U.S. patent application number 12/207165 was filed with the patent office on 2009-08-13 for compositions and methods for increasing protein production.
Invention is credited to Valery Alakhov, Alexander V. Kabanov.
Application Number | 20090203076 12/207165 |
Document ID | / |
Family ID | 35657641 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203076 |
Kind Code |
A1 |
Kabanov; Alexander V. ; et
al. |
August 13, 2009 |
COMPOSITIONS AND METHODS FOR INCREASING PROTEIN PRODUCTION
Abstract
Compositions and methods for increasing protein production are
provided.
Inventors: |
Kabanov; Alexander V.;
(Omaha, NE) ; Alakhov; Valery; (Baie d'Urfe,
CA) |
Correspondence
Address: |
DANN, DORFMAN, HERRELL & SKILLMAN
1601 MARKET STREET, SUITE 2400
PHILADELPHIA
PA
19103-2307
US
|
Family ID: |
35657641 |
Appl. No.: |
12/207165 |
Filed: |
September 9, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10894709 |
Jul 20, 2004 |
7422875 |
|
|
12207165 |
|
|
|
|
Current U.S.
Class: |
435/69.4 ;
435/404; 435/69.1; 435/69.52; 435/91.1 |
Current CPC
Class: |
C12P 21/02 20130101 |
Class at
Publication: |
435/69.4 ;
435/69.1; 435/69.52; 435/91.1; 435/404 |
International
Class: |
C12P 21/02 20060101
C12P021/02; C12N 15/09 20060101 C12N015/09; C12N 15/10 20060101
C12N015/10; C12N 5/00 20060101 C12N005/00 |
Claims
1. A method for producing a protein comprising: a) providing cells
comprising a heterologous nucleic acid encoding a recombinant
protein; and b) incubating the cells in media containing at least
one amphiphilic block copolymer.
2. The method of claim 1, comprising the further step of: c)
replacing said media containing an amphiphilic block copolymer with
media lacking said amphiphilic block copolymer.
3. The method of claim 1, comprising the further step of isolating
the expressed recombinant protein.
4. The method of claim 2, comprising the further step of isolating
the expressed recombinant protein.
5. The method of claim 1, wherein said cells are mammalian
cells.
6. The method of claim 1, wherein said amphiphilic block copolymer
comprises at least one block of poly(oxyethylene) and at least one
block of poly(oxypropylene).
7. The method of claim 6, wherein said amphiphilic block copolymer
is a Pluronic.RTM. copolymer.
8. The method of claim 6, wherein said amphiphilic block copolymer
is a Tetronic.RTM. copolymer.
9. The method of claim 6, wherein said amphiphilic block copolymer
has a hydrophilic-lipophilic balance (HLB) of between 1 and 20.
10. The method of claim 9, wherein said amphiphilic block copolymer
has an HLB of between 8 and 16.
11. The method of claim 7, wherein said Pluronic.RTM. copolymer is
selected from the group consisting of Pluronic.RTM. P123,
Pluronic.RTM. P103, Pluronic.RTM. P85, and Pluronic.RTM. L64.
12. The method of claim 1, wherein said at least one amphiphilic
block copolymer is a mixture of different amphiphilic block
copolymers.
13. The method of claim 1, wherein said amphiphilic block copolymer
is a mixture of a Pluronic.RTM. copolymer and a polycation
conjugated Pluronic.RTM. copolymer.
14. The method of claim 13, wherein said mixture comprises
Pluronic.RTM. P123 and Pluronic.RTM. P123 conjugated to
polyethyleneimine.
15. The method of claim 14, wherein the ratio of Pluronic.RTM. P123
to Pluronic(D P123 conjugated to polyethyleneimine in said mixture
is 9:1 by weight.
16. The method of claim 1, wherein said incubation of the cells in
media comprising at least one amphiphilic block copolymer is for at
least three hours.
17. The method of claim 16, wherein said incubation is for at least
nine hours.
18. The method of claim 6, wherein said amphiphilic block copolymer
is present in the media at a concentration ranging from about
0.0001% to about 5%.
19. The method of claim 18, wherein said concentration ranges from
about 0.1% to about 2%.
20. The method of claim 1, wherein said heterologous nucleic acid
is stably incorporated into said cells.
21. The method of claim 1, wherein said heterologous nucleic acid
encoding a recombinant protein is controlled by the cytomegalovirus
promoter.
22. The method of claim 1, wherein said recombinant protein is
selected from the group consisting of cytokines, enzymes, clotting
factors, vaccines, antibodies, growth factors and hormones,
insulin, hemoglobin, alpha-1-antitrypsin (AAT), lactoferrin, cystic
fibrosis transmembrane conductase (CFTR), human protein C,
anti-viral agents, and interleukins.
23. The method of claim 22, wherein said recombinant protein is
Factor VIII and said amphiphilic block copolymer has a
hydrophilic-lipophilic balance (HLB) of between 1 and 20.
24. A method for producing a protein in a host comprising: a)
providing a cell comprising a heterologous nucleic acid encoding a
recombinant protein; b) incubating the cells in media containing at
least one amphiphilic block copolymer; and c) introducing the cells
into a host.
25. The method of claim 24, wherein the cells in step a) are
obtained from the host and the heterologous nucleic acid was
subsequently incorporated into the cells in vitro.
26. A method for enhancing production an RNA comprising: a)
providing cells comprising a heterologous DNA encoding an RNA; and
b) incubating the cells in media containing at least one
amphiphilic block copolymer.
27. The method of claim 26, wherein said encoded for RNA is an
siRNA.
28. The method of claim 26, wherein said heterologous nucleic acid
encoding a recombinant protein is controlled by the cytomegalovirus
promoter or a polymerase III promoter.
29. A composition comprising stably transformed cells, at least one
amphiphilic block copolymer, and nucleic acid free media.
30. A kit for practicing the method of claim 1 comprising an
amphiphilic block polymer, reagents to transform a cell, and at
least one selection agent to isolate stably transformed cells.
31. The kit of claim 30 further comprising at least one of frozen
stocks of host cells and instruction material.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for increasing
protein and RNA production.
BACKGROUND OF THE INVENTION
[0002] Several publications and patent documents are cited
throughout the specification in order to describe the state of the
art to which this invention pertains. Each of these citations is
incorporated herein by reference as though set forth in full.
[0003] Non-viral gene delivery is a critically important method of
delivering genes to a cell both in vitro and in vivo. To enhance
the transfer of DNA into the cells, polycations and polymer agents
have been employed that can 1) bind and condense DNA, 2) protect
the DNA from degradation, and 3) enhance transport of the DNA into
the cell (Wagner et al. (1990) PNAS, 87:3410-3414; Kabanov et al.
(1993) Bioconj. Chem., 4:448-454; Boussif et al. (1995) PNAS,
92:7297-7301; Tang et al. (1997) Gene Ther., 4:823-832; Pollard et
al. (1998) J. Biol. Chem., 273:7507-7511; Godbey et al. (1999)
PNAS, 96:5177-5181; Merdan et al. (2002) Pharm. Res., 19:140-146).
