICAM-1, ICAM-2, ELAM-1, 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 rim 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.
[0184] Fragments and analogs of naturally-occurring proteins may be
used as well as full-length mature proteins as rim 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.
[0185] The rim selected will vary with the particular target cell
type. For specific targeting to hepatocytes, asialoglycoproteins
(galactose-terminal) are preferred as rim 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, rim 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 non-galactose-bearing
proteins by reductive lactosamination. Other useful
galactose-terminal carbohydrates for hepatocyte targeting include
carbohydrate trees obtained from natural glycoproteins, especially
tri- and tetra-antennary 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, rim species comprising mannose or
mannose-6-phosphate, or complex carbohydrates comprising these
terminal carbohydrate structures may be used.
[0186] 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 rim species. For example, transferrin can be used
as a suitable rim for forming receptor-recognition transfection
complexes to cells expressing 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.
[0187] The nucleic acid-lipid particles may comprise multiple rim
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 rim species, typically with those which lack
membrane fusion activity.
[0188] 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
non-immunoglobulin 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 rim is attached to the polycation
by covalent linkage, frequently by covalent linkage through a
crosslinking agent or by peptide linkage.
[0189] Overall particle charge is an important property of the
particles, as it may affect particle clearance from the blood.
Particles with prolonged circulation half-lives are typically
desirable for therapeutic and diagnostic uses. For instance,
particles which can be maintained from 8, 12, or up to 24 hours in
the bloodstream are particularly preferred. Negatively charged
liposomes and particles are typically, taken up more rapidly by the
reticuloendothelial system (Juliano, Biochem. Biophys. Res. Commun.
63:651 (1975)) and thus have shorter half-lives in the
bloodstream.
C. Pharmaceutical Preparations
[0190] 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.
[0191] 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.
[0192] 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. It is often desirable
to include polyethylene glycol (PEG), PEG-ceramide, or modified
(e.g., ganglioside G.sub.M1-modified) lipids to the particles.
Addition of such components prevents particle aggregation and
provides a means for increasing circulation lifetime and increasing
the delivery of the lipid-nucleic acid particles to the target
tissues. Typically, the concentration of the PEG, PEG-ceramide or
G.sub.M1-modified lipids in the particle will be about 1-15%.
[0193] 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.
[0194] 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.
[0195] The present invention also provides lipid-nucleic acid
particles in kit form. The kit will typically be comprised of a
container which is compartmentalized for holding the various
elements of the kit. 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.
D. Administration of Lipid-Nucleic Acid Particle Formulations
[0196] 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 are carried out in 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.
[0197] 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.
[0198] 1. In Vitro Gene Transfer
[0199] 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 human cells.
[0200] 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 .mu.mol and about 10 mmol. Treatment of the cells with the
nucleic acid-lipid particles is generally carried out at
physiological temperatures (about 37.degree. C.) for periods of
time of from about 1 to 48 hours, preferably of from about 2 to 4
hours.
[0201] 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 10.sup.3 to about 10.sup.5
cells/mL, more preferably about 2.times.10.sup.4 cells/mL. The
concentration of the suspension added to the cells is preferably of
from about 0.01 to 0.2 .mu.g/mL, more preferably about 0.1
.mu.g/mL.
[0202] 2. In Vivo Gene Transfer
[0203] 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 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).
[0204] For in vivo administration, the pharmaceutical compositions
are preferably administered parenterally, i.e., intraarticularly,
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. Pat. 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); Mannino, et al., Biotechniques 6:682-690 (1988); Nicolau,
et al., Crit. Rev. Ther. Drug Carrier 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, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat.
No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871;
Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No.
4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.
[0205] 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, oropharynx, 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.
[0206] The lipid-nucleic acid particles can also be administered in
an aerosol inhale 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 GENE THERAPY, MaryAnn Liebert, Inc.,
Publishers, New York. pp. 70-71 (1994)).
[0207] The methods of the present invention may be practiced in a
variety of hosts. Preferred hosts include mammalian species, such
as humans, non-human primates, dogs, cats, cattle, horses, sheep,
and the like.
[0208] 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 10.sup.8-10.sup.10 particles per
injection.
[0209] 3. Insertion of Functional Copy of a Gene
[0210] Some 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,
.beta.-thalassemia, phenylketonuria, galactosemia, Wilson's
disease, hemochromatosis, severe combined immunodeficiency 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.
[0211] There are several methods for introducing an exogenous
functional gene to compensate for the above genetic defects. In one
approach, cells are removed from a patient suffering from the
disease and contacted with a lipid-vector complex in 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 performed, it is important that the frequency of gene transfer
into cells be high, for example, at least about 1, 5, 10, 25 or 50%
of cells.
[0212] After integration of the vector into the cellular genome,
and optionally, selection, cells are reintroduced into the patient.
In this application, and others discussed 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.
[0213] Alternatively, the lipid-vector complex can be introduced
directly into a patient as a pharmaceutical composition. The
complex is delivered to the tissue(s) 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 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 .mu.g to 10 mg, or 30-300 .mu.g DNA per
patient are typical. Routes of administration include oral, nasal,
gastric, intravenous, intradermal and intramuscular.
[0214] 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. (1984) Methods Enzymol. 101, 414; Hogan et al., Manipulation
of the Mouse Embryo: A Laboratory Manual, C.S.H.L. N.Y. (1986); and
Hammer et al. (1985) Nature 315, 680; Gandolfi et al. (1987) J.
Reprod. Fert. 81, 23-28; Rexroad et al. (1988) J. Anim. Sci. 66,
947-953 and Eyestone et al. (1989) J. Reprod. Fert. 85, 715-720;
Camous et al. (1984) J. Reprod. Fert. 72, 779-785; Heyman et al.
(1987) Theriogenology 27, 5968. However, these methods are
presently more suitable for veterinary applications that human
treatment due to ethical and regulatory constraints in manipulating
human embryos.
[0215] 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 245, 1059-1065 (1989). It encodes a protein
called the cystic fibrosis transmembrane conductance regulator
(CFTR) which is involved in the transfer of chloride ions
(Cl.sup.-) through epithelial cell membranes. Mutations in the gene
cause defects of Cl.sup.-secretion in epithelial cells leading to
the various clinical manifestations. 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 10.sup.8-10.sup.10 particles.
[0216] As another example, defects in the .alpha. or .gamma. globin
genes (see McDonagh & Nienhuis in Hematology of Infancy 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 Group C can be
treated by an analogous strategy (see Walsh et al., J. Clin.
Invest. 94, 1440-1448 (1994)).
[0217] 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.
[0218] 4. Suppression of Gene Expression
[0219] 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. 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.
[0220] 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 selected 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 .alpha. chain (120-180
kDa) and a .beta. chain (90-110 kDa), generally having short
cytoplasmic domains. The three known integrins, LFA-1, Mac-1 and
P150,95, have different alpha subunits, designated CD11a, CD11b and
CD11c, and a common beta subunit designated CD18. LFA-1
(.alpha..sub.L.beta..sub.2) 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
(.alpha..sub.M.beta..sub.2) is distributed on neutrophils and
monocytes, and also binds to ICAM-1 (and possibly ICAM-2). The
third .beta.2 integrin, P150,95 (.alpha..sub.X.beta..sub.2), is
also found on neutrophils and monocytes. The selectins consist of
L-selectin, E-selectin and P-selectin.
[0221] 5. Cells to be Transformed
[0222] 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
non-cycling primary cells, parenchymal 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.
[0223] To the extent that tissue culture of cells may be required,
it is well known in the art. Freshney (1994) (Culture of Animal
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.
[0224] 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., hematopoietic cells, skin
fibroblasts and keratinocytes, hepatocytes, endothelial cells,
muscle cells and lymphocytes, are normally non-dividing. 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. Natl. 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 non-dividing cells (CITE)
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 priot 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.
[0225] 6. Detection of Foreign Nucleic Acids
[0226] 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.
[0227] 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
immunosorbent 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.
[0228] 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.
[0229] 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.beta.-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. Pat.
No. 4,683,202; PCR Protocols A Guide to Methods and Applications
(Innis et al. eds) Academic Press Inc. San Diego, Calif. (1990)
(Innis); Arnheim & Levinson (Oct. 1, 1990), C&EN 36-47; The
Journal Of NlH Research (1991), 3: 81-94; (Kwoh et al. (1989),
Proc. Natl. Acad. Sci. USA, 86: 1173; Guatelli et al. (1990), Proc.
Natl. Acad. Sci. USA, 87: 1874; Lomell et al. (1989), J. Clin.
Chem., 35: 1826; Landegren et al. (1988), Science, 241: 1077-1080;
Van Brunt (1990), Biotechnology, 8: 291-294; Wu and Wallace (1989),
Gene, 4: 560; Barringer et al. (1990), Gene, 89: 117, and Sooknanan
and Malek (1995), Biotechnology, 13: 563-564. Improved methods of
cloning in 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
(NASBA.TM., 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 region later
probed for a specific sequence indicative of a mutation.
[0230] 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 (1981), Tetrahedron Letts., 22(20): 1859-1862, e.g.,
using an automated synthesizer, as described in Needham-VanDevanter
et al. (1984), Nucleic Acids Res., 12: 6159-6168. Purification of
oligonucleotides, where necessary, is typically performed by either
native acrylamide gel electrophoresis or by anion-exchange HPLC as
described in Pearson and Regnier (1983), J. Chrom., 255: 137-149.
