U.S. patent application number 10/957977 was filed with the patent office on 2006-11-16 for transfection reagents.
This patent application is currently assigned to Northwestern University. Invention is credited to Robert C. MacDonald, Li Wang.
Application Number | 20060257464 10/957977 |
Document ID | / |
Family ID | 34426058 |
Filed Date | 2006-11-16 |
United States Patent
Application |
20060257464 |
Kind Code |
A9 |
MacDonald; Robert C. ; et
al. |
November 16, 2006 |
Transfection reagents
Abstract
The present invention provides optimized transfection reagents
comprising mixtures of cationiclipoids. In particular, the present
invention provides DNA delivery vehicles based on identifying the
optimal hydrophobicity of novel cationic phospholipid derivatives
that, alone or in combination, form complexes with DNA (lipoplexes)
and exhibit enhanced transfection activity.
Inventors: |
MacDonald; Robert C.;
(Evanston, IL) ; Wang; Li; (Evanston, IL) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Assignee: |
Northwestern University
Evanston
IL
60208
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20050142179 A1 |
June 30, 2005 |
|
|
Family ID: |
34426058 |
Appl. No.: |
10/957977 |
Filed: |
October 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60508544 |
Oct 3, 2003 |
|
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Current U.S.
Class: |
424/450 ;
435/458 |
Current CPC
Class: |
C12N 15/88 20130101 |
Class at
Publication: |
424/450 ;
435/458 |
International
Class: |
A61K 9/127 20060101
A61K009/127; C12N 15/88 20060101 C12N015/88 |
Goverment Interests
[0002] The present invention was made, in part, under funds from
the National Institutes of Health Grant No. GM 52329. The
government may have certain rights in the invention.
Claims
1. A composition comprising: lipoid transfection reagents
comprising a first cationic lipoid having a head group and a
hydrophobic tail, and a second cationic lipoid, wherein the second
cationic lipoid comprises a hydrophobic tail that is different than
said hydrophobic tail of said first cationic lipid such that said
transfection reagents have a higher transfection efficiency
compared to said reagents that have said first cationic lipoid, but
lack said second cationic lipoid.
2. The composition of claim 1, wherein said hydrophobic tail of
said second cationic lipoid is shorter than said hydrophobic tail
of said first cationic lipoid.
3. The composition of claim 1, wherein said hydrophobic tail of
said second cationic lipoid is longer than said hydrophobic tail of
said first cationic lipoid.
4. The composition of claim 1, wherein said hydrophobic tail of
said second cationic lipoid has a larger cross-sectional area than
said hydrophobic tail of said first cationic lipoid.
5. The composition of claim 1, wherein said hydrophobic tail of
said second cationic lipoid has a smaller cross-sectional area than
said hydrophobic tail of said first cationic lipoid.
6. The composition of claim 1, wherein said hydrophobic tail of
said second cationic lipoid has a different three dimensional shape
than said hydrophobic tail of said first cationic lipoid.
7. The composition of claim 1, wherein said hydrophobic tail of
said second cationic lipoid has a different chemical composition
than said hydrophobic tail of said first cationic lipoid.
8. The composition of claim 1, wherein said first and second
cationic lipoids comprise different short chain fatty acids.
9. The composition of claim 1, wherein said first and second
cationic lipoids comprise different medium chain fatty acids.
10. The composition of claim 1, wherein said first and second
cationic lipoids comprise different long chain fatty acids.
11. The composition of claim 1, wherein said second cationic lipoid
is EDLPC.
12. The composition of claim 1, wherein said first cationic lipoid
is selected from the group consisting of EDOPC, EDMPC, EPOPC,
EDiphytanoyl PC, SDOPC and EC 18C 10PC.
13. The composition of claim 1, wherein said lipoid transfection
reagents further comprise a transfection enhancing agent.
14. The composition of claim 13, wherein said transfection
enhancing agent is selected from the group consisting of
cholesterol, polyamidoamine dendrons, histidylated lipids,
octylglucoside, and phycoerythrin.
15. A method of transfecting a cell comprising, exposing a cell to
the composition of claim 1, wherein said composition further
comprises a nucleic acid molecule.
16. The method of claim 13, wherein said cell is in vivo.
17. The method of claim 13, wherein said cell is in vitro.
18. The method of claim 13, wherein said cell is ex vivo.
19. The method of claim 13, wherein said cells are human dermal
fibroblast cells.
20. The method of claim 13, wherein said cell is a HUAEC cell.
21. The method of claim 13, wherein said cell is a cancer cell.
22. The method of claim 16, wherein said cancer cell is a myeloma
cell.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/508,544, filed Oct. 3, 2003, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention provides optimized transfection
reagents comprising mixtures of cationic lipoids. In particular,
the present invention provides DNA delivery vehicles based on
identifying optimal hydrophobicity of novel cationic phospholipid
derivatives that, alone or in combination, form complexes with DNA
(lipoplexes) and exhibit enhanced transfection activity.
BACKGROUND
[0004] There are approximately four thousand different genetic
diseases, many highly debilitating and frequently resulting in
death at an early age. Because almost all of these diseases involve
a defective protein, conventional treatment is difficult. A direct
approach to treating such diseases involves providing a competent
gene in the proper cells to compensate for the mutation. This
requires some form of effective transfection process. Transfection
is a process whereby a nucleic acid, primarily DNA, is transferred
to a target cell and codes for an expressed protein. Transfection
is implemented typically to modify the gene complement of the
recipient cell for controlled expression of a particular gene. The
means by which "foreign" DNA can be packaged and delivered to a
host cell are many and varied. The most efficient of these make use
of viruses, but viral vectors have shortcomings, not the least of
which is the potential for immune response or disease transmission.
It has become apparent that lipid-like compounds (e.g., lipoids)
can be used to deliver DNA to cells. The lipoids that are most
efficient in delivering DNA to cells are positively charged.
Cationic lipoids are naturally attracted to and spontaneously form
complexes with polyanionic DNA. Such complexes, or "lipoplexes,"
are useful as transfection vehicles both in vitro and in vivo.
Lipoplexes offer several advantages in that they provide a high DNA
packing density, lower immunogeneicity, and are likely to be able
to transport DNA of considerably larger size than the viral
vectors. The possibility of targeting lipidic carriers to specific
cell types also makes them attractive candidates for gene therapy.
However, the delivery of whole genes is not the only form of gene
therapy. Previous research has demonstrated that antisense gene
therapy may be useful to inhibit expression of genes that cause
disease. Additionally, recent research on the RNAi effect has shown
that administration of particular RNA oligonucleotides could be an
especially effective way of silencing genes that are deleterious.
Similarly, it has become newly appreciated that DNA
oligonucleotides engineered for high affinity binding to particular
gene sequences may be useful in gene therapy given the proper
delivery system. These kinds of developments make it clear that
gene therapy is likely to evolve in a variety of different ways and
that different modes will be effective with different diseases.
[0005] To date, the primary approach to improving the transfection
properties of cationic lipids has been the synthesis of new kinds
of cationic amphipaths or the inclusion of non-cationic helper
lipids. While such approaches have met with some success, improved
transfection reagents that provide efficient transfection (e.g.,
efficient nucleic acid uptake, low toxicity) are needed.
DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 shows data demonstrating that the extent of
transfection of HUAEC varies with the ratio of medium chain to long
chain lipids. The cells were seeded in 96-well plates at 24 hours
before transfection at densities to give about 80% confluence at
the time of transfection. Chloroform solutions of EDLPC with EDOPC
and EDLPC with EDMPC were mixed at the different ratios and then
chloroform was removed under N.sub.2 stream and vacuum. The lipid
mixtures were hydrated in HBSS at 1 mg/ml to form liposomes.
