U.S. patent application number 10/278751 was filed with the patent office on 2008-04-17 for compositions and methods for polynucleotide delivery.
Invention is credited to Fred E. Cohen, Nathalie Dubois-Stringfellow, Varavani Dwarki, Michael A. Innis, John E. Murphy, Tetsuo Uno, Ronald N. Zuckermann.
Application Number | 20080089938 10/278751 |
Document ID | / |
Family ID | 21817661 |
Filed Date | 2008-04-17 |
United States Patent
Application |
20080089938 |
Kind Code |
A9 |
Zuckermann; Ronald N. ; et
al. |
April 17, 2008 |
Compositions and methods for polynucleotide delivery
Abstract
This invention relates compositions and methods for increasing
the uptake of polynucleotides into cells. Specifically, the
invention relates to vectors, targeting ligands, and polycationic
agents. The polycationic agents are capable of (1) increasing the
frequency of uptake of polynucleotides into a cell, (2) condensing
polynucleotides; and (3) inhibiting serum and/or nuclease
degradation of polynucleotides.
Inventors: |
Zuckermann; Ronald N.;
(Berkeley, CA) ; Dubois-Stringfellow; Nathalie;
(Berkeley, CA) ; Dwarki; Varavani; (Alameda,
CA) ; Innis; Michael A.; (Moraga, CA) ;
Murphy; John E.; (Oakland, CA) ; Cohen; Fred E.;
(San Francisco, CA) ; Uno; Tetsuo; (San Francisco,
CA) |
Correspondence
Address: |
Novartis Vaccines and Diagnostics, Inc.;Intellectual Property
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20030185890 A1 |
October 2, 2003 |
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Family ID: |
21817661 |
Appl. No.: |
10/278751 |
Filed: |
October 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09620925 |
Jul 21, 2000 |
6468986 |
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10278751 |
Oct 22, 2002 |
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08910647 |
Aug 13, 1997 |
6251433 |
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09620925 |
Jul 21, 2000 |
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60023867 |
Aug 13, 1996 |
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Current U.S.
Class: |
424/484 ;
514/1.1; 514/44R; 530/324; 530/325; 530/326; 530/327 |
Current CPC
Class: |
A61K 38/1816 20130101;
A61P 11/00 20180101; A61K 47/645 20170801; A61P 31/12 20180101;
A61P 19/02 20180101; A61K 48/00 20130101; A61P 25/00 20180101; A61P
3/10 20180101; A61P 29/00 20180101; C07K 7/08 20130101; A61P 35/00
20180101; C12N 15/87 20130101; A61P 3/06 20180101; A61P 31/00
20180101; C07K 14/001 20130101; A61P 7/00 20180101; A61P 17/06
20180101; A61K 48/0041 20130101; A61P 9/10 20180101; A61K 38/2264
20130101; C12N 2810/858 20130101; A61P 7/04 20180101; A61P 7/06
20180101; A61K 48/0008 20130101 |
Class at
Publication: |
424/484 ;
514/008; 530/324; 530/325; 530/327; 530/326; 514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07K 7/08 20060101 C07K007/08; C07K 14/00 20060101
C07K014/00; A61K 9/14 20060101 A61K009/14 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 13, 1997 |
US |
PCT/US97/14465 |
Claims
1. A polycationic agent having the following formula: ##STR36##
wherein n is an integer from 10 to 100; R.sub.1, R.sub.2, and
R.sub.3 for each monomer, ##STR37## are independently selected from
moieties having a molecular weight from 1 to 200 daltons; Ta and Tc
are terminating groups; R.sub.1 is not hydrogen for at least one
monomer; wherein said polycationic agent comprises at least 25%
positively charged monomers, excluding the terminal groups, and
wherein said polycationic agent exhibits a net positive electrical
charge at a physiological pH.
2. The polycationic agent according to claim 1, wherein said
polycationic agent comprises repeating trimers.
3. The polycationic agent according to claim 2, wherein two R.sub.1
groups in each trimer are neutral moieties and one R.sub.1 group in
each trimer is a cationic moiety.
4. The polycationic agent according to claim 1, wherein R.sub.1 is
selected from the group consisting of positively charged,
negatively charged and neutral moieties.
5. The polycationic agent according to claim 1, wherein R.sub.1 is
selected from substituents found on amino acids.
6. The polycationic agent of claim 1, wherein R.sub.1 is selected
from the group consisting of aromatic and aliphatic groups.
7. The polycationic agent according to claim 1, wherein at least
one R.sub.1 is selected from the group consisting of alkylammonium,
aminoalkyl, guanidinoalkyl, amidinoalkyl, aminocyclohexyl,
piperidyl, guanidinobenzyl, amidinobenzyl, pyridylmethyl,
aminobenzyl, alkyphenyl, indolylalkyl, alkoxyphenylalkyl,
halophenylalkyl, and hydroxyphenylalkyl.
8. The polycationic agent according to claim 3, wherein said
cationic moeity is aminoethyl.
9. The polycationic agent according to claim 8, wherein said
neutral moieties are selected from the group consisting of
phenethyl, benzyl, phenylpropyl, (R) alpha-methylbenzyl, (S)
alpha-methylbenzyl, methoxyphenethyl, and chlorophenethyl.
10. The polycationic agent of claim 1, wherein R.sub.1 and R.sub.3
are both hydrogen for at least one monomer.
11. The polycationic agent of claim 10 wherein n is 36.
12. The polycationic agent of claim 10, wherein n is 24.
13. The polycationic agent of claim 10, wherein n is 18.
14. The polycationic agent of claim 10, wherein n is 12.
15. The polycationic agent of claim 8, wherein Ta and Tc are
terminal groups selected from the group consisting of polypeptide,
lipid, lipoprotein, vitamin, hormone, polyakylene glycol, and
saccharide.
16. A composition comprising: (i) a polynucleotide; and (ii) the
polycationic agent of claim 1, wherein said polycationic agent is
capable of mediating entry of polynucleotides into a cell.
17. A pharmaceutical composition comprising: (i) a pharmaceutically
acceptable carrier; (ii) a therapeutically effective amount of
polynucleotides; and (iii) an amount effective to neutralize said
polynucleotides of the polycationic agent of claim 1 wherein said
polycationic agent is capable of mediating entry of polynucleotides
into a cell.
18. A method of complexing polynucleotides with a polycationic
agent comprising: (i) providing a polynucleotide; and (ii)
contacting said polynucleotide with the polycationic agent of claim
1, wherein said polycationic agent is capable of mediating entry of
polynucleotides into a cell.
19. A method of condensing polynucleotides, said method comprising:
contacting a polynucleotide with a condensing amount of the
polycationic agent of claim 1, wherein said condensing amount is an
amount of polycationic agent sufficient to reduce the size of said
polynucleotide.
20. A method of inhibiting serum degradation of polynucleotides,
said method comprising contacting a polynucleotide with the
polycationic agent of claim 1 wherein said polycationic agent is
present in an amount effective to inhibit serum degradation by at
least 10 minutes.
21. A composition comprising: (i) a lipoprotein; (ii) a
polynucleotide binding molecule; and (iii) a polynucleotide,
wherein said composition is capable of increasing the frequency of
polynucleotide uptake into a cell.
22. The composition of claim 21, wherein the lipoprotein is
selected from the group consisting of high density lipoprotein,
intermediate density lipoprotein, low density lipoprotein, and very
low density lipoprotein.
23. The composition of claim 21, wherein the lipoprotein a mutant,
fragment or fusion of the protein selected from the group
consisting of high density lipoprotein, intermediate density
lipoprotein, low density lipoprotein, and very low density
lipoprotein.
24. The composition of claim 21, wherein the lipoprotein is
acetylated low density lipoprotein.
25. A pharmaceutical composition comprising (a) a therapeutically
effective amount of a polynucleotide; (b) a polynucleotide binding
molecule in an amount effective to neutralize the negative charge
of said polynucleotide; and (c) a therapeutically effective amount
of lipoprotein.
26. The pharmaceutical composition of claim 26, wherein said
polynucleotide is a polycationic agent.
27. A method of producing a composition for facilitating entry of a
polynucleotide into a cell said method comprising: (i) providing a
polynucleotide (ii) providing a polynucleotide binding molecule in
an amount effective to neutralize said polynucleotide; (ii)
contacting said polynucleotide with said polynucleotide binding
molecule to form a complex; (iii) providing a lipoprotein; then
(iv) contacting the complex with said lipoprotein.
28. A method of increasing the frequency of polynucleotide uptake
into a cell said method comprising (i) providing a composition that
comprises (a) a therapeutically effective amount of a
polynucleotide; (b) a polynucleotide binding molecule in an amount
effective to neutralize said polynucleotide; and (c) an effective
amount lipoprotein; then (ii) contacting said composition to said
cell.
29. A method of increasing the frequency of polynucleotide uptake
into a cell said method comprising: (i) providing a composition
that comprises (a) a polynucleotide; and (b) the polycationic agent
of claim 1 in an amount effective to neutralize the negative charge
of said polynucleotide; then (ii) contacting said composition to
said cell.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S.
provisional patent application No. 60/023,867, filed Aug. 13, 1996,
which is incorporated herein by reference and to which priority is
claimed under 35 U.S.C. .sctn.120.
DESCRIPTION
[0002] 1. Field of the Invention
[0003] This invention relates to compositions and methods for
increasing the uptake of polynucleotides into cells. Specifically,
the invention relates to vectors, targeting ligands, and
polycationic agents. The polycationic agents are capable of (1)
increasing the frequency of uptake of polynucleotides into a cell,
(2) condensing polynucleotides; and (3) inhibiting serum and/or
nuclease degradation of polynucleotides.
[0004] 2. Background of the Invention
[0005] Polycations, such as polylysine, have been used to
facilitate delivery of nucleic acids to cell interior. Both in
vitro and in vivo applications have taken advantage of this
property. See, for example, Gao et al., 1996, Biochem.
35:1027-1036.
[0006] Polynucleotides, typically DNA, may be taken into a cell by
a receptor-mediated endocytosis pathway, a cellular mechanism which
internalizes specific macromolecules. In general, complexes
designed to be delivered in this fashion contain nucleic acids
encoding the gene of interest and a polycationic agent, which acts
as a DNA-binding carrier and both neutralizes the charge on the
nucleic acids and condenses it.
[0007] Condensation facilitates entry of nucleic acids into cell
vesicle systems by simulating a macromolecular structure. For
example, polylysine condenses DNA into a toroid or doughnut-like
structure. (Wagner et al., 1991, Proc. Natl. Acad. Sci.
88:4255-4259).
[0008] Polycations previously utilized for nucleic acid delivery to
cell interiors include polylysine, protamines, histones, spermine,
spermidine, polyornithine, polyargnine, and putrescine.
[0009] All publications mentioned herein are incorporated herein by
reference for the purpose of disclosing and describing features of
the invention for which the publications are cited in connection
with.
SUMMARY OF THE INVENTION
[0010] An embodiment of the invention is a vector for expression of
polypeptides. The vector of the instant invention comprises: (i) an
Epstein Barr Virus (EBV) origin of replication; (ii) a
polynucleotide encoding an EBV origin binding protein; (iii) an
enhancer; (iv) a promoter; and (v) a terminator. Polynucleotides
encoding a desired polypeptide, such as erythropoietin or leptin
can be inserted into the vector. Also, ribozyme and antisense
polynucleotides can also be inserted into the vector.
[0011] One embodiment of the invention is a composition capable of
targeting a polynucleotide to a specific cell type. The composition
comprises: (i) a lipoprotein; (ii) a polynucleotide binding
molecule; and (iii) a polynucleotide.
[0012] Another embodiment of the invention is a method of
increasing the frequency of uptake of polynucleotides into a cell
by contacting a cell with a composition comprising: (i) a
lipoprotein, (ii) a polynucleotide binding molecule; and (iii) a
polynucleotide.
[0013] Yet another embodiment of the invention is a method of
increasing the frequency of uptake of polynucleotides into a
specific cell type by contacting a population of cells with a
composition comprising (i) a lipoprotein, (ii) a polynucleotide
binding molecule; and (iii) a polynucleotide.
[0014] One embodiment of the invention is a polycationic agent
exhibiting a net positive electrical charge at physiological pH
with the following formula: ##STR1## where Ta and Tc are
terminating groups. A preferred subset of these compounds is the
set where R.sub.2 is hydrogen. Even more preferred are polymers
comprising at least one unnatural amino acid. Also preferred are
polymers where R.sub.2 and R.sub.3 are hydrogen and R.sub.1 is not
hydrogen, also referred to as poly N-substituted glycines or "poly
NSGs."
[0015] Another embodiment is a neutral polymer exhibiting no net
positive or negative electrical charge at physiological pH with the
following formula: ##STR2## where Ta and Tc are terminating groups.
A preferred subset of these compounds is the set where R.sub.2 is
hydrogen. Even more preferred are polymers comprising at least one
unnatural amino acid. Also preferred are polymers where R.sub.2 and
R.sub.3 are hydrogen and R.sub.1 is not hydrogen, also referred to
as poly N-substituted glycines or "poly NSGs."
[0016] The instant polycationic agents and neutral polymers are
capable of neutralizing the electrical charge of nucleic acids. A
subset of these compounds are capable of (1) condensing the
structure of polynucleotides and/or (2) protecting polynucleotides
from serum and/or nuclease degradation.
[0017] Yet another embodiment of the invention are polycationic
agents and neutral polymers that (1) target binding of nucleic
acids to cell surfaces, (2) trigger cell membrane destabilization;
(3) exhibit endosome buffering capacity; (4) trigger endocytosis;
(5) help trigger the release of polynucleotide/lipid complexes from
endosomes or (6) nuclear tropism.
[0018] Another embodiment of the invention is a composition
comprising a polynucleotide of interest and an effective amount of
the polycationic agent to neutralize the charge of the
polynucleotide. Optionally, the composition includes a ligand which
directs the complex to particular cells expressing a ligand-binding
partner, and/or an endosomolytic agent, which serves to cause
disruption of the endosome containing the complex.
[0019] Another embodiment of the invention is a method of
condensing nucleic acids by providing an effective amount of the
polycationic agents or neutral polymers of the invention and
contacting the agent with the desired polynucleotides.
[0020] Also an embodiment of the invention is a method of
inhibiting serum and/or nuclease degradation of nucleic acids by
providing an effective amount of the the polycationic agents or
neutral polymers of the inventions and contacting the agent with
the desired nucleic acids.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic of a two-step monomer assembly
reaction scheme.
[0022] FIG. 2 is a schematic of a three-step monomer assembly
reaction scheme.
[0023] FIG. 3 is a plasmid map of vector pCMVKmITR-EPI.
[0024] FIG. 4 is a plasmid map of vector CMVkm2.
[0025] FIG. 5 is a plasmid map of vector pCMV-KM-cmEPO.
[0026] FIG. 6 is a plasmid map of vector CMVKmLeptinWt.
[0027] FIG. 7A illustrates transfection efficiencies for a diverse
set of polycationic agents. The polycatonic agents were formulated
with DNA at a 2:1, + to - charge ratio and added to either HT1080
(solid bar) or COS (stippled bar) in the presence of 10% serum.
Luciferase activity was analyzed 48 hours post-transfection. Total
cell protein was measured using a Pierce BCA assay and luciferase
activity was normalized against total cell protein.
[0028] FIG. 7B illustrates the effect of oligomer length on
transfection efficiency for polycationic agents having different
numbers of the same repeating trimer motif. For both A and B each
data point represents the average of 2 experiments.
[0029] FIGS. 8(A-C) shows RZ145-1 peptoid-mediated transfection and
transfection mediated by commercially available cationic liposome
preparations. RZ145-1 or the indicated lipid was formulated and
added to cells in the presence (solid bar) or absence (stippled
bar) of 10% serum. Luciferase and total cell protein activity were
measured 48 hours after initial transfection. Cells lines are (FIG.
8A) 293 human embryonic kidney cells, (FIG. 8B) HT1080 human
fibrosarcoma cells, and (FIG. 8C) NIH03T3 mouse fibroblast cells.
Each data point represents the average+strandard error of the mean
of three transfections.
[0030] FIG. 9 illustrates the effect of chloroquine on transfection
with RZ145-1 in (A) 293 cells and (B) HT1080 cells. The cells were
transfected in the presence (black bar) or absence (stippled bar)
of 100 uM chloroquine. Cells were lysed 48 hours post transfection
and luciferase activity and total protein content were
measured.
DETAILED DESCRIPTION
Definitions
[0031] "Lipoproteins" refers to polypeptides that are associated
non-covalently with lipids in the bloodstream and are capable of
binding to cellular receptors. Preferably, lipoproteins are those
involved with transport and storage of lipids. Such proteins
include, for example, chylomicrons, low density lipoprotein (LDL),
very low density lipoprotein (VLDL), intermediate density
lipoprotein (IDL), and high density lipoprotein (HDL). Also,
included in the term are mutants, fragments, or fusions of the
naturally occurring lipoproteins. Also, modifications of naturally
occurring lipoproteins can be used, such as acetylated LDL.
[0032] Mutants, fragments, fusions, or modifications of the
naturally occurring lipoproteins are amino acid sequences that
exhibit substantial sequence identity to naturally occurring
lipoproteins or a fragment thereof. These polypeptides will retain
more than about 80% amino acid identity; more typically, more than
about 85%; even more typically, at least 90%. Preferably, these
polypeptides will exhibit more than about 92% amino acid sequence
identity with naturally occurring lipoproteins or fragment thereof;
more preferably, more than about 94%; even more preferably, more
than about 96%; even more preferably, more than about 98%; even
more preferably, more than about 99%. All of these polypeptides
exhibit receptor binding properties of naturally occurring
lipoproteins. Usually, such polypeptides exhibit at least about 20%
receptor binding of naturally occurring lipoproteins. More
typically, the polypeptides exhibit at least about 40%, even more
typically the polypeptides exhibit at least about 60%; even more
typically, at least about 70%; even more typically, at least about
80%; even more typically, at least about 85%; even more typically,
at least about 90%; even more typically, at least about 95%
receptor binding of the naturally occurring lipoproteins.