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.
[0231] An alternative means for determining the level of expression
of the gene is in situ hybridization. In situ hybridization assays
are well known and are generally described in Angerer et al.
(1987), Methods Enzymol., 152: 649-660. In an in 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.
[0232] 7. Detection of Foreign Gene Products
[0233] 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.
[0234] 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, Wiley/Greene, NY; and Harlow and
Lane (1989), ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor
Press, NY; Stites et al. (eds.) BASIC AND CLINICAL IMMUNOLOGY (4th
ed.) Lange Medical Publications, Los Altos, Calif., and references
cited therein; Goding (1986), MONOCLONAL ANTIBODIES: PRINCIPLES AND
PRACTICE (2d ed.) Academic Press, New York, N.Y.; and Kohler and
Milstein (1975), Nature, 256: 495-497. Such techniques include
antibody preparation by selection of antibodies from libraries of
recombinant antibodies in phage or similar vectors. See, Huse et
al. (1989), Science, 246: 1275-1281; and Ward et al. (1989),
Nature, 341: 544-546. Specific monoclonal and polyclonal antibodies
and antisera will usually bind with a K.sub.D of at least about 0.1
mM, more usually at least about 1 .mu.M, preferably at least about
0.1 .mu.M or better, and most typically and preferably, 0.1 .mu.M
or better.
[0235] 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 murine
antibody) that specifically bind to the labeling antibody.
VII. EXAMPLES
[0236] The following examples are offered solely for the purposes
of illustration, and are intended neither to limit nor to define
the invention. In each of these examples, the term "DNA" or
"plasmid" refers to the plasmid pCMV (pCMV4-CAT).
A. Materials
[0237] Transfecting agents Lipofectin and Lipofectamine were
purchased from Gibco/BRL (Grand Island, N.Y., USA). Transfectam
Reagent was purchased from Promega Corp. (Madison, Wis., USA). The
monocationic lipid DDAB, calcium chloride, L-lysine (free base),
poly L-lysine hydrobromide (Avg. MW 52,000), n-octyl
-D-glucopyranoside (OGP) and DNase I were obtained from Sigma
Chemical Company (St. Louis, Mo., USA). TO-PRO-1 (thiazole orange
monomer) was obtained from Molecular Probes Inc., Eugene, Oreg.,
USA. The plasmid pCMV (GenBank accession # U02451) encoding E.
coli-galactosidase (-gal), a 7.2 kb plasmid DNA reporter gene, was
obtained from Clontech Laboratories, Palo Alto, Calif., USA.
.beta.-gal DNA was propagated and purified using standard
techniques (Sambrook et al., Molecular Cloning, A Laboratory
Manual, Second Ed., Cold Spring Harbor, N.Y. (1989)). Egg
sphingomyclin (SM) and
1,2-dioleoyl-sn-Glycero-3-phosphoethanolamine (DOPE) were purchased
from Avanti Polar Lipids (Alabaster, Ala.).
N-N-diolcoyl-N,N-dimethylammonium chloride (DODAC) was synthesized
and supplied by Steven Ansell of INEX Pharmaceuticals Corp.
(Vancouver, B.C.). TO-PRO-1 was purchased from Molecular Probes
Inc. (Eugene, Oreg.). Dialysis membrane (SPECTRA/POR, mwco:
12.000-14,000) was purchased from Fisher Scientific (Ottawa, ON).
All other chemicals used were reagent grade and all solvents used
were HPLC grade. Radiolabeled DNA was used as a tracer and was
generated by incorporating 3H-dUTP into the plasmid during
bacterial growth, resulting in specific activities of .about.50,000
dpm/g of DNA. All other chemicals used in these Examples were of
reagent grade and all solvents used were HPLC grade. Sterile
distilled water was used throughout all experiments. All materials
were used without further purification.
B. Methods
Bligh and Dyer Extraction Procedure
[0238] Non-cationic lipids, cationic lipids and DNA were
solubilized in chloroform:methanol:water (1:2.1:1) prior to mixing.
This mixture of solvents and water is equivalent to that used in
the preparation of a Bligh and Dyer monophase (Bligh and Dyer, Can.
J. Biochem. Physiol. 37:91-97 (1959)). Typically, DNA was added to
achieve a final concentration of 10 g/mL in solution while lipid
was added at various concentrations. Trace quantities of 3H-plasmid
DNA were added such that 2000 to 4000 dpm were present per 10 g
unlabelled DNA. The reaction mixtures were incubated at room
temperature for 30 min in a total volume of 1 mL. Subsequently the
Bligh and Dyer monophase was partitioned into a two phase system by
the addition of water and chloroform (250 L each). The samples were
mixed by vortexing and the separation of the lower organic and
upper aqueous phases was facilitated by centrifugation at 2000 rpm
for 5 min at room temperature. The aqueous phase was removed and
retained for scintillation counting. The solvent phase was dried
using a stream of nitrogen gas, and the resulting film was
resuspended in SOLVABLE solubilizing agent (Dupont NEN, Boston,
Mass., USA) and incubated at 50.degree. C. for 1 hour. This last
step was necessary to solubilize the dried DNA/lipid complex since
the addition of the scintillation cocktail alone was not sufficient
to dissociate the complex. PICOFLUOR scintillant (Canberra Packard,
Meriden, Conn., USA) was added to all samples and the radioactivity
(3H-DNA) was measured using a Packard TR 1900 Scintillation Counter
(Canberra Packard).
[0239] Assays evaluating the stability of charge-neutralized,
lipid-nucleic acid complexes were done in the presence of varying
concentration of NaCl and OGP. Briefly, cationic lipid-nucleic acid
complexes were prepared under conditions where 100% of the plasmid
was expected to be recovered in the organic phase. NaCl or OGP was
then added to the monophase system and incubations carried out at
room temperature for 15 min. Bligh and Dyer extractions were
performed as described above. The binding of calcium, L-lysine, and
poly-L-lysine to the plasmid was evaluated using a modification of
the above procedure. These nonlipid cationic materials were
dissolved at various concentrations in sterile distilled water and
incubated with the plasmid (10 g/mL final concentration in water)
at room temperature for 30 min in a final volume of 250 L. Reaction
volumes were adjusted to 1 mL with chloroform:methanol (1:2.1) to
produce a monophase. Bligh and Dyer extractions were then performed
as described.
Dye Intercalation Assay
[0240] The fluorochrome TO-PRO-1 was used to evaluate the state of
condensation of the plasmid in the charge-neutralized lipid-nucleic
acid complex. TO-PRO-1 was used in this study due to its stable
intercalation into the plasmid as well as the high sensitivity in
the fluorescence detection compared with the more common
intercalator ethidium bromide (see, Hirons, et al., Cytometry
15:129-140 (1994)). Plasmid was dissolved in either the Bligh and
Dyer monophase or in 100 mM OGP. Poly-L-lysine or DODAC were each
added to 10 g plasmid at a 1:1 charge ratio.
Agarose Gel Electrophoresis
[0241] Complexes involving plasmid and poly-L-lysine were formed at
a nucleic acid concentration of 10 g/mL and a 1:1 charge ratio in
the presence of 100 mM OGP. Complexes involving the cationic lipid
DODAC and plasmid were formed at a plasmid concentration of 10 g/mL
and increasing concentrations of DODAC (10 to 320 nmoles/mL). The
mixtures were incubated at room temperature for 30 min prior to
loading onto a 0.8% agarose gel. Electrophoresis was carried out in
TBE buffer according to standard techniques (Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor, N.Y. (1989)). Nucleic acids were visualized after staining
the gel with ethidium bromide (0.5 g/mL, 20 min) by photography
with UV transillumination.
DNAse I Assay
[0242] To evaluate the protective effect of cationic lipids on DNA,
the complexes formed in the presence of OGP were incubated with
DNase I. Preformed charge-neutralized lipid-nucleic acid complexes
(plasmid/DODAC; 1:1 charge ratio) were mixed with DNase I at a
concentration where plasmid alone was susceptible to degradation at
37.degree. C. for 10 min. The reactions were stopped by the
addition of 25 mM EDTA and the samples were extracted using the
Bligh and Dyer procedure in the presence of 150 mM NaCl. Under
these conditions the charge-neutralized lipid-nucleic acid
complexes dissociate and plasmid can be efficiently recovered in
the aqueous fraction. This DNA was precipitated with 1/10th volume
of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 95% EtOH and
recovered by centrifugation at 14,000 g for 30 min at 4C. The DNA
pellet was resuspended in sterile distilled water and subjected to
electrophoresis on a 0.8% agarose gel.
Example 1
[0243] This example illustrates the encapsulation of a plasmid in a
lipid particle using either a reverse-phase method or a detergent
dialysis method.