Liposomes and plasmid DNA were diluted in OptiMEM to 80 .mu.g/ml
for lipid and to 20 .mu.g/ml for DNA, and liposomes were pipetted
into an equal volume of plasmid DNA solution at a 4:1 weight ratio
and mixed gently. The resultant DNA (plasmid with
.beta.-galactosidase marker gene)--lipid complexes were incubated
at room temperature for 15 min and then added to the cells that
were either in medium lacking serum (black bars) or medium
containing 5% serum (gray bars). The lipoplexes had 35% (lipoid)
excess positive over negative (DNA) charge. Cells were assayed for
.beta.-galactosidase expression 24 hours after transfection. Data
represent the mean.+-.S.D. of a representative experiment performed
in quadruplicate. .about.20,000 fluorescence units corresponded
approximately to 0.1 milliunit of .beta.-galactosidase.
[0007] FIG. 2 shows data demonstrating the change of transfection
with the ratio of EDLPC to EDOPC and the ratio of total lipids to
DNA. Cells were treated with DNA-lipid complex for 2 h in the
absence ("no serum") or presence of serum ("serum") and then washed
with HBSS and supplemented with fresh culture medium. Data
represent the mean.+-.S.D. of a representative experiment performed
in quadruplicate.
[0008] FIG. 3 shows a graph of the membrane fusion of
fluorescence-labeled, cationic lipoplexes with anionic liposomes.
The lipids were labeled with 0.5 mol % each of NBD-PE and Rh-PE and
hydrated at 1 mg/ml in PBS. Lipoplexes were then prepared as for
transfection. Two hundred microliters of the resulting lipoplexes
were titrated with 3-fold mol unlabeled egg PC liposomes containing
20% DOPG. The experiments were done at 37.degree. C. Ex=320 nm,
Em=535 nm. % membrane
fusion=(Fn-F.sub.0)/(F.sub.100-F.sub.0).times.100%, where Fn is the
fluorescence after the addition of anionic lipid, F.sub.0 is the
initial fluorescence of lipoplexes, and F.sub.100 is the
fluorescence when anionic lipid was mixed directly with cationic
lipids in chloroform and then lipoplexes were prepared as
above.
[0009] FIG. 4 shows oligonucleotide distribution of EDOPC and
EDLPC/EDOPC/DNA (60/40/16.7) lipoplexes in HUAECs. Lipoplexes were
labeled with a fluoresce in derivative of a double-stranded
dodecameric oligonucleotide. Cells were incubated with the
resulting lipoplexes in the presence of serum for 2 h and imaged
under a fluorescence microscope at 2 h later after being washed
with HBSS.
[0010] FIG. 5 shows graphs showing the effect of serum on the
composition of lipoplexes. Lipoplexes were treated with 5% serum at
37.degree. C. for different times, and centrifuged at 14,000 rpm
(4.degree. C.) for 1 h. The pellets were collected and extracted
with chloroform. The chloroform phase was applied to a TLC plate,
which was then developed in chloroform/methanol/H.sub.2O (65:25:4).
The separated EDLPC and EDOPC were obtained by extracting SiO.sub.2
of the relevant regions of the TLC plate with
chloroform/methanol/H.sub.2O (10:10:1). EDLPC and EDOPC were
quantified with a phosphate assay.
[0011] FIG. 6 shows a graph showing the effect of serum on the
transfection of HUAEC. Cells were treated with DNA-lipid complex
for 2 h in the absence ("no serum") or presence of 5% normal serum
("normal serum") or 5% delipidated serum ("delipidated serum") and
then washed with HBSS and supplemented with fresh regular culture
medium. Data represent the mean.+-.S.D. of a representative
experiment performed in quadruplicate.
[0012] FIG. 7 shows a mixed lipoid effect in transfection with
tetraalkylammonium compounds. Conditions were identical with those
described in FIG. 1 except that different lipoid mixtures were
used. Ditetradecyldimethyammonium (di C 14 substituted quaternary
ammonium) was mixed with dioctadecylammonium (di C 18 quaternary
ammonium).
[0013] FIG. 8 shows the results of transfecting human dermal
fibroblast cells with EDOPC/EPOPC (one oleoyl chain, which is 18C's
with one double bond, and one palmitoyl, which is 16C's without
double bond) mixtures.
[0014] FIG. 9 shows the results of transfecting human dermal
fibroblast cells with EDOPC/EDiphytanoyl PC (two phytanoyl chains,
16 carbon chains with 4 methyl branches) mixtures.
[0015] FIG. 10 shows the results of transfecting HUAECs with
EDOPC/POEPC, EDOPC/EDiphytanoylPC, and EDOPC/SDOPC (DOPC with an 18
carbon chain instead of an ethyl group on the phosphate oxygen)
mixtures.
SUMMARY OF THE INVENTION
[0016] The present invention provides optimized transfection
reagents comprising mixtures of, for example, cationic lipoids. In
some embodiments, the mixture comprises first and second lipoids
having different fatty acid chains. In some embodiments, the
different fatty acid chains comprise fatty acid chains that differ
in length (e.g., a first chain having 18 carbons and a second chain
having 10, 12 or 14 carbons). The present invention is not limited
by the particular lengths or number of fatty chains in the lipoids.
In some embodiments, the fatty acid chains differ in mean
cross-sectional area (i.e., width). The present invention is not
limited by the nature of the fatty acid chain constituents that
provide difference in cross-sectional area. An example of such a
constituent includes, but is not limited to, branches (e.g., methyl
branches on the fatty acid chain). In some embodiments,
cross-sectional area is altered by mixing a first lipoid with a
second lipoid that differs in that it has a substitution of a long
chain fatty acid in a location where such a chain does not normally
exist (e.g., substitution of an ethyl group of
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) to a long
chain fatty acid (e.g., stearyl) to provide a mixture of EDOPC and
SDOPC).
[0017] In some embodiments, the different fatty acid chains are
contained in a single molecule (e.g., ethylphosphatidylcholine
having an oleoyl and a decanoyl chain). In other embodiments, the
different fatty acid chains are present in different molecules in
the mixture (e.g., a mixture having
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC) and
1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (EDLPC)).
[0018] The present invention is not limited to any particular
mechanism of action and an understanding of the mechanism of action
is not necessary to practice the present invention. However, it is
contemplated that improved transfection is achieved where two or
more (e.g., three, four, . . . ) different fatty acid chains are
provided in the lipoid mixture.
[0019] In some embodiments, the mixture comprises first and second
cationic lipoids. The present invention provides optimized
transfection reagents comprising mixtures of cationic lipoids. In
particular, the present invention provides DNA delivery vehicles
based on identifying the optimal hydrophobicity of novel cationic
phospholipid derivatives that, alone or in combination, form
complexes with DNA (lipoplexes) and exhibit enhanced transfection
activity.