[0033] "Polynucleotide binding molecule" refers to those compounds
that associate with polynucleotides, and the association is not
sequence specific. For example, such molecules can (1) aid in
neutralizing the electrical charge of polynucleotide, or (2)
facilitate condensation of nucleotides, or (3) inhibit serum or
nuclease degradation.
[0034] "Polycationic agent" refers generally to a polymer
comprising positively-charged single units, although some
non-positively charged units may be present in the polymer. The
instant agents exhibit a net positive charge under physiologically
relevant pH. Such agents are capable of neutralizing the charge of
nucleic acids and can exhibit additional properties, such as
condensation and/or serum protection of nucleic acids. Preferably,
the agents comprises both amino acids and NSGs as monomeric units;
also, preferred are agents comprising NSGs as monomeric units.
[0035] "Physiologically relevant pH" varies somewhat between in
vitro and in vivo applications. Typically, the physiological pH is
at least 5.5; more typically, at least 6.0; even more typically, at
least 6.5. Usually, physiologically relevant pH is no more than
8.5; more usually, no more than 8.0; even more usually, no more
than 7.5.
[0036] "Polynucleotide" or "nucleic acid" refers to DNA, RNA,
analogues thereof, peptide-nucleic acids, and DNA or RNA with
non-phosphate containing nucleotides. Additionally, these nucleic
acids may be single-stranded, double-stranded, or chimeric single-
or double-stranded molecules.
[0037] The term "oligomer" includes polymers such as poly NSGs,
produced by the submonomer process described herein and also in
Zuckermann et al., supra. includes polymers, copolymers, and
interpolymers of any length. More specifically, oligomers may
comprise a single repeating monomer, two alternating monomer units,
two or more monomer units randomly and/or deliberately spaced
relative to each other. Regardless of the type of polyamide
produced, the polyamide of the invention may be produced by the
same general procedure which includes repeating a two-step or three
step cycle wherein a new monomer unit is added in each cycle until
an oligomer of desired length is obtained. The oligomer is
preferably 2-100 monomers, more preferably 2-50, or 18-28 monomers
or 24 to 48 monomers in length.
[0038] The term "frequency of uptake of polynucleotides into a
cell" refers to an increase in the amount of polynucleotides
actually taken up by a cell relative to the amount actually
administered to the cell. The frequency of uptake of
polynucleotides into a cell is increased if it is greater than the
frequency of uptake of naked polynucleotides. For example, using in
vitro transfection methods, uptake of naked polynucleotides into
mammalian cells is not usually detectable over background. Some
frequency of uptake, however, can be detected when naked
polynucleotides are delivered in vivo. The frequency of uptake in
vivo and in vitro depends on the tissue type. The frequency of
uptake can be measured by known methods for detecting the presence
of polynucleotides, such as Northern, Southern, or Polymerase Chain
Reaction (PCR) techniques.
[0039] Usually, a composition or compound is capable of increasing
the frequency of polynucleotide uptake into a cell if it induces a
frequency of uptake that is at least 10% greater than the frequency
of naked polynucleotide uptake; more usually, at least 15% greater;
even more usually, 20% greater; even more usually, at least 30%;
and up to 40% to 100% greater, and even 1,000% and 10,000%
greater.
[0040] "Naked polynucleotides" refers to polynucleotides that are
substantially free from any delivery vehicle that can act to
facilitate entry into the cell. For example, polynucleotides are
naked when free from any material which promotes transfection, such
as liposomal formulations, charged lipids, such as Lipofectin.RTM.
or precipitating agents such as Ca.sub.3(PO.sub.4).sub.2.
[0041] "Effective amount to increase the frequency of
polynucleotide uptake into cells" refers to an amount that induces
a frequency of polynucleotide uptake into a cell that is at least
10% greater than the frequency of naked polynucleotide uptake; more
usually, at least 15% greater; even more usually, 20% greater; even
more usually, at least 30%; even more usually, at least 40%.
[0042] "Effective amount to neutralize nucleic acids" refers to the
amount used to neutralize at least 10% of the electrical charge of
the nucleic acid composition; more preferably; the amount refers to
the amount used to neutralize at least 40%; even more preferably,
the amount to neutralize 50% of the electrical charge; even more
preferably, the amount to neutralize 60% of the electrical charge;
even more preferably, the amount to neutralize 70% of the
electrical charge; even more preferably, the amount to neutralize
80% of the electrical charge; and most preferably, at least 90% of
the electrical charge of the nucleic acid composition of
interest.
[0043] "Condensation of nucleic acids" occurs when the polycationic
agent that is combined with nucleic acids, neutralizes the
electrical charge of the nucleic acids and causes it to assume a
reduced structure relative to uncomplexed nucleic acids.
Preferably, condensation reduces the structure of nucleic acids to
a size that can be internalized by structures present on cell
surface membranes. Condensation can be measured by determining the
charge of the nucleic acid/polycationic agent by gel
electrophoresis, for example. Alternatively, an effective amount to
condense nucleic acids can also be measured by the final size of
the polycationic agent/nucleic acid complex.
[0044] "Effective amount to inhibit serum or nuclease degradation
of nucleic acids" refers to the amount used to increase the
half-life of the polynucleotide when exposed to serum and/or
nucleases by at least 5 minutes as compared the uncomplexed nucleic
acids; more preferably, the amount used to inhibit degradation by
at least 10 minutes; even more preferably, the amount used to
inhibit degradation by at least 30 minutes; even more preferably,
the amount used to inhibit degradation by at least 45 minutes; even
more preferably, the amount used to inhibit degradation by at least
60 minutes; even more preferably, the amount used to inhibit
degradation by at least 90 minutes; and more preferably, the amount
used to inhibit degradation by at least 120 minutes.
[0045] A composition containing A is "substantially free of" B when
at least 85% by weight of the total A+B in the composition is A.
Preferably, A comprises at least about 90% by weight of the total
of A+B in the composition, more preferably at least about 95% or
even 99% by weight.
[0046] "Immunogenicity" refers to the ability of a given molecule
or a determinant thereof to induce the generation of antibodies
with binding capacity to the molecule upon administration in vivo,
to induce a cytotoxic response, activate the complement system,
induce allergic reactions, and the like. An immune response may be
measured by assays that determine the level of specific antibodies
in serum, by assays that determine the presence of a serum
component that inactivates the polycationic agent/nucleic acid
complex or conjugated gene delivery vehicle, or by other assays
that measure a specific component or activity of the immune system.
As discussed in more detail below, low immunogencity may be
established by these assays. The terms "low immunogenicity,"
"reduced immunogenicity," "lowered immunogenicity" and similar
terms are intended to be equivalent terms.
[0047] An "origin of replication" is a polynucleotide sequence that
initiates and regulates replication of polynucleotides, such as an
expression vector. The origin of replication behaves as an
autonomous unit of polynucleotide replication within a cell,
capable of replication under its own control. With certain origins
of replication, an expression vector can be reproduced at a high
copy number in the presence of the appropriate proteins within the
cell. Examples of origins are the 2>and autonomously replicating
sequences, which are effective in yeast; and the viral T-antigen,
effective in COS-7 cells.
GENERAL METHODS AND DETAILED DESCRIPTION
Polynucleotides
[0048] Polynucleotides used in the instant invention can be used to
express desired polypeptides, or can be, in themselves,
therapeutic, such as ribozymes or antisense polynucleotides. Such
polynucleotides can be used in in vitro, ex vivo, and in vivo
applications.
[0049] Also, the polynucleotides of the invention can be vectors
that express polypeptides, ribozymes, or antisense molecules.
Vectors contain at least a promoter to initiate transcription
operably linked to the coding region, ribozyme or antisense
molecule. Other components that can be included in the vector are,
for example: (1) a terminator sequence; (2) a sequence encoding a
leader peptide to direct secretion; (3) a selectable marker; and
(4) an origin of replication. An orgin of replication is not
necessary. The polynucleotides to be delivered can be either
replicating or non-replicating. Other components can be added as
desired and convenient.
[0050] The polynucleotides and methods of the invention can be
utilized with any type of host cell. The choice of promoter,
terminator, and other optional elements of an expression vector
will depend on the host cell chosen. The invention is not dependent
on the host cell selected. Convenience and the desired level of
protein expression will dictate the optimal host cell. A variety of
hosts for expression are known in the art and available from the
American Type Culture Collection (ATCC) (Rockville, Md., U.S.A.).
Suitable bacterial hosts suitable include, without limitation:
Campylobacter, Bacillus, Escherichia, Lactobacillus, Pseudomonas,
Staphylococcus, and Streptococcus. Yeast hosts from the following
genera may be utilized: Candida, Hansenula, Kluyveromyces, Pichia,
Saccharomyces, Schizosaccharomyces, and Yarrowia. Aedes aegypti,
Bombyx mori, Drosophila melanogaster, and Spodoptera frugiperda
(PCT Patent Publication No. WO 89/046699; Carbonell et al., 1985,
J. Virol. 56:153; Wright, 1986, Nature 321:718; Smith et al., 1983,
Mol. Cell. Biol. 3:2156; and see generally, Fraser et al., 1989, In
Vitro Cell. Dev. Biol. 25:225).
[0051] Useful mammalian cell types for in vitro applications
include for example, those cell lines available from the American
Type Culture Collection (ATCC), Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), human
embryonic kidney cells, baby hamster kidney cells, mouse sertoli
cells, canine kidney cells, buffalo rat liver cells, human lung
cells, human liver cells, mouse mammary tumor cells, as well as
others.
[0052] Useful cell types for in vivo or ex vivo applications
include, without limitation, any tissue type, such as muscle, skin,
brain, lung, liter, spleen, blood, bone marrow, thymus, heart,
lymph, bone, cartilage, pancreas, kidney, gall bladder, stomach,
intestine, testis, ovary, uterus, rectum, nervous system, eye,
gland, and connective tissue.
[0053] A. In vitro and Ex vivo Vectors
[0054] The polynucleotides encoding the desired polypeptides or
ribozymes, or antisense polynucleotides can be transcribed and/or
translated using the following promoters and enhancers as examples.
The examples include, without limitation: the 422(aP2) gene and the
stearoyl-CoA desaturase 1 (SCD1) gene, which contains suitable
adipocyte-specific promoters, as described in Christy et al., 1989,
Genes Dev. 3:1323-1335. Synthetic non-natural promoters or hybrid
promoters can also be used herein. For example, a T7T7/T7 promoter
can be constructed and used, in accordance with Chen et al., 1994,
Nucleic Acids Res. 22:2114-2120, where the T7 polymerase is under
the regulatory control of its own promoter and drives the
transcription of a polynucleotide sequence, which is placed under
the control of another T7 promoter. The primary determinant for the
fat-specific expression is an enhancer located at about >5 kb
upstream of the transcriptional start site, as described in Ross et
al., 1990, Proc. Natl. Acad. Sci. USA. 87:9590-9594 and Graves et
al., 1991, Genes Dev. 5:428-437. Also suitable for use herein is
the gene for the CCAAT/enhancer-binding protein C/EBP.alpha., which
is highly expressed when 3T3-L1 adioblast commit to the
differentiation pathway and in mature post-mitotic adipocytes, as
described in Birkenmeier et al., 1989, Gene Dev. 3:1146-1156. The
recently isolated transcription factor PPAR.gamma.2, expressed
exclusively in adipocyte tissues, as described in Tontonoz et al.,
1994, Cell 79:1147-1156, can also be used herein.
[0055] Typical promoters for mammalian cell expression include the
SV40 early promoter, the CMV promoter, the mouse mammary tumor
virus LTR promoter, the adenovirus major late promoter (Ad MLP),
and the herpes simplex virus promoter, among others. Other
non-viral promoters, such as a promoter derived from the murine
metallothionein gene, will also find use in mammalian constructs.
Expression may be either constitutive or regulated (inducible),
depending on the promoter. Typically, transcription termination and
polyadenylation sequences will also be present, located 3' to the
translation stop codon. Preferably, a sequence for optimization of
initiation of translation, located 5' to the coding sequence, is
also present. Examples of transcription terminator/polyadenylation
signals include those derived from SV40, as described in Sambrook
et al., 1989, "Molecular Cloning, A Laboratory Manual," second
edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
Introns, containing splice donor and acceptor sites, may also be
designed into the constructs of the present invention.
[0056] Enhancer elements can also be used herein to increase
expression levels of the mammalian constructs. Examples include the
SV40 early gene enhancer, as described in Dijkema et al., 1985,
EMBO J. 4:761, and the enhancer/promoter derived from the long
terminal repeat (LTR) of the Rous Sarcoma Virus, as described in
Gorman et al., 1982b, Proc. Natl. Acad. Sci. USA 79:6777, and human
cytomegalovirus, as described in Boshart et al., 1985, Cell 41:521.
A leader sequence can also be present which includes a sequence
encoding a signal peptide, to provide for the secretion of the
foreign protein in mammalian cells. Preferably, there are
processing sites encoded between the leader fragment and the gene
of interest such that the leader sequence can be cleaved either in
vivo or in vitro.
[0057] Other regulatory regions from viruses can be included in the
polynucleotides of the instant invention to increase transcription
and translation levels or increase the duration of transcription
and translation. For example, the long terminal repeats of HIV can
be included. Alternatively, the inverted terminal repeats of the
Epstein Barr Virus can be used.
[0058] There exist expression vectors that provide for the
transient expression in mammalian cells of DNA encoding the target
polypeptide. In general, transient expression involves the use of
an expression vector that is able to replicate efficiently in a
host cell, such that the host cell accumulates many copies of the
expression vector and, in turn, synthesizes high levels of a
desired polypeptide encoded by the expression vector. Transient
expression systems, comprising a suitable expression vector and a
host cell, allow for the convenient positive identification of
polypeptides encoded by cloned DNAs, as well as for the rapid
screening of such polypeptides for desired biological or
physiological properties. Thus, transient expression systems are
particularly useful for purposes of identifying analogs and
variants of the target polypeptide that have target
polypeptide-like activity.
[0059] B. In vivo Vectors
[0060] For delivery using viral vectors, any of a number of viral
vectors can be used, as described in Jolly, 1994, Cancer Gene
Therapy 1:1-64. For example, the coding sequence of a desired
polypeptide or ribozymes or antisense molecules can be inserted
into plasmids designed for transcription and/or translation in
retroviral vectors, as described in Kimura et al., 1994, Human Gene
Therapy 5:845-852, adenoviral vectors, as described in Connelly et
al., 1995, Human Gene Therapy 6:185-193, adeno-associated viral
vectors, as described in Kaplitt et al., 1994, Nature Genetics
6:148-153 and sindbis vectors. Promoters that are suitable for use
with these vectors include the Moloney retroviral LTR, CMV promoter
and the mouse albumin promoter. Replication competent free virus
can be produced and injected directly into the animal or humans or
by transduction of an autologous cell ex vivo, followed by
injection in vivo as described in Zatloukal et al., 1994, Proc.
Natl. Acad. Sci. USA 91:5148-5152.
[0061] The polynucleotide encoding a desired polypeptide or
ribozyme or antisense polynucleotide can also be inserted into
plasmid for expression of the desired polypeptide in vivo. For in
vivo therapy, the coding sequence can be delivered by direct
injection into tissue, or via oral administration as an aerosol.
Promoters suitable for use in this manner include endogenous and
heterologous promoters such as CMV. Further, a synthetic T7T7/T7
promoter can be constructed in accordance with Chen et al., 1994,
Nucleic Acids Res. 22:2114-2120, where the T7 polymerase is under
the regulatory control of its own promoter and drives the
transcription of polynucleotide sequence, which is also placed
under the control of a T7 promoter. The polynucleotide can be
injected in a formulation that can stablize the coding sequence and
facilitate transduction thereof into cells and/or provide
targeting, as described in Zhu et al., 1993, Science
261:209-211.
[0062] Expression of the coding sequence of a desired polypeptide
or replication of a ribozyme or antisense polynucleotide in vivo
upon delivery for gene therapy purposes by either viral or
non-viral vectors can be regulated for maximal efficacy and safety
by use of regulated gene expression promoters as described in
Gossen et al., 1992, Proc. Natl. Acad. Sci. USA 89:5547-5551. For
example, the polynucleotide transcription and/or translation can be
regulated by tetracycline responsive promoters. These promoters can
be regulated in a positive or negative fashion by treatment with
the regulator molecule.
[0063] For non-viral delivery of the coding sequence of the desired
polypeptide, the sequence can be inserted into conventional vectors
that contain conventional control sequences for high level
expression.
[0064] C. Preferred Vector
[0065] A preferred vector comprises: (1) an (EBV) origin of
replication or a BKV (BK virus), a parvovirus, origin of
replication; (2) a coding region for an EBV or BKV origin binding
protein; (3) at least one inverted terminal repeat; (4) a promoter;
(5) an enhancer; (6) a terminator; (7) optionally, a selectable
marker.
[0066] Preferably, the orgin of replication is EBV ori p; more
preferably, nucleotides 2623 to 4559 of SEQ ID NO:1 are utilized.
The sequence is obtainable from vector pCEP4, commercially
available from Invitrogen, San Diego, Calif., USA.
[0067] Preferably, the coding region encodes the EBV nuclear
antigen A, which binds to EBV ori p; more preferably, the
polynucleotide sequence is nucleotides 14 to 2594 of SEQ ID NO: 1
are utilized. The sequence is obtainable from vector pCEP4,
commercially available from Invitrogen, San Diego, Calif., USA.