Reverse Phase Method
[0244] pCMV4-CAT plasmid was encapsulated in a lipid particle which
was constructed using about 10 mg or 20 mg of lipid. The
encapsulation method involved a modification of the classical
reverse phase method for entrapment. Generally, 1.050 ml of
chloroform: methanol in a 1:2-1 mole % ratio was added to a lipid
film containing 2 .mu.l of .sup.14C-cholesteryl hexadecyl ether
(6.66 .mu.l/.mu.Ci). This was followed by the addition of 220 .mu.l
H.sub.2O and 33 .mu.l .sup.3H-pCMVCAT plasmid (158,000 dpm/.mu.l;
1.5 mg/ml). This combination provided a clear single phase. The
chloroform and most of the methanol were removed under a stream of
nitrogen while vortexing. In some cases, the resulting 250 .mu.l
suspension of encapsulated plasmid was diluted with 1 ml of
H.sub.2O and extruded 5 times through one 400 nm filter followed by
extrusion 5 times through one 200 nm filter. The resulting vesicle
size was approximately 150 to 200 nm in diameter. Liposome sizes
before extrusion varied greatly depending on the lipid
composition.
Detergent Dialysis Method
[0245] pCMVCAT was incubated with DODAC at various DODAC
concentrations in 100 .mu.l of 1 M n-octyl-B-D-glucopyranoside and
400 .mu.l of H.sub.2O for 30 min at room temperature. The resulting
plasmid:DODAC mixture was added to a suspension of approximately 10
mg of lipid containing 1 .mu.l, .sup.14C-cholesteryl hexadecyl
ether; 6.66 .mu.l/.mu.Ci in 100 .mu.l of 1M
n-octyl-.beta.-D-glucopyranoside. The suspension was dialysed
against HBS at pH 7.4 overnight. The resulting encapsulated plasmid
could be used without further sizing.
Example 2
[0246] This example illustrates the level of plasmid "protection"
from the external medium using anion exchange chromatography.
[0247] The extent of encapsulation or protection of the plasmid
from the external medium was assessed by anion exchange
chromatography as follows: a 50 .mu.l aliquot of each sample was
eluted on a DEAE Sepharose CL-6B column and the fractions were
assessed for both .sup.3H-plasmid and .sup.14C-lipid by
scintillation counting. Any exposed negative charges, such as those
present on DNA molecules will bind to the anion exchange column and
will not elute with the .sup.14C-lipid. DNA which has its negative
charge, "protected" or non-exposed will not bind to the anion
exchange resin and will elute with the .sup.14C-lipid.
Alternatively, plasmid DNA was measured using the indicator dye,
PicoGreen.RTM..
Reverse Phase Method
[0248] FIG. 4 presents the results describing the relationship
between the amount of DODAC present in the formulation and the
encapsulation efficiency for POPC:DODAC:PEG-Cer-C.sub.20 (20 mg
total lipid) compositions after extrusion through a 400 nm filter
and a 200 nm filter as measured by anion exchange chromatography.
Lipid was composed of 10% PEG-Cer-C.sub.20 and the remaining
percentage was attributable to POPC and DODAC. An increase in
percent plasmid recovered was observed corresponding to an increase
in DODAC concentration. No plasmid was recovered in the absence of
DODAC while, at a DODAC concentration of 1.5 mole %, 90% of the
plasmid was recovered after extrusion through a 200 nm filter.
Nearly 100% of the plasmid recovered from extrusion through a 200
mn filter was recovered by anion exchange chromatography (FIG. 5)
suggesting that all of the recovered plasmid was encapsulated. This
corresponded to an overall encapsulation efficiency of about 70%.
Lipid recoveries after extrusion and anion exchange chromatography
were 90% after extrusion through a 400 nm filter and 70% after
extrusion through a 200 nm filter (see FIG. 6). Of the 70% lipid
recovered after extrusion through a 200 nm fitter, nearly 100% was
recovered after anion exchange chromatography (FIG. 7). Lipid and
plasmid recovery after extrusion and anion exchange chromatography
were nearly identical. Table 1 illustrates the encapsulation
efficiencies using several different lipid compositions. It is
quite evident that a wide range of lipid compositions may be used.
It is also interesting to note that PEG-Cer does not appear to be
necessary in many of these lipid compositions. TABLE-US-00001 TABLE
1 Some examples of plasmid DNA encapsulation by the modified
reverse phase method. Data are shown only when at least 70% of the
lipid was recovered from anion exchange chromatography. Reverse
Phase Lipid composition % encapsulation EPC-DODAC (98.9:1.1) 98%
DOPE:EPC:DODAC (10:88.9:1.1) 24% DOPE:EPC:DODAC (20:78.9:1.I) 13%
DOPE:EPC:DODAC (40:58.9:1.I) 16% DOPE:EPC:DODAC-(10:85.5:4.5) 78%
DOPE:EPC:DODAC (15:80.5:4.5) 67% DOPE:SM:EPC:DODAC (15:40:40:5) 53%
DOPE:SM:EPC:DODAC (20:37.5:37.5:5) 37% DOPE:EPC:DODAC:PEG-Cer-C8
(25:64:1:10) 83% DOPE:EPC:DODAC:PEG-Cer-C8 (50:39:1:10) 90%
Dialysis Method
[0249] FIG. 8 presents the results describing tile relationship
between the amount of DODAC present in the formulation and the
encapsulation efficiency for DOPE:DODAC:PEG-Cer-C.sub.20 (84:6:10)
as measured by anion exchange chromatography. Table 2 illustrates
the encapsulation efficiencies using several different lipid
compositions. It is quite evident that a wide range of lipid
compositions may be used. It is interesting to note that PEG-Cer
appears to be necessary in these lipid compositions. TABLE-US-00002
TABLE 2 Some examples of plasmid DNA encapsulation by the detergent
dialysis method. Data are shown only when at least 70% of the lipid
was recovered from anion exchange chromatography. Detergent
dialysis Lipid composition % encapsulation amount of DNA comments
DOPE:DODAC:PEG-CER-C.sub.8 64% 50 .mu.g-400 .mu.g/ dialyzed against
(79:6:15) 10 .mu.mole lipid 150 mM NaCl DOPE:DODAC:PEG-CER-C.sub.14
60% 50 .mu.g-400 .mu.g/ dialysed against (84:6:10) 10 .mu.mole
lipid 150 mM NaCl DOPE:DODAC:PEG-CER-C.sub.20 52% 50 .mu.g-400
.mu.g/ dialysed against (84:6:10) 10 .mu.mole lipid 150 mM NaCl
DOPE:DODAC:EPC:PEG-CER-C.sub.8 20% 50 .mu.g-400 .mu.g/ dialysed
against (59:6:20:15) 10 .mu.mole lipid 150 mM NaCl
DOPE:DODAC:DOPC:PEG-CER-C.sub.14 36% 50 .mu.g-400 .mu.g/ dialysed
against (74:6:10:10) 10 .mu.mole lipid 150 mM NaCl
DOPE:DODAC:DOPC:PEG-CER-C.sub.14 17% 50 .mu.g-400 .mu.g/ dialysed
against (64:6:20:10) 10 .mu.mole lipid 150 mM NaCl
DOPE:DODAC:EPC:Chol:PEG-CER-C.sub.14 57% 50 .mu.g-400 .mu.g/
dialysed against (41:9:20:20:10) 10 .mu.mole lipid 150 mM NaCl
DOPE:DODAC:EPC:Chol:PEG-CER-C.sub.14 50% 50 .mu.g-400 .mu.g/
dialysed against (51:9:20:10:10) 10 .mu.mole lipid 150 mM NaCl
DOPE:DODAC:PEG-C.sub.14 22.5% 50 .mu.g-400 .mu.g/ dialysed against.
(80:10:10) 10 .mu.mole lipid 150 mM NaCl DOPE:DODAC:PEG-C.sub.14
22.7% 50 .mu.g-400 .mu.g/ dialysed against (79:11:10) 10 .mu.mole
lipid 300 mM NaCl* DOPE:DODAC:PEG-C.sub.20 57% 50 .mu.g/ dialysed
against (89.4:0.6:10) 10 .mu.mole lipid 5 mM NaCl*
DOPE:DODAC:PEG-CER-C.sub.14 51% 50 .mu.g/ dialysed against
*87:3:10) 10 .mu.mole lipid 50 mM NaCl* *See Example 6.
Example 3
[0250] This example illustrates the serum stability achieved using
plasmid:lipid particles prepared by the methods of Example 1.
[0251] To establish the serum stability of the plasmid-lipid
particles aliquots of the particle mixtures prepared according to
both the reverse phase and dialysis method of Example 1 were
incubated in 80% mouse serum (Cedar Lane) for 30 min at 37.degree.
C. Prior to incubation, the lipid associated plasmid was eluted on
a DEAE Sepharose CL-6B column to remove unencapsulated plasmid.
Following incubation, an aliquot of the incubation mixture was
eluted in HBS on a Sepharose CL-4B column.
[0252] As a control, 1.5 mg of free .sup.3H-pCMVCAT was eluted on a
Sepharose CL-4B column in HBS, pH 7.4 (see FIG. 9A). For
comparison, 1.5 mg of free .sup.3H-pCMVCAT was incubated in 500
.mu.l of mouse serum at 37.degree. C. for 30 min and eluted in the
same manner (FIG. 9B). Note that in FIG. 9A, the free plasmid
eluted in the void volume of the column while, in FIG. 9B, the
plasmid incubated in serum eluted in the included volume suggesting
that the plasmid had been digested by serum enzymes.