[0020] In some embodiments, the first cationic lipoid is a standard
lipoid found in transfection reagents. Such standard lipoids are
those known in the art that are used, for example, to form
liposomes (although the present invention is not limited to the use
of liposome transfection reagents). Liposomes comprise spheres of
lipid bilayers that enclose an aqueous medium. Liposomes can
generally be formed by sonicating a lipid in an aqueous medium, by
resuspension of dried lipid layers in a buffer or by dialysis of
lipids dissolved in an organic solvent or in an aqueous solution
against a buffer of choice. Phospholipids form closed, fluid-filled
spheres when they are mixed with water, in part because the
molecules are amphipathic: they have a hydrophobic
(water-insoluble) tail and a hydrophilic (water-soluble), or
"polar," head. Two fatty acid chains containing from about 16 up to
about 24 carbon atoms generally make up the hydrophobic tail of
most naturally occurring phospholipid molecules. Equivalent
structures may be employed in synthetic lipids. Phosphoric acid
bound to any of several water-soluble molecules composes the
hydrophilic head. When a high enough concentration of phospholipids
is mixed with water, the hydrophobic tails spontaneously herd
together to exclude water, whereas the hydrophilic heads bind to
water. In most instances, the result is a bilayer in which the
fatty acid tails point into the membrane's interior and the polar
head groups point outward. The polar groups at one surface of the
membrane point toward the liposome's interior and those at the
other surface point toward the external environment. As a liposome
forms, any water-soluble molecules that have been added to the
water are incorporated into the aqueous spaces in the interior of
the spheres, whereas any lipid-soluble molecules added to the
solvent during vesicle formation are incorporated into the lipid
bilayer. Phospholipid-related materials that are found in typical
liposomes include, lecithin, lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidylglycerol, sphinogomyelin,
cephalin, cardiolipin, phosphatidic acid, cerebrosides, and
ceramide. Some specific examples of phospholipids include, but are
not limited to, dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol
(DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE),
dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (DOPE-MAL), diheptadecanoyl
phosphatidylethanolamine, dilauroylphosphatilylethanolamine,
dimyristoylphosphatidylethanolamine, distearoyl
phosphatidylethanolamine, beta-linoleoyl-gammapalmitoyl
phosphatidylethanolamine and beta-oleoyl-gammapalmitoyl
phosphatidylethanolamine). Common cationic lipoids found in
liposomes include 1,2-diolelyloxy-3-(trimethylamino) propane
(DOTAP);
N-1-(2,3,-ditetradecyloxy)propyl-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE); N-1-(2,3,-dioleyloxy)propyl-N,N-dimethyl-N-hydroxy
ethylammonium bromide (DORIE); N-1-(2,3-dioleyloxy)
propyl-N,N,N-trimethylammonium chloride (DOTMA); 3.alpha.
N-(N',N'-dimethylaminoethane) carbamoly cholesterol (DC-Chol); and
dimethyldioctadecylammonium (DDAB). Numerous other cationic lipoids
are known in the art.
[0021] In some embodiments, the second cationic lipoid is a lipoid
with a hydrophobic structure that is significantly different from
that of the first lipoid. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, the
mechanism of action of such a hydrophobic structure may disrupt the
packing of the bilayer of a liposome or other structure so that the
lipid organization of the array may be different in the mixture,
thereby enhancing transfection. Differences in physical
organization of lipids of transfection agents may translate into
effects on transfection, as measured by detecting transfection
efficiency with and without the second cationic lipoid or by
comparing the transfection reagents having the second cationic
lipoid to other transfection reagents (see e.g., Example 1 for such
a method). Optimization to determine the optimal composition for
liposomes or other lipoid arrays can be achieved by a variety of
methods. For example, in some embodiments, the second cationic
lipoid has a smaller hydrophobic mass (e.g., shorter hydrophobic
tail or net shorter hydrophobic tails in a molecule with multiple
tails). In some embodiments, the second cationic lipoid is
functionalized to add hydrophilicity (e.g., canceling some of the
hydrophobic mass). For example, in some embodiments, a lipid head
group is made larger or more hydrophilic. In some embodiments, a
lipid tail is functionalized to reduce hydrophobicity. For example,
in some embodiments, the functionalization comprises addition of
one or more polar groups. In some embodiments, a polar fluorophore
is added (e.g., NBD), providing the added feature of fluorescent
detectability. In some embodiments, the second cationic lipoid
comprises a head group and a second component with sufficient
hydrophobicity to allow it to form a bilayer with the first
cationic lipoid, but otherwise with low hydrophobicity.
[0022] In some embodiments, the first or second lipoids are not
cationic. In some embodiments, the second lipoid is configured to
have shorter acyl chains than a first lipoid and/or that alters the
packing of the bilayer of a liposome or other structure) as
compared to the same structure in the absence of a second cationic
lipoid (see, e.g., Example 1).
[0023] In some embodiments, the present invention provides a
composition comprising lipoid transfection reagents, wherein the
reagents comprise a first cationic lipoid (e.g., having a head
group and a lipid tail), and a second cationic lipoid (e.g., having
the same or a different head group and a different, second lipoid
tail), wherein the second cationic lipoid, when combined with said
first cationic lipoid in said reagents, decreases the
hydrophobicity of the reagents compared to said reagents in the
absence of the second cationic lipoid, and wherein said decrease
increases the ability of the reagents to transfect cells. In some
preferred embodiments, the second cationic lipoid comprises either
a short, medium, or long chain fatty acid and the first cationic
lipoid comprises either a short, medium or long chain fatty acid. A
short chain fatty acid is a fatty acid chain having 7 or less
carbons. A medium chain fatty acid is a fatty acid chain having
between 8 and 15 carbons (e.g., laurate, myristate, etc.). A long
chain fatty acid is a fatty acid chain having 16 or more carbons
(e.g., palmitate, stearate, oleate, etc.). The lipid tails may be
saturated or unsaturated.
[0024] In other embodiments, the second lipoid has lipid tails that
may be the same length as those of the first lipoid, but are of a
different shape. Such different shapes arise by incorporating
different carbon chain branches along the lipid tails (e.g., as
when methyl branches are incorporated along the lipid tail, the
result of which is to increase the cross-sectional area (in the
plane of the bilayer) of the lipid). Generally, the chains may be
either shorter and/or fatter than the chains of the first
lipoid.
[0025] The cationic lipoids of the present invention may be of any
form, including, but not limited to, natural or synthetic lipoids
having head groups with one or more (e.g., two) fatty acyl or alkyl
chains attached. Where more than one fatty chain is provided on a
lipoid molecule, the chains may be the same or different. In some
embodiments, the modified bilayer structure is achieved by
lengthening one of the two tails and shortening the other. In some
embodiments, the shortened chain is reduced in size more than the
lengthened chain is increased. In some embodiments, the two chains
are of similar length, but of different cross sectional area. In
some embodiments, the lipoid does not comprise a traditional head
group/tail structure. For example, in some embodiments, cationic
cholesterol derivatives or similar structures may be used. In some
embodiments, the cationic lipoids comprise any type of head group,
including, but not limited to, chemically modified
phosphatidylcholine, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and
sphingomyelin, and derivative thereof. In some preferred
embodiments, the first cationic lipid comprises a dioleoyl
O-ethylphosphatidylcholine. In some preferred embodiments, the
second cationic lipid comprises a dilauroyl
O-ethylphosphatidylcholine. The present invention is not limited by
the ratio of the first and second cationic lipids within the
composition. In some embodiments, the composition further comprises
one or more additional components such as cholesterol or other
lipids. In some embodiments, the composition further comprises a
nucleic acid molecule (e.g., a vector, naked DNA, antisense
oligonucleotides, siRNA, etc.), a protein, a small molecule drug,
or other desired agents.
[0026] The present invention also provides methods for transfecting
cells. In some embodiments, the method comprises exposing a cell to
the composition described above, wherein the composition comprises
a nucleic acid molecule. The cell may reside in vitro (e.g., in
culture), ex vivo, or in vivo. The cell may be isolated or may be
associated with other cells (e.g., in a tissue).