[0068] Fragments and mutants of the preferred origin and binding
protein capable of initiating replication of the vector in the
desired host cell can be utilized. Preferably, the fragments and
mutants will retain more than about 80% sequence identity with
nucleotides 14 to 2594 or 2623 to 4559 of SEQ ID NO: 1 or fragment
thereof; more typically, more than about 85%; even more typically,
at least 90%. Preferably, these polynucleotides exhibit more than
about 92% sequence identity with nucleotides 14 to 2594 or 2623 to
4559 of SEQ ID NO: 1 or fragment thereof; more preferably, more
than about 94%; even more preferably, more than about 96%; even
more preferably, more than about 98%; even more preferably, more
than about 99%.
[0069] Preferably, the inverted terminal repeats are those
sequences found in adenovirus (AV) or adeno-associated virus (AAV);
more preferably, the inverted terminal repeats are those found in
AAV; even more preferably, the polynucleotide sequence is 4938 to
5104 or 7189 to 7355 of SEQ ID NO: 1. The sequence of AAV is
described in Samulski et al., 1987, J. Virol. 61:3096-3101.
[0070] Fragments and mutants of the preferred inverted terminal
repeat capable of initiating replication of the vector in the
desired host cell can be utilized. Preferably, the fragments and
mutants will retain more than about 80% sequence identity with
nucleotides 4938 to 5104 or 7189 to 7355 of SEQ ID NO: 1 or
fragment thereof; more typically, more than about 85%; even more
typically, at least 90%. Preferably, these polynucleotides exhibit
more than about 92% sequence identity with nucleotides 4938 to 5104
or 7189 to 7355 of SEQ ID NO: 1 or fragment thereof; more
preferably, more than about 94%; even more preferably, more than
about 96%; even more preferably, more than about 98%; even more
preferably, more than about 99%.
[0071] Preferably, the cytomegalovirus enhancer/promoter is
utilized; more preferably, the CMV promoter sequence is nucleotide
sequence 5112 to 6734 of SEQ ID NO: 1.
[0072] Mutants and fragments of the preferred enhancer and promoter
capable of initiating transcription and/or translation can be
utilized. Preferably, the fragments and mutants will retain more
than about 80% sequence identity with nucleotides 5112 to 6734 of
SEQ ID NO: 1 or fragment thereof; more typically, more than about
85%; even more typically, at least 90%. Preferably, these
polynucleotides exhibit more than about 92% sequence identity with
nucleotides 5112 to 6734 of SEQ ID NO: 1 or fragment thereof; more
preferably, more than about 94%; even more preferably, more than
about 96%; even more preferably, more than about 98%; even more
preferably, more than about 99%.
[0073] A preferred terminator is the bovine growth hormone poly A
sequence; more preferably, the polynucleotide sequence is
nucleotide 6818 to 7050 of SEQ ID NO:1.
[0074] Mutants and fragments of the preferred terminator capable of
terminating transcription and/or translation can be utilized.
Preferably, the fragments and mutants will retain more than about
80% sequence identity with nucleotides 6818 to 7050 of SEQ ID NO: 1
or fragment thereof; more typically, more than about 85%; even more
typically, at least 90%. Preferably, these polynucleotides exhibit
more than about 92% sequence identity with nucleotides 6818 to 7050
of SEQ ID NO: 1 or fragment thereof; more preferably, more than
about 94%; even more preferably, more than about 96%; even more
preferably, more than about 98%; even more preferably, more than
about 99%.
[0075] The sequence of the preferred vector is shown in SEQ ID NO:
1. Polynucleotides encoding polypeptides, such as erythropoeitin or
leptin, and ribozymes and antisense polynucleotides can be inserted
into the vector.
[0076] D. Examples of Coding Regions, Ribozymes, and Antisense
Molecules
[0077] The following are examples of coding regions, ribozymes, and
antisense molecules that can be used to treat various indications
in mammals. The nucleotide sequence of the genes of interest can be
found, for example, in publically available databases, such as
Genbank. Polynucleotides to be delivered can be used to treat viral
infections or chronic pathogen infection.
[0078] 1. Hemophilia
[0079] Gene replacement by in vivo delivery of polynucleotides can
be effective in treating hemophilia. The following are examples of
polypeptides that can be encoded by the polynucleotides to be
delivered: Factor VIII:C, mutants of Factor VIII:C, preferably
those that are uncleavable. Also, useful to treat hemophilia are
ribozyme and antisense polynucleotides as inhibitors of Tissue
Factor Plasminogen Inhibitor (TFPI).
[0080] The routes of delivery for treating hemophilia include, for
example, intravenous/intrahepatic injection, ex vivo transduction
of stem cells or lymphocytes using retroviral vectors.
[0081] 2. Treatment of Graft Versus Host Disease
[0082] In vivo delivery of polynucleotides encoding prodrugs can be
used for direct ablation to treat graft versus host disease in, for
example, leukemia bone marrow transplantation. Herpes thymidine
kinase in conjunction with gancyclovir can be utilized for this
purpose. Other examples of prodrugs are described in the cancer
section.
[0083] The routes of delivery for treating graft versus host
disease include, for example, ex vivo transduction of T-lymphocytes
using retroviral vectors.
[0084] 3. Vaccines
[0085] In vivo delivery of polynucleotides encoding a desired
antigen can be utilized to induce an immune response. This response
can include both cellular and humoral response. This type of
vaccine can be used to treat cancer as well as infectious diseases.
Further, such treatment can be either prophylactic or therpeutic
immunotherapy. Examples of infectious diseases include, Human
Immunodeficiency Virus (HIV), Hepatitis A, B, C, etc., (HAV, HBV,
HCV, etc.), Human Papiloma Virus (HPV), cytomegalovirus (CMV),
herpes simplex 1 and 2 (HSV), etc. Preferred antigens include
non-structural proteins 3, 4a, and 5b (NS3, NS4, and NS5b) of HCV;
gB2 and gD2 of HSV; env and rev proteins of HIV.
[0086] Also, cancer antigens can be used in vaccines, for both
therapeutic and prophylactic purposes.
[0087] The antigens can be presented in the context of Class I
major histocompatibility antigens, or to induce a cellular
cytotoxic T cell response, or to induce a humoral response
comprising the synthesis of antibodies.
[0088] In addition, an antisense or ribozyme target to a immune
suppressive molecule, IL-10, TGF-.beta., and CTLA-4, for example,
can be useful to be administered with a vaccine.
[0089] The routes of delivery for vaccines include, for example,
intramuscular injection, dendritic cell-based immunization, or oral
immunization by both viral and non-viral vectors.
[0090] 4. Diabetes Mellitus
[0091] Diabetes is another indication that can be treated by in
vivo delivery of a replacement gene. The following are examples of
useful polypeptides to be encoded by the replacement gene: insulin,
insulin-like growth factor I and II (IGF-I and II).
[0092] Also useful for treating diabetes are polynucleotides
encoding IAS-L, found on the surface of B cells in the pancreas, to
protect the cells from immune destruction.
[0093] The routes of delivery for treating diabetes include, for
example, liver-directed, parotid-directed, pancreas-directed,
salivary gland-directed using both viral and non-viral vectors.
[0094] 5. Hyperlipidemia
[0095] Hyperlipidemia can be treated by in vivo delivery of the
following polynucleotides encoding apoproteins or lipoprotein
receptors. A more extensive description of lipoproteins and
apoproteins is provided below.
[0096] The routes of delivery for treating hyperlipidemia include,
for example, liver-directed intravenous administration by both
viral and non-viral vectors.
[0097] 6. Myocardial Ischemia or Infarction
[0098] The following are examples of polynucleotides that are
useful, when delivered in vivo, to treat myocardial ischemia or
infarction: [0099] polynucleotides encoding basic fibroblast growth
factor (bFGF), fibroblast growth factor 5 (FGF-5) and IGF-I.
[0100] The routes of delivery for treating myocardial ischemia or
infarction include, for example, intrapericardial delivery of viral
vector or non-viral vectors.
[0101] 7. Bowel Disease
[0102] The following are examples of polynucleotides that can be
delivered in vivo to treat bowel disease: [0103] (i) ribozymes or
antisense polynucleotides as inhibitors of macrophage/inflammatory
cell recruitment or activation, such as NF.kappa.B; [0104] (ii)
ribozymes or antisense polynucleotides to act as anti-apoptotic
agents, such as inhibitors of interleukin 1b converting enzyme
family; [0105] (iii) polynucleotides encoding complement blockers,
such as decay accelarting factor (DAF), membrane cofactor protein
(MCP); and the fusions of DAF and MCP also known as CAB-2; [0106]
(iv) cyclooxygenase inhibitors; [0107] (v) anti-proliferative
agents, such as, ribozymes, antisense oligonucleotides, antibodies,
protein, or peptides against c-myb, ras/raf, P13 kinase, cyclins;
[0108] (vi) polynucleotides encoding suicide proteins/genes, such
as, herpes thymidine kinase; [0109] (vii) polynucleotides encoding
replacement genes or proteins which maybe deficient or down
regulated during the devleopment of inflammatory bowel disease.
[0110] (viii) polynucleotides encoding I.kappa.B.
[0111] 8. Prostate Cancer and Benign Prostatic Hyperplasia
[0112] The following polynucleotides can be delivered to treat
prostate cancer and benign prostatic hyperplasia: [0113] (i) a
polynucleotide encoding a pro-apoptotic agent, including for
example, fas, fas ligand, fadd, fap-1, tradd, faf, rip, reaper,
apoptin, interleukin-2 converting enzyme; [0114] (ii) a
polynucleotide encoding an anti-angiogenic agent, including, for
example, bFGF soluble receptor and fragments, angiostatin,
transforming growth factor-.beta. (TGF-.beta.), interferon-.alpha.
(IFN.alpha.), proliferin-related protein, a urokinase plasminogen
activator receptor antagonist, platelet factor 4 (PF4),
thrombospondin, a tissue inhibitor of metalloproteinase, and
prolactin; [0115] (iii) a polynucleotide encoding a
immunomodulating agent including, for example, interleukin-2
(IL-2), IFN.alpha., IFN.beta., IFN.gamma., granulocyte
macrophage-colony stimulating factor (GM-CSF), and
macrophage-colony stimulating factor (M-CSF); [0116] (iv) a
ribozyme or antisense polynucleotide as an antiproliferative agent
including, for example, an inhibitor of a signal transduction
pathway, for example, an inhibitor of a signal transduction pathway
mediated by myb, ras, ras superfamily, raf, phosphoinositol
(PI3-kinase), a phosphotyrosine binding (PTB) domain, a SRC
homology-2 (SH2) domain, a SRC homology-3 (SH3) domain, a plextrin
homology (PH) domain, JUN kinase, and a stress activated kinase,
signaling inositol phosphatases; and an inhibitor of a cyclin;
[0117] (v) a ribozyme or antisense polynucleotide as an inhibitor
of a growth factor or inhibitor of a receptor of a growth factor,
including, for example, epidermal growth factor (EGF), TGF-.alpha.,
FGF, TGF-.beta., platelet derived growth factor (PDGF),
keratinocyte growth factor (KGF), or any prostate cell specific
growth factor; [0118] (vi) a polynucleotide encoding a tumor
suppressor gene or a gene down-regulated during the onset of a
hyperplastic condition in the prostate; and [0119] (vii) an
antisense or ribozyme target to a immune suppressive molecule,
IL-10, TGF-.beta., and CTLA-4, for example.
[0120] 9. Anemia, Leukopenia, and Thrombocytopenia
[0121] Anemia can be treated by in vivo delivery of a
polynucleotide encoding erythropoietin, GM-CSF-, G-CSF, M-CSF, and
thrmobopoietin, for example. Examples of delivery routes for this
indication include without limitation: liver-targeted intravenous
administration of viral vectors and non-viral vectors. See the
Examples below.
[0122] 10. Cardiomyopathy
[0123] The following are examples of polynucleotides that can be
delivered in vivo to treat cardiomyopathy: polynucleotides
encoding, IGF-1, L-amino acid decarboxylas, inhibitors of .beta.
adrenergic receptor kinases (BARK), troponin, and adrenergic
receptors.
[0124] Examples of delivery routes for this indication include,
without limitation, pericardial expression of IGF-1, and for the
other genes, intramycardial injection or myocardial trageting via
intracoronary injection or intrapericardial administration of viral
vectors or non-viral vectors.
[0125] 11. Rheumatoid Arthritis
[0126] The following are examples of polynucleotides that can be
delivered in vivo to treat rheumatoid arthritis, polynucleotides
encoding a prodrug, such as herpes thymidine kinase, MMP
inhibitors, fas, and pro-apoptotic proteins, described above, and
interleukin-1 receptor A, interleukin-10, I.kappa.B.
[0127] Also, antisense and ribozyme polynucleotides as inhibitors
of NF.kappa.B.
[0128] Examples of delivery routes for this indication include,
without limitation, intraarticular injection of viral and non-viral
vectors.
[0129] 12. Osteoarthritis and Psoriasis
[0130] The following are examples of polynucleotides that can be
delivered in vivo to treat osteoarthritis and psoriasis:
polynucleotides encoding IGF-1; ribozyme and antisense
polynucleotides as inhibitors of metalloproteinase inhibitors.
[0131] Also, the following are examples of polynucleotides that can
be delivered in vivo to treat osteoarthritis and psoriasis,
polynucleotides encoding a prodrug, such as herpes thymidine
kinase, MMP inhibitors, fas, and pro-apoptotic proteins, described
above, and interleukin-1 receptor A, interleukin-10, I.kappa.B.
[0132] Also, antisense and ribozyme polynucleotides as inhibitors
of NF.kappa.B.
[0133] Examples of delivery routes for this indication include,
without limitation, intraarticular injection.
[0134] 13. Restenosis
[0135] The following are examples of polynucleotides that can be
delivered in vivo to treat restenosis: [0136] (i) polynucleotides
encoding a prodrug, such as thymidine kinase, other examples are
described in the cancer section; [0137] (ii) polynucleotides
encoding tissue factor plasminogen inhibitor (TFPI); [0138] (iii)
polynucleotides encoding c-myb rbz, c-ras rbz, [0139] (iv)
polynucleotides encoding pro-apoptotic agents, described above;
[0140] (v) polynucleotides encoding I.kappa.B.
[0141] Examples of delivery routes for this indication include,
without limitation, intracoronary delivery of viral and non-viral
vectors.
[0142] 14. Cancer
[0143] The gene delivery vectors of the invention are useful in
delivering therapeutic genes for treatment of hyperproliferative
disorders, including malignancy, for treatment of infectious
disease and for treatment of inflammatory diseases, including
autoimmune disease. For instance, the gene therapy vectors can be
used to express cytokines or proteins that convert an inactive or
partially active prodrug into an active drug. In many cases,
conversion of the prodrug into its active form results in a
compound with cytolytic activity.
[0144] a. Prodrug Converting Enzymes
[0145] A number of "suicide genes" which encode different proteins
useful in prodrug conversion can be used in the instant invention.
For instance, nucleoside kinases such as thymidine kinase are
particularly useful. In particular, the HSV-TK system has important
advantages for anti-tumor cell therapy. See PCT publication number
WO 91/02805 entitled "Recombinant Retroviruses Delivering Vector
Constructs to Target Cells" and PCT publication number WO
95/14014091 entitled "Compositions and Methods for Utilizing
Conditionally Lethal Genes" for a description of treatment of
cancer and other diseases by gene delivery vectors expressing
thymidine kinase and other prodrug converting enzymes. HSV-TK
transduced tumor cells can mediate a significant bystander killing
effect on untransduced neighboring cells in vitro and in vivo
(Moolten et al., supra., Freeman et al., 1993, Cancer Res.
53:5274), most commonly as a result of transfer to the toxic
ganciclovir metabolite, GCV triphosphate, between adjacent cells
through intercellular gap junctions (Bi et al., 1993, Human Gene
Therap. 4:725). Endothelial cells in capillary walls are connected
by gap junctions, so a dramatic bystander effect created by
GCV-triphosphate transfer between neighboring endothelial cells and
the massive amplification effects of the clotting cascade and the
tumor to endothelial cell ratio could ensue (Denekamp et al., 1986,
Cancer Topics 6:6; Denekamp et al., 1984, Prog. Appl. Microcir.
4:28). Recent evidence suggests that the occasional transduction of
tumor endothelial cells during intralesional therapy with HSV-TK
retroviral vectors may account for a significant component of the
antitumor activity of the vectors (Ram et al., 1994, J. Neurosurg.
81:256). In addition, the suicide gene is only conditionally
cytotoxic to the target cells (i.e. only when GCV is given).
Consequently, an ex vivo administration method can be be utilized.
For example, in this type of protocol, endothelial cells may be
isolated from tumor biopsies (Medzelewski et al., 1994, Cancer Res.
54:336), induced to proliferate with appropriate mitogens (Ferrara
et al, supra.) and transduced with TK in vitro. Transplanted EC
become incorporated into the neovasculature in days to weeks after
intratumoral injection (Lal et al., 1994, Cancer Gene Therap.
1:322), so GCV treatment would follow a suitable `lag phase` to
allow the transduced EC to integrate functionally in to the tumor
vasculature. The two-step enzyme-prodrug system offers greater
flexibility of delicate clinical management, because cessation of
GCV infusion in the event of (potentially very serious)
complications arising from damage to normal EC, would block
toxicity without the need to block transgene activity in situ.