Serum Stability of Plasmid-lipid Particles Prepared by Reverse
Phase
[0253] The stability of plasmid-lipid particles was assessed by
incubation of a 50 .mu.l aliquot in 500 .mu.l of mouse serum (Cedar
Lane) for 15 min at 37.degree. C. A 500 .mu.l aliquot of the
incubation mixture was eluted in HBS on a Sepharose CL-4B column
(FIG. 10). Comigration of the plasmid and lipid in the void volume
strongly suggests that no plasmid degradation has occurred. Any
serum-degraded plasmid or lipid should have been detected as a peak
at around fraction 35 (see control results in FIG. 9B).
Serum Stability of Plasmid-lipid Particles Prepared by Dialysis
[0254] A 50 .mu.l aliquot of a particle suspension was incubated in
500 .mu.l of mouse serum at 37.degree. C. for 30 min and eluted on
a Sepharose CL-4B column as described above. FIG. 11 shows the
elution profile of the sample after incubation in serum. As can be
seen in FIG. 11A, 94% of the plasmid is recovered in the void
volume suggesting that essentially all of the plasmid recovered
from anion exchange chromatography was encapsulated.
[0255] To demonstrate that this experiment reflects encapsulation
and not inhibition of serum nucleases by lipids in the formulation,
an encapsulated plasmid DNA formulation which had not been treated
by anion exchange chromatography was incubated in mouse serum for
30 min (FIG. 11B). 47% of the encapsulated plasmid DNA was eluted
in the included volume while 53% was eluted in the void volume of
the column. The trapping efficiency as measured by anion exchange
chromatography was 55%.
Example 4
[0256] This example illustrates the in vitro resistance of the
plasmid:lipid particles to DNase I digestion. Complexes formed by
the addition of DOPE:DODAC (50:50) vesicles to plasmid DNA were
compared to the encapsulated formulation
(DOPE:DODAC:PEG-Cer-C.sub.14; 84:6:10). The samples were incubated
in Dnase I, amplified by PCR (Polymerase Chain Reaction) and run on
an agarose gel. The DNA bands were visualized with ethidium
bromide. The complexes were not stable in the DNase I (FIG. 12A) in
the absence of detergent (lane 9) w@e the encapsulated plasmid
(FIG. 12B) was stable (lane 9).
Example 5
[0257] This example illustrates the dependence of plasmid
concentration on encapsulation efficiency.
[0258] pCMVCAT plasmid was encapsulated in the lipid particles by
detergent dialysis as described in Example 1. Encapsulation was
approximately 50%-60% for all concentrations tested (FIG. 13).
Encapsulation efficiency was independent of plasmid concentration
over the range studied.
Example 6
[0259] This example illustrates the dependence of optimal DODAC
concentration for entrapment on NaCl concentration.
[0260] We found that the optimum DODAC concentration for entrapment
was not only dependent oil the lipid composition but also was
dependent on the NaCl concentration of the dialysis buffer. pCMVCAT
plasmid was encapsulated in the lipid particles by detergent
dialysis as described in Example 1. FIG. (14) shows the optimum
DODAC concentration for encapsulation of pCMVCAT plasmid at
different NaCl concentrations. Note that the amount of DODAC
required in the membrane could be controlled in a predictable
manner simply by changing the NaCl concentration during
dialysis.
Example 7
[0261] This example illustrates the size distribution of
plasmid-lipid particles as measured by quasielastic light
scattering using a Nicomp Submicron Particle Sizer (Model 370).
Detergent Dialysis
[0262] Plasmid-lipid particles were prepared by detergent dialysis
as described in Example 1. The lipid composition was
DOPE:DODAC:PEO-Cer-C.sub.20. The particles were sized using a
Nicomp Submicron Particle Sizer. FIG. 15 shows the volume weighted
measurement while FIG. 16 shows the number weighted
measurement.
Example 8
[0263] This example illustrates the size distribution and structure
of the plasmid-lipid particles as measured by cryoelectron
microscopy.
[0264] Cryoelectron microscopy is a relatively nonperturbing
technique which routinely has been used to study liposome shape.
Liposomes are visible in the vitreous ice layer due to the
relatively electron-dense phosphate head groups. The same would
apply to DNA since it consists of many phosphate groups.
[0265] Cryoelectron microscopy was performed as described
previously (Chakrabarti et al., 1992; Wheeler et al., 1994).
Vesicles containing plasmid DNA were enriched from the formulation
by differential centrifugation. A 500 .mu.l aliquot of the
formulation was centrifuged in a microultracentrifuge for 90 min at
60,000.times.g. The supernatant was decanted and the pellet was
resuspended in 100 ml of HBS. A drop of the suspension was placed
on a 700 mesh gold grid, blotted from behind with Whatman No. 50
filter paper to form a thin film and vitrified by plunging into
liquid ethane cooled with liquid nitrogen in a Reichart Jung
Universal Cryo Fixation system (Reichart Corp.). The grid was
transferred to a Zeiss 10C STEM electron microscope equipped with a
Gatan 126 cold stage. The stage and anticontaminator were kept at
120K and 115 k, respectively, with liquid nitrogen. Regions of thin
vitreous ice were observed with an acceleration voltage of 60
kV.
[0266] FIG. 17A is a cryoelectron microscopy picture of an
encapsulated plasmid DNA formulation. For this preparation, 400
.mu.g of plasmid DNA was used. The lipid composition was
DOPE:DODAC:PEG-Cer-C.sub.14 (84:6:10). The small arrows denote
empty liposomes approximately 100 nm in diameter. These are
compared to the lipid particles containing electron-dense centers
(large arrows). These electron-dense centers presumably correspond
to plasmid DNA. These structures were not seen in formulations made
in the absence of DNA FIG. 17B.
Example 9
[0267] This example illustrates the blood clearance of the
plasmid:lipid particles in mice.
Reverse Phase
[0268] Encapsulated plasmid blood clearance was tested in three ICR
mire as a function of percent recovered dose over time. Percent
recovery of free .sup.3H-plasmid was plotted over a similar time
course as a control (see FIG. 18). The encapsulated plasmid
exhibits a clearance rate which is much slower than that of
.sup.3H-plasmid. Additionally, the plasmid:lipid ratio does not
change significantly over the time course of the experiment
confirming that the plasmid clearance rate is associated with the
clearance rate of the lipid carrier itself.
Detergent Dialysis
[0269] Fusogenic particles of pCMVCAT encapsulated in
DOPE:DODAC:PEG-Cer-C.sub.14 or DOPE:DODAC:PEG-Cer-C.sub.20 (84:6:10
mole %) were prepared as follows;
[0270] pCMVCAT (50 .mu.g)(42 of .mu.l of .sub.3H-pCMVCAT; 108
dpm/.mu.I, 1.19 mg/ml) was incubated with DODAC in 100 .mu.l of 1 M
OGP and 400 .mu.l of water for 30 min at room temperature. This
DNA:DODAC complex mixture was added to a suspension of
DOPE-PEG-Cer-C.sub.14 or DOPE:PEG-Cer-C.sub.20 and the particles
were constructed as described in Example 1 (detergent dialysis).
The plasmid:lipid particles for blood clearance studies contained
0.75 .mu.l of .sup.14C-cholesteryl hexadecyl ether (CHE) (6.66
.mu.l/.mu.Ci) in 100 .mu.l of 1 M OGP and 400 .mu.l of water.
Clearance of pCMVCAT encapsulated in DOPE:DODAC:PEG-Cer-C.sub.14
and DOPE:DODAC.PEC-Cer-C.sub.20(84:6:10).
[0271] External "encapsulated" DNA was removed by anion exchange
chromatography using DEAE Sepharose CL-6B prior to injection into
mice. Encapsulation efficiencies were approximately 42% for the
systems containing PEG-Cer-C.sub.20 and 60% for the systems
containing PEG-Cer-Cer.sub.14.
[0272] Three groups of three female ICR mice (20-25 g) were
injected with 200 .mu.l of DNA-encapsulated with the plasmid:lipid
particle. One group of mice was sacrificed and blood was taken at
each of three time points (1, 2 and 5 hours). The plasma was
separated from whole blood by centrifugation in 0.5 ml EDTA coated
Tainer tubes. A 200 .mu.l aliquot of the plasma from each mouse was
assayed for .sup.3H-DNA and .sup.14C-lipid by scintillation
counting.
[0273] FIG. 19A shows the Clearance of DNA encapsulated in a
particle composed of DOPE:DODAC:PEG-Cer-C.sub.20 (84:6:10). The DNA
and lipid were cleared much less rapidly from the circulation than
when the DOPE:DODAC:PEG-Cer-C14 composition was used. Nearly 50% of
the lipid and DNA are present after 2 hour. A significant amount of
DNA and lipid were still present after 5 hours. The amount of DNA
and lipid injected was 1.8 .mu.g and 853 .mu.g, respectively.
[0274] FIG. 19B shows the clearance of DNA encapsulated in particle
composed of DOPE:DODAC:PEG-Cer.sub.14 (84:6: mole %). Both DNA and
lipid are cleared rapidly from the circulation with only about 20%
of the lipid and 10% of the DNA present in the plasma after 1 hour.
The amount of DNA and lipid injected was 2.7 .mu.g and 912 .mu.g,
respectively.
Example 10
[0275] This example illustrates the in vitro transfection of BHK
cells grown in tissue culture.
Example 11
[0276] This example illustrates the in vivo transfection of tissues
in mice.