Definitions
[0027] To facilitate an understanding of the present invention, a
number of terms and phrases are defined below:
[0028] As used herein the term "lipoid" refers to any natural or
synthetic lipid molecules, including non-natural compounds that are
similar to lipids in structure and properties (e.g., they are
amphipaths).
[0029] As used herein the term "transfection efficiency" refers to,
for example, the percentage of target cells, within a population of
target cells, that contain an introduced exogenous nucleic acid
molecule. Transfection efficiency can be determined by transfecting
a nucleic acid molecule encoding a reporter gene into a population
of target cells and determining the percentage of cells having
reporter activity. The term "transfection efficiency" also refers
to the amount of gene product detected following transfection of
the nucleic acid into the cell. This is determined, for example, by
testing an entire cell population for the amount of gene product
produced after a given incubation period. Thus, the term
"transfection efficiency" involves assaying for the relative
expression of the gene product encoded by the introduced nucleic
acid.
[0030] As used herein, the term "liposome" refers to a vesicle
bounded by a lipid bilayer. A "cationic liposome" has a net
positive charge.
[0031] As used herein, the term "short chain fatty acid" refers to
a fatty acid chain having 7 or less carbons.
[0032] As used herein, the term "medium chain fatty acid" refers to
a fatty acid chain having between 8 and 15 carbons (e.g., laurate,
myristate, etc.).
[0033] As used herein, the term "long chain fatty acid" is a fatty
acid chain having 16 or more carbons (e.g., palmitate, stearate,
oleate, etc.).
[0034] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule including, but not limited to
DNA or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N-6-methyladenosine,
aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0035] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide or precursor. The polypeptide can be
encoded by a full length coding sequence or by any portion of the
coding sequence so long as the desired activity or functional
properties (e.g., enzymatic activity, ligand binding, signal
transduction, etc.) of the full-length or fragment are retained.
The term also encompasses the coding region of a structural gene
and the including sequences located adjacent to the coding region
on both the 5' and 3' ends for a distance of about 1 kb or more on
either end such that the gene corresponds to the length of the
full-length mRNA. The sequences that are located 5' of the coding
region and which are present on the mRNA are referred to as 5'
non-translated sequences. The sequences that are located 3' or
downstream of the coding region and which are present on the mRNA
are referred to as 3' non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene which are
transcribed into nuclear RNA (hnRNA); introns may contain
regulatory elements such as enhancers. Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0036] As used herein, the term "gene expression" refers to the
process of converting genetic information encoded in a gene into
RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of
the gene (i.e., via the enzymatic action of an RNA polymerase), and
for protein encoding genes, into protein through "translation" of
mRNA. Gene expression can be regulated at many stages in the
process. "Up-regulation" or "activation" refers to regulation that
increases the production of gene expression products (i.e., RNA or
protein), while "down-regulation" or "repression" refers to
regulation that decrease production. Molecules (e.g., transcription
factors) that are involved in up-regulation or down-regulation are
often called "activators" and "repressors," respectively.
[0037] Transcriptional control signals in eukaryotes comprise
"promoter" and "enhancer" elements. Promoters and enhancers consist
of short arrays of DNA sequences that interact specifically with
cellular proteins involved in transcription (T. Maniatis et al.,
Science 236:1237 [1987]). Promoter and enhancer elements have been
isolated from a variety of eukaryotic sources including genes in
yeast, insect and mammalian cells, and viruses (analogous control
elements, i.e., promoters, are also found in prokaryotes). The
selection of a particular promoter and enhancer depends on what
cell type is to be used to express the protein of interest. Some
eukaryotic promoters and enhancers have a broad host range while
others are functional in a limited subset of cell types (for review
see, S. D. Voss et al., Trends Biochem. Sci., 11:287 [1986]; and T.
Maniatis et al., supra). For example, the SV40 early gene enhancer
is very active in a wide variety of cell types from many mammalian
species and has been widely used for the expression of proteins in
mammalian cells (R. Dijkema et al., EMBO J. 4:761 [1985]). Two
other examples of promoter/enhancer elements active in a broad
range of mammalian cell types are those from the human elongation
factor 1.alpha. gene (T. Uetsuki et al., J. Biol. Chem., 264:5791
[1989]; D. W. Kim et al., Gene 91:217 [1990]; and S. Mizushima and
S. Nagata, Nuc. Acids. Res., 18:5322 [1990]) and the long terminal
repeats of the Rous sarcoma virus (C. M. Gorman et al., Proc. Natl.
Acad. Sci. USA 79:6777 [1982]) and the human cytomegalovirus (M.
Boshart et al., Cell 41:521 [1985]). Some promoter elements serve
to direct gene expression in a tissue-specific manner.
[0038] As used herein, the term "promoter/enhancer" denotes a
segment of DNA which contains sequences capable of providing both
promoter and enhancer functions (i.e., the functions provided by a
promoter element and an enhancer element, see above for a
discussion of these functions). For example, the long terminal
repeats of retroviruses contain both promoter and enhancer
functions. The enhancer/promoter may be "endogenous" or "exogenous"
or "heterologous." An "endogenous" enhancer/promoter is one which
is naturally linked with a given gene in the genome. An "exogenous"
or "heterologous" enhancer/promoter is one which is placed in
juxtaposition to a gene by means of genetic manipulation (i.e.,
molecular biological techniques such as cloning and recombination)
such that transcription of that gene is directed by the linked
enhancer/promoter.
[0039] The presence of "splicing signals" on an expression vector
often results in higher levels of expression of the recombinant
transcript. Splicing signals mediate the removal of introns from
the primary RNA transcript and consist of a splice donor and
acceptor site (J. Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York
[1989], pp. 16.7-16.8). A commonly used splice donor and acceptor
site is the splice junction from the 16S RNA of SV40.
[0040] Efficient expression of recombinant DNA sequences in
eukaryotic cells requires expression of signals directing the
efficient termination and polyadenylation of the resulting
transcript. Transcription termination signals are generally found
downstream of the polyadenylation signal and are a few hundred
nucleotides in length. The term "poly A site" or "poly A sequence"
as used herein denotes a DNA sequence that directs both the
termination and polyadenylation of the nascent RNA transcript.
Efficient polyadenylation of the recombinant transcript is
desirable as transcripts lacking a poly A tail are unstable and are
rapidly degraded. The poly A signal utilized in an expression
vector may be "heterologous" or "endogenous." An endogenous poly A
signal is one that is found naturally at the 3' end of the coding
region of a given gene in the genome. A heterologous poly A signal
is one that is isolated from one gene and placed 3' of another
gene. A commonly used heterologous poly A signal is the SV40 poly A
signal. The SV40 poly A signal is contained on a 237 bp BamHI/BclI
restriction fragment and directs both termination and
polyadenylation (J. Sambrook, supra, at 16.6-16.7).
[0041] Eukaryotic expression vectors may also contain "viral
replicons" or "viral origins of replication." Viral replicons are
viral DNA sequences that allow for the extrachromosomal replication
of a vector in a host cell expressing the appropriate replication
factors. Vectors that contain either the SV40 or polyoma virus
origin of replication replicate to high "copy number" (up to
10.sup.4 copies/cell) in cells that express the appropriate viral T
antigen. Vectors that contain the replicons from bovine
papillomavirus or Epstein-Barr virus replicate extrachromosomally
at "low copy number" (.about.100 copies/cell).