[0146] A number of alternative `suicide genes` in addition to
thymidine kinase may also be useful for cancer gene therapy
(Moolten et al., supra.). Introduction of the bacterial cytosine
deaminase gene (Huber et al., 1993, Cancer Res. 53:4619) into tumor
cells confers sensitivity to the antifungal agent 5-fluorocytosine
(5-FC). Cytosine deaminase converts 5-FC to 5-fluorouracil (5-FU,
Nishiyama et al., 1985, Cancer Res. 45:1753). Since 5-FU is
commonly used chemotherapeutic drug for breast cancer, several
groups have developed cytosine deaminase-based `suicide gene`
therapy models for this disease. Tumor specificity may be further
increased by introducing the c-erbB2 promoter/enhancer elements 5'
to the cytosine deaminase gene, so that the therapeutic transgene
is preferentially transcribed in c-erbB2-overexpressing breast
tumor cells (Harris et al., 1994, Gene Therap. 1: 170). Alkaline
phosphatase has been widely explored as prodrug-activating enzyme
in the related field of antibody directed enzyme-prodrug therapy
(ADEPT). This enzyme has the advantage that it can activate a wide
range of phosphorylated derivatives of anticancer agents (e.g.
mitomycin C, etoposide, etc.) that cannot cross cell membranes
until the charged phosphate group is cleaved off, so a single
enzyme could generate de novo a cocktail of chemotherapeutic agents
within the tumor mass (Senter et al., 1993, Bioconjugate Chem.
4:3). Other suicide genes may encode a polypeptide or polypeptides
(with a corresponding non-cytotoxic agent) such as Herpes Simpex
virus thymidine kinase (gancyclovir or acyclovir), Varicella Zoster
virus thymidine kinase (6 methoxypurine arabino nucleoside; Huber
et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039), E. coli cytosine
deaminase (fluorouracil; Mullen et al., 1992, Proc. Natl. Acad.
Sci. USA. 89:33), E. coli xanthine-guanine phophoribosyl
transferase (thioxanthine; Beshard et al., 1987, Mol. Cell Biol.
7:4139), E. coli or Leishmania purine nucleotide phosphorylase
(various nontoxic purine deoxyadenosine, adenosine, deoxyguanosine,
or guanosine derivatives (Koszalka and Krenitsky, 1979, J. Biol
Chem 254:8185, 1979; Sorscher et al, 1994, Gene Therapy 1:233),
cytochrome pla50 2B1 or cytochrome p450 reductase (e.g.,
3amino-1,2,4 benzotriazine 1,4-dioxide (Walton et al., 1992,
Biochem. Pharmacol. 44:251), cell surface alkaline phosphatase
(e.g., etoposide monophosphate; Senter et al., 1988, Proc. Natl.
Acad. Sci. USA 85:4842, 1988), nitroreductase (e.g., metronidazole
or nitroflirantoin; Hof et al., 1988, Immunitat und Infektion
16:220), N-deoxyribosy transferase (1-deazapurine; Betbeder et al.,
1989, Nucleic Acids Res 17:4217), pyruvate ferrodoxin
oxidoreductase (metronidazol; Upcroft et al., 1990, Int. J.
Parasitolog, 20:489), carboxypepidase G2 (aminoacylate nitrogen
mustards; Antoniw et al., 1990, Brit J. Cancer 62:909),
carboxypeptidase A (methotrexate alpha alanine; Haenseler et al.,
1992, Biochemistry 31:891), .cndot. lactamase (cephalosporin
derivatives; Meyer et al, 1993, Cancer Res. 53:3956; and Vradhula
et al., 1993, Bioconjugate Chemistry 4:334), Actinomycin D
synthetase complex (synthetic pentapeptide lactone precursors; Katz
et al., 1990, J. Antibiotics 43:231), and .cndot.-glucuronidase
(various glucuronide derivatives of toxic drugs such as
doxorubicin; Bosslet et al., 1994, Cancer Res. 54:2151; Haeberlin
et al., 1993, Pharmaceutical Res. 10:1553).
[0147] Any of a variety of other enzymes which convert inactive
prodrugs into active drugs and known to those of skill in the art
can also used in the gene delivery vehicles of the invention. For
example, see PCT publication number WO 95/14014091 entitled
"Compositions and Methods for Utilizing Conditionally Lethal
Genes", and European Patent publication number EP90309430, entitled
"Molecular Chimeras Useful for Cancer Therapy--Comprising
Regulatory Sequences and heterologous enzyme, e.g. Varicella Zoster
Virus Thymidine Kinase" for a description of additional
prodrug/enzyme systems useful for gene therapy. As an additional
example, see PCT Patent Publication No. WO 95/13095 entitled "New
Prodrugs and Enzyme Targeting Molecule Conjugates--Useful in
Antibody Direct Enzyme Prodrug Therapy of e.g. Viral
Infections".
[0148] A variety of tumors may be targeted for treatment by the
gene delivery vehicles of the invention. In general, solid tumors
are preferred, although leukemias and lymphomas may also be treated
if they have developed a solid mass, or if suitable tumor
associated markers exist such that the tumor cells can be
physically separated from nonpathogenic normal cells.
Representative examples of suitable tumors include melanomas,
colorectal carcinomas, lung carcinomas (including large cell, small
cell, squamous and adeno-carcinomas), renal cell carcinomas and
breast adeno-carcinomas. Gene delivery vehicles expressing
thymidine kinase and other prodrug converting enzymes are also
useful in the treatment of autoimmune diseases including rheumatoid
arthritis, osteoarthritis and graft vs. host disease. See e.g. PCT
Patent Publication No. WO 95/14091, entitled "Compositions and
Methods for Utilizing Conditionally Lethal Genes," for a
description of treatment of disease with gene therapy vectors
expressing prodrug converting enzymes.
[0149] b. Cytokines
[0150] A variety of polynucleotides encoding cytokines and immune
system modulators can be delivered by the gene delivery vehicles of
the invention for treatment of a number of different disorders.
Representative examples include cytokines, such as IL-1, IL-2
(Karupiah et al., 1990, J. Immunology 144:290-298; Weber et al.,
1987, J. Exp. Med. 166:1716-1733; Gansbacher et al, 1990, J. Exp.
Med. 172:1217-1224; U.S. Pat. No. 4,738,927), IL-3, IL-4 (Tepper et
al., 1989, Cell 57:503-512; Golumbek et al., 1991, Science
254:713-716, 1991; U.S. Pat. No. 5,017,691), IL-5, IL-6 (Brakenhof
et al., 1987, J. Immunol. 139:4116-4121; WO 90/06370), IL-7 (U.S.
Pat. No. 4,965,195), IL-8, IL-9, IL-10, IL-11, IL-12, IL-13
(Cytokine Bulletin, Summer 1994), IL-14 and IL-15, particularly
IL-2, IL-4, 1L-6, IL-12, and IL-13, alpha interferon (Finter et
al., 1991, Drugs 42(5):749-765; U.S. Pat. No. 4,892,743; U.S. Pat.
No. 4,966,843; WO 85/02862; Nagata et al., 1980, Nature
284:316-320; Familletti et al., 1981, Methods in Enz. 78:387-394;
Twu et al., 1989, Proc. Natl. Acad. Sci. USA 86:2046-2050; Faktor
et al., 1990, Oncogene 5:867-872), beta interferon (Seif et al.,
1991, J. Virol. 65:664-671), gamma interferons (Radford et al., The
American Society of Hepatology 2008-2015, 1991; Watanabe et al.,
PNAS 86:9456-9460, 1989; Gansbacher et al., 1990, Cancer Research
50:7820-7825; Maio et al., 1989, Can. Immunol. Immunother.
30:34-42; U.S. Pat. No. 4,762,791; U.S. Pat. No. 4,727,138), G-CSF
(U.S. Pat. Nos. 4,999,291 and 4,810,643), GM-CSF (WO 85/04188),
tumor necrosis factors (TNFs) (Jayaraman et al., 1990, J.
Immunology 144:942-951), CD3 (Krissanen et al., 1987,
Immunogenetics 26:258-266, 1987), ICAM-1 (Altman et al., 1989,
Nature 338:512-514; Simmons et al., 1988, Nature 331:624-627),
ICAM-2, LFA-1, LFA-3 (Wallner et al., 1987, J. Exp. Med.
166(4):923-932), MHC class I molecules, MHC class II molecules,
B7.1-0.3, .sub.2-microglobulin (Parnes et a., 1981, Proc. Natl.
Acad. Sci. 78:2253-2257), chaperones such as calnexin, MHC linked
transporter proteins or analogs thereof (Powis et al., 1991, Nature
354:528-531, 1991).
[0151] Genes encoding any of the cytokine and immunomodulatory
proteins described herein can be expressed in a gene delivery
vehicle of the invention. Other forms of these cytokines which are
known to those of skill in the art can also be used. For instance,
nucleic acid sequences encoding native IL-2 and gamma-interferon
can be obtained as described in U.S. Pat. Nos. 4,738,927 and
5,326,859, respectively, while useful muteins of these proteins can
be obtained as described in U.S. Pat. No. 4,853,332. As an
additional example, nucleic acid sequences encoding the short and
long forms of mCSF can be obtained as described in U.S. Pat. Nos.
4,847,201 and 4,879,227, respectively.
[0152] Other nucleic acid molecules that encode cytokines, as well
as other nucleic acid molecules that are advantageous for use
within the present invention, may be readily obtained from a
variety of sources, including, for example, depositories such as
the American Type Culture Collection (ATCC, Rockville, Md.), or
from commercial sources such as British Bio-Technology Limited
(Cowley, Oxford England). Representative examples include BBG 12
(containing the GM-CSF gene coding for the mature protein of 127
amino acids), BBG 6 (which contains sequences encoding gamma
interferon), ATCC No. 39656 (which contains sequences encoding
TNF), ATCC No. 20663 (which contains sequences encoding alpha
interferon), ATCC Nos. 31902, 31902 and 39517 (which contains
sequences encoding beta interferon), ATCC No 67024 (which contains
a sequence which encodes Interleukin-1b), ATCC Nos. 39405, 39452,
39516, 39626 and 39673 (which contains sequences encoding
Interleukin-2), ATCC Nos. 59399, 59398, and 67326 (which contain
sequences encoding Interleukin-3), ATCC No. 57592 (which contains
sequences encoding Interleukin-4), ATCC Nos. 59394 and 59395 (which
contain sequences encoding Interleukin-5), and ATCC No. 67153
(which contains sequences encoding Interleukin-6).
[0153] Gene delivery vehicles expressing the above cytokines are
useful in the treatment of a variety of disorders. For example, see
PCT publication number US94/02951 entitled "Compositions and
Methods for Cancer Immunotherapy" for a description of gene therapy
treatment of malignancy.
[0154] 15. Neurological Disorders and Diseases
[0155] Polynucleotides encoding tyrosine hydroxylase can be useful
in treating Parkinson disease.
[0156] For stroke or any acute brain injuries, polynucleotides
encoding IGF-1, bFGF, vascular endothelial growth factor (VEGF) are
useful.
[0157] 16. Pulmonary Disorders
[0158] For treating emphysema, polynucleotides encoding
al-anti-trypsin are useful.
[0159] For treating lung fibrosis, polynucleotides encoding
superoxide dismutase (SOD) are useful.
[0160] For treating cystic fibrosis, polynucleotides encoding CFTR
are useful.
Additional Agents
[0161] Additional agents can be included with the desired
polynucleotides to be delivered. These additional agents can
facilitate endocytosis of the desired nucleic acids or aid binding
of the nucleic acids to the cell surface or both, for example.
[0162] A. Polypeptides
[0163] One example are polypeptides which include, without
limitation: asialoorosomucoid (ASOR); transferrin;
asialoglycoproteins; antibodies; antibody fragments; ferritin;
interleukins; interferons, granulocyte, macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), macrophage colony stimulating factor (M-CSF), stem cell
factor and erythropoietin. Viral antigens, such as envelope
proteins, can also be used. Also, proteins from other invasive
organisms, such as the 17 amino acid peptide from the
circumsporozoite protein of plasmodium falciparum known as RII.
[0164] B. Hormones, Vitamins, Etc.
[0165] Other groups that can be included are, for example:
hormones, steroids, androgens, estrogens, thyroid hormone, or
vitamins, folic acid.
[0166] C. Polyalkylenes, Polysaccharides, Etc.
[0167] Polyalkylene glycols can be included with the desired
polynucleotides. In a preferred embodiment, the polyalkylene glycol
is polyethlylene glycol. In addition, mono-, di-, or polysaccarides
can be included. In a preferred embodiment of this aspect, the
polysaccharide is dextran or DEAE-dextran. Also, chitosan and
poly(lactide-co-glycolide)
[0168] D. Lipids and Liposomes
[0169] The desired polynucleotide can also be encapsulated in
lipids or packaged in liposomes prior to delivery to the subject or
to cells derived therefrom.
[0170] Lipid encapsulation is generally accomplished using
liposomes which are able to stably bind or entrap and retain
nucleic acid. The ratio of condensed polynucleotide to lipid
preparation can vary but will generally be around 1:1 (mg
DNA:micromoles lipid), or more of lipid. For a review of the use of
liposomes as carriers for delivery of nucleic acids, see, Hug and
Sleight, 1991, Biochim. Biophys. Acta. 1097:1-17; Straubinger et
al., in METHODS OF ENZYMOLOGY (1983), Vol. 101, pp. 512-527.
[0171] Liposomal preparations for use in the instant invention
include cationic (positively charged), anionic (negatively charged)
and neutral preparations. Cationic liposomes have been shown to
mediate intracellular delivery of plasmid DNA (Felgner et al.,
1987, Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone et
al., 1989, Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified
transcription factors (Debs et al, 1990, J. Biol. Chem.
265:10189-10192), in functional form.
[0172] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are available under the product line Lipofectin.RTM., from GIBCO
BRL, Grand Island, N.Y. (See, also, Felgner et al., 1987, Proc.
Natl. Acad. Sci. USA 84:7413-7416). Other commercially available
liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boerhinger). Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g., Szoka et al., 1978, Proc. Natl. Acad. Sci. USA 75:4194-4198;
PCT Publication No. WO 90/11092 for a description of the synthesis
of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)
liposomes.
[0173] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0174] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LVs). The various liposome-nucleic acid complexes are prepared
using methods known in the art. See, e.g., Straubinger et al., in
METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al.,
1978, Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos et
al., 1975, Biochim. Biophys. Acta 394:483; Wilson et al., 1979,
Cell 17:77; Deamer and Bangham, 1976, Biochim. Biophys. Acta
443:629; Ostro et al, 1977, Biochem. Biophys. Res. Commun. 76:836;
Fraley et al., 1979, Proc. Natl. Acad. Sci. USA 76:3348); Enoch and
Strittmatter, 1979, Proc. Natl. Acad. Sci. USA 76:145); Fraley et
al., 1980, J. Biol. Chem. 255:10431; Szoka and Papahadjopoulos,
1978, Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder et
al., 1982, Science 215:166.
[0175] E. Lipoproteins
[0176] In addition, lipoproteins can be included with the
polynucleotide to be delivered. Examples of lipoproteins to be
utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants,
fragments, or fusions of these proteins can also be used. Also,
modifications of naturally occurring lipoproteins can be used, such
as acetylated LDL. These lipoproteins can target the delivery of
polynucleotides to cells expressing lipoprotein receptors.
Preferably, if lipoproteins are including with the polynucleotide
to be delivered, no other targeting ligand is included in the
composition.
[0177] If lipoproteins are included with the desired
polynucleotides to be delivered, preferably, the composition
comprises: (1) lipoprotein; (2) polynucleotide; and (3) a
polynucleotide binding molecule.
[0178] Naturally occurring lipoproteins comprise a lipid and a
protein portion. The protein portion are known as apoproteins. At
the present, apoproteins A, B, C, D, and E have been isolated and
identified. At least two of these contain several proteins,
designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.
[0179] A lipoprotein can comprise more than one apoprotein. For
example, naturally occurring chylomicrons comprise A, B, C, and E,
over time these lipoproteins lose A and acquire C and E
apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL
comprises apoprotein B; and HDL comprises apoproteins A, C, and
E.
[0180] The amino acids of these apoproteins are known and are
described in, for example, Breslow, 1985, Annu Rev. Biochem 54:699;
Law et al., 1986, Adv. Exp Med. Biol. 151:162; Chen et al., 1986, J
Biol Chem 261: 12918; Kane et al., 1980, Proc Natl Acad Sci USA
77:2465; and Utermann et al., 1984, Hum Genet 65:232.
[0181] Lipoproteins contain a variety of lipids including,
triglycerides, cholesterol (free and esters), and phopholipids. The
composition of the lipids varies in naturally occurring
lipoproteins. For example, chylomicrons comprise mainly
triglycerides. A more detailed description of the lipid content of
naturally occurring lipoproteins can be found, for example, in
Meth. Enzym. 128 (1986). The composition of the lipids are chosen
to aid in conformation of the apoprotein for receptor binding
activity. The composition of lipids can also be chosen to
facilitate hydrophobic interaction and association with the
polynucleotide binding molecule.
[0182] Naturally occurring lipoproteins can be isolated from serum
by ultracentrifugation, for instance. Such methods are described in
Meth. Enzy., supra; Pitas et al., 1980, J. Biochem. 255:5454-5460;
and Mahey et al., 1979, J. Clin. Invest 64:743-750.
[0183] Lipoproteins can also be produced by in vitro or recombinant
methods by expression of the apoprotein genes in a desired host
cell. See, for example, Atkinson et al., 1986, Annu Rev Biophys
Chem 15:403, and Radding et al., 1958, Biochim. Biophys Acta
30:443.
[0184] Lipoproteins can also be purchased from commercial
suppliers, such as Biomedical Techniologies, Inc., Stoughton,
Mass., USA.