In Vivo Transfection in Lung, Liver and Spleen
[0277] Three groups of four ICR mice were injected via tail vein
with pCMVCAT encapsulated in lipid particles composed of
DOPE:DODAC:PEG-Cer-C.sub.14 (84:6:10) or
DOPE:DODAC:PEG-Cer-C.sub.20, prepared as described in Example 7,
The mice were sacrificed after 2, 4 and 8 days and the lung, liver
and spleen were assayed for CAT activity according to a
modification of Deigh, Anal. Biochem., 156:251-256 (1986). The
amount of plasmid injected was 2.6 .mu.g for the particles
containing PEG-Cer-C.sub.14 and 1.5 .mu.g for the particles
containing PEG-Cer-C.sub.20.
[0278] FIG. 20 shows the results of in vivo transfection achieved
in the lung. As can be seen from this figure, treatment with
DOPE:DODAC:PEG-Cer-C.sub.14 resulted in transfection (based on CAT
activity) up to 4 days. DOPE:DODAC:PEG-Cer-C.sub.20 while resulting
in overall lower levels of CAT activity), provided relatively
constant levels of enzyme activity over 8 days.
[0279] FIG. 21 shows the results of transfection achieved in the
liver. For both formulations, transfection (and CAT activity)
reached a maximum at 4 days.
[0280] FIG. 22 shows the results of transfection achieved in the
spleen wherein the maximum transfection was found for both
formulations to occur after 2 days.
[0281] Examples 12-18 illustrate the formation and characterization
of charge-neutralized lipid-nucleic acid intermediate complexes, in
which the nucleic acid adopts hydrophobic character. In each of
these examples, the term "DNA" or "plasmid" refers to the plasmid
pCMV.beta.. Examples 19 and 20 illustrate the preparation and
characterization of lipid-nucleic acid particles which are suitable
for transfection of cells. Examples 21-23 illustrate the serum
stability and transfecting ability of these lipid-nucleic acid
particles.
Example 12
[0282] This example provides a comparison of cationic lipids and
non-cationic lipids in effecting the formation of hydrophobic
charge-neutralized lipid-nucleic acid complexes.
[0283] LIPOFECTIN.RTM. consists of sonicated unilamellar vesicles
composed of DOTMA and DOPE (50:50 mole ratio, see, Felgner, et al.,
Proc. Natl. Acad. Sci, USA 84:7413-7417 (1987)). The liposomes are
prepared in water and are provided at a total lipid concentration
of 1 mg/mL. DNA (10 .mu.g) was mixed with the liposomes in water,
as described below in Example 2, to provide from 0 to 160 nmoles
total lipid. Each of the mixtures was extracted using the Bligh and
Dyer procedure. Surprisingly, in the presence of LIPOFECTIN.RTM.,
there was a concentration dependent reduction in DNA recovered from
the aqueous phase (see FIG. 23). Addition of 80 nmoles of total
lipid to 10 .mu.g DNA resulted in greater than 95% loss of DNA from
the aqueous phase. This effect could not be achieved using
liposomes prepared from egg phosphatidylcholine/DOPE (50:50 mole
ratio). Thus, the hydrophobic complex which forms and is drawn into
the organic phase is a result of the cationic lipid present in the
complex.
Example 13
[0284] This example provides a comparison of several cationic
lipids in forming hydrophobic, charge-neutralized lipid-nucleic
acid complexes which partition into organic solvents.
[0285] Purified monovalent cationic lipids (DOTMA, DDAB and DODAC)
were each added to DNA in a Bligh and Dyer monophase solvent
system. The resulting mixtures were each partitioned into two
phases by the addition of water and chloroform. Plasmid DNA levels
were determined in the aqueous and organic phases as described
above. The results are presented in FIG. 24, and are consistent
with the results presented in FIG. 23. In particular, there was
found to be a cationic lipid dependent loss of DNA from the aqueous
phase (FIG. 24A). There was no visible evidence of precipitated
material at the aqueous/organic interface and quantification of the
DNA in samples collected to include the interface did not account
for appreciable DNA levels (results not shown). The DNA was found
to be quantitatively transferred to the organic phase (FIG. 24B).
Additionally, greater than 95% of the DNA in the monophase could be
recovered in the organic phase when 40 nmoles monovalent cationic
lipid was added. This value is identical to results presented in
FIG. 23 in which 80 nmoles of LIPOFECTIN.RTM. (50 mol % DOTMA)
resulted in the complete loss of DNA from the aqueous phase. The
results presented in FIG. 24 indicate that the three different
monovalent cationic lipids behave in a similar fashion under the
conditions used.
Example 14
[0286] This example illustrates the influence of multivalent
cationic lipids and cationic nonlipid species on DNA partitioning
into organic solvents.
[0287] LIPOFECTAMINE.RTM. (DOSPA:DOPE, 75:25 mol ratio), and
TRANSFECTAM.RTM. (100% DOGS) were added to DNA (10 .mu.g) as
preformed liposomes, as described in Example 13. The liposomes
contain headgroups derived from spermine and exhibit positive
charges of 5 and 4, respectively at pH<7. As expected,
significantly lower amounts of these lipids (calculated on the
basis of moles) are required to mediate DNA partitioning into the
organic phase (see FIG. 24). Complete partitioning of the DNA into
the organic phase was achieved after addition of approximately 10
nmoles DOSPA and DOGS.
[0288] Previous studies have demonstrated that DNA condenses into
small toroid or rod shaped structures when the DNA phosphate charge
is at least 90% neutralized (see Wilson, et al., Biochemistry
18:2192-2196 (1979). The data presented in FIG. 24 was therefore
expressed as a function of cation/phosphate charge ratio (FIGS. 25A
and 25B). For comparison, results obtained after the addition of
the nonlipid-based monovalent (lysine), divalent (calcium) and
multivalent (poly-L-lysine) cations are included (FIGS. 25C and
5D). The results shown in FIG. 25 demonstrate that for monovalent
cationic lipids, greater than 99% of the DNA partitioned into the
organic phase when a +/- charge ratio >1 was achieved. Similar
results were observed when the polyvalent lipids DOSPA and DOGS
were used, although a slightly greater charge ratio was required to
mediate efficient DNA transfer. However, DNA partitioning into the
organic phase did not occur as a result of simple charge
neutralization. When the DNA was mixed with the nonlipid cations,
at charge ratios up to and in excess of 4, the majority of the DNA
was invariably recovered in the aqueous phase.
Example 15
[0289] This example illustrates that the hydrophobic,
charge-neutralized lipid-nucleic acid complexes formed as described
in Examples 12-14 provide the nucleic acid in an uncondensed
(unprotected) configuration.
[0290] Evaluation of the hydrophobic, charge-neutralized
lipid-nucleic acid complexes was carried out by assessing the
ability of a small fluorescent probe to bind to the nucleic acid in
the complex. This evaluation is similar to an approach using
ethidium bromide (see Gershon, et al., Biochemistry 32:7143-7151
(1993)). TO-PRO-1 is a more sensitive, membrane impermeable,
nucleic acid intercalating dye and therefore, provides a more
stringent test of DNA binding. DNA was mixed with either a
monovalent cationic lipid or poly-L-lysine in the Bligh and Dyer
monophase (FIG. 26A). TO-PRO-1 was then added to a final
concentration of 1 .mu.M and fluorescence was measured at 533 nm
(probe excitation at 509 nm). In the absence of DNA no fluorescence
was observed. However, when plasmid DNA was added (10 .mu.g/mL)
there was a >600 fold increase in fluorescence at 533 nm. When
TO-PRO-1 was added to the DNA/poly-1-lysine mixture, no
fluorescence was observed. This is consistent with the existence of
the DNA in a condensed state due to charge neutralization. In
dramatic contrast, addition of TO-PRO-1 to a hydrophobic
charge-neutralized lipid-nucleic acid complex (plasmid/DODAC
complex), TO-PRO-1 binding was not excluded. This result is
consistent with the concept that DNA within the hydrophobic complex
does not exist as a condensed structure. FIG. 26B shows that
similar results were obtained when TO-PRO-1 was added to plasmid
DNA mixed with either poly-L-lysine or the cationic lipid DODAC in
the presence of 100 mM OGP, a nonionic detergent.
Example 16
[0291] This example illustrates the stability of the hydrophobic,
charge-neutralized lipid-nucleic acid complex in detergent
solutions (FIG. 27) and instability in the presence of added salts
(FIG. 28).
[0292] Plasmid DNA (10 .mu.g) was mixed with 40 nmoles of DODAC in
a Bligh and Dyer monophase as described in Example 12. OGP was
added to achieve concentration up to 20 mM (20 .mu.moles in 1 mL)
prior to separating the sample into two phases. This concentration
was the maximum amount which could be added from a 2 M stock
solution of OGP without disrupting the monophase system. Regardless
of the OGP concentration, greater than 99% of the DNA partitioned
into the organic phase, demonstrating the stability of the
hydrophobic, charge-neutralized complexes.
[0293] The effect of increasing concentrations of NaCl on the
stability of the hydrophobic, charge-neutralized lipid-nucleic acid
complex was also evaluated. As illustrated in FIG. 28, monovalent
cationic lipid binding to DNA was completely inhibited in the
presence of 1 .mu.mole NaCl. At this level, Na.sup.+ is present in
a 25 molar excess relative to the amount of cationic lipid added.