[0042] The term "transgene" as used herein refers to a foreign gene
that is placed into an organism by, for example, introducing the
foreign gene into newly fertilized eggs or early embryos. The term
"foreign gene" refers to any nucleic acid (e.g., gene sequence)
that is introduced into the genome of an animal by experimental
manipulations and may include gene sequences found in that animal
so long as the introduced gene does not reside in the same location
as does the naturally-occurring gene.
[0043] As used herein, the term "vector" is used in reference to
nucleic acid molecules that transfer DNA segment(s) from one cell
to another. The term "vehicle" is sometimes used interchangeably
with "vector." Vectors are often derived from plasmids,
bacteriophages, or plant or animal viruses.
[0044] The term "expression vector" as used herein refers to a
recombinant DNA molecule containing a desired coding sequence and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism.
Nucleic acid sequences necessary for expression in prokaryotes
usually include a promoter, an operator (optional), and a ribosome
binding site, often along with other sequences. Eukaryotic cells
are known to utilize promoters, enhancers, and termination and
polyadenylation signals.
[0045] The terms "overexpression" and "overexpressing" and
grammatical equivalents, are used in reference to levels of mRNA to
indicate a level of expression approximately 3-fold higher than
that typically observed in a given tissue in a control or
non-transgenic animal. Levels of mRNA are measured using any of a
number of techniques known to those skilled in the art including,
but not limited to Northern blot analysis. Appropriate controls are
included on the Northern blot to control for differences in the
amount of RNA loaded from each tissue analyzed (e.g., the amount of
28S rRNA, an abundant RNA transcript present at essentially the
same amount in all tissues, present in each sample can be used as a
means of normalizing or standardizing the mRNA-specific signal
observed on Northern blots). The amount of mRNA present in the band
corresponding in size to the correctly spliced transgene RNA is
quantified; other minor species of RNA which hybridize to the
transgene probe are not considered in the quantification of the
expression of the transgenic mRNA.
[0046] The term "transfection" as used herein refers to the
introduction of foreign DNA into eukaryotic cells.
[0047] The term "stable transfection" or "stably transfected"
refers to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant"
refers to a cell which has stably integrated foreign DNA into the
genomic DNA.
[0048] The term "transient transfection" or "transiently
transfected" refers to the introduction of foreign DNA into a cell
where the foreign DNA fails to integrate into the genome of the
transfected cell. The foreign DNA persists in the nucleus of the
transfected cell for several days. During this time the foreign DNA
is subject to the regulatory controls that govern the expression of
endogenous genes in the chromosomes. The term "transient
transfectant" refers to cells which have taken up foreign DNA but
have failed to integrate this DNA.
[0049] As used herein, the term "cell culture" refers to any in
vitro culture of cells. Included within this term are continuous
cell lines (e.g., with an immortal phenotype), primary cell
cultures, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro.
[0050] As used, the term "eukaryote" refers to organisms
distinguishable from "prokaryotes." It is intended that the term
encompass all organisms with cells that exhibit the usual
characteristics of eukaryotes, such as the presence of a true
nucleus bounded by a nuclear membrane, within which lie the
chromosomes, the presence of membrane-bound organelles, and other
characteristics commonly observed in eukaryotic organisms. Thus,
the term includes, but is not limited to such organisms as fungi,
protozoa, and animals (e.g., humans).
[0051] As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an
artificial environment. In vitro environments can consist of, but
are not limited to, test tubes and cell culture. The term "in vivo"
refers to the natural environment (e.g., an animal or a cell) and
to processes or reaction that occur within a natural
environment.
[0052] The term "test compound" refers to any chemical entity,
pharmaceutical, drug, and the like that can be used to treat or
prevent a disease, illness, sickness, or disorder of bodily
function. Test compounds comprise both known and potential
therapeutic compounds. A test compound can be determined to be
therapeutic by screening using the screening methods of the present
invention. A "known therapeutic compound" refers to a therapeutic
compound that has been shown (e.g., through animal trials or prior
experience with administration to humans) to be effective in such
treatment or prevention.
[0053] The term "sample" as used herein is used in its broadest
sense and includes environmental and biological samples.
Environmental samples include material from the environment such as
soil and water. Biological samples may be animal, including, human,
fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue,
liquid foods (e.g., milk), and solid foods (e.g., vegetables).
DESCRIPTION OF THE INVENTION
[0054] The present invention provides an alternative approach to
improving transfection reagents. The compositions and methods of
the present invention were shown to be unusually effective. In
preferred embodiments, the present invention provides the
combination of two or more cationic lipoids to provide improved
transfection reagents. In some embodiments, the first cationic
lipoid is a standard cationic lipoid used in transfection reagents
and the second cationic lipoid is of the nature where, when
combined with the first cationic lipoid in transfection reagents,
alters the hydrophobicity of the hydrophobic mass either in the
extent of hydrophobicity of the lipoids or in the organization of
the lipoids compared to the reagents in the absence of the second
cationic lipoid, and wherein such change in hydrophobicity
increases the ability of the transfection reagents to transfect
cells.
[0055] In some embodiments, the second cationic lipoid is a lipoid
that has higher water solubility than the first lipoid and/or that
increases the exposure of the hydrophobic core of the lipoid
structure to an aqueous environment (e.g., disrupt the bilayer of a
liposome) as compared to the same structure in the absence of the
second cationic lipoid. The degree of increase in water solubility
and/or increased exposure of the hybrophobic core that finds use in
the present invention can readily be measured by detecting
transfection efficiency with and without the second cationic lipoid
or by comparing the transfection reagents having the second
cationic lipoid to other transfection reagents (see e.g., Example 1
for such a method). Optimization to increase water solubility
and/or increased exposure of the hydrophobic core can be achieved
by a variety a methods. For example, in some embodiments, the
second cationic lipoid has a smaller hydrophobic mass (e.g.,
shorter hydrophobic tail). In some embodiments, the second cationic
lipoid is functionalized to add hydrophilicity (e.g., canceling
some of the hydrophobic mass). In some embodiments, the
functionalization comprises addition of one or more polar groups.
In some embodiments, a polar fluorophore is added (e.g., NBD),
providing the added feature of fluorescent detectability.
[0056] For example, particularly efficient transfection reagents
were produced by the combination of dilauroyl (12 carbon chain) and
dioleoyl (18 carbon chain) homologues of
O-ethylphosphatidylcholine. This mixture transfected DNA into human
umbilical artery endothelial cells (HUAECs) more than 30-fold more
efficiently than either compound separately. A unique advantage of
this kind of combination agent is that transfection can be
optimized either in the presence or absence of serum by adjusting
the component ratio.
[0057] In some embodiments, the second lipoid has chains that are
not significantly different in length from those of the first
lipoid, but the second lipoid has chains that have a larger
cross-sectional area.
[0058] In some embodiments, the second lipoid has chains that are
differently shaped from those of the first lipoid, so as to occupy
space in the bilayer in a different way than those of the first
lipoid.
[0059] Cationic lipids have been widely used for the delivery of
plasmid and antisense DNA into eukaryotic cells; however,
inefficiency of transfection is a major problem confronting their
use in gene therapy. Vascular endothelial cells act as an interface
between circulating blood and various tissues and organs of the
body, and are known to be involved in inflammatory processes such
as leukocyte recruitment, cytokine production (see, e.g., Koning G
A, et al., Endothelium 2002, 9:161-171; Neuhaus T et al., Clinical
Science 2000; 98: 461-470; Stier S et al., FEBS Letters 2000; 467:
299-304; each herein incorporated by reference in their
entireties), and to play a major role in the pathogenesis of
atherosclerosis (see, e.g., Behrendt D, and Ganz P., Am J Cardiol
2002; 90: 40L-48L; Ulrich-Merzenich G, et al., European Journal of
Nutrition 2002; 41: 27-34; each herein incorporated by reference in
their entireties), as well as angiogenesis (see, e.g., Ellis L M.