[0185] Mutants, fragments and fusion of the naturally occurring
apoproteins are useful for delivery of polynucleotides. These
polypeptides will retain more than about 80% amino acid identity;
more typically, more than about 85%; even more typically, at least
90%. Preferably, these polypeptides will exhibit more than about
92% amino acid sequence identity with naturally occurring
lipoproteins or fragment thereof; more preferably, more than about
94%; even more preferably, more than about 96%; even more
preferably, more than about 98%; even more preferably, more than
about 99% sequence identity.
[0186] Such mutants, fragments and fusions can be constructed by
altering the polynucleotides encoding the desired lipoproteins by
recombinant DNA techniques. See, for example, Sambrook et al.,
(1989) Molecular Cloning, A Laboratory Manual, 2d edition (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y.). These
polynucleotides can be inserted into expression vectors and host
cells can be utilized to produce the desired apoprotein.
[0187] In addition, naturally occurring lipoproteins, mutants,
fragments, and fusions can be chemically altered. For example,
acetylated LDL has biological activity. See, for example,
Nagelkerke et al., 1983, J. Biol. Chem. 258(20):12221-12227;
Weisgraber et al., 1978, J. Biol. Chem. 253:9053-9062; Voyta et
al., 1984, J. Cell Biol. 99:2034-2040; Goldstein et al., 1979,
Proc. Natl. Acad. Sci. USA 76:333-337; and Pitas, 1981,
Arterosclerosis 1:177-185.
[0188] Chemically modified lipoproteins can also be purchased from
commercial suppliers, such as Biomedical Techniologies, Inc.,
Stoughton, Mass., USA.
[0189] All of these polypeptides exhibit receptor binding
properties of naturally occurring lipoproteins. Usually, such
polypeptides exhibit at least about 20% receptor binding of
naturally occurring lipoproteins. More typically, the polypeptides
exhibit at least about 40%, even more typically the polypeptides
exhibit at least about 60%; even more typically, at least about
70%; even more typically, at least about 80%; even more typically,
at least about 85%; even more typically, at least about 90%; even
more typically, at least about 95% receptor binding of the
naturally occurring lipoproteins.
[0190] Typically, lipoproteins are present in an amount effective
to increase the frequency of incorporation of polynucleotides into
a cell. Such an amount is sufficient to increase the frequency of
incorporation of polynucleotides into a cell by at least 10%,
compared to the frequency of incoporation of naked polynucleotides;
more usually, at least 15%; even more usually, 20%; even more
usually, at least 30%. The increase can be between 40 to 100%, and
even 1000% and 10000% increase.
[0191] "Polynucleotide binding molecule" refers to those compounds
that associate with polynucleotides, and the association is not
sequence specific. For example, such molecules can (1) aid in
neutralizing the electrical charge of polynucleotide, or (2)
facilitate condensation of nucleotides, or (3) inhibit serum or
nuclease degradation. Optionally, polynucleotide binding molecules
can interact with lipoproteins by either hydrophobic association or
by charge. Polynucleotide binding molecules include, without
limitation, polypeptides, mineral compounds, vitamins, etc.
[0192] Examples of polynucleotide binding molecules include:
polylysine, polyarginine, polyornithine, and protamine. Examples of
organic polycations include: spermine, spermidine, and purtrescine.
Other examples include histones, protamines, human serum albumin,
DNA binding proteins, non-histone chromosomal proteins, coat
proteins from DNA viruses, such as .phi.X174, transcriptional
factors also contain domains that bind DNA and therefore may be
useful as nucleic aid condensing agents. Briefly, transcriptional
factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF,
Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains
that bind DNA sequences.
[0193] Examples of other positively charged moieties include
polybrene, DEAE-dextran, and cationic lipids. Useful cationic
lipids and liposomes are described above. Lipids and liposomes are
not used in this aspect of the invention to encapsulate both
polynucleotide and lipoprotein. The lipoprotein must be exposed to
bind the its cell surface receptor.
[0194] Other synthetic compounds that are capable of binding
negatively charged polynucleotides are useful, such as polymers of
N-substituted glycines and others, as described below.
[0195] In a composition with a lipoprotein, the polynucleotide
binding molecule can be present in an amount effective to
neutralize the polynucleotide. However, the polynucleotide binding
molecule also can be in excess of an effective amount to neutralize
the polynucleotide to be delivered. Such an excess can produce a
net positive electrical charge when complexed with the
polynucleotides to be delivered. The positively charged complex can
then interact with lipoproteins that comprise negatively charged
lipids, such as phospholipids.
[0196] Typically, the polynucleotide binding molecule is in excess
when the amount is 10% greater than the amount to neutralize the
polynucleotide charge; more typically, the amount is 50% greater;
even more typically, 100% greater; even more typically, 150%
greater; even more typically, 200% greater; even more typically,
500% greater; even more typically, 20,000% greater; even more
typically, 22,000% greater; even more typically, 25,000% greater;
even more typically, 30,000% greater; even more typically, more
than 40,000% greater than the amount effective to neutralize the
electrical charge of the desired polynucleotide.
[0197] Polycationic Agents
[0198] Polycationic agents can be included, with or without
lipoprotein, in a composition with the desired polynucleotide to be
delivered.
[0199] Functional Properties
[0200] A. Net Positive Charge
[0201] Polycationic agents typically exhibit a net positive charge
at physiological relevant pH and are capable of neutralizing the
electrical charge of nucleic acids to facilitate delivery to a
desired location. These agents have both in vitro, ex vivo, and in
vivo applications. For example, these polycationic agents can be
used to transfect cells used to produce recombinant proteins.
Alternatively, the instant polycationic agents can be used to
deliver nucleic acids to a living subject either intramuscularly,
subcutaneously, etc.
[0202] Physiological relevant pH varies somewhat between in vitro
and in vivo applications. Typically, physiological pH is at least
5.5; more typically, at least 6.0; even more typically, at least
6.5. Usually, physiologically relevant pH is no more than 8.5; more
usually, no more than 8.0; even more usually, no more than 7.5.
[0203] Preferably, the isoelectric point of the instant
polycationic agents to neutralize nucleic acids is at least 9.
[0204] B. Non-Toxicity and Non-Immunogenic Properties
[0205] The composition of the polycationic agents of the invention
will exhibit the toxicity and immunogenic properties desired. In
vitro cell culture will have different immunogenic constraints than
in vivo mammalian applications.
[0206] The instant polycationic agents can be easily tested for
toxicity. For example, the agents can be added to medium for cells
used in the in vitro assays, such as cos-7, Chinese Hamster Ovary
cells, etc. Alternatively, the agents can be tested in standard
animal tests for safety.
[0207] C. Condensation Properties
[0208] Due to the electric charge, a subset of these polycationic
agents are capable of condensing the desired nucleic acids to a
compact size to facilitate delivery. Typically, condensation
"collapses" polynucleotides or nucleic acids into macromolecular
structures, commonly into a toroid form. The smaller size of
condensed nucleic acids eases delivery by facilitating, for
example, packaging nucleic acids into liposomes and/or reducing
exposure to proteases and/or nucleases.
[0209] The condensed nucleic acids exhibit different properties
compared to "relaxed" nucleic acids, such as (1) a decrease in
intercalation of ethidium bromide or other intercalating dye or (2)
a reduced mobility in gel electrophoresis. Thus, condensation can
be measured by at least two different assays, an intercalating dye
assay or a band shift assay.
[0210] One type of intercalating dye assay uses ethidium bromide.
In this assay, test nucleic acids, conveniently plasmid DNA, are
mixed with polycationic agent in a ratio from about 1:1 to a 1:50
weight/weight ration of plasmid to condensing agent. Following
incubation, ethidium bromide is added to the reaction to a final
concentration of 1 .mu.g/mL. If a nucleic acid such as RNA is used
as the test nucleic acid, acridine orange may be used as the
intercalating dye. The reaction mixtures are transferred into UV
transparent plastic tubes spotted with 1% agarose gel, or placed
upon UV transparent plastic c and illuminated with 260 nm light.
The emission from the DNA-ethidium bromide complex is recorded on
film by a camera equipped with an appropriate UV filter. The
ability of an agent to condense DNA is inversely proportional to
the intensity of the fluorescence in each reaction mixture.
[0211] The more precise test is a band shift assay. Briefly, this
assay is performed by incubating nucleic acids, either labeled or
unlabeled, with various concentrations of candidate condensing
agents. Test nucleic acids, conveniently plasmid DNA, and
condensing agent are mixed at 1:1 to 1:50 w/w ratios. Following
incubation, each sample is loaded on a 1% agarose gel and
electrophoresed. the gel is then either stained with ethidium
bromide or dried and autoradiographed. DNA condensation is
determined by the inability to enter the gel compared to a
non-condensed standard. Sufficient condensation is achieved when at
least 90% of the DNA fails to enter the gel to any significant
degree.
[0212] Condensation can also be measure by directly determining the
size of the complex using a light scattering instrument such as the
a Coulter N4MD submicron analyzer, for example. Polynucleotides and
a condensing agent are incubated at an appropriate ratio, either
alone or in the present of 2% PEG-2000 (Fisher Scientific), and 0.6
M NaCl., and then diluted into 3 mil of water. This dilute solution
is analyzed by the Coulter counter which will detect particles with
a mean size of 0-1,000 nanometers (nm). Condensing agents, such as
poly-L-lysine, typically yield particles with a mean diameter of
approximately 50-200 nm. See Lee et al., 1996, J. Biol. Chem. 271:
8481-8487.
[0213] D. Serum and/or Nuclease Protection Properties
[0214] The instant polycationic agents are capable of protecting
nucleic acids from degradation in serum or from nucleases,
including nucleases present in biological fluids, such as serum,
prostate, synovial fluid, etc. One advantage of this type of
protection is that smaller amounts of the desired nucleic acids are
needed for efficient administration.
[0215] When present in effective amounts, these polycationic agents
can inhibit serum degradation by at least 5 minutes as compared
with uncomplexed nucleic acids; more usually, the amount used is
sufficient to inhibit degradation by at least 10 minutes; even more
usually; the amount used is sufficient to inhibit degradation by at
least 30 minutes; even more usually, the amount used is sufficient
to inhibit degradation by at least 45 minutes; even more usually,
the amount used is sufficient to inhibit degradation by at least 60
minutes; even more usually, the amount used is sufficient to
inhibit degradation by at least 90 minutes; and more usually, the
amount used is sufficient to inhibit degradation by at least 120
minutes.
[0216] Increased serum protection can be measured simply by
incubation of the polycation/polynucleotide complex with mouse
serum, for example. Preferably, the serum will not be heat
inactivated. After incubation, the mixture can be analyzed by gel
electrophoresis to determine the quantity of the polynucleotides
remaining after incubation.
[0217] Alternatively, nucleases can be added to the polycationic
agent/nucleic acid complexes. The resulting mixture can be analyzed
by gel electrophoresis to determine the amount of degradation.
Other biological fluids, such as prostate flud, can also be
tested.
[0218] E. Mediating Entry of Polynucleotides into a Cell
[0219] The polycationic agents can mediate entry of polynucleotides
into a cell. Incorporation of polynucleotides into a cell can be
measured by either protein expression assays or polynucleotide
hybridization techniques, for example.
[0220] One method of detecting frequency of incorporation is to
include a gene that encodes a marker protein, such as luciferase.
Cells that have incorporated the delivered polynucleotides will
express the marker protein. The protein can be detected by standard
immunoassays, or by biological or enzymatic activity, as in the
case of luciferase.
[0221] Alternatively, standard hybridization techniques, such as
Southern or Northern blots or polymerase chain reaction (PCR)
techniques, can be used to detect the presence of the desired
polynucleotides.
[0222] F. Additional Properties
[0223] To facilitate entry of nucleic acids to the interior of
cells, the instant agents can be capable of [0224] (a) binding the
polynucleotide to the cell surface; [0225] (b) cell membrane
destabilization; [0226] (c) triggering endocytosis; [0227] (d)
endosome buffering capacity; [0228] (e) releasing DNA/lipid
complexes from endosomes; or [0229] (f) nuclear tropism. Assays for
detecting these characteristics are standard and known to those
skilled in the art. Physical Properties
[0230] The following physical characteristics are factors to
consider when determining the composition of the polycationic
agents: [0231] (a) distance between the substituents and the
backbone [0232] (b) the total length of the chain; [0233] (b)
hydrophobicity and/or aromacity; [0234] (c) number of hydrogen
bonding groups; and [0235] (c) charge, including [0236] (i) type of
charge group, (ii) density of charge and (iii) position. Other
relevant characteristics include structural flexibility. For
example, a helical conformation of the polycationic agent may be
preferred for some applications.
[0237] Specific dimensions to be considered include [0238] (a) the
distance of phosphate groups in the polynucleotide of interest; and
[0239] (b) the distance of monomer groups in the agents of
interest. Polypeptide Polycationic Agents
[0240] The following are examples of useful polypeptides as
polycationic agents: polylysine, polyarginine, polyornithine, and
protamine. Other examples include histones, protamines, human serum
albumin, DNA binding proteins, non-histone chromosomal proteins,
coat proteins from DNA viruses, such as .phi.X174, transcriptional
factors also contain domains that bind DNA and therefore may be
useful as nucleic aid condensing agents. Briefly, transcriptional
factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF,
Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains
that bind DNA sequences.
[0241] Organic polycationic agents include: spermine, spermidine,
and purtrescine.
[0242] The dimensions and of the physical properties of a
polycationic agent can be extrapolated from the list above, to
construct other polypeptide polycationic agents or to produce
synthetic polycationic agents. Synthetic Polycationic Agents
Synthetic polycationic agents which are useful include, for
example, DEAE-dextran, polybrene. Lipofectin.RTM., and
lipofectAMINE.TM. are monomers that form polycationic complexes
when combined with polynucleotides.
[0243] A preferred group of polycationic agents of the present
invention have the following general formula (I): ##STR3## A
preferred subset of these compounds include compounds having
formula (I) where R.sub.2 is hydrogen. Even more preferred are
polymers comprising at least one natural amino acid. Also preferred
are polymers where R.sub.2 and R.sub.3 are both hydrogen, also
referred to as poly N-substituted glycines or poly NSGs.
[0244] A. Monomers
[0245] The polycationic agent of the invention comprises monomers
with the following structure (II): ##STR4##
[0246] Generally, R.sub.1, R.sub.2, and R.sub.3 are moieties each
with a molecular weight from 1 to 250 daltons. More typically, the
molecular weight is no more than 200; even more typically, no more
than 175.
[0247] Typically, each monomer comprises one hydrogen at R.sub.1,
R.sub.2, or R.sub.3. More, typically, either R.sub.1 and R.sub.3
are both hydrogen, the structure of an L-amino acid; or R.sub.2 and
R.sub.3 are both hydrogen, the structure of a NSG.
[0248] Monomers to be utilized in the polycationic agents can be
either positively or negatively charged. Also, neutral substituents
can also be utilized.
[0249] Degradation sites can be incorporated into the polymer, for
example, by including substituents from a natural amino acid when
R.sub.1 and R.sub.3 are hydrogen. These monomers can be positively
or negatively charged, or neutral.
[0250] As a general rule, a basically charged monomer has a pKa
value for the side chain of at least 7.5. Positively, or basically,
charged monomers include without limitation those containing the
following functional groups: amino, guanidino, hydrazido, and
amidino. These functional groups can be either aromatic or
aliphatic.
[0251] Positively charged monomers comprising hydrogen at R.sub.3
and R.sub.1, can be included in the polycationic agent, for
example, as a degradation site. Such degradation site may aid in
separation of the polycationic agent from the polynucleotide to
permit further processing. For an L-amino acid like monomer, useful
R.sub.2 substitutents are, for example, from those found in
naturally occurring amino acids, such as lysine and arginine. Also,
sidechains from amino acid analogues can be used such as ornithine
and canaline; or modifications of basic amino acids, such as
homoarginine, and modifications of other amino acids such as
guanidinovalinate, and aminoethylcysteine. The substitutents found
in L-amino acids can also be incorporated at the R.sub.1 and
R.sub.3 positions of the instant polycationic agents.
[0252] Naturally occurring amino acids and analogues are designated
D-amino acids to indicate the chirality of these molecules. L-amino
acids can also incorporated as monomers into the polycationic
agents. The substituents of L-amino acids can be, for example, the
same as those named for the D-amino acids.
[0253] Preferable monomers include N-substituted glycine monomers.
Exemplary N-substitutions include alklphenyl, indolylalkyl,
alkoxyphenyl, halophenylalkyl, hydroxyphenylalkyl, as well as the
N-substitutions shown below. ##STR5##
[0254] The positively charged substituents described above can also
be placed at the R.sub.2 or R.sub.3 positions of formulas (I) and
(II).
[0255] The polycationic agents can comprise negatively charged or
neutral monomers. As with the positively charged monomers, D-amino
acid, L-amino acid, and NSGs are preferred to be incorporated as
monomers.
[0256] The following are examples of such monomers: ##STR6##
##STR7##
[0257] B. Polycationic Polymers
[0258] Typically, the polycationic agents exhibit a predicted
isoelectric point of at least 9, excluding the terminal groups.
Further, the agents contain, excluding the terminal groups, at
least 20% positively charged monomers; more typically, at least
25%; more typically, 30%; and preferably, at least 33% positively
charged monomers. Typically, the agents do not comprises greater
than 5% acidic monomers and preferably none.
[0259] The charge density and composition of the polycationic agent
can be altered to accommodate the specific nucleic acid sequence,
type, and other components included with the complex of nucleic
acids and polycationic agent.
[0260] Usually, the length of the polymer is at least 8 monomers;
even more usually, 12 monomers; even more usually, 18 monomers.