Amphiphilic block copolymers have been employed to increase the
transfer of naked DNA in vivo into a variety of tissues including
muscle and skin tissues and tumors (Lemieux et al. (2000) Gene
Ther., 7:986-991; Liaw et al. (2001) Gene Ther., 8:999-1004;
Alakhov et al. (2001) Expert Opin. Biol. Ther., 1:583-602; Gebhart
et al. (2003) Controlled Release Society, Glasgow, Scotland, UK;
Pitard et al. (2002) Human Gene Ther., 13:1767-1775; Batrakova et
al. (2003) J. Pharmacol. Exp. Ther., 304:845-854). Additionally,
amphiphilic block copolymers have been shown to increase the
transfer of polycation-DNA complexes (Nguyen et al. (2000) Gene
Ther., 7:126-138; Gebhart et al. (2002) Bioconj. Chem., 13:937-944;
Astafieva et al. (1996) FEBS Lett., 389:278-280; Kuo, J. H. (2003)
Biotechnol. Appl. Biochem., 37:267-271).
[0004] Transduction of cells with viral vectors, such as adenoviral
and lentiviral, is also increased in the presence of amphiphilic
block copolymers (March et al. (1995) Hum. Gene Ther., 6:41-53;
Feldman et al. (1997) Gene Ther., 4:189-198; Van Belle et al.
(1998) Hum. Gene Ther., 9:1013-1024; Maillard et al. (2000) Gene
Ther., 7:1353-1361; Dishart et al. (2003) J. Mol. Cell. Cardiol.,
35:739-748).
[0005] All of the above studies demonstrate the ability of
amphiphilic block copolymers to assist in the transfer of DNA and
DNA-containing compositions into cells. However, such studies are
silent as to the effect of the polymers on gene expression of genes
already present in the cell.
SUMMARY OF THE INVENTION
[0006] The present invention broadly relates to compositions and
methods for increasing gene expression and protein yield.
[0007] According to one aspect of the invention, a method for
producing a protein is provided comprising the steps of 1)
providing cells comprising a heterologous nucleic acid encoding a
recombinant protein and 2) incubating the cells in media containing
at least one amphiphilic block copolymer. The cells may be
maintained in the media containing at least one amphiphilic block
copolymer or be removed to media which does not contain amphiphilic
block copolymers.
[0008] According to another aspect of the instant invention, the
protein produced by the methods of the instant invention may be
isolated.
[0009] In accordance with another aspect of the instant invention,
the amphiphilic polymer employed in the instant methods is a
copolymer comprising at least one block of poly(oxyethylene) and at
least one block of poly(oxypropylene).
[0010] According to another aspect of the instant invention, the
amphiphilic block copolymer of the instant method is a mixture of
at least two different amphiphilic block copolymers. For example,
the mixture can comprise, without limitation, Pluronic.RTM. P123
and Pluronic.RTM. P127, or Pluronic.RTM. P85 and Pluronic.RTM.
F88.
[0011] In still another embodiment of the instant invention, the
amphiphilic block copolymer of the instant methods is a mixture of
a Pluronic.RTM. copolymer and a polycation conjugated Pluronic.RTM.
copolymer. In a preferred embodiment, the mixture comprises
Pluronic.RTM. P123 and Pluronic.RTM. P123 conjugated to
polyethyleneimine. According to another aspect of the invention,
the cells are incubated in media comprising at least one
amphiphilic block copolymer for at least three hours. In a
particular embodiment, the cells are incubated for at least nine
hours in media comprising at least one amphiphilic block
copolymer.
[0012] According to another aspect of the invention, the
heterologous nucleic acid is stably incorporated into said
cells.
[0013] In still another embodiment of the invention, a method for
producing a protein in a host is provided comprising the steps of
1) providing a cell comprising a heterologous nucleic acid encoding
a recombinant protein, 2) incubating the cells in media containing
at least one amphiphilic block copolymer, and 3) introducing the
cells into a host. In a particular embodiment, the cells are
originally obtained from the host and the heterologous nucleic acid
is incorporated into the cells in vitro.
[0014] In another embodiment of the instant invention, a method is
provided for enhancing production an RNA comprising the steps of 1)
providing cells comprising a heterologous DNA encoding an RNA; and
2) incubating the cells in media containing at least one
amphiphilic block copolymer. In a particular embodiment, the
encoded for RNA is an siRNA.
[0015] In yet another embodiment of the invention, a composition is
provided comprising stably transformed cells, at least one
amphiphilic block copolymer, and nucleic acid free media.
[0016] In yet a further aspect of the invention, kits are provided
for performing the methods described above. Such kits comprise an
amphiphilic block polymer, reagents to transform a cell, and a
selection agent to isolate stably transformed cells. The kits may
further comprise frozen stocks of host cells.
BRIEF DESCRIPTIONS OF THE DRAWING
[0017] FIG. 1 is a graph of the luciferase present in Luc-NIH3T3
cells treated with media alone or media containing 0.03%, 0.1%, or
0.3% Pluronic.RTM. P85. The data is reported as mean.+-.standard
deviation (SD) (n=3). The statistical significance of treated
versus control samples is shown, (*)=p<0.05 and
(**)=p<0.005.
[0018] FIG. 2 is a graph of the ratio of luciferase mRNA to GAPDH
mRNA present in cells treated with media alone or media containing
Pluronic.RTM. L64 or P85. The cells assayed are NIH3T3, Luc-NIH3T3,
CHO, and CHO-luc cells.
[0019] FIG. 3 is a graph of the intensity of fluorescence of
GFP-C166 cells treated with media as a control (1) or treated with
0.1% Pluronic.RTM. P85 for 9 hours (2).
[0020] FIG. 4 is a graph of the fluorescence of cells treated with
media alone or with 0.1% Pluronic.RTM. P85 for 3, 6, or 9 hours.
The data is reported as mean.+-.SD (n=3). The statistical
significance of treated versus control samples is shown,
(**)=p<0.005.
[0021] FIG. 5A is a graph of the luciferase present in Luc-NIH3T3
cells treated with media alone or media containing 0.08%, 0.09%, or
0.1% Pluronic.RTM. L64. FIG. 5B is a graph of the luciferase per mg
of cellular protein present in Luc-NIH3T3 cells treated with media
alone or media containing 0.001%, 0.01%, 0.1%, 1%, or 2%
Pluronic.RTM. P123. FIG. 5C is a graph of the luciferase present in
Luc-NIH3T3 cells treated with media alone or media containing
0.001%, 0.01%, 0.1%, or 1% of a 1:8 mixture of Pluronic.RTM. L61
and Pluronic.RTM. F127. FIG. 5D is a graph of the luciferase
present per mg of cellular protein in Luc-NIH3T3 cells treated with
media alone or media containing 0.03% or 0.1% Pluronic.RTM. L35.
The data are reported as mean.+-.SD (n=3).
[0022] FIG. 6A is a graph of the luciferase present in Luc-NIH3T3
cells treated with media alone or media containing DNA, 0.03% P123,
0.8 .mu.M PEI-P123, P123/P123-PEI, or DNA and P123/P123-PEI. FIG.
6B is a graph of the total cellular protein of the above-identified
cells. The data is reported as mean.+-.SD (n=3).