Am Surg 2003; 69: 3-10; Nam N H, Parang K. Curr Drug Targets 2003;
4: 159-179; Ranieri G, and Gasparini G., Curr Drug Targets Immune
Endocr Metabol Disord 2001; 1: 241-253; Sylven C. Drugs Today
(Barc) 2002; 38: 819-827; each herein incorporated by reference in
their entireties), on which the growth and spread of tumors are
dependent. Hence, they are of considerable interest as a gene
therapy target (see, e.g., Baker AH., J Card Surg 2002; 17:
543-548; Morishita R., Circ J 2002; 66: 1077-1086; each herein
incorporated by reference in their entireties). Even though they
are readily accessible, gene therapy with nonviral vectors of
endothelial tissue has been seriously hampered by the fact that
endothelial cells are very difficult to transfect. According to
Struck et al., Biochemistry 1981, 20:4093-4099, the transfection
efficiency of vascular endothelial cells with cationic lipids was
only 2%. It is known that the cytotoxicity of cationic lipids
increases with the shortening of acyl groups and so cationic lipids
used in transfection invariably have alkyl chains that are 14 or
more carbon long. The present invention provides solutions to such
problems.
[0060] For example, in one embodiment, a short chain cationic
phosphocholine (1,2-dilauroyl-sn-glycero-3-ethylphosphocholine,
EDLPC), when combined with longer chain compounds
(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, EDOPC, or
1,2-dimyristol-sn-glycero-3-ethylphosphocholine, EDMPC)
dramatically enhances (up to 30-fold) the transfection efficiency
of human umbilical artery endothelial cells (HUAECs) even though,
individually, EDLPC, EDOPC or EDMPC are quite weak transfection
reagents. Moreover, transfection efficiency can be adjusted to be
optimal either in the presence or absence of serum by changing the
EDLPC/EDOPC ratio and the ratio of total lipids to DNA. Under
optimal conditions, transfection efficiency can be achieved up to
15% both in the presence and absence of serum. Thus, these
formulations constitute a novel form of cellular transfection
reagent and offer entirely new formulations for optimizing in vivo
gene delivery. At present, only phosphatidylethanolamine and
cholesterol are used as the helper lipids to improve the
transfection properties of cationic lipids. Unlike these prior
methods (although they may be used in conjunction with the present
invention), the present invention employs compounds with different
hydrophobicity-hydrophilicity balance to improve the gene delivery
properties of lipoplexes. The properties of lipoplexes can be tuned
by changing the ratio of the different lipoids (e.g., the ratio of
medium chain to long chain cationic lipoids and the ratio of lipoid
to DNA).
[0061] In some preferred embodiments, one or more agents may be
added to the cationic lipoid mixtures so as to further increase
transfection efficiency. Examples of agents include, but are not
limited to, cholesterol, polyamidoamine dendrons, histidylated
lipids, octylglucoside, phycoerythrin, and non-cationic lipids. In
some preferred embodiments, the cationic lipoid mixtures may be
transfected with additional transfection reagent sytems so as to
further increase transfection efficiency. Examples of transfection
reagent systems include, but are not limited to, LIPOFECTAMINE
(Invitrogen), OPTIFECT (Invitrogen), 293FECTIN (Invitrogen),
OLIGOFECTAMINE (Invitrogen), CELLFECTIN (Invitrogen), LIPOFECTIN
(Invitrogen), DMRIE-C (Invitrogen), EXGEN 500 (Euromedex),
octylglucoside, FUGENE (Roche), EFFECTGENE (Qiagen), and SUPERFECT
(Qiagen).
[0062] In some embodiments, the first or second lipoids are not
cationic but are configured to have a structural impact on the
bilayer into which they are incorporated (e.g., to impact the water
solubility than a cationic lipoid used alone, to increase the
exposure of the hydrophobic core of the lipoid structure to an
acqueous environment, or to disrupt the packing of the bilayer of a
liposome or other structure as compared to the same structure in
the absence of a second cationic lipoid).
Transgenes
[0063] The transfection system described herein is useful to
express any polypeptide of interest or to transfect any nucleic
acid of interest (e.g., siRNAs, antisense oligonucleotides,
expression vectors, etc.).
[0064] The transgene will generally encode a native or recombinant
protein, although the expression of other polypeptides, such as
epitopes or other immunologically active polypeptides, are
contemplated within the scope of this invention. Examples of
proteins that can be expressed using the method of the present
invention are hormones; cytokines, such as growth factors; enzymes;
receptors; oncogenes; polypeptide vaccines, viral proteins, and
structural and secretory proteins.
[0065] The transgene employed in the constructs of the invention
can be cloned sequences that retain intronic regions. If the exonic
structure of the gene is known, the coding exons can be inserted in
the constructs.
[0066] Expression of the polypeptide of interest can be directed by
a promoter homologous to the polypeptide coding sequences (for
example, human glucose-6-phosphate dehydrogenase under the control
of its own transcription promoter sequences). Further, other
homologous or heterologous expression control elements (e.g.,
affecting transcription, translation, or post-translational events)
may be used.
[0067] It should be understood that expression of the transgene in
the mammalian cells of the invention can be stable or transient.
Even transient expression, at a higher than normal level, is useful
for functional studies in the cells or for the production and
recovery of proteins of interest.
Regulatory Sequences
[0068] In addition to selectable markers and transgenes, the
constructs described herein may contain suitable regulatory
elements. Regulatory elements (or control elements) are selected
for use in the host cell of interest; for example, selectable
markers may be included to allow propagation in microorganisms,
(e.g., f1 origin of replication and ampicillin resistance encoding
sequences). Such regulatory elements include, but are not limited
to, transcription promoters, transcription enhancer elements,
transcription termination signals, polyadenylation sequences
(located 3' to the translation stop codon), sequences for
optimization of initiation of translation (located 5' to the coding
sequence), translation termination sequences, secretion signal
sequences, and sequences that direct post-translational
modification (e.g., glycosylation sites). Transcription promoters
can include inducible promoters (where expression of a
polynucleotide sequence operably linked to the promoter is induced
by an analyte, cofactor, regulatory protein, etc.), repressible
promoters (where expression of a polynucleotide sequence operably
linked to the promoter is induced by an analyte, cofactor,
regulatory protein, etc.), and constitutive promoters.
Cells
[0069] The cells (e.g., host cells) employed in this invention
include all eukaryotic cells including mammalian cells (in vivo or
in vitro), cell lines, and cell cultures. The cells can be derived
from mammals, such as mice, rats, or other rodents, or from
primates, such as humans or monkeys. Mammalian germ cells or
somatic cells can be employed for this purpose. It will be
understood that primary cell cultures or immortalized cells can be
employed in carrying out the techniques of the present invention.
The cells may also reside in vivo. Examples of cells used in the
present invention include, but are not limited to, HUAAEC cells,
human dermal fibroblast cells, cancer cells (e.g., myeloma
cells).