More typically, the polycationic agents of the invention will be at
least 24 monomer units in length; more typically, 30 monomer units;
even more typically, 36 monomer units; even more typically, 48
monomer units. The polycationic agent can be up to 50 to 75 to 100
monomer units in length.
[0261] Preferably, the polycationic agent comprises monomers where
all R.sub.2 and R.sub.3 are hydrogen. Even more preferably, where
all R.sub.2 and R.sub.3 are hydrogen, the polycationic agent
comprise repeating trimer units with the following monomer sequence
(from amino to carboxy terminus): (1) neutral monomer, (2) neutral
monomer, and (3) positively charged monomer.
[0262] Preferably, the neutral monomer comprises an aromatic group
at the R.sub.1 position; more preferably, wherein the aromatic
group comprises a single ring; even more preferably, wherein the
aromatic group is a six member ring.
[0263] Typically, the positively charged monomer is aminoalkyl at
the R.sub.1 position; more typically, the aminoalkyl comprises 1-6
carbon molecules; even more typically, the aminoalkyl is
aminoethyl.
[0264] Typically, the polycationic agent comprises between 3 to 20
repeating trimers, trimers having two neutral and one positively
charged R.sub.1 groups are preferred, such as, for example, trimer
shaving the sequence, neutral monomer, neutral monomer, positively
charged monomer. More preferably, the polycationic agent comprises
5 to 18 trimers; preferably 8 to 16 trimers; and even more
preferably, 12 to 16 trimers.
[0265] Optionally, the polycationic agent contains only positively
charged monomers, excluding the terminal groups. Typically, such a
polycationic agent comprises between 24 and 48 monomers; more
typically, 30 to 40 monomers; even more typically, 36 monomers.
[0266] Polycationic agents of the present invention containing only
positively charged monomers typically have guanidinoalkyl
sidechains. Typically, the guanidinoalkyl sidechain comprises 1 to
6 carbon molecules. Preferably, the side chain is guanidino
ethyl.
[0267] C. Neutral Polymers
[0268] A preferred group of neutral polymers of the present
invention have the general formula (I): ##STR8## Preferably,
R.sub.2 is hydrogen. Even more preferred are polymers comprising at
least one natural amino acid. Also preferred are polymers having
formula (I) where R.sub.2 and R.sub.3 are hydrogen, also referred
to as poly N-substituted glycines or poly NSGs.
[0269] Monomers employed in neutral polymers of the present
invention have the same general formula as monomers employed in
cationic polymers of the present invention, i.e.: ##STR9##
[0270] Generally, R.sub.1, R.sub.2, and R.sub.3 are moieties each
with a molecular weight from 1 to 250 daltons. More typically, the
molecular weight is no more than 200; even more typically, no more
than 175.
[0271] Typically, each monomer comprises one hydrogen at R.sub.1,
R.sub.2, or R.sub.3. More, typically, either R.sub.1 and R.sub.3
are both hydrogen, the structure of a L-amino acid; or R.sub.2 and
R.sub.3 are both hydrogen, the structure of a NSG.
[0272] Monomers to be utilized in the neutral agents can be either
positively or negatively charged. Also, neutral substituents can
also be utilized. Neutral polymers exhibit no net positive or
negative charge, excluding the terminal groups.
[0273] Degradation sites can be incorporated into the polymers by
using naturally occuring amino acid substituents in monomers when
R.sub.1 and R.sub.3 are hydrogen.
[0274] Naturally occurring amino acids and analogues are designated
D-amino acids to indicate the chirality of these molecules. L-amino
acids can also incorporated as monomers into the neutral polymers.
The substituents of L-amino acids can be, for example, the same as
those named for the D-amino acids.
[0275] Preferred monomers include N-substituted glycine monomers,
and monomers that are capable of forming hydrogen bonds and/or
ionic bonds with the polynucleotides to be delivered.
[0276] Examples of monomers for the neutral polymers include those
described above and in the Examples below.
[0277] D. Linking Polymers Together
[0278] Polymers can be linked together incorporating terminating
groups or sidechains that permit cross-linking of the polymers. For
example, polymers can be linked by a disulfide bond. Other
terminating groups useful for coupling polymers include, carbonate,
urea, and the like.
[0279] E. Additional Groups to be Incorporated into the Polymer
[0280] Additional components can be included in the polycationic
agents of the instant invention, such as targeting ligands. Such
additional groups can facilitate endocytosis of the desired nucleic
acids or aid binding of the nucleic acids to the cell surface.
[0281] Polypeptides can be incorporated into the polycationic
agents. Examples include, without limitation: asialoorosomucoid
(ASOR); transferrin; asialoglycoproteins; antibodies; antibody
fragments; ferritin; interleukins; interferons, granulocyte,
macrophage colony stimulating factor (GM-CSF), granulocyte colony
stimulating factor (G-CSF), macrophage colony stimulating factor
(M-CSF), stem cell factor and erythropoietin. Viral antigens, such
as envelope proteins, can also be used. Also, proteins from other
invasive organisms are useful, such as the 17 amino acid peptide
from the circumsporozoite protein of plasmodium falciparum known as
RII.
[0282] In addition, lipoproteins can be incorporated into the
polycationic agent, such as low density lipoprotein, high density
lipoprotein, or very low density lipoprotein. Mutants, fragments,
or fusions of these proteins can also be used.
[0283] Other groups that can be incorporated include without
limitation: hormones, steroids, androgens, estrogens, thyroid
hormone, or vitamins, folic acid. Folic acid can be incorporated
into the polycationic agent according, for example, to Mislick et
al., 1995, T.J. Bioconjugate Chem. 6:512.
[0284] Also, the polycationic agents of the instant invention can
be chemically conjugated with polyalkylene glycol. In a preferred
embodiment, the polyalkylene glycol is polyethlylene glycol. PEG
can be incorporated with a polycation agent according, for example,
to Lu et al., 1994, Int. J. Pept. Protein Res. 43:127.
[0285] In addition, the polycationic agent can be chemically
conjugated with mono-, di-, or polysaccaride. In a preferred
embodiment of this aspect, the polysaccharide is dextran.
[0286] These additional groups can be incorporated within the
polycationic agent. For example, R.sub.1, R.sub.2, and R.sub.3 can
be a substituent that is capable of being activated to cross link
with any one of the above groups. For example, a thiol group could
be included to cross link with another group to form a disulfide
bond.
[0287] F. Terminal Groups
[0288] The terminal groups of the instant polycationic agents can
be chosen as convenient. Suitable terminal groups (i.e., Ta and Tc)
include, for example, --NH.sub.2, --OH, --SH, and --COOH. Terminal
groups can be selected to enhance the targeting properties of the
polycationic agent and can be any of the additional groups
described above.
[0289] The additional groups described above can be incorporated at
the terminus of the polycationic agent. For example, the
polycationic agent can be (1) acylated with a variety of carboxylic
acids; (2) sulfonylated with sulfonyl chlorides; or (3) derivatized
with isocyanates or isothiocyanates. Once activated, the terminus
can be reacted with any of the above-mentioned groups, such as a
polypeptide, such as low density lipoprotein, or folic acid.
[0290] One means of adding a terminal group to the polycationic
agent is, for example, is (1) to acylate the amino terminus with
Fmoc-amino-hexanoic acid; and (2) to remove the protecting group,
Fmoc, to generate a primary amine, which can be further
functionalized.
[0291] Alternatively, the amino-terminal groups can include,
without limitation: acyl, such as acetyl, benzoyl; or sulfonyl,
such as dansyl.
[0292] Carboxy terminal groups can include, for example, amide or
alkyl amide.
Synthesis of Polycationic Agents
[0293] Polycationic agents of the present invention can be
synthesized by either solid or solution phase methods. The
following is a solid phase method for the synthesis of NSGs, which
can be generally used for a wide variety of side-chain
substitutents. This method can be performed utilizing automated
peptide synthesis instrumentation to permit rapid synthesis of
polycationic agents of interest. Such instruments are commercially
available from, for example, Applied Biosystems and Milligen.
[0294] A. Two Step Monomer Assembly
[0295] A method of synthesis is to assemble the monomer from two
submonomers in the course of extending a polymer comprising an NSG
monomer. This technique is described in Zuckermann et al., 1992, J
Amer Chem Soc 114(26):10646-10647, and Zuckermann et al., PCT
Patent Publication No. WO 94/06451. The NSGs can also be considered
to be an alternating condensation of copolymer of an acylating
agent and an amine.
[0296] The direction of polymer synthesis with the submonomers
occurs in the carboxy to amino direction. The solid-phase assembly
for each monomer, in the course of polymer formation, eliminates
the need for N.alpha.-protected monomers, as only reactive
side-chain functionalities need to be protected. Each monomer
addition comprises two steps, an acylation step and a nucleophilic
displacement step as shown in FIG. 1.
[0297] Specifically, each cycle of monomer addition consists of two
steps: [0298] (1) acylation of a secondary amine bound to the
support with an acylating agent comprising a leaving group capable
of nucleophilic displacement by an amine and a carbonyl group,
preferably carboxyl. An example is a haloacetic acid; and [0299]
(2) nucleophilic displacement of the leaving group with a
sufficient amount of a submonomer comprising a primary amino group
to introduce a side-chain. The amino group containing submonomer
can be an alkoxyamine, semicarbazide, acyl hydrazide, substituted
hydrazine or the like.
[0300] Acylation can be activitated with carbodiimide or other
suitable carboxylate activation method.
[0301] The efficiency of the displacement is modulated by the
choice of halide, e.g., I>Cl. Protection of aliphatic hydroxyl
groups, carboxylic acids, carboxy, thiol, amino, some heterocycles,
and other reactive side-chain functionalities is preferred to
minimize undesired side reactions. However, the mild reactivity of
some side-chain moieties toward displacement or acylation may allow
their use without protection., e.g., indole, imidazole, and
phenol.
[0302] B. Three Step Monomer Assembly
[0303] NSGs can also be constructed utilizing a three step method
for assembling each monomer as the polymer is extended. The
backbone of the monomer of first extended by acylation step
followed by a nucleophilic displacement. The side chain is
introduced by a second acylation step. The reaction scheme is shown
in FIG. 2.
[0304] The backbone of the monomer is assembled in the first two
steps of the synthesis cycle. The first reaction is an acylation
step where the carbonyl group of the acylating agent reacts with an
amine. The acylating agent comprises a carbonyl group; a backbone,
R.sub.a; and a leaving group, L. Preferably, the carbonyl group is
carboxyl.
[0305] The second step is a nucleophilic displacement of the
leaving group by the first amino group of the displacing agent. The
displacing agent comprises a first and a second amino group and a
backbone, R.sub.d. The first amino group is a primary amine, and
the second step produces a secondary amine.
[0306] The third step is another acylation in which the another
acylating submonomer reacts with the first amino group of the
displacing agent to produce a tertiary amide. The acylation agent
comprises a carbonyl group; an optional linker; and a sidechain.
Preferably, the carbonyl group is carboxyl.
Pharmaceutical Compositions
[0307] The polycationic agent/polynucleotide complexes, whether or
not encapsulated in liposomes, may be administered in
pharmaceutical compositions. The pharmaceutical compositions
comprise a therapeutically effective amount of nucleic acids.
[0308] The term "therapeutically effective amount" as used herein
refers to an amount of a therapeutic agent sufficient to detectably
treat, ameliorate, or prevent a particular disease or condition,
i.e., an amount sufficient to induce a detectable therapeutic or
preventative effect. The effect may include, for example, chemical
markers or antigen levels. Therapeutic effects also include
reduction in physical symptoms, such as decreased body temperature.
The precise effective amount for a subject will depend upon the
subject's size and health, the nature and extent of the
cardiovascular condition, and the therapeutics or combination of
therapeutics selected for administration. Thus, it is not useful to
specify an exact effective amount in advance. However, the
effective amount for a given situation can be determined by routine
experimentation and is within the judgment of the clinician. For
purposes of the present invention, an effective dose will be from
about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the
DNA constructs in the individual to which it is administered.
[0309] A pharmaceutical composition can also contain a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" refers to a carrier for administration of a
therapeutic agent, such as antibodies or a polypeptide, genes, and
other therapeutic agents. The term refers to any pharmaceutical
carrier that does not itself induce the production of antibodies
harmful to the individual receiving the composition, and which may
be administered without undue toxicity. Suitable carriers may be
large, slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, and inactive virus particles.
Such carriers are well known to those of ordinary skill in the
art.
[0310] Pharmaceutically acceptable salts can be used therein, for
example, mineral acid salts such as hydrochlorides, hydrobromides,
phosphates, sulfates, and the like; and the salts of organic acids
such as acetates, propionates, malonates, benzoates, and the like.
A thorough discussion of pharmaceutically acceptable excipients is
available in Remington's Pharmaceutical Sciences (Mack Pub. Co.,
N.J. 1991).
[0311] Pharmaceutically acceptable carriers in therapeutic
compositions may contain liquids such as water, saline, glycerol
and ethanol. Additionally, auxiliary substances, such as wetting or
emulsifying agents, pH buffering substances, and the like, may be
present in such vehicles. Typically, the therapeutic compositions
are prepared as injectables, either as liquid solutions or
suspensions; solid forms suitable for solution in, or suspension
in, liquid vehicles prior to injection may also be prepared.
Liposomes are included within the definition of a pharmaceutically
acceptable carrier.
Delivery Methods
[0312] Once formulated, the compositions of the invention can be
administered (1) directly to the subject; (2) delivered ex vivo, to
cells derived from the subject; or (3) in vitro for expression of
recombinant proteins. The subjects to be treated can be mammals or
birds. Also, human subjects can be treated.
[0313] Direct delivery of the compositions will generally be
accomplished by injection, either subcutaneously,
intraperitoneally, intravenously or intramuscularly or delivered to
the interstitial space of a tissue. The compositions can also be
administered into a tumor or lesion. Other modes of administration
include oral and pulmonary administration, suppositories, and
transdermal applications, needles, and gene guns or hyposprays.
Dosage treatment may be a single dose schedule or a multiple dose
schedule.
[0314] Methods for the ex vivo delivery and reimplantation of
transformed cells into a subject are known in the art and described
in e.g., International Publication No. WO 93/14778 (published Aug.
5, 1993). Examples of cells useful in ex vivo applications include,
for example, stem cells, particularly hematopoetic, lymph cells,
macrophages, dendritic cells, or tumor cells.
[0315] Generally, delivery of nucleic acids for both ex vivo and in
vitro applications can be accomplished by the following procedures,
for example, dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei, all
well known in the art.
[0316] The examples presented below are provided as a further guide
to the practitioner of ordinary skill in the art, and are not to be
construed as limiting the invention in any way.
EXAMPLE 1
Synthesis of Polycationic Agents
[0317] This example describes the synthesis of polycationic agents
with the following structure: ##STR10## where R.sub.3 and R.sub.2
are hydrogen for all monomers. All polymers describe in this
example terminate in an amino and a carboxyl group unless
specified, such as a folate terminating group.
[0318] The polycationic agents described below were synthesized
according to the procedures described in Figliozzi et al., 1996,
Meth. Enzy. 267:437-447, and Zuckermann et al., 1992, J. Amer.
Chem. Soc. 114(26):10646-10647.