DETAILED DESCRIPTION OF THE INVENTION
[0023] In accordance with the present invention, compositions and
methods for the production of a protein are provided. The method of
the instant invention can be employed to increase the production of
protein from a cell in any setting such as, without limitation,
cells in tissue culture on a laboratory scale and cells employed in
large-scale production of recombinant proteins, particularly
proteins having therapeutic value. Notably, the increase in protein
production is observed 1) without the addition of metal ions such
as copper ions and 2) in the presence of media containing protein
derived from a human or animal.
[0024] In a particular embodiment of the invention, increased
protein production is obtained by incubating a cell comprising a
nucleic acid encoding a recombinant protein in the presence of an
amphiphilic polymer. The cell of the instant invention can be
selected from the group consisting of bacteria cells (e.g., E.
coli), insect cells (e.g., SF9, Sf21, High five), yeast cells
(e.g., S. cerevisiae, P. pastoris), and mammalian cells (including
cells typically employed for mass production of recombinant
proteins such as, without limitation, baby hamster kidney cells
(BHK), Chinese hamster ovary cells (CHO), human embryonic kidney
cells (HEK), C127 cells, Cos cells). Preferably, the cell is a
mammalian cell. The cells may be a cell line or may be a part of a
tissue (e.g., a biopsy).
[0025] The cells of the instant method are preferably stably
transformed with a heterologous DNA. In a particular embodiment,
the cells are stably transfected.
[0026] According to one aspect of the invention, the nucleic acid
encoding the recombinant protein is under the control of a
promoter. In a particular embodiment, the promoter is heterologous
to the cell from which the recombinant protein is to be expressed.
In a particular embodiment, the promoter is selected from the group
consisting of the cytomegalovirus immediate early (CMV-IE)
promoter, the simian virus 40 (SV40) early promoter, herpes simplex
virus (HSV) thymidine kinase (tk) promoter, the RSV (Rous sarcoma
virus) promoter, and the Adenovirus major late promoter. In a
preferred embodiment, the promoter is the CMV-IE promoter.
[0027] Additionally, the genes encoding the recombinant protein of
the instant invention may be under the control of a transcription
element present in the cell such as, without limitation, elements
containing the binding sites for the transcription factors
NF-.kappa.B and p53.
[0028] Notably, the promoters of the invention may be the natural
promoters or may be allelic variants or derivatives thereof. For
example, the CMV promoter of the gWIZ plasmid (Gene Therapy
Systems, San Diego, Calif.) contains a modified CMV promoter which
allows for greater expression levels. Additionally, the promoters
may be associated with their natural enhancer elements or with
heterologous enhancer elements. The enhancer elements may be
wild-type, allelic variants, or derivatives thereof.
[0029] The cells to be treated by the methods of the instant
invention can be in a variety of settings. For example, the cells
can be in culture such as in a small scale tissue culture or in
culture conditions conducive to the large scale production of
recombinant proteins. Alternatively, in another embodiment, the
cells are treated ex vivo and then introduced into a patient.
[0030] The present invention also encompasses kits for use in
effecting enhanced expression of a protein or RNA of interest. Such
kits comprise an amphiphilic block polymer, reagents to transform a
cell, and selection media to isolate stably transformed cells. The
kits may further comprise frozen stocks of host cells and
instruction manuals. As used herein, an "instructional material"
includes a publication, a recording, a diagram, or any other medium
of expression which can be used to communicate the usefulness of
the composition of the invention for performing a method of the
invention. The instructional material of the kit of the invention
can, for example, be affixed to a container which contains a kit of
the invention to be shipped together with a container which
contains the kit. Alternatively, the instructional material can be
shipped separately from the container with the intention that the
instructional material and kit be used cooperatively by the
recipient.
I. DEFINITIONS
[0031] The following definitions are provided to facilitate an
understanding of the present invention:
[0032] As used herein, the term "polymer" denotes molecules formed
from the chemical union of two or more repeating units. The term
"block copolymer" most simply refers to conjugates of at least two
different polymer segments, wherein the polymer segments comprise
two or more adjacent units of the same kind.
[0033] As used herein, the term "lipophilic" refers to the ability
to dissolve in lipids.
[0034] As used herein, the term "hydrophilic" means the ability to
dissolve in water.
[0035] As used herein, the term "amphiphilic" means the ability to
dissolve in both water and lipids. Typically, an amphiphilic
compound or substance comprises a hydrophilic portion and a
lipophilic portion.
[0036] The term "polycation" means a polymeric molecule having a
plurality of positive charges distributed thereon. Examples of
polycations include, without limitation, polyamines (e.g.,
spermine, polyspermine, polyethyleneimine, polypropyleneimine,
polybutileneimine, polypentyleneimine, polyhexyleneimine and
copolymers thereof); copolymers of tertiary amines and secondary
amines; partially or completely quaternized amines; polyvinyl
pyridine; quaternary ammonium salts of a polycation; cationic
dendrimers such as polyamidoamines and polypropyleneimines;
aliphatic, heterocyclic or aromatic ionenes; polyamides; protamine
sulfate; polybrene; polylysine; polyarginine; and chitosan.
Polycations may also be a plurality of cationic repeating units of
the formula --N--R.sup.0, wherein R.sup.0 is a straight chain
aliphatic group of 2 to 6 carbon atoms, which may be substituted.
Each --N--R.sup.0-- repeating unit in a polycation can be the same
or different from another --N--R.sup.0-- repeating unit in the
polycation. The polycations may also be branched such as the
products of polycondensation or the condensation reactions between
polyamines containing at least 2 nitrogen atoms and alkyl halides
containing at least 2 halide atoms (including bromide or chloride).
An example of a branched polycation is polyethyleneimine
represented by the formula:
--(NHCH.sub.2CH.sub.2).sub.x[N(CH.sub.2CH.sub.2NH.sub.2)CH.sub.2CH.sub.2]-
.sub.y--, obtainable from, for example, Sigma (St. Louis, Mo.) and
BASF.
[0037] The term "recombinant protein" refers to a protein prepared
by recombinant DNA techniques, wherein generally, DNA encoding the
protein is inserted into a suitable expression vector which is in
turn used to transform a host cell to produce the protein. The
protein may or may not be heterologous, i.e. did not exist as part
of the cell prior to the transformation.
[0038] The term "isolated" as used in connection with an "isolated
protein" refers to a protein that has been sufficiently separated
from other proteins with which it would naturally be associated, so
as to exist in "substantially pure" form. "Isolated" is not meant
to exclude artificial or synthetic mixtures with other compounds or
materials, or the presence of impurities that do not interfere with
the fundamental activity, and that may be present, for example, due
to incomplete purification, addition of stabilizers, or compounding
into, for example, immunogenic preparations or pharmaceutically
acceptable preparations.
[0039] The term "substantially pure" refers to a preparation
comprising at least 50-60% by weight of a given material (e.g.,
nucleic acid, oligonucleotide, protein, etc.). More preferably, the
preparation comprises at least 75% by weight, and most preferably
90-95% by weight of the given compound. Purity is measured by
methods appropriate for the given compound (e.g. chromatographic
methods, agarose or polyacrylamide gel electrophoresis, HPLC
analysis, and the like).