[0070] The transformed cells obtained by some embodiments of the
present invention can be employed for the preparation of continuous
cell lines in which the cells are essentially immortal, or for the
preparation of established cell lines that have the potential to be
subcultured in vitro. Continuous cell lines and established cell
lines can be obtained from a variety of organisms and organs, such
as rodent embryos; primate kidneys; rodent and human tumors; and
fibroblast, epithelial, or lymphoid cells. Cells exhibiting the
highest levels of expression can be cloned, if desired.
EXAMPLES
[0071] The following examples are provided to demonstrate and
further illustrate certain preferred embodiments of the present
invention and are not to be construed as limiting the scope
thereof.
Example 1
EDLPC/EDOPC Transfection Reagents
[0072] Experiments conducted during the development of the present
invention found that attention to the hydrophobic portions of
medium and long-chain cationic lipids synergistically enhance
transfection. It was found that a combination of two cationic lipid
derivatives with the same head group but tails of different chain
lengths behave considerably differently as transfection agents than
the separate molecules. For example, the combination of the
dilauroyl (12 carbon chain) and the dioleoyl (18 carbon chain)
homologues of O-ethylphosphatidylcholine transfected DNA into
primary human umbilical artery endothelial cells (HUAECS) more than
30-fold more efficiently than either compound separately. The
present invention is not limited to a particular mechanism. Indeed,
an understanding of the mechanism is not necessary to practice the
present invention. Nonetheless, these results suggest that the
hydrophobic portions of medium and long-chain cationic lipids is
far more important than previously assumed. An advantage of this
kind of combination agent is that transfection is optimized either
in the presence or absence of serum by adjusting the component
ratio.
[0073] Considering that there are more opportunities to modify and
combine the hydrophobic moieties on cationic lipoids than there are
for variation of the head groups, a study of the transfection
efficiency of lipids with different kinds of tails and different
kinds of combinations of those tails leads to new and improved
nonviral vectors was conducted. A unique advantage of the cationic
phospholipoids for hydrophobic structure modification is that they
allow use of specific enzymes in their synthesis, a feature not
exhibited by the other cationic lipoids described in the literature
because those compounds are not based on a natural product.
[0074] FIG. 1 shows that combining EDLPC with EDOPC enhances by
.about.30-fold of the extent of transfection of HUAECS, compared to
EDLPC or EDOPC alone. The ratio of EDLPC to EDOPC affected
performance, with different ratios optimal, depending upon whether
serum is present or absent. The EDLPC/EDMPC mixture exhibits the
similar pattern to that of EDLPC/EDOPC, but the extent of
transfection is lower than that of EDLPC/EDOPC.
[0075] On the basis of the results presented above, EDLPC/EDOPC
(80/20) and EDLPC/EDOPC (60/40) were chosen to further optimize
transfection; the ratio of lipids to DNA and the amount of DNA were
used as optimization parameters. FIG. 2 depicts the change of
transfection with the ratio of EDLPC to EDOPC and the ratio of
total lipids to DNA. For some formulations, transfection without
serum was better than that in serum; but for others, transfection
in serum was better than that in the absence of serum. The highest
transfection in the absence of serum was obtained when
EDLPC/EDOPC=80/20 and lipid/DNA=4/1, with 0.5 .mu.g DNA/well; under
these conditions the extent of expression was 8.times. higher than
that in the presence of serum. In contrast, the most efficient
transfection in the presence of serum was when EDLPC/EDOPC=60/40
and lipid/DNA=6/1, with 1.0 .mu.g DNA/well, under which condition
the expression was 20.times. that in the absence of serum.
According to X-gal staining, 15% of the cells treated under both of
these conditions were positive. This efficiency of transfection is
more than an order of magnitude higher than has been previously
reported for transfection of these primary cells. These two
formulations were thus used in the subsequent studies. Such assays
can be used to readily determine optimal ratios and optimal
components of the transfection reagents of the present
invention.
[0076] The cell viability and the percentage of cells transfected
for the two formulations were determined using the MTT method and
X-gal staining, respectively (Table 1). Those data revealed that
the low transfection efficiency in the absence of serum for
EDLPC/EDOPC=60/40 and lipid/DNA=6/1 was due to high
cytotoxicity.
[0077] While the present invention is not limited to any particular
mechanism of action and an understanding of the mechanism of action
is not necessary to practice the present invention, it is
contemplated that the medium chain lipid facilitates mixing of the
lipoplex lipid with cellular lipid, which could lead to the
neutralizing of the positive charge of the cationic lipid and
facilitate release of DNA from the complex. Under such
circumstances, EDLPC could facilitate fusion (or at least lipid
mixing) of cationic liposomes with anionic liposomes.
[0078] The fusion of EDLPC/EDOPC (80/20), EDLPC/EDOPC (60/40) and
pure EDOPC lipoplexes were compared to
phosphatidylglycerol-containing (anionic) liposomes. Membrane
fusion was measured using a FRET assay (see, e.g., Struck D K, et
al., Biochemistry 1981; 20: 4093-4099; herein incorporated by
reference in its entirety) that measures reduction of energy
transfer between NBD-PE and Rh-PE in cationic lipids of the
lipoplexes as they fuse with egg-PC liposomes containing 20% DOPG.
From FIG. 3, it is seen that the extent of fusion of EDLPC/EDOPC
(80/20) and EDLPC/EDOPC (60/40) lipoplexes is significantly higher
than that of pure EDOPC. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, these
results indicate that increased transfection efficiency is
associated with membrane fusion characteristics.
[0079] In order to determine if this pattern of fusion is also
observed within cells (e.g., the mixture is more prone to fuse with
endosomal membranes facilitating escape of DNA from endosomal
degradation and nuclei entrance) the intracellular distribution of
fluorescent lipid and oligonucleotide in EDOPC and EDLPC/EDOPC
(60/40) lipoplexes was investigated. It was found that both lipid
and oligonucleotide in EDOPC lipoplexes remained in the cytoplasm
for at least 20 hours, whereas a large amount of the
oligonucleotide from EDLPC/EDOPC (60/40) lipoplexes entered the
nuclei, in particular at the early time point of 2 h, although
lipid in EDLPC/EDOPC (60/40) lipoplexes remained in the cytoplasm
at this and all other time points. FIG. 4 shows oligonucleotide
distribution of EDOPC and EDLPC/EDOPC/DNA (60/40/16.7) lipoplexes
in HUAECSs. Lipoplexes were labeled with a fluorescein derivative
of a double-stranded dodecameric oligonucleotide. Cells were
incubated with the resulting lipoplexes in the presence of serum
for 2 h and imaged under a fluorescence microscope after being
washed in HBSS. As shown in FIG. 4, the results of these
experiments indicated that in the presense of the lipoid mixture
there was an increase in the nuclear distribution of highly
fluorescent oligonucleotides. Similar images were obtained with
fluorescent plasmid DNA, although the fluorescence of the nucleus
was less intense.
[0080] Escape of lipoplexes from endosomes prior to their entry
into lysosomes is important for trangene efficient expression. It
is contemplated that fusion of lipoplexes with endosomal membranes
facilitates DNA release from endosomes into cytoplasm, and thus
increase DNA expression. While the present invention is not limited
to any particular mechanism of action and an understanding of the
mechanism of action is not necessary to practice the present
invention, it is contemplated that this may be one reason that
transfection by the mixtures of lipid is much higher than that of
pure EDOPC.