[0319] All polymers were synthesized using bromoacetic acid and
primary amines. The following are substitutents of the primary
amines to be positioned at R.sub.1 to construct the polycationic
agents: TABLE-US-00001 Cationic Sidechains Other Sidechains
##STR11## P = aminoethyl ##STR12## H = (S)-.alpha.-methylbenzyl
##STR13## Pr = aminopropyl ##STR14## H+ = (R)-.alpha.-methylbenzyl
##STR15## P* = (S)-1-methylethylenediamine ##STR16## Bn = benzyl
##STR17## Q = trimethylaminoethyl ##STR18## Ph = phenethyl
##STR19## G = guanidinoethyl ##STR20## Nm = naphthylmethyl
(R.dbd.H) sN = (S)-.alpha.-methylnaphthylmethyl (R.dbd.CH.sub.3)
##STR21## Gp = guanidinopropyl ##STR22## Py = N-pyrrolidinopropyl
##STR23## P.sup.1 = aminohexyl ##STR24## Chm = cyclohexylmethyl
##STR25## Ff = furfurylmethyl ##STR26## Tmb =
3,4,5-trimethoxybenzyl ##STR27## Me = methoxyethyl ##STR28## Phpr =
phenylpropyl ##STR29## 6-gal = 6-galactosyl ##STR30## Trp =
3'-indolylethyl ##STR31## p-MeOPh = p-methoxyphenylethyl ##STR32##
pClPh = p-chlorophenylethyl ##STR33## Tyr = p-hydroxyphenylethyl
Peptoid-Folic acid conjugates ##STR34## ##STR35## Abbreviation
Description Bn benzyl Chm cyclohexylmethyl Ff furfurylmethyl G
guanidinoethyl Gp guanidinopropyl H (S) alpha-methylbenzyl H+ (R)
alpha-methylbenzyl Me methoxyethyl Nm naphthylmethyl P aminoethyl
P` aminohexyl P* (S)-.alpha.-methylaminoethyl Ph phenethyl Pr
aminopropyl Py N-pyrrolidinopropyl Tmb 3,4,5-trimethoxybenzyl Q
trimethylaminoethyl Phpr phenylpropyl 6-gal 6-galactosyl Trp
N-2-(3-indolylethyl) pMeOph p-methoxyphenethyl pClPh
p-hydroxyphenethyl Tyr p-hydroxyphenethyl sN
(S)-.alpha.-methylnaphthylmethyl
[0320] The polycationic agents synthesized include: TABLE-US-00002
Mol. Name Sequence Length Wt. # charges RZ110-1 (HHP)6 18 2550.8 7
RZ110-2 (HP)9 18 2367.8 10 RZ110-3 (HPP)6 18 2184.8 13 RZ110-4
(HPPP)4HP 18 2123.8 14 RZ110-5 (HHP')6 18 2869.8 7 RZ110-6 (HP')9
18 2871.8 10 RZ110-7 (HP'P')6 18 2856.8 13 RZ110-8 (HP'P'P')4HP' 18
2851.8 14 RZ110-9 (HHP)12 36 5084.6 13 RZ110-10 (HP)18 36 4718.6 19
RZ110-11 (HPP)12 36 4352.6 25 RZ110-12 PP(HPPP)8HP 36 4230.6 27
RZ110-13 (HHP')12 36 5722.6 13 RZ110-14 (HP)18 36 5726.6 19
RZ110-15 (HP'P')12 36 5696.6 25 RZ110-16 P'P'(HP'P'P')8HP' 36
5686.6 27 RZ112-1 (Q)36 36 5181.5 37 RZ112-2 (G)36 36 5130.8 37
RZ112-3 (HP*P*P*)9 36 4529.3 28 RZ112-4 (P*)36 36 4122.8 37 RZ112-5
(HP*P*P*)4HP* 18 2305.2 14 RZ112-6 (P*)18 18 2069.9 19 RZ112-7
(P)18 18 1817.7 19 RZ112-8 (P)36 36 3618.4 37 RZ120-1 (MeMeP)8 24
2658.4 9 R2120-2.sup. (BnBnP)8 24 3170.8 9 RZ120-3 (HHP)8 24 3394.8
9 RZ120-4 (H + H + P)8 24 3394.8 9 RZ120-5 (MeMeP)12 36 3979.2 13
RZ120-6 (BnBnP)12 36 4747.6 13 RZ120-7 (HHP)12 36 5083.6 13 RZ120-8
(H + H + P)12 36 5083.6 13 RZ120-9 (MeMeP)16 48 5299.9 17 RZ120-10
(BnBnP)16 48 6324.5 17 RZ120-11 (HHP)16 48 6772.5 17 RZ120-12 (H +
H + P)16 48 6772.5 17 RZ120-13 (HHP)12 folate 36 5300 13 RZ123-1
(HHPr)12 36 5252 13 RZ123-2 (HHPr)12 36 5252 13 RZ123-3 (HHP)12 36
5084 13 RZ123-4 folate-(HHPr)12 36 5862 13 RZ123-5 (HHGp)12 36 5756
13 RZ123-6 (HHG)12 36 5588 13 RZ124-1 (HHPr)12 36 5252 13 RZ124-2
(sNsNPr)12 36 6452 13 RZ124-3 (NmNmPr)12 36 6116 13 RZ124-4
(PyPyPr) 36 5756 13 RZ124-5 (HHPy)12 36 6069 13 RZ124-6 (Py)36 36
6573 1 RZ124-7 folate-(HHPr)12 36 5862 13 RZ127-1 (PhPhP)12 36 5085
13 RZ127-2 (ChmChmP)12 36 4895 13 RZ127-3 (TmbTmbP)12 36 6912 13
RZ127-4 (FfFfP)12 36 4508 13 RZ136-3 (PhprPhprP)12 36 5419 13
RZ140-2 (6-gal)12-(PhPhP)12 48 7712 13 RZ140-3 (TrpTrpP)12 36 6020
13 RZ144-1 (PhPPh)12 36 5083 13 RZ144-2 (PPhPh)12 36 5083 13
RZ144-3 (pMeoPhpMeoPhP) 36 5803 13 RZ144-4 (pClPhpClPhP)12 36 5910
13 RZ144-5 AMCA-(PhPhP)12 36 5411 12 RZ144-8 (TyrTyrP)12 36 5467 12
RZ144-12 (6gal 6gal P)12 36 6475 13 *RZ145-1 (PhPhP)12 36 5085 13
RZ147-2 (PpMeOPhpMeOPh)12 36 5805 13 *purified
[0321] To summarize the method, Fmoc-Rink amide resin (NovaBiochem,
San Diego, Calif., USA) is used as the solid support. This is the
same resin that is used for the Fmoc synthesis of peptide
C-terminal amides. The polycationic synthesis begins with the
deprotection of the Fmoc group on the resin with 20% (v/v)
piperidine-dimethylformamide (DMF). The amino resin is then
acylated with bromoacetic acid. This is followed by nucleophilic
displacement of the bromide with a primary amine to build the NSG
monomer. The latter two steps are then continued in an iterative
fashion to elaborate the desired oligomer.
[0322] All reactions and washings were performed at room
temperature unless otherwise noted. Washing of the resin refers to
the addition of a wash solvent (usually DMF or dimethylsulfoxide
(DMSO)) to the resin, agitating the resin so that a uniform slurry
is obtained (typically for about 20 seconds), followed by thorough
draining of the solvent from the resin. Solvents were removed by
vacuum filtration through the fritted bottom of the reaction vessel
until the resin appeared dry (typically about 5 seconds). In all
the syntheses, resin slurries were agitated via bubbling argon up
through the bottom of the fritted vessel.
[0323] A fritted reaction vessel was charged with 100 mg (50
.mu.mol) of Fmoc-Rink amide resin with a substitution level
.about.0.50 mml/g resin. Two milliliters of DMF was added to the
resin and this solution was agitated for 1-2 minutes to swell the
resin. The DMF was then drained. The Fmoc group was then removed by
adding 2.0 ml of 20% piperidine in DMF to the resin. This was
agitated for 1 minute and then drained. Another 2 ml of 20%
piperidine in DMF was added to the resin and agitated for 15
minutes and then drained. The resin was then washed with DMF, six
times with 2 ml.
[0324] The deblocked amine was then acylated by adding 850 .mu.l of
0.6 M bromoacetic acid in DMF to the resin followed by 200 .mu.l of
3.2 M N,N'-diisoprooplycarbodiimide (DIC) in DMF. This solution was
agitated for 30 minutes at room temperature and then drained. This
step was repeated a second time. The resin was then washed with
DMF, twice with 2 ml and DMSO, once with 2 ml. This completed one
reaction cycle.
[0325] The second cycle was initiated by the acylating step with
bromoacetic acid and DIC, followed by displacement with the second
amine. This acylation/displacement cycle was repeated until the
desired oligomer was obtained.
[0326] Cleavage of the resin from the polycationic agent is as
follows. The dried resin was placed in a glass scintillation vial
containing a teflon-coated micro stir bar, and approximately 5 ml
of 95% trifluoroacetic acid (TFA) in water was added. The solution
was stirred for 20 minutes and then filtered through an 8-ml
solid-phase extraction (SPE) column fitted with a 20-.mu.m
polyethylene frit into a 50 ml polypropylene conical centrifuge
tube.
[0327] The resin was washed with 1 ml 95% TFA. The combined
filtrates were then lyophilized three times from 1:1
acetonitrile:water. Material was redissolved to a concentration of
5 mM in 5% acetonitrile in water.
Preparation of Guanidinoalkyl-Containing Polymers:
[0328] The guanidinoalkyl sidechains were introduced into the
polymers by post-synthesis modification of aminoalkyl sidechains.
Thus, polymers were synthesized by the submonomer method as
described above except that methoxybenzhydrylanine (MBHA) resin was
used instead of the Rink resin. Wherever a guanidinoalky sidechain
was desired, a mono-Boc-alkanediamine was incorporated in the
displacement step. After elaboration of the polymers, the sidechain
Boc groups were removed by treatment with 95% TFA/water for 20 min
at room temp. (This does not remove the oligomer from the soliud
support). The free amino groups were then guanidinylated by
treatment with 1H-pyrazole-1-carboxamidine (1 M in DMF, 2.times.1
hr, 40.degree. C.). After washing with DMF and methylene chloride,
the oligomer was cleaved from the resin with hydrofluoric acid, and
lyophilized.
Preparation of Folic Acid--Polymer Conjugates:
[0329] Folic acid--polymer conjugates were prepared by adding a
linker to the N-terminus of the resin-bound polymer which was then
acylated with folic acid. Specifically, after elaboration of the
polymer, the N-terminus was acylated with Fmoc-aminohexanoic acid
(0.5 M in DMF, 0.5 M hydroxybenzotriazole, 0.5 M
diisopropylcarbodiimide (DIC), 1.times.1 hr, room temp.). After
Fmoc group removal (20% piperidine/DMF, 1.times.20 min, room
temp.), the free primary amino group was acylated with folic acid
(0.1 M in DMSO, 0.1 M DIC, 1.times.2 hr, 50.degree. C.). After
washing of the resin, the conjugate was cleaved with 95% TFA/water
in the usual fashion.
EXAMPLE 2
Condensation of Polynucleotides
[0330] Polycationic agents were synthesized and isolated to a final
concentration of 5 mM as described in Example 1. Polynucleotides
were condensed with RZ110, RZ112, and RZ120 series compounds
according to the following procedure. [0331] (1) Dilute all
polycationic agents to a final concentration of 3 nanomoles of
positive charge per microliter. [0332] (2) Add 1 .mu.g of DNA to
1-2 .mu.l of diluted polycationic agents. [0333] (3) Adjust volume
to 10 .mu.l. This mixture can be stored overnight at 4.degree. C.
[0334] (4) Add of 5 .mu.l of DNA/polycationic mixture to 2 .mu.l of
5.times.buffer, which does not contain SDS to maintain the complex.
(5.times.buffer=40% sucrose, 0.25% bromphenol blue and 200 mM Tris
Acetate, 4 mM EDTA (PH 7.8). [0335] (5) Adjust volume to 10 .mu.l.
[0336] (6) Run sample on a 1% agarose gel utilizing 75 volts for
1.5 hours.
[0337] Between 1 to 2 .mu.l, all polycationic agents were judged to
retard the migration of DNA into an agarose gel.
EXAMPLE 3
Inhibition of Serum Degradation
[0338] The RZ110, RZ112, and RZ120 series compounds were mixed with
polynucleotide as described in Example 2. Five microliters of the
overnight mixture was added to 5 .mu.l of BalbC mouse serum. The
serum was not heat treated but freeze thawed. The serum,
polycationic agent, and polynucleotide mixture was incubated
typically for 30 minutes at 37.degree. C. The time of incubation
varied between 5 and 60 minutes
[0339] Next, 2 .mu.l of 5.times.buffer containing 0.5% (wt/v) SDS
was added to the incubated mixture. This final solution was loaded
onto a 1% agarose gel and electrophoresed at 75 volts for 1.5
hours.
[0340] All of the compounds tested, i.e., the entire RZ110, 112,
and 120 series, provided significant protection in a direct
comparison. The entire RZ112 series and RZ110-3 and RZ110-8
inhibited serum degradation better than poly-L-lysine.
EXAMPLE 4
Peptoid Mediated in vitro Delivery
[0341] DNA comprising a luciferase gene 1 .mu.g/.mu.l, was diluted
into endotoxin free water. The plasmid DNA was CMVKm luciferase,
which is described in more detail in Example 5.
[0342] The transfection protocol for in vitro delivery was as
follows: [0343] (A) HT1080 cells were used. These cells are
available from American Type Culture Collection, Rockville, Md.,
USA, Accession No. CCL 121. This is a fibrosarcoma. The growth
medium was Dulbecco's Modified Eagle medium (DME) with 10%
heat-inactivated fetal calf serum. [0344] (B) Twenty four hours
prior to transfection, the cells were placed at 5.times.10.sup.4
per well of a 24-well plate in 1 ml of medium. [0345] 1. Feed cells
with 500 .mu.l of DME-10% fetal calf serum (FCS) or 500 .mu.l
Opti-MEM.RTM.. Opti-Mem.RTM. can be purchased from Gibco BRL, Life
Technologies, Inc., Gaithersburg, Md., USA. [0346] 2. Add 200 .mu.l
Opti-MEM.RTM. to each tube. [0347] 3. Add 3 .mu.l of the desired
polycationic agent to the 200 .mu.l of Opti-MEM.RTM.. [0348] 4. Add
2 .mu.l of 1 .mu.g/.mu.l luciferase DNA, mix. [0349] 5. Incubate
mixture for 5 minutes at room temperature. [0350] 6. Add 100 .mu.l
of the polycationic agent/DNA mixture to plate with DME-FCS, 100
.mu.l to cells fed with Opti-MEM.RTM.. [0351] 7. Incubate cells and
polycationic agent/DNA mixture for .about.4 hours at 37.degree. C.
[0352] 8. Change media on all cells to DME-FCS. [0353] 9. DME-FCS
was used as a positive control. As a control, a transfectant, LT1,
was used from Panvera, Inc., Madison, Wis., USA to transfect cells
in serum and cells in Opti-MEM.RTM.. [0354] 10. Cells were tested
for luciferase activity using a Promega Luciferase Assay System
from Promega, Madison, Wis., USA., in accordance with the
manufacturer's directions.
[0355] Results: TABLE-US-00003 Luciferase Name Formula (RLU)
RZ120-1 (MeMeP)8 0 RZ120-2 (BnBnP)8 0.93 RZ120-3 (HHP)8 1.38
RZ120-4 (H + H + P)8 1.5 RZ120-5 (MeMeP)12 0 RZ120-6 (BnBnP)12 1.64
RZ120-7 (HHP)12 2.64 RZ120-8 (H + H + P)12 2.84 RZ120-9 (MeMeP)16 0
RZ120-10 (BnBnP)16 1.42 RZ120-11 (HHP)16 1.94 RZ120-12 (H + H +
P)16 1.32 control LT1 51.96 Experiment #2 RZ110-1 (HHP)6 0.0015
RZ110-2 (HP)9 0.0012 RZ110-4 (HPPP)4HP 0.0004 RZ110-5 (HHP')6
0.0006 RZ110-6 (HP')9 0.0052 RZ110-7 (HP'P')6 0.0005 RZ110-8
(HP'P'P')4HP' 0.0003 RZ110-9 (HHP)12 8.7 RZ110-10 (HP)18 0.0014
RZ110-12 PP(HPPP)8HP 0.0459 RZ110-13 (HHP')12 2.5 RZ110-14 (HP')18
2.2 RZ110-15 (HP'P')12 0.064 RZ110-16 P'P'(HP'P'P')8 0.01 control
LT 1 88.7
EXAMPLE 5
Targeting Ligand
A. Cells, Vector, and Compositions Used.
[0356] In a first experiment, murine endothelial cells (Py-4-1)
which express high levels of acetylated-LDL receptors. The cells
and the LDL receptors are described in Dubois et al., 1991, Exp.
Cell Res. 196:302-313.
[0357] A luciferase-containing plasmid (pCMVkmLUC) was used to
determine if polynucleotides could be delivered and expressed into
endothelial cells when associated with polycationic agents
described in Example 1 with acetylated-LDL (Ac-LDL). A description
of the identification and isolation of endothelial cells based on
their increased uptake of acetylated-low density lipoprotein is in
Voyta et al., 1984, J. Cell Biol. 99: 2034-2040.
[0358] The plasmid used in these experiments pCMVkmLUC, was
constructed by inserting the luc+gene from pSP-luc+ (Promega
Corporation, Madison, Wis.) into the expression vector pCMVkm2.
Briefly, pSP-luc+ was digested with the restriction enzymes
Nhe1-EcoRV (Boehringer Mannheim, Indianapolis, Ind.) and a fragment
of 1691 bp was isolated by standard methods. This fragment was
inserted into pCMVkm2, which had been digested with XbaI and EcoRV
using the Rapid Ligation Kit (Boehringer Mannheim, Indianapolis,
Ind.). The sequence of pCMVkm2 is depicted in SEQ ID NO:2 and
described below. The luc+gene was cloned into pCMVkm2 such that
expression is driven by the CMV immediate early enhancer promoter
and terminated by the bovine growth hormone polyadenylation
signal.
[0359] The luciferase expression was compared to levels obtained
with the same vector delivered in conjunction with lipofectamine,
an agent used commonly to transfect cells in vitro (Hawley-Nelson
et al., 1993, Focus 15:73). The results are presented in the table
below.
B. Method of Transfection:
[0360] Briefly, the cells were plated in 24 well dishes, grown to
approximately 80% confluence, transfected and assayed 24 hours
later for luciferase activity. All transfections were done in serum
containing medium. During transfection mixture preparation,
pCMVkmLUC was first mixed with RZ 112, and the DNA-cationic
polycationic agent complexes were then added to Ac-LDL. Serum
containing medium was then added to the mixtures to adjust the
volume delivered to each well to 0.5 ml.
[0361] Lipofectamine was used as a positive control. No lipoprotein
was added to this positive control. Lipofectamine is a 3:1 (w/w)
liposome formulation of the polycationic lipid
2,3,-dioleylosy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanami-
niumtrifluoroacetate (DOSPA) and the neutral lipid dioleoyl
phosphatidyl-ethanolamine (DOPE) in membrane-filtered water.
Lipofectamine can be purchased from Life Technologies,
Gaithersburg, Md., USA).
C. Luciferase Assay
[0362] Luciferase activity was assayed using the Promega Luciferase
Assay System, Madison, Wis.
D. Results
[0363] Table 1 shows the results of an experiment where the
polycationic agent, RZ-112-2 was compared to lipofectamine to
deliver the luciferase gene to cells comprising the acetylated LDL
receptor. TABLE-US-00004 TABLE 1 Luciferase Activity Ac-LDL
pCMVkmLUC RZ 112-2 pg luc/mg Group (.mu.g) (.mu.g) (nm) protein 1 5
10 2.5 61 2 -- 10 -- 0 3 -- 10 2.5 16 4 0.5 1 0.25 16 5 -- 1 0.25
17 6 0.5 10 2.5 631 7 0.5 10 5 1996 LIPOFECTAMINE CONTROL 8 -- 10
-- 10786 *each number represents the mean of the three wells.