As expected, the complex between DNA and the polyvalent lipid DOSPA
was more stable in the presence of NaCl. In fact, addition of
Na.sup.+ in a 300-fold molar excess relative to DOSPA did not cause
partitioning of the charge-neutralize lipid-nucleic acid complex
into the aqueous phase.
Example 17
[0294] This example illustrates the influence of cationic lipid
binding on DNA migration by agarose gel electrophoresis.
[0295] FIG. 29A shows the gel mobility characteristics of the
charge-neutralized lipid-nucleic acid complexes made in the
presence of OGP compared to that of the poly-L-lysine condensed DNA
control. Lane 2 shows that the nonlipid-based DNA/poly-L-lysine
complexes exhibit significantly reduced mobility in an agarose gel.
This result is consistent with studies which have demonstrated that
DNA condensed with cationic liposomes adopt a macromolecular
structure that does not move within an applied electric field (see,
Bertling, et al., Biotechnol. Appl. Biochem. 13:390-405 (1991)).
This effect may be a consequence of charge neutralization and/or
increases in molecular size. In contrast, when DNA is mixed with
cationic lipids under conditions of Example 12, there is no
indication that the migration of DNA has been altered (see FIG.
29A, lanes 3-5). These studies provide further evidence suggesting
that cationic lipid binding to DNA using the methods of the present
invention does not result in the condensation of DNA. Changes in
DNA mobility were observed, however, when the cationic lipid
concentration was increase beyond cationic lipid to DNA phosphate
charge ratios of 2 (see lanes 6 to 8). For example, addition of 320
nmoles of DODAC resulted in a decrease in DNA migrating into the
gel and a small proportion of the DNA migrating near the top of the
gel. This indicates that condensation of DNA can be achieved with
excess cationic lipids.
Example 18
[0296] This example illustrates the ability of cationic lipids to
protect plasmid DNA from enzymatic digestion.
[0297] To determine the ability of cationic lipids to protect
plasmid DNA from enzymatic digestion, DNase I mediated degradation
of the lipid-nucleic acid complex (plasmid/DODAC complex prepared
as described above) was also evaluated using agarose gel
electrophoresis (see FIG. 29B). In these experiments, plasmid in
OGP solution was mixed with a sufficient amount of DNase I to
generate small DNA fragments after a 10 min incubation at
37.degree. C. (lane 2). Lane 1 shows undigested plasmid as a
control. Using identical conditions, the complexes (plasmid
complexed with the monocationic lipid DODAC) was not protected
against the enzymatic activity of DNase I (lane 4). DNA extracted
from the complex in the absence of DNase I (lane 3) shows intact
DNA. This provides further evidence that the nucleic acids in the
lipid-nucleic acid complexes is in an uncondensed state and is
susceptible to degradation.
Example 19
[0298] This example illustrates the preparation of lipid-nucleic
acid particles of .beta.-gal, DODAC and ESM.
[0299] Cationic lipid DODAC, non-cationic lipid ESM, and nucleic
acid .beta.-gal plasmid were formulated using a detergent dialysis
method according to the "strategy of reverse order" (see FIG. 30)
as follows:
[0300] Individual solutions of DNA (10 .mu.g in 200 .mu.L of 200 mM
aqueous OGP), DODAC (160 nmoles in 400 .mu.L OGP) and ESM (160
nmoles in 400 .mu.L OGP) were prepared. The ESM and DODAC solutions
were each sonicated at low power at 10-20 pulses. The DNA solution
was then added to the ESM solution and the mixture was allowed to
incubate for 0.5 hr at room temperature. The DODAC solution was
added slowly to the DNA/ESM mixture while vortexing the mixture at
low speed. The resultant mixture (1 mL) was placed in a
SPECTRA/POR, mwco: 12-14,000 dialysis tube (Fisher Scientific) and
dialyzed against six changes of 2 L of distilled sterile water over
36 hours. Size distribution of the complexes formed was determined
using quasielastic light scattering (QELS) technique (Nicomp 370
particle sizer operating at a wavelength of 632.8 nm). FIG. 31
shows that two populations of particles were observed, one group
sized from 50 to 150 nm and the second sized 500 to 1000 nm. The
relative numbers of each depended on the type of non-cationic
lipid(s) used, the amount and concentration of the two lipid
components, and the DNA/lipid ratio. About 20-40% of the relative
volume of the mixture were the smaller sized complexes which
accounted for over 90% of the total particle number.
Example 20
[0301] This example illustrates the state of condensation of the
DNA in the lipid-nucleic acid particle.
[0302] The fluorochrome (TO-PRO-1) was used to evaluate the state
of condensation of the DNA in the lipid-nucleic acid particle. A
200 .mu.L aliquot of the lipid-nucleic acid particle (containing 2
.mu.g plasmid DNA prepared with the protocol given in Example 19)
was diluted to 1 mL with 100 mM OGP. TO-PRO-1 was added to make a
final concentration of 1 .mu.M. To measure fluorescence,
spectrofluorometric measurements were performed using a
Luminescence Spectrometer 50B (Perkin Elmer Ltd., Buckinghamshire,
England) with an excitation wavelength of 509 nm and an emission
wavelength of 533 nm. The results are presented in FIG. 32 in which
the values are expressed as arbitrary fluorescence units. As FIG.
32 illustrates, plasmid DNA in lipid-nucleic acid complexes
containing DODAC/ESM is condensed or protected by the lipid
component. Moreover, the detergent (OGP) can dissolve the complex
to uncondense the DNA (see FIG. 32).
[0303] DNA in lipid-nucleic acid particles containing DODAC/DOPE is
partially accessible to TO-PRO-l at a lipid/DNA charge ratio (+/-)
of 4:1, however, at 8:1 DNA is completely protected by the lipid
component. This result suggests that the nucleic acid (DNA) is
partially condensed at the lower charge ratio and fully condensed
at the higher ratio (FIG. 32).
Example 21
[0304] This example demonstrates the stability of lipid-nucleic
acid particles in phosphate-buffered saline and in serum containing
media.
[0305] A lipid-nucleic acid particle formulation was prepared
according to the procedure described in Example 19. Portions of the
formulation (using either ESM or DOPE as the neutral lipid) were
combined with PBS (140 mM NaCl, 10 mM Na.sub.2HPO.sub.4) or
serum-containing medium and incubated for two hours at 37.degree.
C. The resulting complexes were isolated and examined for any
changes in QELS size results or transfection efficiency. No
difference was found for any of the formulations, indicating that
the complexes were not disrupted by either sodium or serum
components. One portion which was incubated with PBS for 10 days
still showed very good transfection efficiency.
Example 22
[0306] This example illustrates the protection of DNA against DNase
I which is afforded by the lipid-nucleic acid particles.
[0307] A lipid-nucleic acid particle formulation of 10 .mu.g DNA,
160 nmoles DODAC and 160 nmoles ESM in 1 mL total volume was
prepared according to the method described in Example 19. The
susceptibility of the DNA in this formulation to degradation by
DNase I was evaluated by mixing the formulation with DNase I in the
presence of OGP (1:1 charge ratio). The level of DNase I was
equivalent to that which degrades uncomplexed DNA within 10 minutes
at 37.degree. C. The reactions were stopped after 10 min by the
addition of 25 mM EDTA. DNA was extracted using the Bligh and Dyer
extraction procedure in the presence of 150 mM NaCl. Under these
conditions the cationic lipid/DNA complex dissociates and the
resulting DNA can be efficiently recovered from the aqueous
fraction. This DNA was precipitated with 1/10th volume of 3M sodium
acetate (pH 5.2) and 2.5 volumes of 95% ethanol and recovered by
centrifugation at 14,000 g for 30 min at 4.degree. C. The DNA
pellet was resuspended in sterile distilled water and subjected to
electrophoresis on a 0.8% agarose gel (Gibco, BRL). The results are
shown in FIG. 33. As FIG. 33 indicates, complexes containing ESM
provide protection of DNA from DNase I degradation.
Example 23
[0308] This example illustrates the in vitro transfection of CHO or
B16 cell lines using lipid-nucleic acid particles prepared by the
method of Example 19.
[0309] In vitro transfection was performed using a 96-well cell
culture plate (Costar, Cambridge, Mass., USA) containing 50%
confluent growth of either Chinese Hamster Ovary (CHO) or murine
melanoma (B16) cell lines. Appropriate amounts (about 6-50 .mu.L)
of the lipid-nucleic acid particle formulation (10 .mu.g DNA/mL)
were premixed with medium containing 10% serum to a final volume of
150 .mu.L. The medium surrounding the cells was removed using a
needle syringe and replaced with the lipid-nucleic acid particles
in 10% serum-containing medium. The cells and complex were
incubated for a further 48 hours at 37.degree. C. The transfection
efficiency was evaluated using .beta.-gal stain or an enzyme
activity assay. Results are presented in FIG. 34.
[0310] The transfection study showed excellent transfection
efficiency with ESM-containing complexes and with DOPE-containing
complexes (not shown). A cationic lipid to DNA charge ratio of 3:1
to 4:1 gave the best in vitro transfection results.
Example 24
[0311] This example illustrates the properties of nucleic
acid-lipid particles prepared as described below in the presence of
100 mM (A) or 20 mM (B) n-octyl .beta.-D-glucopyranoside (OGP).