[0040] "Nucleic acid" or a "nucleic acid molecule" as used herein
refers to any DNA or RNA molecule, either single or double stranded
and, if single stranded, the molecule of its complementary sequence
in either linear or circular form. In discussing nucleic acid
molecules, a sequence or structure of a particular nucleic acid
molecule may be described herein according to the normal convention
of providing the sequence in the 5' to 3' direction. With reference
to nucleic acids of the invention, the term "isolated nucleic acid"
is sometimes used. This term, when applied to DNA, refers to a DNA
molecule that is separated from sequences with which it is
immediately contiguous in the naturally occurring genome of the
organism in which it originated. For example, an "isolated nucleic
acid" may comprise a DNA molecule inserted into a vector, such as a
plasmid or virus vector, or integrated into the genomic DNA of a
prokaryotic or eukaryotic cell or host organism.
[0041] The term "promoters" or "promoter" as used herein can refer
to a DNA sequence that is located adjacent to a DNA sequence that
encodes a recombinant product. A promoter is preferably linked
operatively to an adjacent DNA sequence. A promoter typically
increases an amount of recombinant product expressed from a DNA
sequence as compared to an amount of the expressed recombinant
product when no promoter exists. A promoter from one organism can
be utilized to enhance recombinant product expression from a DNA
sequence that originates from another organism. For example, a
vertebrate promoter may be used for the expression of jellyfish GFP
in vertebrates. In addition, one promoter element can increase an
amount of recombinant products expressed for multiple DNA sequences
attached in tandem. Hence, one promoter element can enhance the
expression of one or more recombinant products. Multiple promoter
elements are well-known to persons of ordinary skill in the
art.
[0042] The term "enhancers" or "enhancer" as used herein can refer
to a DNA sequence that is located adjacent to the DNA sequence that
encodes a recombinant product. Enhancer elements are typically
located upstream of a promoter element or can be located downstream
of or within a coding DNA sequence (e.g., a DNA sequence
transcribed or translated into a recombinant product or products).
Hence, an enhancer element can be located 100 base pairs, 200 base
pairs, or 300 or more base pairs upstream or downstream of a DNA
sequence that encodes recombinant product. Enhancer elements can
increase an amount of recombinant product expressed from a DNA
sequence above the level of expression afforded by a promoter
element. Multiple enhancer elements are readily available to
persons of ordinary skill in the art.
[0043] "Natural allelic variants", "mutants" and "derivatives" of
particular sequences of nucleic acids refer to nucleic acid
sequences that are closely related to a particular sequence but
which may possess, either naturally or by design, changes in
sequence or structure. By closely related, it is meant that at
least about 75%, but often, more than 90%, of the nucleotides of
the sequence match over the defined length of the nucleic acid
sequence referred to using a specific SEQ ID NO. Changes or
differences in nucleotide sequence between closely related nucleic
acid sequences may represent nucleotide changes in the sequence
that arise during the course of normal replication or duplication
in nature of the particular nucleic acid sequence. Other changes
may be specifically designed and introduced into the sequence for
specific purposes, such as to change an amino acid codon or
sequence in a regulatory region of the nucleic acid. Such specific
changes may be made in vitro using a variety of mutagenesis
techniques or produced in a host organism placed under particular
selection conditions that induce or select for the changes. Such
sequence variants generated specifically may be referred to as
"mutants" or "derivatives" of the original sequence.
[0044] A "replicon" is any genetic element, for example, a plasmid,
cosmid, bacmid, phage or virus, which is capable of replication
largely under its own control. A replicon may be either RNA or DNA
and may be single or double stranded.
[0045] A "vector" is a replicon, such as a plasmid, cosmid, bacmid,
phage or virus, to which another genetic sequence or element
(either DNA or RNA) may be attached so as to bring about the
replication of the attached sequence or element.
[0046] An "expression vector" refers to a vector which facilitates
the expression of a polypeptide coding sequence in a host cell or
organism.
[0047] The term "gene" refers to a nucleic acid comprising an open
reading frame encoding a polypeptide, including both exon and
(optionally) intron sequences. The nucleic acid may also optionally
include non-coding sequences such as promoter or enhancer
sequences. The term "intron" refers to a DNA sequence present in a
given gene that is not translated into protein and is generally
found between exons.
[0048] The terms "stably transformed", "stably transfected,"
"stably incorporated" and variations thereof refer to the
incorporation of heterologous DNA into a cell, preferably into the
chromosomes of the cell, where it is expressed for at least the
remainder of the life time of the cell. Preferably, the
heterologous DNA is expressed by future generations of cells
derived from the originally transformed cell. Stable transformation
of a cell may be distinguished from transient expression of
heterologous DNA by a cell by, for example, the length of time the
recipient cell expresses the heterologous DNA. With transient
expression, the cell generally expresses the heterologous protein
for a few days or weeks until the vector containing the
heterologous DNA is lost from the cell. With stable transformation,
the heterologous DNA is expressed for longer periods of time and is
passed to later generations of the cells. The heterologous DNA is
typically a part of an expression vector. The heterologous DNA may
be introduced into the cell by any method such as, without
limitation, microinjection, transfection, lipofection,
transduction, transformation, and electroporation.
[0049] The term "reagents to transform a cell" refers to any
reagents that can be employed to assist the transformation of a
cell by any method such as by microinjection, transfection,
lipofection, transduction, transformation, and electroporation (see
generally, Ausubel et al., eds. Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., (1998)). Specific examples of
such reagents include, without limitation, vectors, buffers, and
solutions comprising CaPO.sub.4, Lipofectamine.TM., and/or
polyethyleneglycol (PEG).
[0050] The term "selection agent" refers to a substance, such as an
antibiotic, that interferes with the growth or survival of a host
cell that has not been successfully transformed with a gene that
confers resistance to the selection agent.
[0051] The term "gene therapy" refers to the transfer of genetic
material (e.g., DNA or RNA) of interest into a host (e.g., a human
or an animal) to treat or prevent a genetic or acquired disease or
condition. The genetic material of interest encodes a product,
particularly a protein, of therapeutic value whose production in
vivo is desired.
[0052] The term "ex vivo gene therapy" refers to the in vitro
transfer of genetic material (e.g., DNA or RNA) of interest into a
cell and then introducing the transformed cells into a host (see,
for example, U.S. Pat. No. 5,399,346). The cells may be isolated
from the host prior to transformation or may be obtained from a
different source such as a different animal or human donor.
[0053] The phrase small, interfering RNA (siRNA) refers to a double
stranded RNA molecule (RNA is usually single stranded) which
inhibits expression of its cognate mRNA (see, e.g. Ausubel et al.,
eds. Current Protocols in Molecular Biology, John Wiley and Sons,
Inc., (1998)). A short hairpin RNA molecule (shRNA) typically
consists of short inverted repeats separated by a small loop
sequence. Generally, one of the inverted repeats is complimentary
to the gene target. Additionally, the shRNA is typically processed
into an siRNA within the cell by endonucleases. siRNAs and shRNAs
specific for a protein of interest can downregulate its expression.