EXAMPLE 6
Comparison of Cells With and Without Acetylated LDL Receptors
A. Cells With Acetylated LDL Receptors
[0364] For this experiment, K1735 mouse, epithelial melanoma cells
were used. These cells express low or non-existent levels of Ac-LDL
receptors. A description of the cells is in J. Natl. Cancer Inst.
69(4): (1982).
B. Methods
[0365] Briefly, the cells were plated in 24 well dishes at 10,000
cells per well in DME with 10% FCS supplemented with 2 mM
L-glutamine. The Py4-1 cells were cultured in 10% CO.sub.2 at
37.degree. C. The K1735 cells were cultured in 5% CO.sub.2 at
37.degree. C. The cells were grown to approximately 50% confluence,
transfected and assayed 24 hours later for luciferase activity. All
transfections were done in serum containing medium.
[0366] During transfection mixture preparation, pCMVkmLUC was first
mixed with RZ 112-2, and the DNA-polycationic agent complexes were
then added to Ac-LDL. Serum containing medium was then added to the
mixtures to adjust the volume delivered to each well to 0.5 ml.
[0367] C. Results TABLE-US-00005 TABLE 2 Luciferase Activity Ac-LDL
pCMVkmLUC RZ 112-2 pg luc/mg protein* Group (.mu.g) (.mu.g) (nm)
Py-4-1 K1735 1 0.5 1 1 1301 24 2 0.5 1 5 2181 0 3 0.5 1 10 373 0 4
0.5 10 5 840 0 5 -- 1 5 327 0 6 -- 1 5 945 ND 10 5 1 5 298 ND
LIPOFECTAMINE CONTROL 7 -- 1 -- 23 0 8 -- 10 -- 2878 960 *each
number represents the mean of three wells.
EXAMPLE 7
Injection of Polynucleotides Encoding Erythropoietin
A. Polynucleotides
[0368] CMVkm2 is the standard vector used in these studies. CMVkm2
is a vector optimized for expression in mammalian cells. The gene
of interest is cloned into a polylinker which is inserted 3' of a
human CMV expression cassette. This cassette contains the human CMV
immediate early promoter/enhancer followed by intron A of the human
CMV immediate early region (Chapman et al., 1991, Nucl. Acids Res.
19:3937-3986). Transcription is terminated by a polyadenylation
site from the bovine growth hormone gene, which has been cloned
immediately 3' of the polylinker. See SEQ ID NO:2 for the CMVkm2
vector.
[0369] The CMV-km-cmEPO vector was constructed from CMVkm2 as
follows. The cynomolgus monkey EPO cDNA was acquired from the ATCC
(Accession No. 67545, Rockville, Md.). This plasmid was cut with
AvrII and BglII and inserted into the XbaI and BamHI sites of the
CMVkm2 vector. The inserted sequence contains the entire coding
region of cmEPO (Genbank accession M18189). See SEQ ID NO:3.
B. Mice
[0370] Immunodeficient severe combined immunodeficiency (SCID) mice
were obtained from Charles River Labs, Wilmington, Mass., USA.
[0371] Intramuscular injections were performed as follows: mice
were anaesthetized with 50 .mu.l of a solution which contained 80
mg/ml ketamine and 4 mg/ml of xylazine. The area surrounding the
anterior tibialis muscle was shaved. Fifty .mu.l of DNA, at a
concentration of 2.7 ug/.mu.l in 0.9% saline solution was injected
into the anterior tibialis muscle of both legs using a 28 gauge
needle. Twenty-four hours after the first injection, a second
injection was performed using the identical protocol. Blood was
taken from the orbital sinus to determine hematocrits on a weekly
basis.
C. Result
[0372] The hematocrit readings on 6 mice which were injected with
plasmid, CMVkm-cmEpo (which expresses the cynomolgus monkeys EPO
cDNA), are shown in Table 3 below. The row marked control shows the
average reading for three uninjected mice. The raw data for the
three control mice is shown in the lower part of Table 3. Mouse 2
in the injected group died between 4 and 5 weeks post-injection.
TABLE-US-00006 TABLE 3 Hematocrit Levels (%) Week # Mouse Week 0
week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8 week 9 week
10 mouse 1 50 63 66.5 57.5 63 63.5 56.5 62.5 54 53.5 54.5 mouse 2
50 64 64 56.5 55.5 mouse 3 50 60 61.5 63 61 56 49.5 53 53.5 54.5 57
mouse 4 50 62 68.5 71.5 67.5 60 62.5 59.5 57.5 53.5 55.5 mouse 5 50
62 62.5 56 61 53.5 58 52.5 54 52.5 48.5 mouse 6 50 66 63.5 62.5 60
58 58 53.5 55.5 52.5 52.5 Control 50 51.5 48 47.5 53 49.5 49.5 50.5
51.5 51 49 control control control control control control control
control control control control week 0 wk 1 wk 2 wk 3 week 4 week 5
week 6 week 7 week 8 week 9 week 10 mouse 1 50 52 48 46.5 52.5 48
49.5 52 51.5 50.5 46.5 mouse 2 50 51 47 47 55 51 50.5 50 52 49 51.5
mouse 3 50 52 49 48.5 52 49.5 48.5 48.5 51 52.5 50
EXAMPLE 8
Injection of Polynucleotides Encoding Leptin
A. Polynucleotides
[0373] The CMV-km2 vector, described above, was used for these
experiments. Either the wild-type or HA version of the leptin
coding region was inserted into the vector. The map of the plasmid
is depicted in FIG. 4 and the sequence of the vector with the wild
type leptin is shown in SEQ ID NO:4.
B. Mice
[0374] Ob/ob mice were obtained from Jackson Labs, Bar Harbor, Me.,
USA. The first of the recessive obesity mutations, the obese
mutation (ob) was identified and described in 1950 by Ingall et
al., 1950, J. Hered. 41:317-318. Subsequently, 5 single-gene
mutations in mice have been observed to produce an obese phenotype,
as described in Friedman et al., 1990, Cell 69:217-220. (More
recently, the mouse obese gene and its human homologue have been
cloned, as described in Zhang et al., 1994, Nature 372:425).
C. Method
[0375] Intramuscular injections were performed as follows: mice
were anaesthetized with the same ketamine solution described above
in the Example 7 and the area surrounding the anterior tibialis
muscle was shaved.
[0376] Fifty microliters of DNA at a concentration of 3.3.
.mu.g/.mu.l in 0.9% saline solution was injected into the anterior
tibialis muscle of both legs using a 28 gauge needle.
[0377] Seventy-two hours after the first injection, a second
injection was performed using the identical protocol.
[0378] Group 1 ob/ob mice were injected with a plasmid (CMVkM
leptin-wt) which encodes the wild-type mouse leptin protein.
[0379] Group 2 ob/ob mice were injected with a plasmid
(CMVkm-leptinHA) which encodes a form of mouse leptin which is
modified with the epitope which is recognized by the antibody
12CA5. The amino acid sequence of the epitope is SYPYDVPDYASLGGPS
(Wilson et al., 1984, Cell 37: 767-778).
[0380] Group 3 ob/ob mice were injected with a solution of 0.9%
saline.
[0381] The mice were weighed each day (see Table 4) and the
proportional weight gain for each mouse during the first eight days
was calculated. The results are shown in Table 5. For any given
day, the weight was subtracted from the weight of the individual
mouse on day 0, and the difference was divided by the weight on day
0. The proportional weight change data from day 8 was analyzed
using an unpaired t-test. When compared with group 3 control mice
the p value from group 2 mice was 0.004. When compared with group 3
control mice, the p value for group 1 mice is 0.0038.
[0382] Note: the mice were not weighed on day 1 and day 2, the
values for these days were extrapolated from day 3. TABLE-US-00007
TABLE 4 Weight of Mice in Grams group 1 day 0 day 3 day 4 day 5 day
6 day 7 day 8 day 9 day 10 day 11 day 12 mouse 1 47 49 49 51 51 51
51 52 52 53 53 mouse 2 48 49 49 51 51 51 51 52 53 52 53 mouse 3 46
48 48 49 49 49 49 50 51 50 51 mouse 4 47 48 48 49 49 49 50 50 50 50
51 mouse 5 49 50 50 51 51 51 52 52 52 52 52 group 2 day 0 day 3 day
4 day 5 day 6 day 7 day 8 day 9 day 10 mouse 1 49 50 50 52 52 52 52
53 54 54 56 mouse 1 43 45 44 45 45 44 45 45 45 46 47 mouse 3 48 49
49 50 50 50 51 52 52 52 52 mouse 4 49 50 50 52 51 51 52 52 52 52 53
mouse 5 46 48 49 49 49 50 50 51 51 51 51 group 3 day 0 day 3 day 4
day 5 day 6 day 7 day 8 day 9 day 10 mouse 1 40 42 42 43 43 45 45
45 45 45 46 mouse 2 48 49 50 50 51 52 52 52 52 53 53 mouse 3 48 50
50 52 52 53 53 55 55 55 55 mouse 4 49 52 52 53 53 54 55 54 54 54 55
mouse 5 43 45 46 47 48 49 50 49 49 48 49
[0383] TABLE-US-00008 TABLE 5 Proportional Change in Weight from
Day 0 of Mice Injected with cDNA for Leptin (gp1), Leptin-HA (gp2)
or Saline day 0 day 1 day 2 day 3 day 4 day 5 day 6 day 7 day 8
Group 1 Mice Below Injected with CMVkM-Leptin-wt mouse 1 0 0.009
0.018 0.028 0.028 0.059 0.059 0.059 0.066 mouse 2 0 0.013 0.026
0.039 0.039 0.085 0.085 0.085 0.085 mouse 3 0 0.006 0.012 0.018
0.018 0.062 0.062 0.062 0.062 mouse 4 0 0.014 0.028 0.042 0.042
0.065 0.065 0.065 0.065 mouse 5 0 0.007 0.014 0.021 0.021 0.042
0.042 0.042 0.06 0 0.007 0.014 0.021 0.021 0.041 0.041 0.041 0.061
Group 2 Mice Below Injected with CMVkM-LeptinHA mouse 1 0 0.006
0.013 0.02 0.02 0.06 0.06 0.06 0.06 mouse 2 0 0.013 0.031 0.046
0.023 0.046 0.046 0.046 0.046 mouse 3 0 0.007 0.014 0.021 0.021
0.04 0.04 0.04 0.062 mouse 4 0 0.006 0.012 0.02 0.02 0.06 0.04 0.04
0.06 mouse 5 0 0.014 0.028 0.043 0.065 0.065 0.065 0.08 0.08 Group
3 Mice Below Injected with Saline mouse 1 0 0.016 0.032 0.05 0.05
0.06 0.06 0.125 0.125 mouse 2 0 0.07 0.014 0.021 0.04 0.04 0.06
0.08 0.08 mouse 3 0 0.013 0.026 0.04 0.04 0.08 0.08 0.1 0.1 mouse 4
0 0.02 0.04 0.06 0.06 0.08 0.08 0.1 0.12 mouse 5 0 0.015 0.03 0.045
0.069 0.09 0.12 0.14 0.14 Average Proportional change in Weight for
Each Group group 1 0 0.009 0.018 0.028 0.028 0.059 0.059 0.059
0.066 group 2 0 0.009 0.018 0.028 0.029 0.054 0.05 0.053 0.061
group 3 0 0.014 0.028 0.042 0.051 0.07 0.08 0.109 0.113
EXAMPLE 9
Peptoid Mediated in vitro Delivery in COS, HT1080, and 293 Cell
Lines
[0384] COS cells (available from the American Type Culture
Collection, Rockville, Md., under Accession No. CRL 1651 and HT1080
cells (available from the American Type Culture Collection,
Rockville, Md., under Accession No. CCL 121) were cultured and
transfected with pCMVkmLUC and various polycationic agents of the
present invention (described in Example 1) according to the
transfection protocol described in Example 4. Luciferase activity
was assayed according to the method described in Example 4. Total
cell protein was measured using a Pierce BCA kit according to
manufacturer's directions.
[0385] The results, shown in FIG. 7A, indicate that the ability of
the polycationic agents to mediate transfection is not dependent on
cell line type. Polycationic agents having a repeating trimer motif
of neutral and cationic sidechains were particularly effective at
mediating transfection.
[0386] Transfection efficiencies for a homologous series of
cationic peptoids were evaluated. Specifically, cationic peptoids
RZ-110-1 (18-mer), RZ-120-3 (24mer), RZ120-7 (36mer), and RZ120-11
(48mer), which have the same repeating (HHP) motif were evaluated
for their ability to transfect COS and HT1080 cells. These
polycationic agents were complexed with pCMVkmLUC at a 2:1, + to -
charge ratio. The concentration of negative charges on DNA was
calculated using 3.03 mmol of phosphate per 1 .mu.g of DNA, on the
basis of the average molecular weight of 330 for each nucleotide.
The formula weight of the polycationic agent was calculated as a
semi-trifluoroacetate salt (50% of amino groups form salt with
TFA), and the concentration of the polycationic agent was
determined on the basis of the weight of the lyophilized peptoid.
Amino groups were formally considered to be fully protonated to
obtain the number of positive charges on the polycationic agent
interest when calculating the + to - charge.
[0387] As shown in FIG. 7B, transfection efficiencies for this
particular series of cationic peptoids were largely independent of
oligomer length for peptoids having 24 or more monomeric units.
[0388] Transfection efficiencies using polycationic agent RZ145-1
and commercially available cationic lipids, DMRIE-C.TM.,
Lipofectin.RTM. and lipofectamine were evaluated. In these
experiments RZ145-1 was complexed with pCMVkmLUC at a 2:1, + to -
charge ratio. Transfection with DMRIE-C.TM. Lipofectin.RTM.,
lipofectamine was conducted according to manufacturer's directions.
The cationic lipids were also employed at a 2:1, + to - charge
ratio. 293 human embryonic kidney cells (Microbix, Toronto,
Ontario, Canada), HT1080 cells, and N1H-3T3 cells (available from
the American Type Culture Collection, Rockville, Md., Accession No.
CRL 1658) were transfected, cultured either in the presence or
absence of 10% serum, then assayed for luciferase production using
the same protocol as described in Example 4. Luciferase was
measured, as described in Example 4, 48 hours after initial
transfection. Total cell protein was measured using a Pierce BCA
kit according to manufacturer's directions.
[0389] The results, shown in FIG. 8, indicate that, in contrast to
Lipofectin.RTM. and lipofectamine, which were respectively 10- and
100-fold less efficient in the presence of serum, gene transfer
mediated by polycationic agent RZ145-1 was insensitive to the
presence of serum.
[0390] Transfection mediated by polycationic polymers, such as
polylysine and histones, is greatly enhanced by addition of
chloroquine to the transfection media. To determine whether
chloroquine affected transfection mediated by polycationic agents
of the present invention, HT1080 and 293 cells were transfected
using RZ145-1 in the presence and absence of chloroquine. As a
control, the same cell lines were transfected with polylysine both
in the presence and absence of chloroquine. The results, shown in
FIG. 9, indicate that the polycationic agent RZ145-1 was equally
effective at mediating transfection both with and without
chloroquine. In contrast, polylysine-mediated transfection in the
absence of chloroquine was 100-fold lower than polylysine mediated
transfection in the presence of chloroquine. In addition, the
results indicate that cationic peptoid mediated transfection is
more efficient than polylysine mediated transfection.
EXAMPLE 10
Preparation of a Stable Formulation of DNA/Polycationic Agent
Complex
A. DNA/Polycatinic Agent Complex Formation (2:1, + to - Change
Ratio)
[0391] All operations were carried out at ambient temperature. DGPW
(diagnosis grade purified water) was used to prepare the stock
solutions. Both the plycatinic agent and DNA samples had low salt
concentrations (i.e., <1 mM) to avoid precipitation.
[0392] (1) Batch Method
[0393] Complexes of polycationic agent RZ145-1 and pCMVkmLUC, as
follows, for up to 250 .mu.g DNA. DNA (i.e., pCMVkmLUC) was diluted
with 30% (v/v) ethanol in water to a concentration of 50 .mu.g/ml
corresponding to 151 .mu.M of negative charge. RZ145-1 was diluted
to 23.2 .mu.M in 30% ethanol in water. To 1 part of the
polycationic agent solution was added 1 part of DNA solution as
quickly as possible with gentle agitation. The DNA solution was
added to the solution of polycationic agent (rather than
vice-versa) to avoid precipitation. Slow addition of the two
solutions was avoided to avoid precipitation and the formation of
large complexes.
[0394] (2) Continuous Method
[0395] For more than 250 .mu.g of DNA, a continuous method for
preparing concentrated formulations of polycationic agent/DNA
complex is preferred. The DNA and peptoid solutions were prepared
as above and placed into separate bottles. Each bottle was
connected to one port of a mixing tee. The bottles were
simultaneously pressurized with 2 to 3 psi to deliver the two
streams to the mixing tee at the same flow rate (e.g., 20 ml/min or
higher).
B. Concentration Step
[0396] Two milliliters of the DNA-polycationic agent complex from
part A was placed in a Centricon.RTM.-100 (Amico Inc. Beverly,
Mass.), and centrifuged at 1000.times.g for 30 minutes or until the
volume of the retentate containing polycationic agentDNA complex
was approximately 50 .mu.L. The filtrate was removed from the
bottom receiver. The retentate was diluted with 2 ml of 5% glucose,
and concentrated to 50 .mu.l again. This operation was repeated
again to produce a concentrated complex solution containing 1 mg/ml
DNA in 5% glucose. This concentration step can be conducted at
either 4.degree. C. or at ambient temperature. The ethanol content
of the final concentrated solution was less than 0.1%. No
precipitation was observed in the concentrated solution.
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