[0312] The protocol involves the preparation of solutions of
pCMV.beta. DNA in OGP and lipid-detergent mixed micelles. DODAC and
the neutral lipid were dissolved in the same concentration of OGP
used to dilute the DNA solution. To ensure that the lipids were
completely dissolved, the mixtures were heated to 50.degree. C. for
5 min and vortexed vigorously. Individual solutions were prepared
with or without neutral lipid. When there was no neutral lipid
involved, the DNA was added to the DODAC solution followed by
gentle vortexing and then incubated at room temperature for 30 min.
When the neutral lipid was present, the detergent solution
containing DNA was mixed with the detergent solution containing the
neutral lipid. This mixture was incubated for 30 min at room,
temperature and then added to detergent solution containing the
cationic lipid DODAC. To remove detergent mixtures were transferred
to dialysis bags and dialyzed against six changes of sterile water
over 72 hrs. The volume of each sample was less than 1 mL.
[0313] nucleic acid-lipid particle formation was evaluated by
measuring changes in 90.degree. light scattering intensity at 600
nm (slit width of 2.5 nm). This wavelength was used because light
scattering from detergent micelles alone was negligible, therefore,
the formation of nucleic acid-lipid particles could be monitored.
This technique was also used to assess the ability of OGP to
solubilize preformed liposomes of DODAC or SM. Multilamellar
liposomes were prepared at a final lipid concentration of 1.0 mM by
hydrating powdered lipid in distilled water at 60.degree. C. The
lipid suspensions were sonicated for 5 min (100 watts, 90% duty
cycle, at 2O kHz) using a probe sonicator (Sonilier Cell Disrupter
350, Branson Sonic Power Co., Danbury, Conn.) to produce
homogeneous suspension. For the lipid dissolution measurement, an
aliquot of the lipid suspension was diluted with distilled water to
a final lipid concentration of 0.2-1.0 mM. This lipid suspension
was titrated with 200 mM OGP and mixed well by pipetting. Light
scattering intensity was measured at room temperature using a
Luminescence Spectrometer 50B (Perkin Elmer).
[0314] In 100 mM OGP there were no significant changes in solution
turbidity observed when DNA was added to DODAC/OGP mixed micelle
solution in the presence and absence of SM. After 3 hrs of dialysis
the solutions became turbid and light scattering increased, a
reflection of increased particle size and/or aggregation. After.
4.5 hrs., a decrease in light scattering was observed for systems
prepared in the absence of SM, a result of the formation of larger
visible aggregates. When the samples were prepared in 20 mM OGP
(FIG. 35B), a concentration close to the critical micelle
concentration of OGP in the absence of added lipids, light
scattering increased at the time when DNA was added to DODAC/OGP
micelles. This increase in turbidity is indicative of spontaneous
particle formation spheroidal mixed micelles. It is therefore
unlikely that lipid vesicles form under conditions where the
detergent concentration is equal or greater than 20 mM. nucleic
acid-lipid particle formation in the presence of 20 mM OGP is
likely not due to DNA-mediated aggregation of cationic liposomes.
We believe that nucleic acid-lipid particle formation is the result
of the hydrophobic lipid-DNA complex adopting a structure that
minimizes lipid acyl chain contact with water.
[0315] The physical characteristics of the nucleic acid-lipid
particles, formed either spontaneously or following detergent
removal, are summarized in Table 3 and FIG. 37. The parameters
evaluated include (i) particle size as estimated by QELS and
electron microscopy, (ii) the observed degree of
aggregation/flocculation, and (iii) an assessment of TO-PRO-1
binding, an intercalating agent that fluoresces when bound to
DNA.
[0316] Since it is believed that nucleic acid-lipid particle
formation is dependent on cationic lipid binding to DNA, particle
characteristics were assessed under conditions where the cationic
lipid to anionic phosphate charge ratio was varied from 1:1 to 8:1.
Under conditions where particle formation occurred following
detergent removal (i.e. lipid and DNA mixtures prepared in 100 Mm
OGP) the resulting particles were large (>2000 nm) and
aggregated (Table 3). This tendency to aggregate was dependent on
the charge ratio.
[0317] After nucleic acid-lipid particle formation, the DNA assumed
a structure that was not accessible to TO-PRO-1 intercalation,
suggesting that the DNA was condensed. It should be noted that a
condensation index of .apprxeq. 1.0 is equivalent to that obtained
when DNA is condensed by the addition of polylysine (Reimer et al.,
1995).
[0318] When low detergent concentrations (20 mM OGP) were used to
promote spontaneous nucleic acid-lipid particle formation there was
no significant change in particle size or aggregation state as a
function of detergent removal, except at charge ratios of 1:1 and
1.5:1 (Table 32), where significant increases in particle size were
observed.
[0319] As shown in FIG. 37A, QELS data indicated that for samples
prepared using the 2:1 charge ratio, the particles were homogeneous
and fit a Gaussian analysis with a mean diameter of 59.+-.38
nm.
[0320] This result is comparable with observations made using
negative stain electron microscopy (FIG. 37B). nucleic acid-lipid
particles were evaluated by electron microscopy (EM) using two
methods. First, the samples were prepared for negative stain EM by
placing a drop of a concentrated nucleic acid-lipid particle
formulation (3 mM lipid) onto a formvar coated nickel grid. After 1
min the sample was carefully drawn away using filter paper and
stained with a 2.5% ammonium molybdate solution. The stained
samples were immediately examined and photographed using a Carl
Zeiss EM10CR electron microscope operated at 80 Kv. Second, nucleic
acid-lipid particles were prepared for freeze-fracture EM, where a
sample of concentrated nucleic acid-lipid particle formulation (15
mM lipid) was mixed with glycerol (25% v/v), frozen in a freon
slush, and subjected to freeze-fracture employing a Balzers BAF
400D apparatus. Micrographs were obtained using a JEOL Model
JEM-1200EX electron microscope.
[0321] Data obtained from freeze-fracture electron microscopic
analysis of the particles (FIG. 37C) indicated that, regardless of
sample concentration (up to 15 mM total lipid), there were only a
few regions on the freeze-fracture replica that exhibited fracture
surfaces typical of membrane bilayer structures. Instead, numerous
bumps were detected on the replica. This is consistent with the
suggestion that particles rather than liposomes were formed using
the procedures described here.
[0322] DNA was accessible to TO-PRO-1 following spontaneous
particle formation and condensation indices of less than 0.05 were
typically measured prior to detergent removal. This result was
unexpected and suggests that particle formation is not a indicator
of whether DNA is condensed. After detergent removal, TO-PRO-1
intercalation was not observed and the resulting DNA condensation
indices were high (.apprxeq.1.0) (Table 3). TABLE-US-00003 TABLE 3
Characteristics of lipid-DNA particles formed with
pCMV.beta./DODAC/SM prepared using 20 mM and 100 mM OGP before and
after dialysis. mean diameter .+-. SD (nm).sup.b (cation/ before
after aggregation condensation anion.sup.a) dialysis dialysis
state.sup.c index.sup.d 100 mM OGP 1:1 ND* >2000 ++ 0.759 2:1 ND
>2000 + 0.927 4:1 ND >2000 + 0.974 8:1 ND >2000 ++ 0.991
20 mM OGP 1:1 71.2 .+-. 37.0 192 .+-. 110 - 0.875 1.5:1 63.1 .+-.
33.8 119 .+-. 76 -- 0.985 2:1 60.8 .+-. 33.3 58.6 .+-. 37.8 --
0.991 4:1 56.7 .+-. 32.0 55.9 .+-. 32.6 -- 0.994 8:1 64.6 + 33.4
66.4 .+-. 35.4 -- 0.989 .sup.aThe charge ratio of cationic lipids
to DNA phosphate groups. .sup.bMean diameter was measured using
QELS techniques as described in the Methods. The # instrument used
to evaluate particle size is accurate only under conditions where
the mean # particle size is less than 1.0 .mu.m. The aggregation
state of the formulations after dialysis # was evaluated
qualitatively through visual inspection of the samples and scored
as # follows: ++ large aggregates that settle out of solution
within 5 min after sample # mixing; + small to medium aggregates
present but the solution retains a # uniform "milky" appearance; -
no obvious aggregates unless viewed by # microscopy-, -- no
aggregates and homogeneous as assessed by QELS. .sup.dDNA
condensation index, a reflection of TO-PRO-1 binding to DNA in the
presence and # absence of lipid binding, was determined as
described in the Methods. .sup.cNID: not detectable because
particles were not formed.
DNA Stability Assay
[0323] To evaluate the protective effect of lipids on DNA, 100
.mu.l of the formulations containing I .mu.g pCMV.beta. DNA were
incubated with 0.67 unit of DNase I at 37.degree. C. for 20 min in
the presence of buffer (0.05 Ni Tris-HCI pH 8.0, 0.01 M MgSO.sub.4,
0.1 mM dithiothreitol) or 20 mM OGP. The enzymatic reactions were
stopped by the addition of 5 .mu.l of 0.5M EDTA and 3 .mu.l of 5M
NaCl. DNA was extracted using a modified Bligh and Dyer extraction
procedure (Reimer et al., 1995). Under these conditions, lipid and
DNA dissociated and the resulting DNA was efficiently recovered in
the aqueous phase. DNA was precipitated with one-tenth volume of
sodium acetate (pH 5.2) and 2.5 volumes of 95% ethanol at
-20.degree. C. for 30 min and recovered by centrifugation at
12,000.times.g for 30 min at 4.degree. C. in a microcentrifuge
(Eppendorf). The DNA pellet was resuspended in 10 .mu.l sterile
distilled water and subjected to electrophoresis on a 0.8% agarose
gel in TBE buffer (89 mM Tris-Borate, 2 mM EDTA, pH 8.0).