Such RNAs are typically expressed from RNA polymerase III promoters
such as, without limitation, the U6 and H1 promoters (see, e.g.,
Myslinski et al. (2001) Nucl. Acids Res., 29:2502-09).
II. AMPHIPHILIC POLYMERS
[0054] Amphiphilic polymers according to the instant invention are
preferably amphiphilic block copolymers. Amphiphilic block
copolymers are exemplified by the block copolymers having the
formulas:
##STR00001##
in which x, y, z, i, and j have values from about 2 to about 800,
preferably from about 5 to about 200, more preferably from about 5
to about 80, and wherein for each R.sup.1, R.sup.2 pair, as shown
in formula (IV) and (V), one is hydrogen and the other is a methyl
group. Formulas (I) through (III) are oversimplified in that, in
practice, the orientation of the isopropylene radicals within the B
block will be random. This random orientation is indicated in
formulas (IV) and (V), which are more complete. Such
poly(oxyethylene)-poly(oxypropylene) compounds have been described
by Santon (Am. Perfumer Cosmet. (1958) 72(4):54-58); Schmolka (Loc.
cit. (1967) 82(7):25-30), Schick, ed. (Non-ionic Suifactants,
Dekker, N.Y., 1967 pp. 300-371). A number of such compounds are
commercially available under such generic trade names as
"lipoloxamers", "Pluronics.RTM.," "poloxamers," and "synperonics."
Pluronic.RTM. copolymers within the B-A-B formula, as opposed to
the A-B-A formula typical of Pluronics.RTM., are often referred to
as "reversed" Pluronics.RTM., "Pluronic.RTM. R" or "meroxapol."
Generally, block copolymers can be described in terms of having
hydrophilic "A" and hydrophobic "B" block segments. Thus, for
example, a copolymer of the formula A-B-A is a triblock copolymer
consisting of a hydrophilic block connected to a hydrophobic block
connected to another hydrophilic block.
[0055] The "polyoxamine" polymer of formula (IV) is available from
BASF under the tradename Tetronic.RTM.. The order of the
polyoxyethylene and polyoxypropylene blocks represented in formula
(IV) can be reversed, creating Tetronic R.RTM., also available from
BASF (see, Schmolka, J. Am. Oil. Soc. (1979) 59:110).
[0056] Polyoxypropylene-polyoxyethylene block copolymers can also
be designed with hydrophilic blocks comprising a random mix of
ethylene oxide and propylene oxide repeating units. To maintain the
hydrophilic character of the block, ethylene oxide can predominate.
Similarly, the hydrophobic block can be a mixture of ethylene oxide
and propylene oxide repeating units. Such block copolymers are
available from BASF under the tradename Pluradot.TM..
[0057] The hydrophobic/hydrophilic properties of a given block
copolymer depends upon the ratio of the number of oxypropylene
groups to the number of oxyethylene groups. For a composition
containing a single block copolymer of
poly(oxyethylene)-poly(oxypropylene), for example, this
relationship, taking into account the molecular masses of the
central hydrophobic block and the terminal hydrophilic blocks, can
be expressed as follows:
n=(H/L)(1.32)
in which H is the number of oxypropylene units and L is the number
of oxyethylene units. In the general case of a block copolymer
containing hydrophobic B-type segments and hydrophilic A-type
segments, the hydrophobic-hydrophilic properties and
micelle-forming properties are related to the value n as defined
as:
n=(|B|/|A|)x(b/a)
where |B| and |A| are the number of repeating units in the
hydrophobic and hydrophilic blocks of the copolymer, respectively,
and b and a are the molecular weights for the respective repeating
units.
[0058] Selecting a block copolymer with the appropriate n value
depends upon the hydrophobic/hydrophilic properties of the specific
agent, or the composite hydrophilic/hydrophilic properties of a
mixture of agents to be formulated. One aspect of the present
invention involves utilizing a mixture of different
block-copolymers of poly(oxyethylene)-poly(oxypropylene) to achieve
a more specific hydrophobic-hydrophilic balance. For example, a
first block copolymer may have an n of 1.0 whereas a second may
have a value of 1.5. If material having an n of 1.3 is desired, a
mixture of one weight portion of the first block copolymer and 1.5
weight portion of the second block-copolymer can be employed.
[0059] Thus, a more generalized relationship for such mixtures can
be expressed as follows:
N=(1.32)[(H.sub.1m.sub.1)/((L.sub.1)(m.sub.1+m.sub.2))+(H.sub.2m.sub.2)/-
((L.sub.2)(m.sub.1+m.sub.2))]
in which H.sub.1 and H.sub.2 are the number of oxypropylene units
in the first and second block copolymers, respectively; L.sub.1 is
the number of oxyethylene units in the first block copolymer;
L.sub.2 is the number of oxyethylene units in the second block
copolymer; m.sub.1 is the weight proportion in the first
block-copolymer; and m.sub.2 is the weight proportion in the second
block copolymer.
[0060] An even more general case of a mixture of K block copolymers
containing hydrophobic B-type block copolymers and hydrophilic
A-type block copolymers, the N value can be expressed as
follows:
k
N = ( b / a ) i = 1 [ ( B i / A i ) , ( m i / M ) ]
##EQU00001##
where |A|.sub.i and |B|.sub.i are the numbers of repeating units in
the hydrophilic (A-type) and hydrophobic (B-type) blocks of the
i-th block copolymer, m is the weight proportion of this block
copolymers, M is the sum of weight proportions of all block
copolymers in the mixture
M = k i = 1 m i ##EQU00002##
and a and b are the molecular weights for the repeating units of
the hydrophilic and hydrophobic blocks of these block copolymers,
respectively.
[0061] If only one block copolymer of
poly(oxyethylene)-poly(oxypropylene) is utilized, N will equal n.
An analogous relationship will apply to compositions employing more
than two block copolymers of poly(oxyethylene)-poly(oxypropylene)
(EO-PO). Where mixtures of block copolymers are used, a value N
will be used, which value will be the weighted average of n for
each contributing copolymer, with the averaging based on the weight
portions of the component copolymers. The value N can be used to
estimate the micelle-forming properties of a mixture of copolymers.
The use of the mixtures of block copolymers enhances solubility and
prevents aggregation of more hydrophobic block copolymers in the
presence of the serum proteins.
[0062] A number of Pluronic.RTM. copolymers are designed to meet
the following formula:
##STR00002##
The ordinarily skilled artisan will recognize that the values of x,
y, and z will usually represent a statistical average and that the
values of x and z are often, though not necessarily, the same. The
characteristics of a number of Pluronic.RTM. copolymers
corresponding to formula (I) are as follows:
TABLE-US-00001 TABLE 1 Hydrophobe CMC Hydrophobe Copolymer Weight
(% w/v) percentage Pluronic .RTM. L61 1750 0.0003 90 Pluronic .RTM.
L64 1750 0.002 60 Pluronic .RTM. F68 1750 4-5 20 Pluronic .RTM. P85
2250 0.005-0.007 50 Pluronic .RTM. F127 4000 0.003-0.005 30
Pluronic .RTM. F108 3250 0.0035-0.007 20
[0063] These critical micelle concentrations (CMC) values were
determined by the surface tension method described in Kabanov et
al. (Macromolecules (1995) 28: 2303-2314).
[0064] These block copolymers can be prepared by the methods set
out, for example, in U.S. Pat. No. 2,674,619 and are commercially
available from BASF under the trademark Pluronic.RTM..
Pluronic.RTM. block copolymers are designated by a letter prefix
followed by a two or a three digit number. The letter prefixes (L,
P, or F) refer to the physical form of each polymer, (liquid,
paste, or flakeable solid). The numeric code defines the structural
parameters of the block copolymer. The last digit of this code
approximates the weight content of EO block in tens of weight
percent (for example, 80% weight if the digit is 8, or 10% weight
if the digit is 1). The remaining first one or two digits encode
the molecular mass of the central PO block. To decipher the code,
one should multiply the corresponding number by 300 to obtain the
approximate molecular mass in daltons (Da). Therefore Pluronic
nomenclature provides a convenient approach to estimate the
characteristics of the block copolymer in the absence of reference
literature. For example, the code `F127` defines the block
copolymer, which is a solid, has a PO block of 3600 Da
(12.times.300) and 70% weight of EO. The precise molecular
characteristics of each Pluronic.RTM. block copolymer can be
obtained from the manufacturer.
[0065] Additional specific poly(oxyethylene)-poly(oxypropylene)
block copolymers which can be used in practicing this invention
include the Pluronic.RTM. and Pluronic.RTM.-R block copolymers of
Table 2.
TABLE-US-00002 TABLE 2 Hydrophobe Hydro- Pluronic- Hydrophobe
Hydro- Pluronic .RTM. Weight phobe % R .RTM. Weight phobe % L31 950
90 10R5 1000 50 L35 950 50 10R8 1000 20 F38 900 20 12R3 1200 70 L42
1200 80 17R1 1700 90 L43 1200 70 17R2 1700 80 L44 1200 60 17R4 1700
60 L61 1750 90 17R8 1700 20 L62 1750 80 22R4 2200 60 L63 1750 70
25R1 2500 90 L64 1750 60 25R2 2500 80 P65 1750 50 25R4 2500 60 F68
1750 20 25R5 2500 50 L72 2050 80 25R8 2500 50 P75 2050 50 31R1 3100
90 F77 2050 30 31R2 3100 80 L81 2250 90 31R4 3100 60 P84 2250 60
P85 2250 50 F87 2250 30 F88 2250 20 L92 2750 80 F98 2750 20 L101
3250 90 P103 3250 70 P104 3250 60 P105 3250 50 F108 3250 20 L121
4000 90 L122 4000 80 L123 4000 70 F127 4000 30
[0066] Other specific poly(oxyethylene)-poly(oxypropylene) block
copolymers which can be included in compositions described herein
are the Tetronic.RTM. and Tetronic.RTM. R nonionic surfactants of
formula (IV) and (V), above, which are tetrafunctional block
copolymers derived from the addition of ethylene oxide and
propylene oxide to ethylenediamine. Tetronic.RTM. and Tetronic.RTM.
R copolymers include, without limitation, those set forth in Table
3.
TABLE-US-00003 TABLE 3 Tetronic .RTM. Form HLB Average MW 304
Liquid 16 1650 701 Liquid 3 3600 704 Liquid 15 5500 901 Liquid 3
4700 904 Liquid 15 6700 908 Solid 31 25000 1107 Solid 24 15000 1301
Liquid 2 6800 1307 Solid 24 18000 90R4 Liquid 7 7240 150R1 Liquid 1
8000
[0067] In selecting copolymers for use in the instant invention,
poly(oxyethylene)-poly(oxypropylene) block units making up the
first segment need not consist solely of ethylene oxide. Nor is it
necessary that all of the B-type segment consist solely of
propylene oxide units. Instead, in the simplest cases, for example,
at least one of the monomers in segment A may be substituted with a
side chain group.
[0068] In addition, the present invention can also be practiced
using diamine-linked polyoxyethylene-polyoxypropylene polymers of
formula:
##STR00003##
wherein the same number and sequence of polyether moieties extend
symmetrically from the second nitrogen, R* is an alkylene of about
2 to about 6 carbons, a cycloalkylene of about 5 to about 8 carbons
or phenylene, R.sup.1 and R.sup.2, either (a) both represent
hydrogen or (b) one represents hydrogen and the other represents
methyl, for R.sup.3 and R.sup.4 either (a) both are hydrogen or (b)
one is hydrogen and the other is methyl; if both of R.sup.3 and
R.sup.4 represent hydrogen, then one of R.sup.5 and R.sup.6
represents hydrogen and the other is methyl; and both of R.sup.5
and R.sup.6 represent hydrogen when R.sup.3 and R.sup.4 each
represent hydrogen. The polyoxyethylene-polyoxypropylene polymers
may be, for example, Pluronic.RTM. or Pluronic.RTM.-R.
[0069] Over 30 Pluronic.RTM. copolymers with different lengths of
hydrophilic ethylene oxide (N.sub.EO) and hydrophobic propylene
oxide (N.sub.PO) blocks are available from BASF Corp. (see, for
example, Table 2). These molecules are characterized by different
hydrophilic-lipophilic balance (HLB) and CMC (Kozlov et al. (2000)
Macromolecules, 33:3305-3313; see, for example, Table 3). The HLB
value reflects the balance of the size and strength of the
hydrophilic groups and lipophilic groups of the polymer (see, for
example, Attwood and Florence (1983) "Surfactant Systems: Their
Chemistry, Pharmacy and Biology," Chapman and Hall, New York) and
can be determined experimentally by, for example, the phenol
titration method of Marszall (see, for example, "Parfumerie,
Kosmetik", Vol. 60, 1979, pp. 444-448; Rompp, Chemistry Lexicon,
8th Edition 1983, p. 1750; U.S. Pat. No. 4,795,643). Notably, as
hydrophobicity increases, HLB decreases.
TABLE-US-00004 TABLE 4 Pluronic .RTM. MW .sup.(a) N.sub.PO .sup.(b)
N.sub.EO .sup.(b) HLB .sup.(a) CMC, .mu.M .sup.(c) L31 1100 17.1
2.5 5 1180 L35 1900 16.4 21.6 19 5260 F38 4700 31 L42 1630 8 L43
1850 22.3 12.6 12 2160 L44 2200 22.8 20.0 16 3590 L61 2000 31 4.5 3
110 L62 2500 34.5 11.4 7 400 L63 2650 11 L64 2900 30 26.4 15 480
P65 3400 17 F68 8400 29 152.7 29 480 L72 2750 7 P75 4150 17 F77
6600 25 L81 2750 42.7 6.2 2 23 P84 4200 43.4 38.2 14 71 P85 4600
39.7 52.3 16 65 F87 7700 39.8 122.5 24 91 F88 11400 39.3 207.8 28
250 L92 3650 50.3 16.6 6 88 F98 13000 44.8 236.4 28 77 L101 3800
58.9 8.6 1 2.1 P103 4950 59.7 33.8 9 6.1 P104 5900 61.0 53.6 13 3.4
P105 6500 56.0 73.9 15 6.2 F108 14600 50.3 265.4 27 22 L121 4400
68.3 10.0 1 1 L122 5000 4 P123 5750 69.4 39.2 8 44 F127 12600 65.2
200.4 22 2.8 10R5 1950 15 10R8 4550 19 12R3 1800 7 17R1 1900 3 17R2
2150 6 17R4 2650 12 17R8 7000 16 22R4 3350 10 25R1 2700 2 25R2 3100
4 25R4 3600 8 25R5 4250 10 25R8 8550 13 31R1 3250 1 31R2 3300 2
31R4 4150 7 .sup.(a) The average molecular weights and HLB provided
by the manufacturer (BASF Co.); .sup.(b) The average numbers of EO
and PO units were calculated using the average molecular weights of
the blocks; .sup.(c) Critical micelle concentration (CMC) values at
37.degree. C. were determined using pyrene probe (Kozlov et al.
(2000) Macromolecules, 33: 3305-3313).
[0070] Preferably, the amphiphilic polymer is a copolymer of
poly(oxyethylene) and poly(oxypropylene), more preferably the
amphiphilic polymer is a Pluronic.RTM. copolymer. Preferably, the
amphiphilic copolymer possesses an HLB of less than or equal to 20,
more preferably an HLB of less than or equal to 16, and most
preferably an HLB of between 8 and 16. Such Pluronics.RTM. include,
for example, Pluronic.RTM. P123, Pluronic.RTM. P103, Pluronic.RTM.
P85, Pluronic.RTM. L64, and others.
[0071] In a particular embodiment of the invention, the amphiphilic
block polymers are present in the media at a concentration ranging
from about 0.0001% to about 5%. In a particular embodiment, the
concentration of the Pluronic.RTM. copolymers ranges from about
0.1% to about 2%.
III. PROTEINS
[0072] The expression product of the invention may be an protein, a
peptide, or polypeptide. The compositions and methods of the
instant invention can be employed to increase the expression of any
protein.
[0073] The increased production of protein by the methods of the
instant invention will prove useful in the field of recombinant
protein expression, particularly in the production of large
quantities of therapeutic proteins, and especially from mammalian
cells. Indeed, for recombinantly produced proteins that are
intended for commercial use, in particular, it is desirable to
obtain a high level of expression of the desired protein from each
host cell. Increasing the amount of desired protein produced per
cell can reduce costs of production due to the decreased volume of
cells that must be grown to obtain a given amount of product, and
also can facilitate purification because the desired product makes
up a larger percentage of the total protein produced by the host
cells.
[0074] Some of the factors that may be considered when selecting a
protein expression system are (1) the success of expression of the
protein in various systems and (2) the requirement for
glycosylation and other post-translational modifications. Each of
these factors can be successfully accounted for by the system
described herein which provides for increased production of active
protein from mammalian cells, wherein post-translational
modifications may occur.
[0075] Exemplary proteins for use in the instant invention include,
without limitation, cytokines, enzymes, clotting factors (e.g.,
Factor VIII, Factor IX, angiostatin, tissue plasminogen activator
(tPA)), vaccines, antibodies (e.g., monoclonal antibodies), growth
factors and hormones (e.g., erythropoietin), insulin, hemoglobin,
alpha-1-antitrypsin (AAT), lactoferrin, cystic fibrosis
transmembrane conductase (CFTR), human protein C, anti-viral
agents, and interleukins (e.g., interleukin-2).
[0076] The following examples provide illustrative methods of
practicing the instant invention, and are not intended to limit the
scope of the invention in any way. While certain of the following
examples specifically recite a specific type of Pluronic.RTM. block
copolymer (e.g., Pluronic.RTM. P85), the use of any amphiphilic
polymer is within the scope of the instant invention.
Example 1
Increased Production of Luciferase
[0077] Luc-NIH3T3 cells were generated by transfecting NIH3T3 cells
with a 5:1 mixture of plasmids gWIZ.TM.-Luc and phCMV1 (both from
Gene Therapy Systems, San Diego, Calif.) using ExGen500 (Fermentas
Inc., Hanover, Md.) as described in Gebhart and Kabanov (J. Contr.
Release (2001) 73:1767-1775). Plasmid gWIZ.TM.-Luc contains the
gene encoding for luciferase under the control of a cytomegalovirus
(CMV) immediate early (IE) promoter/enhancer. Plasmid phCMV1
contains the G418 resistance gene. The luciferase expressing clones
(Luc-NIH3T3) were selected using standard G418 selection procedures
(Ausubel et al. (1992) Short Protocols in Molecular Biology, John
Wiley and Sons) and maintained in Dulbecco's Modified Eagle's
Medium (DMEM) containing 400 .mu.g/ml G418.
[0078] Luc-NIH3T3 cells were seeded in 24-well plates at
5.times.10.sup.4 cells per well. The cells were grown to about 70%
confluency and then exposed to various concentrations of
Pluronic.RTM. P85 for 3 hours. The cells were then washed and
incubated for 24 hours in 0.5 ml of DMEM with 10% fetal bovine
serum (FBS). The cells were then lysed in 100 .mu.l of 1.times.CCLR
(cell culture lysis reagent; Promega, Madison, Wis.) and the
luciferase activity was measured using the Luciferase Assay System
(Promega) as described in Gebhart and Kabanov (J. Contr. Release
(2001) 73:1767-1775). Total cellular protein in lysates was
measured with the BCA protein assay (Pierce, Rockford, Ill.). The
protein levels of the various lysates were within 20%.
[0079] As seen in FIG. 1, the presence of Pluronic.RTM. P85
resulted in a significant increase in the yield of luciferase from
the Luc-NIH3T3 cells. Indeed, treatment with increasing amounts of
Pluronic.RTM. P85 led to increased luciferase production. Notably,
Pluronic.RTM. P85 had no effect on the activity of luciferase as
the luminescence of cellular lysates increased at the same rate as
the protein levels of luciferase.
[0080] The effect of the Pluronic.RTM. copolymers was also tested
on different cell types to demonstrate that the effect was not
limited to NIH3T3 cells. Specifically, CHO (Chinese hamster ovary)
cells stably transfected with the luciferase gene (CHO-Luc) were
generated by the same methods as that used to generate the
Luc-NIH3T3 cells. The cells were incubated for 3 hours with either
Pluronic.RTM. L64 or P85. The mRNA levels of luciferase and the
housekeeping gene D-glyceraldehyde-3-phosphate dehydrogenase
(GADPH) were then obtained by a standard RT-PCR assay. As seen in
FIG. 2, the presence of either Pluronic.RTM. L64 or P85 led to a
significant increase in the production of luciferase compared to
cells not treated with a Pluronic.RTM., regardless of the cell
type. Furthermore, the increase in the ratio of luciferase to GAPDH
production suggests that the effect of the Pluronic.RTM. is
specific to heterologous or foreign promoters.
Example 2
Increased Production of Green Fluorescent Protein
[0081] GFP-C166 cells were generated by transfecting C166 cells
with plasmid pEGFPN1 (Clontech, Palo Alto, Calif.) using ExGen500
(Fermentas Inc., Hanover, Md.) as described in Gebhart and Kabanov
(J. Contr. Release (2001) 73:1767-1775). Plasmid pEGFPN1 contains
the gene encoding for green fluorescent protein (GFP) under the
control of a cytomegalovirus (CMV) immediate early (IE)
promoter/enhancer and contains the G418 resistance gene. The GFP
expressing clones (GFP-C166) were selected using standard
procedures (Ausubel et al. (1992) Short Protocols in Molecular
Biology, John Wiley and Sons) and maintained in Dulbecco's Modified
Eagle's Medium (DMEM) containing 400 .mu.g/ml G418.
[0082] GFP-C166 cells were seeded in 12 well plates at
8.times.10.sup.4 cells per well. The cells were allowed to grow to
about 70% confluency and were then exposed to Pluronic.RTM. P85 for
3, 6, or 9 hours. The cells were then incubated for 24 hours in 1
ml of DMEM with 10% FBS. Following the 24 hour incubation, the
cells were detached by trypsin, washed, and resuspended in
phosphate-buffered saline (PBS) with 1% FBS. 10,000 cells were then
analyzed for GFP fluorescence using a FACStar Plus.TM. flow
cytometer (Becton Dickinson, San Jose, Calif.) operating under
Lysis II (excitation--488 nm; emission filter--530.+-.30 nm).
[0083] As seen in FIG. 3, GFP-C166 cells treated for 9 hours with
Pluronic.RTM. P85 expressed significantly more GFP than cells that
were untreated. Notably, a similar timecourse was seen with L64 on
Luc-NIH3T3 cells. Furthermore, longer treatments yielded greater
GFP production (FIG. 4). Notably, similar effects were noted with
luciferase production wherein luciferase production was greatly
increased after 9 hours of exposure to L64.
[0084] Notably, Pluronic.RTM. P123 was also assayed for the ability
to increase the production of GFP from GFP-C166 cells. As with
Pluronic.RTM. P85, treatment with Pluronic.RTM. P123 resulted in
the increase production of P123, but required slightly higher
concentrations to produce a response with a significant increase
observed at a concentration of about 1%.
Example 3
Effect of Other Pluronic.RTM. Copolymers on Protein Expression
[0085] Pluronic.RTM. copolymers are comprised of different lengths
of hydrophilic (ethylene oxide (EO)) and hydrophobic (propylene
oxide (PO)) blocks (Lemieux et al. (2000) Gene Ther., 7:986-991;
Liaw et al. (2001) Gene Ther., 8:999-1004; Alakhov et al. (2001)
Expert Opin. Biol. Ther., 1:583-602; Gebhart et al. (2003)
Controlled Release Society, Glasgow, Scotland, UK; Pitard et al.
(2002) Human Gene Ther., 13:1767-1775). The Pluronics.RTM. L35,
L61, L64, F88, P85, P103, P123, F127, and a 1:8 mixture of
Pluronic.RTM. L61 and Pluronic.RTM. F127 (Lemieux et al. (2000)
Gene Ther., 7:986-991; Alakhov et al. (1999) Colloids Surfaces B:
Biointerfaces, 16:113-134) were tested for their ability to
increase production of luciferase as described in Example 1. The
Pluronic.RTM. copolymers were tested at a concentration of
0.3%.
[0086] Of the tested Pluronic.RTM. copolymers, the copolymers with
intermediate HLB values (i.e., 9-16) and a relatively large
hydrophobic block (30-69 PO units), such as P123, P103, L64, and
P85, were the most effective at increasing luciferase production
(see, for example, FIG. 1 and FIGS. 5A and 5B). A 1:8 mixture of
Pluronic.RTM. L61 and Pluronic.RTM. F127 (Lemieux et al. (2000)
Gene Ther., 7:986-991; Alakhov et al. (1999) Colloids Surfaces B:
Biointerfaces, 16:113-134), was also effective at increasing
luciferase production (FIG. 5C). Hydrophilic Pluronics.RTM. F127
(HLB 22) and F88 (HLB 28); and Pluronics.RTM. comprising a
relatively short PO block such as L35 (16 PO units) were effective
at increasing luciferase production, but to a lesser extent than,
for example, Pluronic.RTM. P85 (see, for example, FIG. 5D).
[0087] Notably, the pattern of the most active block copolymers
observed is consistent with the increased potency of these
copolymers to incorporate into the hydrophobic portions of cellular
membranes, induce structural changes (e.g., membrane fluidization),
and traverse the membrane to gain access to the cytosol (Batrakova
et al. (2003) J. Pharmacol. Exp. Ther., 304:278-280).
Example 4
Effects of Modification on Ability of Pluronic.RTM. P123 to
Increase Protein Production
[0088] Pluronic.RTM. P123 and polyethyleneimine (PEI) conjugated
Pluronic.RTM. P123 (P123-PEI) have been previously optimized for
the maximal expression of genes delivered in vitro and in vivo
(Gebhart and Kabanov (2001) J. Contr. Release, 13:937-944; Nguyen
et al. (2000) Gene Ther., 7:126-138; Ochietti et al. (2002) Gene
Ther., 9:939-945; Gebhart et al. (2002) Bioconj. Chem.,
13:937-944). P123-PEI was prepared as described in Nguyen et al.
(Gene Ther. (2000) 7:126-138). Briefly, 0.5 mmol of
1,19-carbonyldiimidazole-activated P123 were reacted with 2.5 mmol
of PEI, 2 kDa, in 30 ml of 0.2M carbonate buffer, pH 8.0. After 24
hours the reaction mixture was dialyzed twice and then lyophilized.
The yield of PEI-P123 was 65%.
[0089] Pluronic.RTM. P123, PEI-P123, and a 9:1 (wt) mixture of
Pluronic.RTM. P123 and PEI-P123 (P123/P123-PEI) were tested for
their ability to increase production of luciferase as described in
Example 1. As seen in Table 5, the P123/P123-PEI was the most
effective at increasing production of luciferase, increasing the
amount of expression almost 10-fold.
TABLE-US-00005 TABLE 5 Luciferase Formulation (pg/mg) Media 320
.+-. 14 P123 (0.03%) 545 .+-. 72* P123-PEI (0.8 .mu.M) 650 .+-. 206
P123/P123-PEI (0.8 .mu.M) 2823 .+-. 381** Values are mean .+-. SD
(n = 3). *= p < 0.05; **= p < 0.005.
[0090] The effect of exogenous DNA added to the cells with
Pluronics.RTM. P123 and P123-PEI was also studied. As seen in FIG.
6A, adding DNA (1 .mu.g/ml) to the P123/P123-PEI caused the loss of
the increased production of luciferase seen with the addition of
P123/P123-PEI alone.
[0091] The effect of P123 and P123-PEI on global cellular protein
expression was also investigated. As seen in FIG. 6B, P123 and
P123-PEI, alone or in combination, were ineffective at increasing
the total cellular protein levels present in cellular lysates. This
result further suggests that the specificity of the polymers to
increase expression from heterologous or foreign promoters.
[0092] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
* * * * *