[0081] The present invention is not limited to a particular
mechanism. Indeed, an understanding of the mechanism is not
necessary to practice the present invention. Nonetheless, it is
contemplated that dissociation of DNA from the surface of a lipoid
is caused by neutralization of the lipoid by cellular anionic
lipids. Such neutralization implies fusion or transfer of lipids as
a necessary prerequisite of efficient transfection, and implies
that the DNA must become sufficiently free of the lipid-lipoid
array to be transcribed in the nucleus. Unlike normal cellular
lipids, the combination of cationic lipoids and anionic lipids
gives rise to a variety of non-lamellar phases which may or may not
be capable of retaining a molecule as large as a typical plasmid.
Generally, generation of lipid phases through combination of
cationic and anionic lipids is dependent upon lipids (see, e.g.,
Tarahovsky, Y. S., et al., 2004, Biophysical Journal 87:1054-1064;
herein incorporated by reference in its entirety). For example,
mixtures such as EDOPC-EDLPC, when reconstituted with anionic
lipids such as phosphatidylglycerol, give rise to a highly curved
inverted micellar cubic phase. This phase is characterized by a
cubic array of balls (shells) in which amphipathic molecules are
organized with their polar portion facing a small aqueous core and
their hydrophobic tails facing those of other shells. These phases
have aqueous spaces too small to entrap either a plasmid or other
DNA molecule. Separate experiments have revealed that treatment of
EDOPC-EDLPC lipoplexes with the anionic lipid, phosphatidylserine,
releases more DNA by far that does treatment of EDOPC lipoplexes
with phoshatidylserine. The present invention is not limited to a
particular mechanism. Indeed, an understanding of the mechanism is
not necessary to practice the present invention. Nonetheless, it is
contemplated that there are at least two important effects involved
when certain kinds of lipoid mixtures are used to prepare
lipoplexes. First, the mixed lipoid lipoplex may acquire anionic
lipid from the cell (e.g., perhaps by membrane fusion or molecular
exchange) faster and/or to a larger extent than do lipoplexes
composed of lipoids of a single type. Second, the phase or
3-dimensional array assumed after the cellular anionic lipid and
the lipoplex lipoid may have such a structure as to release faster
and/or to a greater extent its cargo of DNA than do conventional
lipoplexes.
[0082] Serum strongly influences properties of lipoplexes, so
experiments were conducted to examine the effect of serum on the
composition of these two formulations. In FIG. 5, one sees that
during 90 min incubation in serum, for EDLPC/EDOPC=60/40, 20% of
the EDOPC and 10% of the EDLPC were extracted from the lipoplexes;
in the case of EDLPC/EDOPC=80/20, 30% of the EDOPC and 50% of the
EDLPC are extracted. Furthermore, at early times (30 min), which
are contemplated to be more important for endocytosis, the
extraction of EDLPC and EDOPC from EDLPC/EDOPC=80/20 was much
larger than that from EDLPC/EDOPC=60/40.
[0083] Gene expression in delipidated serum was tested (FIG. 6).
Transgene expression of both two formulations decreased
significantly in delipidated serum, in which .about.80% lipids
(including cholesterol, HDL cholesterol, LDL cholesterol and
phospholipids) are absent relative to normal serum. While the
present invention is not limited to any particular mechanism of
action and an understanding of the mechanism of action is not
necessary to practice the present invention, it is contemplated
that this indirectly confirms serum extraction of lipids, since
delipidated serum, with a higher lipid binding capacity than normal
serum, would also extract more lipids from the lipoplex.
[0084] Human dermal fibroblasts are another medically important
cell type through participation in wound healing. It was
contemplated that human dermal fibroblasts would be useful in gene
therapy to accelerate wound healing. It was therefore of interest
to determine if the "mixed lipid" effect also operates in these
primary cells. It was found that the "mixed lipid" effect is more
pronounced than with HUAECs. The response in serum was not as
pronounced, but efforts were not made to optimize the conditions
for this system.
[0085] A human multiple myeloma cell line that is extremely
difficult to transfect was also investigated and the "mixing
effect" was observed, although the transfection efficiency was very
low (1-2%).
[0086] The mixed lipoid effect is not limited to cationic
phospholipoids. As shown in FIG. 7, the effect is seen when a
dimethylammonium with two C14 chains is mixed with EDOPC and when
the C 18 phospholipoid is replaced with a dimethylammonium having
two C 18 chains. The TAP compounds, DOTAP and DMTAP in various
combinations, were investigated with each other and with EDOPC. In
all cases substantial increases at intermediate compositions was
observed. The present invention is not limited to a particular
mechanism. Indeed, an understanding of the mechanism is not
necessary to practice the present invention. Nonetheless, the mixed
lipoid effect appears quite general, as would be anticipated if
some aspect of the hydrophobicity of the lipoplex needs to be
matched to the cell and the transfection conditions. Although other
lipoids can be synthesized to have chain length differences, it is
unlikely that any other such compounds offer the flexibility of
structural variation as the cationic phospholipoids. Thus, while
the present invention is not limited to the use of cationic
lipoids, cationic lipoids are a preferred material. These compounds
offer enormous flexibility in constructing molecules with varied
amount and configuration of hydrophobic moieties.
Example 2
Transfection of Human Dermal Fibroblasts with EDOPC/EPOPC and
EDOPC/EDiphytanoyl PC Transfection Reagents
[0087] FIG. 8 shows that combining EDOPC with EPOPC (one oleoyl
chain, which is 18C's with one double bond, and one palmitoyl
chain, which is 16C's without any double bond) shows little mixing
effect in transfection of human dermal fibroblast cells in the
absence of serum.
[0088] FIG. 9 shows that combining EDOPC with EDiphytanoyl PC (two
phytanoyl chains, 16 carbon chains with 4 methyl branches) shows
marked mixing effect in the transfection of human dermal fibroblast
cells in the absence of serum.
Example 3
Transfection of HUAECs with EDOPC/EPOPC, EDOPC/EDiphytanoylPC,
EDOPC/SDOPC and EDOPC/EC18C10PC
[0089] FIG. 10 shows the results of transfecting HUAECs with
EDOPC/EPOPC, EDOPC/EDiphytanoylPC, EDOPC/SDOPC (DOPC with an 18
carbon chain instead of an ethyl group on the phosphate oxygen),
and EDOPC/EC18C10PC mixtures.
Example 4
Synthesis of New Cationic Phospholipoids (Derivatives of
Phosphatidylcholine) Having Hydrophobic Moieties
[0090] New cationic phospholipids (derivatives of
phosphatidylcholine) are contemplated including, but not limited
to, medium chain cationic PC's with phosphate oxygen alkyl
substituents ranging in length from 2 to 24 C's); lyso cationic
PC's with one long chain (C24) and a phosphate oxygen alkyl
substituent with 2 to 12 C's; cationic PC's with acyl groups having
very much different chain lengths; tetra-acyl cationic PC's with
short acyl chains; and lipoids with very long (>18 carbons) acyl
chains. In some cases, acyl or alky substituents may be branched so
as to effectively increase the number of chains without increasing
the number of attachment points to the hydrophilic cationic head
group.
[0091] All publications and patents mentioned are herein
incorporated by reference. Various modifications and variations of
the described method and system of the invention will be apparent
to those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described
in connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention that are
obvious to those skilled in the relevant fields are intended to be
within the scope of the following claims. TABLE-US-00001 TABLE 1
Cell viability and X-gal staining .mu.g Cell X-gal DNA/ viability
staining EDLPC/EDOPC Lipids/DNA well Serum % % 80:20 4:1 0.5 - 77.5
.about.15 80:20 4:1 0.5 + 94.7 n.d.* 60:40 6:1 1.0 - 40.0 n.d.*
60:40 6:1 1.0 + 82.6 .about.15 *Not determined
* * * * *