[0324] To further define the characteristics of the nucleic
acid-lipid particles produced as a consequence of DNA-cationic
lipid complex formation, we evaluated whether the DNA in the
nucleic acid-lipid particles was protected against the endonuclease
activity of DNase I. This is an important characteristic since we
are developing these systems for in vitro and in vivo DNA transfer.
The results presented in FIG. 38A show that after detergent
removal, DNA within the particle remained intact in the presence of
DNase I (lanes 5 and 7). Interestingly, DNA within particles that
had formed spontaneously in the presence of 20 mM OGP remained
intact in the presence of DNase I even in the absence of detergent
removal (FIG. 38B, lane 5).
Example 25
[0325] This example illustrates that the nucleic acid-lipid
particles prepared as described in Example 24 are useful as plasmid
delivery systems in vitro.
[0326] CHO cells (American Type Tissue Culture, Rockville, Md.)
were plated at 2.times.10.sup.4 cells per well in a 96 well culture
plate (Costar, Cambridge, Mass.) in (.alpha.NMEM supplemented with
5% Fetal Bovine Serum (FBS). The cells were grown for 24 hrs in a
37.degree. C. 5% CO.sub.2 incubator and were 40-50% confluent at
the time of transfection. Media was removed from cells prior to
addition of 100 .mu.l of diluted nucleic acid-lipid particle
formulations prepared from 25 .mu.l nucleic acid-lipid particles
formulation containing 0.3-1.2 .mu.g DNA and 75 .mu.l of
(.alpha.MEM supplemented with 10% FBS. Cells were incubated at
37.degree. C. for 4 hrs, prior to the addition of 100 .mu.l of
.alpha.MEM (10% FBS) containing 100 .mu.g/mL gentamicin sulphate.
The cells were further incubated at 37.degree. C. for two days and
then assayed for .beta.-galactosidase activity. Media was removed
from each well and 30 .mu.l of a cell lysis buffer (0.1% Triton
X-100, 250 mM Na.sub.2HPO.sub.4, pH 8.0) was added. Subsequently,
50 .mu.l of bovine serum albumin (0.5% in phosphate buffer, pH 8.0)
was added to each well followed by the addition of 150 .mu.l of
chlorophenol red galactopyranoside (CPRG, 1 mg/mL in 60 mM
Na.sub.2HPO.sub.4, 1 mM MgSO.sub.4, 10 mM KC1, 50 mM
.beta.-mercaptoethanol). Absorbance at 590 nm was read on a
Titertek Multiscan Type 310C microtiter plate reader (Flow
Laboratories, Mississauga, ONT) at various times and the resulting
optical densities were converted to mU .beta.-galactosidase using a
standard curve obtained for each plate. All assays were evaluated
in at least 3 wells per plate and the values are reported as
means.+-.standard deviations.
[0327] Chinese Hamster Ovary (CHO) cell transfection studies using
nucleic acid-lipid particles prepared using 100 mM OGP are
presented in FIG. 39 and are evaluated by enzyme production
(.beta.-galactosidase activity) as the end product of gene
transfer. Only those systems that were >2000 nm were effective
at transfecting cells. The transfection efficiency for these
systems increased as the cationic lipid to DNA nucleotide phosphate
(charge) ratio increased from 1:1 to 4:1 (FIG. 39A). Unlike results
with preformed liposome-DNA aggregates (see, e.g., Jarnagin et al.,
1992); however, transfection was not affected by the presence of
serum.
[0328] Particle-induced cell toxicity data are shown in FIG. 39B as
a reduction in enzyme activity/well with increasing amounts of
nucleic acid-lipid particle formulation.
[0329] A significant difference between preformed liposome-DNA
aggregates and the nucleic acid-lipid particles concerns the use of
DOPE as a helper lipid required for optimal transfection (Felgner
& Ringold, 1989; Smith et al., 1993; Farhood et al., 1995). As
shown in FIG. 39C, large particles prepared using the detergent
dialysis procedure with DODAC and SM were more effective in
transfecting CHO cells in vitro than particles prepared using DODAC
and DOPE.
Example 26
[0330] This example illustrates the encapsulation of plasmid DNA in
a lipid vesicles by the detergent dialysis method using different
cationic lipids. The dialysis method is as described previously for
DODAC (EXAMPLE 1). The amount of plasmid entrapped with different
mol % of the various cationic lipids was determined by DEAE
Sepharose chromatography (described in EXAMPLE 2). The entrapment
efficiency was similar for all cationic lipids tested with
approximately 50 to 60% of plasmid DNA. The cationic lipid
concentration required in the formulation for optimal plasmid
encapsulation was 6.5 % for DOTMA, DSDAC and DODMA-AN in FIG.
41(a); 8% DODAC and DMRIE in 41(b); DCchol in 41(c).
Example 27
[0331] This example demonstrates the stability of plasmid
containing vesicles prepared with different cationic lipids. The
serum stability and protection of the plasmid form serum nucleases
was determined by the method described in EXAMPLE 3. Stability and
protection was similar for all preparations obtained with the
different cationic lipids. As examples the elution profile for
preparations containing DODAC FIG. 42(a); DOTMA, FIG. 42(b), and
DSDAC, FIG. 42(c) are given after incubation in mouse serum for 30
min at 37.degree. C.
Example 28
[0332] This example demonstrates the encapsulation of plasmid DNA
with the ionizable lipid AL-1 (pK.sub.a=6.6) by the dialysis method
(as described in EXAMPLE 1). AL-1 is positively charged at acidic
pH and neutral at pH >7. Different concentrations of AL-1 were
used in the lipid formulations at pH 4.8 and 7.5 respectively. The
amount of encapsulated DNA was determined using the PicoGreen
assay. Non-encapsulated DNA was removed first by anion exchange
chromatography and the entrapped DNA determined with PicoGreen
after solubilization of the lipid vesicles in detergent.
Encapsulation of plasmid DNA using DODAC is shown as comparison. At
pH 4.8 maximal encapsulation of approximately 75% of plasmid DNA
was achieved with 8% AL-1 similar to the DODAC formulation at pH
7.5. However, no DNA entrapment was obtained with AL-1 at pH 7.5.
FIG. 43. This clearly demonstrates the requirement of positively
charged lipids for DNA entrapment.
Example 29
[0333] This example shows the stability of the plasmid containing
vesicles formed with AL-1 at pH 4.8 and the protection of the
entrapped DNA from degradation by serum nucleases at pH 7.5.
.sup.3H-DNA and .sup.14C-CHE (cholesteryl hexadecyl ether) were
used to follow the DNA and lipid respectively. The vesicles formed
with AL-1 at Ph 4.8 were incubated in mouse serum for 1.5 hr at
37.degree. C. at pH 7.5. The non-encapsulated DNA was not removed
in the preparations used for serum incubation. After incubation in
serum the vesicles were separated on a Sepharose CL6B column. Lipid
and DNA were detected by radioactivity in the different fractions.
FIG. 44. Approximately 60% of the DNA was protected from serum
nucleases. When vesicles formed with AL-1 at pH 7.5 were incubated
in serum virtually all the DNA was degraded and eluted as fragments
separated from lipids. FIG. 45.
Example 30
[0334] Example 30 demonstrates the effect of the PEG-ceramide
concentration on the encapsulation efficiency by the dialysis
method with 7.5% DODAC and DOPE. The non entrapped DNA in the
various formulations with different PEG-C14 concentrations was
separated by DEAE Sepharose CL6B chromatography. DNA and lipid
recovered are shown as a function of % PEG-C14. Best entrapment was
obtained with 10 mol % PEG-C14. FIG. 46. However, a more recent
experiment showed optimum entrapment in the range of 10 to 15 mol %
(data not shown).
VII. Conclusion
[0335] As discussed above, the present invention comprises novel
lipid-nucleic acid complexes and methods of making them. In a
number of embodiments, hydrophobic DNA intermediates can be
isolated and the DNA exists in a non-condensed form as measured by
dye binding and DNase I sensitivity. These complexes can be used in
the preparation of other lipid-nucleic acid particles.
[0336] In further embodiments, the invention provides methods for
preparing serum-stable nucleic acid-lipid particles which are
useful for the transfection of cells, both in vitro and in
vivo.
[0337] The methods described for the preparation and uses of the
various nucleic acid particles can be used with essentially any
nucleic acid which can exist in a lipophilic state when complexed
with an appropriate cationic lipid. Examples of some constructs
include those encoding adenosine deaminase, the low density
lipoprotein receptor for familial hypercholesterolemia, the CFTR
gene for cystic fibrosis, galactocerebrosidase for Gaucher's
disease, and dystrophin or utrophin into muscle cells for
Duchenne's muscular dystrophy.
[0338] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification for all purposes to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference.
[0339] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *